Chronic myelogenous leukemia pathophysiology

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]Associate Editor(s)-in-Chief: Badria Munir M.B.B.S.[2] Mohamad Alkateb, MBBCh [3] "sandbox:SN"

Editor-In-Chief: C. Michael Gibson, M.S., M.D. [4]; Associate Editor(s)-in-Chief:

Overview

Pernicious anemia (also called Addison's anemia) is a type of red blood cell disorder caused by impaired vitamin B12 metabolism. Vitamin B12 is primarily absorbed by the small intestine, after being bound to intrinsic factor secreted by parietal cells of gastric mucosa. When this process is disrupted by conditions like atrophic gastritis, celiac disease, small bowel resection etc, B12 deficiency ensues.

Historical perspective

• Pernicious anemia was first discovered by Thomas Addison, hence it is also known as addison's anemia.
• Loss of life from large volume blood loss in the people fighting in the first world war inspired George Whipple to investigate blood forming components such as arsenic, iron pills etc, but found liver to be the most effective. He bled dogs until they had clinical anemia and fed them cooked liver which showed an improvement in symptoms and hematopoeisis.
• In 1948, Smith, Rickles et al., isolated the anti-pernicious factor from liver extract and named it Vitamin B12. They showed that even small amounts of this factor can be used to treat and to prevent pernicious anemia.

Pathophysiology

Vitamin B12 is an essential vitamin for humans and animals because we cannot synthesise it on our own. B12 is a cofactor in DNA synthesis and other important biochemical reactions. Vitamin B12 deficiency manifests as anemia because hematopoetic stem cells in the bone marrow which are rapidly dividing need B12 for division and DNA production. This process is impaired leading to ineffective hematopoeisis. Vitamin B12 is also necessary for production of myelin which is an important component in the covering sheath of nerves. Deficiency results in improper nerve conduction due to nerve destabilisation.

Physiology

• Vitamin B12 is also called cobalamin because it contains cobalt at the core of its structure. Dietary sources of vitamin B12 include meat, fish and eggs.
• When consumed through its dietary source, B12 is bound to protein till it enters the stomach.
• In the stomach, B12 is uncoupled from its carrier protein due to the presence of gastric acid, which is why vitamin B12 deficiency is so commonly seen among those on chronic antacid medication.
• Once in the stomach, it is then bound to gastric R binder, a glycoprotein secreted by the salivary glands till it reaches the duodenum.
• In the duodenum and jejunum, the pancreatic enzymes digest the gastric R binder and cobalamin is bound to intrinsic factor (IF).
• Intrinsic factor is secreted by the gastric parietal cells. Once bound to IF, vitamin B12 travels up to the ileum where IF is removed and B12 binds with carrier proteins called transcobalamins and this complex is taken up by the liver and bone marrow, among other tissues.
• Inside the cells, the transcobalamin-B12 complex is dissolved and cobalamin is reduced to methylcobalamin which serves as a cofactor and coenzyme in many important biochemical reactions.

The two major reactions involving B12 in the human body are:

• Vitamin B12 in the from of cyanocobalamin is required in the synthesis of methionine. Methionine is produced from homocysteine and is catalysed by the enzyme methionine synthase. This enzyme utilises cyanocobalamin as a cofactor. Deficiency of vitamin B12 causes a decreased production of methionine and buildup of homocysteine. Hyperhomocysteinemia is implicated as a risk factor in cardiovascular disease.
• The Kreb's cycle utilises vitamin B12 in the reaction converting methylmalonyl-CoA to succinyl-CoA. Thus vitamin B12 deficiency causes a buildup of methylmalonic acid, the substrate for the enzyme methylmalonyl coenzyme A mutase. Methylmalonic acid levels are elevated in the urine of people affected with pernicious anemia and other forms of B12 deficiency.

Storage

The human body can store anywhere from 2-5mg of vitamin B12. Most of this is stored in the liver and is recycled via enterohepatic circulation.

Pathogenesis

Pernicious anemia is a type of megaloblastic anemia caused due to improper vitamin B12 absorption by the body. Impaired absorption occurs because of deficiency of intrinsic factor which is produced by the parietal cells of the stomach. The etiology of pernicious anemia can be due to autoimmune causes or genetic disease. In autoimmune disease, the antibodies attack most of the gastric mucosa, but the antrum is spared.

