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Introduction: Clonal hematopoiesis (CH) is defined by acquisition of somatic mutations in hematopoietic stem cells with potential for expansion over time. CH is influenced by exposures to chemotherapy/radiation & has been associated with therapy related myeloid neoplasms (tMN). Peptide receptor radionuclide therapy (PRRT) is a radionuclide therapy and is FDA approved for neuroendocrine tumors (NET). PRRT has been associated with a 2-10% risk of tMN, especially with prior exposure to alkylating agents. A prior study of 13 NET patients found CH prevalence of 64%. We carried out a prospective assessment of CH in NET patients prior to PRRT. Methods: We evaluated pre-PRRT blood samples in 37 NET patients. Genomic DNA from mononuclear cells was analyzed for CH using a custom panel targeting 229 genes to a targeted depth of >1000X. Results: After approval by Mayo Clinic IRB, 45 patients with Stage IV NET were enrolled & CH data was available for 37. Fifty-one percent of patients were female, with a median age of 68yrs (range, 34-84yrs). Types of NET included small bowel (51%), pancreatic (29%), carcinoid (8%), paraganglioma (5%), & other (5%). The median number of therapies prior to PRRT was 1 (range, 0-4) & included somatostatin analogues (SSA)-81%, alkylating agents-30%, & prior radiation-13%. Thirty-six of 37 patients had variants identified, 5 of which were presumed germline (DDX41, CDKN2B, CHEK2, POT1, ERBB2), & 17 (45.9%) had pathogenic variants meeting the operational definition of CH (DNMT3A, TET2, PPM1D, TP53, SF3B1, ASXL1). The median number of pathogenic variants per patient was 1 (range, 0-2), & 6 patients had >1. In patients >60yrs old, 46% had pathogenic variants & 3 patients with pathogenic variants (TET2, TP53, DNMT3A) were Conclusion: To our knowledge this is the largest prospective evaluation of CH in NET. We identified CH to be present in 46% of patients & DNMT3A mutations were most common. This establishes the clonal landscape with mutations involving epigenetic regulators, DNA damage repair, transcription & splicing factors. Prospective longitudinal evaluation will evaluate the association between CH, cytopenias and tMN. Citation Format: Yael Kusne, Terra Lasho, Zaid Elsabbagh, Abhishek Mangaonkar, Rachel Eiring, Timothy Hobday, Jason Starr, Tanios Bekaii-Saab, Thorvardur Halfdanarson, Mrinal Patnaik, Mohamad Sonbol. Prevalence of clonal hematopoiesis in neuroendocrine tumor patients prior to lutetium 177 dotatate therapy (Lu177): A prospective trial of 36 patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5928.

Background: PDAC is a highly morbid disease with no validated biomarkers for first-line (1L) treatment selection. The PurIST single-sample molecular subtyping gene signature, initially described by Rashid et al. (Clin. Cancer Res., 2020), classifies PDAC tumors as basal or classical. Prior work showed that these subtypes are associated with prognosis and that basal subtype patients have significantly lower objective response rate to FOLFIRINOX (FFX) compared to classical subtype patients. This suggests potential implications for treatment with FFX versus gemcitabine and nab-paclitaxel (GnP). Here, we retrospectively demonstrate the clinical validity of PurIST implemented as a laboratory developed test (LDT) on the Tempus Labs sequencing platform using a real-world dataset of advanced PDAC patients. Methods: De-identified PDAC patients were selected from the Tempus Oncology Data Ecosystem under an IRB-approved protocol according to the following inclusion criteria: no systemic treatment, surgically unresectable/metastatic, available RNA-sequencing data from primary or metastatic tissue, and FFX or GnP as 1L systemic therapy with available outcomes. Sequencing was performed by the CAP/CLIA validated Tempus xT assay. The PurIST model was applied to normalized RNA-sequence abundance files to compute a basal or classical subtype label. Predefined statistical analysis parameters included 12-month survival rate and median overall survival (OS) in FFX treated patients. OS was compared using Kaplan-Meier estimates, hazard ratios, and log-rank statistical tests. Results: 258 PDAC patients (64.9 +/- 9.9 yrs, 42% female), were identified for analysis with median OS of 11.7 months (95% CI: 10.6-13.7) and 12-month censorship rate of 12.8%. 151 patients were treated with FFX and 107 with GnP. 42 patients (28%) receiving FFX and 31 patients (29%) receiving GnP were classified as basal. Among FFX treated patients, median OS was 14.4 months (95% CI: 12.5-16.9) in classical patients vs. 9.4 months (95% CI: 8.3-14.5) in basal patients (HR = 1.72, 95% CI = 1.2-2.6, p=0.006). The 12-month survival rate was significantly lower in basal patients receiving FFX vs. classical patients (33.3% vs. 59.4%, p=0.011). In basal patients, no difference in OS was observed between FFX and GnP groups (p=0.6). Classical patients receiving FFX had a 14.4 months median OS vs. 10.8 months for patients receiving GnP (p=0.046). Conclusions: In this real-world cohort, we validate the association between PurIST subtypes and PDAC patient survival when administered FFX or GnP. Among FFX-treated patients, classical patients had significantly better outcomes compared to basal patients. Moreover, classical patients appeared to have improved outcomes with FFX vs. GnP. These findings represent underlying biological PDAC differences and demonstrate the clinical validity of PurIST as a prognostic marker in PDAC patients when performed as an LDT on the Tempus xT platform. Ongoing evaluation of PurIST performance will be continuously monitored through the Tempus Oncology Data ecosystem. Citation Format: Stephane Wenric, James M. Davison, Yun E. Wang, Gregory M. Mayhew, Kirk Beebe, Hyunseok P. Kang, Michael V. Milburn, Vincent Chung, Tanios Bekaii-Saab, Charles M. Perou. Purity Independent Subtyping of Tumor (PurIST): Real-world data validation of a pancreatic ductal adenocarcinoma (PDAC) gene expression classifier and its prognostic implications [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A002.

