scholarly journals Prevalence of Somatic Mutations in Aldosterone-Producing Adenomas in Japanese Patients

2020 ◽  
Vol 105 (11) ◽  
Author(s):  
Kazutaka Nanba ◽  
Yuto Yamazaki ◽  
Nolan Bick ◽  
Kei Onodera ◽  
Yuta Tezuka ◽  
...  
Keyword(s):  
Racial Differences ◽  
Somatic Mutations ◽  
Japanese Patients ◽  
University Hospital ◽  
Female Patients ◽  
Targeted Next Generation Sequencing ◽  
Formalin Fixed Paraffin ◽  
High Prevalence ◽  
Formalin Fixed Paraffin Embedded ◽  
Male Patients

Abstract Context Results of previous studies demonstrated clear racial differences in the prevalence of somatic mutations among patients with aldosterone-producing adenoma (APA). For instance, those in East Asian countries have a high prevalence of somatic mutations in KCNJ5, whereas somatic mutations in other aldosterone-driving genes are rare. Objectives To determine somatic mutation prevalence in Japanese APA patients using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided sequencing approach. Method Patients with a unilateral form of primary aldosteronism who underwent adrenalectomy at the Tohoku University Hospital were studied. Based on CYP11B2 immunolocalization of resected adrenals, genomic DNA was isolated from the relevant positive area of 10% formalin-fixed, paraffin-embedded tissue of the APAs. Somatic mutations in aldosterone-driving genes were studied in APAs by direct Sanger sequencing and targeted next-generation sequencing. Results CYP11B2 IHC-guided sequencing determined APA-related somatic mutations in 102 out of 106 APAs (96%). Somatic KCNJ5 mutation was the most frequent genetic alteration (73%) in this cohort of Japanese patients. Somatic mutations in other aldosterone-driving genes were also identified: CACNA1D (14%), ATP1A1 (5%), ATP2B3 (4%), and CACNA1H (1%), including 2 previously unreported mutations. KCNJ5 mutations were more often detected in APAs from female patients compared with those from male patients [95% (36/38) vs 60% (41/68); P < 0.0001]. Conclusion IHC-guided sequencing defined somatic mutations in over 95% of Japanese APAs. While the dominance of KCNJ5 mutations in this particular cohort was confirmed, a significantly higher KCNJ5 prevalence was detected in female patients. This study provides a better understanding of genetic spectrum of Japanese APA patients.

scholarly journals Double adrenocortical adenomas harboring independent KCNJ5 and PRKACA somatic mutations

2016 ◽  
Vol 175 (2) ◽  
pp. K1-K6 ◽  
Author(s):  
Kazutaka Nanba ◽  
Kei Omata ◽  
Scott A Tomlins ◽  
Thomas J Giordano ◽  
Gary D Hammer ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Gene Mutations ◽  
Molecular Characteristics ◽  
Targeted Next Generation Sequencing ◽  
Dna And Rna ◽  
Adrenocortical Adenomas ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Negative Tumor ◽  
Design And Methods

Objective Co-secretion of cortisol and aldosterone can be observed in adrenal adenomas. The aim of this study was to investigate the molecular characteristics of a co-existing aldosterone- and a cortisol-producing adenoma (CPA) in the same patient. Design and methods Two different adenomas within the same adrenal gland from a 49-year-old female patient with primary aldosteronism (PA) and Cushing's syndrome (CS) were studied. Multiple formalin-fixed paraffin-embedded tumor blocks were used for the analysis. Immunohistochemistry (IHC) was performed using a specific antibody against aldosterone synthase (CYP11B2). DNA and RNA were isolated separately from CYP11B2-positive and -negative tumor regions based on CYP11B2 IHC results. Results CYP11B2 IHC clearly demonstrated that three pieces from one adenoma were positive for CYP11B2 and the remaining three from the other adenoma were negative for CYP11B2. In quantitative real-time RT-PCR, CYP11B2 mRNA was upregulated in CYP11B2-positive tumor specimens (219-fold vs CYP11B2-negative tumor specimens). Targeted next-generation sequencing (NGS) detected novel KCNJ5 gene mutations (p.T148I/T149S, present in the same reads) and a PRKACA gene hotspot mutation (p.L206R) in the CYP11B2-positive and -negative tumors, respectively. Sanger sequencing of DNA from each tumor specimen (CYP11B2-positive tumor, n=3; CYP11B2-negative tumor, n=3) showed concordant results with targeted NGS. Conclusion Our findings illustrate the co-existence of two different adrenocortical adenomas causing the concurrent diagnosis of PA and CS in the same patient. Molecular analysis was able to demonstrate that the two diseases resulted from independent somatic mutations seen in double adrenocortical adenomas.

