Flow Cytometry MRD Research Articles

Monitoring Minimal Residual Disease in Patients with Multiple Myeloma By Tracking Serum M-Protein Using Mass Spectrometry

Objective： We performed this retrospective real-world study to evaluate the clinical utility of blood-based mass spectrometry for longitudinal M-protein monitoring during the disease course of patients of multiple myeloma（MM）. Methods：We initially analyzed serial serum samples of 56 patients with MM by clonal peptide MS Method named EasyM MS, then we compared the MS results with those determined using serum immunofixation electrophoresis (IFE),multiparameter flow cytometry (MFC), next-generation flow cytometry (NGF), to establish the concordance between different disease-monitoring protocols. A sensitivity for MFC MRD negativity of at least 1×10 -4 anda sensitivity for NGF MRD negativity of at least 1×10 -5.Additionally, we determined the optimal cutoff value of EasyM MS negativity to satisfy both the sensitivity and specificity between MS and NGF, and explored the prognostic impact of MS-MRD status. Results：The median diagnostic age of 56 NDMM patients was 56 years old, including 34 males (60.7%). There were 38 patients with IgG type MP and 18 patients with IgA type MP. 47 patients (83.9%) chose to receive proteasome inhibitor - based induction therapy, and a total of 44 patients (78.6%) received first-line ASCT treatment.Among 56 MM patients，38 patients with IgG type MP can further identify IgG subclasses through mass spectrometry. Additionally, 56 patients with MM have successfully used MS to identify their unique clonal peptides of MP. 56 patients provided a total of 447 samples of serum and all sequential serum samples were successfully measured by using the MS method. The initial comparison of the performance of blood-based MS-MRD with that of the conventional M-protein diagnostic method IFE used . Among the 397 comparable results collected at similar timepoints, 70.8% (281/397) of the samples were both M-MRD- and IFE-positive, 3.3% (13/397) were both MS-MRD- and IFE-negative, 25.7% (102/397) were MS-MRD-positive and IFE-negative.Flow cytometry MRD was performed on 218 bone marrow aspirates in this cohort, including 126 analyzed with MFC and 92 analyzed with NGF. MS-MRD detection was also performed on comparable serum acquired at the same time points. The sensitivity of MS-MRD was 100.0% in comparison with both MFC and NGF, with specificity of 1.9% and 22.2%, respectively. Considering the overly high sensitivity of EasyM at a cutoff of dotp >0.9, we used the sensitivity of NGF as the standard for detection (i.e., a detection sensitivity ≤10 -5) and set the EasyM MS negative cutoff value using ROC analysis, determined to be <1.86% of that at first diagnosis. Using these assumptions, we again performed the aforementioned analyses comparing the sensitivity and specificity between EasyM and IFE, MFC, and NGF. Under this scenario, the sensitivity of EasyM remained high in comparison with IFE (88.3%), MFC (85.1%), and NGF (93.2%), but the specificity was also greatly increased to 90.4%, 55.8%, and 93.2%, respectively. We then divided patients into MS-positive and MS-negative groups according to whether they achieved MS-MRD negativity, and median PFS and OS were calculated for each group. The median PFS of the MS-positive and MS-negative groups was 20.6 (95% CI: 16.6, 24.6) and 61.8 (39.7, 83.9) months, respectively . The median OS was 55.7 (95% CI:31.8, 79.5) months for the MS-positive group and not reached (NR) for the MS-negative group ,Both mPFS and mOS were significantly longer among the MS-negative group compared with the MS-positive group (both p < 0.001). Conclusion：EasyM exhibits high sensitivity and minimally invasive technological advantages, and also has important significance in efficacy evaluation and prognosis evaluation. In the future, EasyM MS detection can be used as a minimally invasive MRD monitoring method based on peripheral blood in clinical practice.

Long-Term Remissions with MRD-Guided Acalabrutinib, Venetoclax and Obinutuzumab in Relapsed/Refractory CLL: Follow-up Efficacy and Circulating Tumor DNA Analysis of the CLL2-Baag Trial

Background The phase 2 CLL2-BAAG trial tested a triple combination of acalabrutinib (acala), venetoclax (ven) and obinutuzumab (obi) after an optional debulking with bendamustine in patients (pts) with relapsed/refractory (r/r) CLL. In its primary endpoint analysis, 75.6% of pts achieved undetectable MRD (uMRD) in peripheral blood (PB) at final restaging (RE) after approximately 6 months of the triplet (Cramer, Lancet Haematol. 2022). We now report efficacy data and circulating tumor DNA (ctDNA) analyses with additional follow-up. Methods In the induction phase, obi was started in cycle 1 (days 1, 8, 15) and given 4-weekly in cycles 2-6. Acala was added in cycle 2 and ven in cycle 3. Maintenance treatment with continuous acala and ven and 3-monthly obi was administered until achievement of a deep remission with uMRD <10 -4 in PB or for up to 24 months (mo). MRD was measured centrally by flow cytometry (FCM) in PB and by digital droplet PCR (ddPCR) of patient-specific VDJ rearrangements and CLL-related mutations in blood plasma. uMRD was defined as <1 CLL cell/10,000 leukocytes (<10 -4) and no detected ctDNA. Results In total, 46 pts were included in the trial, 1 pt had to be excluded from the analysis due to a retrospectively noted violation of exclusion criteria. The median number of previous treatments was 1 (range 1-4) and 18 pts (40%) had received a BTK inhibitor (BTKi) and/or ven (BTKi: 8, ven: 7, both: 3) prior to inclusion, 14/44 pts (31.8%) had del(17p) and/or TP53 mutations, 34 (75.6%) had unmutated IGHV. With a median observation time of 34.4 mo (range 12.0-39.2), all 45 pts are off study treatment. The median treatment duration was 14.7 mo (range 6.1-32.9), 2 pts (4.4%) discontinued treatment during the first 6 induction cycles, both due to adverse events (AEs). Following the MRD- and response-guided approach, 25 pts (55.6%) discontinued therapy according to confirmed uMRD and 9 pts (20.0%) completed the maximum of 8 maintenance cycles due to persisting MRD and/or lack of a complete response. As reported, the uMRD rate in PB at RE was 75.6% with 10 pts showing detectable MRD ≥10 -4. In the course of maintenance, 7 of these 10 pts (70%) achieved uMRD, 1 pt had a Richter's transformation (RT) and 2 pts still had detectable MRD after 8 cycles of maintenance ( Figure 1A). With all pts off-treatment, uMRD <10 -4 in PB was achieved in 42 of 45 pts (93.3%) at any time point,including 17 of 18 pts (94.4%) previously exposed to ven and/or a BTKi and 13 of 14 pts (92.9%) with TP53 aberrations. The median time to uMRD in PB was 5.4 months from the start of study treatment. Median progression-free survival (PFS) was not reached, the estimated 30-mo PFS rate for the whole analysis population was 88.2% ( Figure 1B). A similar PFS was shown for pts with TP53 aberrations (30-mo PFS 85.7%) and pts previously exposed to BTKi and/or ven (30-mo PFS 93.3%). The estimated overall survival at 30 mo was 100%. One patient died of COVID-19 18 mo after the end of study treatment. In total, 564 paired FCM/ctDNA samples were available. Overall, 17 MRD recurrences (after uMRD by both methods) occurred. Of these, 11 were first detected by ctDNA, and 3 by FCM (+3 with concordant detection, Figure 1C). Among those with earlier ctDNA detection, more pts appeared to have high-risk genetics (unmutated IGHV in 10/11 pts, complex karyotype in 4/8 evaluated pts), del(11q) and prior exposure to ven/BTKi compared to those with earlier detection by FCM, and MRD recurrences seemed to occur earlier (mean, 7.1 mo after end of treatment). Notably, in 2 of 3 CLL-type progressions (PD, both nodal with normal lymphocyte counts), ctDNA was detected 6 mo/14 mo before MRD by FCM and 7 mo/17 mo before clinical PD, in the third pt ctDNA and FCM MRD recurred at the same time. In a pt with RT (DLBCL), patient-specific VDJ ctDNA was detected at diagnosis while FCM MRD remained negative. At the time of the data cut (02/2023) 50 serious AEs (SAEs) were reported, 33 were of CTC G3, 6 of CTC G4 and 1 CTC G5. The most common SAEs were COVID-19 (n=8), other pneumonias (n=5), infusion-related reactions (n=5) and neutropenias (n=3). Conclusions Time-limited MRD-guided acala, ven and obi led to an estimated 30-mo PFS rate of 88.2% in pts with r/r CLL. Remissions deepen with ongoing maintenance and almost all pts achieve uMRD in PB including pts with TP53 aberrations and those treated with BTKi and/or ven in prior lines. The addition of ctDNA-based analyses to conventional MRD assessment by FCM seems to improve early detection of relapses.

