Unanswered questions in CLL biology

Which are the reasons accounting for the low expression of the B-cell receptor (BCR) in CLL?

Low expression of the BCR is the hallmark of the B-CLL lymphocyte.³ The BCR is a multimeric complex formed by the assembly of surface immunoglobulin (SIg) homodimer and the noncovalently bound heterodimer Ig/Ig (CD79a/CD79b). Both these molecules play a key role for receptor expression and signal transduction through their immunoreceptor tyrosine-based activation motifs (ITAM), by linking the antigen binding Ig chains to intracellular tyrosine kinases of the Src family. These events are transmitted through various signalling pathways into the cell nucleus to induce a cellular response.⁴

Low expression of the BCR in B-CLL cells correlates with a reduced induction of protein tyrosine kinase activity, which results in defective intracellular calcium mobilization and tyrosine phosphorylation,⁵ and lead to impaired responses of B-CLL cells when stimulated through the BCR pathway.⁶

Despite normal transcription and intracellular synthesis, we previously showed that this low expression is accounted for by a defective assembly of the BCR chains resulting in the presence of unprocessed chains and retention of the BCR at the reticulum endoplasmic compartment.⁷ However, the exact mechanism involved in defective assembly of the BCR remains elusive, as it remains to be solved whether the CLL B lymphocyte is an anergic cell and whether BCR underexpression is related to the malignant hit. Although, no definitive proof is presently available concerning the anergic state of CLL B cells, similar underexpression of the BCR and defective signal transduction have been reported in the case of anergic murine B cells.⁸ This could be the consequence of BCR crosslinking by autoantigens, since the BCR of B-CLL can often be autoreactive.^9,10,11 The possibility also exists that BCR retention in the endoplasmic reticulum in CLL B cells could be accounted for by the blockade of its assembly through an interaction with an as yet uncharacterized intracellular protein as reported for the K1 protein from human herpesvirus 8 in a B-lymphoma cell line.¹² We believe that elucidation of the mechanisms implicated in BCR underexpression in B-CLL should be a key step in the comprehension of disease pathogenesis.

Is CLL one disease or two diseases that look alike?

Vast majority of B-CLL cells express CD5 and IgM/IgD and thus have a mantle zone-like phenotype of naive cells, which in normal conditions express unmutated Ig genes.¹³ However, recent studies have shown that 50–70% of CLL harbor somatic mutations of VH genes,^14,15 as if they had matured in a lymphoid follicle. Interestingly, the presence or absence of somatic mutations is associated with the use of particular VH genes. Particular alleles of the V1-69¹⁶ gene and the V4-39 gene are preferentially expressed in an unmutated form, while V4-34 or the majority of VH3 family genes frequently contain somatic mutations.^14,15 The fact that some genes like VH1-69 and VH3-07 recombine this VH segment to particular JH segments and the restricted use of CDR3 sequences by CLLs expressing the VH4-39 gene suggest that the observed differences in BCR structure in B-CLL could result from selection by distinct antigenic epitopes. It is presently unclear whether this putative antigen-driven process could occur prior to leukemic transformation and/or that the precursors were transformed into leukemic cells at distinct maturational stages.^14,15,17

The mutational profile of Ig genes has been shown to be associated to disease prognosis.^18,19,20 These results could favor the idea that CLL could correspond to two different diseases that look alike in morphologic and phenotypic terms. In CLL with mutated Ig genes, the proliferating B cell may have transited through germinal centers, the physiologic site of hypermutation, whereas in CLL with unmutated Ig genes the malignant B cell may derive from a pregerminal center naive B cell.

