Prospective study of cutaneous side-effects associated with the BRAF inhibitor vemurafenib: a study of 42 patients.

BACKGROUND: BRAF inhibitors are being developed for the treatment of metastatic melanoma harboring a V600E mutation. The use of vemurafenib significantly increases progression-free survival (PFS) and overall survival (OS) in this population of patients, but is associated with numerous adverse skin reactions. PATIENTS AND METHODS: We carried out a systematic dermatologic study of 42 patients treated with vemurafenib. We collected detailed dermatologic symptoms, photos and biopsy specimens of the skin lesions which enabled us to classify the side-effects. The management and evolution of the skin symptoms are also reported. RESULTS: All patients presented with at least one adverse skin reaction. The most common cutaneous side-effects consisted in verrucous papillomas (79%) and hand-foot skin reaction (60%). Other common cutaneous toxic effects were a diffuse hyperkeratotic perifollicular rash (55%), photosensitivity (52%) and alopecia (45%). Epidermoid cysts (33%) and eruptive nevi (10%) were also observed. Keratoacanthomas (KA) and squamous cell carcinoma (SCC) occurred in 14% and 26% of the patients, respectively. CONCLUSIONS: These cutaneous side-effects are cause of concern due to their intrinsic potential for malignancy or because of their impact on patients' quality of life. Management of this skin toxicity relies on symptomatic measures and sun photoprotection.

ViralORFeome: an integrated database to generate a versatile collection of viral ORFs.

Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins.

Force spectroscopy of barnase-barstar single molecule interaction.

Results of the single molecule force spectroscopy study of specific interactions between ribonuclease barnase and its inhibitor barstar are presented. Experimental data obtained for the force loading rate ranging 2-70 nN/s are well approximated by a single straight line, from which the dissociation barrier of the width of 0.12 nm and height of 0.75-0.85 × 10(-19)J can be inferred. The measured value of specific interaction does not depend on the NaCl concentration. This apparently contradicts the well-known dependence of the binding energy of this pair on the salt concentration, but such a "contradiction" is explained by the insensitivity of the force spectroscopy data to the relatively long-range electrostatic interaction. The latter essentially contributes to the value of barnase-barstar binding energy revealed by biochemical measurements, and it is exactly this electrostatic interaction which is influenced by the salt concentration.

Study of nitrogen implantation in Ti surface using plasma immersion ion implantation & deposition technique as biocompatible substrate for artificial membranes.

The present investigation reports the modification of Ti substrates by a plasma technique to enhance their physio-chemical properties as biocompatible substrates for the deposition of artificial membranes. For that purpose, nitrogen ions are implanted into Ti substrate using the plasma immersion ion implantation & deposition (PIII&D) technique in a capacitively coupled radio frequency plasma. The plasma was characterized using optical emission spectroscopy, together with radio frequency compensated Langmuir probe, while the ion current towards the substrate was measured during the implantation process using an opto-electronic device. X-ray photoelectron spectroscopy (XPS) was used for chemical analysis of the surface, confirming the presence of δ-TiN. The penetration depth of the nitrogen ions into the Ti substrate was measured using secondary ions mass spectroscopy (SIMS) while the morphological changes were observed using atomic force microscopy (AFM). A calorimetric assay was used to prove that the TiN samples maintain the biocompatibility of the untreated Ti surface with its native oxide layer. The ion implantation increases the load bearing ability of Ti surface by the formation of α-Ti(N) and δ-TiN phases on the sub-surface of Ti, and maintains the bio compatibility of Ti surface. After the plasma treatment a thin layer of chitosan (CH) was deposited in order to provide a moisturizing matrix for the artificial membrane of 1,2-dipalmitoyl-sn-3- phosphor glycerocholine (DPPC). The CH and subsequently the DPPC were deposited on the plasma deposited TiN substrate by using physical vapor deposition. The formation of artificial membranes was confirmed by AFM, measuring the topography at different temperatures and performing force curves.

