An open-label, single-arm, prospective, multi-center, tandem two-stage designed phase II study to evaluate the efficacy of fulvestrant in women with recurrent/metastatic estrogen receptor-positive gynecological malignancies (FUCHSia study).

OBJECTIVE: This study aimed to evaluate fulvestrant efficacy in women with estrogen receptor-positive low-grade gynecological cancers. The primary objective was to determine the response rate. Secondary objectives were progression-free survival, clinical benefit, duration of response, safety, tolerability, and quality of life. METHODS: FUCHSia is an open-label, single-arm, prospective, multi-center phase II study. The study population included patients with recurrent/metastatic low-grade gynecological malignancies with estrogen receptor positivity who received a maximum of two lines of previous hormonal therapy. Patients received fulvestrant (FASLODEX, AstraZeneca) via two intramuscular injections (250 mg/5 mL each) in the gluteal muscle on day 1, day 15, day 29, and then every 28 days thereafter until disease progression, withdrawal from the trial due to any unacceptable adverse event, or withdrawal of patient consent. RESULTS: A total of 15 patients (uterine sarcoma n=4; sex cord-stromal ovarian tumors n=3; endometrial carcinoma n=4; serous ovarian cancer n=4) were enrolled. Median follow-up was 48 weeks (interquartile range (IQR) 26-122) in the uterine sarcoma cohort, 63 weeks (IQR 28-77) for sex cord-stromal tumors, 19 weeks (IQR 17-21) for endometrial carcinoma, and 60 weeks (IQR 40-119) for serous ovarian cancer. One partial response according to Response Evaluation Criteria in Solid Tumors v1.1 was observed in one uterine sarcoma patient. No responses were observed in the other cohorts. However, stable disease was observed in three uterine sarcomas (median duration 12 weeks), three sex cord-stromal tumors (median duration 32 weeks), and four low-grade serous ovarian cancer patients (median duration 20 weeks), leading to a disease control rate of 100% for these tumor types. All patients with endometrial carcinoma showed progressive disease. CONCLUSION: Fulvestrant may control tumor growth in recurrent/metastatic estrogen receptor-positive low-grade gynecological malignancies of specific histology. Further studies are needed to confirm these results.

Proliferative verrucous and homogeneous Leukoplakias exhibit differential methylation patterns.

OBJECTIVE: Proliferative verrucous leukoplakia (PVL) is considered a clinically distinct entity from other oral leucoplakias (OLs) due to its clinical presentation and evolution. However, molecular differences between them remain unclear. We aimed to determine whether there are methylation differences between PVL and other forms of OLs. MATERIALS AND METHODS: Oral biopsies from 12 patients with PVL, eight patients with homogeneous leucoplakia (HL), and 10 healthy individuals were obtained for a genome-wide DNA methylation analysis via the Infinium EPIC Platform. RESULTS: A total of 1815 differentially methylated CpGs were found between PVL and HL, with a prominent state of hypermethylation in HL patients. CpGs covered 813 genes with distinct roles, including cell adhesion, extracellular matrix organization, and cell and synaptic signaling. 43% of these genes had been previously described in cancer and associated with prognosis. We developed a multinomial logistic regression model able to differentiate HL, PVL, and control samples. The model had a cross-validated estimate of 73% and included differentially methylated cancer-related genes between the pathological conditions and the healthy donors, including ADNP, BRCA2, CDK13, GNB1, NIN, NUMB, PIK3C2B, PTK2, SHISA4, THSD7B, WWP1, and ZNF292. It also included CpGs covering differentially methylated genes in HL (MEN1 and TNRC6B) and PVL (ACOXL, ADH1B, CAMTA1, CBFA2T3, CPXM2, LRFN2, SORCS2, and SPN). CONCLUSIONS: PVL and HL present differential methylation patterns that could be linked to their differential clinical behavior. Our findings show the potential of methylation markers and suggest novel diagnostic biomarkers.

