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Chronic lymphocytic leukaemia (CLL) is characterised by an over-production of B cells (lymphocytes) and is a highly variable disease in terms of its clinical progression. Patients with CLL may present with disease which appears to be relatively inactive and offers potential hope for intervention or the disease may manifest as an extremely aggressive cancer, with a poor outcome (Hamblin, 2002). It is the most common form of leukaemia in the Western world with approximately 2,400 cases of CLL being diagnosed in the UK every year (MacMillan Cancer, 2010). The initiation of CLL is thought to be attributable to a single genetic defect of B-lymphocytes, which results in a particular pattern of reaction where the B-cell receptor is stimulated (Hamblin, 2002). CLL rarely affects children and is more often associated with those over the age of 60, while it also appears to be more associated with men rather than women. Excepting stem cell transplantation, which is only applicable for a small number of patients, curative treatment has yet to be defined for the disease (Schweighofer & Wendtner, 2010).
Signs and symptoms
Like many other cancers, the onset of disease is often insidious and there may be no obvious signs or symptoms. In fact, diagnosis of disease is often secondary to a blood test performed as part of a routine health check or prior to an operation. Typical symptomatology includes lethargy, weight loss, night sweats and frequent infections as well as swollen lymph nodes. The frequent infections and enlarged lymph nodes exemplify the underlying problem of CLL - sufferers over-produce immortalised but dysfunctional B lymphocytes, which hinder an effective immune response to invading pathogens. Specifically, hypogammaglobulinaemia predisposes individuals to infectious complications and significantly contributes to the morbidity and mortality experienced by CLL patients (Morrison, 2009). Due to the more novel methods of treating CLL (e.g. purine analogues or monoclonal antibody treatments such as alemtuzumab), individuals can be even more susceptible to infection during the treatment course to pathogens such as cytomegalovirus, herpesvirus and unusual infections such as toxoplasmosis ( Abedalthagafi et al., 2009). Other notable symptoms associated with CLL include autoimmune haemolytic anaemia and idiopathic thrombocytopaenia (ITP) (Diehl & Ketchum, 1998).
Causes and Diagnosis
Risk factors for development of CLL include age, prolonged occupational exposure to benzene (Khalade et al., 2010), gender-specific predisposition in men who had diabetes (Khan et al., 2008) and other various genetic loci which confer enhanced likelihood of developing CLL. These include six recently elucidated low-penetrance risk loci, which were identified as part of a genome-wide study (Di Bernardo et al., 2008) and a novel chromosome region, 13q21.33-q22.2, which may have immune functionality with any mutation in this loci resulting in increased likelihood of developing familial CLL (Ng et al., 2007).
The diagnosis of CLL has evolved considerably in recent years and involves immunological analysis of bone marrow aspirate and biopsy tissue to fully confirm CLL and more crucially, whether the CLL is of the indolent or aggressive form. Bone marrow analysis usually entails flow cytometric evaluation of B lymphocytes with specific over-representation of cells which are monoclonal for the kappa/lambda light chain. Furthermore, immunological phenotyping of B cells confirms the presence of CD5, CD19 to CD24 inclusive, while more recently the expression of CD38 and intracellular zeta-associated protein (ZAP-70) have become important prognostic markers for CLL disease (Matrai, 2005; Wiestner et al., 2003). If possible, bone marrow tissue should be analysed by cytogenetics and a panel of chromosomal abnormalities should be screened using fluorescence in-situ hybridisation (FISH) (Abbott, 2006).
In a recent UK epidemiological study, the incidence and mortality from leukaemias were investigated (Bhayat et al., 2009). Specific aetiological aspects of CLL included an incidence rate of 4.2 per 100,000 person years (compared with 0.49 for acute lymphoblastic leukaemia), females had a lower incidence compared with men and there was no association between socio-economic deprivation status and disease incidence. In terms of survival, women who developed CLL were much less likely to die compared to men (p< 0.001) and this gender-specific difference in mortality was not observed amongst the other leukaemias.
