Data documento
02/05/2024

Título
Establishment and molecular characterization of HCB‑541, a novel and aggressive human cutaneous squamous cell carcinoma cell line

Autor(es)
LAUS, Ana Carolina - GOMES, Izabela Natalia Faria - SILVA, Aline Larissa Virginio da - SILVA, Luciane Sussuchi da - MILAN, Mirella Baroni - TEIXEIRA, Silvia Aparecida - MARTINS, Ana Carolina Baptista Moreno - PEREIRA, Letícia do Nascimento Braga - CARVALHO, Carlos Eduardo Barbosa de - CROVADOR, Camila Souza - PAULA, Flávia Escremin de - NASCIMENTO, Flávia Caroline - FREITAS, Helder Teixeira de - VAZQUEZ, Vinicius de Lima - REIAS, Rui Manuel - OLIVEIRA, Renato José da Silva.

Palavras chave
Cutaneous squamous cell carcinoma - Molecular profile - Cell line establishment - In vivo model

Resumo
Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer that can result in significant morbidity, although it is usually well-managed and rarely metastasizes. However, the lack of commercially available cSCC cell lines hinders our understanding of this disease. This study aims to establish and characterize a new metastatic cSCC cell line derived from a Brazilian patient. A tumor biopsy was taken from a metastatic cSCC patient, immortalized, and named HCB-541 after several passages. The cytokeratin expression profile, karyotypic alterations, mutational analysis, mRNA and protein differential expression, tumorigenic capacity in xenograft models, and drug sensitivity were analyzed. The HCB-541 cell line showed a doubling time between 20 and 30 h and high tumorigenic capacity in the xenograft mouse model. The HCB-541 cell line showed hypodiploid and hypotetraploidy populations. We found pathogenic mutations in TP53 p.(Arg248Leu), HRAS (Gln61His) and TERT promoter (C228T) and high-level microsatellite instability (MSI-H) in both tumor and cell line. We observed 37 cancer-related genes differentially expressed when compared with HACAT control cells. The HCB-541 cells exhibited high phosphorylated levels of EGFR, AXL, Tie, FGFR, and ROR2, and high sensitivity to cisplatin, carboplatin, and EGFR inhibitors. Our study successfully established HCB-541, a new cSCC cell line that could be useful as a valuable biological model for understanding the biology and therapy of metastatic skin cancer.

Citação
Laus AC, Gomes INF, da Silva ALV, da Silva LS, Milan MB, AparecidaTeixeira S, Martin ACBM, do Nascimento Braga Pereira L, de Carvalho CEB, Crovador CS, de Paula FE, Nascimento FC, de Freitas HT, de Lima Vazquez V, Reis RM, da Silva-Oliveira RJ. Establishment and molecular characterization of HCB-541, a novel and aggressive human cutaneous squamous cell carcinoma cell line. Hum Cell. 2024 Apr 3. doi: 10.1007/s13577-024-01054-1. Epub ahead of print. PMID: 38565739.

Data documento
18/10/2024

Título
Gene expression profiles (GEPs) of immuno-oncologic pathways as predictors of response to checkpoint inhibitors in advanced NSCLC

Autor(es)
Pedro De Marchi - Leticia Ferro Leal - Luciane Sussuchi da Silva - Rodrigo de Oliveira Cavagna - Flavio Augusto Ferreira da Silva - Vinicius Duval da Silva - Eduardo Ca da Silva - Augusto O Saito - Vladmir C Cordeiro de Lima - Rui Manuel Reis.

Palavras chave
GEP; Gama interferon; Immunotherapy; Lung-tumor signature; NSCLC; Non-small cell lung cancer; PD-L1; TIS; anti-CTLA-4; anti-PD-(L)1; gene expression profile.

