Biallelic Inactivation Research Articles

MDB-13. GERMLINEELP1 DEFICIENCY SENSITIZES CEREBELLAR GRANULE NEURON PROGENITORS TO SHH MEDULLOBLASTOMA

Abstract BACKGROUND Germline loss-of-function (LOF) variants in Elongator complex protein 1 (ELP1) are the most prevalent predisposing genetic events observed in medulloblastoma (MB), accounting for 30% of the Sonic Hedgehog 3 subtype (SHH-3). Molecularly, ELP1-associated SHH-MBs acquire somatic PTCH1 mutations in >80% of cases, and universal loss-of-heterozygosity of the wild-type ELP1 and PTCH1 alleles through loss of chromosome-arm 9q, resulting in their biallelic inactivation. Notably, germline ELP1 LOF occurs mutually exclusive with somatic/germline TP53 mutations in the SHH-3 subtype, suggesting that genetic perturbation of either ELP1 or TP53 may promote similar tumorigenic consequences in cerebellar granule neuron progenitors (GNPs), the accepted cellular origin of SHH-MB. Despite these findings, the molecular, biochemical, and cellular mechanism(s) by which ELP1-deficiency provokes malignant pathogenesis remain unknown. In this study, we sought to close this knowledge gap and functionally determine why children harboring pathogenic ELP1 germline variants are at an increased risk of developing SHH-MB. METHODS Mice were genetically engineered to mimic heterozygous Elp1 LOF (Elp1HET). We studied the effect of Elp1 LOF in GNPs using a combination of molecular profiling, immunophenotyping, and cellular assays. RESULTS GNPs from Elp1HET mice exhibited a spectrum of molecular and biochemical hallmarks of malignant transformation including increased DNA replication stress, DNA damage, accelerated cell cycle progression, and stalled differentiation. Orthotopic transplantation of Elp1HET GNPs engineered to harbor somatic Ptch1 inactivation yielded SHH-MB-like tumors with compromised p53 signaling, providing an explanation for the specificity of ELP1-associated tumors in SHH-3, and their exclusivity with TP53-mutant tumors. Treatment of ELP1-mutant patient-derived xenografts with an FDA-approved MDM2 inhibitor reactivated p53-dependent apoptosis and extended survival. CONCLUSIONS Collectively, our findings functionally substantiate the role of ELP1 deficiency in predisposition to SHH-3 MB and nominate therapeutic strategies to overcome p53 inhibition as a rational treatment option.

NFS-02. MEDULLOBLASTOMAS WITH ELP1 PATHOGENIC VARIANTS: A WEAKLY PENETRANT SYNDROME WITH A RESTRICTED SPECTRUM IN A LIMITED AGE WINDOW

Abstract BACKGROUND Medulloblastomas (MB) Sonic hedgehog (SHH) subtype are associated with a cancer predisposition syndrome (CPS) in about 15% of cases, the most frequent being related to ELP1 pathogenic variant (PV). The aim of our study is to better evaluate penetrance of this CPS and detail the characteristics of the related tumors. METHODS Twenty-nine ELP1-mutated MB identified in France were retrospectively reviewed. Molecular characteristics of the tumor and clinical features of the patients were collected; whenever possible, the germline DNA from patients and their relatives were sequenced. RESULTS All patients (sex ratio 16/13) presented with SHH-MB, mostly nodular desmoplastic (n=20/28), between 3 and 15 years of age (median 7.3). All mutations are found in the germline when a constitutional DNA sample was available (n=26). Most tumors (93%) showed a biallelic inactivation of PTCH1, other recurrent alterations concerned the TP53 pathway (including 1 somatic TP53 VP) and activation of MYCN/MYCL signaling. We observed that ELP1-mutated MB behave as sporadic cases, with similar distribution within clinical and molecular risk groups of MB, similar outcomes (5y-OS = 85%) and no unusual side effects of treatments. Three patients developed a second tumor (2 high-grade gliomas and 1 thyroid carcinoma) within the irradiation fields. All germline PV were inherited from one asymptomatic parent (11 trio). No specific morphological features were mentioned. Only two index cases from the 29 French patients had a previous familial history of MB (occurring in a cousin), with many asymptomatic carriers. Except the MB, no other cancer was significantly associated with the ELP1 PV. CONCLUSIONS the low penetrance, the ‘at risk’ age window limited to childhood and the narrow tumor spectrum, question the actual benefit of genetic screening in these patients and their family. Our results suggest restricting ELP1 germline sequencing to patients with MB, depending on the parents’ request.

IMG-16. DECIPHERING THE NEUROIMAGING SPECTRUM OF AT/RT: A PICTORIAL REVIEW OF ATYPICAL TERATOID/RHABDOID TUMORS

Abstract BACKGROUND This study presents a detailed pictorial review of Atypical teratoid/rhabdoid tumor (AT/RT), a high-grade central nervous system (CNS) malignancy, emphasizing the relationship between neuroimaging characteristics and clinical, molecular, and histopathological aspects. AT/RT is mainly characterized by the biallelic inactivation of SMARCB1 and occasionally SMARCA4 genes. METHODS The review methodically analyzes neuroimaging findings from various imaging techniques, notably MRI. It documents AT/RT appearances in different brain regions, including the cerebellum, cerebellopontine angle cistern, intraventricular, suprasellar, supratentorial, pineal, spinal canal, and cranial nerves. The study integrates these findings with clinical presentation, molecular subtypes (AT/RT-SHH, AT/RT-TYR, and AT/RT-MYC), and pathogenesis, highlighting the connection between imaging features and molecular subtypes. RESULTS The results indicate distinctive neuroimaging characteristics of CNS AT/RT, varying with tumor location and molecular subtypes. Common imaging features include contrast enhancement and signal intensity variations on MRI, including subtype-specific variations. Advanced neuroimaging plays a key role in distinguishing AT/RT from other pediatric CNS tumors, aiding in biopsy and treatment planning. Notably, imaging findings are linked with genetic changes, particularly the involvement of SMARCB1 and SMARCA4, affecting tumor appearance and behavior. CONCLUSIONS This review underscores the significance of neuroimaging in diagnosing and treating CNS AT/RT. The comprehensive analysis of imaging, combined with molecular and histopathological data, offers valuable insights into AT/RT’s heterogeneous nature. This integrative approach is crucial for a better understanding of AT/RT and for the development of more effective, targeted therapies.

