Heterogeneity of Minimal/Measurable Residual Disease (MRD) Practices in Adult B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) in the United States

Background: MRD testing in BCP-ALL is critical for appropriate patient management, but little is known regarding sample acquisition and testing heterogeneity across clinical practice settings. These factors may impact the quality and reliability of MRD assessment. Methods: Thirty-minute online surveys were conducted in May 2021 with hematologists/oncologists (HEME/ONCs) in the United States in both academic (acad) and community (comm) settings. Respondents were licensed physicians board certified in oncology and/or hematology who treated ≥2 BCP-ALL patients/year or ≥10 patients in the past 5 years, with over 25% of time spent in the clinical setting; pediatric HEME/ONCs were excluded. Survey enrollment is ongoing, with interim results presented here; a related survey for pathologists (PATHs) is underway. Results: HEME/ONC respondents (acad n=40, comm n=57, from 29 states) had been practicing as specialists for a median of between 11-15 years (choices were ranges, eg 6-10, 11-15, min-max was 1-34 years), and typically spent over 75% of their time in the clinic; 94% of respondents had ≥5 BCP-ALL patients/year and 92% ordered MRD tests for ≥5 patients/year. Typical timepoints for MRD testing included the end of induction/suspected complete remission, the end of consolidation, and at suspected disease progression; testing after the end of consolidation was infrequent in both groups (Table). Testing for MRD at the end of consolidation was notably more frequent in the academic setting. In both settings, the HEME/ONC ordering the MRD test generally also performed the bone marrow collection procedure (acad: 78%, comm: 56%). Resources consulted on bone marrow collection best practices included UpToDate (21%), ASH and ASCO (13%), NCCN guidelines (13%), and hematology/oncology journals. About half of practices had defined institutional protocols for bone marrow collection (acad: 55%, comm: 47%), nearly all of which were developed internally. The amount of bone marrow sample collected showed high variability, ranging from 1-10 draws (median=3) and 1-30 mL sample per draw (median=5 mL). While 49% of HEME/ONCs performed <5 draws and extracted ≤6 mL per draw, 22% collected 10 mL/draw, and 10% collected 20 mL/draw; the remaining 18% reported >5 draws and/or >6 mL per draw. In both settings, the first pull was identified and labeled in 35% of procedures; in those cases, the first-pull samples were used primarily for MRD testing in 60% of cases as recommended by NCCN guidelines (vs for morphology assessment and cytogenetic studies). HEME/ONCs typically relied on the expertise of pathologists to choose MRD testing methodology.Survey results indicate that external labs (both national clinical reference labs and commercial labs) were most commonly used for MRD assessments (63%); comm HEME/ONCs were more likely to use external reference labs and acad HEME/ONCs were more likely to use in-house labs. When asked to estimate the frequency with which different MRD methods were used, mean responses were 54% flow cytometry and 40% next-generation sequencing. While all HEME/ONCs indicated that MRD results were presented clearly in lab reports, there was a desire to include more guideline information about MRD interpretation and BCP-ALL treatment. Conclusion: Interim results identified broad heterogeneity in clinical practices affecting sample collection for MRD assessment in Ph- BCP-ALL in the US, indicating several opportunities for harmonization of routine MRD assessment in BCP-ALL. These opportunities include optimization of bone marrow sample collection techniques (volume/draw and identification/use of first pull for MRD), timing/frequency of specimen collection, serial MRD surveillance after consolidation, MRD method chosen, and standardizing reports to include guideline information. There were gaps in awareness of FDA-approved methods of MRD testing for BCP-ALL. Initiatives supporting provider education and harmonization of best practices from professional guideline committees/organizations are needed to optimize outcomes of BCP-ALL patients. Figure 1 Figure 1. Disclosures Hidalgo-Lopez: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Roboz: Janssen: Research Funding; Daiichi Sankyo: Consultancy; MEI Pharma - IDMC Chair: Consultancy; Actinium: Consultancy; AbbVie: Consultancy; Mesoblast: Consultancy; Bayer: Consultancy; Blueprint Medicines: Consultancy; Jazz: Consultancy; Janssen: Consultancy; Astex: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Agios: Consultancy; Astellas: Consultancy; Jasper Therapeutics: Consultancy; Helsinn: Consultancy; Glaxo SmithKline: Consultancy; Novartis: Consultancy; Amgen: Consultancy; AstraZeneca: Consultancy; Otsuka: Consultancy; Pfizer: Consultancy; Roche/Genentech: Consultancy. Wood: Pfizer, Amgen, Seattle Genetics: Honoraria; Juno, Pfizer, Amgen, Seattle Genetics: Other: Laboratory Services Agreement. Borowitz: Amgen, Blueprint Medicines: Honoraria. Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Velasco: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Elkhouly: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Adedokun: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Zaman: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Iskander: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Logan: Amgen, Pfizer, AbbVie: Consultancy; Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding.

