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Background: Paragangliomas (PGLs) and Pheochromocytomas (PCCs) are rare neuroendocrine tumors (NETs). PGLs arise from chromaffin cells in the ganglia of the autonomic nervous system, while PCCs arise from chromaffin cells in the adrenal medulla. The genomic landscapes of PGLs and PCCs have not been well studied. Thus, the aim of this study is to report differences of mutational occurrences and confirm the feasibility of next generation sequencing (NGS) testing circulating tumor DNA (ctDNA) in patients with PGL and PCC. Patients and Methods: Molecular alterations in 46 plasma samples were evaluated using a commercially available ctDNA assay (Guardant360® or Guardant360® CDx) across multiple institutions from 2016-2021. The tests detect single nucleotide variants and indels in 54-83 genes with copy number amplifications and fusions in selected genes. Results: Of the 24 PGL patients, there was a median age of 55 (range:28-78) years and 14 (58%) patients were male. Of the 22 PCC patients, there was a median age of 56 (range:28-86) years and 12 (54.5%) patients were male. Genetic alterations were identified in 16 (67%) PGL and 17 (77%) PCC patients. Of the 16 PGL samples with alterations, TP53 associated genes were most commonly altered (44%), followed by ATM (25%), FGFR2 (19%), APC (13%), BRAF (13%), BRCA1 (13%), CCND2 (13%), FGFR3 (13%), IDH2 (13%), KRAS (13%), PDGFRA (13%), RB1 (13%), TERT(13%), ALK (6%), ARID1A (6%), BRCA2 (6%), CCND1 (6%), CDK6 (6%), CDK12 (6%), EGFR (6%), FGFR1 (6%), KIT (6%), MET (6%), NF1 (6%), NRAS (6%), PIK3CA (6%), PTEN (6%), and ROS1 (6%). Of the 17 PCC samples with alterations, TP53 was most commonly altered (41%), followed by ATM (35%), NF1 (24%), FGFR1 (18%), APC (13%), EGFR (12%), MET (12%), MYC (12%), NOTCH1 (12%), PDGFRA (12%), TSC1 (12%), AR (6%), ARID1A(13%), BRAF (6%), BRCA1 (6%), BRCA2 (6%), CCND1 (6%), CDK6 (6%), CHEK2 (6%), ERBB2 (6%), EZH2 (6%), FGFR2 (6%), IDH2 (6%), KIT (6%), KRAS (6%), NRAS (6%), NTRK1 (6%), NTRK2 (6%), and VHL (6%). 21% of PGL and 41% of PCC patients reported alterations associated with therapies approved in other indications including genes in the MAPK pathway and the homologous recombination repair pathway. Conclusions: Liquid biopsy is a non-invasive method that can provide personalized treatment options for patients. In this study, we found that evaluation of ctDNA was feasible among individuals with advanced PGL and PCC. We report a high rate of homologous recombinant deficiencies that are in need of evaluation in future Percentage refers to frequency of patients with an alteration detected. Citation Format: Lana Khalil, Jill Tsai, Leylah Drusbosky, Olatunji Alese, Maria Diab, Mehmet Akce, Christina Wu, Olumide Babjide Gbolahan, Bassel El-Rayes, Walid Shaib. Genomic landscape of circulating tumor DNA alterations in patients with paraganglioma and pheochromocytoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5790.

