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. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: Histopathology. 2015 May 24;67(5):719–729. doi: 10.1111/his.12712

MED12 somatic mutations in fibroadenomas and phyllodes tumors of the breast

Salvatore Piscuoglio 1, Melissa Murray 1, Nicola Fusco 1,2, Caterina Marchiò 1,3, Florence L Loo 1, Luciano G Martelotto 1, Anne M Schultheis 1, Muzaffar Akram 1, Britta Weigelt 1, Edi Brogi 1, Jorge S Reis-Filho 1
PMCID: PMC4996373  NIHMSID: NIHMS808257  PMID: 25855048

Abstract

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.

Keywords: fibroepithelial tumors, breast, MED12, sequencing, somatic mutations

Introduction

Fibroadenomas (FAs) and phyllodes tumors (PTs) are fibroepithelial tumors of the breast composed of biphasic proliferations of both epithelial and stromal elements.1 FAs are benign and constitute the most frequent type of fibroepithelial tumors,2,3 being characterized by an admixture of epithelial and stromal components that can be arranged in two growth patterns: pericanalicular, characterized by a circumferential stromal proliferation around ductal structures, and intracanalicular, where the proliferating stromal cells compress the ductal structures into clefts.1 PTs are rarer than FAs, accounting for approximately 2.5% of all fibroepithelial tumors of the breast. Although in the majority of cases PTs develop de novo from intralobular or periductal stroma of the breast, there are reports of progression from FAs to PTs and of examples of FAs found in the proximity of PTs.1,4 Histologically, PTs have a cellular stroma and display an intracanalicular growth pattern, which is often more exuberant than that found in FAs and results in leaf-like projections protruding into the dilated luminal spaces of ductal structures.1 PTs are classified as benign, borderline or malignant based on specific histologic criteria,1 however these form a continuum, and grading these lesions is often challenging.5

The Mediator Complex Subunit 12 (MED12) gene (Xq13.1) encodes a member of the Multiprotein mediator complex. MED12 plays role in the regulation of transcription of all RNA polymerase II-dependent genes.6 The kinase/CDK8 module formed by MED12 together with CDK8/CDK19, Cyclin C, and MED13 is often associated with transcriptional repression and functions as a stimulus-specific positive coregulator within the p53 transcriptional program.7 Recent studies have demonstrated that up to 60% of FAs, 80% of benign and borderline PTs, and 40% of malignant PTs harbor somatic mutations in the exon 2 of the MED12 gene.8,9 Transcriptomic analysis of FAs provided additional evidence to support the contention that MED12 exon 2 somatic mutations likely constitute driver genetic alterations in FAs.8 In addition, MED12 somatic mutations have been reported in several tumor types, including hormone-associated lesions, such as uterine leiomyomas, uterine leiomyosarcomas, pelvic/retroperitoneal smooth muscle tumors, prostate cancer and adrenocortical cancer.10-14

Given the histologic similarities between FAs and PTs, and the evidence to suggest that at least a subset of PTs may originate from FAs,1,4 we sought to define whether PTs would harbor mutations affecting exon 2 of the MED12 gene in a way akin to FAs. Furthermore, as a hypothesis generating aim, we characterized the repertoire of MED12 exon 2 mutations in distinct fibroepithelial tumors from patients with multiple lesions to define their potential clonal relatedness.

Materials and Methods

Cases

The files of the Department of Pathology of Memorial Sloan Kettering Cancer Center (MSKCC) were searched for FAs and PTs diagnosed and surgically removed at our Institution between January 1996 and October 2014. The diagnostic slides and tissue blocks of 26 FAs, 25 benign PTs, 9 borderline PTs and 13 malignant PTs from 64 female patients were retrieved. Samples were anonymized prior to analysis, and the study was approved by the MSKCC Institutional Review Board. All cases were independently reviewed by three pathologists (MM, FLL and EB), and classified and graded according to the latest World Health Organization classification (WHO) criteria,1 which are detailed in the Supplementary Methods.

