Genomic epidemiology of SARS-CoV-2 in large university hospital cohort: the UnCoVER-Brazil project.

This work aimed to study the role of different SARS-CoV-2 lineages in the epidemiology of multiple waves of the COVID-19 pandemic in Ribeirão Preto (São Paulo state), with comparison within Brazil and globally. Viral genomic sequencing was combined with clinical and sociodemographic information of 2,379 subjects at a large Brazilian hospital. On the whole 2,395 complete SARS-CoV-2 genomes were obtained from April 2020 to January 2022. We report variants of concern (VOC) and interest (VOI) dynamics and the role of Brazilian lineages. We identified three World Health Organization VOCs (Gamma, Delta, Omicron) and one VOI (Zeta), which caused distinct waves in this cohort. We also identified 47 distinct Pango lineages. Consistent with the high prevalence of Gamma in Brazil, Pango lineage P.1 dominated infections in this cohort for half of 2021. Each wave of infection largely consisted of a single variant group, with each new group quickly and completely rising to dominance. Despite increasing vaccination in Brazil starting in 2021, this pattern was observed throughout the study and is consistent with the hypothesis that herd immunity tends to be SARS-CoV-2 variant-specific and does not broadly protect against COVID-19.

Impact of the Canonical Wnt Pathway Activation on the Pathogenesis and Prognosis of Adamantinomatous Craniopharyngiomas.

CTNNB1 mutations and abnormal ß-catenin distribution are associated with the pathogenesis of adamantinomatous craniopharyngioma (aCP). We evaluated the expression of the canonical Wnt pathway components in aCPs and its association with CTNNB1 mutations and tumor progression. Tumor samples from 14 aCP patients and normal anterior pituitary samples from eight individuals without pituitary disease were studied. Gene expression of Wnt pathway activator (WNT4), inhibitors (SFRP1, DKK3, AXIN1, and APC), transcriptional activator (TCF7), target genes (MYC, WISP2, and, CDH1), and Wnt modulator (TP53) was evaluated by qPCR. ß-Catenin, MYC, and WISP2 expression was determined by immunohistochemistry (IHC). The transcription levels of all genes studied, except APC, were higher in aCPs as compared to controls and TCF7 mRNA levels correlated with CTNNB1 mutation. CDH1 mRNA was overexpressed in tumor samples of patients with disease progression in comparison to those with stable disease. ß-Catenin was positive and aberrantly distributed in 11 out of 14 tumor samples. Stronger ß-catenin immunostaining associated positively with tumor progression. MYC positive staining was found in 10 out of 14 cases, whereas all aCPs were negative for WISP2. Wnt pathway genes were overexpressed in aCPs harboring CTNNB1 mutations and in patients with progressive disease. Recurrence was associated with stronger staining for ß-catenin. These data suggest that Wnt pathway activation contributes to the pathogenesis and prognosis of aCPs.

HPA axis dysregulation, NR3C1 polymorphisms and glucocorticoid receptor isoforms imbalance in metabolic syndrome.

CONTEXT: Metabolic syndrome (MetS) shares several similarities with hypercortisolism. OBJECTIVES: To evaluate hypothalamic-pituitary-adrenal (HPA) axis sensitivity to dexamethasone (DEX), NR3C1 single nucleotide polymorphisms (SNPs), and expression of glucocorticoid receptor (GR) isoforms and cytokines in peripheral immune cells of MetS patients and controls. DESIGN: Prospective study with 40 MetS patients and 40 controls was conducted at the Ribeirão Preto Medical School University Hospital. METHODS: Plasma and salivary cortisol were measured in basal conditions and after 0.25, 0.5, and 1 mg of DEX given at 2300 h. In addition, p.N363S (rs6195), p.ER22/23EK (rs6189-6190), and BclI (rs41423247) SNPs were evaluated by quantitative polymerase chain reaction allelic discrimination. Exons 3 to 9 and exon/intron boundaries of NR3C1 were sequenced. GR isoforms and cytokines (IL1B, IL2, IL4, IL6, IL8, IL10, IFNγ, TNFα) expression were assessed by quantitative polymerase chain reaction. RESULTS: Plasma and salivary cortisol (nmol/L) after 1-mg DEX were higher in MetS patients compared with controls (PF: 70.2 ± 17.3 vs 37.9 ± 2.6, P = .02, and SF: 4.9 ± 1.7 vs 2.2 ± 0.3, P < .0001). After all DEX doses, a lower number of MetS patients suppressed plasma and salivary cortisol compared with controls. The BclI genotypic frequencies (%) differed between patients (CC:56/CG:44) and controls (CC:50/CG:32.5/GG:17.5) (P = .03). The GRß was overexpressed (fold = 100.0; P = .002) and IL4 (fold = -265.0; P < .0001) was underexpressed in MetS. CONCLUSION: MetS patients exhibited decreased HPA sensitivity to glucocorticoid feedback. Moreover, the BclI polymorphism lower frequency, GRß overexpression, and IL4 underexpression might underlie the molecular mechanism of glucocorticoid resistance in MetS. Thus, HPA axis dysregulation might contribute to MetS pathogenesis.

