Checkpoint inhibition in combination with an immunoboost of external beam radiotherapy in solid tumors (CHEERS): study protocol for a phase 2, open-label, randomized controlled trial.

BACKGROUND: While the introduction of checkpoint inhibitors (CPIs) as standard of care treatment for various tumor types has led to considerable improvements in clinical outcome, the majority of patients still fail to respond. Preclinical data suggest that stereotactic body radiotherapy (SBRT) could work synergistically with CPIs by acting as an in situ cancer vaccine, thus potentially increasing response rates and prolonging disease control. Though SBRT administered concurrently with CPIs has been shown to be safe, evidence of its efficacy from large randomized trials is still lacking. The aim of this multicenter randomized phase II trial is to assess whether SBRT administered concurrently with CPIs could prolong progression-free survival as compared to standard of care in patients with advanced solid tumors. METHODS/DESIGN: Ninety-eight patients with locally advanced or metastatic disease will be randomized in a 1:1 fashion to receive CPI treatment combined with SBRT (Arm A) or CPI monotherapy (Arm B). Randomization will be stratified according to tumor histology (melanoma, renal, urothelial, head and neck squamous cell or non-small cell lung carcinoma) and disease burden (≤ or > 3 cancer lesions). The recommended SBRT dose is 24Gy in 3 fractions, which will be administered to a maximum of 3 lesions and is to be completed prior to the second or third CPI cycle (depending on CPI treatment schedule). The study's primary endpoint is progression-free survival as per iRECIST. Secondary endpoints include overall survival, objective response, local control, quality of life and toxicity. Translational analyses will be performed using blood, fecal and tissue samples. DISCUSSION: The CHEERS trial will provide further insights into the clinical and immunological impact of SBRT when combined with CPIs in patients with advanced solid tumors. Furthermore, study results will inform the design of future immuno-radiotherapy trials. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT03511391 . Registered 17 April 2018.

Candidate RNA biomarkers in biofluids for early diagnosis of ovarian cancer: A systematic review.

Ovarian cancer is often diagnosed in an advanced stage and is associated with a high mortality rate. It is assumed that early detection of ovarian cancer could improve patient outcomes. Unfortunately, effective screening methods for early diagnosis of ovarian cancer are still lacking. Extracellular RNAs circulating in human biofluids can reliably be measured and are emerging as potential biomarkers in cancer. In this systematic review, we present 75 RNA biomarkers detectable in human biofluids that have been studied for early diagnosis of ovarian cancer. The majority of these markers are microRNAs identified using RT-qPCR or microarrays in blood-based fluids. A handful of studies used RNA-sequencing and explored alternative fluids, such as urine and ascites. Candidate RNA biomarkers that were more abundant in biofluids of ovarian cancer patients compared to controls in at least two independent studies include miR-21, the miR-200 family, miR-205, miR-10a and miR-346. Amongst the markers confirmed to be lower in at least two studies are miR-122, miR-193a, miR-223, miR-126 and miR-106b. While these biomarkers show promising diagnostic potential, further validation is required before implementation in routine clinical care. Challenges related to biomarker validation and reflections on future perspectives to accelerate progress in this field are discussed.

Custom long non-coding RNA capture enhances detection sensitivity in different human sample types.

Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing.

Contact dermatitis caused by a new rubber compound detected in canvas shoes.

