Chronic lymphocytic leukemia (CLL) DISEASE

Chronic lymphocytic leukemia (CLL) was largely considered to be a disease of slow progression, standard treatment with Chlorambucil and having almost similar prognosis. With the introduction of molecular methods for understanding the disease pathophysiology in CLL there has been a remarkable change in the approach towards the disease. The variation in B-cell receptor response and immunoglobulin heavy chain variable region (IGHV) mutation, genetic aberration and defect in apoptosis and proliferation has had an impact on therapy initiation and prognosis. Early diagnosis of molecular variant is therefore necessary in CLL.

Chronic lymphocytic leukemia is a type of cancer in which the bone marrow makes too many lymphocytes (a type of white blood cell).

Chronic lymphocytic leukemia (also called CLL) is a blood and bone marrow disease that usually gets worse slowly. CLL is the second most common type of leukemia in adults. It often occurs during or after middle age; it rarely occurs in children.

Normally, the body makes blood stem cells (immature cells) that become mature blood cells over time. A blood stem cell may become a myeloid stem cell or a lymphoid stem cell.

A myeloid stem cell becomes one of three types of mature blood cells:

·         Red blood cells that carry oxygen and other substances to all tissues of the body.

·         White blood cells that fight infection and disease.

·         Platelets that form blood clots to stop bleeding.

A lymphoid stem cell becomes a lymphoblast cell and then one of three types of lymphocytes (white blood cells):

·         B lymphocytes that make antibodies to help fight infection.

·         T lymphocytes that help B lymphocytes make antibodies to fight infection.

·         Natural killer cells that attack cancer cells and viruses.

Blood cell development. A blood stem cell goes through several steps to become a red blood cell, platelet, or white blood cell.

In CLL, too many blood stem cells become abnormal lymphocytes and do not become healthy white blood cells. The abnormal lymphocytes may also be called leukemia cells. The lymphocytes are not able to fight infection very well. Also, as the number of lymphocytes increases in the blood and bone marrow, there is less room for healthy white blood cells, red blood cells, and platelets. This may cause infection, anemia, and easy bleeding.

This summary is about chronic lymphocytic leukemia. See the following PDQ summaries for more information about leukemia:

Older age can affect the risk of developing chronic lymphocytic leukemia.

Anything that increases your risk of getting a disease is called a risk factor. Having a risk factor does not mean that you will get cancer; not having risk factors doesn’t mean that you will not get cancer. Talk with your doctor if you think you may be at risk. Risk factors for CLL include the following:

·         Being middle-aged or older, male, or white.

·         A family history of CLL or cancer of the lymph system.

·         Having relatives who are Russian Jews or Eastern European Jews.

Possible signs of chronic lymphocytic leukemia include swollen lymph nodes and tiredness.

Usually CLL does not cause any symptoms and is found during a routine blood test. Sometimes symptoms occur that may be caused by CLL or by other conditions. Check with your doctor if you have any of the following problems:

·         Painless swelling of the lymph nodes in the neck, underarm, stomach, or groin.

·         Feeling very tired.

·         Pain or fullness below the ribs.

·         Fever and infection.

·         Weight loss for no known reason.

Tests that examine the blood, bone marrow, and lymph nodes are used to detect (find) and diagnose chronic lymphocytic leukemia.

The following tests and procedures may be used:

·         Physical exam and history : An exam of the body to check general signs of health, including checking for signs of disease, such as lumps or anything else that seems unusual. A history of the patient’s health habits and past illnesses and treatments will also be taken.

·         Complete blood count (CBC) with differential : A procedure in which a sample of blood is drawn and checked for the following:

o    The number of red blood cells and platelets.

o    The number and type of white blood cells.

o    The amount of hemoglobin (the protein that carries oxygen) in the red blood cells.

o    The portion of the blood sample made up of red blood cells.

Complete blood count (CBC). Blood is collected by inserting a needle into a vein and allowing the blood to flow into a tube. The blood sample is sent to the laboratory and the red blood cells, white blood cells, and platelets are counted. The CBC is used to test for, diagnose, and monitor many different conditions.

·         Immunophenotyping : A laboratory test in which the antigens or markers on the surface of a blood or bone marrow cell are checked to see if they are lymphocytes or myeloid cells. If the cells are malignant lymphocytes (cancer), they are checked to see if they are B lymphocytes or T lymphocytes.