Microdissection and DNA extraction

Representative 8μm-thick sections from formalin-fixed paraffin-embedded histologic blocks of FAs and PTs were subjected to microdissection of tumor and matched normal tissue with a sterile needle under a stereomicroscope as previously described.15,16 Tumor areas were microdissected to enrich for neoplastic cells from the stromal elements (>70%) of the FAs and PTs analyzed. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) according to manufacturer's guidelines.

PCR amplification and Sanger sequencing

The entire MED12 exon 2 was amplified from 10ng of genomic DNA using the AmpliTaq 360 master mix (Life Technologies) as previously described.16 For this, a primer pair that amplifies a 373bp fragment encompassing exon 2 of the MED12 gene was employed (5′-TGTTCTACACGGAACCCTCCTC-3′ (forward) and 5′-CTGGGCAAATGCCAATGAGAT-3′ (reverse)) as described by Lim et al.8 (Supplementary Figure 1). Sequencing reactions were performed on an ABI3730 capillary sequencer using the ABI BigDye Terminator chemistry (v3.1, Life Technologies) according to manufacturer's instructions. To confirm that the MED12 mutations identified in FAs and PTs were somatic events, matched normal DNA was sequenced for all cases included in this study. Sequences of the forward and reverse strands were analyzed using MacVector software (MacVector Inc).16 All analyses were performed in duplicate. Insertions and deletion were manually annotated. Mutation function prediction was performed as previously described17 (Supplementary Methods).

Statistical analysis

The Chi-square test (χ2 test) and the Fisher's exact test were employed for the comparison of non-parametric variables. All statistical analyses were carried out using IBM SPSS Statistics v.20 (IBM). Two-tailed P values <0.05 were considered significant.

Results

Seventy-three tumors from 64 female patients were subjected to Sanger sequencing analysis for the presence of MED12 exon 2 somatic mutations. According to the WHO criteria,1 26 tumors were classified as FAs (36%), 25 benign PTs (34%), 9 borderline PTs (12%) and 13 malignant PTs (18%; Figure 1, Table 1 and Supplementary Tables 1 and 2).

Figure 1. Representative micrographs of a fibroadenoma and phyllodes tumors of the breast included in this study.

Figure 1

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(A) Fibroadenoma. (B) Benign phyllodes tumors of the breast. (C) Borderline phyllodes tumors of the breast. (D) Malignant phyllodes tumors of the breast. Scale bar, 500 μM.

Table 1. MED12 exon 2 mutations identified in 73 fibroepithelial tumors of the breast from 64 female patients.

