bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3713: Higher numbers of cancer stem cells in the peripheral blood of children with B-ALL upon conventional chemotherapy
Authors: Salem Mohamed L.
Published: July 2017

Background: Acute lymphocytic leukemia (ALL) is biologically and clinically considered as a heterogeneous neoplasm of lymphoid progenitor cells in the bone marrow (BM). 15- 20 % of children with ALL who achieve an initial remission, will show relapse. One potential mechanism behind this relapse could be the emergence of cancer stem cells (CSCs) which are considered the driving force of tumourigenesis due to their ability of self-renewal as well as the emergence of immune regulatory cells including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Treg).

Aim: the main aim of this study was to analyze the numbers of CSCs and correlate these numbers with the numbers of blast cells, MDSCs and Treg cells in children with B-ALL before and after induction of chemotherapy.

Materials and Methods: CSCs were defined as CD45dimCD19+CD10+CD34+CD38-, MDSCs were defined as Lin-HLA-DR-CD33+CD11b+ and Treg cells were defined as CD4+CD25+CD127-. The frequencies of these cells were analyzed in the peripheral blood of B-ALL patients before (n= 10) and after (n= 10) induction of chemotherapy using flow cytometry.

Results: Significant increases in the numbers of CSCs were shown in B-ALL patients after induction of chemotherapy as compared to newly diagnosed patients (7.6± 8.3 vs. 2.7± 2.4, P<0.05). The numbers of CSCs in ALL patents before and after induction of chemotherapy inversely correlated with the numbers of the blast cells. Additionally, the numbers of MDSCs and Treg cells were higher and lower, respectively, in patients after induction of chemotherapy as compared to before chemotherapy.

Conclusion: Our results indicate that chemotherapy of B-ALL patients results in emergence of high numbers of CSCs and MDSCs which might be contributing, respectively, to tumor relapse and creation of systemic immune suppression. This pilot study opens a new avenue to investigate the mechanism mediating the emergence of these cells on larger population of B-ALL patients at different treatment stages.

Note: This abstract was not presented at the meeting.

Citation Format: Mohamed L. Salem. Higher numbers of cancer stem cells in the peripheral blood of children with B-ALL upon conventional chemotherapy [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 3713. doi:10.1158/1538-7445.AM2017-3713
Abstract 3720: Biological and clinical relevance of PD-L1 expression in tumor and inflammatory cells in NSCLC
Authors: Bais Carlos , Morehouse Chris , Higgs Brandon W. , Marlon Rebelatto , Steele Keith , Jin Xiaoping , Shi Li , Korolevich Susana , Gupta Ashok , Ranade Koustubh
Published: July 2017

Background: High PD-L1 expression has been shown to be associated with improved clinical outcomes to anti-PD-1/L1 therapies in NSCLC and other indications. However, only a fraction of the PD-L1 high patients (pts) respond. PD-L1 can be induced by IFNG and expressed in tumor cells (TC) and inflammatory cells (IC). Improving our ability to predict patient benefit from anti-PD-1/L1 therapies requires a better understanding of associations between PD-L1 expression in TC and/or IC and outcome. Here we explore the relationship between patterns of IHC PD-L1 expression in TC and IC, gene expression, and clinical outcome.

Methods: CP1108/NCT01693562 was a nonrandomized phase 1/2 trial evaluating durvalumab in pts with advanced NSCLC or other solid tumors. As of 29 APR 2016, 368 previously treated NSCLC pts received durvalumab ≤12 months with a median 18.8 months follow up. Pts with ≥25% TC or IC were scored TC+ and/or IC+, respectively.

Results: TC+ PD-L1 pts (includes IC+ and IC- PD-L1 pts) had improved survival compared to TC- PD-L1 pts. TC+ and IC+ PD-L1 pts had: improved survival compared to TC+/IC-, TC-/IC+, or TC-/IC- PD-L1 pts. However, prevalence of TC+/IC+ was lower than TC+. Twenty-one genes significantly differed between TC+/IC+, TC+/IC-, TC-/IC+, and TC-/IC- PD-L1 patient subsets, the vast majority being well-known IFNG-inducible genes and mostly over-expressed in the TC+/IC+ subset.

Conclusions: TC+ and IC+ PD-L1 pts had the highest levels of IFNG-inducible gene expression, a key biological feature that distinguishes PD-L1 IHC positive from negative pts. Thus, in addition to PD-L1 IHC, the predictive value of IFNG should be investigated in additional relevant studies

Selection CriteriaPrevalence of Biomarker Positive BiomarkerPositive/negative median OSlog-rank pHRcox pTC+158/276 (57%)15.67/7.730.00910.690.046TC+/IC+70/276 (25%)25.63/8.377.46E-060.345.24E-05TC+/IC-88/276 (32%)10.5/13.970.0941.50.032TC-/IC+50/276 (18%)9.07/13.230.830.960.84TC-/IC-68/276 (25%)5.77/14.030.000841.660.012

All comers: median OS: 11.2 months (N=304)

Citation Format: Carlos Bais, Chris Morehouse, Brandon W. Higgs, Rebelatto Marlon, Keith Steele, Xiaoping Jin, Li Shi, Susana Korolevich, Ashok Gupta, Koustubh Ranade. Biological and clinical relevance of PD-L1 expression in tumor and inflammatory cells in NSCLC [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 3720. doi:10.1158/1538-7445.AM2017-3720
Abstract 3722: In vitro induced regulatory T cells for understanding the nature of peripherally induced regulatory cells in relation to cell mediated immunotherapy for cancer
Authors: Hegde Upendra P. , Chakraborty Nitya G.
Published: July 2017

Background: pTregs are appearing to be one of the major obstacles of cellular immunotherapy of cancer. Tumor specific in vivo induced Cytotoxic T lymphocytes (CTL) are rapidly eliminated with concomitant expansion of pTreg cells. In human melanoma in ex vivo condition, tumor antigen specific CTL generated with CD8+ cells persisted for months while ex vivo induced CTL with whole PBL stimulated with Mart-127-35 A2 (tumor antigen) pulsed autologousDC, died within three weeks. Whereas Influenza (Flu 58-66) antigen specific CTL continued to expand and remained functional beyond four weeks.

