# Patent

## Title
Gene expression signature for classification of tissue of origin of tumor samples

## Abstract

The present invention provides a process for classification of cancers and tissues of origin through the analysis of the expression patterns of specific microRNAs and nucleic acid molecules relating thereto. Classification according to a microRNA tree-based expression framework allows optimization of treatment, and determination of specific therapy.

## Background
This application is a Continuation in Part of International Patent Application PCT/IL2011/000849, filed Nov. 1, 2011, which claims priority from 61/415,875, filed Nov. 22, 2010 and is a Continuation in Part of Ser. No. 13/167,489, filed Jun. 23, 2011, which is a Continuation in Part of PCT/IL2009/001212, filed Dec. 23, 2009, and claims priority from 61/140,642, filed Dec. 24, 2008, and Continuation in Part of Ser. No. 12/532,940, filed Sep. 24, 2009, which is a U.S. National Stage of International Patent Application PCT/IL2008/000396, filed Mar. 20, 2008, which claims priority from 60/907,266, filed Mar. 27, 2007, 60/929,244, filed Jun. 19, 2007 and 61/024,565 filed Jan. 30, 2008, the contents of which are incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to methods and materials for classification of cancers and the identification of their tissue of origin. Specifically the invention relates to microRNA molecules associated with specific cancers, as well as various nucleic acid molecules relating thereto or derived therefrom.
BACKGROUND OF THE INVENTION
microRNAs (miRs, miRNAs) are a novel class of non-coding, regulatory RNA genes1-3 which are involved in oncogenesis4 and show remarkable tissue-specificity5-7. They have emerged as highly tissue-specific biomarkers2,5,6 postulated to play important roles in encoding developmental decisions of differentiation. Various studies have tied microRNAs to the development of specific malignancies4. MicroRNAs are also stable in tissue, stored frozen or as formalin-fixed, paraffin-embedded (FFPE) samples, and in serum.
Hundreds of thousands of patients in the U.S. are diagnosed each year with a cancer that has already metastasized, without a clearly identified primary site. Oncologists and pathologists are constantly faced with a diagnostic dilemma when trying to identify the primary origin of a patient's metastasis. As metastases need to be treated according to their primary origin, accurate identification of the metastases' primary origin can be critical for determining appropriate treatment.
Once a metastatic tumor is found, the patient may undergo a wide range of costly, time consuming, and at times inefficient tests, including physical examination of the patient, histopathology analysis of the biopsy, imaging methods such as chest X-ray, CT and PET scans, in order to identify the primary origin of the metastasis.
Metastatic cancer of unknown primary (CUP) accounts for 3-5% of all new cancer cases, and as a group is usually a very aggressive disease with a poor prognosis10. The concept of CUP comes from the limitation of present methods to identify cancer origin, despite an often complicated and costly process which can significantly delay proper treatment of such patients. Recent studies revealed a high degree of variation in clinical management, in the absence of evidence based treatment for CUP11. Many protocols were evaluated12 but have shown relatively small benefit13. Determining tumor tissue of origin is thus an important clinical application of molecular diagnostics9.
Molecular classification studies for tumor tissue origin14-17 have generally used classification algorithms that did not utilize domain-specific knowledge: tissues were treated as a-priori equivalents, ignoring underlying similarities between tissue types with a common developmental origin in embryogenesis. An exception of note is the study by Shedden and co-workers18, that was based on a pathology classification tree. These studies used machine-learning methods that average effects of biological features (e.g., mRNA expression levels), an approach which is more amenable to automated processing but does not use or generate mechanistic insights.
Various markers have been proposed to indicate specific types of cancers and tumor tissue of origin. However, the diagnostic accuracy of tumor markers has not yet been defined. There is thus a need for a more efficient and effective method for diagnosing and classifying specific types of cancers.
SUMMARY OF THE INVENTION
The present invention provides specific nucleic acid sequences for use in the identification, classification and diagnosis of specific cancers and tumor tissue of origin. The nucleic acid sequences can also be used as prognostic markers for prognostic evaluation and determination of appropriate treatment of a subject based on the abundance of the nucleic acid sequences in a biological sample. The present invention provides a method for accurate identification of tumor tissue origin.
The invention is based in part on the development of a microRNA-based classifier for tumor classification. microRNA expression levels were measured in 1300 primary and metastatic tumor paraffin-embedded samples. microRNAs were profiled using a custom array platform. Using the custom array platform, a set of over 300 microRNAs was identified for the normalization of the array data and 65 microRNAs were used for the accurate classification of over 40 different tumor types. The accuracy of the assay exceeds 85%.
The findings demonstrate the utility of microRNA as novel biomarkers for the tissue of origin of a metastatic tumor. The classifier has wide biological as well as diagnostic applications.
According to a first aspect, the present invention provides a method of identifying a tissue of origin of a cancer, the method comprising obtaining a biological sample from a subject, measuring the relative abundance in said sample of nucleic acid sequences selected from the group consisting of SEQ ID NOS: 1-390, any combinations thereof, or a sequence having at least about 80% identity thereto; and comparing the measurement to a reference abundance of the nucleic acid by using a classifier algorithm, wherein the relative abundance of said nucleic acid sequences allows for the identification of the tissue of origin of said sample.
According to one aspect, the classifier algorithm is selected from the group consisting of decision tree classifier, K-nearest neighbor classifier (KNN), logistic regression classifier, nearest neighbor classifier, neural network classifier, Gaussian mixture model (GMM), Support Vector Machine (SVM) classifier, nearest centroid classifier, linear regression classifier and random forest classifier. According to one aspect, the sample is obtained from a subject with cancer of unknown primary (CUP), with a primary cancer or with a metastatic cancer.
According to certain embodiments, the cancer is selected from the group consisting of adrenocortical carcinoma; anus or skin squamous cell carcinoma; biliary tract adenocarcinoma; Ewing sarcoma; gastrointestinal stomal tumor (GIST); gastrointestinal tract carcinoid; renal cell carcinoma: chromophobe, clear cell and papillary; pancreatic islet cell tumor; pheochromocytoma; urothelial cell carcinoma (TCC); lung, head & neck, or esophagus squamous cell carcinoma (SCC); brain: astrocytic tumor, oligodendroglioma; breast adenocarcinoma; uterine cervix squamous cell carcinoma; chondrosarcoma; germ cell cancer; sarcoma; colorectal adenocarcinoma; liposarcoma; hepatocellular carcinoma (HCC); lung large cell or adenocarcinoma; lung carcinoid; pleural mesothelioma; lung small cell carcinoma; B-cell lymphoma; T-cell lymphoma; melanoma; malignant fibrous histiocytoma (MFH) or fibrosarcoma; osteosarcoma; ovarian primitive germ cell tumor; ovarian carcinoma; pancreatic adenocarcinoma; prostate adenocarcinoma; rhabdomyosarcoma; gastric or esophageal adenocarcinoma; synovial sarcoma; non-seminomatous testicular germ cell tumor; seminomatous testicular germ cell tumor; thymoma/thymic carcinoma; follicular thyroid carcinoma; medullary thyroid carcinoma; and papillary thyroid carcinoma.
The invention further provides a method for identifying a cancer of germ cell origin, comprising measuring the relative abundance of SEQ ID NO: 55 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of germ cell origin. According to some embodiments the germ cell is selected from the group consisting of an ovarian primitive cell and a testis cell. According to some embodiments the group of nucleic acid furthers consists of SEQ ID NOS: 29, 62 or a sequence having at least about 80% identity thereto, and the abundance of said nucleic acid sequence is indicative of a testis cell cancer origin selected from the group consisting of seminomatous testicular germ cell and non-seminomatous testicular germ cell.
The invention further provides a method for identifying a cancer origin selected from the group consisting of biliary tract adenocarcinoma and hepatocellular carcinoma, comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 9, 29 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer origin selected from the group consisting of biliary tract adenocarcinoma and hepatocellular carcinoma.
The invention further provides a method for identifying a cancer of brain origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 156, 66, 68 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of brain origin.
According to some embodiments the group of nucleic acid furthers consists of SEQ ID NOS: 40, 60 or a sequence having at least about 80% identity thereto, and wherein the abundance of said nucleic acid sequence is indicative of a brain cancer origin selected from the group consisting of oligodendroglioma and astrocytoma.
The invention further provides a method for identifying a cancer of prostate adenocarcinoma origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 14, 21 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of prostate adenocarcinoma origin.
The invention further provides a method for identifying a cancer of breast adenocarcinoma origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 27, 35, 14, 21, 32, 51, 7, 25, 50, 11, 148, 4, 49, 67 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of breast adenocarcinoma origin.
The invention further provides a method for identifying a cancer of ovarian carcinoma origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 27, 35, 14, 21, 32, 51, 7, 25, 4, 39, 50, 11, 148, 49, 67, 57, 34 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of an ovarian carcinoma origin.
The invention further provides a method for identifying a cancer of thyroid carcinoma origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 14, 21, 32, 51, 7, 11, 148, 4 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of thyroid carcinoma origin.
According to some embodiments the group of nucleic acid furthers consists of SEQ ID NOS: 17, 34 or a sequence having at least about 80% identity thereto, and wherein said thyroid carcinoma origin is selected from the group consisting of follicular and papillary.
The invention further provides a method for identifying a cancer origin selected from the group consisting of lung large cell and lung adenocarcinoma, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 14, 21, 32, 51, 7, 11, 148, 4, 49, 67, 57, 34 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer origin selected from the group consisting of lung large cell and lung adenocarcinoma.
The invention further provides a method for identifying a cancer origin selected from the group consisting of lung large cell and lung adenocarcinoma, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 14, 21, 32, 51, 7, 11, 148, 4, 49, 67, 57, 34 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer origin selected from the group consisting of lung large cell and lung adenocarcinoma.
The invention further provides a method for identifying a cancer of thymic carcinoma origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 14, 21, 32, 51, 7, 50, 4, 39, 3, 34 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of a thymic carcinoma origin.
The invention further provides a method for identifying a cancer origin selected from the group consisting of a urothelial cell carcinoma and squamous cell carcinoma, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 14, 21, 32, 51, 7, 50, 4, 39, 3, 34, 69, 24, 44 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of is indicative of a cancer origin selected from the group consisting of urothelial cell carcinoma and squamous cell carcinoma.
According to some embodiments the group of nucleic acid further consists of SEQ ID NOS: 1, 5, 54 or a sequence having at least about 80% identity thereto, and wherein the abundance of said nucleic acid sequence is indicative of squamous-cell-carcinoma origin selected from the group consisting of uterine cervix squamous-cell-carcinoma and non uterine cervix squamous cell carcinoma.
According to some embodiments the group of nucleic acid further consists of SEQ ID NOS: 11, 23 or a sequence having at least about 80% identity thereto in said sample, and wherein the abundance of said nucleic acid sequence is indicative of a non-uterine cervix squamous cell carcinoma origin selected from the group consisting of anus or skin squamous cell carcinoma; and lung, head & neck, and esophagus squamous cell carcinoma.
The invention further provides a method for identifying a cancer origin selected from melanoma and lymphoma, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 2, 47, 50 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer origin selected from the group consisting of melanoma and lymphoma.
According to some embodiments the group of nucleic acid further consists of SEQ ID NOS: 35, 48 or a sequence having at least about 80% identity thereto, and wherein the abundance of said nucleic acid sequence is indicative of a lymphoma cancer origin selected from the group consisting of B-cell lymphoma and T-cell lymphoma.
The invention further provides a method for identifying a cancer of lung small cell carcinoma origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 20, 45 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of lung small cell carcinoma origin.
The invention further provides a method for identifying a cancer of medullary thyroid carcinoma origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 20, 45, 40, 67, 68 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of medullary thyroid carcinoma origin.
The invention further provides a method for identifying a cancer of lung carcinoid origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 20, 45, 40, 67, 68, 64, 53, 37 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of lung carcinoid origin.
The invention further provides a method for identifying a cancer origin selected from the group consisting of gastrointestinal tract carcinoid and pancreatic islet cell tumor, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 20, 45, 40, 67, 68, 64, 53, 37, 34, 18 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer origin selected from the group consisting of gastrointestinal tract carcinoid and pancreatic islet cell tumor.
The invention further provides a method for identifying a cancer origin selected from the group consisting of gastric and esophageal adenocarcinoma, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 42, 36, 146 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer origin elected from the group consisting of gastric and esophageal adenocarcinoma.
The invention further provides a method for identifying a cancer of colorectal adenocarcinoma origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 42, 36, 146, 20, 43 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of colorectal adenocarcinoma origin.
The invention further provides a method for identifying a cancer origin selected from the group consisting of pancreatic adenocarcinoma and biliary tract adenocarcinoma, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 42, 36, 146, 20, 4351, 49, 16, or a sequence having at least about 80% identity thereto, and wherein the abundance of said nucleic acid sequence is indicative of a cancer origin selected from the group consisting of pancreatic adenocarcinoma or biliary tract adenocarcinoma.
The invention further provides a method for identifying a cancer of renal cell carcinoma origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 2, 66, 68, 19, 29 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of renal cell carcinoma origin.
According to some embodiments the group of nucleic acid further consists of SEQ ID NOS: 36, 147 or a sequence having at least about 80% identity thereto, and wherein the abundance of said nucleic acid sequence is indicative of a chromophobe renal cell carcinoma origin.
According to some embodiments the group of nucleic acid further consists of SEQ ID NOS: 49, 9 or a sequence having at least about 80% identity thereto, and wherein the abundance of said nucleic acid sequence is indicative of a renal cell carcinoma origin selected from the group consisting of clear cell and papillary.
The invention further provides a method for identifying a cancer of pheochromacytoma origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 2, 66, 68, 19, 29, 65, 56 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of pheochromacytoma origin.
The invention further provides a method for identifying a cancer of adrenocortical origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 2, 66, 68, 19, 29, 65, 56, 31, 38, 61 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of adrenocortical origin.
The invention further provides a method for identifying a cancer of gastrointestinal stomal tumor origin, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 2, 66, 68, 19, 29, 65, 56, 31, 38, 61, 14, 45 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer of gastrointestinal stomal tumor origin.
The invention further provides a method for identifying a cancer origin selected from the group consisting of pleural mesothelioma and sarcoma, the method comprising measuring the relative abundance of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 2, 66, 68, 19, 29, 65, 56, 31, 38, 61, 14, 45, 35, 10, 5 or a sequence having at least about 80% identity thereto in said sample; wherein the abundance of said nucleic acid sequence is indicative of a cancer origin selected from the group consisting of pleural mesothelioma and sarcoma.
According to some embodiments the group of nucleic acid further consists of SEQ ID NOS: 3, 40, 15 or a sequence having at least about 80% identity thereto, and wherein said sarcoma is synovial sarcoma.
According to some embodiments the group of nucleic acid further consists of SEQ ID NOS: 3, 40, 15, 12, 58 or a sequence having at least about 80% identity thereto, and wherein said sarcoma is chondrosarcoma.
According to some embodiments the group of nucleic acid further consists of SEQ ID NOS: 3, 40, 15, 12, 58, 36, 26 or a sequence having at least about 80% identity thereto, and wherein said sarcoma is liposarcoma.
According to some embodiments the group of nucleic acid further consists of SEQ ID NOS: 3, 40, 15, 12, 58, 36, 26, 21, 25, 49 or a sequence having at least about 80% identity thereto and wherein said sarcoma is selected from the group consisting of Ewing sarcoma and osteosarcoma.
According to some embodiments the group of nucleic acid further consists of SEQ ID NOS: 3, 40, 15, 12, 58, 36, 26, 21, 59, 39, 33 or a sequence having at least about 80% identity thereto and wherein said sarcoma is selected from the group consisting of rhabdomyosarcoma; and malignant fibrous histiocytoma and fibrosarcoma.
According to another aspect, the present invention provides a method of distinguishing between cancers of different origins, said method comprising:

    
    
        (a) obtaining a biological sample from a subject;
        (b) measuring the relative abundance in said sample of nucleic acid sequences selected from the group consisting of SEQ ID NOS: 1-390 or a sequence having at least about 80% identity thereto; and
        (c) comparing said measurement to a reference abundance of said nucleic acid by using a classifier algorithm;
    
    


wherein the relative abundance of said nucleic acid sequence in said sample allows for distinguishing between cancers of different origins.
According to some embodiments the measurement of the relative abundance of SEQ ID NOS: 372, 233, 55, 200, 201 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from a germ-cell tumor and a cancer originating from the group consisting of non-germ-cell tumors.
According to some embodiments the measurement of the relative abundance of SEQ ID NOS: 6, 30, 13 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from hepatobiliary tumors and a cancer originating from the group consisting of non-germ-cell non-hepatobiliary tumors.
According to some embodiments the measurement of the relative abundance of SEQ ID NOS: 28, 29, 231, 9 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from liver tumors and a cancer originating from biliary-tract carcinomas.
According to some embodiments the measurement of the relative abundance of SEQ ID NOS: 46, 5, 12, 30, 29, 28, 32, 13, 152, 49 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from the group consisting of tumors from an epithelial origin and a cancer originating from the group consisting of tumors from a non-epithelial origin.
According to some embodiments the measurement of the relative abundance of SEQ ID NOS: 164, 168, 170, 16, 198, 50, 176, 186, 11, 158, 20, 155, 231, 4, 8, 46, 3, 2, 7 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from the group consisting of melanoma and lymphoma and a cancer originating from the group consisting of all other non-epithelial tumors.
According to some embodiments the measurement of the relative abundance of SEQ ID NOS: 159, 66, 225, 187, 162, 161, 68, 232, 173, 11, 8, 174, 155, 231, 4, 182, 181, 37 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from brain tumors and a cancer originating from the group consisting of all non-brain, non-epithelial tumors.
According to some embodiments the measurement of the relative abundance of SEQ ID NOS: 40, 208, 60, 153, 230, 228, 147, 34, 206, 35, 52, 25, 229, 161, 187, 179 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from astrocytoma and a cancer originating from oligodendroglioma.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 56, 65, 25, 175, 152, 155, 32, 49, 35, 181, or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from the group consisting of neuroendocrine tumors and a cancer originating from the group consisting of all non-neuroendocrine, epithelial tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 27, 177, 4, 32, 35 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from the group consisting of gastrointestinal epithelial tumors and a cancer originating from the group consisting of non-gastrointestinal epithelial tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 56, 199, 14, 15, 165, 231, 36, 154, 21, 49 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from prostate tumors and a cancer originating from the group consisting of all other non-gastrointestinal epithelial tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 222, 62, 29, 28, 211, 214, 227, 215, 218, 152, 216, 212, 224, 13, 194, 192, 221, 217, 205, 219, 32, 193, 223, 220, 210, 209, 213, 163, 30 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from seminoma and a cancer originating from the group consisting of non-seminoma testis-tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 42, 32, 36, 178, 243, 242, 49, 240, 57, 11, 46, 17, 47, 51, 7, 8, 154, 190, 157, 196, 197, or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from the group consisting of squamous cell carcinoma, transitional cell carcinoma and thymoma, and a cancer originating from the group consisting of non gastrointestinal adenocarcinoma tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 56, 46, 25, 152, 50, 45, 191, 181, 179, 49, 32, 42, 184, 40, 147, 236, 57, 203, 36, or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from breast adenocarcinoma, and a cancer originating from the group consisting of squamous cell carcinoma, transitional cell carcinoma, thymomas and ovarian carcinoma.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 253, 32, 4, 39, 10, 46, 5, 226, 2, 195, 32, 185, 11, 168, 184, 16, 242, 12, 237, 243, 250, 49, 246, 167 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from ovarian carcinoma, and a cancer originating from the group consisting of squamous cell carcinoma, transitional cell carcinoma and thymomas.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 11, 147, 17, 157, 40, 8, 49, 9, 191, 205, 207, 195, 51, 46, 45, 52, 234, 231, 21, 169, 43, 3, 196, 154, 390, 171, 255, 197, 190, 189, 39, 7, 48, 47, 32, 36, 4, 178, 37, 181, 25, 183, 182, 35, 240, 57, 242, 204, 236, 176, 158, 148, 206, 50, 20, 34, 186, 239, 251, 244, 24, 188, 172, 238 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from thyroid carcinoma, and a cancer originating from the group consisting of breast adenocarcinoma, lung large cell carcinoma, lung adenocarcinoma and ovarian carcinoma.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 249, 180, 65, 235, 241, 248, 254, 247, 160, 243, 245, 252, 17, 49, 166, 225, 168, 34 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from follicular thyroid carcinoma and a cancer originating from papillary thyroid carcinoma.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 32, 56, 50, 45, 25, 253, 152, 9, 46, 191, 178, 49, 40, 10, 147, 4, 36, 228, 236, 230, 189, 240, 67, 202, 17 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from breast adenocarcinoma and a cancer originating from the group consisting of lung adenocarcinoma and ovarian carcinoma.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 56, 11, 168, 16, 237, 21, 52, 12, 154, 279, 9, 39, 47, 23, 50, 167, 383, 34, 35, 388, 5, 359, 245, 254, 10, 240, 236, 202, 4, 25, 203, 231, 20, 158, 186, 258, 244, 172, 2, 235, 256, 28, 277, 296, 374, 153, 181 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from lung adenocarcinoma and a cancer originating from ovarian carcinoma.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 161, 164, 22, 53, 285, 3, 152, 191, 154, 21, 206, 174, 19, 45, 171, 179, 8, 296, 284, 18, 51, 258, 49, 184, 35, 34, 37, 42, 228, 15, 14, 242, 230, 253, 36, 182, 293, 292, 4, 294, 297, 354, 377, 189, 30, 386, 249, 5, 274 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from thymic carcinoma and a cancer originating from the group consisting of transitional cell carcinoma and squamous cell carcinoma.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 69, 28, 280, 13, 191, 152, 29, 175, 30, 204, 4, 24, 5, 329, 273, 170, 184, 26, 231, 368, 37, 16, 169, 155, 35, 40, 17 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from transitional cell carcinoma and a cancer originating from the group consisting of squamous cell carcinoma.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 164, 5, 231, 54, 1, 242, 372, 249, 167, 254, 354, 381, 380, 245, 358, 364, 240, 11, 378 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between squamous cell carcinoma cancers originating from the uterine cervix, and squamous cell carcinoma cancers originating from the group consisting of anus and skin, lung, head & neck and esophagus.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 305, 184, 41, 183, 49, 382, 235, 291, 181, 5, 296, 289, 206, 338, 334, 25, 11, 19, 198, 23 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between squamous cell carcinoma cancers originating from the group consisting of anus and skin, and between squamous cell carcinoma cancers originating from the group consisting of lung, head & neck and esophagus.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 4, 11, 46, 8, 274, 169, 36, 47, 363, 231, 303, 349, 10, 7, 3, 16, 164, 170, 168, 198, 50, 245, 365, 45, 382, 259, 296, 364, 314, 12 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from melanoma and a cancer originating from lymphoma.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 11, 191, 48, 35, 228 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from B-cell lymphoma and a cancer originating from T-cell lymphoma.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 158, 20, 176, 186, 148, 36, 51, 172, 260, 265, 67, 188, 277, 284, 302, 68, 168, 242, 204, 162, 177, 27, 65, 263, 155, 191, 190, 45, 59, 43, 56, 266, 14, 15, 8, 7, 39, 189, 249, 231, 293, 2 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from lung small cell carcinoma and a cancer originating from the group consisting of lung carcinoid, medullary thyroid carcinoma, gastrointestinal tract carcinoid and pancreatic islet cell tumor.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 159, 40, 147, 11, 311, 4, 8, 231, 301, 297, 68, 67, 265, 36 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from medullary thyroid carcinoma and a cancer originating from other neuroendocrine tumors selected from the group consisting of lung carcinoid, gastrointestinal tract carcinoid and pancreatic islet cell tumor.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 331, 162, 59, 326, 306, 350, 317, 155, 325, 318, 339, 264, 332, 262, 336, 324, 322, 330, 321, 263, 309, 53, 320, 275, 352, 312, 355, 367, 269, 64, 308, 175, 190, 54, 302, 152, 301, 266, 47, 313, 359, 65, 307, 191, 242, 4, 147, 40, 372, 168, 16, 182, 167, 356, 148, 382, 37, 364, 35 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from lung carcinoid tumors, and a cancer originating from gastrointestinal neuroendocrine tumors selected from the group consisting of gastrointestinal tract carcinoid and pancreatic islet cell tumor.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 263, 288, 18, 286, 162, 225, 287, 206, 205, 296, 258, 313, 377, 373, 256, 153, 259, 265, 303, 268, 267, 165, 15, 272, 14, 202, 236, 203, 4, 168, 310, 298, 27, 29, 34, 228, 3, 349, 35, 26 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from pancreatic islet cell tumors and a Gastrointestinal neuroendocrine carcinoid cancer originating from the group consisting of small intestine and duodenum; appendicitis, stomach and pancreas.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 36, 267, 268, 165, 15, 14, 356, 167, 372, 272, 370, 42, 41, 146 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between adenocarcinoma tumors of the gastrointestinal system originating from:

    
    
        a) the group consisting of gastric and esophageal adenocarcinoma, and
        b) the group consisting of cholangiocarcinoma or adenocarcinoma of the extrahepatic biliary tract, pancreatic adenocarcinoma and colorectal adenocarcinoma.
    
    


According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 42, 184, 67, 158, 20, 186, 284, 389, 203, 240, 236, 146, 204, 43, 176, 202, 49, 46, 38, 363 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from colorectal adenocarcinoma and a cancer originating from the group consisting of adenocarcinoma of biliary tract or pancreas.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 49, 11, 13, 373, 154, 5, 30, 45, 178, 147, 274, 16, 40, 21, 43, 253, 245, 256, 12, 374, 379, 180, 153, 51, 52, 1, 295, 257, 385, 293, 294 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from pancreatic adenocarcinoma, and a cancer originating from the group consisting of cholangiocarcinoma or adenocarcinoma of the extrahepatic biliary tract.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 29, 28, 30, 46, 49, 195, 152, 175, 47, 4, 387, 196, 177, 375, 27, 304, 40, 191, 147, 35, 16, 34, 5, 155, 181, 312, 183, 182, 320, 59, 38, 324, 323, 37, 322, 325, 19, 42, 334, 265, 22 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from:

    
    
        a) renal cell tumors selected from the group consisting of chromophobe renal cell carcinoma, clear cell renal cell carcinoma and papillary renal cell carcinoma, and
        b) the group consisting of sarcomas, adrenal tumors and pleural mesothelioma.
    
    


According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 65, 56, 11, 162, 59, 331, 350, 155, 335, 159, 336, 332, 263, 306, 339, 337, 275, 301, 276, 330, 317, 309, 45, 318, 324, 352, 191, 262, 269, 313, 19, 367, 326, 325, 322, 327, 190, 261, 321, 360, 353, 312, 371, 5, 328, 205, 183, 38, 181, 37, 40, 182, 147, 17, 42, 382, 34, 18, 3 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from pheochromocytoma, and a cancer originating from the group consisting of all sarcoma, adrenal carcinoma and mesothelioma tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 61, 333, 31, 347, 346, 344, 345, 387, 334, 351, 324, 326, 269, 155, 320, 322, 59, 318, 325, 245, 254, 331, 275, 180, 355, 370, 323, 312, 178, 249, 183, 181, 38, 182, 37, 3, 25 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from adrenal carcinoma and a cancer originating from the group consisting of mesothelioma and sarcoma tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 165, 14, 15, 333, 272, 270, 45, 301, 191, 46, 195, 266, 190, 19, 334, 155, 25, 147, 40, 34 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from a gastrointestinal stomal tumor and a cancer originating from the group consisting of mesothelioma and sarcoma tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 13, 30, 361, 280, 362, 147, 40, 291, 387, 290, 299, 152, 178, 303, 242, 49, 11, 35, 34, 36, 206, 16, 170, 177, 17 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from a chromophobe renal cell carcinoma tumor and a cancer originating from the group consisting of clear cell and papillary renal cell carcinoma tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 344, 382, 9, 338, 29, 49, 28, 195, 46, 4, 11, 254 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a renal carcinoma cancer originating from a clear cell tumor and a cancer originating from a papillary tumor.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 49, 35, 17, 34, 25, 36, 168, 170, 26, 4, 190, 46, 10, 240, 43, 39, 385, 63, 202, 181, 37, 5, 183, 182, 38, 206, 296, 1 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from pleural mesothelioma and a cancer originating from the group consisting of sarcoma tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 152, 29, 159, 28, 339, 275, 352, 19, 320, 155, 262, 38, 37, 182, 331, 317, 323, 355, 3, 282, 312, 181, 269, 318, 59, 266, 322, 8, 324, 10, 40, 147, 169, 205, 34, 168, 14, 15, 12, 46, 255, 39, 23, 190, 236, 386, 379, 202 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from a synovial sarcoma and a cancer originating from the group consisting of other sarcoma tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 12, 271, 206, 333, 11, 58, 36, 18, 178, 293, 189, 382, 381, 240, 249, 5, 377, 235, 17, 20, 385, 384, 46, 283 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from chondrosarcoma and a cancer originating from the group consisting of other non-synovial sarcoma tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 295, 205, 25, 26, 231, 183, 42, 254, 168, 64, 14, 178, 15, 39, 36, 154, 265, 174, 384, 67 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from liposarcoma and a cancer originating from the group consisting of other non chondrosarcoma and non synovial sarcoma tumors.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 22, 154, 21, 174, 205, 158, 186, 148, 20, 59, 8, 183, 231 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from:

    
    
        a) the group consisting of Ewing sarcoma and osteosarcoma, and
        b) the group consisting of rhabdomyosarcoma, malignant fibrous histiocytoma and fibrosarcoma.
    
    


According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 155, 179, 43, 208, 278, 17, 385, 174, 5, 52, 257, 366, 48, 49, 12, 25, 169, 34, 35, 23, 384, 189, 377, 265, 294, 293, 292 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from Ewing sarcoma and a cancer originating from osteosarcoma.
According to some embodiments, measurement of the relative abundance of SEQ ID NOS: 33, 268, 267, 333, 276, 319, 306, 320, 334, 323, 300, 281, 59, 339, 316, 176, 348, 352, 349, 67, 357, 315, 343, 342, 355, 340, 344, 10, 341, 331, 20, 277, 318, 158, 265, 284, 36, 183, 40, 63, 147, 43, 289, 52, 190, 4, 5, 39, 169, 208 or a sequence having at least about 80% identity thereto in said sample allows for distinguishing between a cancer originating from rhabdomyosarcoma and a cancer originating from the group consisting of malignant fibrous histiocytoma and fibrosarcoma.
According to some aspects of the invention the biological sample is selected from the group consisting of bodily fluid, a cell line, a tissue sample, a biopsy sample, a needle biopsy sample, a fine needle biopsy (FNA) sample, a surgically removed sample, and a sample obtained by tissue-sampling procedures such as endoscopy, bronchoscopy, or laparoscopic methods. According to some embodiments, the tissue is a fresh, frozen, fixed, wax-embedded or formalin-fixed paraffin-embedded (FFPE) tissue.
According to additional aspects of the invention the nucleic acid sequence relative abundance is determined by a method selected from the group consisting of nucleic acid hybridization and nucleic acid amplification. According to some embodiments, the nucleic acid hybridization is performed using a solid-phase nucleic acid biochip array or in situ hybridization. According to some embodiments, the nucleic acid amplification method is real-time PCR. According to some embodiments, the real-time PCR comprises forward and reverse primers. According to additional embodiments, the real-time PCR method further comprises a probe. According to additional embodiments, the probe comprises a sequence selected from the group consisting of a sequence that is complementary to a sequence selected from SEQ ID NOS: 1-390; a fragment thereof and a sequence having at least about 80% identity thereto.
According to another aspect, the present invention provides a kit for cancer origin identification, the kit comprising a probe comprising a sequence selected from the group consisting of a sequence that is complementary to a sequence selected from SEQ ID NOS: 1-390; a fragment thereof and a sequence having at least about 80% identity thereto.
These and other embodiments of the present invention will become apparent in conjunction with the figures, description and claims that follow.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1F demonstrate the structure of the binary decision-tree classifier, with 45 nodes and 46 leaves. Each node is a binary decision between two sets of samples, those to the left and right of the node. A series of binary decisions, starting at node #1 and moving downwards, lead to one of the possible tumor types, which are the “leaves” of the tree. A sample which is classified to the right branch at node #1 continues to node #2, otherwise it continues to node #11. A sample which is classified to the right branch at node #2 continues to node #4, otherwise it continues to node #3. A sample that reaches node #3, is further classified to either the left branch at node #3, and is assigned to the “hepatocellular carcinoma” class, or to the right branch at node #3, and is assigned to the “biliary tract adenocarcinoma” class.
Decisions are made at consecutive nodes using microRNA expression levels, until an end-point (“leaf” of the tree) is reached, indicating the predicted class for this sample. In specifying the tree structure, clinico-pathological considerations were combined with properties observed in the training set data.
FIG. 2A-2D demonstrates binary decisions at node #1 of the decision-tree. When training a decision algorithm for a given node, only samples from classes which are possible outcomes of this node are used for training. The “non germ cell” classes (right branch at node #1); are easily distinguished from tumors of the “germ cell” classes (left branch at node #1) using the expression levels of hsa-miR-373 (SEQ ID NO: 233, 2A), hsa-miR-372 (SEQ ID NO: 55, 2B), hsa-miR-371-3p (SEQ ID NO: 200, 2C), and hsa-miR-371-5p (SEQ ID NO: 201, 2D). The boxplot presentations comparing distribution of the expression of the statistically significant miR5 in tumor samples from the “germ cell” classes (left box) and “non germ cell” classes (right box). The line in the box indicates the median value. The box contains 50% of the data and the horizontal lines and crosses (outliers) show the full range of signals in this group.
FIG. 3 demonstrates binary decisions at node #3 of the decision-tree. Tumors of hepatocellular carcinoma (HCC) origin (left branch at node #3, marked by squares) are easily distinguished from tumors of biliary tract adenocarcinoma origin (right branch at node #3, marked by diamonds) using the expression levels of hsa-miR-200b (SEQ ID NO: 29, y-axis) and hsa-miR-126 (SEQ ID NO: 9, x-axis).
FIG. 4 demonstrates binary decisions at node #4 of the decision-tree. Tumors originating in epithelial (diamonds) are easily distinguished from tumors of non-epithelial origin (squares) using the expression levels of hsa-miR-30a (SEQ ID NO: 46, y-axis) and hsa-miR-200c (SEQ ID NO: 30, x-axis).
FIG. 5 demonstrates binary decisions at node #5 of the decision-tree. Tumors originating in the lymphoma or melanoma (diamonds) are easily distinguished from tumors of non epithelial, non lymphoma/melanoma origin (squares) using the expression levels of hsa-miR-146a (SEQ ID NO: 16, y-axis), hsa-miR-30a (SEQ ID NO: 46, x-axis) and hsa-let-7e (SEQ ID NO: 2, z-axis).
FIG. 6 demonstrates binary decisions at node #6 of the decision-tree. Tumors originating in the brain (left branch at node #6, marked by diamonds) are easily distinguished from tumors of non epithelial, non brain (right branch at node #6, marked by squares) using the expression levels of hsa-miR-9* (SEQ ID NO: 66, y-axis) and hsa-miR-92b (SEQ ID NO: 68, x-axis).
FIG. 7 demonstrates binary decisions at node #7 of the decision-tree. Tumors originating in astrocytoma (right branch at node #7, marked by diamonds) are easily distinguished from tumors of oligodendroglioma origins (left branch at node #7, marked by squares) using the expression levels of hsa-miR-497 (SEQ ID NO: 60, y-axis) and hsa-miR-222 (SEQ ID NO: 40, x-axis).
FIG. 8 demonstrates binary decisions at node #8 of the decision-tree. Tumors originating in the neuroendocrine (diamonds) are easily distinguished from tumors of epithelial, origin (squares) using the expression levels of hsa-miR-193a-3p (SEQ ID NO: 181, y-axis), hsa-miR-7 (SEQ ID NO: 65, x-axis) and hsa-miR-375 (SEQ ID NO: 56, z-axis).
FIG. 9 demonstrates binary decisions at node #9 of the decision-tree. Tumors originating in gastro-intestinal (GI) (left branch at node #9, marked by diamonds) are easily distinguished from tumors of non GI origins (right branch at node #9, marked by squares) using the expression levels of hsa-miR-21* (SEQ ID NO: 35, y-axis) and hsa-miR-194 (SEQ ID NO: 27, x-axis).
FIG. 10 demonstrates binary decisions at node #10 of the decision-tree. Tumors originating in prostate adenocarcinoma (left branch at node #10, marked by diamonds) are easily distinguished from tumors of non prostate origins (right branch at node #10, marked by squares) using the expression levels of hsa-miR-181a (SEQ ID NO: 21, y-axis) and hsa-miR-143 (SEQ ID NO: 14, x-axis).
FIG. 11 demonstrates binary decisions at node #12 of the decision-tree. Tumors originating in seminiomatous testicular germ cell (left branch at node #12, marked by diamonds) are easily distinguished from tumors of non seminiomatous origins (right branch at node #12, marked by squares) using the expression levels of hsa-miR-516a-5p (SEQ ID NO: 62, y-axis) and hsa-miR-200b (SEQ ID NO: 29, x-axis).
FIG. 12 demonstrates binary decisions at node #16 of the decision-tree. Tumors originating in thyroid carcinoma (diamonds) are easily distinguished from tumors of adenocarcinoma of the lung, breast and ovarian origin (squares) using the expression levels of hsa-miR-93 (SEQ ID NO: 148, y-axis), hsa-miR-138 (SEQ ID NO: 11, x-axis) and hsa-miR-10a (SEQ ID NO: 4, z-axis).
FIG. 13 demonstrates binary decisions at node #17 of the decision-tree. Tumors originating in follicular thyroid carcinoma (left branch at node #17, marked by diamonds) are easily distinguished from tumors of papillary thyroid carcinoma origins (right branch at node #17, marked by squares) using the expression levels of hsa-miR-21 (SEQ ID NO: 34, y-axis) and hsa-miR-146b-5p (SEQ ID NO: 17, x-axis).
FIG. 14 demonstrates binary decisions at node #18 of the decision-tree. Tumors originating in breast (diamonds) are easily distinguished from tumors of lung and ovarian origin (squares) using the expression levels of hsa-miR-92a (SEQ ID NO: 67, y-axis), hsa-miR-193a-3p (SEQ ID NO: 25, x-axis) and hsa-miR-31 (SEQ ID NO: 49, z-axis).
FIG. 15 demonstrates binary decisions at node #19 of the decision-tree. Tumors originating in lung adenocarcinoma (diamonds) are easily distinguished from tumors of ovarian carcinoma origin (squares) using the expression levels of hsa-miR-21 (SEQ ID NO: 34, y-axis), hsa-miR-378 (SEQ ID NO: 57, x-axis) and hsa-miR-138 (SEQ ID NO: 11, z-axis).
FIG. 16 demonstrates binary decisions at node #20 of the decision-tree. Tumors originating in thymic carcinoma (left branch at node #20, marked by diamonds) are easily distinguished from tumors of urothelial carcinoma, transitional cell carcinoma (TCC) carcinoma and squamous cell carcinoma (SCC) origins (right branch at node #20, marked by squares) using the expression levels of hsa-miR-21 (SEQ ID NO: 34, y-axis) and hsa-miR-100 (SEQ ID NO: 3, x-axis).
FIG. 17 demonstrates binary decisions at node #22 of the decision-tree. Tumors originating in SCC of the uterine cervix (diamonds) are easily distinguished from tumors of other SCC origin (squares) using the expression levels of hsa-miR-361-5p (SEQ ID NO: 54, y-axis), hsa-let-7c (SEQ ID NO: 1, x-axis) and hsa-miR-10b (SEQ ID NO: 5, z-axis).
FIG. 18 demonstrates binary decisions at node #24 of the decision-tree. Tumors originating in melanoma (diamonds) are easily distinguished from tumors of lymphoma origin (squares) using the expression levels of hsa-miR-342-3p (SEQ ID NO: 50, y-axis) and hsa-miR-30d (SEQ ID NO: 47, x-axis).
FIG. 19 demonstrates binary decisions at node #27 of the decision-tree. Tumors originating in thyroid carcinoma, medullary (diamonds) are easily distinguished from tumors of other neuroendocrine origin (squares) using the expression levels of hsa-miR-92b (SEQ ID NO: 68, y-axis), hsa-miR-222 (SEQ ID NO: 40, x-axis) and hsa-miR-92a (SEQ ID NO: 67, z-axis).
FIG. 20 demonstrates binary decisions at node #30 of the decision-tree. Tumors originating in gastric or esophageal adenocarcinoma (diamonds) are easily distinguished from tumors of other GI adenocarcinoma origin (squares) using the expression levels of hsa-miR-1201 (SEQ ID NO: 146, y-axis), hsa-miR-224 (SEQ ID NO: 42, x-axis) and hsa-miR-210 (SEQ ID NO: 36, z-axis).
FIG. 21 demonstrates binary decisions at node #31 of the decision-tree. Tumors originating in colorectal adenocarcinoma (diamonds) are easily distinguished from tumors of adenocarcinoma of biliary tract or pancreas origin (squares) using the expression levels of hsa-miR-30a (SEQ ID NO: 46, y-axis), hsa-miR-17 (SEQ ID NO: 20, x-axis) and hsa-miR-29a (SEQ ID NO: 43, z-axis).
FIG. 22 demonstrates binary decisions at node #33 of the decision-tree. Tumors originating in kidney (diamonds) are easily distinguished from tumors of adrenal, mesothelioma and sarcoma origin (squares) using the expression levels of hsa-miR-200b (SEQ ID NO: 29, y-axis), hsa-miR-30a (SEQ ID NO: 46, x-axis) and hsa-miR-149 (SEQ ID NO: 19, z-axis).
FIG. 23 demonstrates binary decisions at node #34 of the decision-tree. Tumors originating in pheochromocytoma (diamonds) are easily distinguished from tumors of adrenal, mesothelioma and sarcoma origin (squares) using the expression levels of hsa-miR-375 (SEQ ID NO: 56, y-axis) and hsa-miR-7 (SEQ ID NO: 65, x-axis).
FIG. 24 demonstrates binary decisions at node #44 of the decision-tree. Tumors originating in Ewing sarcoma (diamonds) are easily distinguished from tumors of osteosarcoma origin (squares) using the expression levels of hsa-miR-31 (SEQ ID NO: 49, y-axis) and hsa-miR-193a-3p (SEQ ID NO: 25, x-axis).
FIG. 25 demonstrates binary decisions at node #45 of the decision-tree. Tumors originating in Rhabdomyosarcoma (diamonds) are easily distinguished from tumors of malignant fibrous histiocytoma (MFH) or fibrosarcoma origin (squares) using the expression levels of hsa-miR-206 (SEQ ID NO: 33, y-axis), hsa-miR-22 (SEQ ID NO: 39, x-axis) and hsa-miR-487b (SEQ ID NO: 59, z-axis).



