Somatic Hits Research Articles

Abstract IA16: RASopathies: the other face of RAS signaling upregulation

Abstract RAS proteins control a major intracellular signaling network that, depending on the cellular context, mediates response to external stimuli to control diverse biological functions. Within this network, signal flow through the RAF-MEK-ERK (MAPK, hereafter) pathway, modulates a wide array of cellular processes as well as early and late developmental programs. Signaling through the RAS-MAPK cascade is tightly controlled, and its enhanced activation has been known for decades to represent a major event in oncogenesis. Unexpectedly, discoveries derived from a massive disease gene hunting effort have more recently established a picture in which the upregulation of this signaling cascade underlies a group of clinically related developmental disorders collectively known as “RASopathies”, which share cardiac defects, reduced postnatal growth, variable cognitive deficits, facial dysmorphism, ectodermal and musculoskeletal anomalies, and variably increased risk for certain malignancies as major features. Based on the relatively high prevalence of some of these disorders (i.e., Noonan syndrome and neurofibromatosis type 1), the dysregulation of this signaling pathway represents one of the most common events affecting development. RASopathies are caused by mutations in genes encoding RAS proteins and structurally/functionally related GTPases, regulators of RAS function, modulators of RAS interaction with effectors or downstream signal transducers of the MAPK backbone. A subset of these genes have previously been implicated in cancer. Many of these RASopathy-causing alleles encode proteins with upregulated functional behavior, but with less activating strength compared to those contributing to oncogenesis. In these genes, a largely non-overlapping spectrum of germline and somatic mutations is generally observed. On the other hand, a few disorders result from a protein with defective function. In these, the implicated proteins negatively control signal flow, and the pathogenic event contributing to oncogenesis is generally represented by a second somatic hit involving the retained wild-type allele. The understanding of the molecular mechanisms involved in the pathogenesis of RASopathies has provided novel and relevant insights in cancer research. The identification of PTPN11 as major disease gene underlying Noonan syndrome represented the basis for the discovery that SHP2, the protein encoded by PTPN11, functions as an oncoprotein in childhood hematologic malignancies when mutated. More recently, genomic sequencing of RASopathies has allowed to identify circuits and players with previously unrecognized regulatory role on RAS function and signaling through the MAPK cascade. Here is provided an overview on the genes implicated in this group of developmental disorders, the molecular basis underlying the differential impact of germline and somatic mutations in development and cancer, the unanticipated molecular mechanisms converging toward the dysregulation of this signaling cascade, and major clinically relevant genotype-phenotype correlations. Citation Format: Marco Tartaglia. RASopathies: the other face of RAS signaling upregulation [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA16.

Somatic Double Inactivation of NF1 Associated with NF1-Related Pectus Excavatum Deformity

Neurofibromatosis type 1 (NF1) is a neurocutaneous genetic disorder with a broad spectrum of associated signs and symptoms, including skeletal anomalies. The association of NF1 with anterior chest wall deformities has been recently reported, especially the pectus excavatum (PE). Over the years, several authors have suggested loss of heterozygosity (LOH) as the possible pathogenic mechanism underlying the development of the typical NF1 skeletal features. Here, we report a NF1 patient with severe chest deformity and harboring the germline heterozygous pathogenic NF1 variant NM_001042492.3: c.4271delC p.(Ala1424Glufs ∗ 4). Through next-generation sequencing (NGS), we investigated the affected cartilage from the PE deformity and identified the additional frameshift variant NM_001042492.3: c.2953delC p.(Gln985Lysfs ∗ 7), occurring as a somatic NF1 second hit mutation. Exome sequencing confirmed the absence of additional variants of potential pathogenic relevance. Western blot analysis showed the absence of wild-type NF1 protein in the cartilage of the patient, consistent with a somatic double inactivation (SDI) of NF1. Taken together, our findings support the role of SDI in NF1-related PE, widening the spectrum of the pathophysiological mechanisms involved in NF1-related skeletal features.

Mismatch repair deficiency testing in Lynch syndrome-associated urothelial tumors.

Lynch syndrome-associated cancer develops due to germline pathogenic variants in one of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6 or PMS2. Somatic second hits in tumors cause MMR deficiency, testing for which is used to screen for Lynch syndrome in colorectal cancer and to guide selection for immunotherapy. Both MMR protein immunohistochemistry and microsatellite instability (MSI) analysis can be used. However, concordance between methods can vary for different tumor types. Therefore, we aimed to compare methods of MMR deficiency testing in Lynch syndrome-associated urothelial cancers. Ninety-seven urothelial (61 upper tract and 28 bladder) tumors diagnosed from 1980 to 2017 in carriers of Lynch syndrome-associated pathogenic MMR variants and their first-degree relatives (FDR) were analyzed by MMR protein immunohistochemistry, the MSI Analysis System v1.2 (Promega), and an amplicon sequencing-based MSI assay. Two sets of MSI markers were used in sequencing-based MSI analysis: a panel of 24 and 54 markers developed for colorectal cancer and blood MSI analysis, respectively. Among the 97 urothelial tumors, 86 (88.7%) showed immunohistochemical MMR loss and 68 were successfully analyzed by the Promega MSI assay, of which 48 (70.6%) were MSI-high and 20 (29.4%) were MSI-low/microsatellite stable. Seventy-two samples had sufficient DNA for the sequencing-based MSI assay, of which 55 (76.4%) and 61 (84.7%) scored as MSI-high using the 24-marker and 54-marker panels, respectively. The concordance between the MSI assays and immunohistochemistry was 70.6% (p = 0.003), 87.5% (p = 0.039), and 90.3% (p = 1.00) for the Promega assay, the 24-marker assay, and the 54-marker assay, respectively. Of the 11 tumors with retained MMR protein expression, four were MSI-low/MSI-high or MSI-high by the Promega assay or one of the sequencing-based assays. Our results show that Lynch syndrome-associated urothelial cancers frequently had loss of MMR protein expression. The Promega MSI assay was significantly less sensitive, but the 54-marker sequencing-based MSI analysis showed no significant difference compared to immunohistochemistry. Data from this study alongside previous studies, suggest that universal MMR deficiency testing of newly diagnosed urothelial cancers, using immunohistochemistry and/or sequencing-based MSI analysis of sensitive markers, offer a potentially useful approach to identification of Lynch syndrome cases.