Method: Peripheral blood and tumors were obtained from patients diagnosed with metastatic melanoma after informed consent. Blood derived dendritic cells, PBL, CD4+ and CD8+ cells were isolated for in vitro co-cultures with Mart-1 or Flu peptide pulsed autologous DC or autologous tumor cells. CD8+ CTL response and CD4+ positive Treg responses were measured. Freshly obtained tumor tissue derived lymphocytes (TIL) were also used for the study.

Results: Our results show that CTL response against Flu survived longer in culture with total PBL than CTL response against Mart-1 in identical culture conditions. CTL response using autologous tumor cells with PBL in culture lasted for a shorter duration but growth of Treg cells were observed. Cytokine analysis with expanding Treg cells showed secretion of IL-10 upon re-stimulation with autologous DC or anti-CD3 ab. Similarly, CD4+ cells isolated from freshly obtained tumor tissue (TIL, representing in vivo condition), suppressed T cell activation and secreted high amount of IL-10 upon re-stimulation.

Conclusion: These findings suggested that in vitro induced iTreg from cultures or pTreg from TILs (in vivo), work identical way, to negatively regulate effector immune response by secreting IL-10. Further analysis with iTregs for their use of metabolic pathways would be important to identify the specific signaling molecules used by autologous (person specific) pTregs , as target molecules, to block the expansion of such pTregs for the better outcome for CTL mediated immunotherapy for cancer.

Citation Format: Upendra P. Hegde, Nitya G. Chakraborty. In vitro induced regulatory T cells for understanding the nature of peripherally induced regulatory cells in relation to cell mediated immunotherapy for cancer [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 3722. doi:10.1158/1538-7445.AM2017-3722
Abstract 3724: PDL1 protein expression and tumor mutation burden in hematologic malignancies: correlation with Hodgkin and high grade lymphoma
Authors: Karim Lina Abdul , Wang Peng , Guzman Jose de , Higgins Brandi , Chahine Joeffrey , Sheehan Christine , Kallakury Bhaskar , Ross Jeffrey S.
Published: July 2017

Background: The success of anti PD–1 therapy in Hodgkin lymphoma has led to clinical trials in other types of lymphomas. However, there is limited data on PD–L1 expression in acute leukemias. In this study, we analyzed PD–L1 protein expression and tumor mutational burden in various hematologic malignancies including leukemias, lymphomas and myelomas.

Methods: In the IHC cohort, formalin fixed paraffin embedded sections from 92 hematologic malignancies including acute myeloid and lymphoblastic leukemia (AML, ALL), Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), low-grade non-Hodgkin lymphoma (LGNHL) and myeloma were immunostained using the FDA approved PD-L1 IHC 22C3 pharmDx assay [monoclonal mouse anti-PD–L1 antibody (DAKO clone 22C3)]. Any perceptible partial or complete membrane staining in all viable tumor cells was scored as follows: <1%-negative, 1-49%-low level and >50%-high level PD-L1 expression. In the second cohort of 2064 cases, comprehensive genomic profiling (CGP) was performed using a hybrid–capture, adaptor ligation assay to a mean depth of >672X. Tumor mutation burden (TMB) was calculated from a minimum of 1.11MB of sequenced DNA as previously described and reported as mutations/MB.

High PD–L1 expression was noted in 100% of HL. DLBCL showed significantly higher PD–L1 positivity compared to LGNHL (p=0.01). No correlation for PD–L1 expression was noted between AML and ALL (p = 0.2). On CGP analysis of the second cohort, TMB was significantly higher in DLBCL as compared to LGNHL (p<0.0001).

Conclusion: The high PD-L1 expression in both HL and DLBCL and high TMB in DLBCL may be linked to enhanced responsiveness to check point inhibitor therapy in these cancers. PD-L1 expression and TMB are relatively infrequent and at low levels in acute leukemias. Additionally, novel anti PD–1/PD–L1 therapeutic strategies are worthy of consideration for both advanced stage refractory/relapsed acute myeloid and lymphoblastic leukemias.

Results: PD-L1 expression and TMB are tabulated below.PD-L1 IHCTMB (mutations/MB)Tumor typenLow PositiveHigh PositiveTotal Positive (%)n>=10.0>=20.0AML21505/21 (24%)50%0%ALL3212012/32 (38%)10%0%HL1001010/10 (100%)368%8%DLBCL10448/10 (80%)25344%15%LGNHL10202/10 (20%)930%0%Myeloma9202/9 (22%)NANANA

Citation Format: Lina Abdul Karim, Peng Wang, Jose de Guzman, Brandi Higgins, Joeffrey Chahine, Christine Sheehan, Bhaskar Kallakury, Jeffrey S. Ross. PDL1 protein expression and tumor mutation burden in hematologic malignancies: correlation with Hodgkin and high grade lymphoma [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 3724. doi:10.1158/1538-7445.AM2017-3724
Abstract 3735: Detection of microsatellite instable colorectal cancers from routine NGS panel sequencing data
Authors: Endris Volker , Kirchner Martina , Kloor Matthias , Volckmar Anna-Lena , Doeberitz Magnus von Knebel , Penzel Roland , Schirmacher Peter , Stenzinger Albrecht
Published: July 2017

