Radiotherapy-based metastasis-directed therapy (MDT) has emerged as a treatment strategy for oligometastatic clear cell renal cell carcinoma (ccRCC). However, optimal integration of MDT with immune checkpoint inhibition (ICI) is unclear, especially in light of the M1 no evidence of disease subgroup analysis of KEYNOTE-564. We undertook an exploratory cohort study of two previously reported trials evaluating either MDT+ICI (NCT02855203) or MDT (NCT03575611) for oligometastatic ccRCC. The objective was primarily to compare RECIST-defined progression-free survival (PFS) and secondarily to compare peripheral immune populations. Among the 150 patients included in the analysis (MDT+ICI: 30; MDT: 120), the MDT+ICI cohort had more metastases (median 3 vs 1) and was slightly younger (median 62 vs 66) than MDT. After a median follow-up time of 34 mo, there was evidence for longer PFS after MDT+ICI vs MDT that did not reach statistical significance (hazard ratio, 0.57; 95% confidence interval: 0.32-1.02; p = 0.058). Interaction testing demonstrated greater PFS benefit with MDT+ICI among patients with previous receipt of systemic therapy. Immediate systemic induction of activated CD8+ T cells (ICOS+) was more common after MDT+ICI, as were decreases in less functional CD8+ T cell subsets. Taken together, this study provides evidence that adding maintenance ICI to MDT improves clinical outcomes for patients with oligometastatic ccRCC. MDT+ICI-evoked immunomodulatory signals are promising and support the observed superiority in clinical outcomes. The A Randomized Trial of Maintenance Systemic Therapy After Radiation for Oligometastatic Renal Cell Carcinoma (ASTROs) trial (NCT06004336) has been initiated to test the hypotheses generated by the present study.

PURPOSE We tested the hypothesis that adding metastasis-directed therapy (MDT) to standard-of-care (SOC) systemic therapy improves progression-free survival (PFS) among patients with oligometastatic disease. METHODS EXTEND was a multicenter randomized phase II trial. Patients with 1-5 metastases were randomly assigned to MDT + SOC versus SOC in one of the six baskets (breast, pancreas, kidney, two prostate baskets, and an other basket) with basket-specific stratification and powering. PFS, the primary end point, was prespecified in the per-protocol set within each basket, across all baskets, and across all baskets excluding the prostate baskets. Exploratory end points included circulating tumor DNA (ctDNA) and immune profiling. RESULTS From 2018 through 2023, 521 patients were screened, 350 were randomly assigned, and 334 were analyzed per protocol (MDT + SOC, n = 166; SOC, n = 168). Radiotherapy was used as MDT for 98% of metastases (370/379). Overall, after a median follow-up of 53 months, PFS was improved with MDT + SOC (hazard ratio [HR], 0.54 [95% CI, 0.41 to 0.72], P < .001). Similarly, PFS was improved when excluding the prostate baskets (HR, 0.60 [95% CI, 0.40 to 0.89]). Within each basket, PFS superiority was identified for the pancreas, prostate, and other baskets, whereas the breast and kidney baskets were inconclusive. At enrollment, detectable ctDNA correlated with shorter PFS and survival; by contrast, ctDNA clearance 3 months postenrollment correlated with improved survival. MDT + SOC-induced systemic immune activation was most pronounced among baskets demonstrating PFS superiority. CONCLUSION The phase II EXTEND trial supports the addition of MDT to SOC for oligometastatic disease. Histology-specific efficacy signals were identified for phase III testing. Translational insights suggest the potential for optimizing the definition of oligometastasis using ctDNA and point to systemic immune responses as a possible mechanism of benefit from MDT.

