Van Nguyen

Phase II trial of bevacizumab and erlotinib as a second-line therapy for advanced hepatocellular carcinoma

Trial registry Clinicaltrials.gov #NCT01180959. Background Early clinical studies of bevacizumab and erlotinib in advanced hepatocellular carcinoma (HCC) have a tolerable toxicity and a promising clinical outcome. We evaluated the efficacy and tolerability of this combination as a second-line therapy for HCC refractory to sorafenib. Methods For this single-arm, Phase II study, we recruited patients with Child–Pugh class A or B liver disease, Eastern Cooperative Oncology Group performance status 0–2, and advanced HCC that was not amenable to surgical or regional therapies and treatment with sorafenib had failed (disease progressed or patient could not tolerate sorafenib). Patients received 10 mg/kg intravenous bevacizumab every 14 days and 150 mg oral erlotinib daily for 28-day cycles until progression. Tumor response was evaluated every two cycles using Response Evaluation Criteria in Solid Tumors. The primary end point was the 16-week progression-free survival rate. Secondary end points included time to progression and overall survival. Results A total of 44 patients were enrolled and had a median follow-up time of 33.8 months (95% confidence interval [CI]: 23.5 months – not defined). The 16-week progression-free survival rate was 43% (95% CI: 28%–59%), median time to progression was 3.9 months (95% CI: 2.0–8.3 months), and median overall survival duration was 9.9 months (95% CI: 8.3–15.5 months). Grade 3–4 adverse events included fatigue (13%), acne (11%), diarrhea (9%), anemia (7%), and upper gastrointestinal hemorrhage (7%). Conclusion Bevacizumab plus erlotinib was tolerable and showed a signal of survival benefit in the second-line setting for patients with advanced HCC. Because standard-of-care options are lacking in this setting, further studies to identify predictors of response to this regimen are warranted.

Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma

Phosphatidylserine (PS) is an upstream immune checkpoint that drives global immunosuppression. Previous work has shown that PS targeting agents can override PS-driven immunosuppression and re-program the tumor microenvironment from immunosuppressive to immunosupportive, break tumor immune tolerance, and elicit potent de novo antitumor T-cell immunity. In the present study, the antitumor effect of the combination of a PS-targeting antibody with antibodies that inhibit the downstream immune checkpoints PD-1 or CTLA-4 antibody in the K1735 mouse melanoma model was examined. Tumor-bearing mice were treated with each antibody alone or the combination at 5 to 10 mg/kg, twice a week. Combination therapy potently suppressed tumor growth and improved overall survival compared to single agent treatment. Flow cytometry revealed that combination therapy induced the highest ratio of tumor-infiltrating immune effector to suppressor cells. Importantly, combination treatment also significantly decreased the levels of myeloid-derived suppressor cells (MDSC) in the spleen. In addition, inhibition of PS and PD-1 or CTLA-4 resulted in significantly more IL-2 and IFNg-secreting splenic CD4+ and CD8+ T cells than any single agent treatment. Finally, combined immune checkpoint blockade did not induce any observable toxicity following multiple treatment doses. In summary, our findings demonstrate that the combination of antibody-mediated PS blockade with an inhibition of established immune checkpoints (e.g., PD-1 and CTLA-4) represents a promising strategy for cancer immunotherapy.

Abstract B27: Targeting of phosphatidylserine by monoclonal antibody ch1N11 enhances the antitumor activity of immune checkpoint inhibitor PD-1/PD-L1 therapy in orthotopic murine breast cancer models

