Healthcare Resource Utilization in Adult Patients with B-Cell Precursor Acute Lymphoblastic Leukemia in First Hematologic Complete Remission with and without Minimal Residual Disease

Abstract Introduction: B-cell precursor acute lymphoblastic leukemia (BCP ALL) is a rare and debilitating hematologic cancer. Among BCP ALL patients in hematologic complete remission (CR), minimal residual disease (MRD) is one of the most important prognostic factors predicting worse survival outcomes. However, to date, limited data in the U.S. have been available to characterize healthcare resource utilization (HRU) in BCP ALL patients with and without MRD. In the present study, a chart review was conducted to assess HRU as measured by the frequency of inpatient stays and outpatient visits among adult BCP ALL patients (MRD-positive and MRD-negative) in first hematologic CR. The chart review study was conducted across National Cancer Institute (NCI) designated cancer centers, academic and community hospitals, and integrated delivery systems. Methods: U.S. hematologists and oncologists were invited to complete an 89-item online case report form, and provided data on patient demographic and disease characteristics, MRD assessment, and HRU over the study period. The study period was defined as the date of first hematologic CR (index date) until the earlier of 12 months post-CR, relapse, or death. Eligible patients were ≥ 18 years of age at diagnosis of BCP ALL, achieved first hematologic CR between April 1, 2015 and March 30, 2017, had no diagnoses for other cancer types, and had chart information available for the entire study period. Patients were characterized based on MRD history over the entire study period. This abstract reports HRU among patients who are in first hematologic CR with MRD, not including care associated with stem cell transplantation. The number of inpatient (IP)/emergency room (ER) days per month (mean) was calculated based on physician-reported dates of admission/discharge and the follow-up period (months) after the first MRD test. The number of outpatient (OP) visits per month was calculated based on the physician-reported number of acute care OP visits and the follow-up period (months) after the first MRD test. Results: Sixty-six U.S. physicians completed the online questionnaire, providing data from a total of 126 patient charts meeting the eligibility criteria. Five patient charts were excluded from the analysis due to inconsistent or missing MRD assessment data. On average, the 63 physicians that provided eligible charts included in the analysis reported being in practice for 13.2 years and half of them have been treating ≥20 adult patients with BCP ALL in first hematologic CR in the last 12 months. At the index date, mean patient age was 48.5 years, 72.7% were male, 74.4% were Caucasian, and 25.6% were Philadelphia chromosome positive. Of the 121 patients included in the analysis, 82 patients were MRD-positive at the first MRD test (42 patients were persistent MRD-positive, 40 subsequently became MRD-negative). The number of IP/ER days per month (mean ± standard deviation [SD]) was 1.90 (± 4.92) vs. 0.52 (± 1.26) and the number of acute care OP visits per month was 0.13 (± 0.44) vs. 0.09 (± 0.37) for patients who were persistent MRD-positive vs. patients who subsequently became MRD-negative (Table 1). Conclusions: This chart review study demonstrated that among adult BCP ALL patients in first hematologic CR with MRD, patients with persistent MRD had more inpatient stays and outpatient visits than those who subsequently became MRD-negative. New therapies that can eradicate MRD among adult ALL patients in hematologic CR may have some cost offsets from the decreased HRU associated with MRD-negative status. Disclosures Rose: Analysis Group, Inc.: Employment; Amgen, Inc.: Consultancy. Shah:Amgen, Inc.: Employment, Equity Ownership. Despiegel:Amgen, Inc.: Employment, Equity Ownership. Tibrewala:Analysis Group, Inc.: Employment; Amgen, Inc.: Consultancy. Chawla:Amgen, Inc.: Consultancy; Analysis Group, Inc.: Employment, Equity Ownership. Cong:Amgen, Inc.: Employment, Equity Ownership.

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Deep Sequencing Approach for Minimal Residual Disease Detection in Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.1388.1388 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1388-1388

Author(s):

Malek Faham ◽

Jianbiao Zheng ◽

Martin Moorhead ◽

Victoria Carlton ◽

Patricia Lee Stow ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Research Funding ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Clonal Evolution ◽

Employment Equity ◽

Equity Ownership ◽

Minimal Residual

Abstract Abstract 1388 Background: The clinical management of patients with acute lymphoblastic leukemia (ALL) relies on accurate prediction of relapse hazard to determine the intensity of therapy and avoid over- or under-treatment.1 The measurement of minimal residual disease (MRD) during therapy has now emerged as the most important predictor of outcome in ALL.2 We developed the LymphoSIGHT platform, a high-throughput sequencing method, which universally amplifies antigen-receptor gene segments and can identify all leukemia-specific sequences at diagnosis, allowing monitoring of disease progression and clonal evolution during therapy. In this study, we determined the sensitivity and specificity of this method, delineated the extent of clonal evolution present at diagnosis, and compared its capacity to measure MRD to that of flow cytometry and allele-specific oligonucleotide PCR (ASO-PCR) in follow-up samples from >100 patients with ALL. Methods: Using the sequencing assay, we analyzed diagnostic bone marrow samples from 100 ALL patients for clonal rearrangements of immunoglobulin (IgH@) and T cell receptor (TRB@, TRD@, TRG@) genes, as well as the extent of clonal evolution present at diagnosis. We assessed the capacity of the sequencing assay to detect MRD using diagnostic samples from 12 ALL patients carrying 13 leukemic IgH clonal rearrangements. Serial dilutions were prepared in normal peripheral blood mononucleated cells, at a range between <1 in 1 million to >1 in 1,000 cells. We also assessed MRD in follow-up samples from 106 ALL patients and analyzed concordance between MRD results obtained by the sequencing assay, flow cytometry and ASO-PCR. Results: In diagnostic bone marrow samples, we detected the presence of a high-frequency clonal rearrangement of at least one receptor (“calibrating receptor”) in all the 100 ALL samples; 94 samples had at least 2 calibrating receptors at diagnosis, with 51 having 3 or more. We also detected a variable degree of clonal evolution: the number of evolved clones in each sample ranged from 0 to 6933, with 39 (37%) samples having 1–50 evolved clones and 17 (16%) >50 (Figure 1). In experiments with mixtures of normal and leukemic cells, the sequencing assay unequivocally and accurately detected leukemic signatures in all dilutions up to a concentration of at least one leukemic cell in 1 million leukocytes. In direct comparisons with established MRD assays performed on follow-up samples from patients with B-ALL, sequencing detected MRD in all 28 samples positive by flow cytometry, and in 35 of the 36 positive by ASO-PCR; it also revealed MRD in 10 and 3 additional samples that were negative by flow cytometry and ASO-PCR, respectively (Figure 2). Conclusions: The sequencing assay is precise, quantitative, and can detect MRD at levels below 1 in 1 million leukocytes (0.0001%), i.e., represents sensitivity 1–2 orders of magnitude higher than standard flow cytometric and ASO-PCR methods. Our assay also allows monitoring of all leukemic rearrangements regardless of their prevalence at diagnosis, which abrogates the risk of false-negative MRD results due to clonal evolution. Finally, the sequencing assay utilizes a set of universal primers and does not require development of patient-specific reagents. These data, together with the results of our comparison with standard MRD assays in clinical samples, strongly support the use of the sequencing assay as a next-generation MRD test for ALL. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Research Funding. Zheng:Sequenta: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta: Employment, Equity Ownership, Research Funding. Carlton:Sequenta: Employment, Equity Ownership, Research Funding.

