Real time-polymerase chain reaction (PCR) improves sensitivity of lymphatic staging in colon cancer patients
Ronit Grinbaum¹, Ali O. Gure⁴, Marina Roycetacher¹, Iris Eisenberg²,

Gerd Ritter⁴, Lloyd J. Old⁴, Herbert R. Freund¹, Tamar Peretz³,

Stella Mitrani-Rosenbaum², Aviram Nissan¹

Departments of Surgery¹, Molecular Biology², and Oncology³, Hadassah-Hebrew University Medical Center Mount Scopus, Jerusalem, Israel,  

Ludwig Institute for Cancer Research⁴, New York, NY, USA.  

INTRODUCTION: One third of patients with node negative colorectal cancer (CRC) will ultimately die of disease recurrence within five years of diagnosis. This may be attributable to the inability of conventional histopathologic techniques to identify nodal micrometastasis.  The aim of the present study was to develop a reproducible technique of molecular processing of sentinel lymph nodes (SLNs) and to evaluate the added value of Real-time PCR in the detection of

micrometastasis in CRC patients.
METHODS: Forty-four SLNs from 35 CRC patients were studied. Enhanced pathologic examination (EPE) including multiple sectioning, H&E and immunohistochemistry staining for cytokeratin was compared to Real-Time PCR for Cytokeratin-20 (CK-20) and for a novel molecular marker, 3.6a. Two lymph nodes obtained from patients without malignant disease and 3 samples of peripheral blood lymphocytes from healthy donors

were used as negative controls.

RESULTS: CK-20 and 3.6a were highly expressed in all 35 primary tumors (positive controls). Of the 44 SLNs, two (4.7%) SLNs were without sufficient RNA for analysis and were excluded from the analysis. All SLNs positive by H&E were also positive by IHC and PCR. Additional 5 SLNs were positive by IHC of which only 2 SLNs were also positive by PCR. Eight additional SLNs negative by EPE were positive by PCR (table 1). None of the 5 negative controls showed amplification of the molecular markers.

CONCLUSION(S): Real-time PCR significantly increases the sensitivity of SLN examination as compared both to H&E and IHC.

Table 1:

Method	n	%positive of all SLNs	Sensitivity	P value
H&E	7/42	16.7%	35%
IHC	12/42	28.6%	60%	0.001
PCR	17/42	40.5%	85%	0.007
ALL POS	20/42	47.6%	100%

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