THE UTILITY OF FACS PURIFICATION OF PLASMA CELLS FOR FISH ANALYSIS IN MONOCLONAL GAMMOPATHIES
(Abstract release date: 05/18/17)
EHA Library. Pereira M. 05/18/17; 182717; PB2003
Dr. Marta Pereira
Contributions
Contributions
Abstract
Abstract: PB2003
Type: Publication Only
Background
Despite the prognostic value of chromosomal aberrations, conventional metaphase karyotyping in monoclonal gammopathies (MG) is often uninformative due to the inherent difficulty of obtaining proliferating plasma cells (PC).
Interphase fluorescence in situ hybridization (FISH) is a simple, quick and effective technique for the detection of cytogenetic aberrations that can overcome this limitation. However, the signal of interest is frequently diluted by the noise of the mixed cellularity of the sample, originating both false negatives and false positives.
Fluorescence-activated cell sorting (FACS) of the target cells enables a focused application of FISH on pathologically significant cells – such as the PC in MG – reducing the confounding noise. This is particularly relevant when the percentage of pathologic cells in the sample is low, such as in monoclonal gammopathy of undetermined significance (MGUS) where, by definition, there are less than 10% PC in the bone marrow.
Aims
This study aims to analyze the utility and effectiveness of FACS purification of PC for the cytogenetic workup of MG by FISH.
Methods
We analyzed all FISH studies performed in our laboratory, in individual patients, on clonal interphase FACS-separated bone marrow PC, between the 1st June 2015 and the 15th September 2016. The probes used in our standard MG panel were del(1p32), amp(1q21), t(4;14) and del(17p13.1) (TP53 gene) and, starting in April 2016, t(14;16). We had previously established 20 000 cells per sample as the minimum (and sufficient) number of cells needed to guarantee the confident application of all 5 probes in our lab.
Results
After the exclusion of samples diluted with peripheral blood, we identified 102 patients with FACS separated purified PC.
An average of 165 393±270 516 PC were separated per patient, and 98 of the cohort (96.1%) had a sufficient number of cells for the hybridization of at least one FISH probe; all 5 probes were applied in 30% of patients, 4 in 50%, 3 in 12% and 2 in 8%; the motives underlying the selection of fewer than all 5 probes in samples with a sufficient number (>20 000) of cells included the individual decision of the assisting physician and, for t(14;16), the date of the study. Considering only those studies performed after the introduction of t(14;16), all 5 probes were used in 67.5% of patients; we were able to apply four or more probes in 80% of patients with 1% or less bone marrow PC according to flow cytometry.
The median age of the 98 patients with a FISH result was 63.6 years old (37.8 to 87.3), and 56.1% were male; 41.8% eventually received a diagnosis of MGUS and 58.2% of multiple myeloma (MM), with an identical median age (64.2±6.9 vs 63.0±10.8 years old, p=NS).
We found that 16.3% (of 92) were positive for t(4;14), 12.2% (of 90) for del(17p13.1), 5.6% (of 90) for del(1p32) and 41.1% (of 90) for amp(1q21); t(14:16) was not identified in any of the 30 patients in whom the probe was used.
The t(4;14) translocation was present in 22.4% of MM and 7.7% of MGUS patients (p=0.055), and del(17p13.1) was found in 18.5% vs 2.8% (p=0.026); on the other hand, both del(1p32) (5.6% vs 5.6%, p=NS) and amp(1q21) (46.3% vs 33.3%, p=NS) were identically distributed across diagnoses.
We observed that 40.4% of MM and 65.8% of MGUS patients were positive for 20% of less of the tested aberrations, while 54.4% vs 34.2% were positive for 20 to 50%, and 5.3% vs 0% were positive for over 50% of the aberrations (p=0.026).
Conclusion
We have found that the application of FISH probes in FACS-separated PC is highly efficient with a robust yield, providing a large enough sample for the application of at least two probes in over 95% of patients, irrespective of bone marrow plasmacytosis; in fact, we obtained an average of 165 000 pure PC per patient, which is more than 8-fold higher than the number we consider invariably sufficient to apply 5 probes, which we achieved in at least 80% of patients.
