Combining immunotherapy with an epidrug in squamous cell carcinomas of different locations: rationale and design of the PEVO basket trial.

Squamous cell carcinomas (SCCs) are among the most frequent solid tumors in humans. SCCs, related or not to the human papillomavirus, share common molecular features. Immunotherapies, and specifically immune checkpoint inhibitors, have been shown to improve overall survival in multiple cancer types, including SCCs. However, only a minority of patients experience a durable response with immunotherapy. Epigenetic modulation plays a major role in escaping tumor immunosurveillance and confers resistance to immune checkpoint inhibitors. Preclinical evidence suggests that modulating the epigenome might improve the efficacy of immunotherapy. We herein review the preclinical and the clinical rationale for combining immunotherapy with an epidrug, and detail the design of PEVOsq, a basket clinical trial combining pembrolizumab with vorinostat, a histone deacetylase inhibitor, in patients with SCCs of different locations. Sequential blood and tumor sampling will be collected in order to identify predictive and pharmacodynamics biomarkers of efficacy of the combination. We also present how clinical and biological data will be managed with the aim to enable the development of a prospective integrative platform to allow secure and controlled access to the project data as well as further exploitations.

Antibodies against the N-terminal domain of annexin A2 in antiphospholipid syndrome.

Annexin A2 (ANXA2, an endothelial cell receptor for plasminogen and tissue plasminogen activator) has been identified as a new autoantigen in antiphospholipid syndrome (APS). The aim of the present study was to evaluate the presence of antibodies against the N-terminal domain of annexin A2 (ANXA2) in primary APS (PAPS). By using a synthetic peptide corresponding to the 31N-terminal amino acids of ANXA2 (ANXA2(N31)) as an antigen, we performed an enzyme-linked immunosorbent assay (ELISA) to measure anti-ANXA2(N31) IgG and IgM antibodies in the serum of PAPS patients (n=19), systemic lupus erythematosus (SLE) patients (n=50) and healthy blood donors (n=106). We did not find any statistically differences between the three groups in terms of IgG and IgM anti-ANXA2(N31) titres. Elevated IgG anti-ANXA2(N31) titres were not observed in the serum of PAPS or SLE patients who had previously tested positive for anti-ANXA2 antibodies. Thus, the ANXA2 N-terminal domain does not appear to be the target antigen for anti-ANXA2 antibodies in APS.

Serum procalcitonin does not differentiate between infection and disease flare in patients with systemic lupus erythematosus.

Systemic erythematosus lupus (SLE) is a common autoimmune disease. Disease flares may mimic infection with fever, inflammatory syndrome and chills, sometimes resulting in a difficult differential diagnosis. Elevated serum procalcitonin (PCT) levels have been reported to be predictive of bacterial infections, but with conflicting results. The value of serum procalcitonin has not been assessed in large series of SLE. We aimed to describe the distribution of PCT levels in SLE patients with and without flares, to assess the factors associated with increased PCT levels, and to determine the positive and negative predictive values of increased PCT for bacterial infection in SLE patients. Hospitalized SLE patients were included in a retrospective study. Serum PCT had been assayed, or a serum sample had been frozen on admission, before treatment modification. Serum PCT, measured by an automated immunofluorometric assay, and SLEDAI were assessed at the same time. Some 53 women (median age: 33.7 years, range 16-76) and seven men (median age: 52.5 years ± 19) were included. The median SLEDAI for patients with flare (n = 16, 28%) was 2 (range: 0-29). Five patients (8%) had systemic infection. Only one patient had increased PCT levels. Men had significantly higher PCT levels than women (0.196 ± 0.23 versus 0.066 ± 0.03, p < 0.01) and a significant correlation was observed between PCT, age, erythrocyte sedimentation rate, and C-reactive protein. We conclude that PCT levels were within the normal range in infected and non-infected SLE patients and there was no ability to differentiate SLE patients with or without bacterial infection.

[Preanalytical guidelines for clinical proteomics investigation of biological fluids]. / Recommandations préanalytiques pour les analyses de protéomique clinique des fluides biologiques.

Research of new diagnosis or prognosis biomarkers is a major challenge for the management of patients with complex pathologies like cancer. Clinical proteomics is one of the recent approaches to identify these biomarkers in biological fluids. Over the last five years, many problems related to the variability and the quality control of these analyses have been observed. This was notably related to the different preanalytical status of each sample. A strong need for standardization of the critical preanalytical phases (collection, transport, processing, storage...) has been therefore recognized. With this goal in mind, working groups of the "Institut national du cancer" (INCa) and the "Société française de biologie clinique" (SFBC) proposed here preanalytical proteomics guidelines for the most common biological fluids: plasma, serum, urine and cerebrospinal fluid. To goal is to provide the basis for the harmonization of the procedures in clinical laboratories and biobanks to allow an optimal use of biological collections.

