Concordance of Molecular Classification Based on Fine Needle Biopsy (FNB) and Surgical Samples
Conditions: Papillary Thyroid Cancer
Sponsor: University of Calgary

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CLINICAL TRIAL PROTOCOL 
 
Protocol Number: Pending 
 
Date: July 11, 2023 
 
Concordance of Molecular Classification Based on Fine Needle Biopsy 
(FNB) and Surgical Samples 
 
Sponsor: Qualisure Diagnostics Inc. 
 
Medical Expert for the Trial: 
Caitlin Yeo, MD, FRCSC 
Title: Clinical Instructor, Endocrine Surgeon, University of Calgary 
Address: North Tower Room 1006, 1403 29th St NW, Calgary, AB, T2N 2T9 
Phone: 403-944-0966 
Fax: 403-944-6008 
Email: Caitlin.Yeo@albertahealthservices.ca 
 
Investigators Responsible for Conducting the Trial 
Caitlin Yeo, Clinical Instructor, Surgery, University of Calgary; Endocrine Surgeon 
Adrian Harvey, Clinical Associate Professor, University of Calgary; Endocrine Surgeon 
Janice Pasieka, Professor, University of Calgary; Endocrine Surgeon 
Shamir Chandarana, Clinical Associate Professor, University of Calgary; Head & Neck Surgeon 
Robert Hart, Professor, University of Calgary; Head & Neck Surgeon 
Wayne Matthews, Associate Professor, University of Calgary; Head & Neck Surgeon 
Martin Hyrcza, Assistant Professor, University of Calgary; Molecular Pathologist 
Moosa Khalil, Clinical Associate Professor, University of Calgary; Pathologist 
Anthony Magliocco, Pathologist; CEO of Protean BioDiagnostics 
Karen Kopciuk, Adjuvant Associate Professor, University of Calgary; Biostatistician, Alberta Health 
Services 
Tasnima Abedin, Biostatistician, Alberta Health Services 
 
Trial Site: Foothills Medical Centre, 3330 Hospital Dr NW, Calgary, AB 
 
 
 
 
 
 

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Signature Page 
 
By signing below, the Principal Investigator agrees to adhere to the protocol in the conduct of this 
study.  Any change in the study must be reviewed by a formal protocol amendment procedure and the 
Principal Investigator will submit all changes, amendments and revisions to the Health Research Ethics 
Board of Alberta Cancer Committee (HREBA.CC).  Any change to the protocol that affects patient selection, 
safety, or changes in the conduct of the trial will require written approval from HREBA.CC and Health 
Canada before implementing the change. 
The Investigator(s) also agree(s) to conduct the study in accordance with the protocol and all applicable 
laws and regulations including, but not limited to, the International Conference on Harmonization (ICH) 
Guideline for Good Clinical Practice (GCP), Division 5 of Health Canada’s Food and Drug Regulations, the 
ethical principles that have their origins in the Declaration of Helsinki, and applicable privacy laws. 
The Principal Investigator also thereby agrees that HREBA.CC will approve all patient informed consent 
forms before the study is initiated.  The investigator will obtain informed consent and document this 
process for all patients enrolled on this study. 
 
 
 
_________________________________ 
 
Principal Investigator (Print) 
 
 
______________________ 
 
Signature 
 
 
________________________ 
Date (dd/mm/yyyy) 
 
 

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STUDY SYNOPSIS 
 
Title 
Concordance of Molecular Risk Stratification 
Based on Fine Needle Biopsy (FNB) and Surgical 
Samples 
Short Title 
Molecular Risk by FNA 
Protocol Number 
HREBA. CC-22-0007 
Phase 
N/A 
Methodology 
Prospective blinded comparison study 
Study Duration 
May 1, 2022 – May 1, 2028 
Study Centres 
Foothills Medical Centre 
Objective(s) 
The primary objective is to determine the 
concordance between Thyroid GuidePx® 
molecular classifications acquired from fine 
needle biopsy (FNB) and matched surgical 
samples. 
Number of participants 
130 
Diagnosis and Main Inclusion Criteria 
Diagnosis: Papillary thyroid cancer 
Main Inclusion Criteria: A diagnosis of papillary 
thyroid cancer based on an FNB interpreted as a 
Bethesda V or VI cytology, candidate for 
resection. 
Study Product 
Thyroid GuidePx® 
Intervention 
None 
Statistical Methodology 
Descriptive statistics will be reported for all study 
variables. Concordance of molecular classification 
between FNB and surgical samples will be 
evaluated by the Cohen kappa statistic. 
Differences in progression free survival will be 
evaluated using the log rank statistic. 
Performance of Thyroid GuidePx® and of the 
American Thyroid Association risk stratification 
index will be evaluated by time-related AUROC, 
sensitivity and specificity. 
 
