Publications by authors named "Li-Hui Tseng"

60 Publications

IDH1 and IDH2 Mutations in Colorectal Cancers.

Am J Clin Pathol 2021 Apr 30. Epub 2021 Apr 30.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Objectives: To elucidate clinicopathologic and molecular characteristics of IDH1 and IDH2 (IDH1/2) mutations in colorectal cancers (CRCs).

Methods: We evaluated IDH1/2 mutations in 1,623 CRCs using a next-generation sequencing assay.

Results: IDH1/2 mutations, predominantly IDH1 p.R132C, were detected in 15 (0.9%) CRCs and in 5 (3.0%) of 167 BRAF p.V600E-mutated CRCs. Three IDH1/2-mutated CRCs were associated with inflammatory bowel disease. They were significantly associated with old age, mucinous or signet ring cell adenocarcinoma, and high-grade histomorphology. Concordance of variant allele frequency between IDH1/2 mutants and other trunk drivers in CRCs and presence of IDH1/2 mutation in the adenoma and early adenocarcinoma indicated IDH1/2 mutations could be trunk drivers suitable for targeted therapy.

Conclusions: IDH1/2 mutations in CRCs were uncommon but enriched in BRAF p.V600E-mutated CRCs and perhaps colitis-associated CRCs. Further studies on IDH1/2-mutated CRCs are needed to clarify their clinicopathologic features and implications for targeted therapy.
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http://dx.doi.org/10.1093/ajcp/aqab023DOI Listing
April 2021

Association between risk factors, molecular features and CpG island methylator phenotype colorectal cancer among different age groups in a Taiwanese cohort.

Br J Cancer 2021 Apr 12. Epub 2021 Apr 12.

Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan.

Background: CpG island methylator phenotype (CIMP) represents a carcinogenesis pathway of colorectal cancer (CRC) and the association between CIMP CRC, molecular features and risk factors in East Asian population is less studied.

Methods: We prospectively enrolled newly diagnosed CRC patients at the National Taiwan University Hospital. Clinicopathological data and risk factors for CRC were collected during interview. The tumour samples were subjected to CIMP, RAS/BRAF mutation and microsatellite instability tests. CIMP-high was determined when ≧3 methylated loci of p16, MINT1, MINT2, MINT31 and MLH1 were identified. Multivariate logistic regression was used to evaluate the association between risk factors and CIMP-high CRC.

Results: Compared with CIMP-low/negative CRC, CIMP-high CRC was associated with more stage IV disease, BRAF V600E mutation and high body mass index (BMI ≧ 27.5 kg/m) in younger patients (age < 50 y), and more right-sided tumour, BRAF V600E mutation, MSI-high and colorectal polyp in elder patients (age ≧ 50 y). Multivariate analyses showed that BMI ≧27.5 kg/m was significantly associated with CIMP-high CRC in younger patients.

Conclusions: We identified distinct clinicopathological features for CIMP-high CRC among different age groups in Taiwan. Our data suggest the association between BMI ≧27.5 kg/m and CIMP-high CRC in patients younger than 50 years.
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http://dx.doi.org/10.1038/s41416-021-01300-5DOI Listing
April 2021

Clonal Origin Evaluated by Trunk and Branching Drivers and Prevalence of Mutations in Multiple Lung Tumor Nodules.

Mol Diagn Ther 2020 08;24(4):461-472

Department of Pathology, Johns Hopkins University School of Medicine, Carnegie 469, Pathology, 600 North Wolfe Street, Baltimore, MD, 21287, USA.

Introduction: Differentiation between intrapulmonary metastasis (IPM) and multiple primary lung cancers (MPLC) in patients with synchronous or metachronous lung tumor nodules is critical but challenging.

Objective: We proposed an algorithm to evaluate clonal origin based on trunk (initiating) versus branching drivers and the prevalence of mutations in lung adenocarcinomas.

Methods: Driver mutations were examined using next-generation sequencing in five trunk driver genes (BRAF, EGFR, ERBB2, KRAS, and NRAS) and three branching driver genes (ATK1, PIK3CA, and TP53).

Results: Mutational profiling supported same clonality and likely same clonality, respectively, in 39 and 14 of 66 pairs of specimens with known identical clonal origin. Discordance of TP53 mutations (branching drivers) was observed in three pairs. Subsequent analyses of 30 pairs of synchronous or metachronous lung tumor nodules revealed different clonality and likely different clonality in 17 and 2 pairs, respectively, including three pairs with similar histomorphology; same clonality and likely same clonality in three and five pairs, respectively, including two pairs with different histomorphology; and inconclusive or noninformative results in three pairs.

Conclusion: While discordance of trunk drivers indicated MPLC in patients with synchronous or metachronous lung tumor nodules, discordance of branching drivers did not exclude IPM. Concordance of uncommon drivers supported IPM, whereas concordance of common drivers did not exclude MPLC. Additional recommendations from official organizations are needed to guide applications of molecular markers in defining clonality of multiple lung tumor nodules.
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http://dx.doi.org/10.1007/s40291-020-00471-wDOI Listing
August 2020

Multiclonal colorectal cancers with divergent histomorphological features and RAS mutations: one cancer or separate cancers?

Hum Pathol 2020 04 12;98:120-128. Epub 2020 Mar 12.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

Detection of coexisting mutations within the same signal transduction pathway, which are expected to be mutually exclusive, raises a concern of laboratory errors. We have previously confirmed the presence of different RAS (KRAS and NRAS) mutations in the adenoma and/or adenocarcinoma subpopulations of colorectal cancers (CRCs). In this study, multiregional analyses by next-generation sequencing were conducted to elucidate the mechanisms underlying multiple RAS mutations seen in 5 CRC specimens. Multiregional analyses were initially conducted in a single tissue block originally submitted for mutational profiling. In 2 specimens, mutational status of the APC gene was not identical, indicating collisional adenoma and adenocarcinoma. In 3 specimens, the same APC mutation was present in different subpopulations with divergent RAS mutations, indicating a common clonal origin. Subsequent comprehensive multiregional analyses of additional adenoma and adenocarcinoma components revealed multiclonal CRCs with divergent histomorphological features and RAS mutations originating from a common APC-mutated founder lineage of adenoma, but from different RAS-mutated founder lineages of adenocarcinoma. These findings are consistent with the stepwise model of colorectal tumorigenesis along with parallel evolution, which affects RAS genes within the mitogen-activated protein kinase pathway and occurs during the progression from adenomas to adenocarcinomas. Evaluation of tumor subpopulations with divergent histomorphological features by pathologists may help identify multiclonal CRCs. Further studies are warranted to evaluate the incidence of multiclonality in CRCs and its impact on clinical outcomes. Perhaps, multiclonal CRCs originating from the same APC-mutated founder lineage of adenoma but from different RAS-mutated founder lineages of adenocarcinomas should be defined and managed as separate CRCs.
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http://dx.doi.org/10.1016/j.humpath.2020.03.002DOI Listing
April 2020

Clinical mutational profiling and categorization of BRAF mutations in melanomas using next generation sequencing.