["Standard" protocol for evaluating the impact of preanalytical variables on peptidic and proteic analytes and standard coding of preanalytical procedures]. / Procédure << standard >> pour tester l'impact des variables préanalytiques sur des analyses peptidiques et protéiques et proposition de codage << standard >> des procédures préanalytiques.

The SFBC Working Group on << Preanalytics and multiplex analyses in proteomics >> is presenting a protocol which will allow harmonization of biospecimen research studies on the impact of different preanalytical variations on peptidic and protein analytes. This protocol is based upon standardization of preanalytical options corresponding to different preanalytical variations and different types of biospecimens (serum, plasma, cedrebrospinal fluid and urine). Application of this protocol will allow, not only harmonization of Biospecimen research, but also elaboration of standard nomenclature of the preanalytical steps.

Procalcitonin at the onset of giant cell arteritis and polymyalgia rheumatica: the GRACG prospective study.

OBJECTIVES: An epidemic pattern has been reported for GCA and PMR. Immunological studies have shown that an unknown antigen activates the dendritic cells of the adventitia and the type 4 toll-like receptors. Procalcitonin (PCT) is an early marker of bacterial infection. The goal of the study was to assess the level of PCT in GCA and PMR at the onset of the disease. METHODS: Patients diagnosed during the 2002-06 period were randomly selected. All the 46 patients fulfilled the ACR or the Hunder criteria, and all blood samples were taken before steroid therapy. RESULTS: PCT was normal in all patients. PCT was slightly increased in men (0.087 +/- 0.023 microg/l) compared with women (0.066 +/- 0.027 microg/l) (P = 0.009), and in PMR (0.092 +/- 0.027 microg/l) compared with GCA (0.068 +/- 0.026 microg/l) (P = 0.018). There was no significant correlation with inflammation markers. CONCLUSIONS: These results are not in favour of a bacterial trigger for GCA or PMR. Increased PCT levels in patients with inflammatory syndrome, GCA-PMR symptoms and negative temporal artery biopsy may rule out the diagnosis of GCA and PMR.

Diagnostic value of an ELISA using a recombinant 54-kDa species-specific protein from Chlamydia pneumoniae.

The aim of this study was to evaluate a 54-kDa recombinant protein encoded by the CPn0980 gene for use in a Chlamydia pneumoniae-specific ELISA. The ability of this affinity-purified protein to detect C. pneumoniae-specific antibodies was evaluated with a panel of 105 serum samples from 62 patients with community-acquired pneumonia. The results of this assay were compared with those obtained with a direct PCR-based detection assay and an outer-membrane complex-based immunoassay. The 54-kDa protein induced specific antibodies following infection of humans, and the recombinant 54-kDa ELISA detected anti-C. pneumoniae IgG and/or IgM antibodies with a sensitivity of 66.7% and a specificity of 79.2% compared with the direct PCR-based detection assay.

Chlamydia pneumoniae IgG serological status and venous thromboembolism: a cross-sectional hospital based study.

OBJECTIVE: To search for a link between Chlamydia pneumoniae serological status and venous thromboembolic disease. METHODS: From March 1992 to October 1999, we conducted a cross-sectional hospital-based study of consecutive unselected outpatients referred to us for clinical suspicion of venous thromboembolism. We compared the Chlamydia pneumoniae serological status with respectively, the venous thromboembolism, the deep vein thrombosis and the proximal deep vein thrombosis status. RESULTS: Among 1193 patients registered for suspected venous thromboembolism, 1010 samples were available (499 negative and 511 positive patients for venous thromboembolism). Seventy-nine patients were Chlamydia pneumoniae positive. Our work failed to demonstrate any clear association between Chlamydia pneumoniae and venous thromboembolism status. Nevertheless, we identified a statistical difference regarding Chlamydia pneumoniae seropositivity and proximal vein thrombosis status (adjusted odds ratio of 1.70, CI95%: 1.05 to 2.77). CONCLUSION: The presence of Chlamydia pneumoniae antibodies might be a minor risk factor for venous thrombosis.

Serological investigation of Chlamydia trachomatis heat shock protein 10.

The humoral immune response to Chlamydia trachomatis 10-kDa heat shock protein (Chsp10) in populations of Russian and French origin was studied by using a recombinant Chsp10 enzyme-linked immunosorbent assay. A physiological but not a serological correlation of Chsp10 exposure with Chsp60 exposure was observed in the Russian population. In the French population studied, there was a significant association between detection of anti-r-Chsp10 immunoglobulin G (IgG) antibodies and chronic genital tract infections. Chsp10 residues 50 to 67 were found to contain an immunodominant although not universal B epitope. Cross-reactions with Chlamydia pneumoniae or Escherichia coli GroES protein are limited but may occur. Our study suggests that detection of anti-Chsp10 IgG antibodies is associated with chronicity of C. trachomatis genital tract infection and does not parallel that of anti-Chsp60 IgG antibodies.