·         FISH (fluorescence in situ hybridization): A laboratory technique used to look at genes orchromosomes in cells and tissues. Pieces of DNA that contain a fluorescent dye are made in the laboratory and added to cells or tissues on a glass slide. When these pieces of DNA bind to specific genes or areas of chromosomes on the slide, they light up when viewed under a microscope with a special light.

·         Flow cytometry : A laboratory test that measures the number of cells in a sample, the percentage of live cells in a sample, and certain characteristics of cells, such as size, shape, and the presence of tumor markers on the cell surface. The cells are stained with a light-sensitive dye, placed in afluid, and passed in a stream before a laser or other type of light. The measurements are based on how the light-sensitive dye reacts to the light.

·         IgVH gene mutation test: A laboratory test done on a bone marrow or blood sample to check for an IgVH gene mutation. Patients with an IgVH gene mutation have a better prognosis.

·         Bone marrow aspiration and biopsy : The removal of bone marrow, blood, and a small piece of bone by inserting a hollow needle into the hipbone or breastbone. A pathologist views the bone marrow, blood, and bone under a microscope to look for abnormal cells.

Bone marrow aspiration and biopsy. After a small area of skin is numbed, a Jamshidi needle (a long, hollow needle) is inserted into the patient’s hip bone. Samples of blood, bone, and bone marrow are removed for examination under a microscope.

Certain factors affect treatment options and prognosis (chance of recovery).

Treatment options depend on:

·         The stage of the disease.

·         Red blood cell, white blood cell, and platelet blood counts.

·         Whether there are symptoms, such as fever, chills, or weight loss.

·         Whether the liver, spleen, or lymph nodes are larger than normal.

·         The response to initial treatment.

·         Whether the CLL has recurred (come back).

The prognosis (chance of recovery) depends on:

·         Whether there is a change in the DNA and the type of change, if there is one.

·         Whether lymphocytes are spread throughout the bone marrow.

·         The stage of the disease.

·         Whether the CLL gets better with treatment or has recurred (come back).

·         Whether the CLL progresses to lymphoma or prolymphocytic leukemia.

·         The patient's general health.

Diagnosis

CLL can be diagnosed if: the clonal B-lymphocyte count is >5,000/cuml of blood (duration of lymphocytosis >2 months). If the lymphocyte count is less than 5,000/cuml in asymptomatic individuals, without organ involvement, it is designated as 'monoclonal B lymphocytosis'.

-Bone marrow lymphocytes >30%

-The immunological profile of CLL lymphocytes is defined by:

• Weak surface membrane immunoglobulin (Ig) levels (most often IgM or both IgM and IgD)
• Monoclonal: expression of either Kappa or lambda
• B-cell antigens CD23, CD19 and CD20 (weak), with co-expression of CD5
• Negative for cyclin D1 and CD10 expression,

No or weak expression of FMC7, CD22 and CD79b.

Differential diagnosis of CLL includes

-Mantle cell Lymphoma

-Follicular lymphoma

-Splenic marginal zone lymphoma

-Hairy cell lymphoma

-B Prolymphocytic Leukemia.

Staging at diagnosis (Rai system) 

0. Lymphocytosis

1. Lymph node enlargement

2. Spleen enlargement

3. Hemoglobin < 11 g/dl

4. Platelets < 100,000/μl

Staging at diagnosis (Binet system)

• Lymphocytosis
• Lymph node enlargement in > 3 areas
• Cytopenia : h0 emoglobin < 10 g/dl or platelets <100,000/μl

Pathophysiology of chronic lymphocytic leukemia

Most of the CLL cells are inert in vitro. Recently, the understanding of the patho-biology in CLL has divided this disease into subgroups and this has had a profound impact on prognosis and treatment.

The three major subdivisions are based upon:

1.  B-cell receptor response and the IGHV mutation, 

2.  Genetic aberration/gene mutation, 

3.  Defects in apoptosis and proliferation. 

B-cell receptor response

The B-cell receptor (BCR) is composed of two immunoglobulins (Ig), heavy and light chains (variable and constant region) and CD79a and CD79b. These contain intracellular activation molecules (SYK and LYN) that transmit signals to intracellular tyrosine kinases [Figure 2]. The ability of these kinases to activate downstream pathways varies in CLL subgroups and is correlated with:

• Ig heavy chain variable region (IGHV) mutational status,
• ZAP-70 and
• CD 38 expression.

