Minimal Residual Disease In Peripheral Blood Research Articles

Using MALDI-TOF mass spectrometry for tracking of minimal residual disease in peripheral blood from patients with multiple myeloma.

e19525 Background: Minimal residual disease (MRD) negativity after completed therapy is associated with longer progression-free survival (PFS) in patients with multiple myeloma (MM). Current standard of care for MRD testing use flow cytometry and/or next generation sequencing (NGS)-based assays applied on bone marrow (BM) aspirate samples. To develop a strategy for MRD tracking in peripheral blood (PB), we were motivated to evaluate MALDI-TOF head-to-head with established bone marrow-based MRD assays. Methods: We used MALDI-TOF mass spectrometry to detect M-proteins in PB. Our cohort included patients who had serum samples available at 2 timepoints including during active disease and within 60 days of MRD results as determined by flow cytometry of BM aspirates. The cohort enrolled 71 patients (26 females, 45 males) with a median age of 61 years (37-78 years). Twenty-seven patients had high-risk cytogenetics at baseline. Patients were classified at diagnosis as ISS1 (n = 38), ISS2 (n = 18) or ISS3 (n = 6). The flow cytometry based MRD assay was performed using MSKCCs 10-color, single-tube method. MALDI-TOF analysis was performed as described by Mills et al. Samples taken during active disease were used to identify the mass/charge ratio of the M-protein at baseline and in follow-up samples. MALDI-TOF results were compared to flow cytometry bone marrow-based MRD results. Results: The median time between diagnosis and the MRD timepoint was 13.4 months (3.4-91 months). MALDI-TOF in PB and flow cytometry BM-based MRD results were concordant for 44/71 (62%) patients (8+/+, 36 -/- respectively) while 27 were discordant (10 +/-, 17-/+). Fifty-four of 71 patients were in complete response (CR) (45/54 in sCR) at the time of MRD. MALDI-TOF was still positive in 13 of these 54 CR patients. In this cohort, the median PFS since MRD assessment was not reached in the 2 subgroups of double negative patients (n = 31) or in patients with a positive result in at least one technique (n = 23) with a median follow-up of 11.2 months (0-34.6 months). Conclusions: In 44/71 (62%) samples, MALDI-TOF of PB results and flow cytometry BM-based MRD results were concordant. MALDI-TOF of PB may be useful for detecting measurable residual disease and for the monitoring of MM patients during maintenance therapy with the future goal to rule out early recurrent disease.

Issue Highlights - March 2019.

This issue features, among others, two reviews and four original manuscripts in the field of clinical cytometry, all of them in the area of leukemia/lymphoma immunophenotyping. These may be summarized as follows: Jevremovic and colleagues reviewed the position of flow cytometry in the diagnosis, prognosis and follow-up of T- and NK-cell lymphoproliferations 1. The diagnosis of these entities is generally based on the demonstration of immunophenotypic abnormalities and demonstration of clonality. The latter studies involve V-beta T-cell receptor repertoire analysis and killer immunoglobulin like receptors (KIR). In this context it is important to realize that a T-cell clone does not need to be synonymous with “malignancy” but could also reflect the transient clonal proliferation of “reactive” T cells. Among the mature T-cell lymphoproliferative disorders, T-cell large granular lymphocytic leukemia (T-LGLL), anaplastic large T-cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), mycosis fungoides or Sezary syndrome (MF/SS), T-cell prolymphocytic leukemia (T-PLL), hepatosplenic T-cell lymphoma (HSTCL) and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) are distinguished. Within the NK-cell disorders, chronic lymphoproliferative disorder of NK cells (CLPD-NKs), extranodal NL/T-cell lymphoma, nasal type, aggressive NL-cell leukemia/lymphoma and NK-cell enteropathy are defined. Nowadays, flow cytometry is a sensitive and specific approach to detect T and NK-cell neoplasms but requires a thorough knowledge of the normal T-cell and NK-cell immune spectrum. Jan W. Gratama, Deputy Editor [Color figure can be viewed at wileyonlinelibrary.com] CD123, the alpha chain of IL3 receptor, is expressed in variable intensities on human leukocytes; the strongest expression is seen on plasmacytoid dendritic cells and basophils, whilst the marker is absent on nucleated red blood cells; a range of intermediate expression levels are seen on T cells, mature B cells, granulocytes and eosinophils. CD123 is expressed on AML blasts and most B-cell precursor ALL blasts whilst it is negative on most T-ALL blasts. The teams of the Laboratory for Medical Immunology at Erasmus MC (Rotterdam, Netherlands) in collaboration with the Dutch Childhood Oncology Group (The Hague, Netherlands) set out to study CD123 expression levels in 846 patients with acute leukemia in comparison to normal cell populations obtained in 1,252 unique bone marrow and peripheral blood samples 6. Immunophenotyping was performed using standardized procedures as developed by the EuroFlow collaborative project 7. Whilst published information on this topic was relatively scarce and difficult to compare due to different methodologies, Bras and colleagues confirmed most observations as they extensively document in this report 6. Importantly, they observed that in paired B-cell precursor ALL, CD123 expression was considerably higher at relapse than at initial diagnosis, whilst in AML, the leukemic stem cells (CD34pos, CD38neg fraction of tumor cells) expressed similar CD123 levels as the remainder of AML blasts. These observations make CD123 an interesting target for antibody-targeted therapy 8. Early positive minimal residual disease (MRD) is a strong predictor of response to treatment in ALL and AML. In an observational study of 125 children with B-cell precursor ALL, Loosveld and colleagues 9 investigated the prognostic value of flow cytometric assessment of MRD in peripheral blood on days 8 and 15 after start of cytoreductive therapy, compared to day 35 MRD on bone marrow using molecular techniques. Children with detectable PB MRD on day 15 and negative day 35 BM results had lower disease-free survival at 4 years follow-up, indicating the prognostic potential of this less invasive, blood-based procedure early after start of therapy as compared to conventional bone marrow studies. Mora and colleagues 10 studied 120 consecutive patients with chronic B-lymphoproliferations (81 within the CLL spectrum and 39 with other monoclonal B-cell disorders) to establish whether CD200, a member of the Ig receptors membrane proteins superfamily, would have added value to the immunological scoring system already developed by Matutes et al. 11. CD200 had already been suggested to be rather specific for B-CLL in earlier studies. This study confirmed the value of CD200 as diagnostic marker and revealed that it's addition to the Matutes score increased the accuracy of that ranking algorithm. Beke Debreceni et al. 12 analyzed the regulation of expression of the adhesion receptor L-selectin (CD62L) in a cohort of untreated patients with B-CLL. Expression of surface-bound CD62L and tumor necrosis factor alpha-converting enzyme (TACE), as well as total phosphatase and protein phosphatase-2A activities, were lower on B-CLL than on normal B cells whilst the reverse was true for soluble CD62L and phosphorylated mitogen-activated protein kinase (pp38MAPK). In separate in vitro experiments confirmed that a phosphatase inhibitor (calyculin A) could reduce the effect of pp38MAPK inhibitor on CD62L shedding. The authors concluded that the reduction of phosphatase activity in B-CLL has resulted in a downstream signaling cascade with subsequent reduction of surface-bound CD62L which is mediated by enhanced phosphorylation of p38MAPK and reduced TACE expression. Jan W. Gratama, Deputy Editor Email: j.w.gratama@erasmusmc.nl

Early (Day 15 Post Diagnosis) Peripheral Blood Assessment of Measurable Residual Disease in Flow Cytometry is a Strong Predictor of Outcome in Childhood B-Lineage Lymphoblastic Leukemia.

