Acute Leukemia with a Focus on WHO Classification
Interpretation of Genetic Designations of the AML Subtypes
- t = translocation. The two numbers in parentheses after t are the numbers of the chromosomes involved in the translocation. - The numbers following "q" in the next set of parenthesis refers to bands on the q or long arm of the chromosome where that translocation created a fusion of two genes that initially had not been in proximity with each other. - The last part, RUNX1-RUNX1T1 is the name of the actual genes involved. That fusion (RUNX1-RUNX1T1) leads to the production of a chimeric protein which disrupts the normal function of a transcription factor complex that regulates normal hematopoiesis. So in other words, a part of chromosome #21 broke off and attached to chromosome #8, resulting in the fusion of genes which resulted in a protein that interfered with the regulation of hematopoietic activity - thus, unchecked proliferation. Also, just to make the correlation with the FAB classification, this particular type of AML corresponds morphologically with the M2 subtype in FAB. Other designations that you may see include the designation inv which means that part of the chromosome was inverted such as in AML with inv(16)(p13.1q22); p indicates the short arm of the chromosome, whereas q indicates the long arm. The image shows a blood smear of the AML with t(8;21)(q22;q22.1);RUNX1-RUNX1T1: subtype, showing myeloblasts (note the abundant cytoplasm in these blasts and azurophilic granules).
A three year old is diagnosed with ALL. Choose the characteristics which would yield the most favorable prognosis for this child. Check all that apply: A white blood count of 65,000/ μL 62 chromosomes Philadelphia Chromosome No CNS involvement
62 chromosomes No CNS involvement feedback: Hyperdiploidy (51-65 chromosomes) and no CNS involvement are both very favorable prognostic factors, as well as the patient's age. Finding the Philadelphia Chromosome, or leukemic involvement in the central nervous system are very unfavorable prognostic factors.
25% blasts are found on the smear of a four-year old. Based on age alone, what is the most likely major type of leukemia? AML ALL ALAL Complete remission for AML is defined as: Please select the single best answer The patient has no recurrence of disease after five yearsThe patient is eligible for consolidation therapyThe patient is showing normal hematopoietic activity following induction therapyThe patient has no disease for three yearsCML
ALL feedback: Acute lymphoblastic leukemia (ALL)is the most common type of leukemia among young children. Following that, AML would be the second most common type. ALAL and CML are more common in elderly adults. The fact that there were more than 20% blasts helps rule out CML and other chronic leukemias.
Occurrence and Common Types of AML
AML in general is much more common in adults than children. Average lifetime risk of AML in the US is approximately 0.5% which translated to approximately 19,520 new cases in 2018. According to the Merck Manual, some of the more common cytogenetic abmormalities of AML include: AML with APL-RARA (t15;17)(q24.1). This is a type of promyelocytic leukemia (FAB category M3). Approximately 13% of AML is this subtype. AML with inv(16)(p13.1;q22) or t(16;16)/CBFB-MY11. Approximately 5% of AML is this subtype. AML with t(8;21)/(q22;q22)/RUNX1-RUNX1T1. This was the example shown on a previous page and occurs in approximately 7% of AML cases. Prognosis related to these and several other subtypes will be discussed in subsequent pages.
A patient is diagnosed with AML with t(9;11)(p21.3;q23.3);MLLT3-KMT2A. What major WHO classification does this belong to? AML with Myelodysplasia related changes AML with recurrent genetic abnormalities AML not otherwise specified Myeloid proliferations related to Down's Syndrome
AML with recurrent genetic abnormalities feedback: When a specific chromosomal/genetic change is specified, that AML most likely falls within the AML with recurrent genetic abnormalities.
Prognostic factors of AML
Accurate assessment of the prognosis of the patient is important for overall management and treatment of AML. Patients can be stratified according to their risk of treatment resistance or treatment-related mortality. This will help guide the type and intensity of treatment. Important prognostic factors are: The WHO subtype Certain cytogentic subtypes have a much better prognosis than others (see subsequent pages). Clinical Factors Age at diagnosis. Increased age is associated with a poorer prognosis, although this is also dependent on the WHO subtype. Prior hematological malignancies. If the patient had a prior hematological malignancy, this carries a substantially poorer prognosis. Other Variables (These variables can be especially important in older patients.) Platelet count Serum creatinine Albumin
Acute Leukemia of Ambiguous Lineage
Acute leukemia of ambiguous lineage (ALAL) was a subtype established in the 2016 WHO classification revision. It is a very rare leukemia, that is further subdivided into five possible subgroups, partially based on chromosomal analysis. ALAL patients present with an acute leukemia, but a specific lineage can not be assigned. Some types of ALAL are immature hematopoietic neoplasms that show no differentiation into lymphoid or myeloid lines (AUL), whereas other types exhibit markers of both AML and ALL. The latter are referred to as mixed phenotype acute leukemia (MPAL). The image to the right shows a case of ALAL. The arrow points to hypolobate neutrophils, emphasizing how unusual some of these cases can be. When immunophenotyping these leukemias, AUL lacks T-cell specific, B-cell specific, and myeloid specific markers. MPAL, on the other hand, shows various immunophenotypes. There are no clinical features that are specific to ALAL. but rather have signs and symptoms similar to AML and/or ALL. ALAL does generally carry a poor prognosis.
