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Sensitive PCR detection of viral nucleic acids plays a critical role in infectious disease research, diagnosis and monitoring. In the context of SARS-CoV-2 detection, recent reports indicate that digital PCR-based tests are significantly more sensitive than traditional qPCR tests. Numerous factors can influence digital PCR reaction sensitivity. In this review, using a model for human HIV infection and the Raindance ddPCR platform as an example, we describe technical aspects that contribute to sensitive viral signal detection in DNA and RNA from tissue samples, which often harbor viral reservoirs and serve as better predictors of disease outcome and indicators of treatment efficacy.

Background & Aims Patient-derived tumor organoids recapitulate the characteristics of colorectal cancer (CRC) and provide an ideal platform for preclinical evaluation of personalized treatment options. We aimed to model the acquisition of chemotolerance during first-line combination chemotherapy in metastatic CRC organoids. Methods We performed next-generation sequencing to study the evolution of KRAS wild-type CRC organoids during adaptation to irinotecan-based chemotherapy combined with epidermal growth factor receptor (EGFR) inhibition. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 protein (Cas9)-editing showed the specific effect of KRASG12D acquisition in drug-tolerant organoids. Compound treatment strategies involving Aurora kinase A (AURKA) inhibition were assessed for their capability to induce apoptosis in a drug-persister background. Immunohistochemical detection of AURKA was performed on a patient-matched cohort of primary tumors and derived liver metastases. Results Adaptation to combination chemotherapy was accompanied by transcriptomic rather than gene mutational alterations in CRC organoids. Drug-tolerant cells evaded apoptosis and up-regulated MYC (c-myelocytomatosis oncogene product)/E2F1 (E2 family transcription factor 1) and/or interferon-α–related gene expression. Introduction of KRASG12D further increased the resilience of drug-persister CRC organoids against combination therapy. AURKA inhibition restored an apoptotic response in drug-tolerant KRAS–wild-type organoids. In dual epidermal growth factor receptor (EGFR)– pathway blockade-primed CRC organoids expressing KRASG12D, AURKA inhibition augmented apoptosis in cases that had acquired increased c-MYC protein levels during chemotolerance development. In patient-matched CRC cohorts, AURKA expression was increased in primary tumors and derived liver metastases. Conclusions Our study emphasizes the potential of patient-derived CRC organoids in modeling chemotherapy tolerance ex vivo. The applied therapeutic strategy of dual EGFR pathway blockade in combination with AURKA inhibition may prove effective for second-line treatment of chemotolerant CRC liver metastases with acquired KRAS mutation and increased AURKA/c-MYC expression., Graphical abstract

Objective To evaluate the prognostic significance of detectable circulating cell-free DNA (cfDNA) BRAF V600E/K mutations in patients with advanced melanoma enrolled in a clinical trial without BRAF-targeted therapy. Patients and Methods BRAF V600E/K mutation status was determined on archived tissue and pretreatment stored plasma from 149 patients with unresectable stage IV melanoma who were enrolled between May 5, 2010 and May 2, 2014 in the North Central Cancer Treatment Group/Alliance N0879 randomized phase 2 clinical trial. Results were reported as presence or absence of cfDNA BRAF V600E/K detection of assay vs tissue. Progression-free survival (PFS) and overall survival (OS) were assessed for patients with and without detectable BRAF mutation. Results In total, 63 of 149 (42.3%) patients had BRAF V600E/K results for tissue and blood, and 20 of 63 (31.7%) patients had tissue-diagnosed mutant BRAF. Of these, 11 of 20 (55.0%) patients had detectable plasma cfDNA BRAF. Among patients with tissue-mutant BRAF V600E/K, PFS and OS were shorter for those with corresponding cfDNA mutations (PFS, 5.8 vs 12.0 months; P=.051; OS, 9.2 vs 27.1 months; P=.054). Our assay demonstrated sensitivity of 55% (95% CI, 0.322 to 0.768), specificity of 97.7% (95% CI, 0.932 to 1.000), positive predictive value of 91.7% (95% CI, 0.760 to 1.000), and negative predictive value of 82.4% (95% CI, 0.719 to 0.928). Conclusion In advanced melanoma, detectable cfDNA BRAF V600E/K mutation is present in about half the patients with stage IV with BRAF-mutant melanoma tumor tissue and appears to confer a poorer prognosis when detectable. Given the poorer prognosis, cfDNA can be used to risk-stratify patients with metastatic melanoma in practice or clinical trials.Trial Registration: clinicaltrials.gov Identifier: NCT00976573

Although some evidence showed the activation of complement systems in COVID-19 patients, proinflammatory status and lectin pathway remain unclear. Thus, the present study aimed to demonstrate the role of MBL and ficolin-3 in the complement system activation and compared to pandemic Influenza A virus H1N1 subtype infection (H1N1pdm09) and control patients. A total of 27 lungs formalin-fixed paraffin-embedded samples (10 from H1N1 group, 6 from the COVID-19 group, and 11 from the control group) were analyzed by immunohistochemistry using anti-IL-6, TNF-alfa, CD163, MBL e FCN3 antibodies. Genotyping of target polymorphisms in the MBL2 gene was performed by real-time PCR. Proinflammatory cytokines such as IL-6 and TNF-alpha presented higher tissue expression in the COVID-19 group compared to H1N1 and control groups. The same results were observed for ICAM-1 tissue expression. Increased expression of the FCN3 was observed in the COVID-19 group and H1N1 group compared to the control group. The MBL tissue expression was higher in the COVID-19 group compared to H1N1 and control groups. The genotypes AA for rs180040 (G/A), GG for rs1800451 (G/A) and CC for rs5030737 (T/C) showed a higher prevalence in the COVID-19 group. The intense activation of the lectin pathway, with particular emphasis on the MBL pathway, together with endothelial dysfunction and a massive proinflammatory cytokines production, possibly lead to a worse outcome in patients infected with SARS-Cov-2. Moreover, 3 SNPs of our study presented genotypes that might be correlated with high MBL tissue expression in the COVID-19 pulmonary samples.

Background & Aims Tight junctions form a barrier to the paracellular passage of luminal antigens. Although most tight junction proteins reside within the apical tight junction complex, claudin-18 localizes mainly to the basolateral membrane where its contribution to paracellular ion transport is undefined. Claudin-18 loss in mice results in gastric neoplasia development and tumorigenesis that may or may not be due to tight junction dysfunction. The aim here was to investigate paracellular permeability defects in stomach mucosa from claudin-18 knockout (Cldn18-KO) mice. Methods Stomach tissue from wild-type, heterozygous, or Cldn18-KO mice were stripped of the external muscle layer and mounted in Ussing chambers. Transepithelial resistance, dextran 4 kDa flux, and potential difference (PD) were calculated from the chambered tissues after identifying differences in tissue histopathology that were used to normalize these measurements. Marker expression for claudins and ion transporters were investigated by transcriptomic and immunostaining analysis. Results No paracellular permeability defects were evident in stomach mucosa from Cldn18-KO mice. RNAseq identified changes in 4 claudins from Cldn18–KO mice, particularly the up-regulation of claudin-2. Although claudin-2 localized to tight junctions in cells at the base of gastric glands, its presence did not contribute overall to mucosal permeability. Stomach tissue from Cldn18–KO mice also had no PD versus a lumen-negative PD in tissues from wild-type mice. This difference resulted from changes in transcellular Cl– permeability with the down-regulation of Cl– loading and Cl– secreting anion transporters. Conclusions Our findings suggest that Cldn18-KO has no effect on tight junction permeability in the stomach from adult mice but rather affects anion permeability. The phenotype in these mice may thus be secondary to transcellular anion transporter expression/function in the absence of claudin-18., Graphical abstract