Background: Sarcopenia is one of the early pathological features of cancer cachexia, yet the effect of sarcopenia on clinical outcomes in patients with advanced HCC is unclear. Our objective was to determine the effect of Sarcopenia on response to systemic therapy with advanced HCC. Methods: Patients with unresectable and advanced liver cancer were retrospectively evaluated between 2010 to 2019. Skeletal muscle area (SMA) was computed with a previously validated computed tomography (CT) body composition AI algorithm. The skeletal muscle index (SMI) was derived from the skeletal muscle area (SMA) (cm2) divided by patient height (m2) calculated from images at the level of L3 on pretreatment CT. Sarcopenia cutoffs for females was less than 39 cm2/m2 SMI or less than 50 cm2/m2 SMI for males. Patients’ demographics, systemic treatment response, toxicity, progression-free survival (PFS) and overall survival (OS) were compared between first-line therapy groups. Kaplan-Meier and Cox proportional hazards regression were used for survival analysis comparisons. Results: A total of 146 patients with advanced HCC were assessed from Mayo Clinic Arizona and Emory University. Seventy-six patients received immunotherapy as first-line treatment and seventy patients received TKI as first-line therapy. Sarcopenia at baseline was assessed in a total of 91 patients at first-line systemic treatment: n=48 with immunotherapy (IO) and n=43 with tyrosine kinase inhibitor (TKI). Sarcopenia was more prevalent in male patients (p=0.009), and in patients with low BMI (p=0.02). When comparing those with sarcopenia at baseline, higher response rate was observed in patients treated with IO without sarcopenia (47% versus 15%, p=0.019), this was not observed in TKI group. Interestingly, median PFS and OS was not significantly changed in patients with or without sarcopenia treated with IO or TKI on first-line therapy. Conclusions: Sarcopenia was associated with reduced response rate (RR) in patients treated with IO, but not in TKI groups. Both didn’t translate into deleterious effect in PFS and or OS. Further research with sufficient adjustments for confounding factors is warranted to better elucidate the prognostic value of sarcopenia in these patients. Citation Format: Gehan Botrus, Pedro Luiz Serrano Uson Junior, Heidi Kosiorek, Mehmet Akce, Isabela Chang, Zhubene Mesbah, Eric Yancey, Daniel Blezek, Jason Klug, Lana Khalil, Puneet Raman, Mohamad Sonbol, Mitesh Borad, Daniel Ahn, Tanios Bekaii-Saab. Prognostic impact of Sarcopenia on clinical outcomes in advanced hepatocellular carcinoma (HCC) treated with systemic therapy [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 6274.

Lipocalin-2 (LCN2) promotes malignant development in many cancer types. LCN2 is upregulated in patients with pancreatic ductal adenocarcinoma (PDAC) and in obese individuals, but whether it contributes to PDAC development is unclear. In this study, we investigated the effects of Lcn2 depletion on diet-induced obesity, inflammation, and PDAC development. Mice with acinar cell–specific expression of KrasG12D were crossed with Lcn2-depleted animals and fed isocaloric diets with varying amounts of fat content. Pancreas were collected and analyzed for inflammation, pancreatic intraepithelial neoplasia (PanIN), and PDAC. We also used a syngeneic orthotopic PDAC mouse model to study tumor growth in the presence or absence of Lcn2 expression. In addition, to understand the mechanistic role of how LCN2 could be mediating PDAC, we studied LCN2 and its specific receptor solute carrier family 22 member 17 (SLC22A17) in human pancreatic cancer stellate cells (PSC), key mediators of the PDAC stroma. Depletion of Lcn2 diminished extracellular matrix deposition, immune cell infiltration, PanIN formation, and tumor growth. Notably, it also increased survival in both obesity-driven and syngeneic orthotopic PDAC mouse models. LCN2 modulated the secretion of proinflammatory cytokines in PSC of the PDAC tumor microenvironment, whereas downregulation of LCN2-specific receptor SLC22A17 blocked these effects. Our results reveal how LCN2 acts in the tumor microenvironment links obesity, inflammation, and PDAC development. Cancer Res; 77(10); 2647–60. ©2017 AACR

Background: The inhibitory immune pathway, consisting of the receptor programmed cell death 1 (PD-1) and its ligands, PD-L1 and PD-L2, plays a vital role in the maintenance of peripheral tolerance. Therapies with monoclonal antibodies targeting PD-1 and its ligands are associated with remarkable response rates in various cancers. Imugene is proposing to develop an anti-PD-1 immunotherapy using a vaccine approach to treat patients with tumors that over-express PD-L1. The hypothesis is that a polyclonal-induced B-cell antibody response will be more effective or as effective with improved safety over current monoclonal antibody therapy. IMU-201, is a B-cell epitope (amino acids 92-110 from PD-1) linked to a promiscuous T cell epitope (amino acid residues 288-302 from measles virus fusion protein) via a 4-amino acid linker (G-P-S-L), combined in an aqueous phase vaccine to be emulsified in Montanide ISA 720 VG, to form the drug product. Study Design: IMU-201-101 is a 2-part open-label dose escalation/dose expansion study of IMU-201. Part 1 will dose escalate IMU-201 as monotherapy and in combination with Standard of Care (SOC) treatment to evaluate safety, tolerability and immunogenicity and assess the optimal biologic dose (OBD) of IMU-201. Part 2 will expand the number treated with IMU-201 at OBD in combination with SOC treatment. OBD is defined as the dose resulting in the best safety/tolerability and immunogenicity and will be determined after all dose cohorts have completed Day 29 of Part 1. A total of approximately 30 adult patients aged 18 years with NSCLC expressing PD-L1 will be enrolled in the study at sites in North America and Australia. In Part 1, three cohorts (25, 125 and 250 µg of IMU-201) of 3-6 participants per cohort are anticipated to establish the Optimum Biological Dose (OBD) of IMU-201 as monotherapy (mOBD). After establishing mOBD, combination dose escalation will combine IMU-201 with SOC treatment which may include ICI, chemotherapy or their combination, to establish combination OBD (cOBD). The starting dose for combination dose escalation will be one dose level below mOBD. The same dosing schedule of IMU-201, DLT criteria, and dose escalation rules will be used to establish mOBD and cOBD. In Part 2, the number of patients treated in combination with IMU-201 at cOBD will be expanded to 12 patients. Patients will receive IMU-201 at Baseline/Day 1, Day 15 and Day 29. Dosing with IMU-201 will continue at Day 64 (± 7 days) and every subsequent 63 days (± 7 days) until progressive disease or complete response. Therapy may continue beyond progression if the treating physician feels that the patient is deriving benefit. Primary Objectives: To evaluate the safety/tolerability and immunogenicity of IMU-201 as monotherapy and in combination with SOC treatment. To identify OBD of IMU-201 as monotherapy and in combination with SOC treatment. To evaluate changes in immunological, biochemical and additional radiological markers of tumor progression in patients treated with IMU-201 as monotherapy and in combination with SOC treatment. To evaluate changes in humoral and cellular immunogenicity data including PD-1 specific antibodies (IgG, IgM), vaccine-specific cytokine levels and regulatory and effector T and B cells. Citation Format: Pravin Kaumaya, Tanios Bekaii-Saab, Tri Phan, Mark T. Marino, Nick Ede, Anthony Good. IMU-201-101 - an open-label, multi-center, dose escalation/expansion, phase 1 study of IMU-201 (PD1-Vaxx), a B-cell immunotherapy, in adults with non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT128.

