scholarly journals Retrospective Analysis of Minimal Residual Disease Testing By High Throughput Immunosequencing Versus High Sensitivity Flow Cytometry and qPCR in B-Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4476-4476
Author(s):  
Omar Fathalla ◽  
Anwar Khan ◽  
Nagehan Pakasticali ◽  
Bijal D. Shah ◽  
Mohammad O Hussaini
Keyword(s):  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Correlation Coefficient ◽  
High Throughput ◽  
Research Funding ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Cancer Center ◽  
Spearman Correlation ◽  
Minimal Residual

Abstract Introduction: Detection of minimal residual disease (MRD) is one of the most powerful predictors of prognosis in patients with B-Lymphoblastic Leukemia (ALL). Standard methods to detect MRD in ALL include high-resolution flow cytometry (FC) (sensitivity 10 -4 to 10 -5) and qPCR for Ph+ ALL (sensitivity 10 -5 to -6). More recently, high-throughput sequencing (NGS) has been leveraged to detect MRD (sensitivity 10 -6). Retrospective, non-clinical trial based real-world data comparing between these methodologies in the literature is limited to absent. We report our experience at a high-volume cancer center comparing NGS, qPCR, and FC. Methods: All cases of ALL with NGS MRD data at the Moffitt Cancer Center were identified. Corresponding FC, qPCR (p190 and p210) and clinical data were collected electronically and via chart review. 10-color flow cytometry was performed on a Gallios System and analyzed on Kaluza (Beckman Coulter, IN). 750,000 events were collected on all cells. Validated lower limit of detection was at least 0.01%. Antibodies included CD15, CD130, CD10, CD58, CD22, CD33, CD13, CD123, CD19, CD20, CD45, CD38, CD34, CD81, CD200 (BD, Biolegend, Beckman Coulter). Quantidex™ BCR-ABL IS Kit (RT qPCR) was used to quantify BCR-ABL (p190 and p210) and ABL transcripts in total RNA in patients with ALL on Applied Biosystems 7500 Fast Dx Real-Time PCR instrument. The results were expressed by normalized BCR-ABL to ABL (control) ratio. clonoSEQ ® (Adaptive Biotechnologies, Seattle, WA) testing was performed which uses multiplex polymerase chain reaction (PCR) and NGS to identify, characterize, and monitor clonotypes of immunoglobulin (Ig) IgH (V-J), IgH (D-J), IgK, and IgL receptor gene sequences, and translocated BCL1/IgH (J) and BCL2/IgH (J) sequences. Statistical analysis was performed by Spearman correlation coefficient and Kaplan-Meier analysis. Results: 95 samples from 54 unique patients were identified that had both NGS and FC data. A subset of patients had qPCR data (n=31 samples). FC+ values ranged from 0.00004 to 0.75. NGS+ values ranged from 8.25E-08 to 0.49. Spearman correlation coefficient showed moderate concordance between NGS and FC at r=0.62 (p<0.001). Spearman correlation coefficient for qPCR vs Flow was r= 0.08 (p=0.68). Three samples were positive by FC (mean tumor burden (MTB= 0.002) but missed by NGS; whereas 31 samples were detected by NGS (MTB= 0.0016) that were missed by flow cytometry. By FC, 9 samples were equivocal of which 7 were definitively designated as MRD+ by NGS. There were 7 qPCR+/NGS- samples (MTB= 2.5 x 10 -5) and 6 qPCR-/NGS+ cases (MTB= 0.012). Overall survival was worse for MRD+ (by NGS, qPCR, or FC) vs MRD(-) (Figure 1). Conclusion: Our study confirms the importance of MRD detection in ALL and shows the robust utility of NGS for MRD detection in routine hematopathology practice. While qPCR, FC and NGS are complementary given that each can potentially detect MRD missed by another method, the data supports the increased sensitivity of NGS over FC. Figure 1 Figure 1. Disclosures Shah: Servier Genetics: Other; Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; BeiGene: Consultancy, Honoraria; Acrotech/Spectrum: Honoraria; Pharmacyclics/Janssen: Honoraria, Other: Expenses; Kite, a Gilead Company: Consultancy, Honoraria, Other: Expenses, Research Funding; Precision Biosciences: Consultancy; Amgen: Consultancy; Pfizer: Consultancy, Other: Expenses; Novartis: Consultancy, Other: Expenses; Bristol-Myers Squibb/Celgene: Consultancy, Other: Expenses; Adaptive Biotechnologies: Consultancy. Hussaini: Stemeline Therapeutics: Honoraria.

