**3. Results and discussion**

diseases (diabetes, uncontrolled hypertension, myocardial infarction within the last 6 months, etc.), and clinically significant hearing impairment (grade 2 or higher). To ascertain ethnicity, women completed a questionnaire about their ancestry; only self-described Russian and Yakut patients with no history of interethnic marriages in the past two generations were recruited. Before the initiation of chemotherapy, venous blood samples were obtained for

chemotherapy and radiotherapy were not allowed. Toxicity of the treatment was described according to standard National Cancer Institute Common Toxicity Criteria version 2.0 [23]. All patients were assessed for the maximal grades of nephrotoxicity, ototoxicity, neurotoxic-

The tumor response was assessed every 2 cycles. After the completion of chemotherapy, the patients were followed-up for disease relapse and survival. Patients with progressive disease were treated with second-line chemotherapy, mostly taxane based. The study protocol and informed consent form were approved by the Ethics Committee of the N. N. Blokhin Cancer

DNA was isolated from the venous blood samples (leukocytes) using a conventional approach including proteinase K treatment with subsequent phenol-chloroform extraction [24]. Some polymorphisms (**Table 1**) were genotyped using a polymerase chain reaction restriction fragment length polymorphism (RFLP)-based technique or determined directly through evalua-

Other polymorphisms were genotyped using a microarray "DNA repair single nucleotide polymorphism detection test" (version 2, Asper Biotech, Tartu, Estonia). The microarray gen-

biotic metabolism. Most of manually genotyped loci were also in the list of the microarray's polymorphisms and served as controls of genotyping efficiency. To check into account the potential mistakes in genotyping with the microarray [25], all polymorphisms, which were associated with any clinical endpoints, were additionally tested using the RFLP method.

the relationship between the variables and alleles/genotypes tested. Correlations between survival and genotype or genetic polymorphism were assessed using the Kaplan-Meier product limit method and the log-rank test. The significance of associations was set at P < 0.05 [26]. The statistical analyses were performed using the Statistica software (version 6.0, StatSoft, Inc., Tulsa, OK, USA) or the IBM SPSS Statistics software package (version 19, SPSS, Inc., IBM Company, IBM Corporation, Armonk, NY, USA), GraphPadInStat (version 3.00, GraphPad

Software, San Diego, CA, USA), and the PowerMarker software (version 3.0) [27].

) on day 1, every 3 weeks, for a maximum of 6 cycles. Intraperitoneal

DNA repair, cell cycle control, apoptosis, and xeno-

test were used to determine

) plus cyclo-

genetic testing. The chemotherapy regimen was intravenous cisplatin (100 mg/m2

ity, emesis, neutropenia, anemia, and thrombocytopenia.

phosphamide (600 mg/m2

312 Ovarian Cancer - From Pathogenesis to Treatment

Research Centre.

**2.2. Genotyping**

tion of their PCR product lengths.

**2.3. Statistical analysis**

1

otypes 228 SNPs in 106 genes involved in1

Alternative variant – "genes which are involved in".

A permutation exact test, a two-sided Fisher exact test, and a χ2

During 2003–2007 years, 104 Russian patients and 87 Yakut patients were enrolled in the study. The median age of patients was 52 and 51 years, respectively. The majority of patients in both groups had stage III disease (72 and 53 women, respectively). Stages I, II, and IV were detected in 14, 6, and 10 Russian patients and 1, 8, and 25 Yakut patients. A total of 21 Russian patients and 11 Yakut patients were receiving adjuvant chemotherapy and were not eligible for evaluation of tumor response (i.e., they had no residual disease after surgery). Overall response rates, comprising complete and partial responders, were 85% in the Russian group and 58% in the Yakut group.

The median progression-free survival (PFS) in the Russian group was 12 months, and the median overall survival (OS) was 55 months. In the Yakut group, both intervals were shorter —8 and 29 months, respectively. However, being adjusted for disease stage, the values became similar to those for the Russian group.

*CYP2E1* genotypes, respectively. Patients with a homozygous *GSTT1* gene deletion (*GSTT1* null) suffered more frequently from nephrotoxicity than carriers of functional *GSTT1* variants (OR = 3.31, 95% CI 1.15–9.54, P = 0.028). Patients who had a 96 bp insertion in the promoter region of the *CYP2E1* were more prone to severe emesis (OR = 4.69, 95% CI 1.31–16.77, P = 0.027). Grade 3 or 4 emesis was also associated with another *CYP2E1* polymorphism—a single nucleotide substitution (9896 C/G) in intron 7. Patients with a heterozygous CG genotype had a higher risk of severe emesis than patients with the homozygous CC genotype (OR = 16.96, 95% CI 2.01–143.16, P = 0.002). In contrast, in Russian patients, *CYP2E1* polymorphisms were not associated with any clinical outcomes and distribution of *GSTT1* genotypes correlated with severity of emesis. The risk of severe emesis was higher in Russian patients with the *GSTT1*-null genotype than in patients with a functional *GSTT1* variant (OR = 4.06, 95% CI 1.40–11.78; P = 0.014). As for the nephrotoxicity in Russian subjects, it was associated with *ERCC1* 19007 T/C or 8092 C/A polymorphisms, and cases of renal dysfunction were more prevalent among patients with the heterozygous genotypes of each locus. Other found genotype-clinical end point associations were also discordant in Russians and Yakuts (**Table 2**). When genotype distributions in both groups were compared significant differences in population genotype frequencies were noted for 10 of 19 polymorphisms studied but only 5 of

Ethnic Differences in Susceptibility to the Effects of Platinum-Based Chemotherapy

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315

Evaluation of the direction/strength of the associations showed that they were not correlated with the differences in population frequencies of corresponding genotypes. For example, the absence of correlation between the *GSTP1* Ile105Val polymorphism and survival in Yakut patients was not simply due to the higher prevalence of Ile/Ile genotype among them because carriers of Ile/Ile genotype did not differ in PFS or OS from those who had genotypes with 1 or 2 Val alleles. Similar results were obtained for the *GSTA1*-69 C/T polymorphism. Its CT and TT genotypes were associated with a risk of anemia in Yakuts; yet the greater frequencies of the CT and TT genotypes in Russians did not result in a higher risk of anemia. Similarly, risk genotypes of *ERCC1* 19007 T/C and ERCC2 Asp312Asn polymorphisms (i.e., heterozygous genotypes in Russians) were not rare among Yakut patients but they did not demonstrate correlations with the corresponding side effects (**Tables 1** and **2**). It seems that only in the case of *CYP2E1* 96 bp insertion polymorphism an effect of allele frequency differences could be proposed but due to small number of individuals with the insertion containing genotypes in Russians the suggestion