Autoimmune causes of pernicious anemia

This is the most common cause of pernicious anemia. In autoimmune pernicious anemia, the body produces antibodies against parietal cells or intrinsic factor.

• Antibodies against parietal cells of the gastric mucosa work to inhibit the H+/K(+)-ATPase which is the proton pump present in the parietal cells. The proton pump serves as an auto antigen and activates the cytotoxic CD4+ T cells which proceed to destroy gastric mucosal cells.
• Intrinsic factor antibodies are present in fewer cases of pernicious anaemia but are highly specific. There are 2 types of IF antibodies. They prevent the binding and absorption of cobalamin in the ileum via its receptor.

Clinical features

• The symptoms of pernicious anemia take months, and often years to manifest. Patients most commonly present with symptoms of anemia like lightheadedness, dizziness, shortness of breath etc. The population affected with pernicious anemia is usually the elderly (>60 years) owing to its insidious onset.
• Pernicious anemia has hematological, gastrointestinal and neurological manifestations.
• Hematological signs are the earliest manifestation of the disease while neurological signs are seen much later.
• Patients with pernicious anemia usually have very low levels of hydrochloric acid in the stomach (achlorhydria) and high levels on gastrin (hypergastrinemia).

Differentiating pernicious anemia from other diseases

Pernicious anemia shares many similarities with other forms of megaloblastic anemia like B12 and folate deficiency.

• Vitamin B12 deficiency due to insufficient intake (eg veganism) has all the features of pernicious anemia like megaloblasts, hypersegmented neutrophils, neuropsychiatric manifestations. But atrophic gastritis is absent, so achlorhydria, parietal cell antibodies or IF antibodies are absent. Intrinsic factor levels are also normal.
• Folic acid deficiency also results in megaloblastic anemia and similar hematological changes as pernicious anemia, but urinary excretion of methylmalonic acid is absent, so are features of pernicious anemia like achlorhydria, antibodies and normal IF levels.
• Ileal resection causes B12 deficiency due to decreased absorption.
• Certain drugs such as methotrexate, azathioprine cause folate deficiency and result in megaloblastic anemia. This is usually seen in patients taking chemotherapy or other chronic conditions such as rheumatoid arthritis.
• Chronic proton pump inhibitor therapy also results in B12 deficiency as vitamin B12 cannot dissociate from its carrier protein in the absence of an acidic environment.
• Long term use of metformin, such as in diabetics, is linked to vitamin B12 deficiency and symptoms similar to pernicious anemia, but this can be differentiated from pernicious anemia as it is seen in diabetics on chronic therapy.

Associated Conditions

People affected with pernicious anemia might have other coexisting autoimmune conditions such as autoimmune thyroiditis, autoimmune diabetes, vitiligo etc. Autoimmune thyroiditis is most commonly seen in patients with pernicious anemia, particularly females. HLA DR3 has been implicated in the development of autoimmune diseases such as pernicious anemia.

Epidemiology and demographics

• Pernicious anemia is a disease of the elderly. The mean age of patients who are symptomatic is >60.
• An exception is the genetic form of the disease which is a congenital deficiency of intrinsic factor and is seen in children <10 years of age.
• Men and women are equally affected
• Prevalence of pernicious anemia is estimated at 0.1% of the population.

Genetics

• Some forms of pernicious anemia are congenital and a genetic link has been postulated because of a higher incidence in certain populations.
• Affected people have a complete or near total absence of intrinsic factor and the presence of antibodies against intrinsic factor.
• The genetic variant is transmitted through an autosomal recessive pattern.

Risk factors

• People who have autoimmune conditions like diabetes mellitus, autoimmune thyroiditis are at higher risk of developing pernicious anemia.

Natural History, Complications and Prognosis

• In most cases, patients affected with pernicious anemia remain asymptomatic for many years.
• Early manifestations include fatigue, shortness of breath, pallor and weakness.
• Long standing untreated pernicious anemia results in irreversible neurological damage such as subacute combined degeneration of the spinal cord.
• Neurological changes are irreversible once they set in and do not resolve with cobalamin supplementation.

Diagnosis

A diagnosis of pernicious anemia is made by a history and physical examination, along with hematological and neurological examination.

