Anaemia with Thrombocytosis


A 49 year old woman presented with weakness and fatigability. On examination, other than pallor of the skin and mucosa there was no other finding.  A haemogram was done that showed a haemoglobin: 6.7g/dL, leucocyte count: 9.1 X 109/L and a platelet: 664 X 109/L.

Anaemia and thrombocytopenia is a feature of

  1. Myeloproloferative neoplasm
  2. Chronic inflammation
  3. Underlying malignancy (Paraneoplastic)
  4. Iron deficiency

A haemogram from a automated haematological counter hides a lot of information. Before more investigations are done it is important to assimilate all the information in the haemogram. The haemogram in the above listed conditions shows:

  1. Myeloproliferative Neoplasm: The myeloproliferative neoplasm are diseases characterised by proliferation of bone marrow. They are distinct from acute leukaemia. According to the 2016 WHO classification myeloproliferative neoplasm include chronic myeloid leukaemia (CML), chronic neutrophilic leukaemia (CNL), polycythaemia vera (PV), progressive myelofibrosis (PMF), essential thrombocytosis (ET), chronic myeloproliferative neoplasm unclassified (CMPN-U) and chronic eosinophilic leukaemia (CEL).  Myeloproliferative diseases associated with anaemia and thrombocytosis are CML, prefibrotic phase of PMF and CMPN-U. Haemogram of patients with myeloproliferative diseases shows leucocytosis with presence of immature leucocyte forms. This is most pronounced in patients with CML. In fact leucocytosis with the presence of immature leucocyte forms is the dominant feature of the haemogram of patients with CML. Anaemia of myloproliferative is normocytic and normochromic.
  2. Chronic Inflammation and Paraneoplastic Diseases: Anaemia and thrombocytosis can also be seen in patients with chronic inflammation and as a paraneoplastic finding. An occasional immature leucocyte form may also be seen in these conditions. This picture may be indistinguishable from that myeloproliferative diseases other than CML. The anaemia is normocytic and normochromic. When the chronic disease or neoplasm is associated with blood loss as may be the case in cancers of the gastrointestinal tract or inflammatory bowel disease, microcytosis due to a coexisting iron deficiency may be seen.
  3. Iron Deficiency: Iron deficiency is associated with microcytic hypochromic anaemia. The degree of microcytosis co-relates with the degree of iron deficiency. The leucocyte counts depends on the cause of iron deficiency. Commonly the leucocyte is normal or slightly decreased. Patients who have iron deficiency because of blood loss due to an inflammatory condition may have leucocytosis. Iron deficiency from blood loss due to helmethiasis may cause eosinophilia.

The differential leucocyte count showed 67% polymorphs, 27% lymphocytes, 2% monocytes and 4% basophils. The erythrocyte indices were MCV 61fl, MCH 15.3pg and MCHC 24.9g/dL. The red cell distribution width was 28.5%. The peripheral smear showed hypochromia, microcytosis, anisocytosis and poikilocytosis.

Of the causes of anaemia and thrombocytosis listed above only iron deficiency is characterised by hypochromic microcytic anaemia. Iron deficiency is also characterised by anisocytosis and poikilocytosis. This manifests as increased red cell distribution on the haemogram.

The serum iron was 27.9 µg/dl, the total iron binding capacity 488 µg/dl with a transferrin saturation 5.7%. The serum ferritin was 8.53 ng/ml. The haemoglobin electrophoresis showed a haemoglobin A2 of 2.8%, the HbA 96% and haemoglobin F 1.2%.

Iron deficiency is diagnosed by documenting low body iron stores and/or impaired iron delivery of iron to the erythroid precursors. The gold standard for depletion of iron stores is absence of stainable iron in the bone marrow. Serum ferritin accurately reflects body iron stores. It has become the preferred method to demonstrate depletion of body iron stores because of the invasive nature of bone marrow aspiration. Levels less than 15ng/ml strongly suggest iron deficiency. Serum ferritin is specific but not sensitive for iron deficiency. Its has a sensitivity of 59% if the cutoff is 15ng/mL and 75% if the cutoff is less than 16ng/ml. The low sensitivity makes the test of limited value to exclude iron deficiency. Ferritin in an acute phase reactant. It has a limited value in the presence of inflammation.

