Molina, Julian R.

Abstract Mutations in the EGFR gene or transcript fusion involving EML4-ALK result in oncogenic activation of these oncogenes driving lung adenocarcinoma growth in tumors carrying such an alteration. For lung adenocarcinoma with these genetic changes, small-molecule tyrosine kinase inhibitors have been highly effective at reducing tumor burden and improve the outcome of the patients. Unfortunately, drug resistance eventually develops in these tumors and challenges remain in controlling lung cancer recurrence after targeted therapy. To overcome recurrence, we initiated a prove-of-principle study to evaluate the feasibility of an integrated strategy utilizing genomic profiles of the tumor and patient-specific tumor xenografts derived (PDX) from biopsies for ex vivo evaluation of antitumor drugs to help guide personalized treatment of lung cancer in patients who have developed resistance to targeted therapy drugs. Our study has three main objectives. 1) Use tumor biopsies obtained at the time of recurrence for oncogene mutation and RNAseq analyses to identify molecular changes in oncogenes and gene pathways that can be potentially targeted. 2) Evaluate drug efficacy and optimize personalized therapy for each patient using PDX models. 3) Assess clinical feasibility and our experience using integrated approaches for lung cancer patients who failed targeted therapy. This study was approved by the Mayo Clinic Institutional Review Board (IRB) and utilizes both clinical and research biopsies. A dedicated nurse study coordinator reviews clinical patient list and history on weekly basis and informs the study team of each potential candidate patient. A total of 30 patients having ALK positive lung cancers have been identified and followed up from nearly 500 potentially ALK positive cases between April 2014 to Sept. 2015. Most patients were never smokers but six were former smokers and two were current smokers. Age at diagnoses ranged from 27-78 years (median = 61). Seven patients (23%) were 45 years or younger at the time of diagnosis. A total of 22 patients are currently being treated with crizotinib while eight are on ceritinib or alectinib. For each biopsy, tumor tissues are obtained using a 20 gauge needle, transferred on ice in preserving media and implanted within one hour into 6-8 week old NOD/SCID mice. Many patients have been on treatment for 2-3 years with stable disease so they do not require biopsy. Using a similar strategy, we also obtained biopsies from patients with EGFR gene mutations in their tumors and have progressed while on targeted therapy. A total of seven cases have been implanted. We will report our experiences in patient selection, clinical follow up, patient consent, PDX development, time from biopsy to tumor establishment, and the results of molecular analyses. Our study enabled us to gain new insights regarding the molecular changes associated with recurring tumors after they have failed targeted therapy as well as clinical experiences on how to utilize state-of-the art approaches and comprehensive genomic information to further improve cancer patient care upon disease progression. Acknowledgements: This work is supported in part by the Hillsberg Award from the National Foundation for Cancer Research and by the Biomarker Discovery Program at the Mayo Clinic Center for Individualized Medicine. Citation Format: Jin Jen, Aaron Mansfield, Patrick W. Eiken, Shawn Stoddard, Karlyn Pierson, Xiaonan Hou, Hong Zheng Ren, Julian Molina, Joanne Yi, Ping Yang. Integrated Approaches to Treating Lung Adenocarcinoma Resistant to Targeted Therapy. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B34.

