Assessment of RAS-RAF-MAPK Pathway Mutation Status in Healthy Skin, Benign Nevi, and Cutaneous Melanomas: Pilot Study Using Droplet Digital PCR.

In the present study, we employed the ddPCR and IHC techniques to assess the prevalence and roles of RAS and RAF mutations in a small batch of melanoma (n = 22), benign moles (n = 15), and normal skin samples (n = 15). Mutational screening revealed the coexistence of BRAF and NRAS mutations in melanomas and nevi and the occurrence of NRAS G12/G13 variants in healthy skin. All investigated nevi had driver mutations in the BRAF or NRAS genes and elevated p16 protein expression, indicating cell cycle arrest despite an increased mutational burden. BRAF V600 mutations were identified in 54% of melanomas, and NRAS G12/G13 mutations in 50%. The BRAF mutations were associated with the Breslow index (BI) (p = 0.029) and TIL infiltration (p = 0.027), whereas the NRAS mutations correlated with the BI (p = 0.01) and the mitotic index (p = 0.04). Here, we demonstrate that the "young" ddPCR technology is as effective as a CE-IVD marked real-time PCR method for detecting BRAF V600 hotspot mutations in tumor biopsies and recommend it for extended use in clinical settings. Moreover, ddPCR was able to detect low-frequency hotspot mutations, such as NRAS G12/G13, in our tissue specimens, which makes it a promising tool for investigating the mutational landscape of sun-damaged skin, benign nevi, and melanomas in more extensive clinical studies.

The Value of ER∝ in the Prognosis of GH- and PRL-Secreting PitNETs: Clinicopathological Correlations.

Pituitary neuroendocrine tumors (PitNETs) are divided into multiple histological subtypes, which determine their clinical and biological variable behavior. Despite their benign evolution, in some cases, prolactin (PRL) and growth hormone (GH)-secreting PitNETs may have aggressive behavior. In this study, we investigated the potential predictive role of ER∝, alongside the clinicopathological classification of PitNETs (tumor diameter, tumor type, and tumor grade). A retrospective study was conducted with 32 consecutive cases of PRL- and mixed GH- and PRL-secreting PitNETs (5 patients with prolactinomas and 27 with acromegaly, among them, 7 patients with GH- and PRL- co-secretion) who underwent transsphenoidal intervention. Tumor specimens were histologically and immunohistochemical examined: anterior pituitary hormones, ki-67 labeling index, CAM 5.2, and ER∝; ER∝ expression was correlated with basal PRL levels at diagnosis (rho = 0.60, p < 0.01) and postoperative PRL levels (rho = 0.58, p < 0.001). In our study, the ER∝ intensity score was lower in female patients. Postoperative maximal tumor diameter correlated with Knosp grade (p = 0.02); CAM 5.2 pattern (densely/sparsely granulated/mixed densely and sparsely granulated) was correlated with postoperative PRL level (p = 0.002), and with ki-67 (p < 0.001). The IGF1 level at diagnosis was correlated with the postoperative GH nadir value in the oral glucose tolerance test (OGTT) (rho = 0.52, p < 0.05). Also, basal PRL level at diagnosis was correlated with postoperative tumor diameter (p = 0.63, p < 0.001). At univariate logistic regression, GH nadir in OGTT test at diagnostic, IGF1, gender, and invasion were independent predictors of remission for mixed GH- and PRL-secreting Pit-NETs; ER∝ can be used as a prognostic marker and loss of ER∝ expression should be considered a sign of lower differentiation and a likely indicator of poor prognosis. A sex-related difference can be considered in the evolution and prognosis of these tumors, but further studies are needed to confirm this hypothesis.

Histological findings in skeletal muscle of SARS-CoV2 infected patient.

Autopsies represent medical procedures through which the causes of patients' deaths are determined or, through tissue sampling and microscopic examination of slides in usual stains or special tests, one can offer the basis for understanding the physiopathological mechanisms that contribute to the patients' death Histological findings of tissue samples from patients who have died of COVID-19 have been mainly orientated to lung, heart, liver, kidney damage with a small percent of them following other organs, but none has, to our knowledge, studied skeletal muscle.

Innovative array-based assay for omics pattern in melanoma.