Mutational Profiling of Peripheral Blood and Bone Marrow Samples Discriminates Reactive Monocytosis from Chronic Myelomonocytic Leukaemia

Background Chronic myelomonocytic leukaemia (CMML) presents a diagnostic challenge to the haematologist. Distinguishing between a reactive monocytosis and clonal expansion is difficult, and current diagnostic criteria allow for a diagnosis of CMML even in the absence of a clonal marker of disease as long as the monocytosis is persistent. This fails to correctly identify patients with prolonged reactive changes, increasing mis-diagnoses. More recently, large sequencing studies have identified somatic mutations in >90% of patients with CMML, providing potential objective evidence to support a diagnosis. To investigate the utility of high throughput sequencing to discriminate clonal disease and therefore improve diagnosis of CMML, we performed mutational analysis on all samples referred for investigation of a monocytosis to the Haematological Malignancy Diagnostic Service (HMDS) over a 2 year period. The aim of this study was to determine the frequency of mutations in this patient group and whether the presence of mutations can predict disease and outcome. Methods Samples from all patients (initial and follow-up) referred to HMDS with a monocytosis or suspected CMML between July 2014-July 2016 were included in the study. Those with a previous history of a myeloid malignancy diagnosed before July 2014 were excluded. 377 samples from 297 patients were processed and reported according to using current gold standard techniques, with targeted sequencing of 27 recurrently mutated genes in myeloid malignancies performed in parallel. Extracted DNA was sequenced using an Illumina MiSeq and analysed using an in-house pipeline. Detected variants were reported down to a minimum variant allele fraction of 5% and coverage of 100X. Low level variants were confirmed by repeat sequencing and SRSF2 regions were infilled using Sanger sequencing. Data from the literature as well as public online databases (dbSNP, COSMIC, ClinVar) and Alamut Visual were evaluated to annotate likely pathogenic variants. Results Of the164 patients who presented with an initial bone marrow sample, 95 had a confirmed diagnosis of CMML, of which 93 had a demonstrable mutation (98%). The spectrum of mutations in this group reflects that reported in the literature with TET2, SRSF2 and ASXL1 being most frequently mutated. A further 15 patients, all with mutations, were diagnosed with an alternative myeloid malignancy. In those without a confirmed diagnosis by conventional means, a somatic mutation was detected in 62% (39/54) of cases. Importantly, those with a mutation had both phenotypic and genotypic features indistinguishable from the CMML group. In particular CD56 overexpression by immunophenotyping was found almost exclusively in patients with a mutation whether a diagnosis was confirmed or not. To date, a follow-up sample has been received from 7 mutation-positive patients, of which 5 were diagnostic. All 5 cases had identical mutational profiles in the paired samples. In 133 patients, a peripheral blood sample was received as the initial specimen and 71% (94/133) of these harboured a mutation. As yet, 94 patients have not had a subsequent bone marrow biopsy so a conventional diagnosis has not been made. In those with a follow-up bone marrow sample (n=39), the mutational profile between paired samples was found to be highly concordant (98%). In addition, the detection of a mutation in the peripheral blood was strongly predictive of a myeloid malignancy in the bone marrow. Of the 30 patients with a detectable mutation, 29 had a confirmed diagnosis, including 20 patients with CMML, 5 with AML and 4 with other chronic myeloid malignancies. The remaining 9 patients without a mutation showed no morphological evidence of disease in the bone marrow (sensitivity 96.7%; CI 82.8%-99.9%, specificity 100%; CI 66.4%-100%). Conclusion This study has shown that patients investigated for a monocytosis commonly harbour somatic mutations which can be detected in the peripheral blood at a high frequency and with high confidence. The presence of a mutation correlates strongly with a CMML phenotype and detection in the peripheral blood is strongly predictive of a bone marrow diagnosis. Screening of peripheral blood samples using a targeted gene panel provides a highly effective tool to diagnose CMML. Follow-up of this patient group is ongoing and updated results will be available for presentation at the meeting. Disclosures Cargo: Celgene: Honoraria, Research Funding; Novartis: Honoraria.

