Pathological images for personal medicine in Hepatocellular carcinoma: Cross-talk of gene sequencing and pathological images

Background: Considering the great heterogeneity of Hepatocellular carcinoma (HCC), more accurate prognostic models are urgently needed. This paper combined the advantages of genomics and pathomics to construct a prognostic model. Methods: First, we collected data from hepatocellular carcinoma patients with complete mRNA expression profiles and clinical annotations from the TCGA database. Then, based on immune-related genes, we used random forest plots to screen prognosis-related genes and build prognostic models. Bioinformatics was used to identify biological pathways, evaluate the tumor microenvironment, and perform drug susceptibility testing. Finally, we divided the patients into different subgroups according to the gene model algorithm. Pathological models were constructed by obtaining HE-stained sections from TCGA in corresponding subgroups of patients. Results: In this study, we constructed a stable prognostic model that could predict overall survival in HCC patients. The signature consisted of six immune-related genes (BX537318.1, TMEM147, CSPG4P12, AC015908.3, CEBPZOS, and SRD5A3). We found increased levels of infiltration of immune cells in the tumor microenvironment in patients with low risk scores, indicating significant antitumor immunity and corresponding to better clinical outcomes. We then screened nine drugs that were more sensitive in the low-risk group than in the high-risk group. Finally, we addressed the complex cellular changes and phenotypic heterogeneity in the HCC microenvironment by combining genomics and pathomics analysis methods. Conclusion: Our study showed that the prognostic evaluation model of HCC based on the immune signaling pathway is feasible and provided a reference value for potential immunotherapy for HCC.     

Profiles of immune infiltration in the tumor microenvironment of hepatocellular carcinoma

BackgroundThus far, few studies have systematically analyzed the profiles of immune cells infiltrated in the tumor microenvironment (TME) of hepatocellular carcinoma (HCC). Therefore, the purpose of our study was to comprehensively analyze the 22 tumor-infiltrating immune cells (TIICs) and the immune subtypes of HCC, as well as the factors associated with the prognosis of HCC patients.MethodsIn this study, we evaluated the abundance of 22 tumor-infiltrating immunocytes of 371 HCC patients from The Cancer Genome Atlas (TCGA) database by using the CIBERSORT algorithm, and defined immune subtypes of HCC according to unsupervised cluster analysis. The immune score of HCC patients was calculated by the prognostic regression model, while the survival analysis was evaluated by the Kaplan-Meier method. In addition, the consistency index of TIICs and principal component analysis (PCA) of immunomodulator genes were estimated.ResultsThe results of this study showed that three distinct immune subtypes of HCC were stratified, and the C1 subtype and C3 subtype were correlated with a good prognosis. The cellular composition of three immune subtypes was different. Moreover, immunomodulator gene and programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) expression in the C1 subtype was significantly higher (P<0.05).ConclusionsThis suggested that the low immune score of HCC patients is associated with better clinical outcomes. In addition, the interaction network of cluster of differentiation CD8+ T cells was mainly concentrated in the C1 subtype. Taken together, this study showed that tumor-infiltrating immune cells can perhaps be an important determinant of clinical outcomes of patients with HCC and may provide biomarkers to reflect the immunotherapy response. Notably, the C1 subtype of HCC may be used as an important predictive factor for immunotherapy response.     

Clinical impact of herpesvirus entry mediator expression in human hepatocellular carcinoma

