In vitro stability of low-concentration ziconotide alone or in admixtures in intrathecal pumps.

OBJECTIVES: Ziconotide is often administered in combination with other analgesics via an intrathecal pump. Studies have established that ziconotide is stable when delivered alone in high concentrations. No stability data are available, however, for ziconotide given in low concentrations and/or with other analgesics as usually occurs in clinical oncology practice. The objective of this study was to assess the in vitro stability of ziconotide alone and combined with other analgesics in intrathecal pumps at 37 °C, as well as in syringes at 5 °C, to evaluate conditions for storing and transporting preparations. MATERIALS AND METHODS: Various ziconotide concentrations (0.1, 0.25, 0.5, and 0.75 µg/mL) were combined with an admixture of ropivacaine (7.5 mg/mL), morphine (7.5 mg/mL), and clonidine (15 µg/mL) in 20-mL intrathecal pumps at 37 °C and in syringes at 5 °C. Solutions of ziconotide alone in concentrations of 0.25, 0.5, 0.75, and 1 µg/mL were introduced into pumps at 37 °C and syringes at 5 °C. Assays were performed using ultra high pressure liquid chromatography. RESULTS: In admixtures, mean ziconotide concentrations decreased linearly to 53.4% (± 3.33%) of baseline after 35 days. When ziconotide was introduced alone in pumps at 37 °C, the residual concentration on day 31 was 35.54% (± 0.04%) with 0.25 µg/mL, 39.37% (± 0.15%) with 0.5 µg/mL, and 44.49% (± 0.18%) with 1 µg/mL. Ziconotide alone or combined with the other analgesics was stable in syringes stored at 5 °C. The preparations complied with the prescriptions, with a mean error of less than 10%, except with the lowest ziconotide concentration (0.1 µg/mL). CONCLUSIONS: At the low ziconotide concentrations studied, the degradation of ziconotide admixed with other drugs was linear and only weakly influenced by the baseline concentration. Linear regression with intrapolation to 30 days showed that the degradation of ziconotide admixed with other drugs was consistent with previously published data.

COVID-19 Infections in Cancer Patients Were Frequently Asymptomatic: Description From a French Prospective Multicenter Cohort (PAPESCO-19).

Background: Cancer patients (CPs) are considered more vulnerable and as a high mortality group regarding COVID-19. In this analysis, we aimed to describe asymptomatic COVID (+) CPs and associated factors. Methods: We conducted a prospective study in CPs and health care workers (HCWs) in 4 French cancer centers (PAPESCO [PAtients et PErsonnels de Santé des Centres de Lutte Contre le Cancer pendant l'épidémie de COvid-19] study). This analysis used data recorded between June 17, 2020 and November 30, 2020 in CPs (first 2 waves, no variants). At inclusion and quarterly, CPs reported the presence of predefined COVID-19 symptoms and had a blood rapid diagnostic test; a reverse transcription polymerase chain reaction (RT-PCR) was done in case of suspected infection. Results: A total 878 CPs were included; COVID-19 prevalence was similar in both CPs (8%) and HCWs (9.5%); of the 70 CPs (8%) who were COVID (+), 29 (41.4%) were and remained asymptomatic; 241/808 of the COVID (-) (29.8%) were symptomatic. 18 COVID (+) were hospitalized (2% of CPs), 1 in intensive care unit (ICU) and 1 died (0.1% of CPs and 2.4% of symptomatic COVID [+] CPs). Only the inclusion center was associated with clinical presentation (in Nancy, Angers, Nantes, and Clermont-Ferrand: 65.4%, 35%, 28.6%, and 10% CPs were asymptomatic, respectively). Conclusions: Seroprevalence of COVID-19 in CPs was similar to that observed in HCWs; mortality related to COVID-19 among CPs was 0.1%. More than 40% of COVID (+) CPs were asymptomatic and one third of COVID (-) CPs had symptoms. Only geographic origin was associated with the presence or absence of symptoms. Social distancing and protective measures must be applied in CPs at home and when hospitalized.

COVID-19 Vaccination Campaign in Cancer Patients and Healthcare Workers-Results from a French Prospective Multicenter Cohort (PAPESCO-19).