BMC Cancer 2019 Jul 5;19(1):665. Epub 2019 Jul 5.

Department of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, 1812 Ashland Ave, Suite 200, Baltimore, MD, 21205, USA.

Background: Analysis of melanomas for actionable mutations has become the standard of care. Recently, a classification scheme has been proposed that categorizes BRAF mutations based on their mechanisms for activation of the MAPK pathway.

Methods: In this analysis BRAF, KIT, NRAS, and PIK3CA mutations were examined by next generation sequencing (NGS) in 446 melanomas in a clinical diagnostic setting. KRAS and HRAS were also analyzed to elucidate coexisting BRAF and RAS mutations. BRAF mutations were categorized into class-1 (kinase-activated, codon 600), class-2 (kinase-activated, non-codon 600) and class-3 (kinase-impaired), based on the newly proposed classification scheme.

Results: NGS demonstrated high analytic sensitivity. Among 355 mutations detected, variant allele frequencies were 2-5% in 21 (5.9%) mutations and 2-10% in 47 (13%) mutations. Mutations were detected in BRAF (42%), NRAS (25%), KIT (4.9%) and PIK3CA (2.7%). The incidence of class-1, class-2 and class-3 mutations were 33% (26% p.V600E and 6.1% p.V600K), 3.1 and 4.9% respectively. With a broader reportable range of NGS, class-1, class-2 and class-3 mutations accounted for 77, 7.4 and 12% of all BRAF mutations. Class-3 mutations, commonly affecting codons 594, 466 and 467, showed a higher incidence of coexisting RAS mutations, consistent with their RAS-dependent signaling. Significant association with old age and primary tumors of head/neck/upper back suggest chronic solar damage as a contributing factor for melanomas harboring BRAF p.V600K or class-3 mutations.

Conclusion: This study categorizes the range, frequency, coexisting driver mutations and clinical characteristics of the three classes of BRAF mutations in a large cohort of melanomas in a clinical diagnostic setting. Further prospective studies are warranted to elucidate the clinical outcomes and benefits of newly developed targeted therapy in melanoma patients carrying each class of BRAF mutation.
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http://dx.doi.org/10.1186/s12885-019-5864-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612071PMC
July 2019

Clinical Validation of Discordant Trunk Driver Mutations in Paired Primary and Metastatic Lung Cancer Specimens.

Am J Clin Pathol 2019 10;152(5):570-581

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD.

Objectives: To propose an operating procedure for validation of discordant trunk driver mutations.

Methods: Concordance of trunk drivers was examined by next-generation sequencing in 15 patients with two to three metastatic lung cancers and 32 paired primary and metastatic lung cancers.

Results: Tissue identity was confirmed by genotyping 17 single-nucleotide polymorphisms within the panel. All except three pairs showed concordant trunk drivers. Quality assessment conducted in three primary and metastatic pairs with discordant trunk drivers indicates metastasis from a synchronous or remote lung primary in two patients. Review of literature revealed high discordant rates of EGFR and KRAS mutations, especially when Sanger sequencing was applied to examine primary and lymph node metastatic tumors.

Conclusions: Trunk driver mutations are highly concordant in primary and metastatic tumors. Discordance of trunk drivers, once confirmed, may suggest a second primary cancer. Guidelines are recommended to establish standard operating procedures for validation of discordant trunk drivers.
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http://dx.doi.org/10.1093/ajcp/aqz077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779251PMC
October 2019

Prediction Model for Audiological Outcomes in Patients With GJB2 Mutations.

Ear Hear 2020 Jan/Feb;41(1):143-149

Department of Otolaryngology, National Taiwan University Hospital, Taipei, Taiwan.

Objectives: Recessive mutations in GJB2 are the most common genetic cause of sensorineural hearing impairment (SNHI) in humans. SNHI related to GJB2 mutations demonstrates a wide variation in audiological features, and there has been no reliable prediction model for hearing outcomes until now. The objectives of this study were to clarify the predominant factors determining hearing outcome and to establish a predictive model for SNHI in patients with GJB2 mutations.

Design: A total of 434 patients confirmed to have biallelic GJB2 mutations were enrolled and divided into three groups according to their GJB2 genotypes. Audiological data, including hearing levels and audiogram configurations, were compared between patients with different genotypes. Univariate and multivariate generalized estimating equation (GEE) analyses were performed to analyze longitudinal data of patients with multiple audiological records.

Results: Of the 434 patients, 346 (79.7%) were homozygous for the GJB2 p.V37I mutation, 55 (12.7%) were compound heterozygous for p.V37I and another GJB2 mutation, and 33 (7.6%) had biallelic GJB2 mutations other than p.V37I. There was a significant difference in hearing level and the distribution of audiogram configurations between the three groups. Multivariate GEE analyses on 707 audiological records of 227 patients revealed that the baseline hearing level and the duration of follow-up were the predominant predictors of hearing outcome, and that hearing levels in patients with GJB2 mutations could be estimated based on these two parameters: (Predicted Hearing Level [dBHL]) = 3.78 + 0.96 × (Baseline Hearing Level [dBHL]) + 0.55 × (Duration of Follow-Up [y]).

Conclusion: The baseline hearing level and the duration of follow-up are the main prognostic factors for outcome of GJB2-related SNHI. These findings may have important clinical implications in guiding follow-up protocols and designing treatment plans in patients with GJB2 mutations.
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http://dx.doi.org/10.1097/AUD.0000000000000742DOI Listing
June 2019

CpG Island Methylator Phenotype May Predict Poor Overall Survival of Patients with Stage IV Colorectal Cancer.

Oncology 2019 12;96(3):156-163. Epub 2018 Dec 12.

Department of Oncology, National Taiwan University Hospital, Taipei City, Taiwan,

Objective: We aimed to study the prognostic role of CpG island methylator phenotype (CIMP) in patients with different stages of colorectal cancer (CRC).

Material And Methods: We analyzed CIMP in stage I-IV CRC specimens from patients who were diagnosed between 2005 and 2013. CIMP status was determined using a 5-gene MethyLight-based assay. The clinicopathologic characteristics were reviewed and the overall survival (OS) was compared between patients with CIMP-high CRC and those with CIMP-low/negative CRC.