Patient ID Sample ID Ethnicity Diagnosis Laterality Type of mutation cDNA change Protein change
Patient 01 MD-01 Black Malignant PT Left wild-type wild-type wild-type
Patient 02 MD-02 White Malignant PT Right wild-type wild-type wild-type
Patient 03 MD-03 Unknown Borderline PT Right wild-type wild-type wild-type
Patient 04 MD-04 White Malignant PT Right SNV c.131G>A p.G44D
MD-60a White Benign PT Right SNV c.131G>T p.G44V
MD-60b White Fibroadenoma Right SNV c.130G>T p.G44C
MD-60c White Fibroadenoma Right SNV c.131G>T p.G44V
MD-60d White Fibroadenoma Right SNV c.131G>A p.G44D
Patient 05 MD-05 a White Malignant PT Left wild-type wild-type wild-type
Patient 06 MD-06 Asian Borderline PT Left SNV c.131G>A p.G44D
Patient 07 MD-07 White Malignant PT Right wild-type wild-type wild-type
Patient 08 MD-08 Asian Malignant PT Left wild-type wild-type wild-type
Patient 09 MD-09 a White Benign PT Left SNV c.130G>A p.G44S
MD-09 b White Benign PT Left SNV c.130G>A p.G44S
MD-09 c White Benign PT Left SNV c.131G>A p.G44D
MD-09 d White Benign PT Left SNV c.131G>A p.G44D
Patient 10 MD-10 Asian Borderline PT Right wild-type wild-type wild-type
Patient 11 MD-11 Black Borderline PT Left Insertion (frameshift) c.112_113insAC p.L39_N40fs
Patient 12 MD-12 White Borderline PT Right Deletion (in-frame) c.115_141del27 p.L39_N47del
Patient 13 MD-13 Black Borderline PT Left SNV c.131G>C p.G44A
Patient 14 MD-14 White Benign PT Left Deletion (in-frame) c.122_139del18 p.V41_N46del
Patient 15 MD-15a Asian Borderline PT Left SNV c.131G>T p.G44V
Patient 16 MD-16 Asian Borderline PT Right SNV c.130G>A p.G44S
Patient 17 MD-17 White Borderline PT Left SNV c.131G>T p.G44V
Patient 18 MD-18 White Benign PT Left Deletion (in-frame) c.123_152del30 p.K42_V51del
Patient 19 MD-19 Unknown Benign PT Left SNV c.130G>A p.G44S
Patient 20 MD-20 White Benign PT Left SNV c.130G>A p.G44S
Patient 21 MD-21 White Benign PT Right SNV c.130G>A p.G44S
Patient 22 MD-22 White Benign PT Right SNV c.131G>T p.G44V
Patient 23 MD-23 Black Benign PT Left Deletion (in-frame) c.141_167del27 p.Q48_H56del
Patient 24 MD-24 White Benign PT Left wild-type wild-type wild-type
Patient 25 MD-25 White Benign PT Left Deletion (in-frame) c.144__167del24 p.P49_H56del
Patient 26 MD-26 White Benign PT Left SNV c.131G>A p.G44D
Patient 27 MD-27 White Benign PT Right SNV c.130G>T p.G44C
Patient 28 MD-28 White Benign PT Right Deletion (in-frame) c.100_129del30 p.D34_Q43del
Patient 29 MD-29 White Benign PT Left wild-type wild-type wild-type
Patient 30 MD-30 White Benign PT Right SNV c.130G>A p.G44S
Patient 31 MD-31 White Benign PT Right SNV c.130G>A p.G44S
Patient 32 MD-32 Asian Benign PT Left Deletion (in-frame) c.114_149del36 p.L39_A50del
Patient 33 MD-33 White Benign PT Right Deletion (in-frame) c.121_138del18 p.V41_N46del
Patient 34 MD-34 White Benign PT Right Deletion (in-frame) c.100_138del39 p.D34_N46del
Patient 35 MD-35 Unknown Benign PT Right Deletion (in-frame) c.116_157del42 p.L39_S52del
Patient 36 MD-36 White Benign PT Right wild-type wild-type wild-type
Patient 37 MD-37 White Fibroadenoma Left Deletion (in-frame) c.121_135del15 p. V41_F45del
Patient 38 MD-38 Unknown Fibroadenoma Right Deletion (in-frame) c.118_159del42 p.N40_G53del
Patient 39 MD-39 White Fibroadenoma Right Deletion (frameshift) c.100_112del13 p.D34_T37fs
Patient 40 MD-40 Unknown Fibroadenoma Left wild-type wild-type wild-type
Patient 41 MD-41 White Fibroadenoma Right SNV c.130G>A p.G44S
Patient 42 MD-42 White Fibroadenoma Right Deletion (in-frame) c.108_122del15 p.T37_V41del
Patient 43 MD-43 White Fibroadenoma Right wild-type wild-type wild-type
Patient 44 MD-44 Black Fibroadenoma Left Deletion (frameshift) c.100_107del8 p.D34_L36fs
Patient 45 MD-45 White Fibroadenoma Right wild-type wild-type wild-type
Patient 46 MD-46 Asian Fibroadenoma Right SNV c.131G>T p.G44V
Patient 47 MD-47 Black Fibroadenoma Right wild-type wild-type wild-type
Patient 48 MD-48 White Fibroadenoma Right wild-type wild-type wild-type
Patient 49 MD-49 Unknown Fibroadenoma Right wild-type wild-type wild-type
Patient 50 MD-50 Unknown Fibroadenoma Right wild-type wild-type wild-type
Patient 51 MD-51 White Fibroadenoma Right wild-type wild-type wild-type
Patient 52 MD-52 White Fibroadenoma Left Deletion (in-frame) c.115_162del48 p.L39_D54del
Patient 53 MD-53 White Fibroadenoma Left Deletion (in-frame) c.138_161del24 p.N47_E55del
MD-54 White Fibroadenoma Right SNV c.130G>A p.G44S
Patient 54 MD-55 White Fibroadenoma Right SNV c.131G>A p.G44D
Patient 55 MD-58 White Fibroadenoma Left SNV c.130G>C p.G44R
Patient 56 MD-56 White Fibroadenoma Right wild-type wild-type wild-type
MD-57 White Fibroadenoma Left Deletion (in-frame) c.117_152del36 p.N40_V51del
Patient 57 MD-59 Unknown Fibroadenoma Right SNV c.131G>A p.G44D
Patient 58 MD-61 White Malignant PT Right wild-type wild-type wild-type
Patient 59 MD-62 Unknown Malignant PT Left wild-type wild-type wild-type
Patient 60 MD-63 White Malignant PT Right wild-type wild-type wild-type
Patient 61 MD-64 White Malignant PT Right wild-type wild-type wild-type
Patient 62 MD-65 White Malignant PT Left wild-type wild-type wild-type
Patient 63 MD-66 White Malignant PT Right wild-type wild-type wild-type
Patient 64 MD-67 Asian Malignant PT Right wild-type wild-type wild-type
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PT, phyllodes tumor; SNV, single nucleotide variant.

