A NOVEL DIGITAL PCR ASSAY FOR MYD88 L265P MUTATION DETECTION IN WALDENSTRÖM MACROGLOBULINEMIA: MINIMAL RESIDUAL DISEASE MONITORING AND CHARACTERIZATION ON CIRCULATING FREE DNA
(Abstract release date: 05/19/16)
EHA Library. Drandi D. 06/09/16; 132934; E1385
Daniela Drandi
Contributions
Contributions
Abstract
Abstract: E1385
Type: Eposter Presentation
Background
MYD88L265P mutation is the earmark of Waldenström Macroglobulinemia (WM) and might represent an ideal marker for minimal residual disease (MRD) monitoring. However, current diagnostic tools, as allele-specific quantitative PCR (ASqPCR), are not sensitive enough for MRD monitoring on peripheral blood (PB), harboring low concentrations of circulating tumor cells. Besides, cell-free DNA (cfDNA) analysis has been shown to be highly promising for mutational studies. However, more sensitive approaches are needed and droplet digital PCR (ddPCR) might thus represent a powerful tool in this setting.
Aims
Here we describe the applications of a new, highly sensitive, ddPCR assay for MYD88L265P detection to assess: 1) its feasibility for MYD88L265P mutation screening and MRD monitoring in bone marrow (BM) and PB; 2) its potential application for mutational studies on cfDNA isolated from plasma.
Methods
BM, PB and plasma samples from a local series of WM and IgG-lymphoplasmacytic lymphomas (LPL) and IgM-MGUS patients (pts) were collected at baseline and during follow-up. 20 healthy subjects were used as negative controls. Genomic DNA (gDNA) and plasma derived cfDNA were extracted, according to the blood or body fluid protocols, by Maxwell RSC automatic system (Promega). MYD88L265P was assessed on gDNA (100ng) and cfDNA (5μl) by a custom ddPCR assay on a QX100 System (Bio-Rad). For comparison ASqPCR was assessed on gDNA (100ng), as described [Xu L, 2013]. MYD88L265P cut-off was settled based on the healthy samples background level. IGH-based MRD analysis was performed as described [Ladetto M, 2000; Drandi D, 2015].
Results
Once optimized, MYD88L265P ddPCR assay sensitivity was compared to ASqPCR on a ten-fold serial dilution standard curves. Whereas ASqPCR confirmed the reported sensitivity of 1.00E−03, ddPCR reached a sensitivity of 5.00E−05. Overall, 137 samples from 77 pts (68 WM, 6 LPL, 3 IgM-MGUS), 86 baseline (64 BM, 22 PB) and 51 follow-up (23 BM and 28 PB), were analyzed. Median values at baseline were: age 67 years (range: 38-88), IgM 2.2 g/l (0.3-10.8), IgG for LPL 1.9 g/l (0.8-3.4), B2M 2.6 mg/l (0.14-7.9), infiltration at BM biopsy 45% (0-90%), BM infiltration by flow cytometry 10% (range: 0-87%). 12 pts had splenomegaly and 15 adenopathies. 63/64 (98.4%) diagnostic BM samples and 19/22 (86.4%) diagnostic PB samples scored positive for MYD88L265P (BM median 4.5%, range: 0.02-72.6%: PB median 0.15%, range: 0.01-27.8%) (all 3 negative PB had a positive BM match). To investigate the concordance between methods, 100 samples (60 BM, 40 PB) were tested by both ASqPCR and ddPCR. Overall a good concordance was observed (p=0.0005) with the majority of discordances found in the follow-up samples (13/60 ddPCR positive-ASqPCR negative, 11/60 ddPCR negative-ASqPCR positive). However, ddPCR was able to detect a higher number of mutated cases in diagnostic samples (38 vs 36). Moreover, to investigate whether MYD88L265P ddPCR could be used for MRD monitoring we compared it to the gold standard IGH-based MRD assay. Preliminary results on baseline and follow-up samples (18 BM, 5 PB) from 5 pts, selected from 33/57 (57.9%) displaying an IGH rearrangement, showed highly superimposable results between methods. Finally, pivotal results on cfDNA from 33 pts showed 1 log higher median levels of MYD88L265P mutation in cfDNA from plasma (0.7%, range 0-25,7%) compared to PB (0.037%, range: 0.01-20.0%).
Conclusion
MYD88L265P ddPCR is a feasible and highly sensitive assay for mutational screening and MRD monitoring in WM, particularly in samples harboring low concentrations of circulating tumor cells (e.g. PB after immunochemotherapy). Moreover, cfDNA from plasma samples represents a promising tissue source and might be an attractive, less invasive, alternative to PB or BM for MYD88L265P detection. Methodological validation against IGH-based MRD detection and flow cytometry, as well as correlations with clinical data, are currently ongoing.
