Significance Of Minimal Residual Disease Research Articles

THE SIGNIFICANCE OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOBLASTIC LEUKAEMIA: A SINGLE CENTRE STUDY

In Acute lymphoblastic Leukemia (ALL) assessment of molecular response to treatment, assessing minimal residual disease (MRD) is a major independent predictor of treatment outcome. Consequently, MRD is implemented in all ALL-treatment protocols to fill up or to redefine stratification of multifactorial risk with optional intensity of customized treatment. Aim: to specify the significance of MRD in the assessment of remission in children with ALL with results discordant between morphology and flow cytometry at the end of induction phase of therapy. Materials and Methods: A descriptive cross-sectional study was conducted at Jin Oncology Center from March 2019 through November 2023. Data were taken out of the records of 58 patients who had ALL less than 16 years old. All patients were less than 16 years old and treated by ukall. They were diagnosed using peripheral blood morphology, bone marrow study and/or flow cytometry when lymphoblasts in peripheral blood or bone marrow aspirate are ≥20% and was confirmed by flow cytometry. On 29th day of induction therapy, bone marrow was examined for morphology and flow cytometry. The presence or absence of MRD was determined, and CD19, CD10 and tdt were tested. By morphologic assessment they were divided patients into: Category 1, C1 (<5% blasts), Category 2, C2 (5-20% blasts), and Category 3, C3 (>20% blasts). Statistical analysis was made using SPSS version 25. P value of less than 0.05 was considered significant. Results: The study involved 58 patients who had ALL. with a median age of 6.5 years, male to females ratio of 1.76:1, mean platelet count of 96.6 x 10⁹/L ,mean hemoglobin of 8.6 g/dL, mean leucocyte count of 74.3 x 10⁹/L), 48 cases (82.7%) of B-cell lineage and 10 cases (17.3%) of T-cell lineage, 94.6% of the B-cell cases were of the common B-ALL and the rest Pro-BALL type, 54.6% of the T-cell ALL was cortical T-ALL and 44.4% Early T-cell ALL. They were tested for MRD by morphology and flow cytometry on day 29. By morphology, 46 patients had remission but by flow only 24 cases. Seventeen cases had residual blasts >5%. In 19 cases there was a discrepancy between the results of morphology and flow. Twenty-five cases (52.08% of B-cell cases) were positive for MRD by flow results. Eight of the ten cases of T-ALL (80%), were positive for MRD by flow cytometry. Among 48 cases of B-ALL, 36 were in C1 category (morphologically in remission), 11 cases were in C2 category and one case in the C3 category. Of cases in C1 category, 17 were MRD +ve and 19 were MRD –ve by flow cytometry. In the C2 category, only 2 out of the 11 cases (18.18%) had discordant results between morphology and flow results. The correlation between morphology and flow results was 100% in the C3 category. Conclusion: MRD should not be the surrogate of morphology but to be used in conjunction in order to give us a more accurate representation of status of remission.

Impact of pretransplant minimal residual disease in patients with multiple myeloma and a very good partial response or better receiving autologous hematopoietic stem cell transplantation.

The prognostic significance of minimal residual disease (MRD) status before autologous hematopoietic stem cell transplantation (autoHCT) in patients with multiple myeloma (MM) has not been clearly elucidated. Retrospective single-center study of adult MM patients who achieved ≥very good partial response (VGPR) after induction therapy from 2015 to 2021 received upfront autoHCT and had available pretransplant MRD status by next-generation flow cytometry. The cohort was divided into pretransplant MRD-negative (MRDneg) and MRD-positive (MRDpos) groups. A total of 733 patients were included in our analysis; 425 were MRDneg and 308 MRDpos at autoHCT. In the MRDpos group, more patients had high-risk cytogenetic abnormalities (48% vs. 38%, respectively; p=.025), whereas fewer patients achieved ≥CR before autoHCT (14% vs. 40%; p<.001). At day 100 after autoHCT, 37% of the MRDpos versus 71% of the MRDneg achieved ≥CR, and at best posttransplant response 65% versus 88% achieved ≥CR, respectively. After a median follow-up of 27.6months (range, 0.7-82.3), the median PFS was significantly shorter for patients in the MRDpos group compared to the MRDneg group: 48.2months (95% confidence interval [CI], 0.3-80.5) versus 80.1months (95% CI, 0.5-80.1), respectively (p<.001). There was no significant difference in overall survival between the two groups (p=.41). Pretransplant MRDpos status was predictive of shorter PFS in multivariate analysis (hazard ratio, 1.80; 95% CI, 1.31-2.46; p<.001). The impact of pretransplant MRD status was retained in most of the examined subgroups. In patients achieving ≥VGPR to induction, pretransplant MRDpos status was associated with a lower CR rate after autoHCT and a shorter PFS.

Clinical Significance of Minimal Residual Disease in Pediatric Patients with TCF3/PBX1+ B-cell Acute Lymphoblastic Leukemia

To explore the consistency of flow cytometry (FCM) method and polymerase chain reaction (PCR) technique in the detection of minimal residual disease (MRD) at different treatment stages in pediatric patients with TCF3/PBX1+ B-cell acute lymphoblastic leukemia (B-ALL) and the correlations between the detection results and prognosis. The clinical data of 64 newly diagnosed pediatric patients with TCF3/PBX1+ B-ALL admitted to the Department of Pediatrics of Peking University People's Hospital from January 2005 to December 2017 were retrospectively analyzed. FCM and PCR methods were used to monitor the MRD level in bone marrow samples from 64 children during the same period of treatment on d33 and d90 respectively, and the detection results were analyzed. There were 37 males and 27 females in the 64 patients, with a median age of 8 years(range 0.8 to 16 years). The complete remission (CR) rate after the first cycle of induction chemotherapy was 98.4% (62/63), with overall CR rate of 100%. 12 patients experienced recurrence, with a median recurrence time of 16.9 (5.3-46.3) months. The median follow-up time of the 64 patients was 77.2 (1.0-184.8) months , and the 5-year overall survival (OS) rate and event-free survival (EFS) rate were 82.8%±4.7% and 75.0%±5.4%, respectively. On d90, the concordance rate of the MRD results from the two methods was 98.4%, and the related kappa value was 0.792 (P < 0.001), which were significantly higher than those on d33. After induction chemotherapy (d33), the 5-year EFS rate of MRD-FCM- group (79.3%±5.3%) was significantly better than that of MRD-FCM+ group (40.0%±21.9%) (P =0.028), there were no significant differences in the 5-year OS rate and EFS rate between MRD-PCR+ group and MRD-PCR- group, and the 5-year EFS rate of MRD-FCM-/PCR- group (85.4%±5.5%) was significantly better than that of MRD-FCM+/PCR+ group (40.0 %±21.9%) (P =0.026). In children with TCF3/PBX1+ B-ALL, the MRD results detected by FCM and PCR methods show good consistency, especially in consolidation therapy period (d90). The MRD level at the end of induction therapy (d33) is an important factor affecting the long-term prognosis, especially the MRD results detected by FCM method, which is significantly associated with prognosis.

