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Simple Summary: Cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss is a key factor in diagnosing meningiomas as Central Nervous System (CNS) WHO grade 3 tumors. Typically, detecting this gene loss involves costly and complex techniques like sequencing or FISH. However, the MTAP gene, which is located near CDKN2A/B on the same chromosome, may provide a more accessible way to detect these losses through a simpler, more affordable test called immunohistochemistry (IHC). This study explored different concentrations and antibodies for MTAP and p16 across two institutions to evaluate their potential as surrogate markers for CDKN2A/B loss. The results showed that p16 expression varied and did not align with either MTAP expression or CDKN2A FISH results. This study suggests that IHC for MTAP is a promising, cost-effective method for identifying high-grade meningiomas, offering a quicker and less expensive alternative to existing techniques. Background: Homozygous cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss is one of the parameters that support the designation of meningiomas as Central Nervous System (CNS) WHO grade 3 tumors. Evaluation of CDKN2A/B by sequencing or Fluorescence in situ hybridization (FISH) is costly and not always readily accessible. An immunohistochemistry (IHC)-based marker for the evaluation of CDKN2A/B loss would provide faster results at a lower cost. Methods: This retrospective study included patients diagnosed with meningioma at our institution between 2016 and 2019. Archival tumor tissue was used for analysis. MTAP immunohistochemistry (IHC) was performed at various dilutions (1:1200, 1:400, 1:200, 1:100) using two different antibodies, and p16 IHC was conducted simultaneously. These analyses were carried out at two different institutions. To determine the sensitivity and specificity of MTAP and p16 as surrogate markers for CDKN2A/B loss, CDKN2A FISH was utilized as the gold standard. Results: Overall, 46/49 tumors showed strong MTAP staining (94%) at institution 1, and 44/49 (90%) showed either faint positive or positive results at institution 2. One grade 3 meningioma that demonstrated homozygous CDKN2A loss by FISH also showed loss of MTAP expression by IHC. One grade 2 meningioma showed regional CDKN2A loss by FISH and variable MTAP expression under different IHC conditions. MTAP expression evaluation was superior at a dilution of 1:100 with the Abnova Anti-MTAP Monoclonal antibody. Conclusions: P16 expression was variable and did not correlate with either MTAP expression or CDKN2A FISH results. MTAP IHC is a promising surrogate marker for the evaluation of CDKN2A status in meningiomas. [ABSTRACT FROM AUTHOR]

The establishment of immunotherapy applying immune checkpoint inhibitors (ICI) has provided an important new option for the treatment of solid malignant diseases. However, different tumor entities show dramatically different responses to this therapy. BCC responds worse to anti-PD-1 ICIs as compared to cSCC. Differential immune checkpoint expression could explain this discrepancy and, therefore, the aim of this study was to analyze activating and inhibitory immune checkpoints in cSCC and BCC tissues. Tissue microarrays of the invasive front as well as the tumor core of BCC and cSCC samples were used to evaluate PD-1, PD-L1, CD28, and CD86 expression and their topographic distribution profiles by chromogenic immunohistochemistry. QuPath was used to determine the labeling index. The expression of PD-1, PD-L1, and CD28 was significantly higher in both the tumor core and the invasive front of cSCC samples as compared to BCC (p < 0.001). In addition, the ratios of PD-L1/CD86 (p < 0.001) and CD28/CD86 (p < 0.001) were significantly higher in cSCC. The invasive front of both tumor entities showed higher expression levels of all immune markers compared to the tumor core in both tumor entities. The significantly higher expression of PD-1, PD-L1, and CD28 in cSCC, along with the predominance of the inhibitory ligand PD-L1 as compared to the activating CD86 in cSCC, provide a potential explanation for the better objective response rates to anti-PD-1 immunotherapy as compared to BCC. Furthermore, the predominant site of interaction between the immune system and the tumor was within the invasive front in both tumor types. [ABSTRACT FROM AUTHOR]

CRNDE is an oncogene expressed as a long non-coding RNA. However, our team previously reported that the CRNDE gene also encodes a micropeptide, CRNDEP. The amino acid sequence of CRNDEP has recently been revealed by other researchers, too. This study aimed to investigate genetic alterations within the CRNDEP-coding region of the CRNDE gene, methylation profiling of this gene, and CRNDEP expression analysis. All investigations were performed on clinical material from patients with ovarian tumors of diverse aggressiveness. We found that CRNDEP levels were significantly elevated in highly aggressive tumors compared to benign neoplasms. Consistently, a high level of this micropeptide was a negative, independent, prognostic, and predictive factor in high-grade ovarian cancer (hgOvCa) patients. The cancer-promoting role of CRNDE(P), shown in our recent study, was also supported by genetic and epigenetic results obtained herein, revealing no CRNDEP-disrupting mutations in any clinical sample. Moreover, in borderline ovarian tumors (BOTS), but not in ovarian cancers, the presence of a single nucleotide polymorphism in CRNDE, rs115515594, significantly increased the risk of recurrence. Consistently, in BOTS only, the same genetic variant was highly overrepresented compared to healthy individuals. We also discovered that hypomethylation of CRNDE is associated with increased aggressiveness of ovarian tumors. Accordingly, hypomethylation of this gene's promoter/first exon correlated with hgOvCa resistance to chemotherapy, but only in specimens with accumulation of the TP53 tumor suppressor protein. Taken together, these results contribute to a better understanding of the role of CRNDE(P) in tumorigenesis and potentially may lead to improvements in screening, diagnosis, and treatment of ovarian neoplasms. [ABSTRACT FROM AUTHOR]

Bone metastasis has been reported in up to 70% of patients with advanced breast cancer. A total of 55.76% of skeletal metastases in women were derived from breast cancer. However, patients with bone metastasis from an occult primary breast cancer are a rare subset of patients. Here, we present the case of a 38-year-old woman who had sternum pain for 4 months. A whole-body PET-CT scan revealed that the FDG uptake of both the sternum and internal mammary node was significantly increased. The final diagnosis of occult breast cancer was established by immunohistochemical (IHC) staining, which is of great significance for identifying the origin of a metastatic tumor despite no visualized lesions of mammary glands. [ABSTRACT FROM AUTHOR]

Background: Timely reporting of molecular biomarkers is critical in guiding optimal treatment decisions in patients with advanced non-small cell lung carcinoma (NSCLC). Any delays along the tissue or treatment pathway may be associated with suboptimal treatment/outcomes and a reduced quality of life. For many centres, biomarkers are tested off-site. Methods: A retrospective chart review of 123 patients with advanced NSCLC seen between 1 June 2021 and 30 June 2022 was conducted. With a focus on core biomarkers (PD L1, EGFR, and ALK), the outcome variables were as follows: total turnaround time (total TAT), divided into pre-laboratory, laboratory, and post-laboratory time intervals, as well as time to treatment decision (TOTD) and time to optimal systemic therapy decision (TOTSD). Results: At first consult, only 20.3% of patients had all core biomarker results available. The median total TAT was significantly longer for non-squamous (non-SCC) than squamous cell carcinoma (SCC) specimens (36.5 versus 22 days, p < 0.001). The median pre-laboratory time for the entire cohort was 5 calendar days. The median laboratory testing time was greater for non-SCC compared to the SCC specimens (23 versus 12 days, p < 0.001). The median time from consult to TOTD was 19 calendar days for the entire cohort. Conclusions: This study emphasizes the need for the expansion of regional resources to meet the clinical needs of advanced NSCLC patients treated at a regional cancer centre which uses an off-site molecular laboratory. [ABSTRACT FROM AUTHOR]

Squamous cell carcinoma of the head and neck (HNSCC) is a malignant cancer disease in humans and animals. There is ample evidence that the high plasticity of cancer cells, i.e., their ability to switch from an epithelial to a mesenchymal, endothelial, and stem cell-like phenotype, chiefly contributes to progression, metastasis, and multidrug resistance of human HNSCCs. In feline HNSCC, the field of cancer cell plasticity is still unexplored. In this study, fourteen feline HNSCCs with a known feline papillomavirus (FPV) infection status were subjected to histopathological grading and subsequent screening for expression of epithelial, mesenchymal, and stem cell markers by immunohistochemistry (IHC) and immunofluorescence staining (IF). Irrespective of the FPV infection status, all tumors except one corresponded to high-grade, invasive lesions and concurrently expressed epithelial (keratins, E-cadherin, β-catenin) and mesenchymal (vimentin, N-cadherin, CD146) proteins. This finding is indicative for partial epithelial-mesenchymal transition (pEMT) events in the lesions, as similarly described for human HNSCCs. IF double staining revealed the presence of CD44/CD271 double-positive cells notably within the tumors' invasive fronts that likely correspond to cancer stem cells. Taken together, the obtained findings suggest that feline HNSCCs closely resemble their human counterparts with respect to tumor cell plasticity. [ABSTRACT FROM AUTHOR]

