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Purpose: The present pilot study investigates the putative role of radiomics from [18F]FDG PET/CT scans to predict PD-L1 expression status in non-small cell lung cancer (NSCLC) patients. Methods: In a retrospective cohort of 265 patients with biopsy-proven NSCLC, 86 with available PD-L1 immunohistochemical (IHC) assessment and [18F]FDG PET/CT scans have been selected to find putative metabolic markers that predict PD-L1 status (< 1%, 1–49%, and ≥ 50% as per tumor proportion score, clone 22C3). Metabolic parameters have been extracted from three different PET/CT scanners (Discovery 600, Discovery IQ, and Discovery MI) and radiomics features were computed with IBSI compliant algorithms on the original image and on images filtered with LLL and HHH coif1 wavelet, obtaining 527 features per tumor. Univariate and multivariate analysis have been performed to compare PD-L1 expression status and selected radiomic features. Results: Of the 86 analyzed cases, 46 (53%) were negative for PD-L1 IHC, 13 (15%) showed low PD-L1 expression (1–49%), and 27 (31%) were strong expressors (≥ 50%). Maximum standardized uptake value (SUVmax) demonstrated a significant ability to discriminate strong expressor cases at univariate analysis (p = 0.032), but failed to discriminate PD-L1 positive patients (PD-L1 ≥ 1%). Three radiomics features appeared the ablest to discriminate strong expressors: (1) a feature representing the average high frequency lesion content in a spherical VOI (p = 0.009); (2) a feature assessing the correlation between adjacent voxels on the high frequency lesion content (p = 0.004); (3) a feature that emphasizes the presence of small zones with similar grey levels inside the lesion (p = 0.003). The tri-variate linear discriminant model combining the three features achieved a sensitivity of 81% and a specificity of 82% in the test. The ability of radiomics to predict PD-L1 positive patients was instead scarce. Conclusions: Our data indicate a possible role of the [18F]FDG PET radiomics in predicting strong PD-L1 expression; these preliminary data need to be confirmed on larger or single-scanner series.

Noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) are low-risk thyroid lesions most often characterised by RAS-type mutations. The histological diagnosis may be challenging, and even immunohistochemistry and molecular approaches have not yet provided conclusive solutions. This study characterises a set of NIFTPs by Matrix-Assisted Laser Desorption/Ionisation (MALDI)–Mass Spectrometry Imaging (MSI) to highlight the proteomic signatures capable of overcoming histological challenges. Archived formalin-fixed paraffin-embedded samples from 10 NIFTPs (n = 6 RAS-mutated and n = 4 RAS-wild type) were trypsin-digested and analysed by MALDI–MSI, comparing their profiles to normal tissue and synchronous benign nodules. This allowed the definition of a four-peptide signature able to distinguish RAS-mutant from wild-type cases, the latter showing proteomic similarities to hyperplastic nodules. Moreover, among the differentially expressed signals, Peptidylprolyl Isomerase A (PPIA, 1505.8 m/z), which has already demonstrated a role in the development of cancer, was found overexpressed in NIFTP RAS-mutated nodules compared to wild-type lesions. These results underlined that high-throughput proteomic approaches may add a further level of biological comprehension for NIFTPs. In the future, thanks to the powerful single-cell detail achieved by new instruments, the complementary NGS–MALDI imaging sequence might be the correct methodological approach to confirm that the current NIFTP definition encompasses heterogeneous lesions that must be further characterised.

ImportanceIdentifying new prognostic features in colon cancer has the potential to refine histopathologic review and inform patient care. Although prognostic artificial intelligence systems have recently demonstrated significant risk stratification for several cancer types, studies have not yet shown that the machine learning–derived features associated with these prognostic artificial intelligence systems are both interpretable and usable by pathologists.ObjectiveTo evaluate whether pathologist scoring of a histopathologic feature previously identified by machine learning is associated with survival among patients with colon cancer.Design, Setting, and ParticipantsThis prognostic study used deidentified, archived colorectal cancer cases from January 2013 to December 2015 from the University of Milano-Bicocca. All available histologic slides from 258 consecutive colon adenocarcinoma cases were reviewed from December 2021 to February 2022 by 2 pathologists, who conducted semiquantitative scoring for tumor adipose feature (TAF), which was previously identified via a prognostic deep learning model developed with an independent colorectal cancer cohort.Main Outcomes and MeasuresPrognostic value of TAF for overall survival and disease-specific survival as measured by univariable and multivariable regression analyses. Interpathologist agreement in TAF scoring was also evaluated.ResultsA total of 258 colon adenocarcinoma histopathologic cases from 258 patients (138 men [53%]; median age, 67 years [IQR, 65-81 years]) with stage II (n = 119) or stage III (n = 139) cancer were included. Tumor adipose feature was identified in 120 cases (widespread in 63 cases, multifocal in 31, and unifocal in 26). For overall survival analysis after adjustment for tumor stage, TAF was independently prognostic in 2 ways: TAF as a binary feature (presence vs absence: hazard ratio [HR] for presence of TAF, 1.55 [95% CI, 1.07-2.25]; P = .02) and TAF as a semiquantitative categorical feature (HR for widespread TAF, 1.87 [95% CI, 1.23-2.85]; P = .004). Interpathologist agreement for widespread TAF vs lower categories (absent, unifocal, or multifocal) was 90%, corresponding to a κ metric at this threshold of 0.69 (95% CI, 0.58-0.80).Conclusions and RelevanceIn this prognostic study, pathologists were able to learn and reproducibly score for TAF, providing significant risk stratification on this independent data set. Although additional work is warranted to understand the biological significance of this feature and to establish broadly reproducible TAF scoring, this work represents the first validation to date of human expert learning from machine learning in pathology. Specifically, this validation demonstrates that a computationally identified histologic feature can represent a human-identifiable, prognostic feature with the potential for integration into pathology practice.

Introduction: Digital pathology can improve the technical and interpretative workflows in nephropathology by creating hub-spoke networks and virtuous collaboration projects among centers in different geographical regions. New high-resolution fast-scanning instruments combined with currently existing equipment were tested in a nephropathology hub to evaluate possible upgrading in the routine processing phases. Methods: The scanning performance of two different instruments (Aperio vs hybrid MIDI II) was evaluated and a comparative quality control check was performed on obtained whole slide images. Results: Both with default and custom settings for light microscopy, MIDI II proved to be faster, with only slightly more time required to prepare the scan and larger final file size as compared to Aperio (p < 0.001). No differences were noted in the number of out-of-focus slides per case (p = 0.75). Regarding immunofluorescence, the new scanner required longer preparation time (p = 0.001) with comparable scanning times and final file size (p = 0.169 and p = 0.177, respectively). Quality control showed differences in 3 quality features related to white background and blurriness (p < 0.001). No major discordances in the final diagnosis were recorded after comparing the report obtained with slides scanned using the two instruments, with only one case (4%) showing minor disagreement. Conclusion: The present report describes the experience of a hub nephropathology center adopting next generation digital pathology tools for the routine assessment of renal biopsies, highlighting the need for a complementary approach towards a philosophy of interoperability.

