The Clinical Validity of Urinary Pellet DNA Monitoring for the Diagnosis of Recurrent Bladder Cancer.

The aim of this study was to evaluate the clinical validity of monitoring urine pellet DNA (upDNA) of bladder cancer (BC) by digital PCR (dPCR) as a biomarker for early recurrence prediction, treatment efficacy evaluation, and no-recurrence corroboration. Tumor panel sequencing was first performed to select patient-unique somatic mutations to monitor both upDNA and circulating tumor DNA (ctDNA) by dPCR. For longitudinal monitoring using upDNA as well as plasma ctDNA, an average of 7.2 (range, 2 to 12) time points per case were performed with the dPCR assay for 32 previously treated and untreated patients with BC. Clinical recurrence based on imaging and urine cytology was compared using upDNA variant allele frequency (VAF) dynamics. A continuous increasing trend of upDNA VAF ≥1% was considered to indicate molecular recurrence. Most (30/32; 93.8%) cases showed at least one traceable somatic mutation. In 5 of 7 cases (71.4%) with clinical recurrence, upDNA VAF >1% was detected 7 to 15 months earlier than the imaging diagnosis. The upDNA VAF remained high after initial treatment for locally recurrent cases. The clinical validity of upDNA monitoring was confirmed with the observation that 26 of 30 cases (86.7%) were traceable. Local recurrences were not indicated by ctDNA alone. The results support the clinical validity of upDNA monitoring in the management of recurrent BC.

Postoperative recurrence detection using individualized circulating tumor DNA in upper tract urothelial carcinoma.

Biomarkers that could detect the postoperative recurrence of upper tract urothelial carcinoma (UTUC) have not been established. In this prospective study, we aim to evaluate the utility of individualized circulating tumor DNA (ctDNA) monitoring using digital PCR (dPCR) as a tumor recurrence biomarker for UTUC in the perioperative period. Twenty-three patients who underwent radical nephroureterectomy (RNU) were included. In each patient, whole exome sequencing by next-generation sequencing and TERT promoter sequencing of tumor DNA were carried out. Case-specific gene mutations were selected from sequencing analysis to examine ctDNA by dPCR analysis. We also prospectively collected plasma and urine ctDNA from each patient. The longitudinal variant allele frequencies of ctDNA during the perioperative period were plotted. Case-specific gene mutations were detected in 22 cases (96%) from ctDNA in the preoperative samples. Frequently detected genes were TERT (39%), FGFR3 (26%), TP53 (22%), and HRAS (13%). In all cases, we obtained plasma and urine samples for 241 time points and undertook individualized ctDNA monitoring for 2 years after RNU. Ten patients with intravesical recurrence had case-specific ctDNA detected in urine at the time of recurrence. The mean lead time of urinary ctDNA in intravesical recurrence was 60 days (range, 0-202 days). Two patients with distal metastasis had case-specific ctDNA in plasma at the time of metastasis. In UTUC, tumor-specific gene mutations can be monitored postoperatively as ctDNA in plasma and urine. Individualized ctDNA might be a minimally invasive biomarker for the early detection of postoperative recurrence.

Circulating Cell-Free DNA as a Biomarker for Prognosis and Response to Systemic Therapy in Patients with Unresectable Hepatocellular Carcinoma.

INTRODUCTION: Systemic therapy provides clinical benefits to a subset of patients with advanced unresectable hepatocellular carcinoma (HCC). However, few biomarkers are available for predicting prognosis and treatment response in patients with advanced HCC undergoing treatment with systemic therapies. This study aimed to examine whether circulating cell-free DNA (cfDNA) containing circulating tumor DNA can act as a therapeutic response and prognostic biomarker in patients with advanced HCC. METHODS: We analyzed longitudinally collected plasma cfDNA of patients with advanced HCC who were naïve to systemic therapy, and assessed their prognostic and predictive values to determine treatment responses. RESULTS: cfDNA concentration positively correlated with entire tumor volume on computed tomography before (p = 0.0231) and at the end (p < 0.0001) of the first-line systemic therapy. The overall survival rate was higher in patients with cfDNA concentrations lower than the median cfDNA level at baseline compared to patients with higher cfDNA concentrations (hazard ratio, 0.2765; 95% confidence interval, 0.08-0.81; p = 0.0197). The ratio of cfDNA at 4 weeks to that at baseline was predictive of radiographic disease response. In patients with progressive disease, cfDNA concentration at 4 weeks increased significantly (p = 0.0245), whereas the concentration remained unchanged in patients with other disease courses (p = 0.9375). CONCLUSION: The baseline plasma cfDNA concentration can be used as a prognostic biomarker in patients with advanced HCC. cfDNA kinetics may also predict the tumor response to therapy and disease progression.

