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please i provide 5 genes as influence genes in childhood acute lymphoblastic leukaemia (discussion influence file, see attached) . please focus on genes.

old order you focused on subtypes acute lumphoblastic leukeamia rather than genes(my results).

please synthesis my new discussion with old discussion ( i have updated old discussion)

if the 371082 can not do my order, i will refund my money.

Discussion
The aim of this project was to investigate gene expression in childhood leukaemia with the use of microarrays of cancer samples obtained from patients. The project also aimed to discover the factors that resist drugs via the use of related biomarkers as well as to investigate disease deterioration for the reasons stated above and finding the genes that can potentially be selected by the therapy. Table 5 shown in the results section already shows some of the biomarkers that are unique and can be associated with acute lymphoblastic leukaemia in terms of response to and resistance to therapy (Litsenburg et al., 2013). A biomarker in this project was used to refer to a biological agent whose presence in a sample is indicative of the presence of a disease, in this case, ALL. Different biomarkers are indicative of different subtypes of ALL in terms of whether the leukaemia is of a B-precursor, T-lineage subtype or ETV6-RUNX1 precursor (Ward et al., 2014).
A lot of haematopoietic differentiation processes have been observed in many studies that have been recently performed by oncologists who have been able to demonstrate that such differentiation processes are characterised by gross epigenetic plasticity (Mcgrath, 2015). The epigenetic changes that occur in this way have, in essence, been found necessary for lineage determination while at the same time interfering with DNA methylation genetically or chemically. Skewing of the results of the epigenetic changes may cause haematopoietic differentiation in the direction of myeloid lineage of leukaemia (Hagopian et al., 2010). It is, however, important to notice that so far the specific control of the lineage-specific signalling pathways that occurs by epigenetic regulation in almost all the subtypes of acute leukaemia has not been clearly established. From the current study, it is clear that there is an extensive differential DNA methylation of many components making up the BCR and TCR signalling pathways in both the B-precursor ALL and T-precursor ALL (Sato et al., 2014). It is possible to suggest from the findings that the lineage-specific signalling pathways in most if not all of the lymphoid leukemic cells may experience regulation at the epigenetic level due to the correlation between the epigenetic changes and the variations in gene expression.
Table 5, 6 and 7 show the existence of a set of recurrent epigenetic variations that are observed across all the subtypes of ALL irrespective of the genetics or epigenetics of the subtype. These observations support the hypothesis that during the process of malignant transformation, a number of epigenetic events are expected to occur (Mcgrath, 2015). Apparently, the DNA methylation characterising all the subtypes indicates the importance of an existence of genes which when silenced can result in the constitution of an event leading to the completion of the transformation process (Pogorzala et al., 2010). In fact, this finding from the current study supports several other previous findings that suggest that most of the recurrent genetic events that characterise both ALL and AML lack the capacity to solely transform a typical haematopoietic progenitor. Also, similar observations have been observed while studying adult AML whereby the set of genes that are targeted for transformation in such a leukaemia is distinct from the genes affected in paediatric ALL despite being similar to the genes affected in myelodysplastic syndromes (Mcgrath, 2015). As such, it is ideological to conclude that such common epigenetic lesions that require establishment during transformation processes are actually lineage-specific and may be used in determining the eventual phenotype of the malignant clones in leukaemia. As can be observed from Table 5 and 6 and linked to the findings of other researchers, about nine of the recurrent epigenetic hits are evident in both ALL and AML cases suggesting their role in leukemic transformation without considering the specific lineage of the leukaemia (Hagopian et al., 2010). Out of these epigenetics, DHRS12 has in most cases been found to be either deleted or under-expressed in most solid malignancies while MCART1 has been linked with the transport of mitochondria suggesting their role in cellular metabolism. Another recurrent epigenetic hit is SPNS2, a homolog of HSpin1 found useful in caspase-independent cell death. Perhaps in the future scientists may venture in using murine models to determine the precise timing and exact mechanisms through which the aforementioned lesions are able to contribute to the leukemogenic process (Ward et al., 2014). An interpretation of the current findings may also imply that the genes that are commonly known to be frequently at the verge of being affected by sequence and copy number variations in paediatric ALL are likely to be targeted by aberrant DNA methylation.

There are various newly developed genetic markers for adult ALL that have been found to exhibit considerable prognostic impact for the patients (Sato et al., 2014). One of these biomarkers is the lymphoid transcription factor gene IKZF1 alterations indicative of an association with high levels of leukemic relapse in many cases of B-ALL. Currently, induction therapy is capable of producing high complete remission rates in ALL although with unsatisfactory long-term survival (Pogorzala et al., 2010). The development of new and less-poisonous agents for chemotherapeutic purposes is highly desirable in an attempt to improve the health outcomes of patients suffering from ALL. Such less toxic agents includetyrosine kinase inhibitors in Ph-positive ALL patients, the use of rituximab in patients that are reportedly CD20-positive of the disease, utilising blinatumomab in cases of precursor B-ALL as well as using nelarabine when managing T-lineage ALL (Litsenburg et al., 2013).
The cytogenetics of the B-precursor ALL has over time faced quite a number of scientific considerations with approximately sufficient documentation. Various specific chromosomal abnormalities have been utilised in risk stratifications of the patients that are presented for treatment (Mcgrath, 2015). In fact, scientific research has been able to establish that the copy number abnormalities (CNA) of the various genes associated with the incidence of B-precursor ALL as well as those that are involved in both haematopoiesis and the control of the cell cycle are similar in B-precursor ALL. Some of the essentially notable gene deletions in the development of B-precursor ALL include PAX, IKZF1 and other genes located at the pseodoautosomal region of the sex chromosomes (Pogorzala et al., 2010). Such deletions elicit a fusion of the P2RY8-CRLF2 genes as well as an over-expression of CRLF2. Particular attention is normally directed at IKZF1 as well as CRLF2 due to the significant roles that these genes play as molecular markers for gene therapy. In fact, a situation whereby the IKZF1 is deleted is illustrative of poor disease prognosis in cases of B-precursor ALL. There is, however, a variable risk associated with deletions that target CRLF2 that is also reliant on other features. Such changes have, however, not resulted in any new form of improved therapeutic procedures for B-precursor ALL. In fact, most other studies relating to the same have involved cohorts and analyses that have been undertaken on independent basis from other genetic changes (Ward et al., 2014). To comprehend the actual relevance of such studies on B-precursor ALL, it is also essential to determine the accuracy in incidence, relationship to each other as well as the main cytogenetic subtypes of the disease. A recently conducted primary study on B-precursor ALL confirmed the importance of using multiplex-dependent probe amplification to exhibit a high throughput method of screening for copy number abnormalities occurring in B-precursor ALL genes with accuracy and reliability (Sato et al., 2014).
