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Chronic
Myeloid Leukaemia
Written
by Dr. Furrat Amen Introduction Chronic myeloid leukaemia (CML) is a malignant blood disorder which involves early haematopoietic cells which become clonally expanded. The disease originates from a single abnormal haematopoietic stem cell which proliferates over months and years so that at diagnosis blood granulocytosis and marrow granulocytopoiesis are apparent. The bone marrow becomes hypercellular. Normal blood cell production is almost completely replaced by leukaemia cells. The incidence is 1-1.5/100000 in the UK, with 700 new cases presenting each year. It may present at any age, although the peak onset is 40-60 years, with a slight male predominance. In the US, the median age of presentation is 53 years. It is very rare in children. The only predisposing factors to have been elucidated include atomic bomb radiation following blasts in Hiroshima, and Nagasaki, Japan, and subsequent to use of irradiation as a treatment of ankylosing spondylitis, in the past. CML may be clinically categorised as follows:
Diagram 1 66% of patients move from the chronic phase to the accelerated phase. The accelerated phase is manifest by an increase in blast cells, increasing anaemia, an increase in platelets. A few months later they move into a frankly blastic phase. The remainder make the transformation directly from chronic to blastic phase immediately (2). In the 1920s, life-expectancy following diagnosis with CML was three months. Now survival from diagnosis is likely to be five to six years, although occasionally patients only survive one year. 3% live for more than 15 years without therapy The blast crisis is myeloid in 80% of patients (like acute myelocytic leukaemia (AML)) and lymphoid in the remainder. Occasionally there is a myelofibrotic phase of the disease which shows intensive marrow fibrosis. Blast cell proliferation is less aggressive and clinically there is splenomegaly and pancytopenia after marrow failure. Signs and Symptoms Many
patients, remain asymptomatic for an extended period before displaying
and signs and symptoms. 20% of patients are asymptomatic at diagnosis.
These include tiredness, weight loss, sweats, bone pain, fever, and
abdominal discomfort that results from splenomegaly (+/- hepatomegaly).
More rarely, patients present with splenic infarction, leukostasis,
gout, retinal haemorrhages, and priaprism. A very high white cell count
(WCC) results in hyperviscosity. Such patients typically have a WCC
in excess of 200,000/microL and present with dyspnoea, neurological
changes, and chest pain. The
genetic finding in this disease is the Philadelphia chromosome (Ph).
The c-abl proto-oncogene on chromosome 9 is translocated to the breakpoint
cluster region (bcr) on chromosome 22. This bcr/abl gene codes for tyrosine
kinase. This in turn inhibits apoptosis and may be the cause of CML. The cytogenetic marker for CML, the Philadelphia chromosome - an abnormally minute chromosome 22, was discovered in 1960 by P. C. Newell and D. A. Hungerford in Philadelphia. In the 1980s, molecular biology techniques detailed the bcr/abl translocation. The translocation that occurs may be written as t(9;22)(q34;q11). The bcr/abl fusion gene that is on the Ph (22q-) is expressed as mRNA. This in turn produces the protein p210, which causes derangement of stem cell function and is the likely cause of chronic phase CML. The aim in the future is to inactivate the bcr/abl gene. P210 bcr/abl can induce malignant transformation in transfected cells and induces leukaemia in transgenic mice. The bcr part of the protein probably localises the abl kinase to inappropriate substrates and causes inappropriate activation of signal transduction and growth pathways. Ph+ cells also show a decreased expression of the adhesion molecule - lymphocyte function antigen 3. This is normally expressed and is important for growth control and differentiation. Using IFNa may restore normal expression of this molecule. Ph+ cells also appear to be protected from apoptosis and do not necessarily proliferate faster than ordinary cells.
Diagram 2 - the philadelphia chromosome 95% of patients with CML show Ph chromosome during karyotypic analysis. In the remainder, a third show the bcr/abl translocation during DNA/RNA analysis. The others may have chronic myelomonocytic leukaemia or myelofibrosis. In experiments in vitro, Ph negative haematopoietic cells were found in CFU-GM colonies grown from CML marrow. This means early in CML, the marrow continues to contain normal haematopoiesis. The aim in therapy is to minimise or eliminate the Ph+ clone, reduce the rate of genetic chance and to postpone the blast crisis. The disease culminates after three or four years in a blast crisis which causes the death of the patient. A smaller proportion of patients die in the first or second years following diagnosis. Subsequently, 25% die per year.
