SETD2 Mutations Research Articles

SETD2 mutations do not contribute to clonal fitness in response to chemotherapy in childhood B cell acute lymphoblastic leukemia

Mutations in genes encoding epigenetic regulators are commonly observed at relapse in B cell acute lymphoblastic leukemia (B-ALL). Loss-of-function mutations in SETD2, an H3K36 methyltransferase, have been observed in B-ALL and other cancers. Previous studies on mutated SETD2 in solid tumors and acute myelogenous leukemia support a role in promoting resistance to DNA damaging agents. We did not observe chemoresistance, an impaired DNA damage response, nor increased mutation frequency in response to thiopurines using CRISPR-mediated knockout in wild-type B-ALL cell lines. Likewise, restoration of SETD2 in cell lines with hemizygous mutations did not increase sensitivity. SETD2 mutations affected the chromatin landscape and transcriptional output that was unique to each cell line. Collectively our data does not support a role for SETD2 mutations in driving clonal evolution and relapse in B-ALL, which is consistent with the lack of enrichment of SETD2 mutations at relapse in most studies.

CT-based radiomics model for the prediction of genomic alterations in renal cell carcinoma (RCC)

Abstract Background Radiogenomics is an emerging tool with applications in screening for molecular biomarkers in diagnostic and prognostic assessment through the extraction of quantitative data from medical images (Shui et al., Front Oncol 2021). In this study, we aim to develop machine learning (ML) models to predict the most common genomic alterations present in our subset of renal cell carcinoma patients (pts). Methods Retrospectively, pts with tissue genomic testing from CT-guided biopsy samples were identified. Genomic testing was done via GEM ExTra assay, a CAP-accredited, CLIA-certified test encompassing tumor whole exome sequencing and whole transcriptome sequencing (TGen; Phoenix, AZ). Biopsy sample collection sites were identified from pre-biopsy contrast CT images, and the lesions were segmented with ITK-SNAP software, from which 510 radiomic features were extracted with Pyradiomics. The Least Absolute Shrinkage and Selection Operator (LASSO) regression was used to select the most relevant features. Logistic regression (LR) and support vector machine (SVM) classifiers were built for the prediction of PBRM1, VHL, and SETD2 gene alterations. Multiple metrics were used to evaluate the predictive performance via leave-one-out cross-validation, including the area under the receiver operating characteristic (AUROC) and the area under the precision-recall curve (AUPRC). Feature importance was evaluated with Shapley additive explanations (SHAP) method. Results A total of 14 RCC pts (10:4 M:F) with genomic testing from CT-guided biopsies were identified. The majority of pts were White (85.7%) and had clear cell histology (71.4%). The most common locations for the CT-guided biopsy were lung (18.2%), soft tissue (13.6%), kidney (9.1%), and bone (9.1%). The most common alterations were seen in PBRM1 (50%), VHL (43.9%), and SETD2 (35.7%) genes. The PBRM1 gene was predicted with the highest AUROC (0.84) and AUPRC (0.88) with the SVM classifier, followed by the SETD2 gene (AUROC=0.78 and AUPRC=0.66) with LR classifier and VHL gene (AUROC=0.56 and AUPRC=0.65) with SVM classifier. Notably, all three models showed good sensitivity in classifying gene mutation status (PBRM1: 0.86; VHL: 0.88; SETD2: 0.89). Among all radiomic features, first-order features, Gray Level Size Zone Matrix features, and Gray Level Dependence Matrix features were found to be the most important features for predictions. Conclusions Using a CT-based radiomics analysis of the biopsy area, we showed that SVM and LR prediction models could predict PBRM1, VHL, and SETD2 mutations with high accuracy. These models may assist in identifying potentially actionable alterations and yield ease in treatment selection for RCC. Further extensive studies are warranted to validate our findings and improve our model. CDMRP DOD Funding: no

Abstract B017: SETD2-loss mediated H3K36me3 causes disruptions in topologically associated domains (TADs) to promote oncogenic expression in clear cell renal cell carcinoma

