In situ evidence of pulmonary endothelial activation in patients with granulomatosis with polyangiitis and systemic sclerosis.

INTRODUCTION: The aim of this study was to investigate the in situ pulmonary endothelial activation in lung lesions of granulomatosis with polyangiitis (GPA) and systemic sclerosis (SScl). METHODS: We examined the endothelial expression of ICAM-1, VCAM-1, and E-selectin using immunohistochemistry on formalin-fixed, paraffin-embedded sections of lung lesions of GPA, interstitial lung disease associated with SScl and controls. RESULTS: A significantly enhanced expression of ICAM-1 and E-selectin was observed in GPA and SScl pulmonary endothelium compared to controls. VCAM-1 was more pronouncedly expressed in GPA compared to SScl. CONCLUSION: These observations are an evidence of in situ pulmonary vascular endothelial activation in lesions of GPA and SScl, adding information to the pathogenic knowledge of both diseases.

Mesothelin expression remodeled the immune-matrix tumor microenvironment predicting the risk of death in patients with malignant pleural mesothelioma.

Background: The combination of immunobiological agents with immune checkpoint proteins is a promising treatment for malignant pleural mesothelioma (MPM). Mesothelin and anti-PD-L1 antibody-drug conjugates specifically target malignant neoplastic cells, inhibit the migration and invasion of neoplastic cells, and restore the immune landscape. In this study, we confirmed the importance of mesothelin and examined the relationship between mesothelin and the immune landscape of the tumor microenvironment (TME) in two MPM cohorts. Methods: The discovery cohort included 82 MPM cases. Tissue microarray slides were generated, and samples were processed for hematoxylin & eosin staining, immunohistochemistry, and immunofluorescence assays. The relationship between mesothelin, biomarkers of histogenesis, histological aggressiveness, PD-L1, immune cells (CD4, CD8, CD20, CD68), and collagen type I and type V fibers was evaluated by quantitative digital analyses. The outcome was the survival time until death from disease recurrence. The exploratory cohort included 87 malignant mesothelioma (MESO) patients from The Cancer Genome Atlas database. Results: Most patients were male (70.7%) with a history of asbestos exposure (53.7%) and with the epithelioid subtype (89%). Surgical resection was performed in 85.4% of patients, and 14.6% received chemotherapy; 59.8% of patients died from disease extension to the mediastinum. Low tumor mesothelin expression was associated with tumor necrosis and nuclear grade 1, whereas high mesothelin expression was significantly associated with the epithelioid histotype and high density of T cells CD8+, macrophages CD68+, and collagen type I fibers. Cox multivariate analysis showed a high risk of death for non-operated patients [hazard ratio (HR), 3.42 (1.15-10.16)] with low tumor mesothelin levels [HR, 2.58 (1.09-6.10)] and high PD-L1 and low infiltration of T cells CD4+ [HR, 3.81 (1.58-9.18)]. In the exploratory cohort, low mesothelin and high COL1A1 and COL5A1 expression were associated with poor overall survival. Conclusion: Tumor mesothelin expression associated with the TME immune landscape predicts the risk of death for patients with MPM and could be a new target for immunotherapy in MPM.

Impact of the COVID-19 Pandemic on Cervical Cancer Screening in São Paulo State, Brazil.

INTRODUCTION: The early identification of precursor lesions followed by appropriate treatment prevents development of cervical cancer and its consequences. OBJECTIVE: The present study evaluated the influence of the COVID-19 pandemic on cervical cancer screening by comparing the quantity of tests to detect cervical cellular changes performed in São Paulo state in 2019, prior to the detection of SARS-CoV-2 in Brazil, to the first (2020) and second (2021) years following its appearance. MATERIALS AND METHODS: Data from Fundação Oncocentro de São Paulo (FOSP), the agency that analyses approximately 220,000 Papanicolaou (Pap) tests annually, were reviewed. RESULTS: A median of 1,835 Pap tests were performed in 55 municipalities in 2019. This was reduced to 815 tests in 2020, a 56% decrease (p = 0.0026). In 2021, the median number was 1,745, a 53% increase over 2020 levels (p = 0.0233). The 26 municipalities with >1,000 tests in 2020 had a median reduction from 4,433 in 2019 to 2,580 in 2020 (p = 0. 0046). The 29 municipalities with <1,000 tests had a median reduction from 951 in 2019 to 554 in 2020 (p < 0.0001). There was a 44% reduction in the number of follow-up cytological evaluations from 2019 to 2020, followed by a 30% increase in the following year. However, the percentage of women with a normal finding or with any abnormality remained unchanged. The findings from a histological evaluation of women in São Paulo city indicated that the percent of cases positive for CIN-1 (p < 0.0410) and CIN-3 (p < 0.0012) increased in 2020 and 2021 as compared to 2019 levels. CONCLUSION: A reduction in testing for cervical cancer in the first year of the COVID-19 pandemic, accompanied by an elevated incidence of precancerous lesions in each of the first 2 years following its initiation, may portend a subsequent increased occurrence of cervical cancer in Brazil.

