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Background: Let-7 is a tumor suppressor microRNA targeting the KRAS lung oncogene. Let-7a downregulation is reversible during the early stages of lung carcinogenesis but is irreversible in cancer cells. The aim of this study is to shed light on the relationship between oncogene (KRAS) mutation and let-7a downregulation in cigarette smoke (CS)-induced lung carcinogenesis. Methods: A total of 184 strain H Swiss albino mice were either unexposed (control) or exposed to CS for 2 weeks (short CS) or 8 months (long CS). After 8 months, the lungs were individually collected. The following end points have been evaluated: (a) DNA methylation of the let-7a gene promoter by bisulphite-PCR and pyrosequencing; (b) let-7a expression by qPCR; (c) KRAS mutation by DNA pyrosequencing; (d) cancer incidence by histopathological examination. Results: let-7a expression decreased by 8.3% in the mice exposed to CS for two weeks (CS short) and by 33.4% (p ≤ 0.01) in the mice exposed to CS for 8 months (CS long). No significant difference was detected in the rate of let-7a-promoter methylation between the Sham-exposed mice (55.1%) and the CS short-(53%) or CS long (51%)-exposed mice. The percentage of G/T transversions in KRAS codons 12 and 13 increased from 2.3% (Sham) to 6.4% in CS short– and to 11.5% in CS long–exposed mice. Cancer incidence increased significantly in the CS long–exposed mice (11%) as compared to both the Sham (4%) and the CS short–exposed (2%) mice. In the CS long–exposed mice, the correlation between let-7a expression and the number of KRAS mutations was positive (R = +0.5506) in the cancer-free mice and negative (R = −0.5568) in the cancer-bearing mice. Conclusions: The effects of CS-induced mutations in KRAS are neutralized by the high expression of let-7a in cancer-free mice (positive correlation) but not in cancer-bearing mice where an irreversible let-7a downregulation occurs (negative correlation). This result provides evidence that both genetic (high load of KRAS mutation) and epigenetic alterations (let-7a irreversible downregulation) are required to produce lung cancer in CS-exposed organisms. [ABSTRACT FROM AUTHOR]

Simple Summary: The molecular heterogeneity of esophageal adenocarcinoma (EAC), a severe malignancy with increasing incidence and low survival rates, misperceives the underlying biology of tumor onset and development. However, advances in high-throughput next-generation sequencing (NGS) technologies have highlighted the potential role of somatic DNA sequence markers for new diagnostic techniques or constitute novel therapeutic targets. Thus, in order to identify a molecular and prognostic signature in EAC patients, we decided to integrate the sequencing of specimens from naïve patients (not treated with chemo-radiotherapy) with histological classification, with the aim of identification of potential biomarkers, and patient stratification. Combining different approaches paves the way for early identification and the selection of better therapy. Esophageal adenocarcinoma (EAC) is a severe malignancy with increasing incidence, poorly understood pathogenesis, and low survival rates. We sequenced 164 EAC samples of naïve patients (without chemo-radiotherapy) with high coverage using next-generation sequencing technologies. A total of 337 variants were identified across the whole cohort, with TP53 as the most frequently altered gene (67.27%). Missense mutations in TP53 correlated with worse cancer-specific survival (log-rank p = 0.001). In seven cases, we found disruptive mutations in HNF1alpha associated with other gene alterations. Moreover, we detected gene fusions through massive parallel sequencing of RNA, indicating that it is not a rare event in EAC. In conclusion, we report that a specific type of TP53 mutation (missense changes) negatively affected cancer-specific survival in EAC. HNF1alpha was identified as a new EAC-mutated gene. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors, including ipilimumab (IPI), achieve a clinical benefit in a small proportion of melanoma patients highlighting the need to investigate predictive biomarkers. In this study, we characterized tumor infiltrating lymphocytes (TILs), focusing on the CTLA-4+ subset, and evaluated their possible predictive significance. We characterized TIL density, cell type, and localization in 40 melanoma lesions from 17 patients treated with IPI. Associations of TILs with IPI timing, tissue localization, and response to IPI were estimated using a linear mixed-effects modelling approach. We found that most of TIL subsets increased in situ upon IPI therapy, with particular reference to FoxP3+ cells. TILs and TIL subsets, such as CD3+, CD45RO+, CTLA-4+, CD4+, CD8+ T cells, CD20+ B cells, and NKp46+ NK cells, showed significantly different spatial distributions in the tumor microenvironment being higher at the invasive margin (IM) as compared to the tumor center (TC) (P value < 0.001 for TIL score and P value < 0.05 for all subsets). Remarkably, high TIL score and density of CD3+, CD8+ T cells, and CTLA-4+ immune cells were significantly associated with a better response to IPI (P values = 0.002, 0.023, 0.007, and 0.001, respectively, for responders vs non-responders). In conclusion, we provide a detailed analysis of CTLA-4+ TIL distribution in melanoma tissues taking into account localization, relationship with CD3+/CD8+ TILs, and changes in response to IPI treatment. We identified that CTLA-4+ TILs may represent a marker of IPI response, alone or with CD3+/CD8+ subsets, although this requires confirmation in larger studies. [ABSTRACT FROM AUTHOR]

Formalin is toxic and has recently been classified as carcinogenic leading to a proposed European formalin ban. But, the pathology use of formalin has however been completely overlooked, and this is proving to be a relevant issue, as no alternative, reliable, tissue fixative is available. Various systems have been proposed to reduce formalin use and exposure; long-term storage and disposal of formalin is also a problem. With this in mind, under vacuum sealing (UVS) systems have been proposed for transportation/storage, however, for how long tissue retains its characteristics (morphological and molecular) is unknown. This study aims to compare histology specimens stored by formalin immersion (FI) and specimens stored after fixation with UVS technique with no additional formalin, at different time periods. Twenty tissue samples (10FI; 10UVS) were stored for different time periods (15 days, 1–2–3–6–12 months) for a total of 120 samples, compared with regard to their morphology, histochemistry, immunoreactivity (24 specific antibodies) and DNA status. All samples showed well-preserved morphology and overlapping staining quality. A significant reduction in immunoreactivity was however identified in the various time periods, particularly for heat pre-treated nuclear antigens, and this commenced earlier (1 month) for FI. UVS storage showed higher DNA content than FI but slightly poorer DNA integrity. These results add important knowledge to the use of UVS in daily practice, as long-term storage of pre-fixed tissue in UVS is not detrimental to the quality of tissue while having the boon of using very little formalin with less operator exposure and lower disposal costs. [ABSTRACT FROM AUTHOR]

Preoperative water-enema computed tomography, requested for staging purposes, confirmed a sub-stenosing lesion with small foci of spontaneously hyperattenuating calcifications (Figure 1) and signs of involvement of the mesorectal fat; no distant metastatic disease was identified. Radiologic imaging: (a) Water-enema contrast-enhanced computed tomography (CT), coronal view, detail showing a large stenosing rectal mass with coarse calcifications (white arrows). (c) Pre-neoadjuvant treatment magnetic resonance imaging (MRI) scan, TSE T2w coronal view for locoregional staging; white arrow heads show the sub-stenosing rectal mass. [Extracted from the article]

The aim of this study was to test alginate beads and silk fibroin non-woven mats as stromal vascular fraction delivery systems to support cell implantation for tissue repair and regeneration, through trophic and immunomodulant paracrine signaling. Furthermore, in vivo scaffold biocompatibility was histologically analyzed in a murine model at different time endpoints, with particular focus on construct-induced vascularization and neoangiogenesis. The fibroin mat induced a typical foreign body reaction, recruiting macrophages and giant cells and concurrently promoted neovascularization of the implanted construct. Conversely, alginate beads triggered a more circumscribed, chronic inflammatory reaction, which decreased over time. The combined in vivo implantation of alginate beads and fibroin mat with stromal vascular fraction promoted vascularization and integration of scaffolds into the surrounding subcutaneous environment. The new blood vessel ingrowth should, hopefully, support engineered cell viability and functionality, as well as the transport of soluble bioactive molecules. Due to their neovascularization properties, stromal vascular fraction administration, using alginate or fibroin scaffolds, is a new, promising, cost-effective tissue engineering approach. [ABSTRACT FROM AUTHOR]

