Chronic Lymphocytic Leukaemia (CLL)

Chronic lymphocytic leukaemia(CLL)is a common adult leukaemia that results from proliferation of neoplastic B- lymphocyte clones. The disease is most frequent in patients over the age of 60 and has a variable clinical course. A variety of genetic aberrations can drive the leukaemia and also initiate the transformation into high grade malignancy.
Diagnosis of CLL is usually straight forward. Most cases are usually suspected by the presence of peripheral blood lymphocytosis of small mature lymphocytes with scant cytoplasm. Smear cells are typically feature of the leukaemic cells due to increased cellular fragility. International Workshop on Chronic Lymphocytic Leukaemia criteria for diagnosis require >5x10^9/L of clonal B lymphocytes. The clonality of the circulating b cells must be confirmed with Immunophenotyping by flow cytometry(1). When a clonal population of B

lymphocytes with an immunophenotype consistent with CLL are identified in association with lymphadenopathy or splenomegaly the diagnosis of small lymphocytic lymphoma(SLL) can be made. Histopathological confirmation of an SLL diagnosis should be made when biopsy is possible(1).
Typically CLL has an indolent progression and patients with the malignancy may survive for many years even without treatment. The “watch and wait” approach is often taken because the usually slow asymptomatic progression of the disease. Meta-analysis of early intervention chemotherapy does not show improved survival which support this conservative approach to treatment. Over time, the cell size, lymphocytosis and proportion of pro-lymphocytes may increase as proliferation activity does. Clinical staging using Rai(2) or Binet(3) staging systems are often used to aid in the decision to initiate treatment. However a proportion of CLL will take an aggressive course resulting in markedly reduced survival times. This can be due to mutations that increase the rate proliferation/reduce the rate of apoptosis or initiate the transformation into diffuse Large B-Cell lymphoma (DLBCL) or B-cell Pro-lymphocytic Leukaemia(B-PLL) or Hodgkins Lymphoma(HL).
Identifying the patients who are at increased risk of rapidly progressing CLL early is necessary in order to maximise their overall survival. Numerous clinical and laboratory markers have been examined to in the prognostication of this disease. Currently clinical staging, haematological and biochemical markers, immunophenotype, cytogenetic and genetic abnormalities are considered in determining the prognosis of a patient with CLL.

What is CLL
How is it diagnosed
CLL
PB – IWCLL update criteria (1)
FLOW – Matutes score
What are it’ s clinical progression pathways
What are the treatments for CLL and what are there advantages and disadvantages
There are clinical and laboratory markers for determining prognosis for CLL
Clinical Staging.
Clinical staging systems of CLL have been in use since the 1970’s. Rai’s system stratifies CLL patients into 5 different stages(0-IV), based upon physical examination (for lymphadenopathy, hepatomegaly or splenomegaly) and basic haematology tests(haemoglobin and platelet count , (2) The system staging is summarised in TABLE XXX.
Rai Lymphocytosis Lymph Node Enlargement Spleen/Liver Enlargement Hemoglobin < 11 g/dL Platelet < 100 x 109/L Survival (years)
________________________________________
0 Yes No No No No > 13
I Yes Yes No No No 8
II Yes ± ± No No 5
III Yes ± ± Yes No 2
IV Yes ± ± ± Yes 1

The Rai system defines low risk disease as stage 0, intermediate risk as I and II and high risk as III and IV. Although the system is simple, and was created prior to immunophenotyping and advanced genetic techniques used today, it still maintains its clinical value and its use is still widespread.
In 1981 Binet proposed a similar system with 3 catergories(4).The presence of thrombocytopaeinia or anaemia results in a C group classification, which indicates the poorest prognosis. In the absence of anaemia or thrombocytopaenia, the number of sites(nodes/organs) with evidence of involvement is used to determine stage A or B. Three or greater sites of involvement is classified as B, which has an intermediate prognosis and less than 3 site of involvement is classified as A, which has the best prognosis.
Binet Lymphocytosis Lymph Node/organ Areas Hemoglobin < 11 g/dL Platelet < 100 x 109/L Survival (years)