Clinical significance of minimal residual disease at the end of induction therapy in childhood acute lymphoblastic leukemia

Clinical significance of minimal residual disease at the end of induction therapy in childhood acute lymphoblastic leukemia

From Generic RNA-Based MRD Assessment Towards Patient-Specific Genomic (DNA-based) MRD in AML and Other Non-B Cell Acute Leukemia

Pediatric acute myeloid (AML) is considered as a heterogeneous disease affecting 15% of children with acute leukemia. Outcome is still unsatisfactory with an overall survival around 70-80% and a relapse rate of 30-40% after primary treatment. The most important factors determining clinical outcome are genetic aberrations, and early response to treatment. Many studies have demonstrated the prognostic significance of minimal residual disease (MRD) monitoring. Although MRD monitoring is now an integral part in the management (including MRD-guided stratification) of Acute Lymphoblastic Leukemia (ALL) in children (generally done by ASO-qPCR on tumor specific IG/TCR targets), its clinical utility in childhood non-lymphoblastic leukemia (such as AML) is currently unknown/not used. Contemporary approaches to MRD detection in AML rely on PCR amplification of usually fixed genetic abnormalities (MRD-PCR) or on flowcytometric profiling of leukemia-associated immunophenotypes (LAIP). Molecular MRD detection is in general more sensitive than flow-based MRD, but the applicability is lower since recurrent mutational targets (e.g. fusion genes) using conventional technology, are present in only 40-50% of pediatric AML patients. This RNA-based MRD is common practice for CBFb-MYH11, RUNX1-RUNXT1, KMT2A-AFF1, -MLLT1, -MLLT3, and NPM1 exon 12 mutations and standard curves necessary for MRD quantification are available for all mentioned genes. Since the introduction of Whole Transcriptome Sequencing (WTS; RNAseq) as standard diagnostics in our laboratory, we identified various additional fusion genes in AML (increasing available molecular targets up to 75%), but also in other non-B cell malignancies such as SCLL, CEL, HES and BPDCN. By designing patient specific primers/probe sets surrounding the breakpoint sequences, we were able to generate 36 RNA-based MRD assays (including FOP-FGFR1, RANBP2-ALK, DEK-NUP214, ETV6-PDGFRB, KMT2A-AFDN) and monitor MRD during/after therapy. Although RNA-based MRD approach allows additional molecular monitoring in these patients, this type of MRD displays several disadvantages: sensitivity of a certain MRD assay can be low due to low expression levels of the fusion gene or low RNA yield and/or quality. Furthermore, quantification can be hampered by lack of available standard curves. Therefore DNA-based MRD is favorable over RNA-based MRD and Whole Exome Sequencing (WES) or Whole Genomic Sequencing (WGS) can identify the genomic breakpoint sequence in respectively genomic exons or introns. In addition, other genomic aberrations (such as insertions, deletions and point mutations) can be identified by these techniques and used for development of DNA-based MRD. By using this approach we were able to generate 17 additional DNA-based MRD assays (including FOP-FGFR1, RANBP2-ALK, RUNX1-CBFA2T3 NPM1-MLF1 UBTF-TD, p53 deletion), all with very high sensitivity (e.g. around 10-4 to 5x10-5) and patient-derived standard curves to quantify MRD. Figure 1 shows an example for DNA-based MRD monitoring of 3 AML patients harboring the recently described poor-outcome UBTF-TD aberration showing no/slow decrease of MRD in all patients. In conclusion, we show that fusion gene detection by WTS, preferably in combination with mutation analysis by WES/WGS, is providing comprehensive diagnostic results to improve the development of highly sensitive patient specific MRD targets to allow better molecular monitoring (and prediction of relapse), eventually leading to treatment stratification of patients in the future. We will present an update of our results and interesting cases at the upcoming conference. Figure 1: By WES, 3 patients with tandem duplications (TD) in exon 13 of the UBTF gene were identified, and patient specific primers and probes were designed for MRD on follow-up DNA samples. MRD was quantified by using a standard curve derived from serial dilutions from the diagnostic DNA. Quantitative range, sensitivity and background were determined according to the ESG-MRD-ALL guidelines. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluation of MRD-status in post-induction period in pediatric patients with acute lymphoblastic leukemia.

The need to study the significance of minimal residual disease (MRD) at the induction therapy in patients with acute lymphoblastic leukemia (ALL) is beyond doubt. This has been confirmed by many years of work by many research groups. The role of MRD in the late stages of treatment and the impact of these values on patients survival requires research and discussion. To evaluate the influence of MRD-status in post-induction period on survival in patients with acute lymphoblastic leukemia. From 2010 to 2022, 135 patients with primary B-ALL enrolled in ALL-IC BFM 2009 protocol. Median age was 5.4 year (range 1-17). Male was 62 (49,5%) and female 73 (54,1%). The diagnosis was based on WHO 2016 criteria. Stratification on prognostic risk groups was carried out according to protocol criteria. Prednisone response evaluated at day 8 of treatment. The 15th, 33th, and 78th (as post-induction) day response was assessed by bone marrow cytology and level of MRD by flow cytometry. 5y-overall survival (OS) for patients with MRD-negative status on day 15 was 94,4±5,4% and 87,0±3,4% for MRD-positive (p=0,5). On day 33 patients with MRD-negative status achieved 5y-OS in 86,7±5,8% and 89,6±3,5% for MRD-positive (p=0,6).5y-OS for patients with MRD-negative status on day 78 was 90.8±4.0%, MRD-positive - 90,4+6.5%. DFS for MRD-negative status was 88.5±4.5%, for MRD-positive - 66.3±11.8% (p=0,1). EFS for MRD-negative patients was 87.2±4,6% and for MRD-positive 66.3±11.8% (p=0,09). We have found a tendency between MRD status on day 78 and the frequency of relapses in patients. At the moment, there are no reliable data on the effect of post-induction MRD status on survival. The assessment of MRD in the post-induction period has prognostic prospects and requires further study.

