Hashimoto Yoko, Masunaga Nanae, Kagara Naofumi, Abe Kaori, Yoshinami Tetsuhiro, Tsukabe Masami, Sota Yoshiaki, Miyake Tomohiro, Tanei Tomonori, Shimoda Masafumi, Shimazu Kenzo
Department of Breast and Endocrine Surgery, Graduate School of Medicine, Osaka University, 2-2-E10 Yamadaoka, Suita 565-0871, Osaka, Japan.
Department of Breast Surgery, Osaka General Medical Center, 3-1-56, Bandai-Higashi, Sumiyoshi-ku, Osaka 558-8558, Osaka, Japan.
Cancers (Basel). 2023 May 6;15(9):2632. doi: 10.3390/cancers15092632.
mutations in breast cancer are one of the mechanisms of resistance to aromatase inhibitors. These mutations are common in metastatic breast cancer; however, these are rare in primary breast cancer. However, these data have been analyzed mainly in formalin-fixed, paraffin-embedded tissue; thus, rare mutations that may be present in primary breast cancer may be overlooked. In this study, we developed a highly sensitive mutation detection method called locked nucleic acid (LNA)-clamp droplet digital PCR (ddPCR) and validated it. The mutation detection sensitivity was substantiated to 0.003%. Then, we used this method to analyze mutations in fresh-frozen (FF) tissues of primary breast cancer. cDNA extracted from the FF tissues of 212 patients with primary breast cancers were measured. Twenty-eight mutations were found in twenty-seven (12.7%) patients. Sixteen (7.5%) patients had Y537S mutations and twelve (5.7%) had D538G mutations. Two mutations with a variant allele frequency (VAF) of ≥0.1% and twenty-six mutations with a VAF of <0.1% were found. By using this LNA-clamp ddPCR, this study demonstrated the presence of minor clones with a VAF of <0.1% in primary breast cancer.
乳腺癌中的突变是对芳香化酶抑制剂耐药的机制之一。这些突变在转移性乳腺癌中很常见；然而，在原发性乳腺癌中却很少见。然而，这些数据主要是在福尔马林固定、石蜡包埋的组织中分析的；因此，原发性乳腺癌中可能存在的罕见突变可能会被忽视。在本研究中，我们开发了一种名为锁核酸（LNA）-夹液滴数字PCR（ddPCR）的高灵敏度突变检测方法并进行了验证。突变检测灵敏度被证实为0.003%。然后，我们使用该方法分析原发性乳腺癌新鲜冷冻（FF）组织中的突变。对从212例原发性乳腺癌患者的FF组织中提取的cDNA进行了检测。在27例（12.7%）患者中发现了28个突变。16例（7.5%）患者有Y537S突变，12例（5.7%）有D538G突变。发现了2个变异等位基因频率（VAF）≥0.1%的突变和26个VAF<0.1%的突变。通过使用这种LNA-夹ddPCR，本研究证明了原发性乳腺癌中存在VAF<0.1%的微小克隆。