DETAILED DESCRIPTION OF THE INVENTION
Identification of the tissue-of-origin of a tumor is vital to its management. The present invention is based in part on the discovery that specific nucleic acid sequences can be used for the identification of the tissue-of-origin of a tumor. The present invention provides a sensitive, specific and accurate method which can be used to distinguish between different tumor origins. A new microRNA-based classifier was developed for determining tissue origin of tumors based on 65 microRNAs markers. The classifier uses a specific algorithm and allows a clear interpretation of the specific biomarkers.
According to the present invention each node in the classification tree may be used as an independent differential diagnosis tool, for example in the identification of different types of lung cancers. The possibility to distinguish between different tumor origins facilitates providing the patient with the best and most suitable treatment.
The present invention provides diagnostic assays and methods, both quantitative and qualitative for detecting, diagnosing, monitoring, staging and prognosticating cancers by comparing the levels of the specific microRNA molecules of the invention. Such levels are preferably measured in at least one of biopsies, tumor samples, fine-needle aspiration (FNA), cells, tissues and/or bodily fluids. The methods provided in the present invention are particularly useful for discriminating between different cancers.
All the methods of the present invention may optionally further include measuring levels of additional cancer markers. The cancer markers measured in addition to said microRNA molecules depend on the cancer being tested and are known to those of skill in the art.
Assay techniques can be used to determine levels of gene expression, such as genes encoding the nucleic acids of the present invention in a sample derived from a patient. Such assay methods, which are well known to those of skill in the art, include, but are not limited to, nucleic acid microarrays and biochip analysis, reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in situ hybridization assays, competitive-binding assays, northern blot analyses and ELISA assays.
According to one embodiment, the assay is based on expression level of 65 microRNAs in RNA extracted from FFPE metastatic tumor tissue.
The expression levels are used to infer the sample origin using analysis techniques such as, but not limited to, decision-tree classifier, K nearest neighbors classifier, logistic regression classifier, linear regression classifier, nearest neighbor classifier, neural network classifier and nearest centroid classifier.
In use of the decision tree classifier the expression levels are used to make binary decisions (at each relevant node) following the pre-defined structure of the binary decision-tree (defined using a training set).
At each node, the expressions of one or several microRNAs are combined together using a function of the form P=exp (β0+β1*miR1+β2*miR2+β3*miR3 . . . )/(1−exp (β0+β1*miR1+β2*miR2+β3*miR3 . . . )), where the values of β0, β1, β2 . . . and the identities of the microRNAs have been pre-determined (using a training set). The resulting P is compared to a probability threshold level (PTH, which was also determined using the training set), and the classification continues to the left or right branch according to whether P is larger or smaller than the PTH for that node. This continues until an end-point (“leaf”) of the tree is reached. According to some embodiments, PTH=0.5 for all nodes, and the value of 30 is adjusted accordingly. According to further embodiments, β0, β1, β2, . . . are adjusted so that the slope of the log of the odds ratio function is limited.
Training the tree algorithm means determining the tree structure—which nodes there are and what is on each side, and, for each node: which miRs are used, the values of β0, β1, β2 . . . and the PTH. These are determined by a combination of machine learning, optimization algorithm, and trial and error by experts in machine learning and diagnostic algorithms.
An arbitrary threshold of the expression level of one or more nucleic acid sequences can be set for assigning a sample to one of two groups. Alternatively, in a preferred embodiment, expression levels of one or more nucleic acid sequences of the invention are combined by a method such as logistic regression to define a metric which is then compared to previously measured samples or to a threshold. The threshold is treated as a parameter that can be used to quantify the confidence with which samples are assigned to each class. The threshold can be scaled to favor sensitivity or specificity, depending on the clinical scenario. The correlation value to the reference data generates a continuous score that can be scaled and provides diagnostic information on the likelihood that a sample belongs to a certain class of cancer origin or type. In multivariate analysis the microRNA signature provides a high level of prognostic information.
In another preferred embodiment, expression level of nucleic acids is used to classify a test sample by comparison to a training set of samples. In this embodiment, the test sample is compared in turn to each one of the training set samples. The comparison is performed by comparing the expression levels of one or multiple nucleic acids between the test sample and the specific training sample. Each such pairwise comparison generates a combined metric for the multiple nucleic acids, which can be calculated by various numeric methods such as correlation, cosine, Euclidian distance, mean square distance, or other methods known to those skilled in the art. The training samples are then ranked according to this metric, and the samples with the highest values of the metric (or lowest values, according to the type of metric) are identified, indicating those samples that are most similar to the test sample. By choosing a parameter K, this generates a list that includes the K training samples that are most similar to the test sample. Various methods can then be applied to identify from this list the predicted class of the test sample. In a favored embodiment, the test sample is predicted to belong to the class that has the highest number of representative in the list of K most-similar training samples (this method is known as the K Nearest Neighbors method). Other embodiments may provide a list of predictions including all or part of the classes represented in the list, those classes that are represented more than a given minimum number of times, or other voting schemes whereby classes are grouped together.
1. DEFINITIONS
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
About
As used herein, the term “about” refers to +/−10%.
Attached
“Attached” or “immobilized”, as used herein, to refer to a probe and a solid support means that the binding between the probe and the solid support is sufficient to be stable under conditions of binding, washing, analysis, and removal. The binding may be covalent or non-covalent. Covalent bonds may be formed directly between the probe and the solid support or may be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Non-covalent binding may be one or more of electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as streptavidin, to the support and the non-covalent binding of a biotinylated probe to the streptavidin. Immobilization may also involve a combination of covalent and non-covalent interactions.
Baseline
“Baseline”, as used herein, means the initial cycles of PCR, in which there is little change in fluorescence signal.
Biological Sample
“Biological sample”, as used herein, means a sample of biological tissue or fluid that comprises nucleic acids. Such samples include, but are not limited to, tissue or fluid isolated from subjects. Biological samples may also include sections of tissues such as biopsy and autopsy samples, FFPE samples, frozen sections taken for histological purposes, blood, blood fraction, plasma, serum, sputum, stool, tears, mucus, hair, skin, urine, effusions, ascitic fluid, amniotic fluid, saliva, cerebrospinal fluid, cervical secretions, vaginal secretions, endometrial secretions, gastrointestinal secretions, bronchial secretions, cell line, tissue sample, or secretions from the breast. A biological sample may be provided by fine-needle aspiration (FNA), pleural effusion or bronchial brushing. A biological sample may be provided by removing a sample of cells from a subject but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods described herein in vivo. Archival tissues, such as those having treatment or outcome history, may also be used. Biological samples also include explants and primary and/or transformed cell cultures derived from animal or human tissues.
Cancer
The term “cancer” is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Examples of cancers include, but are not limited, to solid tumors and leukemias, including: apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, non-small cell lung (e.g., lung squamous cell carcinoma, lung adenocarcinoma and lung undifferentiated large cell carcinoma), oat cell, papillary, bronchiolar, bronchogenic, squamous cell, and transitional cell), histiocytic disorders, leukemia (e.g., B cell, mixed cell, null cell, T cell, T-cell chronic, HTLV-II-associated, lymphocytic acute, lymphocytic chronic, mast cell, and myeloid), histiocytosis malignant, Hodgkin disease, immunoproliferative small, non-Hodgkin lymphoma, plasmacytoma, reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, craniopharyngioma, dysgerminoma, hamartoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastic tumor, adeno-carcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, Leydig cell tumor, papilloma, Sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffin, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angioma sclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma, phyllodes, fibrosarcoma, hemangiosarcoma, leimyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (e.g., Ewing, experimental, Kaposi, and mast cell), neurofibromatosis, and cervical dysplasia, and other conditions in which cells have become immortalized or transformed.
Classification
The term classification refers to a procedure and/or algorithm in which individual items are placed into groups or classes based on quantitative information on one or more characteristics inherent in the items (referred to as traits, variables, characters, features, etc.) and based on a statistical model and/or a training set of previously labeled items. A “classification tree” places categorical variables into classes.
Complement
“Complement” or “complementary” is used herein to refer to a nucleic acid may mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. A full complement or fully complementary means 100% complementary base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. In some embodiments, the complementary sequence has a reverse orientation (5′-3′).
Ct
Ct signals represent the first cycle of PCR where amplification crosses a threshold (cycle threshold) of fluorescence. Accordingly, low values of Ct represent high abundance or expression levels of the microRNA.
In some embodiments the PCR Ct signal is normalized such that the normalized Ct remains inversed from the expression level. In other embodiments the PCR Ct signal may be normalized and then inverted such that low normalized-inverted Ct represents low abundance or low expression levels of the microRNA.
Data Processing Routine
As used herein, a “data processing routine” refers to a process that can be embodied in software that determines the biological significance of acquired data (i.e., the ultimate results of an assay or analysis). For example, the data processing routine can determine a tissue of origin based upon the data collected. In the systems and methods herein, the data processing routine can also control the data collection routine based upon the results determined. The data processing routine and the data collection routines can be integrated and provide feedback to operate the data acquisition, and hence provide assay-based judging methods.
Data Set
As use herein, the term “data set” refers to numerical values obtained from the analysis. These numerical values associated with analysis may be values such as peak height and area under the curve.
Data Structure
As used herein, the term “data structure” refers to a combination of two or more data sets, an application of one or more mathematical manipulation to one or more data sets to obtain one or more new data sets, or a manipulation of two or more data sets into a form that provides a visual illustration of the data in a new way. An example of a data structure prepared from manipulation of two or more data sets would be a hierarchical cluster.
Detection
“Detection” means detecting the presence of a component in a sample. Detection also means detecting the absence of a component. Detection also means determining the level of a component, either quantitatively or qualitatively.
Differential Expression
“Differential expression” means qualitative or quantitative differences in the temporal and/or spatial gene expression patterns within and among cells and tissue. Thus, a differentially expressed gene may qualitatively have its expression altered, including an activation or inactivation, in, e.g., normal versus diseased tissue. Genes may be turned on or turned off in a particular state, relative to another state, thus permitting comparison of two or more states. A qualitatively regulated gene may exhibit an expression pattern within a state or cell type which may be detectable by standard techniques. Some genes may be expressed in one state or cell type, but not in both. Alternatively, the difference in expression may be quantitative, e.g., in that expression is modulated: up-regulated, resulting in an increased amount of transcript, or down-regulated, resulting in a decreased amount of transcript. The degree to which expression differs needs only to be large enough to quantify via standard characterization techniques such as expression arrays, quantitative reverse transcriptase PCR, northern blot analysis, real-time PCR, in situ hybridization and RNase protection.
Epithelial Tumors
“Epithelial tumors” is meant to include all types of tumors from epithelial origin. Examples of epithelial tumors include, but are not limited to cholangioca or adenoca of extrahepatic biliary tract, urothelial carcinoma, adenocarcinoma of the breast, lung large cell or adenocarcinoma, lung small cell carcinoma, carcinoid, lung, ovarian carcinoma, pancreatic adenocarcinoma, prostatic adenocarcinoma, gastric or esophageal adenocarcinoma, thymoma/thymic carcinoma, follicular thyroid carcinoma, papillary thyroid carcinoma, medullary thyroid carcinoma, anus or skin squamous cell carcinoma, lung, head & neck, or esophagus squamous cell carcinoma, uterine cervix squamous cell carcinoma, gastrointestinal tract carcinoid, pancreatic islet cell tumor and colorectal adenocarcinoma.
Non Epithelial Tumors
“Non epithelial tumors” is meant to include all types of tumors from non epithelial origin. Examples of non epithelial tumors include, but are not limited to adrenocortical carcinoma, chromophobe renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, pleural mesothelioma, astrocytic tumor, oligodendroglioma, pheochromocytoma, B-cell lymphoma, T-cell lymphoma, melanoma, gastrointestinal stromal tumor (GIST), Ewing Sarcoma, chondrosarcoma, malignant fibrous histiocytoma (MFH) or fibrosarcoma, osteosarcoma, rhabdomyosarcoma, synovial sarcoma and liposarcoma.
Expression Profile
The term “expression profile” is used broadly to include a genomic expression profile, e.g., an expression profile of microRNAs. Profiles may be generated by any convenient means for determining a level of a nucleic acid sequence, e.g., quantitative hybridization of microRNA, labeled microRNA, amplified microRNA, cDNA, etc., quantitative PCR, ELISA for quantitation, and the like, and allow the analysis of differential gene expression between two samples. A subject or patient tumor sample, e.g., cells or collections thereof, e.g., tissues, is assayed. Samples are collected by any convenient method, as known in the art. Nucleic acid sequences of interest are nucleic acid sequences that are found to be predictive, including the nucleic acid sequences provided above, where the expression profile may include expression data for 5, 10, 20, 25, 50, 100 or more of the nucleic acid sequences, including all of the listed nucleic acid sequences. According to some embodiments, the term “expression profile” means measuring the relative abundance of the nucleic acid sequences in the measured samples.
Expression Ratio
“Expression ratio”, as used herein, refers to relative expression levels of two or more nucleic acids as determined by detecting the relative expression levels of the corresponding nucleic acids in a biological sample.
FDR (False Discovery Rate)
When performing multiple statistical tests, for example in comparing between the signal of two groups in multiple data features, there is an increasingly high probability of obtaining false positive results, by random differences between the groups that can reach levels that would otherwise be considered statistically significant. In order to limit the proportion of such false discoveries, statistical significance is defined only for data features in which the differences reached a p-value (by two-sided t-test) below a threshold, which is dependent on the number of tests performed and the distribution of p-values obtained in these tests.
Fragment
“Fragment” is used herein to indicate a non-full-length part of a nucleic acid. Thus, a fragment is itself also a nucleic acid.
Gastrointestinal Tumors
“gastrointestinal tumors” is meant to include all types of tumors from gastrointestinal origin. Examples of gastrointestinal tumors include, but are not limited to cholangioca. or adenoca of extrahepatic biliary tract, pancreatic adenocarcinoma, gastric or esophageal adenocarcinoma, and colorectal adenocarcinoma.
Gene
“Gene”, as used herein, may be a natural (e.g., genomic) or synthetic gene comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (e.g., introns, 5′- and 3′-untranslated sequences). The coding region of a gene may be a nucleotide sequence coding for an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA or antisense RNA. A gene may also be an mRNA or cDNA corresponding to the coding regions (e.g., exons and miRNA) optionally comprising 5′- or 3′-untranslated sequences linked thereto. A gene may also be an amplified nucleic acid molecule produced in vitro, comprising all or a part of the coding region and/or 5′- or 3′-untranslated sequences linked thereto.
Germ Cell Tumors
“Germ cell tumors” as used herein, include, but are not limited, to non-seminomatous testicular germ cell tumors, seminomatous testicular germ cell tumors and ovarian primitive germ cell tumors.
Groove Binder/Minor Groove Binder (MGB)
“Groove binder” and/or “minor groove binder” may be used interchangeably and refer to small molecules that fit into the minor groove of double-stranded DNA, typically in a sequence-specific manner. Minor groove binders may be long, flat molecules that can adopt a crescent-like shape and thus fit snugly into the minor groove of a double helix, often displacing water. Minor groove binding molecules may typically comprise several aromatic rings connected by bonds with torsional freedom such as furan, benzene, or pyrrole rings. Minor groove binders may be antibiotics such as netropsin, distamycin, berenil, pentamidine and other aromatic diamidines, Hoechst 33258, SN 6999, aureolic anti-tumor drugs such as chromomycin and mithramycin, CC-1065, dihydrocyclopyrroloindole tripeptide (DPI3), 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3), and related compounds and analogues, including those described in Nucleic Acids in Chemistry and Biology, 2nd ed., Blackburn and Gait, eds., Oxford University Press, 1996, and PCT Published Application No. WO 03/078450, the contents of which are incorporated herein by reference. A minor groove binder may be a component of a primer, a probe, a hybridization tag complement, or combinations thereof. Minor groove binders may increase the Tm of the primer or a probe to which they are attached, allowing such primers or probes to effectively hybridize at higher temperatures.
High Expression miR-205 Tumors
“High expression miR-205 tumors” as used herein include, but are not limited, to urothelial carcinoma (TCC), thymoma/thymic carcinoma, anus or skin squamous cell carcinoma, lung, head&neck, or esophagus squamous cell carcinoma and uterine cervix squamous cell carcinoma.
Low Expression 205 Tumors
“Low expression miR-205 tumors” as used herein include, but are not limited, to lung, large cell or adenocarcinoma, follicular thyroid carcinoma and papillary thyroid carcinoma.
Host Cell
“Host cell”, as used herein, may be a naturally occurring cell or a transformed cell that may contain a vector and may support replication of the vector. Host cells may be cultured cells, explants, cells in vivo, and the like. Host cells may be prokaryotic cells, such as E. coli, or eukaryotic cells, such as yeast, insect, amphibian, or mammalian cells, such as CHO and HeLa cells.
Identity
“Identical” or “identity”, as used herein, in the context of two or more nucleic acids or polypeptide sequences mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA sequences, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.
In Situ Detection
“In situ detection”, as used herein, means the detection of expression or expression levels in the original site, hereby meaning in a tissue sample such as biopsy.
K-Nearest Neighbor
The phrase “K-nearest neighbor” refers to a classification method that classifies a point by calculating the distances between it and points in the training data set. It then assigns the point to the class that is most common among its K-nearest neighbors (where K is an integer).
Leaf
A leaf, as used herein, is the terminal group in a classification or decision tree.
Label
“Label”, as used herein, means a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which can be made detectable. A label may be incorporated into nucleic acids and proteins at any position.
Logistic Regression
Logistic regression is part of a category of statistical models called generalized linear models. Logistic regression can allow one to predict a discrete outcome, such as group membership, from a set of variables that may be continuous, discrete, dichotomous, or a mix of any of these. The dependent or response variable can be dichotomous, for example, one of two possible types of cancer. Logistic regression models the natural log of the odds ratio, i.e., the ratio of the probability of belonging to the first group (P) over the probability of belonging to the second group (1−P), as a linear combination of the different expression levels (in log-space). The logistic regression output can be used as a classifier by prescribing that a case or sample will be classified into the first type if P is greater than 0.5 or 50%. Alternatively, the calculated probability P can be used as a variable in other contexts, such as a 1D or 2D threshold classifier.
Metastasis
“Metastasis” means the process by which cancer spreads from the place at which it first arose as a primary tumor to other locations in the body. The metastatic progression of a primary tumor reflects multiple stages, including dissociation from neighboring primary tumor cells, survival in the circulation, and growth in a secondary location.
Neuroendocrine Tumors
“Neuroendocrine tumors” is meant to include all types of tumors from neuroendocrine origin. Examples of neuroendocrine tumors include, but are not limited to lung small cell carcinoma, lung carcinoid, gastrointestinal tract carcinoid, pancreatic islet cell tumor and medullary thyroid carcinoma.
Node
A “node” is a decision point in a classification (i.e., decision) tree. Also, a point in a neural net that combines input from other nodes and produces an output through application of an activation function.
Nucleic Acid
“Nucleic acid” or “oligonucleotide” or “polynucleotide”, as used herein, mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.
A nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs may be included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones, non-ionic backbones and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which are incorporated herein by reference. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within one definition of nucleic acids. The modified nucleotide analog may be located for example at the 5′-end and/or the 3′-end of the nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridine or cytidine modified at the 5-position, e.g., 5-(2-amino) propyl uridine, 5-bromo uridine; adenosine and guanosine modified at the 8-position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. The 2′-OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or CN, wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I. Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g., a hydroxyprolinol linkage as described in Krutzfeldt et al., Nature 2005; 438:685-689, Soutschek et al., Nature 2004; 432:173-178, and U.S. Patent Publication No. 20050107325, which are incorporated herein by reference. Additional modified nucleotides and nucleic acids are described in U.S. Patent Publication No. 20050182005, which is incorporated herein by reference. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on a biochip. The backbone modification may also enhance resistance to degradation, such as in the harsh endocytic environment of cells. The backbone modification may also reduce nucleic acid clearance by hepatocytes, such as in the liver and kidney. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
Probe
“Probe”, as used herein, means an oligonucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. There may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single-stranded nucleic acids described herein. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. A probe may be single-stranded or partially single- and partially double-stranded. The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence. Probes may be directly labeled or indirectly labeled such as with biotin to which a streptavidin complex may later bind.
Reference Value
As used herein, the term “reference value” or “reference expression profile” refers to a criterion expression value to which measured values are compared in order to identify a specific cancer. The reference value may be based on the abundance of the nucleic acids, or may be based on a combined metric score thereof.
In preferred embodiments the reference value is determined from statistical analysis of studies that compare microRNA expression with known clinical outcomes.
Sarcoma
Sarcoma is meant to include all types of tumors from sarcoma origin. Examples of sarcoma tumors include, but are not limited to gastrointestinal stromal tumor (GIST), Ewing sarcoma, chondrosarcoma, malignant fibrous histiocytoma (MFH) or fibrosarcoma, osteosarcoma, rhabdomyosarcoma, synovial sarcoma and liposarcoma.
Sensitivity
“Sensitivity”, as used herein, may mean a statistical measure of how well a binary classification test correctly identifies a condition, for example, how frequently it correctly classifies a cancer into the correct class out of two possible classes. The sensitivity for class A is the proportion of cases that are determined to belong to class “A” by the test out of the cases that are in class “A”, as determined by some absolute or gold standard.
Specificity
“Specificity”, as used herein, may mean a statistical measure of how well a binary classification test correctly identifies a condition, for example, how frequently it correctly classifies a cancer into the correct class out of two possible classes. The specificity for class A is the proportion of cases that are determined to belong to class “not A” by the test out of the cases that are in class “not A”, as determined by some absolute or gold standard.
Stringent Hybridization Conditions
“Stringent hybridization conditions”, as used herein, mean conditions under which a first nucleic acid sequence (e.g., probe) will hybridize to a second nucleic acid sequence (e.g., target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm, may be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.
Substantially Complementary
“Substantially complementary”, as used herein, means that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions.
Substantially Identical
“Substantially identical”, as used herein, means that a first and a second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.
Subject
As used herein, the term “subject” refers to a mammal, including both human and other mammals. The methods of the present invention are preferably applied to human subjects.
Target Nucleic Acid
“Target nucleic acid”, as used herein, means a nucleic acid or variant thereof that may be bound by another nucleic acid. A target nucleic acid may be a DNA sequence. The target nucleic acid may be RNA. The target nucleic acid may comprise a mRNA, tRNA, shRNA, siRNA or Piwi-interacting RNA, or a pri-miRNA, pre-miRNA, miRNA, or anti-miRNA.
The target nucleic acid may comprise a target miRNA binding site or a variant thereof. One or more probes may bind the target nucleic acid. The target binding site may comprise 5-100 or 10-60 nucleotides. The target binding site may comprise a total of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30-40, 40-50, 50-60, 61, 62 or 63 nucleotides. The target site sequence may comprise at least 5 nucleotides of the sequence of a target miRNA binding site disclosed in U.S. patent application Ser. Nos. 11/384,049, 11/418,870 or 11/429,720, the contents of which are incorporated herein.
1D/2D Threshold Classifier
“1D/2D threshold classifier”, as used herein, may mean an algorithm for classifying a case or sample such as a cancer sample into one of two possible types such as two types of cancer. For a 1D threshold classifier, the decision is based on one variable and one predetermined threshold value; the sample is assigned to one class if the variable exceeds the threshold and to the other class if the variable is less than the threshold. A 2D threshold classifier is an algorithm for classifying into one of two types based on the values of two variables. A threshold may be calculated as a function (usually a continuous or even a monotonic function) of the first variable; the decision is then reached by comparing the second variable to the calculated threshold, similar to the 1D threshold classifier.
Tissue Sample
As used herein, a tissue sample is tissue obtained from a tissue biopsy using methods well known to those of ordinary skill in the related medical arts. The phrase “suspected of being cancerous”, as used herein, means a cancer tissue sample believed by one of ordinary skill in the medical arts to contain cancerous cells. Methods for obtaining the sample from the biopsy include gross apportioning of a mass, microdissection, laser-based microdissection, or other art-known cell-separation methods.
Tumor
“Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
Variant
“Variant”, as used herein, referring to a nucleic acid means (i) a portion of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequence substantially identical thereto.
Wild Type
As used herein, the term “wild-type” sequence refers to a coding, a non-coding or an interface sequence which is an allelic form of sequence that performs the natural or normal function for that sequence. Wild-type sequences include multiple allelic forms of a cognate sequence, for example, multiple alleles of a wild type sequence may encode silent or conservative changes to the protein sequence that a coding sequence encodes.
The present invention employs miRNAs for the identification, classification and diagnosis of specific cancers and the identification of their tissues of origin.
1. microRNA Processing
A gene coding for microRNA (miRNA) may be transcribed leading to production of a miRNA primary transcript known as the pri-miRNA. The pri-miRNA may comprise a hairpin with a stem and loop structure. The stem of the hairpin may comprise mismatched bases. The pri-miRNA may comprise several hairpins in a polycistronic structure.
The hairpin structure of the pri-miRNA may be recognized by Drosha, which is an RNase III endonuclease. Drosha may recognize terminal loops in the pri-miRNA and cleave approximately two helical turns into the stem to produce a 60-70 nt precursor known as the pre-miRNA. Drosha may cleave the pri-miRNA with a staggered cut typical of RNase III endonucleases yielding a pre-miRNA stem loop with a 5′ phosphate and  ̃2 nucleotide 3′ overhang. Approximately one helical turn of stem ( ̃10 nucleotides) extending beyond the Drosha cleavage site may be essential for efficient processing. The pre-miRNA may then be actively transported from the nucleus to the cytoplasm by Ran-GTP and the export receptor Ex-portin-5.
The pre-miRNA may be recognized by Dicer, which is also an RNase III endonuclease. Dicer may recognize the double-stranded stem of the pre-miRNA. Dicer may also cut off the terminal loop two helical turns away from the base of the stem loop, leaving an additional 5′ phosphate and a  ̃2 nucleotide 3′ overhang. The resulting siRNA-like duplex, which may comprise mismatches, comprises the mature miRNA and a similar-sized fragment known as the miRNA*. The miRNA and miRNA* may be derived from opposing arms of the pri-miRNA and pre-miRNA. mRNA* sequences may be found in libraries of cloned miRNAs, but typically at lower frequency than the miRNAs.
Although initially present as a double-stranded species with miRNA*, the miRNA may eventually become incorporated as a single-stranded RNA into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Various proteins can form the RISC, which can lead to variability in specificity for miRNA/miRNA* duplexes, binding site of the target gene, activity of miRNA (repress or activate), and which strand of the miRNA/miRNA* duplex is loaded in to the RISC.
When the miRNA strand of the miRNA:miRNA* duplex is loaded into the RISC, the miRNA* may be removed and degraded. The strand of the miRNA:miRNA* duplex that is loaded into the RISC may be the strand whose 5′ end is less tightly paired. In cases where both ends of the miRNA:miRNA* have roughly equivalent 5′ pairing, both miRNA and miRNA* may have gene silencing activity.
The RISC may identify target nucleic acids based on high levels of complementarity between the miRNA and the mRNA, especially by nucleotides 2-7 of the miRNA. Only one case has been reported in animals where the interaction between the miRNA and its target was along the entire length of the miRNA. This was shown for miR-196 and Hox B8 and it was further shown that miR-196 mediates the cleavage of the Hox B8 mRNA (Yekta et al. Science 2004; 304:594-596). Otherwise, such interactions are known only in plants (Bartel & Bartel 2003; 132:709-717).
A number of studies have looked at the base-pairing requirement between miRNA and its mRNA target for achieving efficient inhibition of translation (reviewed by Bartel 2004; 116:281-297). In mammalian cells, the first 8 nucleotides of the miRNA may be important (Doench & Sharp GenesDev 2004; 18:504-511). However, other parts of the microRNA may also participate in mRNA binding. Moreover, sufficient base pairing at the 3′ can compensate for insufficient pairing at the 5′ (Brennecke et al., PloS Biol 2005; 3:e85). Computation studies, analyzing miRNA binding on whole genomes have suggested a specific role for bases 2-7 at the 5′ of the miRNA in target binding but the role of the first nucleotide, found usually to be “A” was also recognized (Lewis et al. Cell 2005; 120:15-20). Similarly, nucleotides 1-7 or 2-8 were used to identify and validate targets by Krek et al. (Nat Genet. 2005; 37:495-500).
The target sites in the mRNA may be in the 5′ UTR, the 3′ UTR or in the coding region. Interestingly, multiple miRNAs may regulate the same mRNA target by recognizing the same or multiple sites. The presence of multiple miRNA binding sites in most genetically identified targets may indicate that the cooperative action of multiple RISCs provides the most efficient translational inhibition.
miRNAs may direct the RISC to down-regulate gene expression by either of two mechanisms: mRNA cleavage or translational repression. The miRNA may specify cleavage of the mRNA if the mRNA has a certain degree of complementarity to the miRNA. When a miRNA guides cleavage, the cut may be between the nucleotides pairing to residues 10 and 11 of the miRNA. Alternatively, the miRNA may repress translation if the miRNA does not have the requisite degree of complementarity to the miRNA. Translational repression may be more prevalent in animals since animals may have a lower degree of complementarity between the miRNA and binding site.
It should be noted that there may be variability in the 5′ and 3′ ends of any pair of miRNA and miRNA*. This variability may be due to variability in the enzymatic processing of Drosha and Dicer with respect to the site of cleavage. Variability at the 5′ and 3′ ends of miRNA and miRNA* may also be due to mismatches in the stem structures of the pri-miRNA and pre-miRNA. The mismatches of the stem strands may lead to a population of different hairpin structures. Variability in the stem structures may also lead to variability in the products of cleavage by Drosha and Dicer.
2. Nucleic Acids
Nucleic acids are provided herein. The nucleic acids comprise the sequences of SEQ ID NOS: 1-390 or variants thereof. The variant may be a complement of the referenced nucleotide sequence. The variant may also be a nucleotide sequence that is substantially identical to the referenced nucleotide sequence or the complement thereof. The variant may also be a nucleotide sequence which hybridizes under stringent conditions to the referenced nucleotide sequence, complements thereof, or nucleotide sequences substantially identical thereto.
The nucleic acid may have a length of from about 10 to about 250 nucleotides. The nucleic acid may have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or 250 nucleotides. The nucleic acid may be synthesized or expressed in a cell (in vitro or in vivo) using a synthetic gene described herein. The nucleic acid may be synthesized as a single-strand molecule and hybridized to a substantially complementary nucleic acid to form a duplex. The nucleic acid may be introduced to a cell, tissue or organ in a single- or double-stranded form or capable of being expressed by a synthetic gene using methods well known to those skilled in the art, including as described in U.S. Pat. No. 6,506,559, which is incorporated herein by reference.







