The Effect of Genetic Polymorphism upon Antineoplastic Sensitivity

Uridine-diphosphoglucuronosyltransferase, UGT

The multigene UGT family, is expressed on the endoplasmic reticulum membrane and nuclear envelope, and catalyzes the glucuronidation of compounds from endogenous and extrinsic sources. This multigene family can be roughly divided into two subfamilies namely UGT1A and UGT2B. The UGT1A gene is located on chromosome 2q37, while the UGT2B gene is on chromosome 4ql3.

UGT1A1 is a major glycosyl transferase with more than 60 mutations and polymorphisms. It’s reactivity differences are mainly caused by the polymorphism in the UGT1A1 gene promoter region, which contains multitude tandem repeats of TA. The genetype of 7 TA tandem repeats named UGT1A1*28 (WT has 6 TA tandem repeats) depresses the expression and activity of UGT1A1. The genotypic frequency of the UGT1A1*28 homozygote is 0.5~23%, however, the frequency among Orientals is lower than Africans and Caucasians. Mutations of G211A are common among Asians (13~23%), which can reduce activity of UGT1A1 by 30% and 60% in the heterozygoty and isozygoty, respectively).^[1]

Irinotecan, a semisynthetic analog of camptothecin is convered by carboxylesterases to form the active metabolite 7-ethyl-10-hydroxy-camptothecin (SN-38). SN-38 acts by inhibiting DNA topoisomerase I. Irinotecan is widely used in chemotherapy for colon carcinoma and lung cancer. SN-38 is conjugated to the inactive SN-38 glucuronide (SN-38G) by UGTs including hepatic UGT1A1, UGT1A6, and UGT1A9 and extrahepatic UGT1A7, so that SN-38 is deactivated. The main reasons for restricting the dosage of irinotecan are diarrhea and myelosuppression, which correlate with the level of SN-38 formed. Consequently, if UGT1A1*28 activity is decreased, SN-38 glucuronidation will decline, increasing SN-38 accumulation and then raising clinical toxicity of irinotecan.^[2]

Ogura et al. ^[3] found that trans-4-hydroxy-TAM (trans-4-HOTAM), one of the TAM metabolites in humans, has a higher affinity toward estrogen receptors (ERs) than the parent drug or other side-chain metabolites. Trans-4-HO-TAM and its geometrical isomer, cis-4-HOTAM were excreted, via N-glucuronidation in humans. Only UGT1A4 catalyzed the N-glucuronidation of 4-HO-TAM among human UGT isoforms. In contrast, all UGT isoforms, except for UGT 1 A3 and UGT 1 A4, catalyzed O-glucuronidation of 4-HO-TAM, which greatly decreased binding affinity for human ERs.

Thiopurine methyttransferase, TPMT

TPMT is a cytosolic drug-metabolizing enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-MP to form a non-bioactive metabolite. Gene mutations of TPMT have an important influence for the bioactivity and toxicity of 6-MP. It has been demonstrated that about 1 in 300 individuals inherit a TPMT deficiency as an autosomal recessive trait, approximately 10% of individuals have intermediate activity due to heterozygosity, while 0.33% are TPMT deficient. Polymorphism of the TPMT gene reduces the metabolic rate of 6-MP conversion to inactive agents. Therefore, patients with a mutated TPMT gene suffer a higher rate of hematologic toxicity when administed 6-MP.^[4]

Wild-type TPMT is named TPMT* 1. The main other forms are TPMT*2 (G238C, Ala80→Pro), TPMT* 3A (G460A Alal54→ Thr and A719G Tyr240→Cys), TPMT*3B (G460A; Alai54 →Thr) and TPMT*3C (A719G Tyr240 →Cys). Three particular TPMT alleles, designated as TPMT*2, TMPT*3A, and TPMT* 3C, have been shown to account for nearly 95% of the observed cases of TPMT deficiency. TPMT*3A is the most common variant allele in Caucasians (5%), whereas TPMT*3C is predominant in East Asia (2%), African and U.S. Blacks. A study of 248 Chinese Han people showed that TPMT*3C accounted for 1.4%, with no TPMT*2, TMPT*3A or TPMT*3B.^[5] There are only two SNPs in *3A, *3B and*3C, and the two common coding SNPs in TPMT result in structural disruption and misfolding of this enzyme. Misfolded proteins can be removed from the cell by degradation, decreasing the amount of TPMT.^[6]

Laking xanthine oxidase activity, hematopoietic cells depend on TPMT metabolizing toxicants, so patients with very low levels of TPMT activity, as in TMPT*3A homozygote, are at greatly increased risk for hematologic toxicity such as myelosuppression when treated with standard doses of 6-MP. Accordingly, when treating these patients, doses of 6-MP should be reduced to 6~10% of the normal dosage. Conversely, patients with high TPMT activity will display drug resistance.^[7]

