Causative Germline Mutation Research Articles

Prevalence and clinical expression of germ line predisposition to myeloid neoplasms in adults with marrow hypocellularity

AbstractSystematic studies of germ line genetic predisposition to myeloid neoplasms in adult patients are still limited. In this work, we performed germ line and somatic targeted sequencing in a cohort of adult patients with hypoplastic bone marrow (BM) to study germ line predisposition variants and their clinical correlates. The study population included 402 consecutive adult patients investigated for unexplained cytopenia and reduced age-adjusted BM cellularity. Germ line mutation analysis was performed using a panel of 60 genes, and variants were interpreted per the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines; somatic mutation analysis was performed using a panel of 54 genes. Of the 402 patients, 27 (6.7%) carried germ line variants that caused a predisposition syndrome/disorder. The most frequent disorders were DDX41-associated predisposition, Fanconi anemia, GATA2-deficiency syndrome, severe congenital neutropenia, RASopathy, and Diamond-Blackfan anemia. Eighteen of 27 patients (67%) with causative germ line genotype were diagnosed with myeloid neoplasm, and the remaining with cytopenia of undetermined significance. Patients with a predisposition syndrome/disorder were younger than the remaining patients and had a higher risk of severe or multiple cytopenias and advanced myeloid malignancy. In patients with myeloid neoplasm, causative germ line mutations were associated with increased risk of progression into acute myeloid leukemia. Family or personal history of cancer did not show significant association with a predisposition syndrome/disorder. The findings of this study unveil the spectrum, clinical expressivity, and prevalence of germ line predisposition mutations in an unselected cohort of adult patients with cytopenia and hypoplastic BM.

Management of patients with germline predisposition to haematological malignancies considered for allogeneic blood and marrow transplantation: Best practice consensus guidelines from the UK Cancer Genetics Group (UKCGG), CanGene-CanVar, NHS England Genomic Laboratory Hub (GLH) Haematological Malignancies Working Group and the British Society of Blood and Marrow Transplantation and cellular therapy (BSBMTCT).

Germline predisposition to haematological cancers is increasingly being recognised. Widespread adoption of high-throughput and whole genome sequencing is identifying large numbers of causative germline mutations. Constitutional pathogenic variants in six genes (DEAD-box helicase 41 [DDX41], ETS variant transcription factor 6 [ETV6], CCAAT enhancer binding protein alpha [CEBPA], RUNX family transcription factor 1 [RUNX1], ankyrin repeat domain containing 26 [ANKRD26] and GATA binding protein 2 [GATA2]) are particularly significant in increasing the risk of haematological cancers, with variants in some of these genes also associated with non-malignant syndromic features. Allogeneic blood and marrow transplantation (BMT) is central to management in many haematological cancers. Identification of germline variants may have implications for the patient and potential family donors. Beyond selection of an appropriate haematopoietic stem cell donor there may be sensitive issues surrounding identification and counselling of hitherto asymptomatic relatives. If BMT is needed, there is frequently a clinical urgency that demands a rapid integrated multidisciplinary approach to testing and decision making involving haematologists in collaboration with Clinical and Laboratory Geneticists. Here, we present best practice consensus guidelines arrived at following a meeting convened by the UK Cancer Genetics Group (UKCGG), the Cancer Research UK (CRUK) funded CanGene-CanVar research programme (CGCV), NHS England Genomic Laboratory Hub (GLH) Haematological Oncology Malignancies Working Group and the British Society of Blood and Marrow Transplantation and Cellular Therapy (BSBMTCT).

Characteristics of familial pancreatic cancer families with additional colorectal carcinoma

Familial pancreatic cancer (FPC) is a rare hereditary tumor entity with broad phenotypic heterogeneity, including colorectal carcinoma (CRC) in some families. The underlying factors for this co-occurrence are still not well evaluated. FPC families in the National Case Collection of Familial Pancreatic Cancer with an additional occurrence of CRC were analyzed regarding the phenotype, genotype and recommendation for a clinical screening program. The total cohort of 272 FPC families included 30 (11%) families with at least one CRC case. The proportion of affected family members with PDAC was 16.1% (73/451) compared to 9.3% of family members with CRC (42/451, p < 0.01). Females were affected with PDAC in 49% (36/73) and CRC in 38% (16/42). The median age of PDAC was 63 compared to 66 years in CRC, whereas 8 (26.6%) of families had an early onset of PDAC and 2 (6.7%) of CRC. Seventeen families had 2 or more affected generations with PDAC and 6 families with CRC. Eleven (9.6%) of affected patients had both PDAC and CRC. Potentially causative germline mutations (2 ATM, 1 CDKN2a, 1 MLH1, 1 PALB2) were detected in 5 of 18 (27.7%) analyzed cases. These findings provide a step forward to include the phenotypic and genotypic characteristics of FPC-CRC families for the genetic counseling and management of these families. Nevertheless, results need to be verified in a larger patient cohort beforehand.

A systematic review and meta-analysis of genetic assessment for women with ovarian cancer: how can we do better?

