Association between telomere length and V(H) gene mutation status in chronic lymphocytic leukaemia: clinical and biological implications

The immunoglobulin V(H) gene mutation status can divide B-cell chronic lymphocytic leukaemia (CLL) into two entities with a different clinical course. Cases with unmutated V(H) genes, considered to evolve from pregerminal centre (GC) cells, have a worse outcome compared to cases showing mutated V(H) genes, that is, post-GC derived. Also, telomere length has been reported to be of prognostic significance in CLL. Interestingly, telomerase becomes activated during the GC reaction and an elongation of the telomeres occurs in GC B cells. We performed telomere length and V(H) gene analysis in a series of 61 CLL cases, in order to investigate if the unique telomere lengthening shown in GC B cells could reflect the telomere status in the two subsets of mutated and unmutated CLL. A novel association was found between V(H) gene mutation status and telomere length, since significantly shorter telomeres were demonstrated in the unmutated group compared to the mutated group (mean length 4.3 vs 6.3 kbp). Shorter telomeres also constituted a subgroup with a worse prognosis than cases with longer telomeres (median survival 59 vs 159 months). Furthermore, the Ig gene sequence data revealed that samples with high mutations frequency (>6%) had long telomeres ( approximately 8 kbp). Thus, both the telomere and V(H) gene mutation status in CLL appear linked, which may reflect the proliferative history of the clonal cells with regard to the GC reaction.     

Prognostic usage of VH gene mutation status and its surrogate markers and the role of antigen selection in chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease with many patients surviving for decades with minimal or no treatment, whereas others succumb rapidly to their disease despite therapy. In recent years, new molecular prognostic factors have emerged in CLL that have significantly improved the subgrouping of the disease. One of the most important molecular predictors, the immunoglobulin V_H gene mutation status, divides CLL into two prognostic groups, depending on the presence or absence of somatic hypermutation, where unmutated V_H genes are associated with considerably worse prognosis than mutated V_H genes. An exception to this appears to be CLL patients utilizing the V_H3-21 gene as they have poor outcome irrespective of mutation status. Surrogate markers for the V_H gene mutation status have been suggested, such as CD38 and ZAP-70 expression. However, the CD38 level was later shown to display poor correlation to the mutation status, although it may still serve as an independent prognostic factor. More promising is the expression levels of ZAP-70, which appears to be both a strong surrogate marker for V_H gene mutation status, although discrepancies have been reported, as well as an independent prognostic marker. Immunoglobulin gene analysis has also indicated the possibility of antigen selection in CLL considering the significant bias in V_H gene usage. Intriguingly, the V_H3-21⁺ group and several other CLL subsets using certain V_H genes was recently reported to display strikingly restricted immunoglobulin gene features, in both their heavy and light chain gene rearrangements, thus further high-lighting the possible role of antigen involvement in CLL development.     

Characterization of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) in Shanghai, China: Molecular and cytogenetic characteristics, IgV gene restriction and hypermutation patterns

The clinical, cytogenetic and molecular features of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), a disease previously considered to be rare in Asia, were examined in consecutive series of 70 cases diagnosed by our laboratory over a 30-month period. Clonal abnormalities were observed in 80% of CLL/SLL cases using a combination of conventional cytogenetic and fluorescence in situ hybridization (FISH) analysis. Those involving 14q32/IGH were the most frequent (24 cases), followed by trisomy 12 and 11q abnormalities. IgV_H gene usage was non-random with over-representation of V_H4-34, V_H3-23 and a previously unreported increase in V_H3-48 gene use. Somatic hypermutation (SHM) of IgV_H germline sequences was observed in 56.5% of cases with stereotyped patterns of SHM observed in V_H4-34 heavy chain complimentary-determining (HCDR1) and framework region CFR2 sequences. These findings in a Chinese population suggest subtle geographical differences in IgV_H gene usage while the remarkably specific pattern of SHM suggest that a relatively limited set of antigens may be involved in the development of this disease worldwide. IgV_H gene mutation status was a significant predictor of initial survival in CLL/SLL. However, an influence of karyotype on prognosis was not observed.     

