Frequent intragenic rearrangements of DPYD in colorectal tumours.

Dihydropyrimidine dehydrogenase is a crucial enzyme for the degradation of 5-fluorouracil (5FU). DPYD, which encodes dihydropyrimidine dehydrogenase, is prone to acquire genomic rearrangements because of the presence of an intragenic fragile site FRA1E. We evaluated DPYD copy number variations (CNVs) in a prospective series of 242 stage I-III colorectal tumours (including 87 patients receiving 5FU-based treatment). CNVs in one or more exons of DPYD were detected in 27% of tumours (deletions or amplifications of one or more DPYD exons observed in 17% and 10% of cases, respectively). A significant relationship was observed between the DPYD intragenic rearrangement status and dihydropyrimidine dehydrogenase (DPD) mRNA levels (both at the tumour level). The presence of somatic DPYD aberrations was not associated with known prognostic or predictive biomarkers, except for LOH of chromosome 8p. No association was observed between DPYD aberrations and patient survival, suggesting that assessment of somatic DPYD intragenic rearrangement status is not a powerful biomarker to predict the outcome of 5FU-based chemotherapy in patients with colorectal cancer.

Molecular patterns in deficient mismatch repair colorectal tumours: results from a French prospective multicentric biological and genetic study.

BACKGROUND: To test the prognostic value of tumour protein and genetic markers in colorectal cancer (CRC) and examine whether deficient mismatch repair (dMMR) tumours had a distinct profile relative to proficient mismatch repair (pMMR) tumours. METHODS: This prospective multicentric study involved 251 stage I-III CRC patients. Analysed biomarkers were EGFR (binding assay), VEGFA, thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) expressions, MMR status, mutations of KRAS (codons 12-13), BRAF (V600E), PIK3CA (exons 9 and 20), APC (exon 15) and P53 (exons 4-9), CpG island methylation phenotype status, ploidy, S-phase, LOH. RESULTS: The only significant predictor of relapse-free survival (RFS) was tumour staging. Analyses restricted to stage III showed a trend towards a shorter RFS in KRAS-mutated (P=0.005), BRAF wt (P=0.009) and pMMR tumours (P=0.036). Deficient mismatch repair tumours significantly demonstrated higher TS (median 3.1 vs 1.4) and TP (median 5.8 vs 3.5) expression relative to pMMR (P<0.001) and show higher DPD expression (median 14.9 vs 7.9, P=0.027) and EGFR content (median 69 vs 38, P=0.037) relative to pMMR. CONCLUSIONS: Present data suggesting that both TS and DPD are overexpressed in dMMR tumours as compared with pMMR tumours provide a strong rationale that may explain the resistance of dMMR tumours to 5FU-based therapy.

Phase I trial of gemcitabine combined with capecitabine and erlotinib in advanced pancreatic cancer: a clinical and pharmacological study.

BACKGROUND: The aim of this phase I trial was to define the maximum tolerated dose (MTD), the dose-limiting toxicity (DLT) and the recommended dose of erlotinib combined with capecitabine and gemcitabine in the treatment of advanced pancreatic cancer (APC). METHODS: Gemcitabine was administered intravenously at 1,000 mg/m(2)/week (days 1, 8 and 15) and oral capecitabine from day 1 to day 21 at 1,660 mg/m(2)/day. Oral erlotinib was administered daily continuously at escalating doses (28-day cycle). Dose levels (DLs) 1, 2, 3 and 4 were 50, 75, 100 and 125 mg/day, respectively. Pharmacokinetic analysis of the three drugs was performed in the first cycle. RESULTS: Nineteen patients were enrolled. At the MTD (DL4; 125 mg/day erlotinib), 100% of patients developed DLT consisting of grade 4 febrile neutropenia and nonhematological grade 3 events (vomiting, diarrhea, stomatitis, rash). The most common toxicities, regardless of grade, were neutropenia, anemia, rash and diarrhea. Erlotinib systemic exposure was significantly related to the administered dose. Of note, toxicity was significantly associated with elevated systemic exposure of capecitabine anabolites. CONCLUSION: When combined concurrently with 1,000 mg/m(2)/week gemcitabine and 1,660 mg/m(2)/day capecitabine, erlotinib can be administered safely at a daily dose of 100 mg in APC patients.

Clinical value of circulating endothelial cells and circulating tumor cells in metastatic breast cancer patients treated first line with bevacizumab and chemotherapy.

