# Patent

## Title
Method for the early detection of pancreatic cancer and other gastrointestinal disease conditions

## Abstract

A method for the early diagnosis of pancreatic cancer and other gastrointestinal disease compares the gene expression patterns from a patient's peripheral blood monocytes-lymphocyte's gene system with either the similar gene expression patterns of a normal person, or with the similar gene expression patterns of a person known to have the condition being screened for. Differences between the patient's gene expression patterns for particular genes and the normal patterns indicates the presence of the condition with the number of differences indicating the probability of the condition. Similarities between the patient's gene expression patterns for those particular genes and the patterns of a person known to have the condition indicates the presence of the condition with the number of similarities indicating the probability of the condition. Particular genes for use in identifying pancreatic cancer are disclosed.

## Background
RELATED APPLICATIONS
This is a continuation-in-part of copending application Ser. No. 10/938,696, filed Sep. 11, 2004, and entitled ‘The Discovery and a Method for the Early Detection of Pancreatic Cancer and other Disease Conditions’, and this application claims the benefit of Provisional Patent Application No. 60/598,477, filed Aug. 3, 2004, entitled “Process for Early Identification of Cancer and Other Disease Conditions,” Provisional Application No. 60/607,088, filed Sep. 5, 2004, entitled “The Discovery and a Method for the Early Detection of Pancreatic Cancer and Other Disease Conditions”, Provisional Patent Application No. 60/664,842, filed Mar. 25, 2005, entitled “A Method for the Early Detection of Pancreatic Cancer and Other Gastrointestinal Disease Conditions,” Provisional Patent Application No. 60/676,670, filed Apr. 30, 2005, entitled “A Method For The Early Detection Of Pancreatic Cancer And Other Gastrointestinal Disease Conditions.” Applicant also makes reference to Disclosure Document No. 532619, filed Jun. 5, 2003 (referred to in parent application Ser. No. 10/938,696), entitled “The Method For A Useful Process for the Early Identification Of Cancer,” Disclosure Document No. 560475, filed Sep. 10, 2004 (referred to in parent application Ser. No. 10/938,696), entitled “The Discovery and a Method for the Early Detection of Pancreatic Cancer and Other Disease Conditions,” Disclosure Documents No. 572656, filed Mar. 6, 2005, entitled “Gene Expression Diagnosis of Pancreatic Cancer (Intraductal Pancreatic Adenocarcinoma) and Other Gastrointestinal Growths and Conditions from Peripheral Blood Lymphocytes,” Disclosure Documents No. 573431, filed Mar. 22, 2005, entitled “Gene Expression Diagnosis of Pancreatic Cancer (Intraductal Pancreatic Adenocarcinoma) and Other Gastrointestinal Growths and Conditions from Peripheral Blood Lymphocytes,” and Disclosure Document No. 574718, filed Apr. 15, 2005, entitled “Gene Expression Diagnosis of Pancreatic Cancer (Intraductal Pancreatic Adenocarcinoma) and Other Gastrointestinal Growths and Conditions from Peripheral Blood Lymphocytes.” All of the above applications and disclosure documents are hereby incorporated herein by reference.