MiR-128-3p as blood based liquid biopsy biomarker in childhood acute lymphoblastic leukemia

BackgroundMinimal residual disease (MRD) is one of the most valuable independent prognostic factors in acute lymphoblastic leukemia (ALL). Bone marrow (BM) aspiration, however, is an invasive process. Previous studies have shown that microRNAs (miR) and extracellular vesicle (EV)-related miRs show different expression profiles at the presence of malignant cells compared to healthy controls. In our previous project, we have reported that two miRs previously described to be overexpressed in blasts were significantly decreased over the first week of the therapy of patients with ALL in the platelet free plasma fraction (PFP) of peripheral blood samples (PB). The aim of the current study was to assess the relation between day 15 flow cytometry (FC) MRD and expression of miR-128-3p and miR-222-3p miRs in exosome-enriched fraction (EEF) of PFP to evaluate whether their expression in EEF correlates with day 15 FC MRD more precisely. MethodsPB was collected from 13 patients diagnosed with pediatric pre-B ALL at 4 time points. Expression of miR-128-3p and miR-222-3p was measured by qPCR in PFP and EEF. ResultsPositive correlation was found between changes of miR-128-3p expression in EEF or PFP by day 8 of chemotherapy and day 15 FC MRD (rEEF = 0.99, pEEF = 1.13E-9 and rPFP = 0.99, pPFP = 4.75E-9, respectively). Furthermore, the decrease of miR-128-3p in EEF by day 15 of treatment also showed a positive correlation with day 15 FC MRD (rEEF = 0.96; pEEF = 4.89E-5). ConclusionOur results show that circulating miRs are potential biomarkers of ALL MRD, asmiR-128-3p level both in PFP and EEF predicts day 15 FC MRD. In addition, the assessment of the EEF gave a more promising result.

Minimal Residual Disease Analysis in Multiple Myeloma

BACKGROUND: Over the years, with changes in treatment approaches, it has been possible to achieve higher complete response (CR) rates with chemotherapies or chemoimmunotherapies in multiple myeloma (MM). However, a subset of patients in CR still relapse owing to the presence of residual tumor cells in the bone marrow not detectable by conventional methods. Residual disease detection by flow cytometry (FCM) has been proven to be highly sensitive and prognostically significant in a number of clinical studies. AIMS AND OBJECTIVES: In this study, we compared FCM minimal residual disease (FCM MRD) in MM cases post-chemotherapy/autologous stem cell transplant with morphology and biochemical methods. We also tried to correlate the pre-therapy stage of the disease and cytogenetics with MRD. MATERIALS AND METHODS: Twenty eight samples from 26 patients were evaluated for MRD on 6 color 3 tube panel over the period of 2 years. RESULTS: MRD was detectable in 19 samples (67.9%). FCM had a sensitivity of 95% compared to immunohistochemistry (IHC). 100% of cases with MRD positivity had abnormalities in at least three surface antigens. The high risk cytogenetics and high risk stage groups had a higher frequency of MRD positivity compared to the low risk groups. CONCLUSION: FCM MRD analysis is able to risk stratify the patients in CR and stringent CR. Routine use of FCM to detect residual disease posttherapy in MM should be implemented.

Short-Term Follow-up of Blinatumomab in Children with B-Cell Precursor Acute Lymphoblastic Leukemia：Study of a Single Center from China

Background: Acute lymphoblastic leukemia (ALL) is the most common hematologic disorders in children. The 5 years overall survival (OS) has reached more than 90%, but still 20% of patients relapsed. Positive of microscopic residual disease (MRD+) is the important factors for pediatric ALL. Blinatumomab is a combination of CD3 and CD19 bispecific antibody. Previous studies have shown that it has a significant advantage in improving the efficacy and survival of refractory /relapsed (R/R) ALL and MRD+ patients. Blinatumomab has been approved in China for R/R ALL indications in adults and children respectively since Aug 2021 and Apr 2022. So far, there is a lack of research data on ALL in Chinese children. This study mainly discusses the short-term efficacy and safety of Blinatumomab for R/R ALL and MRD+ of children with B-ALL in China. Methods: We collected the patients' information of children with R/R or MRD+ B-ALL treated with blinatumomab from Aug 2021 to Jun 2022 in our single institute from China. Retrospectively analyzed were carried out for the short-term efficacy and safety. Results: since Aug 2021, blinatumomab has been introduced to China. 13 cases were enrolled in this study, including R/R ALL 6 cases and MRD + ALL 7 cases, the median onset age of 9.5 (range 5-14) years old, male: Female =8:5, in the R/R-ALL group, there was 1 case of sustained no remission (NR), 1 case of very early recurrence(<18 months) and 1 case of late recurrence(≥36 months), and 3 cases of early recurrence, ALL of which were isolated bone marrow recurrence for the first time. 3 cases were treated with reinduction chemotherapy before blinatumomab. The mean proportion of blast cells in bone marrow (BM) prior therapy was 34.3 (range 0-96) %. The mean flow cytometry MRD was 30.26 (range 0.11-78.3) %. 3 patients were with no dose uphill due to low tumor load. 3 patients were given uphill 5ug/m2 qd d1-7, 15ug/m2 d8-28, of which 1 was treated for 14 days and 2 for 21 days. The investigator evaluated the efficacy of the treatment and stopped the drug. After treatment, bone marrow morphology was complete response (CR) in 3 cases, of which 2 cases had MRD negative (MRD-), the Cytokine release syndrome (CRS) incidence was 83.3%, 2 cases had CRS grade 2, 3 cases CRS grade 1, and the main manifestations were fever and hypotension. The levels of cytokines were significantly increased in patients with CRS, especially in IL-6, IL-8 and IL-10. The median level of IL-6 in CRS was 3081.8 (range 12.6-10405.0). IL-8 214.5 (range 23-408.0) and IL-10 377.7 (range 6.7-1398.0) (normal value ≤ 4.91) were significantly higher than those before blinatumomab and after CRS disappeared. In the MRD+ ALL group, there were 2 cases of chemotherapy drug intolerance, 1 case of inadequate treatment, and 4 cases of continuous MRD+ after standard chemotherapy. In MRD+ALL group, the BM of All cases were CR before treatment, the mean flow MRD value was 0.63 (range 0.01-4.00) %, the blinatumomab was 15ug/m2 (weight < 45kg) or 28ug (weight ≥45kg) for 7-28 days. After treatment, the MRD of 7 cases were all turned negative and the incidence of CRS was 71%. All of which were CRS grade I. When CRS occurs, the median IL-6 level is 76.4 (range 4.4-271.0) (normal value ≤ 5.3), IL-8 18.7 (range 8.0-35.5) (normal value ≤ 20.6), IL-10 447.3 (range 8.9-2570.0) (normal value ≤ 4.91) Conclusion: The CR rate of R/R pediatric B-ALL was 50%, the MRD- rate was 67%. The rate of MRD turned negative was 100% in MRD+ B-ALL group. In this study, the tolerance of blinatumomab in children with B-ALL was good, and the incidence of CRS was about grade 1/2, and no neurological complications occurred. The occurrence of CRS was mainly caused by the increase of cytokines, especially IL-6, IL-8 and IL-10. The IL-6 of CRS in R/R ALL group was significantly higher than that in MRD+ ALL (Fig.12.798, P=0.007).