Microsatellite instability in noncolorectal and nonendometrial malignancies in patients with Lynch syndrome.

Individuals with Lynch syndrome are at increased hereditary risk of colorectal and endometrial carcinomas with microsatellite instability (MSI-H) and mismatch repair-deficiency (dMMR), which make these tumors vulnerable to therapy with immune checkpoint inhibitors. Our aim is to assess how often other tumor types in these individuals share these characteristics. We retrieved the full tumor history of a historical clinic-based cohort of 1745 individuals with Lynch syndrome and calculated the standardized incidence ratio for all tumor types. MSI status, somatic second hit alterations, and immunohistochemistry-based MMR status were analyzed in 236 noncolorectal and nonendometrial malignant tumors. In individuals with Lynch syndrome MSI-H/dMMR occurred both in Lynch-spectrum and in non-Lynch-spectrum malignancies (85% vs 37%, P < .01). MSI-H/dMMR malignancies were found in nearly all non-Lynch-spectrum tumor types. Almost all breast carcinomas had medullary features, and most of them were MSI-H/dMMR. Breast carcinoma with medullary features were shown to be associated with Lynch syndrome (standardized incidence ratio = 38.8, 95% confidence interval = 16.7 to 76.5). In individuals with Lynch syndrome, MSI-H/dMMR occurs in more than one-half of the malignancies other than colorectal and endometrial carcinomas, including tumor types without increased incidence. The Lynch-spectrum tumors should be expanded to breast carcinomas with medullary features. All malignancies in patients with Lynch syndrome, independent of subtype, should be tested for MSI-H/dMMR in case therapy with immune checkpoint inhibitors is considered. Moreover, Lynch syndrome should be considered an underlying cause of all MSI-H/dMMR malignancies other than colorectal and endometrial carcinomas.

Abstract 3522: DICER1 Platform domain missense variants led to tumor susceptibility via failure of precursor miRNA binding

Abstract DICER1 is a key protein in the micro(mi)RNA biogenesis pathway responsible for cleaving precursor (pre)-miRNA stemloops with its RNase IIIa and IIIb domains into mature single stranded miRNAs. DICER1 syndrome is a pleiotropic tumor predisposition syndrome in which clinical manifestations are normally first seen during childhood. This genetic disorder is defined by the presence of inherited or de novo germline pathogenic variants (GPVs) in the DICER1 gene. While many germline loss of function variants fail to generate DICER1 protein, a subset of patients has germline missense variants that are predicted to produce full-length DICER1 protein that cluster in the DICER1 Platform domain, a region that plays a role in binding to the 5’ phosphate of pre-miRNA stemloops. Due to the uncertainty surrounding the pathogenicity of missense variants in DICER1 syndrome, clinical actionability can be limited. To determine how Platform mutations predispose to DICER1 syndrome we characterized a number of these variants using in vitro pre-miRNA binding and cleavage assays. Interestingly, four of these variants displayed impaired pre-miRNA binding and processing, with three of them localizing to a hydrophobic pocket in the Platform domain. In keeping with in vitro results, DICER1 mutants displayed impaired miRNA processing when introduced into mesenchymal stem cells. Furthermore, we identified a second somatic hit in the RNase IIIb domain of DICER1 in thyroid nodular hyperplasia DNA from a patient affected by one of these variants. Taken together, these results identify a key region in the DICER1 protein that is essential for pre-miRNA processing and help the classification of these germline variants as pathogenic to ensure proper clinical management of affected patients. Additionally, these results provide insight on the molecular basis involved in DICER1 syndrome predisposition. Citation Format: Dylan Pelletier, Anne-Laure Chong, Sophie Albert, Marc Fabian, William Foulkes. DICER1 Platform domain missense variants led to tumor susceptibility via failure of precursor miRNA binding. [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 3522.

Abstract 5214: Expanding the spectrum of germline-driven cancers by leveraging population-scale targeted tumor and normal sequencing