BACKGROUND OF THE INVENTION
1. Field
This invention is in the field of methods for diagnosis of disease conditions and particularly diagnosis of pancreatic cancer and other gastrointestinal disease conditions.
2. State of the Art
Pancreatic cancer is a deadly disease which has a mortality rate in the United States of more than 27,000 people a year, Lillemoe, K. D., C. J. Yeo, and J. L. Cameron, Pancreatic cancer: state-of-the-art care. C A Cancer J Clin, 2000. 50(4): p. 241-68. About 85% of those diagnosed with the disease have metastasis or spread of the disease beyond the pancreas and are almost impossible to cure with surgical resection, the only possible method of curing the disease at this time. If the growth is found sooner it may be resected with a much better hope of cure. Only about 15% of the newly diagnosed cases are resectable and the chances of a cure are usually 25% or less. Wiesenauer C. A. et al., Preoperative Predictors of Malignancy in Pancreatic Intraductal Papillary Mucinous Neoplasms. Arch. Surg; 2003 138: p 610-618; Ros, P. R. and K. J. Mortele, Imaging features of pancreatic neoplasms. Jbr-Btr, 2001. 84(6): p. 239-49; Ryu, B., et al., Relationships and differentially expressed genes among pancreatic cancers examined by large-scale serial analysis of gene expression. Cancer Res, 2002. 62(3): p. 819-26; Ito, M., et al., Molecular basis of T cell-mediated recognition of pancreatic cancer cells. Cancer Res, 2001. 61(5): p. 2038-46. Earlier diagnosis is the only hope of allowing earlier successful treatment of pancreatic cancer at this time.
Since the dividing time of the pancreatic cancer cell is around 40 days, the cancer has been present for many months or years before it is detectable by present imaging and other diagnostic methods. Pathway markers have not as yet proved successful in the early diagnosis of pancreatic or other cancers with a high degree of specificity or sensitivity. Lillemoe, K. D., C. J. Yeo, and J. L. Cameron, Pancreatic cancer: state-of-the-art care. C A Cancer J Clin, 2000. 50(4): p. 241-68; Rosty C, Goggins M., Early detection of pancreatic carcinoma. Hematol Oncol Clin North Am, 2002 16(1):37-52.
With the development of tumors, dendritic cells or macrophages note new growth, whether of genetic or epigenetic origin, by recognizing the altered proteins presented on the cancer cell's surface through their receptor channels. The dendritic cells convey these altered protein changes to the lymphocytes with the addition of major histocompatibility complexes. This includes T. lymphocytes CD8 with HCS I and CD4 with HCS II. The B lymphocytes are subsequently programmed by the recognizing T lymphocyte. Zeng, G., MHC Class II-Restricted Tumor Antigens Recognized by CD4+T Cells: New Strategies for Cancer Vaccine Design. J Immunother, 2001. 24(3): p. 195-204; Jonuleit, H., et al., Identification and functional characterization of human CD4(+)CD25(+) T cells with regulatory properties isolatedfrom peripheral blood. J Exp Med, 2001. 193(11): p. 1285-94; Serbina N. V., Pamer E. G. Giving Credit Where Credit Is Due. Science, 2003, 301:1856-1857; and Baxevanis, C. N., et al., Tumor-specific CD4+T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor. J Immunol, 2000. 164(7): p. 3902-12. Through this mechanism, the lymphocytes specifically recognize the new growth and program specifically against it, sending tumor infiltrating lymphocytes or TIL cells to the new growth. These TIL cells may decrease in the area of the tumor as tolerance for the tumor develops. Ryschich, E., et al., Transformation of the microvascular system during multistage tumorigenesis. Int J Cancer, 2002. 97(6): p. 719-25. It has been shown that the CD4-CD25 T lymphocytes contribute to tolerance of developing cancer. Liyanage, U. K., et al., Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol, 2002. 169(5): p. 2756-61. The use of peripheral blood lymphocytes for diagnosis of certain diseases have been proposed and described in Hong M H, X. X., Mai H Q, Cao S M Min H Q, Analysis of gene expression patterns of periphery lymphocytes in patients with nasopharyngeal carcinoma. Ai Zheng, 2002. 21(1): p. 21-4; Xu T et al Microarray analysis reveals differences in gene expression of circulating CD8+ T cells in melanoma patients and healthy donors. Cancer Res. 2004 May 15; 64(10):3661-7; Thomas A M et al. Mesothelin-specific CD8(+) T cell responses provide evidence of in vivo cross-priming by antigen-presenting cells in vaccinated pancreatic cancer patients. J Exp Med. 2004 Aug. 2; 200(3): 297-306; and McLaren P J et al Antigen-specific gene expression profiles of peripheral blood mononuclear cells do not reflect those of T-lymphocyte subsets. Clin Diagn Lab Immunol. 2004 September; 11(5):977-82. Twine & Burczynski. Twine N C, et al. Disease-associated expression profiles in peripheral blood mononuclear cells from patients with advanced renal cell carcinoma. Cancer Res. 2003 Sep. 15; 63(18):6069-75. Burczynski M E, Twine N C et al. Transcriptional profiles in peripheral blood mononuclear cells prognostic of clinical outcomes in patients with advanced renal cell carcinoma. Clinical Cancer Res. 2005 Feb. 1; 11(3):1181-9.
SUMMARY OF THE INVENTION
As indicated above, dendritic cells in a body convey altered protein changes resulting from a change in a body's condition to the lymphocytes with the addition of major histocompatibility complexes. This includes T. lymphocytes CD8 with HCS I and CD4 with HCS II. The B lymphocytes are subsequently programmed by the recognizing T lymphocyte. Because of this, a body's peripheral blood monocyte-lymphocyte's gene system should recognize and continue to react to changes in a body's condition, such as a developing neoplasm. According to the invention, it has been found that this recognition and reaction to changes in a body's condition changes the body's peripheral blood monocyte-lymphocyte's gene system. The state of the body's peripheral blood monocyte-lymphocyte's gene system can be determined by determining the gene expression characteristics of genes of the body's peripheral blood monocyte-lymphocytes. By comparing the monocyte-lymphocyte gene expression characteristics of the monocyte-lymphocyte genes from a group of bodies having a certain known condition with the monocyte-lymphocyte gene expression characteristics of similar peripheral blood monocyte-lymphocytes from a group of bodies known not to have the certain condition, a number of specific genes likely to show different gene expressions between the group known to have the certain condition and the group known not to have the certain condition can be identified. While it may not be possible to pick out one or more particular genes which will always be expressed differently between a body with the certain condition and one without the certain condition, and errors can occur in the determination of individual gene expression characteristics, where the expression characteristics of a number of genes are found likely to be different between the monocyte-lymphocyte genes of a body with the certain condition and a body without the certain condition, an indication is given by a difference in the expression of one or more of the identified genes. The number of identified genes showing a different expression as well as the particular genes showing a different expression provides an indication of the degree of probability of the existence of the condition.
By comparing the monocyte-lymphocyte gene expression characteristics of the monocyte-lymphocyte genes from the group of bodies having the certain known condition with the monocyte-lymphocyte gene expression characteristics of similar peripheral blood monocyte-lymphocytes from the group of bodies known not to have the certain condition, a “normal differential gene expression pattern” typical of a person known not to have the certain condition is developed. This normal differential gene expression pattern will include the gene expression characteristics for a number of the genes likely to have different gene expression characteristics from the expression characteristics of those same genes from a body having the certain condition. Other particular genes that can provide other desired information regarding a body may also be included in the normal differential gene expression pattern, if desired. Once this normal differential gene expression pattern is developed, it can be used to screen or diagnose a patient to determine of the patient has the certain condition. To do this, a “patient differential gene expression pattern” is developed for the patient to be screened for the certain condition. The patient differential gene expression pattern will show the gene expression characteristics for the same genes as included in the normal differential gene expression pattern so that those gene expression characteristics can be compared. Significant differences