This may be made on viewing the blood film. A bone marrow may be performed as a confirmatory test. The WCCis raised at 30-400x109/L, granulocytes are found at all stages of development. This an increase in the number of basophils and eosinophils. Blast (primitive) cells reach a maximum of 10%, and they are not found in normal controls. Haemoglobin may be reduced and red cell morphology remains normal. There may be an increase in nucleated and immature red cells. Platelets may also be increased (300-600x109/L). The marrow shows hypercellularity and immature leukocytes. The megakaryocytes are plentiful, although they are smaller. Cytogenetics shows the Ph chromosome in all dividing cells. The will be an increase in the lactate dehydrogenase (LDH) and the serum urate may be raised. Advanced disease is diagnosed by a blood test. The patients complains of excessive sweating persistent fever. Anaemia, splenic enlargement, splenic infarction, haemorrhage and bone pain. The criteria to be fulfilled for advanced disease are detailed in diagram 3.
Diagram 3 Monitoring It is possible to monitor minimal residual disease following transplant by using quantitative reverse transcriptase PCR (RT-PCR) and florescent in situ hybridisation (FISH). These methods help determine if the patient is Ph-, and patient who are bcr/abl positive - an issue complicated by the fact that it has been recently discovered that bcr/abl may be found in low levels in healthy patients7. It is important to assess residual leukaemia to assess treatment response, to stratify patients into groups based upon their risk of relapse, to assess risk for patient and tailor treatment appropriately eg. change dose of IFa, to assess the need for bone marrow transplant, and to assess the case for infusion of donor lymphocytes post BMT for impending relapse. Cytogenetics
has a sensitivity of 1% and a bone marrow aspirate is required for this
type of monitoring. RT-PCR detects a single bcr/abl+ cell in a background
of 105 - 106 normal cells. This means that even if RT-PCR detects no
residual disease it is possible to have 1000000 malignant cells still
present, which, of course, may contribute to relapse. It is also prone
to false positive. Blood or bone marrow may be used. FISH may be carried
out on peripheral blood and is useful for the monitoring of patient
response to IFNa9. Patients
should be told that people with CML live many years normally before
the disease moves to the advanced stage. The patient also is likely
to have difficulty in remembering what is said by the doctor at the
time of diagnosis, so it is wise to give BACUP leaflets to the patient
at this juncture. Pregnant women should be allowed to continue to term
if diagnosed during pregnancy, and men should have the option of cryopreservation
of their semen before chemotherapy is started. Hydroxyureas is an inhibitor of ribonucleotide reductase, and acts on the myeloid series of cells i.e. neutrophils, basophils and eosinophils. There is a rapid decrease of the leukocyte count in new patients on this therapy. To reduce symptoms many patients are started on drug therapy with hydroxyurea, low dose interferon, or busulfan. Hydroxyurea is often preferred to busulfan because of its less serious side-effects which may have a role to play in extended survival. These drugs are used especially in those over 60 years. Side-effects of busulfan include: pulmonary fibrosis, hepatic veno-occlusive disease, renal failure, myelosuppression, and myeloablation. Some of these effects may be irreversible. These agents are used in an attempt to increase the time to blast crises rather than as cure (see later for interferon) and their use must be a balance between the side-effects and the initial mortality associated with the disease. When symptoms are under control and the leukocyte count has decreased with hydroxyurea, therapy is switched to interferon alpha (IFNa). Hydroxyurea is also useful in controlling the chronic phase when patients are intolerant to IFNa. The dose normally used is 2.0g per day, taken orally, which is reduced to a maintenance dose of 1.0-1.5g per day. Side-effects include rashes, mouth ulcers, gastrointestinal symptoms, macrocytosis and megaloblastoid change in the marrow. IFNa is a naturally occurring glycoprotein which has antiproliferative and antiviral effects. It restores the spleen size to normal in 70-80% of patients. The side-effects are: flu-like symptoms, anorexia, weight loss, depression, alopecia, rashes, neuropathies, autoimmune disorders, and a decline in platelet numbers. Contra-indications to treatment may include hepatic, cardiac and renal failure Nowadays, it is common to use high dose interferon, intensive chemotherapy, and possible stem cell support for patients under 50 years. IFNa results in a complete haematological remission in 50-80% of patients with a cytogenetic response in 60%. Ph+ clones in the bone marrow are suppressed in less than 10% of patients 1. Almost all of the patients with chronic phase complete cytogenetic response to IFa still have residual bcr/abl + cells as detected by RT-PCR. The response to this therapy depends on the compliance, intensity of treatment, duration of the disease, and tumour mass. When interferon was compared to hydroxyurea and/or busulfan, the German CML trial found no difference, but the British, Italian, and Japanese trials showed longer survival on