Background: Preclinical results indicate that metformin can modulate immune cell populations in the tumor microenvironment of solid tumors by diminishing exhaustion of CD8+ tumor infiltrating cells and improving T cell responses. Studies also suggest that metformin could complement PD-1 blockade and potentiate its antitumor activity. Previously we reported the results of a phase II trial with metformin and nivolumab and here we report the findings of correlative analysis of the prospectively collected research samples of 18 patients. Methods: We conducted a phase II trial with nivolumab and metformin in treatment-refractory microsatellite stable (MSS) metastatic colorectal cancer (mCRC). Nivolumab 480 mg IV every 4 weeks and metformin 1000 mg orally twice daily was administered in 28-day cycles following a 14-day metformin only lead-in phase. The primary endpoint was overall response rate (ORR). Secondary endpoints were overall survival (OS) and progression free survival (PFS). Pre-treatment and on-treatment research biopsies and correlative peripheral blood specimens were collected. Paired biopsies obtained at baseline and following treatment with metformin only (n=9) or metformin and nivolumab (n=9) and were stained with a panel of 13 markers using ChipCytometry technology by Canopy Biosciences. Sample was assessed prior to establishing the multiplex assay. 30 out of 36 samples were imaged and analyzed up to 30 Fields of View. Single cell recognition and quantitative biomarker analysis were performed to compare immune cell numbers and population distribution in pre- versus post-treatment samples. Results: As previously reported, no patients had objective response based on RECIST version 1.1 and the study was stopped after the first stage for futility. Median OS and PFS was 5.1 months [95% CI (2-11.7)] and 2.3 months [95% CI (1.7-2.4)], respectively. Multiplex analysis of tissues from patients receiving lead in with metformin alone revealed fewer effector CD4 T cells and effector and effector memory CD8 T cells after treatment vs. baseline biopsy. Biopsy tissue from patients treated with metformin and nivolumab had lower pAMPK and decreased PDL-1 expression vs. baseline. The combination also increased percentages of leukocytes, effector CD4 T cells, effector and effector memory CD8 T cells as well as levels of PDL1-Tim3+ cells. Conclusion: In the setting of MSS mCRC, metformin as a single agent did not enhance effector CD4 and CD8 T cell percentages in clinical samples in our patient cohort. Metformin in combination with nivolumab was associated with increased percentages of effector CD4 and CD8 T cells in biopsy specimens, although these improvements did not translate into enhanced clinical endpoints. Analysis of peripheral blood samples are currently underway to corroborate the findings in the tissue samples. Citation Format: Batoul Farran, Jeffrey M. Switchenko, Lana Khalil, Walid L. Shaib, Brian Olson, Amanda Ruggieri, Christina Wu, Olatunji B. Alese, Maria Diab, Gregory B. Lesinski, Bassel El-Rayes, Mehmet Akce. Correlative analysis of metformin and nivolumab combination in treatment-refractory microsatellite stable (MSS) metastatic colorectal cancer (mCRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3482.

Background: Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality, with dismal 5-year prognosis of 8%, in part due to poor response to available therapies. Thus, new systemic cancer control therapies are in dire need. Semaphorin 4D (SEMA4D) is a soluble and membrane bound glycoprotein that binds its cognate Plexin B1/B2 receptors, expressed on monocytic and granulocytic leukocytes. Prior work has shown that increased expression of SEMA4D and Plexin B in resected PDAC patients is associated with lymph node and distant metastasis, as well as a worse prognosis. Additionally, SEMA4D blockade has conferred improved immune checkpoint blockade response in murine models of colorectal carcinoma and head and neck squamous cell carcinoma via abrogation of myeloid-derived suppressor cell recruitment and function, as well as enhanced T-cell recruitment and activity within the tumor microenvironment. Here we study the effects of SEMA4D blockade in a murine model of PDAC. Methods: C57b/6 mice were orthotopically injected with murine PDAC line (KP2) derived from KRASG12D,TP53Flox/Wt;P48-Cre autochthonous tumors. Mice were treated with Folfirinox (5-FU, irinotecan, oxaliplatin, weekly), immune checkpoint blockade (ICB) (anti-PD1, anti-CTLA-4 mAbs bi-weekly), and anti-SEMA4D mAB (biweekly). Peripheral blood and tumor-infiltrating leukocytes from patients with PDAC undergoing pancreaticoduodenectomy were assessed for Plexin B1 via flow cytometry. Results: Human PDAC demonstrates penetration of Plexin B1 positive leukocytes, most notably tumor-associated macrophages, neutrophils, and monocytes. Mice injected with KP2 developed tumors detectable via high-frequency ultrasound and exhibited longer survival when treated with the combination of Folfirinox, ICB, and anti-SEMA4D antibody, compared to Folfirinox alone, Folfirinox plus ICB, or Folfirinox plus anti-SEMA4D antibody. Conclusions: Plexin B1+ myeloid subsets penetrate human PDAC tumors, and treatment with SEMA4D-blocking antibody improved response to ICB in combination with standard-of-care Folfirinox in preclinical murine studies. Future work will focus on understanding the immune mechanism of improved therapeutic response, as well as further characterization of the prevalence and prognostic ramifications of the SEMA4D-Plexin B axis in PDAC. Citation Format: Luis I. Ruffolo, Katherine M. Jackson, Nicholas Ullman, Alexander Chacon, Alexa Melucci, Paul Burchard, Mary Georger, Rachel Jewell, Peter Prieto, Brian Belt, Crystal Mallow, Elizabeth Evans, Terrence Fisher, Maurice Zauderer, Christina Wu, Brian Olson, Gregory B. Lesinski, David C. Linehan. Targeting the semaphorin 4D-plexin B axis to augment FOLFIRINOX in a murine model of pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B48.

