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There is an unmet need for preclinical models of rare cancers and rare disease sub-types. The National Cancer Institute's Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) is developing quality-controlled, early-passage, clinically-annotated patient-derived tumor xenografts (PDXs), in vitro tumor cell cultures (PDCs), cancer associated fibroblasts (CAFs), and patient-derived organoids (PDOrg) and has focused on addressing unmet needs in the preclinical model space including developing models from adult and pediatric patients with rare cancers. To date, NCI has created and molecularly characterized over 150 preclinical models of rare cancer including indications such as Hurthle cell carcinoma, osteosarcomas, Merkel cell carcinomas, salivary gland cancers, synovial sarcomas, and carcinosarcomas. Rare cancer models developed to date will be reviewed and their histopathologic and molecular characteristics compared to that reported in the clinical setting. A pipeline to identify fusion proteins in these rare cancers such as the Ewing sarcoma EWSR1-FLI1 fusion and NAB2-STAT6 fusions in solitary fibrous tumors (SFT) has been implemented. Four malignant peripheral nerve sheath tumors (MPNST) PDX models are available for researches; these models were developed from patients diagnosed between the ages of 37-68. At the time of model development, two patients were treatment naïve and two had prior radiotherapy. Two of the MPNST PDX models have NF1 oncogenic mutations, three have deep deletions in CDKN2A/B, and three have a mutation in either EED or SUZ12 consistent with the reported molecular characteristics of patients with MPNST. Also of clinical relevance, of two mesothelioma models available, one carries an NF2 driver mutation and the other BAP1 and LATS2 and a PDX model for Hurthle cell carcinoma has wide-spread loss of heterozygosity (LOH 80%). Models for other rare cancers are in development, including four cholangiocarcinoma PDXs with histopathologic confirmation that are currently being expanded for molecular characterization and distribution. Funded by NCI Contract No. HHSN261200800001E Citation Format: Cindy R. Timme, Sergio Y. Alcoser, Devynn Breen, John Carter, Ting-Chia Chang, Alice Chen, Li Chen, Kristen Cooley, Biswajit Das, Emily Delaney, Michelle A. Eugeni, Michelle M. Gottholm-Ahalt, Tara Grinnage-Polley, Jenna Hull, Chris Karlovich, Kimberly Klarmann, Shahanawaz Jiwani, Candace Mallow, Chelsea McGlynn, Justine Mills, Malorie Morris, Michael Mullendore, Dianne Newton, Tia Shearer, Jesse Stottlemyer, Shannon Uzelac, Thomas Walsh, P. Mickey Williams, Yvonne A. Evrard, Melinda G. Hollingshead, James H. Doroshow. Patient-derived models of rare cancers in the National Cancer Institute's patient-derived models repository [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3012.