The results of a certain detailed retrospective study examining the significant gene deletions in patients with B-precursor ALL in the UK elicited comparable outcomes to those of the current study regarding the process of B-cell development, haematopoiesis and cell cycle (Litsenburg et al., 2013). From the findings, only 59% of the entire study population showed the said copy number abnormalities, otherwise, the remaining 41% showed no signs of abnormalities (Sato et al., 2014). Moreover, this study found that the number of simultaneous occurrences of copy number abnormalities in a particular patient was limited. From these findings, it is accurate to pre-conclude that the genomic profiles obtained from cytogenetic data of children with B-precursor ALL are not as complex as they appear to be (Hagopian et al., 2010). Available data from short nucleotide polymer arrays have exhibited infrequencies in the incidence of various recurrent sub-microscopic abnormalities. Regarding karyotypic evolution, it is impossible to determine the exact temporal order in which these series of events occur, neither is it possible to find out the exact drivers of leukemogenesis. Nevertheless, the researchers in this retrospective study were able to examine the relationship between the mentioned abnormalities with demographic and clinical signs, and cytogenetics (Mcgrath, 2015).
Previous studies have also shown heterogeneity in the size of deletions associated with IKZF1 in individual patients of B-precursor ALL. In most cases, the patients experience a deletion of the entire gene which occurs as a visible cytogenetic variation that involves 7p or is sometimes restricted to exons 4 to 7 (Hendrickson & Rimar, 2009). A small number of other deletions that occur in this condition have been found to be biallelic. However, there have been so far no connection between the pattern of exon that is lost and the specific cytogenetic subtype of the disease.
The PAX5 gene may also be characteristically deleted variably in the causation of B-precursor ALL with the deletion ranging from a mere loss of the telomeric exons. Deletions involving the whole PAX5 or part of the gene especially exon 1 can be said to predictably result in reduced expression of PAX5 (Mcgrath, 2015). However, the expression of partial deletions without a possible deletion of exon 1 may be predicted to lead to the expression of mutant alleles Van (Litsenburg et al., 2013). B-precursor ALL has also been previously associated with the expression of ETV6-PAX5 fusion protein.in most cases, the dicentric chromosomes related to ETV6-RUNX1 have been found to exhibit larger deletions of ETV6 as well as an entire loss of PAX5 suggesting that such translocations may not necessarily result in ETV6-PAX5 fusion (Mcgrath, 2015). Often associated with the visible abnormalities of 9p as well as a concurrent loss of PAX5 are the deletions affecting CDKN2A/B, a situation that is quite frequent in B-precursor ALL. Apparently, about eight out of ten of all cases that exhibit intragenic amplification of PAX5 also exhibit the deletion of CDKN2A/B (Pogorzala et al., 2010). When compared to some other forms of deletions characterising the disease condition, RBI deletions can be said to be the most homogeneous.
There are relatively many incidences of copy number abnormalities in ETV6-RUNX1 positive group of patients, a situation that illustrates the possible participation of copy number abnormalities in driving the process of leukemogenesis as opposed to the participation of point mutations in the same. In fact, previous studies have been able to confirm that ETV6-RUNX1 positive leukaemia is only capable of harbouring an unassertive number of point mutations (Anthony et al., 2014). Furthermore, research has established a great association between BTG1 deletions and ETV6-RUNX1 in the causation of the disease subtype. Due the recent association of BTG1 with glucocorticoid receptor auto-induction, it is important to follow up ETV6-RUNX1 positive leukaemia patients so as to find out whether the said deletions impact on their overall survival (Hendrickson & Rimar, 2009).
The level of deletions in high hyperdiploid patients was found in one of the previous studies to be relatively lower than expected. As such, this factor may not be sufficiently used as an artefact in the analysis of copy number abnormalities considering the context of ploidy change. This scenario is made possible by the fact that the genes tested and the reference probes included in the microarray test kit are positioned in chromosomes that are frequently increased in high hyperploidy (Mcgrath, 2015). ETV6-RUNX1 B-precursor ALL subtype in paediatrics were characterised by an increased rate of point mutations, a situations that leads the researcher to pre-conclude that this disease subtype is driven more by point mutations as opposed to gene deletions. Clearly, further research needs to embark on the assessment of the prognostic value of copy number abnormalities with the inclusion of cytogenetic data with an aim of obtaining a clear picture of their relation to the possible outcomes (Sato et al., 2014). The results of yet another study were indicative of a relationship between BCR-ABL1 fusion and IKZF1 deletions with PAX5 and CDKN2A/B deletions being relatively increased in BCR-ABL1 positive groups of individuals.
Ideally, some genes occur in more than one subtype of paediatric ALL indicating the possibility of targeting such a gene when devising a new drug agent against the tumour cells (Sato et al., 2014). For instance, Table 11 clearly shows that genes such as CD9, GNPDA1, INSR, PDLIM1, PSD3, STK32B, VPREB1, and LAT2 are found as biomarkers in the causation of both ETV6-RUNX1 and T-lineage. The same table also shows other sets of genes that are found being involved in the causation or progressive changes that take place during both T-lineage and B-precursor ALL.