Results: Among 450 CRC specimens with successfully determined CIMP statuses, 74 (16.4%) were CIMP-high CRC. Although there was no difference in OS between patients with CIMP-high and CIMP-low/negative CRC across all stages (p = 0.4526), intriguingly, patients with stage IV CIMP-high CRC had significantly worse OS than those with stage IV CIMP-low/negative CRC (p = 0.0047). In a multivariate analysis, CIMP status remained an independent prognostic factor for overall mortality (HR = 5.60, 95% CI: 2.12-14.79, p = 0.0005) in metastatic CRC after adjusting for clinicopathologic variables and anti-cancer therapies.

Conclusion: Our results revealed that the presence of CIMP independently predicts poor OS in patients with stage IV CRC.
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http://dx.doi.org/10.1159/000493387DOI Listing
March 2019

Clinical validation of coexisting driver mutations in colorectal cancers.

Hum Pathol 2019 04 24;86:12-20. Epub 2018 Nov 24.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Mutational profiling is recommended for selecting targeted therapy and predicting prognosis of metastatic colorectal cancer (CRC). Detection of coexisting mutations within the same pathway, which are usually mutually exclusive, raises the concern for potential laboratory errors. In this retrospective study for quality assessment of a next-generation sequencing assay, we examined BRAF, KRAS, and NRAS genes within the mitogen-activated protein kinase (MAPK) pathway and the PIK3CA gene within the phosphatidylinositol 3-kinase (mTOR) pathway in 744 CRC specimens submitted to our clinical diagnostics laboratory. Although coexistence of mutations between the MAPK and mTOR pathways was observed, it rarely occurred within the MAPK pathway. Retrospective quality assessments identified false detection of coexisting activating KRAS and NRAS mutations in 1 specimen and confirmed 2 activating KRAS mutations in 2 specimens and coexisting activating KRAS and NRAS mutations in 2 specimens, but no coexisting activating RAS and BRAF mutations. There were 15 CRCs with a kinase-impaired BRAF mutation, including 3 with a coexisting activating KRAS mutation, which may have therapeutic implications. Multiregional analysis based on different histologic features demonstrated that coexisting KRAS and NRAS mutations may be present in the same or different tumor populations and showed that invasion of adenomas by synchronous adenocarcinomas of different clonal origin may result in detection of coexisting mutations within the MAPK pathway. In this study, we proposed an operating procedure for clinical validation of unexpected coexisting mutations. Further studies are warranted to elucidate the biological significance and clinical implications of coexisting mutations within the MAPK pathway.
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http://dx.doi.org/10.1016/j.humpath.2018.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467705PMC
April 2019

Clinical mutational profiling of 1006 lung cancers by next generation sequencing.

Oncotarget 2017 Nov 20;8(57):96684-96696. Epub 2017 May 20.

Department of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, Maryland, USA.

Analysis of lung adenocarcinomas for actionable mutations has become standard of care. Here, we report our experience using next generation sequencing (NGS) to examine , , , , , , and genes in 1006 non-small cell lung cancers in a clinical diagnostic setting. NGS demonstrated high sensitivity. Among 760 mutations detected, the variant allele frequency (VAF) was 2-5% in 33 (4.3%) mutations and 2-10% in 101 (13%) mutations. A single bioinformatics pipeline using Torrent Variant Caller, however, missed a variety of mutations. Mutations were detected in (36% of tumors), (19%) including 8 (0.8%) within the extracellular domain (4 at codons 108 and 4 at codon 289), (6.3%), and (3.7%). With a broader reportable range, exon 19 deletion and p.L858R accounted for only 36% and 26% of mutations and p.V600E accounted for only 24% of mutations. NGS provided accurate sequencing of complex mutations seen in 19% of exon 19 deletion mutations. Doublet (compound) mutations were observed in 29 (16%) of 187 -mutated tumors, including 69% with two non-p.L858R missense mutations and 24% with p.L858 and non-p.L858R missense mutations. Concordant VAFs suggests doublet mutations were present in a dominant clone and cooperated in oncogenesis. Mutants with predicted impaired kinase, observed in 25% of -mutated tumors, were associated with a higher incidence of concomitant activating mutations. NGS demonstrates high analytic sensitivity, broad reportable range, quantitative VAF measurement, single molecule sequencing to resolve complex deletion mutations, and simultaneous detection of concomitant mutations.
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http://dx.doi.org/10.18632/oncotarget.18042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5722514PMC
November 2017

A panel of 130 autosomal single-nucleotide polymorphisms for ancestry assignment in five Asian populations and in Caucasians.

Forensic Sci Med Pathol 2017 Jun 24;13(2):177-187. Epub 2017 Apr 24.

Department and Graduate Institute of Forensic Medicine, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen Ai Rd, Taipei, 100, Taiwan.

Ancestry informative single-nucleotide polymorphism (AISNP) panels for differentiating between East and Southeast Asian populations are scarce. This study aimed to identify AISNPs for ancestry assignment of five East and Southeast Asian populations, and Caucasians. We analyzed 145 autosomal SNPs of the 627 DNA samples from individuals of six populations (234 Taiwanese Han, 91 Filipinos, 79 Indonesians, 60 Thais, 71 Vietnamese, and 92 Caucasians) using arrays. The multiple logistic regression model and a multi-tier approach were used for ancestry classification. We observed that 130 AISNPs were effective for classifying the ethnic origins with fair accuracy. Among the 130 AISNPs, 122 were useful for stratification between these five Asian populations and 64 were effective for differentiating between Caucasians and these Asian populations. For differentiation between Caucasians and Asians, an accuracy rate of 100% was achieved in these 627 subjects with 50 optimal AISNPs among the 64 effective SNPs. For classification of the five Asian populations, the accuracy rates of ancestry inference using 20 to 57 SNPs for each of the two Asian populations ranged from 74.1% to 100%. Another 14 degraded DNA samples with incomplete profiling were analyzed, and the ancestry of 12 (85.7%) of those subjects was accurately assigned. We developed a 130-AISNP panel for ethnic origin differentiation between the five East and Southeast Asian populations and Caucasians. This AISNP set may be helpful for individual ancestral assignment of these populations in forensic casework.
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http://dx.doi.org/10.1007/s12024-017-9863-8DOI Listing
June 2017

Epithelioid Trophoblastic Tumor Around an Abdominal Cesarean Scar: A Pathologic and Molecular Genetic Analysis.

