Living Medical
eTextbook
A Digital Tool at the Point of Care
From Projects In Knowledge®
Hematology
Insights into Managing Leukemia
& MDS Edition
Chapter 1
Overview of Leukemia
Contributing Writer: Annette F. Skorupa, PhD
Contributing Editor: Jerald P. Radich, MD
This independent CME/CE activity is supported by an educational grant from Bristol-Myers Squibb.
This CME/CE activity has reached its termination date and no longer offers continuing education credit. Please note that expired CME/CE
activities may not contain the most up-to-date information available.
Overview of Leukemogenesis
Leukemias are malignant neoplasms of hematopoietic progenitor cells characterized by overproduction of immature or
differentiated leukocytes depending on the type of leukemia. 1 Accumulated evidence indicates that leukemia cells derive
predominantly from leukemic stem cells. 2,3 These leukemic stem cells confer leukemia when transplanted into nonobese
diabetic severe-combined immunodeficient mice. 4 Unlike normal hematopoietic stem cells, the cells that derive from
leukemic stem cells are not capable of normal differentiation and have reduced capacity for apoptosis. 2
In the bone marrow, leukemic cells crowd out normal hematopoietic cells and suppress normal hematopoiesis, resulting in
anemia, thrombocytopenia, and granulocytopenia that accompany leukemia. 2 In addition, leukemic cells are released into the
circulatory system, from which they commonly gain access to the liver, spleen, lymph nodes, and sometimes metastasize to
gonads or the meninges. 5
Factors that increase the risk for leukemia include a history of treatment with certain antineoplastic agents (eg, procarbazine,
nitrosureas, and epipodophyllotoxins); exposure to ionizing radiation; pre-existing conditions, such as immunodeficiency
disorders, chronic myeloproliferative disorders, and chromosomal disorders; certain viral infections (human T-lymphotrophic
virus 1 and 2, Epstein-Barr virus), and chromosomal translocations.2 Characteristic genetic mutations and/or chromosomal
translocations have been identified for the different types of leukemias. 6 - 1 0
Classification of Leukemias
The 5-year survival rate for the leukemias overall is nearly four times better today than it was half a century ago. 1 1 However,
prognosis varies widely depending on the leukemia type. The leukemias are broadly classified as either acute or chronic,
based on their clinical course and whether the clonal cell type is blast-like or mature. They are also classified as either
lymphoid or myeloid, based on their histologic morphology, which is presumed to depend on the stem cell lineage from
which they originate.2,5 The major types of leukemia, from highest to lowest prevalence in the United States (2004–2008), are
chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), and chronic myeloid
leukemia (CML). 1 2
Acute Leukemias
ALL and Its Subtypes
ALL, predominantly a childhood leukemia, is a heterogeneous disease, with genetic differences underlying the different
subtypes. 1 3 The initial mutation, often TEL/AML1 may occur in the fetus, predisposing the child to leukemia, 13,14 although
other mutations must also occur for the disease to manifest itself. 1 5 Most ALLs (85%) derive from B-cells, but about 15% are
of T-cell origin. 1 3 B-cell ALLs may derive from pre–B-cells, which lack CD10, or from mature B-cells, which bear surface
immunoglobulin. 1 3
AML and Its Subtypes
AML is a notably heterogeneous disease. 9 Some of the subtypes appear to arise from hematopoietic stem cells, whereas
others appear to derive from more mature hematopoietic cells in the leukocyte lineage (see “Morphology”). 1 6 Therefore,
subtyping of AML is based on similarities (morphologic and immunologic) to myeloid cells at various stages of differentiation
as well as on known genetic abnormalities of the clonal cells. The 2008 World Health Organization (WHO) classification of
myeloid neoplasms and acute leukemias provides diagnostic criteria based on blast levels, morphology, cytogenetics,
molecular genetics, and immunologic markers.9,17
Chronic Leukemias
CLL and Its Subtypes
CLL is believed to arise from antigen-experienced B-cells, possibly from anergic self-reactive CD5-positive B-cells in the
marginal zone of secondary lymphoid follicles. 1 8 These B-cells produce polyclonal autoantibodies against blood cell
antigens. However, despite their apparently differentiated state, they retain the ability to proliferate. 18,19 The pathogenesis of
CLL involves dysregulation of normal apoptosis due to factors in the tissue microenvironment, genetic abnormalities, and
silencing tumor suppressor genes through epigenetic mechanisms (eg, DNA methylation).19,20
CML
CML is generally diagnosed in chronic phase, appearing as an expansion of the normal hematopoietic system. Without
CML is generally diagnosed in chronic phase, appearing as an expansion of the normal hematopoietic system. Without
therapy, the disease transforms through an accelerated phase, with increasing immature blasts and new clonal cytogenetic
lesions, to blast phase. The blast cells can have an undifferentiated immunophenotype (25% of cases), lymphoid
immunophenotype (20%–30% of cases), or myeloid immunophenotype (50% of cases). 2 1
Clinical Characteristics
The overall clinical presentation of patients with leukemia is similar regardless of the type. Nonspecific symptoms include
painless swollen lymph nodes (particularly in the neck or armpit), fevers or night sweats, frequent infections, fatigue, greater
tendency for bruising and bleeding, abdominal distension due to an enlarged spleen or liver, weight loss of unknown reason,
and painful bones or joints. While patients with a chronic leukemia may be symptomless, those with an acute leukemia are
more likely to see a physician because of symptoms. Visceral organs besides the spleen and liver that may be affected
include the gastrointestinal system, kidneys, lungs, heart, or testes. Patients with central nervous system involvement may
suffer headaches, vomiting, confusion, difficulty with muscle control, or seizures.5
ALL. Children with ALL often present with cytopenias along with lymphadenopathy, hepatomegaly, or splenomegaly, along
with fever, fatigue, pallor, bleeding, petechiae, bone and/or joint pain, dyspnea, and weight loss. 2 2 Adults with ALL also
display many of these same symptoms. Disseminated intravascular coagulation is present in about 10% of patients at
diagnosis.2 3
AML. Patients with AML present with symptoms stemming from pancytopenia, including weakness, severe fatigue, infections,
and hemorrhagic findings (eg, gingival bleeding, ecchymoses, epistaxis, and menorrhagia). Some patients may present with
signs of extramedullary spread, particularly to cutaneous or gingival sites. The skeleton is a less common site of infiltration at
the time of presentation; these patients present with bone pain (eg, sternal discomfort or tenderness and aching in the long
bones).2 4
CLL. At diagnosis, patients with CLL present with broad symptom severity. Approximately one in five patients with CLL have
no symptoms and turn up with a lymphocytosis finding in a routine blood test. Another one in five patients have serious
symptoms, such as severe weakness, unintended weight loss, fever in the absence of infection, and excessive night sweats.