Significance of WT1 and multiparameter flow cytometry assessment in patients with chronic myelomonocytic leukemia receiving allogeneic hematopoietic stem cell transplantation.

The objective of this study was to investigate the clinical significance of minimal residual disease (MRD) monitoring through Wilms tumor 1 (WT1) gene expression and multicolor flow cytometry (FCM) in patients with chronic myelomonocytic leukemia (CMML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). For this purpose, WT1 gene expression and the CMML-related abnormal immunophenotype were examined using real-time quantitative polymerase chain reaction and FCM, respectively. In total, 59 patients with CMML who underwent allo-HSCT were enrolled in this study. Thirteen cases (22.0%) developed hematological relapse, and 15 patients (25.4%) expired during the follow-up period. Thirty-four patients (37.6%) were positive for WT1 (WT1+), and 44 patients (74.6%) were positive for FCM prior to allo-HSCT. After allo-HSCT, there were 21 WT1+ patients (35.6%) and 10 patients (16.9%) who were positive in FCM (FCM+). Post-transplant WT1+ (post-WT1 0.6+; 50.7% vs. 7.6%, p<.001) and post-transplant FCM+ (post-FCM+; 90.0% vs. 8.8%, p<.001) indicated a higher 3-year cumulative incidence of relapse (CIR) compared with the WT1- or FCM-patients. Multivariate analysis of event-free survival (EFS), overall survival (OS), and CIR showed that the FCM status after transplantation was an independent prognostic factor for relapse (p<.05). Both FCM and WT1 after HSCT were identified as important predictors of recurrence of CMML following transplantation and may be useful in guiding interventions against disease relapse.

Impact of Allogeneic Hematopoietic Stem Cell Transplantation in First Complete Remission and Additional Cytogenetic Aberrations at Diagnosis on Prognosis in 1256 Pediatric Patients with KMT2A-Rearranged Acute Myeloid Leukemia: A Retrospective Study By the I-BFM-SG

IntroductionKMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) is a heterogeneous genetic subgroup with a frequency of about 25% in children with AML. At the 62 nd ASH annual meeting last year, we reported on the differences in outcome of various KMT2A subgroups based on translocation partner and the significance of minimal residual disease (MRD) status during and after induction as a follow-up study of Balgobind et al., Blood 2009. The impact of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1) and the presence of additional cytogenetic aberrations (ACAs) on prognosis have not yet been described for our cohort.MethodsData on allo-HSCT in CR1 and the presence of ACAs of 1256 KMT2A-r de novo pediatric AML patients from 15 AML study groups affiliated with the I-BFM Study Group, diagnosed between 2005 and 2016, were retrospectively collected and studied. Karyotypes were reviewed and classified by two of the authors (RW&CH). Based on translocation partners, patients were classified to the KMT2A high-risk subgroup (6q27, 10p11.2, 10p12, 4q21, and 19p13.3) or non-high-risk subgroup (9p22, 19p13, 19p13.1, 1q21, Xq24, 17q21, 1p32, and 17q12). These two categories have been used to estimate a Cox model. Patients with unknown translocation partners were excluded from these analyses (n=126). Flow cytometry MRD levels at the end of induction course 1 (EOI1) and 2 (EOI2) <0.1% were considered negative, and levels ≥0.1% positive. Kaplan-Meier's methodology was used to estimate disease-free survival (DFS) and overall survival (OS). DFS was calculated from EOI1 for patients in CR to date of relapse or death/last follow-up. OS was calculated from the time of diagnosis to date of death/last follow-up. Two-sided P-values of ≤ .01 were considered statistically significant. Covariates with P-values ≤ .05 in univariate analyses were included in multivariate analyses; allo-HSCT in CR1 was included as a time-dependent covariate in the Cox model. MRD status at EOI2 was excluded from multivariate analyses as therapy could have been adjusted to the MRD status and the number of MRD positive patients was small.ResultsOf 1256 pediatric patients with KMT2A-r AML, data on HSCT in CR1 and ACAs were available for 1186 (94.4%) and 1204 patients (95.9%), respectively; 211 (17.8%) patients received HSCT in CR1 and ACAs were present in 601 (49.9%) patients. Compared with the KMT2A non-high-risk subgroup, patients in the KMT2A high-risk subgroup underwent HSCT in CR1 more often (23.8% vs 15.0%; P < .001). ACAs were borderline significantly more common in the KMT2A high-risk subgroup (54.1% vs 46.4%; P = .015).Univariate analysis of the probability of DFS (Table 1) showed that the KMT2A high-risk subgroup (HR 2.1; 95% CI, 1.7-2.5), age ≥10 years (HR 1.4; 95% CI, 1.2-1.7), and MRD ≥0.1 at EOI1 (HR 1.5; 95% CI, 1.1-1.9) were associated with DFS. HSCT in CR1 was a borderline significant prognostic factor (HR 0.7; 95% CI, 0.6-0.9).In a multivariate analysis for DFS (n=515) (Table 1), the KMT2A high-risk subgroup (HR 2.0; 95% CI, 1.6-2.6), MRD ≥0.1 at EOI1 (HR 1.7; 95% CI, 1.2-2.3), and HSCT in CR1 (HR 0.6; 95% CI, 0.4-0.9) were associated with DFS.Univariate analysis of the probability of OS (Table 1) showed that the KMT2A high-risk subgroup (HR 1.8; 95% CI, 1.5-2.2), age ≥10 years (HR 1.6; 95% CI 1.3-2.0), WBC ≥100 x10 9/L (HR 1.4; 95% CI, 1.1-1.7), the presence of ACAs (HR 1.4; 95% CI, 1.2-1.7), and MRD ≥0.1 at EOI1 (HR 2.1; 95% CI, 1.6-2.7) were associated with OS. HSCT in CR1 was not associated with OS. The effect of HSCT in CR1 was not significantly different between the KMT2A high-risk and non-high-risk subgroups.In a multivariate analysis for OS (n=557) (Table 1), the KMT2A high-risk subgroup (HR 1.9; 95% CI, 1.4-2.5), age ≥10 years (HR 1.5; 95% CI, 1.1-1.9), the presence of ACAs (HR 1.6; 95% CI, 1.2-2.1), and MRD positivity at EOI1 (HR 1.9; 95% CI, 1.4-2.5) were associated with OS.ConclusionsIn this cohort of KMT2A-r pediatric AML patients, the presence of ACAs at diagnosis was independently associated with inferior OS, but not with DFS. This may be due to the exclusion of refractory patients in DFS analysis, who were significantly more common in the group of patients with ACAs. Analysis has yet to be performed to distinguish karyotype complexity. In addition, allo-HSCT in CR1 was an independent predictor of improved DFS, but was not a prognostic factor for OS. [Display omitted] DisclosuresAbrahamsson: wedish Children´s Cancer Foundation. Research grants and 50% senior research position for clinical research on pediatric leukemia: Research Funding. Locatelli: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharamceutical: Speakers Bureau; Takeda: Speakers Bureau.