TABLE 1







SEQ ID NOS of sequences used in the invention


SEQ ID NOs 1-34 are in accordance with Sanger database version 10; 


SEQ ID NOs 35-390 are in accordance with Sanger database version 11;









hairpin SEQ ID NO
miR SEQ ID NO
miR name





 70
 1
hsa-let-7c


 71
 2, 156
hsa-let-7e


 72
 3
hsa-miR-100


 73
 4
hsa-miR-10a


 74
 5
hsa-miR-10b


 75
 6
hsa-miR-122


 76
 7
hsa-miR-125a-5p


 77, 78
 8
hsa-miR-125b


 79
 9
hsa-miR-126


 80
 10
hsa-miR-130a


 81, 82
 11
hsa-miR-138


 83
 12
hsa-miR-140-3p


 84
 13
hsa-miR-141


 85
 14
hsa-miR-143


 86
 15
hsa-miR-145


 87
 16
hsa-miR-146a


 88
 17
hsa-miR-146b-5p


 89
 18
hsa-miR-148a


 90
 19
hsa-miR-149


 91
 20
hsa-miR-17


 92, 93
 21
hsa-miR-181a


 92, 93
 22
hsa-miR-181a*


 94
 23
hsa-miR-185


 95
 24
hsa-miR-191


 96
 25
hsa-miR-193a-3p


 96
 26
hsa-miR-193a-5p


 97, 98
 27
hsa-miR-194


 99
 28
hsa-miR-200a


100
 29
hsa-miR-200b


101
 30
hsa-miR-200c


102
 31
hsa-miR-202


103
 32
hsa-miR-205


104
 33
hsa-miR-206


105
 34
hsa-miR-21


105
 35
hsa-miR-21*


106
 36
hsa-miR-210


107
 37
hsa-miR-214


107
 38
hsa-miR-214*


108
 39
hsa-miR-22


109
 40
hsa-miR-222


110
 41
hsa-miR-223


111
 42
hsa-miR-224


112
 43
hsa-miR-29a


113
 44, 191
hsa-miR-29c


113
 45
hsa-miR-29c*


114
 46
hsa-miR-30a


115
 47
hsa-miR-30d


116
 48
hsa-miR-30e


117
 49
hsa-miR-31


118
 50
hsa-miR-342-3p


119
 51
hsa-miR-345


120
 52
hsa-miR-34a


121
 53
hsa-miR-34c-5p


122
 54
hsa-miR-361-5p


123
 55
hsa-miR-372


124
 56
hsa-miR-375


125
 57, 202
hsa-miR-378


126
 58
hsa-miR-455-5p


127
 59
hsa-miR-487b


128
 60, 208
hsa-miR-497


129, 130, 131
 61
hsa-miR-509-3p


132, 133
 62, 211
hsa-miR-516a-5p


134
 63
hsa-miR-574-5p


135
 64
hsa-miR-652


136, 137, 138
 65
hsa-miR-7


139, 140, 141
 66
hsa-miR-9*


142, 143
 67
hsa-miR-92a


144
 68
hsa-miR-92b


145
 69
hsa-miR-934


149
146
hsa-miR-1201


150
147
hsa-miR-221


151
148
hsa-miR-93



152
hsa-miR-182



153
hsa-let-7d



154
hsa-miR-181b



155
hsa-miR-127-3p



157
hsa-let-7i



158
hsa-miR-106a



159
hsa-miR-124



160
hsa-miR-1248



161
hsa-miR-128



162
hsa-miR-129-3p



163
hsa-miR-1323



164
hsa-miR-142-5p



165
hsa-miR-143*



166
hsa-miR-146b-3p



167
hsa-miR-149*



168
hsa-miR-150



169
hsa-miR-152



170
hsa-miR-155



171
hsa-miR-15a



172
hsa-miR-15b



173
hsa-miR-181c



174
hsa-miR-181d



175
hsa-miR-183



176
hsa-miR-18a



177
hsa-miR-192



178
hsa-miR-193b



179
hsa-miR-195



180
hsa-miR-1973



181
hsa-miR-199a-3p



182
hsa-miR-199a-5p



183
hsa-miR-199b-5p



184
hsa-miR-203



185
hsa-miR-205*



186
hsa-miR-20a



187
hsa-miR-219-2-3p



188
hsa-miR-25



189
hsa-miR-27b



190
hsa-miR-29b



192
hsa-miR-302a



193
hsa-miR-302a*



194
hsa-miR-302d



195
hsa-miR-30a*



196
hsa-miR-30c



197
hsa-miR-331-3p



198
hsa-miR-342-5p



199
hsa-miR-363



200
hsa-miR-371-3p



201
hsa-miR-371-5p



203
hsa-miR-422a



204
hsa-miR-425



205
hsa-miR-451



206
hsa-miR-455-3p



207
hsa-miR-486-5p



209
hsa-miR-498



210
hsa-miR-512-5p



212
hsa-miR-516b



213
hsa-miR-517a



214
hsa-miR-517c



215
hsa-miR-518a-3p



216
hsa-miR-518e



217
hsa-miR-518f*



218
hsa-miR-519a



219
hsa-miR-519d



220
hsa-miR-520a-5p



221
hsa-miR-520c-3p



222
hsa-miR-520d-5p



223
hsa-miR-524-5p



224
hsa-miR-527



225
hsa-miR-551b



226
hsa-miR-625



227
hsa-miR-767-5p



228
hsa-miR-886-3p



229
hsa-miR-9



230
hsa-miR-886-5p



231
hsa-miR-99a



232
hsa-miR-99a*



233
hsa-miR-373



234
hsa-miR-1977



235
hsa-miR-1978



236
MID-00689



237, 369
MID-15684



238
MID-15867



239
MID-15907



240
MID-15965



241
MID-16318



242
MID-16489



243
MID-16869



244
MID-17144



245
MID-18336



246
MID-18422



247
MID-19340



248
MID-19533



249
MID-20524



250
MID-20703



251
MID-21271



252
MID-22664



253
MID-23256



254
MID-23291



255
MID-23794



390
MID-00405



256
hsa-let-7a



257
hsa-let-7b



258
hsa-let-7f



259
hsa-let-7g



260
hsa-miR-106b



261
hsa-miR-1180



262
hsa-miR-127-5p



263
hsa-miR-129*



264
hsa-miR-129-5p



265
hsa-miR-130b



266
hsa-miR-132



267
hsa-miR-133a



268
hsa-miR-133b



269
hsa-miR-134



270
hsa-miR-139-5p



271
hsa-miR-140-5p



272
hsa-miR-145*



273
hsa-miR-148b



274
hsa-miR-151-3p



275
hsa-miR-154



276
hsa-miR-154*



277
hsa-miR-17*



278
hsa-miR-181a-2*



279
hsa-miR-1826



280
hsa-miR-187



281
hsa-miR-188-5p



282
hsa-miR-196a



283
hsa-miR-1979



284
hsa-miR-19b



285
hsa-miR-20b



286
hsa-miR-216a



287
hsa-miR-216b



288
hsa-miR-217



289
hsa-miR-22*



290
hsa-miR-221*



291
hsa-miR-222*



292
hsa-miR-23a



293
hsa-miR-23b



294
hsa-miR-24



295
hsa-miR-26a



296
hsa-miR-26b



297
hsa-miR-27a



298
hsa-miR-28-3p



299
hsa-miR-296-5p



300
hsa-miR-299-3p



301
hsa-miR-29b-2*



302
hsa-miR-301a



303
hsa-miR-30b



304
hsa-miR-30e*



305
hsa-miR-31*



306
hsa-miR-323-3p



307
hsa-miR-324-5p



308
hsa-miR-328



309
hsa-miR-329



310
hsa-miR-330-3p



311
hsa-miR-335



312
hsa-miR-337-5p



313
hsa-miR-338-3p



314
hsa-miR-361-3p



315
hsa-miR-362-3p



316
hsa-miR-362-5p



317
hsa-miR-369-5p



318
hsa-miR-370



319
hsa-miR-376a



320
hsa-miR-376c



321
hsa-miR-377*



322
hsa-miR-379



323
hsa-miR-381



324
hsa-miR-382



325
hsa-miR-409-3p



326
hsa-miR-409-5p



327
hsa-miR-410



328
hsa-miR-411



329
hsa-miR-425*



330
hsa-miR-431*



331
hsa-miR-432



332
hsa-miR-433



333
hsa-miR-483-3p



334
hsa-miR-483-5p



335
hsa-miR-485-3p



336
hsa-miR-485-5p



337
hsa-miR-487a



338
hsa-miR-494



339
hsa-miR-495



340
hsa-miR-500



341
hsa-miR-500*



342
hsa-miR-501-3p



343
hsa-miR-502-3p



344
hsa-miR-503



345
hsa-miR-506



346
hsa-miR-509-3-5p



347
hsa-miR-513a-5p



348
hsa-miR-532-3p



349
hsa-miR-532-5p



350
hsa-miR-539



351
hsa-miR-542-5p



352
hsa-miR-543



353
hsa-miR-598



354
hsa-miR-612



355
hsa-miR-654-3p



356
hsa-miR-658



357
hsa-miR-660



358
hsa-miR-665



359
hsa-miR-708



360
hsa-miR-873



361
hsa-miR-874



362
hsa-miR-891a



363
hsa-miR-99b



364
MID-00064



365
MID-00078



366
MID-00144



367
MID-00465



368
MID-00672



370
MID-15986



371
MID-16270



372
MID-16469



373
MID-16582



374
MID-16748



389
MID-17356 (3651)



375
MID-17375



376
MID-17576



377
MID-17866



378
MID-18307



379
MID-18395



380
MID-19898



381
MID-19962



382
MID-22331



383
MID-22912



384
MID-23017



385
MID-23168



386
MID-23178



387
MID-23751



388
hsa-miR-423-5p









3. Nucleic Acid Complexes

The nucleic acid may further comprise one or more of the following: a peptide, a protein, a RNA-DNA hybrid, an antibody, an antibody fragment, a Fab fragment, and an aptamer.
4. Pri-miRNA
The nucleic acid may comprise a sequence of a pri-miRNA or a variant thereof. The pri-miRNA sequence may comprise from 45-30,000, 50-25,000, 100-20,000, 1,000-1,500 or 80-100 nucleotides. The sequence of the pri-miRNA may comprise a pre-miRNA, miRNA and miRNA*, as set forth herein, and variants thereof. The sequence of the pri-miRNA may comprise any of the sequences of SEQ ID NOS: 1-390 or variants thereof.
The pri-miRNA may comprise a hairpin structure. The hairpin may comprise a first and a second nucleic acid sequence that are substantially complimentary. The first and second nucleic acid sequence may be from 37-50 nucleotides. The first and second nucleic acid sequence may be separated by a third sequence of from 8-12 nucleotides. The hairpin structure may have a free energy of less than −25 Kcal/mole, as calculated by the Vienna algorithm with default parameters, as described in Hofacker et al. (Monatshefte f Chemie 1994; 125:167-188), the contents of which are incorporated herein by reference. The hairpin may comprise a terminal loop of 4-20, 8-12 or 10 nucleotides. The pri-miRNA may comprise at least 19% adenosine nucleotides, at least 16% cytosine nucleotides, at least 23% thymine nucleotides and at least 19% guanine nucleotides.
5. Pre-miRNA
The nucleic acid may also comprise a sequence of a pre-miRNA or a variant thereof. The pre-miRNA sequence may comprise from 45-90, 60-80 or 60-70 nucleotides. The sequence of the pre-miRNA may comprise a miRNA and a miRNA* as set forth herein. The sequence of the pre-miRNA may also be that of a pri-miRNA excluding from 0-160 nucleotides from the 5′ and 3′ ends of the pri-miRNA. The sequence of the pre-miRNA may comprise the sequence of SEQ ID NOS: 1-390 or variants thereof
6. miRNA
The nucleic acid may also comprise a sequence of a miRNA (including miRNA*) or a variant thereof. The miRNA sequence may comprise from 13-33, 18-24 or 21-23 nucleotides. The miRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides. The sequence of the miRNA may be the first 13-33 nucleotides of the pre-miRNA. The sequence of the miRNA may also be the last 13-33 nucleotides of the pre-miRNA. The sequence of the miRNA may comprise the sequence of SEQ ID NOS: 1-69, 146-148, 152-390 or variants thereof
7. Probes
A probe comprising a nucleic acid described herein is also provided. Probes may be used for screening and diagnostic methods, as outlined below. The probe may be attached or immobilized to a solid substrate, such as a biochip.
The probe may have a length of from 8 to 500, 10 to 100 or 20 to 60 nucleotides. The probe may also have a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 or 300 nucleotides. The probe may further comprise a linker sequence of from 10-60 nucleotides. The probe may comprise a nucleic acid that is complementary to a sequence selected from the group consisting of SEQ ID NOS: 1-390 or variants thereof
8. Biochip
A biochip is also provided. The biochip may comprise a solid substrate comprising an attached probe or plurality of probes described herein. The probes may be capable of hybridizing to a target sequence under stringent hybridization conditions. The probes may be attached at spatially defined addresses on the substrate. More than one probe per target sequence may be used, with either overlapping probes or probes to different sections of a particular target sequence. The probes may be capable of hybridizing to target sequences associated with a single disorder appreciated by those in the art. The probes may either be synthesized first, with subsequent attachment to the biochip, or may be directly synthesized on the biochip.
The solid substrate may be a material that may be modified to contain discrete individual sites appropriate for the attachment or association of the probes and is amenable to at least one detection method. Representative examples of substrates include glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses and plastics. The substrates may allow optical detection without appreciably fluorescing.
The substrate may be planar, although other configurations of substrates may be used as well. For example, probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume. Similarly, the substrate may be flexible, such as flexible foam, including closed cell foams made of particular plastics.
The biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two. For example, the biochip may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups. Using these functional groups, the probes may be attached using functional groups on the probes either directly or indirectly using a linker. The probes may be attached to the solid support by either the 5′ terminus, 3′ terminus, or via an internal nucleotide.
The probe may also be attached to the solid support non-covalently. For example, biotinylated oligonucleotides can be made, which may bind to surfaces covalently coated with streptavidin, resulting in attachment. Alternatively, probes may be synthesized on the surface using techniques such as photopolymerization and photolithography.
9. Diagnostics
As used herein, the term “diagnosing” refers to classifying pathology, or a symptom, determining a severity of the pathology (e.g., grade or stage), monitoring pathology progression, forecasting an outcome of pathology and/or prospects of recovery.
As used herein, the phrase “subject in need thereof” refers to an animal or human subject who is known to have cancer, at risk of having cancer (e.g., a genetically predisposed subject, a subject with medical and/or family history of cancer, a subject who has been exposed to carcinogens, occupational hazard, environmental hazard) and/or a subject who exhibits suspicious clinical signs of cancer (e.g., blood in the stool or melena, unexplained pain, sweating, unexplained fever, unexplained loss of weight up to anorexia, changes in bowel habits (constipation and/or diarrhea), tenesmus (sense of incomplete defecation, for rectal cancer specifically), anemia and/or general weakness). Additionally or alternatively, the subject in need thereof can be a healthy human subject undergoing a routine well-being check up.
Analyzing presence of malignant or pre-malignant cells can be effected in vivo or ex vivo, whereby a biological sample (e.g., biopsy, blood) is retrieved. Such biopsy samples comprise cells and may be an incisional or excisional biopsy. Alternatively, the cells may be retrieved from a complete resection.
While employing the present teachings, additional information may be gleaned pertaining to the determination of treatment regimen, treatment course and/or to the measurement of the severity of the disease.
As used herein the phrase “treatment regimen” refers to a treatment plan that specifies the type of treatment, dosage, follow-up plans, schedule and/or duration of a treatment provided to a subject in need thereof (e.g., a subject diagnosed with a pathology). The selected treatment regimen can be an aggressive one which is expected to result in the best clinical outcome (e.g., complete cure of the pathology) or a more moderate one which may relieve symptoms of the pathology yet results in incomplete cure of the pathology. It will be appreciated that in certain cases the treatment regimen may be associated with some discomfort to the subject or adverse side effects (e.g., damage to healthy cells or tissue). The type of treatment can include a surgical intervention (e.g., removal of lesion, diseased cells, tissue, or organ), a cell replacement therapy, an administration of a therapeutic drug (e.g., receptor agonists, antagonists, hormones, chemotherapy agents) in a local or a systemic mode, an exposure to radiation therapy using an external source (e.g., external beam) and/or an internal source (e.g., brachytherapy) and/or any combination thereof. The dosage, schedule and duration of treatment can vary, depending on the severity of pathology and the selected type of treatment, and those of skill in the art are capable of adjusting the type of treatment with the dosage, schedule and duration of treatment.
A method of diagnosis is also provided. The method comprises detecting an expression level of a specific cancer-associated nucleic acid in a biological sample. The sample may be derived from a patient. Diagnosis of a specific cancer state in a patient may allow for prognosis and selection of therapeutic strategy. Further, the developmental stage of cells may be classified by determining temporarily expressed specific cancer-associated nucleic acids.
In situ hybridization of labeled probes to tissue arrays may be performed. When comparing the fingerprints between individual samples the skilled artisan can make a diagnosis, a prognosis, or a prediction based on the findings. It is further understood that the nucleic acid sequences which indicate the diagnosis may differ from those which indicate the prognosis and molecular profiling of the condition of the cells or exosomes may lead to distinctions between responsive or refractory conditions or may be predictive of outcomes.
10. Kits
A kit is also provided and may comprise a nucleic acid described herein together with any or all of the following: assay reagents, buffers, probes and/or primers, and sterile saline or another pharmaceutically acceptable emulsion and suspension base. In addition, the kits may include instructional materials containing directions (e.g., protocols) for the practice of the methods described herein. The kit may further comprise a software package for data analysis of expression profiles.
For example, the kit may be a kit for the amplification, detection, identification or quantification of a target nucleic acid sequence. The kit may comprise a poly (T) primer, a forward primer, a reverse primer, and a probe.
Any of the compositions described herein may be comprised in a kit. In a non-limiting example, reagents for isolating miRNA, labeling miRNA, and/or evaluating a miRNA population using an array are included in a kit. The kit may further include reagents for creating or synthesizing miRNA probes. The kits will thus comprise, in suitable container means, an enzyme for labeling the miRNA by incorporating labeled nucleotide or unlabeled nucleotides that are subsequently labeled. It may also include one or more buffers, such as reaction buffer, labeling buffer, washing buffer, or a hybridization buffer, compounds for preparing the miRNA probes, components for in situ hybridization and components for isolating miRNA. Other kits of the invention may include components for making a nucleic acid array comprising miRNA, and thus may include, for example, a solid support.
The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention.
EXAMPLES
Methods
1. Tumor Samples
1300 primary and metastatic tumor FFPE were used in the study. Tumor samples were obtained from several sources. Institutional review approvals were obtained for all samples in accordance with each institute's institutional review board or IRB equivalent guidelines. Samples included primary tumors and metastases of defined origins, according to clinical records. Tumor content was at least 50% for >95% of samples, as determined by a pathologist based on hematoxylin-eosin (H&E) stained slides.
2. RNA Extraction
For FFPE samples, total RNA was isolated from seven to ten 10-μm-thick tissue sections using the miR extraction protocol developed at Rosetta Genomics. Briefly, the sample was incubated a few times in xylene at 57° C. to remove paraffin excess, followed by ethanol washes. Proteins were degraded by proteinase K solution at 45° C. for a few hours. The RNA was extracted with acid phenol:chloroform followed by ethanol precipitation and DNAse digestion. Total RNA quantity and quality was checked by spectrophotometer (Nanodrop ND-1000).
3. miR Array Platform
Custom microarrays (Agilent Technologies, Santa Clara, Calif.) were produced by printing DNA oligonucleotide probes to: 982 miRs sequences, 17 negative controls, 23 spikes, and 10 positive controls (total of 1032 probes). Each probe, printed in triplicate, carried up to 28-nucleotide (nt) linker at the 3′ end of the microRNA's complement sequence. 17 negative control probes were designed using as sequences which do not match the genome. Two groups of positive control probes were designed to hybridize to miR array: (i) synthetic small RNAs were spiked to the RNA before labeling to verify the labeling efficiency; and (ii) probes for abundant small RNA (e.g., small nuclear RNAs (U43, U24, Z30, U6, U48, U44), 5.8 s and 5 s ribosomal RNA are spotted on the array to verify RNA quality.
4. Cy-Dye Labeling of miRNA for miR Array
One μg of total RNA were labeled by ligation (Thomson et al. Nature Methods 2004; 1:47-53) of an RNA-linker, p-rCrU-Cy/dye (Eurogentec or equivalent), to the 3′ end with Cy3 or Cy5. The labeling reaction contained total RNA, spikes (0.1-100 fmoles), 400 ng RNA-linker-dye, 15% DMSO, 1× ligase buffer and 20 units of T4 RNA ligase (NEB), and proceeded at 4° C. for 1 h, followed by 1 h at 37° C., followed by 4° C. up to 40 min.
The labeled RNA was mixed with 30 μl hybridization mixture (mixture of 45 μL of the 10×GE Agilent Blocking Agent and 246 μL of 2× Hi-RPM Hybridization). The labeling mixture was incubated at 100° C. for 5 minutes followed by ice incubation in water bath for 5 minutes. Slides were Hybridize at 54° C. for 16-20 hours, followed by two washes. The first wash was conducted at room temperature with Agilent GE Wash Buffer 1 for 5 min followed by a second wash with Agilent GE Wash Buffer 2 at 37° C. for 5 min.
Arrays were scanned using an Agilent Microarray Scanner Bundle G2565BA (resolution of 5 μm at XDR Hi 100%, XDR Lo 5%). Array images were analyzed using Feature Extraction 10.7 software (Agilent).
5. Array Signal Calculation and Normalization
Triplicate spots were combined to produce one signal for each probe by taking the logarithmic mean of reliable spots. All data were log 2-transformed and the analysis was performed in log 2-space. A reference data vector for normalization R was calculated by taking the median expression level for each probe across all samples. For each sample data vector S, a 2nd degree polynomial F was found so as to provide the best fit between the sample data and the reference data, such that R≈F(S). Remote data points (“outliers”) were not used for fitting the polynomial F. For each probe in the sample (element Si in the vector S), the normalized value (in log-space) Mi was calculated from the initial value Si by transforming it with the polynomial function F, so that Mi=F(Si).
6. Logistic Regression
The aim of a logistic regression model is to use several features, such as expression levels of several microRNAs, to assign a probability of belonging to one of two possible groups, such as two branches of a node in a binary decision-tree. Logistic regression models the natural log of the odds ratio, i.e., the ratio of the probability of belonging to the first group, for example, the left branch in a node of a binary decision-tree (P) over the probability of belonging to the second group, for example, the right branch in such a node (1−P), as a linear combination of the different expression levels (in log-space). The logistic regression assumes that:




  
    
      ln
      ⁡
      
        (
        
          P
          
            1
            -
            P
          
        
        )
      
    
    =
    
      
        
          β
          0
        
        +
        
          
            ∑
            
              i
              =
              1
            
            N
          
          ⁢
          
            
          
          ⁢
          
            
              β
              i
            
            ·
            
              M
              i
            
          
        
      
      =
      
        
          β
          0
        
        +
        
          
            β
            1
          
          ·
          
            M
            1
          
        
        +
        
          
            β
            2
          
          ·
          
            M
            2
          
        
        +
        ...
      
    
  
  ,




where β0 is the bias, Mi is the expression level (normalized, in log 2-space) of the i-th microRNA used in the decision node, and βi is its corresponding coefficient. βi>0 indicates that the probability to take the left branch (P) increases when the expression level of this microRNA (Mi) increases, and the opposite for βi<0. If a node uses only a single microRNA (M), then solving for P results in:




  P
  =
  
    
      
        e
        
          
            β
            0
          
          +
          
            
              β
              1
            
            ·
            M
          
        
      
      
        1
        +
        
          e
          
            
              β
              0
            
            +
            
              
                β
                1
              
              ·
              M
            
          
        
      
    
    .
  




The regression error on each sample is the difference between the assigned probability P and the true “probability” of this sample, i.e., 1 if this sample is in the left branch group and 0 otherwise. The training and optimization of the logistic regression model calculates the parameters β and the p-values [for each microRNA by the Wald statistic and for the overall model by the χ2 (chi-square) difference], maximizing the likelihood of the data given the model and minimizing the total regression error




  
    
      Σ
      
        
          
            Samples
          
        
        
          
            in
          
        
        
          
            first
          
        
        
          
            group
          
        
      
    
    ⁡
    
      (
      
        1
        -
        
          P
          j
        
      
      )
    
  
  +
  
    
      Σ
      
        
          
            Samples
          
        
        
          
            in
          
        
        
          
            second
          
        
        
          
            group
          
        
      
    
    ⁢
    
      
        P
        j
      
      .
    
  




The probability output of the logistic model is here converted to a binary decision by comparing P to a threshold, denoted by PTH i.e., if P>PTH then the sample belongs to the left branch (“first group”) and vice versa. Choosing at each node the branch which has a probability >0.5, i.e., using a probability threshold of 0.5, leads to a minimization of the sum of the regression errors. However, as the goal was the minimization of the overall number of misclassifications (and not of their probability), a modification which adjusts the probability threshold (PTH) was used in order to minimize the overall number of mistakes at each node (Table 2). For each node the threshold to a new probability threshold PTH was optimized such that the number of classification errors is minimized. This change of probability threshold is equivalent (in terms of classifications) to a modification of the bias β0, which may reflect a change in the prior frequencies of the classes. Once the threshold was chosen β0 was modified such that the threshold will be shifted back to 0.5. In addition, β0, β1, β2, . . . were adjusted so that the slope of the log of the odds ratio function is limited.
7. Stepwise Logistic Regression and Feature Selection
The original data contain the expression levels of multiple microRNAs for each sample, i.e., multiple of data features. In training the classifier for each node, only a small subset of these features was selected and used for optimizing a logistic regression model. In the initial training this was done using a forward stepwise scheme. The features were sorted in order of decreasing log-likelihoods, and the logistic model was started off and optimized with the first feature. The second feature was then added, and the model re-optimized. The regression error of the two models was compared: if the addition of the feature did not provide a significant advantage (a χ2 difference less than 7.88, p-value of 0.005), the new feature was discarded. Otherwise, the added feature was kept. Adding a new feature may make a previous feature redundant (e.g., if they are very highly correlated). To check for this, the process iteratively checks if the feature with lowest likelihood can be discarded (without losing χ2 difference as above). After ensuring that the current set of features is compact in this sense, the process continues to test the next feature in the sorted list, until features are exhausted. No limitation on the number of features was inserted into the algorithm.
The stepwise logistic regression method was used on subsets of the training set samples by re-sampling the training set with repetition (“bootstrap”), so that each of the 20 runs contained somewhat different training set. All the features that took part in one of the 20 models were collected. A robust set of 1-3 features per each node was selected by comparing features that were repeatedly chosen in the bootstrap sets to previous evidence, and considering their signal strengths and reliability. When using these selected features to construct the classifier, the stepwise process was not used and the training optimized the logistic regression model parameters only.
8. K-Nearest-Neighbors (KNN) Classification Algorithm
The KNN algorithm (see e.g., Ma et al., Arch Pathol Lab Med 2006; 130:465-73) calculates the distance (Pearson correlation) of any sample to all samples in the training set, and classifies the sample by the majority vote of the k samples which are most similar (k being a parameter of the classifier). The correlation is calculated on the pre-defined set of microRNAs (the microRNAs that were used by the decision-tree). KNN algorithms with k=1; 10 were compared, and the optimal performer was selected, using k=5. The KNN was based on comparing the expression of all 65 microRNAs in each sample to all other samples in the training database.
9. Reporting a Final Answer (Prediction):
The decision-tree and KNN each return a predicted tissue of origin and histological type where applicable. The tissue of origin and histological type may be one of the exact origins and types in the training or a variant thereof. For example, whereas the training includes brain oligondendrioglioma and brain astrocytoma, the answer may simply be brain carcinoma. In addition to the tissue of origin and histological type, the KNN and decision-tree each return a confidence measure. The KNN returns the number of samples within the K nearest neighbors that agreed with the answer reported by the KNN (denoted by V), and the decision-tree returns the probability of the result (P), which is the multiplication of the probabilities at each branch point made on the way to that answer. The classifier returns the two different predictions or a single prediction in case the predictions concur, can be unified into a single answer (for example into the prediction brain if the KNN returned brain oligondendrioglioma and the decision-tree brain astrocytoma), or if based on V and P, one answer is chosen to override the other.
Example 1
Decision-Tree Classification Algorithm
A tumor classifier was built using the microRNA expression levels by applying a binary tree classification scheme (FIGS. 1A-F). This framework is set up to utilize the specificity of microRNAs in tissue differentiation and embryogenesis: different microRNAs are involved in various stages of tissue specification, and are used by the algorithm at different decision points or “nodes”. The tree breaks up the complex multi-tissue classification problem into a set of simpler binary decisions. At each node, classes which branch out earlier in the tree are not considered, reducing interference from irrelevant samples and further simplifying the decision. The decision at each node can then be accomplished using only a small number of microRNA biomarkers, which have well-defined roles in the classification (Table 2). The structure of the binary tree was based on a hierarchy of tissue development and morphological similarity18, which was modified by prominent features of the microRNA expression patterns. For example, the expression patterns of microRNAs indicated a significant difference between germ cell tumors and tumors of non-germ cell origin, and these are therefore distinguished at node #1 (FIG. 2) into separate branches (FIG. 1A).
For each of the individual nodes logistic regression models were used, a robust family of classifiers which are frequently used in epidemiological and clinical studies to combine continuous data features into a binary decision (FIGS. 2-25 and Methods). Since gene expression classifiers have an inherent redundancy in selecting the gene features, bootstrapping was used on the training sample set as a method to select a stable microRNA set for each node (Methods). This resulted in a small number (usually 2-3) of microRNA features per node, totaling 65 microRNAs for the full classifier (Table 2). This approach provides a systematic process for identifying new biomarkers for differential expression.







TABLE 2







bmicroRNAs used per class in the tree classifier








miR List:
Class





hsa-miR-372 (SEQ ID NO: 55)
Germ cell 



cancer


hsa-miR-372, hsa-miR-122 (SEQ ID NO: 6), hsa-miR-126 
Biliary tract


(SEQ ID NO: 9), hsa-miR-200b (SEQ ID NO: 29)
adenocar-



cinoma


hsa-miR-372, hsa-miR-122, hsa-miR-126, hsa-miR-200b
Hepato-



cellular



carcinoma 



(HCC)


hsa-miR-372, hsa-miR-122, hsa-miR-200c (SEQ ID NO: 30),
Brain tumor


hsa-miR-30a (SEQ ID NO: 46), hsa-miR-146a (SEQ ID NO: 



16), hsa-let-7e (SEQ ID NO: 156), hsa-miR-9* (SEQ ID NO: 



66), hsa-miR-92b (SEQ ID NO: 68)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Brain-


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-222
oligoden-


(SEQ ID NO: 40), hsa-miR-497 (SEQ ID NO: 60)
droglioma


hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Brain-


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-
astrocytoma


222, hsa-miR-497



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Prostate


miR-375 (SEQ ID NO: 56), hsa-miR-7 (SEQ ID NO: 65), hsa-
Adenocar-


miR-193a-3p (SEQ ID NO: 25), hsa-miR-194 (SEQ ID 
cinoma


NO: 27), hsa-miR-21* (SEQ ID NO: 35), hsa-miR-143 



(SEQ ID NO: 14), hsa-miR-181a (SEQ ID NO: 21)



hsa-miR-372
Ovarian 



primitive 



germ cell 



tumor


hsa-miR-372
Testis


hsa-miR-372, hsa-miR-200b, hsa-miR-516a-5p (SEQ ID
Seminoma-


NO: 62)
tous



testicular 



germ cell 



tumor


hsa-miR-372, hsa-miR-200b, hsa-miR-516a-5p
Non 



seminoma-



tous



testicular 



germ cell 



tumor


hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Breast


miR-7, hsa-miR-194, hsa-miR-21*, hsa-miR-143, hsa-miR-
adenocar-


181a, hsa-miR-205 (SEQ ID NO: 32), hsa-miR-345 (SEQ ID
cinoma


NO: 51), hsa-miR-125a-5p (SEQ ID NO: 7), hsa-miR-193a-3p



(SEQ ID NO: 25), hsa-miR-375, hsa-miR-342-3p (SEQ ID



NO: 50)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Ovarian 


miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR-21*, hsa-miR-
carcinoma


143, hsa-miR-181a, hsa-miR-345, hsa-miR-125a-5p, hsa-miR-



193a-3p, hsa-miR-375, hsa-miR-342-3p, hsa-miR-205 (SEQ ID



NO: 32), hsa-miR-10a (SEQ ID NO: 4), hsa-miR-22 (SEQ ID



NO: 39)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Thyroid 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR-
carcinoma


21*, hsa-miR-143, hsa-miR-181a, hsa-miR-205, hsa-miR-345,



hsa-miR-125a-5p, hsa-miR-138 (SEQ ID NO: 11), hsa-miR-93



(SEQ ID NO: 148), hsa-miR-10a (SEQ ID NO: 4)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Thyroid 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR- 
carcinoma


21*, hsa-miR-143, hsa-miR-181a, hsa-miR-205, hsa-miR-345,
follicular


hsa-miR-125a-5p, hsa-miR-138, hsa-miR-93, hsa-miR-10a, hsa-



miR-146b-5p (SEQ ID NO: 17), hsa-miR-21 (SEQ ID NO: 34)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Thyroid 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR- 
carcinoma


21*, hsa-miR-143, hsa-miR-181a, hsa-miR-205, hsa-miR-345,
papillary


hsa-miR-125a-5p, hsa-miR-138, hsa-miR-93, hsa-miR-10a, hsa-



miR-146b-5p, hsa-miR-21



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Breast


miR-375, hsa-miR-7, hsa-miR-194, hsa-miR-21*, hsa-miR-143, 
adenocar-


hsa-miR-181a, hsa-miR-205, hsa-miR-345, hsa-miR-125a-5p,
cinoma


hsa-miR-138, hsa-miR-93, hsa-miR-10a, hsa-miR-193a-3p



(SEQ ID NO: 25), hsa-miR-31 (SEQ ID NO: 49), hsa-miR-92a



(SEQ ID NO: 67)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Lung large 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR-
cell or


21*, hsa-miR-143, hsa-miR-181a, hsa-miR-205, hsa-miR-345,
adenocar-


hsa-miR-125a-5p, hsa-miR-93, hsa-miR-10a, hsa-miR-193a-3p,
cinoma


hsa-miR-31, hsa-miR-92a, hsa-miR-138 (SEQ ID NO: 11), hsa-



miR-378 (SEQ ID NO: 57), hsa-miR-21 (SEQ ID NO: 34)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Ovarian 


miR-375, hsa-miR-7, hsa-miR-194, hsa-miR-21*, hsa-miR-143,
carcinoma


hsa-miR-181a, hsa-miR-205, hsa-miR-345, hsa-miR-125a-5p,



hsa-miR-93, hsa-miR-10a, hsa-miR-193a-3p, hsa-miR-31, hsa-



miR-92a, hsa-miR-138, hsa-miR-378, hsa-miR-21



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Thymoma


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR-



21*, hsa-miR-143, hsa-miR-181a, hsa-miR-205, hsa-miR-345,



hsa-miR-125a-5p, hsa-miR-342-3p, hsa-miR-10a, hsa-miR-22,



hsa-miR-100, hsa-miR-21



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Urothelial


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR-
carcinoma 


21*, hsa-miR-143, hsa-miR-181a, hsa-miR-205, hsa-miR-345,
(TCC)


hsa-miR-125a-5p, hsa-miR-342-3p, hsa-miR-205, hsa-miR-10a,



hsa-miR-22, hsa-miR-100, hsa-miR-21, hsa-miR-934 (SEQ ID



NO: 69), hsa-miR-191 (SEQ ID NO: 24), hsa-miR-29c (SEQ ID



NO: 44)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Squamous 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR-
cell


21*, hsa-miR-143, hsa-miR-181a, hsa-miR-205, hsa-miR-345,
carcinoma 


hsa-miR-125a-5p, hsa-miR-342-3p, hsa-miR-10a, hsa-miR-22,
(SCC)


hsa-miR-100, hsa-miR-21, hsa-miR-934, hsa-miR-191, hsa-



miR-29c



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Uterine 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR-
cervix


21*, hsa-miR-143, hsa-miR-181a, hsa-miR-205, hsa-miR-345,
SCC


hsa-miR-125a-5p, hsa-miR-342-3p, hsa-miR-10a, hsa-miR-22,



hsa-miR-100, hsa-miR-21, hsa-miR-934, hsa-miR-191, hsa-



miR-29c, hsa-miR-10b (SEQ ID NO: 5), hsa-let-7c (SEQ ID



NO: 1), hsa-miR-361-5p (SEQ ID NO: 54)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Anus or 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR-
Skin SCC


21*, hsa-miR-143, hsa-miR-181a, hsa-miR-205, hsa-miR-345,



hsa-miR-125a-5p, hsa-miR-193a-3p, hsa-miR-375, hsa-miR-



342-3p, hsa-miR-205, hsa-miR-10a, hsa-miR-22, hsa-miR-100,



hsa-miR-21, hsa-miR-934, hsa-miR-191, hsa-miR-29c, hsa-



miR-10b, hsa-let-7c, hsa-miR-361-5p, hsa-miR-138, hsa-miR-



185 (SEQ ID NO: 23)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Lung, Head 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR-
& Neck or 


