Cytokeratin-19 and mammaglobin gene expression in circulating tumor cells from metastatic breast cancer patients enrolled in North Central Cancer Treatment Group trials, N0234/336/436/437.

PURPOSE: To investigate the associations between baseline and posttreatment circulating tumor cell (CTC) gene expression and outcome of patients enrolled in four North Central Cancer Treatment Group metastatic breast cancer (MBC) trials in which specimens were shipped (at 4°C) from community-based sites to a reference laboratory (Mayo Clinic, Rochester, MN). EXPERIMENTAL DESIGN: Blood was collected at treating sites from MBC patients before (baseline), during, and at the end of treatment with erlotinib + gemcitabine (N0234), sorafenib (N0336), irinotecan + cetuximab (N0436), or paclitaxel-poliglumex + capecitabine (N0437). CTCs from 10 mL of EDTA blood were enriched with CD45 depletion, 24 to 30 hours postblood collection. Reverse transcription/quantitative PCR was used to determine cytokeratin-19 (CK19) and mammaglobin (MGB1) mRNA levels in CTCs from up to 13 (N0234), 16 (N0336), 18 (N0436), and 39 (N0437) patients. The gene expressions were normalized to ß(2)-microglobulin and calibrated to healthy blood using the 2(-ΔΔCq) algorithm; positivity was defined as 2 or more. RESULTS: CK19+mRNA cells were detected in 56% to 75% and MGB1+mRNA cells in 23% to 38% of 86 patients at baseline. CK19+mRNA cells were detected in 30% to 67% and MGB1+mRNA cells in 14% to 64% of 110 postbaseline serial samples. The presence of baseline CK19+mRNA cells (P = 0.01) but not MGB1+mRNA cells (P = 0.14) was significantly associated with shorter overall survival. A decrease in MGB1+mRNA levels (baseline-week 8) seemed to be associated with clinical response (P = 0.05). CONCLUSIONS: CTC gene expression analysis conducted by a reference laboratory is feasible when blood is collected from treating sites and processed 24 to 30 hours postcollection. The presence of baseline CK19+mRNA CTCs was associated with poor prognosis; a decrease in MGB1+mRNA CTCs may help predict response to therapy of MBC patients.

A promising approach for treatment of tumor-induced bone diseases: utilizing bisphosphonate derivatives of nucleoside antimetabolites.

Despite palliative treatments, tumor-induced bone disease (TIBD) remains highly debilitating for many cancer patients and progression typically results in death within two years. Therefore, more effective therapies with enhanced anti-resorptive and cytotoxic characteristics are needed. We developed bisphosphonate-chemotherapeutic conjugates designed to bind bone and hydrolyze, releasing both compounds, thereby targeting both osteoclasts and tumor cells. This study examined the effects of our lead compound, MBC-11 (the anhydride formed between arabinocytidine (AraC)-5'-phosphate and etidronate), on bone tumor burden, bone volume, femur bone mineral density (BMD), and overall survival using two distinct mouse models of TIBD, the 4T1/luc breast cancer and the KAS-6/1-MIP1alpha multiple myeloma models. In mice orthotopically inoculated with 4T1/luc mouse mammary cells, MBC-11 (0.04 microg/day; s.c.) reduced the incidence of bone metastases to 40% (4/10), compared to 90% (9/10; p=0.057) and 100% (5/5; p=0.04) of PBS- or similarly-dosed, zoledronate-treated mice, respectively. MBC-11 also significantly decreased bone tumor burden compared to PBS- or zoledronate-treated mice (p=0.021, p=0.017, respectively). MBC-11 and zoledronate (0.04 microg/day) significantly increased bone volume by two- and four-fold, respectively, compared to PBS-treated mice (p=0.005, p<0.001, respectively). In mice systemically injected with human multiple myeloma KAS-6/1-MIP1alpha cells, 0.04 and 4.0 microg/day MBC-11 improved femur BMD by 13% and 16%, respectively, compared to PBS (p=0.025, p=0.017, respectively) at 10 weeks post-tumor cell injection and increased mean survival to 95 days compared to 77 days in mice treated with PBS (p=0.047). Similar doses of zoledronate also improved femur BMD (p< or =0.01 vs PBS) and increased mean survival to 86 days, but this was not significantly different than in PBS-treated mice (p=0.53). These results demonstrate that MBC-11 decreases bone tumor burden, maintains bone structure, and may increase overall survival, warranting further investigation as a treatment for TIBD.

Evaluation of a panel of tumor markers for molecular detection of circulating cancer cells in women with suspected breast cancer.

PURPOSE: We examined the feasibility of using molecular characterization of circulating tumor cells as a method for early detection of breast cancer. RESEARCH DESIGN: Women without a prior history of cancer who had a breast abnormality detected on imaging followed by a breast biopsy were enrolled in this study. Density gradient centrifugation and immunomagnetic capture were used to enrich for epithelial cells from approximately 20 mL of blood. Real-time reverse transcription-PCR was used to quantitate the expression levels of the highly breast-specific genes, mammaglobin, gamma-aminobutyric acid type A receptor pi subunit (GABA A(pi)), B305D-C, and B726P in the epithelial cell-enriched samples. RESULTS: The assay was technically feasible in 154 of 199 accrued patients. From their clinical assessment, 100 patients had benign breast disease, 10 patients had ductal carcinoma in situ, and 44 patients had invasive breast cancer. We constructed a diagnostic test that classified patients with mammaglobin levels of at least 32.2 copies/pg beta-actin (units) in their circulating epithelial cells as positive for invasive breast cancer. This resulted in a sensitivity and specificity of 63.3% and 75.0%, respectively. A diagnostic test that classified patients as positive for invasive breast cancer when either mammaglobin levels were >46.3 units or B305D-C levels were >11.6 units increased the sensitivity and specificity to 70.5% and 81.0%, respectively. In the latter test, 12 of the 14 node-positive breast cancer patients were correctly identified. Including GABA A(pi) and B726P in the test did not increase its diagnostic potential. CONCLUSIONS: These results suggest that molecular characterization of circulating epithelial cells using mammaglobin and B305D-C offers potential for early detection of invasive breast cancer.