Chronic lymphocytic leukemia (CLL) was for many years considered an indolent disease caused by the accumulation of small lymphocytes with disturbed apoptosis. Today the comprehension of the pathogenesis has modified this historical view and allowed the condition to be recognized as a dynamic disease, which originates from a wide range of primary and secondary biological and genetic events.

The status of cellular “activation” that follows interaction with antigens and the complex interactions with the microenvironment shape a disease with a heterogeneous clinical course, sometimes preceded by a predisposing condition, called monoclonal B-cell lymphocytosis (MBL), recently defined in its nosographical outline. Thanks to advances in the understanding of the mechanisms that underlie the natural history of CLL, the modern approach to patient management relies on a thorough clinical characterization that includes the assessment of a series of prognostic markers, which proved useful for the proper planning of increasingly effective therapies, some of which were designed to interfere with disease-related mechanisms allowing abnormal cell survival and growth.

 

Definition and epidemiologic overview

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder involving CD5-positive B cells which falls within the malignancies of mature B-cell of the WHO classification (Muller-Hermelink HK, 2008). It is more frequent in males than females (1.5-2.0/ 1) and has an incidence in Western countries between 2-6 cases/ year / 100,000 people, while it is rare in Japan and in Eastern countries, where the incidence is lower than 1 case/ 100,000 inhabitants (Redaelli A, 2004) PubMed (Figure Ia).
The mean age at diagnosis is around 70 years, and the incidence increases from 1 case/year/100,000 in the fifth decade, to 20 cases in the 70-80 year range. Over 40% of CLL cases are diagnosed in patients older than 75 years, while less than 10% are diagnosed before the age of 50 (Brenner H, 2008)PubMed (Figure Ib).

 

Cuneo_Chronic_Lymphocytic_Leukemia_Figura_1

Figure I. a) Incidence in different countries; b) Incidence by age

 

Etiology

The etiology of CLL is unknown. A role for ionizing radiation cannot be ruled out (Richardson DB, 2005)PubMed , although an increased incidence of many forms of leukemia has been demonstrated among survivors of the Chernobyl nuclear disaster, but not CLL (Lin SL, 2009). Some agricultural activities related with the use of pesticides may be associated with an increased incidence of CLL (Caporaso N, 2004) PubMed.

There is evidence of a possible association between CLL and genetic factors. CLL has a low incidence in Oriental populations and ethnic groups who migrate to other countries maintain the same disease incidence of the country of origin. In first-degree relatives of patients with CLL, the risk of developing the disease and other lymphoproliferative syndromes (Hodgkin’s disease and non-Hodgkin’s lymphoma) is higher than that of the general population matched by age and sex (Capalbo S, 2000PubMed ; Goldin LR, 2004PubMed ) and expansions of small B lymphocyte clones with the classical phenotype of CLL and negative for CD38 expression, can be found with a higher frequency than in the general population (Rawstron AC, 2002)PubMed . An analysis of 24 families with more than two affected members documented, next to the classical cytogenetic abnormalities, a high frequency of deletions or gain of genetic material at bands Xp11.1-p21, Xq21-qter, 2p12-14 and 4Q11-21 (Martin AJ, 2002)PubMed. It is possible that, unlike breast cancer, where a gene (BRCA1) has a very important effect, the genetic background of CLL consists of multiple genetic abnormalities with low predisposing potential (Goldin LR, 2007)PubMed .

 

Pathogenesis

The pathogenesis of CLL recognizes several moments (Table I), depending on the peculiar characteristics of the cell of origin, its interaction with hypothetical antigens and the microenvironment and the development of a wide range of genetic lesions.

 

Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_1

 Table I: Pathogenic mechanisms in CLL

 

a) Cell of origin

CLL results from the neoplastic transformation of a B lymphocyte, expressing the surface antigen CD5, corresponding to the B-1 population present in the peritoneal cavity of mice, which shows the ability to produce natural antibodies by a T-independent immune reaction (Darzentas N, 2010)PubMed. The human analogue of this subpopulation has not been identified with certainty. CLL may derive from a B CD5+ lymphocyte of the mantle zone (Dighiero G, 2005PubMed, or from a marginal zone lymphocyte expressing CD5 as a consequence of an abnormal activation state. The gene expression profile of lymphocytes from “unmutated” CLL (vide infra) is similar to CD5+ mature B-lymphocytes, whereas the expression profile of “mutated” CLL lymphocytes reproduce the expression profile of a small subset of CD5+/CD27+ post germinal centre B- (Seifert M, 2012)PubMed. Like lymphocytes of the marginal zone, CLL lymphocytes express a B-cell receptor (BCR) which has structural similarities with antibodies that react against self-antigens and bacterial polysaccharide antigens (Chiorazzi N, 2005)PubMed .

In 50-80% of the cases the CLL cells carry >2% somatic mutations in the sequence of the gene coding for the variable portion of the immunoglobulin heavy chain (IGHV), a process that occurs physiologically in the germinal center in response to T-dependent antigens, thanks to enzymes such as the activation-induced cytidine deaminase (AID). This form of CLL is referred to as “mutated” CLL and may derive from a post-germinal centre B-lymphocyte. Other cases, usually referred to as “unmutated” CLL, display a germline configuration of the IGHV gene (i.e. ≤2% of mutations) and may therefore derive from a lymphocyte that did not encounter the antigen in a germinal centre. However, lymphocytes of “mutated” and “unmutated” CLL have many similarities: a) a global gene expression profile close to that of a memory B-cell; b) the immunophenotype CD23+; CD25+; CD27+, typical of activated lymphocytes which encountered the antigen, with low expression of molecules normally down-regulated in response to cellular activation (CD22; CD79b; IgD). In some cases the unmutated CLL have a pattern of expression of CD69 and CD71 antigens consistent with a temporal proximity to the antigenic stimulus that is more pronounced than “mutated” cases (Damle RN, 2002)PubMed . It can therefore be concluded that the cell of origin of CLL is a memory B-cell that 1) encountered the antigen in a T-dependent response in the germinal center (“mutated” CLL) or, 2) reacted outside the germinal center to an antigen unable to activate the process of somatic hypermutation of the Ig gene (autoantigen, polysaccharide antigen or superantigen) (“unmutated” CLL).