Colorectal adenosquamous carcinoma (ASC) is exceedingly rare, comprising less than 0.1% of all colorectal malignancies, and is characterized by the admixture of glandular and squamous components. Due to its rarity, immunohistochemical and biological profiles have not been well investigated. The clinico-pathologic features of 29 cases of primary colorectal adenosquamous carcinomas, including four synchronous metastases, as well as the immunohistochemical expression of keratin 20, CDX2, keratin 34βE12, keratin 5/6, p63, p40, β-Catenin, Cyclin D1 and mismatch repair protein (MMR) expression in both squamous and glandular components are described. All ASCs showed aggressive clinico-pathologic features; all cases showed at least one aggressive pathologic characteristic (poorly differentiated, vascular invasion, infiltrative growth pattern) and 69% of cases were either stage III or IV. The squamous component was keratin 34βE12 positive in all cases and keratin 5/6 positive in 27 cases, while only 7 cases showed p63 and/or p40 expression. β-Catenin and Cyclin D1 showed different expression, with nuclear staining of Cyclin D1 in the squamous component of all cases (both primary and metastatic lesions) and nuclear staining of β-Catenin predominantly in the glandular component. All but one case showed proficient MMR profile. Sixteen patients (64%) died of their disease with median survival of 10 months. ASC show aggressive clinical outcome and aggressive pathologic characteristics. A peculiar keratin 34βE12 positive profile in the squamous component is seen differing from squamous cell carcinoma and non-intestinal ASC. The staining patterns for β-Catenin and Cyclin D1 between components, supports a possible divergent clonal evolution of the neoplasm. [ABSTRACT FROM AUTHOR]

A rhabdoid colorectal tumor (RCT) is a rare cancer with aggressive clinical behavior. Recently, it has been recognized as a distinct disease entity, characterized by genetic alterations in the SMARCB1 and Ciliary Rootlet Coiled-Coil (CROCC). We here investigate the genetic and immunophenotypic profiling of 21 RCTs using immunohistochemistry and next-generation sequencing. Mismatch repair-deficient phenotypes were identified in 60% of RCTs. Similarly, a large proportion of cancers exhibited the combined marker phenotype (CK7-/CK20-/CDX2-) not common to classical adenocarcinoma variants. More than 70% of cases displayed aberrant activation of the mitogen-activated protein kinase (MAPK) pathway with mutations prevalently in BRAF V600E. SMARCB1/INI1 expression was normal in a large majority of lesions. In contrast, ciliogenic markers including CROCC and γ-tubulin were globally altered in tumors. Notably, CROCC and γ-tubulin were observed to colocalize in large cilia found on cancer tissues but not in normal controls. Taken together, our findings indicate that primary ciliogenesis and MAPK pathway activation contribute to the aggressiveness of RCTs and, therefore, may constitute a novel therapeutic target. [ABSTRACT FROM AUTHOR]

Fassan M, Mastracci L, Grillo F, Zagonel V, Bruno S, Battaglia G, Pitto F, Nitti D, Celiento T, Zaninotto G, Fiocca R & Rugge M (2012) Histopathology 61, 769-776 Early HER2 dysregulation in gastric and oesophageal carcinogenesis Aims: To explore human epidermal growth factor receptor 2 (HER2) status in the histological phenotypes [metaplasia, intraepithelial neoplasia (IEN, i.e. dysplasia), and adenocarcinoma] involved in the morphogenesis of both intestinal-type gastric cancer (GC) and Barrett's adenocarcinoma (BAc). Methods and results: A consecutive series of 275 samples of stomach and oesophagus tissue (representing the whole spectrum of the phenotypic changes involved in gastric and Barrett's carcinogenesis) was studied. HER2 status was assessed by applying two immunohistochemistry (IHC) protocols, using the antibodies 4B5 and CB11. Dual-colour silver chromogenic in-situ hybridization (SISH) was also performed on the same tissue samples. In both oesophageal and gastric samples, the rate of HER2 overexpression rose significantly from low-grade to high-grade IEN to adenocarcinoma ( P < 0.001), with the two IHC protocols showing consistent staining (consistency 95%; k = 0.78; P < 0.001). Intratumour heterogeneity was documented in both GC and BAc (using both IHC protocols). The rate of HER2 amplification (using SISH) increased significantly along with IEN dedifferentiation ( P < 0.001). Neither native nor metaplastic mucosa samples (obtained from either stomach or oesophagus) ever showed HER2 amplification. There was excellent agreement between HER2 amplification and protein overexpression (both IHC protocols: SISH/4B5--consistency 97.8%, k = 0.89, P < 0.001; SISH/CB11-consistency 97.8%, k = 0.91, P < 0.001). Conclusions: There is early involvement of HER2 dysregulation (amplification and protein overexpression) in both gastric (intestinal-type) and Barrett's oncogenesis. [ABSTRACT FROM AUTHOR]

OBJECTIVES:Gastroesophageal reflux disease (GERD)-associated changes in esophageal histology have been reported mainly after short-term medical antireflux therapy, and few individual lesions have been examined. We report detailed histological findings from the LOTUS study, at baseline and at 1 and 3 years after laparoscopic antireflux surgery (LARS) or esomeprazole treatment in patients with chronic GERD.METHODS:LOTUS is a long-term, open, parallel-group, multicenter, randomized, controlled trial conducted in 11 European countries that compared LARS (n=248) with esomeprazole 20–40 mg daily (n=266). Biopsies from the distal esophagus 2 cm above the Z-line and at the Z-line were taken at baseline, and 1 and 3 years. The following lesions were assessed: basal cell hyperplasia (BCH), papillary elongation (PE), intercellular space dilatations (ISDs), intraepithelial eosinophils (EOSs), neutrophils, and necrosis/erosion. A severity score (SS, range 0–2) was calculated by taking the average score of all assessable lesions.RESULTS:All lesions were more severe on Z-line biopsies than at 2 cm, and almost all improved significantly from baseline to 1 and 3 years. The average SS (from 2 cm to Z-line) changed from 0.95 to 0.57 (1 year) and to 0.49 (3 years) on esomeprazole, and from 0.91 to 0.56 (1 year) and to 0.52 (3 years) after LARS (P<0.001 for both treatments at 1 and 3 years, with no significant difference between treatments). The proportions of patients with severe histological changes decreased from approximately 50% at baseline to 11% at 3 years.CONCLUSIONS:Both continuous esomeprazole treatment and laparoscopic fundoplication are associated with significant and similar overall improvement in microscopic esophagitis after 1 year that is maintained at 3 years. [ABSTRACT FROM AUTHOR]

Simple Summary: Esophageal and esophago-gastric junction adenocarcinomas (EA/EGJAs) are a heterogeneous group of cancers. Stage is the most important prognostic factor, while morphology, determined by histologic analysis, has up until now played a minor role. Even new molecular classifications (which should be based on accurate histologic assessment) are a long way off from being used in day to day practice. The reassessment of nearly 300 EA/EGJAs enabled us to re-evaluate morphology and identify a two-tiered grading approach in glandular adenocarcinomas (80%) based on a cut off of 6% of poorly differentiated components (well differentiated versus poorly differentiated). Furthermore, rare, but prognostically significant, variants were recognized with an in-depth morphologic description. On this basis, two morphologic risk groups (lower risk and higher risk) were identified, adding significant prognostic value to the stage. The accurate morphologic description of EA/EGJAs must be a prerequisite for a better understanding of prognosis, molecular events, and response to treatment. Stage significantly affects survival of esophageal and esophago-gastric junction adenocarcinomas (EA/EGJAs), however, limited evidence for the prognostic role of histologic subtypes is available. The aim of the study was to describe a morphologic approach to EA/EGJAs and assess its discriminating prognostic power. Histologic slides from 299 neoadjuvant treatment-naïve EA/EGJAs, resected in five European Centers, were retrospectively reviewed. Morphologic features were re-assessed and correlated with survival. In glandular adenocarcinomas (240/299 cases—80%), WHO grade and tumors with a poorly differentiated component ≥6% were the most discriminant factors for survival (both p < 0.0001), distinguishing glandular well-differentiated from poorly differentiated adenocarcinomas. Two prognostically different histologic groups were identified: the lower risk group, comprising glandular well-differentiated (34.4%) and rare variants, such as mucinous muconodular carcinoma (2.7%) and diffuse desmoplastic carcinoma (1.7%), versus the higher risk group, comprising the glandular poorly differentiated subtype (45.8%), including invasive mucinous carcinoma (5.7%), diffuse anaplastic carcinoma (3%), mixed carcinoma (6.7%) (CSS p < 0.0001, DFS p = 0.001). Stage (p < 0.0001), histologic groups (p = 0.001), age >72 years (p = 0.008), and vascular invasion (p = 0.015) were prognostically significant in the multivariate analysis. The combined evaluation of stage/histologic group identified 5-year cancer-specific survival ranging from 87.6% (stage II, lower risk) to 14% (stage IVA, higher risk). Detailed characterization of histologic subtypes contributes to EA/EGJA prognostic prediction. [ABSTRACT FROM AUTHOR]