High frequency of MED12 somatic mutations in FAs, benign and borderline PTs but not in malignant PTs

MED12 exon 2 somatic mutations were found in 65% of FAs (17/26), 88% of benign PTs (22/25), 78% (7/9) of borderline PTs and 8% (1/13) of malignant PTs. Six distinct somatic single nucleotide variants and nineteen distinct insertions and deletions affecting MED12 were identified in 28 and 19 cases, respectively (Figures 2 and 3, Table 1, Supplementary Table 1). All single nucleotide variants and insertions and deletions were predicted to be pathogenic by multiple mutation effect predictors (Supplementary Table 1). No nonsense single nucleotide variants were found. No associations between the presence of MED12 mutations and age of the patient or tumor size were observed (Student's t-test, P>0.1, Table 2).

Figure 2. MED12 exon 2 mutations identified by Sanger sequencing analysis in fibroepithelial tumors.

Figure 2

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Representative sequence electropherograms (tumor and matched germline) of the 6 single nucleotide variants, and of 3 of the 19 distinct insertions and deletions in exon 2 of MED12 identified by Sanger sequencing in our cohort of 73 fibroepithelial tumors.

Figure 3. Distribution of MED12 mutations in exon 2.

Figure 3

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Domain structure of the MED12 protein, and a close-up view of the amino acid residues in MED12 exon 2 affected by alterations in the fibroadenomas and phyllodes tumors analyzed in this study. The frequency of each alteration is denoted in parentheses. AA, amino acid.

Table 2. Correlation between MED12 exon 2 mutation status and patient age and tumor size in patients with fibroepithelial tumors.

Age (Mean ± SD) MED12 mutant n Age (Mean ± SD) MED12 wild-type n P value
Fibroadenoma 31.8 ± 1.9 17 37.1 ± 3.7 9 0.158
Benign PT 42 ± 2.2 22 36.3 ± 8.1 3 0.404
Borderline PT 49.3 ± 4.5 7 44.5 ± 2.5 2 0.604
Tumor size (cm) (Mean ± SD) MED12 mutant n Tumor size (cm) (Mean ± SD) MED12 wild-type n P value
Fibroadenoma 1.7 ± 0.2 17 2.2 ± 0.3 9 0.168
Benign PT 2.3 ± 0.2 22 2.4 ± 1.3 3 0.899
Borderline PT 5.8 ± 1.2 7 5.8 ± 3.1 2 0.986
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Student's t-tests, two-tailed. PT, phyllodes tumor; SD, standard deviation. Malignant PTs were excluded from this comparison due to small sample size and the low frequency of MED12 mutations.