2 5 Disease progression also varies widely
The remaining patients exhibit the range of symptoms between these two extremes.
from indolent over long periods to rapidly progressive as prolymphocytes increase in the bloodstream. 2 6
Progressive CLL may be characterized by lymphadenopathy, splenomegaly, anemia, thrombocytopenia, lymphocyte doubling
time 2 7 Patients with CLL tend to become immunocompromised, with subnormal immunoglobulin levels. They may also
develop autoantibodies to hematopoietic cells, resulting in autoimmune hemolytic anemia or autoimmune
thrombocytopenia. 27,28
Long-term prognosis has been correlated with the variability present in the immunoglobulin hypervariable region of the
clone, and with the prevalence of cells expressing the surface antigen CD-38 and the intracellular T-cell stimulating signal
ZAP-70. However, routine testing for mutations in the hypervariable region is not currently practical.2 9 Approximately 5% of
patients with CLL progress to diffuse large B-cell lymphoma (Richter's syndrome) per year. 3 0
CML. CML begins as an indolent disease. Patients are usually diagnosed in the chronic stage, often as an incidental finding of
leukocytosis in a complete blood cell count and typically with splenomegaly. 2 1 Patients eventually transition to the
accelerated and blast phases. The transition is marked by a shift to immature myeloid cells in the bone marrow, diminishing
numbers of lymphocytes in bone marrow and peripheral blood, and a rising ratio of myeloid to erythroid cells in the bone
marrow. Symptoms accompanying the transition include fatigue related to anemia, progressive and painful hepatomegaly
and/or splenomegaly, bone pain, bone lesions, and bleeding. Most patients do not live more than 10 years after diagnosis;
the median survival ranges from 4 to 6 years. Once the accelerated phase begins, patients typically live about 1 year, and
survival is limited to a few months for patients in the blast phase.1 0
Demographics—Populations Affected and Incidence
In the United States, about a quarter of a million people are living with leukemia, and in 2010, an estimated 43,050 patients
received a diagnosis of leukemia.1 1 Leukemia constitutes the most common form of cancer in children, accounting for
approximately 27.5% of all cases in this age group. 3 1 However, approximately 90% of those patients with leukemia are adults;
the mean age at diagnosis for all patients is 66 years.3 2 The highest incidence rates for leukemia in adults are seen for white
male patients, 32,33 with blacks, Hispanics, and Asians having lower incidence rates than whites. 3 2
ALL Statistics
ALL comprises about 12% of all leukemias, 3 1 and in persons age 3 4 Children are most vulnerable to ALL between the ages of
2 and 5 years, as shown in Figure 1. The incidence has a bimodal distribution, with a decline after childhood and an increase
in people older than age 50 years, as shown in Figure 2.12,34
Figure 1. Incidence of ALL and Leukemias Overall During Childhood and Adolescence 3 4
Click to Enlarge
SEER Data 2004–2008.
AML Statistics
AML is the second most common type of leukemia in the United States. During 2004 to 2008, 28.2% of all patients with
leukemia in the United States had AML. 1 2 Men are about twice as likely to be affected as women. AML incidence starts to
increase after age 30 years (Figure 2).1 2
CLL Statistics
The incidence of CLL increases throughout adulthood, with many patients diagnosed in their late 60s or early 70s; 12
moreover, the incidence in men is greater than in women (Figure 2). 3 5 Survival with CLL is generally longer than with other
forms of leukemia, which contributes to its relatively greater prevalence in the general population. This disease exhibits a
familial pattern of aggregation, with first-degree relatives having a risk for the disease almost four times higher than that of
the general population. 2 5
CML Statistics
CML comprises about 15% of leukemias, and is most likely to affect older men. The average age at diagnosis is 65 years. 2 1
SEER Data Across Leukemia Types
Figure 2. Incidence of Acute and Chronic Leukemias in Adults by Age and Type of Leukemia 1 2
Click to Enlarge
SEER Data 2004–2008.
Recently Reported Incidence Data
A recently reported epidemiologic analysis found changes in the incidence of leukemia in the United States that may underlie
clinical and/or etiological shifts. 3 6 SEER data reported from 1987 to 2007 were analyzed from 43,970 newly diagnosed adult
cases of AML, ALL, CML, and CLL. Incidence rates were lowest for ALL (7.0) and highest for CLL (54.4), although the rate for
CLL decreased significantly over the study period (annual percentage change [APC] of -0.5 [95% CI: -0.9 to -0.1]). Incidence
rates for CML also declined during this period (APC -1.2; CI: -1.6 to -0.8). For ALL, incidence rates decreased in males (APC
-0.9; CI: -1.9 to 0.2) and slightly increased in females (APC 0.4; CI: -1.0 to 1.7), with this increase most notable in Hispanic
females (APC 4.0; CI: 1.2 to 6.8). Incidence rates increased significantly for males and females with AML from 1987 to 2001;
however, since then the rates have been decreasing. During the study observation period, a statistically significant negative
trend in the male:female ratio for cases of AML was observed ( P = .002). The authors stated that many of these changes in
incidence rates do not reflect changes in screening or diagnostic coding practices but rather from differences in causal
environmental factors during the study observation period.
