Version May 2024
INTRODUCTION — Acquired clonal chromosomal abnormalities are present in the tumor cells of many hematologic malignancies. Certain cytogenetic abnormalities are closely (sometimes uniquely) associated with morphologically or clinically distinct subsets of leukemias or lymphomas and, in many cases, cytogenetic analysis is important for diagnosis, stratifying treatment, estimating prognosis, or distinguishing benign from malignant cell populations. Chromosome banding techniques and fluorescence in situ hybridization (FISH) are the most common clinical tools for karyotypic analysis.
This topic reviews terminology of cytogenetic (chromosomal) abnormalities in hematologic malignancies, clinical settings for analysis, and methods of detection.
Cytogenetic abnormalities in individual diseases are discussed in detail in the appropriate topics. A review of various genetic tools is presented separately. (See "Tools for genetics and genomics: Cytogenetics and molecular genetics".)
DEFINITIONS AND NOTATION — Chromosomal abnormalities are described according to the International System for Human Cytogenetic Nomenclature [1]. The following terminology is used in this topic.
General definitions
●Normal chromosome content – Normal human cells have 46 chromosomes (22 pairs of autosomes and 2 sex chromosomes [X or Y]). The normal male karyotype is 46,XY and the normal female karyotype is 46,XX.
●Chromosome arms – The short arm of a chromosome is designated "p" and the long arm is designated "q." Thus, the short arm of chromosome 9 is designated 9p and the long arm is 9q.
●Chromosome bands – Chromosome bands are alternating light and dark segments that result from various staining procedures (eg, Giemsa staining). The banding pattern of each chromosome is unique.
●Clonality – Clonality refers to a group of cells with the same structural or numerical chromosomal abnormality(ies). An abnormal clone is identified if one of the following is present [1]:
•At least two cells with the same structural rearrangement (eg, translocations, deletions, or inversions) or gain of the same chromosome
•Three cells, each of which shows loss of the same chromosome
One cell with a normal karyotype is considered evidence for the presence of a normal cell line, but one abnormal cell is usually not considered sufficient to define an abnormal clone (because sporadic numerical abnormalities, such as loss of a chromosome, may result from technical artifact). An exception is a single cell with a well-characterized recurring abnormality (eg, the Philadelphia chromosome associated with chronic myeloid leukemia [CML]) or a single cell in a post-treatment sample with one or more aberrations that were observed in the pre-treatment sample. An abnormality in a single cell is also considered to represent a cytogenetic clone if the abnormality is confirmed by another method, such as fluorescence in situ hybridization (FISH) or a molecular technique [1]. (See "Detection of measurable residual disease in acute lymphoblastic leukemia/lymphoblastic lymphoma" and "Acute myeloid leukemia: Induction therapy in medically fit adults", section on 'Remission criteria'.)
●Recurrent abnormalities – A recurring abnormality is a numerical or structural abnormality noted in multiple patients who have a similar disease. Recurrent abnormalities may be characteristic of distinct morphological, immunophenotypic, and/or clinical subtypes of leukemia or lymphoma and may be associated with prognosis.
Cytogenetic/chromosomal abnormalities
●Deletion (del) refers to loss of chromosomal material. Deletions can be an interstitial deletion (in which two breaks, in either the short arm or long arm of the chromosome, cause loss of the intervening material), or due to unbalanced translocations. An example of an interstitial deletion is isolated del(5q) in myelodysplastic syndromes (MDS), in which a variable portion of the long arm of chromosome 5 is lost. (See "Cytogenetics, molecular genetics, and pathophysiology of myelodysplastic syndromes/neoplasms (MDS)", section on 'del(5q)'.)
●Monosomy is loss of an entire chromosome. As an example, monosomy for chromosome 7 is designated -7.
●Trisomy is the gain of an extra chromosome. As an example, trisomy of chromosome 8 is denoted +8.
●Chromosomal translocation (t) is an exchange of genetic material between chromosomes, enabled by a break in the DNA in at least two different chromosomes.
●Reciprocal translocation refers to an exchange in which there is no obvious overall loss of chromosomal material. Standard nomenclature is to designate the involved chromosomes in the first set of parentheses and the specific breakpoints in a second set. As an example, the reciprocal translocation associated with CML is t(9;22)(q34.1;q11.2), which involves a reciprocal translocation between the long arm of chromosome 9 at band q34.1 (9q34.1) and the long arm of chromosome 22 at band q11.2 (22q11.2). The derivative chromosome 22 is the so-called Philadelphia chromosome (figure 1).