Abstract Large case-control and familial studies have established clear cancer-specific risk profiles for several key cancer predisposition genes (CPGs). For example, germline pathogenic variants (PVs) in BRCA1/2 (gBRCA) are associated with increased risk for developing breast, ovarian, pancreatic, and prostate cancers. However, the extent to which gBRCA mutations are involved in mediating the tumorigenesis of other cancer types remains challenging to characterize. We hypothesized that integrating orthogonal features such as selection for biallelic inactivation of the PVs and depletion of canonical somatic drivers among the carriers can enrich the signal for identifying novel gene and cancer type associations. We then extend this framework to identify novel CPGs as well as to understand how tumors arise in patients with PVs in oncogenes. To study this, we leveraged the prospective MSK-IMPACT matched tumor-normal sequencing cohort of 49,291 patients across 77 major cancer types. We study 90 well-known CPGs as well as &amp;gt;300 cancer genes not previously associated with cancer predisposition. Overall, 8% (N=3,964) of patients harbored a PV in high or moderate penetrance CPGs. We identified 90 gene and cancer type associations with enrichment for biallelic inactivation (q&amp;lt;0.05), including 19 novel findings among clinically actionable genes. For example, we find enrichment for biallelic inactivation of BRCA1/2 in unexpected lineages such as hepatobiliary, endometrioid, and ampullary cancers. These tumors were also significantly depleted for somatic gain-of-function driver alterations. Hepatobiliary cancers with gBRCA1 mutations were also enriched for somatic loss of NF1. Among carriers of PVs in oncogenes, we observe two possible mechanisms of first somatic hit towards malignant transformation. We find enrichment for copy number gain or copy neutral loss of heterozygosity of the germline PV in thyroid cancers with a PV in RET. We also find that lung cancers with a germline PV in EGFR frequently developed additional somatic point mutations located in cis with the PV. Investigating genes with no prior association with germline predisposition to cancer, we find evidence for KEAP1 and CIC as likely novel CPGs. Lung (n=8) and thyroid (n=4) cancers with deleterious germline variants in KEAP1 were characterized by loss of the wild-type allele, co-occurring somatic STK11 mutations, and depletion of canonical drivers such as EGFR. We also found biallelic loss of CIC in two patients with Neuroblastoma, each carrying a different germline loss-of-function mutation in CIC. Both tumors were also negative for MYCN and ALK defects. Collectively, our findings expand our understanding of cancer predisposition in cancer, shed new insights into how tumors arise in germline carriers, and provide a framework for identifying new CPGs using population scale tumor-normal paired clinical sequencing data. Citation Format: Miika Mehine, Rebecca Caeser, Yelena Kemel, Daniel Muldoon, Sebastià Franch-Expósito, A. Rose Brannon, Aijazuddin Syed, Ozge Ceyhan-Birsoy, Maksym Misyura, Panieh Terraf, David B. Solit, Marc Ladanyi, Kenneth Offit, Zsofia K. Stadler, Diana L. Mandelker, Yonina R. Murciano-Goroff, Charles M. Rudin, Michael F. Berger, Chaitanya Bandlamudi. Expanding the spectrum of germline-driven cancers by leveraging population-scale targeted tumor and normal sequencing. [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 5214.

Inherited mutations affecting the SRCAP complex are central in moderate-penetrance predisposition to uterine leiomyomas

Uterine leiomyomas (ULs) are benign smooth muscle tumors that are common in premenopausal women. Somatic alterations in MED12, HMGA2, FH, genes encoding subunits of the SRCAP complex, and genes involved in Cullin 3-RING E3 ligase neddylation are mutually exclusive UL drivers. Established predisposition genes explain only partially the estimated heritability of leiomyomas. Here, we examined loss-of-function variants across 18,899 genes in a cohort of 233,614 White European women, revealing variants in four genes encoding SRCAP complex subunits (YEATS4, ZNHIT1, DMAP1, and ACTL6A) with a significant association to ULs, and YEATS4 and ZNHIT1 strikingly rank first and second, respectively. Positive mutation status was also associated with younger age at diagnosis and hysterectomy. Moderate-penetrance UL risk was largely attributed to rare non-synonymous mutations affecting the SRCAP complex. To examine this disease phenotype more closely, we set out to identify inherited mutations affecting the SRCAP complex in our in-house sample collection of Finnish individuals with ULs (n= 860). We detected one individual with an ACTL6A splice-site mutation, two individuals with a YEATS4 missense mutation, and four individuals with DMAP1 mutations: one splice-site, one nonsense, and two missense variants. These individuals had large and/or multiple ULs, were often diagnosed at an early age, and many had family history of ULs. When a somatic second hit was found, ACTL6A and DMAP1 were silenced in tumors by somatic mutation and YEATS4 by promoter hypermethylation. Decreased H2A.Z staining was observed in the tumors, providing further evidence for the pathogenic nature of the germline mutations. Our results establish inactivation of genes encoding SRCAP complex subunits as a central contributor to moderate-penetrance UL predisposition.

Abstract IA026: Novel insights into colorectal pre-cancers from hereditary cancer syndromes: From omics to interception

Abstract It has been an effort of more than three decades to obtain a detailed annotation of the genomic events initially described in the seminal multi-step genetic model of the colorectal adenoma-carcinoma sequence by Vogelstein, Kinzler, and Fearon. Initial genomic studies conducted in colorectal premalignancy interrogated restricted numbers of lesions and assessed only a handful of genomic aberrations. Therefore, the deployment of next-generation sequencing technologies allowed for an updated and expanded understanding of the genomic landscape of premalignancy. This first generation of studies used bulk sequencing (mostly whole exome sequencing of biopsies or crypts) in early adenomas from patients with Familial Adenomatous Polyposis (FAP), identifying a large number of oncogenic mutations (&amp;gt;200 somatic hits in 25 adenomas), early clonal selection, and a mutational rate that overlapped with early-stage carcinomas. In fact, consistent with mathematical models of carcinogenesis, a quarter of the mutational load (all passenger mutations) observed in adenomas was present in adjacent, apparently normal, mucosa. This provided direct evidence that most of the genomic variation present in colorectal cancers is accumulated prior to the acquisition of a driver event in at-risk tissues, with the variation probably secondary to the self-renewal process of stem cells. Moreover, expression profiling of pre-cancers combined with mutational assessment enabled the study of the immune landscape of pre-malignancy as well as the catalogue of mutated neoantigens. Most of the studies on genomic annotation of intestinal carcinogenesis have come mainly from the study of premalignant lesions from hereditary CRC syndrome patients, which are thought to recapitulate the progression of the two major pathways: FAP for chromosomal instability (non-hypermutant) and Lynch Syndrome for mismatch repair deficiency (hypermutant). There are several intrinsic advantages of using hereditary cancer syndromes for molecular characterization of premalignancy: (I) the accelerated pace of carcinogenesis, (II) the abundance of tissue to perform analysis, and (III) frequent and close surveillance of patients. The knowledge generated in hereditary premalignancy has subsequently been translated to sporadic pre-cancers, including interrogation of the application of the consensus molecular sub-classification (CMS) to pre-cancers. The current iteration of -omics studies in pre-cancers fostered by the NCI Pre-Cancer Atlas and the Human Tumor Atlas Network has brought in-depth analysis of premalignancy, leveraging state-of-the-art single-cell sequencing tools. Ultimately, the wealth of genomic knowledge acquired in pre-cancers has started to be translated at the cancer interception level with the design and implementation of clinical trials using immune-modulators and vaccine approaches. Citation Format: Eduardo Vilar-Sanchez. Novel insights into colorectal pre-cancers from hereditary cancer syndromes: From omics to interception [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr IA026.