Background: Well characterized patient derived xenograft models (PDX) are becoming the preferred pre-clinical tool in translational cancer research for biologic understanding of the disease, development of new treatments, and identifying potential therapy predictive and resistant biomarkers. Characterization of PDX models using a multi-omic approach is most desirable, however such efforts can be expensive and technically demanding. Immunohistochemistry (IHC) has become an indispensable ancillary tool in the accurate classification of tumor types, determination of cell of origin, identification of biologic properties like growth and metastatic potential, and evaluation for the presence/absence of therapeutic or prognostic biomarkers. Methods: 43 IHC assays were validated on the Leica Bond RX automated staining platform to identify common inconsistencies in PDX development including markers for classifying carcinomas, lymphomas, sarcomas, murine tumors, and theragnostic biomarkers. Rabbit antibodies are used rather than mouse antibodies to prevent non-specific staining of murine tissue. Results: 1. IHC evaluation of models within NCI's Patient Derived Models Repository (pdmr.cancer.gov) led to re-classification or sub-classification of 12 tumor models in accordance with WHO guidelines. 2. IHC evaluation of theragnostic markers in 8 breast cancer PDX models showed concordant results throughout passaging, suggesting stability of these biomarkers in our models. 3. We observe malignant transformation of murine or transplanted benign human tissue at a rate of 2.5%. On IHC analysis, 52% were human lymphomas, 20% were murine lymphomas, and 28% were other murine tumors. Conclusions: IHC is a rapid, cost-effective tool that can be used for accurate tumor classification, identifying subclonal outgrowth and tumor evolution, assessing stability of biomarkers and identifying malignant transformation of benign tissue. Funded by NCI Contract No. HHSN261200800001E ANTIBODYCLONEVENDORANTIBODYCLONEVENDORAndrogen Receptor[EPR1535(2)]abcamGATA3[EPR16651]abcamB-Catenin[E247]abcamGCDFP-15[EPR1582Y]abcamCD19polyclonalabcamGFAPpolyclonalDAKO/AgilentCD3polyclonalabcamHER2 ErbB2[SP3]abcamCD20[SP32]abcamKi-67[D2H10]Cell SignalingCD34[EP373Y]abcamKu80[EPR3468]abcamCD45polyclonalabcamMGMTMT3.1MilliporeCD56 (NCAM1)[EPR2566]abcamMitochondria Marker (Biotin)MTC02abcamCD68[EPR20545]abcamMyogenin[EPR4789]abcamCDX2[EPR2764Y]abcamNAPSIN A[EPR6252]abcamChromogranin A[SP12]abcamp63polyclonalGeneTexCK7 (purified)[EPR1619Y]abcamPD-1[EPR4877(2)]abcamCK19[EPR1580Y]abcamPD-L1 (CD274)RBT-PDL1LifeSpan BiosciencesCK20[EPR1622Y]abcamProgesterone Receptor[SP2]abcamCytokeratin wide spectrumpolyclonalabcamProstate Specific Antigen (PSA)[EP1588Y]abcamDesmin[Y66]abcamS100[EPR19013]abcamEBV LMP1[D24-G]abcamSmooth Muscle Actin (SMA)polyclonalabcamERG[EPR3864]abcamSynaptophysin[SP11]abcamEstrogen Receptor[SP1]abcamTTF1[SP141]abcamFOXP1monoclonalLifeSpan BiosciencesVimentin[EPR3776]abcamFOXP3(5H10L18)Invitrogen Citation Format: Lindsay Dutko, Gloryvee Rivera, Erin Cantu, Vishnuprabha Rahulkannan, Kelly Benauer, Tiffanie Chase, Emily Delaney, Jesse Stottlemyer, Chelsea McGlynn, Howard Stotler, John Carter, Suzanne Borgel, Michelle M. Gottholm Ahalt, Michelle Eugeni, Melinda Hollingshead, Yvonne Evrard, Chris Karlovich, Biswajit Das, Mickey Williams, James H. Doroshow, Shahanawaz Jiwani. Applications of immunohistochemistry in characterization of patient derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3015.