In favor of this view is the fact that the mutational profile of Ig genes is associated to genetic aberrations. Whereas 11q23 or 17p13 deletions are associated to poor outcome and to an unmutated V_H profile, 13q14 deletion or a normal karyotype are associated to a mutated profile and there is controversy on whether trisomy 12 is associated to an unmutated status.^{2,3,4,5,6,7,8,9,10,11, 12,13,14,15,16,17,18,19,20,21, 22,23} Although, multiple instances of the disease in some families and the low incidence of the disease among individuals of Japanese origin, including those who migrated to Hawaii,²⁴ suggests that genetic influences are much stronger than environmental factors in the pathogenesis of the disease, the nature of this genetic predisposition remains unknown and none of the reported genetic aberrations is constant and frequently occur during evolution.²³ In contrast with what is observed in other B-cell malignancies, where chromosomal translocations associate oncogenes with Ig genes, in CLL the most frequent abnormalities are mutations, deletions or trisomies, whose biological significance remains unknown if we except those affecting the p53 suppressor gene.

Mutated and unmutated CLL patients clearly differ in terms of prognosis and may also differ with respect to oncogenic mechanisms (11q deletions are almost always associated to an unmutated profile). Despite these clinical and molecular differences, recent studies on gene expression profiling of B-CLL cells showed that CLL is characterized by a common gene expression signature, which is irrespective of Ig mutational status and differs from other lymphoid cancers and normal lymphoid subpopulations, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin.^25,26 These results are in agreement with the CLL monotonous phenotypic signature, that is, BCR underexpression.

However, despite sharing a common signature CLLs expressing mutated and unmutated Ig genes differentially express more than 100 genes. Among these differentially expressed genes, overexpression of zeta-chain-associated protein (ZAP-70),²⁷ lipoprotein lipase (LPL), BCL-7a, dystrophin and gravin are observed in the aggressive unmutated cases,^25,26 while stable mutated cases overexpress Wnt3, CTLA-4, NRIP1 nuclear receptor gene, ADAM and the transcription factor TCF7.²⁸ In addition, a nonsupervised hierarchical clustering analysis is able to separate the stable mutated group from the aggressive unmutated one.²⁸ These results suggest that indolent mutated and aggressive unmutated CLLs constitute two variants of the same disease. The striking differences in clinical outcomes of these two variants remain unknown as yet. Whether they correspond to differentiation stages at the moment of malignant transformation or whether unmutated forms of the disease are in a more activated form that favors the proliferative potential of the malignant clone still elude us. Better signalling transduction in the latter, when stimulated through the BCR pathway,⁶ and a positive role of ZAP-70²⁹ in this signalling could also play a role.

What is the normal counterpart of the CLL B lymphocyte?

It has been proposed that the normal counterpart of leukemic cells may be a follicular mantle-zone B1 cell, which normally expresses CD5, CD23, is negative for CD38, coexpresses membrane IgM and IgD and utilizes unmutated Ig V region genes. In contrast, germinal center B cells express CD38, loose IgD, frequently exhibit isotype switch and display somatic mutations in Ig genes.¹³

However, CLL B cells whether displaying mutated or unmutated Ig V genes almost always express IgM and IgD, CD5 and CD23. In addition, CD38 expression paradoxically predominates among unmutated cases and active class switch recombination (CSR) at the RNA, and protein is often observed among CLL B cells.^30,31,32 Constitutive expression of activation-induced cytidine deaminase (AID) predominates among CLL B cells displaying active CSR and unmutated V genes.^33,34 This enzyme known to play a key role in both CSR and somatic hypermutation is not usually expressed in normal circulating B cells, but is constitutively expressed in germinal center B cells. Since expression of AID is induced upon CD40 ligand stimulation, its frequent expression by CLL B cells displaying unmutated V genes challenges the notion that these could correspond to naive B cells.³³

As proposed by Chiorazzi and Ferrarini,¹⁷ the possibility also exists that the normal counterpart could be marginal zone B cells, which have been demonstrated to express both mutated and unmutated V genes. Although, marginal zone B cells do not express CD5, this expression could be the consequence of the activation profile of CLL B cells.³⁵ However, this provocative hypothesis still fails to explain the common gene profiling signature in B-CLL.