MDR-1, but not MDR-3 gene expression, is associated with unmutated IgVH genes and poor prognosis chromosomal aberrations in chronic lymphocytic leukemia

Two P-glycoprotein (P-gp) genes, MDR-1 (ABCB1) and MDR-3 (ABCB4), have been identified in humans. This study was designed to investigate whether associations exist between expression of MDR-1 and MDR-3 P-gp and other markers of poor prognosis and/or prior exposure to therapeutic agents in chronic lymphocytic leukemia (CLL). IgV_H mutational status, gene usage, CD38 positivity, FISH analysis and clinical information were available on all patients. Twenty-one of 101 patients tested showed MDR-3 P-gp positivity. Associations with markers of poor prognosis or prior chemotherapy did not reach statistical significance, but MDR-3 P-gp positive patients had significantly shorter survivals than MDR-3 P-gp negative patients. MDR-1 P-gp expression (18/25) showed a strong association with unmutated IgV_H genes and adverse prognosis cytogenetics (p = 0.015, p = 0.014, respectively), but was independent of prior exposure to chemotherapeutic agents. These results suggest a role for MDR-1 and MDR-3 in chemoresistant disease. This study highlights the value of determining MDR phenotype in CLL patients prior to treatment, to allow the design of novel drug regimens containing agents that reverse MDR function.     

Treatment of chronic lymphocytic leukemia and lymphosarcoma with a new chlorambucil ester of prednisolone (Leo 1031) (NSC-134087)

A new chlorambucil ester of prednisolone (LEO 1031) has been evaluated in patients with advanced lymphocytic lymphosarcoma and chronic lymphocytic leukemia. All 11 patients had been previously treated with radiotherapy and/or combination chemotherapy. Three complete remissions and one partial remission were seen in 4/7 lymphocytic lymphosarcoma patients treated with LEO 1031. In the chronic lymphocytic leukemia group, 3/4 have had benefit. This new drug is considered worthy of further trial.     

Monoclonal origin of B cells producing k, λ and kλ immunoglobulin light chains in a patient with chronic lymphocytic leukemia

Immunoglobulin gene rearrangements can be used as genetic markers of clonality in the study of B-cell populations [4]. We have therefore analysed the structure and expression of heavy and light chain immunoglobulin genes in lymphocytes of a patient with chronic lymphocytic leukemia, where we found both k and λ producing B cells, but in most of the cells both k and λ chains were co-expressed on the same surface membrane. Single rearrangements were observed in μ, JH, k and λ DNA sequences, thus providing strong evidence for the monoclonal origin of the cells bearing different light chains. Moreover, the analysis of Ig sequence RNA showed, in addition to normal μ, k and λ mRNA molecules, high levels of a small λ related RNA sequence. These findings are discussed in relation to a model of B-lymphocyte differentiation which could be either an additional or an alternative hypothesis to the current one of isotypic exclusion.     

V(H)3-48 and V(H)3-53, as well as V(H)3-21, gene rearrangements define unique subgroups in CLL and are associated with biased lambda light chain restriction, homologous LCDR3 sequences and poor prognosis

This study determined IgV(H) gene usage in 228 chronic lymphocytic leukaemia patients to investigate associations between gene usage and other biological or clinical characteristics. V(H)3-48 [N=8] and V(H)3-53 [N=4] gene rearrangements showed biased lambda light chain restriction and were predominantly found in female patients with short lymphocyte doubling time but without adverse prognosis cytogenetics. Overuse of V(L)3-21(Vlambda2-14) gene and highly homologous LCDR3 sequences were found in V(H)3-48 patients. V(H)3-21 gene usage [N=18, 7.9%] was associated with poor prognosis, overuse of V(L)3-21(Vlambda2-14) gene and highly homologous heavy- and light-chain CDR3 sequences, but was not associated with poor prognosis chromosomal aberrations.     

Cytokeratin 19-fragments (CYFRA 21-1) as a novel serum biomarker for response and survival in patients with advanced pancreatic cancer

Background:

CYFRA 21-1 serves as biomarker in several epithelial malignancies. However, its role in pancreatic cancer (PC) has not yet been investigated.

Methods:

Within a prospective single-centre study serial blood samples were collected from patients with confirmed advanced PC. Pre-treatment values and weekly measurements of CYFRA 21-1, carbohydrate antigen 19-9 (CA 19-9) and carcinoembryonic antigen (assessed by Elecsys 2010, Roche Diagnostics) during palliative first-line chemotherapy were obtained. Biomarker data were correlated with objective response (determined by RECIST) as well as time to progression (TTP) and overall survival (OS) using uni- and multivariate analyses.

Results:

Seventy-eight patients were included, 45% of these received treatment in prospective clinical trials. Median TTP was 3.9 months, median OS 7.7 months. Pre-treatment CYFRA 21-1 levels were significantly associated with performance status (P=0.0399) and stage of disease (P=0.0001). Marker values before chemotherapy and at the 2-month staging of all three markers were considered significant predictors for objective treatment response. Pre-treatment CYFRA 21-1 levels, as well as CA 19-9 values, could be applied to define subgroups (categorised by tertiles) with a different OS outcome (CYFRA: 14.8 vs 7.1 vs 4.8 months, CA 19-9: 14.2 vs 7.1 vs 5.2 months; P<0.0001). CYFRA 21-1 and CA 19-9 (both as categorised and as continuous variables) showed a highly significant correlation with TTP and OS at nearly all-time points assessed in univariate analysis. In multivariate analysis, only CYFRA 21-1 and performance status were independent predictors for OS.

