The Role of Toll-like Receptor-4 in Macrophage Imbalance in Lethal COVID-19 Lung Disease, and Its Correlation with Galectin-3.

To the current data, there have been 6,955,141 COVID-19-related deaths worldwide, reported to WHO. Toll-like receptors (TLRs) implicated in bacterial and virus sensing could be a crosstalk between activation of persistent innate-immune inflammation, and macrophage's sub-population alterations, implicated in cytokine storm, macrophage over-activation syndrome, unresolved Acute Respiratory Disease Syndrome (ARDS), and death. The aim of this study is to demonstrate the association between Toll-like-receptor-4 (TLR-4)-induced inflammation and macrophage imbalance in the lung inflammatory infiltrate of lethal COVID-19 disease. Twenty-five cases of autopsy lung tissues were studied by digital pathology-based immunohistochemistry to evaluate expression levels of TLR-4 (CD 284), pan-macrophage marker CD68 (clone KP1), sub-population marker related to alveolar macrophage Galectin-3 (GAL-3) (clone 9C4), and myeloid derived CD163 (clone MRQ-26), respectively. SARS-CoV-2 viral persistence has been evaluated by in situ hybridation (ISH) method. This study showed TLR-4 up-regulation in a subgroup of patients, increased macrophage infiltration in both Spike-1(+) and Spike-1(-) lungs (p < 0.0001), and a macrophage shift with important down-regulation of GAL-3(+) alveolar macrophages associated with Spike-1 persistence (p < 0.05), in favor of CD163(+) myeloid derived monocyte-macrophages. Data show that TLR-4 expression induces a persistent activation of the inflammation, with inefficient resolution, and pathological macrophage shift, thus explaining one of the mechanisms of lethal COVID-19.

Immunohistochemical Evaluation of Aquaporin-4 and its Correlation with CD68, IBA-1, HIF-1α, GFAP, and CD15 Expressions in Fatal Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide. Our understanding of its pathobiology has substantially increased. Following TBI, the following occur, edema formation, brain swelling, increased intracranial pressure, changes in cerebral blood flow, hypoxia, neuroinflammation, oxidative stress, excitotoxicity, and apoptosis. Experimental animal models have been developed. However, the difficulty in mimicking human TBI explains why few neuroprotective strategies, drawn up on the basis of experimental studies, have translated into improved therapeutic strategies for TBI patients. In this study, we retrospectively examined brain samples in 145 cases of death after different survival times following TBI, to investigate aquaporin-4 (AQP4) expression and correlation with hypoxia, and neuroinflammation in human TBI. Antibodies anti-glial fibrillary acid protein (GFAP), aquaporin-4 (AQP4), hypoxia induced factor-1α (HIF-1α), macrophage/phagocytic activation (CD68), ionized calcium-binding adapter molecule-1 (IBA-1), and neutrophils (CD15) were used. AQP4 showed a significant, progressive increase between the control group and groups 2 (one-day survival) and 3 (three-day survival). There were further increases in AQP4 immunopositivity in groups 4 (seven-day survival), 5 (14-dayssurvival), and 6 (30-day survival), suggesting an upregulation of AQP4 at 7 to 30 days compared to group 1. GFAP showed its highest expression in non-acute cases at the astrocytic level compared with the acute TBI group. Data emerging from the HIF-1α reaction showed a progressive, significant increase. Immunohistochemistry with IBA-1 revealed activated microglia starting three days after trauma and progressively increasing in the next 15 to 20 days after the initial trauma. CD68 expression demonstrated basal macrophage and phagocytic activation mostly around blood vessels. Starting from one to three days of survival after TBI, an increase in the number of CD68 cells was progressively observed; at 15 and 30 days of survival, CD68 showed the most abundant immunopositivity inside or around the areas of necrosis. These findings need to be developed further to gain insight into the mechanisms through which brain AQP4 is upregulated. This could be of the utmost clinicopathological importance.

