# Patent

## Title
Compositions, methods and kits for diagnosis of lung cancer

## Abstract

Methods are provided for identifying biomarker proteins that exhibit differential expression in subjects with a first lung condition versus healthy subjects or subjects with a second lung condition. Also provided are compositions comprising these biomarker proteins and methods of using these biomarker proteins or panels thereof to diagnose, classify, and monitor various lung conditions. The methods and compositions provided herein may be used to diagnose or classify a subject as having lung cancer or a non-cancerous condition, and to distinguish between different types of cancer (e.g., malignant versus benign, SCLC versus NSCLC).

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 13/724,823 filed on Dec. 21, 2012 (now allowed), which claims priority and benefit of U.S. Provisional Application No. 61/578,712 filed Dec. 21, 2011, U.S. Provisional Application No. 61/589,920 filed Jan. 24, 2012, U.S. Provisional Application No. 61/676,859 filed Jul. 27, 2012 and U.S. Provisional Application No. 61/725,153 filed Nov. 12, 2012, the contents of each of which are incorporated herein by reference in their entireties.


INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
The contents of the text file named “44549-505001US_ST25.txt”, which was created on Feb. 25, 2013 and is 6 KB in size, are hereby incorporated by reference in their entireties.
BACKGROUND
Lung conditions and particularly lung cancer present significant diagnostic challenges. In many asymptomatic patients, radiological screens such as computed tomography (CT) scanning are a first step in the diagnostic paradigm. Pulmonary nodules (PNs) or indeterminate nodules are located in the lung and are often discovered during screening of both high risk patients or incidentally. The number of PNs identified is expected to rise due to increased numbers of patients with access to health care, the rapid adoption of screening techniques and an aging population. It is estimated that over 3 million PNs are identified annually in the US. Although the majority of PNs are benign, some are malignant leading to additional interventions. For patients considered low risk for malignant nodules, current medical practice dictates scans every three to six months for at least two years to monitor for lung cancer. The time period between identification of a PN and diagnosis is a time of medical surveillance or “watchful waiting” and may induce stress on the patient and lead to significant risk and expense due to repeated imaging studies. If a biopsy is performed on a patient who is found to have a benign nodule, the costs and potential for harm to the patient increase unnecessarily. Major surgery is indicated in order to excise a specimen for tissue biopsy and diagnosis. All of these procedures are associated with risk to the patient including: illness, injury and death as well as high economic costs.
Frequently, PNs cannot be biopsied to determine if they are benign or malignant due to their size and/or location in the lung. However, PNs are connected to the circulatory system, and so if malignant, protein markers of cancer can enter the blood and provide a signal for determining if a PN is malignant or not.
Diagnostic methods that can replace or complement current diagnostic methods for patients presenting with PNs are needed to improve diagnostics, reduce costs and minimize invasive procedures and complications to patients. The present invention provides novel compositions, methods and kits for identifying protein markers to identify, diagnose, classify and monitor lung conditions, and particularly lung cancer. The present invention uses a blood-based multiplexed assay to distinguish benign pulmonary nodules from malignant pulmonary nodules to classify patients with or without lung cancer. The present invention may be used in patients who present with symptoms of lung cancer, but do not have pulmonary nodules.
SUMMARY
The present invention provides a method of determining the likelihood that a lung condition in a subject is cancer by measuring an abundance of a panel of proteins in a sample obtained from the subject; calculating a probability of cancer score based on the protein measurements and ruling out cancer for the subject if the score) is lower than a pre-determined score, wherein When cancer is ruled out the subject does not receive a treatment protocol. Treatment protocols include for example pulmonary function test (PFT), pulmonary imaging, a biopsy, a surgery, a chemotherapy, a radiotherapy, or any combination thereof. In some embodiments, the imaging is an x-ray, a chest computed tomography (CT) scan, or a positron emission tomography (PET) scan.
The present invention further provides a method of ruling in the likelihood of cancer for a subject by measuring an abundance of panel of proteins in a sample obtained from the subject, calculating a probability of cancer score based on the protein measurements and ruling in the likelihood of cancer for the subject if the score in step is higher than a pre-determined score
In another aspect, the invention further provides a method of determining the likelihood of the presence of a lung condition in a subject by measuring an abundance of panel of proteins in a sample obtained from the subject, calculating a probability of cancer score based on the protein measurements and concluding the presence of said lung condition if the score is equal or greater than a pre-determined score. The lung condition is lung cancer such as for example, non-small cell lung cancer (NSCLC). The subject at risk of developing lung cancer
The panel includes at least 4 proteins selected from ALDOA, FRIL, LG3BP, IBP3, LRP1, ISLR, TSP COIA1, GRP78, TETN, PRXD1 and CD14. Optionally, the panel further includes at least one protein selected from BGH3, COIA1, TETN, GRP78, PRDX, FIBA and GSLG1.
The subject has or is suspected of having a pulmonary nodule. The pulmonary nodule has a diameter of less than or equal to 3 cm. In one embodiment, the pulmonary nodule has a diameter of about 0.8 cm to 2.0 cm.
The score is calculated from a logistic regression model applied to the protein measurements. For example, the score is determined as Ps=1/[1+exp(−α−Σi=1Nβi*{hacek over (I)}i,s)], where {hacek over (I)}i,s is logarithmically transformed and normalized intensity of transition i in said sample (s), βi is the corresponding logistic regression coefficient, a was a panel-specific constant, and N was the total number of transitions in said panel.
In various embodiments, the method of the present invention further comprises normalizing the protein measurements. For example, the protein measurements are normalized by one or more proteins selected from PEDF, MASP1, GELS, LUM, C163A and PTPRJ.
The biological sample such as for example tissue, blood, plasma, serum, whole blood, urine, saliva, genital secretion, cerebrospinal fluid, sweat and excreta.
In one aspect, the determining the likelihood of cancer is determined by the sensitivity, specificity, negative predictive value or positive predictive value associated with the score. The score determined has a negative predictive value (NPV) is at least about 80%.
The measuring step is performed by selected reaction monitoring mass spectrometry, using a compound that specifically binds the protein being detected or a peptide transition. In one embodiment, the compound that specifically binds to the protein being measured is an antibody or an aptamer.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a line graph showing area under the curve for a receiving operating curve for 15 protein LC-SRM-MS panels.
FIG. 2 shows six line graphs each showing area under the curve for a receiving operating curve for 15 protein LC-SRM-MS panels for different patient populations and for subjects with large and small PN
FIG. 3 is a graph showing variability among three studies used to evaluate 15 protein panels.
FIG. 4 is a line graph showing area under the curve for a receiving operating curve for a 15 protein LC-SRM-MS panel.
FIG. 5 shows three line graphs each showing area under the curve for a receiving operating curve for a 15 protein LC-SRM-MS panel for a different patient population.
FIG. 6 shows the results of a query of blood proteins used to identify lung cancer using the “Ingenuity”® program.
FIG. 7 is a bar diagram showing Pearson correlations for peptides from the same peptide, from the same protein and from different proteins.
FIG. 8 is a graph showing performance of the classifier on the training samples, validation samples and all samples combined.
FIG. 9 is a graph showing clinical and molecular factors.
FIG. 10 is a schematic showing the molecular network containing the 13 classifier proteins (green), 5 transcription factors (blue) and the three networks (orange lines) of lung cancer, response to oxidative stress and lung inflammation.
FIG. 11 is a graph depicting interpretation of classifier score in terms of risk



DETAILED DESCRIPTION
The disclosed invention derives from the surprising discovery, that in patients presenting with pulmonary nodule(s), protein markers in the blood exist that specifically identify and classify lung cancer. Accordingly the invention provides unique advantages to the patient associated with early detection of lung cancer in a patient, including increased life span, decreased morbidity and mortality, decreased exposure to radiation during screening and repeat screenings and a minimally invasive diagnostic model. Importantly, the methods of the invention allow for a patient to avoid invasive procedures.
The routine clinical use of chest computed tomography (CT) scans identifies millions of pulmonary nodules annually, of which only a small minority are malignant but contribute to the dismal 15% five-year survival rate for patients diagnosed with non-small cell lung cancer (NSCLC). The early diagnosis of lung cancer in patients with pulmonary nodules is a top priority, as decision-making based on clinical presentation, in conjunction with current non-invasive diagnostic options such as chest CT and positron emission tomography (PET) scans, and other invasive alternatives, has not altered the clinical outcomes of patients with Stage I NSCLC. The subgroup of pulmonary nodules between 8 mm and 20 mm in size is increasingly recognized as being “intermediate” relative to the lower rate of malignancies below 8 mm and the higher rate of malignancies above 20 mm [9]. Invasive sampling of the lung nodule by biopsy using transthoracic needle aspiration or bronchoscopy may provide a cytopathologic diagnosis of NSCLC, but are also associated with both false-negative and non-diagnostic results. In summary, a key unmet clinical need for the management of pulmonary nodules is a non-invasive diagnostic test that discriminates between malignant and benign processes in patients with indeterminate pulmonary nodules (IPNs), especially between 8 mm and 20 mm in size.
The clinical decision to be more or less aggressive in treatment is based on risk factors, primarily nodule size, smoking history and age [9] in addition to imaging. As these are not conclusive, there is a great need for a molecular-based blood test that would be both non-invasive and provide complementary information to risk factors and imaging.
Accordingly, these and related embodiments will find uses in screening methods for lung conditions, and particularly lung cancer diagnostics. More importantly, the invention finds use in determining the clinical management of a patient. That is, the method of invention are useful in ruling in or ruling out a particular treatment protocol for an individual subject.
Cancer biology requires a molecular strategy to address the unmet medical need for an assessment of lung cancer risk. The field of diagnostic medicine has evolved with technology and assays that provide sensitive mechanisms for detection of changes in proteins. The methods described herein use a LC-SRM-MS technology for measuring the concentration of blood plasma proteins that are collectively changed in patients with a malignant PN. This protein signature is indicative of lung cancer. LC-SRM-MS is one method that provides for both quantification and identification of circulating proteins in plasma. Changes in protein expression levels, such as but not limited to signaling factors, growth factors, cleaved surface proteins and secreted proteins, can be detected using such a sensitive technology to assay cancer. Presented herein is a blood-based classification test to determine the likelihood that a patient presenting with a pulmonary nodule has a nodule that is benign or malignant. The present invention presents a classification algorithm that predicts the relative likelihood of the PN being benign or malignant.
More broadly, it is demonstrated that there are many variations on this invention that are also diagnostic tests for the likelihood that a PN is benign or malignant. These are variations on the panel of proteins, protein standards, measurement methodology and/or classification algorithm.
As disclosed herein, archival plasma samples from subjects presenting with PNs were analyzed for differential protein expression by mass spectrometry and the results were used to identify biomarker proteins and panels of biomarker proteins that are differentially expressed in conjunction with various lung conditions (cancer vs. non-cancer).
In one aspect of the invention, one hundred and sixty three panels were discovered that allow for the classification of PN as being benign or malignant. These panels include those listed on Table 1. In some embodiments the panel according to the invention includes measuring 1, 2, 3, 4, 5 or more proteins selected from ISLR, ALDOA, KIT, GRP78, AIFM1, CD14, COIA1, IBP3, TSP1, BGH3, TETN, FRI, LG3BP, GGH, PRDX1 or LRP1. In other embodiments the panel includes any panel or protein exemplified on Table 1. For, example the panel includes ALDOA, GRP78, CD14, COIA1, IBP3, FRIL, LG3BP, and LRP1










TABLE 1







Iden-
Number
pAUC
Proteins
















tifier
Proteins
Factor
ISLR
ALDOA
KIT
GRP78
AIFM1
CD14
COIA1





1
9
4.562
0
1
0
1
0
1
1


2
8
4.488
0
1
0
1
0
1
1


3
11
4.451
1
1
0
1
0
0
1


4
11
4.357
1
1
0
1
0
0
1


5
11
4.331
1
1
0
0
0
1
1


6
13
4.324
1
1
0
0
0
1
1


7
10
4.205
1
1
0
1
0
0
1


8
11
4.193
1
1
0
0
0
0
1


9
12
4.189
1
1
0
1
0
0
1


10
12
4.182
1
0
0
0
0
1
1


11
12
4.169
1
1
0
1
0
0
1


12
8
4.107
1
1
0
1
0
1
1


13
13
4.027
0
1
1
1
0
1
1


14
10
3.994
0
1
1
1
0
1
1


15
11
3.979
1
1
1
1
0
1
1


16
10
3.932
1
1
0
1
0
1
1


17
11
3.926
1
1
0
0
0
1
1


18
12
3.913
1
0
1
1
0
0
1


19
12
3.872
0
1
1
1
0
1
1


20
12
3.864
1
1
1
0
0
1
1


21
14
3.853
1
1
0
1
0
1
1


22
9
3.849
1
1
0
1
0
0
1


23
12
3.846
1
1
1
1
0
0
1


24
10
3.829
0
1
1
1
0
1
0


25
10
3.829
0
1
1
1
0
1
1


26
12
3.826
1
0
0
0
1
0
1


27
7
3.804
1
1
0
1
0
1
1


28
10
3.802
0
1
0
1
0
1
1


29
10
3.787
0
1
0
1
0
1
0


30
9
3.779
1
1
0
1
0
1
1


31
11
3.774
0
1
0
1
0
1
1


32
8
3.759
1
1
0
0
0
0
1


33
13
3.758
1
1
0
0
0
1
1


34
11
3.757
1
1
0
1
0
0
0


35
12
3.754
0
1
1
1
0
1
1


36
10
3.750
1
1
0
1
0
1
1


37
11
3.747
0
1
1
1
0
1
1


38
12
3.744
1
0
1
1
0
0
1


39
11
3.742
1
1
0
1
0
1
1


40
9
3.740
1
1
0
1
0
1
1


41
12
3.740
1
1
1
1
0
1
1


42
12
3.739
1
1
0
1
0
1
1


43
9
3.734
1
1
0
0
0
0
1


44
12
3.730
1
1
0
1
0
0
1


45
11
3.725
0
1
1
1
0
1
1


46
12
3.717
0
1
0
0
1
1
1


47
9
3.713
0
1
0
1
0
1
1


48
9
3.713
1
1
1
1
0
1
1


49
10
3.709
0
1
0
1
0
1
1


50
11
3.709
1
1
0
1
0
1
1


51
11
3.701
0
1
1
1
1
1
1


52
12
3.685
1
1
0
1
0
1
1


53
10
3.680
0
0
0
1
0
1
0


54
11
3.676
1
1
1
1
0
0
1


55
9
3.668
0
1
0
1
0
1
1


56
9
3.659
0
0
0
1
0
1
0


57
14
3.657
1
1
0
1
1
1
1


58
10
3.655
1
1
0
1
0
0
1


59
11
3.643
0
1
1
1
0
1
1


60
9
3.643
0
1
0
1
0
1
0


61
8
3.640
1
1
0
1
0
1
0


62
12
3.640
1
1
1
1
0
1
1


63
10
3.638
1
1
0
1
0
0
1


64
12
3.633
1
0
0
1
1
0
1


65
10
3.632
1
1
0
1
0
1
1


66
11
3.627
1
1
0
1
0
1
0


67
10
3.627
1
1
0
0
0
1
0


68
10
3.623
1
1
1
0
0
0
1


69
11
3.619
1
0
0
1
0
1
1


70
6
3.617
1
1
0
1
0
0
1


71
12
3.617
1
0
0
1
0
1
1


72
11
3.613
1
1
0
1
0
1
0


73
11
3.608
1
1
0
1
0
1
0


74
13
3.608
1
1
1
1
0
1
1


75
11
3.605
0
1
1
1
0
1
1


76
11
3.602
0
1
1
1
0
1
1


77
10
3.600
1
1
0
1
0
0
0


78
11
3.596
1
1
0
1
0
0
1


79
10
3.592
1
1
0
1
0
1
0


80
11
3.587
1
0
1
0
0
0
1


81
13
3.584
1
1
0
1
1
1
1


82
8
3.584
0
1
0
1
0
1
0


83
11
3.581
1
1
1
1
0
1
0


84
13
3.578
1
1
0
1
0
1
0


85
9
3.573
1
1
1
0
0
1
1


86
9
3.572
1
1
0
1
0
0
1


87
13
3.571
1
1
1
1
0
1
0


88
10
3.569
1
1
0
1
0
0
1


89
9
3.569
0
1
0
1
0
1
0


90
8
3.559
0
1
0
1
0
1
0


91
10
3.558
0
1
0
1
0
1
0


92
12
3.554
1
1
0
1
0
1
1


93
11
3.552
0
1
0
1
0
1
0


94
12
3.549
0
1
0
1
0
1
0


95
8
3.547
1
1
1
0
0
1
1


96
12
3.545
1
1
1
1
0
1
1


97
8
3.542
1
1
1
0
0
0
0


98
11
3.536
1
1
1
1
0
0
1


99
14
3.530
1
1
1
1
0
1
1


100
9
3.527
1
1
0
1
0
1
1


101
10
3.522
0
1
1
0
1
1
1


102
12
3.509
1
1
0
1
0
1
1


103
5
3.505
0
1
0
0
0
1
0


104
11
3.500
1
1
0
0
1
0
1


105
11
3.497
1
1
1
1
0
0
1


106
9
3.491
1
1
0
0
0
1
0


107
7
3.489
0
1
1
0
0
1
0


108
13
3.486
1
1
1
1
0
1
1


109
11
3.483
1
1
1
1
0
0
1


110
10
3.477
1
1
1
1
0
1
1


111
10
3.473
1
1
0
0
0
1
1


112
15
3.468
1
1
0
1
1
1
1


113
10
3.467
0
1
0
0
1
1
0


114
12
3.467
1
1
0
0
1
1
1


115
13
3.467
1
1
0
1
1
0
1


116
10
3.467
0
1
0
1
0
1
0


117
8
3.465
1
1
0
1
0
0
1


118
10
3.464
0
1
0
1
1
1
1


119
15
3.464
1
1
0
1
1
1
1


120
11
3.462
1
1
0
1
0
1
1


121
9
3.460
1
1
0
0
0
1
0


122
13
3.453
1
1
0
1
0
1
1


123
12
3.449
1
1
1
0
0
1
0


124
10
3.448
1
1
0
1
0
1
0


125
10
3.445
0
1
1
1
0
1
0


126
6
3.441
0
1
0
0
0
1
0


127
11
3.440
1
1
0
1
0
1
0


128
12
3.440
1
1
0
1
1
0
0


129
11
3.439
1
1
0
1
0
1
0


130
10
3.426
0
1
0
0
1
1
0


131
11
3.423
1
1
0
0
0
0
1


132
10
3.420
1
1
0
0
0
1
0


133
10
3.419
1
1
1
1
0
1
0


134
11
3.417
1
1
0
1
1
0
1


135
12
3.414
0
1
0
1
1
1
1


136
10
3.413
0
1
1
1
0
1
0


137
11
3.400
0
1
0
0
1
1
0


138
12
3.398
1
1
0
1
0
1
0


139
13
3.396
1
1
0
1
0
1
0


140
9
3.386
1
1
0
0
0
1
0


141
9
3.373
1
1
0
1
0
1
0


142
12
3.363
1
1
0
0
1
0
1


143
8
3.362
0
1
0
1
0
1
0


144
10
3.360
1
1
0
1
0
1
1


145
9
3.359
1
1
1
0
0
1
0


146
7
3.349
0
1
0
0
0
0
0


147
7
3.348
1
1
0
0
0
1
1


148
9
3.340
1
0
0
0
0
1
0


149
9
3.335
1
1
0
1
0
1
0


150
11
3.333
0
1
1
1
0
1
0


151
9
3.333
0
0
0
1
0
1
0


152
10
3.328
1
1
0
1
0
1
0


153
7
3.315
0
1
0
1
0
1
0


154
11
3.311
1
1
0
1
1
1
1


155
11
3.293
1
1
0
1
0
1
0


156
8
3.292
1
1
0
1
0
0
0


157
9
3.289
0
1
0
1
0
1
0


158
7
3.229
0
1
0
0
0
1
0


159
7
3.229
1
1
0
0
0
1
0


160
7
3.203
1
1
0
1
0
0
0


161
12
3.161
1
1
1
0
1
1
0


162
9
3.138
1
1
0
0
1
0
1


163
13
3.078
1
1
0
0
1
0
1













Iden-
Proteins


















tifier
IBP3
TSP1
BGH3
TETN
FRIL
LG3BP
GGH
PRDX1
LRP1






1
1
0
0
0
1
1
0
0
1



2
1
0
0
0
1
1
0
0
1



3
1
1
1
1
1
0
0
1
1



4
1
1
0
0
1
1
1
1
1



5
0
1
1
1
1
0
1
1
1



6
1
1
1
1
1
1
1
1
1



7
0
1
1
1
1
0
0
1
1



8
0
1
1
1
1
0
1
1
1



9
1
1
1
1
1
0
0
1
1



10
1
1
1
1
1
1
0
1
1



11
1
1
0
0
1
1
1
1
1



12
0
0
0
0
1
1
0
0
1



13
1
1
0
0
1
1
1
1
1



14
1
0
0
0
1
1
0
0
1



15
0
0
0
0
1
1
1
0
1



16
0
0
0
1
1
1
0
0
1



17
1
1
1
1
1
0
0
1
1



18
1
1
0
0
1
1
1
1
1



19
1
0
0
0
1
1
1
1
1



20
0
1
1
1
1
1
0
1
1



21
1
1
1
1
1
1
0
1
1



22
0
1
1
1
1
0
0
0
1



23
1
1
0
0
1
1
1
1
1



24
1
0
0
0
1
1
1
1
1



25
1
0
0
0
1
1
1
0
1



26
1
1
1
1
1
0
1
1
1



27
0
0
0
0
0
1
0
0
1



28
1
0
0
0
1
1
1
1
1



29
1
1
0
0
1
1
1
1
1



30
0
0
0
0
1
1
0
0
1



31
1
0
0
0
1
1
1
1
1



32
0
0
1
1
1
0
0
1
1



33
1
1
1
1
1
1
0
1
1



34
1
1
1
1
1
1
0
1
1



35
1
1
0
0
1
1
1
1
1



36
1
0
0
0
1
1
0
1
1



37
1
1
0
0
1
1
1
1
0



38
1
1
1
1
1
0
0
1
1



39
1
1
0
1
1
1
0
0
1



40
1
0
0
0
1
1
0
0
1



41
1
0
0
1
1
1
0
0
1



42
1
1
0
0
1
1
1
1
1



43
0
1
1
1
1
0
0
1
1



44
1
1
1
1
1
1
0
1
1



45
1
0
0
1
1
1
0
0
1



46
1
1
1
1
1
1
1
1
0



47
1
0
0
0
1
1
0
1
1



48
0
0
0
0
1
1
0
0
1



49
1
0
0
0
1
1
1
0
1



50
0
1
1
1
1
1
0
0
1



51
1
0
0
0
1
1
0
0
1



52
1
1
1
1
1
1
0
0
1



53
1
1
1
1
1
1
0
1
1



54
0
1
1
1
1
0
0
1
1



55
1
0
0
0
1
1
1
0
1



56
1
1
0
0
1
1
1
1
0



57
1
1
1
1
1
0
0
1
1



58
0
1
0
0
1
1
1
0
1



59
1
0
0
0
1
1
1
1
1



60
1
0
1
0
1
1
0
0
1



61
1
0
0
0
1
1
0
0
1



62
0
0
0
1
1
1
0
1
1



63
0
1
1
1
1
1
0
0
1



64
1
1
1
1
1
0
0
1
1



65
1
0
0
0
1
1
0
0
1



66
1
1
1
1
1
1
0
0
1



67
1
1
1
1
1
1
0
0
1



68
0
1
1
1
1
1
0
0
1



69
1
1
1
0
1
1
0
0
1



70
0
0
0
0
0
1
0
0
1



71
1
1
1
1
1
0
0
1
1



72
1
1
0
0
1
1
1
1
1



73
1
1
1
0
1
1
0
1
1



74
1
1
0
0
1
1
0
1
1



75
1
0
0
0
1
1
0
1
1



76
1
0
0
0
1
1
1
0
1



77
1
1
1
1
1
1
0
1
0



78
1
1
1
1
1
0
1
0
1



79
1
1
0
0
1
1
0
1
1



80
1
1
1
1
0
1
0
1
1



81
1
1
1
1
1
1
0
0
1



82
1
1
0
0
1
1
0
1
0



83
1
1
0
0
1
1
1
1
0



84
1
1
1
1
1
1
0
1
1



85
1
0
0
0
1
1
0
0
0



86
0
1
0
0
1
1
0
0
1



87
1
1
0
0
1
1
1
1
1



88
1
1
0
1
1
0
0
1
1



89
1
1
0
0
1
1
0
1
1



90
1
0
0
0
1
1
0
0
1



91
1
0
0
1
1
1
1
1
1



92
0
1
1
1
1
0
1
1
1



93
1
1
0
0
1
1
1
1
1



94
1
1
1
1
1
1
1
1
1



95
1
1
0
0
0
1
0
0
0



96
1
0
0
0
1
1
1
0
1



97
1
1
0
1
0
1
0
0
0



98
1
0
0
0
1
1
1
1
1



99
1
1
0
1
1
1
1
1
0



100
0
1
0
0
1
1
0
0
1



101
1
1
0
0
1
1
0
1
0



102
0
0
1
1
1
1
0
1
1



103
1
1
0
0
0
1
0
0
0



104
1
1
1
1
1
0
1
1
0



105
1
1
0
0
1
1
0
0
1



106
1
1
0
0
0
1
1
1
0



107
1
1
0
0
0
1
0
1
0



108
1
0
0
1
1
1
0
1
1



109
1
0
0
0
1
1
1
0
1



110
1
0
0
0
1
1
0
0
1



111
0
0
1
1
1
1
0
0
1



112
1
1
1
1
1
0
1
1
1



113
1
1
1
1
1
1
0
1
0



114
1
1
1
1
0
1
0
1
1



115
1
1
1
1
1
0
0
1
1



116
1
1
0
0
1
1
1
0
1



117
0
1
0
0
1
1
0
0
1



118
1
0
0
0
1
1
0
0
1



119
1
1
1
1
1
1
1
1
0



120
0
0
0
1
1
1
0
1
1



121
1
1
1
1
0
1
0
1
0



122
1
1
1
1
1
1
1
1
0



123
1
1
0
1
1
1
1
1
0



124
1
1
0
0
1
1
1
1
0



125
1
1
0
0
1
1
0
1
1



126
1
1
0
0
0
1
0
0
0



127
1
1
0
0
1
1
1
0
1



128
1
1
1
1
1
0
0
1
1



129
1
0
0
0
1
1
1
1
1



130
1
1
1
1
0
1
0
1
0



131
1
1
1
1
1
1
1
1
0



132
1
1
0
1
1
1
1
1
0



133
1
0
0
0
1
1
0
0
1



134
0
0
1
1
1
0
0
1
1



135
1
1
0
1
1
1
0
0
1



136
1
1
0
0
1
1
0
1
0



137
1
1
1
1
1
1
0
1
0



138
1
0
1
1
1
1
1
1
1



139
1
1
1
1
1
1
1
1
1



140
1
1
0
0
1
1
1
1
0



141
1
0
0
0
1
1
0
0
1



142
1
1
1
1
1
1
1
1
0



143
1
0
0
0
1
1
0
1
1



144
0
0
0
1
1
1
0
1
0



145
1
1
0
0
1
1
0
0
0



146
1
1
1
1
0
1
0
0
0



147
1
1
0
0
0
1
0
0
0



148
1
1
1
1
0
1
0
1
0



149
1
1
0
0
1
1
0
0
1



150
1
1
0
0
1
1
0
1
1



151
1
1
1
0
1
1
0
0
1



152
1
0
0
0
1
1
1
0
1



153
1
0
0
0
1
1
0
0
1



154
0
0
0
1
1
1
1
0
0



155
1
0
1
0
1
1
0
1
1



156
1
1
0
0
1
1
0
0
1



157
1
1
0
0
1
1
0
1
0



158
1
1
0
0
1
1
0
0
0



159
1
1
0
0
0
1
0
1
0



160
1
0
0
0
1
1
0
0
1



161
1
1
1
1
1
1
0
1
0



162
0
0
1
1
1
1
0
0
0



163
1
1
1
1
1
1
1
1
0





1 = in the panel; 0 = not in the panel.






The one hundred best random panels of proteins out of the million generated are shown in Table 2.

















TABLE 2






Protein 1
Protein 2
Protein 3
Protein 4
Protein 5
Protein 6
Protein 7
Protein 8
Protein 9
Protein 10

























1
IBP3
TSP1
CO6A3
PDIA3
SEM3G
SAA
6PGD
EF1A1
PRDX1
TERA


2
EPHB6
CNTN1
CLUS
IBP3
BGH3
6PGD
FRIL
LRP1
TBB3
ERO1A


3
PPIB
LG3BP
MDHC
DSG2
BST1
CD14
DESP
PRDX1
CDCP1
MMP9


4
TPIS
COIA1
IBP3
GGH
ISLR
MMP2
AIFM1
DSG2
1433T
CBPB2


5
TPIS
IBP3
CH10
SEM3G
6PGD
FRIL
ICAM3
TERA
FINC
ERO1A


6
BGH3
ICAM1
MMP12
6PGD
CD14
EF1A1
HYOU1
PLXC1
PROF1
ERO1A


7
KIT
LG3BP
TPIS
IBP3
LDHB
GGH
TCPA
ISLR
CBPB2
EF1A1


8
LG3BP
IBP3
LDHB
TSP1
CRP
ZA2G
CD14
LRP1
PLIN2
ERO1A


9
COIA1
TSP1
ISLR
TFR1
CBPB2
FRIL
LRP1
UGPA
PTPA
ERO1A


10
CO6A3
SEM3G
APOE
FRIL
ICAM3
PRDX1
EF2
HS90B
NCF4
PTPA


11
PPIB
LG3BP
COIA1
APOA1
DSG2
APOE
CD14
PLXC1
NCF4
GSLG1


12
SODM
EPHB6
C163A
COIA1
LDHB
TETN
1433T
CD14
PTPA
ERO1A


13
SODM
KPYM
IBP3
TSP1
BGH3
SEM3G
6PGD
CD14
RAP2B
EREG


14
EPHB6
ALDOA
MMP7
COIA1
TIMP1
GRP78
MMP12
CBPB2
G3P
PTPA


15
KIT
TSP1
SCF
TIMP1
OSTP
PDIA3
GRP78
TNF12
PRDX1
PTPA


16
IBP2
LG3BP
GELS
HPT
FIBA
GGH
ICAM1
BST1
HYOU1
GSLG1


17
KIT
CD44
CH10
PEDF
ICAM1
6PGD
S10A1
ERO1A
GSTP1
MMP9


18
LG3BP
C163A
GGH
ERBB3
TETN
BGH3
ENOA
GDIR2
LRP1
ERO1A


19
SODM
KPYM
BGH3
FOLH1
6PGD
DESP
LRP1
TBA1B
ERO1A
GSTP1


20
CNTN1
TETN
ICAM1
K1C19
ZA2G
6PGD
EF2
RAN
ERO1A
GSTP1


21
GELS
ENPL
OSTP
PEDF
ICAM1
BST1
TNF12
GDIR2
LRP1
ERO1A


22
KIT
LDHA
IBP3
PEDF
DSG2
FOLH1
CD14
LRP1
UGPA
ERO1A


23
KIT
TSP1
ISLR
BGH3
COF1
PTPRJ
6PGD
LRP1
S10A6
MPRI


24
LG3BP
C163A
GGH
DSG2
ICAM1
6PGD
GDIR2
HYOU1
EREG
ERO1A


25
IBP2
C163A
ENPL
FIBA
BGH3
CERU
6PGD
LRP1
PRDX1
MMP9


26
LG3BP
C163A
TENX
PDIA3
SEM3G
BST1
VTNC
FRIL
PRDX1
ERO1A


27
ALDOA
COIA1
TETN
1433T
CBPB2
CD14
G3P
CD59
ERO1A
MMP9


28
IBP3
TENX
CRP
TETN
MMP2
SEM3G
VTNC
CD14
PROF1
ERO1A


29
SODM
EPHB6
TPIS
TENX
ERBB3
SCF
TETN
FRIL
LRP1
ERO1A


30
LG3BP
IBP3
POSTN
DSG2
MDHM
1433Z
CD14
EF1A1
PLXC1
ERO1A


31
IBP2
LG3BP
COIA1
CNTN1
IBP3
POSTN
TETN
BGH3
6PGD
ERO1A


32
PVR
TSP1
GGH
CYTB
AIFM1
ICAM1
MDHM
1433Z
6PGD
FRIL


33
LYOX
GELS
COIA1
IBP3
AIFM1
ICAM1
FRIL
PRDX1
RAP2B
NCF4


34
KIT
AMPN
TETN
TNF12
6PGD
FRIL
LRP1
EF2
ERO1A
MMP9


35
LG3BP
GELS
COIA1
CLUS
CALU
AIFM1
1433T
CD14
UGPA
S10A1


36
ALDOA
IBP3
TSP1
TETN
SEM3G
ICAM1
EF1A1
G3P
RAP2B
NCF4


37
ALDOA
COIA1
CH10
TETN
PTPRJ
SEM3G
1433T
6PGD
FRIL
ERO1A


38
LG3BP
COIA1
PLSL
FIBA
TENX
POSTN
CD14
LRP1
NCF4
ERO1A


39
LUM
IBP3
CH10
AIFM1
MDHM
6PGD
PLXC1
EF2
CD59
GSTP1


40
SODM
LG3BP
LUM
LDHA
MDHC
GGH
ICAM1
LRP1
TBA1B
ERO1A


41
LG3BP
CD44
IBP3
CALU
CERU
1433T
CD14
CLIC1
NCF4
ERO1A


42
LG3BP
TPIS
COIA1
HPT
FIBA
AIFM1
1433Z
6PGD
CD14
EF2


43
ALDOA
CD44
MMP2
CD14
FRIL
PRDX1
RAN
NCF4
MPRI
PTPA


44
COIA1
CLUS
OSTP
ICAM1
1433T
PLXC1
PTGIS
RAP2B
PTPA
GSTP1


45
KIT
LYOX
IBP3
GRP78
FOLH1
MASP1
CD14
LRP1
ERO1A
GSTP1


46
LG3BP
GGH
CRP
SCF
ICAM1
ZA2G
1433T
RAN
NCF4
ERO1A


47
LG3BP
C163A
BGH3
MMP2
GRP78
LRP1
RAN
ITA5
HS90B
PTPA


48
ALDOA
CLUS
TENX
ICAM1
K1C19
MASP1
6PGD
CBPB2
PRDX1
PTPA


49
IBP3
PDIA3
PEDF
FOLH1
ICAM1
NRP1
6PGD
UGPA
RAN
ERO1A


50
ENPL
FIBA
ISLR
SAA
6PGD
PRDX1
EF2
PLIN2
HS90B
GSLG1


51
LG3BP
COIA1
CO6A3
GGH
ERBB3
FOLH1
ICAM1
RAN
CDCP1
ERO1A


52
GELS
ENPL
A1AG1
SCF
COF1
ICAM1
6PGD
RAP2B
EF2
HS90B


53
SODM
IBP2
COIA1
CLUS
IBP3
ENPL
PLSL
TNF12
6PGD
ERO1A


54
KIT
MMP7
COIA1
TSP1
CO6A3
GGH
PDIA3
ICAM1
LRP1
GSLG1


55
ALDOA
COIA1
TSP1
CH10
NRP1
CD14
DESP
LRP1
CLIC1
ERO1A


56
C163A
GELS
CALU
A1AG1
AIFM1
DSG2
ICAM1
6PGD
RAP2B
NCF4


57
PPIB
LG3BP
IBP3
TSP1
PLSL
GRP78
FOLH1
6PGD
HYOU1
RAP2B


58
KIT
LG3BP
LUM
GELS
OSTP
ICAM1
CD14
EF1A1
NCF4
MMP9


59
KIT
PPIB
LG3BP
GELS
FOLH1
ICAM1
MASP1
GDIR2
ITA5
NCF4


60
IBP3
ENPL
ERBB3
BGH3
VTNC
6PGD
EF1A1
TBA1B
S10A6
HS90B


61
LG3BP
CLUS
IBP3
SCF
TCPA
ISLR
GRP78
6PGD
ERO1A
GSTP1


62
LG3BP
LEG1
GELS
GGH
TETN
ENOA
ICAM1
MASP1
FRIL
NCF4


63
LG3BP
CD44
TETN
BGH3
G3P
LRP1
PRDX1
CDCP1
PTPA
MMP9


64
CALU
ENPL
ICAM1
VTNC
FRIL
LRP1
PROF1
TBB3
GSLG1
ERO1A


65
PPIB
PLSL
TENX
A1AG1
COF1
6PGD
FRIL
LRP1
CLIC1
ERO1A


66
IBP2
IBP3
CERU
ENOA
6PGD
CD14
LRP1
PDGFB
ERO1A
GSTP1


67
COIA1
1433T
CD14
DESP
GDIR2
PLXC1
PROF1
RAP2B
RAN
ERO1A


68
LYOX
OSTP
TETN
SEM3G
ICAM1
ZA2G
FRIL
EREG
RAN
ERO1A


69
LG3BP
IBP3
TSP1
PEDF
FOLH1
MDHM
TNF12
NRP1
S10A6
RAP2B


70
KIT
ALDOA
LG3BP
COIA1
TSP1
A1AG1
BGH3
SEM3G
FOLH1
RAN


71
ALDOA
OSTP
BST1
CD14
G3P
PRDX1
PTGIS
FINC
PTPA
MMP9


72
EPHB6
TETN
PEDF
ICAM1
APOE
PROF1
UGPA
NCF4
GSLG1
PTPA


73
LG3BP
COIA1
ENPL
MMP2
1433T
EF1A1
LRP1
HS90B
GSLG1
ERO1A


74
KIT
IBP3
CYTB
MMP2
1433Z
6PGD
CLIC1
EF2
NCF4
PTPA


75
SODM
LYOX
IBP3
TETN
SEM3G
CD14
PRDX1
PTPA
ERO1A
GSTP1


76
SODM
KPYM
COIA1
MDHC
TCPA
CD14
FRIL
LRP1
EF2
ERO1A


77
PPIB
LG3BP
FIBA
GRP78
AIFM1
ICAM1
6PGD
NCF4
GSLG1
PTPA


78
LG3BP
C163A
PVR
MDHC
TETN
SEM3G
AIFM1
6PGD
EREG
ERO1A


79
GELS
ISLR
BGH3
DSG2
ICAM1
SAA
HYOU1
ICAM3
PTGIS
RAP2B


80
KPYM
TPIS
IBP3
TIMP1
GRP78
ICAM1
LRP1
TERA
ERO1A
MMP9


81
IBP3
HPT
TSP1
GRP78
SAA
MMP12
1433Z
6PGD
CD14
S10A6


82
TENX
A1AG1
ENOA
AIFM1
6PGD
CD14
FRIL
LRP1
RAP2B
CD59


83
ALDOA
KPYM
ISLR
TETN
BGH3
VTNC
LRP1
ITA5
PTPA
MMP9


84
SODM
TENX
ISLR
TETN
VTNC
6PGD
LRP1
EF2
ERO1A
MMP9


85
LG3BP
C163A
COIA1
FOLH1
CD14
LRP1
TBA1B
GSLG1
ERO1A
GSTP1


86
SODM
PVR
COIA1
ISLR
PDIA3
APOE
CD14
FRIL
LRP1
CDCP1


87
ALDOA
PEDF
ICAM1
6PGD
CD14
FINC
RAN
NCF4
GSLG1
PTPA


88
LG3BP
KPYM
GELS
COIA1
IBP3
CD14
EF1A1
PLIN2
HS90B
ERO1A


89
LG3BP
PVR
CLUS
TETN
COF1
SEM3G
DESP
EF2
HS90B
ERO1A


90
LG3BP
COIA1
FIBA
TETN
TFR1
ICAM1
MDHM
CD14
PLXC1
ERO1A


91
PPIB
LG3BP
GELS
CLUS
TENX
ICAM1
SAA
NCF4
PTPA
ERO1A


92
COIA1
TSP1
ISLR
BGH3
SAA
6PGD
LRP1
PROF1
EREG
ERO1A


93
CALU
FIBA
OSTP
ISLR
PDIA3
SEM3G
K1C19
6PGD
HYOU1
RAP2B


94
FIBA
CH10
GRP78
SEM3G
AIFM1
ICAM1
MDHM
FRIL
UGPA
GSTP1


95
COIA1
IBP3
PDIA3
ICAM1
K1C19
CD14
EF1A1
FRIL
PTGIS
PDGFB


96
LG3BP
C163A
COIA1
LDHA
1433T
1433Z
FRIL
LRP1
ERO1A
MMP9


97
LG3BP
GELS
COIA1
GRP78
SEM3G
FRIL
PLXC1
PROF1
S10A1
ERO1A


98
LG3BP
COIA1
ENPL
GRP78
AIFM1
ICAM1
1433Z
CD14
LRP1
ERO1A


99
COIA1
PLSL
NRP1
1433T
CD14
FRIL
LRP1
RAP2B
PDGFB
ERO1A


100
IBP2
COIA1
TETN
DSG2
FOLH1
1433T
CD14
FRIL
LRP1
ERO1A









Preferred panels for ruling in treatment for a subject include the panels listed on Table 3 and 4. In various other embodiments, the panels according to the invention include measuring at least 2, 3, 4, 5, 6, 7, or more of the proteins listed on Tables 2 and 3.