between the patient differential gene expression pattern and the normal differential gene expression pattern indicates that the body from which the patient differential gene pattern was obtained is suffering from the certain condition. It has been found that the peripheral blood monocyte-lymphocytes gene system will begin to change as the condition in the body develops, thereby allowing much earlier diagnosis of the developing condition than with prior art methods of diagnosis. For example, with a developing neoplasm in a patient, such as a pancreatic tumor leading to ductal pancreatic adenocarcinoma, the patient's peripheral blood monocyte-lymphocyte's gene system recognizes and continues to react to the developing neoplasm. The developing changes in the tumor growth will be reflected in statistically significant differences in the peripheral blood monocyte-lymphocyte's gene expression patterns compared to normal peripheral blood monocyte-lymphocyte gene expression patterns in people known not to have the developing neoplasm. The normal differential gene expression pattern is generated from a group of people known not to be suffering from a developing neoplasm. Such group of people may be similar in age and gender, and/or other features, to the patient being screened, although matching age, gender, or other features appears not to be necessary. The comparison of the patient differential gene expression pattern with the normal differential gene expression pattern allows the early diagnosis of the developing neoplasm or disease.
By comparing the monocyte-lymphocyte gene expression characteristics of the monocyte-lymphocytes genes from the group of bodies having the certain known condition with the monocyte-lymphocyte gene expression characteristics of similar peripheral blood monocyte-lymphocytes from the group of bodies known not to have the certain condition, not only is the normal differential gene expression pattern typical of a person known not to be suffering from the certain condition developed, but a “condition differential gene expression pattern” typical of a person known to have the certain condition is also developed. Thus, although it is currently preferred to compare the patient differential gene expression pattern with the normal differential gene expression pattern to determine differences with the differences indicating the existence of the certain condition in the patient being screened, the patient differential gene expression pattern can be compared with the condition differential gene expression pattern to determine similarities between the patterns with the similarities between the patterns, rather than differences between the patterns, indicating the existence of the certain condition in the patient.
In developing the “normal differential gene expression pattern,” the “condition differential gene expression pattern,” and the “patient differential gene expression pattern,” the gene expression characteristics determined for each pattern should be the same gene expression characteristics and such gene expression characteristics should be determined in a similar manner. A currently preferred method of determining the gene expression characteristics is with a gene expression microarray pattern. Such an array provides an indication of whether a gene is expressed neutrally, or whether the gene is over expressed or under expressed. In such case, it is the characteristics of over expression or under expression that are determined and compared.
While there are various ways of preparing the monocyte-lymphocyte genes for determination of the gene expression characteristics, a currently preferred method processes peripheral blood monocyte-lymphocytes isolated from blood drawn from a patient or other body to total RNA, and obtains amplified aRNA or cDNA from the total RNA. The separation of the monocyte-lymphocytes from the blood is preferably begun rapidly, within about two hours of drawing the blood, and more preferably within about twenty to thirty minutes of drawing the blood. The separated mononuclear cells are then preserved before storage or freezing. To determine the gene expressions, the aRNA or cDNA is hybridized to the microarray. The data obtained from the microarray is analyzed with available computer software for that purpose, such as Gene Sight software, with mean paired ratios using Universal Human Reference RNA as a standard, usually with a p value of 0.0001 or 0.00001 of over expressed or under expressed genes.
The separation of the monocyte-lymphocytes from the blood may involve separating and isolating subsets of CD8, CD4, and CD4-CD25 T lymphocytes and B lymphocytes from the blood. The subsets of CD8, CD4, and CD4-CD25 T lymphocytes and B lymphocytes can be obtained through negative selection of the cells which are then processed to total RNA with amplification of polyadenylated messenger RNA to amplified anti-sense aRNA or to cDNA. Use of negatively selected CD8, CD4, CD4-CD25 T lymphocytes and B lymphocytes isolated from the peripheral blood of persons with pancreatic cancer and other disease conditions may provide a specific and more focused early diagnosis of the pancreatic cancer or other disease.
The invention provides identification of a number of particular genes of which the over expression or under expression thereof to a high degree of probability indicate the presence of pancreatic cancer and particularly ductal pancreatic adenocarcinoma.
In a preferred method of preparing the monocyte-lymphocyte genes for determination of the gene expression characteristics, venous blood is drawn from a peripheral vein, usually an anti-cubital arm vein. The blood is drawn into an RNase free, heparinized vacuum tube with a Ficoll gradient. The blood specimens are immediately processed with centrifugation and aspiration of the mononuclear cell layer with sterile RNase free pipettes and RNase free laboratory equipment. This process is started within two hours of the drawing of the blood, and preferably within 20 to 30 minutes of the drawing of the blood. This mononuclear cell layer is then washed and preserved according to a careful and consistent protocol method. Further processing can be continued immediately or the preserved specimen can be stored at −80° C. for later processing. In the further processing, total RNA is extracted from the preserved cells and polyA messenger RNA from the total RNA is amplified to antisense aRNA or cDNA for subsequent hybridization to microarray human slides. Universal Human Reference RNA is similarly processed at that time for use as a reference standard.
The results of these microarray hybridization studies can be analyzed with the Gene Sight software using a Student's Test method. The individual genes are initially matched to the control specimen genes with a pair Mean ratio difference of p 0.00001 or p 0.0001 for selection from Human Slides A and B. The patterns of selection are noted with the Hierarchical Cluster method. The individual genes over expressed and under-expressed in this comparison are recorded. Other methods of analysis can also be used, including the SAM method, a different linear ANOVA test method and other methods.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. In the accompanying drawings, which show the best mode currently contemplated for carrying out the invention:
FIG. 1 is a showing of the results of microarray determination of gene expression characteristics of three patients with known ductal pancreatic adenocarcinoma and seven persons without ductal pancreatic adenocarcinoma used as controls or normals;
FIG. 2, a showing similar to that of FIG. 1 of the results of microarray determination of gene expression characteristics of three patients with known ductal pancreatic adenocarcinoma and seven persons without ductal pancreatic adenocarcinoma used as controls, but to a higher probability of certainty than shown in FIG. 1;
FIG. 3, a showing of the results of microarray determination of gene expression characteristics of four patients with known ductal pancreatic adenocarcinoma and eight persons without ductal pancreatic adenocarcinoma used as controls or normals;
FIG. 4, a showing of the results of microarray determination of gene expression characteristics of two patients with known ductal mucinous pancreatic neoplasm and two persons with ductal pancreatic adenocarcinoma;
FIG. 5, the showing of the results of microarray determination of gene expression characteristics of FIG. 1, executed in color;
FIG. 6, the showing of the results of microarray determination of gene expression characteristics of FIG. 2, executed in color;
FIG. 7, the showing of the results of microarray determination of gene expression characteristics of FIG. 3, executed in color; and
FIG. 8, the showing of the results of microarray determination of gene expression characteristics of FIG. 4, executed in color.



