BIOM3011 Chronic Lymphocytic Leukaemia Notes

Chronic Lymphocytic Leukaemia (CLL)

Introduction

• Chronic Lymphocytic Leukaemia (CLL) is a type of leukaemia characterized by the proliferation of mature-looking lymphocytes (B or T type).
• It is the most common lymphoid leukaemia.
• Primarily affects older adults, with a peak age of onset between 60 and 80 years.
• More prevalent in men, with a male-to-female ratio of approximately 2:1.
• CLL cells infiltrate the peripheral blood (PB), bone marrow (BM), liver, spleen, and lymph nodes due to prolonged lifespan and reduced apoptosis, leading to enlargement of these organs.
• The five-year survival rate for people with CLL is 83% after diagnosis.

Key Learning Objectives

• Outline the clinical and laboratory characteristics of CLL.
• Discuss the genetic mutations involved in CLL.
• Explain the differential diagnosis of CLL, including:
  • B-cell prolymphocytic leukaemia (B-PLL)
  • Hairy cell leukaemia (HCL)
• Explain available treatment plans and monitoring systems for CLL.

Leukaemia Recap Questions

• Question 1: Which of the following is the correct order of maturation stages in granulopoiesis?
  • Answer: d) Myeloid Progenitor → Myeloblast → Promyelocyte → Myelocyte → Metamyelocyte → Band → Granulocyte
• Question 2: How does leukaemia affect the blood?
  • Answer: c) It increases the number of white blood cells but decreases the number of red blood cells and platelets.
• Question 3: What is the main symptom of thrombocytopenia?
  • Answer: c) Bruising and bleeding easily.
• Question 4: Translocation refers to:
  • Answer: a) Part of one chromosome has broken off and is now located on another chromosome.
• Question 5: For which type of leukaemia are tyrosine kinase inhibitors the mainstay of treatment?
  • Answer: b) Chronic myeloid leukaemia.

Chronic Lymphocytic Leukaemia Details

• CLL involves the proliferation of mature looking lymphocytes (B or T type).
• It is the most common type of lymphoid leukaemia.
• CLL predominantly affects older adults, typically between 60-80 years old.
• It is more common in men, with a ratio of approximately 2:1 compared to women.
• CLL cells infiltrate the peripheral blood, bone marrow, liver, spleen, and lymph nodes. This infiltration is due to the prolonged lifespan of these cells and reduced apoptosis, leading to organ enlargement.
• The 5-year survival rate for CLL patients is around 83% after diagnosis.
• CLL can transform into an aggressive lymphoma (Richter’s transformation), such as DLBCL (Diffuse Large B-Cell Lymphoma) or Hodgkin's lymphoma.
• CLL is often incurable due to its chronic and fluctuating nature, despite not usually being aggressive.
• There is a significant overlap between CLL and lymphomas, where the distinction is based on the relative proportions of the tumor in tissues, peripheral blood, and bone marrow.
• Several subtypes of CLL exist, including:
  • Prolymphocytic leukaemia
  • Hairy cell leukaemia
  • Adult T-cell leukaemia
  • Sézary syndrome
  • Large granular lymphocytic leukaemia
  • Splenic lymphoma with villous lymphocytes

Extramedullary Haematopoiesis

• Definition: Haematopoiesis occurs outside the bone marrow.
• Physiologic: Reactivation of HSPCs during fetal development in the liver, which later homes to the bone marrow.
• Pathologic:
  • Can occur in the liver and spleen, leading to hepatomegaly or hepatosplenomegaly.
  • Associated with myeloproliferative neoplasms (MPN) such as essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF).
  • Myelofibrosis progression involves the replacement of HSPCs by fibrotic tissue.

CLL: Risk Factors

• Ionizing radiation (radiotherapy, X-rays, atomic bombs)
• Drugs (alkylating agents)
• Chemicals (benzene, cigarettes, armament waste)
• Immunodeficiency diseases
• Family history of blood and bone marrow cancers
• Age and sex
• Viruses such as HTLV, HIV, and Epstein-Barr virus

CLL: Clinical Symptoms

• 40% of patients are asymptomatic at diagnosis.
• Symptoms include:
  • Fatigue
  • Weight loss
  • Enlargement of lymph nodes (cervical and supraclavicular)
  • Immunosuppression leading to infections and fever
  • Splenomegaly and hepatomegaly
  • Bilateral cervical lymphadenopathy
  • Dermatomal herpes zoster infection