Introduction: Phosphatidylserine (PS) is a phospholipid that resides in the plasma membrane inner leaflet in many types of cells, including both tumor and tumor associated endothelial cells. Conditions that cause cellular stress, including those that occur from oxygen radicals, hypoxia, irradiation, and chemotherapy, cause a dramatic shift in PS localization in both tumor and tumor associated endothelial cells. This change in localization results in PS shifting to the outer plasma membrane, allowing its recognition by components of the tumor microenvironment. Recognition of PS promotes an immunosuppressive environment that encourages tumor growth, in part by promoting the recruitment of myeloid derived suppressor cells, immature dendritic cells, and M2-like macrophages, in addition to inducing production of anti-inflammatory cytokines. Currently the chimeric PS-targeting antibody, bavituximab, is being studied in combination with chemotherapies to treat patients with solid tumors in multiple late-stage clinical trials, where it is believed to help augment the efficacy of chemotherapeutics by blockade of PS-mediated immunosuppression and triggering an Fc-FcR mediated pro-inflammatory response in the tumor microenvironment. The results with PS targeting therapies and chemotherapeutics are encouraging, and the effectiveness of PS targeting therapies in combination with therapies directed towards immune checkpoint regulators warrants further attention. Methods: Immune competent mice bearing established syngeneic EMT-6 or E0771 breast tumors were subjected to treatments comprising of a PS targeting antibody (ch1N11) and an anti-PD-1 antibody (to interrupt the PD-1/PD-L1 signaling axis) either as single or combination therapy. The anti-tumor effects of treatments were determined by measuring primary tumor growth inhibition and specific immunity was determined by tumor re-challenge. Results: In both models, which showed distinct sensitivity to therapy, the combination of ch1N11 with an anti-PD-1 checkpoint blocking antibody had a significantly greater anti-tumor affect than single arm treatments. In the E0771 model, complete tumor regression was observed in 6 of 10 animals treated with combination treatment. Animals with no tumor growth for 30 days post study end were resistant to tumor re-challenge indicating the development of tumor-specific immunity. Conclusions: These results suggest that the combination of PS targeting antibodies in conjunction with checkpoint inhibitors has the potential to block tumor immunosuppression in breast cancer and promote a durable antitumor immune response. Citation Format: Michael J. Gray, Jian Gong, Van Nguyen, Michaela Schuler-Hatch, Chris Hughes, Jeff Hutchins, Bruce Freimark. Targeting of phosphatidylserine by monoclonal antibody ch1N11 enhances the antitumor activity of immune checkpoint inhibitor PD-1/PD-L1 therapy in orthotopic murine breast cancer models. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B27.

Yttrium-90 resin microspheres as an adjunct to sorafenib in patients with unresectable hepatocellular carcinoma.

Purpose The safety and efficacy of the combined use of sorafenib and yttrium-90 resin microspheres (Y90 RMS) to treat advanced hepatocellular carcinoma (HCC) is not well established. We determined the incidence of adverse events with this combination therapy in patients with advanced HCC at our institution and analyzed the treatment and survival outcomes. Materials and methods We reviewed the records of 19 patients with Barcelona Clinic Liver Cancer class B or C HCC who underwent treatment with Y90 RMS (for 21 sessions) while receiving full or reduced doses of sorafenib between January 2008 and May 2010. Therapy response was evaluated using Response Evaluation Criteria in Solid Tumors. We evaluated median overall survival (OS) and progression-free survival (PFS) as well as hepatic and extrahepatic disease PFS and incidence of adverse events. Results The median patient age was 67 years, and portal or hepatic venous invasion was present in eight patients (42%). Ten patients received reduced doses of sorafenib. The median Y90 radiation activity delivered was 41.2 mCi. The partial response of Response Evaluation Criteria in Solid Tumors was observed in four patients (19%). The median hepatic disease PFS was 7.82 months, extrahepatic disease PFS was 8.94 months, OS was 19.52 months, and PFS was 6.63 months. Ninety days after treatment with Y90 RMS, five patients (26%) had grade II adverse events and four patients (21%) had grade III adverse events. Conclusion OS and PFS outcomes were superior to those observed in prior studies evaluating sorafenib alone in patients with a similar disease status, warranting further study of this treatment combination.

Characterization of unresectable cholangiocarcinoma patients treated with or without chemoradiation.

403 Background: Curative treatment for cholangiocarcinoma (CC) is surgical resection. Unfortunately, most CC patients (pts) present with unresectable disease in which gemcitabine plus platinum (GEM-P) chemotherapy is the mainstay of treatment (tx). Advanced CC has a dismal prognosis with 5-year survival reported at 5-10 %. Data regarding chemoradiation (CRT) in pts with unresectable CC (uCC) remains limited. Methods: We retrospectively reviewed uCC pts from 1/1/2009 to 7/31/2013. Primary objective: to evaluate the percentage of pts treated with CRT and the median number of chemotherapy cycles given prior to CRT. Secondary objectives: response to first-line tx, progression free survival (PFS) with or without CRT, overall survival (OS) with or without CRT, and duration of CRT control. Inclusion criteria: uCC diagnosis, received tx, and had follow-up at our institution. Exclusion criteria: pts who received liver-directed therapy other than CRT, mixed histology tumors, and a history of other malignancies. Res...