Better tools are needed for clinical decision-making in oligometastatic clear-cell renal cell carcinoma (ccRCC). Plasma KIM-1 concentrations and circulating tumor DNA (ctDNA) were measured in a clinical trial evaluating metastasis-directed therapy (MDT) for oligometastatic ccRCC (NCT03575611). Shorter systemic therapy-free survival (STFS) was associated with high KIM-1 at baseline (hazard ratio per log10 pg/ml [HR]: 2.45, 95% confidence interval [CI] 1.48-4.08; p < 0.001), the end of radiation therapy (HR 2.56, 95% CI 1.56-4.21; p < 0.001), and 3-mo follow-up (HR 3.22, 95% CI 1.91-5.43; p < 0.001). KIM-1 was also associated with progression-free survival and overall survival. In multivariable analysis, both KIM-1 and ctDNA molecular residual disease status were independently associated with STFS when measured at baseline (KIM-1: HR 1.91, 95% CI 1.03-3.55; p = 0.041; ctDNA: HR 2.47, 95% CI 1.08-5.64; p = 0.03) and 3-mo follow-up (KIM-1: HR 2.22, 95% CI 1.09-4.51; p = 0.03; ctDNA: HR 2.70, 95%CI 1.09-6.67; p = 0.03). After elastic net-informed selection, Weibull regression fitted with four clinical variables plus baseline KIM-1 and ctDNA yielded a prognostic model (K-COMPASS) for STFS that demonstrated favorable discrimination (C index 0.76) and calibration. In conclusion, plasma KIM-1 and ctDNA were independently associated with patient outcomes across time points, and their prognostic value further improved with the addition of clinical variables.

537 Background: Biomarkers do not yet exist to triage patients with oligometastatic ccRCC for de-escalation strategies. Two promising biomarkers are kidney injury molecule-1 (KIM-1) and circulating tumor DNA (ctDNA). Methods: Patients with ccRCC and ≤5 metastases were enrolled on a single-arm phase 2 trial of metastasis-directed therapy (MDT) without systemic therapy (NCT03575611), powered for the primary endpoint of systemic therapy–free survival (STFS). Plasma samples for ctDNA and KIM-1 measurement were collected at baseline and 3-month follow-up. Personalized ctDNA detection panels (≤2000 somatic variants) were constructed from tumor whole-genome sequencing (Myriad Genetics). To create the Kidney Cancer OligoMetastasis Prognostic Assessment Systemic Score (K-COMPASS), we screened 24 candidate variables by elastic net-penalized Cox regression with 5-fold cross-validation repeated 100 times, with performance assessed by C-index. Predictors with nonzero coefficients were refit with Weibull regression for STFS. Results: Among 112 patients with KIM-1 measurements available, median baseline KIM-1 level was 127.0 pg/mL (IQR 71.5–228.0). KIM-1 and ctDNA were associated with STFS at baseline and 3 months in univariable and multivariable analyses (table). Progression-free survival (PFS) and overall survival (OS) were also associated with KIM-1 at baseline (PFS: HR=2.2, 95% CI 1.5–3.3; OS: HR=5.1, 95% CI 2.5–10.2, P <0.001) and at 3 months (PFS: HR=3.5, 95% CI 2.2–5.5; OS: HR=5.0, 95% CI 2.3–10.9) (all P <0.001). K-COMPASS construction selected baseline KIM-1, baseline ctDNA MRD, and 4 clinical variables (prior systemic therapy lines, ECOG performance status, number of metastatic lesions, time from diagnosis to metastasis). The complete model showed strong discrimination (C-index=0.76) and excellent calibration (slope=1). A user-facing K-COMPASS tool is available online (www.trialdesign.org). Conclusions: To our knowledge, this is the first evaluation of KIM-1 in oligometastatic ccRCC and the first to analyze KIM-1 and ctDNA in tandem for RCC. Both biomarkers were strongly and independently associated with outcomes in patients receiving MDT without systemic therapy. Integrating KIM-1 and ctDNA with clinical factors (K-COMPASS) yielded a well-calibrated model with high discrimination to support risk-adapted decision-making. External validation is planned. Univariable and multivariable association of baseline KIM-1 and ctDNA MRD status with systemic therapy free survival. HR (95% CI) Baseline 3-Month Follow-Up Univariable Multivariable Univariable Multivariable KIM-1(per log10[pg/mL]) 2.5 (1.5–4.1) P <0.001 1.9 (1.0–3.6) P =0.041 3.2 (1.9–5.4) P< 0.001 2.2 (1.1–4.5) P =0.028 ctDNA (MRD+ vs MRD–) 2.8 (1.3–5.9) P =0.0089 2.5 (1.1–5.6) P =0.032 4.4 (2.1–9.5) P< 0.001 2.7 (1.1–6.7) P =0.032

