Frequency Of Minimal Residual Disease Research Articles

Zanubrutinib, obinutuzumab, and venetoclax for first-line treatment of mantle cell lymphoma with a TP53 mutation

AbstractTP53-mutant mantle cell lymphoma (MCL) is associated with poor survival outcomes with standard chemoimmunotherapy. Dual Bruton tyrosine kinase and BCL2-inhibition with or without anti-CD20 monoclonal antibody therapy has shown promising activity in TP53-mutant MCL. We conducted a multicenter, phase 2 study of zanubrutinib, obinutuzumab, and venetoclax (BOVen) in untreated patients with MCL with a TP53 mutation. Patients initially received 160 mg zanubrutinib twice daily and obinutuzumab. Obinutuzumab at a dose of 1000 mg was given on cycle 1 day 1, 8, and 15, and on day 1 of cycles 2 to 8. After 2 cycles, venetoclax was added with weekly dose ramp-up to 400 mg daily. After 24 cycles, if patients were in complete remission with undetectable minimal residual disease (MRD) using an immunosequencing assay, treatment was discontinued. The primary end point was met if ≥11 patients were progression free at 2 years. The study included 25 patients with untreated MCL with a TP53 mutation. The best overall response rate was 96% (24/25) and the complete response rate was 88% (22/25). Frequency of undetectable MRD (uMRD) at a sensitivity level of 1 × 10–5 and uMRD at a sensitivity level of 1 × 10–6 at cycle 13 was 95% (18/19) and 84% (16/19), respectively. With a median follow-up period of 28.2 months, the primary end point was met with a 2-year progression-free survival of 72%, and the 2-year disease-specific and overall survival were 91% and 76%, respectively. Common side effects were generally low grade and included diarrhea (64%), neutropenia (32%), and infusion-related reactions (24%). BOVen was well tolerated and met its primary efficacy end point in TP53-mutant MCL. These data support its use and further evaluation of the BOVen regimen in this high-risk population. This trial was registered at www.ClinicalTrials.gov as #NCT03824483.

Relative Telomeric Length of Bone Marrow Cells at Diagnosis and Its Association with Minimal Residual Disease in Juvenile Acute Lymphoblastic Leukemia

Introduction: Identification of prognostic factors at diagnosis, risk stratification and the analysis of minimal residual disease (MRD) may predict treatment outcomes for children diagnosed with Acute Lymphoblastic Leukemia (ALL). A possible prognostic indicator is bone marrow mononuclear cells telomeric length at diagnosis. Currently, the best predictor of treatment response and disease-free survival is the analysis of MRD. Objectives: To investigate the association between bone marrow mononuclear cells telomeric length in children and adolescents with ALL at the time of diagnosis and the assessment of MRD on days 15, 33 and 78 after initiation of treatment. Methods: This study included patients aged 1 to 18 years, diagnosed with ALL and treated at Hospital da Criança de Brasília José Alencar. The telomeric length was determined by real-time polymerase chain reaction (qPCR) from a sample of bone marrow aspirate collected at the diagnosis. MRD was performed by flow cytometry immunophenotyping on days 15, 33, and 78 of chemotherapy. Risk stratification was determined according to specifications from BFM 2009 protocol. Results: 79 subjects were included in the study. Patients with negative MRD on D33 had significantly longer telomeric length (p = 0.02) when compared with those with positive MRD on D33. Patients classified as high risk had a higher frequency of positive MRD on D33 (p<0.01). Only positive DRM on D33 was a predictor of shorter telomeric length at diagnosis. Telomeric length at diagnosis was not related to D15 or D78 MRD results. Conclusion: This study suggests that longer telomeric length may be associated with a better prognosis, since these patients have negative MRD on D33. This result demonstrates that the assessment of the telomeric length of bone marrow cells from pediatric patients with ALL at the time of diagnosis can be a potential prognostic factor.

Modified Delphi Method Identifies Consensus Areas for Routine Minimal Residual Disease Testing in Multiple Myeloma

Modified Delphi Method Identifies Consensus Areas for Routine Minimal Residual Disease Testing in Multiple Myeloma

O-5 Frequency of minimal residual disease as measured by ctDNA in mismatch repair deficient tumors following curative resection

Mismatch repair deficient (MMRd) tumors are highly sensitive to checkpoint blockade (CPB) in patients with metastatic disease, regardless of tumor type. However, the efficacy of CPB in the adjuvant setting is unknown, especially since MMRd is considered a favorable biomarker for most resected tumor types. Circulating tumor DNA (ctDNA) could be used to screen for patients at high risk for recurrence following surgery or adjuvant chemotherapy, and identify patients (pts) that would most benefit from CPB.

The Significance of Flow Cytometry and Positron Emission Tomography and Computed Tomography (PET-CT) in the Assessment of the Minimum Residual Disease in Patients with Multiple Myeloma