●Chromosomal inversion (inv) requires two breaks in the same arm or both short and long arms of the same chromosome with rotation of the intervening material. As an example, inv(16)(p13.1q22) is a distinct category of acute myeloid leukemia (AML). (See "Acute myeloid leukemia: Cytogenetic abnormalities", section on 'inv(16) or t(16;16); CBFB::MYH11'.)
CLINICAL APPLICATIONS
Diagnosis — Cytogenetic analysis is necessary for diagnosis of some hematologic malignancies and it provides key confirmation of other presumptive diagnoses. In general, molecular techniques that define the underlying molecular abnormalities are important for complementing cytogenetic findings, such as fluorescence in situ hybridization (FISH) or reverse transcription polymerase chain reaction (RT-PCR). Importantly, rare cases with cryptic genetic rearrangements (sub-karyotypic) that result in recurring fusion genes can only be diagnosed by molecular techniques, since they are not detectable by conventional karyotypic analysis.
Examples include:
●Detection of t(9;22)(q34.1;q11.2), which results in the BCR::ABL1 fusion, is diagnostic for chronic myeloid leukemia (CML) (figure 1). RT-PCR should be used to document the molecular rearrangement and to monitor response to treatment. (See "Clinical manifestations and diagnosis of chronic myeloid leukemia", section on 'Diagnosis'.)
●Acute myeloid leukemia (AML) with t(8;21)(q22;q22.1), RUNX1::RUNX1T1 can be diagnosed by cytogenetic analysis, and may be confirmed with RT-PCR that documents the RUNX1::RUNX1T1 fusion.
●The presence of the t(14;18)(q32.2;q21.3), IGH::BCL2 can be helpful for distinguishing a follicular lymphoma from a benign, reactive proliferation of lymphoid cells. (See "Clinical manifestations, pathologic features, diagnosis, and prognosis of follicular lymphoma", section on 'Diagnosis'.)
Treatment — Chromosomal abnormalities can aid in treatment planning, since some cytogenetic findings inform the selection of a targeted therapy or predict for response to specific therapies.
As examples:
●Detection of the t(15;17)(q24.1;q21.1); PML::RARA is diagnostic for acute promyelocytic leukemia (APL) and informs treatment with all-trans retinoic acid with arsenic trioxide, rather than intensive chemotherapy. A rapid FISH test may confirm the PML::RARA fusions within two hours. (See "Initial treatment of acute promyelocytic leukemia in adults".)
●In newly-diagnosed multiple myeloma, the presence of various cytogenetic abnormalities (eg, t(14;16)(q32.3;q23), IGH::MAF, t(14;20)(q32.2;q12), IGH::MAFB, del(17p13), t((4;14)(p16;q32.2), IGH::FGFR3/WHSC1/MMSET), +1q) is used to stratify high-risk disease and select therapy. (See "Multiple myeloma: Overview of management".)
Measurable residual disease (MRD) — Detection of MRD in a patient with a morphologic complete remission (CR) has important prognostic and therapeutic implications. Cytogenetic analysis, particularly FISH tests, may provide an important and quick assessment of disease status and treatment response, in combination with morphology observation, flow cytometry and molecular tests.
As examples:
●The presence of post-remission MRD is an important prognostic feature in children with acute lymphoblastic leukemia (ALL) that guides therapy. (See "Clinical use of measurable residual disease detection in acute lymphoblastic leukemia".)
●Detection of MRD in patients in CR after induction therapy for AML is used to stratify treatment in medically-fit adults with intermediate-risk prognosis. (See "Acute myeloid leukemia in younger adults: Post-remission therapy", section on 'Intermediate-risk disease'.)
Prognosis — Cytogenetic analysis provides prognostic information that is independent of other clinical features of some hematologic malignancies. It is particularly useful in detecting complex karyotypes and defining clonal evolution and heterogeneity from single cell level analysis.
As examples:
●For AML, cytogenetic analysis provides important prognostic information that is independent of that associated with other clinical features [2-4]. (See "Acute myeloid leukemia: Risk factors and prognosis", section on 'Cytogenetic and molecular features'.)
