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Introduction VAY736 is an afucosylated, human monoclonal antibody engineered to enhance antibody-dependent cellular cytotoxicity that targets BAFF-R+ B cells for elimination. In preclinical CLL models, VAY736 showed antileukemic activity and, when combined with ibrutinib, significantly reduced disease burden, which may allow some pts to discontinue ibrutinib. Methods This Phase Ib dose-escalation (ESC)/-expansion (EXP) trial (NCT03400176) enrolled pts with CLL who did not achieve a complete response (CR) after >1 year of ibrutinib or had developed a resistance mutation to ibrutinib. Pts received IV VAY736 (ESC: 0.3-9 mg/kg; EXP: 3 mg/kg) once every 2 weeks and oral ibrutinib (420 mg) once daily for up to 8 28-day cycles. Pts achieving undetectable MRD (uMRD) at C9D1 could discontinue ibrutinib at investigator discretion. The study aimed to characterize the safety and tolerability of VAY736 + ibrutinib, assess antitumor activity, PK, and characterize PD profiles. Results By Jul 29, 2022, 39 pts were enrolled (ESC: n=15; EXP: n=24). Table 1 shows pt characteristics, safety, and efficacy data. The overall response at C9D1 for 37 evaluable pts was 40.5% CR + CRi and 16.2% PR (1L: 63.6% CR + CRi and 18.2% PR). At C9D1, 17 pts (45.9%) had uMRD in blood or bone marrow (BM). In the 2-year follow-up period, 16 pts discontinued ibrutinib and were off therapy for 4.9-19.8 months. Frequency of peripheral NKp46+ NK cells increased at least 50% after VAY736 in over 50% of pts. Preliminary coverage-based limiting-cell experiment analysis of RNAseq (CLEAR) data from 10 pts supports peripheral NK cell activation with VAY736. Conclusions VAY736 + ibrutinib appears highly active and has an acceptable safety profile. Multiple pts attained uMRD in blood or BM. Biomarker data suggest NK cell activation with VAY736. More pts will be included in the RNAseq analysis at presentation. Future development of VAY736 for CLL is strongly indicated based on these promising data. Table 1. Patient characteristics, safety, and efficacy results. All patients (N=39) Patient demographics and prior treatment Median age, years (range) 65.0 (39-82) ECOG performance status, n (%) 0 36 (92.3) 1 3 (7.7) No prior regimens excluding ibrutinib, n (%) 12 (30.8) Median number of prior regimens, n (range) 1.0 (0.0-14.0) Median duration of ibrutinib, years (range) 2.95 (0.2-8.3) Patient baseline characteristics Dohner risk by FISH,a n (%) 17p deletion 6 (15.4) 11q deletion 9 (23.1) Trisomy 12 3 (7.7) 13q deletion 10 (25.6) IGHV mutant status, n (%) Non-mutant 32 (82.1) Complex karyotype, n (%) Yes 20 (51.3) Safety Dose-limiting toxicities, n (%) 0 Patients with at least one AE, any grade, n (%) 38 (97.4) Patients with at least one Grade ≥3 AE, n (%) 13 (33.3) Most common (occurring in ≥2 patients) Grade ≥3 AEs, n (%) Neutrophil count decreased 5 (12.8) Lymphocyte count decreased 2 (5.1) Hypophosphatemia 2 (5.1) Lipase increased 2 (5.1) Efficacy 1Lb n=11 R/R n=26 Evaluable patients N=37 Overall response at C9D1 or before discontinuation,c n (%) Complete response 6 (54.5) 8 (30.8) 14 (37.8) Complete response with incomplete marrow recovery 1 (9.1) 0 1 (2.7) Partial response 2 (18.2) 4 (15.4) 6 (16.2) Stable disease 2 (18.2) 8 (30.8) 10 (27.0) Progressive disease 0 5 (19.2) 5 (13.5) uMRD response at C9D1 or before discontinuation,c n (%) Bone marrow uMRD 6 (54.5) 6 (23.1) 12 (32.4) Blood uMRD 7 (63.6) 10 (38.5) 17 (45.9) Blood or bone marrow uMRD 7 (63.6) 10 (38.5) 17 (45.9) Patients elected to discontinue ibrutinib after achieving CR or uMRD, n (%) 16 (43.2) aThe categories were: patients with a 17p deletion; patients with an 11q deletion without a 17p deletion; patients with trisomy 12 without a 17p deletion or an 11q deletion; and patients with a 13q deletion without a 17p deletion, trisomy 12, or an 11q deletion; bPatients with no prior therapies excluding ibrutinib; cFor evaluable patients (N=37). 1L, first line; AE, adverse event; CR, complete response; C, cycle; D, day; ECOG, Eastern Cooperative Oncology Group; FISH, fluorescence in situ hybridization; IGHV, immunoglobulin heavy chain variable region; R/R, relapsed/refractory; uMRD, undetectable minimal residual disease. Citation Format: Kerry Anne Rogers, Pearlly Yan, Ian W. Flinn, Deborah M. Stephens, Thomas J. Kipps, Sarah M. Larson, Laura Martz, Xi Chen, Huabao Wang, Ethan Hopping, Ralf Bundschuh, Alexandra Acosta, Daniela Baldoni, Anwesha Chaudhury, Jeanne Whalen, Nadia B. Hassounah, Nina Orwitz, Janghee Woo, John C. Byrd. Assessing clinical and pharmacodynamic (PD) profiles of patients (pts) with chronic lymphocytic leukemia (CLL) on ianalumab (VAY736) + ibrutinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT021.

Numerous preclinical and clinical studies have demonstrated the significant contribution of inflammation to the development and progression of various types of cancer. Inflammation in the tumor microenvironment mediates complex interactions between innate immunity, adaptive immunity, microbiomes and stroma, and ultimately alters the overall fitness of tumor cells at multiple stages of carcinogenesis. Malignancies are known to arise in areas of chronic inflammation and inflammation in the tumor microenvironment (often called tumor-promoting inflammation) is believed to allow cancer cells to evade immunosurveillance while promoting genetic instability, survival and progression. Among the strongest data suggesting a causal role for inflammation in cancer come from the recent CANTOS trial which demonstrated that interleukin-1β (IL-1β) inhibition with canakinumab leads to a significant, dose-dependent decrease in incident lung cancer. This observation has launched a series of additional clinical studies to understand the role of IL-1β and the inflammasome in cancer, and the clinical utility of IL-1β inhibition in different stages of lung cancer. In this article we will review recent data implicating IL-1β signaling and its upstream regulator NLRP3 in both solid tumor and hematologic malignancies. We will discuss the key preclinical observations and the current clinical landscape, and describe the pharmacologic tools which will be used to evaluate the effects of blocking tumor-promoting inflammation clinically.