scholarly journals Retrospective Analysis of Minimal Residual Disease Testing By High Throughput Immunosequencing Versus High Sensitivity Flow Cytometry in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1625-1625
Author(s):  
Anwar Khan ◽  
Nagehan Pakasticali ◽  
Omar Fathalla ◽  
Taiga Nishihori ◽  
Mohammad O Hussaini
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Correlation Coefficient ◽  
Research Funding ◽  
Residual Disease ◽  
High Sensitivity ◽  
Spearman Correlation ◽  
Color Flow ◽  
Minimal Residual

Abstract Introduction: Detection of minimal residual disease (MRD) is one of the strongest predictors of outcome in multiple myeloma (MM). Until recently, the most commonly available method to detect MRD in clinical practice has been high sensitivity flow cytometry (FC) which can detect MRD with at 10 -5 sensitivity. In recent years, next-generation sequencing (NGS) has become a viable method to assess the MRD in MM patients with a 10 -6 sensitivity. NGS appears to have some advantages over HC-FC by circumventing subjectivity of analysis. However, real-world comparison between these two methodologies in the literature is limited and is important to inform daily hematopathology and oncology ordering practices. Methods: We retrospectively identified all cases of MM with NGS MRD data from bone marrow specimens at the Moffitt Cancer Center and collated corresponding flow MRD data and clinical data (OS, patient demographics) electronically and via chart review. 10-color flow cytometry was performed on a Gallios System and analyzed on Kaluza (Beckman Coulter, IN). Two million events were collected on all cells. Validated lower limit of detection was at least 0.01%. Antibodies included CD28, CD81, CD56, CD138, CD319, CD20, CD19, CD117, CD38, CD45, CD27, CD200 (BD, Biolegend, Beckman Coulter). clonoSEQ ® (Adaptive Biotechnologies, Seattle, WA) testing was performed which uses multiplex polymerase chain reaction (PCR) and NGS to identify, characterize, and monitor clonotypes of immunoglobulin (Ig) IgH (V-J), IgH (D-J), IgK, and IgL receptor gene sequences, and translocated BCL1/IgH (J) and BCL2/IgH (J) sequences Statistical analysis was performed by Spearman correlation coefficient and Kaplan-Meier analysis. Results: 192 samples from 122 unique patients were identified that had both NGS and FC data performed on the same sample. FC+ values ranged from 1x10 -7 to 0.39. NGS+ values ranged from 2.3 x 10 -7 to 0.15. Spearman correlation coefficient showed moderate concordance between NGS and FC at r=0.67 (p<0.001). Six samples were positive by FC (mean tumor burden (MTB)= 0.0007) but missed by NGS; whereas 59 samples were positive by NGS (MTB= 0.002) but missed by flow cytometry. Two cases by FC were equivocal and these were both definitively designated as MRD+ by NGS. Overall survival was worse for MRD+ (by NGS or FC) vs MRD(-) (Figure 1). Conclusion: Our study confirms the importance of MRD detection in MM and shows the robust utility of NGS for MRD detection in routine hematopathology practice. While both FC and NGS are complementary given that each can potentially detect MRD missed by another method, the data supports the increased sensitivity of NGS over FC. Figure 1 Figure 1. Disclosures Nishihori: Novartis: Research Funding; Karyopharm: Research Funding. Hussaini: Stemeline Therapeutics: Honoraria.

scholarly journals Minimal Residual Disease Assessment of Remission after Induction Therapy Is Superior to Morphologic Assessment for Risk Stratification in Childhood Acute Lymphoblastic Leukemia: A Report from the Children's Oncology Group (COG)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 758-758
Author(s):  
Sumit Gupta ◽  
Meenakshi Devidas ◽  
Si Chen ◽  
Cindy Wang ◽  
Mignon L. Loh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Risk Stratification ◽  
Minimal Residual Disease ◽  
Induction Therapy ◽  
Research Funding ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Laboratory Services ◽  
Minimal Residual

