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PURPOSE Recurrent or metastatic adenoid cystic carcinoma (R/M ACC) is a malignant neoplasm of predominantly salivary gland origin for which effective therapies are lacking. We conducted a phase II trial evaluating the multitargeted tyrosine kinase inhibitor lenvatinib in patients with R/M ACC. PATIENTS AND METHODS This study was conducted with a two-stage minimax design. Patients with histologically confirmed R/M ACC of any primary site with radiographic and/or symptomatic progression were eligible. Any prior therapy was allowed except previous lenvatinib. Patients received lenvatinib 24 mg orally per day. The primary end point was overall response rate. Secondary end points were progression-free survival and safety. An exploratory analysis of how MYB expression and genomic alterations relate to outcomes was conducted. RESULTS Thirty-three patients were enrolled; 32 were evaluable for the primary end point. Five patients (15.6%) had a confirmed partial response, 24 patients (75%) had stable disease, two patients (6.3%) discontinued treatment as a result of toxicity before the first scan, and one patient (3.1%) had progression of disease as best response. Median progression-free survival time was 17.5 months (95% CI, 7.2 months to not reached), although only eight progression events were observed. Patients otherwise were removed for toxicity (n = 5), as a result of withdrawal of consent (n = 9), or at the treating physician’s discretion (n = 6). Twenty-three patients required at least one dose modification, and 18 of 32 patients discontinued lenvatinib for drug-related issues. The most common grade 3 or 4 adverse events were hypertension (n = 9; 28.1%) and oral pain (n = 3; 9.4%). Three grade 4 adverse events were observed (myocardial infarction, n = 1; posterior reversible encephalopathy syndrome, n = 1; and intracranial hemorrhage, n = 1). CONCLUSION This trial met the prespecified overall response rate primary end point, demonstrating antitumor activity with lenvatinib in R/M ACC patients. Toxicity was comparable to previous studies, requiring monitoring and management.

Purpose: Prostate cancer translational research has been hampered by the lack of comprehensive and tractable models that represent the genomic landscape of clinical disease. Metastatic castrate-resistant prostate cancer (mCRPC) patient-derived xenografts (PDXs) recapitulate the genetic and phenotypic diversity of the disease. We sought to establish a representative, preclinical platform of PDX-derived organoids that is experimentally facile for high-throughput and mechanistic analysis. Experimental Design: Using 20 models from the LuCaP mCRPC PDX cohort, including adenocarcinoma and neuroendocrine lineages, we systematically tested >20 modifications to prostate organoid conditions. Organoids were evaluated for genomic and phenotypic stability and continued reliance on the AR signaling pathway. The utility of the platform as a genotype-dependent model of drug sensitivity was tested with olaparib and carboplatin. Results: All PDX models proliferated as organoids in culture. Greater than 50% could be continuously cultured long-term in modified conditions; however, none of the PDXs could be established long-term as organoids under previously reported conditions. In addition, the modified conditions improved the establishment of patient biopsies over current methods. The genomic heterogeneity of the PDXs was conserved in organoids. Lineage markers and transcriptomes were maintained between PDXs and organoids. Dependence on AR signaling was preserved in adenocarcinoma organoids, replicating a dominant characteristic of CRPC. Finally, we observed maximum cytotoxicity to the PARP inhibitor olaparib in BRCA2−/− organoids, similar to responses observed in patients. Conclusions: The LuCaP PDX/organoid models provide an expansive, genetically characterized platform to investigate the mechanisms of pathogenesis as well as therapeutic responses and their molecular correlates in mCRPC. Clin Cancer Res; 24(17); 4332–45. ©2018 AACR.