Taken in the context of other data, the obtained results generally supported the role of ethnicity as an additional reason for differences in the outcomes of clinical trials in which the same treatment is used. At the same time, the results of genetic testing suggested that a single genotypic difference was unlikely to account for the observed ethnic variation in toxicity and survival. Moreover, they also suggested that assessing traditionally tested common polymorphisms in GST and DNA repair genes is not enough for relevant description of lesser tolerability of Asians (North/East Asians) to the effects of platinum-containing chemotherapies

In the second part of our study, we systematically investigated the associations between patients' outcomes and SNPs in more than 100 genes using the microarray "DNA repair single nucleotide polymorphism detection test" (version 2) [36, 37]. Like similar genotyping panels, the list of genes tested comprised candidate genes involved in key pathways of cellular

and further studies involving more polymorphic markers are required.

them were associated with clinical outcomes (**Tables 1** and **2**).

requires verification in a larger sample.

More contrast results were obtained in analysis of occurrence of adverse events. To assess the association between genotype and the toxicity of the treatment used, patients were classified as having good or poor tolerance to treatment (grades 3–4 of neutropenia, grades 2–4 of anemia, grades 2–4 of neuropathy, grades 3–4 of emesis, all grade of thrombocytopenia, nephrotoxicity, and ototoxicity were considered as clinically significant toxicities). Comparison of the frequencies of side effects registered in Russian and Yakut patients confirmed higher toxicity of platinum-based regimens for patients of Asian origin than for Europeans. Particularly, Yakut patients suffered more frequently than Russians from nephrotoxicity and severe emesis (P = 0.027 and P = 0.061, respectively), which both were known to be the most common adverse events observed in regimens using cisplatin [28].

Genetic testing of the patients from our groups was performed in two stages. At first, we explored the associations between outcomes of a cisplatin-cyclophosphamide regimen in Russian and Yakut ovarian cancer patients and some most common polymorphisms in several genes [21, 22], among the tested genes, were glutathione S-transferase (GST) genes (*GSTA1, GSTM1, GSTM3, GSTP1* and *GSTT1*), DNA repair genes (*ERCC1, ERCC2* and *XRCC1*) as well as *TP53* and *CYP2E1* genes (**Table 1**). GST and DNA repair genes have been described as important for cisplatin metabolism and activity [29, 30]. GSTs can directly limit the amount of reactive cisplatin species available for interaction with DNA, by catalyzing their binding to tripeptide glutathione. The resulting cisplatin-glutathione conjugates can be further easily excreted from the cell by the GS-X pump transporters [31]. DNA repair proteins remove platinum-DNA adducts, the persistence of which underpins the antitumor potential of platinum drugs. In contrast to GST and the DNA repair proteins, TP53 protein does not seem to directly affect cisplatin metabolism or transformation. At the same time, it has a crucial role in mediating cellular responses to DNA damage, initiating programmed cell death when the effective DNA repair is impossible [29, 30, 32]. There is also no evidence that cisplatin is metabolized by CYP2E1. However, CYP2E1 is a significant potential source of catalytic iron and can serve as a site for generating reactive oxygen species in the presence of cisplatin [33]. It has been proposed that this mechanism underlies cisplatin-induced nephrotoxicity and hepatotoxicity [34, 35].

One of the most significant associations found in the first part of the study was the correlation between the survival time intervals and *GSTP1* Ile105Val polymorphism registered in Russian subjects (P = 0.004 and P = 0.016 for PFS and OS, respectively). Russian ovarian cancer patients with a homozygous Ile/Ile genotype had longer PFS and OS than those ones who carried Ile/ Val and Val/Val genotypes. However, the association was not observed in Yakut patients. PFS and OS of Yakut women with Ile/Ile genotype did not differ from the corresponding time intervals of patients with 1 or 2 Val alleles. In the Yakut group, PFS correlated with *CYP2E1* 7632 T/A polymorphism (P = 0.015), being longer in patients with a homozygous TT genotype than in patients with the heterozygous TA genotype.

Analysis of genotype distribution in the population groups for toxicities revealed that occurrence of nephrotoxicity and severe emesis in Yakut patients correlated with *GSTT1* and *CYP2E1* genotypes, respectively. Patients with a homozygous *GSTT1* gene deletion (*GSTT1* null) suffered more frequently from nephrotoxicity than carriers of functional *GSTT1* variants (OR = 3.31, 95% CI 1.15–9.54, P = 0.028). Patients who had a 96 bp insertion in the promoter region of the *CYP2E1* were more prone to severe emesis (OR = 4.69, 95% CI 1.31–16.77, P = 0.027). Grade 3 or 4 emesis was also associated with another *CYP2E1* polymorphism—a single nucleotide substitution (9896 C/G) in intron 7. Patients with a heterozygous CG genotype had a higher risk of severe emesis than patients with the homozygous CC genotype (OR = 16.96, 95% CI 2.01–143.16, P = 0.002). In contrast, in Russian patients, *CYP2E1* polymorphisms were not associated with any clinical outcomes and distribution of *GSTT1* genotypes correlated with severity of emesis. The risk of severe emesis was higher in Russian patients with the *GSTT1*-null genotype than in patients with a functional *GSTT1* variant (OR = 4.06, 95% CI 1.40–11.78; P = 0.014). As for the nephrotoxicity in Russian subjects, it was associated with *ERCC1* 19007 T/C or 8092 C/A polymorphisms, and cases of renal dysfunction were more prevalent among patients with the heterozygous genotypes of each locus. Other found genotype-clinical end point associations were also discordant in Russians and Yakuts (**Table 2**).

