Chapter 12

MYELOID & LYMPHOID NEOPLASMS

Sr. No. TOPIC Page No.
1 Neoplastic proliferations of white cells 2

2 Acute myeloid leukemia (AML) 2

3 Chronic myeloid leukemia (CML) 7

4 Leukamoid reaction 10

5 Myelodysplastic syndrome (MDS) 11

6 Lymphoid neoplasms 12

7 ALL & CLL 14

8 Multiple myeloma 18

9 Hodgkin’s lymphoma 22

10 Important questions 28

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 NEOPLASTIC PROLIFERATIONS OF WHITE CELLS
WHO classification recognizes all hematopoietic neoplasms into two groups:
myeloid and lymphoid.

WHO classification of Myeloid neoplasms (2016), classifies the neoplasms into four
subcategories:
1. Acute myeloid leukemia (AML)
2. Myeloproliferative neoplasms (MPN)
a) Chronic myeloid leukemia
3. Myelodysplastic syndromes (MDS)
4. MDS/MPN

WHO classifies lymphoid neoplasms into five categories:

1. Precursor B-cell neoplasms (neoplasms of immature B cells)
a) B-cell Acute lymphoblastic leukemia (B-ALL)
2. Peripheral B-cell neoplasms (neoplasms of mature B cells)
a) Chronic lymphocytic leukemia (CLL)
b) Mantle cell lymphoma
c) Follicular lymphoma
d) Marginal zone lymphoma
e) Hairy cell leukemia
f) Plasma cell myeloma
g) Diffuse large B-cell lymphoma
h) Buritt lymphoma
3. Precursor T-cell neoplasms (neoplasms of immature T cells)
a) A-cell Acute lymphoblastic leukemia (A-ALL)
4. Peripheral T-cell and NK-cell neoplasms (neoplasms of mature T cells and NK cells)
5. Hodgkin lymphoma (neoplasms of Reed-Sternberg cells and variants)

Leukemia is the term used for neoplasms that involve the bone marrow and (usually, but not
always) the peripheral blood. Lymphoma is used for proliferations that arise as discrete tissue
masses.

ACUTE MYELOID LEUKEMIA (AML)

FAB Classification
In 1976, a group of French, American and British (FAB) hematologists standardized
morphological classification of acute myeloid leukemia, with subsequent modifications
supplemented by cytochemistry and to some extent immunophenotyping.

Table 1: FAB classification of AML

CATEGORY MORPHOLOGY IMMUNOPHENOTYPING CYTOGENETICS PROGNOSIS
FAB M0 Blasts are agranular and lack They can only be Have complex Complete remission
Minimally differentiated Auer rods; hence have no recognized by karyotype: (CR) rate and survival

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AML morphological features that immunophenotyping abnormalities of chr. 5 are poor
Occur in older patients identify it as myeloid [>20% express or 7, trisomies of 8 and
(median age 60 years) Also, only <3% are MPO panmyeloid antigens 13
and Sudan black B-positive, (CD13, 33, 117)]
thus cannot be identified on T-cell antigen (CD2,
cytochemistry also CD7) positive but
negative for cytoplasmic
CD3.
Stem cell marker: TdT+
Negative for B-lymphoid
marker (CD19, CD10)
FAB M1 >90% myeloblasts without Panmyeloid marker: Trisomies 11 and 13 Chemosensitive and
AML without maturation evidence of maturation CD13, 14, 33 + prognostically
10-20% cases (<10% promyelocytes or favorable
Median age: 45-50 years other mature cells)
Auer rods are variable.
>3% blasts positive for
MPO and SBB
FAB M2 with t(8;21) Prominent Auer rods, Panmyeloid marker: t(8;21)(q22;q22) Favorable prognosis
AML with maturation cytoplasmic globules and CD13, 14, 33 + CR rate: 86-95%
5-12% cases vacuoles Increased expression of
Most are children & young NK-cell associated
adults antigen CD56
Associated with chloromas B-cell antigen CD19+
Stem cell antigen CD34+
FAB M3 Blasts have prominent Panmyeloid marker: t(15;17) Excellent response to
Acute Promyelocytic granules CD13, 33 + therapy all-trans-
leukemia (APL) Bundles of Auer rods seen HLA-DR (stem cell retinoic acid (ATRA)
5-10% cases (“faggot cells”) marker) absent CR rate: >90%
Median age: 30-38 years Stem cell antigen CD34 +
90% patients present with T-cell marker CD2+
hemorrhagic manifestations
secondary to DIC
FAB M4 with abnormal Myeloblasts, monoblasts Panmyeloid marker inv(16)(p13;q22) CR rate: 81-93%
eosinophils and with abnormal eosinophils CD13 +
inversion of chr 16 containing large basophilic Stem cell antigen CD34 +
Acute myelomonocytic granules
leukemia
5-10% cases
Median age: 40-45 years
FAB 5a & 5b and M5a: poorly differentiated CD14, CD4 + Balanced translocation CR rate: 62%
11q23 abnormalities (>80% monocytic) Monoblasts are CD64+ involving 11q23
Acute Monocytic leukemia M5b: well differentiated
(AMOL) (>80% monocytic
2-10% cases predominantly
Wide age range; M5a tend to promonocytes &
occur in younger individuals monocytes)
(<25 years) Monocytic precursors are
Extramedullary disease is positive for fluoride-
more common: cutaneous inhibitable nonspecific
lesions, gum infiltration, esterase
CNS disease, testicular
involvement
FAB M6 M6a: ≥50% erythroid Glycophorin 7 and CD71 Aneuploidy Poor prognosis
Erythroleukemia precursors and 20% + Chr. 5 and 7 CR rate: 10-40%
2-4% cases myeloblasts abnormalities
Bimodal distribution, M6b: ≥80% erythroid
60-70 years and below 20 precursors
Bone pain may be a major PAS positive: coarse
symptom globular or diffuse pattern
FAB M7 Cytoplasmic blebs in the Myeloid surface markers: Children: common Poor prognosis
Acute megakaryocytic blasts suggest diagnosis CD13, 33 + type of AML in CR rate: 43-50%
leukemia (AMgL) BM fibrosis is common Platelet antigen: children with Down’s
Bimodal distribution: CD41, CD42b, CD61, syndrome; trisomies
3-10% of childhood AML factor VIII-related are common (+8, +9,
0.6-1.2% of adult AML antigen + +21)
Adult: complex
karyptypes: -5/del(5q)
or -7/del(7q)

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FAB M2: Auer rods seen FAB M3: Faggot cells

FAB M4: Monoblasts with folded nuclei FAB M6: Erythroblasts (A, B) seen

FAB M7: Megakaryoblasts with cytoplasmic blebs

WHO Classification
Over the last decade, FAB is being increasingly replaced by WHO classification of tumors of
hematopoietic and lymphoid tissues. The WHO classification for AML is based on morphology,
immunophenotyping, cytogenetics and in some circumstances molecular pathology.

Table 2: WHO classification of AML and related precursor neoplasms

CATEGORY SUB-CATEGORY
1. AML with 1. AML with t(8;21)(q22;q22)

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 recurrent genetic 2. AML with inv(16)(p13;q22)
abnormalities 3. Acute promyelocytic leukemia (APL) with t(15:17)(q22;aq12)
4. AML with t(9;11)(p22;q23)
5. AML with t(6;9)(p23;q34)
6. AML with inv(3)(q21;q26.2)
7. AML (megakaryocytic) with t(1;22)(p13;q13)
8. AML with mutated NPM1
9. AML with mutated CEBPA

2. AML with These leukemia increase in incidence with age.

myelodysplasia- May arise from antecedent MDS or de novo with an MDS-related
related changes cytogenetic abnormality.
Blast count should be at least 20% of the nucleated cells in BM
and there must be dysplasia in ≥50% cells of at least two cell
lineages.
3. Therapy- related Therapy-related AML and MDS occur in patients previously
myeloid treated with chemotherapy or radiation therapy.
neoplasms Alkylating drugs and topoisomerase II inhibitors are the most
common drugs that lead to AML/MDS about 5 and 2.5-3 years
after initiation of therapy respectively.
4. AML not Reflects FAB classification with the exception that AML is
otherwise defined by ≥20% blasts in the marrow (rather than ≥30% as in
categorized FAB):
1. AML minimally differentiated
2. AML without maturation
3. AML with maturation
4. Acute myelomonocytic leukemia
5. Acute monoblastic leukemia
6. Acute erythroid leukemia
7. Acute megakaryoblastic leukemia
8. Acute basophilic leukemia
9. Acute panmyelosis with myelofibrosis
10. Myeloid sarcoma (immature myeloid cells in extramedullary
site)
5. Acute leukemia Include cases where blasts lack sufficient lineage-specific antigen
of ambiguous expression to classify them as myeloid or lymphoid
lineage

FAB classification continues to have a place when special techniques are not available or in
making a provisional morphological diagnosis while waiting the results of further tests.

Clinical features
AML is predominantly a disease of the elderly (median age 50 years).

25% of patients present with preleukemic syndrome with anemia and other cytopenias for a few
months or years before overt leukemia develops.

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C/f due to marrow failure:
1. Anemia: producing pallor, lethargy, dyspnoea
2. Bleeding manifestations due to thrombocytopenia include spontaneous bruises, petechiae,
bleeding from gums and other bleeding tendencies. They are more common with AML
FAB M3 (acute promyelocytic leukemia). Patients of M3 may go into DIC.
3. Infections of mouth, throat, skin, respiratory, perianal and other sites
4. Fever

C/f due to organ infiltration:

1. Pain and tenderness of bones are due to bone infarcts or subperiosteal infiltrates by
leukaemic cells.
2. Lymphadenopathy and enlargement of the tonsils
3. Splenomegaly (moderate grade), splenic infarction, subcapsular haemorrhages, and
rarely, splenic rupture
4. Hepatomegaly
5. Gum hypertrophy due to leukemic infiltration of the gingivae is a frequent finding in
myelomonocytic (M4) and monocytic (M5) leukemias
6. Chloroma or granulocytic sarcoma is a localized tumor occurring in the skin or orbit due
to local infiltration of the tissues by leukaemic cells.
7. Meningeal involvement is seen more frequently in ALL
8. Other organ infiltrations: testes, mediastinum

Laboratory diagnosis
CBC:
Total WBC count: ranges from subnormal to markedly elevated

Platelets: reduced to normal

Peripheral smear:
RBC: anemia is severe, progressive and normochromic. Reticulocytosis may be seen.

WBC: Leukocytosis with majority being myeloblasts. The morphological features of M0 to

M7 are given in Table 1.

Bone marrow:
Cellularity: Markedly hypercellular; sometimes dry tap due to pancytopenia or increased
myelofibrosis

M:E ratio: increased

Myeloid cells: myeloblasts should be >20% to diagnose as AML

Erythropoiesis: reduced. Dyserythropoiesis, megaloblasts and ring sideroblasts may be seen.

Megakaryocytes: reduced or absent

Cytochemistry:

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 Myeloperoxidase (MPO): positive; negative in M0
Sudan Black B (SBB): positive
Non-specific esterase (NSE): positive in M4, M5
Acid phosphatase: diffuse positivity in M4, M5 and focal positivity in ALL
Periodic Acid Schiff (PAS): positive in M6 and ALL

Immunophenotyping:

CELL LINEAGE IHC marker

Positive for hematopoietic progenitor cells markers CD34, HLA-Dr, TdT
Positive for myeloid markers CD13, CD33, CD117
Negative for B-lymphoid marker CD19, CD10, CD20
Negative for T-lymphoid marker CD2, CD3, CD7
M4, M5- positive for monoblast markers CD64
M6- positive for erythroblast markers Glycophorin 7, CD71
M7- positive for platelet markers CD41, CD42b, CD61, factor VIII-related
antigen +

Other tests:
1. Serum muramidase: elevated in M4, M5
2. Serum uric acid: elevated

Cytogenetics:
1. M3: t(15;17)(q22;q12)
2. M4E0: inv(16)(p13q22)

CHRONIC MYELOID LEUKEMIA (CML)

Myeloproliferative neoplasms (MPN)

Table: WHO classification of MPN

Myeloproliferative neoplasm
Chronic myelogenous (myeloid) leukemia, Ph chr t(9;22)(q34;q11.2) & BCR-ABL1 positive
Chronic neutrophilic leukemia
Chronic eosinophilic leukemia, NOS
Polycythemia vera
Essential thrombocythemia
Primary myelofibrosis
Mastocytosis
Myeloproliferative neoplasm, unclassified

CHRONIC MYELOID LEUKEMIA (CML)

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Introduction
CML results from reciprocal translocation between part of long arms of chromosomes 9 and 22.
t(9;22)(q34;q11.2) thus formed involves fusion of BCR (breakpoint cluster region) gene on
chromosome 22q11.2 with ABL (Abelson murine leukemia virus) gene on chromosome 9q34.
The resultant gene is called BCR-ABL1 fusion gene and chromosome Philadelphia (Ph)
chromosome. Ph chromosome is seen in 90% cases.

CML is a biphasic or triphasic disease (chronic phase, accelerated phase and blastic phase).

Pathogenesis
BCR-ABL directs the synthesis of BCR-ABL tyrosine kinase.

Tyrosine kinase drives proliferation of pluripotent hematopoietic stem cell. BCR-ABL

preferentially drives the proliferation of granulocytic and megakaryocytic progenitors.

These cells undergo pro-growth and pro-survival (by inhibiting apoptosis) pathways.

Mechanism of progression of CML to blastic phase:

1. Structural alteration in tumor suppressor genes- p53, Rb
2. Inactivation of tumor suppressor protein, phosphatase A2
3. Alterations in oncogenes- RAS, MYC
4. Release of cytokine IL-1b

Clinical features
Age of presentation: 3rd and 4th decade; juvenile CML is seen in children
Both sexes equally affected.
S/s:
1. Anemia: weakness, pallor, dyspnoea and tachycardia
2. Hypermetabolic state: weight loss, lassitude, anorexia, night sweats
3. Splenomegaly: almost always present and frequently massive. In some patients, it may be
associated with acute pain due to splenic infarction.
4. Bleeding tendencies: easy bruising, epistaxis, menorrhagia and haematoma
5. Less common features: gout, visual disturbance, neurologic manifestations and priapism
6. Juvenile CML: is more often associated with lymph node enlargement than
splenomegaly. Other features are frequent infections, hemorrhagic manifestations and
facial rash.

Chronic phase
CBC:
Total WBC count: leukocytosis, >100,000 cells/mm3
Platelets: normal or elevated

Peripheral smear:

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 RBC: mild to moderate normochromia, normocytic anemia. Circulating erythroblasts may be
seen.

WBC: Leukocytosis with neutrophilia with presence of neutrophils, granulocyte precursors

(myelocytes, metamyelocytes and band forms), basophilia, eosinophilia and monocytosis.
Blasts are <10% of the circulating cells.

Bone marrow:
Cellularity: Markedly hypercellular

M:E ratio: increased

Myeloid cells: there are increased number of granulocytic precursors with preponderance of
myelocytes. Precursors of eosinophil and basophil also seen.

Erythropoiesis: normal or mildly decreased in number

Megakaryocytes: increased; small, dysplastic forms may be seen

Histiocytes: A characteristic finding is the presence of scattered macrophages with abundant

wrinkled, green-blue cytoplasm, called sea-blue histiocytes

Cytochemistry:
Neutrophil alkaline phosphatase (NAP) score: reduced (increased in leukamoid reaction)

Other tests:
1. Serum B12: elevated
2. Serum uric acid: elevated

Cytogenetics:
Cytogenetic study on blood or marrow cells shows Ph chromosome positivity in about 90%
cases.

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Accelerated phase
Accelerated phase is defined as increasing anemia, platelet count < 100,000/cmm, blast count in
peripheral blood or marrow between 10-20% and marrow basophils ³20%.

Blastic phase or blast crisis

CML patient is said to be in blast crisis when blood or marrow blasts are >20%. Auer rods are
however not seen.

LEUKAMOID REACTION
Leukaemoid reaction is defined as reactive excessive leucocytosis in peripheral blood resembling
that of leukemia in a subject who does not have leukemia. Differences between leukaemoid
reaction and chronic myeloid leukemia are as follows:

Parameter Leukamoid reaction CML

TLC 25,000-100,000/cmm > 100,000/cmm
DLC 1. Dominant cells PMN’s 1. All maturation stages
2. Immature cells predominantly 2. Immature cells of all stages,
metamyelocytes and myeloblasts and
myelocytes (5-15%), promyelocytes < 10%
myeloblasts and promyelocytes
> 5%
3. Basophils normal 3. Basophilia present
NAP score Elevated Reduced
Philadelphia chr. Absent Present
ABL-BCR fusion Absent Present
gene
Major etiology 1. Infections 1. RNA viruses
2. Intoxication 2. HTLV oncogenesis
3. Disseminated malignancy 3. Genetic factors
4. Severe hemorrhage 4. Radiation
5. Certain drugs and chemicals
Additional 1. Anemia 1. Anemia
haematologic 2. Normal to raised platelet count 2. Normal to raised platelet count
findings 3. Myeloid hyperplasia in BM
3. Myeloid hyperplasia in BM
Organ infiltration Absent May be present
Massive Absent Present
splenomegaly

Leukaemoid reaction may be myeloid or lymphoid; the former is much more common.

Myeloid Leukamoid Reaction

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Causes:
1. Infections: staphylococcal pneumonia, disseminated tuberculosis, meningitis, diphtheria,
sepsis, endocarditis, plague, infected abortions etc.
2. Intoxication: eclampsia, mercury poisoning, severe burns
3. Malignant diseases: multiple myeloma, myelofibrosis, Hodgkin’s disease, bone
metastases
4. Severe hemorrhage and severe hemolysis

Laboratory findings:
1. Leucocytosis: usually moderate, not exceeding 100,000/ l.
2. Proportion of immature cells mild to moderate, comprised by metamyelocytes,
myelocytes (5-15%), and blasts fewer than 5% i.e. the blood picture simulates with that
of CML
3. Infective cases may show toxic granulation and Döhle bodies in the cytoplasm of
neutrophils.
4. Neutrophil (or Leucocyte) alkaline phosphatase (NAP or LAP) score: in the cytoplasm of
mature neutrophils in leukaemoid reaction is characteristically high
5. Cytogenetic studies: negative Philadelphia chromosome in myeloid leukamoid reaction
but positive in cases of CML.
6. Additional features include anemia, normal-to-raised platelet count, myeloid hyperplasia
of the marrow and absence of infiltration by immature cells in organs and tissues.

Lymphoid Leukaemoid Reaction

Causes:
1. Infections: infectious mononucleosis, cytomegalovirus infection, pertussis (whooping
cough), chickenpox, measles, infectious lymphocytosis, tuberculosis.
2. Malignant diseases may rarely produce lymphoid leukaemoid reaction

Laboratory findings: The blood picture is characterised by the following findings:

1. Leucocytosis not exceeding 100,000/cmm.
2. The differential white cell count reveals mostly mature lymphocytes simulating the blood
picture found in cases of CLL.

MYELODYSPLASTIC SYNDROMES (MDS)

Myelodyplastic syndromes are a group of hematological neoplasms characterized by ineffective
and dysplastic haemopoiesis. Ineffective haemopoiesis is apparent from the usual coexistence of
a hypercellular bone marrow and peripheral cytopenia.

The incidence rises exponentially with age.

The predisposing factors include anti-cancer therapy, irradiation and aplastic anemia.

MDS can evolve into AML.

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Blood film and bone marrow aspirate are critical in diagnosis.

Peripheral blood examination:

- Cytopenia: anemia, leucocytopenia and thrombocytopenia
- Dysplastic features:
1. RBC: macrocytosis or dimorphic population as seen in sideroblastic anemia
(hypochromic-microcytic and normochromic-macrocytic)
2. WBC: hypogranular and hypolobated neutrophils
3. Platelets: large and/or hypogranular

Bone marrow examination:

- Hypercellular
- Dysplasia of one, two or three cell lineages
- Erythroblasts: nuclear lobation or fragmentation, binuclearity or multinuclearity,
megaloblastosis
- Granulopoiesis: hypolobation
- Megakaryocytes: small or normal in size, hypolobated or multinucleated
- Perl’s stain: increased iron stores and/or ring sideroblasts

Table: FAB classification for MDS

CATEGORY Peripheral blood criteria Bone marrow criteria

Refractory anemia (RA) or Anemia (RA) Blasts <5%

Refractory cytopenia Anemia &/ Neutropenia &/ Ringed sideroblasts <15%

thrombocytopenia of erythroblasts
(Refractory cytopenia)

Blasts <1%
Monocytes <1 x 109/l
RA with ringed sideroblasts Anemia Blasts <5%
Blasts <1%
Monocytes <1 x 109/l Ringed sideroblasts >15%
of erythroblasts
RA with excess of blasts Anemia Blasts ≥5% but <20%
(RAEB) Blasts >1% but <5%
Monocytes <1 x 109/l
RA with excess of blasts in Anemia Blasts ≥20% but <30%
transformation (RAEB-T) Blasts >5%
Chronic myelomonocytic Monocytes >1 x 109/l Blasts up to 20%
leukemia (CMML) Blasts <1% Promonocytes often
increased

LYMPHOID NEOPLASMS

Definition

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Leukemia is the term used for neoplasms that present with widespread involvement of the bone
marrow and (usually, but not always) the peripheral blood, while lymphoma is the term used for
lymphoid proliferations that arise as discrete tissue masses.

Classification
World Health Organization (WHO) classifies lymphoid neoplasms into five broad categories:

I. PRECURSOR B-CELL NEOPLASMS

1. B-cell acute lymphoblastic leukemia/lymphoma (B-ALL)

II. PERIPHERAL B-CELL NEOPLASMS

1. Chronic lymphocytic leukemia/small lymphocytic lymphoma
2. B-cell prolymphocytic leukemia Lymphoplasmacytic lymphoma
3. Splenic and nodal marginal zone lymphomas Extranodal marginal zone lymphoma
4. Mantle cell lymphoma
5. Follicular lymphoma
6. Marginal zone lymphoma
7. Hairy cell leukemia
8. Plasmacytoma/plasma cell myeloma
9. Diffuse large B-cell lymphoma
10. Burkitt lymphoma

III. PRECURSOR T-CELL NEOPLASMS

1. T-cell acute lymphoblastic leukemia/lymphoma (T-ALL)

IV. PERIPHERAL T-CELL AND NK-CELL NEOPLASMS

1. T-cell prolymphocytic leukemia
2. Large granular lymphocytic leukemia
3. Mycosis fungoides/Sézary syndrome
4. Peripheral T-cell lymphoma, unspecified
5. Anaplastic large-cell lymphoma
6. Angioimmunoblastic T-cell lymphoma
7. Enteropathy-associated T-cell lymphoma
8. Panniculitis-like T-cell lymphoma
9. Hepatosplenic gd T-cell lymphoma
10. Adult T-cell leukemia/lymphoma
11. Extranodal NK/T-cell lymphoma
12. NK-cell leukemia

V. HODGKIN LYMPHOMA
1. Classical subtypes
a) Nodular sclerosis
b) Mixed cellularity
c) Lymphocyte-rich
d) Lymphocyte depletion
2. Lymphocyte predominance

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Salient features:
HL & NHL:
Within this large group of lymphomas, Hodgkin lymphoma is segregated from others, which are
collectively called non-Hodgkin lymphomas (NHLs).

HL NHL
1. Often associated with systemic 1. Less commonly associated
manifestations like fever, night sweats and
weight loss
2. Localized to a single axial group of nodes 2. Involvement of multiple peripheral nodes
(cervical, mediastinal, para-aortic)
3. Waldeyer ring and mesenteric nodes are 3. Commonly involved
rarely involved
4. Spreads by contiguity 4. Noncontiguous spread

5. Extranodal involvement is uncommon 5. Common

6.
6. Morphologically characterized by 7. RS cells absent. Tumour cells of B-cell
neoplastic giant cells called, “Reed neoplasms are positive for pan B-cell
Sternberg (RS) cells”, which are negative markers.
for pan B-cell markers.

Origin:
The vast majority of lymphoid neoplasms (85-90%) are B-cell origin, most of the remainder are
T-cell while rarely NK-cell origin.

Some of the common IHC markers used for lymphoid neoplasms are:

CD2, 3, 5, 7 T cells
CD10, 19, 20 Precursor B cells
CD15, 30 Reed-Sternberg (RS) cells and variants
CD56 NK cells
CD45 Leukocyte common antigen (LCA)

ALL & CLL

ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

FAB classification
Cases of ALL were divided by FAB group on the basis of morphological features, into three
categories designated as L1, L2 and L3 ALL. The distinction between FAB L1 and L2 cases is

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now considered to be of little clinical significance. While FAB L3 category of ALL should be
recognized since it often represents Burkitt lymphoma, which is excluded from precursor B-cell
category and included in mature B-cell category in the WHO classification.

Table: FAB classification of ALL

CATEGORY MORPHOLOGY
L1 Small, homogenous cells with inconspicuous/1-2 nucleoli
L2 Large cells with variable size with 1-2 nucleoli
L3 Large cells, homogenous, finely stippled chromatin with
basophilic vacuolated cytoplasm

Clinical features
The incidence is highest in young children, has a nadir between 25 and 45 years and then rises
again to a peak around 70 years.

90% are B-cell origin while 10% T-cell origin.

B-cell ALL:
Clinical features of bone marrow failure are present: anaemia, neutropenia and thrombocytopenia

Extranodal involvement is early- lymphadenopathy, hepatosplenomegaly, CNS infiltration,

testicular enlargement, cutaneous infiltration

T-cell ALL:

Since the precursor T-cells differentiate in the thymus, this tumour often presents as mediastinal
mass and pleural effusion

Lymphadenopathy, hepatosplenomegaly and CNS involvement are frequent.

Laboratory diagnosis

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CBC:
Total WBC count: ranges from normal to elevated

Platelets: reduced

Peripheral smear:
RBC: anemia
WBC: Leukocytosis with circulating lymphoblasts. Using Romanowsky-stained smears alone,
experienced morphologists are only 70-80% correct in separating a myeloblast from a
lymphoblast.

Table: Morphological and cytochemical differences between myeloblast and lymphoblast

FEATURE MYELOBLAST LYMPHOBLAST

Chromatin Dispersed Partially condensed
Nucleoli Distinct or multiple Variable
Cytoplasm Moderate or abundant, Scant to moderate, granules
often with granules uncommon
Auer rods Seen in 50-60% of AML Absent
Differentiating myeloid Favor AML May be present in ALL as
cells residual normal myelopoesis
Cytochemistry MPO+, SBB+, Nonspecific PAS block positivity
esterase (M4 & M5+)

Bone marrow:
Cellularity: Hypercellular; with increasing number of lymphoblasts
Myeloid precursors: reduced
Erythropoiesis: reduced
Megakaryocytes: reduced or absent

Cytochemistry:
Periodic Acid Schiff (PAS): block positivity in ALL
Acid phosphatase: focal positivity in ALL
Myeloperoxidase (MPO): negative
Sudan Black B (SBB): negative
Non-specific esterase (NSE): negative

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Immunophenotyping:

CELL LINEAGE IHC marker

Positive for hematopoietic progenitor cells markers CD34, HLA-Dr, TdT
Negative for myeloid markers CD13, CD33, CD117
Positive for B-lymphoid marker (B-cell ALL) CD19, CD10, CD20
Positive for T-lymphoid marker (T-cell ALL) CD2, CD3, CD7

Cytogenetics:
B-cell ALL may be Ph chromosome positive.

CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)

Clinical features
It is predominantly a disease of middle and old age.

M:F = 2:1

S/s:
1. Features of anaemia: weakness, fatigue and dyspnoea
2. Superficial lymphadenopathy: lymph nodes are usually symmetrically enlarged, discrete
and non-tender.
3. Hepatosplenomegaly
4. Haemorrhagic manifestations
5. Susceptibility to infections: particularly respiratory tract
6. Less common findings: mediastinal pressure, tonsillar enlargement, disturbed vision, and
bone and joint pains

Laboratory diagnosis
CBC:
Hb: mild to moderate anemia

Total WBC count: elevated

Platelets: normal to moderately reduced

Peripheral smear:
RBC: normocytic, normochromic anemia; mild reticulocytosis

WBC: Leukocytosis with uniform population of mature small lymphocytes with round nuclei,
clumped chromatin, scanty cytoplasm and regular cellular outline. Broken cells known as
‘smear’ or ‘smudge’ cells are characteristic but not pathognomonic, since they are seen in
other conditions also. They are damaged nuclei of fragile malignant lymphocytes.

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 Absolute neutrophil count is normal. Granulocytopenia is seen in advanced cases.

Bone marrow:
Cellularity: Hypercellular; with increasing number of lymphocytes
Myeloid precursors: reduced
Erythropoiesis: reduced
Megakaryocytes: normal or reduced

Other tests: Coomb’s test: positive in 20% cases

Immunophenotyping:
1. Positive for T-cell marker: CD5
2. Positive for pan B-cell markers: CD19, 20

Cytogenetics: Trisomy 12 seen in 25% cases

MULTIPLE MYELOMA

Plasma cell disorders or dyscrasias

Plasma cell disorders are characterised by abnormal proliferation of immunoglobulin-producing
cells, resulting in accumulation of monoclonal immunoglobulin in the serum and urine.

Plasma cell disorders or dyscrasias include:

1. Multiple myeloma
2. Localised plasmacytoma
3. Lymphoplasmacytic lymphoma
4. Waldenström’s macroglobulinemia
5. Heavy chain disease
6. Primary amyloidosis
7. Monoclonal gammopathy of undetermined significance (MGUS)

MULTIPLE MYELOMA (MM)

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Introduction
Multiple myeloma is a plasma cell neoplasm characterized by multifocal involvement of the
skeleton. Late in its course, it may spread to lymph nodes and extranodal sites like skin, spleen,
liver, kidney and lungs.

Pathogenesis
The risk factors for development of myeloma include:
1. Radiation exposure
2. Occupational hazard: farmers, wood workers, leather workers and those working in
petroleum products

The cell of origin for MM is post-germinal center B cell that homes to the bone marrow and
differentiates into plasma cell. Myeloma cells result from hypermutation of Ig genes in these
cells. Common karyotypic abnormalities seen are:
1. t(11;14)(q13;q32)
2. t(4;14)(p16;q32)
3. deletion 13q

Loss of control of cell proliferation occurs due to mutation in cell cycle regulatory genes,
cyclin D1 and D3. Proliferation and survival of myeloma cells is depend on several cytokines,
especially IL-6.

In normal plasma cells, there is a tight balance in the production and coupling of heavy and light
chains, while neoplastic plasma cells produce excess of heavy (IgA, IgG, IgM) or light [kappa
(k) or lambda (l)] chains. In MM, there is excessive production of IgG heavy chain and k light
chain, together called IgGk (‘M’ component or protein).

The free light chains, called Bence-Jones proteins are small enough to be excreted in the urine.

Myeloma derived MIP1a leads to overexpression of NF-kB ligand (RANKL) by marrow

stromal cells, which leads to activation of osteoclasts. Besides, modulators of Wnt pathway
released inhibit osteoblastic function. The net effect is marked bone reabsorption resulting into
hypercalcemia and pathological fracture.

Clinical features
Peak age of incidence: 65 to 70 years
More common in men

The bones most commonly affected in descending order of frequency are:

1. vertebral column
2. ribs
3. skull
4. pelvis
5. femur
6. clavicle

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 7. scapula

S/s due to growth of tumor in the bones:

- chronic bone pain
- pathological fracture
- hypercalcemia can give rise to neurological manifestations like confusion, weakness,
lethargy, constipation, and polyuria

S/s due to production of excessive Ig:

- Renal failure occurs in 50% of these patients. It is due to Bence Jones proteinuria, the
excreted light chains are toxic to renal tubular epithelial cells. Hypercalcemia and
amyloidosis too contribute to renal dysfunction.

S/s due to suppression of normal humoral immunity:

- susceptibility to infections

Diagnosis
X-ray:
Lesions begin in the medullary cavity, erode cancellous bone, and progressively destroy the bony
cortex, often leading to pathologic fractures. On X-ray, these lesions appear as punched-out
defects of 1 to 4 cm diameter.

CBC:
Anaemia
Moderate leukopenia
Moderate thrombocytopenia

Peripheral smear examination:

- normocytic normochromic anaemia
- rouleaux formation is characteristic but not specific. It occurs due to high level of M
proteins causing red cells to stick to one another in a linear fashion

Urine examination:
Positive for proteins and Bence Jones proteins

Protein electrophoresis and immunofixation of serum (or urine):

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Monoclonal Igs are first detected as abnormal protein “spikes” in serum (or urine)
electrophoresis and then further characterized by immunofixation.

Polyclonal IgG in normal serum appears as a broad band (arrow); in contrast, monoclonal IgG in
a patient of multiple myeloma appears as single sharp protein band in the region (arrow head).

The suspected monoclonal Ig is recognised by immunofixation after making it react with specific
antisera. The sharp band in the patient serum for IgG heavy chain and k light chain, indicates the
presence of IgGk (M protein), typical of multiple myeloma. Levels of polyclonal IgG, IgA and
l chains are reduced in MM.

Serum protein electrophoresis (SPEP) by HPLC (or urine):

Normal “M” spike in multiple myeloma

Bone marrow examination:

Marrow contains an increased number of plasma cells, constituting >30% of the cellularity.
Plasma cells have eccentric nucleus with perinuclear clearing due to prominent golgi apparatus.
Plasmablasts and bizarre multinucleate cells may be seen.
Excessive Ig secretion often leads to intracellular accumulations, producing variants of plasma
cells like:
- flame cells with fiery red cytoplasm
- mott cells with multiple grapelike cytoplasmic droplets
- plasma cells with inclusions (fibrils, crystalline rods, globules). When inclusions are
found in the cytoplasm, they are called Russell bodies and when found in the nucleus,
they are called Dutcher bodies.

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 Flame cells Rott cells

Russell bodies Dutcher bodies

Prognosis
Prognosis is variable but generally poor
Median survival rate: 4-6 years
In patients with multiple bony lesions: 6-12 months
Newer cytotoxic drugs like protease inhibitors and thalidomide are bringing hope
Biphosphates reduce bone absorption
Bone marrow transplantation is being tried

HODGKIN’S LYMPHOMA (HL)

Definition
"Hodgkin lymphoma" (HL), encompasses a group of lymphoid neoplasms that:
- arise in a single node or chain of nodes
- spread first to the anatomically contiguous nodes
- are characterized morphologically by the presence of neoplastic giant cells called Reed-
Sternberg (RS) cells or it’s variants, which make up only a minor fraction (1-5 %) of
tumor mass.
- whose predominant component is comprised of non-neoplastic inflammatory cells
(reactive lymphocytes, plasma cells, histiocytes, eosinophils and neutrophils).

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Incidence
HL comprise 20-30% of all malignant lymphomas.

Classification
The WHO classification recognizes five subtypes of HL, which are divided in two groups:
I. Classic HL:
1. Nodular-Sclerosing
2. Mixed-Cellularity
3. Lymphocyte-Rich
4. Lymphocyte-Depleted
II. Lymphocyte-Predominant HL

Basis of grouping:
RS-cells in the first four subtypes have a similar immunophenotype (CD45-, CD5-, CD20-,
CD15+, CD30+); as a result, these subtypes are clubbed together as “Classical forms of HL”.

In lymphocyte-Predominant HL, the RS-cells have a characteristic B-cell immunophenotype

(CD20+, BCL6+) distinct from that of the classical HL subtypes, hence grouped separately.

Etiology and pathogenesis

RS-cells in “Classic forms of HL”, express B-cell markers (CD15 and CD30), thus are believed
to have arisen from germinal center B-cells.

EBV is identified in 70% of Mixed-Cellularity HL, 40% of Lymphocyte-Rich HL and most of

Lymphocyte-Depleted HL. Thus, EBV is thought of playing a causative role, triggering germinal
center B-cells to become neoplastic.

It has been observed that EBV-positive and EBV-negative individuals contain high levels of
activated NF-κB, a transcription factor that normally stimulates B-cell proliferation and protects
B-cells from pro-apoptotic signals. It is now known that several EBV proteins activate NF-κB.

RS-cells secrete a number of cytokines, IL-5, IL-13, TGF-β, which recruit the inflammatory
cells. Hence there is predominance of non-neoplastic inflammatory component in HL.

RS-cells and it’s variants

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 Mummified RS-cell

Classic Reed-Sternberg cell:

- large cell (15-45 μm in diameter)
- bi-lobed nucleus, the two nuclear lobes face each other (“mirror image”) resulting in an
“owl eye” appearance
- nucleus shows presence of a large inclusion-like central nucleolus, surrounded by a clear
halo
- nuclear chromatin is vesicular
- the nuclear membrane is thick and sharply defined
- cell has abundant weakly acidophilic or amphophilic cytoplasm

Lacunar variant:
- large cell (40-50 µm in diameter)
- folded or multi-lobated nucleus with smaller size nucleoli
- surrounded by abundant clear cytoplasm, giving an appearance of the nucleus sitting in
an empty hole (the lacuna)

Mononuclear variant:
- contain only a single round or oblong nucleus with a large inclusion-like nucleolus

Mummified or Zombie cells:

- necrotic RS-cells, characterized by pyknotic nucleus and intense cytoplasmic eosinophilia

Lymphohistiocytic (L&H) or popcorn cell:

- folded multilobed nucleus resembling popcorn kernels
- smaller nucleoli
- moderately abundant cytoplasm

Classic HL, Nodular-Sclerosing type

Incidence: most common form of HL, constituting 65% to 70% of cases

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Clinical features:
- affect adolescents or young adults
- males and females are affected equally
- has propensity to involve the lower cervical or supraclavicular, with mediastinal lymph
nodes
- most present in stage I/II

Morphology:
- there is diffuse effacement of the nodal architecture by collagen bands dividing lymphoid
tissue into circumscribed nodules; and thus, the nomenclature.
- shows “lacunar variant” of the RS-cells. Classic RS-cells are less frequent.
- the non-neoplastic inflammatory component is polymorphous comprised of small
lymphocytes, eosinophils, plasma cells, and macrophages.
- large geographic areas of necrosis are often seen. RS cells may also show necrosis,
presence of mummified variant.

IHC: CD45-, CD5-, CD20-, CD15+, CD30+, EBV-

Prognosis: excellent

Hodgkin Lymphoma, Mixed-Cellularity Type

Incidence: constitute about 20% to 25% of cases

Clinical manifestations:
- biphasic incidence, peaking in young adults and again in adults older than 55 years
- more common in males
- subdiaphragmatic disease is more common
- patients generally present in advanced tumor stage III/IV

Morphology:
- characterized by diffuse effacement of lymph node architecture by a heterogeneous
cellular infiltrate, of small lymphocytes, eosinophils, plasma cells, and macrophages, thus
the nomenclature.
- admixed among them are plentiful number of classic RS-cells and their mononuclear
variant.

IHC: CD45-, CD5-, CD20-, CD15+, CD30+, 70% EBV+

Prognosis: not as favorable as “Nodular-Sclerosing” and “Lymphocyte-Predominant”

Hodgkin Lymphoma, Lymphocyte-Rich Type

Incidence: uncommon form of Classic HL

Clinical features:

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 - Older adults (median age 43 years)
- M >F
- Presents in stage I/II
- Mediastinal involvement is common

Morphology:
- lymph nodes are diffusely effaced, but vague nodularity may be seen
- presence of frequent mononuclear and classic RS- cells
- monomorphic inflammatory infiltrate, comprised of reactive lymphocytes, thus the name

IHC: CD45-, CD5-, CD20-, CD15+, CD30+, 40% EBV+

Prognosis: very good to excellent

Hodgkin Lymphoma, Lymphocyte-Depleted Type

Incidence:
Least common form of HL, amounting to < 5% of cases.

Clinical features:
- Older patients
- More commonly seen in HIV-positive individuals and in developing countries
- constitutional symptoms are common
- peripheral lymphadenopathy is common in “Reticular form” and BM involvement in
“diffuse fibrosis type”
- Subdiaphragmatic disease or organomegaly is common
- presents in advanced stage

Morphology: Characterized by paucity of lymphocytes and a relative abundance of pleomorphic

variants of RS-cells

IHC: CD45-, CD5-, CD20-, CD15+, CD30+, most EBV+

Prognosis: overall outcome is somewhat less favorable than other subtypes

Hodgkin Lymphoma, Lymphocyte-Predominant Type

Incidence: uncommon variant, accounting for approximately 5% of all cases

Clinical features:
- majority of patients are males
- usually younger than 35 years of age
- present with cervical or axillary lymphadenopathy
- mediastinal and bone marrow involvement is rare

Morphology:

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 - characterized by nodal effacement by expanded B-cell follicles
- follicles are comprised by small lymphocytes admixed with variable numbers of
histiocytes
- typical RS-cells are extremely difficult to find. More common are so-called
lymphohistiocytic (L&H) variants ("popcorn cell").

IHC: CD20+, CD15-, CD30-, BCL6+, EBV-

Prognosis:
- excellent
- however, this form of HL is more likely to recur than the classical subtypes
- 3% to 5% of cases transform to diffuse large B-cell lymphoma

Stage
I Involvement of a single lymph node region alone
IE Along with involvement of a single extralymphatic organ or site
II Involvement of two or more lymph node regions on the same side of the diaphragm alone
Along with involvement of limited contiguous extralymphatic organ or tissue
IIE
III Involvement of lymph node regions on both sides of the diaphragm
IIIS Along with spleen
IIIE Along with limited contiguous extralymphatic organ or site
IIIES Involving all the three
IV Multiple or disseminated foci of involvement with one or more extralymphatic organs or
tissues

All stages are further divided on the basis of the absence (A) or presence (B) of the constitutional
symptoms: fever, night sweats, and/or unexplained weight loss of >10%.

Prognosis

HL are now curable in most cases.

Cure rate for stages I and IIA is close to 90%. Even with advanced disease (stages IVA and
IVB), 60% to 70% 5-year disease-free survival is obtained.

Mixed-Cellularity type and Lymphocyte-Deleted type are associated with less favorable
prognosis.

Long-term survivors of chemotherapy and radiotherapy have an increased risk of developing

second cancers:
1. Myelodysplastic syndromes
2. AML
3. lung cancer
4. NHL
5. breast cancer
6. gastric cancer

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 7. sarcoma
8. malignant melanoma

IMPORTANT QUESTIONS

SHORT NOTES
1. Define and classify leukemia. Describe laboratory diagnosis of acute myeloid leukemia.
2. FAB classification of leukemia
3. Chronic myeloid leukemia
4. Classify Lymphomas. Describe the pathology of Hodgkin’s lymphoma.
5. Discuss and describe real classification of non-Hodgkin’s lymphoma.
6. Myelodysplastic syndromes- aetiology, types, pathogenesis and investigations
7. Multiple myeloma

SHORT ANSWERS
Note: Answers only to those questions not addressed in the text or written scattered are given here.

1. Reed Sternberg cell

2. Cytochemistry in AML
3. Peripheral smear findings in AML
4. Peripheral smear findings in CML

The End

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