IGHV3‐21 gene usage is associated with high TCL1 expression in chronic lymphocytic leukemia

T‐cell leukemia/lymphoma protein 1 (TCL1) was recently shown to display an expression pattern in chronic lymphocytic leukemia (CLL) corresponding to molecular subtypes, where poor‐risk patients demonstrated higher expression levels. Here, we examined the mRNA expression pattern of TCL1 in 144 patients with CLL, including 67 immunoglobulin heavy‐chain variable (IGHV) mutated, 58 IGHV unmutated and 19 patients with IGHV3‐21 usage. A higher TCL1 expression level was detected in patients with CLL with unmutated vs. mutated IGHV genes (P < 0.001), whereas no difference was demonstrated within the IGHV3‐21 cohort (i.e., mutated vs. unmutated and stereotyped vs. non‐stereotyped complementarity determining region 3). The IGHV3‐21 subgroup displayed high TCL1 mRNA expression, differing significantly from other IGHV mutated cases (P < 0.001), although 11/19 had mutated IGHV genes. Furthermore, high TCL1 expression levels were associated with significantly shorter overall survival (P < 0.001). Altogether, we show that TCL1 mRNA expression may predict clinical outcome in CLL and that the IGHV3‐21 subset, regardless of mutational status, displays high TCL1 expression.     

V(H) mutation status,CD38 expression level,genomic aberrations,and survival in chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL), biologic risk factors such as immunoglobulin variable heavy chain gene (V(H)) mutation status, CD38 expression level, and genomic aberrations have recently been identified, but the relative prognostic impact of the individual parameters is unknown. In the current study, we analyzed V(H) mutation status by polymerase chain reaction and sequencing (n = 300), genomic aberrations by fluorescence in situ hybridization (+3q, 6q-, +8q, 11q-, +12q, 13q-, t(14q), 17p-) (n = 300), and CD38 expression by triple-color FACS (CD5, CD19, CD38) (n = 157) in a unicentric CLL cohort. The prognostic influence of V(H) mutation rate and CD38 expression level was tested by maximally selected log-rank statistics. A corrected P value (P(cor)) for a cutoff level allowing the best separation of 2 subgroups with different survival probabilities was identified at 97% V(H) homology (95% confidence interval [CI], 96%-98% homology, P(cor) <.001) and at 7% CD38 expression (95% CI, 20%-71% expression, P(cor) =.02). In univariate analyses, unmutated V(H) genes and high CD38 expression levels predicted for shorter survival times. The overall incidence of genomic aberrations was similar in the V(H) unmutated and V(H) mutated subgroups. High-risk genomic aberrations such as 17p- and 11q- occurred almost exclusively in the V(H) unmutated subgroup, whereas favorable aberrations such as 13q- and 13q- as single abnormalities were overrepresented in the V(H) mutated subgroup. In multivariate analysis, unmutated V(H), 17p deletion, 11q deletion, age, WBC, and LDH were identified as independent prognostic factors, indicating a complementary role of V(H) mutation status and genomic aberrations to predict outcome in CLL.     

Clonal evolution in chronic lymphocytic leukemia: acquisition of high-risk genomic aberrations associated with unmutated VH, resistance to therapy, and short survival

In chronic lymphocytic leukemia (CLL), the acquisition of new genomic aberrations during the disease course (clonal evolution) is thought to be an infrequent phenomenon but comprehensive analyses are limited. Genomic aberrations were analyzed by fluorescence in situ hybridization (FISH) at various time points during the disease course of 64 CLL patients. Results were correlated with the mutation status of the immunoglobulin heavy-chain variableregion genes (VH) and clinical characteristics. Following a median observation time of 42.3 months (range 23.2-73) after first genetic study, 11 out of the 64 (17%) patients showed clonal evolution with the following newly acquired aberrations: del(17p13) (n=4), del(6q21) (n=3), del(11q23) (n=2), +(8q24) (n=1), and evolution from monoallelic to biallelic del(13q14) (n=3). Interestingly, clonal evolution only occurred among cases with unmutated VH status. The group with clonal evolution showed a higher rate of progression in Binet stage (82% vs. 28%), a possibly greater need for treatment (91% vs. 62% previously untreated patients received their first therapy), and a higher hazard risk of death (HR = 2.97, 95% CI 1.40-6.27, p=0.004) in multivariable analysis. The estimated median survival time after the occurrence of clonal evolution was 21.7 months. Expansion of the clone with del(17p13) was observed in all patients during treatment, indicating in vivo resistance to therapy. In multivariable Andersen-Gill regression analysis, clonal evolution was identified as an independent prognostic factor for overall survival. Clonal evolution only occurred in CLL with unmutated VH indicating to karyotypic instability as a pathomechanism. Acquisition of genomic aberrations was associated with poor outcome based on multivariable analysis. In vivo resistance to chemotherapy of CLL clones with del(17p13) emphasizes the need for alternative treatment approaches in these patients.     

Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays

Recurrent genomic aberrations are important prognostic parameters in chronic lymphocytic leukemia (CLL). High-resolution 10k and 50k Affymetrix SNP arrays were evaluated as a diagnostic tool for CLL and revealed chromosomal imbalances in 65.6% and 81.5% of 70 consecutive cases, respectively. Among the prognostically important aberrations, the del13q14 was present in 36 (51.4%), trisomy 12 in 9 (12.8%), del11q22 in 9 (12.8%), and del17p13 in 4 cases (5.7%). A prominent clustering of breakpoints on both sides of the MIRN15A/MIRN16-1 genes indicated the presence of recombination hot spots in the 13q14 region. Patients with a monoallelic del13q14 had slower lymphocyte growth kinetics (P=.002) than patients with biallelic deletions. In 4 CLL cases with unmutated VH genes, a common minimal 3.5-Mb gain of 2p16 spanning the REL and BCL11A oncogenes was identified, implicating these genes in the pathogenesis of CLL. Twenty-four large (>10 Mb) copy-neutral regions with loss of heterozygosity were identified in 14 cases. These regions with loss of heterozygosity are not detectable by alternative methods and may harbor novel imprinted genes or loss-of-function alleles that may be important for the pathogenesis of CLL. Genomic profiling with SNP arrays is a convenient and efficient screening method for simultaneous genome-wide detection of chromosomal aberrations.     

B‐cell receptor configuration and adverse cytogenetics are associated with autoimmune hemolytic anemia in chronic lymphocytic leukemia

The development of autoimmune hemolytic anemia (AIHA) in patients with chronic lymphocytic leukemia (CLL) is associated with specific biological features. The occurrence of AIHA was hereby investigated in a retrospective series of 585 CLL patients with available immunoglobulin heavy chain variable (IGHV) gene status. AIHA occurred in 73 patients and was significantly associated with an IGHV unmutated (UM) status (P < 0.0001) and unfavorable [del(17)(p13) and del(11)(q23)] cytogenetic lesions (P < 0.0001). Stereotyped HCDR3 sequences were identified in 29.6% of cases and were similarly represented among patients developing or not AIHA; notably, subset #3 was associated with a significantly higher risk of AIHA than the other patients (P = 0.004). Multivariate analysis showed that UM IGHV, del(17)(p13) and del(11)(q23), but not stereotyped subset #3, were the strongest independent variables associated with AIHA. Based on these findings, we generated a biological risk score for AIHA development according to the presence of none (low risk), one (intermediated risk), or two (high risk) of the independent risk factors. Overall, our data indicate that UM IGHV status and/or unfavorable cytogenetic lesions are associated with the risk of developing secondary AIHA in CLL patients and suggest a possible role of specific stereotyped B‐cell receptor subsets in a proportion of cases. Am. J. Hematol. 2013. © 2012 Wiley Periodicals, Inc.     

NOTCH1 mutations in +12 chronic lymphocytic leukemia (CLL) confer an unfavorable prognosis, induce a distinctive transcriptional profiling and refine the intermediate prognosis of +12 CLL

Trisomy 12, the third most frequent chromosomal aberration in chronic lymphocytic leukemia (CLL), confers an intermediate prognosis. In our cohort of 104 untreated patients carrying +12, NOTCH1 mutations occurred in 24% of cases and were associated to unmutated IGHV genes (P=0.003) and +12 as a sole cytogenetic abnormality (P=0.008). NOTCH1 mutations in +12 CLL associated with an approximately 2.4 fold increase in the risk of death, a significant shortening of survival (P<0.01) and proved to be an independent predictor of survival in multivariate analysis. Analogous to +12 CLL with TP53 disruption or del(11q), NOTCH1 mutations in +12 CLL conferred a significantly worse survival compared to that of +12 CLL with del(13q) or +12 only. The overrepresentation of cell cycle/proliferation related genes of +12 CLL with NOTCH1 mutations suggests the biological contribution of NOTCH1 mutations to determine a poor outcome. NOTCH1 mutations refine the intermediate prognosis of +12 CLL.     