Interrogation of the tumor microenvironment (TME) is crucial to provide insight into biological activity, resistance mechanisms and implementation of rational combination immunotherapies. Semaphorin 4D (SEMA4D, CD100) has broad immunomodulatory effects in the TME. In preclinical models, antibody blockade of SEMA4D promoted immune infiltration and reduced function and recruitment of immunosuppressive myeloid cells within the TME. Importantly, preclinical combinations of anti-SEMA4D with immune checkpoint inhibitors (ICIs) enhanced T cell activity and tumor regression. VX15/2503 (pepinemab), an IgG4 humanized monoclonal antibody targeting SEMA4D, is currently being evaluated in window of opportunity, integrated biomarker trials to characterize immunomodulatory effects in pancreatic (PDAC), colorectal (CRC), and head and neck squamous cell (HNSCC) carcinomas, and melanoma. At present, three biomarker trials are recruiting patients with four resectable indications to investigate novel combinations of pepinemab with ICIs; 1) PDAC and CRC with resectable liver mets (NCT03373188, n=32), 2) HNSCC (NCT03690986, n=36), and 3) metastatic melanoma (NCT03769155, n=36). Prior to surgery, patients enroll in treatment cohorts including combinations of pepinemab with nivolumab and /or with ipilimumab, single agents, or no treatment. Three to seven weeks later, patients will undergo surgery and a substantial surgical section will be collected under the guidance of a pathologist for comparison across treatment groups and with a pre-dose tissue biopsy. Blood will be collected for PK, PD, and additional correlative biomarker assessments. The primary objective is to evaluate the treatment-induced effects on the immune profile in the TME and in peripheral blood. Additional objectives include, extending the previously reported safety profile of single agent pepinemab to ICI combination therapies, as well as exploring pathologic and radiographic responses in the melanoma study. Correlative multiplex flow cytometric flow panels have been established to phenotype cells in the TME and periphery. A multiplex IHC assay utilizing a sequential probe and strip procedure has also been qualified that allows co-localization, spatial orientation, and quantitation of multiple immune markers. Analysis of immune subsets include but are not limited to activated T cells, neutrophils, Treg cells, DCs, monocytes, macrophages, and importantly myeloid-derived suppressor cells (MDSCs). Target engagement and expression of SEMA4D and its cognate receptors will also be evaluated. Seven subjects have been enrolled in these studies as of 7 Jan 2019. These trials will provide the first integrated clinical assessment of anti-SEMA4D antibody activity to reprogram the TME. Citation Format: Gregory B. Lesinski, Terrence L. Fisher, Elizabeth E. Evans, John E. Leonard, Desa Rae Pastore, Crystal Mallow, Ernest Smith, Maurice Zauderer, Conor Steuer, Nabil F. Saba, Michael Lowe, Ragini R. Kudchadkar, Brian Olson, Christina Wu. Integrated biomarker trials of VX15/2503 (pepinemab) in combination with checkpoint inhibitors in window of opportunity studies in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT016.

Pancreatic ductal adenocarcinoma (PDAC) has a prominent fibrotic stroma, which is a result of interactions between tumor, immune and pancreatic stellate cells (PSC). Prior work from our laboratory has defined a role for stroma-derived cytokines such as IL-6 as a significant barrier restraining immunity against PDAC. Our group is pursuing novel approaches to target pathways in the tumor microenvironment (TME), in an effort to improve access of effector immune cells to PDAC and response to immunotherapy. Heat shock protein-90 (Hsp90), is a chaperone protein and a versatile target in pancreatic cancer. Hsp90 regulates a diverse array of cellular processes of relevance to both the tumor and the immune system. However, to date the role of Hsp90 in PSC has not been explored in detail. We hypothesize that targeting Hsp90 can modulate the TME, through its ability to target inflammatory signaling and cytokine production by PSC and enhance the efficacy of immunotherapy. Treatment of immortalized and primary patient PSC with the Hsp90 inhibitor XL888 led to decreased IL-6 at the transcript and protein level in vitro. XL888 directly limited PSC growth, and reduced expression of alpha-SMA, Jak/STAT and MAPK signaling intermediates as determined via immunoblot. Combined therapy with XL888 and anti-PD-1 was efficacious in C57BL/6 mice bearing syngeneic subcutaneous (Panc02) or orthotopic (KPC-Luc) tumors, as compared to treatment with either agent alone. The treatment was well-tolerated, with no difference in body weight observed in either model. Laboratory studies are assessing immune and stromal biomarkers to define the impact on PSC, cytokines and T-cell biomarkers in the TME. Finally, we have completed the dose escalation phase of a Phase Ib/II clinical trial of XL888 (Hsp90i) and pembrolizumab (anti-PD-1) at our institution. Expansion cohorts of patients with metastatic pancreatic cancer (n=16) or colorectal cancer (n=16) are now accruing, with paired biopsy and peripheral blood samples to address the impact of Hsp90 inhibition on anti-PD-1 mediated T cell proliferation, cytokine production, and PSC-derived cytokine signatures. Citation Format: Mohammad Zaidi, Yuchen Zhang, Michael B. Ware, Matthew R. Farren, Hannah Komar, Brian Olson, Ganji P. Nagaraju, Mehmet Akce, Olatunji Alese, Shishir Maithel, Juan Sarmiento, Walid Shaib, Christina Wu, Bassel El-Rayes, Gregory B. Lesinski. Heat shock protein 90 inhibitors alter pancreatic stellate cell cytokine production and enhances the efficacy of immune checkpoint blockade in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4074.