Int J Gynecol Pathol 2017 Nov;36(6):562-567

Departments of Oncology (E.H.-C.H., C.H.) Medical Genetics (L.H.T.) Pathology (K.-T.K.), National Taiwan University Hospital Graduate Institute of Oncology, National Taiwan University College of Medicine (C.H.) National Taiwan University Cancer Center (C.H.) Department of Public Health, Institute of Epidemiology and Preventive Medicine, National Taiwan University (T.-P.L.), Taipei, Taiwan.

Epithelioid trophoblastic tumor (ETT) is a rare chemoresistant gestational trophoblastic neoplasm that typically presents as an intrauterine lesion. To our knowledge, no isolated abdominal wall ETT around a Cesarean scar has been reported. Here we describe a 54-yr-old woman with a complex obstetric history who presented with a solitary abdominal wall tumor adjacent to the abdominal Cesarean section scar. The tumor demonstrated typical morphologic and immunophenotypic features of ETT. The gestational origin of the tumor was confirmed by microsatellite genotyping. The tumor enlarged despite the patient undergoing multiagent chemotherapy. Whole-exome sequencing was performed to explore the mechanisms underlying chemoresistance. The ATP-binding cassette subfamily B member 1 (ABCB1) 3435CC genotype, and a putative deleterious x-ray cross-complementing group 4 (XRCC4) Ala73Pro mutations were found. In conclusion, ETT may present as a solitary abdominal wall lesion and microsatellite genotyping could facilitate the determination of its gestational origin. More studies are required to provide mechanistic insights into the chemoresistance of ETT.
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http://dx.doi.org/10.1097/PGP.0000000000000366DOI Listing
November 2017

RNF43 Is an Early and Specific Mutated Gene in the Serrated Pathway, With Increased Frequency in Traditional Serrated Adenoma and Its Associated Malignancy.

Am J Surg Pathol 2016 10;40(10):1352-9

Departments of *Pathology ‡Oncology ∥Medical Genetics, National Taiwan University Hospital †Graduate Institute of Pathology §Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

RNF43 is an E3 ligase that suppresses the Wnt/β-catenin signaling pathway and is frequently mutated in microsatellite-unstable colorectal carcinoma. To investigate the pathogenetic role of RNF43 in the serrated pathway, we conducted mutation analysis of RNF43 in several types of colorectal neoplasms. RNF43 mutation was found in 2 of 20 (10%) sessile serrated adenomas, 10 of 36 (28%) traditional serrated adenomas, 7 of 37 (19%) traditional serrated adenomas with cytologic dysplasia, and 9 of 31 (29%) BRAF-mutated/microsatellite-stable colorectal carcinomas; however, no mutation was found in 30 tubulovillous/villous adenomas. All mutations were located upstream of the ring finger domain of RNF43 without clustering, which is distinct from the pattern described for microsatellite-unstable colorectal carcinoma. RNF43 mutation was closely associated with BRAF mutation but inversely associated with KRAS mutation in traditional serrated adenoma with or without cytologic dysplasia (P=0.018 and 0.045, respectively). The finding of RNF43 mutation in sessile serrated adenoma and traditional serrated adenoma, but not in tubulovillous/villous adenoma, indicated that RNF43 mutation is an early and specific molecular aberration in the serrated pathway. The frequency of RNF43 mutation was significantly higher in traditional serrated adenoma with or without cytologic dysplasia and BRAF-mutated/microsatellite-stable colorectal carcinoma than sessile serrated adenoma. The unique molecular spectrum of these tumors suggests a stepwise neoplastic progression from sessile serrated adenoma to traditional serrated adenoma and BRAF-mutated/microsatellite-stable colorectal carcinoma, which should be recognized as the traditional serrated pathway to distinguish from the sessile serrated pathway.
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http://dx.doi.org/10.1097/PAS.0000000000000664DOI Listing
October 2016

Heterogeneity of resistance mutations detectable by nextgeneration sequencing in TKI-treated lung adenocarcinoma.

Oncotarget 2016 Jul;7(29):45237-45248

Department of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, Maryland, USA.

EGFR-mutated lung adenocarcinomas routinely develop resistance to tyrosine kinase inhibitors (TKI). To better characterize the relative frequencies of the resistance mechanisms, we analyzed 48 EGFR-mutated TKI-resistant specimens from 41 patients. Next-generation sequencing of post-treatment specimens detected EGFR p.T790M in 31 (79%) of 39 patients, PIK3CA mutations in 10 (26%), EGFR p.S768_V769delinsIL in one, and KRAS p.G12C in one. Five PIK3CA mutations were outside of codons 542, 545, and 1047. Three of four pre-treatment specimens did not carry the PIK3CA mutation found in the post-treatment sample. Small cell carcinoma transformation was identified in four patients; none had p.T790M, including two where p.T790M was identified in the co-existing adenocarcinoma. In p.T790M-mutated specimens, the allele frequency was less than 5% in 24% of cases. p.T790M allele frequency was usually lower than that of the sensitizing mutation indicating that the resistance mutation was present either in a subset of cells or, if the sensitizing mutation was amplified, in a subset of the sensitizing alleles of a dominant clone. Eight patients had multiple resistance mutations, suggesting either multiple separate resistant clones or a single clone harboring multiple resistance mechanisms. PIK3CA mutations appear to be a more significant resistance mechanism than previously recognized.
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http://dx.doi.org/10.18632/oncotarget.9931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5216719PMC
July 2016

Test Feasibility of Next-Generation Sequencing Assays in Clinical Mutation Detection of Small Biopsy and Fine Needle Aspiration Specimens.

Am J Clin Pathol 2016 May;145(5):696-702

From the Departments of Pathology and

Objectives: To evaluate preanalytic factors contributing to failure of next-generation sequencing (NGS) assays.

Methods: AmpliSeq Cancer Hotspot Panel was conducted in 1,121 of 1,152 formalin-fixed paraffin-embedded tissues submitted to a clinical laboratory, including 493 small biopsy or fine needle aspiration (FNA) specimens (44%) and 25 metastatic bone specimens (2.2%).

Results: Single nucleotide mutations and/or insertion/deletion mutations were detected in 702 specimens. Thirty-eight specimens (3.4%) were reported as "no results" due to NGS assay failure. Higher failure rates were observed in specimens submitted for lung cancer panel and melanoma panel (3.1% and 3.7% vs 1.0% colorectal cancer panel), metastatic bone specimens (36% vs 2.6% nonbone specimens), referred specimens (5.0% vs 1.8% in-house specimens), and small biopsy and FNA specimens (5.8% and 3.1% vs 0.7% resection/excision specimens). Test feasibility was higher in in-house specimens than referred specimens (99.1% vs 96.9% in resection specimens, 94.4% vs 87.3% in small biopsy specimens, and 94.3% vs 58.8% in FNA specimens).