Molecular subtypes as a prognostic breast cancer factor in women users of the São Paulo public health system, Brazil.

OBJECTIVE: This study aimed to analyze the prognosis of women with breast cancer by molecular subtypes, sociodemographic variables, and clinical and treatment characteristics. METHODS: This hospital-based retrospective cohort study analyzed 1,654 women over 18 years of age diagnosed with invasive breast cancer from 2000 to 2018. Data were extracted from Brazil's Oncocenter Foundation of São Paulo. The variables analyzed were age, histology, molecular subtypes, clinical staging, treatment type, and diagnosis-to-treatment time. Cox regression analysis was applied to estimate death risk. RESULTS: Women with HER-2-positive (nonluminal) and triple-negative molecular subtypes were more than twice more likely to be at risk of death, with adjusted hazard ratio - HRadj=2.30 (95% confidence interval - 95%CI 1.34-3.94) and HRadj=2.51 (95%CI 1.61-3.92), respectively. A delayed treatment associated with an advanced clinical stage at diagnosis increased fourfold the risk of death (HRadj=4.20 (95%CI 2.36-7.49). CONCLUSION: In summary, besides that interaction between advanced clinical stage and longer time between diagnosis and treatment, HER-2-positive (nonluminal) and triple-negative phenotypes were associated with a worse prognosis. Therefore, actions to reduce barriers in diagnosis and treatment can provide better outcome, even in aggressive phenotypes.

The Immunological Landscape of M1 and M2 Macrophages and Their Spatial Distribution in Patients with Malignant Pleural Mesothelioma.

BACKGROUND: Several tumor-associated macrophages (TAMs) have shown promise as prognosticators in cancer. Our aim was to validate the importance of TAMs in malignant pleural mesothelioma (MPM) using a two-stage design. METHODS: We explored The Cancer Genome Atlas (TCGA-MESO) to select immune-relevant macrophage genes in MPM, including M1/M2 markers, as a discovery cohort. This computational cohort was used to create a multiplex immunofluorescence panel. Moreover, a cohort of 68 samples of MPM in paraffin blocks was used to validate the macrophage phenotypes and the co-localization and spatial distribution of these immune cells within the TME and the stromal or tumor compartments. RESULTS: The discovery cohort revealed six immune-relevant macrophage genes (CD68, CD86, CD163, CD206, ARG1, CD274), and complementary genes were differentially expressed by M1 and M2 phenotypes with distinct roles in the tumor microenvironment and were associated with the prognosis. In addition, immune-suppressed MPMs with increased enrichment of CD68, CD86, and CD163 genes and high densities of M2 macrophages expressing CD163 and CD206 proteins were associated with worse overall survival (OS). Interestingly, below-median distances from malignant cells to specific M2a and M2c macrophages were associated with worse OS, suggesting an M2 macrophage-driven suppressive component in these tumors. CONCLUSIONS: The interactions between TAMs in situ and, particularly, CD206+ macrophages are highly relevant to patient outcomes. High-resolution technology is important for identifying the roles of macrophage populations in tissue specimens and identifying potential therapeutic candidates in MPM.

Primary intestinal type mucinous adenocarcinoma of the ovary diagnosed during pregnancy - Maternal and neonatal treatment and outcomes: A case report.