Anti-semaphorin 4D (SEMA4D, CD100) blocking antibody promotes immune infiltration, reduces immunosuppressive myeloid cells, and enhances T cell activity and tumor growth inhibition in combination with various immunotherapies in preclinical animal models. Clinical trials of immune checkpoint inhibitors (ICI) in combination with pepinemab (VX15/2503), humanized anti-SEMA4D antibody, are currently underway in several cancer indications. SEMA4D exerts multi-faceted effects within the tumor microenvironment by creating a barrier at the tumor-stroma margin to restrict immune cell infiltration and promote immunosuppressive activity of myeloid-derived cells. Blocking antibody to SEMA4D directly enhanced M1/M2 ratio and reduced both expression of chemokines that recruit MDSC and the ability of MDSC to suppress T cell activity. Antibody blockade simultaneously restored the ability of dendritic cells and cytotoxic T cells to infiltrate the TME, increasing ratio of Teffector to Tregulatory cells, in syngeneic tumor models. Importantly, anti-SEMA4D MAb enhanced the activity of co-administered immunotherapies, including antibodies to PD1, CTLA-4, and LAG3, epigenetic modulators, and additional novel immunomodulatory combinations in murine breast, colon, head and neck, and melanoma tumor models. New data will describe development and application of methods to assess immune phenotype and biomarkers in translational and clinical studies. These include flow cytometry and whole slide scans of multiplex IHC panels to examine MDSC, M1/M2 macrophage, monocytes, activated DC, B cells, exhausted, activated and stem-like populations of T cells in clinical samples. Expression of SEMA4D and its receptor PlexinB1 were also evaluated in spatial context and cellular co-localization. In summary, SEMA4D represents a novel target to regulate immune infiltration and mesenchymal suppression, sources of resistance to current immunotherapies. Pepinemab treatment was well tolerated in a Phase I oncology trial (NCT01313065) and is currently being evaluated as single agent or in ICI combinations in: (i) a Phase 1b/2a combination trial of pepinemab with avelumab in ICI naïve or ICI refractory NSCLC (CLASSICAL-Lung) (NCT03268057); (ii) a phase 1 combination trial of pepinemab with nivolumab or ipilimumab in melanoma patients who have progressed on any anti-PD-1/PD-L1 (NCT03425461); (iii) a neoadjuvant integrated biomarker trial in patients with metastatic colorectal, pancreatic (NCT03373188) and head and neck (NCT03690986) cancers treated with pepinemab in combination with nivolumab or ipilimumab; and (iv) a Phase 1/2 trial of pepinemab in children with solid tumors and children and young adults with osteosarcoma (NCT03320330). Clinical trials will evaluate safety, tolerability, efficacy, and biological endpoints, including immunophenotyping tumors and blood. Citation Format: Elizabeth E. Evans, Terrence L. Fisher, Holm Bussler, Crystal Mallow, Christine Reilly, Sebold Torno, Desa Rae Pastore, Maria Scrivens, Alan Howell, Leslie Balch, John E. Leonard, Clint Allen, Paul E. Clavijo, Gregory Lesinski, Christina Wu, Conor Steuer, Nabil F. Saba, Brian Olson, Siwen Hu-Lieskovan, Antoni Ribas, Emily G. Greengard, Ernest S. Smith, Maurice Zauderer. Altered myeloid and lymphoid composition of tumor microenvironment following anti-SEMA4D and immune checkpoint combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1545.

