Combining germline, tissue and liquid biopsy analysis by comprehensive genomic profiling to improve the yield of actionable variants in a real-world cancer cohort.

BACKGROUND: Comprehensive next-generation sequencing is widely used for precision oncology and precision prevention approaches. We aimed to determine the yield of actionable gene variants, the capacity to uncover hereditary predisposition and liquid biopsy appropriateness instead of, or in addition to, tumor tissue analysis, in a real-world cohort of cancer patients, who may benefit the most from comprehensive genomic profiling. METHODS: Seventy-eight matched germline/tumor tissue/liquid biopsy DNA and RNA samples were profiled using the Hereditary Cancer Panel (germline) and the TruSight Oncology 500 panel (tumor tissue/cfDNA) from 23 patients consecutively enrolled at our center according to at least one of the following criteria: no available therapeutic options; long responding patients potentially fit for other therapies; rare tumor; suspected hereditary cancer; primary cancer with high metastatic potential; tumor of unknown primary origin. Variants were annotated for OncoKB and AMP/ASCO/CAP classification. RESULTS: The overall yield of actionable somatic and germline variants was 57% (13/23 patients), and 43.5%, excluding variants previously identified by somatic or germline routine testing. The accuracy of tumor/cfDNA germline-focused analysis was demonstrated by overlapping results of germline testing. Five germline variants in BRCA1, VHL, CHEK1, ATM genes would have been missed without extended genomic profiling. A previously undetected BRAF p.V600E mutation was emblematic of the clinical utility of this approach in a patient with a liver undifferentiated embryonal sarcoma responsive to BRAF/MEK inhibition. CONCLUSIONS: Our study confirms the clinical relevance of performing extended parallel tumor DNA and cfDNA testing to broaden therapeutic options, to longitudinally monitor cfDNA during patient treatment, and to uncover possible hereditary predisposition following tumor sequencing in patient care.

High-efficiency EGFR genotyping using cell-free DNA in bronchial washing fluid.

BACKGROUND: EGFR mutation testing is required for treatment of lung adenocarcinoma using epidermal growth factor receptor-tyrosine kinase inhibitor. However, the amounts of tumor tissue or tumor cells obtained by bronchoscopy are often insufficient. Bronchial washing fluid, obtained by lavage with saline after tumor biopsy or brushing, and the supernatant of bronchial washing fluid are thought to contain cell-free DNA that would be potentially applicable for EGFR testing. METHODS: From among patients with suspected adenocarcinoma or non-small cell lung carcinoma diagnosed from biopsy or surgical specimens at the University of Tsukuba Hospital between 2015 and 2019, cell-free DNAs from 80 specimens of supernatant of bronchial washing fluid (50 with EGFR mutation and 30 with wild type EGFR) and 8 blood serum samples were examined for EGFR mutation using droplet digital PCR. RESULTS: Among the 50 patients harboring EGFR mutation, the rate of positivity for cell-free DNA extracted from supernatant of bronchial washing fluid was 80% (40/50). In nine of the EGFR mutation-positive cases, tumor cells were not detected by either biopsy or cytology, but the mutation was detected in four cases (4/9, 44%). Comparison of the cell-free DNA mutation detection rate between supernatant of bronchial washing fluid and blood serum in six cases showed that mutations were detected from the former in all cases (6/6, 100%), but from the latter in only one case (1/6, 17%). CONCLUSIONS: Using supernatant of bronchial washing fluid samples, the detection rate of EGFR mutation was high, and EGFR mutations were detectable even when no tumor cells had been detectable by biopsy or cytology. Supernatant of bronchial washing fluid might be an effective sample source for EGFR mutation testing.

A Comprehensive ddPCR Strategy for Sensitive and Reliable Monitoring of CAR-T Cell Kinetics in Clinical Applications.

