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Massively parallel sequencing of Phyllodes tumours of the breast reveals actionable mutations, and TERT promoter hotspot mutations and TERT gene amplification as likely drivers of progression
Abstract
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.
... 32 Epidermal growth factor receptor (EGFR) amplification has been reported in up to 80% of squamous MBC, whereas no activating mutations in EGFR were detected. [33][34][35] Conversely, mutations in EGFR have been observed in rare borderline and malignant PT, 36 while EGFR amplifications were detected in only 2-16% of all PT and nearly 20% of malignant PT. 37 Taken together, concerning the prevalence of EGFR molecular alterations, activating mutations are more correlated with malignant PT, while EGFR amplifications are correlated with MBC. Thus, these molecular findings are probably instrumental in the differential diagnosis between MBC and malignant PT. ...
... 38 In PT, a hot-spot TERT promoter mutation (À124 C > T) was found in 52% and TERT amplification in only 4% of cases, respectively. 36 While these alterations were restricted to the mesenchymal component, they were significantly more common in borderline and malignant PT than in benign PT. TERT mutations were observed in 7% of fibroadenomas in one study, 40 with no TERT alterations detected in a series of 100 fibroadenomas analysed by Piscuoglio et al. 36 Furthermore, TERT promoter mutations were observed in a series of primary sarcomas of the breast (non-angiosarcomas), whereas no TERT alteration was detected in angiosarcoma of the breast. ...
... 36 While these alterations were restricted to the mesenchymal component, they were significantly more common in borderline and malignant PT than in benign PT. TERT mutations were observed in 7% of fibroadenomas in one study, 40 with no TERT alterations detected in a series of 100 fibroadenomas analysed by Piscuoglio et al. 36 Furthermore, TERT promoter mutations were observed in a series of primary sarcomas of the breast (non-angiosarcomas), whereas no TERT alteration was detected in angiosarcoma of the breast. 41 Altogether, in the context of those observations, TERT promoter mutations may be useful in the differential diagnosis: (1) between fibroadenoma and PT; (2) between angiosarcoma and malignant PT; and (3) between angiosarcoma and another type of primary breast sarcoma. ...
The diagnosis, histomolecular classes of breast cancers (luminal A, luminal B, HER2-enriched, and basal-like), and accurate prediction of prognosis are commonly determined using morphological and phenotypical analyses in clinical practice worldwide. Therapeutic strategies are mostly based on the disease stage and molecular subclasses of breast cancer. Targeted therapies, such as anti-HER2s, poly-ADP ribose polymerase inhibitors or, to a lesser extent, phosphatidylinositol 3 kinase inhibitors, have substantially improved breast cancer patient prognosis over the past decades. Human epidermal growth factor receptor 2 (HER2) overexpression is widely determined based on immunohistochemistry, while next-generation sequencing (NGS) is currently employed to assess the presence of molecular alterations, including breast cancer gene 1 (BRCA1) and 2 or phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations, which are targets of these new approved therapies. In addition, next-generation sequencing (NGS) can aid the pathologist in challenging situations, such as a diagnostic workup for a metastatic carcinoma in lymph nodes of unknown origin, differential diagnosis of spindle cell tumourtumor in the breast between metaplastic carcinoma, malignant PT and sarcoma, o, as well as determining relatedness between primary breast cancers and recurrences. NGS offers a powerful tool that enables the pathologist to combine morphological analyses together with molecular alterations in challenging diagnostic situations.
... The pathogenesis and underlying genomic landscape of PTs are poorly understood, especially for metastatic disease [9,10]. The application of commercially available, advanced DNA sequencing technologies has enabled standardized investigation of the mutational status of several hundred cancer-related genes in PTs during the last 8 years and has provided new information on this topic [9][10][11][12][13][14][15][16][17][18]. In addition to genetic alterations in classic tumor suppressor genes (TSGs; e.g. ...
... TP53, RB1) or oncogenes (e.g. EGFR), hotspot mutations in PIK3CA [12], the TERT promoter region [18,19], and MED12 [9,14] have frequently been reported. Genetic alterations including loss-of-function alterations in TP53 and RB1 might have a potential driver function in MPTs [11]. ...
... Genetic alterations including loss-of-function alterations in TP53 and RB1 might have a potential driver function in MPTs [11]. It has also been suggested that TERT promoter mutations, either alone [14] or in combination with MED12 mutations [15], play an important role in the etiology and progression of PTs. Moreover, several less-characterized genetic alterations (e.g. in ATRX [16,17], BCORL1 [9], and ZNF217 [11]) whose role in the development and progression of PTs is unknown were identified. ...
Background
The genomic landscape of phyllodes tumors (PTs) of the breast is not well defined, especially in patients with advanced disease. To shed light on this topic, paired primary and progressed tumor samples from two patients with malignant PTs were subjected to next-generation sequencing (NGS) followed by functional analysis of genetic alterations using two prediction tools.
Methods
The DNA of both the primary tumor and distant metastases of Patient 1 and the primary and recurrent tumor of Patient 2 were subjected to molecular profiling. NGS with the FoundationOne® assay was performed in a commercial molecular pathology laboratory. Two in silico prediction tools were used to estimate the pathogenicity of indicated genetic alterations.
Results
In total, 38 genomic alterations were detected, of which 11 were predicted to be probably benign. In Patient 1, 14 aberrations were identified in the primary tumor and 17 in pulmonary metastases, 12 of which were identical. In the primary and recurrent tumor of Patient 2, 17 and 15 sequence variants, respectively, were found, with 13 overlapping findings. Affected genes included seven ( TP53 , TERT , APC , ARID1A , EGFR , KMT2D , and RB1 ) of the top 10 most frequently altered genes in other advanced cancer entities, as well as four actionable therapeutic targets ( EGFR , KIT , PDGFRA , and BRIP1 ). Of note, seven genes coding for receptor tyrosine kinases were affected: three in Patient 1 and four in Patient 2. Several genes (e.g. EPHA3 , EPHA7 , and EPHB1 ) were shown to be altered for the first time in PTs.
Conclusions
The two progressed malignant PTs investigated here share some of the major genetic events occurring in other advanced cancers.
... The transcriptomic features of MBCs also vary according to the predominant histologic component; for instance, MBCs with a predominant spindle cell component are preferentially classified as of claudin-low subtype, whereas MBCs with squamous or chondroid metaplasia are more frequently classified as of basallike or even normal breast-like subtypes than as of claudin-low subtype 8,9 . Somatic TERT promoter mutations (C228T and C250T), associated with telomerase activation, have been reported at a relatively high frequency in human cancers (12% overall) 14 and are associated with disease progression and recurrences [15][16][17] . Although thought to be absent or extremely rare in common forms of breast cancer [18][19][20] , TERT promoter mutations and TERT gene amplifications have been reported in up to 68% of malignant phyllodes tumors of the breast, a potential differential diagnosis of MBCs, and may have a role in the malignant progression in fibroepithelial lesions 15,21,22 . ...
... Somatic TERT promoter mutations (C228T and C250T), associated with telomerase activation, have been reported at a relatively high frequency in human cancers (12% overall) 14 and are associated with disease progression and recurrences [15][16][17] . Although thought to be absent or extremely rare in common forms of breast cancer [18][19][20] , TERT promoter mutations and TERT gene amplifications have been reported in up to 68% of malignant phyllodes tumors of the breast, a potential differential diagnosis of MBCs, and may have a role in the malignant progression in fibroepithelial lesions 15,21,22 . TERT gene amplification has been reported in 13% of adenomyoepitheliomas of the breast, tumors of uncertain malignant potential which have been reported to progress to spindle cell MBCs in a minority of cases 23,24 . ...
... TERT promoter hotspot mutations and TERT gene amplification have been reported in phyllodes tumors of the breast, suggesting that these genetic alterations might be the drivers of the progression from benign to malignant lesions in a subset of patients 15,22,36 . In addition, we have previously demonstrated that TERT somatic genetic alterations in 13% of breast adenomyoepitheliomas and in the carcinomas originating in association with or from these tumors 24 . ...
Metaplastic breast cancers (MBCs) are characterized by complex genomes, which seem to vary according to their histologic subtype. TERT promoter hotspot mutations and gene amplification are rare in common forms of breast cancer, but present in a subset of phyllodes tumors. Here, we sought to determine the frequency of genetic alterations affecting TERT in a cohort of 60 MBCs with distinct predominant metaplastic components (squamous, 23%; spindle, 27%; osseous, 8%; chondroid, 42%), and to compare the repertoire of genetic alterations of MBCs according to the presence of TERT promoter hotspot mutations or gene amplification. Forty-four MBCs were subjected to: whole-exome sequencing (WES; n = 27) or targeted sequencing of 341-468 cancer-related genes (n = 17); 16 MBCs were subjected to Sanger sequencing of the TERT promoter, TP53 and selected exons of PIK3CA, HRAS, and BRAF. TERT promoter hotspot mutations (n = 9) and TERT gene amplification (n = 1) were found in 10 of the 60 MBCs analyzed, respectively. These TERT alterations were less frequently found in MBCs with predominant chondroid differentiation than in other MBC subtypes (p = 0.01, Fisher’s exact test) and were mutually exclusive with TP53 mutations (p < 0.001, CoMEt). In addition, a comparative analysis of the MBCs subjected to WES or targeted cancer gene sequencing (n = 44) revealed that MBCs harboring TERT promoter hotspot mutations or gene amplification (n = 6) more frequently harbored PIK3CA than TERT wild-type MBCs (n = 38; p = 0.001; Fisher’s exact test). In conclusion, TERT somatic genetic alterations are found in a subset of TP53 wild-type MBCs with squamous/spindle differentiation, highlighting the genetic diversity of these cancers.
... A landmark study using exome sequencing reported a high frequency of MED12 mutations in fibroepithelial lesions and other mutations in RARA, FLNA, SETD2, and MLL2 in PTs, as well as additional cancer driver genes, particularly in borderline and malignant PTs [1]. A high frequency of TERT promoter mutations in PTs was also reported [2]. Most subsequent molecular analysis on PTs assessed alterations in MED12, TERT promoter and other very common cancer driver genes [3][4][5][6][7], and few studies have looked at other recurrent mutations. ...
... In an original study, recurrent mutations of SETD2 were found in over 20% of PT regardless of grade [1]. Similar findings were reported in a few smaller studies [2,15,16] and a recent large international collaborative analysis [17], albeit with a lower frequency. The consistent findings of SETD2 mutations in PTs suggested its potential involvement in PT pathogenesis. ...
... Its alterations, mostly with damaging effects functionally, were frequently observed in cancers and display a robust oncogenic property [10]. In PT, its alterations are consistently observed in several studies [1,2,[15][16][17]. Of note, it is one of the genes that were most frequently mutated exclusively in PT, but not fibroadenoma [1]. ...
PurposeSETD2 is one of the key epigenetic regulatory genes involved in histone modifications. Its alterations were potentially oncogenic and commonly found in cancers. Interestingly, SETD2 is one of the most frequent mutated genes found exclusively in phyllodes tumor of the breast (PT). However, little has been done to further characterize SETD2 alterations in PT.Methods
In this study, we examined the alterations of SETD2 gene and protein expression in a large cohort of PTs. Their correlations with SETD2 downstream target, H3K36me3 expression, and clinicopathologic features in PT were also assessed.ResultsSETD2 mutation was found in 15.9% of our cases and was mostly predicted to be damaging mutations. Interestingly, SETD2 mutations were associated with lower H3K36me3 expression, particularly those with damaging mutations (p = .041). Neither SETD2 mutations nor H3K36me3 expression was associated with PT grading and other clinicopathological features. By contrast, the SETD2 protein expression cannot reflect its mutation status and showed a different trend of clinicopathological correlations from H3K36me3.Conclusions
Our findings may suggest a potential involvement of epigenetic regulation via SETD2 alterations and downstream H3K36me3 on PT development. SETD2 mutations may occur early in the pathogenic process of PTs and its loss per se may not be sufficient for progression to malignancy. Exclusive alterations of SETD2 in PT can be used as markers for the diagnosis of fibroepithelial lesions. The association of H3K36me3 with SETD2 mutations may also indicate the value of evaluation of H3K36me3 expression in the diagnosis of fibroepithelial lesions.
... Monoallelic mutations are the predominant form of TERTp mutations [17,[20][21][22]. Mutations in TERTp generate an EST transcription factor binding motif [21], which then binds to GABP, thereby activating hTERT transcription and telomerase activation, which is linked to disease progression and relapse [23][24][25][26] (Fig. 1A). ...
... hTERT promoter mutation and gene amplification are rare in common breast cancers [29,30], and are more common in rare phyllodes neoplasms [24,31]. In a study of 60 metaplastic breast cancers (MBCs) of varying chemical composition, hTERT promoter mutations and gene amplification were less common in MBCs with predominant chondroid differentiation and were mutually exclusive with TP53 mutations [18]. ...
Telomerase reverse transcriptase (TERT/hTERT) serves as the pivotal catalytic subunit of telomerase, a crucial enzyme responsible for telomere maintenance and human genome stability. The high activation of hTERT, observed in over 90% of tumors, plays a significant role in tumor initiation and progression. An in-depth exploration of hTERT activation mechanisms in cancer holds promise for advancing our understanding of the disease and developing more effective treatment strategies. In breast cancer, the expression of hTERT is regulated by epigenetic, transcriptional, post-translational modification mechanisms and DNA variation. Besides its canonical function in telomere maintenance, hTERT exerts non-canonical roles that contribute to disease progression through telomerase-independent mechanisms. This comprehensive review summarizes the regulatory mechanisms governing hTERT in breast cancer and elucidates the functional implications of its activation. Given the overexpression of hTERT in most breast cancer cells, the detection of hTERT and its associated molecules are potential for enhancing early screening and prognostic evaluation of breast cancer. Although still in its early stages, therapeutic approaches targeting hTERT and its regulatory molecules show promise as viable strategies for breast cancer treatment. These methods are also discussed in this paper.