A Novel Germline PAX5 Single Exon Deletion in a Paediatric Patient with B-Cell Leukaemia

Germline predisposition underlies leukaemia development in children in 5-10% of the cases. Over the past years, several somatic drivers for leukaemia, including IKZF1, RUNX1, ETV6 and PAX5, have also been identified as leukaemia predisposing genes. For example, two germline PAX5 missense variants (p.Arg38His and p.Gly183Ser) have been identified in five families with a high incidence of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). The susceptibility to BCP-ALL in these patients is inherited in an autosomal dominant pattern with an incomplete but relatively high penetrance. The leukemic cells in the patients showed loss of the wild-type allele or a somatic mutation, suggesting a second hit is required for leukaemia development in this context. We present a boy who developed BCP-ALL at 5-years-old, with a father who was diagnosed with acute undifferentiated leukaemia at infancy. Because of his family history, germline testing of the patient and his parents was performed using SNP-based copy number analysis and whole exome sequencing (WES). This revealed a PAX5 exon 6 deletion in the child, which was absent in both parents. The PAX5 deletion causes a frameshift with premature stop in exon 7, resulting in a truncated protein with loss of the C-terminal transactivating and inhibitory domains. In contrast to what has been observed in patients with germline PAX5 missense variants, a second somatic hit in PAX5 was not observed. Genotype analysis, using heterozygous SNPs around the PAX5 deletion, revealed that the PAX5 deletion had occurred on the paternal allele. To test whether this deletion was present in (low) mosaic state in the father, we developed a sensitive breakpoint spanning PCR. Also with this method, the PAX5 deletion was not detected in DNA derived from blood and buccal cells of the father. Since we expected a causative germline variant to be inherited from the father, we re-analysed all sequence and copy number variants in the patient-parents' trio datasets for any other aberrations, but no plausible alternative candidates were identified. Since routine diagnostic analysis of PAX5 deletions is usually performed only in leukaemia samples, germline deletions could potentially be missed, particularly in isolated cases. To investigate the incidence of germline PAX5 deletions, we used a multiplex-ligated probe amplification (MLPA) dataset from 2041 ALL patients, from which we selected 303 cases with single-exon or partial (multiple exon) PAX5 deletion. Subsequently, we performed MLPA on 261 DNA samples from bone marrow or peripheral blood at time of remission. No additional case was found, suggesting that germline PAX5 deletions, if recurrent, are very rare. In conclusion, we present a boy with ALL carrying a de novo germline PAX5 exon 6 deletion leading to a truncated protein. Germline PAX5 deletions have not yet been described in ALL patients but since somatic PAX5 aberrations are common in ALL and germline PAX5 variants have been associated with ALL, we consider that this focal germline PAX5 deletion did contribute to leukaemia development in the boy.

Characteristics of Germline CHEK2 Mutated Patients in a Large Cohort of 2322 Myeloid Malignancies

Background Checkpoint kinase 2 (CHEK2) is a critical mediator of the DNA damage response and plays a role in DNA double strand break-induced apoptosis and cell cycle arrest (Bartek, 2003). Deleterious germline CHEK2 variants (CHEK2germ) have been associated with an increased risk for breast, colon, prostate, thyroid and kidney cancers (Cybulski, 2004). Early studies suggested that germline CHEK2 variants also predispose to myeloid malignancies, including myelodysplastic syndromes (MDS) (Janiszewska, 2018). Recently, CHEK2 was identified as the most frequent germline mutated gene in a cohort of 391 adult patients with AML (Yang, 2022). However, more data is warranted to clarify the role of germline CHEK2 mutations in the development of myeloid malignancies. Here we analyzed the prevalence of germline CHEK2 variants in a large series of unselected patients diagnosed with myeloid malignancies and describe their clinical features. Methods Between February 2018 and June 2022, 2322 consecutive adult patients diagnosed at Saint-Louis hospital, Paris with a myeloid malignancy were included in the study. DNA obtained from bone marrow or peripheral blood cells was subjected to capture-based next-generation sequencing of genes recurrently mutated in myeloid malignancies, including CHEK2. In selected patients, germline mutation testing was performed using Sanger sequencing of DNA obtained from skin fibroblasts or sorted CD3-positive cells from peripheral blood. Results We identified a CHEK2 variant in 38 out of 2322 (1.6%) unrelated patients. We focused on the 24 patients with a variant allele frequency (VAF) equal or higher than 40%, highly suggestive of a germline origin, which was subsequently confirmed in all patients with available germline DNA. Of these, 16 variants (Fig1 A) were considered as pathogenic/likely pathogenic (P/LP) according to the American College of Medical Genetics as well as 3 missense variants which were semi-functional in an in-vivo, yeast-based, functional assay (Delimitsou, 2019). The 5 remaining variants were considered of unknown significance (VUS). Finally, 19 CHEK2germ variants were considered causal for the myeloid malignancy representing a frequency of 0.8% (19/2322). Noteworthy, two out of them (10.5%) had a second somatic hit in CHEK2. (Fig1 B) The median age of the 19 CHEK2germ patients was 75 years (42-87) at diagnosis of the hematological disease. According to the revised WHO classification, there were 7 (37%) MDS-MLD, 2 (11% ) MDS-RS, 4 (21%) MDS-EB, 3 (16%) MPN, 2 (11%) AML, and 1 (5%) ICUS. Karyotype was normal in 7 patients (39%), 8 patients had an isolated abnormality, 3 patients had a complex karyotype and one had cytogenetic failure. Additional driver mutations (Fig 1 B) were identified in most patients (18/19, 95%), however no particular association was noticed. Seven patients (37%) had a personal history of cancer (prostate n=4, breast n=1, thyroid n=1, colon n=1). Five patients (26%) had a family history of hematologic disorders (MPN, n=2, multiple myeloma n=2, ICUS n=1) and 7 patients (37%) of non-hematological cancer (breast n=2, brain n=2, lung n=1, ovary n=1, intestinal n=1). Eleven patients were classified as low-risk MDS according to the IPSS classification with a median survival of 5.6 years. They mainly received ESA (N=2) or lenalidomide (N=2). Among the 3 high-risk MDS patients 2 received hypomethylating agent with a survival of 5 and 1.7 years, the remaining one is still alive after sequential bone marrow transplant. One AML patient is still receiving hypomethylating agent with BCL2-inhibitor in complete remission with a follow up of 1.1 year, while the other AML patient died of relapse after 2 sequential bone marrow transplants. Regarding the 3 MPN patients, 1 is still alive after 1.3 year and the 2 others died 11.5 and 3.8 years after diagnosis. Conclusion This is the largest study addressing the frequency (0.8%) and characteristics of carriers of germline CHEK2 pathogenic variants within patients with myeloid disorders. Their presentation is rather heterogenous with a majority of low-risk MDS. They did not appear to have a more complex karyotype or a poorer prognosis and seems to be less frequent than previously suggested. Compared to more frequent genetic predisposition to myeloid malignancies like DDX41, CHEK2germ hematological malignancies did not seem to represent a specific entity, in line with recent debate on CHEK2germ solid cancer. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prevalence of Pathogenic Germline Mutations in Solid Tumor Predisposition Genes Detected By Tumor-Normal Whole Exome Sequencing Analysis Among Adults with Hematologic Malignancies

