Wainman Lauren M, Huang Guohong, Green Donald C, Tsongalis Gregory J, Tafe Laura J, Khan Wahab A, Kaur Prabhjot, Shah Parth S, Karrs Jeremiah X
Dartmouth Hitchcock Medical Center, Lebanon, NH, USA.
Dartmouth Hitchcock Medical Center, Lebanon, NH, USA.
Exp Mol Pathol. 2025 Mar;141:104956. doi: 10.1016/j.yexmp.2025.104956. Epub 2025 Feb 15.
Lymphoplasmacytic lymphoma (LPL) is a B-cell lymphoproliferative disorder typically involving the bone marrow with infiltration by small lymphocytes and plasma cells. Studies have identified MYD88 L265P mutation as a diagnostic marker to distinguish LPL from other small B-cell lymphomas. Detection rates for this mutation have varied depending on the analytic methodology, with previous data suggesting that routine next-generation sequencing (NGS) does not demonstrate the required sensitivity to reliably detect MYD88 L265P. NGS has become part of routine clinical testing because it allows detection of variants across multiple genes. To study the utility of NGS in the detection of MYD88 L265P, we performed droplet digital PCR (ddPCR) and routine NGS on a cohort of 34 cases of lymphoid neoplasms (22 LPL, 4 CLL, 1 MCL, 1 MGUS, 2 plasma cell myeloma, and 4 negative bone marrow cases). We utilized manual review and BAMtools to assess MYD88 L265P in NGS cases. Limit of detection for ddPCR was determined to be 0.4 % variant allele frequency (VAF) with 10 ng DNA input. MYD88 L265P VAF detection by NGS and ddPCR was comparable down to 0.5 % VAF (R = 0.968). Setting an appropriate threshold for detection based on ddPCR results resulted in zero NGS false positives. We found that low tumor content did not impact the detection of MYD88 L265P by NGS. This study demonstrates that NGS can be a sensitive and reliable method for detection of MYD88 L265P with adequate coverage and specific assessment parameters.
淋巴浆细胞淋巴瘤（LPL）是一种B细胞淋巴增殖性疾病，通常累及骨髓，有小淋巴细胞和浆细胞浸润。研究已将MYD88 L265P突变鉴定为区分LPL与其他小B细胞淋巴瘤的诊断标志物。该突变的检测率因分析方法而异，先前的数据表明常规二代测序（NGS）未显示出可靠检测MYD88 L265P所需的灵敏度。NGS已成为常规临床检测的一部分，因为它可以检测多个基因的变异。为了研究NGS在检测MYD88 L265P中的效用，我们对34例淋巴样肿瘤患者（22例LPL、4例慢性淋巴细胞白血病、1例套细胞淋巴瘤、1例意义未明的单克隆丙种球蛋白血症、2例浆细胞骨髓瘤和4例骨髓阴性病例）进行了液滴数字PCR（ddPCR）和常规NGS检测。我们利用人工审查和BAMtools评估NGS病例中的MYD88 L265P。对于输入10 ng DNA的情况，ddPCR的检测限确定为0.4%的变异等位基因频率（VAF）。NGS和ddPCR检测MYD88 L265P的VAF在低至0.5% VAF时具有可比性（R = 0.968）。根据ddPCR结果设置合适的检测阈值可使NGS假阳性为零。我们发现低肿瘤含量不影响NGS对MYD88 L265P的检测。本研究表明，NGS在有足够覆盖范围和特定评估参数的情况下，可成为检测MYD88 L265P的灵敏且可靠的方法。