Current treatment for frontline ccRCC focuses on systemic therapy doublets. Although effective, such combinations exhibit substantial toxicities and healthcare costs. An underutilized option is MRWS, which may facilitate a prolonged systemic therapy-free interval in select patients. Unfortunately, no reliable prognostic markers exist to select patients for MRWS. Although ctDNA assays may guide patient selection, implementation in ccRCC has proven challenging due to limited ctDNA shedding. Therefore, advanced sequencing and bioinformatic pipelines are needed to enhance ctDNA reliability in ccRCC. This phase 2 single-arm trial (NCT03575611) enrolled patients with oligometastatic ccRCC and up to 5 metastases. All patients had either never received systemic therapy or ceased >1 month earlier. Patients were treated with MRWS, consisting of predominately stereotactic radiation therapy to all sites of disease. Subsequent rounds of MRWS were administered if limited progression was observed. The co-primary endpoints were progression free survival (PFS) and systemic therapy free survival (STFS). For the latter, a median STFS of > 24 months (mo) was prespecified as the threshold for success. Individualized ctDNA panels (Myriad Genetics) were created from tumor whole genome sequencing and applied to serial plasma samples. Molecular residual disease (MRD) status was determined based on whether ctDNA was detected (MRD+) or not (MRD-). Between July 2018 to May 2023, 121 oligometastatic ccRCC patients were enrolled. Median follow up was 36 mo (range 13-61 mo). Most patients (72%) had 1 site of metastatic disease and had never received systemic therapy (70%). The median PFS was 18 mo (95% CI: 16-25 mo) and STFS was 34 mo (95% CI: 28-54 mo). The lower bounds of 95% CI of the median STFS exceeded the prespecified 24 mo threshold for success. Two-year PFS and STFS were 40% and 75%, respectively. Median OS was not reached, and 2- and 3-year OS were 94% and 86%, respectively. Nine (8%) patients experienced patients 3+ toxicities at least possibly attributed to MRWS. There were no grade 5 toxicities. MRD+ was detected in 56% of patients at baseline and associated with significantly shorter STFS (HR 2.9, 95% CI 1.4-6.1, P = .003). Patients who were MRD+ and MRD- at baseline exhibited a 27 vs. 54 mo median STFS, respectively. Three months after MRWS, 31% of MRD + patients converted to MRD-. Positive MRD status at 3 month follow up was strongly associated with shorter STFS (HR 4.3, 95% CI 2.0-9.0, P < .001). MRWS exhibited excellent tolerability and facilitated prolonged time off systemic therapy without compromising OS. Our ctDNA approach appears to be a promising baseline prognostic biomarker for STFS and a dynamic marker of MRWS response.

BACKGROUND AND OBJECTIVE:Oligometastatic prostate cancer (omPC) is characterized by limited metastases. We hypothesized that metastasis-directed therapy (MDT) to all sites of omPC combined with androgen deprivation therapy (ADT) would improve clinical outcomes. METHODS:In the multicenter phase 2 EXTEND trial, patients with omPC were randomized 1:1 to ADT versus MDT + ADT in two independently powered and randomized baskets, one using intermittent ADT and one using continuous ADT. The primary endpoint was progression-free survival (PFS). The secondary endpoints included radiologic PFS (rPFS) and castration resistance-free survival (CRFS). Here, the primary results of the continuous ADT basket, the combined analysis of both baskets, and translational immune correlatives are reported. KEY FINDINGS AND LIMITATIONS:From September 2018 through August 2022, 174 patients were randomized and were eligible for the primary analysis. In the continuous ADT basket (N = 87), the median PFS was 47 mo with MDT + ADT versus 22 mo with ADT (hazard ratio [HR], 0.50; one-sided p = 0.036). In the combined analysis, the median PFS was 36 mo with MDT + ADT versus 17 mo with ADT (HR, 0.45; p < 0.001). Radiologic PFS and CRFS were also superior with MDT + ADT. Durable clinical responses after MDT + ADT were associated with systemic Th1-polarizing cytokine upregulation and CD8+ T-cell proliferation. Compared with ADT, MDT + ADT induced greater systemic immune activation, including T-cell receptor expansion/contraction, which we also observed in the independent ORIOLE trial of MDT. The greatest PFS benefit after MDT + ADT was observed in patients with systemic T-cell receptor expansion/contraction. CONCLUSIONS AND CLINICAL IMPLICATIONS:MDT + ADT improves PFS compared with ADT in omPC patients, meriting phase 3 confirmation. Hypothesis-generating immune responses warrant mechanistic validation and future trials with T-cell-targeted immunotherapies.

