Giuseppe Toffoli

Overexpression of folate binding protein in ovarian cancers

The high affinity folate binding protein (FBP) is overexpressed in ovarian cancers. However, its role in the pathogenesis and biological behaviour of these neoplasms is not clearly understood. Using the monoclonal antibody (MAb) MOv 18 and cytofluorimetric analysis, we investigated FBP expression in frozen neoplastic tissues from 136 patients diagnosed with epithelial ovarian cancer. FBP values were compared with clinico‐pathological characteristics (age, stage, histologic grade, histologic type, DNA ploidy, percentage of S‐phase cells, and previous chemotherapeutic treatment). Some amount of FBP overexpression was observed in 122 of the 136 tumours examined. The overall mean value of FBP fluorescence index (FBP FI) was 5.6 (median 2.7; min 0.8 − max, 78.9). By univariate analysis, FBP FI was overexpressed to a higher degree in ovarian neoplasms with high histologic grade, advanced stage, serous histology, aneuploid status, and high percentage of cells in S‐phase. Of the total number (136) of cases, 106 had all the parameters assessed and were thus selected for stepwise selection procedure. The only significant independent variable was the percentage of S‐phase cells, which accounted for about 31% of variance of FBP FI. Our results indicate that FBP is associated with parameters of biological aggressiveness in ovarian cancers. Int. J. Cancer 74:193‐198, 1997.

The role of UGT1A1*28 polymorphism in the pharmacodynamics and pharmacokinetics of irinotecan in patients with metastatic colorectal cancer

PURPOSE UGT1A1*28 polymorphism has been associated with decreased glucuronidation of SN38, the active metabolite of irinotecan. This could increase toxicity with this agent. PATIENTS AND METHODS In a prospective study, 250 metastatic colorectal cancer patients were treated with irinotecan, fluorouracil, and leucovorin as first-line treatment. UGT1A1*28 polymorphism was investigated with respect to the distribution of hematologic and nonhematologic toxicity, objective response rate, and survival. Pharmacokinetics was investigated in a subgroup of patients (71 of 250) who had been analyzed with respect to toxicity and efficacy. RESULTS UGT1A1*28 polymorphism was associated with a higher risk of grade 3 to 4 hematologic toxicity (odds ratio [OR], 8.63; 95% CI, 1.31 to 56.55), which was only relevant for the first cycle, and was not seen throughout the whole treatment period for patients with both variant alleles TA7/TA7 compared with wild-type TA6/TA6. The response rate was also higher in TA7/TA7 patients (OR, 0.32; 95% CI, 0.12 to 0.86) compared with TA6/TA6. A nonsignificant survival advantage was observed for TA7/TA7 when compared with TA6/TA6 patients (hazard ratio, 0.81; 95% CI, 0.45 to 1.44). Higher response rates were explained by a different pharmacokinetics with higher biliary index [irinotecan area under the curve (AUC)x(SN38 AUC/SN38G AUC)] and lower glucuronidation ratio (SN38G AUC/SN38 AUC) associated with the TA7/TA7 genotype and a higher response rate, indicating that the polymorphism is functionally relevant. CONCLUSION The results indicate that UGT1A1*28 polymorphism is of some relevance to toxicity; however, it is less important than discussed in previous smaller trials. In particular, the possibility of a dose reduction for irinotecan in patients with a UGT1A1*28 polymorphism is not supported by the result of this analysis.

Predictive Role of the UGT1A1, UGT1A7, and UGT1A9 Genetic Variants and Their Haplotypes on the Outcome of Metastatic Colorectal Cancer Patients Treated With Fluorouracil, Leucovorin, and Irinotecan

PURPOSE UGT1A1*28 is considered the main pharmacogenetic predictor of the toxicity outcome of irinotecan-treated patients. We evaluated the effect of other UGT1A variants and haplotypes involved in 7-ethyl-10-hydroxycamptothecin (SN-38) glucuronidation on severe toxicity and efficacy of fluorouracil, leucovorin, and irinotecan (FOLFIRI). PATIENTS AND METHODS In addition to UGT1A1*28, UGT1A1*60, UGT1A1*93, UGT1A7*3, and UGT1A9*22 were genotyped in 250 metastatic colorectal cancer patients, and associations with severe hematologic and nonhematologic toxicity, objective response, time to progression (TTP), and overall survival were evaluated. In a subset of 71 patients, pharmacokinetic data were also available. RESULTS UGT1A7*3 was the only marker of severe hematologic toxicity after the first cycle (odds ratio [OR], 3.94; 95% CI, 1.05 to 14.82; P = .04) in a multivariate analysis. It was also associated with glucuronidation ratio (SN-38G area under the curve [AUC]/SN-38 AUC) and biliary index (irinotecan AUC) x (SN-38 AUC/SN-38G AUC). Haplotype I (all the reference sequence alleles but UGT1A9*22) was a predictor of severe hematologic toxicity during the entire course of therapy (OR, 0.39; 95% CI, 0.19 to 0.82; P = .01), together with sex (OR, 2.08; 95% CI, 1.01 to 4.28; P = .05). In addition to UGT1A1*28, haplotype II (all the variant alleles but UGT1A9*22) was associated with a response rate (OR, 8.61; 95% CI, 1.75 to 42.38; P = .01). UGT1A1*28 was the only marker associated with TTP. CONCLUSION We propose that UGT1A variants additional to UGT1A1*28 might improve the prediction of the outcome of colorectal cancer patients treated with FOLFIRI. A UGT1A haplotype-based approach might be an efficacious strategy to achieve treatment individualization of FOLFIRI.

