1. The Chronic Lymphocytic Leukemia (CLL)

2. The Chronic Lymphocytic Leukemias (1)
The group of clonal diseases characterized by proliferation and accumulation of small, mature lymphocytes in blood, bone marrow and lymphoid tissues (lymph nodes, spleen)
Neoplastic lymphocytes belong most often to B-cell lines and they have the special for B-cell antigenes on their surface; exceptionally neoplastic lymphocytes belong to T-cell lines or NK-cell
According to REAL (Revised European-American Lymphoma)/ /WHO classification CLLs belong to the group of:
lymphoproliferative diseases
lymphomas

3. Lymphoproliferative diseases
Primary lymphatic system (central)
bone marrow
thymus
Secondary lymphatic system (peripheral )
spleen
lymph nodes
MALT (The mucosa-associated lymphoid tissue = also called mucosa-associated lymphatic tissue)

4. Clinical stages of lymphomas according to Ann Arbor’s classificationII
III IV
A: no general symptoms
B: general symptoms such as fever, night sweats, weight
loss
Lister T, et al. J Clin Oncol 1989; 7:1630

6. Ann Arbor’s classification is specific for all lymphomas
CLL is classified according to
Rai and Binet classification

7. WHO classification
B-Cell neoplasms - Precursor B-cell neoplasm: B-lymphoblastic leukemia/lymphoma - Mature (peripheral) B-cell neoplasms: B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma B-cell prolymphocytic leukemia Lympfioplasmacytic lymphoma Splenic marginal zone B-cell lymphoma (+ /- villous lymphocytes) Hairy cell leukemia Plasma cell myeloma/plasmacytoma Extranodal marginal zone B-cell lymphoma of MALT type Nodal marginal zone B-cell lymphoma (+/— monocytoid B cells) Follicular lymphoma Mantle-cell lymphoma Diffuse large B-cell lymphoma Mediastinal large B-cell lymphoma Primary effusion lymphoma Burkitts lymphoma/Burkitt cell leukemia
T-cell and NK-cell neoplasms
- Precursor T-cell neoplasm:
T-lymphoblastic lymphoma/leukemia
- Mature (peripheral) T-cell neoplasms:
T-cell prolymphocytic leukemia
T-cell granular lymphocytic leukemia
Aggressive NK-cell leukemia
Adult T-cell lymphoma/leukemia (HTLV1 +)
Extranodal NK/T-cell lymphoma, nasal type
Enteropathy-type T-cell lymphoma
Hepatosplenic gamma-delta T-cell lymphoma
Subcutaneous panniculitis-like T-cell lymphoma
Mycosis fungoides/Sezary syndrome
Anaplastic large-cell lymphoma, T/null cell, primary cutaneous type
Peripheral T-cell lymphoma, not otherwise characterized
Angioimmunoblastic T-cell lymphoma
Anaplastic large-cell lymphoma, T/null cell, primary systemic type

8. The Chronic Lymphocytic Leukemia (1)
Chronic lymphocytic leukemias are derived from:
B-cell line
B-cell chronic lymphocytic leukemia
B-cell chronic prolymphocytic leukemia
Hairy cell leukemia
Splenic marginal zone B-cell lymphoma ( + /- villous lymphocytes)
T-cell line
T-cell chronic lymphocytic leukemia
T-cell chronic prolymphocytic leukemia
T-cell granular lymphocytic leukemia
Chronic lymphocytic leukemias differ form each other in biology, morphology, antigen structure of the cell and in clinical course

9. The B-CLL - definition
B-CLL is a neoplastic disease characterized by proliferation and accumulation of small, mature, long-living lymphocytes in blood, marrow and lymphoid tissues (lymph nodes, spleen)
This lymphocytosis leads to specific clinical and laboratory symptoms of B-CLL
The neoplastic lymphocytes have on their surface the special for B-cell line antigens – CD19, CD20 and also CD5, CD23, and a very weak expression of surface immunoglobulin