Interim Results of a Phase 1/2 Study of Pembrolizumab Combined with Blinatumomab in Patients with Relapsed/Refractory (r/r) ALL

Background: Blinatumomab, a bispecific anti-CD19/CD3 antibody has resulted in significantly improved outcomes in patients (pts) with r/r B-ALL compared to conventional chemotherapy. However, over half of treated pts fail to respond (CR/CRh/PR 44%), and majority of responders eventually relapse without consolidation therapy. There is an unmet need to design novel strategies to improve efficacy. Upregulation of PD-L1 on leukemic blasts and PD-1 on T cells has been shown to associate with resistance to blinatumomab in preclinical and clinical studies. Therefore, blockade of the PD-1 pathway represents a reasonable strategy to augment blinatumomab efficacy. Here we set out to test the combination of pembrolizumab and blinatumomab in a single arm, phase 1/2 trial (NCT03512405). Methods: Pts [≥18 years old (yo); ECOG <1] with CD19+ R/R B-ALL, received standard blinatumomab 9 mcg/day on days 1-7, 28 mcg/day on days 8-28 in cycle 1, and on days 1-28 in subsequent cycles. Pembrolizumab 200mg was given on day 15 (schedule A) or day 22(schedule B) in cycle 1, and on days 1 and 22 in subsequent cycles. Cycle length was 42 days except 35 days for cycle 1 in schedule A. The study completed the schedule A phase 1 portion with primary objectives of safety, tolerability and proceeded with recommended phase 2 dose (RP2D) using the "3+3” design. Results: As of June 15, 2022, 7 pts were enrolled to phase 1 with one unevaluable, and 8 patients to phase 2. All received schedule A dosing. Fourteen evaluable pts received a median of 2 (1-6) cycles of treatment. At baseline, the median age was 53 yr (24-76) with median 2 (1-3) prior lines of regimens, and median 62% (10-95) BM blasts. All except one patient accrued were poor risk. In cycle 1, 64% experienced cytokine release syndrome (CRS) with 1 gr 3 CRS and no gr 4. All CRS except 1 started prior to the day 15 dose of pembrolizumab. Neurologic toxicities were all reversible with only 1 pt with ≥ gr3 AE. All-non-hematologic gr3 toxicities were reversible. No dose limiting toxicity, ≥ gr4 non-hematologic toxicity, or treatment related deaths were seen. The complete remission (CR/CRi) rate is 79% (11/14). Ten of 14 evaluable pts (71%) achieved flow cytometry MRD negative CR/CRi after a median of 1(1-2) cycle. 6/8 patients with ≥50% blast achieved CR/CRi. One patient had >83% blasts after cycle 1 and achieved CR after cycle 2, supporting the positive impact of pembrolizumab. Seven pts in CR received allogeneic transplant (alloHCT) with post-transplant cyclophosphamide (PTCy) as graft versus host disease (GVHD) prophylaxis. Only 1 patient had ≥gr3 acute GVHD which resolved with treatment. With a median follow-up of 9.6 (1.8- 34) months, 6 CRs are ongoing (5 post-transplant) and 10 of 14 evaluable pts are still alive at the data cut-off. PD-1 detection decreased among both responders and non-responders after pembrolizumab administration. Responders after cycle 1 had higher CD8+ T cells which increased further following pembrolizumab administration. Correlative studies are ongoing. Conclusions: The combination of pembrolizumab and blinatumomab in phase 1/2 of this study in pts with r/r B-ALL was deemed safe with a manageable side effect profile. Encouraging anti-leukemic activity has been seen in the majority of treated patients (CR+CRi rate of 79%) and most CR are MRD negative, supporting the rational combination of these 2 agents. The phase 2 portion of the trial is continuing to recruit patients.

Haploidentical Natural Killer Cell Therapy As an Adjunct to Stem Cell Transplantation for Refractory Acute Myeloid Leukemia

Refractory acute myeloid leukemia (AML), defined as failure of 2 cycles of induction therapy at diagnosis or of 1 cycle at relapse, represents a subgroup with poor clinical outcomes. In our transplant cohort, the 5-year overall survival in this subgroup was 16% (Ganapule at al. JGO 2017). Haploidentical natural killer cell (NK) therapy is a strategy that is being explored in refractory malignancies. Our in-vitro and animal model data suggest that exposure to arsenic trioxide (ATO) results in enhanced NK cytotoxicity (Alex AA et al. Front. Immunol 2018). Historically, at our center, patients with refractory AML have been treated with cytoreductive therapy (FLAG ± idarubicin or mitoxantrone + etoposide for 3 to 5 days) followed by 1-week rest and then a reduced-intensity transplant with fludarabine + melphalan conditioning while in peak cytopenia. From February 2019, we initiated a phase II single arm clinical trial (CTRI/2019/02/017505) enrolling patients with refractory AML planned for a stem cell transplant in peak cytopenia. Patients received CD56-positive cells from an HLA haploidentical related family donor (other than the stem cell donor, wherever feasible) following cytoreductive chemotherapy. The NK cell donor preference strategy included presence of KIR ligand mismatch, greater number of KIR B motifs (or the B score), lower donor age, and negative donor specific antibodies tested using flowcytometry crossmatch (Figure 1a). CD56-positive selection was done using CliniMACS prodigy system. This was followed by overnight incubation of the CD56 positive cells in autologous plasma with 2 micromolar ATO and 500 U/mL of interleukin-2. The CD56 positive cells were then infused to the patient 1-day after the completion of cytoreductive chemotherapy. This was followed by a reduced intensity stem cell transplant (Figure 1b). The primary outcome variable was 1-year relapse free survival. From February 2019, 14 patients with median age 28 years (IQR: 15.75-31.5) were enrolled in this trial. Six were females. Six had primary-refractory AML while 8 had relapsed-refractory AML. The cytoreductive chemotherapy was FLAG ± idarubicin (n=7), Mitoxantrone + Etoposide (n=6) and GCLAC (n=1). The median blast percentage on flowcytometry MRD testing prior to NK infusion was 15.9% (IQR: 9.1%-54.5%) (n=11). The median B score for the NK cell donors was 2 (IQR: 1-3). The median age of the NK cell donor was 43 years (IQR: 36-49.5). KIR ligand mismatch with the patient was noted in 2 donors. The median CD56-cell dose infused was 46.16 x 10 6/kg (IQR: 25.06-70.36) (Figure 1c). Pre-defined release criteria, including sterile cultures, and endotoxin negativity were met in all cases. There was no infusion related toxicity. The median blast percentage on flowcytometry MRD testing done following NK cell infusion was 11.9% (IQR: 4.9%-47.6%) (n=8). One patient withdrew consent after NK cell infusion and did not undergo transplant. For the remaining 13 patients, the stem cell donor was HLA matched (n=4), HLA 9/10 matched (n=1) or HLA haplomatched (n=8). The median CD34 cell dose infused was 10 x 10 6/kg (IQR:7.51-11.6). Five (38.5%) patients died of immediate post-transplant complications (sepsis (n=3) on days 1, 2 and 28, cerebral venous sinus thrombosis (n=1) on day 1 in a patient treated with hormonal contraceptives for menorrhagia, and veno-occlusive disease (n=1) on day 15 in a patient undergoing a second transplant) while 2 (15.4%) did not engraft (both subsequently died of infective complications following engraftment post-second transplant). Of the remaining 6 (46.2%) patients who engrafted and survived beyond 1 month of the transplant, the day 28 post-transplant MRD was negative for 5 patients while it was positive in 1 patient (0.13%). On follow up, 2 (15.4%) patients developed disease relapse (on days 54 and 218 respectively) and died. The remaining 4 (30.8%) patients are alive and relapse free at last follow up (mean follow up of surviving patients is 16 months). One patient received a CD34 cell boost on day 96 (cell dose - 8.55 x 10 6/kg) for poor graft function. For the entire cohort, the estimated 1-year event free survival is 28.8% ± 13.1%(Figure 1d). Whereas, acute GVHD was noted in 3 patients (50%; out of 6 evaluable patients) and chronic GVHD was noted in 3 patients (50%; out of the 6 evaluable patients). Thus, haploidentical NK cell therapy as an adjunct to transplant is safe and merits further evaluation in patients with AML. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

Acute Lymphoblastic Leukemia

Acute Lymphoblastic Leukemia

Day 15 and Day 33 Minimal Residual Disease Assessment for Acute Lymphoblastic Leukemia Patients Treated According to the BFM ALL IC 2009 Protocol: Single-Center Experience of 133 Cases.

