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 Targeted Mutational Analysis of Cortisol-Producing Adenomas

2021 ◽  
Author(s):  
Juilee Rege ◽  
Jessie Hoxie ◽  
Chia-Jen Liu ◽  
Morgan N Cash ◽  
James M Luther ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Mutational Analysis ◽  
Cushing Syndrome ◽  
Gene Mutations ◽  
Amplicon Sequencing ◽  
Formalin Fixed Paraffin ◽  
Mutation Profile ◽  
Somatic Gene ◽  
Formalin Fixed Paraffin Embedded ◽  
Aldosterone Producing Adenoma

Abstract Background Somatic gene mutations have been identified in only about half of cortisol-producing adenomas (CPA). Affected genes include PRKACA, GNAS, PRKAR1A, and CTNNB1. Objective To expand our understanding of the prevalence of somatic mutations in CPA from patients with overt Cushing syndrome (OCS) and “subclinical” mild autonomous cortisol excess (MACE), with an immunohistochemistry (IHC)‒guided targeted amplicon sequencing approach using formalin-fixed paraffin-embedded (FFPE) tissue. Method We analyzed FFPE adrenal tissue from 77 patients (n=12 men, 65 women) with either OCS (n=32) or MACE (n=45). Using IHC for 17α-hydroxylase/17,20-lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase (HSD3B2), we identified 78 CPA (32 OCS-CPA and 46 MACE-CPA). Genomic DNA was isolated from the FFPE CPA and subjected to targeted amplicon sequencing for identification of somatic mutations. Results Somatic mutations were identified in 71.8% (56/78) of the CPA. While PRKACA was the most frequently mutated gene in OCS-CPA (14/32, 43.8%), somatic genetic aberrations in CTNNB1 occurred in 56.5% (26/46) of the MACE-CPA. Most GNAS mutations were observed in MACE-CPA (5/7,71.4%). No mutations were observed in PRKAR1A. In addition to the known mutations, we identified one previously unreported mutation in PRKACA. Two patients with MACE harbored two adjacent tumors within the same adrenal gland: one patient had two CPA, and the other patient had a CPA and an aldosterone-producing adenoma (identified by IHC for aldosterone synthase). Conclusion Comprehensive FFPE IHC-guided gene-targeted sequencing approach identified somatic mutations in 71.8% of the CPA. OCS-CPA demonstrated a distinct mutation profile compared to MACE-CPA.

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 Higher EU-TIRADS-Score Correlated with BRAF V600E Positivity in the Early Stage of Papillary Thyroid Carcinoma

2021 ◽  
Vol 10 (11) ◽  
pp. 2304
Author(s):  
Karolina Skubisz ◽  
Joanna Januszkiewicz-Caulier ◽  
Patrycja Cybula ◽  
Elwira Bakuła-Zalewska ◽  
Krzysztof Goryca ◽  
...  
Keyword(s):  
Early Stage ◽  
Braf V600e ◽  
P Value ◽  
Dna And Rna ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ion Proton ◽  
Indolent Disease ◽  
Next Generation Sequencing Ngs ◽  
Formalin Fixed

The data demonstrating a correlation between sonographic markers of malignancy of thyroid cancer (TC) and its genetic status are scarce. This study aimed to assess whether the addition of genetic analysis at the preoperative step of TC patients’ stratification could aid their clinical management. The material consisted of formalin-fixed paraffin-embedded tumor fragments of 49 patients who underwent thyroidectomy during the early stages of papillary TC (PTC). Tumor DNA and RNA were subjected to next-generation sequencing (NGS) on Ion Proton using the Oncomine™ Comprehensive Assay panel. We observed a significant correlation between BRAF V600E and a higher EU-TIRADS score (p-value = 0.02) with a correlation between hypoechogenicity and taller-than-wide tumor shape in analysed patients. There were no other significant associations between the identified genetic variants and other clinicopathological features. For TC patient’s stratification, a strong suspicion of BRAF V600E negativity in preoperative management of TC patients could limit the over-treatment of asymptomatic, very low-risk, indolent disease and leave room for active surveillance.

scholarly journals Prevalence of UL97 gene mutations and polymorphisms in cytomegalovirus infection in the colon associated with or without ulcerative colitis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Satoshi Tamura ◽  
Satoshi Osawa ◽  
Natsuki Ishida ◽  
Takahiro Miyazu ◽  
Shinya Tani ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Gene Mutations ◽  
Pcr Amplification ◽  
Kinase Domain ◽  
Resistance Mutations ◽  
Cmv Infection ◽  
Nested Polymerase Chain Reaction ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ul97 Gene

AbstractCytomegalovirus (CMV) reactivation in the colon is common in patients with severe ulcerative colitis (UC). Ganciclovir (GCV) resistance conferring CMV UL97 gene mutations have been reported in recent years. However, the prevalence of UL97 gene mutations in GCV-naive CMV infection in the colon remains unknown. We investigated the prevalence of CMV UL97 gene mutations in patients with colonic CMV infection associated with or without UC. Twenty-two GCV-naive patients with colonic CMV infection, 15 with UC and 7 with other diseases, were enrolled. Frozen biopsy samples or formalin-fixed paraffin-embedded samples were used for nested polymerase chain reaction (PCR) amplification of the UL97 gene. Sanger DNA sequencing was performed. In comparison with AD169 reference strain, natural polymorphisms were frequently detected in codons N68D (100%), I244V (100%), and D605E (86.4%). Seven polymorphisms were detected infrequently (< 10%) outside the kinase domain. However, no known GCV resistance mutations were found. There seemed to be no difference between the ratio of polymorphisms in patients with and without UC. In conclusions, we did not detect UL97 gene mutations associated with GCV resistance in GCV-naive patients with or without UC. Consistent with previous reports, D605E polymorphism may be used as a genetic marker for CMV in East Asian countries.

