ALBERT

Description: Introduction: Myelodysplastic syndrome (MDS) and acute leukemia (AL) are a clinically diverse and genetically heterogeneous group of hematologic malignancies. Familial forms of MDS/AL have been increasingly recognized in recent years, and can occur as a primary event or secondary to genetic syndromes, such as inherited bone marrow failure syndromes (IBMFS). It is critical to confirm a genetic diagnosis in patients with hereditary predisposition to hematologic malignancies in order to provide prognostic information and cancer risk assessment, and to aid in identification of at-risk or affected family members. In addition, a molecular diagnosis can help tailor medical management including informing the selection of family members for allogeneic stem cell transplantation donors. Until recently, clinical testing options for this diverse group of hematologic malignancy predisposition genes were limited to the evaluation of single genes by Sanger sequencing, which is a time consuming and expensive process. To improve the diagnosis of hereditary predisposition to hematologic malignancies, our CLIA-licensed laboratory has recently developed Next-Generation Sequencing (NGS) panel-based testing for these genes. Methods: Thirty six patients with personal and/or family history of aplastic anemia, MDS or AL were referred for clinical diagnostic testing. DNA from the referred patients was obtained from cultured skin fibroblasts or peripheral blood and was utilized for preparing libraries with the SureSelectXT Enrichment System. Libraries were sequenced on an Illumina MiSeq instrument and the NGS data was analyzed with a custom bioinformatic pipeline, targeting a panel of 76 genes associated with IBMFS and/or familial MDS/AL. Results: Pathogenic and highly likely pathogenic variants were identified in 7 out of 36 patients analyzed, providing a positive molecular diagnostic rate of 20%. Overall, 6 out of the 7 pathogenic changes identified were novel. In 2 unrelated patients with MDS, heterozygous pathogenic sequence changes were identified in the GATA2 gene. Heterozygous pathogenic changes in the following autosomal dominant genes were each identified in a single patient: RPS26 (Diamond-Blackfan anemia 10), RUNX1 (familial platelet disorder with propensity to myeloid malignancy), TERT (dyskeratosis congenita 4) and TINF2 (dyskeratosis congenita 3). In addition, one novel heterozygous sequence change (c.826+5_826+9del, p.?) in the Fanconi anemia associated gene FANCA was identified. . The RNA analysis demonstrated this variant causes skipping of exon 9 and results in a premature stop codon in exon 10. Further review of the NGS data provided evidence of an additional large heterozygous multi-exon deletion in FANCA in the same patient. This large deletion was confirmed using array-CGH (comparative genomic hybridization). Conclusions: This study demonstrates the effectiveness of using NGS technology to identify patients with a hereditary predisposition to hematologic malignancies. As many of the genes associated with hereditary predisposition to hematologic malignancies have similar or overlapping clinical presentations, analysis of a diverse panel of genes is an efficient and cost-effective approach to molecular diagnostics for these disorders. Unlike Sanger sequencing, NGS technology also has the potential to identify large exonic deletions and duplications. In addition, RNA splicing assay has proven to be helpful in clarifying the pathogenicity of variants suspected to affect splicing. This approach will also allow for identification of a molecular defect in patients who may have atypical presentation of disease. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Peripheral blood is the standard tissue source for germline genetic testing in most scenarios. In patients with hematologic malignancies, however, peripheral blood frequently contains tumor- or clonal hematopoiesis-related acquired genetic variants, often occurring in genes that can also cause inherited cancer susceptibility if present in the germline. Thus, an alternative tissue source is necessary. Cultured skin fibroblasts have been used as a potentially ideal alternative because they are free from blood contamination and provide ample DNA yields, advantages that other alternatives such as saliva or nail clippings lack. However, optimal culture methods, expected time from biopsy to sufficient DNA yield, culture failure rate, and limitations of this technique, including the possibility of variants being acquired solely due to the culturing process, are not yet known. Methods We conducted a retrospective cohort study of subjects with cytopenias or hematologic malignancies who underwent skin biopsy and fibroblast culture for germline genetic testing from April 2014 to June 2018. Skin biopsy culture technical data, including time from biopsy to culture set-up, shipment from an outside institution, culture failure, and biopsy size, were abstracted from tissue culture logs. Patient demographics, comorbidities, medication history, and hematologic diagnosis and treatment were abstracted from medical records. Next generation sequencing data from targeted capture of 144 inherited cancer and bone marrow failure predisposition genes obtained for clinical genetic testing purposes were analyzed to identify variants at both germline (40-60%) and subclonal (10-40%) variant allele frequencies (VAF). Pathogenicity was interpreted according to ACMG/AMP guidelines. Fisher's exact tests and logistic regression models were used to assess associations with culture failure. T-tests and linear regression models were used to assess factors associated with mean time to confluency. Results In total, we studied 350 samples from unique patients, including 61 (24%) who carried one or more pathogenic or likely pathogenic cancer susceptibility gene variant(s). Overall, 16 of the 350 (5%) biopsies failed to grow in culture. The median time from skin biopsy to sufficient growth to extract DNA for genetic testing was 27 days (IQR 22-29 days). Culture failure was significantly more likely in samples with a delay in culture initiation for 24 hours post biopsy (OR=4.32; p