Introduction: Childhood acute lymphoblastic leukemia (ALL) is a hematologic malignancy characterized by the acquisition of several genetic lesions in the lymphoid progenitors with subsequent proliferation advantage and lack of maturation. Along the years, it has been repeatedly shown that minimal residual disease (MRD) plays an important role in prognosis and therapy choice. The aim of the current study was to determine the prognostic role of MRD in childhood ALL patients in conjunction with other relevant patient and disease characteristics, thus showing the real-life scenario of childhood ALL.Patients and Methods: The retrospective study includes childhood ALL patients that were treated according to the BFM ALL IC 2009 between January 2016 and December 2018 at the Fundeni Clinical Institute, Bucharest, Romania.Results: None of the variables significantly influenced the induction-related death in our study. None of the variables independently predicted relapse-free survival (RFS) with the highest tendency for statistical significance being represented by poor prednisone response. Non-relapse mortality (NRM) was independently predicted by age, prednisone response, and day 33 flow cytometry-MRD (FCM-MRD). Overall survival (OS) was independently predicted by prednisone response and day 33 FCM-MRD. Event-free survival (EFS) was independently predicted by age, prednisone response, and day 33 FCM-MRD.Conclusion : Prednisone response, day 15 FCM-MRD, day 33 FCM-MRD, and the risk group represent the most important factors that in the current study independently predict childhood ALL prognosis.

Multiparametric in situ imaging of NPM1-mutated acute myeloid leukemia reveals prognostically-relevant features of the marrow microenvironment.

Ancillary testing during the initial workup of acute myeloid leukemia (AML) is largely performed using aspirated materials. We utilized multiplex immunofluorescence (MIF) imaging with digital image analysis to perform an in situ analysis of the microenvironment in NPM1-mutated AML using diagnostic bone marrow biopsy tissues (N = 17) and correlated these findings with diagnostic next-generation sequencing (NGS, N = 17), flow cytometry (FC, N = 14), and first remission (CR1) NPM1-specific molecular MRD (n = 16) data. The total CD3-positive T-cell percentages correlated positively between FC and MIF (r = 0.53, p = 0.05), but were significantly lower by MIF (1.62% vs. 3.4%, p = 0.009). The percentage of mutant NPM1-positive (NPM1c+) cells ranged from 9.7 to 90.8% (median 45.4%) and did not correlate with the NPM1 mutant allele fraction by NGS (p > 0.05). The percentage of CD34+/NPM1c+ cells ranged from 0 to 1.8% (median 0.07%). The percentage of NPM1c+ cells correlated inversely (34% vs. 62%, p = 0.03), while the percentages of CD3-/NPM1c- cells (64% vs. 35%, p = 0.03), and specifically CD3-/CD4-/NPM1c- cells (26% vs. 13%, p = 0.04), correlated positively with subsequent MRD. Discordances between MIF and FC/NGS data suggest that aspirate materials are likely an imperfect reflection of the core biopsy tissue. Furthermore, increased numbers of NPM1 wild-type cells within the microenvironment at diagnosis correlate with the subsequent presence of MRD.

Next Generation Sequence Minimal Residual Disease (NGS-MRD) Predicts Event Free Survival (EFS) Irrespective of Hematopoietic Cell Transplantation (HCT) Conditioning Regimen, Graft Αβ T and B-Cell Depletion or Patient Weight Category in Children with Acute Lymphoblastic Leukemia (ALL)

MRD detected by flow cytometry (FC) or PCR has been associated with key outcomes after HCT for ALL. In a prospective multicenter trial (NCT02646839; Pediatric Blood and Marrow Transplant Consortium [PBMTC] ONC1401), we performed a planned analysis of NGS-MRD to predict outcomes pre- and post-HCT for ALL patients (n=57, median follow-up 523 [range 58-1198] days post-HCT). We evaluated baseline blast samples from 74 patients for dominant BCR/TCR rearrangements and to follow MRD by NGS. Dominant clones were identified in 100% of B-ALL patients, 96.8% (61/63) in BCR and 3.2% in TCR gamma. For T-ALL patients, clones were identified in 62.7% (7/11), with the remaining 37.3% being polyclonal. Patients proceeded to HCT only if they were in morphological remission. Pre-HCT NGS-MRD from bone marrow (BM) was highly predictive of EFS (n=29 P=0.027, Figure 1) and NGS-MRD from peripheral blood (PB) trended similarly (n=27, P=0.17, Figure 2). In BM NGS-MRD negative patients, relapse was exceptionally low with all events due to transplant related mortality (TRM). There did not appear to be a benefit of acute (Figure 3) or chronic graft-vs-host-disease (GVHD) in NGS-MRD- patients. Pre-HCT, 10% of the BM samples were MRD+ by FC, but 35% were MRD+ by NGS. Direct comparison of NGS-MRD in BM and PB with FC MRD pre- and post-HCT showed improvements in positive and negative predictive power. Αβ-T and B-cell depleted haploidentical grafts had similar outcomes to other stem cell sources (Figure 4) with decreased incidence of GVHD [aGVHD > grade 2: n=1 (3.3%) and extensive cGVHD: n=1(3.3%)]. TBI (total body irradiation) based myeloablative conditioning (TBI/TT [Thiotepa]/CY [Cyclophosphamide], TBI/CY, or TBI/VP16; ± anti-thymocyte globulin [ATG]) and non-TBI reduced toxicity (Flu [Fludarabine]/Mel [Melphalan]/TT; ± ATG) had similar EFS (P= 0.31). TRM was very low 8.7% (n=5) in this population (n=57)) and rescue of relapse was high for the duration of follow up to date, resulting in similar OS for MRD- vs. MRD+ patients (P= 0.15), likely due to rescue with cell/immunotherapy. We examined the interaction of obesity, using body mass index (BMI) based on height/weight, pre-HCT, in the context of NGS-MRD on EFS. The BMI was converted to a percentile through population norms for age, gender, and defined thresholds published by the Centers for Disease Control and prevention (CDC). Lean patients (< 85th percentile [%]) overall had better survival than the overweight (OW)/obese (85-94%/≥95%) (Figure 5: P=0.016). Among the lean patients, NGS-MRD was prognostic, with NGS-MRD+ patients having worse EFS. This was also observed with the OW/obese group, where being NGS MRD+ led to dismal survival. Overweight/Obese patients who were pre-HCT MRD- had survival similar lean/MRD+ patients. Thus, both factors, weight category and NGS-MRD influenced EFS (Figure 6: P=0.02).

