Follow-up Bone Marrow Samples Research Articles

Deciphering stage 0 hematogones by flow cytometry in follow-up bone marrow samples of pediatric B-Acute lymphoblastic leukemia cases: A potential mimicker of residual disease after anti CD19 therapy.

CD19 is frequently targeted for immunotherapy in B cell malignancies, which may result in loss of CD19 expression in leukemic cells as an escape mechanism. Stage 0 hematogones (Hgs) are normal CD19-negative very early B cell precursors that can be potentially mistaken for CD19 negative residual leukemic cells by flow cytometry (FCM) in B cell acute lymphoblastic leukemia (BCP-ALL) cases treated with anti CD19 therapy. Our main objective was to characterize and study the incidence of stage 0 hematogones in follow-up bone marrow samples of pediatric BCP-ALL cases. We analyzed the flow cytometry standard files of 61 pediatric BCP-ALL cases treated with conventional chemotherapy and targeted anti-CD19 therapy, for identifying the residual disease and normal B cell precursors including stage 0 Hgs. A non-CD19 alternate gating strategy was used to isolate the B cells for detecting the residual disease and stage 0 Hgs. The stage 0 Hgs were seen in 95% of marrow samples containing CD19+ Hgs. When compared with controls and posttransplant marrow samples, the fraction of stage 0 Hgs was higher in patients receiving anti CD19 therapy (p = 0.0048), but it was not significant when compared with patients receiving chemotherapy (p = 0.1788). Isolated stage 0 Hgs are found in samples treated with anti-CD19 therapy simulating CD19 negative residual illness. Our findings aid in understanding the stage 0 Hgs and its association with CD19+ Hgs in anti CD19 therapy and conventional chemotherapy. This is crucial as it can be potentially mistaken for residual disease in patients treated with anti CD19 therapy.

Final Results of the Phase Ib/II Study Evaluating Enasidenib in Combination with Venetoclax in Patients with IDH2-Mutated Relapsed/Refractory Myeloid Malignancies

BACKGROUND: Theisocitrate dehydrogenase 2 ( IDH2) gene is mutated in ∼10-15% cases of acute myeloid leukemia (AML) and ∼5% cases of myelodysplastic syndromes (MDS). Enasidenib (ENA) is an oral selective IDH2 inhibitor used for treatment of IDH2-mutated (m IDH2) relapsed/refractory (R/R) AML. Preclinical studies showed that m IDH2 AML cells are particularly sensitive to BCL2 inhibition with venetoclax (VEN) and combination of ENA and VEN is more effective than single agents in patient-derived xenograft models of m IDH2 AML. Based on this rationale, we conducted a phase Ib/II trial evaluating the safety and efficacy of ENA plus VEN in patients (pts) with m IDH2 R/R AML or MDS (NCT04092179). METHODS: This single-arm phase Ib/II trial was conducted at the Princess Margaret Cancer Centre and the University of Alberta Hospital in Canada. Pts ≥ 18 years with m IDH2 R/R AML or MDS, adequate organ function and ECOG performance status ≤ 2 were eligible. Participants were not eligible if they had prior exposure to IDH2 or BCL2 inhibitors. Pts were treated continuously by 28-day cycles of VEN 400 mg daily starting with a 3-day ramp-up on cycle 1 day 1 and with ENA 100 mg daily starting on cycle 1 day 15. The primary endpoints were safety, tolerability and recommended phase 2 dose (RP2D) for the phase Ib part and overall response rate (ORR) for the phase II part. ORR included complete remission (CR), CR with partial hematological recovery (CRh), CR with incomplete hematological recovery (CRi), morphologic leukemia-free state (MLFS) and partial remission (PR) as defined by the European Leukemia Network (ELN). Duration of response (DOR), overall survival (OS), pharmacokinetic (PK) analysis for VEN and bone marrow (BM) IDH2 variant allele frequency (VAF) by droplet digital PCR (ddPCR) were also evaluated. RESULTS: From November 2020 to July 2022, 27 pts were enrolled: 12 in the phase Ib and 15 in the phase II. Almost all pts had AML, except one with MDS who progressed on treatment with azacitidine. The data cutoff date was June 30, 2023. The median age was 70 years (range, 23 - 84) ( Table 1). Patients had a median of 1 prior line of treatment (range, 1-2) including 5 (19%) who had previously undergone allogeneic stem cell transplant (HSCT). Fifteen (56%) and 12 (44%) pts had R140Q and R172K/W IDH2 mutation (mut), respectively. The median BM IDH2 VAF at baseline was 23.4 (range 2.3 - 49.9). The most common (≥ 20%) grade 3-5 treatment emergent adverse events (TEAEs), regardless of attribution, were febrile neutropenia (41%), lung infection (22%) and thrombocytopenia (26%). Nausea, vomiting and diarrhea occurred each in 41% of pts, but were primarily grade 1-2 (only 1 pt with grade 3 diarrhea). Hyperbilirubinemia of any grade occurred in 48% pts, including grade 3 in 15%. No case of IDH inhibitor associated differentiation syndrome (IDH-DS) occurred in this study. Grade 3 leukocytosis without IDH-DS occurred in 2 (8%) pts. No patient discontinued ENA because of an adverse event and no treatment-related death was reported. The PK profile of VEN was not altered by ENA, and the RP2D was VEN 400 mg daily plus ENA 100 mg daily. Among 23 pts with R/R AML evaluable for response, the ORR was 70% (16/23), including 57% (13/23) CR ( Figure 1). In pts with R172 mut, the ORR and CR rates were 83% (10/12) and 67% (8/12), respectively, versus 55% (6/11) and 45% (5/11) in pts with R140 mut. m IDH2 VAF at baseline was similar between responders and non-responders. On follow-up BM samples, there was an association between response and decrease in m IDH2 VAF. Clearance of m IDH2 was observed in 47% (7/15) of analyzed responders. The median time to initial response was 1 month (range, 0.9 - 5.7), and the median time to best response was 2.8 months (range, 0.9 - 12.5). The median DOR was 16.6 months (95% CI 5.0 - NR) and pts received a median of 7 cycles of treatment (range, 1 - 32). With a median follow-up of 17.1 months (range, 0.9 - 31.4), 10 (38%) pts were alive, including 9 (35%) in ongoing remission. The median OS was 9.4 months (95% CI, 8.2 - NR) and the 2-year OS rate was 42% (95% CI, 27 - 66). Four pts proceeded to HSCT after achieving remission, of whom 2/4 (50%) are alive in remission on last follow-up. CONCLUSIONS: The combination of ENA and VEN is safe and well tolerated in pts with R/R m IDH2 myeloid malignancies. The ENA-VEN combination compares favorably to ENA single-agent in this patient population. Further studies of targeted combination therapies using IDH2 and BCL2 inhibitors are warranted.

