Digital Pathology and Telepathology in Transplantation: Feasibility With the EHR

Digital pathology and telepathology play an emerging role conveying anatomical pathology diagnostic images in the Electronic Health Record. We sought to focus our attention to an innovative project, while identifying standards and practices between clinicians and the EHR (Electronic Health Record). The project aims at developing a second opinion network, based on telepathology, between two major transplantation centres over two years. The health authorities involved are the Hospital Trust of Verona and of Padua (Italy). In 2015 there were 376 renal and liver transplantations for both centres. We expect to significantly improve the transplantation workflow after combining the digital pathology platform with its proper and timely application in the telepathology network. Firstly it will allow the real time second opinion between pathologists in order to assess the suitability of the donor organs, avoiding the glass-slide transfer, with potential damage or loss. The technical partners delivered two slide scanners and software solutions to enable virtual microscopy and web-based digital slide sharing with storage resources. In addition, the project comprises an online survey which focuses on the accountability of the system, the user perception, and a concordance study for the project outcomes evaluation. The technical transactions between all the main actors and digital slides will be reviewed and updated in order to meet the integration standards and guideline according to IHE (Integrating the Healthcare Enterprise) initiative, Digital Imaging and COmmunications in Medicine (DICOM) and Health Level 7 (HL7). According to the first comparisons, we believe that the efforts to provide this new diagnostic imaging area to the actual EHR developments, will be rewarding and effective for the saving-life transplantation processes

Demographic, Clinical and Hematological Predictors of Carotid Atherosclerotic Plaque Histology

Aims: This retrospective observational study was aimed to identify hematological predictors of histological heterogeneity of carotid atherosclerotic plaques (CAPs). Results: The mean values of all demographic, clinical and haematological parameters did not differ between patients with or without symptomatic CAD. In univariate analysis, intraplaque hemorrhage was associated with male sex (r=0.18; p=0.032), superficial thrombosis with low hemoglobin (r=- 0.18; p=0.033), fibrosis with enhanced RDW (r=0.24; p=0.005), presence of foam cells with high WBC count (r=0.22; p=0.001), neovascularisation with high WBC count (r=0.17; p=0.048), whilst the presence of inflammatory infiltrate was also associated with high WBC count (r=0.17; p=0.043). Conlusions: The results of this retrospective observational study confirm that some traditional and inexpensive hematological parameters such as WBC count, hemoglobin and RDW may help identifying patients with more severe forms of CAD

Primary cutaneous B-cell lymphoma and chronic leg ulcers in a patient with type 2 diabetes

The incidences of type 2 diabetes mellitus and many cancers are rapidly increasing worldwide. Diabetes is a strong risk factor for some cancers (including lymphomas) and is also associated with adverse cancer outcomes. After gastrointestinal tract, the skin is the second most frequent extranodal site involved by non-Hodgkin lymphomas and the cutaneous B-cell lymphomas (CBCLs) range from 25% to 30% of all primary cutaneous lymphomas. The primary cutaneous diffuse large B-cell lymphoma (PCDLBCL) is an aggressive lymphoma with a poor prognosis, representing roughly 20% of all primary CBCLs. Classically, the cutaneous manifestation of this lymphoma is a red or violaceous tumors arising on a leg. To date, despite the large body of evidence suggesting that diabetes is strongly associated with an increased risk of some cancers, very little information is available regarding a possible association between type 2 diabetes and primary cutaneous diffuse large B-cell lymphoma. In this report, we will present the case of a white adult patient with type 2 diabetes with chronic leg ulcers complicated by a primary cutaneous diffuse large B-cell lymphoma

European Society for Organ Transplantation (ESOT)-TLJ 3.0 Consensus on Histopathological Analysis of Pre-Implantation Donor Kidney Biopsy:Redefining the Role in the Process of Graft Assessment