Background: Programmed death-ligand 1 (PD-L1) checkpoint inhibitors represent a mainstay of therapy in head and neck squamous cell cancer (HNSCC). However, little is known about the influence of combined therapy on PD-L1 expression. The study aims to gather evidence on this topic. Methods: A systematic search was carried out in electronic databases Pubmed-MEDLINE and Embase to retrieve studies on the comparison of PD-L1 expression before and after conventional therapy. Data were extracted and a quantitative analysis with pooled odds ratios (ORs) was performed when applicable. Results: Of 5688 items, 15 were finally included. Only a minority of studies assessed PD-L1 with the recommended combined positive score (CPS). The results are highly heterogeneous, with some studies reporting an increase in PD-L1 expression and others reporting a decrease. Three studies allowed for quantitative analysis and showed a pooled OR of 0.49 (CI 0.27–0.90). Conclusions: From the present evidence, a clear conclusion towards an increase or decrease in PD-L1 expression after combined therapy cannot be drawn, but even with few studies available, a trend towards an increase in expression in tumor cells at a cutoff of 1% can be noted in patients undergoing platinum-based therapy. Future studies will provide more robust data on the effect of combined therapy on PD-L1 expression.

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ObjectivesAmyloid light chain (AL)-κ and AL-λ share common histopathologic changes; however, the potential difference in clinical manifestations, histologic findings, and clinical significance between the 2 subtypes remain unclear.MethodsIn a retrospective study, 94 kidney biopsies for AL amyloidosis were evaluated using the composite scarring injury score (CSIS) and amyloid score (AS). Results were then compared between AL-κ and AL-λ.ResultsComparing AS and CSIS between AL-κ and AL-λ, the AS was significantly higher in AL-κ than in AL-λ, with 2 components of AS (capillary wall and vascular amyloid) scoring higher in AL-κ than in AL-λ, while mesangial and interstitial ASs were similar in the 2 cohorts. In addition, the proportion of periodic acid–Schiff strong-staining amyloid in AL-κ was markedly higher than in AL-λ. There was no significant difference in CSIS and its components between the 2 subtypes of AL amyloidosis.ConclusionsOverall, AL-κ presents with higher serum creatinine and a higher AS score than AL-λ at biopsy, which may indicate a worse prognosis and be an important reference for clinical management.

Infectious diseases still threaten the global community, especially in resource-limited countries. An accurate diagnosis is paramount to proper patient and public health management. Identification of many microbes still relies on manual microscopic examination, a time-consuming process requiring skilled staff. Thus, artificial intelligence (AI) has been exploited for identification of microorganisms. A systematic search was carried out using electronic databases looking for studies dealing with the application of AI to pathology microbiology specimens. Of 4596 retrieved articles, 110 were included. The main applications of AI regarded malaria (54 studies), bacteria (28), nematodes (14), and other protozoa (11). Most publications examined cytological material (95, 86%), mainly analyzing images acquired through microscope cameras (65, 59%) or coupled with smartphones (16, 15%). Various deep-learning strategies were used for the analysis of digital images, achieving highly satisfactory results. The published evidence suggests that AI can be reliably utilized for assisting pathologists in the detection of microorganisms. Further technologic improvement and availability of datasets for training AI-based algorithms would help expand this field and widen its adoption, especially for developing countries.

Thyroid fine-needle aspiration (FNA) is a commonly used diagnostic cytological procedure in pediatric patients for the evaluation of thyroid nodules, triaging them for the detection of thyroid cancer. In recent years, greater attention has been paid to thyroid FNA in this setting, including the use of updated ultrasound score algorithms to improve accuracy and yield, especially considering the theoretically higher risk of malignancy of these lesions compared with the adult population, as well as to minimize patient discomfort. Moreover, molecular genetic testing for thyroid disease is an expanding field of research that could aid in distinguishing benign from cancerous nodules and assist in determining their clinical management. Finally, artificial intelligence tools can help in this task by performing a comprehensive analysis of all the obtained data. These advancements have led to greater reliance on FNA as a first-line diagnostic tool for pediatric thyroid disease. This review article provides an overview of these recent developments and their impact on the diagnosis and management of thyroid nodules in children.

Lymphomatous infiltration of kidney parenchyma is a frequent complication of systemic hematologic malignancies and often shows subtle clinical presentation. Diffuse large B-cell lymphoma represents the most frequent form involving the kidney, with advanced stage at diagnosis, poor outcome, and risk for central nervous system relapse if not adequately treated. Kidney biopsy can provide specific and early detection of these cases, helping in the differential diagnosis with more frequent entities. Finally, further hematologic workup (bone marrow biopsy, complete blood cell count, and positron emission tomography) can distinguish secondary involvement of the kidney from the rarer kidney-limited forms, especially in patients without a previous diagnosis of lymphoma. Making a prompt and correct diagnosis directs the management of these cases and may improve the outcome, as described in the present report.

Whole-slide imaging and virtual microscopy are useful tools implemented in the routine pathology workflow in the last 10 years, allowing primary diagnosis or second-opinions (telepathology) and demonstrating a substantial role in multidisciplinary meetings and education. The regulatory approval of this technology led to the progressive digitalization of routine pathological practice. Previous experiences on renal biopsies stressed the need to create integrate networks to share cases for diagnostic and research purposes. In the current paper, we described a virtual lab studying the routine renal biopsies that have been collected from 14 different Italian Nephrology centers between January 2014 and December 2019. For each case, light microscopy (LM) and immunofluorescence (IF) have been processed, analysed and scanned. Additional pictures (eg. electron micrographs) along with the final encrypted report were uploaded on the web-based platform. The number and type of specimens processed for every technique, the provisional and final diagnosis, and the turnaround-time (TAT) have been recorded. Among 826 cases, 4.5% were second opinion biopsies and only 4% were suboptimal/inadequate for the diagnosis. Transmission electron microscopy (TEM) has been performed on 41% of cases, in 22% changing the final diagnosis, in the remaining 78% contributed to the better definition of the disease. For light microscopy and IF the median TAT was of 2 working days, with only 8.6% with a TAT longer than 5 days. For TEM, the average TAT was 26 days (IQR 6–64). In summary, we systematically reviewed the 6-years long nephropathological experience of an Italian renal pathology service, where digital pathology is a definitive standard of care for the routine diagnosis of glomerulonephritides.

