Acute Leukemias and their Diagnosis, Acute Myelocytic and Acute Lymphocytic Leukemia

Sample

1. Blood in EDTA and fresh blood smear needed.
2. Bone marrow aspirate and biopsy are required.

Definition of acute leukemia

1. This is a malignant neoplasm of hematopoietic cells particularly stem cells.
2. There is diffuse infiltration of the bone marrow by these neoplastic cells.
3. These cells enter the blood and may infiltrate the liver, spleen, lymph nodes, and other organs.

Classification of Acute Leukemias

• On the morphology of the cells, these are divided into:

1. Acute lymphocytic leukemia (ALL)
2. Acute myelocytic leukemia (AML)
3. The following diagram shows how these leukemias develop from the blast cells.

Pathophysiology

1. The malignant transformation is at the level of pluripotential cells like stem cell and these are the committed stem cells, called stem cell disorder.
2. There is abnormal proliferation, monoclonal expansion, and decreased apoptosis of these cells leads to the replacement of normal bone marrow cells by these malignant cells or immature cells.
3. Acute Leukemias Criteria:
   1. Acute leukemias are characterized by abnormal differentiation and proliferation of malignantly transformed hematopoietic blast cells.
      1. These cells proliferate in the bone marrow and lead to suppression of the normal cells.
   2. These have little or no maturation and may see mainly blast cells.
   3. Acute Leukemias are diagnosed on the percentage of blast cells in the bone marrow.
   4. 30% or more blast cells are needed to classify the disorder as acute leukemia.
   5. These leukemias are associated with varying degrees of anemia, neutropenia, thrombocytopenia, and or infiltration of the tissues.
      1. The most common site of infiltration is lymph nodes, liver, spleen, CNS, and skin.

Epidemiology of acute leukemias:

1. ALL and AML can occur at any age. But the incidence increases with the increasing age.
2. ALL predominates in the children, while AML accounts for the majority of the cases in adults.
3. AML median age is around 60 years.
4. The peak age for ALL is between 3 to 5 years. These are morphologically different than adult cases.
5. Acute leukemias more common in whites than blacks.
6. Acute leukemias more common in the jews than the non-jews.
7. Acute leukemias are more common in men than in women.

Etiological factors for acute leukemias:

1. The cause of acute leukemias is unknown, this may be hereditary or environmental factors.
   1. The pathogenesis is very complex and this may involve interaction between the host susceptibility, and chromosomal damage secondary to physical or chemicals agents.
2. Chromosomal abnormality:
   1. Deletion:
      1. 5q –  = Myelodysplasia, preleukemia.
      2. Monosomy 7.
   2. Inversion:
      1. inv 16 = AML.
   3. Translocation:
      1. t18:14 = Burkitt’s lymphoma.
      2. t8:21 = AML.
      3. t4:11 = ALL.
      4. t9:22 = CML
3. Oncogenes:
   1. C-abl, C-sis = CML.
   2. C-myc = Burkitt’s lymphoma (L3).
4. Viruses:
   1. There are viruses known to give rise to leukemia in nonhuman species.
   2. The human role of viruses is studied for a long time but no conclusive data is available.
   3. Epstein-Barr virus is seen in the majority of the  Burkitt’s lymphoma.
   4. Human T – leukemia virus  (HTLV-1) is associated with acute leukemia.
5. Inherited factors:
   1. The following examples favor the role of genetic abnormalities:
      1. Increased incidence of acute leukemia in families.
      2. increased incidence of acute leukemia in the monozygotic twins.
         1. If one twin has leukemia, the other is also at risk for leukemia, often before 8 years of age.
         2. Leukemia starts in the next one within a year of the first diagnosis.
   2. Increased incidence of acute leukemias in members of a certain family with high susceptibility, the high increased frequency leukemia in monozygotic twins, an association of acute leukemias genetic disorders, all these factors have established that hereditary abnormalities play role in development of acute leukemia.
   3. There is an increased incidence of AML and ALL in the Down’s syndrome where there is trisomy 21.
      1. 5 to 10% of childhood AML occurs in Down’s syndrome.
   4. There is an increased incidence in Fanconi’s syndrome and Bloom’s syndrome.
      1. Both conditions are hereditary and there is an increased chromosomal breakage.
   5. Ataxia-telangiectasia and congenital agammaglobulinemia have chromosomal abnormalities are associated with acute leukemia.
6. Acquired factors:
   1. Drugs and Chemicals:
      1. Alkylating drugs like nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, and procarbazine.
      2. Exposure to melphalan or nitrogen mustard is associated with acute AML after 5 to 7 years of exposure.
         1. The patient will have pancytopenia, myelodysplasia, and cytogenetic abnormalities (there is a total loss or partial loss of chromosome 5 and 7).
         2. Hodgkin’s lymphoma, Multiple myeloma, and ovarian cancer treatment complication of AML is a well-known example.
         3. Benzen complication of acute leukemia is well-known.
   2. Radiation. The leukemogenic potential of ionizing radiation is recognized for a long time.
      1. Mostly myeloid leukemia takes origin by the radiation.
      2. Fetal exposure to radiation increases the risk of childhood leukemia.