Dihydropyrimidine dehydrogenase, DPD

5-Fluorouracil (5-FU) and its derivatives are widely used in chemotherapy for many types of carcinomas. Approximately 5% of administered 5-FU accounts for its antitumor activity, whereas the other 80%~;95% undergoes catabolism into biologically inactive metabolites that are excreted in the urine and bile. DPD is the initial and rate-limiting enzyme in the catabolism of 5-FU, which dégradâtes the majority of 5-FU in the liver to 5,6-dihydro-5-fluorouracil. DPD activity is completely or partially deficient in 0.1% and 3%~5% of individuals in the general population. ^[8] Patients with deficient levels of DPD activity can develop severe toxic reactions and even death when treated with 5-FU. Tumors expressing low levels of DPD mRNA and DPD activity show a significantly better response to 5FU than those tumors with a high mRNA level and DPD activity. Most importantly, a low intratumoral expression level of the DPD gene appears to be associated with a much longer survival period for 5-FU treated patients.^[9]

The failure of DPD activity is associated with gene polymorphism of DPYD, which is located on chromo-some lq22, with a total of 150,000 bp. DPYD contains 23 extrons, and DPYD*2A is the the most common isoform which is caused by a splice site mutation G—? A transition on exon 14 of the DPYD gene(IVS14+ 1G→A). This change leads to skipping of exon 14 and the synthesis of a truncated inactived protein (frequency is 52%).^[10]

Methylenetetrahydrofolate reductase, MTHFR

MTHFR is a key enzyme for folic acid metabolism, catalyzing the non-reversible reaction of reducing 5.10-methylenetetrahyxrofolate to 5-methyltetrafolate. 5.10-Methylenetetrahyxrofolate participates in the synthesis and repair of DNA, whereas 5-methyltetrafolate takes part in the méthylation of DNA and maintenance of its integrity. The common heterogeneities of MTHFR are C677T (A222V) and A1298C (E429A). The mutation rate of C677T among north-Americans is 35%, while the frequency of homozygosis mutations (TT) and heterozygosis mutations (CT) among Caucasians and Asians are respectively 12~15% and 50%. Sohn et al.^[11] found that the polymorphism of MTHFR C677T restricted the reaction mentioned above, resulting in an increased level of 5,10-methylenetetrahyxro-folate. This reduced activity resulted in strengthening the formation and stability of 5,10-methylenete-trahyxrofolate-thymidylate synthase-5FdUMP trimer, enhancing the drug action. On the other hand, the increased intracellular concentration of 5,10-methylenetetrahyxrofolate provides one carbon units for synthesis of thymidylate and purines, leading to reduced MTX action. An in vitro experiment indicated that colon carcinoma and breast cancer cells with the MTFIFR C677T mutation showed a better response to 5-FU, but a poor response to MTX in the breast cancer cells.

Cytochrome P450, CYP450

CYPs are a family of enzymes involved in oxidation-reduction reactions of many endogenous and exogenous compounds. In mammals, CYPs are generally divided into two groups. Class I participate in the synthesis of some bioactive micromolecular substances, and Class II are associated with the metabolism of many drugs, toxicants and carcinogens. The activity of CYP3A accounts for 50% of the total P450 activity in adult liver, and contributes to catalyzing about 60% of administrated drugs, while CYP2C9, 2C19 and 2D6 metabolize about 30%.

CYP2A6 metabolizes Tegafur, a prodrug, to bioactive 5-FU. Enzyme activity differences are due to genetic polymorphisms. CYP2A6*4, the allele with the whole gene deleted, is high in Orientals, with a frequency of approximately 15%~20% within the population.^[12]

CYP2B6 is involved in the metabolic activation of cyclophosphamide. The enzymatic contribution to its activation is extremely high (80% of the total activity, whereas CYP3A4 contributes only 4% of the total activity), whereas the contribution of CYP2B6 to the activation of ifosfamide is relatively low (20% of the total activity, whereas CYP3A4 contributes 40% of the total activity).^[13]

CYP2C8 is a key enzyme for the detoxification of paclitaxel to form 6a-hydroxypaclitaxel, which is approximately 30 times less toxic than paclitaxel. One study showed that the Japanese have a very rare allele frequency of CYP2C8 (the CYP2C8*5 allele is found in only 0.25% of Japanese, a deletion of adenine 475). Therefore, genotyping of the CYP2C8 gene might have limited utility in predicting adverse effects from paclitaxel in Japanese. In contrast, variant alleles are more frequent in Caucasians and African-Americans (2%~15%).^[14] CYP2D6 plays a role in the conversion of tamoxifen to the more potent antiestrogen, 4-hydroxytamoxifen.^[15]

The CYP3A subfamily’s coding gene is located on 7q21-22, and is comprised of four members, namely CYP3A4, CYP3A5, CYP3A7(expressed in fetal liver), and newly found CYP3A43. A polymorphism of CYP3A5, which can account for the variation in the expression of this gene, is a SNP in intron 3 (A6986G), leading to an inactive CYP3A5*3. Another finding is that of a rare G/A SNP in the intron 7 among African-Americans. This product as an inactive CYP3A5*6, resulting in the depression of CYP3A5 activity. CYP3A4 has a complicated mechanism of expression, regulated by many transcription factors and CYP3A5.^[16]

N-AcetyHransferase, NAT

NAT catalyzes acetylation of many drugs, which are divided into two hypotypes, NATI and NAT2. NATI catalyzes acetylation of aminosalicylic acid and PABA, while NAT2 participates in the metabolism of more than 20 categories of hydrazides, carcinogenic arylamine and heterocyclic amine. The human NAT2 gene is located on 1 lq8 chromosome, and the wildtype allele is called NAT2*4. At present, at least 15 species of NAT2 mutation alleles have been identified.

