Peripheral T-cell lymphomas (PTCL) account for 10 to 15% of all non-Hodgkin lymphomas worldwide.1 Outcome for most PTCL patients is poor after conventional anthracycline-based regimens with 5-year overall survival probabilities of 30 to 40% in most studies.2, 3, 4 High-dose chemotherapy with autologous stem cell transplantation (ASCT) in first CR (CR1) or for refractory disease is widely used, although most patients will still relapse after the procedure. Even when done in CR1, more than half of PTCL patients who undergo ASCT relapse and succumb from their disease,5 likely explained by the persistence of the malignant clone at the time of ASCT.
The detection of molecular MRD in ALK-positive anaplastic large cell lymphoma (ALCL) has been established as a strong predictor of treatment failure and imminent clinical relapse.6 The evaluation of MRD in other PTCL subtypes has not been reported. In this study, we retrospectively investigated the prognostic impact of minimal detectable disease (MDD) on the outcome of 29 PTCL patients undergoing ASCT at our institutions using an innovative multi-parameter flow cytometry approach.
We retrospectively analysed the data from 29 patients with PTCL who underwent ASCT between April 2004 and July 2014 on the Fred Hutchinson Cancer Research Centre (FHCRC, Seattle, WA, USA) protocols for patients with haematological malignancies. Protocols were approved by the Institutional Review Board of the FHCRC and all patients signed consent forms approved by the institutional review board.
Patients with any subtype of mature peripheral T-cell lymphoma were included. Diagnoses were confirmed using 2008 WHO criteria by board-certified haematopathologists as part of routine clinical care. Disease status at ASCT was defined by the standard 1999 and 2007 Cheson criteria, with the latter used when PET/CT data were available (n=22).
Sixteen patients (55%) received BEAM, seven (24%) received 6-grey TBI followed by CyVP-16 whereas six patients (21%) were treated with alternative regimens, including 131I-BC8 (anti-CD45) Ab (n=2), or cyclophosphamide plus TBI (n=4). Clinical follow-up and surveillance imaging studies were performed at the discretion of the referring physician. Stem cell source was peripheral blood in all patients (n=29).
Flow cytometric immunophenotyping of an EDTA anticoagulated bone marrow aspirate specimen was performed before stem cell collection, using a standard bone marrow assay with erythrocyte cell lysis. Flow cytometry was performed on a modified 4-laser, 10-colour Becton Dickinson LSR II flow cytometer (BD Biosciences, San Jose, CA) as previously described.7 Gating reagents, including Ab and fluorochrome combinations have been detailed for standard clinical analysis of T cells at our institution.8 Positive MDD (MDD+) was defined as the identification of an abnormal T-cell population using these different combinations. When indicated, analysis of TCR V-beta repertoire was performed using a modified protocol based on the IO Test Beta Mark (Beckman Coulter, Fullerton, CA) flow cytometry assay.9 We have demonstrated the usefulness of this modified approach to assess TCR V-β clonality in T-cell neoplasms without a priori knowledge of the TCR V-β isoform restriction and details regarding the methodology has been thoroughly described previously.10
Patient characteristics according to the presence (n=7) or absence (n=22) of MDD are detailed in Table 1. In seven patients (21%), FC studies detected pre-ASCT MDD from BM samples (range: 0.01–0.15% of total leukocytes, following erythroid lysis). In two of these patients BM involvement was also identified by morphologic examination. MDD was also evaluated after ASCT at various time points in five patients and remained negative over time (pre-ASCT MDD was negative in all five patients).
We observed in total 8 deaths and 14 relapses after ASCT. The 4-year estimated probability of overall survival (OS) was 48% and the cumulative incidence of relapse (CIR) was 52%. Median follow-up for patients free of relapse after ASCT was 52 months (range: 7–89).
In univariate analysis, pre-ASCT MDD (Figure 1, 85 versus 36%, P<0.001), IPI⩾2 (65 versus 26%, P=0.03), number of prior therapies>1 (69 versus 33%, P=0.04) and absence of CR1 (68 versus 14%) were associated with significantly higher CIR. Regarding OS, pre-ASCT MDD+ (19 versus 89%, P<0.001) and IPI⩾2 (58 versus 100%, P=0.03) significantly impacted survival.
Pre-ASCT was positive in four patients with a negative PET scanner (CR per Cheson 2007 criteria). All four patients relapsed shortly after ASCT (time to relapse: 3–15 months) and all eventually died from the disease (time to death: 7–37 months).
