Analytical Performance of a 15-Gene Prognostic Assay for Early-Stage Non-Small-Cell Lung Carcinoma Using RNA-Stabilized Tissue.

A 15-gene prognostic signature for early-stage, completely resected, non-small-cell lung carcinoma, (which distinguishes between patients with good and poor prognoses) was clinically validated in prior studies. To achieve operational efficiencies, this study was designed to evaluate the assay's performance in RNA-stabilized tissue as an alternative to the fresh-frozen tissue format originally used to develop the assay. The percent concordance between matched tissue formats was 84% (95% Wilson CI, 70%-92%), a level of agreement comparable to the inherent reproducibility of the assay observed within biological replicates of fresh-frozen tissue. Furthermore, the analytical performance of the assay using the RNA-stabilized tissue format was evaluated. When compared to an accredited reference laboratory, the clinical laboratory achieved a concordance of 94% (95% Wilson CI, 81%-98%), and there was no evidence of bias between the laboratories. The lower limit of quantitation for the target RNA concentration was confirmed to be, at most, 12.5 ng/µL. The assay reportable range defined in terms of risk score units was determined to be -4.295 to 4.210. In a large-scale precision study, the assay showed high reproducibility and repeatability. When subjected to a maximal amount of genomic DNA, a potential contaminant, the assay still produced the expected results. The 15-gene signature was confirmed to produce reliable results and, thus, is suitable for its intended use.

A cost-effectiveness analysis of a chemoresponse assay for treatment of patients with recurrent epithelial ovarian cancer.

OBJECTIVE: Clinical validation of a chemoresponse assay was recently published, demonstrating a significant increase in overall survival in recurrent ovarian cancer patients treated with therapies to which their tumor was sensitive in the assay. The current study investigates the cost effectiveness of using the assay at the time of ovarian cancer recurrence from the payer's perspective. METHODS: Using a Markov state transition model, patient characteristics and survival data from the recent clinical study, the cumulative costs over the study horizon (71 months) for both the baseline (no assay) and intervention (assay consistent, hypothetical) cohorts were evaluated. RESULTS: The assay consistent cohort had an incremental cost effectiveness ratio (ICER) of $6206 per life year saved (LYS), as compared to the baseline cohort. Cost-effectiveness was further demonstrated in platinum-sensitive and platinum-resistant populations treated with assay-sensitive therapies, with ICERs of $2773 per LYS and $2736 per LYS, respectively. CONCLUSIONS: The use of a chemoresponse assay to inform treatment decisions in recurrent ovarian cancer patients has the potential to be cost-effective in both platinum-sensitive and platinum-resistant patients.

Analytical performance of a formalin-fixed paraffin-embedded tissue-based 634-probe prognostic assay for predicting outcome of patients with stage II colon cancer.

A formalin-fixed paraffin-embedded tissue-based prognostic assay to assess the risk for recurrence in stage II colon cancer has recently been clinically validated. This study describes the analytical performance and quality control measures of the assay. The reportable range was determined to be [-1.129, 1.414] in risk score units. The accuracy was evaluated with a split sample comparison within the production lab and between the production lab and a reference lab. The concordance between the replicates within the production lab was 79% (95% confidence interval, 64%-91%). There was no evidence of bias, and the concordance was 78% (95% confidence interval, 61%-90%) between the labs. The lab-to-lab concordance was further evaluated by simulating risk scores from the full reportable range. The simulation suggested a higher concordance. The sensitivity study demonstrated that the percentage of tumor tissue did not impact the risk score and that RNA concentration of 9.5 ng/µL was a conservative determination of the analyte lower limit of quantification. From the precision study, the repeatability and reproducibility estimates were 0.1267 and 0.0548 in risk score units, respectively. Furthermore, multifaceted quality control measures were implemented, such as proper tissue processing steps, high-risk and low-risk controls, nontemplate control, and a gene expression-based classifier to evaluate the cDNA amplification kit, a key reagent in the assay. In conclusion, this study demonstrates the strong analytical performance of the assay and further supports its use as an objective standardized prognostic test for stage II colon cancer.