Chronic myelomonocytic leukemia (CMML) is a heterogeneous group of clonal hematopoietic malignancies with variable clinical and molecular features. We analyzed long-term results of allogeneic hematopoietic cell transplantation in patients with CMML and determined clinical and molecular risk factors associated with outcomes. Data from 129 patients, aged 7-74 (median 55) years, at various stages of the disease and transplanted from related or unrelated donors were analyzed. Using a panel of 75 genes somatic mutations present before hematopoietic cell transplantation were identified In 52 patients. The progression-free survival rate at 10 years was 29%. The major cause of death was relapse (32%), which was significantly associated with adverse cytogenetics (hazard ratio, 3.77; P=0.0002), CMML Prognostic Scoring System (hazard ratio, 14.3, P=0.01), and MD Anderson prognostic scores (hazard ratio, 9.4; P=0.005). Mortality was associated with high-risk cytogenetics (hazard ratio, 1.88; P=0.01) and high Hematopoietic Cell Transplantation Comorbidity Index (score ≥4: hazard ratio, 1.99; P=0.01). High overall mutation burden (≥10 mutations: hazard ratio, 3.4; P=0.02), and ≥4 mutated epigenetic regulatory genes (hazard ratio 5.4; P=0.003) were linked to relapse. Unsupervised clustering of the correlation matrix revealed distinct high-risk groups with unique associations of mutations and clinical features. CMML with a high mutation burden appeared to be distinct from high-risk groups defined by complex cytogenetics. New transplant strategies must be developed to target specific disease subgroups, stratified by molecular profiling and clinical risk factors.

Mantle cell lymphoma (MCL) is an aggressive form of B cell non-Hodgkin’s lymphoma and remains incurable under current treatment modalities. One of the main reasons for treatment failure is the development of drug resistance. Accumulating evidence suggests that B cell activating factor (BAFF) and BAFF receptor (BAFF-R) play an important role in the proliferation and survival of malignant B cells. High serum BAFF levels are often correlated with poor drug response and relapse in MCL patients. Our study shows that BAFF-R is expressed on both MCL patient cells and cell lines. BAFF-R knockdown leads to MCL cell death showing the importance of BAFF-R signaling in MCL survival. Moderate knockdown of BAFF-R in MCL cells did not affect its viability, but sensitized them to cytarabine treatment in vitro and in vivo, with prolonged mice survival. Anti-BAFF-R antibody treatment promoted drug-induced MCL cell death. Conversely, the addition of recombinant BAFF (rhBAFF) to MCL cells protected them from cytarabine-induced apoptosis. We tested the efficacy of a humanized defucosylated ADCC optimized anti-BAFF-R antibody in killing MCL. Our data show both in vitro and in vivo efficacy of this antibody for MCL therapy. To conclude, our data indicate that BAFF/BAFF-R signaling is crucial for survival and involved in drug resistance of MCL. Targeting BAFF-R using BAFF-R antibody might be a promising therapeutical strategy to treat MCL patients resistant to chemotherapy.

Hematopoietic cell transplantation (HCT) provides potentially curative treatment for patients with myelofibrosis (MF). HCT outcomes are associated with the Dynamic International Prognostic Scoring System (DIPSS) risk scores. In the present study we analyzed results in 233 patients to determine if the DIPSS plus classification, which adds cytogenetics, thrombocytopenia, and RBC transfusion dependence as risk factors, would better predict post-HCT outcomes than the original DIPSS. Multivariate analysis showed that each risk parameter incorporated into the DIPPS plus model contributed to its predictive power of overall mortality, relapse-free survival, and nonrelapse mortality. The 5-year overall survival (OS), relapse, and treatment-related mortality (TRM) rates for patients with low/intermediate-1 risk MF were 78%, 5%, and 20%, respectively. The 5-year OS, relapse, and TRM rates for patients with high-risk MF were 35%, 28%, and 40%, respectively. The HCT-specific comorbidity index of 3 or greater was associated with higher nonrelapse and overall mortality and reduced relapse-free survival. The relapse incidence was significantly increased in older patients (HR, 3.02; P = .0007). With a median follow-up of 8 years 124 patients (53%) were surviving. The components of the DIPSS plus classification still have prognostic relevance after adjustment by the DIPSS classification. This information should enhance our ability to advise patients when making decisions regarding timing of transplant.

Introduction: The treatment paradigm of chronic lymphocytic leukemia (CLL) has changed significantly with the introduction of targeted oral therapies, including Bruton's tyrosine kinase inhibitors (BTKis) and B-cell lymphoma 2 inhibitors (Bcl-2is). As more therapies enter the clinic, real-world data (RWD) are important to understand changes in the CLL treatment landscape and describe treatment patterns, to guide decisions on treatment strategies. Methods: This retrospective analysis used 2 RWD sources. Patients (pts) diagnosed with CLL and treated with chemotherapy, CD20 monoclonal antibodies (mAbs), BTKis, or Bcl-2is were studied in the Optum Clinformatics® Data Mart (CDM), a de-identified US administrative claims database. Pts in the CDM analysis received at least 1 prescription for or administration of a treatment of interest between 2016 and 2020 Q2, and a diagnosis of CLL within 12 months (mo) of treatment start. Cross-sectional annual cohorts were used to understand changes in treatment mix over time, and longitudinal cohorts were formed to identify treatment patterns. Medication adherence was assessed with persistence at 12 mo using a 60-day gap and proportion of days covered (PDC) at 12 mo. Electronic health records from oncology practices were extracted through December 2020 from the COTA real-world evidence database. Adults diagnosed with CLL/small lymphocytic lymphoma and treated with BTKis and/or Bcl-2is were included. Treatment discontinuation was assessed using physician-documented treatment dates and reasons for discontinuation, and overall duration of treatment with targeted therapy was assessed using Kaplan-Meier analysis. Results: In CDM, annual treatment rate among pts diagnosed with CLL increased from 16% (1788/10911) in 2016 to 26% (2441/9349) in 2020. The proportion of pts treated with BTKis and Bcl-2is increased from 40% (2016) to 65% (2020) and Conclusions: RWD show standard of care shifted from chemoimmunotherapy to targeted therapies. Pts receiving targeted therapies had high rates of discontinuation, most commonly due to toxicity. Most targeted regimens were mono; more BTKi pts were treatment naive compared to Bcl-2i pts. Slow adoption of fixed-duration therapy with the Bcl-2i combination regimen was observed. The duration of Bcl-2i mono was comparable to that of the Bcl-2i combination regimens, and a high number of pts initiating combo regimens continued on mono regimens in contrast to regimens studied in randomized controlled trials. Figure 1 Figure 1. Disclosures Smith: Novartis: Current Employment; Pfizer: Ended employment in the past 24 months. Owusu: Novartis: Current Employment; Roche: Ended employment in the past 24 months. Wormser: Roche: Current equity holder in publicly-traded company; Novartis: Current Employment, Current equity holder in publicly-traded company. Woo: Novartis: Current Employment, Current equity holder in publicly-traded company.

