Yvonne A. Evrard

Monitoring drug-induced gammaH2AX as a pharmacodynamic biomarker in individual circulating tumor cells.

Purpose: Circulating tumor cells (CTC) in peripheral blood of patients potentially represent a fraction of solid tumor cells available for more frequent pharmacodynamic assessment of drug action than is possible using tumor biopsy. However, currently available CTC assays are limited to cell membrane antigens. Here, we describe an assay that directly examines changes in levels of the nuclear DNA damage marker γH2AX in individual CTCs of patients treated with chemotherapeutic agents. Experimental Design: An Alexa Fluor 488–conjugated monoclonal γH2AX antibody and epithelial cancer cell lines treated with topotecan and spiked into whole blood were used to measure DNA damage–dependent nuclear γH2AX signals in individual CTCs. Time-course changes in both CTC number and γH2AX levels in CTCs were also evaluated in blood samples from patients undergoing treatment. Results: The percentage of γH2AX-positive CTCs increased in a concentration-dependent manner in cells treated with therapeutically relevant concentrations of topotecan ex vivo. In samples from five patients, percent γH2AX-positive cells increased post-treatment from a mean of 2% at baseline (range, 0-6%) to a mean of 38% (range, 22-64%) after a single day of drug administration; this increase was irrespective of increases or decreases in the total CTC count. Conclusions: These data show promise for monitoring dynamic changes in nuclear biomarkers in CTCs (in addition to CTC count) for rapidly assessing drug activity in clinical trials of molecularly targeted anticancer therapeutics as well as for translational research. Clin Cancer Res; 16(3); 1073–84

Development of a Validated Immunofluorescence Assay for γH2AX as a Pharmacodynamic Marker of Topoisomerase I Inhibitor Activity

Purpose: Phosphorylated histone H2AX (γH2AX) serves as a biomarker for formation of DNA double-strand break repair complexes. A quantitative pharmacodynamic immunofluorescence assay for γH2AX was developed, validated, and tested in human tumor xenograft models with the use of clinically relevant procedures. Experimental Design: The γH2AX immunofluorescence assay uses a novel data quantitation and image processing algorithm to determine the extent of nuclear-specific γH2AX staining in tumor needle biopsies and hair follicles collected from mice bearing topotecan-responsive A375 xenografts. After method validation with the topoisomerase I (Top1) inhibitor topotecan, the assay was used to compare pharmacodynamic properties of three structurally related indenoisoquinoline Top1 inhibitors. Results: γH2AX response to topotecan was quantified over a 60-fold dose range (0.016-1.0 times the murine single-dose maximum tolerated dose), and significant pharmacodynamic response was measured at the mouse equivalent of the 1.5 mg/m2 clinical dose as well as the lowest dose tested. Responses were within a time window amenable for biopsy collection in clinical trials. These studies enabled characterization of dose and time responses for three indenoisoquinolines, resulting in selection of two for clinical evaluation. γH2AX response to Top1 inhibitors in hair follicles was also observable above a minimal dose threshold. Conclusions: Our γH2AX assay is sufficiently accurate and sensitive to quantify γH2AX in tumor samples and will be used in correlative studies of two indenoisoquinolines in a phase I clinical trial at the National Cancer Institute. Data suggest that hair follicles may potentially serve as a surrogate tissue to evaluate tumor γH2AX response to Top1 inhibitors. Clin Cancer Res; 16(22); 5447–57. ©2010 AACR.

Promise and limits of the CellSearch platform for evaluating pharmacodynamics in circulating tumor cells

Circulating tumor cells (CTCs), which are captured from blood with anti-epithelial cell adhesion molecule (EpCAM) antibodies, have established prognostic value in specific epithelial cancers, but less is known about their utility for assessing patient response to molecularly targeted agents via measurement of pharmacodynamic (PD) endpoints. We discuss the use of CellSearch (Janssen Diagnostics, LLC, Raritan, NJ) CTC isolation technology for monitoring PD response in early phase trials. We present representative data from three clinical trials with the poly(ADP-ribose) polymerase (PARP) inhibitor veliparib (ABT-888) suggesting that CTCs can be used to measure PD effects. However, while often leading to hypothesis-generating information, our experience points to the difficulty in obtaining sufficient EpCAM-expressing CTCs from patients with advanced disease to reach statistically significant conclusions about PD effects from each trial. Overall, the level of phenotypic heterogeneity observed in specimens from patients with advanced carcinomas suggests caution in the use of cell-surface differentiation marker-based methods for isolating CTCs.