A High Throughput Exome Sequencing Approach To Analyse Events Within a Good Responder CML Patient Under Imatinib At Diagnosis and Under Remission

Background Genome sequencing has emerged recently as a technology that can be used to address questions regarding the clonal evolution of cancers. This has the potential to be translated into practical applications in a clinical setting. We have undertaken a case study by carrying out whole exome sequencing of a patient with CML since its clinical outcome varies amongst patients during the progression of disease. Our study is an attempt for a better understanding of why patients differ in their response to different dose of drug regimen and to determine its role in clinical outcome. Our case involves a patient with BCR–ABL positive CML. She has been responding to Imatinib, at 200 mg once a day, a dosage lower than the recommended 400mg /day. She has been taking 200 mg / day irregularly for two years and there were frequent interruptions of regular dosage schedule because of severe symptomatic cytopenias requiring blood transfusions. At the end of 2 years, there has been no progression of disease. Her bone marrow aspirate and biopsy have been normal and the BCR-ABL transcript has been below detectable levels. It was by serendipity that we happened to study the whole exome of this patient before we started her on Imatinib. As her dose response was erratic, we decided to do a longitudinal study. We examined the whole exome of the patient with CML at different points during the progression of disease. The aim of this study is to identify novel polymorphisms or variants which might be associated with the case, there by resisting the disease to progress. We were interested in the process of understanding the variability of the clinical outcome to standard treatment from a genomics perspective. Results The exome sequencing of the bone marrow aspirate was performed at the time of diagnosis and two years post treatment with Imatinib. Matched skin biopsy was used as a control. This study has been approved by the Institutional Ethical Review Board of St. John's Medical college and Hospital. The SNVs of the skin data were used as the control in this experiment to account for germ line mutations. We observe severe genome instability in terms of single nucleotide variants (SNVs) at the time of diagnosis. The counts of SNVs are observed to be drastically reduced in the treated bone marrow sample than at the time of diagnosis. No insertions or deletions were observed in this longitudinal case study. It was also observed that the SNV counts were not dominated in any particular chromosome. The SNVs picked up by exome sequencing were hence contrasted at the stage of diagnosis and post treatment. These SNVs were compared to dbSNP to retain only those which are novel and not previously reported. This finally gave rise to three discreet sets to consider: 1). The SNVs present in the genome of the patient at diagnosis 2). Post treatment, and 3). a subset common to both of them. The non-synonymous coding mutations with single base substitutions were 1,08,436 at the time of diagnosis and reduced to 163 in a stage of remission. The overlaps between these two categories were 169 in number. These hits in respective categories were further filtered based on the score of SNV call, relevance to the disease, presence within domain boundaries and final predicted impact on the function of the coded protein. We have identified novel mutations within the ABL, BCR, Kit and NOTCH genes with high probability of impact on function in the bone marrow sample at diagnosis. These will further be validated experimentally to be confirmed as a probable marker for screening patients. This study will be extended to screen a cohort of patients with similar prognosis as the patient in our case study. The novel mutations will be mapped on the protein structure to show which functional domains could be affected and to provide a structural basis for the deleterious effects of such mutations. We hence attempt to understand this clinical case with an integrated view involving basic experimental biology, bio-informatics and structural biology. These polymorphisms would provide insights on the evolution of CML and eventually allow us to use such readouts as a screen across CML patients to identify cases with better disease prognosis. With the distinct population groups and diverse spectrum of diseases, we believe that our work lays the foundation for larger studies in both CML and other diseases requiring such approaches. Disclosures: No relevant conflicts of interest to declare.

