Armando Diaz

Abstract 627: Establishment and characterization of a panel of cell-based and patient-derived chordoma tumor models

Background: Chordoma is a rare cancer (0.08 per 100k/yr) that originates from the notochord and develops in the skull and spine. Treatment options for chordoma include resection and local radiation therapy; however, recurrence rates following treatment are high and the majority of patients ultimately succumb to the disease. Currently there are no approved chemotherapy options for chordoma, and effective treatment options for patients with recurrent or advanced disease are limited. Recent molecular analyses of chordoma have revealed multiple tractable therapeutic targets, providing rationale for new systemic therapies. However, lack of relevant, validated chordoma models has limited preclinical evaluation. To address this need, the Chordoma Foundation and START have collaborated to establish, bank and characterize chordoma tumor models derived from patient samples and established cell lines. Methods: Chordoma patients undergoing resection or biopsy procedures are identified and consented by the Chordoma Foundation under an IRB-approved protocol; tissue is shipped to START and implanted into immune-deficient mice for patient-derived xenograft (PDX) model establishment and banking. Similarly, models from immortalized chordoma cell lines have been established and banked, along with established PDX chordoma models from START or collaborators. Banked PDX models are anonymously linked to available patient clinical information. All chordoma models are subjected to immunohistochemistry (IHC) to confirm histology and presence of brachyury, a protein highly expressed in chordoma tumors. Following validation, models are used at START for drug sensitivity studies and available to investigators for characterization studies. Results: To date, clinical samples from five chordoma patients have been implanted and two PDX models designated CF322 and CF345 have been established. In addition, xenograft models from the UCH-1 and UCH-2 chordoma cell lines have been established at START. PDX models previously developed at START (ST087) and UCSF (SF8894) have also been validated and banked. Drug sensitivity studies have been initiated these two and the U-CH1 models evaluating agents selected and prioritized through a peer review process established by the Chordoma Foundation. Nominations for additional test agents from academic and industry collaborators are reviewed on a rolling basis and evaluation of selected therapies is funded by the Chordoma Foundation. Conclusion: We have established a collaboration to create, bank and characterize a panel of preclinical chordoma tumor models originating from patient samples and established cell lines. To date we have generated three chordoma models available for drug sensitivity studies. This panel of models is intended to serve as a shared resource to the chordoma research community to enable more rapid preclinical evaluation of therapeutic hypotheses. Citation Format: Michael J. Wick, Melissa Rundle, Lizette Gamez, Armando Diaz, Josh Sommer, Patricia Cogswell, Byron Hann, Joanna Phillips, Kyriakos P. Papadopoulos. Establishment and characterization of a panel of cell-based and patient-derived chordoma tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 627.

Abstract C74: Establishment and characterization of a HER2-positive, TDM1-resistant PDX breast model

Background: Ado-trastuzumab emtansine (T-DM1), a recently approved antibody-drug conjugate (ADC), is approved for treatment of high HER2 expressing (3+), trastuzumab resistant breast and gastric cancers. While this agent is effective, resistance often develops. To better understand the mechanisms for resistance to TDM1 in high HER2 expressing breast cancer we collected tissue from a patient following response and progression to TDM1 and established a xenograft model for preclinical drug screening. The resulting model ST1360B was genomically characterized and screened in vivo using high dose TDM1; anti-tumor activity was compared with two trastuzumab-resistant but TDM-1 sensitive HER2 (3+) breast PDX models designated ST225 and ST340. Methods: The high HER2 expressing (3+) TDM1 resistant breast PDX model ST1360B was established in athymic mice using pleural fluid collected from a thoracentesis. Establishment of the HER2 (3+) ST225 and ST340 models has been previously described. Clinical and PDX tissues were subjected to genomic analysis; for each model, DNA was extracted and subjected to comparative genomic hybridization and exon sequencing of known oncogenes; growth factor receptor and ligand densities was interrogated using immunohistochemistry. Drug sensitivity studies were performed evaluating sensitivity of models to single agent T-DM1, administered intravenously at 10 mg/kg on Day 0; weekly TDM1 dosing at 5 mg/kg was also evaluated in the ST1360B model Study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion. Results: Genomic analysis of ST1360B identified amplified HER2 and TOP2A; interestingly CD44 was found highly amplified, suggesting a possible mechanism for TDM1 resistance in this model. Mutations were not identified in PIK3CA or other relevant genes. In efficacy studies TDM1 was confirmed active towards ST225 and ST340 model including tumor regressions and durable complete responses in both models (TGI>100%). However TDM1 was not effective in ST1360B, with only transient tumor growth inhibition even with weekly drug administration. Conclusion: We have established a model of TDM1 HER2 3+ resistant breast cancer and have characterized the model using comparative genomic hybridization and exon sequencing of known oncogenes, identifying amplified CD44 as a potential mechanism for drug resistance. This model can be utilized to better understand and to develop novel therapies to TDM1 resistance. Citation Format: Michael J. Wick, Teresa L. Vaught, Justin Meade, Monica Farley, Armando Diaz, Anthony W. Tolcher, Drew W. Rasco, Amita Patnaik, Murali Beeram, Kyriakos P. Papadopoulos. Establishment and characterization of a HER2-positive, TDM1-resistant PDX breast model. [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 C74.