BACKGROUND: We investigated whether circulating tumor cells (CTCs) and circulating endothelial cells (CECs) predict clinical outcome of first-line chemotherapy combined with bevacizumab in metastatic breast cancer patients. PATIENTS AND METHODS: In a French substudy of the MO19391 trial, CTC and CEC counts (CellSearch system) at baseline and changes after two cycles of treatment were correlated with time to progression (TtP). RESULTS: CTC and CEC levels were not correlated in the 67 patients included. At baseline, CTC positivity was a significant prognostic marker for TtP at a threshold of 3 CTC/7.5 ml (P < 0.05) but not at 5 CTC/7.5 ml (P = 0.09). Baseline CEC levels (median 17 CEC/4 ml, range 1-769) were associated with age > or =45 years (P = 0.01), elevated lactate dehydrogenase (P < 0.01) and not with TtP at any threshold. Changes of CTC count during treatment were not a surrogate of TtP, with any of the model tested (threshold based or relative decrease in percent). However, increase in CEC count was associated with improved TtP, at the threshold of 20 CEC/4 ml (P < 0.01). CONCLUSION: Bevacizumab combined with first-line chemotherapy may modify the predictive value of CTC during treatment possibly due to impaired tumor cells intravasation through vessels endothelium. Variations in CEC levels appear to be a promising early surrogate marker of TtP under antiangiogenic treatment.

Combination of sunitinib, cetuximab and irradiation in an orthotopic head and neck cancer model.

BACKGROUND: Recent preclinical and clinical studies indicate beneficial effects from combining radiotherapy with either anti-angiogenic drugs or anti-epidermal growth factor receptor (EGFR)-targeting agent. To investigate the effect of combining these approaches, we evaluated in vivo the antitumor efficacy of the anti-angiogenic compound sunitinib, an oral, multi-targeted tyrosine kinase inhibitor that inhibits among others vascular endothelial growth factor (VEGF) receptors-1, -2 and -3, cetuximab, a mAb targeting the EGFR, and irradiation (RT) given alone and in combination. MATERIALS AND METHODS: Investigations were carried out using a VEGF-secreting human head and neck tumor cell line, CAL33, with a high EGFR content, growing as orthotopic xenografts in nude mice. Three days after tumor cell injection, sunitinib (20 mg/kg, p.o.), cetuximab (1 mg/kg, i.p.), both 5 days/week seven doses, and RT (6 Gy, 3 days/week, four doses) were administered alone and in combination during 9 days. RESULTS: Concomitant administration of drugs produced a marked and significant supra-additive decrease, and the addition of RT completely abolished tumor growth. The drug association markedly reduced tumor cell proliferation (Ki67) and the number of the vessels, but enhanced cell differentiation. CONCLUSION: The efficacy of this combination of sunitinib, cetuximab and RT may be of clinical importance in the management of head and neck cancer patients.

Combined effects of bevacizumab with erlotinib and irradiation: a preclinical study on a head and neck cancer orthotopic model.

Clinical benefit has been demonstrated in patients with head and neck tumours receiving an anti-epidermal growth factor receptor (EGFR) agent in combination with radiotherapy (RT). Recent preclinical and clinical studies suggest beneficial effects from combining anti-angiogenic drugs with RT. To investigate the effect of combining these approaches, we evaluated in vivo the anti-tumour efficacy of the anti-angiogenic compound bevacizumab, a highly specific monoclonal antibody directed against the vascular endothelial growth factor (VEGF), erlotinib, an EGFR tyrosine kinase inhibitor, and irradiation given alone and in combination. Investigations were performed using a VEGF-secreting human head and neck tumour cell line, CAL33, with a high EGFR content, injected as orthotopic xenografts into the mouth floor of nude mice. Three days after tumour cell injection, bevacizumab (5 mg kg(-1), 5 days a week, i.p.), erlotinib (100 mg kg(-1), 5 days a week, orally) and irradiation (6 Gy, 3 days a week) were administered alone and in combination for 10 days. As compared with the control, concomitant administration of drugs produced a marked and significant supra-additive decrease in tumour mass; the addition of irradiation almost completely abolished tumour growth. The drug association markedly reduced the number of metastatic nodes and the triple combination significantly reduced the total number of pathologically positive lymph nodes as compared with controls. The RT-induced proliferation, reflected by Ki67 labelling, was reduced to control level with the triple combination. Radiotherapy induced a strong and very significant increase in tumour angiogenesis, which was no longer observed when combined with erlotinib and bevacizumab. The efficacy of the combination of bevacizumab+erlotinib and RT may be of clinical importance in the management of head and neck cancer patients.