Force-clamp spectroscopy with a small dithering of AFM tip, and its application to explore the energy landscape of single avidin-biotin complex.

We have recently developed a new method for directly measuring the spring constant of single molecules and molecular complexes on a real-time basis [L.A. Chtcheglova, G.T. Shubeita, S.K. Sekatskii, G. Dietler, Biophys. J. 86 (2004) 1177]. The technique combines standard force spectroscopy with a small dithering of tip. Changes in the amplitude of the oscillations are measured as a function of the pulling-off force to yield the spring constant of the complex. In this report, we present the first results of combination of this approach with the force-clamp spectroscopy. The standard atomic-force microscope has been supplemented with an electronic unit, which is capable of realizing an arbitrary force function, and permits the force-loading regime to be interrupted at any time. Using this method, the time needed to rupture a single bond can be measured as a function of the force that is required to maintain the complex in a stretched condition. The energy landscape of the avidin-biotin complex is explored and discussed.

Human papillomavirus genotype as a major determinant of the course of cervical cancer.

PURPOSE: To determine whether the prognosis of invasive cancers of the uterine cervix is related to the type of human papillomavirus (HPV) associated with the tumor. PATIENTS AND METHODS: Two hundred ninety-seven patients with invasive cervical cancer were prospectively registered from 1986 to 1994. HPV typing was performed on DNA extracted from frozen tumor specimens by means of Southern blot hybridization (SBH) and polymerase chain reaction (PCR) techniques. The median follow-up was 38 months. RESULTS: HPV sequences were detected in 246 patients (83%): 150 patients had HPV16, 31 patients had HPV18, and 14 patients had one of the intermediate-oncogenic-risk HPV types (HPV31, 33, 35, 52, 58). In 51 patients, HPV type remained undetermined, and in 51 patients, no viral sequences were found. No significant associations were observed between virologic data and tumor stage or node status. The 5-year disease-free survival (DFS) rate was 100% for patients with intermediate-risk HPV-associated tumors, 58% for patients with HPV16-positive tumors, and 38% for patients with HPV18-positive tumors (P = .02). In multivariate analysis, patients with HPV18-associated tumors had a relative risk (RR) of death 2.4 times greater (95% confidence interval [CI], 1.29-4.59) than that for patients with HPV16, and 4.4 times greater (95% CI, 3.48-5.32) than that for patients with a tumor associated with a viral type different from HPV16/18. CONCLUSION: The prognosis for invasive cancers of the uterine cervix is dependent on the oncogenic potential of the associated HPV type. HPV typing may provide a prognostic indicator for individual patients and is of potential use in defining specific therapies against HPV-harboring tumor cells.

Comparison of outcome, clinical, laboratory, and immunological features in 164 children and adults with T-ALL. The Groupe d'Etude Immunologique des Leucémies.

T cell acute lymphoblastic leukemia (T-ALL) is a disease of poor prognosis, usually studied separately in adults or children. Controversial clinical or biological prognostic factors have been reported, and little information is available regarding the frequency and prognostic value of membrane markers identified on blast cells. We report an extensive investigation of the incidence and prognostic value of immunophenotypic, clinical, and laboratory data in T-ALL, performed as a multicenter study in 164 patients. CD7, CD5, and CD2 were the most frequently expressed T cell antigens, and CD2 and CD4 were more frequently observed in children than in adults. MHC class II, CD9, and CD10 were observed in 16, 22, and 21% of the patients, respectively. The male prevalence of T-ALL, and the more frequent presence of a tumoral syndrome in children were confirmed, but mediastinal enlargement and high leukocyte counts were observed in less than half the patients. A poor prognosis was associated with the expression of MHC class II in adults. The presence of a mediastinal mass appeared to be of good prognosis in adults, as well as a leukocyte count lower than 100 x 10(9)/l whatever the age of the patient.

Quantification of cellular adhesion molecules on malignant B cells from non-Hodgkin's lymphoma.