CLL: Pathophysiology

• Genetic predisposition: Increased risk with family history.
• Chromosomal abnormalities are seen in 80% of CLL patients.
• Common chromosomal abnormalities:
  • Deletion of 13q14
  • Trisomy 12
  • Deletion at 11q23 involving the ATM gene
  • 17p deletion involving the TP53 gene
• Over 2,500 somatic mutations can be associated with CLL; approximately 10% of CLL cases are linked to genetic/proto-oncogene variations, such as NOTCH1, TP53, ATM, and SF3B1.
• TP53 mutations (seen in 5% of CLL patients) are associated with poor prognosis and resistance to chemotherapy.
• Several genes and pathways are involved, including BCR, NF-kB, TLR, CDKN2A, NOTCH, MAPK, RAS, BRAF, BIRC3, TRAF3, NFKBIE, MYD88, and more.

CLL: Pathophysiology - Deletion 13q14

• Occurs in 50-60% of CLL cases.
• Leads to the loss of tumor suppressor microRNAs, miR15A and miR16A.
• miR15A and miR16A interact directly with and inhibit BCL2 and other oncogenes like MCL1 and BMI1.
• These microRNAs are crucial for maintaining the balance of apoptosis in B lymphocytes.
• BCL2 protein is increased in nearly all CLL patients.
• Expression of miR15A and miR16A inhibits BCL2, activating apoptosis.
• Loss of miR15A and miR16A results in the activation of BCL2 and inhibition of apoptosis.

CLL: Pathophysiology - TP53 Mutation & del-17p

• TP53 is a tumor suppressor gene, known as the guardian of the genome.
• Involved in cell cycle regulation and DNA Damage Response.
• P53, as a transcription factor, induces the transcription of BCL proteins (BAX, NOXA, BID), which are involved in apoptosis.
• In CLL, TP53 is constitutively deactivated, blocking the mitochondrial apoptotic pathway and conferring an anti-apoptotic advantage to CLL cells.
• Associated with poor outcomes.

CLL: Pathophysiology - c-MYC

• c-MYC is a proto-oncogene involved in cell survival.
• It regulates apoptosis by inducing the expression of pro-apoptotic genes and suppressing the expression of anti-apoptotic genes.
• In CLL, c-MYC is overexpressed, blocking the apoptotic safety mechanism.

CLL: Pathophysiology - NOTCH Mutation

• Associated with poor outcomes and difficulty in treating CLL.
• The NOTCH signaling pathway plays a critical role in cell growth, differentiation, and apoptosis.
• Functionally linked with the B-cell receptor (BCR) signaling pathway.
• In CLL, NOTCH mutations depend on the presence of Notch ligands in the microenvironment and activate processes like cell migration, invasion, and angiogenesis.
• The B-cell receptor (BCR) is crucial for CLL pathogenesis.

CLL: Chromosomal Abnormalities and Prognosis

• Favorable Prognosis:
  • del13q14
  • Trisomy 12
  • Normal karyotype
• Poor Prognosis:
  • 17p deletion
  • TP53 mutation
  • NOTCH mutation
  • Complex karyotype
  • ATM mutation
  • del(11q)
  • SF3B1 mutation
  • BIRC3 mutation

CLL: Classification

• WHO classification groups CLL into B-cell CLL or T-cell CLL.
• B-cell CLL is seen in 95% of patients.
• 1% of people with B-cell CLL have a type called B-cell prolymphocytic leukaemia (PLL).
• T-cell prolymphocytic leukaemia is seen in 1% of patients.
• CLL is further classified based on Rai and International Working Party (Binet) classifications.

CLL: Rai and Binet Classifications for Staging

• Rai Classification:
  • Stage 0: Low risk, good prognosis. Absolute lymphocytosis $≥5 × 10^9/L$ without adenopathy, organomegaly, or cytopenias due to marrow replacement.
  • Stage I & II: Intermediate risk. Enlarged lymph nodes ± enlarged liver or spleen.
  • Stage III & IV: High risk, poor prognosis. Anaemia (Hb <100 g/L) or thrombocytopenia (platelets <$100 × 10^9/L$). Secondary causes of anemia must be treated before staging e.g. iron deficiency
• Binet Classification:
  • Stage A (50-60% of patients): Good prognosis, 0-2 areas of organ enlargement; haemoglobin $≥100 g/L$, and platelets $≥100 × 10^9/L$.
  • Stage B (30% of patients): Poor prognosis, 3-5 areas of organ enlargement; haemoglobin $≥100 g/L$, and platelets $≥100 × 10^9/L$.
  • Stage C (<20% of patients): Poor prognosis, not considered for organ enlargement. Haemoglobin <100 g/L and/or platelets <$100 × 10^9/L$.
  • Organ enlargement refers to lymph nodes >1 cm in neck (including Waldeyer's ring), axillae, groins or spleen, or liver enlargement.