Cholinergic agonists, like levamisole, are a major class of anthelmintic drugs that are known to act selectively on nicotinic acetylcholine receptors (nAChRs) on the somatic muscle and nerves of nematode parasites to produce their contraction and spastic paralysis. Previous studies have suggested that in addition to the nAChRs found on muscle and nerves, there are nAChRs on non-excitable tissues of nematode parasites. We looked for evidence of nAChRs expression in the cells of the intestine of the large pig nematode, Ascaris suum, using RT-PCR and RNAscope in situ hybridization and detected mRNA of nAChR subunits in the cells. These subunits include components of the putative levamisole receptor in A. suum muscle: Asu-unc-38, Asu-unc-29, Asu-unc-63 and Asu-acr-8. Relative expression of these mRNAs in A. suum intestine was quantified by qPCR. We also looked for and found expression of G protein-linked acetylcholine receptors (Asu-gar-1). We used Fluo-3 AM to detect intracellular calcium changes in response to receptor activation by acetylcholine (as a non-selective agonist) and levamisole (as an L-type nAChR agonist) to look for evidence of functioning nAChRs in the intestine. We found that both acetylcholine and levamisole elicited increases in intracellular calcium but their signal profiles in isolated intestinal tissues were different, suggesting activation of different receptor sets. The levamisole responses were blocked by mecamylamine, a nicotinic receptor antagonist in A. suum, indicating the activation of intestinal nAChRs rather than G protein-linked acetylcholine receptors (GARs) by levamisole. The detection of nAChRs in cells of the intestine, in addition to those on muscles and nerves, reveals another site of action of the cholinergic anthelmintics and a site that may contribute to the synergistic interactions of cholinergic anthelmintics with other anthelmintics that affect the intestine (Cry5B)., Graphical abstract Image 1, Highlights • Novel levamisole anthelmintic receptors identified in a nematode parasite intestine. • Molecular techniques and RNAscope show dissimilar distributions of nAChR subunits. • Fluo-3 calcium sensitive dye detects two types of response to agonists. • Evidence of heterogeneous nAChRs present in the intestine. • Intestinal levamisole receptors can explain synergistic anthelmintic interactions.

Graphical abstract, Artificial intelligence (AI) and machine learning have significantly influenced many facets of the healthcare sector. Advancement in technology has paved the way for analysis of big datasets in a cost- and time-effective manner. Clinical oncology and research are reaping the benefits of AI. The burden of cancer is a global phenomenon. Efforts to reduce mortality rates requires early diagnosis for effective therapeutic interventions. However, metastatic and recurrent cancers evolve and acquire drug resistance. It is imperative to detect novel biomarkers that induce drug resistance and identify therapeutic targets to enhance treatment regimes. The introduction of the next generation sequencing (NGS) platforms address these demands, has revolutionised the future of precision oncology. NGS offers several clinical applications that are important for risk predictor, early detection of disease, diagnosis by sequencing and medical imaging, accurate prognosis, biomarker identification and identification of therapeutic targets for novel drug discovery. NGS generates large datasets that demand specialised bioinformatics resources to analyse the data that is relevant and clinically significant. Through these applications of AI, cancer diagnostics and prognostic prediction are enhanced with NGS and medical imaging that delivers high resolution images. Regardless of the improvements in technology, AI has some challenges and limitations, and the clinical application of NGS remains to be validated. By continuing to enhance the progression of innovation and technology, the future of AI and precision oncology show great promise.

Background & Aims We aim to describe the liver immune microenvironment by analyzing liver biopsies from patients with chronic HBV infection (CHB). Host immune cell signatures and their corresponding localization were characterized by analyzing the intrahepatic transcriptome in combination with a custom multiplex immunofluorescence panel. Method Matching FFPE and fresh frozen liver biopsies were collected from immune active patients within the open-label phase IV study GS-US-174-0149. RNA-Seq was conducted on 53 CHB liver biopsies from 46 patients. Twenty-eight of the 53 samples had matched FFPE biopsies and were stained with a 12-plex panel including cell segmentation, immune and viral biomarkers. Corresponding serum samples were screened using the MSD Human V-plex Screen Service to identify peripheral correlates for the immune microenvironment. Results Using unsupervised clustering of the transcriptome, we reveal two unique liver immune signatures classified as immune high and immune low based on the quantification of the liver infiltrate gene signatures. Multiplex immunofluorescence analysis demonstrated large periportal lymphoid aggregates in immune high samples consisting of CD4 and CD8 T cells, B cells and macrophages. Differentiation of the high and low immune microenvironments was independent of HBeAg status and peripheral viral antigen levels. In addition, longitudinal analysis indicates that treatment and normalization of ALT correlates with a decrease in liver immune infiltrate and inflammation. Finally, we screened a panel of peripheral biomarkers and identified ICAM-1 and CXCL10 as biomarkers that strongly correlate with these unique immune microenvironments. Conclusion These data provide a description of immune phenotypes in patients with CHB and show that immune responses are downregulated in the liver following nucleotide analogue treatment. This may have important implications for both the safety and efficacy of immune modulator programs aimed at HBV cure. Lay summary Liver biopsies from patients with chronic hepatitis B were submitted to RNA-Seq and multiplex immunofluorescence and identified two different liver immune microenvironments: immune high and immune low. Immune high patients showed elevated immune pathways, including interferon signaling pathways, and increase presence of immune cells. Longitudinal analysis of biopsies from treatment experienced patients showed that treatment correlates with a marked decrease in inflammation and these findings may have important implications for both safety and efficacy of immune modulator programs for HBV cure., Graphical abstract, Highlights • Two different liver immune microenvironments were identified in patients with chronic hepatitis B: immune high and immune low. • Immune high patients had elevated immune pathway activity and immune cell signatures corresponding to B cells, T cells and macrophages. • Antiviral treatment and normalization of ALT correlates with a marked decrease in liver immune infiltrate and inflammation. • CXCL10 and ICAM-1 were identified as peripheral biomarkers that correlated with these differentiated immune microenvironments.

Hereditary xanthinuria is a rare autosomal recessive disease caused by missense and loss of function variants in the xanthine dehydrogenase (XDH) or molybdenum cofactor sulfurase (MOCOS) genes. The aim of this study was to uncover variants underlying risk for xanthinuria in dogs. Affected dogs included two Manchester Terriers, three Cavalier King Charles Spaniels, an English Cocker Spaniel, a Dachshund, and a mixed-breed dog. Four putative causal variants were discovered: an XDH c.654G > A splice site variant that results in skipping of exon 8 (mixed-breed dog), a MOCOS c.232G > T splice site variant that results in skipping of exon 2 (Manchester Terriers), a MOCOS p.Leu46Pro missense variant (Dachshund), and a MOCOS p.Ala128Glyfs*30 frameshift variant that results in a premature stop codon (Cavalier King Charles Spaniels and English Cocker Spaniel). The two splice site variants suggest that the regions skipped are critical to the respective enzyme function, though protein misfolding is an alternative theory for loss of function. The MOCOS p.Leu46Pro variant has not been previously reported in human or other animal cases and provides novel data supporting this residue as critical to MOCOS function. All variants were present in the homozygous state in affected dogs, indicating an autosomal recessive mode of inheritance. Allele frequencies of these variants in breed-specific populations ranged from 0 to 0.18. In conclusion, multiple diverse variants appear to be responsible for hereditary xanthinuria in dogs.

Highlights • FGFR2 rearrangement frequency in Chinese cases is only 5.45%. • The first genomic aberration profiling of FGFRs in Chinese cases is presented. • Cases with FGFRs rearrangement have distinct clinical phenotype. • Patients without FGFR2 rearrangement may harbor other FGFR alterations., Genomic aberrations (GAs) in fibroblast growth factor receptors (FGFRs) are involved in the pathogenesis of intrahepatic cholangiocarcinoma (ICC), and clinical trials have shown efficacy of FGFR inhibitors in treating ICC patients with FGFR GAs such as FGFR2 rearrangement. To clarify the FGFRs GA profile and corresponding clinicopathological features in Chinese patients with ICC, a total of 257 cases were identified. Fourteen cases (5.45%) were positive for FGFR2 rearrangement. Further analysis on the 110 FGFR2 rearrangement negative cases showed that 13 patients present additional FGFRs GAs, including FGFR3 rearrangement (2.73%), and FGFRs mutations. When compared with patients without FGFRs GAs, those with FGFR2 or FGFR3 rearrangement presented more under the age of 58 years, female sex, HBsAb positivity, CD10 expression, and PD-L1 expression. The clinical characteristics between patients with FGFRs mutation and those without FGFRs GAs were similar, with the exception that cases with FGFRs mutation have more hepatolithiasis. We concluded that FGFR rearrangement is associated with unique clinical phenotypes in ICC.

