A Novel Targeted Sequence for Chromosome 11p15.5 Maternal Loss in SDHD-Related Paragangliomas.

SummarySuccinate dehydrogenase deficiency has been associated with several neoplasias, including renal cell carcinoma (RCC) and those associated with hereditary paraganglioma (PGL)/ pheochromocytoma (PHEO) syndromes, Carney dyad, and Carney triad. Carney triad is a rare multitumoral syndrome characterized by co-existing PGL, gastrointestinal stromal tumor (GIST), and pulmonary chondroma (CHO). We report a case of a 57-year-old male who presented with para-aortic and gastroesophogeal masses, and a right renal superior pole lesion, which were classified as multiple PGLs, a GIST, and a clear cell renal carcinoma, respectively, on pathology following surgical resection. Additionally, a CHO was diagnosed radiologically, although no biopsy was performed. A diagnosis of Carney triad was made. SDHB immunohistochemical staining was negative for the PGL and the GIST, indicating SDH-deficiency. Interestingly, the renal cell carcinoma (RCC) stained positive for both SDHB and SDHA. Subsequent genetic screening of SDH subunit genes revealed a germline inactivating heterozygous SDHA pathogenic variant (c.91 C>T, p.R31X). Loss of heterozygosity was not detected at the tumor level for the RCC, which likely indicated the SDHA variant would not be causative of the RCC, but could still predispose to the development of neoplasias. To the knowledge of the authors this is the first reported case of an SDHA pathogenic variant in a patient with Carney triad complicated by RCC.Learning points The succinate dehydrogenase enzyme is encoded by four subunit genes (SDHA, SDHB, SDHC, and SDHD; collectively referred to as SDHx), which have been implicated in several neoplasias and are classified as tumor suppressor genes. Carney triad is a rare multiple-neoplasia syndrome presenting as an association of PGLs, GISTs, and CHOs. Carney triad is most commonly associated with hypermethylation of SDHC as demonstrated in tumor tissue, but approximately 10% of cases are due to pathogenic SDHx variants. Although SDHB pathogenic variants are most commonly reported in SDH-deficient renal cell carcinoma, SDHA disease-causing variants have been reported in rare cases.

Human chromosome band 16q24 commonly undergoes loss of heterozygosity (LOH) in human hepatocellular carcinoma (HCC). To further localize the region of deletion on 16q24 and to evaluate the genetic role of 17-beta-HSD, which is near 16q24, in HCC, we examined the pattern of loss of heterozygosity in 88 HCC patients. DNAs from 88 pairs of HCCs and corresponding non-tumor parts were prepared. Loss of heterozygosity on chromosomes 16q24 was investigated by 11 sets of microsatellite markers. Mutation analysis of type II 17-beta-HSD was performed by automatic sequencing. LOH on 16q24 for at least 1 locus was found in 43 of the 88 tumor DNAs (49%). Three non-overlapping regions of frequent LOH were defined in these 43 tumors with partial deletions. The first region was between D16S516 loci and D16S507, encompassed by a 1-cM region, defined by the D16S504. The second region was defined by the 17HSDB2 locus between D16S505 and D16S422, encompassed approximately by a 1-cM region. The third region was between D16S520 and D16S413, defined by D16S3048, encompassed approximately by a 4-cM region. Homozygous deletions of any exons in 17HSDB2 gene were identified in 7 of 27 cases (26%). Automated sequencing analysis of 17HSDB2 failed to demonstrate mutations in any of these specimens. Our data suggest that the 17HSDB2 locus is a frequent target of deletion in HCC but the inactivation of 17HSDB2 may not involve sequence mutations. Furthermore, the presence of the other 2 frequent LOH regions suggest that the putative tumor suppressor genes at these locations might be involved in the development of HCC.