<h3>Objectives:</h3> Despite clear consensus guidelines recommending universal germline genetic testing in ovarian cancer patients and a growing appreciation of the significant health implications for patients and their relatives, fewer than 50% of women with ovarian cancer undergo genetic testing. We aim to use systematic review and meta-analysis to assess the status of genetic testing in ovarian cancer and identify strategies that can improve uptake of genetic assessment in this high-risk population. <h3>Methods:</h3> A complete systematic search of online databases (PubMed, EMBASE, MEDLINE, and the Cochrane Library) for studies reporting on genetic testing in ovarian cancer without date restriction was performed. Random effects pooled proportions were calculated using the logit transformation and 95% confidence intervals for individual study proportions were calculated using the Clopper-Pearson exact method. <h3>Results:</h3> The comprehensive search produced 3026 studies, among which 38 met inclusion criteria. Among studies listing mean age of diagnosis, stage, and histology data, the median of the mean age of diagnoses across the studies was 60.5 years. The median proportion of patients with early stage (I/II) ovarian cancer was 22.8%, late stage (III/IV) 70%, and unknown stage 3.6%. The median proportion of patients with serous histology was 74.6%, endometrioid 8.6%, mucinous 6%, clear cell 7%, mixed 3.6%, carcinosarcoma 3.4%, adenocarcinoma 5.8%, and other/unknown histology 7.9%. Twenty-one papers described rates of referral to genetic counseling and found, among 9,629 women, a referral rate of 56% [CI 32-78%]. Twenty-three papers described genetic testing uptake without a specified intervention and found, among 16,412 women, 34% [CI 23-47%] underwent genetic testing (Figure 1). For patients completing genetic testing, 27% [CI 22-32%] had a pathogenic mutation and 7% [CI 3-16%] a variant of uncertain significance. The most successful strategy aimed at improving rates of genetic testing was ‘mainstreaming,' the process of providing counseling and testing in the oncology clinic by trained non-genetics specialists. With mainstreaming, 99% [CI 86-100%] of patients completed genetic testing. Other strategies described in the literature included the use of telemedicine for genetic services (75% underwent genetic testing [CI 43-93%]), embedding a genetic counselor in the clinic (67% [CI 32-90%]), reflex somatic tumor <i>BRCA1/2</i> testing to triage to germline testing (64% [17-94%]), practice recommendation for universal testing of all ovarian cancer patients (42% [CI 15-74%]), and incorporating dedicated referral forms into practice (26% [CI 10-53%]). <h3>Conclusions:</h3> Identifying causative germline mutations in ovarian cancer can impact treatment decisions, inform risk of other malignancies and guide disease prevention and early detection in at-risk relatives. Rates of both referral to genetic counseling and testing remain low, 56% and 34% respectively, despite broad recommendations for universal testing. Mainstreaming, use of telemedicine, embedding genetic counselors in the clinic, and triage via reflex somatic <i>BRCA1/2</i> assessment are all strategies demonstrating success in this patient population and should be considered for those providing care to women with ovarian cancer.

Abstract 3030: Heritability of cancer predisposition gene mutations in 556 neuroblastoma patients with paired parental DNA whole genome sequences

Abstract Background: Neuroblastoma is a childhood cancer that arises from the sympathetic nervous system. Approximately 1% of patients have a family history of disease with causal germline mutations in neuroblastoma predisposition genes, ALK or PHOX2B. Recent genome-wide association studies (GWAS) of sporadic cases have identified over two dozen susceptibility loci, and next-generation sequencing studies estimate that 8-10% of children with cancer harbor a rare pathogenic (P) or likely pathogenic (LP) germline variant in a cancer predisposition gene (CPG). To date, the heritability and clinical significance of germline P/LP variants remains unknown due to a lack of parental germline DNA. Methods: Through the Gabriella Miller Kids First program, we performed whole genome sequencing (WGS) at 30x depth on patient-parent triads (n=457) and dyads (n=99), along with WGS and exome sequencing on matched tumor DNA (n=336) and RNA-sequencing on matched tumor RNA (n=207). Rare variants (allele frequency &amp;lt;0.1% in ExAC, 1000 Genomes, and gnomAD) in a predefined set of CPGs (n=197) were annotated, and pathogenicity was assessed using ClinVar, InterVar, and manual review. Gene-based enrichment was performed by comparison to gnomAD WGS data (n=143,068), excluding TCGA samples. Heritability was evaluated using family-based variant calling. Tumor DNA sequencing was analyzed for second hits or somatic enrichment of the germline P/LP variant. Tumor RNA sequencing was interrogated to identify expressed P/LP variants and preferential allelic expression. Ongoing analyses are utilizing polygenic risk scores from our GWAS study to inform tumor penetrance. Results: We observed 78 P/LP germline variants in CPGs in 75 probands (13.5%). Of the 72 probands with trio data, 95% of the P/LP variants were inherited (54% maternal, 46% paternal). Several CPGs showed enrichment of P/LP variants, including BARD1 (p=0.001; OR 16.16; 95% CI 3.21-50.45). We observed one canonical ALK mutation (R1275Q), but no PHOX2B mutations. For patients with corresponding tumor samples, all germline P/LP variants were observed in the tumor DNA. Germline P/LP variants were associated with high-risk disease as defined by risk group (high vs. low/intermediate, p=0.005), INRG stage (stage M vs stage L1/L2/MS, p=0.024), and MYCN amplification status (amplified vs not amplified, p=0.016). Conclusions: Here we show that approximately 13.5% of patients with neuroblastoma harbor a rare P/LP germline variant in a CPG, and the vast majority of these are inherited. Neuroblastoma patients with P/LP germline variants are more likely to have high-risk disease and several variants suggest potential therapeutic opportunities. Work is ongoing to understand the genetic factors that explain why parents harboring the same P/LP variant did not develop neuroblastoma, and to determine the genetic counseling implications of these data. Citation Format: Emily Blauel, Zalman Vaksman, Alex Lee, Rebecca Kaufman, Laura Egolf, Andrew Olshan, John Maris, Diskin Sharon. Heritability of cancer predisposition gene mutations in 556 neuroblastoma patients with paired parental DNA whole genome sequences [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3030.