Background: Colorectal cancer (CRC) is the second leading cause of cancer death among men and women in the United States, and additional treatment strategies are needed for patients with advanced disease. Prospective therapeutic studies based on tissue-detected genomic targets such as ERBB2 (HER2), BRAF V600E, gene rearrangements/fusions, and microsatellite instability (MSI) have demonstrated clinical benefit for mCRC patients. Although correlative studies and/or retrospective case series utilizing circulating cell-free DNA (cfDNA) next generation sequencing (NGS) have shown that plasma detection of these genomic targets shows promise in mCRC, prospective studies are lacking. We hypothesize that comprehensive genomic profiling of cfDNA will identify actionable targets and improve outcomes for patients with mCRC. Methods: COLOMATE (Colorectal Cancer and Liquid Biopsy Screening Protocol for Molecularly Assigned Therapy; NCT03765736) is a phase II umbrella screening protocol sponsored by the Academic and Community Cancer Research United (ACCRU) consortium. This study utilizes Guardant360™ (Guardant Health, Redwood City, CA) - a blood-based targeted NGS panel - to identify actionable genomic alterations in mCRC patients and to assess the impact of molecularly assigned therapy accordingly under its individual companion studies. Eligible mCRC patients must have progressed on, been intolerant to, or have a contraindication to treatment with a fluoropyrimidine, oxaliplatin, irinotecan, anti-vascular endothelial growth factor (VEGF) monoclonal antibodies (mAbs) and anti-epidermal growth factor receptor (EGFR) mAbs if RAS wild-type. Companion studies are dynamic and currently include those for patients whose tumors are either RAS/BRAF wild-type, HER2 amplified, FGFR altered, RAS mutated, or without any known actionable alteration. Additional companion studies are under active development. The primary objectives of this study are to perform blood-based genomic profiling on patients with treatment refractory mCRC to facilitate accrual to molecularly assigned therapies, and to obtain patient-matched tumor tissue and cell free DNA from peripheral blood to facilitate clinically annotated genomic analyses. Secondary correlative objectives are to explore mechanisms of acquired resistance to molecularly assigned therapy and to explore the correlation between cfDNA mutational burden (allele frequency, copy number) and clinical outcomes such as objective response rate, progression-free survival, and overall survival. Citation Format: Kristen K. Ciombor, Fang-Shu Ou, Andrew Dodge, Tyler Zemla, Christina Wu, Kimmie Ng, Katrina Pedersen, Shumei Kato, Pashtoon M. Kasi, Daniel Ahn, Rebecca Nagy, Richard Lanman, Scott Kopetz, John H. Strickler, Tanios Bekaii-Saab. COLOMATE: Colorectal cancer and liquid biopsy screening protocol for molecularly assigned therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-235.

Background: HER-2 is a transmembrane receptor that is overexpressed in multiple epithelial tumors. We have previously shown that a first generation HER-2 B-cell peptide vaccine targeting epitopes (628-647) and (316-339) was safe and relatively active. We recently developed a novel B-cell epitope specific vaccine (HER-Vaxx) consisting of epitopes derived from the extracellular domain of HER-2 that correspond to amino acid sequences 597-626 (pertuzumab binding site) and 266-296 (trastuzumab binding site). Methods: This first-in-human Phase 1b study evaluated the safety, optimal immunologic/biologic dose (OID/OBD) and immunogenicity of HER-Vaxx. Using a 3+3 dose escalation design, 3 patients (pts) were treated at each dose level (DL). If Results: 49 patients with metastatic and/or recurrent solid tumors and a median of 4 prior lines of chemotherapy received at least one inoculation of HER-vaxx. Common related toxicities include injection site reactions (56% grade 1/2 and 5% grade 3/4). There were no DLTs. The OID/OBD was found to be DL 2 (1.5 mg of each peptide vaccine).A total of 28 patients received ≥ 3 vaccinations including 2 with a partial response and 14 with stable disease. Of those, 6 patients received at least one 6-month boost. The vaccine generated sustained humoral response eliciting HER-2 specific antibodies in the majority of responding patients. Conclusion: We demonstrate that HER-Vaxx is well tolerated and able to generate sustained anti-HER-2 immune response. Given the promise, continuous development of the vaccine is ongoing at the suggested OBD in HER-2 overexpressing tumors. Citation Format: Tanios Bekaii-Saab, Lai Wei, Robert Wesolowski, Daniel Ahn, Christina Wu, Maryam Lustberg, Amir Mortazavi, Bhuvaneswari Ramaswamy, Jay Overholser, Pravin Kaumaya. A Phase Ib of a combination of two chimeric (Trastuzumab-like and Pertuzumab-like) HER-2 B cell peptide vaccine emulsified in ISA 720 and nor-MDP adjuvant in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT017.