●In CML, the presence of additional chromosomal abnormalities (ie, in addition to the Philadelphia chromosome) may indicate blast phase, which is associated with an adverse prognosis. (See "Overview of the treatment of chronic myeloid leukemia", section on 'Advanced disease (accelerated phase/blast phase'.)
EXAMINATION OF TUMOR SPECIMENS
Obtaining and processing the specimen — Chromosomal banding studies can only be performed on specimens that contain viable, dividing cells; thus, specimens immersed in preservatives or fixatives cannot be used. If only fixed tissue is available, fluorescence in situ hybridization (FISH) or molecular techniques can be performed to analyze specific chromosome rearrangements. (See 'FISH' below.)
●Choice of specimen
•Leukemia/myelodysplastic syndromes (MDS) – A bone marrow aspirate is preferred, but peripheral blood may be used if the white blood cell count is >10,000/microL with >10 percent immature myeloid or lymphoid cells. Blood or marrow (5 mL) should be aspirated into a syringe coated with preservative-free heparin and transferred to a tube containing an appropriate culture medium; other anticoagulants (eg, EDTA, which chelates calcium) should be avoided in the collection medium. (See "Bone marrow aspiration and biopsy: Indications and technique", section on 'Materials'.)
If the bone marrow cannot be aspirated ("dry tap"), a bone marrow biopsy specimen can be used and, if present, a leukemia mass (chloroma) can be analyzed. Mitogens (mitosis-inducing substances), such as phytohemagglutinin are generally not used when culturing marrow or blood from patients with acute leukemia because stimulation of division of normal lymphocytes may interfere with the analysis of spontaneously-dividing malignant cells. Once the malignant cells divide readily, a chemical such as demecolcine (Colcemid) is added to arrest mitotic division.
•Lymphomas – Specimens for karyotypic analysis can be obtained from involved lymph nodes, marrow, or blood; tumor masses; or malignant effusions.
T or B cell specific mitogens, such as phytohemagglutinin (PHA) and Poke weed mitogen (PWM), or oligonucleotides DSP30 may be used in culturing cells from lymphoid neoplasms, particularly low-grade T and B cell neoplasms, such as T prolymphocytic leukemia (T-PLL) and chronic lymphocytic leukemia (CLL).
●Transportation and processing – Cytogenetic analysis is generally performed in dedicated laboratories and requires skilled technical expertise and interpretation.
The specimen should be transported without delay at room temperature to the cytogenetics laboratory and processed immediately. Specimens are generally grown in culture for 24 to 48 hours. Well-handled specimens are adequate for cytogenetic analysis in 90 to 95 percent of cases, but there is frequent loss of cell viability and a high proportion of inadequate analyses when overnight delivery services are used, particularly with lymphoid neoplasms.
Methods of detection — Conventional metaphase chromosome analysis is the preferred cytogenetic technique because it enables evaluation of the entire complement of chromosomes. FISH provides important information about specific cytogenetic abnormalities and is a rapid analytical test, but it does not evaluate the entire karyotype.
Metaphase cytogenetic analysis — Conventional metaphase cytogenetic analysis uses various enzymes and histological stains to analyze chromosome banding patterns (figure 2). While technically challenging, it enables examination of the entire tumor genome at the single cell level, which is particularly important for hematologic malignancies that have many potential genetic abnormalities, such as acute myeloid leukemia (AML) or MDS. (See "Tools for genetics and genomics: Cytogenetics and molecular genetics", section on 'Chromosomal analysis'.)
Chromosome analysis often detects primary and secondary chromosome abnormalities. Serial evaluation can reveal clonal evolution of a malignancy. Chromosome analysis provides evidence of a complex karyotype with ≥3 chromosomal abnormalities, or a monosomal karyotype with loss of two autosomal chromosomes, or one autosomal chromosome and ≥1 structural abnormality. Complex karyotypes and monosomal karyotypes are strongly associated with an unfavorable prognosis in AML and MDS. (See "Acute myeloid leukemia: Risk factors and prognosis", section on 'Cytogenetic and molecular features' and "Prognosis of myelodysplastic neoplasms/syndromes (MDS) in adults", section on 'Prognostic factors'.)
Metaphase cytogenetic analysis is important for diagnosis, treatment selection, and prognosis for many types of hematologic malignancies. (See 'Clinical applications' above.)