 

b) Role of antigenic stimulation

The neoplastic clone in CLL shows a preferential usage of some V, D and J families (for example VH1-69; VH4-34), which does not reflect the frequency of these rearrangements in the normal CD5 positive B-lymphocyte population. Since these sequences, assembled during B-lymphocyte maturation in the bone marrow, form the variable portion of the Ig gene expressed on the surface as BCR it can be deduced that some antigens are able to engage the clones expressing this “restricted” set of BCR, favoring their expansion and subsequent transformation. This concept has been strengthened by the demonstration that different CLL patients carry “stereotyped BCRs”, characterized by an identical sequence coding for the “complementarity determining region” (CDR), the portion of the BCR that binds the antigen (Caligaris-Cappio F, 2008PubMed ; Ghiotto F, 2004PubMed ; Tobin G, 2004PubMed ) . Given the complexity of recombination events that lead to the BCR assembly, the probability that two normal B cells randomly have a stereotyped BCR is in the order of 10-12, whereas it was reported that up to 30% of cases of CLL can show this phenomenon (Stamatopoulos K, 2007)PubMed . Bacterial polysaccharide, rheumatoid factor, DNA, cardiolipin, antigens expressed on apoptotic cells are capable of engaging the BCR in CLL (Caligaris-Cappio F, 2008)PubMed and the view emerged that the pathogenesis of the disease is in part driven by antigenic stimulation, often involving autoantigens. An elegant demonstration was provided that the “non-muscle myosin heavy chain”, a protein having a role in cell movement, may be exposed on the surface of apoptotic cells and that most unmutated CLL cells are able to recognize this antigen via their antibodies (Chu CC, 2010)PubMed .

There is also evidence that CLL lymphocytes maintain the ability to respond to antigen, i) undergoing in vivo Ig class switch (Malisan F, 1996)PubMed , ii) developing new IGHV gene mutations (Gurrieri C, 2002)PubMed, iii) expressing the AID enzyme (Oppezzo P, 2003)PubMed , which is important in the process of somatic hypermutation, iv) modifying the gene expression profile and activating the cell cycle (Guarini A , 2008)PubMed. Interestingly, these features are more pronounced in unmutated, CD38+ and ZAP-70+ CLL compared to other CLL cases (Lanham S, 2003PubMed ; Chen L, 2002PubMed ). In fact, the BCR-signaling in unmutated CLL is active, while in mutated forms it is much less active, due to a state of functional anergy probably deriving from prolonged antigenic stimulation and consequent desensitization (Stevenson FK, 2004)PubMed . This condition of “anergy” is associated with a specific expression profile of genes involved in BCR-mediated signaling (Muzio M, 2008)PubMed . BCR signaling that follows antigenic stimulation involves some proteins relevant for the biology of the neoplastic clone (Burger JA, 2011)PubMed and some molecules, such as the Bruton tyrosine kinase (BTK) and the delta subunit of phosphatidylinositol 3-kinase (PI3K delta). Such molecules represent targets for specific treatment of proven efficacy (Burger JA, 2011)PubMed.

 

c) Cellular turn-over

Contrary to previous thinking, CLL can not be considered a disease determined by the accumulation of lymphocytes that do not undergo apoptosis. The pathological cells retain sensitivity to pro-apoptotic stimuli mediated by Fas and by the binding of anti-IgM antibodies that engage the BCR (Chu P, 2002PubMed ; Zupo S, 2002PubMed ) and proliferate in vivo at a rate of 0.1-1% of the entire clone every day (Messmer BT , 2005PubMed ). The rate of cell division and renewal is higher in the CD38+ cell fraction (Calissano C, 2009)PubMed .

 

d) Microenvironment interactions

The lymph nodes in CLL are characterized by an “accumulation” compartment, consisting of small lymphocytes, and by the proliferative compartment consisting of “proliferation centers”, where the cells (mainly paraimmunoblasts and prolymphocytes) show features of activation and undergo cell division. These histological structures, which give a pseudofollicular aspect to the CLL lymph node, are also present in patients with autoimmune diseases (Humby F, 2009)PubMed and recall the role of stimulation by autoantigens in the genesis of CLL. In the proliferation centers prolymphocytes and paraimmunoblasts are in close contact with CD4+ and follicular dendritic cells. In the “accumulation” compartment the small lymphocytes interact with stromal cells, in the context of cell-to-cell interactions, promoting their survival. In fact, the stimulation by CD40 has a role in sustaining survival of the B-lymphocyte clone, as is the case with CD4 lymphocyte interactions, producing anti-apoptotic cytokines (IL4, IFN) (Ghia P, 2000)PubMed . An important role in the regulation of distribution and survival of neoplastic lymphocytes (Stevenson FK, 2004)PubMed is played by a) chemokines and their receptors expressed by leukemic lymphocytes (CXCR3 and CXCR5), b) “nurselike” cells that promote survival and migration of lymphocytes within the marrow spaces through the stromal-derived growth factor (Burger JA,1999)PubMed, c) dendritic cells through CD44 and the induction of Mcl-1, a BCL2-related protein (Pedersen IM, 2002)PubMed. Angiogenesis may also play a role in the acceleration phases of the disease or in more aggressive subgroups, where higher serum levels of VEGF are detectable (Molica S, 2002PubMed ; Maffei R, 2010PubMed ).

