The association between respirable crystalline silica (RCS) and sarcoidosis is becoming more and more apparent. Hua et al. recently coined a new term of silicosarcoidosis to denote patients who exhibit features of both silicosis and sarcoidosis. We present two case reports of patients with occupational exposure to RCS and histopathological features of both silicosis and sarcoidosis. The first patient is a 39 year-old asymptomatic male smoker who worked as an operator of a welding robot that employs manganese-silicate welding wire, with an accidental finding of multiple upper lobe-predominant nodules and mediastinal lymphadenopathy. The second case relates to a 50-year old never-smoker male tile-setter, with multiple bilateral pulmonary nodules and mediastinal lymphadenopathy. Surgical lung biopsy of pulmonary nodules revealed the presence of non-necrotising granulomas (without birefringent particles) and the presence of silicotic nodules. Our first case exemplifies the potential of therapeutic intervention in patients with silosarcoidosis and both cases (with histopathological images included) add towards the growing body of knowledge regarding the phenomenon of concomitant silicosis and sarcoidosis, i.e. silicosarcoidosis.

Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality. Microsatellite instability-high (MSI-H) tumors, resulting from defective DNA mismatch repair (MMR), represent a well-defined subtype with distinctive biological behavior and immunogenicity. In contrast, tumors with elevated microsatellite alterations at tetranucleotide repeats (EMAST) are less well characterized. EMAST can manifest with MSI or arise as an isolated form of instability, delineating discrete phenotypes underpinned by distinct mechanisms. This study aimed to characterize MSI and EMAST status in CRCs. By integrating instability profiles with clinicopathological features and mutational profiles of key driver genes, we aimed to refine molecular classification and advance understanding of CRC tumorigenesis. A total of 332 CRCs were analyzed for MSI and EMAST using established panels. Clinicopathological characteristics were recorded, and mutational profiling of KRAS, BRAF, CTNNB1, PIK3CA, and TP53 was performed. MLH1 expression was assessed using immunohistochemistry. MSS/EMAST-S tumors displayed profiles typical of chromosomally stable CRC, dominated by KRAS and followed by TP53 and PIK3CA mutations. MSI-H/EMAST-H tumors were characterized by frequent BRAF mutations, right-sided location, female predominance, and lower TP53 mutation rate, consistent with the classical hypermutated, immunogenic subtype. In contrast, MSS/EMAST-H tumors exhibited unique features, including enrichment for PIK3CA and CTNNB1 mutations, larger tumor size, and poorer differentiation, suggesting an intermediate phenotype between MSS and MSI-H. MSS/EMAST-L tumors aligned with chromosomally stable, KRAS/Wnt-driven CRC. In conclusion, MSS/EMAST-H tumors represent an underrecognized CRC subtype with intermediate genomic instability and a distinctive molecular profile, with potential implications for prognostic assessment and personalized therapeutic strategies.

AIM:To investigate histopathological changes in the lung tissue of long-COVID patients. METHODS:In this cross-sectional study, transbronchial lung biopsy was performed in long-COVID patients with persisting symptoms and radiological abnormalities. Histopathologic analyses were performed by using hematoxylin-eosin, Martius, Scarlet and Blue, Movat's, thyroid transcription factor 1, CD34, and CD68 staining. RESULTS:Adequate biopsy samples were obtained from 29/32 patients. The median (Q1-Q3) time from disease onset to biopsy was 13 (9-20) weeks. We observed several histopathologic patterns: DAD with vascular abnormalities (VA) (n=8); VA with inflammatory pattern (n=4); inflammatory pattern (n=13), and fibrotic pattern (n=4). VA included capillary thrombi, dilated venules, and dissection of small pulmonary arteries. DAD with VA was detected up to the 9th week from the onset of disease; inflammatory pattern from the 8th to 28th week (4 patients with this pattern biopsied in the 11th-13th week had accompanying VA); and a predominantly fibrotic pattern was found at weeks 8, 10, 48, and 49. CONCLUSION:Our study observed a slow recovery of lung tissue with long-lasting DAD and VA, likely followed by interstitial inflammation or focal fibrosis. These findings might be the underlying cause of the slow recovery of long-COVID patients.