Unlike low serum ferritin, low serum iron is of limited value in diagnosis of iron deficiency. Iron delivery to the haemoglobinizing erythroid precursors is a function of transferrin saturation rather than the serum iron levels. One can have a low serum iron and a low total iron binding capacity as may be seen in anaemia of chronic disease and yet have a normal transferrin saturation. Such patients do not benefit from iron supplementation. Patients with iron deficiency have a low transferrin saturation indicated impaired iron delivery to the developing erythroid cells. Lower the iron saturation higher the probability of iron deficiency being present. Patients are considered to be iron deficient if the transferrin saturation is less than 16%. This patient had a transferrin saturation of 5.7% and a serum ferritin of 8.63ng/ml along with microcytic hypochromic anaemia. A diagnosis of iron deficiency anaemia was made.

The diagnosis of iron deficiency is incomplete without diagnosing the cause of anaemia. Iron deficiency in a 49 year old woman frequently is a result of blood loss that is often menstrual.  This woman had attained menopause and is being evaluated for a gastrointestinal blood loss.

Heme Synthesis


Heme, a porphyrin, is a co-factor in haemoglobin, myoglobin, cytochrome, catalase, heme peroxidase, and endothelial nitric oxide synthase. It has a complex structure with four pyrrole rings with a ferrous iron in the centre that allows it to carry oxygen. The synthesis of heme takes place from glycine and succinyl CoA in eight steps and is extensively studied. Mutations in genes encoding for enzymes involved in heme synthesis result in porphyrias.

Steps in Heme synthesis

About 85% of the heme is synthesised in the developing erythroid cells and almost all the remaining is synthesised in the liver. The control of synthesis differs in erythroid and non-erythroid cells reflecting the exceedingly high heme requirement of the former for haemoglobin synthesis. Heme synthesis takes place in the mitochondria as well as cytosol. The first step, formation of δ-aminolevulenic acid, takes place in the mitochondrial matrix. The next few steps take place in the cytosol. The heme precursor, corpoprophyrinogen III, returns to the mitochondria, is converted to protoporphyrin IX and iron incorporated. The steps in heme synthesis are as follows

  1. Synthesis of δ-aminoleuvelinic acid: Synthesis of δ-aminoleuvelinic acid (ALA) from glycine and succnyl CoA catalysed by ALA synthase (ALAS) is the first step in the synthesis of heme. This is a rate limiting step. ALA synthase is encoded by two genes ALAS1 (OMIM 125290) and ALAS2 (OMIM 301300). ALAS2 codes for the erythroid ALAS and ALAS1 for the non-erythroid (housekeeping) ALAS. The gene ALAS1 is located on chromosome 3p21.1. The product has 12 exons and undergoes is alternate splicing to yield two distinct forms, isoform 1 (640 amino acids) and isoform 2 (657 amino acids). The erythroid specific gene (ALAS2) on X chromosome at Xp11.21. It has 12 exons and also undergo alternate splicing to yield two forms, isoform b (587 amino acids), isoform c (574 amino acids). ALAS is synthesised in the cytosol and transported to the mitochondria. It has a short half life. Heme synthesis is consoled by regulating levels and activity of ALAS (discussed below).
  2. Synthesis of prophobilinogen: ALA moves to the cytosol and is dimerised to prophobilinogen by the action of prophobilinogen synthase (ALA dehydratase). The enzyme is a homo-octomer (made of eight similar units) and needs zinc. The gene (gene ALAD, OMIM 125270) encoding the enzyme is located at 9q32. It has 15 exons. Four isoforms from alternate splicing 361 amino acid, 344 amino acid, 321 amino acid and 304 amino acid are known.
  3. Synthesis of hydroxymethylbilane: Prophobilinogen is converted to hydroxymethylbilane by the action of hydroxymethylbilane synthase. This enzyme is also known as propohbilinogen deaminase. The gene (HMBS OMIM 609806) is located at 11q23.3, has 15 exons. Four alternately spliced forms with 361, 344, 321 and 304 amino acids are known.
  4. Synthesis of uroporphyrinogen: Hydroxymethylbilane is converted to enzymatically to uroporphyrinogen III as well as non-enzymatically to uroporphyrinogen I. The enzymatic conversion is catalysed by the enzyme uroporphyrinogen III synthase. Uroporphyrinogen III synthatase is encoded by a gene (UROS, OMIM, 606938) on 10q25.2-q26 that has 16 exons and encodes for a 265 amino acid protein.
  5. Synthesis of corpoporphyrinogen III: Uroporphyrinogen III is decrboxylated to corpoporphyrinogen III by uroporphyrinogen decarboxylase. The gene (UROD, OMIM 613521) for thes enzyme is at 1p34. It has 10 exons and encodes for a protein 367 amino acid long. This is the last step in the cytosol.
  6. Synthesis of protoporphrinogern IX: Coproporphyrinogen III is converted to propoporphyrinogen IX by a reaction catalysed by corpoporphyrinogen oxidase  in the mitochondria in an oxygen dependent reaction. The gene for corpoporphyrinogen oxidase (COPX, OMIM 612732) is at 3q11.2-q12.1 8 exons. The product has 454 amino acids.
  7. Synthesis of protoporphyrin IX: Propoporphyrin is the final product of the pathway into which iron is incorporated. Protoporphyrin IX is synthesised by the action of protoporphyrinogen oxidase. The gene (PPOX, OMIM 600923) for this enzyme is located at 1q22  and has14 exon. It encodes for a 477 amino acid enzyme.
  8. Synthesis of heme: Ferrochelatse (protoporphyrin ferrochelatase) catalysed the incorporation of iron into protoporphyrin IX. The gene (FECH, OMIM 612386) for ferrochelatse is located at 18q21.31 and has 11 exons. It encodes for a 477 amino acid enzyme.