The incidence above is comparable with another epidemiological study in the Czech Republic where it was calculated at between 5.8 and 6.2 cases per 100,000 for cohorts from 2006 and 2007 (Panovska et al., 2010). This study also reinforces how difficult it is to successfully manage and treat CLL as a significant number were included in clinical trials (presumably because of treatment failures and/or progressive disease). Although dietary factors have been studied, a recent study did not find any association of increased risk of CLL and the role of diet still remains questionable (Tsai et al., 2010).
The stages of CLL have been graded in terms of disease progression and are best described in table 1. Irrespective of the staging system in table 1 (Rai vs Binet), those patients with the most malignant stage only have an expected survival time of 1-2 years, while patients with the most preliminary stage of disease have a median survival time of greater than 10 years. It is important to note that for cases who relapse, the staging systems below have not been validated and depend on the initial site of disease involvement (Abbott, 2006).
Table 1. CLL staging and risk stratification (adapted from Abbott, 2006).
Staging at diagnosis (Rai system)
1. Lymph node enlargement
2. Enlargement of spleen
3. Haemoglobin <11 g/dl
4. Platelets <100,000/Âµl
Staging at diagnosis (Binet system)
Lymph node enlargement in >3 areas
Cytopaenia: haemoglobin <10 g/dl or platelets <100,000/Âµl
High-risk features for CLL
CD38 expression in >30% of lymphocytes
ZAP70 expression in >30% of lymphocytes
Unmutated (germline) IgVH gene
High-risk cytogenetic abnormalities
Rai stage 3 or 4 or Binet stage C
Doubling time of lymphocyte count <12 months
Elevated beta-2 microglobulin
Elevated serum thymidine kinase
Presence of large-cell transformation (Richter's syndrome)
1.6 Treatment and prognosis
One of the most important and unresolved challenges in CLL is the early identification of cases, who may be amenable to therapeutic intervention. Ostensibly, treatment for a patient is dependent on the clinical manifestation of the disease but with CLL, the situation is unique in that a diagnosis of CLL may be confirmed but there may be no clinical symptoms. At this point, diligent monitoring of the patient is all that is required. However, the predicted likelihood of symptom onset and disease progression can be inferred from a number of molecular markers including CD38, ZAP-70 and serum beta-2-microglobulin (table 1). Not only has CD38 expression been associated with a more aggressive form of CLL but it has also been discovered that there is a strong correlation between its expression and treatment failure with fludarabine (Del Poeta et al., 2001). Although intracellular ZAP-70 expression has been studied extensively and appears to be predictive of metastatic disease and death, its accurate quantification by flow cytometry remains problematic (Wiestner et al., 2003). Therefore, its use a clinical prognostic tool is still to be fully justified.
A number of aforementioned symptoms and signs usually dictate the onset of treatment e.g. unexplained weight loss, worsening anaemia, episodes of thrombocytopaenia and/or progressive lymphadenopathy. Those patients who are younger or have entered into the higher stages of disease with prognostic markers of disease, as described above should be considered for aggressive forms of treatment. Currently, only allogeneic stem cell transplantation offers any hope of a long-term cure although laparascopic splenectomy has been used in a case series with some success of long-term cure (Hill et al., 2004).
1.6.1 Chemotherapy-based approaches
Chemotherapeutic treatment of CLL, as with other malignancies, is problematic due to the toxic effects of the drugs and the immune-deficient state of the patient. Historically, a number of crude anti-cancer drugs were used to treat CLL including chlorambucil, cyclophosphamide and vincristine. However, there appears to be no added clinical benefit when a combination of such drugs are used and in recent years, drug discovery focused on the family of drugs known as the purine nucleoside analogues.
Such purine analogues have been shown to be very effective in the treatment of CLL and include fludarabine, pentostatin and 2-chlorodeoxyadenosine and their action is thought to be due to their apoptotic effects on lymphocytes, where they induce DNA fragmentation (Robertson et al., 1993). By removing the immortalised B lymphocyte population, the patient should in theory recover from the CLL and indeed, disease progression and mortality rates have reduced since the clinical inception of these drugs, either singly or in combination with drugs such as chlorambucil (Rai et al., 2000; Keating et al., 1998). Despite the apparent clinical success of these drugs in treating CLL, their myelosuppressive effects on the immune system has resulted in opportunistic infection with pathogens such as herpesvirus, Pneumocystis and other bacterial and fungal microbes (Cheson, 1995; Anaisie et al., 1998).