In this study, we present the design, implementation, and successful use of digital droplet PCR (ddPCR) for the monitoring of chimeric antigen receptor T-cell (CAR-T) expansion in patients with B-cell malignancies treated with different CAR-T products at our clinical center. Initially, we designed a specific and highly sensitive ddPCR assay targeting the junction between the 4-1BB and CD3ζ domains of tisa-cel, normalized with RPP30, and validated it using blood samples from the first tisa-cel-treated patient in Switzerland. We further compared this assay with a published qPCR (quantitative real-time PCR) design. Both assays showed reliable quantification of CAR-T copies down to 20 copies/µg DNA. The reproducibility and precision were confirmed through extensive testing and inter-laboratory comparisons. With the introduction of other CAR-T products, we also developed a corresponding ddPCR assay targeting axi-cel and brexu-cel, demonstrating high specificity and sensitivity with a limit of detection of 20 copies/µg DNA. These assays are suitable for CAR-T copy number quantification across multiple sample types, including peripheral blood, bone marrow, and lymph node biopsy material, showing robust performance and indicating the presence of CAR-T cells not only in the blood but also in target tissues. Longitudinal monitoring of CAR-T cell kinetics in 141 patients treated with tisa-cel, axi-cel, or brexu-cel revealed significant expansion and long-term persistence. Peak expansion correlated with clinical outcomes and adverse effects, as is now well known. Additionally, we quantified the CAR-T mRNA expression, showing a high correlation with DNA copy numbers and confirming active transgene expression. Our results highlight the quality of ddPCR for CAR-T monitoring, providing a sensitive, precise, and reproducible method suitable for clinical applications. This approach can be adapted for future CAR-T products and will support the monitoring and the management of CAR-T cell therapies.

Quantification of Circulating Cell-Free DNA in Idiopathic Parkinson's Disease Patients.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders globally and leads to an excessive loss of dopaminergic neurons in the substantia nigra of the brain. Circulating cell-free DNA (ccf-DNA) are double-stranded DNA fragments of different sizes and origins that are released into the serum and cerebrospinal fluid (CSF) due to cell death (i.e., necrosis and apoptosis) or are actively released by viable cells via exocytosis and NETosis. Using droplet digital polymerase chain reaction (ddPCR), we comprehensively analyzed and distinguished circulating cell-free mitochondrial DNA (ccf mtDNA) and circulating cell-free nuclear DNA (ccfDNA) in the serum and CSF of PD and control patients. The quantitative analysis of serum ccf-DNA in PD patients demonstrated a significant increase in ccf mtDNA and ccfDNA compared to that in healthy control patients and a significantly higher copy of ccf mtDNA when compared to ccfDNA. Next, the serum ccf mtDNA levels significantly increased in male PD patients compared to those in healthy male controls. Furthermore, CSF ccf mtDNA in PD patients increased significantly compared to ccfDNA, and ccf mtDNA decreased in PD patients more than it did in healthy controls. These decreases were not statistically significant but were in agreement with previous data. Interestingly, ccf mtDNA increased in healthy control patients in both serum and CSF as compared to ccfDNA. The small sample size of serum and CSF were the main limitations of this study. To the best of our knowledge, this is the first comprehensive study on serum and CSF of PD patients using ddPCR to indicate the distribution of the copy number of ccf mtDNA as well as ccfDNA. If validated, we suggest that ccf mtDNA has greater potential than ccfDNA to lead the development of novel treatments for PD patients.

Pilot Study by Liquid Biopsy in Gastrointestinal Stromal Tumors: Analysis of PDGFRA D842V Mutation and Hypermethylation of SEPT9 Presence by Digital Droplet PCR.

