Survival of Patients with Acute Lymphoblastic Leukemia

Age is the factor that has been more associated with survival. Younger patients (especial‐ ly those younger than age 50) have a better survival than older patients. Not only age but also gender and race also are related with survival of ALL patients. The girls have showed a better survival than boys, this partly due to boys’ risks for testicular cancer. African-American and Hispanic Individuals have lower survival rates than Caucasian and Asian individual, but this may be due to poorer access to treatment. A very impor‐ tant factor in the clinic that somehow predicts good to bad prognosis of the patient and of course has also been linked to survival is the Initial white blood cell (WBC) count; people diagnosed with a WBC count below 50,000/μL tend to be better than people with higher WBC counts. Even, ALL subtype plays a role very important. For example, pa‐ tients with T-cell ALL tend to have a better prognosis and survival than those with ma‐ ture B-cell ALL (Burkitt leukemia). Nowadays, the identification of chromosome translocations help to prognosis of ALL patients; people who have Philadelphia chromo‐ some-positive ALL tend to have a poorer prognosis, although is important to note that new treatments are helping many of these patients achieve remission.


Introduction
Over the past 50 years, the treatment of patients with acute lymphoblastic leukemia (ALL) has significantly improved. This success is measured by the improved survival of ALL patients from less than 10% in the 1960s to more than 80% in more recent reports. However, many factors influence to a good outcome to treatment and subsequently in the improved survival of patients with ALL.
Age is the factor that has been more associated with survival. Younger patients (especially those younger than age 50) have a better survival than older patients. Not only age but also gender and race also are related with survival of ALL patients. The girls have showed a better survival than boys, this partly due to boys' risks for testicular cancer. African-American and Hispanic Individuals have lower survival rates than Caucasian and Asian individual, but this may be due to poorer access to treatment. A very important factor in the clinic that somehow predicts good to bad prognosis of the patient and of course has also been linked to survival is the Initial white blood cell (WBC) count; people diagnosed with a WBC count below 50,000/μL tend to be better than people with higher WBC counts. Even, ALL subtype plays a role very important. For example, patients with T-cell ALL tend to have a better prognosis and survival than those with mature B-cell ALL (Burkitt leukemia). Nowadays, the identification of chromosome translocations help to prognosis of ALL patients; people who have Philadelphia chromosome-positive ALL tend to have a poorer prognosis, although is important to note that new treatments are helping many of these patients achieve remission.
In Italian children between 1 and 18 years of age with newly diagnosed of ALL, enrolled in the AIEOP-BFM ALL 2000 study, had a 7-year EFS and survival of 80.4% and 91.8%, respectively. However when the children were stratified by minimal residual disease (MRD) their overall 7-year estimates for EFS and survival were 80.7% and 92.8%, respectively [11]. In United Kingdom, children aged 1-18 years survival estimates at 5 years were 87% [12]. In Pakistani population aged > 15 years their median survival was 12.7 months and diseasefree survival was 6.2 months [13].
Currently, due to intensive chemotherapy regimens, the outcome of adult ALL has improved markedly. The complete response rates now are more than 80% [14] and the longterm survival rate is 30%-45% [15]. Based on a study by Stephen Hunger and colleagues, 5year OS rates now above 90% for the first time (83.7% in the period 1990-1994; 90.4% in the most recent period [2000][2001][2002][2003][2004][2005]. This study is based on information on 21,626 ALL patients between 0 and 22 years who were enrolled onto the Children's Oncolocy Group (COG) ALL clinical trials from 1990 to 2005. It is clear that survival improved in all subgroups of ALL (1-9 years), except for infants under the age of 1 year. Besides, 5-year OS also remains significantly lower (81.6%) for children over the age of 10 years [16]. Childhood ALL reflects one of the diagnosis for which the most impressive improvements have been realized [1].
The better results seen among the childhood population as compared to adults with ALL have been attributed to a number of prognostic factors. It is important to consider a number of important differences between younger and older patients with ALL exist. First, the biology of both, underlying disease and the patients' metabolic changes with age are very different between two cohorts [17]. The second major difference is the difference in therapy related toxicity [18]. The third potential cause for a superior outcome in the younger population is relates to the protocols administered. It is important to consider that the treatment protocols from each institution, the dose adjustments and many others factors can increase long-term survival, but the factor toxicity could have a negative effect on long-term.