21*, hsa-miR-143, hsa-miR-181a, hsa-miR-205, hsa-miR-345,
Esophagus 


hsa-miR-125a-5p, hsa-miR-342-3p, hsa-miR-10a, hsa-miR-22,
SCC


hsa-miR-100, hsa-miR-21, hsa-miR-934, hsa-miR-191, hsa-



miR-29c, hsa-let-7c, hsa-miR-361-5p, hsa-miR-10b, hsa-miR-



138, hsa-miR-185



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Melanoma


miR-146a (SEQ ID NO: 16), hsa-let-7e (SEQ ID NO: 2), hsa-



miR-30d (SEQ ID NO: 47), hsa-miR-342-3p



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Lymphoma


miR-146a, hsa-let-7e, hsa-miR-30d, hsa-miR-342-3p



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
B cell 


miR-146a, hsa-let-7e, hsa-miR-30d, hsa-miR-342-3p, hsa-miR-
lymphoma


21*, hsa-miR-30e (SEQ ID NO: 48)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
T cell 


miR-146a, hsa-let-7e, hsa-miR-30a, hsa-miR-30d, hsa-miR-
lymphoma


342-3p, hsa-miR-21*, hsa-miR-30e



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Lung 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-17 (SEQ ID
small cell 


NO: 20), hsa-miR-29c* (SEQ ID NO: 45)
carcinoma


hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Medullary 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-17, hsa-miR-
thyroid


29c*, hsa-miR-222 (SEQ ID NO: 40), hsa-miR-92a (SEQ ID
carcinoma


NO: 67), hsa-miR-92b (SEQ ID NO: 68)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Lung 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-17, hsa-miR-
carcinoid


29c*, hsa-miR-222, hsa-miR-92a, hsa-miR-92b, hsa-miR-652



(SEQ ID NO: 64), hsa-miR-34c-5p (SEQ ID NO: 53), hsa-miR-



214 (SEQ ID NO: 37)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Gastro-


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-17, hsa-miR-
intestinal


29c*, hsa-miR-222, hsa-miR-92a, hsa-miR-92b, hsa-miR-652,
(GI) tract 


hsa-miR-34c-5p, hsa-miR-214, hsa-miR-21 (SEQ ID NO: 34),
carcinoid


hsa-miR-148a (SEQ ID NO: 18)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Pancreas 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-17, hsa-miR-
islet cell 


29c*, hsa-miR-222, hsa-miR-92a, hsa-miR-92b, hsa-miR-652,
tumor


hsa-miR-34c-5p, hsa-miR-214, hsa-miR-21, hsa-miR-148a



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Gastric or


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194 (SEQ ID
Esophageal


NO: 27), hsa-miR-21*(SEQ ID NO: 35), hsa-miR-224 (SEQ ID
Adenocar-


NO: 42), hsa-miR-210 (SEQ ID NO: 36), hsa-miR-1201 (SEQ
cinoma


ID NO: 146)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Colorectal


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR- 
Adenocar-


21*, hsa-miR-224, hsa-miR-210, hsa-miR-1201, hsa-miR-17
cinoma


(SEQ ID NO: 20), hsa-miR-29a (SEQ ID NO: 43)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Pancreas 


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR-
or bile


21*, hsa-miR-224, hsa-miR-210, hsa-miR-1201, hsa-miR-17,



hsa-miR-29a



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Pancreatic


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR- 
adenocar-


21*, hsa-miR-224, hsa-miR-210, hsa-miR-1201, hsa-miR-17,
cinoma


hsa-miR-29a, hsa-miR-345 (SEQ ID NO: 51), hsa-miR-31 



(SEQ ID NO: 49), hsa-miR-146a (SEQ ID NO: 16)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Biliary tract


miR-375, hsa-miR-7, hsa-miR-193a-3p, hsa-miR-194, hsa-miR- 
adenocar-


21*, hsa-miR-224, hsa-miR-210, hsa-miR-1201, hsa-miR-17,
cinoma


hsa-miR-29a, hsa-miR-345, hsa-miR-31, hsa-miR-146a



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Renal cell


miR-146a (SEQ ID NO: 16), hsa-let-7e, hsa-miR-9* (SEQ ID
carcinoma


NO: 66), hsa-miR-92b (SEQ ID NO: 68), hsa-miR-149 (SEQ 
chromo-


ID NO: 19), hsa-miR-200b (SEQ ID NO: 29)
phobe


hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Pheochro-


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-30a,
mocytoma


hsa-miR-149, hsa-miR-200b, hsa-miR-7 (SEQ ID NO: 65), hsa-



miR-375



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Adreno-


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-149,
cortical


hsa-miR-200b, hsa-miR-7, hsa-miR-375, hsa-miR-202 (SEQ ID



NO: 31), hsa-miR-214* (SEQ ID NO: 38), hsa-miR-509-3p



(SEQ ID NO: 61)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Gastro-


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-149,
intestinal


hsa-miR-200b, hsa-miR-7, hsa-miR-375, hsa-miR-202, hsa-
stomal 


miR-214*, hsa-miR-509-3p, hsa-miR-143 (SEQ ID NO: 14),
tumor


hsa-miR-29c*
(GIST)


hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Renal cell


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-149,
carcinoma


hsa-miR-200b, hsa-miR-210 (SEQ ID NO: 36), hsa-miR-221
chromo-


(SEQ ID NO: 147)
phobe


hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Renal cell


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-149, 
carcinoma 


hsa-miR-200b, hsa-miR-210, hsa-miR-221, hsa-miR-31 (SEQ
clear cell


ID NO: 49), hsa-miR-126 (SEQ ID NO: 9)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Renal cell


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-149,
carcinoma 


hsa-miR-200b, hsa-miR-210, hsa-miR-221, hsa-miR-31, hsa-
papilary


miR-126



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Pleural


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-149, 
meso-


hsa-miR-200b, hsa-miR-7 (SEQ ID NO: 65), hsa-miR-375, hsa-
thelioma


miR-202 (SEQ ID NO: 31), hsa-miR-214* (SEQ ID NO: 38),



hsa-miR-509-3p (SEQ ID NO: 61), hsa-miR-143 (SEQ ID



NO: 14), hsa-miR-29c*, hsa-miR-21* (SEQ ID NO: 35), hsa-



miR-130a (SEQ ID NO: 10), hsa-miR-10b (SEQ ID NO: 5)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Sarcoma


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-149,



hsa-miR-200b, hsa-miR-7, hsa-miR-375, hsa-miR-202, hsa-



miR-214*, hsa-miR-509-3p, hsa-miR-143, hsa-miR-29c*, hsa-



miR-21*, hsa-miR-130a, hsa-miR-10b



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Synovial 


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-149,
sarcoma


hsa-miR-200b, hsa-miR-7, hsa-miR-375, hsa-miR-202, hsa-



miR-214*, hsa-miR-509-3p, hsa-miR-143, hsa-miR-29c*, hsa-



miR-21*, hsa-miR-130a, hsa-miR-10b, hsa-miR-100 (SEQ ID



NO: 3), hsa-miR-222 (SEQ ID NO: 40), hsa-miR-145 (SEQ ID



NO: 15)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Chondro-


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-149,
sarcoma


hsa-miR-200b, hsa-miR-7, hsa-miR-375, hsa-miR-202, hsa-



miR-214*, hsa-miR-509-3p, hsa-miR-143, hsa-miR-29c*, hsa-



miR-21*, hsa-miR-130a, hsa-miR-10b, hsa-miR-100, hsa-miR-



222, hsa-miR-145, hsa-miR-140-3p (SEQ ID NO: 12), hsa-miR-



455-5p (SEQ ID NO: 58)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Liposar-


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-149,
coma


hsa-miR-200b, hsa-miR-7, hsa-miR-375, hsa-miR-202, hsa-



miR-214*, hsa-miR-509-3p, hsa-miR-143, hsa-miR-29c*, hsa-



miR-21*, hsa-miR-130a, hsa-miR-10b, hsa-miR-100, hsa-miR-



222, hsa-miR-145, hsa-miR-140-3p, hsa-miR-455-5p, hsa-miR-



210 (SEQ ID NO: 36), hsa-miR-193a-5p (SEQ ID NO: 26)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Ewing 


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-149,
sarcoma


hsa-miR-200b, hsa-miR-7, hsa-miR-375, hsa-miR-202, hsa-



miR-214*, hsa-miR-509-3p, hsa-miR-143, hsa-miR-29c*, hsa-



miR-21*, hsa-miR-130a, hsa-miR-10b, hsa-miR-100, hsa-miR-



222, hsa-miR-145, hsa-miR-140-3p, hsa-miR-455-5p, hsa-miR-



210, hsa-miR-193a-5p, hsa-miR-181a, hsa-miR-193a-3p (SEQ



ID NO: 25), hsa-miR-31 (SEQ ID NO: 49)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Osteo-


miR-146a, hsa-let-7e, hsa-miR-9*, hsa-miR-92b, hsa-miR-149,
sarcoma


hsa-miR-200b, hsa-miR-7, hsa-miR-375, hsa-miR-202, hsa-



miR-214*, hsa-miR-509-3p, hsa-miR-143, hsa-miR-29c*, hsa-



miR-21*, hsa-miR-130a, hsa-miR-10b, hsa-miR-100, hsa-miR-



222, hsa-miR-145, hsa-miR-140-3p, hsa-miR-455-5p, hsa-miR-



210, hsa-miR-193a-5p, hsa-miR-181a, hsa-miR-193a-3p, hsa-



miR-31



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Rhabdomyo


miR-146a, hsa-let-7e, hsa-miR-30a, hsa-miR-9*, hsa-miR-92b, 
sarcoma


hsa-miR-30a, hsa-miR-149, hsa-miR-200b, hsa-miR-7, hsa-



miR-375, hsa-miR-202, hsa-miR-214*, hsa-miR-509-3p, hsa-



miR-143, hsa-miR-29c*, hsa-miR-21*, hsa-miR-130a, hsa-miR-



10b, hsa-miR-100, hsa-miR-222, hsa-miR-145, hsa-miR-140-



3p, hsa-miR-455-5p, hsa-miR-210, hsa-miR-193a-5p, hsa-miR-



181a, hsa-miR-487b (SEQ ID NO: 59), hsa-miR-22 (SEQ ID



NO: 39), hsa-miR-206 (SEQ ID NO: 33)



hsa-miR-372, hsa-miR-122, hsa-miR-200c, hsa-miR-30a, hsa-
Malignant 


miR-146a, hsa-let-7e, hsa-miR-30a, hsa-miR-9*, hsa-miR-92b, 
fibrous 


hsa-miR-30a, hsa-miR-149, hsa-miR-200b, hsa-miR-7, hsa-
histiocy-


miR-375, hsa-miR-202, hsa-miR-214*, hsa-miR-509-3p, hsa-
toma


miR-143, hsa-miR-29c*, hsa-miR-21*, hsa-miR-130a, hsa-
(MFH) or 


miR-10b, hsa-miR-100, hsa-miR-222, hsa-miR-145, hsa-miR-
fibrosar-


140-3p, hsa-miR-455-5p, hsa-miR-210, hsa-miR-193a-5p, hsa-
coma


miR-181a, hsa-miR-487b, hsa-miR-22, hsa-miR-206









Example 2
Expression of miRs Provides for Distinguishing Between Tumors







TABLE 3







miR expression (in florescence units) distinguishing 


between the group consisting of germ-cell 


tumors and the group consisting of all other tumors












fold-

SEQ ID



median values
change
p-value
NO. 
miR name














2.7e+004-5.0e+001
545.73 (+)
 <e−240
233
hsa-miR-373


1.8e+004-5.0e+001
365.93 (+)
 <e−240
55
hsa-miR-372


8.6e+003-5.0e+001
171.72 (+)
 <e−240
200
hsa-miR-371-3p


5.9e+003-5.1e+001
115.94 (+)
7.3e−249
201
hsa-miR-371-5p





+ for all the listed miRs, the higher expression is in tumors from a germ-cell origin.






hsa-miR-372 (SEQ ID NO: 55) is used at node 1 of the binary-tree-classifier detailed in the invention to distinguish between germ-cell tumors and all other tumors.
FIGS. 2A-D are boxplot presentations comparing distribution of the expression of the statistically significant miRs in tumor samples from the “germ cell” class (left box) and “non germ cell” class (right box).







TABLE 4







miR expression (in florescence units) distinguishing between 


the group consisting of hepatobiliary tumors and the group


consisting of non germ-cell non-hepatobiliary tumors














SEQ ID 



median values
fold-change
p-value
NO.
miR name





1.0e+005-5.0e+001
2024.31 (+)
1.1e−123
 6
hsa-miR-122


7.4e+001-8.1e+003
 109.63 (−)
3.6e−010
30
hsa-miR-200c


5.0e+001-1.4e+003
 27.92 (−)
4.8e−010
13
hsa-miR-141





+ the higher expression of this miR is in tumors from a hepatobiliary origin


− the higher expression of this miR is in tumors from a non germ-cell, non-hepatobiliary origin






hsa-miR-122 (SEQ ID NO: 6) is used at node 2 of the binary-tree-classifier detailed in the invention to distinguish between hepatobiliary tumors and non germ-cell non-hepatobiliary tumors.







TABLE 5







miR expression (in florescence units) distinguishing between the group 


consisting of liver tumors and the group consisting of biliary-tract 


carcinomas (cholangiocarcinoma or gallbladder adenocarcinoma)












fold-

SEQ ID



median values
change
p-value
NO.
miR name














6.1e+003-4.1e+002
14.74 (+)
5.5e−005
28
hsa-miR-200a


9.7e+003-9.0e+002
10.74 (+)
2.4e−004
29
hsa-miR-200b


1.9e+003-7.0e+003
 3.67 (−)
8.5e−004
231
hsa-miR-99a


3.3e+003-7.5e+003
 2.28 (−)
6.2e−004
9
hsa-miR-126





+ the higher expression of this miR is in biliary tract carcinomas


− the higher expression of this miR is in liver tumors






hsa-miR-126 (SEQ ID NO: 9) and hsa-miR-200b (SEQ ID NO: 29) are used at node 3 of the binary-tree-classifier detailed in the invention to distinguish between liver tumors and biliary-tract carcinoma.
FIG. 3 demonstrates that tumors of hepatocellular carcinoma (HCC) origin (marked by squares) are easily distinguished from tumors of biliary tract adenocarcinoma origin (marked by diamonds) using the expression levels of hsa-miR-200b (SEQ ID NO: 29, y-axis) and hsa-miR-126 (SEQ ID NO: 9, x-axis).







TABLE 6







miR expression (in florescence units) distinguishing 


between the group consisting of tumors from an 


epithelial origin and the group consisting of tumors 


from a non-epithelial origin












fold-

SEQ ID 



median values
change
p-value
NO.
miR name














1.5e+004-7.7e+001
196.43 (+)
1.5e−300
30
hsa-miR-200c


9.0e+003-5.0e+001
180.07 (+)
1.3e−208
29
hsa-miR-200b


3.9e+003-5.0e+001
 78.09 (+)
2.2e−187
28
hsa-miR-200a


2.7e+003-5.0e+001
 54.64 (+)
7.0e−078
32
hsa-miR-205


2.6e+003-5.0e+001
 51.98 (+)
1.2e−265
13
hsa-miR-141


5.4e+002-9.2e+001
 5.90 (+)
6.3e−048
152
hsa-miR-182


1.1e+003-2.5e+002
 4.35 (+)
4.8e−022
49
hsa-miR-31





+ for all the listed miRs, the higher expression is in tumors from epithelial origins






A combination of the expression level of any of the miRs detailed in table 6 with the expression level of any of hsa-miR-30a (SEQ ID NO: 46), hsa-miR-10b (SEQ ID NO: 5) and hsa-miR-140-3p (SEQ ID NO: 12) also provides for distinguishing between tumors from epithelial origins and tumors from non-epithelial origins. This is demonstrated at node 4 of the binary-tree-classifier detailed in the invention with hsa-miR-200c (SEQ ID NO: 30) and hsa-miR-30a (SEQ ID NO: 46) (FIG. 4). Tumors originating in epithelial (diamonds) are easily distinguished from tumors of non-epithelial origin (squares) using the expression levels of hsa-miR-30a (SEQ ID NO: 46, y-axis) and hsa-miR-200c (SEQ ID NO: 30, x-axis).







TABLE 7







miR expression (in florescence units) distinguishing between 


the group consisting of melanoma and lymphoma


(B-cell, T-cell), and the group consisting of all other 


non-epithelial tumors












fold-

SEQ ID



median values
change
p-value
NO.
miR name














2.0e+003-7.0e+001
28.25 (−)
1.9e−074
164
hsa-miR-142-5p


1.2e+004-6.3e+002
18.86 (−)
6.0e−061
168
hsa-miR-150


5.4e+003-3.1e+002
17.29 (−)
5.6e−060
170
hsa-miR-155


4.2e+003-3.5e+002
12.03 (−)
8.4e−068
16
hsa-miR-146a


5.9e+002-1.4e+002
 4.25 (−)
8.2e−048
198
hsa-miR-342-5p


7.5e+003-1.9e+003
 4.02 (−)
4.8e−056
50
hsa-miR-342-3p


8.9e+002-2.5e+002
 3.53 (−)
6.0e−035
176
hsa-miR-18a


4.4e+003-1.4e+003
 3.28 (−)
8.0e−038
186
hsa-miR-20a


7.9e+002-2.6e+002
 3.03 (−)
7.3e−005
11
hsa-miR-138


6.6e+003-2.3e+003
 2.82 (−)
4.0e−039
158
hsa-miR-106a


4.1e+003-1.4e+003
 2.82 (−)
2.4e−037
20
hsa-miR-17


6.2e+001-5.9e+002
 9.53 (+)
3.7e−027
155
hsa-miR-127-3p


1.2e+003-7.0e+003
 5.71 (+)
1.5e−047
231
hsa-miR-99a


3.9e+002-1.7e+003
 4.25 (+)
6.6e−022
4
hsa-miR-10a


1.0e+004-4.1e+004
 3.91 (+)
3.2e−037
8
hsa-miR-125b


6.5e+002-2.2e+003
 3.37 (+)
2.4e−023
46
hsa-miR-30a


1.9e+003-5.6e+003
 2.98 (+)
1.0e−025
3
hsa-miR-100


2.5e+003-7.1e+003
 2.89 (+)
1.8e−051
2
hsa-let-7e


2.9e+003-8.4e+003
 2.86 (+)
8.1e−047
7
hsa-miR-125a-5p





+ the higher expression of this miR is in the group of non-epithelial tumors excluding melanoma and lymphoma


− the higher expression of this miR is in the group consisting of melanoma and lymphoma






hsa-miR-146a (SEQ ID NO: 16), hsa-let-7e (SEQ ID NO: 2) and hsa-miR-30a (SEQ ID NO: 46) are used at node 5 of the binary-tree-classifier detailed in the invention to distinguish between the group consisting of melanoma and lymphoma, and the group consisting of all other non-epithelial tumors. FIG. 5 demonstrates that tumors originating in the lymphoma or melanoma (diamonds) are easily distinguished from tumors of non epithelial, non lymphoma/melanoma origin (squares) using the expression levels of hsa-miR-146a (SEQ ID NO: 16, y-axis), hsa-miR-30a (SEQ ID NO: 46, x-axis) and hsa-let-7e (SEQ ID NO: 2, z-axis).







TABLE 8







miR expression (in florescence units) distinguishing 


between the group consisting of brain tumors 


(astrocytic tumor and oligodendroglioma) and the group 


consisting of all non-brain, non-epithelial tumors












fold-

SEQ ID 



median values
change
p-value
NO.
miR name














9.1e+003-5.0e+001
182.94 (+)
3.8e−059
159
hsa-miR-124


4.4e+003-5.0e+001
 88.33 (+)
1.1e−125
66
hsa-miR-9*


2.1e+003-6.0e+001
 34.97 (+)
6.0e−035
225
hsa-miR-551b


9.9e+002-5.0e+001
 19.73 (+)
3.0e−116
187
hsa-miR-219-2-3p


6.5e+002-5.0e+001
 12.95 (+)
1.8e−021
162
hsa-miR-129-3p


1.1e+003-1.0e+002
 10.52 (+)
2.0e−034
161
hsa-miR-128


2.3e+003-2.5e+002
 9.45 (+)
2.2e−052
68
hsa-miR-92b


5.2e+002-6.8e+001
 7.61 (+)
6.7e−019
232
hsa-miR-99a*


6.9e+002-9.2e+001
 7.45 (+)
5.5e−023
173
hsa-miR-181c


2.2e+003-3.5e+002
 6.34 (+)
7.4e−007
11
hsa-miR-138


1.2e+005-2.4e+004
 4.78 (+)
1.7e−014
8
hsa-miR-125b


1.8e+003-3.9e+002
 4.70 (+)
7.2e−014
174
hsa-miR-181d


8.5e+002-1.8e+002
 4.64 (+)
2.2e−002
155
hsa-miR-127-3p


1.6e+004-3.5e+003
 4.60 (+)
2.4e−010
231
hsa-miR-99a


8.5e+001-1.1e+003
 13.55 (−)
2.4e−014
4
hsa-miR-10a


7.7e+002-6.6e+003
 8.58 (−)
8.4e−017
182
hsa-miR-199a-5p


5.7e+002-4.7e+003
 8.12 (−)
1.8e−013
181
hsa-miR-199a-3p


2.8e+002-1.9e+003
 6.81 (−)
1.4e−012
37
hsa-miR-214





+ the higher expression of this miR is in the group consisting of brain tumors


− the higher expression of this miR is in the group consisting of all non-brain, non-epithelial tumors






hsa-miR-9* (SEQ ID NO: 66) and hsa-miR-92b (SEQ ID NO: 68) are used at node 6 of the binary-tree-classifier detailed in the invention to distinguish between brain tumors and the group consisting of all non-brain, non-epithelial tumors. FIG. 6 demonstrates that tumors originating in the brain (marked by diamonds) are easily distinguished from tumors of non epithelial, non brain origin (marked by squares) using the expression levels of hsa-miR-9* (SEQ ID NO: 66, y-axis) and hsa-miR-92b (SEQ ID NO: 68, x-axis).







TABLE 9







miR expression (in florescence units) distinguishing between 


astrocytic tumors and oligodendrogliomas












fold-

SEQ ID



median values
change
p-value
NO.
miR name














2.5e+003-2.3e+002
11.10 (+)
5.1e−011
230
hsa-miR-886-5p


4.4e+003-4.9e+002
 9.06 (+)
1.1e−009
228
hsa-miR-886-3p


1.0e+004-1.7e+003
 5.99 (+)
7.7e−008
147
hsa-miR-221


1.3e+004-2.6e+003
 5.03 (+)
2.6e−006
40
hsa-miR-222


3.3e+004-7.3e+003
 4.54 (+)
3.9e−004
34
hsa-miR-21


8.4e+002-2.2e+002
 3.78 (+)
3.7e−006
206
hsa-miR-455-3p


6.0e+002-1.8e+002
 3.30 (+)
1.3e−002
35
hsa-miR-21*


5.8e+003-1.8e+003
 3.15 (+)
2.4e−005
52
hsa-miR-34a


1.1e+003-3.5e+002
 3.04 (+)
1.0e−003
25
hsa-miR-193a-3p


1.6e+002-8.2e+002
 5.17 (−)
1.2e−004
229
hsa-miR-9


4.6e+002-2.3e+003
 5.09 (−)
7.1e−003
161
hsa-miR-128


4.1e+002-1.8e+003
 4.43 (−)
1.3e−002
187
hsa-miR-219-2-3p


3.8e+003-1.3e+004
 3.31 (−)
1.9e−002
179
hsa-miR-195





+ the higher expression of this miR is in astrocytic tumors


− the higher expression of this miR is in oligodendrogliomas






A combination of the expression level of any of the miRs detailed in table 9 with the expression level of hsa-miR-497 (SEQ ID NO: 208) or hsa-let-7d (SEQ ID NO: 153) also provides for classification of brain tumors as astrocytic tumors or oligodendrogliomas. This is demonstrated at node 7 of the binary-tree-classifier detailed in the invention with hsa-miR-222 (SEQ ID NO: 40) and hsa-miR-497 (SEQ ID NO: 208). In another embodiment of the invention, the expression levels of hsa-miR-222 (SEQ ID NO: 40) and hsa-let-7d (SEQ ID NO: 153) are combined to distinguish between astrocytic tumors and oligodendrogliomas.
FIG. 7 demonstrates that tumors originating in astrocytoma (marked by diamonds) are easily distinguished from tumors of oligodendroglioma origins (marked by squares) using the expression levels of hsa-miR-497 (SEQ ID NO: 208, y-axis) and hsa-miR-222 (SEQ ID NO: 40, x-axis).







TABLE 10







miR expression (in florescence units) distinguishing 


between the group consisting of neuroendocrine tumors 


and the group consisting of all non-neuroendocrine, 


epithelial tumors












fold-

SEQ ID



median values
change
p-value
NO.
miR name














3.8e+004-1.5e+002
259.47 (+)
5.3e−086
56
hsa-miR-375


3.6e+003-5.2e+001
 70.47 (+)
4.4e−145
65
hsa-miR-7


1.3e+003-1.8e+002
 6.89 (+)
4.7e−044
175
hsa-miR-183


1.9e+003-4.4e+002
 4.42 (+)
3.5e−025
152
hsa-miR-182


1.2e+003-3.0e+002
 4.16 (+)
5.5e−028
155
hsa-miR-127-3p


5.6e+001-7.0e+003
124.66 (−)
1.4e−023
32
hsa-miR-205


1.5e+002-1.4e+003
 9.25 (−)
1.8e−019
49
hsa-miR-31


3.4e+002-1.4e+003
 4.12 (−)
9.5e−032
35
hsa-miR-21*





+ the higher expression of this miR is in the group consisting of neuroendocrine tumors


− the higher expression of this miR is in the group consisting of all non-neuroendocrine, epithelial tumors






hsa-miR-375 (SEQ ID NO: 56), hsa-miR-7 (SEQ ID NO: 65) and hsa-miR-193a-3p (SEQ ID NO: 25) are used at node 8 of the binary-tree-classifier detailed in the invention to distinguish between the group consisting of neuroendocrine tumors and the group consisting of all non-neuroendocrine, epithelial tumors. FIG. 8 demonstrates that tumors originating in the neuroendocrine (diamonds) are easily distinguished from tumors of epithelial, origin (squares) using the expression levels of hsa-miR-193a-3p (SEQ ID NO: 25, y-axis), hsa-miR-7 (SEQ ID NO: 65, x-axis) and hsa-miR-375 (SEQ ID NO: 56, z-axis).







TABLE 11







miR expression (in florescence units) distinguishing between 


the group consisting of gastrointestinal (GI) epithelial


tumors and the group consisting of non-GI epithelial tumors












fold-

SEQ ID



median values
change
p-value
NO. 
miR name














2.6e+003-7.1e+001
 36.09 (+)
2.5e−127
27
hsa-miR-194


3.9e+003-1.2e+002
 33.26 (+)
1.6e−117
177
hsa-miR-192


2.6e+003-6.7e+002
 3.88 (+)
3.3e−021
4
hsa-miR-10a


5.0e+001-2.1e+004
411.76 (−)
6.5e−045
32
hsa-miR-205





+ the higher expression of this miR is in the group consisting of GI epithelial tumors


− the higher expression of this miR is in the group consisting of non-GI epithelial tumors






hsa-miR-194 (SEQ ID NO: 27) and hsa-miR-21* (SEQ ID NO: 35) are used at node 9 of the binary-tree-classifier detailed in the invention to distinguish between GI epithelial tumors and non-GI epithelial tumors.
FIG. 9 demonstrates that tumors originating in gastro-intestinal (GI) (marked by diamonds) are easily distinguished from tumors of non GI origins (marked by squares) using the expression levels of hsa-miR-21* (SEQ ID NO: 35, y-axis) and hsa-miR-194 (SEQ ID NO: 27, x-axis).







TABLE 12







miR expression (in florescence units) distinguishing between 


prostate tumors and all other non-GI epithelial tumors












fold-

SEQ



median values
change
p-value
ID NO.
miR name














5.1e+003-5.2e+001
96.76 (+)
3.7e−016
56
hsa-miR-375


1.0e+003-5.5e+001
18.27 (+)
4.0e−025
199
hsa-miR-363


6.8e+004-7.2e+003
 9.41 (+)
1.0e−025
14
hsa-miR-143


1.2e+005-1.4e+004
 8.14 (+)
7.8e−022
15
hsa-miR-145


2.8e+003-3.5e+002
 7.89 (+)
1.5e−012
165
hsa-miR-143*


2.1e+004-4.4e+003
 4.76 (+)
2.2e−011
231
hsa-miR-99a


4.6e+002-2.1e+003
 4.58 (−)
8.0e−007
36
hsa-miR-210


2.7e+002-1.1e+003
 3.84 (−)
7.8e−017
154
hsa-miR-181b


1.2e+003-4.3e+003
 3.76 (−)
1.2e−014
21
hsa-miR-181a


5.5e+002-2.0e+003
 3.63 (−)
2.3e−002
49
hsa-miR-31





+ the higher expression of this miR is in prostate tumors


− the higher expression of this miR is in the group consisting of all other non-GI epithelial tumors






hsa-miR-143 (SEQ ID NO: 14) and hsa-miR-181a (SEQ ID NO: 21) are used at node 10 of the binary-tree-classifier detailed in the invention to distinguish between prostate tumors and all other non-GI epithelial tumors.
FIG. 10 demonstrates that tumors originating in prostate adenocarcinoma (marked by diamonds) are easily distinguished from tumors of non prostate origins (marked by squares) using the expression levels of hsa-miR-181a (SEQ ID NO: 21, y-axis) and hsa-miR-143 (SEQ ID NO: 14, x-axis).







TABLE 13







miR expression (in florescence units) distinguishing between


seminiomatous and non-seminiomatous testicular tumors












fold-

SEQ ID



median values
change
p-value
NO.
miR name














4.3e+003-7.6e+002
 5.63 (+)
6.6e−004
152
hsa-miR-182


1.0e+002-2.1e+003
20.46 (−)
6.2e−005
216
hsa-miR-518e


7.8e+001-1.2e+003
15.29 (−)
4.5e−005
212
hsa-miR-516b


6.8e+001-8.2e+002
11.94 (−)
2.2e−005
224
hsa-miR-527


2.1e+002-2.2e+003
10.40 (−)
1.9e−006
13
hsa-miR-141


5.3e+002-5.0e+003
 9.48 (−)
5.0e−004
194
hsa-miR-302d


1.4e+002-1.3e+003
 8.97 (−)
4.1e−006
192
hsa-miR-302a


2.7e+002-2.3e+003
 8.78 (−)
2.9e−003
221
hsa-miR-520c-3p


1.3e+002-1.2e+003
 8.65 (−)
8.3e−004
217
hsa-miR-518f*


3.4e+003-2.9e+004
 5.98 (−)
2.6e−007
205
hsa-miR-451


2.8e+002-1.7e+003
 5.98 (−)
1.1e−002
219
hsa-miR-519d


2.0e+002-1.2e+003
 5.90 (−)
6.8e−005
32
hsa-miR-205


2.0e+002-1.1e+003
 5.59 (−)
5.8e−006
193
hsa-miR-302a*


1.9e+002-1.0e+003
 5.27 (−)
6.7e−003
223
hsa-miR-524-5p


1.5e+002-8.0e+002
 5.22 (−)
5.4e−003
220
hsa-miR-520a-5p


2.2e+002-1.1e+003
 5.21 (−)
4.1e−003
210
hsa-miR-512-5p


3.2e+002-1.4e+003
 4.57 (−)
9.2e−003
209
hsa-miR-498


7.2e+002-3.2e+003
 4.51 (−)
3.1e−002
213
hsa-miR-517a


6.4e+002-2.9e+003
 4.47 (−)
2.9e−002
163
hsa-miR-1323


9.5e+002-4.1e+003
 4.29 (−)
1.3e−004
30
hsa-miR-200c





+ the higher expression of this miR is in seminioma tumors


− the higher expression of this miR is in non-seminioma tumors






A combination of the expression level of any of the miRs detailed in table 13 with the expression level of hsa-miR-200b (SEQ ID NO: 29), hsa-miR-200a (SEQ ID NO: 28), hsa-miR-516a-5p (SEQ ID NO: 211), hsa-miR-767-5p (SEQ ID NO: 227), hsa-miR-518a-3p (SEQ ID NO: 215), hsa-miR-520d-5p (SEQ ID NO: 222), hsa-miR-519a (SEQ ID NO: 218) and hsa-miR-517c (SEQ ID NO: 214) also provides for classification of seminoma and non-seminoma testis-tumors.
hsa-miR-516a-5p (SEQ ID NO: 211) and hsa-miR-200b (SEQ ID NO: 29) are used at node 12 of the binary-tree-classifier detailed in the invention to distinguish between seminoma and non-seminoma testis-tumors.
FIG. 11 demonstrates that tumors originating in seminiomatous testicular germ cell (marked by diamonds) are easily distinguished from tumors of non seminiomatous origins (marked by squares) using the expression levels of hsa-miR-516a-5p (SEQ ID NO: 211, y-axis) and hsa-miR-200b (SEQ ID NO: 29, x-axis).







TABLE 14







miR expression (in florescence units) distinguishing 


between the group consisting of squamous 


cell carcinoma (SCC), transitional cell carcinoma 


(TCC), thymoma and the group consisting of 


non gastrointestinal (GI) adenocarcinoma tumors












fold-

SEQ



median values
change
p-value
ID NO.
miR name














4.6e+004-1.4e+002
321.76 (+)
1.6e−059
32
hsa-miR-205


2.9e+003-4.9e+002
 5.96 (+)
8.6e−015
36
hsa-miR-210


2.5e+003-6.6e+002
 3.82 (+)
2.6e−016
178
hsa-miR-193b


2.5e+003-6.8e+002
 3.67 (+)
1.8e−008
243
MID-16869


3.4e+003-9.7e+002
 3.53 (+)
2.2e−011
242
MID-16489


3.7e+003-1.3e+003
 2.82 (+)
8.2e−004
49
hsa-miR-31


6.4e+003-2.3e+003
 2.78 (+)
1.7e−010
240
MID-15965


1.4e+003-5.2e+002
 2.71 (+)
2.7e−017
57
hsa-miR-378


2.8e+002-2.2e+003
 8.05 (−)
2.9e−023
11
hsa-miR-138


7.3e+002-2.7e+003
 3.70 (−)
1.5e−018
46
hsa-miR-30a


8.6e+002-2.2e+003
 2.60 (−)
4.0e−013
17
hsa-miR-146b-5p


1.8e+003-4.3e+003
 2.44 (−)
1.5e−021
47
hsa-miR-30d


4.5e+002-1.1e+003
 2.38 (−)
2.6e−019
51
hsa-miR-345


4.2e+003-9.6e+003
 2.30 (−)
3.8e−014
7
hsa-miR-125a-5p


1.9e+004-4.3e+004
 2.26 (−)
3.0e−009
8
hsa-miR-125b


9.4e+002-2.1e+003
 2.24 (−)
3.2e−008
154
hsa-miR-181b


7.4e+002-1.6e+003
 2.13 (−)
3.3e−010
190
hsa-miR-29b


6.6e+003-1.3e+004
 2.04 (−)
4.6e−014
157
hsa-let-7i


2.4e+003-4.8e+003
 2.04 (−)
7.9e−010
196
hsa-miR-30c





+ the higher expression of this miR is in SCC, TCC and thymoma


− the higher expression of this miR is in non GI adenocarcinoma






Node 13 of the binary-tree-classifier separates tissues with high expression of miR-205 (SCC marker) such as SCC, TCC and thymomas from adenocarcinomas.
Breast adenocarcinoma and ovarian carcinoma are excluded from this separation due to a wide range of expression of miR-205.
A combination of the expression level of any of the miRs detailed in table 14 with the expression level of hsa-miR-331-3p (SEQ ID NO: 197) also provides for this classification.
hsa-miR-205 (SEQ ID NO: 32), hsa-miR-345 (SEQ ID NO: 51) and hsa-miR-125a-5p (SEQ ID NO: 7) are used at node 13 of the binary-tree-classifier detailed in the invention.







