Spatial transcriptomics (ST) provides unprecedented insights into gene expression patterns while retaining spatial context, making it a valuable tool for understanding complex tissue architectures, such as those found in cancers. Seurat, by far the most popular tool for analyzing ST data, uses the Wilcoxon rank-sum test by default for differential expression analysis. However, as a nonparametric method that disregards spatial correlations, the Wilcoxon test can lead to inflated false positive rates and misleading findings. This limitation highlights the need for a more robust statistical approach that effectively incorporates spatial correlations. To this end, we propose a Generalized Estimating Equations (GEE) framework as a robust solution for differential gene expression analysis in ST. We conducted a comprehensive comparison of the GEE-based tests with existing methods, including the Wilcoxon rank-sum test and z-test. By appropriately accounting for spatial correlations, extensive simulations showed that the GEE test with robust standard error, referred to as the Independent GEE, demonstrated superior Type I error control and comparable power relative to other methods. Applications to ST datasets from breast and prostate cancer showed poor calibration of the p-values and potential false positive findings from the Wilcoxon rank-sum test. Our comparative study based on simulations and real data applications suggests that the Independent GEE test is well-suited for ST data, offering more accurate identification of biologically relevant gene expression changes and complementing the Wilcoxon rank-sum test. We have implemented the proposed method in R package "SpatialGEE", available on GitHub.

Abstract Introduction: Understanding the mechanisms driving evolution of the mucosal field effects to invasive cancer is not possible unless they are analyzed in the context of their geographic distribution in the entire organ. Bladder cancer is an ideal model disease for such studies as it effects an anatomically simple organ permitting the multi-platform analysis of the affected mucosa on the scale of whole organ. Methods: We performed comprehensive multi-platform analyses on nine cystectomies with invasive bladder cancer comprising of 433 mucosal tissue samples analyzed by bulk whole-exome and mRNA sequencing, genome-wide copy number variation and methylation profiling complemented with proteomics, metabolomic and single cell sequencing spatial mapping. The data from multi-platform profiling were geographically annotated to microscopically normal urothelium (NU) and low-grade intraurothelial neoplasia (LGIN; n=243), high-grade intraurothelial neoplasia (HGIN; n=90), and invasive urothelial carcinoma (UC; n=100). Results: We identified ∼16000 non-silent mutations per cystectomy. In two maps the hypermutator phenotypes with ∼48000 and ∼57000 mutations were detected. The mutational analysis identified three types of mutations based on the variant allele frequency and geographic distribution referred to as α, β, and ϒ. Time modeling by a parsimonious time-continuous Markov model incorporating cell migration (immigration) and growth (branching) revealed that bladder carcinogenesis takes approximately three decades and can be divided into dormant and progressive phases. Low selection α mutations were private and most frequent. They continuously developed over 30 years primarily in the dormant phase of carcinogenesis. β mutations clonally expanded regionally and signified the advent of progressive phase of carcinogenesis which lasted five years. ϒ mutations were the ultimate drivers of the progressive phase and emerged 2-3 years before the final progression to invasive bladder cancer. The mutational landscape developed on the background of severely dysregulated urothelial differentiation and increased immune infiltration with T-cell exhaustion. The proteomic and metabolomic changes involved a wide-spread disorganization of glycolipid energy metabolism with downregulation of mitochondrial oxidative phosphorylation. Conclusion: Dysregulated mitochondrial energy metabolism converging on anerobic glycolysis and oxidative phosphorylation with Warburg phenotype emerged as the leading mechanism driving the progression of mucosal field effects to invasive cancer. Citation Format: Bogdan A. Czerniak, Sangkyou Lee, Khanh Ngoc Dinh, Huiqin Chen, Yishan Wang, Jiansong Chen, June Goo Lee, Sung Yun Jung, Nagireddy Putluri, Neema Navai, David McConkey, Charles Chuanhai Guo, Peng Wei, Marek Kimmel. Modeling of bladder cancer evolution from field effects by multi-platform spatial mapping on the whole-organ scale [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5931.

