This document provides information on the diagnosis and management of various types of leukemia. It discusses the main types of leukemia - acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). For AML and ALL, it describes the diagnostic criteria, risk factors, immunophenotyping, cytogenetics, molecular genetics, treatment approaches including induction and consolidation chemotherapy. It also discusses newer targeted therapies like CAR T-cells and CRISPR for leukemia treatment. For CML, it explains the defining Philadelphia chromosome and BCR-ABL fusion gene which causes the disease.

1. DIAGNOSIS AND MANAGEMENT
Presented by - Dr PRIYA A
Guided by – Dr Daruka sir

2. Leukemia
 Leukemia are malignant disorders of the hematopoietic stem cell
compartment, characteristically associated with increased numbers of
white cells in the bone marrow and/or peripheral blood
 leukemia are traditionally classified into four main groups:
• acute lymphoblastic leukemia (ALL)
• acute myeloid leukemia (AML)
• chronic lymphocytic leukemia (CLL)
• chronic myeloid leukemia (CML).

3. Risk factors for leukemia
Ionising radiation
• After atomic bombing of Japanese cities (myeloid leukemia)
• Radiotherapy
• Diagnostic X-rays of the fetus in pregnancy
Cytotoxic drugs
• Especially alkylating agents (myeloid leukemia, usually after
latent period of several years)
• Industrial exposure to benzene

4. Retroviruses
• Adult T-cell leukemia/lymphoma (ATLL) caused by human T-cell
lymphotropic virus 1(HTLV-1, most prevalent in Japan, the Caribbean and
some areas of Central and South America and Africa
Genetic
• Identical twin of patients with leukemia
• Down’s syndrome and certain other genetic disorders
Immunological
• Immune deficiency states (e.g. hypogammaglobulinaemia A)

5. Acute leukemia
• There is a failure of cell maturation in acute leukemia.
• Proliferation of cells that do not mature leads to an accumulation of
primitive cells that take up more and more marrow space at the
expense of the normal hematopoietic elements.
• Acute myeloid leukemia (AML) is about four times more common
than acute lymphoblastic leukemia (ALL) in adults.
• In children, the proportions are reversed, the lymphoblastic variety
being more common.

6. ACUTE MYELOID LEUKEMIA(AML)
 Most common leukemia in india
 More common in males>females, average age >65years
 >20% marrow filled with myeloblast
 Loss of differentiation of myeloblast
 Pancytopenia
 Lymphadenopathy,hepatosplenomegaly : uncommon
 Onset within 2-3 months
 Hyperuricemia
 Skin involvement seen

11. DIAGNOSIS OF AML
 MORPHOLOGICAL FEATURES
- CBC : pancytopenia
increased WBC (bad prognosis)
- Peripheral blood smear : myeloblast (Auer rods m3>m2)

12.  CYTOCHEMISTRY
Positive for Myeloperoxidase and sudan black
 IMMUNOPHENOTYPING
To confirm the diagnosis of AML
 Myeloblast – CD11b,CD13,CD15,CD33,CD117,HLA-DR
 Myelomonocyte - CD11b,CD13,CD14,CD15,CD32,CD33,HLA-DR
 Erythroid – spectrin,ABH ambigens, glycophonin,carbonic anhydrase 1,
HLA-DR
 Promyelocytic – CD13,CD33
 Monocytic – CD11b,11C,CD13,CD14,CD33,CD65,HLA-DR
 Megakaryoblastic – CD34,CD41,CD42,CD61,anti-von Willibrand factor

13.  CYTOGENETICS
- Translocation( 15;17) : PML –RAR alpha best prognosis
- t ( 8;21 ) : RUN –X1 – RUN X1 good prognosis except for m2
- t (16;16 ) : associate with inversion 16
good prognosis except for m4
- any translocations other than ( 16;16 , 8;21, 15;17 ) have bad prognosis
 MOLECULAR GENETICS
- Nucleophosmin Gene mutation(M/C) : good prognosis
- CEBPA( cancer enhancer binding protein) : good prognosis
- FLT3 – ITD : bad prognosis