These pathways can be targeted by small molecule inhibitors, the most promising of which might be SYK inhibitors.

B-cell receptor response and the IGHV mutation 

The B cell response to antigenic stimulation is mediated through the BCR in normal and malignant B-cells.

Each B-cell displays a distinct BCR that is formed through variable combinations of V, D and J segments for the Ig heavy chain and V and J gene segments for the light chain.

CLLs have mutated IGHV genes and unmutated IGHV genes (unmutated IGHVs showing poorer survival).

(IGHV genes = immunoglobulin heavy chain variable region)

Folding and glycosylation defect of the μ and CD79A chains (not of the CD79B chain).

Poor expression of the CD22 molecule in B-cell chronic lymphocytic leukemia cells was also as a result of folding defect arising in CD79A.

Most B-cell chronic lymphocytic leukemia cells express CD5 and IgM/IgD due to which they have a mantle zone-like phenotype of naive cells that., …, express unmutated immunoglobulin genes normally.  Somatic mutations of IGHV genes are seen in Fifty to seventy percent of cases of chronic lymphocytic leukemia., appearing as if they had matured from a lymphoid follicle.

Mutational status of IGHV genes has a profound effect on the prognosis of CLL showing markedly different biological and clinical behaviors.  BCR surface expression is usually weak in CLL. Low expression of the B-cell receptor is the typical presentation of lymphocytes in CLL  (mechanisms remain elusive).

ZAP70 (Zeta-Associated Protein)

Low expression of the B-cell receptor correlates with defective intracellular calcium mobilization and tyrosine phosphorylation due to reduced induction of protein tyrosine kinase activity.  High protein tyrosine kinase activity associated with ZAP70-a receptor (not found in normal circulating B-cells) is usually found in T-cells and natural killer cells detected in most patients with un-mutated chronic lymphocytic leukemia.

ZAP70 expression is associated with advantageous survival of CLL cells because of enhanced access to proliferation centre and increased sensitivity to chemokine migratory signals (presence of ZAP70 is an oncogenic event in CLL, concentrated particularly in lymph node lymphoid cell).

CD38

CD38, which is associated with poor prognosis, predominates in patients with unmutated IGHV genes. 

In chronic lymphocytic leukemia, expression of CD38 on B cells favors -. its growth and survival through sequential interactions between CD38 and CD31 and also between CD100 and plexin B1 (PLXNB1).

Genetic abnormalities

Dohner and colleagues .- in a series of 325 patients with chronic lymphocytic leukemia showed that chromosomal aberrations can be detected in interphase cells by fluorescence in situ hybridization (FISH) technique in 82% of cases. According to them the most frequent aberrations are:

• Deletion on Chromosome 13q (55%),Trisomy 12 (18%), A deletion on Chromosome 11q (16%)., Deletion on chromosome 17p, affecting the TP53 protein, is seen less frequently (7%).

Deletions in band 13q14

'Deletion on Chromosome 13q' is the most frequently found genetic structural aberration in CLL and confers a favorable course.  No inactivation of candidate genes by mutation has been demonstrated. Two microRNA genes, mir-15a and mir-16-1, located in the crucial 13q14 region have been implicated in CLL pathogenesis. A mouse model with a targeted deletion of the mir-15a-mir-16-1 locus recapitulates many features of CLL. This suggests that miR-15a and miR-16-1 have a direct pathogenetic role in CLL.

Deletions at 13q14 occur at high frequencies in other lymphomas and solid tumors, and a recent study has implicated miR-15a and miR-16-1 in the pathogenesis of prostate cancer through their targeting of cyclin D1 and WNT3A, which promote survival and proliferation.

Deletions of ATM (11q22-q23)

Although they are rarely found in early-stage disease, approximately one-quarter of patients with advanced CLL have 11q23 deletions. Correspondingly, patients who have 11q23 deletions have a more rapid disease progression and extensive lymphadenopathy. In stuacminimal consensus region in chromosome bands 11q22.3-q23.1 which also harbors the ataxia telangiectasia-mutated (ATM) gene in almost all cases.