Abstract SETD2 is an RNA Polymerase II (Pol II)-associated histone methyltransferase that catalyzes the co-transcriptional methylation of H3K36me2 to create H3K36me3 marks in gene bodies of actively transcribed genes. SETD2 is one of the most frequently mutated chromatin-modifying genes across different cancer types, with the highest prevalence (13%) in clear cell renal cell carcinoma (ccRCC). ccRCC has been recognized as a VHL loss-driven disease in which VHL is mutated or silenced in up to 80-90% of tumors, and the heterozygous loss of chromosome 3p where VHL resides is identified in >90% of ccRCC. The other three top mutated genes in ccRCC are also located at chromosome 3p, including PBRM1 (~40%), SETD2 (8-13%), and BAP1 (6-10%). Notably, deletion of Vhl alone is insufficient to induce kidney cancer in mice, whereas combined deletion of Vhl and Pbrm1 results in multifocal, transplantable non-metastatic clear cell kidney cancer. Human ccRCC genomics and cancer evolution studies revealed that SETD2 mutations often co-occur with VHL and PBRM1 mutations and SETD2 mutations associate with tumor progression. Previously, using VHLMT PBRM1MT SETD2MT metastatic ccRCC patient-derived cell line and xenograft models we found that H3K36me3 restoration reduced distant metastases of ccRCC in mice, establishing SEDT2 as a metastatic tumor suppressor in kidney cancer. An integrated ATAC-seq, ChIP-seq, and transcriptome analysis uncovered a tumor suppressor model of SETD2 in which loss of SETD2-mediated H3K36me3 creates an epigenetic landscape consisting of widespread open chromatin that amplifies oncogenic transcriptional output through aberrant activation of enhancers. Here, we hypothesize that loss of SETD2-mediated H3K36me3 alters 3D chromosome architecture to increase chromatin accessibility and enhancer-promoter interactions, which in turn activates oncogenic transcription, including pro-metastatic genes such as MMP1, HGF, MYC, and ETV1. Here, we performed chromosome conformation capture (Hi-C) to interrogate 3D chromatin architecture in kidney cancer with defined mutations in VHL, PBRM1, or SETD2 for the first time. Our preliminary global analysis comparing SETD2 mutant and wildtype HiC samples showed disruptions in Topologically Associated Domains (TADs) and increased long-range interaction with loss of SETD2 function. Further understanding of how dysregulated epigenetics conferred by SETD2 loss promotes tumor initiation and metastasis will allow the discovery of novel therapeutic vulnerabilities associated with the loss of SETD2. Citation Format: Amrita M. Nargund, Rui Yang. SETD2-loss mediated H3K36me3 causes disruptions in topologically associated domains (TADs) to promote oncogenic expression in clear cell renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B017.

Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition

Background: The risk of recurrence after nephrectomy for primary clear cell renal cell carcinoma (ccRCC) is estimated in daily practice solely based on clinical criteria. The aim of this study was to assess the prognostic relevance of common somatic mutations with respect to tumor aggressiveness and outcomes of ccRCC patients after definitive treatment. Methods: Primary tumors from 37 patients with ccRCC who underwent radical nephrectomy were analyzed for presence of somatic mutations using a 15-gene targeted next-generation sequencing (NGS) panel. Associations to histopathologic characteristics and outcomes were investigated in the study cohort (n=37) and validated in The Cancer Genome Atlas (TCGA) ccRCC cohort (n=451). Results: VHL was the most frequently mutated gene (51%), followed by PBRM1 (27%), BAP1 (13%), SETD2 (13%), KDM5C (5%), ATM (5%), MTOR (5%), and PTEN (3%). One-third of patients did not have any somatic mutations within the 15-gene panel. The vast majority of tumors harboring no mutations at all or VHL-only mutations (51%) were more frequently of smaller size (pT1-2) and earlier stage (I/II), whereas presence of any other gene mutations in various combinations with or without VHL was enriched in larger (pT3) and higher stage tumors (III) (p=0.02). No recurrences were noted in patients with unmutated tumors or VHL-only mutations as opposed to three relapses in patients with non-VHL somatic mutations (p=0.06). Presence of somatic mutations in PBRM1, BAP1, SETD2, KDM5C, ATM, MTOR, or PTEN genes in 451 TCGA ccRCC patients was associated with a significantly shorter disease-free survival (DFS) compared to those with unaltered tumors (q=0.01). Conclusions: Preliminary findings from this ongoing study support the prognostic value of non-VHL mutations including PBRM1, BAP1, SETD2, KDM5C, ATM, MTOR, and PTEN in primary ccRCC tumors as surrogates of earlier recurrence and potential selection for adjuvant immune checkpoint inhibition.

SETD2 variation correlates with tumor mutational burden and MSI along with improved response to immunotherapy

BackgroundSETD2 protects against genomic instability via maintenance of homologous recombination repair (HRR) and mismatch repair (MMR) in neoplastic cells. However, it remains unclear whether SETD2 dysfunction is a complementary or independent factor to microsatellite instability-high (MSI-H) and tumor mutational burden-high (TMB-H) for immunocheckpoint inhibitor (ICI) treatment, and little is known regarding whether this type of dysfunction acts differently in various types of cancer.MethodsThis cohort study used multidimensional genomic data of 6726 sequencing samples from our cooperative and non-public GenePlus institute from April 1 through April 10, 2020. MSIsensor score, HRD score, RNAseq, mutational data, and corresponding clinical data were obtained from the TCGA and MSKCC cohort for seven solid tumor types.ResultsA total of 1021 genes underwent target panel sequencing reveal that SETD2 mutations were associated with a higher TMB. SETD2 deleterious mutation dysfunction affected ICI treatment prognosis independently of TMB-H (p < 0.01) and had a lower death hazard than TMB-H in pancancer patients (0.511 vs 0.757). Significantly higher MSI and lower homologous recombination deficiency were observed in the SETD2 deleterious mutation group. Improved survival rate was found in the MSKCC-IO cohort (P < 0.0001) and was further confirmed in our Chinese cohort.ConclusionWe found that SETD2 dysfunction affects ICI treatment prognosis independently of TMB-H and has a lower death hazard than TMB-H in pancancer patients. Therefore, SETD2 has the potential to serve as a candidate biomarker for ICI treatment. Additionally, SETD2 should be considered when dMMR is detected by immunohistochemistry.

