The BCR-ABL1 Gene


CML Pathogenesis-600pxBCR-ABL1 is a fusion gene formed as a result of the t(9;22)(q34;q11) chromosomal translocation, the translocation that results in the formation of the Philadelphia chromosome. The Abelson murine leukaemia viral oncogene homolog 1 (ABL1) gene from 9q34 is translocated downstream to a region at 22q11 known as breakpoint cluster (BCR). The fusion gene encodes for a constitutionally active tyrosine kinase that has been shown to drive the expression chronic myeloid leukaemia phenotype. BCR-ABL1 gene has also been on implicated in the pathogens is of acute lymphoblastic leukaemia and in rare cases of acute myeloid leukaemia. The gene has been targeted with unparalleled success by the first tyrosine kinase inhibitor approved in clinical practice, imatinib.

 Molecular Biology of BCR-ABL

The ABL1 proto-oncogene is located on chromosome 9 at q34. Chromosome 22 has the BCR gene at 22q11. The ABL1 gene translocated downstream to the BCR gene as a result of the t(9;22)(q34;q11) translocation. ABL1-BCR translocation also occurs and may express but is of no clinical significance.

 

Molecular Biology of CML

The BCR-ABL1 fusion gene and it’s variants

The breakpoint of the ABL1 gene may be upstream exon 1a, between exon 1a and 1b or downstream exon 1b but it is almost always upstream exon 2. With rare exceptions all transcripts of BCR1-ABL1 gene have exon 2-11 of the ABL1 gene. The BCR breakpoints are variable and determine the size as well as the pathogenic properties of the BCR-ABL1 gene.. The breakpoint on the BCR gene are clustered in three regions known major cluster, minor cluster and micro cluster (Table 1). Depending on the location of breakpoint  on the BCR gene three types of protein are synthesized. The p210 transcript is associated with CML and some patients with Philadelphia positive acute lymphoblastic leukaemia. The shorter p190 transcript is associated with philedelphia positive acute lymphoblastic leukaemia and some patients of chronic myeloid leukaemia. The CML that carry this mutation show monocytosis and have a more aggressive course. The p230 is the largest and the rarest of the BCR-ABL1 transcripts. It is associated with a more indolent course and is found in patients with the rare chronic neutrophilic leukaemia. Atypical transcripts e1a3, e13a3 and e6a2 have been described.

Table 1: The BCR-ABL1 fusion genes

 Major Cluster  Minor Cluster  Micro-Cluster
 Synonym M-Cluster m-cluster µ-cluster
Location exons 12-16 Between alternative exon 2, e2’ and e2 between exons e19 and e20
Protein p210 p190 p230
Associated Leukaemia  CML, e14a2 shown to have thrombocytosis in some studies., Ph+ ALL  Ph + ALL; CML that tends to have monocytosis and an agressive course  Chronic Neutrophilic Leukaemia, Small reports describing patients with a course resembling classical CML

Mutagenicity of BCR-ABL1

ABL1 is a nuclear kinase whose activity is tightly regulated by the cell. BCR-ABL1 translocation results loss of regulation and the kinase is  cosntitutively active. Sustitution of ABL1 at the N termnal by segments of the BCR gene result in the synthesis of a protein that has the capacity to dimerise. Dimerisation transphosphorylates and then aurtophsophorylates the the kinase fully activating it. The precise mechanism how BCR-ABL1 leads to chronic myeloid leukaemia is not known but activation of  phosphatidylinositol kinase, RAS/Mitogen activated protein kinase and JAK/STAT pathway has been demonstrated in BCR-ABL1 positive cells. These pathways are involved in cellular growth and differentiation. The BCR-ABL1 kinase also phosphorylates proteins involved in adhesion and migration and this may have a role in premature release of myeloid cells in circulation. CML cells have a two to sixfold increase in reactive oxygen species and have impaired DNA repair. Reactive oxygen species can induce DNA double strand breaks. The results is additional mutations and these are believed to be responsible for blast crisis and acclerates phase.

