The aim of this study was to investigate the frequency and mutation status of the immunoglobulin heavy variable chain (IGHV) in a cohort of 224 patients from northwest and central region of Spain diagnosed with chronic lymphocytic leukemia (CLL), and to correlate it with cytogenetic abnormalities, overall survival (OS) and time to first treatment (TTFT). 125 patients had mutated IGHV, while 99 had unmutated IGHV. The most frequently used IGHV family was IGHV3, followed by IGHV1 and IGHV4. The regions IGHV3-30, IGHV1-69, IGHV3-23, and IGHV4-34 were the most commonly used. Only 3.1% of the patients belonged to the subfamily IGHV3-21 and we failed to demonstrate a worse clinical outcome in this subgroup. The IGHV4 family appeared more frequently with mutated pattern, similar to IGHV3-23 and IGHV3-74. By contrast, IGHV1-69 was expressed at a higher frequency in unmutated CLL patients. All the cases from IGHV3-11 and almost all from IGHV5-51 subfamily belonged to the group of unmutated CLL.
Chronic lymphocytic leukemia (CLL) is a clinically and biologically heterogeneous disease, with survival times ranging from months to decades [
Over the past decade, several biological markers have become important prognostic factors and may guide treatment decisions. These include immunoglobulin heavy chain variable region (IGHV) mutation status, expression of specific proteins on CLL cells such as CD38 and intracellular zeta-associated protein-70 (ZAP-70), and some cytogenetic abnormalities [
Clonal genomic aberrations can be identified in approximately 80% of CLL patients by fluorescence
The IGHV gene mutation status is one of the most reliable prognostic markers. It defines two different subsets of CLL based on the cutoff value of 98% identity with the closest germ line IGHV genes: mutated CLL (M-CLL) and unmutated CLL (U-CLL). Somatic mutations of IGHV occur in approximately half of the cases and usually present with nonprogressive disease, in contrast to patients with U-CLL who have a more aggressive disease with a shorter progression-free survival, time to first treatment (TTFT), and overall survival (OS) [
In this study, we investigated the frequency and mutation status of IGHV in a cohort of patients from northwest and central region of Spain. We also analyzed the relationship between IGHV mutation status and some other CLL prognostic markers such as the expression of CD38, cytogenetic abnormalities detected by FISH, OS, and TTFT.
A total of 224 unselected patients diagnosed with typical CLL from nine different institutions located in northwest and central region of Spain were studied for IGHV gene usage and mutation status. The diagnosis was based on clinical symptoms, immunophenotypic analysis, blood cell count, and cell morphology, according to the World Health Organization classification of tumors [
Mononuclear cells were isolated using Ficoll density gradient centrifugation from peripheral blood samples containing more than 10% of CLL cells determined by flow cytometry. Genomic DNA was extracted and purified, washed, and lysed according to the manufacturer’s instructions using the DNAzol kit (Molecular Research Center, Cincinnati, OH, USA).
IGHV gene rearrangements were amplified by reverse transcription-PCR according to ERIC recommendations [
Clonality was assessed by size discrimination of PCR products using automatic ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) in conjunction with GeneMapper 4.0 software (Applied Biosystems). To purify amplified products, they were run on 8% polyacrylamide gel, heteroduplexed in cases with polyclonal background, and visualized by ethidium bromide staining. Purified PCR products were then eluted from polyacrylamide gel and sequencing was performed from both directions, at least from 2 PCR reactions, using the BigDye Terminator v1.1 Cycle Sequencing Reaction Kit (Applied Biosystems) on an automated DNA Sequencer analyzer (ABI 3130, Applied Biosystems).
The obtained sequences were aligned to V-Base (
Interphase FISH was performed on peripheral blood samples obtained at diagnosis using commercially available probes for the following regions: 13q14; 12q13; 11q22/ATM; 17pTP53; and 14q32/IGH (Vysis/Abbott Co., Downers Grove, IL, USA). Methods for FISH analysis are described elsewhere [
CD38 expression was analyzed by 4-colour fluorescence—activated cell sorting (FACS) analysis. For data analysis, the Infinicyt software (Cytognos SL, Salamanca, Spain) was used [
Statistical analysis was performed using the SPSS 17.0 software package (SPSS, Chicago, IL, USA). The Fischer’s exact test and the Chi-squared test were used to determine the relationship between categorical variables. Quantitative variables were compared by using the Student’s
We analyzed mutation status of 224 patients with CLL. Based on a cutoff value of 98% nucleotide sequence homology of isolated IGHV gene with that of the germ line counterpart, a total of 125 (55.8%) cases were classified as M-CLL and 99 (44.2%) patients as U-CLL. When we considered 96% as the cutoff value, 102 (45.6%) cases displayed somatic mutations, whereas 124 (54.4%) were unmutated.