Tissue biopsy remains the standard for diagnosing gastrointestinal stromal tumors (GISTs), although liquid biopsy is emerging as a promising alternative in oncology. In this pilot study, we advocate for droplet digital PCR (ddPCR) to diagnose GIST in tissue samples and explore its potential for early diagnosis via liquid biopsy, focusing on the PDGFRA D842V mutation and SEPT9 hypermethylated gene. We utilized ddPCR to analyze the predominant PDGFRA mutation (D842V) in surgical tissue samples from 15 GIST patients, correlating with pathologists' diagnoses. We expanded our analysis to plasma samples to compare DNA alterations between tumor tissue and plasma, also investigating SEPT9 gene hypermethylation. We successfully detected the PDGFRA D842V mutation in GIST tissues by ddPCR. Despite various protocols to enhance mutation detection in early-stage disease, it remained challenging, likely due to the low concentration of DNA in plasma samples. Additionally, the results of Area Under the Curve (AUC) for the hypermethylated SEPT9 gene, analyzing concentration, ratio, and abundance were 0.74 (95% Confidence Interval (CI): 0.52 to 0.97), 0.77 (95% CI: 0.56 to 0.98), and 0.79 (95% CI: 0.59 to 0.99), respectively. As a rare disease, the early detection of GIST through such biomarkers is particularly crucial, offering significant potential to improve patient outcomes.

Fecal Impairment Framework, A New Conceptual Framework for Assessing Fecal Contamination in Recreational Waters.

Fecal pollution of surface water is a pervasive problem that negatively affects waterbodies concerning both public health and ecological functions. Current assessment methods monitor fecal indicator bacteria (FIB) to identify pollution sources using culture-based quantification and microbial source tracking (MST). These types of information assist stakeholders in identifying likely sources of fecal pollution, prioritizing them for remediation, and choosing appropriate best management practices. While both culture-based quantification and MST are useful, they yield different kinds of information, potentially increasing uncertainty in prioritizing sources for management. This study presents a conceptual framework that takes separate human health risk estimates based on measured MST and E. coli concentrations as inputs and produces an estimate of the overall fecal impairment risk as its output. The proposed framework is intended to serve as a supplemental screening tool for existing monitoring programs to aid in identifying and prioritizing sites for remediation. In this study, we evaluated the framework by applying it to two primarily agricultural watersheds and several freshwater recreational beaches using existing routine monitoring data. Based on a combination of E. coli and MST results, the proposed fecal impairment framework identified four sites in the watersheds as candidates for remediation and identified temporal trends in the beach application. As these case studies demonstrate, the proposed fecal impairment framework is an easy-to-use and cost-effective supplemental screening tool that provides actionable information to managers using existing routine monitoring data, without requiring specialized expertize.

Loss of Y chromosome in leukocytes can be regarded as a male-specific age predictor for age group estimation in forensic genetics.

Age prediction is an important field in forensic and aging research. Traditional methods used DNA methylation, telomere shortening, and mitochondrial DNA mutations to conduct age prediction models. Sex chromosomes, like the Y chromosome, have a significant role in aging as previously reported in hematopoietic disease and many non-reproductive cancers. Until now, there is no age predictor based on the percentage of loss of Y chromosome (LOY). LOY has been previously revealed to be correlated with Alzheimer's disease, short survival, and higher risk of cancer. The possible correlation of LOY between normal aging was not fully explored. In this study, we conducted age prediction by measuring LOY percentage by droplet digital PCR (ddPCR), based on 232 healthy male samples, including 171 blood samples, 49 saliva samples, 12 semen samples. The age group of samples ranges from 0 to 99 years, with two individuals in almost every single age. Pearson correlation method was performed to calculate the correlation index. The result indicated a correlation index of 0.21 (p = 0.0059) between age and LOY percentage in blood samples, with the regression formula being y = - 0.016823 + 0.001098x. The correlation between LOY percentage and age is obvious only when the individuals were divided into different age groups (R = 0.73, p = 0.016). In the studied saliva and semen samples, p-values of the correlation are 0.11 and 0.20, respectively, showing no significant association between age and LOY percentage in these two biological materials. For the first time, we investigated male-specific age predictor based on LOY. The study showed that LOY in leukocytes can be regarded as a male-specific age predictor for age group estimation in forensic genetics. This study might be indicative for forensic applications and aging research.

CD4dim CD8bright T Cells Home to the Brain and Mediate HIV Neuroinvasion.

