Cytogenetic and Molecular Evaluation in Myelodysplastic Syndrome and in
Acute and Chronic Leukemia
Peter R. Papenhausen, PhD, Lynn C. Moscinski, MD, and Cameron G.
Binnie, PhD
The advent of molecular cytogenetic techniques has provided a new tool
for which quantitation of residual disease or transplant monitoring may be the major
advantage.
Background: The majority of the presently known nonrandom chromosome changes in
hematologic malignancy were described during the 1970s and 1980s. The last 10 years have
been devoted to the location of oncogenes and tumor suppressor genes altered as a
consequence of those changes. New molecular methodology has helped speed this process,
which has resulted in DNA sequencing of many of the genes involved, permitting molecular
detection of abnormal clones.
Methods: This review examines the most common alteration-based subgroups of
cytogenetics and molecular genetics in hematologic disorders with the exclusion of
lymphoma. Prognosis has been updated to reflect improving treatment protocols.
Results: The versatility of cytogenetics for delineating genetic changes is
difficult to match by molecular testing. Once a clonal anomaly is identified, molecular
methodology can detect residual disease with far greater sensitivity than cytogenetics,
but relies on translocation junction targets that exclude clones characterized by deletion
or trisomy.
Conclusions:Cytogenetic and molecular testing offers independent diagnostic and
prognostic evaluation for most patients with hematologic malignancy.
Introduction
Nonrandom cytogenetic alterations in leukemia provide definitive diagnosis and
prognosis in many cases and have been crucially important in localizing oncogenes and
tumor suppressor genes. DNA sequencing of many of the breakpoint junctions of the commonly
observed translocations in leukemia has provided not only insights into the related
oncogenic mechanisms, but also a target for molecular detection. Most translocations
reported in this review appear to be capable of causing acute disease without any further
genetic changes, although additional changes in chronic myelogenous leukemia (CML) are
generally necessary for blast crisis. Conversely, trisomies or deletions of tumor
suppressor genes in myelodysplastic syndromes (MDS) appear to progressively accumulate in
leukemic clones, with each change increasing the likelihood of acute transformation. It is
also clear, however, that genetic changes occur that cannot be identified by any presently
known methodology. Evidence of this comes from the large percentage of MDS cases and some
acute myeloid leukemia (AML) cases without cytogenetic alterations (approximately 50% and
30%, respectively). Additional evidence stems from the existence of many cases with two
apparently unrelated cytogenetically distinct clones. It is likely that the "common
denominator" in such cases is a submicroscopic alteration since the cells in these
cases can be molecularly confirmed to be monoclonal. These unknown underlying genetic
changes are the probable reason for variable prognosis in single trisomy- or single
deletion-based MDS/AML cases.
Molecular techniques offer specific and highly sensitive diagnostic testing of marrow
aspirates or peripheral blood and are not dependent on dividing cells. Polymerase chain
reaction (PCR)-based testing, available for many translocations, can also be performed
from paraffin-embedded archival samples or frozen sections. The sensitivity of PCR for
detection of residual disease can be as great as one malignant cell in a background of
100,000 normal cells.
This article is intended to be a concise review of cytogenetic and molecular testing in
common hematologic malignancy. There are many nonrandom changes that have not been
included due to low incidence rates.
Myeloid Disorders
Myelodysplasia
Immunophenotyping, cytogenetics, and molecular genetics can all provide valuable and
often complementary diagnostic and prognostic information in hematologic disorders
characterized by overproliferation of various cell lineages. Cytogenetics may be most
valuable in MDS. The multilineage dyspoiesis characteristic of these dyscrasias makes flow
cytometry impractical, and the relative absence of fusion alterations (translocations)
severely limits the use of molecular techniques that target the DNA sequences within the
fusion sites. The five accepted subclasses of MDS, as well as the percentage of cases that
are cytogenetically abnormal, are listed in Table 1. The cytogenetic changes that are most
commonly found in each subgroup are noted in Table 2. The percentage of cases with
abnormal chromosomes tends to increase with the blast percentages.