The incidence of ovarian cancer during pregnancy is low. Most adnexal tumors removed during pregnancy are benign, with ovarian carcinomas found in approximately 1: 10,000-1: 50,000 pregnancies. Literature on this disease is scarce and consists mostly of retrospective studies and case reports. We report the case of a pregnant patient who presented with a primary intestinal-type mucinous adenocarcinoma of the ovary and underwent unilateral salpingo-oophorectomy, with no additional surgical or chemotherapy treatment after the histological diagnosis, despite an infiltrative stromal invasion pattern. To the best of our knowledge, no such case has been previously reported. Conservative treatment in this case of early ovarian carcinoma is possible during pregnancy and should be performed in the Department of Gynecological Oncology and Obstetrics of a tertiary referral hospital. Given the possibility of disease recurrence, such patients require strict clinical oncological surveillance, specialized prenatal care, and assistance from a multidisciplinary team to improve the maternal and perinatal outcomes.

Different morphology, stage and treatment affect immune cell infiltration and long-term outcome in patients with non-small-cell lung carcinoma.

AIMS: Development of effective immune-based therapies for patients with non-small-cell lung carcinoma (NSCLC) depends on an accurate characterization of complex interactions that occur between immune cells and the tumour environment. METHODS AND RESULTS: Innate and adaptive immune responses were evaluated in relation to prognosis in 65 patients with surgically excised NSCLC. Immunohistochemistry and morphometry were used to determine the abundance and distribution of immune cells. We found low numbers of immune cells and levels of cytokines in the tumour environment when compared with surrounding parenchyma. Smoking was associated inversely with the adaptive immune response and directly with innate immunity. We observed a prominent adaptive immune response in squamous cell carcinomas (SCC) but greater innate immune responses in adenocarcinomas and large cell carcinomas. Cox model analysis showed a low risk of death for smoking <41 packs/year, N0 tambour stage, squamous carcinoma, CD4(+) > 16.81% and macrophages/monocytes >4.5%. Collectively, the data indicate that in NSCLC there is not a substantive local immune cell infiltrate within the tumour. CONCLUSION: Although immune cell infiltration is limited in NSCLC it appears to have an impact on prognosis and this may be of relevance for new immunotherapeutic approaches.

Implementing human papillomavirus testing in a public health hospital: challenges and opportunities.

OBJECTIVES: Robust evidence now supports human papillomavirus (HPV) testing as a more effective option to screening and as more sensitive than cytology in detecting high-grade cervical intraepithelial neoplasia . Our goal was to analyze the performance of the Hybrid Capture II (HC2) assay for high-risk HPV (hrHPV) in women undergoing gynecological examination at a public health hospital as part of the evaluation of HPV screening as an alternative or complement to cytology. STUDY DESIGN: This analysis is a subset of a cross-sectional study carried out at a large public hospital serving a predominantly low-resource population. A total of 705 women were enrolled; the sensitivity and specificity of each test were estimated and compared. RESULTS: The analysis identified 272 hrHPV-positive women (mean age 36.3 years) and 433 hrHPV-negative women (mean age 41.2 years). HPV testing showed a significantly increased sensitivity of the HC2 assay versus cytology (84.5 vs. 69.7%; p < 0.0001) but a lower specificity (49.90 vs. 88.78%; p < 0.0001). CONCLUSION: The combination of both methods seems to be useful in improving detection of cervical lesions.

Dissecting and Reconstructing Matrix in Malignant Mesothelioma Through Histocell-Histochemistry Gradients for Clinical Applications.

Background: Malignant pleural mesotheliomas (MM) are known for their heterogenous histology and clinical behavior. MM histology reveals three major tumor cell populations: epithelioid, sarcomatoid, and biphasic. Using a dissecting approach, we showed that histochemical gradients help us better understand tumor heterogeneity and reconsider its histologic classifications. We also showed that this method to characterize MM tumor cell populations provides a better understanding of the underlying mechanisms for invasion and disease progression. Methods: In a cohort of 87 patients with surgically excised MM, we used hematoxylin and eosin to characterize tumor cell populations and Movat's pentachrome staining to dissect the ECM matrisome. Next, we developed a computerized semi-assisted protocol to quantify and reconstruct the ECM in 3D and examined the clinical association between the matricellular factors and patient outcome. Results: Epithelioid cells had a higher matrix composition of elastin and fibrin, whereas, in the sarcomatoid type, hyaluronic acid and total collagen were most prevalent. The 3D reconstruction exposed the collagen I and III that form channels surrounding the neoplastic cell blocks. The estimated volume of the two collagen fractions was 14% of the total volume, consistent with the median estimated area of total collagen (12.05 mm2) for epithelioid MM. Conclusion: Differential patterns in matricellular phenotypes in MM could be used in translational studies to improve patient outcome. More importantly, our data raise the possibility that cancer cells can use the matrisome for disease expansion and could be effectively targeted by anti-collagen, anti-elastin, and/or anti-hyaluronic acid therapies.

