Urothelial Cancer Patients Research Articles

Molecular Subtypes Are Associated With Clinical Benefit in Cisplatin-Treated Metastatic Urothelial Cancer Patients.

Cisplatin-based combination chemotherapy (CHT) is standard of care in metastatic urothelial cancer (mUC); however, no predictive molecular biomarkers are available for clinical use. The aim of this study was to investigate the impact of molecular subtypes in relation to treatment response and survival in patients with mUC treated with first-line CHT. Molecular subtype classification according to the Lund Taxonomy (LundTax) was performed by tumor transcriptomic profiling and immunostaining in a retrospective cohort. Molecular subtypes were investigated in relation to the primary end point overall response rate (ORR) and secondary end points progression-free survival (PFS) and overall survival (OS). Differential gene expression and association to treatment response were explored. Ninety-five patients with mUC were classified into urothelial-like (Uro, 43%), genomically unstable (GU, 26%), basal squamous-like (Ba/Sq, 20%), mesenchymal-like (Mes-like, 8%), and small cell neuroendocrine-like (Sc/NE, 3%) subtypes. Patients with Mes-like tumors had lower ORR (14%) compared with Uro (70%), GU (77%), Ba/Sq (75%), and Sc/NE (67%; odds ratio, 0.06 [95% CI, 0.01 to 0.54], P = .012). Furthermore, patients with Mes-like tumors had significantly shorter PFS (hazard ratio [HR], 5.18 [95% CI, 2.28 to 11.76], P < .001) and OS (HR, 3.19 [95% CI, 1.45 to 7.03], P = .004). Patients with Uro and GU showed the longest survival. In responders, an enrichment of downregulated stromal- and immune-related genes was seen. Downregulation of interferon-induced transmembrane protein 2 was associated with increased ORR and improved OS. This study identifies different CHT responses by LundTax molecular subtypes in patients with mUC, where the Mes-like subtype was associated with lower response rate and shorter survival.

Expert Consensus on the Diagnosis and Treatment of FGFR Gene-Altered Solid Tumors

AbstractThe fibroblast growth factor receptor (FGFR) is a crucial receptor tyrosine kinase involved in essential biological processes, including growth, development, and tissue repair. However, FGFR gene mutations, including amplification, fusion, and mutation, can disrupt epigenetics, transcriptional regulation, and tumor microenvironment interactions, leading to cancer development. Targeting these kinase mutations with small molecule drugs or antibodies has shown clinical benefits. For example, erdafitinib is approved for treating locally advanced or metastatic urothelial cancer patients with FGFR2/FGFR3 mutations, and pemigatinib is approved for treating cholangiocarcinoma with FGFR2 fusion/rearrangement. Effective screening of FGFR variant patients is crucial for the clinical application of FGFR inhibitors. Various detection methods, such as polymerase chain reaction, next-generation sequencing, fluorescence in situ hybridization, and immunohistochemistry, are available, and their selection should be based on diagnostic and treatment decision-making needs. Our developed expert consensus aims to standardize the diagnosis and treatment process for FGFR gene mutations and facilitate the practical application of FGFR inhibitors in clinical practice.

Phase II Clinical Chemoprevention Trial of Weekly Erlotinib before Bladder Cancer Surgery.

We performed a clinical trial in non-muscle invasive urothelial cancer (NMIUC) patients randomized (2:1) to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib or placebo (either orally once weekly x 3 doses prior to scheduled surgery) to assess for a difference in EGFR phosphorylation in tumor adjacent normal urothelium <24 hours post-study dose and tolerance of weekly erlotinib. Thirty-seven volunteers (6 female/31 male, mean age 70, 35 white/2 non-white) with confirmed or suspected NMIUC were enrolled into either erlotinib (n=24; 900 mg-13, 600 mg-11) or placebo (n=13). Immunohistochemical assessment of phosphorylated and total EGFR in adjacent normal urothelium (20 erlotinib; 9 placebo) or tumor (21 erlotinib and 11 placebo subjects) at study end observed no significant difference between those receiving erlotinib or placebo. This was also true for other assessed tissue biomarkers (phosphorylated ERK, ERK, e-cadherin, p53 and Ki67). Adverse events were more common, in a dose-related fashion, in participants receiving erlotinib, e.g. 38% experienced Grade 1 with rare grade 2 diarrhea and skin toxicity vs 8% in placebo. Clinically insignificant, but statistically significant (p=0.001) elevations in serum total bilirubin and creatinine were observed in erlotinib participants. Serum erlotinib and metabolite concentrations (OSI-420) confirmed compliance in all erlotinib subjects and did not significantly differ between the 600 and 900 mg doses. Despite compelling pre-clinical and clinical data for targeted EGFR inhibition in bladder cancer prevention, these data do not support the use of weekly erlotinib to prevent progression of NMI bladder cancer.

Differences in the expression heterogeneity of ADC-related markers between primary tumors and metastatic lymph nodes in advanced urothelial cancers.