Targeting of phosphatidylserine by monoclonal antibodies augments the activity of paclitaxel and anti-PD1/PD-L1 therapy in the murine breast model E0771

The phospholipid lipid phosphatidylserine (PS) normally resides in the inner plasma membrane leaflet in most mammalian cells, including tumor and tumor associated vascular cells. Inducers of cellular stress, such as hypoxia and oxygen radicals encountered in tumors, and treatments by cytotoxic therapies promote PS relocation to the outer leaf of the plasma membrane. In tumors this re-localization allows PS recognition by a number of receptors on myeloid and lymphoid cells in the microenvironment, promoting tumor growth and metastatic disease through the development of an immunosuppressive environment. Currently the PS-targeting antibody bavituximab is being used to treat patients with solid tumors in multiple late-stage clinical trials. Bavituximabs anti-tumor properties are attributed in part through alleviating PS-receptor mediated immunosuppression and assisting in generating an Fc-FcR mediated pro-inflammatory response.

Abstract 2850: Predicting anti-tumor responses to phosphatidylserine targeting antibodies using tumor imaging.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane and becomes exposed on tumor vascular endothelial cells and tumor cells in response to chemotherapy, irradiation and oxidative stresses in the tumor microenvironment. Binding of antibodies targeting PS on the tumor endothelial cells and tumors induces the recruitment of immune cells and engages the immune system to destroy tumor vasculature. The antibodies also enhance anti-tumor immunity by blocking the immunosuppressive action of PS. A chimeric PS-targeting antibody, bavituximab, is being used in combination with chemotherapy to treat patients with solid tumors in Phase II trials. Fully human antibody PGN635 binds PS through the interaction of beta-2-glycoprotein 1 (β2GP1) in the same manner as bavituximab. Using human PC-3 prostate tumor xenografts in SCID mice, we demonstrate that targeting of PS in tumors by PGN635 is enhanced by chemotherapy. Combination of PGN635 with docetaxel inhibited tumor growth compared to the control IgG plus docetaxel group (p<0.05). Near-infrared optical imaging of PS in tumors with PGN650, an F(ab’)2 antibody fragment of PGN635, showed tumor growth inhibition in mice treated with docetaxel correlates with PS expression levels in the tumors. Maximal uptake of the PS imaging was observed when chemotherapy was given 24 hours before the imaging probe. Citation Format: Jian Gong, Richard Archer, Van Nguyen, Christopher C.W. Hughes, Jeff Hutchins, Bruce Freimark. Predicting anti-tumor responses to phosphatidylserine targeting antibodies using tumor imaging. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2850. doi:10.1158/1538-7445.AM2013-2850

Genomic analysis of exceptional responder to regorafenib in treatment-refractory metastatic rectal cancer: A case report and review of the literature

We present the case of a 53-year-old male with metastatic rectal cancer who was treatment resistant to FOLFOX and FOLFOXIRI. Due to a Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, regorafenib was given in the third line setting. Surprisingly, the patient had a prolonged partial response that lasted 27 months. Mutational status was extensively evaluated to identify potential alterations that might play a role as predictive markers for this unusual event. A poorly characterized but nontransforming mutation in Fms-like tyrosine kinase 4 (FLT4) was present in the tumor. Prior to and at the time of clinical progression, we found amplification of fibroblast growth factor receptor 1 (FGFR1) and epidermal growth factor receptor (EGFR), loss of the FLT4 mutation, and gain of KIT proto-oncogene receptor tyrosine kinase (KIT) G961S suggesting potential roles in acquired resistance.We present the case of a 53-year-old male with metastatic rectal cancer who was treatment resistant to FOLFOX and FOLFOXIRI. Due to a Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, regorafenib was given in the third line setting. Surprisingly, the patient had a prolonged partial response that lasted 27 months. Mutational status was extensively evaluated to identify potential alterations that might play a role as predictive markers for this unusual event. A poorly characterized but nontransforming mutation in Fms-like tyrosine kinase 4 (FLT4) was present in the tumor. Prior to and at the time of clinical progression, we found amplification of fibroblast growth factor receptor 1 (FGFR1) and epidermal growth factor receptor (EGFR), loss of the FLT4 mutation, and gain of KIT proto-oncogene receptor tyrosine kinase (KIT) G961S suggesting potential roles in acquired resistance.

Abstract 5116: Phosphatidylserine-targeting antibodies augment anti-tumor activity of PD-1 antibodies and alter immuno-profiles in murine triple negative breast cancers

