1. Lecture 6 and 7
Myelodysplastic syndromes; Chronic lymphocytic leukemia and related lymphoproliferative disorders
Abdulkarim Aldosari

2. Objectives
Describe the etiology, pathogenesis, clinical features and treatment of MDS
Describe laboratory diagnosis of MDS
Differentiate the subtypes of MDS based on morphology and laboratory features
Distinguish between MDS and AML on PB and BM smears
Know the International Prognostic Scoring System (IPSS)
List general features of chronic lymphocytic leukemia
List diagnostic criteria of CLL
Explain differential diagnostic criteria used to characterize lymphoproliferative disorders
Name laboratory methods used to study lymphocytes in lymphoproliferative disorders

3. Myelodysplastic syndromes
The Myelodysplastic syndromes (MDS) are a collection of myeloid malignancies characterized by one or more peripheral blood cytopenias.
MDS are diagnosed in slightly more than 10,000 people in the United States yearly, for an annual age-adjusted incidence rate of approximately 4.4 to 4.6 cases per 100,000 people.
They are more common in men and whites.
The syndromes may arise de novo or secondarily after treatment with chemotherapy and/or radiation therapy for other cancers or, rarely, after environmental exposures.

4. Myelodysplastic syndromes
In Myelodysplastic diseases, the blood stem cells do not mature into healthy cells
Versus myeloproliferative diseases - the total number of blood cells increases, usually mature cells
A heterogeneous group of clonal hematological malignancies
Characterized by PB cytopenias, dysplastic blood cells, propensity to become acute leukemia
Clinical outcome is variable
Outcome is almost always fatal
No cure except for allogeneic stem cell transplantation

5. MDS etiology and pathogenesis
Mostly affect the elderly – 70+ years
Mostly male
Risk factors – exposure to environmental and occupational products
Ammonia, petrochemicals, low dose irradiation, exposure to benzene, smoking, family history
Mechanism that causes MDS not well known
Genetic mutations/defects
Caused by a somatic mutation → a growth advantage of the neoplastic cell

6. MDS etiology and pathogenesis
Which hematopoietic progenitor cell is responsible for the neoplastic clone?
Pluripotent stem cell or an omnipotent stem cell?
Controversial subject > various studies done
Blast cell population in leukemic patients = myeloid or myelomonocytic
Only few cases the blasts are lymphoid
Most studies show that blasts arise from repopulating stem cells committed to myeloid differentiation

7. MDS etiology and pathogenesis
The main biological feature of MDS is increased apoptosis –programmed cell death
Ineffective hematopoiesis seen in MDS – caused by abnormally high rate of intramedullary apoptosis
Factors that are involved in stimulating apoptosis are increased and those which are anti- apoptotic are decreased
TNF-α and TGF-β, Fas-ligand → initiate apoptosis
GM-CSF - anti-apoptotic
TNF-α and TGF-β are both ↑ in MDS patients
GM-CSF ↓ in MDS patients
The balance between growth-stimulatory and growth-inhibitory cytokines may favor apoptosis

8. MDS etiology and pathogenesis
Loss of genetic material is the most common genetic anomaly found in MDS
MDS patients are missing key genes essential for maintaining normal hematopoiesis
No single gene found – focus of research
Characterized mainly by chromosomal deletions
Either sections of a chromosome or the entire chromosome
Versus chromosomal translocations in acute leukemias
Deletions follow a tumor suppressor gene model
Tumor-suppressor genes generally encode proteins that inhibit cell proliferation – (in MDS = apoptosis)
Need mutated allele from both parents – recessive model
Versus oncogene activity of acute leukemias
Missing genes → more genetic anomalies → disease progression

9. MDS progression
Characteristics of MDS changes as it progresses towards leukemia
Blast cells ↑
Leukocytosis replaces leukopenia
Hepatoslenomegaly occurs
Becomes a proliferative PB disorder
Progression due to ↓ in medullary apoptosis, ↓ in apoptosis factors (Fas ligand)
Multistep pathogenesis
Normal → clonal and dysplastic → full blown malignancy → acute

10. Morphology of PB and BM in MDS
Reliable analysis of blast phenotype of MDS difficult
Blasts are not predominant in the BM and PB
Determined using immunophenotyping
Expression of CD34 > myeloid antigens > myeloid blasts
Blast in BM determine type of MDS at initial diagnosis
Different types of blasts and sideroblast according to FAB classification
Type 1 myeloblast - variable size without granules or Auer rods
Type 11 myeloblast– slightly larger with 1-20 granules
Type 111 myeloblast –with ~20 granules, basophilic cytoplasm
Type 1 sideroblast – 1-4 cytoplasmic iron-containing granules = 15-50% erythroblasts
Type 11 sideroblast – 2-10 granules, scattered throughout cytoplasm
Type ringed sideroblast