Abstract 1223: Establishment, characterization and evaluation of a panel of head and neck patient-derived xenograft (PDX) models

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Head and neck cancers account for over five hundred thousand cases worldwide each year. Primary risk factors include tobacco use, alcohol consumption, human papillomavirus (HPV) infection, and Epstein-Barr virus (EBV) infection with certain cancer subtypes disproportionally affecting minorities. Standard treatment for these cancers consists of surgical tumor debulking followed by a platinum-based agent alone or in combination with an EGFR-targeting therapy when relevant. The rate of initial response to treatment is high; however, the majority of patients develop recurrent disease resistant to most secondary chemotherapy treatments, demonstrating the need for identifying additional useful therapies. To better understand pathways involved in head and neck cancers and identify useful novel therapies for this disease, we have established a panel of low passage, patient-derived xenograft (PDX) models representing head and neck cancers and characterized them by activating mutation analysis and receptor expression. Panel drug screening studies were also performed comparing platinum regimens and select targeted therapies and correlating with clinical outcome. Methods: START-PDX head and neck models were established in immune-deficient mice from primary or metastatic patient tissue and once established were confirmed by histologic comparative analysis and linked with patient treatment and outcome data. For each model DNA was extracted and subjected to exon sequencing of known oncogenes and growth factor receptor densities were interrogated using immunohistochemistry. Drug sensitivity studies were performed evaluating models towards chemotherapy and targeted agents. Study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion. Results: To date we have established and validated twenty-five head and neck low passage START-PDX models. High EGFR staining (3+) was reported in over 70% of these models but only 10% reported HER2 and HER3 staining. Activity of platinum regimens was marked and correlated with clinical outcome with over 75% concordance. High EGFR expression did not correlate to cetuximab sensitivity even in models with wildtype K-Ras. However, models found sensitive to erlotinib also demonstrated sensitivity to cetuximab. Conclusion: We have established a panel START-PDX head and neck models and characterized mutation status, receptor expression and benchmarked sensitivity of platinum and select targeted therapies. This panel can be utilized as a valuable screening tool in early and late-stage biomarker discovery and oncology drug development. Citation Format: Michael J. Wick, Teresa Vaught, Lizette Gamez, Armando Diaz, Justin Meade, Monica Farley, Alyssa Moriarty, Jennifer Carlile, Anthony Tolcher, Drew Rasco, Amita Patnaik, Richard Newman, Kyriakos Papadopoulos. Establishment, characterization and evaluation of a panel of head and neck patient-derived xenograft (PDX) models. [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 1223. doi:10.1158/1538-7445.AM2014-1223

Abstract A107: In vivo screen of regorafenib in a panel of low passage START-PDX colorectal models: Working to identify a sensitive patient subpopulation.

Background: Regorafenib is a multi-tyrosine kinase recently approved for unresectable and drug refractory gastrointestinal stromal tumors (GIST) and treatment-refractory colorectal cancers regardless of Ras status. While a subset of colorectal cancer patients have reported benefit from regorafenib therapy, the majority does not benefit and attempts to identify a subpopulation of sensitive colorectal patients have not been successful. To better understand which colorectal cancer patients may respond to regorafenib we screened a panel of low passage colorectal models from the START patient-derived xenograft (PDX) platform and compared results to model characteristics. Methods: PDX colorectal models were established in athymic nude mice from primary or metastatic patient tissue or fluid and once established were confirmed by histologic comparative analysis and linked with patient treatment and outcome data. For each model DNA was extracted and subjected to mutation profiling and growth factor receptor expression was interrogated using immunohistochemistry. Drug sensitivity studies were performed evaluating regorafenib at 25-30 mg/kg once daily for at least twenty-one days. Study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion. Results: To date forty colorectal START-PDX models have been screened against regorafenib. Three models (ST201, ST238 and ST800) reported tumor regressions or partial responses even with activating K-Ras or B-Raf mutations present. An additional 30% of evaluated models reported tumor stasis (T/C= 0-10%) and remaining models demonstrated varying degrees of sensitivity or resistance to regorafenib. High expression of EGFR was reported in two of the most sensitive models and was reduced in the models resistant to regorafenib. Conclusion: We have screened preclinical activity of regorafenib towards a panel of colorectal START-PDX models and identified sensitive and resistant models. Additional genomic and characterization studies are underway to better understand activity of regorafenib with the goal of identifying a sensitive patient subpopulation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A107. Citation Format: Michael J. Wick, Lizette Gamez, Armando Diaz, Teresa L. Vaught, Anthony W. Tolcher, Drew Rasco, Amita Patnaik, Lon Smith, Ron Drengler, Kyriakos P. Papadopoulos. In vivo screen of regorafenib in a panel of low passage START-PDX colorectal models: Working to identify a sensitive patient subpopulation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A107.