The Role of TLR-2 in Lethal COVID-19 Disease Involving Medullary and Resident Lung Megakaryocyte Up-Regulation in the Microthrombosis Mechanism.

Patients with COVID-19 have coagulation and platelet disorders, with platelet alterations and thrombocytopenia representing negative prognostic parameters associated with severe forms of the disease and increased lethality. METHODS: The aim of this study was to study the expression of platelet glycoprotein IIIa (CD61), playing a critical role in platelet aggregation, together with TRL-2 as a marker of innate immune activation. RESULTS: A total of 25 patients were investigated, with the majority (24/25, 96%) having co-morbidities and dying from a fatal form of SARS-CoV-2(+) infection (COVID-19+), with 13 men and 12 females ranging in age from 45 to 80 years. When compared to a control group of SARS-CoV-2 (-) negative lungs (COVID-19-), TLR-2 expression was up-regulated in a subset of patients with deadly COVID-19 fatal lung illness. The proportion of Spike-1 (+) patients found by PCR and ISH correlates to the proportion of Spike-S1-positive cases as detected by digital pathology examination. Furthermore, CD61 expression was considerably higher in the lungs of deceased patients. In conclusion, we demonstrate that innate immune prolonged hyperactivation is related to platelet/megakaryocyte over-expression in the lung. CONCLUSIONS: Microthrombosis in deadly COVID-19+ lung disease is associated with an increase in the number of CD61+ platelets and megakaryocytes in the pulmonary interstitium, as well as their functional activation; this phenomenon is associated with increased expression of innate immunity TLR2+ cells, which binds the SARS-CoV-2 E protein, and significantly with the persistence of the Spike-S1 viral sequence.

Survivin expression in renal cell carcinoma.

Deregulated expression of inhibitors of apoptosis may contribute to cancer by aberrantly extending cell viability and facilitating the insurgence of resistance to chemo- and radiotherapy. In this study, we have investigated, by immunohistochemical technique, the expression and potential prognostic significance of survivin in a series of 49 clear cell type renal cell carcinoma (ccRCC). Survivin expression was significantly associated with poorly differentiated, advanced stages and more aggressive ccRCCs (p < 0.05). Patients with low survivin expression had statistically significant better survival rates than patients with high survivin expression (p < 0.05). This may be relevant for follow-up protocols design and/or alternative therapeutic approaches.

Prognostic significance of multidrug-resistance protein (MDR-1) in renal clear cell carcinomas: a five year follow-up analysis.

BACKGROUND: A large number of renal cancer patients shows poor or partial response to chemotherapy and the mechanisms have not been still understood. Multi-drug resistance is the principal mechanism by which many cancers develop resistance to chemotherapic drugs. The role of the multi-drug resistant transporter (MDR-1/P-glycoprotein), the gene product of MDR-1, and that one of the so-called multi-drug resistance associated protein (MRP), two energy-dependent efflux pumps, are commonly known to confer drug resistance. We studied MDR-1 expression in selected cases of renal cell carcinoma (RCC), clear cell type, with long-term follow-up, in order to establish its prognostic role and its possible contribution in the choice of post-surgical therapy. METHODS: MDR-1 has been studied by standard LSAB-HRP immunohistochemical technique, in paraffin embedded RCC samples. Protein expression has been compared to clinical and histopathological data and to disease specific survival of RCC patients, by Kaplan-Meier curve and Cox multivariate regression analyses. RESULTS: Two groups of RCCs were obtained by esteeming MDR-1 expression and disease specific survival (obtained with Kaplan-Meier curve and Cox multivariate regression analyses): the first one presents low or absent MDR-1 expression and good survival; the second one is characterized by high MDR-1 expression and significant poor outcome (p < 0.05). Afterwards, we have found disease specific survival, adjusted for stages and independent of therapy: this difference of survival rates was statistically significant (p < 0.05). Stage adjusted disease specific survival rate, according to MDR-1 expression and therapy in patients affected by RCC in early stage (stage I), has revealed that the group of patients with high MDR-1 expression and without adjuvant therapy showed poor survival (p < 0.05). Cox multivariate regression analysis has confirmed that, in our cohort of RCC (clear cell type) patients, the strong association between MDR-1 and worse outcome is independent not only of the adjuvant therapy, but also of the other prognostic parameters (p < 0.05). CONCLUSION: In our opinion, the results of this study well prove the relationship between MDR-1 expression and worse clinical prognosis in RCC, because MDR-1 over-expressing RCCs can be considered a group of tumours with a more aggressive behavior. This finding outlines a possible role of MDR-1 as prognostic factor, dependent and independent of multidrug resistance. These results could be useful to predict cancer evolution and to choose the appropriate treatment: this is another step that can stimulate further promising and interesting investigations on broader study population.