The PI3K-Akt signaling pathway modulates cell growth, survival and apoptosis, and this pathway is frequently altered in human cancers, contributing resistance to radiation and chemotherapy treatment. Akt is a target for specific inhibition and recently, a number of small molecules have been developed to improve the pharmacologic properties of known inhibitors like wortmannin and LY294002. However, pan-Akt inhibitors can result in unanticipated side effects due to the lack of specificity for Akt isoforms 1, 2, and 3. Therefore, detection and quantitation of Akt isoforms and their downstream targets for both expression levels and phosphorylation states is crucial for therapeutic drug development. Here we demonstrate application of the Multiple Detection Approach to characterize the PI3K-Akt signaling pathway. This approach uses sequential analysis of proteins separated and immobilized in a capillary, by performing either dual immunoassays or immunoassay with total protein on the Simple Western platform using chemiluminescence detection. Assays with control and LY294002 inhibitor-treated samples were developed. Proteins were first separated based on molecular weight via capillary electrophoresis, followed by immobilization via UV-crosslinking. Next, PI3K-Akt pathway targets were sequentially probed in the same capillary with total and phospho-specific antibodies, to determine the phosphorylated fraction relative to the total fraction. Primary antibodies from the first immunoprobe were removed after the detection step with >95% efficiency, as confirmed by re-probing with the same secondary antibody. Target protein loss was negligible due to covalent immobilization to the capillary wall, which was confirmed with re-probing, thus validating the quantitative data generated using this sequential approach. In addition, total protein normalization was performed in tandem with the immunoassay in the same capillary. This approach enables normalization of phosphorylation levels and/or target abundance in cell line or tissue samples, correcting for change in protein content due to treatment, loading, and/or other systematic errors. These results present the utility of the Multiple Detection Approach to quickly characterize and quantify proteins involved in signaling pathways targeted during development of cancer therapies. Citation Format: Irina G. Kazakova, Crystal Tran, Anita Silver, Jessica Dermody. Novel approach for automated sequential immunoassay for quantitation and characterization of PI3K-Akt pathway proteins [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 6293.

Objective: Based on emerging data on tumor heterogeneity and the evolutionary branching of tumor cells, tumor cells in the lymph node may represent more virulent clones with the inherent capability of metastasis. IHC discordance from original cancer diagnosis to recurrence is documented to occur in up to 20% of cases, raising the question if characterization of these likely more virulent cells would more accurately guide treatment and predict prognosis. Our pilot study sought to determine if crucial clinical information is gained by IHC testing of the surgical breast or lymph node specimens at the time of initial surgery. Methods: Using the cancer registry and oncology records, all invasive breast cancers diagnosed after 2001 with subsequent recurrence were identified. Cases missing all IHC data were disqualified. We then evaluated ER and HER2 of the primary cancer biopsy and recurrence biopsy to identify discordances. Those with discordances who had surgical breast and lymph node specimens available were accessed, tested, and evaluated by our breast cancer pathologist. Results: A total of 128 recurrence cases with partial or complete primary and recurrence IHC data were identified. Of the 95 initially ER positive cases with recurrence IHC available, 13/95 had discordant, or ER negative, recurrence. Additionally, 5/27 initially ER negative tumors, 3/14 initially HER2 positive tumors, and 6/69 initially HER2 negative tumors had discordant recurrence results. In 128 cases, 27 cases were identified to have ER or HER2 discordance from primary biopsy diagnosis to recurrence. Of all cases with original surgical breast or positive lymph node specimen available, 9 markers on 7 patients were performed for our pilot study. One of seven surgical breast specimens and one of two lymph node specimens were concordant with the recurrence, but not the initial biopsy. The tested surgical breast was ER positive, while the surgical lymph node was HER2 positive, concordant to their recurrences, but discordant with initial biopsy. Breast BiopsyRecurrence ConcordantRecurrence DiscordantER Positive98/12782/9513/95 (14%)ER Negative29/12724/275/27 (19%) Total ER Discordance 18/122 (15%)HER2 Positive19/11911/143/14 (22%)HER2 Negative100/11963/696/69 (9%) Total HER2 Discordance 9/83 (11%) Conclusion: Tumor discordance of the original cancer biopsy and recurrence is not uncommon. Our pilot study demonstrated that ER and HER2 discordance occurred in 15% and 11% of cases, respectively. Though our pilot study was limited by small sample size, we found that IHC testing of the surgical breast and lymph node specimen may provide additional clinical information and affect management. Of the two cases that had a positive lymph node available, one was HER2 positive and concordant with the recurrence. Of the seven breast specimens tested, one was ER positive and concordant with the recurrence. Had IHC testing been performed at that time of surgery, adjuvant treatment management would have been altered. Further testing of our IHC discordant recurrence patient population will be pursued to investigate the potential benefits of surgical breast and lymph node IHC testing. Citation Format: Michele M Gage, Martin Rosman, Charles Mylander, Crystal Tran, Rubie S Jackson, Lorraine Tafra. Immunohistochemical (IHC) marker discordance between primary breast cancer biopsy and recurrent cancer: Would IHC testing of the surgical breast or lymph node have altered treatment? [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-10.

