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LEUKEMIA
CHAPTER ONE
Leukemia is a haematological malignancy associated with the Philadelphia chromosome. It accounts for about 3.6 % of all haematological malignancies worldwide.1 In Nigeria CML prevalence remains unknown but studies done in Lagos give an incidence of 90.3 cases annually.
For many years the treatment for CML has included hydroxyurea, interferon alpha with or without cytarabine and busulfan, with hemopoietic cell transplant (HCT) therapy giving a potential of cure.
The introduction of targeted therapy using tyrosine kinase inhibitors has revolutionized the management of CML. During the IRIS trial, Imatinib was compared to treatment with combination treatment with interferon and cytarabine. Due to the superiority of Imatinib demonstrated by this study, it has now become the standard of care. Cases of failure on treatment with imatinib have been documented, with therapeutic failure to imatinib seen in approximately 10% to 15% of patients and can be classified as primary or secondary depending on whether an initial decline in disease levels is observed or not Risk factors for failure on imatinib are yet to be fully established with the Sokal and Hasford scores being used to predict cytogenetic response in some centres 6.
In Nigeria, most of the patients on Imatinib receive the medication via the GIPAP, an international assistance programme which provides Philadelphia chromosome positive (Ph+ve) CML patients with imatinib mesylate (Glivec®) at no cost. There had been no studies done to determine; treatment response, the proportion of patients who fail on imatinib and to describe their clinical-pathologic characteristics.
What is the treatment response of leukemia patients to imatinib therapy, and what are the clinical and haematological characteristics of patients with imatinib failure?
1.4 Broad objective
Broadly we were to determine the hematologic treatment response, proportion of CML patients with hematologic imatinib failure, and to describe the clinical and haematological characteristics of patients with imatinib failure.
The primary objectives were to determine the hematologic treatment response of CML patients receiving imatinib therapy, the proportion of CML patients with hematologic failure and to describe clinical and haematological characteristics of patients with hematologic failure and compare this with those of patients with optimal response.
Our secondary objectives were to determine the possible predictors of imatinib failure in patients with Ph +ve CML and to determine the proportion of patients with imatinib failure in relation to the Sokal and Hasford scores.
2.1 Leukemia
Leukemia is a clonal myeloproliferative disorder associated with the Philadelphia chromosome (Ph), a balanced translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11) producing BCR-ABL oncoprotein as illustrated in figure 1. It is characterized by proliferation of mature granulocytes (neutrophils, eosinophils, and basophils) and their precursors.
Peter C. Nowell and David Hungerford in 1960 at Philadelphia, Pennsylvania, noticed an abnormally small chromosome 22 in the cells of patients with CML, which got named Ph chromosome. This made CML the first cancer shown to be caused by an underlying genetic abnormality.8 Later, in 1973, Janet Rowley reported that Ph chromosome represents a balanced reciprocal translocation between long arms of chromosomes 9 and 22
The chromosome is detected in about 95% of patients, 11 this genetic abnormality results in the formation of a unique gene product (BCR-ABL), which results in a constitutively active tyrosine kinase. It is this deregulated tyrosine kinase that is implicated in the development of CML and is the target of current therapies
BCR-ABL oncogene mediates the development and maintenance of CML through interaction with multiple downstream signalling partners resulting in altered cellular adhesion, activation of mitogenic signalling, defective DNA repair, and inhibition of apoptosis: all these lead to transformation of hematopoietic stem cells.11 The Signaling Pathways of BCR-ABL are illustrated in figure 2.
The Philadelphia (Ph) chromosome is a shortened chromosome 22 that results from the translocation of 3' ABL segments on chromosome 9 to 5' BCR segments on chromosome 22. Breakpoints (arrowheads) on the ABL gene are located 5' of exon a2 in most cases. Various breakpoint locations have been identified along the BCR gene on chromosome 22. Depending on which breakpoints are involved, different-sized segments from BCR are fused with the 3' sequences of the ABL gene. This results in fusion messenger RNA molecules (e1a2, b2a2, b3a2, and e19a2) of different lengths that are transcribed into chimeric protein products (p190, p210, and p230) with variable molecular weights and presumably variable function.
