A SIMPLI (Single-cell Identification from MultiPLexed Images) approach for spatially-resolved tissue phenotyping at single-cell resolution.

Multiplexed imaging technologies enable the study of biological tissues at single-cell resolution while preserving spatial information. Currently, high-dimension imaging data analysis is technology-specific and requires multiple tools, restricting analytical scalability and result reproducibility. Here we present SIMPLI (Single-cell Identification from MultiPLexed Images), a flexible and technology-agnostic software that unifies all steps of multiplexed imaging data analysis. After raw image processing, SIMPLI performs a spatially resolved, single-cell analysis of the tissue slide as well as cell-independent quantifications of marker expression to investigate features undetectable at the cell level. SIMPLI is highly customisable and can run on desktop computers as well as high-performance computing environments, enabling workflow parallelisation for large datasets. SIMPLI produces multiple tabular and graphical outputs at each step of the analysis. Its containerised implementation and minimum configuration requirements make SIMPLI a portable and reproducible solution for multiplexed imaging data analysis. Software is available at "SIMPLI [ https://github.com/ciccalab/SIMPLI ]".

Comparative assessment of genes driving cancer and somatic evolution in non-cancer tissues: an update of the Network of Cancer Genes (NCG) resource.

BACKGROUND: Genetic alterations of somatic cells can drive non-malignant clone formation and promote cancer initiation. However, the link between these processes remains unclear and hampers our understanding of tissue homeostasis and cancer development. RESULTS: Here, we collect a literature-based repertoire of 3355 well-known or predicted drivers of cancer and non-cancer somatic evolution in 122 cancer types and 12 non-cancer tissues. Mapping the alterations of these genes in 7953 pan-cancer samples reveals that, despite the large size, the known compendium of drivers is still incomplete and biased towards frequently occurring coding mutations. High overlap exists between drivers of cancer and non-cancer somatic evolution, although significant differences emerge in their recurrence. We confirm and expand the unique properties of drivers and identify a core of evolutionarily conserved and essential genes whose germline variation is strongly counter-selected. Somatic alteration in even one of these genes is sufficient to drive clonal expansion but not malignant transformation. CONCLUSIONS: Our study offers a comprehensive overview of our current understanding of the genetic events initiating clone expansion and cancer revealing significant gaps and biases that still need to be addressed. The compendium of cancer and non-cancer somatic drivers, their literature support, and properties are accessible in the Network of Cancer Genes and Healthy Drivers resource at http://www.network-cancer-genes.org/ .

Clone competition as a mechanism to reduce tumor formation.

With age, clones carrying somatic mutations in well-known cancer driver genes progressively populate adult tissues, yet cancer transformation is rare. In a recent issue of Nature, Colom et al. showed that competition between mutated clones with different fitness could act as a tumor-protective mechanism.

Immunogenomics of Colorectal Cancer Response to Checkpoint Blockade: Analysis of the KEYNOTE 177 Trial and Validation Cohorts.

BACKGROUND & AIMS: Colorectal cancer (CRC) shows variable response to immune checkpoint blockade, which can only partially be explained by high tumor mutational burden (TMB). We conducted an integrated study of the cancer tissue and associated tumor microenvironment (TME) from patients treated with pembrolizumab (KEYNOTE 177 clinical trial) or nivolumab to dissect the cellular and molecular determinants of response to anti- programmed cell death 1 (PD1) immunotherapy. METHODS: We selected multiple regions per tumor showing variable T-cell infiltration for a total of 738 regions from 29 patients, divided into discovery and validation cohorts. We performed multiregional whole-exome and RNA sequencing of the tumor cells and integrated these with T-cell receptor sequencing, high-dimensional imaging mass cytometry, detection of programmed death-ligand 1 (PDL1) interaction in situ, multiplexed immunofluorescence, and computational spatial analysis of the TME. RESULTS: In hypermutated CRCs, response to anti-PD1 immunotherapy was not associated with TMB but with high clonality of immunogenic mutations, clonally expanded T cells, low activation of Wnt signaling, deregulation of the interferon gamma pathway, and active immune escape mechanisms. Responsive hypermutated CRCs were also rich in cytotoxic and proliferating PD1+CD8 T cells interacting with PDL1+ antigen-presenting macrophages. CONCLUSIONS: Our study clarified the limits of TMB as a predictor of response of CRC to anti-PD1 immunotherapy. It identified a population of antigen-presenting macrophages interacting with CD8 T cells that consistently segregate with response. We therefore concluded that anti-PD1 agents release the PD1-PDL1 interaction between CD8 T cells and macrophages to promote cytotoxic antitumor activity.