Prognostic Significance of Minimal Residual Disease in Patients with Secondary Acute Myeloid Leukemia

BACKGROUNDSecondary acute myeloid leukemia (AML) is defined as AML that develops from a prior myelodysplastic or myeloproliferative disorder, or from exposure to chemotherapy, radiation, or environmental toxins. Patients with secondary AML have inferior prognosis compared to those with de novo AML,1 and are more likely to be elderly with increased comorbidities and worse performance status.2 The detection of minimal residual disease (MRD) also has independent prognostic value in patients with AML.3-6 In elderly AML patients, MRD predicts worse outcomes independent of the poor treatment risk factors associated with increased age.7Due to the similar disease pathogenesis between elderly AML and secondary AML patients, it is likely that MRD testing will have similar implications. However, there is limited data on the prognostic role of MRD testing in secondary AML. METHODSWe retrospectively analyzed de novo and secondary AML patients at Washington University Medical Center who received post-induction, post-consolidation, pre-transplant, or post-transplant MRD testing during their treatment. MRD testing was performed with multiparameter flow cytometry testing using the “different from normal” method at one reference laboratory,8,9 with positive MRD defined as detection of an abnormal myeloblast population above 0.1%. A total of 180 patients were included. Any patient with at least one positive MRD was considered “MRD positive.” Baseline pretreatment characteristics were compared between groups with Mann-Whitney, Chi-square, or Fisher's exact test, when appropriate. We evaluated overall survival (OS) and relapse rates in de novo and secondary AML patients stratified by MRD status using Kaplan-Meier curves and the log rank test, or cumulative incidence function, as appropriate. Multivariate Cox proportional hazards model was also fitted to determine if MRD testing has similar prognostic value across de novo and secondary AML patients. RESULTSOur analysis included 133 patients with de novo and 47 patients with secondary AML. Secondary AML patients were significantly older at age of diagnosis (mean age 59.85 versus 51.17, p<0.001), however had similar rates of MRD positivity (28% versus 29%, p=0.830). There was a trend toward poorer cytogenetic risk profiles and worse Karnofsky Performance Status in secondary AML (Table 1). In all patients, we confirmed that MRD positivity confers significantly worse OS (45.5% versus 70.8%, p=0.020) and relapse rates at 2 years (53% versus 25.5%, p=0.029) (Figure 1). In de novo AML patients, MRD positivity continued to demonstrate significantly worse 2-year OS (44.1% versus 76.3%, p=0.006) and relapse rates (57.6% versus 20.7%, p=0.008). However, in secondary AML patients, there were no differences in OS (p=0.69) or relapse rates (p=0.98) between MRD positive and negative patients. Multivariate analysis demonstrated significant interaction (p=0.042) between MRD status and AML subtypes. MRD positivity emerged as an independent prognostic indicator of worse OS in de novo patients (HR 2.8, 95% CI 1.524-5.405) but not in secondary patients (HR 1.5, 95% CI 0.188-2.336). DISCUSSIONWe confirm that MRD status is a poor prognostic indicator in all AML patients, and this also holds true in patients with de novo AML. However, we found that MRD status has less prognostic value in secondary AML patients. This may be due to the overall poor prognosis seen in secondary AML patients, suggesting that MRD monitoring in this group of patients may be less useful for risk stratification. However, our study is limited by the small sample size, particularly in the number of secondary AML patients who displayed MRD positivity (N=13). In addition, we defined MRD positivity across all time points of testing, which may decrease patient homogeneity. A larger study with increased follow-up is necessary to confirm our findings, as these results have important implications in risk-stratification and treatment of patients with secondary AML. [Display omitted] DisclosuresDiPersio:BMS, Asterias, Amphivena, Bluebird: Other: Travel, Accomodations, Expenses; Hemedicus, DAVA Oncology: Speakers Bureau; Celgene, Bioline, Vasculox, Cellworks, Rivervest: Consultancy; Magenta Therapeutics: Equity Ownership.