Simple Summary: The dysregulated impact of WNT4 and estrogens disrupts uterine homeostasis and function, potentially escalating the risk of endometrial cancer. This research sought to assess aberrations in WNT4 gene expression and WNT4 protein immunoreactivity in clinical samples of endometrial cancer, with a focus on tumor characteristics, clinicopathological association, and estrogen dependence. Analysis of a large patient cohort provided additional support for studying WNT4 expression. Results provide a valuable basis for further investigation of the molecular mechanism of estrogen-induced endometrial carcinogenesis. Background: The inappropriate action of WNT4 and estrogens affects uterine homeostasis and function, and may lead to endometrial cancer (EC). Objective: The aim was to evaluate the alterations of WNT4 gene expression and WNT4 protein immunoreactivity (Ir) in EC, considering tumor characteristics, the clinicopathological association and estrogen dependence. Methods: WNT4 mRNA levels were compared between benign (control) endometrium (n = 8) and endometroid EC (EEC) and non-endometroid EC (non-EEC) samples (n = 28) using the real-time PCR technique. The WNT4-Ir and ERα-Ir were evaluated by immunohistochemistry (IHC). WNT4 mRNA gene and WNT4-Ir were correlated with clinicopathological and blood morphological parameters. Overall survival (OS) was assessed. The bioanalysis was utilized to study WNT4 expression in large patient cohort (n = 549). Results: WNT4 gene expression was decreased in EC samples (specifically in EEC but not in non-EEC) compared to the control. The WNT4 gene expression was also decreased in EC samples categorized by the tumor characteristics. There was no statistical difference in WNT4-Ir or ERα-Ir between the control and EC. There was no correlation between OS and WNT4 gene expression and WNT4-Ir. Bioanalysis showed that WNT4 and ESR1 gene expression alterations tended to be mutually exclusive. An alteration in WNT4 expression was found in different histological tumor types in a large group of EC patients. Conclusions: There is a great need to evaluate the molecular background of EC. Our study suggests that the WNT4 gene has the potential to be a marker of functional estrogen signaling in EEC. [ABSTRACT FROM AUTHOR]

Cancer markers are measurable molecules in the blood or tissue that are produced by tumor cells or immune cells in response to cancer progression. They play an important role in clinical diagnosis, prognosis, and anti-drug monitoring. Although DNA, RNA, and even physical images have been used, proteins continue to be the most common marker. There are currently no specific markers for lung cancer. Metastatic lung cancer, particularly non-small-cell lung cancer (NSCLC), is one of the most common causes of death. SFPQ, YY1, RTN4, RICTOR, LARP6, and HELLS are expressed at higher levels in cells from NSCLC than in control or cells from inflammatory diseases. SFPQ shows the most difference between the three cell types. Furthermore, the cytoplasmic isoform of SFPQ is only found in advanced cancers. We have developed ELISAs to detect SFPQ and the long and short isoforms. Evidence has shown that the short isoform exists primarily in cancers. Furthermore, immunocytometry studies and IHC analysis have revealed that SFPQ levels are consistent with ELISA results. In addition, enhanced DNA methylation in the SFPQ gene may facilitate the SFPQ expression differences between control and cancer cells. Considering this, elevated SFPQ level and the isoform location could serve as a cancer diagnostic and prognostic marker. [ABSTRACT FROM AUTHOR]

The accurate management of testicular germ cell tumors (TGCTs) depends on identifying the individual histological tumor components. Currently available data on protein expression in TGCTs are limited. The human protein atlas (HPA) is a comprehensive resource presenting the expression and localization of proteins across tissue types and diseases. In this study, we have compared the data from the HPA with our in-house immunohistochemistry on core TGCT diagnostic genes to test reliability and potential biomarker genes. We have compared the protein expression of 15 genes in TGCT patients and non-neoplastic testicles with the data from the HPA. Protein expression was converted into diagnostic positivity. Our study discovered discrepancies in three of the six core TGCT diagnostic genes, POU5F1, KIT and SOX17 in HPA. DPPA3, CALCA and TDGF1 were presented as potential novel TGCT biomarkers. MGMT was confirmed while RASSF1 and PRSS21 were identified as biomarkers of healthy testicular tissue. Finally, SALL4, SOX17, RASSF1 and PRSS21 dysregulation in the surrounding testicular tissue with complete preserved spermatogenesis of TGCT patients was detected, a potential early sign of neoplastic transformation. We highlight the importance of a multidisciplinary collaborative approach to fully understand the protein landscape of human testis and its pathologies. [ABSTRACT FROM AUTHOR]

Brain aging has been correlated with high metallothionein I-II (MT-I/II) expression, iron and zinc dyshomeostasis, and Aβ deposition in humans and experimental animals. In the present study, iron and zinc accumulation, the expression of MT-I/II and Aβ42, and their potential association with aging in the feline brain were assessed. Tissue sections from the temporal and frontal grey (GM) and white (WM) matter, hippocampus, thalamus, striatum, cerebellum, and dentate nucleus were examined histochemically for the presence of age-related histopathological lesions and iron deposits and distribution. We found, using a modified Perl's/DAB method, two types of iron plaques that showed age-dependent accumulation in the temporal GM and WM and the thalamus, along with the age-dependent increment in cerebellar-myelin-associated iron. We also demonstrated an age-dependent increase in MT-I/II immunoreactivity in the feline brain. In cats over 7 years old, Aβ immunoreactivity was detected in vessel walls and neuronal somata; extracellular Aβ deposits were also evident. Interestingly, Aβ-positive astrocytes were also observed in certain cases. ICP-MS analysis of brain content regarding iron and zinc concentrations showed no statistically significant association with age, but a mild increase in iron with age was noticed, while zinc levels were found to be higher in the Mature and Senior groups. Our findings reinforce the suggestion that cats could serve as a dependable natural animal model for brain aging and neurodegeneration; thus, they should be further investigated on the basis of metal ion concentration changes and their effects on aging. [ABSTRACT FROM AUTHOR]

In search of novel potential drug candidates that could be used as treatments or prophylactics for memory impairment, an aporphine alkaloid magnoflorine (MAG) isolated from the root of Berberis vulgaris was proven to exhibit beneficial anti-amnestic properties. Its effects on immunoreactivity to parvalbumin in the mouse hippocampus were assessed together with a study on its safety and concentration in the brain and plasma. For this purpose, four experimental groups were created: the MAG10 group—treated with 10 mg MAG/kg b.w. i.p., the MAG20 group—treated with 20 mg MAG/kg b.w. i.p., the MAG50 group—treated with 50 mg MAG/kg b.w. i.p., and a control group—injected with saline i.p. at a volume corresponding to their weight. Our results indicated that the hippocampal fields CA1–CA3 were characterized by an elevated number of parvalbumin-immunoreactive neurons (PV-IR) and nerve fibers in mice at the doses of 10 and 20 mg/kg b.w. (i.p.). No significant changes to the levels of IL-1β, IL-6 or TNF-α were observed for the above two doses; however, the administration of 50 mg/kg b.w. i.p. caused a statistically significant elevation of IL-6, IL-1beta plasma levels and an insignificant raise in the TNF-alpha value. The HPLC–MS analysis showed that the alkaloid's content in the brain structures in the group treated with 50 mg/kg b.w. did not increase proportionally with the administered dose. The obtained results show that MAG is able to influence the immunoreactivity to PV-IR in hippocampal neurons and might act as a neuroprotective compound. [ABSTRACT FROM AUTHOR]