 
 

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BACKGROUND INFORMATION 
Thyroid cancer is the 8th most common cancer by prevalence, and incidence has been increasing by 
>6% per year since 1992.  Papillary thyroid cancer (PTC) accounts for most thyroid cancers, and the rising 
incidence of thyroid cancer can be almost entirely attributed to an increased detection of small PTC’s (1). 
PTC usually has a favorable prognosis with high cure rates. However, approximately 10-15% of PTC’s 
display a more aggressive behavior and are often resistant to conventional adjuvant therapies such as 
radioactive iodine (2,3).  Given the increasing number of PTC cases (and the potential burden on health 
care costs), accurate prognostication is becoming increasingly important. Accurate estimations of 
prognosis and risk of recurrence will avoid unnecessarily extensive surgery, investigations and follow-up 
in individuals with a good prognosis, and will direct more extensive surgery, adjuvant therapies and 
prolonged follow-ups to those who need it.  
 
The American Thyroid Association (ATA) Risk Stratification System 
Treatment decisions are informed by the American Thyroid Association (ATA) Risk Stratification 
System, which estimates the risk of disease recurrence based on clinical and pathological factors. Although 
this system has been retrospectively validated by a number of studies, the proportion of variance 
explained is sub-optimal and ranges from 19%-34% (4, 5). The inability of the ATA system to fully predict 
recurrence may be related to the failure of this system to adequately integrate the risk associated with a 
tumor’s molecular profile. Only one molecular marker, BRAFV600E, is currently incorporated in the ATA Risk 
Stratification System. Another limitation of the ATA Risk Stratification System is that it can only be fully 
implemented after surgery, when all of the clinical and pathological data required to estimate risk are 
available. At that time, significant treatment decisions have already been made and the impact of surgery 
has already been realized by the patient. 
 
Estimating Risk Prior to Treatment: The Current Standard of Care 
In 2015, the ATA reported on criteria that could be used to select patients for more conservative 
surgeries such as thyroid lobectomy. According to the 2015 ATA guidelines, patients with the following 
preoperative criteria are considered low risk: tumor size 1 - 4 cm, clinically node negative, no 
extrathyroidal spread, no family history of thyroid cancer, and no history of radiation therapy to the neck 
(6). These patients could be initially treated with a thyroid lobectomy (as opposed to a total 
thyroidectomy). However, 40 - 60% will require a completion thyroidectomy because the postoperative 
risk stratification (using all clinical and pathological information available after surgery) classified them as 
a higher risk than initially estimated (7-11). In a recent study, 51% of patients required conversion to a 
completion thyroidectomy because additional risk factors were recognized during surgery or after final 
pathology was available (12). This demonstrates the limitations of the clinical preoperative risk 
estimations, and the need for an improved method of preoperative risk estimation. Therefore, thyroid 
cancer surgeons and patients would benefit from additional information that would refine the decision to 
perform lobectomy in appropriate patients while minimizing the need for completion thyroidectomy. 
 