Negative Minimal Residual Disease Associated with Extended Response to Moxetumomab Pasudotox in Patients with Relapsed/Refractory Hairy Cell Leukemia: Long-Term Follow-up of Bone Marrow Immunohistochemistry Analyses from a Phase 1 Study

The clinical significance of minimal residual disease (MRD) in patients with relapsed/refractory hairy cell leukemia (r/r HCL) who have achieved complete response (CR) has not been demonstrated. In other hematologic malignancies, MRD eradication may be associated with prolonged clinical response (Hourigan et al. Leukemia 2017;31:1482-90; Lahuerta et al. J Clin Oncol 2017;epub ahead of print). Moxetumomab pasudotox (formerly CAT-8015), a recombinant immunotoxin fusion protein that specifically binds to B lymphocyte-specific surface antigen CD22, has been shown by flow cytometry to eradicate MRD in a significant (P= 0.04) percentage of patients with r/r HCL (Kreitman et al. ASCO 2015). However, a detailed study of MRD by bone marrow immunohistochemistry (IHC) was not previously presented. We analyzed data from a phase 1 dose escalation study of moxetumomab pasudotox in r/r HCL patients to test the hypothesis that eradication of MRD in bone marrow, assessed by IHC, may be associated with prolonged remission.CAT-8015-1001 (NCT00586924) was a phase 1 study evaluating the safety and efficacy of moxetumomab pasudotox in patients with r/r HCL who previously had received at least 2 courses of treatment, at least 1 of which included a purine analog; initial results from the dose escalation phase in 28 patients have been reported (Kreitman et al. J Clin Oncol 2012;30:1822-8). Moxetumomab pasudotox was administered on days 1, 3, and 5 of each 28-day cycle until 2 cycles past complete remission (CR), disease progression, unacceptable toxicity, or neutralizing antibodies. In the dose escalation phase, 28 patients received moxetumomab pasudotox 5, 10, 20, 30, 40, or 50 µg/kg. An additional 21 patients were enrolled in the 50-µg/kg cohort.Bone marrow samples were obtained at baseline and at multiple time points during and after treatment. A central pathology laboratory performed hematoxylin and eosin and IHC staining for HCL/B-cell antigens CD20, CD79a, Annexin A1, DBA.44, and PAX-5. A blinded independent central pathologist reviewed (BICR) and assessed stained slides as IHC MRD+ or IHC MRD− for each time point.The best BICR IHC MRD response was compared with the best investigator-reported (IR) disease response. Duration of CR, duration of response (DOR), progression-free survival (PFS), and overall survival were evaluated for patients with a best response of BICR IHC MRD+ vs MRD−.Thirty-seven patients were evaluated for BICR IHC MRD; posttreatment bone marrow biopsies were not available for 11 patients, and biopsies from one patient were non-evaluable. The BICR IHC MRD status appears to be associated with the IR response; 17 of 18 BICR IHC MRD− patients had a best response of CR vs 9 of 19 MRD+ patients. Among patients found to be BICR IHC MRD− at 1 or more posttreatment evaluations, median duration of CR and median PFS were not reached after 84 months of follow-up vs 13.08 and 82.07 months, respectively, for patients who were consistently MRD+ at all time points. DOR was 82.7 vs 54.7 months for the BICR IHC MRD− and MRD+ groups, respectively.Cladribine monotherapy and cladribine followed by rituximab eliminated IHC MRD in some newly diagnosed HCL patients (Sigal et. al, Blood 2010;115:1893-6; Ravandi et. al Blood 2011;118:3818-23). Although vemurafenib demonstrated 96% to 100% overall response rates and 35% to 42% CR rates in patients with r/r HCL, all CR patients were IHC MRD+, and duration of MRD+ CRs was 19 months (Tiacci et al. N Engl J Med 2015;373:1733-47). Moxetumomab pasudotox is, to our knowledge, the only non-chemotherapy regimen shown to eradicate MRD in a meaningful percentage of patients with r/r HCL. Here, BICR findings confirmed elimination of IHC MRD in 49% of evaluable patients. MRD− patients experienced a statistically significantly longer duration of CR (P=0.0002) and PFS (P=0.0031) vs MRD+ patients.Based on the results of this retrospective analysis, together with observations made in the vemurafenib study, BICR IHC MRD− vs MRD+ status is associated with extended DOR and PFS in patients with r/r HCL treated with moxetumomab pasudotox. If this finding can be replicated in additional clinical studies, including the phase 3 pivotal study of moxetumomab pasudotox, IHC MRD determinations may be useful in evaluating the efficacy of therapies in the r/r HCL population. [Display omitted] DisclosuresKreitman:NIH: Patents & Royalties: Co-inventor on pending Moxetumomab Pasudotox patent owned by NIH. Robak:Akari Therapeutics Plc: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding. Coutre:Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Janssen: Consultancy; Novartis: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Gilead: Consultancy, Research Funding. Marshall:MedImmune: Employment. Yang:MedImmune: Employment, Equity Ownership, Other: Travel expenses. Pastan:NIH: Patents & Royalties: Pastan is an inventor on several patents on immunotoxins that have all been assigned to the NIH. Yao:MedImmune: Employment, Equity Ownership.

Neoadjuvant Chemotherapy for Breast Cancer: Moving Beyond Pathological Complete Response in the Molecular Age.

This review focuses on neoadjuvant chemotherapy for breast cancer which introduces practical issues for pathologists, including predicting response, optimising specimen handling, size measurement and assessment of residual disease, and recent advances in management of the axilla. The role of neoadjuvant chemotherapy in breast cancer is increasing, and it has become standard of care for high risk Human Epidermal Growth Factor Receptor 2 positive and triple negative breast cancers. The benefits of the neoadjuvant approach extend beyond pathological complete response to tumour downstaging permitting conservative surgical options in the breast and axilla, and assessment of response provides valuable prognostic information to enable escalation and de-escalation of adjuvant therapy to optimise oncological outcomes. Hence histopathologists play a vital role in patient management in the neoadjuvant setting. Optimal patient selection for neoadjuvant chemotherapy requires consideration of pre-treatment histopathological and molecular tumour characteristics. Post chemotherapy, tumour staging can be challenging, and changes in criteria for measurement of primary tumour and metastases in the 7th and 8th editions of the TNM have led to confusion amongst pathologists. This review offers practical guidance on specimen handling and measurement of lesion size. Moving forwards more detailed information on degree of response will be required for adjuvant therapy decision making, and the Residual Cancer Burden is emerging as the preferred method for quantifying residual disease not just within clinical trials but in routine practice. Recent advances in management of the axilla are discussed, including the significance of minimal residual disease in the form of isolated tumour cells and micrometastases which portend a worse prognosis in the neoadjuvant setting. CONCLUSION: Neoadjuvant chemotherapy now forms part of routine breast cancer management, and detailed histopathological assessment and an understanding of the importance of molecular tumour biology is essential for clinical decision making.