Pharmacodynamic Response of the MET/HGF-Receptor to Small Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays

Purpose: Rational development of targeted MET inhibitors for cancer treatment requires a quantitative understanding of target pharmacodynamics, including molecular target engagement, mechanism of action, and duration of effect. Experimental Design: Sandwich immunoassays and specimen handling procedures were developed and validated for quantifying full-length MET and its key phosphospecies (pMET) in core tumor biopsies. MET was captured using an antibody to the extracellular domain and then probed using antibodies to its C-terminus (full-length) and epitopes containing pY1234/1235, pY1235, and pY1356. Using pMET:MET ratios as assay endpoints, MET inhibitor pharmacodynamics were characterized in MET-amplified and -compensated (VEGFR blockade) models. Results: By limiting cold ischemia time to less than two minutes, the pharmacodynamic effects of the MET inhibitors PHA665752 and PF02341066 (crizotinib) were quantifiable using core needle biopsies of human gastric carcinoma xenografts (GTL-16 and SNU5). One dose decreased pY1234/1235 MET:MET, pY1235-MET:MET, and pY1356-MET:MET ratios by 60% to 80% within 4 hours, but this effect was not fully sustained despite continued daily dosing. VEGFR blockade by pazopanib increased pY1235-MET:MET and pY1356-MET:MET ratios, which was reversed by tivantinib. Full-length MET was quantifiable in 5 of 5 core needle samples obtained from a resected hereditary papillary renal carcinoma, but the levels of pMET species were near the assay lower limit of quantitation. Conclusions: These validated immunoassays for pharmacodynamic biomarkers of MET signaling are suitable for studying MET responses in amplified cancers as well as compensatory responses to VEGFR blockade. Incorporating pharmacodynamic biomarker studies into clinical trials of MET inhibitors could provide critical proof of mechanism and proof of concept for the field. Clin Cancer Res; 22(14); 3683–94. ©2016 AACR.

PDX-MI: Minimal Information for Patient-Derived Tumor Xenograft Models

Patient-derived tumor xenograft (PDX) mouse models have emerged as an important oncology research platform to study tumor evolution, mechanisms of drug response and resistance, and tailoring chemotherapeutic approaches for individual patients. The lack of robust standards for reporting on PDX models has hampered the ability of researchers to find relevant PDX models and associated data. Here we present the PDX models minimal information standard (PDX-MI) for reporting on the generation, quality assurance, and use of PDX models. PDX-MI defines the minimal information for describing the clinical attributes of a patients tumor, the processes of implantation and passaging of tumors in a host mouse strain, quality assurance methods, and the use of PDX models in cancer research. Adherence to PDX-MI standards will facilitate accurate search results for oncology models and their associated data across distributed repository databases and promote reproducibility in research studies using these models. Cancer Res; 77(21); e62-66. ©2017 AACR.

Pharmacodynamic analyses in a multi-laboratory network: lessons from the poly(ADP-ribose) assay

Clinical pharmacodynamic assays need to meet higher criteria for sensitivity, precision, robustness, and reproducibility than those expected for research-grade assays because of the long duration of clinical trials and the potentially unpredictable number of laboratories running the assays. This report describes the process of making an immunoassay based on commercially available reagents clinically ready. The assay was developed to quantify poly(ADP-ribose) (PAR) levels as a marker of PAR polymerase inhibitor activity for a proof-of-concept phase 0 clinical trial at the National Cancer Institute (NCI) and subsequent clinical trials. In this publication, we retrospectively examine the measures taken to validate the published PAR immunoassay and outline key lessons learned during the development and implementation of these procedures at both internal and external clinical trial sites; these measures included optimizing PAR measurements in tumor biopsies and peripheral blood mononuclear cells (PBMCs), reagent qualification, analytical validation and assay quality control, instrument qualification and method quality control, and support for external laboratories.