In this prospective, real-life cohort study, we followed 523 cancer patients (CP) and 579 healthcare workers (HCW) from two cancer centers to evaluate the biological and clinical results of the COVID-19 vaccination campaign. Seventy percent of the CP and 90% of the HCW received an mRNA vaccine or the AZD1222 vaccine. Seropositivity was high after the first vaccine among HCW and poor among CP. The second dose resulted in almost 100% seropositivity in both cohorts. Antibody response was higher after the second injection than the first in both populations. Despite at least two doses, 8 CP (1.5%) and 14 HCW (2.4%) were infected, corresponding either to a weak level of antibody or a new strain of virus (particularly the Omicron variant of concern). Sixteen CP and three HCW were hospitalized but none of them died from COVID-19. To conclude, this study showed that two doses of COVID-19 vaccines were crucially necessary to attain sufficient seropositivity. However, the post-vaccination antibody level declines in individuals from the two cohorts and could not totally prevent new SARS-CoV-2 infections.

Irinotecan induces steroid and xenobiotic receptor (SXR) signaling to detoxification pathway in colon cancer cells.

BACKGROUND: Resistance to chemotherapy remains one of the principle obstacles to the treatment of colon cancer. In order to identify the molecular mechanism of this resistance, we investigated the role of the steroid and xenobiotic receptor (SXR) in the induction of drug resistance. Indeed, this nuclear receptor plays an important role in response to xenobiotics through the upregulation of detoxification genes. Following drug treatments, SXR is activated and interacts with the retinoid X receptor (RXR) to induce expression of some genes involved in drug metabolism such as phase I enzyme (like CYP), phase II enzymes (like UGT) and transporters (e.g. MDR1). RESULTS: In this study, we have shown that endogenous SXR is activated in response to SN-38, the active metabolite of the anticancer drug irinotecan, in human colon cancer cell lines. We have found that endogenous SXR translocates into the nucleus and associates with RXR upon SN-38 treatment. Using ChIP, we have demonstrated that endogenous SXR, following its activation, binds to the native promoter of the CYP3A4 gene to induce its expression. RNA interference experiments confirmed SXR involvement in CYP3A4 overexpression and permitted us to identify CYP3A5 and MRP2 transporter as SXR target genes. As a consequence, cells overexpressing SXR were found to be less sensitive to irinotecan treatment. CONCLUSIONS: Altogether, these results suggest that the SXR pathway is involved in colon cancer irinotecan resistance in colon cancer cell line via the upregulation of select detoxification genes.

Phase II study of preoperative radiation plus concurrent daily tegafur-uracil (UFT) with leucovorin for locally advanced rectal cancer.

BACKGROUND: Considerable variation in intravenous 5-fluorouracil (5-FU) metabolism can occur due to the wide range of dihydropyrimidine dehydrogenase (DPD) enzyme activity, which can affect both tolerability and efficacy. The oral fluoropyrimidine tegafur-uracil (UFT) is an effective, well-tolerated and convenient alternative to intravenous 5-FU. We undertook this study in patients with locally advanced rectal cancer to evaluate the efficacy and tolerability of UFT with leucovorin (LV) and preoperative radiotherapy and to evaluate the utility and limitations of multicenter staging using pre- and post-chemoradiotherapy ultrasound. We also performed a validated pretherapy assessment of DPD activity and assessed its potential influence on the tolerability of UFT treatment. METHODS: This phase II study assessed preoperative UFT with LV and radiotherapy in 85 patients with locally advanced T3 rectal cancer. Patients with potentially resectable tumors received UFT (300 mg/m/2/day), LV (75 mg/day), and pelvic radiotherapy (1.8 Gy/day, 45 Gy total) 5 days/week for 5 weeks then surgery 4-6 weeks later. The primary endpoints included tumor downstaging and the pathologic complete response (pCR) rate. RESULTS: Most adverse events were mild to moderate in nature. Preoperative grade 3/4 adverse events included diarrhea (n = 18, 21%) and nausea/vomiting (n = 5, 6%). Two patients heterozygous for dihydropyrimidine dehydrogenase gene (DPYD) experienced early grade 4 neutropenia (variant IVS14+1G > A) and diarrhea (variant 2846A > T). Pretreatment ultrasound TNM staging was compared with postchemoradiotherapy pathology TN staging and a significant shift towards earlier TNM stages was observed (p < 0.001). The overall downstaging rate was 42% for primary tumors and 44% for lymph nodes. The pCR rate was 8%. The sensitivity and specificity of ultrasound for staging was poor. Anal sphincter function was preserved in 55 patients (65%). Overall and recurrence-free survival at 3 years was 86.1% and 66.7%, respectively. Adjuvant chemotherapy was administered to 36 node-positive patients (mean duration 118 days). CONCLUSION: Preoperative chemoradiotherapy using UFT with LV plus radiotherapy was well tolerated and effective and represents a convenient alternative to 5-FU-based chemoradiotherapy for the treatment of resectable rectal cancer. Pretreatment detection of DPD deficiency should be performed to avoid severe adverse events.