The expression of five cellular adhesion molecules (CAMs), CD54, CD58, CD11a, CD29 and CD49d, was studied in 113 B cell non-Hodgkin's lymphomas (NHL) and in normal B cells from 12 control lymph nodes. Rather than reporting the percentage of positive cells, which does not discriminate between NHL subtypes, we quantified the intensity of CAM expression using flow cytometry. Apart from CD49d the expression of all these CAMs was statistically different among the NHL subtypes as defined by the REAL classification. Low grade NHL-small lymphocytic, follicular and mantle cell lymphoma--which are derived from quiescent cells and show an indolent disease course, expressed low levels of CAMs. Conversely, high grade NHL-diffuse large cell lymphoma--which are derived from proliferating cells and are clinically aggressive, expressed high levels of CAMs. These results indicate that in malignant NHL B cell tumour growth and clinical aggressiveness may be related to the adhesive capacities of the tumour cells.

Characteristics of pro-T ALL subgroups: comparison with late T-ALL. The Groupe d'Etude Immunologique des Leucémies.

A group of 30 acute lymphoblastic leukemias (ALL) with the early pro-T phenotype CD7+/cCD3+/CD1-/CD3-/CD4-/CD8-were identified among 103 newly diagnosed ALL with T-lineage markers (T-ALL). Pro T-ALL was more often observed in adults, and showed a lower incidence of hyperleukocytosis than more mature T-ALL. Mediastinal masses and polar acid phosphatase positivity in blast cells were however observed with the same frequency in pro T-ALL and late T-ALL, and rearrangements of both T-cell receptor (TCR) beta and gamma genes were observed in half the pro T-ALL cases tested. The expression of CD34, DR, and myeloid (My) markers was significantly more frequent in pro T-ALL than in late T-ALL, and these three features were strongly linked. TCR gene rearrangements were two to three times more frequent in CD34- and My-pro T-ALL. However, both CD34+ and My+ pro T-ALL showed an incidence of mediastinal masses and polar acid phosphatase positivity similar to this observed in CD34- and My- cases. This supports the assumption that both types of ALL indeed are engaged in the T-lineage, and confirms intracytoplasmic cCD3 as the earliest marker for this lineage. Moreover, CD34 appears to persist up to an early stage of T-cell maturation, where the cells retain myeloid potentiality. Loss of CD34 correlates with TCR-beta gene rearrangement and definitive commitment to the T lineage. Event-free survival analysis suggested a poorer outcome for pro T-ALL in adult patients.

Immunophenotypic patterns and cytogenetic anomalies in acute non-lymphoblastic leukemia subtypes: a prospective study of 432 patients.

This study prospectively analysed the relationships between immunophenotypic and cytogenetic features of blast cells in 432 acute non-lymphoblastic leukemias (ANLL) at presentation. An abnormal karyotype was detected in 232 cases (54%). These abnormalities were related to immunophenotypic markers as detected using a consensual panel of monoclonal antibodies allowing lineage assignment and investigation of myeloid marker expression on blast cells. In univariate analysis, CD9, CD10, CD15, CD34 and TdT expression appeared significantly associated with chromosomal anomalies. Multivariate analysis identified CD34 and CD9 expression as independently predictive of the presence of at least one cytogenetic abnormality (P < 10(-4) and P < 0.03, respectively). Significant associations between immunophenotypic and karyotypic features were observed both within individual FAB subgroups and independently from morphological criteria. Specific features were seen in five ANLL entities: M0 or M1/B lineage antigen positivity/t(9;22) or del(11)(q23); M2/CD13-/t(8;21); M4/CD13+, CD34+, CD36+/inv(16); M4 or M5/lack of B lineage antigen/del(11)(q23) or t(9;11). More practically, and although the relationships demonstrated only represent a fraction of homogeneous immunophenotypic subgroups, identification of such immunophenotypic features should prompt careful karyotypic examination, eventually using molecular biology analysis on non-growing cells.

[HPV contamination of endocavity vaginal ultrasound probes]. / Contamination des sondes d'échographie endovaginale par les papillomavirus humains.