DETAILED DESCRIPTION OF THE INVENTION
The invention, in part, involves obtaining gene expression characteristics of the gene system of monocyte-lymphocytes of the peripheral blood from a body or patient to be tested. This usually includes obtaining peripheral blood from the body or patient to be tested, separating or isolating the monocyte-lymphocytes from the blood, processing the monocyte-lymphocytes to allow determination of gene expression characteristics of the genes, and determining the gene expression characteristics of the genes. In a preferred method of obtaining and processing the monocyte-lymphocytes, a peripheral blood sample is obtained from the patient in the usual manner of obtaining venous blood from a peripheral vein, such as the anti-cubital vein of the arm. Usually 16 ml in two 8 ml tubes is drawn into sterile RNase free vacuum tubes with a Ficoll type gradient and heparin. (Such as the BD Vacutainer CPT tubes with heparin.) These tubes are centrifuged at a centrifugal force of about 1500×g, using, for example, the top of the tubes 17 cm from the center of the center post of the centrifuge, for 20 minutes at 2800 rpm at room temperature. The resulting ‘snow storm’ of monocyte-lymphocytes sits on top of the Ficoll gradient and below the clear plasma layer. Approximately 2 ml of this monocyte-lymphocyte layer is aspirated with a sterile RNase free plastic Pasteur bulb tube and placed in a sterile RNase free 15 ml plastic tube with a screw top.
The cells in the aspirated sample are then washed. Approximately 13 ml of 1×PBS (phosphate buffered saline) solution made with RNase free water, is added to the plastic tube, and centrifuged at 1300 rpm for 15 minutes. The same distance is used for the centrifuge as previously, 17 cm from the center of the center post of the centrifuge. This is done at room temperature. A small white pellet is found at the bottom of the centrifuged 15 ml plastic tube. The supernatant is gently poured from the tube without disturbing the pellet. The small remaining part of the supernatant is very gently aspirated from the tube, again not disturbing the pellet.
The cells in the pellet are now preserved in one of three ways. In method A, two tubes with the pellets are used and 350 μl added of a B-ME (B-Mercapthanol) preservative. (Such as 10 μl of B-ME in 1 ml of Buffer RLT from the Quiagen RNeasy Mini Protect Kit.) Mild vortexing of the lysate with the pellets in the tube is gently done, holding the tube to the side of the rim of the vortexing machine. Allow the cells to be lysated for five or more minutes and draw back and forth through a sterile Rnase free #18 needle and 1 ml sterile Rnase free syringe five times gently. This amount from the two pellets in the two tubes is then transferred to one 1.5 ml Eppendorff sterile RNase free tube. This may then be stored at −80° C. or continued to be processed to total RNA (tRNA). In method B, the sample may instead be placed in a DMSO (dimethyl sulphoxide) solution made up of 500 μl of DMSO, 500 μl of the patient's own serum and 4 ml of RPMI 1640 which is mixed and then 1 ml added to the pellet at the bottom of the 15 ml plastic tube and gently vortex. This may then be slowly frozen to −80° C. for storage or immediately processed to total RNA (tRNA). If it is stored at −80° C., it should be melted rapidly to 37° C. before processing to total RNA. This procedure allows the cells to be negatively selected to lymphocyte subsets of CD8, CD4, CD4-CD25 and B lymphocytes which are then processed to aRNA or to cDNA as described below for microarray pattern recognition. In method C, 100 μl of RNlater (from Qiagen RNeasy Mini Protect Kit) may instead be added to the washed pellet as a preservative to stop enzyme degradation. This is thought to be a high salt solution and the cells in this solution may not be effectively negatively selected for subset analysis. The patterns in this method (Method C) are of the total monocyte-lymphocyte gene expression reaction to the neoplasm.
If the B-ME buffered method of lysate of two tubes of pellets (Method A) is used for further processing to total RNA, an equal amount of 70% ethanol made from pure absolute alcohol with 30% of RNase free non-DEPCA treated sterile water added to the alcohol, is added to the cell containing lysate in the Eppendorff tube. This is gently mixed and then in 700 μl amounts added to a silica gel column. (Such as that supplied by Qiagen in their Mini Protect Kit.) This is then centrifuged at 10,000 rpm (approximately 9,000 g.) for one minute and the flow through discarded. The ethanol bounded total RNA with higher amount of messenger RNA (mRNA) is bound to the silica gel membrane which is then washed and eluted in sterile RNase free water. In more detail, the remaining lysate in the Eppendorff tube is transferred in 700 μl or less volume to the silica column and centrifuged in a microcentrifuge again for one minute at the same speed, 10,000 rpm. The flow through is discarded and 350 μl of a wash solution (Such as that from the Qiagen RNeasy Mini Protect Kit) is placed on the column and again centrifuged for one minute at 10,000 rpm. Following this add 10 μl of DNase1 stock solution from an RNase Free DNAse Set (Invitrogen) to 70 μl RDD buffer. This eliminates the remaining small amount of DNA leaving the enriched mRNA. Mix gently by inverting and add gently to the silica gel column. Let stand for 15 minutes then wash again with 350 μl of a wash solution, microcentrifuging for one minute at 10,000 rpm. Discard the flow through.
Pipette 500 μl of Buffer RPE from the Qiagen Kit to the column and centrifuge for one minute at 10,000 rpm using the same collection tube. Discard the flow through. Pipette another 500 μl of RPE Buffer solution (again to wash the column with ethanol) to the column with a new collection tube and centrifuge again for one minute at 10,000 rpm in a microcentrifuge. If the column is not totally dry, discard the flow through and recentrifuge at 16,000 rpm for one minute. Do not do this last step, if the column is dry.
Transfer the dry silica gel column to a new 1.5 m RNase free collection tube and pipette 30 μl of RNase free sterile water directly onto the silica gel membrane, holding the pipette only one or two millimeters above the membrane. Microcentrifuge the column at 10,000 rpm for one minute. This gives 30 μl of total RNA (tRNA). One may then OD (optical density with UV spectrophotometry) one μl of this, with or without dilution, to determine the concentration or quantity of total RNA (tRNA). One may also run a gel to be sure the bands indicate no degradation of the total RNA.
After the total RNA ( tRNA) is measured for concentration by OD, 3 μg is used for the T7 method of linear amplification. This may very from 500 nanograms to 5 μg, but 3 μg is the preferred amount. The solution is diluted to 10 μl volume, if dilution is necessary, with sterile RNase free water. A 10 fold amplification of the original quantity is desired. Actually the amplification of the messenger RNA to amplified anti-sense RNA (aRNA) is much greater, since the percentage of polyadenylated RNA (AAA messenger RNA) is very small compared to the amount of total RNA in the sample.
1 μg of the first strand synthesis promoter primer is added to the 10 μl of specimen after first thawing, mixing and briefly spinning the primer from the −20° C. frost free refrigerator. This 1 μg is carefully mixed and spun. Then, the specimen is placed in a thermal cycler for incubation at 65° C. for 5 minutes and cooled to 4° C. for denaturing and annealing. This denatures the total RNA and anneals the primer. (If experimentally one is using the Arcturus Kit [Arcturus RiboAmp RNA Amplification Kit # KIT0201] then Primer A is used in this step, according to their directions.)
First strand synthesis solutions (including dNTP, polymerase and buffers for pH) are thawed, mixed and spun for 2 seconds. They are then added to the specimen, stirred, mixed and briefly spun. This specimen is then placed in a thermal cycler for 60 minutes at 42° C. and cooled to 4° C. (If experimentally one is using the Arcturus Kit one mixes 7 μl of 1st Strand Master Mix, then 2 μl of the 1st Strand Enzyme Mix, giving a total of 20 μl.)
After the first strand synthesis a nuclease mix is thawed, mixed and spun briefly. 2 μl is added to the specimen which is briefly spun after being cooled to 4° C. after the last step. The nuclease mix is added to the specimen and spun briefly with a small lab bench centrifuge. This leaves the first strand synthesized specimen. It is then placed in the thermal cycler for 20 minutes at 37° C., 95° C. for 5 minutes and cooled to 4° C. destroying the nuclease after its effect. Spun down briefly. (Experimentally with the Arcturus Kit, one uses the 1st Strand Nuclease Mix as directed.)
1 μl of the second primer is added to this mix after cooling to 4° C. for two or more minutes and spinning briefly. The specimen is then placed in a thermal cycler for 2 minutes at 95° C. and cooled to 4° C. (If experimentally using the Arcturus Kit, Primer B is used for this step of denaturing.)
2nd strand synthesis solutions (dNTP, polymerase and buffers) are thawed, mixed and briefly spun. Then, added to the specimen. This is placed in a thermal cycler for 10 minutes at 25° C., 37° C. for 20 minutes and 70° C. for 5 minutes. Then, cooled to 4° C. (If experimentally one is using the Arcturus Kit 29 μl of 2nd Strand Master Mix is used and then 1 μl of the 2nd Strand Enzyme Mix, giving a total of 53 μl together with the specimen.)
Purification is then carried out by binding cDNA resulting from the previous steps to a column and washing the column with wash buffers of alcohol. Then, the DNA is eluted and taken to the next step. (If one experimentally is using the Arcturus Kit, 250 μl of DNA Binding Buffer is added to the DNA/RNA Purification Column in a collection tube and after several minutes at room temperature centrifuged at full in a microcentrifuge, 16,000×g for one minute to prepare and wet the column, as per directions. Discard the flow through. Then, 200 μl of the DNA Binding Buffer is added to the 2nd strand synthesis specimen very carefully with gentle thorough mixing and pipetted to the previously coated purification column. It is then centrifuged at 100×g for two minutes and then 10,000×g for 1 minute. Discard the flow through. The column is then washed with 250 μl of the Arcturus DNA Wash Buffer to the column and centrifuged at 16,000×g for one minute. If the column is very dry transfer it to a 0.5 ml microcentrifuge tube and place 16 μl of elution buffer onto the center of the column from about 1 to 2 millimeters above the column. Allow to stand for two minutes, then microcentrifuge at 1,000×g for one minute, then 16,000×g for one minute. The flow through contains the purified cDNA.)
The process should be continued immediately by adding transcription solutions (buffers, dNTP and polymerase) and incubating in a thermal cycler with a heated lid at 42° C. for 4 hours then cooling to 4° C. (If one is experimentally using the Arcturus Kit, thaw, mix and spin the IVT Reaction solutions. Then, add 8 μl of the IVT Buffer, followed by 12 μl of the IVT Master Mix and 4 μl of the IVT Enzyme Mix, spinning after thoroughly mixing these together with the 16 μl of cDNA specimen. Place for 4 hours in the thermal cycler as above at 42° C.)
After the above step add a DNA nuclease mix to leave only amplified anti-sense RNA (aRNA) to the 4° C. cooled specimen and place in a thermal cycler for 15 minutes at 37° C. Again, cooling to 4° C. (If experimentally using the Arcturus Kit use 2 μl of DNase Mix thaw, mix and add to specimen, mixing and spinning prior to thermal cycler.)
