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Cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy is recommended for patients with muscle-invasive bladder cancer (MIBC). It has been shown that somatic deleterious mutations in ERCC2, gain-of-function mutations in ERBB2, and alterations in ATM, RB1, and FANCC are correlated with pathological response to NAC in MIBC. The objective of this study was to validate these genomic biomarkers in pretreatment transurethral resection material from an independent retrospective cohort of 165 patients with MIBC who subsequently underwent NAC and radical surgery. Patients with ypT0/Tis/Ta/T1N0 disease after surgery were defined as responders. Somatic deleterious mutations in ERCC2 were found in nine of 68 (13%) evaluable responders and two of 95 (2%) evaluable nonresponders (p = 0.009; FDR = 0.03). No correlation was observed between response and alterations in ERBB2 or in ATM, RB1, or FANCC alone or in combination. In an exploratory analysis, no additional genomic alterations discriminated between responders and nonresponders to NAC. No further associations were identified between the aforementioned biomarkers and pathological complete response (ypT0N0) after surgery. In conclusion, we observed a positive association between deleterious mutations in ERCC2 and pathological response to NAC, but not overall survival or recurrence-free survival. Other previously reported genomic biomarkers were not validated. Patient summary: It is currently unknown which patients will respond to chemotherapy before definitive surgery for bladder cancer. Previous studies described several gene mutations in bladder cancer that correlated with chemotherapy response. This study confirmed that patients with bladder cancer with a mutation in the ERCC2 gene often respond to chemotherapy.

Background: Immune checkpoint inhibitors (ICI) can achieve remarkable clinical responses in urothelial cancer (UC). However, it remains unclear which aspects of the tumor microenvironment (TME) determine a patient’s response. The TME is usually characterized by immune cell density, which ignores cells’ spatial relationships relative to each other. Methods: Using multiplex immunofluorescence data (PanCK, CD20, CD68, CD3, CD8, and FoxP3 antibody panel) of 24 pre-ICI UC transurethral resections retrieved from the NABUCCO trial (NCT03387761), we spatially profiled cancer cells, macrophages, B-cells, and distinct T-cell populations. We first quantified the TME immune cell densities at the tumor and stroma tissue compartments. We then quantified the TME spatial relationships with a novel approach based on fitting a Weibull function to the first nearest neighbor (1-NN) distance distribution, allowing us to uniquely summarize spatial relationships with two parameters (Weibull approach). We compared this approach to conventional techniques (G-functions) that rely upon a predefined distance threshold. We performed a simulation study to identify sources of variation in the spatial relationship parameters. Lastly, we associated the TME parameters with ICI (ipilimumab + nivolumab) response. Results: Unlike the Weibull approach, the G-function quantifications manifested a variable effect size and statistical power in association studies because of its distance threshold dependence. We found that variation in density affected the spatial relationship metrics of rare cell types (i.e., B-cells) but not of abundant cell types (i.e., cancer cells). The spatial relationship metrics from the Weibull approach outperformed immune cell density in ICI response prediction. Specifically, immune cell density did not discriminate between ICI response groups (FDR>10%). In contrast, the spatial relationship between either CD8+ T-cells or macrophages to their closest cancer cell did associate with response to ICI (FDR=1%). Furthermore, non-responding tumors were characterized by CD8+ T-cells close to B-cells (FDR=9%). Importantly, we validated the association between proximity and response from CD8+ T-cells to cancer cells (FDR=1%) and from macrophages to cancer cells (FDR=1%) using data from 25 pre-ICI head and neck squamous cell carcinoma tumors from the IMCISION trial (NCT03003637, arm B, ipilimumab + nivolumab). Conclusion: We created a framework to quantify, study, interpret and analyze spatial relationships in the TME and illustrated its superior clinical relevance compared to density metrics for predicting ICI treatment response. Our findings emphasize the importance of spatial relationships in the TME for response and suggest that proximity between either macrophages or CD8+ T-cells to cancer cells are candidate biomarkers for ICI response. Citation Format: Alberto Gil-Jimenez, Nick van Dijk, Yoni Lubeck, Maurits L. van Montfoort, Dennis Peters, Erik Hooijberg, Annegien Broeks, Joris L. Vos, Charlotte L. Zuur, Bas van Rhijn, Daniel J. Vis, Michiel S. van der Heijden, Lodewyk F. Wessels. Spatial relationships in the tumor microenvironment predict response to immune checkpoint inhibitors in urothelial and head and neck cancer. [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 5786.

