Nodular Lymphocytic Predominant Hodgkin’s Lymphoma


Hodgkin lymphoma (HL) is of two types. Classical (cHL) and nodular lymphocyte predominant (NLPHL). NLPHL is rarer and runs a more indolent clinical course.

Epidemiology

NLPHL accounts for about 5% of all HL.

Age: The disease is characterised by two peaks. The first one in childhood and the second between the ages of 30-40.

Gender: NLPHL shows a male predominance. About three-fourth of the patients are males. Male preponderance is less marked in blacks.

Racial Differences: Black patients are younger, more often female and more often present with axillary involvement. Little is known of NLPHL in other races (Cancer 2015; 121:3472-80).

Familial Susceptibility: Family members of patients with NLPHL at increased risk NLPHL. The standardised incidence ratio in one study was reported to be 19 (J Clin Oncol 2013; 31;938-43).

Histology

The normal architecture of the node is effaced and replaced by large nodules. Occasionally there may be large nodules with diffuse areas. Sometimes uninvolved nodal tissue may be seen. This is usually located peripheral in a sub-capsular area.

Microscopically NLPHL shows the malignant cell, LP cell, in a background mainly made up of small lymphocytes and with a prominent follicular dendritic cell (FDC) network. The follicular dendritic cell meshwork is absent from the diffuse areas. Unlike most other malignancies (and like cHL and T cell/Histolytic rich large B cell lymphoma) the normal reactive cells form the bulk of the enlarged node.

The LP cell has a nucleus that shows complex lobulation. It resembles a exploded kernel of corn and hence the cell is also referred to as the popcorn cell. The nucleolus is smaller than that of the RS cell and lies peripherally and is basophilic. There is a thin rim of cytoplasm.

The infiltrate in a nodule mainly consists of small lymphocytes. Unlike cHL, Eosinophils and plasma cells are occasional or may be absent. Most of the small lymphocytes making up the nodule are CD20+, CD79+ small B lymphocytes. The LP cells is however immediately surrounded by CD20, CD3+ T helper cells that express PD-1 and CD57. Diffuse area have CD4+ T cells and areas between nodes have CD3+ parafollicular T cells.

Varient histological patterns are known, associated with adverse prognosis and should be reported (Am J Surg Pathol 2003;27:1346-56).

Immunophenotype helps in diagnosis and has given clues to the origin of LP cells. The LP cells show a B cell phenotype and express CD20, CD79, CD22, PAX-5 and CD45. They express BCL-6 indicating the germinal centre origin. They do not express BCL-2. They strongly express the B cell transcription factor OCT-2 and its cofactor BOB.1. This distinguishes then from the Reed-Sternberg (RS) cells of cHL. RS cells show a weak expression or do not express these factors. RS cells express CD15, CD30 and fascin that are not expressed by the LP cells. About a fifth of the patients express IgD. These patients tend to be male, present with cervical adenopathy and have a greater risk of having a variant histology.

The normal counterpart of the LP cell appears to be the germinal centre B cell at the cenrtoblastic stage of differentiation.

NLPHL as well as cHL are diseases characterised by malignant cells surrounded by an infiltrate of normal cells. Unlike other cancers, the normal cells form the bulk of the tumour mass in both the cases. The malignant cells affect and are affected by the normal cells surrounding them. LP cells, like normal germinal centre cells, appear to depend on normal immunoglobulin receptor signalling. RS cells depends on other signalling receptors e.g. CD30 and CD40. The growth of normal germinal centre cells depends on The FDC and follicular T cells. These cells also support the growth of LP cells. The LP cell do not produce cytokines at levels seen in the RS cell. B symptoms are less common NLPHL less common than cHL.

 

 

Clinical Presentation

The most common presentation of NLPHL is isolated lymphadenopathy, most often in the cervical, axillary or the inguinal region. The swelling is usually present for a long time and has been growing slowly. About 80% of the patients present with localised disease and less than 20% with stage III/IV (Ann Hematol. 2016; 95: 417–423). B symptoms are uncommon (about 5%). Extranodal disease is very uncommon.

NLPHL runs a more indolent course that cHL. It is characterised by a relapses and transformation to high grade lymphoma diffuse large B cell lymphoma (including T cell/ histiocyte rich large B cell lymphoma). Relapses usually respond to treatment.