Among the different ancillary immunohistochemical tools that pathologists may employ in thyroid nodules, the so-called Hector Battifora’s ‘MEsothelioma’ 1 (HBME-1) staining is one of the most fascinating, since its real identity is currently unknown. In the present review, the different clinical applications of HBME-1 are analysed, with main emphasis on its role in thyroid pathology with overview on less impactful fields, such as haematopathology or mesothelial lesions. Different acceptable or good diagnostic performances were recorded for HBME-1 in thyroid pathology, being used in routine practice as one of the best tools to screen thyroid malignancy both in terms of sensitivity and specificity. From a speculative point of view, after many attempts to hunt the cryptic target antigen of this antibody, its identity still remains elusive. In this setting, the application of high-throughput technologies (mainly in situ proteomics) may be the exact route to improve the knowledge about the pathophysiology of HBME-1 and to finally unveil its true identity.

The GC reaction results in the selection of B cells acquiring effector Ig secreting ability by progressing toward plasmablastic differentiation. This transition is associated with exclusion from the GC microenvironment. The aberrant expansion of plasmablastic elements within the GC fringes configures an atypical condition, the biological characteristics of which have not been defined yet. We investigated the in situ immunophenotypical and transcriptional characteristics of a nonclonal germinotropic expansion of plasmablastic elements (GEx) occurring in the tonsil of a young patient. Compared to neighboring GC and perifollicular regions, the GEx showed a distinctive signature featuring key regulators of plasmacytic differentiation, cytokine signaling, and cell metabolism. The GEx signature was tested in the setting of diffuse large B-cell lymphoma (DLBCL) as a prototypical model of lymphomagenesis encompassing transformation at different stages of GC and post-GC functional differentiation. The signature outlined DLBCL clusters with different immune microenvironment composition and enrichment in genetic subtypes. This report represents the first insight into the transcriptional features of a germinotropic plasmablastic burst, shedding light into the molecular hallmarks of B cells undergoing plasmablastic differentiation and aberrant expansion within the noncanonical setting of the GC microenvironment.

ObjectiveThe International Society of Nephrology/Renal Pathology Society classification is the gold standard for the characterisation of lupus nephritis (LN) on renal biopsy, with therapeutic repercussions. Its recent revision simplified the current class subdivisions, eliminating the S/G forms of class IV, although data on a possible pathogenetic/clinical value of this subdivision are still contradictory.Methods353 renal biopsies from Belimumab International Study in LN were assessed through central pathology review. Univariate logistic models and a decision tree were performed on 314 adequate biopsies to evaluate the impact of histological features on focal/diffuse classes. Removing class I/II (n=6) and ‘pure’ class V (n=34), principal component analysis (PCA) and heatmap were used to explore similarities among III, IVS and IVG biopsies either incorporating or not the mixed classes (+V, n=274). Finally, a method aimed at partitioning the cases into k clusters based on their similarity (KMeans), was used to study features from the cohort of ‘pure’ class III/IVS/IVG cases (n=214) to determine alternative subdivisions based on phenotypic data.ResultsSegmental endocapillary hypercellularity (EH) was prevalent in class III, global EH, wire loops, hyaline thrombi and double contours were hallmarks of class IVG, with IVS cases showing intermediate characteristics. Heatmap and PCA confirmed the segregation of these features among classes, showing better segregation for focal/diffuse LN as compared with the mixed classes (+V). KMeans revealed the presence of two main clusters, membranoproliferative-like (n=83) or vasculitis-like (n=131).ConclusionsThis study reveals new phenotypic forms of LN surpassing the traditional classes as determined by the current classification. Future validation and confirmation are required to confirm these findings.

ObjectiveTo investigate second kidney biopsy as predictor of end-stage kidney disease (ESKD) in active lupus nephritis (LN).MethodsPatients with biopsy-proven LN (International Society of Nephrology/Renal Pathology Society 2003) who had undergone a second kidney biopsy between January 1990 and December 2018 were included. Clinical and histological findings at first and at second biopsy were analysed with Cox proportional hazard models to predict ESKD, defined as start of kidney replacement therapy. Survival curves were calculated with Kaplan-Meier method.ResultsNinety-two patients with LN were included, 87% females, mean follow-up 17.9±10.1 years. Reasons for second kidney biopsy encompassed nephritic flares (n=28, 30.4%), proteinuric flares (n=46, 50%) or lack of renal response (n=18, 19.5%). Class switch from first biopsy occurred in 50.5% of cases, mainly from non-proliferative towards proliferative classes. Class IV remained stable in over 50% of cases. Twenty-five patients (27.2%) developed ESKD, mostly belonging to the nephritic flare group (17/28, 60.7%). Independent predictors of ESKD at second biopsy were activity index (AI; (HR 95% CI) 1.20 (1.03 to 1.41), p=0.022), chronicity index (CI; 1.41 (1.09 to 1.82), p=0.008) and 24h-proteinuria (1.22 (1.04 to 1.42), p=0.013). AI≥2 (log-rank p=0.031), CI >4 (log-rank p=0.001) or proteinuria ≥3.5 g/day (log-rank=0.009) identified thresholds for higher ESKD risk. In a subgroup analysis, glomerular activity and tubular chronicity mostly accounted for AI and CI association with ESKD. No histological or laboratory predictors emerged at first biopsy (95% CI): AI: 0.88 to 1.19; CI: 0.66 to 1.20; proteinuria 0.85 to 1.08.ConclusionsFindings at second but not at first kidney biopsy in patients with persistently active or relapsing LN inform about ESKD development in a long-term follow-up.

