Myopericytoma (MPC) is a rare tumor with perivascular proliferation of pluripotent stem-cell–like pericytes. Although indolent, MPC may be locally aggressive with recurrent disease. The pathogenesis and diagnostic biomarkers of MPC are poorly understood. We discovered that 15% of benign MPCs (thyroid, skin; 3 of 20 samples) harbored BRAF WT/V600E ; 33.3% (1 of 3 samples) of BRAF WT/V600E -MPCs were multifocal/infiltrative/recurrent. Patient-MPC and primary MPC cells harbored BRAF WT/V600E , were clonal and expressed pericytic-differentiation biomarkers crucial for its microenvironment. BRAF WT/V600E -positive thyroid MPC primary cells triggered in vitro (8.8-fold increase) and in vivo (3.6-fold increase) angiogenesis. Anti-BRAF V600E therapy with vemurafenib disrupted angiogenic and metabolic properties (~3-fold decrease) with down-regulation (~2.2-fold decrease) of some extracellular-matrix (ECM) factors and ECM-associated long non-coding RNA (LincRNA) expression, with no effects in BRAF WT -pericytes. Vemurafenib also inhibited (~3-fold decrease) cell viability in vitro and in BRAF WT/V600E -positive thyroid MPC patient-derived xenograft (PDX) mice (n = 5 mice per group). We established the first BRAF WT/V600E -dependent thyroid MPC cell culture. Our findings identify BRAF WT/V600E as a novel genetic aberration in MPC pathogenesis and MPC-associated biomarkers and imply that anti-BRAF V600E agents may be useful adjuvant therapy in BRAF WT/V600E -MPC patients. Patients with BRAF WT/V600E -MPC should be closely followed because of the risk for multifocality/recurrence.
Myopericytoma (MPC) describes rare, nodular tumors characterized by a radial and multilayered perivascular growth of ovoid and spindled-shaped cells with mesenchymal stem-cell–like features, often with associated blood vessels arranged in an irregular, “staghorn” pattern ( 1–3 ). Most MPCs are benign ( 2 ), but some are malignant with metastatic potential and poor survival ( 4 , 5 ). MPCs arise over a wide age range, primarily affecting subcutaneous tissues of the distal extremities ( 2 ), and lesions occasionally present with skin ulceration, pain or tenderness; additionally, symptomatic MPCs arise in the proximal extremities, head region and internal organs ( 1 ). Most MPC are treated surgically, and complete excision should prevent recurrent/persistent disease, although negative surgical margins are difficult to achieve outside of the extremities ( 2 ).
Biomarkers and oncogenic events driving MPC development are not well understood. In this study, we performed a comprehensive genomic and functional validation of associated MPC biomarkers with clinical implications. We have applied a high-throughput genotyping assay on 29 MPCs from available, formalin-fixed paraffin-embedded (FFPE), discarded/unidentified samples, using an Institutional Review Board-approved protocol (Beth Israel Deaconess Medical Center, Boston, MA) to unravel oncogenic events driving pathogenesis ( Supplementary Methods and Supplementary Table 1 , available online). The patient with thyroid MPC provided written informed consent for genetic analysis. For all other patients, we used discarded/unidentified tissue specimens and consent for genotyping test. Disease stage was assessed by radiologic imaging. We also used and immunohistochemistry, primary cell cultures, dynamic functional assays, shRNA, and developed an MPC-patient-derived xenograft (PDX) mouse model (for detailed methods, please see the Supplementary Methods , available online). All animal work was done in accordance with federal, local, and institutional guidelines at the Beth Israel Deaconess Medical Center (Boston, MA), and all experiments were performed with four-month-old Crl:NU(NCr)-Foxn1nu female, athymic, immunodeficient, nude mice (strain code: 490) (Charles River, Wilmington, MA) (n = 5 per group).
Statistical analysis was carried out using GraphPad Prism 6 software (version Prism 6, GraphPad Software Inc., San Diego, CA). Mann-Whitney test was used to analyze the statistical significance of differences between two groups. For categorical data, Fisher’s exact test was used. All reported P values were two sided. Data are reported as the averaged value, and error bars represent the standard deviation of the average for each group in duplicate or triplicate. Results with P values below .05 were considered statistically significant.