The role of survivin in thyroid tumors: differences of expression in well-differentiated, non-well-differentiated, and anaplastic thyroid cancers.

BACKGROUND: Survivin is involved in human cancer and is responsible for aggressive biological behavior and poor clinical outcomes in several human malignancies. Thus, we hypothesized that the upregulation of survivin protein expression may be enhanced in parallel with transition toward a poorly differentiated phenotype in human thyroid carcinomas. METHODS: The expression of survivin was evaluated, using a standard linked streptavidin-biotin horseradish peroxidase technique technique, in a series of 56 human thyroid carcinomas (42 papillary, 4 poorly differentiated, and 10 anaplastic carcinomas) and thyroid carcinoma cell lines at different degrees of differentiation. RESULTS: The cytoplasmic expression of survivin protein was significantly upregulated in all thyroid tumors. A statistically significant association was found between nuclear survivin expression and anaplastic thyroid cancer (mean ± SD: well-differentiated thyroid cancer, 1.22 ± 20.21; non-well-differentiated thyroid cancer, 34.00 ± 25.17; anaplastic thyroid cancer, 56.50 ± 22.10; p<0.001). Nuclear staining of survivin has been shown in poorly differentiated and anaplastic thyroid carcinomas, and this is likely due to the upregulation of the ΔEx3 survivin splicing variant, as shown in poorly differentiated/anaplastic thyroid carcinoma cell lines. Of note, selected thyroid tumors characterized by a mixed population of differentiated and undifferentiated neoplastic cells, likely progressing from well to poorly differentiated and anaplastic phenotypes, exhibited cytoplasmic expression of survivin in differentiated fields and nuclear protein staining in poorly differentiated and anaplastic areas. This expression profile provides substantial added value to conventional clinical markers in predicting anaplastic cancer. The cut-off for distinguishing thyroids that developed ATC from those that remained differentiated was >30% of nuclear survivin expression. The receiver operating characteristic (ROC) area was 0.92, with a p-value of <0.0001. CONCLUSIONS: Upregulation of survivin expression may be a molecular marker of dedifferentiation in thyroid epithelial carcinomas, likely being responsible for survival responses of tumor cells and, thus, favoring progression toward a poorly differentiated phenotype.

The role of EBV in the pathogenesis of Burkitt's Lymphoma: an Italian hospital based survey.