The establishment of an interdisciplinary Inflammatory Bowel Disease (IBD) center is associated with improved healthcare utilization, according to a study published in the European Journal of Clinical Investigation. The study found that after the institution of the IBD center, hospitalizations per patient per year decreased, the number of patients on biologics increased, and there was an improvement in the total number of hospitalizations and length of hospital stay. The findings suggest that the coordinated collaboration of healthcare professionals and the implementation of specialized services can lead to better patient care and outcomes in IBD management. [Extracted from the article]

Aims: The Lynch syndrome (LS) screening algorithm requires BRAF testing as a fundamental step to distinguish sporadic from LS‐associated colorectal carcinomas (CRC). BRAF testing by immunohistochemistry (IHC) has shown variable results in the literature. Our aim was to analyse concordance between BRAFV600EIHC and BRAF molecular analysis in a large, mono‐institutional CRC whole‐slide, case series with laboratory validation. Methods and results: MisMatch repair (MMR) protein (hMLH1, hPMS2, hMSH2, and hMSH6) and BRAFV600EIHC were performed on all unselected cases of surgically resected CRCs (2018–2023). An in‐house validation study for BRAFV600EIHC was performed in order to obtain optimal IHC stains. BRAFVV600EIHC was considered negative (score 0), positive (scores 2–3), and equivocal (score 1). Interobserver differences in BRAFV600EIHC scoring were noted in the first 150 cases prospectively collected. Nine‐hundred and ninety CRCs cases (830 proficient (p)MMR/160 deficient (d)MMR) were included and all cases performed BRAFV600EIHC (BRAFV600EIHC‐positive 13.5% of all series; 66.3% dMMR cases; 3.4% pMMR cases), while 333 also went to BRAF mutation analysis. Optimal agreement in IHC scoring between pathologists (P < 0.0001) was seen; concordance between BRAFV600EIHC and BRAF molecular analysis was extremely high (sensitivity 99.1%, specificity 99.5%; PPV 99.1%, and NPV 99.5%). Discordant cases were reevaluated; 1 score 3 + IHC/wildtype case was an interpretation error and one score 0 IHC/mutated case was related to heterogenous BRAFV600EIHC expression. Among the 12 IHC‐equivocal score 1+ cases (which require BRAF molecular analysis), three were BRAF‐mutated and nine BRAF‐wildtype. Conclusion: BRAFV600EIHC can be used as a reliable surrogate of molecular testing after stringent in‐house validation. [ABSTRACT FROM AUTHOR]

Aims: Patients with Crohn's disease (CrD) have an elevated risk for the development of small bowel adenocarcinomas (SBAs). Actionable isocitrate dehydrogenase 1 (IDH1) mutations have been reported to be more frequent in CrD‐SBAs than in sporadic SBAs. The present study aimed to investigate the clinicopathological and immunophenotypical features, as well as methylation profiles, of IDH1‐mutated CrD‐SBAs. Methods and results: An international multicentre series of surgically resected CrD‐SBAs was tested for IDH1 mutation. Clinicopathological features, immunophenotypical marker expression and O6‐methylguanine‐DNA methyltransferase (MGMT) and long interspersed nuclear element‐1 (LINE‐1) methylation were compared between IDH1‐mutated and IDH1 wild‐type CrD‐SBAs. Ten (20%) of the 49 CrD‐SBAs examined harboured an IDH1 mutation and all the mutated cancers harboured the R132C variant. Compared to IDH1 wild‐type cases, IDH1‐mutated CrD‐SBAs showed significantly lower rates of cytokeratin 7 expression (P = 0.005) and higher rates of p53 overexpression (P = 0.012) and MGMT methylation (P = 0.012). All three dysplastic growths associated with IDH1‐mutated SBAs harboured the same IDH1 variant (R132C) of the corresponding invasive cancer, and all were of non‐conventional subtype (two serrated dysplastic lesions and one goblet cell‐deficient dysplasia). In particular, non‐conventional serrated dysplasia was significantly associated with IDH1‐mutated CrD‐SBAs (P = 0.029). No significant cancer‐specific survival difference between IDH1‐mutated CrD‐SBA patients and IDH1 wild‐type CrD‐SBA patients was found (hazard ratio = 0.55, 95% confidence interval = 0.16–1.89; P = 0.313). Conclusions: IDH1‐mutated CrD‐SBAs, which represent approximately one‐fifth of total cases, are characterised by distinctive immunophenotypical features and methylation profiles, with potential therapeutic implications. Moreover, IDH1‐mutated non‐conventional, serrated dysplasia is likely to represent a precursor lesion to such CrD‐SBAs. [ABSTRACT FROM AUTHOR]

Aims: Very early‐onset inflammatory bowel disease (VEO‐IBD) is a clinical umbrella term referring to IBD‐like symptoms arising in children before the age of 6 years, encompassing both 'pure' IBD, such as ulcerative colitis (UC) and Crohn's disease (CD) and monogenic diseases (MDs), the latter often involving genes associated with primary immunodeficiencies. Moreover, histological features in gastrointestinal (GI) biopsies in MD can also have IBD‐like morphology, making differential diagnosis difficult. Correct diagnosis is fundamental, as MDs show a more severe clinical course and their inadequate/untimely recognition leads to inappropriate therapy. Methods and results: Biopsy samples from the lower and upper GI tract of 93 clinically diagnosed VEO‐IBD children were retrospectively selected in a multicentre cohort and histologically re‐evaluated by 10 pathologists blinded to clinical information. Each case was classified according to morphological patterns, including UC‐like; CD‐like; enterocolitis‐like; apoptotic; eosinophil‐rich; and IBD‐unclassified (IBD‐U). Nine (69%) MD children showed IBD‐like morphology; only the IBD‐U pattern correlated with MD diagnosis (P = 0.02) (available in 64 cases: 51 non‐MD, true early‐onset IBD/other; 13 MD cases). MD patients showed earlier GI symptom onset (18.7 versus 26.9 months) and were sent to endoscopy earlier (22 versus 37 months), these differences were statistically significant (P < 0.05). Upper GI histology was informative in 37 biopsies. Conclusions: The diagnosis of the underlying cause of VEO‐IBD requires a multidisciplinary setting, and pathology, while being one of the fundamental puzzle pieces, is often difficult to interpret. A pattern‐based histological approach is therefore suggested, thus aiding the pathologist in VEO‐IBD reporting and multidisciplinary discussion. [ABSTRACT FROM AUTHOR]

Simple Summary: Low survival rates and a growing incidence characterize esophageal adenocarcinoma (EAC). MicroRNAs (miRNAs) have been linked to the development and progression of cancer, according to earlier research. Our study showed a significant correlation between poor cancer–related survival, tumor recurrence, and advanced disease stages with the overexpression of miR–221 and miR–483–3p. In particular, we have found that in low–risk EAC patients, miR–221 overexpression was linked to a lower survival rate. Therefore, these findings may help define patient stratification and determine targeted treatment for EAC. Alterations in microRNA (miRNA) expression have been reported in different cancers. We assessed the expression of 754 oncology–related miRNAs in esophageal adenocarcinoma (EAC) samples and evaluated their correlations with clinical parameters. We found that miR–221 and 483–3p were consistently upregulated in EAC patients vs. controls (Wilcoxon signed–rank test: miR–221 p < 0.0001; miR–483–3p p < 0.0001). Kaplan–Meier analysis showed worse cancer–related survival among all EAC patients expressing high miR–221 or miR–483–3p levels (log–rank p = 0.0025 and p = 0.0235, respectively). Higher miR–221 or miR–483–3p levels also correlated with advanced tumor stages (Mann–Whitney p = 0.0195 and p = 0.0085, respectively), and overexpression of miR–221 was associated with worse survival in low–risk EAC patients. Moreover, a significantly worse outcome was associated with the combined overexpression of miR–221 and miR–483–3p (log–rank p = 0.0410). To identify target genes affected by miRNA overexpression, we transfected the corresponding mimic RNA (miRVANA) for either miR–221 or miR–483–3p in a well–characterized esophageal adenocarcinoma cell line (OE19) and performed RNA–seq analysis. In the miRNA–overexpressing cells, we discovered a convergent dysregulation of genes linked to apoptosis, ATP synthesis, angiogenesis, and cancer progression, including a long non–coding RNA associated with oncogenesis, i.e., MALAT1. In conclusion, dysregulated miRNA expression, especially overexpression of miR–221 and 483–3p, was found in EAC samples. These alterations were connected with a lower cancer–specific patient survival, suggesting that these miRNAs could be useful for patient stratification and prognosis. [ABSTRACT FROM AUTHOR]

