Chromogenic in situ hybridization for the detection of lambda and kappa immunoglobulin light chains as a potential auxiliary diagnostic technique in canine plasmacytomas

The heterogeneous morphologic features of canine plasmacytomas (PCTs) can make their differentiation from other round cell tumors challenging. Immunohistochemistry (IHC) for lambda (\u3bb) and kappa (\u43a) immunoglobulin (Ig) light chains is often equivocal because of high background staining. The chromogenic in situ hybridization (CISH) technique for light chains has shown higher sensitivity compared to IHC in human plasma cell tumors. Therefore, we aimed to validate automated CISH for light chains in canine tissues and to evaluate its diagnostic potential in canine PCTs, in conjunction with routinely used IHC markers. CISH for light chains demonstrated a clear signal in plasma cell populations of canine control tissues (lymph nodes, lymphoplasmacytic inflammation) showing a polyclonal pattern with a prevalence of \u3bb-producing cells. CISH detected monotypic light chain expression in 33 of 53 (62%) PCTs, 31 expressing \u3bb and 2 expressing \u43a. CISH was more sensitive than IHC for \u3bb light chain (58% vs. 47%, respectively) and more easily interpretable given the absence of confounding background staining. The absence of CISH staining for both \u3bb and \u43a in a considerable subset of tumors may be the result of lower light chain production by neoplastic cells. Multiple myeloma oncogene 1 (MUM1) was expressed by all but 2 PCTs (96%), which showed \u3bb expression by CISH and IHC. The identification of poorly differentiated canine PCTs requires the assessment of a panel of IHC markers, with the potential support of CISH for Ig light chains

Prognostic value of pd-l1, pd-1 and cd8a in canine diffuse large b-cell lymphoma detected by rnascope

Immune checkpoints are a set of molecules dysregulated in several human and canine cancers and aberrations of the PD-1/PD-L1 axis are often correlated with a worse prognosis. To gain an insight into the role of immune checkpoints in canine diffuse large B-cell lymphoma (cDLBCL), we investigated PD-L1, PD-1 and CD8A expression by RNAscope. Results were correlated with several clinico-pathological features, including treatment, Ki67 index and outcome. A total of 33 dogs treated with chemotherapy (n = 12) or chemoimmunotherapy with APAVAC (n = 21) were included. PD-L1 signal was diffusely distributed among neoplastic cells, whereas PD-1 and CD8A were localized in tumor infiltrating lymphocytes. However, PD-1 mRNA was also retrieved in tumor cells. An association between PD-L1 and PD-1 scores was identified and a higher risk of relapse and lymphoma-related death was found in dogs treated with chemotherapy alone and dogs with higher PD-L1 and PD-1 scores. The correlation between PD-L1 and PD-1 is in line with the mechanism of immune checkpoints in cancers, where neoplastic cells overexpress PD-L1 that, in turn, binds PD-1 receptors in activated TIL. We also found that Ki67 index was significantly increased in dogs with the highest PD-L1 and PD-1 scores, indirectly suggesting a role in promoting tumor proliferation. Finally, even if the biological consequence of PD-1+ tumor cells is unknown, our findings suggest that PD-1 intrinsic expression in cDLBCL might contribute to tumor growth escaping adaptive immunity

Deep-phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment

Idiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T-cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in-vitro expandability for potential clinical use. Using mass cytometry (CyTOF) and an unbiased multidimensional analytical approach, we identified two specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene-expression, expandability and function. Treg subpopulation B, predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4 and CD45RO within FOXP3hi, CD127lo Tregs), expresses the IL- 2/STAT5 pathway and cell-cycle commitment genes. Furthermore, in-vitro expanded Tregs become functional and with the characteristics of Treg subpopulation B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2 sensitive and expandable in-vitro, suggesting novel therapeutic approaches such as low dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration

Cell Specific eQTL Analysis without Sorting Cells

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn’s disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus

CD30 cross-reactivity and expression in feline normal tissues and lymphomas

CD30 is a transmembrane glycoprotein of the tumor necrosis factor receptor superfamily included in the diagnostic algorithm of human cutaneous, anaplastic large cell and Hodgkin lymphomas and represents an optimal therapeutic target for CD30+ tumors. Similar diagnostic and therapeutic approaches are largely missing for feline lymphomas. Cross-reactivity of the antihuman CD30 receptor clone Ber-H2 was investigated in feline lymphomas. Comparative analysis of feline and human CD30 identified 61% identity of the amino acid sequence, with 100% identity of the main sequence of the epitope targeted by the antibody (RKQCEPDYYL). CD30 expression in normal feline tissues was restricted to rare lymphoid cells in perifollicular and interfollicular lymph node areas and in the thymic medulla. In feline lymphoma, CD30 was expressed in 4 of 33 (13%) T-cell lymphomas, 3 of 22 (14%) B-cell lymphomas, and 5 of 7 (71%) mixed-cell lymphomas, showing diffuse (1/5) or multifocal (4/5) positivity restricted to neoplastic multinucleated lymphoid cells and binucleated cells consistent with Reed-Sternberg-like cells. Based on the human classification system, cell morphology, expression of multiple markers (mixed cell components), and CD30 positivity, these cases were considered most consistent with classical Hodgkin-like lymphoma (HLL). The other 2 mixed-cell lymphomas were CD30 negative and thus most consistent with either T-cell-rich large B-cell lymphoma (TCRLBCL) or nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). These findings provide multiple data supporting the cross-reactivity of the Ber-H2 anti-CD30 clone in feline tissues and give evidence of the usefulness of CD30 in the diagnostic evaluation of feline lymphoma

Quantitative real time polymerase chain reaction (qRT-PCR) and RNAscope in situ hybridization (RNA-ISH) as effective tools to diagnose feline herpesvirus-1-associated dermatitis

Background: Felid herpesvirus type 1 (FHV-1)-associated dermatitis is characterized by facial and nasal involvement; clinical and histopathological manifestations may overlap with other dermatitides. Objective: To evaluate the realibility of qRT-PCR-2−ΔΔCq and RNAscope in situ hybridization (RNA-ISH) methods to diagnose FHV-1-associated dermatitis, in formalin-fixed paraffin-embedded (FFPE) tissues. Animals: Sixteen FFPE samples from cats with facial dermatitis and four controls were studied. Methods and materials: Based on histopathological features, cases were separated into: Group 1, samples with herpetic dermatitis (four); Group 2, samples with nonherpetic facial dermatitis (six); Group 3, samples with facial dermatitis of ambiguous nature (allergic or viral) (six); and Group 4, samples from healthy cats (four). A relative quantification using the 2−ΔΔCq method was used to estimate the “upregulation” of each FHV-1 target viral gene copies (glycoprotein-B and thymidine-kinase) relative to reference gene. Detection of FHV-1 mRNA was performed using the RNAscope 2.5 detection kit. Results: By 2−ΔΔCq analysis, upregulation of both FHV-1 genes was observed in all samples from Group 1 and two of six from Group 3. No upregulation was identified in samples from groups 2 and 4. Positive mRNA hybridization signal was observed in all cases from Group 1 and two cases of Group 3. No positivity was observed in samples from groups 2 and 4. Conclusions and clinical importance: QRT-PCR 2−ΔΔCq analysis and RNA-ISH can identify the FHV-1 genome as causative agent of the associated dermatitis, even where inclusion bodies are not detectable. Both techniques are functional in retrospective studies, have greater specificity than conventional PCR, and may be proposed for research and diagnostic purposes

Valutazione dell'espressione di CYP1A e HSP70 in Zosterisessor ophiocephalus nell'ambito di un piano di monitoraggio della laguna di Venezia