Genetics of Leukemia
The Philadelphia Chromosome—A Prototype Genetic Lesion
The search for genetic changes that underlie the transformation of normal hematopoietic cells into malignant clones moved
into high gear during the 1960s, following the discovery that mitogens could trigger the proliferation of peripheral blood
lymphocytes. The resulting capability to make mitotic chromosome preparations with ease facilitated the discovery of the
Philadelphia chromosome, which is pathognomonic of CML and was demonstrated in 1973 using chromosome banding
techniques to be a reciprocal translocation of the long arms of chromosomes 22 and 9.37,38 This translocation places the 5'
domains of the BCR gene from chromosome 22 in juxtaposition with the downstream tyrosine kinase domains of the Abl gene
from chromosome 9. The fusion kinase, BCR-ABL, persistently activates a signal transducer and activator of transcription,
which moves into the nucleus and signals for progression of the committed hematopoietic precursor into the cell cycle,
thereby leading to proliferation of the clone.3 8
BCR-ABL has served as a target for low–molecular-weight inhibitors, the first of which was imatinib. These inhibitors block
proliferation of BCR-ABL–expressing cells, and have therefore been successful in treating CML. 3 9
The Genetic Basis of Cell Transformation
The mutations that give rise to the leukemias are often in genes that function to block transcription (thereby blocking
differentiation), activate cell proliferation (eg, by stimulating protein tyrosine kinases), and/or dysregulate the cell cycle or
apoptosis. 4 0 These alterations include random point mutations, gene amplifications, and chromosomal rearrangements. 1 3
Diagnostic Methods
Morphology
ALL. Morphology and histochemistry of peripheral blood and bone marrow are the classic methods of differentiating AML and
ALL. Lymphoid blasts in ALL are larger than a small lymphocyte and have a large (taking up almost the entire cell) round or
convoluted nucleus with fine chromatin and inconspicuous nucleoli. 4 1
AML. In addition to differentiating AML from ALL, the morphology and histochemistry of peripheral blood and bone marrow
are used to identify AML subtypes (see Table). A smear of peripheral blood in AML often reveals dysplastic erythrocytes,
granulocytes, and platelets. The peripheral blood myeloblasts may contain Auer rods, which are needle-shaped cytoplasmic
inclusions. 42,43 Leukemic myeloblasts can also be found in the bone marrow. 1 7
The 2008 revision of the WHO classification of myeloid neoplasms and acute leukemias set the threshold for diagnosis of
AML as 20% of blasts in the blood and/or bone marrow. However, the 2008 version also specifies t(8;21)(q22:q22),
inv(16)(p13.1q22), or t(16:16)(p13.1;q22) as being sufficient for AML diagnosis irrespective of the level of blasts in blood or
bone marrow and t(15:17)(q22;q12) as sufficient for an acute promyelocytic leukemia (APL) diagnosis.1 7
Table. Differentiation between APL, AML, and ALL 9,12,17,41,44
APL
AML
Age
Adults and elderly
predominate
Blood or bone marrow
≥20% blasts
Blast morphology
Hypergranular or hypogranular,
bilobed nuclei
Variable granulation,
Auer rods
Histochemistry
Myeloperoxidase+++ promyelocytes
Myeloperoxidase+
OR
Nonspecific
esterase+
OR
Butyrate
esterase+
Cytogenetics
Children
predominate
≥2 myeloid markers
AND (usually)
markers
Immunohistochemistry
t(15;17)(q22;q12)
[PML-RARA]*
ALL
Large nucleus,
minimal cytoplasm
≥2 lymphoid markers
AND
markers
AND (usually)
TdT-positive
t(8;21)(q22;q22)
OR
inv(16)(p13.1q22)
OR
t(16;16)(p13.1;q22)*
*Cytogenetic criteria sufficient for diagnosis in the absence of ≥20% blasts.
Abbreviations: ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; TdT,
terminal deoxynucleotidyl transferase.
CLL. Peripheral blood smears in CLL often contain "smudge cells," friable white blood cells that appear to have broken on the
slide.3 3 The blood picture generally includes high counts of small, mature-appearing lymphocytes with B-cell
morphology—above 5–10 x 10 9 / L , 26,45 reaching 500 x 10 9 /L in rare cases. 4 5 The condensed chromatin of the small, round
lymphocytes is usually mottled or cracked looking, with a thin rim of cytoplasm. 4 5 Bone marrow with more than 30% of these
types of lymphocytes is diagnostic. The appearance of CLL in the lymph nodes is similar to that of small lymphocytic
29
lymphoma.2 9 In sections of bone marrow, small clumps of CLL cells may appear in proliferation centers or gathered around fat
spaces, or may be more diffuse. 4 5
Evidence of such monoclonal B-cell lymphocytosis (MBL) can be detected in the blood years before the clinical onset of CLL,
although the presence of these cells predicts CLL requiring chemotherapy at a conversion rate of only 1.1% per year.4 6 The age
distribution of people with MBL presages the distribution in people with CLL; younger than age 40, the incidence of the
condition is 1 per 100,000 but is 30-fold higher for individuals older than age 70. 4 6 In the general population, the
incidence of MBL has been estimated to range from 0.12% to 18.2% in studies that varied with respect to methodology and
study subjects. 4 7
CML. In CML, white blood counts are generally above 25 x 10 9 /L and granulocytes of all stages are noted in the differential.
