Role of Cytogenetics in Haematological Malignancy

Dr Samim Reza

Phase B Resident

Department of Haematology

Introduction

• Cytogenetics is the study of chromosomes structure and function
• Chromosome analyses are considered as standard care for daignosis ,management and prognosis prediction of haematological malignances

Types of cytogenetics analysis

• It involves 3 different approches :

Classical chromosomal analysis /karyotyping

FISH using DNA probe

Arrays –1)comprehensive genomic hybridization 2) single neucleotide polymorphism

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Classical Chromosome analysis

• Performed on living, dividing cells on metaphase
• Cells are arrested on metaphase using inhibitors (colcemid,velban)
• Steps: target cells > short term culture > harvesting in hypotonic solution>fixed with mixture of methanol and acetic acid>

Contd…

• dropping of cell suspension on slide and drying> banded with Giemsa and pepsin> banded metaphase are identified and analyzed in high resolution objectives> Image are acquired and software is used to create karyotype.

Cytogenetic nomenclature

• According to international system of cytogenetic nomenclature(ISCN)
• Chromosome are divided into long arm(q) and shot arm(p) by centromere.
• Each arm is divided into region ,band ,sub band which are numbered after p or q
• Total number o chromosome are stated first ,then sex chromosome with comma

• For example a female patient with philadelphia chromosome would have karyotype written as ….
• 46,XX,t(9;22)(q34;q11.2)[18]/46,XX,[2]
• a slash designating a second normal cell line into 2 metaphase

• Basic use : Help to diagnosis congenital ( e.g. t trisomy 21 ) or acquired(translocation 9,22) chromosomal abnormality.

Fluorescence in Situ Hybridization Analysis(FISH)

• It bridges classical Cytogenetics to molecular study
• Here we use DNA probes labeled with radioactive substances(fluorescence)
• Steps : Target DNA is denatured by heat /formamide/salt> reannealed with probe DNA > hybridization with complementary target DNA sequence >

• slide s are washed and counterstained> hybridization is visualaized and image are processed in a software> loss/gain or juxtaposition of sequence can be answered
• Types : single probe to determine the copy number of single locus , Repetive probes for chromosomal enumeration
• Can be performed in metaphase or interphase state

Nomenclature of FISH

• Metaphase FISH prefaced by ‘ish’ and interphase FISH by ‘nuc ish’
• In interphase FISH : nuc ish (ABL1,BCR)*3(ABL1 con BCR* 2)[200] , there are 3 ABL signal and 3 BCR1 signal two of which are connected or fused analysed on 200 interphase

Array Anlysis

• Array can detect smaller( 50-100 bp where CCG can 5-10 mb) ,cryptic anomaly
• Two type ….comprehensive genomic hybridization (CGH) and Single neucleotide polymorphism(SNP)

Common Indications cytogenetics

• To provide evidence of clonality when neoplastic evidence can’t be demonstrated otherwise
• To confirm specific diagnosis e.g APL,Burkit lymphoma
• To permit the classification
• Provide prognostic information

Contd

• Indicate which fusion genes are likely to be present thus giving info to detect MRD
• To distinguish a phenotype switch occurring within a single clone from a therapy related secondary leukaemia
• To determine the causative agent in therapy related AML

FISH can identify –

• Centromeres of a specific chromosome (useful for detecting trisomy or monosomy and chimarism following sex-mismatched bone marrow transplantation; Fig. 8-7, A)

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• Specific oncogenes ,tumour supressor gene using locus-specific probe, useful for detecting translocations

FISH can identify –

• Whole chromosomes (whole chromosome painting, useful in identifying complex chromosomal rearrangements).

Advantages of FISH

• Many more cells can be examined (useful for detecting residual disease)

• Metaphases are not essential, so abnormalities can be detected in non-dividing cells (useful in chronic lymphocytic leukaemia, in which cells rarely divide in culture)

Advantages of FISH

• FISH can be performed in a shorter period of time (may be critical in rapidly confirming a diagnosis of acute promyelocytic leukaemia)

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• Abnormalities that are too subtle to be detected by conventional cytogenetic analysis may be detected (e.g. STIL-TAL fusion in T-lineage ALL or t(12;21)(p12;q22) in B-lineage ALL)

Disadvantages of FISH

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• only those abnormalities that are specifically sought will be found, whereas conventional cytogenetic analysis permits all chromosomes to be evaluated.