Background Approximately 7-10% of cancer patients have pathogenic germline variants (PGVs) in cancer predisposition genes. However, most studies to date have focused on PGVs in patients with solid tumors. Less is known about the prevalence of PGVs in patients with hematologic malignancies (HM). We investigated the prevalence of PGVs in 72 cancer predisposition genes in a cohort of HM cases from our tumor-normal whole exome sequencing (TN-WES) database. Methods We identified 363 unique HM patients with TN-WES performed at our lab (Sema4, Branford, CT) since 2020. Our analysis pipeline calls germline pathogenic/likely pathogenic variants (PGVs) in 72 cancer-predisposing genes (PGV-72) from normal tissue, predominantly buccal samples. These are reported clinically as secondary findings, with the patient's consent. Results The cohort (N=363) was comprised predominantly of patients with lymphoid malignancies (N=299, 82.4%), most commonly myeloma (N=222) and CLL/SLL (N=27). The most common myeloid malignancy was myelodysplastic syndrome (N=25). Of 363 unique HM patients with TN-WES, 33 (9.1%) were found to have PGVs in one of the PGV-72 genes. There was no significant difference between those with lymphoid and myeloid malignancies (8.0% vs. 14.1%, Fisher's exact p=0.15). Among patients with germline findings (N=33), the median age was 67 years (range 23-84). The most commonly mutated genes were APC (N=7), CHEK2 (N=7), MUTYH (N=6), and LZTR1 (N=4). LZTR1 and ATM PGVs were more common in patients with myeloid versus lymphoid malignancies (LZTR1: 4.7% vs 0.3%, p=0.02; ATM: 3.1% vs. 0%, p=0.03). Several founder mutations were identified: all APC variants were p.I1307K; four CHEK2 variants were p.T367Mfs*15 (c.100delC); five MUTYH variants were p.G396D. Two patients had more than one PGV: one patient with myeloma (APC p.I1307K and CHEK2 p.S428F) and one patient with CLL/SLL (CHEK2 p.T367Mfs*15 and HOXB13 p.G84E). One patient with myeloma had a BRCA2 p.S1982Rfs*22 germline variant. All PGVs were heterozygous, and none were associated with a somatic second hit. Conclusions The prevalence of PGVs in 72 cancer predisposition genes was approximately 9% in a set of patients with HM undergoing TN-WES. Most PGVs identified were associated with moderately increased risk for solid tumors. Many of the PGVs identified were founder mutations in APC, CHEK2, and MUTYH, likely reflecting the racial/ethnic composition of our cohort. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Role of MBD4 Mutation in Neoplastic Evolution of Acute Myeloid Leukemia

