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C H A P T E R 56

CONVENTIONAL AND MOLECULAR CYTOGENOMIC BASIS OF

HEMATOLOGIC MALIGNANCIES

Vesna Najfeld

Dedicated to the loving memory of Eta Najfeld, MD, a Holocaust segments within individual cells in a tissue sample. It is based on the
survivor and an amazing mother. ability of single-stranded DNA to anneal to complementary DNA.
In hematologic disorders, the target DNA is the marrow or peripheral
Over the past 60 years the cytogenetic analysis of hematologic blood DNA present in interphase cells or the DNA of metaphase
malignancies has been an area of prolific growth. Chromosome chromosomes that is fixed on a microscope slide. Other biologic
studies and karyotype analysis provide information of both biologic material that may be involved in the leukemic process, such as spleen
and clinical significance. Refinements in cell culture methods and the cells, ascites, and spinal fluid, are particularly useful for FISH studies.
application of chromosome banding techniques have advanced our In lymphoma, the target DNA is present in lymph nodes, and FISH
understanding of disease-specific abnormalities, and molecular studies are performed on touch preparations, frozen sections, or
cytogenetic methods now have made possible the identification of paraffin-embedded tissue.
genes involved at translocation breakpoints in specific chromosomal Fig. 56.3 shows four types of FISH probes that are used alone or
rearrangements. These advances in molecular cytogenetic methods in combination to determine both numeric and structural rearrange-
permit mapping of structural rearrangements within a single gene and ments: (a) centromere enumeration probes, which, as the name
fundamentally contribute to our knowledge of the biology of leuke- implies, are used most frequently in interphase nuclei for detection
mia. This evolution in our understanding of cancer genetics has of numeric chromosome anomalies, (b) whole chromosome painting
resulted in distinct terminology (Table 56.1). Application of conven- probes, which are used only on metaphase cells and are very useful
tional and molecular cytogenetic methods has identified over 600 in delineating complex rearrangements or the origin of a marker,
fusion genes involving over 250 different genes and approximately derivative or ring chromosome, (c) subtelomeric probes, and (d)
1000 recurrent balanced translocations in human cancers. Relevance unique gene loci probes applied to both interphase and metaphase
of these methods has played a pivotal role in the diagnosis, treatment, cells in single, dual, triple, or multiple colors to determine specific
and prognosis of the hematologic malignancies. This chapter discusses chromosomal rearrangements, deletions, or amplifications.
specific cytogenetic events and delineates molecular phenotypes that The four FISH probe strategies are used in probe design for
are important to understand the molecular pathogenesis of hemato- detection of chromosomal translocations in hematologic malignan-
logic malignancies and provides several genetic testing algorithms. cies (Fig. 56.4): (a) standard (conventional strategy), (b) extrasensitive
The remarkable hypothesis put forward by Boveri at the turn of the strategy, (c) dual-fusion strategy, and (d) “breakapart strategy.” The
20th century—namely, that an abnormal chromosome pattern is first application of FISH technology for detection of chromosomal
intimately associated with the malignant phenotype of a tumor translocations in hematologic malignancy was when the BCR-ABL1
cell—has proven correct. Knowledge of the molecular cytogenetic hybrid gene was identified using two-color FISH in interphase cells
phenotype of hematologic malignancies has led to innovative and as well as in metaphase marrow-derived CML cells. In the standard
specifically tailored treatments. The first example of such gene- strategy for interphase evaluation of chromosomal translocation, a
targeted therapy has already been successfully applied to chronic DNA probe comprising sequences mapped proximal to the break-
myelogenous leukemia (CML). point in one of the chromosomes involved in reciprocal translocations
is combined with a differentially labeled DNA probe that includes
sequences mapped distal to the breakpoint in the other chromosome.
METHODS Positive nuclei for the translocation display one dual-color fusion
signal, representing one of the derivative chromosomes generated by
Fig. 56.1 shows the current cytogenomic methods used in detecting the translocation, and two single-color signals, one for each of the
clonal chromosomal, gene or other genomic rearrangements and normal alleles. This standard FISH strategy has been used for detec-
abnormalities in hematologic malignancies. tion of translocations in hematologic disorders at diagnosis.
For detection of minimal residual disease, the conventional
strategy lacks specificity because cells with random spatial
Cytogenetic Analysis co-localization of normal signals with different colors, found at a
frequency from 1% to 10% of scored nuclei, are seen as false positive.
Cells arrested in metaphase are obtained by exposing marrow cells To minimize this problem, an extrasensitive method was developed
sequentially to mitotic inhibitors, hypotonic potassium chloride, and in which a probe for one abnormal chromosome is designed to gener-
fixative. Chromosomes obtained from leukemic marrow are then ate extra, smaller signals in positive nuclei. Hybridization using this
subjected to the most widely used banding method, trypsin-Giemsa strategy results in abnormal cells with co-localization of two signals
banding (Fig. 56.2). The criteria used to define clonal abnormalities in dual colors, an additional two signals in one single color and one
are listed in Table 56.1 and described in the International System for signal in another single color. Application of the extrasensitive probe
Human Cytogenetic Nomenclature, 2013. has been useful in discriminating between BCR-ABL1 fusion-positive
blast crisis of CML and de novo acute lymphoblastic leukemia (ALL).
A dual-fusion strategy was developed not only to minimize false-
Fluorescence In Situ Hybridization Methods positivity but also to detect additional deletions at translocation
breakpoints. The dual-color/dual-fusion strategy includes a probe set
Fluorescence in situ hybridization (FISH) is a molecular method that with DNA sequences that encompasses proximally and distally the
allows detection of the number, size, and location of DNA and RNA translocation breakpoints on both chromosomes involved in the

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