Daniel Chen

Predictive Impact of Circulating Vascular Endothelial Growth Factor in Four Phase III Trials Evaluating Bevacizumab

Purpose: We evaluated the prognostic and predictive use of circulating VEGF-A levels in phase III trials of bevacizumab in colorectal cancer, lung cancer, and renal cell carcinoma. Methods: Baseline plasma samples from 1,816 patients were analyzed for VEGF-A using an ELISA, which recognizes the major isoforms with equivalent sensitivity. HR and 95% confidence intervals (CI) for study end points were estimated using Cox regression analysis. A subset of matched archival tumor samples was analyzed for VEGF-A expression using in situ hybridization. Results: Higher VEGF-A levels showed trends toward adverse prognostic significance in the control arms of multiple trials, reaching statistical significance for overall survival (OS) in AVF2107 (highest vs. lowest 50%: HR = 1.76; 95% CI, 1.28–2.41), AVAiL (HR = 1.52; 95% CI, 1.16–2.00), and AVOREN (HR = 1.67; 95% CI, 1.18–2.36). In predictive analyses, the HRs for progression-free survival were similar across low and high VEGF-A subgroups and favored bevacizumab-containing treatment. In the low VEGF-A subgroups, HRs (95% CIs) were 0.61 (0.43–0.87) in AVF2107, 0.71 (0.43–1.16) in E4599, 0.74 (0.59–0.94) in AVAiL (low-dose), 0.89 (0.70–1.13) in AVAiL (high-dose), and 0.56 (0.40–0.78) in AVOREN. Analyses of OS data have shown similar results. No correlation between primary tumor VEGF-A expression and plasma VEGF-A levels was observed. Conclusions: In this comprehensive evaluation, pretreatment total circulating VEGF-A was prognostic for outcome in metastatic colorectal, lung, and renal cell cancers, but it was not predictive for bevacizumab-based treatment benefit. Clin Cancer Res; 19(4); 929–37. ©2012 AACR.

Identification and Analysis of In Vivo VEGF Downstream Markers Link VEGF Pathway Activity with Efficacy of Anti- VEGF Therapies

Purpose: The aim of this study was to identify conserved pharmacodynamic and potential predictive biomarkers of response to anti-VEGF therapy using gene expression profiling in preclinical tumor models and in patients. Experimental Design: Surrogate markers of VEGF inhibition [VEGF-dependent genes or VEGF-dependent vasculature (VDV)] were identified by profiling gene expression changes induced in response to VEGF blockade in preclinical tumor models and in human biopsies from patients treated with anti-VEGF monoclonal antibodies. The potential value of VDV genes as candidate predictive biomarkers was tested by correlating high or low VDV gene expression levels in pretreatment clinical samples with the subsequent clinical efficacy of bevacizumab (anti-VEGF)-containing therapy. Results: We show that VDV genes, including direct and more distal VEGF downstream endothelial targets, enable detection of VEGF signaling inhibition in mouse tumor models and human tumor biopsies. Retrospective analyses of clinical trial data indicate that patients with higher VDV expression in pretreatment tumor samples exhibited improved clinical outcome when treated with bevacizumab-containing therapies. Conclusions: In this work, we identified surrogate markers (VDV genes) for in vivo VEGF signaling in tumors and showed clinical data supporting a correlation between pretreatment VEGF bioactivity and the subsequent efficacy of anti-VEGF therapy. We propose that VDV genes are candidate biomarkers with the potential to aid the selection of novel indications as well as patients likely to respond to anti-VEGF therapy. The data presented here define a diagnostic biomarker hypothesis based on translational research that warrants further evaluation in additional retrospective and prospective trials. Clin Cancer Res; 19(13); 3681–92. ©2013 AACR.