Cutaneous melanoma remains a major health issue and still an important challenge for research. Thus, omics complex evaluation can provide a more specific molecular classification for this heterogeneous disease. Complex omics analysis based on genomic and proteomic microarrays can identify disease markers that prognosticate disease evolution or can monitor therapies efficacy. Among the technologies that gained momentum in the last years, array-based comparative genomic hybridization offered the possibility to analyze chromosomal numerical aberrations within cutaneous melanomas providing important support for molecular classification of melanoma tumors. This technology can identify new chromosomal alterations and discover new deregulated melanoma genes that can be further used as therapy targets. Integrating genetic profiling with clinical and pathological parameters would lead to seminal improvements in diagnosis, prognosis, and therapy.

Challenges in the Diagnosis and Management of Giant Porokeratosis: A Case Report.

Porokeratosis encompasses a diverse group of dermatoses, both acquired and genetic, marked by a keratinization disorder. Porokeratosis of Mibelli (PKM) presents as solitary plaques or multiple papules/macules with central atrophy and raised hyperkeratotic borders. Here, we present a case of giant porokeratosis (GPK), a rare form often considered a morphological variant of PKM, with unique clinical and histopathological aspects. Our case involves a 29-year-old patient with a 15 × 10 cm irregular plaque on the dorsal aspect of the right hand. The patient was previously prescribed various topical treatments (retinoids, calcineurin inhibitors, and combinations of corticosteroids with vitamin D3 analogs) and systemic retinoids without improvement before presenting to our department. Due to the high risk of neoplastic transformation and the unavailability of imiquimod, the patient was recommended topical 5-fluorouracil treatment. The trajectory of the lesion under treatment revealed a favorable evolution, and the patient was subjected to regular monitoring every three months to assess the ongoing progress. Recognizing GPK as a high-risk variant is crucial for dermatologists, and it requires a personalized approach. Regular monitoring is advised to detect potential malignant transformations promptly. Future research holds promise for diagnostic advancements, refined treatment modalities, and a deeper understanding of the molecular mechanisms underlying malignancy in porokeratosis.

MMP1, MMP9, MMP11 and MMP13 in melanoma and its metastasis - key points in understanding the mechanisms and celerity of tumor dissemination.

BACKGROUND: Matrix metalloproteinase (MMP)1, MMP9, MMP11, and MMP13 are overexpressed in malignant melanoma (MM), being associated with tumor invasive phase, metastases, and more aggressive neoplastic phenotypes. AIM: The main objective of the current study was to correlate the expression of the MMPs with the evolution of MM toward distant metastasis. PATIENTS, MATERIALS AND METHODS: We designed a retrospective cohort study, including 13 patients with metastatic MM. Data concerning age, sex, localization of the primary lesion and metastasis, and histological and immunohistochemical features (intensity of expression and percent of positive cells for MMPs) were statistically processed. RESULTS: The time between the diagnosis of primitive melanoma and the diagnosis of metastasis ranged between 0 and 73 months, with a mean value of 18.3 months. The metastases rich in MMP1- and MMP9-positive cells occurred earlier than the metastases with low levels of positive cells. The mean period until metastasis was shorter for the MMP1-expressing tumors than the ones without MMP1 expression. MMP13 expression in the tumor and its metastasis was significantly linked with the time until the metastasis occurrence. CONCLUSIONS: This study emphasizes the roles of MMP1, MMP9, and MMP13 in the process of metastasis in melanoma and the opportunity to use them as therapeutic targets and surveillance molecules.

Chronic sclerosing sialadenitis of the bilateral submandibular glands in childhood - a diagnostic dilemma.

Chronic sclerosing sialadenitis (CSS), currently included in the group of immunoglobulin G4 (IgG4)-related diseases, is an under-recognized inflammatory lesion that afflicts mostly the submandibular gland of 40-60 years adults. To our knowledge, only one case of CSS located in the submandibular gland has been reported in childhood to date. We present a case of CSS in a 5-year-old male child. He presented with bilateral submandibular swellings that clinically resembled discrete lumps, suspected to be tumors. The completely resected tumors composed predominantly of dense lymphoplasmacytic inflammatory infiltrate rich in IgG4-positive cells [77-90 IgG(+) cells per high-power field; IgG4(+)∕IgG(+) cells ratio of 42.77%]. We discuss the peculiarities of this case, and we also review the literature on CSS.