The deletion of the long arm of chromosome 5 and monosomy 7 deserves special mention
since these alterations have a consistent clinical presentation.1
Characteristics of these are as follows:
Deletion of the Long-Arm
Chromosome 5 (5q– Syndrome)
- Macrocytic anemia resistant to therapy
- Normal or increased platelets
- Morphologically characteristic megakaryocytic abnormalities (micromegakaryocytes;
hypolobulated nuclei)
- Generally mild clinical course
- Only rarely transform to AML, provided 5q- is the only cytogenetic anomaly present
- 5q31 is the minimal common deleted segment suggesting the locus of a tumor suppressor
gene)
Monosomy 7 Syndrome
- Male:female ratio: 2:1
- Age at onset:6 months to 19 yrs (median = 7 yrs)
- Clinical complications:Infection and bleeding
- Background:Usually no exposure to genotoxins, but multiple sibling cases indicate
possible predisposition
- Presenting hematology:Anemia, decreased platelets, normal or elevated white blood
cell count (especially monocytes), marrow hyperplasia
- Myelodysplastic stage:Duration up to 12 yrs (median = 2.2 yrs); may be
categorized as idiopathic myelofibrosis
- Cytogenetics:Percentage of monosomic cells in marrow increases with progression;
additional alterations often arise with transformation
- Subtype of acute leukemia at progression: frequently transforms to AML M1, M2,
and M4
Most MDS-associated aberrations are deletions or trisomies. These types of alterations
exist in almost all types of neoplasia in relation to clonal progression. Specific deleted
segments largely correlate with the loss of tumor suppressor genes.2,3 With
most tumor suppressor genes, a loss of both copies is necessary to produce an effect; with
others, however, a loss of function from a single copy may be sufficient to produce an
effect. A second "hit" in the same suppressor gene may occur as a submicroscopic
mutation undetectable by cytogenetic analysis. At present, it is not known what causes
transformation in the few patients who present with a deletion and no other cytogenetic
change but progress to AML (Fig 1). Most patients who do show clonal karyotypic evolution
transform to AML.
Translocations are rare in MDS and are generally characterized by a brief MDS phase
before blast levels increase to the point where a diagnosis of AML is made. Alterations
involving chromosome 3 correlate with dysmegakaryopoesis and a poor prognosis (Table 3).
Other translocations occur secondary to previous therapy. Prognosis-correlated cytogenetic
subgroups are apparent, although transformation to AML occurs in all groups.
Therapy-Related MDS/AML (t-MDS/t-AML)
The development of MDS or AML secondary to treatment of malignant disease with
cytotoxic drugs or radiation has been recognized for some time.4 Distinct
cytogenetic differences in the developing disease correlate with both separate time
courses and type of therapy administered (Fig 2). The major portion of these differences
involve patients who receive alkylating agents. Most of these patients first present with
trilineal dysplasia within five years of treatment. Abnormalities of chromosomes 5 and/or
7 are noted in more than 70% of the cases leading to refractory AML (median survival = 8
months). Frequently, the same 5q– as seen in refractory anemia (RA) is found, but
additional alterations typify t-MDS (often with the loss of multiple tumor suppressor
genes). A smaller subset of patients develop t-AML following treatment with drugs that
target topoisomerase II. These patients present with little or no MDS phase and are
characterized by translocations involving the MLL or AML1 genes at 11q23 and 21q22,
respectively. These malignancies have been shown to respond to intensive induction
therapy.5,6
Acute Myeloid Leukemia
AML is cytogenetically characterized by many of the same anomalies as noted in the MDS
disorders, indicative of a common derivation, plus a number of specific translocations.
Multiple MDS-related anomalies are typically noted in AML, particularly in conjunction
with loss of all or part of chromosomes 5 and 7. The latter alterations that are so
characteristic of t-AML are also found in de novo disease with the same poor prognosis.
