Abstract: Chronic lymphocytic leukemia (CLL) is a clonal malignancy of mature B cells that displays immense clinical heterogeneity as reflected by the observation that many patients have an indolent disease that will not require intervention for many years while others will present with an aggressive and symptomatic leukemia requiring immediate treatment. Although there is no cure for CLL, the disease is treatable and current standard chemotherapy regimens have been shown to prolong survival. There is no obvious survival advantage to early treatment versus observation but the timing as to when a patient will require treatment is highly unpredictable. Thus, there has been great interest in identifying prognostic markers that can be used to distinguish those patients who may have an aggressive form of CLL and might benefit from early intervention. While clinical staging systems have been used to stratify patients into risk categories, they lack the ability to predict disease progression or response to therapy. Recent advances in our understanding of the biology of CLL have led to the identification of numerous cellular and molecular markers with potential prognostic and therapeutic significance. This review provides a concise overview of prognostic markers in CLL and a discussion of how those markers have impacted the clinical management of the disease.
Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the Western world. It is a disease of older individuals with an incidence that increases exponentially after the age of 50. Thus, as people continue to live longer and the “baby boomer” population ages, the overall prevalence of CLL will continue to rise. Indeed, in the United States in 2010, it is estimated that ~14,990 men and women were diagnosed with CLL and ~4,390 people died as a result of their disease (Altekruse, 2010). CLL is characterized by a clonal proliferation of mature CD5+ CD19+ CD23+ B lymphocytes and weak surface expression of a monoclonal immunoglobulin (Ig) B cell receptor. CLL cells accumulate in the peripheral blood, bone marrow, lymph nodes, and spleen, permitting relatively easy diagnosis from analysis of peripheral blood. The diagnostic criteria have been outlined by the International Workshop on CLL and include the presence of >5,000/µL B lymphocytes in the peripheral blood (Hallek et al., 2008). In the absence of cytopenias, lymphadenopathy, or organomegaly, patients who have <5,000/µL CLL cells are defined as having a monoclonal B cell lymphocytosis, an entity which may progress to frank CLL at a rate of ~1 to 2% per year. Although rare familial forms of CLL have been described (Goldin et al., 2010), the vast majority of patients have no known underlying predisposition and, like most cancers, CLL is thought to arise as a result of acquired genetic mutations.
At the time of presentation, ~25-50% of patients will be asymptomatic and diagnosis is frequently made on routine blood work. CLL is a heterogeneous disease and the majority of patients will have an indolent clinical course that does not require intervention for many years followed by a progressive and often terminal phase lasting 1 to 2 years. Alternatively, some patients may progress rapidly from the time of diagnosis and suffer from complications such as autoimmune hemolytic anemia, thrombocytopenia, and infection. At some point in their clinical course, up to 5% of CLL cases will undergo a Richter’s transformation, whereby the CLL clone evolves into an aggressive high-grade lymphoma. Risk factors for transformation are poorly defined but include an elevated serum lactate dehydrogenase, monoclonal gammopathy, progressive lymphadenopathy, systemic symptoms, and extranodal involvement (Tsimberidou and Keating, 2005).
There is no cure for CLL and early intervention with chemotherapy versus observation has not been proven to improve survival (Dighiero et al., 1998; Shustik et al., 1988). However, appropriate timing of treatment will delay the natural course of the disease and prolong survival. Therefore, the current standard of care is to initiate treatment when a patient has progressive or symptomatic disease (Hallek et al., 2008).
Over the past 10 to 15 years, numerous studies have advanced our understanding of the biology of CLL. Many of these investigations have also led to the identification of various prognostic factors that can be used to stratify patients into risk categories. However, despite ongoing efforts to identify and characterize prognostic risk factors in CLL, it is unclear how these factors have impacted routine clinical practice. This review will summarize the available evidence regarding the different types of prognostic factors in CLL and their relationship to disease management.