11. Morphology of PB and BM in MDS
MDS characterized by multilineage morphological abnormalities
Lineage dysplasia includes:
Dyserythropoiesis
In PB – macrocytic/normochromic anemia in 90% of cases, ↓ retic count, macrocytosis, anisopoikilocytosis, basophilic stippling, dimorphic RBC population (normochromic, hypochromic), Pappenheimer bodies, teardrop (dacryocytes), schistocytes, elliptocytes, Howell-jolly bodies, acanthocytes
In BM – Erythroblasts with asynchronous cytoplasmic maturation, internuclear bridging, nuclear budding, Howell-Jolly bodies, Basophilia, ghosts cells, abnormal ringed sideroblasts (types 1 and 11)

12. Morphology of PB and BM in MDS
Megaloblastoid change Megaloblastoid erythroid precursors with and multinucleation nuclear irregularities and a multinucleated erythroid precursor

13. Morphology of PB and BM in MDS
Characterized by multilineage morphological abnormalities
Lineage dysplasia includes:
Dysgranulocytopoiesis in peripheral blood
neutropenia in 60% of cases
nuclear and cytoplasmic dysplasia in 90% of cases
neutrophilia possible
pseudo-Pelger-Huët and variable degrees of hyposegmentation
monolobulation (pseudo- Stodtmeister anomaly)
variable degrees of hypogranulation
pseudo-Dohle bodies
rare hypergranulation
abnormal chromatin

14. Stodtmeiser Pelger-Huet anomaly
Stodtmeiser anomaly

15. Morphology of PB and BM in MDS
Dysgranulocytopoiesis in bone marrow
Pseudo-Pelger-Huët or Stodtmeiser anomaly,
hypersegmentation
ring formation
chromatin sticks
asynchronous nuclear-cytoplasmic maturation
hybrid myelomonocytic cells
vacuolated monocytes/myeloid precursors
Pelger-Huët anomaly is considered to be the most specific dysplastic marker for the diagnosis of MDS

16. Morphology of PB and BM in MDS
Ring formation
Hypersegmentation with nuclear sticks

17. Morphology of PB and BM in MDS
Characterized by multilineage morphological abnormalities
Lineage dysplasia includes:
Dysmegakaryocytopoiesis
In PB – Thrombocytopenia in 60% of cases, platelet gigantism, ballooning, hypogranulation, rare thrombocytosis associated with 5q-syndrome
In BM – micromegakaryocytes (dwarf or mononuclear megakaryocytes), megakaryocytes with multiple small detached nuclei, or nuclei separated by thin strand, “pawn ball” shape, cytoplasmic vacuoles

18. Morphology of PB and BM in MDS
Discrete nuclear lobes, or multinucleation Micromegakaryocytes Pawn ball megakaryocyte.

19. FAB classification Refractory anemia (RA)
MDS
Peripheral Blood
Bone Marrow
Refractory anemia (RA)
Cytogenetic studies needed to diagnose- 50% of patients have clonal abnormalities
Most difficult to recognize and diagnose. Anemia 90% of cases (normochromic & macrocytic); cytopenias; variable dyserythropoiesis; <1% blasts neutropenia, dysmyelopoiesis & dysmegakaryopoiesis are rare
normal - hypercellular; erythroid hyperplasia with variable dyserythropoiesis; normal myeloid & megakaryocytic lines; <5% blasts; ringed sideroblasts < 15%
Refractory anemia with ringed sideroblasts (RAS)
Ringed sideroblast + lineage dysplasia enough to make diagnosis
Similar to RA; anemia (macrocytic or dimorphic); variable dyserythropoiesis; <1% blasts neutropenia, dysmyelopoiesis & dysmegakaryopoiesis are rare; cytopenias
ringed sideroblasts >15% nucleated cells
Refractory anemia with excess blasts (RAEB)
Anemia (macrocytic normochromic) ovalocytes and dacryocytes; 2 or more of dyserythropoiesis, dysmyelopoiesis with hyposegmentation, hypogranulation; dysmegakaryopoiesis with thrombocytopenia, giant platelets; <5% blasts
hypercellular; dyserythropoiesis, dysmyelopoiesis, and dysmegakaryopoiesis;
5-20% blasts, no Auer rods; ringed sideroblasts variable
Refractory anemia with excess blasts in transformation (RAEB-t)
Can mimic M2 - AML
as in RAEB, but > 5% blasts, may contain Auer rods
as in RAEB, but 20-30% blasts or Auer rods
Chronic myelomonocytic leukemia (CMML)
monocytosis >1.0 x109/L; WBC ≤ 13 x109/L
<5% blasts; dysmyelopoiesis variable
as in RAEB, but ↑ monocytes & promonocytes ≥ 20%; % blasts; variable ringed sideroblast
FAB classification