 

e) Molecular cytogenetic lesions

– microRNA genes and TCL1

To date, the primary genetic lesion able to trigger the transformation process in CLL is not known, but much information is available on some lesions that contribute to this multistep process (Figure II).

 

Cuneo_Chronic_Lymphocytic_Leukemia_Figura_2

Figure II. Antigenic stimulation and molecular-cytogenetic in CLL pathogenesis

 

Two genes encoding for microRNA (i.e. miR-15 and miR-16) have been identified at the 13q14 region, which is deleted in 40-50% of CLL cases. These genes show reduced expression after deletion in CLL (Calin GA, 2002)PubMed with consequent aberrant expression of genes involved in cell cycle progression of B-lymphocytes (Klein U, 2010)PubMed . In fact, deletion of miR-15 and miR-16 in a mouse model was associated with clonal expansion of B lymphocytes, having the biological features of CLL. The lymphoid proliferation was more pronounced and aggressive if the deletion involved, in addition to these microRNA genes, the DLEU2 gene that maps in the same region (Klein U, 2010)PubMed . The concomitant deletion of DLEU7, located within the region of minimal deletion, may contribute to the pathogenesis through the loss/reduction of its physiological inhibition of NF-kB (Palamarchuk A, 2010)PubMed.

CLL is also characterized by a consistent overexpression of TCL1 that maps at 14q32.1, determined by a mechanism of promoter demethylation  (Yuille MR, 2001) and/or by the deregulation of two microRNA genes, miR-29 and miR-181 (Efanov A, 2010)PubMed . It has been documented that transgenic mice for a construct that contains the enhancer of the Ig gene and TCL1 develop a clonal CD5+ B-cell expansion that progressively acquires CLL features (Bichi R, 2002)PubMed. Similarly, the transgenic mouse that overexpresses miR-29 in B lymphocytes can develop CLL (Santanam U, 2010)PubMed. The role of TCL1 in the pathogenesis of CLL is mediated by reduction of the activity of DNA methyltransferase 3A with a consequent decrease of DNA methylation (Palamarchuk A, 2012)PubMed .

TP53, NOTCH1, MYD88, XPO1, KLHL6, SF3B1, BIRC3

Genome sequencing of some patients with CLL (Puente XS, 2011; Fabbri G, 2011) has shown a number of non-silent mutations of numerous genes, able to alter the function of the corresponding proteins. These mutations appear to involve more often loss or gain of genetic material and are typically different from patient to patient. However, some of these lesions are recurrent and are associated with specific clinicobiological features.

In addition to the abnormalities of TP53, here dealt with in a specific paragraph, frameshift mutations of NOTCH1 are present in 10-15% of cases, whereas mutations of XPO1 (exportin 1), MYD88 (myeloid differentiation primary response gene 88) and KLHL6 (kelch-like 6) are detected in <5% of cases. The lesions of NOTCH1 lead to the accumulation of the active isoform of the protein, with consequent activation of NOTCH1-related intracellular signaling. Patients with this aberration generally have a relatively aggressive disease, associated with frequent development of drug resistance and an increased risk of evolution into Richter’s syndrome. NOTCH1 mutations are more frequent in patients with unmutated IGVH and trisomy 12 (Del Giudice I, 2012PubMed ; Balatti V, 2012PubMed ).

Lesions of MYD88, most frequently found in CLL with mutated IGVH genes, are associated in some experimental models with increased secretion of cytokines able to recruit accessory cells (macrophages and T lymphocytes) that play a role in the setting-up of the microenvironment in which CLL cells grow.

The lesions of XPO1 lead to activation of the MAP kinases cascade, while lesions of KLHL6 may involve a protein that has a role in the formation of the germinal center.

Five per cent of untreated CLL display mutations of the SF3B1 gene, which codes for a protein involved in the splicing process (Wang L, 2011)PubMed . These cases are associated with a more advanced stage, unmutated IGVH genes and positivity for ZAP70 (Quesada V, 2011)PubMed. This lesion, like NOTCH1 mutations, is found more frequently in chemorefractory CLL (Rossi D, 2011)PubMed.

BIRC3, a gene that encodes a protein which down-regulates the NF-kB signaling, presents inactivating mutations or deletion in approximately 20% of chemorefractory CLL. The frequency of these mutations is low (<5%) in the early stages of the disease (Rossi D, 2012)PubMed .

It is interesting to note that mutations of TP53, NOTCH1, SF3B1, BIRC3 are in most cases mutually exclusive in CLL refractory to fludarabine, so that it is reasonable to assume that in addition to TP53, the signaling pathways of NOTCH1 and NF-kB and the splicing machinery may play an independent role in the genesis of chemorefractoriness.

Telomeres

Telomeres are composed of repeated DNA sequences that confer stability to the structure of chromosomes. With cell aging and as a result of repeated cycles of cell division a physiological shortening of the telomeres occurs, which is normally limited by the activity of telomerase. Telomeres in CLL lymphocytes are shorter than in normal B lymphocytes of subjects of the same age and sex. Moreover, telomere shortening is most pronounced in unmutated CLL, in which greater telomerase activity is also found (Damle RN, 2004PubMed ; Grabowski P, 2005PubMed ), and is associated with poor prognosis and increased likelihood of developing Richter’s syndrome (Rossi D, 2009)PubMed. These observations indicate how the replicative history of CLL is different depending on the mutational status of Ig genes. Indeed in the most aggressive CLL cases it is reasonable to assume that a proliferative stimulus played a role in the early stages of the natural history of the disease, inducing numerous cycles of activation and replication with shortening, subsequent dysfunction and fusion of telomeres and consequent predisposition to the development of extensive genome damage (Lin TT, 2010)PubMed.