The median progression-free survival (PFS) in the Russian group was 12 months, and the median overall survival (OS) was 55 months. In the Yakut group, both intervals were shorter —8 and 29 months, respectively. However, being adjusted for disease stage, the values became

More contrast results were obtained in analysis of occurrence of adverse events. To assess the association between genotype and the toxicity of the treatment used, patients were classified as having good or poor tolerance to treatment (grades 3–4 of neutropenia, grades 2–4 of anemia, grades 2–4 of neuropathy, grades 3–4 of emesis, all grade of thrombocytopenia, nephrotoxicity, and ototoxicity were considered as clinically significant toxicities). Comparison of the frequencies of side effects registered in Russian and Yakut patients confirmed higher toxicity of platinum-based regimens for patients of Asian origin than for Europeans. Particularly, Yakut patients suffered more frequently than Russians from nephrotoxicity and severe emesis (P = 0.027 and P = 0.061, respectively), which both were known to be the most common

Genetic testing of the patients from our groups was performed in two stages. At first, we explored the associations between outcomes of a cisplatin-cyclophosphamide regimen in Russian and Yakut ovarian cancer patients and some most common polymorphisms in several genes [21, 22], among the tested genes, were glutathione S-transferase (GST) genes (*GSTA1, GSTM1, GSTM3, GSTP1* and *GSTT1*), DNA repair genes (*ERCC1, ERCC2* and *XRCC1*) as well as *TP53* and *CYP2E1* genes (**Table 1**). GST and DNA repair genes have been described as important for cisplatin metabolism and activity [29, 30]. GSTs can directly limit the amount of reactive cisplatin species available for interaction with DNA, by catalyzing their binding to tripeptide glutathione. The resulting cisplatin-glutathione conjugates can be further easily excreted from the cell by the GS-X pump transporters [31]. DNA repair proteins remove platinum-DNA adducts, the persistence of which underpins the antitumor potential of platinum drugs. In contrast to GST and the DNA repair proteins, TP53 protein does not seem to directly affect cisplatin metabolism or transformation. At the same time, it has a crucial role in mediating cellular responses to DNA damage, initiating programmed cell death when the effective DNA repair is impossible [29, 30, 32]. There is also no evidence that cisplatin is metabolized by CYP2E1. However, CYP2E1 is a significant potential source of catalytic iron and can serve as a site for generating reactive oxygen species in the presence of cisplatin [33]. It has been proposed that this mechanism underlies cisplatin-induced nephrotoxicity and

One of the most significant associations found in the first part of the study was the correlation between the survival time intervals and *GSTP1* Ile105Val polymorphism registered in Russian subjects (P = 0.004 and P = 0.016 for PFS and OS, respectively). Russian ovarian cancer patients with a homozygous Ile/Ile genotype had longer PFS and OS than those ones who carried Ile/ Val and Val/Val genotypes. However, the association was not observed in Yakut patients. PFS and OS of Yakut women with Ile/Ile genotype did not differ from the corresponding time intervals of patients with 1 or 2 Val alleles. In the Yakut group, PFS correlated with *CYP2E1* 7632 T/A polymorphism (P = 0.015), being longer in patients with a homozygous TT genotype

Analysis of genotype distribution in the population groups for toxicities revealed that occurrence of nephrotoxicity and severe emesis in Yakut patients correlated with *GSTT1* and

similar to those for the Russian group.

314 Ovarian Cancer - From Pathogenesis to Treatment

hepatotoxicity [34, 35].

adverse events observed in regimens using cisplatin [28].

than in patients with the heterozygous TA genotype.

When genotype distributions in both groups were compared significant differences in population genotype frequencies were noted for 10 of 19 polymorphisms studied but only 5 of them were associated with clinical outcomes (**Tables 1** and **2**).

Evaluation of the direction/strength of the associations showed that they were not correlated with the differences in population frequencies of corresponding genotypes. For example, the absence of correlation between the *GSTP1* Ile105Val polymorphism and survival in Yakut patients was not simply due to the higher prevalence of Ile/Ile genotype among them because carriers of Ile/Ile genotype did not differ in PFS or OS from those who had genotypes with 1 or 2 Val alleles. Similar results were obtained for the *GSTA1*-69 C/T polymorphism. Its CT and TT genotypes were associated with a risk of anemia in Yakuts; yet the greater frequencies of the CT and TT genotypes in Russians did not result in a higher risk of anemia. Similarly, risk genotypes of *ERCC1* 19007 T/C and ERCC2 Asp312Asn polymorphisms (i.e., heterozygous genotypes in Russians) were not rare among Yakut patients but they did not demonstrate correlations with the corresponding side effects (**Tables 1** and **2**). It seems that only in the case of *CYP2E1* 96 bp insertion polymorphism an effect of allele frequency differences could be proposed but due to small number of individuals with the insertion containing genotypes in Russians the suggestion requires verification in a larger sample.

Taken in the context of other data, the obtained results generally supported the role of ethnicity as an additional reason for differences in the outcomes of clinical trials in which the same treatment is used. At the same time, the results of genetic testing suggested that a single genotypic difference was unlikely to account for the observed ethnic variation in toxicity and survival. Moreover, they also suggested that assessing traditionally tested common polymorphisms in GST and DNA repair genes is not enough for relevant description of lesser tolerability of Asians (North/East Asians) to the effects of platinum-containing chemotherapies and further studies involving more polymorphic markers are required.

In the second part of our study, we systematically investigated the associations between patients' outcomes and SNPs in more than 100 genes using the microarray "DNA repair single nucleotide polymorphism detection test" (version 2) [36, 37]. Like similar genotyping panels, the list of genes tested comprised candidate genes involved in key pathways of cellular


response to different drugs [38, 39], including many genes that are related to the cisplatin pathway (platinum pathway) [40]. A total of 213 SNPs from 228 genotyped SNPs were new (i.e., they did not include SNPs from the first stage) and 27 SNPs were associated with one or

Increasing number of polymorphisms yielded an association with tumor response. To assess the association, patients who achieved a complete remission were compared with those with

out it (i.e., patients with partial response, stable and progressive disease). In the Russian group, a significant difference in complete response was observed according to polymorphism in

plete response was higher among carriers of homozygous genotypes AA and GG compared with patients with a heterozygous variant AG. In Yakut patients, the occurrence of complete response was correlated with an allelic status of SNP in *CDKN1B* gene (T/C, rs34330), particu

larly with an allele C. There were no cases of complete remission among patients who carried a homozygous genotype TT at rs34330. The SNP was also associated with PFS in Yakut sub

genotypes. The protein encoded by *CDKN1B* gene participates in regulation of the cell cycle by binding and inhibiting activation of cyclin E-CDK2 or cyclin D-CDK4 complexes, and thus

decreasing the levels of mRNA and CDKN1B protein [41]. Reduced level of *CDKN1B* expres

sion has been associated with a poor outcome in various cancers [42, 43]. In contrast, the *ADH1C* SNP (rs698) has been associated with a risk of alcoholism and ethanol-related cancers [44], but our study was the first to demonstrate an association with a chemotherapy outcome

Genotypes of seven SNPs were associated with differences in PFS in the Russian group (**Table 3**).