ETV6-RUNX1 also denoted as t(12;21) as well as high hyperploidy or the 51 to 65 chromosomes are some of the well-known diagnostic and prognostic biomarkers used in either paediatric or adult ALL. Accurate detection of ETV6-RUNX1 can be achieved through the use of FISH or RT-PCR due to its cytogenetic cryptic nature and it occurs due to chromosomal translocation affecting t(12;21)(p13;q22) (Hendrickson & Rimar, 2009). High hyperploidy on the other hand can be accurately detected through the use of cytogenetics or via the application of locus specific and centromeric FISH probes because the pattern of chromosomal gain is not arbitrary. As a result, about eight chromosomes are isolated that account for about 75% or more gains. The two biomarkers are responsible for about 60% of all cases of paediatric and adolescent ALL but play an insignificant role in adult ALL (Whitlow et al., 2015). In fact, ETV6-RUNX1 is virtually non-existent in adults aged thirty years and above. Patients that are found to have the two biomarkers for ALL have a good prognosis and their survival rates go as high as 90 percent in paediatric ALL. In the past, several research studies have focused on other secondary abnormalities such as IKZF1 deletion, ETV6 deletions as well as RAS pathway mutations with an aim of establishing their prognostic relevance. Nonetheless, no reliable additional biomarkers had been identified to serve as prognostic determinants of the two subtypes of ALL. Other attempts to investigate the possibility of there being additional prognostic markers have been made within high hyperploidy with the assessment of specific trisomies, modal chromosome number as well as some structural abnormalities. Such attempts have, however, been proved futile. Some trisomies have in the recent past, however, been characterised as clinically relevant including +4, +10, +17 and +18 but all of them lack universal applicability. With the current excellent outcomes for patients with ETV6-RUNX1 and high hyperdiploidy while suffering from paediatric ALL, visualising further clinically applicable biomarkers incipient from this risk group is quite difficult.
The most common childhood ALL is the B-cell precursor ALL with the commonest chromosomal rearrangements being the t(12;21)(p13;q22) translocation that generates the ETV6-RUNX1 as the product of gene fusion (Hauri et al., 2013). Ideally, this is an essential gene fusion mechanism but it is insufficient to initialise the development of B-cell precursor ALL. In fact, research has shown the presence of ETV6-RUNX1 fusion in monozygotic twin studies whereby the fusion has been spotted in their foetal blood samples but later fail to develop B-cell precursor ALL (Hagopian et al., 2010). About 40% of all cases of B-ALL have shown the presence of PAX5 mutation, making it an important B-cell somatic identity component. In addition to this identity, it has been established that the commonest recurrent focal deletion regions in the combination of the ETV6-RUNX1 and the malignancy entails PAX5 (9p13.2; 25 %), a pack of deletion that has been associated with the early signs of leukemogenesis (Hendrickson & Rimar, 2009).
It is evident that there are several advances in the treatment attempts for ALL among the young people but the disease continues to be a leading cause of cancer-related deaths. In an attempt to identify a new therapy and detect and manage resistance to therapy among various patients of the disease, it is important to gain a sufficient understanding of both the genetic and epigenetic activities that eventually contribute to leukemogenesis. There are certain numeric and structural chromosomal variations that keep on recurring during the causation of paediatric ALL and are worth mentioning so as to understand the possible biomarkers involved in ALL leukemogenesis. One of the recurring alteration is B-precursor ALL with a high hyperploidy with more than fifty chromosomes and a simple hyperploidy with less than forty-four chromosomal entries (Modak et al., 2014). Other important alterations also exist including t(12;21)(p13;q22), a chromosomal arrangement that codes for ETV6-RUNX1 which is also denoted as TEL-AML1 and t(9;22)(q34;q11) which codes for BCR-ABL1. Moreover, another rearrangement is common in ALL leukemogenesis referred to as the cytokine-like factor 2 alteration denoted as (CRLF2r) while another rearrangement is considered to be the MLLr which occurs specifically at 11q3. The mature B-cell carcinoma also elicits a unique MYC rearrangement that is repeated throughput such lymphomas making it unique characterization of leukemic biomarkers in the disease subtype. The same subtype also produces rearrangements referred to as TLX1 or HOX11), TLX3 (also known as HOX11L2), LYL1, TAL1, as well as MLL rearrangement in T-lineage ALL (T-ALL).
All the above mentioned chromosomal alterations are essential in the determination of leukemic responses to available and newly developed therapeutic agents and procedures. The rearrangements also play a role in determining both the treatment outcomes as well as the specific gene expression profiles. Research has, however, shown that such rearrangements may be necessarily acquired quite a long time before the overt leukaemia is experienced in life. Furthermore, mouse models used in chromosomal studies related to ALL leukemogenesis have shown that these chromosomal alterations may not be solely sufficient to cause leukaemia; hence, they need to need influenced by other genetic events to elicit the disease symptoms. Nonetheless, the recurring chromosomal alterations are necessary for the causation of ALL subtypes as observed by a group of scientists working on mouse models (Modak et al., 2014). There are various sub microscopic genetic changes that occur in the causation of ALL according to the findings obtained through extensive genome wide microarray profiling that involves the consideration of DNA copy number alterations. Various essential pathways in the process of leukemogenesis and its progression are targeted by such alterations and sub microscopic changes including the processes of tumour development, regulation of the entire cell cycle, lymphoid suppression, tumour signalling as well as the responsiveness of the malignancy to therapy. The precise nature of the rearrangements of these processes is dependent on the particular subtype of ALL (Sato et al., 2014). In fact, certain alterations have been found affect the outcome of treatment including the deletion or mutation of IKZF1, a gene that codes for early lymphoid transcription factor known as IKAROS.