111 Background: The booming development of antibody‒drug conjugates (ADCs), which represent a novel, effective and low-toxicity therapeutic approach, has dramatically improved clinical outcomes in urothelial cancers, especially those with advanced disease. However, previous studies focused less on the targeted genes expression pattern and heterogeneity in metastatic tissues, which are more critical to inhibit tumor progression. Here, we aimed to explore the expression of potential ADC-related genes, including HER2, HER3, Nectin4 and Trop2 in advanced urothelial cancers between primary tumors and metastatic lymph nodes (mLN) in pairs. Methods: A large cohort of 306 urothelial cancer patients (292 patients, pN+), including 65 renal pelvis, 38 ureter, 179 bladder and 24 urethral cancer patients (10 patients, pN+), from SYSUCC was enrolled. Paraffin-embedded primary tumors and corresponding mLN sections were subjected to immunohistochemistry (IHC) to detect ADC-related gene expression. HER2 IHC scores of 2/3+ were defined as high expression as previously described. HER3, Nectin4 and Trop2 were evaluated by H-scores (range 0-300, rank 0 to 3+) multiplied by the extent and intensity of the staining, and H-scores &gt;15 were considered positive. Results: A total of 74.8% of advanced urothelial cancer patients were HER2 positive (1-3+), and 49.4% had high HER2 expression. High HER2 expression was detected in 33.8%, 42.2%, 56.5% and 50% of renal pelvis, ureter, bladder and urethral primary tumors, respectively. The rate of high HER2 expression in mLNs slightly decreased to 30.1%, 41.7%, 51.9% and 50%, and the concordance rate of HER2 expression between primary and mLNs was 71.9%. Similarly, 27.7%, 42.1%, 52%, and 66.7% of primary tumors were positive for HER3 expression; 72.3%, 89.5%, 77.7%, and 87.5% were positive for Nectin4; and 89.2%, 94.7%, 83.2%, and 95.8% were positive for Trop2 in renal pelvis, ureter, bladder and urethral cancers, respectively. Overall, 71.9%, 50.7%, and 65.3% of patients had consistent expression of HER3, Nectin4 and Trop2 in mLNs, respectively. Survival analysis indicated that the overexpression of HER2, HER3 and Trop2 in advanced urothelial cancers was associated with poor OS (p=0.001, 0.025 and 0.022, respectively). More importantly, we found that patients with higher HER2 expression in mLNs at primary sites had a worse prognosis (p=0.006). Conclusions: We first investigated the heterogeneity of ADC-related marker expression in primary urothelial tumors and mLNs in a large single cohort, especially in patients who underwent radical lymphadenectomy. Therapy targeting HER2-ADCs might show better homogeneity in advanced disease, but it is more recommended to detect IHC staining both in primary and metastatic tissues if possible.

Concomitant decrease of E- and A-FABP expression predicts worse survival in urothelial bladder cancer patients

Non-muscle invasive bladder cancers (NMIBC) pTa-pT1 are depicted by a high risk of recurrence and/or progression with an unpredictable clinical evolution. Our aim was to identify, from the original resection specimen, tumors that will progress to better manage patients. We previously showed that A-FABP (Adipocyte- Fatty Acid Binding Protein) loss predicted NMIBC progression. Here we determined by immunohistochemistry the prognostic value of E-FABP (Epidermal-Fatty Acid Binding Protein) expression in 210 tumors (80 pTa, 75 pT1, 55 pT2-T4). Thus, E-FABP low expression was correlated with a high grade/stage, the presence of metastatic lymph nodes, and visceral metastases (p < 0.001). Unlike A-FABP in NMIBC, E-FABP low expression was not associated with RFS or PFS in Kaplan–Meier analysis. But patients of the overall cohort with a high E-FABP expression had a longer mOS (53.8 months vs. 29.3 months, p = 0.029). The immunohistochemical analysis on the same NMIBC tissue sections revealed that when A-FABP is absent, a high E-FABP expression is detected. E-FABP could compensate A-FABP loss. Interestingly, patients, whose original tumor presents both low E-FABP and negative A-FABP, had the worse survival, those maintaining the expression of both markers had better survival. To conclude, the combined evaluation of A- and E-FABP expression allowed to stratify patients with urothelial carcinoma for optimizing treatment and follow-up.

Contribution of Matrix Metalloproteinase-2 and Matrix Metalloproteinase-9 to Upper Tract Urothelial Cancer Risk in Taiwan.

Matrix metalloproteinase (MMP)-2 and -9, which degrade type IV collagen, are linked to cancer invasion and metastasis. Gene polymorphisms in MMP-2 and MMP-9 can influence their function, impacting cancer development and progression. This study analyzed the association between polymorphisms MMP-2 rs243865 (C-1306T), rs2285053 (C-735T), and MMP-9 rs3918242 (C-1562T) with serum concentrations of these enzymes in upper tract urothelial cancer (UTUC) patients. We conducted a case-control study with 218 UTUC patients and 580 healthy individuals in Taiwan. Genotyping was performed using PCR/RFLP on DNA from blood samples, and MMP-2 and MMP-9 serum levels and mRNA expressions in 30 UTUC patients were measured using ELISA and real-time PCR. Statistical analysis showed that MMP-2 rs2285053 and MMP-9 rs3918242 genotypes were differently distributed between UTUC patients and controls (p = 0.0199 and 0.0020). The MMP-2 rs2285053 TT genotype was associated with higher UTUC risk compared to the CC genotype (OR = 2.20, p = 0.0190). Similarly, MMP-9 rs3918242 CT and TT genotypes were linked to increased UTUC risk (OR = 1.51 and 2.92, p = 0.0272 and 0.0054). In UTUC patients, TT carriers of MMP-2 rs2285053 and MMP-9 rs3918242 showed higher mRNA and protein levels (p < 0.01). These findings suggest that MMP-2 rs2285053 and MMP-9 rs3918242 genotypes are significant markers for UTUC risk and metastasis in Taiwan.

