Methylation profiling of circulating cell-free DNA (cfDNA) is of great interest as a liquid biopsy assay for the detection and monitoring of cancer and other pathologies. Here we describe ‘circulating cell-free DNA reduced representation bisulfite sequencing’ (cf-RRBS), enabling the use of highly effective RRBS on fragmented plasma cfDNA. This method enriches the CpG-rich RRBS target regions by enzymatic degradation of all off-target DNA rather than by targeted capture, in contrast to previous methods. Critical steps are fully enzymatic in a single-tube, making it rapid, cost-effective, robust, and easily implemented on a liquid-handling station for high-throughput sample preparation. We benchmark cf-RRBS results to those obtained by previous more complex methods and exemplify its use for accurate non-invasive subtyping of lung cancer, a frequent onco-pathology task. cf-RRBS enables any molecular pathology lab to tap into the cfDNA methylome, only making use of off-the-shelf reagents and open-source data analysis tools. One Sentence Summary: Novel methodology enables facile and cost-effective methylation profiling of fragmented plasma DNA, allowing for routine liquid biopsy clinical diagnostics, exemplified here for differential diagnosis of lung cancer subtypes.

The era of immune checkpoint inhibitors has altered the therapeutic landscape in squamous cell cancer of the head and neck (SCCHN). Our knowledge about the tumor microenvironment has fueled the research in SCCHN, leading to several well-known and less-known prognostic and predictive biomarkers. The clinical staging, p16/HPV status, and PD-L1 expression are currently the main tools for assessing the patients’ diagnosis and prognosis. However, several novel biomarkers have been thoroughly investigated, some reaching actual significant clinical contributions. The untangling of the immune infiltrate with the subtyping of tissue-associated tumor infiltrating lymphocytes, tumor-associated macrophages, and circulating blood-based biomarkers are an interesting avenue to be further explored and prospectively assessed. Although PD-L1 expression remains the most important response predictor for immune checkpoint inhibitors, several flaws impede proper assessment such as technical issues, different scoring protocol, and intra-, inter-, and temporal heterogeneity. In addition, the construction of an immune-related gene panel has been proposed as a prognostic and predictive stratification but lacks consensus. Recently, the role of microbioma have also been explored regarding its systemic and antitumor immunity. This review gives a comprehensive overview of the aforementioned topics in SCCHN. To this end, the integration of these clinically advantageous biomarkers via construction of an immunogram or nomogram could be an invaluable tool for SCCHN in future prospects.

Background/Objectives: Gastrointestinal diseases are a major cause of morbidity in common variable immunodeficiency disorder (CVID), clinically often mimicking other conditions including celiac disease and inflammatory bowel disease (IBD). Hence, diagnosis of CVID remains challenging. This study aims to raise awareness and highlight histopathological clues for CVID in intestinal biopsies, emphasizing diagnostic pitfalls for the pathologist/gastroenterologist. Methods: We reviewed 63 (18 duodenal, 23 ileal, 22 colonic) biopsies and case histories from seven CVID patients, obtained over a 31-year period, with attention to active inflammation, intraepithelial lymphocytes, plasma cells, lymphoid hyperplasia, crypt/villous architecture, subepithelial collagen, apoptosis, granulomas, and infections. Clinical information of 41 pathology requests was reviewed. Results: Gastrointestinal symptoms were variable. Histological features included IBD-like (3/7), celiac disease-like (2/7), graft-versus-host disease (GVHD)-like (2/7), lymphocytic sprue/colitis-like (3/7), collagenous colitis-like (2/7), and acute colitis-like (4/7) patterns, often overlapping (2/7) and/or changing over time (3/7). Lymphoid hyperplasia was seen in 3/7 patients; 1/7 had giardiasis; and 5/7 had few plasma cells, usually only in part of the gut (3/5). Clinical information of 12/41 (29%) pathology requests mentioned known/suspected CVID, despite being known in 33/41 (80%). Conclusions: Clinical/histological features of CVID in the gut are diverse, often mimicking IBD, microscopic colitis, celiac disease and/or GVHD, hence the importance of adequate clinical information. Some histological features are atypical of these established entities and may indicate CVID, as may overlapping/changing histological patterns and/or few plasma cells in part of the gut. Awareness of the heterogenous clinical presentation and histopathological indicators of CVID may improve diagnosis.

Non-V600E BRAF mutated colorectal cancer (CRC) is a rare disease entity with specific clinical features. These tumors are less likely to have microsatellite instability than CRC with a V600E BRAF mutation and often harbor a KRAS or NRAS mutation. Notably, median overall survival is longer than in wild-type BRAF CRC. Little is known about treatment possibilities in these patients. We present the case of a 59 year old patient with a rare mutation in BRAF codon 594, who progressed rapidly on all classical therapies but experienced a clear and long lasting response on treatment with Regorafenib. Little is known about therapies that can be effective in the rare non-V600E BRAF mutated CRCs. We present a patient who had a definite response to treatment with Regorafenib. There are no predictive markers that define a subset of CRC patients who benefit most from Regorafenib. The specific features of this non-V600E BRAF mutated CRC may be relevant in the exploration of predictive biomarkers for the efficacy of Regorafenib.