Indian Journal of Cancer Medknow Publications on behalf of Indian Cancer Society ISSN: 0019-509X EISSN: 1998-4774 Vol. 48, Num. 2, 2011, pp. 223-229

Indian Journal of Cancer, Vol. 48, No. 2, April-June, 2011, pp. 223-229

Original Article

Association and treatment response to capecitabine-based chemoradiotherapy with CYP2C9 polymorphism in head and neck cancer

S Paul¹, MC Pant², D Parmar³, J Verma²

¹ Department of Radiotherapy, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India
² Chhatrapati Shahuji Maharaj Medical University (Formerly, King George's Medical University), Lucknow, India
³ Developmental Toxicology and Environmental Epidemiology Division, Indian Institute of Toxicology Research (Council of Scientific and Industrial Research), Lucknow, India

Correspondence Address: S Paul Department of Radiotherapy, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow India sayan_jal@yahoo.com

Code Number: cn11054

DOI: 10.4103/0019-509X.82899

Abstract

Keywords: Chemoradiotherapy, CYP2C9, head and neck squamous cell carcinoma, poor metabolizers, treatment response

Introduction

HNSCC account for approximately 5% of all cancers worldwide. HNSCC is often associated with heavy tobacco and alcohol use. People who use both tobacco and alcohol are at greater risk for developing these cancers. ^[1] Polymorphisms in genes such as cytochrome P450s (CYPs) and glutathione-S-transferases (GSTs), involved in the metabolism and detoxification of alcohol and constituents of tobacco are shown to influence an individual′s susceptibility to cancer. ^[2] Studies including meta- and pooled analysis have shown that polymorphisms in phase I (CYP1A1, CYP2E1) and phase II (GST) enzymes are associated with tobacco induced HNSCC. ^[3]

Mutations in the human genome occur spontaneously at frequencies of about 10 ^-6 per generation due to mis-incorporation or damage of bases during DNA replication. Due to the high conservation of the genetic code through generations, such a mutation can be inherited. However, if a certain mutation is seen in more than 1% of a population group, the variation is considered a polymorphism. Polymorphisms usually occur as point mutations where a single (or a few) base(s) is substituted (substitution), deleted (deletion) or an extra base is inserted (insertion). These point mutations are commonly known as single nucleotide polymorphisms (SNPs). In CYP*2 polymorphism wild type base pair CC(c = cytosine) is replaced either CT(t = thymine) heterozygous mutant type or TT homozygous mutant type. In CYP*3 polymorphism AA(adenine) wild type base pair is replaced by either heterozygous AC or homozygous CC mutant base pair.

CYP2C9 is one of the major drug metabolizing CYPs in human liver and contributes to the metabolism of a number of clinically important drugs. CYP2C9 is also known to be involved in the metabolism of some of the anti-neoplastic drugs such as cyclophosphamide, etoposide, tamoxifen, and ifosfamide. ^[4] A threefold lower intrinsic clearance for cyclophosphamide was observed with recombinant CYP2C9*2 and CYP2C9*3 protein when compared to CYP2C9*1 protein in a yeast expression system. ^[5]

In addition, CYP2C9 enzyme also metabolizes several carcinogenic and mutagenic substrates including heterocyclic aromatic amines and polycyclic aromatic hydrocarbons (PAH). ^[6] It has also been shown that some of the reactions catalyzed by CYP2C9 lead to detoxification of carcinogens. ^[7] Genetic polymorphism has also been reported for CYP2C9. CYP2C9*2 and CYP2C9*3 genotypes account for "poor metabolizer" (PM) phenotype resulting in the slow metabolism of drugs and other substrates metabolized by CYP2C9, and this polymorphism has resulted in generalized poor metabolizing capacity of the individual system that is the person metabolizes all substances poorly as compared to CYP*1 variant. ^[8] Significant differences are known to exist in the distribution of the variant alleles of CYP2C9 in different populations. In general, the polymorphism in CYP2C9 is more frequent in Caucasians compared to Oriental. ^[9] An increased frequency of CYP2C9*2 allele in the patients with lung cancer has been reported. ^[10] Variant alleles of CYP2C9 were reported to increase the risk of distal colorectal adenoma. ^[11]

The present study, therefore, attempted to investigate the association of CYP2C9 genotypes with HNSCC risk and outcome of treatment in the patients receiving chemoradiotherapy.