A Sonic Hedgehog Pathway Score to Predict the Outcome of Resected Non-Small Cell Lung Cancer Patients.

BACKGROUND: Mutations and deregulations in components of the Hedgehog (Hh) pathway have been associated with cancer onset and tumor growth in different malignancies, but their role in non-small cell lung cancer (NSCLC) remains unclear. This study aims to investigate the expression pattern of the main components of the Hh pathway in tumor and adjacent normal tissue biopsies of resected NSCLC patients. METHODS: The relative expression of GLI1, PTCH1, SHH, and SMO was analyzed by quantitative polymerase chain reaction (PCR) in a cohort of 245 NSCLC patients. Results were validated in an independent cohort of NSCLC patients from The Cancer Genome Atlas (TCGA). RESULTS: We found that SMO and GLI1 were overexpressed in the tumor compared with normal-paired tissue, whereas PTCH1 and SHH were underexpressed. In addition, patients with higher expression levels of PTCH1 presented better outcomes. A gene expression score, called the Hedgehog Score, was calculated using a multivariable model including analyzed components of the Hh signaling pathway. NSCLC patients with a high Hedgehog Score had significantly shorter relapse-free survival (RFS) and overall survival (OS) than patients with a low score, especially at stage I of the disease. Similarly, patients in the adenocarcinoma (ADC) subcohort had shorter RFS and OS. Multivariate Cox analysis exhibited that the Hedgehog Score is an independent prognostic biomarker for OS in both the entire training cohort and the ADC subcohort. The Hedgehog Score was validated in an independent cohort of NSCLC patients from TCGA, which confirmed its prognostic value. CONCLUSIONS: Our results provide relevant prognostic data for NSCLC patients and support further studies on the Hh pathway.

Oral cancers preceded by proliferative verrucous leukoplakia exhibit distinctive molecular features.

OBJECTIVE: Proliferative verrucous leukoplakia (PVL) has high rates of malignant transformation into oral squamous cell carcinoma (OSCC), but the clinical and evolutionary pattern of OSCC from PVL (PVL-OSCC) is more favorable than that of OSCC not preceded by PVL (OSCC). Here, we aimed to explore the pathophysiologic differences between PVL-OSCC and OSCC through transcriptomic and DNA methylation analyses. MATERIALS AND METHODS: In this case-control study, oral biopsies from 8 PVL-OSCC and 10 OSCC patients were obtained for global sequencing using RNAseq and a genome-wide DNA methylation analysis via the Infinium EPIC Platform (graphical abstract). RESULTS: One hundred and thirty-three differentially expressed genes (DEGs) were detected, 94 of them upregulated in OSCC. Most of these genes were previously described in cancer and associated with prognosis. The integrative analysis revealed 26 DEGs, corresponding to 37 CpGs, whose promoters were regulated by DNA methylation. Twenty-nine of the CpGs were found as hypermethylated in PVL-OSCC. Only 5 of the genes that were aberrantly methylated and differentially expressed were upregulated in PVL-OSCC patients, whereas 21 were underexpressed. CONCLUSIONS: PVL-OSCC patients presented lower expression of cancer-related genes. Hypermethylation of the promoter region of many genes was also noticed, indicating that DNA methylation could be a regulatory mechanism.

On the Oral Microbiome of Oral Potentially Malignant and Malignant Disorders: Dysbiosis, Loss of Diversity, and Pathogens Enrichment.