• SSc leads to a fibrotic process and EGFR expression is increased in damaged tissues. • EGFR is overexpressed in the esophagus of SSc patients without microscopic esophagitis. • Microscopic esophagitis is associated with increased EGFR expression in all subjects. Systemic sclerosis (SSc) affects the connective tissue and leads to an abnormal fibrotic process in the skin and internal organs. Epidermal Growth Factor Receptor (EGFR) is able to induce cell proliferation and differentiation, and its expression is increased in SSc patients with pulmonary artery hypertension and in skin biopsies in patients with scleroderma. To date, no data on esophageal expression of EGFR are available in SSc patients. We aimed to evaluate whether the pro-fibrogenic pathways of SSc may affect EGFR expression in the esophagus. A retrospective analysis included patients with SSc and control subjects suffering from gastroesophageal reflux symptoms. Endoscopic assessment and histopathologic analyses were performed in all subjects and the presence of microscopic esophagitis was used to distinguish patients with normal esophageal mucosa and subjects with non-erosive reflux disease. EGFR expression was measured in all subjects. A total of 35 patients with SSc were included, while the control group included 67 non-SSc patients. EGFR expression at the Z-line was higher in SSc patients than non-SSc patients in absence of microscopic esophagitis (median 65 %, IQR 56–71 % vs 42 %, IQR 37–54 %, p < 0.001). Microscopic esophagitis was found in 60 % of patients with SSc and 62.7 % of control patients, and EGFR expression was significantly higher in patients presenting microscopic esophagitis both in SSc and non-SSc patients. The EGFR hyperexpression may be due to SSc and/or reflux-related damage in patients with microscopic esophagitis. Further studies are warranted to answer open questions and provide a possible role of EGFR in terms of diagnosis, prognosis, and therapy. [ABSTRACT FROM AUTHOR]

Purpose: Programmed death-ligand 1 (PD-L1) protein plays a central role in the antitumor immune response, and appears to be a predictor of prognosis and efficacy for PD-L1 and programmed death 1 (PD-1) blockade therapy. The immunoregulatory role and prognostic impact of PD-L1 soluble form (sPD-L1) have been investigated in biological fluids of patients with different tumors. In malignant pleural mesothelioma (MPM), circulating sPD-L1 has been recently reported in patients' sera, but no data are available in pleural effusions (PE). In our study, we evaluated the baseline expression levels of sPD-L1 in PE from 84 MPM patients and correlated them with PD-L1-status in matched tumors and patients' overall survival (OS). Methods: sPD-L1 in PE was determined by ELISA and tumor PD-L1 by immunohistochemistry. Association of sPD-L1 with OS was estimated using the Cox regression model. Results: We observed that sPD-L1 was variably expressed in all the PE and tended to be higher (by 30%) in patients with PD-L1-positive tumors (cut-off ≥ 1% stained cells) as compared to patients with PD-L1-negative tumors (geometric mean ratio = 1.28, P value = 0.288). sPD-L1 levels were significantly higher than those of sPD-1 (P value = 0.001) regardless of the MPM histotypes and they were positively correlated (r = 0.50, P value < 0.001). Moreover, high PE sPD-L1 concentrations were associated with a trend towards increased OS (hazard ratio 0.79, 95% CL 0.62–1.01, P value = 0.062). Conclusions: Our study documents the presence of sPD-L1 in PE of MPM patients, and suggests its possible biological and prognostic role in MPM. [ABSTRACT FROM AUTHOR]

Chronic wounds account for 3% of total healthcare expenditure of developed countries; thus, innovative therapies, including Mesenchymal Stem Cells (MSCs) end their exosomes are increasingly considered, even if the activity depends on the whole secretome, made of both soluble proteins and extracellular vesicles. In this work, we prove for the first time the in vivo activity of the whole secretome formulated in a sponge-like alginate wound dressing to obtain the controlled release of bioactive substances. The product has been prepared in a public GMP-compliant facility by a scalable process; based on the murine model, treated wounds healed faster than controls without complications or infections. The treatment induced a higher acute inflammatory process in a short time and sustained the proliferative phase by accelerating fibroblast migration, granulation tissue formation, neovascularization and collagen deposition. The efficacy was substantially supported by the agreement between histological and proteomic findings. In addition to functional modules related to proteolysis, complement and coagulation cascades, protein folding and ECM remodeling, in treated skin, emerged the role of specific wound healing related proteins, including Tenascin (Tnc), Decorin (Dcn) and Epidermal growth factor receptor (EGFR). Of note, Decorin and Tenascin were also components of secretome, and network analysis suggests a potential role in regulating EGFR. Although further experiments will be necessary to characterize better the molecular keys induced by treatment, overall, our results confirm the whole secretome efficacy as novel "cell-free therapy". Also, sponge-like topical dressing containing the whole secretome, GMP- compliant and "ready-off-the-shelf", may represent a relevant point to facilitate its translation into the clinic. [ABSTRACT FROM AUTHOR]

Background: Atopic dermatitis is a multifactorial immune-mediated skin disorder characterized by an alteration of epidermal barrier function and onset of skin lesions, which range from mild erythema to severe lichenification. Treatment consists in hydration with possible use of topical or immunomodulatory corticosteroids, which, however sometimes showed side effects. Recently, the interest in natural compounds has grown significantly and among these, hydroxytyrosol (HT) plays a pivotal role due to its strong and well-known anti-inflammatory activity. Objectives: The aim of this study was to investigate the safety and efficacy of Fenolia® Eudermal Cream 15 (HT-based formulation) on epidermal barrier impaired as consequence of skin injury. Methods: Whit this purpose, morphologic and structural as well as anti-inflammatory evaluations, after treatment with pro-inflammatory mediators (PBS 1 X and LPS) and HT-based formulation on reconstructed human epidermis (RHE) were carried out by qualitative (hematoxylin/eosin- and immunostaining) and quantitative (MTT assay, IL-1α and IL-8 release by ELISA) techniques. Furthermore, HT absorption through the epidermal barrier was evaluated by RP-LC-DAD analysis. Results: A rise in the thickness of the epidermis as well as an appropriate maturation and protein expression (Loricrin, Fillagrin, E-Cadherin and Cytokeratins 5&6) were detected in treated RHE samples. In particular, the HT-based formulation was found to stimulate cell proliferation, as evidenced by the significant increase in Ki67 expression, which suggests the involvement of repair mechanisms, increasing epithelial regeneration and differentiation and improving the epidermal barrier effect. Furthermore, HT-based formulation showed a statistically significant anti-inflammatory activity by reducing both IL-1α and IL-8 release by RHE tissues, greater than the reference drug dexamethasone. Finally, excellent transcutaneous absorption values were found for HT, demonstrating how this new formulation increases the availability of the bioactive compound. Conclusions: In light of these results, Fenolia® Eudermal Cream 15 could be an effective agent to counteract atopic dermatitis. [ABSTRACT FROM AUTHOR]

The nutritional and health properties of algae make them perfect functional ingredients for nutraceutical and cosmeceutical applications. In this study, the Phaeodactylum tricornutum Bohlin (Phaeodactylaceae), a pleiomorphic diatom commonly found in marine ecosystems, was investigated. The in vitro culture conditions used favoured the fusiform morphotype, characterized by a high accumulation of neutral lipids, as detected by fluorescence microscopy after BODIPY staining. These data were confirmed by HPLC-DAD-APCI-MS/MS analyses carried out on the ethanolic extract (PTE), which showed a high content of xanthophylls (98.99%), and in particular of fucoxanthin (Fx, 6.67 g/100 g PTE). The antioxidant activity (ORAC, FRAP, TEAC and β-carotene bleaching) and photostability of PTE and Fx against UVA and UVB rays were firstly evaluated by in vitro cell-free assays. After this, phototoxicity and photoprotective studies were carried out on in vitro reconstructed human epidermidis models. Results demonstrated that PTE (0.1% Fx) and 0.1% Fx, both photostable, significantly (p < 0.05) reduce oxidative and inflammatory stress markers (ROS, NO and IL-1α), as well as cytotoxicity and sunburn cells induced by UVA and UVB doses simulating the solar radiation, with an excellent safety profile. However, PTE proved to be more effective than Fx, suggesting its effective and safe use in broad-spectrum sunscreens. [ABSTRACT FROM AUTHOR]