The abbreviation m-bcr denotes minor breakpoint cluster region, M-bcr major breakpoint cluster region, and μ-bcr a third breakpoint location in the BCR gene that is downstream from the M-bcr region between exons e19 and e20.
Figure 2: Signaling Pathways of p210 BCR–ABL.10
Several regions of BCR–ABL serve as important control elements for RAS, which is at the center of the most prominent signaling pathways in CML. Activation of RAS is mediated through a series of adapter proteins, such as GRB2, CBL, SHC, and CRKL. Adapter proteins also connect p210BCR–ABL to focal adhesion complexes, PI-3 kinase, and other messenger systems such as JAK–STAT kinases. Signaling events downstream of RAS are less well characterized. They appear to involve mainly mitogen-activated protein kinases (MAPKs), preferably the JUN kinase (JNK) pathway. BAP-1 denotes BCR-associated protein 1, GRB2 growth factor receptor–bound protein 2, CBL casitas B-lineage lymphoma protein, SHC SRC homology 2–containing protein, CRKL CRK-oncogene–like protein, JAK–STAT Janus kinase–signal transducers and activators of transcription, FAK focal adhesion kinase, SOS son-of-sevenless, GDP guanosine diphosphate, GTP guanosine triphosphate, SRE stimulated response element, Ser–thr serine–threonine, Y177 a conserved tyrosine residue, GEF GDP– GTP exchange factor, and SH SRC homology domain.
Aetiology: The only known risk factors for CML are high doses of ionizing radiation and occupational exposure to benzene as evidenced by 20-25 fold increase in the incidence of all leukaemia‟s among atomic bomb survivors.13 CML accounts for 0.34% of all cancers, 3.6% of all haematological malignancies and 0.08% of all cancer mortalities. It also accounts for 15% of all adult leukaemias.1, 14. The median age of onset is 45 to 55 years.
In Nigeria CML Prevalence remains unknown, Ministry of health records between 1998 and 2002 estimate a mean of 90.3 cases of CML per 100,000 people in Lagos annually. A retrospective study done on 104 patients treated for CML between April 1990 and August 2000, at Ibom National Hospital, Lagos, Nigeria showed age range of 10-72years with a median of 35 years which is a decade younger than the age of 45 years described among Whites.15
Apart from on average younger patient age group, the other differences in patient characteristics which have been noticed when African patients are compared with patients from other populations include high incidence of cytogenetic abnormalities and long duration of time between diagnosis and onset of treatment.
About 40% of CML patients are asymptomatic and therefore diagnosis is based on abnormal blood count.10 The commonest physical finding is splenomegaly which occurs in about 50% of CML patients, followed by hepatomegaly. Fatigue, anorexia, and weight loss are the commonest symptoms.
The disease is characterized by an overabundance of hematopoietic stem cells and progresses through Chronic, accelerated and blast phases with more than 80% of patients being diagnosed in the chronic phase.11, 14 Median duration of chronic phase is 5-6 years.
The diagnosis of accelerated phase CML may be made when one or more of the following are present:
1. Blasts 10-19% of WBCs in peripheral blood and/or of nucleated bone marrow cells
2. Peripheral blood basophils ≥ 20%
3. Persistent thrombocytopenia (<100 x 10-9/L) unrelated to therapy or persistent thrombocytosis (>1000 x 10-9/l) unresponsive to therapy.
4. Increasing spleen size and increasing WBC unresponsive to therapy.
5. Cytogenetic evidence of clonal evolution.
Blast phase may be diagnosed if one or more of the following are present: Blasts ≥ 20%
Extra medullary blast proliferation
Large foci or clusters of blasts in the bone marrow biopsy17
Peripheral-blood findings include elevated white-cell count (usually greater than 25,000/mm3) elevated platelet count in 30-50% of cases, basophilia and reduced leukocyte alkaline phosphatase activity.
Upon achievement of hematologic response the patient‟s WBC falls to the normal range (4- 10,000/mm3).
The Leucocyte alkaline phosphatase (LAP) score is based on a cytochemistry test that was once often used to test blood samples of patients who were suspected of having CML. Normally the LAP score goes up as the white blood cell (WBC) count goes up. However, people with CML, tend to have low LAP scores in spite of high WBC counts.
