Moving Forward on Tumor Pathology Research Reporting: A Guide for Pathologists From the World Health Organization Classification of Tumors Living Evidence Gap Map by Tumour Type Group.

Evidence-based medicine (EBM) can be an unfamiliar territory for those working in tumor pathology research, and there is a great deal of uncertainty about how to undertake an EBM approach to planning and reporting histopathology-based studies. In this article, reviewed and endorsed by the Word Health Organization International Agency for Research on Cancer's International Collaboration for Cancer Classification and Research, we aim to help pathologists and researchers understand the basics of planning an evidence-based tumor pathology research study, as well as our recommendations on how to report the findings from these. We introduce some basic EBM concepts, a framework for research questions, and thoughts on study design and emphasize the concept of reporting standards. There are many study-specific reporting guidelines available, and we provide an overview of these. However, existing reporting guidelines perhaps do not always fit tumor pathology research papers, and hence, here, we collate the key reporting data set together into one generic checklist that we think will simplify the task for pathologists. The article aims to complement our recent hierarchy of evidence for tumor pathology and glossary of evidence (study) types in tumor pathology. Together, these articles should help any researcher get to grips with the basics of EBM for planning and publishing research in tumor pathology, as well as encourage an improved standard of the reports available to us all in the literature.

Targeted Sequencing of Human Satellite 2 Repeat Sequences in Plasma cfDNA Reveals Potential Breast Cancer Biomarkers.

Liquid biopsies are revolutionizing the detection and management of malignant diseases. While repetitive DNA sequences, such as LINE-1 and ALU are established in cell-free DNA (cfDNA) research, their clinical applications remain limited. In this study, we explore human satellite 2 (HSATII), a prevalent repeat DNA sequence in plasma that exhibits increased levels in cancer patients, thereby positioning it as a potential pan-cancer biomarker. We employed targeted sequencing and copy number variation (CNV) analysis using two primer pairs to assess the differential abundance of HSATII sequences in the plasma of breast cancer patients compared to healthy individuals. PCR amplicons of HSATII from 10 patients and 10 control subjects were sequenced, generating 151 bp paired-end reads. By constructing a pooled reference dataset, HSATII copy ratios were estimated in the patients. Our analysis revealed several significant CNVs in HSATII, with certain sequences displaying notable gains and losses across all breast cancer patients, suggesting their potential as biomarkers. However, we observed pronounced fragmentation of cfDNA in cancer, leading to the loss of longer PCR amplicons (>180 bp). While not all observed losses can be attributed to fragmentation artifacts, this phenomenon does introduce complexity in interpreting CNV data. Notably, this research marks the first instance of targeted HSATII sequencing in a liquid biopsy context. Our findings lay the groundwork for developing sequencing-based assays to detect differentially represented HSATII sequences, potentially advancing the field of minimally-invasive cancer screening.

A New Hierarchy of Research Evidence for Tumor Pathology: A Delphi Study to Define Levels of Evidence in Tumor Pathology.

The hierarchy of evidence is a fundamental concept in evidence-based medicine, but existing models can be challenging to apply in laboratory-based health care disciplines, such as pathology, where the types of evidence and contexts are significantly different from interventional medicine. This project aimed to define a comprehensive and complementary framework of new levels of evidence for evaluating research in tumor pathology-introducing a novel Hierarchy of Research Evidence for Tumor Pathology collaboratively designed by pathologists with help from epidemiologists, public health professionals, oncologists, and scientists, specifically tailored for use by pathologists-and to aid in the production of the World Health Organization Classification of Tumors (WCT) evidence gap maps. To achieve this, we adopted a modified Delphi approach, encompassing iterative online surveys, expert oversight, and external peer review, to establish the criteria for evidence in tumor pathology, determine the optimal structure for the new hierarchy, and ascertain the levels of confidence for each type of evidence. Over a span of 4 months and 3 survey rounds, we collected 1104 survey responses, culminating in a 3-day hybrid meeting in 2023, where a new hierarchy was unanimously agreed upon. The hierarchy is organized into 5 research theme groupings closely aligned with the subheadings of the WCT, and it consists of 5 levels of evidence-level P1 representing evidence types that merit the greatest level of confidence and level P5 reflecting the greatest risk of bias. For the first time, an international collaboration of pathology experts, supported by the International Agency for Research on Cancer, has successfully united to establish a standardized approach for evaluating evidence in tumor pathology. We intend to implement this novel Hierarchy of Research Evidence for Tumor Pathology to map the available evidence, thereby enriching and informing the WCT effectively.

Loss of Key EMT-Regulating miRNAs Highlight the Role of ZEB1 in EGFR Tyrosine Kinase Inhibitor-Resistant NSCLC.

Despite recent advances in the treatment of non-small cell lung cancer (NSCLC), acquired drug resistance to targeted therapy remains a major obstacle. Epithelial-mesenchymal transition (EMT) has been identified as a key resistance mechanism in NSCLC. Here, we investigated the mechanistic role of key EMT-regulating small non-coding microRNAs (miRNAs) in sublines of the NSCLC cell line HCC4006 adapted to afatinib, erlotinib, gefitinib, or osimertinib. The most differentially expressed miRNAs derived from extracellular vesicles were associated with EMT, and their predicted target ZEB1 was significantly overexpressed in all resistant cell lines. Transfection of a miR-205-5p mimic partially reversed EMT by inhibiting ZEB1, restoring CDH1 expression, and inhibiting migration in erlotinib-resistant cells. Gene expression of EMT-markers, transcription factors, and miRNAs were correlated during stepwise osimertinib adaptation of HCC4006 cells. Temporally relieving cells of osimertinib reversed transition trends, suggesting that the implementation of treatment pauses could provide prolonged benefits for patients. Our results provide new insights into the contribution of miRNAs to drug-resistant NSCLC harboring EGFR-activating mutations and highlight their role as potential biomarkers and therapeutic targets.