What Role Does Cyclophosphamide Treatment Play In Neuroblastoma Therapy

Neuroblastoma is a neonatal solid tumour (Wood and Lowis, 2008) arising from early neural crest tissue which populates multiple sites along the sympathetic nervous system (Maris et al, 2007). The aetiology of neuroblastoma is not well understood. Research investigating exposure to environmental factors in utero has not been fully validated but genetic changes are implicated in carcinogenesis (Brodeur, 2003). The incidence of neuroblastoma in the UK is 1 in 6-8 million children (Wood and Lowis, 2008) and accounts for 7-8% of all childhood cancers (Wood and Lowis, 2008). 90% of cases are indentified in children under five years old (Howman-Giles, 2007) and although it is the most common type of cancer in infants under 1 year (Maris, 2010), neuroblastoma are, albeit rarely, recognised in adolescence (Wood and Lowis, 2008).

The median age at diagnosis is 17 months (Maris, 2010) and there are a number of techniques available for identification of neuroblastoma, the most common of which is by 123Iodine-metaiodobenzylguanidine (123I-MIGB) imaging (Wood and Lowis, 2008). C omputed tomography (CT) imaging gives information on location, size and anatomical relationship to neighbouring structures (Wood and Lowis, 2008) and magnetic resonance imaging (MRI) is used to confirm paraspinal tumours (Wood and Lowis, 2008). Elevated urinary catecholamines and their metabolites can indicate the extent of metastasis where a low ratio of vanillylmandelic acid to homovanillic acid indicates low risk disease requiring less intensive therapy (Strenger, 2007). Conversely elevated ferritin and lactate dehydrogenase can indicate advanced and aggressive disease (Wood and Lowis, 2008). Genetic testing for MYCN oncogene amplification, mutations in anaplastic lymphoma kinase (ALK) oncogene (Maris, 2010), diploid DNA content, and chromosomal abnormalities are all adverse prognostic biomarkers involved in sporadic or familial neuroblastoma (1-2% of cases) (Wood and Lowis, 2008 and Castleberry, 1997). The International Neuroblastoma Staging System (INSS) is used to confirm diagnosis after patient evaluation and is based on extent of tumour, involvement of lymph nodes and dissemination (Wood and Lowis, 2008).

Neuroblastoma is stratified into three main risk categories; low, intermediate, and high-risk (Maris et al, 2007). The risk level are related to the 5 year survival rate where low risk correlates to >98% survival, intermediate is 90-95%, and high risk is 40-50% (Maris, 2010). Factors which influence risk level are age of patient, tumour stage in relation to INSS, histological category, and genetic variables (MYNC amplification, tumour genomics, DNA ploidy) (Maris, 2010).

Neuroblastic tumours are a multifaceted group of neoplasms and prognosis depends upon many factors. Survival rate is high in infants <1 year with benign neuroblastoma and infants with localised tumours are often cured (Maris, 2010). If neuroblastoma is detected early, tumours with favourable biological features are treated with reduced therapeutic intensity and if a normal MYCN gene is identified, this correlates with a better prognosis (Cancer Research UK, 2010).

However, neuroblastoma presentation and clinical behaviour is variable where 1% of patients have no detectable tumour (Castleberry, 1997) and more than 50% of patients have extensive metastases and their long term survival rate is only 40% (Wood and Lowis, 2008).

Tumours with adverse prognostic features and recurrences are treated with intensive chemotherapy in combination with cyclophosphamide (Maris, 2010) however, it's action may be affected by different isoforms of cytochrome P450 involved in drug metabolism and bioactivation. Neuroblastomas response to chemotherapy over time seems to worsen despite treatment and this may be due to multidrug resistance (MDR) genes involved in efflux of anticancer drugs (Wood and Lowis, 2008). CD44 negative cell surface glycoprotein expression also correlates with poor survival and older children with metastatic (stage 4) disease also have a poor prognosis.

However, neuroblastomas are a group of enigmatic tumours which can spontaneously regress as with stage 4S which dramatically improves survival rate (Brodeur GM, 2003).