Staging

NLPHL, like cHL is classified by the Ann Arbor staging system with Cotswolds modifications. The stages are summarised below. A more detailed staging can be found here.

  1. Stage I: Involvement of one nodal region, lymphoid structure or one extra-nodal site
  2. Stage II: More than one region involved but disease limited to one side of the diaphragm.
  3. Stage III: Disease on both sides of the diaphragm but limited to the lymphoid system.
  4. Stage IV: Disease disseminated to one or more extra nodal organs.

Patients with fever with hight sweats and significant (>10% in the preceding 6 months) are said to have B symptoms.

The staging workup should include clinical examination, haemogram, ESR and biochemistry. NLPHL is PET avid. PET-CT is better than CT for staging. It is of value in to exclude diseases dissemination in patients where observation or local treatments are being considered. The value is interim PET-CT is NLPHL is uncertain. The bone marrow is very uncommonly involved (about 1-2%). Only patients with advanced disease should be subjected to bone marrow examination.

 

Differential Diagnosis

  1. Lymphocyte Rich Classical Hodgkin lymphoma
  2. T cell/ Histiocyte Rich Large B Cell Lymphoma
  3. Progressively Trasnformed germinal centres
  4. Follicular Lymphoma
  5. Mantle cell Lymphoma

 

Treatment

Early disease (Stage I/IIA)

Patients who have undergone excision biopsy that has resulted in a complete removal of all disease may be observed. Despite a lower progression free survival the patients who are observed do not show an inferior overall survival. This indicates that delaying treatment (radiation, chemotherapy or both as may be appropriate) does not hamper it’s efficacy.

Advanced Disease (Stage IIB, III, IV)

These patients need chemotherapy with the anti-CD20 antibody, rituximab. Three approaches are possible

  1. Classical Hodgkin lymphoma like therapy with Rituximab with ABVD: R-ABVD (Rituximab, doxorubicin, bleomycin, vinblastine and dacarbazine) should be administered to patients needing chemotherapy.
  2. B cell non-Hodgkin Lymphoma like therapy: R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) is the standard treatment for high grade B cell non-hodgkin lymphoma. R-CHOP has been shown to effective in disease control and reducing the risk of transformation. It may be preferred in patients at a high risk of transformation, though there is not comparative trial with R-ABVD. Males and those with variant histology are at a higher risk of transformation. Models for predicting transformation are available.
  3. Single agent Rituximab: Single agent rituximab is indicated in patients with co-morbidities. The risk of relapse remains high.

Treatment of Relapse

Relapses must be rebiopsied to confirm NLPHL and to exclude transformation to a high grade lymphoma. Localized relapses may be treated with radiation. Chemotherapy should be used for other patients. Patients who have a chemosensitive relapse may be considered for allogenic stem cell transplant (Am J Haematol 2017 Oct 3. doi: 10.1002/ajh.24927).

Treatment of Transformation

Patients who undergo transformation are treated with regimen for regimens for high grade B cell lymphoma. The limited data suggests that the outcome is no different from that of de novo large B cell lymphoma.

 

Prognosis

The prognosis of NLHPL is better than conventional HL partially because of a more favourable disease profile – early stage, no B symptoms, no Bulky disease. One study showed a 94% overall survival at 10years (Ann Hematol. 2016; 95: 417–423). The progression free survival was 75% indicating relapses are common but are curable. Progression to diffuse large B cell lymphoma is seen in 5-10% of the patients. Atypical histology increases the risk of relapse (Blood. 2013 Dec 19;122(26):4246-52).

 

 

Advertisements

Myeloproliferative Neoplasm


Myeloproliferative neoplasm are a group of disorders characterized by bone marrow proliferation with increase in the number of blood cells. The proliferation my be limited to one cell line or may involve more than on cell line. They are distinct from acute leukaemia but carry the risk into evolving into leukaemia as a terminal event. This article discusses general aspects of myeloproliferative neoplasm. The disease entities will be discussed elsewhere.

