Droplet-digital PCR reveals frequent mutations in TERT promoter region in breast fibroadenomas and phyllodes tumours, irrespective of the presence of MED12 mutations.
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ABSTRACT: Background
Breast fibroadenoma (FA) and phyllodes tumour (PT) often have variations of gene mediator complex subunit 12 (MED12) and mutations in the telomerase reverse transcriptase promoter region (TERTp). TERTp mutation is usually tested by Sanger sequencing. In this study, we compared Sanger sequencing and droplet-digital PCR (ddPCR) to measure TERTp mutations in FA and PT samples.Methods
FA and PT samples were collected from 82 patients who underwent surgery at our institution from 2005 to 2016. MED12 mutations for all cases and TERTp mutations for 17 tumours were detected by Sanger sequencing. ddPCR was performed to analyse TERTp mutation in all cases.Results
A total of 75 samples were eligible for analysis. Sanger sequencing detected MED12 mutations in 19/44 FA (42%) and 21/31 PT (68%). Among 17 Sanger sequencing-tested samples, 2/17 (12%) were TERTp mutation-positive. In ddPCR analyses, a significantly greater percentage of PT (19/31, 61%) was TERTp mutation-positive than was FA (13/44, 30%; P = 0.0046). The mutation positivity of TERTp and MED12 did not correlate, in either FA or PT.Conclusions
ddPCR was more sensitive for detecting TERTp mutation than Sanger sequencing, being able to elucidate tumorigenesis in FA and PT.
SUBMITTER: Otsuji K
PROVIDER: S-EPMC7852881 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:AimsSomatic mutations in exon 2 of the mediator complex subunit 12 (MED12) gene have been identified in 60% of breast fibroadenomas (FAs). The aim of this study was to define whether phyllodes tumours (PTs) would harbour MED12 somatic mutations in a way akin to FAs.Methods and resultsA collection of 73 fibroepithelial tumours (including 26 FAs, 25 benign PTs, nine borderline PTs and 13 malignant PTs) from 64 patients was retrieved from the authors' institution. Sections from formalin-fixed paraffin-embedded (FFPE) blocks were microdissected to ensure an enrichment in neoplastic stromal elements of >70%. DNA samples extracted from tumour and matched normal tissues were subjected to Sanger sequencing of exon 2 of the MED12 gene. MED12 exon 2 somatic mutations, including 28 somatic single nucleotide variants and 19 insertions and deletions, were found in 65%, 88%, 78% and 8% of FAs, benign PTs, borderline PTs and malignant PTs, respectively. Malignant PTs harboured MED12 exon 2 somatic mutations significantly less frequently than FAs, benign and borderline PTs.ConclusionsAlthough MED12 exon 2 somatic mutations probably constitute the driver genetic event of most FAs, benign and borderline PTs, our results suggest that the majority of malignant PTs may be driven by other genetic/epigenetic alterations.
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Project description:BackgroundPhyllodes tumours are rare fibroepithelial tumours of the breast, that include benign, borderline, and malignant lesions. Although the molecular basis of phyllodes tumours largely remains unknown, a recent exome study identified MED12 mutations as a sole recurrent genetic alteration in fibroadenoma, a common benign fibroepithelial tumour that shares some histological features with the phyllodes tumour.MethodsForty-six phyllodes tumours and 58 fibroadenomas of the breast were analysed for MED12 mutations by using Sanger sequencing.ResultsMED12 mutations were identified in 37 out of the 46 phyllodes tumours (80%). The prevalence of MED12 mutations was similar among benign (15/18, 83%), borderline (12/15, 80%), and malignant tumours (10/13, 77%). MED12 mutations were also identified in 36 of the 58 fibroadenomas (62%). The mutations were frequent among intracanalicular-type (24/32, 75%) and complex-type lesions (4/6, 67%), but were significantly less common among the pericanalicular-type lesions (8/20, 40%). A microdissection-based analysis showed that MED12 mutations were confined to the stromal components in both phyllodes tumours and fibroadenomas.ConclusionsMED12 mutations were frequent among the phyllodes tumours of the breast, regardless of the tumour grade. Phyllodes tumours and fibroadenomas share, at least in part, a common genetic background.
| S-EPMC4430713 | biostudies-literature
Project description:BackgroundMED12 and TERT promoter mutations have been shown to be the most common somatic mutations in phyllodes tumours (PTs). The aims of this study were to determine the frequency of these mutations in recurrent PTs, assess whether TERT promoter mutations could be helpful in distinguishing fibroadenomas (FAs) from PTs and identify novel mutations that may be driving malignant progression.MethodsMED12 and the TERT promoter were Sanger sequenced in 75 primary PTs, 21 recurrences, 19 single FAs and 2 cases of multiple FAs with benign PTs. Whole-exome sequencing was performed on one borderline PT.ResultsRecurrent PTs and multiple FAs showed temporal discordance in MED12 but not TERT. Recurrent samples did acquire TERT mutations, with recurrent benign PTs more likely to have mutations in both genes. TERT mutations were not helpful in differentiating between benign PTs and FAs in cases of multiple FAs/PTs. Exome sequencing revealed a nonsense mutation in RBM15 and Sanger sequencing revealed another three RBM15 mutations in malignant/borderline PTs.ConclusionsThis study has shown that MED12 mutations can be heterogeneous in both synchronous and recurrent PTs unlike TERT mutations. We have also shown that RBM15 mutations may be important in the pathogenesis of borderline/malignant PTs.