Immunotherapy of CLL
In the last decade, one of the most significant steps in the field of immunology is the clinical application of immunomodulators to disease states. This is particularly relevant with chronic diseases such as rheumatoid arthritis (rituximab) and Crohn's disease (infliximab). Unsurprisingly, the treatment of CLL has also benefitted from these new monoclonal antibodies, which offer a safer alternative to some of the drugs listed above.
Alemtuzumab is a monoclonal antibody (mAb) which targets CD52, a marker associated with lymphoma. Its use has been approved for treatment of CLL and its activity appears to be efficacious especially in patients who have relapsed, have refractory CLL and who are considered to be high risk with a poor prognosis (Schweighofer & Wendtner, 2010). The drug appears to be most successful in eradicating residual disease from patients which confers longer-term survival. However, CD52 is also found on T lymphocytes therefore a side-effect of this drug regimen is an enhanced predisposition to infection. To this end, the drug still remains on offer only as part of a clinical trial regimen.
Rituximab targets CD20, a marker previously described which appears to also confer susceptibility to CLL. This mAb only targets B cells and therefore already has an advantage over alemtuzumab in that the susceptibility to infection should be lessened. To that end, rituximab has been approved for clinical treatment and is usually offered in conjunction with fludarabine and cyclophosphamide (FCR). This combination has been used in a number of studies and is considered the most active combination regimen for CLL sufferers with limited co-morbidity (Johnson et al., 2009; Keating et al., 2005).
The most modern mAb which has been approved for use is ofatumumab, another antibody which targets the CD20 marker. In October 2009, this drug was approved by the US Food and Drug Administration (FDA) and is recommended for CLL patients who are refractory to fludarabine and alemtuzumab (Lemery et al., 2010). This drug is extremely efficient at lysing B lymphocytes and does this via antibody and complement-mediated cytotoxicity. However, as with the other mAb discussed, infection is the most prominent side-effect with a number of patients succumbing in the above study. While the expanding field of immunotherapy undoubtedly promises new drugs for the future treatment of CLL, the problem of susceptibility and predisposition to infectious agents remains an important issue, which requires further thought.
1.6.3 Novel targeted chemotherapeutic agents
As described above, the quest to discover drugs which are more specific in their destruction of cells/mediators important in CLL is very important if the patient is to survive opportunistic infection and unwanted immunological side-effects such as neutropaenia.
While the agents of infection are to be feared, in recent years one of the most novel chemotherapeutic drugs used to treat CLL patients is ironically a microbial toxin. A diphtheria toxin has been fused with an interleukin-2 protein to produce DAB(389)-IL2 or denileukin diftitox. The protein binds to the IL-2 receptor on lymphocytes and the endotoxin is then internalised into the cell whereby protein synthesis is stopped and the cell undergoes necrosis. This drug has been used in a phase II clinical trial and while there appeared to be some clinical benefit with moderate toxicity and side effects in CLL patients, there still needs to be further studies done on this drug to enhance the response rate (Frankel et al., 2006).
Expression of Bcl-2 protein is associated with progression of CLL and more importantly enhanced resistance to chemotherapy. Therefore, efforts to target production of this protein have resulted in the production of an anti-sense oligonucleotide, oblimersen (Cheson, 2007). This drug functions by binding to RNA which targets specific proteins for Bcl-2, interfering with the translation process for this protein. This drug has been trialled at Phase 3 and like other drugs for CLL, its effects on tumours is modest when administered as a single drug. However, in conjunction with fludarabine and cyclophosphamide, the clinical benefit rivals that of the treatment regimen currently being used for CLL (O'Brien et al., 2005). Furthermore, the side effects for CLL patients appear to be less severe than for some of aforementioned drugs. While there appear to be a number of drugs (pharmacological or immunological) that provide treatment for CLL, the issue of multi-drug resistance remains a pertinent problem for CLL patients.