Simple Summary: In swine, the diagnosis of enteric diseases is challenging due to simultaneous presence of one or more microbic agents sharing similar clinical signs and pathological lesions. Therefore, the study of microscopic lesions is crucial in establishing the role of pathogens as causative agents in cases of co-infections; this can be confirmed by demonstrating agent–lesion co-localization. Although PCR is more sensitive than IHC, the latter provides a positive result only when the amount of antigen is significant, and therefore is likely the cause of the enteric pathology. Lawsonia intracellularis is the aetiologic agent responsible for Porcine proliferative enteropathy. However, it is complex to determine whether this intracellular bacterium is the cause of clinical disease due to its high prevalence in the field. In this study, we investigate the agreement between PCR and IHC results for L. intracellularis evaluation, and the infection and co-infection by porcine circovirus type 2 and L. intracellularis in the ilea of pigs presented with suspected proliferative enteropathy in Italy. The objective of this study was to employ a diagnostic algorithm, which involves detecting positive farms by stool PCR followed by PCR and histology/immunohistochemistry on ileum samples, for diagnosing Lawsonia intracellularis proliferative enteritis in Northern Italy. The primary aim was to examine the relationship between the gold standard of L. intracellularis diagnostics, namely histology and immunohistochemistry, and PCR in acute and chronic cases of L. intracellularis enteritides. An additional goal was to investigate the coinfection of L. intracellularis with porcine circovirus type 2 (PCV2). Twenty-eight ileum samples, including four from acute cases and 24 from chronic cases, were collected. PCR yielded positive results in 19 cases (four acute and 15 chronic cases). In comparison, immunohistochemistry was positive in 16 cases (four acute and 12 chronic cases), with an observed agreement of 89%. The findings suggest that performing the two tests in series can increase the specificity of the causal diagnosis. PCR may be used as a screening tool to identify the presence of the microorganism, and only positive cases will be examined by histology and immunohistochemistry to confirm the causative role of L. intracellularis. Co-infection with PCV2 was demonstrate in two out of four acute cases and in two out of 24 chronic cases, providing further evidence to support the hypothesis that when the infection starts with ubiquitous pathogens such as L. intracellularis, it may boost the possibility of PCV2 replication, especially in acute cases. As a result, this may trigger a transition from subclinical to clinical forms of PCV2 disease. [ABSTRACT FROM AUTHOR]

Patients with head and neck squamous cell carcinoma (HNSCC) continue to have a rather poor prognosis. Treatment-related comorbidities have negative impacts on their quality of life. TRIM21 is a cytosolic E3 ubiquitin ligase that was initially described as an autoantigen in autoimmune diseases and later associated with the intracellular antiviral response. Here, we investigated the role of TRIM21 as a biomarker candidate for HNSCC in predicting tumor progression and patient survival. We analyzed TRIM21 expression and its association with clinical-pathological parameters in our HNSCC cohort using immunohistochemistry. Our HNSCC cohort included samples from 419 patients consisting of primary tumors (n = 337), lymph node metastases (n = 156), recurrent tumors (n = 54) and distant metastases (n = 16). We found that cytoplasmic TRIM21 expression was associated with the infiltration of immune cells into primary tumors. In addition, TRIM21 expression was significantly higher in primary tumors than in lymph node metastases, and increased TRIM21 expression was correlated with shorter progression-free survival in HNSCC patients. These results suggest that TRIM21 could be a new biomarker for progression-free survival. [ABSTRACT FROM AUTHOR]

Breast cancer is one of the common malignant tumors in women. It seriously endangers women's life and health. The human epidermal growth factor receptor 2 (HER2) protein is responsible for the division and growth of healthy breast cells. The overexpression of the HER2 protein is generally evaluated by immunohistochemistry (IHC). The IHC evaluation criteria mainly includes three indexes: staining intensity, circumferential membrane staining pattern, and proportion of positive cells. Manually scoring HER2 IHC images is an error-prone, variable, and time-consuming work. To solve these problems, this study proposes an automated predictive method for scoring whole-slide images (WSI) of HER2 slides based on a deep learning network. A total of 95 HER2 pathological slides from September 2021 to December 2021 were included. The average patch level precision and f1 score were 95.77% and 83.09%, respectively. The overall accuracy of automated scoring for slide-level classification was 97.9%. The proposed method showed excellent specificity for all IHC 0 and 3+ slides and most 1+ and 2+ slides. The evaluation effect of the integrated method is better than the effect of using the staining result only. [ABSTRACT FROM AUTHOR]

Simple Summary: The role of the blood–brain barrier in intracranial cancer treatment by vascular-disrupting agents remains unclear. Intraindividual comparison of vascular shutdown by a craniofacial metastasis model was performed, on the basis of in vivo MRI imaging, postmortem NanoCT and pathology analyses. The differential VDA therapeutic effects between intracranial and extracranial tumors were observed, with stronger vascular shutdown in the extracranial tumor. In vivo multiparametric MRI may serve as a predictor for the efficacy of VDAs on craniofacial tumors. Vascular-disrupting agents (VDAs) have shown a preliminary anti-cancer effect in extracranial tumors; however, the therapeutic potential of VDAs in intracranial metastatic lesions remains unclear. Simultaneous intracranial and extracranial tumors were induced by the implantation of rhabdomyosarcoma in 15 WAG/Rij rats. Pre-treatment characterizations were performed at a 3.0 T clinical magnet including a T2 relaxation map, T1 relaxation map, diffusion-weighted imaging (DWI), and perfusion-weighted imaging (PWI). Shortly afterward, a VDA was intravenously given and MRI scans at 1 h, 8 h, and 24 h after treatment were performed. In vivo findings were further confirmed by postmortem angiography and histopathology staining with H&E, Ki67, and CD31. Before VDA treatment, better perfusion (AUC30: 0.067 vs. 0.058, p < 0.05) and AUC300 value (0.193 vs. 0.063, p < 0.001) were observed in extracranial lesions, compared with intracranial lesions. After VDA treatment, more significant and persistent perfusion deficiency measured by PWI (AUC30: 0.067 vs. 0.008, p < 0.0001) and a T1 map (T1 ratio: 0.429 vs. 0.587, p < 0.05) were observed in extracranial tumors, in contrast to the intracranial tumor (AUC30: 0.058 vs. 0.049, p > 0.05, T1 ratio: 0.497 vs. 0.625, p < 0.05). Additionally, significant changes in the T2 value and apparent diffusion coefficient (ADC) value were observed in extracranial lesions, instead of intracranial lesions. Postmortem angiography and pathology showed a significantly larger H&E-stained area of necrosis (86.2% vs. 18.3%, p < 0.0001), lower CD31 level (42.7% vs. 54.3%, p < 0.05), and lower Ki67 level (12.2% vs. 32.3%, p < 0.01) in extracranial tumors, compared with intracranial lesions. The BBB functioned as a barrier against the delivery of VDA into intracranial tumors and multiparametric MRI may predict the efficacy of VDAs on craniofacial tumors. [ABSTRACT FROM AUTHOR]