Abstract 2941: Local tumor irradiation combined with α-PDL-1 immune checkpoint inhibition results in local and systemic anti-tumor responses: Successful translation of a mouse model to a human case series

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Tumor irradiation induces innate and adaptive immune responses which, rarely, lead to tumor regression at distant sites, the abscopal effect. We have previously demonstrated that immunotherapy including Toll-like-receptor agonists (CpG) and checkpoint inhibitors (anti-CTLA4) both preclinically and clinically ([NCT00185965][1] & [NCT01769222][2]) can significantly increase the rate of systemic, abscopal responses (Kim, Blood 2012 & Brody, JCO 2010). Here we provide the first report of a preclinical murine model and patient case series following local radiation and systemic anti-PD-L1 ([NCT01375842][3]). Methods: Preclinical modeling was performed in a two-tumor, syngenic, A20, lymphoma BALB/c model combining fractionated single tumor radiation and systemic (i.p.) anti-PD-L1. Patients receiving MPDL3280A, a human mAb containing an engineered Fc-domain, as part of the phase 1 clinical trial with mixed responses or asymptomatic progression of disease were eligible for the addition of local radiation therapy. Murine and human immune responses including cell phenotype and function, specifically assessing expression of PD-L1 and production of IFNγ were determined by standard flow cytometry and time of flight mass cytometry (CyTOF). Results: Fractionated radiation delayed tumor growth at the treated site only, and systemic anti-PD-L1 reduced tumor growth rate at both sites, however despite prolonged survival all mice died by day 38 following either monotherapy (radiation or anti-PD-L1). In contrast, combination local fractionated radiation and systemic anti-PD-L1 flattened tumor growth at both the irradiated and un-irradiated site, and prolonged survival with 50% survival at day 48 post-tumor inoculation. Modulation of PD-L1 expression post-radiation and tumor-specific augmentation of IFNγ secretion correlated with the enhanced anti-tumor activity. Five patients including four with solid tumors received fractionated, non-definitive dose radiation with at least stabilization of systemic progression in all patients and a RECIST partial response at systemic sites in 1 patient, notably with a synovial sarcoma. Transient, grade 1-2 inflammatory adverse events (fevers, flu-like symptoms) occurred with no DLTs or serious immune-related toxicities. Modulation of PD-L1 expression, T cell phenotype and IFNγ secretion was observed and updated clinical and immune response will be presented. Conclusion: We provide the first report of combination local radiotherapy with anti-PD-L1 demonstrating synergy in a preclinical model and clinical activity in a limited case series. The magnitude of the immune response and abscopal response rate in mice and humans provides proof-of-concept that anti-PD-L1 may be an equally if not more potent combination immunotherapy with radiation compared to our experience with CpG and/or anti-CTLA4. Citation Format: Idit Sagiv-Barfi, Amanda Rajapaksa, Debra Czerwinski, Serena Chang, Jonathan Hebb, Cariad Chester, Erin Waller, Gregg Fine, Daniel Chen, Marcin Kowanetz, Bryan Irving, Ronald Levy, Holbrook Kohrt. Local tumor irradiation combined with α-PDL-1 immune checkpoint inhibition results in local and systemic anti-tumor responses: Successful translation of a mouse model to a human case series. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2941. doi:10.1158/1538-7445.AM2014-2941 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00185965&atom=%2Fcanres%2F74%2F19_Supplement%2F2941.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01769222&atom=%2Fcanres%2F74%2F19_Supplement%2F2941.atom [3]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01375842&atom=%2Fcanres%2F74%2F19_Supplement%2F2941.atom

Randomized Phase II Trial of Parsatuzumab (Anti‐EGFL7) or Placebo in Combination with FOLFOX and Bevacizumab for First‐Line Metastatic Colorectal Cancer