Epidermal growth factor receptor (EGFR) status and K-Ras mutations in colorectal cancer.

BACKGROUND: In advanced colorectal cancer, K-Ras somatic mutations predict resistance to mAbs targeting epidermal growth factor receptor (EGFR). Relationships between K-Ras mutations and EGFR status have not been examined so far. We analyzed relationships between K-Ras mutations and EGFR tumoral status based on EGFR germinal polymorphisms, gene copy number and expression. METHODS: Eighty colorectal tumors (stage 0-IV) and 39 normal mucosas were analyzed. K-Ras mutations at codons 12 and 13 were detected by a sensitive enrichment double PCR-restriction fragment length polymorphism (RFLP) assay. EGFR gene polymorphisms at positions -216G>T, -191C>A and 497Arg>Lys were analyzed (PCR-RFLP), along with CA repeat polymorphism in intron 1 (fluorescent genotyping) and EGFR gene copy number (PCR amplification). EGFR expression was quantified by Scatchard binding assay. RESULTS: The number of EGFR high-affinity sites, dissociation constant (Kd), gene copy number, intron 1, -216G>T, -191C>A or 497Lys>Arg genotypes was not different between K-Ras-mutated or K-Ras-non-mutated tumors. No relationship was observed between any of the analyzed EGFR genotypes and EGFR expression. EGFR expression was not related to gene copy number. EGFR gene copy number in tumor and normal tissue was not correlated. The mean value of the tumor/normal mucosa gene copy number ratio was 1.16. CONCLUSIONS: Present data clearly show that EGFR status is independent of K-Ras mutations in colorectal tumors.

Candidate apoptotic and DNA repair gene approach confirms involvement of ERCC1, ERCC5, TP53 and MDM2 in radiation-induced toxicity in head and neck cancer.

INTRODUCTION: Single nucleotide polymorphisms (SNPs) of DNA repair and apoptosis genes have been associated with outcome in head and neck squamous cell carcinoma (HNSCC) patients receiving radiotherapy (RT). Our goal was to conduct a candidate gene study in HNSCC patients receiving RT or chemoRT. METHODS: 122 non-resectable HNSCC patients undergoing RT (N=38) or chemoRT (N=84) between 1992 and 2006 were retrospectively analyzed. ERCC1 Lys259Thr (rs735482), ERCC2 Lys751Gln (rs13181), ERCC5 His46His C>T (rs1047768), XRCC1 Arg399Gln (rs25487), TP53 Arg72Pro (rs1042522) and MDM2 309T>G (rs2279744) were analyzed on tumor DNA. SNP profile was considered to assess RT-related toxicity. RESULTS: All 120 evaluable patients experienced RT-related toxicity at any time. Among them, 83% had G3-4 acute side-effects during RT, mainly dysphagia, mucositis, epithelitis and/or xerostomia (DMEX). 28/105 patients (27%) had early G3-4 toxicity up to 3months after the end of RT. 29/96 patients (30%) had G3-4 late toxicity thereafter. The presence of G allele of MDM2 or Thr allele of ERCC1 was associated with a significantly higher risk of acute and/or early DMEX toxicity. The MDM2 309GG genotype was linked to a higher risk of acute G3-4 dermatitis. The ERCC5 TT genotype was associated with more frequent G3-4 late cervical skin fibrosis or xerostomia. Pro allele of TP53 72 was associated with a higher risk of G3-4 osteoradionecrosis. CONCLUSION: Relevant SNPs in DNA repair (ERCC1 and ERCC5) and apoptosis (MDM2 and TP53) genes might influence the severity of radiation-related side-effects in HNSCC patients. Prospective clinical SNP-based validation studies are needed on these bases.

Gefitinib-trastuzumab combination on hormone-refractory prostate cancer xenograft.