During development, neuroectodermal cells migrate from the neural crest to give rise to the adrenal gland and sympathetic nervous system. In neuroblastoma these neural crest cells fail to respond to normal differentiation signals and maintain a state of dedifferentiation leading to malignancy and variable clinical presentation. The diversity of locations where neuroblastomas arise reflects the migration of these cells (Marieb and Hoehn, 2007, Van Roy et al., 2009).

The exact aetiology of neuroblastoma is unknown; however 22% of all neuroblastomas are the result of a germinal mutation, 1-2% of all neuroblastomas have a family history of neuroblastoma and the remainder are due to spontaneous mutations (Westermann and Schwab, 2002). In the pursuit of understanding the carcinogensis of neuroblastoma scientists have conducted genetic studies to identify neuroblastoma-specific genetic abnormalities (Table 2.1) many of which have been used as indicators of prognosis. The data supporting environmental risk factors for the development of neuroblastoma is very variable however through numerous studies breastfeeding during for the first 6 months of pregnancy has shown a protective role against neuroblastoma (Heck et al., 2009). MYCN is a transcri ption factor, part of the MYC oncogene family where the N stands for neuroblastoma. Myc is responsible for controlling transcri ption of cell growth genes and in neuroblastoma it is amplified resulting in deregulated growth and proliferation. MycN forms a heterodimer with its binding partner Max to initiate transcri ption. The mycN/max heterodimer is favoured (Fig2.1) during mycN amplification and thus mycN genes are overexpressed. (Maris and Matthay, 1999). MYCN amplification is associated with an aggressive tumour and poor prognosis (Brodeur, 2003). Mutated anaplastic lymphoma kinase gene (ALK) which codes for a tyrosine kinase receptor, has been found to explain the majority of hereditary neuroblastomas and 8% of spontaneous neuroblastoma (Mosse 2008). ALK is activated by gene amplification and forms a complex with the hyperphosphorylated ShcC, this alters the activity of the mitogen-activated protein kinase pathway to growth factors (Osajima-Hakomori et al., 2005). Ploidy status is another predictor of prognosis where near-triploidy status gives a favourable prognosis and near-diploidy status a poor prognosis. Triploid DNA content implies a defect with mitosis where diploidy suggests a fault with genome stability that results in chromosomal rearrangements and translocations, which is more detrimental (Van Roy et al., 2009). The Sonic hedgehog, notch and Wnt/?-catenin pathways play a role in stem cell differentiation and renewal in diverse tissues. Changes in these pathways have been associated with embryonal tumourigenesis (Maris et al., 2007). Figure 2.2 illustrates how neurotrophin tyrosine kinase receptors (NTRK) are important regulators of survival, growth and differentiation for neural cells. Two members of this kinase family, TrkA and TrkB and their ligands (NGF and BNDF, respectively) have effects on the growth of neuroblastoma cells. (Maris et al., 2007, Nakagawara and Ohira, 2004) The TrkA/ NGF signalling pathway have a known role in the regulation of apoptosis and this regulation involves p53, p73 and ?p73, which have been previously suggested to be important regulators of neuronal differentiation and apoptosis, therefore these proteins may be associated with the development of neuroblastomas (Nakagawara and Ohira, 2004).

Alteration on the ?-catenin pathway signal has been associated with induction of MYC and other target genes leading to an aggressive neuroblastoma. ?-catenin plays a role in the maintenance and proliferation of neural crest stem cells and other neural progenitors (Maris et al., 2007). Identification and understanding of the common genetic changes provide valuable information on the carcinogenesis of neuroblastoma and may provide a therapeutic target. In the 1950's a wide variety of phosphamides, including isophosphamide and tropophosphamide, were produced in response to a report upon malignant tumours (Gomori, 1948) and this led to lots of preclinical tests to determine therapeutic indexes of the various drugs. Arnold, Bourseaux and Brock reported in their findings that they found cyclophosphamide, an alkylating agent, to have the highest therapeutic index (Arnold et al., 1958). Since those findings it has been in use for over 50 years and has gained popularity, becoming a commonly used cancer drug to treat multiple cancers. However, its parent compound, mustard gas, was used during World War I as a chemical agent to kill soldiers.