In children with acute lymphoblastic leukemia (ALL) low levels of minimal residual disease (MRD) after induction, essentially assessed in the bone marrow, have been shown to be of good prognosis. However, only few studies have tested the peripheral blood for MRD. Here, we report the impact on survival of peripheral blood (PB) MRD assessment by multiparameter flow cytometry (MFC) at early time points of treatment in 125 B-ALL children, compared to Day 35 molecular bone marrow (BM) MRD. Patients were sampled for MFC one week postdiagnosis after a pre-phase of corticotherapy (Day 8), then after one week of chemotherapy (Day 15). The study enrolled 67 boys and 58 girls with a median follow-up of 52 months. Over the duration of the study, 20 patients relapsed and eight died. MFC was performed based on the leukemia-associated immunophenotype at diagnosis, using panels of 10 antibodies. Although, PB MFC-MRD had no prognostic impact at Day 8, Day 15 MRD negativity was associated with a significantly better 4 years DFS (91.6 ± 3% vs. 67.6 ± 9% P = 0.0013). Furthermore, while MFC and molecular data were concordant in most cases, patients with detectable PB MRD on Day 15, yet negative in BM on Day 35 had a significantly lower DFS (P < 0.0001). This study demonstrates that the less invasive procedure of MFC-MRD assessment in PB can be informative for childhood ALL patients at the early point of Day 15 of the treatment schedule. © 2019 International Clinical Cytometry Society.

Obinutuzumab pretreatment abrogates tumor lysis risk while maintaining undetectable MRD for venetoclax + obinutuzumab in CLL

AbstractEarly data on venetoclax-containing regimens for the treatment of chronic lymphocytic leukemia (CLL) show promising results with deep remissions, but are hampered by potential risk for tumor lysis syndrome (TLS). Whether optimal duration of venetoclax treatment can be guided by minimal residual disease (MRD) is currently unknown. To study whether TLS risk can be mitigated in an unfit population by introducing preinduction, and whether MRD-guided duration of venetoclax treatment is a feasible and efficacious approach, we performed the Dutch-Belgian Cooperative Trial Group for Hemato-oncology (HOVON) 139/GIVE trial. The study treatment consists of 4 treatment phases: preinduction (2 cycles obinutuzumab), induction I (6 cycles obinutuzumab and venetoclax), induction II (6 cycles venetoclax), and a randomization phase (group A: maintenance with 12 additional cycles of venetoclax irrespective of MRD; group B: MRD guided venetoclax maintenance with a maximum of 12 cycles). Here we report on a planned interim safety analysis as well as preliminary efficacy and MRD data of the first 30 patients enrolled. Downgrading of TLS risk after preinduction occurred in 25 patients: 3 from high to medium, 3 from high to low, and 19 from medium to low risk. No patient remained high risk. From these 30 patients, peripheral blood MRD data were obtained for 28 patients at the end of induction II (6 months after the last obinutuzumab dose), of whom 26 had undetectable MRD levels, and for 18 patients who reached the 3-month after-randomization point, of whom 16 had undetectable MRD levels. Obinutuzumab preinduction is tolerated well in these unfit patients and results in abrogating high TLS risk in all patients. Preliminary data indicate that efficacy is maintained with a high proportion of patients with undetectable MRD levels after combination treatment.

First Prospective Data on Impact of Minimal Residual Disease on Long-Term Clinical Outcomes after Venetoclax Plus Rituximab Versus Bendamustine Plus Rituximab: Phase III MURANO Study

First Prospective Data on Impact of Minimal Residual Disease on Long-Term Clinical Outcomes after Venetoclax Plus Rituximab Versus Bendamustine Plus Rituximab: Phase III MURANO Study

Residual Abdominal Lymphadenopathy after Intensive Frontline Chemoimmunotherapy Is Associated with Inferior Outcome Regardless of MRD Status in Advanced Chronic Lymphocytic Leukemia (CLL)

Residual Abdominal Lymphadenopathy after Intensive Frontline Chemoimmunotherapy Is Associated with Inferior Outcome Regardless of MRD Status in Advanced Chronic Lymphocytic Leukemia (CLL)

Safety and Efficacy of Venetoclax (VEN) in Combination with Bendamustine (B) Plus Rituximab (R) or Obinutuzumab (G) in Patients (pts) with Previously Untreated Chronic Lymphocytic Leukemia (CLL): Results from a Phase Ib Study (GO28440)

Safety and Efficacy of Venetoclax (VEN) in Combination with Bendamustine (B) Plus Rituximab (R) or Obinutuzumab (G) in Patients (pts) with Previously Untreated Chronic Lymphocytic Leukemia (CLL): Results from a Phase Ib Study (GO28440)

Five-year follow-up of lenalidomide plus rituximab as initial treatment of mantle cell lymphoma

AbstractWe report 5-year follow-up of a multicenter phase 2 study of lenalidomide plus rituximab (LR) as initial treatment of mantle cell lymphoma (MCL). The regimen includes induction and maintenance with the LR doublet. Treatment was continuous until progression, with optional discontinuation after 3 years. The median age of the 38 participants was 65 years, with MCL international prognostic index scores balanced among low, intermediate, and high risk (34%, 34%, and 32%, respectively). Twenty-seven (75%) of the 36 evaluable patients completed ≥3 years of study treatment. At a median follow-up of 64 months (range, 21-78), the 3-year progression-free survival (PFS) and overall survival (OS) were 80% and 90%, respectively, with 5-year estimated PFS and OS of 64% and 77%, respectively. During maintenance, hematologic adverse events (AEs) included asymptomatic grade 3 or 4 cytopenias (42% neutropenia, 5% thrombocytopenia, 3% anemia) and mostly grade 1 or 2 infections managed in the outpatient setting (45% upper respiratory infection, 21% urinary tract infection, 13% sinusitis, 11% cellulitis, 8% pneumonia). Nonhematologic AEs, such as constitutional and inflammatory symptoms, occurred at reduced frequency and intensity compared with induction. A peripheral blood minimal residual disease (MRD) assay (clonoSEQ) showed MRD-negative complete remission in 8 of 10 subjects who had completed ≥3 years of treatment and with available samples for analysis. With longer follow-up, LR continues to demonstrate durable responses and manageable safety as initial induction and maintenance therapy for MCL (ClinicalTrials.gov NCT01472562).