Genetic Counseling for Hereditary Gastric and Pancreatic Cancer in High-Risk Gastrointestinal Cancer Clinics: An Effective Strategy.

The identification of high-risk groups of gastric (GC) and pancreatic adenocarcinoma (PC) due to a hereditary basis could imply a benefit in the affected families by establishing personalized preventive strategies. We aimed at assessing the diagnostic yield of GC/PC hereditary syndromes in individuals evaluated based on specific clinical criteria. In total, 77 unrelated individuals (45 from GC group/32 from PC group) were recruited: 51 (66.2%) cancer diagnosis ≤60 years, 3 (4%) with personal history of GC/PC and other cancer and 23 (29.8%) due to family history. Immunohistochemical analysis of DNA mismatch repair proteins was performed in 38 (49.3%) available tumors, being pathological in one (2%) GC. A genetic analysis was performed if clinical criteria of hereditary syndrome were fulfilled, identifying a mutation in 10/22 (45.5%) families [7/16 (43.7%) with GC and 3/6 (50%) with PC] and 19 (24.7%) fulfilled criteria of familial cancer. Diagnosis of cancer <40 years and personal history of other cancers were independent risk factors of a hereditary syndrome [OR:11.3 (95%IC 1.9–67); p = 0.007 and OR:17.4 (95% IC 2.5–119.9); p = 0.004; respectively]. The selection of patients based on clinical criteria leads to high diagnostic yield, detecting a causative germline mutation in almost half of the cases; therefore, both meticulous genetic counseling and use of multi-gen panels is crucial.

Hereditary vs Familial Pancreatic Cancer: Associated Genetic Syndromes and Clinical Perspective

Pancreatic ductal adenocarcinoma (PDAC) is a disease marked by high rates of mortality; it is mostly incurable at the time of diagnosis. Only about 7% of patients survive 5 years after diagnosis. Diagnosis at a late stage and rapid progression with minimal response to available treatments are the main reasons for this poor outcome. It is crucial to identify individuals at high risk of developing PDAC so preventive and early detection measures can be employed. Approximately 10% to 15% of PDAC cases have a hereditary or familial basis. In the majority of PDAC cases, no main causative gene has been identified, but several known germline pathogenic mutations have been shown to be related to an increased risk of this cancer. The presence of 2 or more patients with pancreatic cancer within the circle of first-degree relatives, without the presence of a causative germline mutation, is defined as familial pancreatic cancer; this accounts for 4% to 10% of PDAC. Based on the growing evidence supporting the benefit of germline genetic testing in patients with PDAC, both the American Society of Clinical Oncology and the National Comprehensive Cancer Network recently updated their guidelines to include recommendations around genetic testing for patients with pancreatic cancer. However, there is no general consensus on the group of patients and individuals who should be studied and screened. We present a demonstrative case and review the available data on hereditary and familial PDAC.

SAT-188 An Unusual Case Of Intra-adrenal Purely Norepinephrine Secreting Pheochromocytoma