Next Generation Sequence Minimal Residual Disease (NGS-MRD) Predicts Outstanding Event Free Survival (EFS) Regardless of Hematopoietic Cell Transplantation (HCT) Preparative Approach or Graft Alpha/Beta Depletion in Children with Acute Lymphoblastic Leukemia (ALL)

MRD detected by flow cytometry (FC) or PCR has been associated with key outcomes after HCT for ALL. In a prospective multicenter trial (NCT02646839; Pediatric Blood and Marrow Transplant Consortium [PBMTC] ONC1401), we performed a planned sub-study of NGS-MRD to predict outcomes pre- and post-HCT for ALL patients (n=57, median follow-up 523 [range 58-1198] days post-HCT). In addition, we performed analyses of relevant clinical factors to assess their relationship to EFS. The larger trial studying the role of KIR favorable haploidentical vs. other transplantation approaches for children diagnosed with ALL, acute myeloid leukemia and myelodysplastic syndrome will be reported separately. We evaluated baseline blast samples from 74 patients for dominant BCR/TCR rearrangements and to follow MRD by NGS. Dominant rearrangements were identified in 100% of B-ALL patients, 96.8% (61/63) in BCR and 3.2% in TCR gamma. For T-ALL patients, rearrangements were identified in 62.7% (7/11), with the remaining 37.3% being polyclonal. Patients proceeded to HCT only if they were in morphological remission. Pre-HCT NGS-MRD from bone marrow (BM) was highly predictive of EFS (n=29 P=0.027, Figure 1) and although NGS-MRD from peripheral blood (PB) did not reach statistical significance due to decreased sample size (n=27, P=0.17, Figure 2), it trended similarly. In BM NGS-MRD negative patients, relapse was exceptionally low with all events due to transplant related mortality (TRM). There did not appear to be a benefit of acute graft-vs-host-disease (aGVHD) in NGS-MRD- patients (Figure 3) or chronic (c)GVHD, but sample size was a limitation. Pre-HCT, 10% of the BM samples were MRD+ by FC, but 35% were MRD+ by NGS. Direct comparison of NGS-MRD in BM and PB with FC MRD pre- and post-HCT showed improvements positive and negative predictive power (data not shown). Alpha/beta depleted haploidentical grafts had similar outcomes to other stem cell sources (Figure 4) with decreased incidence of GVHD [aGVHD &gt; grade 2: n=1 (3.3%) and extensive cGVHD: n=1(3.3%)]. TBI (total body irradiation) based myeloablative conditioning (TBI/TT [Thiotepa]/CY [Cyclophosphamide], TBI/CY, or TBI/VP16; ± anti-thymocyte globulin [ATG]) and non-TBI reduced toxicity (Flu [Fludarabine]/Mel [Melphalan]/TT; ± ATG) had identical EFS (P= 0.31). TRM was very low 8.7%(n=5) in this population (n=57)) and rescue of relapse was high for the duration of follow up to date, resulting in similar OS for MRD- vs. MRD+ patients (P= 0.15), likely due to rescue with cell/immunotherapy. We next examined the interaction of obesity, using body mass index (BMI) based on height/weight pre-HCT, in the context of NGS-MRD on EFS. The BMI score was converted to a percentile through population norms for age and gender and defined thresholds published by the Centers for Disease Control and prevention (CDC). Lean patients (&lt; 85th percentile [%]) overall had better survival than the overweight (OW)/obese (85-94%/≥95%) (Figure 5: P=0.016). But among the lean patients, NGS-MRD still had an effect, with NGS-MRD+ patients had poorer EFS. Same was observed with the OW/obese group, where being NGS MRD+ adds further reduction in survival. Overweight/Obese patients who were pre-HCT MRD- had survival closer to the lean/MRD+ patients. We observed decreased EFS by weight category and NGS-MRD (Figure 6: P= 0.02). In conclusion, NGS-MRD was highly predictive for EFS regardless of HCT preparative approach or graft Alpha/Beta Depletion. NGS improved predictive power for MRD positivity compared to FC. NGS was able identify rearrangements suitable for MRD tracking in all B-ALL and in most of T-ALL samples. In BM NGS-MRD negative patients, relapse was exceptionally low. There did not appear to be a benefit of aGVHD or cGVHD in NGS-MRD- patients. Alpha/beta depleted haploidentical grafts had similar outcomes to other stem cell sources with decreased incidence of severe GVHD. We observed decreased EFS by weight category and NGS-MRD. Lean patients had better survival, after HCT, than OW/Obese patients. But among the lean patients, NGS-MRD+ patients had poorer EFS. Disclosures Abdel-Azim: Adaptive: Research Funding. Dvorak:Jazz Pharmaceuticals: Consultancy; Alexion Inc: Consultancy. Bunin:PRA Health Sciences: Other: Immediate family member employed. Walters:Editas Medicine: Consultancy; TruCode: Consultancy; AllCells, Inc: Consultancy. Cairo:Jazz Pharmaceuticals: Other: Advisory Board, Research Funding, Speakers Bureau; Miltenyi: Other: MTA; Osuka: Research Funding. Kitko:Novartis: Consultancy, Honoraria; Mallinckrodt: Honoraria. Jacob:Adaptive Biotechnologies: Employment, Other: shareholder. Wing:Miltenyi Biotec: Employment. Pulsipher:Medac: Honoraria; Miltenyi: Research Funding; Bellicum: Consultancy; Amgen: Other: Lecture; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Other: Education for employees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Switching Towards Monocytic Lineage and Discordancy between Flow Cytometric and PCR Minimal Residual Disease Results Is a Hallmark Feature of DUX4 Rearranged B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction:Recently we described a subgroup of pediatric patients with B cell precursor acute lymphoblastic leukemia (BCP ALL) with switching from B to monocytic lineage in early phase of the therapy (Slamova et al., 2014). In a limited cohort of patients with switching ALL (swALL), we observed inferior response to treatment with discrepancy of minimal residual disease level (MRD) assessed by flow cytometry (FC) and quantitative polymerase chain reaction (qPCR) of Immunoglobulin-T cell receptor (Ig-TCR) rearrangements. In current Berlin-Frankfurt-Münster (BFM) treatment protocols, FC MRD value at day 15 (d15) and PCR MRD value at day 33 (d33) and week 12 (w12) are used for stratification. Using an extended cohort of patients with available RNA sequencing data (cohort mainly focused on B other cases or swALLs) we aimed to answer following questions:What is the genetic background of swALL? What is the frequency among swALLs of the recently described DUX4 rearranged subgroup?How do B cell oriented FC and PCR MRD correlate in standard protocol timepoints, i.e. day 8 (d8) (peripheral blood, PB) d15, d33 and w12 (bone marrow, BM) of treatment?What is the characteristic MRD response to treatment in swALL?Results:We performed RNA sequencing in 177 patients (median age 6.1 years, range 0-18) treated by several treatment protocols (ALL BFM 95 n=5, ALL IC BFM 2002 n=14, ALL AIEOP BFM 2000 n=17, ALL AIEOP BFM 2009 n=135, Interfant n=3, ALL IC/Interfant n=2, EsPhALL n=1). In 68 patients we observed switching phenomenon by appearance of B/monocytoid population coexpressing B lineage (CD19, CD34) and monocytic lineage (CD33, CD14) markers (median 0.98%, range 0.032-38%). In non swALLs median of this population was 0.059% (range 0.0025-1.1%) and the cells did not form a clear cluster. According to RNAseq data, majority of swALL patients (n=42/68) belong to DUX4 subgroup (chi square p< 0.00001). The distribution into other molecular genetic subtypes is summarized in table 1.Correlation coefficient (Spearman) of all included samples with both available values (n=552) was 0.82 (p<0.0001), Concordance in categorization of positivity and negativity with cut-off 1e-4 was 85%. We observed worse correlation between FC and PCR MRD in patients with swALL (d8 R=0.58, d15 R=0.6, d33 R=0.36, w12 n.s.) compared to non swALL (d8 R=0.83, d15 R=0.91, d33 R=0.69, w12 R=0.37). However, concordance in swALL in categorization of positivity and negativity with cut-off 1e-4 was still ≥80% in each analyzed timepoint apart from d33 with concordance only 44% showing significant discrepancy of both methods (Figure 1a). On the contrary, concordance in non swALL was ≥87% for each analyzed timepoint (d33 with concordance 87% shown in Figure 1b). Poor correlation between B-cell oriented FC MRD and PCR MRD at d33 was also obvious when analyzed DUX4 subgroup separately (R=0.31 (p=0.04), concordance 45%).We observed significantly higher MRD in swALLs compared to non swALLs at all analyzed timepoints: d8 (p=0.0021), d15 (p=0.0088, d33 (p<0.0001) and w12 (p=0.008). Higher MRD levels were also found in DUX4 patients when compared to non DUX4 (all timepoints p<0.05). Interestingly, when compared swALL and non swALLs pts in DUX4 subgroup only, the DUX4 swALLs are those with poorer treatment response (all timepoints p<0.05). With respect to protocolar cut-off values, FC MRD at d15 was above 10% in 18/67 swALL patients (in 13/52 DUX4 patients), d33 PCR MRD was above 0.1% in 33/57 swALLs (24/44 DUX4 pts) and at w12 PCR MRD was above 0.01% in 12/55 swALLs (10/44 DUX4 pts).Conclusions:DUX4 subgroup is the most prevalent genetic subtype among swALLs. SwALLs and/or DUX4 subgroup have poorer treatment response at d15, d33 and w12. However, it remains to be elucidated whether poor initial treatment response is eventually reflected in treatment outcome. In majority of swALL patients B cell phenotype of blasts is preserved at day 15 enabling correct classification. Prominent discrepancy between FC and PCR MRD is present especially at d33 and development of different FC MRD strategies focused on monocytic compartment is needed.Supported by Ministry of Health of the Czech Republic, grant nr. 15-28525A and NV18-03-00343; Czech Science Foundation nr.P302/12/G101, UNCE204012 [Display omitted] DisclosuresBrüggemann:Affimed: Research Funding; Regeneron: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau; Incyte: Consultancy; PRMA: Consultancy. Ritgen:abbvie: Research Funding; Roche: Honoraria, Research Funding.

