[Challenges of cytopathological pancreas diagnostics]. / Herausforderungen der zytopathologischen Pankreasdiagnostik.

The cytologic diagnostics of solid and cystic pancreatic lesions with endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is an integral part of the clinical workup and the decision of a surgical versus a conservative approach. Cystic lesions are increasingly being diagnosed due to improved imaging and represent numerous neoplastic as well as non-neoplastic epithelial and non-epithelial entities, which differ in biological behavior and prognosis. In particular, the differentiation of mucinous and non-mucinous cysts is significant for further clinical management. Regressive cellular changes, gastrointestinal contaminants, and overlapping morphologic changes of reactively altered ductal epithelial cells and cells of well-differentiated neoplasms and preneoplasms are special challenges of cytological diagnostics. For a uniform cytological classification of findings, an internationally developed seven-level classification system has been published and co-published by the World Health Organization (WHO). This classification system takes into account both morphological findings and further procedures on cytological material such as next-generation sequencing and immunocytochemistry and is based on the WHO classification for pancreatic tumors. Against this background, important cytologic diagnostic criteria of various solid and cystic lesions relevant in clinical practice are presented in this article, considering diagnostic possibilities and pitfalls as well as differential diagnoses.

Vesicular Release and Uptake of Circular LSD1-RNAs from Non-Cancer and Cancer Lung Cells.

Lysine-specific demethylase 1 (LSD1) is highly expressed in many cancer types and strongly associated with cancer progression and metastasis. Circular RNAs (circRNAs) are produced by back-splicing and influence the interactive RNA network by microRNA and protein sponging. In the present study, we aimedto identify circRNAs that derive from the LSD1-encoding KDM1A gene, and to investigate their potential to be released and uptaken by lung cancer versus non-cancer epithelial cells. We identified four circLSD1-RNAs by RT-PCR with divergent primers, followed by sequencing. The expression level of circLSD1-RNAs was then studied by quantitative PCR on cellular and extracellular fractions of lung cancer (PC9) and non-cancer primary small airway epithelial (PSAE) cells. Moreover, we established the transgenic overexpression of circLSD1-RNAs. We show that circLSD1-RNAs are primarily located in the cytoplasm, but are packaged and released from lung cancer and non-cancer cells by extracellular vesicles (EVs) and ribonucleoprotein (RNP) complexes, respectively. Proteomics demonstrated a different protein pattern of EV fractions released from PC9 versus PSAE cells. Importantly, released circLSD1-RNAs were differently taken up by PSAE and PC9 cells. In conclusion, our findings provide primary evidence that circLSD1-RNAs participate in the intercellular communication of lung cancer cells with the tumor environment.

[A European comparison of continuing education in pathology]. / Weiterbildung Pathologie im europäischen Vergleich.

In the coming years, the shortage of pathologists will become internationally evident. In addition, the increase in knowledge, technical transformation processes, and the attractiveness of working conditions pose clear challenges for the field of pathology. A bi-directional opening for international mobility of pathologists could be a potential solution.In this analysis, the European training concept of the European Union of Medical Specialists (UEMS) was compared with its implementation in the 27 countries of the EU plus its 4 associated countries with regard to nationally differentiated concepts, type and implementation of the specialist examination, and additional qualifications. Subsequently, questions regarding the recognition of exams, titles, and specialist exams were elicited.The duration of training ranges between 4 and 6 years. The number of cases also varies considerably. Obtaining the specialist title can be done by simply completing the specifications up to a structured examination. In the EU, exams are mutually recognized, but this does not necessarily apply to academic titles and additional qualifications. Increasingly, on-site training centers are also subject to auditing procedures.The European agreements allow a high degree of permeability. However, national regulations pose hurdles for international mobility. The UEMS is therefore focusing on harmonization, including the certification of training centers. The so-called European Pathology Progress Test of the European Society of Pathology (ESP) is a further step towards the development of a future European specialist title. It remains the joint responsibility of residents and institutes to shape the future of the next generation of pathologists from the variety of different concepts.

Corrigendum to "Chaperone-Mediated Autophagy Markers LAMP2A and HSC70 Are Independent Adverse Prognostic Markers in Primary Resected Squamous Cell Carcinomas of the Lung".

[This corrects the article DOI: 10.1155/2020/8506572.].

A prognostic score for non-small cell lung cancer resected after neoadjuvant therapy in comparison with the tumor-node-metastases classification and major pathological response.

