1-Intrudation
1.1 Acute leukaemia:
Acute leukemia occure when have defect in hematopoietic stem cell that lead to immature white blood cell (blat cell)to divided out of control and continue to grow but never mature into normal cell. It usually occur progress quickly and suddenly. The abnormal blst cells are called the leukemia cell. Because its is abnormal cell and immature and cannot carry the normal function of white blood cell. They also crowd out the normal cell lead to prevent of normal cell to work properly and lead to increase of infection. When the bone marrow if filled with blast cell, that lead to leukemia cell to occupy all the surface of bone marrow and little amount of red blood cell and platelet are produce lead to increase the health problem.
.1.1.1 CLASSIFICATION OF ACUTE LEUKEMIAS:
The original classification of the acute leukemia depend on the French-American-British (FAB) divides the AML in to 8 subtypes from M0 to M7 and also divided the ALL in 3 subtype from L1to L3. AML subtypes are arise from common myeloid precursor and all 8 subtype differ from each other in degree of maturation. The characterize of M0 subtype are undifferentiated of blast cell, whereas the M1to M4 is characterize with have granulocytic differentiation with differ degree of maturation.M5 subtypes are mostly monocyte . whereas the M6 subtypes are erythroid and M7 subtypes are megakaryocytic. The FAB classification of .ALL is from L1 to L3 and differentiation based on the morphology, cell size, present of nucleoli and appearance of cytoplasm. About 75% of case of adult ALL have B-cell phenotype and about 25% of case have T-cell phenotype. The FAB classification of AML and ALL classify this two type based on the morphology of blast and cytochemical staining.however. the recent classification and used in nowadyse is proposed by the World Health Organization (WHO) that add addition evaluation of blast cell and depend on tha molecular analysis and flow cytometry. The result present of four methods for evolution of blast cell based on morphology,cytochemistry staning,molecular anlysis and flow cytometry. Not only diffrentiation of AML from ALL also classify the acute leukemia in to subgroup. The new classification of ALL based on immunophenotype by using flow cytometry classify the B-cell ALL in 4 subtupe and T-cell ALL into 2 subtype. And this new classification help in the understand the disease process, prognosis and treatment.
1.1.2 CLINICAL PRESENTATION:
The signs and symptoms of acute lukemia is result from infiltration of blsat cell in bone marrow and extramedullary sites. patient have nonspecifc complaints including fatigue ,lathargy.weakness ,fever and weight loss and all this symptom result from decrease number of red blood cell that lead anemia. Also patiant have bleeding and easy bursing result from thrombocytopenia and reccrunt infection result from neutropenia. Blast also infiltrate other organs like liver,spleen and lymph nodes and lead to hepatosplenomegaly and lymphadenopathy. Infltrite the blast cell in bone marrow lead to bone pain. The physical examination by the doctor see splenomegaly hepatomegally , lymphadenopathy and bone nenderness. Mucosal bleeding ecchymosis hemorrhage as result of thrombocytopenia. Patient with promyloctiv leukemia charactrise of presnt of signs of DIC, and extramedully involvement present more common in ALL patient rather than AML patient. Extramudally invlovemt of ALL is include the central nervous system (CNS), lead to firmness and painless enlargement and affect about 10% of patient of ALL, most patient that have CNS involvemt may be asymptomatic or have some symptom like headache,vomiting nausea, irritability result from intracranial pressure. When make cytological analysis of cerebral spinal fluid it have a lumbar puncture in all patient of ALL . Skin involvement (leukemia cutis) ,it present in approximately 10 % pf patient of AML and most common in monocytic AML (M4 and M5). granulocytic sarcomas is present in 15% of AML patient and present anywhere in the body. Patient with high amount of blast cell thet greater than 50 '' 103 /mm3 that have shgns and symptom of lukocytosis and present most commn in AML patient rather than the ALL patient. High amount of blast cell lead to clumping of this cell in the lung,. brain and lead to hypoxia confusion dyspnea, , and coma and maybe fetal. Also lead to hypervoscosity of blood and lead to heart and kidney problem The patient need to leukapheresis to reduce number of blast cell.