A N/A < 3 No No 12
B 3 or > No No 5
C ± Yes (or low plt) Yes (or low Hb) 2

The 2 classification systems whist similar in assessment criteria, differ in the proportion of patients classified into their respective good, intermediate and poor prognostic groups(5) See table XXX. Both systems require disease progression to classify the respective prognositic groups. If the disease is to be classified as intermediate or poor, the survival times are more predictable. However the good prognostic groups do not separate non/slow progressive CLL from early stages of an aggressive CLL. Up to 1/3 of patients classified as RAI 0 and Binet A will die from the disease within 10 years. A recent study has shown RAI and Binet systems are poor predictors of individual outcomes, demonstrated, by low C-statistics of 0.56 and 0.58
FIGXXX overall survival of CLL patients according to Binet and Rai staging systems(6)

respectively.
?KEEP Good
Rai 0
Binet A Intermediate
Rai I/II
Binet B Poor
Rai III/IV
Binet C
Rai 31% 59% 10%
Binet 63% 30% 7%
The value of the clinical staging systems is to assist in determining when to initiate treatment. Early treatment of Rai 0 or Binet A CLL does not improve long term survival(7) and may in some cases reduce overall survival(5). For this reason a “watch and wait” approach is taken for many patients with good prognostic indicators. International Workshop on Chronic Lymphocytic Leukaemia(IWCLL) 2008 guidelines suggest intermediate and poor prognosis CLL should have criteria additional fulfilled prior to initiating treatment(8). Some of these include features include in the Rai or Binet, such as anaemia, thrombocytopaenia, lymphadenopathy and splenomegaly. Other criteria include; rapidly increasing lymphocytosis, presence of autoimmune anaemia or thrombocytopaenia that is unresponsive to corticosteroids and persistence of “B” symptoms(weight loss, fever, night sweats and fatigue).
Haematological Parameters
One of the first prognostic indicators used in the assessment of CLL was the absolute lymphocyte count. High absolute counts are typically associated with poorer prognosis (9). However there is no consensus on an exact prognostic cut off for lymphocyte counts. Serial Lymphocyte counts are often used to determine the time taken for the lymphocyte count to double (lymphocyte doubling time or LDT). The LDT is of greater value for prognosis and determining treatment initiation because aggressive CLL with high proliferation rate can be differentiated from slow progressing CLL with high white cell counts. IWCLL suggest treatment initiation should be considered when there is an increase of greater than 50% in 2 months a lymphocyte doubling time of less than 6months(1). However absolute lymphocyte count or LDT should not be used as the sole criteria for initiating treatment as the lymphocyte count can be elevated due to other factors such as infection.
Morphology – proportion of Prolymphocytes in peripheral blood correlate with poorer outcome- old publication (BAIN 4)

Biochemical Markers
Biochemical markers are often included in the prognostic assessment of CLL and have the advantages of being easy to measure and simple to perform. The tumour marker concentration can correlate with tumour burden and provide an indication of disease progression, however elevations in early stages of the disease can indicate prognostic information and treatment efficacy.
Lactate dehydrogenase(LDH) is a ubiquitous intracellular enzyme that can be elevated in many diseases including lymphoproliferative diseases (including CLL), haemolysis, myocardial infarction and liver disease.. LDH is released into the plasma during the lysis of cells and is elevated when there is a high turnover of cells. Despite LDH’s lack of specificity, it is frequently used in the assessment of CLL and has high levels(>1.5upper limit of normal) in the early stages of the disease have been found to correlate with poor prognosis and increased risk of Richter’s transformation(10, 11).
B2 Microglobulin (B2M) is a small membrane protein associated with the heavy chains of the MHC1 found on the surface of all cells. Like LDH, increased cell turnover results in increases plasma concentrations. High levels of B2M(2mg/dL)in early stages of CLL (Binet A) correlates with significantly shorter progression free survival(12).
TABLE XXX Comparison between β2-mneg patients versus β2-mpos patients from a study of 222 Binet Stage A CLL patients(12)
Thymidine kinase(TK) is an intracellular enzyme, which is expressed during cell division and normal serum levels are usually very low. In haematological malignancies, including CLL, serum levels can be dramatically increased. The concentration of TK correlates with leukaemic proliferation of CLL patients and increases >15U/L have demonstrated to be a strong predictor of mutational status and aggressive disease progression(13, 14). TK levels have also been shown to correlate treatment response to Fludarabine. One study demonstrated that TK >10U/L were associated with poor response to fludarabine, with only 45% of patients responding and levels < 10U/L were associated with a good response with 83% patient responding(15).