Immune Checkpoint Inhibitors in Tumors Harboring Homologous Recombination Deficiency: Challenges in Attaining Efficacy.

Cancer cells harbor genomic instability due to accumulated DNA damage, one of the cancer hallmarks. At least five major DNA Damage Repair (DDR) pathways are recognized to repair DNA damages during different stages of the cell cycle, comprehending base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR), and non-homologous end joining (NHEJ). The unprecedented benefits achieved with immunological checkpoint inhibitors (ICIs) in tumors with mismatch repair deficiency (dMMR) have prompted efforts to extend this efficacy to tumors with HR deficiency (HRD), which are greatly sensitive to chemotherapy or PARP inhibitors, and also considered highly immunogenic. However, an in-depth understanding of HRD's molecular underpinnings has pointed to essential singularities that might impact ICIs sensitivity. Here we address the main molecular aspects of HRD that underlie a differential profile of efficacy and resistance to the treatment with ICIs compared to other DDR deficiencies.

Predicting immunotherapy response through genomics.

Immune checkpoint inhibitors (ICI) aim to restore the immune system anti-tumor function by blocking two inhibitory axes: CTLA-4/CD28 and PD1/PDL1. ICI is established as a treatment option for multiple cancers, but their remarkable clinical impact is observed only in a fraction of patients. Together with their adverse effects and high cost, it's imperative to identify patients who are likely to benefit from this type of treatment. Genomic features represent promising candidates as predictive biomarkers of response to ICI, with agnostic FDA-approvals of an anti-PD1 drug for tumors with microsatellite instability and tumors with a high mutational burden. Other genomic markers are also emerging to help refine patient selection. In this review, we discuss recent progress in genomic biomarkers development and its challenges, with a focus on alterations in the neoantigen burden, immune, and oncogenic pathways.

Cell-free DNA promotes malignant transformation in non-tumor cells.

Cell-free DNA is present in different biological fluids and when released by tumor cells may contribute to pro-tumor events such as malignant transformation of cells adjacent to the tumor and metastasis. Thus, this study analyzed the effect of tumor cell-free DNA, isolated from the blood of prostate cancer patients, on non-tumor prostate cell lines (RWPE-1 and PNT-2). To achieve this, we performed cell-free DNA quantification and characterization assays, evaluation of gene and miRNA expression profiling focused on cancer progression and EMT, and metabolomics by mass spectrometry and cellular migration. The results showed that tumor-free cell DNA was able to alter the gene expression of MMP9 and CD44, alter the expression profile of nine miRNAs, and increased the tryptophan consumption and cell migration rates in non-tumor cells. Therefore, tumor cell-free DNA was capable of altering the receptor cell phenotype, triggering events related to malignant transformation in these cells, and can thus be considered a potential target for cancer diagnosis and therapy.

Precision oncology: as much expectations as limitations.