Transcriptome Profiling Reveals New Insights into the Immune Microenvironment and Upregulation of Novel Biomarkers in Metastatic Uveal Melanoma.

Metastatic uveal melanoma (mUM) to the liver is incurable. Transcriptome profiling of 40 formalin-fixed paraffin-embedded mUM liver resections and 6 control liver specimens was undertaken. mUMs were assessed for morphology, nuclear BAP1 (nBAP1) expression, and their tumour microenvironments (TME) using an "immunoscore" (absent/altered/high) for tumour-infiltrating lymphocytes (TILs) and macrophages (TAMs). Transcriptomes were compared between mUM and control liver; intersegmental and intratumoural analyses were also undertaken. Most mUM were epithelioid cell-type (75%), amelanotic (55%), and nBAP1-ve (70%). They had intermediate (68%) or absent (15%) immunoscores for TILs and intermediate (53%) or high (45%) immunoscores for TAMs. M2-TAMs were dominant in the mUM-TME, with upregulated expression of ANXA1, CD74, CXCR4, MIF, STAT3, PLA2G6, and TGFB1. Compared to control liver, mUM showed significant (p < 0.01) upregulation of 10 genes: DUSP4, PRAME, CD44, IRF4/MUM1, BCL2, CD146/MCAM/MUC18, IGF1R, PNMA1, MFGE8/lactadherin, and LGALS3/Galectin-3. Protein expression of DUSP4, CD44, IRF4, BCL-2, CD146, and IGF1R was validated in all mUMs, whereas protein expression of PRAME was validated in 10% cases; LGALS3 stained TAMs, and MFGEF8 highlighted bile ducts only. Intersegmental mUMs show differing transcriptomes, whereas those within a single mUM were similar. Our results show that M2-TAMs dominate mUM-TME with upregulation of genes contributing to immunosuppression. mUM significantly overexpress genes with targetable signalling pathways, and yet these may differ between intersegmental lesions.

Balancing neurotrophin pathway and sortilin function: Its role in human disease.

Neurotensin receptor-3 or sortilin is a vacuolar protein sorting 10 protein domain (Vps10p) has been firstly discovered in the human brain, it acts as receptor or co-receptor of the cell and traffics different proteins within the cell. Sortilin deregulation contributes to the development of several diseases, including neurological diseases and cancer. On the other hand, neurotrophins which are a family of proteins essential for the nervous system development, function and plasticity. The first discovered member is the nerve growth factor; other members are brain-derived growth factor, neurotrophin-3 and neurotrophin-4. Nerve growth factor and brain-derived growth factor are the common neurotrophins that have a role in cancer. Neurotrophins initiate their signals through interaction with tyrosine receptor kinases TrkA, TrkB, and TrkC; each member has an affinity for a specific receptor to stimulate cell survival, while the interaction with p75NTR initiates cell apoptosis pathway by forming a complex with sortilin and neurotrophin precursors. A number of therapeutic approaches are emerging to target the neurotrophins pathway as well as sortilin.

Piloting a Deep Learning Model for Predicting Nuclear BAP1 Immunohistochemical Expression of Uveal Melanoma from Hematoxylin-and-Eosin Sections.