10. B-CLL epidemiology
Most common adult leukemia in Europe and North America (in USA incidence of about 3/ population)
predominantly, CLL is a disease of elderly (50-55 years)
40% of leukemias in patients over 60 years old
Morbidity:
Men 2,2-3,69 / / year
Women 0,9-1,59 / / year
/men affect twice as often as women; 2:1 ratio of male to female /
CLL morbidity rapidly increases with age (especially between 50 and 60 years of age)
in 98% of patients the leukemic cells are a monoclonal population of mature B lymphocytes with low-density surface immunoglobulin
death from infections, BM failure, high-grade transformation (Richter's syndrome), kachexia

11. B-CLL etiology & pathogenesis (1)
the cause of CLL is unknown
there is increased incidence in farmers, rubber manufacturing workers and tire repair workers
genetics factors have been postulated to play a role in high incidence of CLL in some families

12. B-CLL etiology & pathogenesis (2)
Cytogenetics - clonal chromosomal abnormalities are detected in approximately 50% of CLL patients
Immunoglobulin genes - monoclonal surface immunoglobulin is expressed by over 90% of patients (60% kappa and 40% lambda light chains)
nearly half of all cases have leukemia cells that express mutated immunoglobulin variable region genes (Ig VH genes) - associated with more indolent disease
Immunologic abnormalities
autoimmune disease (hemolytic anemia and thrombocytopenia, pure red cell aplasia)
hypogammaglobulinemia
cellular immune defects

13. B-CLL clinical symptoms (1)
often none! - 25% of patients are asymptomatic and the diagnosis is typically accidental
unspecific: night sweats, fever, weakness (many patients have fatigue, reduced exercises tolerance or malaise, weight loss)
recurrent infections (bacterial, viral – Herpes Zoster, fungal) – they are the most common cause of death
bleeding and symptoms of anemia and thrombocytopenia
Lymphadenopathy (lymph node enlargement)
at diagnosis - nontender in 80% of patients
later - may become very large
splenomegaly - mild to moderate in 50% of patients
hepatomegaly
some organs infiltration (lungs, pleura, skin and soft tissue)
Blood lymphocytosis does not cause symptoms!

14. B-CLL clinical symptoms (2)
Cervical and axillary limfadenopathy in 60-years old patient with B-CLL

15. B-CLL clinical symptoms (2)
Cervical and axillary limfadenopathy in 70-years old patient with B-CLL

16. B-CLL clinical symptoms (3)
Cervical limfadenopathy in patient with B-CLL

17. B-CLL clinical symptoms (3)
The CLL patient can have splenomegaly

18. B-CLL clinical symptoms (3)
The CLL patient has splenomegaly, which is visble

19. B-CLL laboratory features (1)
Morphology:
Leucocytosis with monoclonal lymphocytosis of greater than 5.000/ul.
anemia
Because of „displacement ”
and/or autoimmunohemolic (10-20% of patients have a positive direct antiglobulin test; AIHA is commonly connected with the presence of warm auto- antibodies IgG class – rapidly increasing fatigue, skin getting yellow, anemia with enlarged reticulocytosis, higher level of bilirubin)
pure red cell aplasia is very rare (selective aplasia of red cell line in bone marrow)
thrombocytopenia
and/or immunologic (about 5% of B-CLL patients have anty-platelet antibodies)
protein electrophoresis – Hipogammaglobulinemia, monoclonal protein in 5% of patients

20. B-CLL laboratory features (2)
Peripheral blood smear:
Lymphocytosis
small, mature, morphologically
normal
Smudge cells
Neutropenia
Because of „displacement ”
and/or autoimmunologic

21. B-CLL laboratory features (3)
Bone Marrow smear (cytological examination)
extensive replacement of marrow element by mature lymphocytes (more than 30%)