20. RAEB-t is eliminated; patients considered to have acute leukemia
Diseases
Blood findings
Bone marrow findings
Comments
Refractory anemia (RA)
Anemia No or rare blasts
Erythroid dysplasia only < 5% blasts < 15% ringed sideroblasts
5-10%
Refractory anemia with ringed sideroblasts (RARS)
Anemia No blasts
Erythroid dysplasia only < 5% blasts ≥ 15% ringed sideroblasts
Makes up about 3% to 11% of MDS cases; also generally good prognosis
Refractory cytopenias with multilineage dysplasia (RCMD)
Cytopenias (bicytopenia or pancytopenia) No or rare blasts No Auer rods < 1 x 109/L monocytes
Dysplasia in ≥ 10% of cells in ≥ 2 myeloid cell lines < 5% blasts No Auer rods < 15% ringed sideroblasts
Makes up about 30% of MDS cases; about 10% of people with RCMD will develop acute leukemia; less favorable prognosis.
Refractory cytopenias with multilineage dysplasia and ringed sideroblasts (RCMD-RS)
Cytopenias (bicytopenia or pancytopenia) No or rare blasts No Auer rods < 1 x 109/L monocytes
Dysplasia in ≥ 10% of cells in ≥ 2 myeloid cell lines < 5% blasts ≥ 15% ringed sideroblasts No Auer rods
Refractory cytopenias with excess blasts (RAEB-1)
Cytopenias < 5% blasts No Auer rods < 1 x 109/L monocytes
Unilineage or multilineage % blasts No Auer rods
20% of MDS cases; About 25% develop acute myeloid leukemia (AML); poorer prognosis
Refractory cytopenias with excess blasts (RAEB-2)
Cytopenias % blasts ± Auer rods < 1 x 109/L monocytes
Unilineage or multilineage % blasts ± Auer rods
20%; Up to 40% may develop AML; the remainder of cases are fatal due to bone marrow failure.
Myelodysplastic syndrome unclassified (MDS-U)
Cytopenias ± Blasts ± Auer rods
Unilineage in granulocytes or megakaryocyte < 5% blasts No Auer rods
Variable; Lacks findings appropriate for classification into any other MDS category
MDS associated with isolated del(5q)
Anemia < 5% blasts Platelets normal or increased
Normal to increased megakaryocytes with hypolobulated nuclei % blasts No Auer rods Isolated del(5q)
< 5%; Affected cells are missing part of chromosome 5; generally good prognosis; progression to acute leukemia occurs in less than 10% of patients.
WHO classification
RA and RARS subdivided according to presence or absence of multilineage dysplasia
RAEB-t is eliminated; patients considered to have acute leukemia
CMML reclassified under a subgroup of myelodysplastic/myeloproliferative disorders
Separate out patients with a 5q deletion syndrome

21. Cytochemical abnormalities of MDS
Cytochemical studies included iron stain, periodic acid-Schiff (PAS), peroxidase, butyrate esterase, chloroacetate esterase, and double esterase stains
↓activity and abnormal positive results of MPO, chloroesterase, alkaline phosphatase, SSB, dual esterase in myeloid and monocytic cells
Especially more difficult to diagnose RARS, RAEB-t, CMML due to major qualitative and quantitative abnormalities, than RA
The iron stain remained most helpful in identifying abnormal ringed sideroblasts, a feature of dyserythropoiesis - supports the diagnosis of MDS
The PAS stain was helpful, if positive, in identifying patients with MDS; when negative did not help distinguish MDS from non-MDS hematologic disorders
The combination of two stains, PAS and iron stain or PAS and double esterase - helpful in excluding MDS – negative results

22. Bone Marrow Histology of MDS
Aspirate and biopsy helpful to estimate:
Cellularity – which cells are dysplastic
Histological changes – displacement of hematopoiesis cells from their usual sites
Abnormal localization of immature precursors (ALIP) – may find clusters of immature myeloid cells away from the BM trabeculae = early indicator of leukemic transformation
Myelofibrosis – Rare occurrence, associated with BM hypoplasia
Useful to determine dysmegakaryocytopoiesis, not so much for dyserythro- or dysgranulopoeisis

23. Cytogenetics and molecular abnormalities of MDS
Clonal abnormalities present in 30-60% of patients with MDS
Most frequent abnormalities:
5q- = deletion of the long arm of chromosome 5
The common deleted region always spans the chromosome band 5q31
Several genes related to hematopoiesis are found on 5q
Genes that encode cytokines and their receptors
More frequent in RA
Clinical features include macrocytic anemia, normal or ↑ platelet, mild leukopenia, monolobular or dwarf megakaryocytes, erythroid hypoplasia
Affects mostly females
Good prognosis – median survival ~8yrs
The prognosis of 5q deletions in MDS is generally favorable if they are not part of complex abnormalities

