Abstract Background Immune checkpoint inhibitor-based combinations represent the standard of care for advanced renal cell carcinoma (RCC). The OMNIVORE trial (NCT03652142) investigated a response-adaptive strategy including treatment discontinuation in early nivolumab responders (Arm A) and salvage ipilimumab addition in non-responders (Arm B). We present long-term outcomes from OMNIVORE with approximately 6 years follow up in Arm A and 2.5 years follow up in Arm B. Methods OMNIVORE was a multi-center investigator-initiated phase II trial in which patients with unresectable, locally recurrent, or metastatic RCC received induction nivolumab monotherapy. Patients achieving a confirmed objective response discontinued nivolumab and entered observation (Arm A), while patients with stable disease or progressive disease received two doses of ipilimumab added to ongoing nivolumab (Arm B). The primary endpoints were the proportion of patients with durable CR/PR at 1 year after nivolumab discontinuation (arm A) and proportion with SD/PD receiving nivolumab who converted to PR/CR after the addition of ipilimumab (arm B). Secondary endpoints included overall survival, treatment-free interval, duration of disease control, progression-free survival (PFS), and toxicity. This analysis characterizes long-term overall survival across the full study cohort and durability of response among patients who discontinued nivolumab following an early objective response. Results Of 83 patients who initiated treatment, 12 (14%) were allocated to Arm A, 57 (69%) were allocated to Arm B, and 14 (17%) were not allocated due to progressive disease or death. The median follow-up among living patients was 59.4 months (range 29.1 to 85.4 months) in Arm A and 31.4 months (range 4.6-62.6 months) in Arm B. The 3-year overall survival rate from nivolumab initiation was 64% (95% CI 51%-74%) in the overall cohort, 83% (95% CI 48%-96%) in Arm A, and 63% (95% CI 47%-76%) in Arm B. Of the 12 Arm A patients, 6 (50%) remained off nivolumab in durable partial or complete response at 1 year following treatment discontinuation, of whom 5 maintained responses beyond 43 months off therapy with all remaining alive at last known follow-up with overall survival ranging from 48.8 to 85.4 months. Of the 6 patients in Arm A who experienced disease progression and resumed treatment, only 1 achieved a durable complete response, and remains on treatment at 83.2 months from nivolumab initiation. All 57 Arm B patients received 2 doses of ipilimumab with maintenance nivolumab for a median treatment duration of 3.7 months (range 1-24.8 months). At the time of last data cutoff, the ORR was 4% (90% CI, 1% to 11%) and median progression-free survival from nivolumab plus ipilimumab initiation was 4.6 months (95% CI 2.7-6.5 months). Conclusions With extended follow-up, the results of OMNIVORE demonstrate that a meaningful subset of patients with metastatic ccRCC who experience early objective response to anti-PD1 may discontinue therapy and sustain prolonged treatment free benefit. Whereas, ccRCC patients who did not experience early objective response to anti-PD1 did not benefit from subsequent treatment with ipilimumab and nivolumab. Correlative studies are underway to identify biomarkers of extreme response to treatment.

Few studies have been published on the existing models of cancer multidisciplinary clinics (MDCs) and no studies have conclusively compared the implications of different MDC models. We aimed to characterize MDC structural elements and reported quality measures through a narrative review of cancer MDCs in the United States. Forty-one unique MDCs were examined (8 breast, 13 gastrointestinal [GI], 8 genitourinary [GU], 4 head and neck [HN], 3 lung, 5 other cancers). MDCs were most commonly weekly (19/41) and evaluated new patients (9/41). All breast and most GU (5/8) and lung (2/3) MDCs were asynchronous whereas all HN MDCs were synchronous. Interdisciplinary discussions most frequently preceded provider visits, except for HN and other cancer MDCs. Medical, radiation, and surgical oncology were almost always included across all MDCs. The 41 unique institutional MDCs were reported across 54 studies (10 breast, 17 GI, 13 GU, 4 HN, 4 lung, 6 other cancers). Outcome measures were investigated by 38/54 (70%) studies, of which 13 reported overall survival and 6 (46%) noted a statistically significant improvement with MDCs. All lung and approximately half of GI (7/17), breast (5/10), and HN (2/4) MDCs examined a process measure. Structure and balance measures were not as widely reported. Current understanding of MDCs is based on disparate reports with significant heterogeneity in MDC structures and reported outcomes. Further investigation is needed to better elucidate the impact of MDCs in cancer care outcomes across different cancer diagnoses.