× 10−6 and P

was seen in patients with the most frequent genotypes AA and CC. SNPs in *NQO1* demon

Thiopurine S-methyltransferase (TPMT) is a cytosolic methylating enzyme with unknown physiological role [45]. However, this enzyme is known to be able to catalyze the S-methylation of some aromatic and heterocyclic compounds, particularly thio-compounds (e.g., 6-mercap

topurine and 6-thioguanine). Discussing the associations revealed between SNPs in *TPMT* and cisplatin ototoxicity, Ross et al. [46] have hypothesized that TPMT can affect cisplatininduced hearing impairment through inactivation of cisplatin-purine compounds that form cytotoxic DNA cross-links, and cause cell death. As might be expected from that data, those patients in our study who had the loss-of-function genotype AG at rs1142345 should demon

strate a better outcome as a result of decreased inactivation of cisplatin-purine compounds, but such a correlation was not observed. Moreover, patients with an AG genotype had even

The tested SNPs in *NQO1* gene are characteristic polymorphic variants affecting functional ability of the corresponding protein — a cytosolic flavoenzyme NAD(P)H: quinone oxidore

ductase 1 (NQO1). *NQO1* polymorphic status has been associated with anticancer chemotherapy

= 9

= 2

shorter PFS than those who carried the functionally normal AA genotype.

= 0.0051); patients with the genotype TT had shorter PFS than patients with CC and CT

= 0.0002). The proportion of patients who achieved a com

Ethnic Differences in Susceptibility to the Effects of Platinum-Based Chemotherapy

> T substitution at rs34330 locus results in

http://dx.doi.org/10.5772/intechopen.73798

= 3 × 10−8) and rs4986998 (C/T)

× 10−11, respectively). The longer PFS


317









more of the assessed clinical end points (**Table 3**).

blocking the transition of the cell into the S-phase. C

(i.e., tumor response) but the mechanism is not obvious.

and rs1800566 (C/T) in *NQO1* genes (P

strated similar correlations with OS.

The most significant SNPs were rs1142345 (A/G) in *TPMT* (P

the *ADH1C* gene (A/G, rs698) (P

jects (P

response to different drugs [38, 39], including many genes that are related to the cisplatin pathway (platinum pathway) [40]. A total of 213 SNPs from 228 genotyped SNPs were new (i.e., they did not include SNPs from the first stage) and 27 SNPs were associated with one or more of the assessed clinical end points (**Table 3**).

Increasing number of polymorphisms yielded an association with tumor response. To assess the association, patients who achieved a complete remission were compared with those without it (i.e., patients with partial response, stable and progressive disease). In the Russian group, a significant difference in complete response was observed according to polymorphism in the *ADH1C* gene (A/G, rs698) (P = 0.0002). The proportion of patients who achieved a complete response was higher among carriers of homozygous genotypes AA and GG compared with patients with a heterozygous variant AG. In Yakut patients, the occurrence of complete response was correlated with an allelic status of SNP in *CDKN1B* gene (T/C, rs34330), particularly with an allele C. There were no cases of complete remission among patients who carried a homozygous genotype TT at rs34330. The SNP was also associated with PFS in Yakut subjects (P = 0.0051); patients with the genotype TT had shorter PFS than patients with CC and CT genotypes. The protein encoded by *CDKN1B* gene participates in regulation of the cell cycle by binding and inhibiting activation of cyclin E-CDK2 or cyclin D-CDK4 complexes, and thus blocking the transition of the cell into the S-phase. C > T substitution at rs34330 locus results in decreasing the levels of mRNA and CDKN1B protein [41]. Reduced level of *CDKN1B* expression has been associated with a poor outcome in various cancers [42, 43]. In contrast, the *ADH1C* SNP (rs698) has been associated with a risk of alcoholism and ethanol-related cancers [44], but our study was the first to demonstrate an association with a chemotherapy outcome (i.e., tumor response) but the mechanism is not obvious.

Genotypes of seven SNPs were associated with differences in PFS in the Russian group (**Table 3**). The most significant SNPs were rs1142345 (A/G) in *TPMT* (P = 3 × 10−8) and rs4986998 (C/T) and rs1800566 (C/T) in *NQO1* genes (P = 2 × 10−6 and P = 9 × 10−11, respectively). The longer PFS was seen in patients with the most frequent genotypes AA and CC. SNPs in *NQO1* demonstrated similar correlations with OS.

Thiopurine S-methyltransferase (TPMT) is a cytosolic methylating enzyme with unknown physiological role [45]. However, this enzyme is known to be able to catalyze the S-methylation of some aromatic and heterocyclic compounds, particularly thio-compounds (e.g., 6-mercaptopurine and 6-thioguanine). Discussing the associations revealed between SNPs in *TPMT* and cisplatin ototoxicity, Ross et al. [46] have hypothesized that TPMT can affect cisplatininduced hearing impairment through inactivation of cisplatin-purine compounds that form cytotoxic DNA cross-links, and cause cell death. As might be expected from that data, those patients in our study who had the loss-of-function genotype AG at rs1142345 should demonstrate a better outcome as a result of decreased inactivation of cisplatin-purine compounds, but such a correlation was not observed. Moreover, patients with an AG genotype had even shorter PFS than those who carried the functionally normal AA genotype.

The tested SNPs in *NQO1* gene are characteristic polymorphic variants affecting functional ability of the corresponding protein — a cytosolic flavoenzyme NAD(P)H: quinone oxidoreductase 1 (NQO1). *NQO1* polymorphic status has been associated with anticancer chemotherapy

**Gene name**

**#rs ID**

**Clinical outcomes\*\***

**PFS**

**R**

*GSTA1* *GSTM1* *GSTM3* *GSTM3*

*GSTP1* *GSTP1* *GSTT1* *ERCC1* *ERCC1* *ERCC2* *ERCC2* *XRCC1* *XRCC1* *XRCC1*

*TP53* *CYP2E1* *CYP2E1* *CYP2E1* *CYP2E1* **Table 2.**

rs2070676 \*The registered associations are indicated by "+" in the corresponding cells.