1.1.3 LABORATORY FEATURES:
At the time of dignosis,in acute leukrmia the number of white blood cell is generally elevated ,but also may be normal or reduced. Approximately less than 20% of patients have WBC count more than 100 '' 103 /mm3. In most case the blast cell is present in Peripheral blood smears and also present of anemia and thrombocytopenia as result from increase number of blast cell in bone marrow,which lead alittle amount of erythroid and megakaryocytic precursore.In the half patient of acute leukemia it have sever thrombocytopenia that is the platlet count is less than 50 '' 103 platelets/mm3. In patient that acute leukemia that have sign and symptom of DIC ,they have also abnormal result of coagulation test that lead to hypofibrinogenemia ,deficiency of coagulation factors and elevated fibrin split products. Serum electrolte is usually normal in acute leukemia patient. Rapid cell turnover lead to elevate the level of Lactate dehydrogenase and uric acid,and lead to acute renal faliure. When use of chemotherapy lead to tumer lysis and lead to tumor lysis syndrome that characterize with hypocalcemia, hyperkalemia,
1.1.4 DIAGNOSIS OF LEUKEMIA:
The dignosis of acute leukemia require the present of blast cell with 30 % or more in bone marrow depend on FAB system,but in new WHO classification it change this to 20% of blast in bone marrow. In acute leukemia the peripheral blood smear may be give some clues. In AML the myeloblast it found with variability in size,the cytoplasm is abundant with pale blue in color and contain azurophilic granules and contain distinct nucleoli. In AML,it charactrize with present of Auer rods in the cytoplasm of myeloblast. In other hand, lymphoblasts is tend to be small, the cytoplasm is scant and present of indistinct nucleoli. It extremely difficult to differentiation of acute leukemia in to myeloid and lymphoblastic based on morphologic characterize of blast cell,must be used addition analysis of blast like phenotype analysis,cytochemical stain,flow cytometry and molecular chromosome abnormalities. The most common cytochemical stain that used to determined the cell lineage are myeloperoxidase, esterases and Sudan black B. About 3% of blast cell that have positive of myeloperoxidase and positve in sudan black that indicate the myeloid origin. Acid phosphatase is demonstrate the activety of T-cell and present in early T-cell and can be diffrentation T-cell from non T-cell. Terminal deoxynucleotidyltransferase (Tdt) is positive in all lymphoblast, while small percentage of meyloblast positve as well. When the morphology and cytochemical stains are equivocal in diagnosis of acute leukemia the Immunophenotyping by flow cytometry will confirm the diagnosis of leukemia. Cytogenetic studies are imoportant because most patient of AML and ALL and 90% of patient with secondary leukemia have chromsomal abnormality. The chromosomal abnormalities help in diffrentaitan between AML and ALL and subtypes. Immunophenotyping and cytogenetic analyses are help in clinical ,prognosis and treatment of disease.
1.2 WILMS' TUMOR GENE (WT1):
wilms tumor gene is act as tumor suppressor gene that encodes transcrption factors that have crucial role in differentiation and cell growth. The expression of WT1 is limited and restricted to some type of tissues including kidney,uterus,gonad ,mesothelium and progenitor cell in most common types of tissues. WT1 is expressed in a time and tissue specific manner including anatomical area associated with hemopoiesis during the embyogenesis, but in adult the expression of WT1 is restricted to Sertoli cells of the testis, podocyte layer of the glomerulus, mesothelial cells, granulosa cells of the ovary and mammary duct and lobule. The WT1 gene is located on the short arm of chromosome 11 and contains 10 exons. Exons 7'10 code for the four zinc-fingers at the carboxy-terminus that bind to promoter regions of DNA. WT1 suppresses transcription of hemopoieticrelated proteins including M-CSF, TGFb, and RAR-a.