Flow cytometry
Cyclic ADP ribose hydrolase(CD38) and zeta chain associated protein kinase 70 (ZAP-70)have been extensively investigated for prognostic use in CLL. Originally both these markers were investigated as potential surrogates for Immunoglobulin VH gene(IVGH) mutation status. At the time routine testing for IVGH mutation status was prohibitively expensive and laborious. Both initially showed strong concordance with IVGH mutation status, however more recent studies have indicated the two markers have prognostic significance independent of IVGH status(16, 17).
CD38 is a transmembrane protein that is normally expressed on lymphocytic progenitor in the bone marrow and germinal centre B-Cells but not on mature lymphocytes. It’s role is believed to be related to regulation of cell proliferation and apoptosis(18). CD38 expression has shown strong association with more nodal sites involved, lower haemoglobin and development of autoimmune manifestations(19). Five year overall survival for CD38+ CLL was found to be 40% compared to 75% for the CD38- group(FIGXXX) (20). The Cut off value used to classify CD38+ cells varies in different studies. Some have used arbitrary cut off of 20%(20) or 30%(21) whilst others have determined a cut off of 7% retrospectively based upon the survival time of prognostic sub-populations(22). Another study has shown CD38 positive population in the peripheral blood is a poor prognostic indicator regardless of whether the percentage is above or below the cut off (19). This study also demonstrated there the considerable variability in the proportions of CD38+ CLL cells in the bone marrow and peripheral blood, with the bone marrow showing 28-97% more CD38+ CLL cells.
FIGXXXKaplan meier Survival curves demonstrating significantly reduced survival of CD38pos(20% or more) compared to CD38neg(less than 20%)(20)

ZAP-70 is an intracellular tyrosine kinase that plays a role in T-cell signalling. Normal B- cell expression is typically low(0-6.5%), however in some more aggressive CLL, ZAP-70 expression can be elevated(23). Initial studies showed very high concordance( with unmutated IGVH(23), however subsequent studies with larger cohorts, showed a weaker association(17, 24). However, the association with aggressive disease and poor prognosis has been repeatedly demonstrated (17, 23, 24). see fig XXX
fig XXX A) Probability of progression of patients with high level (>20%)of ZAP-70 expression vs patients with low level (<20%)ZAP-70 expression. B) Probability of survival over 10 years of ZAP-70 positive patients vs ZAP 70 negative(23).
Compared to
Whilst being a powerful prognostic indicator, ZAP-70 has had limited dissemination in diagnostic laboratories. This is due largely due to high levels of variability in the testing methodology. ZAP- 70 is an intracellular marker so requires cellular permeablisation procedure prior to antibody binding. It is also more labile than other markers such as CD38, and for this reason should be tested with in 24hours(24). The expression of ZAP-70 on CLL is relatively weak compared to the strong expression by T-cells and NK-cells, making it essential to have a well defined population of clonal B-cells, to allow separation from the background of normal B-cells(25).
Combined CD38 and ZAP70

Cytogenetic
Cytogenetics allows identification of several chromosomalaberrations that dramatically influence the prognosis and provide guidance for treatment approaches. Conventional cytogenetics has historically had low sensitivity for detecting aberrations in CLL mainly due to the low mitotic rate and poor yield of metaphase cells. The majority of cytogenetic analysis of CLL involves using FISH probes to identify four prognostically significant aberrations(17p-, 13q-, 11q- or trisomy 12) in interphase cells. Approximately 80% of CLL cases will have one of these aberrations(see table XXX) and the overall survival for each genetic subtype is shown in figXXX. It should be noted that since this study was conducted first line treatment has changed to FCR, which has improved overall survival for most types in the medium term. Long term studies examining the overall survival with FCR treatment are ongoing(26).
Table 4. Genetic aberrations in chronic lymphocytic leukemia (CLL), according to Döhner et al.4

CLL Patients Normal 13q Alone 12q Trisomy 11q Deletion 17p Deletion

% of CLL patients 18 55 16 18 7

% Stage A 53 72 51 25 23
% Stage B 30 20 34 50 41
% Stage C 17 8 15 25 36

Overall Survival(months) 120 132 120 84 30

The most prognostically significant chromosomal aberration is the deletion of the short arm of chromosome 17 (17p-), which is seen in 7% of patients.

