Mahmoud Al-Sheyyab Fathi Balbisi & Jameel Al-Farah 08 / 12 / 2009

1 of 600 children will develop cancer in their childhood. Almost 1/3 of these cases is leukemia (i.e. 30% of child malignancy is leukemia) and it is the commonest type of childhood malignancy. The 2nd common cause of childhood malignancy: Brain tumors. The 3rd: Neuroblastoma. The 4th: Wilm’s tumor. And lastly the 5th is: Rhabdomyosarcoma. Definition: Leukemia is the most common child malignancy clonal expansion .It is blast cell clonal expansion and arrest at a specific stage of lymphoid or myeloid hematopoiesis & differentiation. So, there will be no further differentiation of these blasts but accumulation of these cells according to the underlying clone. Leukemic expansion starts in the bone marrow it is inflected first in the peripheral bone marrow, which causes leukocytosis, and may cause leucopenia in some cases… Any abnormal cell could be a clone for a leukemic transformation. Because of that, there are different types of leukemia, for example: ALL, Pre B, Mature T, Myeloid, promyelocytic, etc. During the process of maturation, there is what we call ((cluster differentiation)), which means that we have CD markers which are antigenic features that appear on the cell surface during a stage of maturation & disappear during the others . Depending on these antigens, we can differentiate between different cell types & stages of development by using antibodies which are specific to these antigens. For instance if the cells had the stem cell markers that would be a leukemia arising from an early stem cell, more over, if it had a B-cell Marker then it is a B-cell leukemia. Hence, each line of cell differentiation can be a site for leukemic clonal expansion.


In the above diagram we can see a blast cell (stem cell) which is precursor for the T-cells, B-cells and myeloid cells. We can also notice that each of these cells have specific antigenic markers that differentiate it from other cells. Antigens are present early during the maturation process some of which will disappear others remain on the surface and others will appear later. You should know that: - Blast cell (stem cell) are CD 34+ e.g. if the leukemic cell are CD 34+ then the clone is very close to the stem cell, hence, very mature. - B-cells are CD 19, 22 and 79a positive (mature B-cells have cell surface immunoglobins). - T-cells are CD 2,3,4,5 and 7 positive (hint; any number less than 8) - Monocytes are CD 14 and 15 positive - DR is a pan cell marker which we use as a marker for blasts. Antigens are important to determine & classify the clone we have and which type of cells we are dealing with. In the normal bone marrow we can see normal cellularity with no predominance of one cell lineage, with variety of all cells. It is abnormal to see predominance of one specific type of cell in the bone marrow, hence, there is clonal expansion and there would be no other cell lineages, other than the leukemic lineage, because there would be no space (this is called arrest of differentiation). Clonal expansion can

occur anywhere along the differentiation process; those cells which undergo leukemic transformation do not mature (this is seen in acute leukemia’s), except in CML there might be a bit of differentiation you would have blasts, neutrophils etc. Leukemia in children is classified into 2 major categories: A) Acute Leukemia: This represents 97% of all leukemia cases. It’s classified into:  Acute lymphoblastic Leukemia (ALL): 80 % --The commonest.  Acute Myeloblastic Leukemia ( AML ): 17% B) Chronic Leukemia: This represents 3% of all leukemia cases. It’s classified into:  Philadelphia positive  Philadelphia negative (Juvenile Chronic Myeloid Leukemia “JCML”). Note: In children, Acute Leukemia has better prognosis because the majority are ALL which has better prognosis than AML. AML is more common in adults. In a peripheral blood smear of a patient with CML. We can see bands, promyelocytes, segments, myeloid which can’t be found in the blood of a normal person. So, we can see all myeloid developmental types in excess numbers. It’s similar to a normal bone marrow in absence of other additional features. Aetiology: • In majority of the cases, the exact cause is unknown, more over; there would be a negative history of exposure to any carcinogens. • Ionizing radiation: has strong relation with leukemia. For example, people who were exposed to nuclear bomb and didn’t die, all of them developed leukemia mainly AML. • Chemicals: like Benzene which can cause Aplastic Anemia. • Drugs- Alkylating Agents: these are used in cases of malignancy or connective tissue disorders like autoimmune diseases. Patients who use these drugs- on the long term- are more prone to develop leukemia. • Genetic causes: cancer has a very strong relation with DNA component & genetics. If the patient is genetically determined to have cancer and was triggered by an acquired factor, then this cells will transform firstly, then it acquires certain features of immortality then it becomes malignant (it can live