\*\*Tumor response and ototoxicity were not included in the table as there were no associations between them and polymorphisms tested.

The associations between polymorphisms and clinical outcomes observed in Russian (R) and Yakut (Y) patient groups (the first stage of the study)\*

.

rs6413432

+

rs2031920

96bp insertion

rs1042522

rs25487

+

+

+

+

rs25489

rs1799782

rs13181

rs1799793

rs3212986

rs11615

gene deletion

+

+

+

+

+

rs1138272

rs1695

+

+

rs7483

rs1799735

gene deletion

rs3957357

**Y**

**R**

**Y**

**R**

**Y**

+

+ +

+

+

**R**

**Y**

**R**

**Y**

**R**

**Y**

**R**

**Y**

**R**

**Y**

316 Ovarian Cancer - From Pathogenesis to Treatment

**OS**

**Anemia**

**Neutropenia**

**Thrombocitopenia**

**Nephrotoxicity**

**Ototoxicity**

**Emesis**


**Gene name**

**#rs ID**

**Clinical outcomes**

**Response**

**R**

> *NBN*

*NQO1* *NQO1* *RAD52*

*TPMT*

rs1142345 \*The designations are the same as in **Table 2**.

**Table 3.**

The associations between polymorphisms and clinical outcomes observed in Russian (R) and Yakut (Y) patient groups (the second stage of the study)\*.

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319

+

rs11226

rs4986998

rs1800566

+ +

+

+

+

rs1063045

**Y**

**R**

**Y**

**R**

**Y**

**R**

**Y**

**R**

**Y**

**R**

**Y**

**R**

**Y**

**R**

**Y**

**R**

**Y**

+

**PFS**

**OS**

**Anemia**

**Neutropenia**

**Thrombocitopenia**

**Nephrotoxicity**

**Ototoxicity**

**Emesis**


**Gene name**

**#rs ID**

**Clinical outcomes**

**Response**

**R**

*ADH1C* *ALDH2*

*APEX1* *CCNH* *COMT* *CDKN1B*

*CYP1A1* *CYP1A2*

*DRD2* *EPHX1* *EPHX1* *ERCC5* *ERCC5*

*GRPR* *GSTA4*

*LIG3* *MSH3* *MSH6* *MUTYH* *MUTYH*

*NAT2*

rs1801280

rs3219489

rs3219484

rs1042821

rs26279

rs1052536

rs405729

rs4986946

+

+

+

+

+

+

+

+

rs17655

rs1047768

rs2234922

rs1051740

rs1079597

+

+ +

+

+

+

rs2470890

rs4646903

rs34330

+

+

+

+

rs4633

+

rs2266690

+

rs1048945

rs4646777

+

rs698

+

**Y**

**R**

**Y**

**R**

**Y**

**R**

**Y**

**R**

**Y**

**R**

**Y**

**R**

**Y**

**R**

+

318 Ovarian Cancer - From Pathogenesis to Treatment

**Y**

**R**

**Y**

**PFS**

**OS**

**Anemia**

**Neutropenia**

**Thrombocitopenia**

**Nephrotoxicity**

**Ototoxicity**

**Emesis**

**Table 3.** The associations between polymorphisms and clinical outcomes observed in Russian (R) and Yakut (Y) patient groups (the second stage of the study)\* . outcome (i.e., individuals with CT and TT genotypes at rs1800566 showed reduced survival compared to CC homozygotes) [47, 48]. The same correlations were observed in our study. Taking into account the key role of NQO1 in preventing the formation of reactive semiquinone radicals and generating reactive oxygen species (ROS) via redox cycling, one can propose that the worse survival of carriers of CT and TT genotypes is at least the consequence of a chronically elevated level of ROS, which results in enhanced ROS-mediated DNA damage, increased genetic instability, and further cancer progression [47].

GSTA4 plays an important role for the detoxification of 4-hydroxynonenal [53], a toxic product of lipid peroxidation, increasing immensely under oxidative stress conditions (e.g., overproduction of ROS), including cisplatin treatment [54, 55]. The role of epoxide hydrolases (EPXHs) in cisplatin-induced toxicity appears to be also related to the effects of oxidative stress. However, in contrast to GSTA4, EPXHs role is inhibitory and results from the abilities of epoxide hydrolases to metabolize epoxyeicosatrienoic acids (EETs) possessing multiple functions, particularly anti-inflammatory effects. The data about relationships between EET hydrolysis and cisplatin toxicity have been mainly obtained from the studies of cisplatin nephrotoxicity [56, 57]. It has been shown that the anti-inflammatory effect of EETs substantially depends on EPHX2, a cytosolic partner of EPHX1. EPHX1 also accepts EETs, although generally to a much lesser extent than EPHX2 [58]. Therefore, a role for EPHX1 in cisplatin toxicity should not be excluded, particularly because of its high expression in kidneys. The association between the SNP rs1051740 in *EPHX1* and nephrotoxicity of the regimen used supports this suggestion.

Ethnic Differences in Susceptibility to the Effects of Platinum-Based Chemotherapy

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Three SNPs in *APEX1* (rs1048945), *MSH3* (rs26279), and *MSH6* (rs1042821) were associated with ototoxicity, thrombocytopenia, and emesis in Russian patients (**Table 3**). *APEX1* gene encodes apurinic/apyrimidinic endodeoxyribonuclease 1 playing an essential role in the DNA base excision repair pathway, where it removes apurinic/apyrimidinic sites produced during the repair of bases modified by ROS, alkylating agents, or ionizing radiation [59]. High *APEX1* expression has been associated with a poor outcome for chemoradiotherapy, poor complete response rate, shorter local relapse-free interval, poorer survival, and high angiogenesis [59]. At the same time, a role for APEX1 in protection against toxicity, particularly neurotoxicity, induced by ionizing radiation, and cisplatin treatment, has also been demonstrated [60–62]. ROS and oxidative DNA damage induced by them were shown to be important components of the deleterious effects of cisplatin on neuronal cells. Taking into account the proposed role of ROS in the mechanism of

cisplatin-induced hearing loss [63], a contribution of APEX1 can also be hypothesized.