Therapeutic targeting of mutant KRAS pathways driving cancers is being actively investigated to identify feedback mechanisms responsible for the development of adaptive resistance to mutant KRAS inhibitors undergoing clinical trials. Here we report RASH3D19 as a mediator of RAS pathway activation through a positive feedback loop involving the KRAS-microRNA signalling axis. KRAS-induced miR-222 represses ETS1 expression and downstream transactivation of miR-301a leading to elevation of its target RASH3D19. RASH3D19 facilitates activation of RAS pathways by promoting dimerization and interaction of EGFR with the SOS2, GRB2, SHP2 and GAB1 complex. Genetic deletion of RASH3D19 in mutant KRAS-expressing cancer cells exhibits growth retardation in vitro, in vivo and sensitized pancreatic ductal adenocarcinoma and colorectal cancer cells, organoids and xenografts to mutant KRAS inhibitors, suppressing feedback reactivation of RAS pathways. Therapeutic targeting of RASH3D19 is expected to lead to tumour debulking and alleviating resistance to KRAS inhibitors in mutant KRAS-expressing cancers.

Current treatment for frontline ccRCC focuses on systemic therapy doublets. Although effective, such combinations exhibit substantial toxicities and healthcare costs. An underutilized option is MRWS, which may facilitate a prolonged systemic therapy-free interval in select patients. Unfortunately, no reliable prognostic markers exist to select patients for MRWS. Although ctDNA assays may guide patient selection, implementation in ccRCC has proven challenging due to limited ctDNA shedding. Therefore, advanced sequencing and bioinformatic pipelines are needed to enhance ctDNA reliability in ccRCC. This phase 2 single-arm trial (NCT03575611) enrolled patients with oligometastatic ccRCC and up to 5 metastases. All patients had either never received systemic therapy or ceased >1 month earlier. Patients were treated with MRWS, consisting of predominately stereotactic radiation therapy to all sites of disease. Subsequent rounds of MRWS were administered if limited progression was observed. The co-primary endpoints were progression free survival (PFS) and systemic therapy free survival (STFS). For the latter, a median STFS of >24 months (mo) was prespecified as the threshold for success. Individualized ctDNA panels (Myriad Genetics) were created from tumor whole genome sequencing and applied to serial plasma samples. Molecular residual disease (MRD) status was determined based on whether ctDNA was detected (MRD+) or not (MRD-). Between July 2018 to May 2023, 121 oligometastatic ccRCC patients were enrolled. Median follow up was 36 mo (range 13-68 mo). Most patients (72%) had 1 site of metastatic disease and had never received systemic therapy (70%). The median PFS was 18 mo (95% CI: 15-22 mo) and STFS was 34 mo (95% CI: 28-54 mo). The lower bounds of 95% CI of the median STFS exceeded the prespecified 24 mo threshold for success. Two-year PFS and STFS were 40% and 75%, respectively. Median OS was not reached, and 2- and 3-year OS were 94% and 87%, respectively. Eight (7%) patients experienced grade 3+ toxicities at least possibly attributed to MRWS. There were no grade 5 toxicities.MRD+ was detected in 56% of patients at baseline and associated with significantly shorter STFS (HR 2.9, 95% CI 1.4-6.1, P=0.003). Patients who were MRD+ and MRD- at baseline exhibited 27 vs. 54 mo median STFS, respectively. Three months after MRWS, 31% of MRD + patients converted to MRD-. Positive MRD status at 3 month follow up was strongly associated with shorter STFS (HR 4.3, 95% CI 2.0-9.0, P<0.001). MRWS exhibited excellent tolerability and facilitated prolonged time off systemic therapy without compromising OS. Our ctDNA approach appears to be a promising baseline prognostic biomarker for STFS and a dynamic marker of MRWS response. Chad Tang, Alexander Sherry, Aaron Seo, Kieko Hara, Haesun Choi, Suyu Liu, Xiaowen Sun, Anya Montoya, Ethan Ludmir, Amishi Y. Shah, Eric Jonasch, Amado J. Zurita, Craig Kovitz, Christopher J. Battey, Sarah Ratzel, Giannicola Genovese, Kanishka Sircar, Jose Karam, Nizar Tannir, Pavlos Msaouel. Phase 2 trial of metastasis directed radiotherapy without systemic therapy (MRWS) for oligometastatic clear cell renal cell carcinoma (ccRCC) and investigation of circulating tumor DNA (ctDNA) as a personalized biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT132.