24. Cytogenetics and molecular abnormalities of MDS
Deletion comprise any region located between band 5q13 and 5q33
Band 5q31 is consistently deleted in most patients

25. Cytogenetics and molecular abnormalities of MDS
Monosomy 7 (-7)
Abnormalities in chromosome 7 occur in about 20 percent of MDS patients
Common in patients who have prior chemotherapy exposure
Associated with a poor prognosis
Monosomy 7 + complex karyotype = short survival rate and ↑ progression to AML
Rarely seen in RA
In children - > risk of bacterial infections independent of neutrophil count

26. Cytogenetics and molecular abnormalities of MDS
Trisomy 8 (+8)
Categorized as intermediate cytogenetic risk group
The most frequent cytogenetic abnormality in de novo MDS in China
Found in 15-20% of MDS
5-10% of MDS with +8 are treatment-related MDS
Present in each FAB subgroup: up to 25-30% of RARS cases have +8; 15-20% of other subgroups have +8

27. Cytogenetics and molecular abnormalities of MDS
Deletion of part of chromosomes 11q, 20q
20q- alone is associated with a good prognosis regarding survival and potential for AML evolution
pathogenic mechanism by which 20q- alters the hematopoietic stem cells is unknown
Anomalies of p17
Where p53 gene reside
Associated with hypolobulated granulocytes with cytoplasmic vacuoles
Mainly refractory anemia with excess of blasts RAEB/ RAEB-t in MDS
Other rare chromosome abnormalities are present in a substantial portion of patients

28. Secondary MDS
After significant exposure to chemotherapy, radiotherapy or other toxic agents Laboratory findings, clinical manifestations, evolution similar to new MDS Higher frequency of chromosomal abnormalities Greater tendency to early leukemic transformation

29. Clinical features of MDS
Symptoms associated with progressive bone marrow failure
Depends on the degree of anemia, neutropenia, thrombocytopenia
Patients without excess blast in BM may be asymptomatic for a long time
Abnormal physical findings are not prominent, non specific
Fatigue and malaise result from anemia.
Signs and symptoms of chronic heart failure may develop in patients with underlying cardiac problems – in the elderly
Petechiae, ecchymosis, and nose and gum bleeding- low platelet count. If underlying dysplastic changes were missed initially - may be mistaken for immune thrombocytopenia.
Fever, cough, dysuria, or shock - serious bacterial or fungal infections associated with neutropenia.

30. MDS in children Rare- less than 5% of all blood cancers in children
The median age at diagnosis in children is 7 years old
RA is the most common subtype of childhood MDS.
About half of all children with MDS are diagnosed with RA
RAEB and RAEB-t subtypes
Symptomatic – fever, pallor, physical weakness/loss of strength
Normocytic, normochromic anemia rather than macrocytic anemia
↑ risk of development of AML associated with Down’s syndrome, Fanconi’s anemia
Children with treatment-related and secondary MDS tend to have less favorable treatment outcomes than children with primary MDS

31. Prognosis Factors that affect prognosis:
The number of blast cells in the bone marrow.
Whether one or more types of blood cells are affected.
Whether the patient has symptoms of anemia, bleeding, or infection.
Whether the patient has a low or high risk of leukemia.
The nature and number of chromosome abnormality
Whether the myelodysplastic syndrome occurred after chemotherapy or radiation therapy for cancer.
The age and general health of the patient.
The presence of abnormal localization of immature precursors (ALIP)

32. Prognostic scoring systems
The International Prognostic Scoring System (IPSS) - used to help assess the severity of MDS
Based on the IPSS score, the patient's history, and observations, a treatment plan is designed to address the MDS
Based on
The presence of one or more low blood cell counts (cytopenias)
BM blast cell count
Cytogenetic results – chromosomal changes in the marrow cells

33. Prognostic scoring systems

34. Diagnostic problems with MDS
Difficult to differentiate from aplastic anemia
Clonal anomaly will confirm MDS
BM smear for dysplastic changes
Differentiate from acute myelofibrosis (AML, M7)
15% of MDS show BM fibrosis – sMDS
Dry tap in BM aspiration, trilineage dysplasia, cytopenias
No organomegaly
Cytogenic abnormalities
From essential thrombocytosis
JAK2 mutation in ET
Cytogenetic analysis – 5q- or other translocation in MDS

35. Diagnostic problems with MDS
CMML to be differentiated from CML
CMML, patients have an elevated number of monocytes in the blood
CMML patients have abnormal looking (dysplastic) cells in their bone marrow
CMML has features of both myelodysplastic syndrome and myeloproliferative disorder
Cytogenetic studies to differentiate
Differentiate from Acute erythroid leukemia
When the total population of BM erythroblasts > 50% of all nucleated cells
Algorithm to help with diagnosis