The protein products of *MSH3* and *MSH6* are essential components of the DNA mismatch repair system (MMR). The presence of MMR is thought to be important in mediating cisplatin and carboplatin cytotoxicity, whereas its deficiency, by contrast, may contribute to desensitization of cancer cells to the drugs [64, 65]. In our study, SNPs in *MSH3* and *MSH6* were not associated with tumor response or survival. Nevertheless, patients with minor alleles of the SNPs rs26279 and rs1042821 were at higher risk of thrombocytopenia and emesis, respectively.

Only one gene from the list above was also present among the genes whose polymorphisms were associated with the adverse reactions in Yakut patients, namely, *EPXH1* gene. However, it was associated with a different side effect (i.e., ototoxicity). Furthermore, the corresponding polymorphisms were also different (A/G, rs2234922 and C/G, rs2260863) (**Table 3**). Higher risk of ototoxicity was observed in patients with the most frequent genotypes AA and CC (OR = 26.26, 95% CI 1.502–458.98, P = 0.0005). Although the role of EPXHs in cisplatin toxicity has been mainly associated with their effects on cisplatin-induced kidney injury, the observed link between the *EPHX1* polymorphism and cisplatin ototoxicity can be due to similarity of mechanisms underlying cisplatin-induced hearing impairment and renal dysfunction [66].

The second gene whose allelic variants were associated with a side effect of chemotherapy in Yakut patients (i.e., severe emesis) was *NBN* (G/A, rs1063045). The risk factor for the development

In addition to *CDKN1B*, SNPs in *CYP1A1* (T/C, rs4646903) and *CYP1A2* (C/T, rs2470890) were found to also be associated with survival, particularly with OS, in Yakut patients (P = 0.007 and P = 0.0072, respectively). In each case the longer OS occurred in patients with homozygous genotypes TT. In contrast to TPMT and NQO1, the role of CYP1A1 and CYP1A2 in cisplatin metabolism or toxicity is difficult to discern. At the same time, it cannot be excluded that the observed associations are related to metabolic pathways of drugs used in the second and subsequent lines of the chemotherapy (taxanes and anthracyclines).

Totally, 16 SNPs were associated with the side effects of chemotherapy. Thirteen such SNPs were revealed in the Russian group and three SNPs in Yakuts (**Table 3**). A total of 6 of 13 SNPs were associated with an incidence of severe neutropenia in Russian patients. A strong association was estimated for SNP rs1052536 in *LIG3*. Carriers of its homozygous genotype CC had more than 20-fold higher risk of grade 3 or 4 neutropenia than patients with other genotypes (OR = 23.211, 95% CI =2.976–181.02, P = 2 × 10−6). However, the most significant was SNP rs3219484 in *MUTYH*. The SNP was represented by only two genotypes, and patients who carried a heterozygous genotype AG had very low (actually unobserved) risk of developing severe neutropenia (OR = 0.013, 95% CI 0.000–0.220, P = 4 × 10−8). *MUTYH* encodes a DNA glycosylase involved in repair of oxidatively damaged DNA, in particular by excising adenines misincorporated opposite 7,8-dihydro-8-oxoguanines. Such mispairs are promutagenic, and if left unrepaired before the next round of replication, they can give rise to CG → AT transversion mutations [49]. The observed association may reflect the substantial contribution of oxidative stress (i.e., ROS) into cisplatin-induced cytotoxicity. On the other hand, an associative grouping of *MUTYH* SNPs together with SNPs from other DNA repair genes, particularly *RAD52* and *ERCC5*, may also indicate a role of MUTYH in the repair of cisplatin-produced DNA lesions (e.g., participation in detection of the lesions) [50].

Another side effect for which multiple associations were found in the Russian group was anemia. SNPs in *NAT2*, *GSTA4*, *CCNH*, and *EPHX1* genes were associated with the toxicity (**Table 3**). The most significant was SNP rs1801280 in *NAT2*. Cases of anemia occurred more frequently among patients with the heterozygous CT genotypes (78.4%) compared with the homozygous variants (OR = 5.945, 95% CI 2.351–15.031, P = 0.00009). This association is of particular interest because there is no information about a role of NAT2 in the metabolism or toxicity of cisplatin or cyclophosphamide (the second drug in our chemotherapy regimen) [51]. One can propose that the association found is due to linkage between the SNP with a functional SNP(s) in other gene(s). Unlike *NAT2*, three other genes (i.e., *GSTA4*, *CCNH*, and *EPHX1*) are more relevant to the effects of intracellular processing of cisplatin. The cyclin encoded by *CCNH* is a part of a TFIIH complex that is an essential component of a nucleotide excision repair pathway, widely accepted as a main player in removing platinum-DNA adducts from DNA molecules [52]. GSTA4 plays an important role for the detoxification of 4-hydroxynonenal [53], a toxic product of lipid peroxidation, increasing immensely under oxidative stress conditions (e.g., overproduction of ROS), including cisplatin treatment [54, 55]. The role of epoxide hydrolases (EPXHs) in cisplatin-induced toxicity appears to be also related to the effects of oxidative stress. However, in contrast to GSTA4, EPXHs role is inhibitory and results from the abilities of epoxide hydrolases to metabolize epoxyeicosatrienoic acids (EETs) possessing multiple functions, particularly anti-inflammatory effects. The data about relationships between EET hydrolysis and cisplatin toxicity have been mainly obtained from the studies of cisplatin nephrotoxicity [56, 57]. It has been shown that the anti-inflammatory effect of EETs substantially depends on EPHX2, a cytosolic partner of EPHX1. EPHX1 also accepts EETs, although generally to a much lesser extent than EPHX2 [58]. Therefore, a role for EPHX1 in cisplatin toxicity should not be excluded, particularly because of its high expression in kidneys. The association between the SNP rs1051740 in *EPHX1* and nephrotoxicity of the regimen used supports this suggestion.

outcome (i.e., individuals with CT and TT genotypes at rs1800566 showed reduced survival compared to CC homozygotes) [47, 48]. The same correlations were observed in our study. Taking into account the key role of NQO1 in preventing the formation of reactive semiquinone radicals and generating reactive oxygen species (ROS) via redox cycling, one can propose that the worse survival of carriers of CT and TT genotypes is at least the consequence of a chronically elevated level of ROS, which results in enhanced ROS-mediated DNA damage,

