Effect of low-frequency KRAS mutations on the response to anti-EGFR therapy in metastatic colorectal cancer.

BACKGROUND: Only patients with wild-type (WT) KRAS tumors benefit from anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (Mabs) in metastatic colorectal cancer (mCRC). Pyrosequencing is now widely used for the determination of KRAS mutation burden and a conservative cut-off point of 10% has been defined. Up until now, the impact of low-frequency KRAS mutations (<10%) on the response to anti-EGFR Mabs has yet to be evaluated. PATIENTS AND METHODS: Tumors from patients receiving anti-EGFR Mabs based on a WT genotype for KRAS, as determined using direct sequencing, have been retrospectively analyzed by pyrosequencing. Patients were categorized as WT (no KRAS mutation) or low-frequency mutation when KRAS mutation was <10% (KRAS low MT). RESULTS: A total of 168 patients treated by anti-EGFR Mabs for mCRC were analyzed. According to pyrosequencing, 138 tumors remained KRAS WT, while 30 tumors were KRAS low MT. In the KRAS low MT and KRAS WT groups, the response rates were 6.7% and 37.0%, respectively, while stabilization amounted to 23.3% versus 32.6% and progression to 70% versus 29% (P < 0.01). Progression-free survival (PFS) was 2.7 ± 0.5 months for KRAS low MT and was 6.0 ± 0.3 months for KRAS WT (P < 0.01). CONCLUSIONS: These results appear to validate consideration of low-frequency KRAS mutation tumors as positive, and justify a large-scale prospective study.

Evoked response activity eigenmode analysis in a convoluted cortex via neural field theory.

Neural field theory of the corticothalamic system is used to explore evoked response potentials (ERPs) caused by spatially localized impulse stimuli on the convoluted cortex and on a spherical cortex. Eigenfunctions are calculated analytically on the spherical cortex and numerically on the convoluted cortex via eigenfunction expansions. Eigenmodes on a convoluted cortex are similar to those of the spherical cortex, and a few such modes are found to be sufficient to reproduce the main ERP features. It is found that the ERP peak is stronger in spherical cortex than convoluted cortex, but in both cases the peak decreases monotonically with increasing distance from the stimulus point. In the convoluted case, cortical folding causes ERPs to differ between locations at the same distance from the stimulus point and spherical symmetries are only approximately preserved.

[Improvement of carbohydrate deficient transferrin measurement by capillary zone electrophoresis using immunosubtraction of immunoglobulins and transferrin]. / Intérêt de l'immunosoustraction des immunoglobulines et de la transferrine dans le dosage de la transferrine désialylée en électrophorèse capillaire.

CDT (Carbohydrate Deficient Transferrin) is considered as the most efficient biomarker of alcohol abuse available for routine use. Among the various methods developed for its measurement, capillary zone electrophoresis (CZE) on the multicapillary analyzer Capillarys2 provides high quality results at high throughput. However, the non CDT specific measurement of protein absorbance at 200 nm may bring abnormal profiles in samples from patients with high polyclonal immunoglobulin level or monoclonal component. We evaluated the automated immunosubtraction procedure developed by the manufacturer in 48 samples with abnormal electrophoretic profiles that potentially could interfere with CZE measurement of CDT. Elimination of the serum immunoglobulins raised the number of interpretable profiles from 19 (40%) to 37 (77%). The immunosubtraction procedure failed in samples with a monoclonal component present at a concentration > 60 g/L and in some samples harbouring a partially degraded C3 fraction. Six samples identified as genetic BC transferrin variants were also included in the study and submitted to an automated transferrin subtraction procedure to ascertain whether the additional peak were actually transferrin glycoforms. After treatment, two samples were classified as homozygote C for transferrin due to the persistence of one of the supposed transferrin peak. In conclusion, immunoglobulin and transferrin subtraction allow a better CDT measurement in most samples with interfering monoclonal components and avoid misclassification of suspected transferrin BC or CD variants.