Progressive myelofibrosis was the first myelroliferative neoplasm to be described. It was described by Gustav Heuck in 1879. This was followed by chronic myeloid leukaemia (CML) by John Hughes Bennett in 1845, polycythaemia vera (PV) described by Louis Henri Vaquez in 1892 and essential thrombocytosis (ET) described by Emil Epstein in 1934. The term myeloproferative disease was coined by Dameshek in 1951 to describe seven conditions that he thought were manifestations of “proliferation of bone marrow to a hitherto undiscovered stimulus” (Blood 1951; 6:372-75). The seven conditions were chronic granulocytic leukaemia (now chronic myeloid leukaemia), polychthaemia vera, agnigeic myeloid metaplasia (now primary myelofibrosis), thrombocytosis (essential thrombocytosis), megakaryocytic leukaemia and erythroleukaemia (including DeGuglielmo’s syndrome). As the malignant nature of these disease was not apparent till recently they were referred to as myeloproliferative disorder. The recognistion of the neoplastic in nature of the diseases has resulted in the change in the name from myelproliferative disorder to myeloprlferative neoplasm.

The WHO 2016 classification of myeloproliferative neoplasm includes the following disease

  1. Chronic myeloid leukaemia (CML)
  2. Chronic neutrophilic leukaemia (CNL)
  3. Polycythaemia vera (PV)
  4. Progressive myelofibrosis (PMF)
  5. Essential thrombocytosis (ET)
  6. Chronic myeloproliferative neoplasm unclassified
  7. Chronic eosinophilia leukaemia NOS
Myeloproliferative disease

Pathogenesis of myeloproliferative diseases

Pathogenesis of Myeloproliferative Neoplasm

The bone marrow proliferates in response to an initiating event (see figure above). This event remains to be discovered. Bone marrow proliferation results in increased blood counts. Increase in erythrocytes results in polycythaemia and features of hyperviscosity. Increased leukocyte counts result in leukostasis. The leucocytes counts that can cause leukostasis are only seen in CML. Thrombocytosis increases the risk of thrombosis. With time a MPN disease progresses. Progression either results in blast transformation or myelofibrosis as the terminal event. CML is more likely to progress to blast crisis and non-CML MPNs to myelofibrosis. Splenomegaly is an early feature of CML. Spleen in enlarged because of infiltration of the spleen by neoplastic haemopoietic cells despite there being no myelofibrosis. In non-CML splenomegaly occurs late and is a result of extra-medullary haematopoiesis due to myelofibrosis.

Clinical Aspects og Myeloproliferative Neoplasm

The myeloproliferative disease have the following common features

  1. Clonal proliferation of bone marrow: The Philadelphia chromosome associated with CML was the first chromosomal abnormality associated with a malignancy. The Philadelphia chromosome is a t(9;22) translocation that results in the formation of the BCR-ABL1 fusion gene. It proved the clonal nature of CML. Mutations proving the clonality of non-CML myeloproliferative neoplasm have been discovered. These include the JAK2, MPL and CALR mutations found in PV, PMF and ET. CNL is characterised by mutations in CSF3R. These mutations are the “undiscovered stimulus” Dameshek wrote about.
  2. Increased Blood Counts: Myeloprolifarative neoplasm have increase leucocytes, erythrocytes and thrombocytes alone or in combinations.
  3. Splenomegaly: Splenomagaly is a future of all myeloproliferative neoplasm. It is most pronounced in CML and PMF. The underlying mechanisms of splenomegaly in the conditions are different. Patients with CML have infiltration. Some develop myelofibrosis. Massive splenomegaly in myeloproliferative neoplasm than CML indicated bone marrow fibrosis.
  4. Overlapping clinical features: The clinical features of myeloproliferative neoplasm overlap. Polycythaemia is only seen in PV. Leucocytosis is a feature of all conditions other than ET. The type of leucocytosis can distinguish entities. Exteme leucocytosis with premature myeloid forms points to CML. But all patients with CML do not have extreme leucocytosis. Neutrophilic leucocytosis is a feature of CNL. Thrombocytosis is a featured shared by myeloproliferative neoplasm. As discussed below myelofibrosis develops in all myeloproliferative diseases. All myeloproliferative neoplasia are at risk of transforming into acute leukaemia. Despite the use of BCR-ABL1 tyrosine kinase inhibitors the risk is of acute leukaemia transformation is highest for CML.
  5. Development of bone marrow fibrosis: As myeloproliferative neoplasia progress bone marrow fibrosis increases. Myelofibrosis develops fastest in progressive myelofibosis. CML often does not develop clinically evident fibrosis but transforms to acute leukaemia as a terminal event. Development of fibrosis results in pancytopenia. Once fibrosis sets in it is not possible to tell the nature of the pre-fibrotic myeloproliferative neoplasm.
  6. Transforamation to acute leukaemia: All myeloproliferative neoplasia have a risk of evolving to acute leukaemia. This is known as blast crisis. The risk is greatest in CML.
  7. A tendency for thrombosis: Myeloproliferative neoplasm increase the risk of thrombosis. The risk is highest in ET and PV.

