In vitro drug responses in primary and metastatic colorectal cancers.

OBJECTIVE: Treatment of primary and metastatic colorectal carcinoma (CRC) based on 5-fluorouracil and folinic acid (5FU + FA), combined with irinotecan (FOLFIRI) or oxaliplatin (FOLFOX), provides response rates approaching 50% and a 20-month overall survival. Approximately 50% of CRC patients fail to respond to one or more drugs in either regimen, in many cases due to inherent or acquired drug resistance. We therefore characterized in vitro drug response and cross-resistance in primary and metastatic CRC lesions. MATERIALS AND METHODS: The in vitro Extreme Drug Resistance Assay (EDRA) identifies extreme drug resistance (EDR) in solid tumors with over 99% accuracy and appears to mimic the clinical experience. We analyzed EDRA results from 4854 freshly resected CRC biopsies, including 1740 primary and 847 liver metastases. RESULTS: Primary and metastatic CRCs responded similarly to single agents 5FU + FA, irinotecan, and oxaliplatin. Primary and metastatic tumors expressing EDR to 5FU + FA demonstrated up to 58% cross-resistance to a variety of chemotherapy agents, with the lowest percentages for oxaliplatin (11% and 8%, respectively) and irinotecan (16% and 14%). Importantly, approximately 20% of tumors showed EDR to either FULFOX or FOLFIRI. CONCLUSION: Overall data analyses indicated that EDRA results obtained at initial diagnosis may be useful in guiding therapy selection for metastatic disease. Pre-testing of tumors before treatment may provide essential drug cross-resistance information for better chemotherapy selection. Prospective clinical trials employing the EDRA are needed to substantiate these data.

Prospective evaluation of an in vitro radiation resistance assay in locally advanced cancer of the uterine cervix: a Southwest Oncology Group Study.

OBJECTIVES: To investigate the feasibility of performing a fresh-tissue, in vitro radiation resistance assay (IVRRA) in a cooperative group setting and to assess the association of IVRRA results with clinical outcomes. METHODS: Women with Stages IIB-IVA carcinoma of the uterine cervix without obvious para-aortic lymphadenopathy on imaging were eligible. Primary tumor biopsies were shipped to a central testing facility where agar-based cell suspensions were exposed to 300 cGy of RT ± cisplatin and cultured for 5 days. ³H-thymidine incorporation was used to determine percent cell inhibition (PCI) of test specimen compared to that of the untreated control. Tumors were considered to exhibit extreme radiation resistance (ERR), intermediate radiation resistance (IRR) or low radiation resistance (LRR) based on a standard data set from 39 previously studied specimens. Standardized doses of external beam radiation and intracavitary brachytherapy, when feasible, in addition to platinum-based chemotherapy were mandated. Progression-free survival (PFS) was the primary endpoint. Clinical response and overall survival (OS) were secondary endpoints. Clinical investigators were blinded to assay data and vice versa. RESULTS: Thirty-six patients were enrolled, but analysis was limited to 17 patients whose specimens were adequate for IVRRA. The median follow-up time among patients still alive at last contact was 40 months (range: 0-56 months). There was no association between IVRRA and response. In the Cox model, IRR/ERR tumors showed worse PFS [HR = 11.2 (95% CI 1.3-96, p = 0.03)] and worse OS [HR=11.7 (95% CI 1.4-99.6, p = 0.03)] compared to LRR tumors when IVRRA was performed with RT alone, but there were no associations between IVRRA and PFS or OS when cisplatin was added to the IVRRA. CONCLUSIONS: IVRRA (RT alone) results correlated with PFS and OS in this prospective trial, but follow-up trials are indicated to address feasibility and to confirm results in an expanded cohort. If confirmed, IVRRA could potentially direct molecular identification of novel targeted therapeutic approaches which might counteract radiation resistance.

Survival among patients with platinum resistant, locally advanced non-small cell lung cancer treated with platinum-based systemic therapy.