The role of dysbiosis in the development and progression of oral potentially malignant disorders (OPMDs) remains largely unknown. Here, we aim to characterize and compare the oral microbiome of homogeneous leucoplakia (HL), proliferative verrucous leukoplakia (PVL), oral squamous cell carcinoma (OSCC), and OSCC preceded by PVL (PVL-OSCC). Fifty oral biopsies from HL (n = 9), PVL (n = 12), OSCC (n = 10), PVL-OSCC (n = 8), and healthy (n = 11) donors were obtained. The sequence of the V3-V4 region of the 16S rRNA gene was used to analyze the composition and diversity of bacterial populations. In the cancer patients, the number of observed amplicon sequence variants (ASVs) was lower and Fusobacteriota constituted more than 30% of the microbiome. PVL and PVL-OSCC patients had a higher abundance of Campilobacterota and lower Proteobacteria than any other group analyzed. A penalized regression was performed to determine which species were able to distinguish groups. HL is enriched in Streptococcus parasanguinis, Streptococcus salivarius, Fusobacterium periodonticum, Prevotella histicola, Porphyromonas pasteri, and Megasphaera micronuciformis; PVL is enriched in Prevotella salivae, Campylobacter concisus, Dialister pneumosintes, and Schaalia odontolytica; OSCC is enriched in Capnocytophaga leadbetteri, Capnocytophaga sputigena, Capnocytophaga gingivalis, Campylobacter showae, Metamycoplasma salivarium, and Prevotella nanceiensis; and PVL-OSCC is enriched in Lachnospiraceae bacterium, Selenomonas sputigena, and Prevotella shahii. There is differential dysbiosis in patients suffering from OPMDs and cancer. To the best of our knowledge, this is the first study comparing the oral microbiome alterations in these groups; thus, additional studies are needed.

Neutrophil Extracellular Traps and Cancer: Trapping Our Attention with Their Involvement in Ovarian Cancer.

Neutrophils, the most abundant circulating leukocytes, play a well-known role in defense against pathogens through phagocytosis and degranulation. However, a new mechanism involving the release of neutrophil extracellular traps (NETs) composed of DNA, histones, calprotectin, myeloperoxidase, and elastase, among others, has been described. The so-called NETosis process can occur through three different mechanisms: suicidal, vital, and mitochondrial NETosis. Apart from their role in immune defense, neutrophils and NETs have been involved in physiopathological conditions, highlighting immunothrombosis and cancer. Notably, neutrophils can either promote or inhibit tumor growth in the tumor microenvironment depending on cytokine signaling and epigenetic modifications. Several neutrophils' pro-tumor strategies involving NETs have been documented, including pre-metastatic niche formation, increased survival, inhibition of the immune response, and resistance to oncologic therapies. In this review, we focus on ovarian cancer (OC), which remains the second most incidental but the most lethal gynecologic malignancy, partly due to the presence of metastasis, often omental, at diagnosis and the resistance to treatment. We deepen the state-of-the-art on the participation of NETs in OC metastasis establishment and progression and their involvement in resistance to chemo-, immuno-, and radiotherapies. Finally, we review the current literature on NETs in OC as diagnostic and/or prognostic markers, and their contribution to disease progression at early and advanced stages. The panoramic view provided in this article might pave the way for enhanced diagnostic and therapeutic strategies to improve the prognosis of cancer patients and, specifically, OC patients.

Liver prometastatic reaction: Stimulating factors and responsive cancer phenotypes.

Cancer is first a localized tissue disorder, whose soluble and exosomal molecules and invasive cells induce a host response providing the stromal components of the primary tumor microenvironment (TME). Once the TME is developed, cancer-derived molecules and cells can more efficiently spread out and a whole-body response takes place, whose pathophysiological changes may result in a paraneoplastic syndrome. Remote organ-specific prometastatic reactions may also occur at this time, facilitating metastatic activities of circulating tumor cells (CTCs) through premetastatic niche development at targeted organs. However, additional signaling factors from the inter-organ communication network involved in the pathophysiology and comorbidities of cancer patients may also regulate prometastatic reaction-stimulating effects of cancer and non-cancer tissue factors. This article provides a conceptual overview of our ongoing clinical research on the liver prometastatic reaction (LPR) of patients with colorectal cancer (CRC), their portal vein- and hepatic artery-driven LPR-Stimulating Factors (LPR-SF), and their resulting LPR-derived Metastasis-Stimulating Factors (LPR-MSF) acting on liver-invading CRC cells. In addition, we also provide new insights on the molecular subtyping of LPR-responsive cancer phenotypes in patients with CRC and melanoma; and on how to investigate and interpret the prometastatic infrastructure in the real pathophysiological context of patients with cancer undergoing surgical procedures and receiving pharmacological treatments with multiple side effects, including those affecting the LPR, its stimulating factors and responsive cancer phenotypes.