However, it is important to notice that somatic gene mutation may not be the only way via which the expression of genes is agitated in lymphoid carcinomas. The rearrangement of DNA methylation mal also play a key role in the disruption of transcriptional regulation since such a process is certainly perturbed during leukemogenesis. Cytosine methylation has been largely studied through genome wide analytic tools leading to the identification of novel subtypes of AML following which important insights into the biology of the disease have been made. The examination of cytosine methylation has been conducted through various previous studies touching on childhood ALL at the promoter regions of certain genes. Other studies have endeavoured to perfrom genome wide analysis with an aim of investigation the methylation in such genes among small patient cohorts in particular subtypes of ALL among paediatrics including MLLr, high hyperploidy as well as ETV6-RUNX1 rearrangements. Most of these studies have been able to link DNA methylation with leukemogenesis with a strong belief that the methylation process of certain genes involved in the disease causation is a hallmark in its progression. As such, the researchers suggested the significance of performing genome wide analysis to involving large patient cohorts so as to determine the role of DNA methylation in leukemic cancer causation.
The number of studies that have specifically included large patient cohorts to interrogate the matter is limited with most of the cohorts utilising genetic methods as opposed to genome wide analysis approaches to investigate the situation. In fact, there is presently no study that has been so far performed with the aim of engaging genome wide analysis to dissect the role of methylation across the spectrum of both B-cell precursor ALL and T-lineage ALL. Consequently, it has not been possible to successfully correlate the status of methylation with the alterations that occur during gene expression as well as the concomitant structural DNA changes. Table 11 in the results section showed a number of genes that were recurrent in the three subtypes of ALL that were investigated during this study. Such recurring episodes in the genetic make-up of the patients are a proof of the cytogenetic alterations that can act as an essential determinant of a leukemic transcriptome (Litsenburg et al., 2013).
The genes in Table 5 including CD3D, TCL1A, HLA-DRA, CD3G, and HLA-DRA are all common in both B-precursor ALL and T-lineage subtype of paediatric ALL. As such, these genes can be considered to be some of the hallmarks in the diagnostics and target therapy for both of these subtypes of paediatric ALL. Incidentally, the recurrence of a particular karyotype in cytogenetics of the B-cell precursors and T-lineage subtypes can be used as regular targets when diagnosing a particular subtype of the ALL disease as well as a point of consideration when designing gene therapy agents (Mcgrath, 2015). The genes in this case can be considered to be the biomarkers of the two subtypes as per the confines of the findings in this research paper. The problem with this generalisation, however, is that it is difficult to isolate the role of a particular gene in a specific subtype of the disease in order to independently diagnose and treat the malignancy. Nevertheless, both of the subtypes can be diagnosed and targeted through gene therapy to eliminate the disease (Sato et al., 2014).
Gene therapy can be employed to eliminate the tumorous cells as identified through the genetic biomarkers identified in Table 5. Genes are carried in chromosomes and they usually act as the basic functional units of heredity in living cells. In fact, the genes represent the specific sequences that carry the instructions necessary for protein synthesis. Much consideration and attention has been so far granted to genes per se but the most important aspect of cytogenetics is the product of the gene which is actually the protein that is responsible for various cellular structures (Bug & Ottmann, 2010). An alteration of the gene itself affects the eventual protein resulting in an undesired protein that may affect the body functioning in a negative manner. The effects of carcinoma are, therefore, linked to the synthesis of wrong proteins or proteins that are dysfunctional, denying the entire system an opportunity to function in the right manner. Since the genes in Table 5 are bound to be altered, the various proteins that they would have synthesised are either not produced or are produced in a dysfunctional nature, affecting the normal functioning of the eventual lymphoid cells in the bone marrow ad elsewhere within the system.
Gene therapy can be applied to the genes in order to correct the defective genes that are responsible for disease development among paediatric ALL patients. Scientists have devised several approaches towards the correction of faulty genes after the identification of such through genetic markers or biomarkers as referred to in this study. The most common of all approaches towards gene correction is through insertion of a functional gene into a non-specific location within the entire genome that replaces the function of the altered gene (Tajuddin et al., 2013). Moreover, homologous recombination may also be used to swap the abnormal gene with a normally acing gene in a process that has been successfully performed in mouse models and in some human trials. At the same time, scientists have been able to successfully repair some altered genes in other diseases affecting the gene function other than lymphoid carcinoma through reverse mutation (Hendrickson & Rimar, 2009). This approach is intended to restore the normal function of the altered gene. In other cases, genetic scientists have suggested an alteration of the gene regulation process involved in a particular gene defect such that the rate at which the gene is turned on or off in a defective gene is regulated.
In a similar manner, Table 6 shows the genes that appeared to be uniquely recurring in the cases of both B-cell precursor ALL and high hyperploid B-precursor ALL among the paediatric patient samples selected for this study. One of the methods mentioned above can be used to restore the function of the defective genes identified through genetic biomarkers AF090939, AF293339 and ATP5L. Such an intervention would add into the possibility of obtaining a therapeutic agent or procedure through which successful elimination of either subtypes of paediatric ALL can be achieved.
In most cases whereby gene therapy is practiced, an apparently normal gene is inserted into the genome in order to replace the apparently abnormal disease-causing gene. A vector is normally used as a carrier molecule to transport the therapeutic gene into the genome of the patient suffering from the disease in question, which in this case is a particular subtype of ALL (Litsenburg et al., 2013). For instance, a viral vector can be used to deliver a therapeutic gene to replace one or all the genes in Table 5, 6 or 7. Viral vectors are some of the most frequently used vectors in gene therapy whose use is made efficient through the alteration of their genome so as to tailor them to carry therapeutic human genome portions to replace the abnormal gene responsible for the lymphoma. Naturally, viruses have evolved to devise their own mechanisms of accessing the human genome and delivering their genomes into those of humans. Eventually, the viruses are known to replace portions of the human genome with their own DNA portions in a pathogenic manner (Bug & Ottmann, 2010). Scientists have, however, taken advantage of this scenario and use it for the benefit of the patient by removing the viral DNA and replacing it with correctional genes aimed at solving a genetic problem in cancer patients. Since the defective genes in this project were identified and highlighted, it is easy to expose them to sequencing, making it easy to come up with corrective gene segments (Sato et al., 2014). Such portions of the corrective DNA intended to have healing effects on the patients can then be packed into the viral vector and passed on to the patient through various devised mechanisms studied in recombinant DNA technology.