Background and Objective: Digital pathology represents an invaluable source of information and a long-term investment with high returns, with the possible deployment of artificial intelligence (AI) tools for both the clinical and research activity. Moreover, the rising nosological complexity of the oncologic diseases, e.g., lung cancer, is stressing the need of integration among different subspecialities (e.g., radiology, molecular biology, and immuno-oncology) for the final characterization of cancer. In this setting, digital pathology can play a pivotal role in the "integration" of these different competencies, and the application of AI for prognostic/predictive purposes can represent a further "third" revolution in pathology. The objective of the present review is to provide an updated overview of the possible role of digital and integrative pathology in detecting gene mutations and, especially, translocations in different types of tumors, focusing on the promising implications that this advancement can have in lung cancer characterization. Methods: A systematic literature search was conducted on different research engines (PubMed, IEEE Xplore, dblp, ACM digital library, Inspec) over a 15-year interval from January 1, 2006 to October 31, 2021 selecting only English-language articles to highlight the possible diagnostic and prognostic role of digital pathology tools in detecting gene mutations and, especially, translocations in different types of tumors. Key Content and Findings: Digital pathology tools already demonstrated a role in the detection of specific mutations and translocations in different types of cancer, both in a targeted (e.g., liver/thyroid carcinoma) and in an agnostic (e.g., MSI) setting. In lung cancer, AI showed the capability of highlighting specific subset of mutations (STK11, EGFR, FAT1, SETBP1, KRAS, and TP53) from whole slide imaging (WSI), with the translocation field representing a promising frontier, with some gene rearrangement (e.g., NTRK) more prone to be detected with this approach as demonstrated in other organs. Conclusions: Digital pathology is becoming a valuable tool to predict gene aberrations in different types of tumors, with a promising role in lung cancer. Digital and integrative tools for the detection of translocations are progressively changing the paradigm of molecular testing, contributing to the switch from a manual to a fully automated workflow, requiring further investigation.

BACKGROUND: Renal involvement in ANCA–associated vasculitis (AAV) is associated with poor outcomes. The clinical significance of arteritis of the small kidney arteries has not been evaluated in detail. METHODS: In a multicenter cohort of patients with AAV and renal involvement, we sought to describe the clinicopathologic characteristics of patients with AAV who had renal arteritis at diagnosis, and to retrospectively analyze their prognostic value. RESULTS: We included 251 patients diagnosed with AAV and renal involvement between 2000 and 2019, including 34 patients (13.5%) with arteritis. Patients with AAV-associated arteritis were older, and had a more pronounced inflammatory syndrome compared with patients without arteritis; they also had significantly lower renal survival (P=0.01). In multivariable analysis, the ANCA renal risk score, age at diagnosis, history of diabetes mellitus, and arteritis on index kidney biopsy were independently associated with ESKD. The addition of the arteritis status significantly improved the discrimination of the ANCA renal risk score, with a concordance index (C-index) of 0.77 for the ANCA renal risk score alone, versus a C-index of 0.80 for the ANCA renal risk score plus arteritis status (P=0.008); ESKD-free survival was significantly worse for patients with an arteritis involving small arteries who were classified as having low or moderate risk, according to the ANCA renal risk score. In two external validation cohorts, we confirmed the incidence and phenotype of this AAV subtype. CONCLUSIONS: Our findings suggest AAV with renal arteritis represents a different subtype of AAV with specific clinical and histologic characteristics. The prognostic contribution of the arteritis status remains to be prospectively confirmed.

AimsIdentification and characterisation of monoclonal gammopathies of renal significance (MGRS) is critical for therapeutic purposes. Amyloidosis represents one of the most common forms of MGRS, and renal biopsy remains the gold standard for their classification, although mass spectrometry has shown greater sensitivity in this area.MethodsIn the present study, a new in situ proteomic technique, matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI), is investigated as an alternative to conventional laser capture microdissection MS for the characterisation of amyloids. MALDI-MSI was performed on 16 cases (3 lambda light chain amyloidosis (AL), 3 AL kappa, 3 serum amyloid A amyloidosis (SAA), 2 lambda light chain deposition disease (LCDD), 2 challenging amyloid cases and 3 controls). Analysis began with regions of interest labelled by the pathologist, and then automatic segmentation was performed.ResultsMALDI-MSI correctly identified and typed cases with known amyloid type (AL kappa, AL lambda and SAA). A ‘restricted fingerprint’ for amyloid detection composed of apolipoprotein E, serum amyloid protein and apolipoprotein A1 showed the best automatic segmentation performance (area under the curve >0.7).ConclusionsMALDI-MSI correctly assigned minimal/challenging cases of amyloidosis to the correct type (AL lambda) and identified lambda light chains in LCDD cases, highlighting the promising role of MALDI-MSI for amyloid typing.

International audience; The interest in implementing digital pathology (DP) workflows to obtain whole slide image (WSI) files for diagnostic purposes has increased in the last few years. The increasing performance of technical components and the Food and Drug Administration (FDA) approval of systems for primary diagnosis led to increased interest in applying DP workflows. However, despite this revolutionary transition, real world data suggest that a fully digital approach to the histological workflow has been implemented in only a minority of pathology laboratories. The objective of this study is to facilitate the implementation of DP workflows in pathology laboratories, helping those involved in this process of transformation to identify: (a) the scope and the boundaries of the DP transformation; (b) how to introduce automation to reduce errors; (c) how to introduce appropriate quality control to guarantee the safety of the process and (d) the hardware and software needed to implement DP systems inside the pathology laboratory. The European Society of Digital and Integrative Pathology (ESDIP) provided consensus-based recommendations developed through discussion among members of the Scientific Committee. The recommendations are thus based on the expertise of the panel members and on the agreement obtained after virtual meetings. Prior to publication, the recommendations were reviewed by members of the ESDIP Board. The recommendations comprehensively cover every step of the implementation of the digital workflow in the anatomic pathology department, emphasizing the importance of interoperability, automation and tracking of the entire process before the introduction of a scanning facility. Compared to the available national and international guidelines, the present document represents a practical, handy reference for the correct implementation of the digital workflow in Europe.

Objective: The American College of Radiology (ACR) and the European Thyroid Association (EU) have proposed two scoring systems for thyroid nodule classification. Here, we compared the ability of the two systems in triaging thyroid nodules for fine-needle aspiration (FNA) and tested the putative role of an approach that combines ultrasound features and cytology for the detection of malignant nodules. Design and Methods: The scores obtained with the ACR and EU Thyroid Imaging Reporting and Data Systems (TIRADS) from a prospective series of 480 thyroid nodules acquired from 435 subjects were compared to assess their performances in FNA triaging on the final cytological diagnosis. The US features that showed the highest contribution in discriminating benign nodules from malignancies were combined with cytology to improve its diagnostic performance. Results: FNA was recommended on 46.5% and 51.9% of the nodules using the ACR and EU-TIRADS scores, respectively. The ACR system demonstrated a higher specificity as compared to the EU-TIRADS (59.0% vs. 52.4%, p = 0.0012) in predicting &gt, TIR3A/III (SIAPEC/Bethesda) nodules. Moreover, specific radiological features (i.e., echogenic foci and margins), combined with the cytological classes improved the specificity (97.5% vs. 91%, p &lt, 0.0001) and positive predictive values (77.5% vs. 50.7%, p &lt, 0.0001) compared to cytology alone, especially in the setting of indeterminate nodules (TIR3A/III and TIR3B/IV), maintaining an excellent sensitivity and negative predictive value. Conclusions: The ACR-TIRADS system showed a higher specificity compared to the EU-TIRADS in triaging thyroid nodules. The use of specific radiological features improved the diagnostic ability of cytology.