... They are responsible for telomerase reactivation, disease progression, and recurrence. Although rare in common forms of breast cancer [20], they have been reported at high frequencies in metaplastic and phyllode malignant breast tumors [21,22]. ...
... However, the absence of hTERT promoter hotspot mutations (C228T and C250T) is consistent with previous studies, since in the common forms of breast tumors, the rate of mutations is less than 1% [43]. When present in tumors, these mutations can stimulate telomerase reactivation, favoring tumor progression [21,22]. ...
HER2-enriched tumors are responsible for 20% of breast tumors and have high rates of immune infiltrates in the tumor stroma that respond favorably to neoadjuvant chemotherapy. In the context of tumors, telomeres control cell death and prevent tumor cells from replicating discontinuously, leading to their immortalization. This study aimed to evaluate the presence of tumor-infiltrating lymphocytes, hTERT expression, hTERT promoter mutation, and leukocyte telomere length in HER2-enriched breast tumors. A total of 103 cases were evaluated, 19 with pathologic complete response. The TILs percentage was above ≥10 in 44 cases (43%) and significantly present in patients ≥50 years of age. hTERT staining positivity was mostly nuclear, significantly present in the non-pCR group, and associated with a lower survival rate. Leukocyte telomeres were elongated for HER2-enriched tumors, and in multivariate analysis, shortening was associated with an increased risk of death. Overall, our results show that the nuclear and cytoplasmic presence of hTERT may indicate a worse prognosis and that leukocyte telomere elongation is a protective factor.
... PCR amplification, Sanger sequencing, and quantitative realtime PCR. For the identification of hotspot somatic mutations in TERT promoter, primer sets that amplify the hotspot sites of the TERT promoter were designed as previously described 22 and are available in our previously published study 23 . PCR amplification was performed from 100 ng of genomic DNA using the AmpliTaq Gold 360 Master Mix Kit (Life Technologies) on a SimpliAmp Thermal Cycler (ThermoFisher) as previously described 23 . ...
... For the identification of hotspot somatic mutations in TERT promoter, primer sets that amplify the hotspot sites of the TERT promoter were designed as previously described 22 and are available in our previously published study 23 . PCR amplification was performed from 100 ng of genomic DNA using the AmpliTaq Gold 360 Master Mix Kit (Life Technologies) on a SimpliAmp Thermal Cycler (ThermoFisher) as previously described 23 . Sequencing was performed using purified PCR fragments (QIAquick PCR Purification Kit, Qiagen) on an ABI 3730 capillary sequencer using the ABI BigDye Terminator chemistry (v3.1, Life Technologies). ...
Background
Focal nodular hyperplasia (FNH) is typically considered a benign tumor of the liver without malignant potential. The co-occurrence of FNH and hepatocellular carcinoma (HCC) has been reported in rare cases. In this study we sought to investigate the clonal relationship between these lesions in a patient with FNH-HCC co-occurrence.
Methods
A 74-year-old female patient underwent liver tumor resection. The resected nodule was subjected to histologic analyses using hematoxylin and eosin stain and immunohistochemistry. DNA extracted from microdissected FNH and HCC regions was subjected to whole exome sequencing. Clonality analysis were performed using PyClone.
Results
Histologic analysis reveals that the nodule consists of an FNH and two adjoining HCC components with distinct histopathological features. Immunophenotypic characterization and genomic analyses suggest that the FNH is clonally related to the HCC components, and is composed of multiple clones at diagnosis, that are likely to have progressed to HCC through clonal selection and/or the acquisition of additional genetic events.
Conclusion
To the best of our knowledge, our work is the first study showing a clonal relationship between FNH and HCC. We show that FNH may possess the capability to undergo malignant transformation and to progress to HCC in very rare cases.
... The pathogenesis of breast fibroepithelial lesions is not completely understood, with a paucity of studies outlining their developmental mechanisms [3,4]. In recent years, there have been new findings from genomic studies of these tumors [5][6][7][8][9][10], with MED12 being the most frequently aberrant gene, where the majority of mutations occur in codon 44 of exon 2 [5,11,12]. Genomic features have also suggested that fibroadenomas may be non-obligate precursors of phyllodes tumors [6,13]. ...
... Sixteen conventional fibroadenomas in our cohort harbored TERT promoter -124 C > T (chr5:1,295,228C > T) and -146 C > T (chr5:1,295,250C > T) mutations (15/16, 94% and 1/16, 6%, respectively), while two cellular fibroadenomas had alterations in the former (see Supplementary material, Table S3). Yoshida et al. similarly reported TERT promoter mutations in fibroadenomas although their proportion was less (4/58, 7%) [57], while four other studies did not detect such alterations among their fibroadenoma samples [8,[58][59][60]. When comparing benign phyllodes tumors and cellular fibroadenomas, mutations in TERT promoter gene showed the greatest difference in mutation frequency (32 vs 4%) (Table 3), while its sensitivity and specificity were 32% and 96%, respectively (AUC 0.641, p = 0.002) ( Table 5 and Fig. 4). ...
Breast fibroepithelial lesions are biphasic tumors which comprise the common benign fibroadenomas (FAs) and the rarer phyllodes tumors (PTs). This study analyzed 262 (42%) conventional FAs, 45 (7%) cellular FAs, and 321 (51%) benign PTs contributed by the International Fibroepithelial Consortium, using a previously curated 16 gene panel. Benign PTs were found to possess a higher number of mutations, and higher rates of cancer driver gene alterations than both groups of FAs, in particular MED12, TERT promoter, RARA, FLNA, SETD2, RB1, and EGFR. Cases with MED12 mutations were also more likely to have TERT promoter, RARA, SETD2, and EGFR. There were no significant differences detected between conventional FAs and cellular FAs, except for PIK3CA and MAP3K1. TERT promoter alterations were most optimal in discriminating between FAs and benign PTs. Our study affirms the role of sequencing and key mutations that may assist in refining diagnoses of these lesions.
... [2][3][4][5] MED12 exon 2 mutations have also been detected in other fibroepithelial neoplasms, including phyllodes tumours (PTs), where these mutations are present in 88% of benign, in 78% of borderline and in 8% of malignant PTs. [5][6][7][8] In addition, our group initially described the presence of telomerase reverse transcriptase (TERT) promoter hotspot mutations in PTs, 7 which have been confirmed to increase in frequency from benign to malignant PTs and to be vanishingly rare in conventional FAs. [9][10][11] FAs comprise histologic variants, including juvenile fibroadenoma, cellular FAs and complex fibroadenomas (CFAs), which display different histologic features and variations in their clinicopathologic characteristics. ...
... [2][3][4][5] MED12 exon 2 mutations have also been detected in other fibroepithelial neoplasms, including phyllodes tumours (PTs), where these mutations are present in 88% of benign, in 78% of borderline and in 8% of malignant PTs. [5][6][7][8] In addition, our group initially described the presence of telomerase reverse transcriptase (TERT) promoter hotspot mutations in PTs, 7 which have been confirmed to increase in frequency from benign to malignant PTs and to be vanishingly rare in conventional FAs. [9][10][11] FAs comprise histologic variants, including juvenile fibroadenoma, cellular FAs and complex fibroadenomas (CFAs), which display different histologic features and variations in their clinicopathologic characteristics. ...
Aims
Here we explore the presence of mediator complex subunit 12 ( MED12 ) exon 2 and telomerase reverse transcriptase ( TERT ) promoter hotspot mutations in complex fibroadenomas (CFAs) of the breast.
Methods
The stromal components from 18 CFAs were subjected to Sanger sequencing of MED12 exon 2 and the TERT promoter hotspot loci. The epithelial and stromal components of two MED12 mutated CFAs were subjected to laser capture microdissection, and Sanger sequencing of MED12 exon 2, TERT promoter and PIK3CA exons 9 and 20, separately.
Results
MED12 exon 2 mutations were identified in the stroma of 17% of CFAs. The analyses of epithelial and stromal components, microdissected separately, revealed that MED12 mutations were restricted to the stroma. No TERT promoter or PIK3CA mutations in exons 9 and 20 were detected in analysed CFAs.
Conclusions
Like conventional fibroadenomas, MED12 exon 2 mutations appear to be restricted to the stromal component of CFAs, supporting the notion that CFAs are stromal neoplasms.
... On the other hand, TERT promoter mutations were rare in FAs and, according to literature, are progressively more frequent in benign, borderline, and malignant categories of phyllodes tumors. Moreover, TERT promoter mutations are the most frequent, occurring in up to 70% of malignant/borderline PTs and found in 50% of benign PTs 21,24 . Therefore, one hypothesis already mentioned is that, since TERT mutations are rare in FAs (0-7%), these mutations could drive the progression of PTs from FAs 14 . ...
Purpose
To determine molecular events involved in the tumorigenesis of phyllodes tumors (PT) and the role of each stromal (SC) and epithelial (EC) cell.
Methods
Frozen breast samples enriched with epithelial and stromal cells from three fibroadenomas and 14 PT were retrieved and laser microdissected. Sanger and polymerase chain reaction-based sequencing of exon 2 MED12 and TERT promoter hotspot mutations were performed; 44K microarray platform was used to analyze gene expression.
Results
All three fibroadenomas (FAs) presented mutations in MED12, but not in TERT, whose mutation was observed in five of the 14 PTs. EC and SC of each affected tumor displayed identical alterations. Of the total differentially expressed genes (DEG) (EC = 1,543 and SC = 850), 984 were EC-eDEGs and 291 were SC-eDEGs. We found a high similarity of diseases and functions enriched by both cell types, but dissimilarity in the number of enriched canonical pathways. Three signaling canonical pathways overlapping with EC and SC were predicted to be activated in one cell type and inactivated in the other, while no overlap in eDEGs was assigned to them. We also identified 13 EC-eDEGs and five SC-eDEGs enriched networks, in which the SC-eDEGs were able to segregate FA from PT samples.
Conclusions
Identical TERT mutations from both SC and ES origins might affect the PTs tumorigenesis. Gene expression differences suggest coordinated molecular processes between these components with determinant differences acquired by SC, able to fully distinguish PTs from FAs lesions.
Key words
Phyllodes Tumor; Fibroadenoma; Epithelial Cells; Stromal Cells
... Yet it is uncertain whether these vaccines will progress to bring authorization for clinical application or not. Approximately 90% telomerase in different malignant cells is reactivated by following steps: i) elevation of hTERT copy numbers, ii) amplification of hTERT gene mainly causing various cancers of thyroid, skin, breast and drug resistance, iii) enhanced TERT protein translation, iv) TERT gene rearrangement and v) several point mutations in TERT gene correlated with poor clinical diagnosis of glioblastoma multiforme, merkel cell carcinoma and phyllodes tumors [50,140,141]. Due to telomerase reactivation, combination therapy that targets both telomerase and antioxidant pathways might be novel therapeutic strategy in the future. ...
Telomerase plays a significant role to maintain and regulate the telomere length, cellular immortality and senescence by the addition of guanine-rich repetitive sequences. Chronic inflammation or oxidative stress-induced infection downregulates TERT gene modifying telomerase activity thus contributing to the early steps of gastric carcinogenesis process. Furthermore, telomere-telomerase system performs fundamental role in the pathogenesis and progression of diabetes mellitus as well as in its vascular intricacy. The cessation of cell proliferation in cultured cells by inhibiting the telomerase activity of transformed cells renders the rationale for culling of telomerase as a target therapy for the treatment of metabolic disorders and various types of cancers. In this article, we have briefly described the role of immune system and malignant cells in the expression of telomerase with critical analysis on the gaps and potential for future studies. The key findings regarding the secrets of the telomerase summarized in this article will help in future treatment modalities for the prevention of various types of cancers and metabolic disorders notably diabetes mellitus.
... Dominant driver mutations that were previously present in the initial tumors are also acquired by metastatic illnesses under increased treatment strain, including modifications to the ERS1 and MAPK (mitogen-activated protein kinase) pathways. using olaparib as an adjuvant [11]. NGS has also made it possible to analyze therapy-resistant breast cancer, which has been shown to have 40 recurring alterations. ...
... Except for the TP53 mutation, which was present in both patients, the gene aberrations of Patients 1 and 2 were significantly different. It has been suggested that TP53 mutation may independently promote the pathogenesis of malignant PTs or promote tumor progression based on MED12 mutation [23,[26][27][28][29][30][31][32][33][34]. TP53 is a classic tumor suppressor gene involved in many malignant tumors, often accompanied by advanced tumor grade and poor prognosis, with high proliferative and invasive ability and genomic instability [35]. ...
Background
The genomic landscape of breast malignant phyllodes tumors (PTs) is not well defined, especially pregnancy-related malignant PTs. To clarify this topic, whole-exome next-generation sequencing (NGS) was performed on tumor samples and paired normal breast tissues from two pregnancy-related malignant PTs, followed by a functional analysis of the genetic alterations.
Methods
DNA from malignant PT samples and matched normal breast tissues of both patients were subjected to molecular profiling. NGS of the whole-exome was performed in a commercial molecular pathology laboratory. Predictive tools were used to estimate genetic variation in somatic and germline genes.