Thyroid GuidePx®: A New Molecular Risk Stratification System 
The shortcomings of the ATA Risk Stratification System to reliably determine prognosis has been 
addressed using a new molecular risk stratification system that comprises the Thyroid GuidePx® gene 
signature. Using a computer software algorithm, transcripts (mRNA) associated with recurrence and 
transcripts associated with absence of recurrence were identified in a training set of 335 patients with 
PTC, then validated in a validation set of 167 patients. Tumors included in the dataset were fresh frozen. 
There were three molecular subtypes identified based on the pattern of expression of prognostic 
genes. These were called Type 1, Type 2 and Type 3. Type 3 PTCs had the most aggressive biology and the 

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highest rate of recurrence. The was subsequently validated in 124 PTC patients from Korea as well as 167 
patients from Edmonton.  
The molecular subtypes were considered in the context of tumor stage. Specifically, tumors measuring 
1 – 4 cm in the absence of lymph node disease (which comprise patients who would be potential 
candidates for thyroid lobectomy) were separately analyzed from patients with more advanced disease. 
Interestingly, Type 3 PTCs had a high recurrence rate regardless of tumor stage. Lobectomy candidates 
with Type 1 and Type 2 PTCs had a recurrence rate of <3% (Figure 1A, B). 
 
 
.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1. Recurrence outcomes for the three molecular subtypes of PTC. A. Five-year 
recurrence rates for each molecular subtype as a function of whether tumor is early or 
advanced. B. Kaplan-Meier curve demonstrating a very low recurrence rate in lobectomy 
candidates with Type 1 and 2 PTCs. The data are based on a total of 742 patients. 
Test Performance 
The NGS-based test that identified the molecular subtype of PTC was called Thyroid GuidePx®. The 
combined molecular and clinical subclassification of PTC using Thyroid GuidePx® was compared against 
the ATA Risk Stratification System. ATA risk stratification groups were determined by two practicing 
clinicians for each case. To calculate time-dependent area under the receiver operating characteristic 
curve (AUROC), a binary classification was applied: low risk versus intermediate/high risk. AUROC was 
calculated based on recurrence outcomes from the TCGA discovery cohort and the Edmonton cohort. The 
best risk stratification performance was achieved by the Thyroid GuidePx® test in conjunction with clinical 
A 
B 

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features (i.e., tumor size and lymph node involvement). Using the smoothROCtime package, the 5-year 
AUROC was 0.80 for Thyroid GuidePx® when considering tumor size and lymph node status, and 0.51 for 
the ATA risk stratification system (Figure 2).  
 
 
 
Figure 2. 5-year AUROC for various dichotomous prognostications. The TCGA test cohort (N 
= 167) and the Edmonton cohort (N = 132) were used. The training set was specifically 
excluded in this analysis. A. ATA risk stratification system (low risk vs. intermediate/high 
risk). B. Low risk defined by Thyroid GuidePx® in conjunction with clinical features (i.e., 
tumor size and lymph node involvement). 
Thyroid GuidePx®: Assay Description 
The Thyroid GuidePx® test is an assay based on targeted hybrid-capture enrichment RNA sequencing 
(RNA-Seq). The test input is RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue or fine 
needle biopsy (FNB) papillary thyroid cancer specimens. Library preparation involves specifically 
amplifying the targeted regions of each of the genes in the Thyroid GuidePx® panel. The panel consists of 
82 genes. The resulting sample libraries are transferred to a MiSeq/MiSeqDx reagent cartridge which 
contains all of the reagents for sequencing. Real-Time Analysis (RTA) software, which is a component of 
the MiSeq/MiSeqDx sequencer, provides base calls and associated quality scores during the sequencing 
run. FASTQ files are generated that contain the sequencing and quality information. The FASTQ file is a 
widely used text-based file format for storing nucleotide sequence for each amplicon and its 
corresponding quality score. The FASTQ files serve as input for sequence alignment algorithms. Aligned 
reads are written to files in BAM format which are subsequently converted to read counts and expression 
values. Gene expression levels are then submitted to the algorithm to classify risk categories.   
The software classifies risk based on the pattern of expression of groups of genes within the 82 gene 
Thyroid GuidePx® panel. Three molecular classes have been defined based on a study with 502 patient 
samples associating the pattern of gene expression with recurrence free survival at 5 years. A high-risk 
group enriched with BRAFV600E mutations; and two lower risk molecular subgroups were identified. 
 