Background: Acquired Uniparental Disomy (aUPD) is relatively common in cancer. Occurrence of aUPD is more frequent in some tumor histologies (e.g., serous ovarian, colorectal) and may be relevant for choice of therapy. The Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov) developed by The National Cancer Institute (NCI) includes patient-derived xenograft (PDX) models from multiple tumor histologies with different passages and lineages. The associated clinical annotation and genomic data make it possible to assess the prevalence of aUPD in the PDMR cohort and the stability of aUPD in different passages and lineages within a PDX model. Methods: High tumor purity in the PDX specimens (after removal of mouse reads representing the stroma) enabled highly accurate assessment of loss of heterozygosity (LOH). Variants called by GATK Haplotype caller from whole exome sequencing (WES) data were used to identify segments of homozygosity using BCFtools/RoH (runs of homozygosity). The RoH segments were then intersected with the bed file for chromosome arms to get %LOH at the arm level. If %LOH on a chromosome arm was >90%, we considered the sample to have aUPD at the arm level. WES was also used to look for associations between DNA damage repair (DDR) pathway alterations and aUPD. Results: We made the following observations: a) aUPD was observed most frequently in chr18q (75/427, 17.6%) and chr3p (69/427, 16%) of PDX models; b) aUPD was observed more frequently in certain tumor histologies, e.g., clear cell renal cell carcinoma (6/8), small cell lung cancer (3/4) and non-small cell lung cancer (25/38); c) extensive aUPD was observed in 4 PDMR models (>50% of evaluated chromosome arms in these models have aUPD); d) aUPD was not observed in some tumor histologies, i.e., synovial sarcoma, uterine endometrioid carcinoma; e) in the vast majority of PDMR models (>90%), aUPD is maintained faithfully across lineages and through multiple passaging; f) subclonal aUPD events were observed in some models across different lineages; g) significant enrichment of double strand DNA break repair (DSBR) pathway alterations was observed in PDMR models without aUPD (p=0.0007, Fisher's exact test) suggesting defects in DSBR are not associated with aUPD; and h) aUPD was rarely observed in MSI-high models (1/30) suggesting mutual exclusivity of mismatch repair (MMR) pathway defects and aUPD. Conclusion: We observed a relatively high frequency of UPD in the PDMR models (at least 1 arm of a chromosome). UPD was more frequently observed in specific chromosomal arms. The frequency of aUPD was higher in some tumor histologies and absent in others. aUPD was stably maintained across passages and lineages, although some heterogeneity was observed. Our data suggest aUPD is not associated with defects in DSBR and MMR pathways. Preclinical drug studies using NCI PDMR models may suggest appropriate therapeutic options for cancers with aUPD. Citation Format: Rajesh Patidar, Li Chen, Chris A. Karlovich, Biswajit Das, Yvonne A. Evrard, Tomas Vilimas, Justine N. McCutcheon, Amanda L. Peach, Nikitha V. Nair, Thomas D. Forbes, Brandie A. Fullmer, Anna J. Lee Fong, Luis E. Romero, Alyssa K. Chapman, Kelsey A. Conley, Robin D. Harrington, Shahanawaz S. Jiwani, Peng Wang, Michelle M. Gottholm Ahalt, Erin N. Cantu, Gloryvee Rivera, Lindsay M. Dutko, Kelly M. Benauer, Vishnuprabha R. Kannan, Suzanne D. Borgel, John P. Carter, Jesse M. Stottlemyer, Tiffanie L. Miner, Devynn R. Breen, Emily T. Delaney, Chelsea A. McGlynn, Candace N. Mallow, Marianne Radzyminski, Shannon N. Uzelac, Sergio Y. Alcoser, Tara L. Grinnage-Pulley, Michelle A. Eugeni, Dianne L. Newton, Melinda G. Hollingshead, Paul M. Williams, James H. Doroshow. Genomic landscape of acquired uniparental disomy in NCI PDMR patient derived xenograft models [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 3554.