A New Method of Artificial-Intelligence-Based Automatic Identification of Lymphovascular Invasion in Urothelial Carcinomas.

The presence of lymphovascular invasion (LVI) in urothelial carcinoma (UC) is a poor prognostic finding. This is difficult to identify on routine hematoxylin-eosin (H&E)-stained slides, but considering the costs and time required for examination, immunohistochemical stains for the endothelium are not the recommended diagnostic protocol. We developed an AI-based automated method for LVI identification on H&E-stained slides. We selected two separate groups of UC patients with transurethral resection specimens. Group A had 105 patients (100 with UC; 5 with cystitis); group B had 55 patients (all with high-grade UC; D2-40 and CD34 immunohistochemical stains performed on each block). All the group A slides and 52 H&E cases from group B showing LVI using immunohistochemistry were scanned using an Aperio GT450 automatic scanner. We performed a pixel-per-pixel semantic segmentation of selected areas, and we trained InternImage to identify several classes. The DiceCoefficient and Intersection-over-Union scores for LVI detection using our method were 0.77 and 0.52, respectively. The pathologists' H&E-based evaluation in group B revealed 89.65% specificity, 42.30% sensitivity, 67.27% accuracy, and an F1 score of 0.55, which is much lower than the algorithm's DCC of 0.77. Our model outlines LVI on H&E-stained-slides more effectively than human examiners; thus, it proves a valuable tool for pathologists.

Direct immunofluorescence on hair follicles--present and future perspectives.

Direct immunofluorescence (DIF) is an important tool for evaluating bullous autoimmune and connective tissue disorders. We report 21 cases of pemphigus vulgaris, bullous pemphigoid and lupus erythematosus that were investigated by performing DIF on scalp hair follicles. The study was done using a simplified technique of preparing the hairs for DIF testing. The anagen hairs tested positive in pemphigus vulgaris patients while the telogen hairs were negative. In bullous pemphigoid and lupus erythematosus cases hair DIF presented negative results.Hair DIF has the potential of taking the place of skin or mucosal DIF in pemphigus patients if performed on anagen hair follicles. The technique used to perform hair DIF is important in obtaining reliable results and eliminating the possibility of generating false-negative testing. Larger studies are needed in order to validate this method.

EDEM1 regulates the insulin mRNA level by inhibiting the endoplasmic reticulum stress-induced IRE1/JNK/c-Jun pathway.

Pancreatic beta cells produce and secrete insulin as a response to rises in blood glucose. Despite the advances in understanding glucose-regulated insulin transcription and translation the mechanisms triggering the synthesis of new insulin molecules are still incompletely described. In this report, we identify EDEM1 as a new modulator of insulin synthesis and secretion. In the presence of EDEM1, INS-1E cells secrete significantly more insulin upon glucose stimulation compared to control cells. We found that overexpression of EDEM1 inhibited the IRE1/JNK/c-Jun pathway, leading to an increase in the insulin mRNA level. Similarly, EDEM1 transduced human islets secreted significantly more insulin upon stimulation. Furthermore, EDEM1 improved insulin secretion restoring normoglycemia and glucose tolerance in diabetic rats. We propose EDEM1 as a regulator of the UPR via IRE1/XBP1s and IRE1/JNK/c-Jun signaling cascades and insulin transcription in pancreatic ß-cells, supporting EDEM1 as a potential target for the treatment of diabetes.

Immunogenicity evaluation after BNT162b2 booster vaccination in healthcare workers.

