Search

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

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.

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.

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.

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.

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.

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.

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.

The overexpression of RRM2 [RR (ribonucleotide reductase) small subunit M2] dramatically enhances the ability of the cancer cell to proliferate and to invade. To investigate further the relevance of RRM2 and CRCs (colorectal cancers), we correlated the expression of RRM2 with the clinical outcome of CRCs. A retrospective outcome study was conducted on CRCs collected from the COH [(City of Hope) National Medical Center, 217 cases] and ZJU (Zhejiang University, 220 cases). IHC (immunohistochemistry) was employed to determine the protein expression level of RRM2, and quantitative real-time PCR was employed to validate. Multivariate logistic analysis indicated that the adjusted ORs (odds ratios) of RRM2-high for distant metastases were 2.06 [95% CI (confidence interval), 1.01–4.30] and 5.89 (95% CI, 1.51–39.13) in the COH and ZJU sets respectively. The Kaplan–Meier analysis displayed that high expression of RRM2 had a negative impact on the OS (overall survival) and PFS (progress-free survival) of CRC in both sets significantly. The multivariate Cox analysis further demonstrated that HRs (hazard ratios) of RRM2-high for OS were 1.88 (95% CI, 1.03–3.36) and 2.06 (95% CI, 1.10–4.00) in the COH and ZJU sets respectively. Stratification analysis demonstrated that the HR of RRM2 dramatically increased to 12.22 (95% CI, 1.62–258.31) in the MMR (mismatch repair) gene-deficient subgroup in the COH set. Meanwhile, a real-time study demonstrated that down-regulation of RRM2 by siRNA (small interfering RNA) could significantly and specifically reduce the cell growth and adhesion ability in HT-29 and HCT-8 cells. Therefore RRM2 is an independent prognostic factor and predicts poor survival of CRCs. It is also a potential predictor for identifying good responders to chemotherapy for CRCs.