Early, On-Treatment Levels and Dynamic Changes of Genomic Instability in Circulating Tumor DNA Predict Response to Treatment and Outcome in Metastatic Breast Cancer Patients.

BACKGROUND: Circulating tumor DNA (ctDNA) offers high sensitivity and specificity in metastatic cancer. However, many ctDNA assays rely on specific mutations in recurrent genes or require the sequencing of tumor tissue, difficult to do in a metastatic disease. The purpose of this study was to define the predictive and prognostic values of the whole-genome sequencing (WGS) of ctDNA in metastatic breast cancer (MBC). METHODS: Plasma from 25 patients with MBC were taken at the baseline, prior to treatment (T0), one week (T1) and two weeks (T2) after treatment initiation and subjected to low-pass WGS. DNA copy number changes were used to calculate a Genomic Instability Number (GIN). A minimum predefined GIN value of 170 indicated detectable ctDNA. GIN values were correlated with the treatment response at three and six months by Response Evaluation Criteria in Solid Tumours assessed by imaging (RECIST) criteria and with overall survival (OS). RESULTS: GIN values were detectable (>170) in 64% of patients at the baseline and were significantly prognostic (41 vs. 18 months OS for nondetectable vs. detectable GIN). Detectable GIN values at T1 and T2 were significantly associated with poor OS. Declines in GIN at T1 and T2 of > 50% compared to the baseline were associated with three-month response and, in the case of T1, with OS. On the other hand, a rise in GIN at T2 was associated with a poor response at three months. CONCLUSIONS: Very early measurements using WGS of cell-free DNA (cfDNA) from the plasma of MBC patients provided a tumor biopsy-free approach to ctDNA measurement that was both predictive of the early tumor response at three months and prognostic.

Trophic transfer of microcystins through the lake pelagic food web: evidence for the role of zooplankton as a vector in fish contamination.

An in situ study was performed to investigate the role of zooplankton as a vector of microcystins (MCs) from Planktothrix rubescens filaments to fish during a metalimnic bloom of P. rubescens in Lake Hallwil (Switzerland). The concentrations of MCs in P. rubescens and various zooplanktonic taxa (filter-feeders and predators) were assessed in different water strata (epi-, meta- and hypolimnion) using replicated sampling over a 24-hour survey. The presence of P. rubescens in the gut content of various zooplanktonic taxa (Daphnia, Bosmina and Chaoborus) was verified by targeting the cyanobacterial nucleic acids (DNA). These results highlighted that cyanobacterial cells constitute a part of food resource for herbivorous zooplanktonic taxa during metalimnic bloom periods. Furthermore, presence of MCs in Chaoborus larvae highlighted the trophic transfer of MCs between herbivorous zooplankton and their invertebrate predators. Our results suggest that zooplanktonic herbivores by diel vertical migration (DVM) act as vectors of MCs by encapsulating grazed cyanobacteria. As a consequence, they largely contribute to the contamination of zooplanktonic predators, and in fine of zooplanktivorous whitefish. Indeed, we estimated the relative contribution of three preys of the whitefish (i.e. Daphnia, Bosmina and Chaoborus) to diet contamination. We showed that Chaoborus and Daphnia were the highest contributor as MC vectors in the whitefish diet (74.6 and 20.5% of MC-LR equivalent concentrations, respectively). The transfer of MCs across the different trophic compartments follows complex trophic pathways involving various trophic levels whose relative importance in fish contamination might vary at daily and seasonal scale.

Quantitative PCR enumeration of total/toxic Planktothrix rubescens and total cyanobacteria in preserved DNA isolated from lake sediments.

The variability of spatial distribution and the determinism of cyanobacterial blooms, as well as their impact at the lake scale, are still not understood, partly due to the lack of long-term climatic and environmental monitoring data. The paucity of these data can be alleviated by the use of proxy data from high-resolution sampling of sediments. Coupling paleolimnological and molecular tools and using biomarkers such as preserved DNA are promising approaches, although they have not been performed often enough so far. In our study, a quantitative PCR (qPCR) technique was applied to enumerate total cyanobacterial and total and toxic Planktothrix communities in preserved DNA derived from sediments of three lakes located in the French Alps (Lake Geneva, Lake Bourget, and Lake Annecy), containing a wide range of cyanobacterial species. Preserved DNA from lake sediments was analyzed to assess its quality, quantity, and integrity, with further application for qPCR. We applied the qPCR assay to enumerate the total cyanobacterial community, and multiplex qPCR assays were applied to quantify total and microcystin-producing Planktothrix populations in a single reaction tube. These methods were optimized, calibrated, and applied to sediment samples, and the specificity and reproducibility of qPCR enumeration were tested. Accurate estimation of potential inhibition within sediment samples was performed to assess the sensitivity of such enumeration by qPCR. Some precautions needed for interpreting qPCR results in the context of paleolimnological approaches are discussed. We concluded that the qPCR assay can be used successfully for the analysis of lake sediments when DNA is well preserved in order to assess the presence and dominance of cyanobacterial and Planktothrix communities.

Relationships between Bacteroides 16S rRNA genetic markers and presence of bacterial enteric pathogens and conventional fecal indicators.