 

Cytogenetic abnormalities

The introduction of FISH technique has allowed for the identification of chromosomal aberrations in approximately 80% of cases of CLL and each patient is now included in a specific group on the basis of a hierarchical cytogenetic classification that assigns importance to the following lesions: 17p- >11q->+12 >13q. The resulting cytogenetic groups show a different incidence depending on the stage of disease, as reported in Table II. An overview of the clinicobiologic correlation of cytogenetic lesions is presented in Table III. Recently, the introduction of an effective technique of mitosis stimulation using oligonucleotides and IL-2 allowed for the identification of chromosome aberrations in approximately 30% of CLL cases with non-informative FISH analysis. The presence of karyotype aberrations predicted for an inferior outcome in this “normal FISH” group (Rigolin GM, 2012)PubMed. Furthermore, this analysis demonstrated that complex karyotypes could be documented in a significant fraction of “normal FISH” cases in association with unfavorable biological and clinical prognostic factors (Haferlach C, 2007)PubMed.

Highly sensitive techniques that are able to scan the entire genome for DNA gains or losses were developed, showing that virtually all cases of CLL may carry a genetic lesion (Grubor V, 2009)PubMed .

Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_2

Table II. Frequency (% of cases) of FISH chromosomal aberrations and IGHV mutational status in >4000 CLL cases enrolled in GCLLSG protocols (*)

 

Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_3

Table III: Clinico-biological significance of recurrent chromosomal aberrations in CLL

 

 

13q

The 13q14 deletion is the most frequent cytogenetic abnormality in CLL and occurs in more than 50% of cases.

This deletion has been described as heterozygous in approximately 75-80% of cases and homozygous in the remaining 20-25%. Patients with homozygous deletion of chromosome 13q14 have a higher lymphocyte growth kinetic compared to patients with heterozygous deletions.

Molecular studies have shown that the commonly deleted region includes a 790 kb region between markers D13S1150 and D13S25; however, no gene in this region, including the above-mentioned DLEU2, shows inactivating mutations in the remaining allele.

In 2002, Calin and colleagues identified a 29 kb deleted region at 13q14, located between exons 2 and 5 of the DLEU2 gene and containing two microRNA coding genes, miR-15A and miR-16-1, the expression of which is significantly deregulated in a fraction of CLL cases. The deletion of these microRNA genes has been confirmed by other researchers who used high resolution CGH arrays in 58 CLL cases (Buhl AM, 2006)PubMed.

+12

Trisomy of chromosome 12 is the most frequent gain of chromosomal material in CLL, being found in about 15% of patients. Some cases of CLL with partial trisomy of chromosome 12, deriving from duplication of the 12q13-12q21.2 segment have been reported. This suggests that this region may contain relevant genes for the pathogenesis of CLL. The CLLU1 gene, upregulated in CLL with aggressive clinical course, is located on the band 12q2252. The MDM2 gene, which maps on 12q14.3-q15, may be upregulated in CLL with trisomy 12. The MDM2 gene product acts as an important regulator of the TP53 tumor suppressor gene and its overexpression results in the functional inactivation of the p53 protein (Austen B, 2007)PubMed. There is an association between +12 and NOTCH1 mutations (49)


11q

This anomaly can be detected in 7-25% of cases, depending on the stage of the disease. The commonly deleted segment includes the ataxia teleangiectasia (ATM) gene which is involved in signal transduction pathways activated in response to DNA strand breaks. The remaining ATM allele is mutated in approximately 30% of CLL with 11q-, and patients with homozygous defects of ATM have a more aggressive disease compared to patients who only have 11q.

Genetic instability is associated with 11q deletion, as demonstrated by the development of additional chromosomal abnormalities by karyotype analysis (Fegan C, 1995)PubMed.

Clinical features frequently associated with 11q- are extensive adenopathies and a treatment-free interval and survival shorter than in other CLL cases (Döhner H, 1997)PubMed. In several clinical trials, the presence of 11q- was associated with lower complete response rates and a short progression-free survival (Catovsky D, 2007PubMed ; Grever MR , 2007PubMed ; Eichhorst BF, 2006PubMed ). However, in younger patients the addition of rituximab to conventional chemotherapy with fludarabine and cyclophosphamide improved the complete response rate and progression-free survival, thus overcoming the adverse prognostic impact of 11q- in CLL (Tsimberidou AM, 2009PubMedHallek M, 2010PubMed ). Allogeneic bone marrow transplantation with reduced-intensity conditioning may offer prolonged disease control in this cytogenetic subset (Sorror ML, 2008)PubMed .

 

17p-/TP53 mutations

The 17p- anomaly is frequently accompanied by additional chromosomal aberrations and complex karyotype and is associated, in virtually all cases, with loss of the TP53 tumor suppressor gene. In more than 70% of CLL with 17p deletion, mutation of the remaining TP53 allele is present. Inactivating mutations of the TP53 gene, detectable by molecular techniques, are present in 2-5% of patients who do not show 17p deletion and are associated with poor prognosis (Dicker F, 2009)PubMed . Expansion of 17p-deleted or TP53-mutated clone after chemotherapy has been reported (Zenz T, 2008)PubMed . A simple cytofluorimetric test to investigate dysfunctions of the p53 pathway has been developed (Carter A, 2004)PubMed.

The clinical course of patients with 17p-/TP53-mutated is severe (Lin SL, 2009; Zenz T, 2008PubMed), especially for patients with intermediate-advanced stage and unmutated IGVH (Tam CS, 2009)PubMed, since the response rate to chemoimmunotherapy, the progression-free survival and overall survival are much worse than in other CLL cytogenetic subgroups. The monoclonal antibody alemtuzumab and high dose steroids can overcome drug resistance in a significant proportion of cases, however virtually all cases relapse soon. Non-myeloablative allogeneic transplantation can have a role in this subset of CLL (Dreger P, 2010)PubMed.

