Atlas of PD-L1 for Pathologists: Indications, Scores, Diagnostic Platforms and Reporting Systems.

BACKGROUND: Innovative drugs targeting the PD1/PD-L1 axis have opened promising scenarios in modern cancer therapy. Plenty of assays and scoring systems have been developed for the evaluation of PD-L1 immunohistochemical expression, so far considered the most reliable therapeutic predictive marker. METHODS: By gathering the opinion of acknowledged experts in dedicated fields of pathology, we sought to update the currently available evidence on PD-L1 assessment in various types of tumors. RESULTS: Robust data were progressively collected for several anatomic districts and leading international agencies to approve specific protocols: among these, TPS with 22C3, SP142 and SP263 clones in lung cancer; IC with SP142 antibody in breast, lung and urothelial tumors; and CPS with 22C3/SP263 assays in head and neck and urothelial carcinomas. On the other hand, for other malignancies, such as gastroenteric neoplasms, immunotherapy has been only recently introduced, often for particular histotypes, so specific guidelines are still lacking. CONCLUSIONS: PD-L1 immunohistochemical scoring is currently the basis for allowing many cancer patients to receive properly targeted therapies. While protocols supported by proven data are already available for many tumors, dedicated studies and clinical trials focusing on harmonization of the topic in other still only partially explored fields are surely yet advisable.

Histology of portal vascular changes associated with idiopathic non-cirrhotic portal hypertension: nomenclature and definition.

Idiopathic non-cirrhotic portal hypertension (INCPH) is a rare vascular liver disease that has attracted new interest in recent years. It is characterised by clinical signs of portal hypertension in the absence of cirrhosis or severe fibrosis and any known cause of portal hypertension. As much uncertainty exists about INCPH pathophysiology, and no definite diagnostic tests are available, liver biopsy is an essential tool for achieving a definite diagnosis. Unfortunately, the histological diagnosis of INCPH is not always straightforward, as the characteristic lesions are unevenly distributed, vary greatly in their severity, are often very subtle, and are not all necessarily present in a single case. Furthermore, specifically for the characteristic portal vessel changes observed in INCPH, the terminology and definition are ambiguous, which adds complexity to the already complex clinicopathological scenario. An international study group of liver pathologists and hepatologists pursued a consensus on nomenclature for the portal vascular lesions of INCPH. Such standardisation may assist pathologists in the recognition of such lesions, and will possibly facilitate further advancement in this field.

Acute-on-chronic liver failure 2018: a need for (urgent) liver biopsy?

INTRODUCTION: 'Acute-on-Chronic-Liver Failure (ACLF)' entered hepatology practice by the end of the 20th century. Although we lack precise and universally agreed definitions, acute decompensation of chronic liver disease with jaundice and deranged clotting, multi-organ failure and high, short-term mortality are hallmarks of the syndrome. Timely recognition and and treatment, including urgent liver transplantation, may save the life of certain patients. The diagnosis and management are mostly based on clinical features, but some have suggested to incorporate histopathology (liver biopsy). This may add to the differentiation between acute and chronic disease, primary and concomitant etiologies, and identify prognostic determinants. Areas covered: A review of the literature on ACLF and the outcome of the discussions at a topical international meeting on specific histopathological aspects of diagnosis and prognosis of the syndrome. Expert commentary: There is a lack of standardized descriptions of histopathological features and there is limited prospective experience with the role of pathology of ACLF. It is important for the clinical hepatologist to understand the potential and limitations of (transjugular) liver biopsy in ACLF and for the pathologist to help address the clinical question and recognise the histopathological features that help to characterize ACLF, both in terms of diagnosis and prognosis.

iPathology cockpit diagnostic station: validation according to College of American Pathologists Pathology and Laboratory Quality Center recommendation at the Hospital Trust and University of Verona.