All stages of granulocyte differentiation are visible on peripheral smear. Most patients have a normochromic normocytic anaemia.
The bone marrow is markedly hyper-cellular, predominantly because of a proliferation of myeloid precursors from myeloblasts to segmented neutrophils, reduced fat content, with increased ratio of myeloid cells to erythroid cells.
Megakaryocytes are increased. Blasts and promyelocytes typically constitute less than 10% of all cells14
Vitamin B12 serum concentration in chronic myeloid leukaemia is approximately 15 times of the normal, it exists in the bound form, and the binding capacity for added B12 is increased.19 Chromosomal analyses are aimed at identifying the Ph chromosome t (9; 22) the hallmark of CML.10
These include cytogenetic detection, Fluorescence In Situ Hybridization (FISH) and molecular techniques. Cytogenetic detection entails Karyotypic analysis best performed from bone marrow material. The finding of a translocation between chromosome 9 and 22, generally the t (9; 22) (q34; q11), confirms the diagnosis. This chromosomal translocation may also be demonstrated by Southern blot analysis, or the Transcripted messenger RNA (mRNA) fusion product may be detected by reverse transcriptase polymerase chain reaction (RT-PCR).
Cytogenetic analysis is the gold standard diagnostic test in CML.
During the ancient times arsenicals were used for the treatment of CML.26 In the first half of 20th century X-ray splenic irradiation for symptomatic relief was the mainstay of therapy.26 For many years busulfan, hydroxyurea, and interferon alpha were used for the treatment of CML. During the chronic phase of CML, cytoreductive therapy (with hydroxyurea or busulfan) is required in most patients to avoid thrombotic complications that can be due to high circulating levels of neutrophils. Hydroxyurea is preferred to busulfan primarily because of its favourable toxicity profile 3. Treatment with either drug has no effect on the rate of progression to blast crisis; therefore, these treatments must be considered palliative11 Allogeneic hematopoietic stem-cell transplantation can cure CML in selected patients. Age <40 years, early disease in chronic phase, and HLA-identical sibling donor, confer better outcome post transplantation.
The IRIS trial (International Randomized study of Interferon and STI571) led to the introduction of targeted therapy using tyrosine kinase inhibitors, the first of which was imatinib mesylate (marketed as Gleevec or Glivec; previously known as STI-571).
Two sets of prognostic factors can be established, namely those that can be identified prior to therapy (baseline factors) and those that can be employed during the treatment (time dependent or response related factors). Most important is accurate identification of the phase of the disease.
In early chronic phase important prognostic information is derived from clinical and laboratory features. Currently, the Hasford score is the best predictor of outcome for patients with CML treated with interferon alpha (IFN-α)
Kantarjian and colleagues in the Anderson Hospital & Tumor institute, Houston, Texas (1985); did a multivariate analysis of the associations of 303 Ph+ CP-CML patient characteristics and therapy with survival. They found that patient characteristics associated with shortened survival were age above 60 years, black race, and the presence of hepatomegaly, splenomegaly, symptoms, weight loss, and poor performance status.
Adverse blood and bone marrow parameters were anaemia, thrombocytosis or thrombocytopenia, a high proportion of peripheral blasts plus promyelocytes or of basophils, a high proportion of marrow blasts or basophils, decreased marrow megakaryocytes and cytogenetic abnormalities in addition to the Philadelphia chromosome.
The Sokal and Hasford Scores have been used for predicting response to therapy.
Hasford risk score7 is a stratification that includes patients‟ age, spleen size, percentage of blasts, eosinophils and basophils in the peripheral blood and platelet count. It was initially used for patients treated with interferon.7, 25 The scoring systems classify patients into three risk groups: low, intermediate, and high and is calculated using of the following equation: (0.6666 × age [0 for <50 years; 1 for older age] +
0.0420 × spleen size [cm below costal margin] + 0.0584 × blasts [%] + 0.0413 × eosinophils [%] + 0.2039 × basophils [0 for <3%; 1 for higher value] + 1.0956 × platelet count [0 for <1500×109/l; 1 for a higher value]) × 1000.
A score of less than 780 is considered to indicate low risk, a score of 780 to 1480, intermediate risk, and a score higher than 1480, high risk.