Abstract Lessons Learned. These negative phase II results for parsatuzumab highlight the challenges of developing an agent intended to enhance the efficacy of vascular endothelial growth factor inhibition without the benefit of validated pharmacodynamic biomarkers or strong predictive biomarker hypotheses. Any further clinical development of anti‐EGFL7 is likely to require new mechanistic insights and biomarker development for antiangiogenic agents. Background. EGFL7 (epidermal growth factor‐like domain 7) is a tumor‐enriched vascular extracellular matrix protein that supports endothelial cell survival. This phase II trial evaluated the efficacy of parsatuzumab (also known as MEGF0444A), a humanized anti‐EGFL7 IgG1 monoclonal antibody, in combination with modified FOLFOX6 (mFOLFOX6) (folinic acid, 5‐fluorouracil, and oxaliplatin) bevacizumab in patients with previously untreated metastatic colorectal cancer (mCRC). Methods. One‐hundred twenty‐seven patients were randomly assigned to parsatuzumab, 400 mg, or placebo, in combination with mFOLFOX6 plus bevacizumab, 5 mg/kg. Treatment cycles were repeated every 2 weeks until disease progression or unacceptable toxicity for a maximum of 24 months, with the exception of oxaliplatin, which was administered for up to 8 cycles. Results. The progression‐free survival (PFS) hazard ratio was 1.17 (95% confidence interval [CI], 0.71–1.93; p = .548). The median PFS was 12 months for the experimental arm versus 11.9 months for the control arm. The hazard ratio for overall survival was 0.97 (95% CI, 0.46–2.1; p = .943). The overall response rate was 59% in the parsatuzumab arm and 64% in the placebo arm. The adverse event profile was similar in both arms. Conclusions. There was no evidence of efficacy for the addition of parsatuzumab to the combination of bevacizumab and chemotherapy for first‐line mCRC.

Randomized Phase II Trial of Parsatuzumab (Anti‐EGFL7) or Placebo in Combination with Carboplatin, Paclitaxel, and Bevacizumab for First‐Line Nonsquamous Non‐Small Cell Lung Cancer

Abstract Lessons Learned. The lack of efficacy associated with anti‐EGFL7 combined with standard bevacizumab and chemotherapy in this phase II trial in non‐small cell lung carcinoma is consistent with the lack of benefit observed in colorectal carcinoma, highlighting the challenge of enhancing the efficacy of VEGF inhibition in unselected populations. Future efforts with agents like anti‐EGFL7 should be guided by advances in pharmacodynamic and predictive biomarker development for antiangiogenic agents. Background. Epidermal growth factor‐like domain 7 (EGFL7) is an extracellular matrix‐associated protein that is upregulated during angiogenesis and supports endothelial cell survival. This phase II trial evaluated the efficacy of the anti‐EGFL7 antibody, parsatuzumab, in combination with bevacizumab plus platinum‐based therapy for advanced or recurrent nonsquamous non‐small cell lung cancer (NS‐NSCLC). Methods. Patients (n = 104) were randomized to either placebo or parsatuzumab (600 mg) in combination with bevacizumab (15 mg/kg) and carboplatin/paclitaxel, administered on day 1 of each 21‐day cycle. Carboplatin and paclitaxel were administered for up to six cycles. Bevacizumab and parsatuzumab/placebo were administered for a maximum of 24 months. Results. The progression‐free survival (PFS) hazard ratio (HR) was 1.7 (95% confidence interval [CI], 1.0–2.8; p = .047). The median PFS was 6.7 months for the parsatuzumab arm versus 8.1 months for the placebo arm. The hazard ratio for overall survival (OS) was 1.1 (95% CI, 0.5–2.2; p = .847). The objective response rate (ORR) was 29% in the parsatuzumab arm and 56% in the placebo arm. Overall safety and tolerability were consistent with the established toxicity profile of bevacizumab. Conclusion. There was no evidence of efficacy for the addition of parsatuzumab to the combination of bevacizumab and chemotherapy for first‐line NS‐NSCLC.

