Bhatia Prateek, Thakur Rozy, Sreedharanunni Sreejesh, Singh Minu, Malhotra Meenakshi, Arora Swati, George Ashish, Trehan Amita
Pediatric Hematology-Oncology Unit, Department of Pediatrics, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Pediatr Blood Cancer. 2025 Feb;72(2):e31443. doi: 10.1002/pbc.31443. Epub 2024 Nov 24.
Digital polymerase chain reaction (PCR) studies for clonal disease monitoring in B-acute lymphoblastic leukemia patients are currently limited due to the heterogeneous nature of mutations, which limit cost-effective assay designs.
In this study, 70 samples (14 relapse and 56 sequential therapy samples) were tested for 13 recurrent mutations identified on deep sequencing in our published cohort (KRAS, NRAS, NT5C2, PMS2, UHRF1, KMT2D, and TP53 genes) via a novel triplex digital PCR assay.
A total of seven major clones of NRAS [five] and NT5C2 [two] were noted in six out of 14 (43%) relapse patients, accounting for 44% of early relapses. In addition, 10 minor clones (PMS2 [two], NRAS [four], NT5C2 [three], and TP53 [one]) were noted in five out of 14 (36%) patients. In the 56 sequential therapy samples, six major clones were noted (NRAS [five], KRAS [one]) in four out of 14 (28.5%) patients, with two increasing in size in maintenance samples, leading to subsequent relapse in both cases. In addition, therapy-acquired minor clones in NT5C2 [four] and PMS2 [one] were seen to emerge in maintenance samples in four out of 14 (28.5%) patients, with concordant detection of such major and minor clones in unpaired relapse samples, indicating the need for their active surveillance during therapy. Overall, digital PCR validated NRAS and NT5C2 major clones in one-third (10 out of 27; 37%) of cases, driving nearly half of early relapses.
由于突变的异质性限制了具有成本效益的检测设计，目前用于监测B急性淋巴细胞白血病患者克隆性疾病的数字聚合酶链反应（PCR）研究受到限制。
在本研究中，通过一种新型的三重数字PCR检测方法，对70个样本（14个复发样本和56个序贯治疗样本）进行检测，以确定在我们已发表队列的深度测序中发现的13个复发性突变（KRAS、NRAS、NT5C2、PMS2、UHRF1、KMT2D和TP53基因）。
在14例（43%）复发患者中的6例中，共发现NRAS的7个主要克隆[5个]和NT5C2的2个主要克隆，占早期复发的44%。此外，在14例（36%）患者中的5例中发现了10个次要克隆（PMS2的2个、NRAS的4个、NT5C2的3个和TP53的1个）。在56个序贯治疗样本中，14例（28.5%）患者中的4例出现了6个主要克隆（NRAS的5个、KRAS的1个），其中2个在维持样本中大小增加，随后均复发。此外，在14例（28.5%）患者中的4例的维持样本中，发现了NT5C2的4个和PMS2的1个治疗获得性次要克隆，在未配对的复发样本中也检测到了这些主要和次要克隆，这表明在治疗期间需要对其进行积极监测。总体而言，数字PCR在三分之一（27例中的10例；37%）的病例中验证了NRAS和NT5C2主要克隆，导致近一半的早期复发。