The Value and Safety of Adjuvant Radiation Therapy After Radical Cystectomy in Locally Advanced Urothelial Bladder Cancer: A Controlled Randomized Study

BackgroundAdjuvant radiotherapy after radical cystectomy in locally advanced bladder cancer was revived after the advancement in precise radiotherapy that decreased the normal pelvic tissue radiation hazards. However, there are still scarce controlled randomized studies addressing this issue. Patients and methodsOne hundred thirty-one cystectomized urothelial bladder cancer patients were enrolled; a hundred and twenty-two were randomized to receive adjuvant radiotherapy (ART) 50 Gy/25 fractions, 4 weeks’ post-cystectomy or cystectomy alone (CY). Sixty-two were included in the ART arm and sixty in the CY arm. Twenty-four ART and 30 CY patients received Neoadjuvant chemotherapy. Eleven patients (9%) had cotenant neo-bladder diversion, 6 in ART, and 5 in CY arms. All ART patients were treated with intensity-modulated radiotherapy (IMRT) with daily verification cone-beam CT (CBCT). The median follow-up was 42.7 months. ResultsThe 3-year adjusted Locoregional relapse-free survival (LRFS) rate was higher in the ART arm, measuring 81% (95%CI: 69-94) compared to 71% (95% CI: 60-80) (p=0.0457). ART significantly improved the locoregional relapse-free rate in the cystectomy bed and the pelvic side wall (p= 0.016 and 0.001, respectively). The overall survival, event-free, and distant metastasis-free survival did not rank to the level of statistical significance in the 2 arms. Even though the acute side effects were slightly higher in ART, the late toxicities were almost equal in the two groups. ConclusionsAdjuvant radiotherapy is safe and quite tolerable after radical cystectomy when using precise radiation techniques. These techniques significantly improved the LRFS but had insignificant improvement on the overall survival. ART did not affect the distant metastasis-free survival. Similar studies are performed in different centers around the world to confirm the value of ART in urothelial bladder cancer.

Analyzing disparities in socioeconomic factors, clinical characteristics, and outcomes in bladder cancer: Insights from the National Cancer Database (NCDB).

4572 Background: Social determinants of health can influence mortality in bladder cancer. We sought to understand the trends and survival differences for urothelial cancer (UC) and non-urothelial cancer (nUC) based on demographic, socioeconomic, clinical, and treatment from the NCDB. Methods: Using the NCDB database with patient-level data extraction from 2004 to 2020, we sought to identify the socioeconomic differences, clinical characteristics and examine survival trends for both UC and nUC bladder cancer. Results: Majority of UC patients (pts) were M (555107, 75.6%) and 178733 were F; nUC pts had 14296 M and 8835 F, median age was 72 years. 669,669 (or 91.3%) of UC pts were White (W), 5.4% (39444) were Black (B) and nUC pts had 19771 (85.5%) W and 2492 (10.8%) B. 41.2% of UC pts were treated at a Comprehensive Community Cancer Program (CCCP) followed by 28.3% at an academic/research program (ARP) with 20.7% at an integrated network cancer program (INCP) compared to nUC pts at 37.5%, 34.3% and 19.2%, respectively. Most UC pts (65.6%) had Medicare followed by 26.9% with private insurance and 3.2% with Medicaid. Distribution of stage at diagnosis for UC pts were: Stage 0 at 36.9%, Stage I at 16.3%, Stage II &amp; III at 11.8% and Stage IV at 3.6%. While most (51.74%) of F UC pts had stage I, only 12.81% of F nUC had stage I. Majority had Charlson-Deyo Score (CDS) of 0 at 512237 (69.8%). Median Overall survival (mOS) in UC for M was 89.26 months (mos) (CI 88.8-89.76) and for F it was 94.88 mos (CI 93.83-95.93), Log-rank p=0.0011; worse for B = 73.03 mos (CI 71.03 – 74.84) vs W = 90.38 mos (CI 89.95-90.81), Log-rank p&lt;0.0001; mOS was highest for ARP at 96.46 mos (CI 95.54-97.45); CDS of 0 at 108.29 mos (CI 107.66-108.85); private insurance at 185.79 mos (CI 183.98-188.16); mOS for nUC was better for M at 15.64 (CI, 15.05 -16.28) vs F at 12.68 (CI, 12.02, 13.31), Log-rank p&lt;0.001. mOS for metastatic involvement for bone: UC: 5.03 (CI, 4.8, 5.39) vs nUC: 5.42 (4.4, 6.34), Log-rank p=0.2007; brain: U: 3.61 (CI 2.99, 4.3) vs nUC: 5.06 (2.5, 7.13); Log-rank p=0.5765; lung: U: 5.09 (CI, 4.76, 5.39) vs nUC: 4.8 (3.68, 5.78); Log-rank p=0.2163; liver: U: 3.32 (CI,3.06, 3.61) vs nUC: 5.42 (CI, 4.11, 6.44); Log-rank p=0.0009; LN: U: 7.1 (CI, 6.6, 7.56) vs nUC: 6.34 (5.36, 6.9); Logrank p=0.0007; Treatment using surgery, radiation, chemotherapy and immunotherapy all showed improvement in U vs nUC (all Log-rank p&lt;0.0001). Conclusions: Women had better survival than men in UC but not in nUC. Factors including lower stages, Whites, private insurance, better CDS scores, treatment at academic/research facility, and lymph node metastases all showed improved survival. These data highlight the importance of bridging the gap between socioeconomic differences to improve outcomes for UC and nUC pts.