Control of heme sythesis

The rate limiting enzyme of heme synthesis is the synthesis of ALA. ALA synthase has a short half life. Heme synthesis is regulated  by controlling the levels and activity of ALA synthase.

  1. Inhibition of ALA synthase: ALA synthase is subject to feedback inhibition by heme and and it’s oxidation product hemin. ALA synthase is synthesised in the cytosol and transported to the mitochondrial matrix. In addition to being an inhibitor of ALA synthase hemin also inhibits the metochondrial transport of the enzyme.
  2. Promotion of ALA synthase activity: Cellular iron and factors promoting erythroid differentiation increase the synthesis of ALAS-2, the enzyme responsible ALA synthesis in erythroid cells. Erythroid specific factors like GATA-1 promote the transcription of the ALAS-2 gene. Untranslated portions of the ALAS-2 mRNA have iron responsive elements (IRE) that promote translation. The activity of ASLS in iron deficient cells is low.

Porphyrias

Porphyrias are inherited diseases resulting from a mutation of genes involved in heme synthesis. With one exception, X-linked porphyria that results from a gain of function mutation of ALAS synthase 2, porphyrias result from a partial deficiency of the enzymes involved in heme synthesis. Enzyme deficiency results in accumulation of substrates for the reaction catalysed by the enzyme encoded by the gene. Symptoms of porphyrias may be intermittant and/or chronic. The symptoms are diverse and include skin changes, photosensitivity, abdominal pain, muscle weakness, CNS disturbance, seizures, hyponatremia, discolouration of urine. Enzyme deficiencies associated with porphyrias as as follows:

  1. ALA synthatase 2: Gains of function mutation in X linked protoporphyria
  2. ALA dehydratase: ALA dehydrate deficient porphyria (ADP). Lead displaces zinc from binding sites inhibiting the function of the  with enzyme. In patients with tyrosinaemia type 1 Succinylacetone (4,6-dioxoheptanoic acid) accumulates in tyrosinaemia type I. It is structurally similar to ALA and a potent inhibitor of ALA dehydratase.
  3. PBG Deaminase deficiency results in acute intermittent porphyria
  4. Uroporphyrin III synthatase deficiency results in congenital erythrocytic porphyria
  5. Uroporphyrin decarboxylase deficiency results in porphyria cutanea tarde. All patients with porphyria cutanea trade do not have a mutation. Only type II has gene mutations. Types I and III are due to mulifactorial effects on the gene.
  6. Coproporphyrin III oxidase deficiency results in hereditary coproporphyria
  7. Protoporphyrin oxidase results in varigate porphyria
  8. Ferrochalase results in erythropoietic porphyria

Further Reading

Porphyrin and Heme Metabolism
Erythroid Heme Biosynthesis and Its Disorders (doi:  10.1101/cshperspect.a011676)

 

Drugs and Eosinophilia


Drugs, prescription and non-prescription,  and nutritional supplements are a common cause of eosinophilia across the world. In regions with a low prevalence of parasitic infestations drugs are the leading cause of eosinophilia.