in another environment than it is supposed to be in and this is a feature of metastasis), lung cancer for instance can metastasize to the bone. Multiple-hits Hypothesis: E ach cancer has several hits, each one has genetic alteration or hits, this is called the multiple-hits hypothesis, the same cell has multiple hits and these hits transform the cell into a malignant one and for this to occur you need time, less likely to occur in a child that’s why old people are more prone to cancer because as they keep on living they would have a higher chance of acquiring multiple hits on the same cell. In general cancer in children is very it is 1 in every 700 children while in adults it is 1 in 3 and the reason for that is that they live long enough to get the multiple hits. Genetic consideration in acute leukemia: • Identical twins have 20% risk of leukemia, so they are at much higher risk. This means that the genetic makeup is the main factor. • Four fold increase in incidence in siblings • Conditions with chromosomal abnormalities like: – Down’s syndrome: 1 in 95 risk > 10 years. – Bloom’s syndrome: 1 in 8 risk >30 years. – Fanconi anemia: 1 in 12 >16 years. • Incidence increased in genetic conditions like: – Agammaglobulinemia. – Poland’s syndrome. – Schwachman – diamond syndrome. Notes:  There are two conditions in Down’s syndrome: a) If the patient acquires leukemia early (since childhood), this is AML (M7; Megakaryocytic leukemia). b) If the patient acquires leukemia later in his life (>2y), this is ALL.  Patients with Down syndrome are more likely to develop cancer, leukemia mainly. They have more risk (250 times greater) than a normal person.  Bloom, Fanconi, Ataxia Telangectasia patients have something common between which is chromosomal instability and breakage syndrome. The chromosomes are febrile and prone to rearrangement and translocation. Certain translocations make the person more prone to develop leukemia.


People with immune deficiency are more prone to develop cancer because as we know that our immune system is important to catch the abnormal cells.

Ataxia Telangectasia which is an autosomal recessive disease associated with chromosomal instability and breakage. Patients that suffer from Ataxia Telangectasia would have dilation of the previously existing small or terminal vessels in the eyes and skin. They also have ataxia (inability to coordinate the voluntary
movement of the muscles).

Main features of Ataxia Telangectasia: • Cerebellar ataxia • Immune defects. • Telangiectases of the conjunctivae and skin. • Predisposition to tumors (lymphoma, leukemia). • Extreme radiation sensitivity. • Autosomal recessive. ____________________ Patients that have Bloom Syndrome suffer from chromosomal instability and breakage and are prone to have AML. Main features of Bloom syndrome (BS): • Extreme intrauterine and postnatal growth retardation. • Chromosomal instability. • Predisposition to leukemia’s, lymphomas, and other tumors. • Immune defects.

• Sunlight-induced erythema of the face. • Hypo- and hyper-pigmented skin areas. • Autosomal recessive.

• Gene locus on chromosome 15. ____________________ Patients with Fanconi Anemia, which is an Autosomal recessive disorder with chromosomal breakage and instability. They have short stature and fingers and high incidence to develop AML (M4 mainly). Main features of Fanconi Anemia (FA): • Growth retardation. • Skeletal defects (e.g., radius and thumb). • Bone marrow failure • Skeletal and kidney malformation. • Localized pigment changes. • Autosomal recessive. • Several gene loci.

Acute leukemia incidence: • 1 : 25000 up to 14 years • Peak incidence occurs at the age between 2-5 years • Accounts for 25 – 30 % of all childhood malignancies.


This table shows us what the doctor was talking about in the beginning of the lecture.