Which are the mechanisms involved in CLL-defective apoptosis?

The accumulation of mature B cells, which have escaped programmed cell death and have undergone cell cycle arrest in the G0/G1 phase, is the hallmark of B-CLL. These cells have a low proliferative activity and recent results support the hypothesis that an 'in vivo' defective apoptosis account for accumulation of B cells in this disease.³⁶ In contrast with 'in vivo' results, apoptosis occurs after 'in vitro' culture, suggesting a role of the microenvironment on the B-CLL cells survival.^37,38 Reports indicating that apoptosis 'in vitro' is prevented by exposure to IL-4^39,40 as well as by stimulation via surface CD40,⁴¹ also favor this view. In vivo, such inhibition may occur in pseudofollicles observed in the lymph nodes and in the cell clusters described in the bone marrow.^37,42 This could be in agreement with a model of selective survival of certain clonal submembers, which would receive survival signals in these particular sites. This mechanism may compensate the clonal decrease that could occur in periphery by apoptosis, and depending on its importance could play a major role in the regulation of the tumoral mass size.

The importance of the antiapoptosis bcl-2 oncogene in B-cell malignancies was established in studies of follicular lymphomas.⁴³ These lymphomas carry the t(14;18), which places bcl-2 on chromosome 18, under the regulation of the Ig heavy-chain enhancer, on chromosome 14, leading to increased bcl-2 expression. In B-CLL cells, translocations of the bcl-2 gene are rare (less than 1% of cases) despite high levels of the bcl-2 protein.⁴⁴ Its role in apoptosis inhibition is not clear since no correlation exists between 'in vitro' apoptosis and the level of bcl-2 expression.

Deregulation of cell cycle regulatory genes may also contribute to the accumulation of malignant cells in early phases (G0/G1) of the cell cycle. In B-CLL cells, elevated levels of the cyclin-negative regulator p27^Kip1 protein are found in a majority of patients.⁴⁵ Given the key role of this protein in cell cycle progression, its overexpression in B-CLL cells could account for the accumulation of B cells in early phases of the cell cycle.

What are the reasons for the high incidence of hypogammaglobulinemia and increased autoimmune disorders in CLL?

Hypogammaglobulinemia occurs in at least 60% of B-CLL cases.⁴⁶ Patients with early forms of the disease tend to have defective-specific antibody responses to infection or immunization.⁴⁷ Hypogammaglobulinemia could result from accumulation of these individual defects. The pathogenesis of hypogammaglobulinemia in B-CLL is poorly understood, as this phenomenon is rare in other B-cell malignancies including acute lymphoblastic leukemia, nodular and diffuse lymphomas, hairy cell leukemia, prolymphocytic leukemia, although it is common in multiple myeloma.⁴⁶ Although, regulatory abnormalities in T cells may play a role in the induction of hypogammaglobulinemia, data concerning helper and suppressive subpopulations are contradictory, and fail to firmly establish their contribution to the development of hypogammaglobulinemia. Alternatively, hypogammaglobulinemia in B-CLL may result as a consequence of dysfunction of nonclonal B CD5-negative B cells. This decrease or inhibition of normal CD5-negative B cells could also explain the classical inability of B-CLL to respond to new antigenic challenges, since Ly1-B cells (the murine counterpart of human CD5 B cells) have been claimed to be unable to respond to exogenous antigens.⁴⁸

A prominent monoclonal immunoglobulin peak, usually of the IgM type, is found in 5% of CLL. With high-resolution agarose gel electophoresis and immunofixation, however, a small amount of a monoclonal component can be identified in the serum or urine of 60% of patients.⁴⁹