The ESOT TLJ 3.0. consensus conference brought together leading experts in transplantation to develop evidence-based guidance on the standardization and clinical utility of pre-implantation kidney biopsy in the assessment of grafts from Expanded Criteria Donors (ECD). Seven themes were selected and underwent in-depth analysis after formulation of PICO (patient/population, intervention, comparison, outcomes) questions. After literature search, the statements for each key question were produced, rated according the GRADE approach [Quality of evidence: High (A), Moderate (B), Low (C); Strength of Recommendation: Strong (1), Weak (2)]. The statements were subsequently presented in-person at the Prague kick-off meeting, discussed and voted. After two rounds of discussion and voting, all 7 statements reached an overall agreement of 100% on the following issues: needle core/wedge/punch technique representatively [B,1], frozen/paraffin embedded section reliability [B,2], experienced/non-experienced on-call renal pathologist reproducibility/accuracy of the histological report [A,1], glomerulosclerosis/other parameters reproducibility [C,2], digital pathology/light microscopy in the measurement of histological variables [A,1], special stainings/Haematoxylin and Eosin alone comparison [A,1], glomerulosclerosis reliability versus other histological parameters to predict the graft survival, graft function, primary non-function [B,1]. This methodology has allowed to reach a full consensus among European experts on important technical topics regarding pre-implantation biopsy in the ECD graft assessment.</p

Pathology Laboratory Archives: Conservation Quality of Nucleic Acids and Proteins for NSCLC Molecular Testing

In the molecular era, proper archival conditions within pathology laboratories are crucial, especially for formalin-fixed paraffin-embedded (FFPE) tissue specimens retrieved years after the original diagnosis. Indeed, improper preservation can impact the integrity of nucleic acids and protein antigens. This study evaluates the quality status of stored FFPE blocks using multilevel omics approaches. FFPE blocks from 45 Non-Small Cell Lung Carcinoma (NSCLC) cases were analyzed. The blocks were collected from six different pathology archives across Italy with distinct environmental characteristics. Nucleic acids’ quantity and quality, as well as protein antigens, were assessed using various techniques, including MALDI-MSI. RNA was quantitatively higher, but more fragmented, compared to DNA. DNA quantity and quality were suitable for molecular analyses in 94.4% and 62.3% of samples, respectively. RNA quantity was adequate across all samples, but it was optimal only in 22.3% of cases. DNA quality started to deteriorate after 6–8 years, whereas RNA quality diminished only after 10 years of storage. These data might suggest a particular DNA susceptibility to FFPE blocks conservation. Immunohistochemical intensity decreased significantly after 6–8 years of storage, and MALDI-MSI analysis revealed that younger tissue blocks contained more unique proteomic signals than the older ones. This study emphasizes the importance of proper FFPE archiving conditions for molecular analyses. Governance should prioritize attention to pathology archives to ensure quality preservation and optimize predictive testing. By elucidating the nuances of FFPE block storage, this research paves the way for enhanced molecular diagnostics and therapeutic insights regarding oncology and beyond

Identification and validation of diagnostic cut-offs of the ELISpot assay for the diagnosis of invasive aspergillosis in high-risk patients

Objective: We investigated the performance of enzyme linked immunospot (ELISpot) assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematologic malignancies. Methods: We prospectively enrolled two cohorts of patients undergoing intensive myelosuppressive or immunosuppressive treatments at high risk for IA. ELISpot was performed to detect Aspergillus-specific T cells producing Interleukin-10. Results: In the discovery cohort, a derived cut-off of 40 spot forming cells (SFCs)/106 PBMCs has shown to correctly classify IA cases with a sensitivity and specificity of 89.5% and 88.6%, respectively. This cut-off is lowered to 25 SFC when considering the subset of possible IA patients, with sensitivity and specificity of 76% and 93%, respectively. The application of the 40 SFCs cut-off to the validation cohort resulted in a positivity rate of 83.3% in proven/probable cases and a negativity rate of 92.5% in possible/non-IA cases. Adopting the 25 SCFs cut-off, the assay resulted positive in 83.3% of proven/probable cases while it resulted negative in 66.7% of possible/non-IA cases. Conclusions: ELISpot shows promises in the diagnosis of IA and the possibility to use two distinct cut-offs with similar diagnostic performances according to patients' different pre-test probability of infection can widen its use in patients at risk