1.2.1 WT1 structured
Wilms' tumour gene 1 (WT1) spans 50 kb genomic DNA and consists of 10
exons which encodes an mRNA transcript of about 3.2 kb. The mRNA
translates into a 429 amino acid protein with a proline and glutamine rich amino
terminus harbouring defined functional domains exerting transcriptional
repression, activation, self-association, RNA-recognition and nuclear
localization signals. The carboxyl terminal part of WT1 contains four Cys2His2
zinc-fingers, encoded by exon 7-10, conferring specific DNA-binding
1.2.2 WT1 in hematopoiesis:
In human hematopoiesis few of the multipotent progenitor cells (1.2% with
equal distribution CD34+ CD38+/CD34+ CD38-) in the bone marrow express
WT1 and during differentiation the expression is rapidly down modulated.
In a murine model, the highest WT1 levels were detected in megakaryocyte /erythrocyte progenitors. In the terminally differentiated blood cells, WT1 seems to be below detection limit, although one paper report WT1 mRNA expression in monocytes, granulocytes and Blymphocytes. WT1 is not essential for reconstitution of hematopoiesis. Since embryonal stem cells (ES) devoid of WT1 can give rise to all types of mature blood cells. Neither was WT1 detected in long-term hematopoietic stem cells (LT-HSC) and in less than 1% of the multipotent progenitor cell. . However, even though WT1 does not seem to be a prerequisite for hematopoiesis, In addition, increased numbers of BFU-E, CFU-GM and CFU-GEMM in the bone
marrow cells. Overexpressing WT1 point to a positive impact on progenitor cell
expansion mediated by WT1 in these cells. In human hematopoietic CD34+ progenitor cells forced expression of WT1 lead to inhibited proliferation, but no alteration in cell cycle phase distribution was observed. The effect of ectopic WT1 expression in differentiating human CD34+ cells appears to be stage-specific; while quiescence is enhanced in primitive cells, cellular differentiation in lineage-committed precursors into the myeloid-monocytic hematopoieticlineage is stimulated . Taken together, available data indicate that WT1 is not critical for hematopoiesis, but that WT1 nevertheless may influence the proliferation, viability and/or differentiation of hematopoietic cells.
1.2.3 WT1 expression in primary leukemia
In leukemic cells the normal maturation is stalled through a differentiation block, which gives raise to clonal expansion of immature leukemic blasts accumulating in bone marrow and blood. This leads to suppression of normal hematopoiesis. WT1 is highly expressed in acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), in myelodysplastic syndromes (MDS), chronic myeloid leukemia in blast crisis and in leukemic cell lines. Eighty percent of the lymphoid leukemias and 90% of the myeloid leukemias, both adult- and childhood, have elevated WT1 expression The majority of AML patients express WT1 at diagnosis and increased amount of WT1 at relapse. When combining four different studies, 66% of ALL cases expressed WT1 at diagnosis, while the prevalence of WT1 in the subgroup adult acute T-lymphoblast leukemia was even lower, approximately 55%. Some reports show no significant correlation between WT1 expression and different myeloid leukemia FAB subtypes, while there are other reports indicating higher WT1 expression in M3 AML and less WT1 transcripts in M5 AML. Depending on analysis method, the WT1 expression in leukemic blast cells have been quantified to be similar or more than ten times as high as detected in the WT1 expressing CD34+ progenitor cells . Thus, a majority of leukemias express high levels of WT1 with no strong correlation to phenotype, suggesting that WT1 may positively affect proliferation and/or viability of the leukemic cells, rather than interfere with specific differentiation mechanisms.