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Treatment of Adults with Minimal Residual Disease (MRD) Positive Acute Lymphoblastic Leukemia with Blinatumomab in a Real-World Setting: Results from the Neuf Study

Blood ◽

10.1182/blood-2019-122931 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2624-2624

Author(s):

Nicolas Boissel ◽

Renato Bassan ◽

Josep-Maria Ribera ◽

Sabina Chiaretti ◽

Robin Foà ◽

...

Keyword(s):

Board Of Directors ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Employment Equity ◽

Advisory Committees ◽

Expanded Access ◽

Expanded Access Program ◽

Equity Ownership ◽

Access Program

Introduction: In November 2015, conditional approval of blinatumomab was granted for adults with relapsed and/or refractory Philadelphia chromosome-negative B-cell precursor acute lymphoblastic leukemia (R/R Ph- BCP-ALL). Prior to country-specific reimbursement, blinatumomab was made available to patients (pts) who met pre-specified criteria via an expanded access program in specific countries: this included both adults and pediatric pts with diagnosis of R/R Ph- BCP-ALL, R/R Ph+ BCP-ALL, or minimal residual disease (MRD)-positive Ph-/Ph+ ALL. Here, we report on adults with MRD+ BCP-ALL enrolled in this expanded access program with reference to their characteristics and both blinatumomab usage and effectiveness. Methods: The retrospective observational study (NEUF) recruited pts who initiated blinatumomab in the available expanded access setting between 2014 and 2016. Pts were followed from blinatumomab initiation until death, entry into a clinical trial, end of follow-up, or the end of the study period (30 June 2017), whichever occurred first. Efficacy analyses were undertaken on a MRD intention-to-treat basis. MRD response was defined as MRD level <10-4 within first cycle and within the first 2 cycles. MRD assessment was undertaken as per local clinical practice, including flow cytometry and polymerase chain reaction (PCR): MRD status was then extracted from the patient medical record. Median disease-free survival (DFS) was defined as time from initiation of blinatumomab until date of relapse (blasts in bone marrow >5% or extramedullary relapse after documented response) or death, whichever occurred first. Adverse events were reported separately, according to local regulations. Results: Out of 373 enrolled pts, 109 MRD positive adult ALLs (83 Ph-; 26 Ph+) were included in Italy (53), France (23), Spain (20), Russia (11), and the UK (2). Most patients (76%, n=83) were Ph-. Forty-one percent (n=45) were female and median age was 43 years (interquartile range [IQR]: 27, 55). In their medical history, 16% (n=17 out of 109 MRD positive patients) had a prior allogeneic hematopoietic stem cell transplant (HSCT). The median number of prior salvage therapies was 0 (IQR: 0.0, 1.0). Among blinatumomab co-medications, almost 90% were treated with dexamethasone, 35% (n=36) as pre-phase and 87% (n=92) as pre-medication. Of the 82 pts with evaluable MRD within two cycles of blinatumomab, 83% (n=66) had a MRD response (Table), including 48 with non-detectable MRD and 18 with MRD <10-4. Following blinatumomab initiation, 68% (n=74 out of 109) of pts proceeded to HSCT: 65 (88%) of these patients had documented complete remission with full/partial/incomplete recovery of peripheral blood counts before transplant. The median time from complete response (CR) to HSCT was 2.4 months (range: 1.6, 5.3), with median follow-up time being 18.5 months (IQR: 14.0, 27.7). The median DFS was 27.6 months (IQR: 7.4, not estimable [NE]). Censoring for HSCT increased DFS to 33.0 months (IQR: 8.9, NE). At 24 months following blinatumomab initiation, overall survival ((OS) was 65% (95% confidence interval [CI]: 52.8, 74.2): when censoring for HSCT, OS was 77.6% (95% CI: 52.8, 88.9); median follow-up time was 4.0 months (IQR: 2.6, 13.7). The Kaplan-Meier estimate of the non-relapse mortality following HSCT post-blinatumomab was 6% (95% CI: 1.9, 16.4) at 3 months and 10% (95% CI: 4.6, 22.3) at 12 months. Conclusions: In this large multi-country, multi-site study, blinatumomab was shown to induce molecular response within two cycles in the majority of patients with evaluable MRD. Furthermore, the median DFS was over two years, while two-thirds of pts were still alive 24 months after initiation. This study demonstrates the real-world effectiveness of blinatumomab and is consistent with results from clinical studies (BLAST). Disclosures Boissel: NOVARTIS: Consultancy. Bassan:Shire: Honoraria; Incyte: Honoraria; Amgen Inc.: Honoraria; Pfizer: Honoraria. Chiaretti:Pfizer: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Foà:Celltrion: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees. Papayannidis:Novartis: Honoraria; Incyte: Honoraria; Teva: Honoraria; Shire: Honoraria; Amgen: Honoraria; Pfizer: Honoraria. Alam:Amgen: Employment, Equity Ownership. Brescianini:Amgen: Employment, Equity Ownership. Pezzani:Amgen: Employment, Equity Ownership. Kreuzbauer:Amgen: Employment, Equity Ownership. Rambaldi:Celgene: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

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Report of a Phase II Trial of Single-Agent BiTE® Antibody Blinatumomab in Patients with Minimal Residual Disease (MRD) Positive B-Precursor Acute Lymphoblastic Leukemia (ALL).