Abstract 1039: PDX models generated from a patient with metastatic colon adenocarcinoma display both spatial and temporal tumor heterogeneity

Background: Patient-derived Xenograft (PDX) models are being widely used in preclinical studies to identify biomarkers of drug response and to enhance our understanding of cancer biology. Since patients with metastatic cancer have both intra-tumor and inter-site heterogeneity, PDX models generated from different tumor sites may provide a way to study tumor heterogeneity. Characterization of the genomic landscape in these models may also provide better insights into treatment response or resistance. It is rare to have multiple PDX models generated from a single patient over multiple time points during a treatment trajectory. Here, we report the genomic profiles of PDX models generated from 4 distinct tissue specimens over a 7-month period from a patient with metastatic colon adenocarcinoma. The first 2 PDX models were generated from circulating tumor cells (CTCs) and a liver biopsy prior to treatment with a combination pan-AKT + MEK inhibitor regimen. A third PDX model was generated from a liver biopsy while on-treatment and a fourth from an adrenal gland resection at progression. Clinically, all reported metastatic sites, except the adrenal gland, responded to the combination therapy. Results: Genomic characterization of the specimens obtained from these 4 PDX models led to the following observations: 1) PIK3CA E545K and KRAS G12D are present in all the specimens tested for all 4 models and are likely truncal driver mutations; 2) exclusive inter-model SNVs (single nucleotide variants) were identified, and may be model-specific variants representing inter-site heterogeneity in the patient; 3) variants involved in known resistance mechanisms to MEK inhibition were not present in any specimens; 4) overexpression of AKT3 has been reported as a resistance mechanism to a pan-AKT inhibitor and was observed in the adrenal tissue from the patient but not in any other PDX model derived from this patient; 5) intra-model and inter-model heterogeneity in whole genome CNV (copy number variant) profiles was observed between individual PDXs obtained from the pre-treatment CTC-derived model and the on-treatment liver biopsy model. Interestingly, one of the PDXs from the CTC-derived model presented a sub-clonal tumor fraction closely related to the on-treatment liver biopsy model. The multiple inter-model CNV profiles in the liver biopsy derived PDX models represent temporal heterogeneity within a tissue. Conclusions: We observed genomic heterogeneity in PDXs generated from specimens from a patient with metastatic colon adenocarcinoma. Both truncal and sub-clonal variants were identified representing various tumor fractions in these models. This case study illustrates how genomic profiling of multiple tumor sites at different times during course of treatment can provide insight into the complexity of tumor heterogeneity and tumor evolution in patients with metastatic disease. Citation Format: Biswajit Das, Chris Karlovich, Corrine E. Camalier, Rajesh Patidar, Li Chen, Vivekananda Datta, William D. Walsh, Sean P. McDermott, Tomas Vilimas, Palmer Fliss, Justine N. McCutcheon, Amanda Peach, Michelle Ahalt-Gottholm, Carrie Bonomi, Kelly Dougherty, John Carter, Shivaani Kummar, Yvonne A. Evrard, Melinda G. Hollingshead, Paul M. Williams, James H. Doroshow. PDX models generated from a patient with metastatic colon adenocarcinoma display both spatial and temporal tumor heterogeneity [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 1039.