New drugs and new combinations of drugs have recently shown promising clinical activity in hormone refractory prostate cancer. We studied the association of gefitinib with trastuzumab on the androgen-refractory prostate cancer cell line DU145 expressing both epidermal growth factor receptor (EGFR) and HER-2. Drug combinations with radiotherapy (RT) were considered along with the analysis of factors linked to cell proliferation and apoptosis. The antitumour effects of gefitinib were more pronounced than those observed with trastuzumab. In mice receiving the gefitinib-trastuzumab combination, reduction in tumour volume was inferior to that predicted by the observed impact of the agents alone. The presence of trastuzumab markedly attenuated the relative increase on p27 expression and the Bax:Bcl2 ratio induced by gefitinib. The combination gefitinib-RT had similar antitumour effects as those predicted by the impact of the individual treatments, whereas the effect of the trastuzumab-RT combination was inferior to that predicted by the individual effects. The present data should be borne in mind when designing new clinical schedules for treatment of hormone-refractory prostate cancer including the use of HER inhibitors.

Chronopharmacokinetics of oral tegafur and uracil in colorectal cancer patients.

Uracil-Ftorafur (UFT) combines the 5-fluorouracil (FU) prodrug tegafur with uracil (at a 1:4 molar ratio), which is a competitive inhibitor of dihydropyrimidine dehydrogenase (DPD), the limiting enzyme of FU catabolism. As a result, sustained FU concentrations are obtained in both plasma and tumor. UFT is an effective alternative to intravenous FU-Leucovorin (LV) in metastatic and adjuvant colorectal cancer treatment. A circadian rhythm for DPD activity has been shown in both human and animal studies, with consequences on FU plasma concentrations in patients receiving FU as a continuous infusion. The chronopharmacokinetics of FU has stimulated clinical trials of chronomodulated delivery schedules for floxuridine and FU infusions, suggesting that such schedules may improve the fluoropyrimidine therapeutic index. Molecular mechanisms responsible for the circadian dependence of FU pharmacodynamics include circadian rhythms in thymidylate synthase activity and DNA synthesis, as recently reported. Chronopharmacology of FU prodrugs is poorly documented. Recently, a feasibility study of chronomodulated administration of the FU oral prodrug capecitabine was reported. To our knowledge, the only study reporting on the time dependency of UFT pharmacokinetics is a phase I study by Muggia et al.

Supra-additive antitumor effect of sunitinib malate (SU11248, Sutent) combined with docetaxel. A new therapeutic perspective in hormone refractory prostate cancer.

PURPOSE: Physiological and molecular findings indicate over-expression of HER proteins and dysregulation of neo-angiogenesis during progression of advanced prostate cancer. The aim of this study was to test a novel rational therapeutic approach by combining docetaxel with an EGFR-targeting agent (cetuximab) and with an anti-angiogenic agent (sunitinib, SUTENT). METHODS: Mice bearing well-established PC3 prostate tumors (mean tumor volume/treatment group approximately 250 mm(3)) were treated every week with vehicle alone (controls), sunitinib (40 mg/kg/day, 5 days/week for 3 weeks, 0.2 ml p.o.), cetuximab (0.2 mg/kg/day, 5 days/week for 3 weeks, 0.2 ml i.p.) and docetaxel (10 mg/kg, 1 day/week for 3 weeks, 0.2 ml i.p.). RESULTS: Each drug, administered as a single-agent, demonstrated comparable and moderate effects on tumor growth with approximately 50 % inhibition at the end of the 3-week dosing schedule. Computed combination ratio (CR) values for tumor growth determined on days 61, 68 and 75 after cell implantation indicated supra-additive effects for the sunitinib-docetaxel (1.53, 1.15 and 1.47, respectively) and sunitinib-cetuximab combinations (1.2, 1.32 and 1.14, respectively), and suggested additive effects only for the sunitinib-cetuximab-docetaxel combination (CR = 1). The effects on tumor growth were accompanied by a parallel diminution in tumor cell proliferation (Ki 67) and tumor vascularization (von Willebrandt factor). There were significantly higher pro-apoptotic effects (caspase-3 cleavage) observed for the sunitinib-docetaxel and sunitinib-docetaxel-cetuximab as compared to the other conditions. CONCLUSION: The supra-additive anti-tumor effect observed with the sunitinib-docetaxel combination might support innovative strategies in the management of advanced prostate cancer.

PK/PD model of indisulam and capecitabine: interaction causes excessive myelosuppression.