A Tumor Sorting Protocol that Enables Enrichment of Pancreatic Adenocarcinoma Cells and Facilitation of Genetic Analyses

At least 10% of the BRCA1/2 tests identify variants of uncertain significance (VUS) while the distinction between pathogenic variants (PV) and benign variants (BV) remains particularly challenging. As a typical tumor suppressor gene, the inactivation of the second wild-type (WT) BRCA1 allele is expected to trigger cancer initiation. Loss of heterozygosity (LOH) of the WT allele is the most frequent mechanism for the BRCA1 biallelic inactivation. To evaluate if LOH can be an effective predictor of BRCA1 variant pathogenicity, we carried out LOH analysis on DNA extracted from 90 breast and seven ovary tumors diagnosed in 27 benign and 55 pathogenic variant carriers. Further analyses were conducted in tumors with PVs yet without loss of the WT allele: BRCA1 promoter hypermethylation, next-generation sequencing (NGS) of BRCA1/2, and BRCAness score. Ninety-seven tumor samples were analyzed from 26 different BRCA1 variants. A relatively stable pattern of LOH (65.4%) of WT allele for PV tumors was observed, while the allelic balance (63%) or loss of variant allele (15%) was generally seen for carriers of BV. LOH data is a useful complementary argument for BRCA1 variant classification.

Introduction: Loss of heterozygosity (LOH) of chromosome arms 1p and 19q in gliomas has diagnostic, prognostic, and therapeutic implications. The deletion of 1p and the codeletion of chromosome arms 1p and 19q correlate with the diagnosis of oligodendroglioma, increased chemosensitivity and improved prognosis. Clinically, LOH is most frequently determined by Fluorescent In Situ Hybridization (FISH) and short tandem repeat (STR) analysis. Each approach suffers from the limitation that the probes/primers interrogate only a small portion of the chromosomes. Small deletions can be misinterpreted as LOH of the entire chromosome arm. We have investigated the use of a single nucleotide polymorphism (SNP) array for identifying LOH in gliomas. Methods: Genomic DNA was extracted from 5 formalin-fixed paraffin-embedded tissue samples following microdissection, and from peripheral blood. Samples were run on an ABI 3100 following multiplex PCR amplification of 5 STRs on chromosome 1p (D1S199, D1S186, D1S162, D1S312, D1S226) and 3 STRs on chromosome 19q (D19S918, D19S112, D19S206). Array analysis was performed using the Affymetrix genome-wide human SNP array 6.0 platform (906,600 SNPs) according to protocol. Data were analyzed with Partek Genomics Suite. Results: Cases 1 and 4 showed no LOH on chromosomes 1p and 19q by STR analysis and SNP array. Cases 2 and 5 showed LOH on chromosomes 1p and 19q by STR analysis and SNP array. Case 3 showed partial centromeric LOH on chromosome 1p by STR analysis which was confirmed by SNP array. Case 1 was a 48 year old male with anaplastic mixed oligoastrocytoma. Additional SNP array findings included a deletion of chromosome arm 13q. Case 4 was a 31 year old female with a low grade glioneural tumor, and no additional findings were made by SNP array. Case 2 was a 47 year old female with anaplastic oligodendroglioma. Additional SNP array findings included losses on chromosomes 5, 6, 9,15, and whole gain of chromosome 11. Case 5 was a 12 year old male with anaplastic oligodendroglioma. SNP array found additional losses on chromosomes 2, 3, 6, 8, 9, 10, and 20. Case 3 was a 47 year old female with an astrocytoma. Analysis of the 3 most centromeric STR loci on 1p demonstrated allelic imbalance that was consistent with LOH. The 2 most telomeric STR loci demonstrated allele ratios consistent with heterozygosity (negative for LOH). SNP array confirmed partial centromeric LOH on 1p and identified losses on chromosomes 2, 6, 10, 13, 16, and 22. Conclusion: Analysis of LOH on chromosome arms 1p and 19q by STR analysis and SNP array were concordant. SNP array identified additional gains or losses in each sample that were not detected by the STR assay. Interestingly, SNP array analysis also identified LOH on chromosome 13q, which contains the BRCA2 gene locus, in cases 1 and 3. If a second-hit can be identified, this may present a novel approach for identifying glioma patients who may potentially benefit from Poly (ADP-Ribose) Polymerase (PARP) inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1781.

The CpG Island Methylator Phenotype and Chromosomal Instability Are Inversely Correlated in Sporadic Colorectal Cancer