Unlike pediatric cases, adult patients with myeloid neoplasia (MN) are less often suspected to harbor germline (GL) driver or predisposition alterations. However, recent discoveries of various genes affected by GL alterations linked to long disease anticipation and incomplete penetrance indicate a growing role of familial predisposition in adult MN including, the impact of heterozygous alleles within recessive traits, biallelic (GL/somatic) or compound heterozygous (GL/GL and GL/somatic) configurations of various genes. Recently, genomic profiling of 3 early-onset AMLs identified MBD4 GL loss as an initiator of 5-methylcytosine (5mC)-dependent hypermutation with a 33-fold higher mutational burden (>95% being C>T in the context of a CG)1. In cases from our institution, a biallelic MBD4 c.1291C>T, p.Arg431* & c.1688T>A p.Leu563* (in trans) tracked with AML in brother and sister2. MBD4 is a known DNA glycohydrolase involved in DNA mismatch repair (MMR) essential for reducing genotoxic stress in normal cells. Loss of function mutation in MBD4 may result in compromised MMR leading to genomic instability. MBD4 binds to 5mC and 5-hydroxymethylcytosine (5hmC), oxidation products of TET2 dioxygenases, and initiates a multi-step MMR around methylated CpG DNA. Therefore, we hypothesized that loss of MBD4 may result in reduced MMR leading to increased genomic instability and acquisition of additional lesions which may facilitate clonal evolution. We first analyzed data from 1,692 MN patients (732 MDS and 960 AML) and found 134 (8%) patients with 22 MBD4 variants. Overall, 20 were non-synonymous missense (in 132 pts), while nonsense and frameshift deletion were also observed in 1 case each. After variant filtering and annotation, we retained 17 recurrent missense in 80 patients and 1 nonsense variant in 1 patient at an odds ratio of >1.5 vs. healthy control, for a total of 81 patients harboring MBD4 VUS (Fig.1A). Among them, 4 variants (p.Arg83His, p.Asp107Tyr, p.Ser135Ala, p.Arg149Ser) mapped within the methyl-CpG-binding domain, whereas 5 variants (p.Arg425Gln, p.Asn461Ser, p.Arg486Lys, p.Asp504His, p.Asp562His) were located in the glycosylase domain (Fig.1B). Of note is that all cases were heterozygous. Comparing MBD4MTvs.MBD4WT cases, there was no significant difference in age (median was 69 vs. 71 yrs), diagnosis, clinical parameters (WBC, ANC, Hb, Plt counts, peripheral and bone marrow blast percentage) and past history of malignancy. When we compared the somatic genotype of MBD4 VUS carriers vs. carriers of MBD4WT, somatic TET2 hits were found with a significantly lower frequency (15 vs. 25%, P=.046), in particular in AML (7 vs. 22%, P=.009) and in cases with TET2 truncations (9 vs. 19%, P=.018). To further investigate the biochemical basis of MBD4 dysfunction, and its connection to TET2 DNA dioxygenases, we knocked out MBD4 in TET2 mutant and in isogenic control cell lines. To test if the DNA hydrolase activity impacts on genomic accumulation of 5hmC in TET2-dependent manner, we assessed the impact of MBD4 loss on DNA methylation using 5mC and 5hmC ratio as read out in MBD4KO and WT cells in TET2MT and isogenic controls. Our results show that the loss of MBD4 resulted in increased 5hmC compared with the isogenic WT cells when accompanied with TET2WT but this effect was absent in TET2KO cells (mean fold change, 1.9±0.4 vs. 0.9±0.1; P=.03), suggesting a cross talk between MBD4 and TET2. Consequently, in agreement with our hypothesis, TET2WT cells with loss of MBD4 were significantly more sensitive to olaparib-mediated PARP1 inhibition. We also performed the RNA-seq analysis of MBD4WT and KO cells in the presence and absence of functional TET2. These results demonstrate that MBD4 variant carrier status may be a predisposition factor for the development of MN. As loss of MBD4 mimicked the loss of TET-dioxygenase function, MBD4 could function downstream of TET2 converting 5mC to 5hmC and to cytosine as a DNA damage repair associated protein (Fig. 1C). Our findings pave the way for consideration of MBD4 as a potential actionable target to regulate DNA methylation in association with TET2 during leukemogenesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EPID-03. PATHOGENIC GERMLINE MUTATIONS IN GLIOMA PATIENTS

Abstract BACKGROUND No consensus germline testing guidelines currently exist for glioma patients. Hence such testing is not routinely performed. The prevalence and type of germline pathogenic variants in these brain tumors remains unknown. DESIGN/ METHODS A retrospective review of patients treated at Baylor College of Medicine with paired tumor/normal sequencing using the Tempus xT tumor/normal matched approach from August 2018- April 2022 was performed. Corresponding clinical data was collected for these patients. RESULTS We identified 152 glioma patients of which 15 (9.8%) had pathogenic germline variants. Pathogenic germline variants were seen in 11/84 (13.1%) of Glioblastoma, IDH wildtype, 3/42 (7.1%) of Astrocytoma, IDH mutant and 1/26 (3.8%) of Oligodendroglioma, IDH mutant and 1p/19q co-deleted patients. Pathogenic variants in BRCA2, MUTYH and CHEK2 were seen more commonly (3/15 or 20% each). BRCA1 variants were seen in 2/15 (13%) patients, with variants in NF1, ATM, MSH2, and MSH3 occurring in one patient (7%) each. Second hit somatic variants were seen in 3/15 patients (20%). A second somatic hit was seen in NF1, MUTYH and MSH2 in one patient (7%) each. Referral to genetics was performed in 6/15 (40%) patients with pathogenic germline variants. Median overall survival was 1.6 years for glioblastoma, IDH wildtype patients with a pathogenic germline variant compared to 1.79 years for glioblastoma, IDH wildtype patients without it (p = 0.67). CONCLUSIONS Although not routinely performed in glioma patients, pathogenic germline variants occurred in ~10% of our patients. Only 40% of these patients were referred to genetics. These findings suggest a possible overlooked opportunity for determination of hereditary cancer syndromes with impact on surveillance as well as potential broader treatment options. Further research to confirm the occurrence and types of pathogenic germline variants in patients with IDH wildtype compared to IDH mutant tumors is necessary.