“Double‐hit” chronic lymphocytic leukemia: An aggressive subgroup with 17p deletion and 8q24 gain

Chronic lymphocytic leukemia (CLL) with 17p deletion (17p‐) is associated with a lack of response to standard treatment and thus the worst possible clinical outcome. Various chromosomal abnormalities (including unbalanced translocations, deletions, ring chromosomes and isochromosomes) result in the loss of 17p and one copy of the TP53 gene. The objective of the present study was to determine whether the type of chromosomal abnormality leading to 17p‐ and the additional aberrations influenced the prognosis in a series of 195 patients with 17p‐CLL. Loss of 17p resulted primarily from an unbalanced translocation (70%) with several chromosome partners (the most frequent being chromosome 18q), followed by deletion 17p (23%), monosomy 17 (8%), isochromosome 17q [i(17q)] (5%) and a ring chromosome 17 (2%). In a univariate analysis, monosomy 17, a highly complex karyotype (≥5 abnormalities), and 8q24 gain were associated with poor treatment‐free survival, and i(17q) (P = .04), unbalanced translocations (P = .03) and 8q24 gain (P = .001) were significantly associated with poor overall survival. In a multivariate analysis, 8q24 gain remained a significant predictor of poor overall survival. We conclude that 17p deletion and 8q24 gain have a synergistic impact on outcome, and so patients with this “double‐hit” CLL have a particularly poor prognosis. Systematic, targeting screening for 8q24 gain should therefore be considered in cases of 17p‐ CLL.     

Interphase Fluorescence In Situ Hybridization Detection of Cytogenetic Abnormalities in B-Cell Chronic Lymphocytic Leukemia

The most frequent chromosomal abnormalities in B-cell chronic lymphocytic leukemia (B-CLL) are deletions on 13q14 and 17p13, trisomy 12, and 14q32 rearrangement. Conventional cytogenetic analysis underestimates the frequency of specific chromosome aberrations in B-CLL because of the low rate of spontaneous mitoses and the poor response to mitogen stimulation. We used interphase fluorescence in situ hybridization (I-FISH) to explore the incidence of chromosomal changes in the peripheral blood cells of B-CLL patients. Probes for 13q14 (D13S319), 17p13 (p53), the centromere of chromosome 12 (CEP12), and 14q32 (IGHC/IGHV) were applied to detect chromosomal aberrations in peripheral blood samples from 83 B-CLL patients (60 men, 23 women). Molecular cytogenetic aberrations were found in 61 cases (73.5%), and 8 patients (9.6%) showed 2 kinds of abnormalities. The most frequent abnormality was deletion of 13q14 (41.0%), followed by +12 (19.3%), deletion of 17p13 (12%), and 14q32 rearrangement (9.6%). FISH results were analyzed for correlation with Binet stages. The percentages of patients who showed abnormalities by FISH were 73.0%, 73.3%, and 80% for Binet stages A, B, and C, respectively, and the percentages of patients with abnormalities who showed 2 anomalies were 7.9%, 27.3%, and 0% for Binet stages A, B, and C, respectively. We noted no consistent pattern among the various Binet stages in the distribution of either the types of FISH-detected anomalies or the numbers of FISH anomalies. I-FISH was found to be a rapid, exact, and sensitive technique for analysis of chromosome aberrations in CLL. FISH could provide accurate information regarding the molecular cytogenetic features of CLL.     

Structural aberrations of chromosomes 17 and 12 in chronic B-cell disorders

OBJECTIVES: Genomic aberrations can now be identified in approximately 80% of chronic lymphocytic leukemia, small lymphocytic lymphoma (CLL/SLL) patients. In the present study, four new structural changes involving chromosomes 17 and 12 in CLL/SLL patients are described. METHODS: Five patients were selected for inclusion in the present report among a total of 92 cases with diagnosis of CLL/SLL. Cytogenetic studies and fluorescence in situ hybridization (FISH) analysis to detect some of the most frequent cryptic aberrations occurring in CLL/SLL patients were performed. Clinical studies are also described. RESULTS: Four cases showed structural rearrangements of chromosome 17. A psu dic(17;2)(p11.2;p21), leading to p53 deletion, was observed in a patient who developed a mixed cellularity Hodgkin's disease coexisting with the CLL/SLL in the same lymph node. Epstein Barr virus was detected in the Reed-Sternberg cells. Two cases had a balanced translocation t(2;17)(p21;q23). Both patients showed trisomy 12 and clonal evolution and one of them also had 11q deletion. In addition, a der(17)t(12;17)(q13;q25) as a part of a complex karyotype, and a complex translocation t(5;12;19) (q15;p11;q13) were also found. Four patients had an adverse clinical outcome and died because of disease progression. CONCLUSIONS: Four unreported nonrandom chromosome aberrations in CLL/SLL patients, one of them who might represent a new recurrent abnormality, are described. These uncommon abnormalities, mostly associated with evolving disease, may have implications for the understanding of genetic events associated with disease progression in this pathology.     

