The M-Band


Monoclonal Gammopathy-02

Figure 1. Each plasma cell produces a different type of antibody. Normal γ globin band is depicted in the left column. The plasma cell numbers are normal and each produces an antibody with a different amino acid structure and electrophoretic mobility. Patients with monoclonal gammopathy have expansion (increase number) of a plasma cell clone (red in the diagram) resulting in the production of a disproportionate large amount of immunoglobulin from one type of plasma cell. This results in the M Band (see below). Patients with polyclonal gammopathy have an expansion (increased number) of plasma cells. This is usually occurs in response to infection/inflammation that result in production of a diversity of antibodies. The diversity is reflected in increase in the γ but as no one clone dominates the sharp M band is not seen.

What is an M-Band?

Immunoglobulins are antigen binding molecules secreted by plasma cells. Immunoglobulins bind antigens and play a role acquired immunity. Plasma cells develop from antigen exposed B-lymphocytes. The process of maturation of lymphocytes involves inducing mutations in region of the immunoglobulin gene that encodes for antigen binding regions, the hypervariable regions. This makes the DNA and consequently the amino acid sequence of the immunoglobulin secereted by a plasma cell unique. This is true even when two plasma cells make antibody against the same antigen or antigenic epitope (see figure 1).

Monoclonal Gammopathy-01

Figure 2. The serum protein separate into many bands on electrophoresis. The albumin is a dark band closest to the anode. This is followed by the α1, α2, β and γ bands. The immunoglobulin are mainly found in the γ globulin band but some may be found in the β globin band. The electrophoretic mobility of a molecule depends on the charge it carries which in turn depends on the amino acid sequence. Amino acid sequence determines the antigen specificity and differs between antibodies resulting in a slight variation in electrophoretic mobility of immunoglobulins and resulting in the γ region being a broad band.


The amino acid sequence determines the charge on the immunoglobulin. The electrophoretic mobility is determined by the charge. Majority of the immunoglobulins move to the γ-globulin fraction of serum proteins, some move with β-globulin. The γ-globulin band is a wide electrophoretic band reflecting the diversity in electrophoretic mobility of immunoglobulins arising from the diversity in amino acid sequences (figure 2).

Monoclonal Gammopathy-03

Figure 3. Patinets of monoclonal gammopathies have an expansion of one clone of plasma cells. This reflects in production of a disproportionally large amount of immunoglobulin with identical electrophoretic mobility resulting in a dense band with in γ globin region


Patients of monoclonal gammopathies have clonal expansion of plasma cells. The cells of a clone have identical DNA and produce identical immunoglobulin molecules. When the clone grows to level that it forms a significant proportion of the plasma cell pool the immunoglobulin it produces forms a significant proportion of the total serum immunoglobulins. The identical electrophoretic mobility of molecules produced by the clone results in a disproportionately large number of immunoglobulin concentrating to a point on electrophoresis forming a band.  This is known as the M band.  Lymphoma cells, notably those of lymphoplasmacytic lymphoma, can secrete immunoglobulin and are associated with an M band for similar reasons.

Diseases associated with an M-Band

The M-Band is a serum marker for plasma cell dycrasias and Waldenström macroglobulinemia. IgM and non-IgM (mainly IgG and IgA) monoclonal bands have differing clinical implications. The former is more commonly associated with lymphoproliferative disease and the latter with plasma cell dycrasias. The presence of an M band only indicates a clonal expansion of immunoglobulin producing cells. It does not indicate malignancy. The diagnosis of malignancy is made by features that suggest end organ damage. The absence of end organ damage indicates a premalignant disease including monoclonal gammopathy of uncertain significance (MGUS), soldering multiple myeloma or smoldering Waldenström macroglobulinemia.  The evidence of end-organ damage includes

  1. non-IgM Monoclonal Gammoathies: CRAB (elevated calcium, renal involvement, anaemia and osteolytic (bone) lesions) creatinine,
  2. IgM Monoclonal Gammapathies: Anemia, constitutional symptoms, hyperviscosity, lymphadenopathy, or hepatosplenomegaly that can be attributed to the underlying lymphoproliferative disorder if diagnosis is Waldenström macroglobulinemia or CRAB (elevated calcium, renal involvement, anaemia and osteolytic (bone) lesions) creatinine if the diagnosis of IgM myeloma

False positive M-Band

The presence of M band indicates presence of a clonal expansion of plasma cells. When end organ damage co-exists with M band a diagnosis of a malignancy (multiple myeloma or Waldenström macroglobulinemia) is made. In the absence of end organ damage the diagnosis of a premalignant disease is made. Proliferation a of plasma cells are seen in infections/inflammation. These are polyclonal and result in s polyclonal gammopath. They do not result in the presence of an M-band.

 

 

Evolution and Spread of HbS


The gene for β globin (OMIM  is present on chromosome 11 (11p15.4) along with other globin genes (ε, γ, γ and δ). This is known as the β-globin cluster . Individuals carrying identical genes on the β-globin gene cluster may not have identical DNA sequences in non-codeing regions of the DNA of the cluster. The non-coding regions include segments of DNA between genes and introns within genes. . Differences in DNA exist between individuals every 1000-2000 bases in the form of single nucleotide polymorphisms (SNPs). Single nucleotide polymorphisms are variations in a single nucleotide that occurs at a specific position in the genome. Many of these differences have no consequences on gene expression because either they do not result in change in amino acid sequence or they occur in regions of DNA that neither code for the gene nor regulate the gene. SNPs evolve by spontaneous mutations over time. The lesser the number of such differences between two individuals closer the individuals are the each other genetically (and in terms of evolution). Fewer differences in SNPs between individuals mean a more recent common ancestor.

One of the meanings of the word haplotype is a pattern of SNPs. A haplotype may be considered as a DNA “environment” in which the gene(s) occurs. This “environment” is created by the sequence of single nucleotide polymorphisms in which the gene(s) exists. As mentioned above differences in SNPs (and hence the “environment” the gene(s) exist in) evolve by spontaneous mutations over period of time. Fewer the differences between the “environments” the genes occurs in the more the likelihood that they come from related individuals.

HbS results from a single base substitution in the codon 6 of the β-globin gene. GAG becomes GTA resulting in substitution of valine for glutamate. This change results in a haemoglobin that crystallizes in hypoxic conditions resulting in a haemolytic anaemia. HbS occurs in diverse population groups including African, Mediterranean, Middle-Eastern and Indian. Is the haplotype of the HbS gene in these regions similar?

The HbS mutation occurs on five different haplotypes four African and one Arab-Indian. The mutation is the same (GAG to GTA on codon 6) but the SNPs are different. The haplotypes are

  1. Senegal: The Senegal HbS haplotype is found in Atlantic West Africa and Portugal
  2. Benin: The Benin HbS haplotype is found Central West Africa, Northern Africa and Mediterranean Europe (Greece, Sicily)
  3. Central African Republic or Bantu: The Central African Republic or Bantu is found in South Central and Eastern Africa
  4. Cameroon: The Cameroon haplotype is found in the Eton ethnic group of eastern Cameroon
  5. Arab-Indian: The Arab-Indian haplotype is the only non-African phenotype of HbS found in the eastern oasis of Saudi Arabia and India.

Origin of Haplotypes

There are two theories about the origin of haplotypes. The first, and the more accepted one, states that the five haplotypes arose from five independent mutations. An alternative hypothesis states that HbS mutation occurred only once and spread to other haplotypes by gene conversion.

