ANTICANCER RESEARCH 30: 2805-2812 (2010) Analysis of Chemotherapeutic Response Heterogeneity and Drug Clustering Based on Mechanism of Action Using an In Vitro Assay SHARA D. RICE, JAMIE M. HEINZMAN, STACEY L. BROWER, PAUL R. ERVIN, NAN SONG, KUI SHEN and DAKUN WANG Precision Therapeutics, Inc., Pittsburgh, PA 15203, U.S.A. Abstract. Background: Cancer chemotherapeutic treatment patient is pre-treated with chemotherapy or radiation and is a complex scientific task. The ChemoFx® Drug Response then the tumor is surgically removed (4, 5). Early detection Marker (DRM) assists physicians in identifying treatment significantly improves treatment success rates for some types protocols likely to be effective for specific patients. Materials of cancer (6, 7). However, recurrent and metastatic forms of and Methods: The ChemoFx® DRM was used to study drug cancer do not demonstrate the same robust treatment response in vitro. Established human cancer cell lines and response rates (8, 9). The presence of inoperable metastases primary cultures of patient tumor specimens were challenged and the differences in response among patients make it with chemotherapeutic agents to observe response of difficult to develop standard treatment protocols for multiple tumor samples and determine whether drugs with metastatic and recurrent disease (10, 11). similar mechanisms of action elicit similar response. Results: Generally, chemotherapeutic agents are indicated for the These studies demonstrated heterogeneous response among treatment of specific cancer types and patient populations patient tumor samples and clustering of drug response with based on the effectiveness in large-population studies (12, similar mechanisms of action. Also highlighted was the 13). However, what is largely unstudied is whether certain reproducibility of ChemoFx DRM and its utility in treatment regimens are likely to be more successful in characterizing tumor response to chemotherapy. Conclusion: specific patients. Clinical studies indicate that a patient who Heterogeneous drug responses observed in vitro were similar does not respond to one chemotherapeutic agent may to those observed clinically. Response characteristics were respond to another (14). Patients may also be generally similar for drugs with similar mechanisms of action, resistant to multiple agents, a condition known as multiple suggesting response heterogeneity is determined at a cellular drug resistance (15). In all, the value of one therapy over and molecular level. another for a specific patient is difficult to determine, short of treating the patient and observing response. For this Cancer is a disease treated with various strategies depending reason, individualized treatment for cancer is becoming more on the type of cancer and the stage of the disease. Generally popular. Physicians are using genetic tests and drug response accepted standards of care have been developed based on markers to guide chemotherapy treatment for their patients large-population studies (1). These standards involve various (16, 17). approaches to chemotherapy, radiation therapy and surgical The ChemoFx® Drug Response Marker (DRM) (Precision resection (2). For some cancers, adjuvant therapy is most Therapeutics, Inc (Precision), Pittsburgh, PA, USA), identi- successful; this involves surgical removal of tumor tissue fies effective agents for solid cancers prior to chemotherapy followed by chemotherapy or radiation (3). In other cancers, treatment (18, 19). The assay involves culturing cells derived neoadjuvant therapy is more efficacious; with this therapy, a from patient tumors and then treating the samples with various chemotherapeutic agents in vitro. The ChemoFx DRM allows individualized testing with a panel of drugs on patient tumor samples to determine the best treatment Correspondence to: Jamie M. Heinzman BS, Precision Thera- regimen for each patient. The goal of the assay is to provide peutics, Inc., 2516 Jane Street, Pittsburgh, PA 15203, U.S.A. Tel: additional information to physicians making therapy +412 4322365, Fax: +412 4883830, e-mail: jheinzman@ ptilabs.com decisions, in order to avoid treating patients with agents that Key Words: ChemoFx DRM, cancer, heterogeneity, chemothera- are not likely to be effective for their cancer. The chemo- peutic response, mechanism of action, chemotherapy drug response therapeutic agents tested in the ChemoFx DRM work marker, in vitro chemosensitivity testing. through a variety of mechanisms of action; a complete list 0250-7005/2010 $2.00+.40 2805 ANTICANCER RESEARCH 30: 2805-2812 (2010) of chemotherapeutic agents by mechanism of action is Table I. Mechanism of action of drugs tested in the ChemoFx DRM. provided in Table I. Chemotherapeutic agents tested in the ChemoFx