Detection of metastatic breast cancer by β-hCG polymerase chain reaction

Abstract

Reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of occult malignancies in breast cancer patients is evolving as a useful diagnostic tool. However, no reliable molecular mRNA markers are available. We developed an RT-PCR plus Southern blot assay using p-hCG @-subunit of human chorionic gonadotropin) gene expression as a tumor marker for detection of breast malignancies metastatic to tumor-draining lymph nodes and blood. Breast carcinoma cell lines, primary breast malignancies and human placenta were used as positive controls for establishing the P-hCG RT-PCR assay. Peripheral blood leukocytes (PBL) from normal volunteer donors, normal breast tissue and lymph nodes from cancer-free patients were used as negative controls. P-hCG RT-PCR was used to assess tumor cell presence in PBL and tumor-draining axillary nodes from patients with AJCC stage I-IV breast cancer. The assay sensitivity and specificity were enhanced by restriction endonuclease digestion of an Sty I site of the RT-PCR cDNA product followed by Southern blot analysis. P-hCG mRNA was expressed in all breast cancer cell lines and 80% of primary breast cancers; it was not expressed in negative Neoplastic cells aberrantly express genes that are silent in their normal counterparts. These genes often encode for proteins that endow neoplastic cells selective advantages with respect to growth and other malignant cell properties, such as metastasis and invasion. Neoplastic cells produce autocrine growth factors or hormones that are not produced or are expressed at low levels by normal cells. Human chorionic gonadotropin (hCG) is a hormone produced by the trophoblast and is critical to sustaining pregnancy (Moyle and Campbell, 1993). hCG and its individual subunits are produced not only in gestational trophoblastic and germ-cell cancers but also in a wide range of non-gonadal tumors, such as gastrointestinal, lung and breast carcinomas (McManus et al., 1976; Madersbacher et al., 1994; Marcillac et al., 1992;Acevedo et al., 1995). We have demonstrated its usefulness as a molecular marker for melanoma (Doi et al., 1996). The pathophysiologic role for the expression of hCG in malignant neoplasms is currently speculative . hCG expression represents recapitulation of fetal cell properties by malignant cells.Immunohistochemical analysis using polyclonal or monoclonal antibodies (MAbs) has demonstrated hCG as a cancer marker (Krichevsky et al., 1995). However, these studies were not always specific, primarily due to the recognition of crossreactive epitopes or subunit chains of related gonadotropins (McManus et al., 1976; Krichevsky et al., 1995). About 7040% of adenocarcinomas arising in the mammary gland stain for the p subunit of hCG (P-hCG) by immunohistochemical techniques (Agnantis et al., 1992). hCG is a dimeric hormone composed of 2 non-covalently linked subunits designated a and P (Bo and Boime, 1992;Boorstein et al., 1982). It belongs to a glycoprotein hormone family, sharing many similarities with luteinizing hormone (LH), follicle stim...

Human Chorionic Gonadotropin Decreases Proliferation and Invasion of Breast Cancer MCF-7 Cells by Inhibiting NF-κB and AP-1 Activation

“…There is evidence that breast cancer tissues and cells synthesize hCG and LH, suggesting paracrine and autocrine actions of these hormones (53)(54)(55). These findings suggest that strategies which can increase the local production of these hormones may work in a fight against this malignancy.…”

Immunohistochemistry and Reverse Transcriptase Polymerase Chain Reaction on Sentinel Lymph Nodes can Improve the Accuracy of Nodal Staging in Breast Cancer Patients

“…RT-PCR has proved to be a highly sensitive method for detecting cancer cells in the regional lymph nodes or SLNs of breast cancer patients (15,16,(34)(35)(36)(37)(38). Since no ideal breast cancer marker has been identified yet, it can be hypothesized that a multimarker RT-PCR panel would increase the sensitivity and specificity of this assay (35)(36)(37).…”

“…Since no ideal breast cancer marker has been identified yet, it can be hypothesized that a multimarker RT-PCR panel would increase the sensitivity and specificity of this assay (35)(36)(37). Several markers have already been investigated (34)(35)(36)(37). CK20 is an epithelial marker which is not expressed in normal lymph nodes (37).…”

“…To our knowledge the results of five studies on RT-PCR examination performed on SLNs of breast cancer patients have been published to date (34)(35)(36)(37)(38). In four of these studies multimarker assays were used (35)(36)(37)(38).…”

True

“…The 734B line is an established subclone of MCF-7. The cell lines JWCI BM-1 and JWCI JM-1 were established from primary invasive ductal carcinoma and characterized as breast cancer cell lines at JWCI (Hoon et al, 1996;Bostick et al, 1998c). All 6 cell lines were grown in RPMI 1640 (Gemini Bioproducts, Calabasas, CA) plus 10% fetal calf serum (heat inactivated), penicillin and streptomycin (GIBCO, Grant Island, NY), in culture flasks and passaged as previously described (Bostick et al, 1998a).…”