TABLE 3






Average (19)
Rule-out (20)
Rule-in (16)








ERO1A
ERO1A
ERO1A



6PGD
6PGD
6PGD



FRIL
FRIL
FRIL



GSTP1
GSTP1
GSTP1



COIA1
COIA1
COIA1



GGH
GGH
GGH



PRDX1
PRDX1
PRDX1



LRP1
CD14
SEM3G



ICAM1
LRP1
GRP78



CD14
LG3BP
TETN



LG3BP
PTPA
AIFM1



PTPA
ICAM1
TSP1



TETN
TSP1
MPRI



GRP78
IBP3
TNF12



AIFM1
FOLH1
MMP9



SEM3G
SODM
OSTP



BGH3
FIBA




PDIA3
GSLG1




FINC
RAP2B





C163A



















TABLE 4






Average (13)
Rule-out (13)
Rule-in (9)








LRP1
LRP1 (
LRP1



BGH3
COIA1
COIA1



COIA1
TETN
TETN



TETN
TSP1
TSP1



TSP1
ALDOA
ALDOA



PRDX1
GRP78
GRP78



PROF1
FRIL
FRIL



GRP78
LG3BP
APOE



FRIL
BGH3
TBB3



LG3BP
ISLR




CD14
PRDX1




GGH
FIBA




AIFM1
GSLG1









A preferred normalizer panel is listed in Table 5.







TABLE 5





Normalizer (6)

















PEDF



MASP1



GELS



LUM



C163A



PTPRJ









The term “pulmonary nodules” (PNs) refers to lung lesions that can be visualized by radiographic techniques. A pulmonary nodule is any nodules less than or equal to three centimeters in diameter. In one example a pulmonary nodule has a diameter of about 0.8 cm to 2 cm.
The term “masses” or “pulmonary masses” refers to lung nodules that are greater than three centimeters maximal diameter.
The term “blood biopsy” refers to a diagnostic study of the blood to determine whether a patient presenting with a nodule has a condition that may be classified as either benign or malignant.
The term “acceptance criteria” refers to the set of criteria to which an assay, test, diagnostic or product should conform to be considered acceptable for its intended use. As used herein, acceptance criteria are a list of tests, references to analytical procedures, and appropriate measures, which are defined for an assay or product that will be used in a diagnostic. For example, the acceptance criteria for the classifier refers to a set of predetermined ranges of coefficients.
The term “average maximal AUC” refers to the methodology of calculating performance. For the present invention, in the process of defining the set of proteins that should be in a panel by forward or backwards selection proteins are removed or added one at a time. A plot can be generated with performance (AUC or partial AUC score on the Y axis and proteins on the X axis) the point which maximizes performance indicates the number and set of proteins the gives the best result.
The term “partial AUC factor or pAUC factor” is greater than expected by random prediction. At sensitivity=0.90 the pAUC factor is the trapezoidal area under the ROC curve from 0.9 to 1.0 Specificity/(0.1*0.1/2).
The term “incremental information” refers to information that may be used with other diagnostic information to enhance diagnostic accuracy. Incremental information is independent of clinical factors such as including nodule size, age, or gender.
The term “score” or “scoring” refers to the refers to calculating a probability likelihood for a sample. For the present invention, values closer to 1.0 are used to represent the likelihood that a sample is cancer, values closer to 0.0 represent the likelihood that a sample is benign.
The term “robust” refers to a test or procedure that is not seriously disturbed by violations of the assumptions on which it is based. For the present invention, a robust test is a test wherein the proteins or transitions of the mass spectrometry chromatograms have been manually reviewed and are “generally” free of interfering signals
The term “coefficients” refers to the weight assigned to each protein used to in the logistic regression equation to score a sample.
In certain embodiments of the invention, it is contemplated that in terms of the logistic regression model of MC CV, the model coefficient and the coefficient of variation (CV) of each protein's model coefficient may increase or decrease, dependent upon the method (or model) of measurement of the protein classifier. For each of the listed proteins in the panels, there is about, at least, at least about, or at most about a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-, -fold or any range derivable therein for each of the coefficient and CV. Alternatively, it is contemplated that quantitative embodiments of the invention may be discussed in terms of as about, at least, at least about, or at most about 10, 20, 30, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more, or any range derivable therein.
The term “best team players” refers to the proteins that rank the best in the random panel selection algorithm, i.e., perform well on panels. When combined into a classifier these proteins can segregate cancer from benign samples. “Best team player” proteins is synonymous with “cooperative proteins”. The term “cooperative proteins” refers proteins that appear more frequently on high performing panels of proteins than expected by chance. This gives rise to a protein's cooperative score which measures how (in)frequently it appears on high performing panels. For example, a protein with a cooperative score of 1.5 appears on high performing panels 1.5× more than would be expected by chance alone.
The term “classifying” as used herein with regard to a lung condition refers to the act of compiling and analyzing expression data for using statistical techniques to provide a classification to aid in diagnosis of a lung condition, particularly lung cancer.
The term “classifier” as used herein refers to an algorithm that discriminates between disease states with a predetermined level of statistical significance. A two-class classifier is an algorithm that uses data points from measurements from a sample and classifies the data into one of two groups. In certain embodiments, the data used in the classifier is the relative expression of proteins in a biological sample. Protein expression levels in a subject can be compared to levels in patients previously diagnosed as disease free or with a specified condition.
The “classifier” maximizes the probability of distinguishing a randomly selected cancer sample from a randomly selected benign sample, i.e., the AUC of ROC curve.
In addition to the classifier's constituent proteins with differential expression, it may also include proteins with minimal or no biologic variation to enable assessment of variability, or the lack thereof, within or between clinical specimens; these proteins may be termed endogenous proteins and serve as internal controls for the other classifier proteins.
The term “normalization” or “normalizer” as used herein refers to the expression of a differential value in terms of a standard value to adjust for effects which arise from technical variation due to sample handling, sample preparation and mass spectrometry measurement rather than biological variation of protein concentration in a sample. For example, when measuring the expression of a differentially expressed protein, the absolute value for the expression of the protein can be expressed in terms of an absolute value for the expression of a standard protein that is substantially constant in expression. This prevents the technical variation of sample preparation and mass spectrometry measurement from impeding the measurement of protein concentration levels in the sample.
The term “condition” as used herein refers generally to a disease, event, or change in health status.
The term “treatment protocol” as used herein including further diagnostic testing typically performed to determine whether a pulmonary nodule is benign or malignant. Treatment protocols include diagnostic tests typically used to diagnose pulmonary nodules or masses such as for example, CT scan, positron emission tomography (PET) scan, bronchoscopy or tissue biopsy. Treatment protocol as used herein is also meant to include therapeutic treatments typically used to treat malignant pulmonary nodules and/or lung cancer such as for example, chemotherapy, radiation or surgery.
The terms “diagnosis” and “diagnostics” also encompass the terms “prognosis” and “prognostics”, respectively, as well as the applications of such procedures over two or more time points to monitor the diagnosis and/or prognosis over time, and statistical modeling based thereupon. Furthermore the term diagnosis includes: a. prediction (determining if a patient will likely develop a hyperproliferative disease) b. prognosis (predicting whether a patient will likely have a better or worse outcome at a pre-selected time in the future) c. therapy selection d. therapeutic drug monitoring e. relapse monitoring.
In some embodiments, for example, classification of a biological sample as being derived from a subject with a lung condition may refer to the results and related reports generated by a laboratory, while diagnosis may refer to the act of a medical professional in using the classification to identify or verify the lung condition.
The term “providing” as used herein with regard to a biological sample refers to directly or indirectly obtaining the biological sample from a subject. For example, “providing” may refer to the act of directly obtaining the biological sample from a subject (e.g., by a blood draw, tissue biopsy, lavage and the like). Likewise, “providing” may refer to the act of indirectly obtaining the biological sample. For example, providing may refer to the act of a laboratory receiving the sample from the party that directly obtained the sample, or to the act of obtaining the sample from an archive.
As used herein, “lung cancer” preferably refers to cancers of the lung, but may include any disease or other disorder of the respiratory system of a human or other mammal. Respiratory neoplastic disorders include, for example small cell carcinoma or small cell lung cancer (SCLC), non-small cell carcinoma or non-small cell lung cancer (NSCLC), squamous cell carcinoma, adenocarcinoma, broncho-alveolar carcinoma, mixed pulmonary carcinoma, malignant pleural mesothelioma, undifferentiated large cell carcinoma, giant cell carcinoma, synchronous tumors, large cell neuroendocrine carcinoma, adenosquamous carcinoma, undifferentiated carcinoma; and small cell carcinoma, including oat cell cancer, mixed small cell/large cell carcinoma, and combined small cell carcinoma; as well as adenoid cystic carcinoma, hamartomas, mucoepidermoid tumors, typical carcinoid lung tumors, atypical carcinoid lung tumors, peripheral carcinoid lung tumors, central carcinoid lung tumors, pleural mesotheliomas, and undifferentiated pulmonary carcinoma and cancers that originate outside the lungs such as secondary cancers that have metastasized to the lungs from other parts of the body. Lung cancers may be of any stage or grade. Preferably the term may be used to refer collectively to any dysplasia, hyperplasia, neoplasia, or metastasis in which the protein biomarkers expressed above normal levels as may be determined, for example, by comparison to adjacent healthy tissue.
Examples of non-cancerous lung condition include chronic obstructive pulmonary disease (COPD), benign tumors or masses of cells (e.g., hamartoma, fibroma, neurofibroma), granuloma, sarcoidosis, and infections caused by bacterial (e.g., tuberculosis) or fungal (e.g. histoplasmosis) pathogens. In certain embodiments, a lung condition may be associated with the appearance of radiographic PNs.
As used herein, “lung tissue”, and “lung cancer” refer to tissue or cancer, respectively, of the lungs themselves, as well as the tissue adjacent to and/or within the strata underlying the lungs and supporting structures such as the pleura, intercostal muscles, ribs, and other elements of the respiratory system. The respiratory system itself is taken in this context as representing nasal cavity, sinuses, pharynx, larynx, trachea, bronchi, lungs, lung lobes, aveoli, aveolar ducts, aveolar sacs, aveolar capillaries, bronchioles, respiratory bronchioles, visceral pleura, parietal pleura, pleural cavity, diaphragm, epiglottis, adenoids, tonsils, mouth and tongue, and the like. The tissue or cancer may be from a mammal and is preferably from a human, although monkeys, apes, cats, dogs, cows, horses and rabbits are within the scope of the present invention. The term “lung condition” as used herein refers to a disease, event, or change in health status relating to the lung, including for example lung cancer and various non-cancerous conditions.
“Accuracy” refers to the degree of conformity of a measured or calculated quantity (a test reported value) to its actual (or true) value. Clinical accuracy relates to the proportion of true outcomes (true positives (TP) or true negatives (TN) versus misclassified outcomes (false positives (FP) or false negatives (FN)), and may be stated as a sensitivity, specificity, positive predictive values (PPV) or negative predictive values (NPV), or as a likelihood, odds ratio, among other measures.
The term “biological sample” as used herein refers to any sample of biological origin potentially containing one or more biomarker proteins. Examples of biological samples include tissue, organs, or bodily fluids such as whole blood, plasma, serum, tissue, lavage or any other specimen used for detection of disease.
The term “subject” as used herein refers to a mammal, preferably a human.
The term “biomarker protein” as used herein refers to a polypeptide in a biological sample from a subject with a lung condition versus a biological sample from a control subject. A biomarker protein includes not only the polypeptide itself, but also minor variations thereof, including for example one or more amino acid substitutions or modifications such as glycosylation or phosphorylation.
The term “biomarker protein panel” as used herein refers to a plurality of biomarker proteins. In certain embodiments, the expression levels of the proteins in the panels can be correlated with the existence of a lung condition in a subject. In certain embodiments, biomarker protein panels comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90 or 100 proteins. In certain embodiments, the biomarker proteins panels comprise from 100-125 proteins, 125-150 proteins, 150-200 proteins or more.
“Treating” or “treatment” as used herein with regard to a condition may refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof.
The term “ruling out” as used herein is meant that the subject is selected not to receive a treatment protocol.
The term “ruling-in” as used herein is meant that the subject is selected to receive a treatment protocol.
Biomarker levels may change due to treatment of the disease. The changes in biomarker levels may be measured by the present invention. Changes in biomarker levels may be used to monitor the progression of disease or therapy.
“Altered”, “changed” or “significantly different” refer to a detectable change or difference from a reasonably comparable state, profile, measurement, or the like. One skilled in the art should be able to determine a reasonable measurable change. Such changes may be all or none. They may be incremental and need not be linear. They may be by orders of magnitude. A change may be an increase or decrease by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more, or any value in between 0% and 100%. Alternatively the change may be 1-fold, 1.5-fold 2-fold, 3-fold, 4-fold, 5-fold or more, or any values in between 1-fold and five-fold. The change may be statistically significant with a p value of 0.1, 0.05, 0.001, or 0.0001.
Using the methods of the current invention, a clinical assessment of a patient is first performed. If there exists is a higher likelihood for cancer, the clinician may rule in the disease which will require the pursuit of diagnostic testing options yielding data which increase and/or substantiate the likelihood of the diagnosis. “Rule in” of a disease requires a test with a high specificity.
“FN” is false negative, which for a disease state test means classifying a disease subject incorrectly as non-disease or normal.
“FP” is false positive, which for a disease state test means classifying a normal subject incorrectly as having disease.
The term “rule in” refers to a diagnostic test with high specificity that coupled with a clinical assessment indicates a higher likelihood for cancer. If the clinical assessment is a lower likelihood for cancer, the clinician may adopt a stance to rule out the disease, which will require diagnostic tests which yield data that decrease the likelihood of the diagnosis. “Rule out” requires a test with a high sensitivity.
The term “rule out” refers to a diagnostic test with high sensitivity that coupled with a clinical assessment indicates a lower likelihood for cancer.
The term “sensitivity of a test” refers to the probability that a patient with the disease will have a positive test result. This is derived from the number of patients with the disease who have a positive test result (true positive) divided by the total number of patients with the disease, including those with true positive results and those patients with the disease who have a negative result, i.e. false negative.
The term “specificity of a test” refers to the probability that a patient without the disease will have a negative test result. This is derived from the number of patients without the disease who have a negative test result (true negative) divided by all patients without the disease, including those with a true negative result and those patients without the disease who have a positive test result, e.g. false positive. While the sensitivity, specificity, true or false positive rate, and true or false negative rate of a test provide an indication of a test's performance, e.g. relative to other tests, to make a clinical decision for an individual patient based on the test's result, the clinician requires performance parameters of the test with respect to a given population.
The term “positive predictive value” (PPV) refers to the probability that a positive result correctly identifies a patient who has the disease, which is the number of true positives divided by the sum of true positives and false positives.
The term “negative predictive value” or “NPV” is calculated by TN/(TN+FN) or the true negative fraction of all negative test results. It also is inherently impacted by the prevalence of the disease and pre-test probability of the population intended to be tested.
The term “disease prevalence” refers to the number of all new and old cases of a disease or occurrences of an event during a particular period. Prevalence is expressed as a ratio in which the number of events is the numerator and the population at risk is the denominator.
The term disease incidence refers to a measure of the risk of developing some new condition within a specified period of time; the number of new cases during some time period, it is better expressed as a proportion or a rate with a denominator.
Lung cancer risk according to the “National Lung Screening Trial” is classified by age and smoking history. High risk—age ≧55 and ≧30 pack-years smoking history; Moderate risk—age ≧50 and ≧20 pack-years smoking history; Low risk—<age 50 or <20 pack-years smoking history.
The term “negative predictive value” (NPV) refers to the probability that a negative test correctly identifies a patient without the disease, which is the number of true negatives divided by the sum of true negatives and false negatives. A positive result from a test with a sufficient PPV can be used to rule in the disease for a patient, while a negative result from a test with a sufficient NPV can be used to rule out the disease, if the disease prevalence for the given population, of which the patient can be considered a part, is known.
The clinician must decide on using a diagnostic test based on its intrinsic performance parameters, including sensitivity and specificity, and on its extrinsic performance parameters, such as positive predictive value and negative predictive value, which depend upon the disease's prevalence in a given population.
Additional parameters which may influence clinical assessment of disease likelihood include the prior frequency and closeness of a patient to a known agent, e.g. exposure risk, that directly or indirectly is associated with disease causation, e.g. second hand smoke, radiation, etc., and also the radiographic appearance or characterization of the pulmonary nodule exclusive of size. A nodule's description may include solid, semi-solid or ground glass which characterizes it based on the spectrum of relative gray scale density employed by the CT scan technology.
“Mass spectrometry” refers to a method comprising employing an ionization source to generate gas phase ions from an analyte presented on a sample presenting surface of a probe and detecting the gas phase ions with a mass spectrometer.
The technology liquid chromatography selected reaction monitoring mass spectrometry (LC-SRM-MS) was used to assay the expression levels of a cohort of 388 proteins in the blood to identify differences for individual proteins which may correlate with the absence or presence of the disease. The individual proteins have not only been implicated in lung cancer biology, but are also likely to be present in plasma based on their expression as membrane-anchored or secreted proteins. An analysis of epithelial and endothelial membranes of resected lung cancer tissues (including the subtypes of adenocarcinoma, squamous, and large cell) identified 217 tissue proteins. A review of the scientific literature with search terms relevant to lung cancer biology identified 319 proteins. There was an overlap of 148 proteins between proteins identified by cancer tissue analysis or literature review, yielding a total of 388 unique proteins as candidates. The majority of candidate proteins included in the multiplex LC-SRM-MS assay were discovered following proteomics analysis of secretory vesicle contents from fresh NSCLC resections and from adjacent non-malignant tissue. The secretory proteins reproducibly upregulated in the tumor tissue were identified and prioritized for inclusion in the LC-SRM-MS assay using extensive bioinformatic and literature annotation. An additional set of proteins that were present in relevant literature was also added to the assay. In total, 388 proteins associated with lung cancer were prioritized for SRM assay development. Of these, 371 candidate protein biomarkers were ultimately included in the assay. These are listed in Table 6, below.













TABLE 6








Sources of

Subcellular
Evidence for




Gene
Tissue
Biomarkers
Location
Presence in


UniProt Protein
Protein Name
Symbol
Biomarkers
in Literature
(UniProt)
Blood







1433B_HUMAN
14-3-3
YWHAB
Secreted,
LungCancers
Cytoplasm.
Literature,



protein

EPI

Melanosome.
Detection



beta/alpha



Note = Identified by








mass spectrometry in








melanosome fractions








from stage I to stage








IV.



1433E_HUMAN
14-3-3
YWHAE
ENDO
LungCancers,
Cytoplasm (By
Literature,



protein


Benign-Nodules
similarity).
Detection



epsilon



Melanosome.








Note = Identified by








mass spectrometry in








melanosome fractions








from stage I to stage








IV.



1433S_HUMAN
14-3-3
SFN
Secreted,
LungCancers
Cytoplasm.
UniProt,



protein

EPI

Nucleus (By
Literature,



sigma



similarity).
Detection







Secreted.








Note = May be








secreted by a non-








classical secretory








pathway.



1433T_HUMAN
14-3-3
YWHAQ
EPI
LungCancers,
Cytoplasm.
Detection



protein


Benign-Nodules
Note = In neurons,




theta



axonally transported








to the nerve








terminals.



1433Z_HUMAN
14-3-3
YWHAZ
EPI
LungCancers,
Cytoplasm.
Detection



protein


Benign-Nodules
Melanosome.




zeta/delta



Note = Located








to stage I to stage IV








melanosomes.



6PGD_HUMAN
6-phosphogluconate
PGD
EPI, ENDO

Cytoplasm (By
Detection



dehydrogenase,



similarity).




decarboxylating







A1AG1_HUMAN
Alpha-1-acid
ORM1
EPI
Symptoms
Secreted.
UniProt,



glycoprotein 1




Literature,








Detection,








Prediction


ABCD1_HUMAN
ATP-binding
ABCD1
ENDO

Peroxisome
Detection,



cassette



membrane;
Prediction



sub-family



Multi-pass membrane




D member 1



protein.



ADA12_HUMAN
Disintegrin and
ADAM12

LungCancers,
Isoform 1: Cell
UniProt,



metalloproteinase


Benign-Nodules,
membrane;
Detection,



domain-containing


Symptoms
Single-pass type I
Prediction



protein 12



membrane








protein.|Isoform 2:








Secreted.|Isoform








3: Secreted








(Potential).|Isoform








4: Secreted (Potential).



ADML_HUMAN
ADM
ADM

LungCancers,
Secreted.
UniProt,






Benign-Nodules,

Literature,






Symptoms

Detection,








Prediction


AGR2_HUMAN
Anterior
AGR2
EPI
LungCancers
Secreted.
UniProt,



gradient



Endoplasmic
Prediction



protein 2



reticulum (By




homolog



similarity).



AIFM1_HUMAN
Apoptosis-inducing
AIFM1
EPI, ENDO
LungCancers
Mitochondrion
Detection,



factor 1,



intermembrane
Prediction



mitochondrial



space. Nucleus.








Note = Translocated








to the nucleus upon








induction of apoptosis.



ALDOA_HUMAN
Fructose-
ALDOA
Secreted,
LungCancers,

Literature,



bisphosphate

EPI
Symptoms

Detection



aldolase A







AMPN_HUMAN
Aminopeptidase N
ANPEP
EPI, ENDO
LungCancers,
Cell membrane;
UniProt,






Benign-Nodules,
Single-pass type II
Detection






Symptoms
membrane protein.








Cytoplasm, cytosol








(Potential).








Note = A soluble








form has also








been detected.



ANGP1_HUMAN
Angiopoietin-1
ANGPT1

LungCancers,
Secreted.
UniProt,






Benign-Nodules

Literature,








Prediction


ANGP2_HUMAN
Angiopoietin-2
ANGPT2

LungCancers,
Secreted.
UniProt,






Benign-Nodules

Literature,








Prediction


APOA1_HUMAN
Apolipoprotein A-I
APOA1

LungCancers,
Secreted.
UniProt,






Benign-Nodules,

Literature,






Symptoms

Detection,








Prediction


APOE_HUMAN
Apolipoprotein E
APOE
EPI, ENDO
LungCancers,
Secreted.
UniProt,






Benign-Nodules,

Literature,






Symptoms

Detection,








Prediction


ASM3B_HUMAN
Acid
SMPDL3B
EPI, ENDO

Secreted (By
UniProt,



sphingomyelinase-like



similarity).
Prediction



phosphodiesterase 3b







AT2A2_HUMAN
Sarcoplasmic/endo-
ATP2A2
EPI, ENDO
LungCancers,
Endoplasmic
Detection



plasmic


Benign-Nodules
reticulum membrane;




reticulum



Multi-pass membrane




calcium



protein.




ATPase 2



Sarcoplasmic








reticulum membrane;








Multi-pass membrane








protein.



ATS1_HUMAN
A disintegrin and
ADAMTS1

LungCancers,
Secreted,
UniProt,



metalloproteinase with


Benign-Nodules,
extracellular space,
Literature,



thrombospondin


Symptoms
extracellular matrix
Prediction



motifs 1



(By similarity).



ATS12_HUMAN
A disintegrin and
ADAMTS12

LungCancers
Secreted,
UniProt,



metalloproteinase with



extracellular space,
Detection,



thrombospondin



extracellular matrix
Prediction



motifs 12



(By similarity).



ATS19_HUMAN
A disintegrin and
ADAMTS19

LungCancers
Secreted,
UniProt,



metalloproteinase with



extracellular space,
Prediction



thrombospondin



extracellular matrix




motifs 19



(By similarity).



BAGE1_HUMAN
B melanoma
BAGE

LungCancers
Secreted
UniProt,



antigen 1



(Potential).
Prediction


BAGE2_HUMAN
B melanoma
BAGE2

LungCancers
Secreted
UniProt,



antigen 2



(Potential).
Prediction


BAGE3_HUMAN
B melanoma
BAGE3

LungCancers
Secreted
UniProt,



antigen 3



(Potential).
Prediction


BAGE4_HUMAN
B melanoma
BAGE4

LungCancers
Secreted
UniProt,



antigen 4



(Potential).
Prediction


BAGE5_HUMAN
B melanoma
BAGE5

LungCancers
Secreted
UniProt,



antigen 5



(Potential).
Prediction


BASP1_HUMAN
Brain acid
BASP1
Secreted,

Cell membrane;
Detection



soluble

EPI

Lipid-anchor.




protein 1



Cell projection,








growth cone.








Note = Associated








with the membranes 








of growth cones








that form the tips of








elongating axons.



BAX_HUMAN
Apoptosis
BAX
EPI
LungCancers,
Isoform Alpha:
UniProt,



regulator


Benign-Nodules
Mitochondrion
Literature,



BAX



membrane;
Prediction







Single-pass membrane








protein.








Cytoplasm.








Note = Colocalizes








with 14- 3-3 proteins








in the cytoplasm.








Under stress








conditions,








redistributes to the








mitochondrion








membrane through the








release from JNK-








phosphorylated 14-3-3








proteins.|Isoform








Beta:








Cytoplasm.|Isoform








Gamma:








Cytoplasm.|Isoform








Delta:








Cytoplasm (Potential).



BDNF_HUMAN
Brain-derived
BDNF

Benign-Nodules,
Secreted.
UniProt,



neurotrophic


Symptoms

Literature,



factor




Prediction


BGH3_HUMAN
Transforming growth
TGFBI

LungCancers,
Secreted,
UniProt,



factor-beta-induced


Benign-Nodules
extracellular space,
Detection



protein ig-h3



extracellular matrix.








Note = May be








associated both with








microfibrils and








with the cell surface.



BMP2_HUMAN
Bone
BMP2

LungCancers,
Secreted.
UniProt,



morphogenetic


Benign-Nodules,

Literature



protein 2


Symptoms




BST1_HUMAN
ADP-
BST1
EPI
Symptoms
Cell membrane;
Detection,



ribosyl



Lipid-anchor,
Prediction



cyclase 2



GPI-anchor.



C163A_HUMAN
Scavenger receptor
CD163
EPI
Symptoms
Soluble CD163:
UniProt,



cysteine-rich type 1



Secreted.|Cell
Detection



protein



membrane;




M130



Single-pass type I








membrane protein.








Note = Isoform 1








and isoform 2 show








a lower surface








expression when








expressed in cells.



C4BPA_HUMAN
C4b-binding
C4BPA

LungCancers,
Secreted.
UniProt,



protein


Symptoms

Detection,



alpha chain




Prediction


CAH9_HUMAN
Carbonic
CA9

LungCancers,
Nucleus.
UniProt



anhydrase


Benign-Nodules,
Nucleus,




9


Symptoms
nucleolus. Cell








membrane;








Single-pass type I








membrane protein. 








Cell projection,








microvillus








membrane;








Single-pass type I








membrane protein.








Note = Found on the








surface microvilli








and in the nucleus,








particularly in








nucleolus.



CALR_HUMAN
Calreticulin
CALR
EPI
Symptoms
Endoplasmic
UniProt,







reticulum lumen.
Literature,







Cytoplasm, cytosol.
Detection,







Secreted, extracellular
Prediction







space, extracellular








matrix. Cell surface.








Note = Also found in








cell surface (T cells),








cytosol and








extracellular matrix.








Associated with the








lytic granules in the








cytolytic T-








lymphocytes.



CALU_HUMAN
Calumenin
CALU
EPI
Symptoms
Endoplasmic
UniProt,







reticulum lumen.
Detection,







Secreted.
Prediction







Melanosome.








Sarcoplasmic








reticulum lumen








(By similarity).








Note = Identified by








mass spectrometry








in melanosome








fractions from








stage I to stage IV.



CALX_HUMAN
Calnexin
CANX
Secreted,
Benign-Nodules
Endoplasmic
UniProt,





EPI, ENDO

reticulum membrane;
Literature,







Single-pass type I
Detection







membrane protein.








Melanosome.








Note = Identified by








mass spectrometry in








melanosome








fractions from








stage I to stage IV.



CAP7_HUMAN
Azurocidin
AZU1
EPI
Symptoms
Cytoplasmic granule.
Prediction







Note = Cytoplasmic








granules of








neutrophils.



CATB_HUMAN
Cathepsin
CTSB
Secreted
LungCancers
Lysosome.
Literature,



B



Melanosome.
Detection,







Note = Identified by
Prediction







mass spectrometry in








melanosome








fractions from








stage I to stage IV.



CATG_HUMAN
Cathepsin
CTSG
Secreted,
Benign-Nodules
Cell surface.
Detection,



G

ENDO


Prediction


CBPB2_HUMAN
Carboxypeptidase
CPB2

LungCancers,
Secreted.
UniProt,



B2


Benign-Nodules,

Detection,






Symptoms

Prediction


CCL22_HUMAN
C-C motif
CCL22

LungCancers,
Secreted.
UniProt,



chemokine 22


Benign-Nodules

Prediction


CD14_HUMAN
Monocyte
CD14
EPI
LungCancers,
Cell membrane;
Literature,



differentiation


Benign-Nodules,
Lipid-anchor,
Detection,



antigen


Symptoms
GPI-anchor.
Prediction



CD14







CD24_HUMAN
Signal
CD24

LungCancers,
Cell membrane;
Literature



transducer


Benign-Nodules
Lipid-anchor,




CD24



GPI-anchor.



CD2A2_HUMAN
Cyclin-dependent
CDKN2A

LungCancers,
Cytoplasm.
Literature,



kinase inhibitor


Benign-Nodules
Nucleus.|Nucleus,
Prediction



2A, isoform 4



nucleolus








(By similarity).



CD38_HUMAN
ADP-ribosyl
CD38
EPI, ENDO
Symptoms
Membrane;
UniProt,



cyclase 1



Single-pass type II
Literature







membrane protein.



CD40L_HUMAN
CD40
CD40LG

LungCancers,
Cell membrane;
UniProt,



ligand


Benign-Nodules,
Single-pass type II
Literature






Symptoms
membrane








protein.|CD40








ligand, soluble








form: Secreted.



CD44_HUMAN
CD44
CD44
EPI
LungCancers,
Membrane;
UniProt,



antigen


Benign-Nodules,
Single-pass type I
Literature,






Symptoms
membrane protein.
Detection,








Prediction


CD59_HUMAN
CD59
CD59

LungCancers,
Cell membrane;
UniProt,



glycoprotein


Benign-Nodules,
Lipid-anchor,
Literature,






Symptoms
GPI-anchor.
Detection,







Secreted.
Prediction







Note = Soluble








form found in a








number of tissues.



CD97_HUMAN
CD97
CD97
EPI, ENDO
Symptoms
Cell membrane;
UniProt



antigen



Multi-pass








membrane








protein.|CD97








antigen subunit








alpha: Secreted,








extracellular space.



CDCP1_HUMAN
CUB
CDCP1

LungCancers
Isoform 1: Cell
UniProt,



domain-containing



membrane;
Prediction



protein 1



Single-pass membrane








protein (Potential).








Note = Shedding








may also lead to








a soluble








peptide.|Isoform








3: Secreted.



CDK4_HUMAN
Cell division protein
CDK4

LungCancers,

Literature



kinase 4


Symptoms




CEAM5_HUMAN
Carcinoembryonic
CEACAM5
EPI
LungCancers,
Cell membrane;
Literature,



antigen-related cell


Benign-Nodules,
Lipid-anchor,
Prediction



adhesion molecule 5


Symptoms
GPI-anchor.



CEAM8_HUMAN
Carcinoembryonic
CEACAM8
EPI
LungCancers
Cell membrane;
Detection,



antigen-related cell



Lipid-anchor,
Prediction



adhesion molecule 8



GPI-anchor.



CERU_HUMAN
Ceruloplasmin
CP
EPI
LungCancers,
Secreted.
UniProt,






Symptoms

Literature,








Detection,








Prediction


CH10_HUMAN
10 kDa
HSPE1
ENDO
LungCancers
Mitochondrion
Literature,



heat shock protein,



matrix.
Detection,



mitochondrial




Prediction


CH60_HUMAN
60 kDa
HSPD1
Secreted,
LungCancers,
Mitochondrion
Literature,



heat shock protein,

EPI, ENDO
Symptoms
matrix.
Detection



mitochondrial







CKAP4_HUMAN
Cytoskeleton-
CKAP4
EPI, ENDO
LungCancers
Endoplasmic
UniProt



associated protein 4



reticulum-Golgi








intermediate








compartment








membrane;








Single-pass membrane








protein (Potential).



CL041_HUMAN
Uncharacterized
C12orf41
ENDO


Prediction



protein C12orf41







CLCA1_HUMAN
Calcium-activated
CLCA1

LungCancers,
Secreted,
UniProt,



chloride


Benign-Nodules
extracellular
Prediction



channel regulator 1



space. Cell membrane;








Peripheral membrane








protein;








Extracellular side.








Note = Protein that








remains attached to








the plasma membrane








appeared to be








predominantly








localized to








microvilli.



CLIC1_HUMAN
Chloride
CLIC1
EPI

Nucleus.
UniProt,



intracellular channel



Nucleus membrane;
Literature,



protein 1



Single-pass membrane
Detection







protein (Probable).








Cytoplasm. Cell








membrane;








Single-pass membrane








protein (Probable).








Note = Mostly in the








nucleus including in








the nuclear membrane.








Small amount in the








cytoplasm and the








plasma membrane.








Exists both as soluble








cytoplasmic protein








and as membrane








protein with probably








a single








transmembrane








domain.



CLUS_HUMAN
Clusterin
CLU
EPI, ENDO
LungCancers,
Secreted.
UniProt,






Benign-Nodules,

Literature,






Symptoms

Detection,








Prediction


CMGA_HUMAN
Chromogranin-A
CHGA

LungCancers,
Secreted.
UniProt,






Benign-Nodules
Note = Neuro-
Literature,







endocrine and
Detection,







endocrine secretory
Prediction







granules.