APAL2020SC Pediatric Acute Leukemia (PedAL) Screening Trial - Developing New Therapies for Relapsed Leukemias

Background: Outcomes for children with relapsed acute myeloid leukemia (AML) and subgroups with relapsed acute lymphoblastic leukemia (ALL) remain poor, and therapeutic options are limited. There is no standard of care for relapsed disease, and data collection after relapse is lacking. In recent years, extensive molecular studies of pediatric leukemias have advanced target identification and clinical trial development. Such studies have shown that pediatric leukemias are biologically distinct from adult leukemias. The current paradigm of requiring new agent studies in adults prior to conducting pediatric trials is thus not appropriate. However, the rarity of relapsed leukemia in children makes the conduct of randomized registration trials for new agents infeasible. Recognizing that impactful progress requires international collaboration, the Leukemia & Lymphoma Society is sponsoring the Pediatric Acute Leukemia (PedAL) Initiative in North America, Australia and New Zealand. In parallel, the European Pediatric Acute Leukemia (EuPAL) Foundation has established a registry for pediatric relapsed leukemia. The principal goal of this international PedAL/EuPAL collaboration is to establish a data-driven developmental therapeutics program for children with relapsed/refractory leukemias. Trial Summary: A central component of the PedAL Initiative is the screening protocol APAL2020SC (NCT04726241), which serves as the entry point for associated therapeutic sub-trials. The primary aims of APAL2020SC are (1) to use clinical and biological characteristics to screen for eligibility for therapeutic sub-trials; and (2) to maintain a longitudinal data registry and biobank for relapsed leukemia. Eligible patients are <22 years old and have one of the following leukemias in first or greater relapse, or primary refractory: AML, myeloid leukemia of Down syndrome, T-ALL, mixed phenotype acute leukemia, and KMT2A-rearranged B-ALL. Additional eligible diagnoses include any B-ALL in second or greater relapse, and de novo or relapsed treatment-related AML. All leukemia specimens are profiled by multiparameter flow cytometry (MFC) at a central lab (Hematologics). Families have the option to submit biospecimens for banking. Patients with myeloid leukemia also have molecular testing through Foundation Medicine. Clinical data collected include demographics, cytomolecular features at diagnosis, prior treatment and post-relapse treatment. Patients are encouraged to submit follow-up bone marrow samples for central MFC. The MFC assay determines lineage and blast percent and quantifies expression of markers relevant to therapeutic sub-trials. Trial Progress: APAL2020SC opened in April 2022. As of July 25, 2023, 148 patients have enrolled and 97 have had leukemia confirmed centrally. The distribution of diagnoses is shown in Table 1. All patients had blast enumeration and target expression assessed by MFC at study entry and when follow-up samples were provided. Of the patients with myeloid leukemia (n=71), targeted DNA/RNA sequencing was successful for 69%. The distribution of AML-associated alterations is shown in Table 2. Of the patients with confirmed leukemia at enrollment, 79% submitted at least 1 follow-up sample, and 77% submitted a sample for biobanking. To date, two therapeutic trials have opened that require enrollment to the screening trial. APAL2020D/ITCC101 is a randomized phase III study comparing fludarabine-cytarabine (FLA) to FLA-venetoclax. APAL2020E/PEPN2113 is a phase 1 study of the E-selectin inhibitor uproleselan in combination with FLA. Pediatric-specific therapeutic trials for menin inhibitors and other new agents are in development. The study is collecting treatment information for children who do not enroll on a subtrial, with the goal of describing current practices and outcomes (Table 2). Conclusion: APAL2020SC will accrue patients with relapsed acute leukemias and treatment-related AML for 5 years. This protocol will promote clinical trial development by engaging industry partners and providing a single entry point to therapeutic studies with biological rationale. APAL2020SC and the EuPAL Foundation registry will generate a large, comprehensive and longitudinal dataset that will inform future trials. The biobank component will support innovative translational research to feed the pipeline of new agents.

Tracing back of relapse clones by Ig/TCR gene rearrangements reveals complex patterns of recurrence in pediatric acute lymphoblastic leukemia.

Relapse remained the major obstacle to improving the prognosis of children with acute lymphoblastic leukemia (ALL). This study aimed to investigate the changing patterns of Ig/TCR gene rearrangements between diagnosis and relapse and the clinical relevance and to explore the mechanism of leukemic relapse. Clonal Ig/TCR gene rearrangements were screened by multiplex PCR amplification in 85 paired diagnostic and relapse bone marrow (BM) samples from children with ALL. The new rearrangements presented at relapse were quantitatively assessed by the RQ-PCR approach targeting the patient-specific junctional region sequence in 19 diagnostic samples. The relapse clones were further back-traced to diagnostic and follow-up BM samples from 12 patients. Comparison of Ig/TCR gene rearrangements between diagnosis and relapse showed that 40 (57.1%) B-ALL and 5 (33.3%) T-ALL patients exhibited a change from diagnosis to relapse, and 25 (35.7%) B-ALL patients acquired new rearrangements at relapse. The new relapse rearrangements were present in 15 of the 19 (78.9%) diagnostic samples as shown by RQ-PCR, with a median level of 5.26 × 10-2 . The levels of minor rearrangements correlated with B immunophenotype, WBC counts, age at diagnosis, and recurrence time. Furthermore, back-tracing rearrangements in 12 patients identified three patterns of relapse clone dynamics, which suggested the recurrence mechanisms not only through clonal selection of pre-existing subclones but also through an ongoing clonal evolution during remission and relapse. Backtracking Ig/TCR gene rearrangements in relapse clones of pediatric ALL revealed complex patterns of clonal selection and evolution for leukemic relapse.

Endleukemia Assay v1: Enabling NGS-Based Comprehensive Routine Molecular Profiling of Leukemias in Routine Clinical Care

Background: Molecular biomarkers provide diagnostic, prognostic, predictive and minimal residual disease (MRD) information and constitute an integral part of diagnostic workup of hematologic neoplasms. Since a variety of different hematologic disorders can often show non-specific clinical symptoms as well as laboratory findings, the initial diagnostic workup needs to be optimized for timely and efficient coverage of a broad range of differential diagnoses. We developed a customized 81-gene panel, EndLeukemia Assay v1, for comprehensive genetic characterization of disorders that include bone marrow (BM) and peripheral blood (PB). The biomarkers on the panel cover genetic alterations in myeloid neoplasms (acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myelodysplastic syndrome/myeloproliferative neoplasms (MDS/MPN), aplastic anemia (AA), acute lymphoblastic leukemia (ALL), hairy cell leukemia, T-large granular lymphocytic leukemia (T-LGL) and T-prolymphocytic leukemia (T-PLL).Materials and Methods: Genomic DNA was isolated from fresh BM aspirate/PB or formalin-fixed paraffin-embedded (FFPE) BM clot specimens. Either entire coding sequences or limited exons/hotspots for 81-gene target genes were captured from 250 ng DNA using the hybridization-based HaloPlexTM target enrichment system (Agilent Technologies, Santa Clara, CA). These genes include A NKRD26, ASXL1, ASXL2, BCOR, BCORL1, BRAF, BRINP3, CALR, CBL, CBLB, CBLC, CEBPA, CREBBP, CRLF2, CSF3R, CUX1, DDX41, DNMT3A, EED, ELANE, ETNK1, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GFI1, GNAS, HNRNPK, HRAS, IDH1, IDH2, IKZF1, IL2RG, IL7R, JAK1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, MAP2K1, MPL, NF1, NOTCH1, NPM1, NRAS, PAX5, PHF6, PIGA, PML, PRPF40B, PTEN, PTPN11, RAD21, RARA, RUNX1, SETBP1, SF1, SF3A1, SF3B1, SH2B3, SMC1A, SMC3, SRSF2, STAG1, STAG2, STAT3, STAT5A, STAT5B, SUZ12, TERC, TERT, TET2, TP53, U2AF1, U2AF2, WT1, and ZRSR2. The panel design incorporated molecular barcodes to improve accuracy and sensitivity. Bidirectional paired-end sequencing was performed using the Illumina Miseq platform (Illumina, San Diego, CA). Variant calling was performed using Agilent SureCall software v3.5 and an in-house developed bioinformatics pipeline. Incorporation of single nucleotide polymorphisms (SNP) allows detection of sample identification errors and improves patient safety.Results: During the validation studies, high concordance was observed for missense mutations, insertions/deletions (indels), and selected copy number aberrations (CNAs) when compared with orthogonal methods. Under standardized conditions, a minimum average coverage depth of 3000x is achieved across the entire target region. The analytical sensitivity of the assay was established at 5% using a per base coverage cut-off of 250x. Of note, >90% of the CEBPA coding sequence, which is challenging to sequence due to a high GC component, was covered at >250x coverage depth. Copy number alterations including KMT2A partial tandem duplications (KMT2A -PTD) and IKZF1 deletions were detected successfully. Initial validation of FFPE tissue shows 100% concordance for all mutation calls for matched fresh and FFPE sample pairs. The ability to provide critical diagnostic and prognostic information across mutation hotspots from FFPE tissue if a fresh PB/BM sample is not available improves patient care. The top 25 mutations in the first 500 patient samples across different clinical indications are shown in the figure below. In addition, efficient panel design allowed uniform testing of follow-up bone marrow samples for the detection of MRD and clonal evolution.Conclusion: EndLeukemia assay v1 allowed comprehensive molecular profiling of a variety of hematologic neoplasms in routine clinical testing. Routine molecular biomarker profiling can be facilitated by incorporating smart design features in clinical NGS panels. [Display omitted] DisclosuresKantarjian:Delta-Fly Pharma: Research Funding; Novartis: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding; Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding.