SETD2 loss in renal epithelial cells drives epithelial-to-mesenchymal transition in a TGF-β-independent manner.

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.

Genomic insights into the genetic basis of eagle-beak jaw, large head, and long tail in the big-headed turtle.

The big-headed turtle (Platysternon megacephalum) is an endemic chelonian species in Asia. Unlike most other turtles in the world, P. megacephalum is characterized with eagle-beak jaw, large head, and long tail. Although these unique characteristics are well recognized, the underlying genetic basis remains largely elusive. Here, we performed comparative genomic analysis between P. megacephalum and other representative species, aiming to reveal the genetic basis of the unique morphological features. Our results revealed that the eagle-beak jaw is most likely enabled by combined effects of expansion of SFRP5, extraction of FGF11, and mutation of both ZFYVE16 and PAX6. Large head is supported by mutations of SETD2 and FGRF2 and copy number variations of six head circumference modulation-related genes (TGFBR2, Twist2, Rdh10, Gas1, Chst11, and SNAP25). The long tail is probably involved in a genetic network comprising Gdf11, Lin 28, and HoxC12, two of which showed a consistent expression pattern with a model organism (mice). These findings suggest that expansion, extraction, and mutation of those genes may have profound effects on unique phenotypes of P. megacephalum.

Genomic profiles of renal cell carcinoma in a small Chinese cohort.

Our aim was to describe the molecular characteristics of Renal Cell Carcinoma (RCC) and develop a small panel of RCC-associated genes from a large panel of cancer-related genes. Clinical data of 55 patients with RCC diagnosed in four hospitals from September 2021 to August 2022 were collected. Among the 55 patients, 38 were diagnosed with clear cell RCC (ccRCC), and the other 17 were diagnosed with non-clear cell RCC (nccRCC), including 10 cases of papillary renal cell carcinoma, 2 cases of hereditary leiomyomatosis and RCC syndrome (HLRCC), 1 eosinophilic papillary RCC, 1 tubular cystic carcinoma, 1 TFE3 gene fusion RCC, and 2 RCC with sarcomatoid differentiation. For each patient, 1123 cancer-related genes and 79 RCC-associated genes were analyzed. The most frequent mutations in a large panel of 1123 cancer-related genes in the overall population of RCC patients were VHL (51%), PBRM1 (35%), BAP1 (16%), KMT2D (15%), PTPRD (15%), and SETD2 (15%). For ccRCC patients, mutations in VHL, PBRM1, BAP1, and SERD2 can reach 74%, 50%, 24%, and 18%, respectively, while for nccRCC patients, the most frequent mutation was FH (29%), MLH3 (24%), ARID1A (18%), KMT2D (18%), and CREBBP (18%). The germline mutation rate in all 55 patients reached 12.7% (five with FH, one with ATM, and one with RAD50). The small panel containing only 79 RCC-associated genes demonstrated that mutations of VHL, PBRM1, BAP1, and SETD2 in ccRCC patients were 74%, 50%, 24%, and 18% respectively, while for the nccRCC cohort, the most frequent mutations were FH (29%), ARID1A (18%), ATM (12%), MSH6 (12%), BRAF (12%), and KRAS (12%). For ccRCC patients, the spectrum of mutations by large and small panels was almost the same, while for nccRCC patients, the mutation spectrum showed some differences. Even though the most frequent mutations (FH and ARID1A) in nccRCC were both demonstrated by large panels and small panels, other less frequent mutations such as MLH3, KMT2D, and CREBBP were not shown by the small panel. Our study revealed that nccRCC is more heterogeneous than ccRCC. For nccRCC patients, the small panel shows a more clear profile of genetic characteristics by replacing MLH3, KMT2D, and CREBBP with ATM, MSH6, BRAF, and KRAS, which may help predict prognosis and make clinical decisions.

Leveraging longitudinal clinical annotations and panel sequencing to characterize dynamics of organotropism in metastatic melanoma.

