High frequency of BRAF mutations in mucinous ovarian carcinoma of Taiwanese patients

In view of the encouraging clinical evidence of inhibitors that can treat some melanoma patients successfully, we aimed to investigate the status of BRAF mutations of primary mucinous ovarian carcinomas (MOC) in Taiwanese women, and apply the emerging paradigm classication of BRAF mutation groups. DNA was extracted from micro-dissected tissue samples using the QIAamp® DNA FFPE Kit. The mutations of activation segment (exon 15), CR3 (conserved regions 3), kinase domain of the BRAF gene were analyzed using the highly sensitive BRAF mutant enriched kit (FemtoPath®) with subsequent Sanger sequencing method. Additionally, we extended our prior data of HER2 aberrations and KRAS mutation into this study in order to compare with the status of BRAF mutation. assess the pairwise comparison of BRAF mutation with the respective KRAS mutation, HER2 mutation and HER2 amplication; (2) evaluate the prevalence of BRAF-based dual, triple and quadruple mutation sets including the co-existing BRAF mutation with KRAS mutations, HER2 mutations and HER2 amplications; (3) determine whether those MOC with wild-type HER2 and KRAS can alternatively activate the MAPK signaling pathway through BRAF mutation; and (4) apply the emerging paradigm classication system for BRAF mutations and predict the potential implication of anti-BRAF therapeutic strategy in MOC.


Abstract Background
In view of the encouraging clinical evidence of BRAF inhibitors that can treat some melanoma patients successfully, we aimed to investigate the status of BRAF mutations of primary mucinous ovarian carcinomas (MOC) in Taiwanese women, and apply the emerging paradigm classi cation of BRAF mutation groups.
Methods DNA was extracted from micro-dissected tissue samples using the QIAamp® DNA FFPE Kit. The mutations of activation segment (exon 15), CR3 (conserved regions 3), kinase domain of the BRAF gene were analyzed using the highly sensitive BRAF mutant enriched kit (FemtoPath®) with subsequent Sanger sequencing method. Additionally, we extended our prior data of HER2 aberrations and KRAS mutation into this study in order to compare with the status of BRAF mutation.

Discussion
Our results showed that 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 ampli cation, HER2 mutation and KRAS mutation were not mutually exclusive, but simultaneously independent. However, they may even have a synergistic effect in tumorigenesis.

Conclusions
The BRAF variant with T599I stands the majority. These ndings suggested that there was a lower potential response to the existing V600 BRAF inhibitors, but may be responsive to dual BRAF plus MEK inhibitors or single MEK inhibitor in MOC. Further studies are warranted to investigate the clinical bene ts of newly targeted therapy in recurrent or advanced stage MOC patients carrying each class of BRAF mutation.

Background
After careful exclusion of metastatic diseases, primary mucinous ovarian carcinoma (MOC) comprised less than 3% of all epithelial ovarian cancers. For years, there has been much discussion that MOC seems to be a unique disease that responds poorly to conventional chemotherapy regimens. Even though long survival and favorable outcome can occur in general with early diagnosis and optimal operation, recurrent and advanced disease are associated with poor prognosis. So far, neither modern guidelines, nor therapeutic consensus existed for the best management of recurrent or advanced MOC. [1][2][3] BRAF, a member of the rapidly accelerated brosarcoma (RAF) kinase family, is able to transduce signals downstream of RAS via the mitogen-activated protein kinase (MAPK) pathway. Under physiologic conditions, this pathway is tightly regulated through a negative feedback loop [4]. Activating mutations of BRAF can autonomously lead to uncontrolled cellular proliferation and cell survival. The anti-BRAF drugs are successfully used in clinical practice for melanoma and achieve favorable responses for low-grade serous ovarian carcinoma [5,6]. Based on that information, we aimed to explore the BRAF mutation status and evaluate whether it can be a potential therapeutic target for patients with recurrent or advanced stage MOC.
Based on the mechanisms for activation of the MAPK pathway, a new model categorized the BRAF mutations into the following: (1) class I variants are V600 mutations, monomer with high level of kinase activity and RAS signaling independence, (2) class II variants are non-V600 mutations, dimer with intermediate to high level of kinase activity and RAS signaling independence, (3) class III variants are non-V600 mutations, dimer with absent or impaired kinase activity and RAS signaling dependence, and (4) unclassi ed variants are other RAF mutations that having unknown function. [7] Currently, we not only investigated the mutational status of BRAF MOC in Taiwanese women, but also extended our prior data of HER2 aberrations and KRAS mutation of all samples tested (n = 20) to this study. After that, we can (1) assess the pairwise comparison of BRAF mutation with the respective KRAS mutation, HER2 mutation and HER2 ampli cation; (2) evaluate the prevalence of BRAF-based dual, triple and quadruple mutation sets including the co-existing BRAF mutation with KRAS mutations, HER2 mutations and HER2 ampli cations; (3) determine whether those MOC with wild-type HER2 and KRAS can alternatively activate the MAPK signaling pathway through BRAF mutation; and (4) apply the emerging paradigm classi cation system for BRAF mutations and predict the potential implication of anti-BRAF therapeutic strategy in MOC.