2. MULTIDRUG RESISTANCE
With regards to cancer, there are several issues to consider in the treatment of the patient. The patient is likely to have a number of tumours; will require cytotoxic treatment for those tumours; will become even more susceptible to infection or other immune-mediated pathology and there is a chance that the drug of choice will not be effective in tumour destruction. A part of the latter problem is attributable to tumour resistance to chemotherapeutic drugs. Whilst there has been much progress in the formulation of drugs needed to combat cancer, for many years the problem of multi-drug resistance in cancer has foiled successful cure of patients and poses a clinical conundrum which has yet to be solved.
2.1 The concept of multidrug resistance
Multi-drug resistance (MDR) is a term which has been used to describe the mechanism of resistance that cells (prokaryotic and eukaryotic) have in response to chemotherapeutic drugs. Specifically, the concept implies that the target cell of interest may be exposed to a number of chemotherapeutic agents but the cells will be able to pump these out before they accumulate in high enough concentrations to have a significant effect. In short, 'drug efflux' underpins this process but in a recent study, it appears that cancer cells may also be able to affect the bioavailability of certain anti-cancer drugs by 'drug influx' (Burger et al., 2005).
Multidrug efflux pumps: the mechanism of action
The mechanism of how multidrug efflux pumps work is best summarised in figure 1.
Figure 1. Mechanism of action of efflux and influx pumps (from Gottesman et al., 2002).
Figure 1 highlights a number of mechanisms which are important in driving multidrug resistance including increased drug efflux and activation of DNA repair. The process of drug expulsion from a cell is mediated by ATP-dependent pumps, which may themselves be over-expressed by cancer cells. The pumps straddle the plasma membrane and due to an energy-driven gradient, drugs which have entered the cell are removed in an energy-efficient manner. If multidrug resistance can be circumvented, the survival of the tumour cell is threatened and this offers an increased chance of host cure.
Multidrug resistance in CLL
The susceptibility of tumours to anti-cancer drugs varies among the different forms of cancer. Although CLL presents as a 'slow' form of cancer, the mortality rate is high and this is largely due to the properties of multidrug resistance which eventually arise. Host immunological factors are thought to be important in immortalising B lymphocytes and many of these signals are driven by bone-marrow progenitor cells and their associated cytokines (O'Hayre et al., 2010). One of these signals (CXCL12) is also thought to be partially responsible for MDR in CLL.
Because MDR is central to CLL and host longevity, it is a very heavily-funded area of research. Cyclosporin A is a potent immunomodulator and has been used in ablating immune responses to transplants therefore it has also been used in CLL treatment. As with other immunomodulators, it has undesirable side-effects so there is much focus on derivatives of this agent as it has been shown to reverse MDR in CLL patient cell lines (Jiang et al., 1995). The mode of action of cyclosporin and its derivatives is thought to affect the efflux pumps, which are influenced by a P-glycoprotein and a multidrug resistance protein (MRP). Other transporter proteins involved in MDR also include lung resistance protein (LRP).
2.3.1 ABCB1 (P-glycoprotein)
Probably the best understood mechanism of MDR action in CLL is over-production of the membrane P-glycoprotein (Pgp) or ABCB1. Pgp is encoded by the MDR-1 (ABCB1) gene and is a transmembrane transporter, which is part of the adenosine triphosphate-binding cassette (ABC) superfamily (Jamroziak et al., 2006). This protein plays a crucial role in both (a) accelerating the efflux of cytotoxic drugs from the lymphocyte and (b) negating the pro-apoptotic effects of such anti-cancer drugs (Svoboda-Beusan et al., 2000). It is understandable therefore that Pgp itself has become the focus of potential treatment intervention.
Pgp is actually expressed in normal host tissue and is important in reducing drug absorption from the digestive tract but it is the over-expression of this protein in tumour cells which confers MDR to a broad range of chemotherapeutic agents used for the treatment of CLL. The protein has been found to be present in some CLL studies in up to 100% of such patients and its importance in disease has been shown by treating CLL patients with drugs which are acted upon by Pgp (Robak et al., 2001).