R‐CHOP/R‐HDAC AND RITUXIMAB MAINTENANCE RESULTS IN HIGH COMPLETE REMISSION RATE, MINIMAL RESIDUAL DISEASE NEGATIVITY, AND EXCELLENT SURVIVAL IN ELDERLY MCL PATIENTS

Introduction: Mantle cell lymphoma (MCL) is an incurable subtype of B-cell non-Hodgkin lymphoma. Implementation of high-dose cytarabine (HDAC) into induction therapy followed by stem-cell transplantation and rituximab maintenance (RM) became standard of care for the younger patients with newly dg. MCL. Treatment of the transplant-ineligible patients is still largely based on CHOP or alkylating agents and RM. Methods: We conducted a multicenter observational study designed by the Czech lymphoma study group (CLSG-MCL-01, GovTrial #NCT03054883), which prospectively analyzed safety and efficacy of alternating 3 + 3 cycles of R-CHOP and R-HDAC (1 or 2 g / m2, 2 doses over 24 hours) for newly diagnosed transplant-ineligible MCL patients. Pathological review of all samples was performed. MRD assessment was implemented in Euro-MRD member CLIP laboratory Prague. Primary objectives included response after induction by PET-CT, and progression-free survival (PFS). Secondary objectives included safety, overall survival (OS), and prognostic significance of PET imaging and minimal residual disease (MRD) after induction. Results: 73 patients were enrolled with median age 70 years. Most patients had intermediate (39.7%) and high-risk (50.7%) disease according to MCL International Prognostic Index (MIPI). Out of the 56 analyzed biopsies 55.4% revealed high proliferation index by Ki-67 (≥ 30%). The overall response rate in the 68 evaluable patients was 95.6% by PET-CT, including 80.9% complete remissions. MRD was evaluated after induction in 54 patients using paired samples of bone marrow (BM) and peripheral blood (PB). Grade 3-4 hematologic and non-hematologic toxicity was documented in 48% and 20.5% patients, respectively. RM was initiated in 59 patients. At the median follow-up of 43.8 months, median PFS and OS were not reached and were 53.9% and 71.3% at 4 years, respectively. For pts on RM 4-year PFS and OS was 61% and 77.2%, respectively. By univariate analysis MIPI, Ki-67 (≥ 30%), bulky disease (≥5 cm), involvement of the spleen, and MRD in PB after induction correlated with PFS. Interestingly, MRD in BM after induction did not correlate with PFS or OS. Multivariate analysis revealed that MIPI, bulky disease ≥5 cm, achievement of PET-negativity, and MRD in PB after induction independently correlated with PFS. Conclusion: Alternating R-CHOP and R-HDAC represents feasible and very effective regimen for elderly/comorbid MCL patients. The observed loss of predictive value of MRD in BM after induction appears to be impacted by rituximab maintenance. Keywords: Ara-C; mantle cell lymphoma (MCL); minimal residual disease (MRD)

Comparative Value of the Assessment of Minimal Residual Disease in Peripheral Blood at Days 8 and 15 By Flow Cytometry in Childhood Acute Lymphoblastic Leukemia

IntroductionAcute lymphoblastic leukemia (ALL) is the most frequent cancer in childhood, but treatments' progress now allowsto obtain prolonged remission or curein over 90% of the patients. Consequently, therapeutic de-escalation is now an objective for future treatment protocols, providing that biomarkers allow to reliablyidentifygood responders. Among such indicators, low levels of Minimal Residual Disease (MRD) obtained early after induction chemotherapy stand out as good candidates. The latter can be investigated usingmultiparameterflow cytometry (MFC) or real-time polymerase chain reaction (RT-PCR) for immunoglobulins or T-cell receptors (IG TCR) rearrangements.In this study we report the impact on survival of two early points of peripheral blood (PB) MRD assessment by MFC at days 8 and 15 on a cohort of 125 children with B-ALL enrolled in the French FRALLE trial and compared to molecular MRD in the bone marrow (BM) at day 35.Patients and methods.The study enrolled 67 boys and 58 girls and the duration of the study allowed for a median follow up of 52,1months. Median age at diagnosis was 57 months old (range 18 to 196), 101 children were between 1 to 10 years old and 24 were older than 10.Complete blood counts (CBC) at diagnosis showed a median of 6.7x109/L leucocytes (range 0.47 - 151x109/L) and 33% blasts (range 0 to 97%). One hundred and eight children had less than 50x109/L leucocytes while 17 had higher counts. EGIL classification at diagnosis allowed to classify patients as three B-I, 94 B-II, 27 B-III and 1 B-IV. Cytogenetic analyses were performed for 118 patients who were partitioned as follows: low risk n=47, intermediate risk n=55 and high risk n=16 (Harrisson CJ et al., BJH, 2010). Eighty-three patients were in the low risk group and 42 in the high-risk group as described by the FRALLE protocol. Seven patients of the 64 tested had an IKZF1 deletion. During the duration of the study, 20 patients relapsed and 8 died.Corticosensitivitywas defined by less than 1x109/L PB blasts on day 8 andchemosensitivity by less than 5% BM blasts on day 21 on BM smears.PB MRD was assessed in MFC with a single five or ten colors tube adapted to each patient's leukemia associatedimmunophenotypeon a backbone of CD45, CD19, CD10 and CD38.Statistical analyzes examined factors impacting disease-free survival (DFS) using Log rank test and Kaplan-Meier using theMedcalc® software (Ostend, Belgium). P values <0.05 were considered significant.ResultsNone of diagnosis features had any significant impact on DFS: age (p=0,95), risk group (p=0,17), EGIL classification (p=0,55), cytogenetics (p= 0,87), leucocyte count (p=0,36) nor IKZF1 deletion (p=0,2). Of the 125 patients, 9 were corticoresistant, 79 corticosensitive and 37 not evaluable because of less than 1x109/L leucocyte at diagnosis.Corticosensitivity had no impact on DFS (p=0,11). Conversely,chemosensitivity had a significant positive impact on DFS (p= 0,009).Day 8 PB MRD did not oultlineany significantly different DFS, whether considering detectable vs undetectable MRD (p=0.65) or MRD levels (logwisefrom >10-1 to <10-4, p=0,22).Conversely, PB MFC at day 15 appeared highly discriminant. Considering notdetectablevs detectable MRD, 4 years DFS was 91,6+3% vs. 67,6+9% p=0,0013 (Figure 1). Further refining the thresholds of MRD logwisedid not modify the significance (p=0.004; Figure 2). Indeed, DFS at 48 months was 61+15 % (n=16) for MRD >10-3, 74+11% ( n=18) for MRD <10-3->10-4 and 92+3% ( n=91) for MRD<10-4.Comparison of PB MFC MRD on day 15 with day 35 BM molecular MRD showed concordance in 72% of the cases (83 negative/negative and 7 positive/positive, 48 months DFS 94.6+2.7% and 38+20% respectively). Eight patients were negative in PB but positive in BM (DFS 62.5+17%).Twenty seven where positive in PB but negative in BM (DFS 83.5+7.6%).These differences were statistically highly significant (p <0.0001).ConclusionThis study demonstrates that even in the good prognosis context of childhood ALL, early MRD retains a highly significant prognostic value. It is of importance that this result was obtained not only on day 35 BM but interestingly, even earlier on day 15 PB. This less invasive procedure can easily be applied, especially for children. It should allow to detectgood responders, with MFC MRD levels below 10-4 for whom a de-escalation of chemotherapy could be considered. Conversely, the detection of blasts by MFC in day 15 PB is worrisome. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Compartment Effect on the Prognostic Significance of MRD Detection in CLL: Impact of Treatment Type and Duration of Follow-up

