Tumor budding is an independent prognostic factor in stage III colon cancer patients: a post-hoc analysis of the IDEA-France phase III trial (PRODIGE-GERCOR).

BACKGROUND: Histological characteristics at the invasive front may reflect tumor aggressiveness; specifically, tumor budding (Bd) is an emerging prognostic biomarker in colon cancer (CC). We explored further the significance of Bd for risk stratification by evaluating survival of stage III CC patients included in the IDEA-France phase III trial. PATIENTS AND METHODS: This post-hoc study was conducted on tissue slides from 1048 stage III CC patients. Bd was scored by central review by the Bd criteria of the 2016 International Tumor Budding Consensus Conference (ITBCC 2016) and classified as Bd1 (0-4 buds/0.785 mm2), Bd2 (5-9 buds), and Bd3 (≥10 buds) categories. Disease-free survival (DFS) and overall survival (OS) were analyzed by the log-rank test. Clinicopathological features and Immunoscore® were correlated with Bd. RESULTS: Overall, Bd1, Bd2, and Bd3 were observed in 39%, 28%, and 33% of CC, respectively. Bd2 and Bd3 were associated with vascular (P = 0.002) and perineural invasions (P = 0.0009). The 3-year DFS and the 5-year OS rates for Bd (1 versus 2-3) were 79.4% versus 67.2% (P = 0.001) and 89.2% versus 80.8% (P = 0.001), respectively. This was confirmed after adjustment for relevant clinicopathological features for DFS [hazard ratio (HR) 1.41, 95% confidence interval (CI) 1.12-1.77, P = 0.003] and OS (HR 1.65, 95% CI 1.22-2.22, P = 0.001). When combined with pTN stage and Immunoscore® subgroups, Bd significantly improved disease prognostication. CONCLUSIONS: Bd demonstrated its independent prognostic value for DFS and OS. Given these findings, Bd as per the ITBCC 2016 should be mandatory in every pathology report in stage III CC patients. Bd and Immunoscore® could play a complementary role in personalized health care in this setting.

Adjuvant FOLFOX +/- cetuximab in full RAS and BRAF wildtype stage III colon cancer patients.

Background: RAS mutations have been shown to confer resistance to anti- epidermal growth factor receptor (EGFR) treatment. We analysed the results of the PETACC8 trial (cetuximab + FOLFOX vs FOLFOX) in full RAS and BRAF wildtype (WT) patients (pts) with resected stage III colon cancer. Patients and methods: Exons 2, 3 and 4 of KRAS and NRAS, and BRAF exons 11 and 15, were sequenced using the Ampliseq colon-lung cancer panel version 2, in PETACC8 trial pts who consented to translational research. The impact of cetuximab on time to recurrence (TTR), disease-free survival (DFS) and overall survival (OS) was investigated in pts with tumours harbouring RAS and BRAF WT, and RAS mutations. The prognostic value of each individual mutation was also tested. Results: Among the 2559 pts analysed, 745 pts (29%) were known to have KRAS exon 2 mutations and 163 pts (6.4%) the BRAF V600E mutation. Of the remaining 1651 pts, 1054 were assessed by NGS, showing that a further 227 pts (21%) had KRAS exon 2, 3, 4 or NRAS exon 2, 3, 4 mutations, and that 46 pts (4.4%) had a newly diagnosed BRAF mutation. Cetuximab added to FOLFOX did not significantly improve TTR, DFS or OS in pts with RAS WT or RAS and BRAF WT tumours (HR 0.77-1.03, all P > 0.05). Cetuximab addition was not either significantly deleterious in RAS mutant pts or in pts with rare RAS or BRAF mutations. In the overall trial population, NRAS and KRAS codon 61 mutations were the only rare mutations with the same pejorative prognostic value as KRAS exon 2 or BRAF V600E mutations. Conclusion: Though not significant, the clinically relevant 0.76 adjusted HR observed for DFS in favour of adding cetuximab to FOLFOX, in full RAS and BRAF WT stage III colon cancer pts, may justify a new randomized controlled trial testing EGFR inhibitors in this setting. Clinical trial number: This is an ancillary study of the PETACC8 trial: EUDRACT 2005-003463-23.