Other chromosomal abnormalities

6q deletion has a 3-7% incidence in CLL patients and is associated with a higher white blood cell count at onset, atypical morphology, CD38+, unmutated IGVH genes in 60% of cases, lower time to treatment and reduced survival compared to CLL with favorable cytogenetic aberrations (13q-, normal karyotype)(Cuneo A, 2004)PubMed . The deleted portion is located around the 6q21 region and using high-resolution allelic typing the allelic loss in 6q has been identified in more than 15% of CLL cases (Novak U, 2002)PubMed.

14q32 translocations involving the IGH gene occur at a 4-9% incidence. Frequent chromosomal partners include 18q21/BCL2 and 19q13/BCL3; other rare partners are 2p12/BCL11A, 2p13, 4p16, 4p31, 5q31, 6p21/CCND3, 7q21/CDK6, 8q11, 9q34 and 17p11. The classic t(11;14)(q13, q32) translocation, identical to that associated with mantle cell lymphoma, has been documented by several groups; however these cases represent an atypical form of CLL, sharing some features with non-nodal leukemic mantle cell lymphoma (Cuneo A, 1997)PubMed. The t(14;19) is associated with an aggressive form of atypical CLL, which is often associated with additional chromosomal abnormalities, especially trisomy 12, and with unmutated IGHV. Cases with t(14;19) and complex karyotypes including abnormalities such as 7q and/ or 6q-, 17p-, 1q rearrangements, may frequently represent cases of leukemic non-Hodgkin lymphomas (Martín-Subero JI, 2007)PubMed. The clinical course of CLL with 14q32 translocations is worse than that of CLL with favorable karyotype (Cavazzini F, 2008PubMed ; Haferlach C,  2010PubMed ).

Clonal evolution in CLL

Modern sequencing techniques have documented that some lesions may represent “primary” genetic changes” driving the expansion of the entire neoplastic clone (13q-, +12, mutations of NOTCH1 or MYD88), whereas other aberrations (17p-/ TP53 mutations, 11q-/ ATM mutations, SF3B1 mutations) are frequently associated with clonal evolution of a pre-existing clone (Puente XS, 2013)PubMed. There is evidence that a fraction of CLL may acquire chromosomal abnormalities during the natural history of the disease. In a prospective study  (Stilgenbauer S, 2007PubMed ; Goldin LR, 2007PubMed ) 11 of 64 patients (17%) followed for a median of 42 months showed clonal evolution with del(17)(p13) in 4 cases, del(6)(q21) in 3 cases, del(11)(q23) in 2 cases, +(8)(q24) in 1 case and evolution of 13q-heterozygous to homozygous in 3 cases. The late acquisition of 11q- in CLL has been associated with disease progression (Cuneo A, 2002)PubMed .

Diagnosis

Nowadays, most cases of CLL are diagnosed in western countries in routine blood tests showing more than 5×109/l lymphocytes in the peripheral blood. A minority of cases at diagnosis shows lymphadenopathy and/or splenomegaly, anemia or thrombocytopenia due to bone marrow invasion by the neoplastic clone, systemic symptoms and, rarely, involvement of extranodal sites (vide infra). In the presence of lymphocytosis, after ruling out reactive conditions (Table IV), laboratory tests are required to establish the correct diagnosis. Indeed CLL must be distinguished from the various forms of chronic lymphoproliferative disorders (Table V) or from the rare condition known as persistent polyclonal B lymphocytosis (Salcedo I, 2002)PubMed, a condition more common in young women smokers that shows a tendency towards familial distribution. Morphological analysis of the peripheral blood smear show small lymphocytes with condensed chromatin (Figure III) and smudge cells (Gumprecht cells), representing lymphocytes damaged during the smear preparation due to their intrinsic fragility determined by reduced expression of vimentin cytoskeleton components (Nowakowski GS, 2010)PubMed. Immunophenotypic analysis allows the establishment of a definite diagnosis in the presence of an expansion of CD19+, CD5+, CD23+ elements: these cells also show weak expression of CD22 and/ or CD79 and weak expression of surface immunoglobulin (sIg) associated with light chains restriction (ratio kappa/lambda >3 or <3) with FMC7 negativity. The immunophenotypic score proposed by Matutes and coworkers (Matutes E, 1994PubMed ; Moreau EJ, 1997)PubMed , assigns 1 point to CD5+, CD23+, CD22/CD79b+ weak, sIg+ weak and FMC7-. Over 90% of CLL score ≥ 3, whereas the vast majority of other lymphoproliferative syndromes score <3 (Table V).

The diagnostic workout may be completed with the investigations shown in Table VI, which are necessary for proper staging, prognostic stratification and therapeutic planning.