DRM, grouped by In the current study, the ChemoFx DRM was used to study mechanism of action. the response characteristics of established cell lines and Mechanism of Action Chemotherapeutic Agent primary cultures of patient tissues from ovarian, lung, breast, and colon cancers. The study aim was two-fold: (i) to Alkylating agent Altretamine, bleomycin, observe drug response profiles of different patient tumor cyclophosphamide, ifosfamide, samples to the same chemotherapeutic agent and (ii) to melphalan, mitomycin Alkylating agent, non-classic Procarbazine, temozolomide determine whether cell lines and patient tumor samples Anthracycline Doxorubicin, epirubicin cultured in vitro respond with similar drug response patterns Anti-metabolite 5-Fluorouracil, gemcitabine, to chemotherapeutic agents with similar mechanisms of Pemetrexed action. This study concentrated on response characteristics Nitrosourea Carmustine, lomustine of platinum, taxane, and anthracycline therapies. Platinum Carboplatin, cisplatin, oxaliplatin Targeted therapy, EGFR Erlotinib Targeted therapy, multiple Sunitinib Materials and Methods kinase inhibitor Taxane Docetaxel, paclitaxel Cell lines. A number of immortalized cell lines were used in these Topoisomerase II inhibitor Etoposide, irinotecan, topotecan studies, including the following: SK-OV-3, MDA-MB-157, MDA- Vinca alkaloid Vinblastine, vincristine, MB-361, BT-474, HCC-1500, HCC-202, AU-565, MDA-MB- Vinorelbine 175VII , MDA-MB-453, ZR-75-1 , BT-20 , HCC-1569, MCF-10A, MDA-MB-468 , MDA-MB-415, MDA-MB-436, BT-549, SK-BR-3, T-47D, HCC-1428, HCC-1143, HCC-1937, HCC-38, MDA-MB- 231 , BT-483, CAMA-1, UACC-812 and HCC-1187 (American tumor type: Mammary Epithelial Growth Medium (MEGM, Lonza, Type Culture Collection (ATCC), Manassas, VA, USA). Cell lines Cologne, Germany), Bronchial Epithelial Growth Medium (BEGM, were maintained in McCoy’s 5A or RPMI 1640 medium Lonza), McCoy’s 5A (Mediatech), RPMI 1640 (Mediatech). The (Mediatech, Herndon, VA, USA) with 10% FBS (Hyclone, Logan, cultures were maintained for 5-28 days in humidified incubators at UT, USA). All cell lines were seeded at 40,000 cells in 25 cm2 37˚C with 5% CO2, until a confluency of at least 30% was attained. flasks and maintained in culture at for 6-8 days in humidified incubators at 37˚C with 5% CO2 until a confluency of approxi- ChemoFx DRM. ChemoFx DRM was performed as described mately 85% was attained. previously (19, 21). Briefly, cultures were plated in Costar 384-well micro titer plates (Corning, Lowell, MA, USA) and incubated for Patient tumor specimens. Primary cultures were established using 24 hours to allow cell attachment. Then, cells were treated in tumor specimens procured for research purposes from the following triplicate with chemotherapeutic agents (untreated cells were used sources: National Disease Research Interchange (NDRI, Phila- as a control). For each chemotherapeutic agent, 10 serially diluted delphia, PA, USA), Cooperative Human Tissue Network (CHTN; drug concentrations were tested. Agents used in this study included Philadelphia, PA, USA), Forbes Regional Hospital (Monroeville, carboplatin (1 μM-500 μM), cisplatin (0.2 μM-100 μM), docetaxel PA, USA), Jameson Hospital (New Castle, PA, USA), Saint (0.1 nM-25 nM), doxorubicin (2 nM-1 μM), epirubicin (0.7 nM-14 Barnabas Medical Center (Livingston, NJ, USA), Hamot Medical μM) and paclitaxel (0.2 nM-0.1 μM). After an incubation period of Center (Erie, PA, USA), Windber Research Institute (Windber, PA, 72 hours, the cells were fixed with anhydrous ethanol (95% fixing USA) and University of Rochester Medical Center (Rochester, NY, grade) and stained with 4’6-diamidino-2-phenylindole (DAPI), a USA). All patient tumor samples were de-identified prior to transfer fluorescent DNA stain for imaging the nucleus (Sigma-Aldrich to Precision’s Research and Development Department, so they were Corp, St Louis, MO, USA). Cells that remained attached after considered exempt (IRB protocol #RD-109). staining were imaged and counted using automated cell quanti- Additionally, analysis was performed on de-identified data from fication systems developed for the 384-well format (21). 361 consecutive primary ovarian cases submitted to Precision for The percentages of cells remaining after drug treatment were commercial testing from October 2006 to February 2009. Drug used to determine survival fraction (SF=average cell countdosex response results had been previously generated and reported to /average cell countcontrol), from which dose–response curves were patients, thus analysis did not jeopardize patient treatment. Data from plotted. Each dose–response curve was assigned a response index the 361 primary ovarian cultures tested with carboplatin, cisplatin, (RI) score ranging from 0