Background: The combination of Programmed Cell Death 1 (PD-1) and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) blockade has dramatically improved the overall survival rate for malignant melanoma. Immune checkpoint blockers (ICBs) limit the tumor's immune escape yet only for approximately a third of all tumors and, in most cases, for a limited amount of time. Several approaches to overcome resistance to ICBs are being investigated among which the addition of epigenetic drugs that are expected to act on both immune and tumor cells. Guadecitabine, a dinucleotide prodrug of a decitabine linked via phosphodiester bond to a guanosine, showed promising results in the phase-1 clinical trial, NIBIT-M4 (NCT02608437). Methods: We used the syngeneic B16F10 murine melanoma model to study the effects of immune checkpoint blocking antibodies against CTLA-4 and PD-1 in combination, with and without the addition of Guadecitabine. We comprehensively characterized the tumor's and the host's responses under different treatments by flow cytometry, multiplex immunofluorescence and methylation analysis. Results: In combination with ICBs, Guadecitabine significantly reduced subcutaneous tumor growth as well as metastases formation compared to ICBs and Guadecitabine treatment. In particular, Guadecitabine greatly enhanced the efficacy of combined ICBs by increasing effector memory CD8+ T cells, inducing effector NK cells in the spleen and reducing tumor infiltrating regulatory T cells and myeloid derived suppressor cells (MDSC), in the tumor microenvironment (TME). Guadecitabine in association with ICBs increased serum levels of IFN-γ and IFN-γ-induced chemokines with anti-angiogenic activity. Guadecitabine led to a general DNA-demethylation, in particular of sites of intermediate methylation levels. Conclusions: These results indicate Guadecitabine as a promising epigenetic drug to be added to ICBs therapy. [ABSTRACT FROM AUTHOR]

The p38 inhibitor SB202190 is a necessary component of the medium used for normal colorectal mucosa cultures. Sato et al. suggested that the primary activity of SB202190 may be EGFR signaling stabilization, causing an increased phosphorylation of Erk1-2 sustaining organoid proliferation. However, the growth of some colorectal cancer (CRC)-derived organoid cultures is inhibited by this molecule via an unknown mechanism. We biochemically investigated SB202190 activity on a collection of 25 primary human CRC organoids, evaluating EGFR, Akt and Erk1-2 activation using Western blot. We found that Erk1-2 phosphorylation was induced by SB202190 in 20 organoid cultures and inhibited in 5 organoid cultures. A next-generation sequencing (NGS) analysis revealed that the inhibition of p-Erk1-2 signaling corresponded to the cultures with BRAF mutations (with four different hits, one being undescribed), while p-Erk1-2 induction was apparently unrelated to other mutations involving the EGFR pathway (Her2, KRAS and NRAS). We found that SB202190 mirrored the biochemical activity of the BRAF inhibitor Dabrafenib, known to induce the paradoxical activation of p-Erk1-2 signaling in BRAF wild-type cells. SB202190 was a more effective inhibitor of BRAF-mutated organoid growth in the long term than the specific BRAF inhibitors Dabrafenib and PLX8394. Overall, SB202190 can predict BRAF-activating mutations in patient-derived organoids, as well as allowing for the identification of new BRAF variants, preceding and enforcing NGS data. [ABSTRACT FROM AUTHOR]

Primary clear cell colorectal carcinoma (CCC) is a very rare entity accounting for only 35 cases reported in the Literature. CCC is neither classified as a distinct entity nor is it defined as a CRC variant because its ontogeny remains unclear. Most of the reported CCC were found in the distal colon in patients with a mean age of 56 years. Histologically, clear cell change is the main morphologic feature and may present in a “pure” form, composed exclusively of clear cells, or in a “composite” form, admixed with other morphologically different components. It is possible to distinguish two biologically different types of CCC, with different clinical-pathologic features, therapeutic management and diagnostic criteria: a) Intestinal CCC consisting of an aggressive neoplasm, affecting mainly adult men, characterized by an intestinal-type immunoprofile (CK20+, CK7-, CEA+, CDX-2+) and b) Müllerian CCC consisting of an indolent carcinoma of the sigmoid-rectum, affecting young women, characterized by a different (CK7+, CK20-, CEA-, CA125 +) immunoprofile. Considerable diagnostic difficulties can arise in distinguishing CCC and primary or secondary clear cell neoplasms, such as metastases from renal carcinoma, lower urinary tract, female genital tract, adrenal gland, mesothelioma, melanoma and primary intestinal PEComa. In this paper we review the Literature with two additional cases in order to define the diagnostic criteria of CCC. [ABSTRACT FROM AUTHOR]

The microenvironment of solid tumours is extremely acidic and this condition arises since the precancerous stage. This acidic milieu could therefore provide a useful target for both prophylactic and therapeutic approaches. In TRAMP transgenic mice, anin vivomodel of prostate adenocarcinoma (AC), oral administration of alkaline water was devoid of unwanted side effects, and when started from an early age was as effective as NaHCO3in significantly delaying tumour progression, while when started when prostate tumours were already present, a nonstatistically significant trend in the same direction was detected. These findings indicate that the use of alkalinizing drugs should be considered for chemoprevention and, in association with standard chemotherapy, for treatment of human prostate AC. [ABSTRACT FROM AUTHOR]

Identifying innovative molecules involved in the tumor immune escape process could help refine the survival stratification of colorectal cancer (CRC) patients. HLA-G, a non-classical HLA molecule, physiologically involved in tolerogenic mechanisms, has recently emerged as a relevant prognostic marker in other tumor types, but ambiguous data are reported in the CRC setting. This study aims to evaluate the HLA-G expression and prognostic potential in a series of stage II/III CRCs. HLA-G expression was evaluated in 100 pT3 CRC cases by means of immunohistochemistry using the 4H84 and MEM-G/2 monoclonal antibodies. We observed heterogeneous expression of HLA-G showing different ranges: 4H84 expression ranged from > 1 to 40%—median 7%; MEM-G/2 expression ranged from 20 to 90%—median 50%. HLA-G positivity (any intensity > 1%) varied according to the antibody employed, identifying: 8 4H84 positive, 34 MEM-G/2 positive, 6 double-positive and 52 negative cases. Correlation with clinico-pathologic data showed a significant association with a poor tumor differentiation in stage III right-sided CRC subgroup (p = 0.043), while no other pathologic variable was significantly associated. Survival analysis revealed a reduced disease-free survival rate (HR 4.304613; p = 0.031) in the subgroup of CRC-related death cases, while no correlations were observed considering the whole series and the overall survival. In conclusion, HLA-G is a promising CRC prognostic marker however much work is still required regarding technical aspects and evaluation of expression. [ABSTRACT FROM AUTHOR]

Simple Summary: Intravascular lymphoma is a neoplasm with tumor cells localized exclusively within blood vessel lumina. Most cases are of B-cell origin; only rare cases of NK or T-cell lineage. Unlike intravascular large B-cell lymphoma, which is a well-recognized entity in the WHO classification, intravascular NK/T-cell lymphoma is not considered a specific entity and there is still a debate about whether it is closer to aggressive NK leukemia or to extranodal NK/T-cell lymphoma. Our aim was to summarize the clinical, pathological, and molecular data on intravascular NK/T-cell lymphoma, which is a challenge both in terms of diagnosis and treatment. Recent molecular studies improved our understanding of the mechanism of lymphomagenesis, showing that multiple genetic events associated with EBV infection are required for the pathogenesis of this aggressive lymphoma. Promising therapeutic results may be offered by immune checkpoint inhibitors due to the high PD-L1 expression, which is possibly related to EBV infection. Intravascular lymphoma is a form of lymphoid malignancy characterized by neoplastic cells growing almost exclusively within the lumina of small- to medium-sized blood vessels. Most cases are of B-cell origin with rare cases of natural killer or T-cell lineage. Extranodal sites are affected, mainly the skin and central nervous system, although any organ may be involved. Intravascular NK/T-cell lymphoma deserves special attention because of its clinicopathologic features and the need for adequate immunophenotyping combined with clonality test for a proper diagnosis. Moreover, intravascular NK/T-cell lymphoma is strongly linked to Epstein–Barr virus (EBV), which is considered to play a role in tumorigenesis and to be responsible for the aggressive behavior of the disease. In this paper, we review the current knowledge on this rare lymphoma and, in particular, the most recent advances about its molecular landscape. The main distinguishing features with other EBV-related entities, such as extranodal NK/T-cell lymphoma, EBV-positive primary nodal T/NK-cell lymphoma, and aggressive NK-cell leukemia, are discussed to help pathologists obtain the correct diagnosis and consequently develop an adequate and prompt therapy response. [ABSTRACT FROM AUTHOR]