Background: The detection of minimal residual disease (MRD) in CLL using a 0.01% cut-off is an independent predictor of progression-free (PFS) and overall survival (OS) after chemo-immunotherapy. There are several new treatments available and using MRD as a trial endpoint could greatly speed up the identification of the best treatment approaches. MRD assessments can be made in the peripheral blood (PB) or bone marrow (BM) but there is substantial variation in the extent to which different treatments deplete disease in the PB, BM, and nodal compartments.Aims: To compare MRD levels in the PB & BM during/after different treatment approaches to determine differential compartment effects and evaluate the impact on predicting PFS/OS.Methods: The level of residual disease was determined using multi-parameter flow cytometry according to ERIC consensus protocols with a limit of quantification of 10-4 / 0.01% or better; "+" and "-" denotes ≥0.01% and <0.01% MRD respectively. MRD assessment in the CAMFlud and CLL 207 (alemtuzumab-based) trials was performed at end of treatment because cessation was determined by MRD status. In the ADMIRE/ARCTIC (FCR-based) trials, the MRD assessment was 3 months after end of treatment. In the CALiBRe (idelalisib) & IcICLLe (ibrutinib±obinutuzumab) trial, MRD assessments were made during treatment.Results: The MRD level in PB & BM samples taken at the same time, either during or within 6 months of treatment, was available in 556 cases of which 85 (15%) showed discordant results at the 0.01% threshold. The frequency of discrepant PB & BM MRD levels varied across treatment and patient groups (table 1). In 322 evaluable cases from the ADMIRE/ARCTIC trials at 3 months after the end FCR-based therapy, 66/322 were BM+PB-, representing 54% of the total MRD+ cases. Discrepant results with >1 log difference between PB & BM MRD levels were most common in refractory patients treated with alemtuzumab. There were no discrepant results in patients undergoing idelalisib or ibrutinib monotherapy although all patients have ≥0.01% MRD to date. A small compartment effect was apparent in patients treated with a combination of ibrutinib and obinutuzumab but the relative difference in PB vs. BM disease level appears to be approximately half that of rituximab in combination with chemotherapy.To determine whether the discrepant results affect the ability of MRD status to predict outcome, we compared the PFS according to BM & PB MRD status. In the CLL202 (fludarabine + alemtuzumab) trial the PFS for BM+PB- cases was similar to BM+PB+ cases (median PFS was 1.6 and 6.5 months respectively) and both were substantially shorter than for BM-PB- cases (median PFS 44.7 months, Log-rank P<0.001). This contrasts with FCR-based therapy in the ADMIRE/ARCTIC trials where the PFS for BM+PB- cases was most similar to BM-PB- cases and both were substantially longer than for BM+PB+ cases (median PFS >66 months vs. 54 months vs. 33 months for BM-PB- vs. BM+PB- vs. BM+PB+ respectively, Log-rank P<0.001). BM MRD was the most sensitive and informative site for predicting outcome, although MRD status in both PB & BM were strong predictors of PFS and OS. All patients with >1% MRD in the PB also had >1% disease in the BM.Summary/Conclusion: The compartment effect varies greatly according to patient group and treatment. Rituximab typically results in 0.7log lower disease in the PB compared to BM. Patients on BCR-pathway inhibitors have similar levels of disease in PB & BM even in combination with obinutuzumab. This suggests either that BCR-pathway inhibitors counteract the compartment effects of anti-CD20 antibody treatment, or that obinutuzumab is more effective in depleting bone marrow disease than rituximab. Peripheral blood MRD status may be appropriate for predicting outcome in some settings but underestimates MRD level in a substantial proportion of patients. Bone marrow remains the most sensitive and reliable source for MRD detection but is not required if there is >1% MRD in the blood. Evaluating the compartment effect of specific treatment combinations will help to identify approaches to achieve maximal MRD response using peripheral blood monitoring. [Display omitted] DisclosuresRawstron:AbbVie: Honoraria; Roche: Honoraria; Celegene: Honoraria; Janssen: Research Funding; BD Biosciences: Other: Remuneration; Gilead: Consultancy, Honoraria, Research Funding; Genzyme: Honoraria; GlaxoSmithKline: Honoraria. Munir:Alexion pharmaceuticals: Honoraria. Brock:AstraZeneca: Equity Ownership; GlaxoSmithKline: Equity Ownership. Hillmen:Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding.

Next-Generation Sequencing Based Minimal Residual Disease Assessment in Peripheral Blood RNA from Multiple Myeloma Patients

