Wolfgang Michael Korn

Transforming Growth Factor-β Receptor Inhibition Enhances Adenoviral Infectability of Carcinoma Cells via Up-Regulation of Coxsackie and Adenovirus Receptor in Conjunction with Reversal of Epithelial-Mesenchymal Transition

Expression of the Coxsackie and Adenovirus Receptor (CAR) is frequently reduced in carcinomas, resulting in decreased susceptibility of such tumors to infection with therapeutic adenoviruses. Because CAR participates physiologically in the formation of tight-junction protein complexes, we examined whether molecular mechanisms known to down-regulate cell-cell adhesions cause loss of CAR expression. Transforming growth factor-β (TGF-β)–mediated epithelial-mesenchymal transition (EMT) is a phenomenon associated with tumor progression that is characterized by loss of epithelial-type cell-cell adhesion molecules (including E-cadherin and the tight junction protein ZO-1), gain of mesenchymal biochemical markers, such as fibronectin, and acquisition of a spindle cell phenotype. CAR expression is reduced in tumor cells that have undergone EMT in response to TGF-β. This down-regulation results from repression of CAR gene transcription, whereas altered RNA stability and increased proteasomal protein degradation play no role. Loss of CAR expression in response to TGF-β is accompanied by reduced susceptibility to adenovirus infection. Indeed, treatment of carcinoma cells with LY2109761, a specific pharmacologic inhibitor of TGF-β receptor types I and II kinases, resulted in increased CAR RNA and protein levels as well as improved infectability with adenovirus. This was observed in cells induced to undergo EMT by addition of exogenous TGF-β and in those that were transformed by endogenous autocrine/paracrine TGF-β. These findings show down-regulation of CAR in the context of EMT and suggest that combination of therapeutic adenoviruses and TGF-β receptor inhibitors could be an efficient anticancer strategy. (Cancer Res 2006; 66(3): 1648-57)

A Phase I Study of a 2-Day Lapatinib Chemosensitization Pulse Preceding Nanoparticle Albumin-Bound Paclitaxel for Advanced Solid Malignancies

Purpose: Systemic chemotherapy fails to access much of the tumor burden in patients with advanced cancer, significantly limiting its efficacy. In preclinical studies, brief high doses of tyrosine kinase inhibitors (TKI) targeting the human epidermal growth factor receptor (HER) family can prime tumor vasculature for optimal chemotherapeutic delivery and efficacy. This study investigates the clinical relevance of this approach. Experimental Design: A phase I clinical study of escalating doses of the HER TKI lapatinib given as a 2-day pulse before a weekly infusion of nab-paclitaxel (100 mg/m2) was conducted in patients with advanced solid tumors. Results: Twenty-five patients were treated. Treatment was associated with grade 1 to 2 toxicities including diarrhea, nausea, rash, neutropenia, neuropathy, fatigue, alopecia, and anemia. The two dose-limiting toxicities were grade 3 vomiting and grade 4 neutropenia, and the maximum tolerated dose of lapatinib was defined as 5250 mg/day in divided doses. Lapatinib concentrations increased with increasing dose. Dynamic Contrast Enhanced Magnetic Resonance Imaging studies in a subset of patients confirmed a decrease in tumor vascular permeability immediately following a lapatinib pulse. Sixty-five percent of evaluable patients experienced a partial or stable response on this therapy, 72% of whom were previously taxane-refractory. Conclusion: A 2-day pulse of high-dose lapatinib given before weekly nab-paclitaxel is a feasible and tolerable clinical regimen, suitable for testing this novel vascular-priming chemosensitization hypothesis developed in preclinical models. (Clin Cancer Res 2009;15(17):5569–75)

Temsirolimus combined with sorafenib in hepatocellular carcinoma: a phase I dose-finding trial with pharmacokinetic and biomarker correlates