Waning of the immune response upon vaccination in SARS-CoV-2 infection is an important subject of evaluation in this pandemic, mostly in healthcare workers (HCW) that are constantly in contact with infected samples and patients. Therefore, our study aimed to establish the specific humoral response of specific IgG and IgA antibodies upon vaccination, during the second year of pandemic and evaluating the booster shot with the same vaccine type. A group of 103 HCW with documented exposure to the virus were monitored for specific IgG and IgA levels prior to vaccination, after the first vaccination round, during the following 8 months and after the booster shot with the same vaccine type. After 8 months post-vaccination the humoral response in both IgG and IgA decreased, 2.4 times for IgG, and 2.7 times for IgA. Although the antibodies levels significantly decreased, no documented infection was registered in the group. After the booster shot, the entire group, displayed IgG increased levels, immediately after booster followed by the increase in specific IgA. IgG levels post-second round of vaccination are statistically higher compared to the first round, while IgA is restored at the same levels. Within the vaccination or booster routine for a multiple waves' pandemic that is generating new virus variants, populational immunity remains an important issue for future implementation of prevention/control measures.

Role of histopathology in the management of the gingival enlargement in a patient on antihypertensive therapy based on calcium channel blockers: a case report.

Periodontal pathology is often represented by increases in gingival volume, with pronounced inflammatory phenomena. These manifestations require a more accurate diagnosis and knowledge of the etiopathogenic factors involved. The periodontal treatment applied must be related with the etiopathogenic circumstances. Periodontal disease sometimes has a complex appearance, with intertwined local and systemic favorable factors that make it difficult to include it in a certain taxonomic form. Also, in general, the adult patients have associated chronic diseases that involve the administration of several drugs, which induce on long-term both therapeutic and side effects. Furthermore, diseases in the oral cavity may occur frequently, which require complex and associated dental and periodontal treatment, also occlusal rebalancing, which is a real interdisciplinary challenge. In this case report, periodontal status is determined by a combination of local and systemic favorable factors. However, the histopathological analysis of the gingival samples revealed inflammation without characteristic fibrous hyperplasia changes of the Amlodipine calcium channel blocker (CCB) administration, the antihypertensive medication of the patient. Thus, Amlodipine does not have a hyperplasic effect on gingival mucosa in all cases. Therefore, even if they are more expensive, investigations must be complex, if necessary, in establishing the involvement of the side effect of the systemic medication in periodontal pathological changes. CCB systemic medication is essential, even vital, for maintain the arterial pressure at normal values, should not be altered without the real indication and to the recommendation from a specialist doctor, and the periodontal treatment must be focused to eliminate the local factors.

Langerhans Cells-Revising Their Role in Skin Pathologies.

Langerhans cells (LCs) constitute a cellular immune network across the epidermis. Because they are located at the skin barrier, they are considered immune sentinels of the skin. These antigen-presenting cells are capable of migrating to skin draining lymph nodes to prime adaptive immune cells, namely T- and B-lymphocytes, which will ultimately lead to a broad range of immune responses. Moreover, LCs have been shown to possess important roles in the anti-cancer immune responses. Indeed, the literature nicely highlights the role of LCs in melanoma. In line with this, LCs have been found in melanoma tissues where they contribute to the local immune response. Moreover, the immunogenic properties of LCs render them attractive targets for designing vaccines to treat melanoma and autoimmune diseases. Overall, future studies will help to enlarge the portfolio of immune properties of LCs, and aid the prognosis and development of novel therapeutic approaches to treating skin pathologies, including cancers.

Characteristics and outcomes of patients with COVID-19 and liver injury: a retrospective analysis and a multicenter experience.

Background and aims. Patients with COVID-19 frequently present abnormal elevated liver function tests of unknown clinical significance. We aimed to investigate the characteristics and factors influencing outcome in patients with confirmed SARS-CoV-2 infection and liver injury on admission.Methods. This is a retrospective observational study of patients hospitalized in two COVID units in Romania. Relevant data on clinical and laboratory parameters and medication administered during the admission were analyzed to identify predictors of a negative outcome. Patients with confirmed COVID-19 and liver function tests (LFTs) above the upper limit of normal were included in the analysis.Results. From 1,207 patients, we identified 134 patients (11%) with abnormal LFTs during hospitalization. The majority of patients had mildly elevated levels and a predominantly cholestatic pattern of liver injury. Patients who received lopinavir/ritonavir were more likely to have increased ALAT levels (p<0.0001). Sixteen patients had pre-existing chronic liver disease, and they were more likely to suffer from severe COVID-19 (p=0.009) and have a negative outcome (p<0.001), but on multivariate analysis, only the severity of COVID-19 was predictive of death (OR 69.9; 95% CI 6.4-761.4).Conclusions. Mild liver injury is relatively common in COVID-19 and possibly influenced by medication. Patients with chronic liver disease are at high risk for negative outcome, but the severity of the infection is the only predictor of death.