BACKGROUND: In 2015 and 2016, female patients in Flanders consulted a dermatologist because they developed skin lesions after wearing a specific brand of canvas shoes. OBJECTIVES: To identify the culprit allergen in the shoes. METHODS: Eighteen young females aged 14-22 years presented with itching and erythematous to purple-coloured eczematous lesions on both feet. They were patch tested by 10 dermatologists with the European baseline series. Some patients underwent testing with additional series. Pieces of the shoe fabrics were tested in 11 of 18 patients. Chemical analysis of the shoe materials was performed. Finally, patients were tested with a thin-layer chromatogram of the shoe extracts and dilutions of the suspected rubber compound. RESULTS: All 18 patients showed positive reactions to thiuram mix. Ten of 11 patients reacted to a piece of shoe fabric. Chemical analysis showed the presence of dimethylthiocarbamylbenzothiazole sulfide (DMTBS). No thiurams were detected. Four patients tested with the chromatogram developed positive reactions to DMTBS. Positive reactions to low concentrations were observed in the 4 patients tested with a DMTBS dilution series; one patient reacted to 0.00001% in acetone. CONCLUSIONS: DMTBS, the culprit allergen, is a component formed during rubber vulcanization that probably cross-reacts with the thiuram mix.

Subpar reporting of pre-analytical variables in RNA-focused blood plasma studies.

Extracellular RNA (cell-free RNA; exRNA) from blood-derived liquid biopsies is an appealing, minimally invasive source of disease biomarkers. As pre-analytical variables strongly influence exRNA measurements, their reporting is essential for meaningful interpretation and replication of results. The aim of this review was to chart to what extent pre-analytical variables are documented, to pinpoint shortcomings and to improve future reporting. In total, 200 blood plasma exRNA studies published in 2018 or 2023 were reviewed for annotation of 22 variables associated with blood collection, plasma preparation, and RNA purification. Our results show that pre-analytical variables are poorly documented, with only three out of 22 variables described in over half of the publications. The percentage of variables reported ranged from 4.6% to 54.6% (mean 24.84%) in 2023 and from 4.6% to 57.1% (mean 28.60%) in 2018. Recommendations and guidelines (i.e., BRISQ, ASCO-CAP, BloodPAC, PPMPT, and CEN standards) have currently not resulted in improved reporting. In conclusion, our results highlight the lack of reporting pre-analytical variables in exRNA studies and advocate for a consistent use of available standards, endorsed by funders and journals.

CiLiQuant: Quantification of RNA Junction Reads Based on Their Circular or Linear Transcript Origin.

Distinguishing circular RNA reads from reads derived from the linear host transcript is a challenging task because of sequence overlap. We developed a computational approach, CiLiQuant, that determines the relative circular and linear abundance of transcripts and gene loci using back-splice and unambiguous forward-splice junction reads generated by existing mapping and circular RNA discovery tools.

IgE-mediated gastroallergic anisakiasis with eosinophilic oesophagitis: a case report.

BACKGROUND: Anisakiasis is an emerging zoonosis caused by the fish parasitic nematode Anisakis infecting the gastrointestinal tract. CASE PRESENTATION: We describe a case of a 58-year-old woman diagnosed with gastro-allergic anisakiasis, in which the patient developed an acute food-induced IgE-mediated hypersensitivity reaction as well as concurrent gastro-intestinal manifestations after consumption of raw fish. The patient presented with epigastric pain, anaphylaxis and acute dysphagia caused by eosinophilic oesophagitis. DISCUSSION: Anisakis allergy should be considered as causative agent in patients presenting with acute urticarial rash, anaphylaxis and/or abdominal manifestations, especially when symptoms occur after consumption of seafood. Moreover, eosinophilic oesophagitis may be a rare but important complication of Anisakis infection. Endoscopic evaluation with esophageal biopsies should therefore be considered if suggestive symptoms are present. Patients with confirmed gastroallergic anisakiasis are advised to properly freeze or cook fish prior to consumption, although caution is advised, since heat-stable allergen proteins have been described. An adrenaline auto-injector should be prescribed.

RNA biomarkers from proximal liquid biopsy for diagnosis of ovarian cancer.