Diagnostic criteria

• The only specific criteria to diagnose pernicious anemia is an intrinsic factor output of less than 200U/h after pentagastrin stimulation, where normal levels would be >2000U/h.

Symptoms

Symptoms of pernicious anemia are summarised below

Hematological symptoms	Gastrointestinal symptoms	Neurological symptoms
Fatigue	Loss of appetite	Parasthesias
Weakness	Weight loss	Depression
Shortness of breath	Nausea	Gait problems
Dizziness	Burning sensation on tongue	Weakness
Tachycardia	Diarrhea	Loss of balance
Lightheadedness	Vomiting	Confusion

Physical examination findings

Most important physical examination findings are the neurological findings of long standing B12 deficiency which leads to subacute combined degeneration of the spinal cord.

• Hematological signs include pallor and icterus.
• Neurological signs: Vitamin B12 deficiency causes nerve demyelination. B12 deficiency also causes a buildup of methylmalonic acid which is toxic to neuronal cells and causes apoptosis..

The main neurological manifestation of pernicious anemia and vitamin B12 deficiency is subacute combined degeneration. The posterior and lateral columns of the spinal cord are affected. Lateral column demyelination manifests as hyperreflexia and spasticity, while posterior column defects are loss of proprioception and vibration sense. Ataxia and loss of tandem gait are also manifestations of posterior column demyelination. Recreational or accidental inhalation of nitrous oxide gas (laughing gas) can precipitate subacute combined degeneration in people with low levels of vitamin B12.

• Gastrointestinal signs: Upto 25% of people affected with pernicious anemia develop glossitis. The tongue appears red, "beefy" and smooth due to atrophy and blunting of the lingual papillae.

Subacute combined degeneration

Laboratory findings

• The first step in diagnosis is a blood vitamin B12 level. Blood levels less than 200 pg/ml are seen in pernicious anemia.
• Intrinsic factor antibodies and Parietal cell antibodies.
• Low intrinsic factor level.
• Gastric mucosal sampling shows parietal cell atrophy with antral sparing.
• Increased level of gastrin.
• Increased levels of homocysteine and methylmalonyl-CoA.
• Decreased folate levels are seen due to "folate trapping" in the form of methyltetrahydrofolate.

Shilling Test

The Shilling test is no longer done to detect an IF deficiency but has historical importance. After a vitamin B12 deficiency is noted, the patient is given radioactively tagged cobalamin to take orally. Soon after this step, the patient is injected with unlabelled cobalamin intramuscularly. Urine is checked for radioactive cobalamin for the next 24 hours. In pernicious anemia, there is an intrinsic factor deficiency, therefore the orally consumed radioactive cobalamin will not be absorbed and can be detected in the urine. In the next step, the patient is given radioactive cobalamin along with intrinsic factor and their urine is checked for traces of radioactive cobalamin. Absence of radioactive cobalamin in the urine points to the deficiency of intrinsic factor in the patients stomach which is the cause of vitamin B12 deficiency. If the cobalamin absorption does not increase even with intrinsic factor supplementation, patient can be given a course of antibiotics as bacterial overgrowth may hinder absorption.

Peripheral smear findings

1. The most obvious peripheral smear finding is megaloblasts and macrocytes.

Megaloblastic anemia results due to the lagging behind of nuclear development when compared to cytoplasmic development. This is known as nuclear-cytoplasmic asynchrony. Such defective cells are destroyed in the bone marrow (intramedullary hemolysis).

1. Decreased number of RBCs (erythopenia)
2. Macrocytosis- the RBCs in pernicious anemia are very large. Macrocytosis is defined as cells that have an MCV >100 femtolitres (normal :80-100fL)
3. Hypersegmented neutrophils : Neutrophils containing ≥ 6 lobes.
4. Poikilocytosis and anisocytosis
5. Low reticulocyte count (reticulopenia)
6. Howell-Jolly bodies

• 

  Atrophic gastritis

• 

  Hypersegmented neutrophil

Treatment

• Standard treatment for pernicious anemia is replacement of cobalamin via intramuscular injection.
• 1000 mcg IM everyday for one week, followed by weekly injections the next month and then monthly once injections.
• Response to treatment is measured by an increase in reticulocyte count within 5 days of starting therapy.
• Patient also experience a sense of wellbeing shortly after beginning therapy.
• If reticulocytosis is not observed within the first week of therapy, other factors such as hypothyroidism, folate deficiency should be considered.
• Intramuscular therapy can be replaced by high dose oral therapy.
• Neurological disease always warrants parenteral treatment.
• Within the first 3-4 weeks of treatment, marrow changes revert and there is resolution in macrocytosis.
• Most patients require lifelong monthly therapy.
• Routine follow up should be done with a CBC every few months.
• A small percentage of patients develop gastric carcinoma, particularly in the elderly. Regular surveillance helps in early detection and treatment.