Modeling extracellular matrix through histo-molecular gradient in NSCLC for clinical decisions.

Lung cancer still represents a global health problem, being the main type of tumor responsible for cancer deaths. In this context, the tumor microenvironment, and the extracellular matrix (ECM) pose as extremely relevant. Thus, this study aimed to explore the prognostic value of epithelial-to-mesenchymal transition (EMT), Wnt signaling, and ECM proteins expression in patients with non-small-cell lung carcinoma (NSCLC) with clinical stages I-IIIA. For that, we used 120 tissue sections from patients and evaluated the immunohistochemical, immunofluorescence, and transmission electron microscopy (TEM) to each of these markers. We also used in silico analysis to validate our data. We found a strong expression of E-cadherin and ß-catenin, which reflects the differential ECM invasion process. Therefore, we also noticed a strong expression of chondroitin sulfate (CS) and collagens III and V. This suggests that, after EMT, the basal membrane (BM) enhanced the motility of invasive cells. EMT proteins were directly associated with WNT5A, and collagens III and V, which suggests that the WNT pathway drives them. On the other hand, heparan sulfate (HS) was associated with WNT3A and SPARC, while WNT1 was associated with CS. Interestingly, the association between WNT1 and Col IV suggested negative feedback of WNT1 along the BM. In our cohort, WNT3A, WNT5A, heparan sulfate and SPARC played an important role in the Cox regression model, influencing the overall survival (OS) of patients, be it directly or indirectly, with the SPARC expression stratifying the OS into two groups: 97 months for high expression; and 65 for low expression. In conclusion, the present study identified a set of proteins that may play a significant role in predicting the prognosis of NSCLC patients with clinical stages I-IIIA.

Proposing Specific Neuronal Epithelial-to-Mesenchymal Transition Genes as an Ancillary Tool for Differential Diagnosis among Pulmonary Neuroendocrine Neoplasms.

Pulmonary neuroendocrine neoplasms (PNENs) are currently classified into four major histotypes, including typical carcinoid (TC), atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC), and small cell lung carcinoma (SCLC). This classification was designed to be applied to surgical specimens mostly anchored in morphological parameters, resulting in considerable overlapping among PNENs, which may result in important challenges for clinicians' decisions in the case of small biopsies. Since PNENs originate from the neuroectodermic cells, epithelial-to-mesenchymal transition (EMT) gene expression shows promise as biomarkers involved in the genotypic transformation of neuroectodermic cells, including mutation burden with the involvement of chromatin remodeling genes, apoptosis, and mitosis rate, leading to modification in final cellular phenotype. In this situation, additional markers also applicable to biopsy specimens, which correlate PNENs subtypes with systemic treatment response, are much needed, and current potential candidates are neurogenic EMT genes. This study investigated EMT genes expression and its association with PNENs histotypes in tumor tissues from 24 patients with PNENs. PCR Array System for 84 EMT-related genes selected 15 differentially expressed genes among the PNENs, allowing to discriminate TC from AC, LCNEC from AC, and SCLC from AC. Functional enrichment analysis of the EMT genes differentially expressed among PNENs subtypes showed that they are involved in cellular proliferation, extracellular matrix degradation, regulation of cell apoptosis, oncogenesis, and tumor cell invasion. Interestingly, four EMT genes (MAP1B, SNAI2, MMP2, WNT5A) are also involved in neurological diseases, in brain metastasis, and interact with platinum-based chemotherapy and tyrosine-kinase inhibitors. Collectively, these findings emerge as an important ancillary tool to improve the strategies of histologic diagnosis in PNENs and unveil the four EMT genes that can play an important role in driving chemical response in PNENs.

Evaluation of the combination of cytology and hybrid capture to safely predict the high-grade lesion status of patients treated with conization with large loop excision of the transformation zone.