9526 Background: Although metastasis plays a major role in the mortality and morbidity of melanoma, patterns of metastasis and drivers of organotropism have not been well characterized clinically. Although previous efforts have been made to examine genomic drivers of organotropism, a longitudinal examination of metastasis dynamics has not been well characterized clinically. Methods: To address these questions, we leveraged a clinico-genomic dataset of patients with metastatic melanoma (Kehl et al. 2021). From this dataset, we assembled a cohort consisting of 26,143 artificial intelligence-annotated imaging reports from 879 patients with metastatic melanoma with an average of 2 years of followup after initial metastatic diagnosis. Annotated metastasis sites included brain, bone, adrenal, liver, lymph node, lung, and mesentery. All patients were evaluated at the Dana-Farber Cancer Institute, and each patient has at least one tumor biopsy sequenced with OncoPanel, a next-generation sequencing panel that identifies mutations in 447 cancer-related genes. Lifetime incidence of metastatic sites was defined as whether a patient ever had a report annotated with that site. Time-to-event analyses on metastatic sites used the acquisition of a metastatic site as the event of interest. For each patient index dates were defined as the date of the first scan with cancer present. We used cumulative incidence models to examine the incidence of each site in our cohort, and both cause-specific hazard models and Fine-Gray modeling to account for competing events and determine the effect of genomic covariates. Results: For lifetime metastasis status, the overall incidence varied by site, with LN being the most common, occurring in ~75% of patients, and mesentery the rarest, occurring in ~12% of patients. For lifetime metastasis status, patients with adrenal metastases were positively enriched for mutations in SETD2 (P &lt; 0.005). Among the significant genes for all sites were many genes encoding epigenetic modifiers. In the time-to-event setting, the order of the cumulative incidence functions for each site matched the lifetime frequency of sites. Through time-to-event analysis we found genes whose mutant status were significantly associated with a change in risk and/or rate of certain metastatic sites. Among other results, we found that having a mutation in SETD2 is significantly positively associated with an increase rate and risk of adrenal metastases (cause-specific HR 2.4, P &lt; 0.005, Fine-Gray HR 2.6, P &lt; 0.005), and NRAS mutations were significantly associated with lower rate and risk of liver metastasis (cause-specific HR 0.59 P &lt; 0.05, Fine-Gray HR 0.58 P &lt; 0.01). Conclusions: By using time-to-event analysis with longitudinal metastatic annotations, we identify potential drivers of organotropism in melanoma patients.

Clinical and genomic characterisation of adenoid cystic carcinoma of the head and neck, lung, and breast.

6094 Background: Adenoid cystic carcinoma (ACC) arises most commonly within the major salivary glands and other exocrine glands. ACC is typically a slow growing disease with high rates of recurrence many years after diagnosis. To inform post-operative surveillance and to provide comparator data for non-randomised studies, we sought to perform clinical and genomic characterisation of a cohort of 450 ACC patients, comparing outcomes between salivary ACC and other primary sites. Methods: 450 patients with ACC underwent clinical review at 4 UK NHS institutions. Electronic records were reviewed to extract clinical and demographic data. In 225 patients, DNA-based next-generation sequencing was performed to identify alterations in the most frequently altered genes in ACC (NOTCH1, ARID1A, KDM6A, SETD2, or TP53). Kaplan-Meier survival analyses calculated the time to first confirmed recurrence (TTFR) and overall survival from diagnosis (OS-diag) and from first confirmed recurrence (OS-rec). Results: Of 450 ACC patients, the primary site was most frequently the major salivary gland (44.7%; 201/450) comprising parotid (86), submandibular (96), sublingual (9) and salivary NOS (12). Other primary sites were sinonasal (99/450; 22.0%), upper aerodigestive (69/450; 15.3%), tracheobronchial (46/450; 10.2%), breast (16/450; 3.6%), skin (10/450; 2.2%), lacrimal (7/450; 1.6%), and other (2/450; 0.4%). Complete survival data were available in 440 patients. Recurrent/metastatic disease was confirmed in 69.8% (314/450). For these patients, the median TTFR was 4.5 yrs and for those with recurrence, the median OS from recurrence (OS-rec) was 4.8 yrs. The median TTFR in major salivary ACC was 5.8 yrs (95% CI 4.3 – 7.0). In comparison, the shortest median TTFR was seen in tracheobronchial (2.9 yrs, 95% CI 1.6 – 6.8; p = 0.019) and sinonasal ACC (3.8 yrs, 95% CI 2.9 – 5.0; p = 0.004). Breast ACC showed a differing survival pattern compared with major salivary ACC. Although the OS-diag in these two groups was not significantly different (9.75 yrs and 11.3 yrs respectively, p = ns), breast ACC showed a significantly increased TTFR (9.8 versus 4.4 yrs, p = 0.067), and a significantly reduced OS-rec (0.75 versus 4.8 yrs, p = &lt; 0.001). KDM6A mutation was seen more frequently in primary sites outside the major salivary glands (p = 0.005). However, there was no relationship between primary tumour site and mutations in NOTCH1, ARID1A, SETD2 or TP53. Conclusions: In this real-world multi-centre UK study, tracheobronchial and sinonasal ACC have significantly shorter TTFR than major salivary ACC. This study suggests that breast ACC has a similar OS from diagnosis to major salivary ACC, but has an increased TTFR and shorter OS following recurrence. This may have implications for follow-up protocols after primary treatment of breast ACC, and initiation of systemic therapy following recurrence.

A phase 2 study of the WEE1 inhibitor AZD1775 in SETD2-deficient advanced solid tumor malignancies.

