1p Loss Research Articles

Abstract IA025: The cell of origin is the dominant constraint in evolution of VHL related neoplasms

Abstract Cancer is an evolutionary process, and an evolutionary framework can be used to decipher cancer initiation, progression and treatment resistance. In the context of VHL disease with repeat evolution of malignancies across different tissue contexts we can examine the factors that constrain tumour evolution. In the largest analysis of VHL-related tumours to date (1321 tumours across 132 patients), we observe the second hits on the background of germline VHL mutation vary and include loss of chromosome 3p, narrow deletion of VHL locus, VHL mutation or methylation. Pheochromocytomas and paragangliomas (PPGL) and solid renal tumours show a higher frequency of 3p loss, while pancreatic neuroendocrine tumors (PNETs) and renal cysts, exhibited a higher frequency of somatic VHL mutations, without 3p loss. Tumour size and progression were contingent on the nature of the second hit, suggesting potential evolutionary "dead-ends" where the tumor initiating event does not involve 3p loss. The type of germline VHL mutation (whether a complete deletion or missense) determined the distribution of cancers and their burden, but beyond this, multiple renal tumours in individual patients did not converge onto the same trajectory. This observation suggests that following malignant transformation evolutionary contingency and chance dominate tumour evolution. Metastases from renal tumours were driven by loss of chromosomes 9p and 14q, reminiscent of sporadic renal tumours, suggesting that acquisition of metastatic competence is associated with a high degree of bottlenecking and highlighting these events as important targets for metastasis prevention. Citation Format: Scott Shepherd, Alex Coulton, Cathy Vocke, Chris Ricketts, Marston Linehan, Samra Turajlic. The cell of origin is the dominant constraint in evolution of VHL related neoplasms [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 IA025.

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.

Embryonic stem cell factor FOXD3 (Genesis) defects in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms, believed to originate from the interstitial cells of Cajal (ICC), often caused by overexpression of tyrosine kinase receptors (TKR) KIT or PDGFRA. Here, we present evidence that the embryonic stem cell factor FOXD3, first identified as 'Genesis' and involved in both gastrointestinal and neural crest cell development, is implicated in GIST pathogenesis; its involvement is investigated both in vitro and in zebrafish and a mouse model of FOXD3 deficiency. Samples from a total of 58 patients with wild-type GISTs were used for molecular analyses, including Sanger sequencing, comparative genomic hybridization, and methylation analysis. Immunohistochemistry and western blot evaluation were used to assess FOXD3 expression. Additionally, we conducted in vitro functional studies in tissue samples and in transfected cells to confirm the pathogenicity of the identified genetic variants. Germline partially inactivating FOXD3 sequence variants (p.R54H and p.Ala88_Gly91del) were found in patients with isolated GISTs. Chromosome 1p loss was the most frequent chromosomal abnormality identified in tumors. In vitro experiments demonstrate the impairment of FOXD3 in the presence of those variants. Animal studies showed disruption of the GI neural network and changes in the number and distribution in the ICC. FOXD3 suppresses KIT expression in human cells; its inactivation led to an increase in ICC in zebrafish, as well as mice, providing evidence for a functional link between FOXD3 defects and KIT overexpression leading to GIST formation.

Genomic Evolution and Transcriptional Changes in the Evolution of Prostate Cancer into Neuroendocrine and Ductal Carcinoma Types.

Prostate cancer is typically of acinar adenocarcinoma type but can occasionally present as neuroendocrine and/or ductal type carcinoma. These are associated with clinically aggressive disease, and the former often arises on a background of androgen deprivation therapy, although it can also arise de novo. Two prostate cancer cases were sequenced by exome capture from archival tissue. Case 1 was de novo small cell neuroendocrine carcinoma and ductal adenocarcinoma with three longitudinal samples over 5 years. Case 2 was a single time point after the development of treatment-related neuroendocrine prostate carcinoma. Case 1 showed whole genome doubling in all samples and focal amplification of AR in all samples except the first time point. Phylogenetic analysis revealed a common ancestry for ductal and small cell carcinoma. Case 2 showed 13q loss (involving RB1) in both adenocarcinoma and small cell carcinoma regions, and 3p gain, 4p loss, and 17p loss (involving TP53) in the latter. By using highly curated samples, we demonstrate for the first time that small-cell neuroendocrine and ductal prostatic carcinoma can have a common ancestry. We highlight whole genome doubling in a patient with prostate cancer relapse, reinforcing its poor prognostic nature.