Clinical Utility 
The more accurate prognostication provided by Thyroid GuidePx® could affect clinicians’ treatment 
decisions. The following are examples of how clinicians may apply the new information derived from 
Thyroid GuidePx®: 
1. Low risk PTC could potentially be treated with a partial thyroidectomy (e.g., lobectomy) or even 
active surveillance, rather than a total thyroidectomy. This has two major benefits in addition to 
time and cost savings. First, there is a reduction in the potential need for life-long replacement of 
AUROC 0.51 
AUROC 0.80 
A 
B 

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thyroid hormone following a lobectomy. Second, there is virtually no risk of the potentially 
devastating complications of a total thyroidectomy such as bilateral recurrent laryngeal nerve 
injury or permanent hypoparathyroidism. 
2. Accurately identifying low risk PTC would also increase the appropriateness of adjuvant 
radioactive iodine (RAI). RAI therapy may result in both transient and long-term side effects such 
as salivary gland dysfunction, ovarian and testicular dysfunction, and bone marrow suppression. 
RAI therapy also has a theoretical risk of secondary malignancy: one estimate is that 100mCi 131I 
induces 56 malignancies per 10,000 patients treated (15). Limiting RAI therapy to those patients 
who would derive improvement in overall or disease-free survival is also cost effective. 
3. Surveillance of PTC can place a strain on patients and healthcare organizations for two reasons: 
PTC is most commonly diagnosed in middle age, and patients often survive for long periods after 
their diagnosis. This means that regular examination and investigations for the early detection of 
recurrence can go on for many years. Follow-up typically involves annual physical examination, 
serum measurement of thyroid stimulating hormone and thyroglobulin, and periodic neck 
ultrasound. Low risk patients can have increased intervals between follow-up examinations and 
investigations, and could potentially be discharged from surveillance. 
4. Accurately identifying patients with a high risk of recurrence is also important. The ATA Risk 
Stratification System is known to be inaccurate at predicting recurrence. In patients classified as 
“low risk” using the 2015 ATA Risk Stratification System, 16% of patients eventually develop 
recurrent disease (16). These patients may be undertreated as a result of this risk classification. 
In patients where a more accurate assignment of high risk is available, more aggressive 
treatments (i.e. total thyroidectomy and RAI) or enrollment in clinical trials may be considered. 
 
Thyroid GuidePx® by Fine Needle Biopsy 
Our studies have so far been done using surgical specimens, and a commercially available assay has 
been produced in this format. However, a recent study by the Institute of Health Economics has 
determined that enhanced cost effectiveness of Thyroid GuidePx® would be achieved by performing the 
test prior to surgery. Implementing a test preoperatively would require that the test performs well using 
samples derived from a fine needle biopsy (FNB).  
Thyroid FNBs are routinely stored in CytoLyt, a methanol based buffered liquid preservative, for up to 
7 days prior to transferring cells to a ThinPrep for cytological evaluation. We and others (17–21) have 
previously determined that quality DNA and RNA can be derived from cells in CytoLyt. Our recent 
feasibility study consisting of 12 patients with PTC demonstrated that performing the Thyroid GuidePx® 
assay on FNBs is feasible. So far, the technical success rate has been 100%, and gene level concordance 
with surgical samples exceeds 0.9. However, reliance on a limited FNB for molecular disease 
characterization implies that the sample is representative of the entirety of the tumor. Genomic and 
transcriptomic heterogeneity has been described in primary tumors and metastases (23). Therefore, it will 
be important to document the concordance between samples acquired by FNB and surgical samples. 
 
TRIAL OBJECTIVES AND PURPOSE 
The general purpose of the study is to determine whether the more limited sample from a fine needle 
biopsy is sufficiently representative of the larger tumor to determine a valid molecular classification using 
the Thyroid GuidePx® test. 
 
Primary Objective 
1. To determine the concordance between Thyroid GuidePx® molecular classifications acquired from 
FNAs and matched surgical samples. 

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Secondary Objectives 
1. To compare the technical success rate in completing a valid Thyroid GuidePx® using FNAs and surgical 
samples. 
2. To compare recurrence outcomes using the ATA risk stratification system vs. patients classified by 
Thyroid GuidePx® using surgical samples. 
3. To compare recurrence outcomes using the ATA risk stratification system vs. patients classified by 
Thyroid GuidePx® using FNA samples. 
 