While the use of endovaginal ultrasound probes is increasing, the risk of contamination of women with endocavity vaginal probes was not assessed. In particular, the clinical significance of detection of human papillomavirus (HPV) infection, the most common sexually transmitted viral infection, on endovaginal ultrasound probes is uncertain. The recommendations of good practice for decontamination of these probes developed by the High Council for Public Health and the Academy of Medicine have not been evaluated. The objective of this article was to review recent publications concluding to the detection of HPV and human cellular DNA after gynecological examination and disinfection of vaginal ultrasound probes.

Premalignant lesions and cancers of the skin in the general population: evaluation of the role of human papillomaviruses.

To evaluate the role of human papillomaviruses (HPV) in the development of premalignant lesions and cancers of the skin in the general population, 314 biopsies obtained from 227 patients with benign neoplasms, premalignant lesions, and cancers of the skin and from 25 patients with squamous cell carcinoma of the lip were analyzed by Southern blot hybridization. DNA probes specific for various cutaneous and genital HPV types were used in hybridizations conducted under nonstringent or stringent conditions. HPV DNA sequences were only detected in eight specimens obtained from six patients: HPV 34 in one case of periungual Bowen's disease, HPV 36 and an as yet uncharacterized HPV in two cases of actinic keratosis, HPV 20 in one case of basal cell carcinoma, an as yet unrecognized HPV in one case of squamous cell carcinoma, and HPV 16 in one case of squamous cell carcinoma of the lip. None of the specimens of cutaneous horn and keratoacanthoma contained detectable HPV DNA. In contrast, HPV DNA sequences, mostly HPV 16, were detected in 13 of 23 cases of anogenital Bowen's disease and invasive Bowen's carcinoma. HPV DNA sequences were not detected in 90 cutaneous samples further analyzed by the polymerase chain-reaction technique, using amplification primers that contain conserved sequences among the genomes of HPV. These results strongly suggest that the known HPV types play only a minor role, if any, in skin carcinogenesis in the general population.

A susceptibility locus for epidermodysplasia verruciformis, an abnormal predisposition to infection with the oncogenic human papillomavirus type 5, maps to chromosome 17qter in a region containing a psoriasis locus.

Epidermodysplasia verruciformis (EV) is a rare genodermatosis characterized by an abnormal susceptibility to infection with a specific group of related human papillomavirus (HPV) genotypes, including the oncogenic HPV5 associated with the skin carcinomas developing in about half of EV patients. EV is usually considered as an autosomal recessive condition. Taking EV as a model to identify a locus underlying the susceptibility to HPV infections, we performed a genome-wide search for linkage with 255 microsatellite genetic markers in three consanguineous EV families comprising six patients, using the homozygosity mapping approach. Homozygosity restricted to affected individuals was observed for a marker of chromosome 17q (D17S784) in two families and a marker about 17 centiMorgan (cM) distal (D17S1807) in the third family. Ten additional microsatellite markers spanning 29 cM in this region were analyzed. Two-point lod score values greater than 3 were obtained for four markers and multipoint linkage analysis yielded a maximum lod score of 10.17 between markers D17S939 and D17S802. Recombination events observed in two families allowed a candidate region for the EV susceptibility locus to be mapped to the 1 cM region defined by these two markers. The EV locus (named EV1) is included in the 17qter region recently found to contain a dominant locus for the susceptibility to familial psoriasis. It has been shown that patients suffering from psoriasis are likely to constitute the reservoir of HPV5. It is thus tempting to speculate that distinct defects affecting the same gene may be involved in the two skin conditions.

Human papillomavirus (HPV) types specific of epidermodysplasia verruciformis detected in warts induced by HPV3 or HPV3-related types in immunosuppressed patients.