Simple Summary: Diseases emerging from wildlife represent a growing public health issue. Cervids share many pathogens with domestic species and humans, representing useful spontaneous models to evaluate host-pathogen balance. Histology and immunohistochemistry can help in fully understanding the pathogenesis of infection in these species, but few studies have been conducted to characterize immune cell markers. This study highlights that lymphocytes and macrophagic subsets in roe and fallow deer lymphoid tissue can be identified by a panel of commercial antibodies developed against humans. A description of the main immune cell distribution was provided. These results may support future investigations on immune cell response and pathogenesis in roe and fallow deer diseases. Roe and Fallow deer are common wild ruminants widely distributed in Italy. Infectious diseases of these species can potentially pose health risks to domestic animals and humans. However, few studies have been conducted in which immune system cells in these species were phenotyped. The aims of this study were to determine the cross-reactivity of a wide anti-human panel of commercial antibodies on formalin-fixed and paraffin-embedded (FFPE) samples and to describe the distribution of roe and fallow deer main immune cell subsets in the lymph nodes and spleen. Twenty retromandibular lymph nodes (RLNs) and spleen samples were collected from 10 roe deer and 10 fallow deer and were tested by a panel of 12 commercial anti-human antibodies. The CD79a, CD20, CD3, Iba-1, MAC387, and AM-3K antibodies were successfully labeled cells in cervine tissue, while the Foxp3 and the CD68 did not show suitable immunostaining. This study supplies the first immunohistochemical description of immune cell subpopulations in non-pathological spleen and RLNs from roe and fallow deer and provides an easily repeatable manual IHC protocol to immunolocalize cervine B-, T-cells, and macrophages subsets in FFPE tissue samples. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer biomarkers are biomolecules synthesized by the tumor cell and have a role in early detection of cancer and determining its stage and progression. HSP27 and Menin are biomolecules involved in tumorigenesis and cancer progression; they are overexpressed in many cancers and could be useful as biomarkers. However, the overexpression of HSP27 and Menin in the serum of prostate cancer (PCa) patients and the correlation they have with their tissue expression, as well as their correlation with the clinical data, have not been explored yet. In this study, we first investigated the correlations between HSP27 and Menin in PCa patients, and then we examined the signification of HSP27/Menin in the diagnosis and prognosis of PCa using ROC analysis. Our results suggest that HSP27/Menin are dependent biomarkers in aggressive PCa and are significantly associated with poor prognosis; they could be used in the diagnosis and clinical decision making of individual PCa patients. The screening of PCa is based on two tests, the total PSA test and the rectal examination. However, PSA is not specific for PCa stage confirmation, leading in false positive result and involving PCa over-diagnosis and over-treatment. HSP27 and Menin have been found to be overexpressed in a wide range of human cancers. Recent studies showed how HSP27 interacts with and stabilizes Menin to lead PCa progression and treatment resistance. The purpose of our study was to evaluate the correlation of HSP27 and Menin molecular expression, and their prognosis value in PCa with respect to clinicopathological features. Elisa was employed to measure serum HSP27 and Menin concentrations in 73 PCa patients and 80 healthy individuals. Immunohistochemistry (IHC) was used to determine HSP27 and Menin tissue expression in 57 tumors and 4 Benign Prostatic Hyperplasia (BPH) tissues. Serum HSP27 expression correlated with its tissue expression in all PCa patients, whereas serum Menin expression correlated only with tissue expression in aggressive PCa patients. Moreover, the results showed a positive correlation between HSP27 and Menin either in serum (r = 0.269; p = 0.021) or in tissue (r = 0.561; p < 0.0001). In aggressive PCa, serum expression of HSP27 and Menin was positively correlated (r = 0.664; R = 0.441; p = 0.001). The correlation between HSP27 and Menin expression in tissue was found only in patients with aggressive PCa (r = 0.606; R = 0.367; p = 0.004). Statistical analysis showed that the expression of both biomarkers was positively correlated with the hormone resistance or sensitivity, tumor aggressiveness, metastasis, Gleason Score, death and did not significantly correlate with age and PSA. Survival was illustrated by Kaplan–Meier curves; increased HSP27 and Menin expression correlated with shorter survival of PCa patients (p = 0.001 and p < 0.0001, respectively). Accuracy in predicting aggressiveness was quantified by the Area Under the Curve (AUC) of Receiver Operating Characteristic (ROC). We demonstrated that the combination of HSP27/Menin was statistically greater than PSA; it achieved an AUC of 0.824 (95% CI, 0.730–0.918; p < 0.0001). However, HSP27/Menin/PSA combination decreased the diagnostic value with an AUC of 0.569 (95% CI, 0.428–0.710; p = 0.645). Our work suggests the potential role of HSP27/Menin as diagnostic and prognostic biomarkers. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is one of the most common protozoan parasites and is widely present in all warm-blooded animals. Although clinical disease is uncommon, some species, including ring-tailed lemurs (Lemur catta), have been found to develop acute and lethal toxoplasmosis. The aim of this study was to describe the pathologic, immunohistochemical, serological, and molecular findings of an outbreak of fatal toxoplasmosis in three captive ring-tailed lemurs in Central Italy in 2009. The animals died acutely within few days. The necropsy was immediately performed; necrotic lesions in the spleen, liver, and kidney, as well as interstitial pneumonia, were found histologically. All animals had high titers of anti–T. gondii-specific antibodies (1:1280 IgM and 1:640 IgG) according to a modified agglutination test (MAT) and immunohistochemistry showed scattered tachyzoites in the target organs. Diagnosis was confirmed by PCR and clonal type II was identified. In addition, the seven co-habiting lemurs were seronegative. This paper reports the first outbreak of acute disseminated toxoplasmosis in captive ring-tailed lemurs in Italy. These findings confirm the high susceptibility of this endangered species to toxoplasma infection, which may be considered a further threat to captive population viability. [ABSTRACT FROM AUTHOR]

Simple Summary: Around 20 years ago, genomic profiling of breast carcinomas identified tumor subtypes with clinical implications and opened the door for a better understanding of breast cancer biology. The commercialization of multigene tests had a significant impact on clinical practice, and yet, controversy exists as to which methodology is best to inform the choice of therapy and existing recommendations are inconsistent and often driven by cost-effectiveness. Here we report data from a cohort of breast cancer patients in which pathological and molecular subtyping are directly compared in a clinical setting. The findings show that some patients with genomic low-risk tumors could receive unnecessary systemic therapy if only following the classical clinical parameters, while others could remain under-treated. This study suggests that to design precise treatment regimens for patients with early breast cancer, the conventional clinicopathological classification should be complemented with the robust prognostic information provided by molecular subtyping. Precise prognosis is crucial for selection of adjuvant therapy in breast cancer. Molecular subtyping is increasingly used to complement immunohistochemical and pathological classification and to predict recurrence. This study compares both outcomes in a clinical setting. Molecular subtyping (MammaPrint®, TargetPrint®, and BluePrint®) and pathological classification data were compared in a cohort of 143 breast cancer patients. High risk clinical factors were defined by a value of the proliferation factor Ki67 equal or higher than 14% and/or high histological grade. The results from molecular classification were considered as reference. Core needle biopsies were found to be comparable to surgery samples for molecular classification. Discrepancies were found between molecular and pathological subtyping of the samples, including misclassification of HER2-positive tumors and the identification of a significant percentage of genomic high risk T1N0 tumors. In addition, 20% of clinical low-risk tumors showed genomic high risk, while clinical high-risk samples included 42% of cases with genomic low risk. According to pathological subtyping, a considerable number of breast cancer patients would not receive the appropriate systemic therapy. Our findings support the need to determine the molecular subtype of invasive breast tumors to improve breast cancer management. [ABSTRACT FROM AUTHOR]

(1) Background: This study aimed to detect feline coronavirus (FCoV) and characterize spike (S) gene mutation profiles in cats suffering from diseases other than feline infectious peritonitis (FIP) using commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) and reevaluating results by sequencing. (2) Methods: In 87 cats in which FIP was excluded by histopathology and immunohistochemistry, FCoV 7b gene and S gene mutation RT-qPCR was performed prospectively on incisional biopsies and fine-needle aspirates of different organs, body fluids, and feces. Samples positive for S gene mutations or mixed FCoV underwent sequencing. (3) Results: In 21/87 cats, FCoV RNA was detectable. S gene mutations were detected by commercial RT-qPCR (and a diagnostic algorithm that was used at the time of sample submission) in at least one sample in 14/21 cats (66.7%), with only mutated FCoV in 2/21, only mixed in 1/21, and different results in 11/21 cats; in the remaining 7/21 cats, RNA load was too low to differentiate. However, sequencing of 8 tissue samples and 8 fecal samples of 9 cats did not confirm mutated FCoV in any of the FCoV RNA-positive cats without FIP. (4) Conclusions: Sequencing results did not confirm results of the commercial S gene mutation RT-qPCR. [ABSTRACT FROM AUTHOR]

In intermediate risk hormone receptor (HR) positive, HER2 negative breast cancer (BC), the decision regarding adjuvant chemotherapy might be facilitated by multigene expression tests. In all, 142 intermediate risk BCs were investigated using the PAM50-based multigene expression test Prosigna® in a prospective multicentric study. In 119/142 cases, Prosigna® molecular subtyping was compared with local and two central (C1 and C6) molecular-like subtypes relying on both immunohistochemistry (IHC; HRs, HER2, Ki-67) and IHC + tumor grade (IHC+G) subtyping. According to local IHC, 35.4% were Luminal A-like and 64.6% Luminal B-like subtypes (local IHC+G subtype: 31.9% Luminal A-like; 68.1% Luminal B-like). In contrast to local and C1 subtyping, C6 classified >2/3 of cases as Luminal A-like. Pairwise agreement between Prosigna® subtyping and molecular-like subtypes was fair to moderate depending on molecular-like subtyping method and center. The best agreement was observed between Prosigna® (53.8% Luminal A; 44.5% Luminal B) and C1 surrogate subtyping (Cohen's kappa = 0.455). Adjuvant chemotherapy was suggested to 44.2% and 88.6% of Prosigna® Luminal A and Luminal B cases, respectively. Out of all Luminal A-like cases (locally IHC/IHC+G subtyping), adjuvant chemotherapy was recommended if Prosigna® testing classified as Prosigna® Luminal A at high / intermediate risk or upgraded to Prosigna® Luminal B. [ABSTRACT FROM AUTHOR]

Papillary renal cell carcinoma (PRCC) is defined by the WHO 2022 classification as a malignant tumor derived from the renal tubular epithelium. However, the WHO 2016 classification subdivided PRCC into two types, with type 1 PRCC showing papillae covered by a single layer of neoplastic cells, and type II PRCC, which can show multiple types of histologies and is more aggressive. The WHO 2022 classification eliminated the subcategorization of PRCC. Here, we present a histopathological case study with a 4-year follow-up diagnosed in 2018 as type I PRCC (WHO 2016) with intra-pyelocalyceal growth pattern in a 59-year-old male patient with a history of Type II diabetes mellitus, left-sided renal–ureteral lithiasis, and benign hypertrophy of the prostate. Microscopically the tumor was composed of small cuboidal cells with inconspicuous nucleoli, arranged on a single layer of tubulo-papillary cores, and scant, foamy macrophages. The tumor had a non-infiltrative, expansive pyelocalyceal growth pattern. Immunohistochemically (IHC), the tumor cells were CK7-intense and diffusely positive, and stained granular for AMACR. Next-generation sequencing (NGS) was performed for the tumor and the normal adjacent tissue for in-depth pathological characterization. To our knowledge, this is the first reported case where a PRCC displays this unique intra-pyelocalyceal growth pattern, mimicking a urothelial cell carcinoma of the renal pelvis system. [ABSTRACT FROM AUTHOR]