Introduction: B-cell activating factor receptor (BAFF-R) enhances the survival and regulation of normal and malignant B cells. VAY736 is a human monoclonal antibody (mAb) targeting BAFF-R that targets BAFF-R+ B cells for elimination by antibody-dependent cell-mediated cytotoxicity (ADCC). VAY736 has anti-leukemia activity in preclinical CLL models that is superior to that of anti-CD20 mAbs (McWilliams EM, et al. Blood Adv 2019;3:447-460). Although Bruton's tyrosine kinase inhibitors (BTKis; acalabrutinib, ibrutinib) are the current standard of care for CLL, the indefinite length of monotherapy required may result in cumulative clinical or economic toxicity and/or acquired treatment resistance. In preclinical models, adding VAY736 to ibrutinib significantly improved survival and reduced disease burden, suggesting that this combination may augment the anti-leukemia response and allow patients (pts) to discontinue ibrutinib. Methods: This dose escalation/expansion trial (NCT03400176) enrolled pts with CLL undergoing either first- or second-line therapy with ibrutinib and whose disease failed to achieve a complete response (CR) after >1 year of therapy or who had CLL with a mutation associated with ibrutinib resistance. Pts received oral ibrutinib (420 mg) once daily and VAY736 IV at 0.3, 1, 3, or 9 mg/kg once every 2 weeks. In the expansion, pts were enrolled into 2 arms depending on whether ibrutinib resistance mutations were present at baseline. Pts received VAY736 + ibrutinib for up to 6 28-day cycles. After 6 cycles, pts with a CR discontinued VAY736 and received ibrutinib for an additional 2 cycles; all other pts continued VAY736 + ibrutinib for those 2 cycles. Pts achieving undetectable minimal residual disease (uMRD) at C9D1 could discontinue ibrutinib at the investigator's discretion. This study aimed to characterize the safety and tolerability of VAY736 + ibrutinib, determine the recommended dose for expansion (RDE), and explore the efficacy of this combination. Results: A total of 32 pts (median age 65 years; ECOG PS 0: 91%) were treated prior to data cutoff (May 10, 2021). Overall, 19 pts completed therapy and 4 discontinued VAY736 + ibrutinib (primarily due to disease progression); 4 pts remain on VAY736 + ibrutinib and 5 pts continue to receive ibrutinib. Of the enrolled pts, 44% had CLL cells with mutations associated with ibrutinib resistance (mainly [71%] BTKC481); the median number of prior regimens was 1 (range: 0.0-14.0); median duration of prior ibrutinib therapy was 3.5 years (range: 0.2-8.3). Baseline cytogenetics were (not mutually exclusive): 19% del(17)(p13.1), 75% unmutated IGHV, 59% stimulated complex karyotypes (≥3 abnormalities), 34% del(11)(q22.3), 44% del(13)(q14), and 6% +12. No dose-limiting toxicities were observed and the RDE was 3 mg/kg, based on pharmacokinetics, exposure-to-response relationship, pharmacodynamics, safety, and in vitro ADCC. Twelve (38%) pts experienced AEs of Grade ≥3, most common (occurring in ≥2 pts) were neutrophil count decreased (n=5), lymphocyte count decreased (n=2), hypophosphatemia (n=2), and elevated lipase (n=2). The overall response at C9D1 for evaluable pts (n=21) was 38% CR, 5% CRi, 14% PR, 24% SD, and 19% PD (Fig. 1A). Thirteen (41%) pts achieved uMRD in blood. Eight pts (42%, 8/19) and 8 pts (42%, 8/19) had uMRD in blood and bone marrow (BM) at end of treatment (EoT), respectively. Among those, 6 pts were elected to discontinue ibrutinib and remained off treatment for 0.2-26.2 months and were still off therapy at the data cutoff. The median percentage change from baseline in MRD was -99.0% (range: -100.0% to -16.7%) and -97.3% (range: -100.0% to 1346.3%) in blood and BM, respectively. None of the pts who enrolled with CLL cells lacking mutations associated with ibrutinib resistance (11/11) developed mutations by C9D1. Although 1 pt with PLCG2 mutation eradicated mutant clones at EoT with VAY736, the response may vary depending on the type of mutation (Fig. 1B). Conclusions: VAY736 + ibrutinib was well tolerated with an acceptable safety profile enabling dose expansion. Clinical activity was observed including multiple pts attaining uMRD status in blood and BM, allowing 6 to discontinue ibrutinib therapy for an extended period. These data provide clinical evidence of the potent anti-leukemia activity of VAY736 and the potential to safely discontinue ibrutinib or other BTKi by VAY736 add-on therapy. Figure 1 Figure 1. Disclosures Rogers: AbbVie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; AstraZeneca: Consultancy; Pharmacyclics: Consultancy; Innate Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Janssen: Research Funding. Flinn: TG Therapeutics: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Gilead Sciences: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Yingli Pharmaceuticals: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; MorphoSys: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; BeiGene: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Karyopharm Therapeutics: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Constellation Pharmaceuticals: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; IGM Biosciences: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Forty Seven: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Agios: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Seagen: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Iksuda Therapeutics: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Juno Therapeutics: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Janssen: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Genentech: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Great Point Partners: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Verastem: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; AbbVie: Consultancy, Other: All Consultancy and Research Funding payments made to Sarah Cannon Research Institute, Research Funding; AstraZeneca: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Novartis: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Loxo: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Roche: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Kite, a Gilead Company: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Takeda: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Celgene: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Calithera Biosciences: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; ArQule: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Curis: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Unum Therapeutics: Consultancy, Other: All consultancy and research funding payments made to Sarah Cannon Research Institute, Research Funding; Forma Therapeutics: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Nurix Therapeutics: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Merck: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Teva: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Incyte: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Infinity Pharmaceuticals: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Acerta Pharma: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Trillium Therapeutics: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Pfizer: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Portola Pharmaceuticals: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Rhizen Pharmaceuticals: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Triphase Research & Development Corp.: Other: All research funding payments made to Sarah Cannon Research Institute, Research Funding; Century Therapeutics: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Hutchison MediPharma: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Vincerx Pharma: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Sarah Cannon Research Institute: Current Employment; Servier Pharmaceuticals: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Yingli Pharmaceuticals: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Seagen: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Servier Pharmaceuticals: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute; Unum Therapeutics: Consultancy, Other: All consultancy payments made to Sarah Cannon Research Institute, Research Funding; Johnson & Johnson: Current holder of individual stocks in a privately-held company; Seattle Genetics: Research Funding. Stephens: TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Adaptive: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Epizyme: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; JUNO: Research Funding; Arqule: Research Funding; AstraZeneca: Consultancy; Abbvie: Consultancy; Mingsight: Research Funding; CSL Behring: Consultancy; Celgene: Consultancy; Innate Pharma: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kipps: Oncternal Therapeutics, Inc.: Current equity holder in publicly-traded company, Patents & Royalties: Cirmtuzumab, Research Funding; Pharmacyclics/AbbiVie: Honoraria, Research Funding; Genentech/Roche: Honoraria; Celgene: Honoraria, Research Funding; VelosBio, Inc.: Research Funding; Janssen, Gilead, Dava Oncology,: Honoraria; Breast Cancer Research Foundation: Honoraria, Research Funding; Md Anderson Cancer Center: Research Funding; Gilead: Honoraria; National Cancer Institute/NIH: Honoraria, Research Funding; OncLive: Honoraria; Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS): Research Funding; California Institute for Regenerative Medicine (CIRM): Research Funding; European Research Initiative on CLL (ERIC): Honoraria; Dava Oncology: Honoraria; Patient Power, LLC: Honoraria; iwNHL: Honoraria; NCCN CLL/SLL Hairy Cell Leukemia Panel Meeting: Honoraria. Larson: TORL biotherapeutics: Current holder of individual stocks in a privately-held company; Abbvie: Research Funding; Bioline: Research Funding; BMS: Research Funding; Celgene: Research Funding; GSK: Research Funding; Janssen: Research Funding; Juno: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Takeda: Research Funding. McGarry: Novartis Institutes for Biomedical Research: Current Employment. Hassounah: Novartis Institutes for Biomedical Research: Current Employment, Patents & Royalties: unrelated . Gou: Novartis Pharma AG: Current Employment. Woo: Novartis: Current Employment, Current equity holder in publicly-traded company. Byrd: Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