Eosinophil counts may also be high, platelet counts may be high or low, and leukocyte alkaline phosphatase is subnormal
(apparently without affecting phagocytic function).2 1 The bone marrow is packed with myeloid cells of all stages,
predominantly myelocytes, with little fat. Myeloblasts and promyelocytes are sparse, but megakaryocytes may be increased. 2 1
Cytogenetics and Fluorescence In Situ Hybridization (FISH)
In traditional karyotyping, cells are blocked in mitosis, fixed, and stained with Giemsa so as to create a light and dark
banding pattern that is distinctive for different chromosome segments.4 8 Using fluorochrome-labeled DNA probes, FISH can
extend chromosomal analysis to interphase cells. 4 9
ALL. Leukemia cells in about 25% of children with ALL are hyperdiploid. 1 3 The most common translocation in ALL is t(12:21),
which occurs in another 25% of children with ALL, but it cannot be detected by karyotyping.1 3 This translocation results in
gene fusion TEL/AML1, and the fusion protein inhibits normal response to the negative regulator TGF-. 13,50 Other recurring
translocations in ALL include t(9;22)—the Philadelphia chromosome, numerous other translocations, missing chromosomes,
and rearrangements of the mixed-lineage leukemia gene. 8,51,52
AML. About 55% of AML cells in newly diagnosed adults are characterized by cytogenetic abnormalities, which remain the
best predictors of outcome. 40,53 AML karyotypes may display rampant heteroploidy as well as deletions, duplications, and
translocations within chromosomes. The presence of any of the reciprocal translocations t(8;21)(q22;q22),
inv(16)(p13.1;q22), and t(16;16)(p13.1;q22) are sufficient for diagnosis of AML.1 7 Individuals with 16q22 breakpoint
abnormalities, such as the pericentric inversion inv(16), a t(15;17) translocation, trisomy 8, and 11q23, and individuals
displaying three or more chromosome abnormalities tend to have worse outcomes.40,54
APL, a subtype of AML, is uniquely characterized by the t(15;17) translocation, which disrupts the retinoic acid receptor
gene on chromosome 17. APL morphology, which is characteristic for hypergranulation in dysplastic promyelocytes, is
usually used as the basis for initial diagnosis to avoid further delays in treatment. This initial diagnosis is followed up with
verification of the t(15;17) translocation because it is necessary to identify: 1) cases with unusual morphology that are
responsive to treatment; 2) rare variant translocations that are resistant to treatment; and 3) the target for disease
44
monitoring.
Amplifications and deletions in AML too small to be detected in karyotype analysis can be detected using molecular
techniques (eg, single-nucleotide-polymorphism array and array CGH platforms), but it is not clear that particular recurring
mutations have a predictable relationship to pathogenesis or outcome. Gene expression profiling is likewise still
experimental. 1 3
CLL. Karyotyping CLL cells has been difficult, because they are difficult to trigger into mitosis, but FISH circumvents this by
analyzing interphase cells.26 Chromosomal abnormalities, including translocations, are generally not seen early in the course
of CLL. The deletion 13q14.3 is eventually seen in more than half of CLL patients. This deletion removes two genes encoding
regulatory micro-RNA genes and may play a role in its pathogenesis.2 9 Other chromosomal abnormalities seen in CLL clones
later in the disease course that confer a particularly bad prognosis include trisomy 12, 11q22–23, 17p13, and 6q21. 16,26,29
Paradoxically, mutations in the immunoglobulin heavy-chain variable (IGHV) gene improve the prognosis for CLL patients, 5 5
and more than 87% of CLL patients have such mutations. Among those who do not, the antigen-binding sites formed by the
variable regions often fall into groups that are structurally similar from one patient to another, an occurrence highly unlikely
to be due to chance. These observations fit a hypothesis that CLL cells are stimulated to proliferate by antigens with
particular 3-dimensional structures.2 9
Using traditional cytogenetic techniques, a novel type of genetic abnormality has been recently reported in patients with
CLL. 5 6 Jumping translocations (JT) are characterized by translocation of a chromosomal fragment onto two or more separate
chromosomes. These JTs are associated with genetic instability and aggressive disease course. The authors report the first (to
their knowledge) cases of JTs in patients with CLL. Using pokeweed mitogen, phorbol 12-myristate 13-acetate, and CpG
oligodeoxynucleotides along with FISH, JTs were observed in eight patients with CLL. Seven of the eight patients had a
deletion of Chr 17p (TP53).