Genomic analysis of spinal meningiomas: correlation with histopathological grade.

Spinal meningiomas are one of the most common primary intradural tumors of the adult spine. Spinal meningiomas typically have a benign course with low rates of recurrence. Recent advances in genomic profiling have provided valuable information on meningioma biology and natural history, but these studies have focused primarily on cranial meningiomas. Chromosomal copy number analysis of meningiomas has been shown to be a valuable molecular profiling technique for distinguishing benign from aggressive tumors. The Integrated Grade for Meningioma is a novel grading scheme that uses mitotic index and copy-number profile to identify aggressive tumors at high risk for recurrence. The integrated grade has been shown to be a better predictor of tumor behavior than WHO grade alone. The objective of this study was to evaluate the chromosomal copy-number profile of spinal meningiomas, and to correlate these findings with the assigned WHO grades. The authors evaluated 94 spinal meningiomas treated surgically at their institution between 2002 and 2022. The histopathological results including WHO grade, mitotic index, presence of atypical features, and MIB-1 index were recorded. Chromosomal copy number as determined by institutional whole-genome DNA copy-number profiling was available for 57 tumors. The WHO grades of the cohort consisted of 81 (86%) WHO grade 1 tumors and 13 (14%) WHO grade 2 tumors. In tumors for which copy-number profiling was available, 44 (77%) of 57 demonstrated loss of 22q/NF2. Notably absent were frequent high-risk copy number alterations including loss of 1p, 3p, 4p/q, 6p/q, 10p/q, 14q, 18p/q, 19p/q, and focal loss of CDKN2A on 9p. Of the 9 WHO grade 2 tumors for which copy-number profiling was available, 6 tumors were reclassified to a lower risk profile (integrated grade 1). This analysis suggests that spinal meningiomas exhibit overwhelmingly indolent biology, as supported by their benign integrated grade. These findings have implications in the surgical management of these patients in relation to the need for complete Simpson grade I resection, as well as the potential avoidance of adjuvant therapy following surgery in the setting of otherwise frequently benign pathology.

LGG-12. CLINICAL AND MOLECULAR FEATURES OF DISSEMINATED PEDIATRIC LOW-GRADE GLIOMA

Abstract Although most pediatric LGG (PLGG) have excellent long-term survival, there is a subset of cases that disseminate throughout the neuraxis (DLGG) that have very poor outcomes. The reason for this aggressive behavior is unknown but we hypothesize that distinct and specific biological mechanisms underlie the metastatic ability. The methylation-based class of diffuse leptomeningeal glioneuronal tumor (DLGNT) is characterized by MAPK pathway activating fusions with chromosome 1p loss, 19q loss, and/or 1q gain. However, it is unknown what proportion of DLGG match the DLGNT group, and which other methylations classes are at risk of metastasis. To improve our understanding of this rare patient population, we created an international DLGG consortium. Data from the first 68 cases shows a broad age distribution and no sex predilection. As expected, DLGG has much worse prognosis than the overall PLGG population. Virtually all DLGG progress at 5 years, compared to a quarter of other PLGG, and DLGG are 5-times more likely to die at 10 years. We observe three patterns of dissemination – 35% present with a localized mass and have secondary dissemination, 50% with disseminated tumor and a clear dominant mass, and 15% with disseminated disease without a dominant mass. In 47 patients with molecular testing, BRAF fusions accounted for 64% of driver alterations. Additional alterations were identified less frequently, including BRAF V600E (9%), FGFR1 alterations (9%), and KRAS mutations (4%). In 25 patients with methylation profiling, 10 (40%) successfully classified with a calibrated score above 0.8, illustrating that many cases do not fit a defined methylation group. The most common methylation classification was pilocytic astrocytoma (5 patients), and surprisingly only two patients classified as DLGNT. In sum this study illustrates the variable clinical behaviour associated with metastasis in PLGG and expands the range of molecular driver alterations, including ones previously unreported in DLGNT.

