Yi Lin Chen

RHD 1227A Is an Important Genetic Marker for RhDel Individuals

Approximately 30% of apparently Rh- Taiwanese people actually were RhD(el), a rare variant of the Rh system that might carry a grossly intact RHD gene. Several studies have indicated that the RhD(el) trait might be generated by multiple molecular mechanisms. In this study, a total of 294 Taiwanese serologically RhD- blood donors were tested for Rh phenotypes and RHD genotypes. Among them, total RHD deletion, partial RHD gene, and RhD(el) were found in 185 (62.9%), 15 (5.1%), and 94 (32.0%), respectively. The 1227A allele and exon 9 of the RHD gene were found in all 94 RhD(el) donors. The Ccee was the most prevalent phenotype in the RhD(el) group (78/94 [83%]), and the ccee phenotype was highly prevalent in the true D- group (87.6%). RHD 1227A can be used as an important and useful genetic marker for RhD(el). It can be detected easily by a simple, rapid, specific sequence primer-polymerase chain reaction method.

BIOMED-2 protocols to detect clonal immunoglobulin and T-cell receptor gene rearrangements in B- and T-cell lymphomas in southern Taiwan

Monoclonal expansions of immunoglobulin (Ig) and T-cell receptor (TCR) genes are, respectively, important markers for B- and T-cell malignancies. We used BIOMED-2 protocols to detect clonality of these genes in B- (BCL) and T-cell lymphoma (TCL) in southern Taiwan. Heteroduplex analysis was used after PCR reactions. Ig/TCR clonality rates were 95% (22 in 23 cases) for BCL and 76% (19 in 25 cases) for TCL. These results reveal overall satisfactory detection rates for both BCL and TCL. None of the four natural killer (NK)/T-cell lymphomas detected showed TCR gene clonality, which suggested that BIOMED-2 protocols distinguished the NK/T-cell lymphoma from TCL. In addition, three of the five tested precursor T-cell lymphoblastic lymphomas showed no TCR gene clonality, probably because the immature T-cells in this type of TCL had not undergone TCR gene rearrangements. We conclude that BIOMED-2 protocols are suitable for detecting Ig and TCR clonalities in B- and T-cell malignancies in southern Taiwan.

High Percentage of JAK2 Exon 12 Mutation in Asian Patients With Polycythemia Vera

We examined the occurrence of JAK2(V617F) and JAK2 exon 12 mutations in a clinical cohort of polycythemia vera (PV) in Taiwan. Of 22 patients with PV, 17 (77%) had the V617F mutation, and all 5 V617F-negative patients (23%) had the exon 12 mutation. We found 3 different exon 12 mutations: 3 N542-E543del, 1 F537-K539delinsL, and 1 novel mutation, I540-E543delinsKK. Patients with V617F showed significantly higher WBC and platelet counts at diagnosis than patients with exon 12 mutations (P = .021 and P = .038, respectively). We report a surprisingly high incidence of exon 12 mutations in Taiwanese patients with PV, a result quite different from reports in the Western literature (P = .001). Our data suggest that exon 12 mutation of JAK2 in patients with PV may have an uneven geographic distribution. A clinical laboratory providing the V617F test alone may risk missing a substantial number of patients with PV in areas with a high incidence of exon 12 mutation.

Human herpesvirus 8-associated lymphoma mimicking cutaneous anaplastic large T-cell lymphoma in a patient with human immunodeficiency virus infection

