(2019). Digital droplet PCR and next-generation sequencing refine minimal residual disease monitoring in acute lymphoblastic leukemia. Leukemia & Lymphoma. Ahead of Print.

Minimal/measurable residual disease (MRD) evaluation has resulted in a fundamental instrument to guide patient management in acute lymphoblastic leukemia (ALL). From a methodological standpoint, MRD is defined as any approach aimed at detecting and possibly quantifying residual neoplastic cells beyond the sensitivity level of cytomorphology. The molecular methods to study MRD in ALL are polymerase chain reaction (PCR) amplification-based approaches and are the most standardized techniques. However, there are some limitations, and emerging technologies, such as digital droplet PCR (ddPCR) and next-generation sequencing (NGS), seem to have advantages that could improve MRD analysis in ALL patients. Furthermore, other blood components, namely cell-free DNA (cfDNA), appear promising and are also being investigated for their potential role in monitoring tumor burden and response to treatment in hematologic malignancies. Based on the review of the literature and on our own data, we hereby discuss how emerging molecular technologies are helping to refine the molecular monitoring of MRD in ALL and may help to overcome some of the limitations of standard approaches, providing a benefit for the care of patients.

Summary: Since 2000, we have investigated 67 consecutive patients with stage I/II follicular lymphoma (FL) for the presence of BCL2/IGH rearrangements by polymerase chain reaction (PCR), real time quantitative PCR (RQ‐PCR) and digital droplet PCR (ddPCR). All patients were treated with involved‐field radiotherapy (IF‐RT) (24–30 Gy). From 2005, patients with minimal residual disease (MRD) after IF‐RT received rituximab (R) (375 mg/m², 4 weekly administrations). The median follow‐up is 82 months (17–196). At diagnosis, 72% of patients were BCL2/IGH+. Progression‐free survival (PFS) was significantly better in patients with undetectable/low levels (<10⁻⁵) of circulating BCL2/IGH+ cells at diagnosis and in those who were persistently MRD− during follow‐up (P = 0·0038). IF‐RT induced an MRD− status in 50% of cases; 16/19 (84%) MRD+ patients after IF‐RT became MRD− after R treatment. A significantly longer PFS was observed in MRD+ patients treated with R compared to untreated MRD+ patients (P = 0·049). In early stage FL, both circulating levels of BCL2/IGH+ cells at diagnosis and MRD status during follow‐up bear prognostic implications. Standard IF‐RT fails to induce an MRD‐negative status in half of patients. Most patients become MRD− following treatment with R and this is associated with a significantly better PFS.

In Ph+ acute lymphoblastic leukaemia (Ph+ ALL), minimal residual disease (MRD) is the most relevant prognostic factor. Currently, its evaluation is based on quantitative real‐time polymerase chain reaction (Q‐RT‐PCR). Digital droplet PCR (ddPCR) was successfully applied to several haematological malignancies. We analyzed 98 samples from 40 Ph+ ALL cases, the majority enrolled in the GIMEMA LAL2116 trial: 10 diagnostic samples and 88 follow‐up samples, mostly focusing on positive non‐quantifiable (PNQ) or negative samples by Q‐RT‐PCR to investigate the value of ddPCR for MRD monitoring. DdPCR BCR/ABL1 assay showed good sensitivity and accuracy to detect low levels of transcripts, with a high rate of reproducibility. The analysis of PNQ or negative cases by Q‐RT‐PCR revealed that ddPCR increased the proportion of quantifiable samples (p < 0.0001). Indeed, 29/54 PNQ samples (53.7%) proved positive and quantifiable by ddPCR, whereas 13 (24.1%) were confirmed as PNQ by ddPCR and 12 (22.2%) proved negative. Among 24 Q‐RT‐PCR‐negative samples, 13 (54.1%) were confirmed negative, four (16.7%) resulted PNQ and seven (29.2%) proved positive and quantifiable by ddPCR. Four of 5 patients, evaluated at different time points, who were negative by Q‐RT‐PCR and positive by ddPCR experienced a relapse. DdPCR appears useful for MRD monitoring in adult Ph+ ALL.