CD4dim CD8bright T cells are a mature population of CD8+ T cells that upon activation upregulate CD4 dimly on their surface. Expression of CD4 on these cells suggests that they can be an additional source of HIV neuroinvasion and persistence in the brain. We used HIV-infected NOD/SCID/IL-2rcγ-/- (NSG) humanized mice to track CD4dim CD8bright T cell homing to the brain and define their role in HIV dissemination into the brain. We report here that CD4dim CD8bright T cells are found in the brain at a median frequency of 2.6% and in the spleen at median frequency of 7.6% of CD3+ T cells. In the brain, 10 to 20% of CD4dim CD8bright T cells contain integrated provirus, which is infectious as demonstrated by viral outgrowth assay. CD4dim CD8bright T cells in the brain exhibited significantly higher expression of the brain homing receptors CX3CR1 and CXCR3 in comparison to their single-positive CD8+ T cell counterpart. Blocking lymphocyte trafficking into the brain of humanized mice via anti-VLA4 and anti-LFA1 antibodies reduced CD4dim CD8bright T cell trafficking into the brain by 60% and diminished brain HIV proviral DNA by 72%. Collectively, our findings demonstrate that CD4dim CD8bright T cells can home to the brain and support productive HIV replication. These studies also reveal for the first time that CD4dim CD8bright T cells are capable of HIV neuroinvasion and are a reservoir for HIV. IMPORTANCE We report here a seminal finding of a novel population of T cells, termed CD4dim CD8bright T cells, that plays a role in HIV neuroinvasion and is a reservoir for HIV in the brain.

Characterization of loss of chromosome Y in peripheral blood cells in male Han Chinese patients with schizophrenia.

BACKGROUND: Schizophrenia (SCZ) has a global prevalence of 1% and increases the risk of mortality, reducing life expectancy. There is growing evidence that the risk of this disorder is higher in males than in females and it tends to develop in early adulthood. The Y chromosome is thought to be involved in biological processes other than sex determination and spermatogenesis. Studies have shown that loss of chromosome Y (LOY) in peripheral blood cells is associated with a variety of diseases (including cancer) and increased all-cause mortality. An analysis of the relationship between LOY and schizophrenia is warranted. METHODS: A total of 442 Chinese males (271 patients with schizophrenia vs. 171 controls) were included in this study. The copy numbers of the Y and X chromosomes were detected by positive droplets targeting the amelogenin gene (AMEL) on the Y chromosome and X chromosome (AMELY and AMELX, respectively), using droplet digital PCR (ddPCR). The LOY percentage was defined as the difference between the concentration of AMELX and the concentration of AMELY divided by the concentration of AMELX, denoted as (X - Y)/X. RESULTS: In the Han Chinese population, the LOY percentage was higher in the schizophrenia group than in the control group (p < 0.05), although there was no significant difference in the presence of LOY between the two groups. A strong correlation was found between the average of the disease duration and the average of the LOY percentage (R2 = 0.506, p = 0.032). The logistic regression analysis implied that the risk of LOY increases by 0.058 and 0.057 per year according to age at onset and duration of disease, respectively (ponset = 0.013, pduration = 0.017). CONCLUSIONS: In the Han Chinese population, the LOY percentage of the disease group was significantly different from that of the control group. The age of onset and duration of schizophrenia might be risk factors for LOY in peripheral blood cells. A larger sample size and expanded clinical information are needed for more in-depth and specific analyses.

Development and application of a sensitive droplet digital PCR-based method to detect tilapia parvovirus.

Tilapia parvovirus (TiPV) causes severe mortality rates in cultured tilapia, resulting in substantial losses to the fish industry. Droplet digital PCR (ddPCR) is a sensitive, accurate, and absolute quantitation method, plus it does not require a standard curve. Herein we report the development and application of a sensitive ddPCR-based method to rapidly detect and quantify TiPV. Optimal annealing temperature was determined to be 59.3°C, and optimal primer and probe concentrations were 900 nmol/L and 250 nmol/L, respectively. Our ddPCR method was highly specific to TiPV and showed no cross-reactivity with other viruses. Further, the detection limit of ddPCR was 0.07 copies/µl, being lower than that of real-time PCR (qPCR, 4.63 copies/µl). We also investigated the ability of ddPCR to detect TiPV in 50 samples and compared the outcome with qPCR data in terms of sensitivity and accuracy. The results showed that the positive detection rate of ddPCR (32%) was higher than that of qPCR (18%). To conclude, our ddPCR method was effective at detecting TiPV in samples with low viral loads. We believe that its application can facilitate the surveillance of sources and transmission routes of TiPV.