Follow the above step with purification of the amplified anti-sense RNA (aRNA) with adherence to a wetted or prepared column and washing with an ethyl alcohol buffered solution. (If experimentally using the Arcturus Kit add 250 μl of RNA Binding Buffer to a DNA/RNA Purification Column, allowing this to stand for several minutes. Then spin the column at 16,000×g in a lab bench microcentrifuge for one minute. Discard the flow through. Next, add 200 μl of this buffer to the specimen gently mixing it and pipette it to the purification column. Microcentrifuge at 100×g for two minutes and 10,000×g for one minute. Wash with 200 μl of RNA Wash Buffer added to the column and microcentrifuge at 10,000×g for one minute. Discard the flow through. Again, add 200 ul of RNA Wash Buffer to the column and centrifuge at 16,000×g to two minutes. Discard the flow through. Be sure the column is very dry. If it is not dry, then centrifuge at 16,000×g for one minute again.)
To the dry column placed in a 0.5 ml microcentrifuge tube add 30 μl of elution solution and allow to stand for two minutes, placing the solution very carefully just above the center of the column, one to two millimeters above the column. After the two minutes, microcentrifuge the column at 1,000×g for one minute, then 16,000×g for one minute. The 30 μl of flow through contains the aRNA. (If one is experimentally using the Arcturus Kit, use RNA Elution Buffer to elute the column.)
The aRNA may now be stored at −80° C. One may measure the concentration with OD, optical density, and run a gel to check for aRNA degradation.
Following the above steps the aRNA may be checked with a bio-analyzer and microarrayed for gene expression. The gene expression patterns will then be analyzed with advanced software to determine the statistically significant expression of the pancreatic cancer and other disease conditions compared to the normal expected patterns of non-diseased control samples. ‘Gene Sight’ software with hierarchical clusters, statistical analysis of microarray ‘SAM’, a linear ANOVA method and other methods were used to analyze the microarray data in this determination of the significantly over expressed and under expressed genes distinguishing intraductal pancreatic adenocarcinoma from age and gender approximated controls without known pancreatic disease.
This method may be modified to increase the availability and reduce the laboratory time and cost of the test with the use of direct linear amplification of smaller amounts of total RNA to cDNA for direct attachment of dyes for microarray with different or hybrid promoters and primers (such as with the NuGene method). Also, this method may be enhanced by use with newer microfluid chips. Even more focused gene patterns may be evaluated with negatively selected combinations of subsets of the T and B lymphocytes for patterns of gene expression of early developing tumors, allowing early resection or destruction of the tumor before metastatic spread of the subsequent cancer.
This method gives the patterns needed for the early diagnosis of the pancreatic cancer. This method describes one useful method of accomplishing the invention claimed in this patent of using the peripheral blood monocyte-lymphocytes for the early diagnosis of pancreatic cancer and other disease conditions.
One hypothesis of this invention is that the peripheral blood lymphocyte microarray gene expression patterns will show a specific and unique identification of an intraductal pancreatic adenocarcinoma patient compared to the microarray gene expression patterns of informed volunteer age and gender approximated controls without known pancreatic disease. To test this hypothesis, peripheral blood mononuclear cells were isolated from the peripheral blood specimens of patients and controls. The isolated mononuclear cells were estimated to consist of about 80% to about 95% lymphocytes. The venous blood was drawn from a peripheral vein, usually an anti-cubital arm vein, of informed and consenting patients with subsequent pathology proven tissue diagnosis of intraductal pancreatic adenocarcinoma. These specimens were from intraductal pancreatic adenocarcinoma patients who did not have treatment with radiation or chemotherapy prior to the obtaining of the blood specimens. The gene expressions from the processed blood specimens were analyzed and compared to similar drawn and processed specimens from age and gender approximated informed volunteer controls without known pancreatic disease.
The blood was drawn into an RNase free, heparinized vacuum tube with a Ficoll gradient. The blood specimens were immediately processed with centrifugation and aspiration of the mononuclear cell layer with sterile RNase free pipettes and RNase free laboratory equipment. This process was started usually within 20 to 30 minutes of the drawing of the blood and certainly within 2 hours. This mononuclear cell layer was then washed and preserved according to a careful and consistent protocol described above. Further processing could be continued immediately or the preserved specimen could be stored at −80° C. for later processing.
As described above, Total RNA was extracted from the preserved cells and polyA messenger RNA from the total RNA was amplified to antisense aRNA or cDNA for subsequent hybridization to microarray human slides. Universal Human Reference RNA was similarly processed at that time for use as a reference standard. The antisense aRNA or cDNA from the preserved cells from a specimen and the Universal Human Reference RNA antisense aRNA or cDNA was hybridized to microarray human microarray slides and the slides processed to show the gene expression characteristics of the genes from the respective blood specimens of patients with pancreatic cancer and controls without pancreatic cancer. The results of these microarray hybridization studies were analyzed with the Gene Sight software using a Student's Test method. As stated above, other methods of analysis were also used, including the SAM method, a different linear ANOVA method and other methods. The results from all methods were compared and the results from different patient's specimens were compared. The individual genes were often matched to the control specimen genes with a pair Mean ratio difference of p 0.00001 (one chance in 10,000 that the indicated over expression, neutral expression, or under expression was random) or p 0.0001 (one chance in 1,000 that the indicated over expression, neutral expression, or under expression was random) for selection with Gene Sight software. The patterns of selection were noted with the Hierarchical Cluster method and the individual genes over and under-expressed in this comparison were recorded. Cross-expression of these genes between methods and specimens were recorded. In the standard microarray gene testing for human gene expression, two slides, human slide A with 9600 genes in duplicate and human slide B with 9600 different genes in duplicate are used. These two slides are used for each gene specimen. The Gene Sight or other software is then used to indicate the results in selected manners, such as in hierarchical clusters. The results obtained in this manner are colored charts showing the expression for individual genes in red if the gene is significantly over expressed, green if the gene is significantly under expressed, and black if there is no significant expression, referred to herein as neutral expression. These gene expression indications and charts for indicating gene expression are well known to those skilled in the art.
FIG. 1 shows the results of microarray determination of gene expression characteristics of three patients with known ductal pancreatic adenocarcinoma and seven persons without ductal pancreatic adenocarcinoma used as controls, and showing only gene expressions that have been determined to be different between patients with ductal pancreatic adenocarcinoma and controls without ductal pancreatic adenocarcinoma, at a p of 0.0001. FIG. 1 shows the results of slide A comparison. The first three columns from the left in FIG. 1, labeled at the bottom of each column as PA, show the gene expressions for the particular genes labeled for each row at the right side of the FIG. 1. The gene labels shown in FIG. 1 are the Image ID Numbers for the genes. Each column labeled PA represents a “patient differential gene expression pattern” as described earlier where the pattern is made up of the particular genes labeled on the right. The seven columns labeled C show the gene expressions for the particular indicated genes for the controls. Each column represents a differential gene expression pattern for a particular control. To generate a “normal differential gene expression pattern,” a combination of all seven individual control column patterns would be generated. Similarly, to generate a “condition differential gene expression pattern,” an average or other combination of all three individual patient column patterns would be generated. When printed in color, the greens and reds of some individual patterns sometimes vary in shade or intensity indicating the measured intensity of the over or under expression. However, in the black and white drawings of FIGS. 1-3, the red is indicated by diagonal lines sloping downwardly to the right and the green is indicated by diagonal lines sloping downwardly to the left. No indications are made to indicate intensity. The black indicates neutral intensity while the white or blank spaces indicate no measurement for that particular gene for that particular sample.
FIG. 1 shows particular genes that exhibit different expression between the patients with ductal pancreatic adenocarcinoma and the controls without ductal pancreatic adenocarcinoma. This data shows distinct patterns of gene expression microarrays with human slides A separating ductal pancreatic adenocarcinoma patients prior to treatment compared to age and gender approximated controls.
FIG. 2 shows the results of microarray determination of gene expression characteristics for the same three patients with known ductal pancreatic adenocarcinoma and the same seven persons without ductal pancreatic adenocarcinoma used as controls, as shown in FIG. 1. However, FIG. 2 shows only gene expressions that have been determined for particular genes to be different between patients with ductal pancreatic adenocarcinoma and controls without ductal pancreatic adenocarcinoma, at a p of 0.00001 rather than 0.0001 (only one chance in 10,000 that the indicated over expression, neutral expression, or under expression was random as opposed to one chance in 1,000). Thus, the difference of these particular genes, which are also included in the genes shown in FIG. 1, between a patient and a normal have a higher degree of indicating the presence of ductal pancreatic adenocarcinoma than the other genes shown on FIG. 1.
FIG. 3 shows the results of microarray determination of gene expression characteristics of four patients with known ductal pancreatic adenocarcinoma and eight persons without ductal pancreatic adenocarcinoma used as controls, and showing only gene expressions that have been determined to be different between patients with ductal pancreatic adenocarcinoma and controls without ductal pancreatic adenocarcinoma, at a p of 0.00001, similar to the p of FIG. 2. FIG. 3 shows the results of slide B comparison. The first four columns from the left in FIG. 3, labeled at the bottom of each column as PA, show the gene expressions for the particular genes labeled for each row at the right side of the FIG. 1. The eight columns labeled C show the gene expressions for the particular indicated genes for the controls. FIG. 3, shows distinct patterns of gene expression microarrays with human slide B separating ductal pancreatic adenocarcinoma patients prior to treatment compared to age and gender approximated controls.
The genes identified by Image ID Numbers in FIGS. 1-3 were further identified using the Oncogenomics and NCBI data reference sources listed as follows:

