Multimarker RT–PCR assay for the detection of minimal residual disease in sentinel lymph nodes of breast cancer patients

Abstract

The presence of metastases in lymph nodes is the most powerful prognostic factor in breast cancer patients. Routine histological examination of lymph nodes has limited sensitivity for the detection of breast cancer metastases. The aim of the present study was to develop a multimarker reverse transcriptase -polymerase chain reaction (RT-PCR) assay for the detection of minimal residual disease in sentinel nodes of breast cancer patients. RNA was extracted from 30 sentinel lymph nodes (SLN) obtained from 28 patients, three primary breast cancers (positive controls), three lymph nodes from patients with benign diseases, and peripheral blood lymphocytes of 10 healthy volunteers (negative controls). RT -PCR was performed using the following markers; cytokeratin (CK)-19, NY-BR-1 and mammaglobin B. RT -PCR results were compared to enhanced histopathologic examination and immunohistochemistry (IHC). All three positive controls showed strong PCR amplification for all three markers. None of the 13 negative controls was amplified by any of the three markers. Among the 30 SLN analysed, breast cancer metastases were detected in six SLNs by routine histology, in eight by IHC and in 15 by RT -PCR. We conclude that a multimarker RT -PCR assay probing for NY-BR-1, mammaglobin-B, and CK-19 is more sensitive compared to enhanced pathologic examination. This method may prove to be of value in breast cancer staging and prognosis evaluation. Although up to 80% of breast cancer patients are identified at an early stage when disease is apparently localised, 30 -35% of nodenegative patients will develop clinically detectable distant metastases within 5 years of treatment of the primary breast carcinoma and ultimately suffer tumour-related mortality (Nemoto et al, 1980). Routine histological examination of lymph nodes has limited sensitivity for the detection of breast cancer metastases. It is usually performed with a single haematoxilyn and eosin (H&E)-stained section through the examined node and therefore small metastases can go undetected. In a landmark study conducted by the International (Ludwig) Breast Cancer Group (International (Ludwig) Breast Cancer Study Group, 1990), serial sectioning of axillary lymph nodes judged to be disease free after routine histological examination revealed micrometastases in 83 (9%) of 921 breast cancer patients. These patients had a poorer disease-free (P ¼ 0.003) and overall (P ¼ 0.002) survival after 5 years' median follow-up than did patients whose nodes remained negative on serial sectioning. A later study performed by the same group (Cote et al, 1999) examined lymph nodes negative for metastases on routine histology from 736 patients by serial sectioning and by immunohistochemistry (IHC) with two anticytokeratins AE-1 and CAM 5.2. Occult nodal metastases were detected by serial sectioning and H&E in 7% of the patients and by IHC in 20%. After median follow-up of 12 years, occult metastases, detected by either method, were associated with significantly poor disease-free and overall survival in pos...

The differentiation antigen NY‐BR‐1 is a potential target for antibody‐based therapies in breast cancer

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Is transplantation of cryopreserved ovarian tissue from patients with advanced-stage breast cancer safe? A pilot study

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Treatment of patients with bone marrow metastases: the present state of the problem

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