IGHV gene usage was identified in 216 patients. The most frequently used IGHV family was IGHV3: 107 (49.5%), followed by IGHV1: 55 (25.5%), IGHV4: 41 (19.0%), IGHV5: 8 (3.7%), IGHV2: 3 (1.4%), and IGHV7: 2 (0.9%), with no expression of IGHV6. Mutation status (≥98% homology) and its relationship with sex and IGHV rearrangements are summarized in Table
Mutation status (≥98% homology) and its relationship with IGHV rearrangements and sex.
IGHV family | Mutated cases | Unmutated cases | Mutated and unmutated | % |
|
Male/female |
---|---|---|---|---|---|---|
IGHV1 | 2 | 5 | 7 | 3.2 | ns | 4/3 |
IGHV1-02 | 3 | 7 | 10 | 4.6 | ns | 5/5 |
IGHV1-03 | 2 | 2 | 4 | 1.9 | ns | 0/4 |
IGHV1-08 | 3 | 2 | 5 | 2.3 | ns | 3/2 |
IGHV1-18 | 2 | 2 | 4 | 1.9 | ns | 4/0 |
IGHV1-46 | 3 | 1 | 4 | 1.9 | ns | 3/1 |
IGHV1-69 | 3 | 18 | 21 | 9.7 | <0.0001 | 15/6 |
Total IGHV1 |
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|
|
|
|
|
IGHV2-01 | 1 | 0 | 1 | 0.5 | ns | 0/1 |
IGHV2-05 | 0 | 1 | 1 | 0.5 | ns | 0/1 |
IGHV2-70 | 0 | 1 | 1 | 0.5 | ns | 1/0 |
Total IGHV2 |
|
|
|
|
|
|
IGHV3 | 3 | 2 | 5 | 2.3 | ns | 3/2 |
IGHV3-07 | 8 | 1 | 9 | 4.0 | ns | 9/0 |
IGHV3-09 | 2 | 2 | 4 | 1.9 | ns | 2/2 |
IGHV3-11 | 0 | 5 | 5 | 2.3 | 0.016 | 4/1 |
IGHV3-13 | 1 | 2 | 3 | 1.4 | ns | 3/0 |
IGHV3-15 | 4 | 2 | 6 | 2.8 | ns | 2/4 |
IGHV3-20 | 1 | 1 | 2 | 0.9 | ns | 1/1 |
IGHV3-21 | 6 | 1 | 7 | 3.2 | ns | 5/2 |
IGHV3-23 | 18 | 1 | 19 | 8.8 | <0.0001 | 12/7 |
IGHV3-30 | 12 | 10 | 22 | 10.0 | ns | 17/5 |
IGHV3-33 | 4 | 3 | 7 | 3.2 | ns | 5/2 |
IGHV3-48 | 1 | 2 | 3 | 1.4 | ns | 3/0 |
IGHV3-49 | 2 | 2 | 4 | 1.9 | ns | 1/3 |
IGHV3-53 | 1 | 0 | 1 | 0.5 | ns | 1/0 |
IGHV3-64 | 0 | 1 | 1 | 0.5 | ns | 1/0 |
IGHV3-72 | 2 | 0 | 2 | 0.9 | ns | 2/0 |
IGHV3-74 | 7 | 0 | 7 | 3.2 | 0.018 | 5/2 |
Total IGHV3 |
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|
|
|
|
|
IGHV4-b | 2 | 2 | 4 | 1.9 | ns | 1/3 |
IGHV4-04 | 5 | 0 | 5 | 2.3 | 0.051 | 3/2 |
IGHV4-30 | 1 | 1 | 2 | 0.9 | ns | 1/1 |
IGHV4-31 | 0 | 1 | 1 | 0.5 | ns | 1/0 |
IGHV4-34 | 14 | 4 | 18 | 8.3 | ns | 11/7 |
IGHV4-39 | 3 | 3 | 6 | 2.8 | ns | 3/3 |
IGHV4-59 | 2 | 0 | 2 | 0.9 | ns | 1/1 |
IGHV4-61 | 3 | 0 | 3 | 1.4 | ns | 2/1 |
Total IGHV4 |
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|
IGHV5-05 | 0 | 1 | 1 | 0.5 | ns | 1/0 |
IGHV5-51 | 1 | 6 | 7 | 3.2 | 0.046 | 7/0 |
Total IGHV5 |
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IGHV7-04 | 0 | 2 | 2 | 0.9 | ns | 0/2 |
Total IGHV7 |
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IGHV3 and IGHV4 regions were found predominantly in the M-CLL cases (72/107,
Among IGHV1 family, IGHV1-69 was also preferentially expressed in U-CLL (18 versus 3 cases
Regarding IGHV3 family, IGHV3-30 was the most common region used (20.6%), followed by IGHV3-23 (19%), with a low representation of IGHV3-7 (4.0%) and IGHV3-21 (3.2%). IGHV3-21 and IGHV3-7 expressed a mutated status in all but one case. Both IGHV3-23 (
IGHV gene segments usage profile of patients with M-CLL and patients with U-CLL (number of cases).