Gender
Survival disparity by the sex of the patient with leukemia has been observed since the nineteen sixties; however, what remains to be fully grasped are the factors responsible for this persisting survival difference between boys and girls. Girls continue to demonstrate survival advantage relative to boys. Studies over the past years have repeatedly shown that after diagnosis of pediatric leukemia, boys present with poorer survival that the girls (Table 2).
In 2012 using a large sample and long-term data could help explain the ongoing variance in leukemia survival comparing boys to girls and found that boys are more likely to die from leukemia ( Table 1). The explanation to the observed disparity in survival by sex, since most of the patients who had T-cell type were boys, and survival was poorer among boys in this study [19]. A biological explanation for sex disparity in leukemia survival, it is plausible to suspect XY chromosomal instability as a possible contribution to abnormal cellular proliferation, thus resulting in a biologically aggressive leukemia among male patients. Also, it might be possible that testosterone or estrogen may play a small role in pediatric leukemia, this partly due to boys' risks for develop testicular cancer [19].

Initial White Blood Cell (WBC) count
Along with age, the initial peripheral blood leukocyte count is another of the firsts identified prognostic factors in every study of ALL. The WBC at diagnosis is a crucial variable for describing the nature of the patient's leukemia and especially the tumor burden. The other measures of the tumor burden are the size of a mediastinal mass, hepatosplenomegaly, and enlargement of lymph nodes. Children with WBC of more than 50x10 9 /L are commonly considered to be at high risk of relapse and receive intensive treatment [1,2]. In retrospective analysis was found that patients with hyperleukocytosis (WBC count >50 10 9 /L) were significantly related to lower survival. Similar findings are the rule in reports from various study groups (Table 1).

Race/ethnicity
Variability in survival outcome across racial and ethnic groups (hereafter referred to as race/ ethnicity) also has been identified in some, but not all, clinical research. Survival rates in Black, Hispanic and Native American children with ALL have been somewhat lower than the rates in White children with ALL (Table 2). This difference may be therapy-dependent [32]. Asian children with ALL fare slightly better than white children [33]. The reason for better outcome in White and Asian children compared with Black, Native American and Hispanic children is at least partially explained by the different spectrum of ALL subtypes. For example, blacks have a higher incidence of T-cell ALL and lower rates of favorable genetic subtypes of ALL. However, these differences do not completely explain the observed racial differences in outcome [33].

Survival by ALL Immunophenotype
The World Health Organization (WHO) classifies ALL as either B lymphoblastic leukemia or T lymphoblastic leukemia [49]. Historically, T-cell ALL patients have had a worse prognosis than other ALL, the relapse rate of T-cell ALL is greater than B-cell ALL cases, and Tcell ALL cases have shown less EFS than B-ALL cases [50,51]. Notably, children and young adults 1 to 4 and 5 to 19 years of age with B-cell ALL had more favorable survival (Approximately 99% and 88%) than those with T-cell ALL (Approximately 84% and 78%). In contrast, survival for T-cell ALL was substantially higher than B-cell ALL among adults 20 to 39, 40 to 59, and 60 years or older of age [52].

Chromosomal translocations in B-cell acute lymphoblastic leukemia (B-cell ALL)
Acute lymphoblastic leukemia is a heterogeneous disease that originates from lymphocyte progenitor cells of B-or T-cell origin.