Disadvantages of FISH

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• only those abnormalities that are specifically sought will be found, whereas conventional cytogenetic analysis permits all chromosomes to be evaluated.

Cytogenetics for AML

• G-banded chromosome analysis should preferably be performed first.

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• In case of a successful normal chromosome analysis with a clear diagnosis of AML by morphology and flowcytometry, additional interphase and metaphase FISH analyses are recommended to exclude cryptic rearrangements eg. KMT2A(MALL) gene rearrangement

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Cytogenetics for AML

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• Depending on the morphology and flow cytometry results, the following FISH probes can be added:

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• a. RUNX1-RUNX1T1 (AML1-ETO) fusion probes

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• b. CBFB rearrangement or CBFB-MYH11 fusion probes: inv(16) and t(16;16) resulting in CBFB-MYH11 fusion can be subtle in cases with suboptimal G-banded chromosomes quality

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• c. KMT2A (MLL) rearrangement probes

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• d. PML-RARA fusion probes: PML-RARA fusion is diagnostic of acute promyelocytic leukemia (APL)

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Cytogenetics for AML

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• MECOM (EVI1) rearrangement probes should be considered when chromosome analysis is suggestive of an inv(3) or t(3;3).

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Cytogenetics in ALL

• In pediatric/young adult B-lineage ALL, G-banded chromosome analysis should be performed simultaneously with interphase FISH analysis using a panel that includes the following probes:
• a. BCR-ABL1 fusion probes
• b. KMT2A (MLL) rearrangement probes
• c. ETV6-RUNX1 fusion probes: for ETV6-RUNX1 fusion, ETV6deletion, and iAMP21 (intrachromosomal amplification of chromosome 21)
• d. Centromeric probes for chromosomes 4 and 10: for trisomies of chromosomes 4 and 10

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Cytogenetics in ALL

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• In adult B-lineage ALL, G-banded chromosome analysis should be performed simultaneously with interphase FISH analysis using the following probes:
• a. BCR-ABL1 fusion probes
• b. KMT2A (MLL) rearrangement probes

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Cytogenetics in ALL

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• MYC rearrangement and/or IGH-MYC fusion probes should be considered in both pediatric and adult ALL, where the morphology and flow cytometry results are suggestive of B-cell ALL (Burkitt leukemia variant)

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• In T-lineage ALL, G-banded chromosome analysis should be performed first. Interphase FISH analysis is optional and could include the following probes:
• a. BCR-ABL1 fusion probes: for BCR-ABL1 fusion and ABL1amplification
• b. KMT2A (MLL) rearrangement probes

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Cytogenetics in ALL

• In both pediatric and adult B-lineage ALL, and depending on the blast cell morphology, flow cytometry, chromosome analysis, and FISH results, additional interphase FISH testing should be considered, including:
• a. CRLF2 rearrangement probes: for P2RY8-CRLF2 fusion and IGH-CRFL2 fusion (Ph-like ALL)¹³
• b. PDGFRB rearrangement probes (Ph-like ALL)¹³
• c. CDKN2A/B (9p21.3) probe: 9p21.3 deletion is common in both B- and T-lineage ALLs, it provides a clonal target for future monitoring of the patient’s disease in the absence of other FISH targets
• d. PAX5 (9p13.2) probe

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Cytogenetics in MDS

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• G-banded chromosome analysis should preferably be performed first, then following probes can be bundled in an MDS FISH panel,¹⁸which should be performed on the diagnostic specimen:
• a. -5/5q- probes
• b. -7/7q- probes
• c. Centromeric probe for chromosome 8: for trisomy 8
• d. 20q- probe

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Cytogenetics in MPN

• CML
• - Bone marrow is the preferred specimen for CML; however, peripheral blood may be used if the level of blasts is >10%.