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.

KIT Gene Mutations and Patterns of Protein Expression in Mucosal and Acral Melanoma

2012 ◽  
Vol 16 (2) ◽  
pp. 135-142 ◽  
Author(s):  
Suzan Abu-Abed ◽  
Nancy Pennell ◽  
Teresa Petrella ◽  
Frances Wright ◽  
Arun Seth ◽  
...  
Keyword(s):  
Protein Expression ◽  
Kinase Inhibitors ◽  
Case Series ◽  
Gene Mutations ◽  
Mucosal Melanoma ◽  
Acral Melanoma ◽  
Formalin Fixed Paraffin ◽  
Kit Gene ◽  
Formalin Fixed Paraffin Embedded ◽  
Optimized Conditions

Background: Recently characterized KIT (CD117) gene mutations have revealed new pathways involved in melanoma pathogenesis. In particular, certain subtypes harbor mutations similar to those observed in gastrointestinal stromal tumors, which are sensitive to treatment with tyrosine kinase inhibitors. Objective: The purpose of this study was to characterize KIT gene mutations and patterns of protein expression in mucosal and acral melanoma. Methods: Formalin-fixed, paraffin-embedded tissues were retrieved from our archives. Histologic assessment included routine hematoxylin-eosin stains and immunohistochemical staining for KIT. Genomic DNA was used for polymerase chain reaction-based amplification of exons 11 and 13. Results: We identified 59 acral and mucosal melanoma cases, of which 78% showed variable levels of KIT expression. Sequencing of exons 11 and 13 was completed on all cases, and 4 (6.8%) mutant cases were isolated. Conclusion: We successfully optimized conditions for the detection of KIT mutations and showed that 8.6% of mucosal and 4.2% of acral melanoma cases at our institution harbor KIT mutations; all mutant cases showed strong, diffuse KIT protein expression. Our case series represents the first Canadian study to characterize KIT gene mutations and patterns of protein expression in acral and mucosal melanoma.

Abstract 057: Cellular and Genetic Causes of Idiopathic Hyperaldosteronism

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Kei Omata ◽  
Fumitoshi Satoh ◽  
Ryo Morimoto ◽  
Sadayoshi Ito ◽  
Yuto Yamazaki ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Clinical Manifestations ◽  
Gene Mutations ◽  
Median Number ◽  
Zona Glomerulosa ◽  
Potential Contribution ◽  
Unilateral Adrenalectomy ◽  
Aldosterone Synthase ◽  
Formalin Fixed Paraffin Embedded ◽  
Idiopathic Hyperaldosteronism

Background: Primary aldosteronism (PA) affects ~10% of hypertensive patients and has unilateral and bilateral forms (30%:70%). Unilateral PA is caused by aldosterone-producing adenomas (APA), which express CYP11B2 (aldosterone synthase) and frequently harbor somatic mutations in aldosterone regulating genes ( KCNJ5>>CACNA1D ). We recently demonstrated that adrenals from normotensive patients present with pockets of cell expressing aldosterone synthase (CYP11B2). These aldosterone-producing foci (APF) have somatic gene mutations similar to those found in APA ( CACNA1D >> KCNJ5 ). Bilateral PA, which is typically treated by mineralocorticoid receptor (MR) blockade rather than surgery, is termed idiopathic hyperaldosteronism (IHA). Its pathobiology is largely unknown but has been thought to be due to zona glomerulosa (ZG) hyperplasia. Methods: We studied 11 IHA patients (7 males, 4 females) who had unilateral adrenalectomy. Immunohistochemistry for CYP11B2 and next generation sequencing (NGS) targeting genes found in APA were performed on formalin fixed paraffin embedded adrenal tissue. Results were compared to previously described cohorts of 53 age-matched normotensive patients (29 males, 24 females) which were evaluated similarly. Results: CYP11B2 expression was absent from intervening ZG cells in 8/11 (73%) IHA adrenals, but all adrenals harbored at least one APF. The median number and size of APF per case were significantly larger in IHA than normotensive controls (6.1 vs 0 APF/cm 2 of adrenal cortex and 0.25 vs. 0.16 mm 2 , respectively; p<0.0001 and p<0.006). In this IHA cohort, NGS identified CACNA1D and KCNJ5 somatic mutations in 44/71 (62%) and 1/71 (1%) of APF, respectively. Interpretations. Diffuse CYP11B2 expression in adrenal ZG cells was only observed in 3/11 IHA cases, arguing against ZG hyperplasia as the major underlying pathobiology. Rather, we demonstrated increased and enlarged APF in IHA adrenals compared to normotensive controls, supporting potential contribution to the clinical manifestations of hyperaldosteronism. The frequent occurrence of aldosterone-dysregulating CACNA1D somatic mutations in APF support CACNA1D as a potential therapeutic target in IHA to complement current MR blockade approaches.

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.

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