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.

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).

 

 

 

The BCR-ABL1 Gene


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

 Molecular Biology of BCR-ABL

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

 

Molecular Biology of CML

The BCR-ABL1 fusion gene and it’s variants

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

Table 1: The BCR-ABL1 fusion genes

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

Mutagenicity of BCR-ABL1

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

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

Targeting the BCR-ABL1 Gene

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

 

Further Reading

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

Erythropoietin and Erythropoietin Receptor


(Post updated on June 21st 2017)

Erythropoietin (EPO), a 34kD 166 amino acid polypeptide, is the main regulator of erythrocyte production. It acts via the erythropoietin receptor (EPOR). 

Importance of Erythropoietin Signalling

No inactivating mutations of EPO signalling pathway are known.  Mice with deletion of EPO or EPOR gene die of anaemia at a gestational age of 12-13 days. They have some haematopoiesis in the yolk sac but none in in foetal liver. CFU-E do not survive in the absence of erythropoietin. Erythropoietin is also essential for survival of the more mature population of BFU-E. EPO reduces apoptosis. The effects are mediated by STAT5 (signal transducer and activator of transcription 5). STAT5 induces the production of the anti-apoptotic protein bcl- x by binding to it’s promoter. As will be discussed below the non-receptor tyrosine kinase Janus kinase 2 (JAK2) activates STAT. Erythropoietin has effect on a range on non-errythroid tissue. These are beyond the scope of this article and is discussed elsewhere (JEM 2013;210:205).



Control of EPO Production


Erythropoietin is produced in response to hypoxia by the interstitial fibroblasts of the kidney. The induction of erythropoietin secretion by hypoxia involves

  1. Hypoxia inducible factor (HIF) that promotes the transcription of genes induced by hypoxia. There are three HIF, HIF1, HIF2 and HIF3
  2. HIF prolyl hydroxylase (PHD) an enzyme that uses oxygen as a substrate and is inactive in hypoxia. It promoted the degradation of HIF.
  3. pVHL (von Hipple-Lindau tumour suppressor gene product) is involved in degradation of HIF

Protein synthesis needs energy. It may appeal to common sense to synthesise proteins only when needed and conserve energy. Increasing protein synthesis is a slow process making it unsuitable for situations where a rapid response is needed. Hypoxia needs a rapid response. The alternate strategy adopted by the body is to synthesise a protein continuously and control the levels of the protein by controlling degradation. A peptide may be made inactive by breaking just one critical bond. Synthesis needs building tens to hundred bonds. Degradation can be stopped almost instantaneously resulting in a rapid rise of the desired molecule. HIFs are synthesised and degraded continuously. The degradation mechanism is hypoxia sensitive allowing for a rapid rise of HIFs and consequently in the transcription of genes under control of HIFs as the partial pressure of oxygen falls. 

There are three HIFs, HIF-1, HIF-2 and HIF-3. HIF-2 controls erythropoietin production. HIF-2 has two units, α and β. The levels of the β subunit are constant. The levels of the α vary inversely with oxygen availability and determine the HIF 2 concentration activity. The α subunit is continuously being synthesized. Synthesis is matched by degradation by a proteolytic system known as proteasome. Proteasome only degrades peptides marked for destruction. Peptides are marked for destruction by tagging them by a multiple molecules of a protein ubiquitin. Ubiquitin is transferred by an enzyme complex known as ubiquitin E3 ligase. This complex consists of pVHL (product of the von Hipple-Lindau tumour suppressor gene), elongins B and C, cullin 2 and ring box 1 (Rbx1). pVHL identifies targets giving the ubiquitin E3 ligase specificity for HIF-1 and HIF-2. HIF needs to be hydroxylated at proline residues before polyubiquitination. The hydroxylation is brought about at proline residues by HIF prolyl hydroxylase (PHD). Oxygen is one of the substrates for PHD. In hypoxic conditions hydroxylation and consequently polyubiquitination does not take place. Proteasomal destruction stops and the levels of HIF-2α levels rapidly rise. HIF-2 translocates to the nucleus where it combines with HIF-2β. The heterodimer (HIF 2) acts on segments of DNA, known as hypoxia response elements, flanking the erythropoietin gene finally leading to erythropoietin synthesis. HIF 2 along with HIF 1, which is regulated by mechanisms identical to those regulating HIF 2, promotes the expression of multiple genes of proteins involved in response to hypoxia.



Mechanism of Action


EPO acts via the EPOR. Binding of erythropoietin has proliferative and anti-apoptotic effects. Receptor activation results in activation of a cascade of enzymes by phosphorylation. These include STATs (a family of seven proteins), Pi3K and RAS/MAPK/Erk. Phosphorylation can either be brought about by the tyrosine kinase activity of the receptor or, as is the case with the erythropoietin receptor, by a non-receptor tyrosine kinase associated with the receptor. The Janus kinase 2 (JAK2) is the non-receptor tyrosine kinase that associates with the erythropoietin receptor. The EPO receptor is a homodimer (a dimmer made from similar monomers). Each monomer associates with a JAK2 molecule.  Binding of EPO to EPOR brings about a conformational change in the receptor bringing the two JAK2 moleclues in proximity that results in transautophosphorylation and activation of the JAK2 kinases. Activated JAK2 phosphorylates tyrosine residues on the receptor forming docking sites for molecules that activate the following pathways:

  1. Dimerization and translocation of STAT5 to the nucleus where it induces transcription of genes involved in proliferation and cell survival. STAT pathway appears to be the most important pathway for EPO action.
  2. Phophoinositide-3-kinase (PI3-K) mediated induction of several anti-apoptotic proteins e.g. Bcl-2 abd BclX. As mentioned above STAT5 is also involved in induction of bcl-x.
  3. Activation of Ras/extracellular-signal-regulated kinase mitogen-activated protein (RAS/Erk/MAP) kinase pathway that sustains proliferation.

The EPO signalling is short lasting and the activated EPOR signal pathway returns to normal levels in 30-60 minutes. The cytoplasmic portion of the receptor is polyubiquitinated and degraded by proteasome. The extracellular portion bound to EPO is internalized and degraded. Regulators of cytokine activity can inhibit EPOR.


Mutations and Therapeutic Manipulation of EPO Signalling


Acquired (somatic) mutations of the JAK2 kinase are associated with myeloproliferative disease. The JAK2V617F mutation is seen in about 95% of the patients of polycythaemia vera and about 50% the patients with essential thrombocytosis and idiopathic myelofibrosis. JAK2V617F causes the JAK2 molecule to be constitutively (continuously, irrespective of EPO binding to EPOR) active eliminating the need for binding of EPO to EPOR for activation pathways stimulated by EPOR. JAK2 exon 12 mutations are found in patients with JAK2V617F negative polycythemia vera. These patients, unlike those with JAK2V617F positive patients, do not show thrombocytosis or leucocytosis. Inherited (germline) mutations in the EPOR, HIF 2α, VHL gene and the PHD gene have been associated with congenital erythrocytosis. The erythrocytosis seen in patients with EPOR mutations is primary, i.e. with low EPO levels. Other mutations result in secondary erythrocytosis, i.e. with inappropriately high EPO levels. Inhibitor of HIF prolyl hydroxylase FG-2216 and FG-4592 are under evaluation for treatment of anaemia associated with chronic kidney disease.