Soluble galectin-3 as a microenvironment-relevant immunoregulator with prognostic and predictive value in lung adenocarcinoma.

Despite the success of therapies in lung cancer, more studies of new biomarkers for patient selection are urgently needed. The present study aims to analyze the role of galectin-3 (GAL-3) in the lung tumor microenvironment (TME) using tumorspheres as a model and explore its potential role as a predictive and prognostic biomarker in non-small cell lung cancer patients. For in vitro studies, lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) primary cultures from early-stage patients and commercial cell lines were cultured, using tumorsphere-forming assays and adherent conditions for the control counterparts. We analyzed the pattern of secretion and expression of GAL-3 using reverse transcription-quantitative real-time PCR (RTqPCR), immunoblot, immunofluorescence, flow cytometry, and immunoassay analysis. Our results using three-dimensional (3D) models of lung tumor cells revealed that soluble GAL-3 (sGAL-3) is highly expressed and secreted. To more accurately mimic the TME, a co-culture of tumorspheres and fibroblasts was used, revealing that GAL-3 could be important as an immunomodulatory molecule expressed and secreted in the TME, modulating immunosuppression through regulatory T cells (TREGS ). In the translational phase, we confirmed that patients with high expression levels of GAL-3 had more TREGS , which suggests that tumors may be recruiting this population through GAL-3. Next, we evaluated levels of sGAL-3 before surgery in LUAD and LUSC patients, hypothesizing that sGAL-3 could be used as an independent prognostic biomarker for overall survival and relapse-free survival in early-stage LUAD patients. Additionally, levels of sGAL-3 at pretreatment and first response assessment from plasma to predict clinical outcomes in advanced LUAD and LUSC patients treated with first-line pembrolizumab were evaluated, further supporting that sGAL-3 has a high efficiency in predicting durable clinical response to pembrolizumab with an area under curve of 0.801 (P = 0.011). Moreover, high levels might predict decreased progression-free survival and OS to anti-PD-1 therapy, with sGAL-3 being a prognosis-independent biomarker for advanced LUAD.

Analgesic Efficacy and Safety of Intrathecal Morphine or Intercostal Levobupivacaine in Lung Cancer Patients after Major Lung Resection Surgery by Videothoracoscopy: A Prospective Randomized Controlled Trial.

Background: Lung resection using video-assisted thoracoscopic surgery (VATS) improves surgical accuracy and postoperative recovery. Unfortunately, moderate-to-severe acute postoperative pain is still inherent to the procedure, and a technique of choice has not been established for the appropriate control of pain. In this study, we aimed to compare the efficacy and safety of intrathecal morphine (ITM) with that of intercostal levobupivacaine (ICL). Methods: We conducted a single-center, prospective, randomized, observer-blinded, controlled trial among 181 adult patients undergoing VATS (ISRCTN12771155). Participants were randomized to receive ITM or ICL. Primary outcomes were the intensity of pain, assessed by a numeric rating scale (NRS) over the first 48 h after surgery, and the amount of intravenous morphine used. Secondary outcomes included the incidence of adverse effects, length of hospital stay, mortality, and chronic post-surgical pain at 6 and 12 months after surgery. Results: There are no statistically significant differences between ITM and ICL groups in pain intensity and evolution at rest. In cough-related pain, differences in pain trajectories over time are observed. Upon admission to the PACU, cough-related pain was higher in the ITM group, but the trend reversed after 6 h. There are no significant differences in adverse effects. The rate of chronic pain was low and did not differ significantly between groups. Conclusions: ITM can be considered an adequate and satisfactory regional technique for the control of acute postoperative pain in VATS, compatible with the multimodal rehabilitation and early discharge protocols used in these types of surgeries.

