Yu-Wei Cheng

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.

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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.

Background/Objective: The prognostic value of specific hot-spot mutations within KRAS, NRAS, and BRAF genes in metastatic colorectal cancer (mCRC) genes remains debatable. This study explores whether certain KRAS, NRAS, and BRAF mutations are associated with the risk of all-cause mortality in mCRC. Methods: We retrospectively analyzed records of 494 patients with mCRC treated at the University of Texas Medical Branch between January 2016 and July 2023. Data on genetic mutations and clinicopathological features were collected for this analysis. We estimated survival probabilities and conducted multivariable Cox proportional hazards regression to evaluate the impact of specific mutations on all-cause mortality risk. Results: KRAS c.35G>T (p.Gly12Val) and c.34G>T (p.Gly12Cys) mutations were significantly associated with an increased risk of all-cause mortality in the overall mCRC population and the treated mCRC subgroup. KRAS c.38G>A (p.Gly13Asp) was significantly associated with an increased risk of all-cause mortality in the treated mCRC subgroup but BRAF c.1799T>A (p.Val600Glu) was significantly associated with an increased risk of all-cause mortality in the overall mCRC population. No significant association was observed between NRAS mutations and mortality risk in mCRC, possibly due to their lower frequency or different biological effects compared to KRAS and BRAF mutations. Conclusions: These findings suggest that specific KRAS [c.35G>T (p.Gly12Val), c.34G>T (p.Gly12Cys), and c.38G>A (p.Gly13Asp)] and BRAF c.1799T>A (p.Val600Glu) mutations may have prognostic value in mCRC. However, given the single-center study design and lack of direct therapeutic implications, larger multicenter studies are needed to substantiate these results and better define the clinical relevance of these mutations.

Background: This study evaluates the performance of the Illumina NextSeq™ 550 and the Element Biosciences AVITI™ next-generation sequencing (NGS) system, in detecting single nucleotide variants (SNVs) and gene fusions. Methods: A set of 66 NGS libraries, consisting of 33 DNA, 24 cDNA, and triplicates of 3 control libraries, were prepared from bone marrow samples targeting 63 genes and related fusions, and initially sequenced using the NextSeq 550 in the Cleveland Clinic's molecular diagnostic laboratory. The same libraries were subsequently sequenced on the AVITI. The resulting data were analyzed using a combination of Cleveland Clinic developed pipelines and ArcherDx virtual machine software. Results: The study found that all 105 SNVs and 39 gene fusions identified by the NextSeq 550 were also detected in the AVITI, demonstrating a high degree of concordance between the platforms. The analyses revealed R2 values of 0.86 for read depth and 0.96 for VAF of the 105 DNA variants, and 0.95 for read depth and 0.97 for fusion percentage of the 39 fusion variants. In the reproducibility studies, the VAF and fusion percentage of all variants were within 2 standard deviations of the mean when the same positive controls were sequenced 3 times on the AVITI. Conclusions: These results indicate that the NextSeq 550 and the AVITI provide comparable performance in terms of accuracy and sensitivity for variant detection. Notably, the AVITI chemistry requires substantially lower PhiX input than the NextSeq 550 needs for this application. This results in substantial cost and efficiency benefits.

Background/Objective: Studies have shown an association between colorectal cancer (CRC) sidedness and gene mutations that may affect CRC clinical behavior. This study examined the association between specific KRAS, NRAS, and BRAF hot-spot mutations and primary CRC sidedness. Methods: We performed a retrospective cohort analysis of 722 patients diagnosed with primary CRC and tested for KRAS, NRAS, and BRAF hot-spot mutations at the University of Texas Medical Branch (UTMB) from January 2016 through July 2023. Multivariable logistic regressions analyses were conducted. Results:KRAS, NRAS, and BRAF hot-spot mutations rates were 37.8%, 4.6%, and 6.1%, respectively. Right-sided primary CRC had the highest prevalence of mutated tumors (64%). KRAS and BRAF hot-spot mutations were significantly different according to tumor sidedness. KRAS p.Gly12Asp, p.Gly12Val, and p.Gly13Asp showed a significantly increased likelihood of right-sided primary CRC compared to KRAS wildtype, 128%, 134%, and 221% higher, respectively. Conversely, KRAS p.Gly12Val and p.Gly13Asp mutations were associated with decreased likelihood of rectal cancer (53% lower) and left-sided tumors (56% lower), respectively. BRAF p.Val600Glu mutation, as opposed to BRAF wildtype, was associated with a 278% higher likelihood of right-sided CRC. No significant associations were observed between NRAS mutations and primary CRC sidedness. Conclusions: In primary CRC, specific mutations in KRAS (p.Gly12Asp, p.Gly12Val, and p.Gly13Asp) and BRAF p.Val600Glu were associated with increased likelihood of right-sided tumors. KRAS p.Gly12Val and p.Gly13Asp mutations were associated with decreased likelihood of rectal cancer and left-sided tumors, respectively. These findings suggest that tumorigenesis and mutational processes differ based on tumor sidedness. Further studies are needed to substantiate these findings.