The identification of tumor cells is pivotal for understanding tumor heterogeneity and the tumor microenvironment. Recent advances in spatially resolved transcriptomics (SRT) have revolutionized the way that transcriptomic profiles are characterized and have enabled the simultaneous quantification of transcript locations in intact tissue samples. SRT is a promising alternative method to study gene expression patterns in spatial domains. Nevertheless, the precise detection of tumor regions within intact tissue remains a great challenge. A common strategy for identifying tumor cells is via tumor-specific marker gene expression signatures, which are highly dependent on marker accuracy. Another effective approach is through aneuploid copy number alterations, as most types of cancer exhibit copy number abnormalities. Here, we introduce a novel computational method, called TUSCAN (TUmor Segmentation and Classification ANalysis in spatial transcriptomics), which constructs a spatial copy number variation profile to improve the accuracy of tumor region identification. TUSCAN combines gene information from SRT data and hematoxylin-and-eosin-staining image to annotate tumor sections and other benign tissues. We benchmark the performance of TUSCAN and several existing methods through the application to multiple datasets from different SRT platforms. We demonstrate that TUSCAN can effectively delineate tumor regions, with improved accuracy compared to other approaches. Additionally, the output of TUSCAN provides interpretable clonal evolution inferences that may lead to novel insights into disease development and potential druggable targets.

Adrenocortical neoplasms with myxoid change are very rare. Herein, we report a case of a 67-year-old man with a history of pancreatic adenocarcinoma status post distal pancreatectomy. During follow-up, PET/CT scan revealed a 2.8 cm tumor involving the left adrenal gland, which was resected. Microscopic examination showed a glandular-appearing lesion involving the adrenal cortex, which raised the differential diagnosis of metastatic pancreatic cancer. Initial immunohistochemical stains showed the lesional cells were negative for CK7, focally positive for pan cytokeratin, and retained SMAD4. Additional evaluation showed the tumor cells were diffusely positive for SF-1, Melan-A, CD56; focally positive for calretinin, and inhibin; and negative for CK20, CDX2, TTF-1, and chromogranin. A Ki67 index was 5 %. While these findings excluded pancreatic adenocarcinoma, they raised the differential diagnosis of adrenal cortical adenoma and/or carcinoma. Final diagnosis was myxoid adrenocortical tumor of uncertain malignant potential (MAT-UMP) following consensus review. In this case report and review, we highlight the diagnostic challenges of MAT-UMP, discuss potential pitfalls associated with this diagnosis, and review literature on this unusual entity.

Consort Diagram for participants in study

MTAP-deficiency in urothelial cancer is associated with poor clinical outcomes but correlates with sensitivity to antifolate chemotherapy. This single-arm phase 2 trial (N=15) assessed the clinical and biological effects of a sequential combination therapy using the immune modulating antifolate agent pemetrexed and anti-PD-L1 agent avelumab in patients with advanced urothelial cancer resistant to prior chemotherapy and/or immunotherapy. The combination regimen treatment resulted in an overall response rate (ORR) of 20% (95% CI: 5%, 42%), a disease control rate (DCR) of 47% (95% CI: 23%, 71%), a median progression-free survival (PFS) of 2.3 months (95% CI: 1.9, 5.5), and a median overall survival (OS) of 13.5 months (95% CI: 3.2. 22.4). To investigate the relationship between clinical outcomes and tumor microenvironment (TME), single-cell RNA sequencing was performed on tumor samples collected from 15 patients at three time points: baseline, after pemetrexed monotherapy (cycle 2, day 1), and following combined treatment (cycle 4, day 1). This analysis profiled 49, 161 cells spanning nine major cell types, including T cells, B cells, plasma cells, myeloid cells, epithelial cells, fibroblasts, and endothelial cells. Correlating TME composition with clinical outcomes revealed that higher proportions of T and NK cells at baseline were positively associated with PFS and clinical benefit. Conversely, elevated levels of proliferating myeloid cells and macrophages correlated with poorer responses to therapy. Pemetrexed monotherapy notably reshape the immune landscape, promoting increased infiltration of type-2 conventional dendritic cells (cDC2). Furthermore, patients with augmented B and plasma cell infiltration following pemetrexed treatment exhibited better clinical outcomes. Resistance to treatment was associated with increased adenosine signaling, suggesting a potentially targetable pathway to enhance treatment efficacy. Preclinical data suggest that targeting adenosine signaling pathway may further enhance the therapeutic effects of pemetrexed and anti-PD-L1 for MTAP-deficient bladder cancer. Additionally, urine methylthioadenosine (MTA), which accumulates due to MTAP deficiency, emerged as a promising biomarker for treatment monitoring. These discoveries unveil the link between TME of MTAP-deficient tumors and response for this clinical trial, suggest potential targetable mechanism to improve efficacy, which better the understanding of MTAP-deficient tumors and design promising strategies to improv clinical translation. These findings underscore the intricate link between the TME and treatment response in MTAP-deficient tumors, offering insights into resistance mechanisms to optimize clinical outcomes in MTAP-deficient urothelial cancer. Amishi Y. Shah, Xinmiao Yan, Rebecca S. Tidwell, Jianfeng Chen, Arlene O. Siefker-Radtke, Sangeeta Goswami, Omar Alhalabi, Yuxue Zhang, Ying Mei, Charles Guo, Miao Zhang, Ana C. Adriazola, Abu Hena Kamal, Nagireddy Putluri, Mark A. Titus, Bogdan Czerniak, Matthew T. Campbell, Linghua Wang, Jianjun Gao. Sequential anti-folate (pemetrexed) and anti-PD-L1 (avelumab) combination therapy in patients with previously treated MTAP-deficient advanced urothelial cancer: Clinical outcomes and immune correlative analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2143.