3104 Background: AZD1775 is an inhibitor of the WEE1 kinase, leading to loss of genome integrity. Pathogenic SETD2 mutations lead to genome-wide loss of histone H3K36 trimethylation (H3K36me3) and hypersensitivity to WEE1 inhibition with a synthetic lethal effect. AZD1775 was studied in 2 cohorts of patients: A) Patients (pts) with locally advanced/metastatic solid tumor malignancy other than clear cell renal cell carcinoma (ccRCC) and B) pts with locally advanced/metastatic ccRCC. Methods: Eligible pts had a locally advanced/metastatic cancer with progression on ≥ 1 prior systemic therapy (including prior tyrosine kinase inhibitor or checkpoint inhibitor for cohort B), and a pathogenic SETD2 mutation by CLIA-certified next generation sequencing panel. Pts received AZD1775 at a starting dose of 300 mg PO daily on days 1-5, 8-12 of each 21-day cycle. The primary objective was to determine the objective response rate (ORR) by RECIST 1.1 criteria in each cohort. Planned sample size was 9 pts in each cohort in stage 1 of a Simon two-stage design. Secondary objectives were to determine the clinical benefit rate (CBR) (defined as partial response or stable disease [SD] &gt; 4 months), duration of response (DOR), and safety. Correlative assays include the evaluation of H3K36me3 mark by immunohistochemistry (IHC) in archival tumor tissue. Results: Between 05/2019 and 10/2021, 18 pts whose tumor harbored a pathogenic mutation in SETD2 were enrolled (9/cohort). The median age was 60 years (range 45 – 74), ECOG PS 0 in 8 (44.4%). One patient remains on treatment; 17 have discontinued (disease progression, n = 9; adverse event, n = 4; patient withdrawal, n = 2; patient death, n = 1). The median duration of treatment was 1.89 months (range 0.75 – 13.5). There were no confirmed objective responses and therefore enrollment was halted following accrual to stage 1. Tumor regression of any magnitude was observed in 5/17 (29%) evaluable pts. Stable disease was the best overall response in 10/17 (59%) evaluable pts, including 3 pts with SD for &gt; 4 months. One patient with RCC remains on treatment with SD for 14+ months duration. The most common adverse events of any grade were nausea (59%), anemia (41%), diarrhea (41%), and neutropenia (41%). Twelve pts (67%) experienced a Grade ≥ 3 adverse event. Evaluation of H3K36me3 using IHC as an exploratory biomarker is underway. Conclusions: AZD1775 given as monotherapy failed to achieve an objective response in SETD2-altered RCC and other solid tumor malignancies. However, a subset of pts derived clinical benefit as manifested by tumor regressions and/or durable stable disease. Evaluation of AZD1775 in combination with other DNA damage-inducing agents that cause replication stress may be warranted, potentially with the integration of the H3K36me3 mark as a biomarker for patient selection. Clinical trial information: NCT03284385 .

Giant juvenile fibroadenomas with and without prominentpseudoangiomatous stromal hyperplasia (PASH)-like change: clinicopathological and molecular characteristics.

Juvenile fibroadenomas (JFA) are biphasic fibroepithelial lesions (FEL) usually occurring in adolescent female patients. Giant (G) JFA, like other FEL, may exhibit prominent pseudoangiomatous stromal hyperplasia (PASH)-like change. We sought to determine clinicopathological and molecular characteristics of GJFA with and without PASH. Archives were searched for cases of GJFA (1985-2020). All were stained for androgen receptor (AR), beta-catenin, CD34 and progesterone receptor (PR). Cases were sequenced using a custom 16-gene panel - MED12 (exons 1 and 2), TERT promoter (-124C>T and -146Ctable>T), SETD2, KMT2D, RARA (exons 5-9), FLNA, NF1, PIK3CA (exons 10, 11 and 21), EGFR, RB1, BCOR, TP53, PTEN, ERBB4, IGF1R and MAP3K1. Twenty-seven GJFA from 21 female patients aged 10.1-25.2 years were identified. Size ranged from 5.2 to 21 cm. Two patients had multiple, bilateral and later recurrent GJFA. Thirteen (48%) cases showed prominent PASH-like stroma. All were positive for stromal CD34, negative for AR and beta-catenin and one case showed focal PR expression. Sequencing showed MAP3K1 and SETD2 mutations in 17 samples, with KMT2D, TP53 and BCOR aberrations in 10 (45%), 10 (45%) and seven (32%) cases, respectively. Tumours with a PASH-like pattern had higher prevalence of SETD2 (P = 0.004) and TP53 (P = 0.029) mutations, while those without PASH had more RB1 mutations (P = 0.043). MED12 mutation was identified in one case. TERT promoter mutation was observed in four (18%), including two recurrences. Gene mutations along more advanced phases of the proposed FEL pathogenetic pathway in GJFA are unusual, and suggest a mechanism for more aggressive growth in these tumours.

Radiogenomic Associations Clear Cell Renal Cell Carcinoma: An Exploratory Study

Introduction: This study investigates how quantitative texture analysis can be used to non-invasively identify novel radiogenomic correlations with clear cell renal cell carcinoma (ccRCC) biomarkers. Methods: The Cancer Genome Atlas-Kidney Renal Clear Cell Carcinoma open-source database was used to identify 190 sets of patient genomic data that had corresponding multiphase contrast-enhanced CT images in The Cancer Imaging Archive. 2,824 radiomic features spanning fifteen texture families were extracted from CT images using a custom-built MATLAB software package. Robust radiomic features with strong inter-scanner reproducibility were selected. Random forest, AdaBoost, and elastic net machine learning (ML) algorithms evaluated the ability of the selected radiomic features to predict the presence of 12 clinically relevant molecular biomarkers identified from the literature. ML analysis was repeated with cases stratified by stage (I/II vs. III/IV) and grade (1/2 vs. 3/4). 10-fold cross validation was used to evaluate model performance. Results: Before stratification by tumor grade and stage, radiomics predicted the presence of several biomarkers with weak discrimination (AUC 0.60–0.68). Once stratified, radiomics predicted KDM5C, SETD2, PBRM1, and mTOR mutation status with acceptable to excellent predictive discrimination (AUC ranges from 0.70 to 0.86). Conclusions: Radiomic texture analysis can potentially identify a variety of clinically relevant biomarkers in patients with ccRCC and may have a prognostic implication.