Background.The minimal residual disease (MRD) status is the single posttreatment predictor of long-term outcomes after treatment in patients with multiple myeloma (MM), regardless of the treatment protocol or the presence of other pre-existing prognostic indicators. Traditionally, the method of evaluating MRD is multicolor flow cytometry (MFC), which has high sensitivity and specificity. Another way of detecting MRD is by the use of positron emission tomography and computed tomography (PET-CT) through the detection of possible patchy bone marrow infiltration or extramedullary involvement with an MRD-negative (MRD-) bone marrow. Aim.To determine the frequency of MRD- status by MFC and PET-CT and evaluate influence of MRD on the progression-free survival (PFS) in multiple myeloma patients. Methods.We analyzed 55 patients with MM (median age 56 years, male/female - 1.4:1). The induction therapy with Bortezomib-based regimens (VD, CVD, VMP, PAD) was used in 37/55 (67.3%) patients, Immunomodulator-based regimens (Thal+D, RD, VRD, PomD) - in 15/55 (27.3%), chemotherapy - in 3/55 (5.4%). Autologous stem cell transplantation (ASCT) was performed 32 (58.2%) patients. For definition of primary tumor cell's phenotype and assessment of MRD we used 5-color flow cytometry with CD38, CD138, CD45, CD19, CD20, CD27, CD56 and CD117 antibodies. MRD status by MCF and/or PET-CT was evaluated after 4-6 cycles of induction therapy or after ASCT, if it (ASCT) was performed. The MFC MRD- response was evaluated as identification less than 0.01% of clonal plasma cells in bone marrow. The PET-CT MRD- response was established if specific accumulation of 18F-fluorodeoxyglucose was absent. Results.The MFC MRD- was reached in 29.1% (16/55): “Bortezomib group” - 35.1% (13/37), “Immunomodulator group” - 13.3% (2/15) (Chi-square=2.472; p>.05). After ASCT MRD- status was achieved in 36.4% (4/11) patients. Maintenance therapy with bortezomib or lenalidomide did not affect the achievement of the MFC MRD- in patients with MRD-positive (MFC MRD+) response. Median PFS in MFC MRD+ group was 21 months, in the MFC MRD- - 66 months (p=.0042). Patients with a low amount residual plasmatic cells after therapy had longer PFS: ˂0.01% - 66 months, 0.01%-0.1% - 48 months at, 0.1%-1% - 22 months, ˃1% - 10.5 months (p=.0002).The PET-CT MRD- was reached in 36.4% (8/22) patients. Median PFS in PET-CT MRD+ group was 20 months, in the PET-CT MRD- - 23 months (p >.05).Integrated assessment of MRD by PET-CT and MFC was performed in 22 patients. Interesting, that median PFS in the MFC MRD- PET-CT MRD- group (n=5) was not achieved, in the other groups (MFC MRD+ PET-CT MRD-, MFC MRD- PET-CT MRD+, MFC MRD+ PET-CT MRD+) was 23 months (n=15) (p=.03). Conclusion .The presence of MRD after induction therapy is unfavorable prognostic factor in patients with multiple myeloma. Currently, multiparameter flow cytometry is the most sensitive approach to detect MRD in bone marrow. The role of PET-CT in the assessment of MRD is not completely understood. However, the combined use of both methods for evaluation of MRD and their prognostic potential is a promising scientific direction. DisclosuresShuvaev:Fusion Pharma LLC: Consultancy.

Frequency of minimal residual disease as measured by ctDNA in mismatch repair deficient tumors following curative resection.

e14520 Background: Mismatch repair deficient (MMRd) tumors are highly sensitive to checkpoint blockade (CPB) in patients with metastatic disease regardless of tumor type. However, the efficacy of CPB in the adjuvant setting is unknown, especially since MMRd is considered a favorable biomarker for most resected tumor types. Circulating tumor DNA (ctDNA) could be used to screen for patients at high risk for recurrence following surgery or adjuvant chemotherapy and identify patients (pts) that would most benefit from CPB. Methods: To assess the frequency of ctDNA in the resected MMRd population, we prospectively screened pts with MMRd tumors who completed standard perioperative chemotherapy and surgery (NCT03832569). DNA from resected tumors and matched postoperative plasma was sequenced for the presence of somatic mutations. Patients were considered to have minimal residual disease (MRD) when mutations were identified in tumor and found to be identical to those in matched plasma DNA. Somatic tissue mutations were assessed using MSK-IMPACT and ctDNA was assessed using FoundationOne, Guardant360 or MSK-ACCESS. Results: A total of 86 pts were screened for the presence of MRD. These represented 7 tumor types with colorectal (63%), endometrial (16%) and esophagogastric (13%) being the most common. The majority of pts were stage III (49%). MRD was detected in 18% of cases (14 of 79). Among the MRD negative group (n=62), only one pt developed disease recurrence. Three samples failed ctDNA analysis for technical reasons. Conclusions: MRD was identified in 18% of resected MMRd tumors using ctDNA analysis, suggesting this to be a feasible tumor agnostic approach to test the efficacy of CPB in a pts at high-risk for recurrence. Future studies will assess the impact of CPB in MRD positive MMRd tumors.[Table: see text]

The Next Step for MRD in Myeloma? Treating MRD Relapse after First Line Treatment in the REMNANT Study

The treatment approach for multiple myeloma (MM) has changed in recent years. After the approval of maintenance treatment after stem cell transplant in younger patients, the paradigm of continuous treatment is now prevailing in all clinical situations of myeloma. However, the best time to initiate relapse treatment is still unclear. With increased frequency of minimal residual disease (MRD) negativity, and the established clinical benefit of this finding, one of the large clinical questions in myeloma is how to approach MRD re-appearance. In this paper, we go through the MRD technology, existing and possible uses of MRD in the clinic, and data for early treatment before we introduce the design of the ongoing REMNANT study; a randomized study with early treatment of MRD relapse after first line treatment.

The Epstein criteria predict for organ-confined prostate cancer but not for minimal residual disease and outcome after radical prostatectomy.

The Epstein criteria (EC) used to select men for active surveillance do not predict biologically insignificant diseases. Minimal residual disease (MRD) is an undetected microscopic disease that remains after radical prostectomy (RP) and is a biological classification associated with the risk of treatment failure. Subtypes of MRD, the 10-year biochemical failure free survival (BFFS), and restricted mean biochemical failure free survival time (RMST) were determined and compared in EC patients treated with RP. Consecutive patients with a Gleason 6 biopsy treated at a single institution were divided into those who did or did not fulfill the EC and underwent RP. One month after surgery, samples were taken for the detection of circulating prostate cells (CPCs) and bone marrow micrometastasis. MRD was defined as negative for both CPCs and micrometastasis; patients were positive for micrometastasis and CPCs separately. BFFS for up to 10 years and RMST were determined for each MRD subgroup for EC positive and negative patients. EC positive men (137/426) were significantly older (p<0.05) and had negative MRD, pT2 (pathologically organ confined) disease (<0.02), and lower frequency of upgrading (p<0.02). Of the EC positive men, 71% were MRD negative, 13% were positive for micrometastasis, and 16% were positive for CPCs with respective 10-year BFFS of 99%, 89%, and 21% (<0.001) (hazard ratio: 1.00, 1.76, 4.03, respectively) with no signficant differences between the 10-year BFFS or RMST for MRD subgroups for EC positive and negative patients. EC predict pT2, MRD negative disease; however, 29% are MRD positive with a high risk of treatment failure.