LC3B and ph-S6K are both expressed in epithelioid and classic renal angiomyolipoma: a rationale tissue-based evidence for combining use of autophagic and mTOR targeted drugs

Background: Targeted drugs to the autophagy processes are emerging in clinical trials. The aim of this work is to assess the magnitude of autophagic expression in renal angiomyolipoma. Methods: Fourteen cases of renal angiomyolipoma were recruited. Anti-LC3B-II and anti-phospho-S6K were detected by Western blot analysis. For immunohistochemical staining, sections were stained with the antibodies LC3B-II and cathepsin-K. LC3B-II was also analyzed by immunofluorescence. We have also carried out electron microscopy analysis on tumor cells. Results: 13 classic and 1 epithelioid renal angiomyolipoma were recruited. The Western-blot LC3B-II analysis shows increasing in protein expression in all cases, however quantitative protein expression ranged from 1 to 15 (mean 5). The autophagosome protein LC3B-I also significantly increased in all tumor extraction. The expression of LC3B-II protein was confirmed in tumoral samples by immunofluorescence. The lysosomal marker cathepsin-K was observed by immunohistochemistry on all tumours. The Western-blot ph-S6K analysis showed significant protein overexpression along all cases after evaluation of the quantitative S6K/Ponceaus ratio. In 6/14 (52%) the expression was high, with a quantitative increase of 653 fold induction in 4 angiomyolipoma compared to normal tissue. At electron microscopy, cancer cells evidenced round or oval electron-dense granules associated with membranes and granules with double membrane. Conclusion: Both autophagic LC3B-II and ph-S6K molecules are over-represented in both epithelioid and classic renal angiomyolipoma and a combined use of inhibitors to the autophagic and mTOR processes may be designed in clinical trials, when enrolling patients affected by tumours in tuberous sclerosis or angiomyolipoma at risk of bledding

True 3q Chromosomal Amplification in Squamous Cell Lung Carcinoma by FISH and aCGH Molecular Analysis: Impact on Targeted Drugs