What are characteristics of acute leukemias as compared to chronic leukemias? (Check all that apply) More than one answer is correct. Please select all correct answers Acute leukemias can lead to death in a few weeks to month, if not treated. Acute leukemias typically show 20% or greater blasts in blood and/or bone marrow Acute leukemias are almost always found in older adults. Acute leukemias rarely exhibit bleeding, bruising, or fever at time of diagnosis.
Acute leukemias can lead to death in a few weeks to month, if not treated. Acute leukemias typically show 20% or greater blasts in blood and/or bone marrow. feedback: Acute leukemias come on suddenly and progress rapidly; if not treated, a patient can die within a few weeks or months if untreated. Patients with chronic leukemias, on the other hand, can possible survive up to several years. A defining characteristic of acute leukemia is 20% or greater blasts. A characteristic of chronic leukemias is that they are most common in older adults, Although patients with chronic leukemias can show bleeding, bruising or fever, these findings are much more commonly seen in acute leukemia.
Signs, symptoms, and background of ALL
Acute lymphoblastic leukemia is the most common pediatric cancer. 60% of all ALL cases occur in children, with a peak incidence at age two to five years old. A second peak occurs in adults over 50. As with AML, chromosomal abnormalities and genetic alterations affect the differentiation and proliferation of precursor cells. Presenting symptoms can include: fatigue pallor infections CNS symptoms easy bruising and bleeding abdominal pain swollen lymph nodes difficulty breathing (especially in T Cell-ALL)
This smear is from a patient with 21% blasts and a diagnosis of acute leukemia. Which of the following is a more specific diagnosis based on the smear? Acute myelocytic leukemia (AML) Acute lymphocytic leukemia (ALL) Acute leukemia of ambiguous lineage (ALAL) Nothing specific can be determined without further testing
Acute myelocytic leukemia (AML) feedback: The arrows point to Auer rods which are specific for blasts of myeloid lineage. Although further testing is needed, Auer rods, if seen, can lead to a fairly certain diagnosis of AML.
This image of a Wright's stained smear is from a patient who presented with fatigue, high white blood count, and anemia. Is this more likely an acute leukemia or a chronic leukemia? Is it more likely to be lymphoblastic or myeloid? Please select the single best answer Acute myeloid leukemiaChronic myeloid leukemiaAcute lymphoblastic leukemiaChronic lymphocytic leukemia
Acute myeloid leukemia feedback: This image shows numerous blasts, indicated by the loose chromatin and nucleoli. Acute leukemias are characterized by 20% or more blasts, so we can guess that this is probably an acute leukemia. It is more likely to be of myeloid origin because the blasts are very large and have very prominent nucleoli and there is one faint Auer Rod in one of the blasts, which also supports myeloid lineage. Lymphoblasts typically are smaller and show less distinct nucleoli. Please note that this would just be the most likely answer, but that further cytochemical, cytogenetic, and other testing would be needed for a definitive diagnosis.
Favorable prognostic factors
Age 3-9 years WBC count <25,000/ μL in adults or <50,000 in children Hyperdiploidy (51-65 chromosomes) T(1/19) and t(12/21) No CNS involvement at diagnosis
The WHO classification of AML can be based on all of the following criteria except: Age of the patient Cytogenetic analysis of the leukemic cells Cell type and morphology of the cells Genetic changes
Age of the patient feedback: Multiple criteria can be used to determine the WHO AML subtype including cytogenetics, actual genes involved, and the cell types and morphology. Although age can figure into the prognosis, it does not determine the diagnosis.
Newer treatments
Although chemotherapeutic drugs are often the mainstay of many forms of AML, newer targeted therapies are constantly being developed. Targeted therapy refers to a drug that targets or interacts specifically with a protein, enzyme, or receptor that is being expressed as a result of the mutation that is causing the AML. An early targeted therapy that was developed was actually to CML in which the translocation known as the Philadelphia Chromosome caused the fusion of the bcr-abl genes which triggered the overexpression of an enzyme known as a tyrosine kinase. This enzyme is pivotal in the unchecked replication of the cells. By inhibiting this tyrosine kinase, the cells can be induced to stop replicating and to differentiate. Newer research shows that some of the mutations in AML can also involve tyrosine kinases, and thus newer generations of these inhibitors may be useful in this disease as well.
Genetic Analysis
Although chromosomal aberrations are the hallmark of ALL, they are not sufficient to generate leukemia. It is usually the genetic changes that are linked to overexpression or underexpression of various genes which regulate proliferation or maturation. It is interesting that a Philadelphia Chromosome (seen in CML) can be found in some cases of ALL. This involves the BCR/ABL1 genes and the tyrosine kinases which are targeted by some CML treatments. Some commonly seen genetic changes include: ETV6-RUNX1 (caused by a translocation of 12;21) IKZF1 (involves a deletion of a key transcription factor important in B cell development) BCR/ABL1 (involves a kinase activating factor) Predisposing factors that can lead to these changes include certain diseases such as Down Syndrome or Fanconi anemia, exposure to radiation, pesticides, or other biological or chemical toxins. However, most cases of ALL appear de novo in previously health individuals.