Perioperative docetaxel, cisplatin, and 5-fluorouracil compared to standard chemotherapy for resectable gastroesophageal adenocarcinoma.

BACKGROUND: Even though the perioperative chemotherapy improves the overall survival (OS) compared to surgery alone in patients with a resectable gastroesophageal adenocarcinoma (GEA), prognosis of these patients remains poor. Docetaxel (D), cisplatin (C), and 5-fluorouracil (F) regimen improves OS compared to CF among patients with advanced GEA. We evaluated the potential interest of a perioperative DCF regimen, compared to standard (S) regimens, in resectable GEA patients. METHODS: We identified 459 patients treated with preoperative DCF or S regimens. The primary endpoint was OS. Propensity scores were estimated with a logistic regression model in which all baseline covariates were included. We then used two methods to take PS into account and thus make DCF and S patients comparable. OS analyses were performed with Kaplan-Meier and Cox models in propensity score matched samples, and inverse probability of treatment weighted (IPTW) samples. RESULTS: In the propensity score matched sample, the p-value from the log rank test for OS was 0.0961, and the 3-year OS rate was 73% and 55% in DCF and S groups, respectively. The multivariate Cox regression underlined a Hazard Ratio of 0.55 (95% CI 0.27-1.13) for DCF patients compared to S patients. The results from IPTW analyses showed that DCF was significantly and independently associated with OS (HR = 0.52; 95% CI 0.40-0.69). CONCLUSIONS: In this retrospective multicenter, hypothesis-generating study, the propensity score analyses underlined encouraging results in favor of DCF compared to S regimens regarding OS. This promising result should be validated in a phase-3 trial.

Multicentre randomised phase III trial comparing Tamoxifen alone or with Transarterial Lipiodol Chemoembolisation for unresectable hepatocellular carcinoma in cirrhotic patients (Fédération Francophone de Cancérologie Digestive 9402).

The FFCD 9402 multicentre phase III trial was designed to compare the effects of the combination of Transarterial Lipiodol Chemoembolisation (TACE) and tamoxifen with tamoxifen alone on overall survival and quality of life in the palliative treatment of hepatocellular carcinoma with cirrhosis. From 1995 to 2002, 138 patients were randomised between the two groups. One hundred and twenty three patients were eligible including 61 in the Tamoxifen group and 62 in the TACE group. Baseline characteristics were similar: Child-Pugh class A: 70%, alcoholic cirrhosis: 76%, Okuda stage I: 71%, multinodular tumour: 70% and segmental portal vein thrombosis: 10%. At 2years, the overall survival was 22% and 25% in the Tamoxifen and TACE groups (P=.68), respectively. Multivariate analysis identified four independent prognostic factors for survival: alpha-fetoprotein (AFP)>400ng/mL (P=.008), abdominal pain (P=.011), hepatomegaly (P=.023) and Child-Pugh score (P=.032). The Spitzer Index level assessing the quality of life during follow-up did not differ between the two groups (P=.70). Amongst patients with stage Okuda I, the 2-year overall survival was 28% in the Tamoxifen group and 32% in the TACE group (P=.58). In this subgroup, two prognostic factors were statistically significant for survival: AFP>400ng/mL (P=.004) and Spitzer Index (P=.013) as shown by multivariable analysis. In conclusion, this study suggests that TACE improves neither the survival nor the quality of life in patients with HCC and cirrhosis.

Treatment of mild chronic hepatitis C with interferon alpha-2b: results of a multi-centre randomized study in 80 patients.