Session topic: E-poster
Keyword(s): Minimal residual disease (MRD), PCR, Waldenstrom's macroglobulinemia
Type: Eposter Presentation
Background
MYD88L265P mutation is the earmark of Waldenström Macroglobulinemia (WM) and might represent an ideal marker for minimal residual disease (MRD) monitoring. However, current diagnostic tools, as allele-specific quantitative PCR (ASqPCR), are not sensitive enough for MRD monitoring on peripheral blood (PB), harboring low concentrations of circulating tumor cells. Besides, cell-free DNA (cfDNA) analysis has been shown to be highly promising for mutational studies. However, more sensitive approaches are needed and droplet digital PCR (ddPCR) might thus represent a powerful tool in this setting.
Aims
Here we describe the applications of a new, highly sensitive, ddPCR assay for MYD88L265P detection to assess: 1) its feasibility for MYD88L265P mutation screening and MRD monitoring in bone marrow (BM) and PB; 2) its potential application for mutational studies on cfDNA isolated from plasma.
Methods
BM, PB and plasma samples from a local series of WM and IgG-lymphoplasmacytic lymphomas (LPL) and IgM-MGUS patients (pts) were collected at baseline and during follow-up. 20 healthy subjects were used as negative controls. Genomic DNA (gDNA) and plasma derived cfDNA were extracted, according to the blood or body fluid protocols, by Maxwell RSC automatic system (Promega). MYD88L265P was assessed on gDNA (100ng) and cfDNA (5μl) by a custom ddPCR assay on a QX100 System (Bio-Rad). For comparison ASqPCR was assessed on gDNA (100ng), as described [Xu L, 2013]. MYD88L265P cut-off was settled based on the healthy samples background level. IGH-based MRD analysis was performed as described [Ladetto M, 2000; Drandi D, 2015].
Results
Once optimized, MYD88L265P ddPCR assay sensitivity was compared to ASqPCR on a ten-fold serial dilution standard curves. Whereas ASqPCR confirmed the reported sensitivity of 1.00E−03, ddPCR reached a sensitivity of 5.00E−05. Overall, 137 samples from 77 pts (68 WM, 6 LPL, 3 IgM-MGUS), 86 baseline (64 BM, 22 PB) and 51 follow-up (23 BM and 28 PB), were analyzed. Median values at baseline were: age 67 years (range: 38-88), IgM 2.2 g/l (0.3-10.8), IgG for LPL 1.9 g/l (0.8-3.4), B2M 2.6 mg/l (0.14-7.9), infiltration at BM biopsy 45% (0-90%), BM infiltration by flow cytometry 10% (range: 0-87%). 12 pts had splenomegaly and 15 adenopathies. 63/64 (98.4%) diagnostic BM samples and 19/22 (86.4%) diagnostic PB samples scored positive for MYD88L265P (BM median 4.5%, range: 0.02-72.6%: PB median 0.15%, range: 0.01-27.8%) (all 3 negative PB had a positive BM match). To investigate the concordance between methods, 100 samples (60 BM, 40 PB) were tested by both ASqPCR and ddPCR. Overall a good concordance was observed (p=0.0005) with the majority of discordances found in the follow-up samples (13/60 ddPCR positive-ASqPCR negative, 11/60 ddPCR negative-ASqPCR positive). However, ddPCR was able to detect a higher number of mutated cases in diagnostic samples (38 vs 36). Moreover, to investigate whether MYD88L265P ddPCR could be used for MRD monitoring we compared it to the gold standard IGH-based MRD assay. Preliminary results on baseline and follow-up samples (18 BM, 5 PB) from 5 pts, selected from 33/57 (57.9%) displaying an IGH rearrangement, showed highly superimposable results between methods. Finally, pivotal results on cfDNA from 33 pts showed 1 log higher median levels of MYD88L265P mutation in cfDNA from plasma (0.7%, range 0-25,7%) compared to PB (0.037%, range: 0.01-20.0%).
Conclusion
MYD88L265P ddPCR is a feasible and highly sensitive assay for mutational screening and MRD monitoring in WM, particularly in samples harboring low concentrations of circulating tumor cells (e.g. PB after immunochemotherapy). Moreover, cfDNA from plasma samples represents a promising tissue source and might be an attractive, less invasive, alternative to PB or BM for MYD88L265P detection. Methodological validation against IGH-based MRD detection and flow cytometry, as well as correlations with clinical data, are currently ongoing.