XBP1 Activation Reduces Severity of Polycystic Kidney Disease due to a Nontruncating Polycystin-1 Mutation in Mice.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in Pkd1 and Pkd2. They encode the polytopic integral membrane proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively, which are expressed on primary cilia. Formation of kidney cysts in ADPKD starts when a somatic second hit mechanism inactivates the wild-type Pkd allele. Approximately one quarter of families with ADPDK due to Pkd1 have germline nonsynonymous amino acid substitution (missense) mutations. A subset of these mutations is hypomorphic, retaining some residual PC1 function. Previous studies have shown that the highly conserved Ire1 α -XBP1 pathway of the unfolded protein response can modulate levels of functional PC1 in the presence of mutations in genes required for post-translational maturation of integral membrane proteins. We examine how activity of the endoplasmic reticulum chaperone-inducing transcription factor XBP1 affects ADPKD in a murine model with missense Pkd1 . We engineered a Pkd1 REJ domain missense murine model, Pkd1 R2216W , on the basis of the orthologous human hypomorphic allele Pkd1 R2220W , and examined the effects of transgenic activation of XBP1 on ADPKD progression. Expression of active XBP1 in cultured cells bearing PC1 R2216W mutations increased levels and ciliary trafficking of PC1 R2216W . Mice homozygous for Pkd1 R2216W or heterozygous for Pkd1 R2216Win trans with a conditional Pkd1 fl allele exhibit severe ADPKD following inactivation in neonates or adults. Transgenic expression of spliced XBP1 in tubule segments destined to form cysts reduced cell proliferation and improved Pkd progression, according to structural and functional parameters. Modulating ER chaperone function through XBP1 activity improved Pkd in a murine model of PC1, suggesting therapeutic targeting of hypomorphic mutations.

Bi-allelic hydroxymethylbilane synthase inactivation defines a homogenous clinico-molecular subtype of hepatocellular carcinoma.

Acute intermittent porphyria (AIP), caused by heterozygous germline mutations of the heme synthesis pathway enzyme HMBS (hydroxymethylbilane synthase), confers a high risk of hepatocellular carcinoma (HCC) development. Yet, the role of HMBS in liver tumorigenesis remains unclear. Herein, we explore HMBS alterations in a large series of 758 HCC cases, including 4 patients with AIP. We quantify the impact of HMBS mutations on heme biosynthesis pathway intermediates and we investigate the molecular and clinical features of HMBS-mutated tumors. We identify recurrent bi-allelic HMBS inactivation, both in patients with AIP acquiring a second somatic HMBS mutation and in sporadic HCC with 2 somatic hits. HMBS alterations are enriched in truncating mutations, in particular in splice regions, leading to abnormal transcript structures. Bi-allelic HMBS inactivation results in a massive accumulation of its toxic substrate porphobilinogen and synergizes with CTNNB1-activating mutations, leading to the development of well-differentiated tumors with a transcriptomic signature of Wnt/β-catenin pathway activation and a DNA methylation signature related to ageing. HMBS-inactivated HCC mostly affects females, in the absence of fibrosis and classical HCC risk factors. These data identify HMBS as a tumor suppressor gene whose bi-allelic inactivation defines a homogenous clinical and molecular HCC subtype. Heme (the precursor to hemoglobin, which plays a key role in oxygen transport around the body) synthesis occurs in the liver and involves several enzymes including hydroxymethylbilane synthase (HMBS). HMBS mutations cause acute intermittent porphyria, a disease caused by the accumulation of toxic porphyrin precursors. Herein, we show that HMBS inactivation is also involved in the development of liver cancers with distinct clinical and molecular characteristics.

Teenage-Onset Colorectal Cancers in a Digenic Cancer Predisposition Syndrome Provide Clues for the Interaction between Mismatch Repair and Polymerase δ Proofreading Deficiency in Tumorigenesis.

Simple SummaryColorectal cancer (CRC) in adolescents and young adults (AYA) is rare. Genetic causes include autosomal recessive and dominant monogenic disorders due to pathogenic variants (PVs) in genes involved in DNA repair. However, the genetic etiology of the majority of AYA-CRC remains unidentified. In two teenage siblings with CRC, we show to our knowledge for the first time that AYA-CRC cases can be caused by digenic inheritance of each a heterozygous pathogenic variant (PV) in the mismatch-repair (MMR) gene PMS2 and the proofreading polymerase (PP) Pol δ gene POLD1. With the aim to elucidate how the constitutional polymerase proofreading defect and the high propensity to MMR deficiency (MMRd) interact, we performed a comprehensive tumor analysis of the two siblings’ tumors. Results indicate that tumorigenesis is initiated by MMRd and the inherited POLD1 PV contributes to fast tumor progression reflected by an ultra-high tumor mutational burden (TMB) and specific mutational signatures.Colorectal cancer (CRC) in adolescents and young adults (AYA) is very rare. Known predisposition syndromes include Lynch syndrome (LS) due to highly penetrant MLH1 and MSH2 alleles, familial adenomatous polyposis (FAP), constitutional mismatch-repair deficiency (CMMRD), and polymerase proofreading-associated polyposis (PPAP). Yet, 60% of AYA-CRC cases remain unexplained. In two teenage siblings with multiple adenomas and CRC, we identified a maternally inherited heterozygous PMS2 exon 12 deletion, NM_000535.7:c.2007-786_2174+493del1447, and a paternally inherited POLD1 variant, NP_002682.2:p.Asp316Asn. Comprehensive molecular tumor analysis revealed ultra-mutation (>100 Mut/Mb) and a large contribution of COSMIC signature SBS20 in both siblings’ CRCs, confirming their predisposition to AYA-CRC results from a high propensity for somatic MMR deficiency (MMRd) compounded by a constitutional Pol δ proofreading defect. COSMIC signature SBS20 as well as SBS26 in the index patient’s CRC were associated with an early mutation burst, suggesting MMRd was an early event in tumorigenesis. The somatic second hits in PMS2 were through loss of heterozygosity (LOH) in both tumors, suggesting PPd-independent acquisition of MMRd. Taken together, these patients represent the first cases of cancer predisposition due to heterozygous variants in PMS2 and POLD1. Analysis of their CRCs supports that POLD1-mutated tumors acquire hypermutation only with concurrent MMRd.

Somatic hits in mismatch repair genes in colorectal cancer among non-seminoma testicular cancer survivors.