The major AML translocations are presented in Table 4. Several of these are clinically
important through their association with distinctive modes of disease presentation and
prognosis7-11 (Table 5). These translocations can also be detected by molecular
genetic techniques. The t(8;21), t(15;17), and inv(16) can be detected by PCR-based
methodologies (more specifically RT-PCR, which looks for the presence of chimeric mRNA
transcripts rather than the DNA translocation directly). The extreme sensitivity of PCR
allows effective monitoring of minimal residual disease. In general, a PCR-negative result
has a good prognosis, while the return to a PCR-positive result may indicate the need for
further treatment. Because of the large number of translocations involving the MLL gene
(11q23), rearrangement of this locus can be achieved only by Southern blot, which is less
sensitive than PCR.
Chronic Myelogenous Leukemia and Other Myeloproliferative Disorders
Four main subtypes of myeloproliferative disorders are generally recognized: chronic
myelogenous leukemia (CML), polycythemia vera, myelofibrosis with agnogenic myeloid
metaplasia, and essential thrombocythemia. These subtypes differ in the proportion of
proliferating cells of each marrow lineage, but all demonstrate a tendency to progress to
AML.
Chronic Myelogenous Leukemia
The specific hallmark translocation t(9;22), commonly known as the Philadelphia
chromosome (Ph1), was the first hematologic cytogenetic anomaly to be
described. The DNA fusion gene (bcr-abl) can be found in virtually all CML
patients, including the 3% to 5% who do not display the typical translocation or a variant
translocation. The CML-related fusion produces a larger protein (210 kDa) than a similar
translocation seen in acute lymphoblastic leukemia (ALL), which involves a different bcr
breakpoint region. The accelerated (or blast) phase of the disease is often
accompanied by additional cytogenetic changes. Trisomy 8, a second Ph1
chromosome, or an isochromosome of the long arm of 17 occurs in 70% of myeloid blast
crisis. Other less consistent changes are observed in lymphoid blast crisis.12
Polycythemia Vera
The most common anomaly is 20q–, which is found in approximately 27% of
chromosomally abnormal patients. The number of patients with this deletion increases with
the duration of disease. Some patients with MDS of the RARS subtype also have this
aberration. Trisomy 8 and/or 9 and interstitial deletion of 13 are also common.13
Myelofibrosis With Agnogenic Myeloid Metaplasia
Trisomy 8 is by far the most frequent anomaly. Monosomy 7, frequently observed in
childhood myeloproliferative disorders (clinically resembling juvenile CML), is a
recognized syndrome with frequent progression to refractory AML. Deletions of long arm 13
and 20 and amplification of long arm 1 are also common.13
Essential Thrombocythemia
Patients with this disorder usually have a normal karyotype except when a t(9;22)
identifies true CML. The risk of transformation to AML is much smaller than in other
subgroups.14
In general, finding cytogenetic anomalies at diagnosis does not imply that conversion
of myeloproliferative disorders to AML is imminent, but further karyotypic evolution has
been found to correlate with disease progression. Complex aberrations, regardless of
presentation time, clearly correlate with adverse prognoses.
Lymphoid Disorders
In general, lymphoid neoplasms are clonally expanded proliferations of cells arrested
at distinct stages of B- or T-cell development. Disorders of the B-cell maturational
cycle, depicted in Fig 3, are far more common than T-cell dyscrasias.
