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Detection of Micrometastases in Peritoneal Washings of Pancreatic Cancer Patients By Quantitative Real Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Kimberly M. Dalal*1,2, Yanghee Woo2, Charles Galanis2, Mithat Gonen3, Peter J. Allen2, Ronald P. Dematteo2, Yuman Fong2, Daniel G. Coit2
1Surgery, David Grant USAF Medical Center, Travis AFB, CA; 2Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY; 3Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY

Introduction:Pancreatic cancer patients with positive (+) peritoneal cytology have a prognosis similar to stage IV patients. We studied the ability of a quantitative RT-PCR assay to detect micrometastases in patients undergoing staging laparoscopy for pancreatic cancer.
Methods:Peritoneal washes were obtained prospectively from 34 consecutive consented patients with pancreatic adenocarcinoma undergoing laparoscopy for staging and 16 patients undergoing laparoscopy for benign disease (negative controls). Half of each sample was sent to pathology for cytologic examination; half underwent RT-PCR analysis for tumor markers. Carcinoembryonic antigen (CEA), cytokeratin 7 (CK7), Kras2, and MUC1 mRNA levels were quantified and normalized to 18s mRNA. Positive controls were patients with visible cytology or peritoneal metastases. The threshold for cycle amplification was <36 cycles. A tumor marker was (+) if the ratio of tumor marker mRNA/18S mRNA was >1.3. Markers and their combinations were evaluated on the basis of their distance, or deviance, from the ideal marker (ie, 100% sensitive and specific).
Results:The median age of the cohort was 67. Thirty-four patients (68%) were female. Pathologic stage for pancreatic cancer patients was: stage 1A-1 (3%); stage IB-1 (3%); stage IIA-5 (15%); stage IIB-13 (38%); stage IIIA-5 (15%); stage IV-9 (26%). The 8 cytology (+) patients were stage IIA-1; stage IIB-2; stage IV-5. CEA had the best profile of sensitivity, specificity, PPV, NPV (Table). Sensitivity was highest for CK7, MUC1, and CK7+MUC1. CEA+Kras2, CEA+Kras2+CK7, CEA+Kras2+MUC1, and CEA+Kras2+CK7+MUC1 had specificity and PPV of 100%. As CEA alone had the smallest deviance, other markers did not provide additional benefit.
Conclusions:RT-PCR using a panel of tumor markers, including CEA, was comparable in sensitivity, specificity, PPV, and NPV to cytology. The clinical significance of “false positive” overexpression of Kras2, CK7, or MUC1 remains to be defined. RT-PCR could represent a more sensitive method for detection of subclinical peritoneal tumor dissemination; this may be useful in improving selection of patients for operative management and clinical trials.

Tumor Marker mRNA True Positive True Negative False Positive False Negative SensitivityTP/(TP+FN) SpecificityTN/(FP+TN) Positive Predictive Value (PPV)TP/(TP+FP) Negative Predictive Value(NPV)TN/(FN+TN) Deviance
CEA 8 38 1 3 0.73 0.97 0.89 0.93 0.07
Kras2 7 20 19 4 0.64 0.51 0.27 0.83 0.37
CK7 6 25 12 2 0.75 0.68 0.33 0.93 0.16
MUC1 6 19 18 2 0.75 0.51 0.25 0.90 0.30
CEA+Kras2 6 39 0 5 0.55 1.00 1.00 0.89 0.20
CK7+MUC1 6 25 12 2 0.75 0.68 0.33 0.93 0.16
CEA+Kras2+CK7 5 37 0 4 0.56 1.00 1.00 0.90 0.19
CEA+Kras2+MUC1 5 40 0 4 0.56 1.00 1.00 0.91 0.19
CEA+Kras2CK7+MUC1 4 39 0 4 0.50 1.00 1.00 0.91 0.25


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