Method of AML treatment
Although it depends on the exact subtype of AML, as well as the patient's age, clinical condition, and other factors, it can generally be said that treatment for AML is done in two phases. The first is known as induction therapy, in which the patient is often given a chemotherapeutic drug to achieve complete remission. The definition of complete remission is revised from time to time, but it can be basically defined as a state of normal hematopoiesis after the induction therapy. Patients should be evaluated after about two weeks of induction therapy by examination of bone marrow aspirate and biopsy. If these do not indicate that the patient is in remission, then they typically would receive another round of induction therapy. Those patients that do show they are in complete remission are often offered consolidation therapy in order to treat any residual disease that has not been detected. Options for such treatment include chemotherapy and allogeneic hematopoietic stem cell transplants.
Molecular Genetics of AML
Although most diagnoses of AML genetic changes are based on whole chromosomal changes seen when doing a karyogram, smaller scale mutations are becoming increasingly important. This is especially true for approximately 40% of cases where no chromosomal change has been identified. The following mutations have been found to be important in AML prognosis and treatment: FLT-3 NPM1 CEBPA KIT Many other mutations are being discovered as well. These gene mutations affect transcription and thus replication either directly, or through epigenetic regulation. As molecular methods advance, gene testing will become increasingly important. Next generation sequencing (high-throughput sequencing) is a method used to detect these mutations. Although expensive and time-consuming, the number of panels are becoming commercially available, and it is likely that this type of testing will be done more widely in the future. Just to stress how important this information is becoming, it has been shown that in the AML groups with recurrent genetic abnormalities, finding the NPM1 and CEBPA mutations has been associated with a very favorable prognosis. It should be noted, however, that in studying the whole genome of AML patients, many mutations were found that were actually silent or unrelated to AML.
Which of the following can be used to treat acute leukemias? (check all that apply) Antimetabolites Monoclonal antibodies Corticosteroids Alkylating agents
Antimetabolites Monoclonal antibodies Corticosteroids Alkylating agents feedback: All of the above can be used for acute leukemia treatment; the actual choice of drug is dependent on the patient's WHO classification, age, and clinical status.
Distinguishing between Acute and Chronic Leukemia
As mentioned previously, one of the important first steps in diagnosis is whether the neoplastic disorder appears to be acute or chronic. To make this determination, count the percentage of blasts in the peripheral blood smear and also in the bone marrow smear. A general guideline is that if 20% or more blasts are found in one or both of these specimens, then it is an acute leukemia. This is an especially important distinction between AML and the chronic myeloid disorders (e.g. myelodysplastic syndromes (MDS) and myeloproliferative disorders (MPD), which may also be referred to as myeloproliferative neoplasms (MPN)). The top image to the right shows numerous blasts in a blood smear of AML. Note the characteristic large nuclei with loose, lacey chromatin, high nuclear/cytoplasmic ratio, and the presence of nucleoli. Also, there are few representatives of other stages of maturation. Contrast this to the smear below which is from a patient with chronic myeloid leukemia (CML). Note that there are fewer blasts, and more mature cells. A number of maturation stages can be seen, such as myelocytes, metamyelocytes. and segmented neutrophils.
Determination of ALL Lineage
As previously mentioned, once it is determined that the patient has 20% or greater blasts, the next step is to establish whether they are lymphoblasts or myeloblasts. Pages 11 and 12 deal with morphological differences as well as using cytochemical stains. Also, histochemical staining for terminal deoxynucleotidyl transferase (TdT) can be useful in determining that the blast is of lymphoid lineage. TdT is an enzyme found in immature (developing) lymphocytes which functions to help synthesize their specific antigen receptors. If it is found to be Acute Lymphoblastic Leukemia, the next important step is to discover whether the lymphocytes involved are T cells or B cells. This can be accomplished by immunophenotyping for "markers" - that is , cell surface proteins that are specific for either B cells or T cells. Flow cytometry uses fluorescently stained antibodies specific for these markers. Using this technique, we can not only determine B or T cell lineages, but we can also count the numbers in a given quantity of blood or bone marrow. These membrane proteins or markers usually have numbers prefaced by "CD" for Cluster of Differentiation. Among the many markers that can be detected, finding CD3 can identify the cell as a T cell and CD19, CD20, and CD22 will indicate B cell lineage. Table 11 shows some examples of CD markers for different cell lines.
Differentiation between Myeloid and Lymphoblastic Leukemias Using Cluster of Differentiation (CD) Markers
As previously mentioned, other tests exist to differentiate between myeloid and lymphoid lines. One way is by the use of monoclonal antibodies to detect surface markers on the cells. This is done using flow cytometry techniques which will be described in more detail later. Cell LineageCD Markers MyeloidCD13, CD33, CD15, CD117 T-CellCD2, CD3, CD5, CD7 B-CellCD19, CD20, CD22, CD79 MegakaryoblasticCD41, CD61
Prognosis of AML
As stated earlier, accurate diagnosis of the specific WHO subtype will help to determine the patient's prognosis, their response to therapy, their likelihood of remission, and their overall chances of survival.
Basics of Laboratory Testing
As stated earlier, laboratory diagnosis begins with finding immature cells on the peripheral blood smear. If blasts are seen, the next step would be to characterize the lineage of the blasts. Bone marrow biopsies and smears are then obtained. If blasts are 20% or greater in the bone marrow and/or peripheral blood, presumptive acute leukemia can be determined. Additional steps that can be taken: Further morphologic evaluation Cytochemical staining Conventional cytogenetic analysis Appropriate molecular genetic and/or FISH studies Obtaining possible CSF sample, skin or other biopsies, depending on patient's clinical situation Steps 1 and 2 were discussed earlier in the sections on "differentiation between myeloid and lymphoid lines" and "distinguishing between acute or chronic leukemia". Steps 3 and 4 will be discussed in the following pages.