Treatment of Myerloproliferative Neoplasm

The goals of therapy of myeloproliferative neoplasia are

  1. Prevention and treatment of complications of increased cell counts
  2. Prevention of progression of MPN
  3. Erradicating the malignant clone

Prevention and treatment of complications of increased cell counts

Acute complication of myeloproliferative neoplasia include Leukostasis hyperviscosity and thrombosis. Polycythaemia is addressed by phlebotomy. Leukostasis is treated by measures to reduce counts (see Hyperleukoytosis and leukocytosis). Thrombocytosis of ET is treated low dose aspirin with or without hydroxyurea. The exact combination depends on the diagnosis and the risk stratification. Low dose aspirin is administered to patients with PV and PMF as they are at risk of thrombosis.

Prevention of progression of MPN

The only MPN where progression can be retarded is CML. The use of BCR-ABL1 tyrosine kinase inhibitors (BCR-ABL1 TKI) like imatinib, nilotinib or dasatinib prevent progression. The longest results are available for imatinib. Eithty five percent of the patients are alive at 10 years compared to a median survival of 45.4 with busulifan and58.2 months with hydroxyurea (Blood. 1993 Jul 15;82(2):398-407).

Eradicating the malignant clone

Supression of the BCR-ABL1 positive clone does not result in eradication of the malignant clone. More than half the patients in deep remission have a relapse of CML in one year off stopping BCR-ABL1 TKI therapy. Allogenic stem cell transplant offers the only possibility of cure in CML. The safety and efficacy of BCR-ABL1 TKIs has reduced the role of allogenic stem cell transplant in CML. It is now used in patients who are poorly controlled with upfront BCR-ABL1 TKI therapy. Allogenic stem cell transplant may be used in selected patients with PV, ET and PMF but does not form a front line therapy.

Evaluation of Splenomegaly


The spleen is a secondary lymphoid organ that lies in intraperitoneally in the left hypochondrium, abuting the diaphragm. It spans from the 9th to 11th rib and weighs between 150-200g. Spleen is supplied by the splenic artery and drains into portal circulation via the splenic vein. It is a part of reticuloendothelial system, immune system and is a site of in utero haematopoiesis. The spleen is enlarged in a diverse set of disease of the above mentioned  systems and in portal hypertension.

Normal Functions of the Spleen

The normal functions of the spleen include

  1. Reticuloendothelial functions: The spleen as a component of the reticuloendothelial system is involved in clearing the blood of ageing or damaged erythrocytes, antibody coated cells and opsonised bacteria. It also removes particles from red cells. The spleen ensures that the red cell in circulation have adequate deformability for passage through microcirculation.
  2. Immune Functions: The spleen is a part of the immune system and plays a role in mounting the immune response . Splenectomy increases the risk of infections particularly with capsulated organisms (see Overwhelming Post-Splenectomy Infection (OPSI)).
  3. Haematopoiesis: Spleen is the site for haematopoiesis in utero. In extrauterine life spleen can become a site of haematopoiesis in disease.

Palpating the Spleen

  1. Palpation of the spleen should start from the right iliac fossa. If this is not done there is a risk of missing a massively enlarged spleen.
  2. Move towards the left costal margin in a direction perpendicular to the margin. Move with each breath. At every position ask the patient to take a deep breath. The tip of the spleen will hit your palpating finger.
  3. If the spleen does not hit your finger move your palpating finger to a position closer to coastal margin, ask the patient to take a deep breath and repeat the procedure described above till your finger hits the costal margin.
  4. If the spleen is felt measure the perpendicular distance between the tip and the left coastal margin. Also note the texture and presence of tenderness.
  5. If the spleen is not felt repeat the procedure with patients lying on right side.
  6. Large spleen can rupture with aggressive palpation. The spleen lies directly under the anterior abdominal wall. One does not need to be aggressive.

