Metastatic Hepatic Angiosarcoma and BRAF Inhibitor Therapy

Context: BRAF mutations lead to constitutive activation of downstream signaling in the Mitogen-activated protein (MAP) kinase pathway, and can serve as a molecular therapeutic target for BRAF inhibitors in melanoma. However, there is a scant data on BRAF mutations in angiosarcoma and its response to BRAF inhibitor treatment. The aim of this study was to evaluate for BRAF mutation in metastatic angiosarcoma and its response to BRAF inhibitor treatment. Methods and results: We retrospectively identified cases of hepatic metastatic angiosarcoma in the departmental archives from 2006 to 2015. Total six cases of metastatic angiosarcoma to the liver were retrieved. Histologically, all tumors were high-grade except one that was low-grade. Four of six were epithelioid type and two were mixed epithelioid and spindle cell types. Immunohistochemical (IHC) stain for BRAF V600E mutation was performed which showed one case positive for BRAF V600E mutation. For the positive case, targeted gene sequencing (total 50-gene panel including BRAF) was followed which confirmed BRAF V600E (c.1799T>A) mutation. The patient with BRAF mutated angiosarcoma received Vemurafenib, a BRAF inhibitor, for treatment and showed a significant response to therapy. Conclusion: BRAF V600E mutations can occur in a subset of metastatic angiosarcoma to the liver, and can serve as a molecular target for treatment with a BRAF inhibitor.


Introduction
Angiosarcoma is a rare aggressive malignancy, accounting for 2-3% of adult soft tissue sarcomas, and resulting in a high mortality with approximately 50% of patients dying within the first year. The most common locations for primary angiosarcoma are the breast, skin, deep tissue, and the liver. The liver is also a common site for metastasis. Surgery, chemotherapy and/or radiation therapy, or combinations are therapeutic options, but these treatment modalities are ineffective.
Recently, molecular targeted therapy has shed light on the treatment of many previously poorly responding tumors. Recent advancements in genomic research of angiosarcoma have revealed potential therapeutic genetic marker(s). Mutations in the Mitogen-activated protein (MAP) kinase pathway are an Methods and results: We retrospectively identified cases of hepatic metastatic angiosarcoma in the departmental archives from 2006 to 2015. Total six cases of metastatic angiosarcoma to the liver were retrieved. Histologically, all tumors were high-grade except one that was low-grade. Four of six were epithelioid type and two were mixed epithelioid and spindle cell types. Immunohistochemical (IHC) stain for BRAF V600E mutation was performed which showed one case positive for BRAF V600E mutation. For the positive case, targeted gene sequencing (total 50-gene panel including BRAF) was followed which confirmed BRAF V600E (c.1799T>A) mutation. The patient with BRAF mutated angiosarcoma received Vemurafenib, a BRAF inhibitor, for treatment and showed a significant response to therapy.
Conclusion: BRAF V600E mutations can occur in a subset of metastatic angiosarcoma to the liver, and can serve as a molecular target for treatment with a BRAF inhibitor.
Keywords: Angiosarcoma, BRAF V600E, BRAF Inhibitor, Vemurafenib, MAP Kinase pathway important pathway in the development of angiosarcoma [1]. MAPs are protein kinases which regulate cell functions including proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis [2]. BRAF is a member of MAP kinase pathway and its mutations can lead to constitutive activation of downstream signaling in the MAP kinase pathway, resulting in increase in cell proliferation and survival [3]. BRAF is seen to be mutated in 8% of human cancers [4,5] and accounts for almost 50% of mutations in melanoma [3]. The majority of the mutations in BRAF result in a substitution for valine with glutamic acid at the 600 position of the BRAF protein (BRAF V600E). BRAF inhibitors like Vemurafenib may be helpful in the management of BRAF mutated tumors. Due to potential therapeutic significance, many studies have attempted to identify BRAF mutation in angiosarcoma. Murali  tumor samples and found 4 tumors harboring BRAF amplification and 1 with activating mutation [1]. But so far there is little evidence indicating angiosarcoma harboring BRAF mutation and regarding the response of angiosarcoma to BRAF V600E inhibitor. The aim of this study was to evaluate for BRAF mutation in angiosarcoma and its response to BRAF inhibitor treatment.

Patients
Index patient: A 40-year-old woman presented to the hospital with complaints of abdominal pain and anemia. The patient was known to have BRCA-1 mutation. The patient also had the history of multiple cancers in the past including papillary thyroid carcinoma status-post thyroidectomy and radioactive iodine therapy (7 years ago), melanoma of left shoulder blade (6 years ago), intraductal papilloma status-post breast lumpectomy (2 years ago). The patient underwent chest/abdomen computed tomography scan revealing multiple lesions in liver and lungs with suspicion for metastatic cancer of unknown primary. Endoscopy revealed a mass lesion in the small intestine. The liver biopsy and concurrent small intestinal biopsy showed high-grade tumor with epithelioid cell morphology ( Figure 1A and 1B) with marked nuclear pleomorphism and high mitotic activity (approximately 5 mitotic figures per 10 high power fields). The tumor was immunoreactive for vimentin, CD31 ( Figure 1C) and ERG ( Figure 1D), while negative for melanoma (S-100, HMB45, SOX-10, Melan-A) and neuroendocrine (synaptophysin, chromogranin) markers. The diagnosis of high-grade angiosarcoma with epithelioid cell type was rendered. As per oncologist request, testing for BRAF V600E mutation was performed by immunohistochemical (IHC) stain on liver biopsy tissue and followed by next-generation sequencing using 50-gene panel (including BRAF). After being managed for angiosarcoma, unfortunately, the patient also developed large cell neuroendocrine carcinoma (grade 3) and underwent left lung lobectomy.