Abstract IA21: NCI's Patient-Derived Models Repository: Generating models from racial and ethnic minorities

The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprising quality-controlled, early-passage, clinically annotated patient-derived xenografts (PDXs) to serve as a resource for public-private partnerships and for academic drug discovery efforts. The first 100 models were released in May 2017, are clinically annotated with pathologic and molecular information available in a publicly accessible database, and are available to the extramural community for research use (pdmr.cancer.gov). The PDMR was established by NCI at the Frederick National Laboratory for Cancer Research (FNLCR) in direct response to discussions with academia and industry; the oncology community9s highest-priority need was identified as better preclinical models that more faithfully reflect the patient9s tumor and are associated with the patient9s treatment history. NCI has focused on collecting specimens from patients with cancers that are under-represented in many other PDX collections such as head and neck, pancreatic, bladder and gynecologic cancers, melanomas, and sarcomas. Recently, NCI has also increased its focus on generation of PDXs from racial and ethnic minorities through new funding opportunities. The initial goal is to have 20-25% of the models in the PDMR from racial and ethnic minorities in diagnoses that are most pertinent to cancer health disparities. The overall goal of NCI is to create a long-term home for at least 1,000 models such that sufficient biologic, clinical, and population diversity is represented to allow researchers to ask questions such as: what is the impact of tumor heterogeneity on target qualification or clinical response; do PDXs more faithfully represent the human tumor for pharmacodynamic assay and predictive marker development; or can an adequately powered preclinical PDX clinical trial lead to better evaluation of therapies for future clinical use? 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: Yvonne A. Evrard. NCI9s Patient-Derived Models Repository: Generating models from racial and ethnic minorities [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr IA21.

Abstract 3840: The National Cancer Institute’s patient-derived models repository (PDMR)

The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprised of quality-controlled, early-passage, clinically-annotated patient-derived xenografts (PDXs) to serve as a resource for public-private partnerships and academic drug discovery efforts. 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, RNASeq), which is available in a publicly accessible database. The PDMR was established by NCI at the Frederick National Laboratory for Cancer Research (FNLCR) in direct response to discussions with academia and industry; the oncology community9s highest priority need was preclinical models that more faithfully reflect the patient9s tumor and are associated with the patient9s treatment history. NCI has focused on generating models to complement existing PDX collections and address unmet needs in the preclinical model space. The PDMR generates the majority of its PDXs by subcutaneous implantation except for those histologies having better success rates in either orthotopic or alternate implant sites. All SOPs and quality-control standards developed by the PDMR as well as those shared by collaborators are posted to a public web site that houses the PDMR database. In May 2017, the public website (https://pdmr.cancer.gov/) went live with its first 100 models from histologies including pancreatic, colorectal, renal, head and neck, and lung squamous cell cancers as well as melanoma and adult soft tissue sarcomas. In early 2018, the PDMR will begin releasing models from gynecological cancers, small cell lung cancer, chondro/osteo sarcomas, lung adenocarcinoma, and squamous cell skin and Merkel cell carcinomas. In addition, wherever available germline sequence and somatic variant calls will be added to the existing molecular characterization data for each model. NCI has also increased its focus on creating PDXs from racial and ethnic minorities through several funding opportunities. The overall goal of NCI is to create a long-term home for at least 1000 models such that sufficient biological and clinical diversity is represented to allow researchers to ask questions regarding the impact of tumor heterogeneity on target qualification or clinical response, whether PDXs more faithfully represent the human tumor for pharmacodynamic assay and predictive marker development, or if adequately powered preclinical PDX clinical trials can lead to better evaluation of therapies for future clinical use. Moving forward the PDMR plans to distribute in vitro, early-passage tumor cell cultures and cancer-associated fibroblasts as well as releasing PDX drug response data for a panel of FNA-approved therapeutic agents. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Michelle M. Gottholm Ahalt, Sergio . Y. Alcoser, Kaitlyn Arthur, Mariah Baldwin, Linda L. Blumenauer, Carrie Bonomi, Suzanne Borgel, Elizabeth Bradtke, Corinne Camalier, John Carter, Tiffanie Chase, Alice Chen, Lily Chen, Donna W. Coakley, Nicole E. Craig, Biswajit Das, Vivekananda Datta, Jordyn Davidson, Margaret R. DeFreytas, Emily Delaney, Michelle A. Eugeni, Raymond Divelbiss, Palmer Fliss, Thomas Forbes, Marion Gibson, Tara Grinnage-Pulley, Sierra Hoffman, Lilia Ileva, Paula Jacobs, Franklyn Jimenez, Joseph Kalen, Catherine Karangwa, Chris Karlovich, Candace Mallow, Chelsea McGlynn, Jenna E. Moyer, Michael Mullendore, Dianne L. Newton, Nimit Patel, Rajesh Patidar, Kevin Plater, Marianne Radzyminski, Lisa Riffle, Larry Rubinstein, Luke H. Stockwin, Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. The National Cancer Institute9s patient-derived models repository (PDMR) [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 986.