[Lentiviral vectors]. / Lentivecteur: l'anti-vecteur rétroviral ?

The recent advent of RNA interference has strongly stimulated the growing interest toward lentiviral vectors. This, plus the occurrence of several adverse effects in a clinical trial in which oncoretroviral vectors were employed, refocused the need for efficient tools allowing a stable in vivo gene transfer. The lentivectors were first developed in 1996 to address the poor efficiency of murine retroviral vectors to transduce arrested cells. Taking advantage of the accumulated experience in retroviral vector design, a rapid evolution of the structural form of the lentivectors converted this gene transfer agent into a very simple and popular tool. This rapidly led to several commercially available solutions. In the present review, more than a simple comparison to oncoretroviral vectors, we aimed at emphasizing the specific points that distinguish lentiviral vectors and confirm them as good and safe candidates for the clinical delivery of therapeutic genes.

Neural field theory of perceptual echo and implications for estimating brain connectivity.

Neural field theory is used to predict and analyze the phenomenon of perceptual echo in which random input stimuli at one location are correlated with electroencephalographic responses at other locations. It is shown that this echo correlation (EC) yields an estimate of the transfer function from the stimulated point to other locations. Modal analysis then explains the observed spatiotemporal structure of visually driven EC and the dominance of the alpha frequency; two eigenmodes of similar amplitude dominate the response, leading to temporal beating and a line of low correlation that runs from the crown of the head toward the ears. These effects result from mode splitting and symmetry breaking caused by interhemispheric coupling and cortical folding. It is shown how eigenmodes obtained from functional magnetic resonance imaging experiments can be combined with temporal dynamics from EC or other evoked responses to estimate the spatiotemporal transfer function between any two points and hence their effective connectivity.

ICE inhibitor YVADcmk is a potent therapeutic agent against in vivo liver apoptosis.

In liver, apoptosis is a physiological process involved in the clearance of injured cells and in homeostatic control [1]. However, in patients with viral fulminant hepatitis or with nonacute liver diseases [2], dramatic liver failure or secondary cirrhosis results from the death of hepatocytes, which express the cell-surface receptor Fas, by apoptosis. To date, treatment of fulminant hepatitis relies mainly on orthotopic liver transplantation, which is limited by immunological complications and graft availability. Unravelling the molecular mechanisms that underlie acute liver failure could allow the design of an appropriate therapy. Ligand-bound Fas and tumour necrosis factor alpha (TNF-alpha) induce hepatic apoptosis in mice [3-6]. In various cell types, Fas- or TNF-alpha-induced apoptosis is blocked by viral proteins (such as p35 and CrmA) as well as by a decoy peptide (YVADcmk) [7-11], suggesting that these mechanisms of apoptosis involve ICE (interleukin-1 beta converting enzyme)-like proteases. Here, we report that, in vivo, pre-treatment of mice with YVADcmk protects them from the lethal effect of anti-Fas antibody and from liver failure induced by injection of TNF-alpha. Remarkably, YVADcmk administration is also highly effective in rescuing mice that have been pretreated with anti-Fas antibody from rapid death, despite extensive hepatic apoptosis. This dramatic curative effect could be of clinical benefit for the treatment of viral and inflammatory liver diseases.

[Alphavirus derived vector, still a relevant system through new mobilisation procedure]. / Les vecteurs dérivés des alphavirus, un système toujours pertinent grâce à de nouveaux modes de vectorisation.