Abstract Background Encoded by ARNT, hypoxia-inducible factor-1β (HIF1β) is a constitutively expressed obligate binding partner for HIF1α and HIF2α, enabling their transcriptional activation of HIF-target pathways, such as angiogenesis. Here, we examine the relation of ARNT expression with hypoxia and immune programs, and its impact on survival outcomes in ccRCC. Methods We included patients (pts) with RNA-seq data from TCGA-KIRC (stages I-IV; n = 529), and 2 first-line clinical trials in metastatic ccRCC involving VEGF-pathway inhibitor alone vs in combination with immunotherapy (Trial 1: n = 741, Trial 2: n = 403). Transcripts per million (TPM) were log2-transformed and scaled per 1 SD change. Buffa Hypoxia Score (BHS) was computed per prior reports (Bhandari et al., 2020): expression for each of the score’s 50 genes per patient was assigned +1, if ≥ median for this gene, or -1 if < median, then summed. The immune signatures, IMmotion150 Angio, Teff and Myeloid, JAVELIN and Tumor Inflammatory Score (TIS) were calculated as means of their genes’ log2-transformed TPMs. Spearman correlation associated ARNT expression, with each of these signatures, and EPAS1 and HIF1A expressions. Multivariable Cox models evaluated the association of ARNT expression with progression-free (PFS) and overall (OS) survival per each full cohort, adjusted for age, sex, stage and sarcomatoid features for KIRC, and for age, sex, IMDC risk, treatment arm and sarcomatoid features for the trials. Likelihood ratio test (LRT) assessed interaction between ARNT and treatment arm in predicting survival. Results Across three cohorts, higher ARNT expression correlated with higher BHS (ρ = 0.23-0.49), higher Angio signature (ρ = 0.14-0.57) and higher TIS (ρ = 0.14-0.21) (p < 0.05). Higher ARNT correlated with higher Teff and JAVELIN signatures in KIRC (Teff: ρ = 0.19, p = 8.3 × 10⁻⁶; JAVELIN: ρ = 0.16, p = 1.8 × 10⁻⁶) and Trial 1 (Teff: ρ = 0.16, p = 1.2 × 10⁻5; JAVELIN: ρ = 0.17, p = 1.8 × 10⁻⁶) but not Trial 2 (ρ = 0.09 for both, p > 0.05). A positive correlation was identified between Myeloid signature and higher ARNT expression in Trial 1 (ρ = 0.17, p = 2.3 × 10⁻⁶) and Trial 2 (ρ = 0.12, p = 0.02), but not KIRC (ρ = 0.05, p = 0.24). A consistent correlation was observed between ARNT and EPAS1 expressions (ρ = 0.19-0.61), and HIF1A (ρ = 0.30-0.51), across all cohorts (p < 0.05). Multivariable Cox models revealed no significant association between ARNT expression and PFS and OS (Table), independent of treatment arm per trial (LRT p > 0.05). Conclusions ARNT expression tracks with hypoxia, angiogenic and immune transcriptional states in ccRCC but is not independently prognostic, suggesting biologic relevance without clear predictive value in current VEGF- and immunotherapy-based regimens.

Abstract Background ABBV-CLS-484 is a first-in-class oral PTPN2/N1 inhibitor with a dual mechanism of action that both increases the immunogenicity of tumor cells and augments the antitumor T cell response. It has monotherapy efficacy in multiple preclinical tumor models, with comparable activity to anti-PD-1 in many cases. Notably, it has good antitumor activity in combination with PD-1 specific antibodies in syngeneic animal models known to be refractory to PD-1 blockade. Methods M20-431 is a first-in-human Phase 1 clinical trial testing the safety and preliminary clinical activity of ABBV-CLS-484 alone and with pembrolizumab in patients with advanced solid tumors whose disease has progressed on standard therapies (NCT04777994). Results To date, ABBV-CLS-484 was tolerable, with transient inflammatory adverse events that rapidly resolve off therapy. Durable anti-tumor activity was observed in dose escalation across several tumor types, including clear cell renal cell carcinoma (ccRCC). In 22 patients with anti-PD-1 exposed, >2nd line ccRCC treated with ABBV-CLS-484 alone or combined with pembrolizumab, the ORR was 18% and the DCR was 41%, with 1 durable CR for >2 years. Preliminary biomarker evaluation demonstrated target binding. Conclusions ABBV-CLS-484 is tolerable, with preliminary evidence of clinical activity in dose escalation. Dose expansion cohorts are now accruing to evaluate efficacy in I/O-experienced ccRCC. In tandem with these clinical cohorts, additional strategies that combine ABBV-CLS-484 with VEGF TKIs, CAR-T therapy, and T cell engagers are being tested in preclinical models.