Chimerism evaluation in measurable residual disease-suspected cells isolated by flow cell sorting as a reliable tool for measurable residual disease verification in acute leukemia patients after allogeneic hematopoietic stem cell transplantation.

The presence of minimal/measurable residual disease (MRD) before or after hematopoietic stem cell transplantation (HSCT) is known as a predictor of poor outcome in patients with acute myeloid (AML) or lymphoblastic (ALL) leukemia. When performed with multiparameter flow cytometry (MFC), assessment of residual leukemic cells after HSCT may be limited by therapy-induced shifts in the immunophenotype (e.g., loss of surface molecules used for therapeutic targeting). However, in such cases, questionable cells can be isolated and tested for hematopoietic chimerism to clarify their origin. Questionable cell populations were detected during the MFC-based MRD monitoring of 52 follow-up bone marrow samples from 37 patients diagnosed with T cell neoplasms (n=14), B cell precursor ALL (n=16), AML (n=7). These cells (suspected leukemic or normal) were isolated by flow cell sorting and tested for hematopoietic chimerism by RTQ-PCR. The origin of cells was successfully identified in 96.15% of cases (n=50), which helped to validate the results of MFC-based MRD monitoring. We believe that a combination of MFC, cell sorting, and chimerism testing may help confirm or disprove MRD presence in complicated cases after HSCT.

DNA-Based Methods for Measurable Residual Disease Detection in NPM1-Mutated Acute Myeloid Leukemia; Establishment of Cut-Offs for qPCR, Digital Droplet PCR and Targeted Deep Sequencing

For detection of measurable residual disease (MRD) in acute myeloid leukemia with NPM1 mutations, RT-qPCR with quantification of leukemic transcripts is currently considered the method of choice; however, MRD can also be determined with DNA-based methods, offering certain advantages. For example, digital droplet PCR (ddPCR) and targeted deep sequencing (deep seq) do neither require standard curves nor reference genes and are thus less labor intense than RT-qPCR. Also, deep seq allows for quantification independently of type of NPM1 mutation. In addition, DNA-based techniques enable MRD assessment of other mutations, beyond the reach of RT-qPCR, which is limited to analyses of highly expressed genes or fusion transcripts (e.g. core binding factor leukemias). With the rapid development of highly sensitive DNA-based techniques for MRD detection, there is a need to establish clinically relevant cut-offs for accurate interpretation of MRD results and risk stratification. Here, we compare and provide MRD cut-offs for three different DNA-based MRD methods for NPM1 mutations: quantitative PCR (qPCR), ddPCR and deep seq. To compare the DNA-based methods with RT-qPCR, we analyzed 110 follow-up peripheral blood (PB) or bone marrow (BM) samples from 32 AML patients harboring NPM1 mutation type A. First, we compared the mere detectability of leukemic signals (without reference to specific MRD cut-off points). We found a high correlation between results from RT-qPCR and the three DNA-based methods (Rs=0.936 for RT-qPCR vs qPCR, Rs=0.774 for RT-qPCR vs ddPCR and Rs=0.743 for RT-qPCR vs deep seq, p<0.001). As expected, RT-qPCR was the most sensitive method. Among the DNA-based methods, qPCR was the most sensitive, detecting leukemic DNA in 95% (55/58) of the RT-qPCR positive samples, compared to 72% (42/58) and 62% (36/58) for ddPCR and deep seq, respectively. Interestingly, the transcript level for a given amount of measurable leukemic DNA (RNA copy number per leukemic DNA molecule) fluctuated considerably between different follow-up samples for certain patients. In some cases, the RNA/DNA ratio exceeded a hundredfold difference between different follow-up time points in both PB and BM. Hence, transcript analysis may be more complex than just a simple measurement of leukemic cell burden, which in turn may influence accurate risk stratification and treatment decisions, if relying on RT-qPCR measurements alone. To select adequate DNA MRD cut-offs, we performed ROC curve analyses for each method at various DNA cut-offs, comparing them with the gold standard RT-qPCR cut-off. In BM, this cut-off can be defined as a less than 3 log reduction of mutated NPM1 transcripts vs diagnosis, separating MRDhigh from MRDlow/undetectable (sometimes inaccurately termed "MRD-positivity" and "MRD-negativity", respectively). In PB, the mere detectability of mutated NPM1 transcripts is considered the relevant cut-off. DNA cut-offs were chosen based on the area under the curve (AUC) for the ROC analyses (Table 1), and influenced by available literature including recommendations of prognostically relevant MRD levels. For qPCR, a cut-off at 0.1% leukemic DNA was judged relevant in BM. For ddPCR and deep seq, 0.05% was chosen to adjust for measuring allelic ratio (variant allele frequency (VAF)) rather than mutant DNA alone. In PB, the selected cut-off was detectable leukemic signal irrespective of DNA method. We next determined the accuracy of the selected cut-offs, for identification of samples with clinically relevant MRD, by comparing them with the gold standard RT-qPCR. In general, the selected DNA cut-off values generated high specificity as well as high positive and negative predictive values (Table 1). The vast majority of all MRD analyses (93% (368/395)) showed concordant results irrespective of MRD method. In BM samples, MRD assessment by the DNA based methods agreed with MRD status as determined by RT-qPCR (MRDhigh high vs MRDlow/undetectable) in 93% (62/67) of the analyses for qPCR, 96% (64/67) for ddPCR, and 97% (65/67) for deep seq. In PB, the agreement was 95% (41/43), 88% (38/43) and 86% (37/43), respectively. In summary, we found strong agreement between different MRD methods and based on this could provide clinically relevant cut-offs for risk stratification. Thus, in BM follow-up samples from AML patients with NPM1 mutation, we propose 0.1% leukemic DNA as cut-off for qPCR and 0.05% VAF for ddPCR and deep seq. Disclosures No relevant conflicts of interest to declare.