Session topic: E-poster
Keyword(s): Minimal residual disease (MRD), PCR, Waldenstrom's macroglobulinemia
Abstract: E1385
Type: Eposter Presentation
Background
MYD88L265P mutation is the earmark of Waldenström Macroglobulinemia (WM) and might represent an ideal marker for minimal residual disease (MRD) monitoring. However, current diagnostic tools, as allele-specific quantitative PCR (ASqPCR), are not sensitive enough for MRD monitoring on peripheral blood (PB), harboring low concentrations of circulating tumor cells. Besides, cell-free DNA (cfDNA) analysis has been shown to be highly promising for mutational studies. However, more sensitive approaches are needed and droplet digital PCR (ddPCR) might thus represent a powerful tool in this setting.
Aims
Here we describe the applications of a new, highly sensitive, ddPCR assay for MYD88L265P detection to assess: 1) its feasibility for MYD88L265P mutation screening and MRD monitoring in bone marrow (BM) and PB; 2) its potential application for mutational studies on cfDNA isolated from plasma.
Methods
BM, PB and plasma samples from a local series of WM and IgG-lymphoplasmacytic lymphomas (LPL) and IgM-MGUS patients (pts) were collected at baseline and during follow-up. 20 healthy subjects were used as negative controls. Genomic DNA (gDNA) and plasma derived cfDNA were extracted, according to the blood or body fluid protocols, by Maxwell RSC automatic system (Promega). MYD88L265P was assessed on gDNA (100ng) and cfDNA (5μl) by a custom ddPCR assay on a QX100 System (Bio-Rad). For comparison ASqPCR was assessed on gDNA (100ng), as described [Xu L, 2013]. MYD88L265P cut-off was settled based on the healthy samples background level. IGH-based MRD analysis was performed as described [Ladetto M, 2000; Drandi D, 2015].
Results
Once optimized, MYD88L265P ddPCR assay sensitivity was compared to ASqPCR on a ten-fold serial dilution standard curves. Whereas ASqPCR confirmed the reported sensitivity of 1.00E−03, ddPCR reached a sensitivity of 5.00E−05. Overall, 137 samples from 77 pts (68 WM, 6 LPL, 3 IgM-MGUS), 86 baseline (64 BM, 22 PB) and 51 follow-up (23 BM and 28 PB), were analyzed. Median values at baseline were: age 67 years (range: 38-88), IgM 2.2 g/l (0.3-10.8), IgG for LPL 1.9 g/l (0.8-3.4), B2M 2.6 mg/l (0.14-7.9), infiltration at BM biopsy 45% (0-90%), BM infiltration by flow cytometry 10% (range: 0-87%). 12 pts had splenomegaly and 15 adenopathies. 63/64 (98.4%) diagnostic BM samples and 19/22 (86.4%) diagnostic PB samples scored positive for MYD88L265P (BM median 4.5%, range: 0.02-72.6%: PB median 0.15%, range: 0.01-27.8%) (all 3 negative PB had a positive BM match). To investigate the concordance between methods, 100 samples (60 BM, 40 PB) were tested by both ASqPCR and ddPCR. Overall a good concordance was observed (p=0.0005) with the majority of discordances found in the follow-up samples (13/60 ddPCR positive-ASqPCR negative, 11/60 ddPCR negative-ASqPCR positive). However, ddPCR was able to detect a higher number of mutated cases in diagnostic samples (38 vs 36). Moreover, to investigate whether MYD88L265P ddPCR could be used for MRD monitoring we compared it to the gold standard IGH-based MRD assay. Preliminary results on baseline and follow-up samples (18 BM, 5 PB) from 5 pts, selected from 33/57 (57.9%) displaying an IGH rearrangement, showed highly superimposable results between methods. Finally, pivotal results on cfDNA from 33 pts showed 1 log higher median levels of MYD88L265P mutation in cfDNA from plasma (0.7%, range 0-25,7%) compared to PB (0.037%, range: 0.01-20.0%).
Conclusion
MYD88L265P ddPCR is a feasible and highly sensitive assay for mutational screening and MRD monitoring in WM, particularly in samples harboring low concentrations of circulating tumor cells (e.g. PB after immunochemotherapy). Moreover, cfDNA from plasma samples represents a promising tissue source and might be an attractive, less invasive, alternative to PB or BM for MYD88L265P detection. Methodological validation against IGH-based MRD detection and flow cytometry, as well as correlations with clinical data, are currently ongoing.