Prevention

• There is no primary preventive measure for pernicious anemia.
• Once sucessfully diagnosed and treated, patients with pernicious anemia are followed up every year for development of stomach cancer, or symptoms of anemia.

Overview

Chronic myeloid leukemia (CML), a myeloproliferative disorder, which is characterized by the uncontrolled expansion of immature bone marrow cells of myeloid origin.The hallmark of CML is the formation of the Philadelphia chromosome resulting from the reciprocal translation (9;22)(q34;q11.2), resulting in a derivative 9q+ and a small 22q- ultimately forms a BCR/ABL fusion gene and production of a BCR/ABL fusion protein. The gene product of the BCR/ABL gene constitutively activates numerous downstream targets including c-myc, Akt and Jun, all of which cause uncontrolled proliferation and survival of CML cells.

Pathogenesis

• The circulating blood cells are produced in bone marrow after a series of events termed as hematopoiesis.
• The bone marrow has an tremendous regenerative ability; it is estimated that 10 trillion red blood cells and 80 to 90 trillion leukocytes are formed per hour at the basal rate.
• In addition to that, while cell numbers are maintained within narrow limits in normal subjects, they can be promptly increased when required.
• Bone marrow primarily has small percentage of pleuripotent stem cells which give rise to various progenitor cells.
• Hematopoeisis occurs in the vertebrae, pelvic bones, metaphysis of long bones such as femur, humerus in basal state.
• However, during certain stressful conditions that require rapid and massive hematopoiesis such as thalassemia it then returns to its former site, liver, spleen and sometimes lymph nodes.
• These hematopoietic stem cells (HSCs are multipotent and have the ability to differentiate into the cells of all 10 blood lineages:
  • Erythrocytes
  • Platelets
  • Neutrophils
  • Eosinophils
  • Basophils
  • Monocytes
  • T and B lymphocytes
  • Natural killer cells
  • Dendritic cells
• This differentiation is mediated through multiple growth factors and cytokines.
• The hematopoietic stem cells (HSCs) and progenitor cells are supported by a stromal cell network that provides cell-cell contact support.
• The stromal network provides two major functions:
• An adhesive framework onto which the developing cells are bound, these cells produce:
  • A variety of adhesion molecules.
  • Hematopoietic Growth factors or cytokines that are thought to support the survival, proliferation, and differentiation of HSCs and progenitors.
• Primitive mesenchymal stromal cells (MSCs) are thought to have the capacity to differentiate into following:
  • Osteolineage cells
  • Chondrocytes
  • Adipocytes
  • Perivascular cells
• Overall Differentiation of myeloid progenitors is mediated through:
  • The production of essential hematopoietic growth factors.
  • Several signaling pathways have come up as integral control devices of HSC fate, such as:
    • Notch
    • Wingless-type (Wnt)
    • Sonic hedgehog (Shh)
    • Smad pathways
• These signaling circuits provide an important structure for our understanding of HSC regulation, alongwith providing information of how the bone marrow micro environment couples and integrates extrinsic with intrinsic factors responsible for HSC differentiation and development of chronic myeloid leukemia.

Genetic Translocation:

• Chronic myeloid leukemia (CML), a myeloproliferative neoplasm, characterized by the presence of the Philadelphia chromosome which is thought to be a definitive diagnostic marker for CML.
• In Philadelphia chromosome translocation, parts of two chromosomes (the 9^th and 22^nd by conventional karyotypic numbering) switch places.
• As a result, part of the BCR ("breakpoint cluster region") gene from chromosome 22 is fused with the ABL ("abelson murine leukemia") gene on chromosome 9.
• This abnormal "fusion" gene generates a protein of p210 .
• Because ABL carries a domain that can add phosphate groups to tyrosine residues (a tyrosine kinase), the BCR/ABL fusion gene product is also a tyrosine kinase.
• The fused BCR/ABL protein interacts with the interleukin 3 beta c receptor subunit.
• The BCR/ABL transcript is continuously active and does not require activation by other cellular messaging proteins that promotes growth and replication through downstream pathways such as:
  • RAS
  • RAF
  • JUN kinase
  • MYC
  • STAT
• In turn BCR/ABL triggers a cascade of proteins which control the cell cycle, speeding up cell division.
• Moreover the BCR/ABL protein inhibits DNA repair, causing genomic instability and making the cell more susceptible to developing further genetic mutations.
• The action of the BCR/ABL protein is the pathophysiologic cause of chronic myelogenous leukemia.

Role of reactive oxygen species:

• Recent studies have demonstarated that BCR/ABL also stimulates the production of reactive oxygen species (ROS), which levels increase with CML progression and this in turn increases BCR/ABL self-mutagenesis.
• Tyrosine kinase inhibitor resistance can also be related to higher ROS production.
• Therefore, ROS-induced self-mutagenesis of BCR/ABL is of prime significance for CML progression.
• These can be dependent on DNA repair, which is modulated by BCR/ABL and can be different in CML stem and progenitor cells.

Altered bone marrow pathway signalling:

• Implication of altered bone marrow signalling on stem cell persistence in the bone marrow niche.
• Recent advancements have been trying to establish the relationship between bone marrow pathway and Wnt pathways and their role to alter the Cdx-Hox axi.

Role of Integrin:

• CML cells present in contact with stroma or fibronectin continue to proliferate, suggesting that failure to adhere through integrin receptors may also underlie the abnormal proliferation of CML progenitors.
• Although, CML progenitors express the same integrin receptors as normal progenitors, they fail to adhere to stroma and fibronectin.
• This indicates that structural or functional abnormalities of these receptors can be integral part of pathogenesis.

Blast crisis:

• Chronic myeloid leukemia (CML) in blast crisis is the transition of CML in chronic or accelerated phase to an acute leukemia.
• It is characterized by:
  • ≥ 30% blasts in the bone marrow or peripheral blood.
  • The development of extramedullary disease outside of the spleen.
• In light of recent changes in the World Health Organization, definition of acute leukemia, the percentage of blasts required for CML in blastic phase may someday be reduced to 20%.
• Consistent with the early stem cell nature of CML, blastic transformation may be:
  • Myeloid
  • Lymphoid
  • Undifferentiated/mixed
• Myeloid blast crisis being about two times more common than lymphoid.

• It is suggested that blast crisis is due to one of following reasons:
  • BCR/ABL is considered to be responsible for progressive genomic instability or epigenetic changes, which occur at the CML stem cell level and/or in later CML progenitor cells.
  • The degree of genomic instability is directly related to the level of BCR/ABL kinase activity.
  • The third is that CML stem cells are the least vulnerable to ABL-targeted therapy and may serve as reservoirs for CML progression.

• All these events concomitantly result in the acquired loss of hematopoietic cell differentiation, resulting in a highly progressive generation of immature blasts in peripheral blood and in bone marrow.
• Various studies have BCR/ABL is implicated in the generation and maintenance of secondary DNA alterations.

Genetic Alterations in Blast crisis:

• Following genetic changes have been observed which play crucial role in progression of disease phase.
  • Duplication of the Ph chromosome, trisomy 8, and isochromosome 17.
  • Alterations in p53 have been found in only a minority of cases.
  • Loss of p16INK4A/ARF has been reported in up to half of patients with CML in lymphoid blast crisis but is rare in the myeloid form.
  • Thus, it is hypothesized that clonal evolution plays integral role in blastic progression and is likely facilitated by the dysregulation of normal apoptotic pathways by BCR/ABL.

• Recent studies have implicated activation of the following pathways
  • LYN
  • AKT
  • STAT5

Gross Pathology

On gross pathology, no distinctive findings are seen in chronic myeloid leukemia.

Microscopic Pathology

Blast cells are seen on peripheral blood smear of patients of chronic myeloid leukemia which are present during blast crisis.

• 

  Blast crisis of chronic myelogenous leukemia (CML). Peripheral blood smear revealing the histopathologic features indicative of a blast crisis in the case of chronic myelogenous leukemia.

References

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