Assessment of erlotinib in chemoresponse assay.

BACKGROUND: There is a need to identify the subset of patients sensitive to epidermal growth factor receptor (EGFR) inhibition prior to using such treatments. MATERIALS AND METHODS: Three non-small cell lung cancer (NSCLC) cell lines (H292, H358, and Calu3) and 34 primary human lung tumor specimens were tested for chemoresponse to erlotinib. RESULTS: The assay distinguished responsiveness to erlotinib among NSCLC cell lines and human lung tumor explants. The H292 cells were responsive, the Calu3 cells were intermediate responsive and the H358 cells were non-responsive. These results were consistent with published tumor growth inhibition by erlotinib in xenografts derived from these cell lines. Out of the 34 patient specimens, 3 (8.8%) were responsive to erlotinib, 7 (20.6%) were intermediate responsive and 24 (70.6%) were non-responsive. CONCLUSION: The in vitro chemoresponse assay profile was similar to that noted for human tumors in clinical trials. Chemoresponse testing may help predict patient response to erlotinib and assist chemotherapy decision-making.

Comparison of angiogenesis-related factor expression in primary tumor cultures under normal and hypoxic growth conditions.

BACKGROUND: A localized hypoxic environment occurs during tumor growth necessitating an angiogenic response or tumor necrosis results. Novel cancer treatment strategies take advantage of tumor-induced vascularisation by combining standard chemotherapeutic agents with angiogenesis-inhibiting agents. This has extended the progression-free interval and prolonged survival in patients with various types of cancer. We postulated that the expression levels of angiogenesis-related proteins from various primary tumor cultures would be greater under hypoxic conditions than under normoxia. METHODS: Fifty cell sources, including both immortalized cell lines and primary carcinoma cells, were incubated under normoxic conditions for 48 hours. Then, cells were either transferred to a hypoxic environment (1% O2) or maintained at normoxic conditions for an additional 48 hours. Cell culture media from both conditions was collected and analyzed via an ELISA-based assay to determine expression levels of 11 angiogenesis-related factors: VEGF, PDGF-AA, PDGF-AA/BB, IL-8, bFGF/FGF-2, EGF, IP-10/CXCL10, Flt-3 ligand, TGF-beta1, TGF-beta2, and TGF-beta3. RESULTS: A linear correlation between normoxic and hypoxic growth conditions exists for expression levels of eight of eleven angiogenesis-related proteins tested including: VEGF, IL-8, PDGF-AA, PDGF-AA/BB, TGF-beta1, TGF-beta2, EGF, and IP-10. For VEGF, the target of current therapies, this correlation between hypoxia and higher cytokine levels was greater in primary breast and lung carcinoma cells than in ovarian carcinoma cells or tumor cell lines. Of interest, patient cell isolates differed in the precise pattern of elevated cytokines. CONCLUSION: As linear correlations exist between expression levels of angiogenic factors under normoxic and hypoxic conditions in vitro, we propose that explanted primary cells may be used to probe the in vivo hypoxic environment. Furthermore, differential expression levels for each sample across all proteins examined suggests it may be possible to build a predictor for angiogenesis-related anticancer agents, as each sample has a unique expression profile. Further studies should be performed to correlate in vitro protein expression levels of angiogenesis-related factors with in vivo patient response.

The ChemoFx assay: an ex vivo chemosensitivity and resistance assay for predicting patient response to cancer chemotherapy.

The ChemoFx Assay is an ex vivo assay designed to predict the sensitivity and resistance of a given patient's solid tumor to a variety of chemotherapy agents. A portion of a patient's solid tumor, as small as a core biopsy, is mechanically disaggregated and established in primary culture where malignant epithelial cells migrate out of tumor explants to form a monolayer. Cultures are verified as epithelial and exposed to increasing doses of selected chemotherapeutic agents. The number of live cells remaining post-treatment is enumerated microscopically using automated cell-counting software. The resultant cell counts in treated wells are compared with those in untreated control wells to generate a dose-response curve for each chemotherapeutic agent tested on a given patient specimen. Features of each dose-response curve are used to score a tumor's response to each ex vivo treatment as "responsive," "intermediate response," or "non-responsive." Collectively, these scores are used to assist an oncologist in making treatment decisions.

Feasibility assessment of a chemoresponse assay to predict pathologic response in neoadjuvant chemotherapy for breast cancer patients.

BACKGROUND: For chemosensitivity and resistance assays to be clinically useful in predicting patient outcome, they should require small amounts of tissue and be highly reproducible and reliable. PATIENTS AND METHODS: Expanded tumor cells from transcutaneous biopsies of breast lesions (n=62) were tested for chemoresponse using the cell-based ChemoFx assay. Pathologic complete response (pCR) was determined on a subset of patients (n=34). Assay score and pCR were determined independently in a blinded manner. Logistic regression models were used to select predictors for response. RESULTS: Tumor cells were successfully isolated from 83.9% of patients. Chemoresponse profiles were robust and reproducible with coefficient of variance of <3%. In a limited initial patient outcome correlation, assay score of docetaxel/capecitabine significantly predicted pCR; the cross-validated model was 75% accurate. CONCLUSION: It is feasible to assess the chemoresponsiveness of small breast lesions using the ChemoFx assay to assist in choosing neoadjuvant chemotherapy for breast cancer patients.

Ex vivo enrichment of malignant carcinoma cells in primary culture.

AIMS: Establishing and maintaining human tumours in primary culture can be challenging. In this application, a short-term primary culture process is desired to ensure cells maintained in culture are representative of the in vivo tumour for the purpose of chemoresponse testing. To ensure the appropriate cells are being grown, the cultures must be evaluated for malignancy. The clinical gold standard determination of malignancy is cytological evaluation by a cytopathologist. METHODS: Fifty human tumour specimens (breast, colon, lung, ovary) were established and maintained in primary culture. Cytospins were prepared upon initiation of culture and again at completion of the culture process. Cytospins were stained (Diff-Quik, Papanicolaou) and evaluated by a cytopathologist for the percentage of malignant cells at both times. RESULTS: An increase in the percentage of malignant cells was noted in 86% (43/50) of the cultures evaluated; 8% (4/50) of the cultures maintained the same percentage of malignant cells throughout the culture period, and 6% (3/50) displayed a decrease in malignant cells. On average, the percentage of malignant cells increased by 37% and was not associated with the length of culture (range 5-28 days). CONCLUSIONS: The described primary culture process enriches for malignant cells, which is desirable for further evaluation such as chemoresponse testing.