OBJECTIVE: The natural history of mild chronic hepatitis C is not well-known and the benefit of treating this form of the disease is not well-defined. We conducted a pilot study to answer this question. DESIGN: Mild chronic hepatitis C was defined by positivity for anti-HCV antibodies, detectable serum HCV RNA by PCR, and a Knodell score < or = 5 on a liver biopsy performed within the previous 6 months. Eighty patients from six centres were randomized into two groups receiving interferon alpha-2b, 3 MU three times a week for 6 months (group 1, n = 39) or no treatment (group 2, n = 41). Sustained response was defined by the loss of detectable serum HCV RNA at 6 months after therapy. RESULTS: The two groups were not different at entry with respect to age, sex ratio, source of infection, disease duration, genotype, viral load and Knodell score. One patient (group 1) was excluded from the study, while two patients in group 1 (5%) and seven in group 2 (17.1 %) did not complete the trial. A sustained response was observed in seven patients (18%) in group 1 versus none in group 2 (P < 0.01). The difference in mean Knodell score remained non-statistically significant between the two groups at the end of the study. Reduction or interruption of interferon was necessary in eight patients (24.2%). CONCLUSIONS: This first randomized controlled study in mild chronic hepatitis C shows a proportion of sustained responders to interferon alpha-2b similar to that observed in active chronic hepatitis C.

Deamidation of asparagine residues in a recombinant serine hydroxymethyltransferase.

Serine hydroxymethyltransferase purified from rabbit liver cytosol has at least two Asn residues (Asn(5) and Asn(220)) that are 67 and 30% deamidated, respectively. Asn(5) is deamidated equally to Asp and isoAsp, while Asn(220) is deamidated only to isoAsp. To determine the effect of these Asn deamidations on enzyme activity and stability a recombinant rabbit liver cytosolic serine hydroxymethyltransferase was expressed in Escherichia coli over a 5-h period. About 90% of the recombinant enzyme could be isolated with the two Asn residues in a nondeamidated form. Compared with the enzyme isolated from liver the recombinant enzyme had a 35% increase in catalytic activity but exhibited no significant changes in either affinity for substrates or stability. Introduction of Asp residues for either Asn(5) or Asn(220) did not significantly alter activity or stability of the mutant forms. In vitro incubation of the recombinant enzyme at 37 degrees C and pH 7.3 resulted in the rapid deamidation of Asn(5) to both Asp and isoAsp with a t(1/2) of 50-70 h, which is comparable to the rate found with small flexible peptides containing the same sequence. The t(1/2) for deamidation of Asn(220) was at least 200 h. This residue may become deamidated only after some unfolding of the enzyme. The rates for deamidation of Asn(5) and Asn(220) are consistent with the structural environment of the two Asn residues in the native enzyme. There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme.

The structure of serine hydroxymethyltransferase as modeled by homology and validated by site-directed mutagenesis.

We describe a model for the three-dimensional structure of E. coli serine hydroxymethyltransferase based on its sequence homology with other PLP enzymes of the alpha-family and whose tertiary structures are known. The model suggests that certain amino acid residues at the putative active site of the enzyme can adopt specific roles in the catalytic mechanism. These proposals were supported by analysis of the properties of a number of site-directed mutants. New active site features are also proposed for further experimental testing.

Purification and characterization of recombinant rabbit cytosolic serine hydroxymethyltransferase.

A rabbit liver cDNA library in phage lambdagt10 was screened using the coding cDNA for human cytosolic serine hydroxymethyltransferase. A clone of 1754 bp was isolated and the nucleotide sequence showed an open reading frame of 1455 bp, which coded for rabbit cytosolic serine hydroxymethyltransferase and was flanked by 12 bp at the 5' end and 287 bp at the 3' end. The full-length cDNA was then cloned into a pET22b vector as a NdeI-EcoRI insert. HMS174(DE3) cells were transformed with this plasmid and, after induction with isopropyl beta-D-thiogalactopyranoside, expressed a catalytically active serine hydroxymethyltransferase. The enzyme was purified and shown to be the expressed rabbit enzyme lacking the first methionine residue. Spectral characteristics of the bound pyridoxal phosphate and kinetic constants for the natural substrates L-serine and tetrahydrofolate were essentially identical to the values obtained previously for the rabbit cytosolic enzyme. The pattern of bands shown by the pure recombinant enzyme on an isoelectric focusing gel containing 6 M urea showed a major band and a minor band representing about 15-20% of the protein. Upon incubation of the recombinant enzyme at pH 7.3 and 37 degreesC, three new bands were observed on isoelectric focusing with the concomitant formation of isoaspartyl residues, as determined by reactivity with protein isoaspartyl methyltransferase. These results are consistent with deamidation of Asn residues to isoaspartyl during the in vitro incubation. The enzyme purified from rabbit liver has previously been shown to contain isoaspartyl residues.

Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate. No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor. These modified folates are not readily available for use in assays for SHMT activity. This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus. The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay. The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S. solfataricus. Replacement of the modified folate with tetrahydrofolate did not support serine synthesis. In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate. The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs. The enzyme contains covalently bound pyridoxal phosphate. Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens.

Determination of serine hydroxymethyltransferase and reduced folate pools in tissue extracts.

Serine hydroxymethyltransferase (SHMT) from all sources tested catalyzes the slow exchange of the pro-2S proton of glycine with solvent protons. In the presence of tetrahydrofolate (H4PteGlun) this exchange rate is increased by about three orders of magnitude. This H4PteGlun-dependent exchange has been developed into a rapid and sensitive assay for both SHMT and H4PteGlun and the one-carbon derivatives of H4PteGlun. The procedure involves incubating [2-3H]glycine, H4PteGlun, and SHMT for 3 min followed by a separation of the exchanged protons in the solvent from the substrate glycine on a small Dowex-50 cation-exchange column at pH 2. In the presence of an excess of H4PteGlun the exchange rate is proportional to nanogram levels of SHMT. In the presence of an excess of SHMT the exchange rate is directly proportional to the concentration of H4PteGlun in the 0.1 to 1 pmol range. The concentration of one-carbon derivatives of H4PteGlun is determined by a preincubation of cell extracts with enzymes that convert each derivative into H4PteGlun. A complete reduced folate pool analysis of a tissue extract can be obtained in less than 2 h once a standard curve has been prepared for H4PteGlun. The method does not distinguish between mono- and polyglutamate forms of the coenzyme.

Site-directed mutagenesis techniques in the study of Escherichia coli serine hydroxymethyltransferase.

The 3340-bp fragment containing the Escherichia coli glyA gene coding for serine hydroxymethyltransferase was reduced in size by PCR, and the 1600-bp fragment obtained was cloned into the vector pBR322 in both orientations (5'-3', and 3'-5'). This DNA manipulation allowed us to perform site-directed mutagenesis by PCR on the glyA gene. To overcome the problem of the presence of wild-type protein in the various mutant enzyme preparations, the E. coli strain GS245 used to express recombinant serine hydroxymethyltransferase was made recA deficient through generalized transduction mediated by phage P1. The new strain was used for the production of a mutant form of the enzyme, in which the pyridoxal 5'-phosphate binding lysine was substituted by a glutamine. The preparation of this mutant form was completely devoid of wild-type enzyme contamination and measurements of its catalytic activity in the transamination reactions of L- and D-alanine confirmed the suggestion that the active site lysine is not the base that removes the alpha-proton from the substrate.

Ciprofloxacin and long-term prevention of spontaneous bacterial peritonitis: results of a prospective controlled trial.

The aim of this prospective double-blind study was to evaluate the value of long-term antibiotic prophylaxis using ciprofloxacin for the prevention of spontaneous bacterial peritonitis (SBP) in 60 cirrhotic patients with low ascitic fluid protein levels (< 15 g/L). The patients were assigned to two groups: group I (n = 28) ciprofloxacin 750 mg per os once a week for 6 months, group II (n = 32) placebo. The two groups were similar for clinical and laboratory characteristics. Twelve patients developed an intercurrent disorder, and 10 patients died during the trial. There were no adverse effects in the treated group. There was a significant decrease in the incidence of SBP (3.6 vs. 22%) (P < .05) and duration of hospitalization (9.3 +/- 4.5 vs. 17.6 +/- 6.2 days) (P < .05) in the treated group as compared with the placebo group. The bacteriological study showed no acquired resistance to ciprofloxacin after 6 months' treatment. These results suggest that long-term preventive antibiotic prophylaxis based on the weekly administration of 750 mg of ciprofloxacin is effective in the prevention of SBP in cirrhotic patients.