CML. More than 90% of CML patients have the Philadelphia chromosome, in which the BCR on chromosome 22 is fused to the
Abelson leukemia virus (ABL) gene on chromosome 9, resulting in an oncogene that codes for a large fusion protein with
uncontrolled tyrosine kinase activity (see “Genetics of Leukemia”). 10 Those patients with CML in whom the Philadelphia
chromosome cannot be identified on cytogenetic analysis of metaphase cells will either have evidence of the BCR-ABL on FISH
or by reverse transcriptase polymerase chain reaction (RT-PCR). Patients for whom evidence of BCR-ABL cannot be found who
otherwise meet diagnostic criteria for CML are diagnosed with chronic myelocytic leukemia. 1 0
Flow Cytometry
Using flow cytometry, thousands of cells per second can be counted and analyzed. The technique is used to differentiate
leukemias and their subtypes using specific probes for cell surface markers. Although molecular techniques (described in
Polymerase Chain Reaction (PCR) Assays and Other Molecular Techniques) are powerful tools, immunophenotyping, such as
that performed using flow cytometry, remains important in diagnosis and monitoring therapeutic response. 5 7
More than 100 CD surface antigens have been identified on hemopoietic cells. Markers important in classifying leukemias
include the stem cell marker CD34; the early myeloid marker CD33, which is expressed in almost all AMLs; the B-cell marker
CD20; the T-cell marker CD3; and the lymphocyte activation marker CD45. 5 7
Flow cytometry can detect one to five leukemic blasts in a background of 10 4 normal cells. 5 8 Flow cytometry is used to detect
minimal residual disease (MRD) 5 9 in patients with acute leukemia after undergoing induction therapy. MRD refers to the
leukemic cells remaining that can trigger relapse.6 0
Some subtypes of acute leukemias have a characteristic immunophenotype involving expression of multiple antigens that can
be used to guide diagnosis. Multiparameter flow cytometry can then be used to measure response to therapy, MRD, and
detect recurrence. 5 7
In CML, flow cytometry is not helpful in the chronic phase, as the predominant cells are mature or maturing myeloid cells,
which have a normal immunophenotype. Flow cytometry may be important in CML to recognize aberrant immature leukemia
blasts as the disease progresses. In CLL, immunotyping at diagnosis of CLL can distinguish it from several types of
lymphoma that also express CD19.2 6 The characteristic phenotype of CLL cells resembles the CD5- and IgM/IgD-positive
phenotype of lymphocytes in the mantle zone of secondary lymphoid follicles with cell surface expression of the B-cell
differentiation antigen CD19, CD22, the activation antigen CD23, and the T-cell antigen CD5.2 6 Flow cytometry can also be
used at diagnosis to study CD34 and ZAP protein levels, both of which have prognostic importance. Flow cytometry is of
lesser use in following disease course in CLL.
The antibody-based immunobead assay is a newer application of flow cytometry for detecting fusion proteins. An antibody to
one component of the fusion protein is attached to beads, which are incubated with leukemia cell lysates, washed, then
incubated with fluorochrome-conjugated antibody to the other element of the fusion protein.6 1 This technique can detect
BCR-ABL (CML and B-cell precursor ALL), PML-RARA (AML/APL), TEL-AML1 (B-cell precursor ALL), E2A-PBX2 (B-cell precursor
ALL), MLL-AF4 (B-cell precursor ALL), AML-ETO (AML), and CBFB-MYH11 (AML). Results can be obtained rapidly—within 3 to
4 hours. The sensitivity and accuracy of this assay is similar to that of cytogenetics and FISH but less than that of PCR,
61
making it sufficient for diagnosis but not for monitoring of MRD.
PCR Assays and Other Molecular Techniques
Molecular techniques are currently utilized for diagnosis, prognostic classification, and response to therapy. The PCR assay is
extremely sensitive because it can amplify very small quantities of DNA or RNA. During diagnosis, PCR assays can be used to
detect important characteristic mutations (eg, FLT3 or NPM in AML), 6 2 or fusion mRNA sequences (eg, BCR-ABL in CML,
PML-RARA in APL). 10,63
Current NCCN guidelines recommend screening for the NPM1, FLT3-ITD, KIT, and CEBPA mutations in patients with AML
where possible, because of the prognostic implications of these mutations.6 3 However, documentation of previously unknown
mutations in patients with AML is growing, increasing the challenge of precise assessment of AML subtype and possibly its
prognosis. A recent report described findings from the use of a multiplex mass-spectroscopy–based panel for AML
genotyping. 6 4 In total, 96 AML cases were screened for oncogene mutations. The multiplex mass spectroscopy panel
revealed recurring and occasionally overlapping mutations, and a mutation frequency of 64% in patients with normal
cytogenetics and a mutation frequency of 27% in patients with abnormal cytogenetics. Using a combination of standard PCR
analysis and the multiplex mass spectroscopy technology, a mutation frequency of 86% was observed for patients with
normal cytogenetics. The broad-based mutation profile capabilities of multiplex mass spectroscopy may enable more precise
definition of prognostic subgroups of patients with AML.
Although pediatric ALL is characterized by both gross and submicroscopic genetic abnormalities, little is known about
epigenetic lesions, such as DNA methylation, in this disease. Figueroa et al reported findings from an integrated epigenetic,
transcriptional, and genetic analysis of 167 pediatric patients with ALL. 6 5 Abnormal patterns of DNA methylation were
detected in genes implicated in leukemogenesis, including TGFB and TNF in the presence of ERG deletion and known and
suspected oncogene transcription factor and tumor suppressor genes. Furthermore, the partial overlap of methylation
signatures with those of normal B-cells shows that these DNA methylation signatures are not indicative of different stages of
B-cell maturation. Analysis showed changes in gene expression of a specific subset of analyzed genes showing recurring
abnormal methylation. These findings suggest that epigenetic gene regulation plays an important role in ALL leukemogenesis
and possibly the disease phenotype.
Other molecular techniques used in leukemia research and sometimes in practice, particularly for AML, include comparative
genomic hybridization array, gene expression array, global gene expression profiling, micro RNA expression profiling, DNA
methylation profiling, and proteomic profiling. 66,67
Case Study
A 40-year-old man presented with fever, infection, leukemia cutis, and thrombophlebitis, and was evaluated for a
hematologic malignancy. The presence of thrombophlebitis was consistent with APL, as was the middle-aged status of the
patient.9 However, the patient’s history listed previous treatment for Ewing sarcoma with cyclophosphamide, vincristine,
doxorubicin, ifosfamide, and etoposide 7 years ago, which raised the suspicion of therapy-related AML.