Peer reviewe

IMMUNOPHENOTYPICAL CHARACTERIZATION OF LUNG ADENOCARCINOMAS: MOLECULAR AND CLINICAL CORRELATIONS

Il carcinoma del polmone \ue8 la neoplasia maligna con incidenza maggiore nonch\ue8 la principale causa di morte per neoplasia al mondo. La classificazione dell'Organizzazione Mondiale della Sanit\ue0 (WHO, 2004) utilizzata fino a poco tempo fa distingueva le neoplasie polmonari in due grandi categorie: i carcinomi a piccole cellule e i carcinomi non a piccole cellule. Tale classificazione risulta ad oggi inadeguata e superata poich\ue9 l\u2019utilizzo di farmaci sempre pi\uf9 specifici e mirati a colpire la biologia del tumore impone una sempre pi\uf9 attenta e precisa classificazione diagnostica delle neoplasie cui far corrispondere una stratificazione prognostica dei pazienti corredata inoltre da dati indicativi di risposta alla terapia. Scopo di questo studio \ue8 quello di distinguere, sulla base dell\u2019immunofenotipo, le differenti entit\ue0 carcinomatose del polmone. Centocinquantanove casi (159) di carcinoma del polmone sono stati selezionati dagli archivi di cinque istituzioni (Anatomia Patologica di Verona, Negrar, Mestre, Trento, Forl\uec). Ogni caso \ue8 stato rivisto da 2 patologi per confermare la diagnosi e per selezionare l\u2019incluso in paraffina significativo su cui effettuare le indagini immunoistochimiche. Abbiamo successivamente classificato i tumori sulla base delle diverse espressioni dei marcatori immunoistochimici ed, in sintesi, il 43% dei casi erano carcinomi "alveolari", il 24% erano carcinomi "bronchiolari", il 16% erano carcinomi a "cellule Clara", il 9% erano di tipo \u201cmetaplastico-enterico", il 4% erano carcinomi sarcomatoidi, il 3 % erano carcinomi neuroendocrini e l'1% erano di tipo \u201cmetaplastico-squamoso". I dati clinici hanno confermato la relazione tra fumo di sigaretta e l'incidenza di adenocarcinoma (82% dei casi), inoltre, il distretto pi\uf9 spesso coinvolto dalla recidiva tumorale \ue8 risultato essere quello dei linfonodi mediastinici (66% dei casi di recidiva), con una sopravvivenza media libera (DFS) da malattia di 9 mesi. In 18 casi la sopravvivenza globale media era di 19,8 mesi, senza significative correlazioni con l\u2019immunofenotipo espresso. In particolare, l'entit\ue0 metaplastico enterica mostrava morfologia prevalentemente mucinosa, K-RAS mutato e prognosi peggiore (DFS media di 5,5 mesi). Tali correlazioni, basate sui profili immunoistochimici delle neoplasie, devono essere verificate ed eventualmente validate su un maggior numero di casi, con un follow-up pi\uf9 lungo, in modo da consentire una eventuale e reale stratificazione prognostica dei pazienti.Lung cancer is the most common cancer worldwide and the leading cause of cancer-related mortality. The lung cancer classification of the World Health Organization (WHO, 2004) can efficiently distinguish small-cell (SCLC) and non-small-cell lung carcinomas (NSCLC) but it results inadequate when facing prognosis and therapeutic decisions, especially for the entity of adenocarcinoma. Knowledge concerning the molecular pathogenesis of this tumor type is far from complete and diagnostic problems are mainly related to the poor reproducibility of morphological and histological criteria. Lung adenocarcinoma represents indeed a heterogeneous group of tumours characterized by different morphological and immunophenotypical features and molecular pathogenesis. Since updated therapeutic approaches are available (e.g. molecular-targeted therapies using tyrosine kinase inhibitors), exact histological classification is required for proper therapy and prognostic estimates. Aim of this study is to distinguish different lung tumour entities outlining a classification on the basis of the neoplastic immunophenotype. One hundred and fifty-nine cases of lung neoplasm were retrieved from the archives of five institutions (Anatomia Patologica of Verona, Negrar, Mestre, Trento, Forl\uec). All the cases were diagnosed as NSCLC carcinomas according to the WHO classification of 2004 and reviewed by two pathologists (MC and AE) to confirm the diagnosis and to select a significative paraffin block containing both tumor and adjacent non-neoplastic lung tissue. For each case multiple (30) paraffine sections were obtained in order to perform immunohistochemistry. The immunohistochemical panel included the following antibodies: CD56 (123C3.D5, Neomarkers), CK20 (IT-KS20.8, Biogenex), CK7 (OV-TL12/30, Biogenex), TTF-1 (8G7G3/1, Dako), NAPSIN A (TMU-A d02, Arp), SP A-1 (PE-10, Dako), CD208 (I10-1112, Beckton Dickinson), CC10 (rabbit, Dako), MUC5 (CLH2, Novocastra), CK5 (XM26, Novocastra), P63 (4A4, Santacruz), VILLINA (CWWB1, Novocastra), CDX2 (CDX2-88, Biogenex), P53 (DO-7, Novocastra), P21 (SX-118, Dako), P16 (JC8, Neomarkers) and DELTA-N-P63 (rabbit, Oncogene). Of 159 patients, 69 underwent surgery in Verona in 2007-2008; of these, clinical data were available for 34 cases and included smoke, type of