Session topic: 14. Myeloma and other monoclonal gammopathies - Clinical
Keyword(s): Monoclonal gammopathy, flow cytometry, FISH, Multiple Myeloma
Abstract: PB2003
Type: Publication Only
Background
Despite the prognostic value of chromosomal aberrations, conventional metaphase karyotyping in monoclonal gammopathies (MG) is often uninformative due to the inherent difficulty of obtaining proliferating plasma cells (PC).
Interphase fluorescence in situ hybridization (FISH) is a simple, quick and effective technique for the detection of cytogenetic aberrations that can overcome this limitation. However, the signal of interest is frequently diluted by the noise of the mixed cellularity of the sample, originating both false negatives and false positives.
Fluorescence-activated cell sorting (FACS) of the target cells enables a focused application of FISH on pathologically significant cells – such as the PC in MG – reducing the confounding noise. This is particularly relevant when the percentage of pathologic cells in the sample is low, such as in monoclonal gammopathy of undetermined significance (MGUS) where, by definition, there are less than 10% PC in the bone marrow.
Aims
This study aims to analyze the utility and effectiveness of FACS purification of PC for the cytogenetic workup of MG by FISH.
Methods
We analyzed all FISH studies performed in our laboratory, in individual patients, on clonal interphase FACS-separated bone marrow PC, between the 1st June 2015 and the 15th September 2016. The probes used in our standard MG panel were del(1p32), amp(1q21), t(4;14) and del(17p13.1) (TP53 gene) and, starting in April 2016, t(14;16). We had previously established 20 000 cells per sample as the minimum (and sufficient) number of cells needed to guarantee the confident application of all 5 probes in our lab.
Results
After the exclusion of samples diluted with peripheral blood, we identified 102 patients with FACS separated purified PC.
An average of 165 393±270 516 PC were separated per patient, and 98 of the cohort (96.1%) had a sufficient number of cells for the hybridization of at least one FISH probe; all 5 probes were applied in 30% of patients, 4 in 50%, 3 in 12% and 2 in 8%; the motives underlying the selection of fewer than all 5 probes in samples with a sufficient number (>20 000) of cells included the individual decision of the assisting physician and, for t(14;16), the date of the study. Considering only those studies performed after the introduction of t(14;16), all 5 probes were used in 67.5% of patients; we were able to apply four or more probes in 80% of patients with 1% or less bone marrow PC according to flow cytometry.
The median age of the 98 patients with a FISH result was 63.6 years old (37.8 to 87.3), and 56.1% were male; 41.8% eventually received a diagnosis of MGUS and 58.2% of multiple myeloma (MM), with an identical median age (64.2±6.9 vs 63.0±10.8 years old, p=NS).
We found that 16.3% (of 92) were positive for t(4;14), 12.2% (of 90) for del(17p13.1), 5.6% (of 90) for del(1p32) and 41.1% (of 90) for amp(1q21); t(14:16) was not identified in any of the 30 patients in whom the probe was used.
The t(4;14) translocation was present in 22.4% of MM and 7.7% of MGUS patients (p=0.055), and del(17p13.1) was found in 18.5% vs 2.8% (p=0.026); on the other hand, both del(1p32) (5.6% vs 5.6%, p=NS) and amp(1q21) (46.3% vs 33.3%, p=NS) were identically distributed across diagnoses.
We observed that 40.4% of MM and 65.8% of MGUS patients were positive for 20% of less of the tested aberrations, while 54.4% vs 34.2% were positive for 20 to 50%, and 5.3% vs 0% were positive for over 50% of the aberrations (p=0.026).
Conclusion
We have found that the application of FISH probes in FACS-separated PC is highly efficient with a robust yield, providing a large enough sample for the application of at least two probes in over 95% of patients, irrespective of bone marrow plasmacytosis; in fact, we obtained an average of 165 000 pure PC per patient, which is more than 8-fold higher than the number we consider invariably sufficient to apply 5 probes, which we achieved in at least 80% of patients.
Session topic: 14. Myeloma and other monoclonal gammopathies - Clinical
Keyword(s): Monoclonal gammopathy, flow cytometry, FISH, Multiple Myeloma
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