 

Haplotypes and Severity of Symptoms

Symptoms of sickle cell anaemia are a consequence of crystallisation of haemoglobin under hypoxic conditions. HbF inhibits sickling. Patients with high HbF have fewer symptoms. The Arab-Indian and the Senegal haplotype are associated with higher HbF levels (17% and 12.4% respectively). In general patients carrying these haplotypes have milder symptoms than the Bantu or Benin haplotypes (Blood 2014; 123: 481)

 

Haplotypes and Human Migrations

Trade, conquests and human migrations (voluntary and slave trade) have disseminated the African haplotypes beyond Africa.

  1. The Mediterranean: Most of the Mediterranean (Greece and Scilly) has the Benin haplotype. This reflects pre-historic migrations from Central West Africa along the then fertile Sahara to North Africa. From here it spread to the Mediterranean via the interactions (Trade and wars) between the two regions. The only exception is Portugal. Portugal has the Senegal haplotype which reflects the trading contacts between Portugal and Atlantic West Africa (Angola and Mozambique).
  2. Americas: Neither the native americans nor the original European settlers to the Americas carried the HbS gene. HbS was imported to the Americas with the slaves from Africa. Jamaica was an important slave import hub and records for where tthe slaves arrived from are available. Jamaica has 73% Benin haplotype, 17% Bantu and 10% Senegal haplotypes. These numbers are close to the actual number of slaves who arrived in Jamaica from regions of Africa where these haplotypes are prevalent. Similarly the distribution of haplotype correspond to the origins of slaves in Baltimore and South Carolina (Mariam Bloom. Understanding Sickle Cell Disease, Page 34).
  3. Arab or Indian: It is not clear if the Arab-Indian haplotype originated in India or Saudi Arabia. But considering that all of tribal India has only one haplotype but the East and West Arabian Peninsula have different haplotypes it is possible that the haplotype originated in India.
  4. Spread to Other Parts: As opposed to the era of slave trade modern migration of people in the recent past have been voluntary. These populations have spread across the world as have those form mediterranean but to a lesser extent. These migrations have introduced the HbS gene in areas where it was not indigenous.

 

Anaemia with Hyperbilirubinaemia


A 49-year-old female presented with dyspnoea on exertion of 1 month duration. Examination reviled pallor and icterus. There was no lymphadenopathy, clubbing, koilonychia, platonychia, petechiae or purpura. There was no oedema of feet. The pulse was 90/min and the blood pressure 130/70 mm of Hg. Examination of the respiratory, cardiac and nervous systems did not show any abnormality. There was no organomegaly.

The haemoglobin was 4.9 g/dL with an erythrocyte count 1.37 x 1012/L, haematocrit of 16%, MCV of 116.78 fL, MCH of 35.77 pg and MCHC 30.63 of g/L.  The leucocytes count was 2800 with 35% neutrophils and 65% lymphocytes. The platelet count was 90 x 109/L. The peripheral smear showed macrocytosis and anisocytosis. Hypersegmented neutrophils were seen. The reticulocyte count was 3%.

The bilirubin was 2.1 mg/dL with a direct bilirubin of 1.8mg/dL and an indirect bilirubin of 0.3mg/dL. The Lactate dehydrogenase was 1417IU (normal 105 – 333 IU/L).

Anaemia and unconjugated hyperbilirubinaemia are characteristic of haemolysis. Does this patient have haemolytic anaemia?

Haemolysis shortens erythrocyte lifespan and results in increases haemoglobin breakdown. Haemoglobin is made of heme and globin. Heme consists of porphyrin ring at the centre of which is iron in the ferrous state. Iron released from catabolism of heme is reused. The porphyrin ring is catabolised to bilirubin. The bilirubin is transported to the liver for conjugation and excretion (see haemoglobin catabolism). Patients of haemolytic anaemia have unconjugated hyperbilirubinaemia because the increased bilirubin production overwhelms the hepatic bilirubin conjugation capacity.

One of the characteristics of megaloblastic anaemia is ineffective erythropoiesis. Ineffective erythropoiesis is defined as a sub-optimal (fewer) production of mature erythrocytes from a proliferating pool of immature erythroblasts. Each immature erythroblast produces less than the optimal number of erythrocytes because of premature death of erythroid precursors including haemoglobinized precursors. The haemoglobin released from haemoglobinized erythroid precursors is catabolised in the same manner as haemoglobin released from lysed erythrocytes (see haemoglobin catabolism). Megaloblastic anaemias are associated with unconjugated hyperbilirubinaemia because of death of haemoglobinized erythroid precursors.

The treatment of haemolytic anaemia and megaloblastic anaemia are different? How does one differentiate megaloblastic anaemia from that because of haemolytic anaemia? Does this patients have a haemolytic anaemia or megaloblastic anaemia?

Haemolytic anaemia is characterised by shortened erythrocyte survival. Erythrocytes survival is estimated by the use of radionucleotides something that is not possible at most centres. In clinical practice, a shortened erythrocyte survival is inferred from a high reticulocyte count. Reticulocytes are erythrocytes that have been produced in the preceding 24 hours. The erythrocytes survival is about 120 days and about 1% of erythrocytes are produced every day. Consistent with this the normal reticulocyte count is 0.5-1.5%.In patients of haemolytic anaemia, ddestruction of erythrocytes is matched by an increased production by the bone marrow. This manifests as reticulocytosis (see reticulocyte count). Megaloblastic anaemia occurs because of decreased production of erythrocytes and this manifests as reticulocytopenia. The difference between haemolytic anaemia and megaloblastic anaemia is the reticulocytosis in the former reticulocytopenia in the latter. This patient had a high reticulcoyte count but after correction both the reticulocyte production index [0.43] and corrected reticulocyte count [1.07%] were low excluding haemolysis. This patient was evaluated for megaloblastic anaemia.

The haemogram has clues to differentiate between haemolytic anaemia and megaloblastic anaemia. These include

  1. A very high MCV: The MCV is very high. Patients with haemolytic anaemia have a mild elevation in MCV. An MCV value >110fL is almost exclusively found in megaloblastic anaemias because of folate and/or B12 deficiency.
  2. Pancytopenia: B12 and folate deficiency impair DNA synthesis impairing erythrpoieis, myelopoiesis and megakaryopoiesis. Nutritional megaloblastic anaemias because of vitamin B12 and/or folate deficiency may show pancytopenia.
  3. Hypersegmented neutrophils (>5% neutrophils with >5lobes) is a feature of megaloblastic anaemia

Other features of megaloblastic anaemia include rise serum transferrin receptor, increased serum iron, serum ferritin and methemalbumin levels. Like haemolytic anaemia the serum haptoglobin is low and the LDH high. LDH levels in megaloblastic anaemia can ve very high.

This patients had a low serum B12 and was treated with parental B12 (1mg alternate day for 5 doses) and was evaluated for cause of vitamin B12 deficiency. As Schilling’s test was not available a diagnosis of pernicious anaemia was made by documenting gastric atrophy and anti-parietal cell antibodies.

Heterozygous β-Thalassaemia 


β-Thalassaemia is an inherited disease characterised by an imbalance between production of α and β globin chains of haemoglobin resulting from impaired production of β chains. The genes responsible for β-thalassaemia carry mutations in areas coding for the β globin gene or regions regulating the expression of this gene. Patients who are homozygous of compound heterozygous for the gene are symptomatic. They manifest as thalassaemia major. Thalassaemia major is a fatal illness where patients suffer the consequences of anaemia, bone marrow hyperplasia and iron overload. Iron overload that results from increased iron absorption and repeated transfusion is the cause of death. The treatment consists of lifelong transfusion with iron chelation or in those who have a matched donor, allogeneic bone marrow transplantation.

Thalassaemia Inheritance

The risk of inheritance of β-thalassaemia in offsprings when both parents are heterozygous is shown on the left. There is a 25% risk of thalassaemia major, 50% risk of heterozygous β-thalassaemia and 25% of the offsprings will be normal. If one of the parents does not carry the thalassaemia gene there is a 50% risk of the offspring carrying heterozygous β-thalassaemia and 50% of the offsprings will be normal.