Autoimmune-associated phenomena are frequently observed in B-CLL. These autotoxic manifestations are mainly directed against hematopoietic cells.⁵⁰ A positive direct antiglobulin test has been reported to be as high as 7–35% of B-CLL patients, depending on the series and stage of disease.^51,52 Nevertheless, autoimmune hemolytic anemia occurs in 10–25% of patients at some time during the course of the disease.^51,53 In most cases, autoantibodies against red blood cells are warm reactive polyclonal IgG, and are not secreted by the malignant clone.51 Immune thrombocytopenia is observed in about 2% of cases.⁵⁰ Pure red cell aplasia and autoantibodies against neutrophils are only rarely observed.^54,55 Whether the appearance of these autoantibodies is the consequence of the dysregulation of the idiotypic network induced by the hypogammaglobulinemia or is the consequence of a defect of T-cell subsets controlling the autoantibody producing B cells remains elusive as yet. This last possibility could explain the higher incidence of autoimmune complications among patients receiving purine analogues therapy, which induce an important T-cell depletion.^56,57 However, this last complication is mainly observed among heavily pretreated patients, receiving purine analogues as salvage therapy.^51,52,56,57

Unanswered questions in CLL prognosis

Is it time to move from Rai and Binet staging systems?

The development of the Rai⁵⁸ and Binet⁵⁹ staging systems has allowed the division of patients with CLL into three prognostic groups: good, intermediate and poor prognosis. Binet's good prognosis group (stage A, 63% of CLL patients with a 10-year survival of 51%) includes twice as many patients as Rai's Stage 0, since it includes all Rai's 0, 2/3 of Rai's I and 1/3 of Rai's II. Rai's stage 0, which includes 31% of CLL patients, displays a 10-year survival 59%. Rai's intermediate prognosis group includes 59% of CLL patients compared to 30% in the Binet's intermediate group.⁶⁰ These staging systems provided a foundation allowing clinicians to build therapeutic strategies.

The low-risk group (Stage A from Binet or Stage 0 from Rai) has a median age at diagnosis of 64 years and an expected survival of >10 years, which is close to the life expectancy of a normal population matched for sex and age.⁶¹ However, over 25% of these indolent cases die of causes related to CLL, 40% progress to advanced stages and 50% ultimately require treatment.²

These results demonstrate that neither the Rai nor the Binet staging system are able to accurately predict which patients among the good prognosis group will shift into progressive disease.^2,62 Lymphocyte doubling time, serum levels of 2-microglobulin and thymidine kinase^63,64 and soluble CD23,⁶⁵ as well as CD38 expression on malignant cells^18,23 can help predict disease activity, but the presence in the leukemic B cells of cytogenetic abnormalities like 11q or 17p deletions^21,22,23 or somatic mutations in the immunoglobulin heavy-chain genes^18,19,20 are better predictors of rapid progression and survival. We could recently confirm, on a retrospective series of 146 patients with a long follow-up, previous reports demonstrating the strong prognostic value of V_H mutational profile.^18,19,20 Our results also showed that the mutational profile of Ig genes is able to delineate prognostic groups within all Binet's stages. Since mutated and unmutated groups display different prognosis when comparing A with B/C cases, both Binet's staging and V_H genes retain their independent prognostic significance in CLL. Indeed, they should be most likely complementary.⁶⁶

The V_H mutational profile has the advantage that it remains constant during evolution, which contrasts genomic aberrations and serum markers. In addition, since 11q23 or 17p13 deletions are associated to poor outcome and to an UM V_H profile in about one-third of unmutated cases, they are included among the unmutated group. Although CD38 expression is associated to poor prognosis,^67,68,69,70 its relationship to Ig mutational status remains controversial.^71,72 Besides the fact that its expression has been demonstrated to change during disease evolution,⁷¹ there are important concerns related to interlaboratory variations, to the definition of the best cutoff value^18,23,69 and to its intensity of expression.⁷³

Which is the best surrogate for Ig mutational profiles?

