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.     

Toll-like receptor signaling pathway in chronic lymphocytic leukemia: distinct gene expression profiles of potential pathogenic significance in specific subsets of patients

Background

Signaling through the B-cell receptor appears to be a major contributor to the pathogenesis of chronic lymphocytic leukemia. Toll-like receptors bridge the innate and adaptive immune responses by acting as co-stimulatory signals for B cells. The available data on the expression of Toll-like receptors in chronic lymphocytic leukemia are limited and derive from small series of patients.

Design and Methods

We profiled the expression of genes associated with Toll-like receptor signaling pathways in 192 cases of chronic lymphocytic leukemia and explored potential associations with molecular features of the clonotypic B-cell receptors.

Results

Chronic lymphocytic leukemia cells express all Toll-like receptors expressed by normal activated B cells, with high expression of TLR7 and CD180, intermediate expression of TLR1, TLR6, TLR10 and low expression of TLR2 and TLR9. The vast majority of adaptors, effectors and members of the NFKB, JNK/p38, NF/IL6 and IRF pathways are intermediately-to-highly expressed, while inhibitors of Toll-like receptor activity are generally low-to-undetectable, indicating that the Toll-like receptor-signaling framework is competent in chronic lymphocytic leukemia. Significant differences were identified for selected genes between cases carrying mutated or unmutated IGHV genes or assigned to different subsets with stereotyped B-cell receptors. The differentially expressed molecules include receptors, NFκB/MAPK signaling molecules and final targets of the cascade.

Conclusions

The observed variations are suggestive of distinctive activation patterns of the Toll-like receptor signaling pathway in subgroups of cases of chronic lymphocytic leukemia defined by the molecular features of B-cell receptors. Additionally, they indicate that different or concomitant signals acting through receptors other than the B-cell receptor can affect the behavior of the malignant clone.     

Analysis of T-cell subsets in B-cell chronic lymphocytic leukemia: a correlation with the stage of disease

T-cell subsets were determined by the Leu monoclonal antibodies in the peripheral blood and/or bone marrow of 52 patients with B-cell chronic lymphocytic leukemia (B-CLL) not on therapy at the time of study. The diagnosis of B-CLL required that the leukemic cells expressed surface receptors for "la-like" antigen, Fc fragment of IgG, mouse red blood cells (MRBC), C3-coated red cells (EAC), and low density of monoclonal surface immunoglobulin. The Leu-3a+/2a+ ratio was applied to define the balance between the helper/suppressor subsets in the residual T-lymphocytes. Most patients showed a decrease in the Leu-3a+/2a+ ratios at all stages of disease. The decrease in ratio was mainly related to a decrease in the Leu-3a+ T-cell subset. The more advanced stages of B-CLL were associated with lower Leu-3a+/2a+ ratio, higher total white cell and percent lymphocyte counts. There was no correlation between the proportion of EAC or MRBC rosetting cells and stages of B-CLL. This analysis further suggests that B-CLL is an immunosuppressed state that becomes more pronounced in the advanced stages and is characterized by a progressive decrease in the Leu-3a+ (helper) T-cell subset.     

B-cell receptor, clinical course and prognosis in chronic lymphocytic leukaemia: the growing saga of the IGHV3 subgroup gene usage

The immunoglobulin heavy chain variable gene (IGHV) mutational status has been recognized as an important predictor of prognosis in chronic lymphocytic leukaemia (CLL) since 1999. More recently, other features of the B-cell receptor, such as stereotypy, have been identified as capable of refining the prognostic potential of IGHV status in the clinical assessment of CLL patients. In this context, different genes belonging to the IGHV3 subgroup, the most frequently used subgroup in CLL, have been shown to denote disease subsets that either display a bad prognosis (i.e. IGHV3-21, IGHV3-23) or are associated with particularly good clinical outcomes, including a highly stable/indolent clinical course, even prone to spontaneous regression (i.e. IGHV3-72, IGHV3-30). The present review focuses on the molecular and biological features of CLL-expressing specific genes belonging to the IGHV3 subgroup that are known to mark disease subsets with completely different clinical courses, and may be possibly related to CLL pathogenesis via antigen and/or superantigen involvement.     

