Abstract Introduction: Androgen Deprivation Therapy (ADT) is a highly effective treatment for prostate cancer. However, resistance to ADT develops in most patients, leading to a lethal state of castration-resistant prostate cancer (CRPC). Senescent prostate cancer (SPC) cells can arise after ADT and contribute to therapy resistance. SPCs can restore replicative function and alter the tumor microenvironment through pro-tumorigenic senescence-associated secretory phenotype (SASP). We aimed to characterize changes in SASP in prostate cancer cell lines and serum samples from patients receiving ADT. Methods: SASP factors (Luminex 100/200 System, xMAP Instruments) were analyzed in patients undergoing ADT. To correlate SASP with the sensitivity to ADT, we sought to evaluate the regulation of SASP in vitro using PCa cell lines. The PC3 (AR resistant) and LNCaP (AR sensitive) cell lines were used in the experiments. We performed an analysis of archival tumor tissue (paired) from 18 patients with or without alterations in DNA repair genes (BRCA1, BRCA2, and CDK12). We performed immunohistochemical (IHC) staining of known SASP markers, such as P16, uPAR, gamma-H2AX, and Lamin B1. Results: In a cohort of patients (n=8) with pre and post-ADT matched status, we demonstrate increased levels of SASP factors such as IL-6 (pre-ADT: 1.54±0.24 pg/ml, post-ADT: 5.23±1.3 pg/ml p=0.001), IL-7 (pre-ADT: 3.11±0.6 pg/ml, post-ADT: 13.5±2.7 pg/ml p=0.01), IL-1 alpha (pre-ADT: 1.06±0.3 pg/ml, post-ADT: 15.3±3.5 pg/ml p=0.001), IL-15 (pre-ADT: 15.6±3.1 pg/ml, post-ADT: 37.1±4.8 pg/ml p=0.002), MIF (pre-ADT: 1036±67.6pg/ml, post-ADT: 2362±55.5 pg/ml p=0.003), and IL-15 (pre-ADT: 5±2.1 pg/ml, post-ADT: 37.5±5.1 pg/ml p=0.002). In vitro, the AR-responsive LNCaP cell line, exposed to AR blockade, showed similar findings in upregulating IL-6, IL-7, and IL-15 but not the PC3 cell line. We also demonstrate measurable differences between the expression of u-PAR, gamma-2AX, p16, and loss of Lamin B1 expression in tumor specimens from patients with advanced castration-resistant prostate cancer (n=18). The expression of p16 was significantly upregulated post-treatment irrespective of DDR status (p=0.0417). Other SASP factors showed a trend in upregulation but were insignificant (u-PAR p=0.056, gamma-H2AX p=0.62, Lamin B1 p=0.72) Conclusion: These results provide evidence of systemic increases in SASP-related cytokines following treatment with ADT. The in vitro results suggest that secretion of SASP is promoted by an AR-mediated mechanism. Preliminary results from tumor specimens suggest increased senescence marker p16 expression induced by therapy. Ongoing experiments are further characterizing the senescence markers in prostate tumor specimens and correlated to treatment effect. Citation Format: Maximilian Schwermann, Matthew Hadfield, Shaolei Lu, Praveen Srinivasan, Vida Tajiknia, William MacDonald, Andrew George, Shengliang Zhang, Leiqing Zhang, Kimberly Meza, Andre De Souza, Dragan Golijanin, Elias Hyams, Galina Lagos, Sheldon Holder, Anthony Mega, John Sedivy, Howard Safran, Wafik El-Deiry, Benedito Carneiro. Androgen deprivation therapy (ADT) and senescence-associated secretory phenotype (SASP) in vitro: Correlation with SASP in tumor specimens as well as in the serum of patients after ADT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2954.

The early detection of cancer is a key goal of the National Cancer Plan formally released by the National Institutes of Health's (NIH) National Cancer Institute (NCI) in April 2023. To support this effort, many laboratories and vendors are developing multi-cancer detection (MCD) assays that interrogate blood and other bodily fluids for cancer-related biomarkers, most commonly circulating tumor DNA (ctDNA). While this approach holds promise for non-invasively detecting early signals of multiple different cancers and potentially reducing cancer-related mortality, there is a dearth of prospective clinical data to inform the deployment of MCD assays for cancer screening in the general adult population. In this review we highlight differing technologies that underpin various MCD assays in clinical development, the importance of achieving adequate performance specifications for MCD assays, ongoing clinical studies investigating the utility of MCD assays in cancer screening and detection, and efforts by the NCI's Division of Cancer Prevention (DCP) to establish a network infrastructure that has the capacity to comprehensively address the scientific and logistical challenges of evaluating blood-based MCD approaches and other cancer screening tools.

Abstract Background/Rationale: The combination of the proteosome inhibitor bortezomib (B) and purine analog clofarabine (C) induced synergy in the NCI-ALMANAC screen of FDA-approved cancer drug combinations; preclinical xenograft studies confirmed this activity and suggested a combination mechanism of action involving greater-than-additive effects on markers of DNA damage response (DDR) and pyroptotic cell death. We conducted a phase 1 clinical trial (NCT02211755) to assess the safety of the B and C combination in patients (pts) with solid tumors and myelodysplastic syndrome (MDS), and to determine the effect of the combination on biomarkers of cell death and DDR. Due to limited MDS pt accrual, this cohort was terminated early, and MDS pt data are not included here.Objectives: The primary objective was to assess safety and maximum tolerated dose (MTD) of B and C in pts with solid tumors and MDS. Secondary objectives were to assess pharmacodynamic (PD) effects using validated assays for cell death and DDR in tumor biopsy tissue and circulating tumor cells (CTCs).Design: The dose escalation followed a 3+3 design, enrolling at least 1 pt from each group (solid tumor and MDS) per dose level until hematologic dose-limiting toxicity (DLT) was observed. Adverse events (AEs) were graded using CTCAE v4.0. Ten planned dose levels (DL) included C ranging from 1 to 10 mg/m2 on D2-5 of cycle 1 and D1-5 from cycle 2 on and B from 0.8 to 1.5 mg/m2 on D1 and D4 of 21-day cycles. Response was assessed by RECIST 1.1 every 2 cycles. Biopsies were performed on the expansion cohort pre-dose, 2-4 hours after C on C1D1, and C2D4; CTCs were collected pre-dose, C1D2, day 1 of subsequent cycles, and at progression.Results: We enrolled 25 pts with advanced solid tumors (15 male, 10 female). Median age was 62 years (range 30-81). DLT occurred in 4 pts (at DL3 [1 pt], DL5 [2], and DL4 [1]) and included Grade 4 neutropenia (3), Grade 3-4 thrombocytopenia (3), and Grade 3 anemia (1). There were no Grade 5 AEs. DL4 (B 1.3 mg/m2 D1 and 4, C 1.5 mg/m2 D1-5, 21-day cycles) was the MTD. Twenty-one pts were evaluable for response; the best response was stable disease (SD) for 10 pts (47.6%). Median duration of response for pts achieving SD was 3.5 months (range 2-7). Of note, 3 out of the 10 colorectal cancer (CRC) pts had SD > 4 months. Four pts with CRC have been enrolled in an expansion cohort for biomarker evaluation; induction of pyroptosis has been measured in a limited number of paired biopsies collected to date. Conclusions: Despite preclinical evidence of synergy, the human equivalent of the efficacious preclinical combination dose could not be achieved clinically, which may account for the lack of antitumor response. Prolonged SD was observed in 3 pts with CRC. PD analyses, including of CTCs and additional cell death biomarkers, are ongoing. Funded by NCI Contract No. HHSN261201500003I. Citation Format: Andre L. De Souza, Naoko Takebe, Alice P. Chen, James H. Doroshow, Ralph E. Parchment, Apurva K. Srivastava, Lawrence Rubistein, Deborah Wilsker, Brandon L. Miller, Murielle Hogu, Geraldine O`Sullivan Coney, Shivaani Kummar, Jeevan Govindharajulu, Robert Meehan, William G. Herrick, Angie Dull, Donna Ketchum, Shaowei Li, Jiuping Ji, Richard Piekarz. Phase 1 trial of bortezomib and clofarabine combination in adults with refractory solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT120.