Diagnosing ALL
As with AML, ALL is first suspected when blasts are seen on the peripheral blood smear. 20% or greater blasts in the blood or bone marrow constitute a diagnosis of acute leukemia, and then establishing that they are lymphoblasts will give an initial diagnosis as ALL. Techniques as described in the section on cytochemical stains can help distinguish myeloblasts from lymphoblasts. In ALL, lymphoblasts can sometimes be very numerous, as much as 90% of the total white blood count. Bone marrow smears and biopsies are usually done next; the bone marrow can typically show between 25-95% lymphoblasts. To further establish a definitive diagnosis, immunophenotyping using flow cytometry and cytogenetic studies can be performed. More information on this will follow under laboratory diagnosis. Also, a lumbar puncture to analyze the CSF is usually done upon initial diagnosis. If the central nervous system is involved, further testing might be done. Coagulation studies and baseline chemistries are also done. The image at the right shows a smear with numerous lymphoblasts.
Prognostic Factors for ALL
As with AML, an accurate assessment of prognosis is important for the treatment and management of ALL. Both clinical factors and cytogenetic changes impact prognosis and treatment. The two most important clinical factors are age and white blood count. The younger the age and the lower the white blood count at the time of initial diagnosis, the better the prognosis. Even including cases with less favorable cytogenetic profiles, the prognosis for children of disease-free survival for five years is >80%. Someone with disease-free survival for five years is considered to be cured. Among cytogenetic findings, hyperdiploidy (~51-65 chromosomes) and the absence of Philadelphia Chromosome are associated with a good prognosis. The bullets below summarizes some of the clinical and genetic prognostic factors.
Blastic Plasmacytoid Dendritic Cell Neoplasm
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare but very aggressive leukemia. Patients show characteristics similar to leukemia and lymphoma; it is found in the bone marrow and blood, but can spread to lymph nodes, spleen, central nervous system, and skin. The skin lesions are characteristic and appear deep purple and numerous. Most patients have been elderly males. This leukemia derives from plasmacytoid dendritic cells and like ALAL has phenotypic ambiguity. It has also been known as blastic NK cell leukemia/lymphoma. The leukemic cells are negative for many of the cytochemical stains such as myeloperoxidase, α-naphthylbutyrate esterase, ND naphthol AS-D chloroacetate esterase. They show complex karyotypes and genetic mutations. Treatment requires high levels of chemotherapy. Because this disease is usually found in very elderly patients who typically can not tolerate such regimens, targeted therapies are being pursued. The image to the right is a bone marrow aspirate of BPDCN which shows medium to large cells with scant cytoplasm, immature chromatin, irregular nuclear contours and prominent nucleoli.
Classification Systems
Both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), along with the other neoplastic blood disorders were originally classified by the French-American-British (FAB) system. The FAB classification system was based primarily on cell staging, cell morphology, and cytochemical staining. As genetic and chromosomal changes causing these disorders were discovered and new techniques for identifying the changes were developed, the World Health Organization (WHO) established a new classification system based on this new information. The WHO classification is based on multiple parameters: cell staging, morphology and cytochemistry are part of the initial diagnosis; chromosomal changes through karyotype analysis, molecular genetic changes, and immunophenotyping of cell surface markers are then used to more specifically and accurately classify them. Initial diagnosis also includes relevant clinical data such as medical history, possible toxic exposures, and sometimes physical exam including imaging studies.
What is the mechanism of action of chemotherapeutic drugs known as alkylating agents? Inducing apoptosis Targeting the product of a mutated gene Breaking DNA strands
Breaking DNA strands feedback: The mechanism of action of alkylating agents, used commonly in the treatment of AML is that the drug breaks DNA strands thus preventing replication. This can affect many rapidly proliferating cells, not just the leukemic cells. The so-called targeted therapies are those developed to interfere with a specific protein that resulted from the genetic mutation. Corticosteroids are known to induce apoptosis.
Following a preliminary diagnosis of ALL, immunophenotyping is performed on the patient's blasts and the diagnosis of T-cell Lymphoblastic leukemia was made. Which marker/s would have been positive? CD3 CD19 CD20 CD22
CD3 feedback: The CD3 marker is found on cells of the T-cell lineage. CD19, CD20, and CD22 are all found on B-cells.
Mutations that cause AML generally are to the genes that regulate which of the following? Cell metabolism Cell replication Export of proteins None of the above
Cell replication feedback: Harmful mutations leading to AML (as well as other neoplastic disorders) generally are those of genes regulating replication and inhibition of replication. Although mutations in genes for cell metabolism and export of proteins can cause other diseases, they are not the causative mutations of AML.
Chromosomal Analysis
Chromosomal analysis can detect a specific translocation or other chromosomal abnormality such as hyperdiploidy or hypodiploidy, and thus help establish the WHO subtype. Hyperdiploidy refers to excess numbers of chromosomes; in ALL we can often see as many as 51-67 chromosomes! Interestingly, this hyperdiploidy is found commonly in childhood ALL, is not associated with substantial genetic abnormality except for the excess chromosomes, and actually renders a good prognosis. Hypodiploidy refers to fewer than 46 chromosomes The image to the right shows hyperdiploidy in a childhood case of ALL (B cell).