Resumo
Background: Immune checkpoint inhibitors (ICIs) revolutionized non-small-cell lung cancer (NSCLC) treatment. However, improving patients selection for this therapy is needed. Gene expression profile (GEP) is a promising biomarker tool. We assessed the predictive value of 48 onco-immune GEPs in an NSCLC real-world scenario. Methods: Retrospective cohort of Brazilian NSCLC patients treated with ICIs in any line. GEP was assessed in FFPE tumor tissue using the nCounter PanCancer IO360 panel, comprising 770 cancer immune genes. Results: The median age of the 135 patients was 61 years old, most male (57.8 %), history of smoking (83.6 %), ECOG-PS 0-1 (88.7 %), clinical stage IV (91.9 %) and adenocarcinoma (65.1 %). First-line ICI in 40 % of cases, alone or in combination with chemotherapy. The median follow-up was 28 months, overall survival after starting immunotherapy (post-immunotherapy survival - PIS) was 17.8 months, and real-world progression-free survival was 5.5 months. The GEP analysis was possible in 66 patients. We found that 14 different GEPs associated with PIS, namely IDO1, PD-L2, Cytotoxicity, Cytotoxic Cells, IFN Downstream, CTLA4, PD-L1, TIGIT, Lymphoid, Immunoproteasome, Exhausted CD8, IFN Gamma, TIS and APM. TIS and IFN-γ were the most significant GEPs associated with favorable outcomes. The median PIS for patients with high TIS expression was 29.2 versus 15.5 months (HR 0.42; 95 %CI; 0.17-0.67; p<0.05) for those with low expression. Similar results were observed for IFN-γ. Conclusions: The TIS (tumor inflammation signature) and IFN-γ signatures constitute predictive biomarkers to identify patients with NSCLC patients who would possibly benefit from ICI therapies.

Citação
Gene expression profiles (GEPs) of immuno-oncologic pathways as predictors of response to checkpoint inhibitors in advanced NSCLC. De Marchi P, Leal LF, da Silva LS, Cavagna RO, da Silva FAF, da Silva VD, da Silva EC, Saito AO, de Lima VCC, Reis RM.Transl Oncol. 2024 Jan;39:101818. doi: 10.1016/j.tranon.2023.101818. Epub 2023 Oct 31.PMID: 37918166

Data documento
13/03/2025

Título
Molecular profile of driver genes in lung adenocarcinomas of Brazilian patients who have never smoked: implications for targeted therapies

Autor(es)
Rodrigo de Oliveira Cavagna - Flávia Escremim de Paula - Gustavo Noriz Berardinelli - Murilo Bonatelli - Iara Santana - Eduardo Caetano Albino da Silva - Gustavo Ramos Teixeira - Beatriz Garbe Zaniolo - Flávio Augusto Ferreira da Silva - Carlos Eduardo Baston Silva - Josiane Mourão Dias - Marcela Gondim Borges Guimarães - Camila Pinto Barone - Alexandre Arthur Jacinto - Rachid Eduardo Noleto da Nóbrega Oliveira - José Elias Miziara - Pedro De Marchi - Miguel A. Molina-Vila - Letícia Ferro Leal - Rui Manuel Reis

Palavras chave
lung adenocarcinoma; molecular profile; driver mutations; never smoker; Latin America.

Resumo
Introduction: Lung cancer in never-smoker (LCINS) patients accounts for 20% of lung cancer cases, and its biology remains poorly understood, particularly in genetically admixed populations. We elucidated the molecular profile of driver genes in Brazilian LCINS. Methods: The mutational and gene fusion status of 119 lung adenocarcinomas from self-reported never-smoker patients, was assessed using targeted sequencing (NGS), nCounter, and immunohistochemistry. A panel of 46 ancestry-informative markers determined patients’ genetic ancestry. Results: The most frequently mutated gene was EGFR (49.6%), followed by TP53 (39.5%), ALK (12.6%), ERBB2 (7.6%), KRAS (5.9%), PIK3CA (1.7%), and less than 1% alterations in RET, NTRK1, MET∆ex14, PDGFRA, and BRAF. Except for TP53 and PIK3CA, all other alterations were mutually exclusive. Genetic ancestry analysis revealed a predominance of European (71.1%), and a higher African ancestry was associated with TP53 mutations. Conclusion: Brazilian LCINS exhibited a similar molecular profile to other populations, except the increased ALK and TP53 alterations. Importantly, 73% of these patients have actionable alterations that are suitable for targeted treatments.