OBJECTIVES: This study aimed to verify whether human papillomavirus (HPV) testing after conization treatment has some potential usefulness for predicting patients' outcome. STUDY DESIGN: One hundred and twenty women were treated for HSIL by conization with large loop excision of the transformation zone (LLETZ). Cytology, colposcopy-guided biopsy, and hybrid capture 2 (HC2) HPV DNA tests were performed before the surgical procedure and every 6 months for 2 years at follow-up. RESULTS: More than 90% of the patients tested positive for high-risk HPV prior to the surgical intervention. Six months after the cervical conization, 74.75% of the patients tested negative for high-risk HPV DNA, and 19.41% were positive. Of the women who were HC2 negative, 72 showed normal cytological smears, 3 ASC-US, 2 LSIL, and 1 HSIL. Of those who were HC2 positive, 8 showed normal smears, 2 ASC-US, 2 ASC-H, 5 LSIL, and 1 case had HSIL, AGC, and squamous cells invasive carcinoma. Clinically, the HSIL case with a negative HPV test did not show any sign of high-grade lesions, and the clinical follow-up did not show residual lesions. CONCLUSIONS: Negative HPV tests correlated with freedom from high-grade disease after 2 years of postconization follow-up, which strongly suggests that negative HPV tests predict the absence of cervical disease.

In Situ Overexpression of Matricellular Mechanical Proteins Demands Functional Immune Signature and Mitigates Non-Small Cell Lung Cancer Progression.

Non-small cell lung carcinoma (NSCLC) is a complex cancer biome composed of malignant cells embedded in a sophisticated tumor microenvironment (TME) combined with different initiating cell types, including immune cells and cancer-associated fibroblasts (CAFs), and extracellular matrix (ECM) proteins. However, little is known about these tumors' immune-matricellular relationship as functional and mechanical barriers. This study investigated 120 patients with NSCLC to describe the immune-matricellular phenotypes of their TME and their relationship with malignant cells. Immunohistochemistry (IHC) was performed to characterize immune checkpoints (PD-L1, LAG-3, CTLA-4+, VISTA 1), T cells (CD3+), cytotoxic T cells (CD8+, Granzyme B), macrophages (CD68+), regulatory T cells (FOXP3+, CD4+), natural killer cells (CD57+), and B lymphocytes (CD20+), whereas CAFs and collagen types I, III, and V were characterized by immunofluorescence (IF). We observed two distinct functional immune-cellular barriers-the first of which showed proximity between malignant cells and cytotoxic T cells, and the second of which showed distant proximity between non-cohesive nests of malignant cells and regulatory T cells. We also identified three tumor-associated matricellular barriers: the first, with a localized increase in CAFs and a low deposition of Col V, the second with increased CAFs, Col III and Col I fibers, and the third with a high amount of Col fibers and CAFs bundled and aligned perpendicularly to the tumor border. The Cox regression analysis was designed in two steps. First, we investigated the relationship between the immune-matricellular components and tumor pathological stage (I, II, and IIIA), and better survival rates were seen in patients whose tumors expressed collagen type III > 24.89 fibers/mm². Then, we included patients who had progressed to pathological stage IV and found an association between poor survival and tumor VISTA 1 expression > 52.86 cells/mm² and CD3+ ≤ 278.5 cells/mm². We thus concluded that differential patterns in the distribution of immune-matricellular phenotypes in the TME of NSCLC patients could be used in translational studies to predict new treatment strategies and improve patient outcome. These data raise the possibility that proteins with mechanical barrier function in NSCLC may be used by cancer cells to protect them from immune cell infiltration and immune-mediated destruction, which can otherwise be targeted effectively with immunotherapy or collagen therapy.

The expression patterns and prognostic significance of pleckstrin homology-like domain family A (PHLDA) in lung cancer and malignant mesothelioma.