An MLS is evaluating this leukemia. Is it more likely to be myeloid or lymphoid? Is it more likely to be acute or chronic? Acute myeloid Chronic myeloid Acute lymphoblastic Chronic lymphocytic
Chronic myeloid feedback: The leukemia is chronic myeloid. It shows varying stages of myelocytic differentiation such as neutrophils, myelocytes, metamyelocytes as well as a blast. Acute myeloid would show more blasts and less of the orderly shift to the left (meaning representatives of each of the stages of maturation). The cells are characteristic of the myeloid lineage rather than the lymphocytic lineage; however, definitive diagnosis would only be done upon further testing.
Which of the following are possible causes of acute leukemia? Check all that apply. Exposure to radiation Environmental toxins No known causes Treatment for other cancers
Exposure to radiation Environmental toxins No known causes Treatment for other cancers feedback: All are possible triggers for acute leukemia; however, there is no known cause for the majority of cases.
FAB Classification
In the former FAB classification, ALL was divided into three types based on cell size, prominence of nucleoli, and amount and appearance of cytoplasm: L1 (found mostly in children) L2 (found mostly in older children and adults) L3 (associated with Burkitt's Lymphoma) The image to the right is a smear from a bone marrow that was diagnosed as ALL L1, often seen in children. Notice the consistently sized lymphoblasts with scant cytoplasm.
Complete WHO ALL Classification
In the old FAB classification systems, leukemia and lymphoma were a point of initial differentiation. A disease was called a lymphoma if it was initially or mostly affecting the lymphatic system (lymph nodes, spleen, etc.), although it could spread to the bone marrow and blood. The disorder was termed leukemia if the major site was the bone marrow and blood, although it could also affect the lymphatic system. However, in the WHO classification, the initial differentiation is based on whether the lineage is that of a B cell or T cell which can be determined by cell surface markers using flow cytometry.
Unfavorable prognositc factors
Karyotype with hypodiploidy (<46 chromosomes) at diagnosis Very high hyperdiploidy - 66-88 chromosomes at diagnosis Presence of Philadelphia Chromosome BCR-ABL1 Other specific translocations associated with an unfavorable outcome Increased age in adults More frequent and severe comorbidities
Causes of Acute Leukemias: Triggers of Genetic Changes
Leukemia is a clonal disease that is caused by mutations and altered expression of genes leading to a malignant transformation of hematopoietic precursors. In most cases, a specific cause of these genetic changes can not be identified. However, we do know that a number of factors could be responsible for causing these genetic changes including: Environmental exposures such as exposures to radiation (especially seen in acute and chronic myeloid leukemias) Chemical and drug exposures such as benzene and organic solvents Immunosuppression such as in organ transplant patients Pre-existing genetic abnormalities as in Down Syndrome, Fanconi's Anemia, etc. Certain viruses (more often seen in chronic leukemias or lymphomas) Treatment of previous cancerous conditions with chemotherapy and/or radiation
Definition and Differentiation of Acute Leukemias from Chronic Leukemias
Leukemia, lymphoma and myeloma are neoplastic proliferative blood cell disorders. These disorders are considered malignant (cancerous) conditions. Leukemias usually originate in the bone marrow but can invade other tissues including the lymphatic system, whereas lymphomas originate in the lymphoid tissues, but can invade other tissues including the bone marrow. Leukemias frequently involve the white blood cells, although red blood cells and platelets can also be involved. Myelomas are a more specific type of disease in that the malignancy involves plasma cells which are specific lymphocytes that have already recognized and responded to antigen, and are often located in the bone marrow. Two major groupings of leukemia are acute leukemias and chronic leukemias.
A large number of blasts are seen on a patient's blood smear but it is difficult to determine their lineage. Therefore, cytochemical stains Myeloperoxidase and Sudan Black are performed. Both were negative. What is the likely lineage? Myeloid Lymphoid Both myeloid and lymphoid Can not be determined
Lymphoid feedback: Myeloid cells stain positive for both Myeloperoxidase and Sudan Black, whereas lymphoid cells stain negative. It is therefore likely to be an acute lymphoblastic leukemia.
Occurrence and Frequency of Acute Leukemia
Lymphomas are much more common neoplastic disorders than acute leukemias. Of the acute leukemias, acute myeloid leukemia (AML) is more common than acute lymphoid leukemia (ALL). Of the two, AML is more common in adults and ALL is more common in children. A third acute leukemia, mixed phenotype acute leukemia (MPAL) is rare and can be found in both age groups. As far as gender differences for all leukemias, males in the US have a slightly higher incidence than females. Although incidence of leukemia has increased over the years, deaths have decreased due to improved treatments and earlier detection. The remainder of this course will be about diagnosis and treatment.
Karyotyping used in the diagnosis of AML refers to: Making images of the patient's chromosomes to detect chromosomal changes or abnormalities Sequencing genes to detect mutations Staining cells to determine cell lineage None of the above
Making images of the patient's chromosomes to detect chromosomal changes or abnormalities feedback: Karyotyping to produce a karyogram is done to be able to analyze chromosomes for abnormalities. Genetic mutations are also important in AML diagnosis, but this process is not known as karyotyping. Staining cells to determine cell lineage is called cytochemical staining.
