Molecular Basis For Novel Tergeted Therapy

Published Date: 02 Nov 2017

Abstract

There has still been no specific treatment found for Chronic lymphocytic leukaemia despite continuous basic and clinical research being made on it. Nevertheless, much information about the disease's genetics, biology, and clinical staging is present which has enabled the patients of the disease to be classified into subtypes on the basis of their cytogenesis, tendency of disease development, or reaction to basic or modern treatment. Moreover, modern drugs or drug-combinations, monoclonal antibodies, stem cell transplantation, or gene therapy is being is being tested by various clinical trials.

Introduction

A tumour of the white blood cell basically formed by the bone marrow is called Leukaemia which arises in the developing blood cells which are unable to mature and start to multiply in an uncontrolled way (Rozman and Monsterrat, 1995). Usually, there are more white blood cells in the patients of leukaemia than normal. Leukaemia can divided into types, and subtypes i.e. it can be either acute or chronic which is formed on the basis of the disease development and progression. Acute leukaemia acts on immature blood cells, unabling them to mature properly, it forms and advances rapidly quickly and should be treated as soon as it is diagnosed, whereas Chronic leukaemia occurs due to the accretion of more mature but abnormal white cells which expands slowly, either during the early stages of disease, or over weeks or months. Furthermore, on the basis of the types of cells in which leukaemia was initiated, it can either be myeloid or lymphoid (Johnston,2009). Myeloid (myelocytic, myelogenous or granulocytic) leukaemia is when leukaemia is initiated in the myeloid cell line whereas lymphocytic (or lymphoblastic or lymphatic) leukaemia is when leukaemia is initiated somewhere in the lymphoid cell line (figure 1, stem cell line).

Four main subtypes of leukaemia are:

Chronic lymphocytic leukaemia (CLL).

Acute lymphocytic leukaemia (ALL).

Chronic myeloid leukaemia (CML).

Acute myeloid leukaemia (AML).

The slow-growing type of leukaemia that acts on developing B-cells (also known as B-lymphocytes) which are specialized white blood cells, making them malignant and leukemic is called Chronic lymphocytic leukaemia (CLL). The job of B-cells is to create immunoglobulin (also called antibodies) which assists the body in the protection against infection and disease (Harris, et al, 1999). Once CLL is diagnosed, around 30 - 50 per cent patients of CLL can continue without any treatment (Chiorazzi et al, 2005) because their CLL remains steady for many months and even years whereas in some patients, the leukemic cells are abnormal and unable to function properly (Jaglowski and Byrd , 2010) causing the cells to expand in an uncontrolled manner and aggregate in the bone marrow, bloodstream, lymph nodes (glands), spleen and other parts of the body causing a disturbance in the normal blood cell production. Subsequently, people with CLL are at a greater risk of developing anaemia, recurrent infections and to bruising and bleeding easily because their bone marrow creates insufficient numbers of red cells, normal white cells and platelets.

Approximately, 35% of all leukaemia cases are reported to be Chronic Lymphocytic Leukaemia (CLL) making it the most widespread type of leukaemia. In the CLL the accretion of mature, neoplastic B cells in the peripheral blood, bone marrow and lymphoid tissue takes place which effects older men more than younger generation and women (Redaelli, 2004). In the CLL, one or more genes which are responsible to encode information that facilitates growth and development of blood cells, undergo mutation which will cause abnormal growth (Koduru et al, 1993). The CLL is also called a clonal blood stem cell disorder (Koduru et al, 1993) because a 'clone' which is a group of alike cells all with the same deficiency is produced when the original mutation is conserved during affected stem cell division and production. The prognosis of CLL was analyzed by the Research investigating gene mutations and it was s seen that it differs from patient to patient and also on the basis of the stage of the disease. Moreover, there is still no information present regarding reaction to chemotherapy and the predictive factors that conclude the prognosis of the disease (Robak, et al, 2009). The CLL is also said to be inherited from family in some cases especially approximately 7 times higher than the population average chances are present if it is a first degree relative (i.e. father, mother, brother, sister or child) of the patient, but still mostly family members are not at risk of CLL because the background chances of developing CLL are very low (Travis et al, 1992), therefore it is not advised to do screening for CLL in every patient just because CLL is present in a family member.