BACKGROUND: Most ovarian cancer patients are diagnosed at an advanced stage and have a high mortality rate. Current screening strategies fail to improve prognosis because markers that are sensitive for early stage disease are lacking. This medical need justifies the search for novel approaches using utero-tubal lavage as a proximal liquid biopsy. METHODS: In this study, we explore the extracellular transcriptome of utero-tubal lavage fluid obtained from 26 ovarian cancer patients and 48 controls using messenger RNA (mRNA) capture and small RNA sequencing. RESULTS: We observed an enrichment of ovarian and fallopian tube specific messenger RNAs in utero-tubal lavage fluid compared to other human biofluids. Over 300 mRNAs and 41 miRNAs were upregulated in ovarian cancer samples compared with controls. Upregulated genes were enriched for genes involved in cell cycle activation and proliferation, hinting at a tumor-derived signal. CONCLUSION: This is a proof-of-principle that mRNA capture sequencing of utero-tubal lavage fluid is technically feasible, and that the extracellular transcriptome of utero-tubal lavage should be further explored in larger cohorts to assess the diagnostic value of the biomarkers identified in this study. IMPACT: Proximal liquid biopsy from the gynecologic tract is a promising source for mRNA and miRNA biomarkers for diagnosis of early-stage ovarian cancer.

Messenger RNA capture sequencing of extracellular RNA from human biofluids using a comprehensive set of spike-in controls.

Comprehensive transcriptome analysis of extracellular RNA (exRNA) purified from human biofluids is challenging because of the low RNA concentration and compromised RNA integrity. Here, we describe an optimized workflow to (1) isolate exRNA from different types of biofluids and (2) to prepare messenger RNA (mRNA)-enriched sequencing libraries using complementary hybridization probes. Importantly, the workflow includes 2 sets of synthetic spike-in RNA molecules as processing controls for RNA purification and sequencing library preparation and as an alternative data normalization strategy. For complete details on the use and execution of this protocol, please refer to Hulstaert et al. (2020).

Charting Extracellular Transcriptomes in The Human Biofluid RNA Atlas.

Extracellular RNAs present in biofluids have emerged as potential biomarkers for disease. Where most studies focus on blood-derived fluids, other biofluids may be more informative. We present an atlas of messenger, circular, and small RNA transcriptomes of a comprehensive collection of 20 human biofluids. By means of synthetic spike-in controls, we compare RNA content across biofluids, revealing a 10,000-fold difference in concentration. The circular RNA fraction is increased in most biofluids compared to tissues. Each biofluid transcriptome is enriched for RNA molecules derived from specific tissues and cell types. Our atlas enables an informed selection of the most relevant biofluid to monitor particular diseases. To verify the biomarker potential in these biofluids, four validation cohorts representing a broad spectrum of diseases were profiled, revealing numerous differential RNAs between case and control subjects. Spike-normalized data are publicly available in the R2 web portal for further exploration.

Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles.

RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.

Psoriasis Vulgaris Exacerbation during Treatment with a PD-1 Checkpoint Inhibitor: Case Report and Literature Review.

OBJECTIVE: The incidence of immune-related adverse events is growing as the use of checkpoint inhibitors is exponentially increasing. Cutaneous adverse events are among the most frequent immune-related adverse events. The purpose of this case report and literature review is to highlight psoriasis as a potential adverse event with need for early recognition. CASE REPORT AND LITERATURE REVIEW: We describe the case of a 65-year-old woman with psoriasis exacerbation while treated with nivolumab (anti-PD-1) for a stage IV melanoma. She had a history of scalp psoriasis but she presented with psoriatic lesions on both lower and upper limbs. Our patient was treated with topical steroids. So far, 34 other cases with an exacerbation of psoriasis during treatment with anti-PDL-1 or PD-1 therapy have been reported in the literature. A broad range of therapies are described, without any available guidelines for this particular condition. CONCLUSION: Psoriasis exacerbation is an established side effect of PD-1/PDL-1 checkpoint inhibitors with 35 reported cases. Early recognition and management are challenging as there are no clear guidelines available. A close collaboration between oncologist and dermatologist is mandatory to manage this immune-related adverse event.