It is encouraging to witness the recent price reduction and expanded access to next generation sequencing platforms, the increasing number of investments and publications on new targets and respective targeted drugs, as well as the worldwide excitement with anti-cancer personalised therapies. This editorial aims to highlight the limitations regarding the small proportion of solid cancers potentially eligible for the use of molecular-based targeted drugs until now. It also covers the expected clinical benefits in refractory patients treated by matched therapies, and detailed cost-effectiveness analysis of the use of DNA sequencing analysis oncology practice in an academic and large-scale community.

Outcomes, toxicity profiles, and prognostic factors of unresectable head and neck cancer treated with chemoradiotherapy

Introduction: Head and neck cancer (HNC) is a heterogeneous group of neoplasms that can have a poor prognosis when diagnosed in advanced stages. The optimized treatment for locally advanced and unresectable lesions is mainly based on radiotherapy associated with chemotherapy (cisplatin 100mg/m²), however, at the expense of a high toxicity index. Objective: Evaluate whether chemoradiotherapy (CRT) ­ the goldstandard treatment for locally advanced head and neck cancer (HNC) ­ is effective in the study population. Methods: This is a retrospective study aimed at determining the efficacy of definitive CRT in patients with unresectable HNC treated between the 2012 and 2018 in a single institution. The following outcomes were evaluated: objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and toxicity profiles. Results: Fifty-two (52) patients diagnosed with HNC between 2012 and 2018 met the inclusion criteria. The ORR was 84.6%, with 50% showing complete response. Median PFS and OS were 35.3 and 52 months, respectively. Analysis of the toxicity profiles revealed that 69.2% of the patients presented grade 3-4 toxicity. Completion of two or more cycles of cisplatin-based therapy (HR 3.57 [95% CI 1.25­10.25]; p p<0.001), grade 3-4 toxicity (HR 0.27 [95% CI 0.09-0.8] ­ p<0.02), and Charlson comorbidity index (CCI) (HR 3.23 [95% CI 1.26­8.29]; p<0.001) were significantly associated with survival. Regarding toxicity, prophylactic low-level laser therapy (HR 0.48 [95% CI 0.27­0.86]; p<0.001 for those without this practice) and body mass index (BMI) (HR 0.27 [95% CI 0.09­0.76]; p<0.01) showed statistical significance. Conclusion: CRT was effective to treat HNC in the study population, with PFS and OS comparable to those reported in larger sample studies and lower toxicity grade. Some clinical characteristics have been identified as prognostic and/or predictive factors.

Glucocorticoid resistance in dialysis patients reduces long-term graft survival after kidney transplantation.

Glucocorticoid (GC) resistance has been observed in chronic kidney disease (CKD) patients on dialysis. It can be evaluated by binding assays based on the dissociation constant (Kd), which is inversely proportional to ligand affinity. CKD patients with GC resistance had increased number of acute rejection episodes. We followed up 26 patients that underwent kidney transplantation to observe whether GC resistance could affect the response to acute rejection episode pulse therapy and the long-term allograft outcome. Using Kaplan-Meier survival curve, GC resistant patients showed lower acute rejection-free survival (p=0.03) and lower kidney allograft survival (p=0.008). No difference was found regarding number of deaths. Multivariate logistic regression showed that high Kd value was an independent predictor of lower kidney allograft survival (p=0.001). There was a negative Spearman correlation between Kd and kidney allograft survival (r=-0.88, p=0.03). In conclusion, our findings indicate the usefulness of binding assay performed previously to kidney transplantation to define GC resistance. In addition, the dissociation constant (Kd) is a reliable and independent predictive marker of higher frequency of acute rejection episodes, lower rejection-free graft survival, poor response of acute rejection episodes to methylprednisolone pulse therapy, and lower kidney allograft survival in a long-term follow-up.

Components of the canonical and non-canonical Wnt pathways are not mis-expressed in pituitary tumors.