ETV6-RUNX1 [t(12;21) cryptic translocation, formerly known as TEL-AML1]
The translocation t(12;21)(p13;q22) is the most frequent chromosomal alteration in childhood Blineage ALL (B-ALL) [54], which involves the fusion of the ETV6 (alias TEL) gene on chromosome 12 to the RUNX1 gene on chromosome 21. It is identified in 20% to 25% of the cases of Bprecursor ALL and is rarely observed in T-lineage ALL [55]. The t(12;21) is most commonly found in children aged 2 to 9 years [56]. Reports generally indicate favorable OS in children with the ETV6-RUNX1 fusion (Table 3); however, the prognostic impact of this genetic feature is modified by factors such as early response to treatment and treatment regimen [57].
The Ph produces a fusion gene on chromosome 22, namely, the breakpoint cluster region Abelson leukemia viral proto-oncogene (BCR-ABL). The translocation can result in 3 fusion protein of different sizes: p190, p210, and p230 [62]. The p190 BCR-ABL fusion gene occurs in about 90% of children with Ph-positive ALL [63] and between 50% and 80% of adults with Ph-positive ALL [64,65].
Ph-positive ALL has an extremely poor prognosis overall (rates of EFS are 30 to 46 percent in children and less than 20 percent in adults) Table 3. However, some investigators suggest that in this type of ALL, the prognosis is influenced by the treatment with glucocorticoids (and intrathecal methotrexate) [66], or by other factors (such as age and leukocyte count at diagnosis) [67,68]. These variations in the response to therapy suggest that Ph-positive ALL is heterogeneous with regard to sensitivity to treatment [39].
A t(4;11)(q21;q23)/MLL-AF4 positive ALL is generally considered as a high risk leukemia, characterized by a poor clinical outcome respect to other cytogenetic risk groups [73]. Moreover, in several studies it has been demonstrated that cytogenetic-molecular risk and WBC count at diagnosis were the main prognostic factors that influenced OS in ALL patients (Table 3). , is a primary and well known chromosome abnormality in childhood B-cell precursor ALL, being present in 3-5% of all such cases [75,76].

Chromosomal translocations in T-cell acute lymphoblastic leukemia (T-cell ALL)
T-cell ALL accounts for about 15% and 25% of ALL in pediatric and adult cohorts respectively [69]. Cytogenetic [81]. Historically, T-cell ALL in children has been associated with a worse prognosis than other sub-types of childhood ALL [82,83].
High expression of TLX1/HOX11 resulting from translocations involving this gene occurs in 5% to 10% of pediatric T-cell ALL cases and is associated with more favorable outcome in both adults and children with T-cell ALL [84][85][86]. Overexpression of TLX3/HOX11L2 resulting from the t(5;14)(q35;q32) translocation occurs in approximately 20% of pediatric T-cell ALL cases and appears to be associated with increased risk of treatment failure [85].

Gene polymorphisms associated to poor survival in ALL patients
It is difficult to define which component of the protocol/regimen is the responsible for the improved outcome of patients with ALL. Antifolates, such as methotrexate (MTX), are competitive inhibitors of folate-dependent enzymes and are widely used in the treatment of many human cancers [87]. In last decades, the MTX has been a key agent for the treatment of ALL and the benefit of high-dose MTX is well established as it significantly increases cure rates and improves patients' prognosis [88]. MTX exerts its cytotoxic effects by competitively inhibiting dihydrofolate reductase (DHFR), the enzyme responsible for converting folates to tetrahydrofolate, the reduced folate carriers which function in the transfer of carbon units. These carbon units are required for de novo purine synthesis and the methylation of uracil to thymine in DNA synthesis [89].
MTX enters the cells and is metabolized into 7-hydroxymethotrexate (7-OHMTX), 2,4-diamino-N 10 -methylpteroic acid (DAMPA) and more active derivatives as methotrexate polyglutamates (MTXPG) with sequential gamma-linkage of 2 to 6 glutamyl residues by the folylpolyglutamate synthetase (FPGS) [88]. MTXPG retained in cells for a longer time result in prolonged MTX antifolate effect [89]. However, accumulation of MTXPG is a critical factor associated with cytotoxicity and response of ALL patients to the therapy [89]. On the other hand, the polyglutamation process competes with deconjugation that converts MTXPG back into MTX by gamma-glutamyl hydrolase (GGH). Long chain MTXPG have higher affinity than MTX for the enzymes involved in de novo purine synthesis such as 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (ATIC) and thymidilate synthase (TS), which results in a reinforcement of MTX inhibition (Figure 1) [88]. Thus, intracellular formation of MTXPG enhances the cytotoxic and antileukemic effect of MTX.
The disease-free survival of childhood ALL has improved steadily the last decades, reaching 80% in the developed countries [17]. Despite the advances, almost 20% of the children either relapse or do not respond to treatment. This seems to be related to various parameters, in-cluding the presence of polymorphisms of drug transporters, receptors, targets, and drugmetabolizing enzymes, hence influencing the efficacy, the toxicity of therapy [91]. Recently, attention has been drawn on genes involved in diverse metabolic pathways, which are known to be polymorphic at various sites and can affect both the susceptibility for leukemia, the treatment outcome and survival in patients with ALL [91].