Conclusions: NGS assays demonstrated clinical utility in solid tumor specimens, including those taken by biopsy or FNA. Preanalytic factors identified by this study that may contribute to NGS assay failure highlight the need for pathologists to revisit tissue processing protocols in order to better optimize cancer mutational profiling.
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http://dx.doi.org/10.1093/ajcp/aqw043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272811PMC
May 2016

BRAF mutation may have different prognostic implications in early- and late-stage colorectal cancer.

Med Oncol 2016 May 31;33(5):39. Epub 2016 Mar 31.

Department of Oncology, National Taiwan University Hospital, No 7, Chung-Shan South Rd, Taipei, 10002, Taiwan.

The prognostic implication of BRAF mutant colorectal cancer remains paradoxical. Records of BRAF mutant and wild-type colorectal cancer patients at all stages were reviewed. Clinicopathologic features, including microsatellite instability, CpG islands methylator phenotype, and overall survival, of these patients were analyzed. Between 2005 and 2013, 428 colorectal cancer patients were enrolled in this study. The overall survival between BRAF mutant and wild-type patients with early-stage (stages I and II) colorectal cancer differed nonsignificantly (P = 0.99). By contrast, in late-stage (stages III and IV) patients, the median overall survival of BRAF mutant patients (N = 25) was significantly poorer than that of BRAF wild-type (N = 207) patients (BRAF mutant: 21.3 months (95% confidence interval [CI] 7.1-35.5); BRAF wild-type: 53.5 months (95% CI 37.5-69.5), P < 0.0001). In early-stage patients, we found that BRAF mutation was significantly associated with CpG island methylator phenotype-positive (P < 0.001), and microsatellite instability-high status (P = 0.0013). Conversely, in late-stage patients, BRAF mutation was significantly associated with CpG island methylator phenotype-positive (P = 0.0015) and the right-side colon (P = 0.014). BRAF mutation may have different prognostic implications in early- and late-stage colorectal cancer.
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http://dx.doi.org/10.1007/s12032-016-0756-6DOI Listing
May 2016

Case report: mismatch repair proficiency and microsatellite stability in gastric cancer may not predict programmed death-1 blockade resistance.

J Hematol Oncol 2016 Mar 24;9:29. Epub 2016 Mar 24.

Department of Oncology, National Taiwan University Hospital, 7, Chun-Shan S Rd, Taipei, 10002, Taiwan.

Background: Anti-programmed death-1 therapy has poor efficacy in mismatch repair-proficient (pMMR) colorectal cancers; however, its efficacy in pMMR gastric cancers remains undetermined. Here, we report the case of a patient with pMMR and microsatellite-stable gastric cancer who exhibited a partial response to salvage anti-programmed death-1 therapy with pembrolizumab.

Case Presentation: Initially, the patient underwent subtotal gastrectomy 4 years ago for early-stage gastric cancer (pT1bN2M0, stage IIA). Immunohistochemical analysis of the tumor revealed strongly positive for HER2/neu. He had received trastuzumab plus pertuzumab, cisplatin, and capecitabine for recurrent tumors since September 2014 for 15 cycles. Disease progression of gastric cancer was found in August 2015. Since September 2015, the patient has received pembrolizumab monotherapy (200 mg as a fixed dose, every 3 weeks) for 3 months and the repeat computed tomography demonstrated a confirmed partial response. The plasma carcinoembryonic antigen also decreased dramatically. Both immunohistochemistry and a polymerase chain reaction-based method revealed that the patient had pMMR gastric cancer.

Conclusions: This case report provides the first report that mismatch repair-proficient and microsatellite-stable gastric cancers can respond well to anti-PD-1 monotherapy and indicates both markers may not sufficiently be predictive of anti-PD-1 therapy resistance in gastric cancer.
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http://dx.doi.org/10.1186/s13045-016-0259-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806434PMC
March 2016

Mutational profiling of colorectal cancers with microsatellite instability.

Oncotarget 2015 Dec;6(39):42334-44

Department of Pathology and The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Microsatellite instability (MSI) is caused by defective mismatch repair in 15-20% of colorectal cancers (CRCs). Higher mutation loads in tumors with mismatch repair deficiency can predict response to pembrolizumab, an anti-programmed death 1 (PD-1) immune checkpoint inhibitor. We analyzed the mutations in 113 CRCs without MSI (MSS) and 29 CRCs with MSI-High (MSI-H) using the 50-gene AmpliSeq cancer panel. Overall, MSI-H CRCs showed significantly higher mutations than MSS CRCs, including insertion/deletion mutations at repeat regions. MSI-H CRCs showed higher incidences of mutations in the BRAF, PIK3CA, and PTEN genes as well as mutations in the receptor tyrosine kinase families. While the increased mutations in BRAF and PTEN in MSI-H CRCs are well accepted, we also support findings of mutations in the mTOR pathway and receptor tyrosine kinase family genes. MSS CRCs showed higher incidences of mutations in the APC, KRAS and TP53 genes, confirming previous findings. NGS assays may be designed to detect driver mutations for targeted therapeutics and to identify tumors with high mutation loads for potential treatment with immune checkpoint blockade therapies. Further studies may be warranted to elucidate potential targeted therapeutics against mutations in the mTOR pathway and the receptor tyrosine kinase family in MSI-H CRCs as well as the benefit of anti-PD-1 immunotherapy in hypermutated MSS CRCs or other cancers.
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http://dx.doi.org/10.18632/oncotarget.5997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747229PMC
December 2015

Frequent BRAF mutation in early-onset colorectal cancer in Taiwan: association with distinct clinicopathological and molecular features and poor clinical outcome.

J Clin Pathol 2016 Apr 23;69(4):319-25. Epub 2015 Oct 23.

Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan Graduate Institute of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan.

Background: Occurrence of early-onset colorectal cancer (EOCRC) under the age of 30 is very rare and the molecular characteristics are poorly understood. A low BRAF mutation rate has been noted in several studies of EOCRC from Western countries.

Aims: To determine the clinicopathological and molecular features of EOCRCs in Taiwan.

Methods: KRAS/BRAF gene mutation, mismatch repair protein immunohistochemistry, microsatellite instability and CpG island methylation phenotype analyses were examined to determine the molecular characteristics of EOCRC.