TRIAL DESIGN 
Trial Schematic 
 
 

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Sample Acquisition and Analysis 
Patients will be invited to participate if they have a preoperative tissue diagnosis of PTC (Bethesda VI) 
or suspicious for PTC (Bethesda V), and they are eligible for partial or total thyroidectomy, as described in 
the inclusion and exclusion criteria. Following consent, patients will undergo surgery as planned by their 
surgeon. Demographic, clinical and pathological data will be acquired from all participants. 
During surgery, when the thyroid gland and the tumor are exposed, the surgeon will perform an FNB 
of the dominant tumor (ie: the lesion identified preoperatively), under direct vision. This most closely 
simulates the in vivo conditions of a preoperative FNB yet limits potential technical downfalls of a 
percutaneous biopsy (eg: inadequate targeting of the lesion). The cellular material derived from the FNB 
will immediately be put in the transport medium (CytoLyt), then refrigerated. Samples will be sent weekly 
to the central laboratory for RNA isolation and RNASeq. At the same time, FNB samples will be examined 
for presence of tumor cells by a trained cytopathologist. 
If more than one nodule is present, the FNB nodule will be marked by the surgeon for the pathologist. 
Under the direction of a collaborating pathologist, a piece of the tumor will be removed and snap frozen, 
and an adjacent piece will be taken for formalin fixation and paraffin embedding (FFPE). One H & E slide 
from the FFPE sample with be stained for H & E, and 3 – 6 unstained sections will be prepared, then sent 
to the central laboratory for microdissection, RNA isolation and RNASeq. This process will follow routine 
specimen processing protocols and will not interfere with standard methods of pathologic diagnosis. 
If patients have signed the optional informed consent form for biobanking, if there is sufficient 
remaining frozen sample, the sample will be retained for the purpose of re-testing (if needed) or to 
support additional studies such as whole transcriptome analysis and mutational analysis. Use of these 
samples for these purposes would require additional ethics approvals. 
A feasibility study consisting of 12 patients has already been completed. Technical success rate in 
acquiring samples and performing the assay was 100%, RNA quantity and quality were excellent in FNBs, 
and there was excellent gene level concordance in assays performed on FNBs and surgical samples. 
RNASeq for Thyroid GuidePx® will be performed at OncoHelix, a clinical laboratory with expertise in 
precision oncology located at the Cumming School of Medicine, University of Calgary. If testing cannot be 
done on a timely basis at OncoHelix, samples will be tested at Protean BioDiagnostics, a CAP/CLIA certified 
lab in Orlando, FL that currently performs the test, commercially. Risk classes will be assigned by software 
designed by Qualisure Diagnostics using the data generated by RNASeq. 
 
Endpoints 
Primary Endpoint: 
1. Concordance, molecular risk classification of papillary thyroid cancer FNBs and matched frozen surgical 
samples  
 
Secondary Endpoints: 
1. 
Technical success rate in completing a valid Thyroid GuidePx® using FNBs 
2. 
Technical success rate in completing a valid Thyroid GuidePx® in FFPE surgical samples. 
3. 
Biochemical recurrence rate at 5 years (thyroglobulin ≥ 1 ng/mL, for patients who had a total 
thyroidectomy only) 
4. 
Structural recurrence rate at 5 years 
5. 
Test performance as a prognostic test, Thyroid GuidePx®: FNB and surgical samples 
a. Specificity: the proportion of patients without structural recurrence predicted by the 
test 
b. Sensitivity: the capability of the test to identify individuals whose disease will recur 
within 5 years (proportion of structural recurrences predicted) 
c. AUROC 

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d. Positive predictive value 
e. Negative predictive value 
6. 
Performance, ATA Risk Stratification 
a. Specificity: the proportion of patients without structural recurrence predicted by the 
test 
b. Sensitivity: the capability of the test to identify individuals whose disease will recur 
within 5 years (proportion of structural recurrences predicted) 
c. AUROC 
d. Positive predictive value 
e. Negative predictive value 
 
Blinding and Limitation of Bias 
Only samples with sufficient RNA quality (DV200>30) will be included in the analysis. Only FNBs 
containing tumor cells (“cytology positive”) will be included in the concordance calculation. RNASeq will 
be performed by a technician who is blinded to any clinical details. The technician will specifically be 
unable to identify matched samples from surgical specimens and FNBs. Molecular classification will be 
performed in a blinded fashion. Data will be monitored by an independent Data Monitoring Committee. 
The Data Monitoring Committee, consisting of at least three individuals including a statistician, will 
monitor data analysis. 
 