RB1 Genetic Alterations in Estrogen Receptor–Positive Breast Carcinomas: Correlation With Neuroendocrine Differentiation

Genetic alterations in the retinoblastoma susceptibility gene (RB1) are present in up to 40% of triple-negative breast cancers (BCs) and frequent in tumors with neuroendocrine differentiation, including small cell neuroendocrine carcinoma. Data on RB1 genetic alterations in estrogen receptor (ER)–positive BCs are scarce. In this study, we sought to define the morphologic, immunohistochemical, and genetic features of ER-positive BCs harboring somatic alterations in RB1, with emphasis on neuroendocrine differentiation. ER-positive BCs with pathogenic RB1 genetic alterations were identified in <1% of cases (N = 55) from a cohort of 6026 BCs previously subjected to targeted next-generation sequencing, including 23 primary BCs (pBCs) and 32 recurrent/metastatic BCs (mBCs). In cases where loss of heterozygosity of the wild-type RB1 allele could be assessed (93%, 51/55), most pBCs (82%, 18/22) and mBCs (90%, 26/29) exhibited biallelic RB1 inactivation, primarily through loss-of-function mutation and loss of heterozygosity (98%, 43/44). Upon histologic review, a subset of RB1-altered tumors exhibited neuroendocrine morphology (13%, 7/55), which correlated with expression of neuroendocrine markers (39%, 9/23) in both pBCs (27%, 3/11) and mBCs (50%, 6/12). Loss of Rb protein expression was observed in BCs with biallelic RB1 loss only, with similar frequency in pBCs (82%, 9/11) and mBCs (75%, 9/12). All cases with neuroendocrine marker expression (n = 9) and/or neuroendocrine morphology (n = 7) harbored biallelic genetic inactivation of RB1 and exhibited Rb loss of expression. TP53 (53%, 29/55) and PIK3CA (45%, 25/55) were the most frequently comutated genes across the cohort. Overall, these findings suggest that ER-positive BCs with biallelic RB1 genetic alterations frequently exhibit Rb protein loss, which correlates with neuroendocrine differentiation in select BCs. This study provides insights into the molecular and phenotypic heterogeneity of BCs with RB1 genetic inactivation, underscoring the need for further research into the potential clinical implications associated with these tumors.

Germline and somatic MUTYH mutations: Clinicopathologic and mutational analysis in a pan-cancer cohort.

10590 Background: MUTYH is a tumor suppressor gene involved in the base-excision DNA repair pathway. Biallelic pathogenic or likely pathogenic germline variants (gPV) in MUTYH are associated with MUTYH-associated polyposis (MAP), inclusive of colorectal carcinoma (CRC) and additional cancer risks. While monoallelic MUTYH gPV carriers do not have the same cancer risk as those with biallelic MAP, further work is needed to fully characterize secondary somatic events, including allelic imbalances. Methods: Using the MSK-IMPACT targeted NGS to detect both MUTYH gPVs and MUTYH somatic variants in patients (pts) with a solid tumor diagnosis, we analyzed clinicopathologic characteristics at cancer diagnosis, ancestry label as defined per cBioPortal, genomic biomarkers, as well as allele-specific copy number estimates using FACETS annotation. Results: Out of 38,907 samples with germline testing available, 587 pts with a solid tumor and presence of MUTYH gPV were identified. Of these, 15 pts had ≥2 MUTYH gPVs, including 4 pts with homozygous MUTYHgPVs (G396D, T179C, p.G480* and c.389-1G&gt;C) and 11 pts with compound heterozygous MUTYHgPVs. In pts of European ancestry (n=403), the MUTYH G396D and T179C variants were predominant, present in 56% and 19% of cases, respectively. All patients with the R241W variant were listed as being of South Asian ancestry (n=6). Concurrent gPV in other genes were observed in 12% (n=69), with high/moderate-penetrance gPVs in 4% (n=24) including BRCA2 (n=6), BRCA1 (n=5), ATM (n=4) and PALB2 (n=3). Among all pts with MUTYH gPVs, 254/587 pts had concurrent tumor samples with FACETS results. Among pts with monoallelic MUTYH gPVs and FACETS annotation available, 24% of evaluable tumors exhibited biallelic MUTYH inactivation due to loss of heterozygosity (LOH). CRC (29% vs 13%; p=0.004) and cholangiocarcinoma (11% vs. 2%; p=0.04) were enriched in the biallelic MUTYH inactivation cohort vs. the cohort of pts with monoallelic MUTYH gPVs. Co-alterations in KRAS G12C and APC were enriched in the biallelic as compared to the monoallelic cohort (9% vs. 3%, p=0.04 and 27% vs. 12%, p=0.005, respectively). In addition, we identified 244 pts for whom FACETS annotation was available without MUTYH gPVs, but with somatic variants at MUTYH. Of these 244 pts, 117 pts had biallelic MUTYH inactivation either from homozygous deletions (n=87), somatic hit + LOH (n=27) or ≥2 somatic hits (n=3). The predominant cancer types with biallelic somatic MUTYH inactivation were breast cancer (16%), non-small cell lung cancer (14%) and CRC (11%). Conclusions: This study represents the largest cohort of pts with MUTYH gPVs and FACETS analysis on concurrent tumor samples, confirming a high prevalence of LOH in pts with monoallelic MUTYH gPVs and enrichment in KRAS G12Cin pts with MUTYH gPVs and biallelic MUTYH inactivation.