EPEN-10. SPINAL EPENDYMOMA WITH MYCN-AMPLIFICATION – A DISEASE OF CHILDHOOD AND YOUNG ADULTHOOD WITH DISMAL PROGNOSIS

Abstract Spinal ependymoma (EPN) with MYCN-amplification (SP-EPN-MYCN) was recently recognized as a distinct tumor type within the 2021 WHO classification of CNS tumors. To date, information about the epidemiological, clinical, and biological features of SP-EPN-MYCN remain sparse. Using DNA-methylation profiling, we collected a cohort of n = 71 (54 primaries, 17 relapses) SP-EPN-MYCNs. Based on the Heidelberg Methylation Brain Tumor Classifier V12.5, 95.6% (68/71) reached a classifier score of ≥ 0.9. In 68/71 cases, focal amplifications of MYCN were detected by copy number variation (CNV) analysis. Two tumors of pediatric patients harbored MYC-amplifications. Other repeated CNVs amongst the primary cases included gains of chromosomes 5 (5/54) and 18 (5/54), loss of chromosome 10 (14/51), and combined 17p loss and 17q gain (5/54). FISH (n = 17) and immunohistochemistry (n = 19) provided positive results for the detection of the MYCN-amplification. Epidemiological data was available for 63% (34/54) of all patients, showing an even sex distribution (17 female patients). Twenty-four percent (8/34) were children and adolescents younger than 20 years. The rest of the cohort consisted of an AYA-population with an overall median age of 31.5 years. Primary lesions were predominantly located in the thoracic (72%) and/or cervical (55%) spinal cord. Two patients showed extra-CNS metastasis to the humerus and the paraspinal musculature, respectively. At the point of analysis, 38% (10/26) of all patients had died, and 10/16 alive patients had already developed disseminated disease. Of patients with available information, 90% (18/20) had relapsed, whereas 10% (2/20) showed stable disease with leptomeningeal spread. Median PFS (n = 17) and OS (n = 25) were 15,5 (12 – 34) and 85 (61 – NA) months. In summary, SP-EPN-MYCN is a disease with dismal prognosis that can disseminate outside of the CNS and affects mainly children and AYA. Currently, comprehensive multi-omics analyses are ongoing.

Tumor-intrinsic features associated with progression-free survival (PFS) in patients (pts) with relapsed and refractory multiple myeloma (RRMM) treated with idecabtagene vicleucel (ide-cel).

8035 Background: Ide-cel, a B-cell maturation antigen chimeric antigen receptor T cell therapy, has demonstrated frequent, deep, and durable responses in pts with RRMM in the KarMMa (NCT03361748), KarMMa-2 (NCT03601078), and KarMMa-3 (NCT03651128) trials. A previous analysis of KarMMa showed comparable efficacy of ide-cel across molecular high-risk/resistance (HR/R) features such as biallelic p53 inactivation, 1q amplification, t(4;14), and CRBN dysregulation, and identified a baseline gene expression pattern (PC4) associated with PFS (Martin N, et al. Hemasphere 2022;6:(S6):1452). We aim to identify additional genomic features associated with ide-cel efficacy in KarMMa and KarMMa-2, and evaluate samples from KarMMa-3 pts as an independent validation cohort. Methods: Transcriptional and genomic profiles were assessed at baseline in CD138+ cells from bone marrow from pts in KarMMa, KarMMa-2 cohort 1, and KarMMa-3. Gene copy number aberrations were evaluated for associations with response. Analyses were post hoc and exploratory, and a P-value, or false discovery rate (FDR), of < 0.05 was used to identify associations of interest. Results: In evaluable pts in KarMMa (n = 70), single copy number loss was observed at 2 loci that associated with PFS, a broad region across 14q (n = 16) and deletion at 1p31.2 (n = 6). These were generally independent and collectively represented 30% (21/70) of pts. Both were associated with shorter median PFS (mPFS) in KarMMa (cohort mPFS, 8.8 mo, n = 128; Munshi et al. N Engl J Med 2021;384:705-16) after FDR correction (del 14q, mPFS, 4.0 mo; del 1p31.2, mPFS, 2.3 mo). No pt with 1p loss achieved a best overall response (BOR) better than partial response. Pts with loss vs non-loss of 14q had similar BOR distribution, but poorer mPFS within each BOR (10.4 vs 29.7 mo for loss vs non-loss in pts with complete response/stringent complete response). Due to small sample size in KarMMa-2 cohort 1 (n = 18), these data were aggregated with those from KarMMa. When combined, previously described associations with HR/R features were maintained except for biallelic p53 inactivation, which showed a stronger association with shorter PFS in the aggregate dataset (n = 83, P = 0.04) than in KarMMa (n = 65, P = 0.09). KarMMa-3 (n = 210) analysis is ongoing and will be used as a validation cohort for these findings. Consistent trends for the association of the PC4 gene signature with PFS were observed in KarMMa-2 but were not statistically significant and limited by sample size. Conclusions: Two novel genomic HR/R features associated with PFS were identified using samples from KarMMa; validation is ongoing using samples from KarMMa-3. The PC4 gene signature was consistent in 2 RRMM cohorts. A modest association between biallelic p53 disruption and PFS emerged with increased sample size and will be studied in KarMMa-3.