Citação
Cavagna RO, Escremim de Paula F, Berardinelli GN, Bonatelli M, Santana I, Albino da Silva EC, Teixeira GR, Zaniolo BG, Mourão Dias J, Ferreira da Silva FA, Baston Silva CE, Guimarães MGB, Barone CP, Jacinto AA, Noleto da Nóbrega Oliveira RE, Miziara JE, De Marchi P, Molina-Vila MA, Leal LF, Reis RM. Oncologist. 2024 Oct 3;29(10):e1419-e1424. doi: 10.1093/oncolo/oyae129. PMID: 38944844

Data documento
26/04/2024

Título
PD L1 expression and microsatellite instability (MSI) in cancer of unknown primary site

Autor(es)
JÚNIOR, João Neif Antonio - PRETO, Daniel D.’Almeida - LAZARINI, Maria Eduarda Zanatta Neder - LIMA, Marcos Alves de - BONATELLI, Murilo - BERARDINELLI, Gustavo Noriz - SILVA, Vinicius Duval da - PINHEIRO, Céline - REIS, Rui Manuel, CÁRCANO, Flavio Mavignier -

Palavras chave
Neoplasms - Unknown primary - Microsatellite instability - Immune checkpoint proteins - Immunotherapy

Resumo
Background Cancer of unknown primary site (CUP) is a heterogeneous group of tumors for which the origin remains unknown. Clinical outcomes might be influenced by regulatory processes in its microenvironment. Microsatellite instability (MSI) is a predictive biomarker for cancer immunotherapy and its status, as well as co-occurrence with PD-L1 expression, is poorly evaluated. We aim to evaluate the expression of PD-L1 and the status of MSI in CUP and their possible associations with clinical–pathological features. Methods The combined positive score (CPS) PD-L1 expression was evaluated by immunohistochemistry. MSI status was assessed using a hexa-plex marker panel by polymerase chain reaction followed by fragment analysis. Results Among the 166 cases, MSI analysis was conclusive in 120, with two cases being MSI positive (1.6%). PD-L1 expression was positive in 18.3% of 109 feasible cases. PD-L1 expression was significantly associated with non-visceral metastasis and a dominance of nodal metastasis. The median overall survival (mOS) was 3.7 (95% CI 1.6–5.8) months and patients who expressed PD-L1 achieved a better mOS compared to those who did not express PD-L1 (18.7 versus 3.0 months, p-value: < .001). ECOG-PS equal to or more than two and PD-L1 expression were independent prognostic factors in multivariate analysis (2.37 and 0.42, respectively). Conclusion PD-L1 is expressed in a subset (1/5) of patients with CUP and associated with improved overall survival, while MSI is a rare event. There is a need to explore better the tumor microenvironment as well as the role of immunotherapy to change such a bad clinical outcome.

Citação
Junior JNA, Preto DD, Lazarini MEZN, de Lima MA, Bonatelli M, Berardinelli GN, da Silva VD, Pinheiro C, Reis RM, Cárcano FM. PD-L1 expression and microsatellite instability (MSI) in cancer of unknown primary site. Int J Clin Oncol. 2024 Mar 25. doi: 10.1007/s10147-024-02494-3. Epub ahead of print. PMID: 38528294.

Data documento
19/03/2025

Título
PD-L1 expression and microsatellite instability (MSI) in cancer of unknown primary site

Autor(es)
João Neif Antonio Junior - Daniel D Almeida Preto - Maria Eduarda Zanatta Neder Lazarini - Marcos Alves de Lima - Murilo Bonatelli - Gustavo Noriz Berardinelli - Vinicius Duval da Silva - Céline Pinheiro - Rui Manuel Reis - Flavio Mavignier Cárcano.

Palavras chave
Immune checkpoint proteins; Immunotherapy; Microsatellite instability; Neoplasms; Unknown primary.