Background: Chemotherapy in combination with targeted agents are standard-of-care options for patients for mCRC with response rates >50% in first line. In the second line setting, efficacy of chemotherapy and targeted agents are much lower with response rates of 5% for FOLFIRI (5-fluorouracil, leucovorin, irinotecan) + bevacizumab (anti-VEGF). New treatment options are urgently needed in particular for the 50 % of patients harboring a KRAS mutation. Polo-like kinase 1 (PLK1) is a serine/threonine kinase, master regulator of the mitotic checkpoint and cell division. PLK1 is overexpressed in CRC and its overexpression is associated with poor prognostic. A genome wide RNAi screen identified PLK1 as a synthetic lethal target in KRAS mutant CRC cells, inducing cell cycle arrest and apoptosis upon inhibition. Onvansertib is an oral, highly selective PLK1 inhibitor that demonstrates single agent and synergistic activity with irinotecan in preclinical CRC models. Additionally, KRAS mutated vs wild-type cells showed higher sensitivity to onvansertib. PLK1 inhibition is a potential target in KRAS-mutated mCRC and onvansertib + FOLFIRI + bevacizumab may provide a new second-line treatment option. Trial design: The primary objective of this single-arm Phase 1b/2 study is to assess the safety and preliminary efficacy of onvansertib in combination with FOLFIRI and bevacizumab in the second line setting for KRAS-mutated mCRC patients. For the Phase 1b segment, a standard 3 + 3 dose-escalation design is used to determine the maximum tolerated dose or recommended phase 2 dose (RP2D) of onvansertib. As of January 24th 2020, enrollment in the second dose level is ongoing. Efficacy will be determined by objective response rate (ORR) according to RECIST v1.1 (primary endpoint), progression-free survival and reduction in KRAS allelic burden in liquid biopsies (secondary endpoints). In the phase 2, based on a one-sided one sample log-rank test with 10% Type I error, there will be at least 90% power to detect an improvement in ORR from 5% to 20% with 26 patients. Exploratory endpoints include genomic studies of circulating tumor cells and ctDNA to evaluate altered pathways that correlate with patient response. Clinical trial identification NCT03829410. Citation Format: Afsaneh Barzi, Heinz-Josef Lenz, Errin Samuëlsz, Maya Ridinger, Mark Erlander, Tanios S. Bekaii-Saab, Daniel H. Ahn. A phase 1b/2 study of onvansertib (PCM-075) in combination with FOLFIRI and bevacizumab for second line treatment of patients with KRAS-mutated metastatic colorectal cancer (mCRC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT235.

Immunotherapy targeting immune checkpoints has become a common approach for many types of cancer but currently there are no reliable biomarkers to predict and monitor treatment response. Immunohistochemical staining of PD-L1, which was initially developed as a companion diagnostic, is problematic due to tumor heterogeneity, variations in specimen preparation and multiple scoring schemes. There is, therefore, a critical need for a better biomarker to guide the use of immunotherapy to improve clinical outcomes. We previously reported that higher levels of Bim and CX3CR1/granzyme B in CD8+ T cells were associated with increased response to PD-1 blockade therapy in patients with melanoma; high levels of Bim were also prognostic of poor survival in melanoma patients. In this study, we investigated the roles of Bim and CX3CR1/granzyme B in patients receiving PD-1 blockade therapy (i.e. pembrolizumab) for small bowel adenocarcinoma as part of planned correlative analysis of the ACCRU clinical trial, NCT02949219. Patients with unresectable or metastatic biopsy-proven small bowel adenocarcinoma (excluding ampulla of Vater and appendix) who had at least one prior line of systemic chemotherapy were eligible for the trial. Pembrolizumab (200 mg) was administered over 30 minutes intravenously every 3 weeks until unacceptable toxicity, disease progression, or patient refusal. All patients underwent peripheral blood collection at baseline prior to initiation of treatment and then after 3 cycles of treatment. Levels of Bim and CX3CR1/granzyme B in circulating T cells were quantified by flow cytometry using patients' peripheral blood mononuclear cells. Data was analyzed via Cox proportional hazards model, Wilcoxon Rank-Sum test, and Kaplan Meier curves using SAS 9.4. A total of 35 eligible patients were included in the analysis. Three patients had confirmed response (all partial responses) and 10 had stable disease as their best overall response. Seven patients had disease progression around the second blood draw. The median time from baseline to the second blood draw was 9 weeks. Lower levels of Bim in CD8+CD11ahi T cells at baseline on the percentage scale were associated with treatment response (median 54.5% vs. 79.5%; p=0.0087) and disease control (partial response plus stable disease; median 71.2% vs. 81.3%; p=0.0104) but higher levels led to worse progression-free survival (hazard ratio=1.05, 95% confidence interval 1.01-1.08; p=0.0051). In addition, positive changes in CX3CR1/granzyme B in CD8+CD11ahi T cells from baseline were associated with better overall survival (median 20.2 months vs. 3.7 months, p=0.0086). The levels of PD-1 and Ki-67 in CD8+CD11ahi T cells were not associated with treatment response or survival. In conclusion, Bim and CX3CR1/granzyme B are biomarkers that associate with response and survival benefits respectively of PD-1 blockade therapy. This preliminary data calls for a larger-scale, prospective study to validate their predictive and prognostic utilities. Citation Format: Mojun Zhu, Henan Zhang, Nathan R. Foster, Haidong Dong, Tanios S. Bekaii-Saab, Brandy L. Jaszewski, Patrick M. Boland, Michael J. Overman, Katrina Pedersen, Robert R. McWilliams, Academic and Community Cancer Research United (AACRU). Bim and CX3CR1/granzyme B in circulating CD8+ T cells are predictive biomarkers for PD-1 blockade therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4467.

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: Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer and demonstrated impressive activity across a broad set of cancer subtypes, even at advanced and metastatic stages of disease. Contrary to treatment with monoclonal antibodies, chimeric B-cell cancer vaccines have the advantage of producing a specific immune response that can potentially induce memory B & T cell responses, while reducing immune evasion and suppression. Methods: We have translated two HER-2 combination peptide vaccines (B-Vaxx) to the clinic in a Phase 1/2b trial to safely deliver curative and transformative cancer immunotherapies to advanced cancer patients. We have created and established the development of a novel B-cell peptide vaccine (Key-Vaxx) with high immunogenicity that binds to human PD-1 and produces tumor inhibition in vivo in an animal model of colon cancer. Here, we describe the transferable CT-26-HER2 neu tumor model in Balb/c was used to test for synergistic effects of anti-PD1 immunization therapy in combination with anti-HER2 immunization therapy to determine whether this combination could increase immunogenicity, enhance anti-tumor responses and provide synergistic benefit in inhibiting tumor growth. Results: We show robust HER-2 and PD-1 antibody responses in all vaccinated mice indicating that the combined vaccination is effective in reducing tumor growth in a Balb/c syngeneic model of colon carcinoma versus either the PD-1 vaccine or more importantly the positive control gold standard that is anti-mouse PD-1 monoclonal antibody. The vaccine combination was found to be safe and did not appear to exhibit toxicity or autoimmunity. Conclusion: Development of a novel PD-1 vaccine in combination with two Chimeric HER-2 peptide vaccine provides synergistic inhibition of tumor growth with no evidence of toxicity or autoimmunity. A clinical trial with the combination is under planning. Citation Format: Tanios Bekaii-Saab, Jay Overholser, Yuhong Yang, Manuel Penichet, Pravin Kaumaya. Development of a novel PD-1 vaccine and in combination with two Chimeric HER-2 peptide vaccine provides synergistic inhibition of tumor growth in a syngeneic Balb/c model challenged with CT26/HER-2 carcinoma cell line [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 1453.

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.