Among eukaryotic cell-expression-systems, the one derived from alphaviruses, including Semliki forest virus (SFV), offers an efficient method for protein production in mammalian cells. Despite this efficacy, twenty years after their discovery alphaviruses vectors remain poorly used. Alphavirus vectors exist as naked RNA vectors or as recombinant particles. The use of costly RNA-based replicons, and the fact that production of recombinant particles is a complex process to carry out, have hampered the attractiveness of the methods. Lastly, the apoptotic signals induced by alphavirus vectors replication leads to a rapid death of the producing cells. This feature, which can be detrimental in vitro, is advantageously exploited for in vivo applications. Besides laboratory applications, alphavirus vectors have been explored in rare phase I clinical trials, for vaccine development and cancer gene therapy, therefore, alphavirus vector will benefit from the advent of new, biosafety-efficient, methods for particles production. Most of the recent advances in the field proposed an heterologous mobilisation of alphavirus replicon. While increasing biosafety aspects, new methods are also simpler regarding the genesis of recombinant particles. In the present review, we overview the alphavirus life cycle with a special attention to the features influencing vector design and utility.

[An anicteric cholectasis and nephrotic syndrome: AL amyloïdosis in a 51 years-old man]. / Une cholestase anictérique associée à un syndrome néphrotique chez un homme de 51 ans: découverte d'une amylose AL.

Amyloidosis is a multiple-organ disease for which the diagnosis is often confusing and thereby delayed. Here, we present an archetypal case illustrating such difficulties. A 51 years-old man presented a mixed dyslipemia in November 2002, in June 2004 he has finally been diagnosed with a primary AL-amyloidosis. Within these two years, the arising of a non-icteric cholestasis and a nephrotic syndrome have triggered the search for a disease related to a multiple-organ protein deposition. Confirmation of the AL-amyloidosis was obtained through an histological examination, including direct immuno-fluorescence. Amyloidosis is a life threatening disease that need to be diagnosed at an early stage, in order to maximise the therapeutic expectations. The average survival after the diagnosis of AL-amyloidosis is 5% at 10 years. Often, treatments are initiated late in the course of the disease, at a time when organ lesion are constituted, severely affecting the prognosis.

Predictors of disease-free survival in colorectal cancer with microsatellite instability: An AGEO multicentre study.

BACKGROUND: A microsatellite instability (MSI) phenotype is found in about 12% of colorectal cancers (CRCs) and is associated with a low recurrence rate after curative surgery. Several studies have identified clinical and pathological factors predictive of recurrence in resected CRC, but not in the MSI subgroup. PATIENTS AND METHODS: This multicentre retrospective study included patients with stage I, II or III MSI CRCs. Disease-free survival (DFS) was calculated with the Kaplan-Meier method. Factors associated with DFS were identified in univariate and multivariate Cox analyses. RESULTS: We studied 521 patients with MSI CRC. Respectively 11%, 51% and 38% of patients were at stage I, II and III. Mean age was 68.7years and 36% of the patients received adjuvant chemotherapy. Median follow-up was 32.8months. The disease recurrence rates were 6% and 21% in stage II and III patients, respectively. The 3-year DFS rate was 77%. In univariate analysis, age, bowel obstruction, lymph node invasion, stage T4, vascular emboli, lymphatic invasion and perinervous invasion were associated with poorer DFS (P<0.05). Three relevant independent predictors of poor DFS were identified in multivariate analysis, namely bowel obstruction (HR=2.46; 95%CI 1.31-4.62, P=0.005), vascular emboli (HR=2.79; 95%CI 1.74-4.47, P<0.001) and stage T4 (HR=2.16; 95%CI 1.31-3.56, P=0.002). CONCLUSIONS: Bowel obstruction, vascular emboli and stage T4 are independently associated with MSI CRC recurrence, suggesting that screening for vascular emboli in routine clinical practice may assist with adjuvant chemotherapy decision-making.

Amphotropic retroviral vectors displaying hepatocyte growth factor-envelope fusion proteins improve transduction efficiency of primary hepatocytes.