TABLE 15







miR expression (in florescence units) distinguishing 


between the group consisting of breast adenocarcinoma 


and the group consisting of SCC, TCC, thymomas and 


ovarian carcinoma












fold-

SEQ ID



median values
change
p-value
NO.
miR name














1.3e+003-5.0e+001
25.95 (+)
4.1e−029
56
hsa-miR-375


2.7e+003-8.3e+002
 3.25 (+)
1.6e−014
46
hsa-miR-30a


4.4e+003-1.4e+003
 3.09 (+)
9.5e−022
25
hsa-miR-193a-3p


1.2e+003-4.1e+002
 2.94 (+)
2.1e−009
152
hsa-miR-182


5.5e+003-2.2e+003
 2.48 (+)
7.3e−014
50
hsa-miR-342-3p


6.6e+002-2.7e+002
 2.48 (+)
6.3e−008
45
hsa-miR-29c*


5.0e+002-2.2e+002
 2.26 (+)
5.8e−007
191
hsa-miR-29c


7.3e+003-3.3e+003
 2.19 (+)
1.3e−004
181
hsa-miR-199a-3p


2.2e+003-1.1e+003
 2.05 (+)
2.0e−006
179
hsa-miR-195


2.2e+002-3.1e+003
13.81 (−)
9.6e−014
49
hsa-miR-31


6.3e+003-4.0e+004
 6.32 (−)
9.3e−008
32
hsa-miR-205


8.9e+001-5.1e+002
 5.72 (−)
5.3e−010
42
hsa-miR-224


1.5e+002-6.3e+002
 4.05 (−)
6.7e−007
184
hsa-miR-203


5.7e+003-1.5e+004
 2.64 (−)
5.8e−018
40
hsa-miR-222


3.8e+003-9.2e+003
 2.41 (−)
1.0e−018
147
hsa-miR-221


4.8e+002-1.1e+003
 2.39 (−)
4.9e−010
236
MID-00689


6.0e+002-1.4e+003
 2.37 (−)
1.2e−010
57
hsa-miR-378


2.3e+002-5.2e+002
 2.24 (−)
2.2e−008
203
hsa-miR-422a


1.2e+003-2.6e+003
 2.22 (−)
1.5e−007
36
hsa-miR-210





+ the higher expression of this miR is in breast adenocarcinoma


− the higher expression of this miR is in SCC, TCC, thymomas and ovarian carcinoma






hsa-miR-193a-3p (SEQ ID NO: 25), hsa-miR-375 (SEQ ID NO: 56) and hsa-miR-342-3p (SEQ ID NO: 50) are used at node 14 of the binary-tree-classifier detailed in the invention. According to another embodiment, hsa-miR-193a-3p (SEQ ID NO: 25), hsa-miR-375 (SEQ ID NO: 56) and hsa-miR-224 (SEQ ID NO: 42) may be used at node 14 of the binary-tree-classifier detailed in the invention.







TABLE 16







miR expression (in florescence units) distinguishing 


between the group consisting of ovarian carcinoma 


and the group consisting of SCC, TCC and thymomas












fold-

SEQ ID



median values 
change
p-value
NO.
miR name














3.1e+003-5.5e+002
 5.57 (+)
1.2e−012
4
hsa-miR-10a


5.1e+003-1.5e+003
 3.41 (+)
1.7e−014
10
hsa-miR-130a


2.5e+002-7.5e+001
 3.39 (+)
1.3e−014
195
hsa-miR-30a*


2.4e+003-8.8e+002
 2.68 (+)
5.5e−009
5
hsa-miR-10b


2.9e+002-1.2e+002
 2.48 (+)
2.5e−012
226
hsa-miR-625


8.8e+003-3.9e+003
 2.28 (+)
7.7e−012
2
hsa-let-7e


1.6e+003-7.3e+002
 2.20 (+)
3.6e−007
46
hsa-miR-30a


1.2e+003-4.6e+004
37.52 (−)
1.0e−033
32
hsa-miR-205


5.0e+001-2.7e+002
 5.42 (−)
4.2e−018
185
hsa-miR-205*


6.0e+001 2.8e+002
 4.63 (−)
1.1e−009
11
hsa-miR-138


5.7e+002-2.4e+003
 4.18 (−)
5.2e−010
168
hsa-miR-150


2.9e+002-8.0e+002
 2.74 (−)
2.5e−003
184
hsa-miR-203


2.9e+002-7.6e+002
 2.62 (−)
2.1e−006
16
hsa-miR-146a


1.4e+003-3.4e+003
 2.49 (−)
1.9e−007
242
MID-16489


9.5e+002-2.3e+003
 2.42 (−)
2.3e−015
12
hsa-miR-140-3p


7.6e+002-1.8e+003
 2.37 (−)
5.2e−006
237
MID-15684


1.1e+003-2.5e+003
 2.23 (−)
9.8e−005
243
MID-16869


2.1e+003-4.6e+003
 2.18 (−)
5.9e−004
250
MID-20703


2.7e+003-5.8e+003
 2.13 (−)
6.5e−012
39
hsa-miR-22


4.3e+002-9.2e+002
 2.13 (−)
1.0e−003
253
MID-23256


1.7e+003-3.7e+003
 2.12 (−)
1.4e−002
49
hsa-miR-31


1.7e+003-3.6e+003
 2.06 (−)
1.3e−003
246
MID-18422


2.1e+003-4.4e+003
 2.04 (−)
1.4e−007
167
hsa-miR-149*





+ the higher expression of this miR is in ovarian carcinoma


− the higher expression of this miR is in SCC, TCC and thymomas






hsa-miR-205 (SEQ ID NO: 32), hsa-miR-10a (SEQ ID NO: 4) and hsa-miR-22 (SEQ ID NO: 39) are used at node 15 of the binary-tree-classifier detailed in the invention.







TABLE 17







miR expression (in florescence units) distinguishing between 


the group consisting of thyroid carcinoma (follicular


and papillary) and the group consisting of breast adenocarcinoma, lung


large cellcarcinoma, lung adenocarcinoma and ovarian carcinoma












fold-

SEQ ID



median values
change
p-value
NO.
miR name














4.1e+003-1.2e+002
33.86 (+)
7.4e−033
11
hsa-miR-138


3.4e+004-6.7e+003
 5.03 (+)
1.4e−009
147
hsa-miR-221


4.9e+003-1.0e+003
 4.74 (+)
1.0e−006
17
hsa-miR-146b-5p


2.2e+004-5.8e+003
 3.71 (+)
2.8e−027
157
hsa-let-7i


3.9e+004-1.1e+004
 3.63 (+)
7.9e−009
40
hsa-miR-222


5.4e+004-1.9e+004
 2.78 (+)
1.5e−014
8
hsa-miR-125b


1.3e+003-4.9e+002
 2.78 (+)
5.0e−003
49
hsa-miR-31


6.9e+003-2.8e+003
 2.48 (+)
1.3e−008
9
hsa-miR-126


8.1e+002-3.4e+002
 2.36 (+)
4.8e−007
191
hsa-miR-29c


1.3e+004-5.7e+003
 2.33 (+)
3.8e−003
205
hsa-miR-451


4.8e+002-2.2e+002
 2.16 (+)
1.3e−003
207
hsa-miR-486-5p


4.9e+002-2.3e+002
 2.12 (+)
8.0e−006
195
hsa-miR-30a*


1.4e+003-6.6e+002
 2.11 (+)
2.7e−011
51
hsa-miR-345


 .5e+003-1.7e+003
 2.10 (+)
1.5e−006
46
hsa-miR-30a


6.4e+002-3.2e+002
 1.97 (+)
1.5e−006
45
hsa-miR-29c*


7.4e+003-4.0e+003
 1.88 (+) 
3.4e−007 
52
hsa-miR-34a


6.1e+003-3.3e+003
 1.88 (+)
5.9e−008 
234
hsa-miR-1977


7.5e+003-4.1e+003
 1.85 (+) 
1.5e−005 
231
hsa-miR-99a


7.6e+003-4.1e+003 
 1.85 (+)
2.7e−004 
21
hsa-miR-181a


1.0e+003-5.5e+002
 1.82 (+) 
5.1e−008 
169
hsa-miR-152


1.1e+004-6.0e+003
 1.79 (+) 
3.7e−008 
43
hsa-miR-29a


5.4e+003-3.0e+003
 1.77 (+)
2.9e−005
3
hsa-miR-100


5.9e+003-3.4e+003
 1.73 (+)
7.3e−007 
196
hsa-miR-30c


2.1e+003-1.2e+003
 1.69 (+) 
4.6e−004 
154
hsa-miR-181b


4.4e+002-2.6e+002
 1.66 (+)
6.4e−003 
390
MID-00405


5.5e+002-3.3e+002
 1.65 (+) 
5.3e−006 
171
hsa-miR-15a


1.3e+003-8.1e+002
 1.60 (+) 
6.0e−003 
255
MID-23794


2.3e+003-1.4e+003
 1.57 (+) 
8.3e−007 
197
hsa-miR-331-3p


1.8e+003-1.1e+003
 1.57 (+) 
1.4e−005 
190
hsa-miR-29b


3.6e+003-2.3e+003
 1.56 (+)
1.4e−003 
189
hsa-miR-27b


6.6e+003-4.3e+003
 1.52 (+)
3.6e−005 
39
hsa-miR-22


9.9e+003-6.5e+003
 1.52 (+)
2.2e−006 
7
hsa-miR-125a-5p


7.8e+002-5.2e+002
 1.51 (+) 
6.2e−006 
48
hsa-miR-30e


4.3e+003-2.8e+003
 1.51 (+) 
1.2e−002 
47
hsa-miR-30d


1.0e+002-2.2e+003
22.05 (−)
2.2e−005 
32
hsa-miR-205


2.1e+002-1.8e+003
 8.83 (−)
4.8e−020 
36
hsa-miR-210


3.2e+002-1.4e+003
 4.34 (−)
8.7e−011
4
hsa-miR-10a


4.9e+002-1.8e+003
 3.59 (−) 
4.6e−014 
178
hsa-miR-193b


1.0e+003-2.8e+003
 2.74 (−) 
8.3e−006
37
hsa-miR-214


2.0e+003-5.5e+003
 2.67 (−)
4.4e−005 
181
hsa-miR-199a-3p


1.1e+003-2.8e+003
 2.65 (−)
3.1e−011 
25
hsa-miR-193a-3p


2.7e+002-7.2e+002
 2.63 (−) 
1.3e−004 
183
hsa-miR-199b-5p


3.1e+003-8.1e+003
 2.57 (−) 
1.6e−005 
182
hsa-miR-199a-5p


5.1e+002-1.2e+003
 2.41 (−) 
4.7e−006 
35
hsa-miR-21*


2.0e+003-4.8e+003
 2.39 (−) 
9.3e−003 
240
MID-15965


3.4e+002-8.0e+002
 2.35 (−) 
3.0e−006 
57
hsa-miR-378


8.2e+002-1.9e+003
 2.25 (−) 
1.2e−003 
242
MID-16489


6.0e+002-1.3e+003
 2.18 (−)
1.4e−011 
204
hsa-miR-425


3.0e+002-6.1e+002
 2.06 (−)
5.3e−006 
236
MID-00689


2.3e+002-4.5e+002
 1.96 (−)
2.0e−006 
176
hsa-miR-18a


2.3e+003-4.3e+003
 1.85 (−)
3.5e−006 
158
hsa-miR-106a


2.4e+003-4.4e+003
 1.79 (−) 
1.9e−010 
148
hsa-miR-93


2.9e+002-5.2e+002
 1.79 (−) 
1.9e−007 
206
hsa-miR-455-3p


1.3e+003-2.3e+003
 1.78 (−) 
7.8e−005 
50
hsa-miR-342-3p


1.4e+003-2.5e+003
 1.75 (−) 
6.0e−006 
20
hsa-miR-17


2.8e+004-4.8e+004
 1.75 (−)
5.1e−006 
34
hsa-miR-21


1.4e+003-2.4e+003
 1.74 (−)
1.1e−004 
186
hsa-miR-20a


2.4e+002-4.1e+002
 1.72 (−)
8.4e−004 
239
MID-15907


3.0e+002-4.8e+002
 1.62 (−)
9.9e−003 
251
MID-21271


2.2e+003-3.6e+003
 1.60 (−)
4.3e−002 
244
MID-17144


3.8e+003-6.1e+003
 1.59 (−)
4.7e−006 
24
hsa-miR-191


1.0e+003-1.6e+003
 1.59 (−) 
2.0e−005 
188
hsa-miR-25


2.1e+003-3.2e+003
 1.57 (−)
1.9e−002
172
hsa-miR-15b


2.6e+003-4.0e+003
 1.56 (−)
9.5e−003
238
MID-15867





+ the higher expression of this miR is in thyroid carcinoma


− the higher expression of this miR is in breast adenocarcinoma, lung large cell carcinoma, lung adenocarcinoma and ovarian carcinoma






FIG. 12 demonstrates binary decisions at node #16 of the decision-tree. Tumors originating in thyroid carcinoma (diamonds) are easily distinguished from tumors of adenocarcinoma of the lung, breast and ovarian origin (squares) using the expression levels of hsa-miR-93 (SEQ ID NO: 148, y-axis), hsa-miR-138 (SEQ ID NO: 11, x-axis) and hsa-miR-10a (SEQ ID NO: 4, z-axis).







TABLE 18







miR expression (in florescence units) distinguishing between 


follicular thyroid carcinoma and papillary thyroid carcinoma












fold-

SEQ ID



median values
change
p-value
NO.
miR name














6.6e+003-7.1e+002
 9.34 (+)
4.5e−011
249
MID-20524


1.7e+003-2.2e+002
 7.80 (+)
1.9e−008
180
hsa-miR-1973


4.5e+002-5.9e+001
 7.58 (+)
8.3e−005
65
hsa-miR-7


2.5e+003-3.8e+002
 6.52 (+)
4.8e−007
235
hsa-miR-1978


2.2e+003-3.6e+002
 6.14 (+)
1.5e−008
241
MID-16318


4.2e+002-7.1e+001
 6.00 (+)
3.0e−004
248
MID-19533


9.6e+002-1.7e+002
 5.76 (+)
1.6e−008
254
MID-23291


9.9e+002-1.9e+002
 5.33 (+)
3.2e−005
247
MID-19340


6.4e+002-1.2e+002
 5.17 (+)
6.8e−009
160
hsa-miR-1248


1.5e+003-3.0e+002
 4.97 (+)
1.1e−006
243
MID-16869


2.7e+003-6.1e+002
 4.48 (+)
1.4e−010
245
MID-18336


5.0e+002-1.2e+002
 4.00 (+)
7.0e−004
252
MID-22664


4.0e+002-2.5e+004
62.88 (−)
6.7e−011
17
hsa-miR-146b-5p


4.4e+002-8.2e+003
18.72 (−)
2.5e−008
49
hsa-miR-31


5.0e+001-9.3e+002
18.69 (−)
5.0e−012
166
hsa-miR-146b-3p


7.6e+001-8.3e+002
10.86 (−)
4.8e−006
225
hsa-miR-551b


3.1e+002-3.3e+003
10.71 (−)
3.2e−007
168
hsa-miR-150


1.1e+004-4.7e+004
 4.40 (−)
3.4e−007
34
hsa-miR-21





+ the higher expression of this miR is in follicular thyroid carcinoma


− the higher expression of this miR is in papillary thyroid carcinoma






FIG. 13 demonstrates binary decisions at node #17 of the decision-tree. Tumors originating in follicular thyroid carcinoma (marked by diamonds) are easily distinguished from tumors of papillary thyroid carcinoma origins (marked by squares) using the expression levels of hsa-miR-21 (SEQ ID NO: 34, y-axis) and hsa-miR-146b-5p (SEQ ID NO: 17, x-axis).







TABLE 19







miR expression (in florescence units) distinguishing between the group


consisting of breast adenocarcinoma and the group consisting of


lung adenocarcinoma and ovarian carcinoma












fold-

SEQ



median values
change
p-value
ID NO.
miR name














6.3e+003 − 6.0e+002
10.55 (+)
8.8e−005
32
hsa-miR-205


1.3e+003 − 1.5e+002
 8.43 (+)
7.9e−006
56
hsa-miR-375


5.5e+003 − 1.7e+003
 3.17 (+)
7.7e−012
50
hsa-miR-342-3p


6.6e+002 − 2.6e+002
 2.52 (+)
2.2e−008
45
hsa-miR-29c*


4.4e+003 − 2.0e+003
 2.23 (+)
2.2e−012
25
hsa-miR-193a-3p


9.5e+002 − 4.3e+002
 2.20 (+)
7.9e−005
253
MID-23256


1.2e+003 − 5.6e+002
 2.15 (+)
4.9e−005
152
hsa-miR-182


3.7e+003 − 1.9e+003
 1.94 (+)
5.3e−005
9
hsa-miR-126


2.7e+003 − 1.4e+003
 1.90 (+)
2.9e−004
46
hsa-miR-30a


5.0e+002 − 2.8e+002
 1.78 (+)
1.1e−004
191
hsa-miR-29c


2.4e+003 − 1.4e+003
 1.72 (+)
1.5e−006
178
hsa-miR-193b


2.2e+002 − 1.3e+003
 5.79 (−)
7.7e−006
49
hsa-miR-31


5.7e+003 − 1.5e+004
 2.69 (−)
3.0e−010
40
hsa-miR-222


1.4e+003 − 3.4e+003
 2.50 (−)
1.6e−006
10
hsa-miR-130a


3.8e+003 − 9.3e+003
 2.41 (−)
1.2e−009
147
hsa-miR-221


9.3e+002 − 2.2e+003
 2.33 (−)
2.2e−002
4
hsa-miR-10a


1.2e+003 − 2.5e+003
 2.09 (−)
1.5e−005
36
hsa-miR-210


1.3e+003 − 2.6e+003
 2.01 (−)
9.9e−005
228
hsa-miR-886-3p


4.8e+002 − 9.4e+002
 1.95 (−)
4.6e−004
236
MID-00689


5.3e+002 − 1.0e+003
 1.92 (−)
6.3e−004
230
hsa-miR-886-5p


1.8e+003 − 3.3e+003
 1.86 (−)
7.1e−005
189
hsa-miR-27b


3.6e+003 − 6.4e+003
 1.79 (−)
6.1e−003
240
MID-15965


2.7e+003 − 4.7e+003
 1.75 (−)
3.5e−005
67
hsa-miR-92a


6.0e+002 − 1.0e+003
 1.73 (−)
3.0e−004
202
hsa-miR-378


8.0e+002 − 1.4e+003
 1.71 (−)
2.9e−004
17
hsa-miR-146b-5p





+ the higher expression of this miR is in breast adenocarcinoma


− the higher expression of this miR is in lung adenocarcinoma and ovarian carcinoma






FIG. 14 demonstrates binary decisions at node #18 of the decision-tree. Tumors originating in breast (diamonds) are easily distinguished from tumors of lung and ovarian origin (squares) using the expression levels of hsa-miR-92a (SEQ ID NO: 67, y-axis), hsa-miR-193a-3p (SEQ ID NO: 25, x-axis) and hsa-miR-31 (SEQ ID NO: 49, z-axis).







TABLE 20







miR expression (inflorescence units) distinguishing between lung


adenocarcinoma and ovarian carcinoma












fold-

SEQ ID



median values
change
p-value
NO.
miR name














1.4e+003 − 5.2e+001
27.96 (+)
3.5e−008
56
hsa-miR-375


5.5e+002 − 6.0e+001
 9.19 (+)
8.1e−009
11
hsa-miR-138


3.2e+003 − 5.7e+002
 5.65 (+)
5.6e−004
168
hsa-miR-150


9.7e+002 − 2.9e+002
 3.35 (+)
2.2e−004
16
hsa-miR-146a


2.3e+003 − 7.6e+002
 2.96 (+)
3.2e−003
237
MID-15684


8.4e+003 − 2.9e+003
 2.88 (+)
1.7e−010
21
hsa-miR-181a


7.0e+003 − 2.6e+003
 2.69 (+)
9.2e−008
52
hsa-miR-34a


2.4e+003 − 9.5e+002
 2.58 (+)
3.2e−007
12
hsa-miR-140-3p


2.1e+003 − 8.8e+002
 2.39 (+)
2.1e−007
154
hsa-miR-18 lb


1.4e+005 − 6.3e+004
 2.28 (+)
1.9e−003
279
hsa-miR-1826


3.2e+003 − 1.4e+003
 2.25 (+)
6.1e−003
9
hsa-miR-126


6.1e+003 − 2.7e+003
 2.24 (+)
2.3e−006
39
hsa-miR-22


4.2e+003 − 2.2e+003
 1.93 (+)
8.9e−005
47
hsa-miR-30d


1.9e+003 − 1.0e+003
 1.90 (+)
3.5e−006
23
hsa-miR-185


2.8e+003 − 1.5e+003
 1.88 (+)
1.4e−003
50
hsa-miR-342-3p


3.6e+003 − 2.1e+003
 1.69 (+)
1.8e−002
167
hsa-miR-149*


9.7e+002 − 5.9e+002
 1.66 (+)
8.7e−005
383
MID-22912


6.8e+004 − 4.2e+004
 1.64 (+)
5.4e−004
34
hsa-miR-21


1.8e+003 − 1.2e+003
 1.55 (+)
5.1e−004
35
hsa-miR-21*


1.4e+003 − 8.9e+002
 1.54 (+)
1.7e−004
388
hsa-miR-423-5p


4.4e+002 − 2.4e+003
 5.38 (−)
1.0e−007
5
hsa-miR-10b


1.9e+002 − 7.2e+002
 3.77 (−)
3.3e−006
359
hsa-miR-708


6.4e+002 − 2.1e+003
 3.27 (−)
5.8e−003
245
MID-18336


1.8e+002 − 5.4e+002
 2.96 (−)
6.6e−003
254
MID-23291


1.7e+003 − 5.1e+003
 2.95 (−)
3.4e−005
10
hsa-miR-130a


2.8e+003 − 8.1e+003
 2.86 (−)
3.7e−003
240
MID-15965


5.1e+002 − 1.3e+003
 2.65 (−)
3.7e−004
236
MID-00689


6.1e+002 − 1.6e+003
 2.63 (−)
1.8e−004
202
hsa-miR-378


1.3e+003 − 3.1e+003
 2.39 (−)
1.2e−002
4
hsa-miR-10a


1.0e+003 − 2.3e+003
 2.30 (−)
1.8e−006
25
hsa-miR-193a-3p


2.6e+002 − 5.6e+002
 2.15 (−)
4.1e−004
203
hsa-miR-422a


3.0e+003 − 6.1e+003
 2.04 (−)
1.8e−002
231
hsa-miR-99a


2.0e+003 − 3.9e+003
 2.01 (−)
3.5e−005
20
hsa-miR-17


3.3e+003 − 6.4e+003
 1.96 (−)
3.5e−005
158
hsa-miR-106a


1.8e+003 − 3.5e+003
 1.88 (−)
3.1e−005
186
hsa-miR-20a


3.2e+003 − 5.9e+003
 1.85 (−)
1.2e−004
258
hsa-let-7f


2.4e+003 − 4.4e+003
 1.85 (−)
2.0e−003
244
MID-17144


2.0e+003 − 3.5e+003
 1.72 (−)
8.7e−004
172
hsa-miR-15b


5.2e+003 − 8.8e+003
 1.69 (−)
5.3e−004
2
hsa-let-7e


4.1e+002 − 6.8e+002
 1.66 (−)
1.2e−002
235
hsa-miR-1978


3.3e+004 − 5.4e+004
 1.66 (−)
3.3e−005
256
hsa-let-7a


2.8e+003 − 4.7e+003
 1.65 (−)
3.5e−003
28
hsa-miR-200a


5.2e+002 − 8.5e+002
 1.62 (−)
1.6e−004
277
hsa-miR-17*


3.7e+002 − 5.8e+002
 1.57 (−)
1.9e−003
296
hsa-miR-26b


1.0e+005 − 1.6e+005
 1.56 (−)
3.7e−002
374
MID-16748


6.0e+003 − 9.1e+003
 1.53 (−)
2.1e−005
153
hsa-let-7d


3.8e+003 − 5.7e+003
 1.50 (−)
4.1e−003
181
hsa-miR-199a-3p





+ the higher expression of this miR is in lung adenocarcinoma


− the higher expression of this miR is in ovarian carcinoma






FIG. 15 demonstrates binary decisions at node #19 of the decision-tree. Tumors originating in lung adenocarcinoma (diamonds) are easily distinguished from tumors of ovarian carcinoma origin (squares) using the expression levels of hsa-miR-21 (SEQ ID NO: 34, y-axis), hsa-miR-378 (SEQ ID NO: 202, x-axis) and hsa-miR-138 (SEQ ID NO: 11, z-axis).







TABLE 21







miR expression (in florescence units) distinguishing between


the group consisting of thymic carcinoma and the group


consisting of TCC and SCC












fold-

SEQ ID



median values
change
p-value
NO
miR name














5.3e+002 − 5.9e+001
 9.00 (+)
5.7e−026
161
hsa-miR-128


7.4e+002 − 9.2e+001
 8.04 (+)
2.2e−007
164
hsa-miR-142-5p


6.8e+002 − 8.8e+001
 7.82 (+)
2.6e−021
22
hsa-miR-181a*


7.1e+002 − 1.2e+002
 6.09 (+)
1.2e−006
53
hsa-miR-34c-5p


9.1e+002 − 1.8e+002
 5.06 (+)
2.2e−008
285
hsa-miR-20b


1.3e+004 − 2.8e+003
 4.59 (+)
7.5e−014
3
hsa-miR-100


1.6e+003 − 3.6e+002
 4.39 (+)
7.1e−007
152
hsa-miR-182


8.7e+002 − 2.0e+002
 4.37 (+)
6.4e−010
191
hsa-miR-29c


3.7e+003 − 9.1e+002
 4.09 (+)
2.6e−014
154
hsa-miR-181b


1.5e+004 − 3.8e+003
 3.82 (+)
4.8e−009
21
hsa-miR-181a


2.1e+003 − 6.4e+002
 3.25 (+)
6.4e−006
206
hsa-miR-455-3p


8.7e+002 − 2.7e+002
 3.23 (+)
1.5e−010
174
hsa-miR-181d


9.4e+002 − 2.9e+002
 3.23 (+)
1.9e−004
19
hsa-miR-149


7.3e+002 − 2.6e+002
 2.80 (+)
2.5e−008
45
hsa-miR-29c*


8.7e+002 − 3.2e+002
 2.69 (+)
2.2e−007
171
hsa-miR-15a


2.7e+003 − 1.0e+003
 2.66 (+)
5.1e−005
179
hsa-miR-195


4.4e+004 − 1.8e+004
 2.46 (+)
8.2e−008
8
hsa-miR-125b


7.3e+002 − 3.2e+002
 2.26 (+)
2.8e−004
296
hsa-miR-26b


2.7e+003 − 1.2e+003
 2.22 (+)
2.4e−003
284
hsa-miR-19b


5.7e+002 − 2.6e+002
 2.17 (+)
7.8e−005
18
hsa-miR-148a


9.1e+002 − 4.4e+002
 2.06 (+)
1.4e−006
51
hsa-miR-345


7.5e+003 − 3.8e+003
 2.00 (+)
1.6e−002
258
hsa-let-7f


1.8e+002 − 4.4e+003
24.66 (−)
3.9e−008
49
hsa-miR-31


5.8e+001 − 1.0e+003
17.65 (−)
1.0e−007
184
hsa-miR-203


2.2e+002 − 1.6e+003
 7.43 (−)
4.5e−018
35
hsa-miR-21*


1.1e+004 − 5.5e+004
 4.97 (−)
1.5e−032
34
hsa-miR-21


6.2e+002 − 2.5e+003
 4.06 (−)
2.0e−008
37
hsa-miR-214


1.6e+002 − 5.8e+002
 3.69 (−)
6.3e−005
42
hsa-miR-224


6.9e+002 − 2.5e+003
 3.58 (−)
2.3e−009
228
hsa-miR-886-3p


4.8e+003 − 1.7e+004
 3.47 (−)
3.9e−009
15
hsa-miR-145


2.7e+003 − 8.2e+003
 3.08 (−)
2.7e−008
14
hsa-miR-143


1.3e+003 − 3.7e+003
 2.93 (−)
6.7e−005
242
MID-16489


2.5e+002 − 7.4e+002
 2.91 (−)
4.3e−006
230
hsa-miR-886-5p


3.5e+002 − 1.0e+003
 2.90 (−)
5.6e−004
253
MID-23256


1.1e+003 − 3.0e+003
 2.82 (−)
7.9e−006
36
hsa-miR-210


2.2e+003 − 5.8e+003
 2.63 (−)
1.2e−007
182
hsa-miR-199a-5p


5.0e+003 − 1.2e+004
 2.48 (−)
3.8e−006
293
hsa-miR-23b


7.5e+003 − 1.8e+004
 2.44 (−)
1.1e−008
292
hsa-miR-23a


2.7e+002 − 6.6e+002
 2.43 (−)
5.4e−003
4
hsa-miR-10a


9.0e+003 − 2.2e+004
 2.43 (−)
5.5e−015
294
hsa-miR-24


3.8e+003 − 8.8e+003
 2.35 (−)
3.0e−004
297
hsa-miR-27a


2.3e+002 − 5.2e+002
 2.28 (−)
1.6e−002
354
hsa-miR-612


1.8e+003 − 3.9e+003
 2.22 (−)
1.1e−006
377
MID-17866


1.6e+003 − 3.3e+003
 2.11 (−)
2.4e−004
189
hsa-miR-27b


6.9e+003 − 1.4e+004
 2.08 (−)
1.5e−004
30
hsa-miR-200c


3.8e+004 − 7.9e+004
 2.05 (−)
1.8e−008
386
MID-23178


1.5e+003 − 3.1e+003
 2.04 (−)
2.6e−002
249
MID-20524


4.6e+002 − 9.3e+002
 2.03 (−)
5.6e−002
5
hsa-miR-10b


2.5e+002 − 5.0e+002
 2.03 (−)
3.2e−005
274
hsa-miR-151-3p





+ the higher expression of this miR is in thymic carcinoma


− the higher expression of this miR is in TCC and SCC






FIG. 16 demonstrates binary decisions at node #20 of the decision-tree. Tumors originating in thymic carcinoma (marked by diamonds) are easily distinguished from tumors of urothelial carcinoma, transitional cell carcinoma (TCC) carcinoma and squamous cell carcinoma (SCC) origins (marked by squares) using the expression levels of hsa-miR-21 (SEQ ID NO: 34, y-axis) and hsa-miR-100 (SEQ ID NO: 3, x-axis).







TABLE 22







miR expression (in florescence units) distinguishing between TCC and


SCC (of anus, skin, lung, head & neck, esophagus or uterine cervix)












fold-

SEQ ID



median values
change
p-value
NO
miR name














2.5e+002 − 5.0e+001
5.05 (+)
7.4e−036
69
hsa-miR-934


9.1e+003 − 2.2e+003
4.14 (+)
1.2e−012
28
hsa-miR-200a


2.1e+002 − 5.3e+001
3.87 (+)
8.4e−007
280
hsa-miR-187


6.0e+003 − 1.9e+003
3.19 (+)
9.8e−008
13
hsa-miR-141


5.6e+002 − 1.8e+002
3.15 (+)
4.7e−013
191
hsa-miR-29c


9.4e+002 − 3.0e+002
3.13 (+)
8.1e−008
152
hsa-miR-182


1.8e+004 − 6.2e+003
2.99 (+)
3.9e−010
29
hsa-miR-200b


3.2e+002 − 1.1e+002
2.81 (+)
8.8e−009
175
hsa-miR-183


3.1e+004 − 1.2e+004
2.65 (+)
8.1e−005
30
hsa-miR-200c


2.1e+003 − 8.1e+002
2.63 (+)
6.2e−020
204
hsa-miR-425


1.2e+003 − 4.8e+002
2.41 (+)
6.3e−007
4
hsa-miR-10a


8.5e+003 − 3.7e+003
2.30 (+)
2.4e−024
24
hsa-miR-191


1.8e+003 − 8.4e+002
2.14 (+)
2.7e−006
5
hsa-miR-10b


3.5e+002 − 1.7e+002
2.09 (+)
2.0e−006
329
hsa-miR-425*


3.4e+002 − 1.6e+002
2.08 (+)
8.0e−005
273
hsa-miR-148b


3.1e+002 − 8.1e+002
2.60 (−)
1.6e−005
170
hsa-miR-155


5.0e+002 − 1.2e+003
2.49 (−)
1.0e−002
184
hsa-miR-203


1.5e+002 − 3.5e+002
2.39 (−)
3.0e−011
26
hsa-miR-193a-5p


1.9e+003 − 4.5e+003
2.35 (−)
1.3e−008
231
hsa-miR-99a


1.2e+002 − 2.7e+002
2.28 (−)
1.6e−003
368
MID-00672


1.4e+003 − 3.2e+003
2.25 (−)
1.3e−005
37
hsa-miR-214


3.9e+002 − 8.7e+002
2.23 (−)
1.5e−004
16
hsa-miR-146a


3.5e+002 − 7.6e+002
2.15 (−)
4.2e−005
169
hsa-miR-152


1.5e+002 − 3.3e+002
2.15 (−)
4.4e−004
155
hsa-miR-127-3p


8.4e+002 − 1.7e+003
2.08 (−)
1.6e−005
35
hsa-miR-21*


7.7e+003 − 1.6e+004
2.07 (−)
5.3e−012
40
hsa-miR-222


4.4e+002 − 9.0e+002
2.06 (−)
1.5e−005
17
hsa-miR-146b-5p





+ the higher expression of this miR is in TCC


− the higher expression of this miR is in SCC






hsa-miR-934 (SEQ ID NO: 69), hsa-miR-191 (SEQ ID NO: 24) and hsa-miR-29c (SEQ ID NO: 191) are used at node #21 of the binary-tree-classifier detailed in the invention to distinguish between TCC and SCC.







TABLE 23







miR expression (in florescence units) distinguishing between SCC


of the uterine cervix and other SCC tumors (anus, skin,


lung, head & neck or esophagus)
















SEQ





fold-

ID



median values
auROC
change
p-value
NO.
miR name















2.4e+002 − 9.2e+001
0.65
2.57 (+)
2.0e−002
164
hsa-miR-







142-5p


1.6e+003 − 7.6e+002
0.85
2.13 (+)
1.7e−005
5
hsa-miR-10b


8.9e+003 − 4.4e+003
0.74
2.01 (+)
2.1e−004
231
hsa-miR-99a


1.2e+003 − 9.8e+002
0.71
1.24 (+)
1.2e−002
54
hsa-miR-







361-5p


3.4e+004 − 2.7e+004
0.71
1.24 (+)
3.9e−004
1
hsa-let-7c


1.3e+003 − 4.3e+003
0.81
3.39 (−)
9.9e−006
242
MID-16489


3.9e+002 − 1.2e+003
0.74
3.10 (−)
2.1e−003
372
MID-16469


1.1e+003 − 3.3e+003
0.84
3.09 (−)
1.3e−008
249
MID-20524


1.7e+003 − 5.2e+003
0.78
3.01 (−)
2.4e−005
167
hsa-miR-149*


2.7e+002 − 8.0e+002
0.79
2.97 (−)
1.4e−004
254
MID-23291


2.2e+002 − 6.2e+002
0.76
2.77 (−)
1.7e−004
354
hsa-miR-612


5.7e+002 − 1.5e+003
0.76
2.65 (−)
7.8e−006
381
MID-19962


2.3e+002 − 6.0e+002
0.79
2.63 (−)
2.1e−005
380
MID-19898


9.8e+002 − 2.4e+003
0.78
2.44 (−)
2.8e−005
245
MID-18336


1.2e+002 − 2.8e+002
0.73
2.34 (−)
5.3e−003
358
hsa-miR-665


6.1e+002 − 1.4e+003
0.70
2.31 (−)
6.1e−003
364
MID-00064


2.9e+003 − 6.7e+003
0.81
2.30 (−)
8.8e−008
240
MID-15965


1.2e+002 − 2.8e+002
0.66
2.26 (−)
1.5e−002
11
hsa-miR-138


1.0e+002 − 2.3e+002
0.77
2.24 (−)
8.9e−005
378
MID-18307





+ the higher expression of this miR is in SCC of the uterine cervix


− the higher expression of this miR is in other SCC tumors






FIG. 17 demonstrates binary decisions at node #22 of the decision-tree. Tumors originating in SCC of the uterine cervix (diamonds) are easily distinguished from tumors of other SCC origin (squares) using the expression levels of hsa-miR-361-5p (SEQ ID NO: 54, y-axis), hsa-let-7c (SEQ ID NO: 1, x-axis) and hsa-miR-10b (SEQ ID NO: 5, z-axis).







