B1 CELLS CHRONIC LYMPHOCYTIC LEUKEMIA (B-CLL) / SMALL LYMPHOCYTIC LYMPHOMA

Table of contents :

Epidemiology Aetiology Pathogenesis Symptoms & signs Laboratory examinations Differential diagnosis Therapy  Prognosis Richter's syndrome prolymphocytic leukemia (PLL) Web resources

Epidemiology : CLL is the most frequent type of leukemia in the Western world and affects mainly elderly individuals, but about a third of patients are < 60 years of age at diagnosis^ref. 95% of all CLLs. Prevalence : 15:100,000. In USA 9730 new cases are expected in 2005.
Aetiology : risk factors :

• male gender
• increasing age
• ethnicity (high in Caucasians, lowest in Asians) and family history : migration studies confirm that ethnic groups retain the risk associated with their origin rather than their new location, favoring a role for heredity. Kindreds with multiple cases of CLL have been well described in the literature and studies in large populations confirm that lymphoproliferative malignancies and especially CLL occur together at a rate that cannot be attributed to chance. Since environmental factors cannot readily explain the familial aggregations, a hereditary factor that affects susceptibility to CLL is likely. The identification of clones that are immunophenotypically identical to CLL in healthy individuals from CLL kindreds (14% to 18%) as well as in the general population (3.5% in age bracket >65 years) suggests a possible precursor condition, but longitudinal studies will be necessary to establish significance in the general population. Family (linkage) and population (candidate gene) studies to date have been too small to identify the specific genes that account for increased susceptibility; larger studies including planned consortia to identify additional high-risk kindreds for genetic studies, as well as the application of advanced technologies such as genomics, cytogenetic, expression, and proteomics, are widely expected to advance understanding over the next few years^ref. Some families show classic findings in support of an autosomal dominant mode of genetic transmission of CLL^ref
• farming and pesticide exposure
• ionizing radiation exposure^ref
• tobacco users and cigarette smokers (OR = 1.6)^ref, and environmental tobacco smoke (ETS) (OR = 2.3)^ref. Approximately 2% of patients with CLL develop lung carcinoma. In a study, 85% of the patients were smokers. These patients had a high risk of a third primary malignancy (including melanoma, basal cell carcinoma, laryngeal carcinoma, and colon carcinoma). Lung carcinoma was diagnosed a decade after CLL. Patients who develop both diseases die of lung carcinoma and not CLL or other solid tumors. CLL and poor performance status limit treatment, particularly for patients with unresectable lung carcinoma^ref
• familal B-CLL : genetic basis in about 10% of patients^ref. The lifetime risk of developing CLL in first-degree relatives of patients with the disease is increased by a factor of 8. The inherited predisposition to CLL may be mediated by a gene with pleiotropic effects as suggested by a 2-fold increase, in relatives of CLL patients, of other B-cell disorders, mostly NHL and HL. In fact, in > 300 families with CLL 12 had multiple myeloma in relatives^ref. By analogy with MGUS, when healthy relatives from CLL families were examined with sensitive flow cytometry methods, monoclonal B-cell lymphocytes are detected with high frequency^ref.
• HLA-B8 is a marker of mild disease while HLA-A2 and the closely associated antigens B12(44) and DR7 are markers for severe disease^ref
• HLA B12, alone or in combination with HLA A2, indicated better prognosis. Relatives HLA identical with the patients showed no evidence of white cell disorder when compared with haplo- or non-identical relatives, or controls. As a group, however, relatives of patients had fewer lymphocytes than relatives of controls^ref
• an increased incidence of lymphoid malignancy has been reported in western Ashkenazi Jews and in particular those of Russian descent^ref.
A genome scan of CLL-prone families found a lod score of one in band 13q22.1. To investigate this finding, 6 CLL families consisting of 65 individuals (CLL affected n=19, unaffected n=46) were selected for genotyping and fine mapped a 23 megabase region in 13q14.2-q22.2. Interphase FISH revealed 13q14 deletion in 85% (11/13) of CLL patients. 4 CLL families shared a 3.68 Mb minimal region in 13q21.33-q22.2. 2 asymptomatic siblings who shared the 13q21.33-q22.2 at risk haplotype exhibited CD5⁺ monoclonal B-cell lymphocytosis (MBL) on flow cytometry. One of these individuals also had 13q14 deletion by FISH. These 2 MBL individuals shared the at risk haplotype with their CLL affected relatives providing further evidence of the relationship between CLL and MBL, and the biologic significance of this novel region. Using direct DNA sequencing analysis 13 genes were screened for germline mutations but no frameshift or nonsense mutations were detected. 11 of the 13 genes in the candidate region were expressed in immune tissues supporting that they have functional relevance in investigations of familial CLL^ref.