Occurrence and prevalence of different bacterial enteric pathogens as well as their relationships with conventional (total and fecal coliforms) and alternative fecal indicators (host-specific Bacteroides 16S rRNA genetic markers) were investigated for various water samples taken from different sites with different degrees of fecal contamination. The results showed that a wide range of bacterial pathogens could be detected in both municipal wastewater treatment plant samples and in surface water samples. Logistic regression analysis revealed that total and human-specific Bacteroides 16S rRNA genetic markers showed significant predictive values for the presence of Escheriachia coli O-157, Salmonella, heat-labile enterotoxin (LT) of enterotoxigenic E. coli (ETEC), and heat-stable enterotoxin for human (STh) of ETEC. The probability of occurrence of these pathogenic bacteria became significantly high when the concentrations of human-specific and total Bacteroides 16S rRNA genetic markers exceeded 10(3) and 10(4) copies/100 mL. In contrast, Clostridium perfringens was detected at high frequency regardless of sampling sites and levels of Bacteroides 16S rRNA genetic markers. No genes related to Shigella spp., Staphylococcus aureus and Vibrio cholerae were detected in any samples analyzed in this study. Conventional indicator microorganisms had low levels of correlation with the presence of pathogens as compared with the alternative fecal indicators. These results suggested that real-time PCR-based measurement of alternative Bacteroides 16S rRNA genetic markers was a rapid and sensitive tool to identify host-specific fecal pollution and probably associated bacterial pathogens. However, since one fecal indicator might not represent the relative abundance of all pathogenic bacteria, viruses and protozoa, combined application of alternative indicators with conventional ones could provide more comprehensive pictures of fecal contamination, its source and association with pathogenic microorganisms.

Quantification of host-specific Bacteroides-Prevotella 16S rRNA genetic markers for assessment of fecal pollution in freshwater.

Based on the comparative 16S rRNA gene sequence analysis of fecal DNAs, we identified one human-, three cow-, and two pig-specific Bacteroides-Prevotella 16S rRNA genetic markers, designed host-specific real-time polymerase chain reaction (real-time PCR) primer sets, and successfully developed real-time PCR assay to quantify the fecal contamination derived from human, cow, and pig in natural river samples. The specificity of each newly designed host-specific primer pair was evaluated on fecal DNAs extracted from these host feces. All three cow-specific and two pig-specific primer sets amplified only target fecal DNAs (in the orders of 9-11 log(10) copies per gram of wet feces), showing high host specificity. This real-time PCR assay was then applied to the river water samples with different fecal contamination sources and levels. It was confirmed that this assay could sufficiently discriminate and quantify human, cow, and pig fecal contamination. There was a moderate level of correlation between the Bacteroides-Prevotella group-specific 16S rRNA gene markers with fecal coliforms (r (2) = 0.49), whereas no significant correlation was found between the human-specific Bacteroides 16S rRNA gene with total and fecal coliforms. Using a simple filtration method, the minimum detection limits of this assay were in the range of 50-800 copies/100 ml. With a combined sample processing and analysis time of less than 8 h, this real-time PCR assay is useful for monitoring or identifying spatial and temporal distributions of host-specific fecal contaminations in natural water environments.

Alternative indicators of fecal pollution: relations with pathogens and conventional indicators, current methodologies for direct pathogen monitoring and future application perspectives.

The ecological and survival characteristics of bacterial, viral and parasitic pathogens vary under environmental conditions, indicating that probably no single indicator organism can predict the presence of all enteric pathogens for all types of waters and different host-associated fecal pollution. If there are true correlations between indicator organisms and pathogens, it is necessary to find out to what extent and under which circumstances these organisms can be used as reliable indicators of fecal pollution. Application of conventional and alternative fecal indicators has greatly enhanced our abilities to predict and reduce health risk associated with the use of surface waters. New molecular-based techniques have shown that combined use of conventional and alternative indicators for fecal pollution increases both the detection sensitivity and specificity of fecal pollution and associated pathogens. In this review, we, therefore, summarize the advantages and limitations of conventional and alternative fecal indicators in terms of predicting pathogen presence as well as current and future methodologies for direct pathogen monitoring in environmental waters. This manuscript is mainly focused on the relationships between microbial fecal indicators and the presence of pathogens, which have not previously been summarized yet and could nicely supplement with recent literature reviews on microbial source tracking.

Application of a direct fluorescence-based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water.

To evaluate the viability and survival ability of fecal Bacteroides spp. in environmental waters, a fluorescence-based live/dead staining method using ViaGram Red+ Bacterial gram stain and viability kit was combined with fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probe (referred as LDS-FISH). The proposed LDS-FISH was a direct and reliable method to detect fecal Bacteroides cells and their viability at single-cell level in complex microbial communities. The pure culture of Bacteroides fragilis and whole human feces were dispersed in aerobic drinking water and incubated at different water temperatures (4 degrees C, 13 degrees C, 18 degrees C, and 24 degrees C), and then the viability of B. fragilis and fecal Bacteroides spp. were determined by applying the LDS-FISH. The results revealed that temperature and the presence of oxygen have significant effects on the survival ability. Increasing the temperature resulted in a rapid decrease in the viability of both pure cultured B. fragilis cells and fecal Bacteroides spp. The live pure cultured B. fragilis cells could be found at the level of detection in drinking water for 48 h of incubation at 24 degrees C, whereas live fecal Bacteroides spp. could be detected for only 4 h of incubation at 24 degrees C. The proposed LDS-FISH method should provide useful quantitative information on the presence and viability of Bacteroides spp., a potential alternative fecal indicator, in environmental waters.