Results
In total, 29 somatic genomic alterations and 18 germline alterations were found in both patients. In Patient 1, 12 aberrations were identified in the tumor tissue, and 9 alterations were identified in matched normal breast tissue. One pathogenic variant in tumor suppressor genes ( TP53 ) was detected in patient 1. In Patient 2, 18 and 10 variants were found in the tumor and matched normal breast tissue, respectively. In Patient 2, pathogenic alterations were identified in two tumor suppressor genes ( PTEN and TP53 ). PTEN and TP53 may be potential drug targets. The functional predictive tools showed that genes of unknown significance for PTs, including FCHO1 in Patient 1, and LRP12 and PKM in Patient 2, were pathogenic. Several genes, including FCHO1 , LRP12 and PKM , were shown for the first time to be altered in malignant PTs. A potentially pathogenic germline variant in PRF1 , was detected in Patient 1.
Conclusion
Our study first demonstrated somatic and germline gene alterations in two malignant PTs during pregnancy and lactation. These two PTs shared major genetic events, including TP53 mutation, which commonly occurs in malignant PTs; additionally, we identified two potential genes for targeted therapy, TP53 and PTEN . One germline mutation in PRF1 was also detected. These results provide clues regarding tumor pathogenesis and precision therapy development.
... 53 Besides MED12, borderline and malignant PTs harbor neurofibromin 1 (NF1), retinoblastoma (RB1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), epidermal growth factor receptor (EGFR), TP53 (p53), and Erb-B2 receptor tyrosine kinase 4 (ERBB4) mutations. 54 Notably, high stromal telomerase reverse transcriptase (TERT) expression and promoter mutation are associated with malignant PTs, which are also commonly seen in other malignancies, including metaplastic carcinoma. 55 Thus, MED12 and RARA mutations are more specific and more likely to be useful in the differential diagnosis of FELs. ...
Context.—
This review article derives from the breast pathology lecture at the Eighth Princeton Integrated Pathology Symposium (PIPS VIII).
Objective.—
To provide a literature review and update on fibroepithelial lesions of the breast with molecular findings and findings regarding the pediatric population.
Data Sources.—
The sources include extensive literature review, personal research, and experience.
Conclusions.—
Given significant differences in prognosis and management of fibroepithelial lesions, we aim to provide readers with pertinent definitions, pathomorphology, molecular findings, and management for each diagnosis, with insights on the pediatric population.
... [15][16][17] PTs, particularly borderline and malignant tumors, subsequently acquire additional mutations like TERT promoter mutations, PIK3CA activating mutations, and EGFR amplification. [18][19][20][21] Incorporating specific molecular changes with the histologic classification of borderline and malignant PT may further improve prognostication and individualize therapy. ...
Objectives:
Phyllodes tumors (PTs) are categorized by the World Health Organization (WHO) as benign, borderline, and malignant. Singapore General Hospital (SGH) nomogram is a recurrence risk assessment tool for PT, which uses cytologic atypia, mitosis, stromal overgrowth, and the surgical margin status. We studied the prognostic significance of WHO classification and its correlation to the SGH nomogram.
Methods:
We identified 270 consecutive cases of PT (195 benign, 49 borderline, 26 malignant). Follow-up was available on 246 cases (mean follow-up of 51 months).
Results:
The recurrence rates were 2% (4 of 176) for benign, 4% (2 of 46) for borderline, and 25% (6 of 24) for malignant (log-rank test P < .0001 for recurrence-free survival). Only five patients with malignant PT experienced distant recurrence. Stromal overgrowth was an independent predictor of recurrence-free survival on multivariable analysis. The mean nomogram scores for benign, borderline, and malignant PT were 20, 20.3, and 32, respectively. The higher than expected score for benign PT was due to positive margins in 39% of cases.
Conclusions:
The WHO three-tiered classification of PT is prognostic. Despite positive margin status, most benign PTs do not recur. Other features of the nomogram help in determining recurrence but are also used for WHO classification.
... Multiple models including replication error, telomere dysfunction, and occurrence of fragile sites in chromosome lead to increased expression (Albertson 2006). Clinically, poor disease outcome in multiple malignancies including breast and thyroid cancers significantly correlated with upregulated hTERT expression due to increased copy number (Piscuoglio et al. 2016;Wang et al. 2016). A study involving a cohort of multiple cancers demonstrated that only 3% of hTERT expressing tumors has gene amplification, thereby suggesting other important mechanisms in deregulating hTERT expression (Zhang et al. 2000). ...
... Apart from insights into pathogenesis, genomic findings may refine grading and diagnostic categorisation of phyllodes tumours. Phyllodes tumour may be differentiated from fibroadenoma with the presence of TERT promoter mutations [38,39] which are rarely encountered in fibroadenoma. Molecular information may help in separating phyllodes tumours from other spindle cell neoplasms, with presence of MED12 mutations supporting the diagnosis of phyllodes tumour [40,41]. ...
Fibroepithelial tumours of the breast are biphasic neoplasms composed of both epithelial and stromal elements, including the common fibroadenoma and the infrequent phyllodes tumour. The admixture of epithelium and stroma in the fibroadenoma shows intra- and pericanalicular patterns, and may display a variety of histological changes. Fibroadenoma variants include the cellular, juvenile, myxoid and complex forms. The cellular fibroadenoma may be difficult to distinguish from the benign phyllodes tumour. Stromal mitotic activity can be increased in fibroadenomas in the young and pregnant patients. Phyllodes tumours, neoplasms with the potential for recurrence, show an exaggerated intracanalicular growth pattern with broad stromal fronded architecture and stromal hypercellularity. They are graded into benign, borderline and malignant forms based on histological assessment of stromal features of hypercellularity, atypia, mitotic activity, overgrowth and the nature of the tumour borders. Classification of phyllodes tumours is imperfect, compounded by tumour heterogeneity with overlapping microscopic features among the different grades, especially in the borderline category. Malignant phyllodes tumours can metastasise and cause death. Determining which phyllodes tumours may behave aggressively has been difficult. The discovery of MED12 mutations in the pathogenesis of fibroepithelial tumours, together with other gene abnormalities in the progression pathway, has allowed refinements in diagnosis and prognosis.
... Some genomic alterations with potential clinical interest have been identified, such as the fusions KIAA1549-BRAF or FGFR3-TACC3 [120,121]. Angiogenesis may play a role in oncogenesis and prognosis, so antiangiogenic treatments also may been explored in this entity [122]. ...
Among all Soft Tissue sarcomas there are some subtypes with low incidence and/or peculiar clinical behaviour, that need to be consider separately. Most of them are orphan diseases, whose biological characteristics imply a clearly different diagnostic and therapeutic approach from other more common sarcoma tumors. We present a brief and updated multidiciplinary review, focused on practical issues, aimed at helping clinicians in decision making. In this second part we review these subtypes: Alveolar Soft Part Sarcoma, Epithelioid Sarcoma, Clear Cell Sarcoma, Desmoplastic Small Round Cell Tumor, Rhabdoid Tumor, Phyllodes Tumor, Tenosynovial Giant Cell Tumors, Myoepithelial Tumor, Perivascular Epithelioid Cell Neoplasms (PEComas), Extraskeletal Myxoid Chondrosarcoma, NTRK-fusions Sarcomas. Most of them present their own radiological and histopathological feautures, that are essential to know in order to achieve early diagnosis. In some of them, molecular diagnosis is mandatory, not only in the diagnosis, but also to plan the treatment. On the other hand, and despite the low incidence, a great scientific research effort has been made to achieve new treatment opportunities for these patients even with approved indications. These include new treatments with targeted therapies and immunotherapy, which today represent possible therapeutic options. It is especially important to be attentive to new and potential avenues of research, and to promote the conduct of specific clinical trials for rare sarcomas.
... Piscuoglio et al. also showed that FAs harbored MED12 exon 2 somatic mutations significantly more frequently than MPTs and concluded that the majority of MPTs might be driven by other genetic/epigenetic alterations [50]. There was an obvious difference in the frequency of TERT mutations between MPTs and FAs, and TERT promoter hotspot mutations might play a role in the progression from the beginning to MPTs [51,52]. In our study, 23 (23/50, 54%) MPT patients had a history of FA relapse, and the high proportion of recurrences of these patients indicates that MPT patients with a history of FAs should be subject to periodic reexaminations. ...
Purpose
The potential recurrence rate of malignant phyllodes tumors (MPTs) of the breast is high, and the prognostic factors are still unclear. We therefore aim to study the factors affecting the outcome of MPTs.
Methods
A retrospective review of MPT patients treated from 2006 to 2020 at our institution was conducted. Univariate and multivariate Cox proportional hazard models were used to examine the influence of different variables on RFS. Moreover, significant prognostic factors were combined to construct the nomogram to predict the probability of relapse occurring in MPT patients. The 5-year and 10-year RFS rates were estimated using the Kaplan–Meier method.
Results
During the study period, 188 MPT patients were identified. The presence of malignant heterologous elements was observed in 23 (12.2%) patients with MPT, and the patients with malignant heterologous elements who received chemotherapy had longer RFS, which could reduce the risk of recurrence (p=0.022). Recurrence occurred in 56/188 (29.8%) patients, of whom 47 experienced local recurrence and 11 experienced distant metastases. The 5-year and 10-year cumulative RFS rates were 77.5% and 70.1%, respectively. Age (p = 0.041), fibroadenoma surgery history (p = 0.004), surgical margins (p =0.001) and malignant heterologous elements (p<0.001) were independent risk factors for postoperative RFS. Subsequently, a nomogram was built, with a C-index of 0.64 (95% CI: 0.629-0.661), to predict the risk of recurrence.
Conclusion
The results of this study showed that younger age, fibroadenoma surgery history, malignant heterologous elements and surgical margins <1 cm predict a higher incidence of recurrence in MPT patients. Patients with malignant heterologous elements treated with chemotherapy could have a reduced risk of recurrence.
... KMT2D (9.7%), CREBBP (9.7%), PIK3CA (9.1%), STK11 (8.6%) and TERT (8.6%); the most common CNAs were homozygous deletions of CDKN2A (10.2%) and CDKN2B (9.7%) and amplifications of CCNE1 (4.3%), ERBB2 (4.3%) and MCL1 (4.3%) [164]. The MSK-IMPACT study reported alterations on the TERT promoter, which is associated with poor prognosis as a mechanism of apoptosis evasion [166,167]. ...
Simple Summary
Patients with cancer of unknown primary site suffer the burden of an uncertain disease, which is characterized by the impossibility to identify the tissue where the tumor has originated. The identification of the primary site of a tumor is of great importance for the patient to have access to site-specific treatments and be enrolled in clinical trials. Therefore, patients with cancer of unknown primary have reduced therapeutic opportunities and poor prognosis. Advancements have been made in the molecular characterization of this tumor, which could be used to infer the tumor site-of-origin and thus broaden the diagnostic outcome. Moreover, we describe here the novel therapeutic opportunities that are based on the genetic and immunophenotypic characterization of the tumor, and thus independent from the tumor type, which could provide most benefit to patients with cancer of unknown primary.
Abstract
Distant metastases are the main cause of cancer-related deaths in patients with advanced tumors. A standard diagnostic workup usually contains the identification of the tissue-of-origin of metastatic tumors, although under certain circumstances, it remains elusive. This disease setting is defined as cancer of unknown primary (CUP). Accounting for approximately 3–5% of all cancer diagnoses, CUPs are characterized by an aggressive clinical behavior and represent a real therapeutic challenge. The lack of determination of a tissue of origin precludes CUP patients from specific evidence-based therapeutic options or access to clinical trial, which significantly impacts their life expectancy. In the era of precision medicine, it is essential to characterize CUP molecular features, including the expression profile of non-coding RNAs, to improve our understanding of CUP biology and identify novel therapeutic strategies. This review article sheds light on this enigmatic disease by summarizing the current knowledge on CUPs focusing on recent discoveries and emerging diagnostic strategies.
... Tumor genomic analysis using the TruSight Oncology 500 Assay (Illumina, San Diego, CA) identified pathogenic variants in p53, mediator-12 (MED12), and the telomerase reverse transcription (TERT) gene promoter, all found commonly in MPTs. 1 No genetic alterations were identified in HIF1-α, other angiogenesisrelated genes, or downstream mitogen-activated protein kinase pathways. A variant of unknown significance within platelet-derived growth factor receptor-α (PDGFR-α) was identified that was not in the region of a known hotspot. ...
... While the complex EGFR V774_C775_insLM is has not been described hitherto, the other complex duplications have been detected in ISP [3,17]. The point mutations H773L and V774M have yet to be described in ISP, though detected in other tumor entities, including adenocarcinoma of the lung [18,19] and the point mutation V774M additionally in sebaceous carcinoma of the skin [20], anaplastic astrocytoma grade III [21], and phyllodes tumors of the breast [22]. The P794L and P772L mutations have also not been reported in ISP, albeit respectively discovered in cases of sebaceous adenoma [20] and adenocarcinoma of the lung [23]. ...
Inverted (Schneiderian) sinonasal papilloma (ISP) is a neoplasm derived from mucosa of the sinonasal tract characterized by local aggressive growth, a tendency to recur and an association with sinonasal carcinoma. The etiology of ISP remains unclear. Recently, identical mutations in exons 19 and 20 of the oncogene EGFR were reported in ISP and ISP-associated sinonasal carcinoma. Nevertheless, it remains unclear whether recurring ISPs show identical EGFR mutations at different time points or whether these mutations are identical throughout the respective ISP sample. We used Sanger sequencing to test 60 formalin-fixed paraffin embedded ISP samples from 40 patients regarding mutations in exons 19 and 20 of EGFR—together with exon 15 of BRAF. Overall, 32 samples of 22 patients showed a mutation in EGFR exon 20, whereas 28 samples of 18 patients showed none. No mutation in EGFR exon 19 was found in any sample. Four samples of four patients showed a BRAF exon 15 mutation. Interestingly, samples of four patients exhibited genetic heterogeneity, enabling us to report this in ISP for the first time.