BACKGROUND: Pleckstrin homology domain family A (PHLDA) genes play important roles in cancer cellular processes, including inhibiting Akt activation, repressing growth factor signaling, inhibiting the negative feedback of EGFR/ErbB2 signaling cells, and inducing apoptosis. However, the prognostic significance of PHLDA in non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MM) remains unclear. The present study investigates the associations between PHLDA expression patterns and their prognostic value in lung adenocarcinoma (LUAD) and MM. METHODS: We analyzed PHLDA family members at the genomic level in silico to explore their mRNA expression pattern and predictive significance in LUAD and MM. We then created a PHLDA-drug interaction network and a protein-protein interaction (PPI) network using different databases. Finally, we immunohistochemically assessed the protein expression of each PHLDA family member on tissue microarrays (TMAs) in both LUAD and MM cohorts with long-term follow-up. RESULTS: While PHLDA1 mRNA expression in both LUAD and MM was lower than that of normal tissue, PHLDA2 mRNA was significantly overexpressed in LUAD, and PHLDA3 mRNA was overexpressed in MM. In NSCLC, both low PHLDA1 mRNA expression and high PHLDA3 mRNA expression correlated with worse overall survival (OS) (P<0.01), whereas high PHLDA2 mRNA expression was associated with better OS (P<0.01). In MM, patients presenting high PHLDA1 and PHLDA2 mRNA expression had poor OS (P=0.01 and P<0.01, respectively). In addition, the PHLDA-drug interaction network indicated that several common drugs could potentially modulate PHLDA expression, and the PPI network suggested that PHLDA1 interacts with Notch family members, whereas PHLDA3 interacts with TP53. Our results also showed that the expression of PHLDA2 and PHLDA3 was significantly higher in LUAD and MM than that of PHLDA1 (P<0.05) and was associated with the risk of death. While patients with PHLDA2 >85.09 cells/mm2 had a low risk of death (P=0.01) and a median survival time of 48 months, those with PHLDA3 <70.38 cells/mm2 had a high risk of death (P=0.03) and a median survival time of 34 months. CONCLUSIONS: We shed light on the role of the PHLDA family as promising predictive biomarkers and potential therapeutic targets in LUAD and MM.

Pulmonary Neuroendocrine Neoplasms Overexpressing Epithelial-Mesenchymal Transition Mechanical Barriers Genes Lack Immune-Suppressive Response and Present an Increased Risk of Metastasis.

Typical carcinoids (TC), atypical carcinoids (AC), large cell neuroendocrine carcinomas (LCNEC), and small cell lung carcinomas (SCLC) encompass a bimodal spectrum of metastatic tumors with morphological, histological and histogenesis differences, The hierarchical structure reveals high cohesiveness between neoplastic cells by mechanical desmosomes barrier assembly in carcinoid tumors and LCNEC, while SCLC does not present an organoid arrangement in morphology, the neoplastic cells are less cohesive. However, the molecular mechanisms that lead to PNENs metastasis remain largely unknown and require further study. In this work, epithelial to mesenchymal transition (EMT) transcription factors were evaluated using a set of twenty-four patients with surgically resected PNENs, including carcinomas. Twelve EMT transcription factors (BMP1, BMP7, CALD1, CDH1, COL3A1, COL5A2, EGFR, ERBB3, PLEK2, SNAI2, STEAP1, and TCF4) proved to be highly expressed among carcinomas and downregulated in carcinoid tumors, whereas upregulation of BMP1, CDH2, KRT14 and downregulation of CAV2, DSC2, IL1RN occurred in both histological subtypes. These EMT transcription factors identified were involved in proliferative signals, epithelium desmosomes assembly, and cell motility sequential steps that support PNENs invasion and metastasis in localized surgically resected primary tumor. We used a two-stage design where we first examined the candidate EMT transcription factors using a whole-genome screen, and subsequently, confirmed EMT-like changes by transmission electron microscopy and then, the EMT-related genes that were differentially expressed among PNENs subtypes were predicted through a Metascape analysis by in silico approach. A high expression of these EMT transcription factors was significantly associated with lymph node and distant metastasis. The sequential steps for invasion and metastasis were completed by an inverse association between functional barrier created by PD-L1 immunosuppressive molecule and EMT transcriptional factors. Our study implicates upregulation of EMT transcription factors to high proliferation rates, mechanical molecular barriers disassembly and increased cancer cell motility, as a critical molecular event leading to metastasis risk in PNENs thus emerging as a promising tool to select and customize therapy.

Bone marrow-derived mononuclear cell therapy in experimental pulmonary and extrapulmonary acute lung injury.

