Drugs and Eosinophilia


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

Clinical Spectrum of Drug Induced Eosinophilia

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

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

Management

The incriminating drug should be withdrawn in symptomatic patients.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

Clinical Features of Megaloblastic Anaemia


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

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

 

Manifestationf o megaloblastic anaemia

Figure 1. Clinical Manifestations of Megaloblastic Anaemia

Haematological Manifestations

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

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

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

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

Neurological Manifestations

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

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

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

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

Other manifestations

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

Response to therapy

Haematological Recovery

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

Neurological Recovery

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

 

 

Classification of β-Thalassaemia


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

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

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

The BCR-ABL1 Gene


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

 Molecular Biology of BCR-ABL

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

 

Molecular Biology of CML

The BCR-ABL1 fusion gene and it’s variants

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

Table 1: The BCR-ABL1 fusion genes

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

Mutagenicity of BCR-ABL1

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

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

Targeting the BCR-ABL1 Gene

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

 

Further Reading

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

Staging of Multiple Myeloma


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

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

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

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

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

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

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

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

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

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

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

The revised staging system is as follows

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

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

Dominant-Negative Phenotype


The human genome has two alleles of any gene. An allele may be dominant, recessive or co-domainant. Only one copy of an allele is needed for a dominant allele to express. A recessive allele expresses only when both the alleles are recessive.

The A and B blood group allele is dominant over the O group but are co-dominant with respect to each other. Individuals having genotype AA or AO express the A blood group antigen. Similarly individuals having genotype BB or BO express the B blood group antigen. An individual having genotype AB express both the blood group antigens. O is a recessive allele and only individuals with genotype OO have blood group O.

The biochemical basis of the ABH system is modification of the H antigen by enzymes encoded by the genes of blood group substances. Addition of N-acetylgalactosemine by the glycosyltransferase encoded by the allele encoding for A blood group converts the H antigen to A group antigen. Similarly the addition of galactose results in the synthesis of B group antigen. The allele for O group has a mutation and does not add any sugar.

Recessive genes are genes with mutations that result in loss of function. This is the case with the O blood group gene or with a thalassaemia gene. If recessive allele is present in the heterozygoyus state the product of the dominant allele, which encodes a functional protein, ensures there is synthesis of functional protein. The levels of encoded proteins in the presence of a mutant and recessive gene may be lower than in an individual without the mutant gene. Normal function is possible even with reduced levels of the encoded protein in almost all individuals and no disease occurs. This is the case with individuals with thalassaemia trait who have lower haemoglobin than normal individuals but this does not cause symptoms related to anaemia.

Some genes that encode for non-functional proteins manifest even when heterozygous with a functioning gene. These genes are known as dominant-negative. These genes usually encode for proteins that are dimers or multimers. The recessive gene has the ability to “poison” the components of the dimer/multimer produced by the normal genes and render them functionless.

Recessive genes encode for proteins that cannot form dimers/multimers. The product of these genes has no effect on the activity of the protein encoded by the normal allele. The levels of proteins may be reduced but all the protein synthesized by the dominant gene is functional. Usually the levels of proteins fall to half that in normal. This is almost always sufficient for normal function.

Dominant-Negative

Figure 1. The dominant negative phenotype

 

The dominant-negative genes have the capacity to form dimers/multimers and also also have the capacity to inhibit the function of the product of the normal gene (figure 1). A patient carrying a dominant-negative allele has reduced levels of functional proteins because of inhibition of function by the product of dominant negative allele. If one considers a patient who has a dominant negative mutation for a protein that is a homodimer, as is the case with factor XI, 75% of the factor XI synthesized will have the monomer produces by the dominant negative gene, Only 25% of the factor XI will be functional. This is consistant with the observation that these patients have factor XI levels between 20-30%. The situations in multimeric proteins and those proteins that are hetrodimers/hetromeltimers is more complex but the principle remains the same.

Primary Cutaneous DLBCL – Leg Type


The first description of a primary cutaneous diffuse large B cell lymphoma was by Willemze et al in 1987 who described a group of elderly women with cutaneous large cell lymphomas with tumours in the legs and a worse prognosis ( Am J Pathol. Feb 1987; 126(2): 325–333).

Primary cutaneous diffuse large B cell lymphoma, leg type is a type of high grade cutaneous B cell lymphomas that was included as a separate entity in the WHO 2008 lymphoma classification. It forms about 20% of all cutaneous B cell lymphomas and about 4% of all cutaneous lymphomas. It is more common in women and the median age of occurrence is the 7th decade.

Pathology

Primary cutaneous large B cell lymphoma is characterized by a monotonous, diffuse, non-epidermotrophic infiltrate that is CD 20 and CD79a positive. and almost always express BCL2, IRF4/MUM1 and FOX-P1. The latter three markers are not expressed in in the primary cutaneous follicular centre cell lymphoma  another type of primary cutaneous B cell lymphoma. BCL6 is usually expressed but CD10 is not. 

Clinical Features

DLBCL-LT is a disease of elderly women (M:F:12-4, median age 70 years). Though called leg type, only 85-90% of the primary cutaneous DLBCL, leg type occur in the legs. The remaining occur at other sites. Patients present with a rapidly growing red or reddish blue nodule on one or both the legs. Patients may have ulceration and may be confused with venous ulcer. Unlike other cutaneous lymphomas primary cutaneous DLBCL, leg type disseminates to non-cutaneous sites.

Treatment

Radiotherapy

As DLBCL-LT has a tendency to disseminate to extra-cutaneous sites than other cutaneous lymphomas radiation is less effective in this disease. . A complete response rate of 88%  with a high (58%) relapse rate has been reported. relapses are in the in field and extra-cutaneous.

Chemotherapy

R-CHOP is the standard first line therapy. Dose reduction may be needed in elderly. Single agent rituximab is also an option but is associated with a high rate of recurrences. Linelidomide has been used in patients with relapse.