Abstract
Trisomies of odd numbered chromosomes are seen in nearly half of patients with multiple myeloma (MM) and typically correlate with a hyperdiploid state and better overall survival (OS). We compared DNA ploidy of monoclonal plasma cells (as a surrogate for the presence of trisomies) assessed simultaneously by PCPRO (plasma cell proliferative index), a novel method that estimates DNA index by multi-parametric flow cytometry to fluorescence in-situ hybridization (FISH) in 1703 patients with plasma cell disorders. The distribution of ploidy was hyperdiploid: 759 (45%), diploid 765 (45%), hypodiploid: 71 (4%), tetraploid/near-tetraploid: 108 (6%).
FISH identified trisomies in 82% (621/756) of patients with hyperdiploidy by PCPRO and no trisomy by FISH was observed in 88% (730/834) of patients without hyperdiploidy. 95% (795/834) of patients without hyperdiploidy on PCPRO had one or less trisomy by FISH. Sensitivity and specificity of PCPRO for detecting hyperdiploidy was 86% (621/725) and 84% (730/865), respectively. Sensitivity increased to 94% (579/618) for patients with more than one trisomy.
Newly diagnosed MM patients with hyperdiploidy on PCPRO (147/275) had better OS compared to non-hyperdiploid patients (median not reached vs. 59 months, p=0.008) and better progression free survival (median: 33 vs. 23 months, p=0.03). Within the hyperdiploidy group, patients with high-hyperdiploidy (DNA index: 1.19–1.50) vs. those with low-hyperdiploidy (DNA index: 1.05–1.18) had superior OS (3 year OS of 88% vs. 68% p=0.03). Ploidy assessment by flow cytometry can provide rapid, valuable prognostic information and also reduces the number of copy number FISH probes required and hence the cost of FISH.
Keywords: ploidy, hyperdiploidy, myeloma, plasma cell disorders, fluorescence in-situ hybridization, trisomies
INTRODUCTION:
A subset of patients with multiple myeloma (MM) and other monoclonal gammopathies exhibit hyperdiploidy with extra copies of odd numbered chromosomes.1–5 Such trisomies are most commonly identified by interphase fluorescence in-situ hybridization (FISH) testing and are associated with a favorable prognosis in MM.2,3,6,7 There is also evidence to suggest that trisomies, in particular trisomies of chromosome 3 and 5 may overcome the impact of high-risk cytogenetics, at least in part3,6, though not all studies support this conclusion.8 However, a major drawback of the current method for evaluating hyperdiploidy by FISH is the need for using multiple probes for chromosomes 3, 5, 7, 9, 11, 15, 17 and 21. This increases both the cost and effort of testing, thus there is a need for a more convenient method to detect hyperdiploidy.
We have developed a flow cytometry based method called Plasma Cell Proliferative Index (PCPRO), which can evaluate hyperdiploidy by determining the DNA index (see Methods section).9 Unlike FISH, this method does not need multiple probes and testing can be completed more rapidly, allowing results to be available within a day. While assessment of ploidy in plasma cell disorders by evaluating DNA index has been used for a long time7,10–15, this method utilizes advanced multi-parametric flow cytometric techniques and allows use of hyperdiploidy as a surrogate marker for the presence of trisomies in the clonal plasma cells. This study compares hyperdiploidy determined by the flow cytometry based PCPRO method to hyperdiploidy (presence of trisomies) detected by FISH.
METHODS:
We evaluated 1703 patients with newly diagnosed or previously treated multiple myeloma and other plasma cell disorders (Table 1) who underwent flow cytometry based evaluation for assessing the DNA index (PCPRO) and had simultaneous productive FISH testing on bone marrow. Patients who did not have monotypic plasma cells for PCPRO evaluation, lacked polytypic plasma cells, (which serve as control for DNA index evaluation) and the rare patients who had co-dominant clones with different ploidy identified, were excluded. Similarly, patients who had insufficient or no plasma cells for successful FISH testing were also excluded from the main analysis. Patients with newly diagnosed, previously untreated multiple myeloma were evaluated as a sub-cohort to determine the prognostic value of hyperdiploidy by PCPRO testing and in this analysis patients who had insufficient plasma cells for FISH were retained. All patients had provided informed consent for review of their medical records for research purposes and this study was approved by the Institutional Review Board.