Anosmia but Not Ageusia as a COVID-19-Related Symptom among Cancer Patients-First Results from the PAPESCO-19 Cohort Study.

Background: Cancer patients may fail to distinguish COVID-19 symptoms such as anosmia, dysgeusia/ageusia, anorexia, headache, and fatigue, which are frequent after cancer treatments. We aimed to identify symptoms associated with COVID-19 and to assess the strength of their association in cancer and cancer-free populations. Methods: The multicenter cohort study PAPESCO-19 included 878 cancer patients and 940 healthcare workers (HCWs). At baseline and quarterly thereafter, they reported the presence or absence of 13 COVID-19 symptoms observed over 3 months and the results of routine screening RT-PCR, and they were systematically tested for SARS-CoV-2-specific antibodies. We identified the symptom combinations significantly associated with COVID-19. Results: Eight percent of cancer patients were COVID-19 positive, and 32% were symptomatic. Among the HCWs, these proportions were 9.5 and 52%, respectively. Anosmia, anorexia, fever, headache, and rhinorrhea together accurately discriminated (c-statistic = 0.7027) COVID-19 cases from cancer patients. Anosmia, dysgeusia/ageusia, muscle pain, intense fatigue, headache, and chest pain better discriminated (c-statistic = 0.8830) COVID-19 cases among the HCWs. Anosmia had the strongest association in both the cancer patients (OR = 7.48, 95% CI: 2.96-18.89) and HCWs (OR = 5.71, 95% CI: 2.21-14.75). Conclusions: COVID-19 symptoms and their diagnostic performance differ in the cancer patients and HCWs. Anosmia is associated with COVID-19 in cancer patients, while dysgeusia/ageusia is not. Cancer patients deserve tailored preventive measures due to their particular COVID-19 symptom pattern.

A universal formula based on cystatin C to perform individual dosing of carboplatin in normal weight, underweight, and obese patients.

PURPOSE: It has recently been shown that it is possible to improve the prediction of carboplatin clearance by adding plasma cystatin C level (cysC), an endogenous marker of glomerular filtration rate, to the other patient characteristics routinely used for carboplatin individual dosing, namely serum creatinine (Scr), actual body weight (ABW), age, and sex. This multicenter pharmacokinetic study was done to evaluate prospectively the benefit of using cysC for carboplatin individual dosing. EXPERIMENTAL DESIGN: The 357 patients included in the study were receiving carboplatin as part of established protocols. A population pharmacokinetic analysis was done using NONMEM program. Seven covariates studied were as follows: Scr, cysC, age, sex, ABW, ideal body weight, and lean body mass. RESULTS: The best covariate equation was as follows: carboplatin clearance (mL/min) = 117.8. (Scr/75)(-0.450). (cysC/1,00)(-0.385). (ABW/65)(+0.504). (age/56)(-0.366). 0.847(sex), with Scr in micromol/L, cysC in mg/L, ABW in kilograms, age in years, and sex = 0 for male. Using an alternative weight descriptor (ideal body weight or lean body mass) did not improve the prediction. This final covariate model was validated by bootstrap analysis. The bias (mean percentage error) and imprecision (mean absolute percentage error) were +1% and 15%, respectively, on the total population, and were of a similar magnitude in each of the three subgroups of patients defined according to their body mass index. CONCLUSION: For the first time, a unique formula is proposed for carboplatin individual dosing to patients, which is shown to be equally valid for underweight, normal weight, and obese patients.

[Therapeutic drug monitoring of 5-fluorouracil after its administration in high-dose protocols]. / Niveau de preuve du suivi thérapeutique pharmacologique du 5-fluorouracile au décours de son administration dans le traitement des cancers des voies aérodigestives supérieures et du cancer colorectal.

