FuncPEP v2.0: An Updated Database of Functional Short Peptides Translated from Non-Coding RNAs.

Over the past decade, there have been reports of short novel functional peptides (less than 100 aa in length) translated from so-called non-coding RNAs (ncRNAs) that have been characterized using mass spectrometry (MS) and large-scale proteomics studies. Therefore, understanding the bivalent functions of some ncRNAs as transcripts that encode both functional RNAs and short peptides, which we named ncPEPs, will deepen our understanding of biology and disease. In 2020, we published the first database of functional peptides translated from non-coding RNAs-FuncPEP. Herein, we have performed an update including the newly published ncPEPs from the last 3 years along with the categorization of host ncRNAs. FuncPEP v2.0 contains 152 functional ncPEPs, out of which 40 are novel entries. A PubMed search from August 2020 to July 2023 incorporating specific keywords was performed and screened for publications reporting validated functional peptides derived from ncRNAs. We did not observe a significant increase in newly discovered functional ncPEPs, but a steady increase. The novel identified ncPEPs included in the database were characterized by a wide array of molecular and physiological parameters (i.e., types of host ncRNA, species distribution, chromosomal density, distribution of ncRNA length, identification methods, molecular weight, and functional distribution across humans and other species). We consider that, despite the fact that MS can now easily identify ncPEPs, there still are important limitations in proving their functionality.

Predictive role of ctDNA in esophageal squamous cell carcinoma receiving definitive chemoradiotherapy combined with toripalimab.

The combination of toripalimab (an anti-PD-1 antibody) with definitive chemoradiotherapy (CRT) demonstrated encouraging efficacy against locally advanced esophageal squamous cell carcinoma (ESCC) in the EC-CRT-001 phase II trial (NCT04005170). The primary endpoint of this trial was the clinical complete response rate (cCR), and the secondary endpoints included overall survival (OS), progression-free survival (PFS), duration of response, and quality of life. The exploratory analyses of EC-CRT-001 include exploring the role of circulating tumor DNA (ctDNA) and blood-based tumor mutational burden (bTMB) in predicting the response and survival. In total, 118 blood and 35 tissue samples from 42 enrolled patients were included in the analyses. We found that ctDNA-negative patients achieved a higher cCR compared to those with detectable ctDNA during CRT (83%, 19/23 vs. 39%, 7/18; p = 0.008) or post-CRT (78%, 21/27 vs. 30%, 3/10; p = 0.017). Patients with detectable ctDNA during CRT had shorter PFS (p = 0.014). Similarly, patients with post-CRT detectable ctDNA had a significantly shorter PFS (p = 0.012) and worse OS (p = 0.004). Moreover, patients with high bTMB levels during CRT had prolonged OS (p = 0.027). In conclusion, ctDNA and bTMB have the potential to predict treatment efficacy and survival in ESCC treated with CRT and immunotherapy.

AHRR and SFRP2 in primary versus recurrent high-grade serous ovarian carcinoma and their prognostic implication.

BACKGROUND: The aim of this study was to analyse transcriptomic differences between primary and recurrent high-grade serous ovarian carcinoma (HGSOC) to identify prognostic biomarkers. METHODS: We analysed 19 paired primary and recurrent HGSOC samples using targeted RNA sequencing. We selected the best candidates using in silico survival and pathway analysis and validated the biomarkers using immunohistochemistry on a cohort of 44 paired samples, an additional cohort of 504 primary HGSOCs and explored their function. RESULTS: We identified 233 differential expressed genes. Twenty-three showed a significant prognostic value for PFS and OS in silico. Seven markers (AHRR, COL5A2, FABP4, HMGCS2, ITGA5, SFRP2 and WNT9B) were chosen for validation at the protein level. AHRR expression was higher in primary tumours (p < 0.0001) and correlated with better patient survival (p < 0.05). Stromal SFRP2 expression was higher in recurrent samples (p = 0.009) and protein expression in primary tumours was associated with worse patient survival (p = 0.022). In multivariate analysis, tumour AHRR and SFRP2 remained independent prognostic markers. In vitro studies supported the anti-tumorigenic role of AHRR and the oncogenic function of SFRP2. CONCLUSIONS: Our results underline the relevance of AHRR and SFRP2 proteins in aryl-hydrocarbon receptor and Wnt-signalling, respectively, and might lead to establishing them as biomarkers in HGSOC.