BACKGROUND: Recent adjuvant chemotherapy trials after resection of stage II and III non-small cell lung cancer (NSCLC) have identified important survival differences among patients with immunohistochemical evidence suggesting platinum resistance. No clinical information exists regarding the impact upon survival of patients treated with platinum agents who exhibit cellular evidence of their tumors' resistance to platinum. We evaluated the utility of the extreme drug resistance (EDR) assay to predict mortality among a consecutive group of stage II through IV NSCLC patients receiving adjuvant or definitive platinum-based chemotherapy after resection or surgical biopsy. METHODS: The Extreme Drug Resistance (EDR) Assay is a clinically validated cellular proliferation assay used to test tumors for chemotherapy drug resistance. Based on response in the EDR assay, tumor specimens from stage II through IV NSCLC patients were segregated into three groups: extreme drug resistant (EDR), intermediate drug resistant (IDR), and low drug resistant (LDR). Patient survival was evaluated after platinum-based chemotherapy. RESULTS: Platinum IDR/EDR was statistically significant in predicting shorter overall survival (29.8 months vs. 15.6 months) among platinum IDR/EDR-resistant patients compared with LDR patients (P = 0.047). Median survival was 16.6 months for patients with IDR/EDR to platinum and any other second agent of doublet therapy compared with patients with LDR to any platinum-based doublet where median survival was not achieved (P = 0.0268). CONCLUSIONS: This is the first study to demonstrate the utility of the EDR assay to predict poor clinical outcome when platinum-based therapy is used to treat patients with biological evidence of tumor resistance to platinum. These data corroborate the finding of recent studies evaluating possible molecular correlates to poor response to specific chemotherapeutic agents.

Association of angiogenesis markers with lymph node metastasis in early colorectal cancer.

HYPOTHESIS: We hypothesized that p53 mutations (mp53) are associated with decreased expression of thrombospondin 1 (TSP-1) and that decreased TSP-1 expression is associated with lymph node metastases. DESIGN: A retrospective study of lymphatic mapping and pathologic determination of angiogenesis markers in primary colorectal cancer. SETTING: Tertiary care cancer institute. PATIENTS: Sixty-one patients with colorectal cancer underwent lymphatic mapping. Lymph nodes that stained negative by hematoxylin-eosin were examined with immunohistochemistry for micrometastases. Primary tumors were analyzed by immunohistochemistry for mp53 and TSP-1 expression. The t test and the Mann-Whitney U test were used to examine the mean difference in TSP-1 expression between tumors. MAIN OUTCOME MEASURES: Mutant p53 expression, TSP-1 expression, and metastatic progression. RESULTS: Thirty-six of the 61 patients (59%) had nodal metastases shown by hematoxylin-eosin or immunohistochemistry in the sentinel node (N2, N1, N1mi, or N0[i+]). Patients with a truly negative sentinel node (pN0[i-][sn]) had significantly higher TSP-1 expression compared with those with some degree of nodal metastases (57.7 vs 30.1; P < .001). Acquisition of mp53 was associated with a decreased mean TSP-1 expression. Tumors without mp53 expression had a mean TSP-1 optical density value of 51.3 while tumors with elevated mp53 had a mean TSP-1 optical density value of 31.8 (P < .03). CONCLUSIONS: Patients with primary colorectal cancer with low TSP-1 expression, with or without detection of mp53 gene product, are more likely to harbor lymph node metastasis than patients with higher expression. Patients with a truly negative sentinel node (pN0[i-][sn]) frequently have higher expression of TSP-1 that may have inhibited metastatic progression. Further studies will investigate the relationship between mp53, TSP expression, and disease progression.

In vitro drug response and molecular markers associated with drug resistance in malignant gliomas.