Coexistence of Trisomy 8 and 13 in a Newly Diagnosed Patient With Diffuse Large B Cell Non-Hodgkin Lymphoma and Acute Myeloid Leukemia Secondary to Primary Myelofibrosis.

Concomitant diagnosis of non-Hodgkin lymphoma (NHL) and acute myeloid leukemia secondary to chronic myeloproliferative neoplasms (MPNs) is rarely reported. Patients with MPNs may have a second neoplasm, and the risk of lymphoid line neoplasms is 2.5-3.5 times for lymphoid line neoplasms. The explanation for this association is the genetic instability of hematopoietic progenitors in MPNs. An 80-year-old Caucasian man, with many comorbidities, presents for physical asthenia, sweating. The right inguinal adenopathy was known one month before the examination. The patient was diagnosed concomitantly with diffuse large B cell lymphoma (DLBCL) and acute myeloid leukemia (AML) secondary to primary myelofibrosis (PMF) and presented trisomy 8, trisomy 13, and triple-negative PMF status. The patient initially received two well-tolerated R mini CHOP series. This type of treatment was selected to treat DLBCL for one unfit patient for intensive chemotherapy due to his age and comorbidities. R mini CHOP administration was followed by severe aplasia that lasted approximately two weeks followed by severe thrombocytosis that reached 4000 x109/L, and Thromboreductin recommendation was mandatory. The result of the treatment was a partial response but with severe adverse events like neutropenia G4, due to the delay of the treatment the patient lost the response. It was mandatory to select another treatment line and the chosen was venetoclax; it was selected for the simultaneous treatment of DLBCL and the underlying AML. It was obtained a significant reduction in the size of the inguinal lymph node block in two weeks of treatment. Severe neutropenia was diagnosed and complicated with sepsis. The evolution is unfavorable with the installation of multiple organ dysfunction. The presence of a complex karyotype (trisomy 8, trisomy 13) in a patient with myeloid metaplasia with triple-negative PMF was associated with blast transformation and severe thrombocytosis. The patient was diagnosed concomitantly with DLBCL, making the therapeutic decision difficult. Venetoclax has been shown to be useful in the treatment of DLBCL but has been associated with severe neutropenia, which has led to infectious complications.

A New Artificial Intelligence-Based Method for Identifying Mycobacterium Tuberculosis in Ziehl-Neelsen Stain on Tissue.

Mycobacteria identification is crucial to diagnose tuberculosis. Since the bacillus is very small, finding it in Ziehl-Neelsen (ZN)-stained slides is a long task requiring significant pathologist's effort. We developed an automated (AI-based) method of identification of mycobacteria. We prepared a training dataset of over 260,000 positive and over 700,000,000 negative patches annotated on scans of 510 whole slide images (WSI) of ZN-stained slides (110 positive and 400 negative). Several image augmentation techniques coupled with different custom computer vision architectures were used. WSIs automatic analysis was followed by a report indicating areas more likely to present mycobacteria. Our model performs AI-based diagnosis (the final decision of the diagnosis of WSI belongs to the pathologist). The results were validated internally on a dataset of 286,000 patches and tested in pathology laboratory settings on 60 ZN slides (23 positive and 37 negative). We compared the pathologists' results obtained by separately evaluating slides and WSIs with the results given by a pathologist aided by automatic analysis of WSIs. Our architecture showed 0.977 area under the receiver operating characteristic curve. The clinical test presented 98.33% accuracy, 95.65% sensitivity, and 100% specificity for the AI-assisted method, outperforming any other AI-based proposed methods for AFB detection.

Brachial and subclavian arteries aneurysms due to tuberous sclerosis complex mechanisms - case report and literature review.

