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.
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Abstract Background: Many patients undergo a next generation sequencing (NGS)-based cancer genome profiling (CGP) test, but it often does not aid in choosing an optimal therapeutic regimen. Therefore, the genomic information should be used for minimal residual disease (MRD) detection, which directly leads to improved post-treatment cancer patient management. Circulating tumor DNA (ctDNA) is a small fraction of DNA present in blood that carries somatic mutations in cancer patients. Our previous studies demonstrated the clinical validity of ctDNA as a tumor marker for MRD detection. However, the variant allele frequency (VAF; reflecting the fraction of somatic mutations) of ctDNA is less than 1%, at which stable detection is difficult for NGS. Instead of NGS, we have established a highly sensitive ctDNA monitoring system for MRD detection using an originally developed library of >1000 digital PCR (dPCR) probes. Patients and Methods: Using a Clinical Institutional Review Board approval from the Iwate Medical University Hospital, we initiated a dPCR assay system, called off-the-shelf (OTS)-Assay, in April 2022 under a partnership with Quantdetect, Inc (Tokyo, Japan). The OTS-Assay has three main components: OTS-Scan, OTS-Select, and OTS-Monitor. The OTS-Scan can provide an original CGP if a patient comes with no CGP results. The OTS-Select chooses matched somatic mutations from our dPCR probe library. The OTS-Monitor quantifies VAFs of ctDNA periodically in the patient plasma with a sensitivity of 0.05% VAF. There is no strict patient eligibility criteria other than the requirement of previous therapy for treatment of an advanced cancer. Results: Between April and November 2022, 26 patients visited Iwate Medical University Hospital for the OTS-Assay. Twenty-five patients were referred from other Departments of Iwate Medical University Hospital. The primary cancer types include esophagus (n=21), colorectal (n=2), pancreas (n=2), and breast (n=1). Twenty patients had CGP results at their first visit. OTS-Select were used for 13 patients and picked an average of 11.4 mutations per patient. Among those, each of eight patients were immediately able to start ctDNA monitoring with one of the 1000 dPCR probes. As of November 2022, there are four patients who received OTS-Monitor more than one time. Any unexpected VAF increase of ctDNA has not been noted, although a substantial decrease of ctDNA VAF was found in one patient who received one cycle of chemotherapy. Conclusion: The OTS-Assay is a practical introduction of our previous clinical research products based on a core technology, which uses an originally developed library of >1000 dPCR probes. The potential of early and accurate MRD detection will improve post-treatment cancer patient management. Citation Format: Satoshi S. Nishizuka, Hayato Hiraki, Masakazu Abe, Akiko Yashima-Abo, Takeshi Iwaya. An introduction of a highly sensitive circulating tumor DNA monitoring system for minimal residual disease detection using a library of 1000 digital PCR probes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5599.
Molecular and physiological processes during cold acclimation (CA) have been investigated using plants incubated under constant low-temperature conditions. However, to comprehensively characterize CA in the field, the effects of day–night temperature cycles and minute-scale random temperature fluctuations must be clarified. Thus, we developed an experimental system that can maintain diurnal cycles and random temperature fluctuations during CA treatments. On the basis of the temperature changes in the field, three CA conditions were applied: conventional CA at 2°C (con-CA), CA with a 10°C day/2°C night cycle (C-CA), and C-CA with random temperature fluctuations only during the day (FC-CA). Because cold-induced Ca 2+ signals help regulate CA, the effects of Ca 2+ signals during the three CA treatments were examined using Ca 2+ channel blockers (LaCl 3 and ruthenium red). The freezing tolerance of Arabidopsis thaliana was similar after the C-CA and con-CA treatments, but it decreased following the FC-CA treatment. The analysis of transcription factors regulating CA processes indicated CBF/DREB1 expression levels tended to be highest for the con-CA treatment, followed by the FC-CA and C-CA treatments. Moreover, the Ca 2+ signals substantially contributed to the freezing tolerance of the plants that underwent the FC-CA and C-CA treatments, while also considerably modulating gene expression in the FC-CA-treated plants. Furthermore, the Ca 2+ signals enhanced CBF/DREB1 expression during the FC-CA treatment, but the Ca 2+ signals derived from intracellular organelles suppressed the expression of CBF2/DREB1C and CBF3/DREB1A during the C-CA treatment. Thus, diurnal temperature cycles and random temperature fluctuations affect CA through different calcium signals, implying that plants regulate CA by precisely sensing temperature changes in the field.
Abstract Introduction: Approximately half of high-risk non-muscle-invasive bladder cancers (NMIBCs) recur after transurethral resection of the bladder tumor (TURBT), whereas conventional urological testing for bladder cancer (BC) patients has been considered invasive and with low sensitivity. Our previous reports have shown that circulating tumor DNA (ctDNA) monitored by digital PCR (dPCR) can predict relapse and evaluate treatment efficacy in digestive tract cancers. In BC patients, tumor-derived genetic mutations detected in urinary DNA are expected to be a diagnostic biomarker. This study evaluates the clinical validity of monitoring tumor-specific gene mutations in ctDNA and urine pellet DNA (upDNA) using dPCR as a biomarker for early relapse prediction and determination of treatment efficacy. Materials and Methods: Thirty-two previously treated and untreated BC patients were enrolled. Tumor DNA was extracted from archived TURBT tissues, and gene mutation analysis by Next Generation Sequencing (NGS) of tumors was performed to select patient-unique somatic mutations for both ctDNA and upDNA monitoring by dPCR. Longitudinal variant allele frequencies (VAFs) of ctDNA and upDNA were monitored by dPCR for up to two years. While dPCR is rapid and has a high sensitivity of less than 0.1% VAF, a mutation-specific primer/probe set is required. Therefore, we established over 1,000 primer/probe sets for frequent mutations in human cancer. This large-scale dPCR primer/probe library allow us to select patient specific mutations for immediate ctDNA monitoring. Clinical recurrence (Crec) based on imaging and urinary cell findings are compared with VAF dynamics of ctDNA and upDNA. Results: The median observation period was 516 days (30-733), and a total of 230 urine samples were collected. The selection of monitoring mutations based on NGS analysis was performed in 93.8% (30/32) cases, with 2.3 (range: 1-6) mutations monitored per case. The primer/probe sets covered 87.5% (28/32) of cases and 87.5% (42/48) of mutations. The VAFs of the upDNA of cases started showing constantly increasing trends 3-12 months earlier than the diagnosis of Crec in 71.4% (5/7) of the cases. The other two Crec cases, which did not show an elevated upVAF, were both pyuria cases. After local treatment, the upDNA VAFs remained high in the Crec cases. Conventional Crec did not seem to be fully reflected by ctDNA alone. Conclusion: Frequent VAF monitoring using upDNA by dPCR enables the prediction of local relapse and the evaluation of treatment efficacy in the management of BC patients. Citation Format: Masakazu Abe, Hayato Hiraki, Takashi Tsuyukubo, Sadahide Ono, Shigekatsu Maekawa, Daichi Tamura, Daiki Ikarashi, Renpei Kato, Tomohiko Matsuura, Mitsugu Kanehira, Ryo Takata, Hiromitsu Fujisawa, Takeshi Iwaya, Masashi Idogawa, Wataru Obara, Satoshi S. Nishizuka. The clinical validity of urinary pellet DNA monitoring of recurrent bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3326.
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.
Abstract 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.
Cold‐induced Ca 2+ 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 Ca 2+ signals only in roots. Ca 2+ signals were captured using Arabidopsis expressing Yellow Cameleon 3.60. In winter, two Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ signal.
Abstract 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.