Purpose: In preclinical models, tumor growth inhibition by anti-semaphorin 4D (SEMA4D, CD100) blocking antibody is enhanced when combined with various immunotherapies. Immune checkpoint combinations with humanized VX15/2503 anti-SEMA4D are currently being evaluated in several clinical trials. Methods: Expanded mechanistic studies in preclinical models investigate the effect of SEMA4D signaling through its Plexin receptors (PLXN) on MDSC function and chemokine secretion in the tumor microenvironment. Humanized VX15/2503 anti-SEMA4D is now also being evaluated as single agent or in combination with other immunotherapies in four clinical trials:: (i) a Phase 1b/2a combination trial of VX15/2503 with avelumab in NSCLC (CLASSICAL-Lung); (ii) a phase 1 combination trial with nivolumab or ipilimumab in melanoma patients who have progressed on any anti-PD-1/PD-L1; (iii) a neoadjuvant integrated biomarker trial in patients with metastatic colorectal and pancreatic cancers treated with VX15/2503 in combination with nivolumab or ipilimumab; and (iv) a Phase 1/2 trial of VX15/2503 in children with solid tumors and children and young adults with osteosarcoma. Results: SEMA4D plays a multi-faceted role within the tumor microenvironment by creating a barrier at the tumor-stroma margin to restrict immune cell infiltration and promoting immunosuppressive activity of myeloid-derived cells. SEMA4D directly enhanced ability of MDSC to suppress T cell proliferation and antibody blockade reversed these effects, both in vitro and in vivo. Furthermore, SEMA4D-PLXN signaling modulates expression of chemokines that recruit MDSC. Importantly, anti-SEMA4D MAb can enhance activity of co-administered immunotherapies in murine colon, head and neck (HNSCC), and melanoma models. For example, anti-SEMA4D plus anti-CTLA-4 results in 100% survival and 90% complete tumor rejection (CR) (p Citation Format: Elizabeth E. Evans, Holm Bussler, Crystal Mallow, Christine Reilly, Sebold Torno, Maria Scrivens, Alan Howell, Leslie Balch, John E. Leonard, Terrence Fisher, Clint Allen, Paul E. Clavijo, Gregory Lesinski, Christina Wu, Siwen Hu-Lieskovan, Antoni Ribas, Emily G. Greengard, Ernest S. Smith, Maurice Zauderer. Shifting the tumor microenvironment with first-in-class semaphorin 4D mab for combination immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1762.

Background: Optimal biomarkers for cancer pts treated with IO are currently lacking. Markers of inflammation, such as neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), and platelet-to-lymphocyte ratio (PLR) are readily available and associated with poor outcomes. We investigated the association between these markers and CO in pts treated with IO. Methods: We conducted a retrospective review of 90 pts with advanced cancer treated on IO-based phase I trials at the Winship Cancer Institute of Emory University between 2009-2017. Baseline NLR, MLR, and PLR were treated as continuous variables and rescaled by their own standard deviation. Overall survival (OS) was measured from the first dose of IO to date of death or hospice referral. Progression-free survival (PFS) was determined from first dose of IO to clinical or radiographic progression or death. We defined clinical benefit (CB) as complete response, partial response, or stable disease. Univariate association (UVA) and multivariable analysis (MVA) were carried out using Cox proportional hazard model or logistic regression model. Baseline covariates included race, gender, ECOG PS, # of prior therapies, Royal Marsden Hospital (RMH) risk group, IO indication, and # of metastatic sites. Results: The median pt age was 63 years and most (59%) were men. The most common histologies were melanoma (33%) and GI cancers (22%). The majority (81%) were RMH good risk. 46% of pts had CB on IO. The median NLR, MLR, and PLR was 3.63, 0.48, and 182.65, respectively. Increased NLR, MLR, and PLR were all associated with worse OS, PFS, and chance of CB (Table 1). NLR, MLR and PLR are highly correlated to each other (Pearson correlation coefficients ≥ 0.8, all p < 0.0001). Conclusion: NLR, MLR, and PLR are strongly associated with CO in pts treated with IO. Prospective validation of these findings are warranted. Table 1: UVA and MVA of NLR, MRL, and PLR with CO OS PFSCBUVAMVAUVAMVAUVAMVAHR (CI)p-valueHR (CI)p-valueHR (CI)p-valueHR (CI)p-valueOR (CI)p-valueOR (CI)p-valueNLR1.37 (1.11-1.70)0.003*1.30 (1.02-1.66)0.031*1.42 (1.18-1.70) *statistically significant Citation Format: Dylan J. Martini, Yuan Liu, Colleen Lewis, Hannah Collins, Mehmet Akce, Haydn Kissick, Bradley C. Carthon, Walid L. Shaib, Olatunji B. Alese, Rathi Pillai, Conor E. Steuer, Christina Wu, David H. Lawson, Ragini Kudchadkar, Bassel El-Rayes, Viraj A. Master, Suresh Ramalingam, Taofeek K. Owonikoko, R Donald Harvey, Mehmet Asim Bilen. Blood based biomarkers and association with clinical outcome (CO) in advanced stage patients (pts) treated with immunotherapy (IO) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2607.