Tyrosine kinase inhibitors targeting the BCR-ABL1 protein induce a remission in most patients of CML. About half the patients who have achieved sustained complete molecular response relapse on discontinuation of the tyrosine kinase inhibitors. This suggests that the stemat least some CML stem cells are not BCR-ABL1 dependent for growth. Experimental observations support this hypothesis.

Targeting the BCR-ABL1 Gene

The BCR-ABL1 gene was the first gene to be targeted by a tyrosine kinase inhibitor, imatinib. Imatinib was followed by dasatinib, nilotinib, Busotinib and Panotinob. Imatanib, Dasatinib and Nilotinib are approved to first line use. Imatinib has resulted in a 85% 8 year survival. Dasatinib and nolitinib are active in imatinib resistant CML and are now approved for first line use. Drug resistance results from mutations in BCR-ABL1 kinase. The T315I mutation or the gatekeeper mutations impaires access of TKIs to the BCR-ABL1 kinase making most drugs inactive. Panotinib can inhibit the T315I mutation.

 

Further Reading

Barnes DJ Melo JV. Molecular Basis of Chronic Myleoid Leukaemia. In Chronic Myeloproliferative Disorders: Cytogenetic and Molecular Anomalies. Bain Barbra J (Ed) 2003.

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Tumour Lysis Syndrome


Rapid tumour lysis can release harmful metabolites at a rate that exceeds the kidney’s excretory capacity resulting in a metabolic syndrome known as tumour lysis syndrome (TLS). TLS is a complex of hyperkalaemia, hyperuricaemia, hypocalcaemia, hyperphosphatamia and lactic acidosis. It is most commonly seen in high-grade malignancies (leukaemia, high grade lymphomas and testicular tumours), usually following therapy but occasionally spontaneously.

 

Definition of TLS

Biochemical changes precede clinical manifestations of TLS. TLS is classified into laboratory TLS (LTSL) or clinical TLS (CTLS). The diagnosis of LTLS requires an abnormality in two or more of the following, occurring within 3 days before or 7 days after initiation of chemotherapy:

  • Uric acid >8 mg/dL or 25% increase from baseline
  • Potassium >6 meq/L or 25% increase from baseline
  • Phosphate >4.5 mg/dL or 25% increase from baseline
  • Calcium <7 mg/dL or 25% decrease from baseline

Clinical TLS is defined as LTLS plus one or more of the following:

  • Increased serum creatinine (1.5 times upper limit of normal)
  • Cardiac arrhythmia or sudden death
  • Seizure

 

Aetiology of TLS

TLS is seen when the renal capacity to excrete products of tumour lysis is overwhelmed. This may happen under the following circumstances:

  1. The metabolites are released too fast as may be seen with tumours that are very sensitive to chemotherapy.
  2. The metabolites are released in a large amount as may happen with a large tumour.
  3. The kidney function is compromised.

The common tumours associated with tumour lysis syndrome are lymphoid malignancies (Burkitt’s lymphoma and other high grade lymphomas, acute lymphoblastic leukaemia) and other acute leukaemias. These tumours have a high proliferation rate and are very sensitive to chemotherapy. As leukaemias do not present with a tumour the malignant cell load in leukaemias is not fully appreciated. The bone marrow is about 4% the body mass. A 72kg patient who has 30% blasts would have about one kg of tumour in the bone marrow. Lymphoid neoplasia also involve the spleen and liver and have a greater cell load than myeloid leukaemia. Lymphoid leukaemias are also more sensitive to chemotherapy than myeloid leukaemias and undergo a more rapid lysis. This makes patients of high grade lymphoid malignancy most prone to TLS.