The integration of the cultural/creative and tourism industries is one way to present different types of regional cultural heritage to the world. This study examined the coupling of intangible cultural heritage and cultural tourism development in Hunan, seeking a pattern for the development of the former under the latter. The study included analyzing the coupling between tourism resources and ICH and exploring modes of revitalizing or developing ICH in the context of cultural tourism. We used methods of GIS spatial technology, including nearest neighbor indexing, kernel density estimation, and queuing analysis, to estimate the kernel density of a buffer of points (high-speed railway stations, and 5A and 4A scenic spots) and lines (high-speed railways and highways). The results show (1) the overall spatial aggregation of Hunan's ICH resources; (2) how tourist attractions drive ICH resources, as illustrated by the coupling between the spatial distribution of ≥4A attractions and ICH; and (3) the pattern of high-speed road and railroad network development, featuring region-wide guidance for the development of Hunan's ICH resources. This research not only provides development patterns and concepts for the study of intangible cultural heritage and tourism development planning in Hunan, but also provides useful references for the combination of the two in other regions. [ABSTRACT FROM AUTHOR]

Cellular senescence (CS) is a major homeostatic biological process, which plays a key role in normal tissue development and provides protection from stressful cell insults. The role of CS in mammary-gland development and breast cancer is not well understood. While there is a lack of experimental data on the role of CS in the development of the pre-pubertal mammary gland, there is evidence for a biphasic senescence response in adult normal-mammary-epithelial cells, where the bypass of the first senescence barrier (M0) seems to be a key step in the development of premalignant lesions, with genetic abnormalities that resemble in situ breast carcinoma. Further, there is accumulating evidence for the role of cellular senescence in breast-cancer response, regarding treatment and patient outcome. Here, we review the current literature on cellular senescence, in epithelial-mammary cells, breast-cancer cells, and breast-tumor-microenvironment-resident cells. Furthermore, we discuss its putative role in breast-cancer response, regarding treatment and disease progression. In addition, we provide preliminary evidence of CS in breast-cancer-microenvironment cells, such as tumor-associated fibroblasts and tumor-infiltrating lymphocytes, by employing the novel GL13 lipofuscin stain, as a marker of cellular senescence. [ABSTRACT FROM AUTHOR]

MALDI imaging is a novel technique with which to study the pathophysiologies of diseases. Advancements in the field of metabolomics and lipidomics have been instrumental in mapping the signaling pathways involved in various diseases, such as cancer and neurodegenerative diseases (Parkinson's). MALDI imaging is flexible and can handle many sample types. Researchers primarily use either formalin-fixed paraffin-embedded (FFPE) or fresh frozen tissue samples to answer their scientific questions. FFPE samples allow for easy long-term storage, but the requirement for extensive sample processing may limit the ability to provide a clear picture of metabolite distribution in biological tissue. Frozen samples require less handling, but present logistical challenges for collection and storage. A few studies, mostly focused on cancer cell lines, have directly compared the results of MALDI imaging using these two tissue fixation approaches. Herein, we directly compared FFPE and fresh frozen sample preparation for murine skin samples, and performed detailed pathway analysis to understand how differences in processing impact MALDI results from otherwise identical tissues. Our results indicate that FFPE and fresh frozen methods differ significantly in the putative identified metabolite content and distribution. The fixation methods shared only 2037 metabolites in positive mode and only 4079 metabolites in negative ion mode. However, both fixation approaches allowed for downstream fluorescent staining, which may save time and resources for samples that are clinically precious. This work represents a direct comparison of the impacts of the two main tissue processing methods on subsequent MALDI-MSI. While our results are similar to previous work in cancer tissue, they provide novel insights for those using MALDI-MSI in skin. [ABSTRACT FROM AUTHOR]

Simple Summary: Around 80% of skin cancer deaths are due to melanoma. An accurate prognosis of melanoma clinical behavior from primary tumors is important for therapeutic patient management, currently based on histopathological features. The aim of our retrospective study was to investigate the clinical significance of IGF2BP3 mRNA and protein expression in melanoma progression and to evaluate which quantification method, RT-qPCR or immunohistochemistry, provides a more reliable prognostic value of IGF2BP3 expression in primary tumors. We found that IGF2BP3 mRNA expression correlated better with clinicopathologic melanoma features than the corresponding proteins and that patients with higher IGF2BP3 mRNA levels were at more risk for earlier development of metastasis, confirming its impact on melanoma survival. Our findings support the use of IGF2BP3 mRNA levels as an independent prognostic biomarker and the implementation of its RT-qPCR analysis for routine melanoma assessment, even for the earliest stages, to improve melanoma clinical outcomes and individualized treatment. Screening for prognostic biomarkers is crucial for clinical melanoma management. Insulin-like growth factor-II mRNA-binding protein 3 (IGF2BP3) has emerged as a potential melanoma diagnostic and prognostic biomarker. It is commonly tested by immunohistochemistry (IHC). Our study retrospectively examines IGF2BP3 mRNA and protein expression in primary melanomas, their correlation with clinicopathologic factors, clinical outcome, and selected miRNAs expression, and their efficiency in predicting melanoma progression and survival. RT-qPCR and IHC on IGF2BP3 expression were performed in 61 cryopreserved and 63 formalin-fixed paraffin-embedded primary melanomas, respectively, and correlated to clinicopathologic factors, distant metastasis-free survival (DMFS), and melanoma -specific survival (MSS). The correlation between RT-qPCR and IHC was significant but moderate. IGF2BP3 mRNA showed a stronger association with clinicopathologic factors (Breslow thickness, ulceration, mitosis rate, growth phase, development of metastasis, and melanoma-specific survival) than its protein counterpart. Interestingly, higher IGF2BP3 mRNA expression was detected in primary melanomas that further metastasized to distant sites and was an independent prognostic factor for the risk of unfavorable DMFS and MSS. RT-qPCR outperformed IHC in sensitivity and in predicting worse clinical outcomes. Therefore, RT-qPCR may successfully be implemented for routine IGF2BP3 assessing for the selection of melanoma patients with a higher risk of developing distant metastasis and dying of melanoma. [ABSTRACT FROM AUTHOR]

Simple Summary: Recurrent ovarian-cancer patients face low 5-year survival rates despite chemotherapy. Oncologists may choose from a variety of guideline-recommended second-line therapeutic options without knowing which one works best. Thus, therapy alterations and adjustments are often required. We analyzed the response that 30 tumor lesions had to certain treatments in our patient-derived ovarian-cancer spheroid model. In addition, we characterized samples by immunohistochemical staining for new druggable molecular targets. Our results might help in tailoring future therapies for individual patients with recurrent ovarian cancer. Recurrent ovarian-cancer patients face low 5-year survival rates despite chemotherapy. A variety of guideline-recommended second-line therapies are available, but they frequently result in trial-and-error treatment. Alterations and adjustments are common in the treatment of recurrent ovarian cancer. The drug response of 30 lesions obtained from 22 relapsed ovarian cancer patients to different chemotherapeutic and molecular agents was analyzed with the patient-derived ovarian-cancer spheroid model. The profile of druggable biomarkers was immunohistochemically assessed. The second-line combination therapy of carboplatin with gemcitabine was significantly superior to the combination of carboplatin with PEGylated liposomal doxorubicin (p < 0.0001) or paclitaxel (p = 0.0007). Except for treosulfan, all nonplatinum treatments tested showed a lesser effect on tumor spheroids compared to that of platinum-based therapies. Treosulfan showed the highest efficacy of all nonplatinum agents, with significant advantage over vinorelbine (p < 0.0001) and topotecan (p < 0.0001), the next best agents. The comparative testing of a variety of treatment options in the ovarian-cancer spheroid model resulted in the identification of more effective regimens for 30% of patients compared to guideline-recommended therapies. Recurrent cancers obtained from different patients revealed profound interpatient heterogeneity in the expression pattern of druggable protein biomarkers. In contrast, different lesions obtained from the same patient revealed a similar drug response and biomarker expression profile. Biological heterogeneity observed in recurrent ovarian cancers might explain the strong differences in the clinical drug response of these patients. Preclinical drug testing and biomarker profiling in the ovarian-cancer spheroid model might help in optimizing treatment management for individual patients. [ABSTRACT FROM AUTHOR]