The prevalence of MED12 exon 2 somatic mutations in FAs (Table 1, Figure 3, Supplementary Table 1) was similar to that recently described by Lim et al.8 In FAs, MED12 exon 2 mutations were found to be similarly frequent in intracanalicular-type (8/8, 100%) and mixed-type FAs (3/3, 100%), but were significantly less prevalent in pericanalicular-type FAs (6/15, 40%) than in intracanalicular-type FAs or the group of intracanalicular-type or mixed-type FAs (Table 3). Of the 17 MED12-mutant FAs, 9 (53%) harbored hotspot point mutations in codon 44 (3 p.G44D, 2 p.G44V, 2 p.G44S, 1 p.G44R and 1 p.G44C). Eight (47%) MED12-mutant FAs displayed insertions or deletions, of which 2 were frameshift and 6 were in-frame (Figure 3, Table 1, Supplementary Table 1).

Table 3. MED12 exon 2 mutation status according to growth patterns in fibroadenomas.

Growth pattern n MED12 wild-type n (%) MED12 mutant n (%) P value
Pericanalicular 15 9 (60%) 6 (40%) 0.007
Intracanalicular 8 0 (0%) 8 (100%)
Intracanalicular 8 0 (0%) 8 (100%) 1.000
Mixed 3 0 (0%) 3 (100%)
Pericanalicular 15 9 (60%) 6 (40%) 0.205
Mixed 3 0 (0%) 3(100%)
Pericanalicular 15 9 (60%) 6 (40%) 0.002
Intracanalicular or mixed 11 0 (0%) 11 (100%)
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Twenty-two (88%) benign PTs and 7 (78%) borderline PTs harbored MED12 exon 2 somatic mutations. Of the 22 MED12-mutant benign PTs, 13 (59%) displayed hotspot point mutations in codon 44 (7 p.G44S, 3 p.G44D, 2 p.G44V and 1 p.G44C), 9 (41%) had in-frame insertions or deletions (Table 1, Figure 3, Supplementary Table 1). In the 7 MED12-mutant borderline PTs, point mutations in codon 44 (1 p.G44D, 1 p.G44S, 2 p.G44V and 1 p.G44A) were found in 5 cases (71%) and in-frame insertions or deletions were found in 2 cases (29%) (Table 1, Figure 3, Supplementary Table 1). By contrast, only 1 of the 13 malignant PTs analyzed (8%) was found to harbor a single nucleotide variant affecting codon 44 (p.G44D), which was considered as likely pathogenic by mutation function predictors.

Although a similarly high prevalence of MED12 exon 2 mutations was found in FAs, benign and borderline PTs (P>0.1, Fisher's exact test), malignant PTs were found to display MED12 exon 2 somatic mutations significantly less frequently than FAs, benign or borderline PTs (P<0.001, Fisher's exact test, Figure 4). These results suggest that whilst the majority of FAs, benign PTs and borderline PTs harbored MED12 exon 2 mutations, only a minority of malignant PTs display these mutations.

Figure 4. Frequencies of MED12 exon 2 mutations in different fibroepithelial tumors of the breast.

Figure 4

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The percentages of cases with and without MED12 exon 2 somatic mutations are depicted. The MED12 exon 2 mutational frequencies in fibroadenomas, benign, borderline and malignant phyllodes tumors of the breast were compared using Fisher's exact tests. PT, phyllodes tumor.

MED12 mutational status in different tumors affecting the same patients

Four of the 64 women part of the study had multiple mammary fibroepithelial lesions (Table 1, Supplementary Table 1). We therefore compared the MED12 mutational status in multiple FAs and/or PTs from the same patient to define whether the lesions would be clonally related. In patient 04, the FAs displayed distinct MED12 exon 2 somatic mutations (p.G44V, p.G44D and p.G44C), the benign PT displayed a p.G44V mutation and the malignant PT harbored a p.G44D mutation. Interestingly, the malignant PT and one of the intracanalicular FAs harbored the p.G44D MED12 mutation, whereas the benign PT and another intracanalicular FA displayed the p.G44V MED12 mutation. In patient 09, two p.G44S and two p.G44D were found in the 4 benign PTs in the right breast, demonstrating that some of the PTs were likely not clonally related. Patients 53 and 56 presented with bilateral FAs; in patient 53, a p.G44S mutation was found in the FA of the right breast, whereas the left breast FA harbored a p.N47_E55del in-frame deletion. In patient 56, the left breast FA harbored a p.N40_V51del in-frame deletion, whereas the right breast FA was MED12 wild-type (Table 1, Supplementary Table 1). Taken together, these results demonstrate that fibroepithelial lesions arising in patients with multiple FAs and/or PTs do not necessarily harbor identical MED12 somatic mutations, even when the lesions are ipsilateral, suggesting that some of these lesions likely originated independently. Furthermore, the presence of a p.G44D in a FA and in a malignant PT affecting the same breast of patient 4 may be interpreted as supportive of a clonal relationship between these two lesions and raises the possibility that in this case the FA may have constituted the substrate for the development of the malignant PT.