Causes of Splenomegaly

The spleen enlarges from the left coastal margin in the direction of the umbilicus. It needs to enlarge 2-3 times before it is palpable. Splenomegaly may be caused be increase in portal venous pressure, infiltrative conditions or when the spleen function needs to increase. Clinically it is useful to classify splenomegaly by size. Massive splenomegaly is enlargement of the spleen beyond the umbilicus. The causes of massive splenomegaly include

  1. Malignant: Chronic myeloid leukaemia, Idiopathic myelofibrois, hairy cell leukaemia, splenic marginal zone lymphoma, chronic lymphocytic leukaemia, prolymphocytic leukaemia
  2. Infections: Tropical splenomegaly, AIDS with Mycobacterium avium complex infections, Kala-azar (visceral leishmaniasis)
  3. Others: β-Thalassaemia major and intermedia, Extrahepatic portal venous obstructions,megaloblastic anaemia, diffuse splenic haemagiosis

The causes of splenomegaly include the above and the following

  1. Portal Hypertension: Cirrhosis, Budd-Chairy syndrome, splenic vein thosmbosis, congestive heart failure, hepatic schistosomiasis
  2. Increased splenic function:
    1. Increased functional demands: Haemolytic anaemia commonly hereditary spherocytosis, autoimmune haemolytic anaemia, β-thalassaemia, early sickle cell anaemia, sickle cell β-thalassaemia,
    2. Infections:
      1. Bacterial: Septicaemia, bacterial endocarditis, splenic abscess, brucellosis, tuberculosis, AIDS with Mycobacterium avium complex infections, secondary syphilis
      2. Viral: Viral hepatitis, infectious mononucleosis, cytomegalovirus,
      3. Parasitic: Malaria , Kala-azar (visceral leishmaniasis), Trypanosomiasis,
      4. Fungal: Histoplasmosis
    3. Immune Disorders:
      1. Autoimmune diseases: Rhumatoid arthritis (Felty’s syndrome), systemic lupus erythrmatosis
      2. Other immune disorders: Immune haemolytic anaemia, immune neutropenia, drug reaction, serum sickness, sarcoidosis
      3. Haemophgocytic lymphohistiocytosis
  3. Infiltrations
    1. Haematological Malignancy:
      1. Myeloid: Chronic myeloid leukaemia, myeloproliferative disease, idiopathic myelofibrosis, polycythaemia vera
      2. Lymphoid: Acute lymphoblastic leukaemia, hairy cell leukaemia, chronic lymphocytic leukaemia, prolymphocytic leukaemia, splenic marginal zone lymphoma, angioimmnoblastic T cell lymphoma
      3. Other: Histiocytosis X, eosinophilic granuloma
    2. Storage disorders:Gaucher disease, Niemann-Pick, Tangier disease, mucopolysachroidosis
    3. Other Infiltrations: Amyloid
  4. Others: Iron deficiency anaemia

 