To achieve gene therapy in this way, scientists have taken advantage of the ability of viruses to invest and manipulate cellular genomes through their specific surface antigens. Since the defective genes in this project were clearly isolated as biomarkers, it would be easy to target them using the viral vectors containing therapeutic genes. The researcher would simply have to infect the target cells with the viruses carrying the therapeutic gene after which the genes will be successfully delivered to the patient’s genome (Mcgrath, 2015). The target cells in this case will be the B cells and T cells with an attempt of eliminating B-cell precursor ALL and T-lineage ALL. Once the viral vectors access the target cells, they integrate with their DNA after the attachment process of viral interaction with human cells and unload their genetic contents into the cellular genome. Normal replication of the genome occurs at the target cells leading to the production of a functional protein from the therapeutic gene inserted via the virus ultimately restoring the function of the defective gene. Many types of viruses have been used in this process of gene therapy including retroviruses, adenoviruses, adeno-associated viruses, and Herpes Simplex Viruses.
Another way of handling the ALL problem identified through the biomarkers in the Table 5 to 11 is through genetic and epigenetic control of cells with genome engineering technologies. This technology is one of the ways through which an extensive variety of applications from basic biology to biotechnology and medicine is made. The recapitulation of causal genetic mutations or epigenetic variations that may be caused by an alteration of the biological function or disease phenotypes has been made rapid and more efficient through the use of animal or cellular models (Ward et al., 2014). All these are done in an attempt to correct the malignancies occurring in the B cells and T lineages in order to restore normality to children suffering from one of the ALL subtypes. Genome editing has been performed in most of these attempts whereby a manipulation technique is used to insert, replace or remove a genome portion via the use of artificially engineered nucleases. During the same process of gene editing, both homologous and non-homologous endogenous mechanisms of human cell repair are harnessed to restore normality in patients with either of the ALL subtypes (Litsenburg et al., 2013). In a better version, genome editing is a scientific strategy of producing stable, permanent and heritable variations to the genetic code of the affected individual with an aim of accomplishing various potential objectives. While making use of the nuclease tools, the entire process of gene editing starts with the researcher stimulating the formation of a double stranded break at the point of the intended genetic insertion (Hendrickson & Rimar, 2009). Since such double stranded breaks are detrimental to the cells when they are left unattended to, the cells use their own mechanisms to repair such a break.
The first mechanism through which such a repair can be accomplished is through the process of homologous recombination that involves correcting the break by filling the space with a homologous pair of chromosomes similar to the one that was destroyed which acts as the template during the repair process (Hagopian et al., 2010). The advantage of the homologous recombination process is that it is free of errors. In normal cases, the template during homologous recombination is considered to be the sister chromatid during the G2 stage of mitosis although it may also be sourced from a DNA fragment that is inserted into the break exogenously. As such, the exogenously inserted gene is purposed to create a knock-in while inserting the desired gene segment to the broken chromosome (Pogorzala et al., 2010). Non-homologous end joining may also occur in the event that the homologous template is missing. This process entails simply reconnecting the broken ends without considering the importance of the genetic material that may have been lost during the breaking of the double stranded break. For this reason, the process of non-homologous end joining to repair damaged DNA is an undesirable process that is prone to errors (Hendrickson & Rimar, 2009). The process also leads to the possibility of small insertions or deletions commonly referred to as indels at the location where the break had occurred. Indels have been found through research to be major causes of frame shifts and gene knockouts making the entire process undesirable.
In most incidences, ALL is considered to be a malignancy of the immature lymphoid progenitors and the commonest progenitors affected by this disease are those of the B cell lineage. Most cases of ALL amongst the paediatrics are sub classified in accordance with gross or sub microscopic genetic variations. In about 75 percent of all cases of all as per some research scientists, aneuploidy or recurring chromosomal alterations characterise the disease causation and progression (Sato et al., 2014). Hyperploidy has been established to be one of the most common frequently occurring variations linked to favourable outcomes which happen in children suffering from ALL with the condition being associated with a gain of at least five chromosomes. Unfortunately, there is a poor understanding regarding the biological basis of the attainment of entire chromosomal additions. Nonetheless, hyperploidy has been associated with dismal prognosis in spite of being characterised by less than 44 chromosomal gains. Apparently, there are plenty of chromosomal rearrangements that occur during B-ALL disease causation and are as such considered to be critical events necessary for ALL leukemogenesis (Ward et al., 2014). Such rearrangements have been found to play a critical role in perturbing the genes that code for regulators of the process of haematopoiesis interfering with the entire control of the process. Furthermore, the genetic rearrangements interfere with the regulation of tumour suppressor expression, expression of oncogenes, and tyrosine kinases although the rearrangements may also require other genetic hits to exhibit the entire leukemic phenotype (Tajuddin et al., 2013).
The last nine year have experienced a gross advancement on the insights of the genetic basis of ALL especially in view of the fact that genome wide profiling while utilising microarrays as well as candidate gene and second generation sequencing techniques have faced great developments (Bug & Ottmann, 2010). Such technological advancements are responsible for the identification and categorisation of ALL into various subtypes some of which were identified via biomarkers as observed in this project. Some changes that have been observed through such technological advancements have included the loss of function gene mutations affecting the genes that regulate the development of the lymphoid resulting in limited maturation characteristics.