Discussion

Somatic mutations in MED12 exon 2 have been recently reported in benign indolent types of stromal tumors, including the majority of leiomyomas,18-23 FAs8 and benign and borderline PTs9, and in a subset of malignant PTs.9 In FAs, which are biphasic tumors with epithelial components, these mutations have been found to be restricted to the stromal elements.8 Here we provide an independent validation of the results reported by Lim et al.8 and Cani et al.9 as we have also identified MED12 exon 2 mutations in 65%, 88% and 78% of FAs, benign and borderline PTs, respectively. In contrast to the high prevalence of MED12 somatic mutations found in FAs, benign and borderline PTs, MED12 exon 2 somatic mutations were found to be significantly less frequent in malignant PTs, being found in only in 8% (1/13) of cases. These observations are consistent with those reported by Cani et al.,9 who observed MED12 somatic mutations in 40% (2/5) of malignant PTs, but in 80% of benign (4/5) and borderline (4/5) PTs.

There is burgeoning evidence to demonstrate that MED12 exon 2 mutations likely constitute genomic drivers of FAs.8 The first 100 amino acids of MED12, which include its entire exon 2, have been shown to be essential for the interaction of MED12 with Cyclin C, and somatic mutations in MED12 exon 2 have been reported to be tumorigenic, decreasing the interaction between MED12 and Cyclin C-CDK8/CDK19 and resulting in loss of Mediator-associated CDK activity24 and in RAD51B overexpression.25 These mutations have also been shown to impact on estrogen signaling. Not only does the Mediator complex interact with estrogen receptors α and β,26 but also MED12 exon 2-mutated FAs have been shown to display dysregulated estrogen signaling.8 Given that breast and uterine stromal cells and uterine smooth muscle cells are estrogen responsive, it is plausible that the high frequency of MED12 exon 2 somatic mutations in these tumors and their relative rarity in stromal tumors originating in other non-estrogen responsive anatomical sites stems from the impact of MED12 mutations on estrogen signaling.8

MED12 somatic mutations appear to be less frequent in malignant than in benign tumors.13,20,27-29 We have observed that MED12 exon 2 somatic mutations were significantly more frequently found in FAs, benign and borderline PTs than in malignant PTs. Consistent with these observations, leiomyosarcomas have also been shown to harbor MED12 mutations at significantly lower frequencies than leiomyomas.29,30 These observations may be reflective of the fact that although some leiomyosarcomas and malignant PTs may originate from leiomyomas and FAs/benign PTs, respectively, which are estrogen dependent lesions, others may originate de novo through an estrogen independent pathway. Consistent with this notion, the expression both ERβ (in the stromal component) and ERα (in the epithelial component) of fibroepithelial lesions of the breast has been reported to be inversely correlated with their degree of aggressiveness.31,32 On the other hand, 60% of the malignant PTs analyzed by Cani et al. lacked MED12 somatic mutations,9 and these cases harbored TERT gene amplification. Further studies are required to define fully the mechanistic interactions between MED12 exon 2 mutations and estrogen signaling in MED12 mutant lesions and the alternative driver genetic alterations in MED12 wild-type malignant PTs.