Background: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Monosomy 3 and BAP1 mutation are strong prognostic factors predicting metastatic risk in UM. Nuclear BAP1 (nBAP1) expression is a close immunohistochemical surrogate for both genetic alterations. Not all laboratories perform routine BAP1 immunohistochemistry or genetic testing, and rely mainly on clinical information and anatomic/morphologic analyses for UM prognostication. The purpose of our study was to pilot deep learning (DL) techniques to predict nBAP1 expression on whole slide images (WSIs) of hematoxylin and eosin (H&E) stained UM sections. Methods: One hundred forty H&E-stained UMs were scanned at 40 × magnification, using commercially available WSI image scanners. The training cohort comprised 66 BAP1+ and 74 BAP1- UM, with known chromosome 3 status and clinical outcomes. Nonoverlapping areas of three different dimensions (512 × 512, 1024 × 1024, and 2048 × 2048 pixels) for comparison were extracted from tumor regions in each WSI, and were resized to 256 × 256 pixels. Deep convolutional neural networks (Resnet18 pre-trained on Imagenet) and auto-encoder-decoders (U-Net) were trained to predict nBAP1 expression of these patches. Trained models were tested on the patches cropped from a test cohort of WSIs of 16 BAP1+ and 28 BAP1- UM cases. Results: The trained model with best performance achieved area under the curve values of 0.90 for patches and 0.93 for slides on the test set. Conclusions: Our results show the effectiveness of DL for predicting nBAP1 expression in UM on the basis of H&E sections only. Translational Relevance: Our pilot demonstrates a high capacity of artificial intelligence-related techniques for automated prediction on the basis of histomorphology, and may be translatable into routine histology laboratories.

Overexpression of HURP mRNA in head and neck carcinoma and association with in vitro response to vinorelbine.

HURP gene encodes the hepatoma upregulated protein (HURP), a microtubule associated protein regulating mitotic spindle dynamics, which promotes chromosomal congression and alignment during mitosis, with a potential role in tumorigenesis. In the present study, HURP mRNA expression was investigated by reverse transcription-quantitative PCR in oropharyngeal squamous cell carcinoma (OPSCC). Primary OPSCC tumors from 107 patients and 48 adjacent normal tissues, as well as 12 respiratory tract cancer cell lines (9 head and neck squamous cell carcinoma, 2 lung cancer and 1 normal bronchial) were utilised in the present study. mRNA expression levels of HURP were higher in malignant OPSCC tissues compared with in normal mucosa (P<1×10-5) and significantly associated with sex and smoking status (P<0.0001). Vinorelbine in vitro toxicity at half-maximal inhibitory concentration (IC50) was measured in the 11 cancer cell lines using an MTT assay. Sensitivity to vinorelbine was significantly correlated with HURP expression (r=0.636; P=0.035). The data indicated that HURP overexpression is frequent in OPSCC tissues and associated with smoking. The correlation between HURP mRNA expression and vinorelbine in vitro response suggests that HURP is a potential modulator of vinorelbine response; therefore, it should be explored for its possible predictive value for the efficiency of vinorelbine treatment in this type of cancer.

Long non-coding RNA dysregulation is a frequent event in non-small cell lung carcinoma pathogenesis.

BACKGROUND: Long non-coding RNAs compose an important level of epigenetic regulation in normal physiology and disease. Despite the plethora of publications of lncRNAs in human cancer, the landscape is still unclear. METHODS: Microarray analysis in 44 NSCLC paired specimens was followed by qPCR-based validation in 29 (technical) and 38 (independent) tissue pairs. Cross-validation of the selected targets was achieved in 850 NSCLC tumours from TCGA datasets. RESULTS: Twelve targets were successfully validated by qPCR (upregulated: FEZF1-AS1, LINC01214, LINC00673, PCAT6, NUTM2A-AS1, LINC01929; downregulated: PCAT19, FENDRR, SVIL-AS1, LANCL1-AS1, ADAMTS9-AS2 and LINC00968). All of them were successfully cross validated in the TCGA datasets. Abnormal DNA methylation was observed in the promoters of FENDRR, FEZF1-AS1 and SVIL-AS1. FEZF1-AS1 and LINC01929 were associated with survival in the TCGA set. CONCLUSIONS: Our study provides through multiple levels of internal and external validation, a comprehensive list of dysregulated lncRNAs in NSCLC. We therefore envisage this dataset to serve as an important source for the lung cancer research community assisting future investigations on the involvement of lncRNAs in the pathogenesis of the disease and providing novel biomarkers for diagnosis, prognosis and therapeutic stratification.

Oral Candida colonization in patients with chronic periodontitis. Is there any relationship? / Colonización oral por Candida en pacientes con enfermedad periodontal crónica. ¿Existe alguna relación?