Introduction: B cell activating factor receptor (BAFF-R) promotes the maturation and survival of normal and malignant B cells. VAY736 is a human investigational Fc-engineered afucosylated monoclonal antibody (mAb) targeting BAFF-R and enhancing NK-cell mediated antibody-dependent cellular cytotoxicity (ADCC). VAY736 demonstrated superior ADCC compared with anti-CD20 mAbs in the clinic and showed promising antileukemic activity in CLL preclinical models (McWilliams EM, et al. Blood Adv 2019;3:447-460). Although Bruton tyrosine kinase inhibitors such as ibrutinib and recently acalabrutinib have become the standard treatment for many patients (pts) with CLL, these agents require indefinite treatment and result in cumulative toxicity. Ibrutinib + VAY736 has synergy in preclinical studies prompting the hypothesis that this combination may deepen responses and allow pts to discontinue ibrutinib. Methods: This Phase Ib dose escalation/expansion trial (NCT03400176) of VAY736 + ibrutinib enrolled pts with R/R CLL who were receiving ibrutinib following relapse from another approved therapy and had either failed to achieve a CR after >1 y of ibrutinib treatment or had developed a mutation associated with resistance to ibrutinib. Pts received VAY736 IV at 0.3, 1, 3, or 9 mg/kg every 2 weeks + oral ibrutinib at 420 mg daily for 6 28-day cycles. After 6 cycles, pts with a CR discontinued VAY736 and received ibrutinib for an additional 2 cycles; a protocol amendment allowed all other pts to continue VAY736 + ibrutinib for an additional 2 cycles. Pts achieving CR and minimal residual disease (MRD)-negativity at Day 1 of Cycle 9 (C9D1) could discontinue ibrutinib at the investigator's discretion. The primary objective was to characterize the safety and tolerability of the combination and to determine the maximum tolerated dose (MTD)/recommended dose for expansion. Results: A total of 15 pts (median age: 65 years; ECOG PS 0: 93%) were treated by the data cutoff (June 9, 2020). Overall, 11 pts completed and 3 discontinued combination treatment (primarily due to disease progression); 1 pt remains on treatment. Most pts (73%) had an ibrutinib resistance mutation at baseline (mainly [82%] BTKC481) and 33% had received ≥4 prior regimens (median: 3, range: 1-5); median duration of prior ibrutinib was 4.1 y (range: 0.2-8.3). Baseline cytogenetics were (not mutually exclusive): 27% del(17)(p13.1), 80% unmutated IGHV, 80% stimulated complex karyotypes (≥3 abnormalities), 60% del(13)(q14), and 7% +12. No dose-limiting toxicities have been observed and the MTD has not been reached. A total of 14 (93%) pts experienced an AE regardless of cause. Four (27%) pts experienced AEs of grade ≥3, including decreased neutrophil count (n=3), hypophosphatemia (n=2), decreased white blood cell count, leukocytosis, increased lymphocyte count, hypertension, hypokalemia, and hypomagnesemia (n=1 each). The overall response at C9D1 was CR in 6 (40%) pts, SD in 4 (27%) pts, PD in 4 (27%) pts, and not assessed in 1 (7%) pt (still on treatment). The mean baseline CLL cells in bone marrow for the CR, SD, and PD groups were 27% (range: 0.8-60.6%), 13% (range: 2.5-27%), and 66% (range: 47-77.9%). Three (20%) pts with CR achieved MRD-negativity and were able to discontinue CLL-directed therapy including ibrutinib; they remained in CR for 1-16 months after ibrutinib discontinuation. The median percentage change from baseline in blood MRD was -92.8% (range: -100%; -16.7%; Figure 1) and in bone marrow MRD was -89.6% (range: -100%; -32.6%). Of the pts who had baseline ibrutinib-resistance mutations and C9D1 assessments, 1 pt (1/6) tested negative for ibrutinib-resistance mutations at C9D1. None of the pts who were ibrutinib-resistance mutation negative at baseline (4/4) developed mutations by C9D1. VAY736 concentration increased with dose, accumulated after repeated dosing in combination with ibrutinib, and achieved linear PK at 3 mg/kg or above. Tissue receptor occupancy was >99% for VAY736 doses of 3 mg/kg or above. Free BAFF was accumulated to steady state with no dose relationship. Conclusions: VAY736 + ibrutinib had an acceptable safety profile and demonstrated promising preliminary activity in pts with R/R CLL on ibrutinib, providing clinical evidence of a potential to discontinue ibrutinib by VAY736 add-on therapy. Further investigation of this combination including in pts on 1st line ibrutinib and other ibrutinib combinations is ongoing. Disclosures Rogers: AstraZeneca: Consultancy, Other: Travel Funding; Janssen: Research Funding; AbbVie: Consultancy, Research Funding; Genentech: Research Funding; Acerta Pharma: Consultancy; Pharmacyclics: Consultancy. Flinn:Juno Therapeutics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; MorphoSys: Consultancy, Research Funding; Curis: Research Funding; Nurix Therapeutics: Consultancy; BeiGene: Consultancy, Research Funding; Loxo: Research Funding; Calithera Biosciences: Research Funding; Celgene: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Vincera Pharma: Consultancy; Roche: Consultancy, Research Funding; Teva: Research Funding; TG Therapeutics: Consultancy, Research Funding; Trillium Therapeutics: Research Funding; Infinity Pharmaceuticals: Research Funding; Incyte: Research Funding; Takeda: Consultancy, Research Funding; IGM Biosciences: Research Funding; Johnson & Johnson: Other; Yingli Pharmaceuticals ≠: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Triphase Research & Development Corp.: Research Funding; Seattle Genetics: Consultancy, Research Funding; Portola Pharmaceuticals: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Constellation Pharmaceuticals: Research Funding; Merck: Research Funding; AstraZeneca: Consultancy, Research Funding; Karyopharm Therapeutics: Research Funding; ArQule: Research Funding; Unum Therapeutics: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; F. Hoffmann-La Roche: Research Funding; Curio Science: Consultancy; Acerta Pharma: Research Funding; Forma Therapeutics: Research Funding; Forty Seven: Research Funding; AbbVie: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding; Rhizen Pharmaceuticals: Research Funding; Agios: Research Funding; Genentech, Inc.: Research Funding; Great Point Partners: Consultancy; Iksuda Therapeutics: Consultancy. McGarry:Novartis: Current Employment. Gou:Novartis: Current Employment. Hassounah:Novartis: Current Employment. Woo:Novartis: Current Employment. Byrd:Leukemia and Lymphoma Society: Other; Trillium: Research Funding; Novartis: Research Funding; Kartos Therapeutics: Research Funding; Janssen: Consultancy; Pharmacyclics LLC, an AbbVie Company, Janssen, Novartis, Gilead, TG Therapeutics: Other; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, Novartis, Janssen: Speakers Bureau; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, BeiGene: Research Funding; Acerta Pharma: Research Funding; Syndax: Research Funding; Vincera: Research Funding.