Deep Digital Sequencing Identifies an AML Subclone with Enhanced in Vitro and in Vivo Growth Properties Associated with Disease Relapse

Abstract 407 Acute myeloid leukemia (AML) is a biologically heterogenous malignancy of hematopoietic cells. All AML samples are comprised of a founding clone and usually one or more subclones that are derived from the founding clone; subclones can gain or lose mutations as they evolve from the founding clone, and often become dominant at relapse1. The clonal architecture of an AML sample can be identified using single nucleotide variants (SNVs) that cluster according to discrete variant allele frequencies (VAFs). To accurately identify clusters with common VAFs, deep digital sequencing must be performed using all of the SNVs present in each genome (hundreds of events). In this report, we studied the subclonal architecture of AML samples from UPN 452198, collected from a 55-year old woman with normal karyotype acute monocytic leukemia (FAB M5) with a high peripheral WBC count (72,700/mm3) at presentation. This bone marrow sample contained a founding clone and 3 subclones at presentation. The SNVs of the founding clone had a mean VAF of 46.4% (i.e. heterozygous mutations found in 92.8% of the cells in the bone marrow sample), including DNMT3A R882H and NPM1 W288 frameshift mutations. The mean VAFs of Subclones 1, 2 and 3 were 31.2%, 12.0% and 2.4%, respectively, and they contained all of the variants in the founding clone, along with additional variants, most notably FLT3 D835H and IDH1 R132H mutations in Subclone 1. The tumor at relapse consisted entirely of Subclone 3, which also contained 42 relapse-specific variants. We designed an oligonucleotide capture reagent to track all 118 de novo and relapse-specific variants, and obtained deep read counts (mean coverage per site: 412 reads/SNV) on the de novo AML sample under different experimental and biological conditions, as follows: 1) We showed that peripheral blood and bone marrow leukemia samples obtained at the same time had nearly identical clonal architectures. We verified this correlation using 4 additional AML samples, suggesting that clonal architecture is preserved in the peripheral blood for many AML samples. 2) We flow-sorted the leukemic peripheral blood sample into blasts, monocytes, and lymphocytes based on side-scatter characteristics and expression of CD45 and CD33. The founding clone and all three subclones were detected in the monocyte population, which was the predominant leukemic cell population in the peripheral blood. By flow cytometry, blasts comprised only 3.3% of the cells, but were strongly enriched for variants in Subclone 3 (mean VAF in sorted blasts 33.9% versus 3.0% in unsorted peripheral blood, p<0.001). Purified lymphocytes, in contrast, contained no leukemia-specific variants, implying that the founding clone for this sample did not contribute to lymphopoiesis. 3) We tested the growth properties of subclones in the de novo sample in vitro and in vivo. We injected 1 million cells from the de novo AML sample into 3 immunodeficient NSG mice, and harvested human AML blasts (co-expression of human CD45 and CD33) 14 weeks later. Although two mice engrafted with the founding clone and Subclone 1 (which comprised the vast majority of the cells in the sample), the 3rd mouse had a tumor composed entirely of the relapse-specific Subclone 3 (which accounted for only 2.4% of the variants in the de novo sample), suggesting that this subclone had a significant growth or engraftment advantage in vivo. In support of this observation, de novo AML cells were strongly enriched for Subclone 3 when grown in the presence of hematopoietic cytokines (SCF, IL3, IL6, TPO and FLT3L) for 7 days on HS27 stroma (VAF at day 7–19.7%; p<0.001) or MS5 stroma (VAF at day 7–22.8%; p<0.001), implying that this clone also had a strong in vitro growth advantage. In summary, a small subclone of AML cells at presentation — that was known to eventually contribute to relapse — had unique growth properties that were revealed using deep digital sequencing of all variants. This approach has allowed us to dissect the evolutionary history of AML clones, and to define their relationship to other hematopoietic cells in a given sample. Similar studies on additional AML samples should allow us to define the mutational profiles of subclones that are destined to contribute to relapse. This data will be essential for improving therapeutic approaches for AML patients. Disclosures: Ley: Washington University: Patents & Royalties.