CNTN1_HUMAN
Contactin-1
CNTN1

LungCancers
Isoform 1: Cell
Detection,







membrane;
Prediction







Lipid-anchor,








GPI- anchor;








Extracellular








side.|Isoform 2:








Cell membrane;








Lipid-anchor,








GPI- anchor;








Extracellular side.



CO4A1_HUMAN
Collagen
COL4A1

LungCancers
Secreted,
UniProt,



alpha-1(IV)



extracellular space,
Detection,



chain



extracellular matrix,
Prediction







basement membrane.



CO5A2_HUMAN
Collagen
COL5A2

LungCancers
Secreted,
UniProt,



alpha-2(V)



extracellular space,
Detection,



chain



extracellular matrix
Prediction







(By similarity).



CO6A3_HUMAN
Collagen
COL6A3
Secreted
Symptoms
Secreted,
UniProt,



alpha-3(VI)



extracellular space,
Detection,



chain



extracellular matrix
Prediction







(By similarity).



COCA1_HUMAN
Collagen
COL12A1
ENDO
LungCancers,
Secreted,
UniProt,



alpha-1(XII)


Symptoms
extracellular space,
Prediction



chain



extracellular matrix








(By similarity).



COF1_HUMAN
Cofilin-1
CFL1
Secreted,
LungCancers,
Nucleus matrix.
Detection,





EPI
Benign-Nodules
Cytoplasm,
Prediction







cytoskeleton.








Note = Almost








completely in








nucleus in cells








exposed to heat








shock or 10%








dimethyl sulfoxide.



COIA1_HUMAN
Collagen
COL18A1

LungCancers,
Secreted,
UniProt,



alpha-1(XVIII)


Benign-Nodules
extracellular space,
Literature,



chain



extracellular matrix
Detection,







(By similarity).
Prediction


COX5A_HUMAN
Cytochrome
COX5A
Secreted,

Mitochondrion
Prediction



c oxidase

ENDO

inner membrane.




subunit 5A,








mitochondrial







CRP_HUMAN
C-reactive
CRP

LungCancers,
Secreted.
UniProt,



protein


Benign-Nodules,

Literature,






Symptoms

Detection,








Prediction


CS051_HUMAN
UPF0470
C19orf51
ENDO


Prediction



protein








C19orf51







CSF1_HUMAN
Macrophage
CSF1

LungCancers,
Cell membrane;
UniProt,



colony-stimulating


Benign-Nodules
Single-pass membrane
Literature,



factor 1



protein (By
Detection







similarity).|Processed








macrophage








colony-stimulating








factor 1:








Secreted,








extracellular space








(By similarity).



CSF2_HUMAN
Granulocyte-
CSF2

LungCancers,
Secreted.
UniProt,



macrophage


Benign-Nodules

Literature,



colony-stimulating




Prediction



factor







CT085_HUMAN
Uncharacterized
C20orf85

LungCancers,

Prediction



protein


Benign-Nodules





C20orf85







CTGF_HUMAN
Connective tissue
CTGF

LungCancers,
Secreted,
UniProt,



growth factor


Benign-Nodules
extracellular space,
Literature,







extracellular matrix
Detection,







(By similarity).
Prediction







Secreted (By








similarity).



CYR61_HUMAN
Protein
CYR61

LungCancers,
Secreted.
UniProt,



CYR61


Benign-Nodules

Prediction


CYTA_HUMAN
Cystatin-A
CSTA

LungCancers
Cytoplasm.
Literature,








Detection


CYTB_HUMAN
Cystatin-B
CSTB
Secreted

Cytoplasm.
Literature,







Nucleus.
Detection


DDX17_HUMAN
Probable
DDX17
ENDO
LungCancers,
Nucleus.
Detection,



ATP-dependent


Benign-Nodules

Prediction



RNA helicase








DDX17







DEFB1_HUMAN
Beta-
DEFB1

LungCancers,
Secreted.
UniProt,



defensin 1


Benign-Nodules

Prediction


DESP_HUMAN
Desmoplakin
DSP
EPI, ENDO
LungCancers
Cell junction,
Detection







desmosome.








Cytoplasm,








cytoskeleton.








Note = Innermost








portion of the








desmosomal plaque.



DFB4A_HUMAN
Beta-
DEFB4A

LungCancers,
Secreted.
UniProt



defensin 4A


Benign-Nodules




DHI1L_HUMAN
Hydroxysteroid
HSD11B1L

LungCancers
Secreted
UniProt,



11-beta-



(Potential).
Prediction



dehydrogenase








1-like protein







DMBT1_HUMAN
Deleted in
DMBT1

LungCancers,
Secreted (By
UniProt,



malignant


Benign-Nodules
similarity).
Detection,



brain tumors 1



Note = Some
Prediction



protein



isoforms may be








membrane-bound.








Localized to the








lumenal aspect of








crypt cells in the small








intestine. In the colon,








seen in the lumenal








aspect of surface








epithelial cells.








Formed in the ducts








of von Ebner gland,








and released into the








fluid bathing the taste








buds contained in the








taste papillae (By








similarity).



DMKN_HUMAN
Dermokine
DMKN

LungCancers
Secreted.
UniProt,








Detection,








Prediction


DPP4_HUMAN
Dipeptidyl
DPP4
EPI
LungCancers,
Dipeptidyl
UniProt,



peptidase 4


Benign-Nodules,
peptidase 4
Detection






Symptoms
soluble form:








Secreted.|Cell








membrane;








Single-pass type II








membrane protein.



DSG2_HUMAN
Desmoglein-2
DSG2
ENDO
Symptoms
Cell membrane;
UniProt,







Single-pass type I
Detection







membrane protein.








Cell junction,








desmosome.



DX39A_HUMAN
ATP-dependent
DDX39A
EPI

Nucleus (By
Prediction



RNA helicase



similarity).




DDX39A







DX39B_HUMAN
Spliceosome
DDX39B
EPI

Nucleus.
Prediction



RNA helicase



Nucleus




DDX39B



speckle.



DYRK2_HUMAN
Dual specificity
DYRK2
ENDO
LungCancers
Cytoplasm.
Literature



tyrosine-



Nucleus.




phosphorylation-



Note = Translocates




regulated



into the nucleus




kinase 2



following DNA








damage.



EDN2_HUMAN
Endothelin-2
EDN2

LungCancers
Secreted.
UniProt,








Prediction


EF1A1_HUMAN
Elongation
EEF1A1
Secreted,
LungCancers,
Cytoplasm.
Detection



factor 1-alpha 1

EPI
Benign-Nodules




EF1D_HUMAN
Elongation
EEF1D
Secreted,
LungCancers

Prediction



factor 1-delta

EPI





EF2_HUMAN
Elongation
EEF2
Secreted,

Cytoplasm.
Literature,



factor 2

EPI


Detection


EGF_HUMAN
Pro-epidermal
EGF

LungCancers,
Membrane;
UniProt,



growth


Benign-Nodules,
Single-pass type I
Literature



factor


Symptoms
membrane protein.



EGFL6_HUMAN
Epidermal growth
EGFL6

LungCancers
Secreted,
UniProt,



factor-like



extracellular space,
Detection,



protein 6



extracellular matrix,
Prediction







basement membrane








(By similarity).



ENOA_HUMAN
Alpha-enolase
ENO1
Secreted,
LungCancers,
Cytoplasm. Cell
Literature,





EPI, ENDO
Benign-Nodules,
membrane.
Detection,






Symptoms
Cytoplasm, myofibril,
Prediction







sarcomere, M-








band. Note = Can








translocate to the








plasma membrane in








either the








homodimeric








(alpha/alpha) or








heterodimeric








(alpha/gamma) form. 








ENO1 is localized to








the M-band.|Isoform








MBP-1:








Nucleus.



ENOG_HUMAN
Gamma-
ENO2
EPI
LungCancers,
Cytoplasm (By
Literature,



enolase


Symptoms
similarity). Cell
Detection,







membrane (By
Prediction







similarity).








Note = Can translocate








to the plasma








membrane in either








the homodimeric








(alpha/alpha) or








heterodimeric








(alpha/gamma)








form (By similarity).



ENOX2_HUMAN
Ecto-NOX
ENOX2

LungCancers
Cell membrane.
UniProt,



disulfide-thiol



Secreted,
Detection



exchanger 2



extracellular space.








Note = Extracellular








and plasma








membrane-associated.



ENPL_HUMAN
Endoplasmin
HSP90B1
Secreted,
LungCancers,
Endoplasmic
Literature,





EPI, ENDO
Benign-Nodules,
reticulum lumen.
Detection,






Symptoms
Melanosome.
Prediction







Note = Identified by








mass spectrometry in








melanosome








fractions from








stage I to stage IV.



EPHB6_HUMAN
Ephrin
EPHB6

LungCancers
Membrane;
UniProt,



type-B



Single-pass type I
Literature



receptor 6



membrane








protein.|Isoform








3: Secreted








(Probable).



EPOR_HUMAN
Erythropoietin
EPOR

LungCancers,
Cell membrane;
UniProt,



receptor


Benign-Nodules,
Single-pass type I
Literature,






Symptoms
membrane
Detection







protein.|Isoform








EPOR-S:








Secreted.








Note = Secreted








and located to








the cell surface.



ERBB3_HUMAN
Receptor
ERBB3

LungCancers,
Isoform 1: Cell
UniProt,



tyrosine-protein


Benign-Nodules
membrane;
Literature,



kinase erbB-3



Single-pass type I
Prediction







membrane








protein.|Isoform








2: Secreted.



EREG_HUMAN
Proepiregulin
EREG

LungCancers
Epiregulin:
UniProt







Secreted,








extracellular








space. Proepiregulin:








Cell membrane;








Single-pass type I








membrane protein.



ERO1A_HUMAN
ERO1-like
ERO1L
Secreted,
Symptoms
Endoplasmic
Prediction



protein

EPI, ENDO

reticulum membrane;




alpha



Peripheral membrane








protein;








Lumenal side.








Note = The








association with








ERP44 is essential








for its retention in 








the endoplasmic








reticulum.



ESM1_HUMAN
Endothelial
ESM1

LungCancers,
Secreted.
UniProt,



cell-specific


Benign-Nodules

Prediction



molecule 1







EZRI_HUMAN
Ezrin
EZR
Secreted
LungCancers,
Apical cell membrane;
Literature,






Benign-Nodules
Peripheral membrane
Detection,







protein;
Prediction







Cytoplasmic side.








Cell projection. Cell








projection,








microvillus








membrane;








Peripheral membrane








protein;








Cytoplasmic side.








Cell projection,








ruffle membrane;








Peripheral membrane








protein;








Cytoplasmic side.








Cytoplasm, cell








cortex.








Cytoplasm,








cytoskeleton.








Note = Localization








to the apical








membrane of parietal








cells depends on the








interaction with








MPP5.








Localizes to cell








extensions and








peripheral processes








of astrocytes (By








similarity).








Microvillar peripheral








membrane protein








(cytoplasmic side).



F10A1_HUMAN
Hsc70-
ST13
EPI

Cytoplasm (By
Detection,



interacting



similarity).|Cytoplasm
Prediction



protein



(Probable).



FAM3C_HUMAN
Protein
FAM3C
EPI, ENDO

Secreted
UniProt,



FAM3C



(Potential).
Detection


FAS_HUMAN
Fatty acid
FASN
EPI
LungCancers,
Cytoplasm.
Literature,



synthase


Benign-Nodules,
Melanosome.
Detection






Symptoms
Note = Identified by








mass spectrometry in








melanosome








fractions from








stage I to stage IV.



FCGR1_HUMAN
High affinity
FCGR1A
EPI
LungCancers,
Cell membrane;
UniProt



immunoglobulin


Benign-Nodules,
Single-pass type I




gamma Fc


Symptoms
membrane protein.




receptor I



Note = Stabilized








at the cell membrane








through interaction








with FCER1G.



FGF10_HUMAN
Fibroblast
FGF10

LungCancers
Secreted
UniProt,



growth factor 10



(Potential).
Prediction


FGF2_HUMAN
Heparin-binding
FGF2

LungCancers,

Literature



growth factor 2


Benign-Nodules,








Symptoms




FGF7_HUMAN
Keratinocyte
FGF7

LungCancers,
Secreted.
UniProt,



growth factor


Benign-Nodules

Literature,








Prediction


FGF9_HUMAN
Glia-activating
FGF9

LungCancers
Secreted.
UniProt,



factor




Literature,








Prediction


FGFR2_HUMAN
Fibroblast
FGFR2

LungCancers,
Cell membrane;
UniProt,



growth


Benign-Nodules
Single-pass type I
Literature,



factor



membrane
Prediction



receptor 2



protein.|Isoform 14:








Secreted.|Isoform








19: Secreted.



FGFR3_HUMAN
Fibroblast
FGFR3

LungCancers
Membrane;
UniProt,



growth



Single-pass type I
Literature,



factor



membrane protein.
Prediction



receptor 3







FGL2_HUMAN
Fibroleukin
FGL2

Benign-Nodules,
Secreted.
UniProt,






Symptoms

Detection,








Prediction


FHIT_HUMAN
Bis(5′-
FHIT

LungCancers,
Cytoplasm.
Literature



adenosyl)-


Benign-Nodules,





triphosphatase


Symptoms




FIBA_HUMAN
Fibrinogen
FGA

LungCancers,
Secreted.
UniProt,



alpha chain


Benign-Nodules,

Literature,






Symptoms

Detection,








Prediction


FINC_HUMAN
Fibronectin
FN1
Secreted,
LungCancers,
Secreted,
UniProt,





EPI, ENDO
Benign-Nodules,
extracellular space,
Literature,






Symptoms
extracellular matrix.
Detection,








Prediction


FKB11_HUMAN
Peptidyl-
FKBP11
EPI, ENDO

Membrane;
UniProt,



prolyl cis-trans



Single-pass
Prediction



isomerase



membrane protein




FKBP11



(Potential).



FOLH1_HUMAN
Glutamate
FOLH1
ENDO
LungCancers,
Cell membrane;
UniProt,



carboxypeptidase 2


Symptoms
Single-pass type II
Literature







membrane








protein.|Isoform








PSMA′:








Cytoplasm.



FOLR1_HUMAN
Folate
FOLR1

LungCancers
Cell membrane;
UniProt



receptor



Lipid-anchor,




alpha



GPI-anchor.








Secreted (Probable).



FOXA2_HUMAN
Hepatocyte
FOXA2

LungCancers
Nucleus.
Detection,



nuclear factor 3-beta




Prediction


FP100_HUMAN
Fanconi
C17orf70
ENDO
Symptoms
Nucleus.
Prediction



anemia-associated








protein of 100 kDa







FRIH_HUMAN
Ferritin
FTH1
EPI
LungCancers,

Literature,



heavy


Benign-Nodules

Detection,



chain




Prediction


FRIL_HUMAN
Ferritin
FTL
Secreted,
Benign-Nodules,

Literature,



light chain

EPI, ENDO
Symptoms

Detection


G3P_HUMAN
Glyceralde-
GAPDH
Secreted,
LungCancers,
Cytoplasm.
Detection



hyde-3-

EPI, ENDO
Benign-Nodules,
Cytoplasm,




phosphate


Symptoms
perinuclear region.




dehydrogenase



Membrane.








Note = Postnuclear








and Perinuclear








regions.



G6PD_HUMAN
Glucose-6-phosphate
G6PD
Secreted,
LungCancers,

Literature,



1-dehydrogenase

EPI
Symptoms

Detection


G6PI_HUMAN
Glucose-6-
GPI
Secreted,
Symptoms
Cytoplasm.
UniProt,



phosphate

EPI

Secreted.
Literature,



isomerase




Detection


GA2L1_HUMAN
GAS2-like
GAS2L1
ENDO

Cytoplasm,
Prediction



protein 1



cytoskeleton








(Probable).



GALT2_HUMAN
Polypeptide N-
GALNT2
EPI, ENDO

Golgi apparatus,
UniProt,



acetylgalactosaminyl-



Golgi stack
Detection



transferase 2



membrane;








Single-pass type II








membrane protein.








Secreted.








Note = Resides








preferentially in








the trans and








medial parts of








the Golgi stack.








A secreted form








also exists.



GAS6_HUMAN
Growth
GAS6

LungCancers
Secreted.
UniProt,



arrest-specific




Detection,



protein 6




Prediction


GDIR2_HUMAN
Rho GDP-dissociation
ARHGDIB
EPI

Cytoplasm.
Detection



inhibitor 2







GELS_HUMAN
Gelsolin
GSN

LungCancers,
Isoform 2:
UniProt,






Benign-Nodules
Cytoplasm,
Literature,







cytoskeleton.|Isoform
Detection,







1: Secreted.
Prediction


GGH_HUMAN
Gamma-glutamyl
GGH

LungCancers
Secreted,
UniProt,



hydrolase



extracellular space.
Detection,







Lysosome.
Prediction







Melanosome.








Note = While its








intracellular location








is primarily the








lysosome, most








of the enzyme








activity is secreted.








Identified by mass








spectrometry in








melanosome








fractions from








stage I to stage IV.



GPC3_HUMAN
Glypican-3
GPC3

LungCancers,
Cell membrane;
UniProt,






Symptoms
Lipid-anchor,
Literature,







GPI-anchor;
Prediction







Extracellular side (By








similarity).|Secreted








glypican-3:








Secreted,








extracellular space








(By similarity).



GRAN_HUMAN
Grancalcin
GCA
EPI

Cytoplasm.
Prediction







Cytoplasmic granule








membrane;








Peripheral membrane








protein;








Cytoplasmic side.








Note = Primarily








cytosolic in the








absence of calcium or








magnesium








ions. Relocates








to granules and other








membranes in








response to elevated








calcium and








magnesium levels.



GREB1_HUMAN
Protein
GREB1
ENDO

Membrane;
UniProt,



GREB1



Single-pass membrane
Prediction







protein (Potential).



GREM1_HUMAN
Gremlin-1
GREM1

LungCancers,
Secreted
UniProt,






Benign-Nodules
(Probable).
Prediction


GRP_HUMAN
Gastrin-releasing
GRP

LungCancers,
Secreted.
UniProt,



peptide


Symptoms

Prediction


GRP78_HUMAN
78 kDa
HSPA5
Secreted,
LungCancers,
Endoplasmic
Detection,



glucose-regulated

EPI, ENDO
Benign-Nodules
reticulum lumen.
Prediction



protein



Melanosome.








Note = Identified by








mass spectrometry in








melanosome








fractions from








stage I to stage IV.



GSLG1_HUMAN
Golgi
GLG1
EPI, ENDO
Benign-Nodules
Golgi apparatus
UniProt



apparatus



membrane;




protein 1



Single-pass type I








membrane protein.



GSTP1_HUMAN
Glutathione
GSTP1
Secreted
LungCancers,

Literature,



S-transferase P


Benign-Nodules,

Detection,






Symptoms

Prediction


GTR1_HUMAN
Solute carrier
SLC2A1
EPI, ENDO
LungCancers,
Cell membrane;
Literature



family 2, facilitated


Benign-Nodules,
Multi-pass membrane




glucose transporter


Symptoms
protein (By




member 1



similarity).








Melanosome.








Note = Localizes








primarily at the








cell surface (By








similarity).








Identified by mass








spectrometry in








melanosome








fractions from








stage I to stage IV.



GTR3_HUMAN
Solute carrier
SLC2A3
EPI

Membrane;
Detection



family 2, facilitated



Multi-pass membrane




glucose transporter



protein.




member 3







H2A1_HUMAN
Histone
HIST1H2AG
Secreted

Nucleus.
Detection,



H2A type 1




Prediction


H2A1B_HUMAN
Histone
HIST1H2AB
Secreted

Nucleus.
Detection,



H2A type 1-B/E




Prediction


H2A1C_HUMAN
Histone
HIST1H2AC
Secreted

Nucleus.
Literature,



H2A type




Detection,



1-C




Prediction


H2A1D_HUMAN
Histone
HIST1H2AD
Secreted

Nucleus.
Detection,



H2A type 1-D




Prediction


HG2A_HUMAN
HLA class
CD74

LungCancers,
Membrane;
UniProt,



II histocompatibility


Benign-Nodules,
Single-pass type II
Literature



antigen gamma chain


Symptoms
membrane protein








(Potential).



HGF_HUMAN
Hepatocyte
HGF

LungCancers,

Literature,



growth


Benign-Nodules,

Prediction



factor


Symptoms




HMGA1_HUMAN
High mobility
HMGA1

LungCancers,
Nucleus.
Literature



group protein


Benign-Nodules,





HMG-I/HMG-Y


Symptoms




HPRT_HUMAN
Hypoxanthine-
HPRT1
EPI

Cytoplasm.
Detection,



guanine




Prediction



phosphoribosyl-








transferase







HPSE_HUMAN
Heparanase
HPSE

LungCancers,
Lysosome membrane;
UniProt,






Benign-Nodules,
Peripheral membrane
Prediction






Symptoms
protein.








Secreted.








Note = Secreted,








internalised and








transferred to late








endo-








somes/lysosomes as a








proheparanase.








In lysosomes, it is








processed into the








active form, the








heparanase. The








uptake or








internalisation of








proheparanase








is mediated by








HSPGs.








Heparin appears to be








a competitor and








retain proheparanase








in the extracellular








medium.



HPT_HUMAN
Haptoglobin
HP

LungCancers,
Secreted.
UniProt,






Benign-Nodules,

Literature,






Symptoms

Detection,








Prediction


HS90A_HUMAN
Heat shock
HSP90AA1
Secreted,
LungCancers,
Cytoplasm.
Literature,



protein

EPI
Symptoms
Melanosome.
Detection



HSP 90-alpha



Note = Identified by








mass spectrometry in








melanosome








fractions from








stage I to stage IV.



HS90B_HUMAN
Heat shock
HSP90AB1
Secreted,
LungCancers
Cytoplasm.
Literature,



protein

EPI

Melanosome.
Detection



HSP 90-beta



Note = Identified by








mass spectrometry in








melanosome








fractions from








stage I to stage IV.



HSPB1_HUMAN
Heat shock
HSPB1
Secreted,
LungCancers,
Cytoplasm.
Literature,



protein

EPI
Benign-Nodules
Nucleus.
Detection,



beta-1



Cytoplasm,
Prediction







cytoskeleton,








spindle.








Note = Cytoplasmic








in interphase cells.








Colocalizes with








mitotic spindles in








mitotic cells.








Translocates to








the nucleus








during heat shock.



HTRA1_HUMAN
Serine protease
HTRA1

LungCancers
Secreted.
UniProt,



HTRA1




Prediction


HXK1_HUMAN
Hexokinase-1
HK1
ENDO
Symptoms
Mitochondrion outer
Literature,







membrane.
Detection







Note = Its








hydrophobic N-








terminal sequence may








be involved in








membrane binding.



HYAL2_HUMAN
Hyaluronidase-2
HYAL2

LungCancers
Cell membrane;
Prediction







Lipid-anchor,








GPI-anchor.



HYOU1_HUMAN
Hypoxia
HYOU1
EPI, ENDO
Symptoms
Endoplasmic
Detection



up-regulated



reticulum lumen.




protein 1







IBP2_HUMAN
Insulin-like
IGFBP2

LungCancers
Secreted.
UniProt,



growth




Literature,



factor-binding




Detection,



protein 2




Prediction


IBP3_HUMAN
Insulin-like
IGFBP3

LungCancers,
Secreted.
UniProt,



growth


Benign-Nodules,

Literature,



factor-binding


Symptoms

Detection,



protein 3




Prediction


ICAM1_HUMAN
Intercellular
ICAM1

LungCancers,
Membrane;
UniProt,



adhesion


Benign-Nodules,
Single-pass type I
Literature,



molecule 1


Symptoms
membrane protein.
Detection


ICAM3_HUMAN
Intercellular
ICAM3
EPI, ENDO
LungCancers,
Membrane;
UniProt,



adhesion


Benign-Nodules,
Single-pass type I
Detection



molecule 3


Symptoms
membrane protein.



IDHP_HUMAN
Isocitrate
IDH2
Secreted,

Mitochondrion.
Prediction



dehydrogenase

ENDO






[NADP],








mitochondrial







IF4A1_HUMAN
Eukaryotic
EIF4A1
Secreted,


Detection,



initiation

EPI, ENDO


Prediction



factor 4A-I







IGF1_HUMAN
Insulin-like
IGF1

LungCancers,
Secreted.|Secreted.
UniProt,



growth


Benign-Nodules,

Literature,



factor I


Symptoms

Detection,








Prediction


IKIP_HUMAN
Inhibitor of
IKIP
ENDO
Symptoms
Endoplasmic
UniProt,



nuclear



reticulum membrane;
Prediction



factor



Single-pass membrane




kappa-B



protein.




kinase-



Note = Isoform 4




interacting



deletion of the




protein



hydrophobic, or








transmembrane








region between








AA 45-63 results in








uniform distribution








troughout the








cell, suggesting








that this region








is responsible








for endoplasmic








reticulum localization.



IL18_HUMAN
Interleukin-18
IL18

LungCancers,
Secreted.
UniProt,






Benign-Nodules,

Literature,






Symptoms

Prediction


IL19_HUMAN
Interleukin-19
IL19

LungCancers
Secreted.
UniProt,








Detection,








Prediction


IL22_HUMAN
Interleukin-22
IL22

LungCancers,
Secreted.
UniProt,






Benign-Nodules

Prediction


IL32_HUMAN
Interleukin-32
IL32

LungCancers,
Secreted.
UniProt,






Benign-Nodules

Prediction


IL7_HUMAN
Interleukin-7
IL7

LungCancers,
Secreted.
UniProt,






Benign-Nodules

Literature,








Prediction


IL8_HUMAN
Interleukin-8
IL8

LungCancers,
Secreted.
UniProt,






Benign-Nodules,

Literature






Symptoms




ILEU_HUMAN
Leukocyte
SERPINB1
Secreted,

Cytoplasm (By
Detection,



elastase inhibitor

EPI

similarity).
Prediction


ILK_HUMAN
Integrin-linked
ILK
Secreted
LungCancers,
Cell junction,
Literature,



protein


Benign-Nodules,
focal adhesion.
Detection



kinase


Symptoms
Cell membrane;








Peripheral membrane








protein;








Cytoplasmic side.



INHBA_HUMAN
Inhibin
INHBA

LungCancers,
Secreted.
UniProt,



beta A chain


Benign-Nodules

Literature,








Prediction


ISLR_HUMAN
Immunoglobulin
ISLR

LungCancers
Secreted
UniProt,



superfamily



(Potential).
Detection,



containing




Prediction



leucine-rich repeat








protein







ITA5_HUMAN
Integrin
ITGA5
EPI
LungCancers,
Membrane;
UniProt,



alpha-5


Benign-Nodules,
Single-pass type I
Literature,






Symptoms
membrane protein.
Detection


ITAM_HUMAN
Integrin
ITGAM
EPI, ENDO
LungCancers,
Membrane;
UniProt,



alpha-M


Benign-Nodules,
Single-pass type I
Literature






Symptoms
membrane protein.



K0090_HUMAN
Uncharacterized
KIAA0090
EPI
Symptoms
Membrane;
UniProt,



protein



Single-pass type I
Prediction



KIAA0090



membrane protein








(Potential).



K1C18_HUMAN
Keratin, type I
KRT18
Secreted
LungCancers,
Cytoplasm,
Literature,



cytoskeletal 18


Benign-Nodules
perinuclear region.
Detection,








Prediction


K1C19_HUMAN
Keratin, type I
KRT19

LungCancers,

Literature,



cytoskeletal 19


Benign-Nodules

Detection,








Prediction


K2C8_HUMAN
Keratin, type II
KRT8
EPI
LungCancers
Cytoplasm.
Literature,



cytoskeletal 8




Detection


KIT_HUMAN
Mast/stem
KIT

LungCancers
Membrane;
UniProt,



cell growth



Single-pass type I
Literature,



factor receptor



membrane protein.
Detection


KITH_HUMAN
Thymidine
TK1

LungCancers
Cytoplasm.
Literature,



kinase, cytosolic




Prediction


KLK11_HUMAN
Kallikrein-11
KLK11

LungCancers
Secreted.
UniProt,








Literature,








Prediction


KLK13_HUMAN
Kallikrein-13
KLK13

LungCancers
Secreted
UniProt,







(Probable).
Literature,








Detection,








Prediction


KLK14_HUMAN
Kallikrein-14
KLK14

LungCancers,
Secreted,
UniProt,






Symptoms
extracellular
Literature,







space.
Prediction


KLK6_HUMAN
Kallikrein-6
KLK6

LungCancers,
Secreted.
UniProt,






Benign-Nodules,
Nucleus, nucleolus.
Literature,






Symptoms
Cytoplasm.
Detection,







Mitochondrion.
Prediction







Microsome.








Note = In brain,








detected in the








nucleus of glial








cells and in the








nucleus and








cytoplasm of neurons.








Detected in the








mitochondrial








and microsomal








fractions of








HEK-293 cells








and released into the








cytoplasm following








cell stress.



KNG1_HUMAN
Kininogen-1
KNG1

LungCancers,
Secreted,
UniProt,






Benign-Nodules,
extracellular
Detection,






Symptoms
space.
Prediction


KPYM_HUMAN
Pyruvate
PKM2
Secreted,
LungCancers,
Cytoplasm.
Literature,



kinase

EPI
Symptoms
Nucleus.
Detection



isozymes



Note = Translocates




M1/M2



to the nucleus in








response to different








apoptotic








stimuli. Nuclear








translocation is








sufficient to








induce cell








death that is caspase








independent,








isoform-specific and








independent of








its enzymatic activity.



KRT35_HUMAN
Keratin, type I
KRT35
ENDO


Detection,



cuticular Ha5




Prediction


LAMB2_HUMAN
Laminin
LAMB2
ENDO
LungCancers,
Secreted,
UniProt,



subunit


Symptoms
extracellular space,
Detection,



beta-2



extracellular matrix,
Prediction







basement membrane.








Note = S-laminin








is concentrated








in the synaptic cleft








of the neuromuscular








junction.



LDHA_HUMAN
L-lactate
LDHA
Secreted,
LungCancers
Cytoplasm.
Literature,



dehydrogenase

EPI, ENDO


Detection,



A chain




Prediction


LDHB_HUMAN
L-lactate
LDHB
EPI
LungCancers
Cytoplasm.
Detection,



dehydrogenase




Prediction



B chain







LEG1_HUMAN
Galectin-1
LGALS1
Secreted
LungCancers
Secreted,
UniProt,







extracellular space,
Detection







extracellular matrix.



LEG3_HUMAN
Galectin-3
LGALS3

LungCancers,
Nucleus.
Literature,






Benign-Nodules
Note = Cytoplasmic
Detection,







in adenomas and
Prediction







carcinomas.








May be secreted by a








non-classical








secretory pathway and








associate with








the cell surface.



LEG9_HUMAN
Galectin-9
LGALS9
ENDO
Symptoms
Cytoplasm (By
UniProt







similarity).








Secreted (By








similarity).








Note = May also








be secreted by a








non-classical








secretory pathway








(By similarity).



LG3BP_HUMAN
Galectin-3-
LGALS3BP
Secreted
LungCancers,
Secreted.
UniProt,



binding


Benign-Nodules,
Secreted,
Literature,



protein


Symptoms
extracellular space,
Detection,







extracellular matrix.
Prediction


LPLC3_HUMAN
Long palate, lung
C20orf185

LungCancers
Secreted (By
UniProt,



and nasal epithelium



similarity).
Prediction



carcinoma-associated



Cytoplasm.




protein 3



Note = According to








PubMed: 12837268








it is cytoplasmic.



LPLC4_HUMAN
Long palate, lung
C20orf186

LungCancers
Secreted (By
UniProt,



and nasal epithelium



similarity).
Prediction



carcinoma-associated



Cytoplasm.




protein 4







LPPRC_HUMAN
Leucine-
LRPPRC
Secreted,
LungCancers,
Mitochondrion.
Prediction



rich PPR

ENDO
Symptoms
Nucleus, nucleoplasm.




motif-



Nucleus inner




containing



membrane.




protein,



Nucleus outer




mitochondrial



membrane.








Note = Seems to








be predominantly








mitochondrial.



LRP1_HUMAN
Prolow-density
LRP1
EPI
LungCancers,
Low-density
UniProt,



lipoprotein


Symptoms
lipoprotein
Detection



receptor-related



receptor-related




protein 1



protein 1 85








kDa subunit:








Cell membrane;








Single-pass type I








membrane protein.








Membrane,








coated pit.|Low-








density lipoprotein








receptor-related








protein 1 515








kDa subunit:








Cell membrane;








Peripheral membrane








protein;








Extracellular side.








Membrane,








coated pit.|Low-








density lipoprotein








receptor-related








protein 1








intracellular domain:








Cytoplasm. Nucleus.








Note = After cleavage,








the intracellular








domain (LRPICD) is








detected both in the








cytoplasm and in the








nucleus.



LUM_HUMAN
Lumican
LUM
Secreted,
LungCancers,
Secreted,
UniProt,





EPI
Benign-Nodules,
extracellular space,
Detection,






Symptoms
extracellular matrix
Prediction







(By similarity).



LY6K_HUMAN
Lymphocyte
LY6K

LungCancers,
Secreted.
UniProt,



antigen


Symptoms
Cytoplasm. Cell
Prediction



6K



membrane; Lipid-








anchor, GPI-anchor








(Potential).



LYAM2_HUMAN
E-selectin
SELE

LungCancers,
Membrane;
UniProt,






Benign-Nodules,
Single-pass type I
Literature,






Symptoms
membrane protein.
Detection


LYAM3_HUMAN
P-selectin
SELP

LungCancers,
Membrane;
UniProt,






Benign-Nodules,
Single-pass type I
Literature,






Symptoms
membrane protein.
Detection


LYOX_HUMAN
Protein-
LOX

LungCancers,
Secreted,
UniProt,



lysine 6-oxidase


Benign-Nodules
extracellular space.
Detection,








Prediction


LYPD3_HUMAN
Ly6/PLAUR
LYPD3

LungCancers
Cell membrane;
Detection,



domain-containing



Lipid-anchor,
Prediction



protein 3



GPI-anchor.



MAGA4_HUMAN
Melanoma-associated
MAGEA4

LungCancers

Literature,



antigen 4




Prediction


MASP1_HUMAN
Mannan-binding
MASP1

LungCancers,
Secreted.
UniProt,



lectin serine


Symptoms

Detection,



protease 1




Prediction


MDHC_HUMAN
Malate
MDH1
Secreted

Cytoplasm.
Literature,



dehydrogenase,




Detection,



cytoplasmic




Prediction


MDHM_HUMAN
Malate
MDH2
ENDO
LungCancers
Mitochondrion
Detection,



dehydrogenase,



matrix.
Prediction



mitochondrial







MIF_HUMAN
Macrophage
MIF
Secreted
LungCancers,
Secreted.
UniProt,



migration


Benign-Nodules,
Cytoplasm.
Literature,



inhibitory


Symptoms
Note = Does not have
Prediction



factor



a cleavable signal








sequence and is








secreted via a








specialized, non-








classical pathway.








Secreted by








macrophages upon








stimulation by








bacterial








lipopolysaccharide








(LPS), or by








M. tuberculosis








antigens.



MLH1_HUMAN
DNA mismatch
MLH1
ENDO
LungCancers,
Nucleus.
Literature



repair


Benign-Nodules,





protein Mlh1


Symptoms




MMP1_HUMAN
Interstitial
MMP1

LungCancers,
Secreted,
UniProt,



collagenase


Benign-Nodules,
extracellular space,
Literature,






Symptoms
extracellular matrix
Prediction







(Probable).



MMP11_HUMAN
Stromelysin-3
MMP11

LungCancers,
Secreted,
UniProt,






Symptoms
extracellular space,
Literature,







extracellular matrix
Prediction







(Probable).



MMP12_HUMAN
Macrophage
MMP12

LungCancers,
Secreted,
UniProt,



metalloelastase


Benign-Nodules,
extracellular space,
Literature,






Symptoms
extracellular matrix
Prediction







(Probable).



MMP14_HUMAN
Matrix
MMP14
ENDO
LungCancers,
Membrane;
UniProt,



metalloproteinase-14


Benign-Nodules,
Single-pass type I
Literature,






Symptoms
membrane protein
Detection







(Potential).








Melanosome.








Note = Identified by








mass spectrometry in








melanosome








fractions from








stage I to stage IV.



MMP2_HUMAN
72 kDa
MMP2

LungCancers,
Secreted,
UniProt,



type IV


Benign-Nodules,
extracellular space,
Literature,



collagenase


Symptoms
extracellular matrix
Detection,







(Probable).
Prediction


MMP26_HUMAN
Matrix
MMP26

LungCancers
Secreted,
UniProt,



metallopro-



extracellular space,
Prediction



teinase-26



extracellular matrix.



MMP7_HUMAN
Matrilysin
MMP7

LungCancers,
Secreted,
UniProt,






Benign-Nodules,
extracellular space,
Literature,






Symptoms
extracellular matrix
Prediction







(Probable).



MMP9_HUMAN
Matrix
MMP9

LungCancers,
Secreted,
UniProt,



metallopro-


Benign-Nodules,
extracellular space,
Literature,



teinase-9


Symptoms
extracellular matrix
Detection,







(Probable).
Prediction


MOGS_HUMAN
Mannosyl-
MOGS
ENDO

Endoplasmic
UniProt,



oligosaccharide



reticulum membrane;
Prediction



glucosidase



Single-pass type II








membrane protein.