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Project description:Phyllodes tumors are rare fibroepithelial neoplasms of the breast, which carry the potential risk of local recurrence and metastasis. Phyllodes tumors share several histological features with fibroadenomas, and no widely accepted markers for distinguishing these lesions have been identified.We analyzed molecular abnormalities related to telomere elongation in tumors, including TERT promoter mutations, as well as loss of expression of ATRX and DAXX, in a total of 104 phyllodes tumors and fibroadenomas.Sequencing analyses showed that TERT promoter mutations were frequent in phyllodes tumors (30/46, 65%), but rare in fibroadenomas (4/58, 7%). Among phyllodes tumors, the mutations were more frequent in borderline tumors (13/15, 87%), but were also common in benign (9/18, 50%) and malignant tumors (8/13, 62%). Remarkably, all but one TERT promoter-mutated tumor also contained MED12 mutations, indicating that these mutations are strongly associated (P=8.4 × 10(-6)). Expression of ATRX and DAXX, as evaluated by immunohistochemistry, was retained in all tumors.Our observations suggest a critical role of TERT promoter mutations, in cooperation with MED12 mutations, in the development of phyllodes tumors. Because TERT promoter mutations are rare among fibroadenomas, their detection may be of potential use in discriminating between phyllodes tumors and fibroadenomas.
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Project description:Phyllodes tumors are rare fibroepithelial tumors with variable clinical behavior accounting for a small subset of all breast neoplasms, yet little is known about the genetic alterations that drive tumor initiation and/or progression. Here, targeted next-generation sequencing (NGS) was used to identify somatic alterations in formalin-fixed paraffin-embedded (FFPE) patient specimens from malignant, borderline, and benign cases. NGS revealed mutations in mediator complex subunit 12 (MED12) affecting the G44 hotspot residue in the majority (67%) of cases spanning all three histologic grades. In addition, loss-of-function mutations in p53 (TP53) as well as deleterious mutations in the tumor suppressors retinoblastoma (RB1) and neurofibromin 1 (NF1) were identified exclusively in malignant tumors. High-level copy-number alterations (CNA) were nearly exclusively confined to malignant tumors, including potentially clinically actionable gene amplifications in IGF1R and EGFR. Taken together, this study defines the genomic landscape underlying phyllodes tumor development, suggests potential molecular correlates to histologic grade, expands the spectrum of human tumors with frequent recurrent MED12 mutations, and identifies IGF1R and EGFR as potential therapeutic targets in malignant cases.Integrated genomic sequencing and mutational profiling provides insight into the molecular origin of phyllodes tumors and indicates potential druggable targets in malignant disease. Visual Overview: http://mcr.aacrjournals.org/content/early/2015/04/02/1541-7786.MCR-14-0578/F1.large.jpg.
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Project description:Fibroepithelial tumors (FTs) of the breast are a heterogeneous group of lesions ranging from fibroadenomas (FAD) to phyllodes tumors (PT) (benign, borderline, malignant). Further understanding of their molecular features and classification might be of clinical value. In this study, we analysed the expression of 105 breast cancer-related genes, including the 50 genes of the PAM50 intrinsic subtype predictor and 12 genes of the Claudin-low subtype predictor, in a panel of 75 FTs (34 FADs, 5 juvenile FADs, 20 benign PTs, 5 borderline PTs and 11 malignant PTs) with clinical follow-up. In addition, we compared the expression profiles of FTs with those of 14 normal breast tissues and 49 primary invasive ductal carcinomas (IDCs). Our results revealed that the levels of expression of all breast cancer-related genes can discriminate the various groups of FTs, together with normal breast tissues and IDCs (False Discovery Rate < 5%). Among FTs, the levels expression of proliferation-related genes (e.g. CCNB1 and MKI67) and mesenchymal/epithelial-related (e.g. CLDN3 and EPCAM) genes were found to be most discriminative. As expected, FADs showed the highest and lowest expression of epithelial- and proliferation-related genes, respectively, whereas malignant PTs showed the opposite expression pattern. Interestingly, the overall profile of benign PTs was found more similar to FADs and normal breast tissues than the rest of tumours, including juvenile FADs. Within the dataset of IDCs and normal breast tissues, the vast majority of FADs, juvenile FADs, benign PTs and borderline PTs were identified as Normal-like by intrinsic breast cancer subtyping, whereas 7 (63.6%) and 3 (27.3%) malignant PTs were identified as Claudin-low and Basal-like, respectively. Finally, we observed that the previously described PAM50 risk of relapse prognostic score better predicted outcome in FTs than the morphological classification, even within PTs-only. Our results suggest that classification of FTs using gene expression-based data is feasible and might provide clinically useful biological and prognostic information.