scholarly journals BRAFV600E-Positive Precursors As Molecular Markers of Bone Marrow Involvement in Pediatric Langerhans Cell Histiocytosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3588-3588
Author(s):  
Ko Kudo ◽  
Rika Kanezaki ◽  
Akie Kobayashi ◽  
Tomohiko Sato ◽  
Takuya Kamio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Somatic Mutations ◽  
Bone Marrow Involvement ◽  
Braf V600e ◽  
Sensitive Assay ◽  
System Disease ◽  
Multiple Organs ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Introduction: The BRAF mutation V600E, the most common somatic mutation in Langerhans cell histiocytosis (LCH), has been reported in approximately 50% of LCH patients and is associated with certain high-risk clinical features. Precursors harboring this mutation can differentiate into Langerhans cells resulting in infiltrates in multiple organs under inflammatory conditions. However, BRAF status in the bone marrow of pediatric LCH patients is unclear. The present study examined somatic mutations in paired tumor and bone marrow samples, using a highly sensitive assay involving next-generation targeted sequencing and droplet digital polymerase chain reaction (PCR) for pediatric LCH patients. Methods: Between 1996 and 2019, in total of 17 Japanese pediatric patients with LCH were enrolled. The male/female ratio was 7/11. Ages of onset of LCH were median 13 months (range 5-193 months). At diagnosis of LCH, 2 patients were positive for risk organ involvement, 15 were negative. We retrospectively performed mutational analyses of 17 LCH cases using formalin-fixed paraffin-embedded LCH tumor specimens to provide templates for PCR-based targeted amplicon sequencing with customized primers to detect mutations in exons 12 and 15 in BRAF, and exons 2 and 3 in MAP2K1. Thereafter, we identified somatic mutations in the 17 paired bone marrow samples via droplet digital allele-specific PCR, targeting BRAF V600E and BRAF exon 12 in-frame deletion 496-500 (Ex12 in-del). Results: We detected BRAF V600E in 11 of 17 tumor samples (65%) and the BRAF Ex 12 in-del in 3 of 17 tumors (18%). We identified BRAF V600E in bone marrow samples in 10 of the 11 cases (90%) with the mutation in the tumor at low variant allele frequency (median 0.25%, range 0.14-7.0%). BRAF Ex 12 in-del was not detected in the bone marrow. Cases with detectable bone marrow involvement included eight patients with multi-system disease affecting multiple organs, one patient with multi-focal bone disease, and one patient with single-system disease. Clinical phenotypes including relapse did not correlate with BRAF V600E upon detection in the bone marrow. Conclusion: We established the sensitive assay based on PCR-based targeted NGS for detecting somatic mutations in LCH even accessible for formalin-fixed, paraffin-embedded clinical specimens. Bone marrow involvement is frequently detectable at the molecular level in pediatric LCH with the BRAF V600E mutation. A prospective study is warranted to evaluate the clinical impact of mutational burden in bone marrow. Disclosures Kudo: Unum Therapeutics: Patents & Royalties. Imai:Juno Therapeutics: Patents & Royalties.

scholarly journals Mosaicism and incomplete penetrance of PCDH19 mutations

2018 ◽  
Vol 56 (2) ◽  
pp. 81-88 ◽  
Author(s):  
Aijie Liu ◽  
Xiaoxu Yang ◽  
Xiaoling Yang ◽  
Qixi Wu ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Male Patient ◽  
Sanger Sequencing ◽  
De Novo ◽  
Digital Pcr ◽  
The Other ◽  
Incomplete Penetrance ◽  
Female Patients ◽  
Targeted Next Generation Sequencing ◽  
Male Patients ◽  
Patients With Epilepsy