PURPOSE: Drug resistance in malignant gliomas contributes to poor clinical outcomes. We determined the in vitro drug response profiles for 478 biopsy specimens from patients with the following malignant glial histologies: astrocytoma (n = 71), anaplastic astrocytoma (n = 39), glioblastoma multiforme (n = 259), oligodendroglioma (n = 40), and glioma (n = 69). EXPERIMENTAL DESIGN: Samples were tested for drug resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, dacarbazine, paclitaxel, vincristine, and irinotecan. Biomarkers associated with drug resistance were detected by immunohistochemistry, including multidrug resistance gene-1, glutathione S-transferase pi (GSTP1), O(6)-methylguanine-DNA methyltransferase (MGMT), and mutant p53. RESULTS: In vitro drug resistance in malignant gliomas was independent of prior therapy. High-grade glioblastomas showed a lower level of extreme drug resistance than low-grade astrocytomas to cisplatin (11% versus 27%), temozolomide (14% versus 27%), irinotecan (33% versus 53%), and BCNU (29% versus 38%). A substantial percentage of brain tumors overexpressed biomarkers associated with drug resistance, including MGMT (67%), GSTP1 (49%), and mutant p53 (41%). MGMT and GSTP1 overexpression was independently associated with in vitro resistance to BCNU, whereas coexpression of these two markers was associated with the greatest degree of BCNU resistance. CONCLUSIONS: Assessment of in vitro drug response and profiles of relevant tumor-associated biomarkers may assist the clinician in stratifying patient treatment regimens.

Chemotherapy resistance and oncogene expression in non-small cell lung cancer.

OBJECTIVES: Empiric chemotherapy for patients with non-small cell lung cancer who have undergone resection is recommended without knowledge of the tumor's specific biologic characteristics, and many patients may not benefit. In vitro chemotherapy resistance is associated with clinical unresponsiveness in some tumors, and in lung cancer, chemotherapy resistance is prevalent. Multiple-agent chemotherapy resistance and association of chemotherapy resistance with molecular markers are described. METHODS: Chemotherapy resistance to doublets--carboplatin and paclitaxel, cisplatin and navelbline, cisplatin and docetaxel, and cisplatin and gemcitabine--was analyzed in 4571 non-small cell lung cancer tumors with the extreme drug resistance assay. Chemotherapy resistance is defined as follows: extreme drug resistance, 1 SD above the median chemotherapy resistance; intermediate drug resistance, between the median and extreme drug resistances; and low drug resistance, 1 SD below the median. Chemotherapy resistance was compared with DNA ploidy measured by flow cytometry, and markers p53 and epithelial growth factor receptor were assayed by immunohistochemistry. RESULTS: Tumors with extreme or intermediate drug resistance were noted in 30% to carboplatin-paclitaxel, in 24% to cisplatin-navelbline, in 42% to cisplatin-gemcitabine, and in 27% to cisplatin-docetaxel. Extreme or intermediate drug resistance to at least one drug occurred in 74% to carboplatin-paclitaxel, in 68% to cisplatin-navelbline, in 88% to cisplatin-gemcitabine, and in 68% to cisplatin-docetaxel. More intermediate plus extreme chemotherapy resistances occurred in aneuploid tumors to etoposide (53% vs 36%, P = .0002) and topotecan (48% vs 36%, P = .0094), with less intermediate or extreme chemotherapy resistance to gemcitabine (88% vs 81%, P = .0345). p53-Positive tumors had more intermediate or extreme resistance to etoposide (57% vs 44%, P = .0009) and doxorubicin (73% vs. 58%, P = .0324) and less intermediate or extreme resistance to cisplatin (44% vs 54%, P = .0125), to carboplatin (47% vs 57%, P = .0129), to taxol (47% vs 57%, P = .0056), and to gemcitabine (78% vs 87%, P = .0108). Fewer epithelial growth factor receptor-positive tumors were extremely drug resistant to cisplatin (13% vs 26%, P = .0074) and carboplatin (13% v. 30%, P = .0008). CONCLUSIONS: Multi-drug chemotherapy resistance in non-small cell lung cancer tumor cultures is common, and associations between molecular markers and in vitro chemotherapy resistance are noted. Clinical validation through integration of such testing into clinical trials seems warranted.

Prevalence of in vitro extreme chemotherapy resistance in resected nonsmall-cell lung cancer.