As opposed to homologous or compound heterozygous β-thalassaemia, heterozygous the β-thalassaemia is asymptomatic. The condition is also known as β-thalassaemia minor (see classification of β-thalassaemia). The terminology reflecting the asymptomatic nature of the disease. Though β-thalassaemia is an asymptomatic disease the diagnosis has clinical implications. These include:

  1. Risk of β-thalassaemia in children: β-Thalassaemia major is inherited in an autosomal recessive manner. If both the parents are heterozygous for β-thalassaemia there is a 25% risk of the child suffering from thalassaemia major (see figure above, left). The most effective way to prevent β-thalassaemia major is to ensure that at least one parents does not carry the β-thalassaemia gene (see figure above, right). Diagnosis of an index case of heterozygous β-thalassaemia should initiate a search for all individuals carrying the β-thalassaemia gene in the family. Patients with heterozygous β-thalassaemia should be discouraged from choosing another heterozygous β-thalassaemia as a life partner. Those who make this choice despite counselling or those who already married should be explained the importance of prenatal diagnosis of β-thalassaemia major on conception and encouraged to undergo the same.
  2. Prevention of unnecessary iron therapy: Iron deficiency anaemia, like thalassaemia, is microcytic and hypochromic. Iron therapy alleviates the anaemia of thalassaemia only if iron deficiency co-exists. Iron therapy is associated with gastrointestinal adverse effects. Some patients with heterozygous β-thalassaemia have increased iron absorption and there have been reports of iron overload in β-thalassaemia trait (Br J Haematol). Diagnosis of heterozygous β-thalassaemia spares the patient unnecessary and sometimes dangerous iron therapy.

Pathophysiology of Heterozygous β-Thalassaemia

Heterozygous β-thalassaemia minor is characterised by an imbalance between the α and β globin chains because of decreased production of β-chains. The clinical manifestations of thalassaemia depend on the degree on imbalance between α chains and non-α (β+γ) chains. Thalassaemia minor, the phenotype of heterozygous β-thalassaemia results when the ratio of α to non-α chains is 2:1 (Cold Spring Harb Perspect Med 2012;2:a011726).

Clinical Features

Patients of heterozygous thalassaemia are asymptomatic. The clinical presentations is that of thalassaemia minor. Diagnosis is usually made incidentally when

  1. A haemogram is performed for another reason or
  2. Screening is performed following detection of a β-thalassaemia patient in the family
  3. Evaluation of anaemia of pregnancy

Though traditionally heterozygous β-thalassaemia are considered to be asymptomatic recent studies have found these patients to have symptoms of mild anaemia. Heterozygous β-thalassaemia may become symptomatic

  1. In pregnancy:The third trimester of pregnancy sees a plasma volume expansion accompanied by an increased production of red cells. In normal women the volume expansion is more than the increase in the number of red cells. Women become anaemic in the third trimester as a result of this discrepancy. Patients with β-thalassaemia trait show a plasma volume expansion but are not able to increase the number of red cells like normal women do. As a consequence women with heterozygous β-thalassaemia become more anaemic than normal women. This anaemia is usually mild and haemoglobin values lower than 8-9g/dL should prompt a search for another cause of anaemia. Iron deficiency anaemia is the commonest anaemia in pregnancy and it mimics thalassaemia. Serum iron and iron binding capacity may not be reliable in pregnancy and a serum ferritin must be performed for diagnosing iron deficiency.
  2. In case of autosomal dominant β-thalassaemia: Some forms of deletion β-thalassaemia result in the formation of an unstable β chain that forms inclusions. These inclusions cause ineffective erythropoietin and a thalassaemia like syndrome. Such patients are said to have a dominant β-thalassaemia and have the clinical picture of thalassaemia intermedia even when heterozygous.
  3. If the co-inherit an overdose of α thalassaemia genes: Manifestations of β-thalassaemia depend on the ratio of α to non-α chains. Thalassaemia minor results when the ration is 1.5-2.5:1 and intermedia when the ratio is about 4:1. Thalassaemia major is seen with higher rations. Some patients have three or even four α globin genes (ααα or αααα). These patients produce more α globin chains. Increase in α chains can push up the ratio of α to non-α chains and result in manifestations of thalassaemia intermedia in heterozygous β-thalassaemia. Similarly co-inheritance of α and β thalassaemias can attenuate the manifestations of thalassaemia.

Laboratory Features

  1. Haemogram: Heterozygous β-thalassaemia is characterised by anaemia, low MCH and low MCV. The MCHC is usually normal. The erythrocytes count is high and there may be a slight increase in the reticulocyte count. The peripheral smear shows microcytosis, hypochromia, poikilocytosis, basophilic strippling and target cells. Co-inheritance of α-thalassaemia attenuates the findings. The red cell indices are normal at birth. Changes associated with heterozygous β-thalassaemia become apparent by 3 months. By 6 months thalassaemic changes are firmly established.
  2. Haemoglobin A2: Haemoglobin A2 (HbA2) is in the range of 3.5-7%. Iron deficiency causes a disproportionate fall in HbA2 in patients with heterozygous β-thalassaemia but does not push the HbA2 levels in the normal range. Heterozygous β-thalassaemia with normal HbA2 is discussed below.
  3. Bone Marrow: The bone marrow shows erythroid hyperplasia with pyknotic normoblasts dominating. There is ineffective erythropoiesis mainly due to destruction of haemoglobinized precursors. Studies have shown approximately 25% decrease in efficiency of erythropoiesis.
  4. Iron Metabolism: Rate of iron absorption is slightly increased. Some cases of iron overload have been reported. Iron deficiency may co-exist the heterozygous β-thalassaemia particularly in pregnancy. Serum ferritin estimations should be performed to diagnose iron deficiency.
  5. Osmotic Fragility: Osmotic fragility is increased particularly after 24 hours of sterile incubation of erhthrocytes. It has been suggested that this be used as a screening test for heterozygous β-thalassaemia but has not gained widespread acceptability.
  6. Globin Chain Synthesis: Heterozygous β-thalassaemia is associated with a α:β ratio of 1.5-2.5:1.

Genotype Phenotype Co-relations

There is a continuous spectrum of changes with mild alleles having less pronounced effect on haematological parameters. Severe alleles have higher HbA2 values. Mild thalassaemia with high HbA2 suggest a promoter mutation.

Interaction between Heterozygous β-thalassaemia and other Haemoglobinopathies

Heterozygous β-thalassaemia is a common disorder and a chance associations may be seen with other haemoglobinopathies or inherited disorders of erythrocytes. Fortunately no deleterious association has been found with most disorders these include glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis  and pyruvate kinase deficiency.

α-Thalassaemia

α-Thalasaemia tends to reduce the α:β globin ratio. The amount of free α globin chain reduces attenuating the manifestations of heterozygous β-thalassaemia.

Sickle Cell Disease

β-Thalassaemia and sickle-cell diseases are common genetic diseases. Co-inheritance of the two is found in Africa, Mediterranean and sporadically through India. The symptoms depend on the relative amounts of HbS and HbA. HbA polymerises less than HbS. High levels of HbA reduce symptoms of sickling.  HbF is excluded from and protects against sickling. The clinical manifestations of patients co-inhereting sickle-cell and β-thalassaemia depend on the type of thalassaemia allele inherited and the HbF levels.

  1. Sickle β-thalassaemia with β0 or severe β+ alleles: Mediterranean forms of β-thalassaemia trait are either β0 severe β+. Patients from this region have severe sickling symptoms and HbA levels <15%.
  2. Sickle β-thalassaemia with mild β+ alleles: African patients of sickle β-thalassaemia inherit mild β+ alleles.  These patients have haemoglobin levels in the range of 20-30%  and mild symptoms . Many do not have symptoms. Diagnosis in some may be made incidentally.
  3. Sickle β-thalassaemia with high HbF: Patients from Indian and Saudi Arabia have mild symptoms despite inheriting severe β alleles because of high levels of HbF.