This manuscript summarizes the first part of the proceedings of the 2023 Dublin ISUP Consensus Conference encompassing the best practice recommendations on the pathology of neoplasms of urachal origin. The rationale for convening this consensus conference was the lack of structured and consented histopathologic recommendations in these rare tumors. Consensus among the meeting participants (n=80) was reached on the following statements: (1) combination of gross, histologic, clinical and imaging findings with exclusion of secondary tumor metastasis are to be used in the diagnosis of urachal carcinoma; (2) the 2022 World Health Organization (WHO) separate criteria for the diagnosis of urachal adenocarcinoma and for nonglandular carcinoma should be applied; (3) specific elements are to be evaluated and recorded in the gross examination of resection specimens containing urachal tumors; (4) sampling considerations for resection specimens containing urachal tumors are advised; (5) participants are against using 5% or 10% cutoff for the extent of intraepithelial carcinoma in urachal mucinous cystic tumor of low malignant potential; (6) use of immunohistochemical markers for the differential diagnosis of urachal adenocarcinomas in transurethral resection (TUR) specimen is considered optional; (7) similar tumor classificatory (nosology) rules for carcinomas arising from bladder mucosa (eg, urothelial carcinoma, squamous cell carcinoma, and neuroendocrine carcinoma) should be applied for nonglandular urachal carcinomas; (8) a new staging approach other than the previously proposed systems should be designed for urachal carcinoma; (9) a system modifying the current Tumor-Node-Metastasis (TNM)/American Joint Committee on Cancer (AJCC) staging system for urinary bladder cancer is considered appropriate for a study in urachal carcinoma; and (10) several histologic elements are to be reported when diagnosing urachal carcinoma in TUR and resection specimens. This report from the Dublin ISUP consensus conference will serve as a practice recommendation for pathologists and as a guide for future standardized reporting protocols and research regarding urachal tumors. In addition, an international database for urachal cancers under the guidance of ISUP is being planned to be established to address pertinent issues in the pathology of urachal cancers.

Multiplatform mutational and gene expression profiling complemented with proteomic and metabolomic spatial mapping were used on the whole-organ scale to identify the molecular profile of bladder cancer evolution from field effects. Analysis of the mutational landscape identified three types of mutations, referred to as α, β, and γ. Time modeling of the mutations revealed that carcinogenesis may span 30 years and can be divided into dormant and progressive phases. The α mutations developed in the dormant phase. The progressive phase lasted 5 years and was signified by expanding β mutations, but it was driven to invasive cancer by γ mutations. The mutational landscape emerged on a background of disorganized urothelial differentiation, activated epithelial-mesenchymal transition, and enhanced immune infiltration with T-cell exhaustion. Complex dysregulation of mitochondrial energy metabolism with downregulation of oxidative phosphorylation emerged as the leading mechanism driving the progression of mucosal field effects to invasive cancer. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