The National Cancer Institute's Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) is performing a large-scale multi-year preclinical study with 39 PDX models of rare cancers (mesothelioma, MPNST, osteosarcoma, Merkel cell carcinoma, etc) treated with 56 novel therapeutic combinations in an exploratory, n-of-4 arm, study design. Combinations that show promising responses (e.g., regression or durable inhibition of tumor growth) will be repeated along with the single agent arms to determine if the response is driven by the combination or only one of the agents. In order to do this in a timely fashion, relatively speaking, the PDX tumors are serially passaged and each passage is treated with a set of 8 combinations plus relevant vehicle control(s) while in parallel enough PDXs are retained to be expanded for the next passage and drug set. Every serial passage undergoes several quality control assessments that serve as go/no-go criteria including pathology assessment, human:mouse DNA content assessment, and low pass whole genome sequencing to determine the average fraction of genome changed compared to the original donor material. If there is a QC failure, the PDX model is restarted from early passage cryo-material (passage 1-2). An additional quality control effort is to bookend the combination studies with the first set of agents to see if tumor response is similar across passages. To date, most of the models have demonstrated a high degree of stability, though a couple of models have moved toward murine content and have been restarted from early passage material so all drug combinations can be tested. DNA and RNA are retained from all passages so a full NGS evaluation can be performed at a later date. This effort has been ongoing for over a year and the first bookend studies are beginning to be tested to determine if response at first and last passage of the study are consistent with each other, given the constraints of the inherent heterogeneity of the models themselves. Single agent studies of drug combinations that demonstrated a response in 30%-50% of the models tested are also underway to determine which combinations have a more than additive effect compared to the single agents. Promising combinations will be moved forward to early phase clinical trials for these rare cancers. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Biswajit Das, Sergio Y. Alcoser, Suzanne Borgel, Devynn Breen, John Carter, Tiffanie Chase, Alice Chen, Lily Chen, Kristen Cooley, Emily Delaney, Raymond Divelbiss, Lyndsay Dutko, Thomas Forbes, Kyle Georgius, Michelle Gottholm-Ahalt, Tara Grinnage-Pulley, Sierra Hoffman, Chris Karlovich, Shahanawaz Jiwani, Justine Mills, Malorie Morris, Michael Mullendore, Dianne Newton, Rajesh Patidar, Gloryvee Rivera, Howard Stotler, Jesse Stottlemyer, Savanna Styers, Debbie Trail, Shannon Uzelac, Thomas Vilimas, Abigail Walke, Thomas Walsh, Nicole Walters, Peng Wang, P. Mickey Williams, Melinda Hollingshead, James H. Doroshow. Quality control efforts in a large-scale, preclinical trial of rare cancer PDXs by the National Cancer Institute's patient-derived models repository (NCI PDMR) [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 5056.

Background: The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR; www.pdmr.cancer.gov) of patient-derived xenografts (PDXs) with clinical annotation and comprehensive genomic characterization using whole exome sequencing (WES) and RNASeq. An in-house data analysis pipeline has been developed and validated to call germline and somatic variants and to perform transcriptional profiling in these models. There is a need to incorporate additional biomarkers into standard data analysis pipeline, including loss of heterozygosity (LOH), microsatellite instability (MSI), structure variants (SVs)/fusions and copy number variation (CNV) for identifying appropriate PDX models for preclinical drug studies. Validation of the methods used for the assessment of these and other genomic biomarkers is a crucial aspect in the development of the PDMR data analysis pipeline. Methods: WGS, WES and RNASeq were conducted on 58 PDX samples and genomic biomarkers were derived from different assays. For LOH calling, a set of ~800,000 heterozygous SNPs was first constructed from a population level genomic database (gnomAD) and a specific list of ~3000 highly heterozygous SNPs from a previous study. LOH regions were detected using Runs of Homozygosity (BCFtools/RoH) based on the genotypes of ~800,000 SNPs. Finally, percent of genomic LOH was calculated as the percent of eligible LOH regions in the whole genome. For MSI calling. mSINGS was used to assign a microsatellite instability score based on the fraction of unstable microsatellite loci. Gene fusions were detected using Tophat-fusion and Fusion-catcher from RNASeq data and Manta from WGS. CNVs were derived from WGS and WES using CNVkit. Results: Genomic biomarkers derived from WES and RNASeq were highly concordant with the ones derived from WGS. Specifically, we found 1) the percent of genomic LOH was highly correlated between WGS and WES across 52 samples with R2=0.99, where LOH% ranged from Conclusions: We observed excellent consistency between WGS, WES and RNASeq data in the assessment of percent of LOH, MSI score, SVs/fusions and CNVs. Our data analysis pipeline can accurately call genomic biomarkers from WES and RNASeq data, which facilitates the molecular characterization and prioritization of PDMR models for preclinical drug treatment. Citation Format: Li Chen, Rajesh Patidar, Biswajit Das, Chris Karlovich, Tomas Vilimas, Corinne Camalier, Vivekananda Datta, Shahanawaz Jiwani, William Walsh, Palmer Fliss, Sean McDermott, Justine N. McCutcheon, Amanda Peach, Michelle Ahalt-Gottholm, Carrie Bonomi, Kelly Dougherty, John Carter, Sergio Y. Alcoser, Tiffanie Chase, Raymond Divelbiss1, Marion Gibson, Kelly Hedger, Candace Mallow, Chelsea McGlynn, Malorie Morris, Marianne Radzyminski, Howard Stotler, Jesse Stottlemyer, Debbie Trail, Yvonne Evrard, Melinda G. Hollingshead, Mickey Williams, James H. Doroshow. Comparison of genomic biomarkers identified by the whole exome, RNASeq and whole genome sequencing pipelines developed for the PDMR [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 3385.