We recently described a positive feedback loop connecting c-MYC, NAMPT, DBC1 and SIRT1 that contributes to unrestricted cancer cell proliferation. Here we determine the relevance of the loop for serrated route intestinal tumorigenesis using genetically well-defined BrafV600E and K-rasG12D mouse models. In both models we show that c-MYC and SIRT1 protein expression increased through progression from hyperplasia to invasive carcinomas and metastases. It correlated with high NAMPT expression and was directly associated to activation of the oncogenic drivers. Assessing functional and molecular consequences of pharmacological interference with factors of the loop, we found that inhibition of NAMPT resulted in apoptosis and reduced clonogenic growth in human BRAF-mutant colorectal cancer cell lines and patient-derived tumoroids. Blocking SIRT1 activity was only effective when combined with a PI3K inhibitor, whereas the latter antagonized the effects of NAMPT inhibition. Interfering with the positive feedback loop was associated with down-regulation of c-MYC and temporary de-repression of TP53, explaining the anti-proliferative and pro-apoptotic effects. In conclusion we show that the c-MYC-NAMPT-DBC1-SIRT1 positive feedback loop contributes to murine serrated tumor progression. Targeting the feedback loop exerted a unique, dual therapeutic effect of oncoprotein inhibition and tumor suppressor activation. It may therefore represent a promissing target for serrated colorectal cancer, and presumably for other cancer types with deregulated c-MYC.

BACKGROUND: Ependymomas (EPNs) are the third most common brain tumor in children. These tumors are resistant to available chemotherapeutic treatments, therefore new effective targeted therapeutics must be identified. Increasing evidence shows epigenetic alterations including histone posttranslational modifications (PTMs), are associated with malignancy, chemotherapeutic resistance and prognosis for pediatric EPNs. In this study we examined histone PTMs in EPNs and identified potential targets to improve chemotherapeutic efficacy. METHODS: Global histone H3 lysine 4 trimethylation (H3K4me3) levels were detected in pediatric EPN tumor samples with immunohistochemistry and immunoblots. Candidate genes conferring therapeutic resistance were profiled in pediatric EPN tumor samples with micro-array. Promoter H3K4me3 was examined for two candidate genes, CCND1 and ERBB2, with chromatin-immunoprecipitation coupled with real-time PCR (ChIP-PCR). These methods and MTS assay were used to verify a relationship between H3K4me3 levels and CCND1 and ERBB2, and to investigate cell viability in response to chemotherapeutic drugs in primary cultured pediatric EPN cells. RESULTS: H3K4me3 levels positively correlate with WHO grade malignancy in pediatric EPNs and are associated with progression free survival in patients with posterior fossa group A EPNs (PF-EPN-A). Reduction of H3K4me3 by silencing its methyltransferase SETD1A, in primary cultured EPN cells increased cell response to chemotherapy. CONCLUSIONS: Our results support the development of a novel treatment that targets H3K4me3 to increase chemotherapeutic efficacy in pediatric PF-EPN-A tumors.

Some early-stage clear cell renal cell carcinomas (ccRCCs) of ≤7 cm are associated with a poor clinical outcome. In this study, we investigated molecular biomarkers associated with aggressive clinical T1 stage ccRCCs of ≤7 cm, which were used to develop a risk prediction tool toward guiding the decision of treatment. Among 1069 nephrectomies performed for ccRCC of ≤7 cm conducted between January 2008 and December 2014, 177 cases with available formalin-fixed paraffin-embedded tissue were evaluated. An aggressive tumor was defined as a tumor exhibiting synchronous metastasis, recurrence, or leading to cancer-specific death. Expression levels of six genes (FOXC2, CLIP4, PBRM1, BAP1, SETD2, and KDM5C) were measured by reverse-transcription polymerase chain reaction (qRT-PCR) and their relation to clinical outcomes was investigated. Immunohistochemistry was performed to validate the expression profiles of selected genes significantly associated with clinical outcomes in multivariate analysis. Using these genes, we developed a prediction model of aggressive ccRCC based on logistic regression and deep-learning methods. FOXC2, PBRM1, and BAP1 expression levels were significantly lower in aggressive ccRCC than non-aggressive ccRCC both in univariate and multivariate analysis. The immunohistochemistry result demonstrated the significant downregulation of FOXC2, PBRM1, and BAP1 expression in aggressive ccRCC. Adding immunohistochemical staining results to qRT-PCR, the aggressive ccRCC prediction models had the area under the curve (AUC) of 0.760 and 0.796 and accuracy of 0.759 and 0.852 using the logistic regression method and deep-learning method, respectively. Use of these biomarkers and the developed prediction model can help stratify patients with clinical T1 stage ccRCC., Graphical Abstract Unlabelled Image

Background & Aims A single point mutation in the Z-variant of alpha 1-antitrypsin (Z-AAT) alone can lead to both a protein folding and trafficking defect, preventing its exit from the endoplasmic reticulum (ER), and the formation of aggregates that are retained as inclusions within the ER of hepatocytes. These defects result in a systemic AAT deficiency (AATD) that causes lung disease, whereas the ER-retained aggregates can induce severe liver injury in patients with ZZ-AATD. Unfortunately, therapeutic approaches are still limited and liver transplantation represents the only curative treatment option. To overcome this limitation, a better understanding of the molecular basis of ER aggregate formation could provide new strategies for therapeutic intervention. Methods Our functional and omics approaches here based on human hepatocytes from patients with ZZ-AATD have enabled the identification and characterisation of the role of the protein disulfide isomerase (PDI) A4/ERP72 in features of AATD-mediated liver disease. Results We report that 4 members of the PDI family (PDIA4, PDIA3, P4HB, and TXNDC5) are specifically upregulated in ZZ-AATD liver samples from adult patients. Furthermore, we show that only PDIA4 knockdown or alteration of its activity by cysteamine treatment can promote Z-AAT secretion and lead to a marked decrease in Z aggregates. Finally, detailed analysis of the Z-AAT interactome shows that PDIA4 silencing provides a more conducive environment for folding of the Z mutant, accompanied by reduction of Z-AAT-mediated oxidative stress, a feature of AATD-mediated liver disease. Conclusions PDIA4 is involved in AATD-mediated liver disease and thus represents a therapeutic target for inhibition by drugs such as cysteamine. PDI inhibition therefore represents a potential therapeutic approach for treatment of AATD. Lay summary Protein disulfide isomerase (PDI) family members, and particularly PDIA4, are upregulated and involved in alpha 1-antitrypsin deficiency (AATD)-mediated liver disease in adults. PDI inhibition upon cysteamine treatment leads to improvements in features of AATD and hence represents a therapeutic approach for treatment of AATD-mediated liver disease., Graphical abstract, Highlights • PDIA4 is upregulated and involved in alpha 1-antitrypsin deficiency (AATD)-mediated liver disease in adults. • Knockdown of PDIA4 by siRNA or inhibition upon cysteamine treatment leads to improvements in features of AATD. • RNA interference against PDIA4 or cysteamine represent approaches for treatment of AATD-mediated liver disease.