Table 1:
Distribution of FISH abnormalities amongst various ploidy groups
| Hyperdiploid(N=759) | Diploid(N=765) | Hypodiploid (N=71) | P-value | |
|---|---|---|---|---|
| Any trisomy | 621/756 (82%) | 96/763 (13%) | 8/71 (11%) | <0.001 |
| One chromosome | 42/621 (7%) | 58/96 (60%) | 7/8 (87%) | |
| 7 | 3 | 3 | 1 | |
| 9 | 15 | 29 | - | |
| 11 | 13 | 6 | 2 | |
| 15 | - | 7 | - | |
| Others | 11 | 13 | 4 | |
| Two chromosomes | 85/621 (14%) | 25/96 (26%) | - | |
| Three or more chromosomes | 494/621 (80%) | 13/96 (14%) | 1/8 (13%) | |
| Any IgH translocation | 121/734 (17%) | 568/751 (76%) | 53/68 (78%) | <0.001 |
| t(11;14) | 19/121 (16%) | 403/568 (71%) | 27/53 (51%) | |
| t(4;14) | 11/121 (9%) | 63/568 (11%) | 13/53 (25%) | |
| t(14;16) | 4/121 (3%) | 32/568 (6%) | 5/53 (9%) | |
| t(14;20) | 2/121 (2%) | 8/568 (1%) | - | |
| t(6;14) | 8/121 (7%) | 10/568 (2%) | 4 (8%) | |
| Unknown partner | 77/121 (64%) | 52/568 (9%) | 4 (8%) | |
| Monosomy 13/17 or deletion 17p/13q | 248/752 (33%) | 296/747 (40%) | 54/68 (79%) | <0.001 |
| Monosomy/deletion 13/17 or any translocation | 314/735 (31%) | 639/750 (85%) | 65/69 (94%) | <0.001 |
Total percentage may exceed 100% due to rounding of percentage values
Plasma Cell Proliferative Index (PCPRO) Assessment:
To assess the DNA index, the bone marrow specimen was spun down and the pellet was lysed using 14 mL of ACK lysing buffer (Thermo Fisher Scientific), followed by two washes with PBS. The cell pellet was then re-suspended in 0.2% BSA/PBS with Azide (BD Pharmingen) and stained with the following antibodies: CD138 PerCPcy5.5, CD19 PE-cy7, CD38 FITC, CD45 APC-H7 (all BD Biosciences) for 15 minutes. Following the wash in Caltag A reagent (Thermo Fisher Scientific), the pellet was re-suspended in Caltag B reagent for permeabilization. Antibodies for cytoplasmic staining were added (Kappa APC and Lambda PE – both from Dako North America Inc.), and the specimen was incubated for 20 minutes. This was followed by the wash step and incubation in the 1000 units/mL RNAse in PBS (Worthington Biochemical Corporation). 21.4 μM working dilution of DAPI (Life Technologies) was added to the cell suspension and incubated at 4°C for 30 minutes. The cell pellet was then re-suspended in 500 μL of PBS. The flow cytometry FCS files were obtained on BD FACSCanto™ II instruments (500,000 events per specimen). The files were analyzed using Kaluza software (Beckman Coulter). Initial broad gates were set on CD138+CD38+ events. The clonal (abnormal) plasma cells were separated from the normal plasma cells using differential expression of CD38, CD19, CD45, kappa, and lambda. DAPI staining on polyclonal plasma cells was used to determine ploidy. A doublet exclusion gate was used and aggregates/doubles were excluded by forward scatter (FSC) height/FSC area plot. The ploidy was calculated by dividing DAPI binding of G0G1 peak between abnormal and normal plasma cell population (DAPI abnormal/DAPI normal). Values of 0.95–1.05 were considered diploid. Values below 0.95 were considered hypodiploid and values between 1.06 – 1.50 were considered hyperdiploid. Values from between 1.51 – 1.7 were considered near-tetraploid and values >1.7 with >10% clonal G2M cells and a visible 4n population were considered tetraploid. Supplementary Figure 1 shows the gating technique as well as an example of two patient samples with hyperdiploid and diploid DNA indices.