BACKGROUND: Validation of digital whole slide images is crucial to ensure that diagnostic performance is at least equivalent to that of glass slides and light microscopy. The College of American Pathologists Pathology and Laboratory Quality Center recently developed recommendations for internal digital pathology system validation. Following these guidelines we sought to validate the performance of a digital approach for routine diagnosis by using an iPad and digital control widescreen-assisted workstation through a pilot study. METHODS: From January 2014, 61 histopathological slides were scanned by ScanScope Digital Slides Scanner (Aperio, Vista, CA). Two independent pathologists performed diagnosis on virtual slides in front of a widescreen by using two computer devices (ImageScope viewing software) located to different Health Institutions (AOUI Verona) connected by local network and a remote image server using an iPad tablet (Aperio, Vista, CA), after uploading the Citrix receiver for iPad. Quality indicators related to image characters and work-flow of the e-health cockpit enterprise system were scored based on subjective (high vs poor) perception. The images were re-evaluated two weeks apart. RESULTS: The whole glass slides encountered 10 liver: hepatocarcinoma, 10 renal carcinoma, 10 gastric carcinoma and 10 prostate biopsies: adenocarcinoma, 5 excisional skin biopsies: melanoma, 5 lymph-nodes: lymphoma. 6 immuno- and 5 special stains were available for intra- and internet remote viewing. Scan times averaged two minutes and 54 seconds per slide (standard deviation 2 minutes 34 seconds). Megabytes ranged from 256 to 680 (mean 390) per slide storage. Reliance on glass slide, image quality (resolution and color fidelity), slide navigation time, simultaneous viewers in geographically remote locations were considered of high performance score. Side by side comparisons between diagnosis performed on tissue glass slides versus widescreen were excellent showing an almost perfect concordance (0.81, kappa index). CONCLUSIONS: We validated our institutional digital pathology system for routine diagnostic facing with whole slide images in a cockpit enterprise digital system or iPad tablet. Computer widescreens are better for diagnosing scanned glass slide that iPad. For urgent requests, iPad may be used. Legal aspects have to be soon faced with to permit the clinical use of this technology in a manner that does not compromise patient care.

TNM staging system of colorectal carcinoma: a critical appraisal of challenging issues.

CONTEXT: Colorectal cancer is the leading cause of morbidity and death among gastrointestinal tumors and ranks fourth after lung, breast, and ovarian cancers. Despite a continuous refinement of the T (tumor), N (node), and M (metastasis) staging system to express disease extent and define prognosis, and eventually to guide treatment, the outcome of patients with colorectal cancer may vary considerably even within the same tumor stage. Therefore, the need for new factors, either morphologic or molecular, that could more precisely stratify patients into different risk categories is clearly warranted. OBJECTIVES: To present the state of the art with regard to the colorectal cancer staging system and to discuss confusing and/or challenging issues, including the assessment of peritoneal membrane involvement, vascular invasion, tumor deposits, and pathologic tumor response to neoadjuvant chemoradiotherapy. DATA SOURCES: Literature review of relevant articles indexed in PubMed (US National Library of Medicine) and primary material from the authors' institutions. CONCLUSIONS: Two emerging needs exist for the TNM system, namely, further stratification of patients with the same tumor stage and incorporation of nonanatomic factors, the latter including molecular and treatment factors. The identification and classification of morphologic features encountered in the pathologic examination of colorectal cancer specimens may be difficult and a source of subjective variability. Enhanced pathologic analysis, agreed-upon standard protocols, and standardization should improve the completeness and accuracy of pathology reports.

Tumor deposits are encountered in advanced colorectal cancer and other adenocarcinomas: an expanded classification with implications for colorectal cancer staging system including a unifying concept of in-transit metastases.

The seventh edition of the TNM Classification of Malignant Tumors is due to be published soon. In the current version dating back to 2002, tumor deposits, which are metastatic lesions commonly encountered in the routine histopathological examination of advanced colorectal cancer specimens, are classified according to their shape with different implications for staging. So distinguished, these lesions are considered either as metastatic lymph nodes (N category) or as vascular invasions (T category). We recently proposed a more comprehensive classification approach that also includes the M category. Relying on two of our independent recent studies, we aim here to provide suggestions for a novel classification of tumor deposits with diverse implications for TNM staging system of colorectal cancer. Furthermore, we show that tumor deposits are not limited to colorectal cancer, but they are common to different adenocarcinoma types.

