Ho, Thai H.

Abstract SETD2 is frequently mutated or deleted in clear cell renal cell carcinoma (ccRCC). Loss of SETD2 could create synthetic lethal dependencies that confer therapeutic vulnerabilities. In this study, we demonstrated that SETD2 deficiency promotes cytoplasmic mitochondrial DNA (mtDNA) leakage, leading to basal activation of cGAS-STING inflammatory signaling and increased apoptotic priming. This inflammatory state upregulated the BH3-only protein NOXA, constrained myeloid cell leukemia 1 (MCL-1) function, and enforced a synthetic lethal dependency on the antiapoptotic protein BCL-xL. Pharmacologic inhibition of BCL-xL further amplified cGAS-STING signaling in SETD2-deficient cells through sublethal mitochondrial outer membrane permeabilization, resulting in increased mtDNA release and robust NOXA induction. Elevated NOXA neutralized the compensatory MCL-1–mediated survival signaling, triggering apoptosis. In contrast, SETD2-proficient ccRCC cells exhibited minimal cGAS-STING activation and failed to induce NOXA following BCL-xL inhibition, rendering them resistant. Genetic ablation of cGAS, STING, IRF3, or NOXA rescued sensitivity to BCL-xL inhibition, confirming that mtDNA-driven innate immune signaling is required for this dependency. In vivo, BCL-xL inhibition suppressed tumor growth and prolonged survival in SETD2-deficient xenograft models. Collectively, these findings establish a mechanistic link between SETD2 loss, mtDNA-driven innate immune activation, and enforced BCL-xL dependence in ccRCC, revealing a therapeutically targetable vulnerability in SETD2-deficient tumors. Significance: SETD2 deficiency, which is associated with worse prognosis in kidney cancer, promotes mitochondrial DNA-driven cGAS-STING signaling and NOXA-mediated apoptotic priming, creating a therapeutically targetable dependency on BCL-xL in kidney cancer.

Abstract Background: In metastatic cancer, Circulating Tumor cells (CTCs) are established prognostic indictors of patients (pts) less likely to respond to new lines of systemic therapy, with poor clinical outcomes, such as shorter progression free survival (PFS) and overall survival (OS). However, CTCs are typically found in specific malignancies (breast, prostate & colon), often in <20% of pts with metastatic disease, and pts without CTCs may also rapidly progress. Recently, an inflammatory pro-tumorigenic macrophage emanating from tumor stroma (i.e. Cancer associated macrophage-like cell [CAML]) was found in >90% of metastatic cancer pts, and whose phagocytic engorgement appears to correlate with poor outcomes, independent of CTCs. As CTCs and CAMLs are isolated in conjunction from a single blood sample, and both are prognostic for outcomes, we evaluated their utilization prior to induction of new systemic therapy in 6 types of metastatic cancer to model pt risk stratification based on 2 year outcomes. Methods: A prospective 2 year blind multi-institutional study was undertaken to model CTCs and CAMLs in prognosticating outcomes prior to induction of a new line of systemic therapy (n=233) in metastatic: Breast (n=60), Prostate (n=40), Pancreas (n=25), Colon (n=28), Renal Cell Carcinoma (RCC) (n=39), and Lung (n=40). Blood was filtered by CellSieveTM filters with subtypes of CTCs & hyper-enlarged CAMLs (≥100µm) enumerated. A machine learning algorithm was trained on this initial data set to develop predictive models which could stratify pt populations by risk for likelihood of PFS & OS over 2 years, including known clinical variables. Results: CTCs were absent in 80% (n=185/233) of pts, and their absence was prognostic for better PFS (HR=1.6, p=0.046), but not OS (HR=1.3, p=0.2045). In parallel, enlarged CAMLs (≥100µm) were found in 28% (n=51/185) of pts without CTCs, and were also prognostic for worse PFS (HR=2.0, p=0.0082) and OS (HR=1.9, p=0.0412). Specifically, CTCs were found in 55% (n=33) breast, 20% (n=8) prostate, 12% (n=3) pancreas, 11% (n=3) colon, 0% (n=0) RCC, and 0% (n=0) lung pts. Enlarged CAMLs were found in 45% (n=27) breast, 20% (n=8) prostate, 44% (n=11) pancreas, 44% (n=17) colon, 18% (n=7) RCC and 20% (n=8) lung pts. Overall, models indicated that ≥1 CTC (n=47) had mPFS=3.9 & mOS=14.9, while pts with 0 CTCs and ≥100µm CAMLs (n=51) had a mPFS=6.9 & mOS=13.8, and pts with 0 CTCs and <100µm CAMLs (n=134) had a mPFS=10.7 & mOS>24 months. Conclusions: These initial models confirm that both CTCs and enlarged CAMLs are prognostic indicators of worse PFS & OS. The simultaneous quantification both CTCs and CAMLs allows for more accurate pan-cancer risk stratification in an array of cancer pt populations. Additional clinical variables incorporated into the models may allow better risk subtyping and possibly forecast optimal treatment regimens. Citation Format: Daniel L. Adams, Steven H. Lin, Massimo Cristofanilli, Carolina Reduzzi, Saranya Chumsri, Martin J. Edelman, Susan Tsai, Raymond C. Bergan, Mohammed Aldakkak, Thai H. Ho, Cha-Mei Tang. Combining circulating tumor cells and cancer associated macrophage-like cells enhances risk stratification models in pan-cancer metastatic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1068.

