Comparison of molecular and immunocytochemical methods for detection of disseminated tumor cells in bone marrow from early breast cancer patients

Abstract

BackgroundDisseminated tumor cells (DTCs) have potential to predict the effect of adjuvant treatment. The purpose of this study was to compare two methods, reverse transcription quantitative PCR (RT-qPCR) and immunocytochemisty (ICC), for detecting breast cancer DTCs in bone marrow (BM) from early breast cancer patients.MethodsWe investigated a subset (n = 313) of BM samples obtained from 271 early breast cancer patients in the “Secondary Adjuvant Taxotere Treatment” (SATT)-trial. All patients in this study had node positive or intermediate/high-risk node negative non-metastatic disease. The DTCs were detected by ICC using AE1-AE3 anti-cytokeratin monoclonal antibodies. Patients with DTCs detected in their BM by ICC after standard adjuvant fluorouracil, cyclophosphamide, epirubicin (FEC) chemotherapy were offered docetaxel treatment. For comparison, 5 × 106 mononuclear cells from the aliquoted BM samples were also analyzed by RT-qPCR using a multimarker (MM) assay based on the tumor cell mRNA markers keratin 19 (KRT19), mammaglobin A (hMAM), and TWIST1. In the MM-assay, a sample was defined as positive for DTCs if at least one of the mRNA markers was positive.ResultsThe MM RT-qPCR assay identified DTCs in 124 (40%) of the 313 BM samples compared with 23/313 (7%) of the samples analyzed by ICC. The concordance between the MM RT-qPCR and ICC was 61% (Kappa value = 0.04) and twelve of the BM samples were positive by both methods. By RT-qPCR, 46/313 (15%) samples were positive for KRT19, 97/313 (31%) for TWIST1, and 3/313 (1%) for hMAM mRNA. There were no statistically significant associations between the individual mRNA markers.ConclusionThe RT-qPCR based method demonstrated more DTC-positive samples than ICC. The relatively low concordance of positive DTC-status between the two different assessment methods suggests that they may be complementary. The clinical relevance of the methods will be evaluated based on future clinical outcome data.Trial registrationClinicalTrials.gov: NCT00248703.

Prognostic significance of KRT19 in Lung Squamous Cancer

“…Furthermore, expression of KRT19 was associated with survival of HCC patients [34]. Importantly, KRT19 can be used as a molecular marker for reverse transcriptase PCR (RT-PCR) and ELISA-mediated detection of disseminated tumor cells in regional lymph nodes (LNs), peripheral blood (PB) and bone marrow from susceptible cancerous patients [12,[35][36]. Recently, KRT19 was also used as a novel marker by circulated tumor cells (CTC) detection to identify tumor cells in breast cancer and colorectal cancer [37][38][39], and its positivity was demonstrated to be a prognostic indicator [40].…”

Gene expression analysis to detect disseminated tumor cells in the bone marrow of triple-negative breast cancer patients predicts metastatic relapse

“…The presence of DTCs in the BM of early-stage breast cancer patients has demonstrated prognostic significance [4–6]. Most assays employ ICC detection of epithelial antigens [14] or PCR-based gene expression [15] but have not been adopted as standard-of-care diagnostics because BM aspirations are not routinely performed on breast cancer patients, current detection assays are technically complex or expensive, and results are not immediately actionable in most breast cancer patients. The present study was designed to address whether multi-marker detection of DTCs and molecular classification of BM could result in a more specific, sensitive, robust, and actionable assay.…”

“…We found the expression of at least one of the 46 genes was detected in all but one Her2-positive patient, whereas the traditional detection of DTCs by ICC methods averages 20–30% in similarly staged breast cancer patients [5, 6]. One explanation for this discrepancy may be that traditional immunocytochemistry methods only measure epithelial antigens such as cytokeratin and EpCAM, which can be frequently downregulated in metastatic tumor cells or cells that revert to a ‘stem cell-like’ phenotype [14, 15]. For example, in our BM cohort, EpCAM expression was detected in only 8% of patient BM and Keratin19 was not significantly expressed in any of the specimens.…”

“…Although DTCs may be tenfold more abundant than circulating tumor cells (CTCs) found in the peripheral blood, they are still exceedingly rare (1–10 DTCs per million mononuclear immunocytes) which makes their individual molecular characterization difficult [13]. Current methods to detect BM DTCs by ICC or single-gene expression biomarkers are technically demanding and lack sufficient sensitivity, specificity, and clinical utility for use in routine clinical practice, particularly where serial monitoring may be required [14, 15]. Therefore, the development of new analytical techniques and biomarkers for the detection, classification, and, in particular, therapeutic targeting of BM DTCs could identify patients at high risk for the development of distant metastatic disease and mitigate its progression.…”

Routine Use of Zoledronic Acid in Early-Stage Breast Cancer

“…DTCs are thought to be present early in the disease course, localize to the skeleton, and reside in a dormant state for many years before developing into incurable metastatic breast cancer. 11 It is estimated that up to half of patients with ESBC have DTCs, 12 and chemotherapy does not clear DTCs from the bone marrow. Preclinical data in a mouse model have shown a profound decrease in circulating tumor cells after a single dose of zoledronic acid compared with controls.…”