The exact worldwide incidence of Burkitt's lymphoma is not known. There are three distinct clinical variants of Burkitt's lymphoma, each manifesting differences in epidemiology, clinical presentation, morphology, biology and genetic features: the endemic (African), the sporadic (non-endemic), and the immunodeficiency-associated form. In particular, we reported data regarding Burkitt's lymphoma incidence in the world and across different European countries. Finally, we described clinic-pathological data of 48 Burkitt's lymphomas occurred in Italy from 2003 to 2013, in 4 different hospitals, two of which located in east side, and the other ones located in the west-coast. Forty Burkitt's lymphomas occurs in children (age range 3-12), and 8 were adulthood Burkitt's lymphomas (age range 18-87). In the pediatric group the Male:Female ratio (M:F) was of 4:1, whereas the group of the adult patients has a M:F of 1:1.67. Immunohistochemical detection of Latent Membrane Protein 1 (LMP1) expression and Epstein-Barr virus Encoded RNA (EBER) In Situ Hybridization (ISH) procedures have been performed. Lymphocyte B monoclonal spread has been demonstrated using a Polymerase Chain Reaction (PCR) based method to amplify Fragment Restriction FR1, FR2 and FR3 immunoglobulin heavy chains DNA fragments. Only 38 cases out of 48 were analyzed for LMP-1 showing various percentage of stained cells in 47.4% of the patients. Considering ISH for EBER detection results: 1 out 2 (50%) adult analyzed cases was positive, with 50% of stained tumor cells (this patient was a 22 years old female, coming from Napoli);15 out 24 (62.5%) children analyzed Burkitt's lymphomas resulted as positive for EBER;the overall positivity has been observed in 16/26 Burkitt's lymphomas (61.53%).Finally, EBV has been detected in children and adult patients, one of them with deregulation of the oncogene c-MYC by chromosomal translocation.

Sudden, unexpected death due to glioblastoma: report of three fatal cases and review of the literature.

Sudden death from an undiagnosed primary intracranial neoplasm is an exceptionally rare event, with reported frequencies in the range of 0.02% to 2.1% in medico-legal autopsy series and only 12% of all cases of sudden, unexpected death due to primary intracranial tumors are due to glioblastomas. We present three cases of sudden, unexpected death due to glioblastoma, with different brain localization and expression. A complete methodological forensic approach by means of autopsy, histological and immunohistochemical examinations let us to conclude for an acute central dysregulation caused by glioblastoma and relative complication with rapid increase of intracranial pressure as cause of death. Although modern diagnostic imaging techniques have revolutionized the diagnosis of brain tumors, the autopsy and the careful gross examination and section of the fixed brain (with coronal section) is still the final word in determining exact location, topography, mass effects and histology and secondary damage of brain tumor and contributed the elucidation of the cause of death. Immunohistochemistry and proteomic analysis are mandatory in such cases. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1218574899466985.

Primary sarcoma of the specialised prostatic stroma: a case report and review of the literature.

Primary sarcoma tumours of the prostate are rare and are classified, according to their histology, as stromal tumours of uncertain malignant potential (STUMP) and stromal prostatic sarcoma (PS; low and high grade). We describe a case of a 71-year-old man that developed progressive urinary obstruction symptoms and was subjected to a transurethral prostatic resection (TURP). Histologically, there is a diffuse proliferation of epithelioid and spindle cells that showed rare atypical mitotic figures. Immunohistochemically, the neoplastic cells express diffusely CD34 and focally progesterone whereas no immunoreactivity was seen for cytocheratin, desmin, S-100, Bcl-2, chromogranin, CD117, and actin smooth muscle. A final diagnosis of low-grade prostatic stromal sarcoma (LG-PS) was made. This is a really rare neoplasm; in the literature, in fact, to our knowledge, only 6 cases are described and all of these were alive and free of disease at followup. Our patient too is free of disease at 15 months from the diagnosis.

The proliferation marker Chromatin Assembly Factor-1 is of clinical value in predicting the biological behaviour of salivary gland tumours.

Salivary gland tumours (SGT) constitute a diagnostically challenging group of neoplasms with frequently unpredictable clinical outcome. The proliferation rate facilitates the identification of aggressive SGT. The Chromatin Assembly Factor-1 (CAF-1) is a major epigenetic regulator of nuclear chromatin organization during DNA replication. It plays a critical function in human tumourigenesis and has been proposed as a new proliferation and prognostic marker for some malignancies. This study focused on the role of CAF-1/p60 protein as a marker of clinical value for SGT. The expression of CAF-1/p60 was evaluated by immunohistochemistry on a retrospective series of 362 surgically excised benign and malignant SGT with different histogenesis and, when available, on fine-needle pre-surgical cytological biopsies. The resulting data were compared with traditional prognostic parameters, including the expression of the routine proliferation marker ki67/MIB1. CAF-1/p60 was detectable in all SGT, with highest degree of expression in metastasizing malignant tumours. Moreover, the cases of benign tumours which progressed to carcinoma during the follow-up, showed significantly higher CAF-1/p60 expression than non-progressing benign SGT, both on histological sections and cytological smears of the primary tumour. Cox's multiple regression analysis selected CAF-1/p60 expression as the best independent predictor of cancer development for benign SGT (p<0.0001), and the best independent predictor of metastasis onset for malignant tumours (p<0.0004). Overexpression of CAF-1/p60, on histological and/or cytological samples, characterizes malignant SGT with aggressive behaviour, irrespective of their specific histotype, and allows the early diagnosis of progression toward malignancy of morphologically benign tumours.