Blood ◽

10.1182/blood.v114.22.840.840 ◽

2009 ◽

Vol 114 (22) ◽

pp. 840-840 ◽

Cited By ~ 5

Author(s):

Max S Topp ◽

Gerhard Zugmaier ◽

Nicola Goekbuget ◽

Peter Kufer ◽

Mariele Goebeler ◽

...

Keyword(s):

T Cells ◽

Phase Ii ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Target Cells ◽

Molecular Response ◽

Intensive Chemotherapy ◽

Employment Equity ◽

Equity Ownership

Abstract Abstract 840 Introduction: In patients with B-precursor ALL, presence of MRD after induction therapy or at any time point later predicts a hematological relapse, despite continued intensive chemotherapy or/and an allogeneic hematological stem cell transplantation (HSCT). Blinatumomab (MT103) targets the CD19 antigen, and is a member of a novel class of bispecific BiTE® antibodies that redirect T cells for lysis of target cells. A phase II study was conducted in collaboration with the German Multicenter Study Group on Adult Lymphoblastic Leukemia (GMALL) in patients with MRD-positive B precursor ALL. Methods: B–precursor ALL patients in complete hematological remission with either persistent or reappeared MRD at any time after consolidation I of front-line therapy were included. One treatment cycle of blinatumomab is a 4-week continuous i.v. infusion, which can be followed by allogeneic HSCT or in case of response by repeated consolidation cycles of blinatumomab with 2-week treatment-free intervals. The dose level at enrollment is 15 μg/m2/day. In patients, who do not respond within four cycles of treatment, the dose can be increased to 30 μg/m2/day. Molecular response is assessed by quantitative PCR of either individual rearrangements of immunoglobulin/TCR-genes or specific genetic aberrations such as bcr/abl or MLL-AF4. Results: Nineteen patients have been treated to date and 16 patients are already evaluable for response. Thirteen of 16 evaluable patients went into molecular complete remission (CR) already after one cycle of blinatumomab. Three patients had a stable MRD level. Of note, 10 of the responding 13 patients had never achieved a molecular CR before blinatumomab treatment despite multiple treatment cycles including tyrosine kinase inhibitors in case of Ph-positive ALL. Two patients in molecular CR had an extra-medullary relapse one in testis and one in cerebro-spinal fluid, both representing immunological niches with limited accessibility for T cells. One patient with stable MRD level had a medullary relapse. All other patients are still relapse-free. None of the patients with molecular CR has shown a medullary relapse to date. The maximum follow-up of molecular CR has been 12 months. Most common adverse events (AEs) included lymphopenia, pyrexia, leucopenia and hypoimmunoglobulinemia. Only one patient had to be discontinued because of a fully reversible epileptical seizure. All other AEs resolved during treatment. Overall, treatment with blinatumomab was well tolerated. Response data of all 21 patients will be presented at ASH. Conclusions: Treatment with blinatumomab converted MRD-positive B–precursor ALL into molecular CR in 13 of 16 evaluable patients with refractory disease as indicated by persistent MRD after intensive chemotherapy. This amounts to a response rate of 81% providing thus the rationale for introducing blinatumomab as a novel agent in the treatment of B–precursor ALL. Disclosures: Zugmaier: Micromet: Employment, Equity Ownership. Goekbuget:Micromet: Consultancy, Research Funding. Kufer:Micromet: Employment, Equity Ownership, Patents & Royalties. Klinger:Micromet: Employment, Equity Ownership. Degenhard:Micromet: Employment, Equity Ownership. Baeuerle:Micromet: Employment, Equity Ownership. Schmidt:Micromet: Employment, Equity Ownership. Nagorsen:Micromet: Employment, Equity Ownership. Riethmueller:Micromet: Consultancy, Equity Ownership. Bargou:Micromet: Consultancy, Equity Ownership, Patents & Royalties.

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Comparison Of Deep Sequencing and Allele-Specific Oligonucleotide PCR Methods For MRD Quantitation In Acute Lymphoblastic Leukemia and Mantle Cell Lymphoma: CALGB 10403 and CALGB 59909 (Alliance)

Blood ◽

10.1182/blood.v122.21.2547.2547 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2547-2547 ◽

Cited By ~ 1

Author(s):

Greg Malnassy ◽

Susan Geyer ◽

Noreen Fulton ◽

Greg Koval ◽

Donna Niedzwiecki ◽

...

Keyword(s):