The genotyping of multiple fibroepithelial lesions occurring in 4 patients constitutes a unique and intriguing aspect of our study. The identification of ipsilateral fibroepithelial lesions harboring distinct MED12 mutations is consistent with the notion that multiple coexisting ipsilateral fibroepithelial tumors can arise independently. The analysis of multiple ipsilateral FAs, benign and malignant PTs also revealed that in one case, identical MED12 mutations were found in a FA and in a malignant PT in the same breast, an observation that is consistent with a clonal relationship between the two lesions and highlights the possibility that, in this case, the FA may have constituted the substrate for the development of the malignant PT.

This study has several limitations. First, the sample size is small, in particular, the cohort of borderline PTs comprises only 9 tumors; despite the small sample size, we have documented a significantly lower prevalence of MED12 exon 2 mutations in malignant PTs than in FAs and benign and borderline PTs. This observation, however, should be considered as hypothesis-generating and warrants the analysis of independent cohorts of malignant PTs. Second, we have deliberately focused on somatic mutations affecting exon 2 of the MED12 gene. Although previous findings demonstrate that the vast majority of MED12 somatic mutations in FAs affect exon 2, we cannot rule out the possibility of somatic mutations affecting other coding sequences of the MED12 gene may be present in breast fibroepithelial lesions. Finally, we employed Sanger sequencing for the detection of MED12 somatic mutations; given that all lesions included in this study were microdissected and considering the sensitivity of Sanger sequencing, subclonal MED12 mutations present in <20% of the clones would not be accurately detected. It should be noted, however, that given the evidence that MED12 somatic mutations are early driver events in fibroepithelial lesions, these mutations would likely be present in the modal populations of MED12 mutant FAs and PTs.

In conclusion, our study demonstrates that somatic mutations in MED12 exon 2 likely constitute driver genetic events of not only FAs but also of benign and borderline PTs. MED12 exon 2 somatic mutations were found to be significantly less frequent in malignant PTs, indicating that genetic and/or epigenetic alterations other than MED12 exon 2 mutations may constitute the driving genetic events in the pathogenesis of malignant PTs.

Supplementary Material

Supp Fig S1

Supplementary Figure 1: Schematic representation of the MED12 somatic mutations reported by Lim et al.8 and Cani et al.9 in relation to the primers employed for Sanger sequencing in this study.

Supp Methods
Supp Table S1

Supplementary Table 1: Clinicopathologic characteristics of the fibroepithelial lesions included in this study, their MED12 exon 2 somatic mutation status, and the predicted pathogenicity of the mutations identified.

Supp Table S2

Supplementary Table 2: Histologic characteristics of the malignant phyllodes tumors included in this study.

Acknowledgments

SP is funded by a Susan G Komen Postdoctoral Fellowship Grant (PDF14298348); CM is funded by the Italian Association of Cancer Research (AIRC, MFAG13310). AMS is funded by a stipend from the German Cancer Aid (Dr. Mildred Scheel Stiftung).

BW, EB and JSR-F conceived the study. MM and EB provided the samples. MM, FLL and EB performed the pathologic review. SP, NF, AMS, CM, LGM and MA carried out experiments and analyzed data. SP, BW and JSR-F wrote the first draft of the manuscript. All authors interpreted the data, and reviewed and approved the final version of the manuscript.

Footnotes

Conflict of interest: The authors have no conflicts of interest to declare

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Fig S1

Supplementary Figure 1: Schematic representation of the MED12 somatic mutations reported by Lim et al.8 and Cani et al.9 in relation to the primers employed for Sanger sequencing in this study.

NIHMS808257-supplement-Supp_Fig_S1.tif (157.6KB, tif)
Supp Methods
NIHMS808257-supplement-Supp_Methods.docx (28KB, docx)
Supp Table S1

Supplementary Table 1: Clinicopathologic characteristics of the fibroepithelial lesions included in this study, their MED12 exon 2 somatic mutation status, and the predicted pathogenicity of the mutations identified.

NIHMS808257-supplement-Supp_Table_S1.docx (47.4KB, docx)
Supp Table S2

Supplementary Table 2: Histologic characteristics of the malignant phyllodes tumors included in this study.

NIHMS808257-supplement-Supp_Table_S2.docx (17KB, docx)

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