BackgroundHerpes virus entry mediator (HVEM), also known as tumour necrosis factor receptor (TNFR) superfamily 14, regulates a variety of physiological and pathological responses in both innate and acquired immunity. Although HVEM is also suggested to be a critical regulator in tumours, actual roles in human cancer are largely unknown. This study aimed to clarify clinical importance of HVEM in human hepatocellular carcinoma (HCC).Patients and methodsWe studied HVEM expression in 150 HCC patients to explore its clinical relevance, and we examined tumour infiltrating T cells and local immune status of them.ResultsHVEM was expressed in HCC cells, while no or only limited expression was observed in normal tissues in the liver. Tumour HVEM expression was significantly correlated with age, serum protein induced by vitamin K absence or antagonist-II (PIVKA-II) level, vascular invasion and tumour node metastasis (TNM) stage. Furthermore, tumour HVEM expression significantly correlated with postoperative recurrence and survival. Importantly, multivariate analysis indicated that the HVEM status had an independent prognostic value. Furthermore, HVEM status was inversely correlated with tumour-infiltrating CD4⁺, CD8⁺ and CD45RO⁺ lymphocytes. In addition, it was also associated with reduced expression of perforin, granzyme B and interferon-γ (IFN-γ). Taken together, tumour-expressing HVEM plays a functionally important role in HCC.ConclusionTumour-expressing HVEM plays a critical role in human HCC, possibly through regulating immune evasion. Therefore, targeting HVEM may be a novel promising therapeutic strategy for HCC.     

CXCL5/CXCL8 is a promising potential prognostic and tumor microenvironment-related cluster in hepatocellular carcinoma

BackgroundImmune checkpoint blockers (ICBs) are increasingly applied to treat patients with advanced HCC. However, the overall survival (OS) of HCC patients is still unsatisfactory, and there is no confirmed immune-related and prognostic gene to identify patients who could clinically benefit from this treatment. The tumor microenvironment (TME) is known to be closely related to immunotherapy and plays a pivotal role in the recurrence and progression of HCC. Our aim is to explore TME-related genes and identify the prognostic value in HCC patients.MethodsESTIMATE, immune, and stromal scores were calculated for HCC patients based on RNA expression data from The Cancer Genome Atlas database. Differential expression analysis was performed to screen the differentially expressed genes (DEGs). A protein-protein interaction (PPI) network was constructed to identify the key DEGs. Univariate and multivariate Cox analyses were adopted to validate hub DEGs associated with clinical prognosis, and a single-sample gene set enrichment analysis (ssGSEA) algorithm was used to dissect the landscape of tumor-infiltrating cells (TIC) in HCC. Finally, the relationship between hub immune-related genes and TIC was explored through difference and correlation analyses.ResultsESTIMATE, immune and stromal scores were all found to be associated with the OS of patients (P<0.05). A total of 1,112 DEGs were identified by comparing low and high score groups of immune and stromal scores. Most of DEGs were enriched in immune-related gene sets by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Additionally, the top 34 genes were included in the protein–protein interaction (PPI) network, and univariate Cox analysis focus on a novel prognosis-related gene cluster CXCL5/CXCL8 (P<0.001). Regarding the immune landscape of HCC, univariable Cox regression analysis showed six immune cells to be associated with OS. Finally, 21 immune cells were commonly determined between high and low expression of CXCL5/CXCL8, suggesting there is a close relationship between expression of CXCL5 and CXCL8 .ConclusionsOur study has revealed that the immune-related gene cluster of CXCL5 /CXCL8 could be a promising prognostic indicator for HCC and a potential novel biomarker to guide the selection of HCC patients for ICB immunotherapy.     

Characterisation of multifocal breast cancer using the 70-gene signature in clinical low-risk patients enrolled in the EORTC 10041/BIG 03-04 MINDACT trial

