Background: Never-smokers and never-drinkers patients (NSND) suffering from oral squamous cell carcinoma (OSCC) are epidemiologically different from smokers drinkers (SD). We therefore hypothesized that they harbored distinct targetable molecular alterations. Patients and methods: Data from The Cancer Genome Atlas (TCGA) (discovery set), Gene Expression Omnibus and Centre Léon Bérard (CLB) (3 validation sets) with available gene expression profiles of HPV-negative OSCC from NSND and SD were mined. Protein expression profiles and genomic alterations were also analyzed from TCGA, and a functional pathway enrichment analysis was performed. Formalin-fixed paraffin-embedded (FFPE) samples from 44 OSCC including 20 NSND and 24 SD treated at CLB were retrospectively collected to perform targeted-sequencing of 2,559 transcripts (HTG EdgeSeq system), and CD3, CD4, CD8, IDO1 and PD-L1 expression analyses by immunohistochemistry (IHC). Enrichment of a six-gene interferon-γ signature of clinical response to pembrozulimab (PD-1 inhibitor) was evaluated in each sample from all cohorts, using the single sample gene set enrichment analysis method. Results: A total of 854 genes and 29 proteins were found to be differentially expressed between NSND and SD in TCGA. Functional pathway analysis highlighted an overall enrichment for immune-related pathways in OSCC from NSND, especially involving T-cell activation. Interferon-γ response and PD1 signaling were strongly enriched in NSND. IDO1 and PD-L1 were overexpressed and the score of response to pembrolizumab was higher in NSND compared to SD, although the mutational load was lower in NSND. IHC analyses in the CLB cohort evidenced IDO1 and PD-L1 overexpression in tumor cells that was associated with a higher rate of tumor-infiltrating T-cells in NSND compared to SD. Conclusion: The main biological and actionable difference between OSCC from NSND and SD lies in the immune microenvironment, suggesting a higher clinical benefit of PD-L1 and IDO1 inhibition in OSCC from NSND.

The yield of pearl millet, a resilient cereal crop crucial for African food security, is severely impacted by the root parasitic weed Striga hermonthica, which requires host-released hormones, called strigolactones (SLs), for seed germination. Herein, we identify four SLs present in the Striga-susceptible line SOSAT-C88-P10 (P10) but absent in the resistant 29Aw (Aw). We generate chromosome-scale genome assemblies, including four gapless chromosomes for each line. The Striga-resistant Aw lacks a 0.7 Mb genome segment containing two putative CARLACTONOIC ACID METHYLTRANSFERASE1 (CLAMT1) genes, which may contribute to SL biosynthesis. Functional assays show that P10CLAMT1b produces the SL-biosynthesis intermediate methyl carlactonoate (MeCLA) and that MeCLA is the precursor of P10-specific SLs. Screening a diverse pearl millet panel confirms the pivotal role of the CLAMT1 section for SL diversity and Striga susceptibility. Our results reveal a reason for Striga susceptibility in pearl millet and pave the way for generating resistant lines through marker-assisted breeding or direct genetic modification.

Given the important role of early detection in improving cancer outcomes, epigenetic alterations, particularly DNA methylation, have emerged as powerful biomarkers for cancer screening and diagnosis. However, methylation profiling of cfDNA (circulating free DNA) presents particular technical challenges because cfDNA is typically present at very low concentrations and is highly fragmented. Bisulfite conversion, although essential for distinguishing methylated from unmethylated cytosines, can further compromise DNA integrity through chemical-induced loss of DNA during purification steps. Inadequate conversion or excessive DNA loss may consequently reduce library complexity, increase DNA duplication rates, and negatively affect mapping efficiency and downstream sequencing quality. Therefore, optimizing bisulfite conversion is particularly important for reliable methylation profiling of low-input cfDNA. The effectiveness of these diagnostic approaches relies on the accurate detection of DNA methylation patterns, which is fundamentally dependent on the efficiency of bisulfite conversion of genomic DNA. This pilot study aims to compare the performance of two widely used bisulfite conversion kits, namely the EpiTect Bisulfite (EpiTect) Kit (Qiagen, Hilden, Germany) and the EZ DNA Methylation (Zymo) Kit (Zymo Research, Irvine, CA, USA), in the context of plasma-derived cfDNA methylation profiling using five samples, including two plasma samples from metastatic colorectal cancer (mCRC) patients collected at the Department of Gastrointestinal Oncology at The University of Texas MD Anderson Cancer Center, in Houston, TX, USA, and two plasma samples from healthy donors (controls) purchased from Innovative Research (Novi, MI, USA), along with one control DNA sample (Corriel NA12878-N88). Our objective was to evaluate and compare their conversion efficiency, mapping efficiency (percentage of mapped and unmapped reads), duplication rate, and distribution of methylated cytosines in CpG, CHG, and CHH contexts. Our results indicate that samples processed using the Zymo kit consistently outperformed those treated with the EpiTect kit across multiple performance metrics. The Zymo-treated samples yielded a higher concentration of cfDNA following bisulfite conversion (125.34 nM vs. 45.60 nM in samples processed with the Epitect kit). Additionally, the Zymo kit achieved a higher average bisulfite conversion efficiency (99.3% vs. 95.7%), as well as a significantly higher average mapping rate (94.86% vs. 72.4%), with fewer unmapped reads and lower duplication rates. Head-to-head comparison of Bismark methylation calls across the same reference regions showed high concordance between the two protocols. The SEPT9 region was strongly hypermethylated with both protocols (100% with EpiTect and approximately 94% with Zymo), whereas the BMP3 region was predominantly unmethylated (approximately 4% with EpiTect and 7% with Zymo). These findings suggest that the Zymo kit demonstrated improved performance under the conditions evaluated for cfDNA methylation analysis compared to the EpiTect protocol. Further investigation using a larger number of samples is needed to validate our findings.

Lung cancer (LC) remains the leading cause of cancer-related mortality worldwide, representing a major public health challenge. Although low-dose computed tomography is currently the standard screening method for high-risk individuals to reduce mortality, its clinical performance is limited by false-positive findings and restricted accessibility. Emerging biomarker-based liquid biopsy offers a promising non-invasive approach for cancer detection. This narrative review provides a critical overview and evaluation of clinically endorsed liquid biopsy applications for guiding treatment in advanced-stage lung cancer, as well as investigational advances in liquid biopsy for early cancer detection. We also address challenges of current liquid biopsy technologies, the integration of artificial intelligence, implementation improvements, and future perspectives for their adoption into routine clinical practice. To complement this narrative review, a structured search of the ClinicalTrials.gov registry was performed to identify clinical trials evaluating liquid biopsy in lung cancer. Current evidence supports the clinical use of liquid biopsy for molecular profiling and treatment selection in advanced non-small cell lung cancer (NSCLC), whereas its application in early detection, screening, and minimal residual disease (MRD)-guided management remains investigational and requires further prospective validation. Overall, liquid biopsy represents a promising complementary approach to advance precision oncology and improve lung cancer management.