Incidental thyroid carcinomas (ITCs) are a fairly frequent finding in daily routine practice, with papillary thyroid microcarcinoma being the most frequent entity. In our work, we isolated incidental cases arising in thyroids removed for other cytologically indeterminate and histologically benign nodules. We retrospectively retrieved cases with available thyroid Fine Needle Aspiration (FNA, 3270 cases), selecting those with an indeterminate cytological diagnosis (Bethesda classes III–IV, 652 cases). Subsequently, we restricted the analysis to surgically treated patients (163 cases) finding an incidental thyroid carcinoma in 22 of them. We found a 13.5% ITC rate, with ITCs representing 46.8% of all cancer histologically diagnosed in this indeterminate setting. Patients received a cytological diagnosis of Bethesda class III and IV in 41% and 59% of cases, respectively. All ITC cases turned out to be papillary thyroid microcarcinomas; 36% of cases were multifocal, with foci bilaterally detected in 50% of cases. We found an overall ITC rate concordant with the literature and with our previous findings. The assignment of an indeterminate category to FNA did not increase the risk of ITCs in our cohort. Rather, a strong statistical significance (p < 0.01) was found comparing the larger size of nodules that underwent FNA and the smaller size of their corresponding ITC nodule.

The germinal center (GC) reaction results in the selection of B-cells acquiring effector Ig secreting ability by progressing towards plasmablastic differentiation. This transition is associated with exclusion from the GC microenvironment. The aberrant expansion of plasmablastic elements within the GC fringes configures an atypical condition, the biological characteristics of which have not been defined yet. We investigated the in situ immunophenotypical and transcriptional characteristics of a non-clonal germinotropic expansion of plasmablastic elements (GEx) occurring in the tonsil of a young patient. Compared to neighboring GC and peri-follicular regions, the GEx showed a distinctive signature featuring key regulators of plasmacytic differentiation, cytokine signaling, and cell metabolism. The GEx signature was tested in the setting of diffuse large B-cell lymphoma (DLBCL) as a prototypical model of lymphomagenesis encompassing transformation at different stages of GC and post-GC functional differentiation. The signature outlined DLBCL clusters with different immune microenvironment composition and enrichment in genetic subtypes.SignificanceThis report represents the first insight into the transcriptional features of a germinotropic plasmablastic burst, shedding light into the molecular hallmarks of B cells undergoing plasmablastic differentiation and aberrant expansion within the non-canonical setting of the GC microenvironment.

Background The current study evaluates the application of histology and in situ proteomics (MALDI-MSI) in Fabry nephropathy (FN), showing investigative and classification role for this coupled approach. Methods A retrospective series of 14 formalin fixed paraffin embedded (FFPE) renal biopsies with diagnosis of FN and 1 biopsy from a patient bearing a galactosidase-alpha (GLA) genetic variant of unknown significance (GVUS, c.376A>G) have been classified for clinical characteristics. Groups were compared for histological differences (following the ISGFN scoring system). Moreover, renal biopsies from these cases have been analyzed with MALDI-MSI as previously described to find proteomic signatures among different mutations and phenotypes. Results Comparison of clinical features revealed lower mean 24 h proteinuria in females (225 mg/24 h) than in males (1477.5 mg/24 h, p = 0.006). As for clinical characteristics, females significantly differed from males only for lower arterial sclerosis, with a mean value of 0.82 vs. 1.05 (p = 0.001). Proteomic analysis demonstrated specific signatures in different subgroups of FN patients. Moreover, MALDI correctly classified cases with undetermined mutation or GVUS. Conclusions The present study demonstrated the feasible application of MALDI-MSI in the analysis of FN FFPE renal biopsies, allowing the detection of putative signatures for phenotypic distinction and demonstrating genetic classification capabilities.

Biopsy site changes in mediastinal lymph nodes (LNs) attributable to prior endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) have not been studied in a systematic manner. Twenty-four contributors from 14 institutions in 5 countries collaborated via social media (Twitter) to retrospectively review consecutive cases of resected mediastinal LNs from patients with prior EBUS-TBNA. Resected LNs were reexamined by submitting pathologists for changes attributable to EBUS-TBNA. Patients who received neoadjuvant therapy were excluded. Cases with suspected biopsy site changes underwent central review by 5 pathologists. A total of 297 mediastinal LN resection specimens from 297 patients (183 male/114 female, mean age: 65 y, range: 23 to 87) were reviewed. Biopsy site changes were most common in station 7 (10 cases) followed by 11R, 4R, and 10R, and were found in 34/297 (11.4%) cases, including displacement of tiny cartilage fragments into LN parenchyma in 26, intranodal or perinodal scars in 7, and hemosiderin in 1. Cartilage fragments ranged from 0.26 to 1.03 mm in length and 0.18 to 0.62 mm in width. The mean interval between EBUS-TBNA and LN resection was 38 days (range: 10 to 112) in cases with biopsy site changes. A control group of 40 cases without prior EBUS-TBNA, including 193 mediastinal LN stations, showed no evidence of biopsy site changes. Biopsy site changes are identified in a subset of resected mediastinal LNs previously sampled by EBUS-TBNA. The location of the abnormalities, temporal association with prior EBUS-TBNA, and the absence of such findings in cases without prior EBUS-TBNA support the contention that they are caused by EBUS-TBNA.