 

Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_4

 Table IV: Conditions associated with reactive lymphocytosis

 

Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_5

Table V: Distinctive laboratory features of chronic lymphoproliferative disorders

 

Cuneo_Chronic_Lymphocytic_Leukemia_Figura_3

Figure III. a) Typical CLL. Small lymphocytes and smudge cells (*); b) Atypical CLL (mixed-cell type) showing small lymphocytes and a large lymphocyte (**)

 

Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_6

Table VI: Diagnostic evaluation in CLL

 

Clinical features

a) General clinical picture

Approximately 70% of patients at diagnosis in western countries display asymptomatic lymphocytosis, with negative physical examination or localized lymphadenopathy (Johnson JB , 2009). Hypogammaglobulinemia may already be present at diagnosis. Painless adenopathy usually occurs in the intermediate stages of disease, involving the main superficial nodal sites. Splenomegaly may be present. Advanced stages are defined by the presence of anemia or thrombocytopenia secondary to bone marrow infiltration. Rai and Binet staging systems  (Rai KR, 1975PubMed ; Binet JL, 1981PubMed ) are presented in Table VII, where the median survival of patients in different stages of disease is also reported. It is important to exclude the autoimmune nature of anemia and thrombocytopenia before assigning a patient to advanced Rai or Binet stages (Oscier D, 2004)PubMed since the prognosis of advanced stages is better if cytopenia is autoimmune (Zent CS, 2008)PubMed. Modern chemoimmunotherapy regimens and better supportive treatment, along with improvement of the general health of the adult population, led to improved survival in CLL as compared with historical data (Shanafelt TD, 2009PubMed ; Abrisqueta P, 2009PubMed ; Kristinsson SY, 2009PubMed ).

 

Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_7

Table VII: Rai and Binet classification

 

Symptoms may derive from massive lymphadenopathy or massive hepatosplenomegaly and/or bone marrow failure with anemia or thrombocytopenia. Weakness without significant anemia may occur. Systemic symptoms, such as fever >38°C without apparent cause, loss of more than 10% of body weight, profuse sweating, itching and muscle pain are present at diagnosis in a minority of cases, but may occur more frequently in advanced stages of disease. The disease may present infectious complications, an expression of immune deficiency linked to disease and/or therapy. Autoimmune hemolytic anemia can occur at any stage of the disease. Immune system dysregulation, sustained by the mechanisms presented in Table VIII, may result in a number of autoimmune manifestations, as summarized in Table IX (Dearden C, 2008)PubMed .

 

Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_8

Table VIII: Immune defects in CLL

 

Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_9

Table IX: Autoimmune complications in CLL

 

b) Disease-related complications and clinical evolution

– Infections

Patients may develop recurrent infections, especially in advanced stages partly due to repeated treatment including purine analogues, alkylating agents and monoclonal antibodies that exert an immunosuppressive effect. The infectious complications are frequently caused by Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus. Herpes zoster infection is quite frequent and, with the introduction of new treatments, opportunistic infections by Legionella pneumoniae, Pneumocystis jirovecii and Listeria monocytogenes are to be taken into account. Cytomegalovirus pneumonia is an emerging problem, as are candidiasis and aspergillus fungal infections (Montserrat E, 2006).

– Second malignancies

The incidence of second malignancies in CLL is 1.2 to 2.2 times the incidence expected in an age-matched population (Hisada M, 2001PubMed ; Tsimberidou AM, 2009bPubMed ), most frequently represented by tumors of the skin, prostate, breast, melanoma, gastrointestinal system and lung. The main risk factors are represented by old age, male sex, high levels of LDH, beta2-microglobulin and creatinine, whereas the type of hematological therapy performed does not seem to play a role (Tsimberidou AM, 2009b)PubMed. The widespread use of purine analogues is not associated with a significantly higher risk of second cancers than expected on the basis of available historical data (Jennifer A, 2009). The risk of developing secondary myelodysplasia or acute myeloid leukemia is low, but may be increased in patients treated with alkylating agents and purine analogues (Cheson BD, 1999)PubMed.

– Histologic transformation

Richter’s syndrome is defined by the appearance of an aggressive lymphoma with histopathological features of diffuse large cell lymphoma, associated with systemic symptoms, serous effusions and cachexia. It may occur in 5-10% of cases (Rossi D, 2008)PubMed, whereas the transformation to prolymphocytic leukemia can be observed in a minority of cases.

 

CLL Therapy

Treatment of CLL is defined in the setting of a structured decision-making process, which must take into account all of the following:
a) when to start treatment
b) age and general clinical condition of the patient
c) clinical features of the disease
d) some biological characteristics
e) results of clinical trials

 

a) When to start treatment

There is convincing evidence (CLL Trialists’ Collaborative Group, 1999)PubMed that treatment of patients with stable and asymptomatic disease in initial or intermediate stage does not prolong survival compared with watchful waiting followed by therapy administered at the time of clinical evolution. This consideration derives from studies conducted when the only available therapy was chlorambucil, however there is no data suggesting that more effective drugs, such as fludarabine, alemtuzumab, bendamustine and a combination of chemotherapy and rituximab may be beneficial in the indolent disease. It is worth noting that treatment in the initial phase exposes the patient to the risk of infection and, in approximately 2% of cases, to the risk of treatment related death, compared with a 49% probability for patients in Binet stage A of remaining asymptomatic with stable disease after 11 years (Dighiero G, 1998)PubMed. Therefore, as opposed to the traditional watchful waiting approach, ongoing randomized clinical trials assign to an early treatment only patients with unfavorable biological risk factors (unmutated IGVH, unfavourable FISH, CD38+) who, on average, require therapy after 2-6 years from diagnosis (Shanafelt TD, 2010PubMed ; Morabito F, 2009PubMed ).
The main criteria for starting therapy (Lin SL, 2009) are listed in Table X.

 

Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_10

Table X: Criteri per iniziare il trattamento nella LLC

 

 

b) Age and clinical conditions

Demographic data show that patients with CLL have a shorter life expectancy compared to the population of the same age (Shanafelt TD, 2009)PubMed and that this difference may not be apparent in cases diagnosed at >75 years of age (Shanafelt TD, 2010)PubMed.

When choosing treatment it is important to consider carefully the patient’s general condition. Nowadays objective assessment tools are available, such as the Cumulative Illness Rating Scale-Geriatric, reproduced in Table XI (Parmelee PA, 1995)PubMed, which, although not designed to test the tolerability to chemotherapy, are widely used in clinical trials. For example, patients with a CIRS> 6 were not enrolled in the study that evaluated the efficacy of FCR compared to FC (Extermann M, 1998PubMed ; Hallek M, 2010PubMed ).