The choice for adjuvant chemotherapy in stage II colorectal cancer is controversial as many patients are cured by surgery alone and it is difficult to identify patients with high risk of recurrence of the disease. There is a need for better stratification of this group of patients. Mass spectrometry imaging could identify patients at risk. We report here the N-glycosylation signatures of the different cell populations in a group of stage II colorectal cancer tissue samples. The cancer cells, compared with normal epithelial cells, have increased levels of sialylation and high-mannose glycans, as well as decreased levels of fucosylation and highly branched N-glycans. When looking at the interface between cancer and its microenvironment, it seems that the cancer N-glycosylation signature spreads into the surrounding stroma at the invasive front of the tumor. This finding was more outspoken in patients with a worse outcome within this sample group., Graphical Abstract, Highlights • N-glycan MS imaging as tool for better cancer stratification. • Increased levels of sialylated and high-mannose glycans in CRC stage II cancer. • Characterization of the interaction between cancer and its direct microenvironment. • Insight into N-glycosylation signature of high-risk patients with short survival., In Brief There is a need for better stratification of patients with stage II colorectal cancer as the treatment option remains controversial. Using MS imaging, we have characterized the N-glycosylation signatures of the different cell populations in stage II CRC tissues. We found, looking at the tumor microenvironment, that the cancer N-glycosylation signature spreads into the surrounding stroma at the invasive front of the tumor. This finding was more outspoken in patients with a worse outcome.

Background & Aims Colorectal cancer (CRC) is thought to arise when the cumulative mutational burden within colonic crypts exceeds a certain threshold that leads to clonal expansion and ultimately neoplastic transformation. Therefore, quantification of the fixation and subsequent expansion of somatic mutations in normal epithelium is key to understanding colorectal cancer initiation. The aim of the present study was to determine how advantaged expansions can be accommodated in the human colon. Methods Immunohistochemistry was used to visualize loss of the cancer driver KDM6A in formalin-fixed paraffin-embedded (FFPE) normal human colonic epithelium. Combining microscopy with neural network-based image analysis, we determined the frequencies of KDM6A-mutant crypts and fission/fusion intermediates as well as the spatial distribution of clones. Mathematical modeling then defined the dynamics of their fixation and expansion. Results Interpretation of the age-related behavior of KDM6A-negative clones revealed significant competitive advantage in intracrypt dynamics as well as a 5-fold increase in crypt fission rate. This was not accompanied by an increase in crypt fusion. Mathematical modeling of crypt spacing identifies evidence for a crypt diffusion process. We define the threshold fission rate at which diffusion fails to accommodate new crypts, which can be exceeded by KRAS activating mutations. Conclusions Advantaged gene mutations in KDM6A expand dramatically by crypt fission but not fusion. The crypt diffusion process enables accommodation of the additional crypts up to a threshold value, beyond which polyp growth may occur. The fission rate associated with KRAS mutations offers a potential explanation for KRAS-initiated polyps., Graphical abstract

Highlights • This finding may provide a novelty insight to help elucidate the mechanisms of the relapses. • Relapsed tumours from stage IA LUAD patients exhibited a weakened immune phenotype. • A targeted 395-gene expression NGS assay helps assess tumour immunity., Patients with early-stage non-small cell lung cancer (NSCLC), even stage IA, are at substantial risk of relapse and death. We explored the distinct features of molecular alterations and immune-related gene expression in Formalin-fixed paraffin-embedded (FFPE) samples from 25 relapsed patients compared with 25 non-relapsed patients through using whole-exome sequencing and an immune oncology panel RNA sequencing platform. Results showed that the chemokine, cytolytic activity and tumour-associated antigen gene signatures exhibited significantly higher expression in non-relapsed tumours from stage IA lung adenocarcinoma (LUAD) than that in relapsed tumours. Besides, Kaplan–Meier survival analysis revealed that the gene signatures of chemokines and tumour-associated antigens were significantly associated with the patients’ disease-free survival (DFS), indicating their prognostic value in early-stage LUAD. Cytolytic activity displayed a similar trend but failed to reach statistical significance. These findings revealed a weakened immune phenotype in relapsed tumours and provide valuable information for improving the treatment management of these high-risk patients. Due to the overall small patient number in this study, these differences should be further validated in a larger cohort.

Burkitt lymphoma (BL) is a malignant tumor in children. Although BL is generally curable, early relapse and refractoriness may occur. Some molecular indicators have been recently suggested for BL diagnosis, but large heterogeneity still exists. This study aimed at providing clinical molecular targets and methods that may help improve diagnosis and treatment of childhood BL. Only children patients were included in the study, and targeted gene sequencing was conducted to identify tumor specific mutations. The mRNA and protein level expression of potential target genes were measured by real-time PCR and immunohistochemistry. The relationship between BL specific gene mutation and differential expression with clinical features was analyzed. The results showed that i) detailed analysis of c-MYC/BCL2/BCL6 gene loci alteration and gene expression would help in accurate diagnosis and treatment determination of childhood BL; ii) loss-of-function mutations in SOCS1 or CIITA gene might be used as malignant markers for BL diagnosis and prognosis; iii) specific mutations of CD79A, MYD88, KLF2, DNMT3A and NFKBIE genes often concurrently existed in BL and showed association with benign clinical outcomes; iv) the high expression of MYC, TCF3 and loss-of-function ID3 genes in tumor may be potential therapeutic targets and could be used for treatment monitoring; and v) four MYC-translocation negative cases were re-defined as high-grade B-cell lymphoma-not otherwise specified (HGBL-NOS) but showed similar clinical outcomes and molecular features to other BL cases in the study, suggesting more studies needed to explore the molecular mechanisms and clinical significance of this provisional tumor entity., Highlights • Detailed analysis of c-MYC/BCL2/BCL6 gene alteration and expression may help in accurate diagnosis and treatment; • The MYC-translocation negative cases (HGBL-NOS) showed similar clinical outcomes and molecular features to other cases; • Loss-of-function mutations of SOCS1 or CIITA gene could be used as malignant markers for diagnosis and prognosis; • Concurrent mutations in CD79A, MYD88, KLF2, DNMT3A and NFKBIE genes associated with benign clinical outcomes; • High expression of MYC, TCF3 and loss-of-function ID3 gene in tumor may be potential therapeutic targets.

Background and Aims Gastrointestinal (GI) manifestations have been increasingly reported in Coronavirus Disease 2019 (COVID-19) patients. However, the roles of the GI tract in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection are not fully understood. We investigated how the GI tract is involved in SARS-CoV-2 infection to elucidate the pathogenesis of COVID-19. Methods Our previously established nonhuman primate (NHP) model of COVID-19 was modified in this study to test our hypothesis. Rhesus monkeys were infected with an intragastric or intranasal challenge with SARS-CoV-2. Clinical signs were recorded after infection. Viral genomic RNA was quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Host responses to SARS-CoV-2 infection were evaluated by examining inflammatory cytokines, macrophages, histopathology and mucin barrier integrity. Results Intranasal inoculation with SARS-CoV-2 led to infections and pathological changes not only in respiratory tissues but also in digestive tissues. Expectedly, intragastric inoculation with SARS-CoV-2 resulted in the productive infection of digestive tissues and inflammation in both the lung and digestive tissues. Inflammatory cytokines were induced by both types of inoculation with SARS-CoV-2, consistent with the increased expression of CD68. Immunohistochemistry and alcian blue/periodic acid-Schiff (AB-PAS) staining showed decreased Ki67, increased cleaved caspase 3 and decreased numbers of mucin-containing goblet cells, suggesting that the inflammation induced by these two types of inoculation with SARS-CoV-2 impaired the GI barrier and caused severe infections. Conclusions Both intranasal and intragastric inoculation with SARS-CoV-2 caused pneumonia and GI dysfunction in our rhesus monkey model. Inflammatory cytokines are possible connections for the pathogenesis of SARS-CoV-2 between the respiratory and digestive systems., Graphical abstract