Treatment of Heterozygous β-Thalassaemia

Heterozygous β-thalassaemia does not need any treatment. A family screening should be carried out to detect other members carrying the thalassaemic β globin gene. Iron therapy should not be administered to patients empirically. Some patient have an increase iron absorption and iron overload has been reported. Iron studies should guide iron therapy. Anaemia can worsen during pregnancy. Folate and iron supplementation may be needed.

 

 

Calreticulin and Myeloproliferative Disease


Myeloproliferative disorders (polycythaemia vera [PV], essential thrombocytosis [ET], progressive myelofibrosis [PMF]) are a group of diseases that are characterised by increased proliferation of blood cells, splenomegaly, myelofibrosis, thrombosis and risk of malignant transformation.  The year 2005 was a landmark year for myeloproliferative diseases. Four groups of scientists identified the presence of JAK2V617F mutations in PV. This mutation is present in about 98% patients with PV. Mutations of exon 12 of the JAK2 gene can be found in 1-2% of the PV. These patients do not show the JAK2V617F mutation. The discovery of these mutations gave a genetic definition PV making diagnosis objective.

PV is diagnosed by the presence primary erythrocytosis in the precession of a JAK2 mutation referred to above. Chronic myeloid leukaemia is diagnosed by demonstrating the BCR-ABL1 translocation. JAK2V617F is also present in 50-60% of ET and PMF. Mutation of the gene MPL is found in 1-2%  patients of ET and 5-10% of the patients with PMF. The presence of these mutation helps make diagnosis. However, The diagnosis of PMF and ET in a large proportion of patients requires exclusion of a reactive disorder and other myeloproliferative diseases because these patients (38-49% of ET and 30-45% of PMF) have no genetic marker.

Two publications have shown that a large proportion of the patients with ET and PMF who do not have JAK have mutation calreticulin (CALR) (N Engl J Med. 2013;369(25):2391-2405,  N Engl J Med. 2013;369(25):2379-2390). In addition to ET and PMF CALR mutations are found in the MDS/MPN overlap disorder and refractory anemia with ring sideroblasts with thrombocytosis (RARS-T). They are rare or absent in other myeloid or lymphoid neoplasms or solid tumors.

Calreticulin (CALR) is a major calcium binding protein. The gene for calreticulin is located on 19p13.2. About a quarter of ET and MF have mutation in the CALR gene. All CALR mutations are localised to exon 9 and generate a 1bp frameshift. As a result of this most or almost all the C terminal negative amino acids and calcium binding sites are lost.  There is a complete loss of the KDEL endoplasmic reticulum binding sequence. These mutations have been identified in the haemopoietic stem cell and progenitor compartments. CALR mutations and JAK2 mutations are mutually exclusive.

CALR mutated myeloproliferative disease have a distinct clinical profile. These patients have a lower haemoglobin, lower leukocyte count, higher platelet count and a lower risk of thrombosis. Patients of PMF carrying a CALR mutation have a longer survival than those carrying JAK2 or MPL mutations. Patients with ET carrying the CALR mutations have a longer survival than those carrying the JALK2 mutation. There is no difference between the survival of ET patients carrying CALR mutations and MPL mutations.

Mutated CALR appears to stimulate STAT pathway. It appears to physically bind with the thrombopoietin receptor to stimulate STAT. The erythropoietin receptor is not needed for this action (Blood. 2015;10.1182/blood-2015-11-681932Blood. 2015;126:LBA-4).

 

 

 

Classification of Lymphoma


Lymphomas are a group of malignancies arising from lymphoid tissue. They have a diverse etiology, pathogenesis, clinical presentation, treatment and outcomes. Morphology alone is insufficient to classify lymphomas but for a long time a pathologist had little other than morphology for diagnosis. By the 1980s many advances that were instrumental in taking lymphoma classification beyond morphology had taken place. These advances included:

  1. Recognition of lymphocyte subtypes, T, B and NK cells and development of immunological and DNA based tests to identify these cells.
  2. Hybridoma technology that made available antibodies which were used initially for lymphoma diagnosis and then in lymphoma treatment
  3. Sanger sequencing made determining the sequence of genes possible
  4. Fluorescent in situ hybridisation (FISH) allowed study the mutations in cells in interphase
  5. Chemotherapy achieved cure in some lymphomas and control in others

These technologies were instrumental in generating information about lymphomas including pathogenesis, genetics, immunophenotype and clinical course. It became apparent that lymphomas are one of the most complex malignancies in terms of pathogeneis diagnosis and treatment. Such is the heterogeneity of lymphomas that one of the aggressive (Burkitts’s lymphoma) and one of the most indolent malignancies (small lymphocytic lymphoma/chronic lymphocytic leukaemia) are both lymphomas.

Historically several lymphoma classifications have came into use. Each specialist looked at lymphomas from a different  and his/her own perspective. To the pathologist it was about defining different histological entities and how these entities related to each other. To the clinician it was about defining entities with distinct treatments and outcomes. To complicate matters similar/same entities were referred to by different names by different groups. The confusion that prevailed highlighted the need for co-operation between experts in the field of lymphoma. The first such attempt of co-operation resulted in  the REAL (Revised European American Lymphoma) classification proposed in 1994 by a group of 19 haematopathologists, the International Lymphoma Study Group. This classification used all available information (including histology, genetics, immunophenotyping and clinical course) to define entities. This approach was adapted by the WHO classifications that followed the REAL classification. The most current classification of lymphomas is the 2008 WHO classification. The milestones in the classification of lymphomas are given in the table below.

Year Classifications Features
1941 Gall and Mallory
  1. First generally accepted classification of lymphoma, defined follicular lymphoma
1947 Jackson Parker
  1. First Classification of Hodgkin Lymphoma
1956 Rapaport (Non-Hodgkin Lymphoma)
  1. Classified lymphomas in to follicular and diffuse and within each category by cell morphology.
  2. Within each category nodular lymphomas had a better outcome.
  3. Continued to regard the origins of large cell lymphomas from non-lymphoid cells
1966 Luke and Buttler
  1. Proposed a classification of Hodgkin lymphoma which from the basis of modern classification.
  2. Recognised nodular sclerosis and mixed cellularity.
  3. Recognized the L&H cell
1974 Kiel Classification (Non-Hodgkin Lymphoma)
  1. Recognised that many lymphomas resemble normal germinal centre.
  2. Classified lymphomas according to lymphocytic differentiation as understood at the time. Suggested the putative normal counterparts of lymphomas.
  3. Classified lymphomas in B and T types
1982 Working Formulation (Non-Hodgkin Lymphoma)
  1. Studied 6 classification schemes in use at the time found none to be superior. Consenseus could not be reached because of lack of agreement between pathologists.
  2. Proposed a formulation to translate amongst schemes.
  3. Stratified outcomes based on outcome of trials conducted in the 1970s. Did not use immunophenotyping.
1994 REAL Classification
  1. Developed by a group of pathologists, international lymphoma study group, that made an attempt to overcome differences and focused on identification of “real” entities by incorporating all (morphology, genetics, immunophenotype and clinical course) knowledge available at the time.
  2. Formed the basis of the currently used WHO classification
2001 and 2008 WHO Classifications
  1. The 2008 WHO lymphoma classification is the current classification
  2. Based on pathology, genetics and clinical outcomes

Classification of Lymphoma

The 2008 WHO classification was a result of international collaboration among pathologists, molecular biologists and clinicians interested in the hematological malignancies. Lymphomas are divided into three groups the

  1. B-cell neoplasm
  2. T and NK cell lymphomas

  3. Hodgkin’s lymphoma.

The non-Hodgkin lymphomas are further divided into into precursor neoplasm and peripheral/mature neoplasm. The peripheral lymphoid tissue have mature lymphocytes (peripheral lymphocytes). The precursor lymphoid cells mature in the bone marrow (B cells) and thymus (T Cells).