Resumo
Background: Cancer of unknown primary site (CUP) is a heterogeneous group of tumors for which the origin remains unknown. Clinical outcomes might be influenced by regulatory processes in its microenvironment. Microsatellite instability (MSI) is a predictive biomarker for cancer immunotherapy and its status, as well as co-occurrence with PD-L1 expression, is poorly evaluated. We aim to evaluate the expression of PD-L1 and the status of MSI in CUP and their possible associations with clinical-pathological features. Methods: The combined positive score (CPS) PD-L1 expression was evaluated by immunohistochemistry. MSI status was assessed using a hexa-plex marker panel by polymerase chain reaction followed by fragment analysis. Results: Among the 166 cases, MSI analysis was conclusive in 120, with two cases being MSI positive (1.6%). PD-L1 expression was positive in 18.3% of 109 feasible cases. PD-L1 expression was significantly associated with non-visceral metastasis and a dominance of nodal metastasis. The median overall survival (mOS) was 3.7 (95% CI 1.6-5.8) months and patients who expressed PD-L1 achieved a better mOS compared to those who did not express PD-L1 (18.7 versus 3.0 months, p-value: < .001). ECOG-PS equal to or more than two and PD-L1 expression were independent prognostic factors in multivariate analysis (2.37 and 0.42, respectively). Conclusion: PD-L1 is expressed in a subset (1/5) of patients with CUP and associated with improved overall survival, while MSI is a rare event. There is a need to explore better the tumor microenvironment as well as the role of immunotherapy to change such a bad clinical outcome.

Citação
unior JNA, Preto DD, Lazarini MEZN, de Lima MA, Bonatelli M, Berardinelli GN, da Silva VD, Pinheiro C, Reis RM, Cárcano FM.Int J Clin Oncol. 2024 Jun;29(6):726-734. doi: 10.1007/s10147-024-02494-3.Epub 2024 Mar 25. PMID: 38528294

Data documento
18/12/2023

Título
SERS sensing for cancer biomarker: Approaches and directions

Autor(es)
Lorena Vázquez-Iglesias - Giovanna Maria Stanfoca Casagrande - Daniel García-Lojo - Letícia Ferro Leal - Tien Anh Ngo - Jorge Pérez-Juste - Rui Manue

Palavras chave
Cancer biomarkers; Invasive and non-invasive biomarkers; Microfluidic devices; Point of care detection; Surface-enhanced Raman scattering (SERS).

Resumo
These days, cancer is thought to be more than just one illness, with several complex subtypes that require different screening approaches. These subtypes can be distinguished by the distinct markings left by metabolites, proteins, miRNA, and DNA. Personalized illness management may be possible if cancer is categorized according to its biomarkers. In order to stop cancer from spreading and posing a significant risk to patient survival, early detection and prompt treatment are essential. Traditional cancer screening techniques are tedious, time-consuming, and require expert personnel for analysis. This has led scientists to reevaluate screening methodologies and make use of emerging technologies to achieve better results. Using time and money saving techniques, these methodologies integrate the procedures from sample preparation to detection in small devices with high accuracy and sensitivity. With its proven potential for biomedical use, surface-enhanced Raman scattering (SERS) has been widely used in biosensing applications, particularly in biomarker identification. Consideration was given especially to the potential of SERS as a portable clinical diagnostic tool. The approaches to SERS-based sensing technologies for both invasive and non-invasive samples are reviewed in this article, along with sample preparation techniques and obstacles. Aside from these significant constraints in the detection approach and techniques, the review also takes into account the complexity of biological fluids, the availability of biomarkers, and their sensitivity and selectivity, which are generally lowered. Massive ways to maintain sensing capabilities in clinical samples are being developed recently to get over this restriction. SERS is known to be a reliable diagnostic method for treatment judgments. Nonetheless, there is still room for advancement in terms of portability, creation of diagnostic apps, and interdisciplinary AI-based applications. Therefore, we will outline the current state of technological maturity for SERS-based cancer biomarker detection in this article. The review will meet the demand for reviewing various sample types (invasive and non-invasive) of cancer biomarkers and their detection using SERS. It will also shed light on the growing body of research on portable methods for clinical application and quick cancer detection.

Citação
SERS sensing for cancer biomarker: Approaches and directions. Vázquez-Iglesias L, Stanfoca Casagrande GM, García-Lojo D, Ferro Leal L, Ngo TA, Pérez-Juste J, Reis RM, Kant K, Pastoriza-Santos I. Bioact Mater. 2023 Dec 31;34:248-268. doi: 10.1016/j.bioactmat.2023.12.018. eCollection 2024 Apr. PMID: 38260819