BACKGROUND Based upon preclinical evidence for improved antitumor activity in combination, this phase I study investigated the maximum-tolerated dose (MTD), safety, activity, pharmacokinetics (PK), and biomarkers of the mammalian target of rapamycin inhibitor, temsirolimus, combined with sorafenib in hepatocellular carcinoma (HCC). PATIENTS AND METHODS Patients with incurable HCC and Child Pugh score ≤B7 were treated with sorafenib plus temsirolimus by 3 + 3 design. The dose-limiting toxicity (DLT) interval was 28 days. The response was assessed every two cycles. PK of temsirolimus was measured in a cohort at MTD. RESULTS Twenty-five patients were enrolled. The MTD was temsirolimus 10 mg weekly plus sorafenib 200 mg twice daily. Among 18 patients at MTD, DLT included grade 3 hand-foot skin reaction (HFSR) and grade 3 thrombocytopenia. Grade 3 or 4 related adverse events at MTD included hypophosphatemia (33%), infection (22%), thrombocytopenia (17%), HFSR (11%), and fatigue (11%). With sorafenib, temsirolimus clearance was more rapid (P < 0.05). Two patients (8%) had a confirmed partial response (PR); 15 (60%) had stable disease (SD). Alpha-fetoprotein (AFP) declined ≥50% in 60% assessable patients. CONCLUSION The MTD of sorafenib plus temsirolimus in HCC was lower than in other tumor types. HCC-specific phase I studies are necessary. The observed efficacy warrants further study.BACKGROUND Based upon preclinical evidence for improved antitumor activity in combination, this phase I study investigated the maximum-tolerated dose (MTD), safety, activity, pharmacokinetics (PK), and biomarkers of the mammalian target of rapamycin inhibitor, temsirolimus, combined with sorafenib in hepatocellular carcinoma (HCC). PATIENTS AND METHODS Patients with incurable HCC and Child Pugh score ≤B7 were treated with sorafenib plus temsirolimus by 3 + 3 design. The dose-limiting toxicity (DLT) interval was 28 days. The response was assessed every two cycles. PK of temsirolimus was measured in a cohort at MTD. RESULTS Twenty-five patients were enrolled. The MTD was temsirolimus 10 mg weekly plus sorafenib 200 mg twice daily. Among 18 patients at MTD, DLT included grade 3 hand-foot skin reaction (HFSR) and grade 3 thrombocytopenia. Grade 3 or 4 related adverse events at MTD included hypophosphatemia (33%), infection (22%), thrombocytopenia (17%), HFSR (11%), and fatigue (11%). With sorafenib, temsirolimus clearance was more rapid (P < 0.05). Two patients (8%) had a confirmed partial response (PR); 15 (60%) had stable disease (SD). Alpha-fetoprotein (AFP) declined ≥50% in 60% assessable patients. CONCLUSION The MTD of sorafenib plus temsirolimus in HCC was lower than in other tumor types. HCC-specific phase I studies are necessary. The observed efficacy warrants further study.

A Multicenter, Open-Label Phase II Clinical Trial of Combined MEK plus EGFR Inhibition for Chemotherapy-Refractory Advanced Pancreatic Adenocarcinoma