14.  BLOOD PICTURE IN AML
 Anaemia
 Thrombocytopenia
 Platelet <50000/microliter
 TLC < 5000/ microliter in 50%
 10% have counts > 1,00,000/microliter
 BONE MARROW PICTURE IN AML
 3-95% leukemic blast cells
 >20% is the arbitrary cut off
 Mpo and Sudan black positive
 Auer rods : m3>m2
 Decrease in granulopoiesis,erythropoiesis,megakaryopoiesis
 Marrow fibrosis is minimal

15. TREATMENT FOR AML
 The first decision must be whether or not to give specific treatment to
attempt to achieve remission.
 This is generally aggressive, has numerous side-effects, and may not be
appropriate for the very elderly or patients with serious comorbidities
 In these patients, supportive treatment can effect considerable
improvement in well-being.
 Low-intensity chemotherapy, such as low-dose cytosine arabinoside or,
recently, azacitidine, is frequently used in elderly and more frail patients
but only induces remission in less than 20% of patients

16.  Specific therapy
- Ideally, whenever possible, patients with acute leukemia should be
treated within a clinical trial.
- The aim of treatment is to destroy the leukaemic clone of cells without
destroying the residual normal stem cell compartment from which
repopulation of the haematopoietic tissues will occur.
- Patient should be prepared as follows before the specific therapy
1. Existing infections identified and treated (e.g. urinary tract
infections, oral candidiasis, dental, gingival and skin infections)
2. Anaemia corrected by red cell concentrate transfusion

17. 3. Thrombocytopenic bleeding controlled by platelet transfusions
4 . If possible, central venous catheter (e.g. Hickman line) inserted
to facilitate access to the circulation for delivery of chemotherapy,
fluids, blood products and other supportive drugs
5. Tumour lysis risk assessed and prevention started: fluids with
allopurinol or rasburicase
6 .Therapeutic regimen carefully explained to the patient and informed
consent obtained
7. Consideration of entry into clinical trial

18. Drugs commonly used in the treatment of acute myeloid
leukemia
Induction Phase : Daunorubicin (IV)
Cytarabine (IV)
Etoposide (IV and oral)
Gentuzumab ozogamicin (IV)
All-trans retinoic acid (ATRA) (oral)
Arsenic trioxide (ATO)
 CONSOLIDATION PHASE : Cytarabine (IV)
Amsacrine (IV)
Mitoxantrone (IV)

19.  RELAPSE PHASE : Fludarabine
Cytarabine
Arsenic trioxide (ATO)
Idarubicin
 Aggressive front line chemotherapy in fit patients
- < 40yrs,without comorbid condition
- 7 + 3 days regimen
7 – cytarabine ( 100-200mg /m2/day)
3 – daunorubicin (60-90mg/day)
- from day 7 : period of strict vigilant monitoring
- from day 14 : bone marrow study
look for remission (molecular> morphological)

20.  Morphological remission
 Blasts - <5%
 Platelet - >100000
 Absolute eosinophilic count >100
 Molecular remission : minimal residual disease is not detected
on flow cytometry
- If there is a morphological remission – Consolidate
- If tehre is no morphological remission – Repeat 7+ 3 regime

24. ACUTE LYMPHOBLASTIC LEUKEMIA
 In acute lymphoblastic leukemia (ALL), the malignant clone arises
from hematopoietic progenitors in the bone marrow or lymphatic
system resulting in an increase of immature nonfunctioning leukemic
cells.
 Infiltration of bone marrow leads to anemia, granulocytopenia, and
thrombocytopenia with the clinical manifestations of fatigue,
weakness, infection, and hemorrhages.

25.  These symptoms are more often the reason a patient first seeks
medical advice rather than consequences of tumor bulk, such as
lymph node enlargement, hepatosplenomegaly caused by leukemic
infiltration, or symptoms of the central nervous system
 ALL is the most frequent neoplastic disease in children with an
early peak at the age of 3–4 years

26.  Patients with some rare congenital chromosomal abnormalities have a
higher risk of development of acute leukemia; e.g., Klinefelter’s
syndrome, Fanconi’s anemia, Bloom’s syndrome, ataxia telangiectasia,
and neurofibromatosis.
 There is a twentyfold increased incidence of leukemia in patients with
Down syndrome, in whom ALL is increased in childhood or AML at an
older age.
 Most common type of ALL is Pre B cell type which has a good
prognosis rest other like Pro B cell type, Pro T cell,Pre T cell type has
bad prognosis

27. Acute Lymphoid Leukemia - Diagnosis
 Bood counts = low
 Peripheral Smear = lymphoblast
 Bone marrow = > 20% blast cells
if the counts are high = very bad prognosis

28. Cytological
 Stains : PAS ( Periodic Acid Schiff ) positive
 Acid phosphatase positive (only for T - Cell all)
Immunophenotyping
 Early pre 6-Cell ALL : CD11,CD19,CD79a,TdT Positive
 If associated with tumor : CO20, CD22 also present
 Pro 6 - Cell ALL CD10 absent
Cytogenetics
1) Children – t(12:21) – good prognosis
2) Adults - t (9:22), t(4:11),t(1:19) has got poor prognosis and poor
response

29. Molecular genetics
 NOTCH-1 and HOX-11 seen in T cell ALL has very good prognosis

32. Acute lymphoid leukemia -Treatment
 Cure rate – Adults – 40-50%
Children – 90%
 Induction
- Steroids + vincristine/ Daunorubicin +/- L-Asperginase( may increase
the risk of pancreatities,thrombosis)
- High dose Methotrexate : good CNS penetration
- Accompanied by intrathecal methotrexate : Prevents CNS relapse

33.  Consolidation
- Repeated cycles of vincristine, Daunorubicin, Methotrexate
Cytarabine
Induction Remission Consolidation
(molecular)
if not achieved
Treatment AHSCT( Autologous Hematopoietic
Cell Transplant)

34.  Maintenance
6- mercaptopurine + methotrexate ends at 2- 3 years
at the end of 3 years patient with no relapse : cured

36. • Chimeric Antigen Receptor (CAR) T cells
- The adoptive transfer of CAR-modified T cells directed against CD19 is a
promising approach to the treatment of CD19+ childhood or adult ALL.
• CRISPR gene editting technique
- The CRISPER gene editing technique is used in humans to remove specfic
genes to allow the immune system to be more activated against cancer.
- Recently,this CRISPER technology has used to insert genes that allow
immune cells to attack cancer cells,potentially leaving normal cells
unharmed and increasing the effectiveness of immunotherapy

37. CHRONIC MYELOID leukemia
 Chronic myeloid leukemia (CML) is a myeloproliferative stem cell
disorder resulting in proliferation of all haematopoietic lineages but
manifesting predominantly in the granulocytic series.
 Maturation of cells proceeds fairly normally.
 The disease occurs chiefly between the ages of 30 and 80 years, with a
peak incidence at 55 years.
 The defining characteristic of CML is the chromosome abnormality
known as the Philadelphia (Ph) chromosome

38.  The break on chromosome 22 occurs in the breakpoint cluster region
(BCR).
 The fragment from chromosome 9 that joins the BCR carries the abl
oncogene, which forms a fusion gene with the remains of the BCR.
 This BCR ABL fusion gene codes for a 210 kDa protein with tyrosine
kinase activity, which plays a causative role in the disease as an
oncogene, influencing cellular proliferation, differentiation and
survival.
 In some patients in whom conventional chromosomal analysis does not
detect a Ph chromosome, the BCR ABL gene product is detectable by
molecular techniques.

39. The disease has three phases:
• A chronic phase :
- in which the disease is responsive to treatment and is easily controlled, which
used to last 3–5 years
- With the introduction of imatinib therapy, this phase has been prolonged to
encompass a normal life expectancy in many patients.
• An accelerated phase (not always seen) :
- in which disease control becomes more difficult

40. Blast crises :
- In this phase the disease will transforms into an acute leukemia,either
Myeloblast(70%) or Lymphoblastic (30%), which is relatively
refractory to treatment
- This is the cause of death in majority of patients, therefore survival is
dictated by the timing of blast crisis,which cannot be predicted.

41. Clinical features
 Symptoms at presentation may include lethargy, weight loss,abdominal
discomfort, gout and sweating, but about 25% of patients are asymptomatic
at diagnosis.
 Splenomegaly is present in 90%; in about 10%, the enlargement is massive,
extending to over 15 cm below the costal margin.
 A friction rub may be heard in cases of splenic infarction.
 Hepatomegaly occurs in about 50%.
 Lymphadenopathy is unusual.

42. DIAGNOSIS OF CML
 Blood picture :-
- decreased hemoglobin
- mild anisopoikilocytosis
- WBC count >25000/microliter
- 50% have count >1,00,000/microliter
- cytochemically abnormal WBC: LOW LAP
- Absolute eosinophil count is increased
- Thrombocytosis : hemorrhage and thrombosis are
very rare
- Basophilia/ tryptase raised

43. - Wright geimsa stain showing bone marrow hypercellularity with
myeloid hyperplasia
 Bone marrow
- marked hypercellularity
- predominant granulopoiesis ( 30:1)
- decreased erythropoiesis
- Dwarf megakaryocytes increased
- Sea blue histiocytes/ pseudo gaucher
cells

44.  Molecular diagnosis
- 95% will have philadelphia chromosome
G banding : sent in T lymphocytes
- 5% can still have BCR-ABL transcripts detectable by PCR
- FISH: is specific for ( 9:22) translocation
- Deletion of 20q, trisomy 8, del 3q, double philadelphia chromosome
has Bad prognosis
 Chemical findings
uric acid - high
LDH - high
histamine - high
angiogenic factor - high

45. PHASES OF AML
 ACCELERATED PHASE
- Persistant or increase in WBC count
- Persistant or increase in splenomegaly
- Persistant thrombocytopenia
- cytogenetic evaluation
- Basophilia in blood > 20%
- Blast in blood/ bone marrow 10- 19% or 5-19%

46.  BLASTIC PHASE
- CML transforming into AML,myeloid sarcoma
- Blast > 20% in blood/bone marrow
- LAP increased
- myeloid > lymphoid

48. Treatment of CML
 Thyrosine kinase inhibitors

49.  Starting with Imatinib lifelong therapy
- Chronic phase : 400mg/day
- Blast/ accelerated phase : 600-800mg/day
 Side effects : Peri orbital edema,hypophosphatemia,diarrhoea
skin rashes and myelosuppression
 Nilotinib and Dasatinib can be started without imatinib also,
indicated if there is any thyrosine kinase domain mutation
- Dasatinib : pulmonary HTN, QT prolongation
- Nilotinib: PVOD,pancreatitis
- Ponatinib : used if there is T3151 mutation

50.  First 2 weeks we look for complete hematological response
- WBC <10,000
- Platelet < 4.5L
- Peripheral smear is normal
- Patient is asymptomatic
 Complete molecular response
- By 3 months
- BCR-ABL transcripts are undetectable by PCR
 Complete cytogenic response
- 6 months
- No cells contains ph chromosome

51.  Response to Imatinib :
- Full blood counts every 2 weeks
- Bone marrow every 6 months to assess cytogenic response
- Quantitative PCR For BCR- ABL 1 transcripts every 3 months
 Treatment of accelerated and blast phase :
- Allogenic transplant ( when patient comes back to chronic
phase)
- 40% response to Imatinib 600mg daily
- Imatinib plus chemotherapy : Blastic phase

54. CHRONIC LYMPHOCYTIC LEUKEMIA
 Chronic lymphocytic leukemia (CLL) is a monoclonal proliferation of
mature B lymphocytes defined by an absolute number of malignant cells
in the blood (5 × 109/mL).
 The presence of malignant B cells under this count in the blood without
nodal, spleen, or liver involvement and absent cytopenias is a precursor of
this disease called monoclonal B-cell lymphocytosis (MBL) with ~1–2%
chance per year of progressing to overt CLL.