Background: Patient-derived tumor xenografts (PDX) are powerful tools to study cancer biology, cancer genomics and developmental therapeutics. A common problem in the development of PDX models is proliferation of atypical lymphocytes at the implantation site, which often overtake or limit the growth of the original tumor. This atypical lymphocyte proliferation has been described as XABLD in our PDX models. In this study, we characterized XABLD cases by morphology, immunophenotyping and genomic profiling. We hypothesize that XABLD tumors are morphologically and phenotypically similar to EBV-driven post-transplant lymphoproliferative disease (PTLD) and diffuse large B cell lymphoma (DLBCL). XABLD is a surrogate model to study EBV-driven PTLD and DLBCL. Materials and Methods: Models were generated from patient tissue collected under NCI Tissue Procurement Protocol (clinicaltrials.gov: NCT00900198) and CIRB Tissue Procurement Protocol 9846 for development of models for NCI’s Patient-Derived Models Repository (https://pdmr.cancer.gov). Specimens were implanted subcutaneously in NOD/SCID/IL2Rg null (NSG) mice and animal health was monitored throughout the study. Tumors in mice with suspected XABLD were harvested and reviewed by histology and immunohistochemical analysis for CD45, B and T cell markers, EBV status, B-cell clonality assay. All samples were also classified by the Lymph2Cx NanoString cell of origin assay and transcriptome profiling. Results: XABLD cases were found to originate from both solid tumor and circulating tumor cell implants. XABLD is a rapidly growing tumor positive for CD45, CD20, and LMP1 stains, 36 of 42 cases are strongly positive for PD-L1 stain. 39 of 42 cases exhibited an activated B cell (ABC) phenotype with evidence of elevated NF-kB signaling. Most cases were monoclonal for IGK/IGH and contained high numbers of tumor infiltrating CD8-positive T-cells with associated high mRNA expression of activated T cell markers. Conclusion: The clinical presentation, morphology and molecular characteristics of XABLD cases were similar to EBV-driven DLBCL. As the XABLD models exhibited frequent PD-L1 expression and marked infiltration of CD8-positive T cells, they may be useful for in vitro evaluation of checkpoint inhibitor response and T cell antitumor activity. Grant Support: This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Citation Format: Tomas Vilimas, Gloryvee Rivera, Brandie Fullmer, Wiem Lassoued, Lindsay Dutko, Amanda Peach, Corinne Camalier, Li Chen, Rajesh Patidar, Suzanne Borgel, John Carter, Howard Stotler, Raymond Divelbiss, Jesse Stottlemyer, Michelle M. Gottholm-Ahalt, Michelle Crespo-Eugeni, Sean McDermott, William Jacob, Liqiang Xi, Pallavi Galera, Yvonne A. Evrard, Melinda G. Hollingshead, Elaine S. Jaffe, Mark Raffeld, Biswajit Das, Chris Karlovich, Vivekananda Datta, James H. Doroshow, P. Mickey Williams. Xenograft-associated B cell lymphoproliferative disease (XABLD) as a surrogate model to study Epstein-Barr virus (EBV) driven B cell Diseases [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 1056.