Whole exome sequencing of low grade serous ovarian carcinoma identifies genomic events associated with clinical outcome

ObjectivesLow-grade serous ovarian carcinoma (LGSOC) is a distinct, rare, ovarian cancer type characterised by younger patient age and intrinsic chemoresistance. Understanding the molecular landscape is crucial for optimising targeted therapy. MethodsGenomic data from whole exome sequencing of tumour tissue was analysed in a LGSOC cohort with detailed clinical annotation. Results63 cases were analysed and three subgroups identified based on single nucleotide variants: canonical MAPK mutant (cMAPKm: 52%, KRAS/BRAF/NRAS), MAPK-associated gene mutation (MAPK-assoc: 27%) and MAPK wild-type (MAPKwt: 21%). NOTCH pathway disruption occurred across all subgroups. Tumour mutational burden (TMB), mutational signatures and recurrent copy number (CN) changes varied across the cohort with co-occurrence of chromosome 1p loss and 1q gain (CN Chr1pq) a recurrent feature. Low TMB and CN Chr1pq were associated with inferior disease-specific survival (HR 6.43; p < 0.001 and HR 3.29, p = 0.011 respectively). Stepwise genomic classification in relation to outcome resulted in four groups (TMB low; CN Chr1pq; MAPKwt/MAPKassoc; cMAPKm). 5 year disease-specific survival was 46%, 55%, 79% and 100% respectively for these groups. The two most favourable genomic subgroups were enriched for the SBS10b mutational signature, particularly the cMAPKm subgroup. ConclusionsLGSOC comprises multiple genomic subgroups with distinct clinical and molecular features. Chr1pq CN arm disruption and TMB represent promising methods to identify individuals with poorer prognosis. Further investigation of the molecular basis for these observations is required. MAPKwt cases represent around a fifth of patients. NOTCH inhibitors represent a candidate therapeutic strategy worthy of exploration across these cases.

Abstract 1414: The role of chromosome arm 3p and human papillomavirus on proliferation, differentiation, and metastasis in squamous cancers