Impact of pathogenic germline variants in HRR genes on gastroesophageal cancer in a large real-world Chinese population: A comprehensive study.

10588 Background: Genetic testing in gastroesophageal cancer has been limited to those with hereditary cancer syndromes, restricting understanding of the impact of germline variants in sporadic cancers. Our study expands this by exploring germline and somatic characteristics in a broad Chinese cohort, enhancing risk assessment and guiding treatment strategies in a wider population. Methods: From October 2017 to April 2023, we retrospectively analyzed 2596 Chinese gastroesophageal cancer (GEC) patients, including 2003 with gastric cancer (GC), 196 with gastroesophageal junction cancer (GEJC), and 397 with esophageal cancer (EC). A 520-gene panel including 62 genes associated with cancer predisposition, was employed for genomic profiling of paired tumor and white blood cell samples. The pathogenicity of germline variants was determined according to ACMG guidelines. Results: In this retrospective cohort, the proportion of patients carrying pathogenic/likely pathogenic (P/LP) germline variants was 5.3% in GC, 5.6% in GEJC, and 5.0% in EC, respectively. Notably, genes associated with HRR exhibited the highest rates of P/LP germline mutations in GEC (94, 3.7%), with the majority occurring in non-BRCA HRR genes (77, 3.0%). The top five genes with the highest mutation rates were ATM, PALB2, FANCA, CHEK2, and RAD54L. Compared to the East Asian population without cancer in the genomAD database, a higher proportion of P/LP germline mutations were found in the CDH1(Inf [6.46-Inf], p &lt; 0.001), PALB2 (5.60 [1.61-13.09], p = 0.002), SDHA (8.99 [1.29-99.42], p = 0.012), ATM (2.15 [1.08-4.09], p = 0.02), and CHEK2 (3.83 [1.06-13.32], p = 0.02) genes in GC. Similarly, significant increases in PALB2 (10.36 [2.32-37.28], p = 0.002) and ATM (4.42 [0.86-14.33], p = 0.036) were also observed in EC and GEJC. The HRR P/LP variant group exhibited a higher incidence of biallelic inactivation (loss of wildtype allele) compared to the B/LB variant group (24.7% vs 6.6%, p&lt;0.001). Moreover, the similar trend was observed for the moderately/uncertainly penetrant gene ATM and FANCA (38.5% vs 0.6%, p &lt; 0.001; 42.9% vs 9.8%, p = 0.025), which suggests that ATM and FANCA may be associated with a high risk. The HRR genes ATM(56 vs 61, p = 0.052), CDH1(33 vs 61, p = 0.003), and FANCI(48 vs 61, p = 0.041) with P/LP variants were associated with a younger age of onset. Carriers of HRR P/LP variants had less prevalent somatic TP53 variants in GC and GEC and higher HRD score compared to non-carriers (p=0.005). Conclusions: Our research delineates the germline P/LP variant landscape in Chinese GEC, revealing the significant role of HRR P/LP variants. The presence of P/LP variants in HRR genes in GEC is linked to distinct disease characteristics and progression, underscoring their importance in guiding personalized treatment approaches and risk evaluation.

Hematologic malignancies in Li-Fraumeni syndrome.

10613 Background: Li-Fraumeni Syndrome (LFS) is a hereditary cancer predisposition syndrome caused by mutations in the tumor suppressor gene TP53. Since its discovery, a growing list of cancers have been described in association with LFS, predominantly solid tumors. However, hematologic malignancies (HMs) cumulatively account for 4-10% of cancer diagnoses in LFS. In this study, we present the largest series of HMs in families with LFS with detailed clinical courses. Methods: This was a two-center retrospective observational cohort study conducted at the University of Utah and the University of Wisconsin-Madison with IRB approval. Cancer genetics clinic registries were reviewed to identify all families with LFS who also had a confirmed diagnosis of a HM. To meet criteria for LFS, the patient had to have a confirmed germline pathologic or likely pathologic TP53 variant. Patients with a TP53 variant of unknown significance (VUS) were also included if they met classical LFS testing criteria. Data was gathered by manual chart review of both the families’ histories and electronic health records. Results: Among the 121 families identified with LFS, 35 (29%) families had one or more HM. Of these, 17 individuals from 16 families (13%) had confirmed germline TP53 mutations. The most frequent HMs found were acute lymphoblastic leukemia (ALL) (n=6), non-Hodgkin lymphoma (NHL) (n=5), myelodysplastic syndromes (MDS) (n=3), and chronic lymphocytic leukemia (CLL) (n=2). Acute myeloid leukemia (AML), Langerhans cell histiocytosis (LCH), and chronic myeloid leukemia (CML) were each diagnosed once. Most cases were identified in adulthood (n=12, 71%). Of the 19 total HMs, the minority (n=5, 26%) were diagnosed post-cytotoxic therapy and only six (35%) individuals had received a diagnosis of LFS prior to HM diagnosis. The MDS/AML cohort (n=4) all exhibited bi-allelic inactivation of TP53. Two of these four patients achieved long term survival after matched-unrelated donor (MUD) hematopoietic stem cell transplant (HSCT). All three patients who underwent MUD donor HSCT had immune-mediated adverse events. Conclusions: In this study we found a significantly higher incidence of HMs in LFS than previously described, with a predominance of lymphoid over myeloid malignancies. The majority of patients had excellent outcomes after standard of care therapy pointing towards more favorable outcomes for patients with LFS and HMs than previously reported. We also report several unusual immune-mediated adverse events in our patients indicating possible immunogenicity of some TP53 variants. Most patients did not have prior exposure to cytotoxic treatments and were diagnosed with the HM prior to their LFS diagnosis. We also show that 38% of individuals with an HM have multiple family members with an HM, consistent with a familial HM pattern. This underscores the importance of detailed family and personal histories in HM patients found to carry a somatic TP53 variant.