Background: Candida can be implicated in the pathology of chronic periodontitis. Aims: To analyze the oral Candida carriage in patients suffering from chronic periodontitis (CP) and its correlation with the severity of this condition. Methods: Microbiological samples were taken from 155 patients using the oral rinse (OR) technique and by using paper points in the periodontal pockets (GPP). These patients were divided into 3 groups: 89 patients without CP (control), 47 with moderate CP, and 19 with severe CP. Samples were cultured in a Candida chromogenic agar for Candida. Species were identified by microbiological and molecular methods. Results: Candida was isolated in the OR of 45 (50.6%), 21 (44.7%), and 11 (57.9%) patients, respectively, and in the GPP of 32 (36%), 14 (29.2%), and 10 (42.6%) patients from the control, moderate CP and severe CP groups, respectively. Candida was isolated more frequently and in a greater burden in OR than in GPP (p<0.01). Candida albicans was the most prevalent species. GPP of patients with CP had poor fungal biodiversity (p<0.01). Conclusions: Colonization by Candida was present in the samples of patients without CP, and with both moderate and severe CP. Nonetheless, patients with severe CP had a higher rate of Candida colonization, especially by C. albicans

Antecedentes: Candida puede estar implicada en la patogenia de la enfermedad periodontal crónica. Objetivos: Analizar la colonización oral por Candida en pacientes con enfermedad periodontal (EP) crónica y su asociación con la gravedad de esta entidad clínica. Métodos: Se tomaron muestras microbiológicas de 155 pacientes mediante enjuagues orales e introduciendo una punta de papel estéril en la bolsa periodontal. Se dividieron los pacientes en tres grupos: 89 pacientes sin EP (control), 47 con EP moderada y 19 con EP grave. Las muestras se cultivaron en un agar cromógeno para Candida y las especies se identificaron mediante métodos microbiológicos tradicionales y moleculares. Resultados: Se aisló Candida en los enjuagues de 45 (50,6%), 21 (44,7%) y 11 (57,9%) pacientes y en las puntas de papel de 32 (36%), 14 (29,2%) y 10 (42,6%) pacientes de los grupos control, con EP moderada y con EP grave, respectivamente. Candida se aisló con mayor frecuencia y mayor carga fúngica en las muestras de los enjuagues que en las de las puntas de papel (p < 0,01). Candida albicans fue la especie más prevalente. Las muestras de la bolsa periodontal de los pacientes con EP presentaron una baja biodiversidad fúngica (p??0,01). Conclusiones: Hubo colonización por Candida en las muestras de pacientes sin EP y con EP tanto moderada como leve. No obstante, los pacientes con EP grave presentaron una mayor colonización por Candida, especialmente por C. albicans

Oral Candida colonization in patients with chronic periodontitis. Is there any relationship?

BACKGROUND: Candida can be implicated in the pathology of chronic periodontitis. AIMS: To analyze the oral Candida carriage in patients suffering from chronic periodontitis (CP) and its correlation with the severity of this condition. METHODS: Microbiological samples were taken from 155 patients using the oral rinse (OR) technique and by using paper points in the periodontal pockets (GPP). These patients were divided into 3 groups: 89 patients without CP (control), 47 with moderate CP, and 19 with severe CP. Samples were cultured in a Candida chromogenic agar for Candida. Species were identified by microbiological and molecular methods. RESULTS: Candida was isolated in the OR of 45 (50.6%), 21 (44.7%), and 11 (57.9%) patients, respectively, and in the GPP of 32 (36%), 14 (29.2%), and 10 (42.6%) patients from the control, moderate CP and severe CP groups, respectively. Candida was isolated more frequently and in a greater burden in OR than in GPP (p<0.01). Candida albicans was the most prevalent species. GPP of patients with CP had poor fungal biodiversity (p<0.01). CONCLUSIONS: Colonization by Candida was present in the samples of patients without CP, and with both moderate and severe CP. Nonetheless, patients with severe CP had a higher rate of Candida colonization, especially by C. albicans.

Toll-like receptor 2 rs4696480 polymorphism and risk of oral cancer and oral potentially malignant disorder.