The Future of ALL Therapy
Many different targeted therapies are in various phases of research, trials and approval. Some examples include: Monoclonal antibodies. These are antibodies which are synthesized to recognize a membrane target expressed by the leukemic cells, thus lessening the side effects often seen with standard chemotherapies. A common target for ALL is the CD22 membrane marker. The monoclonal antibody is conjugated with some type of toxin or enzyme inhibitor, thus delivering it directly to the leukemic cells. Proteosome inhibitors. Proteosomes are the large protein complexes in cells which degrade unneeded or excess proteins. By inhibiting this action, proteins build up in the cell and will eventually kill it. JAK inhibitors. A signaling pathway in cells known as the JAK/STAT pathway is a way that leukemic cells can bypass normal growth and proliferation restrictions. By inhibiting this bypass, the cells will stop proliferating and may go on to mature.
Types of Genetic Changes in Acute Leukemia
Many types of gene mutations, as well as chromosomal abnormalities, are found in acute leukemia. As we will discuss later, these are in fact a basis of classification, treatment and prognosis. Typically, it is not just one gene that is mutated, but multiple genetic changes need to occur to cause the leukemia. We describe the genes affected as: Oncogenes. These can cause neoplastic conditions through a variety of ways such as changing how procarcinogens are metabolized, disabling the person's ability to repair DNA damage, altering the person's immune system or affecting the regulation of cell growth. Protooncogenes. These genes are the upregulators of cell growth and when mutated, can become oncogenes. Tumor suppressor genes. These genes are the downregulators of cell growth, and if altered, can result in unchecked proliferation. The common chromosomal abnormalities seen in acute leukemia that can be visualized on chromosomal analysis include: Translocations. A piece of one chromosome can break off and attach to another chromosome. This is one of the most frequently seen chromosomal abnormalities in acute leukemia. The first translocation that was identified, however, was in Chronic Myeloid Leukemia - the famous Philadelphia Chromosome (later this same translocation was found in other leukemias). Translocations can cause altered expression of genes. For instance, they can position a promoter gene to turn on a gene involved in cell cycle and replication. Inversions. This occurs when part of the chromosome is turned around, causing altered expression of genes as well. Deletions. In a deletion, part of the chromosome breaks off and is lost. If the lost gene(s) are tumor suppressor genes, then replication can continue unchecked. Additions. In this abnormality part of a chromosome is gained or there is an additional chromosome. This can cause problems if a number of oncogenes are added.
A patient sees her physician because of increased fatigue and easy bruising. The CBC shows a white blood cell count of 43,000/μL. The white blood cell differential indicated 23% blasts. What is a likely next step that would be taken? Begin chemotherapy immediately as it is likely an acute leukemia and no time should be lost. Repeat the test in 3-6 months before establishing a diagnosis Obtain bone marrow specimens and do further testing Do as many tests on the blood as possible, including cytochemical stains, immunophenotyping and genetic studies.
Obtain bone marrow specimens and do further testing feedback: The best next step would be to obtain a bone marrow sample for examination and for further testing. It should first be established as to whether it is of lymphoid or myeloid origin before more advanced testing is done.
Important factors which can help determine a patient's prognosis of AML include all of the following EXCEPT: Patient's family history The WHO subtype The patient's platelet count The patient's age
Patient's family history feedback: Although having cancers or leukemias in the patient's family history could possibly make it more likely to develop such a disease, it does not really bear on the prognosis. The WHO subtype and patient's age are two very important prognostic factors. Additionally, in certain subtypes, the platelet count can help inform the prognosis.
Morphology and cytochemical findings are inconclusive on a patient with a suspected acute leukemia. Immunophenotyping is performed and both B-cell markers and myeloid markers are positive. How would you comment on such a finding? It is obviously an error, and needs to be repeated. Such findings are typical of ALL of B-cell lineage. Such findings are typical of acute leukemia of ambiguous lineage (ALAL). Such findings are typical of AML, not otherwise specified.
Such findings are typical of acute leukemia of ambiguous lineage (ALAL). feedback: One of the WHO classification is acute leukemia of ambiguous lineage (ALAL). In this group, blasts can exhibit markers of cells of myeloid and lymphoid lineages, as in this example. Alternatively, blasts can lack either lymphoid or myeloid markers. Such findings are not typical of either AML or ALL.
Which of the following cytochemical stains would yield a positive result in AML? Sudan Black B Periodic Acid Schiff (PAS) Myeloperoxidase
Sudan Black B Myeloperoxidase feedback: Sudan Black B and Myeloperoxidase stains are positive in AML. PAS is useful for erythroleukemia and ALL.
Initial Diagnostic Tests and Samples
The CBC, white blood cell differential, and blood smear examination are what initially alert us to the possibility of a leukemia diagnosis in the laboratory. The laboratory professional will typically note elevated white counts, immature cells and cell morphologies, and possibly abnormal red cell or platelet counts. The white cell differential can give a clue as to whether the leukemia is acute or chronic, and myeloid or lymphocytic. Cell staging and differentiation between myeloid and lymphocytic is an important first step, as is determining if the leukemia is acute or chronic. Further laboratory samples will then be evaluated. Samples will include bone marrow biopsy and aspirations, and less commonly cerebrospinal fluid or tissue samples. Bone marrow morphologic evaluation is typically done by a pathologist; thereafter, flow cytometry, cytogenetic analysis, and genetic studies will be performed for the definitive diagnosis.