BackgroundIn multifocal breast cancer, guidelines recommend basing adjuvant systemic treatment decisions on characteristics of the largest lesion, disregarding multifocality as an independent prognosticator. We assessed the association between multifocal disease and both the 70-gene signature (70-GS), and distant metastasis-free survival (DMFS) in clinical low-risk breast cancer patients enrolled in the European Organisation for Research and Treatment of Cancer 10041/BIG 03-04 Microarray In Node-negative and 1 to 3 positive lymph node Disease may Avoid ChemoTherapy (MINDACT) trial.Patients and methodsThe analysed population consisted of enrolled patients in the MINDACT trial with clinical low-risk disease, defined by a modified Adjuvant! Online cut-off for the 10-year risk of recurrent disease or death. Eligibility criteria of MINDACT dictate that patients with multifocal disease could be included if the different lesions had similar pathological characteristics. The presence of multifocal disease was deducted from the case report form (CRF)-question for sum of diameter for all invasive tumour foci. Clinicopathological characteristics and gene expression of patients with unifocal and multifocal (largest lesion) disease were compared. Subsequently, the association between multifocal disease and the 70-GS was evaluated as well as the association between multifocality and 5-year DMFS.ResultsThe study included 3090 clinical low-risk patients with unifocal and 238 patients with multifocal disease. Apart from a higher prevalence of lobular tumours (21.8% versus 10.8%, by local pathology), we did not observe differences in baseline characteristics between multifocal and unifocal tumours. Patients with multifocal tumours were more likely to be at high genomic risk as compared to patients with unifocal tumours (22.7% versus 17.3%, odds ratio [OR] 1.45, 95% confidence interval [CI] 1.02–2.07, P = 0.038). We did not find a significant association between tumour focality and DMFS (97.1% for unifocal versus 96.9% for multifocal, hazard ratio [HR] = 1.55, 95% CI 0.68–3.46, P = 0.172), nor a signal for a potential interaction between the prognostic effect of the 70-GS and focality of the tumour regarding DMFS.ConclusionIn the group of clinical low-risk MINDACT patients, multifocal tumours were more likely to have a high-risk 70-GS profile compared to unifocal tumours. We did not observe a significant interaction between multifocality and the 70-GS with respect to survival without distant metastasis in these patients.     

Percutaneous thermal ablation for the treatment of colorectal liver metastases and hepatocellular carcinoma: a comparison of local therapeutic efficacy

Aim: This study aimed to compare the local therapeutic efficacy of percutaneous thermal ablation for colorectal liver metastases (CRLM) and hepatocellular carcinoma (HCC).

Methods: One hundred sixty-one CRLM nodules in 101 patients and 122 HCC nodules in 97 patients were treated with thermal ablation. Complications and local efficacy were retrospectively compared.

Results: Major complications were observed in two (2.0%) patients in the CRLM group and one (1.0%) in the HCC group (p?=?1.000). The complete ablation (CA) rate of lesions ≤?3?cm was lower in the CRLM group than in the HCC group (p?=?0.018). After a mean follow-up period of 21.1?±?20.7 months in the CRLM group and 22.1?±?17.6 months in the HCC group, the local tumour progression (LTP) rate of lesions >?3?cm was higher in the CRLM group than in the HCC group (p?=?0.036). The multivariate analysis revealed that only safety margin (≤?0.5?cm/>?0.5?cm) was a significant predictor of LTP in both CRLM and HCC.

Conclusions: To achieve better local tumour control, thermal ablation should be more aggressive for CRLM than for HCC, especially for large tumours in clinical.     

Pan-cancer association of HLA gene expression with cancer prognosis and immunotherapy efficacy

Background The function of major histocompatibility complex (MHC) molecules is to bind peptide fragments derived from genomic mutations or pathogens and display them on the cell surface for recognition by cognate T cells to initiate an immune response.Methods In this study, we provide a comprehensive investigation of HLA gene expression in a pan-cancer manner involving 33 cancer types. We utilised gene expression data from several databases and immune checkpoint blockade-treated patient cohorts.Results We show that MHC expression varies strongly among cancer types and is associated with several genomic and immunological features. While immune cell infiltration was generally higher in tumours with higher HLA gene expression, CD4+ T cells showed significantly different correlations among cancer types, separating them into two clusters. Furthermore, we show that increased HLA gene expression is associated with prolonged survival in the majority of cancer types. Lastly, HLA gene expression is associated with patient response to immune checkpoint blockade, which is especially prominent for HLA class II expression in tumour biopsies taken during treatment.Conclusion We show that HLA gene expression is an important feature of tumour biology that has significant impact on patient prognosis.Subject terms: Tumour immunology, MHC     

Multifocal breast cancer and survival: Each focus does matter particularly for larger tumours