Lymphocyte development begins with the lymphoblast. A mature lymphocyte expresses a antigen receptor complex which consists of two parts, the antigen receptor and associated signal proteins. Immunoglobulins serve as antigen receptors of B cells. Immunoglobulins  have a constant and a variable region. The genome has many DNA segments encoding for the variable region. Antibodies have different antigen specificity because different segments are chosen to form the gene of the variable region. A wide array of antibody   specificity (millions) can be generated from combination of these DNA segments. Antibody specificity can be further diversified by a process known as somatic hypermutation referred to below. Cells that are undergoing antibody editing are precursor B cells. B cell maturation occurs when the process of antibody editing is complete. Mature B cells express a complete antigen receptor, IgD and IgM on the surface. Similarly a mature T cell is a cell that has completed the process of editing its T cell receptor.

Precursor Neoplasm

Precursor cells are cells that have not undergone the B or T cell receptor rearrangement. The malignancies of precursor lymphoid tissue incelude T and B cell lymphoblastic lymphomas and acute lymphoblastic leukaemia.

B lymphoblastic lymphoma/leukaemia is further classified into B-lymphoblastic leukaemia/lymphoma with recurrent genetic anomalies and B-lymphoblastic leukaemia/lymphoma that does not show these anomalies (B-lymphoblastic leukaemia/lymphoma NOS). The recurrent anomalies seen in B-lymphoblastic leukaemia/lymphoma are [gene rearrangements]

  1. t(9;22)(q34;q11.2) [BCR-ABL1]
  2. t(v;11q23) [MLL rearranged]
  3. t(12;21)(p13;q22) [TEL-AML1 (ETV6-RUNX1)]
  4. t(5;14)(q31;q32)[IL3-IGH]
  5. t(1;19)(q23;p13.3)[TCF3-PBX1]
  6. hyperdiploidy
  7. hypodiploidy

 

Neoplasm of the Mature (peripheral) Cells

Neoplasm of mature lymphocytes are classified into B cell neoplasms and T and NK cell neoplasms.

 

Mature B cell neoplasms

Mature B-cell neoplasm arise from B cells that have undergone B cell receptor rearrangement. Though these cells have their immunoglobulin or T cell receptors rearranged and are referred to as mature the process of maturation is not complete. They undergo a final phase of maturation on exposure to antigens that results in increased antibody avidity. This process takes place in the germinal centre. Antibody avidity is increased by inducing mutations in the DNA segments encoding for the variable regions. This process known as somatic hypermutation.  Somatic hypermutation is a considered to be an evidence of a cell that has passed through the germinal centre (and hence been exposed to antigen). Somatic hypermutations result in a spectrum of avidity (both higher and lower than the original cell). Cells producing highest affinity antibodies survive to form memory B cells or mature to antibody secreting plasma cells. The rest undergo apoptosis. Mutations and apoptosis are two phenomena central to malignant transformation. Germinal centre cells are subject to both. It is not surprising that the germinal centre is the site of the largest number of lymphomas. Diffuses large B Cell lymphoma, follicular lymphoma, Hodgkin’s lymphoma classical and nodular lymphocyte predominant and Burkitts’s lymphoma originate in the germinal centre. Together these constitute almost two third of the lymphomas. Most mantle cell lymphomas originate from cells that have yet to enter the germinal centre. Chronic lymphocytic leukaemia, marginal zone lymphomas, plasma cell neoplasms and lymphoplasmacytic lymphomas arise from cells that have passed through the germinal centre.

Diffuse large B cell lymphoma (DLBCL) is a lymphoma composed of B cells where the size of malignant cells is equal to or exceeds the size of a macrophage nucleus. DLBCL is the most common lymphoma across the world. All DLBCLs are aggressive lymphomas. The commonest form of DLBCL lacks any special features and is known as DLBCL NOS (not otherwise specified). There four DLBCL subtypes. EBV positive DLBCL of the elderly is a provisional entity in the 2008 WHO classification.

  1. T Cell/histiocyte rich DLBCL (THRLBCL): THRLBCL is a rare variant of DLBCL that is characterised by scattered large B cells that comprise about 10% of the cells in reactive infiltrate that is abundant in T cells with frequent histiocytes.  It resembles Hodgkin’s lymphoma in having a paucity of malignant cells and an abundance of infiltrate. Some TCRLBCL may be arising from progression of nodular lymphocytic predominant Hodgkin’s lymphoma.
  2. Primary CNS DLBCL: Primary CNS DLBCL forms about 90% of primary CNS lymphomas.
  3. Primary cutaneous DLBCL, leg type: Primary cutaneous DLBCL, leg type is a cutaneous lymphoma most commonly arising in the leg. Unlike other DLBCL women are affected more often than men.
  4. EBV positive DLBCL of the elderly

Other forms of DLBCL include those having special anatomical sites (primary mediastinal B cell lymphoma, intravascular lymphoma), histological features (ALK positive large B cell lymphoma, de novo CD5+ large B cell lymphoma) and pathogenesis (large B cell lymphoma arising out of HHV-8 associated Castleman’s disease, pleural effusion lymphoma)

Follicular lymphomas (FL) arise from germinal centres. They have follicle centre (centerocytes/small cell) and large (centroblasts/transformed) arranged at least in a partially follicular pattern. Eighty percent of the patients have the t(14;18)(q32;q21) translocation that results in fusion of immunoglobulin heavy chain gene with BCL2. FL is divided into three categories according to the number of centrblasts. Grade 1-2 FL have 0-15 centroblasts per high power field, Grade 3A FL has >15 centeroblasts per high power field and 3B FL shows solid sheets of centroblasts. Grade 1-2 and Grade 3A FL are indolent lymphomas and Grade 3B is an aggressive lymphoma to be treated as DLBCL.

Small lymphocytic lymphoma (SLL) is a lymphoma that consists small lymphocytes that co-express CD19 and Cd5. It is the nodal counterpart of chronic lymphocytic leukaemia (CLL) and the entity is referred to as CLL/SLL. Patients having lymph node involvement and <5 X 109/L lymphocytes are classified as SLL. Patients with ≥5 X109/L lymphocytes are said to have CLL. The normal counterpart of SLL is the antigen experienced B cell.

Marginal zone lymphomas (MZL) are indolent lymphomas. They are of three types, nodal MZL, extranodal lymphomas of the mucosa associated lymphoid tissue (MALT) and splenic marginal zone lymphomas (SMZL). They arise from post-germinal memory B lymphocytes in the marginal zone of the germinal follicles. About one third of the patients of SMZL do not have somatic hypermutation of the variable regions of the immunoglobulins. The cell of origin is in these SMZL is not known. MZL are peculiar amongst lymphomas in being related to infection. Gastric MALT lymphomas are associated with H. pylori infection, ocular adnexal MALT lymphoma is associated with Chlaymydia psittaci, immunoproliferative small intestinal disease (IPSID) with Campylobacter jejuni, and cutaneous MALT lymphoma with Borrelia burgdorferi. Hepatitis C infection is associated with splenic marginal zone lymphoma.

Mantle cell lymphomas are lymphomas small to medium sized cells that arise form peripheral B cells of the inner mantle zone. It is associated with the t(11;14)(q13;q32) translocation that results in the formation of the IGH@-CCND1 (Cyclin D1) fusion gene. Cyclin D1 can be detected on almost all mantle cell lymphomas by immunohostochemistry.