Clinical, pathological, and molecular correlates in ferroportin disease: a study of two novel mutations.

BACKGROUND/AIMS: Clinico-pathological manifestations of ferroportin (Fpn) disease (FD) are heterogeneous, with some patients presenting with iron overload predominantly in macrophages ("M" phenotype), others predominantly in hepatocytes ("H" phenotype). This appears to reflect functional heterogeneity of Fpn mutants, with loss-of-function generally resulting in the M type. METHODS: Two unrelated probands with "non-HFE" hemochromatosis were screened for Fpn mutations. Mutants were functionally characterized by immunofluorescence microscopy, evaluation of their ability to bind hepcidin and export iron, and by expressing them in zebrafish. RESULTS: Two novel Fpn mutations were identified: I152F in patient-1, presenting with typical M phenotype; and L233P in patient-2, presenting with ambiguous features (massive overload in both macrophages and hepatocytes). Molecular studies suggested loss of function in both cases. The I152F, normally localized on cell membrane and internalized by hepcidin, showed a unique "primary" deficit of iron export capability. The L233P did not appropriately traffic to cell surface. Loss of function was confirmed by expressing both mutants in vivo in zebrafish, resulting in iron limited erythropoiesis. Clinical manifestations were likely enhanced in both patients by non-genetic factors (HCV, alcohol). CONCLUSIONS: The combination of careful review of clinico-pathological data with molecular studies can yield compelling explanations for phenotype heterogeneity in FD.

Huge renal cyst with parietal renal cell carcinoma, osseous metaplasia and a papillary adenoma: a case report with unique clinicopathological features and literature review.

The unique clinicopathological features of a giant solitary renal cyst with a parietal clear cell carcinoma in contiguity with a focus of osseous metaplasia and a papillary adenoma are reported. Ultrasonography and computed tomography showed a single cyst with a focal wall irregularity. During surgery, a frozen section revealed the presence of a renal cell carcinoma of clear cell type, so a nephrectomy was performed. After extensive pathological sampling of the cyst's wall, a focus of osseous metaplasia in contiguity with the main tumour and a microscopic papillary adenoma were found. Diagnostic implications for the present case are discussed within a pertinent literature review.

Biliary intraepithelial neoplasia: an international interobserver agreement study and proposal for diagnostic criteria.

Cholangiocarcinoma of the intrahepatic and extrahepatic bile ducts develops through a multistep histopathologic sequence. Premalignant or non-invasive neoplastic lesions of bile ducts have been historically called biliary dysplasia or atypical biliary epithelium. To this date, no standard terminology or classification system has been offered for these lesions. In 2005, a conceptual framework and diagnostic criteria for biliary intraepithelial neoplasia (BilIN) were proposed using the livers of patients with hepatolithiasis. We report herein an international interobserver agreement study on the diagnosis of biliary non-invasive neoplastic lesions with the goal to obtain a consensus on the terminology and grading. Seventeen pathologists from the United States, Europe and Asia participated in this study. They shared a digital file containing histological pictures of 30 foci of non-invasive neoplastic lesions selected from the biliary system of patients suffering from primary sclerosing cholangitis, choledochal cyst or hepatolithiasis. In the criteria, we proposed in 2005, BilIN was classified into three categories based on the degree of atypia: BilIN-1, BilIN-2 and BilIN-3. In this study, consensus was reached for the terminology of BilIN and the three-grade classification system. Interobserver agreement on the diagnosis was moderate (kappa-value=0.45). On the basis of the suggestions and opinions obtained from the 17 participants, the original criteria for BilIN were revised. We now propose a new consensus classification of BilIN that may assist in allowing a more uniform terminology for the diagnosis of biliary non-invasive neoplastic lesions. This classification should help to advance clinical and research applications.