PI3K/mTOR inhibition induces tumour microenvironment remodelling and sensitises pS6high uterine leiomyosarcoma to PD-1 blockade.

BACKGROUND: Uterine leiomyosarcomas (uLMS) are aggressive tumours with poor prognosis and limited treatment options. Although immune checkpoint blockade (ICB) has proven effective in some 'challenging-to-treat' cancers, clinical trials showed that uLMS do not respond to ICB. Emerging evidence suggests that aberrant PI3K/mTOR signalling can drive resistance to ICB. We therefore explored the relevance of the PI3K/mTOR pathway for ICB treatment in uLMS and explored pharmacological inhibition of this pathway to sensitise these tumours to ICB. METHODS: We performed an integrated multiomics analysis based on TCGA data to explore the correlation between PI3K/mTOR dysregulation and immune infiltration in 101 LMS. We assessed response to PI3K/mTOR inhibitors in immunodeficient and humanized uLMS patient-derived xenografts (PDXs) by evaluating tumour microenvironment modulation using multiplex immunofluorescence. We explored response to single-agent and a combination of PI3K/mTOR inhibitors with PD-1 blockade in humanized uLMS PDXs. We mapped intratumoural dynamics using single-cell RNA/TCR sequencing of serially collected biopsies. RESULTS: PI3K/mTOR over-activation (pS6high) associated with lymphocyte depletion and wound healing immune landscapes in (u)LMS, suggesting it contributes to immune evasion. In contrast, PI3K/mTOR inhibition induced profound tumour microenvironment remodelling in an ICB-resistant humanized uLMS PDX model, fostering adaptive anti-tumour immune responses. Indeed, PI3K/mTOR inhibition induced macrophage repolarisation towards an anti-tumourigenic phenotype and increased antigen presentation on dendritic and tumour cells, but also promoted infiltration of PD-1+ T cells displaying an exhausted phenotype. When combined with anti-PD-1, PI3K/mTOR inhibition led to partial or complete tumour responses, whereas no response to single-agent anti-PD-1 was observed. Combination therapy reinvigorated exhausted T cells and induced clonal hyper-expansion of a cytotoxic CD8+ T-cell population supported by a CD4+ Th1 niche. CONCLUSIONS: Our findings indicate that aberrant PI3K/mTOR pathway activation contributes to immune escape in uLMS and provides a rationale for combining PI3K/mTOR inhibition with ICB for the treatment of this patient population.

Increased levels of NETosis biomarkers in high-grade serous ovarian cancer patients' biofluids: Potential role in disease diagnosis and management.

Introduction: High-grade serous ovarian cancer (HGSOC) is the second most frequent gynecological malignancy but the most lethal, partially due to the spread of the disease through the peritoneal cavity. Recent evidence has shown that, apart from their role in immune defense through phagocytosis and degranulation, neutrophils are able to participate in cancer progression through the release of neutrophil extracellular traps (NETs) in a process called NETosis. NETs are composed of DNA, histones, calprotectin, myeloperoxidase (MPO) and elastase and the NETosis process has been proposed as a pre-requisite for the establishment of omental metastases in early stages of HGSOC. Nevertheless, its role in advanced stages remains to be elucidated. Therefore, our principal aim is to characterize a NETosis biomarker profile in biofluids from patients with advanced HGSOC and control women. Methods: Specifically, five biomarkers of NETosis (cell-free DNA (cfDNA), nucleosomes, citrullinated histone 3 (citH3), calprotectin and MPO) were quantified in plasma and peritoneal fluid (PF) samples from patients (n=45) and control women (n=40). Results: Our results showed that HGSOC patients presented a higher concentration of cfDNA, citH3 and calprotectin in plasma and of all five NETosis biomarkers in PF than control women. Moreover, these biomarkers showed a strong ability to differentiate the two clinical groups. Interestingly, neoadjuvant treatment (NT) seemed to reduce NETosis biomarkers mainly systemically (plasma) compared to the tumor environment (PF). Discussion: In conclusion, NETosis biomarkers are present in the tumor environment of patients with advanced HGSOC, which might contribute to the progression of the disease. Besides, plasma cfDNA and calprotectin could represent minimally invasive surrogate biomarkers for HGSOC. Finally, NT modifies NETosis biomarkers levels mainly at the systemic level.