Studies validating the prognostic accuracy of the tumor-node-metastases (TNM) classification in patients with lung cancer treated by neoadjuvant therapy are scarce. Tumor regression, particularly major pathological response (MPR), is an acknowledged prognostic factor in this setting. We aimed to validate a novel combined prognostic score. This retrospective single-center study was conducted on 117 consecutive patients with non-small cell lung cancer resected after neoadjuvant treatment at a Swiss University Cancer Center between 2000 and 2016. All cases were clinicopathologically re-evaluated. We assessed the prognostic performance of a novel prognostic score (PRSC) combining T-category, lymph node status, and MPR, in comparison with the eighth edition of the TNM classification (TNM8), the size adapted TNM8 as proposed by the International Association for the Study of Lung Cancer (IASLC) and MPR alone. The isolated ypT-category and the combined TNM8 stages accurately differentiated overall survival (OS, stage p = 0.004) and disease-free survival (DFS, stage p = 0.018). Tumor regression had a prognostic impact. Optimal cut-offs for MPR emerged as 65% for adenocarcinoma and 10% for non-adenocarcinoma and were statistically significant for survival (OS p = 0.006, DFS p < 0.001). The PRSC differentiated between three prognostic groups (OS and DFS p < 0.001), and was superior compared to the stratification using MPR alone or the TNM8 systems, visualized by lower Akaike (AIC) and Bayesian information criterion (BIC) values. In the multivariate analyses, stage III tumors (HR 4.956, p = 0.003), tumors without MPR (HR 2.432, p = 0.015), and PRSC high-risk tumors (HR 5.692, p < 0.001) had significantly increased risks of occurring death. In conclusion, we support 65% as the optimal cut-off for MPR in adenocarcinomas. TNM8 and MPR were comparable regarding their prognostic significance. The novel prognostic score performed distinctly better regarding OS and DFS.

Chaperone-Mediated Autophagy Markers LAMP2A and HSC70 Are Independent Adverse Prognostic Markers in Primary Resected Squamous Cell Carcinomas of the Lung.

LAMP2A and HSC70 are crucial players in chaperone-mediated autophagy (CMA), a targeted, lysosome-dependent protein degradation pathway. Elevated LAMP2A levels, indicative of increased CMA activity, are observed in several malignancies, and CMA downregulation may be exploited therapeutically. We evaluated the impact of LAMP2A and HSC70 in pulmonary squamous cell carcinomas (pSQCC). Antibodies were validated by knockdown and overexpression experiments using three different cell lines. Expression levels in tissue were analyzed by immunohistochemistry in a cohort of 336 consecutive pSQCC using tissue microarrays. There was no significant correlation between the two markers among each other and no association with pathological parameters (TNM categories, grading). However, both high LAMP2A and HSC70 expression were associated with worse outcome, including overall survival (OS; p = 0.012 and p = 0.001) and disease free survival (DFS; p = 0.049 and p = 0.036). In multivariate analysis, both markers and a combination of them were independent adverse prognostic factors for OS (LAMP2Ahigh: HR = 2.059; p < 0.001; HSC70high: HR = 1.987; p < 0.001; LAMP2Ahigh/HSC70high: HR = 1.529; p < 0.001) and DFS (LAMP2Ahigh: HR = 1.709; p = 0.004; HSC70high: HR = 1.484; p = 0.027; LAMP2Ahigh/HSC70high: HR = 1.342, p < 0.001). The negative prognostic impact of high LAMP2A and HSC70 and their variable expression in pSQCC may justify the use of these proteins as potential biomarkers for future CMA-inhibiting therapies.

Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth.

Glucose utilization increases in tumors, a metabolic process that is observed clinically by 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET). However, is increased glucose uptake important for tumor cells, and which transporters are implicated in vivo? In a genetically-engineered mouse model of lung adenocarcinoma, we show that the deletion of only one highly expressed glucose transporter, Glut1 or Glut3, in cancer cells does not impair tumor growth, whereas their combined loss diminishes tumor development. 18F-FDG-PET analyses of tumors demonstrate that Glut1 and Glut3 loss decreases glucose uptake, which is mainly dependent on Glut1. Using 13C-glucose tracing with correlated nanoscale secondary ion mass spectrometry (NanoSIMS) and electron microscopy, we also report the presence of lamellar body-like organelles in tumor cells accumulating glucose-derived biomass, depending partially on Glut1. Our results demonstrate the requirement for two glucose transporters in lung adenocarcinoma, the dual blockade of which could reach therapeutic responses not achieved by individual targeting.