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• - The t(9;22)(q34;q11.2) is detectable in 90–95% of CML cases at diagnosis. The remaining 5–10% of cases have either a variant t(9;22) or a cryptic BCR-ABL1 fusion undetectable by chromosome analysis.

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• - Therefore, both G-banded chromosome analysis as well as interphase FISH analysis using BCR-ABL1 fusion probes should be performed simultaneously at diagnosis.

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Cytogenetics in MPN

• CML
• It is important to establish whether additional chromosome abnormalities are present at diagnosis, including an additional der(22), i(17q), and trisomy 8. These are warning signs that might be associated with inferior overall survival and increased risk of progression to accelerated phase.

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• The CML National Comprehensive Cancer Network (NCCN) guidelines recommend that cytogenetic studies (both G-banded chromosome and BCR-ABL1 fusion FISH analyses) and quantitative RT-PCR BCR-ABL1 fusion testing be performed at diagnosis. If no BCR-ABL1 fusion can be detected, molecular testing for mutations associated with other myeloproliferative conditions is indicated.

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Cytogenetics in MPN

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• Other MPNs
• Bone marrow is the preferred specimen for other MPNs; however, peripheral blood may be used if there is peripheral involvement.

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• With few exceptions, cytogenetic abnormalities are usually not specific in other MPNs. Typical abnormalities of myeloid neoplasms are usually observed and can be useful in demonstrating evidence of clonality.

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Cytogenetics in plasma cell dyscrasia

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• A bone marrow specimen is required for MM.

• For FISH and/or CMA analyses, plasma cell separation is recommended to enrich for the CD138⁺ plasma cell fraction in bone marrow samples with low plasma cell percentages

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Cytogenetics in plasma cell dyscrasia

• G-banded chromosome analysis should be performed (as described above) simultaneously with interphase FISH analysis using a panel that includes the following probes:

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• a. 1q21.3 probe (including CKS1B): for 1q21 copy gain, which has been linked to adverse prognosis

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• b. 13q14.2q14.3 probes (including RB1): 13q14.2q14.3 deletion is common in MM but, when detected only by FISH, it is not predictive of survival in the absence of other adverse cytogenetic abnormalities. However, it provides a clonal target for future monitoring of the patient’s disease in the absence of other FISH targets. 13q deletion detected by G-banded chromosome analysis still retains its prognostic value

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Cytogenetics in plasma cell dyscrasia

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• A bone marrow specimen is required for MM.

• For FISH and/or CMA analyses, plasma cell separation is recommended to enrich for the CD138⁺ plasma cell fraction in bone marrow samples with low plasma cell percentages

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Cytogenetics in plasma cell dyscrasia

• c. IGH rearrangement probes: if IGH is rearranged, including the classical gene disruption as well as deletion of either the 5′ or 3′ region of IGH, then reflex to IGH-FGFR3, IGH-CCND1, and IGH-MAFfusion probes.

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• d. TP53 (17p13.1) probe

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• e. Probes for three of the odd-numbered chromosomes often trisomic in hyperdiploid MM (e.g., chromosomes 5, 9, 11, 15, and 19)

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Cytogenetics in CLL

• To assign the patient into clinically relevant prognostic subgroups, the following panel of FISH probes is recommended:
• a. ATM (11q22.3) probe
• b. Centromeric probe for chromosome 12: for trisomy 12
• c. 13q14.3 probe (including D13S319)
• d. TP53 (17p13.1) probe
• - FISH can also be useful for the differential diagnosis with mantle cell lymphoma (MCL), for which FISH using the IGH-CCND1 fusion probes is recommended.

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Cytogenetics in Lymphoma

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• For all lymphomas, the preferred tissue is lymph node or biopsy material from a suspected lymphoid mass.

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• If fresh material is available, G-banded chromosome analysis is recommended.

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Cytogenetics in Lymphoma

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• Interphase FISH analysis using relevant probes performed on lymph node tissue sections, fine needle aspirate smears, and/or touch imprints should be included.

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• Bone marrow or peripheral blood analysis will not detect clonal chromosomal abnormalities if there is no evidence of infiltration. For FISH analysis, bone marrow smears or core biopsy touch imprints can be used.

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Thank You All