Clinical significance of minimal residual disease in B-lineage acute lymphoblastic leukemia pediatric patients with different fusion gene backgrounds

Objective To study the clinical significance of minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (B-ALL) pediatric patients with different fusion gene backgrounds. Methods A retrospective analysis was performed on the medical data of 441 B-ALL children who were treated from January 2008 to April 2015. Among the 441 children, 336 had negative fusion gene, 79 had positive ETV6-RUNX1 fusion gene, and 26 had positive E2A-PBX1 fusion gene. Flow cytometry was used to detect MRD, and the influence of MRD on day 15 (TP1), day 33 (TP2), and week 12 (TP3) of induction therapy on prognosis was analyzed. Results In patients with negative fusion gene, the positive MRD group had significantly lower overall survival (OS) rate and event-free survival (EFS) rate (P 0.05). Conclusions MRD has the most definite prognostic significance in pediatric B-ALL patients with negative fusion gene, while it has unsatisfactory prognostic significance in those with positive ETV6-RUNX1 or positive E2A-PBX1.

The Prognostic Significance of Minimal Residual Disease in Multiple Myeloma in the Molecular Genetic Groups Msmart 3.0

Assessment of the role of genetic abnormalities and minimal residual disease (MRD) is an active developing area in hematology. The use of genetic methods makes it possible to predict the course of the disease and apply an individualized approach to antimyeloma therapy. At the same time, the identification of MRD after therapy determines possibility of relapse. Aim. To identify the prognostic potential of MRD in patients in the standard and high molecular risk groups. Materials and methods. We analyzed 72 patients with MM (median age was 59 years (range 37-80), male/female - 1.3:1). All patients received initial therapy with proteasome inhibitors and / or immunomodulators. High dose therapy (MEL200) and autologous stem cell transplantation (ASCT) was carried out 50 (69%) patients. Standard cytogenetic and FISH methods were used to stratify patients in risk groups of mSMART 3.0. The standard risk (SR) was established in 52 (72%) patients, the high risk (HR) - in 20 (28%) patients. The MFC MRD status of bone marrow was evaluated after 4-6 cycles of induction therapy or after ASCT with use of 5-colors flow cytometry. MRD-negative status (MRD-) was based on level of clonal plasma cells &amp;lt;10-4 in bone marrow sample. Results. The MRD- was reached in 36% (26/72) patients. The median of OS in MRD+ group was 104 months, in MRD- was 146 months (p=.01). The median of PFS in MRD+ group was 26 months, in MRD- was 70 months (p=.00021). 2-years PFS in MRD+ group was 56%, in MRD- group was 100% (p=.00021). We divided patients into the following groups for evaluation the effect of MRD on survival in risk groups: SR МRD+ 34/72 (47%), SR МRD- 18/72 (25%), HR МRD+ 12/72 (17%) and HR MFC МRD- 8/72 (11%). The median of OS in HR MRD+ group was 72 months, in SR MRD+ - 104 months, in HR MRD- - 146 months, in SR MRD- was not achieved (p=.02). The median of PFS in HR MRD+ group was 24 months, in SR MRD+ - 26 months, in HR MRD- - 68 months, in SR MRD- - 70 months (p=.003). The 2-years PFS in HR MRD+ group was 44%, in SR MRD+ group was 50% and in SR MRD- and HR MRD- groups were 100% (p=.003). Conclusion. The absence of MRD is the most important prognostic factor. The leveling of negative effect of genetic abnormalities become possible when the MRD-negative response status is achieved. Presumably, this is due to the elimination of clonal plasma cells owing to the use of optimal antimyeloma therapy which is based on the risk stratification. Disclosures Martynkevich: Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Shuvaev:Novartis: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.

Outcome of (Novel) Subgroups in 1257 Pediatric Patients with KMT2A-Rearranged Acute Myeloid Leukemia (AML) and the Significance of Minimal Residual Disease (MRD) Status: A Retrospective Study By the I-BFM-SG