Background To develop a radiomics signature for predicting overall survival (OS)/progression-free survival (PFS) in patients with medulloblastoma (MB), and to investigate the incremental prognostic value and biological pathways of the radiomics patterns. Methods A radiomics signature was constructed based on magnetic resonance imaging (MRI) from a training cohort (n = 83), and evaluated on a testing cohort (n = 83). Key pathways associated with the signature were identified by RNA-seq (GSE151519). Prognostic value of pathway genes was assessed in a public GSE85218 cohort. Findings The radiomics-clinicomolecular signature predicted OS (C-index 0.762) and PFS (C-index 0.697) better than either the radiomics signature (C-index: OS: 0.649; PFS: 0.593) or the clinicomolecular signature (C-index: OS: 0.725; PFS: 0.691) alone, with a better calibration and classification accuracy (net reclassification improvement: OS: 0.298, P = 0.022; PFS: 0.252, P = 0.026). Nine pathways were significantly correlated with the radiomics signature. Average expression value of pathway genes achieved significant risk stratification in GSE85218 cohort (log-rank P = 0.016). Interpretation This study demonstrated radiomics signature, which associated with dysregulated pathways, was an independent parameter conferring incremental value over clinicomolecular factors in survival predictions for MB patients. Funding A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Background Pancreatic cancer (PC) is one of the most lethal solid malignancies in the world due to its excessive cell proliferation and aggressive metastatic features. Emerging evidences revealed the importance of posttranscriptional modifications of RNAs in PC progression. However, knowledge about the 5-methylcytosine (m5C) RNA modification in PC is still extremely limited. In this study, we attempted to explore the expression changes and clinical significances of 12 known m5C-related genes among PC patients. Methods A total of 362 normal and 382 tumor specimens from PC patients were examined for candidate m5C-related gene and protein expression by using quantitative PCR (qPCR) and immunohistochemistry (IHC). The proliferation rate of PC cells was detected by MTS assay. Xenograft mouse models were used to assess the role of NSUN6 in PC tumor formation. Findings Through analyzing the four Gene Expression Omnibus (GEO) databases, six m5C-related genes shown significant and consistent alterations were selected for further examination in our 3 independent PC cohorts. Finally, we identified the reduction of NSUN6 as a common feature of all PC sample sets examined. NSUN6 expression correlated with clinicopathologic parameters including T stage, and Ki67+ cell rate. Further assessing the transcriptional profiles of 50 PC tissues, we found biological processes associated with cell proliferation like cell cycle and G2M checkpoint were enriched in NSUN6 lower expression group. Helped by in vitro PC cell lines and in vivo xenograft mouse models, we confirmed the role of NSUN6 in regulating cell proliferation and PC tumor growth. Last but also importantly, we also show the good performance of NSUN6 in evaluating tumor recurrence and survival among PC patients. Interpretation Our data suggested that NSUN6 is an important factor involved in regulating cell proliferation of PC, and highlights the potential of novel m5C-based clinical modalities as a therapeutic approach in PC patients. Funding This study was supported by the National Natural Science Foundation of China (Grant Nos. 81803014, 81802424, and 81802911).

Background: High frequency of MNNG HOS transforming (MET) exon 14 skipping mutation (MET exon 14Δ) has been reported in pulmonary sarcomatoid carcinomas (PSCs). However, the frequencies differ greatly. Our study aims to investigate the frequency of MET alterations and the correlations among MET exon 14Δ, amplification, and protein overexpression in a large cohort of PSCs. MET exon 14Δ, amplification, and protein overexpression were detected in 124 surgically resected PSCs by using Sanger sequencing, fluorescent in situ hybridization (FISH), and immunohistochemistry (IHC) respectively. MET exon 14Δ was identified in 9 (7.3%) of 124 cases, including 6 pleomorphic carcinomas, 2 spindle cell carcinomas and 1 carcinosarcoma. MET amplification and protein overexpression were detected in 6 PSCs (4.8%) and 25 PSCs (20.2%), respectively. MET amplification was significantly associated with overexpression (P

Background/Objectives: Tumour budding and poorly differentiated clusters (PDC) represent forms of tumour invasion. We hypothesised that T-cell densities (reflecting adaptive anti-tumour immunity) might be inversely associated with tumour budding and PDC in colorectal carcinoma. Methods: Utilising 915 colon and rectal carcinomas in two U.S.-wide prospective cohort studies, and multiplex immunofluorescence combined with machine learning algorithms, we assessed CD3, CD4, CD8, CD45RO (PTPRC), and FOXP3 co-expression patterns in lymphocytes. Tumour budding and PDC at invasive fronts were quantified by digital pathology and image analysis using the International tumour Budding Consensus Conference criteria. Using covariate data of 4,420 incident colorectal cancer cases, inverse probability weighting (IPW) was integrated with multivariable logistic regression analysis that assessed the association of T-cell subset densities with tumour budding and PDC while adjusting for selection bias due to tissue availability and potential confounders, including microsatellite instability status. Findings: Tumour budding counts were inversely associated with density of CD3+CD8+ [lowest vs. highest: multivariable odds ratio (OR), 0.50; 95% confidence interval (CI), 0.35–0.70; Ptrend < 0.001] and CD3+CD8+CD45RO+ cells (lowest vs. highest: multivariable OR, 0.44; 95% CI, 0.31–0.63; Ptrend < 0.001) in tumour epithelial region. Tumour budding levels were associated with higher colorectal cancer-specific mortality (multivariable hazard ratio, 2.13; 95% CI, 1.57–2.89; Ptrend < 0.001) in Cox regression analysis. There were no significant associations of PDC with T-cell subsets. Interpretation: Tumour epithelial naïve and memory cytotoxic T cell densities are inversely associated with tumour budding at invasive fronts, suggesting that cytotoxic anti-tumour immunity suppresses tumour microinvasion.

Background & Aims Cancer-associated fibroblasts (CAFs) from pancreatic adenocarcinoma (PDA) present high protein synthesis rates. CAFs express the G-protein–coupled somatostatin receptor sst1. The sst1 agonist SOM230 blocks CAF protumoral features in vitro and in immunocompromised mice. We have explored here the therapeutic potential of SOM230, and underlying mechanisms, in immunocompetent models of murine PDA mimicking the heavy fibrotic and immunosuppressive stroma observed in patient tumors. Methods Large-scale mass spectrometry analyses were performed on media conditioned from 9 patient PDA-derived CAF primary cultures. Spontaneous transgenic and experimental (orthotopic co-graft of tumor cells plus CAFs) PDA-bearing mice were longitudinally ultrasound-monitored for tumor and metastatic progression. Histopathology and flow cytometry analyses were performed on primary tumors and metastases. Stromal signatures were functionally validated through bioinformatics using several published, and 1 original, PDA database. Results Proteomics on the CAF secretome showed that SOM230 controls stromal activities including inflammatory responses. Among the identified secreted proteins, we validated that colony-stimulating factor 1 (CSF-1) (a macrophage growth factor) was reduced by SOM230 in the tumor and plasma of PDA-harboring mice, alongside intratumor stromal normalization (reduced CAF and macrophage activities), and dramatic metastasis reduction. In transgenic mice, these SOM230 benefits alleviate the chemotherapy-induced (gemcitabine) immunosuppressive stroma reshaping. Mechanistically, SOM230 acts in vivo on CAFs through sst1 to disrupt prometastatic CAF production of CSF-1 and cross-talk with macrophages. We found that in patients, stromal CSF-1 was associated with aggressive PDA forms. Conclusions We propose SOM230 as an antimetastatic therapy in PDA for its capacity to remodel the fibrotic and immunosuppressive myeloid stroma. This pharmacotherapy should benefit PDA patients treated with chemotherapies., Graphical abstract

Inflammatory breast cancer (IBC) is the most aggressive and lethal form of breast cancer, characterized by a high infiltration of tumor-associated macrophages and poor prognosis. To identify new biomarkers and to elucidate the molecular mechanisms underlying IBC pathogenesis, we investigated the expression pattern of heparanase (HPSE) and its activator cathepsin L (CTSL). First, we quantitated the HPSE and CTSL mRNA levels in a cohort of breast cancer patients after curative surgery (20 IBC and 20-non-IBC). We discovered that both HPSE and CTSL mRNA levels were significantly induced in IBC tissue vis-à-vis non-IBC patients (p, Highlights • Inflammatory breast carcinomas (IBC) contain high mRNA levels of heparanase and cathepsin L. • Heparanase immunoreactivity correlates with infiltration of M2 tumor-associated macrophages. • Heparanase depletion inhibits SUM149 breast carcinoma cell invasion and MMP2 levels. • The secretome of CD14+ monocytes isolated from IBC axillary tributaries blood upregulates HPSE mRNA in SUM-149 cells. • The master regulator IL-10 evokes HPSE mRNA and protein expression in SUM149 cells.