Introduction: Multiple myeloma (MM) is characterized by the presence of monoclonal protein (M-protein) in serum and/or urine, clonal plasma cell accumulation in bone marrow, and related organ or tissue impairment. MM patients are monitored during and after therapy using immunoglobulin, M-protein and free light chain assays. Minimal residual disease (MRD) assessment of bone marrow samples from MM patients in complete remission (CR) using next-generation sequencing (NGS)-based technology has been shown to have prognostic value, and previous studies have demonstrated evidence of circulating myeloma cells in peripheral blood. In this prospective study, we compared the sensitivity of RNA- and DNA-based MRD assays in peripheral blood (PB) and bone marrow (BM) samples collected from MM patients.Methods: Matched BM and PB samples were obtained from 50 MM patients in various stages of their disease. Diagnostic BM samples were used to identify clonal immunoglobulin sequences (i.e. clonotypes) unique to each MM patient. Matched BM and PB samples were then assessed for the presence of the MM clonotype. Using universal primer sets, we amplified immunoglobulin (IgH) variable (V), diversity (D), and joining (J) gene segments, the incomplete IgH-DJ rearrangement, and IgK receptors. Genomic DNA samples were assessed for all 3 receptors, while RNA samples were only assessed for the functional IgH-VDJ and IgK receptors. Amplified products were sequenced to obtain >1 million reads and analyzed using standardized algorithms for clonotype quantification. Myeloma-specific IgH, IgK, and IgH-DJ clonotypes were identified for each patient based on their high frequency at diagnosis. The presence of the myeloma clonotype was then assessed in BM cells (DNA), PB cells (DNA and RNA), and PB plasma (DNA) samples. Myeloma clonotype levels were calculated as previously described (Martinez-Lopez et al. 2014).Results: High-frequency myeloma clonotypes were identified in the BM of all 50 patients. We performed a sequence-level assessment of each clonotype to determine whether it was also evaluable in RNA. Non-functional clonotypes (e.g. IgH-DJ and kappa deleting element clones, frameshift/nonsense mutations, nonfunctional V or J segments, loss of cysteine in CDR3) were excluded from the analysis. 37 evaluable RNA clonotypes were identified in 28 of the 50 patients (56%).We compared the sensitivity of MRD assessment using DNA and RNA extracted from PB mononuclear cells. 20 clonotypes were qualitatively concordant in both DNA and RNA. 17 clonotypes were discordant, with all 17 clonotypes being detectable in RNA but not in DNA (Figure 1A). Only 4 of 21 (19%) clonotypes detectable in RNA were also detectable in DNA in the PB. Thus, RNA provides a clear sensitivity advantage over DNA analysis in PB cellular samples.We then investigated whether the sensitivity advantage conferred by RNA in PB cells was equivalent or superior to the sensitivity of DNA analysis in BM cells. On a patient level, 23 patients were qualitatively concordant in the PB and BM assays. 5 patients were discordant, with all patients being positive in BM and negative in PB. 4 of the discordant patients were positive at low levels in the BM (1-10 MM clonotype molecules per 1 million cells). On a clonotype level, 28 clonotypes were qualitatively concordant in BM DNA and PB RNA. 9 clonotypes were discordant, with all 9 clonotypes being positive in BM DNA and negative in PB RNA (Figure 1B). Nevertheless, PB RNA detected MRD in 21 of 30 with measurable disease in the BM DNA (ie, 70% sensitivity). These results demonstrate that MRD assessment using PB RNA is more informative than PB DNA in MM; however, despite the sensitivity improvement provided by RNA, MRD assessment in the BM DNA remains the superior sample source for MRD assessment, particularly when achievement of MRD negativity is the goal of therapy.Conclusions: NGS-based MRD assessment of BM in myeloma patients has been shown to have prognostic value. PB-based MRD assessment would improve clinical MRD assessment and monitoring paradigms. Our results demonstrate that RNA provides increased sensitivity for blood-based MRD assessment. Additional assay optimization to improve the fraction of clonotypes evaluable in RNA and to enhance sensitivity compared with BM is necessary before PB MRD monitoring in MM patients can be incorporated into routine clinical practice. [Display omitted] DisclosuresWolf:Telomere Diagnostics: Consultancy; Pharmacyclics: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria. Kong:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Bayes:Adaptive Biotechnologies Corp: Equity Ownership. Carlton:Adaptive Biotechnologies: Employment, Equity Ownership. Martin:Sanofi: Research Funding; Amgen: Research Funding.

Role of peripheral blood minimum residual disease at day 8 of induction therapy in high-risk pediatric patients with acute lymphocytic leukemia.

Risk stratification and treatment intensification, based on minimal residual disease (MRD) mensurement, changed the prognosis of pediatric patients with acute lymphocytic leukemia (ALL). The main aim of this study was to investigate whether peripheral blood (PB) MRD measurement at day 8 (D8) could predict the risk stratification category determined by bone marrow (BM) MRD at day 15 (D15). The study was performed prospectively, in a cohort of 40 children with B-lineage ALL, adopting the protocol of the Brazilian Cooperative Group of the Treatment Childhood Leukemia (GBTLI-2009). MRD was detected by flow cytometry (FC) using a simplifed panel that can reliably identify MRD at early phases of induction therapy. Upon diagnosis, the proportion of low and high-risk patients, was 24:16 (60%:40%). The main result of our study demonstrated the potential of D8 MRD in anticipating of week the risk stratification of high-risk patients as determined by D15 BM MRD. In these patients D8 MRD level of 1% was able to segregate high risk fast responders from high risk slow responders (p = 0.0097). This result could represent an opportunity for early treatment intensification, as already performed in some protocols.

The significance of peripheral blood minimal residual disease to predict early disease response in patients with B-cell acute lymphoblastic leukemia.

Minimal residual disease (MRD) assessment in the bone marrow (BM) postinduction therapy is now standard of care in patients with B-cell acute lymphoblastic leukemia (B-ALL). We examined the use of peripheral blood as a less invasive means of MRD assessment at days 8 and 15 of induction therapy and established the cutoff level that would allow the most accurate prediction of BM MRD postinduction therapy. MRD analysis was performed using 5-color flow cytometry on BM and PB samples from 77 B-ALL patients. BM MRD at diagnosis and day 29 of induction therapy was analyzed using the following antibody combinations: CD45-PC5/CD19-PC7/CD20-PE/CD10-ECD/CD38-FITC/CD13 + CD33-PE/CD10-ECD/CD34-FITC. PB MRD at days 8 and 15 was determined using CD45-PC5/CD19-PC7/CD20-ECD/CD10-PE/CD34-FITC. Day 8 and day 15 PB MRD levels were significantly higher in patients who had persistent BM MRD at day 29. PB MRD <0.01% at day 8 and/or day 15 predicted negative day 29 BM MRD status with 100% sensitivity but poor specificity. ROC curve analysis showed that day 15 PB MRD level of 0.1% yielded the highest sensitivity (78%) and specificity (82%). PB MRD cutoff level of 0.1% at day 15 has the best predictive value in determining positive day 29 BM MRD.

Predictive Power of Early, Sequential MRD Monitoring in Peripheral Blood and Bone Marrow in Patients with Mantle Cell Lymphoma Following Autologous Stem Cell Transplantation with or without Rituximab Maintenance; Interim Results from the LyMa-MRD Project, Conducted on Behalf of the Lysa Group