Clinical Spectrum of Drug Induced Eosinophilia

The spectrum of drug induced eosinophilia extends from an asymptomatic eosinophilia discovered on a routine haemogram to a a serious disorder like drug induced drug reaction with eosinophilia and systemic syndromes (DRESS). Eosinophilia associated with specific organ complications includes

  1. Eosinophilic pulmonary infiltrates associated with the use of sulfadsalazine, nitrofurantoin and non-steroidal anti-inflammatory drugs (NSAID)
  2. Acute interstitial nephritis with eosinophilia  associated with the use of semisynthetic penicillins, cephalosporins, NSAID, sulphonamides, phenytoin, cimetidine and allopurinol
  3. Eosinophilia-myalgia syndrome (EMS) presents with increased eosinophil counts associated with  severe myalgia, neuropathy, skin rash and multi-system complications. The cause of EMS is not known but L-tryptophan has been implemented.
  4. Drug reaction with eosinophilia and systemic symptoms /Drug induced hypersensitivity syndrome (DRESS/DIHS): The syndrome is a form of delayed drug hypersensitivity the presents with fever lymphadenopathy and end organ damage. The spectrum of end-organ damage includes hepetitis, interstitial nephritis, pneumonitis and carditis. The drugs implicated in DRESS/DIHS include
    1. Anti-infective
      1. Antibiotics: Cephalosporins, doxycycline, fluoroquinolone, linezolid, metronidazole, nitrofurantoin, penicillins, tetracycline
      2. Sulfomaides: Sulfasalazine trimethoprim-sulfamethoxozole
      3. Sulfones: Dapsone
      4. Antiviral: Abacavir, Nevirapine
    2. Anti-epileptic: Carbamazepine, lamotrigine, phenobarbital, phenytoin, , valproate
    3. Anti-depressants: Amitriptyline, desimipramine, fluoxetine
    4. Anti-inflammatory: Diclofenac, ibuprofen, naproxen, piroxicam
    5. Antihypertensives: ACE inhibitors, β-blockers, hydrochlorthiazide
    6. Others:  Allopurinol, cyclosporine, ranitidine

Management

The incriminating drug should be withdrawn in symptomatic patients. Asymptomatic eosinophilia does not necessitate discontinuation of therapy. If equally effective therapy is available it is preferable to stop therapy. If this is not the case the drug may be continued with careful monitoring for symptoms.

Clinical Features of Megaloblastic Anaemia


Megaloblastic anaemia is a macrocytic anaemia resulting from the deficiency of vitamin B12 or folic acid characterised by the presence of megaloblasts in the bone marrow. It has haematological and neurological manifestations. The haematological manifestations are seen with folate as well as vitamin B12 deficiency. Folate deficiency in adults does not affect the nervous system.

Cobalamin deficiency is slow and “pure”. Folate deficiency is rapid and “impure”. Deficiecy of vitamin B12 occurs because of loss of intrinsic factor resulting in an isolated defect of B12 absorption. No other nutrients are affected. The body stores of B12 can last months. This results in B12 deficiency being a slow and “pure” deficiency. Symptoms come on slowly, over months. Folate deficiency evolves relatively quickly and is most commonly because of alcoholism or malabsorption. It is associated with other deficiencies and is rapid and “not pure”.

 

Manifestationf o megaloblastic anaemia

Figure 1. Clinical Manifestations of Megaloblastic Anaemia

Haematological Manifestations

Haematological changes resulting from vitamin B12 deficiency and folate deficiency are indistinguishable. Megaloblastic anaemias are macrocytic anaemia but macrocytosis is not specific to megaloblastic anaemia. It is however exceptional for other diseases characterised by macrocytosis to have an mean capsular volume (MCV) > 110fl.  This value can considered the threshold above which an anaemia is unlikely to be anything other than megaloblastic anaemia.

The earliest change in a megaloblastic anaemia is macrocytosis. This precedes changes in erythrocyte indices. Changes in mean capsular haemoglobin (MCH) follow and then the MCV rises. Haemoglobin usually falls after the MCV increases to >97 fl. As the severity of anaemia increases the peripheral smear shows aniscytosis and poikilocytosis, nucleated cells, Howell-Jolly bodies and Cabot’s ring. Microcytes and erythrocyte fragments that represent dyserythropoiesis may be seen. Polychromasia is absent and this distinguishes megaloblastic anaemia from haemolytic anaemia.

The term megaloblatic anaemia is a misnomer. The disease is actually a panmyelosis.  Erythroid, myeloid and megakaryocytic series are affected. Thrombocytopenia and leucopenia (neutropenia and to a lesser extent lymphopenia) usually occur late in the course. It is uncommon for patients with mild anaemia to have platelets and neutrophils but occasionally changes in leucocytes and/or platelets may dominate.