Introduction

In about 15% of all colorectal cancers, microsatellite instability (MSI) caused by defects in the DNA mismatch repair machinery can be detected. MSI driven tumors have a better prognosis compared to microsatellite stable (MSS) tumors and show good response to drugs targeting the PD-1-PD-L1 axis. Current molecular diagnostic procedures for colorectal cancer are primarily focused on the detection of oncogenic mutations in RAS family genes by either classical Sanger sequencing or next-generation sequencing. Routine testingof MSI status for therapeutic purposes is still rare and usually carried out by a different array of methods including PCR and immunohistochemistry.

Experimental procedures

In our routine diagnostic CRC screening, we sequenced more than 600 colorectal tumors with an entity specific customized 180 amplicon NGS panel encompassing HotSpot regions of 31 genes. This panel includes several homopolymeric regions of genes that are known to be affected by MSI. We retrospectively analysed this data set for MSI signatures, including deletion/insertion mutations in 20 different homopolymeric regions (>7) and mutations/deletions of MLH1, MSH3, MSH6 and TGFBR2. Cases with an MSI signature detected by panel sequencing were reevaluated by PCR-based screening (Bethesda panel).

Results

From the 20 homopolymeric regions analyzed, several loci showed deletion/insertion mutations in MSI-positive patients. The highest discriminatory power presented a homopolymeric region within the ACVR2A gene showing a high analytical sensitivity and specificity towards MSI prediction. In combination with the readout of additional homopolymeric regions and the mutation status of MLH1, MSH3, MSH6 and TGFBR2, a sensitive and specific detection algorithm for MSI can be obtained from routine panel sequencing data. In conclusion, using only one assay, this approach facilitates simultaneous detection of mutations in RAS genes (and other druggable targets) and the MSI status in CRC As the assay is tissue sparing and time saving it may be advantageous over classic test approaches that are currently being employed in routine diagnostics.

Note: This abstract was not presented at the meeting.

Citation Format: Volker Endris, Martina Kirchner, Matthias Kloor, Anna-Lena Volckmar, Magnus von Knebel Doeberitz, Roland Penzel, Peter Schirmacher, Albrecht Stenzinger. Detection of microsatellite instable colorectal cancers from routine NGS panel sequencing data [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 3735. doi:10.1158/1538-7445.AM2017-3735
Abstract 3744: Office cervicoscopy in the diagnosis of HPV infection
Authors: Darwish Atef
Published: July 2017

Introduction: Since the association between certain oncogenic (high-risk) strains of HPV and cervical cancer is well established. Searching for HPV became a basic step in all screening programs. Laboratory HPV testing is costly and unavailable in developing countries.

Objective: To compare the diagnostic indices of office cervicoscopy against cytology and punch biopsy for detection of underlying HPV lesions in women with suspicious cervix.

Design: An analytic cross-sectional research design

Setting: Early cervical cancer Detection Unit of the Women's Health University Hospital. Assiut, Egypt.

Patients and methods: A total of 274 non-pregnant women with clinical diagnosis of suspicious cervix were included in this study. After cervical smearing with Ayre's spatula, they were subjected to cervicoscopy utilizing 2.6 mm telescope and a 3.2 mm outer sheath office hysteroscope. Evaluation of the vulva, vagina, ectocervix, endocervical canal and the endometrial cavity were done. The cervix was then painted with saline, acetic acid and Schiller's iodine stains successively as used during colposcopic examination. Abnormal lesions were biopsied using punch biopsy forceps. Cytologic evaluation relied on both classic and non-classic (secondary) criteria for the diagnosis of HPV including "mild koilocytosis," mild dyskeratocytosis," hyperchromatic nuclei, bi- and multinucleation, and cleared cytoplasm. Punch biopsies were taken from every case based on cervicoscopic localization after staining.

Results:. The main complaint was abnormal vaginal discharge in 197 cases (71.4%) . Pap smear sampling was negative for HPV in 188 cases (68.6%) and positive in 86 cases (31.4%). Cervicoscopic findings suggestive of HPV infection were positive in 77 cases ( 28.1% ) and negative in 197 cases (71.9% ). Gold stand histopathology diagnosed HPV in 53 cases only (19.3%). All diagnostic indices of cervicoscopy significantly improved after combination with cytology positive results except false positive results..

Conclusions: Cervicoscopy as a simple, cheap and office procedure can be used as a quick screening tool of HPV infection in women with suspicious cervix. It has the advantage of screening of the rest of the genital tract. In the absence of laboratory HPV testing, cervicoscopy in conjunction with cytology screening of high-risk cases should be encouraged.

Note: This abstract was not presented at the meeting.

