Concordance and Discordance Rates of V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF)V600E Status in Metastatic against Primary Lesion of Melanoma: A Meta-analysis

New effective molecular-targeted agents (1) including combination serine-threonine protein kinase BRAF inhibitor and mitogen-activated protein kinase kinase (MEK) inhibitor (BRAF/MEK inhibitor) therapy have improved overall survival (OS) and progression-free survival (PFS) of patients with metastatic melanoma with BRAF mutations (2), (3), . Observational studies suggest that 40%-60% Caucasians and < 30% Japanese are BRAF mutation positive . BRAF/MEK inhibitors are only approved for BRAF mutation-positive patients because they are only effective against lesions with this mutation . The mutational status of metastatic lesions cannot easily be tested because melanoma metastasizes to subcutaneous and superficial lymph nodes, visceral areas, and deep lymph nodes; therefore, in such cases, whether BRAF/MEK inhibitors are suitable for patients with melanoma is unclear . In clinical practice, primary skin lesions resected during primary treatment are genetically tested to predict the mutational status of inaccessible metastatic lesions. Some cases might have BRAF mutation-positive primary lesions, but have negative metastatic lesions, or vice versa. In the former, BRAF/MEK inhibitors would not be effective, whereas in the latter, the patient loses the opportunity to receive effective treatment. Disagreement in the proportion of BRAF mutations between primary and metastatic lesions varies . A previous study did not address the probability of agreement for metastasis when the primary lesion was BRAF mutation positive or BRAF mutation negative. Moreover, disagreement proportions for all BRAF mutations were included, although molecular-targeted therapy is currently approved only for patients with BRAF. Calculating the mutation probability in metastatic lesions when the primary lesion mutation is known allows the more appropriate use of BRAF/MEK inhibitors. The probability of BRAF-positive [BRAF(+)] metastatic lesions when the primary cancer lesion was BRAF(+) and BRAF-negative [BRAF(−)] metastatic lesions when the primary cancer lesion was BRAF(−) was calculated.

The mutational status of metastatic lesions cannot easily be tested because melanoma metastasizes to subcutaneous and superficial lymph nodes, visceral areas, and deep lymph nodes; therefore, in such cases, whether BRAF/MEK inhibitors are suitable for patients with melanoma is unclear (7) . In clinical practice, primary skin lesions resected during primary treatment are genetically tested to predict the mutational status of inaccessible metastatic lesions.
Some cases might have BRAF V600E/K mutation-positive primary lesions, but have negative metastatic lesions, or vice versa. In the former, BRAF/MEK inhibitors would not be effective, whereas in the latter, the patient loses the opportunity to receive effective treatment. Disagreement in the proportion of BRAF mutations between primary and metastatic lesions varies (8) . A previous study did not address the probability of agreement for metastasis when the primary lesion was BRAF mutation positive or BRAF mutation negative. Moreover, disagreement proportions for all BRAF mutations were included, although molecular-targeted therapy is currently approved only for patients with BRAF V600E/K (8) .
Calculating the mutation probability in metastatic lesions when the primary lesion mutation is known allows the more appropriate use of BRAF/MEK inhibitors. The probability of BRAF V600E -positive [BRAF(+)] metastatic lesions when the primary cancer lesion was BRAF(+) and BRAF V600E -negative [BRAF(−)] metastatic lesions when the primary cancer lesion was BRAF(−) was calculated.

Search methodology and inclusion/exclusion criteria
We searched the PubMed, Cochrane Library, and Igaku Chuo Zasshi databases for observational studies on November 23, 2017. Search criteria with MeSH terms were published elsewhere (9) . One investigator selected articles that potentially met the criteria on the basis of their titles and abstracts. For eligible studies, the same investigator abstracted the data independently using a predefined form. Searched studies used real-time PCR, Sanger sequencing, and BRAF V600E -specific immunohistochemistry to detect mutants. Therefore, we set the outcome of BRAF V600E mutations as those detected by the searched methods and that were predominant in 79%-90% of patients (10), (11) . We included studies investigating BRAF V600E status between primary melanoma and metastatic lesions in the same patients, irrespective of testing methods, and those without BRAF V600E testing were excluded.