Primary effusion lymphoma, a human herpesvirus 8 (HHV8)‐associated lymphoma, is uncommon, and it is usually seen in human immunodeficiency virus (HIV)‐infected patients. It presents as a body cavity‐based lymphomatous effusion, but several cases of the so‐called solid primary effusion lymphoma presenting as solid tumors without associated lymphomatous effusion have been reported. They have similar clinical, histopathological and immunophenotypical features. Most of them have a B‐cell genotype. This suggests the solid variant may represent a clinicopathological spectrum of primary effusion lymphoma. We report a case of HHV8‐associated lymphoma histopathologically and immunophenotypically mimicking cutaneous anaplastic large cell lymphoma. The patient was a 31‐year‐old HIV‐seropositive man presenting with skin nodules over his right thigh. Biopsy of the nodules showed anaplastic large cells infiltrating the dermis. These malignant cells strongly expressed CD3, CD30 and CD43. Cutaneous anaplastic large T‐cell lymphoma was initially diagnosed, but further tests, including immunoreactivity for HHV8 protein and clonal rearrangements of immunoglobulin genes, confirmed the diagnosis of HHV8‐associated B‐cell lymphoma with aberrant T‐cell marker expression. This case provides an example of solid primary effusion lymphoma mimicking cutaneous anaplastic large T‐cell lymphoma and highlights the importance of HHV8 immunohistochemistry and molecular tests in the diagnosis of HHV8‐associated lymphoma with a cutaneous presentation.

Epidermal Growth Factor Receptor Mutation and Anaplastic Lymphoma Kinase Gene Fusion: Detection in Malignant Pleural Effusion by RNA or PNA Analysis.

Analyzing EGFR mutations and detecting ALK gene fusion are indispensable when planning to treat pulmonary adenocarcinoma. Malignant pleural effusion (MPE) is a devastating complication of lung cancer and sometimes the only source for mutation analysis. The percentage of tumor cells in the pleural effusion may be low; therefore, mutant enrichment is required for a successful analysis. The EGFR mutation status in MPE was determined using three methods: (1) PCR sequencing of genomic DNA (direct sequencing), (2) mutant-enriched PCR sequencing of genomic DNA using peptide nucleic acid (PNA-sequencing), and (3) PCR sequencing of cDNA after reverse transcription for cellular RNA (RNA-sequencing). RT-PCR was also used to test cases for ALK gene fusion. PNA-sequencing and RNA-sequencing had similar analytical sensitivities (< 1%), which indicates similar enrichment capabilities. The clinical sensitivity in 133 cases when detecting the common EGFR exon 19 and exon 21 mutations was 56.4% (75/133) for direct sequencing, 63.2% (84/133) for PNA-sequencing, and 65.4% (87/133) for RNA-sequencing. RT-PCR and sequencing showed 5 cases (3.8%) with ALK gene fusion. All had wild-type EGFR. For EGFR analysis of MPE, RNA-sequencing is at least as sensitive as PNA-sequencing but not limited to specific mutations. Detecting ALK fusion can be incorporated in the same RNA workflow. Therefore, RNA is a better source for comprehensive molecular diagnoses in MPE.

Verification of Wild-Type EGFR Status in Non–Small Cell Lung Carcinomas Using a Mutant-Enriched PCR on Selected Cases

EGFR genotyping is required for targeted therapy of lung adenocarcinoma. Because a false-negative result might prevent a patient from receiving appropriate targeted therapies, it is desirable to recheck equivocal results of EGFR genotyping. A cohort of 346 lung cancers was tested with a commercial kit for EGFR mutations; nine of the cases had upward real-time amplification curves at late cycles. They were also investigated using mutant-enriched PCR with peptide nucleic acid-locked nucleic acid (PNA-sequencing). Six of the nine equivocal cases harbored EGFR mutations. These cases likely had a small amount of mutant DNA near the detection limit of the commercial kit. Twenty nonequivocal, wild-type cases were reconfirmed using PNA-sequencing. We noticed a College of American Pathologists proficiency test material that showed a suspicious upward curve and eventually proved to have an H773_V774insPH in exon 20, for which a specific primer was not designed in the commercial kit. Further study using cloned DNA fragments showed that the upward curve most likely resulted from cross-reaction between similar, but nonidentical, sequences. It is desirable to keep the number of false-negative results as low as possible, but rechecking all wild-type cases is impractical. The late upward curves we observed helped identify suspicious cases for rechecking. A second method, such as PNA-sequencing, is recommended to verify wild-type cases.

