Cheng, Yu‐Wei

Abstract Background Atypical hemolytic uremic syndrome (aHUS) is a rare, complement-mediated disorder. Disease-associated variants have been found in complement factor H-related genes and complement regulatory genes. Particularly, homozygous deletions of CFHR1, CFHR3, or CFHR4 have been associated with aHUS. Generally, multiplex ligation-dependent probe amplification (MLPA) is used to detect copy number changes in the CFH and CFHR regions. We have developed a cost-effective droplet digital PCR (ddPCR) method for measuring copy number variations (CNVs) in these genes. Methods A total of 41 DNA samples were analyzed using both ddPCR and MLPA to assess large deletions in the CFHR1, CFHR3, CFHR4 regions. The ddPCR primers were designed to avoid rare single nucleotide polymorphisms to enhance hybridization efficiency and accurate identification of deletions within the CFHR regions. The MLPA probe mix (Probemix P236 CFH Region) targeting CFHR regions was purchased from MRC Holland. Results Multiple heterozygous deletions were observed in the CFHR1, CFHR3, and CFHR4 genes. Homozygous deletions of CFHR1 and CFHR3 were seen in 2 cases, while 2 other cases showed homozygous CFHR1 and heterozygous CFHR3 deletions. All 41 samples demonstrated 100% concordance in copy number results between ddPCR and MLPA, as well as consistent reproducibility in both inter- and intratechnologist evaluations. Conclusions Our findings demonstrate that ddPCR is a robust platform to identify large deletions in the CFHR regions. Future studies are needed to evaluate its ability to distinguish full vs partial deletions and to further establish its clinical diagnostic value.

Abstract Objective β-Catenin–mutated hepatocellular adenomas (HCAs) carry an increased malignant transformation risk and are screened by interpreting glutamine synthetase (GS) and β-catenin by immunohistochemistry (IHC). Our study aims to assess GS and β-catenin interpretation guidelines for applicability and reproducibility in predicting high-risk HCA and other relevant molecular alterations. Methods Hematoxylin and eosin (H&E), β-catenin, GS, and CD34 stains from 75 HCAs were interpreted by three pathologists using Method A (GS interpretation: negative, perivenular patchy, map-like, diffuse, and indeterminate) and Method B criteria (similar GS interpretation scheme based on a recent publication, with and without CD34 expression patterns). Ease of application and interpretation confidence level were assessed. High-risk IHC was defined as nuclear β-catenin and/or diffuse homogeneous GS. Molecular testing was performed on a subset of HCAs and controls. Results There were 57 resections and 18 biopsy specimens examined. Methods A and B (GS only) were rated as easy to apply, with high interpretation confidence (≥90% using both methods). Consensus rate was comparable in biopsy specimens (100% for both methods) and resections (88% for Method A, 93% for Method B). While the same cases were stratified into high-risk GS categories using both systems, clinically significant genetic alterations (TERT promoter, EGFR, MTOR, and TP53) were identified in 25% of cases stratified as not high risk by IHC. Conclusions Both methods have a similar ease of application and level of interpretation confidence, and they also detected β-catenin mutations as expected. Other relevant molecular alterations associated with risk of neoplastic progression and/or bleeding were detected in 25% of HCAs with the non-high-risk IHC phenotype, suggesting the value of molecular testing in this subset.

AbstractBlastic plasmacytoid dendritic cell neoplasm (BPDCN) is recurrently mutated in epigenetic pathway genes. We studied the myeloid‐related genetic mutations in a cohort of five patients with BPDCN and one paired relapse case at our institution and identified a high frequency of biallelic TET2 and canonical ASXL1 (c.1934dupG) mutations. The number of cases is small, but the variant allele fraction (VAF) sums of the TET2 mutations, as well as the persistence of TET2 mutations in a case of relapsed BPDCN, suggest an ancestral/founder nature of TET2 clones in the cases. Further literature review shows a high frequency of biallelic TET2 mutations in reported cases of BPDCN.