Head and neck squamous cell carcinomas (HNSCC) are among the most common cancers worldwide and are associated with a poor prognosis for patients. Among HNSCC, those originating in the hypopharynx have the worst prognosis. The histone demethylase LSD1 has been shown to promote cancer initiation, progression, and relapse through various mechanisms and is upregulated in many cancer tissues. LSD1 physically interacts with SNAIL and is required for SNAIL mediated transcriptional repression. Previous studies of the prognostic value of LSD1 in HNSCC have been limited in their analysis of sub-sites, and a correlation between LSD1 and SNAIL has not been shown in HNSCC patient samples. Here we used a large, representative, and clinically well-characterized cohort of 339 HNSCC patients to investigate the co-expression of LSD1 and SNAIL and their prognostic value in all HNSCC using immunohistochemical staining. Elevated LSD1 expression correlated with advanced tumor stage and poor progression-free survival (PFS) in HNSCC originating in the hypopharynx. Overexpression of the transcription factor SNAIL independently correlated with worse overall survival (OS) and PFS in HNSCC in general and prominently in tumors of the hypopharynx. Furthermore, increased LSD1 expression significantly correlated with elevated SNAIL expression in patient samples. Therefore, the presented data implicates LSD1 and SNAIL as independent prognostic biomarkers. [ABSTRACT FROM AUTHOR]

ROS1 rearrangements are identified in 1–2% of lung adenocarcinoma cases, and reflex testing is guideline-recommended. We developed a decision model for population-based ROS1 testing from a Canadian public healthcare perspective to determine the strategy that optimized detection of true-positive (TP) cases while minimizing costs and turnaround time (TAT). Eight diagnostic strategies were compared, including reflex single gene testing via immunohistochemistry (IHC) screening, fluorescence in-situ hybridization (FISH), next-generation sequencing (NGS), and biomarker-informed (EGFR/ALK/KRAS wildtype) testing initiated by pathologists and clinicianinitiated strategies. Reflex IHC screening with FISH confirmation of positive cases yielded the best results for TAT, TP detection rate, and cost. IHC screening saved CAD 1,000,000 versus reflex FISH testing. NGS was the costliest reflex strategy. Biomarker-informed testing was cost-efficient but delayed TAT. Clinician-initiated testing was the least costly but resulted in long TAT and missed TP cases, highlighting the importance of reflex testing. Thus, reflex IHC screening for ROS1 with FISH confirmation provides a cost-efficient strategy with short TAT and maximizes the number of TP cases detected. [ABSTRACT FROM AUTHOR]

A mature teratoma is a germinal neoplasm that differentiates from embryonic multipotent cells into three germ layers. There may also be glandular tissue. The literature describes a total of 658 cases of ovarian neuroendocrine neoplasms, mainly in women over 40 years of age. The authors, together with a systemic review, present a case of a 16-year-old girl diagnosed with and treated for a neuroendocrine tumor. Case description: A 16-year-old girl visited the Paediatric Gynaecology Outpatient Clinic because of abdominal pains that intensified during menstruation. Standard painkillers and diastolic drugs were ineffective. An ultrasound examination revealed a large tumor with a heterogeneous structure in her right ovary. A sparing operation was carried out. During laparotomy, the lesion was enucleated, leaving healthy tissue. Histopathological examination revealed the typical features of teratoma, as well as the coexistence of a G1 neuroendocrine tumor. Immunohistochemical examination (IHC) showed the presence of markers characteristic for this type of tumor. The patient requires constant monitoring in the Endocrinology and Oncological Gynaecology Clinic. Conclusion: Tissue of neuroendocrine neoplasm within a teratoma is rare in this age group of patients; thus, there are currently no standards for long-term follow-up. This case adds to the body of evidence and demonstrates a possible good prognosis with non-aggressive behavior in G1 neuroendocrine tumors and teratomas in young patients. [ABSTRACT FROM AUTHOR]

Background and Objectives: Colorectal cancer (CRC) can be classified as mismatch-repairdeficient (dMMR) with high levels of microsatellite instability (MSI-H), or mismatch-repair-proficient (pMMR) and microsatellite stable (MSS). Approximately 15% of patients have microsatellite instability (MSI). MSI-H tumors are associated with a high mutation burden. Monoclonal antibodies that block immune checkpoints can induce long-term durable responses in some patients. Pembrolizumab is the first checkpoint inhibitor approved in the EU to treat dMMR-MSI-H metastatic CRC. Materials and Methods: Our study assesses the regional variability of MSI-H colorectal cancer tumors in Romania. Formalin-fixed, paraffin-embedded (FFPE) tissue blocks containing tumor samples from 90 patients diagnosed with colorectal cancer were collected from two tertiary referral Oncology Centers from Romania. Tissues were examined for the expression loss of MMR proteins (MLH1, PMS2, MSH2, MSH6) using immunohistochemistry or MSI status using polymerase chain reaction (PCR), respectively. Results: MSI-H was detected in 19 (21.1%) patients. MSI-H was located more in ascending colon (36.8% vs. 9.9%, p-value = 0.0039) and less in sigmoid (5.3% vs. 33.8%, p-value = 0.0136) than MSS patients. Most patients were stage II for MSI-H (42.1%) as well as for MSS (56.3%), with significant more G1 (40.9% vs. 15.8%, p-value = 0.0427) for MSS patients. Gender, N stage, and M stage were identified as significant prognostic factors in multivariate analysis. MSI status was not a statistically significant predictor neither in univariate analysis nor multivariate analysis. Conclusion: Considering the efficacy of PD-1 inhibitor in metastatic CRC with MSI-H or dMMR, and its recent approval in EU, it is increasingly important to understand the prevalence across tumor stage, histology, and demographics, since our study displayed higher regional MSI-H prevalence (21%) compared to the literature. [ABSTRACT FROM AUTHOR]

Background: To test the value of immunohistochemistry (IHC) staining in prostate biopsies for changes in biopsy results and its impact on treatment decision-making. Methods: Between January 2017–June 2020, all patients undergoing prostate biopsies were identified and evaluated regarding additional IHC staining for diagnostic purpose. Final pathologic results after radical prostatectomy (RP) were analyzed regarding the effect of IHC at biopsy. Results: Of 606 biopsies, 350 (58.7%) received additional IHC staining. Of those, prostate cancer (PCa) was found in 208 patients (59.4%); while in 142 patients (40.6%), PCa could be ruled out through IHC. IHC patients harbored significantly more often Gleason 6 in biopsy (p < 0.01) and less suspicious baseline characteristics than patients without IHC. Of 185 patients with positive IHC and PCa detection, IHC led to a change in biopsy results in 81 (43.8%) patients. Of these patients with changes in biopsy results due to IHC, 42 (51.9%) underwent RP with 59.5% harboring ≥pT3 and/or Gleason 7–10. Conclusions: Patients with IHC stains had less suspicious characteristics than patients without IHC. Moreover, in patients with positive IHC and PCa detection, a change in biopsy results was observed in >40%. Patients with changes in biopsy results partly underwent RP, in which 60% harbored significant PCa. [ABSTRACT FROM AUTHOR]

Breast cancer biology is heterogeneous and patients with the same diagnostic profile have markedly different clinical outcomes. Gene expression profiling and its surrogate IHC markers classified breast cancer into four molecular subtypes with different prognosis, targeted therapies, and/or clinical outcomes. The present study was conducted to investigate breast cancer subtypes among women in NDUTH Bayelsa state and compared them with similar studies in other populations of international and national origin. Archived formalin fixed paraffin embedded tissue blocks were retrieved from January 2010-December 2014 provided 20 cases of microscopically confirmed invasive ductal breast carcinoma that were evaluable for histology and IHC (ER, PR, HER2, and Cytokeratin 5/6) immunostain. All the patients were females and most of them were under 50 years at presentation. The molecular subtypes were luminal A (54.1%), luminal B (25.0%), HER2 (12.5%), basal-like (4.2%), and unclassified (4.2%). Triple negative carcinoma (basal-like and unclassified combined) was 4.2%, ER+ (65%) and (PR + 70%).Breast carcinoma in Bayelsa state women presents at a younger age. We note that the prevalence of the molecular subtypes of breast carcinoma appears to have variation in geographical distribution. The reasons for these differences could be technical, quality of fixation and processing, varying staining techniques, and different criteria in scoring and reporting. This study indicates that the majority of breast cancer in NDUTH lies at the two ends of the molecular spectrum, that is, luminal A (54.1%), sensitive to hormonal therapy and with a good prognosis, and “triple negative” (4.2%) not hormonally sensitive and with a poor prognosis. [ABSTRACT FROM AUTHOR]

