Battling Leukemia: Methods of Diagnosis Expected to Improve
Appold, Karen, Journal of Continuing Education Topics & Issues
Leukemia is a cancer of the white blood cells (WBCs). It results from an uncontrolled proliferation of a clone of abnormal cells. "Over the past 100 years, our understanding of leukemia has evolved that we now recognize it to be a disease of the hematopoietic stem cell," says Ian Chin Yee, MD, FRCPC, chief/chair of hematology, associate professor of medicine, London Health Sciences Centre, University of Western Ontario, London, Ontario, Canada.
All humans have a tiny population of hematopoietic stem cells that function to generate all the normal cellular constituents of blood, including the red blood cell, WBC and platelets. By definition, stem cells have the capacity to self renew and generate multiple other cell lineages. It is believed that leukemia arises when a genetic alteration occurs in the hematopoietic stem cell or very early progenitor cell, Dr. Yee explains. These genetic mutations can occur spontaneously or as a result of viruses, toxic chemicals or radiation. A mutated stem cell becomes leukemic if its growth is uncontrolled. Furthermore, leukemic cells expand at the expense of other normal bone marrow elements, eventually resulting in bone marrow failure and causing anemia, neutropenia and thrombocytopenia.
Leukemia is generally divided into two broad categories--acute leukemia and chronic leukemia. As the term implies, Dr. Yee says acute leukemia describes the very aggressive natural history of this cancer, which usually rapidly progresses over a period of weeks to months and causes death. Chronic leukemias, on the other hand, behave more indolently and usually progress over a period of years.
Leukemias are further subdivided based upon cell type--either lymphoid or myeloid origin. Based on this classification, leukemias can be subdivided into acute myeloblastic leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and chronic lymphoid leukemia (CLL). As the types of leukemia behave and respond differently to treatments, it is important to distinguish these blood cancers, Dr. Yee says.
Tried and True Tests
Because leukemias are essentially a blood disorder, Wendy Brown, RT, technologist, investigational hematology, London Health Sciences Centre, says the hematology laboratory plays a crucial role in diagnosing and differentiating the subtypes of leukemia. Initially, a complete blood count (CBC) will be run to identify changes in the key parameters affected by leukemia, i.e., the WBCs, hemoglobin and platelet counts.
In CLL, an elevated lymphocyte count detected by a hematology analyzer is often the first indication that a patient may have a disease affecting the blood, Brown says. Similarly, in CML, if a persistent increase in neutrophils exists without the presence of infection, further investigation should be performed to rule out a leukemic process.
In acute leukemias, patients often present a severe illness that results in either bleeding or infection. A CBC may show a dramatic increase in the WBC accompanied with very low hemoglobin and platelets. In a subtype of AML affecting mainly promyeloctes, Brown says patients may also have derangement of their coagulation parameters as detected by the prothrombin time and activated partial thromboplastin time. A reduced fibrinogen with elevated D-Dimer, indicating a process of disseminated intravascular coagulation, may also be seen.
Morphologic examination with a microscope is the first critical step in diagnosing and classifying leukemias. For this reason, Brown says the general duty technologist plays a critical role in the initial identification of an abnormal cell population on a blood film. The hallmark of acute leukemia is the presence of very immature cells, so-called blasts.
In contrast, Brown says chronic leukemias are associated with more mature lymphoid or myeloid cells. Because by definition acute leukemia is a proliferation of very immature blast cells, distinguishing ALL from AML can often be challenging and require further testing. Additional morphologic tools include special stains, e.g., the Sudan Black, to identify cells of myeloid origin.
The standard approach to the laboratory diagnosis of ALL involves the morphologic and immunophenotypic evaluation, adds Steven J. Melnick, PhD,MD, chief, Department of Pathology and Clinical Laboratories, Miami (FL) Children's Hospital. The morphologic classification is based on FrenchAmerican-British criteria. In ALL, a consensus that a diagnosis can be established if certain flow cytometric criteria are specified exists. However, in all cases, the final interpretation should be correlated with clinical and morphologic features and possibly other ancillary studies.