	IgVH unmutated B-CLL (U-CLL) (intraclonal variation < 95%) (50%)	IgVH mutated B-CLL (M-CLL) (50%)
intraclonal variation (ongoing SHM)	absent or very low	low
morphology	atypical according to the criteria of Matutes et alref as > 10% (but < 55%) prolymphocytes or > 15% cells with cleaved nuclei and/or lymphoplasmacytoid cells in the peripheral blood of patients whose predominant cell type was a small lymphocyte with coarsely clumped chromatin (dense cells (DC) / Rieder's cell or lymphocyte (a myeloblast whose nucleus with wide and deep indentations suggesting lobulation, which may represent asynchronism of nuclear and cytoplasmic maturation))	typical (Gumprecht shadow or basket cells (BC) are highly suggestiveref1, ref2)
stage at diagnosis	advanced (C)	early (A, B)
cell source	pregerminal center naive B-cells	germinal center exposed B-cells
CD38 : a receptor that induces proliferation and increases survival of CLL cells. CD38 signals start upon interaction with the CD31 ligand expressed by stromal and nurse-like cells. CD38/CD31 contacts up-regulate CD100, a semaphorin involved in sustaining CLL growth. Evidence that nurselike cells express high levels of CD31 and plexin-B1, the high-affinity ligand for CD100, offers indirect confirmation for this model of receptor cross-talk. Elements of variation in the clinical course of CD38+ CLL patients include (1) potential intersection with ZAP-70, a kinase involved in the CD38 signaling pathway in T and NK cells, and (2) the effects of genetic polymorphisms of the receptors involved, at least of CD38 and CD31. Consequently, CD38 together with ZAP-70 appear to be the key elements of a coreceptor pathway that may sustain the signals mediated by the BcR and potentially by chemokines and their receptorsref. Regardless of the size of the CD38+ fraction within a CLL clone, CD38+ subclones are markedly enriched for expression of Ki-67, CD27, CD62L, CD69, ZAP-70, human telomerase reverse transcriptase, and telomerase activity. Although the percentage of cells (~2%) entering the cell cycle, as defined by Ki-67 expression, is small, the absolute number within a clone can be sizeable and is contained primarily within the CD38+ fraction. Despite these activation/proliferation differences, both CD38+ and CD38- fractions have similar telomere lengths suggesting that CD38 expression is dynamic and transientref	high (> 30%)	low (< 30%)
ZAP70ref is a key mediator of TCR signaling and is structurally homologous to Syk, which plays an analogous role in BCR signaling. BCR-mediated activation of ZAP-70 is very inefficient in CLL and lymphoma B-cells and is negligible when compared to activation of Syk. Despite the inefficient catalytic activation, the ability of ZAP-70 to recruit downstream signaling molecules in response to antigen-receptor stimulation appeared relatively preserved. Moreover, ectopic expression of ZAP-70 was found to enhance and prolong activation of several key mediators of BCR signaling, such as the Syk, ERK and Akt kinases, and to decrease the rate of ligand-mediated BCR internalizationref	high (> 20% or ZAP-70/Syk mRNA ratios > 0.25ref) in 86-88% of casesref1, ref2	low (< 20%) in 44% of cases
chromosomal aberrations	+12, del(11q), del(17p)	normal or del(13q14) (which contains 2 small miRNA genes that are turned off in about 60% of CLL cases)
miRNA signatureref	reduced expression of miR-16	reduced expression of miR-16
DNA microarray : the expression of 358 genes differed significantly between ZAP-70+CD38+ with ZAP-70-CD38- subgroups, including genes involved in BcR signaling, angiogenesis and lymphomagenesis. 3 of these genes, that is, IRTA4/FcRH2, angiopoietin 2 and Pim2 were selected for further validating studies in a cohort of 94 B-CLL patients. In healthy individuals, IRTA4/FcRH2 protein expression was associated with a CD19+CD27+ memory cell phenotype. Disease progression may be related to proangiogenic processes and strong Pim2 expressionref	IRTA4/FcRH2 low, 2-fold higher ANGPT2 plasma concentrations, Pim2 overexpression	IRTA4/FcRH2 high
response to immunostimulatory CpG-oligodeoxynucleotides (CpG ODN), related to the magnitude of Akt signalingref	proliferation	G1/S cell cycle arrest and apoptosis
therapy	poor response	good response
survival	average life expectancy of about 8 years and is universally fatal	average survival of 25 years and most people with this form die of other causes rather than of CLL
experimental animal model	IgH and IgL rearrangements in TCL1 mice display minimal levels of somatic mutations and exhibit several molecular features found in the human disease. Like human B-CLL, TCL1 leukemic rearrangements from different mice can be very similar structurally and closely resemble autoantibodies and antibodies reactive with microbial antigens. Antigen-binding analyses confirm that selected TCL1 clones react with glycerophospholipid, lipoprotein, and polysaccharides that can be autoantigens and be expressed by microbes. This (auto)antigen-driven mouse model reliably captures the BCR characteristics of aggressive, treatment-resistant human B-CLLref	?

• a restricted set of variable (V) region genes, specifically V_H1-69 (also known as 51p1) and V_k3-A27 (also known as kv325), which bind to HCV E2 envelope protein, occurs in 10-20% of patients^{ref1, ref2}. Preferential use of the 51p1 gene has also been observed in CLL, with a prevalence of > 20% in particular geographic areas^ref. However, unlike the HCV-associated immunocytomas, the 51p1 V_H sequences in CLL are almost exclusively unmutated and usually have significantly longer CDR3 regions^{ref1, ref2}. V_H1-69 B-CLLs constitute a uniform group of patients that more often present at advanced clinical stages and require early treatment, but their survival does not differ significantly from patients with other unmutated V_H genes^ref.
• cutaneous manifestations of B-CLL comprise a wide spectrum of clinicopathologic presentations. In some cases, onset of skin lesions is triggered by antigenic stimulation, and specific skin infiltrates at sites of previous herpes simplex or herpes zoster infection have been well documented. Specific skin manifestations of B-CLL can also be observed at sites typical for lymphadenosis benigna cutis (nipple, scrotum, earlobe), a Borrelia burgdorferi-associated cutaneous B-cell pseudolymphoma. Histology of solitary erythematous plaques or nodules located on the nipple (4 cases) and scrotum (2 cases) from 6 patients with B-CLL (M : F = 4 : 2; mean age: 67.8) revealed in all cases a dense, monomorphous infiltrate of small lymphocytes showing an aberrant CD20⁺/CD43⁺ phenotype. In all cases monoclonality was demonstrated by PCR analysis of the J_H gene rearrangement. PCR analysis showed in four of the six cases the presence of DNA sequences specific for B.burgdorferi. Infection with B. burgdorferi can trigger the development of specific cutaneous infiltrates in patients with B-CLL^ref.

CLL cells do not undergo SHM in their V region genes

• lymphocytosis =>
• lymphadenopathy =>
• superficial
• deep : phlebothrombosis, lymphedema, obstructive jaundice, hydronephrosis
• hepatosplenomegaly =>
• bone marrow infiltration (anemia, neutropenia, thrombocytopenia) =>
• skin
• gastrointestinal involvement (gastric ulcers/hemorrhages, malabsorption enteritis, miliary pulmonary nodular infiltration, pleural effusion)
• pulmonary apparatus
• central nervous system =>
• autoimmune diseases :
• autoimmune hemolytic anemia (AIHA) occurs in approximately 10-25% of CLL patients at some time during the course of the disease^{ref1, ref2, ref3}. Although the pathogenic autoantibodies occasionally may be produced by the malignant B-cell clone^{ref1, ref2}. in most cases the autoantibodies appear to be made by remnant normal B lymphocyte^{ref1, ref2}. This view is supported by the observation that the antibodies eluted from the red blood cells (RBC) are typically “warm-reactive” polyclonal IgG antibodies that display activity against monomorphic antigens of the rhesus system. On the other hand, the antibodies produced by the B-CLL cells are usually IgM and are always monoclonal. Experiments with SCID mice reconstituted with peripheral blood lymphocytes from a B-CLL patient have also suggested that the anti-RBC antibodies are not related to the malignant clone^ref. A large proportion of these animals developed human IgG anti-RBC autoantibodies that were of polyclonal origin. Collectively, these data have suggested that the anti-erythrocyte autoantibodies in B-CLL occur as a consequence of progressive dilution or inhibition of nonneoplastic B or T lymphocytes whose role is to antagonize the autoreactive Bcell clones^{ref1, ref2}. This model, however, does not provide an explanation for the much higher frequency of AIHA in CLL versus other hematologic malignancies and the lack of correlation between the circulating levels of pathogenic anti-RBC autoantibodies and the duration and severity of the underlying lymphoproliferative disease : the expected frequency of 51p1 in CLL is approximately 15%, which is significantly lower than the frequency observed in the CLL-AIHA patients (47%). A significant correlation between V_H gene usage and AIHA was also observed for the DP-50 gene, which was not present among 75 surveyed sequences^ref, but accounted for 33% of the B-CLL-AIHA sequences^ref. Interestingly, this V_H gene has also been found in 5 of 14 investigated human monoclonal anti-Rh(D) antibodies, indicating that it is also overrepresented in the human anti-Rh(D) response^ref. The antibodies produced by the B-CLL cells could have a similar specificity and could promote the binding of RBCs, sensitized with polyclonal IgG antibodies, to the FcRs on macrophages. A subset of B-CLL cells produces secretory g-chain transcripts, which in 2 of the B-CLL-AIHA patients were predominantly of the g₂ and g₃ H-chain isotype. Antibodies of the IgG₃ subclass seem to play an important role in the initial adherence of sensitized erythrocytes to macrophages because of their greatest affinity for the FcgRIII^ref. Alternatively, the B-CLL antibodies might not bind directly to RBC antigens, but could represent second antibodies that bind to the Fc portion of the anti-RBC antibodies. Initial experiments with a recombinant Fv antibody that corresponds to the CLL Ig of patient HA2 show low-affinity reactivity against a number of self antigens including human IgG. Finally, the B-CLL antibodies could have antiidiotypic activity and could stimulate normal autoreactive B cells to produce anti-RBC antibodies. Antiidiotypic IgG antibodies that crossreact with some anti-Rh(D) antibodies have been detected in eluates from sensitized RBCs and have been implicated in the pathogenesis of AIHA^ref
• infections : reactivation of VZV