Stepwise Analysis of Resection Margin Impact on Survival and Distant Metastasis in Pancreatic Head Ductal Adenocarcinoma.

The prognostic role of tumor cells in pancreatic ductal adenocarcinoma (PDAC) of the pancreatic head with direct microscopic infiltration (DMI) or in close proximity (≤1 mm) to the resection margin (RM) remains unclear. This single-center, retrospective study included specimens from 75 patients who underwent oncological resection of pancreatic head PDAC between February 2013 and July 2020. Two pathologists independently re-measured the distance between tumors and the multiple RMs. The impact of RM involvement for DMI, tumor cells within ≤1 mm, in general, and for individual RMs on overall survival (OS) and development of distant pulmonary (PM) and hepatic (HM) metastasis was analyzed. DMI of RMs was significantly associated with a shorter OS (median 5 vs 19 months, P = .02). The presence of tumor cells within ≤1 mm of RMs yielded a negative impact on OS with a trend toward significance (median 9 vs 21 months, P = .09). DMI and tumor cells within ≤1 mm of the pancreatic transection margin (PRM), individually, had a significant negative impact on OS (median 4 vs 19 months and 6 vs 19 months, P < .05), but not for any other individual RM. RM involvement of ≤1 mm of only the vascular circumferential resection margin (VCRM) resulted in a shorter time to HM development (P = 0.05). DMI of the posterior circumferential resection margin (PCRM) and VCRM, individually, showed shorter time to PM (P < .05). Potential clinical considerations include extended intraoperative evaluation of the PRM (1 mm) and intensified preoperative prediction of R1 resection as a basis for neoadjuvant therapy.

Spatial heterogeneity of tumor cells and the tissue microenvironment in oral squamous cell carcinoma.

PURPOSE: This study describes the morphologic and phenotypic spatial heterogeneity of tumor cells and the tissue microenvironment (TME), focusing on immune infiltration in OSCCs. STUDY DESIGN: Patients with OSCCs and planned surgical tumor resection were eligible for the study. Two biopsies each from the tumor center and the tumor rim were obtained. Immunohistochemical characterization of tumor and immune cells was performed using a panel of immunohistochemical markers. RESULTS: Thirty-six biopsies were obtained from the 9 patients. All patients showed an individual marker expression profile with ITH. Within the same biopsy, the CPS and TPS scores showed relevant variations in PD-L1 expression. Comparisons between the tumor center and rim revealed significant differences in the up/downregulation of p53. Marker expression of patients with recurrences clustered similarly, with the higher expression of FoxP3, IDO, CD4, CD68, and CD163 at the tumor rim. CONCLUSION: OSCCs were found to exhibit relevant ITH involving both tumor cells and TME, suggesting that biomarker analysis of multiple tumor regions may be helpful for clinical decision making and tumor characterization. The analysis of multiple spots within a biopsy is recommended for a reliable determination of PD-L1 expression and other biomarkers, impacting current clinical assessments.

TTF-1 status in early-stage lung adenocarcinoma is an independent predictor of relapse and survival superior to tumor grading.