The principal change during maturation of immature lymphoblasts to B lymphocytes is the
rearrangement of the immunoglobulin genes. The heavy chain locus is located at 14q32, and
the light chain loci are at 2p12(kappa) and 22q11(lambda). All of these loci are involved
in chromosomal alterations that characterize B-cell neoplasia.15 In many cases,
however, the genetic abnormality is too small to be detected by cytogenetics or the
genetic change does not produce a cell surface marker detectable by flow cytometry. The
presence of monoclonal malignant cells can then be established by detection of a molecular
rearrangement of the immunoglobulin genes. Similarly, malignant T-cell disorders, which
are characterized by rearrangement of the T-cell receptor genes (proportional and partial
derivative at 14q11, beta at 7q34, and gamma at 7p13), can be detected by rearrangement of
the loci by cytogenetic or molecular methodology.16 Molecular detection of
immunoglobulin and T-cell receptor gene rearrangement has to this point been performed by
Southern blot. The recent development of PCR-based tests for gene rearrangement promises
to not only increase the sensitivity of detection, but also offer the opportunity of
testing paraffin-embedded specimens.
Acute Lymphoblastic Leukemia
Specific diagnosis/prognosis-associated chromosomal translocations are found in
approximately 40% of B-ALL cases (Tables 6 and 7 -- Please see hard copy of journal for
Table 7). These alterations are clonal and directly involved in leukemogenesis. Similar to
AML, these recurrent alterations result in fusion genes or transcriptional derepression.
Patients with the poor prognosis translocations -- 9;22, 4;11, and 1;19 -- are good
candidates for allogeneic bone marrow transplantation following the first remission.
Although most of the ALL translocations bear a poor prognosis,17 this is not
true of the newly described t(12;21).18 The subtle nature of the terminal band
exchange in this rearrangement and the difficulty of obtaining optimal banded preparations
in ALL have likely led to extensive underdiagnosis. New molecular cytogenetic
(fluorescence in situ hybridization [FISH]) or molecular testing will be important in
detecting this favorable prognostic subgroup.
The hyperdiploid (>50 chromosomes) subgroup is also notable due to the high
incidence of this very favorable prognostic subgroup.19 These cases have a
CALLA (common acute lymphoblastic leukemia antigen)-positive immunophenotype and a DNA
index of 1.14 to 1.20. The invariant nature of specific trisomic chromosomes and tetrasomy
21 facilitates targeted FISH detection methodology, although the pathogenetic mechanism
behind such one-step amplification remains difficult to explain.
Chronic Lymphoproliferative Disorders
As already indicated, neoplastic clones can be detected by monoclonal rearrangements of
immunoglobulin genes (B-cell) or T-cell receptor genes. Cytogenetic alterations at the
site of those genes are the most consistent changes noted in all chronic lymphoid
dyscrasias. An inversion of chromosome 14 involving the T-cell receptor gene located at
q11 and a putative oncogene (often called TCL1) at q32.1 has been reported in adult T-cell
leukemia, T-CLL, and T-PLL (T-prolymphocytic leukemia). TCL1 rearrangement can be observed
in random cells of normal individuals and at increased levels in ataxia telangiectasia, so
rearrangement alone appears insufficient in producing a malignancy.
Many cytogenetic studies have targeted the most common subgroup. B-cell CLL. (T-cell
CLL accounts for less than 2% of all cases.20) The following consensus points
are noteworthy:
- B-mitogen culturing has increased the detection of abnormal clones to 40% to 60% of
cases.
- The most common alteration is trisomy 12, which can be detected in approximately one
third of cytogenetically abnormal cases (greater frequency with FISH).
- Trisomy 12 is associated with a mixed-cell phenotype often evolving to prolymphocytic
leukemia and poorer prognosis.
- Patients characterized by a deletion of long arm 13 (13q–) have typical CLL
morphology and a similar survival as those without clonal changes.21,22
- Those patients with the 14q+ (IgH locus) anomaly or complex karyotypes have the poorest
prognosis.
Numerous cytogenetic studies in multiple myeloma have also generated considerable data.
