High-throughput assessment of the antibody profile in ovarian cancer ascitic fluids

The identification of effective biomarkers for early diagnosis, prognosis, and response to treatments remains a challenge in ovarian cancer (OC) research. Here, we present an unbiased high-throughput approach to profile ascitic fluid autoantibodies in order to obtain a tumor-specific antigen signature in OC. We first reported the reactivity of immunoglobulins (Igs) purified from OC patient ascites towards two different OC cell lines. Using a discovery set of Igs, we selected tumor-specific antigens from a phage display cDNA library. After biopanning, 700 proteins were expressed as fusion protein and used in protein array to enable large-scale immunoscreening with independent sets of cancer and noncancerous control. Finally, the selected antigens were validated by ELISA. The initial screening identified eight antigenic clones: CREB3, MRPL46, EXOSC10, BCOR, HMGN2, HIP1R, OLFM4, and KIAA1755. These antigens were all validated by ELISA in a study involving ascitic Igs from 153 patients (69 with OC, 34 with other cancers and 50 without cancer), with CREB3 showing the highest sensitivity (86.95%) and specificity (98%). Notably, we were able to identify an association between the tumor-associated (TA) antibody response and the response to a first-line tumor treatment (platinum-based chemotherapy). A stronger association was found by combining three antigens (BCOR, CREB3, and MRLP46) as a single antibody signature. Measurement of an ascitic fluid antibody response to multiple TA antigens may aid in the identification of new prognostic signatures in OC patients and shift attention to new potentially relevant targets

Variations of the UNC13D gene in patients with autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is caused by genetic defects decreasing Fas function and is characterized by lymphadenopathy/splenomegaly and expansion of CD4/CD8 double-negative T cells. This latter expansion is absent in the ALPS variant named Dianzani Autoimmune/lymphoproliferative Disease (DALD). In addition to the causative mutations, the genetic background influences ALPS and DALD development. We previously suggested a disease-modifying role for the perforin gene involved in familial hemophagocytic lymphohistiocytosis (FHL). The UNC13D gene codes for Munc13-4, which is involved in perforin secretion and FHL development, and thus, another candidate for a disease-modifying role in ALPS and DALD. In this work, we sequenced UNC13D in 21 ALPS and 20 DALD patients and compared these results with sequences obtained from 61 healthy subjects and 38 multiple sclerosis (MS) patients. We detected four rare missense variations in three heterozygous ALPS patients carrying p.Cys112Ser, p.Val781Ile, and a haplotype comprising both p.Ile848Leu and p.Ala995Pro. Transfection of the mutant cDNAs into HMC-1 cells showed that they decreased granule exocytosis, compared to the wild-type construct. An additional rare missense variation, p.Pro271Ser, was detected in a healthy subject, but this variation did not decrease Munc13-4 function. These data suggest that rare loss-of-function variations of UND13D are risk factors for ALPS development

Synthesis and Evaluation of 99mTc-Labelled Monoclonal Antibody 1D09C3 for Molecular Imaging of Major Histocompatibility Complex Class II Protein Expression

Purpose: It is known that major histocompatibility complex class II protein HLA-DR is highly expressed in B-cell lymphomas and in a variety of autoimmune and inflammatory diseases. Therefore, a radiolabelled fully humanized IgG4 monoclonal antibody (mAb) can provide useful prognostic and diagnostic information. Aims of the present study were to radiolabel an anti-HLA-DR mAb with technetium-99m and to evaluate its binding specificity, tissue distribution and targeting potential. Procedures: For labelling, we compared a direct method, after 2-mercaptoethanol (2-ME) reduction of disulphide bonds, with a two-step labelling method, using a heterobifunctional succinimidyl-6-hydrazinonicotinate hydrochloride chelator. Several in vitro quality controls and in vivo experiments in mice were performed. Results: We obtained highest labelling efficiency (LE, 998%) and specific activity (SA; 5,550 MBq/mg) via the direct method. In vitro quality control showed good stability, structural integrity and retention of the binding properties of the labelled mAb. The biodistribution in mice showed high and persistent uptake in spleen and suggests kidney and liver-mediated clearanc

Thyroid Cancer Imaging In Vivo by Targeting the Anti-Apoptotic Molecule Galectin-3