Oxidative stress in pterygium: relationship between p53 and 8-hydroxydeoxyguanosine.

PURPOSE: Ultraviolet (UV) radiation is known to cause oxidative DNA damage and is thought to be a major factor implicated in the pathogenesis of pterygium, a benign invasive lesion of the bulbar conjunctiva. Among all the photooxidative DNA products, 8-hydroxydeoxyguanosine (8-OHdG) is regarded as a sensitive and stable biomarker for evaluating the degree of DNA damage. The protein p53 is a major cell stress regulator that acts to integrate signals from a wide range of cellular stresses. UV radiation can cause mutations in the p53 tumor suppressor gene that, when inactivated through mutation and loss of heterozygosity, can lead to cell proliferation and genomic instability. In many types of UV-radiation damaged cells, p53 is overexpressed and immunohistochemically detectable. Recent data on tissues exposed to factors inducing oxidative stress have provided evidence of the concomitant presence of increased levels of 8-OHdG and protein p53. To verify a possible significant association between p53 and 8-OHdG, we examined a series of 31 Ecuadorian pterygia for the expression of the two markers. Moreover, we evaluated if clinical variables such as patient's age, gender, geographic location, and disease stage, might play a role affecting the 8-OHdG and p53 immunohistochemical staining results. METHODS: Primary pterygium samples were treated for immunohistochemical evaluations of 8-OHdG and p53 protein. Mouse monoclonal antibodies to 8-OHdG and p53 were used. Statistical analyses were performed using the SPSS 12 statistical software package. RESULTS: In our study, 21 (67.74%) pterygial samples were positive for 8-OHdG staining, 11 (35.48%) specimens were positive for p53 expression, and all negative control samples showed no staining. The staining for 8-OHdG was limited to the nuclei of the epithelial layer. No substantial staining was visible in the subepithelial fibrovascular layers. No differences in the pattern of staining between 8-OHdG and p53 were observed. All samples positive for p53 (11/31, 35.48%) were also positive for 8-OHdG immunostaining, and all specimens negative for 8-OHdG (10/31, 32.26%) were also negative for p53. When analyzed by Fisher's exact test, 8-OHdG expression was significantly associated with p53 positivity (p=0.0049). Student's t-test demonstrated statistically significant association between the expression of p53 and age (p=0.02). The correlation between the two markers and the other clinical variables revealed no statistically significant association. CONCLUSIONS: Although pterygium is a lesion with limited local invasion and an inability to metastasize, the concomitant presence of altered p53 in 8-OHdG-immunoreactive cells could provide evidence of apparent genetic instability, which is in contrast to its benign clinical course.

The predictive value of CD8, CD4, CD68, and human leukocyte antigen-D-related cells in the prognosis of cutaneous malignant melanoma with vertical growth phase.

BACKGROUND: To establish the prognostic value of immune system cells that infiltrate melanoma, the authors evaluated the distribution and density of T lymphocyte subsets, macrophages, and dendritic cells in samples of primary cutaneous melanoma from 47 patients with Stage I and II melanoma according to the American Joint Committee on Cancer staging system. METHODS: Immunohistochemical demonstrations of CD8 and CD4 lymphocytes, CD68 macrophages, human leukocyte antigen-D-related (HLA-DR) cells, S-100 protein, and melanoma-associated antigens Melan A and HMB-45 were performed. The results were derived from independent histopathologic reviews by two pathologists. The low-density, moderate-density, and high-density groups of cells that infiltrated the base of the tumor during the vertical growth phase were compared with the overall survival rate using the Kaplan-Meier method and the log-rank test. Clinical variables (gender, age, tumor location, Clark level, vascular/lymphatic invasion, and thickness) also were analyzed. RESULTS: The CD8 lymphocytes exhibited independent statistically positive significance in survival (log-rank test, 8.49; P = 0.01) between patients in different lymphocyte density groups. There was a difference in 5-year survival among patients in the high-density group (78.8%), the moderate-density group (44.4%), and the low-density group (25.0%). The CD4 lymphocytes, which were less numerous than CD8 cells, had similar distribution. There also was a correlation of HLA-DR cells with overall survival (log-rank test, 5.29; P = 0.02). CD68 cell density was not found to be correlated with survival. CONCLUSIONS: The presence and number of infiltrating CD8 lymphocytes as well as the overall occurrence of HLA-DR cells may be considered independent, favorable prognostic factors in melanoma. The current results may be important for identifying other prognostic factors with which to evaluate disease progression and develop immune therapies for patients with melanoma.