Stopping Rules 
In our surgical samples, inter-batch and intra-batch repeats consistently had a gene level concordance 
of >0.9. Gene level concordance between cytology positive FNB and frozen surgical samples (the “gold 
standard”) will be monitored for each batch of 24 patients. If R<0.9, we will pause to re-evaluate the 
analytical workflow and instrumentation. If concordance is persistently below 0.9 after 72 patients, we 
will stop evaluating FNBs, but we will continue to test surgical samples. Since this is not a high-risk study, 
we do not intend to terminate the trial prematurely. Even if there is low concordance between FNB and 
surgical samples, the data derived from studying outcomes in relation to molecular classification (in 
surgical samples) will be useful. 
 
SELECTION AND WITHDRAWAL OF SUBJECTS  
Inclusion Criteria 
1. Age  ≥ 18 years 
2. A diagnosis of papillary thyroid cancer based on a fine needle biopsy (FNB) interpreted as a Bethesda 
V or VI cytology 
3. Tumor size > 1 cm in maximal diameter 
4. The patient is an operative candidate 
5. The patient has provided consent 
 
Exclusion Criteria 
1. Family history of thyroid cancer 
2. History of radiation to the neck 
3. Unable or unwilling to have a fine needle biopsy  
4. Unwilling to undergo thyroidectomy 
5. Final pathology does not demonstrate papillary thyroid cancer 
6. Cases where there is no clear dominant nodule 
7. Cases where there are multiple nodules that preclude sampling of a defined nodule 
 

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Subject Withdrawal Criteria 
Subjects can discontinue participation at any time by notifying one of the investigators or the research 
staff. Data acquired prior to withdrawal will be used for analysis, but no additional data will be collected 
after withdrawal. At the request of the subject, samples can be withdrawn prior to testing. Subjects will 
be replaced if samples have not been tested by RNASeq (Thyroid GuidePx®). 
 
TREATMENT OF SUBJECTS 
The molecular classification and recurrence risk determined by Thyroid GuidePx® will be disclosed to the 
physician and to the patient. Recurrence risk is based on cumulative validation data including cases from 
Alberta and Australia. Only the results of the frozen surgical sample (the “gold standard”) will be disclosed. 
As is the usual standard, treatment decisions will be made by the physician in collaboration with the 
patient, based on a synthesis of all available clinical information. This clinical trial does not define a 
treatment pathway. Follow up frequency and duration will not be prescribed by protocol and will be 
determined by the treating physician. This is a non-interventional study, and so compliance will not be 
monitored. 
 
 ASSESSMENT OF EFFICACY  
 
A secondary outcome is test performance to stratify recurrence risk. Test performance will be 
compared in samples acquired by FNB and surgical samples, then compared to the current standard of 
care (the ATA risk stratification system). Time to recurrence will be measured from the time of complete 
initial treatment. In patients with microscopic disease remaining after surgery, time to recurrence will be 
measured from the completion of the therapeutic dose of radioactive iodine. Recurrence events will be 
defined based on the 2016 American Thyroid Association guidelines. 
The following criteria will signify a disease-free status after therapy: (i) no clinical evidence of tumor; 
(ii) no evidence of tumor on radioactive iodine imaging and/or cervical ultrasound; (iii) for post-total 
thyroidectomy, unstimulated serum thyroglobulin (Tg) <0.2 ng/ml; for post-lobectomy, unstimulated 
Tg<30. An indeterminate response to therapy will be defined as: (i) no clinical evidence of tumor; (ii) no 
evidence of tumor on radioactive iodine imaging and/or cervical ultrasound; (iii) for post-total 
thyroidectomy, unstimulated Tg 0.2 – 1; for post-lobectomy, Tg>30. Following a total thyroidectomy, a 
biochemical incomplete response is defined as Tg>1 in the absence of structural disease. Tg values are 
only reliable in the absence of interfering antibodies (TgAb) (6).  
Disease will be defined as a true recurrence if a patient previously had an undetectable Tg in the 
absence of TgAb and a negative neck ultrasound examination within 1 year after the previous operation. 
Persistent disease will be defined as Tg greater than the above thresholds, an abnormal neck ultrasound, 
persistently increased levels of TgAb, or presence of distant metastases.  
Patients with persistent disease and/or metastatic disease (non-curative resection) will not be included 
in calculation of recurrence-free survival. 
 