Expression of folate binding protein as a prognostic factor for response to platinum‐containing chemotherapy and survival in human ovarian cancer

Overexpression of the folate binding protein (FBP) is a common feature in epithelial ovarian cancer, but its prognostic significance is not clearly understood. We investigated whether FBP in epithelial ovarian cancer specimens is a predictor of response to chemotherapy and survival. Between 1990 and 1995, 99 patients with epithelial ovarian cancer underwent primary surgery and were treated with chemotherapeutic regimens including platinum derivatives. First‐line chemotherapy was performed in 58 patients with residual disease and in 41 patients without residual disease after primary laparotomy. FBP expression level was determined in frozen specimens by cyto‐fluorimetric assay using the MOv 18 monoclonal antibody (MAb). Association of FBP fluorescence index (FI) with clinical characteristics, response to chemotherapy, and survival was studied by univariate and multivariate analysis.

Effect of methylenetetrahydrofolate reductase 677C-->T polymorphism on toxicity and homocysteine plasma level after chronic methotrexate treatment of ovarian cancer patients.

MTHFR is a critical enzyme that regulates the metabolism of folate and methionine, both of which are important factors in DNA methylation and synthesis. Subjects with the 677C→T variant have impaired remethylation of Hcy to methionine that could determine hyperhomocysteinemia. Remethylation of Hcy into methionine and DNA methylation are also affected by MTX treatment. Thus, a combined effect between MTX and reduced activity of the MTHFR 677C→T polymorphism could occur, leading to toxicity. In a clinical trial, 43 ovarian cancer patients were treated with low doses of MTX. During MTX therapy, 12 patients (27.9%) developed G3/4 WHO toxicity. In these 12 patients, we observed 6 G3/4 thrombocytopenias, 1 G3 neutropenia, 1 G3 anemia, 9 G3 mucositis cases and 1 G4 mucositis case. A significant association was observed between toxicity and TT MTHFR 677 genotype (p < 0.0001). G3/4 toxicity occurred in 10 of 13 (77%), 1 of 17 (6%) and 1 of 13 (8%) patients with the TT, CT and CC MTHFR genotypes, respectively. According to the logistic regression model, patients with the TT genotype had a relative risk of 42.0 (95% CI 4.2–418.6) of developing G3/4 toxicity compared to patients with the CC and CT genotypes. Patients with the TT genotype had Hcy plasma levels after MTX therapy significantly (p = 0.0001) higher than basal levels (mean ± SD = 16.71 ± 4.72 vs. 12.48 ± 3.57 μmol/l); moreover, they also had higher Hcy plasma levels after MTX than patients with other MTHFR 677 genotypes (CC mean ± SD = 9.87 ± 3.61 μmol/l and CT mean ± SD = 11.48 ± 3.13 μmol/l). Finally, significant associations were observed between G3/4 WHO toxicity and higher Hcy plasma levels after MTX treatment (p = 0.0004). In conclusion, our data suggest that the TT MTHFR 677 genotype is associated with marked MTX‐induced hyperhomocysteinemia and could represent a pharmacogenetic marker for toxicity after chronic treatment with low doses of MTX.