The Role of Flow Cytometry
In the past 20 years, Michael Keeney, ART, FIMLS, associate scientist, Lawson Health Research Centre, London Health Sciences Centre, says flow cytometry has played an increasingly important role in identifying specific surface antigens associated with confirming myeloid or lymphoid differentiation and subclassifying leukemia. In CLL, most North American cases arise from an abnormal clone of B cells, i.e., the cells necessary for antibody production. Flow cytometry plays a crucial role in identifying the abnormal lymphocyte population and can demonstrate the clonal origin of the cell by examining the expression of surface kappa or lambda light chains on the affected B cell, confirming its evolution from an abnormal malignant clone of cells.
In acute leukemias, rapid determination of the cell of origin is crucial to allow the appropriate therapy to be started quickly. In a specific subtype of AML, i.e., promyelocytic, flow cytometry is often the first indicator that disease is present and will work in conjunction with cytogenetics to ensure confirmatory testing is done using fluorescence in situ hybridization (FISH). Because acute leukemia is the most common cancer in children, Keeney says a strong emotional dimension often exists for everyone involved in patient treatment and rapid provision of results by the laboratory can significantly assist in the quality of patient care.
Standard morphologic and flow cytometric techniques that identify the molecular abnormality associated with acute and chronic leukemia can often be helpful in diagnosis and prognosis of these diseases, Dr. Yee says. For example, cytogenetic analysis in CML identifies the Philadelphia chromosome, i.e., an abnormal chromosome resulting from the translocation of a portion of DNA between chromosomes 9 and 22. This abnormality is diagnostic of this form of leukemia and was first identified in the 1960s. This was the first definite proof that leukemia was a clonal disease resulting from the proliferation of a small population of leukemic stem cells.
One of the greatest advances in the past decade is the identification of specific genetic abnormalities in different types of leukemia, Dr. Yee says. The genetic abnormality associated with the Philadelphia chromosome in CML has resulted in the development of drugs specifically targeted to the gene product produced by this clonal abnormality. Highly effective drugs, which block the product of this gene, have been effective in treating CML.
In one type of acute leukemia, i.e., acute promyelocytic leukemia, a translocation involving chromosomes 15 and 17 occurs and alters the retinoic acid receptor gene. This genetic abnormality is not only diagnostic of this subtype of leukemia, but is also a target of treatment with retinoic acid combined with conventional chemotherapy. These combination therapies have resulted in improved survival rates of 70 to 80 percent in this type of leukemia. Several other genetic abnormalities have been associated with specific types of leukemia and have been helpful in predicting prognosis for patients and will hopefully lead to other target specific therapies, Dr. Yee says.
Flow cytometry immunophenotyping of acute leukemia provides important prognostic information that is useful therapeutically, particularly in relation to childhood ALL, Dr. Melnick adds. For example, bright expression of CD45 and CD20 are considered poor prognostic indicators and patients may require more aggressive therapy. In B lineage ALL, CD34 is a good prognostic indicator, while CD34 is a poor prognostic indicator in T lineage ALL. DNA ploidy also plays an important role in prognosis. When the DNA index is greater than 1.16, the long-term survival rate is approximately 90 percent. When the DNA index is less than or equal to 1.16, the survival rate is in the 50 to 80 percent range depending upon other various clinical factors.
Genotypic attributes of ALL are also extremely important for prognosis in children, Dr. Melnick says. Therefore, technologies such as FISH and nucleic acid amplification are routinely employed to evaluate ALL. For example, trisomies of certain autosomes such as chromosomes 4, 10, and 21 and certain chromosomal translocations such as t(12;21)(p13;q22) associated with the TEL-AML1 fusion gene are associated with a favorable prognosis in B lineage ALL. Other translocations such as t(1;19)(q23;p13) (E2A/PBX1 fusion), t(4;11)(q21;q23) (MLL/A4F fusion) and t(9;22)(q34;q11) (BCR/ABL fusion) are associated with a poor prognosis.
New Tests Expected to Emerge
The identification of genetic abnormalities also allows laboratorians to monitor for the presence of small populations of leukemic cells, Dr. Yee says. Classic morphologic microscopic examination identifies as many as 1/100 or more leukemic cells. However, flow cytometry has a sensitivity of 1/1,000 to 1/10,000. In contrast, if a specific molecular abnormality can be identified, the use of real time reverse transcription-polymerase chain reaction techniques allow for the identification of a leukemic population in the 1/100,000 to 1 in a million range. This sensitive monitoring may allow for earlier disease detection and predict individuals who will fail to respond and eventually relapse with their disease.