A systematic evaluation of multi-gene predictors for the pathological response of breast cancer patients to chemotherapy.

Previous studies have reported conflicting assessments of the ability of cell line-derived multi-gene predictors (MGPs) to forecast patient clinical outcomes in cancer patients, thereby warranting an investigation into their suitability for this task. Here, 42 breast cancer cell lines were evaluated by chemoresponse tests after treatment with either TFAC or FEC, two widely used standard combination chemotherapies for breast cancer. We used two different training cell line sets and two independent prediction methods, superPC and COXEN, to develop cell line-based MGPs, which were then validated in five patient cohorts treated with these chemotherapies. This evaluation yielded high prediction performances by these MGPs, regardless of the training set, chemotherapy, or prediction method. The MGPs were also able to predict patient clinical outcomes for the subgroup of estrogen receptor (ER)-negative patients, which has proven difficult in the past. These results demonstrated a potential of using an in vitro-based chemoresponse data as a model system in creating MGPs for stratifying patients' therapeutic responses. Clinical utility and applications of these MGPs will need to be carefully examined with relevant clinical outcome measurements and constraints in practical use.

Cell line derived multi-gene predictor of pathologic response to neoadjuvant chemotherapy in breast cancer: a validation study on US Oncology 02-103 clinical trial.

BACKGROUND: The purpose of this study is to assess the predictive accuracy of a multi-gene predictor of response to docetaxel, 5-fluorouracil, epirubicin and cyclophosphamide combination chemotherapy on gene expression data from patients who received these drugs as neoadjuvant treatment. METHODS: Tumor samples were obtained from patients with stage II-III breast cancer before starting neoadjuvant chemotherapy with four cycles of 5-fluorouracil/epirubicin/cyclophosphamide (FEC) followed by four cycles of docetaxel/capecitabine (TX) on US Oncology clinical trial 02-103. Most patients with HER-2-positive cancer also received trastuzumab (H). The chemotherapy predictor (TFEC-MGP) was developed from publicly available gene expression data of 42 breast cancer cell-lines with corresponding in vitro chemotherapy sensitivity results for the four chemotherapy drugs. No predictor was developed for treatment with trastuzumab. The predictive performance of TFEC-MGP in distinguishing cases with pathologic complete response from those with residual disease was evaluated for the FEC/TX and FEC/TX plus H group separately. The area under the receiver-operating characteristic curve (AU-ROC) was used as the metric of predictive performance. Genomic predictions were performed blinded to clinical outcome. RESULTS: The AU-ROC was 0.70 (95% CI: 0.57-0.82) for the FEC/TX group (n=66) and 0.43 (95% CI: 0.20-0.66) for the FEC/TX plus H group (n=25). Among the patients treated with FEC/TX, the AU-ROC was 0.69 (95% CI: 0.52-0.86) for estrogen receptor (ER)-negative (n=28) and it was 0.59 (95% CI: 0.36-0.82) for ER-positive cancers (n=37). ER status was not reported for one patient. CONCLUSIONS: Our results indicate that the cell line derived 291-probeset genomic predictor of response to FEC/TX combination chemotherapy shows good performance in a blinded validation study, particularly in ER-negative patients.

Distinct genes related to drug response identified in ER positive and ER negative breast cancer cell lines.

Breast cancer patients have different responses to chemotherapeutic treatments. Genes associated with drug response can provide insight to understand the mechanisms of drug resistance, identify promising therapeutic opportunities, and facilitate personalized treatment. Estrogen receptor (ER) positive and ER negative breast cancer have distinct clinical behavior and molecular properties. However, to date, few studies have rigorously assessed drug response genes in them. In this study, our goal was to systematically identify genes associated with multidrug response in ER positive and ER negative breast cancer cell lines. We tested 27 human breast cell lines for response to seven chemotherapeutic agents (cyclophosphamide, docetaxel, doxorubicin, epirubicin, fluorouracil, gemcitabine, and paclitaxel). We integrated publicly available gene expression profiles of these cell lines with their in vitro drug response patterns, then applied meta-analysis to identify genes related to multidrug response in ER positive and ER negative cells separately. One hundred eighty-eight genes were identified as related to multidrug response in ER positive and 32 genes in ER negative breast cell lines. Of these, only three genes (DBI, TOP2A, and PMVK) were common to both cell types. TOP2A was positively associated with drug response, and DBI was negatively associated with drug response. Interestingly, PMVK was positively associated with drug response in ER positive cells and negatively in ER negative cells. Functional analysis showed that while cell cycle affects drug response in both ER positive and negative cells, most biological processes that are involved in drug response are distinct. A number of signaling pathways that are uniquely enriched in ER positive cells have complex cross talk with ER signaling, while in ER negative cells, enriched pathways are related to metabolic functions. Taken together, our analysis indicates that distinct mechanisms are involved in multidrug response in ER positive and ER negative breast cells.