6084 Background: R/M ACC is a malignant neoplasm most commonly of salivary gland origin with no standard treatment. The impact of combined PD-1/CTLA-4 checkpoint blockade in R/M ACC is unknown. Methods: In a two-stage minimax phase II trial, pts with progressive R/M ACC (non-salivary primaries allowed) were enrolled and treated with nivolumab 3 mg/kg every 2 weeks plus ipilimumab 1 mg/kg every 6 weeks (1 cycle = 6 weeks). Imaging, using RECIST v1.1 response assessment, was scheduled to be performed approximately every 12 weeks. The primary endpoint was overall response rate (ORR = complete response [CR]+partial response [PR]) per RECIST v1.1. To detect a difference between an unacceptable ORR of 5% and a desirable ORR of 20% (one-sided type I error of 10%, power of 90%), at least 1 in the first 18 pts required an observed response. At least 4 responses of 32 total pts were needed to meet the primary endpoint. Treatment beyond progression of disease (PD) was allowed at the discretion of the investigator. A second cohort of pts with non-ACC salivary cancer is still accruing for separate analysis. Results: From 6/12/2017-6/20/2018, 32 pts were enrolled and evaluable for the primary endpoint. There was 1 confirmed PR in the first 18 pts, therefore enrollment of the second stage continued. ORR was 6% (2/32). One additional pt had an unconfirmed PR (-31% regression before CNS PD). For best overall response, there were 2 PRs, 15 SD, and 11 PD. Four pts never reached a first disease assessment: 3 due to death from clinical PD and 1 was removed for toxicity. Six pts discontinued the trial for toxicities (Grade 4 (G4) neutropenia/sepsis and G3 adrenal insufficiency (1), G2 hypophysitis (2), G3 arthritis > 7 days (1), G3 colitis (1), and G3 hepatitis/G4 creatinine kinase (CK) elevation (1)). The 2 confirmed PRs consisted of -73.1% and -58.4% regressions, with a duration of therapy of 18.4 and 7.8 months, respectively (treatment ongoing for both). Conclusions: The study did not meet its primary endpoint, though the responses observed were dramatic. Paired biopsy and peripheral blood samples will be analyzed to elucidate insights into mechanisms of response and resistance to dual checkpoint blockade. Clinical trial information: NCT03172624.