2.4 Single nucleotide polymorphism (SNP) in the MDR1 gene
Polymorphisms of the MDR1 gene have recently been reported and these have clinical implications in that there may be a considerable impact on the pharmacological impact of anti-cancer drugs. To this end, the discovery of a number of single nucleotide polymorphisms (SNPs) and associated insertion/deletion mutations has resulted in an increasing knowledge of how CLL MDR can arise and is constantly evolving (Ishikawa et al., 2004). If individual SNPs can be determined for specific cases of CLL, this may result in individually-tailored therapy for patients, which may enhance the chances of cure and reduce undesirable side-effects. The most important SNPs associated with CLL disease progression to date include C3435T, G2677T/A and C1236T and these shall be discussed in the following sections.
C3435T (rs1045642) polymorphism
The C3435T polymorphism has recently been found to be associated with an increased susceptibility to other diseases including Parkinson's disease, renal carcinoma and paediatric acute lymphoblastic leukaemia (Lee et al., 2004; Jamroziak et al., 2004; Siegsmund et al., 2002). It has been hypothesised from these and other studies that the C3435T-mutated Pgp has reduced efflux properties and this contributes to toxin-mediated predisposition to the above conditions. Conversely, with regards to CLL, studies have elucidated that there is an increased prevalence of this SNP in CLL patients hence it may be that cells in CLL patients are not able to transport toxins (such as pesticides) from cells as effectively as wild-type Pgp carriers (Jamroziak et al., 2006) resulting in an increased susceptibility to Pgp-transported carcinogens. This may also have direct implications for chemotherapy options in CLL patients.
G2677T/A (rs2032582) polymorphism
As with the C3435T polymorphism, the G2677T/A polymorphism is relevant in predisposing individuals to a number of other diseases including chronic myeloid leukaemia. G2677T/A has been evaluated and confirmed as a prognostic marker for CLL but more importantly, the scant evidence to date suggests that this polymorphism may be associated with higher haemoglobin levels and increased lymphocyte number (Penna et al., 2009). The clinical implications of this polymorphism are yet to be fully elucidated.
2.4.3 C1236T (rs1128503) polymorphism
Although an earlier study found no association between C1236T and multidrug resistance in CLL patients (Goreva et al., 2004), it is the combined effects of C1236T and the aforementioned SNPs that has been explored in greater detail especially with regard to transplantation. These SNPs have been appraised for their effects on cyclosporin (previously discussed) and tacrolimus, agents which are used to abrogate immune responses before and after transplantation (Statz et al., 2010). Therefore, it is likely that C1236T may also play a role in how cyclosporin derivatives (discussed in section 2.3) can target MDR in CLL. However, as with G2677T/A, the evidence is scant at present.
CLL is an insidious disease which primarily affects the elderly, particularly men and it is therefore often another condition which results in the death of that patient. However, in some countries the incidence of CLL is increasing and with omni-present problems with MDR, the outlook may appear bleak for patients who are diagnosed. Nevertheless, there is optimism for increased cure rates in this field.
Molecular biology and immunology are rapidly advancing areas and in recent years, previously hard-to-treat conditions such as rheumatoid arthritis and Crohn's disease have benefitted from clinical application of immunomodulators such as rituximab and infliximab. While the threat of opportunistic infections still poses some problems for CLL patients, generally such infections are easier to treat than B-lymphocytes which acquire MDR over time. The emphasis now appears to be centred on ablating minimal residual disease and it genuinely appears to be quite promising for patients who fall into this category.
RNA-specific drugs offer a tantalising insight into the power of new drug discovery but their use must be carefully monitored. While this therapy may help to control CLL, it may inadvertently affect downstream production of proteins which may be essential in routine cell function resulting in aberrant cell behaviour and this could cause unforeseen disease states.
Finally, MDR is a problem which scientists, medics and clinicians have battled for years. Identification of SNPs may be one of the most important findings of modern science and may help to understand MDR not only in CLL but in a gamut of diseases, where drug resistance is a major issue. It will take concerted efforts from all those listed to elucidate MDR patterns and hopefully fashion person-specific treatments, which result in a cure for such conditions.