INTRODUCTION : Minimal residual disease (MRD) is emerging as an important predictor of clinical outcome in patients with mantle cell lymphoma (MCL). However, its utility in everyday clinical practice remains uncertain since standardized MRD monitoring strategies and response criteria are not yet formally established. To address this question, we conducted the LyMa-MRD project as an ancillary biology study in a prospective phase III trial in MCL (NCI NCT00921414; LyMa Trial).METHODS : The present MRD analysis was performed in a subgroup of randomized patients (n=178) of the 299 MCL patients (<66yrs) enrolled in the LyMa trial (Sept 2008 to Aug 2012). Briefly, all patients were previously untreated and received 4 courses of R-DHAP followed by ASCT using an R-BEAM conditioning regimen (n=257). After ASCT, patients were randomized between observation (obs) (n=120) versus Rituximab maintenance (RM) (n=119). The first planned interim-analysis, with a median follow-up of 40.6 months was presented at ASH 2014 and indicated superior PFS in the RM versus Obs arms (Le Gouill et al. ASH 2014). Sequential MRD monitoring was a predefined secondary objective and was performed throughout. At interim analysis the aim was to determine if peripheral blood (PB) and/or bone marrow (BM) MRD status, pre- and post ASCT, can predict patient outcome in terms of PFS according to RM or Obs. MRD was quantitatively assessed in PB and / or BM after induction, after ASCT by using gold standard EURO-MRD RQ-PCR assays and interpretation guidelines, targeted to clonal immunoglobulin gene rearrangements, in national reference laboratories. MRD data for survival analysis was generated from assays with a minimal sensitivity of at least 10-4. MRD status was assigned according to EURO-MRD interpretation guidelines. MRD negativity at a given time point was defined as absence of RQ-PCR amplification product in a given follow-up sample (minimal assay sensitivity of 10-4).RESULTS: Among the 299 patients enrolled in the LyMa trial, MRD data in PB and / or BM was successfully generated pre- and/or post-ASCT phases, for a total of 178 randomized patients. Of the 61 out of 239 randomized patients without MRD data, 9 are ongoing and other reasons were no MRD target or MRD assay failure. Pre-ASCT MRD in BM and PB was negative in 66% (n=98) and 80% (n=120), of samples, respectively. Post-ASCT, MRD in BM and PB was found negative in 82% (n=122) and 95% (n=162), respectively. MRD status pre-ASCT in either BM or PB was predictive of longer PFS (respectively; p= 0.0451, p= 0.0016). In contrast, PB MRD status, post-ASCT failed to predict PFS while there was a trend for a better PFS for patients achieving MRD negativity in the BM (median PFS: NR vs 43.4 months ; p= 0.07 ). We next investigated patient outcome according to MRD status pre-ASCT and randomization arms. In BM and PB, respectively, 72% and 79% of patients in the obs arm were MRD negative compared to 59% and 80% in the RM arm, respectively. The estimated 3y-PFS for MRD pos/obs, neg/obs, pos/RM, neg/RM patients, according to BM and PB MRD status were: 61,6% (IC95%, 35.4-79.8) vs 83.9% (IC95%, 70.1-91.7) vs 86.2% (IC95%, 67.3-94.6) vs 91.8% (IC95%, 76.3-97.3) (p=0.0110) and 51.8% (IC95%, 24.4-73.6) vs 86.5% (IC95%, 73.5-93.4) vs 80% (IC95%, 50-93.1) vs 92.8% (IC95%, 81.6-97.3) ( p=0.0027), respectively.CONCLUSION : Pre-ASCT MRD status in both BM and PB is an early predictor of PFS in younger MCL patients receiving ASCT. RM provides longer PFS regardless of MRD status pre-ASCT. Early sequential MRD monitoring at the pre-ASCT treatment phase thus offers strong potential for early clinical outcome prediction and MRD-guided, risk-adapted treatment in future MCL trials. Our preliminary results also suggest continued, clinically relevant, direct or indirect Rituximab-mediated anti-tumor activity, in rare residual circulating or 'tissue-resident' MCL cells. DisclosuresThieblemont:St. Louis Hospital, Paris, France: Employment. Casasnovas:Roche: Consultancy, Research Funding; Takeda: Consultancy; Gilead: Consultancy. Ribrag:Pharmamar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abbreviated Regimen with 4 Cycles of Fludarabine, Cyclophosphamide and Rituximab (FCR) in Physically Fit Patients with Chronic Lymphocytic Leukemia Who Achieve Early Complete Remission with Undetectable Minimal Residual Disease: Safety and Efficacy Follow-up Results of a Single Center Experience

Introduction: Chemoimmunotherapy with 6 cycles of fludarabine, cyclophosphamide and rituximab (FCR) is considered standard therapy for physically fit patients with chronic lymphocytic leukemia (CLL). Due to treatment toxicity, some patients are unable to undergo standard 6 cycles of FCR. We evaluated safety and efficacy of abbreviating FCR treatment to 4 cycles in a cohort of 35 untreated physically fit CLL patients who achieved CR with negative minimal residual disease (MRD).Patients and methods: Within 150 physically fit CLL patients treated with FCR on 1st line at our Center, from April 2003 to November 2014, a subgroup of 35 patients interrupted treatment after achieving negative MRD at the end of the 4th cycle. Median age at start of treatment was 62.8 years (34-81). Binet A/B: 24pts and C: 11. CD38 expression was positive (>7% off cells) in 57.1% and negative in 9% of the pts. A bone marrow biopsy was performed at start of treatment and 1 month post 4th cycle. Response was assessed in peripheral blood (PB) or bone marrow (BM). Negative MRD was defined as < 10-4. We used NCI criteria for response modified for the evaluation of MRD by flow cytometry. Progression was defined according to the NCI recommendations. Overall survival (OS) was defined as the time of initiation of therapy until death or last follow-up and progression free survival (PFS) as the time to progression. Data analysis included frequency and contingency tables, survival curves were plotted by the Kaplan Meier method. Treatment schedule: Fludarabine 25 mg/m2 IV day 1-3, cyclophosphamide 250 mg/m2 IV day 1-3, rituximab 375 mg/m2 IV day 3 cycle 1 and day 1 cycles 2-4, in all cycles every 28 days.Results: All 35 patients had negative MRD in PB after one month post 4th cycle. In addition, 28 had bone marrow evaluation showing CR with negative MRD in all of them. No splenomegaly nor hepatomegaly, enlarged lymphadenopathies nor lymphocytosis was observed in all the patients with negative MRD. After a median follow-up of 57 months (7 -141), median PFS was 65.8 months, not being yet reached the median of OS. PFS and OS at 72 months was 46% and 68% respectively. A total of 10 pts ( 3.5%) died: 7 on progressive disease, 3 on secondary neoplasms. Patients who progressed before 24 months had a median of survival of 22 months; median not reached on the group who progressed after 24 months (p=0.0001). Neutropenia grade 3-4 and infectious events were observed in 25.7% and 9.1% during all cycles respectively. Grade 3-4 neutropenia showed to increase over time (Cycle 1: 24%, Cycle 4: 39%). There was no treatment related death.Conclusion: With a long median follow-up, abbreviating treatment to 4 courses of FCR in patients who obtained negative MRD showed durable remissions with high PFS and OS at 72 months, minimizing treatment related toxicity. Sixty five percent of the patients who progressed after 24 months are still alive. Large randomized trials will be necessary to confirm our data. DisclosuresPavlovsky:Bristol Myers Squibb: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Pavlovsky:Novartis: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau.

Molecular Remission One Year Following Reduced-Intensity Allogeneic Hematopoietic Cell Transplantation for Chronic Lymphocytic Leukemia Predicts Relapse-Free and Overall Survival: A Multi-Institutional Landmark Analysis