Syndromic MEN1 Parathyroid Adenomas Consist of Both Subclonal Nodules and Clonally Independent Tumors

Abstract Background Primary hyperparathyroidism with parathyroid tumors is a characteristic presentation in Multiple Endocrine Neoplasia Type 1 (MEN1), historically referred to as "primary hyperplasia." The question of whether these tumors signify a multi-glandular clonal disorder or hyperplasia remains inconclusive. Loss of Menin protein expression serves as a reliable surrogate marker for biallelic inactivation and indicates a mutation in the MEN1 gene. The cyclin-dependent kinase inhibitor 1B (CDKN1B) gene, associated with MEN4, encodes the p27 protein, whose expression is inadequately explored in the context of syndromic MEN1. Aims The aim was to explore the molecular basis of syndromic MEN1 parathyroid adenomas, identifying hyperplasia, multiple independent clones, and the extent of Menin loss. Additionally, p27 expression was assessed to evaluate its potential in indicating a germline mutation, especially in distinguishing MEN4 as a clinical alternative to MEN1. Methods In this investigation, we examined histomorphology and protein expression of Menin and p27 in parathyroid adenomas from 25 patients in two independent, well-characterized MEN1 cohorts. Loss of heterozygosity (LOH) patterns were evaluated using fluorescence in situ hybridization (FISH) in one MEN1-associated parathyroid adenoma. Furthermore, next-generation sequencing (NGS) was conducted on eleven nodules from four MEN1 patients. Results Morphologically, the majority of MEN1 adenomas displayed multiple distinct nodules, characterized by predominantly lost Menin expression and reduced p27 protein expression. FISH analysis indicated that most nodules exhibited MEN1 loss, with or without centromere 11 loss. NGS demonstrated both subclonal evolution and the existence of clonally unrelated tumors. Conclusion Syndromic MEN1 parathyroid adenomas, therefore, consist of multiple clones with subclones, aligning with the current framework of the novel WHO classification of parathyroid tumors (2022). The observed loss of p27 expression in a significant fraction of MEN1 parathyroids emphasizes the need for caution when inferring MEN4 solely based on p27 expression.

Identification of TP53 germline variants in pediatric patients undergoing tumor testing: strategy and prevalence.

TP53 alterations are common in certain pediatric cancers, making identification of putative germline variants through tumor genomic profiling crucial for disease management. We analyzed TP53 alterations in 3123 tumors from 2788 pediatric patients sequenced using tumor-only or tumor-normal paired panels. Germline confirmatory testing was performed when indicated. Somatic and germline variants were classified based on published guidelines. In 248 tumors from 222 patients, 284 tier 1/2 TP53 sequence and small copy number variants were detected. Following germline classification, 86.6% of 142 unique variants were pathogenic or likely pathogenic. Confirmatory testing on 118 patients revealed germline TP53 variants in 28 of them (23 pathogenic or likely pathogenic and 5 of uncertain significance), suggesting a minimum Li-Fraumeni syndrome incidence of 0.8% (23/2788) in this cohort, 10.4% (23/222) in patients with TP53 variant-carrying tumors, and 19.5% (23/118) with available normal samples. About 25% (7/28) of patients with germline TP53 variants did not meet Li-Fraumeni syndrome diagnostic or testing criteria, while 20.9% (28/134) with confirmed or inferred somatic origins did. TP53 biallelic inactivation occurred in 75% of germline carrier tumors and was also prevalent in other groups, causing an elevated tumor-observed variant allelic fraction. Somatic evidence, however, including low variant allele fraction correctly identified only 27.8% (25/90) of patients with confirmed somatic TP53 variants. The high incidence and variable phenotype of Li-Fraumeni syndrome in this cohort highlights the importance of assessing germline status of TP53 variants identified in all pediatric tumors. Without clear somatic evidence, distinguishing somatic from germline origins is challenging. Classifying germline and somatic variants should follow appropriate guidelines.

Abstract PO2-08-11: The MAGIC study: Universal whole genome tumour and germline sequencing of newly diagnosed breast cancer identifies a high proportion of ER+ BRCA-like tumours