Background: In 40% of pheochromocytoma/paraganglioma (PPGL) cases a causal germline mutation in a well-defined gene can be identified. The remainder are sporadic. The most common hereditary syndromes are NF1, MEN2 and VHL. Paragangliomas usually produce exclusively norepinephrine (NE) and are more likely to metastasize than adrenal tumors. Exclusively NE producing adrenal tumors are extremely rare and almost always associated with VHL or SDH syndromes (1).Clinical Case: A 45-year-old South Asian woman with a 5-year history of HTN controlled on losartan presented emergently complaining of chest pressure. Cardiovascular workup was unrevealing. CT chest showed an incidental 3.2 x 2.4 cm lipid-poor left adrenal adenoma. No further follow-up done at that time. Two years later she presented with recurring episodes of chest pressure and uncontrolled HTN on amlodipine, metoprolol and losartan. She denied panic attacks, diaphoresis and other symptoms of anxiety. She denied personal and familial history of clinical features seen in NF1, MEN 2, VHL or SDH. Plasma metanephrines were 26 (<=57 pg/mL) and free normetanephrine 902 (<=148 pg/mL). 24-hour urine metanephrine was 178 (58-203 mcg/24h), normetanephrine 2422 (88-649 mcg/24h) and total metanephrine 2600 (182-739 mcg/24h) confirming the diagnosis of a solely NE secreting PPGL. MRI abdomen showed a well-circumscribed 3.3 cm lipid-poor left adrenal mass. MIBG scan and SPECT CT showed a focal area of intense radiotracer uptake corresponding to a 3.2 x 2.5 cm mass within the left adrenal gland. No extra-adrenal activity was demonstrated. Alpha blockade was started with doxazosin and the patient asked to salt/fluid load. She underwent left adrenalectomy. Pathology revealed a 5 x 2.5 x1.1 cm intra-adrenal pheochromocytoma; Chromogranin (+), synaptophysin (-), MIB1 low reactivity < 5%, S100 positive in sustentacular cells, keratin (-), EMA (-), Inhibin (-). Genetic testing for VHL, SDHD, SDHB, SDHC and MAX has been ordered per guidelines. One month post-operatively the patient had no symptoms of adrenergic overactivity and normal plasma metanephrine levels.Clinical Lessons: This rare case of norepinephrine-only secreting pheochromocytoma is made even more unusual by absence of features of autosomal dominant hereditary syndromes. This may be sporadic or present a novel VHL, SDHD, SDHB, SDHC or MAX germline mutation (2).1.Eisenhofer, G., et al., Distinct gene expression profiles in norepinephrine- and epinephrine-producing hereditary and sporadic pheochromocytomas: activation of hypoxia-driven angiogenic pathways in von Hippel–Lindau syndrome. 2004. 11(4): p. 897.2.Ercolino, T., et al., Uncommon clinical presentations of pheochromocytoma and paraganglioma in two different patients affected by two distinct novel VHL germline mutations. Clinical Endocrinology, 2008. 68(5): p. 762-768.

Abstract PR02: Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer

Abstract Beginning with discovery of a new genetic cause of familial pancreatic ductal adenocarcinoma (PDAC), this work uncovers a mechanism for regulating KRAS intracellular trafficking with implications for hereditary cancer, sporadic cancer, and RASopathy syndromes. PDAC is one of the deadliest solid cancers, with limited treatment options despite intensive research efforts. Familial predisposition to PDAC is thought to occur in ~10% of cases, but causative genes have not been identified in most of these families. Uncovering the genetic basis for PDAC susceptibility has immediate prognostic implications for families and can provide precious mechanistic clues to PDAC pathogenesis. Here, we perform whole-genome sequence analysis in a family with five cases of PDAC and identify a germline nonsense mutation in the member of RAS oncogene family-like 3 (RABL3) gene that has never before been directly associated with hereditary cancer. The truncated mutant allele (RABL3_p.Ser36*) co-segregates with cancer occurrence. To evaluate the contribution of the RABL3 mutant allele in hereditary cancer, we generated rabl3 heterozygous mutant zebrafish and found increased susceptibility to cancer formation in two independent cancer models. Complementary unbiased approaches implicate RABL3 in RAS pathway regulation. RNA-Seq and genome-set enrichment analysis of juvenile rabl3 mutants reveals a KRAS upregulation signature. Furthermore, affinity-purification mass spectrometry for proteins associated with RABL3 or RABL3_p.Ser36* identifies Rap1 GTPase-GDP Dissociation Stimulator 1 (RAP1GDS1, SmgGDS), a chaperone that regulates prenylation of RAS GTPases. Indeed, in vitro studies demonstrate that RABL3_p.Ser36* accelerates KRAS prenylation, and this impact is lost in the absence of H/N/KRAS proteins. Whereas heterozygous rabl3 mutant zebrafish exhibit cancer predisposition, homozygous rabl3 mutant zebrafish develop severe craniofacial, skeletal, and growth defects consistent with human RASopathies, and these defects are partially rescued with the MEK inhibitor trametinib. Finally, we identify additional germline mutations in RABL3 that impact RAS activity in vivo and have a significant burden in a cohort of patients with developmental disorders, suggesting a role in undiagnosed RASopathies. Moreover, RABL3 is upregulated in multiple human PDAC cell lines and knockdown abrogates proliferation, consistent with a broader role for RABL3 in PDAC. The discovered causative RABL3 germline mutation provides new diagnostic opportunities for genetic testing in other cancer families and uncovers an alternative mechanism for dysregulated RAS signaling in development and cancer. This abstract is also being presented as Poster A12. Citation Format: Sahar Nissim, Ignaty Leshchiner, Joseph Mancias, Matthew Greenblatt, Ophélia Maertens, Christopher Cassa, Jill Rosenfeld, Andrew Cox, John Hedgepeth, Julia Wucherpfennig, Andrew Kim, Jake Henderson, Patrick Gonyo, Anthony Brandt, Ellen Lorimer, Bethany Unger, Gad Getz, Shamil Sunyaev, Wade Harper, Karen Cichowski, Alec Kimmelman, Yariv Houvras, Sapna Syngal, Carol Williams, Wolfram Goessling. Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr PR02.

Familial Pancreatic Cancer: Current Perspectives.