Ewing sarcoma is a rare but aggressive type of cancer, primarily occurring in teenagers and young adults, characterized by having a small round cell morphology with positive diffuse membranous CD99 immunostaining of these small round blue cells. Although this cancer is often found in bones, it can also extend into the soft tissue in some cases. A gene fusion of one of the FET family RNA‐binding proteins, including EWSR1 and FUS genes as 5 ^′ partners, and one of the ETS family transcription factors as 3 ^′ partners, is the defining genetic characteristic of essentially all Ewing sarcoma cases. We report a case of a 42‐year‐old male individual with retroperitoneal Ewing sarcoma who underwent chemotherapy treatment following the biopsy diagnosis that revealed the FUS::FLI1 fusion. To the knowledge of the authors, this is the first report of response to chemotherapy in a case of Ewing sarcoma showing a rare FUS::FLI1 fusion.

Background and aims: The percentage of tumor cells (tumor cellularity) in a cancerous tissue has been assumed to correlate with the variant allele fraction (VAF) of an identified pathogenic variant. Many laboratories use the tumor cellularity as part of a quality criteria for specimen processing and clinical reporting. However, a systematic study of such correlation has yet to be shown. We performed a relatively large-scale study to determine whether pathologist-estimated tumor cellularity is correlated with next-generation sequencing (NGS)-derived VAF. Materials and Methods: A total of 1511 non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) specimens, including formalin-fixed paraffin-embedded (FFPE) and fine needle aspirated (FNA) tissues, were analyzed by cancer hotspot NGS. For a given specimen, pathogenic variants of BRAF, EGFR, KRAS, and NRAS were identified and the determined VAFs were correlated with the corresponding tissue tumor cellularity. Results: The coefficient of determination R-squared (R²) values were calculated for each correlation. All R² values were lower than 0.25, indicating poor correlations. Pathogenic variants were found, not uncommonly, in tumor specimens that carried 10% or lower tumor cellularity. There were no apparent differences of R² values between the FFPE and FNA specimens. Conclusion: In both NSCLC and CRC, the lack of linear relationship between tumor cellularity and VAF was found across a wide range of tumor cell percentages. Caution should be used when using tumor cellularity to triage specimens for NGS testing. The tumor cellularity should be considered in relation to the limit of detection of the specific assay for the proper interpretation of a negative test result.

Background: DNA hypermethylation and instability due to inactivation mutations in Ten–eleven translocation 2 (TET2) is a key biomarker of hematological malignancies. This study aims at characterizing two intronic noncanonical splice‐site variants, c.3954+5_3954+8delGTTT and c.3954+5G>A.Methods: We used in silico prediction tools, reverse transcription (RT)‐PCR, and Sanger sequencing on blood/bone marrow‐derived RNA specimens to determine the aberrant splicing.Results: In silico prediction of both variants exhibited reduced splicing strength at the TET2 intron 7 splicing donor site. RT‐PCR and Sanger sequencing identified a 62‐bp deletion at the exon 7, producing a frameshift mutation, p.Cys1298*.Conclusion: This study provides functional evidence for two intronic TET2 variants that cause alternative splicing and frameshift mutation.

Key criteria in the diagnostic workup and risk stratification for myeloproliferative neoplasms (MPN) include molecular testing for JAK2^V617F and other mutant alleles. Multiple methods for quantitatively detecting nucleotide sequence changes exist, but the lower limit of detection can limit identification of the low-level allele fraction of a variant. We evaluated a recently developed blocker displacement amplification (BDA)-based quantitative PCR platform for detection and quantitation of JAK2^V617F variant allele fraction (VAF). Clinical samples were tested using BDA, next-generation sequencing (NGS), and droplet digital PCR (ddPCR) in a head-to-head comparison of sensitivity and specificity in detecting the JAK2^V617F variant. In total, 112 human genomic DNA specimens previously tested for JAK2^V617F gene mutation status with NGS were analyzed, including 12 samples with low-level variants with VAF ≤2%, 6 samples with VAF >2%, and 94 samples with no variant previously identified by NGS. BDA and ddPCR results correlated well across a range of VAFs, with both methods identifying the JAK2^V617F variant down to at least 0.05% VAF. NGS of routine sequencing depth was less sensitive, identifying JAK2^V617F only at 0.6% VAF. BDA can provide a cost-effective alternative means to identify low-level variants using instrumentation commonly found in laboratories.