Burkitts lymphoma (BL) is a lymphoma composed of medium sized cells (nuclei similar to or smaller than histiocytes) that show a diffuse monotonous pattern. The tumour has a very high proliferation index and shows many mitotic figures and a high fraction of apoptosis. It is characterised by translocation that dysregulate the oncogene MYC. These include the t(8;14)(q24;q32) translocation that IGH@ (immunoglobulin heavy chain locus)  to MYC and is the commonest translocation in Burkitt’s lymphoma, the t(2;8)(p12;q24) that translocates the IGK@ (kappa light chain locus) to MYC and t(8;22)(q24;q11) that translocates IGL@ (lambda light chain locus) to MYC. There are two forms of Burkitt’s lymphoma. The Endemic BL occurs in equatorial Africa, affects children and has the EBV genome in majority of the neoplastic cells. The sporadic BL is seen in other parts of the world, is most common in young adults and shows EBV genome only in about 30% of the patients. Sporadic BL is a immunosuppression related malignancy seen in HIV and other forms of immunosuppression.

Lymphoplasmacytic lymphoma is a mature B cell lymphoma that is made of small B lymphocytes and plasmacytoid lymphocytes. These lymphocytes often secret IgM resulting in the syndrome Waldenström macroglobulinaemia. IgM Secretion however in not essential for diagnosis. The normal counterpart of lymphoplasmacytic lymphoma is the post germinal B cell that differentiates into a plasma cell.

Other rarer lymphomas have been described elsewhere (WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues)

 

Mature T-cell and NK neoplasm

The differentiation of T lymphocytes is not understood as well as that of the B lymphomas.  Clinical picture plays a more important role in the diagnosis of T cell/NK cell lymphomas. T cells carry a more diverse set of function than B lymphocytes. These include cytotoxic functions, aiding other cells of the immune system and regulation of immunity. Many subtypes of T cells are recognised. Like B cells, the T cells have a antigen receptor complex. This consists of and antigen receptor and associated signal proteins. The T cell receptor is made of a pair of chains. There are four T cell receptor chains, α, β, δ and γ. These give rise to two types of T cell receptor the αβ  and δγ. Ninty five percent of the T lymphocytes have the αβ receptors and about 5% of the at T cells have δγ receptors. The δγ T cells and NK cells are a part of the innate immune system. Malignancies of these cells are common children and young adults. These include aggressive NK cell leukaemia, systemic EBV positive lymphoproliferative disease of the childhood, most hepatosplenic T cell lymphomas and δγ-T cell lymphoma.

T cells of the adaptive immune system include naive T cells, helper/regulatory T cells, cytotoxic T cells and memory T cells. Regulatory  cells express CD4. Depending on the cytokine secreting profile these cells are of two types Th1 and Th2. Th1 cells produce IL2 and INFγ that mainly help T cells and macrophages. Th2 cells secrete IL-4, IL-5, IL-6 and IL-10 and mainly help B cell. Follicular helper T cells are T cells that help the germinal centre reaction. In addition to the T cell markers they express germinal centre markers BCL6 and CD10. They also express CD57 and PD-1. Regulatory T cells are cells that suppress immune response. They express CD25.

Lymphomas of the T cells of the adaptive immune system are nodal and occur in adults.

Peripheral T cell lymphoma not otherwise specified (PTCL NOS) is a heterogenous group of malignancies of the peripheral T cells. Its is a basket entity that includes peripheral T cell lymphomas that lack any specific features (unlike the ones listed below). It is the commonest peripheral T cell lymphoma. Gene expression profiling has identified two subtypes of PTCL NOS. Lymphomas arising from the Th1 cells and those arising from Th2 cells.

Anaplastic large cell lymphoma (ALCL) is the second most common T peripheral T cell lymphoma. The normal counterpart of ALCL is not known. ALCL has two subtypes depending on the expression of the anaplastic lymphoma kinase (ALK), ALK+ ALCL and ALK -ve ALCL. These have distinct clinical picture.

Angioimmunoblastic T cell lymphoma (AITL) arises from follicular helper T cells. It usually disseminated at presentation.  It is characterised by generalised lymphadenopathy, systemic symptoms and polyclonal hypergammaglobulinaemia. The patients have immune phenomena including circulating immune complexes, cold agglutinins with haemolytic anaemia, rheumatoid factor and anti-smooth muscle antibodies. These are attributed to polyclonal proliferation of B lymphocytes (which are not the malignant lymphocytes).

Adult T cell Leukaemia/lymphoma is a lymphoma composed of highly pleomorphic lymphoid cells. It is seen in Southwest Japan, Caribbean and parts of Central Africa and is caused by the retrovirus HTLV-I. The clinical types include acute, lymphomatous, chronic and smoldering. Patients often have hypercalcaemia and often have immunodeficiency.

Skin unlike other organs has a higher proportions of T cell lymphomas than B cell lymphomas. These include Mycosis fungoides, Sezary syndrome and the primary cutaneous CD30+ T cell lymphoproliferative disorder, primary cutaneous T cell lymphomas, subcutaneous panniculitis like T cell lymphoma.

Other rare T cell lymphomas include T cell prolymphocytic leukaemia, T-cell Large Granular lymphocytic leukaemia, Extranodal NK/T cell lymphoma, nasal type, enteropathy associated T cell lymphoma and hepasplenic T-Cell lymphoma. A complete list is given elsewhere (WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues)

 

Hodgkin lymphoma

Hodgkin’s lymphoma is of two types classical and modular lymphocytic predominant. The uncertainty that surrounded the cell of origin of Hodgkin’s lymphoma was ended when microdissected Reed-Sternberg cells were shown to be of B cell origin. The classical Hodgkin’s lymphoma is further divided into lymphocyte rich, nodular sclerosis, mixed cellularity and lymphocyte depletion types.

 

References

  1. Elaine S. Jaffe, Nancy Lee Harris, Harald Stein, and Peter G. Isaacson. Classification of lymphoid neoplasms: the microscope as a tool for disease discovery. Blood. 2008 Dec 1; 112(12): 4384–4399.
  2. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues

 

Chronic Myeloid Leukaemia



Chronic myeloid leukaemia (CML) is a myeloproliferative disorder characterised by anaemia, leucocytosis and splenomegaly. The natural history of CML is characterised by three phases chronic phase, accelerated phase (AP) and blast phase (BP). CML is characterised by the presence of fusion gene BCR-ABL1 that results from the t(9;22) translocation. This gene encodes for a constitutionally active tyrosine kinase that has been shown to drive the CML stem cells. The diagnosis of CML can not be made in the absence of this gene.  Onset of AP signals a change in the biological behaviour of disease. The disease follows a more agressive course that culminates in blast phase. The blast phase mresembles an acute leukaemia and is a terminal event in the natural history of CML. Inhibitors of BCR-ABL1 prevent the emergence of accelerated and blast phase and have dramatically improved the outcome of CML.

Etiology and Epidemiology

CML constitutes about 15% of all leukaemias. The incidence increase with age and the disease is slightly more common in males. The incidence of the disease does not show geographic variation. The only know etiological factor for CML is exposure to radiation.

Pathogenesis

CML is characterised by the presence of Philedelphia chromosome [t(9;22)(q34;q11)] which results from a reciprocal translocation between the long arms of chromosomes 9 and 22 (figure 1). The ABL1 proto-oncogene is located on chromosome 9 at q34. Chromosome 22 has the BCR gene at 22q11. The ABL1 gene translocates downstream to the BCR gene as a result of the t(9;22)(q34;q11) translocation. This results in the formation of the BCR-ABL1 fusion gene (see The BCR-ABL1 Gene).