437 Background: CheckMate 214 established nivolumab and ipilimumab (NIVO+IPI) as a first line standard of care regimen with superior survival and durable response versus sunitinib (SUN) in advanced RCC. Previous studies have shown that high levels of circulating KIM-1 are associated with worse prognosis and reduction in KIM-1 levels is associated with benefit from adjuvant immunotherapy. In this post-hoc analysis we evaluated whether KIM-1 levels at baseline and after 1 cycle of NIVO+IPI or SUN are associated with treatment outcomes in CheckMate 214. Methods: Patients with advanced RCC were randomized to NIVO+IPI or SUN as previously described. Serum KIM-1 was measured at baseline and 3 wks after first treatment dose using an enzyme based electrochemiluminescence assay. The association between KIM-1 levels and clinical outcomes was evaluated using Kaplan-Meier and Cox proportional hazards analyses. Results: We analyzed serum from 821 patients (75% of the CM 214 ITT population). Median KIM-1 at baseline was 660.4 pg/mL. Across both arms, higher KIM-1 levels were associated with shorter overall survival (OS) independent of IMDC risk group, nephrectomy status, and tumor burden. Benefit for NIVO+IPI versus SUN was seen across KIM-1 tertiles. Decrease in KIM-1 from baseline to C2D1 was strongly associated with progression free survival (PFS) and OS among patients treated with NIVO+IPI (Median PFS 70.8 months vs 4.2 months for patients with >30% decrease vs >30% increase in KIM-1, with median OS 85.4 vs 26.6 months, overall response rate (ORR) 69.3 % vs 13.9%), but not in patients treated with SUN. Conclusions: In CheckMate 214, increased levels of baseline circulating KIM-1 were associated with worse clinical outcomes both in NIVO+IPI and SUN arms. The extent of reduction in serum KIM-1 just 3 wks after single cycle of NIVO+IPI was associated with long term efficacy of this IO doublet. Circulating KIM-1 may be a useful minimally invasive biomarker for monitoring patients on RCC immunotherapy. KIM-1 change at 3 weeks (prior to second dose of NIVO+IPI) and association with outcomes (NIVO+IPI arm). 3 week KIM-1 change N (%) ORR, % (95% CI) mPFS, months (95% CI) mOS, months (95% CI) >30% Decrease 140 (31.7) 69.29 (60.94-76.80) 70.80 (17.84- NA) 85.36 (63.08- NA) >10-30% Decrease 87 (19.7) 36.78 (26.69-47.80) 11.43 (6.28-18.20) 66.14 (40.44-80.10) <10% Change 86 (19.5) 30.23 (20.79-41.08) 15.41 (10.32-20.73) 52.70 (30.26-70.67) >10-30% Increase 56 (12.7) 23.21 (12.98-36.42) 7.13 (4.17-16.79) 40.34 (23.82-58.41) >30% Increase 72 (16.3) 13.89 (6.87-24.06) 4.17 (3.02-8.08) 26.61 (18.79-38.44)

4505 Background: First-line nivolumab plus ipilimumab (NIVO+IPI) provided substantial long-term survival benefits over sunitinib (SUN) in patients (pts) with advanced renal cell carcinoma (aRCC) in the CheckMate 214 trial. We now report final efficacy and safety data in the intent-to-treat (ITT) population and by International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) risk. Methods: Pts with clear cell aRCC were randomized 1:1 to NIVO 3 mg/kg + IPI 1 mg/kg Q3W×4 then NIVO (3 mg/kg or 240 mg Q2W or 480 mg Q4W); or SUN 50 mg once daily for 4 weeks on, 2 weeks off. Efficacy endpoints included overall survival (OS), and independent radiology review committee (IRRC)-assessed progression-free survival (PFS) and objective response rate (ORR) in intermediate/poor-risk (I/P; primary), ITT (secondary), and favorable-risk (FAV; exploratory) pts. Response was assessed using RECIST v1.1. Results: With 9 years median follow-up, OS was improved with NIVO+IPI vs SUN in ITT (HR 0.71) and I/P (HR 0.69) pts. The probability of OS at 108 months was 31% vs 20% in ITT pts and 30% vs 19% in I/P pts, respectively. In pts with FAV risk, the HR for OS improved from 1.45 at first report (Motzer NEJM 2018) to 0.80 at 9 years, showing a delayed benefit with NIVO+IPI vs SUN. OS probabilities at 108 months were 35% vs 22% in FAV pts, respectively (Table). The probability of PFS at 96 months with NIVO+IPI vs SUN was 23% vs 9% in ITT pts, 25% vs 9% in I/P pts, and 13% vs 11% in FAV pts. The probability of remaining in response through 96 months with NIVO+IPI vs SUN was 48% vs 19% in ITT pts, 50% vs 23% in I/P pts, and 36% vs not available (NA) in FAV pts. No new treatment-related deaths occurred in either arm. Additional subgroup analyses will be presented. Conclusions: In the longest and final phase 3 follow-up (9 years) of a first-line checkpoint inhibitor combination in aRCC, milestone rates of OS and PFS and durable response remained higher with NIVO+IPI vs SUN. No new safety signals emerged. NIVO+IPI remains a standard first-line option in aRCC. Clinical trial information: NCT02231749 . ITT I/P FAV Arm; n NIVO+IPI; 550 SUN; 546 NIVO+IPI; 425 SUN; 422 NIVO+IPI; 125 SUN; 124 mOS (95% CI), mo 53 (46–64) 38 (32–44) 47 (35–56) 26 (22–33) 78 (65–92) 67 (56–80) 108-mo OS probabilities (95% CI), % 31 (27–35) 20 (16–23) 30 (26–35) 19 (15–23) 35 (27–44) 22 (15–30) mPFS (95% CI), mo 12 (10–16) 12 (10–15) 12 (9–17) 9 (7–11) 13 (10–18) 29 (23–43) 96-mo a PFS probabilities (95% CI), % 23 (18–27) 9 (5–15) 25 (20–31) 9 (4–15) 13 (6–22) 11 (3–27) ORR per IRRC (95% CI); CR, % 39 (35–44); 12 33 (29–37); 3 42 (38–47); 12 27 (23–32); 3 30 (22–38); 13 52 (43–61); 6 mDOR (95% CI), mo 76 (59–NE) 25 (20–33) 83 (54–NE) 20 (16–26) 61 (23–NE) 33 (25–51) 96-mo a DOR probabilities (95% CI), % 48 (39–55) 19 (10–31) 50 (41–58) 23 (13–36) 36 (17–56) NA b a 96-mo probabilities reported due to small numbers of pts at risk at 108 mo. b No pts remain at risk. CR, complete response; DOR, duration of response; m, median; NE, not estimable.