Introduction: 5-fluorouracil (5-FU)-based chemoradiation (CRT) is a standard neoadjuvant treatment strategy for the treatment of locally-advanced rectal adenocarcinomas (LARC). MEK-1/2 inhibition has been shown to radiosensitize Ras mutant tumors in preclinical models, including colorectal cancer. In this phase 1 trial, we are testing MEK-1/2 inhibition in combination with neoadjuvant CRT for LARC. Methods: This trial is a standard 3+3 design testing 3 dose levels of the MEK-1/2 inhibitor trametinib in combination with CRT: 0.5 mg, 1.0 mg, and 2.0 mg daily, followed by an expansion cohort. All patients first receive a one week lead-in dosing of trametinib monotherapy, and undergo a tumor biopsy at the end of that week to assess pharmacodynamic inhibition by immunohistochemistry (IHC). Then, patients receive standard CRT (5-FU 225 mg/m2 per day by continuous infusion during radiotherapy, and 50.4 Gy in 28 fractions, 5 days/ week) in combination with trametinib (5 days/week). Patients have total mesorectal excision (TME) 6-10 weeks after completion of CRT. Results: 17 patients have been enrolled to date, with current enrollment occurring on the trametinib 2 mg expansion cohort (total 18 patients planned). Of these, 15 patients (10 males, 5 females) have toxicity and surgical response data available. Median age is 53 years (range 35-74 years), and 6 patients have Stage II disease, while 9 patients with Stage III disease. There have been no grade 4-5 toxicities. One dose-limiting toxicity (DLT) of diarrhea occurred in the 2mg dose cohort, attributed mainly to 5FU CRT, but led to trametinib discontinuation. Generalized skin rash led to trametinib discontinuation after 2 weeks of therapy for 1 patient and was held for 3 days for 2 patients. All patients completed TME, and 3 patients had wound infection at 2 weeks after TME, while 2 patients had perineal wound infection at 4 weeks after TME. Three out of 9 (33%) patients at the 2mg dose cohort had a pathologic complete response (pCR), and 2/9 (22%) patients had a near pCR. Adjuvant chemotherapy was given to 10 patients, 4 patients did not receive (3 refused), and 1 patient is pending. Correlative research studies have been performed in the first 12 patients enrolled. Examination of phospho-ERK levels using Vectra quantitative immunohistochemistry in pre-treatment versus post-lead-in trametinib tumor tissue reveals dose-dependent decreases in p-ERK expression in both tumor and stromal tissue as trametinib is increased from 0.5 to 2.0 mg (range 18-46% absolute reduction), with maximal reduction observed in the 2 mg cohort. Additionally, we have performed whole exome sequencing of a custom panel of 279 cancer associated genes (IGNITE platform) in tumor and normal tissue, and have identified high frequency mutations in KRAS, BRAF, and TP53, as well as additional lower frequency mutations in every tumor. One patient was noted to have microsatellite instability. Finally, we will present data correlating degree of change in p-ERK staining, mutational analysis, and mismatch repair status with pathologic response. Conclusions: The combination of trametinib with 5-FU-based CRT for LARC is feasible with promising signs of clinical activity and tolerability. Initial analysis of tumor tissue confirms dose-dependent pharmacodynamic inhibition of ERK-1/2 within 5 days of starting trametinib. Citation Format: Terence M. Williams, Christina Wu, Lai Wei, Emily Chan, Ryan Robb, Sameek Roychowdhury, Amy Webb, Cynthia Timmers, Tanios Bekaii-Saab, Evan Wuthrick. A phase I trial combining MEK-1/2 inhibition in combination with 5-fluorouracil and radiation for locally-advanced rectal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT088. doi:10.1158/1538-7445.AM2017-CT088

Introduction: 5-fluorouracil (5-FU)-based chemoradiation (CRT) is a standard neoadjuvant treatment strategy for the treatment of locally-advanced rectal adenocarcinomas (LARC). MEK-1/2 inhibition has been shown to radiosensitize Ras mutant tumors in preclinical models, including colorectal cancer. In this phase 1 trial, we will test MEK-1/2 inhibition in combination with neoadjuvant CRT for LARC. Methods: This trial is a standard 3+3 design testing 3 dose levels of the MEK-1/2 inhibitor trametinib (GSK1120212) in combination with CRT: 0.5 mg, 1.0 mg, and 2.0 mg daily, followed by an expansion cohort. All patients first receive a one week lead-in dosing of trametinib monotherapy, and undergo a tumor biopsy at the end of that week to assess pharmacodynamic inhibition by immunohistochemistry (IHC). Then, patients receive standard CRT (5-FU 225 mg/m2 per day by continuous infusion during radiotherapy, and 50.4 Gy in 28 fractions, 5 days/ week) in combination with trametinib (5 days/week). Patients have total mesorectal excision (TME) 6-10 weeks after completion of CRT. Results: The trial opened in 2013, and 31 patients have been screened for KRAS/BRAF/NRAS activating mutations, of which 11 (35.4%) tested positive for a mutation. Of these, 9 patients (8 male, 1 female) have been enrolled so far, with 3 patients accrued in each dose level. Regarding mutation status, six patients have KRAS mutations (five G12V, one G13D), two patients have BRAF mutations (one V600E, one D594N), and one patient is unknown (in dose level 1). Age of patients ranges from 38-64 years old (median 55). All patients have successfully completed neoadjuvant therapy and TME (5 low anterior resection, 4 abdominal-perineal resection). No dose-limiting toxicities were observed on dose levels 1 and 2, and additional patients are currently being accrued onto dose level 3 (2.0 mg). One patient in dose level 3 had pathologic complete response (pCR) at the time of TME. Examination of pre-treatment tumor tissue by IHC reveals baseline hyperactivation of ERK-1/2 (p42/44 MAPK) in most tumors with some heterogeneity in levels of phospho-ERK staining, reflecting hyperactivation of the RAS/RAF-MAPK pathway. Qualitatively, ERK-1/2 activity is reduced from pre-treatment to post-trametinib lead-in tumor tissue, particularly in patients on dose level 3. The patient who experienced a pCR had the most significant reduction in ERK-1/2 activity in tumor tissue and tumor-associated stroma. We will also present quantitative data demonstrating ERK-1/2 inhibition using Vectra quantitative immunohistochemistry. Conclusions: The combination of trametinib with 5-FU-based CRT for LARC is feasible with promising signs of clinical activity and tolerability. Ongoing enrollment will further assess safety, tolerability, and determine the maximally-tolerated dose of trametinib. Initial analysis of tumor tissue suggests dose-dependent pharmacodynamic inhibition of ERK-1/2 within 5 days of starting trametinib. Citation Format: Terence M. Williams, Evan Wuthrick, Christina Wu, Ryan Robb, Cynthia Timmers, Tanios Bekaii-Saab. A phase I trial combining MEK-1/2 inhibition in combination with 5-fluorouracil and radiation for KRAS, BRAF, and NRAS mutant locally advanced rectal 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 CT025.

KRAS is the most frequently altered gene in colorectal cancer (CRC), with mutations occurring in 30-40% of colorectal cancer. Although KRAS mutation is associated with poor prognosis and resistance to anti-epidermal growth factor receptor therapy, the mechanism by which it promotes tumor metastasis remains undefined. Our study explored a new hypothesis that targeting the Y-box binding protein-1 (YB-1)-insulin-like growth factor-I receptor (IGF-IR) signaling axis which is downstream of KRAS, with a novel integrin-linked kinase inhibitor, T315, represents a therapeutically relevant strategy to block KRAS mutation-driven tumor progression. In liver metastases from CRC patients, we observed that there was a preponderance (79/108) of over-expression in both YB-1 and IGF-1R by immunohistochemistry. In colon cancer cell lines HCT-116 and SW480, knockdown and over-expression of KRAS affected the protein and mRNA levels of IGF-1R in a dose-dependent manner. But KRAS only affected the protein level and not mRNA of YB-1, thus suggesting that KRAS regulated YB-1 expression at the post-transcriptional level. Manipulation of YB-1 via plasmid overexpression and siRNA affected IGF-1R protein and mRNA levels in a dose-dependent manner, and YB-1 and IGF-1R promoter binding was confirmed via ChIP assay. There was a dose-dependent decrease in YB-1 and IGF-1R protein levels with MEK162, a MEK inhibitor, but not with LY294002m a PI3K inhibitor. This demonstrated that the KRAS regulation of the YB-1- IGFR-1R signaling axis to be via the MEK signaling pathway and not PI3K/Akt. Our novel drug T315 targets YB-1, and was shown to inhibit cell viability and cell migration in a dose-dependent manner. T315 treatment of the colon cancer cells also decreased protein levels of YB-1 and IGF-1R and affected epidermal mesenchymal transition markers such as E-cadherin, vimentin, and snail. These results suggest that a combination of a MEK inhibitor and T315 may be an effective therapeutic strategy to target KRAS mutation driven CRC. Citation Format: Christina Wu, Peng-Chan Lin, Po-Chen Chu, Hsiao-Ching Chuang, Samuel Kulp, Tanios Bekaii-Saab, Ching-Shih Chen. Therapeutic targeting of KRAS mutation-driven tumor progression in colorectal cancer. [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 5424. doi:10.1158/1538-7445.AM2015-5424