The percentage of abnormal cases (20% to 60%) has varied considerably, presumably due to
the slow proliferation of plasma cells that often require extensive searching to identify
the abnormal clone. The following observations are generally accepted23,24: (1)
Survival is longer for patients without a detectable abnormal clone, (2) the most
consistent anomaly is a 14q+ alteration with t(11;14)(q13;q32) predominating, (3) the
often complex abnormal clones are typified by loss of tumor suppressor genes and nonrandom
trisomy but not trisomy 12, and (4) prognosis worsens with increasing proportions of
clonally abnormal cells detected.
Molecular Cytogenetics
In situ hybridization using sequence-specific DNA probes with fluorescence (FISH) or
enzyme-labeling systems is primarily applied for the segmental chromosome analysis at
interphase. The main advantages are (1) no requirement for cell division, (2) many cells
can be evaluated and quantified, as opposed to nonquantitative molecular technology, and
(3) good results can also be obtained from formalin-fixed paraffin embedded or frozen
tissue. The disadvantages are: (1) targeted probes give no information regarding clonal
evolution, and (2) low levels of residual disease (<4%) merge with normal background
levels.
Uses (Interphase)
The primary use involves the detection of residual disease or transplant monitoring
(Fig 4). Cloneidentifying probes may be selected by using the information obtained from a
pretreatment diagnostic karyotype. These may be translocation specific with dual color
merging (Fig 5) with probes specific for DNA sequences adjacent to the breaksites or may
be shown by a split of a probe that spans a specific gene leaving an extra signal (Fig 6).
Alternately, probes may be used that are specific to appropriate alphoid (centromere)
repeats of preidentified clones with monosomy or trisomy.
Additional uses of FISH probes involve the detection of specific oncogene
amplifications or deletions that confer a poor prognosis. For example, the amplified
self-fusion known to occur at the MLL gene site (11q23) may be demonstrated by a probe now
available. This probe may be applied adjunctively to metaphase analysis in complex
rearrangements (Fig 7A-B). Deletion of tumor suppressor genes is readily demonstrable by
loss of a hybridization signal using probes that are specific for the region of the gene
or the gene itself.
Emerging Technology
Specific screening for the loss of a battery of tumor suppressor genes (or gain of
regions of selective advantage) using robotics and FISH (multiplex analysis) will likely
be available in the near future. This technology will probably be better applied to the
analysis of MDS rather than acute leukemia since balanced translocations (the major
oncogenic mechanism in acute leukemia) are not easily detected by FISH.
The new competitive genomic hybridization (CGH) technique has been improved in the last
few years. This methodology uses the principle of competitive hybridization of equal
amounts of whole-tumor DNA and normal-control DNA (each labeled with different-colored
fluorochromes) to normal human metaphases. A fluorescence microscope captures the mixed
color results on each chromosome (Fig 8), and an image analysis system computes the
relative fluorochrome predominance at each chromosomal segment. Gains and losses of test
DNA are reflected in localized color changes. The main drawback of the technique is that
translocations are not detectable. However, there are major advantages of CGH in the
analysis of tumor tissue. CGH can quickly determine whole genomic imbalance without the
need of laborious and often unsuccessful tumor culturing. Table 8 presents a comparison of
genetic testing methodology.
Conclusions
Detection of an acquired clonal cytogenetic change in the bone marrow is diagnostic of
a neoplastic disorder. Typical MDS-correlated cytogenetic anomalies strengthen the
diagnosis. No alteration is completely specific for MDS in the MDS/AML differential.
Deletions and trisomy tend to typify MDS, while balanced translocations are found in
AML or in cases with brief MDS phases preceding AML. Deletions of all or part of
chromosomes 5 and 7 and variable additional changes define a subgroup of MDS/AML with poor
prognosis, frequently arising secondary to previous therapy. Complex karyotypes are
consistent with clonal evolution/transformation. Normal karyotypes do not rule out
neoplasia.
Molecular monitoring of residual disease is now available for most of the
translocation-related leukemias. PCR-based testing is so sensitive that nonproliferative
residual disease may be detected in remission. Conversion to PCR negativity is generally
consistent with prolonged remission.
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