Insulin resistance impairs postprandial glucose uptake through glucose transporter type 4 (GLUT4) and is the primary defect preceding type 2 diabetes. We previously generated an insulin-resistant mouse model with human GLUT4 promoter-driven insulin receptor knockout (GIRKO) in the muscle, adipose, and neuronal subpopulations. However, the rate of diabetes in GIRKO mice remained low prior to 6 months of age on normal chow diet (NCD), suggesting that additional factors/mechanisms are responsible for adverse metabolic effects driving the ultimate progression of overt diabetes. In this study, we characterized the metabolic phenotypes of the adult GIRKO mice acutely switched to high-fat diet (HFD) feeding in order to identify additional metabolic challenges required for disease progression. Distinct from other diet-induced obesity (DIO) and genetic models (e.g., db/db mice), GIRKO mice remained leaner on HFD feeding, but developed other cardinal features of insulin resistance syndrome. GIRKO mice rapidly developed hyperglycemia despite compensatory increases in β-cell mass and hyperinsulinemia. Furthermore, GIRKO mice also had impaired oral glucose tolerance and a limited glucose-lowering benefit from exendin-4, suggesting that the blunted incretin effect contributed to hyperglycemia. Secondly, GIRKO mice manifested severe dyslipidemia while on HFD due to elevated hepatic lipid secretion, serum triglyceride concentration, and lipid droplet accumulation in hepatocytes. Thirdly, GIRKO mice on HFD had increased inflammatory cues in the gut, which were associated with the HFD-induced microbiome alterations and increased serum lipopolysaccharide (LPS). In conclusion, our studies identified important gene/diet interactions contributing to diabetes progression, which might be leveraged to develop more efficacious therapies.

Background: Meningiomas that are progesterone receptor positive have a low recurrence rate and good prognosis compared to those that are progesterone receptor negative. This study aimed to determine the prevalence of expression of progesterone in meningiomas and its association with clinicopathological characteristics. Materials and methods: This was a cross-sectional laboratory-based study that was conducted at Muhimbili National Hospital. The study included 112 formalin-fixed paraffin-embedded tissue blocks of patients who were confirmed to have meningiomas on histological basis from January 2010 to December 2014. Immunohistochemical expression of progesterone receptor was tested using a primary monoclonal progesterone receptor antibody ready to use (IR 068 Dako). The χ2 test was used to determine the association between clinicopathological characteristics and progesterone receptor expression. A 2-tailed P < 0.05 was considered significant. Results: The mean age of the patients was 45.5 ± 3.601 years, and majority (66.1%, n = 74) were in the age group between 31 and 60 years. Also, majority of the patients (60%, n = 67) in this study were females. Over one-third of the cases (34.8%, n = 39) comprised of meningotheliomatous subtype, and majority of the cases (89.3%, n = 100) were of grade I. The prevalence of progesterone expression was 54.5% (n = 61), and only age was associated with progesterone receptor expression (P = 0.043). Conclusion: The finding of high expression of the progesterone receptor for grade I cases in this study indicates that progesterone receptor expression in meningiomas is of prognostic value and may be considered when evaluating patients for management. Lack of expression of progesterone receptor in all the malignant cases is intriguing and needs further studies that can investigate its prognostic role.

Background Emerging evidence indicates that small nucleolar RNAs (snoRNAs) play a central role in oncogenesis. Herein, we systematically evaluated expression profiles of snoRNAs in colorectal cancer (CRC) and investigated their clinical and functional role in this malignancy. Methods We compared expression levels of snoRNAs between cancer and normal tissues using publicly available datasets and identified the most differentially expressed and commonly upregulated snoRNAs in CRC. These results were examined in 489 colorectal tissues to assess their clinical significance, followed by a series of in vitro and in vivo experiments to evaluate the functional role of candidate snoRNAs. Results Using multiple RNA profiling datasets, we identified consistent overexpression of SNORA21 in CRC. In the clinical validation cohorts, the expression level of SNORA21 was upregulated in colorectal adenomas and cancers. Furthermore, elevated SNORA21 emerged as an independent factor for predicting poor survival. Both in vitro and in vivo experiments revealed that CRISPR/Cas9-mediated inhibition of SNORA21 expression resulted in decreased cell proliferation and invasion through modulation of multiple cancer related pathways. Conclusions We systematically identified SNORA21 as a key oncogenic snoRNA in CRC, which plays an important role in cancer progression, and might serve as an important prognostic biomarker in CRC., Highlights • Systematic and comprehensive analysis revealed that SNORA21 was significantly upregulated in CRC. • Elevated SNONA21 expression was related to metastasis and predicted poor prognosis in CRC patients. • SNORA21 is a promising cancer prognostic biomarker and a potential therapeutic target in CRC. Emerging evidence indicates that small nucleolar RNAs (snoRNAs) might play a central role in oncogenesis; however, the clinical significance and functional roles of snoRNAs in colorectal cancer (CRC) remain unclear. Herein, using multiple publicly available datasets, we systematically assessed expression profiles of snoRNAs in CRC and elucidated that the high expression of SNORA21 was associated with distant metastasis and poor overall survival. A series of in vitro and in vivo experiments revealed oncogenic role for SNORA21. Our findings indicate that SNORA21 is a promising cancer prognostic biomarker and a potential therapeutic target in CRC.

Epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of effectiveness of treatment with EGFR tyrosine kinase inhibitors (TKIs). Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKIs therapy. Here, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction. Serial-dilution experiments with genomic DNA harboring EGFR mutations revealed linear performance, with analytical sensitivity ~ 0.01% for each mutation. All 33 EGFR-activating mutations detected in formalin-fixed paraffin-embedded (FFPE) tissue samples by the conventional method were also detected by this multiplex assay. Owing to the higher sensitivity, an additional mutation (T790M; including an ultra-low-level mutation, Highlights • An ultrasensitive multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction was developed. • This multiplex mutation detection of common mutations of EGFR is a feasible alternative. • A rare pretreatment EGFR mutation (T790M) with a mutant allele frequency below 0.1% was detected via this multiplex assay. Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKI therapy. In this article, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction with 0.01% sensitivity. All 33 EGFR-activating mutations detected in FFPE samples by the conventional method were also detected by this multiplex assay. Owing to the higher sensitivity, an additional mutation was detected in the same reaction. Results of this proof-of-principle study on clinical samples indicate clinical utility of multiplex ddPCR for screening for multiple EGFR mutations.

In the United States, the incidence and mortality rates for Merkel cell carcinoma (MCC) have more than tripled in the last 2 decades.1 The main risk factors involved in MCC pathogenesis include ultraviolet light exposure, immunosuppression, and Merkel cell polyomavirus (MCpV).2 The most common sites include the head and neck (53%) and extremities (34%–35%), with trunk and mucosal surfaces representing less than 10% of cases.3 MCC has a high propensity for local recurrence, with a median time between diagnosis and recurrence of 6 months.4 We present a case of a 79-year-old woman who initially presented with 2 MCCs of the nasal bridge and left arm, followed by a third MCC on the right nose 8 years later. We used array comparative genomic hybridization (aCGH) testing to determine whether the most recent tumor was the result of metastasis or represented a delayed primary recurrence of MCC. Through this case, we suggest that using aCGH to distinguish between primary and metastatic disease can play an important role in future MCC management.

Different species of camelids play an important role in the epidemiology of various emerging infectious diseases such as Middle East respiratory syndrome. For precise investigations of the immunopathogenesis in these host species, appropriate immunohistochemical markers are highly needed in order to phenotype distinct immune cells populations in camelids. So far, specific immunohistochemical markers for camelid immune cells are rarely commercially available, and cross-reactivity studies are restricted to the use of frozen dromedary tissues. To bridge this gap, 14 commercially available primary antibodies were tested for their suitability to demonstrate immune cell populations on formalin fixed paraffin-embedded (FFPE) tissue sections of dromedaries, Bactrian camels, llamas, and alpacas in the present study. Out of these, 9 antibodies directed against CD3, CD20, CD79α, HLA-DR, Iba-1, myeloid/histiocyte antigen, CD204, CD208, and CD68 antigen exhibited distinct immunoreaction patterns to certain camelid immune cell subsets. The distribution of these antigens was comparatively evaluated in different anatomical compartments of thymus, spleen, mesenteric, and tracheobronchial lymph nodes. The presented results will provide a basis for further investigations in camelids, especially with respect to the role of the immune response in certain infectious diseases, which harbor a considerable risk to spill over to other species.