The development of retroviral vectors with cell-specific targeting capabilities will be an important step toward successful in vitro gene therapy. This article describes the generation of a retroviral vector with enhanced binding abilities for cells bearing the c-Met receptor: the Madin-Darby canine kidney (MDCK) cell line and primary hepatocytes. The human hepatocyte growth factor (HGF) was displayed on murine amphotropic retroviral vectors by fusion to the viral transmembrane envelope glycoprotein (TM). The resulting chimeric envelope HGF-TM was expressed in an amphotropic packaging cell line producing viral particles that display both HGF-TM and the wild-type envelope. These modified viral particles had a titer equivalent to that of unmodified particles. Modified particles infected MDCK cells more efficiently than did unmodified amphotropic retrovirus. Adding anti-HGF antibodies to the viral vector particle supernatant prior to infection confirmed that the increased infection was mediated by the HGF moiety. The chimeric viruses also infected primary mouse and nonhuman primate fetal hepatocytes more effectively. Furthermore, these cells could be induced to proliferate by the modified HGF-TM viruses. Since exogenous HGF is primarily taken up by the liver, these results may have implications for retroviral vector design for liver-directed human gene therapy.

Preclinical studies for cell transplantation: isolation of primate fetal hepatocytes, their cryopreservation, and efficient retroviral transduction.

Fetal hepatocytes are an attractive target for in utero cellular transplantation. Their use could provide a very efficient way for implanting normal or transduced cells into the livers of affected fetuses. Marking cells with recombinant retroviruses is a powerful tool for evaluating the chimerism of grafted animals. The technique relies on the ex vivo transduction efficiency of the engrafted cells. We have isolated fetal primary hepatocytes from nonhuman primates. The cells were cultured and transduced with a retroviral vector carrying the Escherichia coli beta-galactosidase gene. Optimal gene transfer efficiency was obtained 48-60 hr after plating and was as high as 90%. Cryopreservation had little effect on cell viability and infectivity: The viability of thawed hepatocytes remained high (75-85%) and the infection efficiency was identical to that of freshly isolated cells. Efficient ex vivo retroviral gene transfer into fetal hepatocytes provides an appropriate system for testing allogenic grafting and for modifying immunogenicity of engrafted cells. These results open up new perspectives for cell transplantation through cell banking.

Efficient retroviral-mediated gene transfer into primary culture of murine and human hepatocytes: expression of the LDL receptor.

The ex vivo approach to hepatic gene therapy involves several steps, which include the isolation and culture of hepatocytes, followed by their transduction with a retrovirus. Subsequently, autologous hepatocytes are transplanted. The number of hepatocytes that can be transduced by retroviruses bearing the therapeutic gene is one of the limiting steps that can impair the success of this strategy. We presently describe an experimental approach that leads to improved transduction efficiency in mouse and human hepatocytes in vitro. By using a recombinant retrovirus bearing the Escherichia coli beta-galactosidase gene, we show that addition of growth factors to the cells, namely human hepatocyte growth factor (HGF), allows marked increase in the transduction efficiency in mouse (up to 80%) and human (40%) hepatocytes. Familial hypercholesterolemia (FH) is due to mutation in the low-density lipoprotein (LDL) receptor gene and results in a deficiency in LDL receptors. Transduction of the human LDL receptor cDNA under the transcriptional control of the L-type pyruvate kinase promoter-activator into mouse hepatocytes led to an elevated tissue-specific expression of the human protein. These results suggest that the ex vivo approach remains a promising alternative for hepatic gene therapy.

Detection of rare circulating human colon tumor cells in a nude mouse xenograft model.

A highly sensitive allele specific polymerase chain reaction was developed and applied to the detection of K-ras mutation in human colon tumor cells both in the blood and in tissues. An experimental model of human colon carcinoma cells, carrying a GAT mutation in the 12th codon of the K-ras gene, and grafted into nude mice has been selected for evaluating the occurrence of cells in the course of disseminating into the host. We have found tumor cells circulating in the blood starting 37 days following subcutaneous primary implantation. Occasional micrometastatic deposits could be detected in lymph node draining the xenograft, but no tumor cells were found in lungs and mediastinum. In this experimental model, our results indicate that the mere presence of tumor cells in the blood does not imply the full accomplishment of the multi-step metastatic process.

