Colorectal carcinoma (CRC) is one of the most frequent causes of cancer deaths in industrialized countries. Although surgery alone is the standard approach in localized malignancy, about 50% of early-stage patients present disease relapse. Furthermore, about 30% of CRCs are diagnosed when they are already at an advanced stage. Fluoropyrimidine-based adjuvant chemotherapy is now considered the standard treatment for stage III disease, nevertheless patients with Duke's B colon cancer may also benefit from adjuvant chemotherapy (Mamounas et al., 1999; Freyer et al., 2001).
Since about 90% of patients do not respond to adjuvant therapy based on 5-fluorouracil/folinic acid (also known as leucovorin) (5-FU/FA), in order to improve the clinical outcome of patients with advanced/metastatic CRC, it has become necessary to introduce into common chemotherapy trials new drugs, such as capecitabine, irinotecan, and oxaliplatinum. First line therapy for metastatic CRC involving the combination of 5-FU/leucovorin with irinotecan has been proven to be highly effective than the use of 5-FU/leucovorin alone, and the combination 5-FU/leucovorin with oxaliplatinum has become standard for the first and second line treatment of CRC (Mitchell, 2000; Cassidy et al., 2004; Marcuello et al., 2004).
Although in recent years, there has been a vast improvement in the outcome of patients included in these new trials, present-day protocols are still limited by the unpredictable response to the drug and to its severe toxic effects. The administration of a standard dose of chemotherapy to patients with a hereditary lack of key enzymes needed for metabolism and absorption may have serious toxic results, which may at times be fatal. Likewise, polymorphisms in drug targets can alter the sensitivity of patients to treatment changing the pharmacodynamics of drug response. In a specific population, in fact, whereas certain subjects will respond positively to a particular type of treatment, others may present a modest or marginal response or even no response at all, and others may suffer from adverse drug reactions (ADR), which will be the main cause of the failure of the chemotherapy. The objective of pharmacogenomics is to elucidate the complex genetic network responsible of drug efficacy and toxicity, and to then maximize the therapeutic effects of drug treatment while minimizing ADR. The ultimate goal is to provide new strategies for optimizing the individual's response to drug therapy based on patient's genetic information.
It has been reported, in fact, that the different responses to a particular drug are not only due to the specific clinico-pathological features of the patient (age, gender, diet, or the presence of kidney and/or liver diseases) (Watter and Mc Leod, 2003), or to environmental factors (tobacco, alcohol, professional exposure), but also to the ethnic origins and particular genotype of a single individual. Genes that codify for the metabolism enzymes, receptor proteins, or proteins targets of chemotherapy agents often present different genetic polymorphisms that can influence drug sensitivity, toxicity, and dosing (Table 1). Genetic polymorphism is a difference in DNA sequence among individuals, groups, or populations, and the genetic mutations are a kind of genetic polymorphism. Single nucleotide polymorphisms (SNPs) are the most simple form and most common source of genetic polymorphism in the human genome (90% of DNA polymorphisms), and transitions and transversions represent two types of nucleotide base substitutions resulting in SNPs.
Table 1. Chemotherapeutic drugs: Clinical relevant genes, drug response, toxicity, and survival
One branch of pharmacogenomics, therefore, involves the study of SNPs. SNPs in a gene may bring about the formation of a protein with a different structure and function, and the resulting modification of the ability of the human organism to use and to metabolize a particular drug. SNP analysis is now considered a valid method for helping the oncologist to decide on a more specific, personalized therapeutic approach towards each single patient and to yield crucial information for new drug development. Furthermore, it offers a number of advantages compared with microsatellite analysis, which, in our genome, involves highly polymorphic, frequently repeated sequencing.
SNPs, in fact, are much more numerous in the genome (about 1 for every 800 bp) than microsatellites (Nerber, 1999), show fewer mutations of the germinal line, and are therefore more stable than microsatellites; they are generally found in the codifying or regulatory regions of the gene, and finally are mainly bi-allelic, which facilitates the assessment of their frequency in the population (Sapolsky et al., 1999).
Another equally important branch of pharmacogenomics concerns the simultaneous evaluation of gene transcripts. In fact, the assessment of the transcriptional events, in addiction to SNPs analysis, may be useful to identify biomarkers that predict either adverse pathological responses or the efficacy of the drug tested and then to personalize the treatment for a single patients.