PurposeThe objective of this study is to determine whether the aggregate tumour size of every focus in multifocal breast cancer more accurately predicts 10-year survival than current staging systems which use the largest or dominant tumour size.Patients and methodsThis study examined the original histological reports of 848 consecutive patients with invasive breast cancer treated in New South Wales (NSW), Australia between 1 April 1995 and 30 September 1995. Multifocal tumours were assessed using two estimates of pathologic tumour size: largest tumour focus diameter and the aggregate diameter of every tumour focus. The 10-year survival of patients with multifocal tumours measured in both ways was compared to that with unifocal tumours.ResultsAt a median follow-up of 10.4 years, 27 of 94 patients (28.7%) with multifocal breast cancer have died of breast cancer compared to 141 of 754 (18.7%) with unifocal breast cancer (P = .022). Ten-year survival was not affected by size for tumours measuring 20 mm or less, whether or not dominant tumour size (87.9%) or aggregate tumour size (87.0%) was used for multifocal tumours, compared to unifocal tumours (88.1%). For tumours larger than 20 mm, 10-year survival was 72.1% for unifocal tumours compared to 54.7% (P = .008) for multifocal tumours using dominant tumour size, but this was 69.5% and not significant when multifocal tumours were classified using aggregate tumour size (P = .49). Multivariate analysis also confirmed the above-mentioned results after adjustment for important prognostic factors.ConclusionAggregate size of every focus should be considered along with other prognostic factors for metastasis when treatment is planned. The current convention of using the largest (dominant) lesion as a measure of stage and associated breast cancer survival needs further validation.     

Genetic analysis of multifocal superficial urothelial cancers by array-based comparative genomic hybridisation

The purpose of this study was to investigate the accumulation of genetic alterations during metachronous and/or synchronous development of multifocal low-grade superficial urothelial tumours in the same patient, by using array-based comparative genomic hybridisation (array-CGH) and FGFR mutation analysis. We analysed 24 tumours (pTa-1 G1-2) from five patients. We had previously identified a clonal relationship among the tumours of each patient by microsatellite analysis. This time, unsupervised hierarchical cluster analysis revealed that the tumours from each patient were clustered together independently of the tumours from the other patients. All of the tumours from a single patient showed a set of 2-7 identical regional or whole-arm chromosomal changes. In addition, several individual alterations were also found. Cladistic diagrams revealed that the accumulation of genetic alterations could not be explained by a linear model, and the existence of a hypothetical precursor cell was assumed in four patients. In some cases, FGFR mutation seemed to occur later during multifocal tumour development. Taken together, these findings suggest that low-grade superficial urothelial tumours accumulate minor genetic alterations during multifocal development, although these tumours are genetically stable.     

Immunotherapy in NSCLC patients with brain metastases. Understanding brain tumor microenvironment and dissecting outcomes from immune checkpoint blockade in the clinic

BackgroundBrain metastases are frequent complications in patients with non-small-cell lung cancer (NSCLC) associated with significant morbidity and poor prognosis. Our goal is to give a global overlook on clinical efficacy from immune checkpoint inhibitors in this setting and to review the role of biomarkers and molecular interactions in brain metastases from patients with NSCLC.MethodsWe reviewed clinical trials reporting clinical outcomes of patients with NSCLC with brain metastases as well as publications assessing the tumor microenvironment and the complex molecular interactions of tumor cells with immune and resident cells in brain metastases from NSCLC biopsies or preclinical models.ResultsAlthough limited data are available on immunotherapy in patients with brain metastases, immune checkpoint inhibitors alone or in combination with chemotherapy have shown promising intracranial efficacy and safety results. The underlying mechanism of action of immune checkpoint inhibitors in the brain niche and their influence on tumor microenvironment are still not known. Lower PD-L1 expression and less T CD8⁺ infiltration were found in brain metastases compared with matched NSCLC primary tumors, suggesting an immunosuppressive microenvironment in the brain. Reactive astrocytes and tumor associated macrophages are paramount in NSCLC brain metastases and play a role in promoting tumor progression and immune evasion.ConclusionsDiscordances in the immune profile between primary tumours and brain metastases underscore differences in the tumour microenvironment and immune system interactions within the lung and brain niche. The characterization of immune phenotype of brain metastases and dissecting the interplay among immune cells and resident stromal cells along with cancer cells is crucial to unravel effective immunotherapeutic approaches in patients with NSCLC and brain metastases.     