OBJECTIVES: Thyroid transcription factor 1 (TTF-1) is a well-established independent prognostic factor in lung adenocarcinoma (LUAD), irrespective of stage. This study aims to determine if TTF-1's prognostic impact is solely based on histomorphological differentiation (tumor grading) or if it independently relates to a biologically more aggressive phenotype. We analyzed a large bi-centric LUAD cohort to accurately assess TTF-1's prognostic value in relation to tumor grade. PATIENTS AND METHODS: We studied 447 patients with resected LUAD from major German lung cancer centers (Berlin and Cologne), correlating TTF-1 status and grading with clinical, pathologic, and molecular data, alongside patient outcomes. TTF-1's impact was evaluated through univariate and multivariate Cox regression. Causal graph analysis was used to identify and account for potential confounders, improving the statistical estimation of TTF-1's predictive power for clinical outcomes. RESULTS: Univariate analysis revealed TTF-1 positivity associated with significantly longer disease-free survival (DFS) (median log HR -0.83; p = 0.018). Higher tumor grade showed a non-significant association with shorter DFS (median log HR 0.30; p = 0,62 for G1 to G2 and 0.68; p = 0,34 for G2 to G3). In multivariate analysis, TTF-1 positivity resulted in a significantly longer DFS (median log HR -0.65; p = 0.05) independent of all other parameters, including grading. Adjusting for potential confounders as indicated by the causal graph confirmed the superiority of TTF-1 over tumor grading in prognostics power. CONCLUSIONS: TTF-1 status predicts relapse and survival in LUAD independently of tumor grading. The prognostic power of tumor grading is limited to TTF-1-positive patients, and the effect size of TTF-1 surpasses that of tumor grading. We recommend including TTF1 status as a prognostic factor in the diagnostic guidelines of LUAD.

Propensity Score-Matched Analysis of Minimal Invasive Surgery versus Open Surgery for Colorectal Cancer in Older Patients.

Introduction: Currently, surgeons deal with an older patient cohort, confronting new challenges brought by the raised life expectancy. This population is unrepresented in surgical trials; therefore, the optimal therapy is still a matter of debate. The efficacy of open versus minimal invasive management of colorectal cancer (CRC) in an elderly cohort is not clearly established. The current study assesses the minimal invasive approach in elderly patients undergoing colorectal surgery. Material and Methods: The General Surgery Department database was inquired between 2012 and 2015 using the following filters: age â?¥ 65 and rectal or colon adenocarcinoma. After applying the exclusion criteria, 975 cases were obtained: 842 underwent open surgery (OS) and 133 underwent minimal invasive surgery (MIS). A propensity score matching was performed to reduce patient selection bias. Results: After the propensity score matching, the MIS group had a shorter postoperative hospital stay than the OS group (p = 0.025). From the preoperative variables, the presence of chronic lung disease was significantly higher in the OS group (p = 0.039). The presence of chronic lung disease positively associates with the Clavien-Dindo classification (p 0.001) and with the number of days from surgery to discharge (p = 0.028). Conclusion: The chronological age alone should not be a limit to MIS granting that it showed no inferiority to the OS in terms of postoperative morbidity, correlating with lower postoperative stay in the elderly. Further prospective studies are needed to assess the outcome of MIS in elderly population.

Increased expression of IDO1 is associated with improved survival and increased number of TILs in patients with high-grade serous ovarian cancer.

BACKGROUND: The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays a crucial role in regulating the immune system's response to tumors, but its exact role in cancer, especially in high-grade serous ovarian cancer (HGSOC), remains controversial. We aimed to investigate the prognostic impact of IDO1 expression and its correlation with tumor-infiltrating lymphocytes (TILs) in HGSOC. METHODS: Immunohistochemical (IHC) staining and bioimage analysis using the QuPath software were employed to assess IDO1 protein expression in a well-characterized cohort of 507 patients with primary HGSOC. Statistical evaluation was performed using SPSS, and in silico validation considering IDO1 mRNA expression in bulk and single-cell gene expression datasets was conducted. Additionally, IDO1 expression in interferon-gamma (IFNG) stimulated HGSOC cell lines was analyzed. RESULTS: Our findings revealed that IDO1 protein and mRNA expression serve as positive prognostic markers for overall survival (OS) and progression-free survival (PFS) in HGSOC. High IDO1 expression was associated with a significant improvement in OS by 21 months (p < 0.001) and PFS by 6 months (p = 0.016). Notably, elevated IDO1 expression correlated with an increased number of CD3+ (p < 0.001), CD4+ (p < 0.001), and CD8+ TILs (p < 0.001). Furthermore, high IDO1 mRNA expression and protein level were found to be associated with enhanced responsiveness to pro-inflammatory cytokines, particularly IFNG. CONCLUSIONS: Our study provides evidence that IDO1 expression serves as a positive prognostic marker in HGSOC and is associated with an increased number of CD3+, CD4+ and CD8+ TILs. Understanding the intricate relationship between IDO1, TILs, and the tumor microenvironment may hold the key to improving outcomes in HGSOC.