... TERT amplifications play an important role in the diagnosis of various solid tumors, including breast phyllodes tumors, non-small cell lung cancer, and bladder cancer. TERT amplifications also predict a poor prognosis in breast cancer [40,41], bladder cancer [42], non-small cell lung cancer [43], and acral-lentiginous melanoma [44]. ...
The telomerase reverse transcriptase (TERT) gene, which is repressed in most differentiated human cells, can be reactivated by somatic TERT alterations and epigenetic modulations. Moreover, the recruitment, accessibility, and binding of transcription factors also affect the regulation of TERT expression. Reactivated TERT contributes to the development and progression of cancer through telomere lengthening-dependent and independent ways. In particular, because of recent advances in high-throughput sequencing technologies, studies on genomic alterations in various cancers that cause increased TERT transcriptional activity have been actively conducted. TERT reactivation has been reported to be associated with poor prognosis in several cancers, and TERT promoter mutations are among the most potent prognostic markers in thyroid cancer. In particular, when a TERT promoter mutation coexists with the BRAFV600E mutation, these mutations exert synergistic effects on a poor prognosis. Efforts have been made to uncover the mechanisms of these synergistic interactions. In this review, we discuss the role of TERT reactivation in tumorigenesis, the mechanisms of TERT reactivation across all human cancers and in thyroid cancer, and the mechanisms of interactions between BRAFV600E and TERT promoter mutations.
... Yet it is uncertain whether these vaccines will progress to bring authorization for clinical application or not. Approximately 90% telomerase in different malignant cells is reactivated by following steps: i) elevation of hTERT copy numbers, ii) amplification of hTERT gene mainly causing various cancers of thyroid, skin, breast and drug resistance, iii) enhanced TERT protein translation, iv) TERT gene rearrangement and v) several point mutations in TERT gene correlated with poor clinical diagnosis of glioblastoma multiforme, merkel cell carcinoma and phyllodes tumors [50,140,141]. Due to telomerase reactivation, combination therapy that targets both telomerase and antioxidant pathways might be novel therapeutic strategy in the future. ...
Telomerase plays a significant role to maintain and regulate the telomere length, cellular immortality and senescence by the addition of guanine-rich repetitive sequences. Chronic inflammation or oxidative stress-induced infection downregulates TERT gene modifying telomerase activity thus contributing to the early steps of gastric carcinogenesis process. Furthermore, telomere-telomerase system performs fundamental role in the pathogenesis and progression of diabetes mellitus as well as in its vascular intricacy. The cessation of cell proliferation in cultured cells by inhibiting the telomerase activity of transformed cells renders the rationale for culling of telomerase as a target therapy for the treatment of metabolic disorders and various types of cancers. In this article, we have briefly described the role of immune system and malignant cells in the expression of telomerase with critical analysis on the gaps and potential for future studies. The key findings regarding the secrets of the telomerase summarized in this article will help in future treatment modalities for the prevention of various types of cancers and metabolic disorders notably diabetes mellitus.
Background
Phyllodes tumors (PTs), which account for less than 1% of mammary gland tumors, composed of both epithelial and stromal components. If a malignant heterologous component is encountered, PT is considered malignant. Malignant phyllodes tumors (MPTs) only account for 8% to 20% of PTs. We report a case of MPT with osteosarcoma and chondrosarcoma differentiation and review the literature to discuss the differential diagnosis and therapy.
Case presentation
A 59-year-old Chinese woman come to our hospital because of a palpable mass she had had for 1 months in the left breast. Preoperative core needle biopsy (CNB) was performed on the left breast mass on January 11, 2023. Pathological diagnosis was malignant tumor, the specific type was not clear. Mastectomy and sentinel lymph node biopsy of the left breast was performed. No metastasis was found in 3 sentinel lymph nodes identified by carbon nanoparticles and methylene blue double staining. Heterologous osteosarcoma and chondrosarcomatous differentiation of phyllodes tumor were observed. Immunohistochemistry: spindle tumor cells ER(-), PR(-), HER-2(-), CK-pan(-), CK7(-), CK8(-), SOX10(-), S100(-), and MDM2(-), CK5/6(-), P63(-), P40(-) were all negative. CD34:(+), SATB2(+), P53(90% strong), CD68 (+), Ki-67(LI: about 60%). No ductal carcinoma in situ was found in the breast. Fluorescence in situ hybridization (FISH) indicated USP6 was negatively expressed on formalin-fixed, paraffin-embedded (FFPE) tissue sections.
Conclusion
MPTs are rare, and heterologous differentiation in MPTs is exceedingly rare. It could be diagnosed by pathology when metaplastic carcinoma, primary osteosarcoma, or myositis ossificans were excluded. This case could help clinicians to improve the prognosis and treatment of this disease.
Introduction: Solitary fibrous tumor (SFT) is a fibroblastic tumor with malignant potential that is underpinned by a recurrent inv12(q13q13)-derived NAB2::STAT6 fusion. Breast and axilla are uncommon locations for this entity. Methods: Records of two academic institutions were electronically searched for breast and axillary SFTs. Clinical and pathologic data were reviewed. Literature review for breast or axillary SFTs was performed. Present study and previously reported tumors were stratified using five SFT risk models: original and modified Demicco metastatic risk, Salas local recurrence risk, Salas metastatic risk, and Thompson local recurrence risk. Results: Five patients with breast or axillary SFT were identified. Median age was 49 years, and median follow-up (available for four patients) was 82 months. Three patients showed no evidence of disease, and one developed recurrence. Literature review identified 58 patients with breast or axillary SFT. Median age was 54 years, and median follow-up (available for 35 patients) was 24 months. Thirty-one patients showed no evidence of disease, three developed recurrence, and one developed metastasis. Original and modified Demicco models and Thompson model showed the highest sensitivity; original and modified Demicco models and Salas metastatic risk model demonstrated the highest specificity. Kaplan–Meier models were used to assess recurrence-free probability (RFP). Original and modified Demicco models predicted RFP when stratified by “low risk” and “moderate/intermediate and high risk” tumor, though sample size was small. Conclusions: While many SFTs of breast and axilla remain indolent, a subset may develop recurrence and rarely metastasize. The modified Demicco risk model demonstrated optimal performance characteristics.
A 47-year-old woman was found to have a 4-cm-diameter mass that was palpable at the outer area in the right breast. The mass has been present since adolescence and has shrunk slightly. Mammary gland echography revealed a lobulated multinodular mass, and the echo density of the tumor distal to the nipple was lower than that of other areas. Breast contrast-enhanced MRI revealed an early enhancement in the tumor distal to the nipple. Based on a needle biopsy, the patient was diagnosed with phyllodes tumor with low-grade ductal carcinoma in situ (DCIS). Since the tumor extended to the subareolar area, breast-concerving surgery with resection of the areola tissue directly above the tumor were performed. The pathological diagnosis was fibroadenoma phyllodes with invasive ductal carcinoma and adenomyoepithelioma. The intraductal components similar to a low-grade DCIS extended into the fissure of the phyllodes-like foliate structure and formed an infiltrated area, partly extending outside the tumor. The resected margins were negative. Postoperative whole-breast irradiation was also performed.
In this case, while the fibroadenoma changed over the long-term course, phyllodes-like foliate structure, epithelial cancerization, and myoepithelial proliferation were noted in the interstitium. This case provides an interesting insight on the histogenesis of fibroadenoma and phyllodes.
Fibroepithelial lesions of the breast encompass a broad spectrum of lesions from fibroadenomas and their variants to phyllodes tumors, including their clinical range of benign, borderline, and malignant. Classification of this spectrum of neoplasms has historically and currently been based purely on morphology, although the nomenclature has shifted over the years largely due to the significant histologic overlap that exists primarily within the cellular fibroadenomas to borderline malignant phyllodes tumor categories. A review of the current diagnostic challenge, proposed ancillary studied and their value in prognostic significance, is provided. This article highlights the most recent molecular and genetic findings as well as the limitations of the studies, in the context of practical and available applications for the diagnostician and managerial implications for the clinician.
Phyllodes tumours of the breast are uncommon fibroepithelial neoplasms that pose recurrent classification challenges, in large part due to the multiple histological parameters of stromal hypercellularity and atypia, stromal mitotic count, stromal overgrowth and tumour borders, that are used for grading. While the World Health Organization (WHO) Classification of Breast Tumours provides recommendations on diagnostic features, defining criteria are not always applied in routine practice. Lack of concordance among pathologists in typing and grading further underscores the classification difficulties, especially in the borderline category. Although there has been significant molecular information on phyllodes tumours in recent years which has been diagnostically helpful, it has not been translated into daily clinical practice. In order to refine the classification of phyllodes tumours into one that is simple yet comprehensive, reproducible and prognostically precise, a multipronged approach is needed that leverages on global contributions of the International Fibroepithelial Consortium, support by the International Collaboration on Cancer Classification and Research (IC3 R) in amalgamating evidence translation, and guidance from the International Collaboration on Cancer Reporting (ICCR) for standardised reporting. It is hoped that the evidence generated can be used towards refining the classification of phyllodes tumours for the future.
Context.—:
Low-grade fibromatosis-like metaplastic carcinoma (FLMC) is a very rare subtype of triple-negative metaplastic (spindle cell) breast carcinoma. It is characterized by the proliferation of spindle cells closely resembling fibromatosis, which represents a benign fibroblastic/myofibroblastic breast proliferation. Unlike most triple-negative and basal-like breast cancers, FLMC has a very low potential for metastases, but demonstrates frequent local recurrences.
Objective.—:
To genetically characterize FLMC.
Design.—:
To this end, we analyzed 7 cases by targeted next-generation sequencing for 315 cancer-related genes and performed comparative microarray copy number analysis in 5 of these cases.
Results.—:
All cases shared TERT alterations (6 patients with recurrent c.-124C>T TERT promoter mutation and 1 patient with copy number gain encompassing the TERT locus), had oncogenic PIK3CA/PIK3R1 mutations (activation of the PI3K/AKT/mTOR pathway), and lacked mutations in TP53. TERT was overexpressed in all FLMCs. CDKN2A/B loss or mutation was observed in 4 of 7 cases (57%). Furthermore, tumors displayed chromosomal stability with only few copy number variations and a low tumor mutational burden.
Conclusions—:
We conclude that FLMCs typically show the recurrent TERT promoter mutation c.-124C>T, activation of the PI3K/AKT/mTOR pathway, low genomic instability, and wild-type TP53. In conjunction with previous data of metaplastic (spindle cell) carcinoma with and without fibromatosis-like morphology, FLMC is most likely distinguished by TERT promoter mutation. Thus, our data support the notion of a distinct subgroup within low-grade metaplastic breast cancer with spindle cell morphology and associated TERT mutations.
Background:
Phyllodes tumor (PT) is a relatively rare breast tumor, accounting for <1% of all breast tumors.
Main body:
Adjuvant therapy with chemotherapy or radiation therapy, other than surgical excision, has not been established yet. PT, similar to other breast tumors, is classified as benign, borderline, and malignant according to the World Health Organization classification system, depending on stromal cellularity, stromal atypia, mitotic activity, stromal overgrowth, and tumor border. However, this histological grading system cannot effectively or fully reflect the clinical prognosis of PT. Several studies have investigated prognostic factors for PT as some PTs recur or metastasize to distant sites, and thus, prediction of prognosis is clinically imperative.
Conclusion:
This review discusses clinicopathological factors, immunohistochemical markers, and molecular factors that have been investigated in previous studies to have an impact on the clinical prognosis of PT.
The molecular pathogenesis of breast fibroepithelial tumors continues to be elucidated. Recently, highly recurrent MED12 mutations arising in exon 2 at codon 44 were discovered in fibroadenomas and phyllodes tumors. In addition, a high prevalence of TERT promoter mutations in two hotspots (124 and 126 bp upstream from the translation start site) was discovered in up to 65% of phyllodes tumors. Breast periductal stromal tumors are a potentially distinct category of fibroepithelial lesions that are exceptionally rare with controversial classification and pathogenesis. Herein, we report the first comprehensive molecular genetic workup of a breast periductal stromal tumor that harbored a TERT promoter -124C > T mutation, supporting a relation to phyllodes tumors.
Aims:
Breast phyllodes tumours (PT) are a rare subset of fibroepithelial neoplasms categorised into benign, borderline, and malignant grades according to the World Health Organization (WHO) Classification of Tumours (WCTs). In this report, we developed an evidence gap map (EGM) based on literature cited in the PT chapter of the 5th edition of the breast WCT in order to identify knowledge and research gaps in PT.
Methods:
A framework was first established where the dimensions of the EGM were defined as categories of tumour descriptors, tumour types and evidence levels. Citations were collected into a Microsoft Excel® form and imported into EPPI-reviewer to produce the EGM.
Results:
The EGM showed that "Histopathology" and "Pathogenesis" sections contained the most citations, the majority being of low-level evidence. The highest number of citations considered of moderate-level evidence were found in the "Histopathology" section. There was no high-level evidence cited in this chapter. "Localization", "Etiology" and "Staging" sections had the fewest citations.
Conclusions:
This EGM provides a visual representation of cited literature in the PT chapter of the breast WCT, revealing the lack of high-level evidence citations. There is an uneven distribution of references, probably due to citation practices. Pockets of low-level evidence are highlighted, possibly related to referencing habits, lack of relevant research, or belief that the information presented is standard accepted fact without need for specific citations. Future work needs to bridge evidence gaps and broaden citations beyond those in the latest WCT.
Background:
Breast phyllodes tumours (PTs) are graded as benign, borderline, or malignant by analysis of multiple histological features. PT grading is often inconsistent, likely due to variation in the weighting of grading criteria by pathologists.
Design:
The hierarchy of use of diagnostic criteria was identified using a 20-question survey.