Results: Sixty-six patients with EOCRC at our hospital between 2000 and 2012 were studied. BRAF mutation was detected in 11 of the 59 tumours analysed (19%) and the rate was significantly higher than the overall BRAF mutation rate of colorectal cancer in patients older than 30 years (p<0.001). Clinically, 9 of 11 patients with BRAF-mutated tumours presented with advanced-stage diseases and they presented significantly more frequently with stage IV disease than those with BRAF wild-type tumours (p=0.042). Histologically, BRAF mutation was associated with a poorly differentiated histology, a serrated precursor polyp and focal signet ring cell differentiation (p=0.042, 0.008 and 0.008, respectively). None of the BRAF-mutated tumours was mismatch repair protein-deficient and/or microsatellite instability-high. Overall survival of patients with BRAF-mutated tumours was significantly worse than that of patients with BRAF wild-type tumours, despite adjustment for the disease stages and tumour differentiation.

Conclusions: BRAF mutation was frequent in EOCRCs in Taiwan and was associated with distinct clinicopathological and molecular features.
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http://dx.doi.org/10.1136/jclinpath-2015-203335DOI Listing
April 2016

Clinical detection and categorization of uncommon and concomitant mutations involving BRAF.

BMC Cancer 2015 Oct 24;15:779. Epub 2015 Oct 24.

Departments of Pathology, Johns Hopkins University School of Medicine, Baltimore, USA.

Background: Selective BRAF inhibitors, vemurafenib and dabrafenib, and the MEK inhibitor, trametinib, have been approved for treatment of metastatic melanomas with a BRAF p.V600E mutation. The clinical significance of non-codon 600 mutations remains unclear, in part, due to variation of kinase activity for different mutants.

Methods: In this study, we categorized BRAF mutations according to the reported mutant kinase activity. A total of 1027 lung cancer, colorectal cancer or melanoma specimens were submitted for clinical mutation detection by next generation sequencing.

Results: Non-codon 600 mutations were observed in 37% of BRAF-mutated tumors. Of all BRAF mutants, 75% were kinase-activated, 15% kinase-impaired and 10% kinase-unknown. The most common kinase-impaired mutant involves codon 594, specifically, p.D594G (c.1781A > G) and p.D594N (c.1780G > A). Lung cancers showed significantly higher incidences of kinase-impaired or kinase-unknown mutants. Kinase-impaired BRAF mutants showed a significant association with concomitant activating KRAS or NRAS mutations, but not PIK3CA mutations, supporting the reported interaction of these mutations.

Conclusions: BRAF mutants with impaired or unknown kinase activity as well as concomitant kinase-impaired BRAF mutations and RAS mutations were detected in lung cancers, colorectal cancers and melanomas. Different therapeutic strategies based on the BRAF mutant kinase activity and the concomitant mutations may be worthwhile.
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http://dx.doi.org/10.1186/s12885-015-1811-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619530PMC
October 2015

A Novel Tandem Duplication Assay to Detect Minimal Residual Disease in FLT3/ITD AML.

Mol Diagn Ther 2015 Dec;19(6):409-17

Division of Molecular Pathology, Department of Pathology, Johns Hopkins University School of Medicine, Park SB202, 600 North Wolfe Street, Baltimore, MD, 21287, USA.

Background: Internal tandem duplication (ITD) of the fms-related tyrosine kinase 3 (FLT3) gene is associated with a poor prognosis in acute myeloid leukemia (AML) patients with a normal karyotype. The current standard polymerase chain reaction (PCR) assay for FLT3/ITD detection is not sufficiently sensitive to monitor minimal residual disease (MRD). Clone-specific assays may have sufficient sensitivity but are not practical to implement, since each clone-specific primer/probe requires clinical validation.

Objective: To develop an assay for clinical molecular diagnostics laboratories to monitor MRD in FLT3/ITD AMLs.

Methods: We designed a simple novel assay, tandem duplication PCR (TD-PCR), and tested its sensitivity, specificity, and clinical utility in FLT3/ITD AML patients.

Results: TD-PCR was capable of detecting a single ITD molecule and was applicable to 75 % of ITD mutants tested. TD-PCR detected MRD in bone marrow prior to patient relapse. TD-PCR also identified low-level ITD mutants not only in FLT3/ITD AMLs but also in initial diagnostic specimens that were reportedly negative by the standard assay in patients who progressed with the same ITDs detected by the TD-PCR assay.

Conclusion: Detection of MRD by TD-PCR may guide patient selection for early clinical intervention. In contrast to clone-specific approaches, the TD-PCR assay can be more easily validated for MRD detection in clinical laboratories because it uses standardized primers and a universal positive control. In addition, our findings on multi-clonality and low-level ITDs suggest that further studies are warranted to elucidate their clinical/biological significance.
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http://dx.doi.org/10.1007/s40291-015-0170-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655128PMC
December 2015

Non-p.V600E BRAF Mutations Are Common Using a More Sensitive and Broad Detection Tool.

Am J Clin Pathol 2015 Oct;144(4):620-8

From the Departments of Pathology and

Objectives: To assess the performance of a next-generation sequencing (NGS) platform for the clinical detection of BRAF mutations.

Methods: In this retrospective quality assessment of an NGS assay, we analyzed BRAF mutations within parts of exons 11 and 15 in 835 neoplastic tissues submitted to our molecular diagnostics laboratory.

Results: The NGS assays detected a BRAF mutation in 5.9% of lung adenocarcinomas, 13% of colorectal cancers, and 44% of melanomas. Mutant allele frequencies were less than 20% in 28% of 88 BRAF-mutated specimens. Two lymph node specimens with subcapsular or infiltrative metastasis showed 1% to 2% mutant alleles. There were 26 unique BRAF mutations in exons 11 and 15, including three novel mutations. Mutations were located outside codon 600 in 39% of BRAF-mutated tumors. Lung adenocarcinomas showed significantly higher non-p.V600E mutations (86%) than did colorectal cancers (23%) and melanomas (34%). The three most common BRAF mutations in lung cancers accounted for only 41% of the observed BRAF mutations (p.D594G [18%], p.V600E [14%], and p.G469A [9%]).

Conclusions: The NGS assay demonstrated a high analytic sensitivity and a broad reportable range for clinical detection of BRAF mutations. Elucidating the spectrum of non-p. V600E BRAF mutations in different malignancies is a first step toward understanding their clinical significance.
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http://dx.doi.org/10.1309/AJCP85ATMJOZOUDJDOI Listing
October 2015

Genotyping of 75 SNPs using arrays for individual identification in five population groups.

Int J Legal Med 2016 Jan 22;130(1):81-9. Epub 2015 Aug 22.

Department and Graduate Institute of Forensic Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.