Materials and Methods

A case control study was conducted. One hundred ten males suffering from locally advanced squammous cell carcinoma visiting the OPD of Radiotherapy Department and equal number of controls were included in the study. The cases had squamous cell carcinoma of the oral cavity or pharynx or larynx, which was confirmed by histopathological examinations and were advised a combination treatment of chemoradiotherapy. All the subjects in the study belonged to the same ethnic group (Indo-European community) of North India. Controls were frequency-matched to cases by year of birth in 5-year classes. Based on medical check-up, controls were not found to suffer from any chronic disease.

The protocol was approved by the human ethics committee of the institution where the patients were registered and it conforms to the provisions of the declaration of Helsinki (1995). Informed consent was obtained from the study subjects for inclusion in the study and before the collection of blood samples, and it was also ensured that the subject anonymity was maintained. All study subjects completed a questionnaire covering medical, residential, and occupational history. Information pertaining to dietary habits, family history of disease, smoking, tobacco chewing, and alcohol consumption. Subjects having regular smoking habits and smoking index (cigarettes/day Χ 365 days) of 730 or more were classified as smokers Likewise, smokeless tobacco dose was estimated as ′′chewing year′′ (i.e., CY= frequency of tobacco chewed. kept/day Χ duration of year). Those who had CY of 365 or more were considered as tobacco chewers. ^[12] Similarly, cumulative exposure to alcohol drinking was derived by multiplying the total yearly consumption of alcohol (in l/year) by the duration of habitual alcohol drinking (in years). Those who had cumulative exposure to alcohol about 90 l were considered as regular alcohol users in our study. ^[13]

DNA isolation and CYP2C9 genotyping was done by the following method: 500 μl blood samples collected in citrate containing tubes from the study subjects were processed for the isolation of genomic DNA whole blood using QIAamp DNA mini kit (Qiagen, CA) following the manufacturers' protocol. For identifying the polymorphism in CYP2C9 (CYP2C9*2 and CYP2C9*3), the reaction mixture in 50 μl contained 1Χ buffer (10 mM Tris-HCl pH 8.3, 1.5-3.0 mM of MgCl ₂ , 25 mM KCl), 200 mM of each nucleotide, 200 nM of each of CYP2C9*2 or 2C9*3 primers, 1.5 unit of Taq polymerase (MBI Fermentas, Germany), 100 ng of genomic DNA and sterile milliQ water and was processed for PCR. Amplified PCR products were digested with AvaII or NsiI (MBI Fermentas, Germany) for identifying CYP2C9*2 and CYP2C9*3 respectively. The products were resolved by 3% agarose gel containing ethidium bromide as described earlier. ^[9]

For quality control, randomly 10% of the samples were selected and re-genotyped to confirm the authenticity of the results obtained earlier, and they were found to be in 100% concordance.

We determined whether genotype or allele frequencies of CYP2C9 polymorphism among the cases and controls were in Hardy Weinberg equilibrium (HWE) using the standard chi-square tests. The association between genetic polymorphisms and risk of HNSCC was estimated by calculating crude odds ratio (OR). A P-value of <0.05 was considered statistically significant. The statistical analysis was performed with the SPSS software package (version 11.0 for Windows; SPSS Chicago, IL).

Patients were treated with concurrent chemotherapy with radiotherapy (CT-RT). CT-RT included administration of capcitabine 500 mg twice a day 5 days a week for 7 weeks along with 70 gray of radiation, 2 gray/fraction, 5 days a week. We have used capcitabine as concurrent chemotherapy as concurrent 5 Fu has been used with success in head and neck cancer. In Budach et al., meta analysis, it has been shown to provide maximum benefit in studies using 5 FU as concurrent chemotherapy. Capcitabine in phase 2 trials has been used successfully as concurrent chemotherapy. ^[14],[15]

So, we have used this regimen as it is convenient to administer in a busy set up like us and we have found better compliance with this regime.