Therapeutic drug monitoring is currently investigated in patients receiving the drug by prolonged continuous infusion, either alone or associated with other chemotherapy agents. This arises from an increasing body of evidence that relates plasma fluorouracil concentrations to toxicity or effectiveness. Literature data indicate that threshold levels of exposure, as assessed by the area under the concentration-time curve, are associated with an increased risk of toxicity in patients treated for either a colorectal or a head and neck cancer. Kinetically guided dose adjustment may thus be proposed for the ongoing or the next cycle of chemotherapy. Historical comparisons and the results of a phase III multicenter randomized study in patients with metastatic colorectal cancer showed that dose-adapted fluorouracil resulted in significantly improved objective response rate, a trend to higher survival rate and fewer grade III-IV toxicities. Real-time fluorouracil monitoring may also help identifying patients with higher than expected exposure, thus providing a basis for intensifying supportive care in these patients.

[Not Available]. / Niveau de preuve du suivi thérapeutique pharmacologique du 5-fluorouracile au décours de son administration dans le traitement des cancers des voies aérodigestives supérieures et du cancer colorectal.

Therapeutic drug monitoring is currently investigated in patients receiving the drug by prolonged continuous infusion, either alone or associated with other chemotherapy agents. This arises from an increasing body of evidence that relates plasma fluorouracil concentrations to toxicity or effectiveness. Literature data indicate that threshold levels of exposure, as assessed by the area under the concentration-time curve, are associated with an increased risk of toxicity in patients treated for either a colorectal or a head and neck cancer. Kinetically guided dose adjustment may thus be proposed for the ongoing or the next cycle of chemotherapy. Historical comparisons and the results of a phase III multicenter randomized study in patients with metastatic colorectal cancer showed that dose-adapted fluorouracil resulted in significantly improved objective response rate, a trend to higher survival rate and fewer grade III-IV toxicities. Real-time fluorouracil monitoring may also help identifying patients with higher than expected exposure, thus providing a basis for intensifying supportive care in these patients.

BRAF Non-V600E Mutated Metastatic Colorectal Cancer in a Young Patient: Discussion from a Case Report.

We report the case of a 32-year-old man with a caecal adenocarcinoma with major lymph node extension and peritoneal carcinomatosis, presenting a BRAF-K601E mutation. A triplet (5FU plus oxaliplatin plus irinotecan) combination with bevacizumab achieved tumor control but the disease progressed immediately after cessation and the patient died 8 months after the diagnosis. A short review of BRAF non-V600E mutations shows that outcome and clinical features depend on the mutation.

Predictive factors of oxaliplatin neurotoxicity: the involvement of the oxalate outcome pathway.

PURPOSE: Oxaliplatin displays a frequent dose-limiting neurotoxicity due to its interference with neuron voltage-gated sodium channels through one of its metabolites, oxalate, a calcium chelator. Different clinical approaches failed in neurotoxicity prevention, except calcium-magnesium infusions. We characterized oxalate outcome following oxaliplatin administration and its interference with cations and amino acids. We then looked for genetic predictive factors of oxaliplatin-induced neurotoxicity. EXPERIMENTAL DESIGN: We first tested patients for cations and oxalate levels and did amino acid chromatograms in urine following oxaliplatin infusion. In the second stage, before treatment with FOLFOX regimen, we prospectively looked for variants in genes coding for the enzymes involved (a) in the oxalate metabolism, especially glyoxylate aminotransferase (AGXT), and (b) in the detoxification glutathione cycle, glutathione S-transferase pi, and for genes coding for membrane efflux proteins (ABCC2). RESULTS: In the first 10 patients, urinary excretions of oxalate and cations increased significantly within hours following oxaliplatin infusion, accompanied by increased excretions of four amino acids (glycine, alanine, serine, and taurine) linked to oxalate metabolism. In a further 135 patients, a minor haplotype of AGXT was found significantly predictive of both acute and chronic neurotoxicity. Neither glutathione S-transferase pi nor ABCC2 single nucleotide polymorphisms we looked for were linked to neurotoxicity. CONCLUSION: These data confirm the involvement of oxalate in oxaliplatin neurotoxicity and support the future use of AGXT genotyping as a pretherapeutic screening test to predict individual susceptibility to neurotoxicity.

[Detection of dihydropyrimidine dehydrogenase deficiency before treatment by fluoropyrimidines]. / Dépistage des patients déficitaires en dihydropyrimidine déhydrogénase avant traitement par fluoropyrimidines.