Stage II colorectal cancer (CRC) is a major therapeutic challenge because it is not easy to decide whether patients will benefit from adjuvant chemotherapy or not. This study was designed as a retrospective prognostic study combining standard histopathologic parameters with tumor budding and microsatellite instability. The study included 89 patients on elective treatment for stage II primary colorectal adenocarcinoma from January 2011 to December 2015. Study results indicated that the prognosis of patients with stage II CRC depended on the combination of three factors, (in the order of importance): number of lymph nodes examined; total number of buds per 0.785 mm2 (≥5.5); and positive lymphovascular invasion. There is increasing evidence that tumor biology and non-anatomic characteristics are important in the prognosis and treatment of CRC. One of them is tumor budding which is not yet an integral part of the AJCC staging system. A low number of the lymph nodes examined is associated with high-risk patients. All patients without an adequate number of lymph nodes examined (less than 8 lymph nodes) should a priori be considered a very high-risk group, with a very low survival rate, and chemotherapy should be used.

Microsatellite instability (MSI) has been recognized as an important factor in colorectal cancer (CRC). It arises due to deficient mismatch repair (MMR), mostly attributed to MLH1 and MSH2 loss of function leading to a global MMR defect affecting mononucleotide and longer microsatellite loci. Recently, microsatellite instability at tetranucleotide loci, independent of the global MMR defect context, has been suggested to represent a distinct entity with possibly different consequences for tumorigenesis. It arises as a result of an isolated MSH3 loss of function due to its translocation from the nucleus to the cytoplasm under the influence of interleukin-6 (IL-6). In this study the influence of MSH3 and IL-6 signaling pathway polymorphisms (MSH3 exon 1, MSH3+3133A/G, IL-6-174G/C, IL-6R+48892A/C, and gp130+148G/C) on the occurrence of different types of microsatellite instability in sporadic CRC was examined by PCR–RFLP and real-time PCR SNP analyses. A significant difference in distribution of gp130+148G/C genotypes (p = 0.037) and alleles (p = 0.031) was observed in CRC patients with the C allele being less common in tumors with di- and tetranucleotide instability (isolated MSH3 loss of function) compared to tumors without microsatellite instability. A functional polymorphism in gp130 might modulate the IL-6 signaling pathway, directing it toward the occurrence of microsatellite instability corresponding to the IL-6-mediated MSH3 loss of function.

The lack of pixel-wise annotated images severely hinders the deep learning approach to computer-aided diagnosis in histopathology. This research creates a public database comprised of: (i) a dataset of 82 histopathological images of hematoxylin-eosin stained frozen sections acquired intraoperatively on 19 patients diagnosed with metastatic colon cancer in a liver; (ii) corresponding pixel-wise ground truth maps annotated by four pathologists, two residents in pathology, and one final-year student of medicine. The Fleiss' kappa equal to 0.74 indicates substantial inter-annotator agreement; (iii) two datasets with images stain-normalized relative to two target images; (iv) development of two conventional machine learning and three deep learning-based diagnostic models. The database is available at http://cocahis.irb.hr. For binary, cancer vs. non-cancer, pixel-wise diagnosis we develop: SVM, kNN, U-Net, U-Net++, and DeepLabv3 classifiers that combine results from original images and stain-normalized images, which can be interpreted as different views. On average, deep learning classifiers outperformed SVM and kNN classifiers on an independent test set 14% in terms of micro balanced accuracy, 15% in terms of the micro F-1 score, and 26% in terms of micro precision. As opposed to that, the difference in performance between deep classifiers is within 2%. We found an insignificant difference in performance between deep classifiers trained from scratch and corresponding classifiers pre-trained on non-domain image datasets. The best micro balanced accuracy estimated on the independent test set by the U-Net++ classifier equals 89.34%. Corresponding amounts of F-1 score and precision are, respectively, 83.67% and 81.11%.