Background & Aims Chronic liver inflammation leads to fibrosis and cirrhosis and is associated with an accumulation of intrahepatic TNFα-secreting CD206+ macrophages, which may participate in maintaining chronic liver disease in a GM-CSF-dependent manner. We aimed to elucidate the exact role of GM-CSF in the development and progression of chronic liver disease. Methods Liver immunohistochemistry and serum quantification were performed in patients with viral and non-viral-related liver disease to compare CD206+ monocyte/macrophages, fibrosis and GM-CSF. This was followed by functional validations in vitro and in vivo in humanised mice. Results Using multiplex immunofluorescence and histo-cytometry, we show that highly fibrotic livers had a greater density of CD206+ macrophages that produced more TNFα and GM-CSF in the non-tumour liver regions of patients with hepatocellular carcinoma (n = 47), independent of aetiology. In addition, the absolute number of CD206+ macrophages strongly correlated with the absolute number of GM-CSF-producing macrophages. In non-HCC chronic HCV+ patients (n = 40), circulating GM-CSF levels were also increased in proportion to the degree of liver fibrosis and serum viral titres. We then demonstrated in vitro that monocytes converted to TNFα-producing CD206+ macrophage-like cells in response to bacterial products (lipopolysaccharide) in a GM-CSF-dependent manner, confirming the in vivo normalisation of serum GM-CSF concentration following oral antibiotic treatment observed in HBV-infected humanised mice. Finally, anti-GM-CSF neutralising antibody treatment reduced intrahepatic CD206+ macrophage accumulation and abolished liver fibrosis in HBV-infected humanised mice. Conclusions While the direct involvement of CD206+ macrophages in liver fibrosis remains to be demonstrated, these findings show that GM-CSF may play a central role in liver fibrosis and could guide the development of anti-GM-CSF antibody-based therapy for the management of patients with chronic liver disease. Lay summary Liver fibrosis is a major driver of liver disease progression. Herein, we have shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the development of liver fibrosis. Our findings support the use of anti-GM-CSF neutralising antibodies for the management of patients with chronic liver disease resulting from both viral and non-viral causes., Graphical abstract, Highlights • GM-CSF and TNFα producing CD206+ macrophages accumulate in human fibrotic liver • Serum GM-CSF is increased in HCV+ patients with higher liver fibrosis • GM-CSF drives monocyte to CD206+ macrophage conversion • Anti-GM-CSF therapy suppresses liver fibrosis and CD206+ macrophage accumulation

The histopathological subtype of lung adenocarcinoma (LUAD) is closely associated with prognosis. Micropapillary or solid predominant LUAD tends to relapse after surgery at an early stage, whereas lepidic pattern shows a favorable outcome. However, the molecular mechanism underlying this phenomenon remains unknown. Here, we recruited 31 lepidic predominant LUADs (LR: low-risk subtype group) and 28 micropapillary or solid predominant LUADs (HR: high-risk subtype group). Tissues of these cases were obtained and label-free quantitative proteomic and bioinformatic analyses were performed. Additionally, prognostic impact of targeted proteins was validated using The Cancer Genome Atlas databases (n = 492) and tissue microarrays composed of early-stage LUADs (n = 228). A total of 192 differentially expressed proteins were identified between tumor tissues of LR and HR and three clusters were identified via hierarchical clustering excluding eight proteins. Cluster 1 (65 proteins) showed a sequential decrease in expression from normal tissues to tumor tissues of LR and then to HR and was predominantly enriched in pathways such as tyrosine metabolism and ECM-receptor interaction, and increased matched mRNA expression of 18 proteins from this cluster predicted favorable prognosis. Cluster 2 (70 proteins) demonstrated a sequential increase in expression from normal tissues to tumor tissues of LR and then to HR and was mainly enriched in pathways such as extracellular organization, DNA replication and cell cycle, and high matched mRNA expression of 25 proteins indicated poor prognosis. Cluster 3 (49 proteins) showed high expression only in LR, with high matched mRNA expression of 20 proteins in this cluster indicating favorable prognosis. Furthermore, high expression of ERO1A and FEN1 at protein level predicted poor prognosis in early-stage LUAD, supporting the mRNA results. In conclusion, we discovered key differentially expressed proteins and pathways between low-risk and high-risk subtypes of early-stage LUAD. Some of these proteins could serve as potential biomarkers in prognostic evaluation., Graphical Abstract, Highlights • Proteomic analysis was performed for low-risk and high-risk subtypes of lung adenocarcinoma. • Key proteins and pathways relevant to low-risk and high-risk subtypes were identified. • Candidate proteins were evaluated for prognostic impacts in independent cohorts., In Brief The 1D-LC-MS–based label-free proteomic analysis was performed to analyze the protein expression profiles of early-stage lung adenocarcinoma (LUAD) mainly composed of the low-risk or high-risk subtype. The prognostic impact of certain proteins was validated using other independent cohorts. We discovered key differentially expressed proteins and pathways between low-risk and high-risk subtypes of early-stage LUAD, and some of these proteins could serve as potential biomarkers in prognostic evaluation.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States. Glycans, such as carbohydrate antigen 19-9, are biomarkers of PDAC and are emerging as important modulators of cancer phenotypes. Herein, we used a systems-based approach integrating glycomic analysis of the well-established KC mouse, which models early events in transformation, and analysis of samples from human pancreatic cancer patients to identify glycans with potential roles in cancer formation. We observed both common and distinct patterns of glycosylation in pancreatic cancer across species. Common alterations included increased levels of α-2,3-sialic acid and α-2,6-sialic acid, bisecting GlcNAc and poly-N-acetyllactosamine. However, core fucose, which was increased in human PDAC, was not seen in the mouse, indicating that not all human glycomic changes are observed in the KC mouse model. In silico analysis of bulk and single-cell sequencing data identified ST6 beta-galactoside alpha-2,6-sialyltransferase 1, which underlies α-2,6-sialic acid, as overexpressed in human PDAC, concordant with histological data showing higher levels of this enzyme at the earliest stages. To test whether ST6 beta-galactoside alpha-2,6-sialyltransferase 1 promotes pancreatic cancer, we created a novel mouse in which a pancreas-specific genetic deletion of this enzyme overlays the KC mouse model. The analysis of our new model showed delayed cancer formation and a significant reduction in fibrosis. Our results highlight the importance of a strategic systems approach to identifying glycans whose functions can be modeled in mouse, a crucial step in the development of therapeutics targeting glycosylation in pancreatic cancer., Graphical Abstract, Highlights • Systems approach shows the glycome of KC mouse only partially overlays human PDAC. • α2,6-Sialylation and ST6GAL1 are upregulated in mouse and human pancreatic cancer. • Pancreas-specific ST6GAL1 KO in KC mouse delays cancer formation. • Results show the importance of identifying glycans that can be modeled in mice., In Brief Pancreatic ductal adenocarcinoma is a leading cause of cancer death. Glycans are emerging as important modulators of cancer phenotypes. Herein, we used a strategic systems-based approach integrating glycomics of the KC mouse, modeling early events in transformation, with human data to identify ST6GAL1 (e.g., α-2,6-sialic acid) as a potential cancer promoter. A pancreas-specific ST6GAL1 KO in the KC mouse delayed cancer formation and reduced fibrosis. Our results highlight the importance of identifying glycans whose functions can be modeled in mice.