Abstract A024: Genomic peroxisome proliferator-activated receptor gamma (PPARG) study: A sponsored testing program to identify patients harboring genetic alterations in advanced UC in support of the FX-909 Phase 1 study

Abstract The transcription factor PPARG is associated with the luminal lineage subtype representing ~65% of advanced urothelial cancer (UC) patients. Recurrent genetic alterations in PPARG, fibroblast growth factor receptor 3 (FGFR3), and the heterodimeric binding partner for PPARG, retinoid X receptor alpha (RXRA), are characteristic of this luminal subtype. FX-909, a covalent PPARG inverse agonist, represents a promising first-in-class therapy for patients with advanced UC. FX-909-CLINPRO-1 [NCT05929235] is an open-label Phase 1 study to evaluate the safety, tolerability, pharmacokinetics, biologic activity, and preliminary clinical activity of FX-909. This study includes a Phase 1b expansion cohort, which will enroll advanced UC patients with genetic alterations in PPARG, RXRA, and/or FGFR3. FlareTx is sponsoring a testing program called Genomic PPARG Study (GPS) to enable investigators to identify patients with these relevant genetic alterations. Tumors collected from patients with advanced UC were sequenced using the Tempus xT assay (DNA-seq of 648 genes at 500x coverage; RNA-seq). A clinical report is generated and returned to the physician to guide selection of approved therapies, and if appropriate to support eligibility for inclusion in the study. FlareTx is provided with de-identified genomic data, where assessment of the prevalence of genetic alternations such as copy number variation (CNV), fusions and mutations (single nucleotide variants) in PPARG, RXRA and FGFR3 are undertaken. Additionally, luminal molecular classification is performed using the Robertson method [1]. PPARG mean expression value are normalized by transcripts-per-million (TPM) and reported as Log2[TPM+1]. In the initial data set of 27 patients, 3 patients had PPARG amplification (CN &amp;gt;/= 3), 3 patients had RXRA hotspot mutation (S427F) [2], 6 patients had FGFR3 mutation(s), and 1 patient had both PPARG amplification and a FGFR3 mutation. Twenty of 27 patients tested had sufficient starting material of good quality to also perform whole transcriptome analysis. The mean mRNA expression level of PPARG was 6.77 Log2[TPM+1] (range 2.91 Log2[TPM+1] – 9.22 Log2[TPM+1]). Consistent with the Robertson subtype distribution, 65% of patients were luminal and 35% were non-luminal (basal-squamous and neuronal); 10 of 13 luminal patients had one or more genetic alteration in PPARG, RXRA and/or FGFR3. High PPARG expression was associated with luminal subtype compared to non-luminal subtype (7.90 Log2[TPM+1] vs 4.67 Log2[TPM+1]; p=0.00042). GPS is open at two active Phase 1 sites, with several more planned as the study advances towards the Phase 1B dose expansion stage. GPS runs concurrently with the FX-909 study and is a novel precision strategy to maximize identifying advanced UC patients with actionable genetic alterations to potentially enroll in the clinical study.

Abstract A007: Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer

Abstract Human epidermal growth factor receptor 2 (HER2) is encoded by the ERBB2 gene, and frequently mutated, amplified, and/or overexpressed in urothelial cancer (UC). Promising antibody-drug conjugates have led to new interest in HER2 as a UC clinical target. Patient selection for HER2-targeted therapy typically relies on immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) of tumor tissue, however DNA sequencing can identify ERBB2 genomic alterations. Accurate biomarker-driven patient selection will be critical to optimize the clinical benefit of HER2-targeted therapy. Therefore, we evaluated plasma circulating tumor DNA (ctDNA) for ERBB2/HER2 status determination in UC, as compared to IHC and FISH of metachronous tissue. 411 plasma samples from 236 metastatic UC patients were profiled with targeted sequencing across &amp;gt;50 genes frequently altered in UC, including dense coverage flanking the ERBB2 locus. Using an established bioinformatics workflow, we determined the presence of ERBB2 alterations (mutations, amplifications, and/or structural variants) in the 181 patients with evidence of ctDNA in ≥1 sample. Clinical records were reviewed for the availability of archival tumor tissue; in total, 81 formalin-fixed paraffin-embedded tissue specimens were retrieved from 23 patients with ctDNA ERBB2 alterations and 20 ERBB2-wildtype controls. HER2 IHC was performed with a polyclonal rabbit anti-human Her2 antibody (Dako) and scored according to gastric cancer guidelines. HER2 FISH was performed with the PathVysion HER-2 DNA Probe Kit. Protein-altering ERBB2 mutations were identified in 14% of evaluable patients, with two-thirds at known oncogenic hotspots. ERBB2 copy gain was detected in 8% of patients overall, 9% when excluding low tumor fraction samples. IHC results were assessable for 82 tissue samples from 43 patients, and 33 patients had at least one sample with positive HER2 staining (2+/3+). IHC scores varied in 16/23 patients with ≥2 tissue samples, with the variation leading to a change in classification (HER2-negative [0/1+] versus positive [2+/3+]) in half (8/16). In two patients with mixed variant histology, the urothelial component showed IHC 3+ while the scores were 2+ (plasmacytoid) and 0 (squamous) in the variant regions. Frequent focal staining patterns were observed with both IHC and FISH. ERBB2 alterations in ctDNA were correlated with IHC positivity – when considering the most recent tissue sample per patient, 79% of ctDNA ERBB2-altered patient’s tumors were also positive by IHC. Conversely, 55% of ctDNA ERBB2-wildtype cases were positive by IHC. FISH results were available from 24 samples (20 positive, 4 negative), all from patients with ctDNA ERBB2 amplification. 18/20 samples with FISH positivity were HER2 IHC 2+/3+; the remaining two samples were IHC 1+. Our results demonstrate significant spatial and/or temporal heterogeneity in ERBB2/HER2 status, with implications for the rational implementation of biomarker-directed (HER2-targeted) therapy in UC. Citation Format: Gillian Vandekerkhove, Andrew J. Murtha, David C. Müller, Kimia Rostin, Carlos Vasquez-Rios, Jussi Nikkola, Maria Stephenson, Emily Fung, Jaskirat Atwal, Karan Parekh, Cecily Q. Bernales, Gráinne Donnellan, Gang Wang, Tilman Todenhöfer, Piet Ost, Peter C. Black, Kim N. Chi, Bernhard J. Eigl, Alexander W. Wyatt. Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A007.