Background The prevalence of thyroid nodules increases with age, average 4-7% for the U.S.A. adult population, but it is much higher (19-67%) when sub-clinical nodules are considered. About 90% of these lesions are benign and a reliable approach to their preoperative characterization is necessary. Unfortunately conventional thyroid scintigraphy does not allow the distinction among benign and malignant thyroid proliferations but it provides only functional information (cold or hot nodules). The expression of the anti-apoptotic molecule galectin-3 is restricted to cancer cells and this feature has potential diagnostic and therapeutic implications. We show here the possibility to obtain thyroid cancer imaging in vivo by targeting galectin-3. Methods The galectin-3 based thyroid immuno-scintigraphy uses as radiotracer a specific 99mTc-radiolabeled mAb. A position-sensitive high-resolution mini-gamma camera was used as imaging capture device. Human galectin-3 positive thyroid cancer xenografts (ARO) and galectin-3 knockout tumors were used as targets in different experiments in vivo. 38 mice with tumor mass of about 1 gm were injected in the tail vein with 100 ?Ci of 99mTc-labeled mAb to galectin-3 (30 ?g protein/in 100 ?l saline solution). Tumor images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post injection by using the mini-gamma camera. Findings Results from different consecutive experiments show an optimal visualization of thyroid cancer xenografts between 6 and 9 hours from injection of the radiotracer. Galectin-3 negative tumors were not detected at all. At 6 hrs post-injection galectin-3 expressing tumors were correctly visualized, while the whole-body activity had essentially cleared. Conclusions These results demonstrate the possibility to distinguish preoperatively benign from malignant thyroid nodules by using a specific galectin-3 radio-immunotargeting. In vivo imaging of thyroid cancer may allow a better selection of patients referred to surgery. The possibility to apply this method for imaging and treatment of other galectin-3 expressing tumors is also discussed

Scintigraphic device with high variable collimator

EU pendin

Brevetto Titolarità CNR “Dispositivo scintigrafico con collimazione variabile”

Estensione US ed EU; EU pendin

Super Spatial Resolution (SSR) method for scintigraphic imaging

Super SpatialResolution(SSR)methodforscintigraphicimaging G. Trinci a,b, R.Massari a,b, M.Scandellari a, F.Scopinaro c, A.Soluri a,b,n a Institute ofBiomedicalEngineering,CNR,Rome,Italy b Li-tech S.p.A.,Monterotndo,Rome,Italy c Nuclear Medicine,‘‘Sapienza’’UniversityofRome,Italy a r t i c l e info Article history: Received 26May2010 Received inrevisedform 14 October2010 Accepted 17October2010 Keywords:: Nuclear medicineimaging Super SpatialResolution Spatial Resolution High ResolutionGammaCamera Position SensitivePhotomultiplierTube a b s t r a c t This workdescribesaninnovativepatentedSuperSpatialResolution(SSR)methodappliedto scintigraphicdevices.TheaimofSuperResolution(SR)techniquesistoenhancetheresolutionofan imagingsystem,usinginformationfromseveralimages.SRreconstructionmaybeconsideredasasecond generationproblemofImageRestoration.ItcombinesseveralslightlydifferentLowResolution(LR) images toobtainaHighResolution(HR)image.SRtechniquesare,widely,describedinscientificliterature mainlyforapplicationsinvideocommunication,objectrecognitionandimagecompression. In thispaperwefocustoapplytheSRtasktothescintigraphicimaging.Specifically,itisdescribedasa patentedmethodthatusesaHighResolutionScintigraphicCamera(HRSC)tocollectandprocessasetof scintigraphicimages,inviewofobtainingaveryhighresolutionimage. The HRSCdevice,whichiscurrentlyusedinMedicalImaging,isbasedonaparallelsquareholes collimatorandonaHamamatsuH8500PositionSensitivePhotomultiplierTubes(PSPMT). The SSRmethodisappliedtothesyntheticimagesofthreedifferentphantoms,toverifytheeffective spatialresolutionvalues.Theresultsconfirmthatitispossibletoachieveoptimalspatialresolutionvalues at differentdepths,usefulinsmallobjectandsmallanimalimaging. Our studyconfirmsthefeasibilityofaveryhighresolutionsysteminscintigraphicimagingandthe possibilitytohavegammacamerasusingtheSSRmethod,toperformclinicalapplicationsonpatients. & 2010 ElsevierB.V.Allrightsreserve