BACKGROUND:Preclincal data provide a rationale for cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors alone and in combination with HIF-2α inhibitors in treatment of clear cell renal cell carcinoma (ccRCC), with randomized phase 2 clinical trials currently open exploring the combination of palbociclib with belzutifan vs belzutifan in treatment resistant ccRCC (NCT05468697). However, single agent activity for CDK4/6 inhibitors in ccRCC has not been reported. In this multi-center phase 1b clinical trial (NCT04627064), we investigated the safety and efficacy of monotherapy with abemaciclib, an oral CDK4/6 inhibitor in patients with advanced pretreated RCC. METHODS:Adult patients with advanced RCC with a clear cell component and ECOG status of ≤ 2 progressing after at least 1 prior regimen including immunotherapy and a VEGFR TKI received abemaciclib 200 mg twice daily in 4-week cycles until progression or unacceptable toxicity. The primary objective was to evaluate the objective response rate (ORR) of abemaciclib with a secondary endpoint of safety. First imaging was performed after 8 weeks or 2 cycles. Response was assessed per RECIST 1.1 and toxicity graded per CTCAE v5.0. RESULTS:Eleven patients were enrolled between December 31, 2020 and October 03, 2023. Median age was 62 years (range 54-68); 73% (n = 8) had IMDC intermediate risk disease and 1 patient had translocation RCC with a clear cell component. Median number of prior therapies was 4 (range 1-9). ORR was 0% (0/11; 8 progressive disease, 1 stable disease stopping for clinical progression, 2 not evaluable with clinical progression). About 27% (n = 3) experienced grade ≥3 treatment-related adverse events (diarrhea n = 1, nausea n = 1, neutropenia n = 1). CONCLUSION:In patients with heavily pretreated metastatic RCC, abemaciclib monotherapy had no clinically meaningful activity without new toxicity signals. This data will offer important insight into interpretation of results for ongoing trials exploring CDK4/6 inhibition in combination with HIF-2α inhibitors and immunotherapy.

Tumor-associated macrophage (TAM) infiltration has been shown to modulate response to immune checkpoint inhibitors in various cancers, but its role in metastatic clear cell renal cell carcinoma (mccRCC) remains unclear. Here, we investigated the role of CD163+ TAMs as a potential determinant of clinical outcomes to first-line anti-PD-1 therapy (nivolumab) in patients with mccRCC enrolled in the HCRN GU16-260 trial. Moreover, as recent data suggest that the interaction between TAMs and tumor infiltrating lymphocytes (TILs) promotes T cell exhaustion, we explored the spatial relationship between CD163+ TAMs and CD8+ TILs in different states of exhaustion (ie, terminally exhausted (TE) and non-terminally exhausted (NTE) CD8+ TILs). Pre-treatment tumor samples from 67 patients were analyzed by multiplex immunofluorescence to identify CD163+ TAMs, CD8+PD-1+TIM-3+ and/or LAG-3 + (TE CD8+), and CD8+PD-1+TIM-3−LAG-3− (NTE CD8+) TILs. Associations between the natural log of density of CD163+ TAMs with progression-free survival (PFS) and objective response rate (ORR) were assessed using univariable Cox and logistic regression models, respectively. An optimized cutoff was determined using minimum p value for ORR. For each tumor, the density of TE CD8+ TILs and the density of NTE CD8+ TILs were calculated within a 30 µm radius area centered on CD163+ TAMs (proximal area) and outside of this area (non-proximal area), using the ‘sf’ package within R software. The densities of TE and NTE CD8+ TILs were compared in proximal versus non-proximal areas across all tumor samples using the Wilcoxon signed-rank test. For each CD8+ TIL population (TE and NTE), the enrichment in proximity of CD163+ TAMs was assessed by calculating the difference in densities in proximal and non-proximal areas normalized by the density in the overall tumor area. The level of enrichment in TE CD8+ TILs versus NTE CD8+ TILs in proximity of CD163+ TAMs was compared using the Wilcoxon signed-rank test. The density of CD163+ TAMs, analyzed as a continuous variable was positively associated with ORR (OR: 2.21, 95% CI: 1.33 to 3.69, P = .002) and PFS (HR: 0.77, 95% CI: 0.61 to 0.97, P = .028). At an optimized cutoff, patients with