22. B-CLL laboratory features (4)
Bone Marrow Biopsy (histological examination):
Lymphocyte infiltration
nodular infiltration,
interstitial infiltration,
difussed infiltration
mixed infiltration
Difussed infiltration
(unfavourable
prognostic factor)

23. B-CLL laboratory features (5)
Bone Marrow Biopsy
Interstitial
infiltration

24. B-CLL laboratory features (6)
lymph node finding (histopathological examination)
- diffuse infiltration of small lymphocytes identical to low-grade, small lymphocytic lymphoma

25. B-CLL laboratory features (7)
Immunophenotype:
CD5+/CD19+/CD23+/CD20+,
sometimes also CD38+,
low expression of CD22;
lack expression of CD 10-, CD 103-,
90% of the patient have a very weak expression of surface immunoglobulin (kappa or lambda light chain, IgM, IgD)

26. B-CLL features (8)
Radiological examinations (X-ray, ultrasonography, CT, ...)
Serological examinations (direct and indirect antiglobulin tests)
Biochemical examinations (lactate dehydrogenase, 2-microglobulin)

27. B-CLL laboratory features (9)
Cytogenetic examinations - clonal chromosomal abnormalities are detected in approximately 50% of CLL patients
deletion 13 (13q14.3)
trisomy 12
structural abnormalities of chromosomes 11 (11q-), 14, 17
Genomic aberrations found in approximately 50% of CLL

28. Diagnosis of B-CLL Blood test lymphocytosis ≥ 5G/l (6 weeks)
Morphology
monoclonal population of small mature lymphocyte
B-cell CLL phenotype
clonal CD5+/CD19+ population of lymphocyte
Markers of clonality
κ/λ light chain restriction; cytogenetical abnormalities
Bone marrow infiltration
> 30% of nucleated cells on aspirate
Lymph node
diffuse infiltrate of small lymphocytes

29. RAI’s CLINICAL STAGING SYSTEM
Stage
Clinical Features at Diagnosis
Median Survival (years)
Low risk
Blood lymphocytosis>5G/l,
Bone marrow lymphocytosis>30%
>12,5 I
Intermediate risk
and enlarged lymph nodes 8 II
and enlarged spleen and/or liver 6
III
High risk
and anemia (Hb < 11g/dl)
1,5-2 IV
and thrombocytopenia(< /ul)

30. CLL – Rai stages

31. BINET’s CLINICAL STAGING SYSTEM
Stage
Clinical Features at Diagnosis
Median Survival
(month) A
Blood lymphocytosis>5G/l,
Bone marrow lymphocytosis>30%
and less than 3 areas of palpable lymphoid-tissue enlargement
Without anemia (Hb >= 6,21 mmol/l, 10 g/dl) and thrombocytopenia
> 120 month B
and 3 and more areas of palpable lymphoid-tissue enlargement
60 month C
with anemia (Hgb <10g/dL) or thrombocytopenia (Plt < /uL)
24 month
An area: cervical, axillary left, axillary right,
inquinofemoral left, inquinofemoral right lymph nodes, spleen, liver

32. CLL – Binet’s stages

33. New prognostic indicators in B-CLL (1)
Prognostic factor
Good prognosis
Bad prognosis
Clinical stage according to Binet
Rai A
B, C
I, II, III, IV
Bone marrow infiltration in
- bone marrow biopsy
- cytological examination
Leucocytosis
Prolymphocytes in peripheral blood
Leukemia cell doubling time
Non-Difussed infiltration
<=80% lymphocytes
<= 50 x 109/l
<= 10%
> 12 months
Difussed infiltration
> 80% lymphocytes
> 50 x 109/l
>10%
<= 12 months

34. New prognostic indicators in B-CLL (2)
Prognostic factor
Good prognosis
Bad prognosis
Serum markers:
lactate dehydrogenase activity (LDH)
ß2-mikroglobulin activity
lymphocyte’s thymidine kinase activity
CD23 expression
Normal range
Elevated
Clonal chromosomal abnormalities
Normal karyotype isolated del (13q)
Del (11q)
Del (17p)
CD 38 expression
<= 30 %
> 30%