Hyperspectral imaging (HSI) is demonstrating the growing capability for disease diagnosis and surgical cancer resection. That is mainly due to high spectral resolution of HSI when compared with its color (RGB) counterparts. However, increased spectral resolution is often associated with the loss of spatial resolution. That combined with high cost hinders applicability of HSI. Herein, we propose computational approach that attempts to mimic the HSI. It is using an approximate explicit feature map (aEFM) to augment raw and/or stain normalized RGB images of the hematoxylin and eosin stained histopathological specimen. We demonstrate on two public labeled datasets, related to breast cancer and nuclei, the statistically significant improvement of performance of binary (caner vs. non-cancer) segmentation of augmented RGB images in comparison with the results achieved on their RGB counterparts. For the breast cancer, balanced accuracy is increased from 76.56%±9.05% to 80.42%±9.23% and F1 score from 13.34%±6.46% to 17.33%±6.36%. For nuclei, balanced accuracy is increased from 68.68%±9.25% to 79.99%±8.77% and F1 score from 46.92%±15.10% to 63.31%±14.50%. While  0 constrained nonnegative matrix factorization was used for binary segmentation herein, we conjecture that aEFM based augmentation of RGB images can improve performance of more sophisticated segmentation methods such as deep networks.

The quantity and quality of preoperative material in colorectal cancer is often limiting factor in determination of risk factors and therapy planning. The most important negative prognostic factors are intravascular and perineural invasion, as well as tumor budding. Usually, the only parameter available in preoperative biopsy is tumor budding. However, the growing body of evidence suggests that cancer differentiation based on the poorly differentiated clusters has better prognostic value. The limiting factor in applying of these new parameters is reproducible, simple, cheap and fast method of their determination. In this paper we investigated the prognostic value of lacunarity, determined in preoperative biopsy. Lacunarity is a measure of spatial heterogeneity (inhomogeneity) in an image. It quantifies how objects fill the space, and enables analysis of gaps distribution, homogeneity of gaps, and presence of structures. It was shown that lacunarity and the total number of buds could be combined in a model which clearly divides colorectal cancer patients in low, medium and high risk subgroups. The paper also points out that the quantitative numerical methods are superior to semiquantitative methods, and that individual methods should be combined using algorithms to obtain a more accurate prediction. Because the study described is designed as a pilot study, verification is needed on a larger sample of patients from independent researchers.

There is a need for computer-aided diagnosis (CAD) systems to relieve the workload on pathologists. This seems to be especially important for intraoperative diagnosis during surgery, for which diagnostic time is very limited. This paper presents preliminary results of intraoperative pixel-based CAD of colon cancer metastasis in a liver from phase-contrast images of unstained frozen sections. In particular, two deep learning networks: the U-net and the structured autoencoder for deep subspace clustering, were trained on eighteen phase-contrast images belonging to five patients and tested on eight images belonging to three patients. Spectrum angle mapper was also used in comparative performance analysis. The best result achieved by the U-net yielded balanced accuracy of 83.70%+/- 8%, sensitivity of 94.50%+/- 8%, specificity of 72.9%+/- 8% and Dice coefficient of 45.20%+/- 25.4%. However, factors such as absence of tissue fixation and ethanol-induced dehydration, melting of the specimen under the microscope and/or frozen crystals in the specimen cause variations in quality of phase-contrast images of unstained frozen sections. This, in return, affects reproducibility of diagnostic performance.

