The BRAF (v-raf murine sarcoma viral oncogene homolog B1) gene is located on the long arm of chromosome 7 (7q34) and encodes for an 18-exon cytoplasmic protein, a serine/threonine protein kinase (B-Raf) which can be recruited to the membrane upon stimulation of HER2 receptor. BRAF is a serine/threonine protein kinase, which is an important signal transducer of the HER2 triggered RAS–RAF–mitogen-activated protein kinase kinase (MEK)–extracellular signal regulated kinase (ERK) signaling pathway (also known as RAS/RAF/MEK/ERK pathway or MAPK cascade). Active BRAF then activates MEK1/2 to phosphorylate ERK1/2, which leads to the expression of several downstream transcription factors that regulate cell growth, differentiation, and survival. [4, 16]
We have already known that the early stage MOC usually has an excellent prognosis, but late stage MOC carries a poor outcome. [1] The encouraging success of BRAF inhibitors that can successfully treat some melanoma patients with BRAF (V600) mutations has prompted us to investigate the BRAF status and its therapeutic implication in advanced MOC. However, few studies have characterized its BRAF oncogene status and its response to anti-BRAF drugs has not yet been comprehensively explored in MOC. Even though melanoma and MOC are different tumors, we imagine that the functional consequence and anti-BRAF effect of the tumors harboring BRAF mutations might be similar. [17, 18]
After merging the previously reported raw data of HER2 amplifications, HER2 mutations and KRAS mutations with the new information of BRAF, we identified that their corresponding frequencies are 35% for HER2 amplifications, 35% for HER2 mutations, 60% for KRAS mutations and 80% for BRAF mutations in all 20 MOC Taiwanese patients (Table 1). [9, 10] Our findings also indicated that there was no significant difference in the frequency between BRAF mutations and KRAS mutations when they were compared. However, the frequency of BRAF mutations was significant higher than that of HER2 amplifications and HER2 mutations, respectively. (Fig. 2)
Focusing on the basis of BRAF variant together with HER2 amplifications, HER2 mutations, KRAS mutations within the HER2 triggered MAPK signaling pathway, we divided them into the BRAF-based dual, triple and quadruple sets, respectively. After that, we identified that the BRAF-based dual and triple sets are not uncommon except for the quadruple set (Table 1). Our data indicated that the coexisting mutations of these driver genes indeed occur in MOC, which might cause synergistic effects in tumorigenesis.
In our study, one case showing HER2 amplification, but wild-type HER2, KRAS and BRAF genes indicated that HER2 alone may in fact confer heightened sensitivity to existing anti-HER2 therapies (i.e. trastuzumab, lapatinib) because of dependency on the most upstream receptor tyrosine kinase (RTK) signaling. (Table 1, case no. 19) Moreover, high frequency of HER2 gene amplification (n = 7) combined with HER2 mutation (n = 3; 42.86% & Table 1, case no. 4, 17, 18), with KRAS mutation (n = 2; 28.58% & Table 1, case no. 1, 20) or with BRAF mutation (n = 5; 71.43% & Table 1, case no. 1, 4, 12, 18, 20) can predict unresponsiveness or refractoriness to single HER2 inhibition as a result of the constitutive activation of the MAPK pathway downstream of HER2 signaling. On the other hand, we identified one case with HER2 non-amplification, HER2 wild-type, KRAS wild-type, but alternative BRAF mutation (V600M). (Table 1, case no. 16) It is suggested that the downstream BRAF (V600) mutations alone might be enough to trigger continuous activation of MAPK signaling cascade, leading to tumorigenesis. The existing V600 BRAF inhibitors (i.e. Vemurafenib, dabrafeniband and encorafenib) may have benefit to some Taiwanese patients with primary MOC; however, the clinical evidence that currently exists to substantiate these claims are insufficient.
Previous reports from other countries have shown that MOC has a lower frequency of BRAF mutation (2–20%). [1, 2, 6] Our data revealed that the BRAF missense mutation rate is relatively up to 80% (n = 16/20) using the FemtoPath BRAF Mutation Screen Kit. This kit is a PCR-based test using proprietary primers which can selectively amplify the somatic mutations in activating segment of the BRAF gene, and suppresses the amplification of wild-type BRAF gene in human genomic DNA. [13, 14] Although time-consuming, DNA sequencing techniques are still the current gold standard for mutational testing.
Despite geographical, racial and ethnic differences, the following 5 standpoints explain the reason why the BRAF mutation rate of this study is higher than that of others. (1) We used the H-E (hematoxylin and eosin stain) based microdissection technique to obtain a high percentage of representative tumor parts from formalin-fixed paraffin-embedded (FFPE) tissues, which restricted our analysis to only those tumor cells that express a specific marker or have a specific gene mutation. (2) According to the manufacture’s manual and the previous report of the Stuntmer PCR technology, this highly specific and sensitive mutation enrich technology can detect less than 1% (as little as 20–100 ng) of BRAF V600 variants within exon 15. Additionally, the neighboring mutation sites of V600 (amino acid range 591–620) can also be amplified at the same time. Based on the identical principle of Stuntmer PCR, all of the BRAF mutations detected may share the similar high sensitivity. (3) We used other 7 normal ovarian tissues as negative controls, but none of them revealed BRAF mutations using the same kit. (4) Furthermore, the prior report also demonstrates that a stuntmer can inhibit wild type template replication, thereby allowing for selective amplification of mutants in a non-sequence specific manner. (5) Even after three rounds of PCRs, the original wild-type signal group remained unaltered, demonstrating that the stuntmer does not alter the original sequence of the sample [12–14]. The above-mentioned (1)-(5) evidences support that the possibility of false positive BRAF gene mutations detected using FemtoPath BRAF Mutation Screen Kit is extremely low.
According to the new classification system for BRAF mutations, different classes can predict their matching clinical response to contemporary targeted therapies on the market and have important implications for future anti-BRAF development. [7] In our patient cohort, we detected 4 kinds of known BRAF missense variants, 2 of which were class I (V600E, V600M), 2 were class II (A598V, T599I) and none was class III. Additionally, we identified one novel BRAF variant (S602F) that has never been reported in accordance with the COSMIC database. Even though the biochemical and signaling mechanism of the new BRAF variant (S602F) has not yet been comprehensively studied, its predicted functional effect appeared to be probably damaging in accordance with the Polyphen-2 database. As well, we suspect that the S602F might be categorized as class II BRAF variant, because it is located in the activating segment of BRAF kinase domain.
In summary, BRAF mutation is not uncommon in primary MOC of Taiwanese. When taken together with previous published data, we found that the activating BRAF mutation, HER2 amplification, HER2 mutation and KRAS mutation were not mutually exclusive, but simultaneously independent. However, they may even have a synergistic effect in tumorigenesis. Even though our results are confident and comprehensive, the case number cohort was small. Further exploratory studies should be performed to validate these finding.