ASCT in PTCL is commonly used as consolidation therapy in CR1 and for patients with relapsed or refractory disease.11 However, the prognosis of PTCL patients remains poor with this strategy for most subtypes, due to high relapse rates (56–70%) after ASCT.5 We hypothesised that MDD identified by flow cytometry analysis of BM samples obtained before ASCT could be used to predict post-ASCT outcome. We hence retrospectively analysed the outcome of 29 PTCL patients undergoing ASCT at our institutions. Seven of those patients were MDD+. Our main finding was that pre-ASCT MDD+ detected by flow cytometry correlated with significantly higher CIR and lower OS. Importantly, pre-ASCT MDD was reported in four patients with PET-negative CR, all of whom relapsed quickly after ASCT. In other words, pre-ASCT MDD identified patients in PET-negative CR who might be at higher risk of relapse after ASCT.
Our study investigated pre-ASCT MDD as a potential surrogate for MRD in PTCL. Nonetheless, clear definition of MRD would have required the identification of a positive sample at diagnosis and subsequently at ASCT, using the same flow cytometry approach. Standard flow cytometry data (not using the TCR V-β approach) from bone marrow samples at diagnosis were only available in 13 patients (45%). Abnormal T cells were detected by standard flow cytometry at diagnosis in only two patients (7%) who were subsequently MDD- at ASCT. No abnormal T cells could be identified at diagnosis by standard flow cytometry in 11 patients (38%). In addition, pre-ASCT MDD was defined in our study as the detection of lymphoma cells by flow cytometry from BM samples collected before ASCT. Persistent pre-ASCT BM involvement likely reflects two distinct parameters: disease dissemination, possibly indicating less favourable disease biology, and treatment sensitivity.
The frequency of BM involvement evaluated by morphology at diagnosis is known to vary greatly among the different PTCL subtypes. Although it is frequent in angioimmunoblastic T cell lymphoma (AITL) (60–70%) as well as in NOS PTCL (20–40%), BM involvement is less common in ALCL (15–25%) and occurs rarely in extra-nodal subtypes (<5%).12 In line with these findings, six out of seven patients in our study with pre-ASCT BM involvement by FC fell into the AITL (n=3) and NOS (n=3) PTCL subgroups. Hence, our findings should be interpreted with caution when considering the ALCL and extra-nodal subtypes.
The potential contamination of the autologous graft by lymphoma cells provides another hypothesis explaining the negative prognostic impact of pre-ASCT BM involvement by flow cytometry, although published data have been somewhat equivocal on this point.13, 14, 15
Tempering these encouraging findings, routine approaches using flow cytometry lack an immunophenotypic marker of clonality, highlighting an important drawback in the potential evaluation of MDD. Although most neoplastic T-cells will have clear immunophenotypic abnormalities including the aberrant expression of multiple Ags,16 it can sometimes be difficult to distinguish a reactive T-cell population from the actual PTCL clone by immunophenotyping alone. TCR V-β repertoire analysis can provide a flow cytometry-based evaluation of clonality and overcome this limitation.9, 17 We have demonstrated the feasibility usefulness of this approach to assess TCR V-β clonality in T-cell neoplasms.10 Illustrating those limitations, two patients in this study presented with a T-cell population with equivocal immunophenotyping features, quantified at 0.03%. We chose to retain both cases. In the first case, the T-cell population showed similar immunophenotypic characteristics with that observed at diagnosis at an outside institution. For the other patient, T-cell clonality was confirmed by molecular studies. Of note, both patients quickly relapsed after ASCT (after 7 and 4 months, respectively). TCR rearrangement studies, in particular using high-throughput, next-generation sequencing, should overcome these limitations and stand as reliable and sensitive methods for detection of MRD.18, 19
Despite the limited number of patients and the retrospective nature of our study, we believe our findings to be informative. The correlation between pre-ASCT MDD and relapse post transplant in CR patients should be confirmed in a larger cohort. We previously published our experience with alemtuzumab, used for pre-transplant in vivo purging of a MDD+ PTCL-NOS patient.20 Regular analysis of the collected stem cell product to detect contaminating clonal population in combination with pre-collection BM study might help identify patients at high risk of relapse, avoid unnecessary exposure to high-dose therapy and suggest alternative approaches.
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Gopal AK: Merck: Research Funding; Emergent/Abbott: Research Funding; Cephalon/Teva: Research Funding; BioMarin: Research Funding; Sanofi-Aventis: Honoraria; Millenium: Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Spectrum: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Piramal: Research Funding; Biogen Idec, BMS: Research Funding. Maloney D: Juno Therapeutics: Research Funding; Roche/Genentech: Honoraria; Janssen Scientific Affairs: Honoraria; Seattle Genetics: Honoraria. Till: Roche-Genentech: Research Funding. Shustov A: Celgene: Honoraria and consultancy, Spectrum and BMS: consultancy.
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Gauthier, J., Holmberg, L., Wu, D. et al. Minimal detectable disease confirmed by flow cytometry and poor outcome after autologous stem cell transplantation in peripheral T-Cell lymphomas. Bone Marrow Transplant 51, 1617–1619 (2016). https://doi.org/10.1038/bmt.2016.227