Epidermodysplasia verruciformis (EV) is characterized by an abnormal genetic predisposition to infection with specific types of human papillomavirus (HPV). Specific defects of the cell-mediated immunity and/or of the control of HPV infection in keratinocytes are assumed to be involved in the development of the disease. As a model to test this hypothesis, we have studied the prevalence of EV-specific HPV in skin warts of 56 immunosuppressed patients. All main types of cutaneous HPV (HPV1, 2, 3, 4, 10, and 28) responsible for skin warts in the general population were detected by blot hybridization. EV-specific HPV (HPV5, 20, and 23) were detected in three patients. Four additional patients were found infected with HPV49, first characterized in the course of this study, and found to be related to EV HPV. A most important finding was that HPV5, 20, 23, and 49 were always codetected with HPV3 or the related types HPV10 and 28. None of the specimens showed the typical clinical morphology of EV lesions. In none of these specimens was the specific cytopathic effect of EV recognized; instead that of HPV3 and related types was seen. No evidence for productive EV HPV DNA replication was obtained for the three specimens that could be further analyzed by in situ hybridization. Our data suggest that HPV3 infection favors infection with EV HPV in immunosuppressed patients but that the full expression of EV HPV is usually restricted as in the general population.

Psoriasis: A possible reservoir for human papillomavirus type 5, the virus associated with skin carcinomas of epidermodysplasia verruciformis.

Recent polymerase chain reaction data have shown that most human papillomavirus (HPV) genotypes associated with epidermodysplasia verruciformis (EV) are widespread; however, HPV5 associated with EV skin carcinomas has only rarely been detected in non-EV patients. To identify the reservoir of this virus, we examined 335 sera from different groups of patients for the presence of HPV5 antibodies by an enzyme-linked immunosorbent assay test based on HPV5 virus-like particles. The prevalence of antibodies reacting with HPV5 virus-like particles was found to be significantly higher in psoriatic patients (24.5%) than in other groups (2-5%), including patients with atopic dermatitis and renal transplant recipients. Analysis of scrapings of lesional and uninvolved skin by a nested polymerase chain reaction method, using degenerate EV HPV primers, disclosed HPV DNA in 91.7% of 48 psoriatic skin samples and 35.5% of 31 atopic dermatitis specimens. Eleven EV HPV genotypes, most frequently HPV5 and HPV36, and a putative novel genotype (PsoX1) were identified in psoriasis. Five EV HPV genotypes and two putative novel genotypes (ADX1 and ADX2) were detected in atopic dermatitis patients. HPV5 was not found in atopic dermatitis patients. Using type specific primers, HPV5, HPV36, and HPV1 were found in 89.4%, 84.2%, and 42.1% of specimens from psoriatic patients, whereas HPV36 was detected in 22.5% of specimens from atopic dermatitis patients. HPV16 was never detected. On the whole, 27 HPV5 and 13 HPV36 DNA variants were disclosed after sequencing amplification products. Our data confirm that EV HPV are widespread and point to psoriasis as a reservoir for HPV5. Whether HPV5 is involved in the pathogenesis of psoriasis remains to be determined.

Antibodies to human papillomavirus type 5 are generated in epidermal repair processes.

We reported previously that patients with psoriasis harbored at a very high frequency DNA sequences of the oncogenic human papillomavirus type 5 (HPV5) associated with epidermodysplasia verruciformis. Moreover anti-HPV5 antibodies were detected in 25% of the cases. Our aim was to find out whether keratinocyte hyperproliferation and/or autoimmunity could be responsible for HPV5 expression in psoriasis. We found that epidermal repair in patients with extensive second degree burns (n = 19) is frequently associated with the generation of anti-HPV5 antibodies. In patients with autoimmune bullous diseases (n = 118), a condition in which keratinocyte proliferation is involved in repair mechanisms, the prevalence of anti-HPV5 antibodies (15%-25%) was similar to that reported in psoriasis and significantly higher than that (5%) observed in individuals with no known history of human papillomavirus infection (n = 119). A high detection rate (57.9%) of HPV5 DNA was observed in patients with bullous diseases. Anti-HPV5 antibodies were found in patients with autoimmune connective tissue disorders with cutaneous involvement (n = 40) as frequently as in patients with bullous diseases. HPV5 DNA was detected in one of the 10 patients studied. In contrast, the prevalence of anti-HPV5 antibodies in patients with autoimmune neurological disorders (n = 47) and in patients with common warts (n = 28) or invasive carcinomas of the skin (n = 40) was as low as in the general population. It is worth stressing that a similar prevalence of antibodies against HPV1 was found in all groups studied. Our data strongly suggest that extensive keratinocyte proliferation is a major factor for the generation of anti-HPV5 antibodies and that autoimmunity may contribute to this phenomenon. It remains to be determined whether HPV5 and other human papillomavirus genotypes associated with epidermodysplasia verruciformis contribute to the hyperproliferation of keratinocytes occurring in epidermal repair and in psoriasis.