    
    
        A. Oncogenomics, Pediatric Oncology Branch, CCR, NCI, NIH, DHHS.
        B. National Center for Biotechnology Information (NCBI)
        
            National Library of Medicine (NLM)
            National Institutes of Health (NIH)

Sequence listing for the particular genes, where available, may be found on web sites for these organizations using the identification for the gene provided here. Information regarding the identified genes available on these web sites is incorporated herein by reference.

        
        
    
    


The first four genes listed below were found to be significantly over expressed and the next two genes under expressed in the ductal pancreatic adenocarcinoma patients peripheral blood mononuclear cells, when compared to age and gender approximated controls, constituting a pattern of recognition of pancreatic cancer as compared to the gene expression patterns in those without pancreatic cancer


















Unigene




Gene Symbol
Gene Name
Gene #
Cluster
Image ID #
(Locus Link)







1. SOC
Socius
G #91544
Hs145061
ID #121239



2. C1orf38:
Chromosome 1 open reading frame 38
G #9473
Hs10649
ID #307255



3. BCL6
B cell CLL lymphoma 6 (zinc finger protein 51)
G #8067
Hs155024
ID #201727



4. LTBR
lymphotoxin beta receptor (TNFR superfamily, member 3)
G #4055
Hs1116
ID #811900



5.
Homo Sapiens p38 beta 2 MAP kinase mRNA, completeCDS


ID #84148



6. SLC9A3R2:
solute carrier family 9 (sodium hydrogen exchanger), isoform 3 regulator 2.

Hs440896
ID #178569









When several of the fourteen genes listed below, are over-expressed in the peripheral blood mononuclear cells, they are most likely to express a pattern diagnostic of intraductal pancreatic adenocarcinoma. If two or more of the first 9 genes listed below are found to be over-expressed in the peripheral blood mononuclear cells, one should consider careful evaluation of the patient for the presence of a pancreatic tumor. If three or more of these first listed 14 genes are found to be expressed in an elevated state one should very carefully evaluate the patient for the presence of intraductal pancreatic adenocarcinoma.


















Unigene




Gene Symbol
Gene Name
Gene #
Cluster
Image ID #
(Locus Link)







 1. SOC
Socius
G #91544
Hs145061
ID #121239



 2. C1orf38:
Chromosome 1 open reading frame 38
G #9473
Hs10649
ID #307255



 3. LIMD1:
LIM domain containing 1
G #8994
Hs193370
ID #795770
(757350)


 4. ESTs


Hs47868
ID #244350



 5. UBAP2L (NICE4)
Ubiquitin associated protein 2-Like.
G #9898
Hs8127
ID #245015




(NICE4 protein)






 6. NUP93
Nucleoporin 93 kDA
G #9688
Hs295014
ID #51918



 7. BCL6
B cell CLL lymphoma 6 (zinc finger protein 51)
G #8067
Hs155024
ID #201727



 8. EST


(Hs6716)
ID #564126



 9. LTBR
lymphotoxin beta receptor (TNFR superfamily, member 3)
G #4055
Hs1116
ID #811900



10. MYC
V-myc myctocytomatosis viral oncogene homolog (avain)
G #4609
Hs202453
ID #812965
(417226)


11. RB1CC1
RB1-inducible coiled - coil 1 (Homo Sapiens)
G #9821
Hs151202
ID #23431



12. SELL
selrctin L (lymphocyte adhesion molecule 1)
G #6402
Hs82848
ID #149910



13. LCP2 (SLP-76)
lymphocyte cytosolic protein 2
G #3937
Hs2488
ID #415127
(283715)


14. PRG1
Proteoglycan 1, secretin granule
G #5552
Hs1908
ID #415021
(703581)









The following 7 genes numbered 15 through 21 are most likely to form an under-expressed pattern in the diagnosis of intraductal pancreatic adenocarcinoma, as illustrated on the hierarchical cluster illustration examples of FIGS. 1-3. When one or more of these under-expressed genes is combined with the patterns of the first listed 14 over-expressed genes listed above the patient should be clinically very carefully evaluated for intraductal pancreatic adenocarcinoma.















15.
Homo Sapiens p38 beta2 MAP kinase mRNA, completeCDS

ID #84148


16.
Prostacyclin-stimulating factor human, cultured diplid fibroblast co
Hs119206
ID #302482


17. SLC9A3R2:
solute carrier family 9 (sodium hydrogen exchanger), isoform 3 regulator 2.
Hs440896
ID #178569


18. EST
Transcribed sequence
Hs480744
ID #757160


19.
Transcribed sequences with moderate similarity to protein vet: NP_115737.1 homo sapiens
Hs509207
ID #950395



hypothetical protein MGC 5469




20. EP400:
E1A binding protein p400 (CAGH32)
Hs507307
ID #377000


21. IQSEC1:
IQ motif and Sec7 domain 1
Hs475506
ID #767879









The first nine genes listed in Table 1 were found to be over-expressed in the different specimens and the methods of analysis. Although consistent results between all specimens was not established with regard to the degree of expression, a fairly consistent cross expression of these genes was noted. The remaining 22 genes were cross-expressed between the specimens, but without as great of degree of separation from the control values. The 31 genes in this table are the specific activated over-expressed and under-expressed genes in the peripheral blood mononuclear cells, consisting mostly of lymphocytes, in a specific response to intraductal pancreatic adenocarcinoma. The patterns of expression of these activated genes recorded on microarray will allow the diagnosis of intraductal pancreatic adenocarcinoma.
The 68 genes listed in Table 2 are the specific activated over-expressed and under-expressed genes in the peripheral blood mononuclear cells, consisting mostly of lymphocytes, in a specific response to intraductal pancreatic adenocarcinoma. The patterns of expression of these activated genes recorded on microarray will allow the diagnosis of intraductal pancreatic adenocarcinoma.
The patterns of difference as seen in the Hierarchal Clusters (FIGS. 1-3) between the pancreatic carcinoma patients and the controls reinforces the concept of a unique specific reaction of the various types of lymphocytes in the peripheral blood to the developing tumor, as one would expect from the TIL cell reaction with infiltration of specific lymphocytes to the tumor site. With one questionable exception *, these genes are not the genes noted in the peripheral blood mononuclear cells by Twine & Burczynski. Twine N C, et al. Disease-associated expression profiles in peripheral blood mononuclear cells from patients with advanced renal cell carcinoma. Cancer Res. 2003 Sep. 15; 63(18):6069-75. Burczynski M E, Twine N C et al. Transcriptional profiles in peripheral blood mononuclear cells prognostic of clinical outcomes in patients with advanced renal cell carcinoma. Clinical Cancer Res. 2005 Feb. 1; 11(3):1181-9, with melanoma by Xu. Xu T et al Microarray analysis reveals differences in gene expression of circulating CD8+ T cells in melanoma patients and healthy donors. Cancer Res. 2004 May 15; 64(10):3661-7 or by Hong. Hong M H, X. X., Mai H Q, Cao S M Min H Q, Analysis of gene expression patterns of periphery lymphocytes in patients with nasopharyngeal carcinoma. Ai Zheng, 2002. 21(1): p. 21-4. with naso-pharyngeal carcinoma; mitigating against a universal common generic gene expression reaction of the lymphocytes to all cancers and reinforcing the hypothesis of a specific recognition of the intraductal pancreatic adenocarcinoma by these lymphocytes. This method and concept can be applied to other pancreatic tumors and diseases, as well as to other gastrointestinal diseases. The evaluation of negatively selected subsets of peripheral blood lymphocytes such as CD8, CD4, CD4CD25 T lymphocytes and B lymphocytes may give a more precise and specific reacting gene expression pattern to the growing and changing tumor as it progresses. The use of pattern recognition rather than individual gene expression as a marker offers the advantage of countering the inherent variability in biological samples. Pattern recognition by combining the results of other tests, including proteomic SELDI-TOF patterns. Bhattacharyya S. Siegel E R, Peteresen G M, Chari S T, Suva L J, Haun R S. Diagnosis of pancreatic cancer using serum proteomic profiling. Neoplasia. 2004 September-October 6(5): 674-86 and the use of specific methylation markers Herman J G, Baylin S B Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med. 2003 Nov. 20: 349(21): 2042-54. together, will further focus and elucidate the presence of a developing tumor and its response to modes of therapy. (* Pyridoxal (pyridoxine, vitamin B6) kinase—noted on the original paper of Twine, et al. and on the linear ANOVA test of intraductal pancreatic adenocarcinoma only, but with different Hs. Identification.)