The reduced folate carrier (RFC1/SLC19A1)
Several polymorphisms in enzymes of the folate cycle as well as in the MTX transporters have been described. The reduced folate carrier gene (RFC1) is a major MTX transporter whose impaired function was recognized as a frequent mechanism of antifolate resistance [92]. The most common SNP in RFC1, 80A>G, which results in the amino acid substitution of Arg with His at position 27 of the RFC1 protein, may alter the affinity of the transporter [93]. Several investigators had studied the association of the SNP of RFC G80A and the outcome in ALL. Reports generally indicate association between the G/G and/or A/G genotypes of the G80A polymorphism with a poorer survival in patient's children and adults con ALL. Survival rates in Italian and Mexican population with ALL have been somewhat lower than the rates in European and French-Canadian population ( Table 4).
Several SNPs have been identified in the GGH gene at bases −401C>T, −354G>T, −124T>G, +16T>C, +452C>T, and +1102A>G; these sites comprise both the promoter and the coding region [95,96]. Nevertheless, few SNPs have been associated with catalytic activity of the GGH in B-and T-lineage ALL cells, and greater accumulation of longchain MTX in these cells [97].
The polymorphism +452C>T in the transcribed region of GGH gene alters Thr-127 to Ile-1271, and has been associated with reduced catalytic activity in hyperdiploid B-and T-lineage acute lymphocytic leukemia (ALL) cells, and greater accumulation of longchain MTXPG in these cells [97]. In contrast, all of the promoter polymorphisms enhanced GGH expression and an increased GGH activity may lead to decreased accumulation of MTXPG and to MTX resistance. At least one of these, −401C>T, has been shown to be correlated with decreased accumulation of long-chain MTX-Glu3-5 in rheumatoid arthritis patients treated with MTX [98].
Only polymorphism -354G>T has been associated with survival of children with ALL.
-354GT or -354TT genotypes carrier have better probability of 5-year post-treatment OS compared to -354GG genotypes (p = 0.04) ( Table 4) [99]. This shows the enzyme GGH clearly plays an important role in the metabolism of folates and anti-folates. However, unambiguous demonstration of a direct role of GGH in anti-folate drug resistance has been difficult. Part of the difficulty is that GGH is only one of several factors that can affect anti-folate levels, and its role presumably is directly linked to those of the other enzymes. [100]. However, these studies have demonstrated that polymorphisms in GGH increase promoter activity and an increased GGH activity may lead to decreased accumulation of MTXPG and to MTX resistance [101]

Dihydrofolate Reductase (DHFR)
DHFR is responsible to catalyze the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) [113]. The major mechanism of MTX action involves competitive inhibition of DHFR, leading to the impaired regeneration of THF from DHF; essential for the biosynthesis of purines and thymidylate, thus it also blocks the novo synthesis of DNA [114,115].
Changes in the levels of DHFR expression and consequently in the sensitivity to MTX can also be due to single SNPs, particularly those located in the regulatory elements [105]. The C829T SNP is located at the 223 nucleotide downstream from the stop codon between the first and second polyadenylation sites in the 3'UTR of the DHFR gene, which leads to the stability of mRNA [116]. A previous study reported that the -A317G SNP in the DHFR promoter region results in higher transcriptional activity [106]. Recently demonstrated an association between G/G and T/T genotypes of the -A317G and C829T polymorphisms and reduced survival in pediatric patients with ALL (Table 4).