BACKGROUND: Recent clinical trials suggest that adjuvant chemotherapy provides a survival advantage for patients with completely resected nonsmall-cell lung cancer (NSCLC) yet many patients receive chemotherapy without benefit. Tumor in vitro resistance to antineoplastic agents is highly predictive of clinical unresponsiveness to chemotherapy for some cancers; however, little is known of the prevalence of extreme chemotherapy drug resistance for human NSCLC tumors. Chemoresistance testing may be a way to predict treatment failure, choose alternative agents, and to avoid unnecessary chemotherapy toxicity. This study describes the prevalence of in vitro chemotherapy resistance in NSCLC patient tumor cultures. METHODS: A total of 3,042 NSCLC specimens were cultured in a proliferation assay and tested for resistance to carboplatin, cisplatin, doxorubicin, etoposide, gemcitabine, navelbine, paclitaxel, taxotere, or topotecan. The percentage of cell-growth inhibition measured by 3H-Thymidine uptake was used to determine extreme drug resistance, intermediate drug resistance, or low drug resistance. RESULTS: Extreme drug resistance or intermediate drug resistance to carboplatin was found in 1,056 of 1,565 NSCLC cultures (68%), to cisplatin in 1,409 of 2,227 (63%), to doxorubicin in 1,101 of 1,471 (75%), to etoposide in 1,581 of 2,505 (63%), to gemcitabine in 594 of 823 (72%), to navelbine in 603 of 1,444 (42%), to paclitaxel in 689 of 1,706 (40%), to taxotere in 273 of 521 (52%), and to topotecan in 280 of 896 (31%). CONCLUSIONS: Chemotherapy resistance is prevalent among NSCLC clinical cell cultures. This may account for the small survival seen with empiric adjuvant chemotherapy. The use of viable tumor culture for in vitro chemoresistance testing should be considered when formulating a plan of adjuvant therapy for resected NSCLC. Future trials comparing patient survival after tailored versus empiric adjuvant therapy appear justified.

Gene expression profiling of in vitro radiation resistance in cervical carcinoma: a feasibility study.

OBJECTIVE(S): To determine the feasibility of integrating an in vitro chemo-radiation response assay (IVRRA) with a gene microarray system to investigate the molecular patterns of expression that contribute to radiation resistance in cervical cancer. METHODS: Viable primary untreated cervical cancer specimens were obtained and exposed to gamma irradiation at a dose of 3 Gy in the IVRRA to determine in vitro radiation sensitivity. RNA was purified for microarray analysis with the Affymetrix Human Genome U95A Array carrying more than 12,000 gene probes. Gene expression analysis was performed, and specimen transcript patterns were correlated with radiation response using an iteration analysis model and Pearson's correlation coefficient. RESULTS: A feasibility set of eight tumor specimens was studied. Tumors were classified into 4 extreme (ERR), 2 intermediate (IRR) and 2 low radiation resistance (LRR) categories. An intrinsic radiation response gene set of 54 genes transcripts with 100% accuracy for the classification of each tumor's radiation response category was identified. CONCLUSION(S): Gene sets associated with in vitro radiation response profiles in cervical cancer can be generated using the IVRRA and microarray technology. This has direct applications to the study of the biological pathways contributing to radiation resistance and may lead to the development of alternative treatment modalities. The potential of these technologies for cancers in which radiotherapy is employed warrants further investigation.

A prospective blinded study of the predictive value of an extreme drug resistance assay in patients receiving CPT-11 for recurrent glioma.