Abstract A111: Effects of unconjugated antibody pretreatment to reduce ADC-related toxicity: Preclinical effects of trastuzumab on T-DM1 activity in low passage breast PDX models.

Background: Antibody-drug conjugates (ADC) are comprised of a cytotoxic agent linked to an antibody that targets surface proteins of tumors. Once bound, the cytotoxin enters and destroys the tumor cell while sparing surrounding healthy tissue. Recent approval of the ADC molecules brentuximab (mAb(CD30)-auristatin) for Hodgkins lymphoma and T-DM1 (mAb(trastuzumab)-emtansine) for HER2-positive breast cancer has renewed interest in this agent class leading to development of a number of new molecules. While several of these agents have shown promising activity, low level expression of target proteins on healthy tissue has led to clinical dose-limiting toxicities. Pretreatment with the unconjugated antibody prior to ADC therapy may bind to target proteins on healthy tissue and reduce or prevent ADC-related toxicities. To test this possibility we identified three START-PDX breast models with high HER2 expression: ST225, ST340 (clinically trastuzumab-resistant) and ST910 (T-DM1 sensitive), and tested trastuzumab and T-DM1, alone and in combination, to compare model growth and drug effects. Methods: START-PDX breast models were established in immune-deficient mice from primary or metastatic patient tissue. Once established, models were confirmed by histologic comparative analysis and linked with patient treatment and outcome data. For each study, trastuzumab and T-DM1 were administered alone or in combination at 1:10 or 1:1 trastuzumab: T-DM1 ratios; trastuzumab was administered 24h prior to T-DM1 and all groups were dosed weekly. Study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion. Results: Trastuzumab alone was inactive in all models, while single agent T-DM1 induced tumor regressions including partial and complete responses. Co-administration of trastuzumab at 1:10 or 1:1 had slight to no effect on T-DM1 activity in ST225 or ST340, most likely due to the high inherent activity of T-DM1 towards these models. However, in the ST910 model, trastuzumab: T-DM1 at the 1:1 but not 1:10 combination group showed reduced activity compared with T-DM1 alone, suggesting competitive binding between the naked antibody and ADC at the higher trastuzumab concentration. Conclusion: We have identified trastuzumab refractory, T-DM1-sensitive breast START-PDX models and demonstrated that low level pretreatment with unconjugated trastuzumab prior to T-DM1 therapy did not inhibit its activity, suggesting that sequenced administration of the unconjugated antibody followed by the linked ADC may bind to target proteins on healthy tissue and reduce or prevent ADC-related toxicities without impacting efficacy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A111. Citation Format: Roger Chavez, Michael J. Wick, Teresa L. Vaught, Anthony W. Tolcher, Meredith P. Tolcher, Armando Diaz, Drew Rasco, Amita Patnaik, Gladys Rodriguez, Lon Smith, Kyriakos P. Papadopoulos. Effects of unconjugated antibody pretreatment to reduce ADC-related toxicity: Preclinical effects of trastuzumab on T-DM1 activity in low passage breast PDX models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A111.

Abstract 1733: Development of a novel blood plasma protein test for diagnosis of lung cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: A blood test for detection of lung cancer (LC) could provide survival benefits in high-risk populations. We conducted a retrospective study as the next step of our effort to develop a test for diagnosis of non-small cell lung carcinoma (NSCLC), the most common form of LC, by measuring levels of protein biomarkers in plasma. Materials and Methods: We quantified levels of 106 proteinaceous biomarkers using multiplexed immunoassays of subjects a) diagnosed with stage I/II NSCLC, b) normal non-smoking controls, and c) normal heavy smoking controls (> 10 p.y.). All groups consisted of Caucasian men and women (93 subjects each, ages range 45-87, Ave 59). The specimens were randomized and tested in a blinded manner. A support vector machine (SVM) algorithm, developed and tested in a pilot study, was used to identify differentially expressed biomarkers in the disease and control groups taking into account patients’ gender. Pathway analysis was applied to characterize pathology- and gender-specific patterns of biomarker expression. Results: In a recent pilot study we developed SVM models that classified subjects to NSCLC or normal using 59 plasma markers or subsets thereof, for both genders combined or separately. When data for both genders were analyzed together, SVM classified subjects to NSCLC or normal and identified 4 differentially expressed markers: EGF, sCD40 ligand, IL-8 and MMP-8; sensitivity (SE) 0.93 (95% CI: 0.88-0.96), specificity (SP) 0.87 (95% CI: 0.80-0.92). Considering the sexes separately, the differentially expressed best subset of markers for men (EGF, IL-8, sFAS, MMP-9 and PAI-1) had both SE and SP of 1.0. For women, the best subset of markers (EGF, sCD40 ligand and IL-8) also yielded SE and SP of 1.0. The present study has further confirmed and refined our earlier findings and expanded panels of diagnostic biomarkers for NSCLC with comparable sensitivities and specificities. Analysis of cellular pathways represented by the discriminatory biomarker signatures suggested a role of sex hormone-driven differences of NSCLC plasma biomarkers. Conclusions: The set of diagnostic biomarkers for NSCLC identified in this study holds significant promise for use in the early diagnosis of lung cancer and warrants validation of findings in other ethnic groups and for potentially confounding diseases in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1733. doi:1538-7445.AM2012-1733