Statistical analysis
We integrated the probabilities of BRAF V600E positive and negative in metastatic lesions when primary lesions were BRAF V600E positive and negative, respectively: BRAF(+) metastatic lesion given the primary lesionis BRAF(+), and BRAF(−) metastatic lesion given the primary lesionis To integrate the probabilities and proportions, we used DerSimonian-Laird estimation (random effect model) and fixed-effects models for meta-analysis. Results are shown as forest plots, and publication bias was checked by funnel plots. We used R x64 V.4.4.2 software for statistical analyses and illustrations. All P-values are two-sided, and P < 0.05 indicated statistical significance.
This study was approved by the ethics committee of the School of Medicine, University of Yamanashi (approval number: 1894), in accordance with the ethical guidelines and regulations of the Declaration of Helsinki.
BRAF/MEK inhibitors have changed metastatic melanoma treatment. In the beginning, BRAF inhibitor monotherapy has improved OS and PFS in BRAF V600E/K patients (33) , (34) . Thereafter, randomized controlled trials showed combination therapy with BRAF/MEK inhibitors was superior to BRAF inhibitor monotherapy regarding OS and PFS (2) . Moreover, a lower frequency of adverse events (rash, alopecia, and skin tumors) was observed for combination therapy compared with that for monotherapy (2), (3) . Thus, combination therapy has become a standard approach (7) .
BRAF V600E/K testing in primary lesions is critical for patients with metastasis who may respond to BRAF/MEK inhibitors (35) . Currently, agents are usually administered to patients according to genetic tests performed on primary lesion tissues. Our findings might convince clinicians that metastatic lesions in 82%-87% of patients with BRAF V600E -positive primary lesions are sensitive to BRAF/MEK inhibitors (Figure 1,  Figure 3), whereas 13%-18% would not show an initial response to therapy in clinical trials. Therefore, clinicians might be encouraged to test for BRAF mutations in metastatic lesions, because patients with discrepant results between pri-DOI: 10.31662/jmaj.2020-0016 JMA Journal: Volume 3, Issue 3 https://www.jmaj.jp/ mary and metastatic lesions (14%-18% probability) are often disadvantaged in relation to treatment decisions. Our study had several strengths. We accumulated the re-sults of 20 worldwide studies, including 15 in a previous metaanalysis of disagreement (8) . We focused on BRAF V600E , the most frequent and clinically important mutation in melanoma.  The present study also had some limitations. Selection bias was possible; collated data were from patients who underwent genetic tests, which might decrease the generalizability of the results. Genetic testing methods varied among studies. Ethnic background and stage of melanoma varied and should be considered for treatment decisions after genetic testing. High heterogeneity (I 2 = 72%-80%) was observed in this metaanalysis. Therefore, the agreement and disagreement proportions might vary among regions.
In conclusion, this meta-analysis revealed 82% of patients had BRAF V600E -positive metastatic lesions with BRAF V600E -positive primary lesions and 82% of patients with BRAF V600E -negative primary lesions had BRAF V600E -negative metastatic lesions. The proportion of disagreement between BRAF V600E mutations in primary and metastatic lesions was 13%. Over-reliance on genetic results of primary tumors might prevent patients with discrepant results receiving appropriate treatment for metastatic lesions. Genetic testing for BRAF V600 mutations using metastatic tumor samples is suggested, if available, without invasive biopsies.

Sources of Funding
This work was partly supported by the Japan Society for the Promotion of Science grant (KAKENHI Grant Number JP18K17376) to HY. The funding source had no role in the design of this study and will not have any role during its execution, analyses, interpretation of the data, or decision to submit results.

Author Contributions
Conception/design: Inozume T, Yokomichi H Acquisition of data: All authors Data analysis: Yokomichi H Interpretation: All authors Manuscript writing: Inozume T, Yokomichi H Final approval of manuscript: All authors Hiroshi Yokomichi and Takashi Inozume contributed equally to this work.

Approval by Institutional Review Board (IRB)
This study was approved by the ethics committee of the School of Medicine, University of Yamanashi (approval number: 1894), in accordance with the ethical guidelines and regulations of the Declaration of Helsinki.

Disclaimer
Zentaro Yamagata is one of the Editors of JMA Journal and on the journal's Editorial Staff. He was not involved in the editorial evaluation or decision to accept this article for publication at all.