265 Background: Gemcitabine (G) is the standard treatment for first- line PanC. PanC is one of the most hypoxic solid tumors. TH-302 is an inert prodrug of brominated isophosphoramide mustard and undergoes selective activation in deep hypoxia. As a single agent, tumor responses were reported in patients (pts) with metastatic melanoma, SCLC, and head/neck cancer at TH-302 weekly doses of 480-575 mg/m2. Methods: Eligible pts for the PanC expansion of this phase I/II study ( NCT00743379 ) had ECOG <1, locally advanced or metastatic PanC previously untreated with systemic chemotherapy other than adjuvant G, 5FU, and/or radiation. IV TH-302 was dosed at 240-575 mg/m2 (240 or 340 in expansion) with standard dose G (1000 mg/m2) on days 1, 8 and 15 of a 28-day cycle. Serum protein and microRNA hypoxia biomarkers were analyzed at baseline, start of cycle 3 and end of study. Results: 46 PanC subjects (12 locally advanced, 34 distant mets); median age: 63 (range 41-83); 24 male; ECOG 0/1 in 29/17 pts; RECIST response rate (RR) of 21%, median PFS of 6.1 mo (95%CI 4.8, 7.7) and median survival of 11.4 mo (95%CI 6.0, not reached) were observed. RR was 23% with median survival of 7.4 mo in pts with distant mets. 52% of pts had a >50% decrease in CA19-9. Common adverse events were skin or mucosal toxicity, nausea, fatigue and vomiting; most grade 1/2. Grade 3/4 neutropenia, thrombocytopenia and anemia in 68%, 64%, and 20% of pts respectively. The dose intensities at 240 mg/m2 and 340 mg/m2 were similar and related to hematologic toxicities. Skin toxicities were less common at 240 mg/m2. A TH-302 dose response was present with higher RR and PFS at 340 mg/m2. Initial serum hypoxia biomarkers did not identify a preferential pt population. Conclusions: The activity and clinical benefits of the combination of TH-302 with G in first line PanC are promising as compared to previous studies of G alone. TH-302 adds to the hematologic toxicity of G, but the regimen is well tolerated. The safety and activity provided rationale for comparing TH-302 plus G versus G alone in a randomized phase II trial ( NCT01144455 ) and indicate TH-302 may complement G by penetrating into the hypoxic regions of the PanC tumors where activation induces cytotoxicity. No significant financial relationships to disclose.

Abstract PURPOSE: Previous mutational analyses have failed to identify a predictive or prognostic marker for pulmonary carcinoid tumors (PCT). The heterogeneity of PCTs coupled with a lack of detailed information about the tumor biology, impedes patient stratification into groups based on tumor phenotypes or treatment response. We sought to use high-throughput next-generation sequencing to improve prognostication. METHODS: We stratified 37 patients with resected PCTs as low risk (n = 17) and high risk (n = 20) based on a 5 year long follow up for recurrence. Patients with no recurrence within 5 years from initial treatment were deemed low risk. Macrodissection was followed by genomic DNA isolation and next-generation sequencing was performed using an Illumina Mate Pair library protocol. Sequence reads were mapped to the human genome, and primers spanning the fusion junctions were used for validation polymerase chain reaction. RESULTS: Carcinoids commonly harbor rearrangements (median 3.5, range 1-167). Large chromosomal gains or losses were found in 24 of the cases (64.8%). We developed a prognostic score that included the total amount of genetic alterations (deletions and translocations). ROC analysis revealed an AUC of 0.76. High risk patients had a mean score of 20 and low risk patients had a mean score of 5.45 (p value = 0.03, Welch Two Sample t-test). Further analysis of the specific genetic alterations harbored by high and low risk PCTs and correlation with the pathologic and immunohistochemical information is currently underway. CONCLUSION: Large chromosomal rearrangements and aneuploidy portend a poor prognosis for patients with PCTs. Mate Pair sequencing of PCTs provides valuable prognostic information for this highly heterogeneous group of cancers. Citation Format: Konstantinos Leventakos, Aaron S. Mansfield, Stephen J. Murphy, Sarah H. Johnson, Sarah E. Kerr, Marie Christine Aubry, George Vasmatzis, Julian R. Molina. A prognostic model for pulmonary carcinoid tumors based on large chromosomal alterations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4513.

Abstract Lung cancer accounts for about 27% of the cancer related deaths in the USA annually. Pathologically, it is a very complex disease broadly classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLCs are further classified into squamous cell carcinoma, large cell carcinoma and adenocarcinoma. Achaete-scute homolog 1 (ASCL1), an important transcription factor essential in the development of neuroendocrine cells (NE) in lungs is shown to be specifically expressed in lung NE cancers and 10-20% of adenocarcinomas (AD) with NE differentiation (NED), thus suggesting a role in the pathogenesis of these tumors. Our previous study showed that ASCL1 is a regulator of the RET oncogene in AD with NED. RET is a receptor tyrosine kinase with two isoforms in humans: RET9 (short) and RET51 (long). We performed survival analysis to study implications of RET isoforms in ASCL1+ tumors and found that elevated expression of the long RET mRNA was associated with poor survival. Subsequent in vitro experiments demonstrated that treatment with EGF robustly induced phosphorylation of RET in HCC1833 and H1755 cell lines which have high endogenous levels of ASCL1 and RET but not in VMRC-LCD cell line which has high level of ASCL1 but low level of RET. EGF induced phosphorylation of RET was diminished by gefitinib and by EGFR siRNA. Immunoprecipitation results indicated direct binding between EGFR and RET in presence of EGF. Furthermore, a high throughput drug screening found 8 EGFR inhibitors that were 10 - 250 fold more cytotoxic in ASCL1+ compared with ASCL1- AD cells. These results implicate EGFR as a key regulator of RET activation in ASCL1+ AD and suggest that EGFR inhibitors may be therapeutic for this population of patients. Citation Format: Kaustubh N. Bhinge, Yang Lin, Hamed Rahi, Marie Christine Aubry, Aaron Mansfield, Irina Kovtun, Stephen Murphy, Ping Yang, Dennis Wigle, Joanne (Eunhee) Yi, Aqsa Nasir, Simone Terra, Julian Molina, George Vasmatzis, Farhad Kosari. EGFR-mediated activation of RET in ASCL1+ lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1129.