Symptoms of Chronic Lymphocytic Leukaemia

In many cases, there are no signs of symptoms in the early stages of CLL, as it develops slowly, therefore either during a routine blood test or physical examination, CLL is diagnosed by chance. Some symptoms experienced are:

Recurrent or increased chances of infections due to deficiency of normal white cells and normal antibodies. Constant fatigue, weakness, shortness of breath and pale appearance occurs due to anaemia i.e. deficiency of normal red cells such as. Constant bleeding or bruising for no apparent reason, nose bleeds, bleeding gums and the manifestation of red or purple flat pinhead sized purple spots on the skin, especially on the legs which occurs due to small superficial capillary bleeds known as petechia are symptoms occurring due to deficiency of normal platelets. Aggregation of lymphocytes causes a painless swelling of the lymph nodes (glands) in neck, underarms or in groin. Moreover, while excretion of lymphocytes takes place, there are chances of spleen to be enlarged (splenomegaly) which causes ; discomfort, pain or fullness in the upper left-side of the abdomen, pressure on the stomach causing a feeling of fullness, indigestion and a loss of appetite. Moreover, hepatomegaly can also occur along with excessive sweating during night, fevers and unintentional weight loss (Hallek, 2011).

Diagnosis of Chronic Lymphocytic Leukaemia

There are various diagnosing methods; mostly the CLL is diagnosed by observation of the lymphocytes in the blood sample, as during the CLL a huge amount of lymphocytes can be found. Moreover, another modern technique of diagnosis is immunophenotyping (Hallek, 2008).

Full blood count

Initially a simple blood test called a full blood count or full blood examination (FBC or FBE) or complete blood count (CBC) is done to investigate the CLL therefore once the sample of blood from the patients is taken, it is then sent to the laboratory for investigation under the microscope to observe the number, size and shape of red blood cells, white blood cells and platelets. The lymphocyte count is extremely high in the CLL and for a diagnosis of the CLL, it should be at least 5 x 109L. In severe forms of CLL, anemia and thrombocytopenia (a lower than normal platelet count) can also occur (Correia et al, 2009).

Immunophenotyping

For the confirmation of CLL, Immunophenotyping, or flow cytometry tests are carried out to differentiate it from other similar diseases. In Immunophenotyping, special markers called antigens which are present on the surface of cells and act like flags recognizing the atypical feature of CLL, are used. Antigens are also called 'cluster of differentiation' or CD antigens with a specific number. Certain B-cell antigens like CD19, CD20, CD23 and CD5 and other surface markers are usually expressed on the leukemic cells in the case of CLL (Hardy and Richard, 2008) for the purpose of different ion from other similar diseases such as prolymphocytic leukaemia, hairy cell leukaemia, mantle cell lymphoma and other types of lymphoma (cancer of the lymphatic system).

Cytogenetic Tests

To gain information about the structure and number of chromosomes present which are the structures that carry genes, Cytogenetic tests such as chromosome analysis in which missing, extra or abnormal chromosomes are observed and fluorescent in situ hybridization (FISH) tests are carried out to offer information about the possible course of disease and the best way of approaching treatment (Hallek,2008).

Immunoglobulin Levels

To measure the levels of antibodies in patient blood, a blood sample is done. Continuous infections are likely to occur in people with decreased levels of normal antibodies therefore monthly intravenous immunoglobulin (antibody) treatment can be useful to reduce chances of infections. Regular follow up is kept by repeating blood tests.

Bone Marrow Examination

To verify the diagnosis, course of disease along with the response to treatment of CLL, a sample of bone marrow (biopsy) usually from the back of the iliac crest (hip bone)bone is taken which is then sent to the laboratory for analysis under the microscope. The procedure of biopsy can be carried out either at the haematologist’s rooms or clinic with merely a use of local anaesthesia or sometimes short general anaesthesia in the form of an injection under the skin. Moreover, a mild sedative and a pain-killer can also be given before procedure. Initially the aesthetic injection is inserted and once a mild stinging feeling is felt after a minute or two, a long thin needle enters the skin and outer layer of bone into the bone marrow cavity to pull out a little sample of bone marrow fluid with the help of the syringe placed at the end of the needle - this sample is called a 'bone marrow aspirate'. Then to achieve another small sample of bone marrow for detailed analysis regarding structure of the bone marrow, another slightly larger needle is inserted to draw out the sample called 'bone marrow trephine'. Once the procedure is done, a small dressing or plaster is placed over the area where biopsy was done and it can easily be removed after a day. Paracetamol can be prescribed in case of mild bruising or discomfort, there are less chances of severe complications such as bleeding or infection to occur. It is advised to bring along a family member or friend because there are chances of feeling drowsy after procedure.