Additional cases
We retrospectively identified five more cases of metastatic angiosarcoma to the liver (excluding index patient) in the departmental database from the year 2006 to 2015. The slides were retrieved and reviewed by two independent pathologists. Taking all the cases in the account (including index patient), the male: female ratio was 1:5; and age ranged from 35-88 years. All

Immunohistochemical stain for BRAF mutation V600E
Paraffin-embedded tissue was cut into 4-micron-thick sections, and IHC for BRAF V600E mutation was performed using mouse anti-human BRAF (V600E) clone VE (Spring Biosciences, California, dilution 1:75) by Ventana Discovery XT stainer. The stain result was defined as negative (no stain) or positive (cytoplasmic staining pattern).

Results
Total 6 cases of metastatic angiosarcoma in the liver were examined. Table 1 summarizes the histology and the status of BRAF V600E in these cases. Only one case (index patient) showed positive IHC stain for BRAF V600E (c.1799T>A) mutation ( Figure 2) and was subsequently confirmed by the target gene sequencing. The variants of unknown significance were noted.
She responded well as measured by both imaging study and clinical outcome. All other five cases showed no BRAF mutation by IHC stain (Table 1).

Discussion
Angiosarcoma is a deadly disease with a dismal prognosis. Various therapies have been used for treating metastatic or unresectable angiosarcoma including chemotherapy (paclitaxel), [6] angiogenesis blockers [7] and multikinase inhibitor like Sorafenib [8] but the therapeutic response is quite variable. In this study, we identify a patient with metastatic angiosarcoma in the liver that harbored a BRAF V600E mutation confirmed by IHC study and gene sequencing and showed a great response to treatment with Vemurafenib, an inhibitor to BRAF V600E. The therapeutic strategy of angiosarcoma has heavily relied on our understanding of the genetics of angiosarcoma. The alterations of genes involving angiogenesis, mutation of p53 and the PIK3CA/AKT/mTOR pathway [9,10] and TGF-β signaling [11] are observed in subsets of angiosarcoma. BRAF V600E mutation, a molecular target in metastatic melanoma, colorectal carcinoma, and non-small cell carcinoma of the lung, has not been reported in angiosarcoma [9,12]. However, heterogeneous molecular alterations in angiosarcoma and relatively small samples in these studies do not null the possibility of BRAF mutation in this fatal cancer. Our study demonstrates a positive case of BRAF V600E mutation, suggesting that there may be a small subset of  [13]. It is an oral serine-threonine kinase inhibitor which acts by inhibiting only the active form of the kinase and acts as a selective BRAF inhibitor [13,15]. It blocks downstream processes by blocking ERK phosphorylation in BRAF-mutant cells leading to inhibition of tumor growth and triggers apoptosis [13,14]. It has shown to be effective for BRAF mutant melanoma [16] and colorectal cancer [17]. It also inhibits fluoro-deoxy-glucose uptake in BRAF mutant cells which can be monitored by PET imaging and can act as a marker for assessing metabolic activity of cancer cells [14].
Phase II studies have shown the antitumor effect of Sorafenib in angiosarcoma [8,22] [22]. They also found that progression-free survival with Sorafenib treatment was better in chemotherapy naïve patients [22]. Coquard et al. also did a phase II trial study of Sorafenib in 41 patients with angiosarcoma. In their study progression-free rate was 3.8 % at 9 months in patients with superficial angiosarcoma while 0% in patients with bone/visceral tumors [8]. Majority of the patients in this study had received prior chemotherapy, doxorubicin-based treatment or paclitaxel. Interestingly in this study, they found that Sorafenib was active only in the patients who had received prior chemotherapy in contrary to Maki et al. [8,22].
The advantage of Vemurafenib over Sorafenib is that it has fewer side effects, as it is a selective inhibitor. Toxicities such as arthralgia, rash, photosensitivity, fatigue, and alopecia have been reported with Vemurafenib. Cutaneous squamous cell carcinoma (mainly keratoacanthoma type) is a major complication of the treatment [13,23]. Vemurafenib can have a paradoxical effect by causing activation of the RAF/MEK/ERK pathway in some wild-type BRAF cells by stimulating ERK phosphorylation which may account for the development of keratinocyte proliferation [13,14,23].
In conclusion, our study shows for the first time that a subset of angiosarcoma may harbor BRAF mutation, and is responsive to BRAF inhibitor, Vemurafenib. The finding provides a new therapeutic approach for this deadly disease. However, our study was limited by small sample size. More studies are required to demonstrate clinical validity and effect of BRAF inhibitors in the management of patients with angiosarcoma.