Abstract Background: Wild-type Proviral Insertion Site of Moloney Murine Leukemia Virus 1 Kinase (PIM1) is a constitutively active serine/threonine kinase that inhibits apoptosis and promotes cell cycle progression, proliferation, angiogenesis, invasion, and migration. Due to constitutive activation, PIM1 is regulated at transcription and degradation. We have previously shown PIM1 protein levels are elevated in clear cell renal cell carcinoma (ccRCC) compared to normal kidney and that PIM1 inhibition induces tumor regression in pre-clinical models. Current approved therapies for ccRCC do not inhibit PIM1. We hypothesized that high PIM1 expression will adversely affect clinical outcomes in ccRCC. Methods: Whole transcriptome sequencing (WTS) data in the Caris Life Sciences database were examined for PIM1expression and overall survival (OS; defined as time from diagnosis to last contact or death). We assessed PIM1 in primary and metastatic ccRCC, and the impact on survival in ccRCC and papillary RCC. Differentially expressed genes (DEGs) associated with high PIM1 were also determined. Results: PIM1, in the highest quintile, was associated with a shorter OS (869 vs 1738 days; HR 2.629; CI 1.495 - 4.611; p<0.001; N=147) in ccRCC. PIM1 is enriched in metastatic (median 7.62 transcripts per million (TPM), CI 6.35 - 9.26) vs primary (median 5.07 TPM; CI 4.28 - 6.45; p=0.0025) RCC. Upregulated DEGs associated with high PIM1 are involved in the extracellular matrix. IL-6 expression, known to correlate with worse outcomes, was 6.5-fold higher in PIM1 high vs PIM1 low (13.2 vs 2.1 TPM, p<0.0001) RCC. High PIM1 was associated with poor survival following checkpoint inhibition (HR 3.714, 95% CI 1.339 - 10.303, p=0.007) and a trend towards poorer survival following mTOR inhibition and VEGF inhibition. PIM1 did not affect survival in papillary RCC. Conclusions: Real-word data show that high PIM1 correlates with poorer survival in ccRCC but not in papillary RCC. This effect likely persists independent of treatment received. PIM1 enrichment in metastatic ccRCC, and associated DEGs, suggest that PIM1 influences the extracellular matrix to promote metastases. The correlation between IL-6expression and PIM1 supports our prior data that an IL-6/JAK/STAT/PIM1 pathway is involved in ccRCC. Citation Format: Sheldon L. Holder, Galina Lagos, Benedito A. Carneiro, Anthony Mega, Andre De Souza, Rana R. McKay, Andrew Elliott, Chadi Nabhan, Stephanie L. Graff. An analysis of proviral insertion site of Moloney murine leukemia virus, PIM1, kinase expression and clinical outcomes in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2567.

Abstract Introduction: Approximately 30% of advanced bladder cancer (BCa) tumors exhibit MTAP gene deletion, which has been associated with luminal subtype BCa, poor response to chemotherapy, and shorter progression to metastatic disease (De Souza 2023). MTAP deletion has been proposed as a potential biomarker for response to pemetrexed and an emerging class of PRMT5 and MAT2A inhibitors in development for MTAP-deleted tumors. Here, we describe the first patient-derived 3D model of BCa with MTAP deletion and its translational potential as a drug sensitivity platform. Methods: A patient (pt) with metastatic BCa harboring MTAP deletion underwent a pelvic lymph node biopsy. A fresh tumor sample was processed under collagenase/dispase solution for 30 minutes and filtered through a 70-um cell strainer. Cells were cultures in low attachment plates with MammoCult™ Basal Medium (Stem Cell Technologies). After 5 passages and 3D spheroid formation, immunostaining for cytokeratin (CK) 5 and 6, GATA3, CK20, and Ki67 staining were performed to confirm the urothelial origin. The MTAP-del was confirmed by a western blot of the organoid specimen and compared to other BLCa cell lines (UMUC, RT4, J82, and 5637). The viability assays were performed using the CellTiter-Glo® 3D Cell kit. Next-generation sequencing (NGS) of primary BCa tumor revealed deletion of MTAP, CDKN2A, CDKN2B, and mutations in p53, TERT, and KDM6A. Results: The patient organoid sample was positive for the luminal CK5/6 markers and negative for GATA3. Ki67 and CK20 were primarily expressed in the nucleus of the same sample. The organoid retained morphologic and immunohistochemical features of the patient’s primary bladder tumor, showing the same squamous differentiation and similar Ki-67 proliferative index. The organoid displayed no evidence of the MTAP protein by western blot (similar findings with the MTAP-del cell lines UMUC and RT4). The organoid showed sensitivity to pemetrexed [pemetrexed IC50=0.12 uM (other MTAP-del BLCa IC50: 0.1-0.23 uM)] and resistance to gemcitabine and cisplatin [Gem IC50=55 nM (other BLCa IC50: 1-29 nM), Cis IC50=90.5 uM (other BLCa IC50: 2-17 uM). Conclusions: We describe the first patient-derived MTAP-deleted BCa organoid recapitulating the features of the primary tumor. The organoid displayed sensitivity to pemetrexed as described with MTAP-deleted BCa. The organoid represents a unique platform for testing novel therapeutic strategies for MTAP-deleted BCa. Citation Format: Maximilian Schwermann, Benedito Carneiro, Shaolei Lu, Andre De Souza, Matthew Hadfield, Anthony Mega, Galina Lagos, Sari Khaleel, Dragan Golijanin, Sheldon Holder, Praveen Srinivasan, William MacDonald, Andrew George, Lanlan Zhou, Leiqing Zhang, Wafik El-Deiry. Patient-derived MTAP-deleted bladder cancer organoid model: A unique platform for drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4248.