IFa . Median survival increase by 20months and 8% of patients remained Ph- with a median survival of ten or more years. The effects of combining IFa with cytarabine or homoharringtonin are still undergoing investigation for beneficial effects on prognosis. The use of IFa before bone marrow transplants is still controversial because it is unknown if it has adverse effects on prognosis. The best doses and combinations are still unknown10. A study of 60 patients compared two arms - IFNa with cytosine arabinoside and IFNa alone - in the treatment of late/refractory chronic phase CML. A significantly higher proportion showed complete haematological response on the combination: 56% vs. 28% (p=0.02). A non-significant trend to better cytogenetic response was also shown and longer survival at three years (76% vs 48%, p<0.01) 5. The French CML trial (721 patients) confirmed superiority of the combination and was halted. IFNa is 200 times the cost of conventional chemotherapy. It is normally taken that the acceptable figure is $50,000 per life year gain for emerging treatments to be economically acceptable. As a result it might be maintained that IFa, on a financial basis, should not be used for treating CML (Messori et al). Liberato et al. assessed the any years of life gained according to their quality as assessed by various criteria. They found that life expectancy was increased by 12.5-15.5 months in comparison to conventional treatment. The cost was found to be between $63,000 and $89,000 (depending on the situation) per quality adjusted life year gained - which still exceeds the conventionally accepted limit for the introduction of a new treatment. These studies were contradicted by Katten et all who found that median survival was 69months as opposed to 58 months with hydroxyurea and so the cost was $35000 per life year gained which is regarded, by comparison as inexpensive4. There are several points to make not of in consideration of these results. They assume that IFa prolongs life in comparison with conventional therapy. They do not consider that the effect may be increased by the addition of the cheaper drug - cytarabine. The appropriate dosage to achieve the required effect is, as yet, unknown and so using 3MU as opposed to 5MU has large potential cost savings. The cost charged by manufacturers could be lowered - there appears to be alliances which prevent the companies from lowering prices and operating on a principle of free competition. The threshold of $50000 per life year gain is an arbitrary one and may be raised in western societies where life may have a higher value placed on it, in comparison to the east. IFa could be stopped after two years if no cytogenetic remission is achieved, assuming that the increase in life expectancy is dependant on achieving cytogenetic remission - which is uncertain at the moment. Many therapies induce a cytogenetic response with disappearance of Ph+ cells. Unfortunately, sensitive molecular assays, eg. PCR, often still detect residual CML. With the allogenic stem cell transplant (see below) molecular response may be achieved and a cure is possible. Advanced disease may be treated using hydroxyurea or busulphan. Splenectomy may improve thrombocytopenia and combination chemotherapy may be used. Radiotherapy may be used to treat pain and resistent splenomegaly. Blast
crises is the terminal stage of CML. In Sweden, the state is treated
using mitoxatrone, etoposide, and cytarabine. 56% of patients enter
a second chronic phase with median survival of six months. This second
phase may be slightly prolonged using an autologous stem cell transplant
(occasionally long-term remission may be maintained1. Myeloid transformation
is treated using the drugs used for AML and this results in 30% of patients
entering a chronic phase for months or years. In the case of lymphoid
tranformations, treatment is started using drugs used for adult acute
lymphoblastic leukaemia. 40-60% of this group enter a second chronic
phase and are given craniospinal prophylaxis with five or six sessions
of intrathecal methotrexate. This is currently the only opportunity for cure in CML, and if cure is not possible it increases survival. 60 % of patients are cured. The allogeneic stem cell transplant is performed after a HLA-matched sibling is identified. There is increased mortality associated with the procedure in the first year. The cause of mortality associated with the procedure are: graft versus host disease (GVHD), typical and opportunistic infections, bleeding complications, veno-occlusive diseases of the liver, and pneumonitis. Mortality has been reduced in recent years as a result of better HLA typing techniques, altered radiation schedules, screening for CMV, better antiviral and antimicrobial therapies, and better GVHD prophylaxis. Only initial chemotherapy followed by allogeneic stem cell transplant can irradicate Ph+ cells, but despite this relapses still occur. The German CML trial compared allogeneic stem cell transplant with hydroxyurea and IFa. The drug treatments had advantages in the first four years following diagnosis, after 5.5 years that the transplant had the advantage. This transplant allows use of very high dose chemotherapy and radiotherapy to kill all CML cells and to allow sufficient immunosuppression for the donor marrow to engraft and produce a graft versus leukaemia effect (GVL). In the 1950s, it