Electro-hyperthermia up-regulates tumour suppressor Septin 4 to induce apoptotic cell death in hepatocellular carcinoma

Purpose: Modulated electro-hyperthermia (mEHT) has been shown to be effective against various types of human tumours, including hepatocellular carcinoma (HCC). Here we aimed to investigate the molecular mechanism underlying the cytotoxic effects of mEHT to HCC cells.

Materials and methods: Human liver cancer cell lines, Huh7 and HepG2, were treated with mEHT (42?°C/60?min) three times at 2-day intervals. Growth inhibition and apoptotic induction were evaluated using MTS, microscopic analysis, a clonogenic assay, annexin V/PI staining and a ccK18 ELISA. Global changes in gene expression were examined using RNA sequencing to obtain insights into molecular changes in response to mEHT. For in vivo evaluation of mEHT we used HepG2 HCC xenografts grown in nude mice.

Results: mEHT suppressed HCC cell proliferation and long-term colony formation through induction of apoptosis. The growth inhibitory effects are induced through a subset of molecular changes. Notably the expression level of septin 4 (SEPT4) (involved in pro-apoptotic activity and growth suppression) was up-regulated, whereas a key regulator of invasiveness G-Protein coupled receptor 64 (GPR64) was repressed. Subsequent Western blotting confirmed that the common increase in tumour suppressor SEPT4 in both Huh7 and HepG2 cells is accompanied by the restoration of cyclin-dependent kinase (CDK) inhibitor p21 and decrease in pro-caspase 7 and pro-caspase 3, thereby accelerating apoptotic signalling in HCC cells. Additionally, mEHT significantly inhibited the growth of human HCC xenografts in nude mice.

Conclusions: These findings suggest that apoptotic cell death induced by mEHT is mediated by the up-regulation of tumour suppressor SEPT4 in human HCC cells.     

MicroRNA-193b regulates proliferation,migration and invasion in human hepatocellular carcinoma cells

Background and AimsRecently, some miRNAs have been reported to be connected closely with the development of human hepatocellular carcinoma. However, the functions of these miRNAs in HCC remain largely undefined.MethodsThe expression profiles of miR-193b were compared between HCC tissues and adjacent normal liver tissues using qRT-PCR method. This method was also be used to screen the potential target genes of miR-193b. A luciferase reporter assay was conducted to confirm target association. Finally, the functional effect of miR-193b in hepatoma cells was examined further.ResultsmiR-193b was significantly down-regulated in most of the HCC tissues compared to the matching non-tumoural liver tissues. Furthermore, ectopic expression of miR-193b dramatically suppressed the ability of hepatoma cells to form colonies in vitro and to develop tumours in nude mice. CCND1 and ETS1 were revealed to be regulated by miR-193b directly. By regulating the expressions of these oncogenes, miR-193b induced cell cycle arrest and inhibited the invasion and migration of hepatoma cells.ConclusionsmiR-193b may function as a tumour suppressor in the development of HCC by acting on multiple tumourigenic pathways.     

Frequent genomic imbalances suggest commonly altered tumour genes in human hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is one of the most frequent-occurring malignant tumours worldwide, but molecular changes of tumour DNA, with the exception of viral integrations and p53 mutations, are poorly understood. In order to search for common macro-imbalances of genomic tumour DNA, 21 HCCs and 3 HCC-cell lines were characterized by comparative genomic hybridization (CGH), subsequent database analyses and in selected cases by fluorescence in situ hybridization (FISH). Chromosomal subregions of 1q, 8q, 17q and 20q showed frequent gains of genomic material, while losses were most prevalent in subregions of 4q, 6q, 13q and 16q. Deleted regions encompass tumour suppressor genes, like RB-1 and the cadherin gene cluster, some of them previously identified as potential target genes in HCC development. Several potential growth- or transformation-promoting genes located in chromosomal subregions showed frequent gains of genomic material. The present study provides a basis for further genomic and expression analyses in HCCs and in addition suggests chromosome 4q to carry a so far unidentified tumour suppressor gene relevant for HCC development.     