In addition to *CDKN1B*, SNPs in *CYP1A1* (T/C, rs4646903) and *CYP1A2* (C/T, rs2470890) were found to also be associated with survival, particularly with OS, in Yakut patients (P = 0.007 and P = 0.0072, respectively). In each case the longer OS occurred in patients with homozygous genotypes TT. In contrast to TPMT and NQO1, the role of CYP1A1 and CYP1A2 in cisplatin metabolism or toxicity is difficult to discern. At the same time, it cannot be excluded that the observed associations are related to metabolic pathways of drugs used in the second

Totally, 16 SNPs were associated with the side effects of chemotherapy. Thirteen such SNPs were revealed in the Russian group and three SNPs in Yakuts (**Table 3**). A total of 6 of 13 SNPs were associated with an incidence of severe neutropenia in Russian patients. A strong association was estimated for SNP rs1052536 in *LIG3*. Carriers of its homozygous genotype CC had more than 20-fold higher risk of grade 3 or 4 neutropenia than patients with other genotypes (OR = 23.211, 95% CI =2.976–181.02, P = 2 × 10−6). However, the most significant was SNP rs3219484 in *MUTYH*. The SNP was represented by only two genotypes, and patients who carried a heterozygous genotype AG had very low (actually unobserved) risk of developing severe neutropenia (OR = 0.013, 95% CI 0.000–0.220, P = 4 × 10−8). *MUTYH* encodes a DNA glycosylase involved in repair of oxidatively damaged DNA, in particular by excising adenines misincorporated opposite 7,8-dihydro-8-oxoguanines. Such mispairs are promutagenic, and if left unrepaired before the next round of replication, they can give rise to CG → AT transversion mutations [49]. The observed association may reflect the substantial contribution of oxidative stress (i.e., ROS) into cisplatin-induced cytotoxicity. On the other hand, an associative grouping of *MUTYH* SNPs together with SNPs from other DNA repair genes, particularly *RAD52* and *ERCC5*, may also indicate a role of MUTYH in the repair of cisplatin-produced

Another side effect for which multiple associations were found in the Russian group was anemia. SNPs in *NAT2*, *GSTA4*, *CCNH*, and *EPHX1* genes were associated with the toxicity (**Table 3**). The most significant was SNP rs1801280 in *NAT2*. Cases of anemia occurred more frequently among patients with the heterozygous CT genotypes (78.4%) compared with the homozygous variants (OR = 5.945, 95% CI 2.351–15.031, P = 0.00009). This association is of particular interest because there is no information about a role of NAT2 in the metabolism or toxicity of cisplatin or cyclophosphamide (the second drug in our chemotherapy regimen) [51]. One can propose that the association found is due to linkage between the SNP with a functional SNP(s) in other gene(s). Unlike *NAT2*, three other genes (i.e., *GSTA4*, *CCNH*, and *EPHX1*) are more relevant to the effects of intracellular processing of cisplatin. The cyclin encoded by *CCNH* is a part of a TFIIH complex that is an essential component of a nucleotide excision repair pathway, widely accepted as a main player in removing platinum-DNA adducts from DNA molecules [52].

increased genetic instability, and further cancer progression [47].

320 Ovarian Cancer - From Pathogenesis to Treatment

and subsequent lines of the chemotherapy (taxanes and anthracyclines).

DNA lesions (e.g., participation in detection of the lesions) [50].

Three SNPs in *APEX1* (rs1048945), *MSH3* (rs26279), and *MSH6* (rs1042821) were associated with ototoxicity, thrombocytopenia, and emesis in Russian patients (**Table 3**). *APEX1* gene encodes apurinic/apyrimidinic endodeoxyribonuclease 1 playing an essential role in the DNA base excision repair pathway, where it removes apurinic/apyrimidinic sites produced during the repair of bases modified by ROS, alkylating agents, or ionizing radiation [59]. High *APEX1* expression has been associated with a poor outcome for chemoradiotherapy, poor complete response rate, shorter local relapse-free interval, poorer survival, and high angiogenesis [59]. At the same time, a role for APEX1 in protection against toxicity, particularly neurotoxicity, induced by ionizing radiation, and cisplatin treatment, has also been demonstrated [60–62]. ROS and oxidative DNA damage induced by them were shown to be important components of the deleterious effects of cisplatin on neuronal cells. Taking into account the proposed role of ROS in the mechanism of cisplatin-induced hearing loss [63], a contribution of APEX1 can also be hypothesized.

The protein products of *MSH3* and *MSH6* are essential components of the DNA mismatch repair system (MMR). The presence of MMR is thought to be important in mediating cisplatin and carboplatin cytotoxicity, whereas its deficiency, by contrast, may contribute to desensitization of cancer cells to the drugs [64, 65]. In our study, SNPs in *MSH3* and *MSH6* were not associated with tumor response or survival. Nevertheless, patients with minor alleles of the SNPs rs26279 and rs1042821 were at higher risk of thrombocytopenia and emesis, respectively.

Only one gene from the list above was also present among the genes whose polymorphisms were associated with the adverse reactions in Yakut patients, namely, *EPXH1* gene. However, it was associated with a different side effect (i.e., ototoxicity). Furthermore, the corresponding polymorphisms were also different (A/G, rs2234922 and C/G, rs2260863) (**Table 3**). Higher risk of ototoxicity was observed in patients with the most frequent genotypes AA and CC (OR = 26.26, 95% CI 1.502–458.98, P = 0.0005). Although the role of EPXHs in cisplatin toxicity has been mainly associated with their effects on cisplatin-induced kidney injury, the observed link between the *EPHX1* polymorphism and cisplatin ototoxicity can be due to similarity of mechanisms underlying cisplatin-induced hearing impairment and renal dysfunction [66].

The second gene whose allelic variants were associated with a side effect of chemotherapy in Yakut patients (i.e., severe emesis) was *NBN* (G/A, rs1063045). The risk factor for the development of severe emesis in patients was their heterozygous status at the rs1063045 locus. The protein encoded by *NBN* gene is an important component of the system repairing DNA double-strand breaks that can be induced by different environmental and endogenous agents, including cisplatin. The existing data suggest connections between polymorphic variants of the *NBN* gene and the results of cisplatin-based chemotherapy [67].

