Substantial improvements have been made in the management of metastatic colorectal cancer (mCRC) in the last two decades, but disease monitoring remains underdeveloped. Circulating tumor DNA (ctDNA) ...is a promising prognostic and predictive biomarker; however, ctDNA as a marker for mCRC patients is not well established, and there is still no consensus about how to utilize it most cost-effectively. In this study, we aim to investigate plasma ctDNA levels as a biomarker for therapeutic response of mCRC patients. We performed next-generation sequencing (NGS) by using a 12-gene panel to identify genetic variants in 136 tumor tissue and ctDNA samples from 32 mCRC patients. Genetic variants were detected in approximately 70% of samples, and there was a high concordance (85%) between tumor tissue and plasma ctDNA. We observed ctDNA changes in 18 follow-up patients, including the emergence of new variants. Changes in ctDNA levels significantly correlated with tumor shrinkage (
= 0.041), and patients with a ctDNA decrease >80% after treatment had a longer progression-free survival compared with patients with a ctDNA decrease of <80% (HR, 0.22;
= 0.015). The objective response rate among patients with a ctDNA decrease of >80% was better than those with a ctDNA decrease <80% (OR, 0.026;
= 0.007). In conclusion, this study demonstrates that monitoring of genetic ctDNA variants can serve as a valuable biomarker for therapeutic efficacy in mCRC patients, and that using a moderate-sized 12-gene NGS panel may be suitable for such clinical monitoring.
.
The bacterial genus
Xylella
contains plant pathogens that are major threats to agriculture in America and Europe. Although extensive research was conducted to characterize different subspecies of
...Xylella fastidiosa
(
Xf
), comparative analysis at above-species levels was lacking due to the unavailability of appropriate data sets. Recently, a bacterium that causes pear leaf scorch (PLS) in Taiwan was described as the second
Xylella
species (i.e.,
Xylella taiwanensis
;
Xt
). In this work, we report the complete genome sequence of
Xt
type strain PLS229
T
. The genome-scale phylogeny provided strong support that
Xf
subspecies
pauca
(
Xfp
) is the basal lineage of this species and
Xylella
was derived from the paraphyletic genus
Xanthomonas
. Quantification of genomic divergence indicated that different
Xf
subspecies share ∼87–95% of their chromosomal segments, while the two
Xylella
species share only ∼66–70%. Analysis of overall gene content suggested that
Xt
is most similar to
Xf
subspecies
sandyi
(
Xfs
). Based on the existing knowledge of
Xf
virulence genes, the homolog distribution among 28
Xylella
representatives was examined. Among the 11 functional categories, those involved in secretion and metabolism are the most conserved ones with no copy number variation. In contrast, several genes related to adhesins, hydrolytic enzymes, and toxin-antitoxin systems are highly variable in their copy numbers. Those virulence genes with high levels of conservation or variation may be promising candidates for future studies. In summary, the new genome sequence and analysis reported in this work contributed to the study of several important pathogens in the family Xanthomonadaceae.
The molecular underpinnings of seromucinous borderline tumor (SMBT) – an uncommon ovarian epithelial neoplasm characterized by association with endometriosis, frequent bilateral ovarian involvement, ...and occasional progression to invasive carcinoma – remain poorly understood. Here, we sought to comprehensively characterize the mutational landscape of SMBT and elucidate the clonal relationship between bilateral ovarian SMBTs. We also compared the mutational profiles between SMBTs and concurrent invasive carcinomas. Formalin-fixed, paraffin-embedded tissue specimens were retrieved from 28 patients diagnosed with SMBT. Massively parallel sequencing of 409 cancer-related genes was conducted to identify somatic mutations in 33 SMBT samples and four concurrent invasive carcinoma specimens. TERT promoter mutations were assessed by Sanger sequencing, whereas immunohistochemistry was used as a surrogate tool for detecting deletions or epigenetic silencing of relevant tumor suppressor genes. Twenty-six (92.9%) of the 28 patients were diagnosed with stage I SMBTs. Seven (25%) cases showed bilateral ovarian involvement and 13 (46%) had concomitant endometriosis. Concurrent ovarian carcinomas were identified in three patients, whereas one case had a synchronous endometrial carcinoma. Somatic mutations in the KRAS, PIK3CA, and ARID1A genes were identified in 100, 60.7, and 14.3% of SMBT samples, respectively. In contrast, TERT promoter mutations and DNA mismatch repair deficiencies were absent. Sequencing of paired specimens from patients with bilateral SMBT revealed the presence of at least two shared somatic mutations, suggestive of a clonal relationship. Similarly, we identified shared somatic mutations between SMBT samples and concurrent ovarian carcinoma specimens. Taken together, these findings demonstrated a distinct mutational landscape of SMBT in which (1) KRAS is invariably mutated, (2) PIK3CA is frequently mutated, and (3) TERT promoter mutations and DNA mismatch repair deficiencies are absent. Our findings represent the first extensive characterization of this rare ovarian neoplasm, with potential implications for disease classification and molecular diagnostics.
