Molecular/Cytogenetic

Overview of Molecular and Cytogenetic Testing in Hematology

• Definition: Laboratory techniques used to identify genetic abnormalities (chromosomal and molecular) in blood and bone marrow cells
• Clinical Significance:
  • Diagnosis and Classification: Essential for diagnosing and classifying hematologic malignancies, including leukemias, lymphomas, and myeloproliferative neoplasms
  • Prognosis: Many genetic abnormalities have prognostic significance and can help predict the course of the disease
  • Therapeutic Decision-Making: Some genetic abnormalities are targets for specific therapies
  • Monitoring Treatment Response: Used to detect minimal residual disease (MRD) and monitor treatment response
• Types of Tests:
  • Cytogenetic Analysis (Karyotyping)
  • Fluorescence In Situ Hybridization (FISH)
  • Polymerase Chain Reaction (PCR)
  • Next-Generation Sequencing (NGS)

Cytogenetic Analysis (Karyotyping)

• Principle:
  • Analyzes the structure and number of chromosomes in a cell
  • Cells are cultured, arrested in metaphase, and then stained to visualize the chromosomes
  • Chromosomes are arranged in a standardized format (karyotype)
• Procedure:
  1. Sample Collection: Bone marrow aspirate or peripheral blood
  2. Cell Culture: Cells are cultured to increase the number of cells in metaphase
  3. Chromosome Preparation: Cells are treated with a chemical (e.g., colchicine) to arrest them in metaphase, then lysed and fixed
  4. Staining: Chromosomes are stained with Giemsa or other banding techniques to reveal their structure
  5. Microscopic Examination: A trained cytogeneticist examines the stained chromosomes under a microscope
  6. Karyotype Analysis: Chromosomes are arranged in a standardized format, and any abnormalities (e.g., translocations, deletions, inversions) are identified
• Reporting:
  • Karyotype is described using the International System for Human Cytogenetic Nomenclature (ISCN)
  • Example: 46,XY,t(9;22)(q34;q11.2) indicates a male karyotype with a translocation between chromosomes 9 and 22
• Advantages:
  • Provides a global view of the entire genome
  • Can detect complex chromosomal rearrangements
• Limitations:
  • Requires viable cells and cell culture
  • Time-consuming
  • Limited resolution (cannot detect small deletions or point mutations)
  • Cannot detect abnormalities in non-dividing cells

Fluorescence In Situ Hybridization (FISH)

• Principle:
  • Uses fluorescently labeled DNA probes that bind to specific DNA sequences on chromosomes
  • Allows for the detection of specific chromosomal abnormalities (e.g., translocations, deletions, amplifications) in individual cells
• Procedure:
  1. Prepare a Cell Suspension: Bone marrow aspirate, peripheral blood, or tissue sample
  2. Hybridization: Fluorescently labeled DNA probes are hybridized to the chromosomes on a slide
  3. Washing: Unbound probes are washed away
  4. Microscopic Examination: The slide is examined under a fluorescence microscope, and the number and location of the fluorescent signals are counted
• Types of FISH Probes:
  • Break-Apart Probes: Detect chromosomal translocations by flanking the breakpoint region; a normal cell will show two fusion signals, while a cell with the translocation will show separated signals
  • Fusion Probes: Detect chromosomal translocations by hybridizing to both fusion partners; a normal cell will show separate signals, while a cell with the translocation will show a fusion signal
  • Enumeration Probes: Detect the number of copies of a specific gene or chromosome; used to identify deletions or amplifications
• Advantages:
  • Relatively rapid turnaround time compared to karyotyping
  • Can be performed on non-dividing cells
  • Can detect submicroscopic deletions and translocations
• Limitations:
  • Only detects known chromosomal abnormalities for which probes are available
  • Cannot detect novel or complex chromosomal rearrangements

Molecular Testing (PCR and Next-Generation Sequencing)