Androgen-deprivation therapy (ADT) remains the gold-standard therapy for prostate cancer (PrCa), and although ADT is initially effective, most men progress to castrate-resistant prostate cancer (CRPC) within 2-3 years. Advanced CRPC is challenging to treat because intrinsic tumor heterogeneity and phenotypic plasticity engender short-lived responses and underlie resistance to conventional therapies. Combined PTEN/TP53 alterations represent a major genotype of advanced CRPC (25-30%) and are associated with poor clinical outcomes. Established PrCa cell lines do not accurately represent the heterogeneity of advanced CRPC, and therefore, nonbiased pharmacogenomics screens have not been done. The development of clinically representative, tractable models suitable for high-throughput target identification and validation is crucial for advancing novel CRPC therapies to the clinic. A comprehensive nonbiased high-throughput screen performed on seven cell lines derived from a genetically engineered mouse model (GEMM) of Pten/Tp53 null PrCa identified strongly active compounds, including inhibitors of PI3K/AKT/mTOR signaling, the proteasome, cell cycle regulatory proteins, heat shock proteins, DNA repair signaling, NFKB signaling, MAPK signaling, and several types of epigenetic modifiers. HSP90 inhibitors were one of the most efficacious classes of compounds in the screen, and ganetespib, a clinically used second-generation HSP90 inhibitor with a favorable safety profile, was the most potent. Although HSP90 inhibitors have yet to be successful as single agents, they have not been thoroughly investigated in clinically representative models of advanced PrCa and have shown potential as “network drugs,” prompting our investigations into their utility in polytherapy. We first validated ganetespib as a single agent, where it displayed strong activity against several GEMM-derived and LuCaP PDX-derived organoid models encompassing genotypic, phenotypic, and lineage heterogeneity. These 10 novel LuCaP PDX-derived organoids are representative of the numerous categories of CRPC, including adenocarcinomas with wild-type AR, adenocarcinomas with altered AR, adenocarcinoma with neuroendocrine features, and neuroendocrine disease. Single-agent ganetespib was also strongly inhibitory in vivo, decreasing growth of Pten/Tp53 null endogenous GEMM tumors as well as a human PDX tumor. Mechanistic interrogation of cell lines, organoids, and tumors exposed to ganetespib revealed inhibition of targets from several inter-related networks including AR and pAKT, two central and mutually compensatory growth and survival pathways for PrCa. The efficacy of ganetespib against a diverse group of CRPC organoids and the simultaneous inhibition of PrCa survival signaling suggested it may work well in combination. We performed a proof-of-principle high-throughput matrix screen on organoids derived from a Pten/Tp53 null GEMM and identified docetaxel and etoposide to be synergistic when combined with ganetespib. Preclinical in vivo studies to validate these findings are ongoing. In all, comprehensive data from multiple near-patient models suggest novel contexts for second-generation HSP90-directed intervention against a variety of CRPC genotypes and phenotypes and expand upon the potential of HSP90 inhibitors to simultaneously inhibit oncogenic signaling and compensatory resistance mechanisms. Citation Format: Keith H. Jansson, John B. Tucker, Lauren E. Stahl, John K. Simmons, Caitlyn Fuller, Michael L. Beshiri, Supreet Agarwal, Yasmine Abbey, Lei Fang, Paul G. Hynes, Alilin Aian Neil, Jacob Cawley, Ross Lake, Crystal Tran, Caitlin M. Tice, JuanJuan Yin, Xiahu Zhang, Rajarshi Guha, Shelley Hoover, R. Mark Simpson, Holly Nguyen, Eva Corey, Craig J. Thomas, David Proia, Kathleen Kelly. A high-throughput screen identifies HSP90 inhibitors as potent therapeutics across multiple clinically representative organoid models of advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B018.

Metastatic castration-resistant prostate cancer (CRPC) is a genetically heterogeneous disease. Effective translational research demands a diverse and representative set of preclinical models. A major obstacle in the field is the limited capacity to culture prostate cancer-derived cells in vitro. Few cell lines exist. Those that are available do not well represent the genetic diversity of the disease, nor do they accurately predict in vivo response. Recently, organoid culture techniques developed in the Sawyers and Clevers laboratories have increased our ability to grow metastatic tumor-derived prostate cancer cells in vitro. This represents a great step forward, but is restricted by limited availability of tissue and a success rate of Citation Format: Michael L. Beshiri, Caitlin M. Tice, Crystal Tran, Holly M. Nguyen, Adam G. Sowalsky, Supreet Agarwal, Keith H. Jansson, Kerry McGowen, Aian Neil Alilin, JuanJuan Yin, Fatima H. Karzai, William L. Dahut, Eva Corey, Kathleen Kelly. PDX-derived and patient-derived prostate cancer organoids as a clinically relevant platform for translational research [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B051.