Genotype-Driven Phase I Study of Irinotecan Administered in Combination With Fluorouracil/Leucovorin in Patients With Metastatic Colorectal Cancer

PURPOSE We aimed to identify the maximum-tolerated dose (MTD) of irinotecan in patients with cancer with UGT1A1*1/*1 and *1/*28 genotypes. We hypothesize that the patients without the *28/*28 genotype tolerate higher doses of irinotecan. PATIENTS AND METHODS Patients undergoing first-line treatment for metastatic colorectal cancer (CRC) eligible for treatment with irinotecan plus infusional fluorouracil/leucovorin (FOLFIRI) were screened for the UGT1A1*28/*28 genotype and excluded from the study. Fifty-nine white patients with either the *1/*1 or the *1/*28 genotype were eligible for dose escalation of irinotecan. The starting dose of biweekly irinotecan was 215 mg/m(2) for both genotype groups, whereas the dose of infusional fluorouracil was fixed. Pharmacokinetic data of irinotecan and metabolites were also obtained. Results The dose of irinotecan was escalated to 370 mg/m(2) in patients with the *1/*28 genotype and to 420 mg/m(2) in those with the *1/*1 genotype. Dose-limiting toxicities (DLTs) were observed in two of four of *1/*28 patients at 370 mg/m(2) and in two of three of *1/*1 patients at 420 mg/m(2). No DLTs were observed in 10 *1/*28 patients at 310 mg/m(2) and in 10 *1/*1 patients at 370 mg/m(2); hence these dose levels were the MTD for each genotype group. The most common grade 3 to 4 toxicities were neutropenia and diarrhea. The pharmacokinetics of irinotecan and SN-38 exhibit linear kinetics. CONCLUSION The recommended dose of 180 mg/m(2) for irinotecan in FOLFIRI is considerably lower than the dose that can be tolerated when patients with the UGT1A1*28/*28 genotype are excluded. Prospective genotype-driven studies should test the efficacy of higher irinotecan doses in the FOLFIRI schedule.

C677T and A1298C MTHFR polymorphisms, a challenge for antifolate and fluoropyrimidine-based therapy personalisation

Pharmacogenetics represents an exciting, new promising tool for the individualisation of therapy. Several genetic polymorphisms and haplotypes have been considered in an attempt to optimise therapy with specific drugs but, up to now, their clinical applications remain limited. 5,10-Methylenetetrahydrofolate reductase (MTHFR), a key enzyme of one-carbon metabolism, catalyses the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Two common non-synonymous variants, the C677T (Ala222Val) and A1298C (Glu429Ala), were described for the MTHFR gene and associated with a decreased enzymatic activity and an alteration of intracellular folate distribution. Other MTHFR polymorphisms with marginal impact on enzymatic activity were also reported. Several published clinical studies have investigated the potential predictive role of C677T and A1298C genetic variants on toxicity and efficacy of antifolate and fluoropyrimidine agents, such as methotrexate (MTX), 5-fluorouracil (5-FU) and raltitrexed. Many of these studies show significant associations with MTHFR variants, but others report neither association nor opposite results. A significant interaction between MTHFR polymorphisms and nutrient/environmental factors (i.e. folate status) as well as the ethnicity was reported. Finally, a haplotype approach and the combined analysis of multiple folate pathway gene variants seem to provide a more comprehensive strategy compared to single-locus investigations. The aim of this review is to critically analyse the available data on the importance of MTHFR polymorphisms in modulating the clinical outcome of antifolate and fluoropyrimidine therapies.

Role of folate receptor and reduced folate carrier in the transport of 5‐methyltetrahydrofolic acid in human ovarian carcinoma cells

Folate receptor‐α (FR‐α) is generally over‐expressed in non‐mucinous human ovarian carcinomas. The meaning of FR‐α over‐expression and its role in the 5‐methyltetrahydrofolic acid (N5‐CH3‐H4PteGlu) transport in such tumors is not clear, especially compared with the reduced folate carrier (RFC), the other known folate transporter. In this study, we analyzed molecular FR‐α and RFC expression in 16 ovarian carcinoma tissues and in 5 ovarian carcinoma cell lines using competitive PCR. Co‐expression of the 2 transporters was found both in vivo and in vitro. FR‐α mRNA expression in the cell lines was in good agreement with the corresponding protein expression evaluated by cellular folic acid binding and immunofluorescence analysis, using a specific monoclonal antibody (MAb) (MOv18). Moreover, RFC mRNA expression levels were consistent with the selective cellular binding of N‐hydroxysuccinimide of [3H]‐methotrexate (NHS‐MTX). The 5 ovarian carcinoma cell lines (IGROV‐1, SW‐626, SKOV‐3, OVCAR‐3 and OAW‐42), grown at physiological N5‐CH3‐H4PteGlu concentrations (20 nM) and expressing FR‐α and RFC levels superimposable to those observed in vivo, were used as in vitro cellular model to evaluate the different contribution of FR‐α and RFC to the transport of N5‐CH3‐H4PteGlu. The cytoplasmic N5‐CH3‐[3H]H4PteGlu accumulation observed in each cell line was approximately linear over 4 hr of incubation, but there was no correlation between the rate of folate internalization and FR‐α and RFC expression levels. Furthermore, the selective inhibition of FR‐α and RFC functionality allowed us to distinguish their differential role on the overall N5‐CH3‐[3H]H4PteGlu intracellular delivery. Treatment with the N‐hydroxysuccinimide of folic acid, which blocks FR‐α activity, showed only a partial inhibition (about 20%) of folate internalization in all the cell lines. In contrast, the inhibition of RFC by NHS‐MTX, under conditions that did not affect FR‐α functionality, generally reduced folate accumulation by more than 70%. Only one cell line (IGROV‐1) showed a comparable contribution of the 2 transport systems. Our findings suggest that in ovarian carcinomas, in spite of its over‐expression, FR‐α generally plays a minor role in N5‐CH3‐H4PteGlu transport compared with RFC. Int. J. Cancer 75:125–133, 1998.© 1998 Wiley‐Liss, Inc.