Abstract Background: Identification of germline mutations in hereditary breast cancer (HBC) genes can have profound benefits in the treatment of breast cancer (BC) and manging risk for patient and the family. In addition, sequencing of the tumours can reveal clinically relevant somatic features, such as homologous recombination deficiency (HRD) and mutational signatures, that can guide treatment which are not discernible by germline sequencing alone. We hypothesised that current standard of care where women diagnosed with BC are not routinely offered tumour sequencing is suboptimal as fails to exploit all therapeutic vulnerabilities which can be exploited in the treatment of BC. The MAGIC study is the first prospective trial in Australia of unselected invasive BCs in general oncology practice combining both germline and tumour sequencing. Methods: The MAGIC study included a total of 651 consecutive consented patients presenting with non-metastatic BC between June 2020 to March 2023. It included invasive BC, high-grade DCIS and pleomorphic LCIS. The sequencing was conducted in two phases; for phase one, 157 cases underwent whole genome sequencing (WGS) on both germline and matched BC DNA while for phase two, 494 cases underwent only germline whole exome sequencing. Germline variants were interrogated for pathogenic variants in BRCA1, BRCA2, PALB2, ATM, CHEK2, BARD1, BRIP1, RAD51B, RAD51C, RAD51D, MLH1, MSH2, MSH6, PMS2, CDH1, PTEN, STK11, TP53 and NTHL1. Tumour homologous recombination (HR) repair deficiency was calculated using HRDetect (Nat Med 2017;23:517). For BCs showing a high HRDetect score ( &amp;gt;0.75) all HBC gene promoter CpG islands were assessed for hypermethylation using the Twist NGS Methylation system. Mutational signatures were calculated from somatic mutations identified from whole genome sequenced BCs using the DeconstructSig package in R (Genome Biol. 2016;17:31) and referenced against the COSMIC v2 signature catalogue. Results: Actionable germline variants in any HBC gene were identified in 7.7% of cases (7.8% of invasive and 6.1% of in situ BC) with 5.2% having an actionable mutation in an HR pathway gene (BRCA1, BRCA2, PALB2 and RAD51C). Most cases with pathogenic variants showed bi-allelic inactivation in the tumour. All BRCA1, BRCA2 and PALB2 tumours showed high HRDetect and/or HRD scores consistent with loss of HR repair function, indicating them to be the underlying hereditary driver of the BC. One BC with a pathogenic PMS2 variant showed retention of the wild-type allele with no evidence of a mismatch repair mutational signature suggesting PMS2 was not the driver of this BC. Of the 157 BC in phase one that underwent WGS and did not have a germline pathogenic HBC gene variant, 16% (18/117 invasive and 3/13 DCIS) had an HRDetect score of &amp;gt;0.7 indicative of a homologous recombination repair defect and therefore biologically a “BRCA-like” tumour. Of these 21 BRCA-like cases, only 3 invasive BCs had clear somatic bi-allelic inactivation of an HBC gene that could explain the high HRDetect score (2 BRCA1 and 1 BRCA2). One of the 21 BRCA-like cases had a germline variant of unknown significance (VUS) in an HBC gene (a BRCA2 missense variant in an ER+ tumour. The tumour showed loss of the wild-type allele). Of the BRCA-like cases, 71% were ER positive which included 3 invasive lobular cancers. Conclusion: Tumour sequencing identified three times as many cases that might be eligible for HR repair defect targeted therapy than germline testing alone (16% versus 5.2%). Unlike a previous study (Ann Oncol., 2019 30:1071-1079), the majority of the BRCA-like BCs were ER+ with no evidence of a VUS in a known HBC gene suggesting these are driven by novel germline or somatic mutations in genes involved in DNA HR repair. Citation Format: Ian Campbell, Lisa Devereux, Kirsten Hogg, Luxi Lal, Michelle Sinclair, Lesley Stafford, Magnus Zethoven, Anita Skandarajah, Paul James, Geoffrey Lindeman, Bruce Mann, Dilanka De Silva. The MAGIC study: Universal whole genome tumour and germline sequencing of newly diagnosed breast cancer identifies a high proportion of ER+ BRCA-like tumours [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-08-11.

Abstract PO3-14-08: Metaplastic Breast Cancer: Whole Genome Sequencing Analysis and Emerging Therapeutic Targets

Abstract Background: Metaplastic breast cancer (MpBC) is a rare histologic subtype of triple negative breast cancer (TNBC) with an aggressive behavior and poor outcome. Previous studies by whole-exome and targeted sequencing showed that MpBCs display a dominant mutational signature 3, associated to homologous recombination (HR) deficiency (HRD). Tumors displaying HRD are associated with response to therapies causing double strand breaks. Nonetheless, MpBCs consistently display significantly lower rates of response to neoadjuvant chemotherapy than non-metaplastic TNBCs and effective therapeutic alternatives are an unmet clinical need. Here we sought to assess the HR features of MpBCs using whole-genome sequencing (WGS), and to evaluate the expression in MpBCs of antigens that are targets of novel antibody drug conjugates (ADCs) in common forms of breast cancer. In addition, we sought to assess the efficacy of ADCs and drugs targeting the DNA damage response (DDR) using preclinical MpBC models. Materials and Methods: We subjected 25 primary MpBC to WGS. HRDetect, the ‘gold standard’ tool for assessment of HRD, was utilized. Mutational signatures were inferred using Signal. 31 MpBCs were subjected to RNA-sequencing to assess the gene expression levels ADC targets compared to non-MpBCs from The Cancer Genome Atlas (TCGA). Efficiency of ADCs and DDR drugs was evaluated using the MpBC cell models HCC1806, BT549 and Hs578T in vitroand/or using HCC1806 xenografts in athymic mice in vivo. Results: Although the majority of MpBCs (13/25) displayed a dominant SBS mutational signature 3, only a small subset of cases (3/25) were classified as HRD by HRDetect. These three bona fide HRD cases were found to harbor bi-allelic inactivation of BRCA1 (n=2) or RAD51C (n=1), and displayed various genomic features of HRD, including deletions with microhomology, enrichment in tandem duplications, rearrangement signatures 3 and 5, and high large scale state transitions (LST) and telomeric allelic imbalance (NtAI) scores. Most MpBCs (22/25), however, were not classified as HRD by HRDetect displaying an incomplete HRD phenotype, with only partial genomic features of HRD. 52% of MpBCs (13/25) displayed APOBEC mutagenesis exposure, which was present in the three bona fide HRD MpBCs. RNA-sequencing analyses revealed detectable ERBB2 and TROP2 expression in all MpBCs interrogated, although at lower levels than in non-MpBCs from TCGA. The HCC1806, BT549 and Hs578T MpBC cell models were found to be sensitive to Sacituzumab Govitecan and Trastuzumab Deruxtecan (T-DXd) in cell viability and colony formation assays, and to T-DXd in vivo. These MpBC cell models were also found to be sensitive to DDR compounds, such as ATR, WEE1 and PKMYT1 inhibitors in vitro, alone and in combination with T-DXd or Sacituzumab Govitecan. Conclusions: Most MpBCs display an incomplete HRD genomic scar, except for those cases harboring bi-allelic inactivation of HRD genes. APOBEC mutagenesis is operative in MpBCs and co-exists with HRD. MpBCs express ERBB2 and TROP2, and MpBC cell models are sensitive to T-DXd and Sacituzumab govitecan, as well as DDR compounds, targeting the incomplete HRD phenotype of MpBCs, alone and in combination. Taken together, these findings warrant further studies in patients with MpBCs testing the therapeutic efficacy of agents targeting the incomplete HRD phenotype, as well as the expression of ADC targets in these aggressive rare forms of TNBCs. Citation Format: Fresia Pareja, Andrea Gazzo, Higinio Dopeso, David Brown, Pier Selenica, Yingjie Zhu, Juan Blanco-Heredia, Laxmi Gusain, Xin Pei, Giacomo Montagna, Nour Abuhadra, Hanna Y Wen, Edi Brogi, Nadeem Riaz, Sarat Chandarlapaty, Maurizio Scaltriti, Larry Norton, Simon Powell, Jorge Reis-Filho, Britta Weigelt. Metaplastic Breast Cancer: Whole Genome Sequencing Analysis and Emerging Therapeutic Targets [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-08.