Citation Format: Atef Darwish. Office cervicoscopy in the diagnosis of HPV infection [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 3744. doi:10.1158/1538-7445.AM2017-3744
Abstract 3748: Development of anti-human mesothelin chimeric antigen receptor (CAR) messenger RNA (mRNA) transfected peripheral blood mononuclear cells (CARMA) for the treatment of mesothelin-expressing cancers
Authors: Hung Chien-Fu , Xu Xuequn , Li Linhong , Ma Ying , Jin Qiu , Viley Angelia , Allen Cornell , Natarajan Pachai , Shivakumar Rama , Peshwa Madhusudan V. , Emens Leisha A.
Published: July 2017

CD19-targeted chimeric antigen receptor (CAR)-engineered T/NK-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been less successful in solid cancers. This is partly due to specificity for shared tumor antigens also present on normal host tissues that leads to ‘on-target/off-tumor’ toxicity. We therefore developed a non-viral approach using repeated infusions of mesothelin-specific messenger RNA (mRNA) CAR-transfected T cells to permit prospective control of ‘on-target/off-tumor’ toxicity. Early trials provided preliminary evidence of the safety and anti-tumor activity of this strategy, but the ex vivo selection, activation and expansion of lymphocytes is laborious and expensive. We therefore explored the feasibility of using a rapid, automated, closed system for cGMP-compliant mRNA CAR transfection into freshly isolated peripheral blood mononuclear cells for clinical scale manufacture (CARMA). The resulting cryopreserved cellular product expressed CAR in >95% of cells, and recognized and lysed tumor cells in an antigen-specific manner. Expression of CAR was detectable for 5-7 days in vitro, with a progressive decline of CAR expression related to in vitro cell expansion. In a murine ovarian cancer model, a single intra-peritoneal (IP) injection of CARMA resulted in the dose-dependent inhibition of tumor growth and prolonged the overall survival (OS) of mice. Multiple weekly IP injections of the optimal CARMA dose enhanced disease control and further prolonged OS, both of which improved with an increasing number of injections. No significant off-tumor toxicities were observed. These data support further investigation of serial IP CARMA administration as a potential treatment for ovarian cancer and other mesothelin-expressing tumors involving the peritoneum, and provide preclinical proof of principle of CARMA for solid tumors.

Citation Format: Chien-Fu Hung, Xuequn Xu, Linhong Li, Ying Ma, Qiu Jin, Angelia Viley, Cornell Allen, Pachai Natarajan, Rama Shivakumar, Madhusudan V. Peshwa, Leisha A. Emens. Development of anti-human mesothelin chimeric antigen receptor (CAR) messenger RNA (mRNA) transfected peripheral blood mononuclear cells (CARMA) for the treatment of mesothelin-expressing cancers [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 3748. doi:10.1158/1538-7445.AM2017-3748
Abstract 3762: Superior T cell activity of a membrane-proximal binding antibody when targeting Glypican-3 with an antibody-coupled T-cell receptor (ACTR) armed T cell
Authors: Motz Greg , Shin John , Whiteman Kathleen , Schultes Birgit , Pai Tapasya , Westendorf Lori , Ettenberg Seth , Biechele Travis , Sussman Django , Huet Heather
Published: July 2017

Glypican-3 (GPC3) is a GPI-anchored member of the heparan sulfate proteoglycan family. GPC3 is an oncofetal antigen expressed transiently during fetal development with re-expression during malignant transformation. GPC3 is an ideal tumor target as expression has been found in numerous epithelial malignancies, with highest expression in hepatocellular carcinoma (HCC) and non-small cell lung carcinoma (NSCLC), and normal tissue expression is highly restricted (Baumhoer D., Am J Clin Pathol, 2008.). Adoptive T-cell therapy with single-chain variable fragment (scFv)-derived chimeric antigen receptors (CARs) has transformed cancer therapy, but the broad applicability of this approach has been limited in part by safety concerns due the constitutive expression of a biologically active targeting receptor. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T-cell therapy designed to engage the Fc domain of therapeutic antibodies opsonized to tumor cells to mediate anti-tumor activity. ACTR activity is therefore both regulatable and flexible, providing enhanced therapeutic control and improved safety of the T cell therapy.

Using both HCC and NSCLC tumor cell lines, we tested a panel of wild-type and afucosylated antibodies with similar binding affinities that bound to regions spanning the GPC3 protein across unique epitopes. We found that for GPC3 targeting antibodies, the greatest activity in a Jurkat-NFAT reporter assay was observed for the afucosylated antibody that bound GPC3 most proximal to the membrane. Further, the antibody that bound membrane proximal also had the most potent activity in primary ACTR T cell cytotoxicity and cytokine release assays. The physical distance between T cells and tumor targets has been previously determined to impact T cell activation for both peptide-MHC and CAR-T interactions. Similarly, our results demonstrate a potential relationship between spatial distance of tumor targets and T cells in determining the activity ACTR transduced T cells when targeting GPC3. Our data demonstrate that ACTR T cell activity is antibody-specific and dose-titratable, highlighting both efficacy and improved safety of the ACTR T cell platform when targeting GPC3+ solid tumor malignancies.

Citation Format: Greg Motz, John Shin, Kathleen Whiteman, Birgit Schultes, Tapasya Pai, Lori Westendorf, Seth Ettenberg, Travis Biechele, Django Sussman, Heather Huet. Superior T cell activity of a membrane-proximal binding antibody when targeting Glypican-3 with an antibody-coupled T-cell receptor (ACTR) armed T cell [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 3762. doi:10.1158/1538-7445.AM2017-3762
Abstract 3764: Targeting GPR64 in pediatric sarcomas with CAR transgenic T cells
Authors: Schirmer David , Storz Isabel , Wisskirchen Karin , Feederle Regina , Schmidt Oxana , Abken Hinrich , Mackall Crystall , Protzer Ulrike , Burdach Stefan , Richter Günther H.
Published: July 2017

Introduction: Ewing Sarcoma (ES) is the second most common bone malignancy in children and young adolescents with a high potential of dissemination into lung and bones. Patients with disseminated disease into the bone have an approximate long-term survival rate of only 10%, compelling the search for new therapeutic treatment modalities like engineered T cell therapy. Here the therapeutic potential of chimeric antigen receptor (CAR) transgenic T cells directed against G-protein coupled receptor 64 (GPR64) was examined.