37. Treatment of MDS
Allogeneic stem cell transplantation – only therapy with potential to treat MDS
Since most are elderly – not as practical
Limited to younger patients
<60yrs with a matched sibling
<55yrs unrelated but matched donor
High dose chemotherapy (without transplantation)
Short lived response
Response - < 20% of patients still in remission 2 years after therapy
Advances in pathogenesis – development of novel drugs
IPSS used to tailor therapy

38. For the elderly - IPSS low or intermediate-1 risk category – treat to improve hematopoiesis
For younger patients – IPSS-2 or high risk – treat to improve survival

39. Review questions
The most common dyserythropoietic finding in the bone marrow in MDS
Megaloblastoid development
Impaired hemoglobinization
Pseudo-Pelger- Huet cells
Agranular cytoplasm
Ans. a
Why should you test B12 and folate levels when entertaining a diagnosis of MDS?
Ans.
Megaloblastoid changes are often seen in the erythroid and megakaryocytic lines of MDS and are similar to abnormalities of megaloblastic anemia.
MDS should not be diagnosed if there is evidence of either a B12 or folate deficiency.

40. Chronic lymphocytic disorders
Clonal proliferations of morphologically and immunophenotypically mature B or T lymphocytes
Diagnosis based on morphology, immunology, cytogenetic, molecular and clinical features
Leukemias = diseases affecting the BM and the PB
Lymphomas = disease affecting extramedullary sites
Chronic lymphoid leukemias have various stages
Early stage – small tumor cells, with low proliferation, prolonged survival
Transformative stage – proliferation in extramedullary sites, increase in large immature cells

41. Chronic lymphocytic disorders
Advances in cytogenetics and molecular biology show that hematopoietic neoplasms are associated with unique genotypic profile
Genotype affects the development and the clinical features of the disease
Advances in the development of monoclonal antibodies → identification of unique immunophenotypic profile for most leukemias and lymphomas
Advances have enhanced accuracy and reproducibility of diagnosis

42. Chronic lymphocytic leukemia
A monoclonal disorder characterized by a progressive accumulation of functionally incompetent lymphocytes.
Most common form of leukemia found in adults in Western countries
In some people with CLL, the disease grows and progresses slowly
May take years for symptoms to appear or for treatment to be needed
Some patients may never need treatment for their CLL

43. Chronic lymphocytic leukemia
In other patients the disease grows more quickly and needs treatment sooner.
Some patients die rapidly, within 2-3 years of diagnosis, because of complications from CLL, but most patients live 5-10 years
90% people diagnosed with CLL are over the age of 50
Mostly affects men – incidence is twice that of women
When present - symptoms are related to anemia, thrombocytopenia and neutropenia
Signs and symptoms may develop gradually

44. Chronic lymphocytic leukemia
B lymphocyte neoplasm (B-CLL) – T lymphocyte very rare
CLL is a stage of small lymphocytic lymphoma (SLL) -a type of B-cell lymphoma primarily in the lymph nodes
CLL and SLL are considered the same underlying disease but with different clinical presentation
Included in the lymphoproliferative disorders

45. Chronic lymphocytic leukemia
Characterized by lymphocytosis in PB and BM
Accumulating lymphocytes in CLL → crowding out of other cells
→ neutropenia, anemia, thrombocytopenia
→ Organ infiltration → adenopathy, splenomegaly, hypersplenism
Neoplastic cells of CLL are more fragile than normal lymphocytes > burst open during smear preparation > smudge cells (bare nuclei)
Smudge cells can also be found in reactive lymphocytosis and other neoplasms
Therefore not diagnostic
Reduce smudge cells by mixing a drop of albumin with a drop of blood prior to making smear

46. CLL morphology CLL in BM, with numerous mature- appearing lymphocytes
CLL in PB with smudge cells

47. Chronic lymphocytic leukemia
Characterized by altered humoral immunity (antibody mediated immunity)
Due to suppression of all classes of immunoglobulin (Ig) → hypogammaglobulinemia → increased susceptibility to infections
Production of autoantibodies → autoimmune disease - idiopathic thrombocytopenia or autoimmune hemolytic anemia
15-35% of patients develop autoimmune hemolytic anemia at some time during course of the disease
Immune-mediated destruction of red blood cells and platelets = unique feature that is not present in other kinds of leukemia .

48. Chronic lymphocytic leukemia
51% of B-CLL patients positive for Bence-Jones paraproteinemia
Bence Jones proteins are considered the first tumor marker; made by plasma cells;
The presence of these proteins in a person's urine is associated with a malignancy of plasma cells .

