Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%; P < 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome ( P < 0.05) and improved the sensitivity of pathogen detection ( P < 0.01), with a 20- to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples.

Background: Methods: Results: Conclusion:

There are currently no approved targeted therapies for advanced KRAS mutant (KRASMT) colorectal cancer (CRC). Using a unique systems biology approach, we identified JAK1/2-dependent activation of STAT3 as the key mediator of resistance to MEK inhibitors in KRASMT CRC in vitro and in vivo. Further analyses identified acute increases in c-MET activity following treatment with MEK inhibitors in KRASMT CRC models, which was demonstrated to promote JAK1/2-STAT3-mediated resistance. Furthermore, activation of c-MET following MEK inhibition was found to be due to inhibition of the ERK-dependent metalloprotease ADAM17, which normally inhibits c-MET signaling by promoting shedding of its endogenous antagonist, soluble "decoy" MET. Most importantly, pharmacological blockade of this resistance pathway with either c-MET or JAK1/2 inhibitors synergistically increased MEK-inhibitor-induced apoptosis and growth inhibition in vitro and in vivo in KRASMT models, providing clear rationales for the clinical assessment of these combinations in KRASMT CRC patients.

Purpose: FK506-binding protein like (FKBPL) and its peptide derivative, AD-01, have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here, we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC).

Purpose: Transcriptomic profiling of colorectal cancer (CRC) has led to the identification of four consensus molecular subtypes (CMS1 to 4) that have prognostic value in stage II and III disease. More recently, the Colorectal Cancer Intrinsic Subtypes (CRIS) classification system has helped to define the biology specific to the epithelial component of colorectal tumors; however, the clinical value of these classification systems in the prediction of response to standard-of-care adjuvant chemotherapy remains unknown. Patients and Methods: Using samples from four European sites, we assembled a novel cohort of patients with stage II and III CRC (n = 156 samples) and performed transcriptomic profiling and targeted sequencing and generated a tissue microarray to enable integrated multiomics analyses. We also accessed data from two published cohorts of patients with stage II and III CRC: GSE39582 and GSE14333 (n = 479 and n = 185 samples, respectively). Results: The epithelial-rich CMS2 subtype of CRC benefitted significantly from treatment with adjuvant chemotherapy in both stage II and III disease ( P = .02 and P < .001, respectively), whereas the CMS3 subtype significantly benefitted in stage III only ( P = .001). After CRIS substratification of CMS2, we observed that only the CRIS-C subtype significantly benefitted from treatment with adjuvant chemotherapy in stage II and III disease ( P = .0081 and P < .001, respectively), whereas the CRIS-D subtype significantly benefitted in stage III only ( P = .0034). We also observed that CRIS-C patients with low levels of CD8⁺ tumor-infiltrating lymphocytes were most at risk for relapse in both stage II and III disease (log-rank P = .0031; hazard ratio, 12.18 [95% CI, 1.51 to 98.58]). Conclusion: Patient stratification using a combination of transcriptional subtyping and CD8 immunohistochemistry analyses is capable of identifying patients with poor prognostic stage II and III disease who benefit from adjuvant standard-of-care chemotherapy. These findings are particularly relevant for patients with stage II disease, where the overall benefit of adjuvant chemotherapy is marginal.