Pancreatic cancer (PC) is a highly lethal disease, mostly incurable when detected. Thus, despite advances in PC treatments, only around 7% of patients survive 5-years after diagnosis. This morbid outcome is secondary to multifactorial reasons, such as late-stage diagnosis, rapid progression and minimal response to chemotherapy. Based on these factors, it is of special relevance to identify PC high-risk individuals in order to establish preventive and early detection measures. Although most PC are sporadic, approximately 10% cases have a familial basis. No main causative gene of PC has been identified but several known germline pathogenic mutations are related with an increased risk of this tumor. These inherited cancer syndromes represent 3% of all PC. On the other hand, in 7% of cases of PC, there is a strong family history without a causative germline mutation, a situation known as familial pancreatic cancer (FPC). In recent years, there is increasing evidence supporting the benefit of genetic germline analysis in PC patients, and periodic pancreatic screening in PC high-risk patients (mainly those with a lifetime risk greater than 5%), although there is no general agreement in the group of patients and individuals to study and screen. In the present review, we expose an update in the field of hereditary and FPC, with the aim of describing the current strategies and implications in genetic counseling, surveillance and therapeutic interventions.

Comprehensive Mutational Analysis of Juvenile Myelomonocytic Leukemia Using Whole-Genome Sequencing

Background Juvenile myelomonocytic leukemia (JMML) is a rare and exclusively pediatric myelodysplastic/myeloproliferative neoplasm. This disease is genetically characterized by an extremely small number of somatic mutations (an average of 0.8 mutations/exome/patient). It has been shown that causative somatic and/or germline mutations activating the RAS pathway are located in PTPN11, NF1, NRAS, KRAS, and CBL in 85% of patients with JMML. Furthermore, up to 20% of the patients have additional secondary mutations including SETBP1, and JAK3 mutations. In 2% of the patients, we identified, by RNA sequencing, activating kinase lesions affecting ALK or ROS1. Such findings suggest that other kinase fusions are present in JMML. There is an exceptional scarcity of somatic passenger mutations on the exome, suggesting that a small number of driver mutations drive JMML. However, to date, this hypothesis has not been investigated by whole-genome sequencing. Patients and Methods We performed a whole-genome sequencing (WGS) study in 48 patients with JMML. Bone marrow specimens and in vitro-cultured T cells were used as tumor and germline samples, respectively. Next-generation sequencing was performed using a HiSeq X platform (Illumina). Data analysis was performed by our in-house pipeline. Specifically, the pipeline detects single nucleotide variants (SNVs), copy number variants, somatic loss of heterozygosity (LOH), and chromosomal structural variations (SVs). The study was approved by the institutional review board of Nagoya University Graduate School of Medicine. Results In each patient we detected an average of 28 somatic mutations. These were primarily C-to-T transition in the CpG context, indicating that the mutations occurred by cell division. Besides RAS pathway and known secondary mutations, we observed no significant accumulation of somatic mutations in either coding or non-coding regions. Although we detected RAS pathway mutations in 90% of the patients, all mutations were on exome. However, we identified germline microdeletions affecting CBL and NF1, which had not been identified by exome sequencing. Additionally, we found two LOH events that affected NF1. Bi-allelic inactivation of NF1 is generally observed in patients with JMML; however, no pathogenic SNVs were identified in these two patients. We identified two chromosomal translocations that caused activating kinase lesions (i.e., RANBP2-ALK and TBL1XR1-ROS1). These had been pointed out in our previous RNA sequencing study. Another patient carried a complex SV that affected XPO1 (encoding exportin 1 or chromosome region maintenance 1 protein homolog). Although fusion genes involving XPO1 are reported to be present in lymphoid malignancies, the role of this SV in JMML remains unclear. Conclusions JMML is characterized by driver mutations that are largely present within the exome. However, WGS can still play a role in identifying both coding and non-coding mutations. LOH events without pathogenic SNVs suggest the presence of novel regulatory mechanisms of NF1. Conclusively, JMML is characterized by a paucity of somatic alterations and driver mutations. Hence, current research efforts should focus on RAS pathway mutations and known secondary mutations, many of which can be targeted. Disclosures No relevant conflicts of interest to declare.

Using global DNA-methylation and metabolome profiles to classify variants of unknown significance (VUS) in phaeochromocytoma and paraganglioma tumours

Abstract Background Phaeochromocytomas and paragangliomas (PPGLs) are rare but strongly heritable neuroendocrine tumours. Most patients undergo genetic testing and approximately 40% will have a germline causal mutation. In 10% of patients, genetic analysis identifies uncharacterised mutations, or variants of unknown significance (VUSs). Tests to distinguish between benign and pathogenic mutations would aid clinicians' and patients' decision-making. One test could exploit the biology of a subset of PPGLs. Mutations in genes controlling the Tricarboxylic Acid cycle cause a deranged metabolome, accumulation of oncometabolites and global DNA-hypermethylation. Data show that this stable chemical modification will be greatly elevated in pathogenic over benign mutations, and be present alongside a driver metabolome. Methods Clinical characteristics were compiled for 3 patient groups: causal germline mutations, no identified mutation (sporadic) and VUS. Archival Formalin Fixed Paraffin Embedded (FFPE) material was identified locally and also provided by the international ENS@T collaboration. Tissue analysis was optimised on mouse FFPE tissue. Laser microdisection allowed collection of precise tissue areas. DNA and metabolite extraction was optimised before analysis was undertaken by Liquid Chromatography Mass Spectrometry (LC-MS). Results 93 patients were recruited locally. Clinical characteristics were collected for 25 sporadic and 56 germline cases, including 14 VUS cases. Optimisation experiments in mouse tissue show that discrete areas can be extracted by laser microdissection. LC-MS identifies cytosine, 5'-methylcytosine and 5'-hydroxymethylcytosine in 1 μg of DNA at biologically relevant ratios. TCA cycle metabolites were detected using LC-MS: succinate, fumarate and malate were found in the metabolite extract from the same amount of tissue. Conclusions This study utilises DNA hypermethylation and metabolomics for clinical application. It represents a large repository of PPGL VUS samples nationally. Global DNA-methylation levels and metabolite profiles are shown to be quantifiable from existing FFPE patient samples, using reduced amounts of patient material. Legal entity responsible for the study Guy's and St Thomas' NHS Trust, London, UK; King's College London, UK. Funding National Institute of Health Research, King's College London, Isaac Shapera Research Trust, The Bernice Bibby Research Trust. Disclosure All authors have declared no conflicts of interest.