For studying the treatment response, thorough clinical examination and video laryngoscopy was done 1 month following the completion of all treatment. On the basis of WHO criteria, the treatment outcome was divided into the following categories:

1. Complete response (CR): disappearance of all known lesion(s); confirmed at 4 weeks.
2. Partial response (PR): at least 50% decrease; confirmed at 4 weeks.
3. Stable disease (SD): neither PR nor PD criteria met.
4. Progressive disease(PD): 25% increase; no CR, PR or SD documented before increased disease, or new lesion(s)

Those exhibiting CR are categorized as complete responders while patients exhibiting PR or SD/PD are classified as noncomplete-responders. ^[16]

Observation and Results

Two hundred twenty subjects consisting of 110 cases suffering from HNSCC and one hundred ten healthy controls, were genotyped for CYP2C9*2, CYP2C9*3 polymorphism. The distribution of demographic variables and putative risk factors of HNSCC are summarized in [Table - 1].

Cigarette smoking, tobacco chewing, and daily alcohol use was found to be significantly (P < 0.05) higher in patients as compared to controls [Table - 1].

The frequency of heterozygous genotypes (CT) of CYP2C9*2 polymorphism was found to be higher in the cases than the controls. This increase in frequency resulted in an increased OR in cases, which was found to be statistically significant. The frequency of homozygous genotype (TT) in cases (6.7%) when compared to the controls (6.0%) was found to be similar with no significant difference [Table - 2].

The frequency of heterozygous genotypes (AC) of CYP2C9*3 was found to be increased in the cases (20.9%) when compared to the controls (11.32%) that increased the OR (0.34 CI: 1.79-0.36) in the cases which was statistically significant [Table - 3]. The frequency of homozygous mutant genotype (CC) was found to be higher in cases (4.55%) when compared to controls (2.73%) which slightly increased the risk of HNSCC [Table - 3].

The number of individuals with variant genotypes (homozygous and heterozygous) of CYP2C9*2 was significantly increased in cases (39.62%), who were regular tobacco chewers as compared to the controls (13.33%) with similar habit of tobacco chewing. The increase in the frequency resulted in several fold statistically significant increase in amongst the tobacco chewers. As observed with CYP2C9*2, the frequency of individuals who were regular tobacco chewers with variant (homozygous and heterozygous) genotypes of CYP2C9*3 was also increased in cases (33.96%) when compared to the controls (16.66%). The increase in the frequency resulted in increase in the risk [Table - 4]. Cigarette smoking also increased the risk to HNSCC in the cases with CYP2C9 polymorphism when compared to the smokers in the controls [Table - 5]. The frequency of individuals who were regular smokers and carried variant (homozygous and heterozygous) genotypes of CYP2C9*2 (39.68%) was significantly increased in the cases as compared to the controls (12.5%). As observed with CYP2C9*2 variants, the number of cases with variant (homozygous and heterozygous) genotypes of CYP2C9*3 was also increased amongst smokers as compared to the controls.

The frequency of the individuals with variant (homozygous and heterozygous) genotypes of CYP2C9*2 and who were regular alcohol users significantly increased in the cases (42.18%) when compared to the controls (15.38%). This increase is associated with significant increase in the risk to HNSCC in the cases [Table - 6]. As observed with CYP2C9*2 variants, the number of cases with variant (homozygous and heterozygous) genotypes of CYP2C9*3 also increased amongst alcohol users (28.12%) as compared to the controls (7.69%). A statistically significant increase in risk was also observed in cases amongst the alcohol users with variant genotypes of CYP2C9*3 when compared to the alcohol users in the controls [Table - 6].