Current treatments for triple negative breast cancers rely primarily upon surgical intervention and chemotherapy. Unfortunately, chemotherapies have the propensity to help establish an immunosuppressive condition in part though exposure of phosphatidylserine (PS) in the tumor microenvironment. We demonstrate that treatment of the murine triple negative breast cancer E0771 using a PS-targeting antibody inhibits in vivo growth, and significantly enhances the anti-tumor activity of antibodies directed to the checkpoint inhibitor PD-1. Combinational treatment by antibodies targeting PS and PD-1 increased the number of tumor infiltrating lymphocytes (TILs) to a greater extent than observed with single-arm therapies. Furthermore, combination of PS and PD-1 targeting antibodies provides a distinct survival advantage over treatment by either agent alone, which correlates with an increase in complete tumor regression and the onset of a durable immune mediated response capable of rejecting a secondary challenge by the same tumor. Finally, immune-profiling analysis of tumor RNA using Nanostring revealed that the combination antibody therapy enhanced immune regulatory pathways including MHC class I/II expression, antigen processing and presentation, and leukocyte and macrophage functions. Combined, our data suggest that in triple negative breast cancers antibody therapy blocking PS-associated immunosuppression may further enhance immunotherapies directed at immune checkpoint inhibitors, including those targeting the PDL-1/PD-1 axis. Citation Format: Michael J. Gray, Jian Gong, Ryan N. Parks, Michaela M.S. Hatch, Van Nguyen, Christopher C.W. Hughes, Jeff T. Hutchins, Bruce D. Freimark. Phosphatidylserine-targeting antibodies augment anti-tumor activity of PD-1 antibodies and alter immuno-profiles in murine triple negative breast cancers. [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 5116.

Abstract PR09: Antibody-mediated blockade of phosphatidylserine synergizes with immune checkpoint blockade by inhibiting multiple immune suppressive mechanisms

Background: The expression of phosphatidylserine (PS) on cell surfaces drives immunosuppressive mechanisms associated with tolerogenic cell death. In the tumor microenvironment, PS is exposed on tumor cells and tumor vascular endothelial cells treated with conventional therapies. PS signals through multiple immune cell signaling receptors where it drives the expansion of myeloid-derived suppressor cells (MDSCs), regulatory T cells, M2 macrophages, and stimulates their production of immunosuppressive cytokines (e.g., TGFβ and IL-10), rendering the tumor microenvironment non-responsive to immune activation. PS targeting antibodies have significant anti-tumor effects in multiple tumor xenografts, syngeneic tumors, and genetically engineered preclinical tumor models through mechanisms that re-program the tumor microenvironment from immunosuppressive to immune potentiating. Methods: The combination of PS-targeting and anti-PD1 antibodies was compared to single agent therapy in multiple pre-clinical mouse models, including syngeneic melanoma and breast tumors, and a genetically engineered mouse model (GEMM) of pancreatic cancer. Mice were treated weekly IP at 5 mg/kg with a PS targeting antibody, anti-PD1, or the combination for the duration of each experiment; tumor and spleen tissue microenvironments were immune profiled by FACS, ELISPOT, and immunohistochemistry. Results: In all tumor models examined, the anti-tumor effect of combination therapy was significantly superior to single agent therapy. In B16 and K1735 melanoma tumors, the combination more than doubled the anti-tumor effect of anti-PD-1 alone. Combination therapy significantly inhibited tumor growth by over 90% in E077.1 breast tumors and prolonged animal survival by 30% (n>25 each group) in the GEMM of pancreatic cancer despite the fact that anti-PD1 therapy alone was ineffective in each of these models. Analysis of immune cell subsets in the tumor microenvironment indicated that the combination of antibody-mediated blockade of PS and PD-1 significantly enhanced the effector function of tumor infiltrating CD8+ T cells and increased the ratio of T effector to T regulatory cells, as demonstrated by significant increases in TILs producing IFNγ, TNFα, IL-2, granzyme B, and Ki-67. Combination therapy re-conditioned the tumor microenvironment to favor immune potentiation, as demonstrated by significant decreases in the frequency of MDSC, the ratio of M2 to M1 macrophages, the expression of surface PD-L1, and reductions in TGFβ and IL-10. Furthermore, combination treatment induced systemic tumor specific CD8 T cell immunity, as mice demonstrating complete responses (in the K1735 and E077.1 models) rejected tumor cells upon re-challenge. Splenocytes from these mice had significantly higher numbers of tumor-specific IFNγ-producing cells in ELISPOT assay. Studies were completed without toxicity in any setting. Conclusions: Antibody-mediated PS blockade inhibits PS-mediated immune suppression and stimulates FcRγ activation. This results in immune activation that enables an otherwise non-responsive tumor microenvironment to respond to checkpoint inhibition. This abstract is also presented as Poster B08. Citation Format: Xianming Huang, Jian Gong, Michael Gray, Van Nguyen, Ryan Parks, Chris Hughes, Jeff Hutchins, Rolf Brekken, Bruce Freimark. Antibody-mediated blockade of phosphatidylserine synergizes with immune checkpoint blockade by inhibiting multiple immune suppressive mechanisms. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR09.