Other mutations have been found to severely interfere with the process of tumour suppression as well as the regulation of cell cycle proteins (Jeniffer et al., 2013). Other genetic variations affect the cytokine receptors and the process of kinase signalling resulting in the causation of B-cell precursor ALL. In fact, most of the ALL subtypes are characterised by similar alterations that cause concomitant lesions like those observed in AML resulting in the disruption of haematopoietic development and the process of tumour suppression (Litsenburg et al., 2013). Moreover, problems in key signalling processes and proliferation of cells are important hallmarks in the leukemogenesis of most ALL subtypes (Hendrickson & Rimar, 2009). The most important consideration for this project, however, is that some alterations are found recurring in some of the ALL subtypes, a factor that aids in diagnosis and designing of therapeutic agents and procedures to fight the disease.
t(12;21)(p13;q22) can be said to be the commonest rearrangement in the leukemogenetic process of B-ALL, a rearrangement that codes for ETV6-RUNX1. When a cytogenetic analysis is performed, this alteration in the gene formation is apparently cryptic although it can be freely distinguished using fluorescent in situ hybridisation as well as through other molecular procedures (Gojo et al., 2013). Ideally, ETV6 has been initially classified as part of the ETS family of transcription factors most of which are beleaguered by genetic variations as well as mutations in leukaemia and in other cancers. A unique rearrangement of RUNX1 occurs in AML by binding with CBFB leading to the formation of a core binding transcription complex that is associated with AML leukemogenesis. Such a binding complex is the one that harbours the sequence mutations that characterise both lymphoid and myeloid malignancies (Tajuddin et al., 2013). The normal definitive hamatopoiesis requires both RUNX1 and ETV6 whereby the combination referred to as ETV6-RUNX1 acts as a regular interference agent for gens regulated by RUNX1. In this way, the RUNX1 is changed into a transcriptional repressor (Pogorzala et al., 2010). It is also important to understand that ETV6-RUNX1 plays a role in the causation of an overexpression of erythropoietin receptor denoted as EPOR as well as the instigation of the downstream JAK-STAT signalling process (Modak et al., 2014). Despite the fact that ETV6-RUNX1 does not initiate leukemogenesis on its own, the agent is known to promote self-renewal that occurs in the B cell progenitors. Moreover, the fact that ETV6-RUNX1 has been detected in children during birth suggests that there must be other triggers that cause the transcription complex to induce leukaemia of any subtype (Ward et al., 2014). This aspect has been backed by evidence from research showing the existence of other sub microscopic genetic variations that combine with ETV6-RUNX1 to induce leukaemia. Such genetic alterations have been enlisted as deletions of some B cell transcription factors such as PAX5 and EBF1 as well as the deletion of the second copy of ETV6. The fact that monozygotic twins concordant for ETV6-RUNX1 show unique sub microscopic DNA copy number rearrangements confirms the hypothesis that ETV6-RUNX1 is an initial occurrence in the process of ALL leukemogenesis (Tajuddin et al., 2013). The tiny genetic rearrangements responsible for leukemogenesis are more common in ETV6-RUNX1 ALL as opposed to all other subtypes of acute lymphoid leukaemia in paediatrics. Such rearrangements include deletions that target presumed lymphoid signalling molecules denoted as BTLA, TOX, transcriptional co-activators such as TBL1XR1, glucocorticoid receptor gene called NR3C1 as well as apoptotic regulatory gene known as BTG1 (Ward et al., 2014). It is true to say that such genetic alterations act as hallmarks in the leukemogenetic process, however, the genetic breakpoints affecting most of these alterations are key to aberrant activities of the recombinase activation genes. The researcher recommends further work to be performed with an aim of more critically examining the above mentioned alterations in leukemogenesis (Hagopian et al., 2010).
Studies performed between 2007 and 2008 gave a clear description of DNA copy alterations existing in ALL, and the studies were counter-confirmed using array-based comparative genomic hybridisation while others used single nucleotide polymorphism microarrays (Ward et al., 2014). Most of these studies were designed to carry out cohorts of children patients of ALL while others described the genomics of adolescents and adults with ALL in great detail; some of which are still ongoing and incomplete (Hendrickson & Rimar, 2009). Genomes characterising ALL have been found to harbour lesser structural alterations than many other solid malignancies although more than fifty recurring deletions or amplifications have been singled out in which case most of them involve deletions affecting single of a few genes. Most of the genes that have been identified under these circumstances are genes coding for proteins that play key roles in lymphoid leukemeogenesis such as PAX5, IZKF1, EBF1, and LMO2 (Liu et al., 2011). Other genes, as earlier suggested have been isolated while coding for proteins essential in the regulation and suppression of tumours such as CDKN2A/CDKN2B, PTEN, and RB1 (Pogorzala et al., 2010). Genes whose rearrangement or alteration may interfere with lymphoid signalling may include BTLA, CD200, TOX, and the glucocorticoid receptor NR3C1 while genes affecting both transcriptional regulation and co-activation have been found to include TBL1XR1, ETV6, and ERG (Tajuddin et al., 2013). A number of genes have been found to occur in more than one subtype of ALL as well as in copy number alteration translocation and sequence mutations. Such genes include PAX5, WT1 and PTEN, a scenario that clearly shows that incapability of microarray profiling to detect all the genetic alterations persistent in the leukemogenesis of ALL (Jeniffer et al., 2013). This finding from other research studies indicates a limitation facing the current study regarding its reliance on microarray gene profiling.
The rate at which genetic lesions occur as well as the nature of the genetic lesions is a factor that aids in determining the specific subtype of ALL that an individual may be suffering from (Mcgrath, 2015). For instance, all MLL-rearranged leukaemias are well known for harbouring limited additional structural as well as sequence alterations. On the contrary, ETV6-RUNX1 and BCR-ABL1 ALL have been found to characteristically exhibit more variations. Emerging findings from most experimental set-ups have shown the probability off the participation of most of these alterations in a cooperative manner (Bug & Ottmann, 2010). In one of the experimental set-ups, it was established that the deletion of PAX5 and IKZF1 fastens the rate at which the instigation of leukaemia in retroviral BM transplantation occurs. Furthermore, the same genetic alterations accelerate the process of leukemogenesis in transgenic models of BCR-ABL1 ALL as well as chemical and retroviral prototypes of leukaemia (Litsenburg et al., 2013).
Most of the alterations discussed in this paper are enriched in many subtypes of specific cytogenetic ALL. An exception, however, occurs in the specific rearrangement that affects the ETS family transcription factor ERG which is also an ETS related gene (Hagopian et al., 2010). This gene occurs exclusively in patients that do not have known chromosomal alterations the result of which is its role as an assurance of a unique subtype of B ALL with a unique gene expression profile as well as a positive outcome (Mcgrath, 2015). Deletions affecting ERG entail internal subsets of exons the result of which is a loss in central inhibitory and pointed purviews as well as the expression of an anomalous C terminal ERG segment that remains with the ETS and transactivation purviews plus its role as a competitive inhibitor of wild-type ERG (Litsenburg et al., 2013).