The patient’s peripheral blood cytomorphology was notable for hypergranular and large Auer rod-containing blasts with
bilobed nuclei, promyeloblasts, and rare myelocytes. Hypergranular blast morphology is highly characteristic of APL
(hypergranular form).9,63
Patients with APL are at risk for death due to coagulopathy during standard induction therapy and require initial treatment
with all-trans retinoic acid, which has antithrombotic effects.6 3 Patients presenting with this morphology usually receive a
presumptive diagnosis of APL,6 3 which is to be confirmed using PCR to detect the t(15:17) translocation or FISH to detect the
RNA for the fusion protein containing the promyelocytic leukemia gene (a nuclear regulatory factor located on 15q22) and
the retinoic acid receptor alpha (17q12)9,63 A promising, sensitive immunofluorescent-based method for detecting the fusion
protein may ultimately provide a more rapid diagnosis of APL. 6 8
Although the patient’s presentation was consistent with APL, the t(15;17)(q22;q12) translocation was not detected.
Although the patient’s presentation was consistent with APL, the t(15;17)(q22;q12) translocation was not detected.
Cytogenetic analysis revealed a different translocation (8;16). The diagnosis was therapy-related AML.
Alkylating agents and topoisomerase II inhibitors have been implicated in chemotherapy-related cases of AML. 2 Although the
chromosomal effects of these classes of agents differ, patients generally receive both classes of agents, so agent-specific
diagnostic categories for therapy-related AML have not been developed. 17,69
Approximately 10% of patients with AML have a previous history of malignancy and chemotherapy. 70,71 As survival among
patients with other types of cancers improves, therapy-related AML becomes a concern in their ongoing medical
management.
Therapy-related myelodysplastic syndromes, which have similar biologic characteristics, 9 have also been documented 1 7 , but
the cytogenetics are similar to the de novo diseases. 7 2 Most patients who receive alkylating agents and/or topoisomerase II
inhibitors do not develop therapy-related AML or therapy-related myelodysplastic syndromes (MDS). Those patients who do
may ultimately provide important clues regarding the risk for developing de novo AML or MDS.
Summary
The leukemias are a heterogeneous combination derived from myeloid and lymphoid tissues. The main types—CML, CLL, ALL,
and AML—have been characterized immunophenotypically and genetically to varying degrees, and the genetic basis for some
types (eg, the Philadelphia chromosome in CML) has been extensively studied and has provided the basis for successful drug
discovery. Morphologic, immunohistochemical, immunophenotypic, and/or molecular techniques, form the basis for
diagnosis. Monitoring response to therapy and further follow-up can be assessed using highly sensitive molecular assays.
Subtype classification is evolving as clinical and molecular data continue to accumulate, using an array of sophisticated
molecular techniques.
References
1 . Leukemia & Lymphoma Society. Leukemia. 2011. Accessed 11/1/11 at:
http://www.lls.org/diseaseinformation/leukemia/.
2 . The Merck Manual. Overview of Leukemia. 2011. Accessed 11/1/11 at:
http://www.merckmanuals.com/professional/sec12 /ch152/ch152a.html?qt=leukemia&alt=sh.
3 . Passegue E, Jamieson CH, Ailles LE, Weissman IL. Normal and leukemic hematopoiesis: are leukemias a stem
cell disorder or a reacquisition of stem cell characteristics? Proc Natl Acad Sci U S A. 2003;100(suppl
1):11842-11849.
4 . Misaghian N, Ligresti G, Steelman LS, et al. Targeting the leukemic stem cell: the Holy Grail of leukemia
therapy. Leukemia. 2009;23:25-42.
5 . National Cancer Institute. What You Need to Know About Leukemia. 2008. Accessed 11/1/11 at:
http://www.cancer.gov/cancertopics/ wyntk/leukemia/AllPages#2.
6 . Zenz T, Mertens D, Dohner H, Stilgenbauer S. Importance of genetics in chronic lymphocytic leukemia. Blood
Rev. 2011;25:131-137.
7 . Dohner H, Stilgenbauer S, Benner A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia.
N Engl J Med. 2000;343:1910-1916.
8 . National Cancer Institute. Adult acute lymphoblastic leukemia treatment (PDQ). 2011. Accessed 11/1/11 at:
http://www.cancer.gov/cancertopics/pdq/ treatment/adultALL/HealthProfessional/AllPages.
9 . National Cancer Institute. Adult acute myeloid leukemia treatment (PDQ). 2011. Accessed 11/1/11 at:
http://www.cancer.gov/cancertopics/pdq/treatment/ adultAML/healthprofessional.
10. National Cancer Institute. Chronic myelogenous leukemia treatment (PDQ). 2011. Accessed 11/1/11 at:
http://www.cancer.gov/cancertopics/pdq/ treatment/CML/HealthProfessional/AllPages.
11. The Leukemia & Lymphoma Society. Facts 2010-2011. 2010. Accessed 11/1/11 at:
http://www.lls.org/content/nationalcontent/resourcecenter/
freeeducationmaterials/generalcancer/pdf/facts.pdf.
12. National Cancer Institute. SEER Cancer Statistics Review, 1975-2008; Table 1.10-Age Distribution (%) of
Incidence Cases by Site, 2004-2008. 2011. Accessed 11/1/11 at:
http://seer.cancer.gov/csr/1975_2008/ results_merged/ topic_age_dist.pdf.
13. Pieters R, Carroll WL. Biology and treatment of acute lymphoblastic leukemia. Pediatr Clin North Am.
2008;55:1-20, ix.
14. Hjalgrim LL, Madsen HO, Melbye M, et al. Presence of clone-specific markers at birth in children with acute
lymphoblastic leukaemia. Brit J Cancer. 2002;87:994-999.
15. Plasschaert SL, Kamps WA, Vellenga E, de Vries EG, de Bont ES. Prognosis in childhood and adult acute
lymphoblastic leukaemia: a question of maturation? Cancer Treat Rev. 2004;30:37-51.