Individualized circulating tumor DNA monitoring in head and neck squamous cell carcinoma.

There is no useful biomarker to evaluate treatment response and early relapse in head and neck squamous cell carcinoma (HNSCC). Circulating tumor DNA (ctDNA) is a promising biomarker for detecting minimal residual diseases and monitoring treatment effect. We investigated whether individualized ctDNA analysis could help monitor treatment response and relapse in HNSCC. Mutation analysis of tumor and peripheral blood mononuclear cell (PBMC) DNAs of 26 patients with HNSCC was performed using a custom squamous cell carcinoma (SCC) panel. The identified individualized mutated genes were defined as ctDNA candidates. We investigated whether frequent ctDNA monitoring via digital PCR (dPCR) is clinically valid for HNSCC patients. TP53 was the most frequently mutated gene and was detected in 14 of 24 cases (58.2%), wherein two cases were excluded owing to the absence of tumor-specific mutations in the SCC panel. Six cases were excluded because of undesignable and unusable primer-probes for dPCR. Longitudinal ctDNA was monitored in a total of 18 cases. In seven cases, ctDNA tested positive again or did not test negative, and all seven cases relapsed after initial curative treatment. In 11 cases, after initial curative treatment, ctDNA remained negative and patients were alive without recurrence. Patients who remained negative for ctDNA during follow-up after initial curative treatment (n = 11) had a significantly better prognosis than those who reverted to ctDNA positivity (n = 7; p < 0.0001; log-rank test). Individualized ctDNA monitoring using SCC panel and dPCR might be a novel and promising biomarker for HNSCC.

Cold and exogenous calcium alter Allium fistulosum cell wall pectin to depress intracellular freezing temperatures.

De-methyl esterification of homogalacturonan and subsequent cross-linking with Ca2+ is hypothesized to enhance the freezing survival of cold acclimated plants by reducing the porosity of primary cell walls. To test this theory, we collected leaf epidermal peels from non- (23/18 °C) and cold acclimated (2 weeks at 12/4 °C) Japanese bunching onion (Allium fistulosum L.). Cold acclimation enhanced the temperature at which half the cells survived freezing injury by 8 °C (LT50 =-20 °C), and reduced tissue permeability by 70-fold compared with non-acclimated epidermal cells. These effects were associated with greater activity of pectin methylesterase (PME) and a reduction in the methyl esterification of homogalacturonan. Non-acclimated plants treated with 50 mM CaCl2 accumulated higher concentrations of galacturonic acid, Ca2+ in the cell wall, and a lower number of visible cell wall pores compared with that observed in cold acclimated plants. Using cryo-microscopy, we observed that 50 mM CaCl2 treatment did not lower the LT50 of non-acclimated cells, but reduced the lethal intracellular ice nucleation to temperatures observed in cold acclimated epidermal cells. We postulate that the PME-homogalacturonan-mediated reduction in cell wall porosity is integral to intracellular freezing avoidance strategies in cold acclimated herbaceous cells.

Early dynamics of circulating tumor DNA predict chemotherapy responses for patients with esophageal cancer.