BACKGROUND:Select patients with metastatic clear-cell renal-cell carcinoma can be treated without systemic therapy, yet few studies have explored this population. We investigated the efficacy of metastasis-directed therapy without systemic therapy in oligometastatic clear-cell renal-cell carincoma. METHODS:This investigator-initiated single-arm, phase 2 trial enrolled patients aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0-2, histologically confirmed clear-cell renal-cell carcinoma, and one to five metastases. Patients remained off systemic therapy and underwent metastasis-directed therapy to all disease sites, with additional metastasis-directed therapy for limited progression. Co-primary endpoints were progression-free survival based on Response Evaluation Criteria in Solid Tumours version 1.1 (RECIST 1.1) in the per-protocol population (patients who received radiation to at least one metastatic lesion during their initial local treatment) and systemic therapy-free survival in the intention-to-treat population. Progression-free survival was defined as the interval from enrolment to the first instance of disease progression, according to RECIST 1.1, or clinical progression, or death from any cause. Systemic therapy-free survival was defined as time from enrolment to initiation of systemic therapy or death from clear-cell renal-cell carcinoma. A prespecified 24-month median systemic therapy-free survival was the threshold for success. Safety was analysed in the per-protocol population. This trial is registered with ClinicalTrials.gov, NCT03575611, and is closed to new patient enrolment. FINDINGS:Between July 13, 2018, and May 2, 2023, 121 patients were enrolled and included in the intention-to-treat population, of whom 120 received at least one round of definitive radiotherapy and were included in the per-protocol and safety populations. Median follow-up time for the 121 enrolled patients was 36·3 months (IQR 26·5-51·1). Median progression-free survival was 17·7 months (95% CI 14·9-22·4), and median systemic therapy-free survival time was 34·0 months (28·3-54·1). The median and lower bound of 95% CI of the median systemic therapy-free survival time exceeded the prespecified 24-month target. Eight (7%) of 120 patients had grade 3-4 adverse events at least possibly attributable to metastasis-directed therapy. The most common grade 3 event was pain near the treatment site (four events). The single grade 4 event was hyperglycaemia. There were no treatment-related deaths. INTERPRETATION:Select patients with oligometastatic disease can be managed with serial metastasis-directed therapy with prolonged time off systemic therapy, favourable progression-free survival, and limited adverse events. FUNDING:Cancer Prevention and Research Institute of Texas, US National Cancer Institute, and Myriad Genetics.

Abstract Background: Minimal residual disease (MRD) testing can detect cancer recurrence months to years earlier than the current standard of care, enabling earlier treatment of recurrence and improved patient outcomes. Tumor-informed MRD assays typically utilize formalin-fixed paraffin-embedded (FFPE) tumor tissue, which is available in limited quantities for some patients, for example, following core needle biopsy (CNB), after neoadjuvant treatment or when patients need multiple tests from the same tumor sample. To determine the lower limit of tissue input, we evaluated our MRD assay performance across a range of extracted tumor volumes. Methods: Resected tumors and CNBs were sectioned, H&E stained, and macro-dissected. Tumor gDNA was extracted, quantified, prepared into libraries and sequenced. Sequenced libraries were aligned and evaluated for depth of coverage, variation of coverage, and duplication rate. Somatic calling was performed on matched tumor and normal samples. Up to 1000 target sites were selected for high-depth targeted sequencing of the tumor and normal gDNA for confirmation of somatic variant calls. The positive predictive value (PPV) was computed as the percent of putative somatic variant sites that were present in the tumor capture library and absent in the normal capture library. Results: Extracted tumor volumes varied by almost two orders of magnitude, from 0.06mm3 (equivalent to needle core or fine needle aspirate biopsies) to 5mm3 (achievable with resected tumor). gDNA amount varied linearly (3.6ng to 1549ng) with tumor tissue input, indicating the low tissue to paraffin ratio did not have an adverse effect on yield. Tumor gDNA inputs into library prep ranged from 2.5ng to 100ng. DNA amounts above 100ng into library prep had no discernable benefit. Below 10 ng, depth of coverage and the coefficient of variation in coverage indicated poor-quality libraries. Samples with ~10 ng of gDNA input into library prep showed depth of coverage comparable to higher inputs and saturated the achievable library complexity. Additionally, PPV of somatic calling was consistent across the range of gDNA inputs from 10-100 ng, demonstrating equivalent assay performance. Conclusion: Tumor-informed MRD assays have immense potential for increasingly sensitive treatment response and recurrence monitoring that can inform better treatment decisions. FFPE tumor tissue is a critical input into MRD assays but is a limited resource. This study supports a minimum DNA input of 10 nanograms for a single attempt at extraction, corresponding to a tissue volume of 0.2mm3 or a single 10µm slide with a 20mm2 area, representing one of the lowest tissue input requirements for an MRD assay. Low FFPE tissue requirements expand the patient population that benefit from MRD testing by utilizing samples that have low tumor content, are post-neoadjuvant therapy or do not meet the tumor volume requirements of competing MRD offerings. Citation Format: Matt LaBella, Ashley Acevedo, Ravi Patel, Kiefer Haug, Sangita Ganesh, Elise Buser, Nafei Xu, Shalee Carlson, Kyle Trettin, Sarah Ratzel, Kieko Hara, Pavlos Msaouel, Kanishka Sircar, Chad Tang, Dale Muzzey, Genevieve Gould. Design of high-performance tumor-informed minimal residual disease (MRD) panels from low FFPE tumor input [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 3675.

