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Coronavirus disease (COVID-19), which is caused by the SARS-CoV-2 virus, has become a global pandemic. While susceptibility to COVID-19 is subject to several external factors, including hypertension, BMI, and the presence of diabetes, it is also genetically determined to a significant extent. Infectious agents require iron (Fe) for proper functioning. Carriers of mutations resulting in increased iron concentrations are understood to be at increased risk of COVID-19.We examined HFE genotypes associated with hereditary haemochromatosis (rs1800562 and rs1799945 SNPs) in 617 COVID-19 patients (166 asymptomatic, 246 symptomatic and 205 hospitalised survivors) and 2 559 population-based controls.We found a higher frequency of the minor allele (Tyr282) of the rs1800562 polymorphism (P0.002) in patients compared to controls (8.5% vs. 5.5%). Non-carriers of the minor allele were protected against SARS-Cov-2 infection (OR, 95%CI; 0.59, 0.42 - 0.82). The frequency of minor allele carriers was almost identical across asymptomatic, symptomatic, and hospitalised survivors. The rs1799945 variant did not affect disease severity and its occurrence was almost identical in patients and controls (P between 0.58 and 0.84).In conclusion, our results indicate that presence of the rs1800562 minor allele, which is associated with hereditary haemochromatosis (thus increased levels of plasma Fe), increases susceptibility to SARS-CoV-2.

Lucitanib is a multi-tyrosine kinase inhibitor whose targets are associated with angiogenesis and other key cancer and immune pathways. Its antiangiogenic properties are understood, but lucitanib's immunomodulatory activity is heretofore unknown. Lucitanib exhibited such activity in vivo, increasing CD3 + , CD8 + , and CD4 + T cells and decreasing dendritic cells and monocyte-derived suppressor cells in mouse spleens. Depletion of CD8 + T cells from syngeneic MC38 colon tumor-bearing mice reduced the antitumor efficacy of lucitanib and revealed a CD8 + T-cell-dependent component of lucitanib's activity. The combination of lucitanib and costimulatory immune pathway agonists targeting 4-1BB, glucocorticoid-induced TNFR (GITR), inducible T-cell co-stimulator (ICOS), or OX40 exhibited enhanced antitumor activity compared with each single agent in immunocompetent tumor models. Lucitanib combined with blockade of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein-1 (PD-1) coinhibitory immune pathways also showed enhanced antitumor activity over the single agents in multiple models. In CT26 tumors, lucitanib, alone or combined with anti-PD-1, reduced CD31 + vessels and depleted F4/80 + macrophages. Combination treatment also increased the number of intratumoral T cells. Gene expression in pathways associated with immune activity was upregulated by lucitanib in MC38 tumors and further potentiated by combination with anti-PD-1. Accordingly, lucitanib, alone or combined with anti-PD-1, increased intratumoral CD8 + T-cell abundance. Lucitanib's antitumor and pharmacodynamic activity, alone or combined with anti-PD-1, was not recapitulated by specific vascular endothelial growth factor receptor-2 (VEGFR2) inhibition. These data indicate that lucitanib can modulate vascular and immune components of the tumor microenvironment and cooperate with immunotherapy to enhance antitumor efficacy. They support the clinical development of lucitanib combined with immune pathway modulators to treat cancer.

Supplementary Table S1. Commercial tumor TMAs representing different solid tumor types. Supplementary Table S2. Summary of CD205 expression in human solid tumor types.

Supplementary Figure S1. Representative images of IHC staining for CD205 in different human and cynomolgus tissues, obtained with 2 μg/ml of MEN1309/OBT076 at 20X and 10X magnification. 1) Human donor spleen; moderate to strong membranous/granular cytoplasmic staining of several mononuclear cells present in the white pulp of the spleen (circle); 2) Cynomolgus donor spleen; the staining intensity and distribution pattern was very similar to that described in human samples; 3) Human donor lymph node; a moderate to marked membranous/cytoplasmic staining of mononuclear cells in the cortex, para-cortex and germinal centers of lymphoid follicles was observed; 4) Cynomolgus donor lymph node; positive mononuclear cells presented a moderate to strong membranous/granular cytoplasmic staining; 5) Human donor breast; a concentration-dependent cytoplasmic staining of epithelial cells in glandular alveoli (rectangle) and ducts (circle); 6) Cynomolgus donor breast; the staining was weaker in intensity when compared to human donors, but the pattern was comparable; 7) Human donor colon; membranous/cytoplasmic signal of a variable number of mononuclear cells was observed in the lamina propria and GALT (circle). Minimal to slight cytoplasmic staining of the superficial mucosal epithelium (rectangle); 8) Cynomolgus donor colon; a variable number of mononuclear cells located in lamina propria and GALT (circle) are positive with membranous/cytoplasmic signal; 9) Human donor ureter; a minimal but concentration-dependent staining was observed in mucosal epithelium (rectangle). Rare mononuclear cells with positive signal in lamina propria (circles); 10) Cynomolgus donor ureter; occasional mononuclear cells with membranous/granular cytoplasmic staining was observed in the lamina propria; 11) Human donor cervix; a concentration-dependent cytoplasmic staining of cervical gland epithelium was observed (circle). Cytoplasmic staining of the basal cell layer of mucosal epithelium was also observed (rectangle); 12) Cynomolgus donor cervix; positive mononuclear cells with a membranous/granular cytoplasmic staining were present in the mucosa-associated lymphoid nodules (circle).

Supplementary Table S5: Microscopic IHC staining for CD205 in different cynomolgus tissues after treatment with MEN1309/OBT076. Staining: 1, minimal (+, rare cells with low staining intensity); 2, slight/moderate (++, many cells with medium/high staining intensity); 3, marked (+++, significant numbers of cells with high staining intensity); 0, negative (no stained cells); -, no background present; tm, tissue missing; lp, lesion present; p, background staining present; N/A, not applicable.

Supplementary Figure S3-A. Representative image of IHC staining for CD205 in a tumor mass from HCC-70 xenograft model, 16 days far from the initial treatment with 5 mg/kg of MEN1309/OBT076. The semiquantitative IHC analysis showed a score of 1+ in 30% of cells while the remaining were scored negative (0). Supplementary Figure S3-B. Representative image of IHC staining for CD205 in a tumor mass from PDX-347 model, 16 days far from the initial treatment with 5 mg/kg of MEN1309/OBT076. The semiquantitative IHC analysis showed a score of 2+ in 100% of cells. Red and green lines represent tumor and fibrotic areas.

CD205 is a type I transmembrane glycoprotein and is a member of the C-type lectin receptor family. Analysis by mass spectrometry revealed that CD205 was robustly expressed and highly prevalent in a variety of solid malignancies from different histotypes. IHC confirmed the increased expression of CD205 in pancreatic, bladder, and triple-negative breast cancer (TNBC) compared with that in the corresponding normal tissues. Using immunofluorescence microscopy, rapid internalization of the CD205 antigen was observed. These results supported the development of MEN1309/OBT076, a fully humanized CD205-targeting mAb conjugated to DM4, a potent maytansinoid derivate, via a cleavable N-succinimidyl-4-(2-pyridyldithio) butanoate linker. MEN1309/OBT076 was characterized in vitro for target binding affinity, mechanism of action, and cytotoxic activity against a panel of cancer cell lines. MEN1309/OBT076 displayed selective and potent cytotoxic effects against tumor cells exhibiting strong and low to moderate CD205 expression. In vivo, MEN1309/OBT076 showed potent antitumor activity resulting in durable responses and complete tumor regressions in many TNBC, pancreatic, and bladder cancer cell line–derived and patient-derived xenograft models, independent of antigen expression levels. Finally, the pharmacokinetics and pharmacodynamic profile of MEN1309/OBT076 was characterized in pancreatic tumor–bearing mice, demonstrating that the serum level of antibody–drug conjugate (ADC) achieved through dosing was consistent with the kinetics of its antitumor activity. Overall, our data demonstrate that MEN1309/OBT076 is a novel and selective ADC with potent activity against CD205-positive tumors. These data supported the clinical development of MEN1309/OBT076, and further evaluation of this ADC is currently ongoing in the first-in-human SHUTTLE clinical trial.