Type of cells	% of the cases	FAB subtype
Pre B- cells ALL	75	L1 and L2
B-cell ALL	5	L3
T – cell ALL	20	LI and L2

Acute Leukemias signs and symptoms

1. Appears suddenly.
2. The patient may have weakness and fatigue.
3. The patient may have a low-grade fever.
4. There may be bruising and mild bleeding from the gums.
5. There is bone pain due to the expansion of the marrow.

Diagnosis of Acute Leukemia

1. These are more common in the younger age group.
   1. These are more frequent in children.
   2. Mostly seen before the age of 20 years.
2. Onset is an abrupt and fatal outcome.
   1. There is a fever.
3. There is a rapid development of anemia.
   1. There is normochromic and normocytic anemia.
   2. May see nucleated RBCs.
4. There is thrombocytopenia.
   1. May see petechiae and purpura in the skin and mucous membranes.
5. Leukocytes count is variable.
   1. Mostly leucocyte count is less than 100,000/cmm.

Treatment Golas

1. The main aim of the therapy is to the restoration of bone marrow function and to normality.
2. Complete remission when the patient had full recovery of the peripheral blood smear and bone marrow showing <5% of the blast cells.
3. Induction therapy aims to reduce the total body leukemia cell population roughly from 10¹² cells to below <10⁹.
4. The complication of the chemotherapy:
   1. Cytotoxic drugs lead to:
      1. Tumor lysis syndrome and urate nephropathy.
         1. Hyperuricemia responds rapidly to rasburicase.
         2. Allopurinol, I/V therapy, and alkalinization of the urine reduce the chances of uric acid precipitation in the renal tubules.
      2. Electrolytes imbalance like hyperkalemia, hypocalcemia, and hyperphosphatemia.
      3. There may be a gastrointestinal injury, diarrhea, and mucositis.
      4. There may be thrombocytopenia, bleeding, and neutropenic infection.
      5. Blood products are given to maintain the platelets count >10,000 µL in non-bleeding patients, and hematocrit to keep >25%.
      6. Antibiotics are given if the body temperature is >38.5 °C.
      7. In neutropenic patients when TLC is <500 /µL, antifungal therapy is added for these febrile patients.

Acute Myeloid Leukemia (AML)

1. Pathogenesis:
   1. There are primary AML  which appears de novo.
   2. Secondary AML which develops from:
      1. The myelodysplasia.
      2. Other hematological diseases such as myeloproliferative diseases.
      3. Or following previous treatment with chemotherapy.
2. Age: This is seen during the first month of life and then in the later age group.
   1. 10 to 15% of cases found in the children.
   2. If there is exposure to high energy radiation in early childhood life have increased risk for developing T-cell acute lymphoblastic leukemia.
3. Sex: More common in males than women. Male: female ratio is 3:2.
4. Signs and symptoms:
   1. Mostly these patients present with a varying degree of bone marrow failure.
   2. So clinical symptoms and signs are due to anemia, leukopenia, thrombocytopenia, and infections.
      1. About 1/3 of the patient have bruises and hemorrhages.
      2. About 1/4 of the patients have severe infection involving the lungs, soft tissue, or skin.
      3. Splenomegaly and hepatomegaly are not common and seen in<25% of the cases.
      4. Lymphadenopathy is even less common.
      5. Gingival hypertrophy or skin infiltration by the tumor occurs in 50% of the patients with monocytic leukemia.
      6. Acute promyelocytic leukemias are more commonly seen with severe bleeding from the DIC.
   3. Fever and fatigue are present in most of the patient.
   4. Acute promyelocytic leukemia (M3) causes disseminated intravascular coagulopathy (DIC) and bleeding.
   5. Acute myelomonocytic leukemia (M4) and acute monocytic leukemia (M5) causes gum hypertrophy.
   6. There may be tumor infiltrate of AML called granulocytic sarcoma (Chloroma), skin, and the bone particularly sternum, ribs, and orbit are common sites.  Myeloid sarcomas may involve any organs.
   7. These patients may have respiratory distress due to leukostasis within the pulmonary vasculature.
   8. There may be retinal hemorrhage due to thrombocytopenia.
5. Classification:
   