The first prognostic marker to be used in the clinical management of CLL was the Rai clinical staging system, published in 1975 (Table 1) (Rai et al., 1975). The system uses lymphadenopathy, organomegaly, and cytopenias (anemia and thrombocytopenia) to establish five prognostic groups that can be used to predict median survival: stage 0, >150 months; stage I, 101 months; stage II, 71 months; and stages III or IV, 19 months each. Patients are stratified into three groups representing low risk (stage 0), intermediate risk (stage I and II), and high risk (stage III and IV) disease. This system was later followed by the Binet staging system, published in 1981, which uses the number of involved nodal areas and cytopenias to establish a three group classification, A, B, and C (Table 1) (Binet et al., 1981). Although the Rai staging system tends to be more commonly used in North America while use of the Binet staging is more common in Europe, neither is recommended over the other as standard. Both of these staging systems provide a basic framework for estimating prognosis and are factored into the current International Workshop on CLL guidelines for initiation of treatment. Based on these guidelines, individuals with Rai stage III/IV or Binet stage C would meet the criteria for therapy (Hallek et al., 2008).
Other clinical markers that have been investigated as potential prognostic indicators include age, gender (Catovsky et al., 1989), lymphocyte doubling time (Molica and Alberti, 1987), number of prolymphocytes (Melo et al., 1987), pattern of bone marrow involvement (Han et al., 1984), and percentage of smudge cells (Johansson et al., 2010). Of these prognostic markers, only age and lymphocyte doubling time have been factored into treatment decisions. A lymphocyte doubling time of less than six months is cited as an indication for treatment in the updated guidelines for diagnosis and treatment of CLL (Hallek et al., 2008). While age is not a determinant of when a patient should receive treatment, it may factor into the type of treatment given because co-morbidities that tend to increase with age may limit the amount of chemotherapy-related toxicity that a patient will tolerate.
The Rai and Binet staging systems were the first prognostic markers to affect disease management. These systems are still widely used in clinical practice, but they do not predict disease progression or response to therapy. To fill these voids in the management of CLL, there are numerous ongoing efforts to identify and characterize additional prognostic markers at the cellular and molecular level (Table 2).
Serum levels of β2-microglobulin (β2m), thymidine kinase (TK), and soluble CD23 have all been described as independent prognostic markers in CLL. Elevated β2m levels are associated with high tumor burden and shorter progression-free survival (Gentile et al., 2009; Hallek et al., 1996). In addition, lower β2m levels have been shown to be associated with higher remission rates and longer overall survival following standard fludarabine-based chemotherapy (Wierda et al., 2009). TK is produced by dividing cells and high serum levels are associated with increased CLL proliferation. Elevated TK levels correlate with other markers of adverse prognosis and are an independent predictor of disease progression (Hallek et al., 1999). Soluble CD23 is also produced by CLL cells and is thus a marker of tumor burden that has been shown to be an independent predictor of time to treatment and overall survival (Knauf et al., 1997; Meuleman et al., 2008). While measurement of these serum markers may be achieved easily in most clinical laboratories, cut-off thresholds have not been standardized and test results have yet to alter the management of the disease. Thus, routine testing of these serum markers is not currently recommended.
IgHV Mutational Status
During normal B cell development, immune diversity is achieved when variable (V), diversity, and joining segments of the Ig genes are recombined to give rise to a pool of mature B cells, each expressing a unique Ig B cell receptor. Upon encountering an invading pathogen, B cells enter the germinal center response where somatic hypermutation of the V regions leads to selection of a B cell that produces an antibody with high affinity for its target antigen. In CLL, ~45% of cases have ≥98% sequence homology with the germline Ig heavy chain V region (IgHV) genomic sequence (IgHV unmutated) while ~55% of cases demonstrate somatic hypermutation of IgHV (IgHV mutated) (Hamblin et al., 1999). A major clinical observation from this biological difference is that mutated IgHV is associated with early stage disease and a median survival of 24 years compared with a median survival of 7.9 years for patients with unmutated IgHV (Damle et al., 1999; Hamblin et al., 1999). While the significance of IgHV mutational status as an independent prognostic factor has been confirmed in multiple clinical trials, the value of IgHV mutational status in predicting response to therapy or guiding therapy remains undefined and its routine use is not recommended except in the setting of clinical trials (Hallek et al., 2008).