3053 Background: Recently, early studies have identified circulating stromal cells (CStCs), i.e. Cancer Associated Macrophage-Like cells (CAMLs), in patients (pts) with newly diagnosed invasive cancers, which are phagocytic myeloid immune cells emanating from primary tumors. However, no study has evaluated CAMLs as a pan solid tumor screening tool nor their clinical impact on pt outcomes. We analyzed the blood of untreated newly diagnosed cancer pts (n=441), or in non-malignant conditions (i.e. pancreatic cysts, n=77), or healthy controls (n=100). We found that CAMLs are prevalent (82%) in cancer pt blood, absent in healthy controls (0%) and less common in non-malignant conditions (36%), with correlations to worse clinical outcomes. Methods: Anonymized peripheral blood was taken from n=441 pts before or after confirmation of invasive malignancy [stage I (n=105), stage II (n=125), stage III (n=114), stage IV (n=97) with pathologically confirmed lung (n=84), pancreas (n=61), breast (n=73), prostate (n=65), esophageal (n=34), Renal Cell (n=29), Sarcoma (n=27), or Other (n=68). In addition, anonymized blood was taken from pts with untreated non-malignant conditions including benign breast masses (n=19), Lupus (n=11), liver Cirrhosis (n=6), rising PSA (n=24), or other benign mass (n=17); and from healthy control volunteers (n=100). CAMLs were isolated by CellSieve microfiltration, defined as enlarged polyploid cells with cytokeratin+ and/or CD45/CD14+. Pts were monitored for 0-8 years (median=2.6) for progression free survival (PFS) and overall survival (OS). Results: CAMLs were found in 82% of all cancer pts averaging 5.4 CAMLs, specifically, in 66% of Stage I, 85% Stage II, 84% Stage III, and 92% Stage IV. No CAMLs were found in any healthy controls, but were in 36% of non-malignant pts. CAML sensitivity in cancer vs healthy was 82% (CI95% 78-85%), specificity=100% (CI95% 96-100%), PPV=100% (CI95% 99-100%), NPV=55% (CI95% 50-60%). CAML sensitivity in cancer vs benign was 82% (CI95% 78-85%), specificity=64% (CI95% 32-44%), PPV=93% (CI95% 90-95%), NPV=38% (CI95% 32-44%). CAML presence was significantly associated with worse PFS (HR=2.0, 95%CI 1.4-2.9, p<0.0001) & OS (HR=2.2, 95%CI 1.4-3.3, p=0.0004), with engorged phagocytic CAMLs (>50um), highly significant for worse PFS (HR=3.3, 95%CI 2.5-4.5, p<0.0001) & OS (HR=3.0, 95%CI 2.1-4.3, p<0.0001). Further, in benign pts positive for CAMLs, 2 pts were in situ, 16 had histologies with increased cancer risk (i.e. IPMN, atypical hyperplasia), and 5 pts died within 3 years. Conclusions: CAMLs appear to be a highly sensitive blood based biomarker that can identify invasive carcinoma in all stages of cancer regardless of subtype, but are not found in healthy controls & rare in non-malignant conditions. Further, enlarged phagocytic CAMLs appeared to correlate with shorter PFS and OS prior to treatment induction.