Analysis of a large multi-generational family provides insight into the genetics of chronic lymphocytic leukemia

We report the genetic analysis of a large multi-generational family composed of 144 individuals in which 11 members have been diagnosed with chronic lymphocytic leukaemia (CLL). The observation of a significant over-representation of monoclonal B-cell lymphocytosis (MBL) in unaffected family members strongly supports MBL being a surrogate marker of carrier status. A genome-wide linkage scan of the family using high-density 10K single nucleotide polymorphisms provided no significant evidence for a single gene model of disease susceptibility, inviting speculation that susceptibility to CLL has a more complex basis. The absence of a correlation in IGHV usage between affected family members does however argue strongly against exposure to a single super-antigen in disease development.     

Surface antigen expression and correlation with variable heavy-chain gene mutation status in chronic lymphocytic leukemia

Recent studies have demonstrated that B-cell chronic lymphocytic leukemia (CLL) consists of two clinical entities with either somatically hypermutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy-chain (VH) regions. In view of the fact that the cellular biology of these two subsets of disease is currently unexplored, we performed an extensive analysis of the surface antigen expression and correlated this with the VH gene mutation status in a cohort of 32 CLL patients. Using polymerase chain reaction amplification and nucleotide sequencing, the VH genes were shown to be mutated in 10 cases (31%) and unmutated in 22 (69%). The expression of 27 surface membrane antigens in peripheral blood leukemic cells was analyzed by flow cytometry, measuring both the percentage of positive cells as well as the geometric mean fluorescence intensity (GMF). Most of the surface membrane antigens (CD5, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD45, VD79b, CD80, CD95, CD122, CD124, CD126, CD130, CD154, IgM, and IgD) showed a similar expression pattern in both UM-CLL and M-CLL patients. The similarity of M-CLL and UM-CLL, as demonstrated here for the first time with many protein markers, indicates a considerably homogeneous phenotype in both subsets. Furthermore, CD27 was strongly expressed in all cases, which may suggest a memory cell phenotype for both M-CLL and UM-CLL. More positive cells in the UM-CLL group were observed regarding CD38, but CD38 was not a good predictor of VH gene mutation status. Seventy percent of the M-CLL cases, but only 36% of UM-CLL cases, were Ig-lambda+. The most striking differential expression, however, was observed in the two slicing variants of the common leukocyte antigen CD45, namely CD45RO and CD45RA. CD45RO expression was significantly associated with M-CLL, whereas the GMF intensity of CD45RA tended to be associated with UM-CLL. The role of these CD45 splicing variants in the pathogenesis of CLL deserves further investigation.     

CCR4 expression in a case of cutaneous Richter's transformation of chronic lymphocytic leukemia (CLL) to diffuse large B-cell lymphoma (DLBCL) and in CLL patients with no skin manifestations

Objective: Richter’s transformation of B‐cell chronic lymphocytic leukemia (CLL) to cutaneous diffuse large B‐cell lymphoma (DLBCL) is very rare. We took the advantage of one of these cases to test the hypothesis that the chemokine receptor CCR4 is involved in the homing of CLL cells to skin. Patients and Methods: We evaluated CCR4 expression by flow cytometry in both circulating and skin CD19⁺ leukemic cells from a patient with cutaneous DLBCL. As controls, we used peripheral blood samples from CLL patients without skin manifestations and from elderly healthy donors. Results: We found that both DLBCL cells derived from the original CLL clone and circulating CLL cells from this patient expressed CCR4. Although it was previously reported that CCR4 is not expressed in CLL cells, we found that a low but significant proportion of leukemic cells from CLL patients with no skin manifestations do express CCR4. There was a positive correlation between the expression of CCR4 and the percentage of ZAP‐70 of each sample. Moreover, we consistently observed a higher expression of CCR4 within CD19⁺CD38⁺ and CD19⁺Ki67⁺ subsets compared to CD19⁺CD38^? and CD19⁺Ki67^? lymphocytes from the same sample, respectively. Conclusion: We conclude that the chemokine receptor CCR4 is not a special feature of CLL cells with skin manifestation, but rather it is expressed in a low but significant proportion of peripheral blood CLL cells.     