This adjunct to a prospective phase II blinded study of 48 patients with recurrent malignant glioma evaluated the predictive reliability of an extreme drug resistance (EDR) to identify clinical resistance to irinotecan (CPT-11), using fresh tumor biopsies obtained from recurrent patients immediately prior to their first dose of CPT-11 therapy. In vitro tumor response to SN38 (bioactive species of CPT-11 used in the EDR assay) determined prior to treatment was correlated with objective response, time to tumor progression (TTP) and survival following the administration of CPT-11. SN38 activity was tested in 19 of 29 tumors, with 15 of 18 assay results evaluable for correlation with clinical outcomes. In vitro drug resistance was classified as either extreme, intermediate (IDR), or low (LDR). TTP and survival were estimated by the Kaplan-Meier method, and compared using the Mantel-Haenszel log-rank and Fisher's exact test statistics. In vitro tumor response was bifurcated into either EDR (n = 4) or IDR/LDR (n = 11) categories for comparison with outcomes. Results correlated significantly with both TTP and survival. Median TTP for IDR/LDR cases was 3 months versus 6 weeks for EDR cases (log-rank test; p = 0.0288, hazards ratio = 3.06). A 13-week median survival for EDR cases was significantly shorter compared to 38 weeks for IDR/LDR cases (p = 0.029). Further, 100-day survival favored the IDR/LDR cases (Fisher's exact test; p = 0.008). At last follow-up, two of three survivors were patients who had tumors IDR/LDR to SN38. These prospective data support the notion that patients should avoid agents to which their tumor demonstrates EDR.

Development of an in vitro chemo-radiation response assay for cervical carcinoma.

PURPOSE: To determine if synergistic effects of radiation (RT) and chemotherapy (chemo) on human cervical carcinoma cell lines and fresh tumor explants could be determined using an in vitro assay. EXPERIMENTAL DESIGN: In vitro radiation response was determined for 4 cell lines and 26 fresh tumor explants in an agar-based assay. Cells were exposed to increasing doses of RT with or without cisplatin (CDDP), carmustine (BCNU), buthionine sulfoximine (BSO), or paclitaxel (Tax). Cell suspensions were cultured for 5 days, with [(3)H]thymidine added on day 3 and proliferation was measured. Results were reported as the fraction of proliferation compared to control (FC). For each combination of irradiation and drug, synergy was tested using the Chou analysis, where a combination index (CI) <1 indicated synergistic interaction. In simple correlation analysis, an R value of >0.7 indicated cross-resistance. RESULTS: RT dose-dependent proliferation inhibition was observed for 2 of the 4 cell lines, and for all but 1 of the fresh specimens. Significant heterogeneity of tumor response to RT was seen. Four specimens that were 1 standard deviation below the median FC response after exposure to 300 cGy were classified as extremely radiation resistant. Twenty-one tumors were evaluated for synergistic response using the combination of chemo and RT with a median FC of 0.27 (+/-0.27) for 6.0 Gy of RT alone, 0.22 (+/-0.21) for CDDP alone, and 0.05 (+/-0.08) for the combination. A CI of 0.35 and an R value of 0.09 demonstrated synergy between chemo and RT without cross-resistance. Similar synergy without cross-resistance was found for RT in combination with BCNU, BSO, and TAX. CONCLUSIONS: Heterogeneous RT dose-response relationships in the in vitro assay were demonstrated. Explants were more sensitive to RT than cell lines. Unlike cell lines, fresh tumor cells consistently displayed synergy with RT and chemo. The synergy between RT and BSO suggests that glutathione depletion may enhance the effect of RT. The assay was feasible for examining fresh tumors and may be an important tool for studying RT or drug resistance. Clinical trials to evaluate this assay are indicated.

Extreme drug resistance in primary brain tumors: in vitro analysis of 64 resection specimens.