The C-terminal domain is essential for protective activity of the Bordetella pertussis adenylate cyclase-hemolysin.

The adenylate cyclase-hemolysin of Bordetella pertussis consists of a cell-invasive N-terminal adenylate cyclase domain linked to a C-terminal RTX hemolysin containing extensive glycine-rich repeats. The toxin is an essential virulence factor required in the initial stages of infection. Adenylate cyclase-hemolysin was also shown to be a potent vaccinating antigen inducing protection against B. pertussis colonization of the mouse respiratory tract. This protective activity depends on a posttranslational fatty-acylation modification. We used a set of deletion derivatives of the recombinant adenylate cyclase-hemolysin to localize the protective epitopes on the 1,706-residue toxin. We show that specific anti-adenylate cyclase-hemolysin antibodies present in the sera of B. pertussis-infected mice and humans are directed predominantly against the modification-and-repeat portion of the toxin, contained in the last 800 residues of the adenylate cyclase-hemolysin. These antibodies appear to recognize conformational epitopes present only in a structure formed by the intact C-terminal half of the toxin. There was no correlation between the capacity of the truncated adenylate cyclase-hemolysin derivatives to induce both toxin-neutralizing antibodies upon immunization of mice and protective immunity. However, only the truncated proteins which were recognized by the sera of infected mice and humans and which had their last 800 residues intact had the capacity to induce protection of mice against colonization by B. pertussis. This indicates that the structure of the modification-and-repeat region of adenylate cyclase-hemolysin is critical for its protective activity.

Cloning and sequence of the Bordetella bronchiseptica adenylate cyclase-hemolysin-encoding gene: comparison with the Bordetella pertussis gene.

The cyaA gene from Bordetella bronchiseptica (Bb), encoding the adenylate cyclase-hemolysin (AC-Hly), has been cloned and its complete nucleotide sequence has been determined. The deduced amino-acid sequence was compared to the AC-Hly from B. pertussis (Bp) and the main differences were found in the C-terminal repeat region of the molecule.

Comparison of polymerase chain reaction, culture, and western immunoblot serology for diagnosis of Bordetella pertussis infection.

Polymerase chain reaction (PCR) amplification of the pertussis toxin promoter region was used to detect Bordetella pertussis infection in nasopharyngeal aspirates collected from 24 infants and children infected with pertussis and 13 adult contacts during an epidemiological study. The sensitivity of this PCR assay was approximately one bacterium, and the assay was specific for B. pertussis in tests with other Bordetella species and other respiratory pathogens. The pertussis case definition required a cough with a duration of more than 21 days for infants and children and laboratory confirmation by serology as the primary detection method for infants, children, and adults. The sensitivity of PCR and culture on Bordet-Gengou agar medium was assessed with regard to the case definitions. In the group of infants and children (index cases), the sensitivities of the culture and the PCR were 54.1% (13 of 24) and 95.8% (23 of 24), respectively. In the adult group (household contacts), the sensitivities of the two methods were 15.4% (2 of 13) and 61.5% (8 of 13), respectively. PCR combined with pertussis-specific serology appears to be a useful tool for diagnosis of pertussis especially in epidemiological studies.

CyaC-mediated activation is important not only for toxic but also for protective activities of Bordetella pertussis adenylate cyclase-hemolysin.

Bordetella pertussis adenylate cyclase-hemolysin (AC-Hly), encoded by the cyaA gene, belongs to the RTX family of toxins with extensive glycine-rich repeats in the carboxy-terminal portion. AC-Hly possesses both adenylate cyclase toxic and hemolytic activities that depend on a posttranslational modification mediated by the product of the cyaC gene. An improved system for AC-Hly synthesis and activation in Escherichia coli was developed. The results show that with purified AC-Hly (i) increased expression of the cyaC gene leads to a higher proportion of activated AC-Hly, (ii) the increase in protective activity of the activated recombinant AC-Hly correlates with the increase in its invasive and hemolytic activities, and (iii) the activated recombinant AC-Hly, but not the nonactivated recombinant AC-Hly, is a protective antigen against B. pertussis infection in a murine respiratory model. This suggests that possibly an immunodominant epitope required for protective activity is linked to the CyaC-mediated modification. Surprisingly, the protective and hemolytic activities of activated recombinant AC-Hly were lower than those of AC-Hly produced by B. pertussis, while its invasive activity was higher. This indicates that the modification of AC-Hly in B. pertussis and that in E. coli may differ.