We investigated whether early circulating tumor DNA (ctDNA) changes, measured using digital PCR (dPCR), can predict later chemotherapy responses in esophageal squamous cell cancer (ESCC). We compared the dynamics of ctDNA and tumor volumes during chemotherapy in 42 ESCC. The accuracy of predictions of later chemotherapy responses was evaluated by the ratio of the variant allele frequency of ctDNA (post-/pre-ctDNA) and the total tumor volume (post-/pre-volume) before and after an initial chemotherapy cycle using a receiver-operating characteristic curve analysis. Total positive and negative objective responses (ORs) were defined as either >50 or ≤50% reductions, respectively, in the total tumor volume at the end of first-line chemotherapy. Mutation screening of 43 tumors from 42 patients revealed 96 mutations. The pretreatment dPCR-ctDNA data were informative in 38 patients, using 70 selected mutations (1-3 per patient). The areas under the curve (AUCs) for the post-/pre-volume and post-/pre-ctDNA levels used in predicting the total OR were 0.85 and 0.88, respectively. The optimal cutoff value of post-/pre-ctDNA was 0.13. In 20 patients with post-/pre-volume ≥50%, the total OR could be predicted by the post-/pre-ctDNA with high accuracy; the AUC by post-/pre-ctDNA was higher than that by post-/pre-volume (0.85 versus 0.76, respectively). Patients with low post-/pre-ctDNA (n = 18) had a significantly better overall survival rate than those with high post-/pre-ctDNA (n = 20; P = 0.03). Early ctDNA changes after an initial cycle of chemotherapy predict later responses to treatment with high accuracy in ESCC patients.

Season specificity in the cold-induced calcium signal and the volatile chemicals in the atmosphere.

Cold-induced Ca2+ signals in plants are widely accepted to be involved in cold acclimation. Surprisingly, despite using Arabidopsis plants grown in a growth chamber, we observed a clear seasonal change in cold-induced Ca2+ signals only in roots. Ca2+ signals were captured using Arabidopsis expressing Yellow Cameleon 3.60. In winter, two Ca2+ signal peaks were observed during a cooling treatment from 20 to 0°C, but in summer only one small peak was observed under the same cooling condition. In the spring and autumn seasons, an intermediate type of Ca2+ signal, which had a delayed first peak and smaller second peaks compared with the those of the winter type, was observed. Volatile chemicals and/or particles in the air from the outside may affect plants in the growth chamber. This idea is supported by the fact that incubation of plants with activated carbon changed the intermediate-type Ca2+ signal to the summer-type. The seasonality was also observed in the freezing tolerance of plants cold-acclimated in a low-temperature chamber. The solar radiation intensity was weakly correlated, not only with the seasonal characteristics of the Ca2+ signal but also with freezing tolerance. It has been reported that the ethylene concentration in the atmosphere seasonally changes depending on the solar radiation intensity. Ethylene gas and 1-aminocyclopropane-1-carboxylic acid treatment affected the Ca2+ signals, the shape of which became a shape close to, but not the same as, the winter type from the other types, indicating that ethylene may be one of several factors influencing the cold-induced Ca2+ signal.

Calcium Signaling-Linked CBF/DREB1 Gene Expression was Induced Depending on the Temperature Fluctuation in the Field: Views from the Natural Condition of Cold Acclimation.

Environmental adaptability is essential for plant survival. Though it is well known that a simple cooling or cold shock leads to Ca2+ signals, direct evidence has not been provided that plants use Ca2+ signals as a second messenger in the cold acclimation (CA) process in the field. By developing a technique to analyze Ca2+ signals using confocal cryomicroscopy, we investigated Ca2+ signals under several temperature conditions by combining the start temperature, cooling rate and cooling time duration. In both root and leaf cells, Ca2+ signals rapidly disappeared after cooling stopped, and thereafter under a constant low temperature no Ca2+ signal was observed. Interestingly, under the cooling regime from 2�C to -2�C, non-acclimated plants grown at 23�C hardly showed Ca2+ signals, but cold-acclimated plants at 2�C were able to form Ca2+ signals in root cells. These findings suggest that plants sense temperature decreases with Ca2+ signals while adjusting the temperature sensitivity to their own temperature environment. Furthermore, if the temperature is constant, no Ca2+ signal is induced even during CA. Then, we also focused on the CA under field conditions, rich in temperature fluctuations. In CA under field conditions, the expression patterns of CBF/DREB1 genes were distinctly different from those in artificial CA. Pharmacological studies with Ca2+ channel blockers showed that the Ca2+-induced expression of CBF/DREB1 genes was closely correlated with the amplitude of temperature fluctuation, suggesting that Ca2+ signals regulate CBF/DREB1 gene expression during CA under natural conditions.