Background High-grade non-muscle-invasive-bladder-cancer (NMIBC) has high recurrence rates and potential resistance to intravesical therapies. Anti-PD-L1 immunotherapy with pembrolizumab was approved in January 2020 for treatment of patients with high-risk, BCG refractory NMIBC with carcinoma in situ (CIS) with or without papillary tumors who received adequate BCG therapy and were ineligible for or opted out of radical cystectomy. In this study we report on our single institutional experience using pembrolizumab in BCG refractory patients. Methods Records of patients with NMIBC treated by pembrolizumab from 01/2020–01/2023 at a single institution were retrospectively reviewed for key demographic and clinical information. Kaplan-Meier curves were used to calculate progression free (PFS) and treatment specific survival (TSS), and combined positivity score (CPS) of PD-L1 on immunochemistry was assessed. Results Out of 250 screened records of NMIBC in this time period, 18 records with median age of 74.1 (IQR=67.8 – 81.4), male to female ratio of 3.5:1, and a median follow-up of 17.5 months (IQR= 8.1 – 22.5) met the inclusion criteria. All patients had CIS and were treated with intravesical chemotherapy after they became BCG refractory. At start of pembrolizumab, 1/18 (5.6%) was cTa, 6/18 (33.3%) had CIS, and 11/18 (61.1%) had cT1. After an average of 8.9 cycles (SD=6.3), 72.2% of patients (13/18) stopped treatment. Only five patients (38.5%) are still undergoing treatment with an average of 12.6 cycles (SD=10.4 cycles). Only one patient out of thirteen who stopped treatment had a sustained complete response at 19 cycles. Reasons for discontinuation included: Grade 2 or higher toxicity in 7/13 (53.8%), disease progression in 4/13 (30.8%), , and 1/13 stopped due to disease recurrence. Recurrence-free survival rates at 3-, 6-, and 12-months were 16.7%, 11.1%, and 5.6%, respectively. 6- and 12-month PFS rates were 94% and 77.7%, respectively. Kaplan-Meier methods showed a PFS of 19.5 months (SD=2.4) and a TSS of 26.5months (SD=2.9). Four patients ultimately required radical cystectomy with pathologies showing pTa (n=1), pTis (n=1), pT1 (n=1), and pT4 (n=1). PD-L1 positivity, defined as CPS > 10, was noted for only one patient. Conclusions Our institutional experience using pembrolizumab in the treatment of high risk BCG refractory NMIBC suggests high toxicity leading to early withdrawal from treatment. Similarly, our experience did not confirm previously reported high response rates beyond one year. Additional research is warranted to better identify patients who are likely to benefit from this agent. Ethics Approval This study was approved by institutional review board of the The Miriam Hospital and Lifespan Hospital System in Providence, RI. Study ID: 1047794. All required consents were acquired prior to data collection.

Advanced urothelial carcinoma continues to have a dismal prognosis despite several new therapies in the last 5 years. FGFR2 and FGFR3 mutations and fusions, PD-L1 expression, tumor mutational burden, and microsatellite instability are established predictive biomarkers in advanced urothelial carcinoma. Novel biomarkers can optimize the sequencing of available treatments and improve outcomes. We describe herein the clinical and pathologic features of patients with an emerging subtype of bladder cancer characterized by deletion of the gene MTAP encoding the enzyme S-Methyl-5'-thioadenosine phosphatase, a potential biomarker of response to pemetrexed. We performed a retrospective analysis of 61 patients with advanced urothelial carcinoma for whom demographics, pathologic specimens, next generation sequencing, and clinical outcomes were available. We compared the frequency of histology variants, upper tract location, pathogenic gene variants, tumor response, progression free survival (PFS) and overall survival (OS) between patients with tumors harboring MTAP deletion (MTAP-del) and wild type tumors (MTAP-WT). A propensity score matching of 5 covariates (age, gender, presence of variant histology, prior surgery, and prior non-muscle invasive bladder cancer) was calculated to compensate for disparity when comparing survival in these subgroups. Non-supervised clustering analysis of differentially expressed genes between MTAP-del and MTAP-WT urothelial carcinomas was performed. MTAP-del occurred in 19 patients (31%). Tumors with MTAP-del were characterized by higher prevalence of squamous differentiation (47.4 vs 11.9%), bone metastases (52.6 vs 23.5%) and lower frequency of upper urinary tract location (5.2% vs 26.1%). Pathway gene set enrichment analysis showed that among the genes upregulated in the MTAP-del cohort, at least 5 were linked to keratinization (FOXN1, KRT33A/B, KRT84, RPTN) possibly contributing to the higher prevalence of squamous differentiation. Alterations in the PIK3 and MAPK pathways were more frequent when MTAP was deleted. There was a trend to inferior response to chemotherapy among MTAP-del tumors, but no difference in the response to immune checkpoint inhibitors or enfortumab. Median progression free survival after first line therapy (PFS1) was 5.5 months for patients with MTAP-WT and 4.5 months for patients with MTAP-del (HR = 1.30; 95% CI, 0.64-2.63; P = 0.471). There was no difference in the time from metastatic diagnosis to death (P = 0.6346). Median OS from diagnosis of localized or de novo metastatic disease was 16 months (range 1.5-60, IQR 8-26) for patients with MTAP-del and 24.5 months (range 3-156, IQR 16-48) for patients with MTAP-WT (P = 0.0218), suggesting that time to progression to metastatic disease is shorter in MTAP-del patients. Covariates did not impact significantly overall survival on propensity score matching. In conclusion, MTAP -del occurs in approximately 30% of patients with advanced urothelial carcinoma and defines a subgroup of patients with aggressive features, such as squamous differentiation, frequent bone metastases, poor response to chemotherapy, and shorter time to progression to metastatic disease.