LRP1B-a prognostic marker in tubo-ovarian high-grade serous carcinoma.

Low-density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a member of the LDL receptor family and has often been discussed as a tumor suppressor gene, as its down-regulation is correlated with a poor prognosis in multiple carcinoma entities. Due to the high metastasis rate into the fatty peritoneal cavity and current research findings showing a dysregulation of lipid metabolism in tubo-ovarian high-grade serous carcinoma (HGSC), we questioned the prognostic impact of the LRP1B protein expression. We examined a well-characterized large cohort of 571 patients with primary HGSC and analyzed the LRP1B protein expression via immunohistochemical staining (both in tumor and stroma cells separately), performed precise bioimage analysis with QuPath, and calculated the prognostic impact using SPSS. Our results demonstrate that LRP1B functions as a significant prognostic marker for overall survival (OS) and progression-free survival (PFS) in HGSC on the protein level. High cytoplasmic expression of LRP1B in tumor, stroma, and combined tumor and stroma cells has a significantly positive association with a mean prolongation of the OS by 42 months (P = .005), 29 months (P = .005), and 25 months (P = .001), respectively. Additionally, the mean PFS was 18 months longer in tumor (P = .002), 19 months in stroma (P = .004), and 19 months in both cell types combined (P = .01). Our results remained significant in multivariate analysis. We envision LRP1B as a potential prognostic tool that could help us understand the functional role of lipid metabolism in advanced HGSC, especially regarding liposomal medications.

High EVI1 and PARP1 expression as favourable prognostic markers in high-grade serous ovarian carcinoma.

BACKGROUND: Mechanisms of development and progression of high-grade serous ovarian cancer (HGSOC) are poorly understood. EVI1 and PARP1, part of TGF-ß pathway, are upregulated in cancers with DNA repair deficiencies with DNA repair deficiencies and may influce disease progression and survival. Therefore we questioned the prognostic significance of protein expression of EVI1 alone and in combination with PARP1 and analyzed them in a cohort of patients with HGSOC. METHODS: For 562 HGSOC patients, we evaluated EVI1 and PARP1 expression by immunohistochemical staining on tissue microarrays with QuPath digital semi-automatic positive cell detection. RESULTS: High EVI1 expressing (> 30% positive tumor cells) HGSOC were associated with improved progression-free survival (PFS) (HR = 0.66, 95% CI: 0.504-0.852, p = 0.002) and overall survival (OS) (HR = 0.45, 95% CI: 0.352-0.563, p < 0.001), including multivariate analysis. Most interestingly, mutual high expression of both proteins identifies a group with particularly good prognosis. Our findings were proven technically and clinically using bioinformatical data sets for single-cell sequencing, copy number variation and gene as well as protein expression. CONCLUSIONS: EVI1 and PARP1 are robust prognostic biomarkers for favorable prognosis in HGSOC and imply further research with respect to their reciprocity.

Anti-miR-93-5p therapy prolongs sepsis survival by restoring the peripheral immune response.