The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprised of quality-controlled, early-passage, clinically-annotated patient-derived tumor xenografts (PDXs), in vitro tumor cell cultures (PDCs), cancer associated fibroblasts (CAFs), and patient-derived organoids (PDOrg). NCI has focused on generating models to complement existing PDX collections and address unmet needs in the preclinical model space. These models are offered to the extramural community for research use (https://pdmr.cancer.gov), along with clinical annotation and molecular information (whole exome sequence, gene expression using RNASeq), via a publicly accessible database. Currently, over 200 PDX models, 50 PDC models, and 100 CAF models are available for distribution to the US research community. Approximately 50 PDOrg models will be released in early 2019. As part of its rare cancer initiative, the NCI is also targeting the collection of infrequently-observed tumor histologies to advance both biological investigations and drug development efforts for under-studied malignancies. Comparison of matched models, models where more than one model type are available (e.g., PDX and PDC), demonstrate a high degree of concordance across the model types. Genetic stability across the models is assessed using multiple criteria including genetic assessment of CNVs and presence of driver mutations. Optimal CNV assessment uses whole exome sequence data corrected for cellularity in the patient specimen using germline reads and corrected for cellularity in the PDX specimens by subtraction of the mouse reads. Histomorphologic comparison of PDXs and cell line xenografts (CLX) generated from in vitro PDCs and PDOrgs also overall show a high degree of concordance, though loss of features and dedifferentiation can be observed in some models. Overall these models demonstrate a high degree of conservation at the genetic and pathologic level when compared to the patient tumor. These models can provide researchers the ability to perform high- or mid-throughput screening in 2D or 3D culture followed by targeted selection of PDX models for in vivo studies. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Dianne Newton, Biswajit Das, Sergio Y. Alcoser, Kaitlyn Arthur, Mariah Baldwin, Carrie Bonomi, Suzanne Borgel, John Carter, Tiffany Chase, Alice Chen, Lily Chen, Nikki E. Craig, Vivekananda Datta, Emily Delaney, Raymond Divelbiss, Kelly Dougherty, Thomas Forbes, Kyle Georgius, Joe Geraghty, Marion Gibson, Michelle M. Gottholm-Ahalt, Tara Grinnage-Pulley, Kelly Hedger, Sierra Hoffman, Chris Karlovich, Wiem Lassoued, Shahanawaz Jiwani, Candace Mallow, Chelsea McGlynn, Mallorie Morris, Jenna Moyer, Mike Mullendore, Matt Murphy, Rajesh Patidar, Kevin Plater, Marianne Radzyminski, Nicki Scott, Luke H. Stockwin, Howard Stotler, Jesse Stottlemyer, Savanna Styers, Debbie Trail, Tomas Vilimas, Anna Wade, Abigail Walke, Thomas Walsh, P. Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. Comparison of PDX, PDC, and PDOrg models from the National Cancer Institute’s Patient-Derived Models Repository (PDMR) [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 4524.