With an exception of the IKAROS genetic alteration in B ALL, only few IKZF1 alterations associated with B ALL are reproducibly lowly related to the outcome (Jeniffer et al., 2013). Deletions, alterations or rearrangements that affect PAX5, IKZF1, and EBF1 as important transcriptional factors in the causation of B-ALL have been found in about two-thirds of all patients diagnosed with B-ALL (Pogorzala et al., 2010). Despite the fact that most PAX5 alterations are not linked directly to the outcome, about one-third of all patient cases diagnosed with B-ALL are also found to test positive for PAX5 (Modak et al., 2014). On the contrary, deletions affecting IKZF1 and related sequence mutations are rare and can only be found present in about 15 percent of patients with childhood ALL (Jeniffer et al., 2013). As such, IKZF1 acts as a hallmark in two types of high-risk ALL. Notably and quite important for this project, IKZF1 has been found to possibly encode IKAROS which is the founding member of a group of zinc finger transcription factors necessary for the leukemogenesis of all lymphoid lineages (Ward et al., 2014). The deletions touching on IKZF1 and seen in ALL entail focal and broad deletions that produce a loss of function and internal alterations of the coding exons ranging from four to seven. As such, the N-terminal DNA-binding zinc fingers the result of which is the expression of IK6 which is an overriding negative isoform. Once more, it is essential to notice that the alterations affecting IKZF1 are characteristic in more than 70 percent of all BCR-ABL1 lymphoid leukaemia (Jeniffer et al., 2013). These also include the causation of de novo ALL as well as the progression of chronic myeloid leukaemia to lymphoid blast leukaemia. Such occurrences are linked to undesirable outcomes in patients positive for BCR-ABL1 ALL. In fact, scientists have been able to come up with an association that links the alterations occurring in IKZF1 with negative outcomes of BCR-ABL1− ALL (Bug & Ottmann, 2010).
Next-generation sequencing has had a transformational impact on the comprehension of the pathogenesis of lymphoid malignancies, in general. Various studies on this topic have involved the use of the sequences of whole genomes, exomes, as well as transcriptome (Pogorzala et al., 2010). These regions have been screened for recurrent genetic alterations and mutations, whose effect has been related to major cellular pathways in individuals with T-ALL. The malignancies result into mutations in essential cellular pathways such as transcriptional regulation of differentiation, and antigen receptor signalling (Ward et al., 2014). The tyrosine kinase induced signalling as well epigenetics are also modified by the resulting mutations. These steps are key contributors to the pathogenesis of T-ALL since the altered processes lead to production of undesired outputs. As a result, the patient experiences the pathological effect of the altered processes, and this effect could lead to expression of key signs shown by the patient. The mutations not only lead to alteration of the few stated processes, but also lead to special genes that of significant benefit in the prognosis of T-ALL patients (Hullmann et al., 2010). The genes that are mutated in this condition include NOTCH1, TCF3, MYD88, and BRAFF. Identification of the pathogenesis of T-ALL in terms of genetic alterations is an essential step towards management of the condition in a number of ways. For instance, better diagnostic ways can be identified, such that a combination of tests are done for the improvement of accuracy. Stratification of infected persons can also be made easier, which is a key step in identification of risky regions. Most importantly, therapeutic intervention is improved since the knowledge of affected genes will enable physicians to prescribe the best treatment that will correct the condition (Habibi et al., 2012).
Lymphoid malignancies lead to severe genetic alterations in the somatic cells, which are considered hallmarks for the tumours on most occasions. Chromosomal alterations such as aneuploidy and general rearrangements of the chromosomes may either occur separately or concurrently. Structurally, genetic deletions and additions on a DNA strand may occur, which consequently lead to mutations in the generated sequences. These alterations have been affirmed through various cytogenetic studies as well genome profiling arrays. As a result of mutations, structural alterations on chromosomes are associated with incomplete gene sequencing. T-ALL is mostly accompanied by sequence mutations with minimal cases of deletion of the PHF6 gene, which mostly occurs in young adults. The deletion of PHF6 leads to failure of the gene to be expressed hence T-ALL patients have TLX1/3 and TAL1 rearranged. Therefore, the pathogenesis of T-ALL due to deletion of PHF6 is directly linked to the tumour genesis since the gene is considered a tumour suppressor. It has been hypothesized that the loss of function of the gene due to the associated alterations could be due to the fact that it has a suppressive effect on the development of the tumours. The loss of its functions increases the pathogenicity of the condition since the tumours continue developing in an uncontrolled mode (Hullmann et al., 2010).
T-ALL is also associated with hypodiploidy with less than 44 chromosomes, and it is a condition that reduces the prognosis of the patients. Further research has been ventured into this section with two subtypes of hypodiploidy being described on the basis of their severity. Hypodiploidy is an aneuploidy which has been split into near-haploid and low-haploid cases, having 24-31 and32-39 chromosomes respectively (Habibi et al., 2012). Research works have outlined the effect of hypodiploidy on transcription which has a pathologic effect that allows progression of tumour development during transcription. The effects on submicroscopic genetic alterations and transcriptome signatures are distinct depending on the type of hypodiploidy that occurs in an individual. For instance, near haploid hypodiploidy mostly leads to activation of the Ras signalling in NF1 and IKZF3 which is a gene of the IKAROS family. On the contrary, low haploid cases result into a general negative impact on the tumor suppressing gene TP53. This step is accomplished by the ability of the low hypodiploidism to lead to mutation of the gene such that its expression leads to formation of a gene that is incapable of suppressing tumour development. Analysis of mutations in such a germ line have enabled scientists to come up with therapeutic interventions through carrying out sensitivity tests. Since there is activation of both Ras and P13K signalling, a parallel sensitivity array demonstrated more sensitivity of the tumours to P13K inhibitors as compared to Ras signalling inhibitors such as MEK inhibitors. Therefore, this demonstrates an effective way of developing novel therapeutic intervention through the study of pathogenicity of T-ALL in terms of genetic alterations. Additionally, since low hypodiploidy cases are associated with mutations on the p53 gene, there is a need for children to be screened for the mutational status of the gene (Hagopian et al., 2010).