Metastatic castration-resistant prostate cancer (mCRPC) is a genetically and phenotypically heterogeneous disease. Resistance to contemporary androgen-deprivation therapy (ADT) is inevitable, and occurs via several different mechanisms. One of these is lineage plasticity leading to transdifferentiation of AR-positive adenocarcinoma (ACPC) to the neuroendocrine (NEPC) subtype. The modern era of second-generation ADT has seen a significant increase in the percentage of NEPC, yet little is known about the genetic and epigenetic drivers of this phenotype. Using modified organoid media conditions that we previously developed, we have established a set of mCRPC organoid lines derived from four biopsies (two samples before and two after carboplatin + etoposide treatment, 1 year apart) of a single patient tumor that capture the genetic and phenotypic heterogeneity of the original metastatic tumor. Genomic and histologic analysis show that the original patient tumor is composed of both adenocarcinoma and neuroendocrine subpopulations that are maintained in the organoids at a ratio of approximately 1:4, respectively. The two subpopulations share a common ancestor and a putative early driver event in the homozygous loss of CDKN1B, but our analysis of clonal evolution indicates that the majority NEPC population did not transdifferentiate directly from the minority ACPC population in the metastasis, but rather in the primary tumor or in a prior metastatic lesion. The ACPC subpopulation contains a high copy gain of an upstream regulatory region of the AR gene and is genetically distinct from the NEPC population that lacks this alteration, but uniquely contains a truncating mutation in the ARID1A gene. ARID1A is frequently mutated and/or deleted in several different cancer types. It has been found to be altered in ~2% of prostate cancer patients, but its role is not understood. It is a key component of the BAF complex, which is a known regulator of neural development. Immunofluourescent staining of our organoid cells shows that mutant ARID1A correlates with the NEPC population. Using single-cell RNA-seq and principle component clustering analysis, we have identified five different transcriptionally defined populations: one population that corresponds to the genetically defined ACPC population and four additional transcriptionally defined populations that represent different states of differentiation of the NEPC component, as determined by expression of lineage markers. A full understanding of this model with respect to differentiation of the NEPC population and the specific role of ARID1A in the process will inform the development of mechanism-based treatments to inhibit NEPC formation. Citation Format: Mike L. Beshiri, Caitlin M. Tice, Adam G. Sowalsky, Crystal Tran, Fatima Karzai, William Dahut, Kathy Kelly. A patient-derived organoid model of neuroendocrine prostate cancer transdifferentiation informing the role of the BAF complex component ARID1A [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 5020.

Castrate resistant prostate cancer (CRPC) remains the primary cause of mortality for prostate cancer (PCa) patients. Several therapies are currently approved for use against CRPC, consisting mostly of androgen receptor (AR) signaling inhibitors and toxic anti-microtubule chemotherapeutics that do not score long-term durable responses. Recent deep-sequencing analysis studies have elucidated the complex and heterogeneous landscape of CRPC, highlighting the necessity to develop effective targeted therapies against specific CRPC subtypes or therapies effective against numerous subtypes of CRPC. PTEN and p53 are two of the most frequently altered genes in CRPC, and are associated with therapy resistance and a poor clinical prognosis. The objective of this study was to identify and subsequently validate therapeutic targets against models of advanced PCa, specifically in the absence of PTEN and p53. Using non-biased high throughput screening technology, we identified HSP90 inhibitors to be potent and efficacious against a model of PTEN/p53 null PCa. HSP90 is a critical regulator of prostate cancer cell signaling homeostasis, and recently developed second generation compounds targeting HSP90 are effective against genotypically heterogeneous panels of cancer cell lines and have favorable safety parameters in the clinic. Screening results and subsequent