In SDHD mutation families, paragangliomas and pheochromocytomas usually occur only after paternal transmission of the mutation. This important but unexplained parent-of-origin effect is not due to imprinting of SDHD itself, as was initially suspected, since SDHD is biallelically expressed in several tissues. In clinically affected individuals who possess a paternally inherited SDHD mutation, there is loss of the entire maternal chromosome 11 in tumour DNA, implying that tumorigenesis requires loss of not only maternal (wild type) SDHD but also a further, imprinted, tumor suppressor gene (TSG). We report the second case of an SDHD-related tumor (a pheochromocytoma in a 33 year old woman possessing the common pathogenic mutation, p.Pro81Leu) occurring after maternal transmission. It is the first reported investigation of tumor DNA in this situation. Tumor DNA revealed loss of heterozygosity (LOH) at paternal 11q23 causing loss of the wild-type SDHD allele and also LOH affecting maternal 11p15, including H19. These two LOH regions were separated by a region exhibiting clearly retained heterozygosity, containing SDHAF2 (a recently reported paraganglioma TSG), which therefore appears uninvolved here. This case provides strong molecular evidence that the tumorigenic requirement for maternal 11p15 loss (in addition to inactivation of both SDHD alleles) drives the observed parent-of-origin effect. Thus, SDHD-related tumorigenesis most likely involves a “three-hit” mechanism that includes (as one of the hits) loss of an imprinted (paternally silenced and maternally active) TSG from chromosome 11, such as H19). Tumor formation more commonly results from paternal inheritance of SDHD mutations, as the necessary loss of both the wild type SDHD allele and maternal 11p15 can then occur by a single event (loss of maternal chromosome 11). These findings have important implications regarding the clinical management of carriers of maternally inherited SDHD mutations, who we confirm can develop pheochromocytomas, and the understanding of the parent-of-origin effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3673. doi:1538-7445.AM2012-3673

Background: Carney triad is a rare multiple-neoplasia syndrome presenting as an association of paragangliomas (PGL), gastrointestinal stromal tumors (GIST), and pulmonary chondromas (CHO). Succinate dehydrogenase deficiency has been associated with several neoplasias, including Carney triad, renal cell carcinoma (RCC) and those associated with hereditary PGL/ pheochromocytoma (PHEO) syndromes.Clinical Case: A 57-year-old male diagnosed with hypertension at age 49, presented with a gradual increase in blood pressure over a period of 12 months. For seven years following his diagnosis of hypertension, the patient experienced episodic increases in blood pressure, to a systolic pressure greater than 180 mmHg associated with a tight band sensation around his forehead lasting half a day. Abdominal computed tomography (CT) revealed a left adrenal adenoma, a 5.1 cm para-aortic mass, and a right renal superior pole lesion measuring 2.5 cm, which was suspicious for a carcinoma. 123I-metaiodobenzylguanidine (123I-MIBG) and 18F-fluorodeoxyglucose-positron emission tomography (18F-FDG-PET) scans were performed, which suggested the para-aortic mass to be consistent with a PGL. Additionally, 18F-FDG uptake was noted in the gastroesophageal region and was suspicious for a GIST. The left adrenal mass was not associated with 123I-MIBG or 18F-FDG activity. Chest CT demonstrated a right middle lobe lung lesion suggestive of a CHO, although no biopsy was performed. A diagnosis of Carney triad was made. The patient underwent surgical resection of the PGL and GIST, as well as a partial right nephrectomy. The PGL and GIST were positive for SDHA and negative for SDHB by immunohistochemical (IHC) staining. Pathology from the renal lesion was consistent with a 2.3 cm conventional clear cell renal carcinoma, with positive staining for SDHA and SDHB by IHC. The patient was found to harbor a germline heterozygous pathogenic variant (c.91 C>T, p.R31X) in SDHA which has been previously reported and results in loss of function of SDHA. SHDC hypermethylation was not detected in the PGL, GIST, or RCC. Additionally, DNA sequencing of the RCC did not indicate loss of heterozygosity at the variant region of interest. Although the SDHA disease-causing variant is responsible for the patients Carney triad phenotype, it is unclear if this variant is causative of the RCC.Conclusion: This is a novel presentation of a germline inactivating SDHA pathogenic variant in a patient with Carney triad complicated by RCC. However, an SDHA disease-causing variant was previously reported in a patient with comorbid GIST and RCC. This case provides further support to the increasing evidence that SDHx pathogenic variants may predispose patients to develop renal neoplasms.