Subtypes of minimal residual disease, association with Gleason score, risk and time to biochemical failure in pT2 prostate cancer treated with radical prostatectomy.

IntroductionThe Gleason score is a strong prognostic factor for treatment failure in pathologically organ-confined prostate cancer (pT2) treated by radical prostatectomy (RP). However, within each Gleason score, there is clinical heterogeneity with respect to treatment outcome, even in patients with the same pathological stage and prostate-specific antigen (PSA) at diagnosis. This may be due to minimal residual disease (MRD) remaining after surgery. We hypothesise that the sub-type of MRD determines the risk of and timing of treatment failure, is a biological classification, and may explain in part clinical heterogeneity. We present a study of pT2 patients treated with RP, the subtypes of MRD for each Gleason score and clinical outcomes.Patients and methodsPatients with Gleason ≤6 (G6) or Gleason 7 (G7) pT2 cancer participated in the study. One month after surgery, blood was taken for circulating prostate cell (CPCs); mononuclear cells were obtained by differential gel centrifugation and identified using immunocytochemistry with anti-PSA. The detection of one CPC/sample was defined as a positive test. Touch-preparations from bone-marrow biopsies were used to detect micro-metastasis using immunocytochemistry with anti-PSA. Biochemical failure was defined as a PSA >0.2 ng/mL. Patients were classified as: Group A MRD negative (CPC and micro-metastasis negative), Group B (only micro-metastasis positive) and Group C (CPC positive). Biochemical failure-free survival (BFFS) using Kaplan–Meier and time to failure using Restricted Mean Survival Time (RMST) after 10 years of follow-up were calculated for each group based on the Gleason score.ResultsOf a cohort of 253 men, four were excluded for having Gleason 8 or 9 prostate cancer, leaving a study group of 249 men of whom 52 had G7 prostate cancer. G7 patients had a higher frequency of MRD (69% versus 36%) and worse prognosis. G6 and G7 patients negative for MRD had similar BBFS rates, 98% at 10 years, time to failure 9.9 years. Group C, G6 patients had a higher BFFS and longer time to failure compared to G7 patients (19% versus 5% and 7 versus 3 years). Group B showed similar results up to 5 years, thereafter G6 had a lower BFFS 63% versus 90%.ConclusionsG7 and G6 pT2 patients have different patterns of MRD and relapse. Risk stratification using MRD sub-types may help to define the need for adjuvant therapy. This needs confirmation with large randomised long-term trials.

The Prognostic Significance of Minimal Residual Disease and Possibility of Its Correction in Multiple Myeloma

Introduction.Most patients with multiple myeloma (MM) are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients, the optimal methodology to assess MRD is not clear. The results of previous studies demonstrated the potential of multiparameter flow cytometry (MFC) and (PET-CT) in evaluation of MRD in MM. MRD monitoring should be applied in prospective clinical trials to compare and evaluate the efficacy of different treatment strategies, particularly in the consolidation and maintenance settings. The impact of MRD negativity is important, but further studies are needed to quantify the pharmacoeconomic and quality-of-life differences between early and delayed transplant strategies. Therefore, with the currently available evidence, upfront autologous stem cell transplantation (ASCT) is standard of care regardless of MRD status.Aim.We are aiming to determine the role of MRD and role of autologous stem cells transplantation in MM.Materials and methods.We've recently started a prospective one-center pilot study in subjects with MM. We analyzed 18 transplant-eligible patients with MM (the median age is 57 years, a male/female ratio is 3.5:1).The induction therapy Bortezomib-based only regimens was used in 12/18 (67%) patients, combination of Bortezomib-Immunomodulator-based regimens - in 6/18 (33%). High dose therapy (Mel200) and ASCT is carried out on 100% patients. The standard risk was established on 15 patients, 1 patient has an intermediate risk and 2 patients have high risk according to mSMART 2.0 stratification. The MFC MRD status of bone marrow was evaluated after 4-6 cycles of induction therapy and after ASCT on 5-color flow cytometry with use anti- CD38, CD138, CD45, CD19, CD20, CD27, CD56 and CD117 antibodies. We were based on two levels: MFC MRD- (<10-4) and MFC MRD- (<10-5) for assessing the significance of factors that affect MRD and for identifying the prognostic potential of MRD-negative status. The evaluation of MRD was carried out by genetic (cytogenetic and FISH) analysis of bone marrow plasma cells and PET-CT with 18-FDG before ASCT and on 100 day post ASCT.The results.The MFC MRD- (<10-4) before carrying out an ASCT reached 22.2% (4/18), the MFC MRD- (<10-5) - 0% and was not depended on the variant of pre-transplantation regimen. After the ASCT had been carried out there was a tendency to decrease the tumor burden in bone marrow from 0.65% to 0.1% and to increase the frequency of MFC MRD- (<10-4) status to 44.4% (8/18), of which MFC MRD- (<10-5) was 16.7% (3/18). MRD status was determined before ASCT and after ASCT by MFC and FISH in patients with high risk. The use of maintenance therapy with bortezomib (n = 5) or lenalidomide (n = 13) did not increase the frequency of MRD status. The PFS median in MFC MRD+ (>10-4) group was 23 months, in the MFC MRD- (<10-4) was not achieved; 2-year PFS was 43% and 100%, respectively (p=.04) We compared PFC between MFC MRD+ (>10-4) before ASCT (n = 4) and MFC MRD- (<10-4) after ASCT (n = 6) to assess the effect of ASCT in MM. The median PFS was not reached in both groups; 2-year PFS was 67% and 100%, respectively. The reliable difference between PFS in MFC MRD- (10-4-10-5) group and MFC MRD- (<10-5) was absent: the median of PFS was not achieved in both groups. PET-CT has been tested on 15 patients, PET-CT- response was achieved in 53% (8/15) patients. The PFS median in PET-CT+ group and PET-CT- group was not achieved. The 2-year PFS was higher in PET-CT+ group then PET-CT- probably due to patients with MFC MRD-. The 2-year PFS in «MFC MRD-PET-CT-» group was 100% to 55% in other patients.Conclusion.Carrying out ASCT demonstrated a tendency to increase the percentage of MFC MRD negative responses and improvement of PFS. The use of MFC in evaluation of MRD should be complemented with PET-CT and genetic methods for further analysis of the MFC MRD role status on MM patients. DisclosuresNo relevant conflicts of interest to declare.