• polyadenopathic systemic form (mainly lymph node involvement)
• pure splenomegalic form (no lymphadenomegaly, favourable prognosis, first reported by Binet in 1977)
• dysimmunoglobulinic form
• erythrolytic variant : severe AIHA due to anti-D antibodies
• paraproteinemic or secretory form (5%) : monoclonal Ig in serum
• thrombocytopenic form (autoimmune thrombocytopenia)
• pure medullary form (very rare)
• erythrodermic form (Sezary-like syndrome)
• regional form (single organ or apparatus)
• insect bite-like reaction and true hypersensitivity to mosquito bites

• k (60%) > l (40%)
• the monoclonal B cells must represent the majority of leucocytes with an absolute lymphocyte count >5 · 10⁹ cells/l and < 55% prolymphocytes, which has persisted for at least 1 month^ref
• lymphocytosis > 30%
• hypogammaglobulinemia (80% at diagnosis)
• very high levels of sCD23
• cytomorphology : small mature lymphoid cells, with high nuclear-to-cytoplasm ratio, scant cytoplasm, and round nuclei with highly condensed chromatin and inconspicuous nucleolus. Admixed with these cells may be prolymphocytes and paraimmunoblasts, characterized by larger size and prominent nucleoli


Bone marrow infiltration pattern :
• nodular (10%)
• interstitial (40%)
• diffuse (25%)
• mixed (25%)
• never intrasinusoidal (differential diagnosis with SMZL)
• immunophenotype : pan-T cell marker CD5⁺ (differential diagnosis with MCL !)6⁺10 / CALLA^-11a⁺11c^-19⁺20^loFMC7^lo21⁺22^weak23⁺30^-38^+/-79b^lo sIg (IgM or IgD)^lo. CD5^- B-CLLs represent < 5% of B-CLLs. When compared with CD5⁺ B-CLLs, more cases have advanced disease, splenomegaly, and a cytologically mixed CLL pattern according to the French-American-British (FAB) classification. In addition, CD23 expression is frequently absent, whereas FMC7 positivity and expression of high amounts of SIg are more frequently observed than in CD5⁺ CLL^ref. Thus, CD5^- B-CLL seems to be intermediate between classical CLL and prolymphocytic leukemia^ref. Immunohistochemically, B-CLL differs from SMZL for a lower expression of CD20 and positivity for CD23. CD19/CD8 coexpression has been reported^ref
• atypical CLL :
• CD5^-ref
• CD8⁺ (< 0.5%)^ref
• CD11c⁺
• CD20^high
• FMC7^high
• CD22^high
• CD23^-
• CD56^+ref
• CD154^+ref
• IgL high
Structural and functional features of the B-cell receptor in IgG⁺ chronic lymphocytic leukemia^ref
• cytogenetics :
• trisomy 12 (MDM2; 10-15% in conventional cytogenetics, 15% at FISH) : atypical/mixed-type CLL requiring therapy, reduced survival
• 13q14- (10% in conventional cytogenetics, 53% at FISH) : typical morphology, favorable prognosis
• 11q21- (8% at conventional cytogenetics, 19% at FISH) : typical morphology, massive lymphadenopathy, young age, reduced survival
• 6q- (6% at conventional cytogenetics, 9% at FISH) : artpical morphology, reduced survival
• 11q23- (20% : ATM) => increased TfR expression => more severe disease course, with earlier onset of symptoms, shortened lymphocyte doubling time, poor response to therapy, and shortened survival (may also partially explain why gallium, an atomically iron-like toxic metal that binds to transferrin and the TfR is incorporated into cells and was previously demonstrated to have anti-tumor activity in patients with lymphomas refractory to other chemotherapeutic treatments)^ref
• 17p- (4% at conventional cytogenetics, 8% at FISH : p53) : more common in atypical CLL and advanced stages of CLL, purine analog-refractary
• t(11;14)(q13;q32) (2-3%) : mixed type (CLL/PLL), frequent transformation into PLL, similar to leukemic MCL
• t(14;19)(q32;q13) : very rare, atypical morphology, young age, aggressive course
• molecular biology :
• a signature of microRNA (miRNA) gene expression profiling (based on the development of a microchip containing oligonucleotides corresponding to 245 miRNAs from human and mouse genomes) correlates with diagnosis and prognosis of B-CLL^ref. 2 signatures are found : one correlates with mutations in the variable region of the immunoglobulin gene—a good prognostic indicator, the other correlated with the deletion of chr13q14—a region containing 2 small miRNA genes that are downregulated in about 60% of CLL. There was only one common denominator between these 2 signatures, and that was the downregulation of miR-16, which is in fact the miR found deleted in CLL^ref. miR-15a and miR-16-1 are deleted or down-regulated in the majority of CLLs. miR-15a and miR-16-1 expression is inversely correlated to Bcl2 expression in CLL and both microRNAs negatively regulate Bcl2 at a posttranscriptional level. BCL2 repression by these microRNAs induces apoptopsis in a leukemic cell line model. Therefore, miR-15 and miR-16 are natural antisense Bcl2 interactors that could be used for therapy of Bcl2-overexpressing tumors^ref. A unique microRNA expression signature composed of 13 genes (of 190 analyzed) differentiates cases of B-CLL with low levels of ZAP-70 expression from those with high levels and cases with unmutated IgV_H from those with mutated IgV_H. The same microRNA signature is also associated with the presence or absence of disease progression. A germ-line mutation in the miR-16-1–miR-15a primary precursor causes low levels of microRNA expression in vitro and in vivo and is associated with deletion of the normal allele. Germ-line or somatic mutations are found in 5 of 42 sequenced microRNAs in 11 of 75 patients with CLL, but no such mutations are found in 160 subjects without cancer^ref.
• sequence insertions in the Mcl-1 promoter were found in 37/54 (69%) of the CLL samples. However, insertions were not associated with prognostic markers and were also detected in 38/66 (58%) of normal controls and in normal cells isolated from CLL patients^ref
• abdominal and superficial lymph nodes echography
• chest X-rays
• CBC, SGOT, SGPT, total and direct bilirubin, alkaline phosphatase, gGT, uricemia, glycemia, azotemia, cretininemia, serum protein electrophoresis, b₂-microglobulin, LDH, ESR, PCR, hepatitis mosaic, direct and indirect Coombs tests, haptoglobin, IgG, IgM, IgA, ANA, ENA, ANCA, anti-endomysium, anti-dsDNA antibodies, RF