TLS is rare but has been described in solid tumours like testicular tumours and small cell lung cancers, breast carcinoma and neuroblastomas. Usually low grade malignancies like chronic lymphocytic leukaemia and other low grade lymphomas are not associated with TLS because they do not undergo rapid lysis with therapy.  There have been reports of patients with malignancies that have a negligible risk of TLS on conventional chemotherapy developing TLS when treated with purine analogues like fludarabine and cladribine and with targeted agents. These include:

  1. Treatment of chronic lymphocytic leukaemia and low grade lymphoma with fludarabine
  2. Refractory chronic lymphocytic leukaemia treated with cladribine
  3. Multiple myeloma treated with bortezomib
  4. Chronic myelocytic leukaemia treated with imatinib.
  5. Gastrointestinal stromal tumour  treated with imatinib (Sarcoma. 2007;2007:82012)
  6. Renal Cell carcinoma treated with sunitinib (Invest New Drugs. 2010 Oct;28(5):690-3).

All the above situations are characterised by a rapid tumour response accompanied by a  release of metabolites at a rate the kidneys can not cope up with.

One needs to keep the possibility of TLS in every patient with a large tumour and/or a chemosensitive tumour treated with very active drugs. Many of these patients present with non-specific manifestations.

TLS Pathogenesis

Pathogenesis of Tumour Lysis Syndrome

 

Pathogenesis

Cell lysis releases intracellular contents including potassium, phosphates, purines and lactic acid. Purines are metabolised to uric acid by a pathway needing xanthine oxidase. Uric acid precipitated in the renal tubules causing renal failure. Phosphate released by cells chelates calcium causing hypocalcaemia. Precipitations of calcium phosphate in the kidney adds to the renal failure caused by precipitation of uric acid. Hypocalcaemia causes tetany, muscle cramps, rarely seizures and enhances the arrhythmogenic effect of hyperkalaemia. Lactic acid release causes acidosis. Hyperkalaemia resulting from release of intracellular potassium causes arrhythmias and muscle cramps. Renal failure increases hyperkalaemia and hypocalcaemia enhances the arrhythmogenic effect of hyperkalaemia.

 

Clinical Features

Many patients are diagnosed with only laboratory TLS on tests done as a part of initial evaluation. The symptoms when present are non-specific and include nausea, vomiting, confusion, oliguriua, bronchospasm and seizures. Patients at increased risk of developing TLS include

  1. High volume disease: Patines with high blood counts and/or pronounced organomegaly or elevated LDH are more prone to TLS.
  2. Patients with pretreatment elevation of uric acid
  3. Patients with compromised renal function

 

Treatment

Established TLS is a serious disease. The possibility of TLS must be considered in every patients with high volume disease and preventive measures instituted. These include

  1. Hydration with about 3000ml/day should be started 24 hours before starting chemotherapy. Alkalinization of urine to promote ionisation of uric acid and improve it’s solubility is controversial. The pKa of uric acid is 5.4. About 90% of uric acid exists as sodium urate at a pH of 6.5. Increasing the pH any further will not increase solubility significantly. On the other hand calcium phosphate becomes less soluble with increasing pH. Alkalinization can cause precipitation of calcium phosphate in the tubules. Alkalinization can also worsen hypocalcaemia.
  2. Measure to reduce uric acid: Purines are converted to xanthine. Xanthine is converted to uric acid by xanthine oxidase. Allopurinol inhibits xanthine oxidase and prevents conversion of xanthine to uric acid. Xanthine is more soluble than uric acid and does not precipitate. Allopurinol prevents formation of uric acid. It has no effect on the elimination of uric acid. Urate oxidase converts uric acid to soluble metabolites and may be used in patients who do not respond to allopurinol or those who thought to be at high risk of TLS.
  3. Delaying chemotherapy in patients to give time for correction of defects where possible
  4. Close monitoring of patients early in induction therapy for metabolic defects and instituting therapy to correct these defects at the earliest.

The management of established TLS is the treatment of the metabolic anomalies. Hyperkalaemia is treated with

  1. Potassium restriction
  2. Aggressive hydration with the use of loop diuretics if needed to promote elimination of potassium
  3. Use of cataion exchange resins to decrease absorption of potassium
  4. Other measures include use of calcium gluconate, sodium bicarbonate, glucose-insulin drip, beta-2 agonist aerosols and dialysis.