CLL: Prognostic Factors

• Mutation in the ATM gene is also unfavorable if associated with 11q23 deletion.
• LDH, lactate dehydrogenase; VH, heavy chain variable.

CLL: Diagnosis

• Assessment of medical history, examination, and performance status.
• Full blood count:
  • WBC (lymphocytes may be up to 200), RBC, and Platelets
• Morphology:
  • Smudge or smear cells, normocytic anaemia
• Bone marrow aspirate and trephine biopsy:
  • Increased lymphocytes (25-95%)
• Immunophenotyping of bone marrow (and blood if blasts present):
  • CD19+; CD5+; CD23; κ and λ; CD38; ZAP70
• Cytogenetic analysis by karyotype:
  • del-17p, del-11q23; del-13q14; trisomy 12
• Mutation analysis (PCR, FISH):
  • NOTCH1, TP53, ATM, SF3B1
• Cytochemical analysis:
  • Negative for MPO, SBB, CAE
• CT scan, MRI scan, X-ray:
  • Lymphadenopathy, Splenomegaly
• Lumbar Puncture:
  • Suspicion of CNS involvement

CLL: Differential Diagnosis

B-cell Prolymphocytic Leukaemia (B-PLL)

• Aggressive variant of CLL.
• Late onset – median age 70 years.
• Bad prognosis; median survival rate – 3 years.
• TP53 mutations, MYC abnormalities, and complex karyotypes are often present.
• Splenomegaly without lymphadenopathy; high lymphocyte count. Anemia is a poor prognostic feature.
• Blood film – prolymphocytes with prominent central nuclei & abundance of pale cytoplasm.
• Immunophenotyping: Positive for CD19, CD20; Negative for CD5.

Hairy Cell Leukaemia

• Rare CLL subtype that progresses slowly.
• Male to female ratio: 4:1 (peak incidence: 40-60 years).
• Infections, anaemia or splenomegaly, pancytopenia. Monocytopenia is a distinctive feature. Lymphadenopathy is uncommon.
• Immunophenotyping: Positive for CD11c, CD19, CD25, CD103, CD123; Negative for CD5, CD21.
• Blood film – typical ‘hairy’ cells with oval nuclei and finely mottled pale grey-blue cytoplasm with an irregular edge.

Immunophenotyping of Chronic B-Cell Leukaemias/Lymphomas

• CD103 is positive only in classic hairy cell leukaemia (HCL); a variant form of HCL is negative for CD103 (as well as for CD25, also typically expressed in classical HCL).

CLL: Treatment Strategies

• Depends on the clinical stage of the disease and lymphocyte doubling time.
• Asymptomatic patients: Observation (watchful waiting).
• Intervention is required when symptoms affect quality of life.
• Currently, there is no curative therapy; treatments aim to improve the quality of life.
• Treatment options include:
  • Chemotherapy
  • Radiotherapy
  • Stem cell transplantation (allogeneic)
  • Targeted Therapy (Monoclonal antibodies; CAR-T-cell therapy)
  • Supportive care

CLL Treatment algorithm

• Asymptomatic
  • All: Watch and wait
• Symptomatic
  • Without del(17p13) or TP53 mutation
    • <65 years: FCR
    • >65 years: Obi+CLB or BR
  • With del(17p13) or TP53 mutation
    • Ibrutinib

CLL: Specific treatments

• Chemotherapy: Backbone for induction therapy.
• Radiotherapy
• Stem cell transplantation (allogeneic)
• Targeted Therapy:
  • Monoclonal antibodies
  • CAR-T-cell therapy
• Supportive care

CLL Treatment: Ibrutinib

• Used in the treatment of CLL patients with high-risk genetic characteristics and refractory/relapse CLL; often considered a gold standard therapy.
• Ibrutinib is an irreversible covalent inhibitor of BTK (Bruton's tyrosine kinase).
• BTK is a critical enzyme in the BCR (B-cell receptor) signaling pathway.
• This drug prevents B-cell activation and B-cell mediated signalling.
• Also inhibits the proliferation, adhesion, and migration of CLL lymphocytes that overexpress BTK.
• Promotes apoptosis of CLL cells.
• Also abrogates tumour-protective effects from the microenvironment.