Here, we provide an overview of the known polymorphisms present in the genes which codify for key metabolic factors involved in the action mechanisms of the drugs responsible for significant toxicity from the present-day chemotherapeutic treatment of CRC.
GENETIC POLYMORPHISMS AND DRUG RESPONSE
Fluoropyrimidines, such as 5-FU, are widely used for the treatment of a great many neoplasias, such as CRC. Chemotherapy with 5-FU works by inhibiting thymidilate synthase (TS), a intracellular enzyme critical for the de novo synthesis of DNA.
5-FU, an analog of uracil, is an anticancer prodrug that, after administration, is converted intracellularly into three main active metabolites: 5-fluoro-2-deoxyuridin monophosphate (5-FdUMP), fluorodeoxyuridine triphosphate (FdUTP), and fluorouridine triphosphate (FUTP). Both normal and tumor cells metabolize 5-FU to 5-fluoro-2′-deoxyuridine monophosphate (FdUMP) and 5-fluorouridine triphosphate (FUTP) well-known for their cytotoxic effects.
The toxic effects are mediated by the inhibition of thymidylate synthase through the formation of an extremely stable ternary complex among FdUMP, TS, and the cofactor 5,10-methylene-tetrahydrofolate (5,10-MTHF, CH2THF). The formation of this complex prevents the methylation of the deoxyuridin-5′-monophosphate (dUMP) into deoxythymidine-5′-monophosphate (dTMP) catalyzed by TS (Fig. 1).
Overexpression of TS has been reported in many types of tumors including breast, colon, gastric, and melanoma (Iqbal and Lenz, 2003; Lenz, 2003). In particular, TS expression has been shown to be predictive of response to 5-FU therapy in CRC (Gorlick et al., 1998). For instance, in CRCs, TS overexpression has been found to be significantly associated with a low response to treatment based on 5-FU (Johnston et al., 1995), both as adjuvant (Leichman et al., 1997) and metastatic therapy (Paradiso et al., 2000). Several studies have proposed that genetic polymorphisms of TS gene can affect the response to 5-FU (Gorlick et al., 1998; Iacopetta et al., 2001; Marcuello et al., 2004). TS expression seems to depend on the number of the so-called TSER, tandem repeat polymorphic copies of 28 bp present in the 5′-promoter enhancer region of the gene. TSER polymorphisms, therefore, are involved in the modulation of TS protein levels and can affect the drug response after administration of fluoropyrimidine (Horie et al., 1995; Kawakami et al., 1999). Most Caucasian subjects may be carriers of double (TSER2) or triple (TSER3) repetitions for this type of polymorphism, although there have also been reports of sequences with even more copies. An increase in the number of repeats gives rise to an increase in both mRNA and protein TS levels. Three copies of such repeats (TSER*3) lead to a TS expression which is 2.6 times higher than that produced by the presence of only two copies (TSER*2). Patients with CRCs, which show homozygote triple-tandem repeats (3R/3R), present high levels of intratumoral TS mRNA, elevated levels of TS protein, and a lower rate of response to chemotherapy than subjects with CRCs showing homozygote double-repeats (2R/2R) (Pullarkat et al., 2001). Similar results have been obtained in patients with metastatic CRCs (Aschele et al., 1999). Moreover, a study involving 221 Duke's C stage CRC patients (Iacopetta et al., 2001) has shown that, with regard to survival rate, tumors with 3R/3R genotypes benefit less from chemotherapy than those with 2R/2R and 2R/3R genotypes. A recent meta-analysis of 20 studies (Popat et al., 2004) has made it possible to investigate the association between levels of TS expression and the survival of CRC patients. The results have shown that high levels of TS in patients at any stage of the disease are predictive of outcome. However, the predictive role of TS levels in early-stage CRC patients undergoing chemotherapy is still not fully understood; in fact, whereas in subjects undergoing surgery only high TS levels are an independent prognostic factor for outcome, in those undergoing surgery together with surgery and adjuvant FU, TS expression does not seem to predict outcome (Popat et al., 2004). Another study (Shirota et al., 2001) reports that in patients with advanced CRC treated with 5-FU/oxaliplatin, intratumoral TS levels appear to have an independent predictive value for survival.