The anticancer agent indisulam was evaluated in a dose-escalation study in combination with capecitabine. Severe myelotoxicity was observed after multiple treatment cycles. We hypothesized that capecitabine inhibits the synthesis of CYP2C9, which metabolizes indisulam. The objectives were to develop a pharmacokinetic/pharmacodynamic (PK/PD) model for the combination treatment and to estimate the impact of a drug-drug interaction on the safety of various dose levels. NONMEM was used to develop a PK/PD model, including the impact of capecitabine coadministration on indisulam pharmacokinetics. A simulation study was performed to evaluate the risk of dose-limiting neutropenia. A time-dependent pharmacokinetic drug-drug interaction resulted in increased exposure to indisulam and in increased myelotoxicity. The risk of dose-limiting neutropenia increased with treatment duration and with dose. The excessive myelosuppression after multiple cycles may be explained by a pharmacokinetic interaction between indisulam and capecitabine. The combination of 550 mg/m(2) indisulam and 1,250 mg/m(2) capecitabine twice daily was considered safe.

EGFR in colorectal cancer: more than a simple receptor.

BACKGROUND: Advances in the understanding of tumor biology have led to the development of targeted therapies allowing progress in colorectal cancer treatment. One of the most promising targets is the epidermal growth factor receptor (EGFR). METHOD: The presence and distribution of high- and low-affinity EGFR was investigated retrospectively in a group of 82 colorectal cancer samples (43 normal colon-colon cancer paired samples) using a specific ligand binding assay (Scatchard Analysis). FINDINGS: A large majority of tumor samples exhibited one class of high-affinity binding sites (78%). Eighteen cases (22%) exhibited both high- and low-affinity binding sites. A wide interpatient variability was observed for the site number, with physiologically-relevant high-affinity sites ranging from 7 to 310 fmol/mg protein in tumors and from 6 to 313 fmol/mg protein in normal mucosa. A significant positive correlation was demonstrated between tumor and normal mucosa for the high-affinity Kd values and for the number of high-affinity sites, suggesting a common regulation for both tumor and normal tissue. INTERPRETATION: These observations (i) could explain recently-reported clinically-active EGFR targeting in colorectal tumors apparently negative for EGFR, and (ii) may offer a plausible explanation for the link observed between toxicity in normal tissue (cutaneous rash) and clinical outcome of patients treated with anti-EGFR drugs. Present data extends our understanding of EGFR identity in colorectal cancer which could be useful in reconsidering the predictive tools for the identification of tumors putatively responsive to EGFR targeted therapy.

Analysis of the dinucleotide repeat polymorphism in the epidermal growth factor receptor (EGFR) gene in head and neck cancer patients.

BACKGROUND: Epidermal growth factor receptor (EGFR) overexpression is associated with poor prognosis in head and neck cancer. The first intron of EGFR gene is polymorphic (9-23 CA repeats) and transcription declines when the number of repeats increases. PATIENTS AND METHODS: EGFR polymorphism (fluorescent genotyping) and expression (ligand-binding assay) were analyzed in tumors and normal tissues from 112 patients (100 men, 12 women; mean age 60 years). RESULTS: The number of CA repeats varied from 15 to 22. Allelic distribution was trimodal (predominance of 16, 20 and 18 CA repeats). EGFR concentrations were significantly higher (P=0.02) in homozygous tumors as compared with heterozygous. Considering homozygous tumors, or classifying genotypes as short/long/intermediary (two alleles <17 versus two alleles > or =17 versus others), no relationship was observed between tumoral EGFR genotype and expression. In the 76 tumors exhibiting at least one 16-CA allele, the length of the remaining allele was inversely correlated to EGFR expression (P=0.047). Tumoral EGFR expression, performance status (WHO criteria) and node involvement were independent predictors of specific survival (P <0.01). Tumoral or normal tissue EGFR genotype did not influence survival. CONCLUSIONS: Intron 1 EGFR polymorphism may be implicated in the regulation of EGFR expression in head and neck tumors.

Pharmacological background of EGFR targeting.

Epidermal growth factor receptor (EGFR) signaling pathways play a key role in the regulation of cell proliferation, survival and differentiation. As a consequence, EGFR is one of the best studied ligand-receptor system and specific EGFR inhibition approaches are currently among the most promising and the most advanced in the clinical setting. Monoclonal antibodies (mAbs) and specific tyrosine kinase inhibitors (TKIs) have been developed, among which C225 (Cetuximab) and ZD1839 (Iressa), respectively, are the most advanced. The aim of the present review was not to cover the field of EGFR inhibitors, but to compare at experimental and clinical levels the different key points governing the actions of mAbs and TKIs. In addition, combinations of conventional chemotherapies with EGFR targeting drugs, as well as resistance mechanisms of EGFR targeting, have been reviewed.