Abstract OT2-07-09: Detection and targeting of minimal residual disease in breast cancer to reduce recurrence: The PENN-SURMOUNT and CLEVER trials

Abstract Background: Recurrent breast cancers arise from minimal residual disease (MRD): the pool of disseminated and circulating tumor cells (DTCs and CTCs) that survive in their host following treatment of primary breast cancer. Detection of DTCs in the bone marrow (BM) after treatment is strongly associated with an increased risk of recurrence. Through the analysis of novel genetically-engineered mouse models, we have generated a substantial body of evidence that autophagy and mTOR signaling play key roles in the survival of DTCs. Moreover, administration of agents that block these pathways in mice harboring MRD reduces DTC burden and concomitantly reduces tumor recurrence, providing the rationale for translating these findings to patients (pts). Trial Design: The PENN-SURMOUNT screening study uses a clinically validated IHC assay (DTC-IHC) to identify at-risk pts who harbor DTCs. DTC+ pts are eligible for enrollment on the CLEVER trial, which will determine the feasibility, safety and efficacy of administering hydroxychloroquine (HCQ) and/or everolimus (EVE) in DTC+ patients to target MRD and prevent recurrence. PENN-SURMOUNT is single center, prospective cohort study of pts who have completed therapy for primary breast cancer, are within 5 yrs of diagnosis and are at increased risk for relapse by virtue of nodal positivity, triple negative disease, ER+/Oncotype DX RS ≥ 25, or residual disease after neoadjuvant therapy. Pts undergo screening BM aspirate to test for DTCs following completion of adjuvant chemo and radiotherapy. The primary objective of the study is to determine the incidence and frequency of MRD in pts who have completed primary treatment for breast cancer and to ascertain eligibility for the CLEVER recurrence prevention trial. CLEVER is a randomized, controlled, open label phase II pilot trial. Target enrollment is 60 pts, with 15 pts allocated to each of 4 treatment arms: HCQ (600 mg BID), EVE (10mg daily), combination HCQ/EVE, or control/observation. A cycle is 28 days of continuous dosing. After a 3-month observation period, control pts will be offered HCQ/EVE therapy for 6 cycles; thus, the control group is actually a delayed treatment group and all pts will receive treatment. Pts who demonstrate persistent DTCs after 6 cycles will continue on combination therapy for an additional 6 cycles. The primary endpoint is feasibility of administering HCQ, EVE or the combination in this population. Secondary objectives include safety, efficacy (DTC reduction), and 3-year RFS. The principal translational objective is to assess the utility of a novel DTC assay, "DTC-Flow", for more sensitive detection and response to study therapy, compared to DTC-IHC. Additional translational objectives include determining whether patient DTCs, CTCs, and cell-free circulating plasma tumor DNA (ptDNA) biologically reflect the primary tumor and predict response. As of 5/23/17, 58 patients have been enrolled to PENN SURMOUNT, with a DTC-positivity rate of 22.6%; CLEVER opened in 2/2017; 11 patients are currently enrolled. Contact information: angela.demichele@uphs.upenn.edu Key words: Recurrence, disseminated tumor cells, dormancy, minimal residual disease, autophagy, mTOR, Everolimus, hydroxychloroquine Citation Format: Bayne LJ, Nivar I, Goodspeed B, Wileyto P, Savage J, Shih NNC, Feldman MD, Edwards J, Clark AS, Fox KR, Matro JM, Domchek SM, Bradbury AR, Shah PD, Chislock EM, Belka GK, Wang J, Amaravadi R, Chodosh LA, DeMichele AM. Detection and targeting of minimal residual disease in breast cancer to reduce recurrence: The PENN-SURMOUNT and CLEVER trials [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT2-07-09.

CD34+CD38-CD58- Candidate Leukemia-Initiating Cells Are Clinically Relevant With The Unfavorable Prognosis In Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

▪ BackgroundThe prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) has been greatly improved in the modern era of imatinib. Nevertheless, relapse is still a major cause of treatment failure in human Ph+ALL. Leukemia-initiating cells (LICs) are presumed to be responsible for relapse in leukemia. Therefore, we conducted a study to identify the candidate LICs that are responsible for disease progression and its clinical significance in patients with Ph+ALL. AimsTo investigate the leukemia-initiating and self-renewal capacities of CD34+CD38-CD58- cells and determine the prognostic significance of CD34+CD38-CD58- phenotype in patients with Ph+ALL treated in Peking University Institute of Hematology. MethodsThe leukemia-initiating potential and self-renewal capacity of the sorted CD34+CD38-CD58-, CD34+CD38-CD58+,CD34+CD38+CD58- and CD34+CD38+CD58+ compartments were investigated in vivo using sublethally irradiated and anti-mouse CD122 monoclonal antibody conditioned NOD/SCID mice by intra-bone marrow–injection. Furthermore, we prospectively analyzed whether the identified CD34+CD38-CD58- compartment at diagnosis correlates with minimal residual disease (MRD) after therapy and clinical outcomes in 63 adult patients (18-60 years) with de novo Ph+ALL. ResultsXenotransplantation of the sorted CD34+CD38-CD58- cells led to a repopulation of human B-ALL in primary and secondary recipient mice, which were phenotypically and clonally derived from the original Ph+ALL patients analyzed by flow cytometry, as well as quantitative real-time RT-PCR and fluorescence in situ hybridization for leukemia-specific cytogenetic abnormalities. Furthermore, the candidate CD34+CD38-CD58- LICs phenotype at diagnosis (n=16) significantly correlated with a lower complete remission rate and higher MRD frequency monitored by BCR-ABL mRNA levels in BM of Ph+ALL patients. Additionally, it directly correlated with higher cumulative incidence of relapse (CIR, 60% ± 1.97% vs. 15.51% ± 0.30%, P=0.002) and unfavorable disease-free survival (DFS, 33.75%±12.64% vs. 71.31%±7.17%, P=0.009) at 3-year. The CD34+CD38-CD58- group exhibited a higher rate of BCR-ABL mutations conferring higher level imatinib resistance than the other group (43.75% vs. 17.02%, P=0.04). Multivariate analyses revealed that CD34+CD38-CD58- phenotype at diagnosis was an independent risk factor for relapse (HR=4.35, P=0.009) and DFS (HR=3.38, P=0.008) in adult Ph+ALL. Summary/ConclusionBoth the xenotransplantation data as well as the clinical correlation studies show that CD34+CD38-CD58- compartment enrich for leukemia-initiating cells in adult Ph+ALL. CD34+CD38-CD58- phenotype at diagnosis independently correlates with an adverse prognosis, which promises to be an efficient tool for relapse prediction and risk-stratification treatment in adult Ph+ALL patients. AcknowledgmentsThis work was supported by grants from National Natural Science Foundation of China (grants no. 30800483&81230013) and Beijing Municipal Science and Technology Program (grant no.Z111107067311070). Disclosures:No relevant conflicts of interest to declare.