Summary During development, intestinal epithelia undergo dramatic morphogenesis mediated by mesenchymal signaling to form villi, which are required for efficient nutrient absorption and host defense. Although both smooth-muscle-induced physical forces and mesenchymal cell clustering beneath emerging villi are implicated in epithelial folding, the underlying cellular mechanisms are unclear. Hedgehog (Hh) signaling can mediate both processes. We therefore analyzed its direct targetome and revealed GLI2 transcriptional activation of atypical cadherin and planar cell polarity (PCP) genes. By examining Fat4 and Dchs1 knockout mice, we demonstrate their critical roles in villus formation. Analyses of PCP-mutant mice and genetic interaction studies show that the Fat4-Dchs1 axis acts in parallel to the core-Vangl2 PCP axis to control mesenchymal cell clustering. Moreover, live light-sheet fluorescence microscopy and cultured PDGFRα+ cells reveal a requirement for PCP in their oriented cell migration guided by WNT5A. Therefore, mesenchymal PCP induced by Hh signaling drives cell clustering and subsequent epithelial remodeling.

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy characterized by persistent monocytosis, combined with myeloid proliferation and dysplasia. Here, we report HCT outcomes in 129 patients (pts) with CMML, 7-74 (median 55) years of age, with follow-up extending to 25 years. Patients were conditioned with various intensity regimens and transplanted from related (N=45) or unrelated donors (N= 84). The source of stem cells was marrow in 34, "mobilized" peripheral blood cells in 93, and cord blood in 2 patients. The HCT-CI was 0-11 (median 3). Somatic mutations present pre-HCT were analyzed with a mutation panel sequencing for 75 genes. Fig. 1A and 1B show overall survival by WHO disease criteria and the Spanish cytogenetic classification, respectively. Acute graft-versus-host disease (GVHD) grades II-IV occurred in 74% and chronic GVHD in 43%. Relapse incidence was 32%. Overall and progression-free survival at 10 years was 28% and 29%, respectively. Multivariate regression models showed the following: Blast count >20% at HCT (CMML-T: HR 1.67, CI 0.9-3.2, p=0.13, Fig. 1A), high-risk cytogenetics (HR 1.88, CI 1.2-3.0, p=0.01, Fig. 1B) and high HCT comorbidity index (HCT-CI≥4: HR 1.99, CI 1.2-3.4, p=0.01, Fig. 1 C) negatively impacted survival. There were 52% and 42% of patients who carried mutations in ASXL1 and TET2 and they were not significantly associated with inferior survival (ASXL1: HR=1.6, CI 0.3-7.6, p=0.54, TET2: HR=1.3, CI 0.4-3.9, p=0.63). Mutations in ATRX (HR=17.3, CI 4.1-73, p=0.0005) and WT1 (HR=6.3 CI 1.6-24, p=0.01), however, were associated with inferior survival. The total number of mutations (> 10 mutations, HR=3.5, CI 1.4-7.3, p=0.02) and more mutations in genes regulating epigenetic processes (HR 1.8, CI 1.1-2.8, p=0.01) were associated with relapse. The molecular CMML-specific prognostic scoring system (CPSS-Mol, Elena et al. 2016) was not correlated with relapse or survival. Unsupervised clustering of the correlation matrix revealed distinct associations between mutations and clinical features (Fig. 2), Group 1, proliferative type (mutations in mitotic signaling pathways associated with high WBC/blast counts and high risk disease by prognostic scoring systems), and Group 2, dysplastic type (mutations in genes regulating epigenetic processes including ATRX and WT), which was associated with unfavorable outcome. In summary, a proportion of patients with CMML achieve lasting remissions following HCT. However, relapse and overall mortality remain high. Molecular annotation uncovered distinct subgroups of CMML, and higher number of mutations, in particular, of epigenetic regulators, may confer unfavorable transplant outcomes. Incorporation of mutations in addition to cytogenetics and comorbidity scores should improve transplant prognostication.

Flow cytometry offers great diagnostic opportunities in the vast majority of hematologic and oncologic diseases with multiple cellular and molecular information within an individual cell. We will discuss various applications of flow cytometry, particularly in hematology and oncology, in addition to general principles and limitations of flow cytometry. They include nucleic acid analyses in cancer cells, new methods for assessing rare circulating tumor cells and disease-specific applications in malignancy with emphasis on diagnosis and treatment of hematologic malignancy, including minimal residual disease. With improvement of monoclonal antibodies, fluorescence and laser technology, flow cytometry now offers new avenues of assessing cellular functionality through examination of intracellular compartments. High-throughput quantitative analysis, advancements of in vivo flow cytometry and assessment of minimal residual diseases, as exampled in patient stratification and prediction of leukemia therapeutic response, will further make flow cytometry indispensable in medicine.