OBJECTIVE: To hypothesize that bone marrow-derived mononuclear cell (BMDMC) therapy might act differently on lung and distal organs in models of pulmonary or extrapulmonary acute lung injury with similar mechanical compromises. The pathophysiology of acute lung injury differs according to the type of primary insult. DESIGN: Prospective, randomized, controlled, experimental study. SETTING: University research laboratory. MEASUREMENTS AND MAIN RESULTS: In control animals, sterile saline solution was intratracheally (0.05 mL) or intraperitoneally (0.5 mL) injected. Acute lung injury animals received Escherichia coli lipopolysaccharide intratracheally (40 microg, ALIp) or intraperitoneally (400 microg, ALIexp). Six hours after lipopolysaccharide administration, ALIp and ALIexp animals were further randomized into subgroups receiving saline (0.05 mL) or BMDMC (2 x 10) intravenously. On day 7, BMDMC led to the following: 1) increase in survival rate; 2) reduction in static lung elastance, alveolar collapse, and bronchoalveolar lavage fluid cellularity (higher in ALIexp than ALIp); 3) decrease in collagen fiber content, cell apoptosis in lung, kidney, and liver, levels of interleukin-6, KC (murine interleukin-8 homolog), and interleukin-10 in bronchoalveolar lavage fluid, and messenger RNA expression of insulin-like growth factor, platelet-derived growth factor, and transforming growth factor-beta in both groups, as well as repair of basement membrane, epithelium and endothelium, regardless of acute lung injury etiology; 4) increase in vascular endothelial growth factor levels in bronchoalveolar lavage fluid and messenger RNA expression in lung tissue in both acute lung injury groups; and 5) increase in number of green fluorescent protein-positive cells in lung, kidney, and liver in ALIexp. CONCLUSIONS: BMDMC therapy was effective at modulating the inflammatory and fibrogenic processes in both acute lung injury models; however, survival and lung mechanics and histology improved more in ALIexp. These changes may be attributed to paracrine effects balancing pro- and anti-inflammatory cytokines and growth factors, because a small degree of pulmonary BMDMC engraftment was observed.

Different histological patterns of type-V collagen levels confer a matrices-privileged tissue microenvironment for invasion in malignant tumors with prognostic value.

Previous studies have reported a close relationship between type V collagen (Col V) and tumor invasion and motility in both breast cancer (BC) and lung cancer (LC). The present work aims to determine whether the extracellular-matrix (ECM)-defined microenvironment influences patient clinical outcome and investigate to which extent histological patterns of Col V expression in malignant cells have a prognostic effect in patients. To that end, we examined the expression of Col V in the tissues of 174 primary tumors (MM, N = 82; LC, N = 41; and BC, N = 46) by immunohistochemistry. We found: (1) diffuse strong green birefringence in membrane and cytoplasm individualizing malignant cells in MM; (2) a focal and weak birefringence mainly in cytoplasmic membrane involving groups of malignant cells in LC and BC; (3) higher average H-score of Col V in MM than in LC and BC samples; (4) a direct correlation between Col V histologic pattern and TNM stage IV, status and median overall survival; (5) patients with LC in TNM stage I, and Col V ≤ 41.7 IOD/mm2 had a low risk of death and a median survival time more than 20 months; (6) patients with MM in TNM stage IV and Col V > 41.7 IOD/mm2 presented a high risk of death and a median survival time of just 20 months. These findings suggest that high levels of Col V individualizing malignant cells, as observed in MM, and low levels grouping malignant cells, as observed in LC and BC, confers different immune-privileged tissue microenvironment for tumor invasion with impact on prognosis of the patients.

An integrative histopathologic clustering model based on immuno-matrix elements to predict the risk of death in malignant mesothelioma.

OBJECTIVE: Previous studies have reported a close relationship between malignant mesothelioma (MM) and the immune matricial microenvironment (IMM). One of the major problems in these studies is the lack of adequate adjustment for potential confounders. Therefore, the aim of this study was to identify and quantify risk factors such as IMM and various tumor characteristics and their association with the subtype of MM and survival. METHODS: We examined IMM and other tumor markers in tumor tissues from 82 patients with MM. These markers were evaluated by histochemistry, immunohistochemistry, immunofluorescence, and morphometry. Logistic regression analysis, cluster analysis, and Cox regression analysis were performed. RESULTS: Hierarchical cluster analysis revealed two clusters of MM that were independent of clinicopathologic features. The high-risk cluster included MM with high tumor cellularity, high type V collagen (Col V) fiber density, and low CD8+ T lymphocyte density in the IMM. Our results showed that the risk of death was increased for patients with MM with high tumor cellularity (OR = 1.63, 95% CI = 1.29-2.89, P = .02), overexpression of Col V (OR = 2.60, 95% CI = 0.98-6.84, P = .04), and decreased CD8 T lymphocytes (OR = 1.001, 95% CI = 0.995-1.007, P = .008). The hazard ratio for the high-risk cluster was 2.19 (95% CI = 0.54-3.03, P < .01) for mortality from MM at 40 months. CONCLUSION: Morphometric analysis of Col V, CD8+ T lymphocytes, and tumor cellularity can be used to identify patients with high risk of death from MM.