Background The blockade of the androgen receptor (AR) pathway is an effective treatment for prostate cancer (PCa), but many patients progress to metastatic castration-resistant prostate cancer (mCRPC). Therapies for mCRPC include AR inhibitors (ARi), chemotherapy, PARP inhibitors, and radioligands. Checkpoint inhibitor activity is limited to a small subset of MSI-H mCRPC. AR signaling modulates CD8+ T cell function, but its impact on NK cell (NKc) cytotoxicity is unknown. We investigated the effect of ARi on NKc activation, cytokine secretion, expression of inhibitory receptor NKG2A, and killing of PCa cells in vitro . Methods PCa cell lines (LNCaP, 22Rv1 [ARv7 mutation], DU145[AR-], PC3 [AR-]) were co-cultured with NK-92 cells and treated with ARi (enzalutamide [enza] and darolutamide [daro]) or in combination with anti-NKG2A antibody monalizumab. Immune cell-mediated tumor cell killing assays and multiplexed cytokine profiling were performed. NKc expression of NKG2A and PCa cells expression of HLA-E were investigated by flow cytometry. The AR-negative cell lines (PC3 and DU145) were stably transduced with a functional AR pathway to evaluate the modulation of HLA-E by AR. The activation status of peripheral blood NKc isolated from patients with PCa before and post-initiation of androgen deprivation therapy (ADT) was investigated by flow cytometry. Results ARi significantly increased immune-mediated NK-92 cell killing of PCa cells independent of their sensitivity to androgen signaling. Cytokine analysis revealed that ARi-induced NKc activation is mediated by IFN-γ and TRAIL, as confirmed by blocking antibodies. ARi increased NKG2A expression in NK cells. Immune killing of PCa cells was enhanced with the combination of ARi and monalizumab. ARi also increased the expression of HLA-E, the ligand of the inhibitory NKG2A receptor, on PCa cell lines. Using AR-negative cell lines (PC3 and DU145) and stable transduction of AR, we demonstrate that androgen signaling regulates HLA-E expression. HDAC inhibitors (vorinostat and panobinostat) did not alter the androgen-induced expression of HLA-E in PCa cells. Mirroring the results from NK-92 cells, ADT also activated peripheral blood NK cells isolated from patients with metastatic PCa. Conclusions ARi activates NK cells through upregulating IFN-γ and TRAIL and promotes the killing of PCa cells. This enhanced cytotoxic killing of PCa cells is augmented by monalizumab. ARi upregulates PCa cell’s expression of HLA-E, suggesting a mechanism suppressing the innate immune response against PCa. These results support novel therapeutic strategies for PCa targeting NK activation with the combination of ARi and monalizumab. Graphical Abstract Androgen receptor signaling blockade enhances NK cell-mediated killing of prostate cancer cells and sensitivity to NK cell checkpoint blockade. Nonetheless, ARi can potentially upregulate an NK cell inhibitor ligand (HLA-E), thus suppressing NK cell killing of PCa. This regulation is dependent on a functional AR signal on tumor cell lines. Adding an anti-NKG2a-HLA-E mAb with ARi further enhances the NK cell-mediated killing of PCa.

Background: Inhibition of the androgen pathway remains central to the treatment of prostate cancer, but most patients progress to a lethal state of metastatic castration-resistant prostate cancer (mCRPC). Immune checkpoint inhibitors have a limited role in mCRPC, although androgen receptor (AR) blockade increases CD8+ T cell function and can modulate the antitumor immune response. We investigated the effect of AR inhibitors (ARi) enzalutamide (Enza) and darolutamide (Daro) on NK cell function.Materials and Methods: We evaluated cell viability to investigate the effect of Enza and Daro on PC cells (LNCap, 22Rv1 [ARv7 mutation], DU145, PC3 [AR-]) and NK-92 cells. We performed co-culture experiments with PC and NK cells at a 1:1 ratio and analyzed immune cell-mediated tumor cell killing (ImageXpress Confocal HT). Cytokine profiling (Luminex 200) of culture supernatants was performed following treatment with ARi -/+ and anti-IFN-γ antibodies. In vivo studies were performed in NCr Nude mice harboring subcutaneous PC tumors treated orally with Daro (50mg/kg/bid) and Enza (30mg/kg/bid) for 4 weeks. Results: Treatment of co-cultures of PC plus NK-92 cells with ARi significantly increased immune-mediated PC killing within 24 hours (control: 19%±2.7; Enza: 36%±3; Daro: 41%±4.2). Treatment of NK cells alone with ARi did not impact their viability. ARi increased the secretion of cytokines such as IL-2 (7.6 pg/ml±1.3 vs. 18 pg/ml±2.4, p=0.004), IL-1β (17.8 pg/ml±2.8 vs. 67 pg/ml±12.6, p=0.005), CXCL10 (224.2 pg/ml±16.6 vs. 504 pg/ml±42.4, p=0.017) and GDF-15 (member of the TGFβ superfamily; 8.4 ng/ml±0.43 vs. 14.3 ng/ml±3.2, p=0.042) and decreased VEGF (302.5 pg/ml±62.1 vs. 120 pg/ml±50.4, p=0.01), FGF-basic (7.4 pg/ml±1.6 vs. 3.6 pg/ml±2.1, p=0.007), M-CSF (70.9 pg/ml±14.2 vs. 25.3 pg/ml±4.5, p=0.01) and IL-8 (2645 pg/ml±201 vs. 1821 pg/ml±62.3, p=0.02). ARi increased NK-92 secretion of IFN-γ (15.2 pg/ml±5.4 and 48.2 pg/ml±8.2, p=0.01) and granzyme B (630 pg/ml±32.2 vs. 1024 pg/ml±102.4, p=0.001). IFN-γ upregulation induced by ARi was blocked with IFN-γ mAb in the co-culture experiments and inhibited the NK cell-mediated killing of PCs. Analysis of PC tumors from NCr Nude Mice treated with Daro or Enza showed increased NK cell infiltration in the tumor microenvironment. Results: We report the discovery that ARi promotes the activation of NK cells mediated by IFN-γ, leading to increased killing of PCs. Ongoing validation of these results includes PC organoids and patient-derived samples to understand further the innate immune system response to ARi and direct novel therapeutic strategies. Citation Format: Maximilian Schwermann, Kelsey E. Huntington, Lindsey Carlsen, Lanlan Zhou, Praveen Srinivasan, Andrew George, Laura Jinxuan Wu, Andre De Souza, Anthony E. Mega, Howard P. Safran, Benedito A. Carneiro, Wafik S. El-Deiry. Androgen signaling blockade enhances NK cell-mediated killing of prostate cancer cells (PC) and promotes NK cell tumor infiltration in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1843.