Subtype Pro-B ALL Early pre-B

Profile of antigen expression CD19 ,CD79a ,cCd3¯ CD19 ,CD22 ,CD79a , CD10
+ + + +  +


CD19 ,CD22 ,CD79a ,CD10, cCd3¯,* clg





CD19 , CD22 , CD79a , CD10¯, cCD3¯*, clg , slg , slg¯, slg¯
+ +




B cell

CD19 , CD22 , CD79a , CD10 ,* clg , slg , + slg , or slg+ CD19¯,** CD22¯,CD79a¯, CD10 , CD7 cCD3 ,* clg¯, slg¯
+  +






T cell

The first column (Up---Down) shows the developmental stages of the lymphocytes. The second one shows the antigens that appear/disappear on the cell surface during each developmental stage. As we said before, we can determine the developmental stage depending on the antigens present on the cell surface. Notes:   CD10 is associated with good prognosis. Mature B cell will develop into plasma cell that releases antibodies. If the cell expresses surface immunoglobulin then it is in advance stage of development.


   

Mature B cell has Gamma (γ), Kappa (κ) & Lambda (Λ) surface immunoglobulin then it is in advance stage of development. So…. -If there was cytoplasmic Ig --- Intermediate stage. -If there was surface Ig --- Mature B cell. This is important because by knowing the stage, we know the way of treatment. For example: • Mature B cell leukemia (Burkitt’s ALL, L3) is treated similar to Burkitt’s lymphoma. • Other stages like Pre B are treated according to ALL protocol. T cell markers are CD2, CD3, CD4, CD5, CD6, and CD7. All of them are less than 8! B cell markers are CD19, CD20, CD21, CD22, and CD79a. Myeloid cell markers are CD13 and CD33. $Stem cells marker is CD34.

Clarification & notes:  Constant lineage markers: always positive in all developmental stages. They appear early in development and continue to appear on cell surface in the next stages.  Non-constant lineage markers: they are specific for a lineage and they are not positive in all developmental stages.  Non-lineage specific markers: these markers are not specific for a single lineage. They can be found in multiple lineages. Example: CD10 is positive in B and T lymphocyte lineages.  Megakaryocytic markers are always absent in ALL.


B- lineage

T- lineage

Constant lineage markers Non- constant lineage markers

CD19, CD79a CD20, CD22, cytoplasmic  heavy chain, surface lg

Cytoplasmic CD3 Membrane CD3,CD2 CD5,CD7

Non- lineage specific markers (sometime present ) Myeloid markers (sometime present )


CD10 (CALLA) CD 34 CD 13, CD33

CD13,CD33,CD15 CDw65

Megakaryocytic markers (always absent )



FAB classified leukemia into three major classifications: L1, L2 & L3. Each one of these is characterized by features that differentiate it from the others. This depends on: cell size, nuclear chromatin, nuclear shape, nucleoli, amount of cytoplasm, basophelia of cytoplasm and vacuolation.


Cytologic feature Cell size Nuclear chromatin Nuclear shape

L1 Small cells predominate Homogenous in any one case Regular, occasional clefting or indentation Not visible or small and inconspicuous Scanty Slight or moderate

L2 Large, heterogeneous Variable, heterogeneous in any one case Irregular, clefting or indentation common Variable, often moderately abundant Variable, deep in some Variable

L3 Large, homogeneous Finely stippled and homogeneous Regular oval or round Prominent, one or more Moderately abundant Very deep Often prominent


Amount of cytoplasm Basophilia of cytoplasm Cytoplasmic vacuolation

Acute lymphoblastic leukemia L1

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ALL L3 Characteristics of T-cell ALL: • Massive splenomegaly. • Massive lymphadenopathy. • Large anterior mediastinal mass. • Increased risk for initial CNS involvement or relapse. • Initial leukocyte count ≥50.000/mL • Hemoglobin ≥10 g/dL • L2 morphology. • Focal parancuclear positivity of acid phosphatase staining. • Expression of CD2, CD3, CD5, CD7. ( remember T-cells CD’s are less than 8) • Rearrangements of the T-cell receptor gene in leukemic cells.

