Thomas D. Pfister

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

ATR Inhibitors VE-821 and VX-970 Sensitize Cancer Cells to Topoisomerase I Inhibitors by Disabling DNA Replication Initiation and Fork Elongation Responses

Camptothecin and its derivatives, topotecan and irinotecan, are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress, came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821 confirmed marked antiproliferative synergy with camptothecin and even greater synergy with LMP-400. Single-cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970, enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof of principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials.

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.

Characterization of DNA topoisomerase I in three SN-38 resistant human colon cancer cell lines reveals a new pair of resistance-associated mutations

BackgroundDNA topoisomerase I (Top1) is a DNA unwinding protein and the specific target of the camptothecin class of chemotherapeutic drugs. One of these, irinotecan, acting through its active metabolite SN-38, is used in the treatment of metastatic colorectal cancer. However, resistance to irinotecan represents a major clinical problem. Since molecular alterations in Top1 may result in resistance to irinotecan, we characterized Top1 in three human colon cancer cell lines with acquired resistance to SN-38.MethodsThree SN-38 resistant (20–67 fold increased resistance) cell lines were generated and compared to wild-type parental cells with regards to: TOP1 gene copy number and gene sequence, Top1 expression (mRNA and protein), Top1 enzymatic activity in the absence and presence of drug, and Top1-DNA cleavage complexes in drug treated cells. TOP1 mutations were validated by PCR using mutant specific primers. Furthermore, cross-resistance to two indenoisoquinoline Top1-targeting drugs (NSC 725776 and NSC 743400) and two Top2-targeting drugs (epirubicin and etoposide) was investigated.ResultsTwo of three SN-38 resistant cell lines carried TOP1 gene copy number aberrations: A TOP1 gene copy gain and a loss of chromosome 20, respectively. One resistant cell line harbored a pair of yet unreported TOP1 mutations (R364K and G717R) in close proximity to the drug binding site. Mutant TOP1 was expressed at a markedly higher level than wild-type TOP1. None or very small reductions were observed in Top1 expression or Top1 activity in the absence of drug. In all three SN-38 resistant cell lines Top1 activity was maintained in the presence of high concentrations of SN-38. None or only partial cross-resistance were observed for etoposide and epirubicin, respectively. SN-38 resistant cells with wild-type TOP1 remained sensitive to NSC 743400, while cells with mutant TOP1 was fully cross-resistant to both indenoisoquinolines. Top1-DNA cleavage complex formation following drug treatment supported the other findings.ConclusionsThis study adds to the growing knowledge about resistance mechanisms for Top1-targeting chemotherapeutic drugs. Importantly, two yet unreported TOP1 mutations were identified, and it was underlined that cross-resistance to the new indenoisoquinoline drugs depends on the specific underlying molecular mechanism of resistance to SN-38.

Abstract 2306: Pre-clinical development of 4′-thio-2′-deoxycytidine (TdCyd) as a DNA-demethylating agent for use in treating solid tissue tumors

Targeting cancer epigenetic control of cell growth via DNA methylation has been successful in treating hematologic diseases, such as Decitabine (DAC) and Azacitidine for Myelodysplastic Syndrome including Acute Myelomonocytic Leukemia. This success has not extended to solid tissue tumors. The Division of Cancer Treatment and Diagnositcs of NCI has initiated pre-clinical development of TdCyd as an agent for treating solid tumors after promising early results in a lung adenocarcinoma xenograft model (NCI-H23). IP dosing at 5MKG (0.56 MTD) in nu/nu mice on a Q5D x 3 cycle schedule resulted in tumor stasis with no accompanying weight loss in the mice. A DAC-treated control arm treated at MTD resulted in tumor growth delay but not stasis, and a 10% weight loss was noted. Intratumoral levels of DNMT1 were reduced to undetectable levels in xenografts post administration of TdCyd by ELISA and Western Blot assays, but were unaffected by DAC treatment. Mass Spectrometry analysis demonstrated incorporation of both TdCyd and thiothymidine (TdThd) into H23 DNA. In vitro experiments on a selected panel of cancer cell lines confirmed the conversion of TdCyd to the triphosphate and re-expression of tumor suppressor proteins p15 and p16. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Robert J. Kinders, Melinda Hollingshead, Jaideep Thottassery, William B. Parker, Thomas D. Pfister, Lawrence W. Anderson, Joseph E. Tomaszewski, Jerry M. Collins, James H. Doroshow. Pre-clinical development of 4′-thio-2′-deoxycytidine (TdCyd) as a DNA-demethylating agent for use in treating solid tissue tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2306. doi:10.1158/1538-7445.AM2014-2306