FISH Assessment:
FISH testing was carried out by interphase cytoplasmic immunoglobulin FISH (cIg-FISH) for plasma cell disorders as previously described.3,16
Analysis:
Hyperdiploidy by DNA index evaluation with the PCPRO method was compared to trisomies detected by interphase FISH, which was done simultaneously on the bone marrow. Positive predictive value, negative predictive value, sensitivity and specificity of ploidy assessment by PCPRO compared to trisomy assessment by FISH was assessed for the overall population and sub-groups of patients with loss of genetic material that can lead to a decrease in DNA content. These sub-groups with loss of genetic material primarily include patients with monosomy 13/deletion 13q or monosomy 17/deletion 17p. Receiver operating curve (ROC) analysis was conducted to determine optimal cut-off for DNA index to detect trisomies in the sub-group of patients with monosomy 13/deletion 13q or monosomy 17/deletion 17p. Linear correlation between DNA index and chromosome count was assessed with Spearman’s ρ correlation coefficient.
A subset analysis was also carried out for patients who underwent testing at the time of diagnosis of multiple myeloma, prior to initiating therapy. In this group of patients with newly diagnosed multiple myeloma (NDMM), analysis for overall survival (OS) and progression free survival (PFS) was carried out by the Kaplan Meier method and the log-rank test was used to compare survival curves to evaluate prognostic impact of hyperdiploidy on survival. Within the subgroup of patients with hyperdiploidy, ROC analysis was done to determine the cut-off for DNA index with respect to survival outcomes.
RESULTS:
We identified 1703 unique patients with newly diagnosed or previously treated multiple myeloma or other plasma cell disorders who met inclusion criteria. FISH and PCPRO testing was carried out between May 2012 and Sept 2016. Distribution of the underlying primary diagnosis amongst 1703 patients included in the study cohort was as follows: symptomatic MM (56%, n=958), smoldering MM (6%, n=100), primary light/heavy chain amyloidosis (18.5%, n=317),monoclonal gammopathy of undetermined significance (12%, n=206), Waldenstrom’s Macroglobulinemia (WM)/ Smoldering WM (1%, n=18), plasma cell leukemia (1%, n=17), light/ heavy chain deposition disease (1%, n=14), plasmacytoma (0.5%, n=8) and other diagnoses (4%, n=65). Of these, there were 264 (16%) patients with newly diagnosed MM, who underwent testing before treatment initiation. The distribution of ploidy in the entire cohort was as follows: hyperdiploid: 759 (45%), diploid 765 (45%), hypodiploid: 71 (4%), and near-tetraploid/tetraploid: 108 (6%). The distribution of FISH abnormalities amongst various ploidy groups is shown in Table 1. Five patients had insufficient plasma cells for trisomy analysis. Patients were tetraploidy were excluded for this analysis. Trisomies of odd numbered chromosomes were most common in the hyperdiploid group, observed in 82% of patients. The most common abnormality in the diploid group was translocation of the IgH locus, observed in 76% of patients. In the hypodiploid group, both translocation of the IgH locus and monosomy or deletion of chromosomes 13 or 17 were common and were seen in 78% and 79% of the patients in this group, respectively.
Correlation between hyperdiploidy and trisomies:
Table 2 describes the correlation of ploidy analysis by the PCPRO method and trisomies of odd numbered chromosomes detected by FISH testing. Positive and negative predictive values, as well as sensitivity and specificity are reported for all patients and for the subset of patients with NDMM. Patients with tetraploidy or near-tetraploidy (n=108) on PCPRO testing or those with FISH with insufficient cells for trisomy probes (n=5) were excluded for this analysis as well. Of the remaining 1590 patients, 82% (621/756) of patients with hyperdiploidy on PCPRO analysis were found to have a trisomy by FISH (PPV) and 88% (730/834) of patients who were non-hyperdiploid by PCPRO analysis did not have a trisomy on simultaneous FISH testing (NPV). The NPV increased to 95% (795/834) for patients without hyperdiploidy and presence of only one or no trisomy by FISH. The sensitivity of hyperdiploidy by PCPRO to detect trisomies compared to FISH was 86% (621/725) for any trisomy and increased to 94% (579/618) for the presence of more than one trisomy. As shown in Table 2, when patients with monosomy/deletion of chromosome 13 or 17 were excluded, 81% (407/502) of patients with hyperdiploidy had a simultaneous trisomy on FISH (PPV) and 92% (427/465) of patients without hyperdiploidy were negative for the presence of trisomy on FISH (NPV), which further increased to 98% (455/465) for the presence of one or no trisomy. ROC analysis was conducted to determine optimal cut-offs for DNA index for presence of trisomy on FISH. In patients without presence of monosomy/deletion of chromosome 13 or 17, optimal DNA index cut-off to detect a trisomy was 1.06 (sensitivity: 92%, specificity: 79%), which is the same as the cut-off used for PCPRO testing prior to current study as reported in methods section. In the subset of patients with presence of monosomy/deletion of chromosome 13 or 17 or both, the optimal DNA index cut-off to detect a trisomy was 1.03 (sensitivity: 86%, specificity: 70%).