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In normal cells we have two types of genes: Proto-oncogenes and Tumor suppressor genes. The former induces the cell to proliferate and the later suppresses & regulates the cell cycle. The over-activity of proto-oncogenes and the inactivation of the tumor suppressor genes lead to uncontrolled proliferation of the cells and its transformation into cancer. This happens mainly in translocation. The genes are transformed from one chromosome to another; they become in a new environment that induces or suppress their activity. There are certain translocations that cause leukemia, for example: - T (12:21), T (9:22), T (1:19): lead to L1 or L2. - T (8:14), T (2:8), T (8:22): lead to L3. Notes: - T (1:19) & T (9:22) are associated with poor prognosis leukemia. - T (9:22): (Philadelphia chromosome): BCR (on chromosome 9) is translocated next to ABL (on chromosome 22). When joined together, a new hybrid gene develops. This produces a protein that has high Tyrosine Kinase activity which has growth stimulatory mechanism that transform the cell into leukemic. It can be found in cases of ALL, AML & CML. - L3 (Burkitt’s) = Mature B cells that produce antibodies. Antibodies are composed of one type of heavy chain and two types of light chains: kappa & lambda. Heavy chain is encoded by a gene on ch#14, Kappa on ch#2 and Lambda on ch#22. On ch#8, there are important oncogenes.

Acute Myeloblastic Leukemia (AML) Like ALL, FAB classified AML into different types; from M0 to M7. Each one of these is classified by certain features. The doctor talked briefly about them (Please go back to slides #23&24) ……. Here are the important notes: - M0: very immature myeloid leukemia. - M1 & M2 are Myeloblastic but they are more differentiated. They have more granules in their cytoplasm which contains also Auer Rods.
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- M3: promyelocytic leukemia. Hyper granular & contain heparin and Auer Rods. It can lead to DIC (Disseminated Intravascular Coagulation). - M4: Acute myelomonosytic leukemia. Associated with inversion of ch#16. - M5: Acute monocytic leukemia. Associated with T (9:11) and the patient has two features: gum hypertrophy & cutaneous nodules. - M6 is Erythroleukemia; arising from erythroid elements. - M7 is Megakaryocytic leukemia. Associated with T (1, 22) and common in Down’s syndrome patients. Characterized by cytoplasmic polyps which have both diagnostic and prognostic values.

Markers used in AML: - CD34: stem cell marker. It is positive in immature cells M0, M1, M2. In later developmental stages, it disappears. - DR: positive in both ALL and AML. - CD13 & CD33 are myeloid markers. CD33 is more specific to myeloid and it is positive in all developmental stages. - CD14 & CD15 are monocytic markers. - CD41 (glycoprotein 1b) & CD61 (glycoprotein 2b3a): important glycoproteins for the function of the platelets.

Features Associated with t (4; 11) (q21; Q23) ALL: - Age below 1 year with female sex predominance. - Hyperleukocytosis and high initial CNS involvement. - CD19+, CD10¯ Immunophenotype. - Positivity of myeloid markers: CD15+, Cdw65+ - No hyperdiploidy. - Can differentiate to lymphoid or myeloid pathways in vitro. - Very poor prognosis type of leukemia.

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Signs and symptoms in children with acute leukemia
Signs of anemia Weakness, fatigue, rapid exhaustion, lack of appetite Laboratory: normocytic hypo regenerative anemia or anemia due to bleeding

Signs of infections

Fever (e.g. nasopharynx, anal region), laboratory: reduced absolute number of granulocytes

Sings of bleeding tendency

Purpura, mucosal bleeding, tendency to develop hematomas Laboratory: hypo regenerative thrombocytopenia plasmatic coagulopathy (AML, M3)

Signs of organ infiltration

Bone and join discomfort, hepatomegaly and splenomegaly, generalized lymph node swelling infiltration of thymus Fever of unknown origin, weight loss, night sweats

Signs of systemic disease

How to diagnose leukemia?? The basic investigations required at diagnosis of childhood acute lymphoblastic Leukemia are: Blood Tests CBC, Coagulation Tests, KFT, LFT Immunoglobulin Levels (lgG, A, M, E). Viral serology (EBV, VZV, CMV, Measles, Hepatitis A, B, C). Urine analysis. Bone Marrow Diagnostics.

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Morphology, cytochemistry (PAS, POX, Acid Ph, Ns Esterase). Immunophenotyping. Cytogenetics, DNA content, molecular genetics (to check for translocations). CSF (check for CNS involvement). Cell Count, Cytospin preparation, Protein, Glucose. Diagnostic Imaging. Cranial CT or MRT. Echocardiography. Chest X- Ray / Sonography (Liver, Spleen, Kidney).