Abstract Squamous Cell Carcinoma (SCC) is an aggressive form of cancer that develops in the mucosal epithelium of the digestive and respiratory tracts as well as on the skin. Two major risk factors for development of SCC are carcinogen exposures, such as alcohol and tobacco, and Human Papillomavirus (HPV) infection. Viral- and carcinogen-driven SCCs have differential responses to radiation, chemotherapy, and immunotherapy, with HPV+ SCCs responding better to these treatments than HPV- carcinomas. Analysis of aneuploidy patterns of genomic samples from The Cancer Genome Atlas (TCGA) have shown that squamous cancers cluster together and are characterized by gain of chromosome arm 3q gain and loss of chromosome arm 3p. However, while HPV+ SCCs show similar rates of 3q gain, they have significantly lower rates of 3p loss, suggesting that 3p deletion and HPV infection might have overlapping roles in cancer initiation and/or progression. In addition, the degree to which these two paths of tumor development contribute to differences in disease progression and outcome has been largely unexplored. We have identified a strong correlation between 3p deletion and high epithelial-mesenchymal transition (EMT) gene signature across all cancer types. Interestingly, interferon signaling has been found to be upregulated in non-squamous cancers upon 3p deletion but downregulated in squamous cancers with 3p deletion. SCCs positive for HPV infection show decreased expression of EMT genes and increased expression of genes in interferon signaling and cell cycle checkpoint pathways. Together, the results of these TCGA analyses suggest mechanisms by which chromosome 3p deletion and HPV infection may affect cancer progression and are currently being validated in vitro in a panel of squamous cancer cell lines. Additionally, we will use a targeted CRISPR-Cas9 system developed in our lab to delete chromosome 3p, as well as transfect HPV oncoproteins, in normal keratinocytes to create isogenic models. With these models, we will study the role of individual aneuploidy events in tumorigenesis, tumor development, and metastasis in the context of HPV positive and negative SCC. Citation Format: Samyukta Mallick, Alison M. Taylor. The role of chromosome arm 3p and human papillomavirus on proliferation, differentiation, and metastasis in squamous cancers [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 1414.

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.

Evolutionary characterization of lung adenocarcinoma morphology in TRACERx.

Lung adenocarcinomas (LUADs) display a broad histological spectrum from low-grade lepidic tumors through to mid-grade acinar and papillary and high-grade solid, cribriform and micropapillary tumors. How morphology reflects tumor evolution and disease progression is poorly understood. Whole-exome sequencing data generated from 805 primary tumor regions and 121 paired metastatic samples across 248 LUADs from the TRACERx 421 cohort, together with RNA-sequencing data from 463 primary tumor regions, were integrated with detailed whole-tumor and regional histopathological analysis. Tumors with predominantly high-grade patterns showed increased chromosomal complexity, with higher burden of loss of heterozygosity and subclonal somatic copy number alterations. Individual regions in predominantly high-grade pattern tumors exhibited higher proliferation and lower clonal diversity, potentially reflecting large recent subclonal expansions. Co-occurrence of truncal loss of chromosomes 3p and 3q was enriched in predominantly low-/mid-grade tumors, while purely undifferentiated solid-pattern tumors had a higher frequency of truncal arm or focal 3q gains and SMARCA4 gene alterations compared with mixed-pattern tumors with a solid component, suggesting distinct evolutionary trajectories. Clonal evolution analysis revealed that tumors tend to evolve toward higher-grade patterns. The presence of micropapillary pattern and 'tumor spread through air spaces' were associated with intrathoracic recurrence, in contrast to the presence of solid/cribriform patterns, necrosis and preoperative circulating tumor DNA detection, which were associated with extra-thoracic recurrence. These data provide insights into the relationship between LUAD morphology, the underlying evolutionary genomic landscape, and clinical and anatomical relapse risk.

Molecular Correlates of Aggressive Behavior and Biological Progression in Testicular Sertoli Cell Tumors

Sertoli cell tumor (SCT) is the second most common type of sex cord-stromal tumor in men, and ∼10% exhibit malignant behavior. Although CTNNB1 variants have been described in SCTs, only a limited number of metastatic cases have been analyzed, and the molecular alterations associated with aggressive behavior remain largely unexplored. This study evaluated a series of nonmetastasizing and metastasizing SCTs using next-generation DNA sequencing to further characterize their genomic landscape. Twenty-two tumors from 21 patients were analyzed. Cases were divided into metastasizing SCTs and nonmetastasizing SCTs. Nonmetastasizing tumors were considered to have aggressive histopathologic features if they exhibited ≥1 of the following: size >2.4 cm, necrosis, lymphovascular invasion, ≥3 mitoses per 10 high-power fields, severe nuclear atypia, or invasive growth. Six patients had metastasizing SCTs, and the remaining 15 patients had nonmetastasizing SCTs; 5 nonmetastasizing tumors had ≥1 aggressive histopathologic feature(s). Gain-of-function CTNNB1 or inactivating APC variants were highly recurrent in nonmetastasizing SCTs (combined frequency >90%), with arm-level/chromosome-level copy number variants, loss of 1p, and CTNNB1 loss of heterozygosity occurring exclusively in CTNNB1-mutant tumors with aggressive histopathologic features or size >1.5 cm. Nonmetastasizing SCTs were almost invariably driven by WNT pathway activation. In contrast, only 50% of metastasizing SCTs harbored gain-of-function CTNNB1 variants. The remaining 50% of metastasizing SCTs were CTNNB1-wild-type and harbored alterations in the TP53, MDM2, CDKN2A/CDKN2B, and TERT pathways. These findings suggest that ∼50% of aggressive SCTs represent progression of CTNNB1-mutant benign SCTs, whereas the remaining ones are CTNNB1-wild-type neoplasms that exhibit alterations in genes of the TP53, cell cycle regulation, and telomere maintenance pathways.