Sepsis remains a leading cause of death for humans and currently has no pathogenesis-specific therapy. Hampered progress is partly due to a lack of insight into deep mechanistic processes. In the past decade, deciphering the functions of small noncoding miRNAs in sepsis pathogenesis became a dynamic research topic. To screen for new miRNA targets for sepsis therapeutics, we used samples for miRNA array analysis of PBMCs from patients with sepsis and control individuals, blood samples from 2 cohorts of patients with sepsis, and multiple animal models: mouse cecum ligation puncture-induced (CLP-induced) sepsis, mouse viral miRNA challenge, and baboon Gram+ and Gram- sepsis models. miR-93-5p met the criteria for a therapeutic target, as it was overexpressed in baboons that died early after induction of sepsis, was downregulated in patients who survived after sepsis, and correlated with negative clinical prognosticators for sepsis. Therapeutically, inhibition of miR-93-5p prolonged the overall survival of mice with CLP-induced sepsis, with a stronger effect in older mice. Mechanistically, anti-miR-93-5p therapy reduced inflammatory monocytes and increased circulating effector memory T cells, especially the CD4+ subset. AGO2 IP in miR-93-KO T cells identified important regulatory receptors, such as CD28, as direct miR-93-5p target genes. In conclusion, miR-93-5p is a potential therapeutic target in sepsis through the regulation of both innate and adaptive immunity, with possibly a greater benefit for elderly patients than for young patients.

DNA methylation-based classifier differentiates intrahepatic pancreato-biliary tumours.

BACKGROUND: Differentiating intrahepatic cholangiocarcinomas (iCCA) from hepatic metastases of pancreatic ductal adenocarcinoma (PAAD) is challenging. Both tumours have similar morphological and immunohistochemical pattern and share multiple driver mutations. We hypothesised that DNA methylation-based machine-learning algorithms may help perform this task. METHODS: We assembled genome-wide DNA methylation data for iCCA (n = 259), PAAD (n = 431), and normal bile duct (n = 70) from publicly available sources. We split this cohort into a reference (n = 399) and a validation set (n = 361). Using the reference cohort, we trained three machine learning models to differentiate between these entities. Furthermore, we validated the classifiers on the technical validation set and used an internal cohort (n = 72) to test our classifier. FINDINGS: On the validation cohort, the neural network, support vector machine, and the random forest classifiers reached accuracies of 97.68%, 95.62%, and 96.5%, respectively. Filtering by anomaly detection and thresholds improved the accuracy to 99.07% (37 samples excluded by filtering), 96.22% (17 samples excluded), and 100% (44 samples excluded) for the neural network, support vector machine and random forest, respectively. Because of best balance between accuracy and number of predictable cases we tested the neural network with applied filters on the in-house cohort, obtaining an accuracy of 95.45%. INTERPRETATION: We developed a classifier that can differentiate between iCCAs, intrahepatic metastases of a PAAD, and normal bile duct tissue with high accuracy. This tool can be used for improving the diagnosis of pancreato-biliary cancers of the liver. FUNDING: This work was supported by Berlin Institute of Health (JCS Program), DKTK Berlin (Young Investigator Grant 2022), German Research Foundation (493697503 and 314905040 - SFB/TRR 209 Liver Cancer B01), and German Cancer Aid (70113922).

17ß-estradiol promotes extracellular vesicle release and selective miRNA loading in ERα-positive breast cancer.

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor-positive (ER+) BC, we hypothesized that 17ß-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17ß-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7's miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17ß-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment.

Disclosing quantitative RT-PCR raw data during manuscript submission: a call for action.

Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.

The Immune Checkpoint Landscape in Tumor Cells of Pancreatic Ductal Adenocarcinoma.

Immune checkpoint therapy (ICT) has shown promising potential in the treatment of multiple solid tumors. However, the role of ICT in pancreatic ductal adenocarcinoma (PDAC) remains limited. Patterns of immune checkpoints (ICs) in PDAC represent the basis for establishing a potent ICT. The aim of this study is to create a profile of IC expression and its prognostic relevance in cancer cells of PDAC. Therefore, tumor cells from peripheral and central tissue microarray (TMA) spots from histologically confirmed PDAC of 68 patients after tumor resection were investigated in terms of expressions of TIM3, IDO, B7H4, LAG3, VISTA, and PD-L1 using immunohistochemistry. The presence of the respective ICs was compared to overall survival (OS). The presence of VISTA and PD-L1 significantly correlates with shorter OS (median OS: 22 months vs. 7 months and 22 months vs. 11 months, respectively, p < 0.05). For the presence of TIM3, IDO, B7H4, and LAG3, no difference in OS was observed (p > 0.05). The analysis of OS of combined subgroups for VISTA and PD-L1 (VISTA and PD-L1 neg., VISTA pos. and PD-L1 neg., VISTA neg. and PD-L1 pos., and VISTA and PD-L1 pos.) yielded overall statistical significance difference (p = 0.02). These results suggest that the presence of VISTA and PD-L1 is of prognostic relevance and potentially qualifies them as targets for ICT.