Numerous toxic side-effects, sometimes severe, are regularly reported in patients treated with 5-fluorouracil, and oral fluoropyrimidines, UFT and capecitabine, in metastatic and adjuvant setting. These toxic effects are due to a large interindividual variability of the metabolism, mainly depending on dihydropyrimidine dehydrogenase activity (DPD), the major enzyme of the catabolism of fluoropyrimidines. Thus, the patients with a DPD deficiency are at high risk of early severe acute toxicity, with this kind of drug. These toxic side-effects are potentially lethal. DPD deficiency frequencies, partial or complete, are about 3-5% and 0.2% respectively. They are most often due to a gene polymorphism. Different techniques for the detection of DPD deficiency before treatment have been reported: phenotypic, such as the plasma ratio of dihydrouracil/uracil, or genotypic, such as the detection of DPD gene variants, deleterious for enzyme activity. The pretherapeutic detection of DPD deficiency would permit to avoid almost every early acute toxic side-effects. We must emphasize that it is not merely a genetic result, since the detection of a deficiency most often does not contra-indicate the use of a fluoropyrimidine, but it must be combined with therapeutic advice.

Clinical relevance of different dihydropyrimidine dehydrogenase gene single nucleotide polymorphisms on 5-fluorouracil tolerance.

PURPOSE: Although single nucleotide polymorphisms (SNP) of the dihydropyrimidine dehydrogenase gene (DPYD) have been reported, which affect enzyme activity and the severity of 5-fluorouracil (5-FU) toxicity, no pretherapeutic detection has thus far been developed. We investigated 22 DPYD gene SNPs, their respective incidence, their link with grade 3 to 4 toxic side effects, and their management in practice: 9 were looked for in 487 patients, whereas 13 others were investigated in 171 patients. PATIENTS AND METHODS: SNPs were detected before 5-FU-based treatment in WBC using a Pyrosequencing method. Close clinical and biological follow-up was done. RESULTS: Five different SNPs were found in 187 patients (IVS14 + 1G>A, 2846A>T, 1679T>G, 85T>C, -1590T>C). Three hundred patients had no SNP. Forty-four patients had grade 3 to 4 toxic side effects in either the first or second cycle. Sixty percent of patients with either IVS14 + 1G>A or 2846A>T SNPs and the only patient with 1679T>G SNP experienced early grade 3 to 4 toxicity, compared with 0%, 5.5%, and 15% of those with either -1590T>C, 85T>C SNP, or no SNP, respectively. In cases with grade 3 to 4 toxicity, treatment either had to be quickly stopped, or could be safely continued with an individual dose adjustment. Sensitivity, specificity, and positive and negative predictive values of the detection of these three major SNPs as toxicity predictive factors were 0.31, 0.98, and 0.62 and 0.94, respectively. CONCLUSION: Pretreatment detection of three DPYD SNPs could help to avoid severe toxic side effects. This approach is suitable for clinical practice and should be compared or combined with pharmacologic approaches. In the case of dihydropyrimidine dehydrogenase deficiency, 5-FU administration often can be safely continued with an individual dose adjustment.

A multicenter phase II study of personalized FOLFIRI-cetuximab for safe dose intensification.

We conducted a multicenter proof of concept phase II trial in patients with advanced colorectal cancer receiving FOLFIRI-cetuximab regimens to explore individual drug tailoring using pharmacogenetics and pharmacokinetics (PK) monitoring. Patients were stratified by their pharmacogenetic/phenotypic status: the irinotecan dose was adjusted according to the number of TA tandem repeats in the UGT1A1 promoter, while the 5-fluorouracil (5-FU) dose was initially adjusted according to dihydropyrimidine dehydrogenase (DPD) activity at initial screening (5-FUODPM Tox) followed by PK-guided dose optimization (5-FUODPM Protocol). An advanced cetuximab PK/pharmacodynamics (PD) study was performed but dosage remained unchanged. Eighty-five patients receiving second-line chemotherapy were enrolled. Mean irinotecan doses at 3 months were 247 ± 50, 210 ± 53 and 140 ± 21 mg/m2 for those with 6/6 (33), 6/7 (26), and 7/7 (7) TATA repeats in the UGT1A1 promoter region, respectively. The 5-FU dose was initially reduced in four patients with DPD deficiency, but mean 5-FU dose at 3 months was 2,412 ± 364 mg/m2 (1,615-3,170 mg/m2). Grade 4 toxicities were not encountered and grade 4 neutropenia occurred in 6.8%, 5.9%, and 0% of patients with 6/6, 6/7, and 7/7 UGT1A1 genotypes. The objective response rate was 25.8% among the 85 patients, 57.3% in patients with tumors wild type (WT) for KRAS, and 25% in those whose tumor harbored a mutant-KRAS. Secondary resection of hepatic metastases was performed in 31.7% of patients. Median progression-free survival (PFS) for all 85 patients was 181 days and 200, 132, and 121 days for patients with 6/6, 6/7, and 7/7 UGT1A1 genotypes, respectively; these differences were not statistically different. In parallel, a strong relationship was shown between cetuximab AUC and regimen efficacy. We conclude that personalized drug tailoring when administering in FOLFIRI + cetuximab allows for safe and efficient individual dose intensification.