   French-American-British (FAB) Classification of Acute Myelocytic Leukemia

   FAB type	Characteristic features	Frequency
   M0	Acute myeloblastic leukemia. Blast cells lake cytologic and cytochemical markers of the myeloid series cell. But express myeloid series lineage antigens. 2 to 3% of AML. Sudan black and myeloperoxidase stain are negative. express CD13 and CD33 antigen-positive on the surface.	2 to 3%
   M1	Acute myeloblastic leukemia without differentiation. Undifferentiated myeloblastic. No cytoplasmic granules may see few. Only 3% of the cells are myeloperoxidase positive. Auer rods are common. Acute myelocytic leukemia M1	20%
   M2	Also called AML with maturation. Differentiated myeloblastic (30 to 89%) cells and sparse granules and abundant cytoplasm. Myelocytes and promyelocytes are predominant. Auer rods are often found. Cytogenetics show t(8;21)and (q22;22q). Acute myelocytic leukemia M2	25 to 30%
   M3	Acute Promyelocytic Leukemia. Granules are typical of promyelocytic morphology, these are azurophilic. Blast cells contain prominent granules, nuclei are bilobed, or kidney-shaped. Auer rods are many. These are strongly myeloperoxidase positive. Cytogenetics show t(15;17), t(11;17), and (q22;q11-12). Acute myelocytic leukemia M3	5 to 10% (8 to 15%)
   M4	Acute Myeloid Monoblastic Leukemia. The mixed population of myeloblastic (>30%), and monocytic (>20%) morphology. Auer rods may be seen. Nonspecific esterase and chloroacetate stains are often positive. Acute Myelocytic leukemia M4	20 to 30% (20 to 25%)
   M5	Acute Monoblastic Leukemia. Pure monoblastic cells. Monoblast and promonocytes predominate constitute >80%. Cytogenetics show t(9;11), (p22;q23), and t(11;q23). These are 10% of the AML. Acute myelocytic leukemia M5	10%
   M6	Acute Erythroleukemia. Predominantly immature erythroblast >50%, and can see multinucleated megaloblastoid erythroblast.  Erythroblast cells are strongly PAS-positive. Myeloblasts are 30% or more. Acute erythroleukemia M6	5%
   M7	Acute Megakaryoblastic leukemia. Megakaryocytic  Cells small and large size with shaggy borders that may show some budding. There is a high N/C ratio. Platelet peroxidase and platelets antibodies are often positive. Cytogenetics show t(1;22), and (p13;q13). Acute myelocytic leukemia M7	1 to 2% (1%)

6. Lab. findings : 
   1. WBC count is variable. 25% of the patient has >50,000/cmm, 25% has <5000/cmm and 5 to 10% has count between 5000 to 10,000/cmm.
      1. Count >100,000/cmm is reported in <10% of the patients but these patients have severe CNS or respiratory diseases due to leukostasis.
      2. In these patients, leukapheresis and chemotherapy may be life-saving.
   2. WBC morphology. These are morphologically abnormal and immature cells.
      1. 20% or more of the blast cells in the bone marrow as diagnostic of AML.
         1. Ther blast cells of AML are larger than lymphoblast cells and greater heterogeneity in size and shape.
         2. AML blast cells mostly abundant cytoplasm and often contain cytoplasmic granules.
         3. About 10% of the AML cases show Auer rods and azurophilic granules with Wright’s stain.
      2. The peripheral blood smear may show blast cells in 85 to 90% of the cases.
      3. FAB divide AML into:
         1. M1, M2, and M3 with granulocytic differentiation.
         2. M4, M5 with at least 20% monocytoid appearance.
         3. M6 with a high proportion of erythroblasts.
         4. M7 with predominantly megakaryoblasts.
         5. M0 with minimal myeloid differentiation. This needs immunohistochemical stains for the diagnosis. These cases have myeloid antigens on their surface and myeloperoxidase activity is negative.
   3. Uric acid in 50% of the cases is raised which indicates increased cell turnover.
   4. Lactate dehydrogenase (LDH) about 50% shows a raised level. This is not as common as in ALL.
   5. Bone marrow shows >20% blast cells. Promyelocytes are numerous particularly in M3.
      1. Auer rods can be seen in the cytoplasm.
      2. Cytochemical stains, peroxidase reaction, and Sudan black are positive.
   6. Nucleated RBCs may be seen.
   7. Platelet count may be low and may see count between 30,000 to 100,000/cmm. In some cases may be <20,000/cmm.
   8. Coagulation abnormalities are also seen. PT, PTT, and Thrombin time are prolonged.
7. Treatment: There is a supportive and specific treatment for AML.
   1. Stem cell transplantation, autologous transplantation may be done. But because of the toxicity overall no benefit.
   2. The treatment of patients over 70 years of age of AML is poor because of primary disease resistance and poor tolerability of intensive treatment.
      1. Death in the old people is usually from hemorrhage, infections, and failure of the heart, kidneys, or other organs as compared to the younger patients.
8. Prognosis:
   1. 50% of children and young adults may have a long-term cure.
   2. Recently with the newer advanced chemotherapy, the prognosis in the 60 to 70 years of age is better.
   3. Cytogenetics abnormalities and initial response to the treatment are the major predictors for the prognosis.
   4. AML overall complete remission is 60 to 70% and disease-free survival is 20 to 40%.
      1. <60 years complete remission is 70 to 80% and disease-free survival is 40%.
      2. >60 years complete remission is 45 to 55% and disease-free survival is 5 to 10%.
         