e17066 Background: The blockade of the androgen receptor (AR) pathway is essential for the treatment of prostate cancer (PC). Checkpoint inhibitors have limited activity in immunologically cold prostate tumors. Emerging results suggest that AR signaling modulates CD8+ T cells and modifies its cytotoxic function, but the therapeutic role of NK cells in anti-tumor immune response in PC remains unknown. We investigated the mechanisms of AR inhibitors (ARi) enzalutamide (Enza) and darolutamide (Daro) on the antitumor function of NK cells and their enhancement when combined with NKG2A inhibitor. Methods: We performed co-culture assays with PC cell lines (LNCap, 22Rv1 [ARv7 mutation], DU145, PC3 [AR-]) and NK-92 cells, in a 1:1 ratio, in the presence of ARi and in combination with the anti-NKG2A mAb monalizumab and analyzed immune cell-mediated tumor cell killing (ImageXpress Confocal HT). NK cells expression of NKG2A and PD-1 receptors and PC cell lines expression of HLA-E and PD-L1 were investigated after treatment with Enza and Daro by flow cytometry. Results: ARi significantly increased immune-mediated NK-92 cell killing of PC cell lines at 24hrs (control[C]: 15.8%±3.9; Enza: 40%±4.3; Daro: 36%±3.72, p < 0.0001). This immune enhancement effect was increased by NKG2A blockade with monalizumab ([C]: 31.2%±3.1, [T]: 43.92%±4.52, p = 0.0045). Treatment of the LNCaP cell line (AR+) with ARi increased HLA-E ([MFI values x10³] control[C]: 28.57±2.9; [T]: 37.38 ±1.2 p = 0.0046) and PD-L1 expression ([MFI values x10³] control[C]: 163.1±8.1; [T]: 202.5±11.2 p = 0.003). No significant difference was observed in the surface expression of these markers in the AR- cell lines. NKG2A surface expression on NK cells was also upregulated upon treatment with ARi ([MFI values x10³] control[C]: 52.42±2.9; [T]: 61.4 ±2.2 p = 0.008). Conclusions: Enza and Daro enhance NK cell killing of PC cell lines, and the combination of ARi with checkpoint inhibitor monalizumab promotes NK cell activation. Blockade of AR signaling modulates PC cells' expression of HLA-E and PD-L1 and NK cell expression of NKG2A. These results describe a novel modulatory effect of androgen on NK cell function and provide insights for future NK-cell-based therapeutic approaches for treating PC. Experiments validating these results in patient samples are ongoing.

Background: Molecular alterations in genes regulating homologous recombination (HR) confer sensitivity of mCRPC to poly ADP-ribose polymerase inhibitors (PARPi). Increased activation of thePI3K-AKT-mTOR pathway in mCRPC contributes to tumor progression and represents an important therapeutic target. Preclinical studies have shown that PI3K inhibitors (PI3Ki) impair HR and sensitize cancer cells to PARPi even in the absence of HR gene mutations. We hypothesize that dual PI3K and PARP inhibition may improve clinical outcomes of mCRPC. We describe preliminary results of a phase Ib/II study investigating safety of the combination of copanlisib (pan-class I PI3Ki) and rucaparib (PARP-1, -2 and -3 inhibitor). Methods: Enrollment criteria included progressive mCRPC, prior androgen inhibitors (abiraterone, enzalutamide, and/or apalutamide); prior taxane chemotherapy was allowed. HR defects (HRD) was not required for the phase Ib but was an inclusion criterion for the Phase II component of the trial. The phase I followed a standard 3+3 escalation design. Dose level (DL) 1 consisted of rucaparib 400 mg PO BID continuously and copanlisib 45 mg IV days 1, 8 and 15 every 28 days; in DL -1 the copanlisib was reduced to 45 mg days 1 and 15 only. Adverse events (AE) were graded by CTCAE v5.0. The primary aim of the phase I was to establish the MTD and the recommended phase II dose (RP2D) of copanlisib in combination with rucaparib. Results: Thirteen patients were enrolled with a median age of 64 (55-78) and a median baseline PSA of 11.7 ng/mL (0.015-1939.2). Nine patients (69%) received prior chemotherapy (docetaxel [6], cabazitaxel [3]). Seven patients had HRD (54%) BRCA1 (1), BRCA2 (4), CDK12 (1), and FANCA (1). Treatment-related AEs included grade 2 (G2) leukopenia (46%), G2/G3 anemia (31%), G2/G3 rash (31%). Two dose-limiting toxicities (DLTs) were observed in 5 patients treated in DL 1: G3 rash and G3 AST/ALT elevation attributed to both drugs. Six patients were treated at DL -1 without DLTs; this was identified as the RP2D. Two additional patients have been treated at this dose in Phase II of the study. By RECIST 1.1 criteria, one patient (8%) achieved a partial response (BRCA1 mutation) and three (23%) achieved stable disease resulting in a clinical benefit rate of 30.7%. Median duration of treatment was 12 weeks. One patient harboring a PALB2 VUS mutation remained on treatment for 21.7 months. Of 13 evaluable patients, there were 2 confirmed PSA responses (15%) (one patient without an HRD and one with a BRCA1 mutation). Conclusions: The combination of rucaparib and copanlisib is well tolerated. The RP2D was rucaparib 400mg BID with copanlisib 45mg (D1, D15; 28-day cycle) with signal of efficacy in patients with and without HRD. Clinical trial information: NCT04253262. Citation Format: Matthew J. Hadfield, Andre De Souza, Dragan Golijanin, Roxanne Wood, Adam Olszewski, Sheldon L. Holder, Wafik S. El-Deiry, Rahul Aggarwal, Anthony Mega, Benedito A. Carneiro. BrUOG360: A phase Ib/II study of copanlisib combined with rucaparib in patients with metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT158.

