Detection of Micro Metastasis of Melanoma and Breast Cancer in Paraffin-Embedded Tumor Draining Lymph Nodes by Multimarker Quantitative RT-PCR

Abstract

The invention provides a quantitative realtime RT-PCR assay for detection of metastatic breast, gastric, pancreas or colon cancer cells or metastatic melanoma. The assay allows to predict disease recurrence and survival in patients with AJCC stage I and II, and III disease using multimarker panels. The method for detecting metastatic melanoma cells utilizes panels of markers selected from a group consisting of MAGE-A3, GalNAcT, MART-1, PAX3, Mitf, TRP-2, and Tyrosinase. The method for detecting metastatic breast, gastric, pancreas or colon cancer cells in paraffin-embedded samples utilizes panels of markers selected from a group consisting of C-Met, MAGE-A3, Stanniocalcin-1, mammoglobin, HSP27, GalNAcT, CK20, and β-HCG.

1 - 26 . (canceled) 
     
     
         27 . A method of detecting metastatic melanoma cells in a patient comprising:
 (a) receiving a biological sample from the patient;   (b) amplifying mRNA transcripts encoded by a panel of marker genes, the panel of marker genes comprising GalNAcT and PAX3 marker genes, the mRNA transcripts being obtained from nucleic acid from the biological sample; and   (c) quantifying each of the mRNA transcripts encoded by the GalNAcT and PAX3 marker genes such that detection of GalNAcT and PAX3 indicates the presence of occult melanoma in the patient.   
     
     
         28 . The method of  claim 27  wherein the panel further comprises marker genes selected from a group consisting of MAGE-A3, MART-1, MITF, TRP-2 and Tyrosinase. 
     
     
         29 . The method of  claim 27  wherein the panel of marker genes further comprises MAGE-A3. 
     
     
         30 . The method of  claim 27  wherein the panel of marker genes further comprises MART-1. 
     
     
         31 . The method of  claim 27  wherein the mRNA transcripts are amplified using real-time reverse transcriptase polymerase chain reaction (qRT-PCR). 
     
     
         32 . The method of  claim 27  wherein the biological sample is selected from a group consisting of paraffin-embedded (PE) melanoma tissues, frozen lymph nodes, and PE lymph nodes. 
     
     
         33 . The method of  claim 27  wherein the biological sample is histopathologically negative for melanoma cells. 
     
     
         34 . The method of  claim 33  wherein histopathology of the biological sample is determined by hematoxylin and eosin staining or immunohistochemistry. 
     
     
         35 . A method of detecting metastatic melanoma cells in a patient comprising:
 (a) receiving a biological sample from the patient;   (b) amplifying mRNA transcripts encoded by a panel of marker genes, the panel of marker genes comprising GalNAcT, MAGE-3, and PAX3 marker genes, the mRNA transcripts being obtained from nucleic acid from the biological sample; and   (c) quantifying each of the mRNA transcripts encoded by the GalNAcT, MAGE-3, and PAX3 marker genes such that detection GalNAcT and PAX3 indicates the presence of occult melanoma in the patient.   
     
     
         36 . The method of  claim 35  wherein the panel further comprises marker genes selected from a group consisting of MART-1, MITF, TRP-2 and Tyrosinase. 
     
     
         37 . The method of  claim 35  wherein the panel of marker genes further comprises MART-1. 
     
     
         38 . The method of  claim 35  wherein the mRNA transcripts are amplified using real-time reverse transcriptase polymerase chain reaction (qRT-PCR). 
     
     
         39 . The method of  claim 35  wherein the biological sample is selected from a group consisting of paraffin-embedded (PE) melanoma tissues, frozen lymph nodes, and PE lymph nodes. 
     
     
         40 . The method of  claim 35  wherein the biological sample is histopathologically negative for melanoma cells. 
     
     
         41 . The method of  claim 40  wherein histopathology of the biological sample is determined by hematoxylin and eosin staining or immunohistochemistry. 
     
     
         42 . A method of detecting metastatic breast, gastric, pancreas or colon cancer cells in a patient comprising:
 (a) isolating nucleic acid from a histopathologically negative sample from the patient;   (b) amplifying mRNA transcripts encoded by a panel of marker genes, the panel of marker genes comprising at least one marker gene selected from a group consisting of C-Met, MAGE-A3, Stanniocalcin-1, mammoglobin, HSP27, GalNAcT, CK20 and β-HCG; and   (c) detecting the presence or absence of the marker gene wherein the presence of the marker gene indicates occult disease.   
     
     
         43 . The method of  claim 42  wherein the panel comprises a first combination of C-Met, MAGE-A3, GalNAcT and CK20; a second combination of mammoglobulin, C-Met, GalNAcT, and β-HCG; a third combination of mammoglobulin, β-HCG, HSP27, and C-Met; or a fourth combination of HSP27, CK20, Stanniocalcin-1, and MAGE-A3. 
     
     
         44 . The method of  claim 42  wherein the nucleic acid is mRNA and mRNA transcripts are amplified using qRT-PCR. 
     
     
         45 . The method of  claim 42  wherein histopathology of the sample is determined by hematoxylin and eosin staining or immunohistochemistry.