Abstract A017: PD-L1 as a predictive biomarker for atezolizumab (MPDL3280A; anti-PDL1) in non-small cell lung cancer (NSCLC)

Background: Programmed death ligand-1 (PD-L1), a ligand for PD-1 and B7.1, is broadly expressed on tumor cells (TC) and tumor-infiltrating immune cells (IC) in many human cancers. PD-L1 expression on either TC or IC can negatively regulate antitumor T-cell function within the tumor microenvironment (TME). Consistent with this, the ORR, PFS and OS benefit of atezolizumab (atezo) across PhI and PhII studies appeared to correlate with increasing baseline PD-L1 expression levels on TC and/or IC. Therefore, we explored the biologic reasons for PD-L1 expression on TC and IC, the association with response to atezo and the intrapatient heterogeneity of PD-L1 expression in NSCLC. Methods: Tumor specimens were obtained from patients (pts) prescreened and/or enrolled in NSCLC atezo trials (PhI PCD4989g, PhII POPLAR and FIR [n=1360]) and from pts treated at MSKCC (n=39). Samples included 14 synchronous and 106 metachronous pairs collected in FIR or at MSKCC. Using the SP142 IHC assay, which has been optimized to detect PD-L1 on both TC and IC, PD-L1 expression was scored at 4 levels (TC0-3 and IC0-3) based on increasing expression. A subset of samples was further characterized by histopathologic review and gene expression by RNAseq. CD8 expression (clone C8/144B) was assessed in the tumor center, invasive margin and periphery by IHC. Results: PD-L1 was expressed on IC only, on TC only or on both TC and IC within the TME. Tumors with the highest (TC3 or IC3), moderate/high (TC2/3 or IC2/3) and any (TC1/2/3 or IC1/2/3) PD-L1 expression represented ≈15%, ≈38% and ≈70% of NSCLC, respectively. PD-L1 expression was similar across all paired synchronous and metachronous tissues. At the TC3 or IC3 cutoff, PD-L1 status remained unchanged in 86% of paired synchronous specimens and in 78% of metachronous pairs. Analysis of PD-L1 expression patterns revealed the existence of exclusive TC and IC subpopulations at each PD-L1 expression level, unique to NSCLC and not seen in other cancers, e.g. UBC. Strikingly, TC3 and IC3 tumors represented 2 distinct populations, with Conclusions: These data demonstrated that NSCLC has unique PD-L1 expression patterns. High expression of PD-L1 on TC and/or IC in NSCLC confers sensitivity to atezo, despite exhibiting distinct immunologic profiles. These results further our understanding of how atezo promotes responses in tumors expressing PD-L1 on TC and/or IC and emphasizes the need to assess PD-L1 on both TC and IC in NSCLC. In addition, intrapatient heterogeneity in PD-L1 expression was relatively low in both synchronous and metachronous tissues, indicating that various types of tumor samples (e.g. primary or metastatic, fresh or archival) can be reliably used to assess PD-L1 expression with the SP142 assay. Citation Format: Marcin Kowanetz, Hartmut Koeppen, Wei Zou, Sanjeev Mariathasan, Matthew Hellmann, Mark Kockx, Colombe Chappey, Edward Kadel, Dustin Smith, Natasha Miley, Vincent Leveque, Roel Funke, Alan Sandler, Ian McCaffery, Lukas Amler, Daniel Chen, Priti Hegde. PD-L1 as a predictive biomarker for atezolizumab (MPDL3280A; anti-PDL1) in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A017.

Abstract LB-312: Egfl7 is expressed in a broad range of human tumors with apparent restriction to the endothelium

EGFL7 is a secreted protein expressed by proliferating endothelial cells in growing vessels during normal organ development. Though expression is generally restricted to a small subset of vessels in the adult, elevated expression has been observed under physiological conditions requiring new blood vessel growth, such as wound healing, pregnancy and tumor growth. EGFL7 facilitates new blood vessel formation by supporting endothelial cell adhesion and migration. In addition, EGFL7 can protect endothelial cells from stress-induced apoptosis, such as hypoxia or potentially growth factor withdrawal. A blocking antibody to EGFL7 (anti-EGFL7) has demonstrated increased survival benefits in preclinical models when combined with a blocking antibody to the pro-angiogenic growth factor VEGFA. Anti-EGFL7 is currently being investigated in Phase II clinical trials in NSCLC and mCRC in combination with bevacizumab and chemotherapy. We present here a comprehensive assessment of Egfl7 expression in a broad range of human solid tumors using in situ hybridization (BC n=150, CRC n=134, RCC n=8, LC n=62, LN mets predom. BC n=27, OV n=25) and quantitative PCR (BC n=92 primary, n=20 LN mets synchronous collection, n=12 distant matched metastasis sequential collection; CRC n=85 primary, n=9 distant matched metastasis sequential collection; NSCLC n=77 primary, n=3 distant matched metastasis sequential collection). Egfl7 is expressed in all tumor types interrogated. With the methods utilized in this study, expression appears to be limited to endothelial cells, with no appreciable tumor cell expression. This data is supported by co-expression analysis in preclinical and clinical samples, showing correlation with vascular markers (n=91 xenograft models CD31 r=0.85, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-312. doi:1538-7445.AM2012-LB-312