Supplementary Materials and Methods

Background Colorectal cancer (CRC) is among the most common cancers and cancer causes of death worldwide. CRC screening and early detection is essential to reduce CRC incidence and mortality. CRC screening has been initiated in the Czech Republic in 2000 for persons over 50 and currently offers a faecal occult blood test (FOBT) or screening colonoscopy (CS). The aim of our study was to present complete coverage by examinations in relation to the trends in CRC burden and impact of COVID-19. Methods We defined the complete coverage by examinations as the proportion of persons aged over 50 undergoing examination with CRC early detection potential (FOBT or CS for any indication) during past 3 years. Standardized incidence and mortality rates were used to assess epidemiological trends. The impact of COVID-19 was assessed for 2020 and 2021 by comparing the volume of examinations with 2019. We used national health registries (National Registry of Reimbursed Health Services, Czech National Cancer Registry) as the source of data. Results Complete coverage was increasing over time and reached around 50% in recent years (target population is more than 4 million persons, most of the performed examinations were screening FOBT). However, coverage has decreased to 47.9% in 2020. In 2020 and 2021, the number of tests performed decreased by 16.9% and 5.5%, respectively, compared to 2019. CRC incidence and mortality rates have decreased by more than 20% and almost 30%, respectively, in the last decade. Conclusions Complete coverage has reached a satisfactory level and has likely a positive impact on the epidemiological trends. However, further action is needed to increase coverage, recently affected by COVID-19 pandemic, when non-acute health care may have been neglected. Key messages • The long-term high level of coverage by examinations likely has a positive impact on CRC burden. • The observed decrease in coverage caused by COVID-19 needs to be appropriately compensated.

CD205 is a type I transmembrane glycoprotein and is a member of the C-type lectin receptor family. Analysis by mass spectrometry revealed that CD205 was robustly expressed and highly prevalent in a variety of solid malignancies from different histotypes. IHC confirmed the increased expression of CD205 in pancreatic, bladder, and triple-negative breast cancer (TNBC) compared with that in the corresponding normal tissues. Using immunofluorescence microscopy, rapid internalization of the CD205 antigen was observed. These results supported the development of MEN1309/OBT076, a fully humanized CD205-targeting mAb conjugated to DM4, a potent maytansinoid derivate, via a cleavable N-succinimidyl-4-(2-pyridyldithio) butanoate linker. MEN1309/OBT076 was characterized in vitro for target binding affinity, mechanism of action, and cytotoxic activity against a panel of cancer cell lines. MEN1309/OBT076 displayed selective and potent cytotoxic effects against tumor cells exhibiting strong and low to moderate CD205 expression. In vivo, MEN1309/OBT076 showed potent antitumor activity resulting in durable responses and complete tumor regressions in many TNBC, pancreatic, and bladder cancer cell line–derived and patient-derived xenograft models, independent of antigen expression levels. Finally, the pharmacokinetics and pharmacodynamic profile of MEN1309/OBT076 was characterized in pancreatic tumor–bearing mice, demonstrating that the serum level of antibody–drug conjugate (ADC) achieved through dosing was consistent with the kinetics of its antitumor activity. Overall, our data demonstrate that MEN1309/OBT076 is a novel and selective ADC with potent activity against CD205-positive tumors. These data supported the clinical development of MEN1309/OBT076, and further evaluation of this ADC is currently ongoing in the first-in-human SHUTTLE clinical trial.

Additional file 1: Supplementary Table S1. Most Frequent (≥10% of Patients) Treatment-Related Adverse Events of Any Grade in the Safety Population. Supplementary Table S2. Summary of the Genetic Alterations in Tumor Tissue Samples and Tumor Responses in Patients With DDR Gene Mutation. Supplementary Table S3. Comparison of Genomic Characteristics From Archival and Recently Acquired Tumor Samples. Supplementary Figure S1. Time Profile for Mean (± Standard Deviation) Trough Plasma Concentration of Rucaparib

Background Reduction of risky drinking in women of childbearing age is one strategy that may be employed to prevent fetal alcohol spectrum disorder, a sequela of prenatal alcohol-exposure. Communities differ in risk and protective factors, necessitating culturally informed interventions for maximal efficacy. This manuscript describes the modification of an existing web-based SBIRT intervention to reduce risky drinking among AIAN women of child-bearing age in Southern California into a peer-to-peer based intervention using motivational interviewing. Methods The modification process was iterative and included various community focus groups, interviews, and a final review. Results Intervention modification was required for cultural congruence. Components of the peer-to-peer intervention designed by this project included a flip chart used to guide the motivational interviewing, charts of the financial and physical costs of alcohol consumption, revised baseline and follow-up questionnaires, and guidance regarding the application of motivational interviewing techniques. Conclusions This study may inform the modification of future interventions among AIAN communities. This article is protected by copyright. All rights reserved.

// Christina Krupka 1, 2, 3 , Felix S. Lichtenegger 1, 2, 3 , Thomas Kohnke 1, 2, 3 , Jan Bogeholz 4 , Veit Bucklein 1, 2, 3 , Michael Roiss 1, 2, 3 , Torben Altmann 1, 2, 3 , To Uyen Do 5 , Rachel Dusek 5 , Keith Wilson 5, * , Arnima Bisht 5 , Jon Terrett 6, * , Dee Aud 6, * , Esteban Pombo-Villar 5, * , Christian Rohlff 5 , Wolfgang Hiddemann 1 , Marion Subklewe 1, 2, 3 1 Department of Internal Medicine III, Klinikum of The LMU Munich, Munich, Germany 2 Clinical Cooperation Group Immunotherapy at The Helmholtz Institute Munich, Munich, Germany 3 Laboratory of Translational Cancer Immunology, Gene Center Munich, Ludwig-Maximilians-University, Munich, Germany 4 Department of Hematology, University Hospital Zurich, Zurich, Switzerland 5 Independent consultant Oxford BioTherapeutics Ltd, Abingdon, United Kingdom and San Jose, CA, USA 6 CRISPR Therapeutics, Cambridge, MA, USA * Were employed at Oxford Biotherapeutics at the time the data was generated Correspondence to: Subklewe Marion, email: Marion.Subklewe@med.uni-muenchen.de Keywords: AML, Immunotherapy, CD157, Antibody, Fc-engineering Received: November 01, 2016 Accepted: February 27, 2017 Published: March 09, 2017 ABSTRACT Antibody-based immunotherapy represents a promising strategy to eliminate chemorefractory leukemic cells in acute myeloid leukemia (AML). In this study, we evaluated a novel Fc-engineered antibody against CD157 (MEN1112) for its suitability as immunotherapy in AML. CD157 was expressed in 97% of primary AML patient samples. A significant, albeit lower expression level of CD157 was observed within the compartment of leukemia-initiating cells, which are supposed to be the major source of relapse. In healthy donor bone marrow, CD157 was expressed on CD34 + cells. In ex vivo assays, MEN1112 triggered natural killer (NK) cell-mediated cytotoxicity against AML cell lines and primary AML cells. Compared to its parental analogue, the Fc-engineered antibody exhibited higher antibody dependent cellular cytotoxicity responses. Using NK cells from AML patients, we observed heterogeneous MEN1112-mediated cytotoxicity against AML cells, most likely due to well-documented defects in AML-NK cells and corresponding inter-patient variations in NK cell function. Cytotoxicity could not be correlated to the time after completion of chemotherapy. In summary, we could demonstrate that CD157 is strongly expressed in AML. MEN1112 is a promising antibody construct that showed high cytotoxicity against AML cells and warrants further clinical testing. Due to variability in NK-cell function of AML patients, the time of application during the course of the disease as well as combinatorial strategies might influence treatment results.