Nevertheless, the data so far reported in literature are discordant; although, in fact, TS levels have prognostic value for CRC, this is lower in surgically-treated patients who undergo adjuvant therapy with 5-FU when the TS expression is low, but may be effective for tumors with high TS expression (Takenoue et al., 2000). Moreover, a study conducted by TSUJI has shown that multivariate analysis in the TSER genotype has no prognostic significance, thus indicating that this genotype is not a valid predictor of the efficiency of 5-FU based oral adjuvant chemotherapy for CRC patients (Tsuji et al., 2003). These controversial data might well be justified by the recent identification in an Asian population of a new SNP within the repeat of 28 bp. The presence of double polymorphisms might possibly be more predictive with regard to 5-FU treatment (Kawakami and Watanabe, 2003). It might be interesting to extend this study not only to include the Asian population, but also to other ethnic groups, since allelic variables are often population-specific.
5-FU is inactivated in the liver by dihydropyrimidine dehydrogenase (DPD), which is the first key enzyme involved in the catabolism of the uracil and thymine into β-alanine (Fig. 2). DPD activity is extremely variable in tumoral tissue and this variation might make a difference to the efficiency of 5-FU treatment, since intratumoral drug concentration is one of the most important factors for the determination of the antitumoral effect (McLeod et al., 1998). A recently-published study (Tanaka-Nozaki et al., 2001) reports, in fact, that intratumoral levels of 5-FU are inversely proportional to DPD activity. Patients with low DPD activity show a better response to 5-FU treatment (Salonga et al., 2000), and it has been observed that women are more responsive to adjuvant therapy based on 5-FU since their tumor tissues probably present lower levels of DPD than those of male patients (Yamashita et al., 2002). Deficiency in DPD activity, however, leads to severe toxicity correlated to 5-FU which may even be fatal. The partial or total lack of this enzyme has, in fact, been associated with severe toxicity (mucositis, granulocytopenia, and neuropathy), and in several cases even death, after 5-FU administration (Wei et al., 1996; Milano et al., 1999) in over 3% of these patients carrying heterozygote DPYD allelic variants of the gene DPD. The gene codifying for DPD expression is known, in fact, as DPYD. At least 20 different functional mutations have been reported in DPYD gene and have been associated with a reduced activity of DPD (Innocenti and Ratain, 2002). In the general population, from 3% to 5% of the subjects are heterozygote carriers of mutations leading to DPD inactivation, whereas 0.1% are carriers of homozygote mutations (Ridge et al., 1998). Analysis of the prevalence of various genetic variants of DPD among patients with DPD deficiency has shown that the most common mutation in DPYD is a G–A transition at the invariant GT splice donor site flanking exon 14 (IVS14 + 1G > A) in Caucasian populations; this mutation is responsible for the lack of exon 14 in transcript resulting in production of a truncated mRNA with virtually absent enzyme activity (Wei et al., 1996; van Kuilenburg et al., 2001). This allele is known as DPYD*2A and is one of the variants associated with severe toxicity after 5-FU treatment (Johnson et al., 1999; van Kuilenburg et al., 2001). Recently two new missense mutations have been identified on codon 496 (A substitution into G) in exon 6 and on codon 2,846 (A substitution into T) in exon 22, the latter in a patient with a total lack of DPD (van Kuilenburg et al., 2000). Since the presence of polymorphic sites in the gene is considered as extremely important, a new kit, PGX-5FU StripAssay (Nuclear Laser Medicine, Vienna, Austria), is now used in several biomolecular laboratories for detection of the polymorphism prior to treatment. This kit permits, in fact, a rapid identification of carriers of both homozygote and heterozygote allelic variants of the DPYD*2A.
Treatment of metastatic CRCs now includes the use of another chemotherapeutic agent, capecitabine, which is an oral precursor of 5-FU (Fig. 3) (De Paoli et al., 2004). This orally active tumor-selective fluoropyrimidine reaches a higher intratumoral 5-FU level and is less toxic than 5-FU. With regard to 5-FU, low levels of TS and DPD lead to a better response to capecitabine. In particular, it has been observed that 75% of metastatic colorectal cancer patients, with homozygote double-repeat variants in TS (S/S), respond better to capecitabine administration compared with 8% of those with heterozygote variants (S/L) and 25% of those with triple-repeat homozygote variants (L/L) (Park et al., 2002; Iqbal and Lenz, 2003).