Results:
In all, 213 pathologists from 29 countries responded. 54% reported 10-50 PT cases per year. Criteria considered key to PT diagnosis were: increased stromal cellularity (84.3%), stromal overgrowth (76.6%), increased stromal mitoses (67.8%), stromal atypia (61.5%), stromal fronding (59.0%), periductal stromal condensation (58.0%), irregular tumour borders (46.3%), and/or lesional heterogeneity (33.7%). The importance of grading parameters were: mitotic activity (55.5%), stromal overgrowth (54.0%), stromal atypia (51.9%), increased stromal cellularity (41.7%), and nature of the tumour border (38.9%). 49% would diagnose malignant PT without a full array of adverse features. 89% used the term "cellular fibroepithelial lesion (FEL)" for difficult cases; 45% would diagnose an FEL with stromal fronding (but lacking other PT features) as fibroadenoma (FA), 35% FEL, and 17% PT. 59% deemed clinico-radiological findings diagnostically significant; 68% considered age (≥40 years) important in determining if an FEL was a FA or PT. In FELs from young patients, increased stromal cellularity (83%), fronding (52%), and mitoses (41%) were more common. 34% regarded differentiating cellular FA from PT as a specific challenge; 54% had issues assigning a borderline PT grade.
Conclusion:
Criteria for grading PT lie on a spectrum, leading to interpretive variability. The survey highlights the criteria most used by pathologists, which do not completely align with WHO recommendations.
This chapter encompasses seven uncommon types or patterns of triple-negative breast cancer, namely adenoid cystic carcinoma (AdCC), mucoepidermoid carcinoma (MEC), secretory carcinoma, tall cell carcinoma with reserved polarity (TCCRP), carcinoma with apocrine differentiation, metaplastic carcinoma, and invasive breast carcinoma with medullary pattern. Also included in the discussion for differential diagnosis is the rare subtype acinic cell carcinoma and invasive breast carcinoma with oncocytic pattern. Most of these tumors are basal-like by gene expression and/or immunophenotypic analysis. Although sharing the triple-negative phenotype, these tumors demonstrate remarkably different clinical behaviors, which range along a spectrum from indolent to aggressive, emphasizing the notion that triple-negative and basal-like breast cancers comprise a heterogeneous group. Whereas AdCC, MEC, secretory carcinoma, and TCCRP have well-defined histopathologic features and unique molecular alterations among breast tumors, metaplastic carcinomas represent a morphologically and genetically diverse group of tumors. Due to poor diagnostic reproducibility, similarity to basal-like and BRCA1-associated breast cancers, and outcomes that are likely related to prominent inflammation more so than other histologic features, tumors previously classified as medullary carcinomas or invasive carcinomas with medullary features are now considered a morphologic pattern of invasive breast carcinoma of no special type. Diagnosis of these uncommon triple-negative tumors can be challenging, especially in core needle biopsy specimens.
Fibroepithelial tumors of the breast are a heterogeneous group of biphasic neoplasms with stromal and epithelial components and widely variable clinical behavior, ranging from benign and harmless to frankly malignant with risk of death. Fibroadenomas are benign and are the most prevalent fibroepithelial tumors, being commonly encountered in core needle biopsies and excision specimens. Phyllodes tumors are far less common and comprise a spectrum of neoplasms ranging from benign to malignant, with variable recurrence and metastatic risk. Diagnosis and grading of these tumors can be challenging, and clinical management is controversial and evolving. Periductal stromal tumors are rare phyllodes tumor variants. Lastly, mammary hamartomas are benign epithelial and stromal proliferations that may resemble fibroepithelial tumors.KeywordsFibroepithelialFibroadenomaPhyllodes tumorPeriductal stromal tumorHamartoma
Phyllodes tumour (PT) of breast is a rare biphasic neoplasm. Recent next generation sequencing analyses had revealed novel genetic alterations in PT but lacked a further characterisation of their relationship to different PT features and outcome. Here, using targeted sequencing, we examined a panel of 90 recurrently altered or cancer related genes in 88 PT samples (including 49 benign, 25 borderline and 14 malignant PT). Twenty-three genes showed alterations in at least 8.0% of cases. Alterations were significantly higher with an increasing grade of PT (p=0.033), particularly for copy number alterations. The top ten alterations were TERT promoter (58.0%), MED12 (53.4%), RARA (22.8%), FLNA (19.3%), SETD2 (15.9%), SYNE1 (18.2%), PCLO (15.9%), KMT2D (14.3%), CDKN2A (15.9%) and DNAH11 (14.8%). Alterations in CDKN2A/B, EGFR, TP53, PIK3CA, PTEN and ARID1B (p≤0.039) were associated with a higher grade. Analysing alterations based on common pathways indicated a significant correlation of cell cycle pathway and epigenetic alterations with a higher PT grade (p=0.036 and 0.075 respectively). Interestingly, recurrences were not correlated with tumour grade, but related to the presence of RARA mutation (p=0.011) and the absence of alterations in epigenetic pathway (p=0.031). Analysis of synchronous pair of PT showed more differences in gene mutations with divergent MED12 mutation. By contrast, the recurrent samples showed similar genetic alterations as the primary tumours. In summary, we characterised genetic alterations in PTs of different grades and confirmed the recurrent alterations observed in earlier studies. In addition, current data implicated the roles of cell cycle, epigenetic and RARA changes in PT recurrence and tumourogenesis.
Breast fibroepithelial lesions, which are composed of biphasic epithelial and stromal proliferations, comprise the fascinating spectrum of fibroadenomas and phyllodes tumours. Common difficulties surrounding their diagnosis include distinguishing between cellular fibroadenomas and benign phyllodes tumours, grading phyllodes tumours, classifying fibroepithelial lesions in paediatric patients, and handling surgical margins. Recent molecular advances have provided insights into the pathogenesis of fibroepithelial lesions and may offer diagnostic, prognostic, and therapeutic information. In this review, we briefly revisit the pathological features of fibroadenomas and phyllodes tumours, discuss common diagnostic dilemmas and management implications, and examine their key molecular characteristics.
Telomeres are specialized nucleoprotein structures localized at the ends of eukaryotic chromosomes. Telomere biology is frequently associated with human cancers where dysfunctional telomeres have been proved to participate in genetic instability. Due to end replication problem, there is a loss of nucleotides from telomere after every cell division. Thus, after limited divisions, normal somatic cells cease to divide and undergo senescence and/or apoptosis. This inability of normal cells to continually proliferate is bypassed in immortal or cancer cells via reactivation of telomerase. Telomerase is a highly specialized ribonucleoprotein molecule that promotes addition of telomeric repeats at the 3′ end of the chromosome. Multiple disease-specific studies have unveiled involvement of various genetic and epigenetic mechanisms in regulation of telomerase expression. Further, selective expression of telomerase in cancer cells in comparison with normal somatic cells makes telomerase a suitable therapeutic target. Thus, large numbers of natural and artificial compounds targeting telomerase have been screened in multiple cancers. This chapter focuses on the telomerase structure, function, and its therapeutic implications in cancer.KeywordsTelomereTelomeraseCancerTherapeutics
Phyllodes tumour (PT) is a rare fibroepithelial neoplasm of the breast, histologically categorized into 3 grades, namely benign, borderline and malignant; each with distinct clinical behaviour and implications for management. This article addresses challenges in the diagnosis, grading and management of PT in excisional and core needle biopsy specimens, with a brief review of recent molecular findings.
The genetic alterations in the recurrent breast fibroepithelial tumors are poorly understood. In the present study, we aimed to investigate mediator protein complex subunit 12 (MED12) exon 2 and telomerase reverse transcriptase (TERT) promoter mutations in a series of primary and recurrent fibroepithelial tumors. Sanger sequencing for MED12 exon 2 and TERT promoter was performed in 26 pairs of primary and recurrent fibroepithelial tumors (19 pairs of phyllodes tumors and seven pairs of fibroadenomas). The relationship between the genotypes and clinicopathological variables was also analyzed. MED12 mutation was identified in 19 primary tumors (12 phyllodes tumors and 7 fibroadenomas) and 17 recurrences (14 phyllodes tumors and three fibroadenomas). Most recurrent phyllodes tumors retained the original MED12 variants (17/19). Six recurrent fibroadenomas showed different MED12 variants from their paired primary tumors (6/7). TERT promoter mutation was identified in 13 primary phyllodes tumors (13/19) and 15 recurrent phyllodes tumors (15/19). However, it was only identified in one primary fibroadenoma (1/7). Recurrent phyllodes tumors often retained the original MED12 and TERT promoter mutations, while recurrent fibroadenomas often acquired new MED12 mutations. Our findings suggest that recurrent phyllodes tumors may be “true recurrence,” and TERT mutant “benign fibroepithelial tumors” should be treated as phyllodes tumors.
Aims:
Fibromatosis-like spindle cell carcinomas (FLSCCs) are rare metaplastic breast cancers (MBCs) that are characterised by bland spindle cells in a collagenous stroma. Although some MBCs are highly malignant, FLSCCs have indolent behaviour with low potential for lymph node or distant metastasis. Owing to their rarity, there are limited genomic data on FLSCCs. In this study, we analysed the clinicopathological features and molecular characteristics of four FLSCCs to elucidate the pathogenesis of these rare tumours.
Methods and results:
Four pure FLSCCs were sequenced by DIAN (Hangzhou Lab) using a 324-gene platform (FoundationOne CDx) with licensed technologies. The results showed that most FLSCCs harboured the pathogenic H1047R mutation in PIK3CA (3/4, 75%) and the -124C>T mutation in the telomerase reverse transcriptase (TERT) promoter (3/4, 75%). No copy number variations were observed in any cases in our study.
Conclusions:
Our study showed that PIK3CA and TERT promoter mutations were common genetic features of FLSCCs. These findings contribute to our understanding of FLSCCs biology.
SETD2 encodes an important protein for epigenetic modification of histones which plays an essential role in early development. Variants in SETD2 have been reported in neurodevelopmental disorders including autism spectrum disorder (ASD). However, most de novo SETD2 variants were reported in different large-cohort sequencing studies, mutation pattern and comprehensive genotype-phenotype correlations for SETD2 are still lacking. We have applied target sequencing to identify rare, clinical-relevant SETD2 variants and detected two novel de novo SETD2 variants, including a de novo splicing variant (NM_014159: c.4715+1G>A) and a de novo missense variant (c.3185C>T: p.P1062L) in two individuals with a diagnosis of ASD. To analyze the correlations between SETD2 mutations and corresponding phenotypes, we systematically review the reported individuals with de novo SETD2 variants, classify the pathogenicity, and analyzed the detailed phenotypes. We subsequently manually curate 17 SETD2 de novo variants in 17 individuals from published literature. Individuals with de novo SETD2 variants present common phenotypes including speech and motor delay, intellectual disability, macrocephaly, ASD, overgrowth and recurrent otitis media. Our study reveals new SETD2 mutations and provided a relatively homozygous phenotype spectrum of SETD2-related neurodevelopmental disorders which will be beneficial for disease classification and diagnosis in clinical practice.
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.
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.
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.
ddPCR was more sensitive for detecting TERTp mutation than Sanger sequencing, being able to elucidate tumorigenesis in FA and PT.
Purpose
Phyllodes tumors (PTs) are biphasic tumors accounting for 0.3–1.5% of all breast tumors. Epithelial membrane proteins (EMPs) have been reported in various malignant tumors but their expression in PTs is unclear. In this study, we aimed to evaluate the expression of EMP1, EMP2, and EMP3 in breast phyllodes tumors (PTs), and to investigate their clinical implications.
Methods
In total, 185 PTs were used for constructing a tissue microarray. Immunohistochemical staining for EMP1, EMP2, and EMP3 was performed, and the results were analyzed along with the clinicopathologic parameters.
Results
In total, 185 PTs were included in this study, and comprised 138 benign, 32 borderline, and 15 malignant PTs. In malignant PTs, the epithelial component showed decreased expression of EMP1 (P = 0.027), EMP2 (P = 0.004), and EMP3 (P = 0.032), compared to the benign and borderline PTs. Conversely, stromal component of borderline and malignant PTs showed higher expression of EMP1 (P = 0.027), EMP2 (P = 0.004), and EMP3 (P = 0.032) compared to benign PTs. Expression of EMP1 and EMP3 correlated positively with stromal cellularity and cellular atypia (P < 0.001). In the univariate analysis, stromal EMP3 was associated with shorter disease-free survival (P < 0.001), and shorter overall survival (P = 0.034).
Conclusion
The expression of EMP1, EMP2, and EMP3 is decreased in the epithelial component and is increased in the stromal component of PT with higher histologic grade. Thus, stromal EMP3 expression may serve as an independent prognostic factor in PT.
Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells.
Breast fibroepithelial tumors comprise a heterogeneous spectrum of pathological entities, from benign fibroadenomas to malignant phyllodes tumors. Although MED12 mutations have been frequently found in fibroadenomas and phyllodes tumors, the landscapes of genetic alterations across the fibroepithelial tumor spectrum remain unclear. Here, by performing exome sequencing of 22 phyllodes tumors followed by targeted sequencing of 100 breast fibroepithelial tumors, we observed three distinct somatic mutation patterns. First, we frequently observed MED12 and RARA mutations in both fibroadenomas and phyllodes tumors, emphasizing the importance of these mutations in fibroepithelial tumorigenesis. Second, phyllodes tumors exhibited mutations in FLNA, SETD2 and KMT2D, suggesting a role in driving phyllodes tumor development. Third, borderline and malignant phyllodes tumors harbored additional mutations in cancer-associated genes. RARA mutations exhibited clustering in the portion of the gene encoding the ligand-binding domain, functionally suppressed RARA-mediated transcriptional activation and enhanced RARA interactions with transcriptional co-repressors. This study provides insights into the molecular pathogenesis of breast fibroepithelial tumors, with potential clinical implications.