Conclusions:

CYFRA 21-1 may serve as a valuable tool for monitoring treatment response and assessing prognosis in advanced PC.     

Internal tandem duplications of Flt3 gene (Flt3/ITD) predicts a poor post-remission outcome in adult patients with acute non-promyelocytic leukemia

Despite progress in AML therapy, most patients eventually relapse, even the ones with normal or favorable karyotype. Since survival is poor once relapse occurs, new genetic tools above karyotype at diagnosis are needed to predict leukemia free survival. Recently, Flt3/ITD has been reported as an independent marker for clinical outcome in most studies concerning adult AML patients. To assess the prognostic relevance of activating mutations of Flt3, pretreatment samples of 100 not-M3 AML patients, all of them subjected to an intensive chemotherapy regimen, were analyzed for Flt3/ITD; 25/100 patients had one or more Flt3-ITD. Flt3/ITD patients had higher WBC count (P = 0.005) , a lower incidence of a preceding MDS (P = 0.004) and most of them had a normal karyotype. Flt3/ITD had no impact on CR achievement while karyotype remained the most powerful prognostic factor (HR 2.8 95% CI 1.2 - 6.3). However, post-remission outcome was significantly worsened by the presence of Flt3/ITD. Median RFS of the Flt3/ITD patients was 5 vs. 27 months compared to the patients with wild-type Flt3 (P = 0.0002); moreover, Flt3/ITD patients had a significantly poorer post-remission survival (11 vs. 38 months, P = 0.01). On multivariate analysis, the presence of Flt3-ITD significantly affected relapse free survival and post-remission survival (HR 3.1 and 2.1, respectively). Thus, post-remission outcome highly depends on Flt3 status. Flt3 mutations identify patients at high risk of relapse, who should prospectively receive, according to age, either more aggressive or alternative therapeutic approaches.     

Eradication of minimal residual disease with alemtuzumab in B-cell chronic lymphocytic leukemia (B-CLL) patients: the need for a standard method of detection and the potential impact of bone marrow clearance on disease outcome

The introduction of new therapeutic agents, such as fludarabine phosphate (Fludara) and alemtuzumab (MabCampath, Campath), has made it possible to treat B-cell chronic lymphocytic leukemia (B-CLL) more effectively, compared with alkylating agents. However, although an increasing number of patients are able to achieve complete remission (CR), relapse is almost inevitable, because of the re-emergence of the malignant clone from small numbers of residual malignant cells. This phenomenon has introduced a need for a more sensitive assessment of low-level disease which, in turn, has encouraged the development of therapies aimed at the eradication of all residual disease in CR patients. The eradication of residual disease is associated with improved remission durability and has great potential in offering the possibility of cure. Alemtuzumab is the foundation of many eradication-based treatment approaches because of its ability to achieve clinical remissions and to successfully purge minimal residual disease (MRD) from both blood and bone marrow in B-CLL patients. This article describes and compares polymerase chain reaction (PCR) and flow cytometric methodologies for the assessment of MRD, and presents data demonstrating that alemtuzumab can eliminate residual malignant cells from blood and bone marrow (whether assessed by PCR or flow cytometry) at the highest levels of sensitivity currently available. The ability to clear MRD from bone marrow in patients achieving clinical CR using alemtuzumab is a significant step forward in the treatment of B-CLL, and supports treatment strategies in which alemtuzumab is used in combination with other agents. Purging of MRD from both blood and bone marrow also enables patients to proceed to autologous hematopoietic stem cell transplantation, a strategy that is able to achieve long-term remission.     

Masked t(15;17) APL with the insertion of PML–RARα fusion gene in 4q21

Most cases of acute promyelocytic leukemia (APL) are characterized by the reciprocal translocation t(15;17); however, several complex variant translocations have also been reported. Here we report complex cytogenetic abnormalities without t(15;17) assayed by the G-banding method in a 62-year-old woman with the typical morphology and clinical features of APL. Based on spectral karyotyping and FISH analyses, we confirm the insertion of a cryptic chromosomal segment containing the PML/RARα fusion gene. The patient achieved complete remission after treatment with all-trans retinoic acid (ATRA) alone. Although the mechanism of this cryptic variant insertion is not known, we conclude that the insertion of PML–RARα fusion into 4q21 seems not to alter the effectiveness of treatment with ATRA.