The combination of FA/5-FU together with other drugs such as irinotecan has led to promising results in the treatment of CRCs, particularly in first line therapy of patients with metastatic disease. Irinotecan-based therapy has been approved for the treatment of metastatic CRC, with three-weekly and weekly dosing being the most common schedule.
Moreover, recent data suggested that a synergistic interaction between EGFR tyrosine kinase inhibitor gefitinib (“Iressa”) and irinotecan could be effective in the treatment of colorectal tumor cells that express high levels of EGFR (Koizumi et al., 2004).
Irinotecan (CPT-11) inhibits topoisomerase I, a nuclear protein, which breaks one strand of the DNA, pulls the other strand through the gap, and finally seals the gap. Irinotecan is a prodrug that, in presence of hepatic or gastrointestinal carboxylesterase, is metabolized to its active form 7-ethyl-10-hydroxy camptothecin (SN-38). Irinotecan and SN-38 bind to the topoisomerase DNA complex preventing religation of the single-strand breaks in the DNA molecule and causing DNA double strand breaks, which can lead to apoptosis (Catley et al., 2004; Griffiths et al., 2004).
The dose-limiting toxic effects of irinotecan are myelosuppression and diarrhea. The gene implicated in this toxicity is associated with polymorphisms in the uridine diphosphate (UDP)-glucuronyl-transferase, UGT1A1, which is involved in the glucuronidation of SN-38 (the active metabolite of irinotecan) and important for detoxification of SN-38. SN-38 becomes detoxified by polymorphic hepatic UDP-glucuronosyl-transferase 1A1 (UGT1A1), which glucuronidates and transforms it into the inactive form SN38 glucuronide (SN 38G), which is then excreted in the bile by the small intestine (Fig. 4). In cases of low UGT1A1 activity the accumulation of high levels of SN-38 can cause diarrhea and leucopenia. In the general population, the UGT1A1 gene promoter contains several TA repeats which vary from 5 to 8; more than 30 allelic variants have been identified, both in the promoter region and in the exons. The 6-TA repeats are the most frequent allelic variants and, in fact, it has been shown that the higher the number of repeats, the lower the expression of UGT1A1 will be (Park et al., 2002). A polymorphism in the UGT1A1 promoter, with repeated dinucleotides in the TATA box, brings about a reduction in hepatic expression of UGT1A1. This polymorphism relates to a lower glucuronidation of SN-38, leading to greater irinotecan toxicity (Iyer et al., 1999; Ando et al., 2000; Innocenti and Ratain, 2004). The UGT1A1*28 polymorphism is the most common genetic lesion and consists of an additional TA repeat in the TATA sequence of the UGT1A1 promoter [A(TA)7TAA]. The allelic variant UGT1A1*28 is heterozygous or homozygous for 7 TA repeats and is significantly associated with a resulting reduction of UGT1A1 expression with the consequent decrease of SN38 glucuronidation and a severe increase in toxicity (diarrhea/leucopenia) induced by the use of irinotecan (Fig. 5) (Ando et al., 2000). A prospective study phase 1 conducted on the use of this drug administered every 3 weeks in 20 patients with solid tumors has shown that subjects with the allelic variant UGT1A1*28 presented lower levels of SN-38 glucuronidation compared with patients without the UGT1A1*28 polymorphism (Iyer et al., 2002). Furthermore, patients with heterozygote and homozygote forms of this allelic variant more often showed side-effects such as severe diarrhea and neutropenia. The identification of the UGT1A1*28 polymorphism might help in the screening of patients with lower levels of SN-38 glucuronidation who might be more susceptible to the blood and gastrointestinal toxicity resulting from the use of irinotecan. Although there has been a clear demonstration of the ability of the UGT1A1*28 polymorphism to act as a mediator in the toxicity linked to CPT11, no guidelines regarding the use of the information on the UGT1A1 genotype in the treatment of patients undergoing CPT11 therapy have so far been developed. In a prospective, phase 1 study, the Mayo Clinic is at present assessing the impact of the UGT1A1*28 polymorphism with a combined treatment based on CPT11, capecitabine, and oxaliplatinum. Patients without polymorphisms are given higher doses of this drug combination according to their tolerance, while for subjects who are homozygote or heterozygote for UGT1A1*28 and therefore at higher CPT11 toxicity risk, an assessment is made of the maximum risk-free dose which can be administered. (Goetz et al., 2004).