         Clinical parameters	Good prognosis	Poor prognosis
         Age	<60 years	>60 years
         Onset of AML	Primary	Secondary
         BM response to remission induction	<5% blast cells after one course of chemotherapy	20% blast cells after the first course of chemotherapy
         Cytogenetics	t(15;17), t(8;21), inv(16), NPM mutation	t(6;9), 11q23, abn(3q), deletion of chromosome 5 or 7

 Acute Lymphoblastic Leukemia (ALL)

1. Age:  ALL constitute 20% of adult leukemia and these are more common in the children.
   1. This is most common in childhood. Mostly occur before the age of 4 years. The peak is between 2 to 10 years, it is also called as childhood leukemia.
   2. These are rare after the age of 30 years.
   3. These are most common in children and young adults.
   4. The second peak may be seen in the middle and old age groups.
   5. L3 leukemia is most common in developing countries and may be associated with the infection by the Epstein-Barr virus in the younger age group.
   6. Children with trisomy 21 (Down’s syndrome) have increased risk for the childhood acute lymphocytic and also acute myelocytic leukemias.
2. Signs and symptoms:
   1. S/S of acute AML and ALL are similar, except the onset of ALL is strikingly acute.
   2. Onset is sudden. There is no preleukemic stage.
      1. Symptoms are present only for a few weeks before the diagnosis.
   3. There are fatigue and fever. There is malaise, lethargy, and weight loss.
   4. There may be bleeding and infections.
   5. There is an enlargement of lymph nodes, spleen, and liver is more common than the AML affecting 50% of the adults.
   6. There may be involvement of the central nervous system (CNS) in 5 to 10% of the cases. This shows as leukemic meningitis.
      1. Cranial nerve palsy more common in the 6th and 7th nerves.
      2. There are headaches and papilledema due to meningeal involvement and the obstruction of the outflow of CSF.
   7. Bone pain is due to the infiltration of the leukemic cells.
   8. Retinal hemorrhage is due to thrombocytopenia.
   9. Chest X-ray shows a thymic mass in 10 to 15% of the adults.
3. Classification:
   1. Classification of ALL is based on cytological features like:
      1. Cell size.
      2. Nuclear chromatin pattern.
      3. Nuclear shape.
      4. Presence of nucleoli.
      5. Amount of basophilia in the cytoplasm.
         1. 80% of the ALL are B-lymphocytes origin.
         2. 15 to 20% of cases arise from T-lymphocytes. These cells will express CD2, CD5, and CD7.
            1. T- L ALL are lacking CD19 and CD20.
   2. L1 – ALL account >80% of the cases in children and consists of predominantly small, up to twice the size of small lymphocytes.
   3. L2 – ALL are majority of the adult leukemia. The cells are larger than in the L1 and often heterogeneous in size.
   4. L3 – ALL is the least common form of approximately 3 to 4% of the children and the adults. The cells are morphologically identical to the cells in Burkitt’s lymphoma.

FAB (French-American-British) classification of Acute Lymphocytic Leukemia:

L1	Lymphoblasts with uniform, round nuclei, and scant cytoplasm. Nucleoli are not prominent.
L2	More variable lymphoblast and cytoplasm is abundant. Nucleoli are large and may see one or more nucleoli.
L3	Lymphoblasts with fine nuclear chromatin and blue to deep blue cytoplasm that contains vacuoles. Lymphoblast is large and has prominent nucleoli and these are one or more than one.