The Relation of Clinical Response and Minimal Residual Disease and Their Prognostic Impact On Outcome in Acute Myeloid Leukemia

Abstract 1418▪▪This icon denotes a clinically relevant abstract Introduction:Presence of minimal residual disease (MRD) after induction therapy predicts risk of relapse in AML (San Miguel JF et al 2001). Furthermore, achievement of complete remission (CR) rather than CR with incomplete platelet recovery (CRp) (defined using standard morphologic criteria) after induction therapy is independently associated with shorter relapse-free survival (Walter RB et al 2010). These results suggest a correlation between peripheral blood count recovery at the time of morphologic remission and MRD; however, this has not been documented. Here we address this question and examine whether response and MRD have independent effects on relapse. Patients and methods:We retrospectively analyzed data from 340 adults with AML who achieved CR, CRp or CRi (CR with incomplete peripheral count recovery; cellular marrow with <5% blasts but neutrophil count < 1,000/mL) after induction therapy at University of Washington/Fred Hutchinson Cancer Research Center since 2008. The bone marrows were collected on or closest to the first date of response and MRD was determined by ten-color multiparametric flow cytometry. The impact of MRD and response on relapse was analyzed using Cox proportional regression models. Cytogenetics were classified according to SWOG criteria. Results:Among the 340 patients, 23% of the 247 with CR, 37% of the 65 with CRp and 50% of the 28 with CRi had MRD (p =0.002). Amounts of MRD differed among the 3 groups (p=0.015) and in particular was higher with CRi median 2.6 (interquartile range [IQR] 1.3–3.4), than with CRp median 0.7 (IQR 0.3–1.3) and CR median 0.7 (IQR 0.3–2.4). By univariable Cox proportional regression analysis with death before relapse considered a competing risk, patients with MRD were more likely to relapse than patients without MRD regardless of whether MRD was considered as 0 vs. > 0 (hazard ratio [HR] 1.85; 95% CI 1.25–2.72; p=0.002) or as a numerical variable (HR 1.15 for each 1% increase; 95% CI 1.06–1.25; p <0.001). As expected, the risk of relapse was also higher in patients achieving only CRi (vs. CR: HR 2.95; 95% CI 1.63–5.36; p <0.001) or CRp (vs. CR: HR 2.17; 95% CI 1.37–3.42; p<0.001). In multivariable Cox proportional regression analyses including cytogenetic risk factors, MRD 0 vs. >0 (HR 1.50; 95% CI 1.00–2.23; p=0.047), response (CR vs. CRp and CRi: HR 2.11; 95% CI 1.40–3.17; p<0.001), and cytogenetic factors (intermediate vs. favorable: HR 4.67; 95% CI 1.13–19.2; p=0.033 or unfavorable/unknown prognostic significance vs. favorable: HR 4.59; 95% CI 1.11–19.0; p=0.035) were each independently associated with relapse. Conclusion:CRp and CRi are associated with increased frequency of MRD (0 vs. >0) in comparison to CR while CRi is characterized by higher levels of MRD than CRp or CR. This indicates that failure of blood count recovery is often due to persistent AML rather than toxic effects on normal progenitors and thus should often call for further therapy rather than delay. Clinical response, MRD and cytogenetic factors are independent predictors for relapse in AML patients achieving remission. Disclosures:No relevant conflicts of interest to declare.

Improved Post-Induction Chemotherapy Does Not Abrogate Prognostic Significance of Minimal Residual Disease (MRD) for Children and Young Adults with High Risk Acute Lymphoblastic Leukemia (ALL). A Report From Children's Oncology Group (COG) Study AALL0232