Abstract PL08-02: NCI patient derived models repository

The National Cancer Institute is developing a national repository of patient-derived cancer models (PDMs) comprised of[T] clinically-annotated patient-derived xenografts (PDXs); patient-derived tumor cell cultures (PDCs, including conditionally-reprogrammed tumor cell cultures) prepared from primary and metastatic tumors, circulating tumor cells (CTCs), and/or PDXs; tumor cell lysates, DNA, and RNA; and cancer-associated fibroblast cell lines (CAFs, autologous when possible) to serve as a resource for academic discovery efforts and public-private partnerships for drug discovery. NCI will provide a long-term home for >1000 PDX and PDC models, each produced from tissues and blood supplied by NCI-designated Cancer Centers and NCI-supported clinical trials networks. The effort is targeting the collection of tumors that are less prevalent in current resources, such as: small cell lung cancer, prostate cancer, bladder cancer, pancreatic cancer, head and neck cancers, as well as sarcomas and melanomas. The goals of the project are: (1) to develop a minimum of ∼50 unique patient models (both PDXs and PDCs) per disease such that the size of each molecularly-characterized subgroup is useful for subsequent validation and/or efficacy studies; (2) to perform comprehensive pre-competitive molecular characterization of patient samples and earliest passage PDXs and PDCs that includes the NCI-MPACT mutation panel, WES, RNASeq, copy number determination, histology, growth curves, and pilot proteomic/phospho-proteomic studies; and (3) to make all models and associated pre-clinical and clinical data available through a publicly available website. To date, over 1700 specimens from 1100 patients have been received for the development of PDMs; the overall ‘take9 rate for PDXs originating from solid tumors is 70% with >170 assessable models and another 270 early passage tumors currently in evaluation. As expected, based on collection priorities, tumors of genitourinary, digestive, head and neck, musculoskeletal, respiratory, and skin origin are the major histological sites of origin for our PDX models. In addition, over 90 conditionally-reprogrammed cell lines have been expanded from both 18-gauge needle biopsies and surgical resections, and have passed initial quality control procedures; many of these cell cultures have a matched PDX. Over 150 CAF lines have been developed following repeated (>10) purification steps using flow cytometry and are in the process of quality control procedures that demonstrate complete lack of growth in NOD-SCID gamma IL2 receptor null (NSG) mice; of these CAFs, we have developed matched pairs of PDCs and CAFs from the same patient in 16 cases. To evaluate the potential utility of the NCI PDM Repository, we have prospectively ‘entered9 22 models in a pre-clinical trial for which eligibility (based on actionable mutations) and treatment arms are identical to those in the NCI-MPACT study (NCT01827384). Multiple objective responses (significant improvement in overall survival) have been observed in all arms of the study and in a variety of models. WES and RNASeq analysis have proven essential to explain the therapeutic responses that we have observed. We are also evaluating the relationship of in vitro and in vivo activity for the NCI-MPACT drug panel in the models where concurrent PDCs and PDXs have been produced. A web site has been developed that will provide annotated information on the models (such as DNA sequence, gene expression, prior therapy) to investigators to assist in the distribution of the contents of the repository to the research community. We expect to be able to begin distribution in late spring of 2016. Citation Format: James H. Doroshow, Melinda Hollingshead, Yvonne Evrard, P. Mickey Williams, Vivekananda Datta, Biswajit Das, Chih-Jian Lih, Dianne Newton. NCI patient derived models repository. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL08-02.