BackgroundMutations in the PCDH19 gene have mainly been reported in female patients with epilepsy. To date, PCDH19 mutations have been reported in hundreds of females and only in 10 mosaic male epileptic patients with mosaicism.ObjectiveWe aimed to investigate the occurrence of mosaic PCDH19 mutations in 42 families comprising at least one patient with PCDH19-related epilepsy.MethodsTwo male patients with mosaic PCDH19 variants were identified using targeted next-generation sequencing. Forty female patients with PCDH19 variants were identified by Sanger sequencing and Multiple Ligation Probe Amplification (MLPA). Microdroplet digital PCR was used to quantify the mutant allelic fractions (MAFs) in 20 families with PCDH19 variants.ResultsFive mosaic individuals, four males and one female, were identified in total. Mosaic variant was confirmed in multiple somatic tissues from one male patient and in blood from the other male patient. Among 22 female patients harbouring a newly occurred PCDH19 variant identified by Sanger sequencing and MLPA, Sanger sequencing revealed two mosaic fathers (9%, 2/22), one with two affected daughters and the other with an affected child. Two asymptomatic mosaic fathers were confirmed as gonosomal mosaicism, with MAFs ranging from 4.16% to 37.38% and from 1.27% to 19.13%, respectively. In 11 families with apparent de novo variants, 1 female patient was identified as a mosaic with a blood MAF of 26.72%.ConclusionOur study provides new insights into phenotype-genotype correlations in PCDH19 related epilepsy and the finding of high-frequency mosaicism has important implications for genetic counselling.

Evaluation of the Oncomine Comprehensive Assay v3 panel for the detection of 1p/19q codeletion in oligodendroglial tumours

2021 ◽  
pp. jclinpath-2021-207876
Author(s):  
Rola H Ali ◽  
Mona Alateeqi ◽  
Hiba Jama ◽  
Noor Alrumaidhi ◽  
Ali Alqallaf ◽  
...  
Keyword(s):  
Copy Number ◽  
In Situ Hybridisation ◽  
Fluorescence In Situ Hybridisation ◽  
Ion Torrent ◽  
Robust Detection ◽  
Targeted Next Generation Sequencing ◽  
Formalin Fixed Paraffin ◽  
Custom Made ◽  
Formalin Fixed Paraffin Embedded ◽  
Diffuse Gliomas

AimsAccurate assessment of 1p/19q codeletion status in diffuse gliomas is of paramount importance for diagnostic, prognostic and predictive purposes. While targeted next generation sequencing (NGS) has been widely implemented for glioma molecular profiling, its role in detecting structural chromosomal variants is less well established, requiring supplementary informatic tools for robust detection. Herein, we evaluated a commercially available amplicon-based targeted NGS panel (Oncomine Comprehensive Assay v3) for the detection of 1p/19q losses in glioma tissues using an Ion Torrent platform and the standard built-in NGS data analysis pipeline solely.MethodsUsing as little as 20 ng of DNA from formalin-fixed paraffin-embedded tissues, we analysed 25 previously characterised gliomas for multi-locus copy number losses (CNLs) on 1p and 19q, including 11 oligodendrogliomas (ODG) and 14 non-oligodendroglial (non-ODG) controls. Fluorescence in-situ hybridisation (FISH) was used as a reference standard.ResultsThe software confidently detected combined contiguous 1p/19q CNLs in 11/11 ODGs (100% sensitivity), using a copy number cut-off of ≤1.5 and a minimum of 10 amplicons covering the regions. Only partial non-specific losses were identified in non-ODGs (100% specificity). Copy number averages of ODG and non-ODG groups were significantly different (p<0.001). NGS was concordant with FISH and was superior to it in distinguishing partial from contiguous losses indicative of whole-arm chromosomal deletion.ConclusionsThis commercial NGS panel, along with the standard Ion Torrent algorithm, accurately detected 1p/19q losses in ODG samples, obviating the need for specialised custom-made informatic analyses. This can easily be incorporated into routine glioma workflow as an alternative to FISH.

Quantitative assessment of immune cell populations and associations with clinical outcomes in African-American (AA) versus Caucasian triple-negative breast cancer (TNBC).

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14180-e14180
Author(s):  
Tess O'Meara ◽  
Vesal Yaghoobi ◽  
Kim Blenman ◽  
Vasiliki Pelekanou ◽  
Andrea Silber ◽  
...  
Keyword(s):  
Racial Differences ◽  
Proportional Hazards ◽  
Immune Cell ◽  
Disease Free Survival ◽  
Clinical Information ◽  
Tumor Infiltrating Lymphocytes ◽  
Cox Proportional Hazards ◽  
Cytotoxic T Cell ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded

e14180 Background: Tumor infiltrating lymphocytes (TILs) are powerful prognostic and predictive factors in TNBC. We hypothesized that survival differences in TNBC by race may be caused by differences in the tumor immune microenvironment. We assessed racial differences in the extent and composition of immune infiltration in TNBC and correlated these differences with clinical characteristics and disease-free survival (DFS). Methods: Formalin fixed paraffin embedded TNBC samples and clinical information were collected for n = 43 AA and n = 43 Caucasian cases, matched by diagnosis date and stage. Stromal TILs were assessed on H&E-stained slides. Multiplexed immunofluorescence was performed to quantify CD68 (macrophage), CD8 (cytotoxic T cell) and PD-L1 protein expression in the whole-section, tumor and stromal compartments. Average expression for each marker was calculated over all fields of view. Cox proportional hazards were used to assess associations between DFS, staining markers and clinical variables. Results: Characteristics of AA and Caucasian cases were not significantly different. There were 14 and 8 recurrences in the AA and Caucasian cohorts, respectively (median follow-up 8.7 vs 9.4 yrs). TIL counts (p = 0.031) and overall CD68 expression (p = 0.005) were higher in AA compared to Caucasian patients. 21% percent of AA cases had TIL predominant phenotype versus 3% of Caucasians, but CD8 expression was similar by race. PD-L1 expression was higher in stroma compared to tumor across all patients (median 401 vs 267 au, p = 0.002) and did not differ by race. Higher overall and stromal PD-L1 expression were associated with better DFS in the entire population (median 3501 vs 1895 days, p = 0.0009) and in each race separately. Higher CD68 expression was also associated with better DFS (median 3015 vs 2111 days, p = 0.0004). In multivariate analysis of DFS, stage at presentation remained significant (p = 0.002) in addition to PD-L1 and CD68 expression. Conclusions: AA TNBC had higher TIL counts and CD68 expression but similar CD8 and PD-L1 expression compared to Caucasians. High CD68 and PD-L1 expression were associated with better DFS in both race cohorts.

Next generation sequencing of primary prostate cancer tumors to reveal potentially actionable opportunities for clinical management: The UNC experience.

2019 ◽  
Vol 37 (7_suppl) ◽  
pp. 305-305
Author(s):  
Daniel James Crona ◽  
Anthony Drier ◽  
Jing Daisy Zhu ◽  
Emily Fox Bell ◽  
Margaret Rose Sketch ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Next Generation Sequencing ◽  
Copy Number Variations ◽  
Precision Oncology ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Formalin Fixed Paraffin ◽  
Primary Prostate Cancer ◽  
Formalin Fixed Paraffin Embedded ◽  
Generation Sequencing

305 Background: The Strata trial (NCT03061305) is a multi-institutional precision oncology collaboration structured as an observational protocol that aims to match patients to genomically-guided therapies. Methods: Selected University of North Carolina (UNC) metastatic prostate cancer (mPC) patients were enrolled on this IRB-approved study. Formalin fixed paraffin-embedded primary tumor specimens, without matched germline controls, were sent for targeted next generation sequencing (NGS) to detect actionable variants, including: mutations in 87 genes, copy number variations in 31 genes, and gene fusions in 46 gene drivers. mPC-related genes of particular interest included: AR, ATM, BRCA1/2, ERG, MSH2, MSH6, PTEN, RB1, and TP53. Results: Of the 92 cases sequenced, 5 [5%] failed testing. Of the 87 mPC patients (median age 69 years [47-86]) enrolled: 53 [61%] were white, 28 [32%] were black, 1 [1%] was Asian, and 5 [6%] declined to be identified. NGS data revealed 106 variants in 27 genes: 62 patients (71%) had at least one variant, 21 (24%) had 2 variants, 7 (8%) had 3 variants, and 4 (3%) had 4 variants. Among the 62 patients with at least 1 identified variant, TMPRSS2-ERG fusion occurred most frequently (50%), followed by TP53 (40%), and PTEN (16%). 6% of all sequenced patients had variants in DNA damage repair genes including ATM (3%), BRCA2 (2%) and MSH2 (1%). One patient had a SLC45A3-ERG fusion combined with PTEN deep deletion, which has been associated with a more aggressive phenotype. One patient with a microsatellite-instability high tumor was treated with pembrolizumab. Conclusions: The UNC experience shows that a high proportion of primary prostate cancer tumors from mPC patients have genomic variants, and one patient was treated based on these data. Limited actionability may reflect the landscape of currently FDA approved mPC treatments, and available clinical trials. It may also be due to a short follow-up, and these data could inform treatment planning upon progression.

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