Abstract 1440Improved Post-Induction Chemotherapy Does Not Abrogate Prognostic Significance of Minimal Residual Disease (MRD) for Children and Young Adults with High Risk Acute Lymphoblastic Leukemia (ALL). A Report from Children's Oncology Group (COG) Study AALL0232.Minimal residual disease is one of the strongest prognostic factors in pediatric ALL. COG AALL0232 was a phase 3 randomized trial for patients 1–30 years old with newly diagnosed NCI HR B precursor ALL that used a 2×2 factorial study design comparing dexamethasone (DEX) versus(vs.) prednisone(PRED) during induction, and high dose methotrexate (HD-MTX) vs. Capizzi methotrexate (C-MTX) during interim maintenance 1(IM-1). We previously reported improved event-free survival (EFS) for patients receiving HD-MTX vs. C-MTX (J Clin Oncol 29: 6s, 2011) and for DEX vs. PRED among patients <10 years old randomized to HD-MTX(J Clin Oncol 29: 586s,2011). MRD was measured by 6 color flow cytometry in two central labs (MJB and BLW) to a level of sensitivity of 0.01% at end induction. Patients with >=0.1% MRD at end induction, as well as patients with morphologic slow early response or specific adverse genetic features received intensified therapy including IM-2 and a second delayed intensification, and then had MRD determined at end consolidation, (about 13 weeks post diagnosis). End induction MRD > =0.01% was highly predictive of inferior outcome, though patients with 0.1–1% MRD who received intensive therapy had very low rates of early relapse and a much higher rate of late relapse. [Display omitted] 5 year EFS for end-induction MRD positive (>=0.01%) patients was 63±5% vs. 86±2% for MRD negative patients. However, patients who were MRD positive at end induction who became negative by end consolidation had improved 5y EFS of 79±9%(n=136) compared to 52±14% for those who remained MRD positive(n=52) (p=.0012). Both end induction MRD positive and negative patients benefitted from HD-MTX vs. C-MTX, though the effect was small and did not reach statistical significance for MRD positive patients. By contrast, end-induction MRD was highly predictive of outcome for patients receiving either HD-MTX or C-MTX.5 y EFS as a function of MRD status and IM regimen.End induction MRDCapizziHDMTXP value<.01%84 ± 3%88 ± 2%.04>.01%59 ± 6%67 ± 7%.12P value<.0001<.0001End induction MRD negative patients <10y receiving DEX had better outcome than those getting PRED (5 y EFS 92±3% vs. 87±4% P=.027) while MRD positive patients or those>10y showed no difference. However, DEX patients <10y if anything had a slightly higher rate of end induction MRD positivity than those given PRED (22% vs. 17%, p=.073). In multivariate analysis, end consolidation MRD was the most powerful prognostic factor for the small subset of patients in whom this was assessed. Excluding this, end induction MRD was the most significant variable; age, white blood cell count, day 15 marrow morphology and HD-MTX vs. C-MTX were also significant. We conclude that MRD remains the most powerful prognostic factor even in the context of improved therapy. Additionally, for those patients who were MRD positive at end induction, achieving MRD negative status by end consolidation improved outcome significantly. The higher frequency of MRD in younger patients receiving DEX calls into question the validity of using end induction MRD as a surrogate for outcome when testing novel interventions during induction therapy. Disclosures:Borowitz:BD Biosciences: Research Funding. Wood:BD Biosciences: Research Funding.

High Prognostic Impact of Flowcytometric Minimal Residual Disease Detection In Acute Myeloid Leukemia: Prospective Data From the HOVON/SAKK 42a Study

AbstractAbstract 760Currently, the most important prognostic factors for acute myeloid leukemia (AML) include molecular aberrancies and karyotype of the leukemic blasts. Although these factors have showed to be of utmost importance in upfront risk stratification in current treatment schedules, the treatment outcome of patients within as such defined risk groups is still quite heterogeneous. Therefore, there is an unmet need for therapy-dependent prognostic factors which can be implemented into risk-adapted treatment strategies. Minimal residual disease (MRD) frequency is such a parameter. MRD cells are considered responsible for the outgrowth of AML after treatment, leading to a relapse in 30–40% of the patients in complete remission (CR). In this study, we are the first to report prospective multicenter data on the prognostic impact of MRD frequency in adult AML. In our retrospective study (N.Feller et al. Leukemia 2004), we explored which cut-off points for percentage of MRD cells would define MRD positive (levels above cut-off, MRD+) patients with a relatively poor prognosis, from MRD negative (levels below cut-off, MRD-) patients who showed a longer overall and relapse-free survival (OS and RFS). In search for the most optimal cut-off level which can be used for clinical purpose in risk stratification-directed therapy, we used these cut-offs to evaluate the prognostic value in the current prospective setting. Diagnosis and follow-up samples were collected of 462 patients treated uniformly according to the HOVON/SAKK42a protocol (www.hovon.nl) and MRD frequency was assessed blindly without knowledge of clinical course. MRD detection was accomplished by immunophenotyping by flow cytometry (FCM) through aberrant expression of markers on AML blasts. Together with the expression of normal immature cell markers and/or myeloid lineage markers, this offers a leukemia associated phenotype (LAP). Each LAP was individually designed for each patient in diagnosis bone marrow (BM) or peripheral blood. Subsequently, BM samples obtained during follow-up were analysed for the presence of LAP-positive cells. MRD frequency was expressed as a percentage of leukocytes. The median MRD frequencies of patients in clinical CR after first induction cycle (n=164), second induction cycle (n=182) and consolidation (n=121) were 0.040%, 0.022% and 0.020%, respectively. The cut-off levels for MRD frequency as defined retrospectively were all significant in the identification of patients with adverse (MRD+) and favourable (MRD-) OS and RFS, respectively. After the first cycle, the most significant cut-off was 0.8%, leading to 17 MRD+ patients who showed a median RFS of only 8.6 months, while 147 MRD- patients had a median RFS of >47 months (p=0.003,A). The relative risk of relapse (RR) was 2.9 (95% c.i. 1.4–6.0, p=0.004). After the second induction cycle, a cut-off level of 0.06% was most significant. Above this cut-off, 49 patients showed a median RFS of 7 months, while 133 MRD- patients showed a RFS of more than 47 months (p<0.00001, fig B). The RR was 3.2 (95% c.i. 2.0–5.0, p<0.00001). After consolidation therapy, 11 MRD+ patients with extremely poor prognosis were identified (median RFS 7.3 months vs. >47 months for 110 MRD- patients, p<0.00001, fig C), with a RR of 10.6 (95% c.i. 4.9–22.8, p<0.00001). Multivariate analysis was performed with conventional prognostic factors for AML: cytogenetic risk groups and time to achieve CR. After every cycle of therapy, MRD frequency was an independent prognostic factor for RFS after all cycles (1st cycle: p=0.010, 2nd cycle and consolidation p<0.00001) and for OS after 1st (p=0.023) and 2nd induction cycle (p=0.010). In this prospective multicenter study, already after first induction cycle, MRD detection by FCM was an independent significant factor in the identification of poor prognostic patients. In future treatment studies, risk stratification, e.g. for allogeneic stem cell transplantation, should not only be based on risk estimation determined at diagnosis, but also on MRD frequency as a therapy-dependent prognostic factor. This work was supported by Netherlands Cancer Foundation KWF. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Sequential monitoring of minimal residual disease in acute lymphoblastic leukemia: 7-year experience in a pediatric hematology/oncology unit