Identification, Culture and Targeting of Cancer Stem Cells.

Chemoresistance, tumor progression, and metastasis are features that are frequently seen in cancer that have been associated with cancer stem cells (CSCs). These cells are a promising target in the future of cancer therapy but remain largely unknown. Deregulation of pathways that govern stemness in non-tumorigenic stem cells (SCs), such as Notch, Wnt, and Hedgehog pathways, has been described in CSC pathogenesis, but it is necessary to conduct further studies to discover potential new therapeutic targets. In addition, some markers for the identification and characterization of CSCs have been suggested, but the search for specific CSC markers in many cancer types is still under development. In addition, methods for CSC cultivation are also under development, with great heterogeneity existing in the protocols used. This review focuses on the most recent aspects of the identification, characterization, cultivation, and targeting of human CSCs, highlighting the advances achieved in the clinical implementation of therapies targeting CSCs and remarking those potential areas where more research is still required.

Airway Management of Patients with Suspected or Confirmed COVID-19: Survey Results from Physicians from 19 Countries in Latin America.

Airway management during the COVID-19 pandemic has been one of the most challenging aspects of care that anesthesiologists and intensivists face. This study was conducted to evaluate the management of tracheal intubation in patients with suspected or confirmed COVID-19 infection. This is a cross-sectional and international multicenter study based on a 37-item questionnaire. The survey was available to physicians who had performed intubations and tracheostomies in patients with suspected or confirmed COVID-19 and had provided informed consent to participate. The primary outcome is the preference to use a specific device for tracheal intubation. Secondary outcomes are clinical practice variables, use of video laryngoscopes, difficult airway management, and safety features to prevent cross-infection. This study included 2411 physicians who performed an average of 11.90 and 20.67 tracheal intubations in patients diagnosed or suspected of having COVID-19 disease, respectively. Physicians were mainly from the specialties of Anesthesiology (61.2%) and Intensive Care (7.4%). COVID-19 infection diagnosed by positive PCR or serology in physicians participating in intubation in this study was 15.1%. Respondents considered preoxygenation for more than three minutes very useful (75.7%). The preferred device for tracheal intubation was the video laryngoscope (64.8%). However, the direct laryngoscope (57.9%) was the most commonly used, followed by the video laryngoscope (37.5%). The preferred device to facilitate intubation was the Eschmann guide (34.2%). Percutaneous tracheostomy was the preferred technique (39.5%) over the open tracheostomy (22%). The predicted or unpredicted difficult airway management in these patients was preferably performed with a video laryngoscope (61.7% or 63.7, respectively). Intubation was mostly performed by two or more expert airway physicians (61.6%). The use of personal protective equipment increased the practitioners' discomfort during intubation maneuvers. The video laryngoscope is the preferred device for intubating patients with COVID-19, combined with the Eschmann guide, flexible stylet within the endotracheal tube, or Frova guide to facilitate intubation. The sub-analysis of the two groups of physicians by the level of intubation experience showed a higher use of the video laryngoscope (63.4%) in the experts group and no significant differences between the two groups in terms of cross-infection rates in physicians, in their preference for the use of the video laryngoscope or in the number of intubations performed in confirmed or suspected COVID-19 patients.