Development of computer-aided diagnosis (CAD) systems is motivated by reduction of the workload on the pathologist that is increasing steadily. Among approaches upon which CAD-based systems are built, deep learning (DL) methods seem to be well suited for image analysis in digital pathology. However, DL networks include a large number of parameters and that requires a large annotated training dataset. Unfortunately, probably the biggest problem in digital pathology using machine learning methods is a small number of annotated images. That is especially true in intraoperative tissue analysis which coincides with the topic of the present paper: intraoperative CAD-based diagnosis of metastasis of colon cancer in a liver from hematoxylin-eosin (H&E) stained frozen section. To cope with the insufficiency of training images we adopt a transfer learning approach using the Nested UNet architecture. For better diagnostic performance, the trained model predicted pixels multiple times for different striding levels using the sliding window strategy. Threshold optimization using balanced accuracy score showed the validity of such an approach as balanced accuracy has increased significantly. When compared to often used UNet with VGG16 backbone, Nested UNet model with DenseNet201 backbone performs better on our dataset for both balanced accuracy metric and F1 score.

AIM:To determine the role of immunoglobulin M (IgM) deposits in clinical manifestations, disease outcome, and treatment response of idiopathic and secondary focal segmental glomerulosclerosis (FSGS).METHODS:Kidney biopsy specimens of 171 patients diagnosed with FSGS (primary and secondary) and 50 control patients were retrospectively included in the study. For each patient, clinical and outcome data were obtained and compared to morphological parameters, including immunofluorescence analysis of mesangial IgM and complement 3 (C3) deposits analyzed on kidney biopsy samples.RESULTS:There were significant positive correlations between IgM and C3 deposition in secondary FSGS (P<0.001) and between IgM and mesangial deposits detected by electron microscopy in secondary FSGS (P=0.015), which indicated that higher IgM deposition correlated with higher C3 deposition and mesangial deposits only in secondary FSGS. Patients with secondary FSGS and the deposition of IgM showed inferior renal outcomes at earlier time points in comparison with patients with negative IgM expression (P=0.022).CONCLUSIONS:We detected a positive correlation between IgM and C3 in secondary FSGS. The association between IgM deposition and worse renal outcome in secondary FSGS indicates that IgM may play a role in the progression of this disease.

Introduction: Renal biopsy database in the Department of Pathology, Dubrava University Hospital was created in 2003. The aim of the database is to accumulate computer-based pathologic data concerning all renal biopsy samples examined in the Department. With our database we can track the epidemiology of renal diseases diagnosed by biopsy: incidence and trends in epidemiology. Other than that it serves as an internal database for research programs in our and other departments. Design: We reviewed the database over a 7 years period (from April 2003 to December 2010) and performed the statistical analysis of our data including number of biopsies through years, patients gender, age, number of biopsies per patient, diagnoses and hospitals where the biopsy was performed. Results: During a 7 years period there were 2177 renal biopsy samples (1279 males ; 883 females, 15 donor kidneys, patient’s age range 1-85 years ; median 51). Biopsies came from 11 croatian hospitals, mostly from Dubrava and Merkur University Hospital Zagreb. Six biopsy samples came from Mostar University Hospital. The number of biopsies rapidly grew over the years (from 34 biopsies in 2003. to 542 in 2010.). There were 1289 native kidney biopsies ; out of these the most common diagnoses were IgA nephropathy (223), focal segmental glomerulosclerosis (180) and membranous glomerulonephritis (100). There were 888 renal allograft biopsies and among them the most common diagnoses were acute cellular rejection and acute tubular injury. Conclusion: To our knowledge around 80% of renal biopsy samples from Croatia are examined in our department. We can say that our results reflect the epidemiology of renal diseases requiring biopsy in almost whole Croatia.