Background:
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.
Methods:
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.
Results:
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.
Conclusions:
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.British Journal of Cancer advance online publication, 10 September 2015; doi:10.1038/bjc.2015.326 www.bjcancer.com.
Fifteen per cent of cancers maintain telomere length independently of telomerase by the homologous recombination (HR)-associated alternative lengthening of telomeres (ALT) pathway. A unifying feature of these tumours are mutations in ATRX. Here we show that expression of ectopic ATRX triggers a suppression of the pathway and telomere shortening. Importantly ATRX-mediated ALT suppression is dependent on the histone chaperone DAXX. Re-expression of ATRX is associated with a reduction in replication fork stalling, a known trigger for HR and loss of MRN from telomeres. A G-quadruplex stabilizer partially reverses the effect of ATRX, inferring ATRX may normally facilitate replication through these sequences that, if they persist, promote ALT. We propose that defective telomere chromatinization through loss of ATRX promotes the persistence of aberrant DNA secondary structures, which in turn present a barrier to DNA replication, leading to replication fork stalling, collapse, HR and subsequent recombination-mediated telomere synthesis in ALT cancers.
Small thyroid papillary carcinomas contribute to the worldwide increased incidence of differentiated thyroid cancer observed over the past decades. However, mortality rate did not change over the same period of time, raising questions about the possibility that thyroid cancer patients, especially those with small tumors, are overdiagnosed and overtreated. Molecular prognostic marker able to discriminate aggressive thyroid cancers from those with an indolent course would be of great relevance to better tailor the therapeutic approach and reduce overtreatment. Mutations in the TERT promoter were recently reported to strongly correlate with aggressiveness in advanced forms of thyroid cancer, holding promise for a possible clinical application. The occurrence and potential clinical relevance of TERT mutations in thyroid papillary microcarcinomas (mPTCs) is currently unknown. Here, we aimed to analyze the occurrence of two TERT promoter mutations (-124C>T and -146C>T) and their potential association with unfavorable clinical features in a large cohort of mPTCs.
A total of 431 mPTCs cases were collected from 6 Italian Institutions and TERT promoter mutational status was assessed by a next-generation sequencing approach.
TERT promoter mutations were found in 4.7% of the analyzed mPTCs, showing that even microcarcinomas carry mutations in this gene. Correlation analysis showed that TERT promoter mutations are not associated with aggressive features or clinical outcome in the cohort analyzed.
TERT mutations are present but uncommon in mPTCs. Apparently, in mPTCs the occurrence of TERT mutations is not correlated with unfavorable clinical features.
Bladder cancer is one of the most common malignant tumors worldwide. Bladder cancer stem cells (BCSCs) have been isolated recently but have not been defined yet. Here we sorted BCSCs from bladder tumor tissues or normal bladder stem cells (NBBCs) from adjacent normal bladder tissues. We found that the C228T mutation (chr5, 1, 295, 228 C > T) of TERT promoter frequently occurs in BCSCs, but not exist in NBBCs. Importantly, introducing the C228T mutation in NBBCs causes TERT overexpression and transformation of bladder cancer. Restoration of the C228T mutation to T228C in BCSCs can recover the TERT expression to a basal level and abolish tumor formation. Additionally, the C228T mutation of TERT promoter triggers TERT expression leading to increased telomerase activity. TERT expression levels are consistent with clinical severity and prognosis of bladder cancer.
The unlimited proliferation of cancer cells requires a mechanism to prevent telomere shortening. Alternative Lengthening of Telomeres (ALT) is an homologous recombination-mediated mechanism of telomere elongation used in tumors, including osteosarcomas, soft tissue sarcoma subtypes, and glial brain tumors. Mutations in the ATRX/DAXX chromatin remodeling complex have been reported in tumors and cell lines that use the ALT mechanism, suggesting that ATRX may be an ALT repressor. We show here that knockout or knockdown of ATRX in mortal cells or immortal telomerase-positive cells is insufficient to activate ALT. Notably, however, in SV40-transformed mortal fibroblasts ATRX loss results in either a significant increase in the proportion of cell lines activating ALT (instead of telomerase) or in a significant decrease in the time prior to ALT activation. These data indicate that loss of ATRX function cooperates with one or more as-yet unidentified genetic or epigenetic alterations to activate ALT. Moreover, transient ATRX expression in ALT-positive/ATRX-negative cells represses ALT activity. These data provide the first direct, functional evidence that ATRX represses ALT.
The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in ~40% of genes, with the landscape dominated by cis- and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA–RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the ‘CNA-devoid’ subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome.
Aims
Somatic mutations in exon 2 of the MED12 gene have been identified in 60% of breast fibroadenomas (FAs). The aim of this study was to define whether phyllodes tumors (PTs) would harbor MED12 somatic mutations in a way akin to FAs.
Methods and results
A collection of 73 fibroepithelial tumors (including 26 FAs, 25 benign PTs, 9 borderline PTs and 13 malignant PTs) from 64 patients was retrieved from the authors’ institution. Sections from FFPE blocks were microdissected to ensure an enrichment in neoplastic stromal elements of >70%. DNA samples extracted from tumor 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 significantly less frequently harbored MED12 exon 2 somatic mutations than FAs, benign and borderline PTs.
Conclusions
Although MED12 exon 2 somatic mutations likely 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.
In this report on 303 gliomas we show the highest frequency of TERT promoter mutations in gliobastomas (80%) followed by oligodendrogliomas (70%) and astrocytomas (39%). We observed positive association between TERT promoter and IDH mutations in oligodendroglial tumors (OR = 26.3; 95% CI 2.5-250.2) and inverse association in primary glioblastomas (OR = 0.13; 95% CI 0.03-0.58). Tumors with TERT promoter mutations compared to those without showed increased TERT transcription; we also showed difference in the transcription levels due to the two main mutations. Tumors with TERT promoter mutations had shorter telomeres than those without. The patients with only TERT promoter mutations showed worst survival (median survival 14.6 months) and patients with both IDH and TERT promoter mutations showed best survival (246.5 months). In patients with astrocytoma, the TERT promoter mutations only associated with poor survival (P < 0.0001); IDH mutations and 1p/19q deletions associated with increased survival (P = 0.0004). TERT promoter mutations in low grade gliomas associated with reduced progression free survival (HR 10.2; 95% CI 1.9 - 55.9). While our data affirm the role of TERT promoter mutations in glial tumors, effects on transcription and telomere length emphasise the importance of telomere biology in disease genesis and outcome.
Reactivation of telomerase, the chromosome end-replicating enzyme, drives human cell immortality and cancer. Point mutations in the telomerase reverse transcriptase (TERT) gene promoter occur at high frequency in multiple cancers, including urothelial cancer (UC), but their effect on telomerase function has been unclear. In a study of 23 human UC cell lines, we show that these promoter mutations correlate with higher levels of TERT messenger RNA (mRNA), TERT protein, telomerase enzymatic activity, and telomere length. Although previous studies found no relation between TERT promoter mutations and UC patient outcome, we find that elevated TERT mRNA expression strongly correlates with reduced disease-specific survival in two independent UC patient cohorts (n = 35; n = 87). These results suggest that high telomerase activity may be a better marker of aggressive UC tumors than TERT promoter mutations alone.
Copyright © 2015, American Association for the Advancement of Science.
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 histological 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 (CNAs) 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. Implications: Integrated genomic sequencing and mutational profiling provides insight into the molecular origin of phyllodes tumors and indicates potential druggable targets in malignant disease.
Copyright © 2014, American Association for Cancer Research.
Massively parallel sequencing studies have led to the identification of a large number of mutations present in a minority of cancers of a given site. Hence, methods to identify the likely pathogenic mutations that are worth exploring experimentally and clinically are required. We sought to compare the performance of 15 mutation effect prediction algorithms and their agreement. As a hypothesis-generating aim, we sought to define whether combinations of prediction algorithms would improve the functional effect predictions of specific mutations.
Literature and database mining of single nucleotide variants (SNVs) affecting 15 cancer genes was performed to identify mutations supported by functional evidence or hereditary disease association to be classified either as non-neutral (n = 849) or neutral (n = 140) with respect to their impact on protein function. These SNVs were employed to test the performance of 15 mutation effect prediction algorithms. The accuracy of the prediction algorithms varies considerably. Although all algorithms perform consistently well in terms of positive predictive value, their negative predictive value varies substantially. Cancer-specific mutation effect predictors display no-to-almost perfect agreement in their predictions of these SNVs, whereas the non-cancer-specific predictors showed no-to-moderate agreement. Combinations of predictors modestly improve accuracy and significantly improve negative predictive values.
The information provided by mutation effect predictors is not equivalent. No algorithm is able to predict sufficiently accurately SNVs that should be taken forward for experimental or clinical testing. Combining algorithms aggregates orthogonal information and may result in improvements in the negative predictive value of mutation effect predictions.
Cancer primarily develops because of somatic alterations in the genome. Advances in sequencing have enabled large-scale sequencing studies across many tumor types, emphasizing the discovery of alterations in protein-coding genes. However, the protein-coding exome comprises less than 2% of the human genome. Here we analyze the complete genome sequences of 863 human tumors from The Cancer Genome Atlas and other sources to systematically identify noncoding regions that are recurrently mutated in cancer. We use new frequency- and sequence-based approaches to comprehensively scan the genome for noncoding mutations with potential regulatory impact. These methods identify recurrent mutations in regulatory elements upstream of PLEKHS1, WDR74 and SDHD, as well as previously identified mutations in the TERT promoter. SDHD promoter mutations are frequent in melanoma and are associated with reduced gene expression and poor prognosis. The non-protein-coding cancer genome remains widely unexplored, and our findings represent a step toward targeting the entire genome for clinical purposes.
Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA.
Fibroadenomas are the most common breast tumors in women under 30 (refs. 1,2). Exome sequencing of eight fibroadenomas with matching whole-blood samples revealed recurrent somatic mutations solely in MED12, which encodes a Mediator complex subunit. Targeted sequencing of an additional 90 fibroadenomas confirmed highly frequent MED12 exon 2 mutations (58/98, 59%) that are probably somatic, with 71% of mutations occurring in codon 44. Using laser capture microdissection, we show that MED12 fibroadenoma mutations are present in stromal but not epithelial mammary cells. Expression profiling of MED12-mutated and wild-type fibroadenomas revealed that MED12 mutations are associated with dysregulated estrogen signaling and extracellular matrix organization. The fibroadenoma MED12 mutation spectrum is nearly identical to that of previously reported MED12 lesions in uterine leiomyoma but not those of other tumors. Benign tumors of the breast and uterus, both of which are key target tissues of estrogen, may thus share a common genetic basis underpinned by highly frequent and specific MED12 mutations.
Background:
Plasma-derived cell-free tumor DNA (ctDNA) constitutes a potential surrogate for tumor DNA obtained from tissue biopsies. We posit that massively parallel sequencing (MPS) analysis of ctDNA may help define the repertoire of mutations in breast cancer and monitor tumor somatic alterations during the course of targeted therapy.
Patient and methods:
A 66-year-old patient presented with synchronous estrogen receptor-positive/HER2-negative, highly proliferative, grade 2, mixed invasive ductal-lobular carcinoma with bone and liver metastases at diagnosis. DNA extracted from archival tumor material, plasma and peripheral blood leukocytes was subjected to targeted MPS using a platform comprising 300 cancer genes known to harbor actionable mutations. Multiple plasma samples were collected during the fourth line of treatment with an AKT inhibitor.
Results:
Average read depths of 287x were obtained from the archival primary tumor, 139x from the liver metastasis and between 200x and 900x from ctDNA samples. Sixteen somatic non-synonymous mutations were detected in the liver metastasis, of which 9 (CDKN2A, AKT1, TP53, JAK3, TSC1, NF1, CDH1, MML3 and CTNNB1) were also detected in >5% of the alleles found in the primary tumor sample. Not all mutations identified in the metastasis were reliably identified in the primary tumor (e.g. FLT4). Analysis of ctDNA, nevertheless, captured all mutations present in the primary tumor and/or liver metastasis. In the longitudinal monitoring of the patient, the mutant allele fractions identified in ctDNA samples varied over time and mirrored the pharmacodynamic response to the targeted therapy as assessed by positron emission tomography-computed tomography.
Conclusions:
This proof-of-principle study is one of the first to demonstrate that high-depth targeted MPS of plasma-derived ctDNA constitutes a potential tool for de novo mutation identification and monitoring of somatic genetic alterations during the course of targeted therapy, and may be employed to overcome the challenges posed by intra-tumor genetic heterogeneity.
Registered clinical trial:
www.clinicaltrials.gov, NCT01090960.
The telomerase reverse transcriptase gene (TERT) encodes the reverse transcriptase component of the telomerase complex, which is essential for telomere stabilization and cell immortalization. Recent studies have demonstrated a transcriptional activation role for the TERT promoter mutations C228T and C250T in many human cancers, as well as a role in aggressive disease with potential clinical applications. Although telomerase activation is known in adrenal tumors, the underlying mechanisms are not established. We assessed C228T and C250T TERT mutations by direct Sanger sequencing in tumors of the adrenal gland, and further evaluated potential associations with clinical parameters and telomerase activation. A total of 199 tumors were evaluated, including 34 adrenocortical carcinomas (ACC), 47 adrenocortical adenomas (ACA), 105 pheochromocytomas (PCC; ten malignant and 95 benign), and 13 abdominal paragangliomas (PGL; nine malignant and four benign). TERT expression levels were determined by quantitative RT-PCR. The C228T mutation was detected in 4/34 ACCs (12%), but not in any ACA (P=0.028). C228T was also observed in one benign PCC and in one metastatic PGL. The C250T mutation was not observed in any case. In the ACC and PGL groups, TERT mutation-positive cases exhibited TERT expression, indicating telomerase activation; however, since expression was also revealed in TERT WT cases, this could denote additional mechanisms of TERT activation. To conclude, the TERT promoter mutation C228T is a recurrent event associated with TERT expression in ACCs, but rarely occurs in PGL and PCC. The involvement of the TERT gene in ACC represents a novel mutated gene in this entity.