This letter presents a detailed study of transport in graphene field-effect transistors (GFETs) with various channel lengths, from 5 μm down to 90 nm, using transferred graphene grown by chemical ...vapor deposition. An electron-hole asymmetry observed in short-channel devices suggests a strong impact from graphene/metal contacts. In addition, for the first time, we observe a shift of the gate voltage at the Dirac point in graphene devices as a consequence of gate length scaling. The unusual shift of the Dirac point voltage has been identified as one of the signatures of short-channel effects in GFETs.
The clinical utility of comprehensive genomic profiling (CGP) for guiding treatment has gradually become the standard-of-care procedure for colorectal carcinoma (CRC). Here, we comprehensively assess ...emerging targeted therapy biomarkers using CGP in primary CRC.
A total of 575 primary CRCs were sequenced by ACTOnco® assay for genomic alterations, tumour mutational burden (TMB), and microsatellite instability (MSI).
Eighteen percent of patients were detected as MSI-High (MSI-H), and the remaining cases were classified as microsatellite stable (MSS). Driver mutation prevalence in MSS CRCs were APC (74%), TP53 (67%), KRAS (47%), PIK3CA (21%) and BRAF (13%). The median TMBs for MSI-H and MSS patients were 37.8 mutations per mega base (mut/Mb) and 3.9 mut/Mb, respectively. Forty-seven percent of MSI-H CRC harboured at least one loss-of-function mutations in genes that may hamper immune checkpoint blockade. Among MSS RAS/RAF wild-type CRCs, 59% had at least one actionable mutation that may compromise the efficacy of anti-EGFR therapy. For late-stage CRC, 51% of patients are eligible for standard care actionability and the remaining 49% could be enrolled in clinical trials with investigational drugs.
This study highlights the essential role of CGP for identifying rational targeted therapy options in CRC.
Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is ...the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution analysis in inhibitory tissues.
The type VI secretion system (T6SS) is deployed by many proteobacteria to secrete effector proteins into bacterial competitors for competition or eukaryotic cells for pathogenesis. Agrobacteria, a ...group of soilborne phytopathogens causing crown gall disease on various plant species, deploy the T6SS to attack closely and distantly related bacterial species
and
. Current evidence suggests that the T6SS is not essential for pathogenesis under direct inoculation, but it remains unknown whether the T6SS influences natural disease incidence or the microbial community within crown galls (i.e., the gallobiome). To address these two key questions, we established a soil inoculation method on wounded tomato seedlings that mimics natural infections and developed a bacterial 16S rRNA gene amplicon enrichment sequencing platform. By comparing the
wild-type strain C58 with two T6SS mutants, we demonstrate that the T6SS influences both disease occurrence and gallobiome composition. Based on multiple inoculation trials across seasons, all three strains induced tumors, but the mutants had significantly lower disease incidences. The season of inoculation played a more important role than the T6SS in shaping the gallobiome. The influence of the T6SS was evident in summer, during which two
species and the family
were enriched in the gallobiome induced by the mutants. Further
competition and colonization assays demonstrated the T6SS-mediated antagonism to a
sp. R1 strain isolated from tomato rhizosphere in this study. In conclusion, this work demonstrates that the
T6SS promotes tumorigenesis in infection processes and provides competitive advantages in gall-associated microbiota.