high density of CD163+ TAMs (n = 34, 50.7%) had higher ORR (65% vs. 15%, P < .001) and longer median PFS (16.6 months, 95% CI: 5.5-32.9 vs. 5.5 months, 95% CI: 4.1-10.6, P = .009) compared to patients with low density of CD163+ TAMs (n = 33, 49.3%). The density of CD163+ TAMs was moderately correlated with the density of TE CD8+ TILs (Spearman correlation, r = 0.55) and weakly correlated with the density of NTE CD8+ TILs (r = 0.32). Proximity analysis showed that the density of TE CD8+ TILs was significantly higher in the area proximal to the CD163+ TAMs compared to the non-proximal area (median density: 123.3/mm2 vs. 37.2/mm2; P < .001). Similarly, the density of NTE CD8+ TILs was significantly higher in the area proximal to the CD163+ TAMs compared to the non-proximal area (median density: 127.2/mm2 vs. 66.8/mm2; P < .001). The level of enrichment in proximity of CD163+ TAMs was higher for TE CD8+ TILs compared to NTE CD8+ TILs (0.77 vs. 0.58; p = 0.0011). High levels of CD163+ TAMs are associated with improved outcomes to anti-PD-1 therapy in mccRCC. In addition, exhausted CD8+ TILs preferentially localize in proximity of CD163+ TAMs in ccRCC tissues, supporting that TAM-T cell interactions are critical for driving T cell dysfunction. Taken together, our data are consistent with the hypothesis that the efficacy of PD-1 blockade may be in part mediated by reprogramming TAMs from a pro-tumorigenic to an anti-tumorigenic state.

4539 Background: We previously reported on treatment intensification with the combination of Cabo/Nivo/Ipi in 39 patients (pts) with metastatic RCCdh in a multi-center single arm phase II trial with a starting cabozantinib dose of 40 mg/day (d). Clinical utility was limited with an objective response rate (ORR) of 21% and significant treatment related adverse events (TrAEs) (77% ≥Grade 3 TrAEs). Therefore, we explored the safety and potential efficacy by using a lower starting dose of cabozantinib of 20 mg/d (NCT04413123). Methods: Eligible pts had metastatic RCCdh with ECOG performance status of 0-1 and may have received one line of prior therapy excluding immunotherapy or Cabo. Pts underwent a baseline biopsy and received Nivo 3 mg/kg and Ipi 1 mg/kg intravenously Q3 weeks (W) for 4 cycles followed by Nivo 480 mg IV Q4W. Cabo was given continuously at a dose of 20 mg/d; reductions to 20 mg every other day were allowed; after completion of Ipi, the Cabo dose could be increased to 40 mg/d. The primary endpoint was ORR by RECIST 1.1. Safety was a secondary endpoint. A one-stage design with 20 subjects (for 7 or more responses) would provide 75% power to distinguish an ORR of 40% versus 20% at one-sided alpha of 0.1. Results: 20 pts were enrolled and received at least 1 study drug at 7 sites from Feb. 2023 to Apr. 2024. Following histologic subtypes were included: papillary (n = 11), chromophobe (n = 1), translocation (n = 3), unclassified RCC (n = 2) and other (n = 3). 4 (20%) pts received prior systemic therapy. 10 (50%) pts received all 4 doses of Nivo and Ipi; 13 (65%) pts received maintenance nivolumab. Cabo was increased to 40 mg in 11/13 of these patients. Median follow-up was 9.4 (range 4.6-17.7) months. ORR was 25% (5/20, two-sided 80% CI, 13-41%, Table 1). 6- and 12-month progression free survival rates were 65% and 42% respectively. 11 (55%) pts developed grade 3 or 4 TrAEs (6 were due to elevation in liver function tests) and 1 (5%) had grade 5 TrAE (intraoperative hemorrhage) in setting of disease progression. 5 (25%) required high dose steroids (≥40 mg prednisone or equivalent) of which only 3 (15%) received for hepatitis. All therapy was discontinued due to toxicity in 1 (5%) pt. Conclusions: Although the study did not reach the target of 7 responses to uphold the alternative hypothesis, reduction of the starting dose of Cabo to 20 mg/d in combination with Nivo/Ipi results in numerically lower ≥ grade 3 TrAEs than starting at 40 mg/d (60% vs 77%) and clinical activity in a subset of patients. Clinical trial information: NCT04413123 . Total (N=20) Histology Prior Systemic Therapy N(%) Papillary Chromophobe Translocation Unclassified RCC Other No Yes PR 5 (25) 2 1 0 2 0 4 1 SD 8 (40) 5 0 2 0 1 7 1 PD 7 (35) 4 0 1 0 2 5 2 PR=partial response, SD=stable disease, PD=progressive disease.