Breast cancer--response rates to chemotherapeutic agents studied in vitro.

BACKGROUND: The biological efficacy of, and spectrum of action of, agents used in treatment of breast cancer are important issues in therapy planning. MATERIALS AND METHODS: Techniques used involve monolayer culture and a quantitative microtiter plate-based chemo-response assay. Precision Therapeutics' overall assessability rate is 90% for tumors of all types. In this study, 148 specimens derived from breast cancer were studied. Of these, 111 were additionally studied histopathologically. Ninety-two percent of the 111 specimens were confirmed to be epithelioid in nature and, thus, compatible with cells of breast cancer origin. RESULTS: In vitro chemo-response profiles indicated that individual agents stratified into groups, with cyclophosphamide and fluorouracil demonstrating responses of 69% and 57% respectively; doxorubicin, 45%; and docetaxel, paclitaxel and gemcitabine, 39, 27 and 36%, respectively. CONCLUSION: The spectrum of responsiveness of the individual agents was variable and not completely overlapping, as shown by the Venn diagrams.

Evidence for the isolation, growth, and characterization of malignant cells in primary cultures of human tumors.

Isolation and growth of malignant cells from solid tumors have often met with disappointing results. Consequently, we have developed a cell culture methodology based on ex vivo explantation of tumor tissue, with subsequent monolayer cell outgrowth. In an attempt to assess methods for detection of malignant cells in these cultures, we analyzed and compared the results of cytopathology, growth in soft agar, and detection of telomerase activity with those of standard immunohistochemistry (IHC) techniques for the detection of cytokeratins, tumor marker p53, and proliferation marker Ki-67. The sensitivity of detection of malignant cells was 85% (22/26) for cytopathological examination, 30% (3/10) for soft agar growth, and 100% (12/12) for detection of telomerase activity. From these data, we concluded that both cytopathological examination and assessment of telomerase activity contribute to the detection of malignant cells in primary cultures of human solid tumors, whereas growth in soft agar was not a good indicator of malignant cells. Although not specific for malignant cells per se, IHC detection for epithelial cell cytokeratins showed a high degree of sensitivity (100%, 23/23), whereas the sensitivity for detection of tumor marker p53 and proliferation marker Ki-67 was 30% (7/23) and 70% (16/23), respectively. These data also provide proof that malignant tumor cells, derived from a diverse number of human solid tumors, can be isolated and grown in primary cell culture.

In vitro responses of ovarian cancers to platinums and taxanes.

We describe the in vitro patterns of response of explanted primary and recurrent ovarian cancers to platinum- and taxane-based chemotherapeutics. The chemoresponse assay utilizes cells that grow out from tumor fragments and are then challenged with varied concentrations of chemotherapeutic agents, coupled with a highly quantitative cell counting analysis system. The in vitro response rates for 268 primary cancer explants were 24% and 54% for carboplatin and cisplatin, respectively, and 31% and 25% for docetaxel and paclitaxel, respectively. Recurrent tumors presented lower rates of responsiveness, as expected. Furthermore, the chemotherapies worked on overlapping but distinct populations, even within the same class of drug, with 14% of the carboplatin-sensitive tumors being cisplatin-resistant and 59% of the cisplatin-sensitive tumors being carboplatin-resistant. These in vitro responses compare favorably to published in vivo clinical response rates. The current study serves to demonstrate how an in vitro predictive assay can be used as a surrogate for clinical therapeutic challenge.