New insights into the clinical characteristics of SETD2-mutated acute myeloid leukaemia.

As reported, SETD2 is recurrently mutated in acute myeloid leukaemia (AML), but knowledge about the specifics is limited. We enrolled 530 consecutive newly diagnosed AML patients in our study, and we analysed the distribution pattern and prognostic role of SETD2 mutation in AML. SETD2 mutation was found to affect 6.3% of AML patients, and it frequently co-occurred with IDH2, NRAS and CEBPA mutations. SETD2-mutated patients saw excellent therapeutic responses but failed to gain better survival time than other patients. This could be because of the high recurrence and mortality in SETD2-mutated patients who have additional mutations, such as NRAS mutation.

Abstract 936: Clinicogenomic predictors for the pattern of failure in high-grade glioma

Abstract Background: High-grade gliomas, the most common primary brain tumors, are highly lethal, and treatment options remain limited. Despite advances in genomic technologies, there are few molecular biomarkers to guide precision medicine for high-grade glioma. Here, we aimed to identify the clinicogenomic features associated with its prognosis and recurrence patterns. Materials/Methods: Our single-institution retrospective analysis included 223 patients diagnosed with high-grade gliomas who underwent next-generation sequencing targeting 82 brain tumor-relevant genes. We categorized them according to the 2021 WHO classification, and their clinicopathological statuses, treatment characteristics, survival, and genomic profiles were analyzed. Results: The most common genomic variants were TERT promoter (51%), TP53 (39%), IDH1/2 (18%), NF1 (13%), ATRX (12%), PTEN (11%), PIK3CA (9%), SETD2 (7%), and H3F3A (6%) mutation. Regarding copy number variants, amplification of EGFR (21%), PDGFRA (11%), CDK4 (9%) and loss of CDKN2A/2B (55%), PTEN (29%), 19q (17%), NF2 (14%), and RB1 (10%) were the most common copy number aberrations. At a median follow-up of 18 months (range, 2-54 months), 174 patients (78%) had progression or recurrences. A total of 15.2% of our patients were re-classified under the 2021 WHO classification, and there was a clear separation of overall survival(OS) and progression-free survival(PFS) curves between grade 3 and 4 groups using the revised grading system. On multivariate cox regression analysis, MYCN amplification (HR 6.08, p = 0.002) and SETD2 mutation (HR 0.19, p =0.06) were independent predictors of OS and PFS. Conclusion: The assessment of genomic characteristics in conjunction with the pattern of failure for high-grade glioma aids in identifying patients who are likely to benefit from personalized medicine. The 2021 WHO grading system explains survival differences between grade 3 and 4 tumors better than the 2016 WHO classification dose. We identified SETD2 mutation and MYCN as prognostic biomarkers with potential therapeutic implications in patients with high-grade glioma. Citation Format: Joo Ho Lee, Byung-Hee Kang, Seungbok Lee. Clinicogenomic predictors for the pattern of failure in high-grade glioma [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 936.

Abstract 1226: SETD2 loss in renal epithelial cells induces epithelial to mesenchymal transition in a TGFβ-independent manner

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.

Abstract 2599: Targeting SETD2-inactivated cells with STF-62247 leads to cell cycle arrest

Abstract The SETD2 gene encodes a histone methyltransferase responsible for the trimethylation of histone 3 on lysine 36 (H3K36me3). While this canonic function plays a role in the regulation of cellular processes such as transcription, alternative splicing, and DNA damage reparation, SETD2 is also important for the methylation of other proteins like EZH2, STAT1, and the cytoskeletal proteins actin and α-tubulin. Mutations in SETD2 are found in a broad range of cancers, including Clear Cell Renal Cell Carcinoma (ccRCC). Loss of chromosome 3p followed by the inactivation of the tumor suppressor gene von Hippel-Lindau (VHL) is a focal characteristic of ccRCC, however, additional mutations are common on other genes also found on chromosome 3p, including SETD2. Our previous studies demonstrated the possibility of targeting VHL-deficient cells by blocking the late stages of autophagy with the small molecule STF-62247. We recently identified PIKfyve, a phosphoinositide kinase important for the biosynthesis of phosphatidylinositol 3,5-biphosphate (PI(3,5)P2), as STF-62247’s target. In the mindset of personalized medicine, we hypothesize that other mutations on genes like SETD2 could influence cell response to STF-62247 and other PIKfyve inhibitors such as apilimod. The objectives of this project are to 1) evaluate whether SETD2 expression influences the response to STF-62247 and PIKfyve inhibitors in ccRCC and 2) investigate the mechanism by which STF-62247 is selectively targeting SETD2-inactivated cells. Molecular biology approaches such as CRISPR/Cas9 and the Gateway cloning system were used to repress or reintroduce the gene coding for SETD2 in ccRCC cells like 786-0 and RCC-ER. Western blots were used for the validation of our models. Cell response to STF-62247 and other agents was measured by clonogenic assays, cell counts, and XTT viability assay. Flow cytometry, in addition to western blots, was used to study the cell cycle. Our results indicate that repression of SETD2 in 786-0, especially in 786-0 where VHL was reintroduced (786-0/VHL), sensitized cells to STF-62247 and apilimod. When treated with STF-62247, sensitive cells stopped proliferating and the reintroduction of a truncated version of SETD2 partially rescued them. In RCC-ER, a SETD2-deficient cell line, the reintroduction of SETD2 or VHL increased the viability and proliferation of the cells when treated with STF-62447. That resistance was increased when both genes were reintroduced together. Flow cytometry results showed an increase in S-phase in SETD2-inactivated 786-0/VHL cells treated with STF-62247. Likewise, decreased expression of some proteins related to DNA replication and mitosis, such as CDT1 and Cyclin B1, also indicate alterations in the cell cycle. Altogether, our results put forward the potential of using PIKfyve inhibitors as a new therapeutic approach to treat cancer patients with inactivated SETD2. Citation Format: Mathieu Johnson, Sandra Turcotte. Targeting SETD2-inactivated cells with STF-62247 leads to cell cycle arrest [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 2599.