Hyperphosphataemia is treated with aggressive hydration, phosphate restriction, oral phosphate binders and dialysis if necessary.

Hyperuricaemia is treated with allopurinol, urate oxidase and hydration.

Hyperleukocytosis and Leukostasis


Leukostasis is a oncological emergency seen in patients with leukaemia who present with pronounced leucocytosis. It is seen in about 5-30% of adult acute leukaemia cases. It results from slugging of microcirculation by leucocytes.  It is associated with a mortality of 20-40%.

Pathogenesis of Leukostasis

Manifestations of leukostasis result from impaired circulation in the affected vascular bed. Leukocytosis impairs circulation because of

  1. Increased viscosity
  2. Formation of intravascular leukocyte aggregates (white bland thrombi)
  3. Increased adhesion of blasts to the endothelium

Cells increase the viscosity of blood hampering flow through microcirculation. Pliability of blood cells is important for maintaining blood flow in microvasculature. The biconcave shape of the erythrocytes provides them with deformability allowing smooth passage through the microvasculature. Leukocytes are less pliable than erythrocytes.  Normally the number of leucocytes is a small fraction of the number of erythrocytes. The contribution of leucocytes to blood viscosity in minimal.

Blood viscosity increases with leucocytosis. The increase is related to the leucocyte count, size of the leukocytes and the deformability of the leucocytes. Blasts are less deformable than mature cells. Myeloblasts are larger and less deformable than lymphoblasts. Cells of the monoblastic series are the least deformable. Lymphocytes are the smallest and have the least impact on the viscosity amongst all leucocytes.

Blasts secrete cytokines like IL-1β  and TNF-β. These lead to up regulation of adhesion molecules like ICAM-1, VCAM-1 and E-selectin that increases the adhesion of blasts to the endothelium. Adhesions of blasts to microvasculature further diminish blood flow.

The vascular beds most commonly affected are the lung, CNS and the eye. Tissue hypoxia resulting from impaired circulation is believed to contribute to elevated LDH seen in acute leukaemias. 

Clinical Features

About 5-13% of acute myeloid leukaemia and 10-30% of acute lymphoblastic leukaemia have hyperleukocytosis. The symptoms of leukostasis are related to the ischaemia in the affected circulatory bed. The commonest vascular beds affected and the symptoms attributable to these beds are listed below.

Organ Manifestation
Brain Stupor
Eyes Blurring of vision
Lungs Dyspnoea
Kidney Azoaemia
Heart Arrhythmia
Penis Priapsim

Examination of the fundus shows papilledema, blurred disc margins, dilated blood vessels, and retinal haemorrhages.

The leukocyte count  at which symptoms develop depends on the type of leukaemia. Symptoms develop at the lowest counts in acute myeloid leukaemia. Patients of chronic lymphocytic leukaemia may not have symptoms of hyperleukocytosis at leucocyte counts as high as 400X109/L. Leukostasis is associated with increased morbidity and mortality.

Diagnosis

Leukostasis is clinical diagnosis of exclusion. It should be considered in any patients with lung or CNS symptoms who has hyperleukocyosis. Hyperleukocytosis has been variably defined as a count of 50X109/L or 100X109/L. Symptoms are likely to occur at lower counts in patients with acute myeloblastic leukaemia particularly when there is a monocytoid component. Patients with chronic lymphoblastic leukaemia tolerate counts as high as 400X109/L without symptoms. The conditions that can mimic leukostasis include

  1. Pulmonary infection
  2. Pulmonary embolism
  3. Pulmonary oedema
  4. Pulmonary haemorrhage
  5. Transfusion-related acute lung injury  is blood products have been transfused
  6. CNS infections – meningitis and encephalitis
  7. Conditions causing acute mental status change

The x-ray findings include diffuse interstitial or alveolar infiltrates. It can be normal in early stages. Examination of the fundus is important.