First Description of Hb San Diego (HBB: c.328G>A) in a Chinese Family with Congenital Erythrocytosis

Congenital erythrocytosis is a rare and hereditary disorder of red blood cell (RBC) production that can be caused by high oxygen affinity hemoglobin (Hb) variants. We applied a genetic approach including whole exome sequencing and Sanger sequencing. We identified a heterozygous β-globin gene (Hb San Diego or HBB: c.328G>A) in exon 3 as a causative germline mutation in a Chinese family with congenital erythrocytosis. We concluded that in erythrocytosis with a dominant inheritance and normal or inappropriately high erythropoietin (EPO) levels, the high oxygen affinity Hb variants should be considered. In addition, as a tool for identification of mutations in congenital erythrocytosis, whole exome sequencing improves diagnostic accuracy and provides the opportunity for discovery of novel variants.

APPLICATION OF THE TARGETED MULTIGENE SEQUENCING FOR THE SEARCH OF HEREDITARY BREAST CANCER MUTATIONS IN RUSSIAN PATIENTS

Understanding of the molecular-genetic pathogenesis of hereditary cancer syndrome is extremely important for developing of personal therapeutic approaches and for improving the effectiveness of preventive measures. Today, the optimal solution for the search of causative germ-line mutations in hereditary breast cancer (BC) patients is the next-generation sequencing-based multigene mutational screening. The authors have assembled a targeted panel of 31 genes, based on their potential involvement in the cancer susceptibility and taking into account the frequency of pathogenic alleles in the Russian population. It includes the “canonical” genes of hereditary breast cancer (BRCA1, BRCA2, BRIP1, PALB2, TP53, ATM, NBN), the recently identified “novel” genes (BLM, FANCD2, POLE, FANCM, RAD51C, MRE11A, RECQL, as well as some other genes involved in DNA repair, apoptosis and genome stability maintenance. 94 patients with hereditary BC of unknown genetic etiology were subjected to targeted sequencing. As a result, causative germ-line mutations were identified in 21/94 (22.3%) patients. Importantly, 19 patients harbored rare non-founder BRCA1 and BRCA2 mutations. In the remaining two cases, the functions of the ATM (p.Glu73fs) and POLE (p.Leu1171fs) genes were disrupted. The obtained data are of evident clinical importance; they argue for the expanding of diagnostic panels for monitoring at-risk individuals and for moving the standards of routine clinical diagnostics towards the targeted next-generation sequencing of multigene panels.

3323 Juvenile Polyposis Syndrome Patients Without a Mutation in SMAD4 or BMPR1A: Clinical Presentation and Novel Drivers of Disease

OBJECTIVES/SPECIFIC AIMS: Juvenile Polyposis Syndrome (JPS) is an inherited cancer predisposition syndrome sometimes attributed to a germline mutation in SMAD4 or BMPR1A. However, many patients meet clinical criteria for JPS without having a pathogenic alteration in either gene. Herein, we perform a cross-sectional analysis of JPS patients at a pediatric and adult tertiary referral center to understand potential differences in the clinical presentation and outcomes of patients with or without a known causative gene mutation. Additionally, we conduct whole exome sequencing (WES) on a subset of the pediatric patients to evaluate for novel genomic drivers of disease. METHODS/STUDY POPULATION: Data were abstracted from medical charts using IRB-approved protocols at the Children’s Hospital of Philadelphia (CHOP) and the University of Pennsylvania (Penn). Records were reviewed for patients with a clinical diagnosis of JPS and genetic testing result and seen at either institution in the last 10 years (2008-2018). Patients recruited for sequencing were consented for blood draw through the CHOP IRB protocol, and had whole exome sequencing completed at 70X depth, with data analyzed through institutional pipeline. RESULTS/ANTICIPATED RESULTS: Records were reviewed for 41 patients at CHOP and 19 patients at Penn, for a total of 60 JPS patients. Mean age of CHOP cohort was 11 years: 58.5% male, mean length of follow up 3.9 years. Mean age Penn cohort was 33 years: 47.4% male, mean length of follow up 9.3 years. In the pediatric cohort, 7 patients (17%) had a mutation in BMPR1A (n=6) or SMAD4 (n=1); in the adult cohort, 15 patients (79%) had a mutation in BMPR1A (n=3) or SMAD4 (n=12). The average number of polyps in the pediatric cohort was not significantly higher in patients with a SMAD4 or BMPR1A mutation (9.3 polyps/year of surveillance with a SMAD4 or BMPR1A mutation, vs 5.7 polyps/year; p=0.19). In combined cohort review, all individuals that required gastrectomy and/or colectomy (n=8) as well as all those who developed gastrointestinal cancer (n=3) had a mutation in SMAD4 or BMPR1A. Of the patients who underwent whole exome sequencing (n=13), potential causative germline mutations were identified in four patients (30.8%); all potential drivers identified were within the TNF/BMP pathway. DISCUSSION/SIGNIFICANCE OF IMPACT: This data from a dual-institution review demonstrates that the rate of SMAD4/BMPR1A mutation in JPS is lower in a pediatric cohort compared to an adult cohort. Furthermore, although individuals with JPS may have similar clinical presentations in childhood regardless of whether or not a causative mutation is present, the presence of a mutation in SMAD4 or BMPR1A is associated with a more severe course of disease in adulthood. Further study and a larger cohort will be required to fully validate these findings. Approximately 30% of patients who underwent germline WES had a potential novel driver identified, with further validation underway.