Acoustic neuroma, both familial and sporadic, is clinically and biologically a heterogeneous condition with a wide variation in age of presentation, length of history, and tumor growth rate. In an attempt to correlate this clinical diversity with the underlying molecular pathology, we have analyzed 43 paired blood-tumor DNA samples from patients with acoustic neuromas. Molecular genetic analysis. Molecular genetic research laboratory. Paired blood-tumor DNA samples were obtained from 43 patients (41 sporadic and two patients with neurofibromatosis type 2). Loss of constitutional heterozygosity was looked for in the region of tumor suppressor genes on chromosomes 3p, 5q, 11p, 17p, 17q, and 22. We found loss of heterozygosity exclusively for markers on chromosome 22. Thirty-nine percent of tumors showed allele loss, and in each case the loss of heterozygosity included the region of the neurofibromatosis type 2 (NF2) gene. No loss of heterozygosity was detected in the region of known or putative suppressor genes in chromosomes 3p, 5q, 11p, 17p, and 17q. This study has demonstrated that (1) chromosome 22 allele loss is a frequent event in sporadic acoustic neuroma; (2) the minimal region of loss of heterozygosity in acoustic neuroma includes the NF2 gene; (3) the known tumor suppressor genes investigated (VHL, adenomatous polyposis coli, WT2, p53, and NF1) do not appear to be important in the pathogenesis of acoustic neuroma; and (4) patients with extensive chromosome 22 loss tended to be younger and with a slightly shorter clinical history than those with no detectable allele loss.

Study question Does CRISPR/Cas9 gene editing have a distinct genetic impact in human embryos compared to induced pluripotent stem cells (iPSCs)? Summary answer IPSCs display a difference in repair events, such as template usage, compared to the germline which indicates a distinct genetic impact by CRISPR/Cas9 gene editing. What is known already CRISPR/Cas9 is utilized to induce targeted DNA editing. To introduce specific changes, such as mutational correction, a DNA template is generally administered, stimulating homology-directed repair. Recent human germline editing studies aiming for mutational repair created a moderate amount of embryos which solely carried wild-type alleles. Remarkably, no template use was observed, but instead loss-of-heterozygosity (LOH) was demonstrated. This is the presence of only one (the wild-type) allele, either caused by gene conversion events using the wild type allele or chromosome loss. Up to date, no iPSCs studies have evaluated LOH events when attempting to correct a heterozygous mutation. Study design, size, duration A guide RNA targeting the mutant allele, and a repair template containing the wild-type allele and a synonymous variant to track template usage were designed. For iPSCs targeting, the components were nucleofected after which DNA was extracted from the whole well (n = 3) or from individual colonies (n = 33). For human embryo targeting, the components were injected simultaneously with sperm into donated spare oocytes (n = 32). DNA was extracted from embryos after 3-6 days of in vitro culture. Participants/materials, setting, methods Sperm and renal cells, from which the iPSCs were derived, were donated by a patient with a heterozygous base pair substitution in PLCZ1 (c.136-1G>C) causing fertilization failure. To overcome fertilization failure, assisted oocyte activation was employed during ICSI. For the embryos, next-generation sequencing (NGS), short tandem repeat (STR) analysis and a whole genome single nucleotide polymorphism (SNP) assay were performed. For the iPSCs, NGS and a targeted SNP assay were carried out. Main results and the role of chance The genetic events in embryos originating from mutant sperm (n = 32) displayed following distribution: 19% showed the untargeted mutant allele, 56% showed additional mutagenesis and 25% showed only the wild-type allele. In the latter group, the template was never utilized, pointing to LOH. STR analysis indeed revealed LOH events of different lengths in these embryos. SNP analysis of one embryo, originating from mutant sperm but displaying the wild-type allele, did not demonstrate LOH (with a detection limit of 500bp). These findings suggest the occurrence of gene conversion, which rejects the formerly stated hypothesis that the observed LOH in human embryos can mostly be attributed to chromosome loss. In the iPSCs, whole-well (n = 3) and single colony (n = 33) data demonstrated a similar trend in genetic event distribution compared to the embryos, with the main difference that in 30% of the corrected reads/colonies, template use was observed. LOH was further analysed in nineteen single colonies. In all the colonies (3/3) corrected with the template, no LOH was present. When the colonies displayed additional mutagenesis, 7% (1/13) contained LOH events. In 33% (1/3) of the colonies showing only the wild-type allele (without signs of template use), LOH was observed. Limitations, reasons for caution SNP assays have a higher resolution compared to STR analysis, and enable distinction of LOH events between ‘gene conversion’ and ‘chromosome loss’. Therefore, more embryos and iPSC colonies will be analysed with SNP assays. However, informative SNPs define the resolution of our assay (currently 500 bp from Cas9 cut site). Wider implications of the findings Our results show that template usage seems to differ between human embryos and iPSCs, but that iPSCs do not solely rely on template use as well. This demonstrates that embryo experiments to study the genetic impact of CRISPR/Cas9 cannot fully be replaced by iPSCs experiments. Trial registration number Not Applicable