Treatment

Leukostasis is associated with a mortality of 20-40%. The treatment of leukostatsis is to rapidly reduce the leukocyte count. Three methods of rapidly reducing leucocyte counts are induction chemotherapy, leukocytopheresis or low dose chemotherapy. Each of these methods are supported by theoretical arguments. Induction chemotherapy is the definitive therapy for leukaemia. Whether leukocytopheresis or hydroxyurea add to the benefit of induction chemotherapy is not clear. The use of hydroxyurea and leukocytopheresis is dictated by the experience of the treating centre. The procedures are usually resorted to with the belief that induction therapy with a very high leucocyte count may increase the risk of tumour lysis. 

Leukocytopheresis: Leukocytopheresis is used because it rapidly brings down the leucocyte count without causing lysis of blasts. It has the theoretical advantage of reducing the risk of tumour lysis and reducing mortality. This has never been proven in clinical trials. Two procedures needed about 12-24 hour apart. Leukocytopheresis is indicated in

  1. Symptomatic patients with AML with leucocyte counts more than 50X109/L and ALL with counts more than 150X109/L.
  2. Asymptomatic patients with AML and leucocyte  counts >100 X 109/L
  3. Asymptomatic patients with ALL with leucocyte counts >300 X 109/L
  4. CML patients who are symptomatic with leucocyte counts greater than 150X109/L,
  5. CLL patients who are symptomatic with leucocyte counts greater than 500X109/L. 
  6. It should not be performed in patients with acute promyelocytic leukaemia symptomatic or asymtomatic

The procedure involves insertion of a catheter. This may be associated with an increased risk of bleeding as these patients have thrombocytopenia.

Low Dose Chemotherapy: Like leukocytopheresis the value of low dose chemotherapy has not been proven. The following interventions may be used (with leukocytopheresis)

  1. Acute myeloid leukaemia should be treated with hydroxyurea in a dose of 50-100mg/Kg. It may be administered as a single or multiple doses.
  2. Acute lymphoblastic leukaemia may be treated with steroids with or without vincristine. 

Induction chemotherapy: Induction chemotherapy is the definitive therapy for acute leukaemia. High counts are associated with a higher risk of tumour lysis resulting in an apprehensions of starting chemotherapy in patients with very high leucocyte counts.

Other measures: Cranial irradiation and dexamethasone has been used in patients with CNS symptoms. Blood transfusions can increase viscosity and may worsen symptoms of leukostasis. One needs to be conservative about red cell transfusions till the leukocyte counts become normal. Transfusions in patients with symptoms attributable to anaemia should not be held back. 

 

Leukaemia – The Peripheral Smear


Differentiating Acute and Chronic Leukaemia

Leucocytosis with anaemia is a feature of acute and chronic leukaemia. It is possible to differentiate acute and chronic leukaemia by looking at the peripheral smear. Patients with acute leukaemia often have thrombocytopenia. Patients with chronic lymphocytic leukaemia may have normal or low platelet counts. Patients with chronic myeloid leukaemia have normal or high platelet counts.

Haemoglobin Platelets Peripheral Smear
Acute Leukaemia Low Low Immature forms other than blasts not seen
Chronic Lymphocytic Leukaemia Low or Normal Low or Normal Normal looking lymphocytes
Chronic Myeloid Leukaemia Low or Normal Normal or High Immature leucocytes, all phases of leucocyte maturation seen

The phases of maturation of myeloid cells (from the least to the msot mature) are blasts, promyelocytes, myelocytes, metamyelocytes, band form and mature granulocyte (see Myeloid Precursors Morphlogy). The peripheral smear from patients with acute leukaemia shows blasts (or promyelocytes) and mature neutrophils. Very few cells, if any, with maturity between the two stages that occupy two ends of the spectrum are seen. Patients with with chronic myeloid leukaemia have cells with all stages of maturity between blasts and mature granulocytes. The peripheral smear in patients with acute leukaemia shows mature lymphocytes.

The explanation for the different peripheral smear findings in acute leukaemia and chronic myeloid leukaemia is in the pathogenesis of the two diseases. Chronic myeloid is a myeloprolferative disease. It is a clonal disease. The stem cells of patients with chronic myeloid leukaemia carry the BCR-ABL mutation. This mutation results in clonal expansion. All blood cells in a patient arise from one clone. The release of cells from the bone marrow of patients with CML is not limited to mature granulocytes. Some cells leave the marrow and result in leucocytosis.