Abstract Introduction. For patients with non-small cell lung cancer (NSCLC) to benefit from ALK inhibitors, sensitive and specific detection of ALK genomic rearrangements is needed. ALK break-apart fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard-of-care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false-negative rate. We report here a large series of NSCLC cases assayed by hybrid-capture-based comprehensive genomic profiling (CGP) in the course of clinical care. Materials and Methods. Hybrid-capture-based CGP using next-generation sequencing was performed in the course of clinical care of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements. Results. A total of 47 patients (4.4%) were found to harbor ALK rearrangements, of whom 41 had an EML4-ALK fusion, and 6 had other fusion partners, including 3 previously unreported rearrangement events: EIF2AK-ALK, PPM1B-ALK, and PRKAR1A-ALK. Of 41 patients harboring ALK rearrangements, 31 had prior FISH testing results available. Of these, 20 were ALK FISH positive, and 11 (35%) were ALK FISH negative. Of the latter 11 patients, 9 received crizotinib based on the CGP results, and 7 achieved a response with median duration of 17 months. Conclusion. Comprehensive genomic profiling detected canonical ALK rearrangements and ALK rearrangements with noncanonical fusion partners in a subset of patients with NSCLC with previously negative ALK FISH results. In this series, such patients had durable responses to ALK inhibitors, comparable to historical response rates for ALK FISH-positive cases.

Abstract Purpose: p38 MAPK regulates the production of cytokines in the tumor microenvironment and enables cancer cells to survive despite oncogenic stress, radiotherapy, chemotherapy, and targeted therapies. Ralimetinib (LY2228820 dimesylate) is a selective small-molecule inhibitor of p38 MAPK. This phase I study aimed to evaluate the safety and tolerability of ralimetinib, as a single agent and in combination with tamoxifen, when administered orally to patients with advanced cancer. Experimental Design: The study design consisted of a dose-escalation phase performed in a 3+3 design (Part A; n = 54), two dose-confirmation phases [Part B at 420 mg (n = 18) and Part C at 300 mg (n = 8)], and a tumor-specific expansion phase in combination with tamoxifen for women with hormone receptor–positive metastatic breast cancer refractory to aromatase inhibitors (Part D; n = 9). Ralimetinib was administered orally every 12 hours on days 1 to 14 of a 28-day cycle. Results: Eighty-nine patients received ralimetinib at 11 dose levels (10, 20, 40, 65, 90, 120, 160, 200, 300, 420, and 560 mg). Plasma exposure of ralimetinib (Cmax and AUC) increased in a dose-dependent manner. After a single dose, ralimetinib inhibited p38 MAPK–induced phosphorylation of MAPKAP-K2 in peripheral blood mononuclear cells. The most common adverse events, possibly drug-related, included rash, fatigue, nausea, constipation, pruritus, and vomiting. The recommended phase II dose was 300 mg every 12 hours as monotherapy or in combination with tamoxifen. Although no patients achieved a complete response or partial response,19 patients (21.3%) achieved stable disease with a median duration of 3.7 months, with 9 of these patients on study for ≥6 cycles. Conclusions: Ralimetinib demonstrated acceptable safety, tolerability, and pharmacokinetics for patients with advanced cancer. Clin Cancer Res; 22(5); 1095–102. ©2015 AACR.