Identification of 'Candidatus Liberibacter asiaticus' the Huanglongbing Bacterium in citrus from Colombia.

Candidatus Liberibacter spp is the most prevalent microorganism in the citrus plant, associated with Citrus Huanglongbing (HLB), which is transmitted by the psyllid vector. In Colombia, the vector Diaphorina citri Kugayama has been reported in different regions, but "Ca. Liberibacter asiaticus" (CLas) has only been detected in insect vectors, not in citrus host plants. To identify the presence and quantify the pathogen in citrus tissues, we employed a combined strategy that involved three techniques based on polymerase chain reaction (PCR). First, we used endpoint PCR with specific primers for CLas (OI1-OI2c) to confirm the infection. Second, we used qPCR with specific primers CIT295a - CIT298 designed on 16S rDNA gene regions to quantify the pathogen load. Finally, we employed droplet digital PCR (ddPCR) to determine the copy number of the pathogen in citrus tissues using the ß-subunit of ribonucleotide reductase (RNR) gene (nrdB) that is specific to CLas. We identified the presence of CLas in citrus plants for the first time in Colombia and quantified its titer in the plant tissue. We employed ddPCR and qPCR to provide crucial information for the country's disease management, control strategies, and general crop health.

Using watershed characteristics to enhance fecal source identification.

Fecal pollution is one of the most prevalent forms of pollution affecting waterbodies worldwide, threatening public health and negatively impacting aquatic environments. Microbial source tracking (MST) applies polymerase chain reaction (PCR) technology to help identify the source of fecal pollution. In this study, we combine spatial data for two watersheds with general and host-associated MST markers to target human (HF183/BacR287), bovine (CowM2), and general ruminant (Rum2Bac) sources. Concentrations of MST markers in samples were determined with droplet digital PCR (ddPCR). The three MST markers were detected at all sites (n = 25), but bovine and general ruminant markers were significantly associated with watershed characteristics. MST results, combined with watershed characteristics, suggest that streams draining areas with low-infiltration soil groups and high agricultural land use are at an increased risk for fecal contamination. Microbial source tracking has been applied in numerous studies to aid in identifying the sources of fecal contamination, but these studies usually lack information on the involvement of watershed characteristics. Our study combined watershed characteristics with MST results to provide more comprehensive insight into the factors that influence fecal contamination in order to implement the most effective best management practices.

Detection of Mixed Populations of Clarithromycin-Susceptible and -Resistant Mycobacterium abscessus Strains.

Clarithromycin resistance in Mycobacterium abscessus subsp. abscessus, massiliense, and bolletii occurs through induction of erm(41) or mutations in rrl (23S rRNA) genes. Phenotypic detection of clarithromycin resistance is hindered by the need for extended incubation as well as co-occurrence of mixed populations of M. abscessus with different susceptibility profiles. We developed a quantitative EvaGreen-based droplet digital PCR (ddPCR) scheme for rapid detection of full-length or truncated erm(41) and a probe based ddPCR screening assay for assessment of 23S rRNA rrl mutational resistance. We tested 100 M. abscessus strains, synthetic mixes with different susceptibility profiles, and 13 positive MGIT samples. Truncated and full-length erm(41) genes were detected in 27/100 and 73/100 strains and 4/13 and 9/13 MGIT samples, respectively yielding a sensitivity and specificity of 100%. Clarithromycin resistance mutations in rrl were detected in 26/100 isolates, i.e., A2058G (18/100), A2058C (7/100), and A2059G (1/100), and in 3/13 MGIT samples, i.e., A2058G (2/13) and A2059G (1/13). A screening assay of rrl ddPCR (A2058A/A2058G probes) showed 100% sensitivity in detecting the wild type or A2058G mutation as well as identifying samples requiring further testing. Upon inclusion of additional ddPCR assays, we were able to detect A2058C and A2059G clarithromycin resistance-conferring mutations in the rrl gene. Our ddPCR scheme can differentiate between full-length and truncated erm(41) and identify clarithromycin resistance-conferring mutations in the rrl gene from clinical isolates and positive MGIT samples as well as deconvolute and quantitate mixed populations of M. abscessus with different clarithromycin resistance traits.