818 Background: MSI-high (MSI-H) and dMMR are three times more common in UTUC than bladder cancer. While these features enhance ICI sensitivity in solid tumors, data on ICIs in advanced UTUC with dMMR/MSI-H remain scarce. Methods: We retrospectively reviewed records of patients (pts) with locally advanced (LA) or metastatic with dMMR/MSI-H UTUC treated with ICIs at MD Anderson (2015-2024). Descriptive statistics and the Kaplan-Meier method were used. Results: Twenty-four pts with LA/unresectable (n=8) or metastatic (n=16) disease were treated with ICIs (pembrolizumab, n=17; nivolumab, n=4; atezolizumab, n=3) (Table). Tumor origin was the ureter (10, 41.7%) or renal pelvis (14, 58.3%). Twenty-two (91.7%) pts had Lynch syndrome, mostly (n=20) detected by germline mutation testing ( MSH2 , n=12; MLH1 , n=5; MSH6 , n=3).Twenty-one (87.5%) pts had IHC-proven dMMR(MSH2/MSH6 loss, n=7; PMS2/MLH1 loss, n=5; MLH1 loss, n=4; MSH2 loss , n=3; MSH6 loss, n=3). The most frequent alterations in somatic genetic testing for 22 pts were MSH2 (12 pts), CREBBP (7 pts), ARID1A (7 pts) and NOTCH3 (7 pts). Fourteen (58.3%) pts received prior platinum chemotherapy: cisplatin (n=9) or carboplatin-based (n=5), with only 35.7% showing responses (one complete response [CR] and 4 partial responses [PR]).With a median follow-up of 54.3 months (mo) [95% CI: 19.5 – 87.1], median PFS was 55.8 mo [95% CI: 19.4 – NE]. Milestone PFS probabilities at 12 and 24 mo were 90.7% [95% CI: 79.2% – 1%] and69.2% [95% CI: 51.3% – 93.4%], respectively.At a median follow-up of 51.3 mo [95% CI: 34 – 60.2], median OS was not reached. Median time on treatment was 13.5 mo [IQR: 4 – 25.8]. Eight (33.3%) pts remain progression-free beyond 4 years. ORR was 79.2%, including 16 CR (66.7%) and 3 PR (12.5%), while DCR was 95.8%.Median time to best response was 11.9 mo [IQR: 5.6 – 20]. Four (16.7%) pts were offered surgical consolidation with these outcomes: ypTaN0M0, ypT0N0M0, ypT1N0M0 and mypT1N0 M1-NED. Grade ≥3 immune-mediated toxicity events leading to ICI discontinuation (n=6) included hepatitis (n=3), pancytopenia (n=1), polyendocrinopathy (n=1) and diarrhea (n=1). Conclusions: Single-agentICIs show remarkable efficacy and durable responses in advanced dMMR/MSI-H UTUC, with one-third of pts remaining in remission after over 4 years. Our findings support dMMR/MSI-H as a predictive and prognostic biomarker in UTUC and the use of single-agent ICIs in this subpopulation. Variable Measure Age at dx of advanced disease, median [ICR] 66 [55.8 – 72.3] Sex, male (n, %) 14 (58.3%) Race/Ethnicity, n (%) White 19 (79.2%) Black 1 (4.2%) Hispanic 2 (8.3%) Asian 2 (8.3%) Urothelial carcinoma mixed with variant histology, n (%) 3 (12.5%) Liver mets at ICI start, n (%) 2 (8.3%) Bone mets at ICI start, n (%) 5 (20.8%) Hemoglobin <10 g/dL at ICI start, n (%) 5 (20.8%) Bellmunt score, n (%) 0-1 19 (79.2%) 2 3 (12.5%) 3 2 (8.3%)