49. Cause and physiological process of CLL
No specific cause known
Possibly due to viral infections
B-CLL cells from some patients appear to be a malignant transformation of an antigen-committed B cell responding to the human T-lymphotropic virus type 1 (HTLV-1) infection
HTLV-1 infection seems to precede development of CLL in some patients
May be due to failed apoptosis
B-CLL cells have been shown to have high levels of anti-apoptotic agents (protein BCL2)
The proto-oncogene BCL2 is a known suppressor of apoptosis, resulting in a long life for the involved cells
BCL-2 seen in 80% of B-CLL cells
Presence of p53- tumor suppressor gene in CLL –del of 17p

51. Cause and physiological process of CLL
Deregulation of cell cycle regulatory genes
Abnormality of the regulation of cell proliferation
CLL is characterized by the gradual accumulation of small mature B cells which are in the G0/G1 phase of the cell
Cell Cycle progression governed by interactions between cyclins, cyclin-dependent kinases (CDKs), cyclin-dependent kinase inhibitors
cyclin D1 and D3 mRNA levels correlated positively with lymphocyte doubling time (LDT)
cyclin D1 and D3 mRNA levels were 4 to 6-fold higher in CLL cells than in normal peripheral blood B cells

53. Immunologic features for studying lymphocytes
20% of circulating lymphocytes have surface Igs and are B cells
61-89% are T cells
22% are NK cells
It is not possible to differentiate by morphological features only
Need to differentiate in lymphoproliferative disorders
As lymphocyte matures from pluripotent stem cell → lymphoid tissue – acquire developmental markers – identify subpopulations

54. Immunologic features for studying lymphocytes
Malignant B-cells do not progress normally; do not become plasma cell – arrested development – at earlier stage
Genetic recombination – generate an immature B cell that expresses functional sIg
Characteristic immunophenotype for B-CLL – expression of sIg

55. Chromosomal abnormalities
Most common in B-CLL is an extra chromosome 12 = trisomy 12
Occurs in 15-20% of cases
May occur alone or with other deletions or translocations of chromosome 13q14
Most common chromosomal abnormality – 13q
In 25% of cases
Early clonal abnormality
Suggestive of a loss or inactivation of a tumor suppressor gene
Undetectable at cytogenetic level
Detectable in 50% of CLL cases using molecular probes for the 13q14 region
Presence of multiple chromosomal abnormalities = poor prognosis indicator
Trisomy 12
Del 11q
Del 17p13 (p53)
13q, 14q abnormalities

56. Chromosomal abnormalities

57. Chromosomal abnormalities

58. Clinical course of CLL Median survival for CLL = 4-5yrs
50% of patients live beyond 5yrs
30% have a 10yr survival
Can be indolent, asymptomatic, may not require any treatment
Until lymphocytosis of the PB, BM, lymphadenopathy, splenomegaly, anemia, neutropenia, thrombocytopenia, autoimmune symptoms, and infection develops
This can take up to 10-15yrs to develop after initial diagnosis

59. Clinical course of CLL
On the other hand – 20% of cases have aggressive clinical course
Rapid progression > Death within 1-2yrs
Not clear why the variation in response to the disease
Various prognostic markers used to help predict outcome
CD38, ZAP-70, and mutational status of Ig heavy chain variable region (VH)
ZAP-70 = protein normally expressed near the surface membrane of T cells and natural killer cells. It is part of the T cell receptor, and plays a critical role in T-cell signaling
Aggressive CLL = higher expression of CD38, ZAP-70, “unmutated” Ig VH gene
Clinical and pathologic data have been used to try to predict prognosis, to identify stages and risk groups

60. Staging of CLL Two staging systems are in common use for CLL:
Modified Rai staging in the United States
Has historical precedent and widely used
Binet staging in Europe
Neither is completely satisfactory - both have often been modified.
These CLL staging systems have been unable to provide information regarding disease progression due to its heterogeneity

61. Rai-Sawitsky staging of CLL
Original 5-stage staging system was revised in 1987 to a simpler 3-stage system
The revised system divides patients into low-, intermediate-, and high-risk groups
Low risk (formerly stage 0)
Lymphocytosis in the blood and marrow (≥ 5 x 109/L in PB, ≥ 30% lymphs in BM)
25% of presenting population
Median survival > 12.5yrs

62. Rai-Sawitsky staging of CLL
Intermediate risk (formerly stages I and II)
Lymphocytosis + enlarged lymph nodes - Median survival (Ms) 8.5yrs
Lymphocytosis + enlarged spleen or liver, ± lymphadenopathy – Ms 6yrs
50% of presentation
High risk (formerly stages III and IV)
Lymphocytosis + anemia (Hgb < 11 g/dL), ± enlarged nodes, spleen, liver – Ms 1.5yrs
Lymphocytosis + thrombocytopenia (plts <100 x 109/L), ± anemia, organomegaly – Ms 1.5yrs
25% of all patients