Understanding chemoresistance profiles of brain tumors may aid in more educated selection of chemotherapeutic regimens for clinical trials and patient treatment. Although the literature contains many reports of the application of drug resistance assays, little is known about extreme drug resistance (EDR) in primary brain tumors. We undertook this study to determine chemoresistance profiles for brain tumors. From September 1991 to February 1998, we collected 64 brain tumor specimens from patients admitted to the Johns Hopkins Hospital. Tumors were classified according to the revised World Health Organization system. Brain tumor specimens were tested against 13 different chemotherapeutic agents using an extreme drug resistance assay. Results were reported as percent cell inhibition (PCI) (compared to control cultures). A drug resistance profile (extreme, intermediate, or low) was determined based on statistical comparison to a historical database of tumor specimens tested against the same panel of chemotherapeutic agents. Brain tumor specimens were classified histologically as Grade IV astrocytoma (glioblastoma multiforme, n = 35), Grade II/III astrocytoma (n = 11), oligodendroglioma (n = 6), meningioma (n = 9), hemangiopericytoma (n = 2), and ependymoma (n = 1). A large percentage of glioblastomas displayed extreme drug resistance to paclitaxel (69%, n = 35), SN38 (75%, n = 28), and vincristine (38%, n = 29). The majority of Grade II/III astrocytomas displayed extreme drug resistance to carboplatin (67%, n = 6), cisplatin (60%, n = 10), and paclitaxel (60%, n = 10). In a similar fashion, oligodendrogliomas displayed extreme drug resistance to vincristine (60%, n = 5) and paclitaxel (50% n = 6). Most meningiomas displayed extreme drug resistance to vincristine (75%, n = 8), dacarbazine (63%, n = 8), and 4-HC (50%, n = 8). Through the continued analysis of brain tumor specimens and compilation of data from multiple institutions, chemoresistance profiles could assist in the development of rationale clinical trials and treatment regimens for patients with brain tumors.

Association between in vitro platinum resistance in the EDR assay and clinical outcomes for ovarian cancer patients.

OBJECTIVE: The initial clinical response to platinum is a major determinant of outcome for patients with ovarian cancer. This retrospective study was undertaken to correlate the response and survival of newly diagnosed advanced ovarian cancer patients who received platinum-based therapy with in vitro drug response to cisplatin or carboplatin measured as percentage cell inhibition (PCI) in the in vitro Extreme Drug Resistance (EDR) assay. METHODS: Outcomes for newly diagnosed ovarian cancer patients with tumor specimens submitted in a serial fashion for the EDR assay were studied. EDR assay results for cisplatin and carboplatin were correlated with clinical outcome for 79 evaluable chemotherapy nai;ve cases who presented with advanced (stages IIC, III, and IV) ovarian cancer. Stage IV and suboptimally debulked stage IIIc accounted for 16 cases, while 63 cases were optimally debulked Stage III/IIc. All patients were treated with platinum-based combination chemotherapy at a single institution. In vitro results for patient tumors were classified as low drug resistance (PCI > median), intermediate drug resistance [PCI between the median and 1 standard deviation (SD) below the median], or extreme drug resistance (PCI more than 1 SD below the median). For the purpose of this analysis, in vitro EDR to either cisplatin or carboplatin was considered to represent extreme resistance to platinum (EDRP), while the absence of EDR to either cisplatin or carboplatin was considered to represent low resistance to platinum (LDRP). Patients demonstrating relative in vitro resistance to paclitaxel and non-cross-resistance to cyclophosphamide and/or doxorubicin received cyclophosphamide plus platinum (CP); cyclophosphamide, doxorubicin, and platinum (CAP); or platinum alone in place of paclitaxel plus platinum (TP). Progression-free survival (PFS) and overall survival (OS) were correlated with EDR assay results. RESULTS: Median PFS was 6 months for the 17 cases exhibiting EDRP, compared to 24 months for the 62 cases exhibiting LDRP in vitro [relative risk (RR) 3.78, confidence intervals (CI) 1.82-7.83], adjusted for stage, debulking status, in vitro response to 3-OH-cyclophosphamide, and histological grade. Estimated overall 5-year survival was 19% for patients with tumors showing EDRP, compared to 68% for patients with tumors showing LDRP (RR 2.32, CI 1.06-5.07). Patients treated with CP (n = 20) showed no significant difference in OS compared to patients treated with TP (n = 54), CAP (n = 4), or cisplatin (n = 1) alone. In vitro platinum response remained an independent predictor of PFS and OS in multivariate analyses adjusted for CP versus TP, CAP, or platinum administration, and adjusted for debulking status. Median PFS for all 79 patients was 22 months, with an estimated 5-year survival of 57%. CONCLUSIONS: Patients with tumors demonstrating in vitro EDR to platinum were at significantly increased risk for progression and death when treated with standard platinum-based regimens. Such patients may therefore benefit from entry onto trials with novel agents or combinations.