Diagnosis of CNS disease: This is done by checking CSF sample acquired through lumbar puncture. A) Presence of more than 5 WBCs/mm3. B) Identification of blast cells on cytocentrifuge examination. CNS involvement in leukemia is classified as follows: - CNS1 <5 WBCs/mm3,no blasts on cytocentrifuge slide - CNS2 <5 WBCs/mm3,blasts on cytocentrifuge slide - CNS3 >5 WBCs/mm3,blasts on cytocentrifuge slide C) TdT stains for suspicious cells. If positive …the cells are blasts. D) If a lumbar puncture is traumatic in a patient with peripheral blasts, the following formula can be helpful in defining the presence of CNS leukemia: CNS disease is present if: --CSF WBC is greater blood WBS------CSF RBC is greater than blood RBC----15 | P a g e

How to treat leukemia?? Over years, the treatment of leukemia has developed as follows: - In fifties, docs used only one single agent (steroids). - In sixties, they used combination treatment; CNS- directed therapy, single centers. - In seventies, supportive care became better. Intensification of polychemotherapy, definition of risk factors, single and multicenter trials - During eighties till now, docs use Risk- adapted therapy. This means that intensive treatment is given to patients with high risk. When they get better, they are given less intensive treatment.

The following shows the steps of treatment which are: A) Induction of remission: bringing the number of cells into normal count. This doesn’t mean that the patient is treated because there is high risk of remission. Multiple drugs are used ex, vincrystine, Asparaginase & steroid for 4 weeks. B) CNS prophylaxis: CNS directed treatment (it is given in addition to the previous drugs). It is very important because most of the cases will relapse in the CNS because the drugs in the first step cannot reach the brain (because of BBB). So, these drugs are given intrathecally. C) Consolidation: to maintain the number of the cells less than normal. Here, multiple drugs are used (intrathecally): Adriamycine, Vincrystine, Asparaginase, & Dexamethasone. All these drugs are used in combination for 6 weeks to bring the number of leukemic cells into minimal level. D) Maintenance.

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Prognostic Factors in the Therapy of De Novo ALL:
This table shows us which factors make leukemia associated with a good or bad prognosis. Factor Age Se x White blood count (x10 / I) Response to initial Induction therapy ( bone marrow on day 14) Chromosome count DNA index Chromosomal translocations

Favorable > 1- < 6 years Female <20

Unfavorable < 1 year Male > 100

M1 marrow*

M2,M3 marrow*

> 50 1 . 1 6 t(12;21)

< 45 < 1 .1 6 t(9;22); t(4;11)

*M1 marrow, < 5% blasts and complete regenerations of hematopoiesis; M2 marrow, < 5% -< 25 % blasts and or/ incomplete regeneration of hematopoiesis; M3 marrow, > 25% blasts in the bone marrow

Factors No longer considered Predictive of Outcome: • Enlargement of kidneys. • Testicular disease. • Hgb>10 g/dL or platelets < 100.000/mL • Visible adenopathy. • FAB morphology.
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• PAS positivity. • Labeling index. • Myeloid antigen positivity. • LDH. • Glucocorticoid receptor number.

Prognostic variables in childhood AML: • Favorable: - Chromosomal changes: t (8.21), inv (16), t (9.11), t (15.17). Note: T (15:17) is associated with promyelocytic leukemia. - Remission after one cycle of chemotherapy. - FAB M4 with eosinophilia.  Unfavorable: - Chromosomal changes: monosomy 7. - Initial leukocyte count> 100.000/uL - Secondary AML. - Myelodysplasia associated AML.

Possible Indications for Bone marrow transplant (BMT) in Childhood ALL:  BMT in First Complete Remission: • Philadelphia Chromosome+ (BCR / ABL+ ) ALL • T (4; 11).

• Poor Response to Treatment ( No remission after 4 weeks of induction of therapy)
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 BMT in Second Complete Remission : • Early (< 6 Month After off Therapy) BM Relapse • Late BM Relapse with unfavorable features (BCR / ABL+ ALL; Tel / AML- All; Poor response to relapse therapy after 6 weeks) • All relapses of T- ALL  BMT in > Second Complete Remission • ALL patients.

Done by: Fathi Balbisi and Jameel Al-Farah
Thank you and good luck And congratulations if you reached to this point of the lecture!!


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