Intravenous glutamine decreases lung and distal organ injury in an experimental model of abdominal sepsis.

INTRODUCTION: The protective effect of glutamine, as a pharmacological agent against lung injury, has been reported in experimental sepsis; however, its efficacy at improving oxygenation and lung mechanics, attenuating diaphragm and distal organ injury has to be better elucidated. In the present study, we tested the hypothesis that a single early intravenous dose of glutamine was associated not only with the improvement of lung morpho-function, but also the reduction of the inflammatory process and epithelial cell apoptosis in kidney, liver, and intestine villi. METHODS: Seventy-two Wistar rats were randomly assigned into four groups. Sepsis was induced by cecal ligation and puncture surgery (CLP), while a sham operated group was used as control (C). One hour after surgery, C and CLP groups were further randomized into subgroups receiving intravenous saline (1 ml, SAL) or glutamine (0.75 g/kg, Gln). At 48 hours, animals were anesthetized, and the following parameters were measured: arterial oxygenation, pulmonary mechanics, and diaphragm, lung, kidney, liver, and small intestine villi histology. At 18 and 48 hours, Cytokine-Induced Neutrophil Chemoattractant (CINC)-1, interleukin (IL)-6 and 10 were quantified in bronchoalveolar and peritoneal lavage fluids (BALF and PLF, respectively). RESULTS: CLP induced: a) deterioration of lung mechanics and gas exchange; b) ultrastructural changes of lung parenchyma and diaphragm; and c) lung and distal organ epithelial cell apoptosis. Glutamine improved survival rate, oxygenation and lung mechanics, minimized pulmonary and diaphragmatic changes, attenuating lung and distal organ epithelial cell apoptosis. Glutamine increased IL-10 in peritoneal lavage fluid at 18 hours and bronchoalveolar lavage fluid at 48 hours, but decreased CINC-1 and IL-6 in BALF and PLF only at 18 hours. CONCLUSIONS: In an experimental model of abdominal sepsis, a single intravenous dose of glutamine administered after sepsis induction may modulate the inflammatory process reducing not only the risk of lung injury, but also distal organ impairment. These results suggest that intravenous glutamine may be a potentially beneficial therapy for abdominal sepsis.

Usefulness of complementary next-generation sequencing and quantitative immunohistochemistry panels for predicting brain metastases and selecting treatment outcomes of non-small cell lung cancer.

To demonstrate the usefulness of complementary next-generation sequencing (NGS) and immunohistochemistry (IHC) counting, we analyzed 196 patients with non-small cell lung cancer who underwent surgical resection and adjuvant chemotherapy. Formalin-fixed, paraffin-embedded samples of adenocarcinoma (ADC), squamous cell carcinoma, and large cell carcinoma were used to prepare tissue microarrays and were examined by protein H-score IHC image analysis and NGS for oncogenes and proto-oncogenes and genes of tumor suppressors, immune checkpoints, epithelial-mesenchymal transition factors, tyrosine kinase receptors, and vascular endothelial growth factors. In patients with brain metastases, primary tumors expressed lower PIK3CA protein levels. Overexpression of p53 and a higher PD-L1 protein H-score were detected in patients who underwent surgical treatment followed by chemotherapy as compared with those who underwent only surgical treatment The absence of brain metastases was associated with wild-type sequences of genes EGFR, CD267, CTLA-4, and ZEB1. The combination of protein overexpression according to IHC and mutation according to NGS was rare (ie, represented by a very low percentage of concordant cases), except for p53 and vascular endothelial growth factor. Our data suggest that protein levels detected by IHC may be a useful complementary tool when mutations are not detected by NGS and also support the idea to expand this approach beyond ADC to include squamous cell carcinoma and even large cell carcinoma, particularly for patients with unusual clinical characteristics. Conversely, well-pronounced immunogenotypic features seemed to predict the clinical outcome after univariate and multivariate analyses. Patients with a solid ADC subtype and mutated genes EGFR, CTLA4, PDCD1LG2, or ZEB1 complemented with PD-L1 or p53 protein lower expression that only underwent surgical treatment who develop brain metastases may have the worst prognosis.