Abstract Background: The decision to perform cytoreductive nephrectomy (CN) and timing of CN in patients (pts) with metastatic renal cell carcinoma (mRCC) and primary who respond to immune checkpoint therapy (ICT) is a matter of debate. In CheckMate-214, nivolumab plus ipilimumab (N+I) yielded a 35% objective response rate in the primary; however, there is a paucity of pathological data from nephrectomy specimens (Nx) after treatment with N+I. We sought to investigate the radiologic and pathological responses and correlate the findings with outcomes in pts receiving 1L N+I followed by CN. Methods: We reviewed the medical records and Nx of pts with mRCC who received N+I followed by CN at our institution (2016-2021). H&E slides were reviewed to determine the % of residual viable malignant cells. Pathological response was defined as tumor viability ≤20%, and pathological complete response (pCR) defined as absence of residual cancer. Radiologic response was defined as ≥30% reduction in primary size. Progression-free survival (PFS) and overall survival (OS) were determined from date of CN. Results: 22 pts (median age 59) are included: 18 (81.8%) males, 19 (86%) Caucasian, 20 (91%) had clear-cell histology, 20 (91%) had ECOG Performance Status 0-1, 10 (45%) had intermediate-risk disease and 12 (55%) had poor-risk disease by IMDC. Median ICT duration before CN was 6.5 months (mo) (range, 2-19 mo). 17 (77%) completed 4 cycles of N+I, and 16 (73%) received N maintenance before CN. 5 pts (23%) had grade 3/4 immune-related adverse effects; 1 pt did not complete 4 cycles of N+I due to hepatitis. There were no therapy-related deaths. 12 pts (54.5%) had progressive disease (PD); 3 pts (14%) died of RCC. Median PFS was 22 mo, median OS not reached (NR). Pts with pathological response had greater radiologic reduction in primary size compared to pts without pathological response (median reduction 38% vs. 15% p-value 0.01), smaller final primary pathological size (median 5.7 cm vs. 10.65 cm p-value 0.002), and lower pathological yT stage (pT0: 1, pT1: 6, pT2: 2 pT3: 3, pT4: 0 vs. pT0: 0, pT1: 1, pT2: 0, pT3: 8, pT4: 1 p-value 0.045); ICT duration was similar between groups (median 5.5 mo vs. 7 mo p-value 0.733). 6 pts achieved low viability within 5 mo of ICT. In 9 pts with optimal radiopathological response [viability ≤20% and ≥30% primary size reduction], PFS was longer compared to PFS in pts with suboptimal radiopathological response: median PFS NR vs. 17 months (HR 0.23, 95% CI 0.076 - 0.741; p-value 0.041). In 13 pts with suboptimal response, 10 (77%) had PD, the most common sites of PD were lymph nodes [6 pts (60%)] and brain [3 pts (30%)]. Conclusions: In this small cohort, N+I yielded a low pCR in Nx of pts with mRCC. Radiologic response but not ICT duration was associated with low tumor viability. Citation Format: Leticia Campos Clemente, Omar Alhalabi, Matthew T. Campbell, Guillermo Corredor Alonso, Kieko Hara, Pavlos Msaouel, Andrew C. Johns, Chad Tang, Jose A. Karam, Priya Rao, Nizar M. Tannir. An integrated analysis of radiologic and pathological responses in patients with metastatic renal cell carcinoma who presented with primary renal tumor in situ and received first-line nivolumab plus ipilimumab followed by cytoreductive nephrectomy [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 7523.