Droplet digital PCR or quantitative PCR for in-depth genomic and functional validation of genetically altered rodents.

Gene targeting and additive (random) transgenesis have proven to be powerful technologies with which to decipher the mammalian genome. With the advent of CRISPR/Cas9 genome editing, the ability to inactivate or modify the function of a gene has become even more accessible. However, the impact of each generated modification may be different from what was initially desired. Minimal validation of mutant alleles from genetically altered (GA) rodents remains essential to guarantee the interpretation of experimental results. The protocol described here combines design strategies for genomic and functional validation of genetically modified alleles with droplet digital PCR (ddPCR) or quantitative PCR (qPCR) for target DNA or mRNA quantification. In-depth analysis of the results obtained with GA models through the analysis of target DNA and mRNA quantification is also provided, to evaluate which pitfalls can be detected using these two methods, and we propose recommendations for the characterization of different type of mutant allele (knock-out, knock-in, conditional knock-out, FLEx, IKMC model or transgenic). Our results also highlight the possibility that mRNA expression of any mutated allele can be different from what might be expected in theory or according to common assumptions. For example, mRNA analyses on knock-out lines showed that nonsense-mediated mRNA decay is generally not achieved with a critical-exon approach. Likewise, comparison of multiple conditional lines crossed with the same CreERT2 deleter showed that the inactivation outcome was very different for each conditional model. DNA quantification by ddPCR of G0 to G2 generations of transgenic rodents generated by pronuclear injection showed an unexpected variability, demonstrating that G1 generation rodents cannot be considered as established lines.

Reliable and robust droplet digital PCR (ddPCR) and RT-ddPCR protocols for mouse studies.

Droplet digital PCR (ddPCR) is a recent method developed for the quantification of nucleic acids sequences. It is an evolution of PCR methodology incorporating two principal differences: a PCR reaction is performed in thousands of water-oil emulsion droplets and fluorescence is measured at the end of PCR amplification. It leads to the precise and reproducible quantification of DNA and RNA sequences. Here, we present quantitative methods for DNA and RNA analysis using Bio-Rad QX100 or QX200 systems, respectively. The aim of these methods is to provide useful molecular tools for validating genetically altered animal models such as those subject to CRISPR/Cas9 genome editing, as well for expression or CNV studies. A standard procedure for simultaneous DNA and RNA extraction adapted for mouse organs is also described. These methods were initially designed for mouse studies but also work for samples from other species like rat or human. In our lab, thousands of samples and hundreds of target genes from genetically altered lines were examined using these methods. This large dataset was analyzed to evaluate technical optimizations and limitations. Finally, we propose additional recommendations to be included in dMIQE (Minimum information for publication of quantitative digital PCR experiments) guidelines when using ddPCR instruments.

Development and evaluation of a molecular based protocol for detection and quantification of Cryptosporidium spp. in wastewater.