Serrated adenocarcinoma morphology in colorectal mucinous adenocarcinoma is associated with improved patient survival

Colorectal mucinous adenocarcinoma (MAC) and serrated adenocarcinoma (SAC) share many characteristics, including right-side colon location, frequent mucin production, and various molecular features. This study examined the frequency of SAC morphology in MACs. We assessed the correlation of SAC morphology with clinicopathological parameters, molecular characteristics, and patient prognosis. Eighty-eight colorectal MACs were collected and reviewed for SAC morphology according to Makinens criteria. We sequenced KRAS and BRAF, assessed CpG island methylator phenotype (CIMP) frequency, and analyzed DNA mismatch repair enzyme levels using immunohistochemistry in tumor samples. SAC morphology was observed in 38% of MACs, and was associated with proximal location (P=0.001), BRAF mutation (P=0.042), CIMP-positive status (P=0.023), and contiguous traditional serrated adenoma (P=0.019). Multivariate analysis revealed that MACs without both SAC morphology and CIMP-positive status exhibited 3.955 times greater risk of cancer relapse than MACs having both characteristics or either one (P=0.035). Our results show that two MAC groups with distinct features can be identified using Makinens criteria, and suggest a favorable prognostic role for the serrated neoplastic pathway in colorectal MAC.

TRIg: a robust alignment pipeline for non-regular T-cell receptor and immunoglobulin sequences

BackgroundT cells and B cells are essential in the adaptive immunity via expressing T cell receptors and immunoglogulins respectively for recognizing antigens. To recognize a wide variety of antigens, a highly diverse repertoire of receptors is generated via complex recombination of the receptor genes. Reasonably, frequencies of the recombination events have been shown to predict immune diseases and provide insights into the development of immunity. The field is further boosted by high-throughput sequencing and several computational tools have been released to analyze the recombined sequences. However, all current tools assume regular recombination of the receptor genes, which is not always valid in data prepared using a RACE approach. Compared to the traditional multiplex PCR approach, RACE is free of primer bias, therefore can provide accurate estimation of recombination frequencies. To handle the non-regular recombination events, a new computational program is needed.ResultsWe propose TRIg to handle non-regular T cell receptor and immunoglobulin sequences. Unlike all current programs, TRIg does alignments to the whole receptor gene instead of only to the coding regions. This brings new computational challenges, e.g., ambiguous alignments due to multiple hits to repetitive regions. To reduce ambiguity, TRIg applies a heuristic strategy and incorporates gene annotation to identify authentic alignments. On our own and public RACE datasets, TRIg correctly identified non-regularly recombined sequences, which could not be achieved by current programs. TRIg also works well for regularly recombined sequences.ConclusionsTRIg takes into account non-regular recombination of T cell receptor and immunoglobulin genes, therefore is suitable for analyzing RACE data. Such analysis will provide accurate estimation of recombination events, which will benefit various immune studies directly. In addition, TRIg is suitable for studying aberrant recombination in immune diseases. TRIg is freely available at https://github.com/TLlab/trig.

Obtaining long 16S rDNA sequences using multiple primers and its application on dioxin-containing samples

BackgroundNext-generation sequencing (NGS) technology has transformed metagenomics because the high-throughput data allow an in-depth exploration of a complex microbial community. However, accurate species identification with NGS data is challenging because NGS sequences are relatively short. Assembling 16S rDNA segments into longer sequences has been proposed for improving species identification. Current approaches, however, either suffer from amplification bias due to one single primer or insufficient 16S rDNA reads in whole genome sequencing data.ResultsMultiple primers were used to amplify different 16S rDNA segments for 454 sequencing, followed by 454 read classification and assembly. This permitted targeted sequencing while reducing primer bias. For test samples containing four known bacteria, accurate and near full-length 16S rDNAs of three known bacteria were obtained. For real soil and sediment samples containing dioxins in various concentrations, 16S rDNA sequences were lengthened by 50% for about half of the non-rare microbes, and 16S rDNAs of several microbes reached more than 1000 bp. In addition, reduced primer bias using multiple primers was illustrated.ConclusionsA new experimental and computational pipeline for obtaining long 16S rDNA sequences was proposed. The capability of the pipeline was validated on test samples and illustrated on real samples. For dioxin-containing samples, the pipeline revealed several microbes suitable for future studies of dioxin chemistry.