799 Background: Metastasis-directed therapy (MDT) for oligometastatic cancer is a concept utilized for prostate and kidney cancer. Clinical research in MDT for oligometastatic urothelial carcinoma (UC) remains sparse especially in the modern era where systemic therapy advancements have substantially improved patient’s outcomes overall. We explored our institutional experience of patients with oligometastatic carcinoma of the bladder and upper tract undergoing MDT utilizing radiotherapy (RT). Methods: Patients were retrospectively identified with oligometastatic bladder or upper tract cancer from January 2016 to July 2024 with five or less sites of metastases. Those with equivalent dose of RT (EQD2 10 ) ≥ 45Gy to metastases were included. Progression free survival (PFS) and overall survival (OS) were evaluated using Kaplan Meier from time of diagnosis to metastatic disease. Cox proportional hazards analysis was conducted to determine covariates associated with survival endpoints. Results: 60 patients with oligometastasis were included with 8 patients excluded due to a RT dose EQD2 10 < 45Gy. 52 patients were in final analysis. Most were men (67%) with a median age 68 years (range, 35-91). Most had bladder primary (79%) with the remaining including upper tract. Majority had pure UC (85%) and the remainder were UC subtypes with variant histology including small cell (8%), squamous (6%), sarcomatoid (2%). Median number of metastases was 1 site, while 23% had 3+ sites. Bony metastases (27%) were most common site, then retroperitoneal nodes (18%) and lung (17%). Most received ≥2 systemic therapy cycles before MDT (62%) with 8% without any therapy prior to MDT. Most commonly used therapy included ddMVAC (21%), Gem/Cis (20%), pembrolizumab (15%), and EV (12%). MDT was delivered to all metastases in 71%, while the remaining had MDT to select sites. Most common MDT dose was 30Gy in 3 fractions (21%) followed by 50Gy in 4 fractions (17%). Median follow up from metastatic diagnosis was 19 months (range, 3-106 months). Median PFS and OS was 21 months (95% CI 8-33 months), and 39 months (95% CI, 14-64 months), respectively. At last follow up, 31 patients were alive (60%). Most common first recurrence was distant from site of MDT (96%) while 2 patients had in-field recurrence in pelvic bones. On univariate analysis, those with 1 vs 2+ sites had improved PFS with MDT (p=0.03, 95% CI 1.1-6.0). Univariate showed no association with MDT to all sites vs select, age, or # lines of systemic therapy. Conclusions: As systemic therapy has improved for patients with bladder and upper tract cancers, MDT may serve as an effective adjunct to improve cancer control. Baseline characteristics. Characteristic (n=52) No % Median Age (yrs) 68 Histology UC 44 85% UC subtype with variant histology 7 15% Site of Primary Bladder 41 79% Upper Tract 11 21% Lines of therapy before MDT 0 4 8% 1 17 33% 2+ 31 60%

Preneoplastic and precursor lesions are important to recognize and report, as they can influence clinical management decisions. The International Society of Urological Pathology (ISUP) organized a consensus meeting in Florence, Italy, in September 2024 focused on preneoplastic and precursor lesions of the genitourinary organs. Working group 2 was assigned the topic of bladder and a group of pathologists and clinicians was convened. They developed a 46 question premeeting survey for the ISUP membership assessing flat, papillary, squamous, and glandular entities and clinical issues to determine use of terminology, reporting practices, and areas that needed to be addressed at the consensus meeting. The premeeting survey results showed consistency in the terminology used by pathologists, similarities in reporting practices, and highlighted areas of uncertainty with respect to whether certain entities could be classified as precursors/preneoplastic. The results enabled the working group to conduct focused literature reviews and to develop a presentation and set of in-meeting polling questions to address the problematic topics from the survey results. Overall, 14/18 in-meeting polling questions achieved consensus. The surveys and in-person voting demonstrated a strong preference to use existing terminology such as dysplasia, verrucous squamous, and papillary hyperplasia, to grade glandular and squamous dysplasia and to judiciously use immunohistochemistry to classify lesions. Pathologists expressed highly variable opinions with respect to questions about quantification, management recommendations, and inclusion of newer entities as precursors/preneoplastic lesions.