ALKS 4230, an engineered cytokine designed for selective binding to the intermediate affinity interleukin-2 receptor (IL-2R), is being evaluated as a monotherapy and in combination with pembrolizumab in patients with advanced solid tumors. The selectivity of ALKS 4230 is achieved through stable fusion of circularly permuted IL-2 to IL-2Rα, with the goal of driving antitumor responses via selective activation of CD8+ T cells and NK cells, while avoiding activation of regulatory T cells (Tregs), which express the high affinity IL-2R. High levels of vascular endothelial growth factor (VEGF) are associated with an immunosuppressive tumor microenvironment and diminished response to high-dose IL-2. Lucitanib is an anti-angiogenic, multi-tyrosine kinase inhibitor targeting VEGFR1-3, PDGFRα/β, and FGFR1-3. We evaluated the antitumor efficacy and mechanism of the combination of RDB 1462, the mouse ortholog of ALKS 4230, and lucitanib in the MC38 mouse syngeneic tumor model. C57BL/6 mice were subcutaneously implanted with MC38 cells and treated with RDB 1462 (1.5 mg/kg and 3 mg/kg once every 3 days for 3 weeks), lucitanib (10 mg/kg daily for 28 days), or the combination of both molecules. After 11 days of treatment, lucitanib monotherapy resulted in 100% tumor growth inhibition (TGI), while 1.5 mg/kg and 3 mg/kg RDB 1462 monotherapy resulted in 25% and 51% TGI, respectively. Lucitanib monotherapy extended survival, with a mean survival time (MST) of 39 days compared to 15 days for vehicle, with no complete tumor regressions. MSTs were 18-22 days for RDB 1462 monotherapy groups, 60 days for the lower dose combination arm, and >60 days for the higher dose combination. Durable complete tumor regressions were observed in 1/20 mice treated with either dose of RDB 1462 monotherapy and 5/10 mice treated with the lower dose of RDB 1462 in combination with lucitanib. 10/10 mice treated with the higher dose of RDB 1462 in combination with lucitanib exhibited complete responses on Day 60. Flow cytometric analyses revealed that RDB 1462 treatment induced significant peripheral CD8+ T cell expansion compared to vehicle (p The combination of an intermediate affinity IL-2R-selective cytokine and an angiogenesis inhibitor resulted in durable dose-dependent antitumor efficacy in the MC38 mouse tumor model. Citation Format: Jared E. Lopes, Rachel L. Dusek, Liliane Robillard, Minh Nguyen, Bruce Roth, Bryan Vought, Arthur Liberzon, Raymond J. Winquist, Heather C. Losey. The combination of a mouse ortholog of ALKS 4230, a selective agonist of the intermediate affinity IL-2 receptor, and the angiogenesis inhibitor lucitanib enhances antitumor activity [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 2202.

The preoperative assessment of structural and functional changes in renal tumors using contrast-enhanced pulse inversion harmonic imaging (CEUS) and contrast-enhanced computed tomography (CECT).All consecutive patients referred to two tertiary hospitals for surgery on suspicion of a malignant renal lesion, who had been examined under the predefined study protocol using CEUS and CECT, were prospectively included in the study. All renal lesions suspected of being malignant were subjected to histopathological examination. Lesions expected to be benign were followed up according to the study protocol. The accuracy of CEUS and CECT with the final histology or follow-up results and the statistically significant difference between the two imaging techniques was calculated.Over a period of 3 years (2008-2011), 68 of 93 patients examined met the study criteria. The prevalence of malignant tumors in the study was 72%. Fifty four (79%) patients underwent surgery and had a histologically confirmed renal tumor (clear cell carcinoma 45, urothelial papillocarcinoma 4, angiomyolipoma 1, oncytoma 3, xanthogranulomatous pyelonephritis 1) and 14 (21%) patients underwent regular follow-up. Specificity, sensitivity and area under the curve (AUC) reached 57.9%, 98% and 0.779 for CEUS and 52.6%, 98% and 0.753 for CECT.The results show that both imaging methods can reliably rule out malignant disease due to absence of enhancement. Taking into consideration that CEUS can be carried out without severe risk or discomfort, it is time to reconsider CEUS as the method of choice for diagnosis, while CECT should be reserved for staging.

Find fulltext at Masaryk University(opens in a new window)|View at Publisher| Export | Download | More... Klinicka Onkologie Volume 28, Issue 1, 2015, Pages 30-43 Estimating cancer incidence, prevalence, and the number of cancer patients treated with anti-tumor therapy in 2015 and 2020 analysis of the Czech national cancer registry (Article) Dusek, L.ab , Pavlik, T.ab, Majek, O.ab, Buchler, T.c, Muzik, J.ab, Maluskova, D.ab, Koptikova, J.ab, Bortlicek, Z.ab, Abrahamova, J.c a Institute of Biostatistics and Analyses, Faculty of Medicine and Faculty of Science, Masaryk University, Kamenice 126/3, Brno, Czech Republic b Institute of Health Information and Statistics of the Czech Republic, Prague, Czech Republic c Department of Oncology, Thomayer Hospital, Prague, Czech Republic View references (23) Abstract Background: Cancer burden in the Czech population ranks among the highest worldwide, which introduces a strong need for a prospective modelling of cancer incidence and prevalence rates. Moreover, a prediction of number of cancer patients requiring active anti-tumor therapy is also an important issue. This paper presents the stage-specific predictions of cancer incidence and prevalence, and the stage- and region-specific patients requiring active anti-tumor therapy for the most common cancer diagnoses in the Czech Republic for years 2015 and 2020. The stage-specific estimates are also presented with regard to the treatment phase as newly diagnosed patients, patients treated for non-terminal recurrence, and patients treated for terminal recurrence. Patients and Methods: Data of the Czech National Cancer Registry from 1977 to 2011 has been used for the analysis, omitting the records of patients diagnosed as death certificate only or at autopsy. In total, 1,777,775 incidences have been considered for the estimation using a statistical model utilizing solely the population-based cancer registry data. All estimates have been calculated with respect to the changing demographic structure of the Czech population and the clinical stage at diagnosis. Results: Considering year 2011 as the baseline, we predict 89%, 15%, 31% and 32% increase in prostate, colorectal, female breast and lung cancer incidence, respectively, in 2020 resulting in 13,153,9,368,8,695, and 8,604 newly diagnosed cancer patients in that year, respectively. Regarding cancer prevalence in 2020, the estimated increase is 140%, 40%, 51 %, and 17% for prostate, colorectal, female breast and lung cancer, respectively, meaning that more than 100,000 prevalent female breast cancer patients as well as more than 100,000 prevalent prostate cancer patients are expected in the Czech Republic The estimated numbers of patients requiring active anti-tumor therapy for prostate, colorectal, female breast and lung cancer in the Czech Republic in 2020 are 23,652,14,006,14,759 and 8,272; respectively. Conclusions: The analysis documents a serious increase in cancer incidence and prevalence in the Czech Republic in years 2015 and 2020 when compared to the situation in 2011. Regarding the estimated numbers of patients requiring active anti-tumor therapy, the model confirms a continuous increase that must be accounted for in the future planning of health care in the Czech Republic.