3039 Background: Due to low shedding and low tumor-mutation burden, metastatic clear cell renal cell carcinoma (ccRCC) is poorly modeled by current cell-free circulating tumor DNA (ctDNA) assays. Second generation tumor-informed ctDNA approaches may demonstrate improved sensitivity in ccRCC, where somatic variants identified by whole-genome sequencing (WGS) of tumor tissue are tracked in probe panels to monitor molecular residual disease (MRD) in patient plasma. However, intertumoral genomic heterogeneity of input tissue may hinder optimal panel design. Here we evaluated the concordance of ctDNA results using panels designed from primary nephrectomy or metastatic tumors in ccRCC. Methods: In five patients with oligometastatic ccRCC (≤5 lesions of metastatic disease; median of 1, range 1-4), 13 primary tumor, 24 metastatic lesion subsites and matched buffy coat samples were collected. Baseline plasma was collected prior to radiotherapy. Samples were tested with Myriad Genetics High-Definition MRD assay. Briefly, tumor and normal DNA were whole-genome sequenced (median coverage 30x). A sub-panel with up to 1000 somatic variants was designed for each lesion subsite; variants could be common across sub-panels. Sites were enriched using hybridization capture from patient plasma-derived cfDNA and sequenced to high depth. A statistical model of variant allele counts was used to assess the presence or absence of ctDNA (MRD status) and infer quantitative tumor fraction. Results: Median time from nephrectomy or most recent metastasis sample collection to baseline plasma collection was 146 months (71-169) and 32 months (7-48), respectively. Median panel probe count per patient was 4,311 (3,400-5,000) and per tumor sample was 1,545 (102-2,158). Confident calls were emitted for tumor fractions ranging from 0.0004% to 0.027%. Baseline MRD status was fully concordant across sub-panels derived from primary tumor lesion subsites and metastases in three of five patients (3/5; 60%). Of the patients with discordant calls, a single metastasis-derived sub-panel (comprising 20%-25% of metastatic sub-panels) called MRD discordantly in each. Of the three patients MRD positive at baseline, only one was alive at last follow up. All patients MRD negative at baseline are still alive. Conclusions: A tumor-informed, WGS MRD assay for ccRCC showed comparable performance between probe capture panels designed from primary tumor and metastatic lesion subsites despite differences in anatomical location and time from plasma collection. This result suggests that intertumoral genomic heterogeneity may not be consequential in MRD assays that leverage large panels to detect ctDNA variants, obviating the need for repeat biopsies of metastatic sites. Work is underway to validate this MRD assay in a larger cohort of patients with ccRCC before and after treatment.

"YIA24-001: Spatial Evolution of Immune Cell Dysfunction During Development of Oligometastatic Renal Cell Carcinoma: Secondary Analysis of a Prospective Trial" published on 05 Apr 2024 by National Comprehensive Cancer Network.

Background: Thymic squamous cell carcinoma and type B3 thymoma are primary neoplasms of the anterior mediastinum that are sometimes difficult to differentiate from one another histologically. However, only a few immunohistochemical markers are available for the differential diagnosis. The purpose of this study was to discover a novel marker for differentiating between thymic squamous cell carcinoma and type B3 thymoma.Methods: We used histological samples of thymic carcinomas (n = 26) and type B3 thymomas (n = 38) which were resected between 1986 and 2017. To search for candidates of differential markers, gene expression levels were evaluated in samples using promoter analysis by cap analysis of gene expression (CAGE) sequencing.Results: Promoter level expression of CALML5 genes was significantly higher in thymic carcinomas than in type B3 thymomas. We further validated the results of the CAGE analysis in all 26 thymic carcinomas and 38 type B3 thymomas by immunohistochemistry (IHC). CALML5 was strongly expressed in the cytoplasm in 19 of 26 cases with thymic carcinoma, whereas positivity at the protein level was shown in two of 38 type B3 thymomas. Thus, the sensitivity (73.1%) and specificity (94.7%) of CALML5 as markers for immunohistochemical diagnosis of thymic carcinoma were extremely high.Conclusion: We identified CALML5 as a potential marker for differentiating thymic squamous cell carcinoma from type B3 thymoma. It is assumed that future clinical use of CALML5 may improve the diagnostic accuracy of differentiating between these two diseases.