Table 2:
Correlation of ploidy by PCPRO vs. FISH to detect trisomies
| Positive predictive value (any trisomy) | Negative predictive value (any trisomy) | Negative predictive value (>1 trisomy) | Sensitivity (any trisomy) | Sensitivity (>1 trisomy) | Specificity (any trisomy) | |
|---|---|---|---|---|---|---|
| All patients in cohort (N=1590) | ||||||
| Entire cohort | 82% (621/756) | 88% (730/834) | 95% (795/834) | 86% (621/725) | 94% (579/618) | 84% (730/865) |
| Excluding monosomy 13/del 13q or monosomy 17/del17p | 81% (407/502) | 92% (427/465) | 98% (455/465) | 91% (407/445) | 97% (376/386) | 82% (427/522) |
| Newly diagnosed multiple myeloma (N=264) | ||||||
| All patients with NDMM | 89% (124/140) | 81% (84/104) | 92% (96/104) | 86% (124/144) | 94% (117/125) | 84% (84/100) |
| Excluding monosomy 13/del 13q or monosomy 17/del17p | 93% (79/85) | 82% (36/44) | 96% (42/44) | 91% (79/87) | 97% (75/77) | 82% (36/44) |
In the subset of patients with newly diagnosed MM, the PPV, NPV, sensitivity and specificity of ploidy assessment by PCPRO compared to FISH were even higher (Table 2). In this group, 89% (124/140) of patients with hyperdiploidy on PCPRO had a trisomy on FISH (PPV), while 81% (84/104) of patients without hyperdiploidy did not have a trisomy (NPV), which increased to 92% (96/104) for presence of one or no trisomy. The sensitivity was 86% for any trisomy and 94% for more than one trisomy. The specificity in this cohort was 84% (84/100).
Chromosome count using FISH results was estimated by adding a point for trisomy or trisomy/tetrasomy, adding two points for tetrasomy and subtracting one point for monosomy 13/17 or deletion of one of the arms of these chromosomes. We observed a strong linear correlation between DNA index and chromosome count, with Spearman’s ρ correlation coefficient of 0.75, p <0.001. (Supplementary Figure 2)
Newly Diagnosed MM:
There were 275 patients with NDMM who were diagnosed from 4/2/2012 to 12/03/2015 and underwent PCPRO testing. Along with 264 patients (of 1703 total) in the main analysis, there were eleven additional patients who underwent PCPRO testing in whom FISH data was not available (insufficient plasma cells on FISH, n=10; FISH testing not done, n=1). Survival analysis was conducted in all 275 patients with PCPRO data at diagnosis. Distribution of ploidy in this group was as follows: hyperdiploid: 147 (53%), diploid: 98 (36%), hypodiploid: 10 (4%), near-tetraploid: 3 (1%) and tetraploid: 17 (6%). Median follow-up from start of treatment was 49 months. All patients, except one in whom treatment details were not known, received novel agent based therapy. First-line treatment was transplant based in 39% (108/275) of the patients.