440 Background: ATLAS (NCT03397394) evaluated the efficacy/safety of the PARP inhibitor (PARPi) rucaparib in patients (pts) with previously treated locally advanced/unresectable UC or mUC. Methods: Pts with measurable disease who had progressed after 1–2 prior regimens (ie, platinum-based chemotherapy [PBC] and/or immune checkpoint inhibitors [ICI]) were enrolled regardless of tumor homologous recombination deficiency (HRD) status. Prior PARPi was not allowed. Pts received rucaparib 600 mg PO BID. Baseline tumor tissue or archival tissue ≤6 mo without intervening therapy was required; serial circulating tumor DNA samples were collected. Primary endpoint was investigator-assessed objective response rate (RECIST v1.1) in the intent-to-treat and HRD-positive (defined as genomic loss of heterozygosity ≥10%) populations. Key secondary endpoints: progression-free survival (PFS) and safety. Clinical benefit rate (CBR) was defined as complete or partial response or stable disease (SD) lasting ≥16 weeks. Results: As of Oct 7, 2019, 97 pts were enrolled (median age 66 y [range, 39–87]); most were men (n=76, 78.4%) and had ECOG PS 1 (n=65, 67.0%). Sixty-six pts (68.0%) had both prior PBC and ICI. Twenty pts (20.6%) were HRD-positive, 30 (30.9%) were HRD-negative and 47 (48.5%) had unknown HRD status; 4 pts had a deleterious BRCA1/2 alteration. Median time on treatment was 54 d (range, 2–224). There were no confirmed responses. Of 96 evaluable pts, 27 (28.1%) had a best response of SD; CBR was 12.5% and median PFS was 1.8 mo. No relationship was observed between HRD status and clinical activity. Treatment was discontinued by 93 pts (95.9%), mainly due to radiologic or clinical progression (73.1%). Most frequent any grade treatment-emergent (any cause) adverse events were asthenia/fatigue (n=56, 57.7%), nausea (n=40, 41.2%), and anemia (n=34, 35.1%). Conclusions: Single agent rucaparib did not show activity in pts with previously treated advanced UC and enrollment was suspended at the first interim analysis. The safety profile was consistent with that observed in pts with ovarian cancer. Next generation sequencing–based characterization of the genomic landscape of mUC will be presented. Clinical trial information: NCT03397394.

Background: Lucitanib is a small molecule inhibitor of multiple tyrosine kinases, including some that promote angiogenesis. Lucitanib monotherapy has demonstrated potent tumor growth inhibition in xenograft models of various carcinomas, mediated predominately through the inhibition of angiogenesis. In addition to its role in angiogenesis, vascular endothelial growth factor (VEGF) and other angiogenic factors are thought to dampen antitumor immune responses by promoting inhibitory immune subsets, suppressing dendritic cell maturation, and altering lymphocyte development and trafficking. In these studies, we further characterized lucitanib’s kinase selectivity and investigated the antitumor efficacy of lucitanib in combination with anti-PD-1 therapy. Methods & Results: The inhibitory profile of lucitanib was evaluated against 376 wild-type kinases in functional enzymatic assays. At 500 nM, lucitanib demonstrated >35% inhibition of 5.1% (19/376) of the kinases evaluated. The IC50 values of lucitanib against VEGF receptors 1-3 (VEGFR1-3), platelet-derived growth factor receptors alpha/beta (PDGFRα/β), and fibroblast growth factor receptors 1-3 (FGFR1-3) ranged from 8.96 to 95.7 nM. In addition, inhibition was observed against DDR1, CSF1R, RET, and KIT. Cell-based kinase assays confirmed that lucitanib is a potent and selective VEGFR1-3, PDGFRα/β, and FGFR1-3 inhibitor. Lucitanib antitumor activity was evaluated in combination with an anti-PD-1 antibody in a panel of syngeneic murine models. The combination treatment was generally well tolerated. In the H22 hepatocellular carcinoma model, the tumor growth inhibition in animals treated with lucitanib (10 mg/kg once daily), anti-PD-1 (5 mg/kg biweekly), or the combination was 58.6%, 50.4%, and 81.4%, respectively, following 16 days of treatment. The median survival time (MST) for lucitanib and anti-PD-1 treated animals was 35.0 and 46.5 days, respectively, whereas the MST had not been reached for the combination group when the study was terminated on day 63. Importantly, the combination treatment resulted in 2/10 mice that were tumor free at the end of the study; however, there were no tumor-free animals in the groups treated with the single agents. Antitumor efficacy was also greater with the combination of lucitanib and anti-PD-1 therapy than with treatment with the single agents in the MC38 and CT26 syngeneic murine models of colon cancer. Conclusions: Lucitanib demonstrated potent and selective targeting of VEGFR1-3, PDGFRα/β, and FGFR1-3 and enhanced antitumor activity when combined with PD-1 inhibition in multiple syngeneic models. Results from these studies support the clinical development of combined lucitanib and anti-PD-1 treatments to further evaluate the safety and efficacy of the combination in multiple tumor types. Citation Format: Rachel L. Dusek, Liliane Robillard, Thomas C. Harding, Andrew D. Simmons, Minh Nguyen. Enhancement of anti-PD-1 antitumor efficacy in syngeneic preclinical models by the angiogenesis inhibitor lucitanib [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 1214.