Other Tests

A wide combination of blood tests and imaging tests are carried out once the diagnosis of CLL is verified to determine the stage, extent of the spread of disease in the body and side effects on other organs such as the kidneys, liver and other vital organs. Moreover, the finest treatment can also be determined by these tests and it can also be used as a standard for future reference to differentiate that how well the patients are progressing.

Other Blood Tests

• Kidney function tests

• Liver function tests

Prognostic Factors in Chronic Lymphocytic Leukaemia

To estimate the hazard of development, an important prognostic marker; scientific staging residue along with beta 2-microglobulin can be incorporated into nomograms (Wierda, El at, 2007). Depending on the number of lymphoid areas affected by the disease (lymph nodes, spleen or liver), and the red cell and platelet counts in the circulating blood, usually, to measure the prognosis in CLL, the Rai and Binet staging systems (Molica et al,1975) have been used to form three major subgroups (good, intermediate or poor prognosis) of patients. Early form of disease is placed in stage A in which the patient does not have any symptoms and can survive without treatment. Progressive forms of disease are placed in Stage B and C which usually requires treatment (see table 1). By analyzing the various biomarkers that have been acknowledged, a molecular profile can be created, where the most essential is cytogenetic analysis by fluorescent in situ hybridization (FISH), mutational status of the immunoglobulin heavy-chain variable gene (IgVH), use of IgVH, and expression of 70-kDa zeta-associated protein (ZAP70) and CD38. With the help of FISH, del13q, del11q, trisomy 12, del17p, and del6q are few of the chromosome abnormalities that can be detected in more than 80% of CLL patients (Krober et al, 2002). In 50% of the patients, del13q,14 can be found and it usually has a good prognosis. In approximately 20% of patients, two microRNA clusters, mir-15a and mir-16-1, are present in the deleted region of 13q14.7 Del11q and trisomy 12. Despite del17p being rare i.e. only 10%, still this abnormality does not give good prognosis, it spreads rapidly and has a short survival time ((Bosch et al, 2006). It is essential to reassess these markers once cytogenetic changes take place in the CLL. Somatic hyper mutations in IgVH has occurred in fifty percent of CLL patients who have experienced a slow clinical course and as compared to patients without somatic hyper mutations, a longer survival time (Haublim et al, 1999). A biased repertoire of V genes with over-representation of certain Ig gene segments, such as IGHV1-69, IGHV4-34, IGHV3-7, and IGHV3-21 are being used by the CLL cells and this was determined once the examination of variable region sequences was done (Fais et al, 1998). Both mutated IGHV3-21 and regular IGHV3-21 impart rapid spread of disease in patients with CLL (Mauerer et al, 2005). Surrogate markers such as expression of ZAP70 and CD38 determine the IgVH mutational status despite being no association between ZAP70 expression and IgVH mutational status (Rassenti et al,2004) whereas in some cases ZAP70 is better to determine progression of disease rather than mutation status, but this theory still remains contentious. Various other biological characteristics having poor prognostic implications such as del17p, del11q or use of IGHV3- 21 are used once a contradictory case is introduced (Krober, 2006). A 13- gene signature which occurs simultaneous with the ZAP70 status, unmutated IgVH expression and disease progression altered microRNA expression have been shown by the MicroRNA array analysis, as the regulation of expression of genes that facilitate apoptosis and cell cycle progression is done by the micro RNA expression (Calin et al, 2004). There is still no confirmation regarding the mutation status of CD38 and IgVH as CD38 expression may vary over time (Montillo et al, 2005). del17p and del11q, IgVH unmutated status, use of the IGHV3-21 gene segment, and expression of either ZAP70 or CD38 are few of high-risk characteristics exhibiting disease progression. Studies are still being carried out on the impact of these biomarkers therefore is it essential to understand the working of these biomarkers in clinical practice. The prognostic consequence of some of these parameters is likely to be altered once therapy is enhanced as some groups react better to modern treatment combinations.