MPRI_HUMAN
Cation-independent
IGF2R
EPI, ENDO
LungCancers,
Lysosome membrane;
UniProt,



mannose-6-phosphate


Symptoms
Single-pass type I
Literature,



receptor



membrane protein.
Detection


MRP3_HUMAN
Canalicular
ABCC3
EPI
LungCancers
Membrane;
Literature,



multispecific



Multi-pass membrane
Detection



organic anion



protein.




transporter 2







MUC1_HUMAN
Mucin-1
MUC1
EPI
LungCancers,
Apical cell membrane;
UniProt,






Benign-Nodules,
Single-pass type I
Literature,






Symptoms
membrane protein.
Prediction







Note = Exclusively








located in the apical








domain of the plasma








membrane of








highly polarized








epithelial cells.








After endocytosis,








internalized and








recycled to the








cell membrane.








Located to microvilli








and to the tips of








long filopodial








protusions.|Isoform 5:








Secreted.|Isoform 7:








Secreted.|Isoform 9:








Secreted.|Mucin-1








subunit beta:








Cell membrane.








Cytoplasm. Nucleus.








Note = On EGF








and PDGFRB








stimulation,








transported to








the nucleus through








interaction with








CTNNB1, a








process which








is stimulated by








phosphorylation.








On HRG stimulation,








colocalizes with








JUP/gamma-catenin








at the nucleus.



MUC16_HUMAN
Mucin-16
MUC16

LungCancers
Cell membrane;
UniProt,







Single-pass type I
Detection







membrane protein.








Secreted,








extracellular space.








Note = May be








liberated into








the extracellular








space following the








phosphorylation








of the intracellular C-








terminus which








induces the proteolytic








cleavage and








liberation of the








extracellular domain.



MUC4_HUMAN
Mucin-4
MUC4

LungCancers,
Membrane;
UniProt






Benign-Nodules
Single-pass membrane








protein (Potential).








Secreted.








Note = Isoforms








lacking the Cys-








rich region, EGF-like








domains and








transmembrane








region are secreted.








Secretion occurs by








splicing or








proteolytic








processing. Mucin-








4 beta chain:








Cell membrane;








Single- pass








membrane








protein.|Mucin-








4 alpha chain:








Secreted.|Isoform








3: Cell membrane;








Single-pass membrane








protein.|Isoform








15: Secreted.



MUC5B_HUMAN
Mucin-5B
MUC5B

LungCancers,
Secreted.
UniProt,






Benign-Nodules

Detection,








Prediction


MUCL1_HUMAN
Mucin-like
MUCL1

LungCancers
Secreted (Probable).
UniProt,



protein 1



Membrane (Probable).
Prediction


NAMPT_HUMAN
Nicotinamide
NAMPT
EPI
LungCancers,
Cytoplasm (By
Literature,



phosphoribosyl-


Benign-Nodules,
similarity).
Detection



transferase


Symptoms




NAPSA_HUMAN
Napsin-A
NAPSA
Secreted
LungCancers

Prediction


NCF4_HUMAN
Neutrophil cytosol
NCF4
ENDO

Cytoplasm.
Prediction



factor 4







NDKA_HUMAN
Nucleoside
NME1
Secreted
LungCancers,
Cytoplasm.
Literature,



diphosphate


Benign-Nodules,
Nucleus.
Detection



kinase A


Symptoms
Note = Cell-cycle








dependent nuclear








localization which








can be induced by








interaction with








Epstein-barr viral








proteins or by








degradation of the








SET complex by








GzmA.



NDKB_HUMAN
Nucleoside
NME2
Secreted,
Benign-Nodules
Cytoplasm.
Literature,



diphosphate

EPI

Nucleus.
Detection



kinase B



Note = Isoform 2








is mainly








cytoplasmic and








isoform 1 and








isoform 2 are








excluded from








the nucleolus.



NDUS1_HUMAN
NADH-ubiquinone
NDUFS1
Secreted,
Symptoms
Mitochondrion
Prediction



oxidoreductase

ENDO

inner




75 kDa subunit,



membrane.




mitochondrial







NEBL_HUMAN
Nebulette
NEBL
ENDO


Prediction


NEK4_HUMAN
Serine/threonine-
NEK4
ENDO
LungCancers
Nucleus
Prediction



protein kinase Nek4



(Probable).



NET1_HUMAN
Netrin-1
NTN1

LungCancers,
Secreted,
UniProt,






Benign-Nodules
extracellular space,
Literature,







extracellular matrix
Prediction







(By similarity).



NEU2_HUMAN
Vasopressin-
AVP

LungCancers,
Secreted.
UniProt,



neurophysin 2-


Symptoms

Prediction



copeptin







NGAL_HUMAN
Neutrophil
LCN2
EPI
LungCancers,
Secreted.
UniProt,



gelatinase-associated


Benign-Nodules,

Detection,



lipocalin


Symptoms

Prediction


NGLY1_HUMAN
Peptide-N(4)-(N-
NGLY1
ENDO

Cytoplasm.
Detection,



acetyl-beta-




Prediction



glucosaminyl)as-








paragine amidase







NHRF1_HUMAN
Na(+)/H(+)
SLC9A3R1
EPI
Benign-Nodules
Endomembrane
Detection



exchange



system;




regulatory



Peripheral membrane




cofactor



protein. Cell




NHE-RF1



projection,








filopodium.








Cell projection,








ruffle. Cell projection,








microvillus.








Note = Colocalizes








with actin in








microvilli-rich








apical regions of the








syncytiotrophoblast.








Found in microvilli,








ruffling membrane and








filopodia of HeLa








cells. Present in lipid








rafts of T-cells.



NIBAN_HUMAN
Protein
FAM129A
EPI

Cytoplasm.
Literature,



Niban




Detection


NMU_HUMAN
Neuromedin-U
NMU

LungCancers
Secreted.
UniProt,








Prediction


NRP1_HUMAN
Neuropilin-1
NRP1

LungCancers,
Cell membrane;
UniProt,






Benign-Nodules,
Single-pass type I
Literature,






Symptoms
membrane
Detection,







protein.|Isoform
Prediction







2: Secreted.



ODAM_HUMAN
Odontogenic
ODAM

LungCancers
Secreted (By
UniProt,



ameloblast-associated



similarity).
Prediction



protein







OSTP_HUMAN
Osteopontin
SPP1

LungCancers,
Secreted.
UniProt,






Benign-Nodules,

Literature,






Symptoms

Detection,








Prediction


OVOS2_HUMAN
Ovostatin
OVOS2
ENDO

Secreted (By
UniProt,



homolog 2



similarity).
Prediction


P5CS_HUMAN
Delta-1-pyrroline-5-
ALDH18A1
ENDO

Mitochondrion
Prediction



carboxylate synthase



inner membrane.



PA2GX_HUMAN
Group 10 secretory
PLA2G10

Symptoms
Secreted.
UniProt



phospholipase A2







PAPP1_HUMAN
Pappalysin-1
PAPPA

LungCancers,
Secreted.
UniProt,






Benign-Nodules,

Literature,






Symptoms

Prediction


PBIP1_HUMAN
Pre-B-cell
PBXIP1
EPI

Cytoplasm,
Prediction



leukemia



cytoskeleton.




transcription



Nucleus.




factor-



Note = Shuttles




interacting



between the




protein 1



nucleus and the








cytosol. Mainly








localized in the








cytoplasm,








associated with








microtubules.








Detected in








small amounts








in the nucleus.



PCBP1_HUMAN
Poly(rC)-
PCBP1
EPI, ENDO

Nucleus.
Detection,



binding



Cytoplasm.
Prediction



protein 1



Note = Loosely








bound in the








nucleus. May








shuttle between








the nucleus and








the cytoplasm.



PCBP2_HUMAN
Poly(rC)-
PCBP2
EPI

Nucleus.
Detection,



binding



Cytoplasm.
Prediction



protein 2



Note = Loosely








bound in the








nucleus. May








shuttle between








the nucleus and








the cytoplasm.



PCD15_HUMAN
Protocadherin-
PCDH15
ENDO

Cell membrane;
UniProt,



15



Single-pass type I
Detection







membrane








protein (By








similarity).|Isoform








3: Secreted.



PCNA_HUMAN
Proliferating
PCNA
EPI
LungCancers,
Nucleus.
Literature,



cell nuclear


Benign-Nodules,

Prediction



antigen


Symptoms




PCYOX_HUMAN
Prenylcysteine
PCYOX1
Secreted
LungCancers,
Lysosome.
Detection,



oxidase 1


Symptoms

Prediction


PDGFA_HUMAN
Platelet-derived
PDGFA

LungCancers
Secreted.
UniProt,



growth factor




Literature,



subunit A




Prediction


PDGFB_HUMAN
Platelet-derived
PDGFB

LungCancers,
Secreted.
UniProt,



growth factor


Benign-Nodules,

Literature,



subunit B


Symptoms

Detection,








Prediction


PDGFD_HUMAN
Platelet-derived
PDGFD

LungCancers
Secreted.
UniProt,



growth factor D




Prediction


PDIA3_HUMAN
Protein
PDIA3
ENDO
LungCancers
Endoplasmic
Detection,



disulfide-isomerase



reticulum lumen (By
Prediction



A3



similarity).








Melanosome.








Note = Identified by








mass spectrometry in








melanosome








fractions from








stage I to stage IV.



PDIA4_HUMAN
Protein
PDIA4
Secreted,

Endoplasmic
Detection,



disulfide-

EPI, ENDO

reticulum lumen.
Prediction



isomerase



Melanosome.




A4



Note = Identified by








mass spectrometry in








melanosome








fractions from








stage I to stage IV.



PDIA6_HUMAN
Protein
PDIA6
Secreted,

Endoplasmic
Detection,



disulfide-

EPI, ENDO

reticulum lumen (By
Prediction



isomerase



similarity).




A6



Melanosome.








Note = Identified by








mass spectrometry in








melanosome








fractions from








stage I to stage IV.



PECA1_HUMAN
Platelet endothelial
PECAM1

LungCancers,
Cell membrane;
UniProt,



cell adhesion


Benign-Nodules,
Single-pass type I
Literature,



molecule


Symptoms
membrane protein.
Detection


PEDF_HUMAN
Pigment
SERPINF1

LungCancers,
Secreted.
UniProt,



epithelium-


Symptoms
Melanosome.
Literature,



derived



Note = Enriched
Detection,



factor



in stage I
Prediction







melanosomes.



PERM_HUMAN
Myeloperoxidase
MPO
Secreted,
LungCancers,
Lysosome.
Literature,





EPI, ENDO
Benign-Nodules,

Detection,






Symptoms

Prediction


PERP1_HUMAN
Plasma
PACAP
EPI, ENDO

Secreted (Potential).
UniProt,



cell-



Cytoplasm.
Detection,



induced



Note = In
Prediction



resident



(PubMed: 11350957)




endoplasmic



diffuse granular




reticulum



localization in




protein



the cytoplasm








surrounding the








nucleus.



PGAM1_HUMAN
Phosphoglycerate
PGAM1
Secreted,
LungCancers,

Detection



mutase 1

EPI
Symptoms




PLAC1_HUMAN
Placenta-specific
PLAC1

LungCancers
Secreted
UniProt,



protein 1



(Probable).
Prediction


PLACL_HUMAN
Placenta-specific
PLAC1L

LungCancers
Secreted
UniProt,



1-like protein



(Potential).
Prediction


PLIN2_HUMAN
Perilipin-2
ADFP
ENDO
LungCancers
Membrane;
Prediction







Peripheral membrane








protein.



PLIN3_HUMAN
Perilipin-3
M6PRBP1
EPI

Cytoplasm.
Detection,







Endosome membrane;
Prediction







Peripheral membrane








protein;








Cytoplasmic








side (Potential).








Lipid droplet








(Potential).








Note = Membrane








associated on








endosomes.








Detected in the








envelope and the core








of lipid bodies and in








lipid sails.



PLOD1_HUMAN
Procollagen-
PLOD1
EPI, ENDO

Rough endoplasmic
Prediction



lysine, 2-



reticulum membrane;




oxoglutarate 5-



Peripheral membrane




dioxygenase 1



protein;








Lumenal side.



PLOD2_HUMAN
Procollagen-
PLOD2
ENDO
Benign-Nodules,
Rough endoplasmic
Prediction



lysine, 2-


Symptoms
reticulum membrane;




oxoglutarate 5-



Peripheral membrane




dioxygenase 2



protein;








Lumenal side.



PLSL_HUMAN
Plastin-2
LCP1
Secreted,
LungCancers
Cytoplasm,
Detection,





EPI

cytoskeleton.
Prediction







Cell junction.








Cell projection.








Cell projection,








ruffle membrane;








Peripheral membrane








protein;








Cytoplasmic side (By








similarity).








Note = Relocalizes








to the immunological








synapse between








peripheral blood T








lymphocytes








and antibody-








presenting cells








in response to








costimulation through








TCR/CD3 and








CD2 or CD28.








Associated with the








actin cytoskeleton at








membrane ruffles (By








similarity).








Relocalizes to actin-








rich cell projections








upon serine








phosphorylation.



PLUNC_HUMAN
Protein
PLUNC

LungCancers,
Secreted (By
UniProt,



Plunc


Benign-Nodules
similarity).
Prediction







Note = Found in








the nasal mucus








(By similarity).








Apical side of airway








epithelial cells.








Detected in








nasal mucus








(By similarity).



PLXB3_HUMAN
Plexin-B3
PLXNB3
ENDO

Membrane;
UniProt,







Single-pass type I
Detection,







membrane protein.
Prediction


PLXC1_HUMAN
Plexin-C1
PLXNC1
EPI

Membrane;
UniProt,







Single-pass type I
Detection







membrane protein








(Potential).



POSTN_HUMAN
Periostin
POSTN
Secreted,
LungCancers,
Secreted,
UniProt,





ENDO
Benign-Nodules,
extracellular space,
Literature,






Symptoms
extracellular matrix.
Detection,








Prediction


PPAL_HUMAN
Lysosomal
ACP2
EPI
Symptoms
Lysosome membrane;
UniProt,



acid



Single-pass membrane
Prediction



phosphatase



protein;








Lumenal side.








Lysosome lumen.








Note = The








soluble form arises by








proteolytic








processing of








the membrane-








bound form.



PPBT_HUMAN
Alkaline phosphatase,
ALPL
EPI
LungCancers,
Cell membrane;
Literature,



tissue-nonspecific


Benign-Nodules,
Lipid-anchor,
Detection,



isozyme


Symptoms
GPI-anchor.
Prediction


PPIB_HUMAN
Peptidyl-
PPIB
Secreted,

Endoplasmic
Detection,



prolyl cis-

EPI, ENDO

reticulum lumen.
Prediction



trans



Melanosome.




isomerase



Note = Identified by




B



mass spectrometry in








melanosome








fractions from








stage I to stage IV.



PRDX1_HUMAN
Peroxiredoxin-
PRDX1
EPI
LungCancers
Cytoplasm.
Detection,



1



Melanosome.
Prediction







Note = Identified by








mass spectrometry in








melanosome








fractions from








stage I to stage IV.



PRDX4_HUMAN
Peroxiredoxin-
PRDX4
Secreted,

Cytoplasm.
Literature,



4

EPI, ENDO


Detection,








Prediction


PROF1_HUMAN
Profilin-1
PFN1
Secreted,
LungCancers
Cytoplasm,
Detection





EPI

cytoskeleton.



PRP31_HUMAN
U4/U6
PRPF31
ENDO

Nucleus speckle.
Prediction



small nuclear



Nucleus, Cajal body.




ribonucleo



Note = Predominantly




protein



found in speckles and




Prp31



in Cajal bodies.



PRS6A_HUMAN
26S
PSMC3
EPI
Benign-Nodules
Cytoplasm
Detection



protease



(Potential).




regulatory



Nucleus




subunit 6A



(Potential).



PSCA_HUMAN
Prostate
PSCA

LungCancers
Cell membrane;
Literature,



stem cell



Lipid-anchor,
Prediction



antigen



GPI-anchor.



PTGIS_HUMAN
Prostacyclin
PTGIS
EPI
LungCancers,
Endoplasmic
UniProt,



synthase


Benign-Nodules
reticulum membrane;
Detection,







Single-pass membrane
Prediction







protein.



PTPA_HUMAN
Serine/threonine-
PPP2R4
ENDO
Symptoms

Detection,



protein




Prediction



phosphatase 2A








activator







PTPRC_HUMAN
Receptor-type
PTPRC
Secreted,
LungCancers
Membrane;
UniProt,



tyrosine-protein

EPI, ENDO

Single-pass type I
Detection,



phosphatase C



membrane protein.
Prediction


PTPRJ_HUMAN
Receptor-type
PTPRJ
EPI
LungCancers,
Membrane;
UniProt,



tyrosine-protein


Symptoms
Single-pass type I
Detection,



phosphatase eta



membrane protein.
Prediction


PVR_HUMAN
Poliovirus
PVR

Symptoms
Isoform Alpha:
UniProt,



receptor



Cell membrane;
Detection,







Single-pass type I
Prediction







membrane








protein.|Isoform








Delta: Cell membrane;








Single-pass type I








membrane








protein.|Isoform








Beta:








Secreted.|Isoform








Gamma:








Secreted.



RAB32_HUMAN
Ras-related
RAB32
EPI

Mitochondrion.
Prediction



protein Rab-32







RAGE_HUMAN
Advanced
AGER
Secreted
LungCancers,
Isoform 1: Cell
UniProt,



glycosylation


Benign-Nodules
membrane;
Literature



end product-



Single-pass type I




specific



membrane




receptor



protein.|Isoform








2: Secreted.



RAN_HUMAN
GTP-
RAN
Secreted,
LungCancers,
Nucleus.
Detection,



binding

EPI
Benign-Nodules
Cytoplasm.
Prediction



nuclear



Melanosome.




protein Ran



Note = Becomes








dispersed throughout








the cytoplasm








during mitosis.








Identified by mass








spectrometry in








melanosome








fractions from








stage I to stage IV.



RAP2B_HUMAN
Ras-related
RAP2B
EPI

Cell membrane;
Prediction



protein



Lipid-anchor;




Rap-2b



Cytoplasmic








side (Potential).



RAP2C_HUMAN
Ras-related
RAP2C
EPI

Cell membrane;
Prediction



protein



Lipid-anchor;




Rap-2c



Cytoplasmic








side (Potential).



RCN3_HUMAN
Reticulocalbin-
RCN3
EPI
Symptoms
Endoplasmic
Prediction



3



reticulum lumen








(Potential).



RL24_HUMAN
60S
RPL24
EPI


Prediction



ribosomal








protein L24







S10A1_HUMAN
Protein
S100A1

Symptoms
Cytoplasm.
Literature,



S100-A1




Prediction


S10A6_HUMAN
Protein
S100A6
Secreted
LungCancers
Nucleus
Literature,



S100-A6



envelope.
Detection,







Cytoplasm.
Prediction


S10A7_HUMAN
Protein
S100A7

LungCancers
Cytoplasm.
UniProt,



S100-A7



Secreted.
Literature,







Note = Secreted
Detection,







by a non-classical
Prediction







secretory pathway.



SAA_HUMAN
Serum
SAA1

Symptoms
Secreted.
UniProt,



amyloid A




Literature,



protein




Detection,








Prediction


SCF_HUMAN
Kit ligand
KITLG

LungCancers,
Isoform 1: Cell
UniProt,






Symptoms
membrane;
Literature







Single-pass type I








membrane protein (By








similarity).








Secreted (By








similarity).








Note = Also exists as a








secreted soluble








form (isoform 1








only) (By








similarity).|Isoform








2: Cell membrane;








Single-pass type I








membrane protein (By








similarity).








Cytoplasm,








cytoskeleton








(By similarity).



SDC1_HUMAN
Syndecan-1
SDC1

LungCancers,
Membrane;
UniProt,






Benign-Nodules,
Single-pass type I
Literature,






Symptoms
membrane protein.
Detection


SEM3G_HUMAN
Semaphorin-3G
SEMA3G

LungCancers
Secreted (By
UniProt,







similarity).
Prediction


SEPR_HUMAN
Seprase
FAP
ENDO
Symptoms
Cell membrane;
UniProt,







Single-pass type II
Literature,







membrane protein.
Detection







Cell projection,








lamellipodium








membrane;








Single-pass type II








membrane protein.








Cell projection,








invadopodium








membrane;








Single-pass type II








membrane protein.








Note = Found in








cell surface








lamellipodia,








invadopodia and on








shed vesicles.



SERPH_HUMAN
Serpin H1
SERPINH1
Secreted,
LungCancers,
Endoplasmic
Detection,





EPI, ENDO
Benign-Nodules
reticulum lumen.
Prediction


SFPA2_HUMAN
Pulmonary
SFTPA2
Secreted
LungCancers,
Secreted,
UniProt,



surfactant-associated


Benign-Nodules
extracellular space,
Prediction



protein A2



extracellular matrix.








Secreted,








extracellular








space, surface film.



SFTA1_HUMAN
Pulmonary
SFTPA1
Secreted
LungCancers,
Secreted,
UniProt,



surfactant-associated


Benign-Nodules,
extracellular space,
Prediction



protein A1


Symptoms
extracellular matrix.








Secreted,








extracellular








space, surface film.



SG3A2_HUMAN
Secretoglobin
SCGB3A2

LungCancers,
Secreted.
UniProt,



family 3A member 2


Benign-Nodules

Prediction


SGPL1_HUMAN
Sphingosine-1-
SGPL1
ENDO

Endoplasmic
UniProt,



phosphate



reticulum membrane;
Prediction



lyase 1



Single-pass type III








membrane protein.



SIAL_HUMAN
Bone
IBSP

LungCancers
Secreted.
UniProt,



sialoprotein




Literature,



2




Prediction


SLPI_HUMAN
Antileukopro-
SLPI

LungCancers,
Secreted.
UniProt,



teinase


Benign-Nodules

Literature,








Detection,








Prediction


SMD3_HUMAN
Small nuclear
SNRPD3
Secreted
Benign-Nodules
Nucleus.
Prediction



ribonucleo








protein Sm D3







SMS_HUMAN
Somatostatin
SST

LungCancers
Secreted.
UniProt,








Literature,








Prediction


SODM_HUMAN
Superoxide
SOD2
Secreted
LungCancers,
Mitochondrion
Literature,



dismutase


Benign-Nodules,
matrix.
Detection,



[Mn], mitochondrial


Symptoms

Prediction


SORL_HUMAN
Sortilin-
SORL1
EPI
LungCancers,
Membrane;
UniProt,



related


Symptoms
Single-pass type I
Detection



receptor



membrane protein








(Potential).



SPB3_HUMAN
Serpin B3
SERPINB3

LungCancers,
Cytoplasm.
Literature,






Benign-Nodules
Note = Seems to
Detection







also be secreted








in plasma by








cancerous cells








but at a low level.



SPB5_HUMAN
Serpin B5
SERPINB5

LungCancers
Secreted,
UniProt,







extracellular space.
Detection


SPON2_HUMAN
Spondin-2
SPON2

LungCancers,
Secreted,
UniProt,






Benign-Nodules
extracellular space,
Prediction







extracellular








matrix (By similarity).



SPRC_HUMAN
SPARC
SPARC

LungCancers,
Secreted,
UniProt,






Benign-Nodules,
extracellular space,
Literature,






Symptoms
extracellular matrix,
Detection,







basement membrane.
Prediction







Note = In or








around the basement








membrane.



SRC_HUMAN
Proto-oncogene
SRC
ENDO
LungCancers,

Literature



tyrosine-protein


Benign-Nodules,





kinase Src


Symptoms




SSRD_HUMAN
Translocon-
SSR4
Secreted,

Endoplasmic
UniProt,



associated protein

ENDO

reticulum membrane;
Prediction



subunit delta



Single-pass type I








membrane protein.



STAT1_HUMAN
Signal transducer and
STAT1
EPI
LungCancers,
Cytoplasm.
Detection



activator of


Benign-Nodules
Nucleus.




transcription



Note = Translocated




1-alpha/beta



into the nucleus in








response to








IFN-gamma-induced








tyrosine








phosphorylation








and dimerization.



STAT3_HUMAN
Signal
STAT3
ENDO
LungCancers,
Cytoplasm.
Prediction



transducer


Benign-Nodules,
Nucleus.




and


Symptoms
Note = Shuttles




activator of



between the




transcription



nucleus and the




3



cytoplasm.








Constitutive nuclear








presence is








independent of








tyrosine








phosphorylation.



STC1_HUMAN
Stanniocalcin-
STC1

LungCancers,
Secreted.
UniProt,



1


Symptoms

Prediction


STT3A_HUMAN
Dolichyl-
STT3A
EPI
Symptoms
Endoplasmic
Literature



diphosphooligosac-



reticulum membrane;




charide--protein



Multi-pass membrane




glycosyltransferase



protein.




subunit STT3A







TAGL_HUMAN
Transgelin
TAGLN
EPI
LungCancers
Cytoplasm
Literature,







(Probable).
Prediction


TARA_HUMAN
TRIO and
TRIOBP
ENDO

Nucleus.
Detection,



F-actin-



Cytoplasm,
Prediction



binding



cytoskeleton.




protein



Note = Localized








to F-actin in a








periodic pattern.



TBA1B_HUMAN
Tubulin
TUBA1B
EPI
LungCancers

Detection



alpha-1B chain







TBB2A_HUMAN
Tubulin
TUBB2A
EPI
LungCancers,

Detection,



beta-2A chain


Benign-Nodules

Prediction


TBB3_HUMAN
Tubulin
TUBB3
EPI
LungCancers,

Detection



beta-3 chain


Benign-Nodules




TBB5_HUMAN
Tubulin
TUBB
EPI
LungCancers,

Detection



beta chain


Benign-Nodules




TCPA_HUMAN
T-complex
TCP1
EPI

Cytoplasm.
Prediction



protein 1 subunit








alpha







TCPD_HUMAN
T-complex
CCT4
EPI

Cytoplasm.
Detection,



protein 1



Melanosome.
Prediction



subunit



Note = Identified by




delta



mass spectrometry in








melanosome








fractions from








stage I to stage IV.



TCPQ_HUMAN
T-complex protein 1
CCT8
Secreted,

Cytoplasm.
Prediction



subunit theta

EPI





TCPZ_HUMAN
T-complex protein 1
CCT6A
Secreted,

Cytoplasm.
Detection



subunit zeta

EPI





TDRD3_HUMAN
Tudor
TDRD3
ENDO

Cytoplasm.
Prediction



domain-



Nucleus.




containing



Note = Predominantly




protein 3



cytoplasmic.








Associated with








actively translating








polyribosomes and








with mRNA stress








granules.



TENA_HUMAN
Tenascin
TNC
ENDO
LungCancers,
Secreted,
UniProt,






Benign-Nodules,
extracellular space,
Literature,






Symptoms
extracellular matrix.
Detection


TENX_HUMAN
Tenascin-X
TNXB
ENDO
LungCancers,
Secreted,
UniProt,






Symptoms
extracellular space,
Detection,







extracellular matrix.
Prediction


TERA_HUMAN
Transitional
VCP
EPI
LungCancers,
Cytoplasm, cytosol.
Detection



endoplasmic


Benign-Nodules
Nucleus.




reticulum



Note = Present in




ATPase



the neuronal hyaline








inclusion bodies








specifically found in








motor neurons from








amyotrophic








lateral sclerosis








patients. Present








in the Lewy bodies








specifically found in








neurons from








Parkinson








disease patients.



TETN_HUMAN
Tetranectin
CLEC3B

LungCancers
Secreted.
UniProt,








Literature,








Detection,








Prediction


TF_HUMAN
Tissue
F3

LungCancers,
Membrane;
UniProt,



factor


Benign-Nodules,
Single-pass type I
Literature






Symptoms
membrane protein.



TFR1_HUMAN
Transferrin
TFRC
Secreted,
LungCancers,
Cell membrane;
UniProt,



receptor

EPI, ENDO
Benign-Nodules,
Single-pass type II
Literature,



protein 1


Symptoms
membrane protein.
Detection







Melanosome.








Note = Identified by








mass spectrometry in








melanosome








fractions from








stage I to stage








IV.|Transferrin








receptor protein








1, serum form:








Secreted.



TGFA_HUMAN
Protransforming
TGFA

LungCancers,
Transforming
UniProt,



growth


Benign-Nodules
growth factor
Literature



factor alpha



alpha: Secreted,








extracellular








space.|Protransforming








growth factor alpha:








Cell membrane;








Single-pass type I








membrane protein.



THAS_HUMAN
Thromboxane-
TBXAS1
EPI, ENDO
LungCancers,
Membrane;
Prediction



A


Benign-Nodules,
Multi-pass




synthase


Symptoms
membrane protein.



THY1_HUMAN
Thy-1
THY1
EPI
Symptoms
Cell membrane;
Detection,



membrane



Lipid-anchor,
Prediction



glycoprotein



GPI-anchor (By








similarity).



TIMP1_HUMAN
Metalloproteinase
TIMP1

LungCancers,
Secreted.
UniProt,



inhibitor 1


Benign-Nodules,

Literature,






Symptoms

Detection,








Prediction


TIMP3_HUMAN
Metalloproteinase
TIMP3

LungCancers,
Secreted,
UniProt,



inhibitor 3


Benign-Nodules
extracellular space,
Literature,







extracellular matrix.
Prediction


TLL1_HUMAN
Tolloid-like protein
TLL1
ENDO

Secreted
UniProt,



1



(Probable).
Prediction


TNF12_HUMAN
Tumor
TNFSF12

LungCancers,
Cell membrane;
UniProt



necrosis


Benign-Nodules
Single-pass type II




factor



membrane




ligand



protein.|Tumor




superfamily



necrosis factor ligand




member 12



superfamily member








12, secreted form:








Secreted.



TNR6_HUMAN
Tumor
FAS

LungCancers,
Isoform 1: Cell
UniProt,



necrosis


Benign-Nodules,
membrane;
Literature,



factor


Symptoms
Single-pass type I
Prediction



receptor



membrane




superfamily



protein.|Isoform




member 6



2: Secreted.|Isoform








3: Secreted.|Isoform








4: Secreted.|Isoform








5: Secreted.|Isoform








6: Secreted.



TPIS_HUMAN
Triosephosphate
TPI1
Secreted,
Symptoms

Literature,



isomerase

EPI


Detection,








Prediction


TRFL_HUMAN
Lactotransferrin
LTF
Secreted,
LungCancers,
Secreted.
UniProt,





EPI, ENDO
Benign-Nodules,

Literature,






Symptoms

Detection,








Prediction


TSP1_HUMAN
Thrombospondin-
THBS1

LungCancers,

Literature,



1


Benign-Nodules,

Detection,






Symptoms

Prediction


TTHY_HUMAN
Transthyretin
TTR

LungCancers,
Secreted.
UniProt,






Benign-Nodules
Cytoplasm.
Literature,








Detection,








Prediction


TYPH_HUMAN
Thymidine
TYMP
EPI
LungCancers,

Literature,



phosphorylase


Benign-Nodules,

Detection,






Symptoms

Prediction


UGGG1_HUMAN
UDP-glucose:
UGGT1
Secreted,

Endoplasmic
Detection,



glycoprotein

ENDO

reticulum lumen.
Prediction



glucosyltransferase



Endoplasmic




1



reticulum-Golgi








intermediate








compartment.



UGGG2_HUMAN
UDP-glucose:
UGGT2
ENDO

Endoplasmic
Prediction



glycoprotein



reticulum lumen.




glucosyltransferase



Endoplasmic




2



reticulum-Golgi








intermediate








compartment.



UGPA_HUMAN
UTP--glucose-1-
UGP2
EPI
Symptoms
Cytoplasm.
Detection



phosphate








uridylyltransferase







UPAR_HUMAN
Urokinase
PLAUR

LungCancers,
Isoform 1: Cell
UniProt,



plasminogen


Benign-Nodules,
membrane;
Literature,



activator


Symptoms
Lipid-anchor,
Prediction



surface receptor



GPI-anchor.|Isoform








2: Secreted








(Probable).



UTER_HUMAN
Uteroglobin
SCGB1A1

LungCancers,
Secreted.
UniProt,






Benign-Nodules,

Literature,






Symptoms

Detection,








Prediction


VA0D1_HUMAN
V-type proton
ATP6V0D1
EPI


Prediction



ATPase subunit d 1







VAV3_HUMAN
Guanine nucleotide
VAV3
ENDO


Prediction



exchange factor








VAV3







VEGFA_HUMAN
Vascular
VEGFA

LungCancers,
Secreted.
UniProt,



endothelial


Benign-Nodules,
Note = VEGF12
Literature,



growth


Symptoms
1 is acidic and
Prediction



factor A



freely secreted.








VEGF165 is








more basic, has








heparin-binding








properties and,








although a signicant








proportion








remains cell-








associated, most








is freely secreted.








VEGF189 is








very basic, it is








cell-associated








after secretion








and is bound avidly








by heparin and the








extracellular matrix,








although it may








be released as a








soluble form by








heparin, heparinase or








plasmin.



VEGFC_HUMAN
Vascular
VEGFC

LungCancers,
Secreted.
UniProt,



endothelial


Benign-Nodules

Literature,



growth factor C




Prediction


VEGFD_HUMAN
Vascular
FIGF

LungCancers
Secreted.
UniProt,



endothelial




Literature,



growth factor D




Prediction


VGFR1_HUMAN
Vascular
FLT1

LungCancers,
Isoform Flt1:
UniProt,



endothelial


Benign-Nodules,
Cell membrane;
Literature,



growth


Symptoms
Single-pass type I
Detection,



factor



membrane
Prediction



receptor 1



protein.|Isoform








sFlt1: Secreted.



VTNC_HUMAN
Vitronectin
VTN
ENDO
Symptoms
Secreted,
UniProt,







extracellular
Literature,







space.
Detection,








Prediction


VWC2_HUMAN
Brorin
VWC2

LungCancers
Secreted,
UniProt,







extracellular space,
Prediction







extracellular matrix,








basement membrane








(By similarity).



WNT3A_HUMAN
Protein
WNT3A

LungCancers,
Secreted,
UniProt,



Wnt-3a


Symptoms
extracellular space,
Prediction







extracellular matrix.



WT1_HUMAN
Wilms
WT1

LungCancers,
Nucleus.
Literature,



tumor


Benign-Nodules,
Cytoplasm (By
Prediction



protein


Symptoms
similarity).








Note = Shuttles








between nucleus and








cytoplasm (By








similarity).|Isoform








1: Nucleus








speckle.|Isoform








4: Nucleus,








nucleoplasm.