Abstract Cancer Associated Macrophage Like cells (CAMLs) are antigen presenting circulating stromal cells found in the blood of cancer patients (pts). Recently, PD-L1 expression on CAMLs was described in breast (BC), lung (LC) and renal cell carcinomas (RCC), appearing to parallel the inflammatory PD-L1 state of the tumor microenvironment, and acting as a possible predictive biomarker for PD-L1/PD-1 immunotherapeutics (IMTs). We monitored CAML PD-L1 from 48 pts in an array of solid tumors, prior PD-L1/PD-1 IMTs induction, to evaluate CAML’s ability to predict response by progression free survival (PFS) and overall survival (OS). Three single blind multi-year prospective studies were run to test PD-L1 CAML expression against PFS & OS, prior to induction of therapy in metastatic (m) LC (n=24), mBC (n=16) or mRCC (n=8). We recruited pts with pathologically confirmed progressing disease after at least one failed prior line of therapy. Blood samples (7.5 mL) were taken prior to start of new therapeutic regimes, i.e. pembrolizumab (n=36) or nivolumab (n=12). Whole blood was filtered by CellSieve microfilters to collect CAMLs, stain for PD-L1, and then scored as a binary high or low, to evaluate PFS & OS hazard ratios (HRs) by censored univariate & multivariate analysis at 24 months. CAMLs were found in 98% of all tested samples. Overall, pts with high CAML PD-L1 expression treated with IMT had significantly better PFS (HR=2.9, 95%CI=1.4–6.1, p=0.0067) and better OS (HR=2.7, 95%CI=1.2–6.1, p=0.0317) vs patients with low CAML PD-L1. These data suggests that PD-L1 expression in circulating CAMLs appears to predict pts with increased benefit to PD-L1/PD-1 IMTs in an array of tumor subtypes, though larger studies are needed to validate these findings. Private Funding by Creatv MicroTech, Inc. and supported grants by NCI-NIH (R43CA206840), and the Defense Advanced Research Projects Agency (DARPA)(W911NF-14-C-0098).

Despite advancements in the early-stage detection and expansion of treatments for prostate cancer (PCa), patient mortality rates remain high in patients with aggressive disease and the overtreatment of indolent disease remains a major issue. Prostate-specific antigen (PSA), a standard PCa blood biomarker, is limited in its ability to differentiate disease subtypes resulting in the overtreatment of non-aggressive indolent disease. Here we assess engorged cancer-associated macrophage-like cells (CAMLs), a ≥50 µm, cancer-specific, polynucleated circulating cell type found in the blood of patients with PCa as a potential companion biomarker to PSA for patient risk stratification. We found that rising PSA is positively correlated with increasing CAML size (r = 0.307, p = 0.004) and number of CAMLs in circulation (r = 0.399, p < 0.001). Over a 2-year period, the presence of a single engorged CAML was associated with 20.9 times increased likelihood of progression (p = 0.016) in non-metastatic PCa, and 2.4 times likelihood of progression (p = 0.031) with 5.4 times likelihood of death (p < 0.001) in metastatic PCa. These preliminary data suggest that CAML cell monitoring, in combination with PSA, may aid in differentiating non-aggressive from aggressive PCas by adding biological information that complements traditional clinical biomarkers, thereby helping guide treatment strategies.