Targeted Sequencing Predicts the Development of Myeloid Malignancies and Clinical Outcome in Patients with Unexplained Cytopenia

The high frequency of somatic mutations in myelodysplastic syndromes (MDS) provides an additional biomarker modality for MDS diagnosis. This has significant potential in the investigation of cytopenias particularly in cases with <5% blasts when the diagnosis of MDS relies heavily on morphological assessment, which is limited by interobserver bias. However, consideration of mutations in the diagnostic workup has been complicated by reports of frequent somatic mutations and clonal expansion in aging healthy individuals involving genes recurrently mutated in myeloid malignancies. As a result, the presence of a somatic mutation is not a core criterion in the diagnosis of MDS. There is a clear and unmet need to understand the clinical and diagnostic implications of acquired mutations along the axis of clinical presentation from cytopenias to confirmed myeloid disease. This study aimed to investigate the clinical significance of detecting somatic mutations in an unselected cohort of cytopenic patients by correlating baseline mutation analysis with clinical phenotype and outcome. All patient samples referred for unexplained cytopenia or suspected MDS to the Haematological Malignancy Diagnostic Service in the UK over a 2-year period (July 2014-2016) were included. Samples were analysed using established diagnostic assays (morphology, flow cytometry, cytogenetics) and in parallel subjected to targeted sequencing of 27 genes commonly mutated in myeloid malignancies. Outcome data was captured including serial blood count data, subsequent diagnoses and overall survival. A subgroup of 37 mutation negative samples underwent extended genotyping of 126 genes. A total of 2089 samples passed QC for targeted sequencing. Of these, 538 had a confirmed diagnosis (26%), the majority of which were diagnosed with a myeloid malignancy (449/538; 83%). Mutations were commonly identified in the latter with ≥1 somatic mutation +/or karyotypic abnormality detected in 91% of patients. Importantly, mutations were also detected in 28% of non-diagnostic (ND) samples (412/1496). The spectrum of mutations was similar between ND and MDS samples, though varied in frequency. In ND samples TET2, SRSF2 and DNMT3A mutations predominated (47%, 22%, 19% respectively) while SF3B1, TET2 and ASXL1 were most frequently mutated in MDS (25%, 24%, 24% respectively). The number of mutations detected per sample and the variant allele fraction (VAF) was also significantly lower in ND samples (median no. of mutations 1 vs 2; median VAF 17.7% vs 35.1%; p<0.0001) suggesting that clonal expansion and/or additional mutations are required for overt dysplasia. To determine the clinical significance of mutations, correlation was made with subsequent diagnoses and clinical outcome. Follow-up bone marrow samples were received in 205 ND samples and 82 of these had a confirmed diagnosis of which 61 had a myeloid malignancy. The presence of a mutation at baseline was strongly associated with a subsequent myeloid diagnosis (NDmut vs NDunmut ;13% vs 0.5%; p<0.0001) though was rarely seen in those with an isolated mutation (1 vs >1 mutation; p<0.0001). BCOR, EZH2, RUNX1 and SRSF2 mutations were found to be most predictive. The mutations in the NDmut samples also had a VAF comparable to MDS (median 31.2% vs 35.1%; p=0.14). Extended genotyping of NDunmut patients who underwent follow-up sampling identified additional rare mutations involving DDX41, SMC1A and PHF6. Importantly the presence of a mutation impacted significantly on survival in the ND group (odds ratio=1.59; p<0.0001). The mutations with the greatest impact on survival differed from those that predicted a subsequent diagnosis and involved ASXL1, BCOR, IDH2 and TP53. Certain mutations can therefore predict increased mortality which is unrelated to having a subsequent diagnosis. By analysing a large unselected cohort of cytopenic patients, this study has demonstrated the power of mutation analysis to predict both a subsequent myeloid diagnosis and clinical outcome in those without a confirmed diagnosis. While standard techniques will identify some of these patients over time, crucially, those at highest risk can remain undetected. Identifying these patients would ensure close clinical follow-up and provide an opportunity for early intervention. Ultimately this will facilitate the development of predictive models and refine diagnostic algorithms and this work is ongoing. Disclosures Cargo: Celgene: Research Funding. Evans:Diaceutics: Honoraria; Novartis: Honoraria. Papaemmanuil:Celgene: Research Funding.

Chemotherapy-Free Combination of Obinutuzumab and Ibrutinib in First LINE Treatment of Follicular Lymphoma. the Alternative Study By the German Low Grade Lymphoma Study Group (GLSG)

Chemotherapy-Free Combination of Obinutuzumab and Ibrutinib in First LINE Treatment of Follicular Lymphoma. the Alternative Study By the German Low Grade Lymphoma Study Group (GLSG)

Flow cytometry immunophenotypic findings in chronic myelomonocytic leukemia and its utility in monitoring treatment response.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm, characterized by persistent monocytosis. Due to the lack of unique surface markers expressed by neoplastic monocytes and the frequent CD34-negative blast immunophenotype, the diagnostic value of flow cytometric immunophenotyping (FCI) in CMML is rarely studied. In this study, by using a multicolor FCI assay, we assessed bone marrow (BM) immunophenotypical alterations in 118 CMML patients and follow-up BM samples in 35 of these patients. The median BM monocytes as determined by FCI were 14% (1-63%), correlated with morphologic count (P = 0.0004). FCI alterations in monocytes were observed in 96% and granulocytes in 83% of cases. The percentage of CD34(+) myeloblasts by FCI was low [median 0.6% (0.02-12.6%)], but exhibiting frequent aberrancies [median 6 (2-12)]. CD34(+) B-cell precursors were absent in 93% of cases. In 35 patients with follow-up BM samples assessed, the CD34(+) myeloblasts showed persistent FCI aberrancies in all 29 patients treated with hypomethylating agents and 3 patients on observation, but became normal in 3 patients following stem cell transplant. In conclusion, CMML exhibit numerous FCI alterations in monocytes, granulocytes, and more profound/frequent in CD34(+) myeloblasts. These findings provide solid evidence for using FCI as an ancillary test in CMML diagnosis and also, in assessment of treatment responses.