Assessment of renal function is essential in the choice of therapy, and raised creatinine levels and/or a creatinine clearance <60-70 ml/min, should impose caution in the use of intense combination regimens. Equally important for appropriate treatment selection is the patient’s medical history, with special reference to infections, especially in previously treated patients.

 

Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_11

Table XI: Cumulative Illness Rating Scale (from (Parmelee PA, 1995)PubMed)

 

c) Clinical features of disease

The patient may require treatment for a clear expansion of the disease in the bone marrow, or a prevailing progressive lymph node involvement. In the treatment decision-making it is important to keep in mind that the effectiveness of certain drugs is reduced in the presence of large lymph node masses (Hillmen P, 2007)PubMed and that, on the contrary, some chemoimmunotherapic combinations are able to produce satisfactory responses in CLL associated with adenopathies (Grever MR, 2007)PubMed . Sometimes CLL patients require therapy for conditions indirectly related to the disease, especially autoimmune manifestations (see Table IX), the most frequent being represented by autoimmune hemolytic anemia (AHA) or autoimmune thrombocytopenia. The therapeutic approach of AHA, which may occur at diagnosis or, more frequently, during the course of the disease and in pre-treated patients (Mauro FR, 2000)PubMed, is centered on the use of steroids, which produce global responses in 90% of cases with disappearance of hemolysis in 60-70% of cases. A proposal for a therapeutic algorithm is schematically shown in Figure IV (Dearden C, 2008)PubMed.

 

Cuneo_Chronic_Lymphocytic_Leukemia_Figura_4

Figure IV. Treatment of autoimmune hemolytic anemia in CLL
d) Biological features

Generally speaking, knowledge of prognostic factors is useful in guiding physician and patient on therapeutic strategies.

At the time of first-line treatment or relapse, determination of 17p deletion and TP53 mutation appears to be necessary in young patients to plan an appropriate therapy, while the demonstration of 11q deletion may suggest chemoimmunotherapy as the preferred option (Hallek M, 2010b)PubMed. In clinical practice the final choice of the induction therapy or the total number of cycles to be administered is frequently the result of a complex evaluation combining the patient’s general condition, the degree of aggressiveness of the disease and the tolerance to treatment.

 

e) Results of clinical trials

The efficacy and tolerability of treatment regimens most commonly used in current clinical practice are summarized in Table XII (first line) and in Table XIII (subsequent treatment lines).

The results of clinical trials have to be transferred to real-world patients taking into account that, as a rule, the population with CLL has a higher median age (Figure V) and a greater number of comorbidities (Figure VI).

 

Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_12

Table XII: Summary of efficacy and tolerability of the main CLL first line regimens
Cuneo_Chronic_Lymphocytic_Leukemia_Tabella_13

Table XIII: Efficacy and tolerability of second line treatment in CLL

 

Cuneo_Chronic_Lymphocytic_Leukemia_Figura_5

Figure V. CLL frequency in the US population, stratified by age groups, compared with the median age of CLL patients enrolled in clinical trials.

 

 

Cuneo_Chronic_Lymphocytic_Leukemia_Figura_6

Figure VI. Average number of comorbidities in CLL patients stratified by age.

 

Over the past years numerous studies tested various drug combinations, demonstrating that, despite a prolongation of PFS, there was no significant impact on survival (Table XII). In more recent years, however, the adjunct of rituximab to the combination of fludarabine and cyclophosphamide was shown to improve survival in young patients compared to the best standard of care (Hallek M, 2010)PubMed, thus reproducing the results achieved by the introduction of rituximab in the therapy of indolent and aggressive lymphomas (Schulz H, 2007; Coiffier B, 2010).

Even though chemoimmunotherapy combinations such as FCR may be effective and relatively well tolerated in the elderly without significant comorbidities, there is no evidence deriving from prospective studies that they prolong survival in this subset of patients (Hallek M, 2010)PubMed. Control of symptoms and reduction of disease burden may be achieved with less aggressive therapies and may be convenient in the elderly population, especially in the presence of comorbidities (Eichhorst BF, 2009)PubMed.  However a retrospective study showed improved survival using fludarabine and rituximab in elderly patients with respect to previous treatment protocols employing fludarabine or chlorambucil alone (Woyach JA, 2013)PubMed.

First-line therapy

The main options for first-line therapy are summarized in Figure VII. Nowadays the standard of care in young and fit patients and in a minority of elderly patients without significant comorbidity is represented by the combination of fludarabine (F), cyclophosphamide (C), and rituximab (R), according to the FCR scheme (Hallek M, 2010)PubMed. This combination improved all outcome measures including survival as compared with the best standard, represented by the FC combination which, in turn, was shown to be superior to F and C single agents in previous studies, as summarized in Table XII. The goal of treatment in the young patients should be, whenever possible, achievement of complete remission according to NCI clinical criteria (Hallek M, 2008)PubMed, a goal reached in 44-70% of cases treated by FCR in clinical trials (Keating MJ, 2005; Hallek M, 2010bPubMed). FCR therapy requires close hematologic monitoring and, in general, anti-infective and antiviral prophylaxis. In case of neutropenia or haematological toxicity at the time of subsequent cycle administration, a 20% dose reduction is mandatory.

 

Cuneo_Chronic_Lymphocytic_Leukemia_Figura_7

Figure VII. First line treatment options for CLL patients

 

It is worth noting that in two studies describing the effectiveness of FCR (Keating MJ, 2005PubMed ; Hallek M, 2010PubMed):

– the median age of patients was 58-61 years, with only 11-13% of patients >70 years;
– 74% of patients completed the planned therapy with 6 cycles in the above studies;
– survival advantage in patients >70 years was not documented (Hallek M, 2010)PubMed.