Comparison of Real-time Quantitative Polymerase Chain Reaction and Eight-color Flow Cytometry in Assessment of Minimal Residual Disease in Adult Acute Lymphoblastic Leukemia

BackgroundMinimal residual disease (MRD) is an important prognostic maker in acute lymphoblastic leukemia (ALL). However, few data comparing the measurement of adult ALL MRD using different methods in daily practice are available. We conducted an analysis comparing the importance of flow cytometry (FCM) and real-time quantitative polymerase chain reaction (PCR) in the assessment of MRD in adult ALL. Patients and MethodsFifty-six consecutive adult patients with both Philadelphia-negative and -positive ALL treated according to an intensive protocol were enrolled in the study. Bone marrow samples were acquired on day 26 and during week 11 of treatment. MRD evaluation was performed using 8-color FCM and PCR of immunoglobulin or T-cell receptor gene clonal rearrangements and BCR-ABL1, KMT2A-AF4 and E2A-PBX1 fusion genes. ResultsOn day 26, both FCM and PCR seemed to have good discrimination sensitivity for overall survival (P = .001 to .008) and progression-free survival (P = .03 to .04) prediction for both Philadelphia-positive and -negative cases. The most sensitive method in week 11 was PCR including all results > 0 considered to indicate MRD positivity (P = .002 for overall survival and P = .02 for progression-free survival). PCR with other cutoffs was not sufficiently sensitive in week 11. Moreover, no FCM+ samples were found in week 11. The subanalysis of the Philadelphia-negative patients showed similar results. ConclusionOur analysis showed that both FCM and PCR MRD assessment methods are sensitive for survival prediction during induction. However, we believe FCM could not be sufficiently sensitive in later phases of treatment.

SIGNIFICANCE OF ETV6-RUNX1 FUSION GENE TRANSCRIPT DETECTION IN PEDIATRIC B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA WITH TRANSLOCATION t(12;21)(p13;q22)

Introduction. Translocation t(12;21)(p13;q22) is one of the most common structural genetic abnormalities in childhood acute lymphoblastic leukemia (ALL). It cannot be detected by conventional G-banding, so a reverse-transcriptase polymerase chain reaction (RT-PCR) or fluorescent in situ hybridization are used for this purpose. The aim of the study was to evaluate the prognostic significance of qualitative and quantitative detection of ETV6-RUNX1 fusion gene transcript at various time points in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Materials and methods. ETV6-RUNX1 fusion gene transcript was revealed by both reverse-transcriptase PCR and quantitative real-time PCR (RQ-PCR) in 34 out of 166 (20.5 %) children with BCP-ALL. Qualitative ETV6-RUNX1-positivity at days 36 and 85 led to unfavorable outcome (lower event-free survival –EFS and higher cumulative incidence of relapse – CIR). While ETV6-RUNX1 status at day 15 did not allow to divide patients with different outcomes. By ROC curve analysis we determined threshold levels (TL) for ETV6-RUNX1/ABL1 ratio at days 0, 15, 36 and 85. Afterwards we adjusted obtained results to 10-fold scale. Results. So practically applicable TL were as follows 500.0 %, 1 %, 0.1 % и 0.01 % for days 0, 15, 36 and 85, respectively. EFS and CIR were both worse in patients with ETV6-RUNX1/ABL1 ratio equal or above defined TL. Moreover, initial ratio ≥500,0 % corresponded to delayed blast clearance at days 15 and 36. We showed good qualitative (84.8 %) and quantitative (R 2 = 0.953) concordance between ETV6-RUNX1/ABL1 ratio and MRD data obtained by flow cytometry at days 15, 36, 85. Of note, defined TL for ETV6-RUNX1/ABL1 at days 15, 36, 85 were equal to prognostically important levels for flow cytometry MRD. Conclusion. Thus, qualitative detection and quantitative value of ETV6-RUNX1 fusion gene transcript showed prognostic significance in the course of treatment in children with BCP-ALL. Based on these results we propose standardization approaches for Moscow – Berlin ALL study group.

Evaluation of Minimal Residual Disease As a Surrogate Endpoint for Event Free Survival in Childhood B-Lineage Acute Lymphoblastic Leukemia

IntroductionThe need to optimize clinical evaluation of new drugs stimulates researchers and regulatory bodies to consider novel endpoints that facilitate assessment of the efficacy of a new drug earlier in time than do traditional endpoints. To be a useful marker of efficacy, an early endpoint must be an accurate surrogate for the true clinical endpoint.Minimal residual disease (MRD) is a strong prognostic factor for Event Free Survival (EFS) in children with newly diagnosed acute lymphoblastic leukemia (ALL) and is used routinely to assess treatment response and stratify treatment intensity. However, it is not known whether or not early MRD response is an accurate surrogate endpoint for EFS in evaluating the efficacy of treatment interventions.This study addresses for the first time in childhood ALL the formal validation of surrogacy of MRD levels at the end of induction treatment by a meta-analytic approach on individual data from two large phase III trials with a randomized question on type of steroids in induction (dexamethasone 10mg/m2/day vs prednisone 60mg/m2/day).Material and MethodsWe performed a meta-analysis of individual data of 2955 B-ALL patients from AIEOP-BFM-ALL2000 (NCT00613457, NCT00430118), and 945 and 930 high risk B-ALL patients randomized for steroids after being allocated either to Capizzi or High-dose Methotrexate in COG AALL0232 (NCT00075725; separately considered due to the significant quantitative interaction between Methotrexate regimens and type of steroid). The trials included evaluation of MRD at day +33 (PCR-MRD) and +29 (flow-cytometry MRD), respectively, with a sensitivity of at least 10-4. The three categories MRD level (negative, low positive i.e. <5x10-4 and positive ≥5x10-4), was assessed as surrogate for the EFS endpoint (time to event defined as resistance at the end of induction, relapse, death in remission, second malignancy).A two-level modelling approach was used to estimate the association between MRD and EFS and between the treatment effect on MRD (proportional odds model) and on EFS (proportional hazard model). The quality of the surrogate at the individual level was assessed on the basis of the bivariate Plackett copula model, with a parameter representing the global odds ratio. The quality of the surrogate at the trial level was assessed on the basis of the coefficient of determination R2trial from a linear regression through the estimated treatment effects.ResultsThe main results on MRD and EFS by trial and treatment are in table 1.The meta-analytic approach shows that MRD at the end of induction is a poor surrogate for treatment effect on EFS (Figure 1) at the trial level, with R2trial=0.09 (95% CI: 0-0.29), while, at the individual level, it shows a considerable prognostic association with EFS, after adjusting for treatment, with a 3.9 odds ratio of failure for patients with higher compared to lower MRD levels (95% CI: 3.4-4.4). Additional sensitivity analyses on relevant subgroups generally confirmed the previous findings both at the trial and patient level association.ConclusionsUsing a meta-analytic approach, we found that MRD, in 3 categories defined according to standard cut-points, is a poor surrogate for EFS at the trial level, thus indicating that the effect of the randomized steroids (dexamethasone vs. prednisone in induction) on the MRD level at the end of induction does not reliably predict the effect of the intervention on EFS. In contrast, the analysis shows a strong and highly significant association between end induction MRD level and EFS time for individual patients, regardless of treatment, confirming the prognostic effect of early MRD response on clinical outcome.This study shows, for the first time, the limitation of a strong prognostic factor in being a surrogate in the context of front line ALL treatments. The impact of type of steroid on MRD distribution at the end of induction is relatively limited and subsequent treatment complexity and intensity, partly tailored on MRD itself as a key criterion used to modulate the intensity of post-induction therapy, may dilute a potential surrogacy. These data suggest that clinicians and regulatory bodies should be cautious in using early MRD response in the context of complex multiagent therapy as an early surrogate endpoint to evaluate the effect of a randomized treatment intervention on long-term EFS. [Display omitted] [Display omitted] DisclosuresMoricke:JazzPharma: Honoraria, Other: financial support of travel costs. Biondi:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees; Cellgene: Other: Advisory Board.