Simple Summary: In this manuscript, we offer an integrated molecular analysis of 44 combined large cell neuroendocrine carcinomas (CoLCNECs) in order to deepen the knowledge about these rare histotypes and to clarify their relationship with lung cancers. In the present state of research, molecular studies are still scant, consisting of small and heterogeneous cohorts, and the genomic landscape is poorly characterized. This study shows that CoLCNECs constitute a standalone group of neuroendocrine neoplasm, with three different molecular profiles, two of which overlap with pure LCNEC or adenocarcinoma. CoLCNECs can be considered an independent histologic category with specific genomic and transcriptomic features, different and therefore not comparable to other lung cancers. Indeed, in addition to a histological re-evaluation of lung cancer classification, our study may help to develop a new diagnostic approach for novel and personalized treatments in CoLCNECs. Background: Combined large cell neuroendocrine carcinoma (CoLCNEC) is given by the association of LCNEC with adeno or squamous or any non-neuroendocrine carcinoma. Molecular bases of CoLCNEC pathogenesis are scant and no standardized therapies are defined. Methods: 44 CoLCNECs: 26 with adenocarcinoma (CoADC), 7 with squamous cell carcinoma (CoSQC), 3 with small cell carcinoma (CoSCLC), 4 with atypical carcinoid (CoAC) and 4 napsin-A positive LCNEC (NapA+), were assessed for alterations in 409 genes and transcriptomic profiling of 20,815 genes. Results: Genes altered included TP53 (n = 30), RB1 (n = 14) and KRAS (n = 13). Targetable alterations included six KRAS G12C mutations and ALK-EML4 fusion gene. Comparison of CoLCNEC transcriptomes with 86 lung cancers of pure histology (8 AC, 19 ADC, 19 LCNEC, 11 SCLC and 29 SQC) identified CoLCNEC as a separate entity of neuroendocrine tumours with three different molecular profiles, two of which showed a non-neuroendocrine lineage. Hypomethylation, activation of MAPK signalling and association to immunotherapy signature specifically characterized each of three CoLCNEC molecular clusters. Prognostic stratification was also provided. Conclusions: CoLCNECs are an independent histologic category. Our findings support the extension of routine evaluation of KRAS mutations, fusion genes and immune-related markers to offer new perspectives in the therapeutic management of CoLCNEC. [ABSTRACT FROM AUTHOR]

The metachronic onset of diffuse large B-cell lymphoma (DLBCL) after classic Hodgkin lymphoma (cHL) is a rare event affecting patients' outcomes. However, although several studies have investigated the prognostic role of this event, little is known about a hypothetical common origin of the two different neoplastic cells. Aims: To investigate a possible relationship between DLBCL and cHL, in this retrospective study of 269 patients with newly diagnosed cHL treated at Bari University Hospital (Italy) between 2007 and 2020, we analyzed data from 4 patients (3 male and 1 female) with cHL who subsequently developed DLBCL. Methods: Gene expression profile analysis, assessed by NanoString Lymphoma Subtype Assay, was performed to identify the cell of origin in the DLBCL cases, in addition to Hans's algorithm. Results: Using Hans's algorithm, all DLBCL cases showed a germinal center-B-Cell subtype. The gene expression profile evaluated by the NanoString Lymphoma Subtype Assay revealed two cases of the GCB molecular subtype, while the others were unclassified. After first-line chemotherapy, 1 patient achieved complete remission, 3 were non-responders (2 died of lymphoma within 6 months, whereas the other achieved complete remission after autologous and allogeneic stem cell transplantation and is still alive). Conclusions: The origin of the second neoplastic cell in patients with DLBCL with a previous history of cHL remains controversial, although the different immunophenotypic characteristics suggest that it may mainly arise de novo in a subject with a possible individual predisposition to develop lymphoid neoplasms. [ABSTRACT FROM AUTHOR]

Background: Total mesorectal excision (TME) is the gold standard to treat locally advanced rectal cancer. This monocentric retrospective study evaluates the results of laparotomic, laparoscopic and robotic surgery in "COMRE GROUP" (REctalCOMmittee). Methods: 327 selected stage I-II-III patients (pts) underwent TME between November 2005 and April 2020 for low or middle rectal cancer; 91 pts underwent open, 200 laparoscopic and 36 robotic TME. Of these, we analyzed the anthropomorphic, intraoperative, anatomopathological parameters and outcome during the follow up. Results: The length of hospital stay was significantly different between robotic TME and the other two groups (8.47 ± 3.54 days robotic vs. 11.93 ± 5.71 laparotomic, p < 0.001; 8.47 ± 3.54 robotic vs. 11.10 ± 7.99 laparoscopic, p < 0.05). The mean number of harvested nodes was higher in the laparotomic group compared to the other two groups (19 ± 9 laparotomic vs. 15 ± 8 laparoscopic, p < 0.001; 19 ± 9 laparotomic vs. 15 ± 7 robotic, p < 0.05). Median follow-up was 52 months (range: 1–169). Overall survival was significantly shorter in the open TME group compared with the laparoscopic one (Chi2 = 13.36, p < 0.001). Conclusions: In the experience of the "COMRE" group, laparoscopic TME for rectal cancer is a better choice than laparotomy in a multidisciplinary context. Robotic TME has a significant difference in terms of hospital stay compared to the other two groups. [ABSTRACT FROM AUTHOR]

Background: Microscopic esophagitis (ME) is common in patients with non-erosive reflux disease (NERD), and dilation of intercellular spaces (DIS) has been regarded as the potential main mechanism of symptom generation. We aimed to compare these histological abnormalities in healthy volunteers (HVs) and patients with erosive esophagitis (EE), NERD, and functional heartburn (FH). Methods: Consecutive patients with heartburn prospectively underwent upper endoscopy and impedance-pH off-therapy. Twenty EE patients and fifty-seven endoscopy-negative patients (NERD), subclassified as 22 with pH-POS (positive for abnormal acid exposure), 20 with hypersensitive esophagus (HE; normal acid/symptom association probability [SAP]+ or symptom index [SI]+), and 15 with FH (normal acid/SAP-/SI-/ proton pump inhibitor [PPI] test-), were enrolled. Twenty HVs were also included. In each patient/control, multiple specimens ( n = 5) were taken from the distal esophagus and histological alterations were evaluated. ME was diagnosed when the global histological score was >0.35. Results: The prevalence of ME was higher ( p < 0.0001) in EE (95 %), pH-POS (77 %), and HE (65 %) NERD patients than in FH patients (13 %) and HVs (15 %). Also, basal cell hyperplasia ( p < 0.0023), DIS ( p < 0.0001), and papillae elongation ( p < 0.0002) showed similar rates of prevalence in the above populations ( p < 0.0001). ME, including each histological lesion, had similar low frequencies in FH and HVs ( p = 0.9990). Considering the histological abnormalities together, they permitted us to clearly differentiate EE and NERD from FH and HVs ( p < 0.0001 and p < 0.0001, respectively). Conclusions: The lack of ME in the esophageal distal biopsies of FH patients indicates a limited role of these histological abnormalities in symptom generation in them. ME can be considered as an accurate and reliable diagnostic marker for distinguishing FH patients from GERD patients and has the potential to be used to guide the correct therapy. [ABSTRACT FROM AUTHOR]

Background: Standardized criteria for assessing microscopic esophageal lesions are required to test their utility as markers of gastroesophageal reflux disease (GERD).Aims: To finalize draft criteria for assessing microscopic esophageal lesions associated with gastroesophageal reflux and to test them for interobserver agreement.Methods: An international group of gastrointestinal pathologists was convened to finalize, using a consensus-based approach, draft criteria for recognizing microscopic esophageal lesions. Finalized criteria were retested for interobserver variability by four of the pathologists using 120 digitized esophageal biopsy slides from patients with GERD.Results: The finalized criteria included further clarification on lesion definitions and new guidance on how to select the area for assessing each lesion. This latter refinement was guided by the high interobserver agreement observed when draft criteria were previously applied to biopsies where the assessment area was preselected. When finalized criteria were applied in the current study to digitized biopsies without a preselected assessment area, the pairwise agreement was 73-97% for basal cell hyperplasia, papillary elongation, intraepithelial eosinophil, neutrophil and mononuclear cell numbers, and active/healed erosions, with slightly lower agreement (64%) for dilated intercellular spaces (DIS). When a combined severity score was applied, the level of agreement was 77%. The mean kappa ranged from fair to high (0.26-0.77) for individual lesions and was high for the combined score (0.64).Conclusions: These levels of agreement are comparable with or higher than those for other accepted histologic definitions. Further steps include clinical validation of these criteria by correlating microscopic lesions with clinical variables such as esophageal acid exposure. [ABSTRACT FROM AUTHOR]