Analysis of Exosomal Cargo Provides Accurate Clinical, Histologic and Mutational Information in Non-Small Cell Lung Cancer.

Lung cancer is a malignant disease with high mortality and poor prognosis, frequently diagnosed at advanced stages. Nowadays, immense progress in treatment has been achieved. However, the present scenario continues to be critical, and a full comprehension of tumor progression mechanisms is required, with exosomes being potentially relevant players. Exosomes are membranous vesicles that contain biological information, which can be transported cell-to-cell and modulate relevant processes in the hallmarks of cancer. The present research aims to characterize the exosomes' cargo and study their role in NSCLC to identify biomarkers. We analyzed exosomes secreted by primary cultures and cell lines, grown in monolayer and tumorsphere formations. Exosomal DNA content showed molecular alterations, whereas RNA high-throughput analysis resulted in a pattern of differentially expressed genes depending on histology. The most significant differences were found in XAGE1B, CABYR, NKX2-1, SEPP1, CAPRIN1, and RIOK3 genes when samples from two independent cohorts of resected NSCLC patients were analyzed. We identified and validated biomarkers for adenocarcinoma and squamous cell carcinoma. Our results could represent a relevant contribution concerning exosomes in clinical practice, allowing for the identification of biomarkers that provide information regarding tumor features, prognosis and clinical behavior of the disease.

Oral microbiome in Proliferative Verrucous Leukoplakia exhibits loss of diversity and enrichment of pathogens.

OBJECTIVES: Oral microbiome plays an important role in oral diseases. Among them, proliferative verrucous leucoplakia (PVL) is an uncommon form of progressive multifocal leukoplakia with a worryingly rate of malignant transformation. Here, we aimed to characterize the oral microbiome of PVL patients and compare it with those of healthy controls. MATERIAL AND METHODS: Oral biopsies from ten PVL patients and five healthy individuals were obtained and used to compare their microbial communities. The sequence of the V3-V4 region of 16S rRNA gene was used as the taxonomic basis to estimate and analyze the composition and diversity of bacterial populations present in the samples. RESULTS: Our results show that the oral microbial composition and diversity are significantly different among PVL patients and healthy donors. The average number of observed operational taxonomic units (OTUs) was higher for healthy donors than for PVL, proving a loss of diversity in PVL. Several OTUs were found to be more abundant in either group. Among those that were significantly enriched in PVL patients, potential protumorigenic pathogens like Oribacterium sp. oral taxon 108, Campylobacter jejuni, uncultured Eubacterium sp., Tannerella, and Porphyromonas were identified. CONCLUSION: Oral microbiome dysbiosis was found in patients suffering from PVL. To the best of our knowledge, this is the first study investigating the oral microbiome alterations in PVL and, due to the limited number of participants, additional studies are needed. Oral microbiota-based biomarkers may be helpful in predicting the risks for the development of PVL.

Differentially methylated genes in proliferative verrucous leukoplakia reveal potential malignant biomarkers for oral squamous cell carcinoma.