35. New prognostic indicators in B-CLL (3)
Prognostic factor
Good prognosis
Bad prognosis
The mutational status of immunoglobulin variable region of heavy chain genes (IgvH)
mutated
unmutated
ZAP–70 expression
low (< 20%)
high ( > 20 %)
Survivin
absence
presence

36. New prognostic indicators in B-CLL (4) - summary
clinical stage
bone marrow histology (diffuse replacement carries worst prognosis)
leukemia cell doubling time (less than 1 year - worse prognosis)
percentage of prolymphocyte
high cell-surface expression of CD38
ZAP-70 expression
serum level of: B2-microglobulin; thymidine kinaze, LDH, sCD23
IgVH mutational status
genetic features - FISH cytogenetic
low-risk: normal kariotype; isolated del(13q)
high-risk: del(17p0, del(11q), trisomy 12

37. CLL : ZAP-70 ZAP70 is an intracellular protein which is strongly correlated with the VH status in CLL

38. CLL – treatment (1) We have to remember:
B-CLL – indolent lymphoma, but incurable
Elderly patients – risk of additional diseases
Course of the disease can be very long, indolent for many years, patient can die because of another reason which is not connected to B-CLL.
Decision about treatment depends on clinical stage, prognostic factors and patient’s condition
Indications to treatment:
III/IV stage according to Rai’s classification
Progressive disease (rapidly increasing lymphadenopathy, infections, general symptoms)
leukemia cell doubling time <6 (12) months
rapidly increasing organomegaly
Secondary anemia, neutropenia, thrombocytopenia because of bone marrow infiltration
Richter’s syndrome

39. CLL – treatment (2) Watch and wait Monotherapy
Glucocorticoids (autoimmunological complications)
alkylating agents (Chlorambucil, Cyclophosphamide)
purine analogues (Fludarabine, Cladribine, Pentostatin)
Combination chemotherapy
Chlorambucil/Cyclophosphamide + Prednisone
purine analog (Fludarabine) + Cyclophosphamide +/- Mitoxantrone
CVP, CHOP (Cyclophosphamide, Doxorubicin, Vincristin, Prednisone)
Monoclonal antibodies (monotherapy and in combination)
Alemtuzumab (anti-CD52) = CAMPATH
Rituximab (anti-CD20) = Mabthera
antiCD23 etc.
monoclonal antibodies conjugated with radionuclides = Ibritumomab tiuxetan = Zevalin
Splenectomy (hypersplenism)
Radiotherapy (massive lymphadenopathy)

40. CLL – treatment (3) Hematopoietic stem cell transplantation
autologous - still no cure with auto-SCT
allogenic with reduced intensity conditioning
Even RIC-SCT is still a risky procedure - indicated only in high-risk disease
Can allo-SCT cure CLL? - YES
New and novel agents
Oblimersen – bcl2-directed antisense oligonucleotide
Lenalidomide
Flavopiridol
Anti-CD23
Anti-CD40
Vaccine strategies
Supportive therapy (allopurinol, G-CSF, blood and platelet transfusion, immunoglobulins, antibiotics)

41. Response criteria (NCI working group 1996)
Complete response (for at least 2 months)
clinical features – normal
morphology – normal (Hb>11 g/dl; Pt> /ul, lymphocytes <4000 G/l; neutrofiles >1500 G/l))
bone marrow - lymphocytosis less than 30%
Partial response
Stable Disease
Progressive Disease

42. Richter’s Syndrome
is always the transformation of CLL into an aggressive Lymphoma – diffuse large cell lymphoma (DLCL) or Hodgkin‘s lymphoma
usually evolves after a long indolent course -
can occur as 1st manifestation of CLL: Primary Richter‘s - but still CLL
has a poor prognosis