BACKGROUND: Histology is generally considered as a tool of limited value in the diagnosis of gastro-esophageal reflux disease (GERD). AIM: To reevaluate the diagnostic role of histological alterations in GERD, using multiple biopsy sites and an appropriate control group. METHODS: We studied 135 patients with typical and atypical symptoms of GERD. They underwent upper GI endoscopy and Los Angeles classification was used for grading cases with mucosal breaks. Biopsies were taken at the Z-line, 2 and 4 cm above it. Microscopic esophagitis was identified by necrosis/erosion, neutrophil/eosinophil intraepithelial infiltration, basal cell hyperplasia, elongation of papillae, dilation of intercellular spaces and a score (range: 0–2) was given for each lesion. Twenty-four-hour esophageal pH monitoring was performed in each patient. Twenty subjects without reflux symptoms, and with normal endoscopy and pH testing were considered as controls. RESULTS: Histological alterations were found in 100 of 119 GERD patients (84%) and in 3 of 20 controls (15%) with a significant difference ( p < 0.00001). Histology was abnormal in 96% of patients with erosive esophagitis and in 76% of patients with nonerosive reflux disease (NERD). The sum of scores of microscopic lesions found in all biopsy sites ranged from 0 to 22 and we identified a cut-off value (score 2) that distinguished efficiently controls from GERD patients. CONCLUSIONS: In contrast with previous reports on the marginal role of histology in patients with GERD, our study shows that this technique can be a useful diagnostic tool, particularly in patients with NERD, when biopsies are taken at two sites including Z-line and 2 cm above it. [ABSTRACT FROM AUTHOR]

Pathology archives are a treasure trove of paraffin embedded tissue spanning many years and covering a wide variety of tissues and diseases. The possibility of using old archival formalin fixed paraffin embedded (FFPE) tissues for diagnostic updates and research projects is a widespread need and it requires archives of stable, well-preserved samples. Immunohistochemistry performed on old archival paraffin blocks may give unreliable results, in particular for some antigens, such as Ki67. In consideration of this phenomenon, our aim is to comprehensively test and identify methods which may be used to obtain Ki67 immunohistochemical reactions of good quality from old archival FFPE blocks. Various methods were tested in order to evaluate their possible efficacy in increasing Ki67 immunointensity in a collection of 40-year-old, archival blocks including re-embedding, with deeper sectioning of tissue from the block and increasing heat-based pretreatment times (20 cases) and re-processing (20 cases). All reactions were performed using an automated immunostainer and Ki67 stained immunosections compared using a visual colour-based scale (the first immunostained section was considered as baseline). The combination of deep sectioning (1000 µM) and prolonged heat-based pretreatment (64 min) markedly increased immunoreactivity for Ki67. Re-embedding and reprocessing did not have a significant effect. Large tissue samples showed heterogeneity of Ki67 immunoexpression between the periphery of the sample and the central area. In conclusion, the study defines a useful protocol to increase antigen retrieval applicable to dated archival tissues. [ABSTRACT FROM AUTHOR]

Keywords: BRAF; colorectal carcinoma; Lynch syndrome; MLH1 methylation; NTRK EN BRAF colorectal carcinoma Lynch syndrome MLH1 methylation NTRK 335 336 2 07/11/23 20230801 NES 230801 Ethics statement For this study, approval and/or informed consent were not required. BRAF, colorectal carcinoma, Lynch syndrome, MLH1 methylation, NTRK Lynch syndrome-related colorectal carcinomas are NTRK-negative. [Extracted from the article]

Lung fibrosis has specific computed tomography (CT) findings and represents a common finding in advanced COVID-19 pneumonia whose reversibility has been poorly investigated. The aim of this study was to quantify the extension of collagen deposition and aeration in postmortem cryobiopsies of critically ill COVID-19 patients and to describe the correlations with qualitative and quantitative analyses of lung CT. Postmortem transbronchial cryobiopsy samples were obtained, formalin fixed, paraffin embedded and stained with Sirius red to quantify collagen deposition, defining fibrotic samples as those with collagen deposition above 10%. Lung CT images were analyzed qualitatively with a radiographic score and quantitatively with computer-based analysis at the lobe level. Thirty samples from 10 patients with COVID-19 pneumonia deceased during invasive mechanical ventilation were included in this study. The median [interquartile range] percent collagen extension was 6.8% (4.6–16.2%). In fibrotic compared to nonfibrotic samples, the qualitative score was higher (260 (250–290) vs. 190 (120–270), p = 0.036) while the gas fraction was lower (0.46 (0.32–0.47) vs. 0.59 (0.37–0.68), p = 0.047). A radiographic score above 230 had 100% sensitivity (95% confidence interval, CI: 66.4% to 100%) and 66.7% specificity (95% CI: 41.0% to 92.3%) to detect fibrotic samples, while a gas fraction below 0.57 had 100% sensitivity (95% CI: 66.4% to 100%) and 57.1% specificity (95% CI: 26.3% to 88.0%). In COVID-19 pneumonia, qualitative and quantitative analyses of lung CT images have high sensitivity but moderate to low specificity to detect histopathological fibrosis. Pseudofibrotic CT findings do not always correspond to increased collagen deposition. [ABSTRACT FROM AUTHOR]

The free online trading of herbal mixtures useful for various purposes facilitates the circulation of dangerous herbs or plant parts. This is the case, for example, of the illegal trade in seeds of Peganum harmala (Pgh), which contain alkaloids capable of inhibiting monoamine oxidase (MAO) and are therefore used in hallucinogenic preparations, such as the psychedelic drink ayahuasca. The precise identification of these seeds and their distinction from other very similar but not dangerous seeds are necessary for forensic purposes and represents an advance in avoiding the adulteration of mixtures. In this work, we show the qualitative identification of Pgh seeds by optical and electron microscopy and the parallel development of a real-time qPCR test, which reveals, in a species-specific manner, the presence of Pgh DNA up to quantities lower than 1 pg. In addition to the species specificity and high sensitivity, the reaction accurately quantifies the presence of seeds or parts of seeds of Pgh in complex herbal mixtures, thus giving an indication of the danger or otherwise of the product. [ABSTRACT FROM AUTHOR]

Background: The primary objective of the study is to describe the cellular characteristics of bronchoalveolar lavage fluid (BALF) of COVID-19 patients requiring invasive mechanical ventilation; the secondary outcome is to describe BALF findings between survivors vs non-survivors.Materials and Methods: Patients positive for SARS-CoV-2 RT PCR, admitted to ICU between March and April 2020 were enrolled. At ICU admission, BALF were analyzed by flow cytometry. Univariate, multivariate and Spearman correlation analyses were performed.Results: Sixty-four patients were enrolled, median age of 64 years (IQR 58-69). The majority cells in the BALF were neutrophils (70%, IQR 37.5-90.5) and macrophages (27%, IQR 7-49) while a minority were lymphocytes, 1%, TCD3+ 92% (IQR 82-95). The ICU mortality was 32.8%. Non-survivors had a significantly older age (p = 0.033) and peripheral lymphocytes (p = 0.012) were lower compared to the survivors. At multivariate analysis the percentage of macrophages in the BALF correlated with poor outcome (OR 1.336, CI95% 1.014-1.759, p = 0.039).Conclusions: In critically ill patients, BALF cellularity is mainly composed of neutrophils and macrophages. The macrophages percentage in the BALF at ICU admittance correlated with higher ICU mortality. The lack of lymphocytes in BALF could partly explain a reduced anti-viral response. [ABSTRACT FROM AUTHOR]

Esophageal squamous cell carcinoma represents the most common histotype of epithelial neoplasm occurring within esophageal mucosa worldwide. Despite the comprehensive molecular characterization of this entity, to date no significant targeted therapy has been introduced into clinical practice. In this review, we describe the molecular landscape of esophageal squamous cell carcinoma based on the most recent literature. Moreover, we focus on other rare variants and on the relationship with head and neck squamous cell carcinomas. [ABSTRACT FROM AUTHOR]