Telomeres and prognosis in patients with chronic lymphocytic leukaemia

In the present study, telomere length, telomerase activity, the mutation load of immunoglobulin variable heavy chain (IGHV) genes, and established prognostic factors were investigated in 78 patients with chronic lymphocytic leukaemia (CLL) to determine the impact of telomere biology on the pathogenesis of CLL. Telomere length was measured by an automated multi-colour flow-FISH, and an age-independent delta telomere length (ΔTL) was calculated. CLL with unmutated IGHV genes was associated with shorter telomeres (p?=?0.002). Furthermore, we observed a linear correlation between the frequency of IGHV gene mutations and elongation of telomeres (r?=?0.509, p?相似文献   

Monoclonal B‐cell lymphocytosis is closely related to chronic lymphocytic leukaemia and may be better classified as early‐stage CLL

The World Health Organization classification uses a cut‐off point of 5·0 × 10⁹/l cells with a chronic lymphocytic leukaemia (CLL)‐phenotype in peripheral blood to discriminate between monoclonal B‐lymphocytosis (MBL) and B‐CLL. This study analysed 298 MBL patients by multi‐parameter flow cytometry, chromosome banding analysis (CBA)/fluorescence in situ hybridization (FISH), and IGHV mutation status and compared them with 356 CLL patients. In MBL, CBA more frequently revealed a normal karyotype and FISH identified less frequently del(6q), del(13q) (as sole alterations), and del(17)(p13). Within the MBL cohort, a shorter time to treatment (TTT) was found for ZAP‐70‐positivity, 14q32/IGH‐translocations (CBA), del(11)(q22·3) (FISH) and unmutated IGHV status. Higher CD38 and ZAP‐70 expression, del(11)(q22·3) (FISH), trisomy 12 (FISH), and 14q32/IGH‐translocations (CBA) were correlated with a shorter TTT in the combined cohort (MBL + CLL); a sole del(13)(q14) (FISH) correlated with longer TTT. Regarding overall survival, unmutated IGHV status and ‘other’ alterations (CBA) had an adverse impact. There was no correlation between the concentration of CLL‐cells and TTT or overall survival. Multivariate analysis confirmed a negative impact on TTT for del(11)(q22·3)/ATM, trisomy 12 (both by FISH), and 14q32/IGH‐translocations by CBA. These data emphasize a close relationship between MBL and CLL regarding clinically relevant parameters and provide no evidence to strictly separate these entities by a distinct threshold of clonal B‐cells.     

Molecular and clinical features of chronic lymphocytic leukaemia with stereotyped B cell receptors: results from an Italian multicentre study

A fraction of chronic lymphocytic leukaemia (CLL) cases carry highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable ( IGHV ) genes and heavy-chain complementarity determining region-3 (HCDR3), often associated with a restricted selection of IGVK/L light chains. Such 'stereotyped' BCR occur more frequently in CLL with unmutated (UM) than mutated (M) IGHV genes. We analysed 1426 IG rearrangements (from 1398 CLL cases) by a clustering driven by HCDR3 similarities. Molecular findings were correlated to time-to-treatment (TTT) and presence of known prognosticators. Sixty-nine clusters (319 IG-rearrangements, 22·4%) with stereotyped BCR were identified. Among 30 confirmed clusters (≥3 IG-rearrangements/cluster), we found 14 novel clusters, of which 11 had M IG rearrangements (M clusters) and predominantly (8/11) used IGHV3 subgroup genes. Recurrent cluster-biased amino acid changes were found throughout IGHV sequences of these 'M clusters'. Regarding clinical outcome: (i) UM CLL from the IGHV1-2/1-3/1-18/1-46/7-4-1/IGKV1-39 cluster had poorer prognosis than UM/M cases, or UM cases using the same IGHV genes but not in clusters; (ii) M CLL from the IGHV3-21/IGLV3-21 cluster had TTT similar to UM CLL, and shorter than M CLL expressing IGHV3-21 but not in cluster. Altogether, our analysis identified additional molecular and clinical features for CLL expressing stereotyped BCR.     