Evidence for a nonallelic heterogeneity of epidermodysplasia verruciformis with two susceptibility loci mapped to chromosome regions 2p21-p24 and 17q25.

Epidermodysplasia verruciformis is a rare genodermatosis associated with a high risk of skin cancer. This condition is characterized by an abnormal susceptibility to specific related human papillomavirus genotypes, including the oncogenic HPV5. Epidermodysplasia verruciformis is usually considered as an autosomal recessive disease. We recently mapped a susceptibility locus for epidermodysplasia verruciformis (EV1) to chromosome 17qter within the 1 cM interval between markers D17S939 and D17S802. We report here the genotyping for 10 microsatellite markers spanning 29 cM around EV1 in two consanguineous epidermodysplasia verruciformis families from Colombia (C2) and France (F1) comprising five patients and two patients, respectively. Using homozygosity mapping, linkage with 17qter markers was observed for family C2 only. Multipoint linkage analysis yielded maximum multipoint LOD-score values above 10 between markers D17S1839 and D17S802 encompassing the EV1 locus. A genome-wide search performed in family F1 yielded evidence for linkage between epidermodysplasia verruciformis and the chromosomal 2p marker D2S365. Nine additional microsatellite markers spanning 15 cM in this region were analyzed. Assuming an autosomal recessive inheritance with a complete penetrance, the expected maximum two-point LOD-score value of 1.8 was obtained for three markers and multipoint linkage analysis yielded a maximum LOD-score value of 3. 51 between markers D2S2144 and D2S392. Haplotype analysis allowed to map a candidate region for a second epidermodysplasia verruciformis susceptibility locus (EV2) within the 8 cM interval between markers D2S171 and D2S2347 of the 2p21-p24 region. In contrast, linkage with 2p markers was excluded for family C2 and for the three families in which we mapped EV1 previously. The disclosure of two susceptibility loci for epidermodysplasia verruciformis provides evidence for a nonallelic heterogeneity in this disease.

A possible vertical transmission of human papillomavirus genotypes associated with epidermodysplasia verruciformis.

Epidermodysplasia verruciformis (EV) is characterized by an abnormal genetic susceptibility to a group of related human papillomavirus (HPV) genotypes, including the oncogenic HPV5 and HPV8. The mode of transmission of these viruses remains unknown. In view of the rare incidence of EV, we had a unique opportunity to perform a virologic study of the amniotic fluid and placenta from an EV patient infected with HPV5, HPV8, several other EV HPV, and HPV3. The child was born by cesarean section and the amniotic fluid specimen was taken prior to rupture of membranes. Analysis of the amniotic fluid and placenta specimens by a nested polymerase chain reaction method, using degenerate EV HPV primers or type-specific HPV primers, disclosed the presence of the variants of EV HPV5, HPV8, HPV24, and HPV36, and of HPV3 detected in the skin lesions of the patient. HPV5, HPV8, HPV24, and HPV3 were also detected in the placenta. No viral sequences were detected in peripheral blood mononuclear cells collected 2 y and 6 mo before cesarean section, rendering an hematogenous transmission unlikely. The same HPV variants were also detected in cervical scrapes taken from the patient, which may suggest an ascending infection of the placenta. This first report of the detection of EV HPV in amniotic fluid, placenta, and cervical scrapes from an EV patient renders vertical transmission of EV HPV likely.