Background: The implementation of a fully digital workflow in any anatomic pathology department requires a complete conversion to a tracked system. Ensuring the strict correspondence of the material submitted for the analysis, from the accessioning to the reporting phase, is mandatory in the anatomic pathology laboratory, especially when implementing the digital pathology for primary histological diagnosis. The proposed solutions, up to now, rely on the verification that all the materials present in the glass slide are also present in the whole slide images (WSIs). Although different methods have already been implemented for this purpose (e.g., the 'macroimage' of the digital slide, representing the overview of the glass slide), the recent introduction of a device to capture the cut surface of paraffin blocks put the quality control of the digital workflow a step forward, allowing to match the digitized slide with the corresponding block. This system may represent a reliable, easy-to-use alternative to further reduce tissue inconsistencies between material sent to the lab and the final glass slides or WSIs. Methods: The Anatomic Pathology of the Gravina Hospital in Caltagirone, Sicily, Italy, has implemented the application of the BlocDoc devices (SPOT Imaging, Sterling Heights, USA) in its digital workflow. The instruments were positioned next to every microtome/sectioning station, with the possibility to capture the 'normal' and the polarized image of the cut surface of the blocks directly by the technician. The presence of a monitor in the BlocDoc device allowed the technician to check the concordance between the cut surface of the block and the material on the corresponding slide. The link between BlocDoc and the laboratory information system, through the presence of the 2D barcode, allowed the pathologists to access the captured image of the cut surface of the block at the pathologist workstation, thus enabling the direct comparison between this image and the WSI (thumbnail and 'macroimage'). Results: During the implementation period, more than 10.000 (11.248) blocks were routinely captured using the BlocDoc. The employment of this approach allowed a drastic reduction of the discordances and tissue inconsistencies. The implementation of the BlocDoc in the routine allowed the detection of two different types of 'errors,' the so-called 'systematic' and 'occasional' ones. The first type was intrinsic of some specific specimens (e.g., transurethral resection of the prostate, nasal polypectomies, and piecemeal uterine myomectomies) characterized by the three-dimensional nature of the fragments and affected almost 100% of these samples. On the other hand, the 'occasional' errors, mainly due to inexperience or extreme caution of the technicians in handling tiny specimens, affected 98 blocks (0.9%) of these samples and progressively reduced with the rising confidence with the BlocDoc. One of these cases was clinically relevant. No problems in the recognition of the 2D barcodes were encountered using a laser cassette printer. Finally, rare failures have been recorded during the period, accounting for

Fine needle aspiration (FNA) is the reference standard for the diagnosis of thyroid nodules. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been successfully used to discriminate the proteomic profiles of benign and malignant thyroid FNAs within the scope of providing support to pathologists for the classification of morphologically borderline cases. However, real FNAs provide a limited amount of material due to sample collection restrictions. Ex vivo FNAs could represent a valuable alternative, increasing sample size and the power of statistical conclusions. In this study, we compared the real and ex vivo MALDI-MSI proteomic profiles, extracted from thyrocyte containing regions of interest, of 13 patients in order to verify their similarity. Statistical analysis demonstrated the mass spectra similarity of the proteomic profiles by performing intra-patient comparison, using statistical similarity systems. In conclusion, these results show that post-surgical FNAs represent a possible alternative source of material for MALDI-MSI proteomic investigations in instances where pre-surgical samples are unavailable or the number of cells is scarce. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-020-03088-4.

Immune checkpoint inhibitors for blocking the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis are now available for squamous cell carcinoma of the head and neck (HNSCC) in relapsing and/or metastatic settings. In this work, we compared the resulting combined positive score (CPS) of PD-L1 using alternative methods adopted in routine clinical practice and determined the level of diagnostic agreement and inter-observer reliability in this setting. The study applied 5 different protocols on 40 tissue microarrays from HNSCC. The error rate of the individual protocols ranged from a minimum of 7% to a maximum of 21%, the sensitivity from 79% to 96%, and the specificity from 50% to 100%. In the intermediate group (1 &le, CPS &lt, 20), the majority of errors consisted of an underestimation of PD-L1 expression. In strong expressors, 5 out of 14 samples (36%) were correctly evaluated by all the protocols, but no protocol was able to correctly identify all the &ldquo, strong expressors&rdquo, The overall inter-observer agreement in PD-L1 CPS reached 87%. The inter-observer reliability was moderate, with an ICC of 0.774 (95% CI (0.651, 0.871)). In conclusion, our study showed moderate interobserver reliability among different protocols. In order to improve the performances, adequate specific training to evaluate PD-L1 by CPS in the HNSCC setting should be coordinated.

Nephropathy represents a major complication of Fabry Disease and its accurate characterization is of paramount importance in predicting the disease progression and assessing the therapeutic responses. The diagnostic process still relies on performing renal biopsy, nevertheless many efforts have been made to discover early reliable biomarkers allowing us to avoid invasive procedures. In this field, proteomics offers a sensitive and fast method leading to an accurate detection of specific pathological proteins and the discovery of diagnostic and prognostic biomarkers that reflect disease progression and facilitate the evaluation of therapeutic responses. Here, we report a review of selected literature focusing on the investigation of several proteomic techniques highlighting their advantages, limitations and future perspectives in their application in the routine study of Fabry Nephropathy.

Cytological specimens may be the ideal source of nucleic acids for the molecular characterization of tumors in the next generation sequencing (NGS) era. Liquid‐based shaking of core needle biopsy samples could enhance the diagnostic yield, allowing additional material to be examined in parallel with histopathology.

BACKGROUND Bronchoalveolar lavage (BAL) in patients with severe coronavirus disease 2019 (COVID‐19) may provide additional and complementary findings for the management of these patients admitted to intensive care units (ICUs). This study addresses the cytological features of the infection and highlights the more influential inflammatory components. The correlation between pathological variables and clinical data is also analyzed. METHODS The authors performed a retrospective analysis of the cytopathological features of BAL in 20 COVID‐19 patients and 20 members of a matched cohort from a critical ICU who had acute respiratory distress syndrome caused by other pulmonary conditions. RESULTS A comparison of the controls (n = 20) and the COVID‐19 patients (n = 20) revealed that the latter had a higher neutrophil count (median, 63.8% of the cell count) with lower percentages of macrophages and lymphocytes. An increase in the expression of CD68‐positive, monocytic multinucleated giant cells (MGCs) was reported; megakaryocytes were not detected on CD61 staining. Perls staining showed isolated elements. In situ RNA analysis demonstrated scattered chromogenic signals in type II pneumocytes. An ultrastructural analysis confirmed the presence of intracytoplasmic vacuoles containing rounded structures measuring 140 nm in diameter (putative viral particles). In COVID‐19 patients, the clinicopathological correlation revealed a positive correlation between lactate dehydrogenase values and MGCs (r = 0.54). CONCLUSIONS The analysis of BAL samples might be implemented as a routine practice for the evaluation of COVID‐19 patients in ICUs in the appropriate clinical scenario. Additional studies using a larger sample size of patients who developed COVID‐19 during the second wave of the epidemic in the autumn of 2020 are needed to further support our findings., An analysis of the cytological features of bronchoalveolar lavage samples can provide useful information for the management of coronavirus disease 2019 patients in the intensive care unit.