Novel antibody reagents for characterization of drug- and tumor microenvironment-induced changes in epithelial-mesenchymal transition and cancer stem cells

The presence of cancer stem cells (CSCs) and the induction of epithelial-to-mesenchymal transition (EMT) in tumors are associated with tumor aggressiveness, metastasis, drug resistance, and poor prognosis, necessitating the development of reagents for unambiguous detection of CSC- and EMT-associated proteins in tumor specimens. To this end, we generated novel antibodies to EMT- and CSC-associated proteins, including Goosecoid, Sox9, Slug, Snail, and CD133. Importantly, unlike several widely used antibodies to CD133, the anti-CD133 antibodies we generated recognize epitopes distal to known glycosylation sites, enabling analyses that are not confounded by differences in CD133 glycosylation. For all target proteins, we selected antibodies that yielded the expected target protein molecular weights by Western analysis and the correct subcellular localization patterns by immunofluorescence microscopy assay (IFA); binding selectivity was verified by immunoprecipitation−mass spectrometry and by immunohistochemistry and IFA peptide blocking experiments. Finally, we applied these reagents to assess modulation of the respective markers of EMT and CSCs in xenograft tumor models by IFA. We observed that the constitutive presence of human hepatocyte growth factor (hHGF) in the tumor microenvironment of H596 non-small cell lung cancer tumors implanted in homozygous hHGF knock-in transgenic mice induced a more mesenchymal-like tumor state (relative to the epithelial-like state when implanted in control SCID mice), as evidenced by the elevated expression of EMT-associated transcription factors detected by our novel antibodies. Similarly, our new anti-CD133 antibody enabled detection and quantitation of drug-induced reductions in CD133-positive tumor cells following treatment of SUM149PT triple-negative breast cancer xenograft models with the CSC/focal adhesion kinase (FAK) inhibitor VS-6063. Thus, our novel antibodies to CSC- and EMT-associated factors exhibit sufficient sensitivity and selectivity for immunofluorescence microscopy studies of these processes in preclinical xenograft tumor specimens and the potential for application with clinical samples.

The root causes of pharmacodynamic assay failure

Robust pharmacodynamic assay results are valuable for informing go/no-go decisions about continued development of new anti-cancer agents and for identifying combinations of targeted agents, but often pharmacodynamic results are too incomplete or variable to fulfill this role. Our experience suggests that variable reagent and specimen quality are two major contributors to this problem. Minimizing all potential sources of variability in procedures for specimen collection, processing, and assay measurements is essential for meaningful comparison of pharmacodynamic biomarkers across sample time points. This is especially true in the evaluation of pre- and post-dose tumor biopsies, which suffer from high levels of tumor insufficiency due to variations in biopsy collection techniques and significant specimen heterogeneity within and across patients. Developing methods to assess heterogeneous biopsies is necessary in order to evaluate a majority of tumor biopsies collected for pharmacodynamic biomarker studies. Improved collection devices and standardization of methods are being sought in order to improve the tumor content and quality of tumor biopsies. In terms of reagent variability, we have found that stringent initial reagent qualification and quality control of R&D-grade reagents is critical to minimize lot-to-lot variability and prevent assay failures, especially for clinical pharmacodynamic questions, which often demand assay performance that meets or exceeds clinical diagnostic assay standards. Rigorous reagent specifications and use of appropriate assay quality control methodologies help to ensure consistency between assay runs, laboratories and trials to provide much needed pharmacodynamic insights into the activity of investigational agents.

Abstract B24: Ataxia-telangiectasia and Rad3-related (ATR) phosphorylation as a pharmacodynamic biomarker of ATR activation in solid tumor tissue models