The T6SS is widespread among proteobacteria and used for interbacterial competition by agrobacteria, which are soil inhabitants and opportunistic bacterial pathogens causing crown gall disease in a wide range of plants. Current evidence indicates that the T6SS is not required for gall formation when agrobacteria are inoculated directly on plant wounding sites. However, in natural settings, agrobacteria may need to compete with other bacteria in bulk soil to gain access to plant wounds and influence the microbial community inside crown galls. The role of the T6SS in these critical aspects of disease ecology have remained largely unknown. In this study, we successfully developed a
oil
noculation method coupled with
locker-mediated enrichment of
terial 16S rRNA gene amplicon
uencing, named SI-BBacSeq, to address these two important questions. We provided evidence that the T6SS promotes disease occurrence and influences crown gall microbiota composition by interbacterial competition.
During neoplastic development, a multitude of changes in genome-encoded information are progressively selected to confer growth and survival advantages to tumor cells. microRNAs-mRNAs regulatory ...networks, given their role as a critical layer of robust gene expression control, are frequently altered in neoplasm. However, whether and how these gene perturbations impact metabolic homeostasis remains largely unresolved.
Through targeted miRNA expression screening, we uncovered an oral squamous cell carcinoma (OSCC)-associated miRNAome, among which miR-31-5p was identified based on extent of up-regulation, functional impact on OSCC cell migration and invasion, and direct regulation of the rate-limiting enzyme in peroxisomal β-oxidation, ACOX1.
We further found that both miR-31-5p and ACOX1 underpin, in an antagonistic manner, the overall cellular lipidome profiles as well as the migratory and invasive abilities of OSCC cells. Interestingly, the extracellular levels of prostaglandin E2 (PGE2), a key substrate of ACOX1, were controlled by the miR-31-5p-ACOX1 axis, and were shown to positively influence the extent of cell motility in correlation with metastatic status. The promigratory effect of this metabolite was mediated by an elevation in EP1-ERK-MMP9 signaling. Of note, functional significance of this regulatory pathway was further corroborated by its clinicopathologically-correlated expression in OSCC patient specimens.
Collectively, our findings outlined a model whereby misregulated miR-31-5p-ACOX1 axis in tumor alters lipid metabolomes, consequently eliciting an intracellular signaling change to enhance cell motility. Our clinical analysis also unveiled PGE2 as a viable salivary biomarker for prognosticating oral cancer progression, further underscoring the importance of lipid metabolism in tumorigenesis.
BackgroundImmunological checkpoint blockade is effective in treating various malignancies. Identifying predictive biomarkers to assist patient selection for immunotherapy has become a priority in ...both clinical and research settings.MethodsMutations in patients who responded to immunotherapy were identified through next-generation sequencing. Relationships among protein kinase, DNA-activated, catalytic polypeptide (PRKDC) mutations, mutation load and microsatellite instability (MSI) were analyzed using datasets from The Cancer Genome Atlas. These relationships were validated by conducting an in vitro study and by using tissue samples from 34 patients with gastric cancer. The CT26 animal model was used to evaluate the role of PRKDC as a predictive biomarker and the efficacy of the DNA-PK inhibitor.ResultsFrom the published literature, we found that among patients whose tumors harbored PRKDC mutations, 75%, 53.8%, and 50% of those with lung cancer, melanoma, and renal cell carcinoma, respectively, responded to immunotherapy. Most of these mutations were truncating and located in functional domains or in a destabilizing PRKDC protein structure. Additional analysis showed that a PRKDC mutation was significantly associated with a high mutation load in cervical cancer, colon adenocarcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, gastric adenocarcinoma and endometrial cancer. Patients with gastric cancer or colon cancer harboring PRKDC mutations were also highly associated with MSI-high status. Finally, we found that knockout PRKDC or DNA-PK inhibitor (PRKDC encodes the catalytic subunit of DNA-dependent protein kinase) enhanced the efficacy of the anti-programmed cell death protein one pathway monoclonal antibody in the CT26 animal model.ConclusionsPRKDC is not only a predictive biomarker but also a drug target for immune checkpoint inhibitors.
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