Abstract 5273: The E3 ubiquitin ligase SIAH2 promotes clear cell renal cell carcinoma epithelial-mesenchymal transition via regulation of SETD2 stability

Abstract Introduction &amp; Objective: SETD2, a major histone H3K36 trimethyltransferase, has been shown to play an important role in multiple biological processes. Mutations in SETD2 have been evidenced in several types of cancer, including clear cell renal cell carcinoma (ccRCC). Previous studies reported the potential involvement of SETD2 in driving ccRCC tumorigenesis. However, the mechanism by which SETD2 causes cell metastasis remains poorly understood. Methods: Genome-wide CRISPR-Cas9-based screens and immunoprecipitation-mass spectrometry were used to identify candidates for regulating SETD2 stability. Immunohistochemistry staining of the ccRCC tissue array and bioinformatics analysis of TCGA database were used to investigate the clinical relevance of SETD2 in ccRCC. The gene expression was detected by qRT-PCR, and the protein level was detected by western blotting. Wound healing and transwell migration assay were used to explore the cell migration ability. SIAH2 was deleted from ccRCC cell lines using CRISPR/Cas9-mediated gene editing. Furthermore, we used BALB/c-nude mice to construct xenograft mouse models and pulmonary metastasis models. Moreover, RNA-seq, H3K36me3 CUT&amp;Tag and RNA N6-methyladenosine-IP were performed to uncover the mechanism. Results: SETD2 mutant or low expression was correlated with poor prognosis in patients with ccRCC based on TCGA data and tissue array. Through the CRISPR/Cas9 screening system and immunoprecipitation-mass spectrometry, we identified the E3 ubiquitin ligase SIAH2 that regulates the stability of SETD2. Then, we verified that the protein stability of SETD2 was downregulated by SIAH2 in a dose-dependent manner and further confirmed that the SIAH2 promotes SETD2 degradation in a proteasome-dependent pathway. In addition, we constructed a dominant negative mutant of SIAH2 and proved the degradation dependent on its enzymatic activity. Moreover, we showed that SIAH2 knockout increased the protein level of SETD2. In SETD2 stable knockdown ccRCC cell lines, loss of SETD2 enhanced the capability of cell migration, while knockdown of SIAH2 resulted in the rescue of SETD2 deficient effect. Moreover, SETD2 ablation significantly decreases H3K36me3 peaks of E-cadherin, further reducing the m6A signal and stability of E-cadherin mRNA. Conclusion: In this study, we firstly revealed the mechanism that E3 ubiquitin ligase SIAH2 interacted with SETD2 and promoted the ubiquitination degradation of SETD2. Moreover, SETD2 ablation in ccRCC cells enhanced EMT through impaired epigenetic regulation of E-cadherin, suggesting SIAH2/SETD2/E-cadherin axis as a potential therapeutic target for ccRCC. Citation Format: Mengxue Yu, Lingao Ju, Gang Wang. The E3 ubiquitin ligase SIAH2 promotes clear cell renal cell carcinoma epithelial-mesenchymal transition via regulation of SETD2 stability. [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 5273.