Frontline Inotuzumab Ozogamicin in Combination with Low-Intensity Chemotherapy (mini-hyper-CVD) for Older Patients with Acute Lymphoblastic Leukemia (ALL)

▪Background: Elderly patients with ALL have a significantly poor outcome. This is primarily due to poor tolerance of intensive chemotherapy. Addition of targeted non-myelosuppressive therapy to effective low-intensity chemotherapy might improve outcome. CD22 expression occurs in >90% of patients with ALL. Inotuzumab ozogamicin (IO) is a CD22 monoclonal antibody bound to a toxin, calecheamicin, and has shown single-agent activity in relapsed/refractory ALL (Kantarjian et al. Lancet Oncology 2012).Methods: Patients ≥60 years with newly-diagnosed B-cell ALL were eligible for the chemotherapy with mini-hyper-CVD (cyclophosphamide and dexamethasone at 50% dose reduction, no anthracycline, methotrexate at 75% dose reduction, cytarabine at 0.5 g/m2 x 4 doses) in combination with IO given on Day 3 of each of the first 4 courses. Rituximab and intrathecal chemotherapy were given for first 4 courses. The first 6 pts received 1.3 mg/m2 for cycle 1 followed by 0.8 mg/m2 for subsequent cycles; Pts 7 onwards received 1.8 mg/m2 for Cycle 1 followed by 1.3 mg/m2 for subsequent cycles.Results: Thirty-four patients (21 men, 13 women) have been treated so far. Patient characteristics and outcome are summarized in Table 1. Median age is 69 years (range 60-79). Median follow-up is 19 months. Of the 31 patients evaluable for response (3 started in CR), 30 (97%) achieved CR/CRp (25 CR, 5 CRp). All patients achieving CR have also achieved flow-cytometry MRD negative status, in 80% at the time of CR achievement. Median time to platelet and neutrophil recovery was 23 days (18-91), and 16 days (12-49) after induction, and was 22 days (14-64), and 17 days (13-49) after subsequent cycles. Grade 3-4 toxicities ≥10% included prolonged thrombocytopenia (n=27; 79%), infections during consolidation (n=25; 73%), infections during induction (n=18; 52%), hyperglycemia (n=17; 50%), hypokalemia (n=12; 35%), hyperbilirubinemia (n=8; 24%), increased ALT/AST (n=7; 21%), and hemorrhage (n=6; 18%). Of note, veno-occlusive disease (VOD) was observed in 4 patients (11%); 1 in CR and negative MRD, G2 VOD at C3D57; 1 in CR and negative MRD, G5 VOD at C2D40; 1 in CR and negative MRD, G2 VOD at D43 post matched related allogeneic stem cell transplantation with conditioning of fludarabine and busulfan after 4 cycles of mini-hyper-CVD + IO; and 1 in CR and negative MRD, G2 VOD at C2D70. At the last follow-up, 23 (68%) are alive and 22 (65%) in CR. Eleven patients (32%) died: 1 had primary refractory ALL and died after the first salvage; 2 relapsed after receiving 2 and 3 courses only due to prolonged myelossuppression and died of disease progression; and 8 died in CR from sepsis (n=4), gunshot wound (n=1), VOD (n=1), dementia (n=1), and unknown cause (n=1). Two patients (6%) received allogeneic stem cell transplantation. Four patients (12%) are currently receiving consolidation chemotherapy with a median of 5 cycles (1-8); 16 patients (47%) are receiving POMP maintenance chemotherapy. The 2-year progression-free survival and overall survival rates were 87% and 70%, respectively. The mini-hyper-CVD (n=34) appears superior to the historical HCVAD +/- rituximab (n=46) in similar patient population (2-year OS, 70% and 38%, respectively; Figure 1).Conclusions: The combination of IO with low-intensity mini-hyper-CVD chemotherapy is safe and shows encouraging results (97% CR/CRp) in the frontline setting in older patients with ALL. These results appear to be better than those achieved with a chemotherapy alone only approach and may become the new standard of care for frontline treatment of older patients with ALL.Table 1Patient characteristics and outcomeCharacteristicMedian (range) / No. (%) N=34Age (yrs)69 [60-79]Male21 (62)Performance Status (ECOG) ≥ 24 (12)WBC at DX3.5 [0.6-111.0]WBC at DX ≥ 502 (6)KaryotypeDiploid Complex Misc IM11 (32) 15 (44) 5 (15) 3 (9)CD22 Positivity97 [72-100]CD20 ≥ 2020 (65)ResponseCR25/31 (81)CRp5/31 (31)Cytogenetic CR19/19 (100)Negative MRD, D2120/25 (80)Negative MRD, Overall33/33 (100)ORR30/31 (97)No response1/31 (3)Early death0 [Display omitted] DisclosuresO'Brien:Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Cortes:Teva: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; ARIAD Pharmaceuticals Inc.: Consultancy, Research Funding.

FLT3-ITD As a Target for Minimal Residual Disease Monitoring in Early T-Cell Precursor (ETP) Acute Lymphoblastic Leukemia