Cyclophosphamide is an ester of merochlorethamin (Pharmacogenomics Knowledge Base, 2007). It is administered in the body as an inactive enantiomer. It requires activation by hydroxylation at its C4 position which is mediated by cytochrome P450 enzymes in the liver (Fernandes et al., 2011). Many cytochrome P450 enzyme isoforms exist for example CYP2B6, CYP2C9, CYP2C19. Different polymorphisms exist (LeBlanc and Waxman, 1990); there is a difference in the sequence of the protein. With CYP2B6*6 the rate of hydroxylation is increased. Upon hydroxylation 4-hydroxycyclophosphamide is formed, this renders the cyclophosphamide soluble, hence is can get transported out of the hepatocyte and enter the cancerous cell where is becomes in equilibrium with aldophosphamide. Within the cancerous cell aldophosphamide undergoes decomposition, forming phosphoramide mustard and acrolein (Pharmacogenomics Knowledge Base, 2007). Phosphoramide mustard produces a highly electrophilic aziridinium species that forms DNA cross-links (Whitley and Day, 2011). Acrolein, an aldehyde, on the other hand is neurotoxic (Roy and Waxman, 2006) in high concentrations and causes inhibition of the cytochrome P450 enzymes in three ways.

Firstly, by the depletion of growth hormone, which is required for the expression of some sex dependent isoforms of cytochrome P450.

Secondly, inhibition through the direct action of acrolein upon cytochrome P450 enzymes can be broken down into two further actions. The first is it causes degradation of the cytochrome P450 mRNA, the second is upon the flavoenzymes which are responsible for donating H+ to the cytochrome P450. This reaction is paramount for the C4 hydroxylation of cyclophosphamide.

Lastly, acrolein can also act upon the hypothalmic-pituatry gonadal axis. This causes hepatic alkaline phosphatase and tyrosine-alphaketoglutarate transaminase activity to increase resulting in dysregulation of cytochrome P450 production. Acrolein can be detoxified by conjugation with glutathiones or by action of aldophosphamide dehydrogenase. There are 2 different isoforms of cyclophosphamide that exist: R-(+)-N-dechoroethlcyclophosphamide (R-type) and S-(-)-N-decholoroethylcyclophosphamide (S-type). Both show different properties. R-type has a higher rate of clearance, lower volume of body distribution (hence a lower plasma concentration) and is more effective to leukemic cells compared to the S-type. The R-type shows a higher therapeutic index against solid tumours. Phosphoramide mustard (PM) forms a crosslink between two guanylic acids in deoxyribonucleic acid (DNA) (Dong et al., 1995). It does this by creating a link between the two strands which are separated by a base e.g. G-X-C on the 5' strand and C-Y-G on the 3' strand, respectively (Dong et al., 1995). The order of the bases must be as specified above, otherwise interstrand crosslinks do not form between the guanine moieties allowing DNA replication to continue. PM binds to the N7 (Brookes and Lawley, 1961, Mattes et al., 1986) and forms the first adduct, 7-(N`-(2-chloroethyl)-2-aminoethyl]-guanine by alkylation. This is then hydroxylated giving a conformational change allowing the crosslink to occur with a G - PM - G construct. The formation of the construct prevents DNA replication from occurring because ribosomes cannot traverse the length of the DNA strand and thus prevents cancerous cells from proliferating.