Iron deficiency or β-thalassaemia trait result in microcytosis and hypochromia and may incidentally co-exist with megaloblastic anaemia. Co-existence of either of these diseases with megaloblastic anaemia may mask macrocytosis of megaloblastic anaemia. Presence of hypersegmented neutrophils in a patients with normocytic normochromic anaemia should raise the suspicion of a megaloblastic anaemia co-existing with Iron deficiency or β-thalassaemia trait.

Neurological Manifestations

Cobalamine deficiceny causes neurological dysfunction. Folate deficiency causes symptoms only in children. Children with inborn errors of folate metabolism may have myelopathy, brain dysfunction and seizures.

The neurological manifestations of B12 deficiency are a result of a combination of upper motor neuron manifestations from subacute combined degeneration of the spinal cord, sensory and lower motor neuron manifestations from peripheral neuropathy and neurophychiatratic manifestations. Subacute combined degeneration of the spinal cord (SACD) is a degerative disease of the spinal cord involving the posterior and lateral column (corticospinal and spinoceribellar tracts) that starts in the cervical and the thoracic region.

The earliest neurological manifestations are impaired sense of vibration and position and symmetric dysesthasia that involve the lower limb. This is frequently associated with sensory ataxia. With progression spastic paraparesis develops. The patients have brisk knee reflexes, reflecting an upper motor neuron involvement and depressed ankle reflex, reflecting a peripheral neuropathy. Bladder involvement is unusual. Some patients may have optic atrophy.

Neuropsychiatric manifestation include memory loss, depression, hypomania, paranoid psychosis with auditory and visual hallucinations.

Other manifestations

Skin and nails can show pigmentations. Mucosa of the villi undergoes megalobkastic change resulting in temporary malabsorption.

Response to therapy

Haematological Recovery

  • Day 1: Feeling better
  • Day2-3: Reticulocytosis appears
  • Day 7-10: Peak retuculocytosis
  • Day 15 onwards: Neutrophilic hypersegmentation disappears
  • Day 56 (8 weeks): Blood counts become fully .normal

Neurological Recovery

Neurologic improvement begins within the first week also and is typically complete in 6 weeks to 3 months. Its course is not as predictable as hematologic response and may not be complete.

 

 

Classification of β-Thalassaemia


β-Thalassaemia is a term applied to describe heterozygous group of diseases that are characterised by a decrease in the production of β globin  chain. Over 200 mutations in the β-globin gene and promoter regions that cause β-thalassaemia have been recognised. Thalassaemic alleles that produce no β-chain are designated β0 and those producing some β chain are designated as β+.  Before the genetic basis of thalassaemia was understood the disease was classified according to the clinical presentation and natural history of the disease. The genetic defects need to be determined for prenatal diagnosis but the clinical patterns remains relevant for clinical management of β-thalassaemia.

Based of the severity of disease three patterns of disease have been identified, thalassaemia major, thalassaemia minor and thalassaemias intermedia

  1. Thalassaemia Major: Thalasaemia major is a transfusion dependent anaemia that usually appears early in life, often in the first year. Anaemia is associated with splenomegaly, skeletal deformities and growth retardation. Iron overload develops by the end of second decade unless chelation is used. Unless treated with blood transfusion and chelation or allogeneic stem cell transplant, it is a fatal illness. There is a severe impairment of β-chain synthesis. Genetically these patients may be β0β0, β0β+ or β+β+.
  2. Thalassaemia Minor: patients with thalassaemia minor are asymptomatic. They are diagnosed when a complete haemogram is performed as a part of antenatal care or as a pert of investigations of another illness. Genetically they me be β0β or β+β.
  3. Thalassaemia Intermedia: Thalassaemia intermedia has a clinical presentation between that of thalassaemia major and thalassaemia minor. It is a very heterogeneous condition. The patient is not transfusion dependent but anaemic with a low and stable haemoglobin. Transfusion may be needed during periods of stress like infection and pregnancy. Advancing age is also associated with transfusion requirement. This may in part be due to hyperplenism associated with splenomegaly. The genetics of thalassaemia intermedia are complex. It may result from a mild β chain defect or because of interaction of β chain defects with other defects of haemoglobin synthesis

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.