This mutation is associated with particularly poor outcome and resistance to front-line treatment with fludarabine, cyclophosphamide and rituximab(FCR)(26). In clinical trials, treatment of del(17p) CLL with FCR only achieve complete remission in 5% of patients and overall survival is only 38% at 3years (27). The key gene effected by the 17p deletion is tumour protein p53(TP53), which has an integral role in the induction of apoptosis or cell cycle arrest following DNA damage(28). 80-90% of CLL patients with the 17p deletion have also been shown to have TP53 mutation on the remaining copy, thus disabling the apoptotic pathway in the malignant cells. Interphase FISH is suitable for identifying del(17p) however it does not detect mutations in TP53, which can have the same poor prognosis and treatment refractoriness(29). Between 13.5-26% of CLL cases have dysfunctional/non-functional TP53 gene which is considerable more than the 7% which have

del(17p).
FIG XXX 3 year overall survival of genetic sub-groups treated with FCR(26).
Deletion of the long arm of Chromosome 11 (11q-) is present in 25% of CLL cases and is associated with bulky lymphadenopathy, rapid progression and reduced overall survival (30) .Chromosome 11 harbours ataxia telangiectasia mutated gene(ATM) which encodes for the proximal DNA damage response kinase involved in the p53 apoptosis pathway(31). The residual ATM gene is mutated in 36% of cases with del(11q) which has been shown to have a significantly shorten survival time (median OS 83months) when compared to del(17p) with residual wild type ATM gene(median OS 134months) (31).
Trisomy 12 occurs in approximately 16% of CLL. The role this has in the pathogenesis of CLL is unclear and the prognostic impact is intermediate although recent CLL8 trials have shown good response to FCR with 100% overall response rate and 96% overall survival (27) . Cases with Trisomy 12 typically express high levels of CD20(32), which is the target site of rituximab, which may explain the improved treatment outcomes under this regime.
Deletion of the long arm of chromosome 13 is present in 55% of CLL cases. If present and in the absence of other poor prognostic aberrations, it is associated with a very good prognosis(26, 30, 33). In the deleted region genes encoding micro RNAs ; miR-15a and miR-16-1 are located. Whilst idt is unclear how these contribute to the pathogenesis of CLL, Mouse studies show deletion of these genes results in Monoclonal B-cell Lymphocytosis, CLL and Lymphoma(34).
Interphase FISH has proved very useful in detecting these prognostically significant recurrent abnormalities. However this technique will not detected any aberrations that are do not affect the hybridisation sites of the probes, so rarer and potentially prognostically significant mutations are likely to go undetected. Conventional cytogenetics in the past had proved limited due to the poor mitotic rate of CLL cells. Recent development of immunostimulation techniques using CpG-oligonucleotide DSP30 and IL-2, can improve the metaphase cell yield and improve sensitivity (35, 36) . Whilst this technique is unlikely to supplant FISH, it could provide an option for investigating CLL that do not common aberrations.

Mutated or unmmutated IGVH
IVGDuring normal B-cell development, IVGH genes undergo somatic rearrangement and hypermutation in order to generate diversity in antibody paratope. Cases of CLL can have either mutated (has rearrangement) or unmutated( rearrangement has not occurred) IGVH genes. Unmutated cases orginated from cells that probably underwent malignant transformation prior to entering the germinal centre whilst in mutated cases, the transformation probably occurred after passing through the germinal centre(6). There is a considerable difference in overall survival when the 2 groups are compared. A study of mutation status in Binet A CLL patients showed a median survival of 7.9 years in the unmutated subtype compared to24years in the mutated(37).
Kaplan-Meier Curve comparing Binet A CLL patients with mutated and unmutated IVGH genes.
Biologically it is unclear how the mutation status of CLL has such a marked impact on the progression of the disease. Other poor prognostic indicators are also more frequently seen in the unmutated IVGH. Unfavourable chromosomal aberrations 17p- and 11q- are more frequent in the unmutated sub-type(22) whilst favourable prognostic aberration 13q- is typically only seen in mutated sub-types.
Testing
mutation analysis is a PCR based analysis that compares the variable sequences of the IVGH genes with the germline or reference sequence.
-somatic mutation of IgVh gene is marker of B cell maturation in the follicular germinal center
Mutated incorporation of VH3-21 chain – poor prognosis
IGHV1