Glutathione S-transferases, GST

GSTs promote the conjugaction of reduced glutathione and electrophilic substrates, enhancing elimination of drugs, such as chlorambucil and cyclophosphamide, and correlating with antineoplastic resistance.

Polymorphisms in Drug Transporters

P-glycoprotein (PGP), encoded by the MDR1 gene (ABCB1), is the best-characterized ATP-binding cassette (ABC) transporter, which can transport a large range of drugs out of the cell (lipophilic drugs, e.g ADM, MMC, VCR and Vp-16), decreasing intracellular drug concentration and thereby weakening the cytotoxic action of the drug. At least 29 MDR1 gene polymorphisms have been found. Among these, 2 SNPs —G2677T/A of extron 21 and C3435T of extron 26, have been widely investigated, showing that the two polymorphisms contribute to down regulating P-glycoprotein expression. The principal polymorphism of ABCG2, 421C→A (Q141K), in Chinese and Japanese has a 3.1% gene frequency, and is concerned with metabolism of irinotecan.

Thymidyiate synthase, TS

TS fonctions as a critical enzyme in the de novo synthesis of thymidyiate. Therefore this enzyme is a target of many antineoplastic agents such as 5-FU and AL-IMTA. Using a methylene carbon from 5,10-MTHF, TS catalyzes the formation of dTMP from dUMP and then dUMP is converted to thymidine triphosphate, which is required for DNA synthesis and repair.

TYMS, the coding gene for TS, located on 18p, displays genetic polymorphism. A polymorphism of a 28-bp sequence in the 5’ promotor enhancer region of the TS gene, termed thymidylate-synthase promoter-enhancer region (TSER), is correlated with the regulation of TS expression. In all ethnic populations, 2 and 3 tandem repeats of this 28-bp sequence are predominant (TSER*2, TSER*3). TS mRNA and protein are expressed more in the TSER*3 allele. A study reported that a G>C variation within the tandem repeats of the TSER*3 genotype affected the transcriptional activation of TYMS.^[17] TSER*2 homozygotes respond better to 5-FU based chemotherapy and have a better prognosis than TSER*2/*3 heterozygotes and TSER*3 homozygotes do, but develop toxic reactions more readily. Lecomte et al.^[18] reported that in colorectal cancer patients who underwent adjuvant chemotherapy based on 5-FU, the frequency of a 3~4 grade toxic reaction was 43, 18 and 3%, respectively in patients with the 2R/2R, 2R/3R and 3R/3R genotypes (P<0.01).

One study of 121 cases of colorectal cancer patients found that TSER*3 isozygotes accounted for 29%, TSER*2 isozygotes 16%, and heterozygotes 55%. Patients who were TSER*2 isozygotes obtained longer survival than those who were TSER*3 isozygotes.^[19] Another investigation of 211 Duke’s C colon carcinoma patients showed that 29% of the subjects were TSER*3 isozygotes and insensitive to chemotherapy, whereas TSER*2 isozygotes and heterozygotes received longer survival after chemotherapy(R=0.005).^[20] The same conclusion was reached following capecitabine chemotherapy. ^[21] Accordingly, before treating progressive colon cancer patients, expression of TS should be evaluated in order to select an adequate drug regimen. People with a lower expression of TS would more sensitive to 5-FU than those with high expression. Patients with the high expression of both TS and DPD are insensitive to 5-FU, and therefore should be treated with other drugs such as irinotecan or oxaliplatin.^[22] Another polymorphism occurs as a 6bp deletion in the 3’ untranslated region, 447 bp upstream from the stop codon.^[23] This polymorphism was shown to enable one to predict a response to 5-FU containing combination therapy of colorectal carcinomas cases.

TSER is variable among races, as TSER*2 occurs in Chinese at a lower frequency than in Europeans, Americans and South-Asians. Therefore pharmacogenetic polymorphism should be viewed more carefully when coming to conclusions based on foreign clinical trials.^[24]

Polymorphisms in DNA repairase

Nucleotide excision repair (NER) serves to repair DNA after undergoing damage, and decreases the tumor drug sensitivity. The general process occurs as follows: at the beginning of the repair, XPC binds with HR23B recogniting the DNA damage, and then, XPD works as an evolutionary conserved DNA unwindase, participating in nucleotide excision repair and genetic transcription, while ERCC1 removes the impaired nucleotide promptly and efficiently. Polymorphism of XPC results from an AT nucleotide insertion or deletion in intron 9, C751A (Lys→Gln) of the XPD gene and is associated with the functional change of nucleotide excision repair.^[25] XRCC1 is an important element of the base excision repair and single-strand break restoration systems of which the polymorphism is caused by a 194 codon Arg→Trp mutation.