ZA2G_HUMAN
Zinc-alpha-
AZGP1

LungCancers,
Secreted.
UniProt,



2-


Symptoms

Literature,



glycoprotein




Detection,








Prediction


ZG16B_HUMAN
Zymogen granule
ZG16B

LungCancers
Secreted
UniProt,



protein 16 homolog B



(Potential).
Prediction









190 of these candidate protein biomarkers were shown to be measured reproducibly in blood. A moderately powered multisite and unbiased study of 242 blood samples from patients with PN was designed to determine whether a statistically significant subpanel of proteins could be identified to distinguish benign and malignant nodules of sizes under 2 cm. The three sites contributing samples and clinical data to this study were the University of Laval, University of Pennsylvania and New York University.
In an embodiment of the invention, a panel of 15 proteins effectively distinguished between samples derived from patients with benign and malignant nodules less than 2 cm diameter.
Bioinformatic and biostatistical analyses were used first to identify individual proteins with statistically significant differential expression, and then using these proteins to derive one or more combinations of proteins or panels of proteins, which collectively demonstrated superior discriminatory performance compared to any individual protein. Bioinformatic and biostatistical methods are used to derive coefficients (C) for each individual protein in the panel that reflects its relative expression level, i.e. increased or decreased, and its weight or importance with respect to the panel's net discriminatory ability, relative to the other proteins. The quantitative discriminatory ability of the panel can be expressed as a mathematical algorithm with a term for each of its constituent proteins being the product of its coefficient and the protein's plasma expression level (P) (as measured by LC-SRM-MS), e.g. C×P, with an algorithm consisting of n proteins described as: C1×P1+C2×P2+C3×P3+ . . . +Cn×Pn. An algorithm that discriminates between disease states with a predetermined level of statistical significance may be refers to a “disease classifier”. In addition to the classifier's constituent proteins with differential expression, it may also include proteins with minimal or no biologic variation to enable assessment of variability, or the lack thereof, within or between clinical specimens; these proteins may be termed typical native proteins and serve as internal controls for the other classifier proteins.
In certain embodiments, expression levels are measured by MS. MS analyzes the mass spectrum produced by an ion after its production by the vaporization of its parent protein and its separation from other ions based on its mass-to-charge ratio. The most common modes of acquiring MS data are 1) full scan acquisition resulting in the typical total ion current plot (TIC), 2) selected ion monitoring (SIM), and 3) selected reaction monitoring (SRM).
In certain embodiments of the methods provided herein, biomarker protein expression levels are measured by LC-SRM-MS. LC-SRM-MS is a highly selective method of tandem mass spectrometry which has the potential to effectively filter out all molecules and contaminants except the desired analyte(s). This is particularly beneficial if the analysis sample is a complex mixture which may comprise several isobaric species within a defined analytical window. LC-SRM-MS methods may utilize a triple quadrupole mass spectrometer which, as is known in the art, includes three quadrupole rod sets. A first stage of mass selection is performed in the first quadrupole rod set, and the selectively transmitted ions are fragmented in the second quadrupole rod set. The resultant transition (product) ions are conveyed to the third quadrupole rod set, which performs a second stage of mass selection. The product ions transmitted through the third quadrupole rod set are measured by a detector, which generates a signal representative of the numbers of selectively transmitted product ions. The RF and DC potentials applied to the first and third quadrupoles are tuned to select (respectively) precursor and product ions that have m/z values lying within narrow specified ranges. By specifying the appropriate transitions (m/z values of precursor and product ions), a peptide corresponding to a targeted protein may be measured with high degrees of sensitivity and selectivity. Signal-to-noise ratio is superior to conventional tandem mass spectrometry (MS/MS) experiments, which select one mass window in the first quadrupole and then measure all generated transitions in the ion detector. LC-SRM-MS.
In certain embodiments, an SRM-MS assay for use in diagnosing or monitoring lung cancer as disclosed herein may utilize one or more peptides and/or peptide transitions derived from the proteins set forth in Table 6. In certain embodiments, the assay may utilize peptides and/or peptide transitions from 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 345 or more, or 371 or more biomarker proteins. In certain embodiments, two or more peptides may be utilized per biomarker proteins, and in certain of these embodiments three or more of four or more peptides may be utilized. Similarly, in certain embodiments two or more transitions may be utilized per peptide, and in certain of these embodiments three or more; four or more; or five or more transitions may be utilized per peptide. In one embodiment, an LC-SRM-MS assay for use in diagnosing lung cancer may measure the intensity of five transitions that correspond to selected peptides associated with each biomarker protein. The achievable limit of quantification (LOQ) may be estimated for each peptide according to the observed signal intensities during this analysis. For examples, for sets of target proteins associated with lung cancer see Table 12.
The expression level of a biomarker protein can be measured using any suitable method known in the art, including but not limited to mass spectrometry (MS), reverse transcriptase-polymerase chain reaction (RT-PCR), microarray, serial analysis of gene expression (SAGE), gene expression analysis by massively parallel signature sequencing (MPSS), immunoassays (e.g., ELISA), immunohistochemistry (IHC), transcriptomics, and proteomics.
To evaluate the diagnostic performance of a particular set of peptide transitions, a ROC curve is generated for each significant transition.
An “ROC curve” as used herein refers to a plot of the true positive rate (sensitivity) against the false positive rate (specificity) for a binary classifier system as its discrimination threshold is varied. A ROC curve can be represented equivalently by plotting the fraction of true positives out of the positives (TPR=true positive rate) versus the fraction of false positives out of the negatives (FPR=false positive rate). Each point on the ROC curve represents a sensitivity/specificity pair corresponding to a particular decision threshold. FIGS. 7 and 9 provide a graphical representation of the functional relationship between the distribution of biomarker or biomarker panel sensitivity and specificity values in a cohort of diseased subjects and in a cohort of non-diseased subjects.
AUC represents the area under the ROC curve. The AUC is an overall indication of the diagnostic accuracy of 1) a biomarker or a panel of biomarkers and 2) a ROC curve. AUC is determined by the “trapezoidal rule.” For a given curve, the data points are connected by straight line segments, perpendiculars are erected from the abscissa to each data point, and the sum of the areas of the triangles and trapezoids so constructed is computed. In certain embodiments of the methods provided herein, a biomarker protein has an AUC in the range of about 0.75 to 1.0. In certain of these embodiments, the AUC is in the range of about 0.8 to 0.8, 0.9 to 0.95, or 0.95 to 1.0.
The methods provided herein are minimally invasive and pose little or no risk of adverse effects. As such, they may be used to diagnose, monitor and provide clinical management of subjects who do not exhibit any symptoms of a lung condition and subjects classified as low risk for developing a lung condition. For example, the methods disclosed herein may be used to diagnose lung cancer in a subject who does not present with a PN and/or has not presented with a PN in the past, but who nonetheless deemed at risk of developing a PN and/or a lung condition. Similarly, the methods disclosed herein may be used as a strictly precautionary measure to diagnose healthy subjects who are classified as low risk for developing a lung condition.
The present invention provides a method of determining the likelihood that a lung condition in a subject is cancer by measuring an abundance of a panel of proteins in a sample obtained from the subject; calculating a probability of cancer score based on the protein measurements and ruling out cancer for the subject if the score) is lower than a pre-determined score, wherein when cancer is ruled out the subject does not receive a treatment protocol. Treatment protocols include for example pulmonary function test (PFT), pulmonary imaging, a biopsy, a surgery, a chemotherapy, a radiotherapy, or any combination thereof. In some embodiments, the imaging is an x-ray, a chest computed tomography (CT) scan, or a positron emission tomography (PET) scan.
The present invention further provides a method of ruling in the likelihood of cancer for a subject by measuring an abundance of panel of proteins in a sample obtained from the subject, calculating a probability of cancer score based on the protein measurements and ruling in the likelihood of cancer for the subject if the score in step is higher than a pre-determined score
In another aspect the invention further provides a method of determining the likelihood of the presence of a lung condition in a subject by measuring an abundance of panel of proteins in a sample obtained from the subject, calculating a probability of cancer score based on the protein measurements and concluding the presence of said lung condition if the score is equal or greater than a pre-determined score. The lung condition is lung cancer such as for example, non-small cell lung cancer (NSCLC). The subject at risk of developing lung cancer
The panel includes at least 4 proteins selected from ALDOA, FRIL, LG3BP, IBP3, LRP1, ISLR, TSP COIA1, GRP78, TETN, PRXD1 and CD14. Optionally, the panel further includes at least one protein selected from BGH3, COIA1, TETN, GRP78, PRDX, FIBA and GSLG1.
The subject has or is suspected of having a pulmonary nodule. The pulmonary nodule has a diameter of less than or equal to 3 cm. In one embodiment, the pulmonary nodule has a diameter of about 0.8 cm to 2.0 cm.
The score is calculated from a logistic regression model applied to the protein measurements. For example, the score is determined as Ps=1/[1+exp(−α−Σi=1Nβi*{hacek over (I)}i,s)], where {hacek over (I)}i,s is logarithmically transformed and normalized intensity of transition i in said sample (s), βi is the corresponding logistic regression coefficient, a was a panel-specific constant, and N was the total number of transitions in said panel.
In various embodiments, the method of the present invention further comprises normalizing the protein measurements. For example, the protein measurements are normalized by one or more proteins selected from PEDF, MASP1, GELS, LUM, C163A and PTPRJ.
The biological sample such as for example tissue, blood, plasma, serum, whole blood, urine, saliva, genital secretion, cerebrospinal fluid, sweat and excreta.
In one aspect, the determining the likelihood of cancer is determined by the sensitivity, specificity, negative predictive value or positive predictive value associated with the score. The score determined has a negative predictive value (NPV) is at least about 80%.
The measuring step is performed by selected reaction monitoring mass spectrometry, using a compound that specifically binds the protein being detected or a peptide transition. In one embodiment, the compound that specifically binds to the protein being measured is an antibody or an aptamer.
In specific embodiments, the diagnostic methods disclosed herein are used to rule out a treatment protocol for a subject, measuring the abundance of a panel of proteins in a sample obtained from the subject, calculating a probability of cancer score based on the protein measurements and ruling out the treatment protocol for the subject if the score determined in the sample is lower than a pre-determined score. In some embodiments the panel contains at least 4 proteins selected ALDOA, FRIL, LG3BP, IBP3, LRP1, ISLR, TSP, COIA1, GRP78, TETN, PRXD1 and CD14
Optionally, the panel further comprises one or more proteins selected from ERO1A, 6PGD, GSTP1, GGH, PRDX1, CD14, PTPA, ICAM1, FOLH1, SODM, FIBA, GSLG1, RAP2B, or C163A or one or more proteins selected from LRP1, COIA1, TSP1, ALDOA, GRP78, FRIL, LG3BP, BGH3, ISLR, PRDX1, FIBA, or GSLG. In preferred embodiments, the panel contains at least TSP1, LG3BP, LRP1, ALDOA, and COIA1. In more a preferred embodiment, the panel contains at least TSP1, LRP1, ALDOA and COIA1.
In specific embodiments, the diagnostic methods disclosed herein are used to rule in a treatment protocol for a subject by measuring the abundance of a panel of proteins in a sample obtained from the subject, calculating a probability of cancer score based on the protein measurements and ruling in the treatment protocol for the subject if the score determined in the sample is greater than a pre-determined score. In some embodiments the panel contains at least 4 proteins selected ALDOA, FRIL, LG3BP, IBP3, LRP1, ISLR or TSP1 or ALDOA, FRIL, LG3BP, IBP3, LRP1, ISLR, TSP COIA1, GRP78, TETN, PRXD1 and CD14. Optionally, the panel further comprises one or more proteins selected from ERO1A, 6PGD, GSTP1, COIA1, GGH, PRDX1, SEM3G, GRP78, TETN, AIFM1, MPRI, TNF12, MMP9 or OSTP or COIA1, TETN, GRP78, APOE or TBB3.
In certain embodiments, the diagnostic methods disclosed herein can be used in combination with other clinical assessment methods, including for example various radiographic and/or invasive methods. Similarly, in certain embodiments, the diagnostic methods disclosed herein can be used to identify candidates for other clinical assessment methods, or to assess the likelihood that a subject will benefit from other clinical assessment methods.
The high abundance of certain proteins in a biological sample such as plasma or serum can hinder the ability to assay a protein of interest, particularly where the protein of interest is expressed at relatively low concentrations. Several methods are available to circumvent this issue, including enrichment, separation, and depletion. Enrichment uses an affinity agent to extract proteins from the sample by class, e.g., removal of glycosylated proteins by glycocapture. Separation uses methods such as gel electrophoresis or isoelectric focusing to divide the sample into multiple fractions that largely do not overlap in protein content. Depletion typically uses affinity columns to remove the most abundant proteins in blood, such as albumin, by utilizing advanced technologies such as IgY14/Supermix (Sigma St. Louis, Mo.) that enable the removal of the majority of the most abundant proteins.
In certain embodiments of the methods provided herein, a biological sample may be subjected to enrichment, separation, and/or depletion prior to assaying biomarker or putative biomarker protein expression levels. In certain of these embodiments, blood proteins may be initially processed by a glycocapture method, which enriches for glycosylated proteins, allowing quantification assays to detect proteins in the high pg/ml to low ng/ml concentration range. Exemplary methods of glycocapture are well known in the art (see, e.g., U.S. Pat. No. 7,183,188; U.S. Patent Appl. Publ. No. 2007/0099251; U.S. Patent Appl. Publ. No. 2007/0202539; U.S. Patent Appl. Publ. No. 2007/0269895; and U.S. Patent Appl. Publ. No. 2010/0279382). In other embodiments, blood proteins may be initially processed by a protein depletion method, which allows for detection of commonly obscured biomarkers in samples by removing abundant proteins. In one such embodiment, the protein depletion method is a GenWay depletion method.
In certain embodiments, a biomarker protein panel comprises two to 100 biomarker proteins. In certain of these embodiments, the panel comprises 2 to 5, 6 to 10, 11 to 15, 16 to 20, 21-25, 5 to 25, 26 to 30, 31 to 40, 41 to 50, 25 to 50, 51 to 75, 76 to 100, biomarker proteins. In certain embodiments, a biomarker protein panel comprises one or more subpanels of biomarker proteins that each comprise at least two biomarker proteins. For example, biomarker protein panel may comprise a first subpanel made up of biomarker proteins that are overexpressed in a particular lung condition and a second subpanel made up of biomarker proteins that are under-expressed in a particular lung condition.
In certain embodiments of the methods, compositions, and kits provided herein, a biomarker protein may be a protein that exhibits differential expression in conjunction with lung cancer. For example, in certain embodiments a biomarker protein may be one of the proteins associated with lung cancer set forth in Table 6.
In other embodiments, the diagnosis methods disclosed herein may be used to distinguish between two different lung conditions. For example, the methods may be used to classify a lung condition as malignant lung cancer versus benign lung cancer, NSCLC versus SCLC, or lung cancer versus non-cancer condition (e.g., inflammatory condition).
In certain embodiments, kits are provided for diagnosing a lung condition in a subject. These kits are used to detect expression levels of one or more biomarker proteins. Optionally, a kit may comprise instructions for use in the form of a label or a separate insert. The kits can contain reagents that specifically bind to proteins in the panels described, herein. These reagents can include antibodies. The kits can also contain reagents that specifically bind to mRNA expressing proteins in the panels described, herein. These reagents can include nucleotide probes. The kits can also include reagents for the detection of reagents that specifically bind to the proteins in the panels described herein. These reagents can include fluorophores.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention
EXAMPLES
Example 1
Identification of Lung Cancer Biomarker Proteins
A retrospective, case-control study design was used to identify biomarker proteins and panels thereof for diagnosing various lung diseases in pre-defined control and experimental groups. The first goal of these studies was to demonstrate statistically significant differential expression for individual proteins between control and experimental groups. The second goal is to identify a panel of proteins which all individually demonstrate statistically significant differential expression between control and experimental groups. This panel of proteins can then be used collectively to distinguish between dichotomous disease states.
Specific study comparisons may include 1) cancer vs. non-cancer, 2) small cell lung cancer versus non-small cell lung cancer (NSCLC), 3) cancer vs. inflammatory disease state (e.g., infectious granuloma), or 4) different nodule size, e.g., <10 mm versus ≧10 mm (alternatively using 10, 15 or 20 mm cut-offs depending upon sample distributions).
Data for each subject consisted of the following:
Archived plasma samples from subjects previously enrolled in Institute Review Board (IRB)-approved studies was used to identify biomarker proteins and biomarker panels for distinguishing lung malignancies from non-malignancies. Plasma samples were originally obtained by routine phlebotomy, aliquotted, and stored at −80° C. or lower. Sample preparation, assignment of subject identification codes, initial subject record entry, and specimen storage were performed as per IRB study protocols. Sample eligibility is based on clinical parameters, including the subject, PN, and clinical staging parameters. Parameters for inclusion and exclusion are set forth in Table 7.







TABLE 7





Inclusion Criteria
















Sample
Sample eligibility will be based on clinical parameters,


Inclusion
including the following subject, nodule and clinical


Criteria
staging parameters:



Subject









age ≧40



any smoking status, e.g. current, former, or never



co-morbid conditions, e.g. COPD



prior malignancy—only skin carcinomas—squamous



or basal cell









Nodule









radiology









size ≧4 mm and ≦30 mm



solid, semi-solid or non-solid



any spiculation or ground glass opacity









pathology









malignant - e.g. adenocarcinoma, squamous,



or large cell



benign - inflammatory (e.g. granulomatous,



infectious) or non-inflammatory (e.g. hamartoma)



confirmed by biopsy, surgery or stability of lung



nodule for 2 years or more.









Clinical stage









Primary tumor: ≦T1 (e.g. 1A, 1B)



Regional lymph nodes: N0 or N1 only



Distant metastasis: M0 only








Sample
Subject








Exclusion
prior malignancy within 5 years of lung nodule diagnosis








Criteria
Nodule









size data unavailable



for cancer or benign nodule, no pathology or follow-up



CT data available









Clinical stage









Primary tumor: ≧T2



Regional lymph nodes: ≧N2



Distant metastasis: ≧M1









The assignment of a sample to a control or experimental group, and its further stratification or matching to other samples within and between these groups, is dependent on various clinical data about the subject. This data includes, for example, demographic information such as age, gender, and clinical history (e.g., smoking status), co-morbid conditions, PN characterization, and pathologic interpretation of resected lesions and tissues (Table 8).









TABLE 8








1.
Enrollment Data



a.
Demographics—age, birth date, gender, ethnicity



b.
Measurements—Height (cm) and weight (kg)



c.
Smoking history—never, former, or current with pack-




year estimation



d.
Medical history—details of co-morbid conditions, e.g.




chronic obstructive pulmonary disease (COPD),




inflammatory or autoimmune diseases, endocrine




(diabetes), and cardiovascular



e.
Medication history—current medications, dosages and 




indications



f.
Radiographic data and nodule characteristics









1) nodule size in millimeters (width × height × length)



2) location, e.g. right or left and upper, lower or



middle



3) quality, e.g. solid, semi-solid, ground glass,



calcified, etc.










2.
Diagnostic Evaluation Data



a.
Primary diagnosis and associated reports (clinical




history, physical exam, and laboratory tests report)



b.
Pulmonary Function Tests (PFTs), if available



c.
Follow-up CT scans—subsequent nodule evaluations by




chest CT



d.
PET scan



e.
Clinical Staging



f.
Biopsy procedures









1) FNA or TTNA



2) bronchoscopy with transbronchial or needle biopsy



3) surgical diagnostic procedures, e.g. VATS and/or



thoracotomy










3.
Radiology Report(s)



4.
Pathology Report(s)



5.
Blood Sample Collection Information



6.
Reporting of Adverse Events



a.
AEs resulting from center's SOC, e.g. procedural




morbidity.








Subject
demographics—e.g. age, gender, ethnicity



smoking status—e.g. never-, former- (“ex-”) or current- 



smoker; pack-years



clinical history—e.g. co-morbid conditions, e.g. COPD,



infection


Nodule
size—e.g. planar (width × height × length) and volume



dimensions



appearance—e.g. calcifications, ground glass appearance,



eccentricity


Pathology
primary lung vs. systemic disorder



malignancy status—malignant vs. benign (vs. indeterminate)



histopathology—e.g. small cell lung cancer (SCLC) vs. non-



small cell lung cancer (NSCLC-adenocarcinoma, squamous



carcinoma, large cell carcinoma); other types, e.g.



hematologic, carcinoid, etc.



immunologically quiescent, e.g. hamartoma, vs. inflammatory,



e.g. granulomatous and/or infectious, e.g. fungal









The study design and analytical plan prioritizes the control:experimental group pairings set forth in Table 9. Additional clinical and molecular insights may be gained by selective inclusion of phenotypes, e.g. effect of smoking, in the assignment of experimental and control groups. Demographic information available in the clinical database will enable further refinements in sample selection via the stratification or matching of samples in the case-control analyses with respect to clinical parameters, e.g., age and nodule size.







TABLE 9







Assignment of Experimental and Control Groups


to Achieve Proteomic Analysis Objectives












Experimental



Analysis
Objective
Group
Control Group





1
Differentiate
A. Cancer
Any non-



cancer from benign
nodule
malignant



lung nodule

(benign)





phenotype with





nodule ≧4 mm in





diameter


2
Differentiate
A. Cancer
Non-malignant



cancer from non-
nodule
(non-benign) lung



malignant

disorder, e.g.



(inflammatory,

granulomatous



infectious)

(fungal) disease,



lung nodule

with nodule









LC-SRM-MS is performed to identify and quantify various plasma proteins in the plasma samples. Prior to LC-SRM-MS analysis, each sample is depleted using IgY14/Supermix (Sigma) and then trypsin-digested. Samples from each control or experimental group are batched randomly and processed together on a QTrap 5500 instrument (AB SCIEX, Foster City, Calif.) for unbiased comparisons. Each sample analysis takes approximately 30 minutes. Peak areas for two transitions (native and heavy label) are collected and reported for all peptides and proteins. The data output for each protein analyzed by LC-SRM-MS typically yields four measurements consisting of two transition measurements from each of two peptides from the same protein. These measurements enable an inference of the relative abundance of the target protein, which will be used as its expression level in the bioinformatics and statistical analyses.
Identification of biomarker proteins having differential expression levels between the control and experimental groups yields one or more novel proteomic profiles. For example, biomarker proteins are identified with expression levels that differ in subjects with PNs who are diagnosed with NSCLC versus those without an NSCLC diagnosis, or in subjects with PNs who are diagnosed with NSCLC versus an inflammatory disorder. Panels of biomarker proteins are also identified which can collectively discriminate between dichotomous disease states.
Analyses may be (a priori) powered appropriately to control type 1 and type 2 errors at 0.05 and to detect inter-cohort differences of 25% per analyte. The diagnostic power of individual proteins is generally assessed to distinguish between two cohorts, assuming a one-sided paired non-parametric test is used. This provides a lower bound on the sample size required to demonstrate differential expression between experimental and control groups. Multiple testing effects apply for the identification of panels of proteins for assessing diagnostic efficacy, which requires larger sample sizes.
The sequence of steps for determining statistical significance for differential expression of an individual protein includes the following: 1) assessing and correlating the calibrated values of transitions of a single protein (a quality control measure); 2) comparing paired analysis of groups to control for other influences using the Mann-Whitney U-test (rank sum) to determine statistical significance; and 3) determining its significance based on a pre-defined significance threshold. Transitions within a protein that are not correlated across samples (e.g., Pearson correlation <0.5) will be deemed unreliable and excluded from the analysis.
Comparison of calibrated samples between two cohorts, e.g., cancer and non-cancer, requires pairing or matching using a variety of clinical parameters such as nodule size, age and gender. Such pairing controls for the potential influence of these other parameters on the actual comparison goal, e.g. cancer and non-cancer. A non-parametric test such as the Mann-Whitney U-test (rank sum) will then be applied to measure the statistical difference between the groups. The resulting p value can be adjusted using multiple testing corrections such as the false discovery rate. Permutation tests can be used for further significance assessments.
Significance will be determined by the satisfaction of a pre-defined threshold, such as 0.05, to filter out assays, with the potential use of higher threshold values for additional filtering. An additional significance criterion is that two of three replicate assays must individually be significant in order for the assay, e.g., single protein, to be significant.
Panels of proteins that individually demonstrate statistically significant differential expression as defined above and which can collectively be used to distinguish dichotomous disease states are identified using statistical methods described herein. This requires developing multivariate classifiers and assessing sensitivity, specificity, and ROC AUC for panels. In addition, protein panels with optimal discriminatory performance, e.g., ROC AUC, are identified and may be sufficient for clinical use in discriminating disease states.
The sequence of steps for determining the statistical significance of the discriminatory ability of a panel of proteins includes 1) developing multivariate classifiers for protein panels, and 2) identifying a protein panel with optimal discriminatory performance, e.g. ROC AUC, for a set of disease states.
A multivariate classifier (e.g., majority rule) will be developed for protein panels, including single protein assays deemed to be significant. The sensitivity and specificity of each classifier will be determined and used to generate a receiver operating characteristics (ROC) curve and its AUC to assess a given panel's discriminatory performance for a specific comparison, e.g. cancer versus non-cancer.
Protocol
1. Review clinical data from a set of subjects presenting with lung disease.
2. Provide plasma samples from the subjects wherein the samples are either benign, cancerous, COPD or another lung disease.
3. Group the plasma samples that are benign or cancerous by PNs that are separated by size of the nodule.
4. Target a pool of 371 putative lung cancer biomarker proteins consisting of at least two peptides per protein and at least two LC-SRM-MS transitions per peptide. Measuring the LC-SRM-MS transitions in each specimen along with 5 synthetic internal standards consisting of 10 transitions to compare peptide transitions from the plasma to the synthetic internal standards by LC-SRM-MS mass spectroscopy.
5. Quantitate the intensity of each transition.
6. Normalize the quantitated transitions to internal standards to obtain a normalized intensity.
7. Review the measured peptide transitions for correlations from the same peptide, rejecting discordant transitions.
8. Generate an ROC for each transition by comparing cancerous with benign samples. (ROC compare specificity (true positive) to (1-sensitivity) false positive).
9. Define the AUC for each transition. (An AUC of 0.5 is a random classifier; 1.0 is a perfect classifier).
10. Determine an AUC cut-off point to determine transitions that are statistically significant.
11. Define the transitions that exceed the AUC cutoff point.
12. Combine all pairings of significant transitions.
13. Define a new AUC for each transition pair by means of logistical regression.
14. Repeat pairing combinations into triples, quad, etc.; defining a new AUC based upon the logistical regression of combined transitions until a panel of biomarker transitions with combined desired performance (sensitivity & specificity) have been achieved.
15. The panel of biomarker transitions is verified against previously unused set of plasma panels.
Example 2
Diagnosis/Classification of Lung Disease Using Biomarker Proteins
Plasma samples will be obtained from one or more subjects presenting with PNs to evaluate whether the subjects have a lung condition. The plasma samples will be depleted using IgY14/Supermix (Sigma) and optionally subjected to one or more rounds of enrichment and/or separation, and then trypsinized. The expression level of one or more biomarker proteins previously identified as differentially expressed in subjects with the lung condition will be measured using an LC-SRM-MS assay. The LC-SRM-MS assay will utilize two to five peptide transitions for each biomarker protein. For example, the assay may utilize one or more of the peptide transitions generated from any of the proteins listed in Table 6. Subjects will be classified as having the lung condition if one or more of the biomarker proteins exhibit expression levels that differ significantly from the pre-determined control expression level for that protein.
Example 3
Blood-Based Diagnostic Test to Determine the Likelihood that a Pulmonary Nodule (PN) is Benign or Malignant
A panel of 15 proteins was created where the concentration of these 15 proteins relative to the concentration of 6 protein standards is indicative of likelihood of cancer. The relative concentration of these 15 proteins to the 6 protein standards was measured using a mass spectrometry methodology. A classification algorithm is used to combine these relative concentrations into a relative likelihood of the PN being benign or malignant. Further it has been demonstrated that there are many variations on these panels that are also diagnostic tests for the likelihood that a PN is benign or malignant. Variations on the panel of proteins, protein standards, measurement methodology and/or classification algorithm are described herein.
Study Design
A Single Reaction Monitoring (SRM) mass spectrometry (MS) assay was developed consisting of 1550 transitions from 345 lung cancer associated proteins. The SRM-MS assay and methodology is described above. The goal of this study was to develop a blood-based diagnostic for classifying PNs under 2 cm in size as benign or malignant. The study design appears in Table 10.







TABLE 10







Study Design










Small (<2 cm)
Large (>2 cm)














Laval
UPenn
NYU
Laval
UPenn
NYU
















Benign
14
29
29
13
21
15


Malignant
14
29
29
13
21
15


Batches
1
2
2
1
2
1










72 vs 72 (94% power)
49 vs 49 (74% power)









The study consisted of 242 plasma samples from three sites (Laval, UPenn and NYU). The number of benign and malignant samples from each site are indicated in Table 10. The study consisted of 144 plasma samples from patients with PNs of size 2 cm or less and of 98 samples from patients with PNs of size larger than 2 cm. This resulted in an estimated power of 94% for discovering proteins with blood concentrations of 1.5 fold or more between benign and malignant cancer samples of size 2 cm or less. Power is 74% for PNs of size larger than 2 cm.
This study was a retrospective multisite study that was intended to derive protein biomarkers of lung cancer that are robust to site-to-site variation. The study included samples larger than 2 cm to ensure that proteins not detectable due to the limit of detection of the measurement technology (LC-SRM-MS) for tumors of size 2 cm or less could still be detected in tumors of size 2 cm or larger.
Samples from each site and in each size class (above and below 2 cm) were matched on nodule size, age and gender.
Sample Analysis
Each sample was analyzed using the LC-SRM-MS measurement methodology as follows:
1. Samples were depleted of high abundance proteins using the IGy14 and Supermix depletion columns from Sigma-Aldrich.
2. Samples were digested using trypsin into tryptic peptides.
3. Samples were analyzed by LC-SRM-MS using a 30 minute gradient on a Waters nanoacuity LC system followed by SRM-MS analysis of the 1550 transitions on a AB-Sciex 5500 triple quad device.
4. Raw transition ion counts were obtained and recorded for each of the 1550 transitions.
It is important to note that matched samples were processed at each step either in parallel (steps 2 and 4) or back-to-back serially (steps 1 and 3). This minimizes analytical variation. Finally, steps 1 and 2 of the sample analysis are performed in batches of samples according to day of processing. There were five batches of ‘small’ samples and four batches of ‘large’ samples as denoted in Table 10.
Protein Shortlist
A shortlist of 68 proteins reproducibly diagnostic across sites was derived as follows. Note that each protein can be measured by multiple transitions.
Step 1: Normalization
Six proteins were identified that had a transition detected in all samples of the study and with low coefficient of variation. For each protein the transition with highest median intensity across samples was selected as the representative transition for the protein. These proteins and transitions are found in Table 11.







TABLE 11







Normalizing Factors









Protein 
Peptide 
Transition 


(Uniprot ID)
(Amino Acid Sequence)
(m/z)












CD44_HUMAN
YGFIEGHVVIPR 
272.2



(SEQ ID NO: 1)



 


TENX_HUMAN
YEVTVVSVR (SEQ ID NO: 2)
759.5


 


CLUS_HUMAN
ASSIIDELFQDR 
565.3



(SEQ ID NO: 3)



 


IBP3_HUMAN
FLNVLSPR (SEQ ID NO: 4)
685.4


 


GELS_HUMAN
TASDFITK (SEQ ID NO: 5)
710.4


 


MASP1_HUMAN
TGVITSPDFPNPYPK 
258.10



(SEQ ID NO: 6)









We refer to the transitions in Table 11 as normalizing factors (NFs). Each of the 1550 transitions were normalized by each of the six normalizing factors where the new intensity of a transition t in a sample s by NF f, denoted New(s,t,f), is calculated as follows:

New(s,t,f)=Raw(s,t)*Median(f)/Raw(s,f)

where Raw(s,t) is the original intensity of transition t in sample s; Median(f) is the median intensity of the NF f across all samples; and Raw(s,f) is the original intensity of the NF f in sample s.
For each protein and normalized transition, the AUC of each batch was calculated. The NF that minimized the coefficient of variation across the 9 batches was selected as the NF for that protein and for all transitions of that protein. Consequently, every protein (and all of its transitions) are now normalized by a single NF.
Step 2: Reproducible Diagnostic Proteins
For each normalized transition its AUC for each of the nine batches in the study is calculated as follows. If the transition is detected in fewer than half of the cancer samples and in fewer than half of the benign samples then the batch AUC is ‘ND’. Otherwise, the batch AUC is calculated comparing the benign and cancer samples in the batch.
The batch AUC values are transformed into percentile AUC scores for each transition. That is, if a normalized transition is in the 82nd percentile of AUC scores for all transitions then it is assigned percentile AUC 0.82 for that batch.
Reproducible transitions are those satisfying at least one of the following criteria:
1. In at least four of the five small batches the percentile AUC is 75% or more (or 25% and less).
2. In at least three of the five small batches the percentile AUC is 80% or more (or 20% and less) AND the remaining percentile AUCs in the small batches are above 50% (below 50%).
3. In all five small batches the percentile AUC is above 50% (below 50%).
4. In at least three of the four large batches the percentile AUC is 85% or more (or 15% and less).
5. In at least three of the four large batches the percentile AUC is 80% or more (or 20% and less) AND the remaining percentile AUCs in the large batches are above 50% (below 50%).
6. In all four large batches the percentile AUC is above 50% (below 50%).
These criteria result in a list of 67 proteins with at least one transition satisfying one or more of the criteria. These proteins appear in Table 12.











TABLE 12







Percentage





Occurrence
Occurrence





Across131
Across 131

Uniprot


Protein (Uniprot)
Panels
Panels
Protein Names
Accession No.



















G3P_HUMAN
113
86%
Glyceraldehyde-3-phosphate
P04406





dehydrogenase; Short name = GAPDH;






Alternative name(s):






Peptidyl-cysteine S-nitrosylase GAPDH



FRIL_HUMAN
107
82%
Recommended name:
P02792





Ferritin light chain






Short name = Ferritin L subunit



HYOU1_HUMAN
69
53%
Recommended name:
Q9Y4L1





Hypoxia up-regulated protein 1






Alternative name(s):






150 kDa oxygen-regulated protein






Short name = ORP-150






170 kDa glucose-regulated protein






Short name = GRP-170



ALDOA_HUMAN
66
50%
Recommended name:
P04075





Fructose-bisphosphate aldolase A






EC = 4.1.2.13






Alternative name(s):






Lung cancer antigen NY-LU-1






Muscle-type aldolase



HXK1_HUMAN
65
50%
Recommended name:
P19367





Hexokinase-1






EC = 2.7.1.1






Alternative name(s):






Brain form hexokinase






Hexokinase type I






Short name = HK I



APOE_HUMAN
63
48%
Recommended name:
P02649





Apolipoprotein E






Short name = Apo-E



TSP1_HUMAN
63
48%
Recommended name:
P07996





Thrombospondin-1



FINC_HUMAN
62
47%
Recommended name:
P02751





Fibronectin






Short name = FN






Alternative name(s):






Cold-insoluble globulin






Short name = CIG






Cleaved into the following 4 chains:






1. Anastellin






2. Ugl-Y1






3. Ugl-Y2






4. Ugl-Y3



LRP1_HUMAN
58
44%
Recommended name:






Prolow-density lipoprotein receptor-






related protein 1






Short name = LRP-1






Alternative name(s):






Alpha-2-macroglobulin receptor






Short name = A2MR






Apolipoprotein E receptor






Short name = APOER






CD_antigen = CD91






Cleaved into the following 3 chains:






1. Low-density lipoprotein receptor-related






protein 1 85 kDa subunit






Short name = LRP-85






2. Low-density lipoprotein receptor-related






protein 1 515 kDa subunit






Short name = LRP-515






3. Low-density lipoprotein receptor-related






protein 1 intracellular domain






Short name = LRPICD



6PGD_HUMAN
50
38%
Recommended name:
P52209





6-phosphogluconate dehydrogenase,






decarboxylating



S10A6_HUMAN
47
36%
Recommended name:
P06703





Protein S100-A6






Alternative name(s):






Calcyclin






Growth factor-inducible protein 2A9






MLN 4






Prolactin receptor-associated protein






Short name = PRA






S100 calcium-binding protein A6



CALU_HUMAN
45
34%
Recommended name:
O43852





Calumenin






Alternative name(s):






Crocalbin






IEF SSP 9302



PRDX1_HUMAN
45
34%
Recommended name:
Q06830





Peroxiredoxin-1






EC = 1.11.1.15






Alternative name(s):






Natural killer cell-enhancing factor A






Short name = NKEF-A






Proliferation-associated gene protein






Short name = PAG






Thioredoxin peroxidase 2






Thioredoxin-dependent peroxidereductase 2



RAN_HUMAN
45
34%
Recommended name:
P62826





GTP-binding nuclear protein Ran






Alternative name(s):






Androgen receptor-associated protein 24






GTPase Ran






Ras-like protein TC4






Ras-related nuclear protein



CD14_HUMAN
43
33%
Recommended name:
P08571





Monocyte differentiation antigen CD14






Alternative name(s):






Myeloid cell-specific leucine-rich






glycoprotein






CD_antigen = CD14






Cleaved into the following 2 chains:






1. Monocyte differentiation antigen CD14,






urinary form






2. Monocyte differentiation antigen CD14,






membrane-bound form



AMPN_HUMAN
41
31%
Recommended name:
P15144





Aminopeptidase N






Short name = AP-N






Short name = hAPN






EC = 3.4.11.2






Alternative name(s):






Alanyl aminopeptidase






Aminopeptidase M






Short name = AP-M






Microsomal aminopeptidase






Myeloid plasma membrane glycoprotein






CD13






gp150






CD_antigen = CD13



GSLG1_HUMAN
36
27%
Recommended name:
Q92896





Golgi apparatus protein 1






Alternative name(s):






CFR-1






Cysteine-rich fibroblast growth factor






receptor






E-selectin ligand 1






Short name = ESL-1






Golgi sialoglycoprotein MG-160



1433Z_HUMAN
32
24%
Recommended name:
P63104





14-3-3 protein zeta/delta






Alternative name(s):






Protein kinase C inhibitor protein 1






Short name = KCIP-1



IBP3_HUMAN
31
24%
Recommended name:
P17936





Insulin-like growth factor-binding protein 3






Short name = IBP-3






Short name = IGF-binding protein 3






Short name = IGFBP-3



ILK_HUMAN
31
24%
Recommended name:
Q13418





Integrin-linked protein kinase






EC = 2.7.11.1






Alternative name(s):






59 kDa serine/threonine-protein kinase






ILK-1






ILK-2






p59ILK



LDHB_HUMAN
30
23%
Recommended name:
P07195





L-lactate dehydrogenase B chain






Short name = LDH-B






EC = 1.1.1.27






Alternative name(s):






LDH heart subunit






Short name = LDH-H






Renal carcinoma antigen NY-REN-46



MPRI_HUMAN
29
22%
Recommended name:
P11717





Cation-independent mannose-6-phosphate






receptor






Short name = CI Man-6-P receptor






Short name = CI-MPR






Short name = M6PR






Alternative name(s):






300 kDa mannose 6-phosphate receptor






Short name = MPR 300






Insulin-like growth factor 2 receptor






Insulin-like growth factor II receptor






Short name = IGF-II receptor






M6P/IGF2 receptor






Short name = M6P/IGF2R






CD_antigen = CD222



PROF1_HUMAN
29
22%
Recommended name:
P07737





Profilin-1






Alternative name(s):






Profilin I



PEDF_HUMAN
28
21%
Recommended name:
P36955





Pigment epithelium-derived factor






Short name = PEDF






Alternative name(s):






Cell proliferation-inducing gene 35 protein






EPC-1






Serpin F1



CLIC1_HUMAN
26
20%
Recommended name:
O00299





Chloride intracellular channel protein 1






Alternative name(s):






Chloride channel ABP






Nuclear chloride ion channel 27






Short name = NCC27






Regulatory nuclear chloride ion channel






protein






Short name = hRNCC



GRP78_HUMAN
25
19%
Recommended name:
P11021





78 kDa glucose-regulated protein






Short name = GRP-78






Alternative name(s):






Endoplasmic reticulum lumenal Ca(2+)-






binding protein grp78






Heat shock 70 kDa protein 5






Immunoglobulin heavy chain-binding






protein






Short name = BiP



CEAM8_HUMAN
24
18%
Recommended name:
P31997





Carcinoembryonic antigen-related cell






adhesion molecule 8






Alternative name(s):






CD67 antigen






Carcinoembryonic antigen CGM6






Non-specific cross-reacting antigen






NCA-95






CD_antigen = CD66b



VTNC_HUMAN
24
18%
Recommended name:
P04004





Vitronectin






Alternative name(s):






S-protein






Serum-spreading factor






V75






Cleaved into the following 3 chains:






1. Vitronectin V65 subunit






2. Vitronectin V10 subunit






3. Somatomedin-B



CERU_HUMAN
22
17%
Recommended name:
P00450





Ceruloplasmin






EC = 1.16.3.1






Alternative name(s):