surgery, adjuvant therapy, relapse, disease free survival (DFS), type of chemotherapy and overall survival (OS). We classified the tumors on the basis of the immunophenotype. In summary, 43% of the cases were \u201calveolar\u201d carcinomas, 24% were \u201cbronchiolar\u201d carcinomas, 16% were carcinomas with \u201cClara cells features\u201d, 9% were \u201cmetaplastic-enteric type\u201d, 4% were sarcomatoid carcinomas, 3% were neuroendocrine carcinomas and 1% were \u201cmetaplastic-squamous type\u201d. The mean age was 64, 113 were males (70%) and 46 were females (30%). The mean tumor size was 3,3 cm. The cases with available clinical data were 34, of which 17 were \u201calveolar\u201d carcinomas, 8 were \u201cbronchiolar\u201d carcinomas, 4 were carcinomas with \u201cClara cells features\u201d, 3 were \u201cmetaplastic-enteric type\u201d and 2 were sarcomatoid carcinomas. Six patients (18%) never smoked, 9 patients (26%) quit smoking and 19 patients (56%) are currently smokers. Pathological stage according to Cancer staging (6th edition) was IA in 12 cases (35%), IIA in 6 cases (18%), IIB in 6 cases (18%), IIIA in 6 cases (18%) and IV in 4 cases (11%). Seven patients (20%) underwent neoadjuvant radiotherapy. The disease recurred in 12 patients (35%) with a mean DFS of 9 months and a predilection (66%) of recurrency in mediastinal lymphnodes. In seven patients (20%) we did not observe any recurrency while in 15 patients (45%) the data were not available. Surgery type, regarding the resection margins was R0 in 26 cases (76%) and R1 in 8 cases (24%). In 18 cases OS was available with a mean period of 19,8 months. The goal of our classification is not only to provide the most accurate diagnosis but also to manage the tissue in a way that immunohistochemical and/or molecular studies can be performed to obtain predictive and prognostic data that will lead to improvement in patient outcomes. It is well known that immunohistochemistry is a practical and powerful method for diagnosis and research, since it is simple, not time consuming, less expensive than molecular investigations, feasible on paraffin sections, amenable on small biopsy and cytology samples and permits correlations with morphology at both the cellular and subcellular levels. Particularly, the immunohistochemical panel including CK7, CK20, CK5, TTF-1, NAPSIN-A, CC-10, SP-A, CD208, CDX-2, VILLIN, MUC-5 provides a useful tool in distinguishing different subtypes of adenocarcinomas. Available clinical data confirmed the well-known relationship between smoking and incidence of adenocarcinoma (82% of cases); moreover, the sites most often involved by tumor recurrence resulted to be mediastinal lymphnodes (66% of cases with recurrency) with a mean DFS of 9 months. In 18 cases OS was available with a mean period of 19,8 months without correlation to the histotype. The metaplastic enteric type entity of adenocarcinoma, related to \u201cbronchiolar\u201d derivation, shows mainly mucinous morphology, frequently harbour KRAS mutations and worse prognosis (mean DFS of 5,5 months with mediastinal lymphnodes recurrency). Among cases with available clinical data the follow-up period resulted probably not long enough in order to establish adequate and statistically significant relationships between different immunophenotypes and prognosis. Difficulties in clinical data collection have been determined mainly by patients\u2019 compliance, different modalities and locations of post-operative care and by the fact that the cases were retrieved from the archives of different institutions. Although highly promising, these studies should be further validated in future investigation. Such correlations need in fact more cases with longer follow-up period to be reliably verified and to allow stratification of patients with different NSCLC entities on the basis of molecular-immunophenotypical features

Digital diagnostic cytopathology: has the pandemic brought us closer?

: The Covid-19 pandemic has acted as a powerful change driver in the field of pathology and has had relevant consequences on the practice of cytopathology, in terms of changes in workload, rates of malignancy, and performance of cytology. At the same time, regulatory authorities have relaxed their requirements for the deployment of digital pathology for remote diagnostic reporting. However, most of these improvements concerned digital histopathology. Data from literature search show that experiences in digital cytopathology during the pandemic concerned mainly educational and academic activities. From a broader point of view, when searching for all published literature on digital pathology, only a minority of papers deal with cytopathology, but a noticeable increase in publications has been seen in the last ten years, with a continuous trend toward this increase with a maximum of papers in 2021. Indeed, the pandemic has led to greater awareness of the opportunity of digital for cytopathology as well