1.2.4 Expression levels of WT1 correlate to poor clinical prognosis
In acute leukemias increased WT1 levels have prognostic significance and are
associated to poor prognosis In adult MDS and AML, WT1 mRNA levels in bone marrow at diagnosis may be used as a predictor of clinical outcome, since several studies show a positive correlation between high WT1 expression and a worse long-term outcome , although not confirmed in all studies . In pediatric ALL the prevalence of WT1 overexpression is lower than in AML or in adult AML and ALL. In fact, in childhood ALL the WT1 expression levels are very variable and both abnormally high and very low WT1 expression is associated with increased risk of relapse. During and after treatment, quantification of WT1 mRNA in peripheral blood (PB) is used as a measurement of minimal residual disease (MRD) in myeloid leukemias and in myelodysplastic syndrome . Persistent high WT1 level is regarded as a prognostic predictor of poor outcome with a significant lower complete remission rate and worse overall survival .Moreover, WT1 levels rise as progression occurs in acute leukemias and myelodysplastic syndromes and at relapse of the disease after clinical remission. Altogether, the positive correlation between WT1 expression and poor clinical
1.2.5 WT1 mutations in leukemia:
Not only high WT1 expression, but also mutations in the WT1 gene are found
in leukemias. The first report of WT1 mutations, associated with development
of AML,they found WT1 mutations in 15% of AML cases and in one
biphenotypic leukemia and also noted that the patients with WT1 mutations
were more refractory to chemotherapy with worse disease-free and overall
survival rate. Since then there is a growing amount of data on WT1 mutations detected in various leukemias. Large cohorts of cytogenetically normal (CN) AML cases confirm the frequency of about 10% mutated WT1 in adult AML. WT1
mutations are in general not found in conjunction with cytogenetic abnormalities such as t(15;17)(q22;q12), t(8;21)(q22;q22), inv(16)(p13q22) ort(16;16)(p13;q22)202, which have a relatively good prognosis. Other molecular markers with established prognostic significance, as mutations in NPM1 (nucleophosmin 1), CEBP& and MLL (myeloid/lymphoid or mixed-lineageleukemia) more or less frequently coincide with WT1. Analysis of almost 2500 patients provide evidence of aggressive forms of the AML disease with increased risk of relapse and death associated with WT1 mutation. WT1 mutations in adult CN-AML appears as an independent unfavourable prognostic predictor, regarding inferior cytogenetic remission rate and higher frequency of resistance to chemotherapy. In 58% of CN-AML patients, WT1 mutations was concurrent with a possible worse clinical outcome. In all childhood AML 12% WT1 mutations, but the incidence in pediatric CN-AML is as high as 22% with elevated expression levels of mutated WT1 as compared to wild-type cases. WT1 mutations found in AML are mostly heterozygous, with a remaining WT1 wild-type allele. They are missense mutations, deletions and insertions, resulting in truncated WT1 protein with loss of DNA-binding. The mutational 'hotspots' are in exon 7 and 9, but rare mutations are also reported in exon 1, 2, 3. Frameshift mutations in exon 7 are the most predominant.They generate a truncated WT1 protein lacking the four
DNA-binding zinc-fingers and with loss of the nuclear localization signal
(NLS). Other recurrent type of mutations in AML is point mutations in exon 9,
conferring amino acid substitutions at similar positions as in Denys Drash
syndrome (DDS)116, where the substitutions confer impaired DNA-binding.
Frameshift mutations are less frequent than the point mutations in exon 9. These
may lead to a WT1 protein deficient of zinc-finger 3 and 4.
1.3 aim of stydy
Many leukemias, including up to 80% of acute myeloid leukemias (AMLs), have been demonstrated to express WT1.2-10 Mutations of WT1have been found in approximately 5% to 10% of AMLs, similar to the level seen in Wilms tumors, and sporadically in a variety of other leukemias.11-18 Manipulation of WT1 expression in leukemia cells results in alterations in differentiation and growth potential.19-28 In the treatment of leukemia, expression of WT1 in the peripheral blood and bone marrow has been suggested to be an indicator of clinical relapse. Attempts have been made to correlate WT1expression with progression of disease and prognosis in several types of hematologic malignancies. The role of the Wilms tumor 1 gene (WT1) in acute leukemias has been underscored by mutations found in acute myeloid leukemia identifying patients with inferior survival. Furthermore, aberrant expression of WT1 in acute myeloid leukemia was associated with an increased risk of relapse. No larger studies have performed a combined approach including WT1 mutation and expression analyses in acute T-lymphoblastic leukemia. We have previously reported high expression of wild-type WT1 in fresh leukemia cells regardless of the disease type, 9-11 an inverse correlation between WT1 expression levels and prognosis,9 increased expression of WT1 at relapse in acute leukemia,12 and growth inhibition of leukemic cells by WT1 antisense oligomers.13 These results suggested that WT1 plays an important role in leukemogenesis and may have an oncogenic function rather than a tumor-suppressor gene function in hematopoietic progenitor cells.