• mantle cell lymphoma (MCL), which is also a tumor of CD5⁺ B cells and which has a much worse prognosis than CLL
• follicular lymphoma (FL) in leukemic phase; it is important to stress that t(14;18)(q32;q21) or t(18;22)(q21;q11) translocations have been previously reported in CLL and are not confined to FL^ref. B-CLL has not the intraclonal variation of immunoglobulin V_H genes seen in FL^ref , implying that cells showing somatic mutations are no longer under the influence of mutator enzymes and have therefore passed through the GC^ref
• splenic marginal zone lymphoma with villous lymphocytes, of which a small proportion are reportedly CD5^+ref
• nodal marginal zone lymphoma
• hairy cell leukemia
• prolymphocytic leukemia (B cell or T cell)
• lymphoplasmacytic lymphoma
• Sézary syndrome
• smoldering adult T cell leukemia/lymphoma

Binet's staging, 1981ref	criteria		Rai's staging, 1975ref	median survival, years
A (80% at diagnosis)	no lymphadenomegaly	lymphocytosis > 15,000/ml in peripheral blood or > 40% of bone marrow cells	0	>10
	1 or 2 lymphoid areas involved (cervical, axillary, inguinofemoral, or liver+spleen)	lymphocytosis + lymphadenomegaly	I	7
B	>= 3 lymphoid areas involved	lymphocytosis + hepatomegaly and/or splenomegaly	II	< 5
C (Fisher-Evans syndrome)	autoimmune hemolytic anemia (AIHA) < 10 g/dl in females, < 11 g/dl in males (from activation of non-neoplastic clones) independently from the number of lymphoid areas involved	lymphocytosis + autoimmune hemolytic anemia (AIHA) (HGB > 11 g/dl; from activation of non-neoplastic clones)	III	1.5-2
	autoimmune thrombocytopenia < 100,000/ml (from activation of non-neoplastic clones) independently from the number of lymphoid areas involved	lymphocytosis + autoimmune thrombocytopenia (PLT > 100,000/ml; due to activation of non-neoplastic clones)	IV	1.5-2

• clinical patient characteristics such as age, gender and performance status
• laboratory parameters reflecting the tumor burden or disease activity such as :
• absolute lymphocyte count
• LDH elevation
• bone marrow infiltration pattern
• lymphocyte doubling time (LDT)^{ref1, ref2, ref3}
• serum markers such as :
• soluble CD23
• ß₂-microglobulin (ß₂-MG)
• thymidine kinase (TK)^{ref1, ref2}
• cytogenetics :
• genomic aberrations^ref can be identified in about 80% of CLL cases by fluorescence in-situ hybridization (FISH) of interphase cell nuclei ("Interphase-Cytogenetics") with a disease-specific comprehensive probe set^ref :

	low-risk					high-risk
	13q-	13q-single	VH mutated	6q-	+12q	17p-	11q-	VH unmutated
single center cohort of CLL patients distributed over all stages	55%	36%	44%	7%	16%	7%	18%	56%
CLL1 trial of the GCLLSG for untreated Binet A patients with no classical indication for treatment	59%	40%	59%	2%	13%	4%	10%	41%
CLL4 trial (randomized F vs FC) of the GCLLSG for untreated Binet B/C patients up to 65 years of age with indication for treatment	53%	34%	31%	9%	11%	3%	21%	69%
CLL3 trial (early myeloablative radio-chemotherapy and autologous transplantation) of the GCLLSG for Binet B/C patients up to 60 years of age with maximum one line of prior therapy	52%	27%	32%	6%	12%	3%	22%	68%
CLL2H trial (subcutaneous alemtuzumab) of the GCLLSG for fludarabine-refractory patients with indication for treatment	48%	14%	19%	9%	18%	27%	32%	81%

Genomic aberrations provide insights into the pathogenesis of the disease since they point to loci of candidate genes (17p13: p53; 11q22-q23: ATM) and identify subgroups of patients with distinct clinical features. Specific genomic aberrations have been associated with disease characteristics such as marked lymphadenopathy (11q deletion) and resistance to treatment (17p deletion). Moreover, such aberrations define specific subgroups that differ in the rate of disease progression as determined by the time from diagnosis to first treatment and the overall survival time of CLL^ref.
Probabilities of disease progression as assessed by the treatment-free interval in the 5 dominant categories of genomic aberrations. The median treatment-free intervals for the 17p deletion (n = 23), 11q deletion (n = 56), 12q trisomy (n = 47), normal karyotype (n = 57), and 13q deletion (single abnormality; n = 117) groups were 9, 13, 33, 49, and 92 months, respectively :

Estimated survival probabilities from the date of diagnosis in 325 CLL patients divided into the 5 categories defined in a hierarchical model of genomic aberrations in CLL. The median survival times for the 17p deletion (n = 23), 11q deletion (n = 56), 12q trisomy (n = 47), normal karyotype (n = 57), and 13q deletion (as single abnormality; n = 117) groups were 32, 79, 114, 111, and 133 months, respectively.
Relation of V_H mutation status and genomic aberrations in 300 CLL cases^ref :

	VH mutated	VH unmutated	P-value at Fisher’s exact test
clonal aberrations	80%	84%	0.37
13q14 deletion appear to have a more favorable outcomeref	65%	48%	0.004
13q deletion single	50%	26%	< 0.001
trisomy 12	15%	19%	0.44
11q23 deletion have been associated with a shorter median survivalref1, ref2, ref3, ref4, ref5, ref6	4%	27%	< 0.001
17p deletion	3%	10%	0.03
17p or 11q deletion	7%	35%	< 0.001