Survival outcomes:
Patients with newly diagnosed MM with hyperdiploidy at diagnosis based on DNA index had superior survival outcomes, both for OS and PFS (Figures 1a–b). Median OS in patients in patients with hyperdiploidy (n=147) was not reached vs. 59 months in those with non-hyperdiploidy (n=128) by DNA index, p=0.008. Similarly, median PFS in patients with hyperdiploidy vs. non-hyperdiploidy by DNA index was: 33 vs. 23 months, p=0.03. In the cohort of patients with hyperdiploidy, the DNA index cut-off of 1.19 was selected based on ROC analysis to divide patients into two groups: high-hyperdiploidy (DNA index of 1.19–1.50) and low-hyperdiploidy (DNA index 1.06–1.18). Two patients could not be categorized due to the presence of more than one hyperdiploid clone. Patients with high-hyperdiploidy (n=77) had superior OS compared with low-hyperdiploidy (n=69), though median OS was not reached in either group. OS at 1, 3 and 5 years in the high-hyperdiploidy and low-hyperdiploidy groups was 99%, 88% and 75% vs. 86%, 68% and 64%, respectively, p=0.03. Median PFS in the high vs. low-hyperdiploidy group was 41 vs. 30 months, p=0.10. (Figure 1c–d)
Figure 1: Survival analysis in newly diagnosed multiple myeloma based on hyperdiploidy by PCPRO.
Figure 1a: Overall Survival (OS) based on presence or absence of hyperdiploidy
Hyperdiploidy present, N=147, median OS: not reached
Non-hyperdiploidy, N= 128, median OS: 59 months
P=0.008
Figure 1b: Progression Free Survival (PFS) based on presence or absence of hyperdiploidy
Hyperdiploidy present N=147, median PFS: 33 months
Non-hyperdiploidy N= 128, median PFS: 23 months
P=0.03
Figure 1c: OS based on presence high-hyperdiploidy (DNA index 1.19–1.50) vs. low-hyperdiploidy (DNA index 1.06–1.18)
High hyperdiploid N=77, median OS: not reached
Low hyperdiploid, N=69, median OS: not reached
P=0.03
Figure 1d: PFS based on presence high-hyperdiploidy (DNA index 1.19–1.50) vs. low-hyperdiploidy (DNA index 1.06–1.18)
High hyperdiploid N=77, median PFS: 41 months
Low hyperdiploid, N=69, median PFS: 30 months
2 patients had more than one clone, so DNA index not categorized for those patients (Figures 2c–d)
DISCUSSION
In this study, we have compared hyperdiploidy assessment by PCPRO, which estimates the DNA index by multi-parametric flow cytometry to that assessed by routine cIg-FISH testing. We observed that DNA index assessment by PCPRO has high positive and negative predictive value for detecting hyperdiploidy in all patients with plasma cell disorders, including patients with newly diagnosed myeloma, where such prognostic information is of most value.17 The correlation further increased when common genetic abnormalities that result in loss of DNA content were excluded. In our cohort, 82% of patients with hyperdiploidy on PCPRO had trisomies on FISH. When PCPRO testing did not indicate hyperdiploidy, 88% of patients did not have a trisomy on FISH testing, which increased to 92% after excluding patients with deletion or monosomy of chromosomes 13 or 17.
FISH testing plays a critical role in determining the prognosis of patients with multiple myeloma and other plasma cell disorders, both at diagnosis and later in the disease course.18,1,3,16,19,20 Presence of hyperdiploidy as defined by trisomies of odd numbered chromosomes is associated with a better prognosis, while the presence of certain IgH translocations or other abnormalities such as 17p deletion are associated with inferior survival. Although FISH testing is widely available, one major drawback is that multiple probes are required to test for various FISH abnormalities and the FISH assay is expensive, time consuming and labor intensive. Increasingly, additional probes are being used as we learn more about the biology and risk assessment of plasma cell disorders. For example, in recent years we have added probes for assessment of MYC translocation and gain of 1q and 1p loss. Although this information may provide additional prognostic data, it increases both the time and effort of testing, resulting in delays and added costs. A large number of probes being tested can also lead to incomplete testing when insufficient plasma cells are present for testing all probes. Therefore, there is a need for a more rapid and cost-effective method to assess these genetic abnormalities. Such a method can also useful provide prognostic information in resource deficient geographic regions, where FISH testing may not be as widely available as flow cytometry.