Germline Mutations in CTNNB1 Associated With Syndromic FEVR or Norrie Disease.

Germline and somatic mutations in CTNNB1 have been found in different types of human diseases. This follow-up study aimed to identify causative germline mutations in CTNNB1 and their associated ocular phenotypes through a comparative analysis of whole-exome sequencing data. Annotated sequence variations in CTNNB1 were selected from in-house data from whole-exome sequencing of genomic DNA prepared from leucocytes of 3280 unrelated probands with different forms of eye diseases. Potentially pathogenic variants in CTNNB1 were analyzed by multistep bioinformatics analyses. Clinical data from probands with pathogenic variants in CTNNB1 were collected, and potential genotype-phenotype correlations were analyzed. Eleven rare variants that potentially affect the coding regions of CTNNB1 were detected in 11 of the 3280 samples, and four variants were considered to be potentially pathogenic. All four mutations, namely, c.999delC (p.Tyr333*), c.1104delT (p.His369Thrfs*2), c.1738_1742delinsACA (p.Leu580Thrfs*28), and c.1867C>T (p.Gln623*), were heterozygotes and considered to have a germline origin. Three of the four mutations are de novo mutations, and the status of the remaining mutation is unavailable. All four probands had the same class of closely related ocular diseases: one proband had FEVR, and three probands had Norrie-like retinopathy. The molecular results indicated that three probands showed systemic anomalies, as demonstrated by a follow-up survey, but relevant information for the remaining proband was unavailable. The data suggest that germline truncating mutations in CTNNB1 cause autosomal dominant syndromic FEVR or Norrie disease. Patients with mutations in CTNNB1, KIF11, or NDP may have similar or overlapping phenotypes, but this phenomenon needs to be studied further.

Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes.

BackgroundCarriers of mutations in hereditary cancer predisposition genes represent a small but clinically important subgroup of oncology patients. The identification of causal germline mutations determines follow-up management, treatment options and genetic counselling in patients’ families. Targeted next-generation sequencing-based analyses using cancer-specific panels in high-risk individuals have been rapidly adopted by diagnostic laboratories. While the use of diagnosis-specific panels is straightforward in typical cases, individuals with unusual phenotypes from families with overlapping criteria require multiple panel testing. Moreover, narrow gene panels are limited by our currently incomplete knowledge about possible genetic dispositions.MethodsWe have designed a multi-gene panel called CZECANCA (CZEch CAncer paNel for Clinical Application) for a sequencing analysis of 219 cancer-susceptibility and candidate predisposition genes associated with frequent hereditary cancers.ResultsThe bioanalytical and bioinformatics pipeline was validated on a set of internal and commercially available DNA controls showing high coverage uniformity, sensitivity, specificity and accuracy. The panel demonstrates a reliable detection of both single nucleotide and copy number variants. Inter-laboratory, intra- and inter-run replicates confirmed the robustness of our approach.ConclusionThe objective of CZECANCA is a nationwide consolidation of cancer-predisposition genetic testing across various clinical indications with savings in costs, human labor and turnaround time. Moreover, the unified diagnostics will enable the integration and analysis of genotypes with associated phenotypes in a national database improving the clinical interpretation of variants.