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Project description:Exon 2 of MED12, a subunit of the transcriptional mediator complex, has been frequently mutated in uterine leiomyomas and breast fibroadenomas; however, it has been rarely mutated in other tumors. Although the mutations were also found in uterine leiomyosarcomas, the frequency was significantly lower than in uterine leiomyomas. Here, we examined the MED12 mutation in phyllodes tumors, another biphasic tumor with epithelial and stromal components related to breast fibroadenomas. Mutations in MED12 exon 2 were analyzed in nine fibroadenomas and eleven phyllodes tumors via Sanger sequencing. A panel of cancer- and sarcoma-related genes was also analyzed using Ion Torrent next-generation sequencing. Six mutations in fibroadenomas, including those previously reported (6/9, 67%), and five mutations in phyllodes tumors (5/11, 45%) were observed. Three mutations in the phyllodes tumors were missense mutations at Gly44, which is common in uterine leiomyomas and breast fibroadenomas. In addition, two deletion mutations (in-frame c.133_144del12 and loss of splice acceptor c.100-68_137del106) were observed in the phyllodes tumors. No other recurrent mutation was observed with next-generation sequencing. Frequent mutations in MED12 exon 2 in the phyllodes tumors suggest that it may share genetic etiology with uterine leiomyoma, a subgroup of uterine leiomyosarcomas and breast fibroadenoma.
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Project description:Phyllodes tumours (PTs) are breast fibroepithelial lesions that are graded based on histological criteria as benign, borderline or malignant. PTs may recur locally. Borderline PTs and malignant PTs may metastasize to distant sites. Breast fibroepithelial lesions, including PTs and fibroadenomas, are characterized by recurrent MED12 exon 2 somatic mutations. We sought to define the repertoire of somatic genetic alterations in PTs and whether these may assist in the differential diagnosis of these lesions. We collected 100 fibroadenomas, 40 benign PTs, 14 borderline PTs and 22 malignant PTs; six, six and 13 benign, borderline and malignant PTs, respectively, and their matched normal tissue, were subjected to targeted massively parallel sequencing (MPS) using the MSK-IMPACT sequencing assay. Recurrent MED12 mutations were found in 56% of PTs; in addition, mutations affecting cancer genes (eg TP53, RB1, SETD2 and EGFR) were exclusively detected in borderline and malignant PTs. We found a novel recurrent clonal hotspot mutation in the TERT promoter (-124 C>T) in 52% and TERT gene amplification in 4% of PTs. Laser capture microdissection revealed that these mutations were restricted to the mesenchymal component of PTs. Sequencing analysis of the entire cohort revealed that the frequency of TERT alterations increased from benign (18%) to borderline (57%) and to malignant PTs (68%; p < 0.01), and TERT alterations were associated with increased levels of TERT mRNA (p < 0.001). No TERT alterations were observed in fibroadenomas. An analysis of TERT promoter sequencing and gene amplification distinguished PTs from fibroadenomas with a sensitivity and a positive predictive value of 100% (CI 95.38-100%) and 100% (CI 85.86-100%), respectively, and a sensitivity and a negative predictive value of 39% (CI 28.65-51.36%) and 68% (CI 60.21-75.78%), respectively. Our results suggest that TERT alterations may drive the progression of PTs, and may assist in the differential diagnosis between PTs and fibroadenomas. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Project description:WG-DASL expression of breast fibroepithelial lesions Breast fibroepithelial lesions are biphasic tumors and include fibroadenomas and phyllodes tumors. Fibroadenomas are clinically benign while phyllodes tumors are more unpredictable in biological behavior, with potential for recurrence. Differentiating the tumors may be challeneging when they have overlapping clinical and histological features especially on the core biopsies
2016-04-29 | GSE78071 | GEO
Project description:BackgroundThis study was performed to compare the mammographic, sonographic, and magnetic resonance imaging (MRI) characteristics of phyllodes tumors and fibroadenomas, which may resemble each other.MethodsPreoperative mammograms, B-mode and Doppler sonograms, and dynamic breast MRIs of 72 patients with pathologically proven fibroadenomas and 70 patients with pathologically proven phyllodes tumor were evaluated in this retrospective study. Statistical significance was evaluated using chi-square and Fisher's exact tests. Correlations in lesion size among radiological methods were examined by Pearson's correlation analysis.ResultsThe features that differed on mammogram were size, shape, and margin of the mass. Sonograms showed significant differences in size, shape, margin, echo pattern, and vascularization of the mass. Pearson's correlation analysis showed strong agreement among radiological methods in terms of assessment of size. Tumor size ? 3 cm, irregular shape, microlobulated margins, complex internal echo pattern, and hypervascularity were significant findings of phyllodes tumors. Internal cystic areas on MRI were frequently associated with phyllodes tumors.ConclusionMammographic, sonographic, and MRI findings of fibroadenomas and phyllodes tumors could help radiologists to ascertain imaging-histological concordance and guide clinicians in their decision making regarding adequate follow-up or the necessity of biopsy.
| S-EPMC4881274 | biostudies-literature