▪Introduction: Chronic lymphocytic leukemia (CLL) cells typically carry one or more clonally rearranged immunoglobulin (Ig) genes. The specific sequence of an Ig rearrangement, or clonotype, can serve as a marker of minimal residual disease (MRD) in blood or bone marrow samples. The clonoSEQ assay (Adaptive Biotechnologies Corp) employs a next-generation sequencing (NGS) based MRD quantification method that uses consensus primers to amplify and then sequence the entire repertoire of Ig genes in a clinical sample. Each Ig clonotype, including the one marking a patient's CLL, can then be individually quantified with high precision. We previously demonstrated the utility of this NGS approach for predicting relapse-free survival in a single-institution cohort of CLL patients undergoing reduced-intensity allogeneic hematopoietic cell transplantation (RIC alloHCT). In this landmark analysis, we now assess the prognostic utility of molecular MRD quantification one year following RIC alloHCT in a multi-institution cohort.Methods: We retrospectively evaluated the outcomes and MRD status of patients surviving without relapse to one year after RIC alloHCT for CLL. Patients were assessable for MRD based on availability of a tumor specimen from which to determine the CLL-associated Ig heavy chain (IgH) clonotypes and a cryopreserved peripheral blood mononuclear cell (PBMC) aliquot obtained one year post-HCT. Forty-six patients underwent conditioning with total lymphoid irradiation and anti-thymocyte globulin (TLI/ATG) and 56 were conditioned with fludarabine and busulfan (Flu/Bu) regimens. Clonal IgH rearrangements were identified by NGS-based IgH profiling in CLL-bearing blood or marrow samples obtained prior to HCT and these clonotypes were quantified in the IgH repertoire of PBMC samples from one year (+/- 90 days) post-HCT.Results: One hundred two patients were assessable for outcomes beyond the one year post-HCT landmark. Amongst 97 patients with a diagnostic sample available, the IgH clonotype calibration rate was 90%. Five archived diagnostic samples yielded insufficient DNA to proceed with clonotype identification. 87/102 (85%) patients had paired tumor and one year PBMC samples suitable for MRD quantification. Patients receiving TLI/ATG or Flu/Bu conditioning had similar clinical characteristics. Median relapse-free survival (RFS) from the one year landmark was 4.7 years with TLI/ATG and 6.8 years with Flu/Bu (p=0.08; Figure 1A), and overall survival was not significantly different (Figure 1B). Acute GVHD (grades 2-4) occurred in 15.6% with TLI/ATG and 37.5% with Flu/Bu. Chronic GVHD occurred in 53% and 77%, respectively, with less extensive grade chronic GVHD using TLI/ATG (39%) compared with Flu/Bu (73%). Amongst patients alive and in remission one year after RIC alloHCT, peripheral blood MRD <10-6 was associated with more favorable outcomes, with just 5.3% relapsing within 7 years post transplant, compared with 59% relapse in those with ≥10-6 MRD (HR 11, 95% CI 4.3-28, p<0.0001; Figure 1C). MRD negativity one year post-HCT was also associated with improved long term overall survival (HR 2.6, 95% CI 1.1-6.4, p=0.03; Figure 1D).Conclusions: In this non-randomized retrospective analysis of outcomes after RIC alloHCT for high-risk CLL, use of TLI/ATG or Flu/Bu conditioning regimens were associated with similar RFS, though a trend toward earlier relapse with TLI/ATG is observed. Flu/Bu was associated with substantially higher rates of acute and chronic GVHD and higher rates of death in remission. Irrespective of conditioning regimen used, molecular MRD quantification at one year post-HCT provides strong positive and negative predictive value for subsequent relapse. Identification of MRD positive patients at risk for relapse may permit the implementation of post-HCT immune modulation or maintenance treatment to reduce relapse risk for those in need, but spare MRD negative patients from the potential for additional toxicity. [Display omitted] DisclosuresLogan:Pharmacyclics: Consultancy; Amgen: Consultancy; Jazz Pharmaceuticals: Consultancy. Herrera:Genentech: Research Funding; Sequenta, Inc.: Research Funding; Pharmacyclics: Research Funding. Rezvani:Pharmacyclics: Research Funding. Kong:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder. Miklos:Pharmacyclics: Research Funding.

Minimal Residual Disease (MRD) As Exploratory Endpoint in a Phase 1 Study of the Anti-CD123 Mab CSL362 Given As Post-Remission Therapy in Adult Acute Myeloid Leukemia (AML)

BackgroundAML patients (pts) who achieve a morphologic complete remission (CR) may have MRD in bone marrow (BM) and/or peripheral blood detected as patient-specific leukemia-aberrant immunophenotype by multiparameter flow cytometry (MFC) or as abnormal transcripts of AML gene mutations/translocations by quantitative RT-PCR. Presence of MRDafter induction and/or consolidation therapy identifies pts at high risk of relapse, with increasing MRD levels indicating impendingrelapse in individual pts. Although standardized testing and validated endpoints for MRD determination across institutions are lacking, major cancer treatment centers can diagnose and track MRD reliablywhen assessed in serial BM samples.CD123 (IL3-Rα) is overexpressed by AML blasts and leukemia stem cells (LSC), suggesting that agents designed to target and kill CD123+ AML LSC and blasts may eliminate MRD and delay or prevent relapse. Retrospective clinical data suggest that conversion of AML pts from MRD + to MRD negative (-) status may confer clinical benefit; however this has not been fully characterized in prospective clinical trials.MethodsCSL362 is a fully humanized, anti-CD123 monoclonal antibody, Fc domain-engineered to bind with high affinity to CD16 on natural killer (NK) cells. CSL362 activates antibody-dependent cell-mediated cytotoxicity (ADCC) of CD123+ cells. The first-in-human Phase 1 study of CSL362 assessed safety, PK and targeted bioactivity. An exploratory objective was to track changes in MRD and to correlate changes with maintenance of CR.Eligible pts had CD123+ de novo or secondary AML in 1st or 2nd CR or CRp who recovered from induction with or without consolidation therapy, had risk factors prognostic for early relapse, and were not candidates for hematopoietic stem cell transplantation. CSL362 was infused IV over 3 hours once every 14 days for up to 6 doses. Sequential pt cohorts were treated at ascending dose levels from 0.3 to 12.0 mg/kg. The final safety evaluation visit was at week (wk) 16, and each center reported remission status at wk 24 from first dose. BMwas assayed for MRD at baseline as well as in wk 5 and wk 12 during therapy according to individual site's laboratory standards using MFC and/or PCR. Changes in MRD were compared to the baseline result for each pt.ResultsEnrollment completed with 30 pts total, in 6 dose level cohorts, treated at 4 hospitals. Safety, PK and biomarker results showing an acceptable safety profile, and targeted bioactivity of CSL362 were reported at the ASH 2014 Annual Meeting [Blood 124 (21); 120]. At baseline, 12 pts were MRD+ (5 by MFC only, 4 by PCR only and 3 by both methods); 11 of 12 had a follow up evaluation. Molecular mutations in post-remission BM identified by PCR were FLT3 (3 pts), NPM1 (3 pts) and MLL (1 pt).Of 4 pts MRD+ by MFC only, 1 converted to MFC- and maintained CR at wk 24. Of 4 pts MRD+ by PCR only, 1 converted to PCR- and maintained CR at wk 24; a 2nd pt, who remained PCR+ on study also maintained CR at wk 24. Of 3 pts MRD+ by both MFC and PCR, 1 converted to MFC-, PCR follow-up was not done but the pt maintained CR at wk 24. The 7 pts MRD+ who did not convert to MRD- relapsed prior to wk 24. [See table].Table 1MRD+ at baselineConverted to MRD - at Wk 5Converted to/Maintained MRD - at Wk 12Maintained CR at Wk 244 + by MFC only1214 + by PCR only (1 FLT3, 1 MLL, 2 NPM1)01 (NPM1)2(NPM1; MLL)3 + by MFC & PCR (2 FLT3, 1 NPM1)by MFC 3by PCR 0by MFC 1by PCR 01 (Flt3)DiscussionConversion from MRD + to MRD - in 4 of 11 pts, with CR maintained at wk 24, is hypothesis generating; MRD conversion suggests possible eradication of residual leukemia cells while on study. Maintenance of morphologic CR was more likely for patients who converted to a MRD-state than those who remained MRD+ during therapy. Multiple factors may affect AML response to CSL362 immunotherapy, including cytogenetic abnormalities, specific molecular mutations, and each pt's immune system status. Preclinical data suggest the activity of CSL362 requires adequate numbers and function of NK cells and expression of the target receptor (CD123) on LSC and MRD cells. This observation would provide validation for the use of MRD response as efficacy endpoint and indicator of benefit for CSL362 and, perhaps, other AML therapies. With further efforts to standardize AML MRD assessments across laboratories, MRD response may become useful as a surrogate endpoint for clinical drug development in AML. DisclosuresDeWitte:CSL Behring: Employment. Dasen:CSL Behring: Employment. Bensen-Kennedy:CSL Behring: Employment. Walter:AstraZeneca, Inc.: Consultancy; Pfizer, Inc.: Consultancy; Amphivena Therapeutics, Inc.: Consultancy, Research Funding; Covagen AG: Consultancy; Seattle Genetics, Inc.: Research Funding; Amgen, Inc.: Research Funding.