Retinoblastoma protein expression predicts response to bacillus Calmette-Guérin immunotherapy in patients with T1G3 bladder cancer.

OBJECTIVES: Bacillus Calmette-Guérin (BCG) immunotherapy is regarded as the current treatment of choice for stage T1 grade 3 (T1G3) bladder cancer (BC), though its efficacy is limited by high recurrence and progression rate. Identification of molecular prognosticators that might be helpful in discriminating between responders and nonresponders to BCG treatment is therefore of major clinical importance; thus we focused on the cell-cycle related retinoblastoma protein (pRB), which had been already investigated in bladder cancer. The goal of our study was specifically to address whether its expression predicts the outcomes of BCG treatment for patients with T1G3 disease. MATERIALS AND METHODS: To address this issue, paraffin-embedded specimens of 27 patients having undergone transurethral resection of T1G3 BC and intravesical instillations of BCG (induction + 1 year maintenance) were immunostained with pRB monoclonal antibody. Patients in whom the bladder muscle was not clearly visible, and healthy, as well as patients with TaG3 tumors or with concomitant carcinoma in situ were excluded. Mean follow-up was 60 months (range 15-135). RESULTS: Thirteen tumors showed normal (1% to 50% labeling index) while 14 showed altered pRB expression, consisting of no expression (0% labeling index) in six and overexpression (>50% labeling index) in eight. Recurrence occurred in 10 (37%) patients and mean time to recurrence was 22.8 months (range 6-48). Recurrence rate was 57% in patients with altered and 15% in those with normal pRB expression, with a statistically significant difference in disease-free survival (P = 0.037). Progression occurred in five (18.5%) patients and mean time to progression was 24 months (range 6-48). Progression rate was 36% in patients with altered and 0% in patients with normal pRB expression, with a statistically significant difference in progression-free survival (P = 0.018). CONCLUSIONS: In this homogeneous population of T1G3 bladder tumors, altered pRB expression predicted recurrence and progression after BCG treatment. These findings outline the potential role of pRB immunostaining in predicting T1G3 BC response to BCG immunotherapy.

Altered p53 and pRb expression is predictive of response to BCG treatment in T1G3 bladder cancer.

BACKGROUND: The aim of this study was to determine the prognostic value of p53 and retinoblastoma protein (pRb) expression in patients with stage T1 grade 3 (T1G3) bladder cancer (BC) treated by transurethral resection of bladder tumour (TURBT) and intravesical instillations of bacillus Calmette-Guerin (BCG). MATERIALS AND METHODS: p53 and pRb expression were independently recorded within a homogeneous series of 27 patients. Fisher exact test and the log-rank test were carried out, along with Kaplan-Meier survival analysis. RESULTS: Sixteen tumours showed altered p53 expression, while 14 showed altered pRb expression. Overall, 7 tumours showed normal expression of both markers, 10 altered expression of one of the two markers, and 10 altered expression of both markers. Only altered pRb expression was an independent predictor of both recurrence (p=0.037) and progression (p=0.018); altered expression of both markers was a strong predictor (p=0.001) of progression. CONCLUSION: This is the first study demonstrating that altered p53 and pRb expression are predictive of T1G3 BC response to BCG treatment. These findings provide grounds for inclusion and prospective validation of these markers in the decision-making process for treating BC.