Single nucleotide polymorphism (SNP) typing offers promise to forensic genetics. Various strategies and panels for analyzing SNP markers for individual identification have been published. However, the best panels with fewer identity SNPs for all major population groups are still under discussion. This study aimed to find more autosomal SNPs with high heterozygosity for individual identification among Asian populations. Ninety-six autosomal SNPs of 502 DNA samples from unrelated individuals of five population groups (208 Taiwanese Han, 83 Filipinos, 62 Thais, 69 Indonesians, and 80 individuals with European, Near Eastern, or South Asian ancestry) were analyzed using arrays in an initial screening, and 75 SNPs (group A, 46 newly selected SNPs; groups B, 29 SNPs based on a previous SNP panel) were selected for further statistical analyses. Some SNPs with high heterozygosity from Asian populations were identified. The combined random match probability of the best 40 and 45 SNPs was between 3.16 × 10(-17) and 7.75 × 10(-17) and between 2.33 × 10(-19) and 7.00 × 10(-19), respectively, in all five populations. These loci offer comparable power to short tandem repeats (STRs) for routine forensic profiling. In this study, we demonstrated the population genetic characteristics and forensic parameters of 75 SNPs with high heterozygosity from five population groups. This SNPs panel can provide valuable genotypic information and can be helpful in forensic casework for individual identification among these populations.
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http://dx.doi.org/10.1007/s00414-015-1250-yDOI Listing
January 2016

Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal cancers.

Mod Pathol 2015 Oct 31;28(10):1390-9. Epub 2015 Jul 31.

Departments of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.

Activating mutations in downstream genes of the epidermal growth factor receptor (EGFR) pathway may cause anti-EGFR resistance in patients with colorectal cancers. We present performance characteristics of a next-generation sequencing assay designed to detect such mutations. In this retrospective quality assessment study, we analyzed mutation detected in the KRAS, NRAS, BRAF, and PIK3CA genes by a clinically validated next-generation sequencing assay in 310 colorectal cancer specimens. Tumor cellularity and mutant allele frequency were analyzed to identify tumor heterogeneity and mutant allele-specific imbalance. Next-generation sequencing showed precise measurement of mutant allele frequencies and detected 23% of mutations with 2-20% mutant allele frequencies. Of the KRAS mutations detected, 17% were outside of codons 12 and 13. Among PIK3CA mutations, 48% were outside of codons 542, 545, and 1047. The percentage of tumors with predicted resistance to anti-EGFR therapy increased from 40% when testing for only mutations in KRAS exon 2 to 47% when testing for KRAS exons 2-4, 48% when testing for KRAS and NRAS exons 2-4, 58% when including BRAF codon 600 mutations, and 59% when adding PIK3CA exon 20 mutations. Right-sided colorectal cancers carried a higher risk of predicted anti-EGFR resistance. A concomitant KRAS mutation was detected in 51% of PIK3CA, 23% of NRAS, and 33% of kinase-impaired BRAF-mutated tumors. Lower than expected mutant allele frequency indicated tumor heterogeneity, while higher than expected mutant allele frequency indicated mutant allele-specific imbalance. Two paired neuroendocrine carcinomas and adjacent adenomas showed identical KRAS mutations, but only PIK3CA mutations in neuroendocrine carcinomas. Next-generation sequencing is a robust tool for mutation detection in clinical laboratories. It demonstrates high analytic sensitivity and broad reportable range, and it provides simultaneous detection of concomitant mutations and a quantitative measurement of mutant allele frequencies to predict tumor heterogeneity and mutant allele-specific imbalance.
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http://dx.doi.org/10.1038/modpathol.2015.86DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618462PMC
October 2015

Cetuximab Might Be Detrimental to Metastatic Colorectal Cancer Patients with KRAS Codon 12 Mutations.

Anticancer Res 2015 Jul;35(7):4207-14

Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan, R.O.C. Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan, R.O.C. Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan, R.O.C.

Background: Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies benefit patients with wild-type KRAS exon 2 metastatic colorectal cancer (mCRC). However, their effect in KRAS-mutant mCRC remains unclear.

Patients And Methods: This was a retrospective study enrolling 163 patients with unresectable KRAS-mutant mCRC diagnosed at the National Taiwan University Hospital between 2007 and 2011.

Results: The median overall survival (mOS) was 29.5 months in patients who had never used cetuximab and 19.0 months in those who had (p=0.040). The mOS was 32.0 months in patients with mutant KRAS codon 12 who had never used cetuximab and 17.5 months in those who had (p=0.017). In patients with mutant KRAS codon 13, the mOS was not significantly different. Univariate and multivariate Cox proportional hazards analysis revealed that absence of cetuximab treatment was an independent prognostic factor for longer mOS in patients with unresectable KRAS-mutant mCRC.

Conclusion: Cetuximab usage might be detrimental to patients with mCRC with mutant KRAS codon 12.
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July 2015

Granulosa cell-derived induced pluripotent stem cells exhibit pro-trophoblastic differentiation potential.

Stem Cell Res Ther 2015 Feb 27;6:14. Epub 2015 Feb 27.

Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan.

Introduction: Human induced pluripotent stem cells (hiPSCs) have been derived from various somatic cell types. Granulosa cells, a group of cells which surround oocytes and are obtained from the (normally discarded) retrieved egg follicles of women undergoing infertility treatment, are a possible cell source for induced pluripotent stem cell (iPSC) generation. Here, we explored the possibility of using human granulosa cells as a donor cell type for iPSC reprogramming, and compared granulosa cell-derived iPSCs (iGRAs) with those derived from other cell sources, to determine the potential ability of iGRA differentiation.

Methods: Granulosa cells were collected from egg follicles retrieved from women undergoing infertility treatment. After short-term culture, the granulosa cells derived from different patients were mixed in culture, and infected with retroviruses encoding reprogramming factors. The resulting iPSC clones were selected and subjected to microsatellite DNA analysis to determine their parental origin. IGRAs were subjected to RT-PCR, immunofluorescence staining, and in vitro and in vivo differentiation assays to further establish their pluripotent characteristics.

Results: Microsatellite DNA analysis was used to demonstrate that hiPSCs with different parental origins can be simultaneously reprogrammed by retroviral transfection of a mixed human granulosa cell population obtained from multiple individuals. The iGRAs resemble human embryonic stem cells (hESCs) in many respects, including morphological traits, growth requirements, gene and marker expression profiles, and in vitro and in vivo developmental propensities. We also demonstrate that the iGRAs express low levels of NLRP2, and differentiating iGRAs possess a biased differentiation potential toward the trophoblastic lineage. Although NLRP2 knockdown in hESCs promotes trophoblastic differentiation of differentiating hESCs, it does not result in exit from pluripotency. These results imply that NLRP2 may play a role in regulating the trophoblastic differentiation of human pluripotent stem cells.