Treatment of CLL

Chronic lymphocytic leukaemia is still a life threatening despite advancements made in its treatment strategies (Awan, et al, 2010). A research was done on pre-treated CLL patients in 1989, whose results revealed a clinical testing which was done on fludarabine as monotherapy with an efficient response data and toxicity outline (Keating et al, 1989). Therefore we can conclude that for effective CLL treatment to be done, the purine analogue fludarabine is very helpful as the p53-mediated apoptotic pathway is initiated by Fludarabine treatment ( Dohner et al, 1995).

First Line Therapy

For a severe form of CLL, usually chemotherapy with incorporation of alkylating agents was the first line of treatment for patients (Robak, 2007), but some alkylating agents such as chlorambucil, cyclophosphamide and bendamustine were dismissed for younger healthy patients once purin nucleoside analogues (PNAs) were created.

Alkylating Agents

The most commonly used treatment for CLL since ages was either chlorambucil and cyclophosphamide alone or to develop response rate, their combination with corticosteroids. An overall response rate (ORR) of 60–90% along with a complete response rate (CR) in around 20% of all patients was achieved when initially chlorambucil was used, and this result was similar to PNAs when Jaksic et al. [1997] discovered an ORR of 89% when chlorambucil at 15 mg/day was used. When six cycles of fludarabine (25 mg/m2 for 5 days intravenously every 28 days) were compared to chlorambucil during a multicentre phase III trial carried out by the German CLL Study Group (GCLLSG) (Eichhors et al,2009) on patients >65 years (0.4 mg/kg body weight, increasing to 0.8 mg/kg every 15 days) then it was observed that fludarabine (72% vs. 51%, P = 0.003 and 7% vs. 0%, P = 0.011, respectively)exhibited a greater ORR and CR rates with no change in progression free survival (PFS) or overall survival (OS) (19 months vs. 18 months, P = 0.7 and 46 months vs. 64 months, P = 0.15, respectively). An alternative for chlorambucil can be cyclophosphamide due to alike chemistry (Robak et al, 2002) and sometimes replaces chlorambucil as a single agent when chlorambucil is not being tolerated at a dose of 2–3 mg/kg/day orally or 20 mg/kg intravenously every 2–3 weeks. Moreover, the combination of chlorambucil and prednisone (Raphael et al, 1991) was also considered better than the combination of cyclophosphamide with vincristine, doxorubicin and prednisone (COP, CHOP, CVP regimens) as evidence says in the meta-analysis published by the CLL Trialists Collaborative Group. On the other hand, high dosage of chlorambucil was proven more useful over CHOP in terms of ORR (89.5% vs. 75%, P < 0.001) and OS (68 months vs. 47 months, P < 0.005) (Jaksie et al, 1997) by Jaksic et al.

Purine Nucleoside Analogues (PNAs)

PNAs such as Fludarabine, cladribine and pentostatin are basically cytotoxic agents with antitumor characteristics because they are structurally alike to adenosine and deoxyadenosine (Robak, 2002) so therefore by inhibiting DNA primers and DNAligase, they cause a disturbance in the DNA synthesis mechanism. Moreover, there has been evidence to support that when it comes to response and time to progression in previously untreated CLL patients, PNAs are more efficient than alkylating agents, but there is still doubt regarding their impact on survival. A common example of an active alkylating agent is, Fludarabine which can be used both as a single-agent and as part of combination with other cytotoxic drugs. Sorensen et al observed a 32% overall response rate (ORR) in formerly treated or relapsed CLL patients when Fludarabine was administered at 25 mg/m2 per day for 5 days as a single-agent with haematological (43%) toxicity, 22% infections, and neurotoxicity in 14% of patients (Sorensen et al, 1997). Moreover, the functioning of fludarabine alone or in combination with prednisone in 174 patients having severe form of CLL was observed by Keating et al. (1998) who analyzed an ORR and CR of 78% and 29%, respectively once the treatment was done. It took 31 months for patients with response, whereas the overall median survival was 74 months. Randomised trials verified by a meta-analysis published by Steurer et al. (2006) also gave comparable results. As a result, an efficient and tolerable oral formulation was created that was corresponding to the intravenous administration (Ross et al,2004).