PCPRO testing quantitates the overall DNA index of plasma cells using multi-parametric flow cytometry. Hyperdiploidy increases the DNA index, but loss of genetic material can lower this value. In plasma cell disorders, common abnormalities resulting in loss of genetic material include monosomy or deletion of chromosomes 13 and 17. Therefore, we conducted sub-group analyses after excluding patients with these known abnormalities. We further identified cut-offs for DNA index by ROC analysis for patients with monosomy or partial deletion of chromosomes 13 and 17, which may further increase sensitivity of trisomy detection in this sub-group. These results suggest that an algorithmic approach to testing which integrates both FISH and PCPRO testing may be ideal to provide prognostic information and optimize the number of FISH probes tested. Such an algorithm is described in Figure 2, with a step wise approach incorporating results from PCPRO analysis (data available within one day) followed by FISH testing and a lower cut-off for hyperdiploidy assessment in patients with loss of genetic material as determined by ROC analysis in this sub-group. This sample algorithm may be adapted to suit institutional needs based on laboratory workflow.
Figure 2:
An example algorithmic approach for evaluation of hyperdiploidy with PCPRO and FISH testing
Hyperdiploidy was observed in 53% of patients with newly diagnosed myeloma in our cohort, which is similar to prior observations.1,2,5 In the subset of patients with newly diagnosed MM in our study, presence of hyperdiploidy by PCPRO testing was associated with better survival outcomes. This is consistent with prior observations of hyperdiploidy assessed with FISH or older flow cytometry techniques to assess DNA content.1,2,12,13 While association of hyperdiploidy with better OS is well known, previous studies have not evaluated the cumulative effect of multiple trisomies. We observed that amongst the hyperdiploidy group, patients with high hyperdiploidy (DNA index of 1.19 or greater) had better overall survival (3 year OS 88% vs. 68%, p=0.03) and numerically higher PFS, though the difference did not reach statistical significance (41 vs. 30 months, p=0.10). This suggests a cumulative protective effect of multiple trisomies.
PCPRO testing has several advantages, including a rapid turn-around time with results often obtained within one day. Moreover, the majority of testing is automated and requires less technician support than FISH testing. DNA index testing to evaluate ploidy status in multiple myeloma has been described before.7,11,13 A strength of our study compared to prior reports is comparison of ploidy status by DNA index vs. FISH testing in a very large cohort of patients who underwent simultaneous testing for both DNA index and FISH. Limitations of PCPRO testing include the lack of detection of individual trisomies, and the need for a fresh sample for flow cytometry as antigen shedding limits testing on aged specimens. Hyperhaploidy, although rare, has been described as a poor prognostic factor in case series of patients with myeloma. Very rarely, patients with plasma cell disorders may have a double hyperhaploid clone which may mimic a hyperdiploid clone on PCPRO (Jess F. Peterson, P.T.G., R.P.K, L.B.B, et al.; manuscript submitted November 2018). 21 PCPRO is unable to identify double hyperhaploid clones, but this is a very rare abnormality and patients often have deletion of chromosome 17, which can be identified with a combined approach. Another limitation of our study is that data on presence of gain1q or deletion 1p was not available for all patients, and therefore has not been included in the analysis.
In conclusion, evaluation of hyperdiploidy by DNA index measurement using MFC has a high sensitivity to detect hyperdiploidy in plasma cell disorders, especially when used in an algorithmic approach with FISH testing. Implementation of such an approach can significantly decrease the effort, time, and cost of testing, while still providing relevant prognostic information.
Supplementary Material
ACKNOWLEDGEMENTS:
This publication was made possible by CTSA Grant Number UL1 TR002377 from the National Center for Advancing Translational Sciences (NCATS), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NIH.
CONFLICTS OF INTEREST/FINANCIAL DISCLOSURES: SS: Honoraria/consultancy: Janssen; AD: Consultant for Takeda; Research funding from Celgene, Takeda, Janssen; GlaxoSmithKline, Alnylam, and Pfizer; MAG: Honoraria/consultancy from Ionis, Alnylam, Prothena, Celgene, Janssen, Specytrum, Annexon, Apellis, Amgen, Medscape, Abbvie, Research to Practice, Physcians Education Resource and Teva; PK: Research funding from Celgene, Takeda; MQL: Research Funding from Celgene; NL: Membership on an entity’s Board of Directors or advisory committees: Takeda; SKK: Research Funding and membership on an entity’s Board of Directors or advisory committees: AbbVie, Celgene, Janssen KITE, Merck. Membership on an entity’s Board of Directors or advisory committees: Oncopeptides, Takeda. Research funding from Novartis and Roche; DD: Consulting from Takeda/Millenium, Alexion Pharmaceuticals, Rigel Pharmaceuticals and Janssen. Remaining authors: None.
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