TABLE 1







31 LISTED GENES













Gene Symbol
Gene Name
Gene #
Unigene Cluster
Image ID #
(Locus Link)





 1. SOC
Socius
G #91544
Hs145061
ID #121239



 2. C1orf38:
Chromosome 1 open reading frame 38
G #9473
Hs10649
ID #307255



 3. LIMD1:
LIM domain containing 1
G #8994
Hs193370
ID #795770
(757350)


 4. ESTs


Hs47868
ID #244350



 5. UBAP2L (NICE4)
Ubiquitin associated protein 2-Like.
G #9898
Hs8127
ID #245015




(NICE4 protein)






 6. NUP93
Nucleoporin 93 kDA
G #9688
Hs295014
ID #51918



 7. ZNF313
Zinc finger protein 313
G #55905
Hs144949
ID #487165



 8. PRUNE
Prune Homolog (Drosphila, Homo Sapiens)
G #149428
Hs78524
ID #364324




(HTCD37 TcD37 homolog 58497)

(Hs78524)




 9. FLJ12584
Hypothetical protein FLJ12584
G #80210
Hs471610
ID #269293



*10. PDXK
pyrdoxal (pyridoxine, vitamin B6)
G #8566
Hs284491
ID #590640




(C21orf124 chromosome 21 open reading frame 124)






 11. MYC
V-myc myctocytomatosis viral oncogene homolog (avain)
G #4609
Hs202453
ID #812965
(417226)


 12. RB1CC1
RB1-inducible coiled - coil 1 (Homo Sapiens)
G #9821
Hs151202
ID #23431



 13. NKG7
natural killer cell group 7 sequence
G #4818
Hs10306
ID #71606



 14. SELL
selectin L (lymphocyte adhesion molecule 1)
G #6402
Hs82848
ID #149910



 15. LCP2 (SLP-76)
lymphocyte cytosolic protein 2
G #3937
Hs2488
ID #415127
(283715)


 16. BCL6
B cell CLL lymphoma 6 (zinc finger protein 51)
G #8067
Hs155024
ID #201727



 17. EST


(Hs6716)
ID #564126



 18. LTBR
lymphotoxin beta receptor (TNFR superfamily, member 3)
G #4055
Hs1116
ID #811900



 19. PRG1
Hematopoetic proteoglycan 1, secretory granule
G #5675
Hs1908
ID #415021
(703581)


 20. Serpine 1:
(PAL1): Plasminogen - activation inhibitor type 1
G #24617
Hs82085
ID #589458



 21. EDN1:
Endothelin
G #1906
Hs2271
ID #549409



 22.
(Homo sapiens chromosome associated protein - ECLAP-E


ID #66638




mRNA complete c)






 23. PBEF1:
Human pre-B cell enhancing factor (PBEF) mRNA,
10135
Hs154968
ID #488548




complete cds






 24. FPR1:
formyl peptide receptor 1
G #2357
Hs753
ID #773236



 25. FLJ 31978:
hypothetical protein FLJ 31978
G #144423
Hs12381
ID #809504



 26. BNC2
basonuclin 2
G #54796
Hs103853
ID #344036



 27. MAST3 (KIAA0561)
microtubule associated serine/threonine kinase 3
G #23031
Hs173864
ID #203350



 28. LOC113330
hypothetical gene supported by NM 005631


ID #113330



 29. (LOC487647
similar to hypothetical protein FLJ21128)


ID #487647



 30. EST (ZGC86904)
ZGC86904 (dario revio) (ZDB-GENE-040625)

Hs64906
ID #415186



 31. EST
moderately similar to WW domain binding protein 1

Hs7709
ID #998681




(M. musculus) (cbp A curved DNA binding protein)
















TABLE 2







The 68 genes in this table are the specific activated over-expressed and under-expressed genes in the peripheral blood mononuclear cells,


consisting mostly of lymphocytes, in a specific response to intraductal pancreatic adenocarcinoma. 


The patterns of expression of these activated genes recorded on microarray will allow the diagnosis of intraductal pancreatic adenocarcinoma.












Gene Symbol
Gene Name
Gene #
Unigene Cluster
Image ID #
(Locus Link)





 1. BCL6
B-cell CLL/lymphoma-6 (Zinc finger protein 51)
G #604
Hs155024
ID #201727



 2. SOC
Socius
G #91544
Hs145061
ID #121239



 3. C1orf38:
Chromosome 1 open reading frame 38
G #9473
Hs10649
ID #307255



 4. LIMD1:
LIM domain containing 1
G #8994
Hs193370
ID #757350



 5. ESTs


Hs47868
ID #244350



 6. UBAP2L (NICE4)
Ubiquitin associated protein 2-Like.
G #9898
Hs8127
ID #245015




(NICE4 protein)






 7. IGA9
Integrin, alpha 9
G #3680
Hs222
ID #898288




(weakly similar to CCAAT Box - BINDing transcript factor 1)






 8. NR6A1
Nuclear receptor subfamily 6, group A, number 1
G #2649
Hs195161
ID #743582



 9. LMCD1
LIM and cysteine-rich domain 1
G #29995
Hs279943
ID #786550



10. SYN2
Synapsin II
G #6854
Hs445503
ID #51254



11. FGD4
FYVE, Rho GEF and PH domain containing 4
G #121512
Hs409311
ID #730270



12.
Transcribed sequences
G #23283
Hs12700
ID #30959



13. ESTs
(Moderately similar to Munc 13 [H. Sapiens])

Hs112921
ID #1055608



14. AP1S2
Adaptor-related protein complex 1, Sigma 1 subunit 2
G #8905
Hs40368
ID #813756



15. PB1
Polybromo 1
G #55193
Hs173220
ID #813644



16. PARD3
Par-3 partitioning defective 3 homolog (C. elegans)
G #56288
Hs98872
ID #753026




(Transcribed sequence)






17. CDNA
FLJ20913F13cloneADSEOO630

Hs7063
ID #49110



18. EST
Transcribed locus

Hs21169
ID #30102



19. ZD73D05
Full length insert cDNA clone ZD73D05

Hs134314
ID #346281



20. KIAA0789:
KIAA0789 gene product.
G #9671
Hs158319
ID #33621



21. E2IG4
hypothetical protein estradiol-induced
G #25987
Hs8361
ID27098



22. C18orf11
Chromosome 18 open reading frame 11
G #64762
Hs12727
ID #37671



23. ESTs
transcribed locus.

Hs537583
ID #626765



24. RAMP:
RA-regulated nuclear matrix associated protein
G #51514
Hs126774
ID #41815O



25. CLDN1:
claudin 1
G #9076
Hs7327
ID #664975



26. FAM38B:
Family with sequence similarly 38 member B.
G #63895
Hs293907
ID #302766



27. CDNA
FLJ144273 fis, clone TOVAR 2001281
G #389011
Hs142074
ID #241900



28. ESTs


Hs28501
ID #139883



29. VNN3
vanin 3
G #55350
Hs183656
ID #120544



30. EST


Hs45033
ID #485827



31. SLC1A7
Solute carrier family 1 (glutamate transporter) member 7
G #6512
Hs104637
ID #276515



32. ALCAM:
Activated leukocyte cell adhesion molecule
(G #214?)
Hs10247
ID #686180






[Hs150693?]




33. SLC22A3:
solute carrier family 22 (extraneuronal monamine
G #6581
HS242721
ID #127120




transporter, member 3






34. CD44?


Hs169610
ID #530788



35. EST


Hs6716
ID #564126



36. LTBR:
lymphotoxin beta receptor (TNFR superfamily, member 3)
G #4055
Hs1116
ID #811900



37. MRNA
cDNA DKFZp434D0818 (from cloneDKFZp434D0818)

Hs5855
ID #308478



38. CD59:
antigen p18-20 (antigen identification by monoclonal
G #966
Hs278573
ID #208001




antibodies 16.3A5, EJ16, EJ30, EL32 andG344)






39. BBX:
Bobby sox homolog (Drosophila)
G #56987
Hs35380
ID #503691



40. NR6A1:
Nuclear receptor subfamily 6, group A, member1
G #2649
Hs195161
ID #258666



41.
Homo Sapiens p38 beta2 MAP kinase mRNA, completeCDS


ID #84148



42.
Prostacyclin-stimulating factor human, cultured diplid fibroblast co

Hs119206
ID #502482



43. SLC9A3R2:
solute carrier family 9(sodium hydrogen exchanger),
G #9351
Hs440896
ID #178569




isoform 3 regulator 2.






44. EST
Transcribed sequence

Hs480744
ID #757160



45.
Transcribed sequences with moderate similarity to protein vet:

Hs509207
ID #950395




NP_115737.1 homo sapiens







hypothetical protein MGC 5469






46. EP400:
E1A binding protein p400 G#57634 Hs507307 (CAGH32) protein

AA427519
ID #377000




EP400: E1A binding protein p400






47. IQSEC1:
IQ motif and Sec7 domain 1G#9922 Hs475506

AA418726
ID #767879




IQSEC1: IQmotif and Sec7 domain 1.






48 LPHN2:
Lactrophilin 2
G #23266
Hs24212
ID #346583



49. PHF10:
PHD finger protein 10 Homo Sapiens
G #55274
Hs435933
ID #138589



50. ZCCHC11:
Zinc finger, CCHC domain containing 11
G #23318
Hs528341
ID #785963



51. FLJ14775:
Hypothetical protein FLJ14775
G #84923
Hs103555
ID194023



52. TRIM37:
Tripartite motif-containing 37 Homo Sapiens
G #4591
Hs80667
ID #305520



53. DHX34:
DEAH (Asp-Glu-Ala-His) box polypeptide 34
G #9704
Hs151706
ID #739990



54. EST


Hs22031
ID #129624



55. EST
Transcribed sequence

Hs226284
ID #124052



56. FLJ00133
FLJ00133 protein
G #25992
Hs471834
ID #341641



57. MGC15407
similar to RIKEN cDNA4931428D14
G #112942
Hs23128
ID #364873



58. SPTAI:
spectrin, alpha, erthrocytic 1 (elliptocytosis 2)
G #6078
Hs418378
ID #204774



59. SLC26A2:
solute carrier family 26 (sulfate transporter) member 2
G #1836
Hs302738
ID #322537



60. NVP160
nucleoporin 160 kDa
G #23279
Hs22559
ID #33299]



61.



ID #48772]



62. FPR1:
formyl peptide receptor 1
G #2357
Hs753
ID #773236



63. Serpine 1:
(PAL1): Plasminogen - activation inhibitor type 1
G #24617
Hs82085
ID #589458



64. EDN1:
Endothelin
G #1906
Hs2271
ID #549409



65. FLJ 31978:
hypothetical protein FLJ 31978
G #144423
Hs12381
ID #809504



66. PRGI:
Hematopoetic protoglycan core protein
G #5552
Hs1909
ID #415021
(703581)


67. SMC2L1:
structural maintenance of chromosome 2
G #10592
Hs119023
ID #66638




(Homo sapiens chromosome associated protein - 







ECLAP-E mRNA complete c)






68. PBEF1:
Human pre-B cell enhancing factor (PBEF) mRNA, complete cds
G #10135
Hs154968
ID #488548









Once particular genes are identified to look at for particular conditions, i.e., the particular genes are identified for a normal differential gene expression pattern for a paticular condition or for a condition differential gene expression pattern for a particular condition which identifies the particular genes to be looked at for either differences or similarities to determine if a person is suffering from the particular condition, microarrays can be configured to provide only information on the particularly identified genes, i.e., to create the patient differential gene expression pattern. Thus, for screening for pancreatic cancer, a microarray could be configured to determine the expression for the patient to be screened of the particular genes identified in Table 1 or in Table 2 and a patient could be screened by looking at the expression for only those particular genes. The patient differential gene expression pattern would be generated by the microarray and then compared with either the normal differential gene expression for differences or the condition differential gene expression pattern for similarities to determine if the patient was likely to be suffering from the condition.
To show that different conditions, even when similar, can be identified, patient differential gene expression patterns were obtained for two patients having known intraductal mucinous pancreatic neoplasm. The patient differential gene expression patterns for these two patients are shown in FIG. 4 in the last two columns labeled IMPN. The gene expression patterns of the two patients known to have ductal pancreatic adenocarcinoma are labeled PA. These two intraductal pancreatic adenocarcinoma patients with intraductal mucinous pancreatic neoplasms were analyzed with a consistent method on human slide B with Significant Analysis of Microarray (SAM) with the Students T Test and a p value of 0.0001 using Gene Sight software. The consistent patterns of over and under expression of the eleven identified genes allow the establishment of significant patterns characteristic of this intraductal pancreatic cancer state as compared to the other pancreatic conditions. This indicates that the method of the invention can be used to distinguish between different pancreatic conditions, so could be used to distinguish between pancreatic disease conditions including islet cell tumors, other growths, and inflammatory states.
While the invention has been described with specific reference to pancreatic disease conditions, and expression patterns for specific genes have been identified for use in screening patients for pancreatic disease conditions, the method of the invention can be used for screening various other bodily conditions and in generating patterns for specific genes for use with other bodily conditions.
Whereas the invention is here illustrated and described with reference to embodiments thereof presently contemplated as the best mode of carrying out the invention in actual practice, it is to be understood that various changes may be made in adapting the invention to different embodiments without departing from the broader inventive concepts disclosed herein and comprehended by the claims that follow.

## Claims

1. A method of screening a human patient for increased likelihood of pancreatic cancer comprising: obtaining peripheral blood monocyte-lymphocytes sample from the human patient; isolating mRNA from the sample; determining the expression level of mRNA for Socius, Chromosome 1 open reading frame 38 (C 1 orf38), LIM domain containing 1, Ubiquitin associated protein 2- like, Nucleoporin 93 kDa, zinc finger protein 313, Prune Homolog, and FLJ12584 in the sample; comparing the expression of Socius, Chromosome 1 open reading frame 38 (Clorf38) ̃ LIM domain containing 1, Ubiquitin associated protein 2- like, Nucleoporin 93 kDa, zinc finger protein 313, Prune Homolog, and FLJ12584 in the sample to the expression of Socius, Chromosome 1 open reading frame 38 (C 1 orf38), LIM domain containing 1, Ubiquitin associated protein 2- like, Nucleoporin 93 kDa, zinc finger protein 313, Prune Homolog, and FLJ12584 in control peripheral blood monocytes-lymphocytes samples from human subjects known not to have pancreatic cancer; and determining the human patient patients with increased expression of Socius, Chromosome 1 open reading frame 38 (C 1 orf38), LIM domain containing 1, Ubiquitin associated protein 2- like, Nucleoporin 93 kDa, zinc finger protein 313, Prune Homolog, and FLJ12584 relative to control peripheral blood monocyte-lymphocytes samples indicates the patient is more likely to have pancreatic cancer.