Abstract GS03-04: Novel Mechanisms of CDH1 Inactivation in Breast Invasive Lobular Carcinoma Unveiled by the Integration of Artificial Intelligence and Genomics

Abstract Background: Invasive lobular carcinoma (ILC) of the breast is the second most common histologic subtype of breast cancer (BC), following invasive ductal carcinoma of no special type (IDC-NST). The hallmark histologic feature of ILC is cellular discohesiveness, the result of bi-allelic inactivation of CDH1, and represents an important genotypic-phenotypic correlation in BC. Although most ILCs harbor CDH1 loss-of function mutations associated to loss-of-heterozygosity (LOH) of the wild type-allele, a subset of ILCs lack these alterations despite displaying a typical lobular phenotype. Here, we sought to identify alternative molecular mechanisms converging on CDH1 inactivation by employing an integrative artificial intelligence (AI) and genomics approach. Materials and Methods: A genomics-driven AI-based algorithm using hematoxylin and eosin (H&amp;E) whole slide images (WSIs) as input, previously developed to detect bi-allelic CDH1 mutations (inactivating mutation associated to LOH) in BC was employed. WSIs of 1,057 BCs including ILCs (n=187) and non-lobular BCs (n=870) previously subjected to FDA-cleared tumor/normal targeted sequencing were subjected to analysis with the AI-based algorithm. Cases predicted to harbor CDH1 bi-allelic mutations by the AI-model but lacking CDH1 bi-allelic mutations by targeted sequencing were assessed through targeted sequencing data re-analysis, CDH1 gene promoter methylation evaluation and/or whole genome sequencing analysis. Results: AI-based analysis WSIs corresponding to 1,057 BCs resulted in the identification of 34 cases found to lack CDH1 bi-allelic mutations by targeted sequencing but predicted to harbor these genetic alterations by the AI-based model. CDH1 gene promoter methylation assessment revealed CDH1 promoter methylation in 18 cases. Targeted sequencing data reanalysis revealed other genetic mechanisms of CDH1 inactivation including CDH1 homozygous deletions (n=3), intragenic deletion with LOH (n=1), and likely pathogenic non-coding CDH1 alterations associated with LOH (n=2). WGS analysis of an ILC revealed a novel deleterious CDH1 fusion stemming from translocation t(13;16), resulting in loss of the 5’UTR, transcription start site and exons 1 and 2 of CDH1, associated with complete loss of E-cadherin protein expression. Taken together, we identified alternative/novel mechanisms of bi-allelic CDH1inactivation in 74% (25/34) cases analyzed. Conclusions: By applying an AI-based algorithm trained to detect a genetic alteration (i.e., CDH1 bi-allelic mutations), we were able to identify alternative epigenetic and genetic molecular mechanisms of CDH1 inactivation in ILCs, including novel non-coding CDH1 genetic alterations and a new inactivating CDH1 fusion gene. These findings indicate that molecular mechanisms affecting a single gene or process converging on the same phenotype can be unveiled by the integration of AI and genomics, highlighting the robustness of this approach for the discovery of novel biology. Citation Format: Fresia Pareja, Higinio Dopeso, Yikan Wang, Andrea Gazzo, David Brown, Pier Selenica, Jan Bernhard, Fatemeh Derakhshan, Edaise M. da Silva, Lorraine Colon-Cartagena, Thais Basili, Antonio Marra, Jillian Sue, Qiqi Ye, Arnaud Da Cruz Paula, Selma Yeni, Xin Pei, Hunter Green, Kaitlyn Gill, Yingjie Zhu, Matthew Lee, Ran Godrich, Adam Casson, Britta Weigelt, Nadeem Riaz, Hanna Y Wen, Edi Brogi, Matthew Hanna, Diana Mandelker, Jeremy Kunz, Brandon Rothrock, Sarat Chandarlapaty, Christopher Kanan, Gerard Oakley III, David Klimstra, Thomas Fuchs, Jorge Reis-Filho. Novel Mechanisms of CDH1 Inactivation in Breast Invasive Lobular Carcinoma Unveiled by the Integration of Artificial Intelligence and Genomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-04.