Peritumoral endothelial indoleamine 2, 3-dioxygenase expression is an early independent marker of disease relapse in colorectal cancer and is influenced by DNA mismatch repair profile.

Targeting immune checkpoint molecules has become a major new strategy in the treatment of several cancers. Indoleamine 2,3-dioxygenase (IDO)-inhibitors are a potential next-generation immunotherapy, currently investigated in multiple phase I-III trials. IDO is an intracellular immunosuppressive enzyme and its expression/activity has been associated with worse prognosis in several cancers. The aim of this study was to investigate the expression pattern of IDO in colorectal cancer (CRC). In a cohort of 94 CRC patients, primary tumors (PTs) with corresponding tumor-draining lymph nodes (TDLNs, n = 93) and extranodal/distant metastases (n = 27) were retrospectively analyzed by immunohistochemical staining for IDO, CD8 and Foxp3. 45 MSS and 37 MSI-H tumors were selected to compare IDO expression, as these tumors are considered to have different immunogenicity. A highly consistent expression pattern of IDO was observed in the PT, TDLNs and metastases, indicating that immune resistance may be determined very early in the disease course. IDO was expressed both by tumoral cells and host endothelial cells and these expressions were highly correlated (p < 0.001). IDO expression was observed more frequently in the MSI-H subset compared with the MSS subset (43% vs 22% for tumoral expression (p = 0.042) and 38% vs 16% for endothelial expression (p = 0.021)). Endothelial IDO expression was demonstrated to be a negative prognostic marker for recurrence free survival independent of disease stage and DNA mismatch repair (MMR) status (HR 20.67, 95% CI: 3.05-139.94; p = 0.002). These findings indicate that endothelial IDO expression in primary CRC, in addition to the MMR profile, may be helpful in disease stratification.

Long non-coding RNAs in cutaneous melanoma: clinical perspectives.

Metastatic melanoma of the skin has a high mortality despite the recent introduction of targeted therapy and immunotherapy. Long non-coding RNAs (lncRNAs) are defined as transcripts of more than 200 nucleotides in length that lack protein-coding potential. There is growing evidence that lncRNAs play an important role in gene regulation, including oncogenesis. We present 13 lncRNA genes involved in the pathogenesis of cutaneous melanoma through a variety of pathways and molecular interactions. Some of these lncRNAs are possible biomarkers or therapeutic targets for malignant melanoma.

Rapidly growing gastric metastasis of Merkel cell carcinoma, an unusual cause of melena.

Merkel cell carcinoma (MCC) is an uncommon, highly aggressive neuroendocrine skin carcinoma that has a tendency for local recurrence and metastatic disease. We report a rare case of recurrent melena in a 77-year-old Caucasian male. Three years earlier, the patient had undergone a radical resection of a para-umbilical MCC. A repeat esophagogastroduodenoscopy proved necessary to identify rapidly proliferating gastric metastasis of MCC as the cause of bleeding.

Intramuscular hepatitis B immunoglobulins for reinfection control after liver transplantation: a cost-saving alternative.

AIM: We explore the effectiveness and cost-effectiveness of intramuscular versus intravenous hepatitis B immunoglobulins (HBIG-IV vs HBIG-IM) to prevent reinfection with the hepatitis B virus after orthotopic liver transplantation. PATIENTS & METHODS: Overall, 14 patients had orthotopic liver transplantation in 2003-2013 at Ghent University Hospital for HBV-related liver disease. On average 32 months after transplantation patients switched from high-dose HBIG-IV to low-dose HBIG-IM, always in combination with a nucleos(t)ide analog. RESULTS: Seven patients were switched so far. No significant differences between HBIG-IV and HBIG-IM were found in HBsAg and hepatitis B virus-DNA. CONCLUSION: Switching patients from HBIG-IV to HBIG-IM can be done safely if well monitored. Net yearly savings for the healthcare payer were €5000 for each patient switched to HBIG-IM.