Experimental procedure: Two different monoclonal antibodies (mAb) directed against the extracellular region of GPR64 were generated and characterized. Subsequently, retroviral constructs containing second generation CARs together with the scFv fragments of the respective mAbs were designed. Constructs consisting of different spacers and co-stimulatory elements were compared (dIgG1-CD28-CD3z vs. CD8a-4-1BB-CD3z). Primary lymphocytes were transduced and tested in vitro via flow cytometry, ELISpot and xCelligence assay as well as in immunodeficient xenograft mice.

Results: Antibodies specifically stained ES cells as determined by flow cytometry and immune histology. The signal intensity was reduced after RNAi mediated down-regulation of GPR64 in ES cell lines confirming specificity of the isolated mAbs. Following sequence determination of those mAbs two different CAR constructs were designed. Retroviruses containing such CARs transduced primary lymphocytes with high efficiency. The CAR transgenic T cells were enriched for CD8+CAR+ cells via microbead isolation and demonstrated strong proliferative capacities in vitro. Furthermore, target structures were specifically recognized as determined by ELISpot and xCelligence assays but dIgG1-CD28 CAR T cells derived from one hybridoma revealed hints of early exhaustion via auto-activation.

Conclusion: CAR transgenic T cells targeting GPR64 show a promising approach to transfer the success of CARs in hematological malignancies to solid tumors. The cells generated in this study show strong specificity towards GPR64 and are able to control tumor cell growth. Since GPR64 expression is not restricted to ES but also up-regulated in a number of carcinomas derived from prostate, kidney or lung, GPR64-specific CARs may also be a future treatment option for other tumor entities.

Citation Format: David Schirmer, Isabel Storz, Karin Wisskirchen, Regina Feederle, Oxana Schmidt, Hinrich Abken, Crystall Mackall, Ulrike Protzer, Stefan Burdach, Günther H. Richter. Targeting GPR64 in pediatric sarcomas with CAR transgenic T cells [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 3764. doi:10.1158/1538-7445.AM2017-3764
Abstract 3776: Examination of ERCC1 status in circulating tumor cells as a prognostic tool for patients with nasopharyngeal carcinoma
Authors: Hui Edwin P. , Ma Brigette BY , Chan KC Allen , Chan Charles ML , Wong SC Cesar , To Ka Fai , Loong Herbert HF , Mo Frankie KF , Ngan Roger KC , Chan Anthony TC
Published: July 2017

Investigation of the expression pattern of DNA-repair protein excision repair cross-complementation group 1 (ERCC1) has been reported to allow selection of patients with non-small cell lung cancer who are likely to benefit from cisplatin-based therapy. Recent evidence suggests that ERCC1 expression may also find prognostic use in patients with nasopharyngeal carcinoma (NPC). We evaluated ERCC1 expression and genotype from NPC tissues and peripheral blood mononuclear cells of patients with NPC. ERCC1 expression was detected in 61/77 cases (79.2%) with varying intensities, where high ERCC1 expression significantly associated with worse relapse-free survival (RFS) (HR 2.34, 1.06-5.16, p=0.036). In addition, the presence of ERCC1 C118T genotype was significantly associated with favorable RFS and overall survival (OS) in a subgroup of patients with undetectable post-treatment plasma EBV DNA. These findings support a prognostic role for ERCC1 examination in NPC. However, the invasive nature of obtaining biopsy samples for tumor marker studies is a major hindrance of this approach. To facilitate the investigation of ERCC1 expression in a noninvasive manner, we have developed a negative selection immunomagnetic method for isolating circulating tumor cells from patient blood. Cell line spike-in experiments reveal a mean recovery rate of 66% for NPC cells with greater than 99% removal of non-targeted blood cells. Specificity of CTC identification was confirmed by detection of the expression of the Epstein-Barr virus encoded small RNA (EBER) in the CTCs. This method also allowed the simultaneous analysis of the expression of multiple protein markers including CD45, cytokeratin and ERCC1. It is expected that the development of noninvasive methodologies for tumor marker studies will facilitate their clinical application for improved patient care and monitoring in the future.

Note: This abstract was not presented at the meeting.

Citation Format: Edwin P. Hui, Brigette BY Ma, KC Allen Chan, Charles ML Chan, SC Cesar Wong, Ka Fai To, Herbert HF Loong, Frankie KF Mo, Roger KC Ngan, Anthony TC Chan. Examination of ERCC1 status in circulating tumor cells as a prognostic tool for patients with nasopharyngeal carcinoma [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 3776. doi:10.1158/1538-7445.AM2017-3776
Abstract 3811: Defining the relevant genome in solid tumors
Authors: Hamidi Habib R. , Bandla Santoshi , Khazanov Nick , William Paul , Bonevich Nikki , Zurenko Chris , Anstead Sarah , Taylor Chris , Richmonds Reuban , Galimberti David , Kopp Ken , Cyanam Dinesh , Hogan Michael , Mittal Vinay , Sadis Seth
Published: July 2017