Tissue microarray (TMA) methodology allows the concomitant analysis of hundreds of tissue specimens arrayed in the same manner on a recipient block. Subsequently, all samples can be processed under identical conditions, such as antigen retrieval procedure, reagent concentrations, incubation times with antibodies/probes, and escaping the inter-assays variability. Therefore, the use of TMA has revolutionized histopathology translational research projects and has become a tool very often used for putative biomarker investigations. TMAs are particularly relevant for large scale analysis of a defined disease entity. In the course of these exploratory studies, rare subpopulations can be discovered or identified. This can refer to subsets of patients with more particular phenotypic or genotypic disease with low incidence or to patients receiving a particular treatment. Such rare cohorts should be collected for more specific investigations at a later time, when, possibly, more samples of a rare identity will be available as well as more knowledge derived from concomitant, e.g., genetic, investigations will have been acquired. In this article we analyze for the first time the limits and opportunities to construct new TMA blocks using tissues from older available arrays and supplementary donor blocks. In summary, we describe the reasons and technical details for the construction of rare disease entities arrays. [ABSTRACT FROM AUTHOR]

Liver tumours are among the leading causes of cancer-related death worldwide and hepatocellular carcinoma (HCC) accounts for the vast majority of liver tumours. When detected at an early stage of disease, patients might still be eligible for surgical-based curative treatments. However, currently only small portion of HCC affected patients are diagnosed at an early stage. For late stage HCC no treatment option exists beside the multi-tyrosine kinase inhibitor Sorafenib. Thus new molecular targets and treatment options for HCC are urgently needed. Nevertheless, despite some improvements in diagnosis and patient management, the biology of liver tumour remains inadequately understood, mainly because these tumours have shown to harbour a highly complex genomic landscape. In addition, one major obstacle delaying the identification of new molecular targets in biomedical research is the necessity to validate them using a large collection of tissue specimens. Tissue microarray (TMA) technology allows the prompt molecular profiling of multiple tissue specimens and is therefore ideal to analyze presumptive candidate biomarkers in a fast an effective manner. The use of TMA has substantial benefits over standard techniques and represents a significant advancement in molecular pathology. For example, TMA technology reduces laboratory work, offers a high level of experimental uniformity and provides a judicious use of precious tissue. On the other hand, one potential limitation of using TMA is that the small cores sampled may not be representative of whole tumors. This issue is very critical in particularly heterogeneous cancers such as HCC. For liver focused studies, it is ideal to evaluate the staining patters of a determined marker over the structure of an entire acinus and to define staining in as many as possible anatomical regions. In this review we analyze the limits and opportunities offered by the usage of TMA technology in HCC research. In summary, TMA has revolutionized the histopathological analysis and will be of great help to further advance the knowledge in the field of hepatocarcinogenesis research. [ABSTRACT FROM AUTHOR]

Advances in spectral deconvolution technologies are rapidly enabling researchers to replace or enhance traditional epifluorescence microscopes with instruments capable of detecting numerous markers simultaneously in a multiplexed fashion. While significantly expediting sample throughput and elucidating sample information, this technology is limited by the spectral width of common fluorescence reporters. Semiconductor nanocrystals (NC's) are very bright, narrow band fluorescence emitters with great potential for multiplexed fluorescence detection, however the availability of NC's with facile attachment chemistries to targeting molecules has been a severe limitation to the advancement of NC technology in applications such as immunocytochemistry and immunohistochemistry. Here we report the development of simple, yet novel attachment chemistries for antibodies onto NC's and demonstrate how spectral deconvolution technology enables the multiplexed detection of 5 distinct NC-antibody conjugates with fluorescence emission wavelengths separated by as little as 20 nm. [ABSTRACT FROM AUTHOR]

An adult female osprey (Pandion haliaetus) was found weak and unable to fly in Auburn, Alabama in August 2019. The bird was captured and submitted to the Southeastern Raptor Center of the Auburn University College of Veterinary Medicine for evaluation. On presentation, the bird was thin with a body condition score of approximately 1.5 out of 5. The bird died during the examination and was submitted for necropsy. At the necropsy, there was a severe loss of muscle mass over the body, and the keel was prominent. The liver and spleen were moderately enlarged with pale tan to red foci randomly scattered throughout the parenchyma. A histopathologic observation revealed multifocal to coalescing areas of necrosis and hemorrhage with intralesional protozoans in the liver, spleen, lungs, kidney, sciatic nerve, esophagus, cerebrum, heart, and proventriculus. Immunohistochemistry using anti-Toxoplasma gondii-specific antibodies showed a strong positive labeling of the parasite. Semi-nested PCR, specific for the B1 gene of T. gondii, successfully identified T. gondii. This is the first confirmed case of T. gondii infection in an osprey. [ABSTRACT FROM AUTHOR]

In order to determine the frequency of SARS-CoV-2 placental and fetal involvements, we analyzed placentas of 197 women positive for infection at delivery and fetal tissues in cases of pregnancy loss in women positive by SARS-CoV-2 PCR (N = 2) and COVID-19 serology (N = 4), using in situ hybridization (ISH), immunohistochemistry (IHC) and, in selected cases, RT-PCR of tissue homogenates. The virus was identified in situ, accompanied by intervillositis, in 2 of 197 placentas (1.02%). In three more cases, SARS-CoV-2 was detected by tissue PCR without in situ localization and placental inflammation. There were no maternal mortality or association of placental infection with the clinical severity of COVID-19. All tested neonates born to SARS-CoV-2-positive women (N = 172) were negative for the virus. There were three pregnancy losses among 197 infected women and in two cases available fetal tissues were negative for SARS-CoV-2. In one of four fetal autopsies performed in women with positive COVID-19 serology, the mother-to-child transmission (MTCT) could be inferred based on positive SARS-CoV-2 nucleocapsid IHC in fetal pulmonary endothelium. Placental involvement by SARS-CoV-2 is rare, but may be underestimated due to its transient nature. MTCT is even rarer, supporting the protective role of placenta in SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo. [ABSTRACT FROM AUTHOR]

Immune-checkpoint inhibitors (ICIs) play a key role in the treatment of advanced stage colorectal cancer (CRC) patients featuring a deficient DNA mismatch repair (dMMR) system or a high microsatellite instability (MSI-H) profile. However, beyond the established role in CRC patients, ICIs have highly proven efficacy in other solid tumors featuring MSI-H/dMMR status represented by endometrial, gastric, ovarian, prostatic, and pancreatic carcinomas (EC, GC, OC, PrC, and PaC). Our aim was to compare the concordance rates among the Idylla™ MSI test, TapeStation 4200, and immunohistochemical (IHC) analysis in assessing MSI-H/dMMR status in EC, GC, OC, PrC, and PaC patients. The Sanger sequencing-based Titano MSI test was used in discordant cases. One hundred and eighty-five cases (n = 40 PrC, n = 39 GC, n = 38 OC, n = 35 PaC, and n = 33 EC) were retrospectively selected. MMR protein expression was evaluated by IHC. After DNA quality and quantity evaluations, the IdyllaTM and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Remarkably, compared to IHC, the Idylla™ platform achieved a global concordance rate of 94.5% (154/163) for the microsatellite stable (MSS)/proficient MMR (pMMR) cases and 77.3% (17/22) for the MSI-H/dMMR cases. Similarly, a global concordance rate of 91.4% (149/163) and 68.2% (15/22) for MSS/pMMR and MSI-H/dMMR cases was also identified between IHC and the TapeStation 4200 microfluidic system. In addition, a global concordance of 93.1% (148/159) and 69.2% (18/26) for MSS/pMMR and MSI-H/dMMR cases was observed between the Idylla™ and TapeStation 4200 platforms. Discordant cases were analyzed using the Titano MSI kit. Overall, our data pinpointed a central role for molecular techniques in the diagnostic evaluation of dMMR/MSI-H status not only in CRC patients but also in other types of solid tumors. [ABSTRACT FROM AUTHOR]