In addition to molecular testing, exciting developments are also seen in flow cytometric functional as says, Keeney says. Because of its quantitative and qualitative nature, flow cytometry can be used to evaluate cell treatments affecting gene expression. In the example of CML, abnormal phosphorylation of signal transducer and activator of transcription 5 (Stat5) is present. This abnormal phosphorylation is inhibited by treatment with the tyrosine kinase inhibitor imatinib mesylate. A recent paper describes an assay which can detect and quantify cellular response to this drug and suggests that it could be used successfully as a measure of Bcr/Abl activity1.
The diversity of immunophenotypic and genotypic attributes of ALL that underlie morphology have yielded many important associations which have prognostic importance and have contributed greatly to the present understanding of ALL, Dr. Melnick says.
However, the understanding of the molecular mechanisms that underlie these features has given rise to other techniques than those described that are important for the prognosis of ALL.
Novel technologies that will likely be used in the near future for the diagnosis and prognostication of ALL include drug resistance assays which may be performed using flow cytometry methodologies, microarray technology which has been used to identify novel genes of prognostic importance in ALL and may be used to identify relevant patterns of gene expression and proteomics which involve the study of proteins and their interactions, Dr. Melnick says.
These methodologies are among those that fall into the evolving field of cytomics, considered to be the science of cell-based analysis that integrate genomics and proteomics with dynamic functions of cells and tissues, Dr. Melnick says. This burgeoning field is of particular relevance to acute leukemia and other hematopoietic malignancies because of the relative ease in which the cells of inters may be separated for analysis.
In summary, Keeney says the future for diagnostic testing and disease monitoring is a promising field and advances in molecular and cellular analysis will have an increasingly important role to play in this group of diseases.
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In the following, choose the one best answer for each question.
1 Leukemia arises when a genetic alteration occurs in the hematopoietic stem cell or very early progenitor cell.
2 Which is not a subdivision of leukemia?
A. Acute myeloblastic leukemia
B. Acute lymphoblastic leukemia
C. Chronic myeloid leukemia
D. Chronic lymphoid leukemia
E. All of the above are correct
3 In acute myeloblastic leukemia, an elevated lymphocyte count detected by a hematology analyzer is often the first indication that a patient may have a disease affecting the blood.
4 In chronic leukemias, patients often present a severe illness that results in either bleeding or infection.
5 Morphologic examination with a microscope is the first critical step in diagnosing and classifying leukemias.
6 The hallmark of chronic leukemia is the presence of very immature cells. In contrast, acute leukemias are associated with more mature lymphoid or myeloid cells.
7 In a specific subtype of acute myeloblastic leukemia, i.e., promyelocytic, flow cytometry is often the first indicator that disease is present and will work in conjunction with cytogenetics to ensure confirmatory testing is done using fluorescence in situ hybridization (FISH).
8 Acute leukemia is the most common cancer in children.
9 Technologies such as FISH and nucleic acid amplification are routinely employed to evaluate chronic lymphoid leukemia.
10 Novel technologies that will likely be used soon for the diagnosis and prognostication of acute lymphoblastic leukemia include:
A. Drug resistance assays
B. Microarray technology
D. All of the above
E. A and C only
(1.) Jacobberger JW, Sramkoski RM, Frisa PS, et al. Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell-based measure of Bcr/Abl kinase activity. Cytometry A. 2003;54(2):75-88.
Karen Appold is a freelance medical writer and editor based in suburban Philadelphia. To learn more about her services, visit www.WriteNowServices.com. Contact her at firstname.lastname@example.org.…
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Publication information: Article title: Battling Leukemia: Methods of Diagnosis Expected to Improve. Contributors: Appold, Karen - Author. Magazine title: Journal of Continuing Education Topics & Issues. Volume: 11. Issue: 1 Publication date: January 2009. Page number: 22+. © 2007 American Medical Technologists. COPYRIGHT 2009 Gale Group.
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