16. Delhommeau F, Dupont S, Della Valle V, et al. Mutation in TET2 in myeloid cancers. N Engl J Med.
2009;360:2289-2301.
17. Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO)
classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood.
2009;114:937-951.
18. Chiorazzi N. Cell proliferation and death: forgotten features of chronic lymphocytic leukemia B cells. Best
Pract Res Clin Haematol. 2007;20:399-413.
19. Chen J, McMillan NA. Molecular basis of pathogenesis, prognosis and therapy in chronic lymphocytic
leukaemia. Cancer Biol Ther. 2008;7:174-179.
20. Plass C, Byrd JC, Raval A, Tanner SM, de la Chapelle A. Molecular profiling of chronic lymphocytic leukaemia:
genetics meets epigenetics to identify predisposing genes. Br J Haematol. 2007;139:744-752.
21. Quintas-Cardama A, Cortes JE. Chronic myeloid leukemia: diagnosis and treatment. Mayo Clin Proc.
2006;81:973-988.
22. MedlinePlus. Acute lymphoblastic leukemia.
23. Sarris A, Cortes J, Kantarjian H, et al. Disseminated intravascular coagulation in adult acute lymphoblastic
leukemia: frequent complications with fibrinogen levels less than 100 mg/dL. Leukemia Lymphoma.
1996;21:85-92.
24. Schiffer CA, Stone RM. Acute myeloid leukemia in adults. In: Kufe DW, Pollock RE, Weichselbaum RR, et al,
eds. Holland-Frei Cancer Medicine. 6th ed. 2003.
25. Rai KR, Keating MJ. Chronic lymphocytic leukemia. In: Kufe DW, Pollock RE, Weichselbaum RR, et al, eds.
Holland-Frei Cancer Medicine. 2003.
26. Dighiero G, Hamblin TJ. Chronic lymphocytic leukaemia. Lancet. 2008;371:1017-1029.
27. Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic
leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National
Cancer Institute-Working Group 1996 guidelines. Blood. 2008;111:5446-5456.
28. Ward JH. Autoimmunity in chronic lymphocytic leukemia. Curr Treat Options Oncol. 2001;2:253-257.
29. Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. N Engl J Med. 2005;352:804-815.
30. Tsimberidou AM, Keating MJ. Richter syndrome: biology, incidence, and therapeutic strategies. Cancer.
2005;103:216-228.
31. Leukemia & Lymphoma Society. Disease information and support. 2011. Accessed 11/1/11 at:
http://www.lls.org/#/diseaseinformation/ getinformationsupport/factsstatistics/leukemia/.
32. National Cancer Institute. SEER Stat Facts Sheet: Leukemia. 2011. Accessed 11/1/11 at:
http://seer.cancer.gov/statfacts/html/leuks.html.
33. Burke VP, Startzell JM. The leukemias. Oral Maxillofac Surg Clin North Am. 2008;20:597-608.
34. National Cancer Institute. SEER Cancer Statistics Review 1975-2008. Table 28.13. Age-Specific Rates and
Counts for the Top 5 Cancer Sites by Single Year of Age at Diagnosis. 2011. Accessed 11/1/11 at:
http://seer.cancer.gov/csr/1975_2008/ results_single/sect_28_table.13_2pgs.pdf.
35. National Cancer Institute. Chronic lymphocytic leukemia treatment (PDQ® ). Patient information. 2011.
Accessed 11/1/11 at:
http://www.cancer.gov/cancertopics/pdq/ treatment/CLL/Patient.
36. Ross JA, Johnson KJ, Cerhan JR, Blair CK, Soler JT, Nguyen PL. Significant recent declines in adult leukemia
incidence rates in the United States. Blood (ASH Annual Meeting Abstracts). 2009;114:Abstract 861.
2010;116:Abstract 873.
37. Rowley JD. Letter: deletions of chromosome 7 in haematological disorders. Lancet. 1973;2:1385-1386.
38. Smith KA, Griffin JD. Following the cytokine signaling pathway to leukemogenesis: a chronology. J Clin Invest.
2008;118:3564-3573.
39. Druker BJ. Translation of the Philadelphia chromosome into therapy for CML. Blood. 2008;112:4808-4817.
40. Haferlach T. Molecular genetic pathways as therapeutic targets in acute myeloid leukemia. 2008:400-411.
http://asheducationbook.hematologylibrary.org/ cgi/reprint/2008/1/400.pdf.
41. Garcia-Manero G, Kantarjian HM, Schiffer CA. Adult acute lymphocytic leukemia. In: Kufe DW, Pollock RE,
Weichselbaum RR, et al, eds. Holland-Frei Cancer Medicine. 6th ed. 2003.
42. Leukemia Library. AML Atlas, Facts, and Current Treatments. 2009. Accessed 11/1/11 at:
http://www.meds.com/leukemia/ facts/facts.html.
43. University of Virginia. March 11, 2011. Accessed 11/2/11 at:
http://www.medicine.virginia.edu/clinical/ departments/medicine/divisions/
hematology/hess-images/leukemias/ acute-myelogenous-leukemia- m3-auer-rods-100x.jpg/view.
44. Lo-Coco F, Ammatuna E. The biology of acute promyelocytic leukemia and its impact on diagnosis and
treatment. Hematology Am Soc Hematol Educ Program. 2006:156-161, 514.
treatment. Hematology Am Soc Hematol Educ Program. 2006:156-161, 514.
45. Hsi ED. The leukemias of mature lymphocytes. Hematol Oncol Clin North Am. 2009;23:843-871.
46. Rawstron AC, Bennett FL, O'Connor SJ, et al. Monoclonal B-cell lymphocytosis and chronic lymphocytic
leukemia. N Engl J Med. 2008;359:575-583.