63. Binet staging of CLL Stage A Hemoglobin ≥ 10 g/dL, no anemia
Platelets ≥ 100 × 109/L, no thrombocytopenia
Fewer than 3 lymph node areas involved
Stage B
Hemoglobin and platelet levels as in stage A
3 or more lymph node areas involved
Ms 5yrs
Stage C
Hemoglobin < 10 g/dL
Platelets < 100 × 109/L, or both
Ms 2yrs
After successful treatment of immune cytopenias, CLL may be down-staged

64. Staging of CLL
Staging systems do not predict whether patient’s clinical course is more likely to be slow moving or progressive
Most reliable predictive factor for slow growing CLL
blood lymphocyte doubling time (LDT) greater than12 months
Non-diffuse pattern of BM lymphocyte infiltration + a Rai stage of 0,1 or 11 (low or intermediate)
Progressive clinical course and shorter survival
Short LDT – less than 12 months
diffuse lymphocyte infiltration of the BM + Rai of 111 or 1V (high risk)
↑ β2-microglobulin, CD23 in serum
Presence of chromosomal abnormalities

65. Transformation of CLL Prolymphocytic transformation Richter’s syndrome
Low grade, slow progression
Richter’s syndrome
Diffuse large-cell lymphoma
Rapid progression
Accounts for 5% of all deaths in CLL
p53 present in 40% of patients -associated with resistance to chemotherapy
Acute leukemia
Unusual in CLL
Terminal transformation
When there is a proliferation of a new population of immature cells
Appearance of chromosomal changes not previously present
More malignant clone → resistance to therapy
Poor prognosis

66. Clinical features of CLL
Patients > 50yrs presenting with lymphadenopathy, lymphocytosis
CBC with differential shows absolute lymphocytosis
> 5000 B-lymphocytes/µL
Lymphocytosis must persist for longer than 3 months
30% lymphocytosis of the BM consisting of morphologically mature appearing lymphocytes
Lymphocytes with hyper-clumped nuclear chromatin pattern, smudge cells
Positive sIg, CD19, CD20, CD5, CD23, CD43
Cytopenia caused by clonal bone marrow involvement establishes the diagnosis of CLL regardless of the peripheral B-lymphocyte count
Clonality must be confirmed by flow cytometry.

67. Other features of CLL
Varies from no treatment to the use of chemotherapeutic agents with or without radiation therapy
Prognosis – 50% 5yr survival; 30% ≤ 10yr survival
Differential diagnosis – ALL, PLL, SLL, HCL, Sezary syndrome, LGL, ATCL, SCCL, Waldenstrom’s macroglobulinemia, viral infection

68. Clinical features and differential diagnosis of CLL
Microscopic examination of PB smear is indicated to confirm lymphocytosis
Morphologically identical to SLL
Presence of smudge cells = artifacts from lymphocytes damaged during the slide preparation.
Large atypical cells, cleaved cells, and prolymphocytes
may account for up to 55% of peripheral lymphocytes.
If >55%, prolymphocytic leukemia (B-cell PLL) is a more likely diagnosis

69. Clinical features and differential diagnosis of CLL

70. Diagnosis of CLL
CLL can be diagnosed morphologically via immunophenotyping – immunoperoxidase and flow cytometry
Using monoclonal abs to detect specific surface antigens (cluster differentiation CD antigens)
The typical CLL phenotype is CD5+, CD23+, FMC7-, weak expression of surface Ig (sIg) and weak or absent expression of membrane CD22 and CD79b
CD 19, CD 20, CD79a, CD5, CD23, CD45, faint CD11 = B-CLL
Characteristic for B-CLL = detection of a predominance of sIg with κ or λ light-chain restriction = monoclonality

71. Diagnosis of CLL
Peripheral blood flow cytometry - most valuable test to confirm a diagnosis of CLL
Confirms the presence of circulating clonal B-lymphocytes
expressing CD5, CD19, CD20(dim), CD 23, and an absence of FMC-7 staining
Bone marrow aspiration and biopsy with flow cytometry is not required in all cases of CLL.
may be necessary in selected cases to establish the diagnosis and to assess other complicating features such as anemia and thrombocytopenia
Lymph node biopsy - if lymph node(s) begin to enlarge rapidly in a patient with known CLL
to assess the possibility of transformation to a high-grade lymphoma
When such transformation is accompanied by fever, weight loss, and pain, it is termed Richter syndrome

72. Diagnosis of CLL
Molecular testing now exists that may help predict prognosis or clinical course - not necessary for the diagnosis or staging of CLL
At present, these tests are not recommended for routine use, although this may change with further research.
Chromosomal evaluation using FISH can identify certain chromosomal abnormalities of CLL with prognostic significance.
Patients with del(17p) tend to have a worse prognosis, as well as resistance to therapy with alkylating agents and purine analogues.
Patients with del(11q) also have a worse prognosis and bulky lymphadenopathy at presentation.