A Rare Case of Chronic Myelogenous Leukemia and Plasma Cell Myeloma in the Same Patient

Dear Editor CML and plasma cell myeloma (PCM) arise from pluripotent stem cells and lymphoid cells, respectively [1]. Since different cell lines give rise to CML and PCM, development of both malignancies in one patient is uncommon; only 17 such cases have been reported worldwide [2, 3, 4, 5]. Here, we attempt to address questions about the mechanisms of co-existence of these two diseases. A 76-yr-old Korean male was referred to our hospital in April 2010 with thrombocytosis and leukocytosis with basophilia. Radiographic examination revealed splenomegaly. Using bone marrow (BM) analysis and quantitative reverse-transcription PCR, the patient was diagnosed as having chronic phase BCR-ABL1-positive CML. After three weeks of imatinib mesylate (IM) therapy, the patient's leukocyte and platelet counts normalized. Three years later, examination of the patient's BM revealed normal cellularity, but with plasma cells accounting for 17.5% of all nucleated cells (Table 1). Hypercalcemia, uremia, anemia, and lytic bone lesions were not observed. Serum electrophoresis and immunofixation electrophoresis revealed immunoglobulin (Ig)A lambda type monoclonal protein. The patient was diagnosed as having asymptomatic PCM. No chemotherapy was administered for PCM, but IM therapy achieved maintenance of the CML major molecular response status. Clinical and laboratory findings are summarized in Table 1. Table 1 Laboratory findings at diagnosis of CML and PCM No symptoms or blood tests indicated the presence of PCM at the time of CML diagnosis. However, since it is possible that a PCM clone was present at the time of CML diagnosis, we retrospectively performed immunohistochemical staining of a BM specimen. This staining was positive for CD138 and IgA lambda. We also analyzed Ig heavy chain gene rearrangements (InVivoScribe Technologies, San Diego, CA, USA) in initial and follow-up BM samples. DNA samples from both time points showed gene rearrangement at the same diversity region, indicating that the same PCM clone was present at CML diagnosis. When we reviewed the previously reported cases of CML and PCM in the same patient (Table 2), we first asked whether the malignancies occurred spontaneously, or whether treatment of the first neoplasm induced the second malignancy. Among the 17 cases, six patients were diagnosed first as having CML, and then PCM. Five of those six patients received IM, while one received busulfan. The mean interval before discovery of the second malignancy was 37.7 months (range, 3 to 88 months). IM has been reported to have both stimulatory and inhibitory effects on PCM cell proliferation [6]. Studies have found no evidence that IM treatment increases the occurrence of any secondary malignancies compared with the lifetime risk of cancer in the general population [7, 8]. There is no clear evidence that IM used for CML treatment caused PCM; however, because IM becomes widely used as an effective target therapy in other cancers as well as CML, close investigation of this issue is necessary. Table 2 Reported cases of CML and PCM occurring in the same patient Another five of the 17 patients were diagnosed as having PCM first, and then CML. Two of them were not treated for PCM until their CML diagnosis. The remaining three patients received melphalan, dexamethasone, and other drugs, which have not been reported to cause CML. Two patients with PCM underwent radiation therapy (RT) before their CML diagnosis. In general, irradiation is known to cause leukemia. One study calculated the theoretical probability of an individual exposed to radiation acquiring the t(9;22) translocation to be 0.007/Gy, with a minimum lapse of 5.1 yr [9]. Since the intervals between irradiation and CML development in these cases were less than two years, it seems unlikely that RT induced CML. Concurrent development of CML and PCM was identified in six of 17 patients, and two patients developed CML before being treated for PCM. Therefore, 8 patients (47%) had a secondary malignancy without having received therapy, so it is difficult to conclude that treatment of the first neoplasm caused a secondary malignancy. Another important question is whether the two cancers have the same origin. Since CML arises from pluripotent hematopoietic stem cells, it may be possible that CML cells can transform into malignant plasma cells. No Philadelphia chromosomes or BCR-ABL1 transcripts were found when our patient was diagnosed as having PCM, suggesting that the myeloma cells did not evolve from pre-existing CML cells. We have described a patient with both CML and PCM. The mechanism of co-existence is unclear. To identify more cases, it is important for clinicians and pathologists to be aware that co-existence of CML and PCM is rare, but possible.

B acute lymphoblastic leukemia with t(14;19)(q32;p13.1) involving IGH/EPOR: a clinically aggressive subset of disease

B acute lymphoblastic leukemia (B-ALL) with t(14;19)(q32;p13.1), in which IGH and EPOR are juxtaposed, has been reported rarely. We describe the clinicopathological features of six patients, three men and three women, with a median age of 39 years. Initial and follow-up bone marrow samples were examined from each patient. The clinical, morphologic, and immunophenotypic results were compared with data obtained from conventional cytogenetic analysis and by using home-brew fluorescence in situ hybridization (FISH) probes for IGH at 14q32 and EPOR at 19p13.1. The bone marrow specimens were hypercellular (median 90%; range 80–100%), with a median blast count of 90% (range 60–93%). Immunophenotypic analysis performed by flow cytometry demonstrated a stable, precursor B-cell immunophenotype. The t(14;19)(q32;p13.1) was present in all cases with morphologic evidence of disease. The translocation was stable and appeared morphologically subtle on conventional karyotypic analysis. Detection was facilitated using FISH, which confirmed IGH/EPOR rearrangement in all cases. All patients received aggressive multiagent chemotherapy as part of a variety of regimens. Four of six patients achieved an initial complete remission, but all relapsed. At last follow-up, five of six patients had died of disease (median survival, 12 months after diagnosis). We conclude that B-ALL associated with t(14;19)(q32;p13.1) is a distinctive form of disease that is associated with younger patient age and an aggressive clinical course.

Identification Of Low Risk Group In Infants With Acute Lymphoblastic Leukemia By Flow Cytometric Minimal Residual Disease Measurement At Day 15 Of Interfant-99 and Interfant-06 Protocols Treatment

BackgroundAcute lymphoblastic leukemia (ALL) in infants is a relatively rare disease with peculiar biological features and worse outcome in comparison to ALL in older children. Infant ALL is characterized by a high frequency of MLL gene rearrangements, mainly CD10-negative B-cell precursor ALL (BCP-ALL) immunophenotype and high tumor burden at diagnosis. Even with new therapeutic approaches event-free survival (EFS) in this subgroup of patients does not exceed 50%. Although flow cytometric (FCM) minimal residual disease (MRD) detection at day 15 of remission induction is well established for patients' stratification in older children treated with the AIEOP-BFM-2009 protocol, the prognostic value of FCM MRD in infant ALL is not fully known yet.Aim of the present study was to evaluate the prognostic significance of FCM MRD measurement in infants with ALL treated with Interfant-99 and Interfant-06 protocols in AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica) centers in Italy. Patients and methodsBetween May 1999 and December 2011, 120 consecutive infants aged 0 to 365 days with newly diagnosed ALL were treated in AIEOP centers with the Interfant99 and the on-going Interfant-06 protocols. Among these patients, 51 (42.5%) with available day 15 follow-up bone marrow samples were included in this study on FCM MRD. In 39 (76.5%) cases, different types of MLL gene rearrangements were identified by fluorescence in situ hybridization (FISH), while 12 (23.5%) patients had germline MLL. MRD detection was performed by 4-6-color FCM. Median follow-up time was 3.5 years (range: 1 month – 7.5 years). Outcome was estimated by evaluating the probability of EFS and the cumulative incidence of relapse (CIR). Analysis of prognostic relevance of FCM MRD in combination with other criteria used for stratifying patients enrolled in the Interfant-06 protocol was performed with the Cox model on the cause-specific hazard of relapse. Results and discussionWe classified infants according to the AIEOP-BFM day 15 stratification into three risk groups: 14 patients (27.5%) were considered at standard risk (SR: MRD less than 0.1%), 9 patients (15.7%) at high risk (HR: MRD 10% or more), and the majority of infants (29, 56.9%) at intermediate risk (IR: MRD 0.1% to 10%). As the 14 SR patients had 3-year EFS and CIR significantly better than other patients, we considered two major groups of patients with different outcome: SR group (MRD<0.1%) with 3-year EFS 77.9% (standard error, SE, 11.3) and CIR 14.9% (SE 10.2), and non-SR group with 3-year EFS 32.0% (SE 8.5) and CIR 58.0% (SE 8.8, p=0.0104 and p=0.0085, respectively). Half of SR group (7 of 14 cases) had germline MLL. 4 out of 7 MLL-positive SR-patients were in continuous complete remission (CCR) In contrast, the majority of infants in the non-SR group carried various types of MLL rearrangements. Only 5 cases in the non-SR group were MLL germline and only two of them are still in CCR. We evaluated the prognostic impact of day 15 MRD in MLL-positive cases (n=39). In this cohort of patients, we also observed a difference, although not statistically significant, between SR and non-SR groups both in 3-year EFS (57.1%, SE 18.7 and 30.9%, SE 9.2, respectively; p=0.3630) and in 3-year CIR (28.6%, SE 18.9 and 60.9%, SE 9.5, respectively; p=0.1733). We evaluated the suitability of MLL negativity and of day 15 FCM MRD <0.1% as single criterion for the identification of low-risk patients. Each factor, when separately analyzed in a Cox model, was significantly correlated with a reduction in the risk of relapse, as shown in Table 1, left panel. Nevertheless, as day 15 FCM MRD levels are strictly related to MLL status, the Cox model which analyzes jointly the two factors, is unable to identify the one independently impacting on the risk of relapse (Table 1, right panel). Thus, although being a strong prognostic factor by itself, day 15 FCM MRD stratification did not confer an advantage in relapse prediction when considered in combination with MLL status, which is the only low-risk group criterion in the Interfant-06 stratification. ConclusionDay 15 FCM MRD proved to be a suitable variable predicting treatment failure and can be used as an alternative or in combination with Interfant-06 stratification criteria to identify SR patients. [Display omitted] Disclosures:Popov:Alexion: Research Funding.

Comparison of High-Throughput Sequencing and Flow Cytometry for Measuring Minimal Residual Disease in Pediatric Acute Lymphoblastic Leukemia: A Children's Oncology Group Cohort

AbstractAbstract 1440 Background:Measurement of minimal residual disease (MRD) during and after induction therapy has emerged as the most important predictor of outcome in pediatric acute lymphoblastic leukemia (ALL). Despite this, over 1/3 of relapses occur in patients who are MRD negative. In addition, ∼50% of children that have detectable MRD do not relapse. The Children Oncology Group (COG) trials use flow cytometry (FC) with a sensitivity of 10−4 for MRD detection and subsequent intensification of therapy in MRD+ patients. A more sensitive tool for monitoring MRD could lead to the identification of more patients who are likely to relapse, while a more specific assay could prevent unwarranted therapy intensification. To this end, we are employing the LymphoSIGHT platform developed by Sequenta Inc., which utilizes high-throughput sequencing for identification of clonal gene rearrangements in the B-cell repertoire and subsequent MRD measurement. In this blinded pilot study (COG AALL12B1), we compared the ability of the sequencing assay to measure MRD to that of FC in diagnostic and post-induction samples from 6 ALL patients. Methods:Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity (D), and joining (J) gene segments from genomic DNA in diagnostic and follow-up bone marrow samples from 6 ALL patients. Amplified products were sequenced to obtain >1 million reads per sample and were analyzed using algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the diagnostic sample. The presence of the tumor-specific clonotype was then monitored in post-induction samples. Absolute quantification was performed by normalizing the patient’s reads to internal reference DNA. We then analyzed concordance between MRD results obtained by sequencing and FC. Results:We detected a high-frequency IgH clonal rearrangement in 5/6 diagnostic ALL samples. MRD was assessed in the 5 post-induction samples from these patients (Table 1). Deep coverage of all MRD samples was obtained, with each original IgH molecule generating ∼20 sequencing reads, ensuring the detection of a single leukemic cell if present in the sample. Leukemic clones were detected in 4/5 follow-up samples (Table 1). In the positive samples, the number of detected leukemic molecules ranged from 12 to over 6,000 and the MRD level ranged from 0.008% to 0.3%. MRD results were concordant with FC in 3 of 5 patients and were consistent with the patient’s clinical courses. In one patient we detected MRD at 0.008%, a level below the sensitivity of FC, which was negative. In another sample, FC detected MRD of 0.01–0.1%, but no leukemic clones were detected by the sequencing assay despite the fact that the sample contained sufficient cell input (almost 2 million cells). The patient remained in continuous remission. Evaluation of additional paired diagnostic and post-induction samples and their association with clinical outcomes is ongoing. Conclusions:We show the application of a high-throughput sequencing method for MRD detection in childhood ALL. IgH clonal rearrangements were detected in 5/6 (83%) of samples using the sequencing assay. The absence of a clonal rearrangement in 1/6 of patients was anticipated and is likely to be mitigated by the presence of a clonal rearrangement in another immunoglobulin or T cell receptor gene. Experiments are ongoing to assess the presence of clonal rearrangements in these receptors (i.e., IgH D-J, IgK, TRB@, TRD@ or TRG@) in the diagnostic samples. In 3/5 patients there was concordance between FC and sequencing-based MRD detection. In one patient, sequencing detected MRD at a level below the threshold of FC. The last patient was negative by sequencing but positive by FC and has not relapsed. Further analysis of the sensitivity and specificity of the sequencing platform compared to FC using additional paired diagnostic and post-induction samples is ongoing.Table 1Comparison of Day 29 (end of induction) MRD results using sequencing and FC methodsSampleNumber of input cellsNumber of input leukemic clone moleculesSequencing MRD (%)Flow cytometry MRD (%)Clinical outcome1162,945120.008<0.01CR25,789,0336,3560.1>0.1Relapse4291,352480.020.01-0.1CR51,901,076000.01-0.1CR61,968,5115,8550.3>0.1CR Disclosures:Faham:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Comparison of Minimal Residual Disease Detection Using High-Throughput Sequencing and Allele-Specific Oligonucleotide PCR Methods in Pediatric B-Lineage Acute Lymphoblastic Leukemia.

AbstractAbstract 2532▪▪This icon denotes a clinically relevant abstract Background:The clinical management of patients with pediatric B-lineage acute lymphoblastic leukemia (B-ALL) relies on combinations of multiagent anticancer drugs and risk-stratified treatment. The prognostic significance of minimal residual detection (MRD) in pediatric B-ALL has been demonstrated in multiple cohorts. Allele-specific oligonucleotide PCR (ASO-PCR) amplification of immunoglobulin or T-cell receptor rearrangements, a method for MRD detection, requires the development of patient-specific reagents and cannot detect clonal evolution. ASO-PCR also has limited coverage, with clonal rearrangements being detected in only 90% of patients. We developed the sequencing-based LymphoSIGHT platform to address these limitations. Here we report the results of a pilot study of MRD detection using both the sequencing assay and ASO-PCR in paired diagnostic and end-of-induction samples from 7 B-ALL patients. Analysis of 82 additional patients is ongoing. Methods:Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity, and joining gene segments from genomic DNA in diagnostic and follow-up bone marrow samples. Amplified products were sequenced to obtain >1 million reads (20× coverage per B-cell), and were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified in the diagnostic sample of each patient based on high-frequency within the B-cell repertoire. The presence of the tumor-specific clonotype was then assessed in the end-of-induction sample. A quantitative and standardized measure of MRD level among all leukocytes in the sample was determined using internal reference DNA. Following identification of IgH clonal rearrangements and MRD assessment using the sequencing assay, we examined the MRD results obtained at Boston Children’s Hospital using ASO-PCR. Among the 7 patients analyzed to date, 6 patients were in complete remission at the time of the second sample; 1 patient had persistent evidence of disease. Sequencing was performed blinded to all clinical and ASO-PCR information on these patients. Results:With the sequencing platform, we detected a high-frequency IgH clonal rearrangement in all 7 diagnostic ALL samples. The leukemic clonotype that was identified at diagnosis was detected in the end-of-induction sample in each of the 7 patients. The quantitative range of the leukemic sequence in MRD samples ranged over 5 orders of magnitude. MRD results were concordant between sequencing and ASO-PCR in 5 of 7 patients. The detected MRD level differed by > 10 fold in 2 patients. In patient 1, sequencing detected high MRD, while low MRD was detected by ASO-PCR; this patient relapsed 1 year later while still on therapy. In patient 6, sequencing detected low MRD, while ASO-PCR detected high MRD. This patient remains in complete remission after 7 years. In patient 7, sequencing and ASO-PCR concordantly detected low MRD; this patient relapsed after completion of therapy. Conclusions:Results from the application of a high-throughput sequencing method for MRD detection in childhood B-ALL are shown. The sequencing assay does not require development of patient-specific reagents, which will reduce cost and laboratory turnaround time. This data, along with the laboratory workflow improvements, support the use of the sequencing assay as a next-generation MRD test for B-ALL. Analysis of samples from 82 patients is ongoing.Table 1Comparison of MRD results using sequencing and ASO-PCR methodsPatientNumber of input cellsNumber of input leukemic clone moleculesSequencing MRD (%)ASO-PCR MRD (%) (High MRD ≥ 0.1%)Clinical outcome1163,33317,1705.30.008Relapse22,043,8609,958,326∼10018.2Induction failure32,052,6512,4640.066.5Alive and well41,298,291670.0010.001Alive and well5984,91810,0360.50.2Alive and well6863,757474,93827.528.1Relapse731,536730.10.03Relapse Disclosures:Faham:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Fang:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Comparative Analysis of Different Approaches to Measure Treatment Response in Acute Myeloid Leukemia

In acute myeloid leukemia (AML), initial treatment response by morphologic analysis of bone marrow predicts long-term outcome. Response can now be assessed by minimal residual disease (MRD) monitoring with flow cytometry or polymerase chain reaction (PCR). We determined the relation among the results of these approaches and their prognostic value. In the multicenter AML02 study, follow-up bone marrow samples from 203 children and adolescents with newly diagnosed AML were examined by flow cytometry (n = 1,514), morphology (n = 1,382), and PCR amplification of fusion transcripts (n = 508). Results were correlated with treatment outcome. Among 1,215 samples with less than 5% leukemic myeloblasts by morphology, 100 (8.2%) were MRD positive (≥ 0.1%) by flow cytometry, whereas 96 (57.5%) of the 167 samples with ≥ 5% blasts were MRD negative. Virtually all (308 of 311; 99.0%) MRD-negative samples by PCR were also MRD negative by flow cytometry. However, only 19 (9.6%) of the 197 PCR-positive samples were flow cytometry positive, with analyses of AML1-ETO and CBFβ-MYH11 accounting for most discrepancies, whereas eight of 13 MLL-positive samples had detectable MRD by flow cytometry. MRD by flow cytometry after induction 1 or 2 predicted lower event-free survival and higher relapse rate (P < .001) and was an independent prognostic factor in a multivariable analysis; prediction was not improved by morphologic information or molecular findings. In childhood AML, morphologic assessment of treatment response has limited value if MRD is measured by flow cytometry. MLL fusion transcripts can provide prognostic information in some patients, whereas monitoring of AML1-ETO and CBFβ-MYH11 transcripts is largely uninformative.

Flow cytometry and IG/TCR quantitative PCR for minimal residual disease quantitation in acute lymphoblastic leukemia: a French multicenter prospective study on behalf of the FRALLE, EORTC and GRAALL

Minimal residual disease (MRD) quantification is widely used for therapeutic stratification in pediatric acute lymphoblastic leukemia (ALL). A robust, reproducible, sensitivity of at least 0.01% has been achieved for IG/TCR clonal rearrangements using allele-specific quantitative PCR (IG/TCR-QPCR) within the EuroMRD consortium. Whether multiparameter flow cytometry (MFC) can reach such inter-center performance in ALL MRD monitoring remains unclear. In a multicenter study, MRD was measured prospectively on 598 follow-up bone marrow samples from 102 high-risk children and 136 adult ALL patients, using IG/TCR-QPCR and 4/5 color MFC. At diagnosis, all 238 patients (100%) had at least one suitable MRD marker with 0.01% sensitivity, including 205/238 samples (86%) by using IG/TCR-QPCR and 223/238 samples (94%) by using MFC. QPCR and MFC were evaluable in 495/598 (83%) samples. Qualitative results (<0.01% or ≥0.01%) concurred in 96% of samples and overall positivity (including <0.01% and nonquantifiable positivity) was concurrent in 84%. MRD values ≥0.01% correlated highly (r(2)=0.87) and 69% clustered within half-a-log(10). QPCR and MFC can therefore be comparable if properly standardized, and are highly complementary. MFC strategies will benefit from a concerted approach, as does molecular MRD monitoring, and will contribute significantly to the achievement of 100% MRD informativity in adult and pediatric ALL.

Leukemia Initiating Cells: New Markers for Minimal Residual Disease Monitoring in B-Precursor Acute Lymphoblastic Leukemia

