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Chronic Lymphocytic Leukemia: A Contemporary Perspective on Diagnosis and Assessment Part 1: Diagnosis, Staging, and Prognosis
Compliments of Bayer HealthCare Pharmaceuticals Inc.

2. Diagnosis: NCI vs IWCLL guidelines for CLL
Variable
NCI
IWCLL
Diagnosis
Lymphocytes (x 109/L)
≥5; ≥ B-cell Marker (CD19, CD20, CD23) + CD5
≥10 + B phenotype or bone marrow involved <10 + both of above
Atypical cells (%)
<55
Not stated
Duration of lymphocytosis
None required
Bone marrow lymphocytes (%)
≥30
>30
Staging
Modified Rai, correlate with Binet
In 1988, the National Cancer Institute–sponsored Working Group (NCI-WG) on CLL published guidelines on diagnosis and response criteria in CLL primarily designed for the conduct of clinical trials. A year later, the International Workshop on CLL (IWCLL) published general practice recommendations for CLL.
In 1996, the original NCI guidelines were revised by Cheson et al and are currently in use although new guidelines are anticipated in the near future.
Some differences between the 2 sets of CLL guidelines are apparent:
A diagnosis of CLL requires a lymphocyte count of 5 x 109/L according to NCI guidelines and 10 x 109/L in the IWCLL guidelines. IWCLL criteria do allow diagnosis with a lower lymphocyte count if the lymphocytes are B cells and the bone marrow is involved.
The revised NCI guidelines suggest lymphocytosis lower than the “greater than 5,000” that appeared in the original guidelines to help distinguish CLL from small lymphocytic non-Hodgkin’s lymphoma. The new guidelines state that retesting at 4 weeks is rarely necessary.
Whereas <55% atypical cells is indicative of CLL, the cut-off value for PLL is controversial.
The IWCLL guidelines recommend that the Rai and Binet staging systems be integrated, and the revised NCI guidelines suggest the use of the modified Rai classification, which reduces the number of groups at risk from 5 to 3.
1. Cheson BD, et al. Blood. 1996;87:
1. Cheson BD, et al. Blood. 1996;87:

3. Diagnosis: NCI vs IWCLL guidelines for CLL
Variable
NCI
IWCLL
Response criteria CR
Physical exam
Normal
Symptoms
None
Lymphocytes (x 109/L) ≤4
<4
Neutrophils (x 109/L)
≥1.5
>1.5
Platelets (x 109/L)
>100
Hemoglobin (g/dL)
>11 (untransfused)
Not stated
Bone marrow lymphs (%)
<30, no nodules
Normal, allowing nodules or focal infiltrates PR
Physical exam (nodes and/or liver, spleen)
≥50% decrease
Downshift in stage
Plus ≥1 of:
>11 or 50%
improvement
Duration of CR or PR
≥2 months
A complete remission according to NCI criteria requires that bone marrow contain fewer than 30% CLL lymphocytes; the criteria recommend that the clinical significance of lymphoid nodules be assessed prospectively.
The criteria for the confirmation of a complete remission are similar for both sets of guidelines although the IWCLL allows focal infiltrates or nodules in bone marrow while the NCI group specifies “no nodules.”
The criteria for partial remission according to IWCLL are limited to a downshift in clinical stage, whereas the NCI group provides more specific hematologic requirements similar to those specified for a complete response.
1. Cheson BD, et al. Blood. 1996;87:
1. Cheson BD, et al. Blood. 1996;87:

4. Staging: Rai and Binet staging systems for CLL
Clinical staging systems for CLL
Stage
Value
Rai
Binet
Median survival
Lymphocytosis (>15,000/mm3) -
150 months (12.5 years)
Lymphocytosis plus nodal involvement I A
<3 node groups
months (8.5-9 years)
Lymphocytosis plus organomegaly II B
>3 node groups
60-71 months (5-6 years)
Anemia (RBCs)
III Hgb <11 g/dL C
Hgb <10 g/dL
19-24 months (1.5-2 years)
Lymphocytosis plus thrombocytopenia (platelets)
IV PLT <100,000/mm3
PLT <100,000/mm3
The clinical staging of CLL is usually done according to the staging of Rai or Binet. The Rai system is more common in the US, whereas the Binet system is prevalent in Europe.
Both systems classify the disease stages according to platelet count, hemoglobin count, and the size of lymph nodes, spleen, and liver.
The original Rai staging system included 5 stages of disease progression. However, it was modified by Rai into 3 categories: Low risk (Rai stage 0), Intermediate risk (Rai stages I and II), and High risk (Rai stages III and IV). This revision is the preferred version according to the modified NCI guidelines.
The predictive nature of both systems is limited by the wide-ranging nature of the clinical course of CLL, which can be unpredictable at the early stage of the disease.
1. Rai KR, et al. Blood. 1975;46: Binet JL, et al. Cancer. 1981;48: Binet JL, et al. Cancer. 1977;40:
1. Rai KR, et al. Blood. 1975;46:
2. Binet JL, et al. Cancer. 1981;48:
3. Binet JL, et al. Cancer. 1977;40:

5. Comparison of CLL and PLL
B-CLL
CLL-PLL
CLL
PLL
slg + ++
CD19
CD20
CD5
-/+
Panel A shows the typical morphology of B-CLL: nuclear chromatin is condensed, nucleoli are inconspicuous, and there is a thin rim of cytoplasm. In addition to the intact CLL cells, there are 2 smudge cells, commonly seen in CLL. These represent naked nuclei and are an artifact of smear preparation.
Panel B demonstrates the typical morphology of a blood smear showing prolymphocytoid transformation of CLL, referred to as CLL/PLL. In addition to a background of typical B-CLL cells, there are larger lymphocytes with finer chromatin, prominent single nucleoli, and more abundant blue cytoplasm. These latter cells are prolymphocytes, which can be found within the growth centers typically seen in histologic sections of B-CLL lymph nodes.
Morphologic features and immunophenotypic characteristics can be used to distinguish between CLL and PLL cells. In some cases, both types of cells are found, and their relative proportions help indicate the diagnosis.
CLL cells are small lymphocytes. In general, they are smaller than 2 red blood cells, and nucleoli are absent. The nuclear outline is regular and smooth, and the chromatin is characteristically clumped. The nuclear/cytoplasmic ratio is high. Immunophenotypically, CLL cells have reduced sIg, and expression of CD20 is characteristically dim. CD19 and CD5 coexpression is typical.
In contrast, PLL cells are larger than CLL cells. Usually, these lymphocytes are bigger than 2 red blood cells, and each cell has a prominent nucleolus. The nuclear/cytoplasmic ratio is lower in PLL cells than in CLL cells. Like CLL cells, PLL cells express CD19, but, in contrast to CLL cells, PLL cells express bright CD20 and bright slg, and CD5 expression is variable.
Courtesy of Randy Gascoyne, MD. 1. Bennett JM, et al. J Clin Pathol. 1989;42:
1. Bennett JM, et al. J Clin Pathol. 1989;42:

6. Immunophenotype scoring system
Scoring system for B-CLL
Membrane marker
Points 1
Smlg
Weak
Moderate/strong
CD5
Positive
Negative
CD23
FMC7
CD79b (SN8)
Until recently, B-CLL cells were differentiated from other B-cell lymphoproliferative diseases by immunophenotyping with 5 cell membrane protein markers: SmIg (Surface Membrane-bound Immunoglobulin), CD5 (T1 antigen), CD23 (The Fc Receptor for IgE), FMC7 (a specific conformation of CD20, possibly multimeric and possibly associated with membrane cholesterol), and CD22 (gp135; a B-cell adhesion molecule).
CD79b (a signal transduction molecule that associates with SmIg) is now preferred to CD22, a change that has significantly increased the ability to discriminate between B-CLL and other B-cell disorders.
An immunophenotyping scoring system was developed that gives a value of 1 or 0 according to whether it is typical or atypical for CLL. Total scores range from 5 (typical of CLL) to 0 (atypical of CLL).
1. Matutes E, et al. Leukemia. 1994;8: Moreau EJ, et al. Am J Clin Pathol. 1997;108:
1. Matutes E, et al. Leukemia. 1994;8:
2. Moreau EJ, et al. Am J Clin Pathol. 1997;108:

7. Prognosis: histologic bone marrow patterns
Nodular (low risk)
Interstitial (low risk)
Diffuse (high risk)
The different bone marrow patterns probably reflect variations in amount of lymphoid accumulation during the natural course of the disease
Histologic patterns of trephine biopsies from bone marrow reveal patterns that can be useful in assessing patient risk.
Nodular patterns are made up of mature lymphocytes. The nodules are larger-than-normal lymphoid follicles and lack clear centers. There is no interstitial infiltration. Fat cells are preserved. Nodular patterns are associated with low-risk disease.
Interstitial patterns show some degree of replacement of normal hematopoietic tissue by mature lymphocytes, but fat cells and bone marrow structure are preserved. Interstitial patterns are also associated with low-risk disease.
Diffuse patterns show diffuse lymphoid infiltration with massive replacement of normal hematopoietic tissue as well as replacement of fat cells. Diffuse patterns are associated with high-risk disease.
Biopsy patterns may reflect variations in the amount of lymphoid accumulation during the course of the disease.
Courtesy of Randy Gascoyne, MD. 1. Montserrat E, et al. Cancer. 1984;54:
1. Montserrat E, et al. Cancer. 1984;54:

8. Prognosis: lymphocyte doubling time
Survival time according to LDT (all stages)
1.0
0.9
0.8
Doubling time ≤12 months
0.7
Doubling time >12 months
0.6
Probability of survival
0.5
0.4
0.3
0.2
0.1
Lymphocyte doubling time (LDT) is clearly related to prognosis in patients with CLL.
In a study of 100 untreated patients, LDT correlated partially with clinical stage and with bone marrow patterns, but it also had a clear prognostic significance by itself.
Patients with an LDT 12 months were likely to have a poor prognosis, whereas those with an LDT >12 months had a good prognosis, with a long treatment-free period and survival.
0.0 20 40 60 80
100
120
140
160
Months
1. Montserrat E, et al. Br J Haematol. 1986;62:
1. Montserrat E, et al. Br J Haematol. 1986;62:

9. Prognosis with serum markers: the effect of 2-microglobulin on survival in untreated CLL
1.0
Pts
Died
2M
445 53
<2.1
429 95
183
175 67
>4.0
0.8
0.6
Proportion surviving
0.4
0.2
Serum 2-microglobulin, soluble CD23, and serum thymidine kinase are independent predictors of progression-free survival in CLL.
In a large set of patients treated independently over a 17-year period, follow-up according to 2-microglobulin status showed that survival is improved with lower levels of 2-microglobulin, whereas those with high 2-microglobulin levels have the poorest survival outcome.
0.0 2 4 6 8 10 12 14 16 18
Years
1. Keating M. Unpublished data. 2. Hallek M, et al. Leuk Lymphoma. 1996;22:
3. Sarfati M, et al. Blood. 1996;88:
4. Fayad L, et al. Blood. 2001;97:
1. Keating M. Unpublished data.
2. Hallek M, et al. Leuk Lymphoma. 1996;22:
3. Sarfati M, et al. Blood. 1996;88:
4. Fayad L, et al. Blood. 2001;97:

10. Genetic abnormalities in CLL
Genetic abnormality
Incidence (%)
Median survival (months)
Clinical correlation
13q14
55-62
Typical morphology Mutated VH genes Stable disease
+ 12
16-30
Atypical morphology Progressive disease
del 11q23 18
79-117
Bulky lymphadenopathy Unmutated VH genes Progressive disease Early relapse post autograft
p53 loss/mutation 7
32-47
Atypical morphology Unmutated VH genes Advanced disease Drug resistance
Because of the limitations of the Rai and Binet systems in predicting the progression of CLL, other prognostic criteria are being considered; for instance, advanced disease stage, male gender, CD38 expression >30%, and atypical morphology predict relatively poor outcomes in CLL. Additionally, karyotyping and molecular biology techniques reveal that the behavior of certain genetic markers in CLL may offer insights into the molecular mechanism of the disease and predict treatment outcome.
In a study of 205 patients with CLL, 69% were found to have an abnormal karyotype. Genetic abnormalities included:
structural abnormality of chromosome 13q14
trisomy 12
11q23 deletion
17p13 abnormalities; loss or mutation of the p53 gene
13q14 deletion carries a better prognosis than deletion of 11q13 or 17p13.
Deletion of 11q23 is associated with bulky lymphadenopathy and a high incidence of residual disease following autologous transplantation.
17p13 abnormalities that result in mutation or loss of the p53 gene correlate with resistance to purine analogs.
1. DÖhner H, et al. N Engl J Med. 2000;343: Oscier DG, et al. Blood. 2002;100:
1. Döhner H, et al. N Engl J Med. 2000;343:
2. Oscier DG, et al. Blood. 2002;100:

11. Effect of genetic abnormalities on survival1
In CLL
Effect of genetic abnormalities on survival1
Effects of genetic abnormalities on survival in patients with CLL (N=325)1
Döhner et al evaluated 325 cases of CLL to assess the frequency and clinical relevance of genomic aberrations.1 Of the 325 patients, 248 had received no prior treatment, 39 had received 1 chemotherapeutic agent, and 38 had received 2 or more chemotherapeutic regimens before the cytogenetic analysis was conducted.1
Of the 325 patients, 268, or 82%, exhibited abnormalities. The primary endpoint for this study was survival from time of diagnosis. All cases were evaluated by interphase cytogenetics. On the basis of regression analysis, the investigators constructed a hierarchical model of 5 genetic categories for evaluation as prognostic factors: 17p deletion; 11q deletion but not a 17p deletion; 12q trisomy but not a 17p or 11q deletion; normal genome; and 13q deletion as the sole aberration. Of the 325 patients, 300 could be assigned to one of these 5 subgroups; 25 with various chromosomal abnormalities could not.
This slide illustrates the percentage of surviving patients by genetic aberration over 168 months. Median survival times for the groups were: 17p deletion, 32 months; 11q deletion, 79 months; 12q trisomy, 114 months; normal genome, 111 months; and 13q deletion as the only abnormality, 133 months. As the slide shows, patients with 17p deletions had by far the worst prognosis.1
1. Döhner H, et al. N Engl J Med. 2000;343:
1. Döhner H, et al. N Engl J Med. 2000;343:

12. Prognosis: effect of VH gene mutations on survival
100
Unmutated VH gene Median = 117 months
Mutated VH gene Median = 293 months 90 80 70 60
Percent surviving (%) 50 40 30 20 10
In this patient population, median survival times were significantly worse for patients with unmutated VH genes than for those with mutated VH genes (117 months vs 293 months, respectively; P=.001). However, this comparison may be skewed because in this population a greater number of patients with unmutated VH genes had advanced stage disease. 25 50 75
100
125
150
175
200
225
250
275
300
325
Months
1. Hamblin TJ, et al. Blood. 1999;94:
1. Hamblin TJ, et al. Blood. 1999;94:

13. Prognosis: VH gene/p53 concordance
VH gene/p53 multivariate analysis
p53 loss/mutation Median = 47 months
1.0
0.9
Unmutated VH gene Median = 119 months
Mutated VH gene Median = 310 months
0.8
0.7
0.6
Probability of survival (%)
0.5
0.4
0.3
0.2
0.1
Two multivariate analyses comprising over 500 patients with B-CLL demonstrated that VH gene mutation status and p53 loss or mutation are independent prognostic factors.
In the group of patients with mutated VH genes, median survival was 310 months compared with 119 months for the patients with unmutated VH genes. In patients with p53 loss or mutation, median survival was only 47 months.
In most cases, the average clinical laboratory is not equipped to determine VH gene status; in addition, the technique required to make this determination is prohibitively expensive to be part of the standard workup for CLL patients. For this reason, finding a surrogate for IgVH mutational status has become an important goal. 38 76
114
152
190
228
266
304
342
380
Months
1. Krober A, et al. Blood. 2002;100:
2. Crespo M, et al. N Engl J Med. 2003;348:
3. Oscier DG, et al. Blood. 2002;100:
1. Krober A, et al. Blood. 2002;100:
2. Crespo M, et al. N Engl J Med. 2003;348:
3. Oscier DG, et al. Blood. 2002;100:

14. Prognosis: effect of CD38 expression on survival
100
CD38 ≥30% Mean = months 90 80
CD38 <30% Mean = 288 months 70
N=162 P=.008 60
Percent surviving (%) 50 40 30 20 10
Patients with a low level of CD38 expression tend to have a more favorable clinical course than those with a high level.
In a study where the positivity status for CD38 was set at 30% of examined cells staining for CD38, median survival among those with CD38 expression <30% was 288 months (24 years), compared with approximately 163 months (13.5 years) for those with CD38 expression >30%.
Some investigators have suggested using CD38 expression as a correlate for the VH gene status. However, in about a third of patients, the CD38 level does not predict the VH mutation status.
100
200
300
400
500
Months
1. Orchard JA, et al. Lancet. 2004;363:
1. Orchard JA, et al. Lancet. 2004;363:

15. Probability of survival (%)
Prognosis: effect of ZAP-70 on survival of patients with Binet stage A CLL
100
<20% ZAP-70-positive cells 90 80
N=44 P=.01 70 60
Probability of survival (%) 50
≥20% ZAP-70-positive cells 40 30 20 10
Zeta-associated protein 70 (ZAP-70) is a 70 kDa zeta-chain CD-3 receptor-associated protein tyrosine kinase usually active in T-lymphocytes. B-CLL cells that have non-mutated immunoglobulin V genes also express ZAP-70 RNA. Normal B-cells usually lack ZAP-70 and use the syk family of protein tyrosine kinases instead. The role of ZAP-70 in B-CLL with non-mutated IgV genes is unknown although it may enhance the B-cell receptor signal.
Kaplan-Meier estimate of the probability of survival at 10 years was 90% among patients with a low level of ZAP-70 expression. The median survival was 90 months among the patients with a high level of ZAP-70 expression, whereas it was not reached among patients with a low level of ZAP-70 expression (P=.01). 4 8 12 16 20 24 28 32 36
Years after diagnosis
1. Crespo M, et al. N Engl J Med. 2003;348:
1. Crespo M, et al. N Engl J Med. 2003;348:

16. Prognosis with serum markers: effect of sCD23 level on survival
1.0
0.9
0.8
0.7
0.6
Probability
0.5
0.4
0.3
≤574 U/mL >574 U/mL
0.2
0.1
The membrane-bound CD23 antigen (Fc receptor for IgE) sheds soluble (s) CD23 into the serum. Soluble CD23 is selectively elevated in the serum of patients with CLL.
A prospective study of 153 patients with CLL assessed the predictive value of serum sCD23 level on overall survival and on disease progression in early-stage patients.  
With a median follow-up of 78 months, patients with sCD23 levels above the median value (574 U/mL) had a significantly worse prognosis than those with lower values, with a median survival of 53 months vs over 100 months (P=.0001).
0.0 20 40 60 80
100
120
Months
1. Sarfati M, et al. Blood. 1996;88:
1. Sarfati M, et al. Blood. 1996;88: