This document discusses the use of molecular cytogenetics techniques in the diagnosis and classification of hematological malignancies. It begins by outlining the indications for cytogenetic analysis in hematological neoplasms. It then describes several cytogenetic analysis methods including conventional cytogenetics, fluorescence in situ hybridization (FISH), multicolor FISH (mFISH), multicolor banding (mBAND), and array-based comparative genomic hybridization. The document discusses the approach to cytogenetic analysis in several hematological malignancies and lists some common translocations in lymphomas. It provides an overview of recurrent chromosomal abnormalities seen in several leukemias and lymphomas.

1. Molecular Cytogenetics
in Hematological
malignancy
Dr.Nikhil

2. Skeleton
• Indications of Cytogenetics in haematological neoplasm.
• Methods of cytogenetic analysis.
• Conventional Cytogenetic principle.
• FISH principle.
• mFISH , mBAND , Array based comparative genomic hybridization.
• Approach of Cytogenetics in Hematological malignancies.
• Translocations in lymphomas.

3. Indications of cytogenetic study
• Unknown cause of cytopenia.
• WHO classification of AML and ALL
• Risk stratification of ALL.
• MDS diagnosis ,Classification.
• Lymphoma diagnosis ,classification and staging work-up.
• Following up of treatment response.

4. Methods of Cytogenetic analysis
• Conventional Cytogenetics.(karyotyping)
• Fluorescence in situ hybridization (FISH).
• Multicolor FISH.
• Multicolor banding with high resolution.
• Array based comparative genomic hybridization.

5. Methods of cytogenetic analysis
• Classical cytogenetic is carried out on a cell that has entered into mitosis
Arrested in metaphase
• Individual chromosomes - recognized by their size
• Banding pattern following stain (e.g Geimsa staining [G-Banding] or
staining with fluorescent dye.
• Alternating dark and light bands are numbered from centromere toward
telomere to facilitate description of any abnormalities detected.

6. Conventional cytogenetics

7. G-banding Human male karyotype

8. Methods of Cytogenetic analysis
• Cytogenetic analysis can be carried out on following type of tissues
1.Skin fibroblasts or phytohemagglutinin (PHA ) Stimulated lymphocytes (to study
constitutional abnormalities ).
2. Bone marrow cells.
3.Blood cells .
4. Cells isolated from lymph nodes or other organs suspected of infiltrated by
lymphoid or other neoplasm.
5.Cells isolated from serous effusions

9. Methods of cytogenetic analysis
• In studying suspected haematological neoplasms
• There are two reasons for seeking to detect constitutional abnormalities
• 1)Constitutional abnormality underlying a haematological neoplasm.
2)Irrelevant and previously undetected constitutional chromosomal
abnormality that has to be recognised.
To distinguish from an acquired chromosomal abnormality associated with a
neoplastic process.

10. Methods of cytogenetic analysis
• A bone marrow aspirate for cytogenetic analysis should be
• Anticoagulated by the addition of preservative free heparin or tissue culture
medium containing heparin.
• The specimen can be stored at room temperature for some hours or at 4 °C if
delay in analysis is expected.
• with the exception of samples from patients with suspected lymphoid malignancy
• which should be stored at room temperature as disease cells may die very rapidly
at low temperatures.

11. Methods of cytogenetic analysis
• If samples are being sent to central laboratory :
• Detailed clinical and haematological information
• Central laboratory is aware if there is clinical urgency in obtaining the
results and so that appropriate techniques are used.

12. Methods of Cytogenetics
• FISH (Flouroscent insitu hybridization )bridges classical cytogenetic
analysis and molecular diagnostic techniques.
• Chromosomes can be stained and visualised but the technique is also
dependent on recognition of specific DNA sequences
• By means of a fluorescent probe that can anneal to a specific DNA
sequence.

13. • FISH can be carried out on metaphase preparations or cells in interphase.
• FISH probes may identify the following :
• Centromeres of a specific chromosome ( useful for detecting trisomy or
monosomy.)
• Specific oncogenes (locus specific probe useful for detecting
translocations).
• whole chromosome Paimting probes.
Methods of Cytogenetics

14. • Advantages of FISH in comparison with conventional chromosomal
analysis include the following :
• Many more cells can be examined.
• Metaphases are not essential. So abnormalities can be detected in non
dividing cells. So Useful in chronic lymphocytic leukemia , in which cell
rarely divide in culture.
• FISH can be performed in a shorter period of time(May be critical in
rapidly confirming a diagnosis acute promyelocytic leukemia.)
Methods of cytogenetics

15. • Abnormalities that are too subtle to be detected by conventional cytogenetic
analysis may be detected example STIL-TAL Fusion in T-lineage ALL or
t(12;21) in B-Lineage ALL.
The main disadvantage of FISH is that only those abnormalities that are
specifically sought will be found , whereas conventional cytogenetics permits
all chromosomes to be evaluated.
Methods of cytogenetics

16. Centromere specific probe
• For determination of numerical aberrations, for identification of
centromere in Marker chromosomes.

17. Locus specific DNA probes
• For detection of structural rearrangements

18. Chromosome painting probes
• Determination of structural rearrangements( translocations and deletion of
large extent )

19. Multicolor FISH
Allows in one hybridization experiment distinguish according to different
colour of every pair of autosomes and sex chromosomes and then it is
possible to make analysis of whole genome and every structural and
numerical rearrangement.

20. mFISH

21. mFISH
Analysis of complex chromosomal rearrangements in bone marrow cells of
patients with hematological malignancies will bring us detailed information
about involvement of specific chromosomes or their regions into genetic
rearrangements

23. mBAND
Multicolor banding with high resolution.
Enables determination of exact breakpoints of chromosomal aberrations with
higher resolution than classical banding.

24. Array- Based comparative Genomic
hybridization(aCGH)
• New tool to search for recurrent gains and loss of chromosomal regions
throughout the genome
• Very high resolution of copy number changes at DNA level.
• Recently it is utilised in diagnostics of leukemias and the results revealed a
large spectrum of genomic imbalances , including the novel recurrent
deletions and amplifications.

26. The impact of conventional and
molecular cytogenetic analysis
in onco-hematology
• help to specify diagnosis
• help to determine the prognosis
• help monitor effectiveness of treatment

27. Hematological malignacies
Acute myeloid
leukemia[AML]
Acute Lymphoblastic
leukemia[ALL]
Chronic Myeloid
Leukemia[CML]
Chronic lymphocytic
leukemia [CLL]
Myelodysplastic
syndromes [MDS]
Non-hodgkin
lymphoma[NHL]

28. Recurrent chromosomal abnormalities in
Hematological malignancies
• Abnormalities important for classification (Leukemias)
• AML t(8;21), t(15;17), inv(16), t(9;11), inv(3),
• t(6;9), t(1;22), -5/5q-, -7/7q-
• ALL t(4;11), t(1;19), t(v;11q23), t(12;21)
• CML t(9;22)
• MDS -Y, del(11q); del(5q), del(12p), del(20q);
• del(7q), +8, +19, i(17q); -7, inv(3)

29. Recurrent chromosomal abnormalities in
Hematological malignancies
• Lymphomas:
• Diffuse large B-cell lymphoma t(14;18)
• Burkitt lymphoma t(8;14)
• Follicular lymphoma t(14;18)
• Mantle cell lymphoma t(11;14)
• Marginal zone lymphoma t(11;18), t(1;14), t(14;18)

30. Acute leukemias

31. Leukemia
• Definition :Malignant disease of the bone marrow stem cell.

32. • Approach for diagnosis of Acute leukemias
• Clinical features , complete blood count , Peripheral blood smear , bone
marrow aspirate smear.
Assessment of morphology and percentage of blasts
>20 % Blasts <20% Blasts
AML ,B-ALL , T-ALL ,Mixed phenotypic AML with recurrent genetic abnormalities
Leukemia.

33. Case 1
• A 35 year old boy presented with one day history of bleeding gum ,
subconjuctival bleed and pupuric rash.
• Investigations revealed following results : Hb 6.4 g/dl , TLC 26,500/mm3 ,
Platelet 35,000 mm3. Prothrombin time 20 seconds with a control of 13
seconds , partial thromboplastin time -50 seconds. Fibrinogen 10 mg/dl
• Peripheral smear suggestive of Acute myeloblastic leukemia .
• Most likely cytogenetic abnormality in this case ?

34. Acute myeloid leukemia
• Heterogenous group of malignant disease of haemopoeisis
• Accumulation of immature myeloid cells (Myeloblasts) in the bone marrow
• Diagnosed in all age groups
• Most commonly affects the people older than 60 years (Median age 64-68 years)
• Specific chromosomal aberrations with clear prognostic significance 
stratification of therapy according to cytogenetic findings

35. FAB Classification of AML
• M0 –Minimally differentiated AML
• M1-AML without differentiation
• M2-AML with maturation
• M3-Acute promyelocytic leukemia
• M4-Acute myelomonocytic leukemia
• M5-Acute monocytic leukemia
• M6-Acute ertyhtroleukemia
• M7-Acute megakaryocytic leukemia

36. Acute myeloid leukemia
• Clinical findings :
• AML may develop at any age , but is more in common in adults
• Symptoms : Anemia , neutropenia and thrombocytopenia

37. Acute leukemias
• Anemia
• Fever
• Fatigue
• Bleeding
• Gum hypertrophy
• Hepatosplenomegaly
• Lymphadenopathy
Mediastinal mass

40. Lab findings in AML
• Hemoglobin: is decreased (5 to 9 g/dl)
• Peripheral smear :
• RBC :Normocytic normochromic anemia
• Total WBC count : markedly raised.
• Differential count : More than 20% Myeloid blasts
• Auer rods : Azurophilic needle like structures of varying length and
width

44. Bone marrow findings in AML
Cellularity : hypercellular due to proliferation of blasts.
Erythropoeisis : markedly suppressed.
Myelopoeisis : Myeloblasts constitute more than 20 % of cells
Megakaryopoeisis : gradually decreased.

45. Cytogenetic findings in AML
Chromosomal
aberrations
Genes FAB category Prognosis
t(8;21)(q22;q22) RUNX1-RUNX1T1 M2 Favourable
inv(16)(p13;1q22)or
t(16;16)(p13.1;q22)
CBFB-MYH1 M4 Favourable
t(15;17)(q22;12) PML-RARA M3 Good
t(9;11)(p22;q23) MLLT3-MLL M4,M5 Intermediate
t(6;9)(p23;q34) DEK-NUP 214 - Poor
Inv(3)(q21q26.2) or
t(3;3)(q21.3;q26.2)
GATA 2,MECOM - Poor
t(1;22)(p13;q13) RBM15-MKL1 - Variable
AML with mutated NPM1 - Favourable
AML with mutated
CEBPA
- Favourable

46. Acute myeloid leukemia
• Conventional Cytogenetics.
• Normal karyotype Aberrant karyotype
• Insufficient metaphases
Targeted FISH
Subtype specific FISH: PML/RARA, or other molecular cytogenetic
,CBFB etc. Techniques.

47. Case 2
• A 4 year old boy admitted with a history of abdominal pain and fever for
two months ,maculopapular rash for ten days and dry cough, dyspnea and
wheezing for three days.
• On examination liver and spleen were enlarged 4cm and 3 cm respectively
below the costal margin.
• Hb -10.0 g/dl
• Leukocyte count 70 x 109 /L

48. • Bone marrow examination revealed a cellular marrow comprising of 45 %
Blasts and 34% Eosinophil precursors.
• The blasts stained negative for myeloperoxidase and non specific esterase
• Positive for CD19,CD 10, CD 22 and CD20
• What is the diagnosis ?
• Most likely cytogenetic abnormality in this case ?

49. Acute lymphoblastic leukemia
• Accumulation of immature lymphoid cells in the bone marrow and mostly
also in peripheral blood.
• Most common malignancy in children representing 25 % of pediatric
cancer.
• 80 % of pediatric leukemias
• Heterogenous disease –distinct therapeutic and prognostic implications
• Chromosomal aberrations – found upto 90 % of cases

50. Clinical findings in ALL
• Age and sex : ALL is the most common hematological malignancy of children.
• Most commonly found between 1 and 5 years of age and second peak in adults
found between 30 and 40 years.
• Hepatosplenomegaly.
• Lymphadenopathy : 75% of patients.
• Mediastinal mass
• CNS involvement : may spread to meninges.
• Testicular enlargement

51. Lab findings in ALL
• Hemoglobin : progressively decreases and may be reduced even to 3g/dl.
• Total WBC count : Total white cell count is markedly raised ranging from 20 x
109 /L to 200 X 109/ L
• Peripheral smear :
• RBCs : Normocytic normochromic
• WBCs : Total count is usually markedly increased. Few may present with
pancytopenia. Blasts may replace normal myeloid series and can cause
neutropenia.

53. • Lymphoblasts : minimum requirement for diagnosis by WHO criteria is 20%
blasts or more.
• Platelets : Thrombocytopenia
• Bone Marrow :
• Cellularity : Bone marrow is markedly hypercellular due to proliferation of blasts
which replace normal hematopoietic cells.
• Erythropoiesis and myelopoiesis : Reduced
• Megakaryopoiesis : Reduced
Lab findings in ALL

54. Cytogenetic findings in Acute lymphoblastic
leukemia
Chromosomal abnormality Molecular abnormality Prognosis
Hyperdiploidy (>50) - Favourable
Hypodiploidy - Unfavourable
t(12;21)(p13;q22) TEL/AML1 Favourable
t(1;19)(q23;p13.3) PBX2/E2A Unfavourable
t(9;22)(q34;q11.2) BCR/ABL Unfavourable
t(v;11),(v;q23) MLL/AF4 Unfavorable
t(8;14)(q24;q32) MYC/IGH No effect

55. Risk stratification in ALL
Low-Risk Intermediate risk High-Risk Very High Risk
Hyperdiploidy Age 1-10 years E2A/PBX fusion BCR/ABL fusion with
high TLC
TEL/AML1 fusion TLC 50,000/mm
without genetic risk
factors
T-ALL MLL rearrangement
Age <1year, >10 year
And TLC
>50,000/cu.mm without
genetic risk factors

56. Acute lymphoblastic leukemia
• Conventional Cytogenetics.
Normal karyotype
Insufficient metaphases
B-ALL FISH Aberrant karyotype
• RUNX1
• Hyperdiploidy
• MLL
Targeted FISH
T-ALL or other molecular cytogenetic
TCR genes (TP16(9p21),ABL1(9q34) Techniques.

57. Case 3
• A 60 yr old man with fatigue , weight loss and heaviness in left hypochondrium
for 6 months.
• The hemogram showed Hb.10 g/dl TLC 5 lakhs/mm3 , Platelet count 4
lakhs/mm3 .
• DLC neutrophil 55% , lymphocytes 4 % , monocytes 2 % , basophils 6%,
metamyelocytes 10 % , myelocytes 18 % , promyelocytes 2 % and blast 3%.
• What is the diagnosis ?
• The most likely cytogenetic abnormality in this case is ?

58. Chronic myeloid leukemia
• 15-20 % of all cases
• Mainly in Adults(Median Age 65 years)
• Tri-phasic disease :
CP - chronic phase (relatively benign, 3-9 years)
Acute Phase - More malignant accelerated phase
• Blast Crisis - Terminal blast crisis
• Blasts increase rapidly (crowd out healthy cells)

59. • One of the best-studied malignancies - has served as a paradigm for
elucidation how genetic changes cause cells to become malignant:
Philadelphia chromosome t(9;22)(q34;q11) - BCR/ABL fusion
• One of the first malignancies in which a therapy targeting the underlying
molecular defect has improved the clinical outcome of patients: Gleevec™
(Novartis) used for therapy
• t(9;22)(q34;q11) in 90-95% of patients
• BCR/ABL fusion gene.

60. Clinical findings
• Commonest leukemia and 25-30 % of all leukemias in India.
• Age : fifth and sixth decades of life.
• Males are more commonly affected than females.
• Onset : Gradual with vague nonspecific symptoms
• Anemia , fatigue , weakness , weight loss , anorexia and fullness of abdomen due to
splenomegaly.
• Rarely present with bleeding.
• Leukostatic symptoms : Due to sludging in the pulmonary or cerebral vessels –Dyspnea ,
drowsiness .

61. Lab findings
• Hemoglobin : less than 11 g/dl.
• Peripheral smear :
• RBCs: Normocytic normochromic.
• WBCs : Leuocytosis. Total leucocyte count usually exceeds 100 x 109 /L
shift to left.Predominant cells are neutrophils and myelocytes.
Blasts are usually less than 10%
Basophils and eosinophilia usually seen.
• Platelets : range from normal to increased. Thrombocytosis.

62. Lab findings
• Bone marrow findings :
• Cellularity : Hypercellular
• M: E ratio exceeds 20 :1
• Erythropoiesis : decreased
• Megakaryocytes : Normal or increased dwarf megakaryocytes : small form of
megakaryocytes.
Sea blue histiocytes (Gaucher cells/pseudogaucher cells ) : scattered large storage
histiocytes with wrinkled green-blue cytoplasm

64. Pseudo-gaucher cells ,sea blue histiocytes
Scattered amongst marrow
cells are macrophages with
linear striations or granular
cytoplasm (pseudo gaucher
cells ) some with sea blue
colored granules resembling
histiocytosis.

65. Chronic myeloid leukemia
• Conventional Cytogenetics.
• Normal karyotype Aberrant karyotype
• Insufficient metaphases
• Normal karyotype Without t(9;22) Targeted FISH or other molecular
cytogenetic techniques.mFISH , mBAND
array comparative genomic hybridization.
FISH : BCR/ABL1

68. FISH in CML

69. Case 4
• A 80 year old Asymptomatic man present with a total leucocyte count of 1
lakh and with 80 % lymphocytes ?

70. Chronic lymphocytic leukemia
• Tumor composed of monomorphic small B lymphocytes in the peripheral
blood , bone marrow and lymphoid organs (spleen and lymph nodes )
• Age : 50 -60 years
• Present with non specific symptoms –fatigue , weight loss , anorexia.
• Generalized lymphadenopathy
• Few cases hepatosplenomegaly

71. Chronic lymphocytic leukemia
• Cytogenetics in CLL :
• Conventional cytogenetics is difficult.
• As only few dividing neoplastic lymphocytes.
• FISH can detect abnormalities in non dividing cells.
• No single cytogenetic abnormality specific for CLL.
• Common abnormalities include 13q-, 11q-,trisomy 12 , 17p-
• 11q- and 17p- are related with poor outcome.

72. Laboratory findings
• Hb usually below 13 g/dl , it may decrease below 10g/dl
• Peripheral smear
• RBCs: Normocytic normcohromic
• WBC : Lymphocytosis. Lymphocytes are mature in majority of cases.
• Smudge cells or basket cells : disintegrated lymphocytes represent the
spread out nuclearmaterial. Due to rupture of neoplastic lymphoid cells
while making the peripheral smear due to its fragile nature

73. • Bone marrow : hypercellular marrow
Infiltrated by lymphocytes
Erythropoeisis: Normal
Myelopoeisis : Normal
Megakaryopoeisis : Normal

74. Myelodysplastic syndromes
• Heterogeneous group of acquired clonal stem cell disorder characterized
by progressive cytopenias , dysplasia in one or more cell lines, ineffective
hematopoeisis and high risk of transformation to AML

75. Myelodysplastic syndromes
• Role of cytogenetics :
• Presumptive diagnosis of MDS in the presence of persistent unexplained cytopenia
• Absence of morphological features.
• Specific Cytogenetic abnormality is present :
• Unbalanced : -7, or del7q- or del5q , -13q del or 13q, del11q , del 12p, del 9q
• Balanced : t(11;16),t(3;21),t(1;3),t(2;11)
• Abnormality of >3 chromosomes have poor prognosis and increased risk of
progression to AML

76. Other Hematologic malignancies
• Burkitt lymphoma.
• Diffuse large B-cell lymphoma
• Follicular lymphoma
• Mantle cell lymphoma
• Marginal Zone lymphoma

77. B-Cell Leukemias/Lymphomas
B-Cell lymphoblastic leukemia
1.Burkitt lymphoma t(8;14), t (2;8), t(8;22)
2.Mantle cell lymphoma t( 11;14)
3.Follicular lymphoma t(14;18)(bcl-6)
4.Diffuse large B-cell lymphoma
5.Small lymphocytic leukemia
t(14;18)(bcl-2)
t(11;18)
T-cell leukemia/lymphomas
Anaplastic large cell leukemia t(7;11)

79. Minimal residual disease
• Minimal residual disease (MRD) is the name given to small numbers <0.01 %
of leukaemic cells (cancer cells from the bone marrow) that remain in the person
during treatment, or after treatment when the patient is in remission (no
symptoms or signs of disease).
• It is the major cause of relapse in cancer and leukemia.
• very sensitive molecular biology tests are available, based
on DNA, RNA or proteins.
• These can measure minute levels of cancer cells in tissue samples, sometimes as
low as one cancer cell in a million normal cells.

80. Recent advances in Molecular Cytogenetics for
Hematological malignancies
• Gene Expression Profiling Technique of gene expression profiling (GEP)
enables
• study of thousands of genes expressed in a tumor simultaneously.
• In the context of hematologic malignancies, GEP has been used to classify
tumors into various genetic subtypes.
• This technique is based on DNA microarray.
• In the field of Hematology GEP has been best utilized for diffuse large B
cell lymphoma.
• .

81. Recent advances in Molecular Cytogenetics for
Hematological malignancies
• Now GEP is being used for stratification of cases of various hematologic
neoplasm like plasma cell disorders,
• chronic lymphoproliferative disorders and lymphoma, into high and low
risk groups.
• However, at present availability of GEP is restricted to few research
centers only limiting its wide use in clinical practice

82. PCR
• The applications of PCR in hematologic malignancies include
• Detection of fusion genes
• Detection of mutations
• The best example of fusion gene is BCR-ABL1 in CML. The fusion gene
produces mRNA transcript which can be quantified using real time RT-PCR thus
allowing determination of response to treatment .(Major molecular response—as
defined by >3 log reduction in bcr-abl transcript), monitoring of residual disease
and treatment failure (2-5 fold increase in BCR-ABL1 transcripts)..

83. Next Generation Sequencing (NGS)
• Sanger sequencing (now called first generation sequencing) could sequence only a single DNA fragment at a time by
capillary electrophoresis.
• In NGS millions of DNA fragments can be sequenced simultaneously. Thus, it is a high throughput parallel sequencing
technique and allows thousands of genomes to be studied in a short time.
• Through NGS one can study not only the genome but also the transcriptome (from RNA) and epigenome (DNA
methylation sites). The commonly used NGS methods include:
• Genomics—whole genome sequencing, exome sequencing, targeted sequencing
• Transcriptomics—mRNA sequencing
• Epigenomics—CHIP sequencing (chromatin immunoprecipitation) to study DNA–protein interactions.
• NGS is unraveling several novel mutations in genes involved in hematologic malignancies. The list of such mutated
genes like IDH1, IDH2, DNMT3A, and SF3B1 is continuously increasing .

84. Disease Genetic abnormality Current genetic
technology
Clinical utility
CML Philadelphia chromosome
only
Conventional
Cytogenetics
To diagnose CML
Bcr-abl gene
rearrangement
FISH To diagnose CML
AML Chromsomal aberrations Conventional karyotyping Prognostication of AML
patient
Selection of optimal
therapy
ALL Chromosomal aberrations
(Translocations)
Conventional
Karyotpye/FISH
For prognostication
CLL Chromsomal aberrations
( Deletions/translocations)
FISH Prognostication
MDS Various chromosomal
deletions and
translocations
FISH Diagnosis of MDS and
assessment of prognosis
Summary

85. References
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• 3.Mckenzie S. Clinical laboratory hematology 3rd ed. New jersey : Pearson Education 2015 .p.895-
897.
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Biochemistry Laboratory diagnostics. Available from : Charles university
[Accessed 22 Oct 2019].
• 5. Kawathalkar S. Essentials of Clinical Pathology 2nd ed. New Delhi: Jaypee Brothers Medical
publishers (p) Ltd;2018 .p.213-16.

86. References
• 6.Nayak R. Essentials in Hematology and clinical pathology 2nd ed. New
Delhi: Jaypee brothers medical publishers (p) Ltd 2017.p.158-70.
• 7. Prakash G, Kaur A, Malhotra P, Khadwal A, Sharma P, Suri V, Varma
N, Varma S. Current Role of Genetics in Hematologic Malignancies.
Indian J Hematol Blood Transfus 2016;32(1):18-31.

87. • THANK YOU