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

Pancreatic stellate cells (PSC) are a subset of pancreatic cancer-associated fibroblasts. These cells provide prosurvival signals to tumors; however, little is known regarding their interactions with immune cells within the tumor microenvironment. We hypothesized that factors produced by human PSC could enhance myeloid-derived suppressor cell (MDSC) differentiation and function, which promotes an immunosuppressive microenvironment. Primary PSC cell lines (n = 7) were generated from human specimens and phenotypically confirmed via expression of vimentin, α-smooth muscle actin (α-SMA), and glial fibrillary acidic protein (GFAP). Luminex analysis indicated that PSC but not human fetal primary pancreatic fibroblast cells (HPF; negative controls) produced MDSC-promoting cytokines [interleukin (IL-6), VEGF, macrophage colony-stimulating factor (M-CSF) ] and chemokines (SDF-1, MCP-1). Culture of peripheral blood mononuclear cells [peripheral blood mononuclear cell (PBMC), n = 3 donors] with PSC supernatants or IL-6/granulocyte macrophage colony-stimulating factor (GM-CSF; positive control) for 7 days promoted PBMC differentiation into an MDSC (CD11b+CD33+) phenotype and a subpopulation of polymorphonuclear CD11b+CD33+CD15+ cells. The resulting CD11b+CD33+ cells functionally suppressed autologous T-lymphocyte proliferation. In contrast, supernatants from HPF did not induce an MDSC phenotype in PBMCs. Culture of normal PBMCs with PSC supernatants led to STAT3 but not STAT1 or STAT5 phosphorylation. IL-6 was an important mediator as its neutralization inhibited PSC supernatant-mediated STAT3 phosphorylation and MDSC differentiation. Finally, the FLLL32 STAT3 inhibitor abrogated PSC supernatant-mediated MDSC differentiation, PSC viability, and reduced autocrine IL-6 production indicating these processes are STAT3 dependent. These results identify a novel role for PSC in driving immune escape in pancreatic cancer and extend the evidence that STAT3 acts as a driver of stromal immunosuppression to enhance its interest as a therapeutic target. Cancer Res; 73(10); 3007–18. ©2013 AACR.

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.

Background: Targeting MEK is of great interest in the development of novel agents for treatment of a variety of malignancies. Resistance to MEK inhibitors has delayed the development of novel agents, and better strategies are needed to overcome acquired resistance. Preclinical studies performed at the University of Colorado in KRAS mutant cell lines have shown that several members of the Wnt pathway are overexpressed in cell lines resistant to the MEK inhibitor, selumetinib. Gene set enrichment analysis and synthetic lethal screens demonstrated that genes involved in the canonical and non-canonical Wnt pathways were upregulated in selumetinib-resistant CRC cell lines, whereas the combination of selumetinib and cyclosporin A (CsA), a WNT pathway modulator, demonstrated antitumor activity in patient-derived xenograft (PDX) models. We are conducting an NCI CTEP-approved Phase I/IB trial of the combination of selumetinib and CsA. Biomarkers of response to therapy are being co-developed in a preclinical trial of MEK/WNT inhibition in PDX models. We hypothesize that this combination will be safe, potentially effective in patients with mCRC and that upregulation of FZD2 may predict for sensitivity. Methods: This is a dose-escalation phase I trial investigating the combination of selumetinib and CsA in patients with solid tumors with an expansion cohort in patients with mCRC (n = 20). The expansion cohort will utilize a “run-in” of MEK inhibition alone to evaluate adaptive mechanisms of resistance that may identify those patients most likely to respond to the combination. An adaptive statistical design will be used to assess both KRAS/NRAS wild-type and mutant CRC to determine whether either or both subsets should be studied further in phase II trials. Results: As of January 15, 2016, a total of 18 patients have been enrolled and treated with selumetinib and CsA in the escalation phase. One DLT was reported in cohort 1 (G3 hypertension), and three DLTs (G3 hypertension, rash and elevated creatinine) were reported in cohort 2 (n = 12). Grade 1 or 2 nausea (67%) and rash (50%) were reported as the most common AEs. One unconfirmed partial response was noted (CRC KRAS mut), and 9 patients exhibited stable disease as their best response. One patient maintained stable disease for over 9 months. Disease control rate was 56% in the escalation cohort. Preclinical studies are currently being performed using next-generation MEK and WNT inhibitors in CRC PDX models. Conclusions: The combination of selumetinib and CsA is well tolerated with evidence of preliminary anti-tumor activity. Selumetinib at 75mg/kg BID and CsA at 2mg/kg was selected as the recommended phase 2 dose. The dose expansion in patients with metastatic CRC is underway. Preclinical studies and tissue acquisition for correlative studies are ongoing. Citation Format: Anna Capasso, Arvind Dasari, A. Craig Lockhart, Mark Stein, Hanna Sanoff, James Lee, Aaron Hansen, Tanios Bekaii-Saab, Sarah Rippke, James Yao, Funda Meric-Bernstam, S Gail Eckhardt, Christopher h. Lieu. A phase IB study of the combination of AZD6244 hydrogen sulfate (selumetinib) and cyclosporin A (CsA) in patients with advanced solid tumors with an expansion cohort in metastatic colorectal cancer (mCRC). [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 CT146.

Background: Nivo is an inhibitory antibody against programmed death receptor-1 (PD-1), a regulator of antitumor immunity. Nivo is approved for treatment of unresectable or metastatic melanoma and disease progression (PD) following ipilimumab or, in BRAF V600 mutation–positive melanoma, following a BRAF inhibitor, and for metastatic squamous non-small cell lung cancer (NSCLC) following PD during or after platinum-based chemotherapy. Nivo and other immune checkpoint inhibitors are also being investigated in other tumor-types. nab-P is a novel taxane formulation and does not require prophylaxis with immunosuppressive steroids. It has demonstrated superior efficacy over control regimens in phase III studies of MBC, pancreatic cancer, and NSCLC. This open-label, 6-arm, multicenter phase I trial will evaluate the safety of Nivo with nab-P in 3 cancer types (2 arms/disease): MBC, advanced NSCLC (+ carboplatin), and advanced pancreatic cancer (± gemcitabine). The study design for the MBC portion is described below. Methods: Eligibility criteria include histologically/cytologically confirmed HER2-negative MBC; 1 prior chemotherapy for MBC, including an anthracycline unless clinically contraindicated; no relapse < 12 months after taxane adjuvant therapy; measurable disease by RECIST v1.1; ECOG performance status 0-1; adequate organ function; and preexisting peripheral neuropathy grade < 2. Patients (pts) with MBC will be treated in 2 arms: nab-P 100 mg/m2 on days 1, 8, and 15 of each 28-day cycle plus Nivo 3 mg/kg on days 1 and 15 starting at cycle 3 or nab-P 260 mg/m2 on day 1 of each 21-day cycle plus Nivo 5 mg/kg on day 15 starting at cycle 3. Pts will be treated until PD or allowed to continue treatment beyond RECIST v1.1–defined PD if they continue to meet study eligibility; do not have rapid PD or clinical deterioration or unacceptable toxicities; and can benefit from continuation of study treatment in the treating physician's opinion and will not delay an imminent intervention to prevent serious complications of PD. The primary endpoints of the study are the number of pts with dose-limiting toxicities (DLTs) in each treatment arm (part 1) and the percentage of pts with grade 3/4 treatment-emergent adverse events (TEAEs) or treatment discontinuation due to a TEAE (parts 1 and 2). Part 1 of the study will assess whether the starting dose of Nivo is deemed safe (≤ 1 DLT in 6 pts); otherwise, the Nivo dose will be de-escalated and assessed in a new cohort at the next lower dose level. The Nivo dose in combination with nab-P deemed safe in a treatment arm may be further assessed in part 2 of the study, with enrollment expanded to an additional ∼ 14 pts/arm (total of 20 Nivo-treated pts/arm). Secondary study endpoints include TEAEs leading to dose reduction, delay, interruption, or treatment discontinuation; progression-free survival; overall survival; disease control rate; overall response rate; and duration of response (per RECIST v1.1). Exploratory endpoints include tumor-associated PD-L1 expression, modulation of immune activation in the tumor and peripheral blood in response to Nivo treatment, Nivo serum levels, and development of anti-globulin antibodies. ClinicalTrials.gov identifier NCT02309177. Citation Format: Waterhouse D, Gutierrez M, Bekaii-Saab T, DeRosa W, Wainberg Z, George B, Duval Fraser C, Ko A, Pierce DW, Stergiopoulos S, Soliman H. nab-paclitaxel (nab-P) plus nivolumab (Nivo) in human epidermal growth factor receptor 2 (HER2)–negative recurrent metastatic breast cancer (MBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-01-07.