Although blocking the androgen receptor (AR) pathway is a core treatment strategy in early prostate cancer, most patients progress to metastatic castration-resistant prostate cancer(mCRPC). Immunotherapy with PD-1/PD-L1 inhibition has limited activity in mCRPC. AR signaling modulates CD8+ T cells and antitumor immune responses in mCRPC. In a companion presentation (Schwermann et al., submitted AACR 2023), we demonstrate that AR blockade activates NK cell killing of prostate cancer (PC) cells. We hypothesize this activation requires IFN-γ and TRAIL pathways. We investigated the mechanisms of AR inhibitors (ARi)enzalutamide (Enza) and darolutamide (Daro) on the antitumor function of NK cells and potential enhancement with checkpoint inhibitors. Material and Methods: We performed ATP-based CellTiterGlo viability assays to investigate the effects of Enza and Daro on PC cells (LNCap, 22Rv1 [ARv7mutation], DU145, PC3 [AR-]) and NK-92 cells. We performed co-culture experiments with PC, NK, and T cells, at a 1:1 ratio, using IFN-γ blocking mAb (10 μg/ml), RIK-2 (TRAIL blocking mAb- 10 μg/ml) and analyzed immune cell-mediated tumor cell killing (ImageXpress Confocal HT). The NK cell immune checkpoint inhibitor monalizumab that targets NKG2A was combined with ARi. Results: Co-cultures of PC plus NK-92 cells with ARi significantly increased immune-mediated PCkilling within 24hrs (control[C]: 20%±1.9; Enza: 40%±4.3; Daro: 36%±3.72, p=0.0001). This immune enhancement effect was abolished by IFN-γ mAb. The combination of TRAIL inhibitory mAb with Enza decreased NK immune-mediated killing by 16±2.4% ([C]: 40%±4.3; Enza+RIK-2:25.6%±2.1, p=0.002). Treatment of PC cells plus NK cells with Enza, IFN-γ mAb, and TRAIL-blocking RIK-2 significantly reduced NK cell-mediated killing of PCs (control[C]: 20%±1.9, [T]:3.2%±2.6, p=0.003). ARi immune enhancement effect was increased by NKG2A blockade with monalizumab ([C]: 31.2%±3.1, [T]: 43.92%±4.52, p=0.0045). Conclusions: ARi promotes NK cell killing of PC cells via IFN-γ gamma and TRAIL. NK cell activation is enhanced by the combination of ARi and the checkpoint inhibitor monalizumab. Validation of these results in patient-derived organoids paired with tumor samples from patients with PC is ongoing to further understand the innate immune response and direct novel therapeutic strategies. Citation Format: Maximilian Schwermann, Lindsey Carlsen, Kelsey E. Huntington, Lanlan Zhou, Andrew George, Praveen Srinivasan, Vida Tajiknia, Arielle De La Cruz, Andre De Souza, Anthony E. Mega, Howard P. Safran, Benedito A. Carneiro, Wafik S. El-Deiry. IFN-γ and TRAIL involvement in androgen signaling-mediated NK cell killing of prostate cancer cells and further enhancement by NKG2A checkpoint inhibition with monalizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1822.

The precursor nature of papillary urothelial hyperplasia of the urinary bladder is uncertain. In this study, we investigated the telomerase reverse transcriptase (TERT) promoter and fibroblast growth factor receptor 3 (FGFR3) mutations in 82 patients with papillary urothelial hyperplasia lesions. Thirty-eight patients presented with papillary urothelial hyperplasia and concurrent noninvasive papillary urothelial carcinoma, and 44 patients presented with de novo papillary urothelial hyperplasia. The prevalence of the TERT promoter and FGFR3 mutations is compared between de novo papillary urothelial hyperplasia and those with concurrent papillary urothelial carcinoma. Mutational concordance between papillary urothelial hyperplasia and concurrent carcinoma was also compared. The TERT promoter mutations were detected in 44% (36/82) of papillary urothelial hyperplasia, including 23 (23/38, 61%) papillary urothelial hyperplasia with urothelial carcinoma and 13 (13/44, 29%) de novo papillary urothelial hyperplasia. The overall concordance of TERT promoter mutation status between papillary urothelial hyperplasia and concurrent urothelial carcinoma was 76%. The overall FGFR3 mutation rate of papillary urothelial hyperplasia was 23% (19/82). FGFR3 mutations were detected in 11 patients with papillary urothelial hyperplasia and concurrent urothelial carcinoma (11/38, 29%) and 8 patients with de novo papillary urothelial hyperplasia (8/44, 18%). Identical FGFR3 mutation status was detected in both papillary urothelial hyperplasia and urothelial carcinoma components in all 11 patients with FGFR3 mutations. Our findings provide strong evidence of a genetic association between papillary urothelial hyperplasia and urothelial carcinoma. High frequency of TERT promoter and FGFR3 mutations suggests the precursor role of papillary urothelial hyperplasia in urothelial carcinogenesis.

4571 Background: p53 target and cell cycle inhibitor CDKN1A/p21(WAF1) was initially not found to be mutated in cancer. TCGA analysis identified CDKN1A mutations are present but rare with frequencies of < 1%, but enrich in bladder cancer (̃8%). Truncating WAF1 mutations are associated with sensitivity to cisplatin and are associated with truncating Rb mutations in bladder cancer (RW+). We hypothesized RW+ bladder cancers may represent a unique subgroup with sensitivity to therapeutics. Methods: A total of 1104 urothelial tumors underwent molecular profiling at Caris Life Sciences (Phoenix, AZ) utilizing NGS of DNA (592 Gene Panel, NextSeq, or WES, NovaSeq) and RNA (NovaSeq, WTS). Wilcoxon, Fisher’s exact were used for statistical significance (p value without and q value with multi comparison correction). Immune cell fraction (QuanTIseq) and pathway analysis (ssGSEA) were assessed by mRNA analysis. Immune epitope prediction was performed using the NetMHCpan v4.0 method in the Immune Epitope Database. Results: Concurrent truncating mutation (frameshift, nonsense) for RB1 and WAF1 were detected in 47 tumors (RW+, 4.25%) and tumors with wild-type status for both RB1 and WAF1 genes were classified as RW- group (54.08%). Tumors harboring only one RB1 or WAF1 mutation were excluded for further analysis. When compared to RW- group, RW+ tumors showed lower mutation rate of TP53 (54.5% vs 80.9%, q < 0.05), ARID1A (23.5% vs 38.3%, p < 0.05), and PIK3CA (18.4% vs 31.9%, p < 0.05). Interestingly, RW+ was mutually exclusive with FGFR3 mutation (18.0% vs 0%, p < 0.05). We further evaluated RNA expression of DNA repair and checkpoint arrest pathways. Notably, E2F pathway (Normalized Enrichment Scores, NES: 0.89 vs 0.86, q < 0.01) and DNA G2M checkpoint (NES: 0.89 vs 0.86, q < 0.01) were found to be the most enriched in RW+ with respect to RW- group. In addition, mRNA levels of FANCC/A, CHEK1, WEE1, CDC25A/C, PALB2 and BRCA1/2 were found to be overexpressed in RW+ group (q < 0.05). RW+ tumors also displayed a distinct immunological profile: They were associated with higher PD-L1 status (63.8% vs 37.3%, q < 0.01), higher median TMB (11 mut/Mb vs 8 mut/Mb, q < 0.01) and with less frequent loss of heterozygosity for HLA-DPA1 (51.1% vs 66.7%, p < 0.05), with more high-binding-affinity neoantigen load (4.78 vs 3.89, p < 0.05) to MHC proteins, consistent with the significantly more myeloid dendritic cells in in RW+ group (0.3 vs 0.04, q < 0.001). Conclusions: Concurrent truncating mutation in RB1 and WAF1 (RW+) bladder carcinomas have fewer p53, ARID1A, and PIK3CA mutations but are enriched for E2F targets, G2/M checkpoint genes, FANCC/A, CHEK1, WEE1, CDC25A/C, PALB2 and BRCA1/2 and have a distinct immunological profile. The findings suggest therapeutic strategies for RW+ bladder cancers including Chk1/Wee1, PARP inhibitors, -/+ immunotherapy that may impact on clinical outcomes.