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.

Abstract P5-14-08: Machine learning based histopathology images analysis reveals cancer stemness in TNBC patient with 17p loss

Abstract Background: The area of computational pathology has made huge progresses due to advances in artificial intelligence (AI) and machine learning technologies. It has been applied to many research and translational tasks which provide great improvement on medical diagnosis and treatment. Cancer stem-like cells (CSC) have been consistently reported for its key role in Triple negative breast cancer (TNBC) . Given the large amount of existing H&amp;E stained histological slides of TNBC, digital identification of CSC could benefit the evaluation of tumor status and prediction of patients’ response to chemotherapy. Here we proposed an AI framework based on Convolutional Neural Network (CNN) to predict CSC from the histological images of TNBC patient. And our preliminary work suggested that chromosome 17p loss, a common genetic variations in breast cancer, is linked to cancer stemness. Methods: A modified GoogleNet model was adopted as our CNN classifier. Consecutive breast cancer tissue microarrays (TMA), which stained with H&amp;E ,SOX2, OCT4 and NANOG antibodies respectively by IHC, were used as training dataset for the CNN model. Gene expression data from the TCGA and METABRIC datasets were used to identify gene signatures associated with CSC. The connectivity map (CMAP) and Cancer Cell Line Encyclopedia (CCLE) were used for screening compounds that target stemness in cancer cell lines with chromosomal 17p loss. HS578T and EO771 cells with or without heterozygous 17p loss (11b in EO771) were used for in vitro experiments. Female immunodeficient nude (Nu/J) mice were used for animal studies. Results: The well trained GoogleNet model was applied to TNBC patient diagnosis images in TCGA BRCA dataset. By analyzing patient genomic alteration on chromosomal level, we found that loss of chromosome 17p associate with high cancer stemness in TNBC. Flow cytometry assays also demonstrated higher ALDH1 activity and higher CD44+/CD24−/low cell population in HS578T cells with 17p loss. RNA-seq of HS578T cells revealed that most CSC marker genes were located in the unregulated differentially expressed genes (DEGs) of 17p loss cells. We next compared the cytotoxicity of chemotherapy drugs including doxorubicin, paclitaxel, docetaxel and cisplatin on 17p loss and 17p intact HS578T cells, 11b loss and 11b intact EO771 cells, in terms of IC50 value. The IC50 value of indicated drug on 17p loss HS578T cells with were 3-6 fold higher than their IC50 on 17p intact HS578T cells. Similar result was observed in EO771 cells. Next, 17p loss and 17p intact HS578T cells were orthotopically implanted into the Nu/J mice. Under the doxorubicin treatment, mice bearing 17p loss HS578T derived tumors had larger and heavier tumors in compare to mice bearing 17p intact tumors. Next, we did a computational drug screening to identify compounds that can target the cancer stemness in 17p loss cells. Screened out compounds were tested for their cytotoxicity on 17p loss and 17p intact HS578T cells and FK866 showed the most pronounced efficacy on inhibiting the viability of 17p loss cells, compared to 17p intact cells. Followup experiments demonstrated that FK866 can decrease the CSC features induced by doxorubicin, both in vitro and in vivo. FK866 also potentiates the effect of doxorubicin on treating TNBC cells with 17p loss, which provide a drug combination potential for TNBC patient with 17p loss. Conclusions: A CNN based model was developed to identify CSC from TNBC histopathology images. The images analysis combined with patient genomic data revealed that chromosome 17p loss associated with cancer stemness in TNBC. This result was confirmed using assays on TNBC cells with or without 17p loss. A computational drug screening was performed to identify candidates that targeting stemness in 17p loss cells. FK866 was identified and it potentiates the anti-tumor effect of doxorubicin on treating TNBC cells with 17p loss. Our study provides a novel strategy on applying AI to precision treatment for cancers. Citation Format: tianhan dong, jiannan liu, yuanzhang Fang, Ziyu Liu, Xiongbin Lu, kun huang. Machine learning based histopathology images analysis reveals cancer stemness in TNBC patient with 17p loss [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-14-08.