Introduction Outcome of KMT2A-rearranged (KMT2A-r) pediatric AML (pAML) is in general poor with a 5-year probability of event-free survival (5y-pEFS) and overall survival (5y-pOS) of 44% and 56%, respectively (Balgobind et al., 2009). However, over the past decades, the heterogeneity of KMT2A-r pAML has emerged, showing differences in outcome between subgroups based on translocation partners. The predictive value of MRD in KMT2A-r pAML is undefined. This retrospective study aimed to confirm the outcome of pediatric KMT2A subgroups (Balgobind et al., 2009) in a more recent era and to study the significance of MRD status during and after induction. Methods Outcome and MRD data of 1257 KMT2A-rde novo pAML patients from 15 AML groups affiliated with the I-BFM-AML study group, diagnosed between 2005 and 2016 were retrospectively collected. Patients were assigned to KMT2A subgroups, or to the KMT2A-other group in case of unknown translocation partner. Flow cytometry MRD levels &amp;lt;0.1% were considered negative, and levels ≥0.1% positive. Kaplan-Meier methods were used to estimate probabilities of disease-free survival (pDFS), pEFS and pOS. Cox regression analyses were performed to study the independent impact of KMT2A subgroups and potentially prognostic factors: white blood cell count (WBC), age and MRD status. Results The 1257 patients were assigned to 13 KMT2A subgroups, or the KMT2A-other group. Two novel subgroups were identified: t(X;11)(q24;q23) (n=21, 2%) and t(1;11)(p32;q23) (n=12, 1%). The median age was 2.5 years (range, 0-18.9). The median WBC was 21.4 x 109/L (range, 0.2-727). Overall complete remission rate was 91%. The 5y-pEFS was 46% [SE, 2%] and the 5y-pOS was 62% [SE, 2%]. Differences across subgroups in 5y-pEFS (Figure 1) ranged from 24% [SE, 5%] to 76% [SE, 9%], and in 5y-pOS from 25% [SE, 13%] to 92% [SE, 8%] (both p&amp;lt;0.0001). The median follow-up time of patients at risk was 5 years. The subgroups t(10;11)(p12;q23) (HR 1.7, p&amp;lt;0.0001), t(6;11)(q27;q23) (HR 1.9, p&amp;lt;0.0001), t(4;11)(q21;q23) (HR 2.9, p=.003) and t(10;11)(p11.2;q23) (HR 2.7, p&amp;lt;0.0001), WBC of &amp;gt;100 x 10^9/L (HR 1.3, p=.006), and age &amp;gt;10y (HR 1.3, p=.005) were revealed as independent predictors of poor EFS. These factors also predicted OS. MRD data after induction course one were available for n=635 (MRD-positivity (range, 0.1-94) n=126, 20%) and after course two for n=527 (MRD-positivity (range, 0.1-88) n=51, 10%). In the four KMT2A poor-risk subgroups, MRD-positivity was not significantly more common after induction course one (p=.0232) or two (p=.066), compared with the other subgroups. MRD-positivity was associated with inferior 5y-pDFS after both induction course one (36% [SE, 4%] vs 48% [SE, 2%]; p=.002) and course two (28% [SE, 6%] vs 49% [SE, 2%]; p&amp;lt;0.0001) (Figure 2). Within the t(9;11)(p22;q23) subgroup, MRD-positivity after induction course one, and within the t(10;11)(p12;q23) subgroup after course two, was associated with inferior 5y-pDFS (36% [SE, 8%] vs 56% [SE, 4%]; p=.004, and 0% [SE, 0%] vs 35% [SE, 5%]; p&amp;lt;0.0001, respectively). After induction course one, the subgroups t(10;11)(p12;q23) (HR 1.7, p&amp;lt;0.0001) and t(10;11)(p11.2;q23) (HR 4.0, p&amp;lt;0.0001), and MRD-positivity (HR 1.5, p=.003) were revealed as independent predictors of poor DFS. After induction course two, the subgroups t(10;11)(p12;q23) (HR 1.8, p&amp;lt;0.0001), t(4;11)(q21;q23) (HR 4.9, p=.008) and t(10;11)(p11.2;q23) (HR 3.2, p&amp;lt;0.0001), MRD-positivity (HR 2.0, p&amp;lt;0.0001), and age &amp;gt;10y (HR 1.5, p=.002) were revealed as independent predictors of poor DFS. Within the group of patients with MRD-negativity after induction course two, the subgroups t(10;11)(p12;q23) and t(10;11)(p11.2;q23) were independent predictors of poor EFS (5y-pEFS 35%, HR 1.7, p=.003 and 5y-pEFS 18%, HR 2.7, p=.004, respectively). Conclusion Outcome for KMT2A-r pAML patients has improved slightly, but similar subgroups were identified as poor risk (Balgobind et al., 2009), including t(10;11)(p12;q23), t(10;11)(p11.2;q23) and t(6;11)(q27;q23). In our study, t(4;11)(q21;q23) was poor risk as well. These subgroups should be considered for high-risk pAML therapy protocols. The favorable risk of t(1;11)(q21;q23) could not be confirmed in our cohort. MRD status is highly predictive of outcome within KMT2A subgroups. In MRD-negative patients after induction course two, both t(10;11) KMT2A subgroups were associated with poor outcome. Disclosures Guest: Syndax Pharmaceuticals: Consultancy. Locatelli:Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceeutical: Speakers Bureau. Rubnitz:AbbVie Inc.: Research Funding. Kaspers:Helsinn Healthcare: Ended employment in the past 24 months; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Janssen R&amp;D: Ended employment in the past 24 months; AbbVie: Ended employment in the past 24 months.

Comparison of salvage chemotherapy regimens and prognostic significance of minimal residual disease in relapsed/refractory acute myeloid leukemia

We compared the outcomes of salvage chemotherapy in 146 patients with relapsed (57.5%) or refractory (42.5%) AML who received CLAG-M (51%), MEC (39%) or CLAG (10%). Minimal residual disease (MRD) was assessed by flow cytometry. Bivariate, Kaplan–Meier, and Cox regression analyses were conducted. Complete remission (CR) rate of 46% (CLAG-M 54% versus MEC/CLAG 40%, p = .045) was observed with MRD-negative CR of 33% (CLAG-M 39% versus MEC/CLAG 22%, p = .042). Median overall survival (OS) was 9.7 months; the longest OS occurred with CLAG-M (13.3, 95%CI 2.4–24.3) versus MEC (6.9, 95%CI 2.9–10.9) or CLAG (6.2, 95%CI 2.4–12.6) (p = .025). When adjusted for age, gender, relapsed/refractory AML, poor risk AML, MRD, chemotherapy and transplant, CLAG-M (HR 0.63, 95% CI 0.40–0.98, p = .042), MRD-negativity (HR 0.15, 95% CI 0.07–0.30, p < .001) and transplant (HR 0.22, 95% CI 0.13–0.39, p < .001) were associated with higher OS. Our findings confirm that CLAG-M is a reasonable salvage regimen for RR-AML followed by transplant.

Bone Marrow Stromal Cells Drive Key Hallmarks of B Cell Malignancies.

All B cell leukaemias and a substantial fraction of lymphomas display a natural niche residency in the bone marrow. While the bone marrow compartment may only be one of several sites of disease manifestations, the strong clinical significance of minimal residual disease (MRD) in the bone marrow strongly suggests that privileged niches exist in this anatomical site favouring central elements of malignant transformation. Here, the co-existence of two hierarchical systems, originating from haematopoietic and mesenchymal stem cells, has extensively been characterised with regard to regulation of the former (blood production) by the latter. How these two systems cooperate under pathological conditions is far less understood and is the focus of many current investigations. More recent single-cell sequencing techniques have now identified an unappreciated cellular heterogeneity of the bone marrow microenvironment. How each of these cell subtypes interact with each other and regulate normal and malignant haematopoiesis remains to be investigated. Here we review the evidences of how bone marrow stroma cells and malignant B cells reciprocally interact. Evidently from published data, these cell–cell interactions induce profound changes in signalling, gene expression and metabolic adaptations. While the past research has largely focussed on understanding changes imposed by stroma- on tumour cells, it is now clear that tumour-cell contact also has fundamental ramifications for the biology of stroma cells. Their careful characterisations are not only interesting from a scientific biological viewpoint but also relevant to clinical practice: Since tumour cells heavily depend on stroma cells for cell survival, proliferation and dissemination, interference with bone marrow stroma–tumour interactions bear therapeutic potential. The molecular characterisation of tumour–stroma interactions can identify new vulnerabilities, which could be therapeutically exploited.