Inhibitors of checkpoint kinases, such as ATR, Chk1 or Wee1, in combination with cytotoxic agents could enhance therapeutic efficacy compared to monotherapy, and these combination approaches are currently being extensively explored. The presence of replicative stress or deregulated S-phase in cancer has been recognized as a rationale for the use of ATR and Chk1 inhibitors with chemotherapy and efforts are underway to define genetic determinants that sensitize cancer cells to ATR inhibition. Pharmacodynamic (PD) biomarkers of drug activity are valuable tools in clinical trials using targeted agents to determine whether each investigational agent is acting upon its intended target, the expected intracellular signaling pathways are modulated, and the dosage regimen and sequencing are optimal. In addition, investigations of the potential effects of cell cycle distribution within the tumor (in animal models) on drug efficacy may point to alternative dosing regimens. The first clinical trials evaluating inhibitors of the ataxia telangiectasia and Rad3-related kinase (ATR) in combination with cytotoxic agents are enrolling patients. The current method for quantifying ATR inhibition used in preclinical in vitro and in vivo models is via indirect measurement of downstream effector proteins, such as phosphorylated Chk1, rather than measurement of direct target engagement. We propose that an immunofluorescence microscopy assay of ATR autophosphorylation status as a biomarker of DNA damage activation combined with measurement of the downstream effector proteins will reveal more specific information about target engagement and intended PD effect than would be obtained from a single-biomarker assay. Here we propose that the measurement of ATR pT1989, Chk1 pS345 and γH2AX by validated quantitative immunofluorescence assays will be useful in PD evaluation of tumor responses in clinical trials as a biomarker for ATR pathway activation or inhibition. In addition, evaluation of the variability of expression of activated ATR in tumor models of different histologies may provide valuable insight into tumor types likely to be sensitive to ATR inhibitors. Citation Format: Deborah Wilsker, Allison M. Marrero, Angie Dull, Thomas D. Pfister, Scott M. Lawrence, John Carter, Michelle Gottholm-Ahalt, Melinda Hollingshead, James Doroshow, Ralph E. Parchment, Robert J. Kinders. Ataxia-telangiectasia and Rad3-related (ATR) phosphorylation as a pharmacodynamic biomarker of ATR activation in solid tumor tissue models. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B24.

Abstract LB-B18: Epithelial to mesenchymal transition in human tumor biopsies: Quantitative, histopathological proof of the existence of EMT in vivo by immunofluorescence microscopy

Epithelial-mesenchymal transition (EMT) is a dynamic process whereby epithelial cells acquire mesenchymal properties. Despite the clinical significance of the acquired mesenchymal properties for metastasis and drug resistance, histopathological evidence of transitional cells in patient samples is lacking and EMT remains an unproven clinical hypothesis. We previously developed and validated a multiplex immunofluorescence assay (IFA) that quantifies the levels of EMT biomarkers (E-Cadherin, Vimentin, β-catenin) in snap-frozen, formalin-fixed, paraffin-embedded (FFPE) tumor tissue (Navas et al, AACR 2013). Building upon that method, we now report a precise, quantitative and unbiased IFA method of tissue analysis (EMT-IFA) using Definiens® software to quantify co-expression of the epithelial marker E-cadherin (E) and mesenchymal marker Vimentin (V) at the cellular level in FFPE clinical biopsies. FFPE human tumor xenografts and cell lines serve as calibrators and reference materials for establishing initial image acquisition parameters for segmented tumor regions of interest. Flanking HE 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B18.

Abstract 5082: Impact of HGF knockin microenvironment on epithelial-mesenchymal transition and cancer stem cells in a non-small cell lung cancer xenograft model

We previously reported the generation of rabbit monoclonal antibodies to twelve EMT (epithelial-to-mesenchymal transition) transcription factors and cancer stem cell (CSC) markers for the development of pharmacodynamic assays to inform clinical trials of new anticancer therapies (Pfister et al., AACR 2013). Here we demonstrate the functional utility of some of these reagents in detecting HGF-induced changes in EMT and CSC biology in a xenograft tumor model. Initial antibody characterization was performed in vitro and a subset [including SNAIL, SLUG, SOX9, Goosecoid (GSC), NANOG and CD133] was selected for further testing of functional utility in FFPE tissues by quantitative multiplex IFA. The antibodies were applied to xenograft tissues derived from the non-small cell lung cancer tumor line, NCI-H596, implanted in hHGFscid/scid, hHGFki/scid or hHGFki/ki mice to examine HGF-induced changes in EMT factors, CSC markers, as well as pY1235-MET expression in vivo. H596 tumors grown in either hHGFki/scid or hHGFki/ki mice exhibited enhanced EMT particularly in tumor microenvironments adjacent to mouse stroma containing the HGF knockin gene, compared to those in hHGFscid/scid mice. By quantitative immunofluorescence, H596 tumors showed increased Vimentin:E-cadherin ratio when grown in hHGFki/scid (P Citation Format: Tony Navas, Thomas D. Pfister, Scott M. Lawrence, Apurva K. Srivastava, Robert J. Kinders, Suzanne Borgel, Sergio Alcoser, Melinda G. Hollingshead, Lindsay M. Dutko, Brad A. Gouker, Donna Butcher, Elinor Ng-Eaton, Naoko Takebe, Young H. Lee, Donald P. Bottaro, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow. Impact of HGF knockin microenvironment on epithelial-mesenchymal transition and cancer stem cells in a non-small cell lung cancer xenograft model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5082. doi:10.1158/1538-7445.AM2015-5082