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haematologica | 2021; 106(6)
Impact of translocations in mutated IGHV CLL at diagnosis
The karyotype of the patient is another genetic factor that influences disease course and response to therapy. The complexity of the karyotype is prognostic with three or more unrelated abnormalities predictive of a more adverse prognosis.4-12 Furthermore, a complex kary- otype has been associated with unmutated IGHV, CD38 expression and deletion of 17p or mutated TP53.5,9,11,13-16 Additional studies showed that in addition to complex karyotypes, translocations, particularly unbalanced translocations, were associated with a poor prognosis, reflected as both an increased risk of requirement for treatment and a shortened overall survival.5,9,17-19 Translocations have also been associated with a complex karyotype and with deletions of 17p, and in some cases with del(11q),9 but not in others.20 While these studies are informative, the relationship of translocations to mutat- ed versus unmutated IGHV detected early in the course of the disease is unclear.
Traditionally metaphase cytogenetic studies of CLL have been minimally informative, as CLL cells do not readily divide spontaneously in culture, nor do they respond to traditional B-cell mitogens. The advent of CpG oligodeoxynucleotide stimulation of CLL cells has facilitated detection of cytogenetic abnormalities in up to 80% of CLL patients.17,21-26 As a result of this increased detection of karyotypic abnormalities, the significance of translocations and of a complex karyotype at disease diagnosis and during disease progression can be assessed more accurately. As CLL progresses, the karyotype fre- quently evolves.27,28 Therefore, the significance of kary- otypic complexity and/or translocations at disease pres- entation is not clear. We examined CpG-stimulated kary- otypes completed within 1 year of diagnosis of CLL and correlated the presence of a translocation and of com- plexity with disease prognosticators such as IGHV muta- tional status, sex, age, Rai stage, FISH abnormalities and with time to first treatment (TFT).
Methods
Patients and samples
All patients had immunophenotypically defined B-cell CLL as outlined by the International Workshop on CLL Revised CLL Guidelines.29 Peripheral blood or bone marrow was collected from patients after having obtained written informed consent in accordance with the Declaration of Helsinki and under a proto- col approved by the institutional review board of The Ohio State University (Columbus, OH, USA).
Cytogenetic analyses
Cells were stimulated with CpG oligodeoxynucleotides and analyzed according to standard laboratory procedures (described in detail in the Online Supplementary Methods). FISH using probes for D13S319, D12Z3, ATM, TP53, BCL6, MYC, MYB (Abbott Molecular, Des Plaines, IL, USA) and SEC63 (Empire Genomics, Buffalo, NY, USA) were done according to the manufacturers’ recommendations.
Patients’ inclusion criteria
Patients with CLL who had cytogenetic analyses between December 2006 and December 2013 and were within 1 year of diagnosis were identified from The Ohio State University Wexner Medical Center databases. Balanced and unbalanced translocations and insertions were identified. Inversions were
considered as balanced translocations, and “adds” as unbal-
anced translocations. A complex karyotype was defined as ≥3
independent aberrations on metaphase cytogenetics. The asso-
ciation of complex karyotype defined as ≥5 independent aber-
rations with outcome was also examined. Baseline characteris-
tics were obtained from the patients’ charts. White blood cell
(WBC) count and b -microglobulin concentration, determined 2
within 90 days of diagnosis were used.
Statistical analyses
The patients’ demographic and genetic characteristics were described using mean and standard deviation or median and range for continuous variables, and frequency and percentage for categorical variables. Associations between translocations or cytogenetic complexity with FISH abnormalities were tested using Fisher exact tests. Cases with both balanced and unbal- anced translocations were classified together for statistical analyses.
TFT was calculated from the date of diagnosis until the date of first treatment or last follow-up, censoring patients who had not started treatment at the date last seen; patients who died prior to starting treatment were censored at the date last seen. Kaplan-Meier curves were used to estimate TFT probability, and Cox proportional hazard models were used to examine the association between potential risk factors and TFT. Table 1 shows variables considered for modeling. WBC counts were log-transformed.
Significant risk factors from univariable Cox models were included in the multivariable Cox model. Due to missing values in IGHV mutation status (n=37, 11%), WBC count (n=83, 25%), and b2-microglobulin (n=146, 44%), a multiple imputation pro- cedure was applied to obtain combined results from 15 multiply imputed datasets. Using stepwise backward selection, variables that reached statistical significance after adjusting for all other covariates remained in the final model. To evaluate for potential effect modification, pairwise interactions among all variables in the final model were further tested, and TFT curves were gen- erated.
Among patients who started chemoimmunotherapy, overall survival from the start of therapy was determined. Univariable analyses were performed testing for risk factors associated with overall survival, but multivariable analyses were precluded because of the limited number of deaths. The analyses were conducted using Stata 14, SAS 9.4 and the statistical signifi- cance was set as P<0.05.
Results
Patients’ characteristics
Diagnostic samples meeting the above criteria were identified for 329 patients. The patients’ clinical data are presented in Table 1. The median age of the patients was 60 years (range, 34-88), 36.8% were female, and 90.2% had Rai stage 0-2.
Unbalanced translocations are more frequent in complex karyotypes, but balanced translocations are more frequent in non-complex karyotypes
Translocations occurred in 85 (25.8%) patients: 29 with balanced, 48 with unbalanced and eight with both a balanced and an unbalanced translocation. Defining complexity as ≥3 unrelated aberrations, 16.1% of patients had complex karyotypes, while 7.6% had ≥5 aberrations. The distributions of karyotype complexity,
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