Current approaches to the chemotherapy of B-cell chronic lymphocytic leukemia: a review

Standard therapy has not substantially improved the outcome of patients with chronic lymphocytic leukemia (CLL). However, an increased understanding of the biology and immunology of CLL, and the availability of several new and active chemotherapy agents (eg, fludarabine, 2'-deoxycoformycin [DCF], 2-chlorodeoxyadenosine [CDA]) has stimulated enthusiasm for clinical trials. DCF induces CRs or PRs in 25% of heavily treated patients. Fludarabine has been associated with a response rate of greater than 50% in previously treated patients, and greater than 70% in untreated patients, with almost a third of these achieving a CR. Currently, phase I and II clinical trials are evaluating combinations of these drugs with each other or with conventional agents (eg, fludarabine/chlorambucil [CLB]/prednisone [P]; DCF/CLB/P; fludarabine/DCF; fludarabine/P) in previously treated patients. To facilitate comparison of these regimens, each study is adhering to the NCl-Working Group guidelines for eligibility and response criteria, and toxicity assessment. A collaborative phase III trial will then compare the most promising of these regimens with "standard" chemotherapy in previously untreated patients. The widespread availability of these clinical trials will allow clinicians ready access to the new treatments.     

Analysis of CLLU1 expression levels before and after therapy in patients with chronic lymphocytic leukemia

Objective: Chronic lymphocytic leukemia (CLL) is incurable, but therapy leading to eradication of minimal residual disease (MRD) in CLL is associated with improved clinical outcomes. CLL upregulated gene 1 (CLLU1) is solely upregulated in CLL patient samples. We hypothesized that CLLU1 could be used to monitor for residual disease in CLL patient samples after therapy. Methods: We examined whether the CLLU1 real‐time quantitative PCR (RQ‐PCR) could detect small numbers of CLL cells in mixtures of normal peripheral blood mononuclear (PBMC) cells. We then performed a retrospective analysis on time‐matched cryo‐preserved specimens from patients who achieved MRD‐negative remissions that underwent serial marrow biopsies for evaluation of residual disease by 4‐color flow cytometry. RNA from PBMC samples collected at the time of the marrow assessments was analyzed for CLLU1. Nine patients underwent a total of 46 paired blood and marrow evaluations (median 5 assessments per patient). Results: CLLU1 RQ‐PCR on PBMCs of healthy donors reconstituted with varying amounts of CLL cells demonstrated leukemia cells could be reliably detected with high sensitivities depending on the CLLU1 expression level. Analysis of time‐matched samples assessed for CLLU1 levels in the blood by RQ‐PCR and residual disease of the marrow determined by 4‐color flow cytometry revealed a correlation coefficient of 0.96 (P < 0.0001). Conclusion: The CLLU1 RQ‐PCR is a sensitive and specific assay for detecting residual CLL cells after therapy. Assessment of blood CLLU1 levels can be used as a reliable marker of tumor burden and has the potential to complement currently used techniques for MRD monitoring in patients with CLL.     

ATM gene alterations in chronic lymphocytic leukemia patients induce a distinct gene expression profile and predict disease progression

Background

The genetic characterization of chronic lymphocytic leukemia cells correlates with the behavior, progression and response to treatment of the disease.

Design and Methods

Our aim was to investigate the role of ATM gene alterations, their biological consequences and their value in predicting disease progression. The ATM gene was analyzed by denaturing high performance liquid chromatography and multiplex ligation probe amplification in a series of patients at diagnosis. The results were correlated with immunoglobulin gene mutations, cytogenetic abnormalities, ZAP-70 and CD38 expression, TP53 mutations, gene expression profile and treatment-free interval.