Immune checkpoint inhibitors (ICIs) improve overall survival in patients with metastatic urothelial cancer (mUC). To identify predictive markers of response, whole-genome DNA (n=70) and RNA-sequencing (n=41) were performed using fresh metastatic biopsies prior to treatment with pembrolizumab. PD-L1 combined positivity score failed, while tumor mutational burden and APOBEC mutagenesis modestly predicted response. Using gene expression analysis, we defined the T cell-to-stroma enrichment (TSE) score, a signature-based metric that captures the relative abundance of T cells and stromal cells. Patients with a positive and negative TSE score show progression free survival rate at 6 months of 67% and 0%, respectively. The significant predictive value of the TSE score was validated in two independent ICI treated cohorts of patients with mUC (IMvigor210) and muscle-invasive UC (ABACUS). The TSE score represents a clinically applicable marker that may select patients with metastatic and primary UC who do not benefit from ICI treatment.

Preoperative immunotherapy with anti-PD1 plus anti-CTLA4 antibodies has shown remarkable pathological responses in melanoma1 and colorectal cancer2. In NABUCCO (ClinicalTrials.gov: NCT03387761 ), a single-arm feasibility trial, 24 patients with stage III urothelial cancer (UC) received two doses of ipilimumab and two doses of nivolumab, followed by resection. The primary endpoint was feasibility to resect within 12 weeks from treatment start. All patients were evaluable for the study endpoints and underwent resection, 23 (96%) within 12 weeks. Grade 3-4 immune-related adverse events occurred in 55% of patients and in 41% of patients when excluding clinically insignificant laboratory abnormalities. Eleven patients (46%) had a pathological complete response (pCR), meeting the secondary efficacy endpoint. Fourteen patients (58%) had no remaining invasive disease (pCR or pTisN0/pTaN0). In contrast to studies with anti-PD1/PD-L1 monotherapy, complete response to ipilimumab plus nivolumab was independent of baseline CD8+ presence or T-effector signatures. Induction of tertiary lymphoid structures upon treatment was observed in responding patients. Our data indicate that combined CTLA-4 plus PD-1 blockade might provide an effective preoperative treatment strategy in locoregionally advanced UC, irrespective of pre-existing CD8+ T cell activity.

Patients (pts) with stage III (cT3-4aN0M0 or cT1-4aN1-3M0) urothelial cancer (UC) have a poor prognosis. In NABUCCO cohort 1, 24 stage III UC pts were treated with ipilimumab (ipi) plus nivolumab (nivo) followed by radical surgery (day 1: ipi 3 mg/kg; day 22: ipi 3 mg/kg + nivo 1 mg/kg; day 43: nivo 3 mg/kg). 14/24 (58%) of pts showed a pathological response (ypT0N0 or ypTisN0/ypTaN0). Currently, there are no good biomarkers to assess response before surgery, potentially leading to overtreatment and unnecessary surgical complications. Here, we investigated whether detection of circulating tumor DNA (ctDNA) in plasma and urine by the RaDaR™ personalized liquid biopsy assay was associated with treatment response and outcomes. EDTA-plasma and urine supernatant were collected before start of treatment (day 1; “baseline”), before each subsequent treatment cycle (day 22 and 43) and before radical surgery. WES was performed on tumor FFPE and peripheral blood germline DNA to identify somatic variants for designing patient-specific, multiplex PCR-based NGS RaDaR™ panels. Plasma and urine ctDNA was analyzed using these panels to determine ctDNA detection and its estimated variant allele frequency (eVAF). Tissue somatic variants were detected in all patients, a median of 48 variants was used per RaDaR™ panel (range: 43-51). ctDNA was detected in 50/94 plasma samples (53%) and in 74/93 urine samples (80%). Detection levels were higher in urine with a median eVAF of 1.98% (range: 0.00057%-35.85%) vs. 0.049% (range: 0.00026%-18.94%) in plasma. ctDNA was detected in baseline plasma in 10/14 (71%) responding pts (median eVAF: 0.325%) and in 8/10 (80%) non-responders (median eVAF: 0.107%). Changes in ctDNA levels reflected clinical responses. After treatment with ipi plus nivo, ctDNA was undetectable in 13/14 (93%) responding pts, and in only 4/10 (40%) of non-responders (p=0.0088). Of the 17 pts with undetectable ctDNA before surgery, 13 (76%) had a pathological response and 16/17 (94%) pts remained recurrence-free after a median follow-up of 34 months. Urine ctDNA was detected at baseline in 12/14 (86%) responding pts (median eVAF: 9.634%) and in 8/10 (80%) non-responders (median eVAF: 2%). After treatment with ipi plus nivo, urine ctDNA was detected in 8/14 (57%) responding pts (median eVAF: 0.87%), and in 8/10 (80%) non-responders (median eVAF: 0.162%). No association was observed between urine ctDNA detection and response (p=0.39). Detection of plasma ctDNA by RaDaR™ after neoadjuvant treatment was associated with pathological response and clinical outcome. In contrast, ctDNA detection in urine was not associated with outcomes. Absence of plasma ctDNA pre-surgery may predict complete response to ipi plus nivo at surgery and may be helpful in guiding clinical decisions in stage III UC, in particular to select pts for bladder-sparing strategies. Citation Format: Jeroen van Dorp, Christodoulos Pipinikas, Nick van Dijk, Greg Jones, Alberto Gil-Jimenez, Giovanni Marsico, Maurits L. van Montfoort, Sophie Hackinger, Linde Braaf, Kirsten McLay, Daan van den Broek, Bas W. Van Rhijn, Nitzan Rosenfeld, Michiel S. van der Heijden. Predicting pathological response after ipilimumab plus nivolumab in stage III urothelial cancer by liquid-biopsy assessment of plasma and urine ctDNA using the RaDaR assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1273.