The randomized first-line trials, including the CRYSTAL trial, the OPUS trial, and the PRIME trial, have demonstrated the significant efficacy of cetuximab or panitumumab in patients with v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) wild-type tumors. The addition of an antiepidermal growth factor receptor (anti-EGFR)-directed monoclonal antibody to chemotherapy for these patients significantly improved progression-free survival, response rates, and R0 resection rates to a greater extent than overall survival compared with patients who received chemotherapy alone. However, 2 recent randomized phase 3 trials, the MRC COIN trial and the Nordic VII trial, reported an unexpected lack of benefit from the addition of cetuximab to chemotherapy in the first-line setting. In addition, recent retrospective analyses performed on a pooled data set from major clinical trials added more complexity, reporting an unexpected association of KRAS G13D mutation with a better clinical outcome compared with patients who had other KRAS mutations in the first-line and salvage settings, whereas the other independent analysis failed to demonstrate a benefit from panitumumab in patients with the same KRAS G13D mutation. The anti-EGFR monoclonal antibody-associated skin toxicity and the controversial strategies of management also are discussed. In this review, the authors analyze the previous randomized clinical trials and more critically re-evaluate recent trials and subgroup analyses to derive 3 factors that need to be taken into consideration regarding the addition of EGFR-directed monoclonal antibodies to chemotherapy: the preclinical data on mechanisms of action between chemotherapy and anti-EGFR antibodies along with mechanisms of resistance to anti-EGFR antibodies, the role of cross-over events in overall survival data, and the significant dose reductions of chemotherapeutic agents when combined with anti-EGFR agents. Cancer 2013;119:1941–1950. © 2013 American Cancer Society.

DNA methyltransferase 3A (DNMT3A) regulates diverse epigenetic processes, and DNMT3A mutations occur frequently in myelodysplastic syndromes (MDS), including in founding clones of MDS samples. Most DNMT3A mutations affect Arg882 (R882) in the catalytic domain of DNMT3A, and are found almost exclusively in a heterozygous state. To resolve the relationship between the genetic and epigenetic architectures of R882H+ MDS, we engineered primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) to carry heterozygous DNMT3A R882H and performed temporally resolved, genome-wide regulatory mapping via DNase-seq combined with RNA-seq during erythroid differentiation in vitro, and in an in vivo transplantation model. Compared with isogenic controls, heterozygous R882H HSPCs cells exhibited markedly impaired erythroid differentiation, accumulation of early myeloid progenitors, and diverse maturation defects. Transplantation of R882H HSPCs into W41 NSG mice revealed both impaired erythroid differentiation and preferential survival of mutant alleles in multiple hematopoietic lineages compatible with an early progenitor defect. Regulatory profiling of DNMT3A R882H heterozygous cells during differentiation via combined DNase- and RNA-seq revealed global and sequential alterations in the regulatory landscapes in mutant cells, most prominently decommissioning of thousands of regulatory regions normally found in primitive cells that mark gene loci destined for expression during later differentiation stages. Decommissioned regulatory elements in R882H heterozygotes were concentrated around genes involved in both regulation of erythropoiesis and cell-cycle control, biasing HSPC differentiation away from erythropoiesis. Similar findings were observed in CD34+-selected bone marrows from 33 patients with MDS, comparing heterozygous DNMT3A R882H and wild type. Collectively, our results indicate that DNMT3A R882H mutation reprograms early myeloid regulatory landscapes by preferentially targeting elements that control genes destined to be expressed at later stages of differentiation, resulting in a combined phenotype of impaired myeloid differentiation, impaired erythroid maturation, and preferential survival of R882H+ cells. The results provide novel mechanistic insights into the chromatin programming of erythroid differentiation and its connection with MDS. Disclosures No relevant conflicts of interest to declare.

While overexpression of several aquaporins (AQPs) has been reported in different types of human cancer, the role of AQPs in carcinogenesis has not been clearly defined. Here, by immunochemistry, we have found expression of AQP5 protein in 62.8% (59/94) of resected colon cancer tissue samples as well as association of AQP5 with liver metastasis. We then demonstrated that overexpression of human AQP5 (hAQP5) induces cell proliferation in colon cancer cells. Overexpression of wild-type hAQP5 increased proliferation and phosphorylation of extracellular signal-regulated kinase-1/2 in HCT116 colon cancer cells whereas these phenomena in hAQP5 mutants (N185D and S156A) were diminished, indicating that both membrane association and serine/threonine phosphorylation of AQP5 are required for proper function. Interestingly, overexpression of AQP1 and AQP3 showed no differences in extracellular signal-regulated kinase-1/2 phosphorylation, suggesting that AQP5, unlike AQP1, may be involved in signal transduction. Moreover, hAQP5-overexpressing cells showed an increase in retinoblastoma protein phosphorylation through the formation of a nuclear complex with cyclin D1 and CDK4. Small interfering RNA analysis confirmed that hAQP5 activates the Ras signaling pathway. These data not only describe the induction of hAQP5 expression during colorectal carcinogenesis but also provide a molecular mechanism for colon cancer development through the interaction of hAQP5 with the Ras/extracellular signal-regulated kinase/retinoblastoma protein signaling pathway, identifying hAQP5 as a novel therapeutic target.

Human aquaporin 5 (AQP5) has been shown to be overexpressed in multiple cancers, such as pancreatic cancer and colon cancer. Furthermore, it has been reported that ectopic expression of AQP5 leads to many phenotypic changes characteristic of transformation. However, the biochemical mechanism leading to transformation in AQP5-overexpressing cells has not been clearly elucidated. In this report, the overexpression of AQP5 in NIH3T3 cells demonstrated a significant effect on Ras activity and, thus, cell proliferation. Furthermore, this influence was shown to be mediated by phosphorylation of the PKA consensus site of AQP5. This is the first evidence demonstrating an association between AQP5 and a signaling pathway, namely the Ras signal transduction pathway, which may be the basis of the oncogenic properties seen in AQP-overexpressing cells.

