In patients with invasive breast cancer, fluorescence in situ hybridization (FISH) testing for HER2 typically demonstrates the clear presence or lack of ERBB2 (HER2) amplification (i.e., groups 1 or 5). However, a small subset of patients can present with unusual HER2 FISH patterns (groups 2–4), resulting in diagnostic confusion. To provide clarity, the 2018 CAP/ASCO HER2 testing guideline recommends additional testing using HER2 immunohistochemistry (IHC) for determining the final HER2 status. Despite this effort, the genomic correlates of unusual HER2 FISH groups remain poorly understood. Here, we used droplet digital PCR (ddPCR) and targeted next-generation sequencing (NGS) to characterize the genomic features of both usual and unusual HER2 FISH groups. In this study, 51 clinical samples were selected to represent FISH groups 1–5. Furthermore, group 1 was subdivided into two groups, with groups 1A and 1B corresponding to cases with HER2 signals/cell ≥6.0 and 4–6, respectively. Overall, our findings revealed a wide range of copy number alterations in HER2 across the different FISH groups. As expected, groups 1A and 5 showed the clear presence and lack of HER2 copy number gain, respectively, as measured by ddPCR and NGS. In contrast, group 1B and other uncommon FISH groups (groups 2–4) were characterized by a broader range of HER2 copy levels with only a few select cases showing high-level gain. Notably, these cases with increased HER2 copy levels also showed HER2 overexpression by IHC, thus highlighting the correlation between HER2 copy number and HER2 protein expression. Given the concordance between the genomic and protein results, our findings suggest that HER2 IHC may inform HER2 copy number status in patients with unusual FISH patterns. Hence, our results support the current recommendation for using IHC to resolve HER2 status in FISH groups 2–4.

在浸润性乳腺癌患者中，HER2 的荧光原位杂交 (FISH) 检测通常可以明确显示是否存在ERBB2 ( HER2 ) 扩增（即第 1 组或第 5 组）。然而，一小部分患者可能会出现异常的HER2 FISH 模式（第 2-4 组），从而导致诊断混乱。为明确起见，2018 年 CAP/ASCO HER2 检测指南建议使用 HER2 免疫组织化学 (IHC) 进行额外检测以确定最终 HER2 状态。尽管做出了这些努力，但异常HER2的基因组相关性对 FISH 组仍然知之甚少。在这里，我们使用液滴数字 PCR (ddPCR) 和靶向下一代测序 (NGS) 来表征常见和不常见HER2 FISH 组的基因组特征。在这项研究中，选择了 51 个临床样本来代表 FISH 组 1-5。此外，第 1 组被细分为两组，1A 组和 1B 组分别对应于HER2信号/细胞≥6.0 和 4-6 的病例。总体而言，我们的研究结果揭示了不同 FISH 组中HER2的广泛拷贝数变化。正如预期的那样，第 1A 组和第 5 组显示出明显存在和缺乏HER2分别通过 ddPCR 和 NGS 测量的拷贝数增益。相比之下，1B 组和其他不常见的 FISH 组（第 2-4 组）的特点是HER2拷贝水平范围更广，只有少数选定病例显示出高水平增益。值得注意的是，这些HER2拷贝水平增加的病例也通过 IHC 显示 HER2 过表达，从而突出了HER2拷贝数和 HER2 蛋白表达之间的相关性。鉴于基因组和蛋白质结果之间的一致性，我们的研究结果表明 HER2 IHC 可能会告知具有异常 FISH 模式的患者的HER2拷贝数状态。因此，我们的结果支持当前使用 IHC 解决 FISH 组 2-4 中 HER2 状态的建议。