Despite the success of immune checkpoint inhibitors (ICI) for the treatment of renal cell carcinoma (RCC), many patients do not receive durable clinical benefit. Therefore, an understanding of resistance to ICIs is critical for the treatment of this disease. Using scRNA-seq, we previously found increased tissue-resident ZNF683 + (Hobit) SLAMF7+ CD8+ exhausted T cells (T-exh-SLAMF7) in human RCC resistant to PD-1 blockade in the HCRN GU16-260 trial. A T-exh-SLAMF7 gene expression signature (GES) was associated with worse clinical outcomes with PD-1 blockade in multiple validation cohorts. Here, through bulk RNA-seq of RCC biospecimens from 90 patients enrolled in this trial, we identified higher tertiary lymphoid structures (TLS) in patients responsive to PD-1 blockade and investigated the interplay between TLS and T-exh-SLAMF7 in shaping therapeutic responses. Bulk RNA sequencing was performed on tumor samples from 90 RCC patients enrolled in the HCRN GU16-260 clinical trial. GES scores were calculated for each sample by z-scoring all genes and then computing the average expression of the genes comprising each signature of interest. To validate TLS presence at the protein level, 19 FFPE RCC tumor samples were analyzed using a 7-plex multiplex immunofluorescence panel targeting DAPI, CD20, CD3, CD21, CD4, PD-1, and FOXP3. TLS were manually quantified based on the colocalization of B and T cell markers in organized structures. Patients were stratified by high versus low TLS and T-exh-SLAMF7 GES scores (≥ or < median) for downstream analyses of clinical response and progression-free survival. Patients with complete/partial response had a higher (≥ median) TLS GES score compared to patients with progressive disease (P = .0004). Similarly, high TLS signature scores were associated with improved progression-free survival (PFS; HR = 2.08, 95% CI: 1.28–3.4, P = .0032), indicating significantly higher risk of progression in patients with low TLS scores. We confirmed that tumors with a high TLS GES score had a higher number of TLS detected by multiplex immunofluorescence (P = .028). Finally, we analyzed the interplay between TLS and tissue-resident exhausted CD8+ T cells. We divided patients into four categories based on median split TLS and T-exh-SLAMF7 GES score values: TLS high SLAMF7 low (n = 28), TLS high SLAMF7 high (n = 15), TLS low SLAMF7 high (n = 28), and TLS low SLAMF7 low (n = 15). Patients with both high TLS and low T-exh-SLAMF7 GES scores had substantially improved PFS compared to all other patients (HR = 0.45, 95% CI: 0.26-0.79, P = .0052), with 60.7% PFS at 12 months compared to 25.9% in the remaining groups. These findings support a paradigm where both high TLS and low T-exh-SLAMF7 cells are required for optimal response to PD-1 blockade in RCC. Ongoing studies will use spatial transcriptomics and functional assays to evaluate the interaction between TLS and T-exh-SLAMF7 cells and to define the roles of SLAMF7 and Hobit in regulating CD8+ T cell effector functions within the RCC tumor microenvironment. Note: Encore Presentation; recently published in Cancer Discovery (PMID: 39992403)

BACKGROUND:Treatment-free survival (TFS; time spent free of systemic anticancer therapy) is increasingly used to support traditional endpoints. TFS was previously evaluated in patients with advanced melanoma treated with nivolumab plus ipilimumab. This analysis compared TFS for nivolumab plus relatlimab and nivolumab monotherapy in patients with advanced melanoma. METHODS:Data were from 714 patients in the phase 2/3 RELATIVITY-047 trial (ClinicalTrials.gov identifier: NCT03470922). TFS was defined as the difference in restricted mean event times between the Kaplan-Meier curves for time to protocol therapy cessation and time to subsequent systemic anticancer therapy initiation or death. TFS was further partitioned into time with and time without grade ≥3 treatment-related adverse events (TRAEs). Subgroup analysis based on tumor BRAF mutational status and tumor programmed death ligand 1 (PD-L1) expression level was conducted. Between-treatment group differences were calculated with bootstrapped 95% CIs. RESULTS:At 48 months from randomization, Kaplan-Meier estimates of overall survival were 52% and 43% for patients in the nivolumab plus relatlimab and nivolumab groups, respectively; 38% and 33% of patients in these respective groups were free of subsequent systemic therapy. The 48-month mean TFS was 2.9 months (95% CI 1.0 to 4.9) longer with nivolumab plus relatlimab than with nivolumab (9.7 vs 6.8 months, respectively). Mean TFS represented 20% and 14% of the 48-month period after initiating nivolumab plus relatlimab and nivolumab, respectively. Considering only time without grade ≥3 TRAEs, the 48-month mean TFS was 2.6 months (95% CI 0.8 to 4.5) longer with nivolumab plus relatlimab than with nivolumab (9.1 vs 6.5 months, respectively). The 48-month mean total TFS was consistently longer with nivolumab plus relatlimab than with nivolumab in the BRAF mutant (9.4 vs 6.5 months), BRAF wild-type (9.9 vs 6.9 months), PD-L1 ≥1% (12.3 vs 7.7 months), and PD-L1<1% (7.9 vs 6.2 months) subgroups. CONCLUSIONS:This analysis demonstrated TFS benefit during the 48 months since initiating nivolumab plus relatlimab compared with nivolumab alone in patients with advanced melanoma. A direct comparison between nivolumab plus relatlimab and nivolumab plus ipilimumab is needed to determine the differences between the regimens in TFS and those in traditional endpoints.