OBJECTIVES: The aim of this study was to identify the possible association between TLR polymorphisms and an increased risk of developing head and neck cancer, including oral (OSCC) and laryngeal squamous cell carcinoma (LSCC), and oral potentially malignant disorders, such as oral lichenoid disease (OLD), including oral lichen planus (OLP) and oral lichenoid lesions (OLL). DESIGN: This case-control study included 40 OSCC, 35 LSCC, 175 OLD (129 OLP and 46 OLL) patients and 89 healthy controls, all of them from the Basque Country, Spain. Genetic polymorphisms in TLR1, TLR2, TLR4, TLR6, TLR9, and TLR10 were genotyped by TaqMan® assays or pyrosequencing. RESULTS: The chi-square analysis showed that the variant A of the SNP TLR2-rs4696480 polymorphism significantly increased the risk of OSCC (p=0.03) and OLL (p=0.02). CONCLUSIONS: The TLR2-rs4696480 polymorphism may be relevant to OSCC and OLL susceptibility in this population encouraging further studies on the TLR2 pathway and its possible association with this group of oral potentially malignant disorders and oral cancer. This may also prove the use of TLR polymorphisms as risk markers for oral and laryngeal cancer.

Development and validation of molecular methodologies to assess PALB2 expression in sporadic breast cancer.

OBJECTIVES: Recent reports have included PALB2 (Partner and localizer of BRCA2) in the growing list of hereditary cancer genes. PALB2 mutations confer a moderate breast cancer risk in heterozygotes and Fanconi anemia in biallelic mutation carriers. PALB2 protein co-localizes with BRCA2 and BRCA1 in nuclear structures and enables error-free homologous recombination repair of double-stranded DNA breaks. This important contribution could be severely diminished if affected by epigenetic mechanisms such as promoter CpG island methylation. The aim of our study was to develop molecular methodologies in order to assess accurately PALB2 expression in breast cancer tissues. DESIGN AND METHODS: DNA and RNA were extracted from 91 sporadic fresh-frozen breast tissues with known histopathological data. DNA was subjected to sodium bisulfite conversion reaction and the CpG island of the PALB2 promoter was analyzed by pyrosequencing. RNA was converted to cDNA and analyzed by a newly developed and validated RT-qPCR assay based on a hydrolysis probe (TaqMan) in the Light Cycler. RESULTS: PALB2 promoter was not methylated in any of the samples tested. 87 out of 91 (95.6%) primary tumors were positive for PALB2 expression, as checked at the mRNA level. When levels of PALB2 mRNA were compared to histopathological data (tumor size, grade, lymph node involvement, metastasis, hormone receptors and HER2 overexpression), no significant statistical correlation was found. CONCLUSIONS: DNA methylation is an unlike mechanism for PALB2 transcriptional regulation. PALB2 mRNA expression does not seem be a promising prognostic biomarker for sporadic breast cancer.

Polymorphisms in alcohol and tobacco metabolism genes in head and neck cancer in the Basque Country.

BACKGROUND: The Basque Country has one of the highest rates of head and neck squamous cell carcinoma (HNSCC) in Europe, although tobacco and alcohol consumption are not high when compared to other European countries where HNSCC incidence is lower. Our aim was to determine the role of genetic variation with regard to the metabolism of alcohol and carcinogens from tobacco smoke in the Basque Country. METHODS: Fourteen polymorphisms in alcohol or tobacco metabolism genes were genotyped in 84 HNSCC patients and 242 healthy individuals from the Basque Country. RESULTS: ADH1B histidine allele (rs1229984), CYP2E1 rs3813867 heterozygous genotype, and GSTT1 deletion conferred protection against HNSCC (OR: 0.318 [0.04-0.75], OR: 0.13 [0.02-0.94], and OR: 0.12 [0.02-0.60], respectively) while GSTP1 (rs1695) Val/Val genotype was related to an increased risk (OR: 4.12 [1.11-15.31]). Regarding alcohol and tobacco habits, GSTT1 deletion was associated with tobacco usage, while the 3 polymorphisms tested in ALDH2 were associated with alcohol consumption. However, genotypic distributions of these 7 SNPs did not differ from those observed for other Caucasian populations where HNSCC incidence is lower. CONCLUSIONS: The identified genotypic variations in alcohol and tobacco metabolizing genes only by themselves do not seem to be responsible for the higher incidence of HNSCC observed in the Basque Country.

Role of cytochrome P-450 genetic polymorphisms in oral carcinogenesis.