We developed the first translational model to date of multifocal and infiltrative thyroid MPC ( Figure 1 ; Supplementary Figures 1 and 2 , available online). The initial pathologic diagnosis in October 2010, reported by the referring international institution, was undifferentiated (anaplastic) thyroid carcinoma with two consequent cycles of chemotherapy given to the patient. By patient request, outside review of the pathology was performed at the Massachusetts General Hospital (Boston) the following month, and the diagnosis was amended to reflect a multifocal and infiltrative left thyroid MPC ( Figure 1 ; Supplementary Figures 1 and 2 , available online), with no clinically suspicious lymph nodes or distant metastases. Completion thyroidectomy was performed in June 2011 ( Supplementary Figure 1F , available online). Histologic evaluation confirmed residual thyroid MPC ( Supplementary Figure 1 , A-D , available online).
The MPC was about two-fold more metabolically active (standardized uptake value [SUV] = 4.5) compared with the non-tumoral thyroid hyperplastic nodules (SUV = 2.4) ( Figure 1A4 ); but 18-FDG (Fluorodeoxyglucose) PET (positron emission tomography)/CT (computed tomography) did not note any non-regional tissue involvement ( Supplementary Figure 1D , available online). Twelve months post-operatively, imaging revealed persistent disease in the thyroid bed ( Figure 1A5 ; Supplementary Figure 1 , G-J , available online). MPC lacked features of malignancy (increased number of mitoses, necrosis, vascular invasion), showed apparent differentiation towards pericyte lineage ( Figure 1 , B2-B7) and was characterized by perivascular growth ( Figure 1 , B5 and B6, and 1C2). A higher vascular density (CD31+) was found in the thyroid MPC (mean vessels/field = 20.2, SD = 0.4 vessels/field) compared with the adjacent normal thyroid (mean = 5.2 vessels/field, SD = 0.2, P = .007) ( Figure 1C2 ). MPC cells are arranged circumferentially around smaller vessels ( Figure 1 , B5 and B6, and 1C2; Supplementary Figure 2B5 , available online). The ratio of MPC cells to endothelial cells is about 3:1, quantified based on the number of platelet-derived growth factor receptor beta (PDGFRB)–positive MPC cells and CD31-positive endothelial cells ( Supplementary Methods , available online). MPC biomarkers (alpha-smooth muscle actin [αSMA], PDGFRB, NG2 [neuron-glial antigen 2], and extracellular-matrix [ECM] molecules, eg, CollagenIA1 [COLIA1]), as well as desmin and p16/Ink4A immunoexpression are described in Figure 1 , C3-C7; Supplementary Figure 2 , B1-B19 , and Supplementary Table 2 , available online.
As MPC is a poorly characterized “orphan” disease, we performed in-depth genotyping. We analyzed 29 FFPE tissues (20 benign MPC, four benign intravascular MPC, and five malignant MPC) ( Supplementary Table 1 , available online) by Mass Spectrometry genomic technology, which interrogates about 1000 mutations in 112 validated oncogenes and tumor suppressors. Results were validated by performing Sequenom ( 6 ) ( Figure 1 , D1-D2; and Supplementary Figure 2 , C1-C4 , available online), pyrosequencing and Sanger sequencing (data not shown). We found that 3/20 (15%) benign MPCs (ie, one thyroid and two cutaneous MPCs) from three different patients ( Figure 1 , D1-D2; Supplementary Figure 2 , C1-C4 , and Supplementary Table 1 , available online) harbored the heterozygous BRAF WT/V600E mutation in exon-15 hot-spot T1799A of the BRAF gene sequence. None of the four benign intravascular MPCs or five malignant MPCs harbored BRAF WT/V600E . Two out of three (66.6%) BRAF WT/V600E -MPCs (thyroid, skin) derived from two different patients’ infiltrated adjacent soft tissue; two of four (50%) multifocal MPCs harbored BRAF WT/V600E , compared with 1/25 (4%) unifocal MPCs with wild type (WT) BRAF (P = .04) ( Supplementary Table 1 , available online). One out of three (33.3%) MPCs (ie, thyroid) with BRAF W/V600E showed recurrent/persistent disease after one year of follow-up ( Figure 1A5 ; Supplementary Figure 1 , G-J , available online). Furthermore, our analysis of X-chromosome inactivation and methylation profile from the female patient with thyroid BRAF W/V600E -MPC ( Figure 1D1 and Figure 2 , A1 and A2) revealed that this tumor was monoclonal compared to the adjacent, uninvolved thyroid tissue ( Figure 2A3 ) ( 7 ).