INTRODUCTION: Canonical and non-canonical Wnt pathways are involved in the genesis of multiple tumors; however, their role in pituitary tumorigenesis is mostly unknown. OBJECTIVE: This study evaluated gene and protein expression of Wnt pathways in pituitary tumors and whether these expression correlate to clinical outcome. MATERIALS AND METHODS: Genes of the WNT canonical pathway: activating ligands (WNT11, WNT4, WNT5A), binding inhibitors (DKK3, sFRP1), ß-catenin (CTNNB1), ß-catenin degradation complex (APC, AXIN1, GSK3ß), inhibitor of ß-catenin degradation complex (AKT1), sequester of ß-catenin (CDH1), pathway effectors (TCF7, MAPK8, NFAT5), pathway mediators (DVL-1, DVL-2, DVL-3, PRICKLE, VANGL1), target genes (MYB, MYC, WISP2, SPRY1, TP53, CCND1); calcium dependent pathway (PLCB1, CAMK2A, PRKCA, CHP); and planar cell polarity pathway (PTK7, DAAM1, RHOA) were evaluated by QPCR, in 19 GH-, 18 ACTH-secreting, 21 non-secreting (NS) pituitary tumors, and 5 normal pituitaries. Also, the main effectors of canonical (ß-catenin), planar cell polarity (JNK), and calcium dependent (NFAT5) Wnt pathways were evaluated by immunohistochemistry. RESULTS: There are no differences in gene expression of canonical and non-canonical Wnt pathways between all studied subtypes of pituitary tumors and normal pituitaries, except for WISP2, which was over-expressed in ACTH-secreting tumors compared to normal pituitaries (4.8x; p = 0.02), NS pituitary tumors (7.7x; p = 0.004) and GH-secreting tumors (5.0x; p = 0.05). ß-catenin, NFAT5 and JNK proteins showed no expression in normal pituitaries and in any of the pituitary tumor subtypes. Furthermore, no association of the studied gene or protein expression was observed with tumor size, recurrence, and progressive disease. The hierarchical clustering showed a regular pattern of genes of the canonical and non-canonical Wnt pathways randomly distributed throughout the dendrogram. CONCLUSIONS: Our data reinforce previous reports suggesting no activation of canonical Wnt pathway in pituitary tumorigenesis. Moreover, we describe, for the first time, evidence that non-canonical Wnt pathways are also not mis-expressed in the pituitary tumors.

ZAC1 and SSTR2 are downregulated in non-functioning pituitary adenomas but not in somatotropinomas.

INTRODUCTION: There are few data regarding ZAC1 expression in clinically non-functioning pituitary adenomas (NFPA). Because somatotropinomas and NFPA behave differently with respect to tumor shrinkage during somatostatin analogs (SA) therapy, we sought to compare the ZAC1 and somatostatin receptor (sstr) types 1, 2, 3 and 5 mRNA expression in these two pituitary adenoma subtypes and in normal human pituitaries. METHODS: ZAC1 and SSTR mRNA expression levels were evaluated using real-time RT-PCR (TaqMan) in 20 NFPA and compared with the expression levels in 23 somatotropinomas and five normal pituitaries. The NFPA invasiveness was evaluated using magnetic resonance imaging with Hardy's modified criteria. Ki-67 and p53 were evaluated using immunohistochemistry. RESULTS: A total of 20 patients with NFPA [6 males, median age 56 years (range: 30-78)], 23 with acromegaly [12 males, median age 43 years (range: 24-57)] and five normal pituitaries [4 males, median age 48 years (range: 36-54)] were included. Four of the patients (20%) had Hardy's grade 2 tumors; all of the others had Hardy's grade 3 tumors. The Ki-67 median expression was 2.35 (range: 0.2-9.23), and only four of the tumors (20%) were positive for p53. The ZAC1 mRNA expression was significantly lower in NFPA than in somatotropinomas and in normal pituitaries (p<0.001 for both), as well as the SSTR2 (p=0.001 and 0.01, respectively). The SSTR3 expression was higher in the NFPA than in the somatotropinomas and in the normal pituitaries (p=0.03 and 0.02, respectively). No correlation was found between the ZAC1 mRNA expression and the tumor invasiveness, Ki-67 and p53. CONCLUSION: ZAC1 and SSTR2 are underexpressed and SSTR3 is overexpressed in NFPA compared to those in somatotropinomas and in normal pituitaries, which might explain the lack of tumor shrinkage that is observed in response to commercially available SA therapy in patients with NFPA.

