Dottorato in Medicina Molecolare

Minimal residual disease (MRD) detection by real-time quantitative PCR (RQ-PCR) in Mantle Cell Lymphoma (MCL): preliminary analysis of patients enrolled in the FIL study “MCL0208”

 INTRODUCTON

Mantle cell lymphoma (MCL) is an aggressive neoplasia accounting for 6-8% of all non-Hodgkin's lymphomas and is characterized by the translocation t(11;14)(q13;q32) and the overexpression of cyclin D1. Despite considerable therapeutic progress in the last years, MCL remains a disease difficult to manage, characterized by a poor prognosis in the medium-long term. High-dose chemotherapy, supported by autologous stem cell transplantation (ASCT) is the current standard of care for younger patients, generally providing high response rates and long progression-free survival (PFS), but relapse eventually occurs and patients usually die because of disease progression (Dreyling, Leukemia 2014). However, nowadays MCL has revealed as a highly heterogeneous disease, with some cases extremely aggressive and refractory and others characterized by a better outcome, with stable post-therapy remissions. Therefore, there is urgent need to adapt therapy to the pleomorphic presentation of the disease. The chance to personalize the treatment on the specific characteristics of each patient is made possible by the availability of some validated prognostic tools, such as the MIPI (MCL international prognostic index) and the Ki-67 proliferative index, as well as of early predictors of treatment response, like minimal residual disease (MRD) analysis (Dreyling, Annals of Oncology 2014).

MRD analysis by allele-specific oligonucleotide (ASO) PCR is able to detect very low levels of residual tumor cells (up to 1 tumor cell out of 100000 healthy cells) in patients achieving complete clinical response (CR) after treatment. This tool, currently applicable to about 90% of MCL patients is an effective early predictor of outcome, showing independent prognostic value in large patients series and demonstrating superior than the CR achievement in multivariate analysis (Pott, Blood 2010). Moreover, MRD prospective assessment is able to early identify patients with increasing risk of upcoming relapse, monitoring those patients experiencing “MRD reappearance” (as to say, again positive MRD results) and thus prone to relapse in the course of the next years): these “high-risk” patients could be ideal subjects to receive pre-emptive treatments, aimed at avoiding a more challenging full-blown relapse. Thus, in the near future MRD analysis might be used to stratify MCL patients into different risk classes, to whom offer a personalized treatment, as already happening in other hematological tumors, such as acute lymphoblastic leukemia (Zugmaier G, Blood 2015).

 PATIENTS AND METHODS

 MCL0208 trial enrolled 302 adult patients with newly diagnosed MCL, according to the WHO criteria. Upon completion of the diagnostic phase, eligible patients entered in the treatment phase consisting in an induction phase (3 cycles of R-CHOP, given every 21 days) followed by a consolidation phase (high-dose cyclophosphamide with rituximab (R-CTX), 2 cycles of high-dose Cytarabin with rituximab (R-Ara-C), BEAM and ASCT). Patients who achieved complete or partial response (after ASCT) were randomized between maintenance with lenalidomide or observation.

MRD has been assessed by both qualitative nested PCR and quantitative real time PCR on BM and PB samples at different time points: 1) At Diagnosis; 2) After R-CHOP and high-dose cyclophosphamide; 3) First stem cell collection; 4) Second stem cell collection (performed only if the first stem cell harvest will result PCR positive or if patients lack a molecular marker); 5) Prior to ASCT; 6) Prior to randomization; 7) During maintenance/observation at 6-12-18-24-30 months (both arms).

Nested PCR and RQ-PCR for MRD monitoring (Bcl-1/IgH rearrangements - MCL).

Qualitative PCR amplification of the t(11;14) translocation was performed using a seminested PCR, in the first amplification a sense primer (5′-GAAGGACTTGTGGGTTGC-3′) and in the second PCR reaction a sense primer (5′-GCTGCTGTACACATCGGT-3′). Both amplifications were performed with a region (JH) consensus primer (5′- ACCTGAGGAGACGGTGACC-3′). The nature of the rearrangements was confirmed by direct sequencing. RQ-PCR was performed with a MTC forward primer (5’-AGCTTTCTAAAAAGTGGTTTTGTT-3’) together with a specific ASO primer (designed in N insertion) and MTC probe (5’-TGAATATGACATTCTGAAACAGAAAA-3’). All standard curves were performed in an AbiPrism 7900HT sequence detector system (LifeTechnologies).[Ladetto BBMT2006; Pott MMB2013]

Nested PCR and RQ-PCR for MRD monitoring (IgH VDJ rearrangements - MCL).

The technique was based on amplification and direct-sequencing of gDNA using consensus sense primers derived from the leader and first framework region (FR1) and a consensus antisense primer derived from the FR4. Nested PCR amplification was performed using ASO primers designed on the Complementarity Determining Region 2 (CDR2) and CDR3 for each IgH sequence identified. For RQ-PCR analysis, specific consensus reverse TaqMan probe was derived from the FR3. Standard curves were prepared by serial 10-fold dilutions, starting from 500 ng, obtained from the diagnostic tissue diluted in polyclonal DNA derived from equivalent amounts of 5 healthy donors.[Ladetto JCO2010]

For all cases, the calculation of MRD was based on comparative cycle threshold analysis between follow-up samples and standards according to Euro-MRD criteria (Van der Velden Leukemia 2007). RNase P gene was used as a reference standard gene for DNA quality and normalization [Pott MMB2013]. 

Droplet Digital PCR (DDPCR)

 ddPCR was performed with the QX100 Droplet Digital PCR system (Bio-Rad Laboratories, Hercules, CA). gDNA samples were loaded in triplicate with the use of either the manufacturer-recommended 100 ng gDNA dose, or an increased amount of 500 ng (aiming at greater sensitivity).

The 20 µL ddPCR reaction included 10 µL of 2x ddPCR Master Mix (Bio-Rad Laboratories), 1 µL of 20x primers and probe (final concentration, 500 nmol/L and 200 nmol/L), and 5 µL of gDNA. Of note, ddPCR experiments used the same primers and probes used in qPCR, with the identical nucleotide sequence, although MGB or BHQ-1 quenchers were used instead of TAMRA (Primmbiotech, West Roxbury, MA). Droplets were generated by a QX100 droplet generator device, and end point PCR was performed on a T100 Thermal Cycler (Bio-Rad Laboratories) following the manufacturer’s recommendations. PCR products were loaded into the QX100 droplet reader and analyzed by QuantaSoft version 1.2 (Bio-Rad Laboratories). Each experiment included a positive control sample (diagnostic sample gDNA) and a negative control (pool of PB mononuclear cells from 10 healthy donors, or MCF-7 gDNA). The final tumor load was calculated as a mean of all available technical replicates that were considered reliable when giving reproducible amplification after application of a Poisson correction.

 RESULTS

The multicenter, randomized phase III FIL- MCL0208 expected to enroll 300 adult patients ( <65 years) with MCL. At the time (October 2015) 42 centers are active (41 Italian and Portuguese 1) and 302 patients have been enrolled in the study.

   Of all the 302 patients enrolled we received at least a diagnostic sample of PB or BM (mostly both)   adequate for molecular studies.

265 cases (88%) did have a valid molecular marker (BCL-1, IGH or both). The vast majority of cases without a molecular marker showed a BM infiltration inferior to 3%.

We have collected 221 samples at the first restaging, 205 at the first leukapheresis, 42 at the second leukapheresis, 177 at restaging pre-ASCT, 168 post- ASCT, 107 at the first follow-up (FU), 81 at second FU, 43 at third FU, 30 at fourth FU, 20 at fifth FU and 12 at sixth FU.