ATM mutations have been shown to be present in 12% of all patients with CLL and in approximately one-third of the cases with a 11q23 deletion. The ATM protein kinase is a central component of the DNA damage pathway and mediates cellular responses to DNA double-strand breaks (DSBs). ATM deficiency leads to ataxia-telangiectasia, which is characterized by extreme sensitivity to irradiation, genomic instability and a predisposition to lymphoid malignancies. ATM activates cell cycle checkpoints, can induce apoptosis in response to DNA breaks and functions directly in the repair of DNA DSBs by maintaining DNA ends in repair complexes.

Trisomy 12

Trisomy 12 is among the more frequent aberrations in CLL (10-20%),. the genes implicated in the pathogenesis of CLL with Trisomy 12 are unknown. A previously described association with poor outcome has not been confirmed. Incidence of Trisomy 12 does not increase with advanced stage or progression to refractory disease.

Deletions in band 17p13 or TP53 mutations

Deletion of 17p13 is found in 4-9% of CLLs at diagnosis or at initiation of the first treatment.17p13 deletion usually encompasses most of the short arm of Chromosome 17p, the deletion always includes band 17p13, where the tumor suppressor TP53 (which encodes p53) is located. Among CLL cases that have monoallelic 17p13 deletions, the majority show mutations in the remaining TP53 allele (>80%). Among cases without 17p13 deletion, TP53 mutations are much rarer  [Figure 4].

TP53 mutations in chronic lymphocytic leukemia

TP53- tumor suppressor gene, is a transcription factor activated by strand breaks in DNA which can trigger apoptosis or cell cycle arrest.

The Genetic lesions associated with deletions of the short arm of Chromosome 17 (del17p13)). encodes the TP53 -. gene. The long arm of Chromosome 11 (del11q23), which encodes the ataxia telangiectasia mutated (ATM) gene can also result in a loss of function of TP53.

Patients with a 17p13 deletion or TP53 mutation have a poor outcome and are candidates for experimental strategies with novel agents and stem cell transplantation. 17p13 deletion is invariably associated with loss of TP53 as confirmed by fluorescence in situ hybridization (FISH) ('+' in [Figure 4] denotes a TP53 deletion). Multiple genomic aberrations target the p53 pathway in CLL.

Recurrent translocations

Recurrent translocations are rare in CLL in contrast to other types of leukemia or B-cell lymphoma in which specific and recurrent Ig locus-associated translocations deregulate known oncogenes. However, IGHV-mutated CLLs, in which the precursors were exposed to SHM (Somatic Hyper Mutation) and, in a fraction of cases, also had undergone class switching. The lack of Ig-associated translocations may support the notion that CLL is derived from post-GC B-cells, in which SHM and class switching are silenced.

Microenvironment

CLL cells interact with and seem to shape their microenvironment, which consists of T-cells, stromal cells and soluble factors.  CLL cells rapidly undergo apoptosis when they are removed from patients. This process can be prevented by adding a number of cytokines or other cell types to the CLL cell culture.

In the lymph node, the microenvironment provides anti-apoptotic signals and proliferative stimuli, resulting in the formation of proliferation centers of CLL cells (pseudo follicles) that are not found in other lymphomas.

CLL cells seem to recruit accessory cells and thereby create a microenvironment that supports their own survival.

There is an increase of CD3+ T-cells, most of which are CD40L+CD4+, and cluster in and around pseudo follicles. CLL cells that are in close proximity and in contact with activated CD4+ T-cells show expression of the cell surface marker CD38. This is of interest because CD38 has been linked to the proliferation of CLL cells. The presence of high numbers of CD38+ CLL cells in the blood is associated with a poor prognosis. 

High-risk features for chronic lymphocytic leukemia 

1.  CD38 expression in > 30% of lymphocytes

2.  ZAP70 expression in > 30% of lymphocytes

3.  Unmutated (germ line) IgVH gene

4.  High-risk cytogenetic abnormalities

a.  14q changes

b.  11q changes

c.   17p depletion

d.  Trisomy 12

5.  Rai Stage 3 or 4 or Binet Stage C

6.  Doubling time of lymphocyte count <12 months

7.  Elevated beta-2 microglobulin

8.  Elevated serum thymidine kinase

Presence

The following stages are used for chronic lymphocytic leukemia:

Stage 0

In stage 0 chronic lymphocytic leukemia, there are too many lymphocytes in the blood, but there are no other symptoms of leukemia. Stage 0 chronic lymphocytic leukemia is indolent (slow-growing).