Purpose: On the basis of preclinical evidence of synergistic activity between MEK and EGFR inhibitors in pancreatic ductal adenocarcinoma (PDAC), we evaluated the safety and efficacy of selumetinib, a MEK1/2 inhibitor, plus erlotinib in patients with previously treated advanced PDAC. Experimental Design: In this single-arm phase II trial, eligible patients received the combination of erlotinib 100 mg plus selumetinib 100 mg daily in 3-week cycles. Study assessments included measurement of clinical outcomes, with a primary endpoint of overall survival, and exploration of potential molecular predictors of treatment benefit. Results: Forty-six patients were enrolled and received a median of two cycles (range, 1–7). Although no objective responses were observed, 19 patients (41%) showed evidence of stable disease for ≥6 weeks, and 13 of 34 patients (38%) had a CA19-9 decline ≥50%. Median progression-free survival was 1.9 months [95% confidence interval (CI), 1.4–3.3 months], with a median overall survival of 7.3 months (95% CI, 5.2–8.0 months). Common adverse events included rash, diarrhea, and nausea/vomiting. Patients with tumors exhibiting an epithelial phenotype (demonstrated by a high level of E-cadherin expression) were more likely to be sensitive to study treatment. Tumor-derived DNA was detectable in plasma from the majority of patients using next-generation digital DNA sequencing, and its relative abundance correlated with tumor burden. Conclusions: A therapeutic strategy of dual targeted inhibition of the MEK and EGFR pathways shows modest antitumor activity in pancreatic cancer. Specific molecular subtypes may derive greatest benefit from this combination. Further exploration, both with more potent MEK inhibitors and in molecularly enriched patient subsets, is warranted. Clin Cancer Res; 22(1); 61–68. ©2015 AACR.

A phase 1 trial of imatinib, bevacizumab, and metronomic cyclophosphamide in advanced colorectal cancer

Background:This phase 1 clinical trial was conducted to determine the safety, maximum-tolerated dose (MTD), and pharmacokinetics of imatinib, bevacizumab, and metronomic cyclophosphamide in patients with advanced colorectal cancer (CRC).Methods:Patients with refractory stage IV CRC were treated with bevacizumab 5 mg kg−1 i.v. every 2 weeks (fixed dose) plus oral cyclophosphamide q.d. and imatinib q.d. or b.i.d. in 28-day cycles with 3+3 dose escalation. Response was assessed every two cycles. Pharmacokinetics of imatinib and cyclophosphamide and circulating tumour, endothelial, and immune cell subsets were measured.Results:Thirty-five patients were enrolled. Maximum-tolerated doses were cyclophosphamide 50 mg q.d., imatinib 400 mg q.d., and bevacizumab 5 mg kg−1 i.v. every 2 weeks. Dose-limiting toxicities (DLTs) included nausea/vomiting, neutropaenia, hyponatraemia, fistula, and haematuria. The DLT window required expansion to 42 days (1.5 cycles) to capture delayed toxicities. Imatinib exposure increased insignificantly after adding cyclophosphamide. Seven patients (20%) experienced stable disease for >6 months. Circulating tumour, endothelial, or immune cells were not associated with progression-free survival.Conclusion:The combination of metronomic cyclophosphamide, imatinib, and bevacizumab is safe and tolerable without significant drug interactions. A subset of patients experienced prolonged stable disease independent of dose level.

Subtype-Specific MEK-PI3 Kinase Feedback as a Therapeutic Target in Pancreatic Adenocarcinoma

Mutations in the KRAS oncogene are dominant features in pancreatic ductal adenocarcinoma (PDA). Because KRAS itself is considered “undruggable,” targeting pathways downstream of KRAS are being explored as a rational therapeutic strategy. We investigated the consequences of MAP–ERK kinase (MEK) inhibition in a large PDA cell line panel. Inhibition of MEK activated phosphoinositide 3-kinase in an EGF receptor (EGFR)-dependent fashion and combinations of MEK and EGFR inhibitors synergistically induced apoptosis. This combinatorial effect was observed in the epithelial but not mesenchymal subtype of PDA. RNA expression analysis revealed predictors of susceptibility to the combination, including E-cadherin, HER3, and the miR200-family of microRNAs, whereas expression of the transcription factor ZEB1 was associated with resistance to the drug combination. Knockdown of HER3 in epithelial-type and ZEB1 in mesenchymal-type PDA cell lines resulted in sensitization to the combination of MEK and EGFR inhibitors. Thus, our findings suggest a new, subtype-specific, and personalized therapeutic strategy for pancreatic cancer. Mol Cancer Ther; 12(10); 2213–25. ©2013 AACR.