Follicular thyroid carcinoma (FTC) exhibits frequent loss of heterozygosity (LOH) on chromosomes 10q and 3p, suggesting involvement of tumor suppressor genes. We screened 14 FTC (10 Hurthle cell carcinomas and 4 nonoxyphilic FTC), 14 papillary thyroid carcinomas, and 7 follicular adenomas for LOH on chromosome arms 1p, 3p, 3q, 10p, 10q, 11p, 11q, 13q, 17p, and 17q. LOH was more frequent in FTC than in follicular adenoma or papillary thyroid carcinoma. In FTC, rates of LOH on 3p (86%), 17p (72%), and 10q (57%) were higher than the average rate of LOH (33%; P < 0.05). Most frequently involved were 3p21-25 and 17p13.1-13.3, the sites for the VHL (3p25-26) and p53 (17p13.1) tumor suppressors. We, therefore, characterized these genes by dideoxy fingerprinting and DNA sequencing. Two FTC had mutations in p53, but only 1 of these exhibited LOH at 17p. No VHL gene mutations were found. Thus, neither p53 nor VHL genes play a significant role in the pathogenesis of differentiated thyroid cancer. LOH on 17p, but not on 3p or 10q, was correlated with mortality. Accordingly, 3p and 10q LOH may represent early, and 17p LOH late, events in FTC development. The data suggest the presence of novel tumor suppressor genes on chromosomes 3p and 17p that may be important in the pathogenesis of FTC.

BRCA germline pathogenic variants represent the most common inherited mechanism predisposing individuals to breast cancer, while germline pathogenic variants in one of the mismatch repair (MMR) genes represent the most common colon cancer-predisposing inherited syndrome, known as the Lynch syndrome (LS). Individuals who harbor pathogenic germline variants for both syndromes are extremely rare. Germline testing is now done routinely for patients with breast cancer and MMR testing is recommended for nearly all patients diagnosed with colon or rectal cancer (Benson et al in NCCN clinical practice guidelines in oncology (NCCN guidelines) colon cancer (Version 4.2019-November 8, 2019). www.NCCN.org, Gradishar et al in NCCN clinical practice guidelines in oncology (NCCN guidelines) breast cancer (Version 3.2019-September 6, 2019).www.NCCN.org). We report a patient with germline mutations in both BRCA2 and the MMR gene MLH1 who developed breast cancer. The breast cancer showed loss of heterozygosity (LOH) in BRCA2 (the molecular hallmark of cancers related to inheritance of a BRCA alteration) and was also deficient in mismatch repair gene protein expression (dMMR), the hallmark of LS-related cancers. We discuss the possible mechanisms of transformation that would explain the finding that the tumor showed both BRCA2 LOH and was dMMR, each of which would generally be considered a gatekeeper event for transformation of normal cells to malignancy. This report describes a patient with molecularly diagnosed breast and ovarian cancer syndrome (BRCA2) and LS. Next generation sequencing (NGS) and immunohistochemical (IHC) testing demonstrated her breast cancer to show BRCA2 LOH and to be dMMR. The patient presented represents the first reported case where both next generation sequencing (NGS) for BRCA LOH and MMR IHC testing of her breast cancer were performed and underscores the importance of using NGS including the reported mutational allelic frequency (MAF) and IHC use to predict the likely responsiveness to the recently approved PARP inhibitors and checkpoint inhibitor therapies (Robson et al in N Engl J Med 377:523-533, 2017, Lemery et al in 377(15):1409-1412, https://doi.org/10.1056/NEJMp1709968, 2017), key because the gatekeeper transforming event for tumors related to inherited cancer syndromes is loss of normal tumor suppressor gene (TSG) protein expression.