Stage I

In stage I chronic lymphocytic leukemia, there are too many lymphocytes in the blood and the lymph nodes are larger than normal.

Stage II

In stage II chronic lymphocytic leukemia, there are too many lymphocytes in the blood, the liver orspleen is larger than normal, and the lymph nodes may be larger than normal.

Stage III

In stage III chronic lymphocytic leukemia, there are too many lymphocytes in the blood and there are too few red blood cells. The lymph nodes, liver, or spleen may be larger than normal.

Stage IV

In stage IV chronic lymphocytic leukemia, there are too many lymphocytes in the blood and too fewplatelets. The lymph nodes, liver, or spleen may be larger than normal and there may be too few red blood cells.

After chronic lymphocytic leukemia has been diagnosed, tests are done to find out how far the cancer has spread in the blood and bone marrow.

Staging is the process used to find out how far the cancer has spread. It is important to know the stageof the disease in order to plan the best treatment. The following tests may be used in the staging process:

·         Chest x-ray : An x-ray of the organs and bones inside the chest. An x-ray is a type of energy beam that can go through the body and onto film, making a picture of areas inside the body, such as the lymph nodes.

·         MRI (magnetic resonance imaging): A procedure that uses a magnet, radio waves, and a computer to make a series of detailed pictures of areas inside the body, such as the brain andspinal cord. This procedure is also called nuclear magnetic resonance imaging (NMRI).

·         CT scan (CAT scan): A procedure that makes a series of detailed pictures of areas inside the body, taken from different angles. The pictures are made by a computer linked to an x-ray machine. A dye may be injected into a vein or swallowed to help the organs or tissues show up more clearly. This procedure is also called computed tomography, computerized tomography, or computerized axial tomography.

·         Blood chemistry studies : A procedure in which a blood sample is checked to measure the amounts of certain substances released into the blood by organs and tissues in the body. An unusual (higher or lower than normal) amount of a substance can be a sign of disease in the organ or tissue that makes it.

·         Antiglobulin test : A test in which a sample of blood is looked at under a microscope to find out if there are any antibodies on the surface of red blood cells or platelets. These antibodies may react with and destroy the red blood cells and platelets. This test is also called a Coomb's test.

Genotype-specific therapy

The first risk-adapted treatment for patients with CLL has been developed for patients with 17p13 deletions who have a very poor prognosis with alkylator- and purine analogue-based chemo-immunotherapy.There are evidences that several 'biological' agents, such as alemtuzumab, corticosteroids, lenalidomide and flavopiridol act independently of functional p53 in CLL, therefore the current treatment approaches in clinical trials use these agents before going for allogeneic stem cell transplantation.

Translating biological insights into treatment

The clinical course is generally indolent in the majority of patients and therefore clinical endpoints are reached slowly. In spite of this, the growing number of approaches that act differently from classical chemotherapy show great promise and some of them are particularly promising for CLL (for example, SYK inhibition).

Conclusions and perspectives

CLL can be divided into subtypes (IGHV-unmutated and mutated) that have distinct biological and clinical characteristics. IGHV-mutated CLLs derive from post-GC B-cells, and that IGHV-unmutated CLLs stem from B-cells that have been activated by antigens. The dependence and interaction of CLL cells with the microenvironment is of pivotal importance and is being used as a drug target in preliminary studies.

Specific aberrations (11q23 and 17p13 deletions) and gene mutations (TP53 and ATM) help to define distinct biological and clinical subgroups of CLL. The most common genetic lesion-deletion 13q14-may serve as a model of how deregulated non-coding RNAs contribute to cancer initiation, and this may become a 'druggable' target in the future.

Overall, CLL may serve as a model for how microenvironmental stimuli, antigenic drive and genetic deregulation are combined in cancer pathogenesis. Importantly, there are a growing number of agents that act on specific biological targets and therefore open of large-cell transformation (Richter's syndrome)

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