A landscape of germ line mutations in a cohort of inherited bone marrow failure patients

AbstractBone marrow (BM) failure (BMF) in children and young adults is often suspected to be inherited, but in many cases diagnosis remains uncertain. We studied a cohort of 179 patients (from 173 families) with BMF of suspected inherited origin but unresolved diagnosis after medical evaluation and Fanconi anemia exclusion. All patients had cytopenias, and 12.0% presented ≥5% BM blast cells. Median age at genetic evaluation was 11 years; 20.7% of patients were aged ≤2 years and 36.9% were ≥18 years. We analyzed genomic DNA from skin fibroblasts using whole-exome sequencing, and were able to assign a causal or likely causal germ line mutation in 86 patients (48.0%), involving a total of 28 genes. These included genes in familial hematopoietic disorders (GATA2, RUNX1), telomeropathies (TERC, TERT, RTEL1), ribosome disorders (SBDS, DNAJC21, RPL5), and DNA repair deficiency (LIG4). Many patients had an atypical presentation, and the mutated gene was often not clinically suspected. We also found mutations in genes seldom reported in inherited BMF (IBMF), such as SAMD9 and SAMD9L (N = 16 of the 86 patients, 18.6%), MECOM/EVI1 (N = 6, 7.0%), and ERCC6L2 (N = 7, 8.1%), each of which was associated with a distinct natural history; SAMD9 and SAMD9L patients often experienced transient aplasia and monosomy 7, whereas MECOM patients presented early-onset severe aplastic anemia, and ERCC6L2 patients, mild pancytopenia with myelodysplasia. This study broadens the molecular and clinical portrait of IBMF syndromes and sheds light on newly recognized disease entities. Using a high-throughput sequencing screen to implement precision medicine at diagnosis can improve patient management and family counseling.

Abstract IA15: Genetic predisposition to hematopoietic malignancies

Abstract There is a growing appreciation for the impact of baseline genetic variants in the development of hematopoietic malignancies, and the germline risk factors that make an individual more susceptible to developing bone marrow-derived cancers are being defined through highly informative families and next-generation sequencing with increasing frequency. At the University of Chicago, we use a rigorous pipeline for clinical testing of individuals comprising next-generation sequencing-based molecular testing to detect point mutations as well as microarrays to detect chromosomal rearrangements. Overall, we are now able to identify the causative germline mutation in about 20% of cases, a level commensurate with other cancer risk clinics, but hinting that additional predisposition alleles remain to be discovered. Many of our patients/families also contribute to research by banking samples for future studies designed to identify new predisposition syndromes. This presentation will include a discussion of the new insights into hematopoietic biology that can be appreciated by studying these emerging predisposition syndromes. Citation Format: Lucy A. Godley. Genetic predisposition to hematopoietic malignancies [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA15.

Abstract 4272: Mutations in RABL3 alter RAS prenylation and are associated with hereditary pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest solid cancers with limited treatment options despite intensive research efforts. Familial predisposition to PDAC is thought to occur in ~10% of cases, but causative genes have not been identified in most of these families. Uncovering the genetic basis for PDAC susceptibility has immediate prognostic implications for families and can provide precious mechanistic clues to PDAC pathogenesis. Here, we perform whole-genome sequence analysis in a family with high incidence of PDAC and identify a germline nonsense mutation in the member of RAS oncogene family-like 3 (RABL3) gene that has never before been directly associated with hereditary cancer. The truncated mutant allele (RABL3_p.Ser36*) co-segregates with cancer occurrence. To evaluate the contribution of the RABL3 mutant allele in hereditary cancer, we generated rabl3 heterozygous mutant zebrafish and found increased susceptibility to cancer formation in two independent cancer models. Complementary unbiased approaches implicate RABL3 in RAS pathway regulation. RNA-Seq and genome-set enrichment analysis of juvenile rabl3 mutants reveals a KRAS upregulation signature. Furthermore, affinity-purification mass-spectrometry for proteins associated with RABL3 or RABL3_p.Ser36* identifies Rap1 GTPase-GDP Dissociation Stimulator 1 (RAP1GDS1, SmgGDS), a chaperone that regulates prenylation of RAS GTPases. Indeed, in vitro studies demonstrate that RABL3_p.Ser36* accelerates KRAS prenylation, and this impact is lost in the absence of H/N/KRAS proteins. Whereas heterozygous rabl3 mutant zebrafish exhibit cancer predisposition, homozygous rabl3 mutant zebrafish develop severe craniofacial, skeletal, and growth defects consistent with human RASopathies, and these defects are partially rescued with the MEK inhibitor trametinib. Our findings support a gain-of-function rather than a null function typically associated with premature protein truncations. The discovered causative RABL3 germline mutation provides new diagnostic opportunities for genetic testing in other cancer families and uncovers an alternative mechanism for dysregulated RAS signaling in development and cancer. Note: This abstract was not presented at the meeting. Citation Format: Sahar Nissim, Ignaty Leshchiner, Joseph D. Mancias, Matthew B. Greenblatt, Ophélia Maertens, Christopher A. Cassa, Jill A. Rosenfeld, Andrew G. Cox, John Hedgepeth, Julia Wücherpfennig, Andrew J. Kim, Jake E. Henderson, Patrick Gonyo, Anthony Brandt, Ellen Lorimer, Bethany Unger, Jeremy W. Prokop, Jeremy W. Heidel, Xiao-Xu Wang, Chinedu I. Ukaegbu, Gad Getz, Shamil R. Sunyaev, J. Wade Harper, Karen Cichowski, Alec C. Kimmelman, Yariv Houvras, Sapna Syngal, Carol Williams, Wolfram Goessling. Mutations in RABL3 alter RAS prenylation and are associated with hereditary pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4272. doi:10.1158/1538-7445.AM2017-4272