Histone‐lysine N‐methyltransferase SETD2 (SETD2), the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is often mutated in clear cell renal cell carcinoma (ccRCC). SETD2 mutation and/or loss of H3K36me3 is linked to metastasis and poor outcome in ccRCC patients. Epithelial‐to‐mesenchymal transition (EMT) is a major pathway that drives invasion and metastasis in various cancer types. Here, using novel kidney epithelial cell lines isogenic for SETD2, we discovered that SETD2 inactivation drives EMT and promotes migration, invasion, and stemness in a transforming growth factor‐beta‐independent manner. This newly identified EMT program is triggered in part through secreted factors, including cytokines and growth factors, and through transcriptional reprogramming. RNA‐seq and assay for transposase‐accessible chromatin sequencing uncovered key transcription factors upregulated upon SETD2 loss, including SOX2, POU2F2 (OCT2), and PRRX1, that could individually drive EMT and stemness phenotypes in SETD2 wild‐type (WT) cells. Public expression data from SETD2 WT/mutant ccRCC support the EMT transcriptional signatures derived from cell line models. In summary, our studies reveal that SETD2 is a key regulator of EMT phenotypes through cell‐intrinsic and cell‐extrinsic mechanisms that help explain the association between SETD2 loss and ccRCC metastasis.

12051 Background: The combination of ipilimumab and nivolumab (Ipi-Nivo) is a standard of care regimen in many malignancies. However, immune-related adverse events (irAE) are common and are of great concern, especially when treating the elderly population. This subgroup of patients may be at greater risk for serious irAEs and be less able to tolerate the potential treatment side effects. We sought to retrospectively evaluate our experience treating elderly patients ≥ 80 years with Ipi-Nivo to characterize adverse events. Methods: A retrospective chart review of the electronic medical records of all patients treated with Ipi-Nivo at the Mayo Clinic Health System was performed. Patients with any solid tumor who received their first dose of Ipi-Nivo at the age of ≥ 80 were included. Records were analyzed for baseline characteristics, cancer type, toxicity, and response to therapy. Differences between continuous and categorical variables were assessed using the students’ t-test and the chi-squared test, respectively. Overall survival (OS) was estimated using the Kaplan-Meier method. Cox proportional hazard models assessed the relative contribution of factors to survival after treatment. P values < 0.05 were considered statistically significant. All analyses were performed using SAS V.9.4. Results: Forty-six patients met inclusion criteria, 13 females and 33 males. Median age of first dose was 83 (Range: 80 - 94). Sixty-seven percent had an ECOG performance status (PS) 0-1 and 33% had an ECOG PS 2-3. Thirteen (28.3%) patients received 4 cycles of Ipi-Nivo. Cancers included: 18 (40%) melanomas, 10 (21%) mesothelioma, 9 (19.5%) lung cancers, and 9 (19.5%) other types. Thirty-one patients (67%) developed at least one irAE. Of the irAEs, 8 (17.4%) were grade 1, 20 (43.5%) were grade 2, 14 (30.4%) were grade 3, and 3 (6.5%) were grade 4. Systemic steroids were used in 29 (63%) patients. Twenty-six (56.5%) patients were hospitalized for any cause, and 12 (26.1%) were hospitalized due to an irAE. Of the 26 patients hospitalized during treatment, 19 (73%) died within 1 year of discharge; 11 (42%) died within < 1 month of discharge and 3 (12%) died within 1-3 months of discharge. Median OS from the date of first Ipi-Nivo dose was 14.6 months in the ECOG PS 0-1 and 6.5 months in the ECOG PS 2-3. ECOG PS of 0-1 was associated with a significantly decreased risk of mortality (HR [hazard ratio] 0.33, 95 % CI [confidence interval]: 0.15-0.70; p=0.0025) compared to ECOG PS of 2-3. Conclusions: This is the first analysis to evaluate the outcomes of Ipi-Nivo in patients ≥ 80 years. Approximately 60% of the patients required systemic steroids and a quarter were hospitalized with grade 3-4 irAEs. As expected, patients with ECOG PS 2-3 did significantly worse than ECOG PS 0-1. Ipi-Nivo should be used with caution in patients ≥ 80 years.