55.  CLL is a heterogeneous disease in terms of natural history, with some
patients presenting asymptomatically and never requiring therapy,
• Whereas others present with symptomatic disease, require multiple
lines of therapy, and eventually die of their disease
 CLL is primarily a disease of older adults, with a median age at diagnosis
of 71 years
 The male:female ratio is 2:1; however, as patients age, the ratio becomes
more even, and over the age of 80, the incidence is equal between men
and women

56.  The morphology, immunophenotype, and gene expression pattern of
CLL cells are that of a mature B cell, and so it has been presumed that
the initiating cell is a mature lymphocyte, perhaps memory B
 Monoclonal B cell count > 5000cells/microliter– CLL
 Monoclonal B cell count < 5000cells/microliter – monoclonal B cell
Lymphocytosis
- Bone marrow not involved, perpheral blood is involved
 CD markers ; CD19,CD20,CD21,CD22,CD79a,CD79b,S1gm
 On mature B cell CD5,CD23 is strongly expressed

57. CYTOGENETIC ABNORMALITIES
 The most well-characterized abnormalities include del(13) (q14.3),
trisomy 12, del(11)(q22.3), and del(17)(p13.1)
 The presence of sole del(13)(q14.3) is associated with more indolent
disease, prolonged survival, and good response to traditional therapies.

58.  GENE MUTATIONS
- Whole genome and whole exome sequencing
have identified the most common mutations in
CLL to be in SF3B1, NOTCH1, MYD88, ATM,
and TP53
- Mutations of the tumor suppressor TP53 are
found in ~5% of CLL in previously untreated
early stage disease and up to 40% in later
stages.
- TP53 mutations are associated with a poor
prognosis and expected lack of response to
DNA-damaging therapies.

59.  Clinical features
- Asymptomatic (80%0
- Painless lymphadenopathy
- warm antibody AIHA( auto immune hemolytic anaemia)
- Splenomegaly ( only as apart of SLL)
- Spontaneous regression – 1-2%
- Pancytopenia rare

60.  Peripheral blood smear
- small, mature lymphocytes
with scanty bluish cytoplasm
and moderate condensation
- smudge cells

61.  Fish – cytogenetics in CLL
1. 13q deletion assosiated with mutated Ig heavy chain –Good
prognosis
2. Trisomy 12,Deletion 11q, Deletion 17p – Bad prognosis
 Coombs test - To detect warm antibody AIHA

63. Treatment of CLL

64.  First line of treatment
FCR regime : Fludarabine, Cyclophosphamide,Rituximab
BR regime : Bendamustine,Rituximab
< 60 year FCR regime, >60 year BR regime
 Antiapoptotic Therapies
- BCL2 is another promising target in CLL.
- Venetoclax is an orally bioavailable, selective BCL2 inhibitor.
- It is currently Food and Drug Administration (FDA) approved for
marketing in patients with relapsed or refractory CLL who have the
del(17)(p13.1).

65. Immune Therapies
 Current immune therapies include allogenic stem cell transplantation,
chimeric antigen receptor (CAR) T-cell therapy, and oral
immunomodulatory agents such as lenalidomide
 Stem cell transplantation is currently considered the only standard curative
approach to CLL.
 Because most CLL patients are older and many have significant comobidity
myeloablative transplants results in extensive morbidity and mortality, making
them prohibitive in many individuals
 Reduced intensity conditioning (RIC) allogeneic transplants have been
successfully incorporated into the treatment of patients up to ~75 years in age but
still have a ≥50% frequency of chronic graft-versus-host disease.

67. HAIRY CELL leukemia
 This is a rare chronic B-cell lymphoproliferative disorder.
 The male-to-female ratio is 6 : 1 and the median age at diagnosis is 50 years.
Presenting symptoms are general ill health and recurrent infections.
 Splenomegaly occurs in 90% but lymph node enlargement is unusual.
 Severe neutropenia, monocytopenia and the characteristic hairy cells in the
blood and bone marrow are typical.
 These cells usually have a B-lymphocyte immunotype but they also
characteristically express CD25 and CD103.
 Recently, all patients with hairy cell leukemia have been found to have a
mutation in the BRAF gene.

69.  Immunophenotyping :
Key markers : CD11c, CD25, CD103, CD123
Others – CD19, CD20, CD22
TREATMENT
 treatment of choice : Pentostatin / cladribine
 CD20 involvement : Rituximab
 BRAF inhibitor : Vemurafenib

70. THANK YOU