TABLE 24







miR expression (in florescence units) distinguishing between anus or


skin SCC and upper SCC tumors (lung, head & neck or esophagus)
















SEQ





fold-

ID



median values
auROC
change
p-value
NO.
miR name















3.2e+002 − 5.0e+001
0.78
6.38 (+)
3.0e−006
305
hsa-miR-31*


4.3e+003 − 8.0e+002
0.80
5.39 (+)
1.8e−006
184
hsa-miR-203


8.6e+002 − 2.5e+002
0.78
3.49 (+)
1.8e−006
41
hsa-miR-223


1.7e+003 − 5.4e+002
0.80
3.12 (+)
3.5e−006
183
hsa-miR-







199b-5p


9.4e+003 − 3.5e+003
0.70
2.73 (+)
2.4e−003
49
hsa-miR-31


8.7e+003 − 3.2e+003
0.86
2.71 (+)
3.6e−007
382
MID-22331


1.9e+003 − 7.1e+002
0.87
2.68 (+)
1.7e−008
235
hsa-miR-1978


2.4e+002 − 9.2e+001
0.83
2.55 (+)
9.6e−009
291
hsa-miR-222*


6.8e+003 − 2.9e+003
0.74
2.31 (+)
7.4e−004
181
hsa-miR-







199a-3p


1.5e+003 − 6.7e+002
0.88
2.28 (+)
7.1e−007
5
hsa-miR-10b


5.3e+002 − 2.4e+002
0.75
2.21 (+)
1.4e−004
296
hsa-miR-26b


3.4e+002 − 1.6e+002
0.74
2.19 (+)
7.7e−005
289
hsa-miR-22*


1.3e+003 − 6.0e+002
0.71
2.13 (+)
1.2e−003
206
hsa-miR-







455-3p


7.9e+003 − 3.8e+003
0.84
2.11 (+)
4.2e−006
338
hsa-miR-494


2.9e+002 − 1.4e+002
0.73
2.08 (+)
1.1e−004
334
hsa-miR-







483-5p


2.8e+003 − 1.3e+003
0.82
2.07 (+)
4.5e−006
25
hsa-miR-







193a-3p


1.1e+002 − 3.3e+002
0.77
3.03 (−)
2.3e−005
11
hsa-miR-138


1.3e+002 − 3.1e+002
0.65
2.29 (−)
1.5e−002
19
hsa-miR-149


9.7e+001 − 2.1e+002
0.75
2.16 (−)
4.0e−005
198
hsa-miR-







342-5p


1.1e+003 − 1.8e+003
0.83
1.63 (−)
1.1e−006
23
hsa-miR-185





+ the higher expression of this miR is in anus or skin SCC


− the higher expression of this miR is in upper SCC tumors






hsa-miR-10b (SEQ ID NO: 5), hsa-miR-138 (SEQ ID NO: 11) and hsa-miR-185 (SEQ ID NO: 23) are used at node 23 of the binary-tree-classifier detailed in the invention to distinguish between anus or skin SCC and upper SCC tumors.







TABLE 25







miR expression (in florescence units) distinguishing between


melanoma and lymphoma (B-cell or T-cell) tumors
















SEQ




au-
fold-

ID



median values
ROC
change
p-value
NO.
miR name















1.7e+003 − 3.0e+002
0.89
 5.81 (+)
2.8e−010
4
hsa-miR-10a


1.9e+003 − 6.0e+002
0.80
 3.13 (+)
7.9e−005
11
hsa-miR-138


1.7e+003 − 5.7e+002
0.94
 2.98 (+)
2.3e−011
46
hsa-miR-30a


2.5e+004 − 8.8e+003
0.87
 2.83 (+)
1.1e−009
8
hsa-miR-125b


6.2e+002 − 2.3e+002
0.94
 2.74 (+)
9.2e−011
274
hsa-miR-151-







3p


9.2e+002 − 3.4e+002
0.87
 2.70 (+)
1.9e−007
169
hsa-miR-152


1.6e+003 − 6.0e+002
0.77
 2.60 (+)
2.0e−004
36
hsa-miR-210


4.8e+003 − 1.9e+003
0.90
 2.56 (+)
2.1e−011
47
hsa-miR-30d


1.2e+003 − 5.5e+002
0.88
 2.26 (+)
2.5e−008
363
hsa-miR-99b


2.4e+003 − 1.1e+003
0.85
 2.24 (+)
1.4e−006
231
hsa-miR-99a


6.5e+003 − 3.0e+003
0.80
 2.17 (+)
2.2e−005
303
hsa-miR-30b


6.4e+002 − 3.0e+002
0.86
 2.14 (+)
2.9e−008
349
hsa-miR-532-







5p


2.1e+003 − 1.0e+003
0.86
 2.08 (+)
1.6e−006
10
hsa-miR-130a


5.4e+003 − 2.6e+003
0.81
 2.06 (+)
8.3e−006
7
hsa-miR-125a-







5p


3.6e+003 − 1.8e+003
0.82
 2.05 (+)
2.5e−006
3
hsa-miR-100


7.9e+003 − 3.9e+003
0.69
 2.04 (+)
1.5e−002
16
hsa-miR-146a


1.6e+002 − 2.2e+003
0.93
13.84 (−)
1.1e−014
164
hsa-miR-142-







5p


7.2e+002 − 7.5e+003
0.93
10.40 (−)
1.1e−013
170
hsa-miR-155


2.0e+003 − 1.4e+004
0.90
 7.18 (−)
2.2e−010
168
hsa-miR-150


1.7e+002 − 7.0e+002
0.91
 4.14 (−)
5.2e−011
198
hsa-miR-342-







5p


2.2e+003 − 8.3e+003
0.97
 3.83 (−)
6.2e−019
50
hsa-miR-342-







3p


9.1e+002 − 2.6e+003
0.86
 2.87 (−)
1.3e−008
245
MID-18336


2.3e+002 − 6.4e+002
0.77
 2.74 (−)
2.4e−004
365
MID-00078


1.9e+002 − 5.2e+002
0.78
 2.68 (−)
4.7e−004
45
hsa-miR-29c*


2.8e+003 − 6.6e+003
0.74
 2.34 (−)
1.6e−003
382
MID-22331


3.4e+003 − 7.9e+003
0.85
 2.30 (−)
1.2e−004
259
hsa-let-7g


3.8e+002 − 8.5e+002
0.75
 2.25 (−)
4.3e−003
296
hsa-miR-26b


7.5e+002 − 1.6e+003
0.78
 2.16 (−)
2.3e−004
364
MID-00064


6.3e+002 − 1.3e+003
0.79
 2.08 (−)
7.9e−005
314
hsa-miR-361-







3p


2.7e+003 − 5.4e+003
0.80
 2.05 (−)
7.5e−007
12
hsa-miR-140-







3p





+ the higher expression of this miR is in melanoma


− the higher expression of this miR is in lymphoma






FIG. 18 demonstrates binary decisions at node #24 of the decision-tree. Tumors originating in melanoma (diamonds) are easily distinguished from tumors of lymphoma origin (squares) using the expression levels of hsa-miR-342-3p (SEQ ID NO: 50, y-axis) and hsa-miR-30d (SEQ ID NO: 47, x-axis).







TABLE 26







miR expression (in florescence units) distinguishing between B-cell


lymphoma and T-cell lymphoma
















SEQ





fold-

ID



median values
auROC
change
p-value
NO.
miR name





8.3e+002 − 2.8e+002
0.74
2.96 (+)
3.7e−005
 11
hsa-miR-138


6.7e+002 − 2.8e+002
0.72
2.37 (+)
2.2e−003
191
hsa-miR-29c


1.2e+003 − 5.9e+002
0.76
2.02 (+)
1.4e−003
 48
hsa-miR-30e


6.7e+002 − 1.8e+003
0.79
2.77 (−)
1.1e−006
 35
hsa-miR-21*


1.5e+003 − 3.9e+003
0.68
2.68 (−)
2.6e−003
228
hsa-miR-886-3p





+ the higher expression of this miR is in B-cell lymphoma


− the higher expression of this miR is in T-cell lymphoma






hsa-miR-30e (SEQ ID NO: 48) and hsa-miR-21* (SEQ ID NO: 35) are used at node 25 of the binary-tree-classifier detailed in the invention to distinguish between B-cell lymphoma and T-cell lymphoma.







TABLE 27







miR expression (in florescence units) distinguishing between lung


small cell carcinoma and other neuroendocrine tumors selected from


the group consisting of lung carcinoid, medullary thyroid carcinoma,


gastrointestinal tract carcinoid and pancreatic islet cell tumor
















SEQ




au-
fold-

ID



median values
ROC
change
p-value
NO.
miR name















1.2e+004 − 1.2e+003
0.99
 9.68 (+)
3.3e−021
158
hsa-miR-106a


7.3e+003 − 7.9e+002
1.00
 9.17 (+)
3.4e−022
20
hsa-miR-17


1.4e+003 − 1.6e+002
0.99
 8.53 (+)
8.2e−022
176
hsa-miR-18a


5.8e+003 − 7.0e+002
1.00
 8.38 (+)
7.4e−021
186
hsa-miR-20a


1.1e+004 − 1.5e+003
0.98
 7.71 (+)
1.7e−022
148
hsa-miR-93


4.7e+003 − 6.7e+002
0.89
 6.99 (+)
1.0e−008
36
hsa-miR-210


2.2e+003 − 3.7e+002
0.95
 5.87 (+)
2.8e−016
51
hsa-miR-345


8.9e+003 − 1.8e+003
0.95
 4.96 (+)
1.6e−010
172
hsa-miR-15b


8.2e+003 − 1.8e+003
0.98
 4.68 (+)
6.3e−020
260
hsa-miR-106b


1. le+003 − 2.4e+002
0.91
 4.62 (+)
7.7e−010
265
hsa-miR-130b


8.0e+003 − 1.8e+003
0.94
 4.33 (+)
2.7e−013
67
hsa-miR-92a


4. le+003 − 9.8e+002
0.98
 4.15 (+)
2.6e−019
188
hsa-miR-25


1.1e+003 − 3.4e+002
0.98
 3.40 (+)
7.9e−016
277
hsa-miR-17*


2.5e+003 − 8.3e+002
0.99
 2.96 (+)
1.8e−011
284
hsa-miR-19b


5. le+002 − 1.8e+002
0.74
 2.84 (+)
6.1e−004
302
hsa-miR-301a


7.9e+002 − 2.9e+002
0.91
 2.78 (+)
8.7e−010
68
hsa-miR-92b


9.9e+002 − 4.3e+002
0.69
 2.28 (+)
5.1e−002
168
hsa-miR-150


2.5e+003 − 1.1e+003
0.70
 2.24 (+)
4.5e−003
242
MID-16489


1.4e+003 − 6.6e+002
0.91
 2.12 (+)
1.1e−009
204
hsa-miR-425


5.0e+001 − 1.6e+003
0.91
31.23 (−)
4.5e−009
162
hsa-miR-129-







3p


1.1e+002 − 1.6e+003
0.91
14.13 (−)
8.6e−009
177
hsa-miR-192


7.6e+001 − 7.9e+002
0.91
10.42 (−)
1.5e−008
27
hsa-miR-194


5.5e+002 − 5.0e+003
0.92
 9.14 (−)
1.7e−009
65
hsa-miR-7


7.1e+001 − 5.7e+002
0.78
 8.02 (−)
5.8e−005
263
hsa-miR-129*


2.5e+002 − 1.6e+003
0.80
 6.30 (−)
3.5e−005
155
hsa-miR-127-







3p


1.5e+002 − 9.1e+002
0.96
 6.05 (−)
3.5e−015
191
hsa-miR-29c


3.3e+002 − 2.0e+003
0.93
 5.99 (−)
3.3e−013
190
hsa-miR-29b


1.7e+002 − 9.9e+002
0.99
 5.76 (−)
1.3e−020
45
hsa-miR-29c*


1.2e+002 − 6.6e+002
0.75
 5.60 (−)
8.0e−004
59
hsa-miR-487b


1.8e+003 − 8.0e+003
0.90
 4.44 (−)
1.6e−012
43
hsa-miR-29a


1.3e+004 − 4.9e+004
0.88
 3.87 (−)
3.3e−006
56
hsa-miR-375


1.6e+002 − 5.5e+002
0.95
 3.37 (−)
9.6e−011
266
hsa-miR-132


4.0e+003 − 1.2e+004
0.82
 2.98 (−)
9.4e−006
14
hsa-miR-143


7.8e+003 − 2.3e+004
0.85
 2.89 (−)
6.1e−006
15
hsa-miR-145


1.2e+004 − 3.4e+004
0.79
 2.83 (−)
4.3e−005
8
hsa-miR-125b


4.5e+003 − 1.2e+004
0.97
 2.70 (−)
1.7e−014
7
hsa-miR-







125a-5p


1.9e+003 − 5.0e+003
0.89
 2.67 (−)
3.6e−010
39
hsa-miR-22


2.5e+003 − 5.7e+003
0.79
 2.25 (−)
8.7e−004
189
hsa-miR-27b


1. le+003 − 2.4e+003
0.64
 2.18 (−)
4.1e−002
249
MID-20524


2.2e+003 − 4.8e+003
0.72
 2.14 (−)
8.5e−003
231
hsa-miR-99a


9.6e+003 − 2.0e+004
0.82
 2.12 (−)
1.3e−003
293
hsa-miR-23b


5.1e+003 − 1.0e+004
0.80
 2.01 (−)
6.3e−005
2
hsa-let-7e





+ the higher expression of this miR is in lung small cell carcinoma


− the higher expression of this miR is in other neuroendocrine tumors






hsa-miR-17 (SEQ ID NO: 20) and hsa-miR-29c* (SEQ ID NO: 45) are used at node #26 of the binary-tree-classifier detailed in the invention to distinguish between lung small cell carcinoma and other neuroendocrine tumors.







TABLE 28







miR expression (in florescence units) distinguishing between medullary


thyroid carcinoma and other neuroendocrine tumors selected from the


group consisting of lung carcinoid, gastrointestinal tract carcinoid


and pancreatic islet cell tumor
















SEQ




au-
fold-

ID



median values
ROC
change
p-value
NO.
miR name















4.4e+003 − 5.5e+001
0.84
79.70 (+)
1.5e−007
159
hsa-miR-124


4.0e+004 − 4.9e+003
0.98
 8.07 (+)
1.6e−015
40
hsa-miR-222


1.9e+004 − 2.8e+003
0.98
 6.85 (+)
4.8e−016
147
hsa-miR-221


1.1e+003 − 2.0e+002
0.70
 5.55 (+)
1.1e−003
11
hsa-miR-138


3.2e+002 − 7.8e+001
0.83
 4.12 (+)
7.6e−007
311
hsa-miR-335


5.8e+003 − 1.5e+003
0.86
 3.91 (+)
1.3e−006
4
hsa-miR-10a


6.3e+004 − 1.7e+004
0.83
 3.61 (+)
3.9e−006
8
hsa-miR-125b


1.1e+004 − 3.2e+003
0.79
 3.43 (+)
5.5e−005
231
hsa-miR-99a


4.3e+002 − 2.0e+002
0.78
 2.10 (+)
2.8e−004
301
hsa-miR-







29b-2*


7.9e+003 − 3.8e+003
0.82
 2.06 (+)
4.4e−005
297
hsa-miR-27a


1.4e+002 − 4.0e+002
0.95
 2.95 (−)
7.5e−011
68
hsa-miR-92b


1.1e+003 − 2.8e+003
0.87
 2.50 (−)
3.2e−006
67
hsa-miR-92a


1.8e+002 − 3.7e+002
0.76
 2.07 (−)
2.0e−003
265
hsa-miR-130b


4.4e+002 − 9.0e+002
0.75
 2.04 (−)
2.1e−003
36
hsa-miR-210





+ the higher expression of this miR is in medullary thyroid carcinoma


− the higher expression of this miR is in other neuroendocrine tumors






FIG. 19 demonstrates binary decisions at node #27 of the decision-tree. Tumors originating in medullary thyroid carcinoma (diamonds) are easily distinguished from tumors of other neuroendocrine origin (squares) using the expression levels of hsa-miR-92b (SEQ ID NO: 68, y-axis), hsa-miR-222 (SEQ ID NO: 40, x-axis) and hsa-miR-92a (SEQ ID NO: 67, z-axis).







TABLE 29







miR expression (in florescence units) distinguishing between


lung carcinoid tumors and GI neuroendocrine tumors


selected from the group consisting of gastrointestinal


tract carcinoid and pancreatic islet cell tumor
















SEQ




au-
fold-

ID



median values
ROC
change
p-value
NO.
miR name















4.0e+003 − 9.9e+001
0.90
40.08 (+)
1.9e−010
331
hsa-miR-432


6.0e+003 − 1.5e+002
0.86
39.24 (+)
4.6e−008
162
hsa-miR-129-







3p


6.3e+003 − 1.9e+002
0.87
34.16 (+)
7.8e−009
59
hsa-miR-487b


1.3e+003 − 5.5e+001
0.88
23.36 (+)
2.9e−010
326
hsa-miR-409-







5p


1.1e+003 − 5.0e+001
0.88
21.14 (+)
5.2e−010
306
hsa-miR-323-







3p


1.0e+003 − 5.5e+001
0.87
18.59 (+)
1.5e−009
350
hsa-miR-539


7.9e+002 − 5.6e+001
0.84
14.25 (+)
1.4e−008
317
hsa-miR-369-







5p


1.0e+004 − 7.2e+002
0.86
13.95 (+)
3.2e−007
155
hsa-miR-127-







3p


1.7e+003 − 1.2e+002
0.86
13.60 (+)
2.1e−008
325
hsa-miR-409-







3p


1.6e+003 − 1.2e+002
0.88
13.10 (+)
4.2e−009
318
hsa-miR-370


9.5e+002 − 7.3e+001
0.81
13.03 (+)
3.1e−006
339
hsa-miR-495


9.5e+002 − 7.4e+001
0.84
12.92 (+)
5.7e−007
264
hsa-miR-129-







5p


6.4e+002 − 5.0e+001
0.91
12.84 (+)
1.6e−013
332
hsa-miR-433


6.5e+002 − 5.7e+001
0.88
11.52 (+)
5.1e−011
262
hsa-miR-127-







5p


5.6e+002 − 5.2e+001
0.90
10.76 (+)
2.7e−012
336
hsa-miR-485-







5p


2.0e+003 − 1.9e+002
0.86
10.44 (+)
4.2e−008
324
hsa-miR-382


7.9e+002 − 7.8e+001
0.83
10.20 (+)
1.3e−007
322
hsa-miR-379


6.0e+002 − 5.9e+001
0.89
10.15 (+)
9.6e−012
330
hsa-miR-431*


4.7e+002 − 5.0e+001
0.90
 9.41 (+)
6.1e−012
321
hsa-miR-377*


1.3e+003 − 1.4e+002
0.80
 9.40 (+)
1.5e−005
263
hsa-miR-129*


4.7e+002 − 5.0e+001
0.86
 9.35 (+)
1.8e−008
309
hsa-miR-329


4.9e+002 − 5.3e+001
0.79
 9.24 (+)
3.1e−005
53
hsa-miR-34c-







5p


1.1e+003 − 1.2e+002
0.83
 9.05 (+)
6.4e−007
320
hsa-miR-376c


1.1e+003 − 1.2e+002
0.86
 8.81 (+)
2.3e−008
275
hsa-miR-154


6.5e+002 − 8.4e+001
0.83
 7.73 (+)
8.1e−007
352
hsa-miR-543


9.9e+002 − 1.3e+002
0.82
 7.49 (+)
3.2e−007
312
hsa-miR-337-







5p


6.2e+002 − 8.8e+001
0.86
 7.10 (+)
3.0e−008
355
hsa-miR-654-







3p


3.5e+002 − 5.0e+001
0.91
 7.05 (+)
3.2e−013
367
MID-00465


6.0e+002 − 1.0e+002
0.84
 5.76 (+)
5.9e−007
269
hsa-miR-134


3.2e+003 − 8.5e+002
0.91
 3.84 (+)
1.2e−011
64
hsa-miR-652


3.2e+002 − 1.1e+002
0.83
 2.84 (+)
2.3e−005
308
hsa-miR-328


2.6e+003 − 9.4e+002
0.74
 2.78 (+)
1.1e−003
175
hsa-miR-183


2.8e+003 − 1.0e+003
0.87
 2.73 (+)
3.0e−006
190
hsa-miR-29b


3.9e+003 − 1.6e+003
0.88
 2.49 (+)
6.9e−010
54
hsa-miR-361-







5p


4.1e+002 − 1.7e+002
0.67
 2.44 (+)
2.1e−002
302
hsa-miR-301a


4.0e+003 − 1.7e+003
0.79
 2.41 (+)
5.9e−004
152
hsa-miR-182


4.0e+002 − 1.7e+002
0.88
 2.39 (+)
4.7e−007
301
hsa-miR-







29b-2*


8.7e+002 − 3.7e+002
0.77
 2.36 (+)
6.8e−005
266
hsa-miR-132


7.7e+003 − 3.3e+003
0.82
 2.34 (+)
5.4e−006
47
hsa-miR-30d


3.7e+002 − 1.6e+002
0.70
 2.32 (+)
5.8e−003
313
hsa-miR-338-







3p


3.3e+002 − 1.5e+002
0.66
 2.16 (+)
1.3e−002
359
hsa-miR-708


5.5e+003 − 2.5e+003
0.68
 2.16 (+)
4.2e−002
65
hsa-miR-7


2.1e+003 − 9.9e+002
0.78
 2.13 (+)
7.0e−005
307
hsa-miR-324-







5p


1.2e+003 − 5.9e+002
0.81
 2.02 (+)
1.6e−004
191
hsa-miR-29c


3.5e+002 − 1.9e+003
0.88
 5.36 (−)
1.0e−007
242
MID-16489


6.5e+002 − 1.9e+003
0.76
 2.96 (−)
4.9e−004
4
hsa-miR-10a


1.3e+003 − 3.6e+003
0.84
 2.79 (−)
1.9e−006
147
hsa-miR-221


2.2e+003 − 5.9e+003
0.81
 2.75 (−)
8.5e−006
40
hsa-miR-222


2.6e+002 − 6.8e+002
0.76
 2.56 (−)
7.4e−004
372
MID-16469


3.5e+002 − 8.9e+002
0.71
 2.56 (−)
4.7e−003
168
hsa-miR-150


1.5e+002 − 3.7e+002
0.83
 2.55 (−)
4.8e−005
16
hsa-miR-146a


1.9e+003 − 4.7e+003
0.82
 2.40 (−)
1.7e−005
182
hsa-miR-199a-







5p


1.3e+003 − 3.0e+003
0.79
 2.35 (−)
1.2e−004
167
hsa-miR-149*


2.1e+002 − 4.8e+002
0.84
 2.26 (−)
3.1e−005
356
hsa-miR-658


1.2e+003 − 2.8e+003
0.74
 2.25 (−)
1.4e−003
148
hsa-miR-93


1.4e+003 − 3.1e+003
0.70
 2.23 (−)
1.9e−002
382
MID-22331


8.0e+002 − 1.8e+003
0.83
 2.21 (−)
2.9e−005
37
hsa-miR-214


4.4e+002 − 8.9e+002
0.79
 2.01 (−)
2.1e−004
364
MID-00064


2.1e+002 − 4.2e+002
0.78
 2.01 (−)
1.1e−003
35
hsa-miR-21*





+ the higher expression of this miR is in lung carcinoid tumors


− the higher expression of this miR is in GI neuroendocrine tumors






hsa-miR-652 (SEQ ID NO: 64), hsa-miR-34c-5p (SEQ ID NO: 53) and hsa-miR-214 (SEQ ID NO: 37) are used at node 28 of the binary-tree-classifier detailed in the invention to distinguish between lung carcinoid tumors and GI neuroendocrine tumors.







TABLE 30







miR expression (in florescence units) distinguishing between


pancreatic islet cell tumors and GI neuroendocrine carcinoid


tumors selected from the group consisting of small intestine


and duodenum; appendicitis, stomach and pancreas
















SEQ




au-
fold-

ID



median values
ROC
change
p-value
NO.
miR name















1.1e+002 2.3e+003
0.80
20.91 (+)
2.8e−004
263
hsa-miR-129*


5.0e+001 4.8e+002
0.72
 9.61 (+)
6.6e−003
288
hsa-miR-217


1.9e+002 1.6e+003
0.90
 8.54 (+)
6.8e−006
18
hsa-miR-148a


5.2e+001 4.3e+002
0.68
 8.34 (+)
2.7e−002
286
hsa-miR-216a


2.5e+002 1.8e+003
0.74
 7.22 (+)
4.4e−003
162
hsa-miR-129-







3p


9.9e+001 6.6e+002
0.74
 6.65 (+)
2.3e−003
225
hsa-miR-551b


5.0e+001 3.0e+002
0.75
 6.04 (+)
5.4e−003
287
hsa-miR-216b


1.9e+002 7.1e+002
0.92
 3.75 (+)
7.3e−007
206
hsa-miR-455-







3p


3.4e+003 1.3e+004
0.79
 3.65 (+)
2.5e−003
205
hsa-miR-451


2.6e+002 8.9e+002
0.83
 3.43 (+)
2.8e−004
296
hsa-miR-26b


2.6e+003 8.7e+003
0.91
 3.29 (+)
3.6e−004
258
hsa-let-7f


1.6e+002 5.2e+002
0.78
 3.25 (+)
3.2e−003
313
hsa-miR-338-







3p


2.4e+003 6.6e+003
0.85
 2.71 (+)
5.0e−005
377
MID-17866


7.6e+003 1.9e+004
0.88
 2.45 (+)
1.2e−005
373
MID-16582


2.7e+004 6.4e+004
0.80
 2.42 (+)
1.0e−003
256
hsa-let-7a


4.7e+003 1.1e+004
0.89
 2.36 (+)
1.8e−004
153
hsa-let-7d


2.8e+003 6.4e+003
0.86
 2.28 (+)
2.2e−003
259
hsa-let-7g


2.3e+002 4.8e+002
0.69
 2.11 (+)
1.0e−002
265
hsa-miR-130b


3.1e+003 6.4e+003
0.75
 2.09 (+)
7.5e−003
303
hsa-miR-30b


9.7e+002 1.0e+002
0.81
 9.40 (−)
5.4e−004
268
hsa-miR-133b


1.0e+003 1.1e+002
0.80
 9.22 (−)
5.4e−004
267
hsa-miR-133a


1.9e+003 2.3e+002
0.93
 8.37 (−)
2.1e−006
165
hsa-miR-143*


9.2e+004 1.1e+004
0.94
 8.18 (−)
3.7e−008
15
hsa-miR-145


5.3e+002 6.6e+001
0.91
 8.05 (−)
7.0e−006
272
hsa-miR-145*


3.8e+004 5.2e+003
0.96
 7.30 (−)
3.7e−009
14
hsa-miR-143


2.0e+003 3.1e+002
0.88
 6.35 (−)
6.2e−006
202
hsa-miR-378


1.4e+003 2.9e+002
0.88
 4.99 (−)
8.1e−006
236
MID-00689


6.4e+002 1.4e+002
0.88
 4.74 (−)
7.9e−006
203
hsa-miR-422a


3.6e+003 9.2e+002
0.82
 3.91 (−)
2.4e−004
4
hsa-miR-10a


1.1e+003 3.0e+002
0.78
 3.78 (−)
4.4e−003
168
hsa-miR-150


3.6e+002 1.1e+002
0.81
 3.23 (−)
3.4e−004
310
hsa-miR-330-







3p


6.7e+002 2.1e+002
0.95
 3.16 (−)
4.6e−007
298
hsa-miR-28-3p


2.2e+003 7.2e+002
0.74
 3.09 (−)
1.4e−002
27
hsa-miR-194


2.1e+004 7.5e+003
0.91
 2.72 (−)
7.6e−005
29
hsa-miR-200b


2.2e+004 8.5e+003
0.87
 2.57 (−)
2.8e−006
34
hsa-miR-21


1.7e+003 6.7e+002
0.74
 2.56 (−)
8.0e−003
228
hsa-miR-886-







3p


3.8e+003 1.5e+003
0.77
 2.50 (−)
3.6e−003
3
hsa-miR-100


5.3e+002 2.5e+002
0.94
 2.14 (−)
4.3e−007
349
hsa-miR-532-







5p


5.0e+002 2.4e+002
0.82
 2.06 (−)
8.5e−004
35
hsa-miR-21*


3.3e+002 1.6e+002
0.77
 2.01 (−)
5.7e−003
26
hsa-miR-193a-







5p





+ the higher expression of this miR is in pancreatic islet cell tumors


− the higher expression of this miR is in GI neuroendocrine carcinoid tumors






hsa-miR-21 (SEQ ID NO: 34), and hsa-miR-148a (SEQ ID NO: 18) are used at node 29 of the binary-tree-classifier detailed in the invention to distinguish between pancreatic islet cell tumors and GI neuroendocrine carcinoid tumors.







TABLE 31







miR expression (in florescence units) distinguishing between gastric or


esophageal adenocarcinoma and other adenocarcinoma tumors of the


gastrointestinal system selected from the group consisting of


cholangiocarcinoma or adenocarcinoma of extrahepatic biliary


tract, pancreatic adenocarcinoma and colorectal adenocarcinoma
















SEQ





fold-

ID



median values
auROC
change
p-value
NO.
miR name















6.7e+001 6.2e+002
0.74
9.14 (+)
4.6e−008
267
hsa-miR-133a


6.3e+001 5.5e+002
0.74
8.73 (+)
3.9e−008
268
hsa-miR-133b


5.9e+002 2.5e+003
0.75
4.26 (+)
3.9e−007
165
hsa-miR-143*


2.8e+004 7.9e+004
0.71
2.82 (+)
4.5e−004
15
hsa-miR-145


1.3e+004 3.2e+004
0.68
2.55 (+)
1.3e−003
14
hsa-miR-143


5.1e+002 1.3e+003
0.71
2.53 (+)
8.2e−004
356
hsa-miR-658


3.1e+003 7.2e+003
0.72
2.33 (+)
2.2e−004
167
hsa-miR-149*


1.4e+003 3.1e+003
0.69
2.22 (+)
7.2e−004
376
MID-17576


6.5e+002 1.4e+003
0.71
2.20 (+)
3.0e−004
372
MID-16469


1.5e+002 3.2e+002
0.69
2.14 (+)
3.0e−004
272
hsa-miR-145*


1.4e+003 2.9e+003
0.74
2.11 (+)
3.8e−004
370
MID-15986


3.6e+002 5.5e+001
0.83
6.57 (−)
5.4e−008
42
hsa-miR-224


4.0e+002 1.5e+002
0.73
2.61 (−)
1.1e−004
41
hsa-miR-223


1.2e+003 9.0e+002
0.67
1.28 (−)
1.2e−002
146
hsa-miR-1201





+ the higher expression of this miR is in gastric or esophageal adenocarcinoma


− the higher expression of this miR is in other adenocarcinoma tumors of the gastrointestinal system






FIG. 20 demonstrates binary decisions at node #30 of the decision-tree. Tumors originating in gastric or esophageal adenocarcinoma (diamonds) are easily distinguished from tumors of other GI adenocarcinoma origin (squares) using the expression levels of hsa-miR-1201 (SEQ ID NO: 146, y-axis), hsa-miR-224 (SEQ ID NO: 42, x-axis) and hsa-miR-210 (SEQ ID NO: 36, z-axis).







TABLE 32







miR expression (in florescence units) distinguishing between colorectal


adenocarcinoma and cholangiocarcinoma or adenocarcinoma of


biliary tract or pancreas
















SEQ





fold-

ID



median values
auROC
change
p-value
NO.
miR name















2.1e+002 5.4e+002
0.69
2.55 (+)
4.0e−003
42
hsa-miR-224


1.8e+002 4.2e+002
0.70
2.28 (+)
1.2e−003
184
hsa-miR-203


3.2e+003 6.2e+003
0.77
1.91 (+)
5.1e−007
67
hsa-miR-92a


3.1e+003 5.6e+003
0.81
1.81 (+)
4.6e−007
158
hsa-miR-106a


1.8e+003 3.2e+003
0.81
1.81 (+)
1.3e−007
20
hsa-miR-17


1.8e+003 3.2e+003
0.76
1.80 (+)
7.9e−005
186
hsa-miR-20a


1.1e+003 1.9e+003
0.76
1.75 (+)
1.4e−005
284
hsa-miR-19b


1.6e+003 2.6e+003
0.70
1.67 (+)
3.0e−003
389
MID-17356


3.1e+002 5.1e+002
0.75
1.63 (+)
2.1e−005
203
hsa-miR-422a


4.5e+003 7.2e+003
0.67
1.60 (+)
5.6e−003
240
MID-15965


6.9e+002 1.1e+003
0.76
1.59 (+)
1.7e−005
236
MID-00689


1.1e+003 1.6e+003
0.68
1.53 (+)
2.5e−003
146
hsa-miR-1201


9.1e+002 1.4e+003
0.69
1.49 (+)
5.2e−004
204
hsa-miR-425


6.5e+003 9.3e+003
0.77
1.44 (+)
1.2e−005
43
hsa-miR-29a


4.5e+002 6.4e+002
0.75
1.44 (+)
7.3e−006
176
hsa-miR-18a


9.1e+002 1.3e+003
0.72
1.41 (+)
1.4e−004
202
hsa-miR-378


1.8e+003 5.3e+002
0.69
3.39 (−)
2.0e−003
49
hsa-miR-31


2.0e+003 8.2e+002
0.82
2.39 (−)
2.2e−008
46
hsa-miR-30a


3.7e+002 2.5e+002
0.66
1.47 (−)
1.3e−002
38
hsa-miR-214*


1.3e+003 9.0e+002
0.73
1.41 (−)
2.2e−003
363
hsa-miR-99b





+ the higher expression of this miR is in colorectal adenocarcinoma


− the higher expression of this miR is in other cholangiocarcinoma or adenocarcinoma tumors of biliary tract or pancreas






FIG. 21 demonstrates binary decisions at node #31 of the decision-tree. Tumors originating in colorectal adenocarcinoma (diamonds) are easily distinguished from tumors of cholangiocarcinoma or adenocarcinoma of biliary tract or pancreas origin (squares) using the expression levels of hsa-miR-30a (SEQ ID NO: 46, y-axis), hsa-miR-17 (SEQ ID NO: 20, x-axis) and hsa-miR-29a (SEQ ID NO: 43, z-axis).







TABLE 33







miR expression (in florescence units) distinguishing between


cholangiocarcinoma or adenocarcinoma of extrahepatic biliary


tract and pancreatic adenocarcinoma
















SEQ





fold-

ID



median values
auROC
change
p-value
NO.
miR name















1.1e+003 3.4e+003
0.81
3.06 (+)
1.5e−003
49
hsa-miR-31


1.4e+002 3.3e+002
0.71
2.36 (+)
1.1e−002
11
hsa-miR-138


1.7e+003 3.0e+003
0.70
1.77 (+)
1.7e−002
13
hsa-miR-141


1.1e+004 1.8e+004
0.70
1.65 (+)
1.5e−002
373
MID-16582


8.4e+002 1.4e+003
0.69
1.63 (+)
9.6e−002
154
hsa-miR-181b


4.3e+002 7.0e+002
0.69
1.62 (+)
5.1e−001
5
hsa-miR-10b


9.3e+003 1.5e+004
0.68
1.61 (+)
7.4e−002
30
hsa-miR-200c


2.7e+002 4.2e+002
0.72
1.58 (+)
1.3e−002
45
hsa-miR-29c*


1.0e+003 1.5e+003
0.66
1.47 (+)
1.1e−001
178
hsa-miR-193b


6.6e+003 9.0e+003
0.75
1.36 (+)
1.2e−002
147
hsa-miR-221


4.7e+002 6.4e+002
0.70
1.36 (+)
4.0e−002
274
hsa-miR-151-3p


5.4e+002 7.3e+002
0.66
1.34 (+)
2.4e−002
16
hsa-miR-146a


1.1e+004 1.5e+004
0.71
1.32 (+)
3.7e−002
40
hsa-miR-222


3.8e+003 4.9e+003
0.71
1.30 (+)
8.4e−002
21
hsa-miR-181a


6.0e+003 6.8e+003
0.66
1.14 (+)
6.3e−002
43
hsa-miR-29a


5.9e+002 3.3e+002
0.74
1.81 (−)
2.1e−002
253
MID-23256


1.9e+003 1.1e+003
0.66
1.70 (−)
8.0e−002
245
MID-18336


4.5e+004 2.7e+004
0.73
1.68 (−)
7.4e−003
256
hsa-let-7a


2.7e+003 1.8e+003
0.65
1.51 (−)
9.2e−002
12
hsa-miR-140-3p


1.4e+005 9.3e+004
0.75
1.47 (−)
5.4e−003
374
MID-16748


8.9e+004 6.1e+004
0.66
1.45 (−)
2.9e−002
379
MID-18395


4.7e+002 3.3e+002
0.69
1.41 (−)
6.6e−002
180
hsa-miR-1973


6.0e+003 4.3e+003
0.68
1.40 (−)
2.6e−002
153
hsa-let-7d


4.4e+002 3.2e+002
0.75
1.39 (−)
7.1e−002
51
hsa-miR-345


6.1e+003 4.4e+003
0.70
1.38 (−)
3.9e−002
52
hsa-miR-34a


3.3e+004 2.4e+004
0.73
1.37 (−)
1.4e−002
1
hsa-let-7c


2.0e+004 1.5e+004
0.68
1.36 (−)
2.8e−002
295
hsa-miR-26a


3.9e+004 2.9e+004
0.77
1.35 (−)
3.3e−003
257
hsa-let-7b


6.1e+003 4.8e+003
0.66
1.26 (−)
6.5e−002
385
MID-23168


1.3e+004 1.0e+004
0.67
1.21 (−)
9.0e−002
293
hsa-miR-23b


2.4e+004 2.1e+004
0.68
1.18 (−)
2.6e−002
294
hsa-miR-24





+ the higher expression of this miR is in pancreatic adenocarcinoma


− the higher expression of this miR is in cholangiocarcinoma or adenocarcinoma of extrahepatic biliary tract






hsa-miR-345 (SEQ ID NO: 51), hsa-miR-31 (SEQ ID NO: 49) and hsa-miR-146a (SEQ ID NO: 16) are used at node #32 of the binary-tree-classifier detailed in the invention to distinguish between cholangio cancer or adenocarcinoma of extrahepatic biliary tract and pancreatic adenocarcinoma.







