Identification of novel accessible proteins bearing diagnostic and therapeutic potential in human pancreatic ductal adenocarcinoma

Pancreas ductal adenocarcinoma (PDAC) remains a deadly malignancy with poor early diagnostic and no effective therapy. Although several proteomic studies have performed comparative analysis between normal and malignant tissues, there is a lack of clear characterization of proteins that could be of clinical value. Systemically reachable ("potentially accessible") proteins, suitable for imaging technologies and targeted therapies represent a major group of interest. The current study explores potentially accessible proteins overexpressed in PDAC, employing innovative proteomics technologies. In the discovery phase, potentially accessible proteins from fresh human normal and PDAC tissues were ex vivo biotinylated, isolated and identified using 2D-nano-HPLC-MS/MS method. The analysis revealed 422 up-regulated proteins in the tumor, of which 83 (including protein isoforms) were evaluated as potentially accessible. Eleven selected candidates were further confirmed as up-regulated using Western blot and multiple reaction monitoring protein quantification. Of these, transforming growth factor beta-induced (TGFBI), latent transforming growth factor beta binding 2 (LTBP2), and asporin (ASPN) were further investigated by employing large scale immunohistochemistry-based validations. They were found to be significantly expressed in a large group of clinical PDAC samples compared to corresponding normal and inflammatory tissues. In conclusion, TGFBI, LTBP2, and ASPN are novel, overexpressed, and potentially accessible proteins in human PDAC. They bear the potential to be of clinical value for diagnostic and therapeutic applications and merit further studies using in vivo models. Peer reviewe

Peripheral T-cell lymphoma with t(6;14)(p25;q11.2) translocation presenting with massive splenomegaly

Recurrent chromosomal translocations associated to peripheral T-cell lymphomas (PTCL) are rare. Here, we report a case of PTCL, not otherwise specified (NOS) with the karyotype 46,Y,add(X)(p22),t(6;14)(p25;q11) and FISH-proved breakpoints in the IRF4 and TCRAD loci, leading to juxtaposition of both genes. A 64-year-old male patient presented with mild cytopenias and massive splenomegaly. Splenectomy showed diffuse red pulp involvement by a pleomorphic medium- to large-cell T-cell lymphoma with a CD2+ CD3+ CD5− CD7− CD4+ CD8+/− CD30− TCRbeta-F1+ immunophenotype, an activated cytotoxic profile, and strong MUM1 expression. The clinical course was marked by disease progression in the bone marrow under treatment and death at 4months. In contrast with two t(6;14)(p25;q11.2)-positive lymphomas previously reported to be cytotoxic PTCL, NOS with bone marrow and skin involvement, this case was manifested by massive splenomegaly, expanding the clinical spectrum of PTCLs harboring t(6;14)(p25;q11.2) and supporting consideration of this translocation as a marker of biological aggressiveness

Infusion of third-party mesenchymal stem cells after liver transplantation: a phase-1, open-label, clinical study

peer reviewedBackground: Mesenchymal stromal cells (MSC) are multipotent bone mar- row progenitors that have demonstrated significant immunosuppressive effects in various in vivo and in vitro studies. This study aimed to be the first evaluation of the safety and tolerability of MSC infusion after liver transplantation in a prospective, controlled phase-1 study. Methods: 10 liver transplant recipients under standard immunosuppression (TAC-MMF-low dose steroids until day 30) received 1.5–3 9 106/kg third party MSC on post-operative day 3 ` 2. These patients were prospectively compared to a group of 10 control liver recipients. Primary endpoints were MSC infusion toxicity, and incidence of cancer and opportunistic infections at month 6. Secondary endpoints were patient and graft survivals and rejection at month 6, as well as the effects of MSC on recipients’ immune function and on immunohistology of at month 6 graft biopsies. Results: No MSC infusional toxicity was observed. Both groups were comparable in terms of donor and recipient characteristics. There was no difference in primary end-points between control and MSC groups. No patient developed de novo cancer. There was no statistical difference in patient and graft survivals or in rejection rates. There was no graft rejection in the MSC group. Month-6 graft biopsies were not different according to Banff and fibrosis scores. Discussion: This phase 1 study showed excellent tolerability and safety of a single infusion of third-party MSC after liver transplantation. There were no graft safety issues and no excess of immunosuppression after MSC injection. Further analyses of consequences of MSC injection on the immune profile are needed. The possibility of avoiding calcineurin-inhibitors with repeated MSC injections as main immunosuppressive therapy and/of tolerance induction by MSC infusion should be investigated by further studies. This study is in part supported by an ESOT Senior Clinical Research Grant and by the University of Liege

