Minimal Residual Disease Monitoring By 8-Color Flow Cytometry in Mantle Cell Lymphoma Is Complementary to Q-PCR Monitoring and Will Facilitate Pre-Emptive Treatment: An EU-MCL and Lysa Study
Authors: Cheminant Morgane , Schmit Stephanie , Touzart Aurore , Derrieux Coralie , Delfau-Larue Marie-Hélène , Thieblemont Catherine , Ribrag Vincent , Ysebaert Loic , Jardin Fabrice , Cheze Stephane , Lefrère Francois , Delarue Richard , Pott Christiane , Hoster Eva , Dreyling Martin , Asnafi Vahid , Hermine Olivier , Macintyre Elizabeth 
Published: October 2019

Abstract
Introduction: Mantle Cell Lymphoma (MCL) is characterized by frequent blood and bone marrow involvement. It has been demonstrated that use of Minimal Residual Disease (MRD) quantification in blood and/or bone marrow might be helpful in patient management. Gold standard MRD is based on Q-PCR clone specific amplification of IgH VDJ or IgH-BCL1 rearrangements, but these are relatively complex and time consuming and over half of the positive results are in a grey zone of borderline positivity. Flow cytometry (FCM) is more rapid and better adapted to individual patient management if quantitatively reproducible, but insufficiently sensitive when only 4 colors are used. We therefore developed a universal, 8-color, EuroFlow inspired, FCM strategy, which we compared with classical Q-PCR MRD in 61/97 patients included in (and 1 treated according to) the EU-MCL Younger and Elderly prospective trials who underwent Q-PCR MRD monitoring at Necker Hospital.
Method: Q-PCR MRD from IgH VDJ (n=92) or BCL1-IgH (n=5) was performed prospectively from ficolled blood (PB) or bone marrow, from which residual material was cryopreserved in DMSO for FCM quantitation, using 10 antibodies labelled with 8 fluorochromes for positive and negative (CD45, CD19, CD5, LAIR1, CD11a, IGK, IGL, CD3, CD14 and CD56) gating, after diagnostic phenotyping of fresh material, using the same panel and a EuroFlow B lymphoid screening tube. Sensitivity of both techniques was at least 0.01% (1E-04). FCM was only considered positive if above 0.01%, whereas Q-PCR results were considered positive below quantifiable range (BQR) if borderline, above sensitivity, within Euro-MRD criteria for MRD positivity. BQR samples were separated based on the number of positive, triplicate samples. The objectives were to compare the two techniques and to determine their suitability for regular screening, with a view to pre-emptive treatment on molecular or phenotypic (MRD) relapse. Two patients were treated with Rituximab at MRD relapse, prior to clinical relapse, as proof of principle.
Results: A total of 302 blood or bone marrow samples from 62 patients were quantified. Overall, 79% (42/53) of samples positive at or above 0.01% by PCR were also positive by FCM, compared to 29% (19/65) of those below 0.01%, but with at least 2 positive triplicates and virtually none of those with only 1 or no results above sensitivity (1%, 2/184). Quantification of the paired MRD results positive with PCR and/or FCM were significantly correlated (r2=0.74, P<0.0001).
Amongst the 62 patients, 30 have relapsed and 19 have died. Nine relapsing patients (including one off protocol patient treated and monitored at initial and second MRD relapses) had sufficient MRD points to assess the capacity of PB Q-PCR or FCM to predict future clinical relapse sufficiently to justify pre-emptive treatment at MRD relapse. Clinical relapse was preceded by MRD relapse in 9/10 relapses by Q-PCR and 7/9 by FCM. Six of the 9 relapsing patients had achieved Q-PCR negativity in at least one PB sample. The mean latency for prediction by Q-PCR, when considering any increase in positivity to at least 2 positive triplicates as positive, was 11.3 months (range 1-24mths) and 5.4 months (0.5-11) when only results above 0.01% were considered positive. The equivalent latency by FCM was slightly shorter, at 6.5 months (0.5-21) Pre-emptive treatment of 2 patients at MRD relapse, prior to clinical relapse allowed re-establishment of molecular complete remission and a durable second remission in at least one with sufficient follow-up (Cf Fig.).
Figure 1 Figure 1.
Conclusion: Eight color flow cytometry is a promising alternative to classical clone-specific Q-PCR strategies in monitoring therapy in MCL, with an excellent correlation (29/31, 94%) for MRD levels of at least 0.1% and acceptable correlation at 0.01-0.1% (13/22, 59%). While less sensitive at very low levels on cryopreserved material, FCM may clarify the clinical relevance of low-level borderline positivity; however it remains to be determined prospectively which technique will have greater prognostic value in patient management. FCM sensitivity will be improved by prognostic testing of fresh whole blood or bone marrow, and this pilot data clearly justifies such studies. Finally, MRD relapse precedes clinical relapse by several months, justifying pre-emptive treatment, monitored by prospective FCM and IgH Q-PCR within clinical trials.

Disclosures
Dreyling: Roche: Honoraria, Research Funding.