Somatic, Genetic and Epigenetic Changes in Nephrogenic Rests and Their Role in the Transformation to Wilms Tumors, a Systematic Review.

To review somatic genetic changes in nephrogenic rests (NR), which are considered to be precursor lesions of Wilms tumors (WT). This systematic review is written according to the PRISMA statement. PubMed and EMBASE were systematically searched for articles in the English language studying somatic genetic changes in NR between 1990 and 2022. Twenty-three studies were included in this review, describing 221 NR of which 119 were pairs of NR and WT. Single gene studies showed mutations in WT1 and WTX, but not CTNNB1 to occur in both NR and WT. Studies investigating chromosomal changes showed loss of heterozygosity of 11p13 and 11p15 to occur in both NR and WT, but loss of 7p and 16q occurred in WT only. Methylome-based studies found differential methylation patterns between NR, WT, and normal kidney (NK). Over a 30-year time frame, few studies have addressed genetic changes in NR, likely hampered by technical and practical limitations. A limited number of genes and chromosomal regions have been implicated in the early pathogenesis of WT, exemplified by their occurrence in NR, including WT1, WTX, and genes located at 11p15. Further studies of NR and corresponding WT are urgently needed.

Rapidly progressive diffuse leptomeningeal glioneuronal tumor in an adult female: illustrative case.

Diffuse leptomeningeal glioneuronal tumor (DLGNT) is a rare brain tumor only recently classified by the World Health Organization in 2016 and has few reports on its incidence in adults. The authors describe a case of DLGNT presenting in a 47-year-old female with seizures, cranial neuropathies, and communicating hydrocephalus with rapid clinical progression. Workup demonstrated progressive leptomeningeal enhancement of the skull base, cranial nerves, and spine, and communicating hydrocephalus. Elevated serum rheumatological markers and early response to systemic corticosteroids and immunosuppressant therapy complicated the diagnosis. Multiple biopsy attempts were required to obtain diagnostic tissue. Pathology demonstrated hypercellularity surrounding leptomeningeal vessels with nuclear atypia, staining positive for GFAP, Olig2, S100, and synaptophysin. Molecular pathology demonstrated loss of chromosome 1p, BRAF overexpression but no rearrangement, and H3K27 mutation. Repeat cerebrospinal fluid (CSF) diversion procedures were required for hydrocephalus management due to high CSF protein content. This report describes a rare, aggressive, adult presentation of DLGNT. Leptomeningeal enhancement and communicating hydrocephalus should raise suspicion for this disease process. Biopsy at early stages of disease progression is essential for early diagnosis and prompt treatment. Further study into the variable clinical presentation, histological and molecular pathology, and optimal means of diagnosis and management is needed.

Cytogenetic Abnormalities for Predicting the Risk of Metastases in Choroidal and Ciliary Body Melanoma.