Long-term histological follow-up of genetically modified myoblasts grafted into the brain.

Although primary muscle cells have been used as intracerebral vehicles for transgene expression in the past, data concerning their long-term survival after grafting into the brain, and the reaction of the host tissue to their implantation are lacking. In order to study these aspects, we have implanted, into the brain, primary muscle cells infected ex vivo with recombinant retroviruses carrying the E. coli LacZ gene. The muscle cells were delivered stereotaxically into different areas of the brain of adult rats and the grafts were analyzed up to 105 days after implantation. Intraventricular implantations did not lead to surviving grafts. In contrast, myoblasts developed when they were grafted into gray or white matter regions. They appeared numerous during the first weeks, but decreased dramatically in number over time. Over months, the grafts appeared to fill up with collagen. Astrocytes elaborated a continuous glia limitans surrounding the implant. Blood vessels coming from the host tissue were found within the grafts. The blood-brain barrier was permanently disrupted within the transplants. beta-Galactosidase activity was abundant during the first weeks, but decreased to a very low level subsequently. This decrease paralleled that of the number of muscle cells. In conclusion, myoblasts transplanted into the adult brain survived only temporarily, which implies a transient transgene expression. In addition, before being eliminated, muscle cells were surrounded by a glia limitans, which may limit exchanges with the host tissue. Altogether, these results suggest that intracerebral transplantation of myoblasts may possibly provide a relevant vehicle only for short-term delivery of a gene product.

Sutton's disease (periadenitis mucosa necrotica recurrens).

Sutton's disease is characterized by giant necrotizing ulcers around minor salivary glands and is of unknown cause. We report a case, review the medical literature, and discuss the treatment of this affliction.

[Adenocarcinomas of the ethmoid sinuses. Epidemiological data]. / Les adénocarcinomes de l'ethmoïde.

OBJECTIVE: The relationship between adenocarcinomas of the ethmoid (ADKE) and wood-dust exposure has been well established. Sino-nasal cancer in wood-workers has been added to the list of occupational disorders in France, as prescribed disease number 47-Bq. PATIENTS AND METHODS: Our data set consisted of 207 cases with sino-nasal cancer (from January 1985 to January 2001). Among these cases, 67.1% were adenocarcinoma. A wood dust exposure has been reported in 96.4% cases. The mean duration of wood dust exposure was 30 years. The mean latency between the end of the exposure and the diagnostic was 10.6 years. RESULTS: Our epidemiological data confirmed those from the biomedical literature. The occupations at greatest risk are furniture workers, sawmill workers, carpentry workers, and other wood product workers. Two components of exposure - duration and average level - contributed independently to the overall elevated risk. The risk is greater among men who were employed in jobs with the highest wood dust exposure and increases with the duration of exposure. CONCLUSIONS: The preinvasive stages of ADKE (mucostasis/cuboïd metaplasia/dysplasia) are still an unverified hypothesis. ADKE were observed in workers who use "hard" woods. The chemical nature of the carcinogenic factor(s) in wood dust is not known. The factors responsible for induction of ADKE in hard wood-workers probably exist in the wood-dust itself. Tannins were suggested as possible contributing agents to induction of ADKE. In addition to wood dust, exposures may include formaldehyde. Given these facts, it should be possible to define preventive measures, so that incidence of ADKE in professional wood and leather workers should decrease.

[Malignant tumors of the ethmoid region. Neurosurgical techniques]. / Tumeurs malignes de la région ethmoïdale. Techniques neurochirurgicales.

We describe the main neurosurgical approaches in use for removing cancers of the ethmoid region: a) the classical subfrontal procedure usually combined with a transfacial approach, b) the sub-fronto-orbito-nasal approach (SFON) now performed in a majority of procedures as long as the tumor does not present with a large intra-cranial and/or maxillary extension. The advantages and drawbacks of both techniques are discussed. The progressive simplification of techniques for anterior cranial base reconstruction is explained and warranted.