Peripheral blood minimal residual disease may replace bone marrow minimal residual disease as an immunophenotypic biomarker for impending relapse in acute myeloid leukemia.

As relapses are common in acute myeloid leukemia (AML), early relapse prediction is of high importance. Although conventional minimal residual disease (MRD) measurement is carried out in bone marrow (BM), peripheral blood (PB) would be an advantageous alternative source. This study aims to investigate the specificity of leukemia-associated immunophenotypes used for MRD detection in blood samples. Consistency of PB MRD as compared with BM MRD was determined in flow cytometric data of 205 paired BM and PB samples of 114 AML patients. A significant correlation was found between PB and BM MRD (r=0.67, P<0.001), while median PB MRD percentage was factor 4-5 lower compared with BM MRD. Primitive blast (CD34+/CD117+/CD133+) frequency was significantly lower in PB (median factor 23.7), indicating that PB MRD detection is more specific than BM. Cumulative incidence of relapse 1 year after induction therapy was 29% for PB MRD-negative and 89% for PB MRD-positive patients (P<0.001). Three-year OS was 52% for MRD-negative and 15% for MRD-positive patients (P=0.034). Similar differences were found after consolidation therapy. As PB MRD appeared to be an independent predictor for response duration, the highly specific PB MRD assay may have a prominent role in future MRD assessment in AML.

T-Lymphoblastic Leukemia (T-ALL) Shows Excellent Outcome, Lack of Significance of the Early Thymic Precursor (ETP) Immunophenotype, and Validation of the Prognostic Value of End-Induction Minimal Residual Disease (MRD) in Children’s Oncology Group (COG) Study AALL0434

▪Background: Although outcomes for children and young adults with T-ALL continue to improve, relapse on therapy continues to be a major cause of treatment failure. COG AALL0434 is a phase III study for patients with T-ALL that used a standard 4-drug induction followed by response-based risk stratification at Day 29 with Intermediate and High-risk patients randomized to receive or not receive six 5-day courses of nelarabine during consolidation, delayed intensification and maintenance. All patients are also randomized to receive either Capizzi or high-dose methotrexate during interim maintenance and all patients except those who were Low-risk received cranial irradiation (1200 cGY for CNS1 or 2 and 1800 cGy for CNS3). Risk stratification incorporates MRD detection by flow cytometry at Day 29 using the following cutoffs: Low-risk < 0.1%, Intermediate-risk < 1%, and High-risk > 1%. Recently, the immunophenotype has been associated with a particularly poor outcome in T-ALL, a finding that requires confirmation in a large prospective trial using contemporary therapy.Methods: 1144 children with T-ALL enrolled on COG AALL0434 between January 22, 2007 and June 30, 2014 were categorized at the time of study enrollment as ETP (n=130; 11.3%), Near-ETP (ETP but with elevated CD5; n=195; 17%) or Not-ETP (n=819; 71.6%) by flow cytometry. MRD was assessed by 8-9 color flow cytometry at Day 8 in peripheral blood (PB) and Day 29 in bone marrow (BM). Flow cytometry to characterize ETP status and MRD was performed in a single central laboratory. Event-free survival (EFS) and Overall survival (OS) were estimated by Kaplan-Maier curve analysis.Results: Despite significantly higher (P<0.0001) rates of induction failure (>25% blasts by morphology at end induction) for ETP (7.8%) and Near-ETP (6.7%) than Not-ETP (1.1%), all 3 immunophenotypic groups showed excellent 5-year EFS and OS that were not statistically different: ETP (87.0%; 93.0%), Near-ETP (84.4%; 91.6%), and Not-ETP (86.9%; 92.0%). In all 3 groups, most events occurred within 12 months from diagnosis and plateaued after 2 years, although events generally occurred earlier for ETP and Near-ETP than Not-ETP. There were no relapses in ETP patients later than 12 months post diagnosis. Initial white blood cell count (WBC) greater than 200,000/microliter was seen in 9.6% of ETP and 30% of Not-ETP/Near-ETP (P<0.0001) and was associated with inferior EFS and OS for Near-ETP (EFS p=0.0003) and Not-ETP (EFS p=0.012), but not ETP. Day 29 MRD > 0.01% was associated with inferior EFS (76.3% vs. 89.0%; p=0.0001) and OS (86.6% vs. 93.8%; p=0.0008) for the total cohort and was detected in 81.4%, 64.8%, and 30.5% of ETP, Near-ETP and Not-ETP patients, respectively. Interestingly, there was no difference in EFS or OS for Day 29 MRD <0.01% vs. 0.01%-1.0%, suggesting significance largely for MRD > 1.0%. Day 29 MRD >0.01% was also associated with inferior EFS (76.6% vs. 90.8%; p=0.0001) and OS (84.3% vs. 94.0%; p=0.0064) in Not-ETP and inferior EFS (80.2% vs. 94.0%; p=0.0073) in Near-ETP, but no difference was seen for EFS or OS in ETP. Day 8 PB MRD > 0.01% was associated with inferior EFS (80.3% vs. 92.0%; p=0.017) but not OS and lost significance in the subset having Day 29 MRD < 0.01%, suggesting Day 8 does not add prognostic information to Day 29 MRD.Conclusions: AALL0434 is the largest study of T-ALL ever conducted and shows excellent outcomes for T-ALL. In particular, ETP is found to have an outcome identical to non-ETP patients. WBC count > 200,000/microliter is associated with inferior outcome in non-ETP T-ALL. BM MRD > 1% at Day 29 is able to identify a subset of non-ETP T-ALL patients having inferior outcomes. PB Day 8 MRD does not provide unique prognostic information. DisclosuresNo relevant conflicts of interest to declare.