V_H mutation status and genomic aberrations are 2 separate genetic parameters of prognostic relevance, but they appear to be correlated. Unfavorable aberrations (11q-, 17p-) occur more frequently in V_H unmutated tumors, and favorable aberrations (13q-, 13q- single) occur more frequently in the V_H mutated subgroup^{ref1, ref2, ref3}. This unbalanced distribution of genomic aberrations emphasizes the different biological background of the CLL subgroups with mutated or unmutated V_H and could in part explain their different clinical course. On the other hand, about 66% of the V_H-unmutated CLL cases show no unfavorable genomic aberrations, indicating a differential influence of these factors.
Incidence of genomic aberrations and V_H mutation status in one large single center fluorescence in situ hybridization (FISH) study compared with preliminary results from prospective multicenter trials of the German Chronic Lymphocytic Leukemia (CLL) Study Group (GCLLSG) for different clinical situations.
• gene abnormalities (p53 and ATM)
• CLL unmutated cases showed a strikingly higher level of CLLU1 expression (several hundred fold) compared with mutated ones, which in turn showed a median 5.7-fold up-regulation compared with normal B cells.
• the mutation status of the variable segments of immunoglobulin heavy chain genes (V_H), or surrogate markers for these factors (CD38, ZAP-70, LPL, etc.)^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9} (Orchard J, Davis Z, Ibbotson R, Richards S, Catovsky D, Oscier D. Comparison of ZAP-70, IgVH gene mutational status and CD38 in CLL patients requiring therapy: preliminary report from the UK CLL IV trial [abstract]. Blood. 2003;102:#105). Although early studies of the IgH gene in CLL failed to demonstrate somatic mutation^{ref1, ref2, ref3, ref4}, more recent analyses have shown that a substantial (20-50%) percentage of cases show somatic mutations, suggesting that CLL may arise from either pregerminal center naive B-cells, or germinal center exposed B-cells^{ref1, ref2, ref3, ref4, ref5}. These studies also suggest that the presence or frequency of somatic mutation correlates with immunophenotype^ref, the usage of variable region gene (V_H) families^ref and the presence of specific chromosomal abnormalities^ref. Fais et al^ref reported somatic mutation in 50% of the IgM⁺ expressing cases, with the highest incidence noted in the V_H3 family, compared with V_H1 and V_H4. Oscier et al^ref noted that cases with trisomy 12 lacked somatic mutation, whereas cases with 13q14 deletion showed significant levels of somatic mutation. The existence of CLL lymphocytes having undergone somatic mutation suggests that a subset of CLL is derived from memory B cells that have passed through the germinal center stage of B-cell differentiation. Taken together, the above observations indicate that CLL is more heterogeneous than previously thought and may develop either from ontogenically naive B lymphocytes or from a more mature antigen-exposed memory B cells. The mutation status of the V_H genes is one of the most important molecular genetic parameters defining pathogenic and prognostic subgroups of CLL^{ref1, ref2} :
• pregerminal center, unmutated (V_H homology > 98%) (50%) : unfavorable course with rapid progression (estimated median survival time = 79 months)
• germinal center, mutated (V_H homology < 98%) (50%) : slow progression and long survival (estimated median survival time = 152 months)
However, genome-wide gene expression profiling studies revealed a surprisingly homogeneous pattern of gene expression in both subtypes of CLL with only a limited set of genes being differentially expressed in the subgroups^{ref1, ref2}. Most importantly, it could be demonstrated that the V_H mutation status is clinically highly relevant^{ref1, ref2}. Survival curves for patients distributed over all stages (n = 300) and separately for patients diagnosed with Binet stage A disease (n = 189) from the largest published cohort^ref.
• independent of the mutation status, the usage of specific V_H genes such as V_H3-21 may be associated with an inferior outcome^ref. IGHV3-21-using CLL is a distinct entity with restricted Ig gene features and poor prognosis, more frequently encountered in Northern than Southern Europe. To further investigate this subset and its geographical distribution in the context of a country (Italy) with both continental and Mediterranean areas, 37 IGHV3-21 CLLs were collected out of 1076 cases enrolled by different Institutions from Northern or Central-Southern Italy. 18/37 cases, identified as homologous(hom)HCDR3-IGHV3-21 CLLs, were found almost exclusively (16/18) in Northern Italy, while 19 non-homHCDR3-IGHV3-21 cases were similarly distributed throughout Italy. Clinically, poor survivals were documented for IGHV3-21 CLLs as well as for subgroups of mutated and homHCDR3-IGHV3-21 CLLs. Negative prognosticators CD38, ZAP-70, CD49d, CD79b were expressed at higher levels in homHCDR3 than non-homHCDR3-IGHV3-21 cases. Differential gene expression profiling (GEP) of 13 IGHV3-21 vs. 52 non-IGHV3-21 CLLs, identified, among 122 best-correlated genes, TGFB2 and VIPR1 / PACAPR2 as down- and up-regulated in IGHV3-21 CLL cases, respectively. Moreover, GEP of 7 homHCDR3 vs. 6 non-homHCDR3-IGHV3-21 CLLs yielded 20 differentially expressed genes, WNT16 being that expressed at the highest levels in homHCDR3-IGHV3-21 CLLs. Altogether, IGHV3-21 CLLs, including those with homHCDR3, had a peculiar global phenotype in part explaining their worse clinical outcome^ref
To make the estimation of prognosis based on the V_H status accessible to the routine hematology laboratory, surrogate markers for the V_H status were identified :
• originally, a correlation was observed between the V_H mutation status and CD38 expression of the CLL cells pointing to CD38 expression as a prognostic marker^ref
• based on genome-wide gene expression studies other surrogate markers such as ZAP-70 expression were identified and validated^{ref1, ref2}. ZAP-70 expression appears to strongly correlate with V_H mutation status and was therefore a strong prognostic marker in a pivotal study^ref.
However, for both CD38 and ZAP-70, subsequent studies have yielded controversial results with regard to their validity as a surrogate marker for V_H and prognostic indicator. The facts that ... :
• divergent results have been obtained in different laboratories (CD38 and ZAP-70)
• the expression level may change over time (CD38)
• a careful separation of T cells is necessary (ZAP-70)
• different cut-off values to distinguish "positive" from "negative" cases were defined (CD38 and ZAP-70)
• approximately 10–30% of cases show discordant status for CD38 or ZAP-70 as compared to V_H in all series described
... indicate that these markers may not be as reliable as initially thought for routine diagnostics^{ref1, ref2, ref3, ref4} (Orchard J, Davis Z, Ibbotson R, Richards S, Catovsky D, Oscier D. Comparison of ZAP-70, IgV_H gene mutational status and CD38 in CLL patients requiring therapy: preliminary report from the UK CLL IV trial [abstract]. Blood. 2003;102:#105). Furthermore, the relationship of the V_H mutation status to other biological disease characteristics of potential pathogenic relevance such as ongoing V_H hypermutation as well as VDJ diversification, telomere length, ability for BCR signaling, expression of specific genes such as AID, and genomic aberrations are currently undergoing examination. Levels of F-actin-capping protein b subunit (CAPZB), 14-3-3b protein / YWHAB, and laminin-binding protein precursor / lectin, galactoside-binding, soluble, 3 (LGALS3) were significantly increased in mutated B-CLL relative to unmutated B-CLL. In addition, primary sequence data from tandem mass spectrometry showed that nucleophosmin was present as several protein spots in M-CLL but was not detected in unmutated B-CLL samples, suggesting that several post-translationally modified forms of nucleophosmin vary between these 2 sample groups. No specific differences were found between CD38⁺ and CD38^- patient samples using the same approach^ref.
IgG⁺ CLL is phenotypically similar to mutated IgM⁺IgD⁺ CLL (M-CLL) and variably expressed CD38 (4 of 14). ZAP-70, a tyrosine kinase preferentially expressed in unmutated CLL, was found in only 2 of 14 cases. The ability to signal via surface IgM (sIgM) varies between the main subsets of CLL and is associated with expression of ZAP-70. In IgG⁺ CLL, 9 of 14 responded to engagement of sIgG with no apparent requirement for expression of CD38 or ZAP-70. However, signal capacity correlated with intensity of sIgG expression. Most switched immunoglobulin variable region genes were somatically mutated without intraclonal variation, and no case expressed activation-induced cytidine deaminase. Derivation from a postgerminal center B cell is, therefore, likely, and a relationship with M-CLL is suggested. This is supported by a shared biased usage of the V4-34 gene. Similar bias in normal B cells developed with age, providing an expanded population for transforming events. However, conserved sequences detected in the CDR3 of V4-34-encoded gamma chains were not found M-CLL, indicating no direct path of isotype switch from M-CLL. IgG⁺ CLL is likely to arise from an age-related expanded pool of B cells, on a path parallel to M-CLL, and perhaps with a similar clinical course^ref
• longer survival in individuals heterozygous for 1513AC P2X₇ SNP, expecially in patients with mutated V_H genes^ref. P2X₇ receptor triggers IL-1 maturation and exteriorization^ref
• ongoing CSR occurs in only a fraction of the CLL clone, as only small proportions of CD5⁺CD19⁺ cells express surface IgG or IgA and lack IgM and IgD.