The function of arginine 363 as the substrate carboxyl-binding site in Escherichia coli serine hydroxymethyltransferase.

Both the highly conserved Arg363 and Arg372 residues of Escherichia coli serine hydroxymethyltransferase were changed to alanine and lysine residues. Each of the four mutant proteins were purified to homogeneity and characterized with respect to spectral properties of the enzyme-bound pyridoxal phosphate and kinetic properties with substrates and substrate analogs. The R372A and R372 K mutant enzymes exhibited spectra and kinetic properties close to those of the wild-type enzyme. The R363 K mutant enzyme exhibited only 0.03% of the catalytic activity of the wild-type enzyme and a 15-fold reduction in affinity for glycine and serine. The R363A mutant enzyme did not bind serine and glycine and showed no activity with serine as the substrate. Both R363 K and R363A enzymes bound amino acid esters at the active site and catalyzed the retro-aldol cleavage of serine ethyl ester and serinamide. The catalytic activity of the R363 K and R363A enzymes with the serine ethyl ester were about 0.006% and 0.1% of wild-type enzyme activity with serine, respectively. The R363A mutant enzyme catalyzed the half transamination of D-alanine methyl ester and L-alanine methyl ester at rates similar to the rates of transamination of D-alanine and L-alanine by the wild-type enzyme. The results are interpreted to show that R363 is the binding site of the amino acid substrate carboxyl group and that forming an ion pair between R363 and the substrate carboxyl group is an important feature in catalysis by serine hydroxymethyltransferase. Evidence is also provided that R363 may play a role in the substrate-induced open to closed conformational change of the active site.

Evaluation of efficacy of liver transplantation in alcoholic cirrhosis by a case-control study and simulated controls.

To assess the efficacy of liver transplantation in patients with alcoholic cirrhosis, we compared 2-year survival of 169 liver transplantation patients in 12 French centres with survival of two control groups treated conservatively. The matched group was 169 patients of similar age, cirrhosis severity, and bleeding history; the simulated group was 169 patients whose theoretical survival was determined in a cohort of 797 patients with alcoholic cirrhosis. The probability of survival to 2 years in transplanted patients was 73 (95% confidence interval 67-79%) versus 67% (59-75) in the matched and 67% (63-71) in simulated controls. When prognostic factors were taken into account, transplantation was associated with survival (r = 0.527; p = 0.069). Patients with severe liver disease (high-risk group) benefited most for 2-year survival: 64% (42-86) vs 41% (23-59) in the matched and 23% (19-27) in the simulated control groups (p < 0.01). There was no difference for patients at low and at medium risk. Liver transplantation increases the 2-year survival of patients with severe alcoholic cirrhosis. In patients with less severe disease, further studies should be done by non-randomised controlled studies with longer follow-up or by randomised trials.

Function of the active-site lysine in Escherichia coli serine hydroxymethyltransferase.