Validation of the International Tumor Budding Consensus Conference (ITBCC) 2016 recommendation in squamous cell carcinoma of the lung-a single-center analysis of 354 cases.

There are no universally accepted grading systems in pulmonary squamous cell carcinoma (pSQCC). Recently, tumor budding, cell nest size, and spread through airspaces (STAS) have been proposed as grading scheme candidates. Tumor budding is a well-established independent prognostic factor in colorectal cancer. The International Tumor Budding Consensus Conference (ITBCC) provided consensus on scoring in 2016, albeit for colorectal cancers. Here, we aimed to validate the ITBCC method in pSQCC and evaluate additional proposed grading parameters. We analyzed a fully clinico-pathologically annotated Western single-center cohort of 354 consecutive primary resected pSQCC (resected 2000-2013). Patients with SQCC of other organs were excluded to reliably exclude lung metastases. We assessed conventional grading, keratinization, STAS, and tumor budding according to ITBCC recommendations, and correlated them with clinico-pathological parameters and survival. Tumor budding was low (0-4 buds/0.785 mm2) in 41%, intermediate (5-9 buds/0.785 mm2) in 30%, and high (≥10 buds/0.785 mm2) in 29% of cases (mean bud count = 7.45 (H&E), min = 0, max = 84). Cell nests of 1, 2-4, 5-15, >15 cells were present in 68%, 20%, 5%, 7%, respectively. We detected STAS in 33% of cases, desmoplasia in 68%. Tumor budding assessed as continuous and categorized variables was highly concordant between hematoxylin and eosin (H&E) and pancytokeratin (AE1/AE3) stained slides (P < 0.001) and significantly associated with tumor size, UICC/AJCC pT, pN, stage (all P < 0.001) and presence of mediastinal lymph node metastases (H&E: P = 0.028). Tumor budding was a significant prognostic parameter for overall, disease-specific, and progression-free survival (PFS) (all P < 0.001). ITBCC tumor budding categories were independent prognostic factors for overall survival (HR = 1.581; 95% CI 1.186-2.108; P = 0.002), disease-specific survival (HR = 1.710; 95% CI 1.111-2.632; P = 0.015), and PFS (HR = 1.457; 95% CI 1.123-1.890; P = 0.005). STAS or conventional tumor grade had no prognostic value. In conclusion, we confirm tumor budding as an independent prognostic marker in pSQCC and validate the ITBCC 2016 scoring recommendations in pSQCC.

Comparison of the 7th and 8th Edition of the UICC/AJCC TNM Staging System in Primary Resected Squamous Cell Carcinomas of the Lung-A Single Center Analysis of 354 Cases.

Background: The AJCC/UICC TNM (tumor, node, metastasis) classification is a standardized system for the description of anatomical extent and stage grouping of solid malignant tumors and is regularly updated. We aimed at testing the new 2017 8th edition of the TNM classification (TNM8) compared to the former 2009 7th edition (TNM7), in pulmonary squamous cell carcinomas (pSQCC). Methods: We analyzed a clinico-pathologically well-annotated Western single-center cohort of 354 consecutive pSQCC, resected 2000-2013, without previous neoadjuvant therapy. Patients with a clinical history of SQCC of other organs were excluded to reliably exclude lung metastases. Patients in whom TNM was unclear due to multiple tumor nodules were excluded. We reevaluated all pathological records and slides and retrospectively validated pleural invasion for all cases. Raw data of our cohort are provided as Supplementary Material. Results: The stage distribution according to TNM7 was as follows: IA (2009): 59 (16.7%), IB: 75 (21.2%), IIA: 71 (20.1%), IIB: 53 (15.0%), IIIA: 79 (22.3%), IIIB: 7 (2.0%), IV: 10 (2.8%). Staging the cases according to TNM8, 7/354 (2.0%) cases were down-staged, 154 (43.5%) were upstaged; most pronounced between stages IIA(TNM7) and IIB(TNM8), and IIB(TNM7) and IIIA(TNM8). Both staging systems showed significant prognostic impact for overall survival, disease free and disease specific survival and time to recurrence, without significant differences regarding goodness-of-fit criteria (Akaike Information Criterion and Schwarz Bayesian Criterion). Conclusion: In conclusion, we show a significant stage migration between tumors staged using TNM7 and TNM8, without benefit regarding prognostication in our cohort of primary resected pSQCC.