Infusion of third-party mesenchymal stem cells after liver transplantation: a phase-1, open-label, clinical study

peer reviewedBackground: Mesenchymal stromal cells (MSC) are multipotent bone mar- row progenitors that have demonstrated significant immunosuppressive effects in various in vivo and in vitro studies. This study aimed to be the first evaluation of the safety and tolerability of MSC infusion after liver transplantation in a prospective, controlled phase-1 study. Methods: 10 liver transplant recipients under standard immunosuppression (TAC-MMF-low dose steroids until day 30) received 1.5–3 9 106/kg third party MSC on post-operative day 3 ` 2. These patients were prospectively compared to a group of 10 control liver recipients. Primary endpoints were MSC infusion toxicity, and incidence of cancer and opportunistic infections at month 6. Secondary endpoints were patient and graft survivals and rejection at month 6, as well as the effects of MSC on recipients’ immune function and on immunohistology of at month 6 graft biopsies. Results: No MSC infusional toxicity was observed. Both groups were comparable in terms of donor and recipient characteristics. There was no difference in primary end-points between control and MSC groups. No patient developed de novo cancer. There was no statistical difference in patient and graft survivals or in rejection rates. There was no graft rejection in the MSC group. Month-6 graft biopsies were not different according to Banff and fibrosis scores. Discussion: This phase 1 study showed excellent tolerability and safety of a single infusion of third-party MSC after liver transplantation. There were no graft safety issues and no excess of immunosuppression after MSC injection. Further analyses of consequences of MSC injection on the immune profile are needed. The possibility of avoiding calcineurin-inhibitors with repeated MSC injections as main immunosuppressive therapy and/of tolerance induction by MSC infusion should be investigated by further studies. This study is in part supported by an ESOT Senior Clinical Research Grant and by the University of Liege

Targeting the T cell receptor β-chain constant region for immunotherapy of T cell malignancies

Mature T cell cancers are typically aggressive, treatment resistant and associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy (normal) T cells. Unlike B cell depletion, pan–T cell aplasia is prohibitively toxic. We report a new targeting strategy based on the mutually exclusive expression of T cell receptor β-chain constant domains 1 and 2 (TRBC1 and TRBC2). We identify an antibody with unique TRBC1 specificity and use it to demonstrate that normal and virus-specific T cell populations contain both TRBC1+ and TRBC2+ compartments, whereas malignancies are restricted to only one. As proof of concept for anti-TRBC immunotherapy, we developed anti-TRBC1 chimeric antigen receptor (CAR) T cells, which recognized and killed normal and malignant TRBC1+, but not TRBC2+, T cells in vitro and in a disseminated mouse model of leukemia. Unlike nonselective approaches targeting the entire T cell population, TRBC-targeted immunotherapy could eradicate a T cell malignancy while preserving sufficient normal T cells to maintain cellular immunity

Combined analysis of HPV DNA, p16, p21 and p53 to predict prognosis in patients with stage IV hypopharyngeal carcinoma

We examined p16, p21 and p53 expression in combination with the presence of human papillomavirus (HPV) DNA as molecular markers to predict survival in patients with stage IV hypopharyngeal squamous cell carcinoma (HSCC).Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