701 Background: In patients with clear cell renal cell carcinoma (ccRCC), progressive local dysfunction in the tumor immune microenvironment (TIM) has been shown to play a significant role in clinical outcomes and response to therapy. Patients with oligometastatic ccRCC may particularly benefit from localized treatment such as radiation therapy. However, immune cell dysfunction in TIM and its influence on patient outcomes in oligometastatic ccRCC has yet to be defined. To address this unmet need we conducted spatially resolved immune profiling on longitudinally collected patient samples from our ongoing prospective phase II trial (NCT03575611) of radiation therapy in oligometastatic ccRCC. This methodology offers a unique multidimensional evaluation of TIM in relation to histological architecture. Methods: Thirty-seven FFPE tumor samples from 32 patients with ccRCC (primary, 21; oligometastatic, 16) were obtained to construct a tissue microarray (TMA) and assess tumor immune activation biomarkers using the nanoString GeoMx Digital Spatial Profiler (DSP). We used the pan-cytokeratin, CD3, CD68, and SYTO13 as morphology biomarkers to profile, in each TMA core, a panel of 49 protein immune biomarkers in each of these compartments (DSP areas of illumination/AOI): tumor (panCK+), T-cell (CD3+), and macrophage (CD68+). In addition, DSP Immunofluorescence images were used to calculate cell density, and cell-to-cell distances of T cell and Macrophage. A median of 7 AOIs (range 3-9) were assessed in each tissue. Comparisons were made between primary and metastatic tissues by Mann-Whitney test with correction false discovery rate of 1%. Regression models were used to evaluate association between protein counts within each compartment and clinical outcomes. Results: In T-cells, CD127 was significantly higher in primary ccRCC tissue (p=0.00009) while FOXP3 and LAG3 were significantly increased in oligometastatic tissue (p=0.001). Macrophages in oligometastatic tissue expressed higher CD163 and ARG1 (p=0.01 and 0.02, respectively), compared to those in primary tissue. Average distances between macrophage and the nearest T cell were significantly shorter in oligometastatic tumor tissue compared to those in primary ccRCC (22.9 vs 28.8 µm, p=0.04). Higher PD-1 (R 2 =0.26) with lower CD127 (R 2 =0.22) and CD27 (R 2 =0.21) expression in T cells in primary tumor predicted earlier time to first diagnosis of metastases after nephrectomy (all p<0.05). Conclusions: Spatially resolved immune profiling revealed that the TIM in metastatic tumor harbored more T cells exhibiting markers of suppression with closer spatial interaction to macrophages compared with primary tumors. Furthermore, TIM with T cell exhaustion in primary tumors predicted earlier diagnosis of metastasis after nephrectomy.

Supplementary Figures S1-S7 and figure legends

OBJECTIVES Programmed death ligand 1 and 2 (PD-L1 and PD-L2) bind programmed death 1 (PD-1). PD-L1 is an established predictive biomarker of response to immunotherapies targeting PD-1 and PD-L1 in lung adenocarcinoma (LUAD). However, the clinical relevance of PD-L2 expression in patients with LUAD remains unclear; we aimed to examine this aspect using LUAD specimens. MATERIALS AND METHODS PD-L2 expression status was immunohistochemically evaluated in 980 surgically resected LUAD specimens. PD-L2 expression status was classified based on the tumor proportion score (TPS) as negative (<1%), weakly positive (1-49%), or strongly positive (≥50%). Correlations between PD-L2 and PD-L1 expression status, clinicopathological features, driver oncogene alterations (EGFR, KRAS, ALK, ROS1, and RET), and prognosis were also analyzed. RESULTS PD-L2 expression was negative in 720 (73%) of 980 LUADs, weakly positive in 190 (19%), and strongly positive in 70 (7%). The concordance rate between PD-L1 and PD-L2 expression was 60%. Male sex, smokers, tumors > 3 cm in size, high-grade tumors, tumors without EGFR mutation or ALK fusion, and tumors with KRAS mutation were more common in patients with PD-L2-positive tumors (TPS ≥ 1%) than in patients with PD-L2-negative tumors (TPS < 1%). PD-L2 expression was not associated with overall survival (OS) or relapse-free survival (RFS). However, positive PD-L2 expression tended to be associated with better OS/RFS in PD-L1-positive patients and worse OS/RFS in PD-L1-negative patients. CONCLUSIONS PD-L2-positive LUADs showed biologically aggressive characteristics. PD-L2 expression status was not associated with survival outcomes, but tended to show contrasting prognostic impacts based on PD-L1 expression status.