Choroidal melanoma (CM) and ciliary body melanoma (CBM) are the two most common subtypes of uveal melanoma. Starting from the observation that CBM tends to have a higher metastatic potential than CM, we hypothesized that specific cytogenetic abnormalities could be associated with tumor location - reflecting distinct genetic signatures that would drive the risk of distant spread. Chromosomal alterations were investigated by molecular cytogenetic techniques in 217 and 97 patients with CM and CBM, respectively. Cox proportional hazards regression analysis was used to identify the independent predictors of distant metastasis. Patients with CBM had larger tumor sizes (P < 0.001), higher disease stages (P < 0.001), and more frequently showed distant metastasis (P = 0.002) than those with CM. On analyzing the entire study cohort, we found that specific chromosomal alterations - including chromosome 8p loss (P < 0.001), 1p loss (P < 0.001), and monosomy 3 (P < 0.005) - were independent predictors of distant metastasis. Based on a decision-tree learning algorithm, we identified three specific subgroups of patients with uveal melanoma at high risk of distant spread. Monosomy 3 occurred significantly more frequently in patients with T3 CBM tumors. Specific cytogenetic abnormalities - including chromosome 8p loss, 1p loss, and monosomy 3 - are independent risk factors for distant metastasis in uveal melanoma. Larger tumor size at presentation and monosomy 3 contribute to a higher metastatic risk in patients with CBM.

Single-cell profiling of multiple myeloma reveals molecular response to FGFR3 inhibitor despite clinical progression.

Genomic characterization of cancer has enabled identification of numerous molecular targets, which has led to significant advances in personalized medicine. However, with few exceptions, precision medicine approaches in the plasma cell malignancy multiple myeloma (MM) have had limited success, likely owing to the subclonal nature of molecular targets in this disease. Targeted therapies against FGFR3 have been under development for the past decade in the hopes of targeting aberrant FGFR3 activity in MM. FGFR3 activation results from the recurrent transforming event of t(4;14) found in ∼15% of MM patients, as well as secondary FGFR3 mutations in this subgroup. To evaluate the effectiveness of targeting FGFR3 in MM, we undertook a phase 2 clinical trial evaluating the small-molecule FGFR1-4 inhibitor, erdafitinib, in relapsed/refractory myeloma patients with or without FGFR3 mutations (NCT02952573). Herein, we report on a single t(4;14) patient enrolled on this study who was identified to have a subclonal FGFR3 stop-loss deletion. Although this individual eventually progressed on study and succumbed to their disease, the intended molecular response was revealed through an extensive molecular characterization of the patient's tumor at baseline and on treatment using single-cell genomics. We identified elimination of the FGFR3-mutant subclone after treatment and expansion of a preexisting clone with loss of Chromosome 17p. Altogether, our study highlights the utility of single-cell genomics in targeted trials as they can reveal molecular mechanisms that underlie sensitivity and resistance. This in turn can guide more personalized and targeted therapeutic approaches, including those that involve FGFR3-targeting therapies.

Classification of HER2-negative breast cancers by ERBB2 copy number alteration status reveals molecular differences associated with chromosome 17 gene aberrations.

Recently, HER2-negative breast cancers have been reclassified by protein expression into 'HER2-low' and 'HER2-zero' subgroups, but the consideration of HER2-low breast cancer as a distinct biological subtype with differing prognoses remains controversial. By contrast, non-neutral ERBB2 copy number alteration (CNA) status is associated with inferior survival outcomes compared to ERBB2 CNA-neutral breast cancer, providing an alternative approach to classification. Here, we investigated the molecular landscape of non-metastatic HER2-negative BCs in relation to ERBB2 CNA status to elucidate biological differences. Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) TCGA-BRCA datasets (n = 1875) were analyzed. Nearly two-fifths of the cohort harbored ERBB2 CNAs (39.4%), which were significantly enriched within hormone receptor-negative (56.1%) than within hormone receptor-positive BCs (35.5%; p < 0.0001). Globally, CNAs across the genome were significantly higher in ERBB2 non-neutral compared to neutral cohorts (p < 0.0001). Notably, genetic aberrations on chromosome 17 - BRCA1, NF1, TP53, MAP2K4, and NCOR1 - were widespread in the ERBB2 non-neutral cases. While chromosome 17q arm-level alterations were largely in tandem with ERBB2 CNA status, arm-level loss in chromosome 17p was prevalent regardless of ERBB2 gain, amplification, or loss. Differential gene expression analysis demonstrated that pathways involved in the cell cycle, proteasome, and DNA replication were upregulated in ERBB2 non-neutral cases. Classification of HER2-negative BCs according to ERBB2 CNA status reveals differences in the genomic landscape. The implications of concurrent aberrations in other genes on chromosome 17 merit further research in ERBB2 non-neutral BCs.