4566 Background: We previously identified that 9p21-loss (containing MTAP and CDK2NA genes) occurs in 25% of metastatic urothelial cancer (mUC) and is associated with worse survival and increased visceral metastasis. Additionally, we found significantly increased expression of the TGFβ-SMAD3 pathway in 9p21-loss UC, which is well studied to induce epithelial mesenchymal transition (EMT) and lead to metastasis. We hypothesize that the TGFβ signaling pathway mediates visceral metastasis in 9p21-loss UC. Methods: We included mUC patients at MD Anderson Cancer Center (MDACC) who had MTAP testing completed (surrogate for 9p21-loss) and received chemotherapy, immune checkpoint therapy (ICT), and/or antibody drug conjugate between 2012 and 2022. Survival and metastasis were compared between MTAP deficient patients and MTAP proficient patients. The Memorial Sloan Kettering-Metastatic Events and Tropisms (MSK-MET) cohort of mUC patients and IMVigor210 dataset were assessed for genomic alterations. Mouse models bearing CDKN2A-MTAP double knock out (DKO) MB49 tumors and wild type (WT) MB49 tumors, as well as corresponding in vitro models were used for mechanistic studies. Results: 298 mUC patients at MDACC were identified with 27% (n = 81) MTAP deficient. MTAP deficient patients experienced significantly worse overall survival (16.2 vs 21.1 months; p = 0.002; HR 1.61; 95% CI 1.2-2.2), progression-free survival (3.9 vs 5.8 months; p < 0.001; HR 1.75; 95% CI 1.3-2.4) and increased visceral metastasis (62% vs 39%; p = 0.001) with lung predominance (44% vs 26%; p = 0.003) compared to MTAP proficient patients. We also found in the MSK-MET cohort that 26% (186/714) of patients with mUC to the bladder had lung metastasis and those with lung metastasis had significantly increased frequency of CDK2NA deletion compared to patients without lung metastasis (30% vs 18%; OR 2.0; 95% CI 1.3-3.0). Our preclinical data also demonstrated that CDKN2A-MTAP DKO mice resulted in significantly larger primary bladder tumors and worse survival compared to mice bearing WT MB49 tumors. Additionally, mice bearing orthotopic CDKN2A-MTAP DKO MB49 tumors readily developed lung metastasis. Analysis of the IMvigor210 dataset (N = 298) showed that 9p21-loss UC patients had significantly increased expression of TGFβ and EMT pathway genes. These data are consistent with data from our DKO MB49 tumor models. We are currently assessing the impact of a TGFβ inhibitor on mitigating CDKN2A-MTAP DKO-mediated metastasis. Conclusions: Our clinical and pre-clinical data demonstrates that 9p21-loss mUC leads to worse survival and increased visceral metastasis, especially to the lung. We found that the TGFβ signaling pathway may play a role in the development of metastasis. Moving forward, we plan to prospectively investigate the therapeutic potential of TGFβ inhibition in patients with 9p21-loss UC.

All Adverse Events in the MIBC cohort regardless of attribution

Germ cell tumors of the testis (GCTs) provide an ideal tumor model to investigate the cellular versus genetic origin of cancers. In this single institutional study, we evaluated 38 patients with bilateral GCT, including tumors that occurred simultaneously (synchronous) and those occurring at different times (metachronous). For nine of these patients, DNA was isolated from the right and left GCT to determine the genomic and epigenetic differences between tissues using whole-exome sequencing (WES) and reduced representation bisulfite sequencing (RRBS). We found that seminomas and non-seminomas are molecularly distinct based on DNA methylation and not due to synchronous or metachronous disease. In addition, we did not observe conservation of genetic mutations in right and left GCT in either synchronous or metachronous disease. Our data suggest a cellular origin for bilateral GCT.

Grade heterogeneity is common in papillary urothelial carcinoma (PUC) of the urinary bladder. Mixed-grade PUC is generally treated as high-grade if the high-grade component accounts for ≥5 % of the tumor. However, the clinical behavior of predominantly low-grade PUC with a minor high-grade (<5 %) component remains uncertain. We retrospectively searched our pathology files and identified 52 cases of non-invasive mixed-grade PUC (predominantly low-grade PUC with a minor high-grade (<5 %) component). These patients' clinical outcomes were compared with those of 2 other cohorts, 52 cases of pure low-grade PUC and 50 high-grade PUC. During follow-up, 27 patients with mixed-grade PUC developed cancer recurrence in an average of 25 months, and 12 patients experienced progression to high-grade PUC, including invasive UC in 3 patients, in an average of 17 months. For comparison, 35 patients with low-grade PUC experienced cancer recurrence in an average of 17 months, but only 1 (2 %) experienced progression to high-grade non-invasive PUC; 34 patients with high-grade PUC experienced recurrence in an average of 13 months, and 10 (20 %) experienced progression to invasive UC. There was no significant difference in cancer recurrence in the 3 cohorts; cancer grade and stage progression rates in patients with mixed-grade PUC were significantly higher than those in those with low-grade PUC, but stage progression rate in patients with mixed-grade PUC was significantly lower than that in high-grade PUC. In conclusion, patients with mixed-grade PUC had a significantly worse clinical outcome than those with pure low-grade PUC but a significantly better outcome than those with high-grade PUC. Our results suggest that mixed-grade PUC should be treated as a distinct category from low-grade PUC.