Nonstochastic pairing of immunoglobulin heavy and light chains expressed by chronic lymphocytic leukemia B cells is predicated on the heavy chain CDR3

We analyzed the immunoglobulin (Ig) variable heavy (IGHV) and variable light chain genes used by leukemia cells of 258 unrelated patients with chronic lymphocytic leukemia (CLL) found to express unmutated Ig heavy chains (IgH) encoded by a 51p1 allele of IGHV1-69 among 1846 CLL patients examined. We found each had at least 98% homology to an identified germline IGKV or IGLV gene. Within the 258 IgH, we identified heavy chain CDR3 (HCDR3) motifs encoded by certain unmutated IGHD and IGHJ genes with restricted reading frames. Frequent and restricted use of particular IGKV and IGLV genes revealed nonstochastic pairing of disparate Ig light chains (IgL) with IgH that had restricted HCDR3 motifs designated CLL69A, -B, -C, and -D. Eighty-six percent (19/22) of CLL cases that expressed motif CLL69B encoded by IGHD2-2/IGHJ6 had distinctive IgL encoded by IGKV1-39. Similarly, 83% (5/6) of samples with motif CLL69D encoded by IGHD2-2/IGHJ6 expressed IGKV3-11, 100% (25/25) with motif CLL69A encoded by IGHD3-16/IGHJ3 used IGKV3-20, and 77% (10/13) with motif CLL69C encoded by IGHD3-3/IGHJ6 expressed IGLV3-9. This study reveals nonstochastic pairing of IgH with particular IgL that is predicated upon Ig HCDR3 structure, providing compelling evidence for selection of antibodies expressed in CLL by conventional antigens.     

A new genetic abnormality leading to TP53 gene deletion in chronic lymphocytic leukaemia

The analysis of chromosomal abnormalities provides significant prognostic information in patients with chronic lymphocytic leukaemia (CLL), a disease with a highly heterogeneous clinical course. Chromosomal abnormalities commonly found are trisomy 12, del(13)(q14), del(11)(q22-23), del(17)(p13) and del(6)(q21). Translocations are present in some patients and affect regions recurrently involved in CLL. This report describes the clinical and pathological characteristics of four CLL patients showing a new recurrent chromosomal abnormality dic(8;17)(p11;p11), that implied loss of the TP53 gene in all cases. In addition, TP53 gene was mutated in three out of four patients. Mechanically, Low Copy Repeats (LCR) in 17p12 and 8p11 may explain the origin of the translocation by non-allelic homologous recombination (NAHR). Isolated dic(8;17)(p11;p11) in patients with mutated IGHV genes status may not have the same prognostic impact as other mutations or deletions affecting the TP53 gene. Larger series are needed to better evaluate the clinical impact of this chromosomal aberration during the course of the disease.     

Genetics and risk-stratified approach to therapy in chronic lymphocytic leukemia

The clinical staging systems developed by Rai and Binet have remained the mainstay for clinical decision-making in patients with chronic lymphocytic leukemia (CLL). However, there is substantial heterogeneity in the course of the disease. In recent years molecular and cellular markers have helped to predict the prognosis of patients with CLL. Ig V(H) status and genomic aberrations subdivide CLL into distinct clinical subgroups. Fluorescence in-situ hybridization (FISH) can identify genomic aberrations in approximately 80% of CLL cases. The most frequent aberrations are deletions in 13q, 11q, or 17p, and trisomy 12. Apart from providing insights into the pathogenesis, genomic aberrations identify subgroups of patients with distinct clinical pictures: lymphadenopathy (11q-) or resistance to therapy (17p-). Deletions at 11q and particularly 17p are associated with rapid disease progression or inferior survival. Patients with these genetic abnormalities may be candidates for clinical trials investigating alternative treatments and stem-cell transplantation.     