Lo scopo del presente studio \ue8 stato valutare l\u2019espressione di alcuni bioindicatori, comunemente utilizzati in programmi di biomonitoraggio, in diversi siti della Laguna di Venezia che, per la loro collocazione e per le caratteristiche idrologiche, possono essere esposti a diversi livelli di inquinamento. La specie monitorata \ue8 stata il pesce bentonico Zosterisessor ophiocephalus (Teleostei: Gobiidae), animale particolarmente adatto al biomonitoraggio in quanto stanziale, che vive a stretto contatto con il fondo ed \ue8 facilmente reperibile nella Laguna di Venezia. Dieci soggetti (5 maschi e 5 femmine) per sito sono stati pescati in 3 diversi siti (Porto Marghera, Caroman, Val di Brenta) nel periodo tra marzo ed aprile 2008. Parallelamente 5 individui maschi di Z. ophiocephalus pescati nel sito di Porto Marghera, sono stati stabulati in vasche con acqua di mare per un periodo sufficiente a detossificarsi, al fine di consentire una comparazione non solo tra i diversi siti di campionamento, ma anche rispetto ad un controllo detossificato. I biomarcatori selezionati e analizzati sono stati: l\u2019espressione della citocromo P4501A (CYP1A) e delle Heat Shock Protein 70 (HSP70). Il livello di espressione di tali marcatori \ue8 stato valutato in campioni di fegato, immediatamente congelati in azoto liquido e conservati a -80\ub0C, con metodiche di Real Time PCR e Western blot. Inoltre, organi quali fegato, ovaio, testicolo, milza e rene sono stati fissati in paraformaldeide al 4% ed inclusi in paraffina per la valutazione immunoistochimica. I livelli di espressione genica e di espressione proteica sono stati correlati con i diversi siti di pesca e con gli animali di controllo. I dati ottenuti, seppur preliminari, confermano l\u2019utilit\ue0 del CYP1A come biomarcatore di inquinamento ambientale e vanno a sostegno dei dati riportati in letteratura, secondo cui il sito di Porto Marghera \ue8 quello maggiormente impattato della Laguna, poich\ue9 influenzato dalla presenza di un\u2019estesa area industriale e della citt\ue0 di Venezia. Inoltre, l\u2019espressione di CYP1A \ue8 risultata sesso-specifica, in quanto i livelli pi\uf9 elevati di espressione sono stati evidenziati nei maschi. Tale risultato potrebbe essere spiegato dal fatto che i maschi di questa specie si occupano della costruzione del nido sui fondali e delle cure parentali; di conseguenza potrebbero essere pi\uf9 esposti ai contaminanti presenti nel sedimento

Felis catus Papillomavirus Types 1, 2, 3, 4, and 5 in Feline Bowenoid in Situ Carcinoma: An In Situ Hybridization Study

Several studies based on histopathology or molecular investigations suggest a causal relation between Felis catus papillomavirus (FcaPV-2) infection and bowenoid in situ carcinoma (BISC) in cats. Nevertheless, data on distribution of viral DNA for different F. catus papillomavirus types (FcaPV-1, 2, 3, 4, 5) in precancerous skin lesions are lacking. In this study, incisional and excisional skin biopsies from 18 cats with BISC were investigated for the presence of FcaPV DNA by quantitative polymerase chain reaction (qPCR) and chromogenic in situ hybridization (CISH) using specific probes to detect each of the FcaPVs that have been identified so far. By qPCR analysis, 15 of 18 samples were positive for FcaPV-2, 2 were positive for FcaPV-4, and 1 sample was negative for all FcaPVs studied. Two cases were positive for FcaPV-5 by qPCR only. FcaPV-1 and FcaPV-3 were not detected by either method. CISH positivity for FcaPV-2 and FcaPV-4 was 100% concordant with qPCR. FcaPV-2 CISH signal was observed as nuclear dots within grouped neoplastic keratinocytes in 12 BISCs and in the perilesional skin of 9 biopsies. In 3 of these 9 cases, the signal was not observed within the BISC. FcaPV-4 CISH positivity was detected only within BISCs in 2 cases. The overall rate of concordance for FcaPV detection between PCR and CISH was 97.8%. This study suggests that CISH is a reliable method to detect FcaPV-2 and FcaPV-4 infection in cats and provides useful information on the type, rate, and localization of infected cells

Localization and genotyping of canine papillomavirus in canine inverted papillomas

Numerous canine papillomaviruses (CPVs) have been identified (CPV1–23). CPV1, 2, and 6 have been associated with inverted papillomas (IPs). We retrieved 19 IPs from 3 histopathology archives, and evaluated and scored koilocytes, inclusion bodies, giant keratohyalin granules, cytoplasmic pallor, ballooning degeneration, and parakeratosis. IHC targeting major capsid proteins of PV was performed, and CPV genotyping was achieved by PCR testing. Tissue localization of CPV DNA and RNA was studied by chromogenic and RNAscope in situ hybridization (DNA-CISH, RNA-ISH, respectively). IPs were localized to the limbs (50%), trunk (30%), and head (20%), mainly as single nodules (16 of 19). In 15 of 19 cases, immunopositivity was detected within the nuclei in corneal and subcorneal epidermal layers. PCR revealed CPV1 in 11 IPs and CPV2 DNA in 3 IPs. Overall, 14 of 17 cases were positive by both DNA-CISH and RNA-ISH, in accord with PCR results. A histologic score >5 was always obtained in cases in which the viral etiology was demonstrated by IHC, DNA-CISH, and RNA-ISH. IHC and molecular approaches were useful to ascertain the viral etiology of IPs. Although IHC is the first choice for diagnostic purposes, ISH testing allows identification of PV type and the infection phase. RNA-ISH seems a promising tool to deepen our understanding of the pathogenesis of different PV types in animal species