The marrow of a patient of acute leukaemia has two clone one malignant one normal. The malignant clone can not differentiate beyond the stage of a blast (or promyelocyte). It slowly effaces the normal clone. The mature granulocytes seen in the peripheral smear arise from the normal clone and the blasts from the malignant clone. The normal clone releases cells only when they mature to the stage of band cell or beyond. The malignant clone can not mature beyond the stage of a blast. The stages between blasts and band forms/mature granulocytes are not seen in peripheral smear of acute leukaemia.

Cluster of Differentiation


Table 1 Cluster of differentiation was proposed as a system to classify monoclonal antibodies. The antibodies, their cluster of differentiation and their linage is listed in the table above

The technique for production of monoclonal antibodies invented by Cesar Milstein and Georges J. F. Köhler in 1975 allowed production of antibodies that could identify an array of antigens on leucocyte surfaces. The monoclonal antibodies gave insights into the process of maturation of leucocytes allowing recognition of stages of differentiation that were not evident by morphology-based methods. Immunophenotyping, the classification cells according to antigens they express, has highlighted the inhomogeneity of morphological subtypes of leukaemia and lymphomas. This has allowed recognition of new disease entities and development of specific management protocols for some of these. Today the impact of immunophenotyping is seen across oncopathology. Few diagnoses are made without the use of immunophenotyping. The period few years after the development of the first anti-leucocyte monoclonals was a period of confusion. In a reflection of the ease of mastering the hybridoma technology, the number of monoclonal antibodies proliferated resulting in chaos about the identity of antigen they characterized (table 1). The CD4 antigen was recognized by the OKT4 (Ortho Diagnostics) and the Leu-3 (Becton Dickson) monoclonal antibodies. It was known as the Leu 3 and T4 antigen. The cluster of differentiation nomenclature was proposed at the 1st International Workshop and Conference on Human Leukocyte Differentiation Antigens at Paris in 1982 as a system of classification of monoclonal antibodies directed against leucocyte surface antigens. Following adoption of the cluster of differentiation nomenclature OKT4 and Leu3 would be called anti-CD4 antibodies, as would any new antibody directed against the same antigen. The antigen defined by these is referred to as CD4. Over 200 antigens are now recognized. Table 2 gives the lineage specificity of some markers. Some CD antigens are designated with suffix ‘w’, e.g. CDw12. These are antigens that have been identified by only one antibody or by up to two antibodies generated in the same lab (Bull. WHO 1994; 72:807-808).

Table 2 Some commonly used lineage specific markers</h5.

The complete list of CD antigens is available at the Human Cell Differentiation Molecules (HCDM) site.

Auer Rods


Auer rods are needle shaped azurophilic intracytoplasmic inclusion bodies described by John Auer in 1906. They are 0.1-2μ wide and 3-6μ long and are formed by fusion of lysosomes. They contain peroxidase and lysosomal enzymes. They are seen in acute leukaemia (myeloblastic, promyelocytic, monomyelocytic and monocytic), myelodysplastic syndromes (RAEB-2) and chronic monomyeloid leukemia. Myelodysplastic syndromes, according to the WHO classification are disorders cytopenias and <20% blasts in the bone marrow. Patients with 10-19% blasts are classified as RAEB–2. Documenting Auer rods is important in examination of bone marrow smears of patients suspected to have MDS as a patient is classified as RAEB-2 even with <10% blast if Auer rods are present. Of the Romanovsky stains there are reports of Leishman’s stain being suboptimal for staining for Auer rods. The prognosis of patients with myelodysplastic syndromes worsens with increasing blasts. Patients with Auer rods have a worse prognosis than similar patients without Auer rods (Am J Clin Pathol 124:191; 2005). A lymphoid leukaemia can be excluded in patients with Auer rods.