Monoclonal Antibodies

C-1 Rituximab

A 297-amino acid transmembrane phosphoprotein which resides on more than 90% of mature B-cell leukaemia and lymphomas in the form of a cell-surface antigen is CD20, whereas chimeric anti-CD20 monoclonal antibody is also present called, Rituximab which by various mechanisms such as: antibody-dependent cell-mediated cytotoxicity, complement-dependent lysis and induction of apoptosis, performs against leukaemia cells. But for efficient performance of rituximab, its dosage should be kept in mind, at conventional doses (375 mg/m2 weekly), it gives poor results in CLL whereas evidence suggests that high doses are more effectual (Byrd,et al, 2005). When the effect of the rituximab combination with PNAs was observed by Byrd et al. (2005) an ORR rate of 90% with 47% CR along with a 77% ORR and 28% CR was observed in patients treated simultaneously with rituximab plus fludarabine and a sequential regimen respectively during a phase II randomised study. Moreover, in the rituximab group, PFS and OS were also considerably elevated (P < 0.0001 and P < 0.0006, respectively). Moreover, an ORR rate of 95%, a CR rate of 70% and 80 months of median time to progression was observed by Keating et al. [2005] when he analyzed this relationship in the FC combination on 224 non treated CLL patients. A phase III trial was done by the GCLLSG on 817 patients by Hallek et al. (2009) which produced progressive CLL randomised to FC or FCR as first-line therapy. In the FCR arm, CR rate was considerably elevated (45% vs. 23%) along with PFS (43 months vs. 32 months). A revision of results showing differences in PFS (64.9% vs. 44.7%, P < 0.001) and OS (87.2% vs. 82.5%, P = 0.012) which were approving FCR arm were reviewed by Hallek et al.( 2009) in ASH2009. Despite being myelotoxic, FCR caused less complications such as infection or toxic deaths as compared to others, nevertheless, in elderly patients it should be administered carefully as it can cause myelosuppression. An alternative for the aforementioned problem is decreasing dose of fludarabine and cyclophosphamide and enhancing doses of rituximab (Foon et al, 2009). Moreover, greater ORR, CR rates an median duration of response are obtained by combinations of rituximab plus PNAs, with or without cyclophosphamide, results, such as pentostatin and rituximab and rituximab plus high-dose methylprednisolone are well thought-out during conditions like limited myeloid reserve as compared to PNA alone. The combinations can also be used as a first-line therapy in younger patients suffering from symptomatic and/or progressive CLL.

C-2 Alemtuzumab

A small cell-surface glycoprotein residing on normal and malignant B and T lymphocytes, compared with other blood cell types is, CD52 (Fabion et al,1993), whereas a humanised anti-CD52 monoclonal antibody that repairs complement and decreases normal lymphocytes and lymphoma cells is, Alemtuzumab (Campath 1-H®) (Hale et al,1983) which has been analyzed evaluated in refractory disease, front-line therapy, and as a fraction of combination. Osterborg et al analyzed the efficiency of alemtuzumab in 1996 in non treated CLL patients who also gave results of a phase II trial (Lundian et al,2002). A 19% CR and 68% PR was gained by Hillmen et al(2007)when 38 non treated patients were given alemtuzumab subcutaneously, during a prospective randomised phase III trial (CAM 307), whereas, an ORR rate of 83% for alemtuzumab and 55% for chlorambucil was achieved when alemtuzumab (30 mg iv three times a week for a maximum of 12 weeks) and chlorambucil (40 mg/m2 orally every 28 days up to a maximum of 12 cycles) were compared. A 42% decreased risk of development or death was observed by Alemtuzumab due to greater PFS (HR = 0.58; P = 0.0001). Furthermore, alemtuzumab is considered effective for patients having genetic abnormalities (p53 mutations and 17p deletions) as it is a poor cytogenetic risk for CLL patients. For the purpose of verification, Lozanski et al(2004), performed a randomised study on 21 patients having chromosome 17p deletion to be treated either with chlorambucil or alemtuzumab for the purpose of comparison, as a result it was seen that the alemtuzumab group exhibited both an increase in ORR and PFS rate with no differences in OS due to the small sample size. Moreover, an ORR rate of 40% and median PFS of 9 months was observed when alemtuzumab was administered during relapse. Positive results were also achieved by the combination therapy of alemtuzumab with cytotoxic agents, specifically fludarabine (Kennedy et al, 2002). The combination therapy of alemtuzumab and fludarabine cyclophosphamide- rituximab (CFAR regimen) was analyzed by Wierda et al. (2008) in 63 patients having severe CLL, he observed that the ORR was 94% and CR was 69%, whereas ORR was 77% and CR was 54%, in patients (n = 13) with 17p deletion and after 16 months follow up, no change in time of advancement and survival was seen. Moreover, the management guidelines for the use of alemtuzumab in CLL considers this treatment routine as myelosuppressive (Osterborg et al,2009) therefore, for controlled clinical trials, a combination therapy should only be on hand.