Abstract PO3-24-06: Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers

Abstract Background Pathogenic BRCA1 and BRCA2 (BRCA1/2) germline mutations contribute to hereditary breast cancer and are linked with increased risk of other cancers. Paradoxically, bi-allelic inactivation of BRCA1/2 (bBRCA1/2) is embryonically lethal. Although TP53 loss can at least in part mitigated the deleterious impact of Brca1 deficiency in mice, this is insufficient in human cells to compensate for decreased proliferative capacity. What other compensatory mechanisms facilitate oncogenesis remains unclear. Methods We performed an integrative analysis of cancer genomes from bBRCA1/2 tumors and CRISPR/Cas9 screens from cellular models to identify novel gene candidates that facilitate synthetic viability. First, we analyzed cancer genomes from ovarian and breast cancer derived from TCGA and identified genomic loci, which were enriched for recurrent copy number alterations (CNAs) in bBRCA1/2 tumors. We subsequently identified genes from these CNA loci that had corresponding changes in gene expression. Then, we used high-throughput genome-wide CRISPR/Cas9 knockout screens in BRCA1/2−/− cell lines (BRCA1−/- in RPE1 cells, BRCA2−/− in DLD1 cells) to identify which of these genes increased cell proliferation in vitro. Lastly, we experimentally validated these synthetic viable interactions using an orthogonal MCF12a system to evaluate changes in colony formation with a clonogenic cell survival assay. Results In human cancer genomes, we identified that bBRCA1/2 tumors harbored recurrent large-scale genetic deletions significantly more frequent than histologically matched control tumors. We identified 148 cytobands (23 distinct genomic loci) frequently deleted in bBRCA1/2 ovarian cancers (N=92), and 90 cytobands (15 distinct genomic loci) in bBRCA1/2 ER+ breast cancers (N=30). Subsequent computational analysis of gene expression data identified transcriptionally decreased genes in the frequently deleted loci. Cross-referencing with CRISPR/Cas9 screens in BRCA1/2−/− cells, we ultimately identified 277 and 218 genes that were altered in human tumors and putatively enhanced viability in BRCA1/2-null models but not in isogenic wild-type cell lines in BRCA1 and BRCA2 models, respectively. We subsequently selected 17 genes that either increased viability in BRCA1, BRCA2, or both contexts for experimental validation. Of these, about 40% (7 of 17) were experimentally validated (RIC8A, GNA12, ATMIN, IPO7, ATXN2, KDM1A, and NUP98) and had evidence of synthetic viability in this cellular context. Interestingly, RIC8A and GNA12 interact with each other, are both involved in G protein-coupled signaling pathway, and are synthetically viable with both BRCA1−/− and BRCA2−/− cells. In a re-analysis of a recently published genomic dataset on metastatic breast cancer, we found that homologous recombination deficient tumors developed additional loss of function mutations in RIC8A in the metastatic setting. Conclusions This study provides insights into the oncogenesis of BRCA1/2 malignancies and describes a high-throughput framework to identify synthetic viability interactions and causal driver genes affected by large-scale CNAs in human cancers. Citation Format: Yingjie Zhu, Xin Pei, Ardijana Novaj, Jeremy Setton, Fatemeh Derakhshan, Pier Selenica, Niamh McDermott, Sonali Sinha, Atif Khan, Simon Powell, Jorge Reis-Filho, Nadeem Riaz. Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-06.

Atypical Teratoid/Rhabdoid Tumor with Retained SMARCB1 (INI1) Expression and Rare SMARCA4 Gene Mutation: A Case Report of a Pediatric Patient

Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive tumors of the central nervous system (CNS), accounting for 1–3% of all pediatric CNS tumors. In general, AT/RTs are associated with biallelic inactivation of SMARCB1, resulting in the loss of expression of the integrase interactor 1 (INI1) protein. In this report, we describe the clinical course of an infant patient who presented with fatigue, postprandial vomiting, and disability of left side movement. Histological examination revealed classical features indicative of rhabdoid tumors, yet an atypical immunohistochemical profile with preserved INI1 expression was observed. Molecular diagnostics further elucidated the presence of a heterozygous frameshift variant, SMARCA4 c.2693del, p.(Asn898Thrfs*12), underscoring the distinctive genetic foundations of the case. Surgical resection of the tumor was administered with subsequent chemotherapy to the patient, but the condition worsened dynamically, and a decision was made to give the patient palliative therapy. We report on a patient with AT/RT caused by a rare mutation of the SMARCA4 gene and an aggressive course of disease to provide more information and characteristics of these tumors.

A cohort study of CNS tumors in Multiple Endocrine Neoplasia Type 1.

Multiple Endocrine Neoplasia Type-1 (MEN1) is thought to increase the risk of meningioma and ependymoma. Hereby, we aimed to describe the frequency, the incidence and specific clinical and histological features of CNS tumors in the MEN1 population (except pituitary tumors). The study population included patients harboring CNS tumors diagnosed with MEN1 syndrome after 1990 and followed-up in the French MEN1 national cohort. Standardized incidence rate (SIR) was calculated based on the French Gironde CNS tumors registry. Genomic analyses were performed on somatic DNA from 7 CNS tumors including meningiomas and ependymomas from MEN1 patients, then in 50 sporadic meningiomas and ependymomas. Twenty-nine CNS tumors were found among the 1498 symptomatic patients (2%) (incidence=47.4/100'000 person-years; SIR=4.5), including 12 meningiomas (0.8%) (incidence=16.2/100'000; SIR=2.5), 8 ependymomas (0.5%) (incidence=10.8/100'000; SIR=17.6), 5 astrocytomas (0.3%) (incidence=6.7/100'000; SIR=5.8), and 4 schwannomas (0.3%) (incidence=5.4/100'000; SIR=12.7). Meningiomas in MEN1 patients were benign, mostly meningothelial, with 11 years earlier onset compared to the sporadic population and an F/M ratio of 1/1. Spinal and cranial ependymomas were mostly classified WHO grade 2. A biallelic MEN1 inactivation was observed in 4/5 ependymomas and 1/2 meningiomas from the MEN1 patients, whereas MEN1 deletion in one allele was present in respectively 3/41 and 0/9 sporadic meningiomas and ependymomas. Incidence of each CNS tumor was higher in the MEN1 population than in the French general population. Meningiomas and ependymomas should be considered part of the MEN1 syndrome, but somatic molecular data are missing to conclude for astrocytomas and schwannomas.

Abstract NG01: Germline-mediated immunoediting sculpts breast cancer subtypes and metastatic proclivity

Abstract NG01: Germline-mediated immunoediting sculpts breast cancer subtypes and metastatic proclivity

A glycolytic metabolite bypasses “two-hit” tumor suppression by BRCA2

Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.