Background: HCC is the sixth most common cancer and the third most common cause of cancer death worldwide. Sorafenib treatment improves survival in advanced disease, but no therapy has demonstrated significant activity after progression on sorafenib. Increased methylation of genes implicated in tumorigenesis has been described in HCC and has been associated with outcome and etiology. We evaluated the therapeutic and biologic effects of SGI-110, a hypomethylating agent in patients with HCC. SGI-110, a dinucleotide of decitabine and deoxyguanosine, increases decitabine exposure by protecting it from deamination due to slow release on subcutaneous (SC) injection. PK and PD results of an open-label, phase 2 study in patients with HCC are presented here. Methods: Adults with histologically confirmed, advanced-stage HCC who had received sorafenib, had evidence of disease progression, and ECOG PS 0-1 were enrolled. SGI-110 (SC) was given on D1-5 of a 28-day cycle. Blood samples were taken for PK/PD analysis and, when possible, paired tumor biopsies were taken for analysis of global DNA (LINE-1) and gene-specific methylation and gene expression. Patients were imaged every 8 weeks and allowed to continue treatment after radiologic but not clinical progression. End points include disease control rate (DCR) at 16 weeks, overall response rate, progression-free survival, and overall survival. Results: 50 HCC patients (43M/7F; median age 60 years [range 32-82]; ECOG PS 0/1: 21/29) were enrolled. The initial dose of SGI-110 was 60 mg/m2 (4 patients treated), but due to grade 4 neutropenia, the dose was decreased to 45 mg/m2 for subsequent patients. SGI 110 was well tolerated at 45 mg/m2; myelosuppression was the major adverse event. Full PK was available from 16 patients (3F/13M). The PK profile for SGI-110 after 45 mg/m2 showed protracted conversion to deliver active metabolite decitabine with exposure window lasting beyond 8-hr and mean(SD) decitabine AUC exposures of 94(22) ng*hr/mL that were comparable to those achieved in AML/MDS after 60 mg/m2. Potent LINE-1 demethylation was observed in blood (-35.6%, n = 27) and in tumor (-12.9%, n = 10) DNA; significant demethylation (-27.4%, n = 6) was also observed on promoter of tumor suppressor gene MZB1 which is frequently hypermethylated and silenced in HCC. Conclusions: SGI-110 was well tolerated at a dose of 45 mg/m2 administered SC on D1-5 of a 28-day cycle. PK was consistent with that seen in another solid tumor study and provided more persistent decitabine exposures compared with PK in hematologic malignancies. The PD changes in blood and tumor LINE-1 and MZB-1 methylation are promising and consistent with the desired biologic effect of SGI-110. Analysis for clinical efficacy is ongoing. Citation Format: Anthony El-Khoueiry, Mary F. Mulcahy, Tanios Bekaii-Saab, Richard Kim, Crystal Denlinger, Rakesh Goel, Shweta Gupta, Simone Jueliger, Aram Oganesian, Harold Keer, John Nemunaitis. Pharmacodynamic (PD) and pharmacokinetic (PK) results of the second-generation hypomethylating agent, SGI-110, in patients with hepatocellular carcinoma (HCC) after progression on sorafenib. [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 2947. doi:10.1158/1538-7445.AM2015-2947

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

Cholangiocarcinoma (CC) responds poorly to chemo- and immunotherapy and is nearly always fatal within one year. In recent years we have gained important insight into the signaling pathways that drive this cancer. These cancers are characterized by deregulated mitogen-activated protein kinase (MAPK), PI3 kinase/Akt and a dependence on the IL-6 axis of signal transduction. Importantly, we previously reported clinical responses for CC patients receiving single agent MEK inhibitors, indicating these agents have activity in this disease. We hypothesized that dual targeting of these pathways in CC using MEK162 and buparlisib would lead to potent antitumor and immunomodulatory activity, possibly circumventing resistance to single agent MEK162. In a panel of n = 7 human CC cell lines with diverse genetic profiles, constitutive phosphorylation of ERK (7/7) and Akt (2/7) was observed. Human CC cell lines displayed variable sensitivity to the growth inhibitory and pro-apoptotic effects of single agent MEK162 or buparlisib. CC cell lines with basal AKT phosphorylation (WITT, Mz-Cha-1) showed greater sensitivity to growth inhibition by buparlisib (IC50 10-20 μM), as compared to CC lines lacking pAKT (HuCCT1, HuH28, IC50 > 20μM). Immunoblot analysis confirmed decreased phosphorylated ERK (pERK) in the HuCCT1 and SNU-478 CC cell lines following treatment with MEK162. Culture supernatants from four separate human CC cell lines displayed significant reductions in IL-6, VEGF, and GM CSF in a concentration-dependent manner after treatment with MEK162. Immunomodulatory effects of MEK162 were also evident, independent of its tumor-intrinsic effects upon CC cell lines. Namely, it significantly reduced IL 6/GM-CSF driven MDSC differentiation (HLA-DRlo CD11b+ CD33+) from healthy normal donor PBMC in vitro. These observations were not due to cytotoxic activity as treatment with MEK162 for 72 hours did not reduce viability of bulk human PBMCs. Finally, to evaluate the effect of this treatment combination, in vivo studies were conducted in athymic mice bearing Mz-Cha1 or SNU-478 xenografts. Tumor bearing mice received daily oral administration of MEK162 (30 mg/kg), buparlisib (25 mg/kg), or both agents combined. Vehicle treated animals served as negative controls. Inhibition of tumor growth was observed following administration of single agent MEK162 or buparlisib as compared to control animals. This effect was further enhanced in the combination treated animals. Body weight of animals indicated this regimen was well-tolerated. Together, these data suggest that dual PI3K and MEK inhibition can target CC with varying genotypes and represents a promising therapeutic regimen with potential for direct antitumor activity and immunomodulation in CC. Citation Format: Jennifer Yang, Omar Elnaggar, Thomas Mace, Matthew Farren, Gregory Young, Patrice Lee, Kaitlin Keenan, Zheng Che, Jacob Kaufman, Denis Guttridge, David Carbone, Cynthia Timmers, Tanios Bekaii-Saab, Gregory Lesinski. Combined inhibition of MEK and PI3K elicits anti-tumor activity in human cholangiocarcinoma. [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 2656. doi:10.1158/1538-7445.AM2015-2656