Cell Separation System for Fully Automated Clinical Scale Separation of CD133+ Cells From Bone Marrow Aspirates.

Abstract 1188 An increasing number of clinical trials are enrolling patients in studies designed to examine the safety and efficacy of autologous stem cells for cardiac repair. Recent reports suggest that patients receiving CD133+ bone marrow cells after myocardial infarction, or as a treatment for ischemic cardiomyopathy, may benefit from an increase in global left ventricular function. Today the clinical scale enrichment of CD133+ cells has to be performed as a complex procedure involving numerous manual handling steps as well as a semi-automated magnetic separation process. We have developed a fully automated clinical scale process to purify CD133+ cells out of human bone marrow aspirates. The whole process was performed in a closed system, containing appropriate adaptors and tubing material, suitable for sterile connection of the bone marrow sample and required solutions, respectively. For the whole separation process, the total processing time was reduced from about 4.5 h (previous process) to 2.5 h. In this context, erythrocyte reduction, generation of autologous plasma, labeling time as well as the conditions for immunomagnetic separation of the CD133+ cells and the automatic monitoring of the whole process by a newly developed camera were optimized. To determine the reproducibility and stability of the process, CD133+ cells were separated from bone marrow aspirates with an initial volume of about 60 mL (n=10). The intitial frequency of CD133+ cells amounted to 0.34% (range: 0.11% to 0.66%) and the number of isolated CD133+ cells was 7.9×105 (range: 3.7×105 to 1.9×106). The yield was 47% (range: 23.9% to 50.9%) and the average viability of the separated CD133+ cells achieved 90% (range: 69.9% to 96.9%). The separation process typically achieved a >3.0 log depletion of CD133 negative cells, i.e. 99.9% of CD133 negative cells were removed. The log depletion of different cell types were: 4.0 for CD3+ cells (i.e. 99.99% removal), 3.1 for CD19+ cells, 3.4 for CD56+ cells, 3.2 for CD14+ cells, and 3.7 for CD15+ cells (n=3, respectively). After separation the CD133+ cells were automatically resuspended in 6 mL of clinical grade isotonic NaCl solution. For storage or transport of the cells, the NaCl solution could be automatically supplemented with 10% autologous plasma, generated out of the bone marrow sample during the separation process. The described cell separation system provides a safe and easy way to purify CD133+ cells from bone marrow aspirates within 2.5 h without any intermediate manual steps. The cell preparation in a closed sterile system facilitates a fast and robust enrichment of CD133+ cells. After separation the CD133+ cells are available in small volume and can be formulated for further use e.g. according to requirements for use in regenerative medicine. Disclosures: Essl: Miltenyi Biotec GmbH: Employment. Stuth:Miltenyi Biotec GmbH: Employment. Huppert:Miltenyi Biotec GmbH: Employment. Balshüsemann:Miltenyi Biotec GmbH: Employment. Bauer:Miltenyi Biotec GmbH: Employment. Miltenyi:Miltenyi Biotec GmbH: Employment.

Correlation between prognosis and bone marrow chromosomal patterns in children with acute nonlymphocytic leukemia: similarities and differences compared to adults

The chromosomal complement of 28 children with the diagnosis of acute nonlymphocytic leukemia (ANLL) were examined. An abnormal cytogenetic pattern was found in 50% of these patients, which is similar to the results in adults with ANLL. Unlike the reports in adult patients, however, no specific chromosomal changes were found. This observation may imply that the etiology and mechanisms by which abnormal clones develop in ANLL could differ significantly between children and adults. Those patients with chromosomal abnormalities in their initial bone marrow sample had a median survival of 7.1 mo, whereas those with a normal diploid pattern in their bone marrow had a median survival of 20.5 mo (1-sided, p = 0.04). If all metaphases were abnormal, the median survival was only 3 mo.