• A) among all 300 patients estimated median survival times were: 17p- 30 months, 11q- 70 months, V_H  98% 89 months. and V_H < 98% not reached (54% survival at 152 months)
• B) in Binet A patients only (n = 189) estimated median survival times were: 17p- 36 months, 11q- 68 months, V_H  98% 86 months months, and VH < 98% not reached (52% survival at 152 months).

• according to V_H mutation status :


• according to genomic aberrations :

• Richter's syndrome (RS)) (4-10%, high malignancy; Richter MN. Generalized reticular cell sarcoma of lymph nodes associated with lymphatic leukemia. Am.J.Pathol. 6:285-292 (1928)) :
• DLBCL (most) : despite DLBCL is generally thought to arise from the B-CLL clone, approximately 50% of the patients had genetically unrelated cancers^ref
• Hodgkin’s disease variants (0.4%) arising probably only in V_H mutated CLL^ref : presenting features included B-symptoms (67%), lymph node enlargement (79%), splenomegaly (43%), hepatomegaly (29%), hypercalcemia (6%), infection (6%), and mental status changes (6%). The median age was 72 years (range, 49-81 years), and there was a male preponderance (78%). The median time from CLL to HL diagnosis was 4.6 years (range, 0-12.9 years). 14 patients (78%) had been previously treated for CLL/SLL. 10 patients (56%) had received >1 prior therapy. The median b₂-m level was 4.5 mg/L, and the median LDH level was 610 IU/L. EBV was positive by in situ hybridization for EBV-encoded RNA in 3 of 4 tested patients. 14 patients (78%) received chemotherapy. The overall response rate was 44% (complete response rate, 19%). The median overall survival duration was 0.8 years (range, 0.03 years-6.7(+) years). The median failure-free survival (FFS) duration was 0.4 years. The rates of response, survival, and FFS in patients with Hodgkin transformation of CLL/SLL were inferior to those reported in patients with de novo HL and were similar to those in patients with Richter syndrome^ref
Approximately 50% have an age > 60 years, HGB level < 11 g/dL, received > 2 prior therapies, LDH levels > 1.5 time the ULN, and tumour size >5 cm. Approximately 40% of patients with RS have platelet counts < 100,000/ml, their time to transformation was > 5 years, and b₂-m levels were 3 times the ULN. 21% had a PST >1. Most subjects had prior treatment, including chemotherapy alone (61%), chemotherapy + rituximab (36%) and immunotherapy alone with rituximab and alemtuzumab (3%). The large cells of RS may arise through transformation of the original CLL clone or represent a new neoplasm. RS may be triggered by viral infections, such as HHV-4 / EBV.
Laboratory examinations :
• cytogenetics : trisomy 12 and chromosome 11 abnormalities, as well as multiple genetic defects, have been described in patients with RS. These abnormalities may cause CLL cells to proliferate and, by facilitating the acquisition of new genetic abnormalities, to transform into RS cells. The therapeutic strategies for RS typically include therapies developed for NHL or acute lymphoblastic leukemia. The reported response rates with these therapies are 5% to 43%, and the median survival duration ranges from 5 to 8 months. The median OS of patients with RS is 9.1 months (95% confidence interval, 7.8 to 11 months), and the median FFS duration is 7.1 months (95% confidence interval, 5.1 to 10.4 months)
• molecular analysis of the IgH confirmed that the CLL and the RS had a different clonal origin. Subsequent molecular analyses of stored samples showed that the clone with transforming capacity already appeared 2 years before the clinical appearance of the RS. Alemtuzumab promoted the uncontrolled growth of the latest clone by eradicating the initial B-CLL clone efficiently, and by inducing a strong T cell depletion with consequent impairment of the immunosurveillance. EBV was negative^ref
Therapy : while ORRs to various therapeutic strategies can vary between 5% and 43%, median survival for RS patients with cytotoxic therapy is 5 to 8 months. Cytoreductive therapy consisting of chemotherapy and immunotherapy, followed by allogeneic HSCT, as postremission therapy. As part of a program aiming to cure RS, clinical trial of oxaliplatin, fludarabine, and cytarabine in combination with rituximab are running and recommend postremission therapy, including allogeneic HSCT in patients with available donors^ref. PET/CT can detect Richter's transformation of CLL to DLBCL with a high sensitivity and a high negative predictive value^ref. Some patients underwent subsequent allo-HSCT in remission (7%), as salvage (6%) or as auto-HSCT in remission (2%). There were no significant differences between the chemotherapy and chemotherapy/rituximab groups for either mean complete response (11% vs. 13%, respectively) or mean overall response (34% vs. 47%, respectively). The combination of these treated RS patients gave a complete response of 12% and an overall response of 39%, with a median FFS of 7.1 months (95% confidence interval [CI], 5-10 months). Median OS was 9.1 months (95% CI, 8-11 months) for the full group of 143 RS patients. When divided according to subsequent transplantation, the patients that received allo-HSCT in remission showed significantly (P =.004) greater survival than both the therapy responders without SCT and those that received HSCT. In multivariate analysis, of the large range of significant factors for response and survival in the previously treated patients, the significant risk factors for poor survival were PST > 1 (P =.006), LDH level > 1.5 times the ULN (P =.003), platelet count below 100,000 (P =.012), tumour size > 5 cm (P =.022) and > 2 prior therapies (P =.024). However, when the SCT was included in this multivariate analysis, platelet count lost its significance, and the absence of allo-SCT in remission became a significant risk factor for poor survival (P =.002). As the use of chemotherapy with or without immunotherapy followed by allo-SCT resulted in improved survival when compared with the other therapies used in this trial, allogenic HSCT should be offered to all RS patients with available donors as post-remission therapy
• B-cell prolymphocytic leukemia (B-PLL) (5-10%; 60% of all PLLs)