TABLE 34







miR expression (in florescence units) distinguishing between kidney tumors


selected from the group consisting of chromophobe renal cell carcinoma, clear cell renal cell


carcinoma and papillary renal cell carcinoma and other tumors selected from the group


consisting of sarcoma, adrenal (pheochromocytoma, adrenocortical carcinoma) and


mesothelioma (pleural mesothelioma)


















SEQ






fold-

ID













median values
auROC
change
p-value
NO.
miR name
















5.0e+001
4.8e+003
0.94
96.12 (+) 
7.6e−042
29
hsa-miR-200b


5.0e+001
2.3e+003
0.94
45.03 (+) 
3.3e−044
28
hsa-miR-200a


5.0e+001
7.7e+002
0.82
15.36 (+) 
1.1e−015
30
hsa-miR-200c


1.2e+003
1.1e+004
0.96
9.73 (+)
8.6e−041
46
hsa-miR-30a


1.2e+002
1.1e+003
0.74
9.21 (+)
1.1e−008
49
hsa-miR-31


1.9e+002
1.7e+003
0.94
8.87 (+)
8.6e−039
195
hsa-miR-30a*


7.5e+001
5.0e+002
0.74
6.58 (+)
1.1e−009
152
hsa-miR-182


5.0e+001
2.5e+002
0.76
5.07 (+)
9.8e−011
175
hsa-miR-183


2.2e+003
8.3e+003
0.92
3.81 (+)
5.0e−033
47
hsa-miR-30d


1.5e+003
5.1e+003
0.83
3.52 (+)
2.5e−016
4
hsa-miR-10a


7.3e+001
2.3e+002
0.75
3.15 (+)
6.4e−011
387
MID-23751


3.2e+003
9.5e+003
0.89
2.95 (+)
1.1e−025
196
hsa-miR-30c


1.4e+002
4.0e+002
0.76
2.80 (+)
2.1e−012
177
hsa-miR-192


9.8e+001
2.5e+002
0.79
2.52 (+)
1.7e−015
375
MID-17375


9.0e+001
2.2e+002
0.75
2.43 (+)
2.1e−012
27
hsa-miR-194


1.2e+002
2.8e+002
0.76
2.40 (+)
6.5e−013
304
hsa-miR-30e*


6.7e+003
1.6e+004
0.75
2.37 (+)
1.6e−012
40
hsa-miR-222


2.5e+002
5.9e+002
0.69
2.33 (+)
9.5e−006
191
hsa-miR-29c


4.5e+003
9.8e+003
0.74
2.20 (+)
1.4e−011
147
hsa-miR-221


4.5e+002
9.8e+002
0.61
2.19 (+)
7.6e−003
35
hsa-miR-21*


2.8e+002
6.1e+002
0.71
2.15 (+)
3.7e−007
16
hsa-miR-146a


2.4e+004
4.9e+004
0.64
2.07 (+)
1.2e−003
34
hsa-miR-21


2.3e+003
4.7e+003
0.66
2.06 (+)
5.4e−004
5
hsa-miR-10b


1.2e+003
1.2e+002
0.85
9.53 (−)
2.1e−018
155
hsa-miR-127-3p


1.2e+004
1.6e+003
0.89
7.57 (−)
4.9e−023
181
hsa-miR-199a-3p


3.7e+002
5.0e+001
0.86
7.45 (−)
1.2e−019
312
hsa-miR-337-5p


1.0e+003
1.4e+002
0.82
7.21 (−)
1.7e−015
183
hsa-miR-199b-5p


1.7e+004
2.6e+003
0.86
6.48 (−)
1.4e−017
182
hsa-miR-199a-5p


2.9e+002
5.0e+001
0.86
5.73 (−)
3.5e−019
320
hsa-miR-376c


3.4e+002
6.5e+001
0.86
5.23 (−)
2.6e−016
59
hsa-miR-487b


4.9e+002
9.4e+001
0.82
5.18 (−)
9.7e−016
38
hsa-miR-214*


2.4e+002
5.0e+001
0.86
4.83 (−)
2.2e−017
324
hsa-miR-382


2.1e+002
5.0e+001
0.83
4.27 (−)
6.6e−017
323
hsa-miR-381


5.0e+003
1.2e+003
0.81
4.22 (−)
2.7e−013
37
hsa-miR-214


2.1e+002
5.0e+001
0.86
4.21 (−)
8.5e−018
322
hsa-miR-379


2.1e+002
5.0e+001
0.83
4.14 (−)
1.2e−015
325
hsa-miR-409-3p


2.5e+002
6.7e+001
0.86
3.76 (−)
2.8e−016
19
hsa-miR-149


2.6e+002
7.5e+001
0.71
3.51 (−)
2.8e−007
42
hsa-miR-224


3.2e+002
9.9e+001
0.79
3.25 (−)
1.3e−011
334
hsa-miR-483-5p


3.0e+002
1.5e+002
0.79
2.08 (−)
9.5e−012
265
hsa-miR-130b


2.0e+002
1.0e+002
0.76
2.00 (−)
4.8e−009
22
hsa-miR-181a*





+ the higher expression of this miR is in kidney tumors


− the higher expression of this miR is in sarcoma, adrenal and mesothelioma tumors






FIG. 22 demonstrates binary decisions at node #33 of the decision-tree. Tumors originating in kidney (diamonds) are easily distinguished from tumors of adrenal, mesothelioma and sarcoma origin (squares) using the expression levels of hsa-miR-200b (SEQ ID NO: 29, y-axis), hsa-miR-30a (SEQ ID NO: 46, x-axis) and hsa-miR-149 (SEQ ID NO: 19, z-axis).







TABLE 35







miR expression (in florescence units) distinguishing between pheochromocytoma


(neuroendocrine tumor of the adrenal) and all sarcoma, adrenal carcinoma and mesothelioma


tumors
















SEQ





fold-

ID



median values
auROC
change
p-value
NO.
miR name
















5.0e+001
1.5e+004
0.96
295.36 (+) 
6.7e−067
65
hsa-miR-7


5.0e+001
9.8e+003
0.91
196.58 (+) 
5.0e−036
56
hsa-miR-375


1.3e+002
4.0e+003
0.85
29.73 (+) 
3.2e−009
11
hsa-miR-138


5.0e+001
1.0e+003
0.94
20.53 (+) 
1.5e−021
162
hsa-miR-129-3p


2.7e+002
4.0e+003
0.84
15.11 (+) 
3.0e−008
59
hsa-miR-487b


1.5e+002
2.2e+003
0.81
14.54 (+) 
7.4e−008
331
hsa-miR-432


7.0e+001
8.7e+002
0.84
12.45 (+) 
9.4e−011
350
hsa-miR-539


9.6e+002
1.2e+004
0.80
12.36 (+) 
8.3e−005
155
hsa-miR-127-3p


5.8e+001 
6.8e+002 
0.80
11.61 (+) 
 1.2e−0083
35
hsa-miR-485-3p


5.0e+001 
5.7e+002 
0.87
11.48 (+) 
2.7e−008
159
hsa-miR-124


5.3e+001 
5.6e+002 
0.86
10.67 (+) 
2.3e−014
336
hsa-miR-485-5p


5.0e+001 
5.2e+002 
0.83
10.38 (+) 
1.2e−012
332
hsa-miR-433


5.0e+001 
5.1e+002 
0.94
10.28 (+) 
1.1e−029
263
hsa-miR-129*


5.0e+001 
4.8e+002 
0.82
9.55 (+) 
9.6e−008
306
hsa-miR-323-3p


1.2e+002 
1.2e+003 
0.79
9.22 (+) 
1.0e−005
339
hsa-miR-495


5.8e+001 
5.2e+002 
0.80
9.01 (+) 
4.4e−006
337
hsa-miR-487a


1.6e+002 
1.4e+003 
0.80
8.75 (+) 
4.8e−006
275
hsa-miR-154


7.1e+001 
6.1e+002 
0.90
8.56 (+) 
6.0e−013
301
hsa-miR-29b-2*


5.3e+001 
4.5e+002 
0.78
8.54 (+) 
3.7e−005
276
hsa-miR-154*


5.3e+001 
4.1e+002 
0.83
7.77 (+) 
4.7e−009
330
hsa-miR-431*


9.8e+001 
7.4e+002 
0.81
7.56 (+) 
1.3e−006
317
hsa-miR-369-5p


6.4e+001 
4.8e+002 
0.80
7.56 (+) 
1.4e−007
309
hsa-miR-329


2.4e+002 
1.8e+003 
0.90
7.28 (+) 
1.4e−009
45
hsa-miR-29c*


1.5e+002 
1.1e+003 
0.79
7.24 (+) 
1.5e−005
318
hsa-miR-370


2.2e+002 
1.5e+003 
0.80
6.74 (+) 
1.5e−005
324
hsa-miR-382


1.3e+002 
8.6e+002 
0.76
6.53 (+) 
3.1e−004
352
hsa-miR-543


2.3e+002 
1.5e+003 
0.89
6.44 (+) 
2.6e−008
191
hsa-miR-29c


9.6e+001 
6.2e+002 
0.79
6.40 (+) 
1.1e−005
262
hsa-miR-127-5p


1.5e+002 
9.6e+002 
0.77
6.39 (+) 
2.3e−004
269
hsa-miR-134


5.4e+001 
3.3e+002 
0.90
6.03 (+) 
2.1e−012
313
hsa-miR-338-3p


2.2e+002 
1.3e+003 
0.84
5.80 (+) 
4.8e−008
19
hsa-miR-149


5.0e+001 
2.7e+002 
0.82
5.32 (+) 
7.5e−012
367
MID-00465


1.1e+002 
5.6e+002 
0.78
5.27 (+) 
5.3e−005
326
hsa-miR-409-5p


1.8e+002 
9.6e+002 
0.76
5.26 (+) 
3.1e−004
325
hsa-miR-409-3p


1.8e+002 
9.2e+002 
0.76
5.25 (+) 
8.2e−004
322
hsa-miR-379


5.0e+001 
2.5e+002 
0.79
5.05 (+) 
4.6e−008
327
hsa-miR-410


8.1e+002 
4.0e+003 
0.97
4.95 (+) 
3.4e−011
190
hsa-miR-29b


7.6e+001 
3.7e+002 
0.93
4.85 (+) 
7.6e−019
261
hsa-miR-1180


6.4e+001 
2.8e+002 
0.79
4.43 (+) 
1.0e−005
321
hsa-miR-377*


5.0e+001 
2.0e+002 
0.81
4.09 (+) 
2.9e−009
360
hsa-miR-873


5.3e+001 
2.1e+002 
0.88
4.08 (+) 
1.2e−012
353
hsa-miR-598


3.3e+002 
1.3e+003 
0.73
4.01 (+) 
3.0e−003
312
hsa-miR-337-5p


6.6e+001 
2.6e+002 
0.77
3.96 (+) 
8.8e−005
371
MID-16270


2.1e+003 
7.2e+003 
0.85
3.51 (+) 
6.2e−005
5
hsa-miR-10b


6.7e+001 
2.3e+002 
0.68
3.40 (+) 
2.6e−002
328
hsa-miR-411


6.9e+003 
2.2e+004 
0.77
3.17 (+) 
4.3e−004
205
hsa-miR-451


1.3e+003 
1.1e+002 
0.90
12.33 (−) 
2.9e−008
183
hsa-miR-199b-5p


5.5e+002 
1.1e+002 
0.86
4.75 (−) 
2.4e−006
38
hsa-miR-214*


1.5e+004 
3.2e+003 
0.84
4.48 (−) 
2.9e−005
181
hsa-miR-199a-3p


5.9e+003 
1.3e+003 
0.85
4.47 (−) 
6.8e−005
37
hsa-miR-214


7.8e+003 
1.8e+003 
0.80
4.23 (−) 
5.9e−004
40
hsa-miR-222


1.8e+004 
4.5e+003 
0.87
4.13 (−) 
9.0e−006
182
hsa-miR-199a-5p


5.2e+003 
1.3e+003 
0.80
4.11 (−) 
6.4e−004
147
hsa-miR-221


7.0e+002 
1.9e+002 
0.85
3.72 (−) 
4.7e−007 
17
hsa-miR-146b-5p


3.1e+002 
8.9e+001 
0.72
3.48 (−) 
2.3e−003
42
hsa-miR-224


2.7e+003
8.0e+002
0.81
3.42 (−)
3.8e−005
382
MID-22331


2.6e+004
7.9e+003
0.79
3.35 (−)
1.1e−004
34
hsa-miR-21


4.2e+002
1.4e+002
0.74
3.08 (−)
1.3e−003
18
hsa-miR-148a


6.3e+003
2.1e+003
0.83
3.03 (−)
2.6e−005
3
hsa-miR-100





+ the higher expression of this miR is in pheochromocytoma


− the higher expression of this miR is in sarcoma, adrenal carcinoma and mesothelioma tumors






FIG. 23 demonstrates binary decisions at node #34 of the decision-tree. Tumors originating in pheochromocytoma (diamonds) are easily distinguished from tumors of adrenal, mesothelioma and sarcoma origin (squares) using the expression levels of hsa-miR-375 (SEQ ID NO: 56, y-axis) and hsa-miR-7 (SEQ ID NO: 65, x-axis).







TABLE 36







miR expression (in florescence units) distinguishing between adrenal carcinoma


and mesothelioma or sarcoma tumors












median values
auROC
fold-change
p-value
SEQ ID NO.
miR name
















5.0e+001
2.6e+003
0.98
51.10 (+)  
1.3e−040
61
hsa-miR-509-3p


5.4e+001
1.3e+003
0.76
24.55 (+) 
4.9e−007
333
hsa-miR-483-3p


5.0e+001
1.2e+003
0.99
24.01 (+) 
8.1e−066
31
hsa-miR-202


6.4e+001
1.4e+003
0.95
21.83 (+) 
2.6e−024
347
hsa-miR-513a-5p


5.0e+001
6.0e+002
0.96
12.08 (+) 
9.3e−030
346
hsa-miR-509-3-5p


1.9e+002
2.2e+003
0.92
11.82 (+) 
2.2e−016
344
hsa-miR-503


5.0e+001
5.1e+002
0.98
10.25 (+) 
3.8e−033
345
hsa-miR-506


6.1e+001
5.9e+002
0.96
9.70 (+)
1.2e−026
387
MID-23751


3.1e+002
2.7e+003
0.71
8.66 (+)
6.0e−005
334
hsa-miR-483-5p


1.4e+002
1.1e+003
0.91
7.79 (+)
1.1e−015
351
hsa-miR-542-5p


2.0e+002
1.2e+003
0.72
5.77 (+)
8.5e−005
324
hsa-miR-382


1.0e+002
5.5e+002
0.75
5.44 (+)
3.1e−007
326
hsa-miR-409-5p


1.4e+002
7.2e+002
0.73
5.31 (+)
2.7e−004
269
hsa-miR-134


7.9e+002
3.9e+003
0.69
4.98 (+)
8.9e−003
155
hsa-miR-127-3p


2.1e+002
1.0e+003
0.68
4.93 (+)
6.4e−003
320
hsa-miR-376c


1.6e+002
7.8e+002
0.69
4.84 (+)
2.6e−003
322
hsa-miR-379


2.2e+002
1.0e+003
0.72
4.53 (+)
4.9e−005
59
hsa-miR-487b


1.5e+002
6.6e+002
0.69
4.49 (+)
1.3e−003
318
hsa-miR-370


1.7e+002
7.8e+002
0.71
4.45 (+)
2.9e−004
325
hsa-miR-409-3p


1.1e+003
4.7e+003
0.92
4.19 (+)
3.9e−011
245
MID-18336


2.9e+002
1.1e+003
0.84
3.79 (+)
1.8e−007
254
MID-23291


1.4e+002
5.0e+002
0.70
3.71 (+)
1.5e−004
331
hsa-miR-432


1.5e+002
5.3e+002
0.69
3.60 (+)
1.6e−003
275
hsa-miR-154


3.1e+002
1.1e+003
0.90
3.48 (+)
7.2e−011
180
hsa-miR-1973


1.7e+002
5.4e+002
0.63
3.22 (+)
6.9e−002
355
hsa-miR-654-3p


1.1e+003
3.5e+003
0.86
3.14 (+)
8.6e−009
370
MID-15986


1.8e+002
5.4e+002
0.64
3.07 (+)
4.5e−002
323
hsa-miR-381


2.9e+002
8.9e+002
0.63
3.07 (+)
5.8e−002
312
hsa-miR-337-5p


1.8e+003
5.6e+003
0.88
3.03 (+)
1.0e−008
178
hsa-miR-193b


1.4e+003
4.2e+003
0.81
3.02 (+)
1.6e−006
249
MID-20524


1.7e+003
9.5e+001
0.96
18.32 (−) 
1.3e−015
183
hsa-miR-199b-5p


1.9e+004
1.7e+003
0.95
10.80 (−) 
9.7e−014
181
hsa-miR-199a-3p


6.5e+002
6.1e+001
0.97
10.75 (−) 
2.6e−016
38
hsa-miR-214*


2.4e+004
2.5e+003
0.97
9.43 (−)
1.9e−015
182
hsa-miR-199a-5p


7.1e+003
9.0e+002
0.96
7.89 (−)
1.2e−011
37
hsa-miR-214


7.5e+003
1.5e+003
0.90
4.87 (−)
1.8e−012
3
hsa-miR-100


2.5e+003
7.6e+002
0.83
3.37 (−)
3.6e−006
25
hsa-miR-193a-3p


1.3e+003
4.3e+002
0.80
3.05 (−)
2.5e−006
169
hsa-miR-152





+ the higher expression of this miR is in adrenal carcinoma


− the higher expression of this miR is in sarcoma and mesothelioma tumors






hsa-miR-202 (SEQ ID NO: 31), hsa-miR-509-3p (SEQ ID NO: 61) and hsa-miR-214* (SEQ ID NO: 38) are used at node 35 of the binary-tree-classifier detailed in the invention to distinguish between adrenal carcinoma and sarcoma or mesothelioma tumors.







TABLE 37







miR expression (in florescence units) distinguishing between GIST and


mesothelioma or sarcoma tumors












median values
auROC
fold-change
p-value
SEQ ID NO.
miR name
















2.4e+002
5.6e+003
0.97
23.39 (+) 
4.2e−033
165
hsa-miR-143*


4.9e+003
1.0e+005
0.97
21.41 (+) 
1.7e−025
14
hsa-miR-143


8.1e+003
1.5e+005
0.99
18.42 (+) 
4.2e−026
15
hsa-miR-145


5.0e+001
7.9e+002
0.87
15.77 (+) 
1.5e−010
333
hsa-miR-483-3p


6.2e+001
8.4e+002
0.98
13.54 (+) 
1.5e−037
272
hsa-miR-145*


1.6e+002
1.6e+003
0.99
9.58 (+)
2.7e−024
270
hsa-miR-139-5p


1.8e+002
1.8e+003
0.96
9.49 (+)
2.7e−019
45
hsa-miR-29c*


6.1e+001
5.8e+002
0.95
9.48 (+)
7.9e−028
301
hsa-miR-29b-2*


1.9e+002
1.5e+003
0.94
7.89 (+)
1.9e−015
191
hsa-miR-29c


1.2e+003
6.3e+003
0.96
5.12 (+)
8.8e−014
46
hsa-miR-30a


1.9e+002
7.3e+002
0.93
3.84 (+)
6.6e−013
195
hsa-miR-30a*


2.4e+002
8.7e+002
0.92
3.66 (+)
1.8e−008
266
hsa-miR-132


6.1e+002
2.2e+003
0.91
3.52 (+)
4.2e−008
190
hsa-miR-29b


1.9e+002
6.5e+002
0.82
3.50 (+)
1.5e−006
19
hsa-miR-149


2.6e+002
9.0e+002
0.82
3.47 (+)
4.8e−005
334
hsa-miR-483-5p


9.3e+002
1.9e+002
0.70
5.00 (−)
2.1e−003
155
hsa-miR-127-3p


3.4e+003
7.1e+002
0.88
4.74 (−)
2.3e−007
25
hsa-miR-193a-3p


7.0e+003
1.9e+003
0.79
3.64 (−)
5.5e−004
147
hsa-miR-221


9.8e+003
2.8e+003
0.78
3.54 (−)
1.1e−003
40
hsa-miR-222


3.2e+004
9.8e+003
0.75
3.26 (−)
1.1e−003
34
hsa-miR-21





+ the higher expression of this miR is in GIST


− the higher expression of this miR is in sarcoma and mesothelioma tumors






hsa-miR-29C* (SEQ ID NO: 45) and hsa-miR-143 (SEQ ID NO: 14) are used at node 36 of the binary-tree-classifier detailed in the invention to distinguish between GIST and sarcoma or mesothelioma tumors.







TABLE 38







miR expression (in florescence units) distinguishing between chromophobe renal cell 


carcinoma tumors and clear cell or papillary renal cell carcinoma tumors












median values
auROC
fold-change
p-value
SEQ ID NO.
miR name
















8.8e+001
2.1e+003
0.99
23.68 (+) 
4.7e−017
13
hsa-miR-141


3.0e+002
5.7e+003
0.99
18.81 (+) 
8.4e−012
30
hsa-miR-200c


6.6e+001
9.8e+002
0.99
14.85 (+) 
7.5e−019
361
hsa-miR-874


5.0e+001
7.4e+002
0.97
14.80 (+) 
1.0e−014
280
hsa-miR-187


5.0e+001
7.2e+002
0.98
14.47 (+) 
4.7e−018
362
hsa-miR-891a


5.3e+003
7.4e+004
0.98
13.97 (+) 
5.3e−017
147
hsa-miR-221


7.6e+003
9.0e+004
0.97
11.89 (+) 
1.4e−015
40
hsa-miR-222


5.3e+001
5.1e+002
0.98
9.66 (+)
1.2e−017
291
hsa-miR-222*


1.4e+002
1.1e+003
0.94
8.01 (+)
2.7e−010
387
MID-23751


7.4e+001
5.4e+002
0.97
7.32 (+)
4.4e−015
290
hsa-miR-221*


1.1e+002
5.6e+002
0.93
4.97 (+)
3.2e−010
299
hsa-miR-296-5p


3.2e+002
1.5e+003
0.90
4.90 (+)
3.1e−007
152
hsa-miR-182


8.4e+002
3.3e+003
0.73
3.89 (+)
6.3e−003
178
hsa-miR-193b


5.4e+003
1.7e+004
0.92
3.26 (+)
2.2e−007
303
hsa-miR-30b


1.1e+003
3.5e+003
0.74
3.20 (+)
8.1e−003
242
MID-16489


6.2e+003
3.3e+002
0.85
18.53 (−) 
4.3e−006
49
hsa-miR-31


6.1e+002
5.0e+001
0.90
12.13 (−) 
2.1e−007
11
hsa-miR-138


1.8e+003
2.2e+002
0.98
8.38 (−)
6.7e−014
35
hsa-miR-21*


7.9e+004
1.0e+004
0.95
7.54 (−)
3.1e−013
34
hsa-miR-21


3.7e+003
5.4e+002
0.92
6.79 (−)
3.1e−009
36
hsa-miR-210


9.8e+002
1.7e+002
0.97
5.71 (−)
3.1e−013
206
hsa-miR-455-3p


1.0e+003
2.5e+002
0.91
4.07 (−)
4.7e−008
16
hsa-miR-146a


6.0e+002
1.7e+002
0.89
3.64 (−)
7.1e−007
170
hsa-miR-155


7.5e+002
2.1e+002
0.78
3.48 (−)
1.6e−003
177
hsa-miR-192


8.6e+002
2.5e+002
0.86
3.39 (−)
6.6e−006
17
hsa-miR-146b-5p





+ the higher expression of this miR is in chromophobe renal cell carcinoma tumors


− the higher expression of this miR is in clear cell or papillary renal cell carcinoma tumors






hsa-miR-210 (SEQ ID NO: 36) and hsa-miR-221 (SEQ ID NO: 147) are used at node #37 of the binary-tree-classifier detailed in the invention to distinguish between chromophobe renal cell carcinoma tumors and clear cell or papillary renal cell carcinoma tumors.







TABLE 39







miR expression (in florescence units) distinguishing between clear cell and papillary renal 


cell carcinoma tumors












median values
auROC
fold-change
p-value
SEQ ID NO.
miR name
















1.2e+002
5.7e+002
0.89
4.81 (+)
2.3e−005
344
hsa-miR-503


1.6e+003
5.9e+003
0.81
3.65 (+)
5.8e−003
382
MID-22331


1.8e+003
6.4e+003
0.94
3.54 (+)
1.1e−005
9
hsa-miR-126


1.7e+003
5.7e+003
0.82
3.45 (+)
3.0e−003
338
hsa-miR-494


1.1e+004
1.3e+003
0.87
8.35 (−)
3.1e−004
29
hsa-miR-200b


8.7e+003
1.3e+003
0.81
6.61 (−)
3.0e−002
49
hsa-miR-31


5.1e+003
9.5e+002
0.92
5.30 (−)
5.0e−005
28
hsa-miR-200a


2.1e+003
5.1e+002
1.00
4.10 (−)
1.1e−009
195
hsa-miR-30a*


1.9e+004
5.0e+003
1.00
3.70 (−)
4.5e−010
46
hsa-miR-30a


5.3e+003
1.6e+003
0.86
3.39 (−)
6.9e−004
4
hsa-miR-10a


7.6e+002
2.3e+002
0.76
3.23 (−)
2.0e−002
11
hsa-miR-138


6.4e+002
2.0e+002
0.79
3.17 (−)
7.4e−003
254
MID-23291





+ the higher expression of this miR is in renal clear cell carcinoma tumors


− the higher expression of this miR is in papillary renal cell carcinoma tumors






hsa-miR-31 (SEQ ID NO: 49) and hsa-miR-126 (SEQ ID NO: 9) are used at node 38 of the binary-tree-classifier detailed in the invention to distinguish between renal clear cell and papillary cell carcinoma tumors.







TABLE 40







miR expression (in florescence units) distinguishing between pleural mesothelioma


and sarcoma tumors












median values
auROC
fold-change
p-value
SEQ ID NO.
miR name
















1.2e+002
1.7e+003
0.78
13.97 (+)  
1.7e−006
49
hsa-miR-31


4.3e+002 
2.1e+003
0.89
5.01 (+)
2.0e−011
35
hsa-miR-21*


5.9e+002 
1.6e+003
0.84
2.75 (+)
2.1e−008
17
hsa-miR-146b-5p


2.5e+004 
6.9e+004
0.89
2.71 (+)
4.8e−010
34
h`sa-miR-21


2.4e+003 
6.1e+003
0.77
2.57 (+)
2.3e−005
25
hsa-miR-193a-3p


1.2e+003
3.1e+003
0.75
2.49 (+)
5.9e−005
36
hsa-miR-210


4.6e+002
1.1e+003
0.70
2.33 (+)
9.7e−004
168
hsa-miR-150


2.9e+002
6.8e+002
0.75
2.33 (+)
1.4e−004
170
hsa-miR-155


3.2e+002
7.3e+002
0.76
2.25 (+)
1.4e−005
26
hsa-miR-193a-5p


1.1e+003
2.4e+003
0.76
2.13 (+)
1.7e−004
4
hsa-miR-10a


5.1e+002
1.0e+003
0.70
2.03 (+)
1.1e−003
190
hsa-miR-29b


9.0e+002
1.8e+003
0.77
1.99 (+)
1.5e−005
46
hsa-miR-30a


2.6e+003
4.9e+003
0.71
1.90 (+)
8.9e−003
10
hsa-miR-130a


2.8e+003
5.2e+003
0.69
1.88 (+)
1.6e−003
240
MID-15965


4.3e+003
7.3e+003
0.67
1.71 (+)
1.5e−002
43
hsa-miR-29a


5.0e+003
8.2e+003
0.72
1.65 (+)
1.1e−004
39
hsa-miR-22


3.8e+003
5.9e+003
0.64
1.57 (+)
2.9e−002
385
MID-23168


9.9e+002
1.5e+003
0.66
1.55 (+)
1.4e−002
63
hsa-miR-574-5p


7.5e+002
1.2e+003
0.66
1.53 (+)
2.8e−002
202
hsa-miR-378


2.7e+004
7.4e+003
0.84
3.62 (−)
3.3e−007
181
hsa-miR-199a-3p


1.1e+004
3.1e+003
0.81
3.45 (−)
3.2e−006
37
hsa-miR-214


2.8e+003
8.3e+002
0.85
3.36 (−)
6.7e−008
5
hsa-miR-10b


2.9e+003
9.1e+002
0.74
3.19 (−)
2.0e−004
183
hsa-miR-199b-5p


3.0e+004
1.1e+004
0.80
2.84 (−)
6.0e−005
182
hsa-miR-199a-5p


8.4e+002
3.8e+002
0.78
2.22 (−)
5.7e−005
38
hsa-miR-214*


1.2e+003
7.0e+002
0.75
1.69 (−)
2.8e−004
206
hsa-miR-455-3p


7.0e+002
4.4e+002
0.75
1.61 (−)
2.9e−003
296
hsa-miR-26b


5.0e+004
3.2e+004
0.71
1.58 (−)
4.3e−003
1
hsa-let-7c





+ the higher expression of this miR is in pleural mesothelioma tumors


− the higher expression of this miR is in sarcoma tumors






hsa-miR-21* (SEQ ID NO: 35) hsa-miR-130a (SEQ ID NO: 10) and hsa-miR-10b (SEQ ID NO: 5) are used at node 39 of the binary-tree-classifier detailed in the invention to distinguish between pleural mesothelioma tumors and sarcoma tumors.







TABLE 41







miR expression(in florescence units) distinguishing between synovial sarcoma and other 


sarcoma tumors












median values
auROC
fold-change
p-value
SEQ ID NO.
miR name
















5.1e+001
1.3e+003
0.89
25.03 (+) 
2.9e−009
152
hsa-miR-182


5.0e+001
1.1e+003
0.92
21.59 (+) 
9.2e−009
29
hsa-miR-200b


5.0e+001
9.7e+002
0.88
19.35 (+) 
5.9e−007
159
hsa-miR-124


5.0e+001
6.4e+002
0.92
12.81 (+) 
1.5e−008
28
hsa-miR-200a


1.3e+002
8.9e+002
0.88
7.00 (+)
8.2e−005
339
hsa-miR-495


1.6e+002
1.1e+003
0.89
6.94 (+)
1.6e−005
275
hsa-miR-154


1.1e+002
7.3e+002
0.87
6.53 (+)
2.8e−004
352
hsa-miR-543


1.4e+002
8.8e+002
0.91
6.51 (+)
6.1e−006
19
hsa-miR-149


3.3e+002
2.0e+003
0.86
6.22 (+)
9.6e−005
320
hsa-miR-376c


1.0e+003
6.2e+003
0.84
6.05 (+)
1.5e−003
155
hsa-miR-127-3p


9.7e+001
5.2e+002
0.84
5.40 (+)
1.7e−004
262
hsa-miR-127-5p


7.8e+002
4.2e+003
0.86
5.33 (+)
2.5e−004
38
hsa-miR-214*


9.5e+003
5.0e+004
0.84
5.29 (+)
7.3e−003
37
hsa-miR-214


2.9e+004
1.4e+005
0.87
4.90 (+)
4.7e−003
182
hsa-miR-199a-5p


1.4e+002
6.4e+002
0.81
4.56 (+)
1.9e−003
331
hsa-miR-432


1.1e+002
5.0e+002
0.84
4.43 (+)
2.7e−004
317
hsa-miR-369-5p


2.1e+002
8.9e+002
0.78
4.19 (+)
9.6e−003
323
hsa-miR-381


1.9e+002
7.7e+002
0.81
3.96 (+)
2.7e−003
355
hsa-miR-654-3p


5.6e+003
2.1e+004
0.91
3.79 (+)
9.6e−006
3
hsa-miR-100


1.7e+002
6.5e+002
0.86
3.76 (+)
2.8e−004
282
hsa-miR-196a


4.1e+002
1.5e+003
0.80
3.55 (+)
6.0e−003
312
hsa-miR-337-5p


2.6e+004
9.1e+004
0.86
3.52 (+)
7.4e−003
181
hsa-miR-199a-3p


1.6e+002
5.5e+002
0.80
3.41 (+)
6.5e−003
269
hsa-miR-134


1.9e+002
6.4e+002
0.79
3.32 (+)
8.3e−003
318
hsa-miR-370


3.2e+002
1.0e+003
0.78
3.08 (+)
7.6e−003
59
hsa-miR-487b


2.1e+002
6.4e+002
0.87
3.02 (+)
7.1e−007
266
hsa-miR-132


2.2e+002
6.3e+002
0.78
2.92 (+)
1.1e−002
322
hsa-miR-379


4.4e+004
1.2e+005
0.92
2.83 (+)
1.6e−004
8
hsa-miR-125b


2.4e+002
6.1e+002
0.78
2.58 (+)
8.4e−003
324
hsa-miR-382


2.4e+003
6.0e+003
0.80
2.45 (+)
1.8e−003
10
hsa-miR-130a


9.2e+003
8.4e+002
0.96
10.95 (−) 
2.1e−010
40
hsa-miR-222


6.8e+003
6.7e+002
0.96
10.19 (−) 
7.9e−010
147
hsa-miR-221


1.8e+003
3.6e+002
0.88
4.92 (−)
2.1e−005
169
hsa-miR-152


7.9e+003
1.7e+003
0.72
4.72 (−)
2.8e−002
205
hsa-miR-451


2.7e+004
5.9e+003
0.84
4.59 (−)
4.4e−005
34
hsa-miR-21


5.9e+002
1.4e+002
0.83
4.32 (−)
9.2e−004
168
hsa-miR-150


5.5e+003
1.3e+003
0.92
4.15 (−)
2.2e−007
14
hsa-miR-143


9.5e+003
2.9e+003
0.93
3.30 (−)
2.7e−007
15
hsa-miR-145


3.5e+003
1.0e+003
0.91
3.30 (−)
1.6e−003
12
hsa-miR-140-3p


1.0e+003
3.5e+002
0.78
2.92 (−)
6.1e−003
46
hsa-miR-30a


1.0e+003
3.6e+002
0.82
2.86 (−)
4.1e−004
255
MID-23794


5.8e+003
2.0e+003
0.88
2.82 (−)
1.9e−005
39
hsa-miR-22


1.1e+003
4.0e+002
0.77
2.74 (−)
3.9e−003
23
hsa-miR-185


5.8e+002
2.4e+002
0.80
2.45 (−)
6.8e−003
190
hsa-miR-29b


7.1e+002
3.1e+002
0.79
2.27 (−)
4.9e−003
236
MID-00689


6.7e+004
3.2e+004
0.85
2.10 (−)
2.2e−004
386
MID-23178


7.7e+004
3.7e+004
0.80
2.08 (−)
2.9e−003
379
MID-18395


7.8e+002
3.8e+002
0.78
2.07 (−)
7.4e−003
202
hsa-miR-378





+ the higher expression of this miR is in synovial sarcoma tumors


− the higher expression of this miR is in other sarcoma tumors






hsa-miR-100 (SEQ ID NO: 3) hsa-miR-145 (SEQ ID NO: 15) and hsa-miR-222 (SEQ ID NO: 40) are used at node 40 of the binary-tree-classifier detailed in the invention to distinguish between synovial sarcoma tumors and other sarcoma tumors.







TABLE 42







miR expression (in florescence units) distinguishing between chondrosarcoma and other 


non synovial sarcoma tumors












median values
auROC
fold-change
p-value
SEQ ID NO.
miR name
















2.9e+003
2.2e+005
1.00
75.69 (+) 
2.1e−022
12
hsa-miR-140-3p


1.5e+002
5.1e+003
0.91
35.23 (+) 
8.5e−015
271
hsa-miR-140-5p


1.1e+003
1.6e+004
0.98
14.49 (+) 
6.1e−015
206
hsa-miR-455-3p


5.0e+001
5.5e+002
0.71
11.03 (+) 
3.1e−003
333
hsa-miR-483-3p


9.5e+001
1.1e+003
0.88
11.01 (+) 
1.2e−006
11
hsa-miR-138


9.2e+001
8.2e+002
0.87
8.87 (+)
6.3e−012
58
hsa-miR-455-5p


1.1e+003
4.7e+003
0.91
4.37 (+)
1.5e−006
36
hsa-miR-210


3.6e+002
1.4e+003
0.83
3.98 (+)
3.1e−004
18
hsa-miR-148a


1.5e+003
3.6e+003
0.72
2.36 (+)
2.3e−002
178
hsa-miR-193b


1.3e+004
2.8e+004
0.84
2.13 (+)
1.5e−004
293
hsa-miR-23b


2.7e+003
5.5e+003
0.80
2.05 (+)
5.8e−004
189
hsa-miR-27b


3.4e+003
6.7e+002
0.70
5.01 (−)
1.1e−004
382
MID-22331


6.6e+002
1.7e+002
0.81
3.91 (−)
1.2e−004
381
MID-19962


3.2e+003
8.5e+002
0.83
3.76 (−)
1.9e−004
240
MID-15965


1.5e+003
4.2e+002
0.79
3.47 (−)
8.0e−004
249
MID-20524


2.9e+003
9.0e+002
0.85
3.27 (−)
1.3e−005
5
hsa-miR-10b


2.9e+003
1.0e+003
0.78
2.92 (−)
6.9e−005
377
MID-17866


7.1e+002
2.7e+002
0.75
2.62 (−)
1.3e−003
235
hsa-miR-1978


7.0e+002
2.8e+002
0.81
2.48 (−)
4.4e−005
17
hsa-miR-146b-5p


1.3e+003
5.7e+002
0.71
2.36 (−)
2.7e−002
20
hsa-miR-17


4.5e+003
1.9e+003
0.73
2.36 (−)
8.2e−003
385
MID-23168


1.1e+004
5.0e+003
0.74
2.16 (−)
4.8e−003
384
MID-23017


1.1e+003
5.4e+002
0.79
2.04 (−)
3.2e−004
46
hsa-miR-30a


1.6e+004
8.1e+003
0.83
2.02 (−)
3.0e−004
283
hsa-miR-1979





+ the higher expression of this miR is in chondrosarcoma tumors


− the higher expression of this miR is in other non-synovial sarcoma tumors






hsa-miR-140-3p (SEQ ID NO: 12) and hsa-miR-455-5p (SEQ ID NO: 58) are used at node 41 of the binary-tree-classifier detailed in the invention to distinguish between chondrosarcoma tumors and other non-synovial sarcoma tumors.