Management of urothelial cancer in patients with chronic kidney disease receiving platinum-based chemotherapy.

Despite recent advances in the management of urothelial cancer (UC), cisplatin-based combination chemotherapy regimens remain critical. However, their use can be complicated in patients with chronic kidney disease (CKD), which is not uncommon in UC patients. Based on the Galsky criteria for cisplatin ineligibility, most patients with CKD will be excluded from receiving cisplatin-based chemotherapy altogether. For patients with borderline kidney function, several strategies-such as the use of split-dose cisplatin, dose reductions,or extra hydration-may facilitate the use of cisplatin, but theseneedto be prospectively validated. This review highlights the critical need for a multidisciplinary team, including onco-nephrologists, to help manage renal complications and optimize delivery of cancer care in complex UC patients with CKD.

Comparing survival in trial- versus routine-care advanced urothelial cancer patients on immune checkpoint blockade.

Although recent trials involving first-line immune checkpoint inhibitors have expanded treatment options for patients with advanced urothelial carcinoma (aUC) who are ineligible for standard cisplatin-based chemotherapy, there exists limited evidence for whether trial efficacy translates into real-world effectiveness for patients seen in routine care. This retrospective cohort study compares differences in overall survival (OS) between KEYNOTE-052 trial participants and routine-care patients receiving first-line pembrolizumab monotherapy. A routine-care patient cohort was constructed from the Flatiron Health database using trial eligibility criteria and was weighted to balance EHR and trial patient characteristics using matching-adjusted indirect comparisons. The routine-care cohort was older, more likely to be female, and more often cisplatin-ineligible due to renal dysfunction. ECOG performance status was comparable between the cohorts. Median OS was 9 months (95% CI 7-16) in the weighted routine-care cohort and 11.3 months (9.7-13.1) in the trial cohort. No significant differences between the Kaplan-Meier OS curves were detected (p = 0.76). Survival probabilities were similar between the weighted routine-care and trial cohorts at 12-, 24-, and 36- months (0.45 vs. 0.47, 0.31 vs. 0.31, 0.26 vs. 0.23, respectively). Notably, routine care patients had modestly lower survival at 3 months compared to trial participants (0.69 vs. 0.83, respectively). Our results provide reassurance that cisplatin-ineligible aUC patients receiving first-line immunotherapy in routine care experience similar benefits to those observed in trial patients.

Development and Validation of an Inflammatory Prognostic Index to Predict Outcomes in Advanced/Metastatic Urothelial Cancer Patients Receiving Immune Checkpoint Inhibitors.

Immune checkpoint inhibitors (ICIs) improve overall survival (OS) in advanced/metastatic urothelial cancer (a/mUC) patients. Preliminary evidence suggests a prognostic role of inflammatory biomarkers in this setting. We aimed to develop a disease-specific prognostic inflammatory index for a/mUC patients on ICIs. Fifteen variables were retrospectively correlated with OS and progression-free survival (PFS) in a development (D, n = 264) and a validation (V, n = 132) cohort of platinum-pretreated a/mUC pts receiving ICIs at L2 or further line. A nomogram and inflammatory prognostic index (U-IPI) were developed. The index was also tested in a control cohort of patients treated with chemotherapy only (C, n = 114). The strongest predictors of OS were baseline platelet/lymphocyte (PLR) and neutrophil/lymphocyte (NLR) ratios, and lactate dehydrogenase (LDH), NLR, and albumin changes at 4 weeks. These were used to build the U-IPI, which can distinctly classify patients into good or poor response groups. The nomogram scoring is significant for PFS and OS (p < 0.001 in the D, V, and combined cohorts) for the immunotherapy (IO) cohort, but not for the control cohort. The lack of a baseline systemic inflammatory profile and the absence of early serum inflammatory biomarker changes are associated with significantly better outcomes on ICIs in a/mUC pts. The U-IPI is an easily applicable dynamic prognostic tool for PFS and OS, allowing for the early identification of a sub-group with dismal outcomes that would not benefit from ICIs, while distinguishing another that draws an important benefit.

Abstract LB105: Sensitive urothelial cancer detection via high volume urine DNA analysis

Abstract Background: The detection of cancer-associated DNA in urine samples has significant potential in urothelial cancer (UC) detection and surveillance. Every year millions of patients with urinary symptoms undergo cystoscopy, an examination of the bladder using a flexible scope. Only 10% of these invasive examinations result in a cancer diagnosis. Cystoscopy is also used in post-operative surveillance, with many UC patients requiring life-long cystoscopies. Methods: We developed a high-volume urine DNA collection kit and laboratory platform and prospectively analyzed 433 urine samples collected from diagnostic and post-operative timepoints from 167 UC patients and 95 negative controls from January 2021 to September 2023. We used a hybridization capture assay targeting the coding regions of 21 commonly mutated genes in urothelial tumors, as well as 8 frequently copy number altered loci. Samples were sequenced using Illumina NovaSeq instruments. Mutations, copy number alterations, and chromosomal rearrangements were computationally identified. Key Findings: In the diagnostic settings our assay achieved a sensitivity of 100% for pT1+ bladder cancer, 97% for pTa bladder cancer, and 91% for upper tract urothelial cancer (UTUC) (100% for pT1+ or high grade) in an all-comers UC cohort, at a specificity of 93%. Post-operative urine DNA surveillance informed by preoperative urine mutations detected 28/30 (93%) residual cancer cases found in cystoscopy or surgery, with two pTa low grade recurrences missed. Using a secondary, highly stringent urine tumor DNA detection threshold (100% specificity for UC), we show that urine DNA testing in the diagnostic setting may allow up to 68% of UC patients to be scheduled directly to operating room procedures, reducing hospital workload and expediting care. In addition, we demonstrate the feasibility of urine DNA for detecting underlying Lynch syndrome in UC patients by identifying three UTUC patients who carried germline defects in mismatch repair genes MSH2 or MSH6, indicative of Lynch syndrome. In two of these patients our assay also found a somatic second hit and high mutation load. Conclusions and Clinical Implications: These results show promise in urine DNA based cancer diagnostics even in patients with low grade tumors. The assay is designed to offer a non-invasive and cost-effective alternative for cystoscopies. Citation Format: Jussi Nikkola, Lauri Ryyppö, Juuso Vuorinen, Heini Kallio, Hanna Selin, Pyry Jämsä, Jonne Åkerla, Tarmo Pekkarinen, Antti Kaipia, Matti Nykter, Thea Veitonmäki, Matti Annala. Sensitive urothelial cancer detection via high volume urine DNA analysis [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 LB105.