Complete WHO AML Classification
The WHO classification of AML includes many more individual diagnostic categories than the old FAB list of M0-M7. The major categories of AML are: AML with recurrent genetic abnormalities AML with myelodysplasia-related changes AML not otherwise specificied (NOS)- (several of the subcategories of this group are on the FAB M0-M7 list) Myeloid sarcoma Myeloid proliferation related to Down Syndrome CHART
All of the following are classification criteria in the WHO classification system of ALL EXCEPT: Surface markers to determine lymphoblast lineage The amount and appearance of the cytoplasm of the lymphoblasts Chromosomal abnormalitiesGenetic changes
The amount and appearance of the cytoplasm of the lymphoblasts feedback: The amount and appearance of the cytoplasm of the lymphoblasts was an important diagnostic criteria in the former FAB system; however, surface markers, chromosomal changes, and genetic mutations are now essential diagnostic criteria of the WHO system.
true or false The World Health Organization (WHO) classification system relies solely on cell staging, cell morphology and cytochemical stains.
false feedback: It is the FAB system that is based solely on cell morphology, staging, and cytochemical stains. The WHO system is based on additional information such as genetic mutations, karyotyping of chromosomes, and immunophenotyping.
Common Cytogenetic Abnormalities and their Prognosis
The following are among the more common cytogenetic abnormalities: Those with a favorable prognosis: hyperdiploidy (51-65 chromosomes in leukemia cells) t(12;21)/TEL-AML1 (ETV6-RUNX1) Those with an unfavorable prognosis: t(9;22)/BCR-ABL1; (Philadelphia Chromosome positive) hypodiploidy (<46 chromosomes in leukemia cells)
A patient is diagnosed with AML with t(9;11)(p21.3;q23.3);MLLT3-KMT2A. What does the MLLT3-KMT2A refer to? The chemotherapy regimen that the patient needs The chromosomal translocation The terminal end of the chromosome The genes that were affected
The genes that were affected feedback: The information in italics following the translocation information refers to the actual genes that are affected by the translocation. The translocation itself is written as t(9;11).
A 6-year old is diagnosed with ALL. Immunophenotyping indicated it was of B-cell lineage. Chromosomal analysis showed hyperdiploidy with 56 chromosomes. What is the most likely prognosis? The leukemia is untreatable The leukemia is treatable but unfavorable prognosis The leukemia is treatable with a favorable prognosis
The leukemia is treatable with a favorable prognosis feedback: Young age, B-cell lineage, and hyperdiploidy are all favorable prognostic factors.
Differentiation between Myeloid and Lymphoid Lines
The major WHO categories of acute leukemias are: Acute Myeloid Leukemia (AML) Acute Lymphoblastic Leukemia (ALL) - comprised of Acute B Lymphoblastic and Acute T Lymphoblastic Acute Leukemia of Ambiguous Lineage Blastic Plasmacytoid Dendritic Cell Neoplasm The first two - AML and ALL are by far the most common; the second two are rare. Therefore, this course will cover AML and ALL thoroughly, with only mention of the other two. Typically, when a white blood cell differential indicates a substantial percentage of blasts, the first thing necessary is to determine whether they are myeloblasts or lymphoblasts. Although it might seem at first like distinguishing between myeloid or lymphocytic cells is quite obvious, that is not always the case when it comes to neoplastic disorders. The first step is to look for obvious clues. Acute leukemias are characterized by a ''leukemic gap', which refers to the presence of both blasts and mature cells, while representative cells in the intermediate stages are absent. So one way to obtain a clue is to look at the mature cells. If most of the mature cells seen are lymphocytes, then it could possibly be ALL. If neutrophils or other cells in the myeloid line are seen, then it is more likely to be an AML. One clue that is almost foolproof is the presence of Auer Rods. Auer Rods are red staining, elongated needle-like inclusions that can be seen in the cytoplasm of AML myeloblasts. They result from the fusion of azurophilic (red staining) granules. If Auer Rods are seen, you can be certain that the leukemia is of myeloid origin. However, if they are not seen, you can not strictly rule out myeloid leukemias as they aren't seen in everyone or all the time. (Note: the presence of Auer Rods indicates that the cell is of myeloid lineage. but doesn't define it as acute. Most likely it is AML, but Auer Rods can occasionally be seen in other neoplastic disorders of myeloid lineage.)
true or false An essential point of differentiation in the WHO classification system of ALL is whether the disorder is a leukemia or lymphoma.
false feedback: The WHO classification is based on chromosomal and genetic changes which can be found in disorders that can present as either a leukemia or lymphoma.
Complete remission for AML is defined as: The patient has no recurrence of disease after five years The patient is eligible for consolidation therapy The patient is showing normal hematopoietic activity following induction therapy The patient has no disease for three years
The patient is showing normal hematopoietic activity following induction therapy feedback: In AML, a patient is in complete remission if they show normal hematopoietic activity two weeks after induction therapy. They would now be eligible to start consolidation therapy. A patient is designated as cured if they have no recurrence for five years.