History and Physical Examination

  1. Fever: Fever is a feature of splenomegaly due to infections, inflammations or malignancy, particularly haematological malignancy. Usually the fever is low grade. High grade fever suggests splenic abscess.
  2. Painful splenemegaly: The nature of pain associated with splenomegaly varies with the cause of splenomegaly.
    1. An enlargement spleen from any cause can cause a dragging pain in the left upper quadrant.
    2. Acute pain left upper quadrant pain is a feature of is a feature of splenic infarct and splenic abscess. Sickle Cell anaemia is associated with small fibrotic spleen because of repeated splenic infarcts. Early in disease the spleen enlarges. Patients may present with acute pain from splenic infarcts. Enlarged spleen from any cause is predisposed to infarction. Acute pain in the left upper quadrant is also a feature of acute splenic abscess.
    3. Splenic vein thrombosis can cause splenomegly and pain in left upper quadrant or epigastric region. It may also cause generalised abdominal pain.
    4. Pancreatitis presents with abdominal pain and can cause painful splenomegaly secondary to splenic vein thrombosis.
    5. Alcohol induced pain is an uncommon but unique feature of Hodgkin lymphoma. Spleen is a common site of involvement by Hodgkin lymphoma. Such patients may have alcohol induced pain in an enlarged spleen.
  3. Pallor: Pallor in a patient with splenomegaly suggests a diagnosis of haemolytic anaemia, haemolymphatic malignancy and infective endocarditis.
  4. Clubbing: Clubbing with splenomegaly is a feature of infective endocarditis and cirrhosis of the liver.
  5. Skin rash: Skin rash in a patient with splenomegaly is seen in systemic lupus erthomatosis, infective endocarditis, lymphoma (angioimmuniblastic T Cell lymphoma, mycosis fungiodes, skin involvement with lymphoma) and drug reaction.  Each of these conditions have a distinct type of rash.
  6. Skin Pigmentation: Hyperpigmantation suggests be seen in hemachromatosis or megaloblastic anaemia. The patients with megaloblastic anaemia may also have knuckle pigmentation.
  7. Jaundice: Jaundice with enlarged spleen is a feature of haemolytic anaemia. The jaundice is usually achloruric. Patients with haemolytic anaemia are predisposed to gallstones. Obstruction of the biliary system from a calculus dislodged from the gall bladder can cause obstructive jaundice with abdominal pain and signs of acute inflammation. Splenomegaly with jaundice is a feature of advanced cirrhosis. Patients with advanced cirrhosis almost always have ascites.
  8. Lymphadenopathy: The enlargement of lymph nodes and spleen is a feature of lymphoid malignancies or diseases that stimulate the lymphoid systems viz. infections and autoimmune diseases and lymphoid malignancy.
  9. Joint symptoms: Arthropathy with splenomegaly suggests the diagnosis of rheumatoid arthritis, systemic lypus erythrmatosis or haematochromatosis.
  10. Oral symptoms: infectious mononucleosis is charecterized by pharyngitis and generalised lymphadenopathy. Bleeding gums and/or gum hypertrophy suggests a diagnosis of leukaemia. Lymphoma can cause tomsillar enlargement. Amyloid is charectetized by macroglossia.
  11. Evidence of Portal Hypertension and Liver Cell Failure: Patients with portal hypertension often have history of haemetemesis. Examination may reveal periumbilical veins (capital medusae), anterior abdominal or flank veins. Patients with evidence liver cell failures with portal hypertension (e.g. jaundice, ascites, spider angiomas, asterxis etc. see Portal Hypertension) have cirrhosis. When the jugular venous pressure is high a diagnosis of congestive cardiac failure should be considered.

Laboratory Evaluation

Haemogram; The haemogram is the most important laboratory test in evaluating a patient with splenomegaly. The significance of findings on haemogram is described in the table below.

Haemogram Finding Conditions
Pancytopenia Hypersplenism, Lymphoma (splenic marginal zone lymphoma), Hairy cell leukaemia, Myelofibrosis, systemic lupus erythrmotosis
Neutrophilic Leucocytosis Acute infections, inflammation
Leucocytosis with premature white cells Chronic myeloid leumaemia, Myeloproliferative disease, Myeloproliferative/Myelodysplastic overlap, Acute lymphoblastic leukaemia
Leucoerythroblastic anaemia Idiopathic myelofibrosis, Bone marrow infiltration
Polycythaemia Polycythaemia vera
Atypical Lymphocytes Infectious mononucleosis
Thrombocytosis Myeloproliferative disease (Chronic myeloid leukaemia, idiopathic myelofibrosis, polycythaemia vera), chronic infections like tuberculosis
Parasites Malaria, bartonelosizs, babesiosis

Other investigations are dictated by the clinical presentations. Commonly performed investigations include biochemistry, microbiology, echocardiography, endoscopy and biopsy of any lymph node or any other mass. Other investigation may be performed as indicated

Imaging

Imaging is an important aspect of evaluation of the spleen but is beyond the scope of this article. Several good reviews exist e.g Singapore Med J 56(3):133-144.

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.

 

 

Calreticulin and Myeloproliferative Neoplasm


Myeloproliferative neoplasm (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.