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

Prior studies by our group have shown that single-agent MEK inhibitors have clinical activity in cholangiocarcinoma (CC), including objective responses. Yet no relationship was found between clinical benefit, and oncogenic driver mutations such as KRAS or BRAF. Strategies to expand upon MEKi have focused on combinations with agents targeting resistance, such as the PI3K/Akt pathway. CC patients treated with MEKi also had reduced pro-inflammatory cytokines and weight gain with restoration of muscle mass. These findings are clinically significant, and led us to hypothesize that MEKi act via tumor-extrinsic mechanisms to modulate immunity and limit cancer cachexia, while overcoming tumor-intrinsic resistance. We used the colon-26 adenocarcinoma model to evaluate the effect of single and combined treatment with MEK162 (30mg/kg) and buparlisib (25mg/kg) on muscle wasting, tumor growth, and immune modulation. This murine model depends on interleukin-6 (IL-6) and recapitulates the cancer cachexia syndrome. Single agent MEK162 inhibited growth initially, but after 14 days, tumor volume was comparable between MEKi and vehicle treated mice, possibly from acquired MEK162 resistance. Despite these data, reduced serum IL-6 and splenic Gr1+CD11b+ cells were evident compared to control mice (mean = 358 pg/mL to 43 pg/mL, and 13.8% to 8.4%, respectively), while body weight from MEK162 treated mice was spared (mean = 24.0g to 20.5g in control mice). In addition, markers of muscle catabolism, including the E3 ubiquitin ligases Atrogin-1 (from 1 to 0.043 in combo) and MuRF1 (from 1 to 0.03 in combo), and the autophagy gene, Bnip3, were all reduced in tibialis anterior muscles from tumor-bearing mice treated with MEK162. Similar to our clinical data, these results suggest MEK162 modulates immune biomarkers, and acts as an anti-cachexia agent. Since cross talk between MAPK and PI3K/AKT pathways promotes resistance to monotherapy, we next investigated the effects of MEK162 combined with buparlisib in the colon-26 model. As before, single agent MEK162 had a modest growth inhibitory effect, yet rescued body weight. Consistent with in vitro studies, single agent buparlisib was cytostatic, but not as effective as MEK162 in rescuing weight loss. However, dual treatment with MEK162 + buparlisib significantly inhibited tumor growth. This combination also significantly reduced splenic MDSC (mean = 37% to 9% in control mice) and increased CD4+ and CD8+ (mean = 24% to 6%, and 7.7% to 2%, respectively) T cells. These results suggest that dual inhibition of MEK and PI3K has efficacy, while MEKi may have under-appreciated tumor-extrinsic mechanisms of activity that can be leveraged to benefit patients with advanced malignancy. This treatment combination will be evaluated in CC patients in the setting of a Phase II clinical trial. Citation Format: Jennifer Yang, Erin Talbert, Omar Elnaggar, Priyani Rajasekera, Thomas Mace, Matthew Farren, Zheng Che, Benjamin Swanson, Gregory Young, Ericka Haverick, Cynthia Timmers, Mark Bloomston, Tanios Bekaii-Saab, Denis Guttridge, Gregory Lesinski. Dual targeting of MEK and PI3K pathways can act via tumor-intrinsic mechanisms to overcome resistance and tumor-extrinsic mechanisms to modulate immunity and limit cancer cachexia. [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 4695. doi:10.1158/1538-7445.AM2015-4695

Background: Cachexia occurs in more than 50% of cancer patients. Cachexia is characterized by severe loss of weight and skeletal muscle that is not reversed by nutritional support, and contributes significantly to morbidity and mortality. The development of effective therapies for cancer cachexia is clearly warranted. AR-42 is a novel class I/IIB HDAC inhibitor that was developed in our laboratories and currently in Phase I/IB trials at The Ohio State University. Here, we report the anti-cachectic activity of AR-42 in two murine models of cancer cachexia. Methods: Experiments were conducted using the colon-26 adenocarcinoma (C-26) and Lewis lung carcinoma (LLC) tumor mice models of cancer cachexia. AR-42 was administered by oral gavage at 50 mg/kg every other day starting at day 6 after tumor cell inoculation. Serum samples (n = 3) and muscle tissues (n = 8) from each group were sent for RNAseq and small RNAseq (OSU), cytokine/chemokine profiling assays [Eve Technologies, Alberta, Canada] and metabolomic analyses [Metabolon, Research Triangle Park, NC]. Results: In the C-26 model, AR-42 significantly attenuated cachexia-induced losses of skeletal muscle mass and body weight, with minimal effects on C-26 tumor growth, and prolonged survival time relative to mice treated with vehicle only or with other HDAC inhibitors (vorinostat and romidepsin). Metabolomic and gene expression analyses revealed that the effects of AR-42 were associated with its ability to maintain metabolic and gene expression profiles in skeletal muscle to levels comparable to those in muscle from tumor-free mice. Analysis of RNAseq data of relevant genes suggests that AR-42 modulates major pathways involved in muscle wasting, mitochondrial function, lipolysis, and cytoskeletal integrity. The main mechanism of action is thought to be through down-regulating FOXO1 (> 5-fold) leading to suppression of MuRF1 and Atrogin-1. Additional mechanistic validation is underway and full results will be available at the meeting. AR-42-induced abrogation of cachexia and rescue of muscle weight was additionally confirmed in the LLC model. Conclusion: Our results suggest AR-42 induces unique protective effects on cancer-induced muscle wasting and lipolysis, and our findings support further evaluation of AR-42 as a potential treatment for cancer cachexia. Citation Format: Yu-Chou Tseng, Samuel Kulp, I-Lu Lai, Wei He, Ralf Bundschuh, David Frankhouser, Pearlly Yan, Denis Guttridge, Guido Marcucci, Ching-Shih Chen, Tanios Bekaii-Saab. Preclinical investigation of the novel histone deacetylase (HDAC) inhibitor AR-42 in the treatment of cancer-induced cachexia. [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 5540. doi:10.1158/1538-7445.AM2014-5540