A three year old has been diagnosed with ALL. Chromosomal analysis performed on this child's lymphocytes showed 53 chromosomes. A true statement about this case is: This child has hypodiploidy This finding is associated with a good prognosis This child has Down's Syndrome This most likely was a mistake
This finding is associated with a good prognosis feedback: Although it seems quite abnormal to have duplications of chromosomes, cases of ALL with excess chromosomes often have a good prognosis. Excess numbers of chromosomes (greater than 46) is called hyperdiploidy, not hypodiploidy. Although Down Syndrome is associated with a chromosomal aberration, and people with Down's syndrome are more susceptible to ALL, having 53 chromosomes is not specific to Down's syndrome. It is unlikely that seeing extra chromosomes in a chromosomal analysis is a mistake.
Types of AML treatments
Treatment of AML is extremely complex, and is based on the genetic changes, the patient's age, and other prognostic factors. In general, possible treatment regimens consist of: chemotherapeutic agents target therapies directed at the specific genetic change stem cell transplantation The aim of chemotherapeutic drugs is to stop the cell cycle, and thus halt the proliferation of the leukemic cells, or in some cases, directly kill rapidly proliferating cells. Table 8 shows a few examples of such drugs. Keep in mind that chemotherapeutic drugs are used not only to treat AML, but other neoplastic conditions such as chronic leukemias and solid tumor cancers as well; and, in so doing, these treatments can actually cause AML.
Cytogenetic Analysis
Two-thirds of patients with AML have leukemic blasts containing chromosomal abnormalities. These abnormalities differ among the various subtypes, as indicated in the WHO classification on previous pages. Cytogenetic analysis helps to inform important clinical, prognostic, and treatment decisions. Also, the diagnosed patient can then be monitored for remission and relapse using chromosomal and molecular analysis. The analysis of chromosome morphology is known as karyotyping. Karyotyping is performed by growing the patient's white blood cells in special media to induce cell division and replication. Cells are then treated to stop cell division at the metaphase stage of replication. The cells are then lysed to release the chromosomes and then stained to show the banding. A picture of the stained chromosomes is taken. The chromosomes are counted, sorted by chromosome number and paired, and then analyzed for their structure and banding Newer methods include spectral karyotyping such as multiplex fluorescence in situ hybridization (M-FISH), which is particularly useful in more complex rearrangements. Various automated methods can also be used for performing the karyotypes and visualizing the chromosomes. The top image shows a normal karyotype (karyogram) of a male. The lower right image shows a karyotype (karyogram) of a male with AML with a translocation of chromosomes 6 and 9 (short arrows) with a further chromosomal change of an additional chromosome 8 (long arrow).
Treatment of ALL
Types of treatment for ALL include systemic chemotherapy, prophylactic CNS chemotherapy (sometimes CNS radiation), and supportive care. For some patients, immunotherapy, targeted therapy, stem cell transplantation and/or radiation therapy might be used. For Philadelphia Chromosome (+) patients, a targeted therapy of a tyrosine kinase inhibitor can be used. The method of treatment is similar to AML treatment. It begins with induction therapy to induce complete remission (defined as <5% blast cells in the bone marrow, an absolute neutrophil count of >1000/uL, a platelet count of 100,000 and no need for blood transfusion ). The success of initial induction treatment is often indicative of positive overall survival. Typical chemotherapy drugs to induce remission are vincristine, corticosteroids, and an anthracycline. After induction, some patients may go on to receive an allo-stem cell transplant. Others will go on to receive more chemotherapy in an intensification/consolidation phase. Following the intensification/consolidation phase, patients may then be put on a maintenance chemotherapy for up to 3 years. ALL treatment is very complex; it depends on the patient's initial diagnosis, age, general health and initial induction therapy response. There are numerous drugs that are used, as well as radiation, and on the horizon are immunotherapies and targeted therapies.
Signs and Symptoms of AML
Unlike chronic neoplastic disorders, symptoms of AML may be present for only a few days or weeks before the patient is diagnosed. Although they can vary, the most common presenting symptoms of AML are those caused by crowding out of normal hematopoietic lines such as: Anemia Thrombocytopenia Granulocytopenia Typical symptoms associated with anemia are fatigue, pallor, dyspnea, and others. Thrombocytopenia manifests as mucosal bleeding, easy bruising, and heavy periods. Occasionally, patients can have more serious effects such as spontaneous hemorrhage. Granulocytopenia leads to a greater risk of infections, recurrent infections, and fevers. Much less common is leukemia cutis, which is the infiltration of the epidermis, dermis, or subcutis by leukemic cells. This condition causes nodules or papules on the skin. Leukemic cell infiltration of other organs is also possible but less common or severe than in ALL; however, the liver, spleen, lymph nodes, joints and meninges can occasionally be affected.
true or false Adults rarely get ALL; it is typically only found in children.
false feedback: Although ALL is the most common pediatric cancer, it can also be seen in adults of all ages, with a peak incidence over 50 years old.
true or false Acute leukemia of ambiguous lineage (ALAL) is a common leukemia in which the lineage can not be specified or which shows features of both lymphoid and myeloid lines.
false feedback: Although it is true that ALAL has either non-specific or mixed lineage, it is quite a rare leukemia.
true or false When studying the genome of an acute leukemia patient, mutations can be found that are unrelated to the leukemia.
true feedback: If sequencing the whole genome, many mutations unrelated to the leukemia can be found. When doing molecular testing on acute leukemia patients, the focus is generally on certain specific gene mutations known to cause leukemia.