Molecular Biology of CML

 

The expression of the ABL1 tyrosine kinase is tightly regulated. The t(9;22)(q34;q11) results in the N terminal segment of the ABL1 gene being replaced by that of the  BCR gene. This results in the ABL1 tyrosine kinase being constitutively expressed. The size of the N terminal amino acids contributed by BCR determine the length and the clinical properties of the fusion gene.

A model of pathogenesis of CML has to account for three features of the disease

  1. Uncontrolled proliferation of leucocytes accompanied sometimes with the proliferation of platelets
  2. Progression from a relatively stable phase of proliferation, the chronic phase, to a agressive  phase charecterized by increasing leucocytes counts, anaemia and falling platelet counts ultimately culminating in a acute leukaemia like picture, the blast phase. Unlike the chronic phase that shows myeloid and sometimes platelet proliferation, the blast phase may show a myeloid, lymphoid or rarely megakaryocytic lineage.
  3. Failure of tyrosine kinase inhibitors to errdicate the malignant clone despite pronounced and prolonged supression of the BCR-ABL1 positive clone.

The precise mechanism how BCR-ABL1 leads to chronic myeloid leukaemia is not known. Activation of phosphatidylinositol kinase (PI3K), RAS/Mitogen activated protein kinase (RAS/MAPK) and JAK/STAT pathway has been demonstrated in BCR-ABL1 positive cells. These pathways have been implicated in malignant transformation of cells and are believed to be responsible for the malignant phenotype of CML. Progression of CML from chronic phase to accelerated phase and eventually blast phase marks change in the disease that makes it progressively less responsive to drugs inhibiting BCR-ABL1 kinase. Cells with BCR-ABL1 fusion gene have an increase in the reactive oxygen species predisposing them to DNA damage. Progression from chronic phase to acclerated phase and eventually blast phase is associated with the cell accquiring additional mutations. This is known as clonal evolution. Reactive oxygen species induced DNA damage is believed to result in clonal evolution which results in progression to accelerated phase and blast phase resulting in treatment failure.

CML Pathogenesis-600px

Imatinib, an inhibiter of ABL1 tyrosine kinase has been in use for over a decade. Treatment of CML patients with Imatinib results in normalisation of blood counts, regression of splenomegaly and a decrease in the number of cells showing BCR-ABL1 fusion gene. The BCR-ABL1 kinase expressing cells are suppresses to undetectable levels in about 40% of the patients creating an impression that disease has been eradicated. If treatment is discontinued in such patients cells expressing BCR-ABL1 fusion reappear in 50-60% of such patients. These observations have been interpreted as suggesting that the CML stem cells have mechanisms that survive inhibition by Imatinib and other inhibitors of ABL1 tyrosine kinase. Studies to identify and target these pathways to erradicate CML stem cells with an goal to curing CML are undertaken. Some of the pathways that have shown a promise are Alox5pathway, the sonic hedgehog pathway (SHH), the Wnt/β-catenin pathway, the JAK/STAT pathway, the TGF-Beta/FOXO/BCL-6 pathway (Stem Cells International Volume 2013 (2013),  Article ID 724360, 12 pages)

Clinical Manifestations

Presentations

The manifestation of CML included

  1. Anaemia
  2. Fatigue, weight loss, fever, night sweats because of a hyper metabolic state induces by high leucocyte counts
  3. Bone pain
  4. Abdominal pain, early satiety and fullness because of splenomegaly. The pain is usually a tugging pain but sharp pain may indicate a splenic infarct. Splenic rupture though described is a rare event.
  5. Leucostasis because of very high white blood cell counts may present with neurological deficits, respiratory insufficiency or priapism.
  6. Incidental discovery for a haemogram performed for another reason is becoming a common presentation in populations that have a good healthcare system.

Splenomegaly is commonly seen. Historically CML is associated with massive splenomegaly (splenomegaly below the umbilicus). Today this may be seen only in communities with suboptimal health care and diagnosis is delayed. Hepatomegaly may be seen in some patients.

CML-Phases

Stages of Disease

CML has three stages, chronic Phase, accelerated phase and blast phase. Before the introduction of definitive therapy (Initially interferon, currently inhibitors of BCR-ABL1 kinase) every patient progressed from chronic phase to accelerated phase and finally to blast phase. The blast phase was terminal. The WHO definitions of the phases are listed in table 1. Acclerated phase definition is defined differently by different authors. Definitions may differ in the details but recognise the following

  1. Increasing WBC counts with appearance of new anomalies. These may be basophilia, eosinophilia or increase in the immature forms particularly blasts and promyelocytes. Progression of patients on BCR-ABL1 kinase inhibitors is accompanied by appearance mutations in BCR-ABL1.
  2. Increasing splenomegaly

The TKIs have dramatically reduced the rate of progression to accelerated phase or blast crisis. 4.6% for Dasatinib (DASSISION trial 5 year follow up), 3.5% for nilotinib (ENESTnd)and 8% imatinob (IRIS trial 8 Year follow up). Patients who do not achieve an early response to TKIs have a higher risk of progression.

Investigation

Haemogram

A complete haemogram should be performed in all patients. Patients of CML have leucocytosis with shift to the left and often have eosinophilia and/or basophilia. Platelet counts are often slightly increased but may be normal high or occasionally low. There is mild to moderate anaemia. The differential count of peripheral blood depends on the phase of disease. Patients with chronic phase have <10% blasts and ≤ 20% basophilis. Patients with accelerated phase have 10-19% blasts or >20% basophilic. Patients with blast crisis have ≥20% blasts.

Bone Marrow

Bone marrow studies are not needed for diagnosis of CML. This can be made by demonstration of BCR-ABL1 on peripheral blood. Bone marrow studies are essential for determining the phase of disease. The bone marrow of a patients of chronic phase of CML shows a high myeloid to erythroid ratio with a normal myeloid maturation. Dysplasia is not a feature of CML and suggests the diagnosis of myeloproliferative disease/mydlodysplasia overlap.  The chronic phase is characterised by <10% blasts. The megakaryocytes are smaller with reduced lobulation and may be increased in number. Bone marrow of patients from accelerated phase show myeloid hyperplasia with myelodysplasia and a blast percentage between 10-19%. Megakaryocytes may be seen in clusters or sheets. Bone marrow from patients with blast phase shows ≥20% blasts.

Risk Scores

Risk scores help in prognosticating the patients and should be performed at diagnosis. The three scores are listed in table 2. Hasford and Sokal scores are prognostic scores for predicting outcomes of CML patients. The  EUTOS score was developed to predict the outcome of patients receiving imatinib at 18 months of therapy and was reported to perform better than Sokal and Hasford scores. It has not been validated by other investigators.

CML Prognostic Scores

Table 2. CML Prognostic (Risk) Scores

 

Treatment

The approval of imatinib in May 2001 by the US FDA saw CML become the first disease to benefit from targeted therapy. The 8 year survival of patients in chronic phase has improved from 6% in 1975 to 42%-65% from 1983-2000 and 87% for patients diagnosed after 2001 (Blood 119:1981-87;2012). Before the introduction of imatinib the treatments used for patients of CML included (in chronological order) splenic radiation, arsenic, busulfan, hydrourea and a combination of interferon and cytarabine. With the exception of interferon and cytarabine none of the other therapies suppress the BCR-ABL1 positive clone.  The IRIS trial established imatinib, an inhibitor of BCR-ABL1 tyrosine kinase as the first line treatment for chronic myeloid leukaemia. DASSISION trial and the ENESTen trials established dasatinib and nilotinib as front line therapy.
 The aims of therapy is prevent progression of disease to accelerated phase and blast phase. This can be achieved by
  1. Inducing a haematological remission
  2. Inducing a molecular remission
  3. Eradicating the CML stem cell

Tyrosine kinase inhibitors (TKIs) are the first line of treatment in all patients of CML. The use of imatinib in pregnancy is associated with increase in malformations. These include malformations of the skeleton, respiratory system, kidney and gastrointestinal the gastrointestinal tract. The risk of exompholous is increased by almost 1000 times. Imatinib and other TKIs are contraindicated in pregnancy. The treatment of pregnant woman is challenging. Interferon may be used after the period of organogenesis. Women who have not completed their family are encouraged to do so early and should be switched to TKI. Women in the childbearing age on TKI should be made to understand that contraception is mandatory.