Conclusions: These findings provide a means of generating iPSCs from multiple granulosa cell populations with different parental origins. The ability to generate iPSCs from granulosa cells not only enables modeling of infertility-associated disease, but also provides a means of identifying potential clinical interventions through iPSC-based drug screening.
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http://dx.doi.org/10.1186/s13287-015-0005-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430911PMC
February 2015

Split hand/foot malformations with microdeletions at chromosomes 7 and 19 detected using array comparative genomic hybridization.

Taiwan J Obstet Gynecol 2015 Feb;54(1):92-4

Department of Obstetrics and Gynecology, College of Medicine and Hospital, National Taiwan University, Taipei, Taiwan. Electronic address:

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http://dx.doi.org/10.1016/j.tjog.2014.01.006DOI Listing
February 2015

Immunohistochemical study of endometrial high-grade endometrioid carcinoma with or without a concurrent low-grade component: implications for pathogenetic and survival differences.

Histopathology 2015 Oct 31;67(4):474-82. Epub 2015 Mar 31.

Seirei Mikatahara General Hospital, Hamamatsu, Japan.

Aims: To compare the clinical and pathogenetic differences between high-grade (HG) endometrioid carcinomas with and without concurrent low-grade (LG) components.

Methods And Results: The expression of ARID1A, PTEN, p53 and mismatch repair (MMR) proteins in HG endometrioid carcinomas without (n = 19) or with (n = 22) concurrent LG endometrioid carcinomas was studied by immunohistochemistry. Microsatellite instability (MSI) was also tested in 31 cases. The frequencies of ARID1A loss, PTEN loss, MMR deficiency or MSI and aberrant p53 expression were 58%, 37%, 37% and 47% in pure HG tumours, and 77%, 45%, 55% and 32% in HG tumours with concurrent LG components (P = 0.07 for ARID1A; P > 0.1 for other proteins). Pure HG tumours had a higher frequency of the type II phenotype (positive for ARID1A, PTEN and MMR proteins; aberrant p53 expression) than HG tumours with concurrent LG components (21% versus 5%) (P = 0.2). The 5-year overall survival rate was worse for pure HG tumours (61.7%) than for HG tumours with concurrent LG components (93.3%) (P = 0.07).

Conclusions: HG endometrioid carcinomas are heterogeneous in pathogenesis: some arise from LG endometrioid carcinomas via the type I pathway, whereas others may arise de novo through either the type I pathway or the type II pathway, and have a different prognosis. Thus, HG endometrioid carcinomas should be subclassified properly and treated accordingly.
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http://dx.doi.org/10.1111/his.12664DOI Listing
October 2015

Challenges posed to pathologists in the detection of KRAS mutations in colorectal cancers.

Arch Pathol Lab Med 2015 Feb;139(2):211-8

From the Departments of Pathology (Drs Dudley, Tseng, Rooper, Chen, Gocke, Eshleman and Lin, Mr Harris, and Ms Haley) and Oncology (Drs Gocke and Eshleman), Johns Hopkins University School of Medicine, Baltimore, Maryland; the Department of Pathology, Massachusetts General Hospital, Boston (Dr Dudley); and the Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan (Dr Tseng).

Context: Detection of KRAS mutation is mandatory to predict response to anti-epidermal growth factor receptor monoclonal antibodies in patients with metastatic colorectal cancers.

Objective: To demonstrate challenges posed to pathologists in the clinical detection of KRAS mutations in colorectal cancers.

Design: In this retrospective analysis for quality assessment of the pyrosequencing assay, we survey the characteristics of 463 formalin-fixed, paraffin-embedded neoplastic tissues submitted for KRAS mutation detection during a 26-month period.

Results: The KRAS mutation was detected in 39.2% of tumors. This included 2 tumors with complex pyrograms (GGT>GAG at codon 12 and GGC>GTT at codon 13, as resolved by a Pyromaker software program) and 3 tumors with an indeterminate percentage of mutant alleles (defined as 4% to 5% and confirmed by a next-generation sequencing platform). Among the 25 specimens (5.5%) with fewer than 20% tumor cells, 22 were resected after chemotherapy/radiation. Significant depletion of tumor cells was observed in rectal cancers resected after neoadjuvant therapy (31.0%) versus those without previous treatment (0%) (P = .01). We also explore other specimens with low tumor cellularity and potential causes of discrepancy between the estimated tumor cell percentage and detected mutant allele frequency, such as intratumor heterogeneity of KRAS mutation.

Conclusions: Neoadjuvant therapy may deplete tumor cells and confound the molecular diagnosis of KRAS mutations. Accurate detection of specimens with poor tumor cellularity requires the appropriate selection of neoplastic tissues, evaluation of tumor cellularity, use of assays with high sensitivity, and prospective quality assessment.
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http://dx.doi.org/10.5858/arpa.2013-0649-OADOI Listing
February 2015

Lymph node metastases of melanoma: challenges for BRAF mutation detection.

Hum Pathol 2015 Jan 7;46(1):113-9. Epub 2014 Oct 7.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, 21287 USA. Electronic address:

Detection of B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutations is required to predict response to BRAF or mitogen-activated protein kinase kinase 1 and 2 inhibitors in metastatic melanoma. Lymph node (LN) specimens carrying melanoma cells intermingled with abundant lymphocytes often contain low tumor cellularity. This study is aimed to examine challenges in the clinical detection of BRAF mutations in LN specimens with metastatic melanoma and to illustrate characteristic features of p.V600E and non-p.V600E mutations. In this retrospective study for quality assessment of the pyrosequencing assay, we compared characteristics of 53 LN and 135 non-LN formalin-fixed, paraffin-embedded specimens with metastatic melanoma submitted for BRAF mutation detection over a 40-month period. LN specimens showed a significantly higher incidence of p.V600E mutations than non-LN specimens (49% versus 22%, P < .01) but a significantly lower tumor cellularity, particularly in the case of subcapsular or infiltrative metastases. Mutant allele-specific imbalance of the p.V600E mutation was predominantly present in specimens with distant organ metastases (79% versus 27% in LN metastases versus 13% in primary cutaneous tumors or adjacent soft tissue, P < .001). p.V600K was detected in 23% of men older than 60 years old, compared with 6% in women older than 60 years old and 2% in both men and women younger than 60 years old (P < .001). LN specimens with low tumor cellularity due to numerous adjacent lymphocytes may pose a challenge to clinical detection of BRAF mutations of melanoma. The higher incidence of p.V600E mutations in LNs may prompt further studies to elucidate if the p.V600E mutation in primary tumors is associated with a higher risk of LN metastasis.
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http://dx.doi.org/10.1016/j.humpath.2014.09.014DOI Listing
January 2015