Abstract Background: Minimal residual disease (MRD) assessment after initial therapy is integral to modern risk stratification in both precursor B and T lineage acute lymphoblastic leukemia (B-ALL and T-ALL). While MRD is used to determine depth of remission, remission is still defined, both in clinical practice and clinical trials, according to morphological assessment. We aimed to determine the outcomes of children, adolescents and young adults with discordant assessments of remission by morphology vs. by MRD, and in doing so, the extent to which morphologic assessment of remission contributes to risk assessment in this population. Methods: We identified a cohort of patients age 1-30.99 years enrolled on frontline COG trials for B-ALL [standard risk (SR): AALL0331; high risk (HR) AALL0232] and T-ALL (AALL0434) that underwent bone marrow assessment of remission at the end of induction therapy (Day 29). Morphologic response was assessed by local centers and was categorized according to traditional criteria: M1 (<5% leukemic blasts - remission) vs. M2 (5-25%) vs. M3 (>25%). MRD was measured by flow cytometry at one of two central laboratories. We determined predictors of MRD discordance and compared event free survival (EFS) between those with discordant vs. concordant morphology/MRD remission assessments. Results: Day 29 remission assessments and central MRD data were available on 9,350 patients, 7,857 (84%) with B-ALL (AALL0331: N=5049; AALL0232: N=2808) and 1,493 (16%) with T-ALL. Table 1 shows the distribution of end induction marrow morphology vs. flow cytometry results. Few patients with M2/M3 marrows had discordant low MRD values. For example, of 84 patients with M3 morphology, only 2 (2.4%) had MRD <5%. Of 202 patients with M2/M3 morphology, 23 (11.4%) had MRD<1% and 9 (4.5%) had MRD<0.1%. Subsequent analyses of discordance were thus restricted to patients with M1 morphology but flow cytometry consistent with failure to achieve remission (MRD>=5%). Using this definition, discordance was uncommon among subjects with B-ALL (66/7,748; 0.9%) but significantly more common in T-ALL (97/1,400; 6.9%; p<0.0001). Among subjects with B-ALL and M1 morphology, significant predictors of discordance (MRD>=5%) in multivariable regression included variables traditionally associated with poor response: age >=10 years [odds ratio (OR)=1.7, 95th percentile confidence interval (CI) 1.1-2.8; p=0.03), presenting white blood cell count >=50,000/microliter (OR=2.1, CI 1.3-3.6; p=0.004), and unfavorable compared to favorable cytogenetics (OR=31, CI 8.9-109; p<0.0001). In B-ALL, subjects with end induction M1 morphology but discordant MRD (>=5%) had modestly superior 5-year EFS when compared to those with M2 morphology and MRD >=5% (33.1%±6.2% vs. 22.0%±6.9%; p=0.03), but EFS was significantly inferior to those with M1 morphology and concordant MRD (<5%) (33.1%±6.2% vs. 86.8%±0.4%; p<0.0001) (Figure 1). In T-ALL, the 5-year EFS of subjects with M1 morphology/discordant MRD was not significantly different from those with M2 morphology and MRD >=5% (80.3%±7.3% vs. 62.7%±13.5%; p=0.13); outcomes of both groups were superior to their equivalents with B-ALL, in keeping with known slower disease clearance kinetics in T-ALL. Conclusions: Patients in morphologically defined remission but with MRD >=5% have outcomes similar to those who fail to achieve morphological remission. These results suggest that, in addition to measuring depth of remission, MRD should replace morphology in defining remission in subjects with ALL, with consequent implications for risk stratification, treatment assignment and eligibility for experimental agents. Disclosures Loh: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding. Borowitz:HTG Molecular: Consultancy; Bristol-Myers Squibb: Research Funding; MedImmune: Research Funding; BD Biosciences: Research Funding. Wood:Juno: Other: Laboratory Services Agreement; Pfizer: Honoraria, Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Seattle Genetics: Honoraria, Other: Laboratory Services Agreement.