Decades of cancer research including comprehensive molecular profiling combined with the development of a broad array of targeted therapies have created the opportunity to transform cancer care by implementing precision oncology based approaches. An important element of this system is the widespread availability of robust and cost-effective multi-variate profiling methods to characterize relevant cancer associated molecular alterations in the future. Current commercially available multi-variate profiling methods vary dramatically with regard to the number of cancer genes that are interrogated. Given that many large scale and detailed molecular profiling studies have been completed, the landscape of somatic alterations in solid tumors is reasonably well-known. Furthermore, the specific gene variants that are relevant to application of targeted therapies are also a matter of record. Therefore, we set out to define the number of relevant cancer genes for precision oncology research based on the currently available empirical evidence. To define recurrent somatic alterations in solid tumors, we created a compendium of variant calls from > 15,000 exomes, defined focal amplifications and deletions from > 30,000 arrays, and defined recurrent fusions from several thousand RNAseq profiles. Statistical approaches were implemented to define genes containing recurrent missense mutations (i.e., hotspots), enriched in truncating mutations or subject to recurrent copy number gain/loss or translocation. This gene set was then used to comprehensively search approved cancer drug labels, clinical practice guidelines, and clinical trials to identify records containing published evidence that specific recurrent somatic gene variants were used as part of the indication statement of an approved targeted therapy, were recommended for testing as part of clinical practice for therapeutic decisions, or were used as enrollment criteria in clinical trials. The relevant cancer genome thus defined consists of < 100 genes. These results suggest that targeted multi-variate profiling approaches may be sufficient to support precision oncology goals in the future.

Citation Format: Habib R. Hamidi, Santoshi Bandla, Nick Khazanov, Paul William, Nikki Bonevich, Chris Zurenko, Sarah Anstead, Chris Taylor, Reuban Richmonds, David Galimberti, Ken Kopp, Dinesh Cyanam, Michael Hogan, Vinay Mittal, Seth Sadis. Defining the relevant genome in 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 3811. doi:10.1158/1538-7445.AM2017-3811
Abstract 3813: MPF as a monitoring biomarker for a mesothelin-targeted therapy against malignant mesothelioma and other cancer
Authors: Yu Yunkai , Hassan Raffit , Zhang Jingli , Onda Masanori , Pastan Ira , Cao Liang
Published: July 2017

Introduction: Tumor antigen mesothelin had been shown to be elevated in mesothelioma, ovarian, pancreatic, and lung cancer. The mesothelin gene encodes a precursor protein of 69 kDa, which is cleaved into a 31 kDa soluble megakaryocyte potential factor (MPF), and a cell membrane-bound 40 kDa mesothelin. There is a high degree of interest in targeting mesothelin for mesothelioma and other cancers with various antibody-based agents, and some successes in early stage clinical trials with some of the agents. For the development of these targeted therapeutics, specific biomarkers would be needed for monitoring treatment responses, and detecting and evaluating tumor progression. The current test that detects mesothelin is not suitable due to the antigen-binding from the therapeutic antibodies. There is an urgent need to develop and validate an alternative test to support these clinical trials and for disease management.

Methods: Using ECLIA technology and proprietary antibody reagents, we developed a new test for the cleaved MPF polypeptide. We further conducted comprehensive analytical and clinical validation of the test according to the guidelines for tumor antigen tests. Using samples from a retrospective clinical trial with an anti-mesothelin immunotoxin and the standard 1st line chemotherapeutic agents for newly diagnosed malignant mesothelioma patients, we examined the effectiveness of the test for treatment monitoring of the investigational therapy.

Results: Our data gives a strong validation for the analytical and clinical performances of MPF test. While no other current test has been proven to be able to monitor response of mesothelioma to systemic therapies, our data provides the proof of clinical effectiveness for MPF test. In the patients with the elevated tumor antigen, there is a specific and highly significant reduction of MPF at the end of each treatment cycle for a total of 6 cycles in the patients with objective response. Later, about half of these patients with objective response and MPF reduction regained the levels of MPF at the time of disease progression.

Conclusion: The study indicates that MPF test is effective in monitoring the treatment response to systemic therapies for mesothelioma patients with elevated MPF. It may also provide pivotal information on the expression of target mesothelin protein when tumors progress.

Citation Format: Yunkai Yu, Raffit Hassan, Jingli Zhang, Masanori Onda, Ira Pastan, Liang Cao. MPF as a monitoring biomarker for a mesothelin-targeted therapy against malignant mesothelioma and other cancer [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 3813. doi:10.1158/1538-7445.AM2017-3813
Abstract 3819: Identification and quantification of isoforms of eukaryotic initiation factor 4E as biomarker in Mnk inhibitor-treated mouse model by capillary-based immunoassay platform
Authors: Ke Zhiyuan , Wang Sifang , Manoharan Vithya , Vuddagiri Susmitha , Ong Esther , Lim Sharon , Ong Sin Tiong , Sangthongpitag Kanda , Kassoum Nacro , Hill Jeffrey , Lee May Ann
Published: July 2017

Elevated levels of the phosphorylated m7G cap binding protein, eukaryotic initiation factor 4E (eIF4E) are associated with neoplasia. An observed oncogenic signaling effect upon activation of Erk1/2 or p38 MAPKs in cells is the phosphorylation of eIF4E specifically at Serine 209 by downstream kinase MAP kinase-interacting kinase 1 and 2 (Mnks). Therefore, inhibiting Mnks could be a potential therapeutic approach that selectively targets cancer cells without introducing toxicity to normal cells. To measure the target engagement and efficacy of Mnk inhibitors, we have established a sensitive method to quantitatively detect phospho-eIF4E and non-phospho-eIF4E. Here, we report using NanoPro 100 system, an automated capillary-based immunoassay platform that could separate the phosphorylated and non-phosphorylated eIF4E isoforms based on different isoelectric points. Primary antibodies against both non-phospho eIF4E and Serine 209 phosphorylated eIF4E were used for identification and verification of eIF4E isoforms in protein samples obtained from cell lysate, blood samples and tumor samples. Two major isoforms of eIF4E with isoelectric point at 5.38 for phospho-eIF4E and 5.88 for non-phospho-eIF4E were identified in eIF4E-overexpressing K562 cells. siRNA knockdown of eIF4E expression reduced both isoforms, while treatment with Mnk inhibitor only reduced phospho-eIF4E level. The measurement and quantification of the relative changes of phosphorylated eIF4E by this method can potentially be used in monitoring the efficacy of Mnk inhibitors in xenograft and future clinical trials.

