No abstract

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.

The National Cancer Institute has developed a Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) comprised of quality-controlled, early passage, and clinically-annotated patient-derived tumor xenografts (PDXs), organoids (PDOrgs), cell cultures (PDCs), and cancer associated fibroblasts (CAFs) available with genomic data to the extramural community for research use. Models are developed by the NCI PDMR in decreasing order of complexity, specifically 1) patient material can be used to develop PDXs, PDOrgs, PDCs, and CAFs, 2) PDX material for PDOrgs and PDCs, and 3) PDOrg material for PDCs, but a PDC is never used to develop a PDOrg or PDX. Eleven pairs of 22 matched PDCs have been developed in parallel from either patient, PDX, patient-derived organoid, or PDX-derived organoid tumor material and sequenced by WES and RNASeq. Genetic stability was assessed using multiple approaches including microsatellite instability (MSI) generated from MSISensor2, percentage of genomic loss of heterozygosity (LOH) using a set of ~800k heterozygous SNPs from a population level genomic database (gnomAD), pairwise Spearman correlation based on BIN level copy number (CN)/RNA expression profiles, and OncoKB annotated oncogenic/likely oncogenic variants. No systematic differences were observed within PDC pairs derived from different origins or compared to their patient and/or PDX material in MSI, LOH% and RNA expression profile but pairwise Spearman correlation (0.66-0.88) in CNV profiles were somewhat variable, likely due to low sequencing depth. In one PDC pair (299254), 3 out of 12 OncoKB annotated Indels and CNV showed opposite level of variant allele frequencies/CN when comparing a model derived from patient material to one developed from a PDX-derived organoid, possibly driven by a lineage-specific subclonal outgrowth when compared to patient and PDX data. Phenotypic characteristics of matched PDCs also overall show no major differences, though variability in growth rates and the ability to form spheroids in serum-free medium were noted. In one pair (919269), the PDC derived from patient material was able to form a cell line xenograft (CLX) in NSG mice but not the PDC developed from a patient-derived organoid. Overall, these models demonstrate a high degree of concordance at the genetic and phenotypic level when compared to the originating patient and/or PDX tumor. Though further characterization (e.g., preclinical drug testing) may be needed to define differences between matched PDC pairs, lack of access to patient tissue or failure to generate tumor cell cultures from one source of material should not hamper development of preclinical in vitro models from other patient-derived model types as long as the source-of-origin is clearly defined. Funded by NCI Contract No. HHSN261200800001E Citation Format: Cindy R. Timme, Ting-Chia Chang, Sergio Y. Alcoser, Gareth Bliss, Carrie Bonomi, Suzanne Borgel, John Carter, Alice Chen, Li Chen, Kevin Cooper, Biswajit Das, Kelly Dougherty, Lindsay Dutko, Marion Gibson, Michelle M. Gottholm-Ahalt, Tara Grinnage-Pulley, Shahanawaz Jiwani, Keegan Kalmbach, Chris Karlovich, Kimberly Klarmann, Tiffanie Chase, Michael Mullendore, Matthew Murphy, Kevin Plater, Gloryvee Rivera, Jessica Steed, Luke Stockwin, Yvonne A. Evrard, Mickey Williams, Dianne L. Newton, Melinda G. Hollingshead, James H. Doroshow. Comparing twenty-two matched patient-derived cell lines developed from either patient, PDX, or organoid tumor cell material [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 36.

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.