Tyrosine Kinase Inhibitors (TKI)

Many inhibitors of BCR-ABL1 tyrosine kinase have been developed. Out of these imatinib, dasatinib and nilotinib are approved for use in the first line setting. Dasatinib and nilotinib can be used for patients who relapse on imatinib. Busotinib and ponatinib are approved for use relapsed CML. The TKIs drugs used in CML are listed in the table below.

 Dose Indications Adverse Effects
Imatinib
  1. Chronic Phase CML: 400mg
  2. CML AP/BP: 400mg od the doses may be increased to 600mg-800mg/day. The 800mg dose is administered in two daily doses
  3. Ph+ALL: 600mg od
  4. PDGFRA mutated MDS/MPD: 400mg/day
  5. GIST: 400mg/day
  6. HES/CEL: 400mg od
  1.  CML First line therapy for chronic, accelerated and blast phase
  2. Ph+ve ALL
  3. Gastrointestinal stromal tumour
  4. MDS/MPD with PDGFA gene rearrangements
  5. Hypereosainophilic syndrome/chronic eosinophilic leukaemia
  1. Skin depigmentation
  2. Nausea, vomiting
  3. Periorbital oedema
  4. Fluid retention manifesting as effusions and oedema
  5. Myalgias
  6. Diarrhoea
  7. Insomnia, deprtesson
Dasatinib
  1. Chronic Phase: 100mg od
  2. Accelerated and blast phase: 140mg od
  1. CML first line and second line therapy
  2. Ph+ve ALL resistance or intolerance to prior tyrosine kinase therapy
  1. Myelosupression
  2. Bleeding
  3. Fliud retention manifestion as oedema and pleural effusion
  4. Diarrhoea, nauseas vomiting
  5. Fatigue
  6. Insomnia, depression
  7. Elevated transaminases
  8. Hypocalcaemia, hypophosphataemia
  9. QTc prolongation
Nilotinib
  1. CML Chronic Phase 300mg bd
  2. CML chronic phase resistant/intolerant to other TKIs: 400mg bd
  3. AP/BC: 400mg bd
CML first line and second line therapy

 

  1. Myelosupression
  2. Fatigue, asthenia, anorexia
  3. Prolonged QTc
  4. Electrolyte anomalies – hypophosphatemia, hypokalemia, hypocalcaemia, hyponatraemia
  5. Elevated transaminases
Bosutinib
  1. CML chronic phase: 500 mg orally once daily with food. Escalation to 600 mg daily in patients who do not respond.
  2. Dose in Hepatic impairment: Reduce 200 mg daily
CML failure or intolerance to firstling therapy
  1. Diarrhoea
  2. Nausea/vomiting
  3. Abdominal pain
  4. Myelosupression
  5. Skin rash
  6. Elevated Transaminases
  7. Fliud retention manifestion as oedema and pleural effusion
  8. Fatigue
Ponatinib
45mg reduce dose to 30mg in patients taking strong CYP3A inhibitors
  1. CML first line and second line therapy
  2. Ph+ve ALL resistance or intolerance to prior tyrosine kinase therapy
  3. The only TKI active against the T315I BCR-ABL1 mutation
  1. Arterterial thrombosis including myocardial infraction. Toxicity may be seen in up to 10% of the patients
  2. Cardiac toxicity – arrhythmias, pericardial effusions
  3. Elevated transaminases,  liver failure
  4. Pancrititis
  5. Hypertension
  6. Fluid retention,
  7. Gastrointestinal perforation
  8. Tumour lysis syndrome
Omacetaxine (Non TKI, inhibitor of protein synthesis)
 1.25mg/m2 SC bd for 14 consecutive days every 28 days till haematological response. Continue as 1.25mg/m2 sc bd for seven days every 28 days after as maintenance CML chronic or acclerated phase resistant to two more TKI
  1. Myelosupression – thrombocytopenia, neutropenia and anaemia
  2. Impaired glucose tolerance with hyperglycaemia in upto 11%
  3. Diarrhoea, nausea, vomiting abdominal pain
  4. Fatigue asthenia, arthralgia peripheral oedema
  5. Injection site reactions

A newly diagnosed patients should be initiated on treatment with Imatinib (400mg od), dasatinib (100mg od)  or nilotinib (300mg bd). Dasatinib and nilotinib. Busotinib and  ponatinib can be used in patients progressing on these the first line drugs. Ponatinib is indicated in the T315I mutations. Patients carrying this mutation do not respond to any of the other TKI.

Monitoring Therapy

Patients are monitored for response by clinical examination, haemogram and determining the levels of the BCR-ABL1 transcript. The BCR-ABL1 transcript is measured by RT-PCR. The response definitions are given in table below

Response Definitions

Complete Haematological Response
  1. WBC counts < 10 X 109/L
  2. Platelet Count < 450 X 109/L
  3. No immature myeloid cells on the peripheral blood differential count
  4. Basophils less than 5%
  5. no splenomegaly
Cytogenetic Response
  1. Complete (CCyR) 0% Ph+
  2. Partial Cytogenic Response  (PCyR) 1-35%
  3. Major Cyogenic response 36%-65%
  4. Minor Cytogenic Response 66%-95%
  5. No Cytogenic Response >95%
Molecular Response
  1. Early molecular response (EMR) – BCR-ABL1 transcripts ≤10% by QPCR (IS) at 3 and 6 months.
  2. Major molecular response (MMR) – BCR-ABL1 transcripts 0.1% by QPCR (IS) or ≥3-log reduction in BCR-ABL1 mRNA from the standardized baseline, if QPCR (IS) is not available.
  3. Complete molecular response (CMR) – no detectable BCR-ABL mRNA by QPCR (IS) using an assay with
  4. a sensitivity of at least 4.5 logs below the standardized baseline. CMR is variably described, and is best defined by the the assay’s level of sensitivity (eg. MR 4.5).
Relapse Resistance
  1. Any sign of loss of response (defined as hematologic or cytogenetic relapse)
  2. 1-log increase in BCR-ABL transcript levels with loss of MMR (two consecutive samples to be tested)
  3. loss of CCyR

The haematological and cytogenetic milestones in the treatment of CML have been described by the NCCN and ELN.  Secondary resistance to TKIs results from point mutations. Mutations analysis of the kinase domain of ABL should be performed in case of failure to achieve or loss of a milestone. Mutations that result in resistance to the first line TKIs are characterised and mutational analysis serves as a guide to choose second line therapy. The T315I mutation imparts resistance to all TKIs except ponatinib.

The TKI treatment needs to be continued lifelong. Attempts to withdraw treatment in patients who have achieved prolonged deep molecular response have shown that the disease returns in a majority of the patients on discontinuation of treatment.

Allogenic stem cell transplant was the mainstay of treatment of CML before the introduction of TKIs. The availability of many TKIs has diminished the role of allogenic stem cell transplant. Allogenic stem cell transplant is indicated only in patients who progress on TKIs.

Prognosis

Introduction of imatinib altered the prognosis of CML by converting it form a progressive ad fatal disease to a chronic disease. The 8 year survival of CML on imatinib is 85%. Those progressing on imatinib can be treated with other TKIs.