Citation Format: Zhiyuan Ke, Sifang Wang, Vithya Manoharan, Susmitha Vuddagiri, Esther Ong, Sharon Lim, Sin Tiong Ong, Kanda Sangthongpitag, Nacro Kassoum, Jeffrey Hill, May Ann Lee. Identification and quantification of isoforms of eukaryotic initiation factor 4E as biomarker in Mnk inhibitor-treated mouse model by capillary-based immunoassay platform [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 3819. doi:10.1158/1538-7445.AM2017-3819
Abstract 3822: Expression of androgen receptor and CD24 correlates with improved 5-year survival in Pakistani patients with invasive breast cancer
Authors: Riaz Nazia , Idress Romana , Habib Sadia , Hussain Azhar , Lalani El-Nasir
Published: July 2017

Introduction: Androgen receptor (AR) expression has been shown to correlate with improved survival in luminal A, B and triple negative (TN) breast cancer (BCa). Cancer stem cells (CSC) with CD44+/CD24- and ALDH1+ phenotype is associated with pro-invasive gene signature in BCa. Aim of our study was to evaluate prognostic significance of AR expression in relation to CD44, CD24 and ALDH1 in invasive BCa.

Methodology: Immunohistochemical expression of AR, CD44, CD24 and ALDH1 was evaluated in 180 FFPE blocks of BCa cases from one institution. Clinico-pathological details and follow-up data were collected from medical charts. Data was analyzed on SPSS 19. Five-year survival was estimated by Kaplan Meier.

Results: Median age of patients was 55 years (range: 28-87). Fifteen percent of patients were diagnosed with stage I, 52.2% with stage II, 25% with stage III and 7.8% with stage IV disease. Mean duration of follow-up was 4.5 years (SD+ 2.7) and 29% mortalities were attributed to BCa. AR was expressed in 63.9%, ER in 61%, PR in 51% & HER-2/neu in 30% of tumors. CD44, CD24 and ALDH1 were expressed in 60%, 43.3% & 28.3% of tumors respectively.

AR expression was associated (p-value<0.001) with grade II tumors expressing ER, PR and CD24 but lacking ALDH1. Multivariable analysis confirmed AR (adjusted HR 0.43, 95% CI 0.19-0.95) and PR (adjusted HR: 0.20, 95% CI: 0.07-0.60) to be independently associated with better outcome.

Five year survival of patients with AR+ versus AR- expressing tumors, showed that patients with AR+ tumors had significantly better outcome (p-value=0.03) with survival advantage of 39.6 months. Survival advantage decreased by 9.6 months in tumors expressing CD44+/CD24-/AR- (p-value=0.01) phenotype and by 12 months in patients whose tumors were phenotypically ALDHI+ /AR- (p-value=0.12).

We stratified TN tumors into 2 groups: A. TN/CD24+/AR+; B. TN/CD24-/AR- and found that group A tumors had a trend towards better 5 survival (p-value=0.09).

Conclusions: Three individual markers (CD44, CD24, AR) may afford prognostic information. Combinations of CD44, CD24 and AR permitted patient stratification into 3 risk categories: poor risk CD24-/AR-; intermediate risk CD44+/CD24-/AR- and good risk CD24+/AR+. Stratification of TN group showed that loss of AR and CD24 was associated with poorest outcome. These results underscore the relevance and importance of AR and CD24 as prognostic markers in invasive BCa.

Citation Format: Nazia Riaz, Romana Idress, Sadia Habib, Azhar Hussain, El-Nasir Lalani. Expression of androgen receptor and CD24 correlates with improved 5-year survival in Pakistani patients with invasive breast cancer [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 3822. doi:10.1158/1538-7445.AM2017-3822
Abstract 3823: High intrinsic DPD activity matters too
Authors: Chamorey Emmanuel , Francois Eric , Etienne-Grimaldi Marie-Christine , Viotti Julien , Peyrade Frederic , Bozec Alexandre , Ferrero Jean-Marc , Schiappa Renaud , Milano Gerard
Published: July 2017

Introduction: Fluoropyrimidines (FP) are of major use in treating cancer. Dihydropyrimidine dehydrogenase (DPD) is the main enzyme responsible for FP catabolism. Low DPD activity screening has proven its predictive clinical value in identifying patients at risk for toxicity. However, high DPD activity may translate to decreased FP activation and efficacy.

Patients and methods: One hundred and forty-three patients (monocentric retrospective study) underwent a pretreatment assessment of DPD activity in lymphocytes between 01/01/2004 and 20/04/2016. Included patients were male or female, >18 years, FP-treatment-based. Cut-off for DPD activity was assessed using smoothing spline curves.