CHAPTER ONE
Leukemia is a haematological malignancy associated with the Philadelphia chromosome. It accounts for about 3.6 % of all haematological malignancies worldwide.1 In Nigeria CML prevalence remains unknown but studies done in Lagos give an incidence of 90.3 cases annually.
For many years the treatment for CML has included hydroxyurea, interferon alpha with or without cytarabine and busulfan, with hemopoietic cell transplant (HCT) therapy giving a potential of cure.
The introduction of targeted therapy using tyrosine kinase inhibitors has revolutionized the management of CML. During the IRIS trial, Imatinib was compared to treatment with combination treatment with interferon and cytarabine. Due to the superiority of Imatinib demonstrated by this study, it has now become the standard of care. Cases of failure on treatment with imatinib have been documented, with therapeutic failure to imatinib seen in approximately 10% to 15% of patients and can be classified as primary or secondary depending on whether an initial decline in disease levels is observed or not Risk factors for failure on imatinib are yet to be fully established with the Sokal and Hasford scores being used to predict cytogenetic response in some centres 6.
In Nigeria, most of the patients on Imatinib receive the medication via the GIPAP, an international assistance programme which provides Philadelphia chromosome positive (Ph+ve) CML patients with imatinib mesylate (Glivec®) at no cost. There had been no studies done to determine; treatment response, the proportion of patients who fail on imatinib and to describe their clinical-pathologic characteristics.
What is the treatment response of leukemia patients to imatinib therapy, and what are the clinical and haematological characteristics of patients with imatinib failure?
1.4 Broad objective
Broadly we were to determine the hematologic treatment response, proportion of CML patients with hematologic imatinib failure, and to describe the clinical and haematological characteristics of patients with imatinib failure.
The primary objectives were to determine the hematologic treatment response of CML patients receiving imatinib therapy, the proportion of CML patients with hematologic failure and to describe clinical and haematological characteristics of patients with hematologic failure and compare this with those of patients with optimal response.
Our secondary objectives were to determine the possible predictors of imatinib failure in patients with Ph +ve CML and to determine the proportion of patients with imatinib failure in relation to the Sokal and Hasford scores.
2.1 Leukemia
Leukemia is a clonal myeloproliferative disorder associated with the Philadelphia chromosome (Ph), a balanced translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11) producing BCR-ABL oncoprotein as illustrated in figure 1. It is characterized by proliferation of mature granulocytes (neutrophils, eosinophils, and basophils) and their precursors.
Peter C. Nowell and David Hungerford in 1960 at Philadelphia, Pennsylvania, noticed an abnormally small chromosome 22 in the cells of patients with CML, which got named Ph chromosome. This made CML the first cancer shown to be caused by an underlying genetic abnormality.8 Later, in 1973, Janet Rowley reported that Ph chromosome represents a balanced reciprocal translocation between long arms of chromosomes 9 and 22
The chromosome is detected in about 95% of patients, 11 this genetic abnormality results in the formation of a unique gene product (BCR-ABL), which results in a constitutively active tyrosine kinase. It is this deregulated tyrosine kinase that is implicated in the development of CML and is the target of current therapies
BCR-ABL oncogene mediates the development and maintenance of CML through interaction with multiple downstream signalling partners resulting in altered cellular adhesion, activation of mitogenic signalling, defective DNA repair, and inhibition of apoptosis: all these lead to transformation of hematopoietic stem cells.11 The Signaling Pathways of BCR-ABL are illustrated in figure 2.
The Philadelphia (Ph) chromosome is a shortened chromosome 22 that results from the translocation of 3' ABL segments on chromosome 9 to 5' BCR segments on chromosome 22. Breakpoints (arrowheads) on the ABL gene are located 5' of exon a2 in most cases. Various breakpoint locations have been identified along the BCR gene on chromosome 22. Depending on which breakpoints are involved, different-sized segments from BCR are fused with the 3' sequences of the ABL gene. This results in fusion messenger RNA molecules (e1a2, b2a2, b3a2, and e19a2) of different lengths that are transcribed into chimeric protein products (p190, p210, and p230) with variable molecular weights and presumably variable function.