Prevention of 5-fluorouracil-induced early severe toxicity by pre-therapeutic dihydropyrimidine dehydrogenase deficiency screening: Assessment of a multiparametric approach.

5-Fluorouracil (5-FU)-based treatments can lead to early-onset severe (4%-5%) even fatal (0.3%) toxicities in patients with dihydropyrimidine dehydrogenase (DPD) deficiency. This multicenter prospective cohort study aimed to assess the clinical benefit of pretherapeutic screening for DPD deficiency using a multiparametric approach. Two parallel cohorts of patients treated with 5-FU-based chemotherapy for colorectal carcinoma were compared in a prospective nonrandomized study. In arm A, patients had DPD deficiency screening before treatment, whereas in arm B no pretherapy screening was performed. Dosing was based on 5-FU administration guidelines of each institution. DPD deficiency screening was performed using a combined multiparametric approach (5-FUODPM Tox). The frequency of early grade 4-5 toxic events potentially induced by 5-FU was compared in the two groups. At total of 1,142 patients (n = 1,116 evaluable) were enrolled. In arm A, out of 718 evaluable patients, nine grade 4 early toxicities potentially related to 5-FU were reported in nine patients (1.2%) with no toxic death despite one complete DPD deficiency and 24 partial deficiencies. The 24 patients with partial deficiency had safe pharmacokinetics (PK)-monitored 5-FU. In arm B, among 398 evaluable patients, 17 grade 4-5 toxic early events potentially related to 5-FU were reported in 12 patients (4.2%). The incidence of early severe toxicity was significantly higher in arm B (P = .0019), confirming the positive impact of pretherapeutic DPD assessment. The percent of patients with a toxicity grade 3 or higher observed in arm A was 10.8% (n = 78) compared to 17.55% (n = 69)  in arm B (P = .0497). The percentage of death was reduced from 2.5/1,000 in arm B to 0 in arm A. The time to occurrence of all grade ≥3 toxicities was determined in both arms and the difference between the two arms was significant (P = .047). Overall, one patient with complete DPD deficiency confirmed retrospectively died within 13 days from grade 5 multivisceral toxicity. Enrollment was prematurely closed after external experts' decision. In conclusion, multiparametric pretherapeutic DPD deficiency screening significantly lowered the risk of early severe toxicity and avoided an early toxic death. This approach should be used for safe administration of 5-FU-based treatments.

Tumor quantification of several fluoropyrimidines resistance gene expression with a unique quantitative RT-PCR method. Implications for pretherapeutic determination of tumor resistance phenotype.

Pretherapeutic determination of tumor resistance to chemotherapy is a main challenge, hindered by the low number of mechanisms characterized at the same time, the small size of the clinical specimens and the heterogeneity of the techniques or the lack of true quantification. The aim of the present study was to determine in real time quantitative RT-PCR, tumor cell expression of several transcripts involved in cancer cell resistance with a unique cDNA sample from a tumor biopsy. The technique had to be suitable in clinical practice for determination of several factors involved in resistance to a given drug family, for example, fluoropyrimidines resistance factors: thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine kinase (TK), dihydrofolate reductase (DHFR), folylpolyglutamate synthetase (FPGS). A frame-shifted artificial construct was designed specifically to work within the same conditions. We validated our technique by quantifying expressions of these 5 genes starting from tissue samples of colorectal carcinoma and the surrounding normal mucosa of 33 different patients. That real time quantitative RT-PCR technique using the frame-shifted artificial construct as a standard provided a real comparison and quantification of different resistance factors. Tumor resistance phenotype determination based on that approach will be investigated in a control study.

Gemcitabine aerosol: in vitro antitumor activity and deposition imaging for preclinical safety assessment in baboons.