Tumor analysis of MMR genes in Lynch-like syndrome: Challenges associated with results interpretation.

Up to 70% of suspected Lynch syndrome patients harboring MMR deficient tumors lack identifiable germline pathogenic variants in MMR genes, being referred to as Lynch-like syndrome (LLS). Previous studies have reported biallelic somatic MMR inactivation in a variable range of LLS-associated tumors. Moreover, translating tumor testing results into patient management remains controversial. Our aim is to assess the challenges associated with the implementation of tumoral MMR gene testing in routine workflows. Here, we present the clinical characterization of 229 LLS patients. MMR gene testing was performed in 39 available tumors, and results were analyzed using two variant allele frequency (VAF) thresholds (≥5% and ≥10%). More biallelic somatic events were identified at VAF ≥ 5% than ≥10% (35.9% vs. 25.6%), although the rate of nonconcordant results regarding immunohistochemical pattern increased (30.8% vs. 20.5%). Interpretation difficulties question the current utility of the identification of MMR somatic hits in the diagnostic algorithm of suspected LS cases.

Mono- and Biallelic Inactivation of Huntingtin Gene in Patient-Specific Induced Pluripotent Stem Cells Reveal HTT Roles in Striatal Development and Neuronal Functions.

Mutations in the Huntingtin (HTT) gene cause Huntington's disease (HD), a neurodegenerative disorder. As a scaffold protein, HTT is involved in numerous cellular functions, but its normal and pathogenic functions during human forebrain development are poorly understood. To investigate the developmental component of HD, with a specific emphasis on understanding the functions of wild-type and mutant HTT alleles during forebrain neuron development in individuals carrying HD mutations. We used CRISPR/Cas9 gene-editing technology to disrupt the ATG region of the HTT gene via non-homologous end joining to produce mono- or biallelic HTT knock-out human induced pluripotent stem cell (iPSC) clones. We showed that the loss of wild-type, mutant, or both HTT isoforms does not affect the pluripotency of iPSCs or their transition into neural cells. However, we observed that HTT loss causes division impairments in forebrain neuro-epithelial cells and alters maturation of striatal projection neurons (SPNs) particularly in the acquisition of DARPP32 expression, a key functional marker of SPNs. Finally, young post-mitotic neurons derived from HTT-/- human iPSCs display cellular dysfunctions observed in adult HD neurons. We described a novel collection of isogenic clones with mono- and biallelic HTT inactivation that complement existing HD-hiPSC isogenic series to explore HTT functions and test therapeutic strategies in particular HTT-lowering drugs. Characterizing neural and neuronal derivatives from human iPSCs of this collection, we show evidence that HTT loss or mutation has impacts on neuro-epithelial and striatal neurons maturation, and on basal DNA damage and BDNF axonal transport in post-mitotic neurons.

Abstract 1202: Signatures of selection for biallelic inactivation in tumor suppressor genes across cancer types

Abstract Background: The canonical model of tumor suppressor gene (TSG)-mediated oncogenesis, Knudson’s “two hit hypothesis,” posits that loss of both alleles is necessary for TSG inactivation. Many key TSGs accordingly exhibit near universal biallelic loss in associated cancers (e.g., APC in colorectal cancer and RB1 in retinoblastoma). However, because the majority of large-scale cancer genomics studies have utilized non-allele-specific copy number analysis methods, neither the extent of biallelic inactivation across TSGs, nor patterns of selective pressure for biallelic inactivation of TSGs, nor the functional and translational consequences of biallelic inactivation, are well-understood. Methods: Here, through allele-specific analysis of sequencing data from 48,179 cancer patients, we define the prevalence, selective pressure for, and functional consequences of biallelic inactivation across TSGs. We classify TSGs according to their patterns of selection for or against biallelic inactivation across cancer types. Finally, we investigate in detail the association of biallelic status with gene expression and clinical outcome for two TSGs, KEAP1 (in lung adenocarcinoma) and APC (in lung and prostate adenocarcinoma). Results: TSGs largely assort into distinct classes associated with either pan-cancer (Class I) or lineage-specific (Class II) patterns of biallelic loss, while some TSGs, mostly transcription factors, were predominantly monoallelic (Class III/IV). We discover that gene expression and co-mutation patterns indicate selection for biallelic losses in noncanonical contexts, including APC in lung and prostate adenocarcinomas. Selection for biallelic inactivation is also significantly elevated among variants of unknown significance (VUS) of several TSGs, including in KEAP1 in lung adenocarcinoma, nominating these VUS as worthy of in depth functional characterization. Patients with KEAP1 VUS also show similarly poor survival and response to chemoimmunotherapy to patients with known KEAP1 driver mutations, indicating that KEAP1 VUSs phenocopy established KEAP1 oncogenic alleles, and that zygosity, rather than variant classification, is prognostic of therapeutic response. Conclusion: TSGs vary significantly in their patterns of biallelic inactivation, and can be classified according to whether and how consistently, across cancer types, they are selected for loss of both copies. Additionally, taking into account the zygosity of TSG alteration events is often necessary to understand their phenotypic and clinical consequences, and can be useful in identifying novel driver events. Citation Format: Mark R. Zucker, Maria A. Perry, Arielle Elkrief, Anton Safonov, Rohit Thummalapalli, Samuel I. Gould, Debyani Chakravarty, Rose Brannon, Marc Ladanyi, Pedram Razavi, Mark T. Donoghue, Yonina R. Murciano-Goroff, Francisco J. Sánchez-Rivera, Yuan Chen, Ronglai Shen, Sarat Chandarlapaty, David B. Solit, Nikolaus Schultz, Michael F. Berger, Adam J. Schoenfeld, Jason Chang, Ed Reznik, Chaitanya Bandlamudi. Signatures of selection for biallelic inactivation in tumor suppressor genes across cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1202.