Background: For patients with metastatic urothelial carcinoma (mUC) that has progressed on or after platinum-based chemotherapy (cisplatin or carboplatin) and/or immune checkpoint inhibitors, there are currently no standard treatments, emphasizing the urgent unmet need for additional treatment options. Based on analysis of The Cancer Genome Atlas bladder cancer dataset, ≈60% of muscle invasive bladder tumors have homologous recombination (HR) deficiency (HRD), as defined by high genomic loss of heterozygosity (LOH) or a deleterious mutation in a DNA repair pathway gene. Poly(ADP-ribose) polymerase (PARP) inhibitors such as rucaparib exert their antitumor activity through synthetic lethality in cancer cells with HRD. Tumors with HRD have been shown to be sensitive to PARP inhibitors in breast, ovarian, and prostate cancers, and PARP inhibitors are approved for the treatment of patients with breast or ovarian cancer. Although there are little data on their activity in mUC, we hypothesize that PARP inhibition may have antitumor activity in this tumor type. The ATLAS (NCT03397394) trial is evaluating the efficacy and safety of the PARP inhibitor rucaparib as monotherapy for patients with locally advanced (unresectable) urothelial carcinoma or mUC previously treated with 1-2 anticancer treatments for advanced/metastatic disease. Methods: Eligible patients must have received 1-2 prior treatments (eg, platinum-based chemotherapy, immune checkpoint inhibitor, and/or clinical trial agent) and have radiographic progression, measurable disease (RECIST version 1.1), and adequate organ function. Tumor HRD is not required as rucaparib may potentially benefit patients with HR-proficient or HR-deficient tumors; however, fresh tumor or recently obtained archival tissue is mandatory for central HRD profiling. Prior PARP inhibitor treatment is an exclusion criterion. All patients will receive rucaparib monotherapy (600 mg BID) until disease progression or other reason for discontinuation. The primary endpoint is confirmed objective response rate (investigator assessed per RECIST version 1.1) in the intent-to-treat and HRD-positive (signature based on tumor genomic LOH) populations. Secondary endpoints include response duration, progression-free survival, overall survival, safety, and pharmacokinetics. Exploratory endpoints include evaluating plasma and tumor samples for biomarkers associated with response and resistance to rucaparib. The study has >90% power to reject the null hypothesis (P=0.10) at a one-sided 5% significance level if the true response rate for rucaparib is 20%. ATLAS is currently enrolling at ≈65 sites in 6 countries (France, Germany, Italy, Spain, United Kingdom, and United States), with a target enrollment of 200 patients. Citation Format: Petros Grivas, Min Yuen Teo, Nicholas Vogelzang, Ajjai Alva, Yousef Zakharia, Nabil Adra, Alexandra Drakaki, Arif Hussain, Rafael Morales-Barrera, Andrea Necchi, Alejo Rodriguez-Vida, Susan Feyerabend, Sumati Gupta, Debra H. Josephs, Yohann Loriot, Sandy Srinivas, Kenton Wride, Daleen Thomas, Rachel Dusek, Andrew D. Simmons, Dale Nepert, Simon Chowdhury. ATLAS: A Phase II, open-label study of rucaparib in patients with locally advanced or metastatic urothelial carcinoma [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 CT179.

The mechanisms by which the p53 tumor suppressor acts remain incompletely understood. To gain new insights into p53 biology we used high throughput sequencing to analyze global p53 transcriptional networks in primary mouse embryo fibroblasts in response to DNA damage. Chromatin immunoprecipitation sequencing reveals 4785 p53 bound sites in the genome located near 3193 genes involved in diverse biological processes. RNA sequencing analysis shows that only a subset of p53 bound genes is transcriptionally regulated yielding a list of 432 p53 bound and regulated genes. Interestingly we identify a host of autophagy genes as direct p53 target genes. While the autophagy program is regulated predominantly by p53 the p53 family members p63 and p73 contribute to activation of this autophagy gene network. Induction of autophagy genes in response to p53 activation is associated with enhanced autophagy in diverse settings and depends on p53 transcriptional activity. While p53 induced autophagy does not affect cell cycle arrest in response to DNA damage it is important for both robust p53 dependent apoptosis triggered by DNA damage and transformation suppression by p53. Together our data highlight an intimate connection between p53 and autophagy through a vast transcriptional network and indicate that autophagy contributes to p53 dependent apoptosis and cancer suppression. © 2013 by Cold Spring Harbor Laboratory Press.

Background The MYC family of proteins promotes neuroblastoma tumorigenesis at least in part through the induction of aerobic glycolysis by promoting the transcription of key glycolytic enzymes, such as LDHA. FX11 is a selective inhibitor of LDHA that has demonstrated preclinical efficacy in adult cancers. Herein, we hypothesized that FX11 would inhibit aerobic glycolysis and block growth of neuroblastoma cells. Methods We surveyed 3 MYCN–single copy and 5 MYCN-amplified neuroblastoma cell lines to correlate C-MYC/N-MYC protein levels with LDHA expression. Cell viability was measured with FX11 using a tetrazolium-based assay. Cell cycle analysis using propidium iodide with flow cytometry was performed to evaluate for growth arrest. Immunoblotting demonstrated PARP and Caspase 3 cleavage as evidence of apoptosis. Results LDHA is frequently expressed in both MYCN--amplified and MYCN-single copy cell lines. N-MYC and C-MYC protein levels did not correlate with LDHA protein expression. FX11 inhibits aerobic glycolysis and growth in three MYCN-amplified and one MYCN–single copy neuroblastoma cell lines. FX11 induces modest G1 cell cycle arrest with selective induction of apoptosis. Conclusion Small molecule LDHA inhibition is capable of blocking aerobic glycolysis and growth of neuroblastoma cell lines in vitro and merits further in vivo evaluation of its preclinical efficacy in neuroblastomas.

Neuroblastoma arises from the neural crest, the precursor cells of the sympathoadrenal axis, and differentiation status is a key prognostic factor used for clinical risk group stratification and treatment strategies. Neuroblastoma tumor-initiating cells have been successfully isolated from patient tumor samples and bone marrow using sphere culture, which is well established to promote growth of neural crest stem cells. However, accurate quantification of sphere-forming frequency of commonly used neuroblastoma cell lines has not been reported. Here, we show that MYCN-amplified neuroblastoma cell lines form spheres more frequently than non-MYCN-amplified cell lines. We also show that sphere formation is directly sensitive to cellular differentiation status. 13-cis-retinoic acid is a clinically used differentiating agent that induces a neuronal phenotype in neuroblastoma cells. Induced differentiation nearly completely blocked sphere formation. Furthermore, sphere formation was specifically FGF-responsive and did not respond to increasing doses of EGF. Taken together, these data suggest that sphere formation is an accurate method of quantifying the stemness phenotype in neuroblastoma.

To analyze the accuracy of ultrasound in assessing pelvic and intra-abdominal spread in patients with ovarian cancer.This prospective study enrolled all consecutive patients referred to a single gynecological oncology center for suspected ovarian cancer. We analyzed only data from patients with histologically confirmed primary ovarian cancer who were evaluated following predefined preoperative ultrasound, intraoperative and pathology protocols. We evaluated the agreement of depth of infiltration of the rectosigmoid wall, tumor spread in different peritoneal compartments and presence of metastatic retroperitoneal and inguinal lymph nodes, as determined at ultrasound, with intraoperative and histopathological findings.In total, 578 patients were enrolled between March 2008 and January 2013, of whom 394 met the study inclusion criteria and were analyzed; 74% of these suffered from advanced-stage cancer. Our results showed excellent agreement between ultrasound and histology in assessment of rectosigmoid wall infiltration (kappa value, 0.812; area under the receiver-operating characteristics curve, 0.898). The overall accuracy in evaluating different peritoneal compartments, retroperitoneal and inguinal lymph nodes and depth of rectosigmoid wall infiltration was 85.3%, 84.8%, 99.7% and 91.1%, respectively. Ultrasound showed high sensitivity only in the assessment of rectosigmoid wall infiltration (83.1%), peritoneal spread into the pelvis (81.4%) and omentum (67.3%), and inguinal metastatic lymph nodes (100%). The specificity of ultrasound in detection of all evaluated parameters was 90%.This is the largest imaging study to date on ovarian cancer staging. Ultrasound can be used as the method of choice to plan rectosigmoid wall resection and dissection of infiltrated inguinal lymph nodes. In assessing different peritoneal and retroperitoneal compartments, ultrasound was accurate and highly specific. However, similar to other modern imaging techniques, it had relatively low sensitivity, further supporting the role of comprehensive surgical staging. Copyright © 2016 ISUOG. Published by John WileySons Ltd.