The major problem associated with T-ALL is the inability to sufficiently treat it, a situation that is activated by various predisposing factors, including relapse (Gojo et al., 2013). Since T-ALL involves chromosomes that have acquired abnormalities, it has been established that tumour heterogeneity brought about by genetic changes is a major basis of treatment and relapse. These alterations in an individual’s genomic composition continue from diagnosis up to relapse, according to polymorphism microarray profiling studies. During the diagnosis period, the patient’s blood may harbour relapse acquired lesions, although at low proportions (Whitlow et al., 2015). In diagnosis-relapse samples are most likely to show mutations on co-activator of transcription as well as acetyltransferase binding protein. These will be the relapse acquired lesion which targets various genes such as IKZF1, which is associated with high risk tumours (Hullmann et al., 2010). Mutations on the binding protein CREB are pathogenic to both histones and B-cell lymphoma through their bone marrow infiltrations and acetylation respectively. Therefore, relapse acquire mutations are also pathogenic in terms of having detrimental effects on other parts of the genome such as the histones (Gojo et al., 2013).
T-ALL is also characterized by abnormal activation of NOTCH1 mutations, which gives a clue of the impact of the mutations in the pathogenesis of the disease through NOTCH signaling (Bug & Ottmann, 2010). As a result, therapeutic interventions have been developed to target the pathway, and they include gamma secretase inhibitors which are small molecules that efficiently inhibit oncogenes generated through the NOTCH pathway (Habibi et al., 2012). This signaling pathway is highly evolutionary and it involves a cascade of events which lead to the transduction of signals on the periphery of cells into the expression of genes in the nucleus. Therefore, an alteration in these pathway can lead to formation of abnormal T-cells, a situation that may activate progression into T-ALL. The pathway involves the use of surface receptors which are four related proteins (Hagopian et al., 2010). The proteins are both heterodimeric and are structurally transmembrane proteins made up of two subunits. Normal T cells from the NOTCH signalling pathway come as a result of NOTCH1 which is involved in the commitment of hematopoietic progenitors onto the T-cell lineage hence supporting cell growth and proliferation (Habibi et al., 2012). On the contrary, T-ALL develops when there is chromosomal translocation which is induced by the NOTCH1 gene, a step that is activated by mutations. NOTCH1 mutations have gross effects in cellular pathways such as transcription, regulation of cellular development, and metabolism. In terms of metabolism, NOTCH1 signalling has a direct impact on the expression of genes as proteins that regulate various anabolic reactions. Impact on cell growth is evident through the direct transcriptional up regulation of MYC (Ward et al., 2014).
Conclusion and Recommendation
Acute lymphoblastic leukaemia is one of the most prevalent cancers among children, and is a leading cause of death among the same population. In the case of acute leukaemia, cancerous cells grow rapidly and can spread through the body in a span of weeks or months. Blast cells or immature cells lack the capability to perform normal functions, and remain immature. In time, the number of these cells circulating in the body rises. In contrast, cancerous cell growth in chronic leukaemia tends to be slower. Blast cells can be seen, but fewer circulate and a lot of cells are mature, with some functional capability. Although chronic leukaemia cells stay circulating in the blood for longer, their numbers rise gradually. ALL can be sub classified into different categories depending on the various cell types affected by the malignancy. Leukaemia can also be classified according to the type of cells affected. Lymphoid leukaemia refers to abnormal lymphocytes, a type of white blood cell. When the abnormal cells are monocytes or granulocytes in the bone marrow, the cancer is called myeloid leukaemia.
Thus, depending on both disease chain and type of cell affected, leukaemia can be divided into four types including Acute Lymphoid Leukaemia (ALL), Acute Myeloid Leukaemia (AML), Chronic Lymphoid Leukaemia (CLL), and Chronic Myeloid Leukaemia (CML). For the purposes of this project, ALL was differentiated into three subtypes including B-cell precursor ALL, T-lineage ALL and ETV6-RUNX1 ALL, all of which are subcategorised based on the genetic alterations that occur with the affected cells. This project found that there are a number of genes unique in particular subtypes of ALL identified through biomarkers assessing the various genes involved. CD3D, TCL1A, HLA-DRA, CD3G are the unique markers associated with the causation of B-cell precursor ALL. The findings also demonstrate genes of comparison between B-precursor ALL with miscellaneous karyotype and T lineage subtype in paediatric acute lymphoblastic leukaemia. Other genes symbolised by AF090939, AF293339, and ATP5L were among the top 10 genes of comparison between B-precursor ALL with miscellaneous karyotype subtype and High hyperdiploid B-precursor ALL subtype in paediatrics. The third set of findings entailed a group of genes of comparison between B-precursor ALL with miscellaneous karyotype and ETV6-RUNX1 B-precursor ALL subtype in paediatric whose symbols included DSC3, ARHGEF4, CLIC5, TUSC3, and SDC2. The research was also able to highlight the influenceable genes of molecular interaction between B-precursor ALL with miscellaneous karyotype and ETV6-RUNX1 B-precursor ALL subtype among children patients. Moreover, there were influenceable genes of molecular interaction between B-precursor ALL with miscellaneous karyotype subtype and High hyperdiploid B-precursor ALL subtype in paediatrics. The samples examined also proved the existence of influenceable genes of molecular interaction between B-precursor ALL with miscellaneous karyotype and T lineage subtype in paediatric acute lymphoblastic leukaemia.
Having considered all the possible biomarkers that characterise the occurrence either of the three possible subtypes of ALL, the researcher recommends further research to compare the various genetic alterations with those that occur in other subtypes of ALL. Any similarities in the occurrence of such alterations identified via the biomarkers would create a platform for more accurate diagnosis for specific subtypes of ALL for a more serious treatment plan.

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