We evaluated minimal residual disease (MRD) in 91 children with acute lymphoblastic leukemia (ALL) by PCR amplification of clonal rearrangements, immunoglobulin (IgH; VDJ rearrangement, CDR3 region) and T-cell receptor (TCRδ). Sequential monitoring of MRD was performed at different time points during and after chemotherapy and was correlated to patient outcome. In total, 792 bone marrow samples were assessed for MRD at the end of induction, and during and after treatment completion. MRD positivity at the end of induction was detected in 12% of patients and was associated with high incidence of relapse, 54.55% (p = 0.0002), at 5 years. On the other hand, 88% of patients were MRD-negative at the end of induction and the relapse rate at 5 years was very low, 5%. The frequency of MRD decreased to 16% in the first 6 months of chemotherapy; however, the incidence of relapse in MRD-positive patients remained high, 42.8%. After treatment completion (24–36 months from diagnosis), 32% patients were MRD-positive and the relapse rate was 36.5% (p = 0.0009). Our results indicated that monitoring of MRD constituted an essential prognostic marker, and detection of MRD particularly at the end of induction and after treatment completion was strongly predictive for patient outcome.

Leukemic Stem Cell Assessment in Remission Bone Marrow of Acute Myeloid Leukemia Patients Is a New Prognostic Parameter.

Abstract 399In acute myeloid leukemia (AML), relapses originate from the outgrowth of therapy surviving leukemic blasts know as minimal residual disease (MRD). Accumulating evidence shows that leukemia initiating cells or leukemic stem cells (LSCs) are responsible for persistence and outgrowth of AML. Monitoring LSCs during and after therapy might thus offer accurate prognostic information. However, as LSCs and hematopoietic stem cells (HSCs) both reside within the immunophenotypically defined CD34+CD38- compartment, accurate discrimination between LSCs and HSCs is required. We previously showed that within the CD34+CD38- stem cell compartment, LSCs can be discriminated from HSC by aberrant expression of markers (leukemia associated phenotype, LAP), including lineage markers like CD7, CD19 and CD56 and the novel LSC marker CLL-1 (van Rhenen, Leukemia 2007, Blood 2007). In addition, we reported that flowcytometer light scatter properties add to even better detection of LSCs, allowing LSCs detection in AML cases lacking LAP (ASH abstract 1353, 2008). Using this gating strategy, we determined LSC frequency in 64 remission bone marrow samples of CD34+ AML patients. A stem cell compartment was defined as a minimum of 5 clustered CD34+CD38- events with a minimal analyzed number of 500,000 white blood cells. After first cycle of chemotherapy, high LSC frequency (>1 × 10-3) clearly predicted adverse relapse free survival (RFS, figure 1a). LSC frequency above cut-off led to a median RFS of 5 months (n=9), while patients with LSC frequency below cut-off (n=22) showed a significantly longer median RFS of >56 months (p=0.00003). In spite of the relatively low number of patients, again a high LSC frequency (>2 × 10-4) after the second cycle and after consolidation therapy predicted worse RFS: after second cycle, median RFS was 6 months (n=9) vs. >43 months for patients with LSC frequency below cut-off (p=0.004). After consolidation, these figures were 6 months (n=7) vs. >32 months (n=6, p=0.03). Although total blast MRD (leukemic blasts as % of WBC) is known to predict survival (N.Feller et al. Leukemia 2004), monitoring LSCs as compared to total blast MRD has two major advantages: the specificity is higher (van Rhenen et al. Leukemia 2007) and well-known LSC makers like CLL-1, CD96 and CD123 can in principle be used for LSC monitoring, but not for total blast MRD detection since these markers are also expressed on normal progenitor cells. On the other hand, LSCs constitute only a small fraction of all leukemic blasts and therefore monitoring total blast MRD may have the advantage of a higher sensitivity. We thus tested the hypothesis that even more accurate prognostic information could be obtained by combining LSC frequency with total blast MRD. Total blast MRD after first cycle was predictive for survival with borderline significance (p=0.08): a cut-off of 0.3% resulted in two patient groups with median RFS of 9 months vs. >56 months. Figure 1b shows the result of the combined data of LSC and MRD frequency after first cycle therapy. We used the terms LSC+ and MRD+ for cell frequencies above cut-off and LSC- and MRD- for those below cut-off. We could clearly identify that apart from LSC+/MRD+ patients, LSC+/MRD- patients too have very poor prognosis, while MRD+/LSC- patients show an adverse prognosis as compared to LSC-/MRD- patients. These results from the first study on the in vivo fate of LSCs during and after therapy, strongly support the hypothesis that in CD34+ AML the leukemia initiating capacity originates from the CD34+CD38- population and is important for tumor survival and outgrowth. These results show that LSC frequency might be superior in predicting prognosis of AML patients in CR as compared to MRD total blast frequency, while the combination of both may offer the most optimal parameter to guide future intervention therapies.This work was supported by Netherlands Cancer Foundation KWF. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Caspase-cleaved cytokeratin 18 fragment (M30) as marker of postoperative residual tumor load in colon cancer patients

Background Soluble cytokeratin 18 (CK18; M65) and a caspase-cleaved fragment of CK18 (M30) have been used as biomarkers, corresponding to tumor cell death and apoptosis, respectively. Methods In the present study, M30 was quantified for the first time in serum samples of colon cancer patients pre- and postoperatively as well as during chemotherapy. Minimal residual disease (MRD) was assessed preoperatively by detection of pan-cytokeratin antibody A45-B/B3-positive cells in bone marrow aspirates. Results Out of 46 patients, those with colon tumors of stages I and IV had significantly elevated M30 serum concentrations compared to controls (n = 23). In 31 colon cancer patients, M30 determinations were performed prior to and seven days after tumor surgery. A group of 24 patients exhibited a significant decrease of M30 in response to tumor removal, in contrast to seven patients who revealed either persistent or higher M30 levels postoperatively. The frequency of MRD was not significantly different for patients with decreasing (4/24) and persisting (3/7) M30. However, M30 correlated significantly with the increased number of recurrences within 36 months in the group with persisting M30 (4/7 versus 2/24, p = 0.032; hazard ratio 8.3, p = 0.016). In a group of patients (n = 10) receiving capecitabine/oxaliplatin chemotherapy (CapOx), transient increases in M30 did not correlate with responses. Conclusion The data obtained within the present limited pilot study in colon cancer patients demonstrate that perioperative changes of M30 may indicate systemic residual tumor load and increased risk of recurrence warranting further evaluation of this marker of apoptosis in a larger prospective clinical trial.