FISH — FISH analysis uses fluorochrome-labeled DNA probes (figure 3), which are hybridized to either metaphase chromosomes or interphase nuclei. This is a rapid and sensitive technique for detecting recurring numerical and structural chromosomal abnormalities.
●Applications – FISH is most useful when analyzing specific abnormalities associated with a particular malignancy.
•Diagnosis – FISH can be used as the primary method of confirming the diagnosis of certain hematologic malignancies with distinctive morphology or clinical presentation, such as acute promyelocytic leukemia (APL) or chronic myeloid leukemia (CML). However, in most clinical cases, FISH serves as a complement to conventional cytogenetic analysis because it may not detect additional chromosomal abnormalities or multiple clones (picture 1) [5-7]. (See "Clinical manifestations, pathologic features, and diagnosis of acute promyelocytic leukemia in adults".)
FISH probes are available for detection of all of the common recurring chromosomal abnormalities in hematologic malignancies, including chromosome gains or losses, deletions and translocations. As an example, different probes from the three principal BCR breakpoint regions can distinguish different chromosome 22 breakpoints in the Philadelphia chromosome/BCR-ABL1 fusion gene of CML. (See "Molecular genetics of chronic myeloid leukemia", section on 'Detecting the Philadelphia chromosome or its products'.)
FISH, in conjunction with morphology and immunophenotyping, can define the lineage and clarify the origin of tumor stem cells and cell populations in certain leukemias and lymphomas.
•Measurable residual disease (MRD) or relapse – When complete cytogenetic analysis has identified chromosomal abnormalities in the malignant cells of a particular patient, FISH can be used to detect MRD, relapse, and/or assess treatment efficacy [5-8]. In some cases, FISH is more sensitive than morphological and conventional cytogenetic analyses to detect the Philadelphia chromosome in CML. (See "Initial treatment of chronic myeloid leukemia in chronic phase", section on 'Treatment response'.)
•Allele loss – FISH can detect allele loss in tumor cells, such as deletion of the TP53 gene in CLL and other malignancies [9].
●Technique – FISH uses the ability of single-stranded DNA to sensitively and specifically anneal to complementary DNA [5]. The target DNA is the nuclear DNA of interphase cells or the DNA of metaphase chromosomes that are affixed to a glass microscope slide. Importantly, FISH does not require live cells (eg, from bone marrow or blood smears or freshly-prepared touch preparations), as it can be used with cytospin slides or fixed and sectioned tissue. (See "Tools for genetics and genomics: Cytogenetics and molecular genetics", section on 'Fluorescence in situ hybridization'.)
Commercially available FISH probes are typically bacterial artificial chromosome (BAC) clones (usually several hundred kilobases long) that are directly labeled with fluorochrome and hybridized to the target DNA. With the development of dual- and triple-pass filters, most laboratories can hybridize and detect two to three probes simultaneously.
In general, there are three types of FISH probes that are used to detect chromosomal abnormalities:
•Centromere-specific probes, which are unique to the repetitive sequences that flank the centromere of each human chromosome, can identify monosomy, trisomy, and other aneuploidies.
•Locus-specific probes are useful for detecting translocations, inversions, deletions, and gain or loss of gene copy number that involve specific chromosomal loci. Because of its high sensitivity and specificity, this technique can be used to detect MRD. The probes are usually BAC clones that flank the entire (or partial) genomic region of the relevant gene (eg, BCR or ABL1), labeled in single- or dual-color.
•Chromosome-specific libraries, which paint the entire chromosome, chromosome arm, or specific chromosomal bands, are useful in identifying structural rearrangements (eg, translocations) or marker chromosomes (eg, rearranged chromosomes of unidentified origin).
Other techniques — Various molecular techniques can complement cytogenetics, but none fully replaces chromosome analysis. Examples of complementary molecular techniques include:
●Microarray techniques enable high-resolution genome-wide genotyping using single nucleotide polymorphisms (SNPs) and can complement cytogenetic and FISH analysis [10]. Microarrays can detect genetic imbalances, including focal and cryptic deletions and duplications and loss of heterozygosity (LOH) that occurs without concurrent changes in the gene copy number (eg, somatic mitotic recombination; referred to as copy-neutral LOH, or uniparental disomy) [11,12]. (See "Tools for genetics and genomics: Gene expression profiling".)