Clinical and Dosimetric Predictors for Postoperative Cardiopulmonary Complications in Esophageal Squamous Cell Carcinoma Patients Receiving Neoadjuvant Chemoradiotherapy and Surgery.

BACKGROUND: Neoadjuvant chemoradiotherapy followed by esophagectomy is the standard treatment for patients with locally advanced esophageal squamous cell carcinoma (ESCC). This study explored correlations of clinical factors and dose-volume histogram (DVH) parameters with postoperative cardiopulmonary complications and predicted their risk by establishing a nomogram model. METHODS: Clinical and DVH parameters of ESCC patients who underwent trimodality treatment from 2002 to 2020 were collected. Postoperative cardiopulmonary complications were recorded. Logistic regression analysis was applied, and a nomogram model was constructed. Area under the receiver operating characteristic (AUC) curve, calibration curve, and decision curve analyses were performed to evaluate the performance of the nomogram. RESULTS: Of the 307 ESCC patients enrolled in this study, 65 (21.2%) experienced pulmonary complications and 57 (18.6%) experienced cardiac complications. The following six risk factors were identified as independent risk factors for pulmonary complications by multivariate logistic regression analyses in the integrated model: male sex (odds ratio [OR], 3.26; 95% confidence interval [CI], 1.27-9.70; P = 0.021), post-radiation therapy (RT) forced expiratory volume in 1 s (FEV1) (OR, 0.51; 95% CI 0.28-0.90; P = 0.023), mean lung dose (MLD) (OR, 1.13; 95% CI 1.01-1.28; P = 0.041), and pre-RT monocyte (OR, 8.36; 95% CI 1.23-11.7; P = 0.03). The AUC of this integrated model was 0.705 (95% CI 0.64-0.77). The paclitaxel and cisplatin (TP) concurrent chemotherapy regimen was the independent predictor of cardiac complication (OR, 2.50; 95% CI 1.22-5.55; P = 0.016). CONCLUSIONS: For ESCC patients who underwent trimodality treatment, male sex, post-RT FEV1, MLD, and pre-RT monocyte were confirmed as significant predictors of postoperative pulmonary complications. A nomogram model including six risk factors was further established. The independent predictor of cardiac complication was TP concurrent chemotherapy.

The seen and the unseen: Molecular classification and image based-analysis of gastrointestinal cancers.

Gastrointestinal cancers account for 22.5% of cancer related deaths worldwide and represent circa 20% of all cancers. In the last decades, we have witnessed a shift from histology-based to molecular-based classifications using genomic, epigenomic, and transcriptomic data. The molecular based classification revealed new prognostic markers and may aid the therapy selection. Because of the high-costs to perform a molecular classification, in recent years immunohistochemistry-based surrogate classification were developed which permit the stratification of patients, and in parallel multiple groups developed hematoxylin and eosin whole slide image analysis for sub-classifying these entities. Hence, we are witnessing a return to an image-based classification with the purpose to infer hidden information from routine histology images that would permit to detect the patients that respond to specific therapies and would be able to predict their outcome. In this review paper, we will discuss the current histological, molecular, and immunohistochemical classifications of the most common gastrointestinal cancers, gastric adenocarcinoma, and colorectal adenocarcinoma, and will present key aspects for developing a new artificial intelligence aided image-based classification of these malignancies.