Liver stellate cells and aminoterminal peptide of type III procollagen in chronic hepatitis C treated with interferon.

BACKGROUND/AIMS: Fibrogenesis plays a crucial role in development of cirrhosis, and liver stellate cells, activated to myofibroblasts expressing alpha-smooth muscle actin, are responsible for deposition of fibrous matrix; aminoterminal peptide of type III procollagen is a serum marker of active fibrogenesis. Interferon can slow ongoing fibrogenesis in chronic viral hepatitis, but it remains unclear whether the drug acts by a direct effect on stellate cells or by inhibiting the necro-inflammatory process. The aim of this study was to evaluate, in selected cases of chronic hepatitis C, whether changes in stellate cell expression induced by interferon correlated with changes in serum levels of procollagen or with clinical response to the therapy, in order to further investigate the mechanism of interferon's effect on fibrogenesis. METHODOLOGY: We studied 30 patients with chronic hepatitis C, treated with interferon and followed for more than 6 months. Before and after-treatment evaluation included histological scores for portal activity, lobular activity and fibrosis; immunohistochemical scores for alpha-actin expression by activated stellate cells in liver biopsy; and radioimmunoassay for procollagen in serum. According to the clinical response to interferon, the patients were subdivided into sustained responders, delayed relapsers, early relapsers and non-responders. RESULTS: We found that alpha-actin scores, portal and lobular activity scores and procollagen levels were all significantly lower after the treatment in responder patients, whereas in non-responders the after-interferon values were not different from the basal values. Moreover we found that the patients who were still clinical responders at the time of after-therapy evaluation, but relapsed subsequently (delayed relap-sers), still showed a pattern of "low fibrogenesis", like the sustained responders. On the contrary, the patients who had already relapsed at the time of after-interferon evaluation (early relapsers) showed a pattern of "high fibrogenesis", like the non-responders. CONCLUSIONS: Since all the patients had received a similar treatment for a similar period, our results suggest that fibrogenesis activity in a defined time is related to the clinical response present in that time, and is influenced by short-term variations in necro-inflammatory activity induced by interferon, rather than by interferon itself.

Clinical and pathologic findings in hemochromatosis type 3 due to a novel mutation in transferrin receptor 2 gene.

BACKGROUND & AIMS: Although most patients with hereditary hemochromatosis are homozygous for a single mutation of the HFE gene on chromosome 6p, accumulating evidence indicates that the disease is genetically heterogeneous. Type 3 hemochromatosis, recently described in 4 families, is linked to mutations of the gene encoding transferrin receptor 2 on chromosome 7q22. Here we report data from a family carrying a new mutation of the transferrin receptor 2 gene. METHODS: Detailed clinical and histopathologic documentation was available for most family members. The entire coding sequence and exon/intron boundaries of the transferrin receptor 2 gene were analyzed by direct sequencing. RESULTS: A 12-nucleotide deletion in exon 16, causing the loss of 4 amino acids (AVAQ 594-597 del), was detected at the homozygous state in the 3 patients with histologically proven iron overload. The deletion segregated with the disease within the family and was not found in 100 healthy controls. Some clinical and pathologic characteristics, such as low penetrance in the premenopausal woman, and early iron deposition in periportal hepatocytes resembled those of classic, HFE-related hemochromatosis. CONCLUSIONS: Our data support the role of the transferrin receptor 2 gene in hemochromatosis type 3 as well as its critical involvement in the maintenance of iron homeostasis in humans.