Since the ability to sequence Ig V genes is not available in most laboratories, a valid and easily performed surrogate assay is desirable. Damle et al⁷⁴ suggested that CD38 determination might be a useful alternative. However, correlation of Ig mutational status with CD38 expression remains controversial,^22,23 although its independent prognostic value is demonstrated. In addition, a clear correlation between mutational profiles and serum levels of thymidine kinase has also been established.⁶⁴ Crespo et al⁷⁵ proposed that detection of ZAP-70, by simple and convenient multiparameter flow-cytometric test, is highly correlated to the presence of an unmutated profile of Ig genes (91% sensitiviy). If confirmed in other series, these results should constitute an important contribution in the search of surrogate markers of Ig mutational profile.

Is it possible to identify smouldering CLL?

The association of Binet's stage A with V_H MT profile isolates a group accounting for more than 40% of CLL patients, with very good prognosis (12-year survival of 75%, median progression free survival of 13 years and <10% CLL related deaths). The survival of this group of patients should be very close to that of a sex- and age-matched normal population. However, progression is observed for one-third of these patients and CLL-related death for 10% of them. Since the possibility that a second malignant hit could occur exists, it is unclear whether early identification of the small group of A mutated patients, who will display disease progression during evolution, is possible.

Unanswered questions in CLL treatment

When should a CLL patient receive treatment?

Once the diagnosis of CLL has been made, the treating physician is faced with the decision of not only how to treat the patient, but when to initiate therapy. A subset of patients are considered as having smoldering CLL; they include those with Rai stage 0 or Binet A expressing mutated Ig genes and no deleterious chromosomal aberration (40% of all CLL patients). For these cases, a wait and watch police is justified. On the other hand, wide recognition of unmutated cases among Binet's stage A (or Rai's indolent forms) should provide means of testing the putative benefits for early treatment, in the frame of prospective randomized trials.

By contrast, there is consensus that most patients with Binet Stage B or C, or with Rai Stage III or IV, and patients with Rai Stages I or II with progressive disease⁷⁶ whose life expectancy does not exceed 7 years should be considered for early treatment. Although, most patients with Rai stages I and II with progressive disease and Rai' stages III and IV or Binet's B and C require treatment at presentation, some patients can still be monitored without therapy until they exhibit evidence of progressive or symptomatic disease. In addition, the prognostic differences observed among B/C patients according to V_H mutational status (median survival of 120 months for B and C patients expressing mutated Ig genes as compared to 78 for patients expressing unmutated ones) raise the question whether therapy could be tailored for these patients.

Which is the best treatment?

Chlorambucil given daily or intermittently, alone or combined to corticosteroids, is the most commonly used drug. It often provides a period of relief from any symptoms, even in advanced disease. However, several randomized trials have failed to demonstrate its ability to improve survival.^2,77,78,79 Results from the ICCI CLL-01 trial⁸⁰ showed that high dose continuous chlorambucil could improve survival, although these results need to be further confirmed.

A meta-analysis of 10 randomized trials involving 2035 patients with advanced CLL, in which chlorambucil was compared with several combination chemotherapy regimens which in their majority contained CHOP, was carried out.⁷⁹ Although improvement in response rates was observed for combination chemotherapy regimens, in none of these trials this improvement translated into improved survival.

The MD Anderson group has shown in important nonrandomized studies that the purine analogue fludarabine is able to obtain response rates never reported before for a drug in CLL.⁸¹ In this series, the association of corticosteroids to fludarabine did not improve survival and was associated to increased toxic manifestations. Since then, three randomized studies have compared fludarabine⁸² or cladribine⁸³ to chlorambucil or fludarabine to two variants from CHOP.⁵² Purine analogues were demonstrated to induce higher response rates when compared to chlorambucil and CAP and similar response rates than CHOP. The CALGB trial⁸² aimed also to compare fludarabine and chlorambucil as single agents to the association of fludarabine and chlorambucil. Unfortunately, this last arm has been discontinued because of increased toxicity.