Staging of Multiple Myeloma


Cancer is a heterogenous disease in terms of survival. Cancer staging is a method to classify patients according to prognosis. More advanced stages are associated with a worse prognosis. Patients with a poorer prognosis need to treated more aggressive. Non-haematological cancers are stages by the TNM staging. This system relies on the size of primary tumour, number of regional nodes involved and the presence or absence of distant metastasis to stage cancers. This scheme of of things is inappropriate for haematological cancers because

  1. The T Stage: Either it may be difficult to define the primary or in case of lymphoma the lymph node disease may be the primary
  2. The N Stage: Lymph node are not involved except in lymphoma where they are the primary site.
  3. The M Stage: Other than lymphomas haematological malignancies are “disseminated” at presentation with either the disease in the blood, as is the case with leukaemia, or in the bone marrow in case of multiple myeloma

These differences have resulted in evolution of staging/prognostic systems distinct from the TNM when one deals with haematological malignancies.

Multiple myeloma is a plasm cell neoplasm. It is characterised by a monoclonal protein in the serum and/or the urine, osteolytic lesions, anaemia, hyercalcaemia and renal failure. In the 1960s and 1970s these and other features of the disease were found to predict prognosis. Durie and Salmon in 1975 proposed the first staging system for multiple myeloma using type and amount of the monoclonal protein, haemoglobin, serum calcium and serum creatinine. They defined three stages of multiple myeloma I, II and , III. The tumour load increased as the stage increased. Each stage was further divided into substage A and B depending on the serum creatinine. The Durie-Salmon staging system is as follows:

    1. Stage I (All of the below)
      1. Hemoglobin value >10 g/dL
      2. Serum calcium value normal or =12 mg/dL
      3. Bone x-ray, normal bone structure (scale 0) or solitary bone plasmacytoma only
      4. Low M-component production rate (IgG < 5 g/dL; IgA < 3 g/dL; Bence Jones protein <4 g/24 hr)
    2. Stage II – Neither stage I nor stage III
    3. Stage III  One or more of the following:
      1. Haemoglobin < 8.5g/dL
      2. Serum calcium > 12 mg/dL
      3. Advanced lytic bone lesions (scale 3)
      4. High M-component production rate – IgG  >7 g/dL; IgA >5 g/dL; Bence Jones protein >12 g/24 h

Durie-Salmon sub classifications (either A or B)
A: Relatively normal renal function (serum creatinine value <2.0 mg/dL)
B: Abnormal renal function (serum creatinine value =2.0 mg/dL)

This system was widely adapted but the assesment of osteolytic lesions is subjective resulting in a poor reproducibility. Several attempts to improve the system that did not gain widespread acceptance were proposed.

After the introduction of the Duris-Salmon system other prognostic factors emerged. These included serum albumin, C-reactive protein, proliferation indices for bone marrow plasma cells (flowcytometery and S-phase fraction), bone marrow plasma cells and serum β2-microglobulin levels. Serum β2-microglobulin emerged as a good predictor of prognosis. 2005 the international staging system for multiple myeloma was proposed. The ISS stage was determined by only two objectively assessable parameters, serum β2-microglobulin and serum albumin. The staging system was as follows (J Clin Oncol May 20, 2005 vol. 23 no. 15 3412-3420)

  1. Stage I, Serum β2-microglobulin less than 3.5 mg/L and serum albumin ≥ 3.5 g/dL
  2. Stage II, neither stage I nor III
  3. Stage III, Serum β2-microglobulin ≥ 5.5 mg/L

The median survivals by stage are as follows: stage I 62months, Stage II 44 months and stage III 29 months.

Chromosomal studies, conventional karyotyping and interphase chromosomal studies using fluorescent In-situ hybridisation (FISH), identified translocations that adversely affect the out come of myeloma. These included del(17p), translocation t(4;14)(p16;q32) and translocation t(14;16)(q32;q23). In 2015 the chromosomal translocation were incorporated in the ISS and a revised system (R-ISS) was proposed (JCO.2015.61.2267)

The revised staging system is as follows

  1. R-ISS I: Serum β2-microglobulin level < 3.5 mg/L and serum albumin level ≥ 3.5 g/dL), no high-risk chromosome anomalies – [del(17p) and/or t(4;14) and/or t(14;16)] and LDH level less than the upper limit of normal range.
  2. R-ISS II: Nether R-ISS I nor R-ISS II
  3. R-ISS III: ISS stage III (serum β2-microglobulin level > 5.5 mg/L) with either high-risk chromosomal anomalies or high LDH level

Median OS not reached R-ISS, 83 months for R-ISS II and 43 months for R-ISS III. The improved median survival for all stages in R-ISS is a reflection of the efficacy of drug therapy of myeloma.