5020 Background: Encouraging pathological complete response (pCR) rates were observed in trials testing neoadjuvant pembrolizumab or atezolizumab in urothelial cancer (UC). In cT3-4N0 tumors, pCR to atezolizumab was only 17% and restricted to tumors showing characteristics of preexisting T cell immunity. In NABUCCO, we aimed to increase response to pre-operative checkpoint blockade, particularly in high risk patients (pts), by combining ipi plus nivo in stage III UC. We previously reported pCR in 46% and downstaging to no remaining invasive disease in 58% (ESMO2019). Here, we present biomarker analyses and updated clinical follow-up (FU) data. Methods: Twenty four stage III (cT3-4aN0 or cT2-4aN1-3) UC pts who were unfit to receive cisplatin-based chemotherapy or refused, were treated with ipi 3 mg/kg (day 1), ipi 3 mg/kg + nivo 1 mg/kg (day 22), and nivo 3 mg/kg (day 43), followed by resection. The primary endpoint was feasibility (resection < 12 weeks). Efficacy (pCR), safety and biomarker analysis were secondary endpoints. Whole-exome sequencing (WES) was done on baseline tumor samples and local lymph node (LN) metastases showing no response. RNA-seq and multiplex immunofluorescence (mIF) for immune cell markers were done pre- and post-therapy. Results: After a median FU of 15.6 months, 2 pts relapsed (both non-pCR); 1 of these 2 pts died of metastatic disease. Tumors showing complete response (CR, for biomarker analysis defined as pCR, CIS or pTa) had a significantly higher tumor mutational burden than non-CR tumors. CR to ipi+nivo was independent of baseline CD8 T-cell presence. There was no difference between CR and non-CR tumors in baseline immune gene signatures, such as interferon gamma and T-effector signatures. Surprisingly, exploratory gene expression analysis revealed that non-CR was associated with a baseline B cell immune signature, particularly immunoglobulins and genes involved in B cell receptor signaling. CD20 positive cells (by mIF) and presence of tertiary lymphoid structures (TLS) at baseline were also associated with non-CR. Upon treatment with ipi+nivo, early and mature TLS increased significantly in responding tumors. A subset of pts showed CR in the bladder, but non-CR in a local LN tumor focus. WES revealed that these LN metastases were genetically different from the primary tumor bulk. Conclusions: At 15.6 months follow-up, recurrence after pre-operative ipi+nivo was low. Pathological complete response was not restricted to tumors exhibiting preexisting T cell immunity. Clinical trial information: NCT03387761 .