Ferroxidase



DSG2_HUMAN
22
17%
Recommended name:
Q14126





Desmoglein-2






Alternative name(s):






Cadherin family member 5






HDGC



KIT HUMAN
22
17%
Recommended name:
P10721





Mast/stem cell growth factor receptor Kit






Short name = SCFR






EC = 2.7.10.1






Alternative name(s):






Piebald trait protein






Short name = PBT






Proto-oncogene c-Kit






Tyrosine-protein kinase Kit






p145 c-kit






v-kit Hardy-Zuckerman 4 feline sarcoma






viral oncogene homolog






CD_antigen = CD117



TBB3_HUMAN
22
17%
Recommended name:
Q13509





Tubulin beta-3 chain






Alternative name(s):






Tubulin beta-4 chain






Tubulin beta-III



CH10_HUMAN
21
16%
Recommended name:
P61604





10 kDa heat shock protein, mitochondrial






Short name = Hsp10






Alternative name(s):






10 kDa chaperonin






Chaperonin 10






Short name = CPN10






Early-pregnancy factor






Short name = EPF



ISLR_HUMAN
21
16%
Immunoglobulin superfamily containing
O14498





leucine-rich repeat protein



MASP1_HUMAN
21
16%
Recommended name:
P48740





Mannan-binding lectin serine protease 1






EC = 3.4.21.—






Alternative name(s):






Complement factor MASP-3






Complement-activating component of Ra-






reactive factor






Mannose-binding lectin-associated serine






protease 1






Short name = MASP-1






Mannose-binding protein-associated serine






protease






Ra-reactive factor serine protease p100






Short name = RaRF






Serine protease 5






Cleaved into the following 2 chains:






1. Mannan-binding lectin serine protease 1






heavy chain






2. Mannan-binding lectin serine protease 1






light chain



ICAM3_HUMAN
20
15%
Recommended name:
P32942





Intercellular adhesion molecule 3






Short name = ICAM-3






Alternative name(s):






CDw50






ICAM-R






CD_antigen = CD50



PTPRJ_HUMAN
20
15%
Recommended name:
Q12913





Receptor-type tyrosine-protein






phosphatase eta






Short name = Protein-tyrosine phosphatase






eta






Short name = R-PTP-eta






EC = 3.1.3.48






Alternative name(s):






Density-enhanced phosphatase 1






Short name = DEP-1






HPTP eta






Protein-tyrosine phosphatase receptor






type J






Short name = R-PTP-J






CD_antigen = CD148



A1AG1_HUMAN
19
15%
Recommended name:
P02763





Alpha-1-acid glycoprotein 1






Short name = AGP 1






Alternative name(s):






Orosomucoid-1






Short name = OMD 1



CD59_HUMAN
18
14%
Recommended name:
P13987





CD59 glycoprotein






Alternative name(s):






1F5 antigen






20 kDa homologous restriction factor






Short name = HRF-20






Short name = HRF20






MAC-inhibitory protein






Short name = MAC-IP






MEM43 antigen






Membrane attack complex inhibition






factor






Short name = MACIF






Membrane inhibitor of reactive lysis






Short name = MIRL






Protectin






CD_antigen = CD59



MDHM_HUMAN
18
14%
commended name:
P40926





Malate dehydrogenase, mitochondrial



PVR_HUMAN
18
14%
Recommended name:
P15151





Poliovirus receptor






Alternative name(s):






Nectin-like protein 5






Short name = NECL-5






CD_antigen = CD155



SEM3G_HUMAN
18
14%
Recommended name:
Q9NS98





Semaphorin-3G






Alternative name(s):






Semaphorin sem2



CO6A3_HUMAN
17
13%
Collagen alpha-3(VI) chain
P12111


MMP9_HUMAN
17
13%
Recommended name:
P14780





Matrix metalloproteinase-9






Short name = MMP-9






EC = 3.4.24.35






Alternative name(s):






92 kDa gelatinase






92 kDa type IV collagenase






Gelatinase B






Short name = GELB






Cleaved into the following 2 chains:






1. 67 kDa matrix metalloproteinase-9






2. 82 kDa matrix metalloproteinase-9



TETN_HUMAN
17
13%
Recommended name:
P05452





Tetranectin






Short name = TN






Alternative name(s):






C-type lectin domain family 3 member B






Plasminogen kringle 4-binding protein



TNF12_HUMAN
17
13%
Recommended name:
O43508





Tumor necrosis factor ligand superfamily






member 12






Alternative name(s):






APO3 ligand






TNF-related weak inducer of apoptosis






Short name = TWEAK






Cleaved into the following 2 chains:






1. Tumor necrosis factor ligand superfamily






member 12, membrane form






2. Tumor necrosis factor ligand superfamily






member 12, secreted form



BST1_HUMAN
16
12%
Recommended name:
Q10588





ADP-ribosyl cyclase 2






EC = 3.2.2.5






Alternative name(s):






Bone marrow stromal antigen 1






Short name = BST-1






Cyclic ADP-ribose hydrolase 2






Short name = cADPr hydrolase 2






CD_antigen = CD157



COIA1_HUMAN
16
12%
Recommended name:
P39060





Collagen alpha-1(XVIII) chain






Cleaved into the following chain:






1. Endostatin



CRP_HUMAN
16
12%
Recommended name:
P02741





C-reactive protein






Cleaved into the following chain:






1. C-reactive protein(1-205)



PLSL_HUMAN
16
12%
Recommended name:
P13796





Plastin-2






Alternative name(s):






L-plastin






LC64P






Lymphocyte cytosolic protein 1






Short name = LCP-1



BGH3_HUMAN
15
11%
Recommended name:
Q15582





Transforming growth factor-beta-induced






protein ig-h3






Short name = Beta ig-h3






Alternative name(s):






Kerato-epithelin






RGD-containing collagen-associated






protein






Short name = RGD-CAP



CD44_HUMAN
15
11%
Recommended name:
P16070





CD44 antigen






Alternative name(s):






CDw44






Epican






Extracellular matrix receptor III






Short name = ECMR-III






GP90 lymphocyte homing/adhesion






receptor






HUTCH-I






Heparan sulfate proteoglycan






Hermes antigen






Hyaluronate receptor






Phagocytic glycoprotein 1






Short name = PGP-1






Phagocytic glycoprotein I






Short name = PGP-I






CD_antigen = CD44



ENOA_HUMAN
15
11%
Recommended name:
P06733





Alpha-enolase






EC = 4.2.1.11






Alternative name(s):






2-phospho-D-glycerate hydrolyase






C-myc promoter-binding protein






Enolase 1






MBP-1






MPB-1






Non-neural enolase






Short name = NNE






Phosphopyruvate hydratase






Plasminogen-binding protein



LUM_HUMAN
15
11%




SCF_HUMAN
15
11%
Recommended name:
P21583





Kit ligand






Alternative name(s):






Mast cell growth factor






Short name = MGF






Stem cell factor






Short name = SCF






c-Kit ligand






Cleaved into the following chain:






1. Soluble KIT ligand






Short name = sKITLG



UGPA_HUMAN
15
11%
Recommended name:
Q16851





UTP--glucose-1-phosphate






uridylyltransferase






EC = 2.7.7.9






Alternative name(s):






UDP-glucose pyrophosphorylase






Short name = UDPGP






Short name = UGPase



ENPL_HUMAN
14
11%
Recommended name:
P14625





Endoplasmin






Alternative name(s):






94 kDa glucose-regulated protein






Short name = GRP-94






Heat shock protein 90 kDa beta member 1






Tumor rejection antigen 1






gp96 homolog



GDIR2_HUMAN
14
11%
Recommended name:
P52566





Rho GDP-dissociation inhibitor 2






Short name = Rho GDI 2






Alternative name(s):






Ly-GDI






Rho-GDI beta



GELS_HUMAN
14
11%
Recommended name:
P06396





Gelsolin






Alternative name(s):






AGEL






Actin-depolymerizing factor






Short name = ADF






Brevin



SODM_HUMAN
14
11%
Recommended name:
P04179





Superoxide dismutase [Mn], mitochondrial



TPIS_HUMAN
14
11%
Recommended name:
P60174





Triosephosphate isomerase






Short name = TIM






EC = 5.3.1.1






Alternative name(s):






Triose-phosphate isomerase



TENA_HUMAN
13
10%
Recommended name:
P24821





Tenascin






Short name = TN






Alternative name(s):






Cytotactin






GMEM






GP 150-225






Glioma-associated-extracellular matrix






antigen






Hexabrachion






JI






Myotendinous antigen






Neuronectin






Tenascin-C






Short name = TN-C



ZA2G_HUMAN
13
10%
Recommended name:
P25311





Zinc-alpha-2-glycoprotein






Short name = Zn-alpha-2-GP






Short name = Zn-alpha-2-glycoprotein



LEG1_HUMAN
11
 8%
Recommended name:
P09382





Galectin-1






Short name = Gal-1






Alternative name(s):






14 kDa laminin-binding protein






Short name = HLBP14






14 kDa lectin






Beta-galactoside-binding lectin L-14-I






Galaptin






HBL






HPL






Lactose-binding lectin 1






Lectin galactoside-binding soluble 1






Putative MAPK-activating protein PM12






S-Lac lectin 1



FOLH1_HUMAN
9
 7%
Recommended name:
Q04609





Glutamate carboxypeptidase 2






EC = 3.4.17.21






Alternative name(s):






Cell growth-inhibiting gene 27 protein






Folate hydrolase 1






Folylpoly-gamma-glutamate






carboxypeptidase






Short name = FGCP






Glutamate carboxypeptidase II






Short name = GCPII






Membrane glutamate carboxypeptidase






Short name = mGCP






N-acetylated-alpha-linked acidic






dipeptidase I






Short name = NAALADase I






Prostate-specific membrane antigen






Short name = PSM






Short name = PSMA






Pteroylpoly-gamma-glutamate






carboxypeptidase



PLXC1_HUMAN
9
 7%




PTGIS_HUMAN
9
 7%
Recommended name:
Q16647





Prostacyclin synthase






EC = 5.3.99.4






Alternative name(s):






Prostaglandin I2 synthase









Step 3: Significance and Occurrence
To find high performing panels, 10,000 trials were performed where on each trial the combined AUC of a random panel of 15 proteins selected from Table 12 was estimated. To calculate the combined AUC of each panel of 15 proteins, the highest intensity normalized transition was utilized. Logistic regression was used to calculate the AUC of the panel of 15 across all small samples. 131 panels of 15 proteins had combined AUC above 0.80, as shown in FIG. 1. (The significance by study separated into small (<2.0 cm) and large (>2.0 cm) PN are shown in FIG. 2). The resilience of the panels persisted despite site based variation in the samples as shown in FIG. 3. The panels are listed in Table 13.















TABLE 13







AUC
P1
P2
P3
P4
P5
P6
P7
P8





0.8282
CD59
CALU
LDHB
ALDOA
DSG2
MDHM
TENA
6PGD


0.8255
CD59
TSP1
KIT
ISLR
ALDOA
DSG2
14332
CD14


0.8194
S10A6
ALDOA
PVR
TSP1
CD44
CH10
PEDF 
APOE


0.8189
ALDOA
LEG1
CALU
LDHB
TETN
FOLH1
MASP1
1433Z


0.8187
PVR
CD59
CRP
ALDOA
GRP78
DSG2
6PGD
CD14


0.8171
AMPN
IBP3
CALU
CD44
BGH3
GRP78
1433Z
6PGD


0.8171
CALU
CH10
ALDOA
BST1
MDHM
VTNC
APOE 
CD14


0.8165
LDHB
CO6A3
CD44
A1AG1
GRP78
DSG2
MDHM
VTNC


0.8163
TPIS
CD59
S10A6
CALU
ENPL
CH10
ALDOA
DSG2


0.8163
LEG1 
AMPN
S10A6
CALU
ISLR 
ENOA
VTNC
6PGD


0.8161
AMPN
S10A6
TSP1
MPRI
VTNC
LUM
6PGD 
APOE


0.8159
ALDOA
AMPN
TSP1
BGH3
GRP78
PTPRJ
MASP1
CERU


0.8159
ALDOA
CO6A3 
MPRI
SEM3G
CERU
LUM
APOE
CD14


0.8159
AMPN
CALU
ISLR 
SODM
CERU 
LUM
6PGD
APOE


0.8159
CALU
PEDF
CRP
GRP78
VTNC 
1433Z
CD14
FRIL


0.8157
TPIS 
LEG1
S10A6
LDHB
TSP1
ENPL
MDHM
6PGD


0.8155
CALU
CRP
ALDOA
SODM
SEM3G
1433Z
FRIL 
G3P


0.8153
CALU
MPRI
ALDOA
PEDF
DSG2
CERU
APOE
G3P


0.814 
LEG1
COIA1 
AMPN 
S10A6
TSP1 
MPRI
PEDF 
GRP78


0.8138
TSP1
KIT
CERU
6PGD
APOE
CD14
FRIL
G3P


0.8132
S10A6
COIA1 
AMPN
TSP1
PEDF
ISLR
PTPRJ
CERU


0.8128
TPIS 
LEG1
AMPN
S10A6
IBP3
CALU
DSG2 
PTPRJ 


0.8128
TPIS 
AMPN
TSP1
PEDF
A1AG1
MPRI
ALDOA
VTNC


0.8124
ALDOA
CALU
LDHB 
PLSL
PEDF 
MASP1
6PGD 
APOE


0.8124
AMPN 
S10A6
TSP1 
ENOA
GRP78
6PGD
APOE
FRIL


0.812
IBP3
TSP1
CRP
A1AG1
SCF
ALDOA
PEDF 
DSG2


0.8106
COIA1
CALU
CD44
BGH3
ALDOA
TETN
BST1
LUM


0.8106
TSP1
PLSL
CRP
ALDOA
GRP78
MDHM
APOE
FRIL


0.8099
CD59
CALU
ENPL
CD44
ALDOA
TENA
6PGD
FRIL


0.8097
AMPN
S10A6
IBP3
A1AG1
MPRI
ALDOA
GRP78
FRIL


0.8093
ALDOA
S10A6
TSP1
ENPL
PEDF
A1AG1
GRP78
APOE


0.8093
PVR
IBP3
LDHB
SCF
TNF12
LUM
1433Z
FRIL


0.8093
CALU
LDHB
CO6A3
PEDF
CH10
BGH3
PTPRJ
ALDOA


0.8087
ALDOA
AMPN
ENPL
KIT
MPRI
GRP78
LUM
1433Z


0.8087
CD59
S10A6
IBP3
TSP1
ENPL
SODM
MDHM
6PGD


0.8083
ALDOA
AMPN
S10A6
IBP3
PLSL
CRP
SCF
MPRI


0.8081
PVR
IBP3
TSP1
CRP
ALODA
SODM
MDHM
TNF12


0.8081
S10A6
LDHB
ENPL
PLSL
CH10
CERU
FRIL
G3P


0.8081
IBP3
LDHB
PEDF
MPRI
SEM3G
VTNC
APOE
CD14


0.8079
ALDOA
AMPN
CALU
PLSL
PEDF
CH10
MASP1
TNF12


0.8077
S10A6
IBP3
LDHB
MDHM
ZA2G
FRIL
G3P
HYOU1


0.8077
CD59
S10A6
LDHB
TSP1
CD44
ISLR
CERU
1433Z


0.8077
AMPN
CALU
LDHB
TSP1
PLSL
CD44
ALDOA
TETN


0.8075
TPIS
AMPN
S10A6
TSP1
CH10
COIA1
CERU
ZA2G


0.8073
CALU
PEDF
MPRI
ISLR
BGH3
ENOA
CERU
1433Z


0.8071
TPIS
CALU
CO6A3
KIT
DSG2
MASP1
6PGD
APOE


0.8071
LEG1
COIA1
TSP1
CD44
MPRI
ALODA
FOLH1
TNF12


0.8065
AMPN
S10A6
CALU
CO6A3
TSP1
PLSL
KIT
MASP1


0.8063
S10A6
TSP1
A1AG1
BGH3
ZA2G
1433Z
FRIL
G3P


0.8063
CALU
KIT
ENOA
6PGD
APOE
CD14
G3P
ICAM3


0.8061
AMPN
MPRI
GRP78
DSG2
TENA
APOE
CD14
FRIL


0.8059
TPIS
IBP3
TSP1
PEDF
TNF12
1433Z
6PGD
APOE


0.8059
CALU
LDHB
PLSL
CRP
PEDF
SEM3G
MDHM
APOE


0.8058
ALDOA
TSP1
PLSL
CD44
KIT
CRP
ISLR
TNF12


0.8058
TPIS
TSP1
MPRI
ISLR
ALODA
PEDF
GRP78
SEM3G


0.8054
ALDOA
S1046
CALU
CRP
A1AG1
VTNC
TENA
ZA2G


0.8054
TPIS
CO6A3
TSP1
MPRI
DSG2
TNF12
FRIL
G3P


0.8054
CALU
LDHB
DSG2
1433Z
CD14
FRIL
G3P
HYOU1


0.805
CALU
MPRI
ENOA
FOLH1
LUM
ZA2G
APOE
CD14


0.8048
PVR
S10A6
IBP3
PEDF
ALDOA
BST1
MDHM
VTNC


0.8048
AMPN
CALU
CH10
DSG2
TNF12
CERU
6PGD
APOE


0.8046
ALDOA
LDHB
TSP1
KIT
ISLR
DSG2
MASP1
1433Z


0.8046
ALODA
COIA1
CD59
IBP3
PTPRJ
SEM3G
CERU
CD14


0.8046
PVR
CD59
S10A6
PLSL
PEDF
CH10
SCF
BST1


0.8046
COIA1
IBP3
MASP1
DSG2
TENA
ZA2G
1433Z
APOE


0.8042
BGH3
CD59
CALU
LDHB
CO6A3
SODM
TENA
APOE


0.8042
IBP3
TSP1
ENPL
CH10
CD14
FRIL
G3P
HYOU1


0.8042
IBP3
TSP1
KIT
ZA2G
6PGD
APOE
CD14
FRIL


0.804
TPIS
BGH3
S10A6
LDHB
CO6A3
CH10
PEDF
TENA


0.804 
CALU
LDHB
BGH3
TETN
FOLH1
TNF12 
VTNC
FRIL


0.8038
TPIS
PVR
COIA1 
CALU
SCF
MPRI
ALDOA
ENOA


0.8036
S10A6
TPIS
COIA1
CD59
CO6A3
TSP1
MPRI
ALDOA


0.8036
LEG1
CD59
AMPN
CALU
CH10
GRP78
SEM3G
TETN


0.8036
AMPN
S10A6
TSP1
ENPL
PEDF
SODM
FOLH1
6PGD


0.8036
S10A6
CALU
MASP1
A1AG1 
MPRI
ALDOA
VTNC
TENA


0.8036
IBP3
CALU
PLSL
CD44
KIT
CERU
6PGD
CD14


0.8036
TSP1
PLSL
FOLH1
COIA1 
TNF12
VTNC
6PGD
FRIL


0.8034
ALDOA
BGH3
CD59
TSP1
KIT
CH10
SODM
VTNC


0.8034
S10A6
CALU
LDHB
TSP1
GRP78
1433Z 
6PGD
G3P


0.8032
S10A6
CALU
TSP1
KIT
CH10
PEDF
GRP78
SEM3G


0.8032
TSP1
MASP1
CRP
ALDOA
GRP78
TETN
TNF12
1433Z 


0.803 
AMPN
TSP1
KIT
MPRI
SEM3G
TETN
DSG2
1433Z 


0.803 
CALU
CO6A3
PLSL
A1AG1 
ALDOA
GRP78 
6PGD
APOE


0.8028
COIA1
CD59
AMPN
TSP1
KIT
ISLR
ALDOA
MDHM


0.8024
S10A6
CD44
SCF
MPRI
ISLR
ALDOA
APOE
FRIL


0.8024
S10A6
TSP1
ALDOA
SODM
ENOA
BST1
FRIL
HYOU1


0.8024
IBP3
TSP1
SCF
ALDOA
SODM
DSG2
VTNC
1433Z 


0.802
ALDOA
TSP1
PLSL
CD44
CH10
A1AG1
ENOA
TETN


0.802
LEG1
CALU
LDHB
TSP1
CH10
ALDOA
MDHM
APOE


0.802
CD59
IBP3
TSP1
A1AG1
MPRI
PTPRJ
6PGD
APOE


0.802
IBP3
TSP1
CRP
BST1
TNF12
VTNC
1433Z
FRIL


0.8018
LEG1
S10A6
IBP3
CALU
TSP1
MASP1 
A1AG1
SCF


0.8018
COIA1
CD59
AMPN
CALU
MASP1
BST1
VTNC
CERU


0.8018
AMPN
ALDOA
SODM
GRP78
MDHM
VTNC
6PGD
FRIL


0.8018
LDHB
CO6A3
ALDOA
SEM3G
DSG2
6PGD
APOE
FRIL


0.8016
S10A6
LDHB
SCF
MPRI
ALDOA
PEDF
ENOA
SEM3G


0.8016
LDHB
CO6A3
TSP1
1433Z 
APOE
CD14
FRIL
G3P


0.8014
ALDOA
PEDF
MPRI
ISLR
FOLH1
TNF12
MASP1
CERU


0.8014
COIA1
PEDF
CRP
A1AG1
ENOA
CERU
FRIL
G3P


0.8014
CD59
IBP3
TSP1
KIT
MASP1
ENOA
TNF12
CD14


0.8014
LDHB
KIT
SCF
BGH3
SEM3G
VTNC
1433Z
FRIL


0.8013
PVR
AMPN
LDHB
CD44
DSG2
TETN
MDHM
FRIL


0.8013
S10A6
LDHB
TSP1
ISLR
LUM
G3P
HYOU1
ICAM3


0.8013
CALU
A1AG1
MPRI
ALDOA
PEDF
DSG2
VTNC
ZA2G


0.8013
TSP1
ENPL
KIT
SODM
SEM3G
DSG2
TETN
LUM


0.8013
TSP1
PLSL
ISLR
ALDOA
ENOA
MDHM
APOE
G3P


0.8011
ALDOA
AMPN
CO6A3 
SEM3G
APOE
CD14
FRIL
G3P


0.8011
TPIS
BGH3
AMPN
S10A6
CALU
LDHB
KIT
TENA


0.8011
COIA1
IBP3
TSP1
A1AG1
TETN
DSG2
6PGD
FRIL


0.8011
AMPN
S10A6
IBP3
CALU
KIT
SCF
ALDOA
APOE


0.8011
IBP3
A1AG1
PEDF
SEM3G
MDHM 
TNF12 
VTNC
1433Z


0.8009
ALDOA
BGH3
AMPN
LDHB
TSP1
PLSL
MPRI 
ISLR 


0.8009
LEG1
COIA1
IBP3
CH10
MASP1
SCF
ALDOA
TNF12


0.8009
AMPN
ENPL
ALDOA
TETN 
FOLH1
BST1
ZA2G
6PGD 


0.8009
CALU 
CO6A3
ENPL
ALDOA
GRP78
PTPRJ 
VTNC
APOE


0.8009
TSP1
CH10
PTPRJ
TETN
TNF12
VTNC
TENA
1433Z


0.8007
CD59 
S10A6
IBP3
CO6A3
TSP1
KIT
ISLR
GRP78


0.8007
AMPN 
TSP1
KIT
SCF
TETN
ZA2G
1433Z
6PGD 


0.8007
S10A6
IBP3
TSP1
CD44
PEDF
A1AG1
PTPRJ
SODM


0.8007
CALU 
CO6A3
TSP1
CH10 
SCF
BGH3
ALDOA
ENOA


0.8007
ENPL 
CD44
MASP1
GRP78
1433Z
CD14
FRIL
G3P


0.8005
TPIS
LEG1
LDHB
TSP1
MASP1
A1AG1
MPRI
ALDOA


0.8005
PEDF
CRP
ISLR
ALDOA
GRP78
PTPRJ
ZA2G 
6PGD


0.8003
ALDOA
S10A6
CALU
CRP
BGH3 
TETN
6PGD
CD14


0.8003
AMPN
TSP1 
A1AG1
MPRI 
ISLR 
ALDOA
MASP1
LUM


0.8003
CO6A3
TSP1 
SCF
MPRI
ISLR 
FOLH1
1433Z
APOE


0.8001
S10A6
IBP3
TSP1
KIT
TETN 
COIA1 
CERU
6PGD 


0.8001
S10A6
CALU
CH10
ISLR
ALDOA
SODM
PTPRJ
MDHM


0.8001
IBP3 
TSP1
ENPL
CH10 
CRP
ISLR
ALDOA
SODM


0.8001
IBP3
TSP1
PTPRJ
ALDOA
BST1
LUM
1433Z
APOE


0.8001
LDHB 
TSP1
MPRI
GRP78
SEM3G
LUM
ZA2G
FRIL






AUC
P9
P10
P11
P12
P13
P14
P15






0.8282
APOE
FRIL
G3P
HYOU1
LRP1
RAN
HXK1



0.8255
FRIL
HYOU1
LRP1
PROF1 
TBB3
FINC
CEAM8



0.8194
FRIL
G3P
HYOU1
LRP1
TBB3
CLIC1
RAN



0.8189
APOE
G3P
HYOU1
PRDX1
PROF1
ILK
HXK1



0.8187
FRIL
G3P
PRDX1
ILK
FINC
GSLG1
HXK1



0.8171
CD14
FRIL
G3P
LRP1
TBB3
FINC
RAN



0.8171
FRIL
G3P
ICAM3
PRDX1
PROF1
PVR
HXK1



0.8165
1433Z
FRIL
G3P
S10A6 
FINC
GSLG1
HXK1



0.8163
6PGD
FRIL
G3P
HYOU1
ICAM3
PRDX1
FINC



0.8163
APOE
G3P
LRP1
UGPA
RAN
CEAM8
HXK1



0.8161
CD14
FRIL
G3P
LRP1
PROF1
RAN
CEAM8



0.8159
6PGD
FRIL
G3P
HYOU1
LRP1
PRDX1
CEAM8



0.8159
FRIL
G3P
LRP1 
TBB3
FINC
GSLG1
HXK1



0.8159
CD14
FRIL
G3P
PRDX1
CLIC1
ILK
HXK1



0.8159
G3P
TBB3
ILK
GELS
FINC
RAN
GSLG1



0.8157
APOE
FRIL 
G3P
HYOU1
CLIC1
ILK
HXK1



0.8155
HYOU1
LRP1
PRDX1
PROF1
FINC
RAN
GSLG1



0.8153
HYOU1
PLXC1
PRDX1
ILK
CEAM8
HXK1
BST1



0.814 
CERU
FRIL
G3P
PLXC1 
PRDX1
ILK
HXK1



0.8138
HYOU1
PLXC1
RAN
CEAM8
HXK1
BST1
MMP9



0.8132
6PGD
CD14
FRIL 
HYOU1
FINC
GSLG1
BST1



0.8128
BST1
6PGD
G3P
HYOU1
ILK
FINC 
HXK1



0.8128
1433Z 
APOE
FRIL
G3P
LRP1
PTGIS
RAN



0.8124
CD14
FRIL
G3P
GDIR2
FINC
GSLG1
HXK1



0.8124
GDIR2
LRP1
CLIC1
FINC
GSLG1
HXK1
BST1



0.812
1433Z 
APOE
FRIL
LRP1
PRDX1
PROF1
FINC



0.8106
1433Z
6PGD
FRIL
G3P
HYOU1
PRDX1
CLIC1



0.8106
G3P
PRDX1
UGPA
ILK
CEAM8
GSLG1
HXK1



0.8099
G3P
HYOU1
PRDX1
PROF1
FINC
GSLG1
HXK1



0.8097
G3P
HYOU1
LRP1
PTGIS
ILK
FINC
MMP9



0.8093
CD14
FRIL
G3P
LRP1
PLXC1
CLIC1
GSLG1



0.8093
G3P
GDIR2
PRDX1
UGPA
CLIC1
FINC
HXK1



0.8093
SEM3G
MASP1
G3P
HYOU1
FINC
CEAM8
HXK1



0.8087
6PGD
CD14
FRIL
HYOU1
TBB3
CLIC1
FINC



0.8087
FRIL
G3P
HYOU1
LRP1
FINC
CEAM8
HXK1



0.8083
GRP78
CERU
CD14
FRIL
LRP1
FINC
CEAM8



0.8081
TENA
FRIL
G3P
HYOU1
PROF1
RAN
HXK1



0.8081
HYOU1
ICAM3
PLXC1
CLIC1
ILK
FINC
GSLG1



0.8081
FRIL
G3P
HYOU1
S10A6
CEAM8
GSLG1
HXK1



0.8079
LUM
6PGD
APOE
FRIL
HYOU1
RAN
HXK1



0.8077
LRP1
PTGIS
CLIC1
FINC
RAN
GSLG1
MMP9



0.8077
FRIL
G3P
HYOU1
LRP1
ILK
GSLG1
HXK1



0.8077
APOE
CD14
FRIL
G3P
LRP1
PRDX1
GSLG1



0.8075
6PGD
FRIL
G3P
LRP1
UGPA
ILK
HXK1



0.8073
6PGD
FRIL
G3P
HYOU1
LRP1
PRDX1
FINC



0.8071
CD14
FRIL
G3P
LRP1
AMPN
RAN
HXK1



0.8071
APOE
FRIL
HYOU1
LRP1
PTGIS
CLIC1
AMPN



0.8065
ALDOA
APOE
FRIL
G3P
TBB3
RAN
HXK1



0.8063
LRP1
PROF1
TBB3
UGPA
CLIC1
AMPN
RAN



0.8063
LRP1
PLXC1
PROF1
FINC
RAN
HXK1
MMP9



0.8061
G3P
LRP1
PLXC1
PROF1
PVR
FINC
CEAM8



0.8059
CD14
FRIL
G3P
LRP1
TBB3
RAN
GSLG1



0.8059
G3P
HYOU1
PRDX1
TBB3
ILK
RAN
HXK1



0.8058
APOE
CD14
FRIL
G3P
HYOU1
RAN
HXK1



0.8058
FRIL
G3P
HYOU1
PROF1
GELS
PVR
RAN



0.8054
6PGD
FRIL
G3P
HYOU1
ILK
GSLG1
HXK1



0.8054
HYOU1
ICAM3
PLXC1
TBB3
GELS
RAN
BST1



0.8054
PLXC1
PRDX1
PROF1
FINC
CEAM8
GSLG1
MMP9



0.805
G3P
HYOU1
ICAM3
PRDX1
UGPA
ILK
HXK1



0.8048
CD14
FRIL
G3P
HYOU1
PTGIS
FINC
RAN



0.8048
FRIL
G3P
LRP1
PRDX1
UGPA
RAN
CEAM8



0.8046
FRIL
G3P
GDIR2
HYOU1
RAN
GSLG1
HXK1



0.8046
FRIL
G3P
LRP1
PRDX1
FINC
GSLG1
MMP9



0.8046
FRIL
G3P
CLIC1
ILK
AMPN
FINC
HXK1



0.8046
CD14
FRIL
G3P
ICAM3
AMPN
FINC
HXK1



0.8042
G3P
HYOU1
S10A6
ILK
FINC
RAN
HXK1



0.8042
ICAM3
LRP1
PRDX1
PROF1
GELS
FINC
GSLG1



0.8042
GDIR2
HYOU1
LRP1
PRDX1
PROF1
CLIC1
HXK1



0.804
FRIL
G3P
HYOU1
LRP1
PRDX1
ILK
GSLG1



0.804 
G3P
GDIR2
PRDX1
CLIC1
GELS
FINC
HXK1



0.8038
MASP1
APOE
FRIL
G3P
PRDX1
FINC
HXK1



0.8036
ENOA
6PGD
FRIL
G3P
GDIR2
LRP1
PRDX1



0.8036
APOE
G3P
HYOU1
ICAM3
RAN
CEAM 8
HXK1



0.8036
APOE
FRIL
G3P
HYOU1
LRP1
HXK1
MMP9



0.8036
FRIL
G3P
PROF1
PTGIS
FINC
CEAM8
HXK1



0.8036
FRIL
G3P
HYOU1
PRDX1
FINC
CEAM8
HXK1



0.8036
G3P
LRP1
PRDX1
PROF1
GELS
FINC
RAN



0.8034
TENA
6PGD
G3P
HYOU1
LRP1
TBB3
ILK



0.8034
HYOU1
ICAM3
PROF1
ILK
GELS
AMPN
FINC



0.8032
MASP1
6PGD
CD14
FRIL
G3P
HYOU1
ILK



0.8032
APOE
CD14
G3P
HYOU1
PVR
RAN
HXK1



0.803 
APOE
FRIL
G3P
TBB3
UGPA
PVR
RAN



0.803 
CD14
FRIL
G3P
HYOU1
ICAM3 
PRDX1
RAN



0.8028
CERU
LUM
ZA2G
APOE
FRIL
LRP1
MMP9



0.8024
G3P
HYOU1
PRDX1 
GELS
FINC
CEAM8
HXK1



0.8024
LRP1
PROF1 
CLIC1
GELS
FINC
CEAM8
GSLG1



0.8024
APOE
FRIL
G3P
LRP1
PRDX1
UGPA
PTPRJ



0.802
TENA
APOE
FRIL
G3P
TBB3
AMPN
GSLG1



0.802
FRIL
G3P
HYOU1
ILK
PVR
GSLG1
PTPRJ



0.802
FRIL
G3P
LRP1
ILK
RAN
CEAM8
MMP9



0.802
G3P
GDIR2
HYOU1
LRP1
PRDX1
TBB3
FINC



0.8018
ALDOA
SEM3G
VTNC
FRIL
G3P
LRP1
CLIC1



0.8018
6PGD
APOE
CD14
FRIL
HYOU1
PROF1
GSLG1



0.8018
G3P
HYOU1
LRP1
PTGIS
GELS
FINC
RAN



0.8018
G3P
HYOU1
ICAM3
PROF1
FINC
PTPRJ
HXK1



0.8016
APOE
FRIL
G3P
HYOU1
PRDX1
CLIC1
GSLG1



0.8016
HYOU1
PROF1
UGPA
CLIC1
RAN
CEAM8
PTPRJ



0.8014
6PGD
FRIL
G3P
HYOU1
PRDX1 
FINC
HXK1



0.8014
GDIR2
LRP1
S10A6
GELS
FINC
GSLG1
HXK1



0.8014
FRIL
G3P
PRDX1
UGPA
FINC
PTPRJ
HXK1



0.8014
G3P
HYOU1
LRP1
PRDX1
PROF1
FINC
HXK1



0.8013
G3P
LRP1
PRDX1
ILK
FINC
HXK1
MMP9



0.8013
LRP1
PROF1 
UGPA
ILK
FINC
PTPRJ
HXK1



0.8013
6PGD
FRIL
G3P
CLIC1
S10A6
ILK
PVR



0.8013
APOE
FRIL
G3P
HYOU1
CLIC1
RAN
HXK1



0.8013
GDIR2
LRP1
PTGIS
FINC
RAN
HXK1
MMP9



0.8011
GDIR2
HYOU1
ICAM3
PRDX1
FINC
HXK1
MMP9



0.8011
6PGD
APOE
G3P
LRP1
PROF1
GELS
MMP9



0.8011
GDIR2
HYOU1
LRP1
CLIC1
S10A6
PVR
GSLG1



0.8011
G3P
ICAM3
LRP1
GELS
FINC
RAN
CEAM8



0.8011
G3P
HYOU1
PRDX1
FINC
GSLG1
PTPRJ
HXK1



0.8009
APOE
FRIL
LRP1
PVR 
FINC
RAN
PTPRJ



0.8009
CERU
APOE
CD14
FRIL
TBB3
ILK
FINC



0.8009
CD14 
FRIL
CLIC1
S10A6
ILK
FINC
MMP9



0.8009
CD14
G3P
TBB3
CLIC1
GELS
RAN
HXK1



0.8009
6PGD
FRIL
G3P
HYOU1
RAN
HXK1
MMP9



0.8007
MDHM
CD14
FRIL
G3P
HYOU1
GSLG1
HXK1



0.8007
APOE 
G3P
GDIR2
LRP1 
PRDX1
TBB3
RAN



0.8007
CERU 
APOE
FRIL
ICAM3
LRP1
UGPA
GSLG1



0.8007
TETN
LUM
APOE
FRIL
G3P
RAN
HXK1



0.8007
GDIR2
ICAM3
LRP1 
PRDX1
PROF1
FINC
HXK1



0.8005
ENOA
FRIL 
G3P
LRP1
UGPA
ILK
FINC



0.8005
G3P
HYOU1
PRDX1
TBB3 
FINC
RAN
CEAM8



0.8003
FRIL
G3P
CLIC1
FINC
GSLG1
HXK1
MMP9



0.8003
6PGD 
APOE 
FRIL
ICAM3
TBB3
GSLG1
BST1



0.8003
G3P
HYOU1
ICAM3
PRDX1
UGPA
RAN
HXK1



0.8001
CD14
FRIL
G3P
PROF1
FINC
HXK1
MMP9



0.8001
VTNC
FRIL
G3P
CLIC1 
ILK
AMPN
HXK1



0.8001
1433Z
G3P
HYOU1
LRP1 
PRDX1 
PROF1
CEAM8



0.8001
G3P
HYOU1
LRP1
PTGIS
TBB3
PVR
RAN



0.8001
G3P
ICAM3
PROF1
TBB3 
FINC
RAN
GSLG1









To calculate the combined AUC of each panel of 15 proteins, the highest intensity normalized transition was utilized. Logistic regression was used to calculate the AUC of the panel of 15 across all small samples. 5 panels of 15 proteins had combined AUC above 0.80.
Finally, the frequency of each of the 67 proteins on the 131 panels listed in Table 13 is presented in Table 12 both as raw counts (column 2) and percentage (column 3). It is an important observation that the panel size of 15 was pre-selected to prove that there are diagnostic proteins and panels. Furthermore, there are numerous such panels. Smaller panels selected from the list of 67 proteins can also be formed and can be generated using the same methods here.
Example 4
A Diagnostic Panel of 15 Proteins for Determining the Probability that a Blood Sample from a Patient with a PN of Size 2 cm or Less is Benign or Malignant
In Table 14 a logistic regression classifier trained on all small samples is presented.