Methods
Originally, we had 21 MOC tissue specimens left over from previous studies. However, except for one case missing residual DNA and lacking of enough extra tumor component, the characteristics of all remaining study materials of all 20 cases of MOC were described in our previous report, including tissue retrieval and DNA preparation. [2,[8][9][10] Additionally, 7 normal ovarian tissues are used as negative controls. The research was conducted according to International Conference on Harmonization guidelines and complied with all applicable regulations for protection of human subjects of research, including review and approval by the Institutional Review Board, Chung-Shan Medical University Hospital Taichung, Taiwan.
In this study, we used the FemtoPath BRAF Mutation Screen Kit, also named Medaysis Ultra-Sensitive BRAF Mutation Detection Kit. The Medaysis Inc. US is the delegation agent of the HongJing Inc. TW in the United States. Since the Medaysis business model should use its own brand in US, Femtopath's products need to be changed to Medaysis labels in their own brand names. This Kit applies a CloDiA™ PCR method by means of novel and proprietary mutation enrichment technology, in which two types of skills are involved -Unindel™ PCR and Stuntmer™ PCR. [11,12] Unindel™ PCR is designed to identify a broad range of insertions/deletions (universal insertions/deletions) in the target region. Stuntmer™ PCR can detect a broad range of point mutations in sequence before and after V600 hot spots (amino acid range 591-620) in exons 15 of human BRAF gene. [13,14] Brie y, the sample nucleic acid with the mutation sequence is preferentially ampli ed over the wild type sequence by the self-competitive primers. PCR products were sequenced by the Sanger sequencing technique.
The pathogenicity associated with each BRAF mutations were identi ed in accordance with data of the Catalogue of Somatic Mutations in Cancer (COSMIC). The functional effect of novel BRAF missense mutations can be calculated using the Web software server Polymorphism Phenotyping v2 (PolyPhen-2), which can predict the possible impact of amino acid substitutions on the stability and function of human proteins using structural and comparative evolutionary considerations. [15] McNemar's test was used to assess the signi cance of the difference between 2 paired dichotomous oncogenomic status, including (1) BRAF mutation rates vs. KRAS mutation rates; (2) BRAF mutation rates vs. HER2 mutation rates; and (3) BRAF mutation rates vs. HER2 ampli cation rates, individually. Data were analyzed using standard statistical software, version 9.0 (SPSS, Inc., Chicago, IL). The test was 2sided and the signi cance level was 0.05.