1. Lab. Findings:
   1. WBC count is variable from very high to low count.
   2. There are anemia and thrombocytopenia.
2. Bone marrow: Very few leucocytes are seen.
   1. Difficult to find normal WBCs or RBCs in the bone marrow.
   2. Characteristically there are blast cells.
   3. Auer rods are absent.
3. Flow cytometry studies (differentiation of the antigens over the surface of these abnormal cells) provide rapid diagnosis.
   1. 80% of the ALL arises from the B-lymphocytes and will show CD molecules of CD19, CD20, or both.
   2. Few cases of the ALL may show CALA (common ALL antigen) which is CD10.
   3. Roughly 20% of the CD10-positive cases may show cµ and are called pre-B – ALL.
4. Special stains:
   1. 1. Sudan’s black and peroxidase reactions must be negative to support the diagnosis of ALL.
      2. PAS stain shows clumpy positivity by glycogen in all ALL except the L3 which is negative..
      3. Cloroectate esterase and lysozyme stains negative in all ALL.
      4. α-naphthyl acetate esterase may be positive in T-lymphoblast.
      5. All cases of ALL contain TdT (terminal deoxynucleotidyl transferase) enzyme and this fairly reliable marker of the ALL.
      6. ALL – L3 often stains with oil red O because of the presence of neutral lipids present in the vacuoles.
   2. Morphology of the cells:
      

      L1	Small uniform blast cells
      L2	Larger and more variable size of the cells
      L3	1. Uniform cells with basophilic and sometimes vacuolated cytoplasm, typical of Burkitt’s lymphoma. 2. Philadelphia chromosomes-positive in acute lymphoblastic leukemia and t(8;14) found.

5. Treatment:
   1. The aim is to restore the normal function of the bone marrow by multiple chemotherapy drugs.
   2. Typically four drugs e.g. vincristine, prednisone, daunorubicin, L-asparaginase, and cyclophosphamide are used for induction and remission.
   3. 80 to 90% of the adult enters complete remission.
   4. around 30 to 50% will be alive free of disease for 3 years.
6. Prognosis:
   1. Patients with count >30,000/cmm have a shorter duration of the remission than patients with a lower count.
   2. Older age >60 years is not a good favorable factor.
   3. Another factor which matters the prognosis are:
      1. Circulating blast cells %.
      2. The degree of bone marrow involvement.
      3. The presence of splenomegaly, hepatomegaly, and lymphadenopathy.
      4. LDH level.
      5. Involvement of the CNS at the time of diagnosis.
      6. The time needed to get the remission for 4 to 6 weeks.
   4. ALL overall 80 to 90% have complete remission and disease-free survival is 30 to 40%.
      1. T-ALL 90 to 95% have complete remission and disease-free survival is 60 to 65%.
      2. Precursor B-Cell ALL has complete remission of 75 to 85% and disease-free survival is 30 to 40%.
      3. ≥60 years complete remission of 75 to 80% and disease-free survival is 10 to 25%.

Difference between Acute Myelocytic Leukemia and Acute Lymphocytic Leukemia

Characteristic Features	Acute Myelocytic Leukemia	Acute Lymphocytic Leukemia
Origin of the cells	Myeloid series cells	Lymphoid  series cells
Features of Blast cells	Large in size,	Small in size
	Cytoplasm is moderate	Cytoplasm is scanty
	Chromatin is fine and lacy	Chromatin is dense
	Nucleoli are prominent and >2	Nucleoli are indistinct and <2
Auer rods	Present	Absent
Sudan black and peroxidase	Positive	Negative
Bone marrow	A mixed population of myeloid cells and blast cells	Mainly blast cells and very few  WBCs or RBCs
Leukocyte Alkaline phosphatase (ALP)	Positive  (differentiate CML from the leukemoid reaction.	Negative
Periodic Acid-Schiff (PAS)	Positive in Erythroblast in M6 leukemia	Positive (block patterns) in L1 and L2 Negative in L3

Prognosis of the ALLand AML:

Type of leukemia	Complete remission	Disease-free survival
ALL
ALL (overall types)	80 to 90%	30 to 40%
Precursor B cell ALL	75 to 85%	30 to 40%
pH+  ALL	70 to75%	0 to 10%
Burkitt’s lymphoma	75%	70%
≥60 years	75 to80%	10 to 25%
AML
AML  (overall types)	60 to 70%	20 to40%
Acute promyelocytic leukemia	90%	70 to 75%
<60 years	70 to 80%	40%
≥60 years	45 to 55%	5 to 10%

Test value for the layman:

1. A blood examination is advised if the patient has a high TLC.
2. If the patient has anemia.
3. If the patient has enlarged lymph nodes.
4. In the case of patients has a fever and weakness.

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