La biopsie de surface : Indications et modalités pratiques

La biopsie cutanée de surface a pour objet d’échantillonner la couche cornée de l’épiderme ou stratum corneum, formée de cellules matures, différenciées appelées « cornéocytes ». Le principe est d’appliquer à la surface de la peau une lamelle plastique flexible et transparente enduite d’une substance adhésive. Sous l’effet d’une pression exercée sur cette lamelle, la substance adhésive va s’étaler à la surface de la couche cornée et se lier de manière étroite aux cornéocytes. Les forces de cohésion entre la substance adhésive et son support sont supérieures aux forces de cohésion entre les cornéocytes de la couche cornée. Quand une traction est exercée sur la lamelle plastique flexible pour la détacher de la peau, la couche cornée se détache des couches profondes de l’épiderme. La couche cornée reste solidaire du support utilisé. La profondeur d’échantillonnage de la couche cornée dépend du type de substance adhésive et du type de supports utilisés : plus le pouvoir adhésif est fort, plus le clivage de la couche cornée est profond. Deux modalités pratiques existent : la biopsie au cyanoacrylate et le D-squame. En ce qui concerne la biopsie de surface à la colle cyanoacrylate, la lamelle de support en polyéthylène et la substance adhésive (cyanoacrylate liquide à durcissement rapide et non en gel) sont mises en contact au moment de la réalisation du prélèvement : une ou 2 gouttes de colle cyanoacrylate sont déposées sur une lamelle en polyéthylène qui est alors apposée à la surface de la peau. Avant de déposer la colle, il est conseillé de dégraisser la languette de plastique avec de l’alcool. Par contre, il est déconseillé de nettoyer la peau avant le prélèvement sauf au niveau du visage où il faut la démaquiller si nécessaire. Il est important de veiller à déposer la colle de façon excentrique sur la surface de la lamelle plastique afin de ménager une zone indemne de colle qui permettra de la décoller, mais aussi d’identifier le prélèvement au laboratoire. Pendant environ 30 secondes une pression est exercée pour étaler la colle et permettre le déroulement d’une réaction chimique entre la couche cornée et la colle. La lamelle est ensuite doucement retirée ; si le prélèvement tend à se détacher du support, il convient de ne pas insister à tirer par cette extrémité, mais de plutôt terminer de la décoller par l’autre extrémité. Le D-squame consiste en un disque plastique préalablement recouvert d’une substance adhésive (support auto-adhésif) et il suffit de l’appliquer directement à la surface de la peau en exerçant également une pression. La profondeur de couche cornée échantillonnée est plus importante avec la biopsie de surface au cyanoacrylate (épaisseur de 3 à 5 cellules). Les prélèvements sont transmis sans autre manipulation au laboratoire où ils seront colorés afin d’être examinés au microscope. Ces biopsies de surface peuvent être réalisées sur toute la surface corporelle, mais en tenant compte de quelques spécificités. Au niveau des zones pileuse ou chevelues, la biopsie de surface est douloureuse, surtout au cyanoacrylate, et de mauvaise qualité en raison de la mauvaise adhésion avec la couche cornée. Il convient donc de raser ces zones avant de réaliser les biopsies de surface. D’autre part, la cohésion entre les cornéocytes au niveau des paumes et des plantes est importante. Le prélèvement risque d’être plus douloureux tout en donnant un échantillon dont l’épaisseur de couche cornée sera probablement irrégulière. Dans ces localisations à forte cohésion cellulaire, retirer la languette en effectuant un mouvement tournant facilitera la manœuvre. Cependant, certaines pathologiques diminuent la cohésion cellulaire et rendent la biopsie de surface possible. Ce type de biopsie ne peut cependant pas être appliqué sur des lésions érodées où la couche cornée est absente ni sur des lésions suintantes où les sérosités limiteront l’adhésion du support aux cornéocytes. Pour optimaliser le rendement de ces biopsies, il peut être conseillé de réaliser 2 prélèvements afin d’augmenter la représentativité des échantillons. Les indications de la biopsie de surface sont évidemment limitées aux pathologies qui entrainent des modifications de la couche cornée. Les indications les plus fréquentes sont les dermatoses squameuses (ichtyoses et xéroses), érythémato-squameuses et spongiotiques (eczéma, dermatite atopique, dermatite de contact, dermatite séborrhéique, pityriasis rosé de Gibert) et parakératosiques (psoriasis). Parmi les infections cutanées, les pathogènes infiltrant la couche cornée peuvent être observés qu’il s’agisse de champignons, de bactéries ou de parasites. Les dermatomycoses accessibles à la biopsie cutanée de surface sont les teignes, les dermatophytoses, la candidose et le pityriasis versicolor. Parmi les infections bactériennes superficielles, citons l’érythrasma. Enfin, la présence de demodex et d’oxyures, ainsi que la gale peuvent être diagnostiqués. Cependant, pour détecter les parasites de la gale, il convient de réaliser impérativement 2 biopsies de surface successives : la 1ère détache le toit du sillon et la 2ème récolte le parasite. Ces indications sont valables pour les 2 techniques. Les biopsies de surface à la colle cyanoacrylate peuvent également fournir des orientations diagnostiques en cas de pathologie tumorale. Les indications sont les kératoses séborrhéiques, les kératoses actiniques et les lésions pigmentées. Cependant, dans ce cas, un diagnostic formel ne peut être apporté : la biopsie de surface permettra d’orienter le diagnostic vers une lésion maligne ou bénigne selon la présence ou non de mélanocytes ou de cellules pigmentées atypiques. Par exemple, la présence de cornéocytes pigmentés est en faveur d’une lésion pigmenté bénigne. La biopsie cutanée de surface apporte des informations sur l’état in vivo de la couche cornée ; elle représente donc une alternative rapide, simple, non invasive, peu couteuse à la biopsie-punch. Cependant, elle ne permet pas un diagnostic formel et précis ; elle apporte des arguments complémentaires à l’observation clinique afin de le renforcer ou de le confirmer. Comme toujours en dermatopathologie, la confrontation anatomo-clinique reste indispensable. Le matériel nécessaire à la réalisation de ces biopsies de surface (D-squames) peut vous être transmis sur simple demande au laboratoire de dermatopathologie du CHU de Liège. Contact : 04/366.24.07 ou 04/366.24.0