Emerging Drugs for CLL

For the cure of CLL patients, various modern therapies such as immunomodulating agents, novel PNAs, bcl-2 inhibitors, protein kinase inhibitors and small molecule signal transduction inhibitors that influence the genetic and epigenetic tumour pathway generation are being introduced which are then tested with the help of preclinical studies and early phases of clinical trials. Following mentioned are few novel drugs being introduced.

6.1- Anti-CD20 mAbs

Another modern third generation (mAb) drug obtained from the humanization of the parenteral B-Ly1 mouse antibody is, Obinutuzumab (GA-101) which is given to patients with CD20+ malignant disease in a phase I/IIa trial in the form of a single agent (Salles et al, 2008), and it has a high affinity for the CD20 epitope and can bind to it along with exhibiting superior caspase-independent apoptosis induction than rituximab.

6.2- Lumiliximab

Another novel drug that is a genetically engineered macaque-human chimeric anti- CD23 IgG1_ mAb which can stimulate various antiproliferatives functions such as apoptosis is Lumiliximab, which has an affinity to bind with CD23. The safety, efficacy and pharmacokinetics of lumiliximab was analyzed by Byrd et al(2007) in 46 patients with refractory/ relapsed CLL during a phase I trial. Byrd et al gave a dosage of 6 regimens for 4 weeks together with the lowest dose of 125 mg/m2/week up to the highest dose of 500 mg/m2 3 times per week for 4 weeks and in 91% of the patients, observed a decrease in absolute lymphocyte count and lymphadenopathy.

6.3- Anti-CD40

Another anti-CD40 mAb drug that has the ability to stimulate cytotoxicity against the CLL cells is called, Dacetuzumab. A 5% ORR was observed by Furman et al(2010) on 12 patients during a phase I study with maximum doses given between 3 and 8 mg/kg/week.

6.4- Bcl-2 Family Inhibitors

A synthetic, 18-base, single-strand phosphorothioateDNAoligonucleotide intended to decrease the bcl-2 mRNA expression, is Oblimersen, which was administered at doses 3 to 7 mg/kg/day as 5- day constant intravenous infusion to 40 relapsed/refractory CLL patients every 3 weeks during the initial phase I/II study (OiBrien et al, 2005). A combination of FC and oblimersen gave a noteworthy 5-year survival advantage (HR 0.60; P = 0.038) to the patients treated by it (OiBrien et al, 2009).

6.5- Obatoclax (GX15-070)

Obatoclax (GX15-070) is an agent that can encourage the release of cytochrome C from mitochondria isolated from leukaemia cells by having the ability to put a standstill to various anti-apoptotic bcl-2 family proteins. 4% of ORR (one patient achieved PR) was analyzed by OˇıBrien et al. during the phase I trial (OiBrien et al, 2009).

Conclusion

The CLL is a highly multifaceted disease which has been focused on for the past 10-15 years resulting in modern advancements in its treatment. Initially, the first line of treatment of progressive and symptomatic CLL patients were alkylating agents which then advanced to being purine-analogues especially in combination to be more efficient particularly in formerly treated, unmanageable, relapsed or young patients without any associated morbidity. Another novel advancement in treatment was, Monoclonal antibodies such as Alemtuzumab which was considered first-line treatment in fludarabine-refractory patients. Moreover, Haematopoietic stem cell transplantation, non-myeloablative allogeneic transplant is also treatment options due to decreased toxicity especially for elderly CLL patients. There are still attempts being made to specifically identify high-risk patients so they can gain suitable treatment for themselves.