Session topic: E-poster
Keyword(s): Minimal residual disease (MRD), PCR, Waldenstrom's macroglobulinemia
Type: Eposter Presentation
Background
MYD88L265P mutation is the earmark of Waldenström Macroglobulinemia (WM) and might represent an ideal marker for minimal residual disease (MRD) monitoring. However, current diagnostic tools, as allele-specific quantitative PCR (ASqPCR), are not sensitive enough for MRD monitoring on peripheral blood (PB), harboring low concentrations of circulating tumor cells. Besides, cell-free DNA (cfDNA) analysis has been shown to be highly promising for mutational studies. However, more sensitive approaches are needed and droplet digital PCR (ddPCR) might thus represent a powerful tool in this setting.
Aims
Here we describe the applications of a new, highly sensitive, ddPCR assay for MYD88L265P detection to assess: 1) its feasibility for MYD88L265P mutation screening and MRD monitoring in bone marrow (BM) and PB; 2) its potential application for mutational studies on cfDNA isolated from plasma.
Methods
BM, PB and plasma samples from a local series of WM and IgG-lymphoplasmacytic lymphomas (LPL) and IgM-MGUS patients (pts) were collected at baseline and during follow-up. 20 healthy subjects were used as negative controls. Genomic DNA (gDNA) and plasma derived cfDNA were extracted, according to the blood or body fluid protocols, by Maxwell RSC automatic system (Promega). MYD88L265P was assessed on gDNA (100ng) and cfDNA (5μl) by a custom ddPCR assay on a QX100 System (Bio-Rad). For comparison ASqPCR was assessed on gDNA (100ng), as described [Xu L, 2013]. MYD88L265P cut-off was settled based on the healthy samples background level. IGH-based MRD analysis was performed as described [Ladetto M, 2000; Drandi D, 2015].
Results
Once optimized, MYD88L265P ddPCR assay sensitivity was compared to ASqPCR on a ten-fold serial dilution standard curves. Whereas ASqPCR confirmed the reported sensitivity of 1.00E−03, ddPCR reached a sensitivity of 5.00E−05. Overall, 137 samples from 77 pts (68 WM, 6 LPL, 3 IgM-MGUS), 86 baseline (64 BM, 22 PB) and 51 follow-up (23 BM and 28 PB), were analyzed. Median values at baseline were: age 67 years (range: 38-88), IgM 2.2 g/l (0.3-10.8), IgG for LPL 1.9 g/l (0.8-3.4), B2M 2.6 mg/l (0.14-7.9), infiltration at BM biopsy 45% (0-90%), BM infiltration by flow cytometry 10% (range: 0-87%). 12 pts had splenomegaly and 15 adenopathies. 63/64 (98.4%) diagnostic BM samples and 19/22 (86.4%) diagnostic PB samples scored positive for MYD88L265P (BM median 4.5%, range: 0.02-72.6%: PB median 0.15%, range: 0.01-27.8%) (all 3 negative PB had a positive BM match). To investigate the concordance between methods, 100 samples (60 BM, 40 PB) were tested by both ASqPCR and ddPCR. Overall a good concordance was observed (p=0.0005) with the majority of discordances found in the follow-up samples (13/60 ddPCR positive-ASqPCR negative, 11/60 ddPCR negative-ASqPCR positive). However, ddPCR was able to detect a higher number of mutated cases in diagnostic samples (38 vs 36). Moreover, to investigate whether MYD88L265P ddPCR could be used for MRD monitoring we compared it to the gold standard IGH-based MRD assay. Preliminary results on baseline and follow-up samples (18 BM, 5 PB) from 5 pts, selected from 33/57 (57.9%) displaying an IGH rearrangement, showed highly superimposable results between methods. Finally, pivotal results on cfDNA from 33 pts showed 1 log higher median levels of MYD88L265P mutation in cfDNA from plasma (0.7%, range 0-25,7%) compared to PB (0.037%, range: 0.01-20.0%).
Conclusion
MYD88L265P ddPCR is a feasible and highly sensitive assay for mutational screening and MRD monitoring in WM, particularly in samples harboring low concentrations of circulating tumor cells (e.g. PB after immunochemotherapy). Moreover, cfDNA from plasma samples represents a promising tissue source and might be an attractive, less invasive, alternative to PB or BM for MYD88L265P detection. Methodological validation against IGH-based MRD detection and flow cytometry, as well as correlations with clinical data, are currently ongoing.
Session topic: E-poster
Keyword(s): Minimal residual disease (MRD), PCR, Waldenstrom's macroglobulinemia
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