Structure-activity relationship of verapamil analogs and reversal of multidrug resistance

We studied the relationship between the chemical structure and multidrug resistance (MDR) reversal activity of racemic verapamil (VER) and 14 VER analogs (VAs). The LoVo-R human colon carcinoma cell line was used as an experimental model. This cell line exhibited a typical MDR phenotype and overexpressed the MDR1 gene products. Key structural features were identified as being related to MDR reversal and cytotoxic activity. In particular, we demonstrated that the methoxy groups in the VER molecule structure [1.7-Bis-(3.4-dimethoxyphenyl)-3-methylaza-7-cyan-8-methyl-n onane] prevented cytotoxicity when the VAs were used alone, whereas the 7-cyan-8-methyl groups were important for MDR reversal activity and interaction with P-glycoprotein (P-gp). Among the VAs tested, the most active compounds were gallopamil, R-isomer of VER (R-VER), and nor-VER, which potentiated doxorubicin (DOX) cytotoxicity by 52.3 +/- 7.2 (n = 3 +/- SD), 38.9 +/- 6.4 (n = 4 +/- SD), and 35.4 +/- 4.3 (n = 3 +/- SD) times, respectively. The reversal activity of these compounds was similar to that of VER, which enhanced DOX cytotoxicity by 41.3 +/- 5.0 (n = 3 +/- SD) times. The potentiation of DOX cytotoxicity was associated with an increase in DOX uptake in LoVo-R cells and with an increased [3H]azidopine P-gp photolabeling inhibition. Some compounds that had a high reversal potency (i.e. R-VER and nor-VER) showed a lower calcium antagonist activity than VER, and seem useful candidates for the treatment of MDR in cancer patients.

Tumor response is predicted by patient genetic profile in rectal cancer patients treated with neo-adjuvant chemo-radiotherapy

The aim of the study was the identification of a pharmacogenetic profile predictive of the tumor regression grade (TRG), considered as tumor response parameter, after neo-adjuvant treatment in rectal cancer patients. A total of 238 rectal cancer patients treated in a neo-adjuvant setting by a fluoropyrimidines-based chemo-radiotherapy (RT) were genotyped for 25 genetic polymorphisms in 16 genes relevant for treatment-associated pathways. Two polymorphisms were associated with TRG in a multivariate analysis: hOGG1-1245C>G, which can affect radiosensitivity and MTHFR-677C>T, which is involved in fluoropyrimidines action. Patients bearing at least one variant allele had a lower chance to get TRG⩽2 (OR=0.46 95% CI 0.23–0.90, P=0.024; and OR=0.48 95% CI 0.24–0.96, P=0.034; respectively). An association trend was observed for ABCB1-3435C>T, which is responsible for the multi-drug resistance (odds ratio (OR)=1.96, 95% confidence interval (CI) 0.98–3.95, P=0.057). Exploratory classification and regression tree (CART) analysis highlighted high-order gene–gene and gene–environment interactions and a genetic signature associated with differential response, with hOGG1-1245C>G as the most predictive factor. Other significant variables were: ABCB1-3435C>T, MTHFR-677C>T, ERCC1-8092C>A, ABCC2-1249G>A, XRCC1-28152G>A, XRCC3-4541A>G and patients gender. On the basis of CART results, patients were categorized into three groups according to tumor response probability: intermediate and high profiles had a higher probability to get TRG⩽2 as compared with low profiles (OR=4.12 95% CI 1.46–11.65, P<0.001 and OR=12.44, 95% CI 5.52–28.04, P<0.0001, respectively). This study evidences a major role of hOGG1-1245C>G and MTHFR-677C>T polymorphisms in the tumor response of rectal cancer patients treated with chemo-RT in neo-adjuvant setting, and shows the relevance of gene–gene and gene–environment interactions for complex phenotypes as tumor response.