The Cancer Genome Atlas (TCGA) has used the latest sequencing and analysis methods to identify somatic variants across thousands of tumours. Here we present data and analytical results for point mutations and small insertions/deletions from 3,281 tumours across 12 tumour types as part of the TCGA Pan-Cancer effort. We illustrate the distributions of mutation frequencies, types and contexts across tumour types, and establish their links to tissues of origin, environmental/carcinogen influences, and DNA repair defects. Using the integrated data sets, we identified 127 significantly mutated genes from well-known (for example, mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase, Wnt/β-catenin and receptor tyrosine kinase signalling pathways, and cell cycle control) and emerging (for example, histone, histone modification, splicing, metabolism and proteolysis) cellular processes in cancer. The average number of mutations in these significantly mutated genes varies across tumour types; most tumours have two to six, indicating that the number of driver mutations required during oncogenesis is relatively small. Mutations in transcriptional factors/regulators show tissue specificity, whereas histone modifiers are often mutated across several cancer types. Clinical association analysis identifies genes having a significant effect on survival, and investigations of mutations with respect to clonal/subclonal architecture delineate their temporal orders during tumorigenesis. Taken together, these results lay the groundwork for developing new diagnostics and individualizing cancer treatment.
Reactivation of telomerase has been implicated in human tumorigenesis, but the underlying mechanisms remain poorly understood. Here we report the presence of recurrent somatic mutations in the TERT promoter in cancers of the central nervous system (43%), bladder (59%), thyroid (follicular cell-derived, 10%) and skin (melanoma, 29%). In thyroid cancers, the presence of TERT promoter mutations (when occurring together with BRAF mutations) is significantly associated with higher TERT mRNA expression, and in glioblastoma we find a trend for increased telomerase expression in cases harbouring TERT promoter mutations. Both in thyroid cancers and glioblastoma, TERT promoter mutations are significantly associated with older age of the patients. Our results show that TERT promoter mutations are relatively frequent in specific types of human cancers, where they lead to enhanced expression of telomerase.
Introduction. Phyllodes tumours are rare fibroepithelial lesions. Accurate preoperative pathological diagnosis allows correct surgical planning and avoidance of reoperation. Treatment can be either wide local excision or mastectomy to achieve histologically clear margins. Discussion. The exact aetiology of phyllodes tumour and its relationship with fibroadenoma are unclear. Women aged between 35 and 55 years are commonly involved. The median tumour size is 4 cm but can grow even larger having dilated veins and a blue discoloration over skin. Palpable axillary lymphadenopathy can be identified in up to 10-15% of patients but <1% had pathological positive nodes. Mammography and ultrasonography are main imaging modalities. Cytologically the presence of both epithelial and stromal elements supports the diagnosis. The value of FNAC in diagnosis of phyllodes tumour remains controversial, but core needle biopsy has high sensitivity and negative predictive value. Surgical management is the mainstay and local recurrence in phyllodes tumours has been associated with inadequate local excision. The role of adjuvant radiotherapy and chemotherapy remains uncertain and use of hormonal therapy has not been fully investigated. Conclusion. The preoperative diagnosis and proper management are crucial in phyllodes tumours because of their tendency to recur and malignant potential in some of these tumours.
The molecular foundations of lower-grade gliomas (LGGs)-astrocytoma, oligodendroglioma, and oligoastrocytoma-remain less well characterized than those of their fully malignant counterpart, glioblastoma. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) likely represent initiating pathogenic events. However, while IDH mutations appear to dramatically alter cellular epigenomic landscapes, definitive downstream transformative mechanisms have not been characterized. It remains likely, therefore, that additional genomic abnormalities collaborate with IDH mutation to drive oncogenesis in LGG. We performed whole exome sequencing in 4 LGGs, followed by focused resequencing in an additional 28, and found a high incidence of mutations in the ATRX gene (α thalassemia/mental retardation syndrome X-linked). ATRX forms a core component of a chromatin remodeling complex active in telomere biology. Mutations in ATRX have been identified in multiple tumor types and appear to cause alternative lengthening of telomeres (ALT), a presumed precursor to genomic instability. In our samples, ATRX mutation was entirely restricted to IDH-mutant tumors, closely correlated with TP53 mutation and astrocytic differentiation, and mutually exclusive with 1p/19q codeletion, the molecular hallmark of oligodendroglioma. Moreover, ATRX mutation was highly enriched in tumors of so-called early progenitor-like transcriptional subclass (~85%), which our prior work has linked to specific cells of origin in the forebrain subventricular zone. Finally, ATRX mutation correlated with ALT, providing a mechanistic link to genomic instability. In summary, our findings both identify ATRX mutation as a defining molecular determinant for a large subset of IDH-mutant gliomas and have direct implications on pathogenic mechanisms across the wide spectrum of LGGs.
As next-generation sequencing projects generate massive genome-wide sequence variation data, bioinformatics tools are being developed to provide computational predictions on the functional effects of sequence variations and narrow down the search of casual variants for disease phenotypes. Different classes of sequence variations at the nucleotide level are involved in human diseases, including substitutions, insertions, deletions, frameshifts, and non-sense mutations. Frameshifts and non-sense mutations are likely to cause a negative effect on protein function. Existing prediction tools primarily focus on studying the deleterious effects of single amino acid substitutions through examining amino acid conservation at the position of interest among related sequences, an approach that is not directly applicable to insertions or deletions. Here, we introduce a versatile alignment-based score as a new metric to predict the damaging effects of variations not limited to single amino acid substitutions but also in-frame insertions, deletions, and multiple amino acid substitutions. This alignment-based score measures the change in sequence similarity of a query sequence to a protein sequence homolog before and after the introduction of an amino acid variation to the query sequence. Our results showed that the scoring scheme performs well in separating disease-associated variants (n = 21,662) from common polymorphisms (n = 37,022) for UniProt human protein variations, and also in separating deleterious variants (n = 15,179) from neutral variants (n = 17,891) for UniProt non-human protein variations. In our approach, the area under the receiver operating characteristic curve (AUC) for the human and non-human protein variation datasets is ∼0.85. We also observed that the alignment-based score correlates with the deleteriousness of a sequence variation. In summary, we have developed a new algorithm, PROVEAN (Protein Variation Effect Analyzer), which provides a generalized approach to predict the functional effects of protein sequence variations including single or multiple amino acid substitutions, and in-frame insertions and deletions. The PROVEAN tool is available online at http://provean.jcvi.org.
The rate at which nonsynonymous single nucleotide polymorphisms (nsSNPs) are being identified in the human genome is increasing dramatically owing to advances in whole-genome/whole-exome sequencing technologies. Automated methods capable of accurately and reliably distinguishing between pathogenic and functionally neutral nsSNPs are therefore assuming ever-increasing importance. Here, we describe the functional analysis Through hidden Markov models (FATHMM) software and server: a species-independent method with optional species-specific weightings for the prediction of the functional effects of protein missense variants. Using a model weighted for human mutations, we obtained performance accuracies that outperformed traditional prediction methods (i.e., SIFT, PolyPhen, and PANTHER) on two separate benchmarks. Furthermore, in one benchmark, we achieve performance accuracies that outperform current state-of-the-art prediction methods (i.e., SNPs&GO and MutPred). We demonstrate that FATHMM can be efficiently applied to high-throughput/large-scale human and nonhuman genome sequencing projects with the added benefit of phenotypic outcome associations. To illustrate this, we evaluated nsSNPs in wheat (Triticum spp.) to identify some of the important genetic variants responsible for the phenotypic differences introduced by intense selection during domestication. A Web-based implementation of FATHMM, including a high-throughput batch facility and a downloadable standalone package, is available at http://fathmm.biocompute.org.uk.
We describe a computational method that infers tumor purity and malignant cell ploidy directly from analysis of somatic DNA alterations. The method, named ABSOLUTE, can detect subclonal heterogeneity and somatic homozygosity, and it can calculate statistical sensitivity for detection of specific aberrations. We used ABSOLUTE to analyze exome sequencing data from 214 ovarian carcinoma tumor-normal pairs. This analysis identified both pervasive subclonal somatic point-mutations and a small subset of predominantly clonal and homozygous mutations, which were overrepresented in the tumor suppressor genes TP53 and NF1 and in a candidate tumor suppressor gene CDK12. We also used ABSOLUTE to infer absolute allelic copy-number profiles from 3,155 diverse cancer specimens, revealing that genome-doubling events are common in human cancer, likely occur in cells that are already aneuploid, and influence pathways of tumor progression (for example, with recessive inactivation of NF1 being less common after genome doubling). ABSOLUTE will facilitate the design of clinical sequencing studies and studies of cancer genome evolution and intra-tumor heterogeneity.
Cancer is a disease driven by genetic variation and mutation. Exome sequencing can be utilized for discovering these variants and mutations across hundreds of tumors. Here we present an analysis tool, VarScan 2, for the detection of somatic mutations and copy number alterations (CNAs) in exome data from tumor-normal pairs. Unlike most current approaches, our algorithm reads data from both samples simultaneously; a heuristic and statistical algorithm detects sequence variants and classifies them by somatic status (germline, somatic, or LOH); while a comparison of normalized read depth delineates relative copy number changes. We apply these methods to the analysis of exome sequence data from 151 high-grade ovarian tumors characterized as part of the Cancer Genome Atlas (TCGA). We validated some 7790 somatic coding mutations, achieving 93% sensitivity and 85% precision for single nucleotide variant (SNV) detection. Exome-based CNA analysis identified 29 large-scale alterations and 619 focal events per tumor on average. As in our previous analysis of these data, we observed frequent amplification of oncogenes (e.g., CCNE1, MYC) and deletion of tumor suppressors (NF1, PTEN, and CDKN2A). We searched for additional recurrent focal CNAs using the correlation matrix diagonal segmentation (CMDS) algorithm, which identified 424 significant events affecting 582 genes. Taken together, our results demonstrate the robust performance of VarScan 2 for somatic mutation and CNA detection and shed new light on the landscape of genetic alterations in ovarian cancer.
To define a predictive model for clinical behaviour of breast phyllodes tumours (PT) using histological parameters and surgical margin status.
Cases of breast PT diagnosed in the Department of Pathology Singapore General Hospital between January 1992 and December 2010 were stratified into benign, borderline and malignant grades based on a combination of histological parameters (stromal atypia, hypercellularity, mitoses, overgrowth and nature of tumour borders). Surgical margin status was assessed. Clinical follow-up and biostatistical modelling were accomplished.
Of 605 PT, 440 (72.7%) were benign, 111 (18.4%) borderline and 54 (8.9%) malignant. Recurrences, which were predominantly local, were documented in 80 (13.2%) women. Deaths from PT occurred in 12 (2%) women. Multivariate analysis revealed stromal atypia, overgrowth and surgical margins to be independently predictive of clinical behaviour, with mitoses achieving near significance. Stromal hypercellularity and tumour borders were not independently useful. A nomogram developed based on atypia, mitoses, overgrowth and surgical margins (AMOS criteria) could predict recurrence-free survival at 1, 3, 5 and 10 years. This nomogram was superior to a total histological score derived from adding values assigned to each of five histological parameters.
A predictive nomogram based on three histological criteria and surgical margin status can be used to calculate recurrence-free survival of an individual woman diagnosed with PT. This can be applied for patient counselling and clinical management.
Recent advances in sequencing technology make it possible to comprehensively catalog genetic variation in population samples, creating a foundation for understanding human disease, ancestry and evolution. The amounts of raw data produced are prodigious, and many computational steps are required to translate this output into high-quality variant calls. We present a unified analytic framework to discover and genotype variation among multiple samples simultaneously that achieves sensitive and specific results across five sequencing technologies and three distinct, canonical experimental designs. Our process includes (i) initial read mapping; (ii) local realignment around indels; (iii) base quality score recalibration; (iv) SNP discovery and genotyping to find all potential variants; and (v) machine learning to separate true segregating variation from machine artifacts common to next-generation sequencing technologies. We here discuss the application of these tools, instantiated in the Genome Analysis Toolkit, to deep whole-genome, whole-exome capture and multi-sample low-pass (∼4×) 1000 Genomes Project datasets.
We describe a simple and reproducible method to measure absolute telomere length (aTL) using quantitative real-time polymerase chain reaction (qPCR). This method is based on the Cawthon method for relative measurement of telomere length (TL) but modified by introducing an oligomer standard to measure aTL. The method describes the oligomer standards, the generation of the standard curve and the calculations required to calculate aTL from the qPCR data. The necessary controls and performance characteristics of the assay are described in detail and compared relative to other methods for measuring TL. Typical results for this assay for a variety of human tissue samples are provided as well as a troubleshooting schedule. This method allows high throughput measurement of aTL using small amounts of DNA making it amenable for molecular epidemiological studies. Compared to the traditional relative TL qPCR assays, the aTL method described in this protocol enables a more direct comparison of results between experiments within and between laboratories.