Tumor suppressor genes (TSGs) protect normal cells from tumorigenesis (Lasko et al., 1991; Sherr, 2004). Except in cases of haploinsufficiency, heterozygosity for a non-functional TSG allele protects a cell from tumor formation because the functional TSG allele produces a functional protein. Loss of heterozygosity (LOH) is a mechanism by which the remaining wild type tumor suppressor allele is lost, resulting in tumor formation (Lasko et al., 1991; Sherr, 2004). Loss of TSG expression may also occur by epigenetic silencing. The probability of a second hit follows a Poisson distribution with the number of tumors and time of incidence being variable in heterozygous carriers (Shao et al., 1999). Many TSGs have been identified. Such genes play roles in many cellular functions including cell cycle checkpoint control, mitogenic signaling pathways, protein turnover, DNA damage, hypoxia and other stress responses (Sherr, 2004). Surprisingly, the SdhB, SdhC, and SdhD subunits of the metabolic enzyme succinate dehydrogenase (SDH), have also been identified as TSGs for neuroendocrine tumors such as paraganglioma (PGL) and pheocheomocytoma (PHEO). PGLs are rare (1:300,000) tumors of neuroectodermal origin derived from paraganglia, a diffuse neuroendocrine system dispersed from the base of the skull to the pelvic floor (Baysal, 2002). PGLs are highly vascularized tumors that can originate in either the sympathetic or parasympathetic nervous systems (Baysal, 2002; Pacak et al., 2001). Patients with PGL tumors that secrete catecholamines present symptoms of catecholamine excess including palpitations. The predominant clinical features of nonchromaffin PGLs are cranial nerve palsies and tinnitus; however, a small proportion of these nonchromaffin PGLs secrete catecholamines (Dluhy, 2002). A hereditary PGL predisposition is involved in at least 30% of cases (Maher & Eng, 2002; Bryant et al., 2003). Individuals with familial predisposition display at least 40% penetrance and a more severe presentation than those with the sporadic form of the disease. Extra-adrenal pheochromocytomas are estimated to be malignant in 40% of cases (Young et al., 2002). There is currently no effective cure for malignant PGL.

e16296 Background: Therapeutic resources are often limited in rare tumors. NGS (Next Generation Sequencing) represents a valuable tool that can provide biological and prognostic information and guide therapeutic options. Here we describe a case series of the University Hospital of Verona, a center of excellence for neuroendocrine and pancreatic neoplasms. Methods: Between October 2020 and December 2022, 75 patients (pts) with a neuroendocrine neoplasm (NEN) or mixed neuroendocrine non-neuroendocrine neoplasia (MiNEN) underwent NGS analysis with an in-house assay CORE panel (covering 174 genes; 42 pts) or FoundationOne CDx panel (33 pts). We reported ACMG/AMP class IV-V molecular alterations as single nucleotide variations (SNVs), copy number variations (CNVs) including amplifications, deletions, fusions and loss of heterozygosity (LOH). Results: We included 53 well-differentiated neuroendocrine tumors (NETs), 17 poorly differentiated neuroendocrine carcinomas (NECs) and 5 MiNENs. The majority originated from gastro-entero-pancreatic tract (n=67, 89%). The most frequent gene alterations were TP53 (n=30; 12%: 28 SNV and 2 LOH); MEN1 (n=14, 5%: all SNV), CDKN2A (n=12; 5%: 2 SNV, 4 CNV LOH and 5 CNV loss), DAXX (n=12, 5%: all SNV) and RB1 (n=11, 4%: 8 SNV, 4 CNV LOH and 5 CNV loss). In MiNEN pts 3 druggable alterations were found: 1 TRIM-BRAF fusion, 1 SCL4A4-ROS1 fusion and 1 ERBB2 amplification. All MiNENs were MSS; median TMB (mTMB) was 4.86 mut/Mb. Among NET pts the most often altered genes were MEN1 (n=14), DAXX (n=12), TP53 (n=10), PTEN (n=7), CDKN2A (n=5), NF1 (n=5), TSC2 (n=5). The most prevalent mutations in NEC pts involved TP53 (n=17), RB1 (n=9), KRAS (n=7), CDKN2A (n=6), CDKN2B (n=5), CCNE1 (n=5), APC (n=4), SMAD4 (n=3). The mutational rate in NETs was 2.7 with a mTMB of 6.5; the mutational rate in NECs was 5.3 with a mTMB of 7.3 mut/Mb. In NET and NEC 23 actionable mutations were reported. In pancreatic NENs, DAXX mutation was present in 29% of NET G3 and absent in NEC cases. Among G1-G2 NET pts treated with PRRT in 2nd or further lines with a follow-up longer than 60 months (n=17), 6 (35%) were DAXX mutated and all (100%) were still alive at 5 years, compared to 7/11 (64%) in the DAXX wild type (wt) group. Focusing on NET with Ki67% between 3% and 55% (n=45), TP53 wt pts (n=35, 78%) showed a better median overall survival (mOS) than TP53 mutated (n=10, 22%) pts (131.3 vs 56.7 months; p= 0.0433). Conclusions: Our data support the role of NGS in pancreatic rare histologies. This work highlighted the negative prognostic role of TP53 status in G2-G3 NETs, the potential predictive role of DAXX in G1-G2 NET treated with PRRT and its contribution to the differential diagnosis between NET and NEC. Our findings suggest that the pathogenesis in NET is mostly mediated by defects in tumor suppressor genes, while the role of oncogene alterations seem to acquire more importance with increasingly poor differentiation.