Background. The 'early T-cell precursor’ (ETP) subtype of T-ALL comprises up to 15% of T-ALL and has been reported to be associated with high risk of relapse. In addition to properties of T cell development, gene expression profile and immunophenotype of ETP-ALL show stem cell and early myeloid features. Consistently, this leukemia subgroup shows lower frequencies of prototypical T-ALL lesions and a higher prevalence of mutations typically associated with AML, including RAS and FLT3 mutations. In particular, FLT3-ITD was identified in up to 35% of adult ETP-ALL but data on its prevalence in pediatric ETP-ALL are lacking.In agreement with its stem-cell signature, ETPs frequently lack Immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements, the most used and sensitive targets for MRD monitoring. As a consequence, alternative markers are required to extend the application of molecular MRD to most ETP-ALL patients.Aim. We explored the prevalence of FLT3-ITD mutation in a large series of pediatric ETP-ALL enrolled in two consecutive protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP), and we evaluated the potential use of FLT3-ITD as an alternative DNA marker for MRD monitoring.Methods. Out of 439 T-ALL patients enrolled in Italy into the AIEOP-BFM ALL2000 and AIEOP ALLR2006 consecutive protocols, 145/168 Early-T ALL (TI/II) patients were screened for FLT3-ITD occurrence. Among Early-T patients, 34 were defined as ETP according to immunophenotype, and 31 of them were screened for FLT3-ITD. Twenty-two ETP patients enrolled in Italy into the ongoing AIEOP-BFM ALL2009 were also screened, together with T-ALL cases without IG/TR molecular markers only for the technical validation of the method. PCR screening and RQ-PCR for FLT3-ITD were performed as previously reported (Nakao, Leukemia 1996;10:1911; Beretta, Leukemia 2004;18:1441). Parallel MRD analysis for IG/TR on the same samples, and flow cytometry-MRD were performed by standard procedures. EuroMRD guidelines were applied for performance and interpretation of RQ-PCR.Results. Among ALL2000/R2006 and ALL2009 ETP cases, 4/31 (12.9%) and 3/22 (13.6%) were FLT3-ITD positive, respectively; 5/7 were PPR, and all 7 were stratified as high risk. For ALL2000/R2006 patients, IG/TR MRD monitoring was feasible in 2 cases, and both were MRD-HR; 3/4 cases are alive in CCR, and one died after HSCT. Overall, the FLT3-ITD marker was detected in 12 T-ALL cases; only 4 of them had valuable IG/TR markers, while 8/12 (66%) did not present a suitable IG/TR MRD marker. FLT3-ITD MRD monitoring was performed on 11/12 FLT3-ITD positive T-ALL cases. Mean length of the ITD was 44 nucleotides (nts) (range 24-71), with a mean of 7 randomly inserted nts (range 1-26). Standard curves performed by 10-fold dilutions in DNA from PB Healthy Donor, showed a quantitative range of at least 5.0E-04 in all cases and 1.0E-04 in 5/11. Sensitivity of the assay was at least 1.0E-04 in all tested cases, and 1.0E-05 in 7/11.A comparison between IG/TR and FLT3-ITD was feasible in 3 out of 4 cases (1 is ongoing); all 3 cases were monitored by 2 IG/TR markers. At day15 and day33 of the Induction therapy, when MRD was very high (10-1 to 10-3 range), the IG/TR and FLT3-ITD were fully comparable, with less than 2 times difference. At day78 (after IB Induction block) 1 case was fully negative for both markers, 1 was slightly positive by FLT3-ITD (although not quantifiable and at the limit of the sensitivity) and negative for both IG/TR. The latter case was highly positive for both IG/TR (5.0E-03) but low positive (<1.0E-04) by FLT3-ITD. In this case, the IG/TR and FLT3-ITD were concordant and finally both were negative at subsequent time points.Conclusions. This is the first report on FLT3-ITD prevalence in a consecutive series of children with ETP-ALL, which resulted to be 14.8% (4/31), a value lower than that reported in adult ETP-ALL. The limited number of cases does not allow to draw conclusions on the prognostic impact of FLT3-ITD in ETP-ALL, although 3 out of 4 patients are alive in CCR. Although available in a limited subset, FLT3-ITD can be used as a marker for sensitive molecular MRD monitoring in ETP-ALL, when IG/TR markers are not available (about 2/3 of cases). The results of MRD monitoring in a limited set of cases suggests that ETP patients might respond well to IB Induction therapy. As already known for AML, caution for false negative results is required when only FLT3-ITD is monitored. DisclosuresNo relevant conflicts of interest to declare.

Flow-Cytometry MRD Evaluation in Children with ALL; Can MRD Level on Day 33 Serve As a Surrogate Marker for Relapse?

Treatment-response assessment via quantification of MRD has become a corner-stone for risk stratification in childhood ALL. Although MRD has been shown to retain independent prognostic significance, the relationship with other prognostic variables has been incompletely explored. Moreover, determination of the most reliable treatment time-point for FCM-MRD evaluation that would serve as the best surrogate marker in predicting relapse, is still an issue of debate. To determine the utility of sequential measurements of FCM-MRD, with specific emphasis on day 33 and its correlation with known prognostic factors and outcome, we retrospectively analyzed the results of FCM-MRD study/data of 825 bone marrow samples of 133 children with ALL, at different treatment time-points, during induction and early consolidation (days 15, 33, 78, week 22-24, at start and at the end of maintenance therapy). All patients were homogeneously treated on BFM-based protocols and prognostic groups were defined by sex, WBC, prednisone response, risk-group allocation, immunophenotype and ETV6/RUNX1-presence. We investigated: a) the relationship between relapse probability and known prognostic factors like age, sex, WBC, immunophenotype, prednisone-response, ETV6/RUNX1 translocation, CDKN2A/2B deletion, hyperdiploidy, protocol risk-group, MRD(constant variable) and MRD positivity(≥0.1% vs <0.1%)(Kaplan-Meier and lïgrank test), b) the correlation between MRD(+) and outcome (OS and EFS, Kaplan-Meier). Additionally, multivariable Cox-models were used to determine the impact of MRD and WBC, sex, age, prednisone-response, on OS and EFS.Patients with MRD≥0.1% at the end of induction (day 33) were estimated with 5-year RFS rates of only 60.0±12.8%, compared to MRD low(<0.1%)/negative patients that enjoyed superior 5-year RFS rates of 91.3±3.0%. Both days 15 and 33 of FCM-MRD positivity maintained a significant prognostic impact on the incidence of relapse within all subgroups. MRD(+)d15 and MRD(+)d33 detection combined, appeared to have a statistically significant impact on survival probability (RFS 65.9%±10.6, p<0.001). Additionally, FCM-MRD on day 33 was notably more strongly associated with used prognostic factors, while we were able to demonstrate that FCM-MRD log-reduction rates between days 15 and 33 can also be added as an important relapse prediction marker, apart from absolute MRD quantifications.In our study, MRD(+)d15 and MRD(+)d33 in combination, strongly and statistically significant correlated with survival probability. Additionally, we propose the FCM-MRD log-reduction rate between days 15 and 33 and end-induction evaluation(day 33) as better predictors of survival and relapse probability, than only day 15 FCM-MRD estimations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Minimal Residual Disease-Based Risk Stratification in Chinese Childhood Acute Lymphoblastic Leukemia by Flow Cytometry and Plasma DNA Quantitative Polymerase Chain Reaction

Minimal residual disease, or MRD, is an important prognostic indicator in childhood acute lymphoblastic leukemia. In ALL-IC-BFM 2002 study, we employed a standardized method of flow cytometry MRD monitoring for multiple centers internationally using uniformed gating, and determined the relevant MRD-based risk stratification strategies in our local patient cohort. We also evaluated a novel method of PCR MRD quantitation using peripheral blood plasma. For the bone marrow flow MRD study, patients could be stratified into 3 risk groups according to MRD level using a single time-point at day-15 (Model I) (I-A: <0.1%, I-B: 0.1–10%, I-C: >10%), or using two time-points at day-15 and day-33 (Model II) (II-A: day-15<10% and day-33<0.01%, II-B: day-15≥10% or day-33≥0.01% but not both, II-C: day-15≥10% and day-33≥0.01%), which showed significantly superior prediction of relapse (p = .00047 and <0.0001 respectively). Importantly, patients with good outcome (frequency: 56.0%, event-free survival: 90.1%) could be more accurately predicted by Model II. In peripheral blood plasma PCR MRD investigation, patients with day-15-MRD≥10−4 were at a significantly higher risk of relapse (p = 0.0117). By multivariate analysis, MRD results from both methods could independently predict patients’ prognosis, with 20–35-fold increase in risk of relapse for flow MRD I-C and II-C respectively, and 5.8-fold for patients having plasma MRD of ≥10−4. We confirmed that MRD detection by flow cytometry is useful for prognostic evaluation in our Chinese cohort of childhood ALL after treatment. Moreover, peripheral blood plasma DNA MRD can be an alternative where bone marrow specimen is unavailable and as a less invasive method, which allows close monitoring.