Radial and longitudinal strain assessment in the carotid artery wall using speckle tracking

The promise of immunotherapy for cancer is underscored by the recent efficacy of checkpoint inhibitors, which hinder the ability of tumors to escape attack by the immune system. Patients most likely to benefit from such therapy include those whose tumors are inflamed and who express the PD-L1 checkpoint protein. It is imperative that alternative therapies are developed for patients whose tumors do not exhibit these characteristics. Using our proprietary OGAP® system, we identified a novel membrane cancer target, OX001L. OX001L expression in certain cancers is associated with poor prognosis and reduced survival. IHC studies showed substantial prevalence of OX001L across multiple tumor types, with a majority of the OX001L positive samples scoring negative for PD-L1. In non-small cell lung cancer, OX001L expression was significantly increased in tumors lacking abundant intratumoral PD-1 positive T-cell infiltrate or PD-L1 expression compared to inflamed or PD-L1 positive tumors (p Citation Format: Angelo Kaplan, Nickolas Attanasio, To Uyen T Do, Sudha Swaminathan, Arnima Bisht, San Lin Lou, Jason Allen, Robert Boyd, James E. Ackroyd, Gleb Feldman, Christian Rohlff, Rachel L. Dusek. Preclinical development of a novel antibody-drug conjugate targeting “cold” tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2772.

e15148Background: Evasion of the host’s immune system by tumor cells is a well-established mechanism of tumor establishment and progression. Using proteomic analysis of membrane-associated proteins...

TPS4592Background: There are limited treatment options for pts with mUC that has progressed during or after platinum-based chemotherapy and/or immune checkpoint inhibitors (ICIs), emphasizing the n...

Dsg1 (desmoglein 1) is a member of the cadherin family of Ca2+-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal growth factor receptor–Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.

To examine prospectively the accuracy of ultrasound in predicting rectosigmoid tumor infiltration in patients with epithelial ovarian cancer.Patients referred for a suspicious pelvic mass between 2012 and 2014 were examined by ultrasound following the standard protocol for assessment of tumor infiltration. Of the 245 patients examined, 191 had proven ovarian cancer and underwent primary surgery and were included in the analysis. Patients with apparently benign or inoperable disease were excluded. Rectosigmoid infiltration was evaluated by histopathology or according to perioperative findings. Clinical, pathological and laboratory parameters were analyzed as factors potentially affecting the sensitivity and specificity of sonography.The sensitivity of ultrasound in detecting rectosigmoid infiltration in patients with ovarian cancer was 86.3%, with specificity of 95.8%, positive predictive value of 92.6%, negative predictive value of 91.9% and overall accuracy of 92.1%.Ultrasound is a highly accurate method for detecting rectosigmoid tumor infiltration in ovarian cancer patients, and thus, can be used for planning adequate management, including patient consultation, surgical team planning, suitable operating time and postoperative care. Copyright © 2016 ISUOG. Published by John WileySons Ltd.

Although a number of cell adhesion proteins have been identified as caspase substrates, the potential role of differentiation-specific desmosomal cadherins during apoptosis has not been examined. Here, we demonstrate that UV-induced caspase cleavage of the human desmoglein 1 cytoplasmic tail results in distinct 17- and 140- kDa products, whereas metalloproteinase-dependent shedding of the extracellular adhesion domain generates a 75-kDa product. In vitro studies identify caspase-3 as the preferred enzyme that cleaves desmoglein 1 within its unique repeating unit domain at aspartic acid 888, part of a consensus sequence not conserved among the other desmosomal cadherins. Apoptotic processing leads to decreased cell surface expression of desmoglein 1 and re-localization of its C terminus diffusely throughout the cytoplasm over a time course comparable with the processing of other desmosomal proteins and cytoplasmic keratins. Importantly, whereas classic cadherins have been reported to promote cell survival, short hairpin RNA-mediated suppression of desmoglein 1 in differentiated keratinocytes protected cells from UV-induced apoptosis. Collectively, our results identify desmoglein 1 as a novel caspase and metalloproteinase substrate whose cleavage likely contributes to the dismantling of desmosomes during keratinocyte apoptosis and also reveal desmoglein 1 as a previously unrecognized regulator of apoptosis in keratinocytes.

Genetically modified (GM) rice (LibertyLink, event LLRICE62) that is tolerant to glufosinate ammonium (Liberty) herbicide was compared with a near-isogenic (NI) conventional medium-grain brown rice (cultivar, Bengal) and a commercially milled long-grain brown rice in diets for growing-finishing pigs. The GM and NI rice were grown in 2000. The GM rice was from fields treated (GM+) or not treated (GM-) with glufosinate herbicide. The GM- and NI rice were grown using herbicide regimens typical of southern United States rice production practices. The four rice grains were similar in composition. Growing-finishing pigs (n = 96) were fed fortified rice-soybean meal diets containing the four different rice grains from 25 to 106 kg BW. Diets contained 0.99% lysine initially (growing phase), with lysine decreased to 0.80% (early finishing phase) and 0.65% (late finishing phase), when pigs reached 51 and 77 kg, respectively. The percentage of rice in the four diets was constant during each of the three phases (72.8, 80.0, and 85.8% for the growing, early-finishing, and late-finishing phases, respectively). There were six pen replicates (three pens of barrows and three pens of gilts) and four pigs per pen for each dietary treatment. All pigs were slaughtered at the termination of the study to collect carcass data. At the end of the 98-d experiment, BW gain, feed intake (as-fed basis), and feed:gain ratio did not differ (P > 0.05) for pigs fed the GM+ vs. conventional rice diets, but growth performance traits of pigs fed the GM+ rice diets were superior (P < 0.05) to those of pigs fed the GM- rice diet (ADG = 0.86, 0.79, 0.81, and 0.85 kg/d; ADFI = 2.41, 2.49, 2.37, and 2.45 kg/d; feed:gain = 2.80, 3.17, 2.95, and 2.89 for GM+, GM-, NI, and commercially milled rice, respectively). Carcass traits (adjusted for final BW) did not differ (P = 0.10) among treatments (hot carcass yield = 73.5, 72.6, 72.6, and 73.2%; 10th-rib backfat = 23.0, 22.7, 21.3, and 23.8 mm; LM area = 38.6, 38.0, 38.2, and 38.1 cm(2); carcass fat-free lean = 50.5, 50.5, 51.2, and 50.0%). Gilts grew slower (P < 0.05) and were leaner (P < 0.05) than barrows. Responses to type of rice did not differ between barrows and gilts, with no evidence of a diet x gender interaction (P = 0.50) for any trait. The results indicate that the glufosinate herbicide-tolerant rice was similar in composition and nutritional value to conventional rice for growing-finishing pigs.

Desmoglein 1 (Dsg1) is a component of desmosomes present in the upper epidermis and can be targeted by autoimmune antibodies or bacterial toxins, resulting in skin blistering diseases. These defects in tissue integrity are believed to result from compromised desmosomal adhesion; yet, previous attempts to directly test the adhesive roles of desmosomal cadherins using normally non-adherent L cells have yielded mixed results. Here, two complementary approaches were used to better resolve the molecular determinants for Dsg1-mediated adhesion: (1) a tetracycline-inducible system was used to modulate the levels of Dsg1 expressed in L cell lines containing desmocollin 1 (Dsc1) and plakoglobin (PG) and (2) a retroviral gene delivery system was used to introduce Dsg1 into normal human epidermal keratinocytes (NHEK). By increasing Dsg1 expression relative to Dsc1 and PG, we were able to demonstrate that the ratio of Dsg1:Dsc1 is a critical determinant of desmosomal adhesion in fibroblasts. The distribution of Dsg1 was organized at areas of cell-cell contact in the multicellular aggregates that formed in these suspension cultures. Similarly, the introduction of Dsg1 into NHEKs was capable of increasing the aggregation of single cell suspensions and further enhanced the adhesive strength of intact epithelial sheets. Endogenous Dsc1 levels were also increased in NHEKs containing Dsg1, providing further support for the coordination of these two desmosomal cadherins in regulating adhesive structures. These Dsg1-mediated effects on intercellular adhesion were directly related to the presence of an intact extracellular domain as ETA, a toxin that specifically cleaves this desmosomal cadherin, inhibited adhesion in both fibroblasts and keratinocytes. Collectively, these observations demonstrate that Dsg1 promotes the formation of intercellular adhesion complexes and suggest that the relative level of Dsg and Dsc expressed at the cell surface regulates this adhesive process.

