MARCELO LUIZ BALANCIN
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- The expression patterns and prognostic significance of pleckstrin homology-like domain family A (PHLDA) in lung cancer and malignant mesothelioma(2021) BALDAVIRA, Camila M.; MACHADO-RUGOLO, Juliana; PRIETO, Tabatha G.; BASTOS, Daniel R.; BALANCIN, Marcelo; AB'SABER, Alexandre M.; YAEGASHI, Lygia B.; SOUZA, Paola C.; FARHAT, Cecilia; TAKAGAKI, Teresa Y.; NAGAI, Maria Ap; CAPELOZZI, Vera L.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/mm(2) had a low risk of death (P=0.01) and a median survival time of 48 months, those with PHLDA3 <70.38 cells/mm(2) 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(2021) PRIETO, Tabatha Gutierrez; BALDAVIRA, Camila Machado; MACHADO-RUGOLO, Juliana; FARHAT, Cecilia; OLIVIERI, Eloisa Helena Ribeiro; SA, Vanessa Karen de; SILVA, Eduardo Caetano Abilio da; BALANCIN, Marcelo Luiz; SABER, Alexandre Muxfeldt Ab; TAKAGAKI, Teresa Yae; LIMA, Vladmir Claudio Cordeiro de; CAPELOZZI, Vera LuizaTypical 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.
- Mesothelin expression remodeled the immune-matrix tumor microenvironment predicting the risk of death in patients with malignant pleural mesothelioma(2023) QUALIOTTO, Aline Nery; BALDAVIRA, Camila Machado; BALANCIN, Marcelo; AB'SABER, Alexandre; TAKAGAKI, Teresa; CAPELOZZI, Vera LuizaBackgroundThe 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.MethodsThe 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.ResultsMost 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.ConclusionTumor 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.
conferenceObject Pleckstrin Homology-Like (PHLDA) Domain Family Members Immunoexpression as Prognostic Marker in Lung Cancer and Mesothelioma(2021) BALDAVIRA, C. Machado; PRIETO, T.; BALANCIN, M.; EHER, E.; FERNEZLIAN, S.; SOUZA, P.; NAGAI, M.; CAPELOZZI, V.- A semi-automated microscopic image analysis method for scoring Ki-67 nuclear immunostaining(2023) FERNEZLIAN, S. M.; BALDAVIRA, C. M.; SOUZA, M. L. F. de; FARHAT, C.; VILHENA, A. F. de; PEREIRA, J. C. N.; CAMPOS, J. R. M. de; TAKAGAKI, T.; BALANCIN, M. L.; AB'SABER, A. M.; CAPELOZZI, V. L.Nuclear proliferation marker MIB-1 (Ki-67) immunohistochemistry (IHC) is used to examine tumor cell proliferation. However, the diagnostic or prognostic value of the Ki-67 nuclear staining intensity and location, defined as nuclear gradient (NG), has not been assessed. This study examined the potential association between Ki-67 NG and cell cycle phases and its effect on the prognosis of pulmonary typical carcinoid (PTC) tumors. We propose a method for classifying the NG of Ki-67 during the cell cycle and compare the results between PTC, pulmonary adenocarcinoma (PAD), and breast ductal carcinoma (BDC). A literature review and objective analysis of IHC-stained paraffin sections were used to determine the Ki-67 labeling index and composed a stratification of the NG into NG1, NG2, and NG3/4 categories. A semi-automated image analysis protocol was established to determine the Ki-67 NG in PTC, PAD, and BDC. High intraobserver consistency and moderate interobserver agreement were achieved in the determination of Ki-67 NG in tumor specimens. NG1 and NG2 were lower in PTC than in PAD and BDC. Cox multivariate analysis of PTC after adjusting for age and number of metastatic lymph nodes showed that Ki-67 NG1 and NG2 significantly predicted clinical outcomes. The semi-automated method for quantification of Ki-67 nuclear immunostaining proposed in this study could become a valuable diagnostic and prognostic tool in PTC.
- The Immunological Landscape of M1 and M2 Macrophages and Their Spatial Distribution in Patients with Malignant Pleural Mesothelioma(2023) LABERIANO-FERNANDEZ, Caddie; BALDAVIRA, Camila Machado; MACHADO-RUGOLO, Juliana; TAMEGNON, Auriole; PANDURENGAN, Renganayaki Krishna; AB'SABER, Alexandre Muxfeldt; BALANCIN, Marcelo Luiz; TAKAGAKI, Teresa Yae; NAGAI, Maria Aparecida; CAPELOZZI, Vera Luiza; PARRA, Edwin RogerSimple Summary Identifying biomarkers to guide immunotherapy regimens remains an unmet clinical need in malignant pleural mesothelioma. A potential source of such markers is tumor-associated macrophages (TAMs), which contribute to the immunosuppressive microenvironment of mesothelioma. By examining distinct subsets of pleural macrophages to identify their gene signatures and protein expression, we found that TAMs preferentially contribute to M2a and M2b phenotypes, and M2a, M2b, and M2c more specifically contributed to immune tolerance. CD206, ARG1, CD274, CD163, and MRP8-14 are potential therapeutic targets in this disease.Abstract 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.
- Dissecting and Reconstructing Matrix in Malignant Mesothelioma Through Histocell-Histochemistry Gradients for Clinical Applications(2022) BALANCIN, Marcelo Luiz; BALDAVIRA, Camila Machado; PRIETO, Tabatha Gutierrez; MACHADO-RUGOLO, Juliana; FARHAT, Cecilia; ASSATO, Aline Kawassaki; VELOSA, Ana Paula Pereira; TEODORO, Walcy Rosolia; AB'SABER, Alexandre Muxfeldt; TAKAGAKI, Teresa Yae; CAPELOZZI, Vera LuizaBackgroundMalignant 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. MethodsIn 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. ResultsEpithelioid 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 mm(2)) for epithelioid MM. ConclusionDifferential 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.
- Different histological patterns of type-V collagen levels confer a matrices-privileged tissue microenvironment for invasion in malignant tumors with prognostic value(2020) BALANCIN, Marcelo Luiz; TEODORO, Walcy Rosolia; BALDAVIRA, Camila Machado; PRIETO, Tabatha Gutierrez; FARHAT, Cecilia; VELOSA, Ana Paula; SOUZA, Paola da Costa; YAEGASHI, Lygia Bertalha; AB'SABER, Alexandre Muxfeldt; TAKAGAKI, Teresa Yae; CAPELOZZI, Vera LuizaPrevious 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.