4569 Background: HER2-directed therapy with trastuzumab deruxtecan is now FDA approved for UC with high HER2 expression. The interaction between HER2 expression, clinical phenotype and genomic tumor make-up is poorly understood in UC. Methods: We reviewed the records of 209 patients with UC and available HER2 expression status treated at MD Anderson Cancer Center between 2021 and 2024. Immunohistochemical (IHC) staining for HER2 and PD-L1 was conducted using 4B5 Ventana PATHWAY and 22C3 pharmDx antibodies, respectively. Formalin-fixed paraffin embedded metastatic or primary UC tumors were sequenced using the MD Anderson Mutation Analysis Precision Panel (MDA MAPP), a 610-gene high-throughput next generation sequencing (NGS) CLIA assay. Chi-square and wilcoxon tests were used to test correlations between HER2 expression and clinico-genomic features. P-values were adjusted for false-detection rate < 0.25. Results: We included 209 patients with available HER2 status (see Table). HER2 and PD-L1 had significant inverse correlation (p=0.0062). HER2 3+ status was significantly associated with bladder primary tumors compared to other sites (renal pelvis, ureter, and urethra) (p=0.0261). HER2 expression correlated with ERBB2 amplifications (0/1+: 0%, 2+: 2%, 3+: 26%, p<0.0001), but not with ERBB2 mutations. Of 13 patients with ERBB2 amplification, 12 were HER2 3+, and 1 was HER2 2+. HER2 also correlated with HELQ missense mutations (0/1+: 0%, 2+: 0%, 3+: 11%, p=0.0002), CDK12 alterations (0/1+: 0%, 2+: 0%, 3+: 20%, p=0.0003), and BCR missense mutations (0/1+: 0%, 2+: 0%, 3+: 11%, p=0.0002). Of 6 HER2 3+ patients with CDK12 amplification, all had ERBB2 amplification. Conclusions: Our study confirms prior observations of HER2 inverse correlation with PD-L1, but positive correlation with bladder origin. Beyond the known association with ERBB2 amplification, we identified molecular correlations between high HER2 expression, missense mutations of HELQ and BCR , and CDK12 alterations. The observed co-occurrence of CDK12 and ERBB2 amplifications may be related to their proximity (<267 kb apart) on chromosome 17q12. Only 26% of HER2 3+ patients had ERBB2 amplifications, highlighting the importance of IHC testing, not just NGS testing, to determine therapy candidacy for trastuzumab deruxtecan. HER2 Expression Status (n) 0/1+ (105) 2+ (58) 3+ (46) p value Primary tumor - Bladder, n (%) 74 (70) 43 (74) 41 (89) 0.0260 Primary tumor - Other, n (%) 31 (30) 15 (26) 5 (11) PD-L1 positive score, median % [IQR] 6 [1-30] 2 [1-10] 1.5 [0.75-5] 0.0062 ERBB2 amplifications, n (%) 0 (0) 1 (2) 12 (26) <0.0001 ERBB2 mutations, n (%) 10 (10) 8 (14) 9 (20) 0.2030 HELQ missense, n (%) 0 (0) 0 (0) 5 (11) 0.0002 CDK12 amplifications, n (%) 0 (0) 0 (0) 6 (13) <0.0001 CDK12 alterations, n (%) 0 (0) 0 (0) 9.2 (20) 0.0003 BCR missense, n (%) 0 (0) 0 (0) 5 (11) 0.0002