High Frequency ◽

Lymphoblastic Leukemia ◽

Cell Receptor ◽

Employment Equity ◽

Sequencing Platform ◽

Sequencing Method ◽

Mantle Cell ◽

Allele Specific ◽

Equity Ownership

Abstract Background The assessment of minimal residual disease (MRD) is a key component of prognosis and monitoring in acute lymphoblastic leukemia (ALL) and mantle cell lymphoma (MCL). Allele-specific oligonucleotide (ASO)-PCR can be used to assess MRD; however, this technique requires preparation of clonotype-specific primers for each patient, which is laborious and time-consuming. We demonstrated the utility of sequencing-based MRD assessment in ALL (Faham et al., Blood 2012). This quantitative approach relies on amplification and sequencing of immunoglobulin and T-cell receptor gene segments using consensus primers and can address some of the limitations associated with traditional MRD detection. Here we compared the ability of the sequencing and ASO-PCR methods to identify clonal cancer gene rearrangements at diagnosis and evaluated the concordance of MRD detection in bone marrow (BM) or blood samples from 37 patients (pts) with ALL and 22 pts with MCL entered onto prospective CALGB treatment trials, 10403 and 59909 (Alliance), respectively . Methods Using the quantitative ASO-PCR and sequencing assays, we analyzed diagnostic blood and BM samples from ALL pts for clonal rearrangements of immunoglobulin (IGH-VDJ, IGH-DJ, IGK) and T cell receptor (TRB, TRD, TRG) genes. We then assessed MRD at the IGH and/or TRG locus in 84 follow-up samples from ALL pts. Similarly, we analyzed samples from 22 MCL pts for immunoglobulin (IGH-VDJ, IGH-DJ, IGK) clonal gene rearrangements and measured MRD at the IGH and/or IGK locus in 114 follow-up samples. Sensitivity of the ASO-PCR vs sequencing was 1 X 10 4-5 vs 1 X 106, respectively. Concordance between sequencing and ASO-PCR MRD assessment on serial samples collected during and post-treatment was evaluated across all pts and within disease groups using concordance correlation coefficients for repeated measures (CCC-RM). Concordance in identification of detectable vs. undetectable MRD by both methods was also evaluated using Kappa statistics. Results Using the sequencing platform, high frequency clonal rearrangements were observed in at least two receptors in 97% and 95% of pts with ALL and MCL, respectively. Selected ALL samples were known to have IGH-VDJ or TRG clonal rearrangements by ASO-PCR; however, sequencing revealed additional clonal rearrangements in 36/37 (97%) pts with ALL. Good concordance was observed with identification of MRD positive vs. negative between the methods (K=0.62; p<0.0001), where better agreement was observed within the ALL (K=0.63) than within the MCL (K=0.59) cohorts. Across both diseases, the sequencing method proved a more sensitive measure and CCC-RM was 0.82, reflecting the fact that 28 pts with undetectable MRD by ASO-PCR methods had low detectable MRD measures by the sequencing approach. The sequencing platform also provided new insights into kinetics of relapse. In one ALL case (Fig 1), we observed three high frequency IGH-VDJ clones, two of which are related based on VDJ replacement model. Dramatic reduction in frequency of one clone (clone 3, Fig 1) was observed following initial induction chemotherapy in both BM and blood. Only this single clone was monitored using ASO-PCR, and the patient appeared to be in molecular remission. However, the other two high frequency clones were chemo-resistant, and sequencing MRD monitoring revealed no reduction in clone frequency in either the BM or blood. The patient experienced early relapse at day 56, which may have resulted from the expansion of a clone carrying one or both of the clonal sequences that was not monitored by ASO-PCR. Thus, the sequencing method can be used to monitor response to treatment at the individual clone level. Conclusions This study demonstrates concordance between identification of detectable MRD in ALL and MCL by sequencing and ASO-PCR Methods. The sequencing approach offers improvements over ASO-PCR including the ability to monitor multiple clonal sequences, faster turnaround time (results in 1 week), and greater sensitivity. The clinical significance of this greater sensitivity remains to be tested prospectively and must be correlated with clinical results. Nevertheless, the sequencing method represents an alternative approach for clinical MRD monitoring which could fundamentally improve the ability to monitor disease progression and recurrence in patients with lymphoid malignancies. Disclosures: Carlton: Sequenta, Inc.: Employment, Equity Ownership. Weng:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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Time Points, Dynamics and Clinical Impact of Minimal Residual Disease (MRD) Monitoring in AML with NPM1 mutation: A Study on 428 Patients

Blood ◽

10.1182/blood.v128.22.1710.1710 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1710-1710

Author(s):

Torsten Haferlach ◽

Claudia Haferlach ◽

Wenke Worseg ◽

Karolína Perglerová ◽

Wolfgang Kern ◽

...

Keyword(s):

Median Time ◽

Molecular Level ◽

Residual Disease ◽

Treatment Intervention ◽

Quantitative Real Time Pcr ◽

Employment Equity ◽

Npm1 Mutation ◽

Equity Ownership ◽

Minimal Residual

Abstract Introduction: Investigation of minimal residual disease (MRD) using NPM1 as a target has been proven to be of importance in AML. Guidelines for best schedules and implication on clinical use need to be defined. Aims: To better define the clinical impact and to suggest strategies for MRD monitoring in AML with NPM1 mutation. Patients and Methods: Between 2005 and 2015 we investigated 428 AML patients (pts) with NPM1 mutation at diagnosis and at a minimum of 2 follow-up time points. All pts had to achieve at least once a complete molecular remission (CMR) to be considered for this study. Sensitivity for MRD detection was at least 1:10,000. The median age of the cohort was 57 years (range: 18-85 yrs) and comprised of 198 males and 230 females. 3,039 samples (median number of samples per pts: 7, range: 2-35) were studied during course of disease. Molecular techniques applied included gene scan, sequencing and quantitative real-time PCR at diagnosis and quantitative real-time PCR during follow-up. Median time between 2 investigations was 2.8 months (mo; range: 0.3-71.0 mo). All pts were treated with standard protocols according to genotype and age. Allogeneic bone marrow or stem cell transplantation was performed in 136 pts (31.8%). Results: NPM1 type A mutation was the most frequent mutation type (317/428, 74.1%), followed by type B and D (36/428, 8.4% and 23/428, 5.4%), respectively. 25 other NPM1 types occurred at frequencies between 0.2 and 3.7%, in total demonstrating the expected distribution of NPM1 mutation types in an adult AML cohort. Subgroups of these pts were analyzed for FLT3-ITD (n=421) and mutations in DNMT3A (n=236). 122/421 (29%) pts showed a FLT3-ITD. In 96/236 (41%) DNMT3A was mutated. Further in 33/235 (14%) both genes were mutated. 103/235 (44%) screened for all three genes had a sole NPM1 mutation. All sole NPM1 mutated study pts achieved the CMR after a median of 4.1 mo (range: 1.0-8.6 mo). The presence of an additional DNMT3A mutation (CMR after a median of 4.4 mo, range 1.0-8.7) or a FLT3-ITD (CMR after a median of 2.7 mo, range 1.0-8.7) or both mutations (CMR after a median of 4.1 mo, range 1.1-7.9 mo) had no influence on time to achieve CMR. After achievement of CMR an increase of NPM1 ratio was detected in 185/428 (43%) pts. The median time to loss of CMR was 5.1 mo (range: 0.4-88 mo). In more detail, 42/185 of these patients also had FLT3-ITD, 53/109 had DNMT3A mutations and 13/109 had mutations in both genes. Patients with a DNMT3A mutation showed more often loss of CMR (40/60, 67%), while FLT3-ITD and FLT3-ITD/DNMT3A mutated patients showed no significant influence on loss of CMR ratio (46% and 48%, respectively) maybe due to number of cases. In 152/185 molecular relapses further follow up samples after loss of CMR were available. The median time between detected loss of CMR and the next follow-up sample was 2.0 mo. Due to treatment intervention 46/152 patients achieved a second CMR and 27/152 a decrease in NPM1 ratio. However, in 79/152 a further increase leading to clinical relapse was observed. The increase after loss of CMR was in median 13-fold between first and second sample after CMR was lost. Importantly, keeping periods between two MRD samplings at an interval of 3 mo allowed the detection of nearly all cases of first relapse at the molecular level. Addressing the sensitivity levels of the assays applied to bone marrow (BM) versus peripheral blood (pB) samples showed a 1.6 fold higher sensitivity for BM samples (median copies of reference gene, 14,628 vs 9,363). Due to the comparable sensitivities pB can be investigated until a first increase on the molecular level is detectable, followed by BM sampling for confirmation 4 weeks later. Conclusions: 1) NPM1 has proven to be a good marker for MRD monitoring in AML. 2) Time to CMR is short with a median of 4.1 mo. 3) An increase of NPM1 in all cases is followed by relapse after a median of 5.1 mo, if no treatment intervention has been initiated before. 4) Time intervals for MRD should be no longer than 3 mo, pB can be used. 5) Transplantation should already be planned after first molecular increase is detected. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Worseg:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment.

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Vaso-Occlusive Crises and Costs of Sickle Cell Disease from a Commercial Payer's Perspective

Blood ◽

10.1182/blood-2019-124920 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3464-3464

Author(s):

George J Joseph ◽

Dominick Latremouille-Viau ◽

Vikash Kumar Sharma ◽

Patrick Gagnon-Sanschagrin ◽

Menaka Bhor ◽

...

Keyword(s):

Healthcare Costs ◽

Medical Service ◽

Cell Disease ◽

Employment Equity ◽

Consulting Services ◽

Analysis Group ◽

Equity Ownership ◽

The Mean ◽

Insured Patients

Background: Episodes of acute pain caused by vaso-occlusive crises (VOCs) are a frequent and debilitating complication associated with sickle cell disease (SCD) and represent the most common cause for emergency room (ER) visits and inpatient (IP) stays. VOCs are due to a complex pathophysiology including multicellular adhesion. The purpose of this study was to characterize VOCs and assess the costs of SCD for patients with commercial insurance using an Excel-based model. Methods: Patients with SCD aged ≥16 years were identified in the IBM Truven MarketScan commercial databases (01/01/2000-06/30/2018). The index date was randomly selected among potential calendar dates to have ≥12 months of continuous health plan enrollment before (pre-index period) and after (follow-up period) that date. Patients with Medicare Supplemental coverage or stem cell transplant (SCT) were excluded. Data were analyzed at the state level for 23 key states with the highest concentration of patients with SCD and at the national level to be incorporated into an Excel-based model. The key variables in the model were age, gender, state of residence, and SCD clinical characteristics measured during the 12-month pre-index period. The following model inputs were assessed during the subsequent 12-month follow-up period: number, type, and setting (i.e., IP, ER, or outpatient [OP]) of VOC episodes; and total all-cause and SCD-related healthcare costs. Costs were reported in 2018 USD from a payer's perspective. We defined a VOC episode requiring medical services in claims data as follows: medical service claims with a VOC-related diagnosis occurring within 3 days of each other, IP re-admission within 14 days of a previous IP stay (both with VOC-related diagnoses), or any follow-up medical services with VOC-related diagnoses in the 7 days following an initial VOC diagnosis. A complicated VOC type was defined as a VOC episode with a diagnosis of priapism, splenic sequestration, acute hepatic sequestration, or acute chest syndrome. Variables were stratified by annual number of VOCs (i.e., 0, 1, ≥2 VOCs) and medical service setting (i.e., IP, ER, or OP). Results: A total of 16,092 commercially-insured patients with SCD from all US states were included in this study: mean age was 36.7 years, and 61.4% were females. In total, 27.7% had Hb-SS, 23.4% Hb-SC, 25.8% Hb-thalassemia, and 23.1% had an unspecified SCD type. The five states that contributed the highest number of patients with SCD were New York (n=1,711; 10.6%), Texas (n=1,593; 9.9%), Florida (n=1,397; 8.7%), Georgia (n=1,382; 8.6%), and California (n=966; 6.0%). In a given year, 64.7% of patients did not have any VOC episodes, 14.0% had only 1 VOC, and 21.2% had ≥2 VOCs (10.1% had ≥4 VOCs). Among patients with ≥1 VOC, the mean number of VOC episode was 3.3 (7.3% were complicated VOCs); among those with ≥2 VOCs, this figure was 4.8 (6.9% were complicated VOCs). The model showed that VOC episodes were distributed as follows: 37.4% in an IP setting, 26.4% in an ER, and 36.2% in an OP setting (Figure 1). The mean duration of a VOC episode was 11.7 days in an IP setting, 2.3 days in an ER setting, and 1.9 days in an OP setting (Figure 2). Total annual all-cause healthcare costs for patients with 0, 1, and ≥2 VOCs were $15,747, $27,194, and $64,555, respectively (Figure 3). Total annual SCD-related healthcare costs for patients with 0, 1, and ≥2 VOCs were $8,885, $21,323, and $60,624, respectively, representing 56.4%, 78.4%, and 93.9% of total annual all-cause healthcare costs, respectively (Figure 3). Conclusions: Among commercially-insured patients with SCD in the US, over one-third of patients experienced VOCs. The model showed that the contribution of SCD-related costs to annual total healthcare costs increases with the number of VOCs per year. Disclosures Joseph: Amgen: Equity Ownership; Novartis: Employment, Equity Ownership; Pfizer: Equity Ownership; Cigna: Equity Ownership. Latremouille-Viau:Novartis: Other: I am an employee of Analysis Group, Inc., which provided paid consulting services to Novartis for the conduct of this study. Sharma:NOVARTIS HEALTHCARE PVT. LTD.: Employment. Gagnon-Sanschagrin:Novartis: Other: I am an employee of Analysis Group, Inc., which provided paid consulting services to Novartis for the conduct of this study. Bhor:Novartis: Employment, Equity Ownership. Khare:HEALTHCARE PVT. LTD.: Employment. Singh:NOVARTIS HEALTHCARE PVT. LTD.: Employment. Serra:Novartis: Other: I am an employee of Analysis Group, Inc., which provided paid consulting services to Novartis for the conduct of this study. Davidson:Novartis: Other: I am an employee of Analysis Group, Inc., which provided paid consulting services to Novartis for the conduct of this study. Guerin:Novartis: Other: I am an employee of Analysis Group, Inc., which provided paid consulting services to Novartis for the conduct of this study. Shah:GBT: Research Funding; Alexion: Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau.

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Sensitive Monitoring of Minimal Residual Disease Status in CEBPA-Mutated Acute Myeloid Leukemia Using Amplicon Deep-Sequencing

Blood ◽

10.1182/blood.v118.21.2517.2517 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2517-2517

Author(s):

Alexander Kohlmann ◽

Vera Grossmann ◽

Annette Fasan ◽

Elisa Stopp ◽

Tamara Alpermann ◽

...

Keyword(s):

Deep Sequencing ◽

Residual Disease ◽

Normal Karyotype ◽

Initial Diagnosis ◽

Employment Equity ◽

Clone Size ◽

Equity Ownership ◽

Conventional Methods ◽

Biallelic Mutations

Abstract Abstract 2517 Introduction: CEBPA (CCAAT/enhancer binding protein alpha) encodes a member of the basic region leucine zipper (bZIP) transcription factor family essential for myeloid differentiation. CEBPA mutations occur predominantly in AML with a normal karyotype and CEBPA mutated AML has been included as provisional entity in the WHO classification. Cases with biallelic mutations were reported as being associated with a favorable clinical outcome, thus patients are spared from allogeneic transplantation in first CR. Screening for CEBPA mutations in patients with AML is often performed applying a combination of fragment length analysis, DHPLC and subsequent direct sequencing using Sanger technique (conventional methods). Study Design: Next-generation amplicon deep-sequencing (454 Life Sciences, Branford, CT) is a more sensitive quantitative detection method than Sanger sequencing and thus was used to analyze 144 samples from 29 CEBPA mutated AML patients with a normal karyotype. For a longitudinal analysis starting at diagnosis and following the course of treatment bone marrow (n=134) or peripheral blood (n=10) samples were obtained between 5/2006 and 6/2011. The sequencing assay targeted the complete coding region of CEBPA, covered with 4 amplicons, and was performed using genomic DNA extracted from mononuclear cells. In median, 711 reads per amplicon were generated using the NGS assay, thereby allowing a sensitive quantitative assessment of the CEBPA mutational burden in order to monitoring minimal residual disease (MRD). In median, 4 time points per patient (range: 2–9) were included with a median time span of 9.5 months (range: 1–45 months). The median sampling interval was 2 months (range: 0.3–45 months). Results: First, we evaluated the concordance of mutation detection by comparing data from NGS and conventional methods using the samples at initial diagnosis. In all 29 AML patients NGS concordantly detected the mutations known from conventional methods, i.e. in total 26 frame-shifts, 15 in-frame alterations, 8 missense, and 2 nonsense mutations. Further, at initial diagnosis, deep-sequencing detected the mutations with a median burden of 44% sequencing reads (range 3%–88%) and thus already allowed a quantitative assessment of the mutational load. There was no difference observed for 6 patients with monoallelic vs. 21 cases with biallelic mutations (excluding 2 cases with homozygous alterations). We next investigated the distribution of clones and their underlying kinetics of clone size reduction during subsequent high-dose chemotherapy cycles. Overall, 26/29 cases were evaluable and the clone size was assessed by NGS at the second analysis point during course of disease–in median 63 days from time of diagnosis (range 10–215 days): (i) In 16/26 cases, deep-sequencing was not able anymore to detect the mutations as observed at diagnosis. 14 of these 16 negative cases stayed in complete molecular remission till the end of follow-up (median follow-up 6.5 months, range 1–34.2; 2/14 cases with allogeneic stem cell transplantation). (ii) Interestingly, in 4/26 cases residual disease with clones ranging from 8%–50% was indicative of non-response to treatment. In this subgroup 3/4 patients were characterized by resistant disease or early relapse (1 case excluded due to short follow-up). (iii) In the remainder group of 6/26 patients with mutations still detectable in a range of 0.12%–3.7%, complete molecular remission status was achieved at subsequent time points. However, in this group also 3 relapses were observed including 2 cases with allogeneic stem cell transplantation. Of note, in 3/6 cases from the latter group, NGS had outperformed conventional methods and was able to still detect residual clones enabling a superior monitoring of therapy response. In all cases with biallelic mutations both clones responded in parallel with similar kinetics. Moreover, 5 patients were investigated following relapse of AML or non-response to therapy. In all 5/5 analyses including 2 monoallelic and 3 biallelic alterations the same mutations as harbored at initial diagnosis remained detectable. Conclusion:CEBPA mutations provide increasing clinical utility for the detection of MRD. We here demonstrated that deep-sequencing is a suitable unbiased and robust method to accurately detect and quantify CEBPA aberrations enabling an individualized monitoring of disease status and treatment efficacy. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Grossmann:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Stopp:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Minimal Residual Disease Monitoring In Acute Lymphoblastic Leukemia Patients Using Immune Receptor Sequencing

Blood ◽

10.1182/blood.v122.21.1381.1381 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1381-1381

Author(s):

Malek Faham ◽

Maddalena Paganin ◽

Giulia Fabbri ◽

Elisa Magrin ◽

Emanuela Giarin ◽

...

Keyword(s):

T Cell Receptor ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Cell Receptor ◽

Clinical Relapse ◽

Employment Equity ◽

Childhood All ◽

Lymphoid Malignancies ◽

Equity Ownership ◽

Minimal Residual

Abstract Background The assessment of residual tumor cells persisting after therapy, or minimal residual disease (MRD), is a central component of accurate disease prognosis in acute lymphoblastic leukemia (ALL) (Pui et al, JCO 2011). MRD assessment has recently been shown to be useful for monitoring disease before and after stem cell transplantation and during salvage therapy for early detection of an imminent relapse (Bruggemann et al., SemOncol 2012). Allele-specific oligonucleotide (ASO)-PCR can be used to assess MRD; however, this technique requires preparation of clonotype-specific primers for each individual which is laborious and time-consuming. We recently demonstrated the utility of sequencing-based MRD assessment in lymphoid malignancies (Faham et al., Blood 2012). This quantitative approach, termed the LymphoSIGHT™ platform, relies on amplification and sequencing of immunoglobulin and T-cell receptor gene segments using consensus primers and can address some of the limitations associated with traditional MRD detection techniques. This sequencing platform has a sensitivity to detect one cancer cell per million leukocytes in peripheral blood and bone marrow samples. In this retrospective study, we evaluated the ability of the sequencing and ASO-PCR methods to detect MRD prior to clinical relapse in 17 patients with childhood ALL. Methods Using the sequencing assay, we analyzed bone marrow and/or peripheral blood samples collected at the diagnostic and relapse time points from 17 childhood ALL patients. Diagnostic and relapse samples were assessed for clonal rearrangements of immunoglobulin (IGH-VDJ, IGH-DJ, IGK) and T cell receptor (TRB, TRD, TRG) genes. Following identification of leukemia specific clonotype(s), we measured the corresponding MRD levels in 66 follow-up samples that were collected prior to clinical relapse. We analyzed the time from MRD positivity to clinical relapse for each patient using the sequencing and ASO-PCR methods. Results Sequencing detected the presence of a high-frequency clonal rearrangement of at least one receptor (“calibrating receptor”) in all the 17 childhood ALL patients; all patients had at least 2 calibrating receptors at diagnosis and/or relapse, 15 patients had at least 3 calibrating receptors, and 6 patients had 4 or more. The IGH-VDJ and TRG assays were the most frequent gene rearrangement: at least one IGH-VDJ and/or TRG clonal rearrangement was detected in 13 ALL patients. TRD was the third most informative receptor, with clonal rearrangements being detected in 11 patients. In the majority of patients, most leukemic clones identified at diagnosis were also present at high levels in the relapse sample. We analyzed the time from MRD positivity to clinical relapse in the patient cohort. In 2 patients (Figure 1, Patients G and P), we observed MRD positivity by sequencing 16 and 6.5 months prior to relapse, respectively. ASO-PCR also detected MRD positivity in Patient P at the 6.5 month time point. In 6 patients there was a sample collected within 1-3 months prior to relapse, and sequencing detected MRD in 4 of these patients (67%, Figure 1 Patients B, A, R, I), while ASO-PCR detected MRD in 2 of the 6 patients (33%, Figure 1 Patients B, R). Conclusions The clinical value of monitoring MRD by ASO-PCR for assessment of treatment response and outcome has been established in multiple lymphoid malignancies. This preliminary study provides further support for the sequencing-based MRD monitoring in ALL patients. This new approach offers improvements over ASO-PCR in the ability to monitor multiple clonal sequences and in the sensitivity to detect the leukemic cell. Thus, the sequencing method represents a new potential approach for MRD monitoring. However a larger number of patients must be analyzed to support its clinical application. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Pepin:Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership.

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Clinical Impact of Minimal Residual Disease (MRD) Monitoring in AML with PM-Rara, CBFB-MYH11, and RUNX1-RUNX1T1: A Study on 600 Patients

Blood ◽

10.1182/blood.v126.23.228.228 ◽

2015 ◽

Vol 126 (23) ◽

pp. 228-228

Author(s):

Torsten Haferlach ◽

Manja Meggendorfer ◽

Susanne Schnittger ◽

Annette Fasan ◽

Wolfgang Kern ◽

...

Keyword(s):

Bone Marrow ◽

Residual Disease ◽

Treatment Strategies ◽

Molecular Techniques ◽

Allogeneic Bone ◽

Employment Equity ◽

First Relapse ◽

Equity Ownership ◽

Minimal Residual

Abstract Introduction: The cure rate in AML is dependent on patient´s (pts) age and performance, cytogenetics, early blast clearance and sustainable first complete remission. Investigation of minimal residual disease (MRD) is possible by multiparameter flow cytometry and molecular techniques. Recent findings have further depicted a broad spectrum of molecular markers in AML in 99% of pts (TCGA, NEJM, 2013). This broadens the portfolio of targets for MRD assessment and will hopefully help to better individualize treatment strategies. We here focused - as a paradigm - on the three hallmarks for molecular MRD studies in AML. Aims: To better define the clinical impact and to suggest strategies for MRD monitoring in AML with PML-RARA, CBFB-MYH11, and RUNX1-RUNX1T1. Patients and Methods: Between 2005 und 2015 we at diagnosis investigated 321 PML-RARA, 134 CBFB-MYH11, and 145 RUNX1-RUNX1T1 AML pts. Individual follow-up time points during their course of disease were studied in 2657, 1047, and 890 samples, respectively. Thus, the combined number of investigated samples is 4,594. Molecular techniques applied comprised quantitative real-time PCR and nested PCR. Median age in PML-RARA was 52 years (yrs) (2-86 yrs), in CBFB-MYH11 53 yrs (21-81 yrs), and in RUNX1-RUNX1T1 52 yrs (10-83 yrs). Median time between 2 investigations was 3.0 months (mo) in PML-RARA, 2.1 mo in CBFB-MYH11, and 2.8 mo in RUNX1-RUNX1T1 pts (range for all 0.1-40.4 mo), respectively. All pts were treated with standard protocols according to genotype and age. Allogeneic bone marrow or stem cell transplantation was performed in 85 pts (14%). Results: 294/321 pts (92%) with PML-RARA achieved complete molecular remission (CMR) after a median of 2.9 mo (range: 0.8-9.7 mo). In contrast, in CBFB-MYH11 CMR was reached in 89/134 pts (66%) after a median of 7.4 mo (range: 1.6-16.8 mo), and in RUNX1-RUNX1T1 CMR was reached in 75/145 pts (51%) after a median of 4.7 mo (range: 1.0-11.5 mo). Of note, some of the CBFB-MYH11 pts never reached CMR, always showing low level signals. 95% (278/294) of PML-RARA pts that achieved CMR stayed in first CMR and did not relapse within a median follow-up of 32.6 mo (range: 1.2-134.5 mo). 5% (16/294) relapsed at a median interval after CMR of 8.1 mo. However, a second CMR was reached in 12/16 pts after relapse. Five of these 12 pts suffered from second relapses, whereof 4 pts achieved a third CMR. Third relapses occurred in 2/4 pts. 69/89 (78%) of pts with CBFB-MYH11 stayed in first CMR and never relapsed during a median follow-up of 10.4 mo (range: 1.6-47.1 mo). 20/89 relapsed after 4.0 mo of CMR, whereof 11 achieved second CMR. 3/11 relapsed again. 63/75 (84%) of pts with RUNX1-RUNX1T1 stayed in first CMR and never relapsed during a median follow-up of 10.1 mo (range: 1.0-65.8 mo). However, 12/75 relapsed after a median time of CMR of 5.3 mo. 4/12 achieved another CMR. In 85 patients (10 PML-RARA, 42 CBFB-MYH11, and 33 RUNX1-RUNX1T1) allogeneic bone marrow or stem cell transplantation (Tx) was performed, and 72/85 (85%) were rescued by Tx. However, two patients each with PML-RARA and RUNX1-RUNX1T1 relapsed, respectively, and 9 in CBFB-MYH11 positive AML after Tx. Patients did not experience first relapse later than 50.3 mo in CMR in PML-RARA, later than 30.7 mo in CBFB-MYH11, and later than 35.7 mo in RUNX1-RUNX1T1. Additionally, keeping periods between two MRD samplings at a maximum of 3 mo allowed the detection of nearly all cases of first relapse due to the molecular hint. Addressing the sensitivity levels of the assays applied to bone marrow (BM) versus peripheral blood (pB) samples showed a 1.4 fold higher sensitivity for BM samples (median copies of reference gene, 13,204 vs 9,240). Due to the comparable sensitivities pB can be investigated until a first hint of relapse, followed by BM sampling for confirmation. Conclusions: 1) MRD by molecular techniques reliably defines pts risks in AML with PML-RARA, CBFB-MYH11, and RUNX1-RUNX1T1, respectively. 2) Clinical decisions are reliable within screening intervals of 3 mo using pB. 3) Relapses in first CMR are not detected later than 50.3 mo in PML-RARA, 30.7 mo in CBFB-MYH11, and 35.7 mo in RUNX1-RUNX1T1 AML, respectively. 4) Pts after relapse can be rescued by transplantation in the majority of cases. 5) As the availability of other molecular markers in AML has dramatically increased, more individualized treatment strategies based on specific MRD monitoring are achievable in nearly every patient in the near future. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Fasan:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Highly Sensitive Detection of Minimal Residual Disease in Acute Lymphoblastic Leukemia Using Next-Generation Sequencing of Immunoglobulin Heavy Chain Variable Region

Blood ◽

10.1182/blood.v118.21.2540.2540 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2540-2540

Author(s):

Malek Faham ◽

Tom Willis ◽

Martin Moorhead ◽

Victoria Carlton ◽

Jianbiao Zheng ◽

...

Keyword(s):

Flow Cytometry ◽

Next Generation Sequencing ◽

Lymphoblastic Leukemia ◽

Patient Specific ◽

Next Generation ◽

Conventional Pcr ◽

Employment Equity ◽

Equity Ownership ◽

Generation Sequencing

Abstract Abstract 2540 Background: Minimal Residual Disease (MRD) assessment is increasingly used for treatment stratification since it is a strong predictor of outcome in Acute lymphoblastic leukemia (ALL). The most widely used MRD assays include flow cytometric detection of aberrant immunophenotypes and PCR amplification of patient-specific antigen-receptor sequences. The latter approach has proven to provide reliable clinical information but requires the development of patient-specific reagents which is laborious, time-consuming, and generates assays with variable sensitivities. In addition, this methodology may miss clonal changes that can occur during the course of the disease, such as the emergence of subclones as well as genetic evolution. To overcome these limitations, we developed a universal amplification assay with a sequencing readout that eliminates the need for patient-specific reagents, allows the assay to detect leukemic cells that have genetically evolved, and has a higher sensitivity than conventional tests. Methods and Results: To amplify all the IgH sequences, we developed a PCR assay to amplify all alleles of all the V and J segments with very low amplfication bias. Amplified molecules were then subjected to clonal sequencing to obtain >1 million reads to measure the frequency of the different IgH clonotypes in the sample. It should be noted that current next generation sequencing costs of this deep sequencing are similar to those of an MRD test conducted by flow cytometry. We tested the sensitivity of the method by in serial dilutions of genomic DNA from a leukemia sample known to carry two IgH clonotypes with into genomic DNA obtained from peripheral blood sample from a healthy donor. The material from the dilution series was then sequenced and analyzed to measure the level of these clones. The leukemic clonotypes could be readily detected even when diluted 1 million fold. To directly compare the our method to established MRD assays in ALL, we studied diagnostic and follow-up samples from 10 ALL patients whose MRD levels have been previously assessed by both real-time PCR amplification of IgH genes and flow cytometry. The results of these tests were not disclosed until completion of the deep sequencing analysis. The follow up samples were collected during (n = 3) or at the end of remission induction therapy (n = 4), or during continuation therapy (n = 3). Samples were processed similarly to identify the leukemia-specific sequence in the diagnostic samples and determine the level of these sequences in the follow up samples. The sequencing-based method identified all 5 samples that were MRD-positive according to flow cytometry and PCR (Figure 2), with highly concordant estimates of MRD levels. Notably, among the remaining 5 samples, scored as MRD-negative by both flow cytometry and conventional PCR, the sequencing method detected residual leukemic sequences at a very low level (∼10−6) in one of the samples. The other 4 samples were MRD-negative by all three methods. Studies with a larger cohort of ALL samples are ongoing. Contrary to conventional PCR-based MRD testing, the sequencing technology allows for the detection of leukemic clones that evolve by V replacement or other mechanisms. In this study, we identified clonotypes in several of the diagnostic samples that appeared to be the result of V replacement. These and other newly appearing related clones can be monitored in subsequent samples using the generic amplification and sequencing assay. Conclusions: We developed a highly sensitive and specific MRD detection method based on next-generation sequencing of IgH genes. This method has substantial advantages over conventional PCR MRD in that it eliminates the need for patient-specific reagents, can follow genetic evolution, and has potential for higher sensitivity. Disclosures: Faham: Sequenta Inc: Employment, Equity Ownership. Willis:Sequenta Inc: Employment, Equity Ownership. Moorhead:Sequenta Inc: Employment, Equity Ownership. Carlton:Sequenta Inc: Employment, Equity Ownership. Zheng:Sequenta Inc: Employment, Equity Ownership.

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