The Minimal Residual Disease in Non-Hodgkin's Lymphomas: From the Laboratory to the Clinical Practice

Abstract

Minimal residual disease (MRD) in non-Hodgkin's lymphomas (NHLs) still represents matter of interest and debate: indeed, the new available treatments offer higher rates of complete responses and MRD negativity than in the past, with a positive impact on the long-term survival. Furthermore, the introduction of more sensitive and accurate molecular techniques, such as digital PCR (ddPCR) and the next generation sequencing techniques (NGS), increased the possibility of identifying molecular targets to be followed after therapy (such as rearrangement of immunoglobulins, fusion genes, or mutations). This review focused on how molecular biology can help to detect MRD in different types of NHLs and how MRD can change the clinical practice in 2019. In follicular lymphoma (FL), contamination of the grafts and molecular disease persistence after transplantation represent a negative prognostic factors. The combination of Rituximab or Obinutuzumab with Bendamustine seems to be the most effective way to clear MRD in FL patients receiving chemo-immunotherapy (further studies are in progress), and also ⁹⁰Yttrium-Ibritumomab-Tiuxetan offers a deep clearance of molecular disease. Finally, molecular MRD can further stratify PET-negative cases, with subjects both PET- and MRD-negative presenting the best outcome. In aggressive lymphomas, MRD has a relevant prognostic power and can represent the platform for immunotherapy (such as CAR-T). In diffuse large B-cell lymphoma (DLBCL), the assessment of MRD in the plasma (where cell-free DNA and exosomes circulate) seems to be more predictive than the bone marrow analysis or peripheral blood mononuclear cells. Finally, NGS technologies could be more useful than the classical "patient allele-specific PCR" because they can identify any possible clone emerging during the treatment or follow-up, even if different from that identified at diagnosis, thus predicting relapse. After all, the present available molecular approaches can move MRD from the bench side to the clinical practice.

Keywords: MRD; NGS; NHL; PCR; QT-PCR; digital PCR; lymphoma; minimal residual disease.

Figure 1

The figure depicts an example of qualitative PCR for IGH rearrangement (according to the BIOMED strategy). In (A) is represented a B clone in a polyclonal context (MRD-positive); in (B) the IGH rearrangement appears as polyclonal (MRD-negative). Qualitative PCR has been performed by Genescan method (fluorescent PCR followed by the capillary electrophoresis on a automatic DNA sequencer).

Figure 2

The figure represents a comparison of results coming from two different labs. RQ-PCR for BCL2/JH rearrangement has been performed. As reported, the sensitivity of the test reached 1 × 10⁻⁵, and the quantitative ranges 1 × 10⁻⁴ and 5 × 10⁻⁵, respectively. The tested sample, MRD-positive at the first follow-up, became MRD-negative at the second control, then positive but not quantifiable (at the limit of detection), and finally MRD-positive again. In the bottom panel, are represented the real plots from MRD1 and MRD4.

Figure 3

The figure represents a ddPCR Fluorescent Amplitude Plot. The droplets contained into the red circle correspond to 1 × 10⁻⁴ BCL2/JH-positive cell line (limit of detection). The results were analyzed on the base of FAM fluorescence BCL2/JH-linked (Y-axis: channel 1) and corrected by the unspecific background fluorescence (X-axis: channel 2). The lines identified the threshold amplitudes of positive vs. negative signals (ch1: 3000 RFU), and specific vs. unspecific signals (ch2: 1000 RFU). Experimental session details: the experimental session was set up using three replicates of unknown samples (plasma cfDNA extracted by QIAamp Circulating Nucleic Acid Kit – Qiagen, Milan, Italy), six replicates of negative pooled samples, two replicate of diluted positive cell line (DOHH2) and two replicate of a No Target Control (NTC) sample. The cfDNA sample was tested also for housekeeping gene. All replicates reached >10,000 droplets, the cut off for defined as technically valid a ddPCR analysis. Patient was MRD-negative.

Figure 4

The figure represents a case where MRD was tested by NGS. In the part above the IGH clones found by the HashClone software [(91) BMC Bioninformatics], are detailed; in the bottom part the IGH frequencies clones describing MRD monitoring in diagnostic and follow-up samples are depicted. As reported, MRD at the follow-up became negative; BC = positive control; H₂O = negative control (water). MRD was performed using primer annealing IGH framework region 1 and JH loci, and MiSeq Illumina platform was used for the sequencing.