INTRODUCTION: Tuberous sclerosis complex (TSC) is a rare autosomal dominant condition characterized by cutaneous, cerebral, and other multiorgan involvement. Aneurysms due to TSC pathogenic mechanism are rarely present, mainly aortic, renal, or intracranial and very few associated with peripheral circulation. A TSC patient, aged 31 years, who developed brachial and subclavian arteries aneurysms is presented. The question of a random association of the aneurysms with TSC versus aneurysms within pathogenic released mammalian target of rapamycin (mTOR) pathway effect was raised. CASE PRESENTATION: Patient's file, available from the age of six months, was analyzed for demonstration of the TSC diagnosis. Patient was examined, and cerebral magnetic resonance imaging (MRI) was repeated. Surgery and angiographic reports and images were reviewed. Pathology of the aneurysmal wall available from surgery was reexamined and special stainings and immunohistochemistry markers were applied. Genetic characterization of the patient was performed. Definite TSC was diagnosed based on major criteria [ungual fibromas, shagreen patch, cortical tubers, subependymal nodules (SENs), subependymal giant cell astrocytoma (SEGA)], minor criteria (confetti skin lesions, dental enamel pits, gingival fibromas), genetic result showing heterozygous variant in exon 8 of TSC1 gene (c.733C>T-p.Arg245*). Pathology analysis revealed markedly thickened aneurysmal wall due to smooth muscle cells (SMCs) proliferation in media and neoformation vessels with similar characteristics in the aneurysmal wall. DISCUSSIONS AND CONCLUSIONS: This is a rare case with aneurysms related to TSC, with an exceptional peripheral localization. Pathology exam is the key investigation in demonstrating the TSC-related pathogenic mechanism. A literature review showed 73 TSC cases presenting aneurysms published until now.

The prognostic significance of the clinical and histological parameters in primary cutaneous melanoma patients.

Cutaneous melanoma is the most aggressive form of skin cancer and its incidence is unfortunately increasing. In the last decades, a progressive increase of new cases of diagnosed thin melanoma has been noted. This may be due to earlier detection, better surveillance, improved diagnostic criteria or increased exposure to sunlight. Despite the fact that Breslow tumor thickness has the strongest proven prognostic significance, there are still thin melanomas that metastasize and thick melanomas with favorable evolution. Therefore, the identification of strong predictive factors for survival is mandatory, particularly for patients with thin melanoma.

Role of immunohistochemistry in the diagnosis and staging of cutaneous squamous-cell carcinomas (Review).

Non-melanoma skin cancer (NMSC) is the most common type of neoplasm affecting Caucasian individuals, with squamous-cell carcinoma (cSCC) being the second most common type of NMSC after basal-cell carcinoma. The immunohistochemical study of cSCC is of particular importance, especially for the diagnosis of its rare forms, for which accurate and early diagnosis is crucial for survival. In the present review of the literature, the potentially significant value of immunohistochemical markers were highlighted to more accurately assess the biological behaviour, the prognosis of cSCC and to optimize case management. The immunohistochemical markers were classified from a pathophysiological point of view in order to present the mechanism by which carcinogenesis occurs with its subsequent evolution and therefore, to develop a more accurate novel risk staging criteria for this type of neoplasm.

COVID­19 vaccination and IgG and IgA antibody dynamics in healthcare workers.

Given the current outbreak of coronavirus disease 2019 (COVID­19) and the development and implementation of mass vaccination, data are being obtained by analyzing vaccination campaigns. In the present study, 69 healthcare workers who were exposed to patients with severe acute respiratory syndrome coronavirus­2 were monitored for specific immunoglobulin (Ig)G and IgA levels at different time periods. Prior to vaccination, after the first round of vaccination at 21 days (when the second dose of vaccine was administrated) and 24 days after the second round of vaccination, with an mRNA­based vaccine. The basal IgG and IgA levels in previously infected subjects and non­infected subjects notably differed. Vaccination increased the IgG and IgA levels after the first dose in most subjects from both groups, the levels of which further increased following the second round of vaccination. The associations between IgG and IgA levels following the first and second rounds of vaccination demonstrated that in the entire vaccination group, regardless of prior exposure to the infectious agent, the increment and levels of IgG and IgA were similar. Thus, the levels upon vaccination were statistically similar irrespective of the starting base line prior to vaccination. In the present study, seroconversion was achieved in all subjects following the second round of vaccination, with similar antibodies levels.