TABLE 43







miR expression (in florescence units) distinguishing between liposarcoma 


and other non chondrosarcoma and non synovial sarcoma tumors












median values
auROC
fold-change
p-value
SEQ ID NO.
miR name
















1.9e+004
1.2e+005
0.93
6.18 (+) 
1.6e−011
295
hsa-miR-26a


4.2e+003
1.8e+004
0.73
4.20 (+)
8.1e−003
205
hsa-miR-451


1.5e+003
5.9e+003
0.84
3.94 (+) 
6.5e−006
25
hsa-miR-193a-3p


2.4e+002
8.8e+002
0.88
3.70 (+) 
7.5e−007
26
hsa-miR-193a-5p


6.1e+003
2.0e+004
0.88
3.24 (+)
2.2e−005
231
hsa-miR-99a


2.3e+003
5.9e+003
0.75
2.60 (+)
1.9e−003
183
hsa-miR-199b-5p


3.1e+002
7.9e+002
0.79
2.54 (+)
1.7e−004
42
hsa-miR-224


2.9e+002
7.4e+002
0.71
2.54 (+)
9.9e−003
254
MID-23291


4.2e+002
1.0e+003
0.71
2.38 (+)
1.5e−002
168
hsa-miR-150


3.2e+002
7.7e+002
0.77
2.36 (+)
1.1e−004
64
hsa-miR-652


4.8e+003
1.1e+004
0.84
2.27 (+)
5.4e−006
14
hsa-miR-143


1.4e+003
3.0e+003
0.76
2.20 (+)
2.7e−004
178
hsa-miR-193b


7.9e+003
1.7e+004
0.78
2.13 (+)
1.1e−004
15
hsa-miR-145


4.6e+003
9.7e+003
0.79
2.12 (+)
9.8e−004
39
hsa-miR-22


1.4e+003
3.1e+002
0.79
4.49 (−)
1.8e−004
36
hsa-miR-210


2.4e+003
9.0e+002
0.71
2.60 (−)
1.2e−002
154
hsa-miR-181b


5.9e+002
2.6e+002
0.75
2.29 (−)
4.0e−003
265
hsa-miR-130b


6.5e+002
3.0e+002
0.75
2.16 (−)
3.2e−003
174
hsa-miR-181d


1.2e+004
5.6e+003
0.79
2.14 (−)
2.0e−004
384
MID-23017


3.3e+003
1.6e+003
0.80
2.04 (−)
6.6e−004
67
hsa-miR-92a





+ the higher expression of this miR is in liposarcoma tumors


− the higher expression of this miR is in other non-chondrosarcoma and non-synovial sarcoma tumors






hsa-miR-210 (SEQ ID NO: 36) and hsa-miR-193a-5p (SEQ ID NO: 26) are used at node 42 of the binary-tree-classifier detailed in the invention to distinguish between liposarcoma tumors and other non-chondrosarcoma and non-synovial sarcoma tumors.







TABLE 44







miR expression (in florescence units) distinguishing between Ewing sarcoma or


osteosarcoma; and rhabdomyosarcoma, malignant fibrous histiocytoma (MFH) or


fibrosarcoma












median values
auROC
fold-change
p-value
SEQ ID NO.
miR name
















1.9e+002
1.2e+003
0.87
6.62 (+)
1.1e−006
22
hsa-miR-181a*


1.1e+003
6.4e+003
0.91
5.68 (+)
8.7e−009
154
hsa-miR-181b


3.7e+003
2.1e+004
0.93
5.67 (+)
2.9e−010
21
hsa-miR-181a


4.2e+002
1.8e+003
0.85
4.19 (+)
3.5e−006
174
hsa-miR-181d


2.9e+003
9.4e+003
0.72
3.27 (+)
1.2e−002
205
hsa-miR-451


1.8e+003
4.7e+003
0.78
2.63 (+)
2.9e−003
158
hsa-miR-106a


1.1e+003
2.8e+003
0.78
2.52 (+)
2.9e−003
186
hsa-miR-20a


2.0e+003
4.9e+003
0.81
2.45 (+)
9.2e−005
148
hsa-miR-93


1.1e+003
2.6e+003
0.77
2.32 (+)
5.1e−003
20
hsa-miR-17


6.0e+002
1.3e+002
0.71
4.54 (−)
1.1e−002
59
hsa-miR-487b


4.9e+004
1.7e+004
0.84
2.86 (−)
2.9e−005
8
hsa-miR-125b


3.4e+003
1.3e+003
0.72
2.70 (−)
9.4e−003
183
hsa-miR-199b-5p


8.1e+003
3.5e+003
0.76
2.34 (−)
1.1e−003
231
hsa-miR-99a





+ the higher expression of this miR is in Ewing sarcoma or osteosarcoma tumors


− the higher expression of this miR is in rhabdomyosarcoma, malignant fibrous histiocytoma (MFH) or fibrosarcoma tumors






hsa-miR-181a (SEQ ID NO: 21) is used at node 43 of the binary-tree-classifier detailed in the invention to distinguish between Ewing sarcoma or osteosarcoma tumors and rhabdomyosarcoma, malignant fibrous histiocytoma (MFH) or fibrosarcoma tumors.







TABLE 45







miR expression(in florescence units) distinguishing between Ewing sarcoma and


osteosarcoma












median values
auROC
fold-change
p-value
SEQ ID NO.
miR name
















1.6e+002
1.1e+003
1.00
6.60 (+)
3.7e−006
155
hsa-miR-127-3p


1.4e+003
8.5e+003
0.97
5.85 (+)
8.9e−004
179
hsa-miR-195


2.8e+003
1.4e+004
0.86
4.90 (+)
1.4e−002
43
hsa-miR-29a


1.4e+003
6.5e+003
1.00
4.58 (+)
1.1e−004
208
hsa-miR-497


1.7e+002
7.6e+002
0.88
4.42 (+)
1.0e−003
278
hsa-miR-181a-2*


4.0e+002
1.6e+003
0.86
4.05 (+)
6.0e−003
17
hsa-miR-146b-5p


3.4e+003
8.9e+003
0.81
2.64 (+)
1.4e−002
385
MID-23168


8.0e+002
2.1e+003
0.77
2.60 (+)
1.5e−002
174
hsa-miR-181d


1.6e+003
4.1e+003
0.82
2.55 (+)
1.3e−002
5
hsa-miR-10b


2.2e+003
4.9e+003
0.84
2.19 (+)
7.1e−003
52
hsa-miR-34a


2.5e+004
5.4e+004
0.97
2.16 (+)
2.7e−004
257
hsa-let-7b


2.5e+002
5.2e+002
0.88
2.12 (+)
2.1e−003
366
MID-00144


4.5e+002
9.4e+002
0.84
2.06 (+)
6.2e−003
48
hsa-miR-30e


1.3e+003
5.0e+001
0.96
25.44 (−) 
7.9e−005
49
hsa-miR-31


1.2e+004
2.0e+003
0.89
5.72 (−)
1.4e−003
12
hsa-miR-140-3p


3.8e+003
7.6e+002
0.94
4.92 (−)
5.2e−005
25
hsa-miR-193a-3p


1.8e+003
4.4e+002
0.89
4.09 (−)
3.3e−003
169
hsa-miR-152


3.7e+004
1.2e+004
0.89
3.00 (−)
3.2e−003
34
hsa-miR-21


8.1e+002
2.7e+002
0.83
2.96 (−)
1.7e−003
35
hsa-miR-21*


1.7e+003
6.7e+002
0.88
2.55 (−)
4.2e−003
23
hsa-miR-185


2.1e+004
8.2e+003
0.82
2.53 (−)
1.7e−002
384
MID-23017


4.3e+003
1.7e+003
0.84
2.52 (−)
3.8e−003
189
hsa-miR-27b


5.1e+003
2.3e+003
0.80
2.18 (−)
3.0e−002
377
MID-17866


9.6e+002
4.4e+002
0.78
2.17 (−)
3.0e−002
265
hsa-miR-130b


3.7e+004
1.8e+004
0.82
2.07 (−)
3.3e−003
294
hsa-miR-24


1.8e+004
8.8e+003
0.86
2.03 (−)
9.0e−003
293
hsa-miR-23b


3.0e+004
1.5e+004
0.80
2.02 (−)
1.6e−002
292
hsa-miR-23a





+ the higher expression of this miR is in Ewing sarcoma tumors


− the higher expression of this miR is in osteosarcoma tumors






FIG. 24 demonstrates binary decisions at node #44 of the decision-tree. Tumors originating in Ewing sarcoma (diamonds) are easily distinguished from tumors of osteosarcoma origin (squares) using the expression levels of hsa-miR-31 (SEQ ID NO: 49, y-axis) and hsa-miR-193a-3p (SEQ ID NO: 25, x-axis).







TABLE 46







miR expression (in florescence units) distinguishing between rhabdomyosarcoma


and malignant fibrous histiocytoma (MFH) or fibrosarcoma












median values
auROC
fold-change
p-value
SEQ ID NO.
miR name
















5.0e+001
4.1e+003
0.96
81.34 (+) 
1.9e−007
33
hsa-miR-206


5.7e+001
4.3e+003
0.89
74.89 (+) 
1.8e−004
268
hsa-miR-133b


5.9e+001
3.9e+003
0.88
66.65 (+) 
3.2e−004
267
hsa-miR-133a


5.0e+001
1.3e+003
0.89
25.89 (+) 
3.9e−006
333
hsa-miR-483-3p


5.3e+001
5.2e+002
0.85
9.90 (+)
1.3e−004
276
hsa-miR-154*


5.8e+001
5.6e+002
0.85
9.63 (+)
1.2e−004
319
hsa-miR-376a


5.7e+001
5.1e+002
0.86
9.00 (+)
4.8e−005
306
hsa-miR-323-3p


2.5e+002
1.8e+003
0.84
7.01 (+)
2.8e−003
320
hsa-miR-376c


2.6e+002
1.7e+003
0.82
6.52 (+)
3.9e−003
334
hsa-miR-483-5p


3.1e+002
1.9e+003
0.87
6.22 (+)
5.1e−004
323
hsa-miR-381


1.0e+002
6.3e+002
0.85
6.19 (+)
5.4e−004
300
hsa-miR-299-3p


1.3e+002
7.9e+002
0.82
6.18 (+)
1.4e−003
281
hsa-miR-188-5p


4.1e+002
2.3e+003
0.86
5.73 (+)
1.4e−003
59
hsa-miR-487b


1.5e+002
8.4e+002
0.85
5.68 (+)
8.1e−004
339
hsa-miR-495


3.7e+002
1.7e+003
0.79
4.57 (+)
3.1e−002
316
hsa-miR-362-5p


2.0e+002
9.2e+002
0.80
4.49 (+)
2.4e−003
176
hsa-miR-18a


2.9e+002
1.3e+003
0.82
4.39 (+)
1.4e−003
348
hsa-miR-532-3p


1.8e+002
7.8e+002
0.85
4.27 (+)
4.0e−004
352
hsa-miR-543


4.0e+002
1.7e+003
0.81
4.18 (+)
2.3e−002
349
hsa-miR-532-5p


1.9e+003
7.8e+003
0.87
4.14 (+)
4.9e−004
67
hsa-miR-92a


5.7e+002
2.4e+003
0.86
4.13 (+)
9.2e−004
357
hsa-miR-660


1.3e+002
5.6e+002
0.78
4.13 (+)
4.2e−003
315
hsa-miR-362-3p


2.3e+002
8.6e+002
0.81
3.73 (+)
2.8e−003
343
hsa-miR-502-3p


2.0e+002
7.2e+002
0.84
3.64 (+)
1.5e−003
342
hsa-miR-501-3p


2.3e+002
8.5e+002
0.82
3.62 (+)
6.7e−003
355
hsa-miR-654-3p


1.9e+002
6.7e+002
0.79
3.56 (+)
1.3e−002
340
hsa-miR-500


2.4e+002
8.4e+002
0.80
3.56 (+)
7.9e−003
344
hsa-miR-503


2.2e+003
7.6e+003
0.78
3.53 (+)
7.2e−003
10
hsa-miR-130a


2.6e+002
8.8e+002
0.80
3.35 (+)
3.7e−003
341
hsa-miR-500*


2.6e+002
7.9e+002
0.79
3.06 (+)
7.3e−003
331
hsa-miR-432


9.3e+002
2.7e+003
0.77
2.90 (+)
1.4e−002
20
hsa-miR-17


4.3e+002
1.2e+003
0.86
2.90 (+)
1.0e−003
277
hsa-miR-17*


2.4e+002
6.7e+002
0.83
2.77 (+)
7.0e−003
318
hsa-miR-370


1.6e+003
4.5e+003
0.78
2.75 (+)
1.4e−002
158
hsa-miR-106a


4.3e+002
1.1e+003
0.83
2.67 (+)
3.0e−003
265
hsa-miR-130b


1.0e+003
2.7e+003
0.86
2.63 (+)
7.1e−004
284
hsa-miR-19b


8.6e+002
2.1e+003
0.82
2.43 (+)
8.6e−003
36
hsa-miR-210


6.1e+003
6.8e+002
0.90
8.92 (−)
1.8e−004
183
hsa-miR-199b-5p


1.9e+004
4.5e+003
0.83
4.15 (−)
8.0e−004
40
hsa-miR-222


1.1e+003
3.1e+002
0.90
3.55 (−)
5.6e−005
63
hsa-miR-574-5p


1.1e+004
3.2e+003
0.82
3.52 (−)
2.2e−003
147
hsa-miR-221


5.9e+003
1.8e+003
0.80
3.25 (−)
2.0e−003
43
hsa-miR-29a


5.2e+002
1.6e+002
0.82
3.19 (−)
5.4e−003
289
hsa-miR-22*


5.1e+003
1.7e+003
0.82
3.04 (−)
7.0e−003
52
hsa-miR-34a


8.1e+002
2.9e+002
0.76
2.81 (−)
1.4e−002
190
hsa-miR-29b


1.2e+003
4.5e+002
0.86
2.67 (−)
4.7e−003
4
hsa-miR-10a


3.7e+003
1.5e+003
0.86
2.43 (−)
1.3e−003
5
hsa-miR-10b


7.0e+003
2.9e+003
0.85
2.39 (−)
1.5e−003
39
hsa-miR-22


1.6e+003
6.9e+002
0.78
2.25 (−)
1.5e−002
169
hsa-miR-152


2.9e+003
1.3e+003
0.76
2.19 (−)
2.8e−002
208
hsa-miR-497





+ the higher expression of this miR is in rhabdomyosarcoma tumors


− the higher expression of this miR is in MFH or fibrosarcoma tumors






FIG. 25 demonstrates binary decisions at node #45 of the decision-tree. Tumors originating in Rhabdomyosarcoma (diamonds) are easily distinguished from tumors of malignant fibrous histiocytoma (MFH) or fibrosarcoma origin (squares) using the expression levels of hsa-miR-206 (SEQ ID NO: 33, y-axis), hsa-miR-22 (SEQ ID NO: 39, x-axis) and hsa-miR-487b (SEQ ID NO: 59, z-axis).







TABLE 47







β values of the decision tree classifier


The classification at node 11 is based on the gender of subject rather than on beta values;


accordingly, no data is provided for this node. PTH = 0.5 for all nodes










miR 3
miR 2
miR 1



















SEQ
miR

SEQ
miR

SEQ
miR
β0



β3
ID NO
hsa-
β2
ID NO
hsa-
β1
ID NO
hsa-
intercept
Node


























2.3127
55
miR-372
−23.3111
1








2.3127
6
miR-122
−26.9408
2





−1.379
9
miR-126
1.8567
29
miR-200b
−3.8519
3





−1.2306
46
miR-30a
1.9582
30
miR-200c
−8.2646
4


−0.88435
46
miR-
−1.7697
2
let-7e
1.1979
16
miR-146a
17.4706
5




30a





1.7188
68
miR-92b
1.5475
66
miR-9*
−32.5621
6





2.0005
208
miR-497
−1.1606
40
miR-222
−9.5521
7


1.6602
56
miR-
1.2404
65
miR-7
−1.0267
25
miR-193a-3p
−23.053
8




375





1.1414
35
miR-21*
2.0115
27
miR-194
−29.3207
9





0.9879
14
miR-143
−1.5458
21
miR-181a
1.244
10





−1.256
211
miR-516a-5p
−1.942
29
miR-200b
21.3416
12


−1.0128
51
miR-
1.1064
32
miR-205
−1.1455
7
miR-125a-5p
10.3775
13




345


0.82076
56
miR-
0.93196
50
miR-342-3p
1.9505
25
miR-193a-3p
−40.666
14




375


−0.91632
32
miR-
0.61098
4
miR-10a
−1.8153
39
miR-22
26.2937
15




205


−1.119
4
miR-
1.5494
11
miR-138
−1.3023
148
miR-93
9.4008
16




10a





−1.4509
17
miR-146b-5p
−1.801
34
miR-21
42.5529
17


−1.3119
67
miR-
−0.63021
49
miR-31
1.7974
25
miR-193a-3p
0.52521
18




92a


1.6447
34
miR-
−1.3077
202
miR-378
0.9662
11
miR-138
−20.7179
19




21





−2.0444
34
miR-21
1.0814
3
miR-100
15.0039
20


1.734
69
miR-
0.22547
191
miR-29c
1.5137
24
miR-191
−31.6015
21




934


1.6178
54
miR-
0.86212
1
let-7c
1.41
5
miR-10b
−44.3141
22




361-




5p


−1.8652
23
miR-
1.3275
5
miR-10b
−0.32773
11
miR-138
7.6168
23




185





1.5521
47
miR-30d
−1.7146
50
miR-342-3p
2.4904
24





−1.3096
45
miR-29c*
1.9063
20
miR-17
−10.0563
26


−0.63749
67
miR-
−1.5907
68
miR-92b
1.5531
40
miR-222
−2.3904
27




92a


1.0197
53
miR-
−0.65807
37
miR-214
1.9688
64
miR-652
−22.027
28




34c-




5p





−1.3936
18
miR-148a
1.8457
34
miR-21
−11.4697
29


−0.50909
146
1201
−0.79749
36
miR-210
−1.3059
42
miR-224
21.7628
30


−1.3361
46
miR-
1.6268
43
miR-29a
0.95763
20
miR-17
−17.747
31




30a


−1.8214
51
miR-
0.62041
16
miR-146a
1.0661
49
miR-31
−2.3716
32




345


1.0224
46
miR-
−2.0172
19
miR-149
0.48415
29
miR-200b
−4.226
33




30a





0.87847
56
miR-375
2.1394
65
miR-7
−29.6828
34


−0.057027
38
miR-
0.76095
61
miR-509-3p
2.1832
31
miR-202
−23.6445
35




214*





1.9413
14
miR-143
1.2571
45
miR-29c*
−41.4047
36





−0.63202
36
miR-210
2.2247
147
miR-221
−25.1227
37





2.3043
9
miR-126
−0.19797
49
miR-31
−24.5409
38


1.7948
35
miR-
−1.0484
5
miR-10b
1.014
10
miR-130a
−20.7495
39




21*


−0.77759
15
miR-
−1.0289
40
miR-222
1.9198
3
miR-100
−6.0971
40




145





1.6244
58
miR-455-5p
1.6462
12
miR-140-3p
−38.5059
41





1.9298
26
miR-193a-5p
−0.84091
36
miR-210
−10.7873
42








2.3127
21
miR-181a
−30.4778
43





−1.0974
49
miR-31
−2.0358
25
miR-193a-3p
31.0975
44


1.8651
487
miR-
1.0201
59
miR-487b
−0.91078
39
miR-22
−17.5516
45




206
















TABLE 48







Using fine-needle aspiration (FNA), pleural effusion or bronchial


brushing for the identification of cancer tissue of origin










Class identified
Biopsy Site
Histological Type
Sampling Method





lung-small
Lymph Node
Neuroendocrine;
percutaneous FNA




Small



UpperSCC
Lung
Non-small;
percutaneous FNA




squamous



UpperSCC
Lung
Non-small;
percutaneous FNA




adenocarcinoma



lung-small
Lung
Neuroendocrine;
percutaneous FNA




Small



lung-adeno
Lung
Non-small;
percutaneous FNA




adenocarcinoma



UpperSCC
Lung
Non-small;
percutaneous FNA




squamous



lung-small
Lymph Node
Neuroendocrine;
transbronchial FNA




Small



lung-small
Lung
Neuroendocrine;
transbronchial FNA




Small



lung-adeno
Lung pleura
Non-small;
Pleural effusion




adenocarcinoma



lung-adeno
Lung pleura
Non-
Pleural effusion




small; 





adenocarcinoma



Lung, small
Lung
Neuroendocrine;
bronchial brushing




Small



Lung, small
Lung
Neuroendocrine;
bronchial brushing




Small



Lung, small
Lung
Neuroendocrine;
bronchial brushing




Small



Lung, small
Lung
Neuroendocrine;
bronchial brushing




Small



Lung, small
Lung
Neuroendocrine;
bronchial brushing




Small









The foregoing description of the specific embodiments so fully reveals the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
It should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
REFERENCES


    1. Bentwich, I. et al. Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet (2005).
    2. Farh, K. K. et al. The Widespread Impact of Mammalian MicroRNAs on mRNA Repression and Evolution. Science (2005).
    3. Griffiths-Jones, S., Grocock, R. J., van Dongen, S., Bateman, A. & Enright, A. J. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 34, D140-4 (2006).
    4. He, L. et al. A microRNA polycistron as a potential human oncogene. Nature 435, 828-33 (2005).
    5. Baskerville, S. & Bartel, D. P. Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. Rna 11, 241-7 (2005).
    6. Landgraf, P. et al. A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing. Cell 129, 1401-14 (2007).
    7. Volinia, S. et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA (2006).
    8. Lu, J. et al. MicroRNA expression profiles classify human cancers. Nature 435, 834-8 (2005).
    9. Varadhachary, G. R., Abbruzzese, J. L. & Lenzi, R. Diagnostic strategies for unknown primary cancer. Cancer 100, 1776-85 (2004).
    10. Pimiento, J. M., Teso, D., Malkan, A., Dudrick, S. J. & Palesty, J. A. Cancer of unknown primary origin: a decade of experience in a community-based hospital. Am J Surg 194, 833-7; discussion 837-8 (2007).
    11. Shaw, P. H., Adams, R., Jordan, C. & Crosby, T. D. A clinical review of the investigation and management of carcinoma of unknown primary in a single cancer network. Clin Oncol (R Coll Radiol) 19, 87-95 (2007).
    12. Hainsworth, J. D. & Greco, F. A. Treatment of patients with cancer of an unknown primary site. N Engl J Med 329, 257-63 (1993).
    13. Blaszyk, H., Hartmann, A. & Bjornsson, J. Cancer of unknown primary: clinicopathologic correlations. Apmis 111, 1089-94 (2003).
    14. Bloom, G. et al. Multi-platform, multi-site, microarray-based human tumor classification. Am J Pathol 164, 9-16 (2004).
    15. Ma, X. J. et al. Molecular classification of human cancers using a 92-gene real-time quantitative polymerase chain reaction assay. Arch Pathol Lab Med 130, 465-73 (2006).
    16. Talantov, D. et al. A quantitative reverse transcriptase-polymerase chain reaction assay to identify metastatic carcinoma tissue of origin. J Mol Diagn 8, 320-9 (2006).
    17. Tothill, R. W. et al. An expression-based site of origin diagnostic method designed for clinical application to cancer of unknown origin. Cancer Res 65, 4031-40 (2005).
    18. Shedden, K. A. et al. Accurate molecular classification of human cancers based on gene expression using a simple classifier with a pathological tree-based framework. Am J Pathol 163, 1985-95 (2003).
    19. Raver-Shapira, N. et al. Transcriptional Activation of miR-34a Contributes to p53-Mediated Apoptosis. Mol Cell (2007).
    20. Xiao, C. et al. MiR-150 Controls B Cell Differentiation by Targeting the Transcription Factor c-Myb. Cell 131, 146-59 (2007).

## Claims

1. A method of identifying a tissue of origin of a cancer sample, said method comprising:
(a) obtaining a biological sample from a subject in need thereof, wherein the sample is of a cancer selected from the group consisting of cancer of unknown primary (CUP), primary cancer, and metastatic cancer;
(b) measuring the level of nucleic acids consisting of SEQ ID NOS: 1; 2 or 156; 3-7; 9-12; 14-21; 23-27; 29-40; 42; 43; 44 or 191; 45-47; 49-51; 53-56; 57 or 202; 58; 59; 60 or 208; 61; 62 or 211; 64-69; and 146-148, and optionally at least one control nucleic acid, in the biological sample and comparing said level to a reference level using a classifier algorithm, wherein the tissue of origin of the cancer is identified in the subject if the level of the nucleic acids in the biological sample is above a reference threshold, wherein the measuring comprises contacting the sample with probes to the nucleic acids, wherein each probe consists of the complement of one of SEQ ID NOS: 1; 2 or 156; 3-7; 9-12; 14-21; 23-27; 29-40; 42; 43; 44 or 191; 45-47; 49-51; 53-56; 57 or 202; 58; 59; 60 or 208; 61; 62 or 211; 64-69; and 146-148, or of the optional at least one control nucleic acid, and wherein the probes are attached to a solid substrate; and
(c) identifying the tissue of origin of the sample based on the level of the nucleic acids measured in step (b).

2. The method of claim 1, wherein the classifier algorithm is selected from the group consisting of: decision tree classifier, K-nearest neighbor classifier (KNN), logistic regression classifier, nearest neighbor classifier, neural network classifier, Gaussian mixture model (GMM), Support Vector Machine (SVM) classifier, nearest centroid classifier, linear regression classifier and random forest classifier.

3. The method of claim 1, wherein the cancer is selected from the group consisting of adrenocortical carcinoma; anus or skin squamous cell carcinoma; biliary tract adenocarcinoma; Ewing sarcoma; gastrointestinal stomal tumor (GIST); gastrointestinal tract carcinoid; renal cell carcinoma: chromophobe, clear cell and papillary; pancreatic islet cell tumor; pheochromocytoma; urothelial cell carcinoma (TCC); lung, head & neck, or esophagus squamous cell carcinoma (SCC); brain: astrocytic tumor, oligodendroglioma; breast adenocarcinoma; uterine cervix squamous cell carcinoma; chondrosarcoma; germ cell cancer; sarcoma; colorectal adenocarcinoma; liposarcoma; hepatocellular carcinoma (HCC); lung large cell or adenocarcinoma; lung carcinoid; pleural mesothelioma; lung small cell carcinoma; B-cell lymphoma; T-cell lymphoma; melanoma; malignant fibrous histiocytoma (MFH) or fibrosarcoma; osteosarcoma; ovarian primitive germ cell tumor; ovarian carcinoma; pancreatic adenocarcinoma; prostate adenocarcinoma; rhabdomyosarcoma; gastric or esophageal adenocarcinoma; synovial sarcoma; non-seminomatous testicular germ cell tumor; seminomatous testicular germ cell tumor; thymoma thymic carcinoma; follicular thyroid carcinoma; medullary thyroid carcinoma; and papillary thyroid carcinoma.

4. The method of claim 3, wherein a level of SEQ ID NOS: 55 above the reference threshold indicates a cancer of germ cell origin selected from the group consisting of an ovarian primitive cell and a testis cell, and further wherein a level of SEQ ID NOS: 29 and 62 above the reference threshold indicates a testis cell cancer origin selected from the group consisting of seminomatous testicular germ cell and non-seminomatous testicular germ cell.

5. The method of claim 3, wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 9 and 29 above the reference threshold indicates a cancer origin selected from the group consisting of biliary tract adenocarcinoma and hepatocellular carcinoma.

6. The method of claim 3, wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 156, 66 and 68 above the reference threshold indicates a cancer of brain origin, and further wherein a level of SEQ ID NOS: 40 and 60 above the reference threshold indicates a brain cancer origin selected from the group consisting of oligodendroglioma and astrocytoma.

7. The method of claim 3, wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 14 and 21 above the reference threshold indicates a cancer of prostate adenocarcinoma origin; wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 27, 35, 14, 21, 32, 51, 7, 25, 50, 11, 148, 4, 49 and 67 above the reference threshold indicates a cancer of breast adenocarcinoma origin; wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 27, 35, 14, 21, 32, 51, 7, 25, 4, 39, 50, 11, 148, 49, 67, 57 and 34 above the reference threshold indicates a cancer of an ovarian carcinoma origin; wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 14, 21, 32, 51, 7, 11, 148, 4, 49, 67, 57 and 34 above the reference threshold indicates a cancer of lung large cell or lung adenocarcinoma origin; and wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 20 and 45 above the reference threshold indicates a cancer of lung small cell carcinoma origin.

8. The method of claim 3, wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 14, 21, 32, 51, 7, 11, 148 and 4 above the reference threshold indicates a cancer of thyroid carcinoma origin, and further wherein a level of SEQ ID NOS: 17 and 34 above the threshold indicates that the thyroid carcinoma origin is follicular or papillary.

9. The method of claim 3, wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 14, 21, 32, 51, 7, 50, 4, 39, 3 and 34 above the reference threshold indicates a cancer of a thymic carcinoma origin; or wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 14, 21, 32, 51, 7, 50, 4, 39, 3, 34, 69, 24 and 44 above the reference threshold indicates a cancer of urothelial cell carcinoma or squamous cell carcinoma origin, and further wherein a level of SEQ ID NOS: 1, 5 and 54 above the reference threshold indicates that the squamous-cell-carcinoma origin is uterine cervix squamous-cell-carcinoma or non-uterine cervix squamous cell carcinoma; or further wherein a level of SEQ ID NOS: 11 and 23 above the reference threshold indicates that the non-uterine cervix squamous cell carcinoma origin is selected from the group consisting of: a) anus or skin squamous cell carcinoma, and b) lung, head & neck, and esophagus squamous cell carcinoma.

10. The method of claim 3, wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 20, 45, 40, 67 and 68 above the reference threshold indicates a cancer of medullary thyroid carcinoma origin; wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 20, 45, 40, 67, 68, 64, 53 and 37 above the reference threshold indicates a cancer of lung carcinoid origin; and wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 20, 45, 40, 67, 68, 64, 53, 37, 34 and 18 above the reference threshold indicates a cancer of gastrointestinal tract carcinoid or pancreatic islet cell tumor origin.

11. The method of claim 3, wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 42, 36 and 146 above the reference threshold indicates a cancer of gastric or esophageal adenocarcinoma origin; wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 42, 36, 146, 20 and 43 above the reference threshold indicates a cancer of colorectal adenocarcinoma origin; wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 56, 65, 25, 27, 35, 42, 36, 146, 20, 43, 51, 49 and 16 above the reference threshold indicates a cancer of pancreatic adenocarcinoma or biliary tract adenocarcinoma origin; wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 2, 66, 68, 19 and 29 above the reference threshold indicates a cancer of renal cell carcinoma origin, and further wherein a level of SEQ ID NOS: 36 and 147 above the reference threshold indicates a chromophobe renal cell carcinoma origin, or further wherein a level of SEQ ID NOS: 49 and 9 above the reference threshold indicates that the renal cell carcinoma origin is clear cell or papillary.

12. The method of claim 3, wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 2, 66, 68, 19, 29, 65 and 56 above the reference threshold indicates a cancer of pheochromacytoma origin; wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 2, 66, 68, 19, 29, 65, 56, 31, 38 and 61 above the reference threshold indicates a cancer of adrenocortical origin; wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 2, 66, 68, 19, 29, 65, 56, 31, 38, 61, 14 and 45 above the reference threshold indicates a cancer of gastrointestinal stomal tumor origin; wherein a level of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 55, 6, 30, 46, 16, 2, 66, 68, 19, 29, 65, 56, 31, 38, 61, 14, 45, 35, 10 and 5 above the reference threshold indicates a cancer of pleural mesothelioma or sarcoma origin, and further wherein a level of SEQ ID NOS: 3, 40 and 15 above the reference threshold indicates that the sarcoma is synovial sarcoma, or further wherein a level of SEQ ID NOS: 3, 40, 15, 12 and 58 above the reference threshold indicates that the sarcoma is chondrosarcoma, or further wherein a level of SEQ ID NOS: 3, 40, 15, 12, 58, 36 and 26 above the reference threshold indicates that the sarcoma is liposarcoma, or further wherein a level of SEQ ID NOS: 3, 40, 15, 12, 58, 36, 26, 21, 25 and 49 above the reference threshold indicates that the sarcoma is Ewing sarcoma or osteosarcoma; or further wherein a level of SEQ ID NOS: 3, 40, 15, 12, 58, 36, 26, 21, 59, 39 and 33 above the reference threshold indicates that the sarcoma is selected from the group consisting of: a) rhabdomyosarcoma, and b) malignant fibrous histiocytoma and fibrosarcoma.

13. The method of claim 1, wherein the biological sample is selected from the group consisting of a bodily fluid, a cell line, a tissue sample, a biopsy sample, a needle biopsy sample, a fine needle biopsy (FNA) sample, a surgically removed sample, and a sample obtained by tissue-sampling procedures such as endoscopy, bronchoscopy, or laparoscopic methods.

14. The method of claim 13, wherein the tissue is a fresh, frozen, fixed, wax-embedded or formalin-fixed paraffin-embedded (FFPE) tissue.

15. The method of claim 1, wherein the level of the nucleic acids is determined by nucleic acid hybridization.

16. The method of claim 15, wherein nucleic acid hybridization is performed using a solid-phase nucleic acid biochip array.

17. A kit for performing the method of claim 1 comprising probes to nucleic acids consisting of SEQ ID NOS: 1; 2 or 156; 3-7; 9-12; 14-21; 23-27; 29-40; 42; 43; 44 or 191; 45-47; 49-51; 53-56; 57 or 202; 58; 59; 60 or 208; 61; 62 or 211; 64-69; and 146-148, and optionally at least one control nucleic acid, wherein each probe consists of the complement of one of SEQ ID NOS: 1; 2 or 156; 3-7; 9-12; 14-21; 23-27; 29-40; 42; 43; 44 or 191; 45-47; 49-51; 53-56; 57 or 202; 58; 59; 60 or 208; 61; 62 or 211; 64-69; and 146-148, or of the optional at least one control nucleic acid, wherein the probes are attached to a solid substrate.

18. A method of treating a cancer selected from the group consisting of cancer of unknown primary (CUP), primary cancer, and metastatic cancer, comprising:
(a) identifying a tissue of origin of a cancer sample, said method comprising:
(i) obtaining a biological sample from a subject in need thereof, wherein the sample is of a cancer selected from the group consisting of cancer of unknown primary (CUP), primary cancer, and metastatic cancer;
(ii) measuring the level of nucleic acids comprising SEQ ID NOS: 1; 2 or 156; 3-7; 9-12; 14-21; 23-27; 29-40; 42; 43; 44 or 191; 45-47; 49-51; 53-56; 57 or 202; 58; 59; 60 or 208; 61; 62 or 211; 64-69; and 146-148, in the biological sample and comparing said level to a reference level using a classifier algorithm, wherein the tissue of origin of the cancer is identified in the subject if the level of the nucleic acids in the biological sample is above a reference threshold, wherein the measuring comprises contacting the sample with probes comprising the complements of SEQ ID NOS: 1; 2 or 156; 3-7; 9-12; 14-21; 23-27; 29-40; 42; 43; 44 or 191; 45-47; 49-51; 53-56; 57 or 202; 58; 59; 60 or 208; 61; 62 or 211; 64-69; and 146-148; and
(iii) identifying the tissue of origin of the sample based on the level of the nucleic acids measured in step (ii); and

(b) administering a cancer treatment to the subject according to the tissue of origin of the cancer identified in step (a).

19. The kit of claim 17, wherein the probes are attached to a solid substrate through a linker.