Abstract 2528 Background:Minimal residual disease(MRD) is currently the most powerful prognostic indicator in childhood ALL as well as in adult ALL. Using neonatal NOD/SCID/IL2rγnull xenotransplantation model, we previously demonstrated that CD34+CD38+CD19+ cells as well as CD34+CD38−CD19+ cells have the capacities to initiate B-ALL in vivo and to self-renew, that is, CD34+CD19+ cells are leukemia initiating cells(LICs) in human B-precursor ALL (B-ALL)(Kong Y et al. Leukemia 2008; 22: 1207–1213). Nevertheless, immunophenotypic differences between B-ALL initiating cells and normal progenitor B cells have not been clearly clarified. Especially, whether the LICs identified in xenotransplantation assay are clinically useful for routine MRD monitoring in B-ALL patients is largely unknown. Objective:To compare phenotypic characteristics of LICs in B-ALL patients to that of normal progenitor B cells in healthy donors. To evaluate clinical significances of LICs as novel MRD markers in predicting relapses in human B-precursor ALL. Materials and methods:To identify new markers for MRD detection in B-ALL, we compared phenotypic characterization of LICs from 40 patients with newly diagnosed B-ALL and 40 patients with relapsed B-ALL to that of normal progenitor B cells from 40 healthy donors by seven-color flow cytometry (FCM). Comparative analysis was performed at mean fluorescence intensity (MFI) of CD38, CD45, CD58 and CD123 on CD34+CD19+ cells among the above three groups. Subsequently, the most promising markers were examined in detail for their usefulness as MRD markers by seven-color FCM at different time points in 823 patients (including pediatric and adult patients) with B-ALL from January 2010 to June 2011 at Peking University Institute of Hematology. A total of 1,050,000 events were routinely collected. More than 0.001% of LICs with aberrant highly expression of CD123 and/or CD58 in bone marrow samples detected by seven-color FCM were defined as FCM positive (FCM+). FCM positivity at any time was defined as MRD positive (FCM MRD+), all others cases were defined as MRD negative (FCM MRD−). Real-time quantitative polymerase chain reaction (RQ-PCR) was applied concurrently to evaluate MRD in Philadelphia chromosome positive ALL (Ph+ ALL) patients. The value of BCR/ABL equal to 0 was defined as RQ-PCR negative (RQ-PCR-). The results of MRD studies by the two methods were recorded independently. Results:MFI of CD58(3.46±1.85 vs.3.82±1.83 vs.1.35±0.77, p<0.001) and CD123(6.37±3.47 vs.7.56±3.90 vs. 3.66±1.94, p<0.001) was significantly higher on LICs from newly diagnosed B-ALL and relapsed B-ALL than that of their normal counterparts. Meanwhile, MFI of CD38(8.09±6.36 vs.11.23±8.59 vs. 37.89±7.91, p<0.05) and CD45(7.42±5.14 vs.9.91±7.72 vs.13.50±8.20, p<0.05) was significantly lower on LICs than that of their normal counterparts. Further ANOVA analysis by LSD method demonstrated MFI of CD38, CD45, CD58 and CD123 on LICs differed significantly from that of normal progenitor B cells, whereas the phenotypic differences on LICs from newly diagnosed B-ALL or relapsed B-ALL showed no statistically significance.In the sequential MRD follow up, 185 samples from110 cases of B-ALL were detected as FCM MRD+. Among them, LICs from 56 cases(51%) with aberrant highly CD58 expression, 106 cases(96%) with aberrant highly CD123 expression and 40 cases(36%) with simultaneously aberrant highly expressions of CD58 and CD123. Twenty-two cases relapsed during MRD monitoring, of them 19 cases were detected as FCM MRD+ at a median time of 57 days (13–90days) before their recurrence. The left 3 relapsed cases did not monitor MRD status for about 3 months as we expected. MRD analysis was performed concurrently by both RQ-PCR and FCM in 85 follow-up bone marrow samples from 23 Ph+ ALL patients. A good correlation was found between RQ-PCR and FCM (Spearman r=0.883, p< 0.001). Conclusions:Mean fluorescence intensity of CD38, CD45, CD58 and CD123 antigens distinguished B-ALL initiating cells from their normal counterparts, CD34+CD19+ normal progenitor B cells. The B-ALL initiating cells with aberrant highly expression of CD123 and/or CD58 detected by seven-color FCM may serve as promising markers for MRD monitoring in B-ALL. Disclosures:No relevant conflicts of interest to declare.

Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods

Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring clone-specific genomic markers (53 follow-up bone marrow samples from 28 children with B-cell precursor acute lymphoblastic leukemia). Cell populations (presumed leukemic and non-leukemic) were flow-sorted during standard flow cytometry-based minimal residual disease monitoring and explored by PCR and/or fluorescence in situ hybridization. We found good concordance between flow cytometry and genomic analyses in the individual flow-sorted leukemic (93% true positive) and normal (93% true negative) cell populations. Four cases with discrepant results had plausible explanations (e.g. partly informative immunophenotype and antigen modulation) that highlight important methodological pitfalls. These findings demonstrate that with sufficient experience, flow cytometry is reliable for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia, although rare cases require supplementary PCR-based monitoring.

Qualitative and Quantitative Concordance of Minimal Residual Disease Data Assessed by Multicolor Flow Cytometry and PCR of Fusion Gene Transcripts In Childhood B-Cell Precursor Acute Lymphoblastic Leukemia

AbstractAbstract 1720Minimal residual disease (MRD) monitoring by flow cytometry (FC) or polymerase chain reaction (PCR) is a strong tool for risk-adapted treatment in childhood acute lymphoblastic leukemia (ALL). Monitoring of IgH/TCR rearrangements by real-time quantitative PCR (RQ-PCR) is well-standardized but very laborious and costly approach. Hence other methods - FC and PCR of fusion gene transcripts (FGt) – can be used for long-term MRD monitoring. Aim.To evaluate qualitative and quantitative concordance between MRD data assessed by FC and PCR of FGt in children with B-cell precursor ALL (BCP-ALL) during treatment. Methods.Concurrent detection of MRD by multicolor FC, RQ-PCR and RT-PCR was performed in 184 follow-up bone marrow samples from 46 children with BCP-ALL. Among them 21 patients (pts) carried ETV6-RUNX1 FGt, 3 pts – TCF3-PBX, while in other 22 pts various types of MLL-rearrangements were detected. 18 pts had CD10(-) and 28 pts – CD10(+) BCP-ALL. 62 samples were obtained during remission induction while 122 – during consolidation/intensification. 6–8-color FC was used. FGt CN was measured by RQ-PCR according to recommendations with normalization to ABL. MRD value was calculated as previously described. Results.Sensitivity of FC MRD detection varied from 10-4 to 10-5. RQ-PCR sensitivity ranged from 5×10-5 to 1×10-5. 65 of 184 samples were MRD-negative by both methods, 13 (7.07%) - were negative by FC but positive by RT-PCR, while in one sample (0.54%) tumor cells were detected by FC but not by PCR. Remaining 105 samples were MRD-positive by both methods. High qualitative concordance (92.39%) between FC and RT-PCR data was found. Similar concordance was observed in MLL-rearranged (91.60%), ETV6-RUNX1-positive (95.45%) and TCF3-PBX-positive (88.89%) cases was observed (p=0.650). Samples with and without normal lymphoid regeneration were analyzed separately, because presence of normal B-cell precursors (BCP) in follow-up samples is known to be an obstacle for FC data analysis. Qualitative concordance in BCP-negative and BCP-positive samples was very similar (92.31% and 92.47% respectively, p=0.814). Concordance of data assessed by two methods in samples obtained during remission induction and during consolidation/intensification was also very close (90.32% and 92.62% respectively, p=0.799). Among remission induction samples high qualitative concordance was observed in both day 15 and day 36 (end of induction therapy) samples (91.30% and 89.19% respectively, p=0.859). These results are in contrast with the previously shown data that at the end of remission induction concordance between FC and molecular techniques is relatively low [G. Gaipa et al, ASH-2008]. As the flow cytometric data analysis in CD10(+) and CD10(-) BCP-ALL patients bases on different approaches, these types of leukemia were also analyzed separately. High qualitative concordance was found in samples from both CD10(+) and CD10(-) BCP-ALL (89.66% and 94.85% respectively, p=0.295). Despite high qualitative concordance between FC and RT-PCR data, low quantitative concordance between FC and RQ-PCR results was found (R2=0.291). Significant quantitative difference in FC and RQ-PCR data could be associated with variability of FG expression during treatment that does not correspond to the cells' number. Moreover, percentage of tumor blasts among all nucleated cells is calculated during FC MRD detection, while MRD value in RQ-PCR of FGt is corresponded to the initial FGt and control gene levels. FC appears to be better for the quantitative MRD assessment however FGt detection by RT-PCR is more appropriate for MRD qualitative detection due to higher sensitivity. Hence, FC is more applicable for MRD monitoring during early treatment phases, when the precise MRD value is essential. In contrast, PCR of FGt is more useful for later time-points where MRD-positivity corresponds to poor outcome. Conclusion.Tandem application of FC and PCR of FGt seems to be a useful tool for long-term MRD monitoring in childhood BCP-ALL. Disclosures:No relevant conflicts of interest to declare.