Serine hydroxymethyltransferase has a conserved lysine residue (Lys-229) that forms the internal aldimine with pyridoxal 5'-phosphate. In other pyridoxal 5'-phosphate enzymes investigated so far, this conserved lysine residue also plays a catalytic role as a base that removes the alpha-proton from the amino acid substrate. Three mutant forms of Escherichia coli serine hydroxymethyltransferase (K229Q, K229R, and K229H) were constructed, expressed, and purified. The absorbance spectra, rapid reaction kinetics, and thermal denaturation of the mutant analogs were studied. Only the K229Q mutant serine hydroxymethyltransferase resembled the wild-type enzyme. The results indicate that Lys-229 plays a critical role in expelling the product by converting the external aldimine to an internal aldimine. In the absence of Lys-229, ammonia can also catalyze the same function at a much slower rate. However, Lys-229 apparently is not the base that removes the alpha-proton from the amino acid substrate. The K229Q mutant enzyme could catalyze one turnover of either serine to glycine or glycine to serine at rates approaching those of the wild-type enzyme. After one turnover, the mutant enzyme could not expel the product and bind new substrate. The K229Q mutant enzyme can also transaminate D-alanine, which, like the hydroxymethyltransferase activity, also requires removing the alpha-proton from the substrate. The absorbance spectra of the K229R and K229H serine hydroxymethyltransferases showed that their pyridoxal 5'-phosphate could not readily form an external aldimine with substrates, suggesting that Lys-229 in the wild-type enzyme may never bear a positive charge, further evidence that it is not the base that removes the alpha-proton.

[Primary sclerosing cholangitis and autoimmune cochlear deafness: a new syndrome?]. / Cholangite sclérosante primitive et surdité endocochléaire auto-immune: nouveau syndrome?

Association of autoimmune cochlear hearing loss with primary sclerosing cholangitis is reported in two patients. Endocochlear sensorineural hearing loss was associated with the presence of anti-cochlear antibodies in the serum directed against the walls of vessels in the stria vascularis. The hearing loss appeared at the same time or shortly after the diagnosis of cholangitis. This association, which has never been described, may reinforce the theory of the role of immunologic factors in the pathogenesis of primary sclerosing cholangitis, possibly linked to or initiated by vasculitis.

[Comparative study of results of hepatic transplantation between 2 groups of patients: alcoholic cirrhosis versus non-alcoholic cirrhosis]. / Etude comparative des résultats de la transplantation hépatique entre deux groupes de malades: cirrhose alcoolique versus cirrhose non alcoolique.

Among the patients treated for alcoholic cirrhosis, only a small group could be candidates for liver transplantation (LT). The aim of this multicentric study was to analyse the results of LT in a group of 75 patients with alcoholic cirrhosis (AC) compared with a group of 61 patients with non-alcoholic cirrhosis (NAC). Results were similar in both groups concerning survival rate and quality of life. However the ability to go back to a normal professional life was less in the AC group. The reported recurrence of alcoholic intoxication, which was around 26%, was much lower for patients who interrupted alcohol consumption during at least 3 months before L.T.

Comparative ultrastructural study of rat livers preserved in Euro-Collins or University of Wisconsin solution.

University of Wisconsin solution greatly lengthens the time liver storage is possible compared with all previous solutions used. To test whether this improvement is related to better preservation of the endothelial cell, which is thought to be the most vulnerable cell type in cold storage, we compared time-related ultrastructural changes in rat livers stored in this solution or in Euro-Collins solution. Rat livers were harvested after combined arterial and portal perfusion with the cold-storage solution. They were then preserved for different lengths of time in the same solution at 4 degrees C before being perfusion-fixed and processed for light and electron microscopy. The first preservation damage was noted in endothelial cells; the time course of the lesions was similar in both solutions. After 2 hr of storage, enlarged and ruptured fenestrae with many gaps were observed. Swollen at 4 hr, the endothelial cells became stringlike at 10 hr, leading to stripped sinusoidal walls. Hepatocytes appeared better preserved in University of Wisconsin solution. The amount of glycogen, maintained near the control level at 24 hr in the latter, decreased dramatically between 0 and 4 hr in Euro-Collins solution, as ultrastructurally observed and biochemically confirmed. Furthermore, sinusoidal obstruction by blebs originating from the hepatocytes and quantified by image analysis on electron micrographs was markedly delayed. It was significantly less pronounced in University of Wisconsin solution at 24 hr than in Euro-Collins solution at 2 hr (p less than or equal to 0.05). Our findings confirm that endothelial cells are highly susceptible to preservation damage and show that University of Wisconsin solution does not improve preservation during storage.(ABSTRACT TRUNCATED AT 250 WORDS)