Modulation of intratumoural myeloid cells,the hallmark of the anti-tumour efficacy induced by a triple combination: tumour-associated peptide,TLR-3 ligand and α-PD-1

Background Anti-programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) monoclonal antibodies (mAbs) show remarkable clinical anti-tumour efficacy. However, rational combinations are needed to extend the clinical benefit to primary resistant tumours. The design of such combinations requires the identification of the kinetics of critical immune cell populations in the tumour microenvironment.Methods In this study, we compared the kinetics of immune cells in the tumour microenvironment upon treatment with immunotherapy combinations with different anti-tumour efficacies in the non-inflamed tumour model TC-1/A9. Tumour-bearing C57BL/6J mice were treated with all possible combinations of a human papillomavirus (HPV) E7 long peptide, polyinosinic–polycytidylic acid (PIC) and anti-PD-1 mAb. Tumour growth and kinetics of the relevant immune cell populations were assessed over time. The involvement of key immune cells was confirmed by depletion with mAbs and immunophenotyping with multiparametric flow cytometry.Results The maximum anti-tumour efficacy was achieved after intratumoural administration of HPV E7 long peptide and PIC combined with the systemic administration of anti-PD-1 mAb. The intratumoural immune cell kinetics of this combination was characterised by a biphasic immune response. An initial upsurge of proinflammatory myeloid cells led to a further rise in effector CD8⁺ T lymphocytes at day 8. Depletion of either myeloid cells or CD8⁺ T lymphocytes diminished the anti-tumour efficacy of the combination.Conclusions The anti-tumour efficacy of a successful immunotherapy combination in a non-inflamed tumour model relies on an early inflammatory process that remodels the myeloid cell compartment.Subject terms: Oncology, Cancer     

A deep convolutional neural network for segmentation of whole-slide pathology images identifies novel tumour cell-perivascular niche interactions that are associated with poor survival in glioblastoma

Background Glioblastoma is the most aggressive type of brain cancer with high-levels of intra- and inter-tumour heterogeneity that contribute to its rapid growth and invasion within the brain. However, a spatial characterisation of gene signatures and the cell types expressing these in different tumour locations is still lacking.Methods We have used a deep convolutional neural network (DCNN) as a semantic segmentation model to segment seven different tumour regions including leading edge (LE), infiltrating tumour (IT), cellular tumour (CT), cellular tumour microvascular proliferation (CTmvp), cellular tumour pseudopalisading region around necrosis (CTpan), cellular tumour perinecrotic zones (CTpnz) and cellular tumour necrosis (CTne) in digitised glioblastoma histopathological slides from The Cancer Genome Atlas (TCGA). Correlation analysis between segmentation results from tumour images together with matched RNA expression data was performed to identify genetic signatures that are specific to different tumour regions.Results We found that spatially resolved gene signatures were strongly correlated with survival in patients with defined genetic mutations. Further in silico cell ontology analysis along with single-cell RNA sequencing data from resected glioblastoma tissue samples showed that these tumour regions had different gene signatures, whose expression was driven by different cell types in the regional tumour microenvironment. Our results further pointed to a key role for interactions between microglia/pericytes/monocytes and tumour cells that occur in the IT and CTmvp regions, which may contribute to poor patient survival.Conclusions This work identified key histopathological features that correlate with patient survival and detected spatially associated genetic signatures that contribute to tumour-stroma interactions and which should be investigated as new targets in glioblastoma. The source codes and datasets used are available in GitHub: https://github.com/amin20/GBM_WSSM.Subject terms: CNS cancer, Cancer microenvironment, Machine learning