MP41-14 PROGNOSTIC IMPACT AND GENOMIC BACKGROUNDS OF RENAL PARENCHYMAL INFILTRATION OR MICRONODULAR SPREAD IN CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023MP41-14 PROGNOSTIC IMPACT AND GENOMIC BACKGROUNDS OF RENAL PARENCHYMAL INFILTRATION OR MICRONODULAR SPREAD IN CLEAR CELL RENAL CELL CARCINOMA Hajime Tanaka, Yuki Fukawa, Kouhei Yamamoto, Kousuke Tanimoto, Akira Takemoto, Hisashi Hasumi, Akihiro Hirakawa, Yudai Ishikawa, Shohei Fukuda, Yuma Waseda, Soichiro Yoshida, Minato Yokoyama, Steven C. Campbell, and Yasuhisa Fujii Hajime TanakaHajime Tanaka More articles by this author , Yuki FukawaYuki Fukawa More articles by this author , Kouhei YamamotoKouhei Yamamoto More articles by this author , Kousuke TanimotoKousuke Tanimoto More articles by this author , Akira TakemotoAkira Takemoto More articles by this author , Hisashi HasumiHisashi Hasumi More articles by this author , Akihiro HirakawaAkihiro Hirakawa More articles by this author , Yudai IshikawaYudai Ishikawa More articles by this author , Shohei FukudaShohei Fukuda More articles by this author , Yuma WasedaYuma Waseda More articles by this author , Soichiro YoshidaSoichiro Yoshida More articles by this author , Minato YokoyamaMinato Yokoyama More articles by this author , Steven C. CampbellSteven C. Campbell More articles by this author , and Yasuhisa FujiiYasuhisa Fujii More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003279.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The efficacy of adjuvant immune checkpoint inhibition has been reported recently for locally advanced clear cell renal carcinoma (ccRCC); however, further exploration is required to define optimal candidates for this strategy, who are at high risk for recurrence. In this study, we defined renal parenchymal infiltration or micronodular spread (RPI/MNS) in ccRCC and investigated its prognostic impact and genomic backgrounds. METHODS: We retrospectively analyzed 333 M0 ccRCC patients treated with radical nephrectomy (approval # M2019-172 and M2021-272). Two pathologists evaluated the whole tissue sections of surgical specimens and performed the focused analysis on the tumor interface with normal renal parenchyma. Recurrence-free survival (RFS) was analyzed as an endpoint. Genomic DNA was extracted from tumor tissue in eligible cases (n=51), and variants were screened using a panel of 440 cancer-related genes. RESULTS: Overall, ≥pT3a, WHO/ISUP grade 3-4, and microvascular invasion were observed in 107 (32%), 138 (41%), and 199 (60%) patients, respectively. Histological assessment revealed the variations of the tumor interface with normal renal parenchyma and identified the specific pattern at high risk for recurrence. This finding was defined as RPI/MNS, in whichcancer cells infiltrated or spread into the renal parenchyma by involving normal renal tissue and/or by forming micronodules. RPI/MNS was finally identified in 40 patients (12%). During the median follow-up of 6.0 years, 67 patients (20%) developed recurrence. On multivariable analysis, ≥pT3a, grade 3-4, and RPI/MNS were independently associated with shorter RFS; RPI/MNS showed the highest hazard ratio (4.63) followed by grade 3-4 (2.03) and ≥pT3a(1.99). 2y-RFS rates were 38% and 95% in patients with and without RPI/MNS, respectively. The genomic analysis revealed significantly different patterns of representative gene mutations in ccRCC (figure). Patients with RPI/MNS (n=18) had significantly more frequent mutations in SETD2 (28% vs. 6%) and TSC1 (17% vs. 0%) in comparison to those without RPI/MNS (n=33). CONCLUSIONS: RPI/MNS had a strong impact on recurrence after surgery with the background of aggressive genomic features in ccRCC. Adjuvant therapy may be recommended for patients showing RPI/MNS. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e560 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hajime Tanaka More articles by this author Yuki Fukawa More articles by this author Kouhei Yamamoto More articles by this author Kousuke Tanimoto More articles by this author Akira Takemoto More articles by this author Hisashi Hasumi More articles by this author Akihiro Hirakawa More articles by this author Yudai Ishikawa More articles by this author Shohei Fukuda More articles by this author Yuma Waseda More articles by this author Soichiro Yoshida More articles by this author Minato Yokoyama More articles by this author Steven C. Campbell More articles by this author Yasuhisa Fujii More articles by this author Expand All Advertisement PDF downloadLoading ...

Somatic mutation landscape in a cohort of meningiomas that have undergone grade progression

BackgroundA subset of meningiomas progress in histopathological grade but drivers of progression are poorly understood. We aimed to identify somatic mutations and copy number alterations (CNAs) associated with grade progression in a unique matched tumour dataset.MethodsUtilising a prospective database, we identified 10 patients with meningiomas that had undergone grade progression and for whom matched pre- and post-progression tissue (n = 50 samples) was available for targeted next-generation sequencing.ResultsMutations in NF2 were identified in 4/10 patients, of these 94% were non-skull base tumours. In one patient, three different NF2 mutations were identified in four tumours. NF2 mutated tumours showed large-scale CNAs, with highly recurrent losses in 1p, 10, 22q, and frequent CNAs on chromosomes 2, 3 and 4. There was a correlation between grade and CNAs in two patients. Two patients with tumours without detected NF2 mutations showed a combination of loss and high gain on chromosome 17q. Mutations in SETD2, TP53, TERT promoter and NF2 were not uniform across recurrent tumours, however did not correspond with the onset of grade progression.ConclusionMeningiomas that progress in grade generally have a mutational profile already detectable in the pre-progressed tumour, suggesting an aggressive phenotype. CNA profiling shows frequent alterations in NF2 mutated tumours compared to non NF2 mutated tumours. The pattern of CNAs may be associated with grade progression in a subset of cases.