Nowadays, platinum agents (cisplatin, carboplatin, and oxaliplatin) used in the treatment of CRCs are generally associated with 5-FU. Platinum-based chemotherapy drugs produce crosslinks between the two complementary strands of the DNA double helix (Fig. 6). The ability of cells to repair this damage is an important factor which determines response to these drugs. In vitro assays have been shown that high levels of enzymes involved in the DNA repair system lead to drug resistance, whereas low enzyme levels produce a better response to treatment with cisplatin (Hoeijmakers, 1993). Resistance to platinum compounds takes place through several mechanisms including enhanced DNA repair capacity (DRC), which is genetically determined and modulates cancer susceptibility. The repair system based on nucleotide excision (NER), which is implicated in the repair of DNA lesions, includes a great many genes, aptly-named as the excision repair cross-complementation group 1 and the Xeroderma Pigmentosum Group D (ERCC1 and XPD), which codify proteins involved in the removal of the cisplatin–DNA adducts. Recent studies have been shown that platinum chemotherapy response may be influenced by genetic variations not only in the genes whose proteic products are implicated in the nucleotide excision repair system, but also in those codifying for proteins involved in other DNA repair pathways, such as the X-ray cross-complementing group 1 protein (XRCC1) (Lunn et al., 1999; Stoehlmacher et al., 2001; Park et al., 2003).
The X-ray cross-complementing group 1 protein (XRCC1) acts as a scaffold to form a quaternary complex with the β polymerase DNA, the DNA ligase III, and the polymerase polyADP-ribose, and is important for the repair of single-strand DNA, for the nucleotide excision repair, in oxidative damage and in the adducts formed subsequent to treatment with alkylating agents. XRCC1 binds to the single-strand DNA interruptions and is able to recruit proteins involved in repair (Wood et al., 2001).
Various polymorphisms in the gene XRCC1 have been proved to be associated with a different response to platinum agents. Three polymorphisms of XRCC1 have been described, on codons 194, 280, and 399. For instance, codon 399 is particularly involved in treatment response (Shen et al., 1998). A polymorphism within codon 399 of the XRCC1 gene causes the substitution of adenine with a cytosine, and in the protein the substitution of arginine with a glutamine. Patients with a homozygote or heterozygote genotype for glutamine (Arg399Gln) would seem to be more subject to a lack of response to treatment with 5-FU/oxaliplatinum, which leads to a reduction of DNA repair possibility (Lunn et al., 1999; Stoehlmacher et al., 2001). A confirmation that there is a link between an XRCC1 polymorphism (Arg399Gln) and clinical outcome has been demonstrated in a study where patients at advanced stages of colorectal cancer were treated with platinum; the presence of an allelic variant (both the Gln/Gln and the Gln/Arg genotypes), proved to be more common in non-responsive subjects (Stoehlmacher et al., 2001).
Xeroderma Pigmentosum Group D (XPD) is a helicase component of the TFIIH transcription factor complex which takes part in DNA unwinding and is implicated in the excision repair mechanisms of the DNA for the removal of platinum-DNA lesions. Several studies (Spitz et al., 2001) have been reported the existence of a polymorphism on codon 751, with A/C substitution, leading to a substitution of a Lys with a Gln in the protein (Lys751Gln). These data have been confirmed by a retrospective study conducted on 69 patients with metastatic CRC treated with 5-FU and oxaliplatinum. Homozygous subjects with an Lys751/Lys751 genotype showed a better response to chemotherapy and a longer survival rate compared with patients with heterozygote XPD Lys/Gln or homozygote Gln/Gln genotypes (Park et al., 2001; Lenz, 2003).
Nevertheless, further studies are necessary in order to confirm these data and to establish the real importance of polymorphism in the gene XPD with regard to resistance to platinum agents.