Oxaliplatin-Fluoropyrimidine Chemotherapy Plus Bevacizumab in Advanced Neuroendocrine Tumors: An Analysis of 2 Phase II Trials.

Objectives This study aimed to determine the safety and effectiveness of bevacizumab (B) plus FOLFOX or CAPOX in advanced neuroendocrine tumors (NETs) by performing a combined analysis of 2 separate prospective phase II studies. Methods In the FOLFOX/B study, patients received chemotherapy without scheduled breaks in 3 cohorts: carcinoid, pancreatic NET, and poorly differentiated neuroendocrine carcinomas. In the CAPOX/B study, NET subtypes were pooled, and patients were treated with 4 cycles of CAPOX/B followed by optional maintenance therapy. Primary end points were radiographic response rate (RR) after 12 cycles (FOLFOX/B), progression-free survival (PFS) (CAPOX/B), and toxicity (both). Results Seventy-six patients (FOLFOX/B, n = 36; CAPOX/B, n = 40) were included. In FOLFOX/B, RR for carcinoid at 12 cycles 3/22 (13.6%), median PFS 19.3 months; RR for pancreatic NET at 12 cycles 4/12 (41.7%), median PFS 21 months; RR 1/2 (50%) in poorly differentiated neuroendocrine carcinoma; pooled RR 25% and median PFS 21 months (1-year PFS 68%). In CAPOX/B (pooled NET), RR 18% and median PFS 16.7 months (1-year PFS 65%). Predictable toxicity was observed. Conclusions Neither study met its primary end point, but radiographic responses and prolonged disease stability in previously progressing patients suggest that selected patients with NET may benefit from oxaliplatin–fluoropyrimidine chemotherapy plus bevacizumab and that the combination may warrant further study.

Hybrid minimally invasive Ivor Lewis esophagectomy after neoadjuvant chemoradiation yields excellent long-term survival outcomes with minimal morbidity.

There is a clear survival benefit to neoadjuvant chemoradiation prior to esophagectomy for patients with stages II–III esophageal cancer. A minimally invasive esophagectomy approach may decrease morbidity but is more challenging in a previously radiated field and therefore patients who undergo neoadjuvant chemoradiation may experience more postoperative complications.

Abstract 5244: Next-generation sequence analysis of cell-free DNA in patients with chemotherapy-refractory advanced pancreatic adenocarcinoma (PDAC) treated with selumetinib (AZD6244) and erlotinib