TABLE 14







Transition

Normalized





column

By column
Logistic




SEQ ID

SEQ ID
Regression


Protein
Transition
NO.
Normalized By
NO
Coefficient




















ALDOA_HUMAN
ALQASALK_401.25_
 7
YGFIEGHVVIPR_
1
-1.96079



617.40

462.92_272.20




 


BGH3_HUMAN
LTLLAPLNSVFK_
 8
YEVTVVSVR_
2
2.21074



658.40_804.50

526.29_759.50




 


CLIC1_HUMAN
LAALNPESNTAGL
 9
ASSIIDELFQDR_
3
0.88028



DIFAK_

465.24_565.30





922.99_256.20






 


CO6A3_HUMAN
VAVVQYSDR_
10
ASSIIDELFQDR_
3
-1.52046



518.77_767.40

465.24_565.30




 


COIA1_HUMAN
AVGLAGTFR_
11
YGFIEGHVVIPR_
1
-0.76786



446.26_721.40

462.92_272.20




 


FINC_HUMAN
VPGTSTSATLTGLTR_
12
FLNVLSPR_
4
0.98842



487.94_446.30

473.28_685.40




 


G3P_HUMAN
GALQNIIPASTGAAK_
13
TASDFITK_
5
0.58843



706.40_815.50

441.73_710.40




 


ISLR_HUMAN
ALPGTPVASSQPR_
14
FLNVLSPR_
4
1.02005



640.85_841.50

473.28_685.40




 


LRP1_HUMAN
TVLWPNGLSLDIPAG
15
YEVTVVSVR_
2
-2.14383



R_855.00_400.20

526.29_759.50




 


PRDX1_HUMAN
QITVNDLPVGR_
16
YGFIEGHVVIPR_
1
-1.38044



606.30_428.30

462.92_272.20




 


PROF1_HUMAN
STGGAPTFNVTVTK_
17
TASDFITK_
5
-1.78666



690.40_503.80

441.73_710.40




 


PVR_HUMAN
SVDIWLR_
18
TASDFITK_
5
2.26338



444.75_702.40

441.73_710.40




 


TBB3_HUMAN
ISVYYNEASSHK_
19
FLNVLSPR_
4
-0.46786



466.60_458.20

473.28_685.40




 


TETN_HUMAN
LDTLAQEVALLK_
20
TASDFITK_
5
-1.99972



657.39_330.20

441.73_710.40




 


TPIS_HUMAN
VVFEQTK_
21
YGFIEGHVVIPR_
1
2.65334



425.74_652.30

462.92_272.20




 


Constant (C0)




21.9997









The classifier has the structure



  
    Probability
    =
    
      
        exp
        ⁡
        
          (
          W
          )
        
      
      
        1
        +
        
          exp
          ⁡
          
            (
            W
            )
          
        
      
    
  




  
    W
    =
    
      
        C
        0
      
      +
      
        
          ∑
          
            i
            =
            1
          
          15
        
        ⁢
        
          
            C
            i
          
          *
          
            P
            i
          
        
      
    
  



Where C0 and Ci are logistic regression coefficients, Pi are logarithmically transformed normalized transition intensities. Samples are predicted as cancer if Probability ≧0.5 or as benign otherwise. In Table 14 the coefficients Ci appear in the sixth column, C0 in the last row, and the normalized transitions for each protein are defined by column 2 (protein transition) and column 4 (the normalizing factor).
The performance of this classifier, presented as a ROC plot, appears in FIG. 4. Overall AUC is 0.81. The performance can also be assessed by applying the classifier to each study site individually which yields the three ROC plots appearing in FIG. 5. The resulting AUCs are 0.79, 0.88 and 0.78 for Laval, NYU and UPenn, respectively.
Example 5
The Program “Ingenuity”® was Used to Query the Blood Proteins that are Used to Identify Lung Cancer in Patients with Nodules that were Identified Using the Methods of the Present Invention
Using a subset of 35 proteins (Table 15) from the 67 proteins identified as a diagnostic panel (Table 13), a backward systems analysis was performed. Two networks were queried that are identified as cancer networks with the identified 35 proteins. The results show that the networks that have the highest percentage of “hits” when the proteins are queried that are found in the blood of patients down to the level of the nucleus are initiated by transcription factors that are regulated by either cigarette smoke or lung cancer among others. See also Table 16 and FIG. 6.
These results are further evidence that the proteins that were identified using the methods of the invention as diagnostic for lung cancer are prognostic and relevant.











TABLE 15





No.
Protein
Protein Name
Gene Symbol
Gene Name



















1
6PGD_HUMAN
6-phosphogluconate
PGD
phosphogluconate dehydrogenase




dehydrogenase, decar-




boxylating


2
AIFM1_HUMAN
Apoptosis-inducing
AIFM1
apoptosis-inducing factor, mito-




factor 1, mitochondrial

chondrion-associated, 1


3
ALDOA_HUMAN
Fructose-bisphosphate
ALDOA
aldolase A, fructose-bisphosphate




aldolase A


4
BGH3_HUMAN
Transforming growth
TGFBI
transforming growth factor, beta-




factor-beta-induced

induced, 68 kDa




protein ig-h3


5
C163A_HUMAN
Scavenger receptor
CD163
CD163 molecule




cysteine-rich type 1




protein M130


6
CD14_HUMAN
Monocyte differentia-
CD14
CD14 molecule




tion antigen CD14


7
COIA1_HUMAN
Collagen alpha-
COL18A1
collagen, type XVIII, alpha 1




1(XVIII) chain


8
ERO1A_HUMAN
ERO1-like protein
ERO1L
ERO1-like (S. cerevisiae)




alpha


9
FIBA_HUMAN
Fibrinogen alpha chain
FGA
fibrinogen alpha chain


10
FINC_HUMAN
Fibronectin
FN1
fibronectin 1


11
FOLH1_HUMAN
Glutamate carboxy-
FOLH1
folate hydrolase (prostate-specific




peptidase 2

membrane antigen) 1


12
FRIL_HUMAN
Ferritin light chain
FTL
ferritin, light polypeptide


13
GELS_HUMAN
Gelsolin
GSN
gelsolin (amyloidosis, Finnish






type)


14
GGH_HUMAN
Gamma-glutamyl
GGH
gamma-glutamyl hydrolase (con-




hydrolase

jugase, folylpolygammaglutamyl






hydrolase)


15
GRP78_HUMAN
78 kDa glucose-
HSPA5
heat shock 70 kDa protein 5 (glu-




regulated protein

cose-regulated protein, 78 kDa)


16
GSLG1_HUMAN
Golgi apparatus protein
GLG1
golgi apparatus protein 1




1


17
GSTP1_HUMAN
Glutathione S-
GSTP1
glutathione S-transferase pi 1




transferase P


18
IBP3_HUMAN
Insulin-like growth
IGFBP3
insulin-like growth factor binding




factor-binding protein 3

protein 3


19
ICAM1_HUMAN
Intercellular adhesion
ICAM1
intercellular adhesion molecule 1




molecule 1


20
ISLR_HUMAN
Immunoglobulin super-
ISLR
immunoglobulin superfamily




family containing leu-

containing leucine-rich repeat




cine-rich repeat protein


21
LG3BP_HUMAN
Galectin-3-binding
LGALS3BP
lectin, galactoside-binding,




protein

soluble, 3 binding protein


22
LRP1_HUMAN
Prolow-density lipo-
LRP1
low density lipoprotein-related




protein receptor-related

protein 1 (alpha-2-macroglobulin




protein 1

receptor)


23
LUM_HUMAN
Lumican
LUM
lumican


24
MASP1_HUMAN
Mannan-binding lectin
MASP1
mannan-binding lectin serine pep-




serine protease 1

tidase 1 (C4/C2 activating com-






ponent of Ra-reactive factor)


25
PDIA3_HUMAN
Protein disulfide-
PDIA3
protein disulfide isomerase family




isomerase A3

A, member 3


26
PEDF_HUMAN
Pigment epithelium-
SERPINF1
serpin peptidase inhibitor, clade F




derived factor

(alpha-2 antiplasmin, pigment






epithelium derived factor), mem-






ber 1


27
PRDX1_HUMAN
Peroxiredoxin-1
PRDX1
peroxiredoxin 1


28
PROF1_HUMAN
Profilin-1
PFN1
profilin 1


29
PTPA_HUMAN
Serine/threonine-
PPP2R4
protein phosphatase 2A activator,




protein phosphatase 2A

regulatory subunit 4




activator


30
PTPRJ_HUMAN
Receptor-type tyrosine-
PTPRJ
protein tyrosine phosphatase,




protein phosphatase eta

receptor type, J


31
RAP2B_HUMAN
Ras-related protein
RAP2B
RAP2B, member of RAS onco-




Rap-2b

gene family


32
SEM3G_HUMAN
Semaphorin-3G
SEMA3G
sema domain, immunoglobulin






domain (Ig), short basic domain,






secreted, (semaphorin) 3G


33
SODM_HUMAN
Superoxide dismutase
SOD2
superoxide dismutase 2, mito-




[Mn], mitochondrial

chondrial


34
TETN_HUMAN
Tetranectin
CLEC3B
C-type lectin domain family 3,






member B


35
TSP1_HUMAN
Thrombospondin-1
THBS1
thrombospondin 1



















TABLE 16





Gene

Lung Cancer PubMed



Name
Protein
Associations
Sample Publications







NFE2L2
nuclear
92
Cigarette Smoking Blocks the Protective


(NRF2)
factor
transcription
Expression of Nrf2/ARE Pathway . . .



(erythroid-
factor
Molecular mechanisms for the regulation



derived 2)-
protecting
of Nrf2-mediated cell proliferation in non-



like 2
cell from
small-cell lung cancers . . .




oxidative stress


EGR1
early
38
Cigarette smoke-induced Egr-1 upregulates



growth
transcription
proinflammatory cytokines in pulmonary



response
factor
epithelial cells . . .




involved
EGR-1 regulates Ho-1 expression induced




oxidative stress
by cigarette smoke . . .





Chronic hypoxia induces Egr-1 via activa-





tion of ERK1/2 and contributes to pulmo-





nary vascular remodeling.





Early growth response-1 induces and en-





hances vascular endothelial growth factor-





A expression in lung cancer cells . . .









Example 6
Cooperative Proteins for Diagnosing Pulmonary Nodules
To achieve unbiased discovery of cooperative proteins, selected reaction monitoring (SRM) mass spectrometry (Addona, Abbatiello et al. 2009) was utilized. SRM is a form of mass spectrometry that monitors predetermined and highly specific mass products of particularly informative (proteotypic) peptides of selected proteins. These peptides are recognized as specific transitions in mass spectra. SRM possesses the following required features that other technologies, notably antibody-based technologies, do not possess:

    
    
        Highly multiplexed SRM assays can be rapidly and cost-effectively developed for tens or hundreds of proteins.
        The assays developed are for proteins of one's choice and are not restricted to a catalogue of pre-existing assays. Furthermore, the assays can be developed for specific regions of a protein, such as the extracellular portion of a transmembrane protein on the cell surface of a tumor cell, or for a specific isoform.
        SRM technology can be used from discovery to clinical testing. Peptide ionization, the foundation of mass spectrometry, is remarkably reproducible. Using a single technology platform avoids the common problem of translating an assay from one technology platform to another.

SRM has been used for clinical testing of small molecule analytes for many years, and recently in the development of biologically relevant assays [10].

    
    


Labeled and unlabeled SRM peptides are commercially available, together with an open-source library and data repository of mass spectra for design and conduct of SRM analyses. Exceptional public resources exist to accelerate assay development including the PeptideAtlas [11] and the Plasma Proteome Project [12, 13], the SRM Atlas and PASSEL, the PeptideAtlas SRM Experimental Library (www.systemsbiology.org/passel).
Two SRM strategies that enhance technical performance were introduced. First, large scale SRM assay development introduces the possibility of monitoring false signals. Using an extension of expression correlation techniques [14], the rate of false signal monitoring was reduced to below 3%. This is comparable and complementary to the approach used by mProphet (Reiter, Rinner et al. 2011).
Second, a panel of endogenous proteins was used for normalization. However, whereas these proteins are typically selected as “housekeeping” proteins (Lange, Picotti et al. 2008), proteins that were strong normalizers for the technology platform were identified. That is, proteins that monitored the effects of technical variation so that it could be controlled effectively. This resulted, for example, in the reduction of technical variation due to sample depletion of high abundance proteins from 23.8% to 9.0%. The benefits of endogenous signal normalization has been previously discussed (Price, Trent et al. 2007).
The final component of the strategy was to carefully design the discovery and validation studies using emerging best practices. Specifically, the cases (malignant nodules) and controls (benign nodules) were pairwise matched on age, nodule size, gender and participating clinical site. This ensures that the candidate markers discovered are not markers of age or variations in sample collection from site to site. The studies were well-powered, included multiple sites, a new site participated in the validation study, and importantly, were designed to address the intended use of the test. The careful selection and matching of samples resulted in an exceptionally valuable feature of the classifier. The classifier generates a score that is independent of nodule size and smoking status. As these are currently used risk factors for clinical management of IPNs, the classifier is a complementary molecular tool for use in the diagnosis of IPNs.
Selection of Biomarker Candidates for Assay Development
To identify lung cancer biomarkers in blood that originate from lung tumor cells, resected lung tumors and distal normal tissue of the same lobe were obtained. Plasma membranes were isolated from both endothelial and epithelial cells and analyzed by tandem mass spectrometry to identify cell surface proteins over expressed on tumor cells. Similarly, Golgi apparatus were isolated to identify over-secreted proteins from tumor cells. Proteins with evidence of being present in blood or secreted were prioritized resulting in a set of 217 proteins. See Example 7: Materials and Methods for details.
To ensure other viable lung cancer biomarkers were not overlooked, a literature search was performed and manually curated for lung cancer markers. As above, proteins with evidence of being present in blood or secreted were prioritized. This resulted in a set of 319 proteins. See Example 7: Materials and Methods for Details.
The tissue (217) and literature (319) candidates overlapped by 148 proteins resulting in a final candidate list of 388 protein candidates. See Example 7: Materials and Methods.
Development of SRM Assays
SRM assays for the 388 proteins were developed using standard synthetic peptide techniques (See Example 7: Materials and Methods). Of the 388 candidates, SRM assays were successfully developed for 371 candidates. The 371 SRM assays were applied to benign and lung cancer plasma samples to evaluate detection rate in blood. 190 (51% success rate) of the SRM assays were detected. This success rate compares favorably to similar attempts to develop large scale SRM assays for detection of cancer markers in plasma. Recently 182 SRM assays for general cancer markers were developed from 1172 candidates (16% success rate) [15]. Despite focusing only on lung cancer markers, the 3-fold increase in efficiency is likely due to sourcing candidates from cancer tissues with prior evidence of presence in blood. Those proteins of the 371 that were previously detected by mass spectrometry in blood had a 64% success rate of detection in blood whereas those without had a 35% success rate. Of the 190 proteins detected in blood, 114 were derived from the tissue-sourced candidates and 167 derived from the literature-sourced candidates (91 protein overlap). See Example 7: Materials and Methods and Table 6.
Typically, SRM assays are manually curated to ensure assays are monitoring the intended peptide. However, this becomes unfeasible for large scale SRM assays such as this 371 protein assay. More recently, computational tools such as mProphet (Reiter, Rinner et al. 2011) enable automated qualification of SRM assays. A complementary strategy to mProphet was introduced that does not require customization for each dataset set. It utilizes correlation techniques (Kearney, Butler et al. 2008) to confirm the identity of protein transitions with high confidence. In FIG. 7 a histogram of the Pearson correlations between every pair of transitions in the assay is presented. The correlation between a pair of transitions is obtained from their expression profiles over all 143 samples in the discovery study detailed below. As expected, transitions from the same peptide are highly correlated. Similarly, transitions from different peptide fragments of the same protein are also highly correlated. In contrast, transitions from different proteins are not highly correlated and enables a statistical analysis of the quality of a protein's SRM assay. For example, if the correlation of transitions from two peptides from the same protein is above 0.5 then there is less than a 3% probability that the assay is false. See Example 7: Materials and Methods.
Classifier Discovery
A summary of the 143 samples used for classifier discovery appears in Table 17. Samples were obtained from three sites to avoid overfitting to a single site. Participating sites were Laval (Institut Universitaire de Cardiologie et de Pneumologie de Quebec), NYU (New York University) and UPenn (University of Pennsylvania). Samples were also selected to be representative of the intended use population in terms of nodule size (diameter), age and smoking status.
Benign and cancer samples were paired by matching on age, gender, site and nodule size (benign and cancer samples were required to have a nodule identified radiologically). The benign and cancer samples display a bias in smoking (pack years), however, the majority of benign and cancer samples were current or past smokers. In comparing malignant and benign samples, the intent was to find proteins that were markers of lung cancer; not markers of age, nodule size or differences in site sample collection. Note that cancer samples were pathologically confirmed and benign samples were either pathologically confirmed or radiologically confirmed (no tumor growth demonstrated over two years of CT scan surveillance).







TABLE 17







Clinical data summaries and demographic analysis for discovery and validation sets.












Discovery
Validation
















Cancer
Benign
P value
Cancer
Benign
P value

















Sample

72
71

52
52



(total)









Sample
Laval
14
14
1.00†
13
12
0.89†


(Center)
NYU
29
28

6
9




UPenn
29
29

14
13




Vanderbilt
0
0

19
18



Sample
Male
29
28
1.00†
25
27
0.85†


(Gender)
Female
43
43

27
25



Sample
Never
5
19
0.006†
3
15
0.006†


(Smoking
Past
60
44

38
29



History)
Current
6
6

11
7




No data
1
2

0
1



Age
Median
65
64
0.46‡
63
62
0.03‡



(quartile
(59-72)
(52-71)

(60-73)
(56-67)




range)








Nodule
Median
13
13
0.69‡
16
15
0.68‡


Size (mm)
(quartile
(10-16)
(10-18)

(13-20)
(12-22)




range)








Pack-year§
Median
37
20
0.001‡
40
27
0.09‡



(quartile
(20-52)
 (0-40)

(19-50)
 (0-50)




range)





†Based on Fisher's exact test.


‡Based on Mann-Whitney test.


§No data (cancer, benign): Discovery (4, 6),Validation (2, 3)






The processing of samples was conducted in batches. Each batch contained a set of randomly selected cancer-benign pairs and three plasma standards, included for calibration and quality control purposes.
All plasma samples were immunodepleted, trypsin digested and analyzed by reverse phase HPLC-SRM-MS. Protein transitions were normalized using an endogenous protein panel. The normalization procedure was designed to reduce overall variability, but in particular, the variability introduced by the depletion step. Overall technical variability was reduced from 32.3% to 25.1% and technical variability due to depletion was reduced from 23.8% to 9.0%. Details of the sample analysis and normalization procedure are available in Example 7: Materials and Methods.
To assess panels of proteins, they were fit to a logistic regression model. Logistic regression was chosen to avoid the overfitting that can occur with non-linear models, especially when the number of variables measured (transitions) is similar or larger than the number of samples in the study. The performance of a panel was measured by partial area under the curve (AUC) with sensitivity fixed at 90% (McClish 1989). Partial AUC correlates to high NPV performance while maximizing ROR.
To derive the 13 protein classifier, four criteria were used:

    
    
        The protein must have transitions that are reliably detected above noise across samples in the study.
        The protein must be highly cooperative.
        The protein must have transitions that are robust (high signal to noise, no interference, etc.)
        The protein's coefficient within the logistic regression model must have low variability during cross validation, that is, it must be stable.

Details of how each of these criteria were applied appear in Example 7: Materials and Methods.

    
    


Finally, the 13 protein classifier was trained to a logistic regression model by Monte Carlo cross validation (MCCV) with a hold out rate of 20% and 20,000 iterations. The thirteen proteins for the rule-out classifier are listed in Table 18 along with their highest intensity transition and model coefficient.







TABLE 18







The 13 protein classifier.












SEQ ID



Protein
Transition
NO
Coefficient





Constant(α)


36.16


 


LRP1_HUMAN
TVLWPNGLSLDIPAGR_
15
-1.59



855.00_400.20




 


BGH3_HUMAN
LTLLAPLNSVFK_
 8
 1.73



658.40_804.50




 


COIA1_HUMAN
AVGLAGTFR_
11
-1.56



446.26_721.40




 


TETN_HUMAN
LDTLAQEVALLK_
20
-1.79



657.39_330.20




 


TSP1_HUMAN
GFLLLASLR_
22
 0.53



495.31_559.40




 


ALDOA_HUMAN
ALQASALK_
 7
-0.80



401.25_617.40




 


GRP78_HUMAN
TWNDPSVQQDIK_
23
 1.41



715.85_260.20




 


ISLR_HUMAN
ALPGTPVASSQPR_
14
 1.40



640.85_841.50




 


FRIL_HUMAN
LGGPEAGLGEYLFER_
24
 0.39



804.40_913.40




 


LG3BP_HUMAN
VEIFYR_
25
-0.58



413.73_598.30




 


PRDX1_HUMAN
QITVNDLPVGR_
16
-0.34



606.30_428.30




 


FIBA_HUMANN
SLFEYQK_
26
 0.31



514.76_714.30




 


GSLG1_HUMAN
IIIQESALDYR_
27
-0.70



660.86_338.20









Validation of the Rule-Out Classifier
52 cancer and 52 benign samples (see Table 17) were used to validate the performance of the 13 protein classifier. All samples were independent of the discovery samples, in addition, over 36% of the validation samples were sourced from a new fourth site (Vanderbilt University). Samples were selected to be consistent with intended use and matched in terms of gender, clinical site and nodule size. We note a slight age bias, which is due to 5 benign samples from young patients. Anticipating a NPV of 90%, the 95% confidence interval is +/−5%.
At this point we refer to the 13 protein classifier trained on 143 samples the Discovery classifier. However, once validation is completed, to find the optimal coefficients for the classifier, it was retrained on all 247 samples (discovery and validation sets) as this is most predictive of future performance. We refer to this classifier as the Final classifier. The coefficients of the Final classifier appear in Table 21.
The performance of the Discovery and Final classifiers is summarized in FIG. 8. Reported are the NPV and ROR for the Discovery classifier when applied to the discovery set, the validation set. The NPV and ROR for the Final classifier are reported for all samples and also for all samples restricted to nodule size 8 mm to 20 mm (191 samples).
NPV and ROR are each reported as a fraction from 0 to 1. Similarly, the classifier produces a score between 0 and 1, which is the probability of cancer predicted by the classifier.
The discovery and validation curves for NPV and ROR are similar with the discovery curves superior as expected. This demonstrates the reproducibility of performance on an independent set of samples. A Discovery classifier rule out threshold of 0.40 achieves NPV of 96% and 90%, whereas ROR is 33% and 23%, for the discovery samples and the validation samples, respectively. Final classifier rule threshold of 0.60 achieves NPV of 91% and 90%, whereas ROR is 45% and 43%, for all samples and all samples restricted to be 8 mm-20 mm, respectively.
Applications of the Classifier
FIG. 9 presents the application of the final classifier to all 247 samples from the discovery and validation sets. The intent of FIG. 9 is to contrast the clinical risk factors of smoking (measured in pack years) and nodule size (proportional to the size of each circle) to the classifier score assigned to each sample.
First, note the density of cancer samples with high classifier scores. The classifier has been designed to detect a cancer signature in blood with high sensitivity. As a consequence, to the left of the rule out threshold (0.60) there are very few (<10%) cancer samples, assuming cancer prevalence of 25% [16, 17].
Third is the observation that nodule size does not appear to increase with the classifier score. Both large and small nodules are spread across the classifier score spectrum. Similarly, although there are a few very heavy smokers with very high classifier scores, increased smoking does not seem to increase with classifier score. To quantify this observation the correlation between the classifier score and nodule size, smoking and age were calculated and appear in Table 19. In all cases there is no significant relationship between the classifier score and the risk factors. The one exception is a weak correlation between benign classifier scores and benign ages. However, this correlation is so weak that the classifier score increases by only 0.04 every 10 years.







TABLE 19







Correlation between classifier scores and clinical risk factors.











Age
Nodule Size
Smoking
















Benign
0.25
−0.06
0.11



Cancer
0.01
−0.01
0.06










This lack of correlation has clinical utility. It implies that the classifier provides molecular information about the disease status of an IPN that is incremental upon risk factors such as nodule size and smoking status. Consequently, it is a clinical tool for physicians to make more informed decisions around the clinical management of an IPN.
To visual how this might be accomplished, we demonstrate how the cancer probability score generated by the classifier can be related to cancer risk (see FIG. 11)
At a given classifier score, some percentage of all cancer nodules will have a smaller score. This is the sensitivity of the classifier. For example, at classifier score 0.8, 47% of cancer patients have a lower score, at classifier score 0.7, 28% of cancer patients have a lower score, at classifier score 0.5, only 9% are lower and finally at score 0.25, only 4% are lower. This enables a physician to interpret a patient's classifier score in terms of relative risk.
The Molecular Foundations of the Classifier
The goal was to identify the molecular signature of a malignant pulmonary nodule by selecting proteins that were the cooperative, robustly detected by SRM and stable within the classifier. How well associated with lung cancer is the derived classifier? Is there a molecular foundation for the perturbation of these 13 proteins in blood? And finally, how unique is the classifier among other possible protein combinations?
To answer these questions the 13 proteins of the classifier were submitted for pathway analysis using IPA (Ingenuity Systems, www.ingenuity.com). The first step was to work from outside the cell inwards to identify the transcription factors most likely to cause a modulation of these 13 proteins. The five most significant were FOS, NRF2, AHR, HD and MYC. FOS is common to many forms of cancer. However, NRF2 and AHR are associated with lung cancer, response to oxidative stress and lung inflammation. MYC is associated with lung cancer and response to oxidative stress while HD is associated with lung inflammation and response to oxidative stress.
The 13 classifier proteins are also highly specific to these three networks (lung cancer, response to oxidative stress and lung inflammation). This is summarized in FIG. 10 where the classifier proteins (green), transcription factors (blue) and the three merged networks (orange) are depicted. Only ISLR is not connected through these three lung specific networks to the other proteins, although it is connected through cancer networks not specific to cancer. In summary, the modulation of the 13 classifier proteins can be tracked back to a few transcription factors specific to lung cancer, lung inflammation and oxidative stress networks.
To address the question of classifier uniqueness, every classifier from the 21 robust and cooperative proteins was formed (Table 20). Due to the computational overhead, these classifiers could not be fully trained by Monte Carlo cross validation, consequently, only estimates of their performance could be obtained. Five high preforming alternative classifiers were identified and then fully trained. The classifier and the five high performing alternatives appear in Table 20. The frequency of each protein appears in the tally column, in particular, the first 11 proteins appear in 4 out of the 6 classifiers. These 11 proteins have significantly higher cooperative scores than the remaining proteins. By this analysis it appears that there is a core group of proteins that form the blood signature of a malignant nodule.







TABLE 20







The classifier and the high performing alternatives; coefficients


for proteins on the respective panels are shown.























Coop-




Panel
Panel
Panel
Panel
Panel
Protein
erative


Protein 
Classifier
110424
130972
126748
109919
60767
Tally
Score


















Constant
36.16
27.72
27.69
23.47
21.32
23.17
—
—


ALDOA
−0.8
−0.67
−0.87
−0.83
−0.64
−0.68
6
1.3


COIA1
−1.56
−1.04
−1.68
−1.37
−0.94
−1.2
6
3.7


TSP1
0.53
0.53
0.39
0.42
0.47
0.41
6
1.8


FRIL
0.39
0.45
0.39
0.41
0.41
0.41
6
2.8


LRP1
−1.59
−0.84
−1.32
1.15
−0.84
−0.87
6
4.0


GRP78
1.41
1.14
1.31
−0.34
0.78
0.6
6
1.4


ISLR
1.4
1.03
1.08
0.75
0.74

5
1.4


IBP3

−0.23
−0.21
−0.38
−0.33
−0.54
5
3.4


TETN
−1.79
−1.23
−1.99
−1.26


4
2.5


PRDX1
−0.34
−0.38


−0.36
−0.4
4
1.5


LG3BP
−0.58

−0.61

−0.38
−0.48
4
4.3


CD14


0.99
1.08

1.4
3
4.0


BGH3
1.73

1.67
−0.83


3
1.8


KIT




−0.31
−0.56
3
1.4


GGH




0.44
0.52
3
1.3


AIFM1


−0.51



1
1.4


FIBA
0.31





1
1.1


GSLG1
−0.7





1
1.2


ENPL






0
1.1


EF1A1






0
1.2


TENX






0
1.1









This result suggests that there is a core group of proteins that define a high performance classifier, but alternative panels exist. However, changes in panel membership affect the tradeoff between NPV and ROR.
Example 7
Materials and Methods
Assay Development Candidates Sourced from Tissue
Patient samples obtained from fresh lung tumor resections were collected from Centre Hospitalier de l'Université de Montréal and McGill University Health Centre under IRB approval and with informed patient consent. Samples were obtained from the tumor as well as from distal normal tissue in the same lung lobe. Plasma membranes of each pair of samples were then isolated from the epithelial cells of 30 patients (19 adenocarcinoma, 6 squamous, 5 large cell carcinoma) and endothelial cells of 38 patients (13 adenocarcinoma, 18 squamous, 7 large cell carcinoma) using immune-affinity protocols. Golgi apparatus were isolated from each pair of samples from 33 patients (18 adenocarcinoma, 14 squamous, 1 adenosquamous) using isopycnic centrifugation followed by ammonium carbonate extraction. Plasma membrane isolations and Golgi isolations were then analyzed by tandem mass spectrometry to identify proteins overexpressed in lung cancer tissue over normal tissue, for both plasma membranes and Golgi.
Assay Development Candidates Sourced from Literature
Candidate lung cancer biomarkers were identified from two public and one commercial database: Entrez (www.ncbi.nlm.nih.gov/books/NBK3836), UniProt (www.uniprot.org) and NextBio (www.nextbio.com). Terminologies were predefined for the database queries which were automated using PERL scripts. The mining was carried out on May 6, 2010 (UniProt), May 17, 2010 (Entrez) and Jul. 8, 2010 (NextBio), respectively. Biomarkers were then assembled and mapped to UniProt identifiers.
Evidence of Presence in Blood
The tissue-sourced and literature-source biomarker candidates were required to have evidence of presence in blood. For evidence by mass spectrometry detection, three datasets were used. HUPO9504 contains 9504 human proteins identified by tandem mass spectrometry [13]. HUPO889, a higher confidence subset of HUPO9504, contains 889 human proteins [18]. The PeptideAtlas (November 2009 build) was also used. A biomarker candidate was marked as previously detected if it contained at least one HUPO889, or at least two HUPO9504 peptides, or at least two PeptideAtlas peptides.
In addition to direct evidence of detection in blood by mass spectrometry, annotation as secreted proteins or as single-pass membrane proteins [19] were also accepted as evidence of presence in blood. Furthermore, proteins in UniProt or designation as plasma proteins three programs for predicting whether or not a protein is secreted into the blood were used. These programs were TMHMM [20], SignalP [21] and SecretomeP [22]. A protein was predicted as secreted if TMHMM predicted the protein had one transmembrane domain and SignalP predicted the transmembrane domain was cleaved; or TMHMM predicted the protein had no transmembrane domain and either SignalP or SecretomeP predicted the protein was secreted.
SRM Assay Development
SRM assays for 388 targeted proteins were developed based on synthetic peptides, using a protocol similar to those described in the literature [15, 23, 24]. Up to five SRM suitable peptides per protein were identified from public sources such as the PeptideAtlas, Human Plasma Proteome Database or by proteotypic prediction tools [25] and synthesized. SRM triggered MS/MS spectra were collected on an ABSciex 5500 QTrap for both doubly and triply charged precursor ions. The obtained MS/MS spectra were assigned to individual peptides using MASCOT (cutoff score ≧15) [26]. Up to four transitions per precursor ion were selected for optimization. The resulting corresponding optimal retention time, declustering potential and collision energy were assembled for all transitions. Optimal transitions were measured on a mixture of all synthetic peptides, a pooled sample of benign patients and a pooled sample of cancer patients. Transitions were analyzed in batches, each containing up to 1750 transitions. Both biological samples were immuno-depleted and digested by trypsin and were analyzed on an ABSciex 5500 QTrap coupled with a reversed-phase (RP) high-performance liquid chromatography (HPLC) system. The obtained SRM data were manually reviewed to select the two best peptides per protein and the two best transitions per peptide. Transitions having interference with other transitions were not selected. Ratios between intensities of the two best transitions of peptides in the synthetic peptide mixture were also used to assess the specificity of the transitions in the biological samples. The intensity ratio was considered as an important metric defining the SRM assays.
Processing of Plasma Samples
Plasma samples were sequentially depleted of high- and medium-abundance proteins using immuno-depletion columns packed with the IgY14-Supermix resin from Sigma. The depleted plasma samples were then denatured, digested by trypsin and desalted. Peptide samples were separated using a capillary reversed-phase LC column (Thermo BioBasic 18 KAPPA; column dimensions: 320 μm×150 mm; particle size: 5 μm; pore size: 300 Å) and a nano-HPLC system (nanoACQUITY, Waters Inc.). The mobile phases were (A) 0.2% formic acid in water and (B) 0.2% formic acid in acetonitrile. The samples were injected (8 μl) and separated using a linear gradient (98% A to 70% A over 19 minutes, 5 μl/minute). Peptides were eluted directly into the electrospray source of the mass spectrometer (5500 QTrap LC/MS/MS, AB Sciex) operating in scheduled SRM positive-ion mode (Q1 resolution: unit; Q3 resolution: unit; detection window: 180 seconds; cycle time: 1.5 seconds). Transition intensities were then integrated by software MultiQuant (AB Sciex). An intensity threshold of 10,000 was used to filter out noisy data and undetected transitions.
Plasma Samples Used for Discovery and Validation Studies
Aliquots of plasma samples were provided by the Institut Universitaire de Cardiologie et de Pneumologie de Quebec (IUCPQ, Hospital Laval), New York University, the University of Pennsylvania, and Vanderbilt University (see Table 17). Subjects were enrolled in clinical studies previously approved by their Ethics Review Board (ERB) or Institutional Review Boards (IRB), respectively. In addition, plasma samples were provided by study investigators after review and approval of the sponsor's study protocol by the respective institution's IRB as required. Sample eligibility for the proteomic analysis was based on the satisfaction of the study inclusion and exclusion criteria, including the subject's demographic information, the subject's corresponding lung nodule radiographic characterization by chest computed tomography (CT), and the histopathology of the lung nodule obtained at the time of diagnostic surgical resection. Cancer samples had a histopathologic diagnosis of either non-small cell lung cancer (NSCLC), including adenocarcinoma, squamous cell, large cell, or bronchoalveolar cell carcinoma and a radiographic nodule of 30 mm or smaller. Benign samples, including granulomas, hamartomas and scar tissue, were also required to have a radiographic nodule of 30 mm or smaller and either histopathologic confirmation of being non-malignant or radiological confirmation in alignment with clinical guidelines. To ensure the accuracy of the clinical data, independent monitoring and verification of the clinical data associated with both the subject and lung nodule were performed in accordance with the guidance established by the Health Insurance Portability and Accountability Act (HIPAA) of 1996 to ensure subject privacy.
Study Design
The objective of the study design was to eliminate clinical and technical bias. Clinically, cancer and benign samples were paired so that they were from the same site, same gender, nodule sizes within 10 mm, age within 10 years, and smoking history within 20 pack years. Up to 15 pairs of matched cancer and benign samples per batch were assigned iteratively to processing batches until no statistical bias was demonstrable based on age, gender or nodule size.
Paired samples within each processing batch were further randomly and repeatedly assigned to positions within the processing batch, until the absolute values of the corresponding Pearson correlation coefficients between position and gender, nodule size, and age were less than 0.1. Afterwards, each pair of cancer and benign samples was randomized to their relative positions. To provide a control for sample batching, three 200 μl aliquots of a pooled human plasma standard (HPS) (Bioreclamation, Hicksville, N.Y.) were positioned at the beginning, middle and end of each processing batch, respectively. Samples within a batch were analyzed together.
Logistic Regression Model
The logistic regression classification method [27] was used to combine a panel of transitions into a classifier and to calculate a classification probability score between 0 and 1 for each sample. The probability score (Ps) of a sample was determined as Ps=1/[1+exp(−α−Σi=1Nβi*{hacek over (I)}i,s)], where {hacek over (I)}i,s was the logarithmically transformed (base 2), normalized intensity of transition i in sample s, βi was the corresponding logistic regression coefficient, α was a classifier-specific constant, and N was the total number of transitions in the classifier. A sample was classified as benign if Ps was less than a decision threshold. The decision threshold can be increased or decreased depending on the desired NPV. To define the classifier, the panel of transitions (i.e. proteins), their coefficients, the normalization transitions, classifier coefficient α and the decision threshold must be learned (i.e. trained) from the discovery study and then confirmed using the validation study.
Discovery of the Rule-Out Classifier
A summary of the 143 samples used for classifier discovery appears in Table 17 and processed as described above.
Protein transitions were normalized as described above. Transitions that were not detected in at least 50% of the cancer samples or 50% of the benign samples were eliminated leaving 117 transitions for further consideration. Missing values for these transitions were replaced by half the minimum detected value over all samples for that transition.
The next step was finding the set of most cooperative proteins. The cooperative score of a protein is the number of high performing panels it participates in divided by the number of such panels it could appear on by chance alone. Hence, a cooperative score above 1 is good, and a score below 1 is not. The cooperative score for each protein is estimated by the following procedure:
One million random panels of 10 proteins each, selected from the 117 candidates, were generated. Each panel of 10 proteins was trained using the Monte Carlo cross validation (MCCV) method with a 20% hold-off rate and one hundred sample permutations per panel) to fit a logistic regression model and its performance assessed by partial AUC [28].
By generating such a large number of panels, we sample the space of classifiers sufficiently well to find some high performers by chance. The one hundred best random panels (see Table 2) out of the million generated were kept and for each of the 117 proteins we determined how frequently each occurred on these top panels. Of the 117 proteins, 36 had frequency more than expected by chance, after endogenous normalizers were removed. (Table 22) The expected number of panels on which a protein would appear by chance is 100*10/117=8.33. The cooperative score for a protein is the number of panels it appears on divided by 8.33.