Microarray-based genomic copy number analysis is particularly useful to detect genomic imbalances in multiple myeloma and CLL, because conventional chromosome analysis often results in a normal karyotype or yields inadequate results due to the low mitotic activity or low-level involvement by neoplastic plasma cells. (See "Multiple myeloma: Clinical features, laboratory manifestations, and diagnosis", section on 'Cytogenetics'.)
●Polymerase chain reaction (PCR) is a highly sensitive technique for amplification of DNA or RNA that can detect genetic changes associated with chromosomal abnormalities. PCR is particularly useful for assessing response to therapy and persistence of MRD. (See "Polymerase chain reaction (PCR)".)
●Next-generation sequencing (NGS) panels can detect gene fusions or mutations for routine clinical laboratory evaluation of leukemias and lymphomas. (See "Next-generation DNA sequencing (NGS): Principles and clinical applications".)
Molecular techniques to detect genetic abnormalities are discussed in separately. (See "Tools for genetics and genomics: Cytogenetics and molecular genetics".)
Comparison with molecular techniques — Chromosome banding and FISH can examine cytogenetic abnormalities at the single cell level, whereas clinically-available molecular techniques generally examine populations of cells. However, cytogenetic techniques have certain disadvantages. Chromosome banding only detects genetic abnormalities that involve large regions of chromosomes (rather than small pieces of DNA), while FISH is less sensitive than molecular techniques for detecting MRD. Cytogenetic analysis should be complemented by morphology and molecular techniques for diagnosis of hematologic malignancies.
●Technical challenges – Conventional cytogenetic analysis of tumors is often technically difficult because of the complexity of the chromosomal pattern and the presence of multiple abnormal clones.
●Sensitivity – Cytogenetic techniques are considerably less sensitive for detecting MRD, compared with molecular techniques (table 1). As an example, the lower limit for detecting a cytogenetic abnormality by FISH is 1 to 5 percent of cells, whereas reverse transcription (RT)-PCR or NGS panels have sensitivity of 1 cell per 10,000 or greater [2].
●Smaller genetic abnormalities – Cytogenetic techniques can detect abnormalities that involve large regions of chromosomes (eg, numerical or structural chromosome abnormalities), but they don't detect point mutations or small regions of gene deletion (<10 Mb) or amplification.
Molecular methods, such as PCR and NGS, are described separately. (See "Tools for genetics and genomics: Cytogenetics and molecular genetics", section on 'Polymerase chain reaction'.)
SUMMARY
●Chromosomal abnormalities in hematologic malignancies – Acquired clonal chromosomal abnormalities are present in malignant cells of many hematologic malignancies. Certain cytogenetic abnormalities are closely or even uniquely associated with distinct morphologic or clinical disease subsets. (See 'Introduction' above.)
●Clinical applications – Cytogenetic studies can confirm certain diagnoses (eg, chronic myeloid leukemia [CML], acute promyelocytic leukemia [APL]), assess prognosis (eg, acute myeloid leukemia [AML]), detect measurable residual disease (MRD; eg, AML, acute lymphoblastic leukemia [ALL]), and identify disease transformation (eg, blast phase CML). (See 'Clinical applications' above.)
●Specimen acquisition – Cytogenetic evaluation of leukemia can use bone marrow, blood, or a tumor mass, while evaluation of lymphomas can use involved lymph nodes, marrow, blood, tumor masses, or malignant effusions. Conventional cytogenetic studies can be performed only on specimens that contain viable dividing cells; specimens immersed in preservatives or fixatives cannot be used for chromosome banding analysis. Specimens should be collected into a syringe coated with preservative-free heparin or in another container, promptly transferred to appropriate culture medium, and transported without delay at room temperature to the cytogenetics laboratory. (See 'Obtaining and processing the specimen' above.)
●Methods of detection – Cytogenetic abnormalities may be detected by:
•Metaphase cytogenetic analysis – While technically challenging, conventional metaphase cytogenetic analysis can examine the entire tumor genome at the single cell level, which is especially important for hematologic malignancies with many potential genetic abnormalities (eg, AML). (See 'Metaphase cytogenetic analysis' above.)
•Fluorescence in situ hybridization (FISH) – FISH is a complement to conventional cytogenetic analysis that can rapidly and sensitively detect recurring numerical and structural abnormalities. It is most valuable for identifying abnormalities that are associated with a particular disease, but it may not detect additional abnormalities or multiple clones. (See 'FISH' above.)
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