ASSESSMENT OF SAFETY 
The only departure from standard clinical care is the intraoperative procurement of sample by FNB. 
However, in comparison to the planned surgical procedure, this risk is small. The main risk is a small risk 
of clinically significant bleeding, estimated at <1%. As there will not be any intervention associated with 
this study, there is no plan for monitoring adverse events. 
 
STATISTICS 

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Optimization of RNASeq Conditions for Samples in CytoLyt 
RNA will be isolated from FNBs in CytoLyt as per SOP. RNA quantity will be assessed by NanoDrop and 
and quality will be quantified by DV200. RNASeq will be performed in CytoLyt samples and matched frozen 
samples at varying read depths. In paired CytoLyt and FFPE tissues, assay metrics will be evaluated as a 
function of input tissue storage method, RNA quality (DV200) and storage time. Gene expression will be 
compared in paired CytoLyt and frozen tissues, then correlated between conditions. The most important 
outcome, molecular class assignment, will be compared in paired samples.  
The following performance metrics will be evaluated for each experiment, based on the College of 
American Pathologists (CAP) checklist of requirements specific to next generation sequencing: 
Assay Metrics 
a) RNASeq Metrics:  
(i) Read counts (total reads, unique reads, duplicate reads, rRNA reads, strand specificity);  
(ii) Coverage (number of reads that cover a given genomic position): mean coverage, mean 
coefficient of variation, 5’/3’ coverage, gaps in coverage, GC bias 
(iii) Expression correlation (measured expression levels vs. known levels of ERCC internal standards) 
b) Individual Gene Detection: 
(i) Analytical sensitivity: fraction of cases in which each gene in the ERCC internal standard is 
correctly identified. 
(ii) Analytical specificity: ability to identify the absence of the genes comprising the ERCC internal 
standard 
(iii) Accuracy: sensitivity + specificity 
 
Comparison of Molecular Risk Stratification (Thyroid GuidePx®) Determined in FNB Specimens and in 
Surgical Specimens  
Patients with a final pathology that does not demonstrate papillary thyroid cancer will be excluded 
from concordance analysis. Similarly, FNB samples that are cytology negative will be excluded from 
concordance analysis. The molecular class assignment will be compared between samples obtained by 
FNB and matched frozen surgical specimens. Surgical samples will be considered the gold standard. The 
kappa statistic will be used to determine the significance of concordance between the two sample types. 
Risk strata will be assigned in a blinded fashion using Thyroid GuidePx® and the ATA Risk Stratification 
System. Structural and biochemical recurrence rates at 5 years will be compared for each risk class. A 
binary test result, conditional on disease status (structural recurrence), will be used to assess test 
accuracies. Structural recurrence represents the gold standard. A 2-sided McNemar test for two 
dependent proportions will be used to evaluate the sensitivity and specificity of each method of risk 
stratification, with Holm’s adjustment for multiplicity for assessing each accuracy measure separately. 
 
Performance of Thyroid GuidePx®  
From the studies described above, the performance of Thyroid GuidePx® will be assessed separately 
in FNB samples and surgical samples. The performance characteristics will be assessed as follows: 
1. Time-dependent AUROC (area under the receiver operator curve) (24). For the purpose of the 
calculations, the threshold level for a positive test is a designation of intermediate or high risk 
(since it is anticipated that an intermediate risk assignment would result in significantly different 
clinical decisions). 
2. Sensitivity: calculated as cumulative sensitivity at 5 years, as described by Kamarudin and 
Kolamunnage-Dona (24) 
3. Specificity: calculated as dynamic specificity, as described by Kamarudin and Kolamunnage-Dona 
(24) 