The cell surface antigen Lymphocyte antigen 75 (LY75, CD205, DEC-205) is over-expressed in several tumor histotypes. It is a type I C-type lectin receptor (CLR), normally expressed on various APC subsets, characterized by a cytoplasmic domain containing protein motifs crucial for endocytosis and internalization upon ligation. These features make the antigen ideal to be exploited as a target for a novel ADC. MEN1309 is a humanized IgG1 antibody directed against the cell surface antigen Ly75, conjugated through a cleavable linker to a potent maytansinoid microtubule disruptor, DM4. In this study, we evaluated the in vitro and in vivo (xenografts and PDX) efficacy of MEN1309 in different tumor histotypes. A PK/PD relationship was also investigated in tumor-bearing mice. IHC demonstrated high prevalence of Ly75 in human pancreatic, triple negative breast, and bladder cancers, as well as in diffuse large B-cell lymphoma. In vitro experiments showed that cytotoxic activity of MEN1309 was in nM/sub nM range against several lymphoma, pancreatic, bladder and triple-negative breast cancer (TNBC) cell lines. Moreover, MEN1309 exhibited high cell-killing ability against cells having either strong as well as low to moderate antigen expression. In vivo, MEN1309 at 2.5-5 mg/kg (schedule varying from single dose, q7dx3, or q21dx3) showed an impressive antitumor activity, resulting in complete and long lasting responses in most of the xenograft models representing lymphoma, TNBC, bladder and pancreatic cancers, expressing the antigen at high but also at low levels. No treatment related toxicity in terms of change of body weight and death events were detected. Moreover, the administration of (i) isotype control-DM4, (ii) the non-conjugate antibody IgG1 and (iii) the free toxin DM4 (at a dosage corresponding to the equimolar concentration linked at 10 mg/kg ADC) showed little to no therapeutic efficacy on tumor growth. In TNBC patient-derived xenograft (PDX) model (coming from a heavily pre-treated patient and expressing high level of the antigen Ly75), MEN1309 (5 mg/kg q21dx3) showed a complete tumor regression. Finally, in the pancreatic adenocarcinoma xenograft model HPAFII, the pharmacokinetics profile in serum of MEN1309 at 5 mg/kg was characterized and it was qualitatively correlated, using immunofluorescence, with the occurrence of phosphorylation of Serine 10 of H3 Histone in cancer cells, as a pharmacodynamic (PD) marker of DM4 activity on microtubules. Initial ADC exposure was noteworthy and was followed by a relatively fast decline. In parallel with the decay of the serum ADC concentrations there was a progressive increase in the number of positive cells showing the PD marker for mitotic arrest. Overall, our data suggest that MEN1309 is a selective and potent novel antitumoral ADC and it deserves to enter into aPhase I study for a variety of Ly75 positive tumor histotypes. Citation Format: Mario Bigioni, Giuseppe Merlino, Cristina Bernadó Morales, Rossana Bugianesi, Attilio Crea, Rosanna Manno, Joaquin Arribas, Rachel Dusek, Nickolas Attanasio, Keith Wilson, Christian Rohlff, Monica Binaschi. MEN1309, a novel antibody drug conjugate (ADC) targeting Ly75 antigen, induces complete responses in several xenografts of solid tumors [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 2630. doi:10.1158/1538-7445.AM2017-2630

Ly75 (CD205, DEC-205) is a type I transmembrane glycoprotein and a C-type lectin receptor involved in antigen uptake and processing, mainly expressed by antigen presenting cells (APC). The short cytoplasmic tail contains motifs for amino acid-based endocytosis, making this receptor an ideal target antigen for an antibody drug conjugate (ADC)-based antitumoral therapy. MEN1309 is a novel fully humanized ADC which binds to Ly75 with high affinity as shown by ELISA and FACS analysis. The antibody is conjugated to a maytansinoid DM4, a potent tubulin inhibitor, through a cleavable linker.The ability of Ly75 to internalize the antibody after binding was determined using an immunoflourescence assay that showed a rapid, efficient, and near complete internalization over a one hour time course.The expression of Ly75 mRNA and protein was investigated in human cancer cell lines derived from different histotypes and revealed high expression in pancreas, bladder, triple negative breast cancer (TNBC) cells and in diffuse large B-cell lymphoma (DLBCL). Indeed, MEN1309 shows a powerful (pM range) in vitro cytotoxic activity on different cancer cell lines expressing Ly75, whereas it exerts a weaker effect on antigen-negative cells. Besides the mechanism of action (MoA) of MEN1309 as an ADC, the putative efficacy of the antibody to drive an ADCC response was investigated through in vitro binding and functional assays. In spite of a high binding affinity of MEN1309 to FcγRIIIa, no ADCC response was observed, suggesting that the high internalization rate of the antigen could hamper the triggering of NK responses.Moreover, in order to characterize the functional role of Ly75 in cancer cell lines, its expression was downregulated by siRNA demonstrating an inhibition of the proliferation rate in cells from different histotypes.Finally, we investigated if some cancer cell lines could show a higher expression of two intergenically spliced forms derived from Ly75 and DCL-1 genes recently reported in literature. We found that the intergenically spliced forms were expressed on average 30 fold less than CD205 mRNA in all the cancer cell lines analyzed, suggesting that these variants derive just from an intergenic readthrough without a specific transcriptional regulation. Citation Format: Alessandro Bressan, Alessio Fiascarelli, Giuseppe Merlino, Corrado Carrisi, Daniela Bellarosa, Rachel Dusek, Rahel Awdew, Sudha Swaminathan, Arnima Bisht, To Uyen T. Do, San Lin Lou, Dee Aud, Jonathan Terrett, Keith Wilson, Christian Rohlff, Monica Binaschi. Characterization of the novel antibody drug conjugate MEN1309 and its target antigen Ly75 [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 3646. doi:10.1158/1538-7445.AM2017-3646