Thus, the efficacy and tolerability of a scheme that reduced the doses of FC while increasing the dose of rituximab (FCR-lite) was tested, showing efficacy and manageable toxicity (Foon KA, 2009)PubMed .

The combination that includes mitoxantrone, in the R-FCM scheme, proved very effective, though the need of careful monitoring and management of side effects might be an issue outside experienced centres (Bosch F, 2009)PubMed .

The combination of bendamustine and rituximab proved very effective and relatively safe (Fischer K, 2012)PubMed and a randomized trial comparing this regimen with FCR in fit patients is ongoing.

In elderly patients and in all circumstances in which the goal of therapy is to achieve control of the disease, chlorambucil retains an important role due to its limited toxicity and manageability. Recent data show increased efficacy in terms of responses with the addition of rituximab (Hillmen P, 2009). Bendamustine and fludarabine are more effective than chlorambucil, but this advantage was not associated with longer survival (Catovsky D, 2007PubMed ; Eichhorst BF, 2009PubMed; Knauf WU, 2009PubMed ). The adjunct of rituximab to fludarabine improved the quality and duration of responses (Byrd JC, 2005)PubMed .

In patients with 17p- the best therapeutic approach is not established. FCR improves progression-free survival compared to FC, but only 18% of patients were free of progression at 3 years in one study (Hallek M, 2010)PubMed. Treatment with alemtuzumab, associated or not with dexamethasone or high dose methylprdnisolone, has shown efficacy in these patients, which however invariably show relapse and disease progression (Pettitt AR, 2012)PubMed. The efficacy and tolerance of the combination of fludarabine and alemtuzumab as first-line therapy in high-risk patients were good (Mauro FR, 2008). Bone marrow transplantation is an option in these patients, especially within clinical trials (Dreger P, 2010)PubMed.

 

Second-line therapy

The efficacy and tolerability of the most commonly used second-line schemes are summarized in Table XIII. Nowadays it is reasonable to repeat the first-line treatment if remission lasted at least 12 months (Eichhorst B, 2010)PubMed or at least 24 months if modern chemoimmunotherapy protocols were used as first line treatment, Hallek M, 2010)PubMed. One randomized study demonstrated a greater efficacy of FCR compared to FC in patients pretreated with alkylating agents (82% of cases) and fludarabine (18% of cases) (Robak T, 2010)PubMed , making this combination ideal for patients receiving monotherapy as first-line.

Combination of fludarabine and alemtuzumab was effective and well tolerated (Elter T, 2005)PubMed and proved more effective than fludarabine alone (Elter T, 2011)PubMed in a cohort of pretreated patients, 21% of whom received fludarabine as first-line.

In pretreated patients the addition of oblimersen, a BCL2 antisense oligonucleotide, to FC was shown to increase response rates and to prolong progression-free survival and overall survival in patients who achieved at least a partial response and were sensitive to fludarabine (O’Brien S, 2009)PubMed .

Ofatumumab demonstrated efficacy and a good safety profile in heavily pre-treated patients and is approved for clinical use in patients refractory to fludarabine and alemtuzumab or in patients refractory to fludarabine with bulky disease (Wierda WG, 2010)PubMed .

High-dose methylprednisolone in combination with rituximab has shown efficacy in pretreated patients with 17p deletion (Castro JE, 2009)PubMed.

New drugs targeting the cell cycle such as flavopiridol proved effective but dosage and schedule of administration need to be better defined (131). Immunomodulatory agents such as lenalidomide in combination with rituximab showed promising activity (132: sostituire la vecchia voce con Badoux XC, Keating MJ, Wen S, Wierda WG, O’Brien SM, Faderl S, Sargent R, Burger JA, Ferrajoli A. Phase II study of lenalidomide and rituximab as salvage therapy for patients with relapsed or refractory chronic lymphocytic leukemia. J Clin Oncol. 2013 Feb 10;31(5):584-91).

Allogeneic bone marrow transplantation has been shown to induce prolonged clinical responses (Dreger P, 2010)PubMed , with progressive negativization of minimal residual disease in a fraction of cases through an immune-mediated effect (Sorror ML, 2008)PubMed . Five-year survival was about 50% with a 20% non-relapse mortality. Toxicity included ≥grade 2 acute graft versus host disease (GVHD) in 50% of cases and extensive chronic GVHD in 50% of cases, both in patients transplanted from HLA-matched siblings and from HLA-identical unrelated donors, with a median GVHD duration of 25 months (Sorror ML, 2008)PubMed. Possible candidates for a transplant approach are young patients refractory to fludarabine containing regimens or relapsed within 12 months after chemoimmunotherapy, and untreated patients with 17p deletion, possibly in the context of clinical trials (Dreger P, 2007)PubMed.

Excellent results were recently described using the BTK inhibitor ibrutinib as single agent, which attained a high overall response rate, with an impressive 75% progression free survival at 26 months (Byrd JC, 2013)PubMed. This oral agent interferes with BCR signaling, causes mobilization of the lymphocytes from the lymph nodes with lymph node shrinkage and initial increase of lymphocytosis in the peripheral blood. The majority of patients progressing under treatment had unfavourable chromosome lesions (i.e. 17p- or 11q-). The concurrent publication of frequent and durable responses using another agent interfering with BCR signalling, i.e. the PI3K delta inhibitor idelalisib in combination with rituximab and/or bendamustine (Coutre SE, 2012) opens a new scenario for the employment of a mechanism-driven treatment of relapsed/refractory CLL (Foà R, Guarini A. A mechanism-driven treatment for chronic lymphocytic leukemia? N Engl J Med. 2013 Jul 4;369(1):85-7).

 

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