• Polymerase Chain Reaction (PCR):
  • Principle: Amplifies specific DNA sequences, allowing for the detection of gene mutations and fusion genes
  • Procedure:
    1. DNA Extraction: Extract DNA from bone marrow aspirate or peripheral blood
    2. Amplification: Use PCR to amplify the target DNA sequence using specific primers
    3. Detection: Detect the amplified DNA product using gel electrophoresis, real-time PCR, or other methods
  • Real-Time Quantitative PCR (RQ-PCR):
    • Allows for the quantification of the amplified DNA product
    • Used for monitoring minimal residual disease (MRD) in leukemia
  • Advantages:
    • High sensitivity and specificity
    • Relatively rapid turnaround time
  • Limitations:
    • Can only detect known mutations or fusion genes for which primers are available
    • Cannot detect novel or unknown genetic abnormalities
• Next-Generation Sequencing (NGS):
  • Principle: Massively parallel sequencing technology that allows for the simultaneous sequencing of millions of DNA fragments
  • Procedure:
    1. DNA Extraction: Extract DNA from bone marrow aspirate or peripheral blood
    2. Library Preparation: Prepare a DNA library by fragmenting the DNA and adding adaptors
    3. Sequencing: Sequence the DNA fragments using an NGS platform
    4. Data Analysis: Align the sequencing reads to a reference genome and identify gene mutations, insertions, deletions, and other genomic alterations
  • Advantages:
    • Can detect both known and novel mutations
    • Can detect multiple mutations simultaneously
    • Can be used to identify gene expression changes
  • Limitations:
    • More complex and expensive than PCR-based methods
    • Requires bioinformatics expertise for data analysis

Key Genetic Abnormalities in Myeloid Neoplasms

Here’s a table summarizing the key genetic abnormalities and associated myeloid neoplasms (as defined by the WHO classification):

Genetic Abnormality	Myeloid Neoplasm	Clinical Significance
t(9;22)(q34.1;q11.2); BCR-ABL1	Chronic Myeloid Leukemia (CML)	Diagnostic hallmark of CML; target for tyrosine kinase inhibitors (TKIs)
JAK2 V617F Mutation	Polycythemia Vera (PV), Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF)	Diagnostic and prognostic marker; target for JAK2 inhibitors (e.g., ruxolitinib)
t(15;17)(q24.1;q21.1); PML-RARA	Acute Promyelocytic Leukemia (APL)	Diagnostic hallmark of APL; target for all-trans retinoic acid (ATRA) and arsenic trioxide (ATO)
inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11	Acute Myeloid Leukemia (AML) with inv(16)	Favorable prognosis
t(8;21)(q22;q22); RUNX1-RUNX1T1	Acute Myeloid Leukemia (AML) with t(8;21)	Favorable prognosis
NPM1 Mutation	Acute Myeloid Leukemia (AML) with mutated NPM1	Favorable prognosis in the absence of FLT3-ITD
FLT3-ITD Mutation	Acute Myeloid Leukemia (AML)	Adverse prognosis
del(5q)	Myelodysplastic Syndrome (MDS) with isolated del(5q)	Good response to lenalidomide
SF3B1 Mutation	MDS with Ringed Sideroblasts (MDS-RS)	Better prognosis; may be responsive to luspatercept

Specific Genetic Abnormalities

• BCR-ABL1 Fusion Gene (Chronic Myeloid Leukemia)
  • Created by the reciprocal translocation t(9;22)(q34.1;q11.2), resulting in the Philadelphia chromosome (Ph chromosome)
  • Results in a fusion protein with constitutive tyrosine kinase activity, driving uncontrolled cell proliferation
  • Detected by:
    • Cytogenetic analysis (karyotyping)
    • FISH (fluorescence in situ hybridization)
    • RT-PCR (reverse transcription polymerase chain reaction): Used to quantify BCR-ABL1 transcript levels and monitor minimal residual disease (MRD)
• JAK2 V617F Mutation (MPNs)
  • A point mutation in the JAK2 gene (Janus kinase 2)
  • Leads to constitutive activation of the JAK-STAT signaling pathway, resulting in increased cell proliferation
  • Found in:
    • Polycythemia Vera (PV): >95% of cases
    • Essential Thrombocythemia (ET): ~50-60% of cases
    • Primary Myelofibrosis (PMF): ~50-60% of cases
  • Detected by:
    • PCR-based assays

Key Terms

• Cytogenetic Analysis (Karyotyping): The study of chromosomes and their abnormalities
• FISH (Fluorescence In Situ Hybridization): A technique that uses fluorescent probes to detect specific DNA sequences on chromosomes
• PCR (Polymerase Chain Reaction): A technique to amplify specific DNA sequences
• Next-Generation Sequencing (NGS): A high-throughput sequencing technology
• Mutation: A change in the DNA sequence of a gene
• Translocation: The transfer of genetic material from one chromosome to another
• Deletion: The loss of a portion of a chromosome
• Inversion: The reversal of a segment of a chromosome
• Fusion Gene: A gene formed by the fusion of two separate genes due to a chromosomal translocation or deletion
• Immunophenotyping: Technique that uses antibodies to identify cell surface and intracellular markers,
• Minimal Residual Disease (MRD): Small numbers of residual cancer cells that remain after treatment