To provide a translational application for our study, we established early and late passages of primary cells cultured in vitro from human thyroid BRAF WT/V600E -MPC. These cells also harbored BRAF WT/V600E and expressed pericyte lineage-specific differentiation biomarkers ( Figure 2A1 ). BRAF WT/V600E is the most frequently mutated oncogenic kinase. Vemurafenib is the first orally available selective inhibitor of BRAF V600E approved by the US Food and Drug Administration for the treatment of BRAF WT/V600E -melanoma ( 8–11 ). We tested the effects of vemurafenib on BRAF WT/V600E - MPC cells and BRAF WT pericytes ( Figure 2 , A-G). Vemurafenib substantially reduced phospho(p)ERK1/2 and pMEK1/2 protein levels in BRAF WT/V600E -MPC cells as compared with controls ( Figure 2C ). As a result, this treatment statistically significantly ( P < .001) suppressed BRAF WT/V600E -MPC cell viability, with no effect on the growth of BRAF WT -pericytes ( Figure 2B ), suggesting its high specificity for the BRAF V600E vs BRAF WT . Furthermore, we hypothesized that BRAF WT/V600E plays a role in MPC angiogenic and metabolic properties; we found that BRAF WT/V600E -MPC cells substantially grew as large cell aggregates on Matrigel ( Supplementary Figure 3A , available online), and, when cocultured with human microvascular endothelial cells, stastistically significantly (8.8-fold, P = .002) triggered in vitro angiogenesis as compared with controls ( Figure 2 , D1-D2). Vemurafenib treatment statistically significantly (about 3-fold, P = .002) disrupted this effect ( Figure 2 , D1 and D2); additionally, treatment with shRNA that targeted BRAF V600E statistically significantly reduced MPC cell adhesion and migration ( Supplementary Figure 4C , available online) with no effect in BRAF WT -pericytes (data not shown). Remarkably, BRAF WT/V600E -MPC cells also statistically significantly ( P = .002) increased vascular density in MPC-PDX mice ( Supplementary Figure 5 , available online), and vemurafenib therapy statistically significantly (3-fold, P = .007) suppressed both BRAF WT/V600E -MPC cell viability ( Figure 2G ; Supplementary Figure 5 , available online) and vascular density/angiogenesis (3.6-fold, P = .002) ( Supplementary Figure 5 , available online) without any obvious toxicity. Subsequently, we found that COL1A1 (5-fold, P = .007), PDGFRB (1.3-fold increase, P = .02), integrin-β1 (ITGβ1) (1.7-fold increase, P = .02), ID2 (DNA-binding protein inhibitor) (6.5-fold increase, P = .002), and the long intergenic non-coding RNA (LincRNA) ID2 (2.8-fold increase, P = .02) were statistically significantly (with moderate or high copy number) BRAF V600E -dependent in MPC cells as compared with control cells with BRAF WT ( Figure 2F ; Supplementary Table 3 , available online). Their expression levels ( ID2 = 2.2-fold; COL1A1 = 1.5-fold; PDGFRB = 1.5-fold; ITGβ1 = 1.5-fold; and ID2 LincRNA = 1.6) were statistically significantly reduced by vemurafenib treatment as compared with BRAF WT -pericytes ( Figure 2F ). Furthermore, MPC cell adhesion ( P = .002, Supplementary Figure 3B , available online) and migration ( P = .002, Figure 2E ) were also statistically significantly reduced by vemurafenib treatment compared with BRAF WT -pericytes. MPCs continue to be under recognized, resulting in inappropriate treatment and patient anxiety ( 12 ). Benign MPCs are generally indolent tumors ( 1 ); however, when they involve internal organs, they may be locally aggressive, with recurrent/persistent disease because of difficulty of complete excision. We discovered that BRAF V600E is a distinct genetic alteration seen in benign MPCs, often multifocal and infiltrative. Patients with multifocal MPCs present a treatment dilemma, often requiring multiple surgeries for lesions that may be quite painful, even if not malignant. BRAF V600E is a prognostic biomarker of tumor recurrence and aggressiveness ( 13–24 ) and also facilitates tumorigenesis ( 25–29 ).