AIM: To characterize gemcitabine aerosol, its in vitro activity against lung cancer cells, its deposition, and tolerance in a non-human primate model. METHODS: In vitro cytotoxicity of nebulized gemcitabine against NCI-H460 and A549 lung cancer cells was tested using a growth inhibition assay and compared with non-nebulized gemcitabine. The (99m)Tc-DTPA-radiolabeled gemcitabine aerosol was characterized by cascade impaction and the gemcitabine mass/(99m)Tc activity relationship was established for further quantitative nuclear imaging. Nine weekly inhalations at a target dose of 1 mg/kg body weight of gemcitabine were performed in three baboons using dynamic scintigraphic acquisitions for continuous monitoring of gemcitabine delivery during inhalation. Gemcitabine plasma concentrations were measured during the first inhalation. RESULTS: Growth inhibition assays for both NCI-H460 and A549 cells did not differ between nebulized and non-nebulized gemcitabine. Aerosol characterization showed a particle mass median aerodynamic diameter of 3.7+/-0.8 microm and a linear relationship between gemcitabine mass (y) and (99m)Tc activity (x) (y=0.82x - 10(-5), R (2)=0.88). No toxicity was observed after nine weekly inhalations of a mean dose of gemcitabine of 11.1 mg (88% of the target dose) as assessed from scintigraphic data. A dose-dependent peak plasma concentration of gemcitabine (20-74 ng/ml) was observed by the tenth minute of inhalation. CONCLUSIONS: We have characterized a gemcitabine aerosol suitable for intrathoracic airway deposition and demonstrated that jet nebulization does not alter the cytotoxic properties of the drug. In a primate model, we have developed a scintigraphic procedure for the monitoring of aerosol deposition, and we have demonstrated the safety of nine weekly aerosol administrations of gemcitabine.

An accurate dihydrouracil/uracil determination using improved high performance liquid chromatography method for preventing fluoropyrimidines-related toxicity in clinical practice.

An accurate and improved HPLC method was set up to measure both dihydrouracil (UH2) and uracil (U) in plasma, and to assess their ratio. Analytes retention time, separation and peak purity were greatly optimized with a Hypercarb column and a diode array detector. U and UH2 limits of quantification were 1.25 and 0.625 ng/mL. U and UH2 within-day precisions were 0.9-2.3% and 0.7-5.6%. Between-day precisions were 1.3-5.3% and 1.3-7.1%. Accuracy was 0.1-6.1%. UH2/U ratio between-day variability was low, but ratio decreased from 02:00 p.m. This method is now used in practice to detect patients at risk of fluoropyrimidine toxicity and to individually adapt the dosage.

Relevance of different UGT1A1 polymorphisms in irinotecan-induced toxicity: a molecular and clinical study of 75 patients.

PURPOSE: We wanted to assess polymorphisms in the uridine diphosphoglucuronosyl transferase 1A1 (UGT 1A1) gene: the TATA box polymorphism and UGT 1A1 G71R and Y486D mutations in the coding sequence, the main mutations characterizing Gilbert's syndrome, as predictors of severe toxic event occurrence after irinotecan (CPT-11) administration. Therefore, we set up a rapid, sensitive, and reliable technique in routine practice to detect before CPT-11 treatment, the at-risk patients. EXPERIMENTAL DESIGN: Seventy-five patients with advanced colorectal cancer and treated with CPT-11 and 5-fluorouracil, entered the study. We used the Pyrosequencing technology a real-time sequencing method, to detect the UGT 1A1 TATA box polymorphisms and mutations in the coding regions. Patients were also assessed for both biochemical and clinical evaluation and tolerance to treatment. RESULTS: No G71R and Y486D mutations were found in our population. Frequencies for UGT 1A1 TATA box polymorphisms were 41, 47, and 9% for wild-type 6/6, heterozygous 6/7, and Gilbert's syndrome 7/7, respectively. Tolerance to treatment decreased with increased number of TA repeat with 71% of the patients in 7/7 group who experienced grade 3/4 toxicity. CONCLUSIONS: The method we set up is suitable for the detection of UGT 1A1 polymorphism in routine practice before irinotecan treatment. It could help to detect the patients homozygous or heterozygous for Gilbert's syndrome, at-risk of CPT 11-induced toxicity, and thus could help to individualize the dose to optimize efficacy and limit toxicity.