Biological and clinical characterization of recurrent 14q deletions in CLL and other mature B‐cell neoplasms

14q‐deletions have been repeatedly described in mature B‐cell neoplasms, but not yet characterized in a larger cohort. Based on chromosome banding analysis, the present study identified 47 del(14q) cases in 3054 mature B‐cell neoplasms (1·5%) (chronic lymphocytic leukaemia [CLL]: 1·9%; CLL/prolymphocytic leukaemia [PL]: 9·0%; others: 0·2%). Interphase fluorescence in situ hybridization was performed with probes for 14q22.1, 14q24.1, 14q32.33, and IGH@ (14q32.3). The del(14q) had heteregeneous size but showed a breakpoint cluster at the centromeric site in 14q24.1 (62% of cases). At the telomeric side, the most frequent breakpoint was within the IGH@ locus (14q32.3) between IGH@ 3′‐flanking and IGHV (IgVH) probes (45%). In 16 cases (34%), breakpoints occurred within 14q24.1 and 14q32.3. Eighty‐one percent of del(14q) cases showed 1–3 additional cytogenetic alterations (in 45%, +12), and 56% were IGHV‐unmutated. In all cases (16/16) with breakpoints in 14q24.1 and 14q32.3, a B‐CLL immunophenotype was found. Clinical follow‐up in 32 del(14q) patients was compared to 383 CLL and CLL/PL patients without del(14q). While 3‐year‐overall survival did not differ significantly, time to treatment was significantly shorter in the del(14q) cohort (21·0 months vs. 80·1 months, P = 0·015). In conclusion, the del(14q) is a rare recurrent alteration in diverse mature B‐cell neoplasms, shows variable size but distinct clustering of breakpoints, and is associated with short time to treatment.     

Four novel non-random chromosome rearrangements in B-cell chronic lymphocytic leukaemia: 6p24-25 and 12p12-13 translocations, 4q21 anomalies and monosomy 21

Nine patients with previously unreported chromosome changes were identified among 209 B-cell chronic lymphocytic leukaemia (CLL) cases: three patients had a translocation involving 6p24-25; three had a 12p12-13 translocation; two had 4q21 involvement (one with coexisting 6p anomaly); and two had monosomy 21. Interphase fluorescence in situ hybridization (FISH) detected some cryptic aberrations (+12, 6q-, 17p-, 11q-) in those patients with 6p translocations, whereas only a cytogenetically undetected 13q14 deletion was found in the remaining cases. Atypical morphology was noted in six cases, including both cases with monosomy 21, two cases with 6p and 4q21 anomaly and one case with 12p involvement. Four of these cases also had more than one phenotype deviation with respect to the classical CLL phenotype. Disease progression after 21-51 months (median 41) was noted in two cases with 6p and 4q21 involvement and in one case with 12p anomaly and monosomy 21. We arrived at the following conclusions: (i) 6p24-25 and, possibly, 4q21 lesions represent non-random events in CLL, occurring in association with other well-known unbalanced rearrangements; (ii) 12p rearrangements and monosomy 21 may possibly represent early chromosome defects that are not associated with the classical DNA gains and losses known to be present in the majority of CLL; and (iii) atypical morphology and immunophenotype as well as disease progression were frequently observed in these cases     

Abnormal serum free light chain ratios are associated with poor survival and may reflect biological subgroups in patients with chronic lymphocytic leukaemia

The measurement of immunoglobulin serum free light chains (sFLC) has prognostic significance in plasma cell dyscrasias but its role in chronic lymphocytic leukaemia (CLL) is unknown. This retrospective study from three UK hospitals analysed sFLC in 181 untreated/pre-treatment CLL patients and 78 treated CLL patients, with samples taken later in their disease. An abnormal sFLC ratio was significantly associated with poor overall survival for the 181 untreated/pre-treatment patients ( P  = 0·0001) and for all patients ( P  = 0·002), irrespective of cause of death. Using multivariate analysis ( n  = 194), four independent prognostic variables for overall survival were identified namely Zap-70 ( P  = 0·0001), β2M ( P  = 0·01), IGHV mutation status ( P  = 0·017) and an abnormal sFLC ratio ( P  = 0·024). For CLL patients with unmutated IGHV genes, elevated κ/λ ratios were adversely prognostic. For patients with mutated IGHV genes, reduced κ/λ ratios were adversely prognostic and associated with the poor prognostic IGHV3-21 , IGHV3-48 and IGHV3-53 subgroups, suggesting an abnormal sFLC ratio may reflect biological subgroups within CLL. Abnormal sFLC ratios need to be studied prospectively in CLL patients and the biological rationale for their abnormality investigated.