[Role of p16(INK4A) in detection of human papillomavirus DNA and stratification of cancer-specific mortality on the basis of long term follow-up data of 34 patients with invasive penile cancer]. / Bedeutung von p16(INK4A) hinsichtlich des Nachweises von HPV-DNA und in der Stratifizierung der tumorspezifischen Mortalität bei 34 Patienten mit invasivem Peniskarzinom in Langzeit-Nachbeobachtung.

BACKGROUND: Human papillomavirus (HPV) infection plays an important role in the pathogenesis of penile squamous cell carcinoma (PSCC) in about one third of the affected patients. Initial data indicate that HPV status could facilitate optimised risk stratification and individualised targeted therapy. The polymerase chain reaction (PCR) assay is the reference method for detection of HPV DNA. It is unclear if alternatives such as in situ hybridisation (ISH) or various surrogate markers (defined as immunohistochemical detection of p16INK4a, histological subtype, tumour invasion front, koilocytosis) are sufficient to determine HPV status METHODS: In this single centre study on 34 patients with PSCC, multiplex nested PCR and ISH were conducted for HPV detection and identification of HPV DNA genotypes. Various histological criteria and p16INK4a were determined by central review. The influence of different criteria on cancer-specific mortality (CSM) was investigated with the Cox proportional hazards models (FU: 92 mo.). Furthermore, the discriminative qualities of various tumour invasion patterns (i. e., pT-classification 7th vs. 8th ed.) for CSM prediction were compared. RESULTS: Pursuant to PCR assay, HPV DNA was detected in 26 % of patients (n = 9). ISH and the examined histological criteria were of inadequate quality in the prediction of HPV status (p > ;0.3). Test parameters of p16INK4a were calculated as follows: sensitivity 66.7 %, specificity 84 %, positive predictive value 60 %, negative predictive value 87.5 %, overall agreement 79.4 % (Area-Under-Curve: 0.753, p = 0.026). None of the examined HPV criteria significantly influenced CSM. In comparison to the 7th pT-edition, the 8th version was superior in CSM prediction (c-indices 70.2 % vs. 72.9 %). In addition to penile corpora invasion, infiltration of the urethra had no independent predictive value. Regrouping of the corpora invasion patterns, as proposed by us, resulted in an increase in discriminative quality (c-index 77.9 %). CONCLUSIONS: In contrast to ISH and the examined histological criteria, p16INK4a allows a reasonable prediction of HPV status. Neither HPV DNA nor its surrogate markers independently impacted CSM. As urethral tumour invasion does not independently influence CSM, the recent pT classification can be considered useful for prognosis.

Expression Analysis of Autophagy Related Markers LC3B, p62 and HMGB1 Indicate an Autophagy-Independent Negative Prognostic Impact of High p62 Expression in Pulmonary Squamous Cell Carcinomas.

Autophagy is involved in maintaining cellular homeostasis under stress conditions. It also plays an important role in various diseases including cancer. Pulmonary squamous cell carcinomas (pSQCC) at present lack targetable molecular alterations, and demand alternative therapeutic options. We assessed the expression levels of autophagy related proteins LC3B, p62, and HMGB1 in 271 primary resected pSQCC by immunohistochemistry, in correlation with clinical and pathological parameters, as a rationale for a potential autophagy directed therapy. LC3B, p62, and HMGB1 staining showed various patterns. LC3Bhighp62low levels, suggested to indicate intact activated autophagy, were associated with prolonged disease specific survival (DSS) and LC3Bhighp62high levels, indicating activated but late stage impaired autophagy, with shorter DSS (p = 0.024). p62high expression regardless of LC3B, however, showed an even stronger association with shorter DSS (p = 0.015) and was also an independent negative prognostic factor in multivariate analysis (HR = 2.99; 95% CI 1.38⁻6.52; p = 0.006). HMGB1 expression correlated neither with the expression of LC3B and p62, nor with patients' outcome. Different states of autophagy characterized by distinct p62 and LC3B expression patterns may be linked to patient's prognosis in pSQCC. Our results, however, point also to an autophagy independent role of p62 with an even more pronounced prognostic impact compared to autophagy related p62.

Adverse prognostic value of PD-L1 expression in primary resected pulmonary squamous cell carcinomas and paired mediastinal lymph node metastases.