A follow-up study was also carried out in 94 patients to investigate the effect of treatment on the patients with different genotypes of CYP2C9 [Table - 7]. Sixteen patients were loss to follow up or did not complete treatment. The response to chemo-radiotherapy was based on WHO criteria. Amongst the patients with wild type genotype of CYP2C9 (CYP2C9*1), 73.52% completely responded to the treatment of chemoradiotherapy while 26.47% showed PR or NR. Amongst the patients with variant genotypes of CYP2C9*2, only 32.35% could be categorized as complete responders while 67.64% were found to be PR or non-responders. Likewise, among the PMs with CYP2C9*3 genotypes, only 29.16% Completely responded to the treatment while 70.85% could be categorized as PR or non-responders [Table - 7]. Interestingly, among two cases who carried compound heterozygous genotype of CYP2C9*2/*3 (cases with both the heterozygous i.e., CYP2C9*1/*2 and CYP2C9*1/*3) of CYP2C9, 2 (50%) did not respond to the treatment while only 1 (50%) was found to be complete responder [Table - 7].

Discussion

The data of the present study has shown that functionally important polymorphism of CYP2C9 exists in North Indian population. The frequency of the variant genotypes (CYP2C9*1/*2 and CYP2C9*3) was found to be higher (20% and 14.7%, respectively) than that reported in South Indian (7% and 1%) population. ^[17] This could be partly attributed to the population structure of India comprising a mixture of endogamous ethnic groups. ^[18] The frequency of the CYP2C9*2 genotypes in our control population was higher than that observed in other Asian populations (Chinese and Japanese) but was comparable to the Caucasians. ^[9] It was observed that the CYP2C9*3 polymorphism was more common in North Indian population. The frequency of the variant genotype CYP2C9*3 was relatively higher (14.7%) when compared to the Chinese population (0.01%) and the Caucasians (7%). ^[9]

A relatively higher prevalence of cases with variant genotypes of CYP2C9*2 or *3 have clearly indicated that individuals inheriting PM genotypes of CYP2C9 are at increased risk to develop HNSCC. The association of CYP2C9 polymorphism has not been studied with HNSCC. London et al., in 1996 have reported an increased frequency of CYP2C9*2 allele in the cases suffering from lung cancer. Since CYP2C9 is involved in the detoxification of carcinogenic substrates, the increased risk observed in cases with PM genotypes of CYP2C9 in our study could be attributed to their poor detoxifying ability. ^[11] Our study has indicated that the risk to HNSCC was increased in cases who were tobacco or alcohol users, suggesting that interaction of CYP2C9 genotypes with tobacco or alcohol increases the susceptibility to HNSCC. Though tobacco chewing has been demonstrated to be the major risk factor in HNSCC as compared to cigarette smoking. Polycyclic aromatic hydrocarbons are known to be generated both during cigarette smoking and tobacco chewing. The increased risk observed in cases using tobacco with PM genotypes of CYP2C9 could be explained by higher affinity of CYP2C9 for benzo (a) pyrene. CYP2C9 variant alleles have also been associated with altered metabolism of alkylating agents that are well-established mutagens. ^[19] A similar increase in risk in cases drinking alcohol with CYP2C9 PM genotypes have suggested that alcohol possibly interacts with CYP2C9 genotypes in increasing the risk to HNSCC. Though not much data is available on interaction of CYP2C9 genotypes and alcohol, ethanol is known to inhibit CYP2C9 activity. ^[20] The increase in risk could thus be possibly explained by inhibition of CYP2C9 detoxifying ability in alcohol users. Our study has further shown that chemotherapeutic response is modified in patients with PM genotypes of CYP2C9. A higher percentage of non-responders were observed in the cases with PM genotypes of CYP2C9.

The present study has demonstrated that a several fold increase in the risk to HNSCC in the cases with variant genotypes (PMs) of CYP2C9. The risk is even more in cases who were tobacco or alcohol users. We have come to the conclusion that CYP2C9 genotypes may have a role in interacting with environmental risk factors in modifying the susceptibility to HNSCC. Furthermore, it is also possible that PMs of CYP2C9 can modify the treatment outcome in cases receiving chemoradiotherapy. This is a very preliminary study and with best of our knowledge, this kind of study has never been done before. Further studies with larger sample size may establish this preliminary result in future and the genetic polymorphism of CYP2C9 may play an important role in head and neck cancer susceptibility and treatment response.

References

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