These above-mentioned comparative trials clearly conclude to significant differences for response rates and progression-free survival between the treatments. However, these advantages did not translate into improved survival. The absence of plateau on survival curves suggests that these conventional treatments are not curative, even considering the small proportion of patients which behave as long-term survivors with a stable or smoldering residual disease.^52,81,82 Interestingly, this also occurred among patients included in the pivotal study from Minot et al,⁸⁴ using splenic irradiation as treatment. Actually, recurrences of the disease are observed in almost every patient, leading to second and subsequent treatments that obscure the interpretation of long-term results. Despite the absence of survival improvement in these trials, the overall survival of CLL patients has globally improved from 5 to 8 years for stage B patients and from 2 to 5 years for stage C patients.^52,77,85 This consistent improvement could be due to subsequent treatment provided to patients failing to respond initial treatment.

The humanized monoclonal anti-CD52 antibody (CAMPATH-1, alemtuzumab) has important activity against B-CLL cells. Initial studies demonstrated high activity of this drug in previously treated and even refractory CLL patients.⁸⁶ In front line, either in an intravenous or a subcutaneous schedule, encouraging results have also been reported.^87,88 Thus, this monoclonal has a promising activity in refractory CLL and could play an important role in front-line therapy in the frame of randomized trials aiming at obtaining complete molecular response, as a consolidation of a good response to chemotherapy.

Anti-CD20 (Rituximab, MabThera) as single agent shows limited efficacy in CLL. However, in combination with chemotherapy and particularly fludarabine it appears to act synergistically and to achieve high rates of response including in some cases molecular responses.⁸⁹ The combination of anti-CD20 and anti-CD52 has been reported to provide high response rates in refractory patients.⁹⁰ Although, both monoclonal antibodies have been demonstrated to be effective and should play an important role in refractory cases, this needs to be substantiated in randomized trials and it is presently unclear whether they are able to improve survival.

Which is the place for intensification procedures in CLL treatment?

Evaluations of treatments called intensification procedures, which aim for a complete molecular remission (no evidence of molecular markers of the malignant clone after treatment), are in progress. The rationale of these treatments is based on the speculation that increasing the quality of response should provide a longer time to progression interval, and the assumption that it is the best way to increase overall survival. They include purine analogs with or without other drugs followed by autologous bone-marrow transplantation or monoclonal antibodies or both. Some patients have entered a sustained molecular remission with such intense treatments, but it is unknown whether the treatment cures the disease or just delays a relapse.⁹¹

Increasing number of patients are presently included for autologous bone marrow transplantation (AutoBMT). Mortality related to it is acceptable (<5%). Although true molecular remission can be achieved, it mainly depends on initial disease stage, residual disease before AutoBMT, response to previous treatment and quality of the graft. An European trial on an important series of heterogeneous patients showed a 3-year survival of 78% and a relapse rate of 45%.⁹² Randomized clinical trials are needed to answer whether this treatment is able to improve outcome of CLL patients.

Conventional allogeneic bone marrow transplantation (ABMT) can probably be curative in some cases, but only 10% of patients with CLL are eligible for this treatment, which has a mortality rate >40% mortality, 20% relapse rate and 40% prolonged remissions, which may in some cases correspond to cures.⁹³ ABMT in which the patient's marrow is not ablated by high-dose chemotherapy is another option under evaluation.^94,95

AutoBMT compares favorably to ABMT in terms of treatment-related toxicity, mortality and overall survival. However, a lower incidence of relapse is observed with allotransplants, suggesting a graft vs leukemia allogenic effect and the possibility that some of these patients might have achieved cure.⁹²

Which is the best treatment for relapsing and refractory patients?

Fludarabine is the best option for patients refractory to alkylating agents,⁹⁶ and mini-CHOP⁵² or anti-CD52⁸⁶ for patients who are refractory to fludarabine. There is evidence that these patients with a poor prognosis may benefit from intensification strategies if their general health permits. Depending on age and coexisting diseases, intensification procedures are justified for these patients.

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