GSK-3β is a serine/threonine kinase implicated in tumorigenesis and chemotherapy resistance. GSK-3β blockade downregulates the NF-κB pathway, modulates immune cell PD-1 and tumor cell PD-L1 expression, and increases CD8 + T cell and NK cell function. We report a case of adult T-cell leukemia/lymphoma (ATLL) treated with 9-ING-41, a selective GSK-3β inhibitor in clinical development, who achieved a durable response. A 43-year-old male developed diffuse lymphadenopathy, and biopsy of axillary lymph node showed acute-type ATLL. Peripheral blood flow cytometry revealed a circulating clonal T cell population, and CSF was positive for ATLL involvement. After disease progression on the 3rd line of treatment, he started treatment with 9-ING-41 monotherapy in a clinical trial (NCT03678883). CT imaging after seven months showed a partial response. Sustained reduction of peripheral blood ATLL cells lasted 15 months. Treatment of patient-derived CD8 + T cells with 9-ING-41 increased the secretion of IFN-γ, granzyme B, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In conclusion, treatment of a patient with refractory ATLL with the GSK-3β inhibitor 9-ING-41 resulted in a prolonged response. Ongoing experiments are investigating the hypothesis that 9-ING-41-induced T cell activation and immunomodulation contributes to its clinical activity. Further clinical investigation of 9-ING-41 for treatment of ATLL is warranted.

128 Background: mCRPC with alterations in genes associated with homologous recombination (HR) DNA repair (e.g., BRCA1/2) is sensitive to poly ADP-ribose polymerase inhibitors (PARPi). Preclinical studies showed that PI3K inhibitors (PI3Ki) impairs HR and sensitize cancer cells to PARPi even in the absence of HR gene mutations. These results support our hypothesis that dual PI3K and PARP inhibition may improve clinical outcomes in progressive mCRPC. We describe preliminary results of a phase Ib/II study investigating safety of the combination of copanlisib (pan-class I PI3Ki) and rucaparib (PARP-1, -2 and -3 inhibitor). Methods: Enrollment criteria included progressive mCRPC, prior androgen inhibitors (abiraterone, enzalutamide, and/or apalutamide); prior taxane chemotherapy was allowed. HR-deficiency was not required for the phase Ib. The phase I followed a standard 3+3 escalation design. Dose schema: rucaparib (continuous oral administration twice daily) 400mg (dose level [DL] -1, 1), 500mg (DL 2) or 600mg (DL 3,4) and intravenous copanlisib (45mg D1, D15 (DL -1, -2); 45mg, D1, D8, D15 (DL 1, 2, 3); 60mg, D1, D8, D15 (DL 4); 28-day cycle). Adverse events (AE) were graded by CTCAE v5.0. The primary aim of the phase I was to establish the MTD and the recommended phase II dose (RP2D) of copanlisib in combination with rucaparib. Results: Eleven pts were enrolled with a median age of 63 (55-78) and median PSA of 12 ng/mL (0.018–2,101). Seven pts (63%) received prior chemotherapy (docetaxel [7], cabazitaxel [3]). Pathogenic HR mutations included BRCA1 (1), BRCA2 (3), CDK12 (1), and FANCA (1). Treatment-related AE included grade 2 (G2) leukopenia (30%), G2 anemia (20%), G2 rash (20%). Two dose-limiting toxicities (DLTs) were observed in DL 1: G3 rash and G3 AST/ALT elevation attributed to both drugs. Six pts were treated at DL -1 without DLTs. The RP2D was rucaparib 400mg BID with copanlisib 45mg (D1, D15; 28-day cycle). There were 2 confirmed PSA50 responses among 7 evaluable pts (28%). One pt had BRCA2 loss and 1 had PALB2 VUS (ongoing PSA response for 14 mo). Three stable disease and 1 partial response were observed among 6 pts evaluable by RECIST 1.1. Conclusions: The combination of rucaparib and copanlisib is well tolerated. The RP2D was rucaparib 400mg BID with copanlisib 45mg (D1, D15; 28-day cycle) with signal of efficacy. Enrollment in a phase 2 expansion cohort in HR-mutated mCRPC is ongoing. Clinical trial information: NCT04253262.

Introduction: Small cell carcinoma of the urinary tract (SCCUT) is a rare finding with poor clinical course. This study sheds light on the molecular subtype and identifies risk factors in patients diagnosed with SCCUT. Methods: Immunohistochemical expression of immunotherapy target programmed death ligand 1 (PD-L1) and luminal (GATA3), basal (p63), and p53 markers are assessed in patients diagnosed with SCCUT. Univariate analysis identified risk factors. Overall survival (OS) is computed using the Kaplan–Meier method. Results: Tissue was available for 70.2% (33/47). All showed a high PD-L1 expression phenotype. p53 is seen in 93.9% (31/33), mostly as overexpression, GATA3 in 45.5% (15/33), and p63 in 57.6% (19/33). For the entire cohort ( n = 47), 1-year survival was 59.6%, and the median OS was 17 months. Univariate analysis shows that chemotherapy [hazard ratio (HR) = 0.29, 95% confidence interval (CI) = 0.14–0.61, p = 0.001], radical surgery (HR = 0.37, 95% CI = 0.18–0.76, p = 0.007), and diagnosis of non-pure SCCUT (HR = 0.44, 95% CI = 0.22–0.86, p = 0.02) are favorable prognostic features. Metastasis had negative associations with survival (HR = 2.1, 95% CI = 1.1–4.2, p = 0.03). Conclusions: In this series, pure and mixed SCCUT are characterized by p53 overexpression and a high PD-L1 phenotype. Histology of non-pure SCCUT is a positive prognosticator, and radical cystectomy or chemotherapy can improve OS. These findings demonstrate that SCCUT may be eligible for PD-L1 immunotherapy.

