H. Ben Hsieh

Multiple Biomarker Expression on Circulating Tumor Cells in Comparison to Tumor Tissues from Primary and Metastatic Sites in Patients with Locally Advanced/Inflammatory, and Stage IV Breast Cancer, Using a Novel Detection Technology

Patients with locally advanced/inflammatory breast cancer (LABC/IBC) face a high likelyhood of recurrence and prognosis for relapsed, or de novo stage IV metastatic breast cancer (MBC) remains poor. Estrogen (ER) and HER2 receptor expression on primary or MBC allow targeted therapies, but an estimated 10–18% of tumors do not exhibit these biomarkers and survival in these cases is even poorer. Variations in discordance rates for the expression of ER and HER2 receptors have been observed between primary and metastatic tumors and such discordances may lead to suboptimal treatment. Circulating tumor cells (CTCs) are considered the seeds of residual disease and distant metastases and their characterization could help guide treatment selection. To explore this possibility, we used multiple biomarker assessment of CTCs in comparison to primary and metastatic tumor sites. Thirty-six patients with LABC/IBC, or stage IV MBC were evaluated. Blood samples were procured prior to initiating or changing therapy. CTCs were identified based on presence of cytokeratin and nucleus staining, and the absence of CD45. A multimarker assay was developed to simultaneously quantify expression of HER2, ER, and ERCC1, a DNA excision repair protein. Novel fiber-optic array scanning technology (FAST) was used for sensitive location of CTCs. CTCs were detected in 82% of MBC and 62% LABC/IBC cases. Multiplex marker expression was successfully carried out in samples from18 patients with MBC and in 8 patients with LABC/IBC that contained CTCs. In MBC, we detected actionable discordance rates of 40 and 23%, respectively for ER and HER2 where a biomarker was negative in the primary or metastatic tumor and positive in the CTCs. In LABC/IBC, actionable discordances were 60 and 20% for ER and HER2, respectively. Pilot trials evaluating the effectiveness of treatment selections based on actionable discordances between biomarker expression patterns on CTCs and primary or metastatic tumor sites may allow for a prospective assessment of CTC-based individualized targeted therapies.

ERCC1 expression in circulating tumor cells (CTCs) using a novel detection platform correlates with progression-free survival (PFS) in patients with metastatic non-small-cell lung cancer (NSCLC) receiving platinum chemotherapy.

PURPOSE To utilize a novel circulating tumor cell (CTC) technology to quantify ERCC1 expression on CTCs and determine whether ERCC1 expression levels predict efficacy of platinum-based chemotherapy in patients with metastatic non-small-cell lung cancer (NSCLC). EXPERIMENTAL DESIGN ERCC1 expression was measured in 17 metastatic NSCLC patients who received platinum-based therapy and had ≥2 intact CTCs with acceptable ERCC1 expression assay results. ERCC1 levels were determined from average expression on individual CTCs in each sample. Progression-free survival (PFS) was calculated from the date of therapy initiation. RESULTS PFS decreased with increasing ERCC1 expression (p<0.04, F-test, linear regression). Lack of ERCC1 expression was associated with longer PFS (266 days versus 172 days, log-rank, p<0.02) in a Kaplan-Meier analysis using ERCC expression level of 1 as a cutoff (range 0-30). The difference in survival was statistically significant with a hazard ratio of 4.20 (95% CI 1.25-14.1, p<0.02, log-rank). PFS was also observed to decrease with increased cytokeratin (CK) expression (p<0.01 long-rank (Cox regression) and F-test (linear regression)). The hazard ratio is 4.38 (95% CI 1.76-10.9) for each log-change in CK value until progression was noted on imaging. CONCLUSION Low expression of ERCC1 on CTCs correlates with PFS in patients with metastatic NSCLC receiving platinum-based therapy.

A new method for high speed, sensitive detection of minimal residual disease

Investigations of rare cell types in peripheral blood samples, such as tumor, fetal, and endothelial cells, represent an emerging field with several potentially valuable medical applications. Peripheral blood is a particularly attractive body fluid for the detection of rare cells as its collection is minimally invasive and can be repeated throughout the course of the disease. Because the number of rare cells in mononuclear cells can be very low (1 in 10 million), a large number of cells must be quickly screened, which places demanding requirements on the screening technology. While enrichment technology has shown promise in managing metastatic disease, enrichment can cause distortions of cell morphology that limit pathological identification, and the enrichment targeting adds additional constraints that can affect sensitivity. Here, we describe a new approach for detecting rare leukemia cells that does not require prior enrichment. We have developed an immunocytochemical assay for identification of leukemia cells spiked in peripheral blood samples, and a high‐speed scanning instrument with high numerical aperture and wide field of view to efficiently locate these cells in large sample sizes. A multiplex immunoassay with four biomarkers was used to uniquely identify the rare cells from leukocytes and labeling artifacts. The cytometer preserves the cell morphology and accurately locates labeled rare cells for subsequent high resolution imaging. The sensitivity and specificity of the approach show promise for detection of a low number of leukemia cells in blood (1 in 10 million nucleated cells). The method enables rapid location of rare circulating cells (25 M cells/min), no specific enrichment step, and excellent imaging of cellular morphology with multiple immunofluorescent markers. The cell imaging is comparable to other imaging approaches such as laser scan cytometry and image flow cytometry, but the cell analysis rate is many orders of magnitude faster making this approach practical for detection of rare cells.

Sensitive Characterization of Circulating Tumor Cells for Improving Therapy Selection

For metastatic disease, biomarker profiling of distant metastases is done only when feasible because biopsy of metastases is invasive and associated with potential morbidity without proven benefit. So although biomarker expression may differ in distant metastases, treatment with targeted therapies is almost always based on biomarker targets derived from a patient’s primary breast tumor, usually excised years before development of metastatic disease. This work addresses measurement of biomarker expression on circulating tumor cells (CTCs) as a source of current biomarker expression. CTCs are rapidly located on a planar substrate with a sensitive detection instrument using Fiber Array Scanning Technology. The instrument targets abundant cytokeratins rather than EpCAM. The assay includes quantitative measurement of expression levels of 3 breast cancer markers (HER2, ER and ERCC1) that predict efficacy of treatment. We have observed high discordance rates in cancer markers between CTC and tissue. Multiplex testing may allow for personalized therapy for patients.