Several studies have shown preferential bias of IgV region usage in CLL with associated prognostic significance. For example, patients whose CLL cells use V4-34 tend to follow an indolent course while patients whose CLL cells use V3-21 have aggressive disease, independent of IgHV mutational status (Ghia et al., 2005; Tobin et al., 2004). In general, more than 20% of CLL patients carry stereotypical B cell receptors within their CLL clone (Stamatopoulos et al., 2007). While routine analysis of V region usage is not currently recommended, the biased and recurrent nature of the usage suggests that the CLL clones in these cases may be antigen-driven. If an effective means for interrupting this selection process could be developed, then V region usage could become a useful prognostic tool.
CD38 is a transmembrane glycoprotein that is normally expressed at high levels in B cell precursors, germinal center B cells, and plasma cells, with low expression on circulating B cells. In CLL, a high level of surface CD38 expression was initially found to correlate with unmutated IgHV status (Damle et al., 1999). The threshold for defining CD38 positivity has been controversial, and although most studies now use 30%, it has been demonstrated that CD38 expression in CLL is transient and variable during the clinical course of the disease (Hamblin et al., 2000; Hamblin et al., 2002). Moreover, discordance between IgHV mutation status and CD38 expression is observed in ~28% of patients (Hamblin et al., 2002). Thus, CD38 cannot be used as a reliable surrogate marker for IgHV mutational status. Nonetheless, CD38 expression is associated with shorter time to first treatment, poor response to therapy, and shorter progression free survival (Hamblin et al., 2002; Ibrahim et al., 2001; Jelinek et al., 2001). CD38 expression is easily measured at diagnosis by flow cytometry and although its expression is considered to be an independent prognostic factor, no trial to date has shown CD38 expression status to change the management of CLL.
Zeta-associated protein 70 (ZAP-70) is an intracellular protein that is normally expressed in T cells and transmits signals from the T cell receptor to downstream pathways. Aberrant expression of ZAP-70 in a subset of CLL patients was discovered by gene expression profiling and subsequently identified as a marker that correlated with unmutated IgHV and poor outcome (Crespo et al., 2003; Rosenwald et al., 2001). However, much like CD38 expression, there is measurable discordance between IgHV mutation status and ZAP-70 expression in ~25% of patients (Krober et al., 2006). Additional investigations demonstrated that ZAP-70 is an independent predictor of outcome and may be a better predictor of time to treatment initiation than CD38 and IgHV mutation status (Rassenti et al., 2004; Rassenti et al., 2008). ZAP-70 may be measured by flow cytometry, but unlike CD38, its expression is relatively stable over time (Poulain et al., 2007). ZAP-70 expression has been used in combination with CD38 and IgHV mutation status as a predictor of aggressive disease (Morilla et al., 2008; Schroers et al., 2005), and while it has been suggested that these patients may benefit from close surveillance (Rassenti et al., 2008), there are no specific treatment recommendations for patients who are ZAP-70+. From a biological standpoint, CD38+ ZAP-70+ IgHV unmutated CLL cells are more responsive to B cell receptor stimulation than their mutated IgHV counterpart (Deaglio et al., 2007). This leads to subsequent activation of a signaling cascade involving LYN, SYK, ERK, and NF-κB with enhanced proliferation of CLL cells. Thus, if pharmacologic targeting of these signaling pathways could be achieved, then CD38, ZAP-70, and IgHV mutation status may become very important clinical markers.
Approximately 80% of individuals with CLL have acquired chromosomal abnormalities within their malignant clone and can be categorized into five prognostic groups accordingly: deletion 13q (median survival, 133 months); deletion 11q (median survival, 79 months); trisomy 12 (median survival, 114 months); normal cytogenetics (median survival, 111 months); and deletion 17p (median survival, 32 months) (Döhner et al., 2000). Reciprocal chromosome translocations are described but rare in CLL. A complex cytogenetic karyotype can be identified in ~16% of patients and is commonly associated with poor prognostic features including CD38 expression and unmutated IgHV (Haferlach et al., 2007). In addition to clinical investigations into the prognostic significance of karyotypic changes in CLL, the cloning and characterization of genes affected by these abnormalities is the subject of intense investigation.