SAGE analysis highlights the putative role of underexpression of ribosomal proteins in GH-secreting pituitary adenomas.

BACKGROUND: Although the molecular pathogenesis of pituitary adenomas has been assessed by several different techniques, it still remains partially unclear. Ribosomal proteins (RPs) have been recently related to human tumorigenesis, but they have not yet been evaluated in pituitary tumorigenesis. OBJECTIVE: The aim of this study was to introduce serial analysis of gene expression (SAGE), a high-throughput method, in pituitary research in order to compare differential gene expression. METHODS: Two SAGE cDNA libraries were constructed, one using a pool of mRNA obtained from five GH-secreting pituitary tumors and another from three normal pituitaries. Genes differentially expressed between the libraries were further validated by real-time PCR in 22 GH-secreting pituitary tumors and in 15 normal pituitaries. RESULTS: Computer-generated genomic analysis tools identified 13,722 and 14,993 exclusive genes in normal and adenoma libraries respectively. Both shared 6497 genes, 2188 were underexpressed and 4309 overexpressed in tumoral library. In adenoma library, 33 genes encoding RPs were underexpressed. Among these, RPSA, RPS3, RPS14, and RPS29 were validated by real-time PCR. CONCLUSION: We report the first SAGE library from normal pituitary tissue and GH-secreting pituitary tumor, which provide quantitative assessment of cellular transcriptome. We also validated some downregulated genes encoding RPs. Altogether, the present data suggest that the underexpression of the studied RP genes possibly collaborates directly or indirectly with other genes to modify cell cycle arrest, DNA repair, and apoptosis, leading to an environment that might have a putative role in the tumorigenesis, introducing new perspectives for further studies on molecular genesis of somatotrophinomas.

CTNNB1 gene mutations, pituitary transcription factors, and MicroRNA expression involvement in the pathogenesis of adamantinomatous craniopharyngiomas.

Genes involved in formation/development of the adenohypophysis, CTNNB1 gene, and microRNAs might be implicated in the craniopharyngioma pathogenesis. The objective of this study is to perform the molecular analysis of HESX1, PROP1, POU1F1, and CTNNB1 genes and evaluate a panel of miRNA expression in craniopharyngioma. We also verified whether the presence of CTNNB1 mutation is associated with clinical findings and miRNA expression. The study included 16 patients with adamantinomatous craniopharyngioma (nine children and seven adults; eight females and eight males; 6-55 years, median 15.5 years). DNA, RNA, and cDNA were obtained from craniopharyngioma and normal pituitaries. DNA was also extracted from peripheral blood of healthy subjects. All genes were amplified by polymerase chain reaction and direct sequenced. Relative quantification of miRNA expression was calculated using the 2(-ΔΔCt) method. We found no mutations in HESX1, PROP1, and POU1F1 genes and four polymorphisms in PROP1 gene which were in Hardy-Weinberg equilibrium and had similar allelic frequencies in craniopharyngioma and controls. We found seven different mutations in CTNNB1 in eight of 16 patients. Younger patients presented more frequently CTNNB1 mutation than adults. We observed hyperexpression of miR-150 (1.7-fold); no different expression of miR-16-1, miR-21, and miR23a; and an underexpression of miR-141, let-7a, miR-16, miR-449, miR-145, miR-143, miR-23b, miR-15a, and miR-24-2 (ranging from -7.5 to -2.5-fold; p = 0.02) in craniopharyngioma. There was no association between tumor size or the recurrence and the presence of CTNNB1mutations. miR-16 and miR-141 were underexpressed in craniopharyngioma presenting CTNNB1 mutations. miR-23a and miR24-2 were hyperexpressed in patients who underwent only one surgery. Mutations or polymorphisms in pituitary transcription factors are unlikely to contribute to the adamantinomatous craniopharyngioma pathogenesis, differently of CTNNB1 mutations. Our data suggest the potential involvement of the deregulation of miRNA expression in the craniopharyngioma pathogenesis and outcome and also that the miRNA could modulate the Wnt signaling pathway in craniopharyngioma tumorigenesis.