PURPOSE While immune checkpoint inhibition (ICI) has transformed the management of many advanced renal cell carcinomas (RCCs), the determinants of effective antitumor immunity for chromophobe RCC (ChRCC) and renal oncocytic tumors remain an unmet clinical and scientific need. METHODS Single-cell transcriptomic and T-cell receptor profiling was performed on tumor and adjacent normal tissue of patients with ChRCC and renal oncocytic neoplasms. Using machine learning, the cellular origin of renal oncocytic neoplasms was evaluated, with analysis of associated oncogenic pathways. Using immunohistochemistry, immune infiltration was analyzed in renal oncocytic neoplasms in comparison with clear cell RCC (ccRCC). Immune checkpoint expression, clonal expansion, and tumor specificity were compared between ChRCC and ccRCC. Using the International Metastatic RCC Database Consortium data set, clinical outcomes of patients with metastatic ChRCC (mChRCC) treated with first-line systemic regimens were compared with those of patients with ccRCC. RESULTS We validated α-intercalated cells as the cellular origin of renal oncocytic neoplasms. We identified a downregulation of HLA class I molecules with enrichment of potentially targetable pathways including mammalian target of rapamycin and ferroptosis in ChRCC. The tumor microenvironment of ChRCC showed markedly decreased immune infiltration, with a pronounced depletion in tumor-infiltrating CD8 + T cells. ChRCC-infiltrating CD8 + T cells demonstrated lower immune checkpoint expression, diminished clonal expansion, and decreased tumor specificity. Clinical analysis identified poor survival outcomes selectively among patients with mChRCC treated with immune-based therapies. CONCLUSION Immunogenomic analysis of ChRCC revealed profound depletion of T cells, with an immune phenotype marked by a lack of expression of immune checkpoints and poor tumor specificity, suggesting that the few T cells in these tumor types are likely nonspecific bystanders. This immune-cold environment hinders an effective response to immunotherapy and underscores the need for ChRCC-tailored treatments designed to improve tumor-specific T-cell infiltration into the microenvironment.

598 Background: PD-1 directed immune checkpoint inhibitors are effective in many tumor types, including kidney cancers. HHLA2 is a negative immune checkpoint, generally expressed on PD-L1 negative clear cell renal cell carcinomas (ccRCC) (Bhatt, et al. 2021). Therapeutics targeting either HHLA2 or its inhibitory receptor KIR3DL3 are in Phase I trials and predictive biomarkers are needed to help direct treatment. We previously reported that efficacy of nivolumab correlated with tumor PD-L1 status in patients with kidney cancer treated in the first line setting (GU16- 260; Atkins, et al. 2022). We hypothesize that HHLA2 expression in combination with PD-L1 expression may provide a clinically significant biomarker for resistance to PD-1 blockade in patients with advanced ccRCC. Methods: HHLA2 expression in tumor cells (TC) was assessed by immunohistochemistry coupled with image analysis algorithms in 63 pre-treatment primary ccRCC tissues from patients enrolled in the HCRN GU16-260 trial. TC PDL-1 positivity had been previously evaluated in this cohort (Atkins, 2022). TC HHLA2 expression in combination with TC PD-L1 expression was correlated with progression-free survival (PFS) and objective response rate (ORR). Results: Consistent with our prior reports, HHLA2 expression did not overlap with PD-L1 expression on tumor cells. TC HHLA2 expression in combination with TC PD-L1 expression identified three groups of patients with different clinical outcomes with regards to ORR (trend test p-value = 0.016) and PFS (trend test p-value = 0.024) (Table). Patients with positive (>0%) TC PD-L1 expression had the best outcomes (ORR=62.5%, median PFS=24.7 months); patients with negative TC PD-L1 expression and low (<61%) TC HHLA2 expression had intermediate outcomes (ORR=38.5%, median PFS=10.6 months) and patients with negative TC PD-L1 expression and high (≥61%) TC HHLA2 expression had the worst outcomes (ORR=12.5%, median PFS=3.5 months). Conclusions: Further investigation of the HHLA2/PD-L1 combined biomarker is warranted as it may be helpful in identifying patients that do not respond to anti-PD-1 therapy but could benefit from agents targeting the HHLA2/KIR3DL3 pathway. HHLA2 and PDL-1 category Number of patients(63 total) ORR PFS (median, months) TC PD-L1 >0% 16 62.5% 24.7 TC PD-L1 = 0% & TC HHLA2 <61% (low) 39 38.5% 10.6 TC PD-L1 = 0% & TC HHLA2 ≥61% (high) 8 12.5% 3.5 Trend test p-value 0.016 0.024