Epidemiology : male-to-female ratio 4:1, age > 60 years; 20-fold lower incidence than B-CLL; a relatively rare form of chronic lymphoproliferative disease, first described by Galton in 1974^ref. Although PLL is considered to be distinct from CLL by many criteria, the delineation between the 2 is not clearcut because some cases of CLL can transform into PLL^ref and most PLL cases studied appeared to evolve from the B-CLL clone^ref. Furthermore, mixed forms of CLL-PL have been identified in which the number of prolymphocytes is 10-55%^ref
Symptoms & signs : massive splenomegaly and only rarely lymphadenopathy
Laboratory examinations :
• k (70%) > l (30%)
• according to French-American-British (FAB) diagnostic criteria, the hallmark of the disease is the presence of > 55% (or > 15,000/ml) circulating morphologically identified prolymphocytes^ref. It is a distinct clinicopathological entity characterized by massive splenomegaly without lymph node enlargement, frequent marked hyperleukocytosis (> 150,000/ml in 60%; the majority of which are prolymphocytes), and associated anemia and thrombocytopenia. Morphologically, prolymphocytes are large cells with condensed nuclear chromatin and a prominent central nucleolus. The vast majority of PLL are from the B-lineage origin and express high density monotypic sIg, usually IgM with or without IgD, and the CD19, CD20, and CD22 B cell marker (Catovsky D: Prolymphocytic leukemia, in Catovsky D, Foa R (eds): The Lymphoid Leukemia. London, UK, Butterworths, 1990, p 438).
• immunophenotype : CD5^-/+(30%)19⁺20⁺FMC7⁺21⁺22⁺23^-30^- sIg^hi
Prognosis is often poor (median survival = 24 months)
• acute leukemias (rare) :
• B cell acute lymphoblastic leukemia (B-ALL) (rare) : there is genetic relatedness to the B-CLL clone in some cases and unrelatedness in others^ref
• multiple myeloma : there is genetic relatedness to the B-CLL clone in some cases and unrelatedness in others^ref
• acute myeloid leukemia (due to chemotherapy)