Significance of minimal residual disease in pediatric mixed phenotype acute leukemia: a multicenter cohort study.

The rarity of mixed phenotype acute leukemia (MPAL) has precluded adequate data to incorporate minimal residual disease (MRD) monitoring into therapy. Fluidity in MPAL classification systems further complicates understanding its biology and outcomes; this includes uncertainty surrounding the impact of shifting diagnostic requirements even between iterations of the World Health Organization (WHO) classification. Our primary objective was to address these knowledge gaps. To do so, we analyzed clinicopathologic features, therapy, MRD, and survival in a centrally-reviewed, multi-center cohort of MPAL uniformly diagnosed by the WHO classification and treated with acute lymphoblastic leukemia (ALL) regimens. ALL induction therapy achieved an EOI MRD negative (<0.01%) remission in most patients (70%). EOI MRD positivity was predictive of 5-year EFS (HR=6.00, p<0.001) and OS (HR=9.57, p=0.003). Patients who cleared MRD by EOC had worse survival compared to those EOI MRD negative. In contrast to adults with MPAL, ALL therapy without transplantation was adequate to treat most pediatric patients. Earlier MRD clearance was associated with better treatment success and survival. Prospective trials are now necessary to validate and refine MRD thresholds within the pediatric MPAL population and to identify salvage strategies for those with poor predicted survival.

Clinical Significance of Minimal Residual Disease Monitoring by Multi-parameter Flow Cytometry before Allogeneic Hematopoietic Stem Cell Transplantation for Prognosis of Acute Leukemia Patients

To investigate the clinical significance of minimal residual disease (MRD) monitoring by multiparameter flow cytometry (MFC) before allogeneic hematopoietic stem cell transplantation (allo-HSCT) in acute leukemia. 81 cases of patients with AL treated with allo-HSCT in Department of Hematology, the First Affiliated Hospital of Chongqing Medical University form July 2015 to July 2018 was selected and retorspectively analyed. of which 79 patients were in CR and two patients were in non-CR. The CR group was further divided into two groups of MRD+ and MRD- based on the MRD level prior to HSCT. Among 81 patients, there were statistically significant differences in the three-year overall survival(OS) (CR 82.2%: NCR 0%), cumulative relapse incidence(RI) (CR 17.7%; NCR 100%) and leukemia-free survival rate(LFS) (CR 42.3%: NCR 0%) between CR and NCR group(P<0.05). Among 79 CR patients, MRD was negative in 30 patients while positive in 49 patients, there was significant differences in the three-year overall survival between MRD- and MRD+ group. The results of univariate analysis showed that the MRD+ group showed lower LFS compared with that of MRD- group （10.5% vs 36.2%）(P＜0.001，95%CI). MRD- patients shows longer LFS as compared with that of MRD+ patients, therefore, MRD monitoring by MFC before allo-HSCT is very important for the prognosis of the AL patients.

Clinical Significance of Minimal Residual Disease at the End of Remission Induction Therapy in Childhood Acute Lymphoblastic Leukemia.

BACKGROUND:Detection of minimal residual disease (MRD) in the early phase of therapy is the most powerful predictor of relapse risk in children with acute lymphoblastic leukaemia (ALL).AIM:We aimed to determine the significance of MRD at the end of remission induction therapy in the prediction of treatment outcome in children with ALL.METHODS:Sixty-four consecutive patients aged 1-14 years with newly diagnosed ALL were enrolled in this study from January 2010 to October 2017. All patients were treated according to the ALL IC BFM 2002 protocol. MRD was detected at the end of remission induction therapy (day 33) by multiparameter 6-colour flow cytometry performed on bone marrow specimens with a sensitivity of 0.01%.RESULTS:Overall, 42.2% of patients had detectable MRD on day 33 of therapy. MRD measurements were not significantly related to presenting characteristics but were associated with a poorer blast clearance on day 8 and 15 of remission induction therapy. Patients with negative MRD status on day 33 had a 5-year event-free survival of 94.6% compared with 76.1% for those with positive MRD status (P = 0.044).CONCLUSION:MRD levels at the end of remission induction therapy measured by multiparameter flow cytometry have clinical significance in childhood ALL. High levels of MRD are strongly related to poor treatment outcome.

Clinical implication of minimal residual disease monitoring by 10-color flow cytometry in multiple myeloma

目的采用十色流式细胞术监测多发性骨髓瘤（MM）患者治疗后骨髓微小残留病（MRD），探讨其对MM患者预后的预测价值。方法回顾性分析2015年7月至2017年7月苏州大学附属第一医院收治的150例MM患者的临床资料，十色流式细胞术检测MRD水平。结果①诱导治疗结束后采用十色流式细胞术监测MRD的87例MM患者中，MRD阴性者34例（39.1%）。自体造血干细胞移植后1年内行十色流式细胞术监测MRD的69例患者中，MRD阴性者34例（49.3%），诱导治疗结束和移植后MRD阴性患者的无进展生存（PFS）期优于MRD阳性患者（未达到对21个月，P=0.022；未达到对18个月，P<0.001）。②根据移植前后MRD的动态改变将MM患者分为MRD持续阴性、阳性转阴性、持续阳性、阴性转阳性四组，四组的2年PFS率分别为83%、82%、44%、0（P=0.002）。③多因素分析显示诱导治疗结束后的MRD水平是影响PFS的独立预后因素[P=0.002，HR=4.808（95%CI 1.818~12.718）]。结论治疗后MRD转阴提示更好的临床预后，MRD水平对于MM患者的预后价值优于血清学疗效评估，可以联合R-ISS分期及细胞遗传学异常评估患者预后。