was discovered in experiments that patients with AML that received a syngeneic transplant relapsed. Only 30% of patients will have a matching sibling, and so other treatments may be necessary instead. The patients who are 40-60 years old have poorer outcome with this treatment. The optimal timing for bone marrow transplant (BMT) is within one year of diagnosis in the chronic phase. Poor prognostic indicators are: older age, prior busulfan treatment, a male recipient with a female donor, considerable GVHD, and an increased time interval between diagnosis and transplant. Relapse is still a major cause of treatment failure. The only cure in this situation is a second BMT which has only a 25% chance of producing long term remission of the disease. A safer alternative is to infuse donor T cells to induce a GVL effect, as was successfully demonstrated by H. Kolb in 1990. Remission rates were found to be 60-80% with the majority showing no Ph+ cells by PCR. The logical next step is to pose the question - why not use donor T cells as a primary treatment instead of a BMT. The reason is that there is no immunosuppression and a relatively normal immune system is likely to reject the cells before it is possible to initiate a GVL reaction. Therefore, trials are underway to use low dose immunosuppressive chemotherapy in this setting. Matched Unrelated Donor Transplant These transplants may have an increased long term survival profile, but also have increased mortality, in comparison to allogeneic transplants. This is due to an increase in late infections, graft failure and GVHD. 10% relapse rate occurs in those patients that survive in excess of five years. Relapse is decreased by an increase in GVHD as this is associated with a GVL. The GVL effect is used in order to induce complete molecular remission and this may be performed by using donor leukocyte infusions in early relapses, should they occur after transplant. The complications of such treatment must be balanced - GVHD and graft failure increases. The balance must also be borne in mind when depleting a graft of T-cells. T-cells implicated in GVHD and GVL and the number required to produce GVL without significant GVHD is currently unknown. It has been found, however, that remission is possible with a low dose (1x107kg) of donor T cells. Complications of GVHD include skin, liver and intestinal effects. Autologous transplant In the 1970s, Spiers and his colleagues, at the Hammersmith Hospital, London, postulated that they could harvest chronic phase stem cells found at diagnosis. Then, these would be stored in liquid nitrogen and when progression occurred chemotherapy would produce remission and the chronic phase cells would be reinfused. The aim was to have some sort of 'perpetual reinduction of the chronic phase of the disease. This does not work because the disease becomes resistant to chemotherapy. To have any chance for cure it would be necessary to institute cytoreductive treatment eg. transplant conditions must be achieved with busulphan, cyclophosphamide and total body irradiation and even then GVL is required. In chronic phase CML, a transplant, unpurged of Ph+ cells, produces recovery in 75% of patients with a proportion of Ph- cells. Benign and malignant progenitor cell coexist in the bone marrow allowing for Ph - cells to be maintained for a long time. A study of 200 patients conducted in eight separate centres showed five year survival rates of 50% 1. There is no prospect for cure. The majority of those who receive the transplant have a haemolytic or cytogenetic relapse. The alternative is a purged autograft. Early relapse after an autologous transplant is probably due to reinfusion of Ph+ progenitor cell contamination of the graft. This theory is supported by the fact that the time to relapse is shortened in identical twin transplants and sibling T-cell depleted transplants where the GVL effect does not occur. As a result it is recommended to use a purged transplant for patients in current practice. It is possible to use IFa and collection of Ph - progenitor cells from blood or bone marrow after intensive chemotherapy. A Scandinavian study showed that some patients achieved cytogenetic remission of in excess of eight years in a group of patients who were less than 55 years old. These patients were treated with high dose IFa and intensive chemotherapy and then had harvest and transplant of stem cells. Sibling allografts or autotransplant were completed in 213 patients and these had 92 month median survival as opposed to 42 months for matched controls1. Idarubicin and cytarabine may be given with subsequent peripheral stem cell collection. The cells collected, however, appear to be of poor quality and the number of colony forming units an CD34+ cells are low. Alternatively, it is possible to mobilise Ph- progenitor cells using recombinant G-CSF alone in patients treated with interferon. The mortality rate is 5%. There is no GVHD and no GVL. Ex vivo purging is possible using: gamma-interferon and mafosfamide, ex vivo marrow culture, and antisense oligodeoxynucleotide against bcr/abl or c-myb. Autografting
should be considered when there is no compatible sibling, the patient
is too old for a sibling autograft, but fit enough for intensive chemotherapy
(i.e.all those over 60 years who are fit and some between 50-60 years)