OBJECTIVES: To explore the pathophysiology of proliferative verrucous leucoplakia (PVL) through a methylated DNA immunoprecipitation and high-throughput sequencing (MeDIP-seq) case-control study. MATERIALS AND METHODS: Oral biopsies from ten PVL patients and five healthy individuals were obtained and used to compare their epigenetic patterns. Network biology methods and integrative analyses of MeDIP-seq and RNAseq data were applied to investigate functional relations among differentially methylated genes (DMGs). The value of selected genes as malignant biomarkers was evaluated in a large cohort of oral squamous cell carcinoma (OSCC) patients from TCGA. RESULTS: A total of 4647 differentially methylated regions were found, with a prominent state of hypermethylation in PVL patients. At the gene level, differentially methylated regions (DMRs) covered 826 genes with distinct roles, including transcription factors and binding proteins with functions in cell adhesion, migration, proliferation, regulation of transcription, bone morphogenesis, and cell signalling. Network analysis revealed three major hubs, two of them collecting proteins related to the response of the patients to PVL and treatment and one hub collecting proteins related to PVL and cancer. The integrative analysis revealed 8 genes (ARTN, CD8A, GATA3, HOXD10, MYO7A, OSR2, PLCB1, and SPOCK2) significantly upregulated in PVL compared to control and 5 genes (ANKRD6, DLG2, GPX3, PITX2, and ZNF736) significantly downregulated. The status of de-regulation found for PVL patients was concordant with what was found for OSCC samples compared to normal adjacent tissue. CONCLUSION: Our findings show the potential of methylation markers in PVL and suggest novel OSCC diagnostic biomarkers which may boost the development of novel epigenetic-based therapies.

Analysis of the Gut Microbiota: An Emerging Source of Biomarkers for Immune Checkpoint Blockade Therapy in Non-Small Cell Lung Cancer.

BACKGROUND: The human gut harbors around 1013-1014 microorganisms, collectively referred to as gut microbiota. Recent studies have found that the gut microbiota may have an impact on the interaction between immune regulation and anti-cancer immunotherapies. METHODS: In order to characterize the diversity and composition of commensal microbiota and its relationship with response to immune checkpoint blockade (ICB), 16S ribosomal DNA (rDNA) sequencing was performed on 69 stool samples from advanced non-small cell lung cancer (NSCLC) patients prior to treatment with ICB. RESULTS: The use of antibiotics and ICB-related skin toxicity were significantly associated with reduced gut microbiota diversity. However, antibiotics (ATB) usage was not related to low ICB efficacy. Phascolarctobacterium was enriched in patients with clinical benefit and correlated with prolonged progression-free survival, whereas Dialister was more represented in patients with progressive disease, and its higher relative abundance was associated with reduced progression-free survival and overall survival, with independent prognostic value in multivariate analysis. CONCLUSIONS: Our results corroborate the relation between the baseline gut microbiota composition and ICB clinical outcomes in advanced NSCLC patients, and provide novel potential predictive and prognostic biomarkers for immunotherapy in NSCLC.

3D printing novel in vitro cancer cell culture model systems for lung cancer stem cell study.

Two-dimensional (2D) in vitro cell cultures and laboratory animals have been used traditionally as the gold-standard preclinical cancer model systems. However, for cancer stem cell (CSC) studies, they exhibit notable limitations on simulating native environment, which depreciate their translatability for clinical development purposes. In this study, different three-dimensional (3D) printing platforms were used to establish novel 3D cell cultures enriched in CSCs from non-small cell lung cancer (NSCLC) patients and cell lines. Rigid scaffolds with an elevated compressive modulus and uniform pores and channels were produced using different filaments. Hydrogel-based scaffolds were printed with a more irregular distribution of pores and a lower compressive modulus. As a 3D model of reference, suspension spheroid cultures were established. Therein, cancer cell lines exhibited enhanced proliferation profiles on rigid scaffolds compared to the same cells grown on either hydrogel scaffolds or tumor spheres. Meanwhile, primary cancer cells grew considerably better on hydrogel scaffolds or in tumor sphere culture, compared to cells grown on rigid scaffolds. Gene expression analysis confirmed that tumor spheres and cells seeded on hydrogel scaffolds significantly overexpress most of stemness and invasion promoters tested compared to control cells grown in 2D culture. A different phenomenon was observed within cells growing on the rigid scaffolds, where fewer significant variations in gene expression were detected. Our findings provide strong evidence for the advantageous usage of 3D printed models, especially those which use GelMA-PEGDA hydrogels as the primary scaffold material, for studying lung CSCs. The results demonstrated that the 3D printed scaffolds were better to mimic tumor complexity and regulate cancer cell behavior than in vivo 2D culture models.