Immunohistochemical assessment of programmed cell death (PD)-ligand 1 (PD-L1) expression in lung cancer in the context of therapeutically targeting the PD1/PD-L1 axis is still controversially discussed. This includes the comparability of antibody clones, prognostic value, and discrepancies between primary tumors and metastases. We assessed tumoral PD-L1 expression using clones E1L3N and SP142 in 372 primary resected pulmonary squamous cell carcinomas, including 40 paired N2 lymph node metastases, in relation with clinico-pathological parameters. PD-L1 expression was negative (<1%) in 163/372 (44%, E1L3N) or 231/370 patients (62%, SP142). Positivity of 1-<50% was observed in 135 (36%, E1L3N) or 92 patients (25%, SP142) and ≥50% in 74 (20%, E1L3N) or 47 patients (13%, SP142). PD-L1 staining correlated significantly between both antibodies (r=0.781; P<0.001). Scores correlated significantly between full-slide sections (N=40) and tissue microarrays, and between primaries and N2 metastases (P<0.001 all). CD8+ tumor infiltrating lymphocyte counts positively correlated with PD-L1 expression (P<0.001). PD-L1 ≥50% showed the best prognostic discrimination using the split-sample validation method. It was associated with shorter disease-specific survival in the observation group (E1L3N: P=0.035, SP142: P=0.002) and validation group (E1L3N: P=0.024, SP142: P=0.101) and shorter time to recurrence (observation group: E1L3N: P=0.056, SP142: P<0.001; validation group: E1L3N: P=0.036, SP142: P=0.247). Multivariate analysis showed that PD-L1 expression ≥50% determined by clone E1L3N was an independent prognostic factor in the observation group regarding disease-specific survival (HR=2.768; 95% CI=1.149-6.666; P=0.023) and time to recurrence (HR=2.164; 95% CI=1.056-4.436; P=0.035) and in the validation group (disease-specific survival: HR=1.978; 95% CI=0.928-4.214; P=0.077 and time to recurrence: HR=1.571; 95% CI=0.838-2.944; P=0.159). High PD-L1 expression was associated with adverse prognosis in pulmonary squamous cell carcinoma. Clone E1L3N was more sensitive than SP142 and superior regarding prognostication. PD-L1 expression correlated significantly between primary tumor and N2 metastases, rendering mediastinal lymph node metastases adequate for immunohistochemical assessment.

Yip1 domain family, member 6 (Yipf6) mutation induces spontaneous intestinal inflammation in mice.

Using an environmentally sensitized genetic screen we identified mutations that cause inflammatory colitis in mice. The X-linked Klein-Zschocher (KLZ) mutation created a null allele of Yipf6, a member of a gene family believed to regulate vesicular transport in yeast, but without known functions in mammals. Yipf6 is a five transmembrane-spanning protein associated with Golgi compartments. Klein-Zschocher mutants were extremely sensitive to colitis induced by dextran sodium sulfate (DSS) and developed spontaneous ileitis and colitis after 16 mo of age in specific pathogen-free housing conditions. Electron microscopy, gene expression, and immunocytochemistry analyses provided evidence that impaired intestinal homeostasis stemmed from defective formation and secretion of large secretory granules from Paneth and goblet cells. These studies support a tissue- and organ-specific function for Yipf6 in the maintenance of intestinal homeostasis and implicate the orthologous human gene as a disease susceptibility locus.

MyD88 signaling in nonhematopoietic cells protects mice against induced colitis by regulating specific EGF receptor ligands.

Toll-like receptors (TLRs) trigger intestinal inflammation when the epithelial barrier is breached by physical trauma or pathogenic microbes. Although it has been shown that TLR-mediated signals are ultimately protective in models of acute intestinal inflammation [such as dextran sulfate sodium (DSS)-induced colitis], it is less clear which cells mediate protection. Here we demonstrate that TLR signaling in the nonhematopoietic compartment confers protection in acute DSS-induced colitis. Epithelial cells of MyD88/Trif-deficient mice express diminished levels of the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG), and systemic lipopolysaccharide administration induces their expression in the colon. N-ethyl-N-nitrosourea (ENU)-induced mutations in Adam17 (which is required for AREG and EREG processing) and in Egfr both produce a strong DSS colitis phenotype, and the Adam17 mutation exerts its deleterious effect in the nonhematopoietic compartment. The effect of abrogation of TLR signaling is mitigated by systemic administration of AREG. A TLR→MyD88→AREG/EREG→EGFR signaling pathway is represented in nonhematopoietic cells of the intestinal tract, responds to microbial stimuli once barriers are breached, and mediates protection against DSS-induced colitis.