Abstract LB113: Immunogenicity of Atezolizumab plus personalized neoantigen vaccination (PGV-001) in patients with urothelial cancer in adjuvant vs metastatic setting

Abstract Background: Urothelial cancer (UC) of the genitourinary tract, most commonly arising from the bladder, has historically been a challenging area for the development of new therapeutics. Immune checkpoint blockade inhibitors (CBIs) induce durable responses only in a subset of patients with UC, potentially due to lack of neoantigen primed T cells. To build on this hypothesis, we designed a study to evaluate safety and immunogenicity of a combination treatment with atezolizumab and PGV-001, a personalized genomic neoantigen vaccine. Having previously reported clinical outcomes, here we describe analysis of immunogenicity induced by the treatment. Methods: In this phase I trial (NCT03359239) UC patients were enrolled to receive PGV-001 with atezolizumab in the adjuvant (undergone radical cystectomy or nephroureterectomy with a high risk of recurrence) or metastatic setting. Each PGV001 vaccine included up to 10 neoantigen (predicted using OpenVax pipeline) synthetic long peptides (SLPs). Each subject received 10 PGV-001 vaccines and 1200 mg i.v. atezolizumab every 3 weeks for up to 12 months (adjuvant) or 24 months (metastatic). To investigate T cell immunity, ex vivo IFNγ ELISPOT and T cell expansion assays were performed using 15mer overlapping peptide (OLP) pools corresponding to each neoantigen SLP. Results: Samples from 9 subjects (3 adjuvant and 6 metastatic) were evaluable for ex vivo ELISPOT analysis. In 100% of the subjects, at least 1 neoantigen was found to elicit an immune response. Overall, out of 87 evaluated neoantigens, 47% were immunogenic, with the immunogenic peptides inducing an average of 16.7 fold increase in ex vivo ELISPOT response over baseline. While 39% (11/28) of the peptides in adjuvant cohort were immunogenic vs 51% (39/50) in metastatic cohort, it is not possible to determine statistically significant difference in response between metastatic and adjuvant cohort. The T cell expansion assay was performed for 6 subjects, 3 each from the adjuvant and metastatic cohorts. Among 57 evaluated neoantigens, 46% and 54% of the peptides elicited a CD8+ and CD4+ T cell targeted immune response, respectively. A greater proportion of neoantigens induced both CD8+ and CD4+ T cell responses in subjects treated in the metastatic setting vs adjuvant setting. Upon epitope mapping, for 5 evaluable subjects, 141 OLPs were analyzed and a similar number of OLPs were found to be immunogenic for CD8+ (31.2%, 44/141) and CD4+ response (30%, 43/141). Conclusions: These data indicate that vaccination with personalized neoantigens in combination with systemic anti-PD-L1 can induce neoantigen-focused T cell responses in both adjuvant and metastatic disease settings. However, the single arm nature of the trial, and sample size, preclude definitive assessment of the contribution of the components of the combination therapy. Future studies will investigate the efficacy of this approach on a larger scale with pre- and on-treatment analyses of treatment-driven immunomodulation. Citation Format: Mansi Saxena, Jonathan Anker, Julia Kodysh, Timothy O'Donnell, Marcia Meseck, Olivia Hapanowicz, Scot Niglio, Hardik Shah, Yayoi Kinoshita, Rachel Brody, Alex Rubinsteyn, Robert Sebra, Nina Bhardwaj, Mathew Galsky. Immunogenicity of Atezolizumab plus personalized neoantigen vaccination (PGV-001) in patients with urothelial cancer in adjuvant vs metastatic setting [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 LB113.

Cisplatin-Resistant Urothelial Bladder Cancer Cells Undergo Metabolic Reprogramming beyond the Warburg Effect.

Advanced urothelial bladder cancer (UBC) patients are tagged by a dismal prognosis and high mortality rates, mostly due to their poor response to standard-of-care platinum-based therapy. Mediators of chemoresistance are not fully elucidated. This work aimed to study the metabolic profile of advanced UBC, in the context of cisplatin resistance. Three isogenic pairs of parental cell lines (T24, HT1376 and KU1919) and the matching cisplatin-resistant (R) sublines were used. A set of functional assays was used to perform a metabolic screening on the cells. In comparison to the parental sublines, a tendency was observed towards an exacerbated glycolytic metabolism in the cisplatin-resistant T24 and HT1376 cells; this glycolytic phenotype was particularly evident for the HT1376/HT1376R pair, for which the cisplatin resistance ratio was higher. HT1376R cells showed decreased basal respiration and oxygen consumption associated with ATP production; in accordance, the extracellular acidification rate was also higher in the resistant subline. Glycolytic rate assay confirmed that these cells presented higher basal glycolysis, with an increase in proton efflux. While the results of real-time metabolomics seem to substantiate the manifestation of the Warburg phenotype in HT1376R cells, a shift towards distinct metabolic pathways involving lactate uptake, lipid biosynthesis and glutamate metabolism occurred with time. On the other hand, KU1919R cells seem to engage in a metabolic rewiring, recovering their preference for oxidative phosphorylation. In conclusion, cisplatin-resistant UBC cells seem to display deep metabolic alterations surpassing the Warburg effect, which likely depend on the molecular signature of each cell line.