Abstract B2: Patterns of serum biomarkers for lung cancer are different for men and women

Introduction: Lung cancer has one of the highest mortality rates among all human cancers. Higher mortality has been noted in the past in men with the history of smoking but no significant differences in mortality exist in never smoking men and women. Some gender differences to date have been linked to genes located on sex chromosomes. Methods: We quantified levels of 57 cytokines, chemokines and growth factors using multiplexed immunoassays to determine patterns of serum biomarkers in men and women with non-small cell lung cancer (NSCLC; 250 men, 116 women) and compared these with serum marker patterns in subjects with asthma (AST; 68 men, 132 women) and normal controls (NOR; 250 men, 116 women). Data were reduced using inter-pathology comparisons with statistical significance determined using the Kruskall-Wallis U test with p Results: The markers showed unique gender- and disease specific patterns. For unisex analysis of NSCLC, we identified 36 markers with the 25% change cutoff threshold and 32 markers with the 50% cutoff. For women, we found 32 markers with the 25% cutoff and 30 with the 50% cutoff. For men, 39 markers were found at the 25% cutoff and 37 at the 50% cutoff. Expression of four markers was unique for women with NSCLC compared to NOR: IL-8 and serum amyloid P (downregulated), serum amyloid A and C-reactive protein (all upregulated). Five markers were unique for men with NSCLC compared to NOR: Insulin (downregulated), matrix metalloproteinases-7 and -8, resistin and hepatocyte growth factor (all upregulated). Three markers showed opposite patterns of expression; VEGF was downregulated in women and upregulated in men with NSCLC compared to NOR. Leptin was upregulated in women and downregulated in men and MIP-1α were upregulated in men and downregulated in women with NSCLC compared to NOR. Conclusion: Our study has revealed surprisingly large qualitative and quantitative differences in biomarker expression patterns and levels in serum specimens from men and women suffering from NSCLC. The markers appear to be products of genes residing on several chromosomes and are not limited to sex chromosomes. Our findings may have implications for the diagnosis of non-small cell lung cancer, early detection of the disease through simple blood tests, characterization of disease progression and differentiation among the pathologies. It may find application in discovery and development of novel therapeutic strategies for human diseases. Citation Information : Clin Cancer Res 2010;16(7 Suppl):B2

Abstract C15: A novel panel of serum biomarkers distinguishes asthma from non‐small cell lung cancer

Purpose: Asthma affects an increasing proportion of the population and may predispose sufferers to lung cancer. Frequent misdiagnosis of asthma and lack of early detection tests for lung cancer might be mitigated by better diagnostic tests. We used multiplexed immunoassays and mass spectrometry to identify serum biomarkers capable of distinguishing asthma from non‐small cell lung cancer, the most common type of lung cancer. Methods: Sera from normal subjects (NO; n = 300), patients with asthma (AST; n=180), and non‐small cell lung cancer (LC; n = 334) were acquired from commercial vendors. Levels of 58 cytokines, chemokines and growth factors were quantified using multiplexed immunoassays. For biomarker discovery, specimens were digested with trypsin and analyzed by liquid chromatography electrospray ionization MS (LC ESI MS/MS). Proteins were identified using Mascot search software. Validation of select biomarkers identified by MS was achieved by immunodetection of target proteins in serum specimens. Data were reduced using inter‐pathology comparisons with statistical significance determined using Student9s t‐test. Results: Multiplexed immunoassays identified 29 analytes with significant (t Conclusion: We have identified a group of serum biomarkers having high inter‐pathology discrimination power that are capable of differentiating AST and LC from normal controls and that may potentially be used in diagnostic tests for early detection of lung pathologies. Our experimental strategy is widely applicable to discovery and validation of biomarkers for diverse human diseases, response to a therapy, or pre‐selection of patients for clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C15.