73. Mantle cell -derived from a subset of center cells in the mantle region of lymphoid follicles. MCL represents 2-10% of all non-Hodgkin lymphomas

74. Clinical features and differential diagnosis of CLL
Patients with < 5000 B-lymphocytes/µL + lymphadenopathy and (-) cytopenias more likely = small lymphocytic lymphoma (SLL)
diagnosis should be confirmed by lymph node biopsy
Patients with a clonal B-cell population < 5000/µL (-) lymphadenopathy or organomegaly, cytopenia, or other disease-related symptoms = monoclonal B- lymphocytosis (MBL)
MBL progress to CLL at a rate of 1-2% per year

75. Differential diagnosis of CLL
ALL – morphological differences of the proliferating population
Smooth, nuclear chromatin of lymphoblasts in ALL versus, dense nuclear chromatin of CLL
TdT (+) for ALL, TdT (-) for CLL
B-prolymphocytic leukemia (B-PLL) – > 55-75% prolymphocytes
<10% in CLL
If 11-55% = Prolymphoid transformation = mixed cell type of CLL/PLL
CD24/CD22, ± CD10
Strong expression of sIg and CD20
(+) FMC-7
antigen expressed on mature human B cells used in immunophenotypic analysis and differential diagnosis of lymphomas and leukemias
Sezary syndrome –
a type of cutaneous T-cell lymphoma CTCL) which belongs to a larger group of disorders known as non-Hodgkin’s lymphomas
Sezary cells = malignant T lymphocytes, mature memory T cells
Mycosis fungoides in the skin
pleomorphic abnormal T cells with convoluted nucleus
CD2, CD3, CD4, CD5
CD7 (-), CD8 (-)

76. Differential diagnosis of CLL
Hairy cell leukemia - abnormal growth of B cells
The cells look "hairy" under the microscope because they have fine projections coming from their surface
CD19/20/24; CD22 CD25; CD103
CD5(-)
Pancytopenia
Tartrate resistant acid phosphatase TRAP (+)
T-granular lymphocytosis
Lymphocytosis
Anemia; neutropenia; thrombocytopenia
Large cell size, moderate cytoplasm with prominent vacuoles
CD2, CD3, CD8, CD57 (HNK-1)

77. ALOT, acute leukemia orientation tube; AML, acute myeloid leukemia; BC, blast crisis; BCP, B-cell precursor; BM, bone marrow; CLL, chronic lymphocytic leukemia; CLPD, chronic lymphoproliferative disorders; CML, chronic myeloid leukemia; CSF, cerebrospinal fluid; FL, follicular lymphoma; HCL, hairy cell leukemia; LN, lymph node; LST, lymphoid screening tube; MCL, mantle cell lymphoma; MDS, myelodysplastic syndrome; MPD, myeloproliferative disorders; PCD, plasma cell disorders; PCST, plasma cell screening tube; PNH, paroxysmal nocturnal hemoglobinuria; SST, small sample tube.

78. Other methods for diagnosing CLL
Molecular probes – rearrangements of B-cell Ig and T-cell receptor genes Cytogenetics – chromosomal abnormalities Cytochemistry – Electron microscopy

79. Treatment Chemotherapy Radiation therapy Stem cell transplantation
The most common treatment for CLL is the chemotherapy drug fludarabine.
Radiation therapy
an alternative or used in conjunction with other therapy
Stem cell transplantation
Only used to treat the occasional young patient with aggressive CLL, since most patients with CLL live so long that the risk of transplant can seldom be justified.
Investigational therapy – clinical trials
Clinical trials currently available to UCSF patients include Flavopiridol for CLL cases that don't respond to standard treatment. Flavopiridol kills CLL so quickly that blood salt and acid-base abnormalities arise, potentially causing kidney failure

80. Review questions
What are the consequences of the accumulation of the lymphocytes in the peripheral blood and bone marrow in a patient with CLL?
A) Neutropenia
B) Anemia
C) Thrombocytopenia
D) All of the above
E) None of the above
Ans.: D

81. Review questions
A cell that could suggest that lymphocytes in CLL are not normal is a:
A) Smudge cell
B) Lymphoblast
C) Monoblast
D) All of the above
E) None of the above
Ans. A

82. Review questions
Which type of MDS demonstrates a mild decreased white blood cell (WBC) count (3.9 ´ 10/L), increased erythropoiesis and normal numbers of blast cells in the marrow, and <15% sideroblasts?
A) Refractory anemia with ringed sideroblasts (RARS)
B) Refractory anemia (RA)
C) Refractory anemia with excess blasts (RAEB)
D) Chronic myelomonocytic leukemia (CMML)
E) None of the above
Ans. B

83. Review questions
What is the purpose of transfusing granulocytes to a patient with RA?
A) Prevent hemorrhages
B) Prophylaxis to infection
C) Increase blood volume
D) Decrease plasma viscosity
E) None of the above
Ans.: B