scholarly journals Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies

Blood ◽  
2015 ◽  
Vol 125 (26) ◽  
pp. 3996-4009 ◽  
Author(s):  
Jacques J. M. van Dongen ◽  
Vincent H. J. van der Velden ◽  
Monika Brüggemann ◽  
Alberto Orfao
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Minimal Residual Disease ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Pcr Analysis ◽  
Advantages And Disadvantages ◽  
Minimal Residual

Abstract Monitoring of minimal residual disease (MRD) has become routine clinical practice in frontline treatment of virtually all childhood acute lymphoblastic leukemia (ALL) and in many adult ALL patients. MRD diagnostics has proven to be the strongest prognostic factor, allowing for risk group assignment into different treatment arms, ranging from significant treatment reduction to mild or strong intensification. Also in relapsed ALL patients and patients undergoing stem cell transplantation, MRD diagnostics is guiding treatment decisions. This is also why the efficacy of innovative drugs, such as antibodies and small molecules, are currently being evaluated with MRD diagnostics within clinical trials. In fact, MRD measurements might well be used as a surrogate end point, thereby significantly shortening the follow-up. The MRD techniques need to be sensitive (≤10−4), broadly applicable, accurate, reliable, fast, and affordable. Thus far, flow cytometry and polymerase chain reaction (PCR) analysis of rearranged immunoglobulin and T-cell receptor genes (allele-specific oligonucleotide [ASO]-PCR) are claimed to meet these criteria, but classical flow cytometry does not reach a solid 10−4, whereas classical ASO-PCR is time-consuming and labor intensive. Therefore, 2 high-throughput technologies are being explored, ie, high-throughput sequencing and next-generation (multidimensional) flow cytometry, both evaluating millions of sequences or cells, respectively. Each of them has specific advantages and disadvantages.

scholarly journals Marked Variability in Reported Minimal Residual Disease Lower Level of Detection of 4 Hematolymphoid Neoplasms: A Survey of Participants in the College of American Pathologists Flow Cytometry Proficiency Testing Program

2015 ◽  
Vol 139 (10) ◽  
pp. 1276-1280 ◽  
Author(s):  
Michael Keeney ◽  
Jaimie G. Halley ◽  
Daniel D. Rhoads ◽  
M. Qasim Ansari ◽  
Steven J. Kussick ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
Plasma Cell Myeloma ◽  
Minimal Residual

Context Flow cytometry is often applied to minimal residual disease (MRD) testing in hematolymphoid neoplasia. Because flow-based MRD tests are developed in the laboratory, testing methodologies and lower levels of detection (LODs) are laboratory dependent. Objectives To broadly survey flow cytometry laboratories about MRD testing in laboratories, if performed, including indications and reported LODs. Design Voluntary supplemental questions were sent to the 549 laboratories participating in the College of American Pathologists (CAP) FL3-A Survey (Flow Cytometry—Immunophenotypic Characterization of Leukemia/Lymphoma) in the spring of 2014. Results A total of 500 laboratories (91%) responded to the supplemental questions as part of the FL3-A Survey by April 2014; of those 500 laboratories, 167 (33%) currently perform MRD for lymphoblastic leukemia, 118 (24%) for myeloid leukemia, 99 (20%) for chronic lymphocytic leukemia, and 91 (18%) for plasma cell myeloma. Other indications include non-Hodgkin lymphoma, hairy cell leukemia, neuroblastoma, and myelodysplastic syndrome. Most responding laboratories that perform MRD for lymphoblastic leukemia reported an LOD of 0.01%. For myeloid leukemia, chronic lymphocytic leukemia, and plasma cell myeloma, most laboratories indicated an LOD of 0.1%. Less than 3% (15 of 500) of laboratories reported LODs of 0.001% for one or more MRD assays performed. Conclusions There is major heterogeneity in the reported LODs of MRD testing performed by laboratories subscribing to the CAP FL3-A Survey. To address that heterogeneity, changes to the Flow Cytometry Checklist for the CAP Laboratory Accreditation Program are suggested that will include new requirements that each laboratory (1) document how an MRD assay's LOD is measured, and (2) include the LOD or lower limit of enumeration for flow-based MRD assays in the final diagnostic report.

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