Thymidylate Synthase (TS)
The TS is a key enzyme in the nucleotide biosynthesis and important target of several chemotherapeutics. TS provides the only source for de novo thymidylate production by catalyzing the methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) [107]. TS is efficiently inhibited by the uracil analog, 5-fluorouracil (5-FU) and MTX, used for many years as a treatment for a variety of cancers.
The most common polymorphism in TS is a unique double (2R) or triple (3R) 28-bp tandem repeat sequence in the 5' untranslated region (5'-UTR) of the TS gene also called TS enhancer region (TSER), immediately upstream from the initiation site, which influences protein expression in cancer cells [117]. The presence of a triple versus double 28-bp repeat in the enhancer region has been associated with an increased TS expression both in in vivo and in vitro studies [118,119].

Methylenetetrahydrofolate Reductase (MTHFR)
The MTHFR is a key folate enzyme that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate in the folic acid cycle, and is interrupted by methotrexate (MTX), a critical chemotherapy agent in ALL therapy [120].
Despite the fact that several MTHFR polymorphisms have been described thus far, only two polymorphisms, C677T and A1298C, have been intensively investigated. The C-to-T transi- tion at the nucleotide position 677 in exon 4 of MTHFR generates an alanine-to-valine substitution at amino acid 222 [121]. As a result, carriers of the MTHFR 677TT genotype possess a thermolabile enzyme of reduced activity [122]. The second most studied polymorphism in MTHFR is an A-to-C transversion substitution at nucleotide 1,298 (exon 7) that results in an amino acid substitution of glutamate for alanine at codon 429 [123]. Once this amino acid substitution takes place at the S-adenosylmethionine regulatory domain of the MTHFR, the A1298C polymorphism also generates an enzyme with a decreased activity [123]. Other investigators have reported that A1298C and C677T polymorphisms in MTHFR gene are associated with disease outcomes and survival both in children and adults (table 4).
The overall survival rate of MTHFR 677TT and 1298CC carriers was lower than that of patients carrying MTHFR C or A alleles respectively. A limited amount of evidence has been reported on the influence of MTHFR polymorphisms on survival.

Methylenetetrahydrofolate Dehydrogenase (MTHFD1)
MTHFD1 is an enzyme involved in folate metabolism, which plays an important role in the generation of the 5,10-methylene-THF and 10-formyl-THF. The last two are the donor cofactors for de novo purine and pyrimidine biosynthesis and, thus, for the biosynthesis of DNA [124]. The G to A substitution at position 1958 of the MTHFD1 gene, causing an alanine to glycine substitution at codon 653 located within the 10-formyl-THF synthetase enzyme domain, which reduces the enzyme's activity [124].
A analysis of 201 children treated with methotrexate showed that patients with the MTHFD1 A1958 variant had a remarkably lower probability of 5-year post-treatment survival, compared to subjects with no event-predisposing genotypes (45.0% vs 95.0%, p=0.0002) [112].
Recent studies have shown that in ALL patients, high expression of MRP1, is a highly significant indicator of poor response to chemotherapy and poor overall survival in both in children and adults (Table 5).

Summary and future directions
Several clinical and biological features have been associated with the improved survival of patients with ALL, including age, sex, WBC, race/ethnicity, immunophenotype, recurrent chromosomal abnormalities, and genetics polymorphisms. The application of risk-stratified therapy utilizing these prognostic factors has resulted in long-term event-free survival in up to 80-85% of patients with ALL. Further improvement in outcome will require, in part, the discovery of novel prognostic factors, (such as, genetic variation in the folate pathway, transport of drugs, as well as miRNAs expression) to identify the 15-20% of patients who are not cured with current therapies. Recent advances in our understanding of underlying leukemia biology, including the identification of prognostically distinctive subsets of patients, and of host pharmacogenomics may allow for more precise risk stratification and more targeted, individualized treatment planning that will lead to higher survival of the patients with ALL.