The early detection of pancreatic ductal adenocarcinoma (PDAC) is a complex clinical obstacle yet is key to improving the overall likelihood of patient survival. Current and prospective carbohydrate biomarkers carbohydrate antigen 19-9 (CA19-9) and sialylated tumor-related antigen (sTRA) are sufficient for surveilling disease progression yet are not approved for delineating PDAC from other abdominal cancers and noncancerous pancreatic pathologies. To further understand these glycan epitopes, an imaging mass spectrometry (IMS) approach was used to assess the N-glycome of the human pancreas and pancreatic cancer in a cohort of patients with PDAC represented by tissue microarrays and whole-tissue sections. Orthogonally, these same tissues were characterized by multiround immunofluorescence that defined expression of CA19-9 and sTRA as well as other lectins toward carbohydrate epitopes with the potential to improve PDAC diagnosis. These analyses revealed distinct differences not only in N-glycan spatial localization across both healthy and diseased tissues but importantly between different biomarker-categorized tissue samples. Unique sulfated biantennary N-glycans were detected specifically in normal pancreatic islets. N-glycans from CA19-9–expressing tissues tended to be biantennary, triantennary, and tetra-antennary structures with both core and terminal fucose residues and bisecting GlcNAc. These N-glycans were detected in less abundance in sTRA-expressing tumor tissues, which favored triantennary and tetra-antennary structures with polylactosamine extensions. Increased sialylation of N-glycans was detected in all tumor tissues. A candidate new biomarker derived from IMS was further explored by fluorescence staining with selected lectins on the same tissues. The lectins confirmed the expression of the epitopes in cancer cells and revealed different tumor-associated staining patterns between glycans with bisecting GlcNAc and those with terminal GlcNAc. Thus, the combination of lectin-immunohistochemistry and lectin-IMS techniques produces more complete information for tumor classification than the individual analyses alone. These findings potentiate the development of early assessment technologies to rapidly and specifically identify PDAC in the clinic that may directly impact patient outcomes., Graphical Abstract, Highlights • N-glycan structures localize to distinct regions in normal and PDAC tumor tissue. • N-glycan composition differs between biomarker-stratified PDAC subtypes. • Modeling with N-glycan IMS and biomarker data improves tumor/normal discrimination. • Multiplexed IMS and lectin staining potentiates new PDAC classification strategies., In Brief N-glycosylation is an attractive target for PDAC biomarker discovery because of its well-understood roles in oncogenesis, cancer maintenance, and metastasis. Using MALDI-IMS, we observed distinct histopathology localized differences in N-glycosylation distributions between healthy and cancerous tissues. We combined the tumor-to-normal ratio of N-glycan changes determined from IMS with biomarker IHC data into modeling which improved PDAC identification over models utilizing either data set individually. This multiplexed approach potentiates the development of cross-disciplinary biomarker panels for pancreatic cancer detection.

Highlights • NF-κB2 and RELB levels were upregulated in CRC and surrounding stroma compared to non-neoplastic tissue and stroma. • RELB protein expression in the tumour had prognostic value for overall survival. • Tumour RELB gene expression in the tumour was associated withTTP and NF-κB2 gene expression was prognostic for survival and TTP. • NFKB2 rs12769316 and rs7897947 were associated with lymph node infiltration and rs12769316 correlated with relapse status. • Rs7897947 G allele was associated with reduced risk for developing CRC., The Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) family of transcription factors plays an important role in immune responses and cancer development and progression. We have focused on NF-κB2 and RELB of the alternative pathway of NF-κB, which remains largely underexplored in colorectal cancer (CRC). We found that NF-κB2 and RELB protein levels were upregulated in tumour and surrounding stromal tissue compared to distant non-neoplastic tissue (NN) and associated stroma (p

Background & Aims: \ud Cholangiocarcinomas (CCA) are resistant to chemotherapy, so new therapeutic agents are needed. We performed a screen to identify small-molecule compounds that are active against CCAs. Levels of microRNA 21 (MIR21 or miRNA21) are increased in CCAs. We investigated whether miRNA21 mediates resistance of CCA cells and organoids to HSP90 inhibitors.\ud \ud Methods: \ud We performed a high-throughput screen of 484 small-molecule compounds to identify those that reduced viability of 6 human CCA cell lines. We tested the effects of HSP90 inhibitors on cells with disruption of the MIR21 gene, cells incubated with MIR21 inhibitors, and stable cell lines with inducible expression of MIR21. We obtained CCA biopsies from patients, cultured them as organoids (patient-derived organoids). We assessed their architecture, mutation and gene expression patterns, response to compounds in culture, and when grown as subcutaneous xenograft tumors in mice.\ud \ud Results: \ud Cells with IDH1 and PBRM1 mutations had the highest level of sensitivity to histone deacetylase inhibitors. HSP90 inhibitors were effective in all cell lines, irrespective of mutations. Sensitivity of cells to HSP90 inhibitors correlated inversely with baseline level of MIR21. Disruption of MIR21 increased cell sensitivity to HSP90 inhibitors. CCA cells that expressed transgenic MIR21 were more resistant to HSP90 inhibitors than cells transfected with control vectors; inactivation of MIR21 in these cells restored sensitivity to these agents. MIR21 was shown to target the DnaJ heat shock protein family (Hsp40) member B5 (DNAJB5). Transgenic expression of DNAJB5 in CCA cells that overexpressed MIR21 re-sensitized them to HSP90 inhibitors. Sensitivity of patient-derived organoids to HSP90 inhibitors, in culture and when grown as xenograft tumors in mice, depended on expression of miRNA21.\ud \ud Conclusions: \ud miRNA21 appears to mediate resistance of CCA cells to HSP90 inhibitors by reducing levels of DNAJB5. HSP90 inhibitors might be developed for the treatment of CCA and miRNA21 might be a marker of sensitivity to these agents.

We investigated the hypothesis that the varying treatment efficacy of adjuvant 5-fluorouracil (5FU) in stage III colon cancer is linked to the TP53 mutational status. ABCSG-90 was a prospective randomized trial in which effect of adjuvant 5FU was studied in stage III colon cancer patients. Tumor material of 70% of these patients (389/572) was available for analysis of the biomarker TP53 using a TP53-gene-specific Sanger sequencing protocol. Median follow-up was 88 months. TP53 mutation frequency was 33%. A significant interaction between TP53 status, outcomes and nodal category was found (P = 0.0095). In the N1 category, TP53 wildtype patients had significantly better overall survival than TP53 mutated (81.0% vs. 62.0% overall survival at 5 years; HR = 2.131; 95% CI: 1.344–3.378; P = 0.0010). In the N2 category, the TP53 status did not affect survival (P = 0.4992). In TP53 wildtype patients, the prognostic significance of N category was significantly enhanced (P = 0.0002). In TP53 mutated patients, survival curves of N1 and N2 patients overlapped and nodal category was no longer prognostic. The biomarker TP53 independently predicted effect of adjuvant 5FU in N1 colon cancer patients. TP53 was not predictive in N2 patients, in whom 5FU is known to have no effect., Highlights • The TP53 status was found to be an independent predictive marker for the effect of adjuvant 5FU in stage III colon cancer. • In the N1 category, patients with wildtype TP53 experienced a significant survival benefit from adjuvant 5FU. • In TP53 mutant patients survival curves of N1 and N2 patients overlapped and nodal category was no longer prognostic. Postoperative chemotherapy is recommended for all patients with lymph node positive colon cancer. In colon cancer, mutations in the TP53 gene are present in more than 30% of tumors. We found that the most commonly used postoperative chemotherapy resulted in a marked survival benefit for patients with normal TP53 status while it was associated with significant survival disadvantage in TP53 mutant patients. Overall the survival disadvantage of the mutated patients almost balanced the survival benefit of the TP53 normal patients. This might be an explanation why chemotherapy resulted in less progress in survival of colon cancer than we would have expected.

The overall 5-year survival for melanoma is 91%. However, if distant metastasis occurs (stage IV), cure rates are, Highlights • A seven-miRNA panel (MELmiR-7) detected the presence of melanoma with high sensitivity (93%) and specificity (≥ 82%). • In serially collected stage IV specimens, members of the ‘MELmiR-7’ panel confirmed tumour progression in 100% of cases. • The ‘MELmiR-7’ panel is superior to currently used serological markers for melanoma progression, recurrence, and survival.