Fine-needle aspiration biopsies (FNA) represent the gold standard to exclude the malignant nature of thyroid nodules. After cytomorphology, 20–30% of cases are deemed “indeterminate for malignancy” and undergo surgery. However, after thyroidectomy, 70–80% of these nodules are benign. The identification of tools for improving FNA’s diagnostic performances is explored by matrix-assisted laser-desorption ionization mass spectrometry imaging (MALDI-MSI). A clinical study was conducted in order to build a classification model for the characterization of thyroid nodules on a large cohort of 240 samples, showing that MALDI-MSI can be effective in separating areas with benign/malignant cells. The model had optimal performances in the internal validation set (n = 70), with 100.0% (95% CI = 83.2–100.0%) sensitivity and 96.0% (95% CI = 86.3–99.5%) specificity. The external validation (n = 170) showed a specificity of 82.9% (95% CI = 74.3–89.5%) and a sensitivity of 43.1% (95% CI = 30.9–56.0%). The performance of the model was hampered in the presence of poor and/or noisy spectra. Consequently, restricting the evaluation to the subset of FNAs with adequate cellularity, sensitivity improved up to 76.5% (95% CI = 58.8–89.3). Results also suggest the putative role of MALDI-MSI in routine clinical triage, with a three levels diagnostic classification that accounts for an indeterminate gray zone of nodules requiring a strict follow-up.

Background and Aims The introduction of effective direct-acting antiviral agents (DAAs) has allowed safe transplantation of kidneys from HCV positive (HCV+) donors into HCV negative (HCV-) recipients. Some studies demonstrated an increased risk for rejection as well as polyomavirus infection in these situations (Molnar et al, Am J Transplant. 2019 Nov;19(11):3046-3057), possibly due to the development of an inflammatory milieu in the setting of HCV seroconversion and persistent viremia (Durand et al, Am J Transplant. 2019 Nov;19(11):2969-2970). With this concern for HCV-associated renal injury and increased risk for rejection, this retrospective study explores the histologic findings of allograft biopsies from HCV+ donors. Method Fifty renal allograft biopsies from 28 recipients of HCV+ donors evaluated at Vanderbilt University Medical Center between September 2018 and December 2019 were retrospectively reviewed with recording of clinical and histological features, and final diagnoses. All recipients were transplanted under a clinical protocol and were treated with DAA therapy. In the same period of time, 30 renal allograft biopsies from 24 recipients of HCV- donors were selected having the same post-transplant interval as the study cohort. Categorical variables are expressed in percentage and compared using Pearson’s chi-squared test. Continuous variables are expressed as mean±SD and compared used Student t-test. A p-value of Results The complete results of the comparison between the two groups (HCV+ and HCV- donors) are demonstrated in Figure. The differences reaching statistical significance include higher frequency of males and tubulointerstitial fibrosis (IFTA) in the HCV+ group (p = 0.038 and 0.044, respectively) and the higher incidence of DSA positivity and acute tubular injury in the control group (p = 0.001 and 0.029). Notably, no significant differences were noted in terms of antibody-mediated events (microcirculation inflammation, transplant glomerulopathy, C4d positivity, or diagnostic ABMR), or T-cell mediated events including borderline or diagnostic T-cell mediated rejection (Banff 2013). The two groups also demonstrated the same incidence of polyomavirus nephropathy and de novo/recurrent glomerulonephritis. Interestingly, two of the HCV+ cases showed mesangial IgA deposits on immunofluorescence and electron microscopy on the first biopsy, of which one showed persistence of deposits, 18 and 44 days after transplant respectively. Once case demonstrated a de novo focal proliferative IgM-dominant glomerulonephritis on the 7th biopsy, 266 days after transplantation. Conclusion Kidneys from HCV+ donors represent a valuable resource in the era of DAA therapy. This reported experience demonstrates similar frequency of rejection and viral infection affecting allografts from HCV positive and negative donors. The presence of immune complex deposits and glomerulonephritis in some of these cases could represent either a donor-derived injury or an acquired post-seroconversion feature. In conclusion, HCV+ donors appear to be a safe resource for transplantation. Further studies with post-transplant intervals will provide additional characterization of their impact.

Background and Aims Whole-slide imaging (WSI) and virtual microscopy are useful tools implemented in the routine pathology workflow in the last 10 years, allowing second-opinion on definitive or frozen-section diagnosis (telepathology) and demonstrating a substantial role in multidisciplinary (MDT) meetings and education (Griffin et al, Histopathology. 2017;70(1):134-145). The FDA approval of the clinical employment of this technology led to the progressive digitalization of routine pathological practice. In this setting, a recent work explored the usefulness of this technology applied to renal biopsies, stressing the possibility to create integrate networks to share cases for diagnostic and research purposes (Barisoni et al, Clin Kidney J. 2017;10(2):176–187). Here is discussed the 5-years experience of an Italian Renal Pathology service in which telepathology is a definitive standard of care. Method Renal biopsies were collected in the Anatomic Pathology Department of San Gerardo Hospital, Monza, Italy from 14 different Italian Nephrology centers (9 from the North, 1 from the Center and 4 from the South of Italy) from January 2015 to December 2019. After the routing processing of the tissue, biopsies were scanned with Aperio CS2 device for light microscopy and Aperio ScanScope FL for immunofluorescence (Leica Biosystem, Illinois, USA, Figure 1A). Digital slides were then imported in the Spectrum platform along with the final report and additional pictures (eg. electron micrographs and special stains) such as shown in Figure 1B. Results In 5 years, 704 renal biopsies (interval 110-160) were scanned and analysed with a turnaround time of around 2-3 working days. The most frequent diagnosis was represented by MN (16%, 115 cases, interval 18-28) followed by IgA nephropathy (12%, 84 cases, interval 12-21), FSGS (9%, 66 cases, interval 8-18), MCD (8%, 54 cases, interval 7-13), paucimmune crescentic GN (7%, 48 cases, interval 6-13), diabetic nephropathy (6%, 45 cases, interval 6-12), lupus nephritis (5%, 37 cases, interval 5-11), arterionephrosclerosis (5%, 35 cases, interval 5-13), amyloidosis (5%, 32 cases, interval 3-7) and tubulointerstitial nephritis (4%, 28 cases, interval 4-6). Only 4% of cases (28, interval 4-10) were suboptimal for the diagnosis and the remaining 19% (132 cases, interval 20-36) were characterized by rarer diseases, as detailed in Figure 1C. Conclusion In renal pathology the assessment of biopsies with different techniques (light and electron microscopy and immunofluorescence) is mandatory and can represent an obstacle in the routine employment of digital pathology. In the present experience we demonstrated the feasibility and sustainability of the digital switch in renal pathology routine, thanks to appropriate devices and platform. This led to store cases for teaching and MDT meetings, allowing to maintain the same short turnaround time for the diagnosis. Moreover, the employment of WSI technique can allow to couple the proteomic features of renal tissue with the classic morphological aspects of the diseased parenchyma (L’Imperio et al Proteomics Clin Appl. 2016 Apr;10(4):371-83). This approach, along with the possibility of a multicentric enrollment of patients, led to the publication of 6 scientific papers in highly impacted journals, further confirming the positive role of digital pathology in this field.