Patients with U-CLL seemed to have a more aggressive disease. Despite establishing homology in 98 or 96%, U-CLL patients presented with a higher initial white blood cell count (WBC) (median 31.5 × 109/L versus 21.2 × 109/L;
The estimated median OS of the group with 98% or greater IGHV homology was 215 months, while the estimated median OS time of the subgroup with IGHV homology less than 98% was 117 months (
Kaplan-Meier survival curve comparing OS and TTFT between patients with M-CLL (125 cases) and U-CLL (99 cases). (a) Median OS for U-CLL: 117 months; median OS for M-CLL 215 months. The difference is significant at the
Regarding subfamily usage, it is noteworthy that patients with IGHV1-69 had a higher probability to be treated (
We failed to correlate the use of IGHV3-21 region with any feature, probably due to the low representation of this subfamily.
Finally, it is remarkable that all patients who expressed IGHV5-51 were males (
OS was significantly worse in patients expressing IGHV1-69 (
Patients with 13q deletion belonged significantly more frequently to the mutated group (
Relationship between genomic aberrations, CD38, and mutation status.
FISH | Mutation status | |||
---|---|---|---|---|
M-CLL | U-CLL |
| ||
11q deletion | Present | 4 | 17 | 0.0001 |
Absent | 121 | 82 | ||
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13q deletion | Present | 61 | 20 | 0.0001 |
Absent | 64 | 79 | ||
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17p deletion | Present | 0 | 10 | 0.0001 |
Absent | 126 | 88 | ||
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||||
12q trisomy | Present | 10 | 22 | 0.0003 |
Absent | 115 | 77 | ||
|
||||
CD38* | Present | 20 | 36 | 0.0001 |
Absent | 46 | 19 |
Data for the expression of CD38 was available for 121 patients. Fifty-six patients were CD38 positive and 65 were negative at a cutoff level for CD38 of 30%. A significant different expression of CD38 between patients with M-CLL (20) and those with U-CLL (36) was observed (
In patients with CLL, the presence of a correlation between unmutated IGHV status and poor survival has been well established in several studies [
The aim of our study was to investigate the frequency and mutation status of IGHV in a cohort of 224 patients from northwest and central region of Spain and to correlate it with cytogenetic abnormalities, CD38 expression, OS, and TTFT.
We found 56% cases of M-CLL and 44% of U-CLL when we established homology cutoff value on 98%. These findings are consistent with previous publications in Western countries [
IGHV gene usage family distribution was also comparable to those observed in Western countries [
With regard to the IGHV3 family, IGHV3-30 was the most frequently used segment followed by IGHV3-23. By contrast, IGHV3-7 was present in a relatively low frequency (4%) and was seen preferentially in M-CLL (8/9). Similar observations have been reported previously in Mediterranean countries and Serbian patients [
IGHV3-21 has been reported to be overrepresented in Scandinavian patients with a lower frequency in Southern European countries and has been associated with poor prognosis independent of mutational status [
IGHV1-69 is the most frequently used gene in IGHV1 gene family of Western populations, exhibiting in most of the cases a germ line profile [
IGHV4 family has been reported to be overused in the mutated subgroup of CLL [
Regarding the remaining genes, we found a low representation of IGHV5, IGHV2, and IGHV7 gene families and lack of expression of IGHV6, as also described in other studies, regardless of their ethnic origin [
Del(13q), del(11q), +12, del(17p), and IGH rearrangement have been proved to be the most common and prognostically relevant chromosomal changes in CLL. As previously reported [
CD38 has also been reported as an independent predictor of prognosis for CLL, and its high expression has been correlated to unfavorable outcomes. It is controversial whether high levels of CD38 expression may be used as a surrogate marker of mutation status [
Our data confirm that IGHV mutation status is one of the most significant molecular predictors for CLL prognosis. The identification of IGHV gene segments can provide additional information regarding clinical course of CLL. The use of IGHV3-21 had a low incidence in our series. IGHV3-11 identified a group of patients with poor prognosis. IGHV5-51 revealed a group of patients with a trend to a worse outcome. In addition, OS and TTFT were significantly related to IGHV mutation status. Further studies of larger series, preferably in the context of prospective clinical trials, need to be performed to validate our data.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The study was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias 02/1041, FIS 09/01382, FIS 09/01543, and PI12/00281; RD12/0036/0069 from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness & European Regional Development Fund (ERDF) “Una manera de hacer Europa”; and Caja de Burgos-Banca Cívica. A. Rodrígues was fully supported by an Ayuda Predoctoral FIS de Formación en Investigación by the Spanish Fondo de Investigaciones Sanitarias. M. Hernández-Sánchez was partially supported by a grant from the “Fundación Española de Hematología y Hemoterapia.” Partially supported by grants from “Proyectos de Investigación Biomédica del SACYL” 106/A/06, the authors thank all the physicians from the Spanish hospitals who contributed with the clinical data; Eva Lumbreras, María Pozo, Teresa Prieto, María Ángeles Hernández, Ana Simón, Ana Díez, and Almudena Martín, from “Centro de Investigación del Cáncer, Salamanca”; and Alicia Antón from IBSAL-Hospital Universitario de Salamanca for the technical assistance.