TABLE 21









Official
Cooper-






Protein
Gene
ative
Partial
Coeffi-



Category
(UniProt)
Name
Score
AUC
cient CV
Transition





Classifier
TSP1_HUMAN
THBS1
1.8
0.25
0.24
GFLLLASLR_








495.31_559.40


 


Classifier
COIA1_HUMAN
COL18A1
3.7
0.16
0.25
AVGLAGTFR_








446.26_721.40


 


Classifier
ISLR_HUMAN
ISLR
1.4
0.32
0.25
ALPGTPVASSQPR_








640.85_841.50


 


Classifier
TETN_HUMAN
CLEC3B
2.5
0.26
0.26 
LDTLAQEVALLK_








657.39_330.20


 


Classifier
FRIL_HUMAN
FTL
2.8
0.31
0.26
LGGPEAGLGEYLFER_








804.40_913.40


 


Classifier
GRP78_HUMAN
HSPA5
1.4
0.27
0.27 
TWNDPSVQQDIK_








715.85_260.20


 


Classifier
ALDOA_HUMAN
ALDOA
1.3
0.26
0.28
ALQASALK_401.25_








617.40


 


Classifier
BGH3_HUMAN
TGFBI
1.8
0.21
0.28
LTLLAPLNSVFK_








658.40_804.50


 


Classifier
LG3BP_HUMAN
LGALS3BP
4.3
0.29
0.29
VEIFYR_








413.73_598.30


 


Classifier
LRP1_HUMAN
LRP1
4.0
0.13
0.32
TVLWPNGLSLDIPAGR_








855.00_400.20


 


Classifier
FIBA_HUMAN
FGA
1.1
0.31
0.35
NSLFEYQK_








514.76_714.30


 


Classifier
PRDX1_HUMAN
PRDX1
1.5
0.32
0.37
QITVNDLPVGR_








606.30_428.30


 


Classifier
GSLG1_HUMAN
GLG1
1.2
0.34
0.45
IIIQESALDYR_








660.86_338.20


 


Robust
KIT_HUMAN
KIT
1.4
0.33
0.46



Robust
CD14_HUMAN
CD14
4.0
0.33
0.48



Robust
EF1A1_HUMAN
EEF1A1
1.2
0.32
0.56



Robust
TENX_HUMAN
TNXB
1.1
0.30
0.56



Robust
AIFM1_HUMAN
AIFM1
1.4
0.32
0.70



Robust
GGH_HUMAN
GGH
1.3
0.32
0.81



Robust
IBP3_HUMAN
IGFBP3
3.4
0.32
1.82



Robust
ENPL_HUMAN
HSP90B1
1.1
0.29
5.90



Non-Robust
ERO1A_HUMAN
ERO1L
6.2





Non-Robust
6PGD_HUMAN
PGD
4.3





Non-Robust
ICAM1_HUMAN
ICAM1
3.9





Non-Robust
PTPA_HUMAN
PPP2R4
2.1





Non-Robust
NCF4_HUMAN
NCF4
2.0





Non-Robust
SEM3G_HUMAN
SEMA3G
1.9





Non-Robust
1433T_HUMAN
YWHAQ
1.5





Non-Robust
RAP2B_HUMAN
RAP2B
1.5





Non-Robust
MMP9_HUMAN
MMP9
1.4





Non-Robust
FOLH1_HUMAN
FOLH1
1.3





Non-Robust
GSTP1_HUMAN
GSTP1
1.3





Non-Robust
EF2_HUMAN
EEF2
1.3





Non-Robust
RAN_HUMAN
RAN
1.2





Non-Robust
SODM_HUMAN
SOD2
1.2





Non-Robust
DSG2_HUMAN
DSG2
1.1

















Coefficient
Coefficient

Predicted




(Discovery)
(Final)

Concen-



SEQ
alpha =
alpha =
Tissue
tation


Category
ID NO
36.16
26.25 
Candidate
(ng/ml)





Classifier
22
 0.53
 0.44

510


Classifier
11
-1.56
-0.91

35


Classifier
14
 1.40
 0.83

—


Classifier
20
-1.79
-1.02

58000


Classifier
24
 0.39
 0.17
Secreted,
12






Epi, Endo



Classifier
23
 1.41
 0.55
Secreted,
100






Epi, Endo



Classifier
 7
-0.80
-0.26
Secreted,
250






Epi



Classifier
 8
 1.73
 0.54
Epi
140


Classifier
25
-0.58
-0.21
Secreted
440


Classifier
15
-1.59
-0.83
Epi
20


Classifier
26
 0.31
 0.13

130000


Classifier
16
-0.34
-0.26
Epi
60


Classifier
27
-0.70
-0.44
Epi, Endo
—


Robust




8.2


Robust



Epi
420


Robust



Secreted,
61






Epi



Robust



Endo
70


Robust



Epi, Endo
1.4


Robust




250


Robust




5700


Robust



Secreted,
88






Epi, Endo



Non-Robust



Secreted,
—






Epi, Endo



Non-Robust



Epi, Endo
29


Non-Robust




71


Non-Robust



Endo
3.3


Non-Robust



Endo
—


Non-Robust




—


Non-Robust



Epi
180


Non-Robust



Epi
—


Non-Robust




28


Non-Robust




—


Non-Robust



Endo
32


Non-Robust



Secreted,
30






Epi



Non-Robust



Secreted,
4.6






Epi



Non-Robust



Secreted 
7.1


Non-Robust



Endo
2.7









The 36 most cooperative proteins are listed in Table 22.













TABLE 22









Official
Cooper-






Protein
Gene
ative
Partial
Coeffi-



Category
(UniProt)
Name
Score
AUC
cient CV
Transition





Classifier
TSP1_HUMAN
THBS1
1.8
0.25
0.24
GFLLLASLR_








495.31_559.40


 


Classifier
COIA1_HUMAN
COL18A1
3.7
0.16
0.25
AVGLAGTFR_








446.26_721.40


 


Classifier
ISLR_HUMAN
ISLR
1.4
0.32
0.25
ALPGTPVASSQPR_








640.85_841.50


 


Classifier
TETN_HUMAN
CLEC3B
2.5
0.26
0.26 
LDTLAQEVALLK_








657.39_330.20


 


Classifier
FRIL_HUMAN
FTL
2.8
0.31
0.26
LGGPEAGLGEYLFER_








804.40_913.40


 


Classifier
GRP78_HUMAN
HSPA5
1.4
0.27
0.27 
TWNDPSVQQDIK_








715.85_260.20


 


Classifier
ALDOA_HUMAN
ALDOA
1.3
0.26
0.28
ALQASALK_401.25_








617.40


 


Classifier
BGH3_HUMAN
TGFBI
1.8
0.21
0.28
LTLLAPLNSVFK_








658.40_804.50


 


Classifier
LG3BP_HUMAN
LGALS3BP
4.3
0.29
0.29
VEIFYR_








413.73_598.30


 


Classifier
LRP1_HUMAN
LRP1
4.0
0.13
0.32
TVLWPNGLSLDIPAGR_








855.00_400.20


 


Classifier
FIBA_HUMAN
FGA
1.1
0.31
0.35
NSLFEYQK_








514.76_714.30


 


Classifier
PRDX1_HUMAN
PRDX1
1.5
0.32
0.37
QITVNDLPVGR_








606.30_428.30


 


Classifier
GSLG1_HUMAN
GLG1
1.2
0.34
0.45
IIIQESALDYR_








660.86_338.20


 


Robust
KIT_HUMAN
KIT
1.4
0.33
0.46



Robust
CD14_HUMAN
CD14
4.0
0.33
0.48



Robust
EF1A1_HUMAN
EEF1A1
1.2
0.32
0.56



Robust
TENX_HUMAN
TNXB
1.1
0.30
0.56



Robust
AIFM1_HUMAN
AIFM1
1.4
0.32
0.70



Robust
GGH_HUMAN
GGH
1.3
0.32
0.81



Robust
IBP3_HUMAN
IGFBP3
3.4
0.32
1.82



Robust
ENPL_HUMAN
HSP90B1
1.1
0.29
5.90



Non-Robust
ERO1A_HUMAN
ERO1L
6.2





Non-Robust
6PGD_HUMAN
PGD
4.3





Non-Robust
ICAM1_HUMAN
ICAM1
3.9





Non-Robust
PTPA_HUMAN
PPP2R4
2.1





Non-Robust
NCF4_HUMAN
NCF4
2.0





Non-Robust
SEM3G_HUMAN
SEMA3G
1.9





Non-Robust
1433T_HUMAN
YWHAQ
1.5





Non-Robust
RAP2B_HUMAN
RAP2B
1.5





Non-Robust
MMP9_HUMAN
MMP9
1.4





Non-Robust
FOLH1_HUMAN
FOLH1
1.3





Non-Robust
GSTP1_HUMAN
GSTP1
1.3





Non-Robust
EF2_HUMAN
EEF2
1.3





Non-Robust
RAN_HUMAN
RAN
1.2





Non-Robust
SODM_HUMAN
SOD2
1.2





Non-Robust
DSG2_HUMAN
DSG2
1.1

















Coefficient
Coefficient

Predicted




(Discovery)
(Final)

Concen-



SEQ
alpha =
alpha =
Tissue
tation


Category
ID NO
36.16
26.25 
Candidate
(ng/ml)





Classifier
22
 0.53
 0.44

510


Classifier
11
-1.56
-0.91

35


Classifier
14
 1.40
 0.83

—


Classifier
20
-1.79
-1.02

58000


Classifier
24
 0.39
 0.17
Secreted,
12






Epi, Endo



Classifier
23
 1.41
 0.55
Secreted,
100






Epi, Endo



Classifier
7
-0.80
-0.26
Secreted,
250






Epi, Endo



Classifier
8
 1.73
 0.54
Epi
140


Classifier
25
-0.58
-0.21
Secreted
440


Classifier
15
-1.59
-0.83
Epi
20


Classifier
26
 0.31
 0.13

130000


Classifier
16
-0.34
-0.26
Epi
60


Classifier
27
-0.70
-0.44
Epi, Endo
—


Robust




8.2


Robust



Epi
420


Robust



Secreted,
61






Epi



Robust



Endo
70


Robust



Epi, Endo
1.4


Robust




250


Robust




5700


Robust



Secreted,
88






Epi, Endo



Non-Robust



Secreted,
—






Epi, Endo



Non-Robust



Epi, Endo
29


Non-Robust




71


Non-Robust



Endo
3.3


Non-Robust



Endo
—


Non-Robust




—


Non-Robust



Epi
180


Non-Robust



Epi
—


Non-Robust




28


Non-Robust




—


Non-Robust



Endo
32


Non-Robust



Secreted,
30






Epi



Non-Robust



Secreted,
4.6






Epi



Non-Robust



Secreted 
7.1


Non-Robust



Endo
2.7









The set of 36 cooperative proteins was further reduced to a set of 21 proteins by manually reviewing raw SRM data and eliminating proteins that did not have robust SRM transitions due to low signal to noise or interference.
Proteins were iteratively eliminated from the set of 21 proteins until a classifier with the optimal partial AUC was obtained. The criteria for elimination was coefficient stability. In a logistic regression model each protein has a coefficient. In the process of training the model the coefficient for each protein is determined. When this is performed using cross validation (MCCV), hundreds of coefficient estimates for each protein are derived. The variability of these coefficients is an estimate of the stability of the protein. At each step the proteins were trained using MCCV (hold out rate 20%, ten thousand sample permutations per panel) to a logistic regression model and their stability measured. The least stable protein was eliminated. This process continued until a 13 protein classifier with optimal partial AUC was reached.
Finally, the 13 protein classifier was trained to a logistic regression model by MCCV (hold out rate 20%, twenty thousand sample permutations). The thirteen proteins for the rule-out classifier are listed in Table 18 along with their highest intensity transition and model coefficient.
Selection of a Decision Threshold
Assuming the cancer prevalence of lung nodules is prev, the performance of a classifier (NPV and ROR) on the patient population with lung nodules was calculated from sensitivity (sens) and specificity (spec) as follows:




  
    
      
        
          NPV
          =
          
            
              
                (
                
                  1
                  -
                  prev
                
                )
              
              *
              spec
            
            
              
                prev
                *
                
                  (
                  
                    1
                    -
                    sens
                  
                  )
                
              
              +
              
                
                  (
                  
                    1
                    -
                    prev
                  
                  )
                
                *
                spec
              
            
          
        
        ,
      
    
    
      
        (
        1
        )
      
    
  
  
    
      
        
          PPV
          =
          
            
              prev
              *
              sens
            
            
              
                prev
                *
                sens
              
              +
              
                
                  (
                  
                    1
                    -
                    prev
                  
                  )
                
                *
                
                  (
                  
                    1
                    -
                    spec
                  
                  )
                
              
            
          
        
        ,
      
    
    
      
        (
        2
        )
      
    
  
  
    
      
        ROR
        =
        
          
            prev
            *
            
              (
              
                1
                -
                sens
              
              )
            
          
          +
          
            
              (
              
                1
                -
                prev
              
              )
            
            *
            
              spec
              .
            
          
        
      
    
    
      
        (
        3
        )
      
    
  




The threshold separating calls for cancer or benign samples was then selected as the probability score with NPV ≧90% and ROR ≧20%. As we expect the classifier's performance measured on the discovery set to be an overestimate, the threshold is selected to be a range, as performance will usually degrade on an independent validation set.
Validation of the Rule-Out Classifier
52 cancer and 52 benign samples (see Table 17) were used to validate the performance of the 13 protein classifier. Half of the samples were placed in pre-determined processing batches analyzed immediately after the discovery samples and the other half of samples were analyzed at a later date. This introduced variability one would expect in practice. More specifically, the three HPS samples run in each processing batch were utilized as external calibrators. Details on HPS calibration are described below.
Calibration by HPS Samples
For label-free MS approach, variation on signal intensity between different experiments is expected. To reduce this variation, we utilized HPS samples as an external standard and calibrated the intensity between the discovery and validation studies. Assume that {hacek over (I)}i,s is the logarithmically transformed (base 2), normalized intensity of transition i in sample s, {hacek over (I)}i,dis and {hacek over (I)}i,val are the corresponding median values of HPS samples in the discovery and the validation studies, respectively. Then the HPS corrected intensity is

Ĩi,s={hacek over (I)}i,s−{hacek over (I)}i,val+{hacek over (I)}i,dis 

Consequently, assume that the probability for cancer of a clinical sample in the validation study is predicted as prob by the classifier. Then the HPS corrected probability of cancer of the clinical sample is calculated as follows:





  
    probability
    corrected
  
  =
  
    1
    
      1
      +
      
        e
        
          -
          
            S
            corrected
          
        
      
    
  




where

Scorrected=S−SHPS,val+SHPS,dis 

and





  S
  =
  
    ln
    ⁢
    
      
        prob
        
          1
          -
          prob
        
      
      .
    
  




Here SHPS,dis and SHPS,val were the median value of S of all HPS samples in the discovery and validation studies, respectively.
Statistical Analysis
All statistical analyses were performed with Stata, R and/or MatLab.
Depletion Column Drift
We observed an increase of signal intensity as more and more samples were depleted by the same column. We used transition intensity in HPS samples to quantify this technical variability. Assuming Ii,s was the intensity of transition i in a HPS sample s, the drift of the sample was defined as




  
    
      drift
      S
    
    =
    
      median
      ⁡
      
        (
        
          
            
              I
              
                i
                ,
                s
              
            
            -
            
              
                I
                ^
              
              s
            
          
          
            
              I
              ^
            
            s
          
        
        )
      
    
  
  ,




where Îi was the mean value of Ii,s among all HPS samples that were depleted by the same column and the median was taken over all detected transitions in the sample. Then the drift of the column was defined as

driftcol=median(drifts>0)−median(drifts<0).

Here the median was taken over all HPS samples depleted by the column. If no sample drift was greater or less than zero, the corresponding median was taken as 0. The median column drift was the median of drifts of all depletion columns used in the study.
Identification of Endogenous Normalizing Proteins
The following criteria were used to identify a transition as a normalizer:

    
    
        Possessed the highest median intensity of all transitions from the same protein.
        Detected in all samples.
        Ranked high in reducing median technical CV (median CV of transition intensities that were measured on HPS samples) as a normalizer.
        Ranked high in reducing median column drift that was observed in sample depletion.
        Possessed low median technical CV and low median biological CV (median CV of transition intensities that were measured on clinical samples).

Six transitions were selected and appear in Table 23.

    
    









TABLE 23







Panel of endogenous normalizers.














Median
Median




SEQ
Technical
Column


Normalizer
Transition
ID NO
CV (%)
Drift (%)





PEDF_HUMAN
LQSLFDSPDFSK_692.34_593.30
28
25.8
 6.8


 


MASP1_HUMAN
TGVITSPDFPNPYPK_816.92_258.10
6
26.5
18.3


 


GELS_HUMAN
TASDFITK_441.73_710.40
5
27.1
16.8


 


LUM_HUMAN
SLEDLQLTHNK_433.23_499.30
29
27.1
16.1


 


C163A_HUMAN
INPASLDK_429.24_630.30
30
26.6
14.6


 


PTPRJ_HUMAN
VITEPIPVSDLR_669.89_896.50
31
27.2
18.2


 



Normalization by Panel of

25.1
 9.0



Transitions





 



Without Normalization

32.3
23.8









Data Normalization
A panel of six normalization transitions (see Table 23) were used to normalize raw SRM data for two purposes: (A) to reduce sample-to-sample intensity variations within same study and (B) to reduce intensity variations between different studies. For the first purpose, a scaling factor was calculated for each sample so that the intensities of the six normalization transitions of the sample were aligned with the corresponding median intensities of all HGS samples. Assuming that Ni,s is the intensity of a normalization transition i in sample s and {circumflex over (N)}i the corresponding median intensity of all HGS samples, then the scaling factor for sample s is given by Ŝ/Ss, where




  
    S
    s
  
  =
  
    median
    ⁡
    
      (
      
        
          
            N
            
              1
              ,
              s
            
          
          
            
              N
              ^
            
            1
          
        
        ,
        
          
            N
            
              2
              ,
              s
            
          
          
            
              N
              ^
            
            2
          
        
        ,
        ...
        ⁢
        
          
        
        ,
        
          
            N
            
              6
              ,
              s
            
          
          
            
              N
              ^
            
            6
          
        
      
      )
    
  




is the median of the intensity ratios and Ŝ is the median of Ss over all samples in the study. For the second purpose, a scaling factor was calculated between the discovery and the validation studies so that the median intensities of the six normalization transitions of all HGS samples in the validation study were comparable with the corresponding values in the discovery study. Assuming that the median intensities of all HGS samples in the two studies are {circumflex over (N)}i,dis and {circumflex over (N)}i,val, respectively, the scaling factor for the validation study is given by





  R
  =
  
    median
    ⁡
    
      (
      
        
          
            
              N
              ^
            
            
              1
              ,
              dis
            
          
          
            
              N
              ^
            
            
              1
              ,
              val
            
          
        
        ,
        
          
            
              N
              ^
            
            
              2
              ,
              dis
            
          
          
            
              N
              ^
            
            
              2
              ,
              val
            
          
        
        ,
        ...
        ⁢
        
          
        
        ,
        
          
            
              N
              ^
            
            
              6
              ,
              dis
            
          
          
            
              N
              ^
            
            
              6
              ,
              val
            
          
        
      
      )
    
  




Finally, for each transition of each sample, its normalized intensity was calculated as

Ĩi,s=Ii,s*R*Ŝ/Ss 

where Ii,s was the raw intensity.

Isolation of Membrane Proteins from Tissues
Endothelial plasma membrane proteins were isolated from normal and tumor lung tissue samples that were obtained from fresh lung resections. Briefly, tissues were washed in buffer and homogenates were prepared by disrupting the tissues with a Polytron. Homogenates were filtered through a 180-μm mesh and filtrates were centrifuged at 900×g for 10 min, at 4° C. Supernatants were centrifuged on top of a 50% (w:v) sucrose cushion at 218,000×g for 60 min at 4° C. to pellet the membranes. Pellets were resuspended and treated with micrococcal nuclease. Membranes from endothelial cells were incubated with a combination of anti-thrombomodulin, anti-ACE, anti-CD34 and anti-CD144 antibodies, and then centrifuged on top of a 50% (w:v) sucrose cushion at 280,000×g for 60 min at 4° C. After pellets were resuspended, endothelial cell plasma membranes were isolated using MACS microbeads, treated with potassium iodide to remove cytoplasmic peripheral proteins.
Epithelial plasma membrane proteins from normal and tumor lung tissue samples were isolated from fresh lung resections. Tissues were washed and homogenates as described above for endothelial plasma membrane proteins preparation. Membranes from epithelial cells were labeled with a combination of anti-ESA, anti-CEA, anti-CD66c and anti-EMA antibodies, and then centrifuged on top of a 50% (w:v) sucrose cushion at 218,000×g for 60 min at 4° C. Epithelial cell plasma membranes were isolated using MACS microbeads and the eluate was centrifuged at 337,000×g for 30 minutes at 4° C. over a 33% (w:v) sucrose cushion. After removing the supernatant and sucrose cushion, the pellet was resuspended in Laemmli/Urea/DTT.
Isolation of Secreted Proteins from Tissues
Secreted proteins were isolated from normal and tumor lung tissue samples that were isolated from fresh lung resections. Tissues were washed and homogenized using a Polytron homogenization. The density of the homogenates was adjusted to 1.4 M with concentrated sucrose prior to isolating the secretory vesicles by isopycnic centrifugation at 100,000×g for 2 hr at 4° C. on a 0.8 and 1.2 M discontinuous sucrose gradient. Vesicles concentrating at the 0.8/1.2 M interface were collected and further incubated for 25 minutes with 0.5 M KCl (final concentration) to remove loosely bound peripheral proteins. Vesicles were recuperated by ultracentrifugation at 150,000×g for one hour at 4° C. and then opened with 100 mM ammonium carbonate pH 11.0 for 30 minutes at 4° C. Secreted proteins were recovered in the supernatant following a 1-hour ultracentrifugation at 150,000×g at 4° C.
Preparation of IgY14-SuperMix Immunoaffinity Columns
Immunoaffinity columns were prepared in-house using a slurry containing a 2:1 ratio of IgY14 and SuperMix immunoaffinity resins, respectively (Sigma Aldrich). Briefly, a slurry (10 ml, 50%) of mixed immunoaffinity resins was added to a glass chromatography column (Tricorn, GE Healthcare) and the resin was allowed to settle under gravity flow, resulting in a 5 ml resin volume in the column. The column was capped and placed on an Agilent 1100 series HPLC system for further packing (20 minutes, 0.15M ammonium bicarbonate, 2 ml/min). The performance of each column used in the study was then assessed by replicate injections of aliquots of HPS sample. Column performance was assessed prior to beginning immunoaffinity separation of each batch of clinical samples.
IgY14-Sumermix Immunoaffinity Chromatography
Plasma samples (60 μl) were diluted (0.15M ammonium bicarbonate, 1:2 v/v, respectively) and filtered (0.2 μm AcroPrep 96-well filter plate, Pall Life Sciences) prior to immunoaffinity separation. Dilute plasma (90 μl) was separated on the IgY14-SuperMix column connected to an Agilent 1100 series HPLC system using a three buffers (loading/washing: 0.15M ammonium bicarbonate; stripping/elution: 0.1M glycine, pH 2.5; neutralization: 0.01M Tris-HCl, 0.15M NaCl, pH 7.4) with a load-wash-elute-neutralization-re-equilibration cycle (36 minutes total time). The unbound and bound fractions were monitored using a UV absorbance (280 nm) and were baseline resolved after separation. Only the unbound fraction containing the low abundance proteins was collected for downstream processing and analysis. Unbound fractions were lyophilized prior to enzymatic digestion.
Enzymatic Digestion of Low Abundance Proteins
Low abundance proteins were reconstituted under mild denaturing conditions (200 μl of 1:1 0.1M ammonium bicarbonate/trifluoroethanol v/v) and allowed to incubate (30 minutes, room temperature, orbital shaker). Samples were then diluted (800 μl of 0.1M ammonium bicarbonate) and digested with trypsin (Princeton Separations; 0.4 μg trypsin per sample, 37° C., 16 hours). Digested samples were lyophilized prior to solid-phase extraction.
Solid-Phase Extraction
Solid phase extraction was used to reduce salt and buffer contents in the samples prior to mass spectrometry. The lyophilized samples containing tryptic peptides were reconstituted (350 μl 0.01M ammonium bicarbonate) and allowed to incubate (15 minutes, room temperature, orbital shaker). A reducing agent was then added to the samples (30 μl 0.05M TCEP) and the samples were incubated (60 minutes, room temperature). Dilute acid and a low percentage of organic solvent (375 μl 90% water/10% acetonitrile/0.2% trifluoroacetic acid) were added to optimize the solid phase extraction of peptides. The extraction plate (Empore C18, 3M Bioanalytical Technologies) was conditioned according to manufacturer protocol. Samples were loaded onto the solid phase extraction plate, washed (500 μl 95% water/5% acetonitrile/0.1% trifluoroacetic acid) and eluted (200 μl 52% water/48% acetonitrile/0.1% trifluoroacetic acid) into a collection plate. The eluate was split into two equal aliquots and each aliquot was taken to dryness in a vacuum concentrator. One aliquot was used immediately for mass spectrometry, while the other was stored (−80° C.) and used as needed. Samples were reconstituted (12 μl 90% water/10% acetonitrile/0.2% formic acid) just prior to LC-SRM MS analysis.
Inclusion and Exclusion Criteria
Plasma samples were eligible for the studies if they were (A) obtained in EDTA tubes, (B) obtained from subjects previously enrolled in IRB-approved studies at the participating institutions, and (C) archived, e.g. labeled, aliquotted and frozen, as stipulated by the study protocols. The samples must also satisfy the following inclusion and exclusion criteria:

    
    
        1) Inclusion Criteria:
        2) Sample eligibility was based on clinical parameters, including the following subject, nodule and clinical staging parameters:
        
            a) Subject
            
                i) age ≧40
                ii) any smoking status, e.g. current, former, or never
                iii) co-morbid conditions, e.g. COPD
                iv) prior malignancy with a minimum of 5 years in clinical remission
                v) prior history of skin carcinomas—squamous or basal cell
            
            
            b) Nodule
            
                i) Radiology
                
                    (1) size ≧4 mm and ≦70 mm (up to Stage 2B eligible)
                    (2) any spiculation or ground glass opacity
                
                
                ii) pathology
                
                    (1) malignant—adenocarcinoma, squamous, or large cell
                    (2) benign—inflammatory (e.g. granulomatous, infectious) or non-inflammatory (e.g. hamartoma)
                
                
            
            
            c) Clinical stage
            
                i) Primary tumor: ≦T2 (e.g. 1A, 1B, 2A and 2B)
                ii) Regional lymph nodes: NO or N1 only
                iii) Distant metastasis: MO only
            
            
        
        
        3) Exclusion Criteria
        
            a) Subject: prior malignancy within 5 years of IPN diagnosis
            b) Nodule:
            
                i) size data unavailable
                ii) for cancer or benign SPNs, no pathology data available
                iii) pathology—small cell lung cancer
            
            
            c) Clinical stage
            
                i) Primary tumor: ≧T3
                ii) Regional lymph nodes: ≧N2
                iii) Distant metastasis: ≧M1
            
            
        
        
    
    


Power Analysis for the Discovery Study
The power analysis for the discovery study was based on the following assumptions: 1) The overall false positive rate (α) was set to 0.05. 2) {hacek over (S)}idák correction for multiple testing was used to calculate the effective αeff for testing 200 proteins, i.e., αeff=1−200√{square root over (1−α)}. 3) The effective sample size was reduced by a factor of 0.864 to account for the larger sample requirement for the Mann-Whitney test than for the t-test. 4) The overall coefficient of variation was set to 0.43 based on a previous experience. 5) The power (1−β) of the study was calculated based on the formula for the two-sample, two-sided t-test, using effective αeff and effective sample size. The power for the discovery study was tabulated in Table 24 by the sample size per cohort and the detectable fold difference between control and disease samples.







TABLE 24







Cohort size required to detect protein


fold changes with a given probability.










Detectable Protein Fold Difference












Cohort Size
1.25
1.5
1.75
2














20
0.011
0.112
0.368
0.653


30
0.025
0.277
0.698
0.925


40
0.051
0.495
0.905
0.992


50
0.088
0.687
0.977
0.999


60
0.129
0.812
0.994
1


70
0.183
0.902
0.999
1


80
0.244
0.953
1
1


90
0.302
0.977
1
1


100
0.369
0.99
1
1









Power Analysis for the Validation Study
Sufficient cancer and benign samples are needed in the validation study to confirm the performance of the rule-out classifier obtained from the discovery study. We are interested in obtaining the 95% confidence intervals (CIs) on NPV and ROR for the rule-out classifier. Using the Equations in the Selection of a Decision Threshold section herein, one can derive sensitivity (sens) and specificity (spec) as functions of NPV and ROR, i.e.,

sens=1−ROR*(1−NPV)/prev,

spec=ROR*NPV/(1−prev),

where prev is the cancer prevalence in the intended use population. Assume that the validation study contains NC cancer samples and NB benign samples. Based on binomial distribution, variances of sensitivity and specificity are given by

var(sens)=sens*(1−sens)/NC 

var(spec)=spec*(1−spec)/NB 

Using the Equations in the Selection of a Decision Threshold section herein, the corresponding variances of NPV and ROR can be derived under the large-sample, normal-distribution approximation as





  
    
      var
      ⁡
      
        (
        NPV
        )
      
    
    =
    
      
        
          
            NPV
            2
          
          ⁡
          
            (
            
              1
              -
              NPV
            
            )
          
        
        2
      
      ⁡
      
        [
        
          
            
              var
              ⁡
              
                (
                sens
                )
              
            
            
              
                (
                
                  1
                  -
                  sens
                
                )
              
              2
            
          
          +
          
            
              var
              ⁡
              
                (
                spec
                )
              
            
            
              spec
              2
            
          
        
        ]
      
    
  
  ,
  
    

  
  ⁢
  
    
      var
      ⁡
      
        (
        ROR
        )
      
    
    =
    
      
        
          prev
          2
        
        *
        
          var
          ⁡
          
            (
            sens
            )
          
        
      
      +
      
        
          
            (
            
              1
              -
              prev
            
            )
          
          2
        
        *
        
          
            var
            ⁡
            
              (
              spec
              )
            
          
          .
        
      
    
  




The two-sided 95% CIs of NPV and ROR are then given by ±zα/2√{square root over (var(NPV))} and ±zα/2√{square root over (var(ROR))}, respectively, where zα/2=1.959964 is the 97.5% quantile of the normal distribution. The anticipated 95% CIs for the validation study were tabulated in Table 24 by the sample size (NC=NB=N) per cohort.







TABLE 24







The 95% confidence interval (CI) of NPV as a function of cohort


size. The corresponding 95% CI of ROR is also listed. The prevalence


was set at 28.5%. The expected NPV and ROR were set to values in


the discovery study, i.e., 90% and 52%, respectively.










95% CI of
95% CI of


Cohort Size
NPV (± %)
ROR (± %)












10
12.5
22.1


20
8.8
15.7


30
7.2
12.8


40
6.2
11.1


50
5.6
9.9


60
5.1
9.0


70
4.7
8.4


80
4.4
7.8


90
4.2
7.4


100
3.9
7.0


150
3.2
5.7


200
2.8
5.0









Calculation of Q-Values of Peptide and Protein Assays
To determine the false positive assay rate the q-values of peptide SRM assays were calculated as follows. Using the distribution of Pearson correlations between transitions from different proteins as the null distribution (FIG. 7), an empirical p-value was assigned to a pair of transitions from the same peptide, detected in at least five common samples otherwise a value of ‘NA’ is assigned. The empirical p-value was converted to a q-value using the “qvalue” package in Bioconductor (www.bioconductor.org/packages/release/bioc/html/qvalue.html). Peptide q-values were below 0.05 for all SRM assays presented in Table 6.
The q-values of protein SRM assays were calculated in the same way except Pearson correlations of individual proteins were calculated as those between two transitions from different peptides of the protein. For proteins not having two peptides detected in five or more common samples, their q-values could not be properly evaluated and were assigned ‘NA’.
Impact of Categorical Confounding Factors







TABLE 25







Impact of categorical confounding factors on classifier score.












Cancer
p-value
Benign
p-value
















Gender
# Female
70
0.786*
68
0.387*



Median score
0.701

0.570



(quartile range)
(0.642-0.788)

(0.390-0.70) 



# Male
54

55



Median
0.736

0.621



(quartile range)
(0.628-0.802)

(0.459-0.723)


Smoking
# Never
8
0.435**
34
0.365**


Status
Median score
0.664

0.554



(quartile range)
(0.648-0.707)

(0.452-0.687)



# Past
98

73



Median
0.703

0.586



(quartile range)
(0.618-0.802)

(0.428-0.716)



# Current
17

13



Median score
0.749

0.638



(quartile range)
(0.657-0.789)

(0.619-0.728)





*p-value by Mann-Whitney test


**p-value by Kruskal-Wallis test






Impact of Continuous Confounding Factors







TABLE 26







Impact of continuous confounding factors on classifier score.












Coefficient of




Correlation
linear fit (95% CI)
p-value















Age
All
0.198
0.003
0.002





 (0.001-0.005)



Cancer
0.012
0.000
0.893





(−0.003-0.003)



Benign
0.248
0.004
0.006





 (0.001-0.007)


Nodule
All
−0.057
−0.002
0.372


size


(−0.005-0.002)



Cancer
−0.013
0.000
0.889





(−0.005-0.004)



Benign
−0.055
−0.001
0.542





(−0.006-0.003)


Pack-
All
0.154
0.001
0.019


year


 (0.00-0.002)



Cancer
0.060
0.000
0.520





(−0.001-0.001)



Benign
0.108
0.001
0.254





 (0.00-0.002)









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## Claims

1. A method of determining the likelihood that a pulmonary nodule in a subject is not lung cancer, comprising:
(a) contacting a blood sample obtained from the subject with a proteolytic enzyme to produce peptide fragments from a panel of proteins present in the blood sample, wherein the panel comprises ALDOA, FRIL, LG3BP, TSP1, and COIA1;
(b) combining the produced peptide fragments from the panel from step (a) with labeled, synthetic peptide fragments which correspond to the produced peptide fragments from the panel;
(c) performing selected reaction monitoring mass spectrometry to measure the abundance of the peptide fragments from step (b);
(d) calculating a probability of lung cancer score based on the peptide fragment measurements of step (c); and
(e) ruling out lung cancer for the subject if the score in step (d) is lower than a pre-determined score.

2. The method of claim 1, wherein said panel further comprises at least one protein selected from the group consisting of PEDF, MASP1, GELS, LUM, C163A and PTPRJ.

3. The method of claim 1, wherein when lung cancer is ruled out, the subject does not receive a treatment protocol.

4. The method of claim 3, wherein said treatment protocol is a pulmonary function test (PFT), pulmonary imaging, a biopsy, a surgery, a chemotherapy, a radiotherapy, or any combination thereof.

5. The method of claim 4, where said imaging is an x-ray, a chest computed tomography (CT) scan, or a positron emission tomography (PET) scan.

6. The method of claim 1, wherein said pulmonary nodule has a diameter of less than or equal to 3 cm.

7. The method of claim 1, wherein said pulmonary nodule has a diameter of about 0.8 cm to 2.0 cm.

8. The method of claim 1, wherein said score is calculated from a logistic regression model applied to the peptide fragment measurements.

9. The method of claim 1, wherein said score is determined as Ps=1/[1+exp(−α−Σi=1Nβi*{hacek over (I)}i,s)], where is logarithmically transformed and normalized intensity of transition in said sample (s), βi is the corresponding logistic regression coefficient, α was a panel-specific constant, and N was the total number of transitions in said panel.

10. The method of claim 1, wherein the determining the likelihood that a pulmonary nodule is not lung cancer is determined by the sensitivity, specificity, negative predictive value or positive predictive value associated with the score.

11. The method of claim 1, wherein said score determined in step (d) has a negative predictive value (NPV) of at least about 80%.

12. The method of claim 1, wherein the selected reaction monitoring mass spectrometry is performed using a compound that specifically binds the peptide fragments being detected.

13. The method of claim 12, wherein the compound that specifically binds to the peptide fragments being measured is an antibody or an aptamer.

14. The method of claim 1, wherein the subject is at risk of developing lung cancer.

15. The method of claim 1, wherein the proteolytic enzyme is trypsin.