Background. Prolonged cold ischemia (CI) is a risk factor for the development of delayed graft function that predicts reduced 5-year kidney transplant survival. CI results in caspase-3 activation, tubular injury, and apoptosis. Autophagy, a highly conserved pathway that permits recycling of nutrients within the cell during stress, is linked to apoptosis. We hypothesized that CI during kidney preservation would induce autophagy. We sought to determine apoptosis and autophagic flux in CI. Methods. Autophagic flux and apoptosis were examined in kidneys of wild-type and green fluorescent protein (GFP)-microtubule-associated protein1 light chain 3 (LC3) transgenic mice that were subjected to 48 hr of CI. Autophagic flux was determined by performing experiments with and without bafilomycin A1. Results. CI alone significantly increased the number of apoptotic cells/hpf, caspase-3/7 activity, and protein expression of autophagy markers LC3 II and autophagy-related protein 5. To determine the effect of inhibiting autophagic flux on apoptosis, kidneys of wild-type and GFP-LC3 transgenic mice were subjected to 48 hr of CI in the presence of lysosomal inhibitor bafilomycin A1. The combination of CI and bafilomycin A1 suppressed autophagic flux and significantly reduced the number of apoptotic cells/hpf, caspase-3/7 activity, LC3 II (both by immunoblot and in GFP-LC3 transgenic mice), and autophagy-related protein 5 protein expression. Conclusion. In summary, we have shown that autophagy and autophagic flux are reduced in cold ischemic kidneys treated with bafilomycin A1. Reduced autophagy and autophagic flux were associated with a significant reduction in apoptotic cell death, which may provide a therapeutic rationale for including bafilomycin A1 in University of Wisconsin solution during organ preservation.

Background. We have previously shown that cold ischemia (CI) results in massive increases in caspase-3 activity, tubular apoptosis, and brush border injury (BBI) in mouse kidneys. During hibernation, the 13-lined ground squirrel (GS) cycles through repeated CI during torpor, followed by warm ischemia/reperfusion (WI) during interbout arousal (IBA). We sought to determine whether CI and WI during hibernation caused caspase-3 activation, tubular apoptosis, acute tubular necrosis, or BBI, and reduced renal function. We also determined whether protection was dependent on the stage of hibernation. Methods. Radiotelemeters were implanted in 1-year-old GS, and core body temperature was remotely monitored. GS kidneys at various stages of hibernation were subjected to ex vivo CI. Results. Tubular apoptosis was not detected and caspase-3-like activity was not different between hibernating and summer kidneys. Despite prolonged CI followed by WI and reperfusion, acute tubular necrosis and apoptosis did not occur in hibernating kidneys. BBI was absent in torpid kidneys but significantly increased in IBA kidneys and associated with an increase in caspase-3-like activity, suggesting that IBA kidneys are more susceptible to injury than summer or torpid kidneys. Renal function and urine concentrating ability diminished during torpor but returned during IBA. Conclusions. Despite BBI, IBA kidneys clear serum creatinine and concentrate urine. Kidneys from both summer and hibernating animals tolerated ex vivo CI, confirming that protection from apoptotic and necrotic cell death is independent of the stage of hibernation. An understanding of how renal protection occurs during hibernation may help in understanding the pathophysiology of delayed graft function.

Inflammation contributes to the pathogenesis of acute kidney injury (AKI). IL-33 is a proinflammatory cytokine, but its role in AKI is unknown. Here we observed increased protein expression of full-length IL-33 in the kidney following induction of AKI with cisplatin. To determine whether IL-33 promotes injury, we administered soluble ST2 (sST2), a fusion protein that neutralizes IL-33 activity by acting as a decoy receptor. Compared with cisplatin-induced AKI in untreated mice, mice treated with sST2 had fewer CD4 T cells infiltrate the kidney, lower serum creatinine, and reduced acute tubular necrosis (ATN) and apoptosis. In contrast, administration of recombinant IL-33 (rIL-33) exacerbated cisplatin-induced AKI, measured by an increase in CD4 T cell infiltration, serum creatinine, ATN, and apoptosis; this did not occur in CD4-deficient mice, suggesting that CD4 T cells mediate the injurious effect of IL-33. Wildtype mice that received cisplatin and rIL-33 also had higher levels of the proinflammatory chemokine CXCL1, which CD T cells produce, in the kidney compared with CD4-deficient mice. Mice deficient in the CXCL1 receptor also had lower serum creatinine, ATN, and apoptosis than wildtype mice following cisplatin-induced AKI. Taken together, IL-33 promotes AKI through CD4 T cell-mediated production of CXCL1. These data suggest that inhibiting IL-33 or CXCL1 may have therapeutic potential in AKI.