Assessment of Therapeutic Effects of Combined Treatment With Cisplatin and Anti-mouse Programmed Death (PD)-1 Antibody in a Mouse Urothelial Cancer Model.

The therapeutic impact of combination treatment with an immune checkpoint inhibitor (ICI) and chemotherapeutic agent on patients with urothelial cancer (UC) remains controversial. Therefore, the present study investigated differences in the therapeutic effects of combination therapy with cisplatin plus anti-mouse programmed death (PD)-1 antibody according to the dose of cisplatin using the mouse bladder tumor model MBT2. The effects of treatment with two different doses cisplatin and/or anti-mouse PD-1 antibody on tumor growth after the subcutaneous injection of MBT2 cells were compared. Infiltrating patterns of lymphocytes into tumors after treatment were assessed using immunohistochemical staining. MBT2 tumor volumes were significantly larger in mice receiving high-dose cisplatin alone than in those receiving low-dose cisplatin alone. Combination treatment with cisplatin plus anti-mouse PD-1 antibody exerted significantly stronger growth inhibitory effects on MBT2 tumors than treatment with either agent alone, irrespective of cisplatin doses; however, no significant differences were observed in MBT2 tumor volumes between mice receiving anti-mouse PD-1 antibody plus high-dose cisplatin and those receiving anti-mouse PD-1 antibody plus low-dose cisplatin. Furthermore, CD8+ to CD3+ and CD8+ to CD11b+ T-lymphocyte ratios in MBT2 tumors were both significantly higher in the low-dose cisplatin alone group than in the high-dose cisplatin alone group, whereas no significant differences were noted in either ratio between the two different combination treatment regimens. When combined with ICI, a lower dose of cisplatin may achieve favorable antitumor effects in UC patients by preventing lymphocyte exhaustion.

Abstract 5627: PPARG-high circulating monocytes exhibit an immunosuppressive phenotype in urothelial cancer patients treated with anti-PD1

Abstract Background: PPARG is expressed in a variety of immune cells such as monocytes, macrophages, and lymphocytes, where it plays a role in their maturation and function. PPARG has been implicated in immune mediated resistance but to date, a comprehensive analysis of PPARG expression in peripheral blood mononuclear cells (PBMCs) in urothelial cancer (UC) patients is lacking. In this study, we identified the immunophenotypes associated with PPARG expression in UC patients treated with anti-PD1 therapy. Methods: We performed single-cell RNA sequencing of PBMCs from 5 UC patients and 3 normal healthy volunteers (NHV). Real-world samples (Discovery Life Sciences) were collected from patients who received prior Pembrolizumab. Time post-treatment to collection ranged from 46 to 307 days. An average of 9,500 cells were recovered per donor reflecting an ~80% QC pass rate. Count matrices were generated using 10x Genomics Cell Ranger pipeline (v7.0.0; GRCh38 reference). Downstream data processing, analysis and visualization were done on Cellenics software. Results: We evaluated 75,725 cells and identified 9 distinct cell populations. The proportion of T-cells was significantly decreased in UC samples compared to NHVs (40% vs 60%, p=0.03). Highest PPARG expression was observed on Classical Monocytes (CMs) cell population. PPARG expression was higher on CMs in UC compared to NHV samples (1.74 vs 0.11 logcounts; padj&amp;lt;0.001). Unsupervised clustering of all monocytes revealed two distinct CM clusters. In cluster A, 95% of the cells originated from UC samples, while most cells in cluster B (81%) originated from NHV samples. PPARG was the top differentially expressed gene upregulated in cluster A (PPARG-high) vs cluster B (PPARG-low) (1.89 vs 0.23 logcounts; padj&amp;lt;0.001). PPARG-high CMs are characterized by a gene signature inclusive of GPRIN3, RGCC, MAFB and IRF2BP, which have been shown to be upregulated in M2 polarized macrophages [1]. Moreover, pathway analysis using GSEA highlighted that PPARG-high CMs are enriched for TREM1 signaling genes (padj&amp;lt;0.001), whose high expression has been associated with immunosuppressive phenotypes in myeloid cells [2] and shorter survival of patients across other cancers [3]. Conclusion: PPARG-high circulating CMs in UC patients exhibit a transcriptomic profile associated with immunosuppression and M2 macrophage polarization. FX-909, a first-in-class PPARG inverse agonist, is being evaluated in the clinic and the study includes an exploratory biomarker approach to assess the potential effect of PPARG inhibition on the CM immune-profile of advanced UC patients enrolled in the Ph1 study [4].