Results
In this research, we found 4 cases (20%) with wild type BRAF and 16 cases (80%) with BRAF somatic missense mutations in all 20 cases tested. Of the 16 BRAF mutants, 1 had double missense mutations (T599I and S602F); 1 had a single missense mutation with an additional silent mutation (T599I and V600V); as well as 14 had single missense mutation including T599I (n = 9), A598V (n = 1), V600E (n = 1), V600M (n = 1) and S602F (n = 2). The missense mutation (S602F) is novel. All data of BRAF mutation analysis were based on the Catalogue of Somatic Mutations in Cancer (COSMIC) database. The locations and types of BRAF mutations are presented in Table 1 and Fig. 1. Neither insertion, nor deletion of BRAF gene was detected. On the other hand, no BRAF mutations were detected in the 7 normal ovarian tissues. In addition, our previously published raw data of HER2 ampli cations, HER2 mutations and KRAS mutations of all samples tested are integrated in Table 1.  (Fig. 2) We identi ed that the dual set with BRAF and KRAS mutations occurred in 10 cases (50%), the dual set with BRAF mutation and HER2 ampli cation occurred in 4 cases (20%), as well as the dual set with BRAF and HER2 mutations occurred in in 5 cases (25%). Additionally, the triple set with BRAF mutation, KRAS mutation and HER2 ampli cation occurred in 2 case (10%), the triple set with BRAF mutation, KRAS mutation and HER2 mutation occurred in 1 case (5%), as well as the triple set with BRAF mutation, HER2 mutation and HER2 ampli cation occurred in 2 case (10%). Nonetheless, the quadruple set with BRAF mutation, HER2 ampli cation, HER2 mutation and KRAS mutation occurred in none (0%). (Table 1) Adopting the newly classi cation scheme for BRAF mutations to MOC, we discovered 2 cases of class I BRAF mutants including V600E (n = 1) and V600M (n = 1); as well as 12 cases of class II BRAF mutants including A598V (n = 1), T599I (n = 10) and dual T599I/S602F (n = 1). No class III BRAF mutants were found. However, we identi ed 3 cases with the novel unclassi ed BRAF mutants, including single S602F (n = 2) and the dual T599I/S602F (n = 1) that are repeatedly counted. (Fig. 3)

Discussion
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-RAFmitogen-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 ampli cations, HER2 mutations and KRAS mutations with the new information of BRAF, we identi ed that their corresponding frequencies are 35% for HER2 ampli cations, 35% for HER2 mutations, 60% for KRAS mutations and 80% for BRAF mutations in all 20 MOC Taiwanese patients (Table 1). [9,10] Our ndings also indicated that there was no signi cant difference in the frequency between BRAF mutations and KRAS mutations when they were compared. However, the frequency of BRAF mutations was signi cant higher than that of HER2 ampli cations and HER2 mutations, respectively. (Fig. 2) Focusing on the basis of BRAF variant together with HER2 ampli cations, 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 identi ed 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 ampli cation, 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. ( 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 ampli cation of wild-type BRAF gene in human genomic DNA. [13,14] Although timeconsuming, 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-xed para n-embedded (FFPE) tissues, which restricted our analysis to only those tumor cells that express a speci c marker or have a speci c gene mutation. (2) According to the manufacture's manual and the previous report of the Stuntmer PCR technology, this highly speci c 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 ampli ed 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 ampli cation of mutants in a non-sequence speci c 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][13][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 classi cation 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 identi ed 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 ampli cation, 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 con dent and comprehensive, the case number cohort was small. Further exploratory studies should be performed to validate these nding.

Conclusions
Despite possible underestimation in some other studies, our solid data demonstrated that BRAF gene mutation rate was 80% of primary MOC in Taiwanese patients, using the FemtoPath BRAF Mutation Screen Kit. One novel BRAF mutation (S602F) has been identi ed in this study. Unlike melanoma and papillary thyroid carcinoma, the most common BRAF mutation of MOC is the non-V600 class II variant with T599I (n = 11/20; 55%) in Taiwanese. So far, there are no effective targeted treatments available for patients who have tumors or diseases harboring non-V600 BRAF mutations. [5,19] Our results highlight the importance of developing new anti-BRAF therapeutic options for such patients harboring non-V600 BRAF mutations. Alternatively, it indicated that the potential treatment strategy of recurrent or metastatic MOC might favor dual BRAF plus MEK inhibitor or single MEK inhibitor rather than the existing anti-BRAF V600 class I inhibitors. Further studies are warranted to investigate the clinical bene ts of class-speci c therapy in BRAF-altered metastatic or advanced stage MOC patients.   legend BRAF mutations are categorized into class-1 (high kinase activity, V600), class-2 (high or intermediate kinase activity, nonV600), class-3 (impaired BRAF kinase activity) and unclassi ed (unknown BRAF kinase activity). The "n" means number of cases existed. The symbol " †"means one case with dual missense mutations (T599I and S602F) of BRAF that are counted twice in class 2 and unclassi ed categories, disparately.