Background: We have previously reported that adjuvant treatment with a therapeutic vaccine targeting the mutated Ras oncogene product generated mutation-specific T cell responses associated with a trend toward improved survival in patients with post-operative residual disease (R1 resections) but no improvement in the overall population1. Initial analysis of 90 pretreatment plasma samples using matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) showed the potential to predict improved RFS and OS for treatment with GI-4000/gemcitabine, but not placebo/gemcitabine. Methods: We have developed a novel technique, combining methods used in recent advances in learning theory (‘deep learning’) with newly-refined MS techniques that allow exploration deeper into the proteome to create diagnostic tests. Using 500,000 laser shot Deep MALDI spectra2 more than 700 mass spectral features were identified. A subset of these was used to create many multivariate classifiers that were filtered for performance and combined using dropout regularization. This method allows the use of smaller training sets and so left a test set with which performance of the signature could be independently assessed. This new methodology was used to create a test (BDX-001) to identify patients likely to benefit from the addition of GI-4000 to gemcitabine. Results: Using BDX-001 for stratification, subjects who are BDX-001(+) demonstrated a 499 day advantage in median OS when treated with GI-4000/gemcitabine vs. placebo/gemcitabine. Additionally, these subjects demonstrated a 351 day improvement in median RFS. BDX-001 did not predict response for placebo/gemcitabine treated subjects. These results were obtained using only test set data, and although the small sample size prohibited statistical significance, it should give an unbiased test performance estimate to be validated independently. Conclusions: BDX-001 is a test developed using novel proteomic and learning theory methods that appears to predict treatment response to GI-4000 in resected pancreas cancer patients, potentially identifying patients with improved RFS and OS in the GI-4000/gemcitabine arm. We plan to prospectively validate BDX-001 as a companion diagnostic in a future study of GI-4000 in pancreas cancer. References 1. Richards et al, ESMO GI. Annals of Oncology, June 2012 23 (suppl 4) 2. Duncan et al, ASMS 2013, http://asms.inmerge.com/Proceedings/2013Proceedings.aspx. Citation Format: Donald A. Richards, Peter Muscarella, Tanios Bekaii-Saab, Lalan S. Wilfong, Vic Velanovich, Julian Raynov, Patrick J. Flynn, William E. Fisher, Samuel H. Whiting, Constana Timcheva, Tom Holmes, Claire Coeshott, Alicia Mattson, Heinrich Roder, Joanna Roder, Allen Cohn, Timothy C. Rodell. A proteomic signature predicts response to a therapeutic vaccine in pancreas cancer; analysis from the GI-4000-02 trial. [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 5314. doi:10.1158/1538-7445.AM2014-5314

BRCA 1/2 dysfunction sensitizes cells to inhibition of PARP enzymatic activity, due to the persistence of DNA breaks normally repaired by homologous recombination (HR), thus cancer pts carriers of germ line BRCA deleterious mutations have been targeted for treatment with PARP inhibitors. An example of HR repair is the FA pathway where BRCA 1/2 collaborate with several other genes resulting in FancI and FancD2 activation and nucleus repair foci formation. We have developed a triple stain immunofluorescence based method to evaluate FA pathway functionality (foci formation) (FancD2/DAPI/Ki67) (FATSI) in paraffin embedded tumor tissues, and hypothesized that among patients with sporadic tumors, a significant proportion with tumoral dysfunctional FA pathway can be identified. A two steps clinical trial (NCT01017640) was designed to 1- screen solid tumor patients for absence of FA tumor foci formation, and 2- evaluate safety and recommended doses in these pts of the PARPi veliparib, either alone or in combination with chemotherapy (MMC). 607 pts were consented, 532 had their tumor tissue screened, 142 (27%) had absent foci. All major solid tumors were represented in FA dysfunction, notably 36 of 105 (34%) colorectal and 40 of 135 (30%) breast cancers. 52 pts received 192 cycles of veliparib, 29 pts as monotherapy in escalating dose cohorts (starting at 50 mg PO BID continuously), and 23 following MMC 10 mg-sqm every 4 weeks (40mg-sqm cumulative cap) at 50 mg BID at increased duration cohorts (1, 2 and 3 wks) every 4 wks (100 and 200 mg BID for 3 wks in subsequent cohorts). Recommended dose levels are: veliparib 300 mg BID as monotherapy (2DLT/3 pts at 400 BID:G3 fatigue) and MMC/veliparib 10mg-sqm/200 mg BID three weeks duration. Analysis for germ line mutations (BRCA1 and 2 sequencing and for the 5 most common BRCA1 large rearrangements) in PBMC among 50 pts showed 7 pts (3 breast, 1 ampullary, 2 ovarian) with BRCA abnormalities: 6 known deleterious mutations and 1 polymorphism. Five RECIST criteria responses occurred (1CR, 4 PR) (1 lung, 2 breast, 1 ovarian, 1 endometrial), all in the combination arm. Two breast cancer pts (39 and 13 cycles) and 1 ovarian (6 cycles) on monotherapy, plus 1 breast (15 cycles), 1 colon (7 cycles) on the combination, had prolonged stability. rH2AX (n=49) and PAR (n=17) analysis in PBMC suggests veliparib dose dependency (blunting of rH2AX induction, lower PAR level and slower recovery for higher doses). The trial was amended to enroll an expanded cohort of 10 FATSI negative colorectal patients who will undergo fresh biopsy for whole exome sequencing (RNAseq) and FA/DNA repair gene panel analysis prior to treatment at the recommended dose in the combination arm, with the goal of identifying the specific genetic defect mediating the FA dysfunction, as well as partner mutations to be targeted in subsequent trials. Supported by R01CA152101 & N01CM62207 Citation Format: Miguel A. Villalona-Calero, Wenrui Duan, Weiqiang Zhao, Konstantin Shilo, Jiuping Ji, Jennifer Thurmond, Adam Norris, Jeffrey Rose, Rachel Layman, John Marshall, Tanios Bekaii-Saab, Alice Chen. Phase I trial of veliparib or mitomycin (MMC) + veliparib in patients with sporadic solid tumors screened for somatic deficiency in the Fanconi Anemia (FA) pathway. [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 LB-139. doi:10.1158/1538-7445.AM2013-LB-139

Background: Pancreatic cancers produce multiple immune-suppressive factors that can be measured in peripheral blood. Elevated levels of myeloid derived suppressor cells (MDSC) induced by tumor derived factors are associated with inhibition of immune responses in gastrointestinal malignancies (Mundy-Bosse, et al., CII, 2011). We hypothesized that levels of MDSC and pro-MDSC cytokines would be elevated in pancreatic adenocarcinoma patients with progressive disease. Methods: Plasma and immune cells from peripheral blood were obtained from 30 pancreatic adenocarcinoma patients. Group A consisted of 15 patients undergoing chemotherapy (11 stage III/IV, 4 stage II). Group B consisted of 15 chemonaive patients (10 stage III/IV, 5 stage II) and group C consisted of 9 normal patients. The nonparametric Wilcoxon test was used to compare the cytokines in the plasma between the various groups (A/B vs C, A vs B). The Bonferroni method was used to adjust for data obtained for 27 cytokines via BioPlex assay. Pathway analysis on cytokine profiles obtained from the BioPlex analysis was performed using IPA. A S100A9 ELISA was performed on chemonaive patients and the Wilcoxon test was used to compare levels between patients with different cancer stages. MDSC (HLADRneg/low, CD33+, CD11b+, CD15+/− and CD14+/−) were phenotyped via flow cytometry. Results: BioPlex analysis was performed on all subjects (Groups A, B and C). Increases in the levels of pro-MDSC cytokines were observed for A/B vs C (IL4 (p=0.005), IL5 (p=0.046), IL10 (p=0.046), GCSF (p=0.027). Increases were also observed in levels of pro-inflammatory cytokines (e.g. RANTES p Conclusions: Levels of pro-MDSC cytokines are increased in the circulation of pancreatic adenocarcinoma patients. MDSC chemotaxic factors such as S100A9 increase with stage. This study suggests that MDSC in peripheral blood may be a predictive biomarker of chemotherapy failure in pancreatic cancer patients. Citation Format: Joseph Markowitz, Bonnie K. Paul, Taylor R. Brooks, Lai Wei, Jeff Pan, Katherine L. Martin, Eric Luedke, Bethany L. Mundy-Bosse, Gregory B. Lesinski, Tanios Bekaii-Saab, William E. Carson. Immune-suppressive myeloid cells are induced during disease progression in patients with advanced pancreatic adenocarcinoma. [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 456. doi:10.1158/1538-7445.AM2013-456