3131 Background: Currently, patient selection criteria for clinical testing of MET inhibitors are limited. Robust studies selecting patients based on MET protein expression, MET gene amplification, or mutations have not met their efficacy goals. Development of microscopy-based assays to quantify levels of phospho-MET (pMET) in tumors has been hampered by poor antibody specificity. Here, we present the development and validation of a robust, highly specific multiplex immunofluorescence assay (IFA) that measures pY1235-MET and total MET in tumor tissue. Methods: This assay utilizes antibodies to pY1235-MET (NCI-23111), total MET (D1C2), and plasma membrane (PM) marker Na+/K+-ATPase, each conjugated to a different Alexa Fluor dye. We used tumor tissue from crizotinib-treated SNU5 xenograft models to demonstrate pY1235-MET assay fitness-for-purpose and cross-platform assay concordance with our validated pMET ELISA. In addition, this IFA was validated by phospho-peptide competition using custom tissue microarrays (TMA) derived from patients with colorectal carcinoma (CRC). Finally, we developed quantitative algorithms to assess pY1235 MET levels in the plasma membrane and nucleus using PM and DAPI masks, respectively. Patient-derived xenograft models (PDX) were obtained from NCI’s Patient-Derived Models Repository (www.pdmr.cancer.gov). Results: The prevalence of high pY1235-MET expression in CRC patient specimens was greater than expected; of the 64 TMA cores evaluated, 29 (45%) and 19 (29%) had high pY1235-MET and total MET levels, respectively, as defined by mean marker area of ≥ 30 μm2/cell. To address the potential utility of pY1235-MET as a diagnostic biomarker, we examined 15 CRC PDX models by pMET ELISA and IFA. Two CRC tumor models were positive for pY1235-MET expression in both assays. The pY1235-MET IFA results and gene expression data were used to select PDX models for ongoing preclinical trials of potent MET inhibitors. Conclusions: This novel pY1235-MET IFA will enable clinicians to address the utility of activated MET as a biomarker for patient selection and/or prediction of response in clinical trials of MET inhibitors. Funded by NCI Contract No. HHSN261200800001E.

Background: Patient-derived tumor xenografts (PDX) are powerful tools to study cancer biology, cancer genomics and developmental therapeutics. A common problem in the development of PDX models is proliferation of atypical lymphocytes at the implant site, which often overtake or limit the growth of the original tumor. This atypical proliferation has been described as Xenograft-Associated B cell Lymphoproliferative Disease (XABLD) in our PDX models. In this study, we characterized XABLD cases by morphology, immunophenotyping and genomic profiling. We hypothesize that XABLD tumors are morphologically and phenotypically similar to EBV-driven lymphoma of the elderly and may function as a surrogate model for that lymphoma. Materials and Methods: Models were generated from patient tissue collected under NCI Tissue Procurement Protocol (clincialtrials.gov: NCT00900198) and CIRB Tissue Procurement Protocol 9846 for development of models for NCI's Patient-Derived Models Repository (https://pdmr.cancer.gov). Specimens were implanted subcutaneously in NOD/SCID/IL2Rg null (NSG) mice and animal health was monitored throughout the study. Tumors in mice with suspected XABLD were harvested and reviewed by histology and immunohistochemical analysis for CD45, B and T cell markers and EBV status. All samples in this study were classified by the Lymph2Cx NanoString cell of origin assay and transcriptome profiling. Results: XABLD-associated mice had rapidly growing CD45-positive tumors at the implantation site. Histopathological features were consistent with EBV-driven diffuse large B-cell lymphoma (DLBCL) primarily of polymorphous subtype. All XABLD specimens were diffusely positive for CD20 and EBNA, and most cases contained tumor infiltrating CD8-positive T-cells. Out of 42 cases, 36 were PD-L1-positive and 26 were PD-1-positive by IHC. 39 cases exhibited an activated B cell (ABC) phenotype, which is predominant in EBV-positive DLBCL. Conclusion: XABLD development has been seen across multiple patient histologies from both solid tumor and circulating tumor cells tissues of origin. The clinical presentation, morphology and molecular characteristics of XABLD cases were similar to EBV-driven DLBCL. As DLBCL is an aggressive disease with limited treatment options, our early-passage XABLD models may be useful in the preclinical evaluation of new therapies for EBV-positive DLBCL. Grant Support: This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Citation Format: Tomas Vilimas, Gloryvee Rivera, Brandie Fullmer, Wiem Lassoued, Lindsay Dutko, William Walsh, Amanda Peach, Corinne Camalier, Li Chen, Rajesh Patidar, Suzanne Borgel, John Carter, Howard Stotler, Raymond Divelbiss, Jesse Stottlemyer, Margaret Defreytas, Michelle M. Gottholm-Ahalt, Michelle A. Crespo-Eugeni, Sean McDermott, Yvonne A. Evrard, Melinda G. Hollingshead, Biswajit Das, Chris Karlovich, Vivekananda Datta, James H. Doroshow, P. Mickey Williams. Xenograft-associated B cell lymphoproliferative disease as a surrogate model to study Epstein-Barr virus (EBV) driven lymphoma of the elderly [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 1038.