Phosphorylation pathway has been identified as an important step in membrane trafficking for AQP5. We generated stably transfected BEAS-2B human bronchial epithelial cells with various over-expression constructs on permeable support. In stable cells with wild-type AQP5 and S156A (AQP5 mutant targeting PKA consensus sequence), AQP5 expression was predominantly polarized to the apical membrane, whereas stable cells with N185D (AQP5 mutant targeting second NPA motif), mainly localized to the cytoplasm. Treatment with H89 and/or chlorophenylthio-cAMP (cpt-cAMP) did not affect membrane expression of AQP5 in any of three stable cells. In cells with wild-type AQP5 and N185D, AQP5s were phosphorylated by PKA, while phosphorylation of AQP5 was not detected in cells with S156A. These results indicate that, in AQP5, serine156 may be phosphorylated by PKA, but membrane expression of AQP5 may not be regulated by PKA phosphorylation. We conclude that AQP5 membrane targeting can include more than one mechanism besides cAMP dependent phosphorylation.

N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2'-deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.

Gastroesophageal cancer is a significant global problem that frequently presents at an incurable stage and has very poor survival with standard chemotherapy approaches. This review will examine the epidemiology and molecular biology of gastroesophageal cancer and will focus on the key deregulated signaling pathways that have been targeted in the clinic. A comprehensive overview of clinical data highlighting successes and failures with targeted agents will be presented. Most notably, HER2-targeted therapy with the monoclonal antibody trastuzumab has proven beneficial in first-line therapy and has been incorporated into standard practice. Targeting the VEGF pathway has also proven beneficial, and the VEGFR-targeted monoclonal antibody ramucirumab is now approved for second-line therapy. In contrast to these positive results, agents targeting the EGFR and MET pathways have been evaluated extensively in gastroesophageal cancer but have repeatedly failed to show benefit. An increased understanding of the molecular predictors of response to targeted therapies is sorely needed. In the future, improved molecular pathology approaches should subdivide this heterogeneous disease entity to allow individualization of cancer therapy based on integrated and global identification of deregulated signaling pathways. Better patient selection, rational combinations of targeted therapies and incorporation of emerging immunotherapeutic approaches should further improve the treatment of this deadly disease.

Overexpression of several aquaporins has been reported in different types of human cancer but the role of AQPs in human carcinogenesis has not yet been clearly defined. Here, we demonstrate that ectopic expression of human AQP5 (hAQP5), a water channel expressed in lung, salivary glands, and kidney, induces many phenotypic changes characteristic of transformation both in vitro and in vivo. Furthermore, the cell proliferative ability of AQP5 appears to be dependent upon the phosphorylation of a cAMP-protein kinase (PKA) consensus site located in a cytoplasmic loop of AQP5. In addition, phosphorylation of the PKA consensus site was found to be phosphorylated preferentially in tumors. These findings altogether indicate that hAQP5 plays an important role in human carcinogenesis and, furthermore, provide an attractive therapeutic target.

The aquaporins (AQP) are water channel proteins playing a major role in transcellular and transepithelial water movement. Recently, the role of AQPs in human carcinogenesis has become an area of great interest. Here, by immunohistochemistry (IHC), we have found an expression of AQP5 protein in 35.3% (IHC-score: > or = 1, 144/408) of the resected NSCLC tissue samples. Cases with AQP5-positive status (IHC-score: > or = 2) displayed a higher rate of tumor recurrence than negative ones in NSCLC (54.7% vs. 35.1%, p = 0.005) and worse disease-free survival (p = 0.033; OR = 1.52; 95%CI: 1.04-2.23). Further in vitro invasion assay using BEAS-2B and NIH3T3 cells stably transfected with overexpression constructs for full length wild-type AQP5 (AQP5) and its two mutants, N185D which blocks membrane trafficking and S156A which blocks phosphorylation on Ser156, showed that AQP5 induced cell invasions while both mutants did not. In BEAS-2B cells, the expression of AQP5 caused a spindle-like and fibroblastic morphologic change and losses of cell-cell contacts and cell polarity. Only cells with AQP5, not either of two mutants, exhibited a loss of epithelial cell markers and a gain of mesenchymal cell markers. In a human SH3-domains protein array, cellular extracts from BEAS-2B with AQP5 showed a robust binding activity to SH3-domains of the c-Src, Lyn, and Grap2 C-terminal. Furthermore, in immunoprecipitation assay, activated c-Src, phosphorylated on Tyr416, showed a stronger binding activity to cellular extracts from BEAS-2B with AQP5 compared with N185D or S156A mutant. Fluorescence in situ hybridization (FISH) analysis failed to show evidence of genomic amplification, suggesting AQP5 expression as a secondary event. Based on these clinical and molecular observations, we conclude that AQP5, through its phosphorylation on Ser156 and subsequent interaction with c-Src, plays an important role in NSCLC invasion and, therefore, may provide a unique opportunity for developing a novel therapeutic target as well as a prognostic marker in NSCLC.

Affiliation 1 is incorrect. It should be: Department of Otolaryngology - Head and Neck Surgery, Johns Hopkins University, Baltimore, Maryland, United States of America

Gastroesophageal cancer is a significant global problem that frequently presents at an incurable stage and has very poor survival with standard chemotherapy approaches. This review will examine the epidemiology and molecular biology of gastroesophageal cancer and will focus on the key deregulated signaling pathways that have been targeted in the clinic. A comprehensive overview of clinical data highlighting successes and failures with targeted agents will be presented. Most notably, HER2-targeted therapy with the monoclonal antibody trastuzumab has proven beneficial in first-line therapy and has been incorporated into standard practice. Targeting the VEGF pathway has also proven beneficial, and the VEGFR-targeted monoclonal antibody ramucirumab is now approved for second-line therapy. In contrast to these positive results, agents targeting the EGFR and MET pathways have been evaluated extensively in gastroesophageal cancer but have repeatedly failed to show benefit. An increased understanding of the molecular predictors of response to targeted therapies is sorely needed. In the future, improved molecular pathology approaches should subdivide this heterogeneous disease entity to allow individualization of cancer therapy based on integrated and global identification of deregulated signaling pathways. Better patient selection, rational combinations of targeted therapies and incorporation of emerging immunotherapeutic approaches should further improve the treatment of this deadly disease.

The aquaporins represent a family of transmembrane water channel proteins that play a major role in trans-cellular and transepithelial water movement. Most tumors have been shown to exhibit high vascular permeability and interstitial fluid pressure, but the transport pathways for water within tumors remain unknown. Here, we tested 10 non-small cell lung cancer cell lines of various origins by reverse transcriptase-polymerase chain reaction and Western blot analysis and identified clear expression of aquaporin 1 (AQP1) in seven cell lines. We next examined the distribution of the AQP1 protein in several types of primary lung tumors (16 squamous cell carcinomas, 21 adenocarcinomas, and 7 bronchoalveolar carcinomas) by immunohistochemical staining. AQP1 was overexpressed in 62% (13 of 21) and 75% (6 of 8) of adenocarcinoma and bronchoalveolar carcinoma, respectively, whereas all cases of squamous cell carcinoma and normal lung tissue were negative. Forced expression of full-length AQP1 cDNA in NIH-3T3 cells induced many phenotypic changes characteristic of transformation, including cell proliferation-enhancing activity by the MTT assay and anchorage-independent growth in soft agar. Although further details on the molecular function of AQP1 related to tumorigenesis remain to be elucidated, our results suggest a potential role of AQP1 as a novel therapeutic target for the management of lung cancer.