Results

Mutational screening of the ATM gene identified point mutations in 8/57 cases (14%). Multiplex ligation probe amplification analysis identified six patients with 11q deletion: all of them had at least 20% of deleted cells, analyzed by fluorescent in situ hybridization. Overall, ATM point mutations and deletions were detected in 14/57 (24.6%) cases at presentation, representing the most common unfavorable genetic anomalies in chronic lymphocytic leukemia, also in stage A patients. Patients with deleted or mutated ATM had a significantly shorter treatment-free interval compared to patients without ATM alterations. ATM-mutated cases had a peculiar gene expression profile characterized by the deregulation of genes involved in apoptosis and DNA repair. Finally, definition of the structure of the ATM-mutated protein led to a hypothesis that functional abnormalities are responsible for the unfavorable clinical course of patients carrying these point mutations.

Conclusions

ATM alterations are present at diagnosis in about 25% of individuals with chronic lymphocytic leukemia; these alterations are associated with a peculiar gene expression pattern and a shorter treatment-free interval.     

Prognostic significance of the heterogenous expression of IgG Fc receptors in B-cell chronic lymphocytic leukemia

Summary Receptors for the Fc part of IgG (FcR) are expressed in three forms on peripheral blood lymphocytes. The presence of the releasable form (FcR_REL.) as well as of the two nonreleasable forms with lower (FcR_LOW) and higher (FcR_HIGH) cellular avidity was correlated with survival in 63 patients with B-cell chronic lymphocytic leukemia (B-CLL). High percentage of cells with FcR_LOW as well as high absolute number of cells carrying the two nonreleasable forms of FcR were connected to unfavorable prognosis. Combining these three parameters, an FcR constellation was defined which pointed to a favorable prognosis (in 24 patients) when all three parameters were low, but detected short survivors when all three data were high (in 14 patients). The FcR constellation was capable of identifying patients with better or worse prognosis within groups that were homogeneous regarding some other known prognostic factors. FcR constellation as a prognostic factor was shown to be independent of age, sex, and Rai and Binet stages, but it was found to be connected with the total tumor mass score (TTM). The three forms of FcR on B cells might reflect stages of B-cell activation. Differences in FcR constellations between patients with B-CLL would thus correspond to differently activated B-cell clones with variable prognosis.     

Quantification improves the prognostic value of CD38 expression in B-cell chronic lymphocytic leukaemia

Recent studies have shown that CD38 expressed as a percentage of the antigen positivity can predict prognosis and disease progression in patients with B-cell chronic lymphocytic leukaemia (B-CLL). The present study showed that quantification of CD38 expressed as antibody-binding capacity (ABC) improves the prognostic value of the percentage of CD38 positivity in B-CLL. In a cohort of 81 patients with B-CLL, a level of CD38 expression of > or = 30% and an ABC value of 250 proved statistically valid cut-off points to predict disease progression (% CD38: P=0.0027; ABC: P < 0.0001). There was a positive and significant correlation between the percentage of CD38 expression and ABC (r=0.7; P < 0.0001). There was a better discrimination of survival using ABC rather than percentage CD38 positivity (P < 0.0001 compared with P=0.0027). Only ABC predicted for survival in patients under 60 years of age (P=0.0076) or with stage A disease (P=0.0195). Both percentage CD38 and ABC discriminated between time to first treatment for all patients but only ABC predicted time to treatment for stage A patients (P=0.0004). In conclusion, CD38 positivity is an important prognostic factor in B-CLL. However, quantification of CD38 is superior to the percentage positivity and should be used clinically in conjunction with other variables of predictive value to identify B-CLL patients that are likely to progress.     

Subjects with chronic lymphocytic leukaemia‐like B‐cell clones with stereotyped B‐cell receptors frequently show MDS‐associated phenotypes on myeloid cells