Molecular biomarker analyses using circulating tumor cells

Background: Evaluation of cancer biomarkers from blood and other accessible tissues could significantly enable biomarker assessment by providing a relatively noninvasive source of representative tumor material. Circulating tumor cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard. Methodology/Principal Findings: Using a tumor-cell spike-in model system we evaluated CTC capture on different CTC technology platforms, including CellSearch® and two biochip platforms, and used the isolated CTCs to develop and optimize assays for molecular characterization of CTCs. We report similar performance for the various platforms tested in capturing CTCs, and find that capture efficiency is dependent on the level of EpCAM expression. We demonstrate that captured CTCs are amenable to biomarker analyses such as HER2 status, qRT-PCR for breast cancer subtype markers, KRAS mutation detection, and EGFR staining by immunofluorescence (IF). We demonstrate that cell surface expression of EGFR can be quantitated in CTCs from metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients and in the majority of patients (89%) found concordance with HER2 status from patient tumor tissue, while in a subset of patients (11%), HER2 status had changed from the primary tumor at diagnosis. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients despite their more aggressive phenotype. This may be explained by our findings that the basal-like molecular subtype of breast cancer has low EpCAM expression and a more mesenchymal phenotype and CTCs will likely not be efficiently captured using EpCAM alone in tumors that arise from this subtype. Conclusions/Significance: Our data suggests that molecular characterization from captured CTCs is possible and can inform us of the patients’ current biomarker status. In this regard, CTCs hold significant promise as a source of tumor material to facilitate clinical biomarker evaluation. However, limitations exist from a purely EpCAM-based capture system and addition of antibodies to mesenchymal markers in addition to EpCAM could further improve CTC capture efficiency to enable routine biomarker analysis from CTCs.

Abstract 5768: Predictive diagnostics from circulating tumor cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Tumor tissue collection is essential for biomarker assessment but can be difficult in tumor types such as non small-cell lung cancer (NSCLC) where often no surgery is performed and diagnosis is done with biopsies yielding only very limited tissue quantities. Also, in cases where primary tissue is available, the samples may not be representative of patients current disease, i.e. in the case of hormone refractory prostate cancer (HRPC). Rationale: Evaluation of cancer biomarkers (both predictive and pharmacodynamic) from blood and other biological material could significantly enable our ability to generate biomarkers by providing an accessible and relevant source of samples that can be analyzed. Successful assessment of biomarkers that do not require access to tumor “tissue” could improve the inclusion of biomarker evaluations in clinical development and across our oncology practice. Results: We report on our efforts to evaluate Circulating Tumor Cells (CTC) for utility in tumor biomarker assessment. These efforts included evaluating multiple technologies for isolation and molecular analysis of CTCs from blood. By spiking tumor cells into blood as a model system for CTCs we have developed assays for biomarker evaluation in CTCs including: Fluorescence in situ hybridization (FISH), Immunofluoresecnce (IF), qRT-PCR and mutation detection. Validation of these assays in patient blood is ongoing and early data suggests that biomarker assessment is indeed possible from CTCs. Using blood samples from metastatic, HER2+ breast cancer patients; we were able to evaluate the HER2 status from CTCs using immunofluorescence and/or FISH. 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 5768.