Background: Monitoring molecular events in response to treatment with targeted agents is particularly challenging in pancreatic cancer due to its poor accessibility for biopsies and high stroma content. Recent advances in digital sequencing of rare DNA species allows for detection of circulating tumor-derived free DNA (ctDNA) in plasma of patients with cancer. Here, we explored the potential of gaining information on the genomic composition of pancreatic cancers in patients treated with a combination of the MEK inhibitor selumetinib (AZD6244) and the EGFR kinase inhibitor, erlotinib. Methods: 46 patients were enrolled into this study at two study centers. Treatment consisted of erlotinib 100 mg + AZD6244 100 mg daily in 3-week cycles, with tumor evaluation by CT scan every 2 cycles. Primary objective was overall survival (OS). Correlative endpoints included assessment of expression of EMT-related proteins, and next-generation sequencing analysis of ctDNA. Plasma “on treatment” samples were uniformly obtained at the 6 week time point. Results: Our preclinical studies had demonstrated that inhibition of MEK leads to enhanced signaling through EGFR with hyperactivation of a parallel RAS effector pathway (PI3K), supporting a therapeutic strategy of combined target inhibition in PDAC to overcome this negative feedback loop. Disease control rate was 58% (0 PR; 19 with stable disease (SD) > 6 weeks, 12 with SD > 12 weeks, including 12 (26%) minor responses). 13/34 patients (38%) demonstrated CA19-9 decline > 50%. Assessment of E-cadherin expression in pre-treatment FFPE biopsy material revealed that high expression of the protein was associated with greater likelihood of CA19-9 decline. Paired pre- and on-treatment plasma samples were available for digital sequencing of ctDNA using the Guardant360 assay for 32 patients. Sequence variants likely originating from the tumor (based on allele frequency and absence in the above-mentioned germ-line variants) were found in 27 (84%) pre-treatment and 25 (78%) on-treatment samples. Most frequently mutated gene in pre-therapeutic plasma samples, in which circulating tumor fraction is > 0.4%, was KRAS (85%), followed by TP53 (60%), ATM (30%), and CDKN2A (15%). Relative change in allele frequency for those alleles that were present in pre- and on-treatment biopsies were positively correlated with differences in RECIST measurements. Conclusions: In summary, dual targeting of EGFR/MEK signaling shows antitumor activity in PDAC in a subset of patients, in particular those with high levels of E-cadherin expression, which is in agreement with our preclinical findings. Digital sequencing of ctDNA provides information about genomic alterations in the majority of patients and holds the potential of providing early information on response to therapy. Supported by CTEP and NIH R21 CA149939. Citation Format: Andrew H. Ko, Tanios Bekaii-Saab, Ryan Courtin, Olga K. Mirzoeva, Sharvina Ziyeh, Robin K. Kelley, Elizabeth Ditto, Anna Ong, Regina Linetskaya, Margaret Tempero, Alan P. Venook, Amirali Talasaz, Wolfgang Michael Korn. Next-generation sequence analysis of cell-free DNA in patients with chemotherapy-refractory advanced pancreatic adenocarcinoma (PDAC) treated with selumetinib (AZD6244) and erlotinib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5244. doi:10.1158/1538-7445.AM2015-5244

Abstract 3494: Experimentally validated modeling for optimizing therapeutic combinations of oncolytic adenoviruses and MEK inhibitor

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Oncolytic adenoviruses are therapeutic anti-cancer agents that selectively replicate in and lyse cancer cells motivating their use in targeted gene therapy. Successful entry of adenoviruses into target cells depends on attachment of the adenovirus fiber-knob protein to the Coxsackie-and Adenovirus Receptor (CAR), which is often down-regulated in highly malignant cells. In a previous study, we demonstrate that MEK inhibition may counter this effect by up-regulating CAR expression. In this study we optimize infection by investigating the combinatorial effect of oncolytic adenovirus infection and MEK inhibition through data-driven models and model-driven experiments. To motivate and test our hypotheses, we use HCT116 colon cancer cell line treated with MEK inhibitor, CI1040, and infected with oncolytic adenoviruses, ONYX-015, Delta-24 and Delta-24RGD. We developed a nonlinear ordinary differential equation model that characterizes the dynamics of these cancer cells subject to ONYX-015 infection modulated by CI1040. Resulting simulations offer predictions that may help guide experimental design and inform optimal timing of CI1040 treatment and ONYX-015 infection to minimize cancer growth. Experimental results demonstrate that HCT116 cells treated with CI1040 for 2 days and infected with adenoviruses have stronger cytopathic effects, greater virus uptake, and greater production when compared to the DMSO control. We also found that G1 cell cycle arrest, caused by CI1040 treatment, is associated with increased virus production. To assess the role of release from cell cycle arrest as a mechanism leading to enhanced production of oncolytic adenoviruses, we synchronize cells by density arrest for 48 hours, release and infect with adenoviruses. The results showed a more potent cell killing effect and virus production on cells arrested in G1 and released. We expanded our analysis to a panel of 21 pancreatic cell lines, as this tumor type is a possible target for adenoviral therapies. Enhanced virus replication and cell killing were observed in different pancreatic cell lines, suggesting that combination therapy of CI1040 and oncolytic adenoviruses have potential implications for the design of future clinical trials and improved oncolytic adenoviruses therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3494.