Background: Modulation of cytidine methylation in DNA continues to be actively investigated as a target for chemotherapy. The primary source of methyl groups is methionine, via S-adenosyl-methionine (SAM) as the direct methyl donor for DNA methyl transferases, e.g., DNMT1. Methods were developed using stable isotopes and mass spec detection to examine the flux of the DNMT reaction and modification to this flux by cytidine analogs such as decitabine, an established hypomethylation agent, and 5-aza-4’-thio-2’-deoxy-cytidine (AzaTdC), currently under development at NCI. Methods: For in vitro studies; A549, H23, HCT116, H522, and OVCAR3 cell lines grown in RPMI1640 media supplemented with 10% FBS were exposed to D3-methionine(20uM) and either decitabine or AzaTdC (NSC77586) at 0 to 5uM in the media for 0 to 48hr. For in vivo studies; mice were maintained under standard ACUC protocols and practices. Tumors were grown SC in the flank area. Stable labeled methionine, decitabine and AzaTdC were administered as an oral bolus. At set times, mice were sacrificed and tumors, tissues and plasma samples collected. DNA was extracted and purified from cells and tissues using PureLink Genomic DNA kits (Invitrogen) according to manufacturer’s instruction. The purified DNA was digested to nucleosides for analysis. The isotopic enrichment in 5-methyl-deoxy-cytidine (mdC) was determined by analysis using HPLC coupled to a QE mass spectrometer (Thermo Scientific). Results: D3-methionine was found to be an efficient precursor via SAM for the methylation of cytidine in DNA. Fortification of basal media (100uM methionine) with 20uM D3-methionine yielded D3-mdC enrichment of 5-15% in 24 hr, depending on cell line. All cell lines were found to have some enrichment. Treatment of cells with either decitabine or AzaTdC caused a concentration dependent reduction of the enrichment for mdC in DNA. In mice, after an oral dose of D3-methionine, enrichment of methionine and SAM in plasma peaked within 1hr and fell to 5% of peak levels within 5hr. Methionine and SAM enrichment in tissues followed similar time curves. At 50mg/kg of D3-methionine, no perturbation was observed for endogenous levels of either methionine or SAM. Enrichment of mdC in DNA was detectable in 1hr, maximal in 5hr, and remained constant for up to 48hr. When mice were treated with 1-2 mg/kg of either decitabine or AzaTdC along with D3-methionine, the level of enrichment of mdC in DNA was reduced by 20-70% in tumors, bone marrow and intestine. Conclusions: Stable labeled methionine can be efficiently used to monitor the flux of methyl groups into mdC in DNA, both in vitro and in vivo. Changes in the flux of the methylation reaction can be used to assess the extent and time course for inhibition caused by cytidine analogs. These techniques may have relevance for improvement of doses and schedules to produce epigenetic modulation. Funded by NCI Contract No. HHSN261200800001E Citation Format: Lawrence Anderson, Eva Majerova, Kimberly D. Hill, John Carter, Jesse Stottlemyer, Howard Stotler, Melinda G. Hollingshead, Jerry M. Collins. Effects of epigenetic agents on methylation of DNA in vitro and in vivo, as measured with stable isotopically labeled methionine [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 4351. doi:10.1158/1538-7445.AM2017-4351