Next-generation sequencing has drawn the genetic landscape of hepatocellular carcinoma and several signaling pathways are altered at the DNA level in tumors: Wnt/β-catenin, cell cycle regulator, epigenetic modifier, histone methyltransferase, oxidative stress, ras/raf/map kinase and akt/mtor pathways. Hepatocarcinogenesis is a multistep process starting with the exposure to different risk factors, followed by the development of a chronic liver disease and cirrhosis precede in the vast majority of the cases the development of HCC. Several lines of evidence have underlined the pivotal role of telomere maintenance in both cirrhosis and HCC pathogenesis. TERT promoter mutations were identified as the most frequent genetic alterations in hepatocellular carcinoma with an overall frequency around 60%. Moreover, in cirrhosis, TERT promoter mutationsare observed at the early steps of hepatocarcinogenesis since they are recurrently identified in low-grade and high-grade dysplastic nodules. In contrast, acquisition of genomic diversity through mutations of classical oncogenes and tumor suppressor genes (TP53, CTNNB1, ARID1A…) occurred only in progressed HCC. In normal liver, a subset of HCC can derived from the malignant transformation of hepatocellular adenoma (HCA). In HCA, CTNNB1 mutations predispose to transformation of HCA in HCC and TERT promoter mutations are required in most of the cases as a second hit for a full malignant transformation. All these findings have refined our knowledge of HCC pathogenesis and have pointed telomerase as a target for tailored therapy in the future.
Copyright © 2015 Elsevier Masson SAS. All rights reserved.
The identification of specific genetic alterations as key oncogenic drivers and the development of targeted therapies are together transforming clinical oncology and creating a pressing need for increased breadth and throughput of clinical genotyping. Next-generation sequencing assays allow the efficient and unbiased detection of clinically actionable mutations. To enable precision oncology in patients with solid tumors, we developed MSK-IMPACT, a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes in formalin-fixed, paraffin-embedded tumors. Barcoded libraries from patient-matched tumor and normal samples were captured, sequenced, and subjected to a custom analysis pipeline to identify somatic mutations. Sensitivity, specificity, reproducibility of MSK-IMPACT were assessed through extensive analytical validation. We tested 284 tumor samples with previously known point mutations and insertions/deletions in 47 exons of 19 cancer genes. All known variants were accurately detected, and there was high reproducibility of inter- and intrarun replicates. The detection limit for low-frequency variants was approximately 2% for hotspot mutations and 5% for nonhotspot mutations. Copy number alterations and structural rearrangements were also reliably detected. MSK-IMPACT profiles oncogenic DNA alterations in clinical solid tumor samples with high accuracy and sensitivity. Paired analysis of tumors and patient-matched normal samples enables unambiguous detection of somatic mutations to guide treatment decisions.
Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
We screened promoter region of the telomerase reverse transcriptase (TERT) for activating somatic mutations in 188 tumors from patients with clear cell renal cell carcinoma (ccRCC). Twelve tumors (6.4%) carried a mutation within the core promoter region of the gene. The mutations were less frequent in high grade tumors compared to low grade tumors (odds ratio (OR) = 0.15, 95% CI = 0.03-0.72, p = 0.02). Multivariate analysis for cause specific survival showed statistically significant poor outcome in patients with TERT promoter mutations (hazard ratio (HR) = 2.90, 95% CI = 1.13–7.39, p = 0.03). A common polymorphism (rs2835699) within the locus seemed to act as a modifier of the effect of the mutations on patient survival as the non-carriers of the variant allele with the TERT promoter mutations showed worst survival (HR = 3.34, 95% CI = 1.24-8.98, p = 0.02). We also measured relative telomere length (RTL) in tumors and difference between tumors with and without the TERT promoter mutations was not statistically significant. Similarly, no difference in patient survival based on RTL in tumors was observed. Our study showed a relatively low frequency of TERT promoter mutations in ccRCC. Nevertheless, patients with the mutations, particularly in the absence of the rs2853669 variant showed the worst disease-specific survival. Thus, it is possible that the TERT promoter mutations define a small subset of tumors with an aggressive behavior. © 2014 Wiley Periodicals, Inc.
Background:
Patients seeking a second opinion or continuation of care at our hospital will routinely have their pathology reviewed prior to initiating treatment. To assess the relevance of this review in patients with breast cancer, we compared original pathology reports submitted during the referral with second-review reports issued at our institution. We also assessed compliance with College of American Pathologists (CAP) requirements regarding inclusion of scientifically validated data elements (SVDE) in these pathology reports.
Methods:
We retrospectively studied all 1,970 breast pathology referral cases reviewed during one calendar year. The variables studied were histologic classification; tumor grade, necrosis, size, margin status, lymphatic/vascular invasion, dermal involvement, and biomarker profile (ER, PR, and Her-2). Each variable was rated as "agree," "disagree," "missing information," or "not applicable."
Results:
A significant discrepancy, defined as a disagreement that affected patient care, was found in 226 cases (11.47%). Additionally, in 418 resection cases (31.6%), some CAP-checklist specific required information was missing. The most common areas of significant discrepancy were histologic category (66 cases; 33%) and biomarker reporting (50 cases; 25%). The most problematic diagnostic categories were intraductal lesions, lobular carcinoma, metaplastic carcinomas, and phyllodes tumors. Most disagreements in the biomarker-profile category were interpretive, but in 20% of discrepant cases, findings were supported by repeat immunohistochemical analysis.
Conclusions:
Our results confirm the value and utility of obtaining a second opinion to optimize patient care. Changes in diagnoses obtained after second review should be interpreted and reported in a collaborative fashion, noting the benefit of a review from second pair of experienced eyes. Our results support the use of second review to ensure inclusion of CAP-required data elements in pathology reports.
Human telomerase reverse transcriptase (TERT) encodes a rate-limiting catalytic subunit of telomerase that maintains genomic integrity. TERT expression is mostly repressed in somatic cells with exception of proliferative cells in self-renewing tissues and cancer. Immortality associated with cancer cells has been attributed to telomerase over-expression. The precise mechanism behind the TERT activation in cancers has mostly remained unknown. The newly described germline and recurrent somatic mutations in melanoma and other cancers in the TERT promoter that create de novo E-twenty six/ternary complex factors (Ets/TCF) binding sites, provide an insight into the possible cause of tumor-specific increased TERT expression. In this review we discuss the discovery and possible implications of the TERT promoter mutations in melanoma and other cancers.
Although a few cancer genes are mutated in a high proportion of tumours of a given type (>20%), most are mutated at intermediate frequencies (2-20%). To explore the feasibility of creating a comprehensive catalogue of cancer genes, we analysed somatic point mutations in exome sequences from 4,742 human cancers and their matched normal-tissue samples across 21 cancer types. We found that large-scale genomic analysis can identify nearly all known cancer genes in these tumour types. Our analysis also identified 33 genes that were not previously known to be significantly mutated in cancer, including genes related to proliferation, apoptosis, genome stability, chromatin regulation, immune evasion, RNA processing and protein homeostasis. Down-sampling analysis indicates that larger sample sizes will reveal many more genes mutated at clinically important frequencies. We estimate that near-saturation may be achieved with 600-5,000 samples per tumour type, depending on background mutation frequency. The results may help to guide the next stage of cancer genomics.
The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in similar to 40% of genes, with the landscape dominated by cis-and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA-RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the 'CNA-devoid' subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome.
Clonal evolution is a key feature of cancer progression and relapse. We studied intratumoral heterogeneity in 149 chronic lymphocytic leukemia (CLL) cases by integrating whole-exome sequence and copy number to measure the fraction of cancer cells harboring each somatic mutation. We identified driver mutations as predominantly clonal (e.g., MYD88, trisomy 12, and del(13q)) or subclonal (e.g., SF3B1 and TP53), corresponding to earlier and later events in CLL evolution. We sampled leukemia cells from 18 patients at two time points. Ten of twelve CLL cases treated with chemotherapy (but only one of six without treatment) underwent clonal evolution, predominantly involving subclones with driver mutations (e.g., SF3B1 and TP53) that expanded over time. Furthermore, presence of a subclonal driver mutation was an independent risk factor for rapid disease progression. Our study thus uncovers patterns of clonal evolution in CLL, providing insights into its stepwise transformation, and links the presence of subclones with adverse clinical outcomes.
Detection of somatic point substitutions is a key step in characterizing the cancer genome. However, existing methods typically miss low-allelic-fraction mutations that occur in only a subset of the sequenced cells owing to either tumor heterogeneity or contamination by normal cells. Here we present MuTect, a method that applies a Bayesian classifier to detect somatic mutations with very low allele fractions, requiring only a few supporting reads, followed by carefully tuned filters that ensure high specificity. We also describe benchmarking approaches that use real, rather than simulated, sequencing data to evaluate the sensitivity and specificity as a function of sequencing depth, base quality and allelic fraction. Compared with other methods, MuTect has higher sensitivity with similar specificity, especially for mutations with allelic fractions as low as 0.1 and below, making MuTect particularly useful for studying cancer subclones and their evolution in standard exome and genome sequencing data.
Whole genome and exome sequencing of matched tumor-normal sample pairs is becoming routine in cancer research. The consequent increased demand for somatic variant analysis of paired samples requires methods specialized to model this problem so as to sensitively call variants at any practical level of tumor impurity.
We describe Strelka, a method for somatic SNV and small indel detection from sequencing data of matched tumor-normal samples. The method uses a novel Bayesian approach which represents continuous allele frequencies for both tumor and normal samples, while leveraging the expected genotype structure of the normal. This is achieved by representing the normal sample as a mixture of germline variation with noise, and representing the tumor sample as a mixture of the normal sample with somatic variation. A natural consequence of the model structure is that sensitivity can be maintained at high tumor impurity without requiring purity estimates. We demonstrate that the method has superior accuracy and sensitivity on impure samples compared with approaches based on either diploid genotype likelihoods or general allele-frequency tests.
The Strelka workflow source code is available at ftp://strelka@ftp.illumina.com/.
csaunders@illumina.com
Pathologists can distinguish benign phyllodes tumors, which very rarely metastasize, from malignant phyllodes tumors, which metastasize in approximately one fourth of patients. However, whether these same histologic criteria can be used to predict the likelihood that a phyllodes tumor will locally recur after breast conserving therapy remains controversial.
Since few patients with malignant phyllodes tumors have been treated with breast conserving surgery in any individual series, the literature was reviewed using a Medline search.
After local excision, 21% (111/540), 46% (18/39), and 65% (26/40) of patients with benign, borderline, and malignant phyllodes tumors, respectively, recurred in the breast. Following wide local excision, 8% (17/212), 29% (20/68), and 36% (16/45) of patients with benign, borderline, and malignant phyllodes tumors recurred in the breast.
Malignant phyllodes tumors are much more likely than benign phyllodes tumors to recur in the breast after breast conserving surgery. This high rate of local recurrence of borderline and malignant phyllodes tumors suggests that wide local excision is less than optimal therapy, and challenges us to look for methods to improve local tumor control.
Next-generation DNA sequencing (NGS) projects, such as the 1000 Genomes Project, are already revolutionizing our understanding of genetic variation among individuals. However, the massive data sets generated by NGS--the 1000 Genome pilot alone includes nearly five terabases--make writing feature-rich, efficient, and robust analysis tools difficult for even computationally sophisticated individuals. Indeed, many professionals are limited in the scope and the ease with which they can answer scientific questions by the complexity of accessing and manipulating the data produced by these machines. Here, we discuss our Genome Analysis Toolkit (GATK), a structured programming framework designed to ease the development of efficient and robust analysis tools for next-generation DNA sequencers using the functional programming philosophy of MapReduce. The GATK provides a small but rich set of data access patterns that encompass the majority of analysis tool needs. Separating specific analysis calculations from common data management infrastructure enables us to optimize the GATK framework for correctness, stability, and CPU and memory efficiency and to enable distributed and shared memory parallelization. We highlight the capabilities of the GATK by describing the implementation and application of robust, scale-tolerant tools like coverage calculators and single nucleotide polymorphism (SNP) calling. We conclude that the GATK programming framework enables developers and analysts to quickly and easily write efficient and robust NGS tools, many of which have already been incorporated into large-scale sequencing projects like the 1000 Genomes Project and The Cancer Genome Atlas.
Invasive lobular carcinoma (ILC) is the most frequent special type of breast cancer. The majority of these tumours are of low histological grade, express hormone receptors, and lack HER2 expression. The pleomorphic variant of ILCs (PLCs) is characterized by atypical cells with pleomorphic nuclei and is reported to have an aggressive clinical behaviour. Expression profiling studies have demonstrated that classic ILCs preferentially display a luminal phenotype, whereas PLCs may be of luminal, HER2 or molecular apocrine subtypes. The aims of this study were two-fold: to determine the transcriptomic characteristics of lobular carcinomas and to define the genome-wide transcriptomic differences between classic ILCs and PLCs. To define the transcriptomic characteristics of ILCs, minimizing the impact of histological grade and molecular subtype on the analysis, we subjected a series of grade- and molecular subtype-matched ILCs and invasive ductal carcinomas (IDCs) to genome-wide gene expression profiling using oligonucleotide microarrays. Hierarchical clustering analysis demonstrated that ILCs formed a separate cluster and a supervised analysis revealed that 5.8% of the transcriptionally regulated genes were significantly differentially expressed in ILCs compared to grade- and molecular subtype-matched IDCs. ILCs displayed down-regulation of E-cadherin and of genes related to actin cytoskeleton remodelling, protein ubiquitin, DNA repair, cell adhesion, TGF-beta signalling; and up-regulation of transcription factors/immediate early genes, lipid/prostaglandin biosynthesis genes, and cell migration-associated genes. Supervised analysis of classic ILCs and PLCs demonstrated that less than 0.1% of genes were significantly differentially expressed between these tumour subtypes. Our results demonstrate that ILCs differ from grade- and molecular subtype-matched IDCs in the expression of genes related to cell adhesion, cell-to-cell signalling, and actin cytoskeleton signalling. However, classic ILCs and PLCs are remarkably similar at the molecular level and should be considered as part of a spectrum of lesions.