Colorectal cancer's (CRC) ability to invade local tissues and lymph nodes and generate distant metastases is the key for TNM classification. Aspartate-β-hydroxylase (ASPH), a transmembrane protein that catalyzes Notch receptors and ligand activation, is involved in tumor invasion. Because Notch is involved in gut homeostasis, it could be a target for CRC therapy. ASPH mRNA and protein expression, promoter methylation and gene copy numbers were evaluated using the TCGA and CPTAC human CRC datasets. Using digital pathology, ASPH was scored in the luminal area (LM), center tumor (CT) and invasive margin (IM) of 100 human CRCs. The effect of ASPH targeting on invasiveness and viability was tested by siRNA knockdown and small molecule inhibitors (SMI). Bioinformatics analysis showed increased expression of ASPH mRNA and protein in CRC, paired with a decreased methylation profile. ASPH genetic gain or amplification was frequent (56%), while deletion was rare (0.03%). Digital pathology analysis showed that ASPH exerted its pathological activity in the invasive margin of the tumor, affecting invasive front morphology, tumor budding and patients' overall survival. In vitro, ASPH targeting by siRNA or SMI reduced cell invasion and growth and caused Notch-1 downregulation. This study demonstrates that ASPH targeting by specific inhibitors could improve CRC treatment strategies. [ABSTRACT FROM AUTHOR]

Standard treatments of chronic skin ulcers based on the direct application of dressings still present several limits with regard to a complete tissue regeneration. Innovative strategies in tissue engineering offer materials that can tune cell behavior and promote growth tissue favoring cell recruitment in the early stages of wound healing. A combination of Alginate (Alg), Sericin (SS) with Platelet Lysate (PL), as a freeze-dried sponge, is proposed to generate a bioactive wound dressing to care skin lesions. Biomembranes at different composition were tested for the release of platelet growth factors, cytotoxicity, protective effects against oxidative stress and cell proliferation induction. The highest level of the growth factors release occurred within 48 h, an optimized time to burst a healing process in vivo; the presence of SS differently modulated the release of the factors by interaction with the proteins composing the biomembranes. Any cytotoxicity was registered, whereas a capability to protect cells against oxidative stress and induce proliferation was observed when PL was included in the biomembrane. In a mouse skin lesion model, the biomembranes with PL promoted the healing process, inducing an accelerated and more pronounced burst of inflammation, formation of granulation tissue and new collagen deposition, leading to a more rapid skin regeneration. [ABSTRACT FROM AUTHOR]

Background: B cell receptor Immunoglobulin (BcR IG) repertoire of Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of quasi-identical BcR IG. These are observed in approximately 30% of patients, defined as stereotyped receptors and subdivided into subsets based on specific VH CDR3 aa motifs and phylogenetically related IGHV genes. Although relevant to CLL ontogeny, the distribution of CLL-biased stereotyped immunoglobulin rearrangements (CBS-IG) in normal B cells has not been so far specifically addressed using modern sequencing technologies. Here, we have investigated the presence of CBS-IG in splenic B cell subpopulations (s-BCS) and in CD5+ and CD5− B cells from the spleen and peripheral blood (PB). Methods: Fractionation of splenic B cells into 9 different B cell subsets and that of spleen and PB into CD5+ and CD5− cells were carried out by FACS sorting. cDNA sequences of BcR IG gene rearrangements were obtained by NGS. Identification of amino acidic motifs typical of CLL stereotyped subsets was carried out on IGHV1-carrying gene sequences and statistical evaluation has been subsequently performed to assess stereotypes distribution. Results: CBS-IG represented the 0.26% average of IGHV1 genes expressing sequences, were detected in all of the BCS investigated. CBS-IG were more abundant in splenic and circulating CD5+ B (0.57%) cells compared to CD5− B cells (0.17%). In all instances, most CBS IG did not exhibit somatic hypermutation similar to CLL stereotyped receptors. However, compared to CLL, they exhibited a different CLL subset distribution and a broader utilization of the genes of the IGHV1 family. Conclusions: CBS-IG receptors appear to represent a part of the "public" BcR repertoire in normal B cells. This repertoire is observed in all BCS excluding the hypothesis that CLL stereotyped BcR accumulate in a specific B cell subset, potentially capable of originating a leukemic clone. The different relative representation of CBS-IG in normal B cell subgroups suggests the requirement for additional selective processes before a full transformation into CLL is achieved. [ABSTRACT FROM AUTHOR]

Standard treatment for locally advanced rectal adenocarcinoma (LARC) includes a combination of chemotherapy with pyrimidine analogues, such as capecitabine, and radiation therapy, followed by surgery. Currently no clinically useful genomic predictors of benefit from neoadjuvant chemoradiotherapy (nCRT) exist for LARC. In this study we assessed the expression of 8,127 long noncoding RNAs (lncRNAs), poorly studied in LARC, to infer their ability in classifying patients' pathological complete response (pCR). We collected and analyzed, using lncRNA-specific Agilent microarrays a consecutive series of 61 LARC cases undergoing nCRT. Potential lncRNA predictors in responders and non-responders to nCRT were identified with LASSO regression, and a model was optimized using k-fold cross-validation after selection of the three most informative lncRNA. 11 lncRNAs were differentially expressed with false discovery rate < 0.01 between responders and non-responders to NACT. We identified lnc-KLF7-1, lnc-MAB21L2-1, and LINC00324 as the most promising variable subset for classification building. Overall sensitivity and specificity were 0.91 and 0.94 respectively, with an AUC of our ROC curve = 0.93. Our study shows for the first time that lncRNAs can accurately predict response in LARC undergoing nCRT. Our three-lncRNA based signature must be independently validated and further analyses must be conducted to fully understand the biological role of the identified signature, but our results suggest lncRNAs may be an ideal biomarker for response prediction in the studied setting. [ABSTRACT FROM AUTHOR]

The article presents a case study of a 76-year-old man who was diagnosed with ectopic thyroid. Topics include detection of the presence of cholelithiasis and sludge, gallbladder wall thickening, and pericholecystic fluid in an ultrasound during the procedure of diagnosis; detection of a an aerobic gram-negative bacillus called citrobacter in blood culture; and occurrence of ectopic thyroid from the base of the tongue to the inferior margin of the cricoid cartilage and intra-abdominal parts.

Background: Claudin-18 (CLDN18) is a highly specific tight junction protein of the gastric mucosa. An isoform of CLDN18, the Claudin 18.2, has recently emerged as an innovative drug target for metastatic gastric cancer.Methods: We investigated the immunohistochemical profile of CLDN18, p53, p16, E-cadherin, MSH2, MSH6, MLH1, PSM2, HER2, and PDL-1 in a large series of 523 primary gastric carcinomas (GCs; n = 408) and gastro-oesophageal carcinomas (GECs; n = 115) and 135 matched and synchronous nodal metastases. The status of HER2 and EBER by means of chromogenic in situ hybridisation (CISH) was also evaluated.Results: High membranous CLDN18 expression was present in 150/510 (29.4%) primary cases and in 45/132 (34.1%) metastases. An abnormal expression (i.e. nuclear and/or cytoplasmic) was observed in 115 (22.5%) primary cases and in 33 (25.0%) metastases. A 38.8% of the cases showed significant CLDN18 intratumoural variability among the different tissue microarray cores obtained from the same tumour. Positive membrane CLDN18 expression was statistically associated with non-antral GCs (p = 0.016), Lauren diffuse type (p = 0.009), and with EBV-associated cancers (p < 0.001).Conclusions: CLDN18 is frequently expressed in gastric and gastro-oesophageal cancers; further studies should investigate the prognostic significance of CLDN18 heterogeneity in order to implement its test into clinical practice. [ABSTRACT FROM AUTHOR]

The article presents a case study of an 86-year-old woman who was suffering from intestinal bleeding and anemia. Topics discussed include presence of polypoid mass in small intestine revealed from abdominal computed tomography, dialation of submucosal glands and proliferation of stroma was observed from histological testing, and diagnosis of adenomyoma along small bowel was reported.

The section was shown to all our pathology trainees; first-year trainees thought of perhaps an unknown helminth or synthetic fibers, while our fifth-year trainee also suggested a fruit peel of some sort, for example, tomato. For comparison (Figure 2), a tomato peel was fixed in formalin, processed in paraffin, microtome cut, and stained, demonstrating the unknown organic ream as indeed tomato in origin. The case strand shows only the yellow cuticle as the underlying small portion, with 2 to 3 layers of thick-walled epidermal cells[2] seen in the test peel (Figure 2C), which has probably been digested. [Extracted from the article]

Thyroid ectopia is a rare finding below the diaphragm. It is characterized by normal thyroid parenchyma in unusual locations with preserved thyroid marker immunoreactivity. In this article, we present the first known case of thyroid tissue in the periappendiceal fat and discuss possible ethiopathogenic theories. [ABSTRACT FROM AUTHOR]