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma (RCC) and accounts for majority of the deaths from renal cancer. SETD2, the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is frequently mutated in ccRCC. Studies have shown that SETD2 mutations are often subclonal and the mutation rate dramatically increases from 25% in primary ccRCC to more than 60% in metastatic ccRCC, suggesting SETD2 loss plays an important role in renal cancer metastasis. However, mechanisms underpinning how SETD2 mutation drives invasion and metastasis in ccRCC is largely unknown. Here, by performing RNA-seq and ATAC-seq in a non-tumorigenic renal epithelial cell line isogenic for SETD2 loss-of-function, we found that SETD2 inactivation induces dramatic changes in the transcriptome and drastically enhances chromatin accessibility, which allows binding of a variety of transcription factors. We also discovered that SETD2 loss induces a TGFβ-independent EMT program, which is a major pathway linked to metastasis. Correlation of RNA-seq with ATAC-seq identifies SOX2, OCT2 and PRRX1 as key transcription factors promoting the SETD2 loss-driven EMT program. Furthermore, we found that this distinct EMT program is in part triggered by secreted factors, which suggests that SETD2 mutation promotes a metastasis-permissive tumor microenvironment through paracrine signaling. In summary, our findings reveal that SETD2 is a key regulator of EMT phenotypes that may explain the role of SETD2 mutations in ccRCC metastasis and further uncover potential therapeutic targets for SETD2-mutant ccRCC patients. Citation Format: Tianchu Wang, Ryan Wagner, Ryan Hlady, Sungho Kim, Xiaoyu Pan, Thai Ho, Keith Robertson. SETD2 loss in renal epithelial cells induces epithelial to mesenchymal transition in a TGFβ-independent manner [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 1226.

695 Background: Recent results from COSMIC 313 trial and updated findings from other contemporary trials have added to the existing body of evidence for 1L mRCC. Therefore, we present the most up-to-date results from our living, interactive systematic review (LISR) to facilitate the choice of optimal therapy in 1L mRCC setting. Methods: This LISR and network meta-analysis is maintained using a novel living evidence synthesis (LIvE) framework. The framework facilitates the identification of new or updated studies using an automated ‘living’ search which is followed by screening and extraction in a semi-automated fashion within a machine learning assisted graphical user interface. Relevant data is parsed and analyzed to compute mixed treatment comparisons and certainty of evidence is adjudicated using a rule-based algorithm. Detailed methods have been published before (PMID: 33824031). Results: As of October 1st 2022, this LISR includes 16 clinical trials (30 references). Mixed treatment comparisons showed statistically significant PFS benefit with lenvatinib-pembrolizumab (LenPem; rank 1) when compared to cabozantinib-nivolumab (CabNivo; rank 3; hazard ratio [HR]: 0.69, 95% confidence interval: 0.52-0.93), CaboNivo-ipilimumab (CaboNivoIpi; rank 4; HR: 0.62; 0.43-0.89), avelumab-axitinib (AveAxi; rank 5; HR: 0.56; 0.43-0.75), PemAxi (rank 6; HR: 0.55; 0.42-0.72), atezolizumab-bevacizumab (AteBev; rank 7; HR: 0.46; 0.35-0.59), and NivoIpi (rank 8; HR: 0.45; 0.35-0.59). No significant differences were observed between CaboNivoIpi and other combination therapies for PFS improvement. Similarly, for OS, LenPem (rank 1), PemAxi (rank 2) and CaboNivo (rank 3) were ranked as potentially more efficacious treatment options than other counterparts. However, no statistically significant difference was observed for OS benefit with mixed treatment comparisons. The odds of achieving an ORR were higher with LenPem (rank 1; odds ratio [OR]: 2.44; 1.43-4.17), and CaboNivo (rank 2; OR: 1.77; 1.04-3.03) when compared to CaboNivoIpi (rank 6). NivoIpi (rank 1), LenPem (rank 2), CaboNivoIpi (rank 3) were ranked potentially as more efficacious treatments for achieving a CR. In terms of grade 3 or higher treatment related adverse events, NivoIpi (rank 3) was ranked as potentially the safest option among other combinations while CaboNivoIpi (rank 12), LenPem (rank 11), and CaboNivo (rank 10) were more likely to increase the risk of toxicity compared to most other treatments. Conclusions: Triplet combination of CaboNivoIpi may not provide additional survival benefit compared to other immunotherapy combinations and may increase toxicity. Patient-level considerations such as treatment toxicity, and quality of life should be factored in when opting intensified 1L therapies in mRCC patients.