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Sample Size Calculation 
We will compare the molecular risk assignment from surgical specimens and matched FNBs from 109 
patients. A minimum level of concordance that is clinically acceptable is 80%. Sample size calculations 
were based on the Lin’s Kappa statistic for agreement between the two measures for three molecular 
subgroups (25). A two-sided hypothesis was applied with a significance level of 0.05, and power was fixed 
at 90%. Molecular subgroup sizes were fixed at 23% (Type 1), 49% (Type 2), and 28% (Type 3). If the null 
hypothesis is that the level of agreement between FNB and surgical samples is 0.6, then we would be able 
to detect a level of agreement of 0.8 or higher with 109 patients. A concordance of 0.90 would be 
detectable with 40 patients assuming the same null level of agreement. 
About 25% of patients with Bethesda V cytology and about 5% of patients with a Bethesda VI cytology 
will have benign disease. In addition, in approximately 5% of cases, RNASeq quality is insufficient in FFPE 
samples. Based on this, we plan a drop-out rate of 20%. Therefore, the trial is designed to include 130 
patients. 
 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
The clinical trial site will permit trial-related monitoring, audits, REB review, and regulatory 
inspection(s), providing direct access to source data/documents. 
Clinical trial audits are performed to provide assurance that the rights, safety and wellbeing of patients 
are properly protected, to assess compliance with the protocol, processes and agreements, ICH Good 
Clinical Practice standards and applicable regulatory requirements, and to assess the quality of data. 
Any domestic regulatory agency may come at any time to audit or inspect the study site. This audit 
consists of interviews with the principal investigator and study team, review of documentation and 
practices, review of facilities, equipment and source data verification. 
The investigator will grant to any domestic regulatory agency direct access to paper and/or electronic 
documentation pertaining to the clinical study (e.g. CRFs, source documents such as hospital patient 
charts and investigator study files). All site facilities related to the study conduct could be visited during 
an audit (e.g. laboratory, outpatient department). The investigator agrees to co-operate and provide 
assistance at reasonable times and places with respect to any auditing activity. 
 
 
QUALITY CONTROL AND QUALITY ASSURANCE  
The Principal Investigator will ensure that all study members are following the protocol and complying 
with regulatory and Good Clinical Practice standards. All clinical trial sites will permit trial-related 
monitoring, audits, REB review, and regulatory inspection(s), providing direct access to source 
data/documents. 
The Data Monitoring Committee (DMC) will meet during the conduct of the trial as follows: 1. Upon 
trial initiation to finalize the DMC Charter; 2. After 24 participants have been enrolled; 3. After 72 
participants have been enrolled; and 4. after completion of enrollment. 
 
 
ETHICS 
The responsible investigator will ensure that this study is conducted in agreement with the Declaration 
of Helsinki. This study will be conducted per Canadian and international standards of Good Clinical Practice 
for all studies including the ICH Harmonized Tripartite Guideline on Good Clinical Practice. Applicable 
government regulations, NNHPD clinical trial guidelines and Alberta Health Services research policies and 
procedures will also be followed. 

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This protocol and any amendments will be submitted to the HREBA-CC for formal approval to conduct 
the study. The decision of the HREBA-CC concerning the conduct of the study will be made in writing to 
the investigator.  
Disclosure of molecular risk classification from surgical samples (the “gold standard”) is justifiable, 
since there are substantial outcomes data from surgical samples. Moreover, when this trial is launched, 
the test will be commercially available as a Laboratory Developed Test (LDT). 
 
 
DATA HANDLING AND RECORD KEEPING 
Once the study is completed, all data for this study will be retained for 15 years, stored as per the 
protocol at the Calgary Centre for Clinical Research. 
 
FINANCING AND INSURANCE 
The trial will be funded by Qualisure Diagnostics, Inc., supplemented by peer-reviewed grants. 
Insurance will be provided by the University of Calgary and Alberta Health Services, as well as Qualisure 
Diagnostics. 
 
PUBLICATION POLICY  
The findings from this clinical study will be published in a peer-reviewed journal with all named 
investigators as co-authors. All funding sources will be acknowledged.  
 
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