Matrix metalloproteinases (MMPs) have been studied historically for their role in tissue invasion and metastasis. The tumor microenvironment also produces MMPs that may influence other tumor behaviors, as recent mouse models suggest in the intestine and other sites. We examined MMP9 expression in 642 cases of human colorectal cancer (CRC) in relation to clinicopathological features and mutations. MMP9 antibody (Ab) unambiguously stained mesenchymal cells in 104 cases (16.2%) and gave weaker stromal staining in another 12.8%. High stromal MMP9 expression correlated most strongly with the CpG island methylator phenotype (CIMP-high) in tumors (multivariate odds ratio – OR – 2.45, 95% confidence interval – CI – 1.41 to 4.27, p=0.0015) and also with absence of tumor KRAS mutations (multivariate OR 0.54, 95% CI 0.33 to 0.87, p=0.011). The latter association suggested KRAS allelic status as one reason for heterogeneity in CRC cell line responses to MMP9. Four out of 6 KRASWT cell lines proliferated significantly more in the presence of exogenous MMP9, while 4 of 5 KRASMut lines failed to respond. This led us to ask if stromal MMP9 expression might predict the outcome of treatment with anti-EGFR mAb cetuximab, allowing KRASWT tumors to bypass mAb blockade of EGFR-KRAS pathway activation. We retrospectively evaluated MMP9 expression by immunohistochemistry on primary tumors and progression-free survival (PFS) in 86 patients who had received chemotherapy combined with cetuximab as first-line or salvage therapy. Tumors from 29 patients (33.8%) harbored mutant KRAS. PFS of patients with MMP9high;KRASWT tumors (median 3.5 months) was lower than that of patients with low or no stromal MMP9 expression and KRASWT tumors (median 5.8 months, 95% CI for the hazard ratio 1.135-2.179). Patients with KRASMut tumors showed no significant difference in PFS with respect to MMP9 expression (2.55 months and 2.25 months in the MMP9high and MMP9neg/low subgroups, respectively). Thus, low stromal MMP9 expression in KRASWT tumors predicted a better outcome with cetuximab treatment than high MMP9 expression. In CRC cell lines that grow faster in response to MMP9, this factor activates ERBB2 and ERK/JUN signaling. Together, these findings suggest that stroma-derived MMP9 may help tumors bypass common mutational mechanisms for constitutive growth factor pathway activation and confer resistance to anti-EGFR therapy through cell-extrinsic activation of the ERBB2/ERK/JUN pathway. Stromal MMP9 expression may thus predict cetuximab response in KRASWT CRCs and have value in selecting patients for treatment. This proffered talk is also presented as Poster A26.

Cancer cells communicate extensively with their stroma, exchanging signals that influence growth and metastasis. Intestinal sub-epithelial myofibroblasts (ISEMFs) direct organogenesis and constitute the colorectal cancer (CRC) microenvironment. ISEMFs from the mouse small intestine and colon express regional characteristics and different cytokines. In line with these regional patterns, ApcMin mice develop adenomas principally in the small intestine but not in the colon; conversely, humans develop tumors mainly in the colon and rarely in the small bowel. We hypothesize that intestinal ISEMFs influence tumor regionality through cell-cell communication. Murine ileal ISEMFs supported growth of human CRC cells markedly better than those originating in the colon in NOD/SCID xenografts and in vitro. We traced this effect to a single secreted factor, matrix metalloproteinase 9 (MMP9), which is exclusively mesenchymal in expression and substantially enriched in native ileal over colonic ISEMFs. Actively cycling intestinal epithelial progenitors and Lgr5+ stem cells showed selectively reduced bromodeoxyuridine uptake in Mmp9-/- mouse ileum, revealing that Mmp9 deficiency impairs epithelial self-renewal. Moreover, ApcMin mice developed fewer ileal adenomas upon treatment with an Mmp9 inhibitor or on the Mmp9-/- genetic background. MMP9 immunohistochemistry revealed expression in 106 of 642 (16.5%) human CRC samples; expression was confined to stromal fibroblasts and considerably enriched among CRCs that lack characteristic KRAS mutations and harbor the CpG island methylator phenotype. Gene expression in epithelial cells interacting with ileal fibroblasts pointed to activation of cytoskeletal remodeling and integrin ß4 signaling pathways, signatures absent in cells cultured with colonic ISEMFs. Biochemical assays verified that stromal MMP9 activates epithelial integrin ß4 and, in turn, ErbB and c-Met receptor tyrosine kinases (RTKs) in adjoining epithelial, including human CRC, cells. This activation is mediated by MMP9 cleavage of the integrin ligand laminin alpha4. Activation of epithelial integrin ß4 and RTKs and the paucity of KRAS mutations in CRCs with stromal MMP9 expression together suggest that potent MMP9-mediated microenvironment signals can help bypass common mutational mechanisms of constitutive growth factor pathway activation in CRC. These results define a novel and therapeutically tractable pathway of tumor-stromal interaction in early CRC pathogenesis. 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 3106. doi:10.1158/1538-7445.AM2011-3106

Aquaporins (AQPs) have previously been associated with increased expression in solid tumors. However, its expression in hematologic malignancies including CML has not been described yet. Here, we report the expression of AQP5 in CML cells by RT-PCR and immunohistochemistry. While normal bone marrow biopsy samples (n = 5) showed no expression of AQP5, 32% of CML patient samples (n = 41) demonstrated AQP5 expression. In addition, AQP5 expression level increased with the emergence of imatinib mesylate resistance in paired samples (p = 0.047). We have found that the overexpression of AQP5 in K562 cells resulted in increased cell proliferation. In addition, small interfering RNA (siRNA) targeting AQP5 reduced the cell proliferation rate in both K562 and LAMA84 CML cells. Moreover, by immunoblotting and flow cytometry, we show that phosphorylation of BCR-ABL1 is increased in AQP5-overexpressing CML cells and decreased in AQP5 siRNA-treated CML cells. Interestingly, caspase9 activity increased in AQP5 siRNA-treated cells. Finally, FISH showed no evidence of AQP5 gene amplification in CML from bone marrow. In summary, we report for the first time that AQP5 is overexpressed in CML cells and plays a role in promoting cell proliferation and inhibiting apoptosis. Furthermore, our findings may provide the basis for a novel CML therapy targeting AQP5.