Non-seminoma testicular cancer survivors (TCS) have an increased risk of developing colorectal cancer (CRC) when they have been treated with platinum-based chemotherapy. Previously we demonstrated that among Hodgkin lymphoma survivors (HLS) there is enrichment of rare mismatch repair (MMR) deficient (MMRd) CRCs with somatic hits in MMR genes. We speculate that this phenomenon could also occur among other cancer survivors. We therefore aim to determine the MMR status and its underlying mechanism in CRC among TCS (TCS-CRC). Thirty TCS-CRC, identified through the Dutch pathology registry, were analysed for MMR proteins by immunohistochemistry. Next-generation sequencing was performed in MMRd CRCs without MLH1 promoter hypermethylation (n = 4). Data were compared with a male cohort with primary CRC (P-CRC, n = 629). MMRd was found in 17% of TCS-CRCs vs. 9% in P-CRC (p = 0.13). MMRd was more often caused by somatic double or single hit in MMR genes by mutation or loss of heterozygosity in TCS-CRCs (3/30 (10%) vs. 11/629 (2%) in P-CRCs (p < 0.01)). MMRd CRCs with somatic double or single hit are more frequent in this small cohort of TCS compared with P-CRC. Exposure to anticancer treatments appears to be associated with the development of these rare MMRd CRC among cancer survivors.

Exome sequencing reveals a distinct somatic genomic landscape in breast cancer from women with germline PTEN variants

Germline PTEN variants (PTEN hamartoma tumor syndrome [PHTS]) confer up to 85% lifetime risk of female breast cancer (BC). BCs arising in PHTS are clinically distinct from sporadic BCs, including younger age of onset, multifocality, and an increased risk of second primary BCs. Yet, there is no previous investigation into the underlying genomic landscape of this entity. We sought to address the hypothesis that BCs arising in PHTS have a distinct genomic landscape compared to sporadic counterparts. We performed and analyzed exome sequencing data from 44 women with germline PTEN variants who developed BCs. The control cohort comprised of 497 women with sporadic BCs from The Cancer Genome Atlas (TCGA) dataset. We demonstrate that PHTS-derived BCs have a distinct somatic mutational landscape compared to the sporadic counterparts, namely second somatic hits in PTEN, distinct mutational signatures, and increased genomic instability. The PHTS group had a significantly higher frequency of somatic PTEN variants compared to TCGA (22.7% versus 5.6%; odds ratio [OR] 4.93; 95% confidence interval [CI] 2.21 to 10.98; p<0.001) and a lower mutational frequency in PIK3CA (22.7% versus 33.4%; OR 0.59; 95% CI 0.28 to 1.22; p= 0.15). Somatic variants in PTEN and PIK3CA were mutually exclusive in PHTS (p= 0.01) but not in TCGA. Our findings have important implications for the personalized management of PTEN-related BCs, especially in the context of more accessible genetic testing.

Clinical and molecular features of pediatric cancer patients with Lynch syndrome.

The association of childhood cancer with Lynch syndrome is not established compared with the significant pediatric cancer risk in recessive constitutional mismatch repair deficiency syndrome (CMMRD). We describe the clinical features, germline analysis, and tumor genomic profiling of patients with Lynch syndrome among patients enrolled in pediatric cancer genomic studies. There were six of 773 (0.8%) pediatric patients with solid tumors identified with Lynch syndrome, defined as a germline heterozygous pathogenic variant in one of the mismatch repair (MMR) genes (three with MSH6, two with MLH1, and one with MSH2). Tumor analysis demonstrated evidence for somatic second hits and/or increased tumor mutation burden in three of four patients with available tumor with potential implications for therapy and identification of at-risk family members. Only one patient met current guidelines for pediatric cancer genetics evaluation at the time of tumor diagnosis. Approximately 1% of children with cancer have Lynch syndrome, which is missed with current referral guidelines, suggesting the importance of adding MMR genes to tumor and hereditary pediatric cancer panels. Tumor analysis may provide the first suggestion of an underlying cancer predisposition syndrome and is useful in distinguishing between Lynch syndrome and CMMRD.

OTHR-22. Malignant mesothelioma (MM) as second cancer in childhood brain tumor survivors: the first child with neurofibromatosis type 2 and concurrent MM

Abstract We report two cases of malignant mesothelioma (MM) in childhood brain tumor survivors. A 40-year-old man, who was treated at the age of ten with chemotherapy and craniospinal radiotherapy (up to 54.5 Gy) for a pineal secreting non-germinomatous germ cell tumor, had persistent fever, fatigue, and weight loss. A total body CT scan revealed several pulmonary and abdominal wall nodularities. The histological examination on a biopsy diagnosed a biphasic MM. The karyotype and array-CGH of peripheral blood were normal; a FISH of tumor cells showed a breakage of locus EWSR1 (22q12.2). In this case, environmental exposure was identified (asbestos in public water tanks). His MM followed an aggressive course and resulted in death after three months. A twelve-year-old boy affected by severe neurofibromatosis type 2 (NF2) already treated with several neurosurgical exeresis and chemotherapy lines (hydroxycarbamide, bevacizumab, sirolimus), developed multiple abdominal lesions, associated with pleural effusion and weight loss. The CT scan showed the thickening of the left pleura and multiple peritoneal nodules. The cytological examination on thoracentesis diagnosed an epithelioid MM. The karyotype on paracentesis-obtained cells revealed monosomy of chromosomes 14 and 22, loss of Y, and trisomy of 6, 10, 11, 16, and 20. NGS is currently ongoing to exclude germline or somatic second hits. Importantly, no exposure to asbestos was found. Less than 2% of MMs occur below 45 years of age; it is a rare secondary tumor in cancer survivors and environmental or genetic contributing causes are often detectable on a careful investigation. Variants in NF2 are considered to be the second most common after those in BAP1. To our knowledge, this is the first child with NF2 and concurrent MM reported to date. Compared to the expected prognosis, his MM has followed a mild course, and one year after diagnosis he is still alive.