Prognosticating of Relapse Risk Based on Multiparameter Flow Cytomeric Assessment of Minimal Residual Disease in Patients with Acute Myeloid Leukemia

Abstract Objective: To investigate the prognosticating of relapse risk based on assessment of minimal residual disease(MRD) in patients with acute myeloid leukemia(AML).Methods: Multiparameter flow cytometry (MPFC) analysis were used to detect the leukemia-associated aberrant immunophenotype (LAIP)of the pretreated patients with AML and assess the level of MRD after remission induction(Post-Ind MRD) and consolidation therapy(Post-Cons MRD).Results: The results showed that definite LAIP could be detected in about 94.3%of patients (115/122) with AML (except APL). Only one LAIP was identified in 15 cases (13.0%), but in other 100 cases (87.0%), two or more LAIP were identified. The most frequent LAIP identified was cross-lineage antigen expression (40.9%). The percentages of asynchronous antigen expression, antigen over-expression and antigen lack expression were 20.9%, 27.0% and 34.8% respectively. MRD frequency was monitored in 41 CR patients with AML after remission induction chemotherapy and 2 or more cycles of consolidation chemotherapy. 24 patients were Post-Ind MRD+ and 17 patients were Post-Ind MRD−. The percentages of relapse in Post- Ind MRD+ and Post-Ind MRD− were 75.0%(18/24) and 29.4%(5/17) respectively after consolidation chemotherapy. The relapsed free survival(RFS) of the patients with Post-Ind MRD+ and Post-Ind MRD− was 49.06±6.53 months and 11.92±1.64 months(P<0.0001) respectively. 18 patients were Post-Cons MRD+ and 23 patients were Post-Cons MRD−. The percentages of relapse in Post-Cons MRD+ and Post-Cons MRD− patients were 100%(18/18) and 21.7%(5/23) respectively after consolidation chemotherapy. The RFS of the patients with Post-Cons MRD+ and Post-Cons MRD− was 41.74±5.52 months and 10.06±1.72 months (P<0.0001) respectively.Conclusions: The level of post-Ind MRD and post-Cons MRD identified in the patients with AML was highly associated with their RFS. MRD measured by MPFC provided prognostic information in AML patients.

High Leukemic Stem Cell Frequency in Remission Bone Marrow Predicts Poor Outcome in Acute Myeloid Leukemia

Relapses of acute myeloid leukemia (AML) originate from the outgrowth of therapy surviving leukemic blasts known as minimal residual disease (MRD). We and others have shown that immunophenotypical detection of MRD in remission bone marrow (BM) is a strong prognostic factor for survival. However, some patients relapse despite low MRD levels, indicating the presence of remaining, but undetectable leukemic stem cells (LSCs). These LSCs are proposed to give rise to AML and because we previously found a correlation between stem cells at diagnosis and MRD (van Rhenen et al., CCR 2005), they might be responsible for relapse. Therefore, we investigated whether LSCs could be detected in remission BM and whether monitoring remaining LSCs might predict clinical outcome more accurately as compared to the immunophenotypically defined “total blast” MRD. We defined LSCs by FACS analysis by the use of aberrant lineage markers, as well as the novel LSC marker CLL-1 (van Rhenen et al., Leukemia 2007 and Blood 2007) in 22 patients with CD34+ leukemia. In patients with more than 20% leukemia associated phenotype (LAP) positive CD34+ CD38− stem cells, 37 follow-up remission BM samples were collected (17 after first cycle, 13 after second cycle and 7 after third cycle of chemotherapy) and analysed for LAP expression. A stem cell compartment was defined as a minimum of 5 clustered CD34+ CD38− events when a minimum of 500,000 white blood cells (WBCs) were measured. In cases of less than 5 events, LSC frequency was set at <1/500,000 (< 2 × 10−4) LSCs/total WBC. In 10 follow-up samples, no CD34+CD38− compartment could be detected. In the other samples, the markers CD7 (n=9), CD11b (n=2), CD19 (n=3), CD56 (n=1) and CLL-1 (n=12) were used to detect LSCs. In addition, in 4 of these cases, CD45 expression, cell size (forward light scatter, FSC) and cell granularity (sideward light scatter, SSC) characteristics were used as a secondary gating strategy to exclude malignant cells in the aberrant marker negative fraction (M.Terwijn, accompanying abstract for this meeting). After the first cycle of chemotherapy (n=17), high LSC frequency (> 1 × 10−3) clearly predicted overall survival (OS), relapse free survival (RFS) and disease free survival (DFS). Regarding OS, LSC frequency above this cut-off lead to a median survival of 8 months (n=7) vs. >29 months in the group above cut-off (n=10, p=0.028). Median RFS in the high LSC frequency group was 8 months vs. >27 months (p=0.019) and for DFS, these numbers were 6 months vs. >27 months, respectively (p=0.025). After the second and third cycle of chemotherapy a cut-off of 2 × 10−4 was used. Again, a high LSC number predicted OS, RFS and DFS (p=0.035, 0.029 and 0.029, respectively) with a median RFS of 6 months (n=6) versus >30 months (n=7) in patients with a low LSC frequency. Although only limited number of patients was followed after the third cycle, trends were similar: 3/3 patients with a high LSC frequency relapsed within 6 months after CR, while only 1/4 patients with a low LSC frequency relapsed within 6 months. The remaining 3 patients had a RFS of 11, 21 and 31 months. In contrast to LSC frequency, “total blast” MRD frequency did not predict prognosis after the first, second or third cycle of chemotherapy, which likely is due to the small sample size. LSC frequency is superior in predicting prognosis of AML patients in CR as compared to MRD total blast frequency. The prognostic value of remaining LSCs supports the hypothesis that the leukaemia initiating capacity in patients with AML originates from this population. The identification of LSCs in remission BM and the possibility to distinguish these from HSCs will allow monitoring differential effects of present and future therapies.This work was supported by Netherlands Cancer Foundation KWF.