The abbreviation m-bcr denotes minor breakpoint cluster region, M-bcr major breakpoint cluster region, and μ-bcr a third breakpoint location in the BCR gene that is downstream from the M-bcr region between exons e19 and e20.
Figure 2: Signaling Pathways of p210 BCR–ABL.10
Several regions of BCR–ABL serve as important control elements for RAS, which is at the center of the most prominent signaling pathways in CML. Activation of RAS is mediated through a series of adapter proteins, such as GRB2, CBL, SHC, and CRKL. Adapter proteins also connect p210BCR–ABL to focal adhesion complexes, PI-3 kinase, and other messenger systems such as JAK–STAT kinases. Signaling events downstream of RAS are less well characterized. They appear to involve mainly mitogen-activated protein kinases (MAPKs), preferably the JUN kinase (JNK) pathway. BAP-1 denotes BCR-associated protein 1, GRB2 growth factor receptor–bound protein 2, CBL casitas B-lineage lymphoma protein, SHC SRC homology 2–containing protein, CRKL CRK-oncogene–like protein, JAK–STAT Janus kinase–signal transducers and activators of transcription, FAK focal adhesion kinase, SOS son-of-sevenless, GDP guanosine diphosphate, GTP guanosine triphosphate, SRE stimulated response element, Ser–thr serine–threonine, Y177 a conserved tyrosine residue, GEF GDP– GTP exchange factor, and SH SRC homology domain.
Aetiology: The only known risk factors for CML are high doses of ionizing radiation and occupational exposure to benzene as evidenced by 20-25 fold increase in the incidence of all leukaemia‟s among atomic bomb survivors.13 CML accounts for 0.34% of all cancers, 3.6% of all haematological malignancies and 0.08% of all cancer mortalities. It also accounts for 15% of all adult leukaemias.1, 14. The median age of onset is 45 to 55 years.
In Nigeria CML Prevalence remains unknown, Ministry of health records between 1998 and 2002 estimate a mean of 90.3 cases of CML per 100,000 people in Lagos annually. A retrospective study done on 104 patients treated for CML between April 1990 and August 2000, at Ibom National Hospital, Lagos, Nigeria showed age range of 10-72years with a median of 35 years which is a decade younger than the age of 45 years described among Whites.15
Apart from on average younger patient age group, the other differences in patient characteristics which have been noticed when African patients are compared with patients from other populations include high incidence of cytogenetic abnormalities and long duration of time between diagnosis and onset of treatment.
About 40% of CML patients are asymptomatic and therefore diagnosis is based on abnormal blood count.10 The commonest physical finding is splenomegaly which occurs in about 50% of CML patients, followed by hepatomegaly. Fatigue, anorexia, and weight loss are the commonest symptoms.
The disease is characterized by an overabundance of hematopoietic stem cells and progresses through Chronic, accelerated and blast phases with more than 80% of patients being diagnosed in the chronic phase.11, 14 Median duration of chronic phase is 5-6 years.
The diagnosis of accelerated phase CML may be made when one or more of the following are present:
1. Blasts 10-19% of WBCs in peripheral blood and/or of nucleated bone marrow cells
2. Peripheral blood basophils ≥ 20%
3. Persistent thrombocytopenia (<100 x 10-9/L) unrelated to therapy or persistent thrombocytosis (>1000 x 10-9/l) unresponsive to therapy.
4. Increasing spleen size and increasing WBC unresponsive to therapy.
5. Cytogenetic evidence of clonal evolution.
Blast phase may be diagnosed if one or more of the following are present: Blasts ≥ 20%
Extra medullary blast proliferation
Large foci or clusters of blasts in the bone marrow biopsy17
Peripheral-blood findings include elevated white-cell count (usually greater than 25,000/mm3) elevated platelet count in 30-50% of cases, basophilia and reduced leukocyte alkaline phosphatase activity.
Upon achievement of hematologic response the patient‟s WBC falls to the normal range (4- 10,000/mm3).
The Leucocyte alkaline phosphatase (LAP) score is based on a cytochemistry test that was once often used to test blood samples of patients who were suspected of having CML. Normally the LAP score goes up as the white blood cell (WBC) count goes up. However, people with CML, tend to have low LAP scores in spite of high WBC counts.