Organized proteomic heterogeneity in colorectal liver metastases and implications for therapies

Introduction : Tumor heterogeneity is a major obstacle for developing effective anti-cancer treatments. Recent studies have pointed at large stochastic genetic heterogeneity within cancer lesions, where no pattern seems to exist that would enable a more structured targeted therapy approach. Aim : Because to date no similar information is available at the protein (phenotype) level, we aimed at characterising the proteomic heterogeneity in human colorectal carcinoma (CRC) liver metastases. Methods & Results : We employed MALDI imaging-guided proteomics and explored the heterogeneity of extracellular distribution of over 1000 proteins we found unexpectedly that all liver metastasis lesions displayed a reproducible, zon- ally delineated, pattern of functional and therapeutic biomarker heterogeneity. Peritumoral region featured elevated lipid metabolism and protein synthesis, the rim of the metastasis displayed increased cellular growth, movement and drug metabolism whereas the center of the lesion was characterized by elevated carbohydrate metabolism and DNA- repair activity. From the aspect of therapeutic targeting zonal expression of known and novel biomarkers was evident, reinforcing the need to select several targets in order to achieve optimal coverage of the lesion. Finally we highlight two novel antigens, LTBP2 and TGFBI, whose expression is a consistent feature of CRC liver metastasis. Conclusions : proteome heterogeneity has a distinct, organized, pattern. This particular hallmark can now be used as a part of the strategy for developing rational therapies based on multiple sets of targetable antigens