CLL Treatment: Anti-CD20 Monoclonal Antibodies

• Based on selective B-cell depletion.
• Examples: Rituximab, Ofatumumab, Obinutuzumab.
• These antibodies induce complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and apoptosis.
• Often used in combination with chemotherapy in CLL treatment.
• Rituximab is often combined with fludarabine, bendamustine, or cyclophosphamide.
• Obinutuzumab is often combined with chlorambucil.
• Chemoimmunotherapy is commonly used for CLL patients without high-risk genetic mutations.

CLL Treatment: Venetoclax

• Used in the treatment of refractory/relapsed CLL with deletion 17p.
• Selective inhibitor; blocks the function of anti-apoptotic protein BCL2.
• BCL2 is a family of proteins that regulate apoptosis.
• Pro-apoptotic proteins include BAD, BIK, NOXA, BMF, PUMA, BIM, BID.
• Anti-apoptotic proteins include BCL2, BCL-XL, BCL-W, and MCL-1.
• Venetoclax restores apoptosis in tumor cells.
• It is metabolized by CYP3A4 and CYP3A5 and is a substrate for P-glycoprotein.
• Combination with anti-CD20 mAbs improves efficacy.

CLL: Treatment - Emerging Monoclonal Antibodies

Monoclonal antibodies target specific antigens on cancer cells to trigger immune responses.

• CD20
  • Rituximab
  • Ofatumumab
  • Obinutuzumab (GA101)
  • Ocrelizumab
  • Veltuzumab
  • AME-133
• CD22
  *Alemtuzumab
  *Epratuzumab
  *Inotuzumab ozogamicin
  *Moxetumomab pasudotox
• CD19
  *Blinatumomab
• CD23
  *Lumiliximab
• CD74
  *Milatuzumab

CLL: Treatment - Targets

• Key targets include NOTCH1 signalling, BCR signalling, and inflammatory pathways.
• Examples of drugs targeting these pathways or key proteins involved:
  • Ibrutinib
  • Acalabrutinib
  • Venetoclax
  • Idelalisib

CLL: Supportive Therapy

• General supportive therapy includes:
  • Painkillers
  • Blood transfusions
  • Antibiotics
  • Nutritional support

Quiz Time Questions And Answers

• Question 1: A 69-year-old woman is referred with painful joints, morning stiffness, lymphadenopathy, and splenomegaly. A full blood count shows absolute lymphocytosis, and the blood film shows mature small lymphocytes with scanty cytoplasm, round nuclei, coarsely clumped chromatin, and smudge cells. The most likely diagnosis is:
  • Answer: d) Chronic Lymphocytic Leukaemia
• Question 2: Which of the following is ONE of the indications to start treatment for chronic lymphocytic leukaemia?
  • Answer: d) Rapid doubling time of white blood cells
• Question 3: Staging of chronic lymphocytic leukemia is based upon which of these laboratory and clinical findings?
  • Answer: a) Platelets, b) Haemoglobin, c) Number of lymph nodes involved

Further Reading

• Mihalyova et al., (2018): Venetoclax: A new wave in hematooncology
• Bosch and Dalla-Favera (2019): Chronic lymphocytic leukaemia: from genetics to treatment
• Robak (2012): Emerging Monoclonal Antibodies and Related Agents for the Treatment of Chronic Lymphocytic Leukemia
• Robak, Smolewski and Robak (2015): Emerging immunological drugs for chronic lymphocytic leukemia
• Delgado et al., (2020): Chronic lymphocytic leukemia: from molecular pathogenesis to novel therapeutic strategies
• Puiggros, Blanco and Espinet (2014): Genetic Abnormalities in Chronic Lymphocytic Leukemia: Where We Are and Where We Go
• Gaidano and Rossi (2017): The mutational landscape of chronic lymphocytic leukemia and its impact on prognosis and treatment
• Ring and Zenz (2020): Genetics of “high-risk” chronic lymphocytic leukemia in the times of chemoimmunotherapy
• Brown et al., (2017): The Role of Rituximab in Chronic Lymphocytic Leukemia Treatment and the Potential Utility of Biosimilars
• Cenariu et al., (2021): Extramedullary Hematopoiesis of the Liver and Spleen - PMC