Abstract Understanding the distinct genetic features of the primary tumor and metastases in advanced prostate cancer (PCa) has clinical relevance and potential therapeutic implications. Studies utilizing differential gene expression analysis between primary tumors and sites of metastases continue to characterize the evolution of PCa from castration-sensitive (CSPC) through metastatic castration-resistant PCa (CRPC). This study aimed to investigate the genomic features of a cohort of patients (pts) with PCa with primary and/or metastatic tumors submitted for genomic analysis. We hypothesize that unique genomic features are associated with the site of metastases and can reveal potential site-specific drivers of disease progression as well as therapeutic targets. We performed a retrospective analysis of tumor biopsies by next-generation sequencing (NGS) from 45 patients with PCa. Targeted DNA sequencing of more than 500 cancer-associated genes was performed in paraffin-embedded tumor samples. All 45 tumors [(primary tumors (17), metastatic sites (28)] had DNA sequencing data available. Among these, 22 were additionally subjected to transcriptome profiling by RNA-seq. This subset comprised: 8 prostate and 14 metastatic samples [lymph node (7), bone (3), and visceral (4)]. Visceral sites included liver (1), colon (1), stomach (1), and bladder (1). The median age of pts was 66 years (range 50-84). The median prostate-specific antigen (PSA) level at diagnosis was 65 ng/mL (range 0.5-3,403). Among the 24 pts with Gleason score available, 17 (70%) had grade group 5 (3 - Gleason 10; 14 - Gleason 9). Primary tumors analyzed by NGS included CSPC (12) and CRPC (6). Most metastatic tumors were CSPC at biopsy (15 - CSPS; 12 - CRPC). Among the cohort, frequent gene alterations affected homologous repair deficiency genes (29.6%), as well as PTEN (20.3%) TMPRSS2 (25.9%), and FOXA1 (5%) genes. Differential gene expression and pathway analysis of RNA-seq data was performed using Bioconductor on the R statistical computing environment. Comparisons between primary and metastatic gene expression profiles revealed shared features among sample groups, including transcriptional changes to interferon-alpha and -gamma, cell cycle regulators and Myc targets, whereas others were distinct to the sites of metastasis. Bone metastases exhibited regulatory changes to JAK/STAT signaling and angiogenesis, as well as alterations in DNA repair. Visceral tumor samples were the most diverse cohort in terms of global transcriptome state, but were notable for MTOR and androgen signaling. Lymph node biopsies showed pronounced TNF-α signaling via NFkB and oxidative phosphorylation. Our results exemplify the cellular and molecular diversity between primary tumors and metastatic sites and highlight potential pathways and transcriptome profiles unique sites of metastases. Additional analysis on expanded cohorts of pts is ongoing. Citation Format: Luke B. Soliman, Andre L. De Souza, Praveen Srinivasan, Matthew Danish, Dragan J. Golijanin, Ali Amin, Anthony E. Mega, Wafik S. El-Deiry, Paul Bertone, Benedito A. Carneiro. Differential transcriptomic profiling of primary tumors and metastatic sites in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2195.

Abstract Chromosome 9p21 deletion occurs in 25% of urothelial carcinomas (UC). 9p21 includes the MTAP gene that encodes methylthioadenosine phosphorylase, a critical enzyme in the alternate pathway of adenine synthesis. MTAP-deleted UC tumors are very sensitive to pemetrexed-induced blockage of purine synthesis. MTAP is commonly co-deleted with CDKN2A, an immediately adjacent gene associated with sensitivity to CDK4/6 inhibitors. We describe clinical and molecular features of a 9p21-deleted subgroup of UC to better inform targeted therapy approaches. We retrospectively analyzed 46 patients (pts) with UC treated at our institution. All tumor specimens were submitted to next-generation sequencing (NGS). Patient demographics, clinic-pathological and genomic features, and clinical outcomes were summarized with descriptive statistics. MTAP/CDKN2A co-deletion was defined as a surrogate for 9p21 deletion. Fifteen (32%) pts with 9p21-deleted UC were identified. The median age was 68 years-old (range 54-79), 80% of pts were former or active smokers. Fifty-three percent (8 of 15) of these tumors showed squamous differentiation. Other histology variants were papillary differentiation (6), micropapillary (1), and sarcomatoid (1). Sites of tumor specimens submitted to NGS included bladder (11), lymph node (1), abdominal wall (1), renal pelvis mass (1), and bone (1). All pts had metastatic disease involving bone (9), lung (8), lymph nodes (7), liver (3), abdominal wall (2), penis (2), peritoneum (1), and pararenal mass (1). Most pts (8) underwent curative surgery and later developed metastases; seven pts presented with de novo metastases. Among 9 pts with progression-free survival (PFS) data available, median PFS was 2 months (range 1-17). Median overall survival was 6 months (range 1-35). Median PD-L1 expression on tumor cells was 1% (range 1-20); 4 tumors were PD-L1 (-). All evaluable patients (14) had stable microsatellite tumors. The most common genomic alterations co-occurring with 9p21 deletion affected CDKN2B (100%), TERT (87%), KDM6A (60%), TP53 (40%), and FGFR3 (20%). The best response associated with immune checkpoint inhibitors among 7 patients with 9p21 deletion was a disease control rate of 28.5%. Our cohort with UC and 9p21 deletion had predominantly squamous cell differentiation. Thus, MTAP is always co-deleted with CDKN2A and is a potential biomarker for pemetrexed-based combinations. Molecular characterization of 9p21 deleted UC may in the future inform targeted therapeutic approaches. Citation Format: Andre L. De Souza, Praveen Srinivasan, Luke B. Soliman, Dragan J. Golijanin, Ali Amin, Howard Safran, Anthony E. Mega, Wafik S. El-Deiry, Benedito A. Carneiro. Clinical and genomic features of advanced urothelial carcinoma with 9p21 deletion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1205.