validation efforts in vitro identified ganetespib as the most potent HSP90 inhibitor, with efficacy that translated into established human PCa cells lines. Ganetespib also displayed strongly inhibitory activity against PTEN/p53 null progenitor cells, a plastic subpopulation of PCa believed to be partly responsible for therapy resistance. In vivo, ganetespib completely inhibited progression of PIN to invasive adenocarcinoma in the Pten/Tp53 null PCa mouse model, leading to a significant reduction in tumor weight. Expanding from the mouse model to clinically predictive PDX-derived LuCaP models of human PCa, we found that ganetespib displayed a range of activity against a genotypically diverse array of 11 LuCaP organoids ex vivo. Furthermore, the PTEN null/p53 altered LuCaP 136 displayed a robust response to ganetespib in vivo, with ganetespib causing a significant reduction in tumor growth. Mechanistic interrogation in vitro, ex vivo, and in vivo revealed ganetespib induced effects to be multifactorial and model specific, with unifying trends being inactivation of PI3K signaling and modulation of cell cycle regulatory proteins. In all, these data indicate that inhibition of HSP90 is a novel therapeutic to treat advanced PCa via multifactorial perturbation of growth regulatory pathways. Citation Format: Keith H. Jansson, John Tucker, Lauren Stahl, Caitlyn Fuller, Michael Beshiri, John K. Simmons, Supreet Agarwal, Lei Fang, Aian Neil Alilin, Ross Lake, Crystal Tran, Paul G. Hynes, Jacob Cawley, JuanJuan Yin, Xiaohu Zhang, Rajarshi Guha, Shelley Hoover, R. Mark Simpson, Holly Nguyen, Eva Corey, Craig Thomas, David Proia, Kathleen Kelly. High-throughput screen identifies HSP90 as a potential therapeutic target in models of advanced CRPC [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 3065. doi:10.1158/1538-7445.AM2017-3065

A major obstacle in the field of prostate cancer research is the limited capacity to culture prostate cancer-derived cells in vitro. There are relatively few established prostate cancer cell lines in use today. Those that are available are amenable to in vitro drug assays and mechanistic studies, but do not well-represent the genetic diversity of the disease, nor do they accurately predict in vivo response. Recently, organoid culture techniques have been developed for prostate cancer by the laboratories of Hans Clevers and Charles Sawyers that have increased our ability to grow metastatic tumor-derived prostate cancer cells in vitro. This represents a great step forward, but we are still restricted by the limited availability of biopsy tissue and the relatively low success rate (~20%) in establishing new organoid lines. The LuCaP series of patient-derived xenografts (PDXs) represents a well-characterized set of prostate cancer specimens that is genetically diverse, reflective of a range of treatment histories, and available to the research community. We have modified the organoid culture technique based upon systematic comparisons and analysis for organoid growth of the LuCaP PDXs. Our modified conditions represent an improvement upon the original growth conditions. We have successfully cultured 21 out of 24 LuCaPs attempted, for at least one generation, and 16 out of 24 are able to grow long-term over several generations. This includes 3 of the newest LuCaPs (167, 170.2, and 189.3) that we have genomically characterized. Comparison of the PDX and various organoid generations showed significant conservation of genomic changes ranging from 75-90% for nonsynonymous point mutations. The modified organoid culture method immediately and significantly increases the number of prostate cancer lines that are available by making the LuCaP PDXs accessible to in vitro culture. In addition to the PDXs, our method has improved the success for culturing metastatic samples grown from biopsies. We show here that 2 out of 3 patient-derived organoid lines that we have established, grow well in the modified conditions but poorly in the original culture conditions, the third grows equally well in both conditions. Citation Format: Mike L. Beshiri, Crystal Tran, Adam G. Sowalsky, Holly M. Nguyen, Supreet Agarwal, Caitlin M. Tice, Eva Corey, Kathy Kelly. Optimization of prostate cancer organoid culture methods to grow PDX and human biopsy tissue [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 4838. doi:10.1158/1538-7445.AM2017-4838