To date, SARS-CoV-2 pandemic has caused more than 188 million infections and 4.06 million deaths worldwide. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein has been regarded as an important target for vaccine and therapeutics development because it plays a key role in binding the human cell receptor ACE2 that is required for viral entry. However, it is not easy to detect RBD in Western blot using polyclonal antibody, suggesting that RBD may form a complicated conformation under native condition and bear rare linear epitope. So far, no linear epitope on RBD is reported. Thus, a monoclonal antibody (mAb) that recognizes linear epitope on RBD will become valuable. In the present study, an RBD-specific rabbit antibody named 9E1 was isolated from peripheral blood mononuclear cells (PBMC) of immunized rabbit by RBD-specific single B cell sorting and mapped to a highly conserved linear epitope within twelve amino acids 480CNGVEGFNCYFP491 on RBD. 9E1 works well in Western blot on S protein and immunohistochemistry on the SARS-CoV-2 infected tissue sections. The results demonstrated that 9E1 can be used as a useful tool for pathological and functional studies of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Simple Summary: Advances have been made in the study of NSCLC tumors using in vivo models, such as patient-derived xenografts (PDXs). However, the number of PDX models that can represent the heterogeneity of NSCLC between different individuals is still limited. We successfully established nine PDX mice, from which lung adenocarcinoma tumors bearing the KRAS-G12C mutation were the most frequently grafted. We show that the most aggressive tumors have a greater implantation capacity, and the success of their implantation is indicative of a poor prognosis. By using H-score to quantify cell proliferation and mesenchymal markers, we show that PDX tumors evolved towards a more proliferative and mesenchymal phenotype associated with higher protein levels of Ki67, vimentin, and ezrin, suggesting that the evaluation of their combined expression could be used as a prognostic marker to study disease progression. These PDX models provide a valuable platform for NSCLC translational research. Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. The high mortality is very often a consequence of its late diagnosis when the cancer is already locally advanced or has disseminated. Advances in the study of NSCLC tumors have been achieved by using in vivo models, such as patient-derived xenografts. Apart from drug screening, this approach may also be useful for study of the biology of the tumors. In the present study, surgically resected primary lung cancer samples (n = 33) were implanted in immunodeficient mice, and nine were engrafted successfully, including seven adenocarcinomas, one squamous-cell carcinoma, and one large-cell carcinoma. ADC tumors bearing the KRAS-G12C mutation were the most frequently engrafted in our PDX collection. Protein expression of vimentin, ezrin, and Ki67 were evaluated in NSCLC primary tumors and during serial transplantation by immunohistochemistry, using H-score. Our data indicated a more suitable environment for solid adenocarcinoma, compared to other lung tumor subtypes, to grow and preserve its architecture in mice, and a correlation between higher vimentin and ezrin expression in solid adenocarcinomas. A correlation between high vimentin expression and lung adenocarcinoma tumors bearing KRAS-G12C mutation was also observed. In addition, tumor evolution towards more proliferative and mesenchymal phenotypes was already observed in early PDX tumor passages. These PDX models provide a valuable platform for biomarker discovery and drug screening against tumor growth and EMT for lung cancer translational research. [ABSTRACT FROM AUTHOR]

Emerging evidence supports a significant association between human cytomegalovirus (HCMV) and human malignancies, suggesting HCMV as a human oncomodulatory virus. HCMV gene products are found in >90% of breast cancer tumors and seem to be correlated with more aggressive disease. The definitive diagnosis of HCMV relies on identification of virus inclusions and/or viral proteins by different techniques including immunohistochemical staining. In order to reduce biases and improve clinical value of HCMV diagnostics in oncological pathology, automation of the procedure is needed and this was the purpose of this study. Tumor specimens from 115 patients treated for primary breast cancer at Akershus University Hospital in Norway were available for the validation of the staining method in this retrospective study. We demonstrate that our method is highly sensitive and delivers excellent reproducibility for staining of HCMV late antigen (LA), which makes this method useful for future routine diagnostics and scientific applications. [ABSTRACT FROM AUTHOR]

The development of a compliant neural probe is necessary to achieve chronic implantation with minimal signal loss. Although fiber-based neural probes fabricated by the thermal drawing process have been proposed as a solution, their long-term effect on the brain has not been thoroughly investigated. Here, we examined the mechanical interaction of thermally drawn fiber implants with neural tissue through computational and histological analyses. Specifically, finite element analysis and immunohistochemistry were conducted to evaluate the biocompatibility of various fiber implants made with different base materials (steel, silica, polycarbonate, and hydrogel). Moreover, the effects of the coefficient of friction and geometric factors including aspect ratio and the shape of the cross-section on the strain were investigated with the finite element model. As a result, we observed that the fiber implants fabricated with extremely softer material such as hydrogel exhibited significantly lower strain distribution and elicited a reduced immune response. In addition, the implants with higher coefficient of friction (COF) and/or circular cross-sections showed a lower strain distribution and smaller critical volume. This work suggests the materials and design factors that need to be carefully considered to develop future fiber-based neural probes to minimize mechanical invasiveness. [ABSTRACT FROM AUTHOR]

Simple Summary: The DNA sequencing of cancer provides information about specific genetic changes that could help with treatment decisions. Tumors from different tissues change over time and acquire new genetic changes particularly with treatment. Our study analyzed the gene changes of 171 advanced cancer patients. We predicted that tumors gain new mutations but that TP53 mutations (guardian of the human genome) are conserved as the tumor progresses from primary to metastatic sites and across tissue types. We analyzed the primary and metastatic site gene changes in 25 tissue types and conducted in-depth analysis of colon and lung cancer sites for substantial changes. TP53 site specific mutations were different across tissue types and suggest different molecular changes. Other genetic changes that occur together with TP53 as drivers collectively alter how cells respond to signals which are important to tumor treatments. Molecular profiling with next generation sequencing (NGS) delivers key information on mutant gene sequences, copy number alterations, gene-fusions, and with immunohistochemistry (IHC), is a valuable tool in clinical decision making for patients entering investigational agent trials. Our objective was to elucidate mutational profiles from primary versus metastatic sites from advanced cancer patients to guide rational therapy. All phase I patients (n = 203) with advanced cancer were profiled by commercially available NGS platforms. The samples were annotated by histology, primary and metastatic site, biopsy site, gene mutations, mutation count/gene, and mutant TP53. A molecular profile of each patient was categorized into common and unique mutations, signaling pathways for each profile and TP53 mutations mapped to 3D-structure of p53 bound to DNA and pre/post therapy molecular response. Of the 171 patients analyzed, 145 had genetic alterations from primary and metastatic sites. The predominant histology was adenocarcinoma followed by squamous cell carcinoma, carcinoma of unknown primary site (CUPS), and melanoma. Of 790 unique mutations, TP53 is the most common followed by APC, KRAS, PIK3CA, ATM, PTEN, NOTCH1, BRCA2, BRAF, KMT2D, LRP1B, and CDKN2A. TP53 was found in most metastatic sites and appears to be a key driver of acquired drug resistance. We highlight examples of acquired mutational profiles pre-/post- targeted therapy in multiple tumor types with a menu of potential targeted agents. Conclusion: The mutational profiling of primary and metastatic lesions in cancer patients provides an opportunity to identify TP53 driver 'pathways' that may predict for drug sensitivity/resistance and guide rational drug combinations in clinical trials. [ABSTRACT FROM AUTHOR]

Simple Summary: Renal collecting duct carcinoma (CDC) is rare, but very aggressive, variant histology of kidney cancers. Besides surgery, the other therapeutic options, such as pharmacological or radiation therapy, have a poor impact on survival. Therefore, there is an urgent need to identify novel targets that can open up new avenues for alternative treatments. From this perspective, the aim of our study was to assess the HER2 protein expression by immunohistochemistry (IHC) and the gene copy number by fluorescence in-situ hybridization (FISH) in a cohort of 26 CDC. According to the 2018 ASCO/CAP guidelines, 2/26 CDC cases (8%) were HER2-positive. The HER2 protein is a well-established target of anti-HER2 mAbs or kinase inhibitors already used for breast and gastric cancer treatment. Thus, this study provides evidence that supports future biomarker-driven clinical trials that could address the lack of therapy, which is still an unmet clinical need for CDC patients. Collecting duct carcinoma (CDC) is rare and aggressive histology of kidney cancers. Although different therapeutic approaches have been tested, the 2-year survival remains very poor. Since CDC exhibits overlapping features with urothelial carcinoma, the analysis of shared molecular alterations could provide new insights into the understanding of this rare disease and also therapeutic options. We collected 26 CDC cases, and we assessed HER2 protein expression by immunohistochemistry (IHC) and gene amplification by fluorescence in-situ hybridization (FISH) according to 2018 ASCO/CAP HER2-testing recommendations. Six out of twenty-six (23%) tumors showed HER2 positive staining. In particular, 3+ score was present in 2/6 cases (33%), 2+ in 3/6 cases (50%) and 1+ in 1/6 cases (17%). The 6 HER2+ tumors were also analyzed by FISH to assess gene copy number. One out of six CDC with IHC 3+ was also HER2 amplified, showing an average HER2 copy number ≥4.0 (10.85) and a HER2/CEP17 ratio ≥ (5.63), while the 5/6 cases were HER2 negative. Based on the 2018 ASCO/CAP guidelines overall, 2/26 CDC cases (8%) were HER2+. The present study provides evidence for testing, in future studies, HER2 to assess its clinical value as a novel target for the treatment of this highly malignant cancer. [ABSTRACT FROM AUTHOR]