An increasing body of evidence suggests the potential occurrence of antigen encounter by the cell of origin in chronic lymphocytic leukaemia (CLL) and CLL‐like monoclonal B‐cell lymphocytosis (MBL). However, the scenario in which this event might occur remains unknown. In order to gain insight into this scenario we investigated the molecular, cytogenetic and haematological features of 223 CLL‐like (n = 84) and CLL (n = 139) clones with stereotyped (n = 32) versus non‐stereotyped (n = 191) immunoglobulin heavy chain variable region (IGHV) amino acid sequences. Overall, stereotyped CLL‐like MBL and CLL clones showed a unique IGHV profile, associated with higher IGHV1 and lower IGHV3 gene family usage (P = 0·03), longer IGHV complementary determining region 3 (HCDR3) sequences (P = 0·007) and unmutated IGHV (P < 0·001) versus non‐stereotyped clones. Whilst the overall size of the stereotyped B‐cell clones in peripheral blood did not appear to be associated with the CLL‐related cytogenetic profile of B‐cells (P > 0·05), it did show a significant association with the presence of myelodysplastic syndrome (MDS)‐associated immunophenotypes on peripheral blood neutrophils and/or monocytes (P = 0·01). Altogether our results point to the potential involvement of different selection forces in the expansion of stereotyped vs. non‐stereotyped CLL and CLL‐like MBL clones, the former being potentially favoured by an underlying altered haematopoiesis.     

Clinical signification of natural killer activity in B-cell chronic lymphocytic leukemia

The natural killer (NK) activity of peripheral blood mononuclear cells (PBMC) and lymphocytes with the capacity to form stable rosettes with neuraminidase-treated sheep red blood cells (E+) was studied in 28 previously untreated patients (11 at stage 0, 10 at stage I and 7 at stages II and III, according to Rai's classification) and 7 treated patients with B-cell chronic lymphocytic leukemia (B-CLL), all of them at stage 0 according to Rai's classification after treatment, and in 15 healthy controls. The mean NK activities of PBMC and E+ lymphocytes from untreated patients were significantly decreased (p less than 0.001) when compared with those of PBMC and E+ lymphocytes, respectively, from healthy controls. However, PBMC and E+ cells from treated patients demonstrated NK activity similar to that of the corresponding cellular populations of controls (p greater than 0.05). Furthermore, there were no significant differences among the NK activities of E+ lymphocytes from untreated B-CLL patients in the different clinical stages 0, I, II and III, according to Rai's classification (p less than 0.05). These results demonstrate that the very low or undetectable levels of NK activity present in PBMC and E+ cell populations from previously untreated patients with B-CLL, regardless of the clinical stage of the disease, can be modified by systemic therapy with alkylating agents. Moreover, the NK activity of PBMC and E+ lymphocytes from some treated patients that have achieved the stage 0 according to Rai's classification after chemotherapy can be found within the range of the lytic activity shown by PBMC and E+ cells from normal donors.     

Genetic modification of primary chronic lymphocytic leukemia cells with a lentivirus expressing CD38

Studies of the role of individual genes in chronic lymphocytic leukemia (CLL) have been hampered by the inability to consistently transfect primary tumor cells. Here, we describe a highly efficient method of genetically modifying primary CLL cells using a VSVG pseudotyped lentiviral vector. We transduced CD38 negative CLL cells with a lentiviral vector encoding CD38 which caused increased surface CD38 expression in all the samples tested (n=17) with no evidence of plasmacytoid differentiation. The mean percentage of positive cells expressing CD38 was 87%±8.5% and the mean cell viability 74%±17%. This high level of transduction of all the CLL cell samples tested demonstrates the utility of this technique which should prove applicable for the introduction and analysis of other genes in these non-dividing cells.     

Cryptochrome-1 expression: a new prognostic marker in B-cell chronic lymphocytic leukemia

Chronic lymphocytic leukemia is an adult-onset leukemia with a heterogeneous clinical behavior. When chronic lymphocytic leukemia cases were divided on the basis of IgV_H mutational status, widely differing clinical courses were revealed. Since IgV_H sequencing is difficult to perform in a routine diagnostic laboratory, finding a surrogate for IgV_H mutational status seems an important priority. In the present study, we proposed the use of Cryptochrome-1 as a new prognostic marker in early-stage chronic lymphocytic leukemia. Seventy patients (Binet stage A, without treatment) were included in the study. We correlated Cryptochrome-1 mRNA with well established prognostic markers such as IgV_H mutations, ZAP70, LPL or CD38 expression and chromosomal abnormalities. High Cryptochrome-1 expression correlated with IgV_H unmutated samples. In addition, Cryptochrome-1 was a valuable predictor of disease progression in early-stage chronic lymphocytic leukemia, therefore it can be introduced in clinical practice with the advantage of a simplified method of quantification.