Deletion 13q is found in ~55% of patients, making it the most common cytogenetic abnormality in CLL (Döhner et al., 2000). Recent investigations into the minimally deleted region at chromosome 13q14 have revealed the presence of a long non-coding RNA called DLEU2 which contains a microRNA (miR) cluster that expresses miR-15a and miR-16-1 (Calin et al., 2005). While current evidence suggests a role for these miRs in regulating the expression of genes important for proliferation (CCND1, CCND3, and CHK6) and apoptosis (BCL2) (Klein and Dalla-Favera, 2010), the mechanism by which they directly contribute to the pathogenesis of CLL is not clear.
Deletion 11q is identified in ~18% of CLL patients and is associated with several adverse prognostic factors including extensive lymphadenopathy, unmutated IgHV, advanced disease at diagnosis, poor response to treatment, and shorter progression free survival (Döhner et al., 2000; Döhner et al., 1997). At the molecular level, a critical DNA damage response gene, ATM, is normally located within the deleted region at chromosome 11q23. Although there is limited evidence supporting a specific role for the deletion of ATM in the pathogenesis of the CLL, the inability to signal and repair DNA damage due to this deletion might contribute to CLL by allowing the accumulation of mutations during cell proliferation. It has recently been demonstrated that inhibition of poly (ADP-ribose) polymerase (PARP) induces selective killing of ATM-deficient lymphoid tumor cells (Weston et al., 2010), thus providing a potential means for specifically targeting this relatively poor prognostic indicator. A subgroup analysis of the data from the CLL4 Trial revealed that patients with deletion 11q had improved overall survival when treated with fludarabine and cyclophosphamide versus fludarabine alone (Catovsky et al., 2007; Stilgenbauer et al., 2008). Based on this data, the current recommendation is to use regimens containing both fludarabine and cyclophosphamide in patients with a known deletion 11q.
Trisomy 12 is found in ~16% of CLL, and ~18% of CLL patients have normal cytogenetics (Döhner et al., 2000). However, the molecular genetic defects associated with these risk categories are unknown. For trisomies in general, it is often assumed that there is a gene dosage effect of a candidate oncogene on the additional chromosome, but such a gene has yet to be identified on chromosome 12. It is also conceivable that there are molecular mutations of genes that have yet to be identified in patients with trisomy 12 as well as with normal cytogenetics. As intermediate prognostic indicators, these findings do not yet alter disease management beyond standard of care.
Deletion 17p is found in ~7% of CLL patients, is often associated with unmutated IgHV, and confers the highest risk (Dicker et al., 2009; Döhner et al., 2000). While the deletion frequently encompasses most of short arm of chromosome 17, the minimally deleted region always involves 17p13, the locus of the TP53 gene encoding the tumor suppressor p53 (Zenz et al., 2008). In addition, the majority of CLL patients with monoallelic deletions of 17p have point mutations in the remaining TP53 allele, thus completely inactivating a critical component of the DNA damage response pathway. It is well-established that CLL patients with p53 inactivation respond poorly to conventional fludarabine or alkylating agent-based regimens, possibly because both agents require p53-dependent pathways to induce cell death (Laurenti et al., 2011; Rosenwald et al., 2004; Rossi et al., 2009; Turgut et al., 2007). Patients who do not respond to fludarabine have a median overall survival of ~10 months (Keating et al., 2002b). Allogeneic stem cell transplant has been found to induce long-term disease-free survival in CLL patients with deletion 17p (Schetelig et al., 2008). However, given the age of diagnosis and frequent presence of co-morbidities, transplant is not often an option for these patients. This has led to a search for non-p53 dependent agents for use in the management of CLL with deletion 17p. The anti-CD52 antibody alemtuzumab has demonstrated efficacy in such patients with overall response rates of 30-40% and median survival of 16-19 months (Keating et al., 2002a; Lozanski et al., 2004; Stilgenbauer et al., 2009). Ofatumumab is a novel anti-CD20 antibody that has also been shown to be active in fludarabine-resistant CLL with response rates of >45% and median survival of 13-15 months (Wierda et al., 2010). It should be noted that these phase II studies administered these antibodies as monotherapy and future trials investigating combination therapy with other agents such as bendamustine may further improve survival in this high-risk subgroup. Hence, it is clear that deletion 17p is a poor prognostic factor in CLL that predicts resistance to fludarabine-based regimens, and it is recommended that these patients be treated with alternative regimens in the setting of clinical trials.