e14039 Background: CD205 is a type I C-type lectin receptor normally expressed on various APC and some leukocyte sub-populations, characterized by a cytoplasmic domain containing protein motifs crucial for endocytosis and internalization. These features make CD205 ideal to be exploited as target of a novel ADC. MEN1309/OBT076 is a humanized IgG1 antibody against CD205, conjugated through a cleavable linker to a potent maytansinoid microtubule disruptor, DM4. Indeed, impressive results were obtained in high and low CD205 expressing xenografts for solid tumours and NHL. In vitro experimental model describing the by-stander effect of MEN1309 was implemented. Methods:A retrospective analysis to evaluate the CD205 expression on archival NHL biopsies was performed in the CRO-National Cancer Institute, Aviano IT. An immunohistochemistry (IHC) was performed on 1 slide per case, using a commercial antibody NCL-L-DEC205 (clone 11A10, Leica Biosystems). All slides were visually evaluated and a validated staining scoring system was applied, ranging from negative score 0 to strong immunostaining score 3+. Percentages (from 0 to 100%) of negative and positive tumor cells (scored as follows: 1-25% = score 1; 26-50% = score 2; 51-75% = score 3; 76-100% = score 4) were reported for each individual slide.Evaluation of correlations between CD205 expression and clinical characteristics is ongoing. Results: 100 cases of Diffuse Large B Cells Lymphomas (DLBCL) and 33 of Follicular Lymphomas (FL) were analysed. 60% of DLBCL cases were positive for CD205 and 39% had more than 25% of positive cells (score ≥2), while the 16% showed 2+/3+ staining intensity. 79% of FL cases were CD205 positive and 66% had more than 25% of positive cells, while the 27% showed 2+/3+ staining intensity. Conclusions: CD205 is heterogeneously expressed in the majority of DLBCL and FL cases. Data on the correlation between CD205 expression and clinical features will be discussed.MEN1309 has shown anti-tumour activity in vitro and in vivo even in low antigen expressing tumours. Hence CD205 expression among NHL, even if heterogeneous, makes, both DLBCL and FL, promising settings in which to explore MEN1309 activity in an upcoming FIH trial.

Tyrosine phosphorylation of junctional components has been proposed as a mechanism for modulating cell-cell adhesion. Although a correlation exists between the tyrosine phosphorylation of the adherens junction protein beta-catenin and loss of classical cadherin-mediated adhesion, the effects of tyrosine phosphorylation on the function of the adherens junction and desmosome-associated protein plakoglobin is unknown. In the present study, we investigated the effects of epidermal growth factor receptor (EGFR) tyrosine kinase activation on the subcellular distribution of plakoglobin and its association with its junctional binding partners. Long term epidermal growth factor (EGF) treatment of A431 cells revealed a modest decrease in the cytoskeleton-associated pool of plakoglobin (Pg) and a corresponding increase in the cytosolic pool of Pg. After short term EGF treatment, plakoglobin was rapidly phosphorylated, and tyrosine-phosphorylated Pg was distributed predominantly in a membrane-associated Triton X-100-soluble pool, along with a co-precipitating high molecular weight tyrosine-phosphorylated protein identified as desmoglein 2. Analysis of deletion and point mutants defined the primary EGFR-dependent targets as one or more of three C-terminal tyrosine residues. Whereas phosphorylated Pg remained associated with the desmoglein tail after both short and long term EGFR activation, no phosphorylated Pg was found associated with the N-terminal Pg-binding domain (DPNTP) of the intermediate filament-associated protein, desmoplakin. Together these results are consistent with the possibility that EGF-dependent tyrosine phosphorylation of Pg may modulate cell-cell adhesion by compromising the link between desmosomal cadherins and the intermediate filament cytoskeleton.

Studies using intensity-modulated radiation therapy (IMRT) in the treatment of head and neck tumors have shown to decrease acute and late radiation toxicity. However, the high conformity of this technique can increase the risk of recurrence due to geographic miss. The aim of this study was to analyze whether the results of IMRT met the theoretical expectations concerning treatment efficacy.From a total of 185 patients (152 males and 33 females, mean age 58±10.36 years) 176 were evaluable and were studied. Eighty-nine (48.1%) patients had surgical treatment and 50 of them were scheduled for concomitant cisplatin chemotherapy. Irradiation was performed using IMRT, a sliding window with 9 fields in a Varian 2100 C/D linear accelerator, X-ray beam, 6 MeV. The prescribed dose in the planning treatment volume (PTV1), i.e., the area of the primary tumor and nodal area, was 66 Gy/2.2 Gy-70 Gy/2.12 Gy. In the PTV2 (the area at high risk) the dose was 60Gy/2 Gy-59.4 Gy/ 1.8 Gy, and in the PTV 3 (the area treated with prophylactic irradiation) the prescribed dose was 54 Gy/1.8 Gy/50.4 Gy/1.53 Gy.The 3-year overall survival (OS) and relapse-free survival (RFS) of IMRT-treated patients, most of whom were in stages III and IV (158 out of 177), were 50 and 57%, respectively. Using postoperative radiotherapy/chemoradiotherapy 3-year locoregioncal control was achieved in 75% of the cases as compared with 35% in non-operated patients.The worst outcomes were found in oral cavity and hypopharyngeal tumors, and the best in laryngeal and oropharyngeal tumors. Better results were found in surgically treated patients, and in lower disease stages. Despite the high conformity of dose distribution and efforts to spare healthy tissues, most cases of locoregional relapse occurred in areas receiving the full radiation dose. If dividing relapses into cases of persistence and local recurrence, the former predominated.

To identify major factors in the under- and overestimation of cervical and myometrial invasion by endometrial cancer at preoperative staging by ultrasound.This prospective study involved all patients with histologically confirmed endometrial cancer referred consecutively for surgical staging between January 2009 and December 2011. All patients underwent transvaginal ultrasound examination, obtaining metric and perfusion data, and the results were compared with final histology: myometrial invasion was defined at histology in the final pathology report as being eitheror ≥ 50%, while cervical stromal invasion was reported as being either present or absent, and sonographic over-/underestimation was determined relative to these.Enrolled prospectively into the study were 210 patients. The proportion of cases with sonographic underestimation, relative to final histology, of myometrial invasion (i.e. false-negative estimation of no or superficial invasion 50%) and of cervical invasion (i.e. false-negative finding of absence of stromal invasion) was comparable: 8.6% (n = 18) and 10.5% (n = 22), respectively. Myometrial invasion was overestimated by ultrasound (i.e. false-positive estimation of deep invasion ≥ 50%) in 15.7% (n = 33) of cases, and cervical invasion was overestimated (i.e. false-positive finding of presence of stromal invasion) in 4.8% (n = 10) of cases. These outcomes correspond to positive and negative predictive values of 67.6% (95% CI, 57.7-76.6) and 83.3% (95% CI, 74.9-89.8), respectively, for the subjective assessment of myometrial invasion, and 60.0% (95% CI, 38.2-79.2) and 88.1% (95% CI, 82.5-92.4), respectively, for that of cervical stromal invasion. The staging error in subjective assessment was not related to body mass index (BMI), to the position of the uterus in the pelvis or to image quality. Cervical and myometrial invasion were more often underestimated in well-differentiated endometrial cancers that were smaller in size, with thick minimum tumor-free myometrium and lower perfusion, and more often overestimated in moderately and poorly differentiated cancers that were larger in size, with thin minimum tumor-free myometrium and richer perfusion.The accuracy of subjective assessment of myometrial and cervical invasion by ultrasound was significantly influenced by tumor size, density of tumor vascularization, tumor vessel architecture and histological grading, while it was not significantly affected by BMI, uterine position and image quality.

Desmosomes and hemidesmosomes are adhesive junctions important for maintaining adherence within epithelial tissues – a function reflected in the Greek root desmo , meaning bound. Desmosomes facilitate adhesion between adjacent epithelial cells, whereas hemidesmosomes, named for their ultrastructural resemblance to half a desmosome, mediate adhesion between basal cells of epithelial tissues and the substratum. These junctions are functionally alike in their ability to couple the intermediate filament cytoskeleton to sites of cell–cell or cell–substratum contact at the plasma membrane. However, these organelles differ dramatically in their molecular composition and specialized functions.