47. Shim YK, Middleton DC, Caporaso NE, et al. Prevalence of monoclonal B-cell lymphocytosis: a systematic
review. Cytometry B Clin Cytom. 2010;78 (suppl 1):S10-S18.
48. Tefferi A, Dewald GW, Litzow ML, et al. Chronic myeloid leukemia: current application of cytogenetics and
molecular testing for diagnosis and treatment. Mayo Clin Proc. 2005;80:390-402.
49. National Human Genome Research Institute. Fluorescence in situ hybridization. 2010. Accessed 11/1/11 at:
http://www.genome.gov/10000206.
50. Ford AM, Palmi C, Bueno C, et al. The TEL-AML1 leukemia fusion gene dysregulates the TGF-beta pathway in
early B lineage progenitor cells. J Clin Invest. 2009;119:826-836.
51. Mullighan CG, Downing JR. Global genomic characterization of acute lymphoblastic leukemia. Semin
Hematol. 2009;46:3-15.
52. Faderl S, O'Brien S, Pui CH, et al. Adult acute lymphoblastic leukemia: concepts and strategies. Cancer.
2010;116:1165-1176.
53. Mardis ER, Ding L, Dooling DJ, et al. Recurring mutations found by sequencing an acute myeloid leukemia
genome. N Engl J Med. 2009;361:1058-1066.
54. Estey E. The role of cytogenetics in the treatment of AML. 2009. Accessed 11/1/11 at:
http://www.meds.com/leukemia/current/curr1.html.
55. Landgren O, Albitar M, Ma W, et al. B-cell clones as early markers for chronic lymphocytic leukemia. N Engl J
Med. 2009;360:659-667.
56. Miller C, Racke F, Lozanski G, McFaddin A, Byrd JC, Heerema NA. Novel jumping translocations (JT) associated
with genetic instability and aggressive disease in chronic lymphocytic leukemia (CLL). J Clin Oncol.
2011;29(suppl):abstr. 6533.
57. Peters JM, Ansari MQ. Multiparameter flow cytometry in the diagnosis and management of acute leukemia.
Arch Pathol Lab Med.2011;135:44-54.
58. Paietta E. Assessing minimal residual disease (MRD) in leukemia: a changing definition and concept? Bone
Marrow Transplant. 2002;29:459-465.
59. Coustan-Smith E, Campana D. Immunologic minimal residual disease detection in acute lymphoblastic
leukemia: a comparative approach to molecular testing. Best Pract Res Clin Haematol. 2010;23:347-358.
60. Hokland P, Ommen HB. Towards individualized follow-up in adult acute myeloid leukemia in remission.
Blood.2011;117:2577-2584.
61. Dekking E, van der Velden VH, Bottcher S, et al. Detection of fusion genes at the protein level in leukemia
patients via the flow cytometric immunobead assay. Best Pract Res Clin Haematol. 2010;23:333-345.
62. Kern W, Haferlach C, Haferlach T, Schnittger S. Monitoring of minimal residual disease in acute myeloid
leukemia. Cancer. 2008;112:4-16.
63. NCCN Clinical Practice Guidelines in Oncology. Acute Myeloid Leukemia. 2011; v2.2011. Accessed 11/1/11
at:
http://www.nccn.org/professionals/physician_gls/ pdf/aml.pdf.
64. Dunlap J, Corless CL, Fleming WH, et al. High-throughput mutation analysis in acute myeloid leukemia
(AML). J Clin Oncol. 2011;29(suppl):abstr 6529.
65. Figueroa ME, Chen SE, Andersson AK, et al. Integrated genetic and epigenetic analysis of childhood acute
lymphoblastic leukemia reveals a synergistic role for structural and epigenetic lesions in determining disease
phenotype. Blood (ASH Annual Meeting Abstracts). 2010;116:abstr 537.
66. Gulley ML, Shea TC, Fedoriw Y. Genetic tests to evaluate prognosis and predict therapeutic response in acute
myeloid leukemia. J Mol Diagn. 2010;12:3-16.
67. Estey E. High cytogenetic or molecular genetic risk acute myeloid leukemia. Hematology Am Soc Hematol
Educ Program. 2010;2010:474-480.
68. Dimov ND, Medeiros LJ, Kantarjian HM, et al. Rapid and reliable confirmation of acute promyelocytic
leukemia by immunofluorescence staining with an antipromyelocytic leukemia antibody: the M. D. Anderson
Cancer Center experience of 349 patients. Cancer. 2010;116:369-376.
69. Smith SM, Le Beau MM, Huo D, et al. Clinical-cytogenetic associations in 306 patients with therapy-related
myelodysplasia and myeloid leukemia: the University of Chicago series. Blood. 2003;102:43-52.
70. Tallman MS, Kwaan HC. Intravascular clotting activation and bleeding in patients with hematologic
malignancies. Rev Clin Exp Hematol. 2004;8:E1.
71. Sahin FI, Yilmaz Z, Karakus S, Boga S, Akcali Z, Demirhan B. t(8;16) AML developed subsequent to breast
cancer therapy. Hematology. 2006;11:153-155.
72. Vardiman JW. The World Health Organization (WHO) classification of tumors of the hematopoietic and
lymphoid tissues: an overview with emphasis on the myeloid neoplasms. Chem Biol Interact. 2010;184:16-20.
Copyright © 1997-2013, Projects In Knowledge, Inc. All rights reserved.
PROJECTS IN KNOWLEDGE is a registered trademark of Projects In Knowledge, Inc. LIVING MEDICAL ETEXTBOOK is a trademark of Projects In Knowledge, Inc.
PROJECTS IN KNOWLEDGE • 290 W. Mt. Pleasant Avenue, Suite 3150 • Livingston, New Jersey 07039 (973) 890-8988