e16618 Background: Adenocarcinomas of the urinary tract, including urachal (UA) and non-urachal (NUA) subtypes, are rare tumors lacking standard systemic treatments. We aim to define the efficacy of targeted therapies post-chemotherapy (chemo) progression. Methods: We retrospectively reviewed records of 62 and 45 patients (pts) with advanced UA and NUA, respectively, at MD Anderson from 2000 to 2023. Advanced disease was defined as Sheldon stages IVA/IVB for UA and TNM stage IV for NUA. Two radiologists assessed responses using RECIST version 1.1. Results: First-line (1L) chemo was received by 57 UA pts and 39 NUA pts. NUA pts had either bladder (19, 48.7%) or urethral (20, 51.3%) origin. Chemo + EGFRi strategies included either cetuximab or panitumumab, while Chemo + VEGFi ones included bevacizumab. Visceral metastasis was present in 45 (78.9%) and 23 (59%) pts in UA and NUA, respectively. The most frequent metastatic sites were lung (24, 53.3%), peritoneum (21, 46.7%), liver (7, 15.6%) and bone (6, 13.3%) in UA, versus lung (11, 47.8%), bone (7, 30.4%) and liver (4, 17.4%) in NUA. Median OS (mOS) from 1L chemo was 17 mo [95% CI: 11.8 - 26] and 16.9 mo [95% CI: 8.1 – 23.3], respectively. Chemo responses were mainly in 1L, with ORRs of 22% and 25% with 5-FU based regimens in UA and NUA, respectively (Table). Subsequent chemo + EGFRi or VEGFi showed ORRs of 5.3% and 12.5% in UA but no responses in NUA. Genomic sequencing for 25 UA and 12 NUA pts showed frequent alterations in TP53 , MYC , KRAS , SMAD4 and GNAS in UA, versus TP53 , KRAS , PTPRD in NUA. All three KRAS mutations in NUA were G12D. Twelve UA pts had KRAS alterations, mostly amplification (n = 3), G12D (n = 3) and G12V (n = 2). mOS from metastatic disease was lower for UA pts with KRAS alterations (n = 12) than those without (n = 13) (17.7 mo vs. 29.4 mo, HR = 2.4 [95% CI: 1-5.9], p = 0.02). Conclusions: Beyond active frontline 5-FU based regimens, subsequent chemo with either EGFRi or VEGFi shows limited efficacy in advanced UA. Non-5-FU based chemo shows more activity in NUA, aligning genomically with urothelial cancer. The prognostic value of KRAS alterations and the role of KRAS as a promising therapeutic target in UA, genomically resembling colorectal cancer, merits further study. Responses 1L Chemo Subsequent Tx 5-FU based Non-5-FU based 5-FU based chemo Non-5-FU based chemo Chemo + EGFRi EGFRi mono Chemo + VEGFi UA(n=57) CR 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) PR 11 (22%) 0 (0%) 0 (0%) 0 (0%) 1 (5.3%) 0 (0%) 1 (12.5%) SD 23 (46%) 2 (28.6%) 2 (33.3%) 5 (41.7%) 8 (42.1%) 3 (50%) 2 (25%) PD 13 (26%) 5 (71.4%) 4 (66.7%) 7 (58.3%) 7 (36.8%) 3 (50%) 5 (62.5%) NE 3 (6%) 0 (0%) 0 (0%) 0 (0%) 3 (15.8%) 0 (0%) 0 (0%) Total (n) 50 7 6 12 19 6 8 NUA(n=39) CR 0 (0%) 1 (14.3%) 0 (0%) 0 (0%) 0 (0%) * 0 (0%) PR 8 (25%) 0 (0%) 0 (0%) 3 (25%) 0 (0%) * 0 (0%) SD 11 (34.4%) 1 (14.3%) 1 (20%) 3 (25%) 2 (40%) * 1 (20%) PD 10 (31.2%) 2 (28.5%) 2 (40%) 5 (41.7%) 3 (60%) * 3 (60%) NE 3 (9.4%) 3 (42.9%) 2 (40%) 1 (8.3%) 0 (0%) * 1 (20%) Total 32 7 5 12 5 * 5

The Dublin ISUP Consensus Conference covered the proceedings on the best practice recommendations on nonurachal glandular lesions of the urinary bladder, bladder diverticular cancers, and molecular features of bladder and urachal glandular lesions. The conference proceedings on urachal neoplasms (except for their molecular features) are published elsewhere. The rationale for convening this conference was the lack of structured and consented pathologic recommendations in these rare lesions. Consensus by participants was reached on the following statements: (1) intestinal metaplasia with dysplasia is considered to be a precursor to primary bladder adenocarcinoma; (2) dysplasia arising from cystitis glandularis should be reported in terms of focality (focal or nonfocal) and grade (low or high); (3) the term "adenocarcinoma" should only be used for carcinomas showing pure (nonurothelial) morphology and should not be used interchangeably in urothelial carcinoma with "glandular differentiation" because of the pathobiological differences and management implications; (4) the different histologic subtypes of bladder adenocarcinoma should be specified in the report; (5) immunohistochemistry has an ancillary role in the work up of bladder adenocarcinoma versus gastrointestinal or Müllerian-type adenocarcinomas; (6) lymphovascular invasion should be included as a parameter when reporting bladder adenocarcinoma; (7) representative or targeted sampling will be sufficient for bladder diverticulum resection specimens; and (8) molecular analysis in genomic profiling should be performed only in advanced or metastatic bladder and urachal adenocarcinomas for targetable therapy. This report on glandular (nonurachal) lesions of the bladder from the Dublin ISUP consensus conference will serve as a best practice recommendation and as a guide for future research on these relatively rare lesions.