ROS or ROS-mediated lesions is of particular interest. First, it can point to the higher potential of ROS in total cisplatin-related cytotoxicity [66, 73]. Second, it has been proposed that populations from different geographic regions possess a difference in efficiency of coupling mitochondrial oxidation with phosphorylation, with more heat production and lower ROS generation in North/Northeastern Asians [74, 75]. Consequently, we can expect in Asians lower ability to utilize extra ROS and higher sensitivity to effects of platinum-based drugs. However, because of the relatively small sample sizes and limited number of markers tested,

Ethnic Differences in Susceptibility to the Effects of Platinum-Based Chemotherapy

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323

In summary, comprehensive exploration of genotypes of polymorphisms in more than 100 genes in ovarian cancer patients from Russian and Yakut ethnic groups, receiving cisplatinbased chemotherapy, revealed pronounced differences in associative spectra between them. Taken in the context of absence of correlations between the associations and polymorphic genotype frequencies, the differences suggest a potential for distinct ethnic-related molecular mechanisms determining the sensitivity of patients to platinum drug effects. The mechanisms are thought to be associated with activity of different metabolic enzymes, including those involved in processing the reactive oxygen species. These genetic findings and differential responses to platinum-based chemotherapy between ethnic groups suggest that future genetic testing may be invaluable not only in predicting chemotherapy response but also in deciding the most appropriate chemotherapy regimen. It may be possible to identify in detail the susceptibility differences to chemotherapy sensitivity at the molecular level and harness

This study was supported by grants from the Programs "Fundamental Researches for Development of Biomedical Technologies" and "Molecular and Cell Biology" of the Russian

, Vera Gorbunova3

2 Institute of Modern Information Technologies in Medicine, Russian Academy of Sciences,

1 Department of Molecular Bases of Human Genetics, Institute of Molecular Genetics,

3 Department of Chemotherapy, N.N.Blokhin Cancer Research Centre, Ministry of

and Svetlana Limborska1

Academy of Sciences and Russian Foundation for Basic Research.

\*, Alexey Moisseev2

\*Address all correspondence to: khrunin@img.ras.ru

Healthcare of the Russian Federation, Moscow, Russia

Russian Academy of Sciences, Moscow, Russia

further studies are required to confirm this hypothesis.

this for therapeutic gains.

**Acknowledgements**

**Author details**

Andrey Khrunin1

Moscow, Russia

Intergroup comparison of genotypes generated with the microarrays revealed substantial differences in population frequencies of alleles and genotypes for many polymorphic markers. More than half of all markers differed significantly in the occurrence of their allelic variants in Russian and Yakut patients.

The proportion of significant genotype frequency differences resembled the results obtained in the first part of the study where a smaller number of polymorphic markers was involved (**Table 2**). Furthermore, the results of the comparisons of population-related associative spectra were also the same: there were no identical correlations for any of significant polymorphisms. All associations between the polymorphic markers and clinical outcomes were specific for each of the ethnic group studied.

These findings are generally compatible with the results of the HapMap project studying of the toxicity of platinum compounds (i.e., cisplatin and carboplatin) to lymphoblastoid cell lines from three groups of racially different individuals [19]. One can propose that the failure to detect common associations/commonly associated polymorphisms in our two groups was due to distinctive ethnic-related characteristics in the molecular mechanisms determining the sensitivity of patients to platinum drugs. Hence the difference in platinum drug sensitivity might not exclusively depend on the difference in variant frequencies of given polymorphisms. Another, but not exclusive, explanation of the findings could be a limitation of the number of polymorphisms tested and a possible omission of other potentially important markers. The latest may be mainly due to the misunderstanding of molecular phenotype(s) of the particular drug(s) [68]. The more relevant is the molecular phenotype, the higher is the potential to optimize the use of a particular drug. For some drugs, such as fluorouracil, irinotecan, and mercaptopurine, some relevant variants (i.e., *DPYD\*2A*, *DPYD* 2846T/A, and *TYMS* 2R/3R; *UGT1A1\*28* and *UGT1A1\*6*; *TPMT \*2, TPMT \*3A*, and *TPMT\*3C*) have been established but for other ones, including platinum-containing agents, they are less apparent [68, 69].

The importance of DNA repair, particularly nucleotide excision repair, for platinum cytotoxicity is widely accepted [64]. However, the overall contribution of even the most common genetic variants to predictions of response to platinum-based therapy is not yet well established [70, 71]. In principle, the situation with other "canonical" pathways affecting mainly cisplatin pharmacokinetics could be described the same way [72]. Therefore, the role of additional mechanisms that are not directly related to cisplatin cellular processing has also been proposed [73]. The results of our study overrepresented with the associations with polymorphisms in genes for different metabolic enzymes (*TPMT, NQO1, EPXH1,* etc.) supports the suggestion (the associations would remain significant even if they were adjusted with the Bonferroni method). The abundance of associations with genes involved in processing of ROS or ROS-mediated lesions is of particular interest. First, it can point to the higher potential of ROS in total cisplatin-related cytotoxicity [66, 73]. Second, it has been proposed that populations from different geographic regions possess a difference in efficiency of coupling mitochondrial oxidation with phosphorylation, with more heat production and lower ROS generation in North/Northeastern Asians [74, 75]. Consequently, we can expect in Asians lower ability to utilize extra ROS and higher sensitivity to effects of platinum-based drugs. However, because of the relatively small sample sizes and limited number of markers tested, further studies are required to confirm this hypothesis.

In summary, comprehensive exploration of genotypes of polymorphisms in more than 100 genes in ovarian cancer patients from Russian and Yakut ethnic groups, receiving cisplatinbased chemotherapy, revealed pronounced differences in associative spectra between them. Taken in the context of absence of correlations between the associations and polymorphic genotype frequencies, the differences suggest a potential for distinct ethnic-related molecular mechanisms determining the sensitivity of patients to platinum drug effects. The mechanisms are thought to be associated with activity of different metabolic enzymes, including those involved in processing the reactive oxygen species. These genetic findings and differential responses to platinum-based chemotherapy between ethnic groups suggest that future genetic testing may be invaluable not only in predicting chemotherapy response but also in deciding the most appropriate chemotherapy regimen. It may be possible to identify in detail the susceptibility differences to chemotherapy sensitivity at the molecular level and harness this for therapeutic gains.