Whole genome sequencing investigations of CLL have re-identified mutations associated with the genes affected by chromosomal aberrations such TP53 and ATM, as well as other novel genes, which may contribute to pathogenesis and may be of prognostic importance. Some of these genes include notch 1 (NOTCH1), splicing factor 3b, subunit 1(SF3B1), myeloid differentiation primary response gene 88(MYD88), exportin 1(XPO1), protection of telomeres protein 1(POT1) and Chromodomain helicase binding protein 2(CHD2)(38, 39). Some of these have suspected roles in pathogenesis, but most are currently unclear.
NOTCH1 has been repeatedly identified in whole genome investigations of CLL. This gene encodes a ligand activated transcription factor , that targets multiple genes involved in proliferation and apoptosis including TP53, MYC and molecules of the NF-kappaB pathway(40). NOTCH1 mutations occur in approximately 10% of CLL cases and their frequency increases in advanced disease phases, particularly in Richters Syndrome(41). Multivariate analysis identified NOTCH1 as an independent prognostic marker(Hazard ratio 2.15) with a similar impact on survival time as the TP53 mutation. see fig XXX
fig XXX Comparison of overall survival of CLL cases with NOTCH1 mutation (NOTCH1 M) and TP53 disruption(TP53 DIS) with germline.
SF3B1 gene encodes a core component of cell splicosome and is frequently mutated by missense mutations. It has been detected in both mutated and unmutated IGHV, which suggests it is a possible independent risk factor. It has been associated with an earlier need for treatment(42) and fludarabine refractoriness(40).
MYD88 is an intracellular adapter involved in the activation of NF-Kappa B. Mutation of MYD88 is predominantly seen in conjunction with mutated IGHV and del(13q)(40).
Comprehensive Prognostic Index
Prognostic markers should aid clinician in determining the best clinical course for a patient. In a heterogeneous disease like CLL, which may have numerous and often discordant prognostic markers, interpreting the data and determining the best course of action can be especially difficult. Multiparameter prognostic tools, which collate this data and stratify patients based on overall risk, can simplify the decision making process. A prognostic index has recently been developed, which used multivariate analysis to identify the strongest independent prognostic factors of CLL(43). These independent factors were then ranked bases on Hazard ratio and assigned a risk score(see TABLE XXX)
Independent factor HR* (95% CI†)
P Risk score
Chromosomal aberration Del(17p) 6.0 (4.2 – 8.6) <.001 6
s-TK >10.0 U/L 2.1 (1.5-2.9) <.001 2
s-β2m >3.5 mg/L 2.3 (1.4-3.6) .001 2
s-β2m >1.7 and ≤3.5 mg/L 1.7 (1.1-2.7) .01 1
IGHV MS Unmutated 1.9 (1.5-2.5) <.001 1
ECOG PS >0 1.7 (1.3-2.1) <.001 1
Chromosomal aberration Del(11q) 1.4 (1.03-2.0) .03 1
Sex Male 1.3 (1.01-1.6) .026 1
Age >60 y 1.3 (1.04-1.7) .045 1

The total risk score is then used to determine the risk group (Low, Intermediate, High or Very High)
Risk Score 5y OS%
Low 0-2 95.2
Intermediate 3-5 86.9
High 6-10 67.6
Very High 11-14 18.7

Testing at QEII
At PathWest QEII a selection of different tests are provided to assist in determining of prognosis of CLL. LDH and B2M are routinely tested at a diagnosis and during monitoring of patients with CLL. The lymphproliferative disorder immunophenotyping panel includes markers used for the diagnosis of CLL and CD38 which is used for prognostic purposes. As noted previously, CD38 expression in CLL cells correlates with poor prognosis and decreased overall survival. ZAP-70 is not offered due to the technical difficulties of providing the test.
Cytogenetics testing is routinely limited to interphase FISH for 4 prognostically important chromosomal aberrations. These fish probes include break apart probes for deletions 17p, 13q and 11q. For trisomy 12 a centromeric probe is used. Routine karyotyping is not routinely performed due to the low diagnostic yield. Of all the different tests, 17p/TP53 status has the most significant impact on patient treatment. The 17q deletion is associated with a very aggressive clinical course and poor response to treatment. Common treatment for progressive CLL, fludarabine, cyclophosphamide and rituximab(FCR) is unlikely to be effective in cases of 17p- CLL and alternatives such as Alemtuzumab must be used. Microarray has been considered as an alternative to FISH, however presently it is considerably more expensive and laborious.
IGHV mutation analysis is not currently performed at PathWest. Although the mutation status of the IVGH has a strong association with prognosis, it’s impact on treatment is not fully established. The testing also relies on extraction of mRNA and conversion to cDNA by reverse transcriptase, amplification by PCR then sequencing. The short viability of the sample, the high cost of testing , the high level of technical expertise required and considerable analysis time are some of the reasons why this testing is currently not offered. However with the rapid reduction in the cost of next generation sequencing and the improving data analysis tools, this testing may become feasible in the near future.