Results: Median patient follow-up was 30 months [CI95%: 27.3-36.2], mean age 63+/-3 years, females accounted for 53%, 90% of patients had a good performance status (0 or 1). Fifty-eight percent of FP indications (65% 5-FU and 35% capecitabine) were adjuvant or neoadjuvant and 42% were prescribed for local or metastatic recurrences. Tolerance was poor for 43% of patients and 49% needed dose reduction. Objective response (complete and partial) was observed in 50% of patients, stable disease in 38% and progression in 12%. No significant correlation was observed between DPD activity and response to treatment. Mean DPD activity (pmol min−1 mg−1 protein) was 0.21+/-0.1 (quartile: [0.001-0.14-0.20-0.28-0.48]). DPD activity analyzed as a continuous variable was significantly linked to overall survival (OS) (p=0.042) but not with progression-free survival (PFS). The higher the DPD activity, the lower the OS. DPD activity analyzed as a binary variable (cut-off at 0.30) was significantly linked to overall survival (p=0.012) but also with PFS (p=0.016). A Cox regression model for OS and PFS adjusted for age, sex, type of cancer, type of administered FP, and associated surgical or radiotherapy treatment was performed. After adjustment, DPD activity remained significantly linked to OS (p=0.03) and PFS (p=0.021).

Conclusions: DPD activity screening could lead to a two-pronged approach: FP dose reduction in the event of low DPD and dose increase for high DPD. Such a strategy needs to be prospectively validated under personalized DPD-based treatment.

Note: This abstract was not presented at the meeting.

Citation Format: Emmanuel Chamorey, Eric Francois, Marie-Christine Etienne-Grimaldi, Julien Viotti, Frederic Peyrade, Alexandre Bozec, Jean-Marc Ferrero, Renaud Schiappa, Gerard Milano. High intrinsic DPD activity matters too [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 3823. doi:10.1158/1538-7445.AM2017-3823
Abstract 3829: TCIRG1, T-cell immune regulator 1, functions as a metastatic enhancing gene in liver tumorigenesis
Authors: Yang Hee Doo , Eun Jung Woo , Shen Qingyu , Kim Hyung Seok , Kim Sang Yean , Nam Suk Woo
Published: July 2017

Recurrence and metastasis are a major challenge in the management of hepatocellular carcinoma (HCC) patients after HCC resection. In order to identify metastatic molecular signature, we performed comparative gene expression profiling analysis with recurred HCC tissues from HCC patients who underwent partial or total hepatectomy and non-metastatic primary HCC. From this, we were able to recapitulate molecular signatures associate with HCC recurrence, and found that TCIRG1 (T-Cell Immune Regulator 1) was one of the aberrantly overexpressed gene elements in recurred HCC patients with total hepatectomy. We then observed that TCIRG1 is significantly overexpressed in TCGA-LIHC (Liver Hepatocellular Carcinoma Dataset of The Cancer Genome Atlas), and high expression of TCIRG1 was significantly associated with poor prognosis of HCC patients in 5-year disease-free. TCIRG1 knockdown suppressed tumor cell growth and proliferation in Huh7 and SNU475, liver cancer cell lines. From flow cytometric analysis for cell cycle analysis and cell death, we observed that TCIRG1 knockdown caused a significant increase of G1/S phase, and also induced programmed cell death processing in liver cancer cells. TCIRG1 knockdown also reduced the metastatic potential of HCC cells by selectively regulating epithelial-mesenchymal transition (EMT) regulatory proteins, E-cadherin, N-cadherin, Vimentin, Fibronectin, Snail and Slug, in liver cancer cells. In addition, sustained expression of TCIRG1 in liver cancer cell line reduced tumor growth rate in mouse xenograft model. Moreover, targeted-disruption of TCIRG1 significantly attenuated the metastatic potential of ras-transformed NIH-3T3 cells in vitro and in vivo. In conclusion, our findings suggest that TCIRG1 functions as metastatic enhancing gene by modulating cellular growth, death, and EMT in liver cancer cells providing TCIRG1 as a therapeutic target for the treatment of liver malignancy and metastasis.

Citation Format: Hee Doo Yang, Jung Woo Eun, Qingyu Shen, Hyung Seok Kim, Sang Yean Kim, Suk Woo Nam. TCIRG1, T-cell immune regulator 1, functions as a metastatic enhancing gene in liver tumorigenesis [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 3829. doi:10.1158/1538-7445.AM2017-3829
Abstract 3843: Autologous resconstitution of human cancer and immune system in vivo
Authors: Fu Juan , Sen Rupashree , Masica David L , Karchin Rachel , Pardoll Drew , Kim Young
Published: July 2017

Correlative studies from checkpoint inhibitor trials have indcated that better understanding of human leukocytic trafficking into the human tumor microenvironment can expedite the translation of future immune-oncologic agents. In order to directly characterize signaling pathways that can regulate human leukocytic trafficking into the tumor, we have developed a completely autologous xenotransplanation method to reconstitute the human tumor immune microenvironment in vivo. When we analyzed the TCGA database of human head and neck squamous cell carcinoma(HNSCC), we found that STAT3 signaling was associated with worse prognostic mesenchymal subtye. We silenced STAT3 signaling in the tumor compartment in these autologously reconstituted humanized mice, and we noted increased tumor infiltrating lymphocytes and slower tumor growth rate. We also used this novel agents that can alter endogenous leukocytic infiltration into the tumor. Taken together, we present a valuable method to study individialized human tumor microenvironments in vivo without confound allgeneic responses.

Citation Format: Juan Fu, Rupashree Sen, David L Masica, Rachel Karchin, Drew Pardoll, Young Kim. Autologous resconstitution of human cancer and immune system in vivo [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 3843. doi:10.1158/1538-7445.AM2017-3843