[Malignant ethmoid-sphenoidal tumors. 130 cases. Retrospective study]. / Les tumeurs malignes ethmoïdo-sphénoïdales. Cent trente cas. Etude rétrospective.

We report on 130 ethmoidal cancers. 96 (74%) were adenocarcinomas (ADKE). 110 were operated upon between 1984 and 1996: 9.1% T1 + T2, 27.7% T3, 36.2% T4a, 27% T4b. Neoadjuvant chemotherapy was administered in 93 patients (76 ADKE). Combined surgical route was performed 103 times, sub-fronto-orbito-nasal (SFON) route 7 times. Post-operative radiotherapy was performed in 36 patients. Complete clinical and radiological response to chemotherapy was noted in 21.5% of cases (23% of ADKE). Post-operative mortality concerned one patient who died from a pulmonary embolism during the third post-operative week. Morbidity included: 3 transient clinical rhinorrheas, 5 meningitis (one of which was responsible for heavy psycho-intellectual disability), 4 deep suppurations associated with osteitis of the bone flap and two superficial suppurations. 44 patients had a local recurrence (10 ADKE). No recurrence appeared in complete chemoresponders. Systematic preservation of intra-orbital contents did not increase the risk of local failure. Eleven patients (4 ADKE) developed cervical nodes and/or systemic metastasis. Death occurred after a mean of three months following the diagnosis of metastasis. Survival rate was: 60% at 3 years, 51.5% at 5 years, 32.5% at 10 years. ADKE survival rate was: 55% at 3 years, 51.5% at 5 years, 23% at 10 years. Survival ws related to tumoral extension: 75% at 5 and 10 years for T3, 45% at 5 years and 38% at 10 years for T4a, 40% at 3 years and null at 5 years for T4b, 5 and 10 years survival rate of complete chemoresponders are 100% whatever the tumour. Prognosis remained poor for epidermoid carcinomas (survival rate: 36% at 3 years, 0% at 5 years) and for melanomas (mean survival: 19.6 months). Post-operative radiotherapy should be indicated for large tumors T3, T4a and T4b).

[Use of endoscopy in pituitary surgery]. / Utilisation de l'endoscopie dans la chirurgie pituitaire.

Endoscopy has changed many of surgical procedures concerning nasal and paranasal cavities. It has been proposed for transnasal removal of pituitary adenomas. The authors report their experience concerning four pituitary macroadenomas operated with the neurosurgical team of Sainte-Anne hospital. The endonasal trans-septal route seemed to be more anatomical and less traumatic than the rhinoseptal sublabial route. The 30 degrees rigid Hopkins endoscope was a good help for controlling the absence of any tumor remnant in the supra and parasellar regions after complete removal of the tumor performed through the endoscope or with the operating microscope. The authors discuss the advantages and limitations of such endoscopic procedures in the light of recent literature.

[Drainage after thyroid surgery: 264 patients]. / Drainage dans la chirurgie thyroïdienne.

OBJECTIVES: A prospective study was conducted in 1996-1997 in 100 patients who underwent thyroid surgery and who were randomly assigned to receive drainage or not. No statistical difference in complication rate was observed. The aim of the present retrospective study was to assess the consequences of this attitude in patients undergoing surgery since that time and to determine the number of postoperative complications, length of hospital stay, and type of thyroidectomy where cervical drains still appear to be indicated. PATIENTS AND METHODS: Total or partial thyroid surgery was performed in 264 patients between June 1997 and October 2000. Neck dissection was associated with 24 patients. RESULTS: Cervical drains were used in 29 patients (10.9%). Postoperative complications were comparable to those commonly reported. CONCLUSION: Except for neck dissection and mediastinal extension, thyroidectomy can be safely performed without drainage. This attitude reduces the overall hospital stay.