Infections caused by protozoan parasites are a major public health concern globally. These infections are commonly diagnosed during water-borne outbreaks, necessitating accurate and highly sensitive detection procedures to assure public health protection. Current molecular techniques are challenged by several factors, such as low parasite concentration, inefficient DNA extraction methods, and inhibitors in environmental samples. This study focused on the development and validation of a molecular protocol for DNA extraction, efficient protozoan (oo)cyst recovery and quantification of protozoan parasites from wastewater using droplet digital polymerase chain reaction (ddPCR). Five DNA extraction methods, including commercial kits, custom phenol-chloroform, and in-house modified methods, were evaluated. The efficiency of each method was assessed via spectrophotometric analysis and ddPCR amplification using specific primers. Lastly, the developed protocol was evaluated for the detection and quantification of Cryptosporidium parvum in wastewater from different regions in South Africa. The conventional phenol-chloroform extraction method yielded the highest DNA concentration of 223 (±0.71) ng/µl and detected the highest number of Cryptosporidium parvum (1807 (±0.30) copies/ddPCR reaction) compared to other methods evaluated in this study. Additionally, the phenol-chloroform method demonstrated high sensitivity in extracting DNA from as few as one cyst/L of Cryptosporidium parvum, corresponding to 5.93 copies/ddPCR reaction. It was also observed that analysis of both the filtered supernatant and pellets after centrifugation improves the recovery efficiency of oocysts from wastewater by 10.5%, resulting in a total recovery of 64.1%. This optimized protocol was successfully applied to measure protozoan concentration in wastewater from different regions in South Africa. The improved DNA extraction and quantification method proposed in this study would be effective in monitoring protozoan concentration in the environment, which will help in instituting mitigation measures to reduce water-borne infections.

Longitudinal analysis of cell-free mutated KRAS and CA 19-9 predicts survival following curative resection of pancreatic cancer.

BACKGROUND: Novel biomarkers and molecular monitoring tools hold potential to improve outcome for patients following resection of pancreatic ductal adenocarcinoma (PDAC). We hypothesized that the combined longitudinal analysis of mutated cell-free plasma KRAS (cfKRASmut) and CA 19-9 during adjuvant treatment and follow-up might more accurately predict disease course than hitherto available parameters. METHODS: Between 07/2015 and 10/2018, we collected 134 plasma samples from 25 patients after R0/R1-resection of PDAC during adjuvant chemotherapy and post-treatment surveillance at our institution. Highly sensitive discriminatory multi-target ddPCR assays were employed to screen plasma samples for cfKRASmut. cfKRASmut and CA 19-9 dynamics were correlated with recurrence-free survival (RFS) and overall survival (OS). Patients were followed-up until 01/2020. RESULTS: Out of 25 enrolled patients, 76% had undergone R0 resection and 48% of resected PDACs were pN0. 17/25 (68%) of patients underwent adjuvant chemotherapy. Median follow-up was 22.0 months, with 19 out of 25 (76%) patients relapsing during study period. Median RFS was 10.0 months, median OS was 22.0 months. Out of clinicopathologic variables, only postoperative CA 19-9 levels and administration of adjuvant chemotherapy correlated with survival endpoints. cfKRASmut. was detected in 12/25 (48%) of patients, and detection of high levels inversely correlated with survival endpoint. Integration of cfKRASmut and CA 19-9 levels outperformed either individual marker. cfKRASmut outperformed CA 19-9 as dynamic marker since increase during adjuvant chemotherapy and follow-up was highly predictive of early relapse and poor OS. CONCLUSIONS: Integrated analysis of cfKRASmut and CA 19-9 levels is a promising approach for molecular monitoring of patients following resection of PDAC. Larger prospective studies are needed to further develop this approach and dissect each marker's specific potential.

Novel Quantification of Extracellular Vesicles with Unaltered Surface Membranes Using an Internalized Oligonucleotide Tracer and Applied Pharmacokinetic Multiple Compartment Modeling.