BACKGROUND:Donation after cardiac death (DCD) kidneys suffer a high incidence of delayed graft function attributable to warm ischemia and cold ischemia (CI). Neither the mechanism of injury nor type of cell death has been described. Clinical studies suggest that perfusion storage (PS) of DCD kidneys may reduce injury although the mechanism of protection is unknown. In a porcine model of DCD, we hypothesized that DCD kidneys have increased caspase-1 due to warm ischemia (WI) and increased caspase-3 and apoptosis due to CI. METHODS:Male Yorkshire pigs subjected to cardiac death were perfused with cold University of Wisconsin solution. The perfused kidneys were removed and stored in cold University of Wisconsin solution for 24 hr. Kidney biopsies were obtained before cardiac death and at 0 and 24 hr of CI. RESULTS:There was an increase in caspase-1 activity due to WI before cold preservation. CI was associated with a massive increase in apoptosis, caspase-3/7 activity, and caspase-3 protein expression. Next, we hypothesized that PS would protect against apoptosis. We compared DCD kidneys subjected to static versus PS for 24 hr. PS significantly reduced proximal tubular apoptosis and was associated with increased B-cell lymphoma-extra large, and hypoxia-inducible transcription factor-1α. CONCLUSIONS:These findings suggest that in DCD kidneys, WI preferentially activates caspase-1, whereas CI activates caspase-3 and causes apoptosis. PS may protect DCD kidneys through activation of antiapoptotic pathways involving B-cell lymphoma-extra large and hypoxia-inducible transcription factor-1α.

Childhood infection with polyomaviruses leads to a life-long latent infection of renal and urinary tract epithelia. Replication in the reno-urinary epithelium is associated with viral cytopathic changes such as nuclear inclusions and decoy cells. During the 2005-2009 period, cytological urine analysis was performed in 154 samples (94 male and 60 female) from patients with kidney transplantation (n = 19), simultaneous pancreas-kidney transplantation (SPKT) (n = 9) and simultaneous kidney and liver transplantation (n = 2). Urine samples were analyzed monthly following transplantation according to the protocol. The period from transplantation to the first occurrence of decoy cells in the urine and the period of decoy cell persistence in the urine were assessed. The presence of decoy cells (< 10 and > 10 decoy cells) and red blood cells (< 20 E, 20-100 E and > 100 E) per cytospin smear was semiquantitatively determined, along with analysis of inflammatory cells (neutrophilic granulocytes) and fungi. In patients with decoy cells detected, their sensitivity, specificity, and negative and positive predictive value for BK virus nephropathy were calculated. Correlation of the study parameters was estimated by use of Kruskal-Wallis test (Statistica 7.1, StatSoft Inc., Tulsa, USA). Decoy cells were found in 30 patients (20 male and 10 female), age median 40 (range 16-69) years, at a mean of day 115 (range day 5-747) post transplantation, whereas their presence was recorded for a mean of 141 (range 77-771) days. Immunohistochemical staining of kidney biopsy sample for polyomavirus (SV40 large T-antigen) yielded positive reaction in 2/30 (7%) patients. Erythrocyturia was present in 29/30 patients with decoy cells. The number of decoy cells per cytospin smear generally ranged less than 10 in 25/30 patients, whereas more than 10 decoy cells per cytospin smear were only recorded in 5/30 patients. Immunohistochemistry produced positive finding for BK virus in one patient with SPKT and simultaneous kidney and liver transplantation each, which was statistically significantly more common as compared with patients with kidney transplantation alone (p = 0.0244). Immunohistochemical positivity for BK virus was more significant in cases with more than 10 decoy cells detected in cytospin smear (p = 0.013). In BK nephropathy, the finding of urinary decoy cells showed a 100% sensitivity, 84% specificity, 100% negative predictive value and 6% positive predictive value. BK virus nephropathy remains a significant post transplantation complication.