Oral cancer is one of the most frequent head and neck cancers, and epidemiological studies have shown that smoking is a major risk factor in this pathology. However, as not all smokers develop oral cancer, some individuals must be more susceptible to develop this disease. This individual susceptibility has been related to different genetic variants in metabolizing enzymes. The cytochrome P-450 (CYP) family of enzymes metabolizes tobacco-related carcinogens producing reactive metabolites, which could cause DNA damage. Because of their functional role in the metabolism of tobacco-related compounds, the genetic polymorphisms found in the genes that code for CYP enzymes have been suggested to modulate oral cancer risk and contribute to individual susceptibility. In this review, we analyze and update the available evidence in the literature regarding the polymorphisms of CYP genes in relation to the susceptibility of developing oral cancer.

Alcohol-dehydrogenase (ADH1B) Arg48His polymorphism in Basque Country patients with oral and laryngeal cancer: preliminary study.

Oral and laryngeal cancer has a high incidence in the Basque Country (Spain), the main risk factors in this pathology being regular consumption of tobacco and alcohol. However, since not all the individuals exposed to these risk factors develop cancer, the individual genetic susceptibility should be investigated in this population. The aim of this study was to determine the distribution of alcohol dehydrogenase-1B polymorphism (Arg48His; rs1229984) in our region and analyze its association with the risk of oral and laryngeal squamous cell carcinoma. Samples from 87 patients with oral or laryngeal cancer and 242 healthy controls were analyzed. Multivariate logistic regression analysis showed that the combined Arg/His and His/His genotypes were associated with a reduced risk of head and neck squamous cell carcinoma (odds ratio: 0.203; 95% confidence interval: 0.052-0.796). In conclusion, the histidine allele was associated with a reduced risk of oral and laryngeal cancer in the Basque Country.

DNA methylation epigenotypes in breast cancer molecular subtypes.

INTRODUCTION: Identification of gene expression based breast cancer subtypes is considered as a critical means of prognostication. Genetic mutations along with epigenetic alterations contribute to gene expression changes occurring in breast cancer. So far, these epigenetic contributions to sporadic breast cancer subtypes have not been well characterized, and there is only a limited understanding of the epigenetic mechanisms affected in those particular breast cancer subtypes. The present study was undertaken to dissect the breast cancer methylome and deliver specific epigenotypes associated with particular breast cancer subtypes. METHODS: Using a microarray approach we analyzed DNA methylation in regulatory regions of 806 cancer related genes in 28 breast cancer paired samples. We subsequently performed substantial technical and biological validation by Pyrosequencing, investigating the top qualifying 19 CpG regions in independent cohorts encompassing 47 basal-like, 44 ERBB2+ overexpressing, 48 luminal A and 48 luminal B paired breast cancer/adjacent tissues. Using all-subset selection method, we identified the most subtype predictive methylation profiles in multivariable logistic regression analysis. RESULTS: The approach efficiently recognized 15 individual CpG loci differentially methylated in breast cancer tumor subtypes. We further identify novel subtype specific epigenotypes which clearly demonstrate the differences in the methylation profiles of basal-like and human epidermal growth factor 2 (HER2)-overexpressing tumors. CONCLUSIONS: Our results provide evidence that well defined DNA methylation profiles enables breast cancer subtype prediction and support the utilization of this biomarker for prognostication and therapeutic stratification of patients with breast cancer.

HPV infection and oral carcinogenesis

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Oral cancer and polymorphism of ethanol metabolising genes.

Oral cancer is the sixth most common cancer worldwide and a major health problem in some parts of the world. Epidemiological studies have shown that habitual alcohol consumption could be a risk factor in oral carcinogenesis, although the true involvement of alcohol is unknown. Via alcohol dehydrogenase (ADH) and cytochrome P450 oxidase (CYP) alcohol is metabolized to acetaldehyde, a highly toxic compound, which plays an important role in carcinogenesis. Subsequently, and during the metabolizing process, acetaldehyde becomes acetate by acetaldehyde dehydrogenase (ALDH). Therefore, acetaldehyde levels are determined mainly by the action of ADH, CYP and ALDH. Recently, several studies have found that certain polymorphisms of genes encoding these enzymes confer a higher or lower metabolic activity and therefore different risk for certain malignancies such as oral cancer. In this review, we analyze the polymorphisms of alcohol metabolising enzymes in relation susceptibility to an oral cancer.