PURPOSE: We developed an accessible method for labeling small extracellular vesicles (sEVs) without disrupting endogenous ligands. Using labeled sEVs administered to conscious rats, we developed a multiple compartment pharmacokinetic model to identify potential differences in the disposition of sEVs from three different cell types. METHODS: Crude sEVs were labeled with a non-homologous oligonucleotide and isolated from cell culture media using a commercial reagent. Jugular vein catheters were used to introduce EVs to conscious rats (n = 30) and to collect blood samples. Digital PCR was leveraged to allow for quantification over a wide dynamic range. Non-linear mixed effects analysis with first order conditional estimation - extended least squares (FOCE ELS) was used to estimate population-level parameters with associated intra-animal variability. RESULTS: 86.5% ± 1.5% (mean ± S.E.) of EV particles were in the 45-195 nm size range and demonstrated protein and lipid markers of endosomal origin. Incorporated oligonucleotide was stable in blood and detectable over five half-lives. Data were best described by a three-compartment model with one elimination from the central compartment. We performed an observation-based simulated posterior predictive evaluation with prediction-corrected visual predictive check. Covariate and bootstrap analyses identified cell type having an influence on peripheral volumes (V2 and V3) and clearance (Cl3). CONCLUSIONS: Our method relies upon established laboratory techniques, can be tailored to a variety of biological questions regarding the pharmacokinetic disposition of extracellular vesicles, and will provide a complementary approach for the of study EV ligand-receptor interactions in the context of EV uptake and targeted therapeutics.

Differential tissue specific expression of Kif23 alternative transcripts in mice with the human mutation causing congenital dyserythropoietic anemia type III.

Kinesin Family Member 23 (KIF23), a cell cycle regulator, has a key task in cytokinesis. KIF23 over-expression in cancer has been associated with tumor growth, progression, and poor prognosis, indicating a potential to be a cancer biomarker. A mutation in KIF23 (c.2747C > G, p.P916R) was shown to cause congenital dyserythropoietic anemia, type III (CDA III). To-date, fifteen KIF23 transcripts have been annotated, but their expression is poorly investigated. We hypothesized that tissue specific expression of a particular transcript can be critical for CDA III phenotype. In this study, we quantified expression of alternative Kif23 transcripts in a mouse model with human KIF23 mutation and investigated its association with a regulator of alternative splicing, serine/arginine-rich splicing factor 3 (Srsf3). We confirmed presence of an additional exon 8 in both human and mouse KIF23 transcripts. A transcript lacking exons 17 and 18 was ubiquitously expressed in mice while other isoforms were common in human tissues however in bone marrow of knock-in mice a transcript without exon 18 was prevalent as it was in bone marrow of a CDA III patient. We conclude that the possibility that the tissue specific expression of KIF23 alternative transcripts influence the CDA III phenotype cannot be neglected.

Comparison of the Clinical Validity of Droplet Digital PCR to ARMS-PCR for BRAF V600E Mutation Detection in Thyroid Nodules.

OBJECTIVES: Droplet digital PCR (ddPCR) has been reported to have a superior validity over PCR with amplification-refractory mutation system (ARMS-PCR) for detecting the BRAF V600E mutation in thyroid nodule fine-needle aspiration (FNA) samples using cytological diagnosis as the reference. However, the added value of ddPCR on surgical decision-making remains to be illustrated when the technique is combined with FNA cytology. METHODS: A total of 277 consecutive patients with thyroid nodules were subjected to FNA cytology and BRAF V600E testing with ARMS-PCR. Within this patient cohort, 90 patients underwent surgical intervention with pathological diagnosis available. BRAF V600E testing with ddPCR was performed retrospectively using FNA frozen DNA specimens. The clinical validity and utility of ddPCR in comparison with ARMS-PCR were compared using surgical pathology as the reference. RESULTS: Overall, 101 BRAF V600E mutations were detected by ddPCR, including five ARMS negative patients, four of whom were confirmed to have papillary thyroid cancer (PTC) by surgical pathology. Of the 90 patients with surgical pathology, which is considered the gold standard, ddPCR BRAF V600E testing yielded a sensitivity of 91.3% and specificity of 100% for PTC diagnosis, higher than that of ARMS (sensitivity 83.1%, specificity 100%). However, ddPCR only identified one more candidate patient for surgical intervention than ARMS when the techniques were combined with cytology. CONCLUSIONS: This study highlighted the superior performance of ddPCR over ARMS in BRAF V600E detection from thyroid nodule FNA samples. Further studies are needed to evaluate the cost-effectiveness of replacing ARMS-PCR with ddPCR for surgical decision-making.