Chemotherapy is key in treating and preventing further recurrence of neuroblastoma. Cyclophosphamide is one of the main chemotherapeutic agents and is used in over 50% of paediatric cancer patients, including those with neuroblastoma where it is given as part of combination therapy(McCune et al., 2009). The course of treatment depends on what risk group patients fall into as cyclophosphamide use depends on the progression of neuroblastoma (see table 1). Low risk patients are observed or undergo surgery if necessary to remove the tumour. However, if only less than half the tumour can be removed, then a short course of combinational chemotherapy (via oral tablets or intravenous injection) is given (see table 2). This treatment is shown to cure 90% of low risk patients( Matthay et al., 1998). In certain circumstances some patients have a recurrence of the tumour transferring them from the low to intermediate group. These patients, alongside those primarily diagnosed as intermediate risk are subject to 4- 8 cycles of chemotherapy (Maris et al., 2007). The first course is the same as course 3 (see table 4.2), followed by courses 2 and 1. Finally, course 4 is replaced by cyclophosphamide and doxorubicin. Cyclophosphamide use is very common here; as given in 3 out of 4 courses. This is largely due to its effects on tumour cells; in that cyclophosphamide kills and prevents the tumour from growing (U.S.National Institute of Health, 2010a). For chemotherapy in high risk patients, cyclophosphamide is again, commonly used. However, courses of chemotherapy are given in high doses, including other agents not previously used, such as: ifosfamide, vincristine and toptecan. A pilot study by COG (Children's Oncology Group) considered substituting cyclophosphamide and topotecan for a combination of vincristine, cyclophosphamide and doxorubicin. Although both cycles have different combinations of drugs, cyclophosphamide is consistent in both, emphasising its importance in the treatment of neuroblastoma (Maris et al., 2007). Apart from chemotherapy treatment other measures taken include surgery to remove the primary tumour, myeloablative consolidation therapy, radiotherapy, stem cell transplantation and finally treating minimal residual disease to avoid any relapses(Children`s Neuroblastoma Cancer Foundation, 2011). Recurrent neuroblastoma treatment depends on the type of neuroblastoma primarily treated for, but treatment is often experimental as there are currently no standard treatment procedures. One favoured treatment is the combination of topotecan and cyclophosphamide where there are currently various methods in use with varying doses (De Ioris et al., 2011, Kushner et al., 2011, London et al., 2010 and Saylors et al., 2001). Some treatments also include the addition of other chemotherapeutic drugs(Kushner et al., 2011). This combination has been shown to increase progression free survival, but not overall survival (London et al., 2010). Generally, the higher the dose of cyclophosphamide, the better the patient's outcome however, this is associated with more severe side effects (McCune et al., 2009 and Kushner et al., 1994). Common side effects include hair loss, neutropenia, anaemia, immunodeficiency and bleeding can occur if platelet counts are low ( Takimoto,2008) and the extent of these side effects depends on the patient ( Sweetman, 2007). Serious problems associated with cyclophosphamide are disruption of the menstrual cycle due to ovarian failure which can cause temporary or permanent sterility. It also has teratogenic effects, thus it is not given to pregnant women (Ozolins, 2010). Furthermore, a possible serious effect is bladder toxicity from the metabolite acrolein, which can lead to cystitis and in the long-term transitional cell carcinoma of the bladder (Monach et al., 2010 and Perry, 2007).

Currently there is no cure for neuroblastoma. The cancer possesses a broad spectrum of clinical behaviour (Weinstein, 2003) and the molecular events that occur affect the response to treatment and rate of tumour growth are largely unknown (Weinstein, 2003). It is therefore vital that research continues so that we can understand better how the cancer 'behaves' in order to generate more therapeutic targets, leading to more effective . Clinical trials are being undertaken to investigate the effect of cyclophosphamide with other drugs and treatment types. One clinical trial at Stanford University aims to improve the effectiveness of cyclophosphamide chemotherapy using the drug isotretinoin (National Cancer Institute, 2007). During cell crisis, cell populations proliferate to vast numbers resulting in the shortening of telomere ends subsequently causing cell death. Cancer cells have the remarkable ability to regenerate their telomere ends, avoiding crisis and becoming 'immortal'. Mutated variants of proliferating cells promote the genes encoding telomerase, resulting in abnormally high levels of the enzyme (hTERT). This environment lengthens the telomeres despite vast proliferation (Weinberg, 2006). The drug isotretinoin has been found to regulate telomerase activity in tumourigenic cells preventing telomerase regeneration and controlling disordely cell proliferation. Studying the effect of isotretinoin on NB4 cell lines proved effective however, clinical trials are required (Pendino et al., 2001). The clinical trial may lead to reduction of tumour size to the point where it can be easily excised through surgery, but it remains to be seen whether the proposed treatment will succeed.