6. Linomide is currently undergoing research as to its simulation of GVL mechanisms. A study, using this therapy in minimal residual disease, found that it might increase the time patients remained Ph - and reduce the number of tumour cells. Antisense oligonucleotides have the bcr/abl gene as their target. Transcription of bcr/abl would be reduced. This is thought to have applications in ex vivo purging of autologous stem cell transplants. Better vectors and delivery of the treatment will be necessary before this becomes a feasible option. Bacterial superantigens (SAgs) are involved in the pathophysiology of food poisoning and gram positive septic shock. These SAgs are related in structure to proteins which are able to activate a large proportion of T lymphocytes. An example are staphylococcal enterotoxins (SEs) which stimulate 10-15% of human T cells which express a T cell receptor sequence - Vb. Part of the SE molecule has a high affinity for the MHC class II and is presented to T-cells as unprocessed antigen on the exterior of the peptide binding cleft. In response to this, CD8+ and CD4+ proliferate and produce cytokines such as IFgamma, IL2, TNF. They also have a powerful effect of direct cytotoxicity. T-cells, directed using SE, lyse HLA DR+ tumour targets cells, but also lyse normal cells which are HLA-DR+, such as activated T cells, B cells, and monocytes. It is possible to raise monoclonal antibodies (mAbs) against myeloid surface antigens such as CD13, CD15 and CD33. Subsequently, T-cells may be directed by SAgs to lyse leukaemic cells which are antigen positive - their natural specificity for HLA-DR+ cells is deleted and specificity to myeloid cells is introduced by tumour reactive mAbs. SE molecules are then fused to protein A, and so the fusion product so formed will bind in turn to the Fc portion of IgG monoclonal antibodies. The next step is to try this therapy in vivo using a recombinant SAg-mAb fusion protein, although it has already been found that AML cell lines are more sensitive than CML in this setting3. Homoharringtonine, a plant alkaloid found in the chinese tree Cephalotaxis harringtonia shows promise in the treatment of chronic, late stage and refractory phases of CML. In a 71 patient study of late chronic phase disease, there was complete haematological response in 72% of patients, and 22% showed a major cytogenetic response. The advantage of this substance was its comparative lack of side-effects - only nausea, vomiting, diarrhoea and myelosuppression were apparent. Selection of benign progenitors and expansion ex vivo for reinfusion is another possibility. Bone marrow mononuclear cells from CML patients have a primitive population of progenitor cells which are Ph- and Ph+ cells with a long term culture initiating capability. The CD34+DR- fraction of resting bone marrow is rich in Ph- progenitors. It is possible to identify and select benign progenitors by expression of surface antigens. Early in the disease, it may be possible to isolate enough progenitors for transplant. Selection may be achieved using the differential responses in culture of normal and malignant progenitor cells to MIP-1a and stem cell factor. Tyrosine kinase inhibitors may be used, as the abnormal bcr/abl gene product has the effect of increasing tyrosine kinase activity over the wild type c-abl. Specific tyrosine kinase inhibitors have been identified which selectively inhibit, in vitro, the proliferation of CFU-GM - the CML progenitors. This approach could also be extended to purging grafts ex vivo. Gene therapy may allow retroviral transduction of normal Ph- cells with drug resistance genes so that selective therapy may be applied after an autologous transplant. This would spare the patient the progression which normally follows such transplants by destroying all the Ph+ cells. Immunomodulation is possible using T-cells. The p210 bcr/abl fusion region and peptide binding to MHC class I alleles has been demonstrated. It has been possible to generate peptide specific CD4 T-cell lines that recognise bcr/abl expressing cells. This in turn means that therapy may be possible using T-cell mediated recognition of tumour associated antigens. IL
2 activated natural killer cells suppress progenitor growth from CML
and leave normal marrow unaffected. This has applications in ex vivo
expansion, in vivo immunotherapy, and in vitro purging. It is also possible
to use dendritic cells which are generated in culture, from a patient
with CML, to simulate autologous T-cells to produce anti-proliferative
and cytotoxic effects against CML cells, whilst maintaining low levels
of activity against normal bone marrow cells. Conclusion There are many varieties of treatment for patients who present with CML. Unfortunately, only the allogenic stem cell transplant offers a chance of cure in these patients - and this is associated with the best outcome in those with matched siblings - this group comprises only 30%. There is much hope in this field however, with better understanding of the molecular genetics, more sensitive testing to detect disease early, and a multitude of new treatments on the horizon from workers worldwide - the future seems to be bright. References 1.
Simonsson B. 2.
Goldman J. 3.
Totterman TH, et al. 4.
Goldman JM. 6.
Singer IO, et al. 7.
Enright H, et al. 8.
Quaglino D, et al. 9.
Cross NC. 10.
Richards SM. 11.
Verfaillie CM. |
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