• chlorambucil 0.1 mg/kg/day PO daily is the oldest and best-known treatment for CLL. Emetogenic potential : level 1. 66 second malignancies in chlorambucil arm over 72 months. 48 second malignancies in observation arms over 67 months. It can induce partial remissions (PR) in 60–70% of previously untreated patients, but no significant complete remissions (CR)^ref
• chlorambucil+prednisone :
• chlorambucil 0.3 mg/kg/day PO days 1-5, prednisone 40 g/m2/day PO days 1-5. Repeat cycles every 28 days. Emetogenic potential days 1-5 level 1^ref
• chlorambucil 30 mg/m² PO day 1, prednisone 80 mg/day PO days 1-5. Repeat cycle every 14 days. Severe/life-threatening hematologic toxicity (25%), infection (7%), fever (2%), severe vomiting (3%). Emetogenic potential : day 1 level 1. Infectious mortality : 2%^ref
• alkylator/anthracycline combinations are frequently used with similar and perhaps better responses to those seen with chlorambucil, but no change in survival^ref
• CVP (cyclophosphamide, vincristine, prednisone)
• ChOP (cyclophosphamide, doxorubicin, vincristine, prednisone)
• purine analogues are nucleoside analogues that inhibit DNA polymerase and ribonucleotide reductase, promoting apoptosis^ref.
• fludarabine has been the most widely tested and used nucleoside analogue in CLL.
• the efficacy of fludarabine was studied in a large North American intergroup trial in which more than 500 patients were randomized to receive fludarabine, chlorambucil, and fludarabine and chlorambucil. Overall response (OR) favored the fludarabine group at 63% (20% CR + 43% PR) versus 37% (4% CR + 33% PR)^ref. The median duration of remission and the median progression-free survival (PFS) in the fludarabine group were 25 months and 20 months, respectively, whereas both values were 14 months in the chlorambucil group (P < 0.001 for both comparisons)
• Leporrier et al also reported their results in patients with previously untreated CLL treated with either fludarabine or an anthracycline-containing regimen (CAP [cyclophosphamide, doxorubicin, prednisone] or ChOP (cyclophosphamide, doxorubicin, vincristine, prednisone)). 938 patients (651 stage B and 287 stage C) were randomized in 73 centers. Compared to ChOP and fludarabine, CAP induced lower overall remission rates (58.2%; ChOP, 71.5%; fludarabine; 71.1%; P < .0001 for each), including lower clinical remission rates (CAP, 15.2%; ChOP, 29.6%; fludarabine, 40.1%; P = .003). By contrast, median survival time did not differ significantly according to randomization (67, 70, and 69 months in the ChOP, CAP, and fludarabine groups, respectively)^ref.
• fludarabine 25-30 mg/m²/day IV days 1-5 (phase 1/2 trial, 2 dose levels). Repeat cycle every 28 days. Grade 4 neutropenia (56%), FUO/minor infection (13%), pnemonia+/-septicemia (9%), thrombocytopenia (25%), stomatitis (2%), neuropathy (4%), nausea (3%), diarrhea (2%) => infectious mortality (3%). Emetogenic potential : days 1-5 level 1^ref
An important observation in all these trials is the higher percentage of CRs seen in the fludarabine treated groups. Fludarabine is well tolerated with major side effects being hematologic and immunologic toxicities. Also seen is autoimmune hemolytic anemia (AIHA) and, at very high doses, significant neurotoxicity. The issue of treating patients with fludarabine who have a history of AIHA or ITP, either primary or secondary to previous purine analogue therapy, is a topic of continued controversy (Montillo M, Tedeschi S, O’Brien SM, Lerner S, Morra E, Keating MJ. Autoimmune phenomena against hematopoietic cells and myelosuppression in CLL treated with fludarabine - including regimens as front-line therapy [abstract]. VIII International Workshop on CLL Programme and Abstract Book. 1999;54. Abstract P091; Hall R, Bolan S, Orchard J, Oscier D, Myint H, Hamblin TJ. Autoimmune phenomena following treating with fludarabine [abstract]. VIII International Workshop on CLL Programme and Abstract Book. 1999;58, Abstract P103). The decision to treat these patients should be done with extreme caution and close monitoring.
• cladribine (2-CdA) : there is evidence that produces similar responses as fludarabine in both previously treated and untreated populations. Juliusson and Liliemark reported a 31% CR and 27% PR in 52 patients with previously treated CLL, with a median PFS of 23 months for CR patients and 16 months for PR patients. Toxicities were similar to those observed with fludarabine^ref. Recently, Robak et al reported their experience with 2-CdA on 378 patients with CLL. 194 patients were previously untreated and 184 had relapsed or refractory disease. Results showed a CR of 45.4% and a PR of 82.5% in the previously untreated group with a median survival of 19.4 months, and a CR of 12.5% and PR of 48.4% in the previously treated group with a median survival of 16.3 months^ref. Ckadruvrube 0.12 mg/kg/day IV continuous infusion days 1-5. Repeat cycle every 28 days. Grade 3-4 neutropenia (9%), severe infections (13%), minor infections and FUO (34%), thrombocytopenia (23%) => infectious mortality (7%), hemorrhagic mortality (6%). Emetogenic potential days 1-5 level 1^ref
• cladribine (2-CdA) + prednisone: cladribrine 0.12 mg/kg/day IV over 2 hours days 1-5, prednisone 30 mg/m2/day PO days 1-5, repeat cycle every 28 days for 3 courses, grade 3-4 neutropenia (9%), anemia grade 1-2 (6%), grade 3-4 (2%), FUO/infection (56%), thrombocytopenia (9%), eosinophilia (9%), autoimmune hemolytic anemia (AIHA) (6%: related-deaths in 3% of patients), hepatic (6%), skin reaction (6%), CNS (2%), diarrhea (2%), nausea/vomiting (2%), neuropathy (2%) => herpes zoster reactivation in 21% of patients, herpes simplex infections in 11% of patients; URI or pneumonia in 30% of patients. Emetogenic potential : days 1-5 level 1^ref
The accumulating 2-CdA data is provocative but not much different from the fludarabine data. Unfortunately, crossresistance is seen among all the nucleoside analogues, so a sequential treatment algorithm is unlikely. Reports of synergism between alkylators and nucleoside analogues by alkylators inducing DNA damage and nucleoside analogues inhibiting DNA repair prompted trials combining these 2 modalities^ref (Koel U, Li L, Nowak B. Fludarabine and cyclophosphamide: synergistic cytotoxicity associated with inhibition of interstrand cross-link removal [abstract]. Proc Am Ass Cancer Res. 1997;38:2). Early reports of the combination of fludarabine + cyclophosphamide showed higher OR rates than fludarabine alone in previously treated patients with alkylators or fludarabine^{ref1, ref2, ref3} (O’Brien SM, Kantarjian H, Beran M. Fludarabine and cyclophosphamide therapy in chronic lymphocytic leukemia [abstract]. Blood. 1996;88:480). Fludarabine and cyclophosphamide produced an increase in OR and CR, and it improved PFS in patients with previously untreated CLL compared with fludarabine alone and was not associated with an increase in infectious toxicity^ref.
• cyclophosphamide 300 mg/m²/day IV over 1 hour days 1-3, fludarabine 30 mg/m²/day IV over 30 minutes days 1-3. Repeat cycle every 28-42 days. Neutropenia grade 3 (75%), grade 4 (48%), fever/infection (40%), thrombocytopenia (19%), nausea/vomiting (6%), fatigue/aches (2%), rash (2%) => documented sepsis or pneumonia (25%), FUO or neutropenic fever (25%), 6 atypical infections were reported, herpes zoster infection (5$), reactivation of herpes simplex (8%), 29% of patients at the 300 mg/m2 dose level required a dose reduction of cyclophosphamide^ref
• fludarabine + cyclophosphamide + filgrastim:  in a multicenter Phase II study conducted by ECOG, 36 patients received cyclophosphamide 600 mg/m² intravenous (iv) day 1 and fludarabine 20 mg/m² IV days 1 through 5, followed by filgrastim 5 mg/kg subcutaneous starting approximately day 8. 15 of 36 (41.7%) evaluable patients achieved complete response and 8 (22.2%) achieved a partial response, giving an OR rate of 63.9% (90% CI: 48.4%, 77.2%) (Flinn IW, Jemiai Y, Bennett JM, et al. Fludarabine and cyclophosphamide achieves high complete response rate in patients with previously untreated chronic lymphocytic leukemia: E1997 [abstract]. Blood. 2001;98:633a)
• Preliminary results from a North American Intergroup trial comparing this combination regimen to single-agent fludarabine revealed the combination produced a superior CR rate compared to fludarabine alone, 23% versus 6%, respectively
• Alternative regimens using 3-day schedules of fludarabine and cyclophosphamide have produced encouraging results. The German CLL Study Group noted a higher CR rate with fludarabine and cyclophosphamide than with fludarabine alone (20.2% vs 8.6%) (Eichhorst B, Busch R, Hopfinger G, et al. Fludarabine + cyclophosphamide induces higher remission rates and longer progression free survival than fludarabine alone in first line therapy of advanced chronic lymphocytic leukemia: results of a Phase III study (CLL4 Protocol) [abstract]. Blood. 2003;102:72a). While these CR rates were disappointingly low in both arms, the difference in response translated into a longer PFS in the combination arm. In a single center Phase II arm study, CR rates were not found to be substantially increased. However, the quality of these CRs was improved, since only 8% of CR patients demonstrated residual CD5⁺ cells in the marrow compared with 33% after fludarabine and prednisone^ref.
Taken together these studies suggest that the combination of fludarabine and cyclophosphamide improves CR in patients with previously untreated CLL. However, it is not clear as to what regimen might serve as the best backbone by which to add new agents such as rituximab. Numerous other fludarabine combination regimens have been tested with promising results in Phase II trials^{ref1, ref2, ref3}.
• single-agent antibodies :
• anti-CD20 mAbs : rituximab has afforded excellent responses in patients with B-cell lymphomas. Early disappointing results in small lymphocytic leukemia (SLL)/CLL from the pivotal trial led to skepticism about its ultimate utility in CLL. However, subsequent studies examining alternative schedules, doses, and patient populations have clearly demonstrated rituximab’s efficacy in CLL. One explanation for the meager response rates in patients with SLL is the low serum trough levels seen in these patients, which are known to be inversely related to response rates. To overcome the apparent altered pharmacokinetics, the antibody has been given thrice weekly for 4 weeks at standard doses (375 mg/m²) as well as very large single doses weekly for 4 doses. The thrice weekly regimen has resulted in 45% OR rate in pat