The excision repair cross-complementing gene (ERCC1) is a highly conserved enzyme that, together with the Xeroderma Pigmentosum Group F (XPF), is implicated in the repair mechanisms of the crosslinking intrastrands caused by the platinum agent, by means of the nucleotide excision repair pathway. The gene ERCC1 contains a known polymorphism on codon 118 (exon 4) which causes the substitution of a C with a T. Although this does not lead to amino acid substitution, it does cause a variation in the expression levels. In patients with metastatic CRC treated with 5-FU/oxaliplatinum, it has, in fact, been observed that the presence of C in homozygosity leads to higher expression levels of the protein and to a better outcome (mean survival rate = 15.3 months) than subjects with heterozygote or homozygote T, who show a mean survival rate of, respectively, 7 and 11.1 months (Shirota et al., 2001; Park et al., 2003). Nevertheless, how a silent polymorphism is able to affect mRNA expression levels is still not clearly understood.
The enzymes involved in platinum response until now mentioned are implicated in the DNA repair pathway. In addition, the glutathione S-transferase (GST) is a phase II metabolic enzyme involved in metabolic drug inactivation. GST is a multigene family of enzymes involved in detoxification by means of the conjugation of glutathione with a wide range of chemotherapy agents. GSTs are extremely polymorphic, especially those of the GSTPI subclass, which directly participate in the detoxification of platinum compounds. Glutathione S-Transferase 1 (GSTP1) belongs to the GST family of enzymes which help to protect against a vast number of compounds, such as carcinogens, pesticides, antitumoral agents such as platinum and environmental pollutants (Cotton et al., 2000; Sheehan et al., 2001). By conjugating with glutathione, GSTP1 is able to inactivate these compounds and transform them into more soluble molecules which are able to exit more easily through the membrane of the target cell. Moreover, GSTP1 is normally expressed in the epithelial tissues and is overexpressed in CRCs. This gene presents an SNP on codon 105 (A into C) which leads to the replacement of an isoleucine with a valine in the protein (Watson et al., 1998) causing a reduction of GSTP1 enzyme activity. A study conducted on 107 patients with metastatic CRC treated with 5-FU/oxaliplatinum has been shown an association between genotype and survival rate. In fact patients with a homozygote Val105/Val105 genotype had a mean survival rate of 25 months, those with heterozygote Ile/Val genotypes of 13 months, and those with homozygote Ile/Ile genotypes of about 8 months. The reduced expression of GSTP1 in patients with homozygote valine genotype led to a lower level of enzyme detoxification after treatment with oxaliplatinum (Stoehlmacher et al., 2002).
The recently-published studies of pharmacogenomics applied to CRC which we have consulted have brought about a more rational approach towards therapeutic monitoring aimed at avoiding both under- and over-dosing. The field of pharmacogenomics opens up new prospects for the treatment of CRC, since it permits a rapid genetic analysis able to predict individual response to a specific drug and a resulting personalized therapeutic approach leading to a more positive treatment outcome.
It would be useful, for example, to identify CRC patients carrying polymorphisms in the genes TS and DPD before beginning chemotherapy with 5-FU, in order to make a more individual therapeutic choice based on non-TS-DPD-directed anticancer drugs such as irinotecan or oxaliplatinum, possibly used in association with 5-FU (Lenz, 2003).
On the other hand, Lenz reports that patients with polymorphisms of the genes implicated in DNA repair mechanisms, for example, ERCC1, might benefit from alternative types of treatment not based on the use of platinum agents, whereas those with low levels of ERCC1 expression might undergo traditional therapy with 5-FU associated with oxaliplatinum.
Then, in the light of what we said, it may be useful to characterize the alterations, not in a single gene but in all genes, nowadays known, responsible of drug efficacy and toxicity.
Present-day techniques provide rapid, cheap methods which make it possible to identify in the genes the presence of already-known polymorphisms whose products interfere with the mechanisms of drug action and at the same time to identify new polymorphisms. Although such techniques are evolving rapidly, they cannot yet be considered sufficiently sensitive or accurate for them to be used in routine clinical practice. Future improvements in pharmacogenomic techniques will certainly make it possible to perform the genotyping of patients with CRCs or other neoplasias in order to choose a specific type of chemotherapy for each individual patient. Only in this way will it be possible to predict before beginning such treatment any individual resistance or toxicity towards a specific drug in order to reduce to the minimum any adverse effects and to adapt therapy according to the subject's individual genetic differences. It goes without saying that it is not enough to establish the functional significance of genetic polymorphisms involved in CRC; additional, rigorous, well-designed, large-scale prospective clinical trials with long periods of follow-up are also necessary, so that such genotypical features may lead to the identification of new therapeutic populations and the widespread use of the results.