Determining how immune checkpoint inhibitors (ICI) alter functional characteristics of lymphocytes can improve our understanding of ICI. HERV-H LTR-associating 2 (HHLA2, B7-H7) is an immune checkpoint expressed by many tumors. Multiple HHLA2:KIR3DL3 blocking antibodies are currently in Phase I clinical trials and enrolling cancer patients (NCT05958199, NCT06240728), but little is known about how different subsets of NK cells are affected by HHLA2 expression. Our group previously found that in addition to an activating receptor, TMIGD2, HHLA2 has an inhibitory receptor, KIR3DL3. The KIR family of receptors is expressed in NK cells as well as T cells. Through co-culture experiments of CD56+ NK cells in the presence of K562 cells, we sought to investigate the mechanisms of NK cell engagement. Additionally, we generated transgenic K562 cell lines, over-expressing HHLA2, validated through flow cytometry, to maximize our ability to glean insight into HHLA2-KIR3DL3 mediated activation of NK cells. To this end, we generated a high quality scRNA-seq dataset of more than 100, 000 cells, comprising of more than 90, 000 NK cells, with approximately 30, 000 cells either cultured alone, co-cultured with K562 or K562 over-expressing HHLA2 tumor cells, allowing unprecedented granularity. In conjunction with published NK cell states and newly identified ones through this study, we recapitulate important known cytokines, growth factors and other regulators underlying NK cell maturation and substates that are differentially affected by treatment, in abundance, expression, and regulation. Furthermore, we shed some light on the complex interplay of positive and negative regulators, including CD48 - 2B4 and CD56 related interactions in CCL4 and XCL2 high NK cells, as well as TGFb related pathways. Uncovering NK cell activity modulators, such as amphiregulin, NKG7, FAM49B, and TXNIP, we then focused on NK cells in the context of KIR3DL or TMIGD2 expression. We add to previous efforts to understand immune checkpoint inhibition by the HHLA2-KIR3DL3 axis and in a concerted effort with blocking antibodies, provide evidence of enhanced NK cell efficacy. Better understanding of how HHLA2 pathway affects the function of different subsets of lymphocytes could determine which patients may best benefit from targeting this pathway. Nahuel Perrot, Nikolaos Kalavros, Deepthi Chowbene, Shoushuo Wang, Yered Pita-Juarez, Antonella Arruda de amaral, David McDermott, Gordon Freeman, Ioannis Vlachos, Kathleen Mahoney. Investigating the transcriptomic signature of HHLA2-mediated suppression of NK cell activity [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 3229.

BACKGROUND:Retrospective analyses of studies of IO-containing combinations for advanced renal cell carcinoma (RCC) suggest that depth of response is associated with overall survival but have methodological limitations. We investigated the relationship of week 12 depth of response as a continuous variable with overall survival. METHODS:Pooling data from patients with treatment-naïve advanced RCC enrolled in randomized IO-containing frontline advanced RCC trials submitted to the US Food and Drug Administration that included week 12 imaging assessment, we developed 36-month overall survival prediction models based on week 12 depth of response (reduction from baseline in target lesion diameter) using Cox proportional hazards with natural spline in an IO combination group and a sunitinib group. To avoid guarantee-time bias, only patients in follow-up at the week 12 scan were included. Overall survival was defined from the week 12 imaging date. RESULTS:Among the 4 trials that met our inclusion criteria, 1364 patients in the IO combination group and 1267 patients in the sunitinib group had week 12 imaging. Depth of response and 36-month overall survival were correlated throughout the entire range of depth of response in both treatment groups, with no plateau in overall survival as depth of response approached complete response. Across this range, estimated 36-month overall survival was higher in the IO combination group. CONCLUSIONS:Deeper response was associated with better 36-month overall survival in this pooled exploratory analysis of treatment-naïve patients with advanced RCC treated with IO combination or sunitinib. Further work characterizing the relationship between depth of response and overall survival at the trial level may advance understanding of the utility of depth of response as a pharmacodynamic response biomarker or early endpoint in signal-seeking trials and to facilitate efficient drug development.