Abstract 5219: Loss of chromosome Y is unrelated to the composition of the tumor microenvironment and patient prognosis in muscle-invasive urothelial bladder cancers

Abstract Background: Loss of chromosome Y (LOY) occurs commonly in cancers of male patients. It was recently proposed to be associated with cancer aggressiveness and altered T-cell function in bladder cancer and it was found to be linked to poor prognosis in a TCGA bladder cancer patient cohort of 300 patients based on a combined analysis of RNAs from Y-chromosome genes. In this study, we aimed to clarify the clinical relevance of LOY in a large cohort of urothelial bladder cancer patients by using fluorescence in-situ hybridization (FISH). Design: X and Y chromosomes were analyzed by FISH on a tissue microarray (TMA) containing 1,819 urothelial carcinomas of the urinary bladder from male patients including 487 patients who had undergone cystectomy for muscle-invasive disease and for which follow-up data were available. Data of tumor microenvironment parameters were obtained in a previous study. Results: LOY was found in 26.0% of 1,479 analyzable cancers. In the subset of non-invasive cancers, the frequency of LOY increased from pTaG2 (14.5%) to pTG3 (28.6%) but these differences were not significant (p=0.1015). In muscle-invasive cancers, the frequency of LOY increased slightly from pT2 (25.4%) to pT4 cancers (32.8%) but this association was again not significant (p=0.1868). Within pT2-4 cancers, LOY was associated with venous invasion (p=0.0010) but unrelated to pT, pN, and L-status, as well as to overall, recurrence-free, and cancer-specific survival. Muscle-invasive urothelial carcinomas with and without LOY did not show significant differences in the number of CD8 positive lymphocytes, the fraction of CD8 positive intraepithelial lymphocytes, the number of macrophages and dendritic cells, as well as the fraction of t helper and t regulatory cells. Conclusion: FISH is the gold standard for determining LOY. That LOY was neither associated with histopathological parameters of cancer aggressiveness and patient prognosis nor with any parameters describing the tumor microenvironment strongly argues against a driving role of LOY for bladder cancer progression and cancer associated immune reaction. The slight increase of LOY from pTaG2 to pT4 cancers is consistent with a continuous (but clinically irrelevant) loss of the dispensable Y chromosomes during tumor evolution over time. Citation Format: Martina Kluth, Henning Plage, Sebastian Hofbauer, Kira Furlano, Sarah Weinberg, Antonia Franz, Annika Fendler, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Niclas C. Blessin, Maximilian Lennartz, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Stefan Koch, Nico Adamini, Ronald Simon, Guido Sauter, Joachim Weischenfeldt, Tobias Klatte, Sarah Minner, David Horst, Thorsten Schlomm, Dr. med Henrik Zecha Zecha. Loss of chromosome Y is unrelated to the composition of the tumor microenvironment and patient prognosis in muscle-invasive urothelial bladder cancers [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 5219.

Abstract 6006: Modeling antibody drug conjugate sensitivity using urothelial carcinoma patient-derived models

Abstract Introduction: Enfortumab Vedotin (EV), an antibody-drug conjugate (ADC) targeting Nectin-4, has emerged as a new standard-of-care for patients with metastatic urothelial cancer (UC). Promising results have also been observed in bladder cancer (BLCA) patients with ADCs targeting HER2. To identify predictive biomarkers of ADC sensitivity, we characterized a large and expanding biobank of patient-derived organoid (PDO) and xenograft (PDX) models for Nectin-4 and HER2 expression, and Nectin-4 and HER2-targeted ADC sensitivity. Methods: We generated 19 PDOs and 34 PDXs that reflect the genomic and biologic heterogeneity of human UC. A subset of these models was further characterized using a multiplatform approach, including bulk and single-cell DNA and RNA sequencing and immunoblot for Nectin-4 and HER2 expression. We leveraged these models to study the biologic determinants of sensitivity to EV and the HER2-targeted ADC trastuzumab deruxtecan (T-DXd). PDX models were treated with EV (5 mg/kg IV on day 1, 3, 6) or T-DXd (10 mg/kg IV on day 1, 21, 42) or vehicle only as control. We also studied sensitivity to T-DXd and EV, as well as to their cytotoxic payloads (exatecan and vedotin) in vitro using paired PDO models. Results: We observed high degree of variability of HER2 and Nectin-4 expression across UC PDOs and PDXs. In PDO models, sensitivity to EV correlated strongly with Nectin-4 expression, whereas HER2 expression was not strongly correlated with sensitivity to T-DXd. We also noted a poor correlation between Nectin-4 expression in PDOs and PDXs derived from the same patient. A subset of models, for example, SMBO-104 (ERBB2 wildtype with low HER2 expression), exhibited significantly greater sensitivity to EV than to T-DXd. Conversely, T-DXd was significantly more active than EV in SMBO-170, a model with ERBB2 amplification (~40 copies) and HER2 3+ expression. Sensitivity to exatecan, an analogue of the T-DXd payload deruxtecan, as measured by inhibition of cell proliferation and induction of apoptosis, was more predictive of T-DXd sensitivity than HER2 expression level. Conclusion: In this study, we generated and biologically characterized UC patient-derived models with the goal of identifying predictive biomarkers of sensitivity to Nectin-4 and HER2-targeted ADCs. We observed significant heterogeneity of HER2 and Nectin-4 expression across models. Models were identified in which T-DXd was superior to EV, and vice versa. Sensitivity to the T-DXd cytotoxic payload was more predictive of PDX response than HER2 expression levels, with significant tumor regression observed with T-DXd in BLCA models with low HER2 expression. Our data suggest that clinical trials of HER2-targeted ADCs such as T-DXd should not be restricted to patients with HER2 overexpression and that novel biomarkers will be needed to identify those UC patients most likely to benefit from FDA-approved and investigational ADCs. Citation Format: Xinran Tang, Ziyu Chen, Jasmine Thomas, Karan Nagar, John Christin, Naryan Rustgi, Sizhi Gao, Elisa de Stanchina, Jonathan Coleman, Michael Shen, Hikmat Al-Ahmadie, Gopa Iyer, Kwanghee Kim, David Solit. Modeling antibody drug conjugate sensitivity using urothelial carcinoma patient-derived models [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 6006.