S100A9 is a pro-inflammatory protein originally found in macrophages which is felt to be important in promoting inflammatory responses and cancer cell growth. The protein is found in cancer cells of gastric, colon, breast cervical and thyroid cancer. Generally, S100A9 is a cytoplasmic, cell surface and secreted protein that promotes cancer cell growth via inflammatory mechanisms and migration of immune cells (e.g. PBMCs) to the cancer microenvironment via non-covalent interactions with glycosaminoglycans on endothelial surfaces. Previously, this protein was reported in the stroma of pancreatic cancers but not in pancreatic cancer cells themselves. This report iilustrates S100A9 up-regulation on the surface of p53 mutant pancreatic cancer cell lines BxPC3 and AsPC1 as determined by immunoblot analysis and flow cytometry. In this study, pentamidine a known inhibitor of S100B, inhibited S100A9 dependent cell growth in BxPC3 cell lines (IC50 = 20.1 +/- 10.3 μM). Pentamidine treated BxPC3 cells (20 μM for 96 hrs) exhibited 30.1% apoptotic cell death as measured by Annexin/PI statining vs less than 3% for control treated cells. Given that S100A9 was found in other cancer models to promote recruitment of pro-infammatory cells (e.g. PBMCs) and subsequent support of cancer cell growth, we studied the effect of pentamidine on PBMC migration. Pentamidine also blocked the migration of PBMC from a metastatic pancreatic cancer patient (67%) to a similar degree as an Anti-s100A9 (68%) Ab or an anti-s100A8/A9 (65%) antibody. S100A8/A9 antibody is known to bind to helix 1 of S100A8 and helix 4 of S100A9, areas of the protein considered important for target interactions. It is likely that pentamidine indiscriminately binds to both the S100A8/A9 heterodimer as well as the homodimer in addition to other S100 proteins. S100A9 levels in BxPC3 cells as detected via Western analysis also increase with 20 μM pentamidine treatment, but not 10 fold as reported in the literature for S100B. As p53 was also shown previously to bind to the S100A9 promoter and S100A9 is not as robustly expressed in mutant p53 cell lines, p53 independent pathways are clearly important for cancer cell survival. This study suggests that S100A9 is up-regulated in pancreatic cancer and that inhibition causes pancreatic cancer cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 540. doi:10.1158/1538-7445.AM2011-540

Interferon-alpha (IFNα) can promote anti-tumor immunity through its actions on immune cells. We have shown in murine models that a class of immune suppressor cells known as myeloid-derived suppressor cells (MDSC) have inhibitory effects on IFN signal transduction and gene regulation. Interleukin (IL)-6 and IL-10 are produced by tumor cells in cancer patients and can regulate the survival and function of MDSC. This study evaluated relationships between plasma IL-6, IL-10, circulating MDSC subsets and IFNα-induced signal transduction in 40 patients with gastrointestinal (GI) malignancies. Plasma IL-6 and IL-10 levels were significantly higher in patients versus normal donors. CD33+HLADR−CD11b+CD15+ and CD33+HLADR−/lowCD14+ MDSC were also elevated in patients versus normal donors (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5517. doi:10.1158/1538-7445.AM2011-5517

Background: Hepatocellular carcinoma (HCC) is on the rise in the US and the rest of the world. HCC responds poorly to systemic chemotherapy. EGFR 1 and 2 are both overexpressed in HCC. We reported that HER2/NEU (EGFR2) somatic mutations in HCC may predict response to EGFR-targeted agents. Lapatinib is an oral inhibitor of EGFR and HER2/NEU with evidence of activity in a number of tumor types. This trial was designed to determine the efficacy of lapatinib in HCC patients. Methods: A Fleming phase II design resulting in a single stage of 25 patients was utilized with a 90% power to detect a true response rate >20%. Adequate hematologic, renal and hepatic function were required. Eligible pts included Child’s A or favorable B group and ECOG PS of 0-1. One prior treatment regimen was allowed. Lapatinib dose was 1,500 mg/d orally without interruption. One cycle was 28 days and radiological assessment was done every 8 weeks. Tumor and blood specimens were analyzed for expression of EGFR/EGFRP, HER2/NEU/CEP17, status of downstream signal pathway molecules (including PTEN, P-Akt, cyclin D, p27 and p70S6K, p53 and others) and correlated to response. Results: 26 pts with HCC were accrued with the following characteristics: Males (17 pts), ECOG PS 0/1 (12/14 pts), median age of 58 yrs (range 29-83). A median of 2 cycles were administered (range 1-12). The most common toxicities were: diarrhea (69%) and nausea ( 54% ). Grade 3/4 toxicities were noted in 3 patients including diarrhea, rash and acute renal failure. There was no evidence of cardiac dysfunction. In 25 patients with HCC, no objective responses were observed. However, 8 (31%) patients had stable disease (SD) including 2 (7.6%) with SD lasting > 6 months. Survival data is being analyzed. The overall goal of correlative studies underway is to attempt to understand the molecular biology of this tumor and guide directed therapy for subsets of patients with hepatocellular carcinoma that may benefit from EGFR inhibition. Tissue and blood specimens were available on >95% of patients. No mutations in EGFR (exons 18-21) were found, consistent with our previous findings, although three single nucleotide polymorphisms (SNP) were found that correlate with SNPs in the NCBI SNP database. We did not find evidence of HER2/NEU somatic mutations. HER2/NEU copy number may be elevated in one patient with stable disease using FISH and is currently being confirmed by IHC staining. Studies are currently underway to assess the expression of EGFR/EGFR-P protein expression and the genes that regulate the cell cycle and apoptosis, which are either downstream of or cross-talk with the EGFR signaling pathway. KRAS mutational status is also underway as it is hypothesized that KRAS mutations will correlate with resistance to EGFR directed-treatment. Conclusions: Lapatinib is well-tolerated and may have some activity, mainly stabilization of disease, in HCC. Source of support: NCI-NO1-CM-62207.