MicroRNAs (miRs) are small non-coding RNA molecules (~22 nucleotides) that have the capacity to regulate the expression of a multitude of other genes through translational repression or mRNA transcript degradation. The clinical relevance to CLL became evident when it was discovered that an expression signature composed of 13 miRs was associated with other known prognostic factors such as ZAP-70 expression and IgHV mutation status and could be used to predict the time from diagnosis to initial treatment (Calin et al., 2005; Calin et al., 2004). It was subsequently demonstrated that expression of miR-150, miR-223, and miR-29b/c correlates with mutated IgHV and a favorable clinical course, albeit with some discordance (Fulci et al., 2007). Similarly, low expression of miR-223 and miR-29c was found to correlate with unmutated IgHV, ZAP-70 expression, and disease progression (Stamatopoulos et al., 2009). Most recently, Rossi et al. (2010) developed a composite mortality risk score called 21FK by combining miR-21 expression, fluorescence in situ hybridization (FISH), and karyotype. Patients were given 1 point each for miR-21 expression and 17p deletion and 0 point each for low miR-21 and normal FISH and/or karyotype. Survival was significantly higher in patients with a 21FK score of 0 compared with scores of 1 or 2, and the 21FK score had the lowest p-value in multivariate analysis with ZAP-70, CD38, and IgHV mutation status. While these studies provide a foundation for future investigations, miR expression signatures have yet to change the management of CLL. The functional contribution of miR dysregulation to the pathogenesis of CLL is also an area of active investigation but is beyond the scope of this review and is discussed elsewhere (Calin and Croce, 2009). This is a rapidly evolving field and continued research on this subject is likely to have an increasing influence on the prognosis and management of CLL.
Additional Molecular Markers
In addition to the more commonly studied prognostic markers described above, numerous investigations into the pathophysiology of CLL have contributed to a rapidly growing list of genes, small molecules, and biologic profiles of potential prognostic significance (Table 3). While many of these factors have been found to correlate with clinical features and outcome in small cohorts, much work is needed to further define their role in both the biology and clinical management of CLL. Advances in technology such as array-based expression profiling, comparative genomic hybridization, single nucleotide polymorphism analysis, and epigenetic profiling continue to provide a means for generating a wealth of information. However, the data is being generated at a speed that is faster than it can be analyzed, highlighting the need for complementary advances in computational bioinformatics. Over the next several years, as whole genome sequencing approaches are applied to CLL, additional markers of potential clinical significance are likely to be identified. However, given that the overall goal of CLL research is cure, the utility of all this information will ultimately come from investigations that are focused on understanding the biology of the disease. Insight into the precise mechanisms that contribute to the development and progression of CLL hold the prospect of identifying novel targets for therapeutic intervention.
An estimation of survival or time to treatment in patients with CLL may be achieved based on numerous clinical, cell-based, and molecular prognostic markers. However, with the exception of deletion 17p, there is little evidence to suggest that these markers influence clinical management. A biological observation that can be made from studies of prognostic markers in CLL is that the clinical heterogeneity of the disease is paralleled and perhaps surpassed by a complex molecular heterogeneity. The ongoing characterization of old and new prognostic markers will continue to provide insight into the mechanisms that contribute to the pathogenesis of CLL. Thus, as new therapeutic agents are investigated, it will be important to determine the response to therapy within individual molecular subgroups. Only then will the value of prognostic markers in CLL be determined. In the meantime, decisions regarding the initiation of treatment should be made based on clinical features of the disease and clinical judgment. The current standard of care is to initiate treatment when a patient has progressive or symptomatic disease (Hallek et al., 2008). Thus, the use of prognostic markers in the management of CLL remains to be validated by clinical trials.
The authors report no conflicts of interest.
Matthew P. Strout, M.D., Ph.D., is at the Yale Cancer Center and Department of Internal Medicine Section of Hematology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
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[Discovery Medicine; ISSN: 1539-6509; Discov Med 11(57):115-123, February 2011.]