BRAF WT/V600E -induced MPC cell adhesion, migration, and angiogenesis associated with upregulation of molecules (eg, COL1A1, PDGFRB, ID2) ( 30–33 ) crucial for ECM remodeling, angiogenesis, and for autocrine and paracrine communication in the tumor microenvironment ( Supplementary Figure 6 , available online), which ultimately may lead to MPC aggressiveness. High doses of vemurafenib therapy were effective to inhibit BRAF V600E -MPC cell viability and angiogenesis, suggesting that this therapy blocked BRAF V600E -dependent pro-migratory pathways and so diminished pro-angiogenic capabilities of MPC cells.
Please see the Supplementary Results (available online) for additional findings that may be of interest.
Collectively, using anti-BRAF V600E therapy as a surgical adjuvant may provide a novel advancement in the therapeutic strategy and treatment of locally aggressive multifocal BRAF V600E -positive MPCs or possibly serve as a therapeutic alternative for cases in which surgical options are limited by location and extent of disease, or in medically poor surgical candidates.
Our study is limited by sample size (29 available MPCs), precluding optimal evaluation of MPC pathological features with/without BRAF V600E . However, our results were substantiated by our integrated in vitro and in vivo approaches. It is also possible that MPC heterogeneity reduced the sensitivity for detection of the BRAF V600E mutation in some cases; therefore, we cannot exclude the possibility that the percentage of BRAF V600E -positive MPC cases is higher.
In conclusion, our results demonstrate a subset of MPC harbor BRAF V600E that drives tumor development. We report the first MPC arising in the thyroid. Anti-BRAF V600E therapy effectively suppresses viability in the only currently available MPC short-term cell culture that harbors the BRAF V600E mutation. BRAF V600E plays a role in the MPC microenvironment ( Supplementary Figure 6 , available online) and might ultimately lead to aggressive behavior. Finally, we report a multiplex panel of diagnostic markers for MPCs (eg, PDGFRB, NG2, αSMA, peritumoral-fibronectin, vimentin, CollagenIA1).
We propose that genetic testing for the BRAF WT/V600E -mutation is part of the pathologic evaluation for multifocal and infiltrative MPCs, and patients with BRAF WT/V600E - positive MPCs should be periodically re-evaluated for recurrence, especially with available targeted drug therapies.
Carmelo Nucera (Principal Investigator, Human Thyroid Cancers Preclinical and Translational Research at the Beth Israel Deaconess Medical Center/Harvard Medical School) was awarded grants by the National Cancer Institute/National Institutes of Health (1R21CA165039-01A1 and 1R01CA181183-01A1) the American Thyroid Association (ATA), and ThyCa:Thyroid Cancer Survivors Association Inc. for Thyroid Cancer Research. Carmelo Nucera was also recipient of the Guido Berlucchi “Young Investigator” research award 2013 (Brescia, Italy). Beth Israel Deaconess Medical Center was recipient of a research grant by Roche (2011). CP was awarded a grant by the Tuberous Sclerosis Complex Research Program (TSCRP) of the USA Department of Defense (DOD) (W81XWH-13-1-0262).
The study funders had no role in the design of the study, the collection, analysis, or interpretation of the data, the writing of the manuscript, nor the decision to submit the manuscript for publication.
We thank Drs. Yutaka Kawakami (Keio University, Tokyo, Japan) for providing HIV-U6 vectors. We also thank Drs. Shiva Gautam (statistical consultant), Alexander Gimelbrant, Janice Nagy, Gideon Bollag, Riccardo Taulli and Francesca Ianni, as well as Mrs. Sudeepa Syamala, Mrs. Mei Zheng, Miss Nina Hu, Mr. Bhavik Padmani and Neal Smith for technical assistance. We thank Professor William Aird for critical suggestions.