Background and Aims ANCA-associated vasculitis is the most common cause of rapidly progressive glomerulonephritis (GN) and can lead to ESRD or death, even after aggressive immunosuppressive therapies (Jones et al, N Engl J Med, 363(2010), 211-220). The histological assessment of renal tissue can help in predicting the outcome and a 4-tiered classification based on glomerular lesions has been proposed to stratify these cases (Berden et al, J Am Soc Nephrol, 21(2010), 1628-1636). Subsequent studies, however, failed to confirm its predictive value (Tanna et al, Nephrol Dial Transplant, 30(2015), 1185-1192), stressing the need of new prognostic markers of the disease. The present study investigates whether additional histological features can improve the performance of the current classification in predicting the outcome of these patients. Method A retrospective series of biopsy-proven paucimmune crescentic GN has been collected between January 2012 and June 2018 from two Italian centers (San Gerardo Hospital, Monza and Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma). After the exclusion of patients with negative/unavailable serum ANCA testing, lacking clinical data, less than 12 months of follow-up, less than 5 glomeruli per biopsy and coexisting glomerular diseases, a final cohort of 52 cases was selected. Demographic and clinical data at the time of the diagnosis are reported in Figure, panel A. Renal biopsies have been reviewed by two experts in renal pathology and scored for multiple glomerular, tubulointerstitial and vascular lesions, as well as classified following the 4-tiered schema. The outcome of interest was time to need for RRT or death, whatever occurred first. Cox proportional hazards regression models were constructed with time to composite event, loss to follow-up or censoring (06/30/2019). Time at risk started at the date of renal biopsy. Histologic predictors were collapsed into binary variables (0-1 = low; 2-3 = high) and tested in univariate models, with the association expressed as hazard ratios (HRs) and 95% confidence intervals (CI); those showing a significant association with the outcome ( Figure, panel B) were included in a multivariable model together with a variable representing the Berden class. The prognostic performance of models including only the Berden class or the Berden class plus additional predictors was assessed using Harrell’s c-statistic. Results During a follow-up of 1,828 person-months, 13 composite events developed (8 deaths, 5 RRT), corresponding to an incidence rate of 7.1 (95% CI 4.2, 12.2) per 1,000 person-months. Of the tested predictors, Bowman’s capsule rupture (BCR, Figure, panel C) was significantly associated with the outcome (p = 0.023). Compared with a model including only Berden class (c = 0.67), the addition of this parameter significantly improved the prognostic performance (c = 0.76). In the multivariable model including Berden class, BCR remained significantly associated with the outcome (HR 3.61, 95% CI 1.15, 11.34; p = 0.028). Conclusion The present study demonstrated an improved performance of Berden classification in predicting patients’ outcome after the implementation of BCR. This can allow a better stratification of these cases, leading to tailored therapeutic approaches. Further investigations on a larger prospective cohort are required to confirm these results.

Background and Aims Circulating donor-derived cell-free DNA (dd-cfDNA) has recently been proposed as an early non-invasive marker for renal allograft rejection (Bloom et al, J. Am. Soc. Nephrol. 2017;28, 2221–2232). Recent studies demonstrated its potential role in the detection of any kind of rejection with better performance in detecting antibody-mediated rejection (ABMR) (Huang et al, Am J Transplant. 2019;19:1663–1670). However, at the current cutoff value for a positive test, the test seems to be affected by low sensitivity for T-cell mediated rejection (TCMR) and false positive results in the setting of non-rejection related damage. In the present study, the performance of dd-cfDNA is assessed on a retrospective series of transplant renal biopsies. Method Sixty-three renal biopsies from 52 allograft recipients with available dd-cfDNA results, evaluated between November 2017 and December 2019 at Vanderbilt University Medical Center, Nashville, Tennessee, were reviewed. Clinical and histological features along with the final diagnosis were recorded. Renal allograft rejection was classified following Banff 2013 criteria. Pearson’s chi-squared test was used to compare cases positive and negative for dd-cfDNA. Receiving operator curve (ROC) analysis was used to assess the performance of the test in detecting any rejection and ABMR. A p-value of Results The patients were 53±12.5 years old, 43% were men, 51% and 49% were Caucasian and African-American, respectively. The median value of dd-cfDNA was 2.6% for ABMR and 2.4% for any kind of rejection, statistically different from the group without rejection (0.56%, p = 0.002 and 0.0003, respectively). TCMR failed to demonstrate a statistically significant difference in dd-cfDNA levels vs controls (1.8% vs 0.56%, p = 0.627). The comparison of groups with positive and negative dd-cfDNA test is reported in Figure (panel A). Positive patients demonstrated a higher rate of findings associated with ABMR such as DSA positivity, moderate microcirculation inflammation, and transplant glomerulopathy, in addition to diagnostic ABMR and chronic/active ABMR. Moreover, a significant difference was found between the two groups in the rate of any kind of rejection. ROC analysis for any ABMR (Figure, panel B) demonstrated an AUC of 0.76 (95% CI 0.61-0.91) with a sensitivity (Sn) of 92.3%, a specificity (Sp) of 60%, a positive predictive value (PPV) of 37.5% and a negative predictive value (NPP) of 96.8%. ROC analysis for any kind of rejection (Figure, panel C) demonstrated an AUC of 0.84 (95% CI 0.74-0.94) with a Sn of 94.1%, a Sp of 65.2%, a PPV of 50% and a NPP of 96.8%. Among the patients with positive dd-cfDNA and without rejection (n=16, 51%), allograft biopsy demonstrated borderline TCMR in 4, acute tubular injury in 8, chronic pyelonephritis in 1, mild interstitial fibrosis in 1, and 2 with minimal histological abnormalities. Notably, none of the cases with a positive test were diagnosed with polyomavirus nephropathy. Conclusion Similar to previous studies, dd-cfDNA represents a sensitive test for detection of ABMR, with high performance in any rejection setting with the Banff 2013 criteria. However, the high rate of false positive results and the presence of undetected T-cell mediated events (e.g. borderline cases) at the current recommended cut-off value for positivity limits its employment as a surrogate for the renal biopsy.