All stages of granulocyte differentiation are visible on peripheral smear. Most patients have a normochromic normocytic anaemia.
The bone marrow is markedly hyper-cellular, predominantly because of a proliferation of myeloid precursors from myeloblasts to segmented neutrophils, reduced fat content, with increased ratio of myeloid cells to erythroid cells.
Megakaryocytes are increased. Blasts and promyelocytes typically constitute less than 10% of all cells14
Vitamin B12 serum concentration in chronic myeloid leukaemia is approximately 15 times of the normal, it exists in the bound form, and the binding capacity for added B12 is increased.19 Chromosomal analyses are aimed at identifying the Ph chromosome t (9; 22) the hallmark of CML.10
These include cytogenetic detection, Fluorescence In Situ Hybridization (FISH) and molecular techniques. Cytogenetic detection entails Karyotypic analysis best performed from bone marrow material. The finding of a translocation between chromosome 9 and 22, generally the t (9; 22) (q34; q11), confirms the diagnosis. This chromosomal translocation may also be demonstrated by Southern blot analysis, or the Transcripted messenger RNA (mRNA) fusion product may be detected by reverse transcriptase polymerase chain reaction (RT-PCR).
Cytogenetic analysis is the gold standard diagnostic test in CML.
During the ancient times arsenicals were used for the treatment of CML.26 In the first half of 20th century X-ray splenic irradiation for symptomatic relief was the mainstay of therapy.26 For many years busulfan, hydroxyurea, and interferon alpha were used for the treatment of CML. During the chronic phase of CML, cytoreductive therapy (with hydroxyurea or busulfan) is required in most patients to avoid thrombotic complications that can be due to high circulating levels of neutrophils. Hydroxyurea is preferred to busulfan primarily because of its favourable toxicity profile 3. Treatment with either drug has no effect on the rate of progression to blast crisis; therefore, these treatments must be considered palliative11 Allogeneic hematopoietic stem-cell transplantation can cure CML in selected patients. Age <40 years, early disease in chronic phase, and HLA-identical sibling donor, confer better outcome post transplantation.
The IRIS trial (International Randomized study of Interferon and STI571) led to the introduction of targeted therapy using tyrosine kinase inhibitors, the first of which was imatinib mesylate (marketed as Gleevec or Glivec; previously known as STI-571).
Two sets of prognostic factors can be established, namely those that can be identified prior to therapy (baseline factors) and those that can be employed during the treatment (time dependent or response related factors). Most important is accurate identification of the phase of the disease.
In early chronic phase important prognostic information is derived from clinical and laboratory features. Currently, the Hasford score is the best predictor of outcome for patients with CML treated with interferon alpha (IFN-α)
Kantarjian and colleagues in the Anderson Hospital & Tumor institute, Houston, Texas (1985); did a multivariate analysis of the associations of 303 Ph+ CP-CML patient characteristics and therapy with survival. They found that patient characteristics associated with shortened survival were age above 60 years, black race, and the presence of hepatomegaly, splenomegaly, symptoms, weight loss, and poor performance status.
Adverse blood and bone marrow parameters were anaemia, thrombocytosis or thrombocytopenia, a high proportion of peripheral blasts plus promyelocytes or of basophils, a high proportion of marrow blasts or basophils, decreased marrow megakaryocytes and cytogenetic abnormalities in addition to the Philadelphia chromosome.
The Sokal and Hasford Scores have been used for predicting response to therapy.
Hasford risk score7 is a stratification that includes patients‟ age, spleen size, percentage of blasts, eosinophils and basophils in the peripheral blood and platelet count. It was initially used for patients treated with interferon.7, 25 The scoring systems classify patients into three risk groups: low, intermediate, and high and is calculated using of the following equation: (0.6666 × age [0 for <50 years; 1 for older age] +
0.0420 × spleen size [cm below costal margin] + 0.0584 × blasts [%] + 0.0413 × eosinophils [%] + 0.2039 × basophils [0 for <3%; 1 for higher value] + 1.0956 × platelet count [0 for <1500×109/l; 1 for a higher value]) × 1000.
A score of less than 780 is considered to indicate low risk, a score of 780 to 1480, intermediate risk, and a score higher than 1480, high risk.
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