Introduction: Women carrying BRCA1 or BRCA2 germline mutations have a 45-80% lifetime risk of developing breast cancer (BC). BRCA1 and BRCA2 are perceived as bona fide tumor suppressor genes, whereby bi-allelic inactivation in tumor cells is required for tumorigenesis. Recent studies have indicated that loss of heterozygosity (LOH) of the wild-type allele of BRCA1 may be heterogeneous and constitute a late event. Therefore, additional somatic events prior to full BRCA1/2 inactivation may be required for tumorigenesis. Given that the somatic events that result in the development of BRCA1/2-BCs and their chronology are not understood, here we sought to define the genomic landscape of BRCA1/2-BCs and whether LOH of BRCA1/2 wild-type allele and/or mutations affecting additional tumor suppressor genes would be clonal or subclonal in these cancers. Methods: We retrieved 29 BRCA1-BCs and 10 BRCA2-BCs from the Pathology Departments of the authors' institutions. DNA extracted from microdissected tumor and normal breast samples was subjected to targeted capture massively parallel sequencing using either the MSK-IMPACT assay or an assay targeting all exons of 254 genes recurrently mutated in BC or related to DNA repair. Somatic single nucleotide variants, small insertions and deletions and copy number alterations affecting genes present in both sequencing assays (111 genes) were defined using state-of-the-art bioinformatics algorithms. ABSOLUTE and FACETS were employed to define clonal (i.e. present virtually in 100% of the cancer cells of a given case) and subclonal mutations and the presence of LOH of the BRCA1 and BRCA2 wild-type alleles. Results: Our analysis revealed bi-allelic inactivation of BRCA1 in 28 of 29 BRCA1-BCs (93% harbored LOH of the BRCA1 wild-type allele and 3% harbored a second somatic BRCA1 pathogenic mutation). The only BRCA1-BC lacking bi-allelic inactivation of BRCA1 was an estrogen receptor-positive lobular carcinoma, lacking genomic features consistent with homologous recombination DNA repair defects, diagnosed at 62 years of age. Bi-allelic inactivation of BRCA2 was found in all cases (100% of harbored LOH of the BRCA2 wild-type allele). A clonal somatic 'second hit' resulting in bi-allelic inactivation of BRCA1 or BRCA2 was detected in 76% and 100% of BRCA1-BCs and BRCA2-BCs, respectively. In BRCA1-BCs, TP53 mutations were detected in 76% of cases, and these mutations were found to be clonal in 58% of cases. The repertoire of somatic mutations affecting BRCA1-BCs included clonal somatic mutations or homozygous deletions of known tumor suppressor genes, such as PTEN, RB1, CDKN2A and NF1. In contrast, only 10% of the BRCA2-BCs harbored TP53 somatic mutations. Though clonal somatic mutations in several cancer genes were detected, 40% of BRCA2-BCs had no mutations affecting the cancer genes analyzed. Conclusions: Bi-allelic inactivation of BRCA1 and BRCA2 are frequent events in BRCA1-BCs and BRCA2-BCs, respectively. In a subset of BRCA1-BCs, however, the second 'hit' appeared to be subclonal, whereas mutations affecting TP53 and other tumor suppressor genes were clonal, supporting the notion that at least in a subset of these tumors, loss of the wild-type allele of BRCA1 may be preceded by inactivation of another tumor suppressor gene. Citation Format: Geyer FC, Burke KA, Macedo GS, Piscuoglio S, Ng CK, Martelotto LG, Papanastatiou AD, De Filippo MR, Schultheis AM, Brogi E, Robson M, Wen YH, Weigelt B, Schnitt SJ, Tung N, Reis-Filho JS. The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S2-02.