The potential of circulating tumor DNA (ctDNA) analysis to serve as a real-time "liquid biopsy" for children with central nervous system (CNS) and non-CNS solid tumors remains to be fully elucidated. We conducted a study to investigate the feasibility and potential clinical utility of ctDNA sequencing in pediatric patients enrolled on an institutional clinical genomics trial. A total of 240 patients had tumor DNA profiling performed during the study period. Plasma samples were collected at study enrollment from 217 patients and then longitudinally from a subset of patients. Successful cell-free DNA extraction and quantification occurred in 216 of 217 (99.5%) of these initial samples. Twenty-four patients were identified whose tumors harbored 30 unique variants that were potentially detectable on a commercially-available ctDNA panel. Twenty of these 30 mutations (67%) were successfully detected by next-generation sequencing in the ctDNA from at least one plasma sample. The rate of ctDNA mutation detection was higher in patients with non-CNS solid tumors (7/9, 78%) compared to those with CNS tumors (9/15, 60%). A higher ctDNA mutation detection rate was also observed in patients with metastatic disease (9/10, 90%) compared to non-metastatic disease (7/14, 50%), although tumor-specific variants were detected in a few patients in the absence of radiographic evidence of disease. This study illustrates the feasibility of incorporating longitudinal ctDNA analysis into the management of relapsed or refractory patients with childhood CNS or non-CNS solid tumors.

Background Pediatric papillary thyroid carcinoma (PTC) is clinically and biologically distinct from adult PTC. We sequenced a cohort of clinically annotated pediatric PTC cases enriched for high-risk tumors to identify genetic alterations of relevance for diagnosis and therapy. Methods Tumor DNA and RNA were extracted from FFPE tissue and subjected to next-generation sequencing (NGS) library preparation using a custom 124-gene hybridization capture panel and the 75-gene Archer Oncology Research Panel, respectively. NGS libraries were sequenced on an Illumina MiSeq. Results Thirty-six pediatric PTC cases were analyzed. Metastases were frequently observed to cervical lymph nodes (29/36, 81%), with pulmonary metastases less commonly found (10/36, 28%). Relapsed or refractory disease occurred in 18 patients (18/36, 50%). DNA sequencing revealed targetable mutations in 8 of 31 tumors tested (26%), most commonlyBRAFp.V600E (n = 6). RNA sequencing identified targetable fusions in 13 of 25 tumors tested (52%):RET(n = 8),NTRK3(n = 4), andBRAF. Mutually exclusive targetable alterations were discovered in 15 of the 20 tumors (75%) with both DNA and RNA analyzed. Fusion-positive PTC was associated with multifocal disease, higher tumor staging, and higher American Thyroid Association risk levels. BothBRAFV600E mutations and gene fusions were correlated with the presence of cervical metastases. Conclusions Targetable alterations were identified in 75% of pediatric PTC cases with both DNA and RNA evaluated. Inclusion of RNA sequencing for detection of fusion genes is critical for evaluation of these tumors. Patients with fusion-positive tumors were more likely to have features of high-risk disease.

Abstract BACKGROUND The role of plasma cell-free DNA (cfDNA) as a cancer biomarker for tracking treatment response and detecting early relapse has been well described for solid tumors outside the central nervous system (CNS). However, the presence of a blood-brain barrier complicates the application of plasma cfDNA analysis for patients with CNS malignancies. METHODS cfDNA was extracted from plasma of pediatric patients with CNS tumors utilizing a QIAmp® MinElute® kit and quantitated with Qubit 2.0 Fluorometer. Extensive genomic testing, including targeted DNA and RNA solid tumor panels, exome and transcriptome sequencing, as well as copy number array, was performed on matched tumor samples as part of the Texas KidsCanSeq study. An Archer® Reveal ctDNA28 NGS kit was then used for assaying the sensitivity of detecting tumor-specific mutations in the plasma of these patients. RESULTS A median of 10.7ng cfDNA/mL plasma (Interquartile range: 6.4 – 15.3) was extracted from 78 patients at time of study enrollment. Longitudinal samples from 24 patients exhibited a median yield of 7.7ng cfDNA/mL plasma (IQR: 5.9 – 9.1). An initial cohort of 6 patients was identified with 7 somatic variants covered by the Archer® Reveal kit. Four of seven mutations identified in matched tumor specimens were detected in patient plasma at variant allele frequencies ranging from 0.2–1%. CONCLUSIONS While challenging, detection of cfDNA in the plasma of pediatric patients with CNS tumors is possible and is being explored in a larger patient cohort along with pilot studies investigating cerebrospinal fluid as an additional source for tumor-specific cfDNA.

A liquid drop may spread faster on surfaces when surfactants are added. Here we show that after some time the spreading in such systems can, under certain conditions, spontaneously reverse to retraction and the droplet pulls itself back, receding from areas it has just recently wetted, elevating its center of mass in a jerklike motion. The duration from drop placement to the onset of retraction ranges from hours to less than a second primarily as a function of surfactant concentration. When the retraction is asymmetric, it results in drop motion, and when it is symmetric, the mass of the drop collects itself on its spot. This phenomenon, which was predicted theoretically in 2014, is apparently a general one for drops with surfactants; however, other factors, such as evaporation and contamination, prevented its observance so far.

Background: Papillary thyroid carcinomas (PTC) comprise 90% of thyroid cancers in children. Pediatric PTC is known to be clinically and biologically different than adult PTC, with pediatric tumors more likely to present with metastases and less likely to have the typical alterations found in adult tumors, including BRAF V600E mutations and RET alterations. While the prognosis for pediatric PTC is generally good, some patients with metastatic and/or refractory disease have poorer outcomes and novel treatment approaches are needed. Given the relative lack of genomic information available for pediatric PTC, we sought to perform integrated DNA and RNA-based sequencing of a cohort of clinically annotated tumors in order to identify genetic alterations that might inform improved diagnostic and therapeutic approaches for these patients. Methods: Potential cases were identified through review of the Texas Children’s Hospital Pathology archives for the years 2005-2016. Clinical review of each patient chart was performed, with metastatic and treatment-refractory tumors prioritized for analysis. Nucleic acids (DNA/RNA) were extracted from FFPE tumor samples with sufficient tissue available. Tumor DNA was captured using a custom-designed next-generation mutation panel that includes the entire coding regions and select noncoding regions of 124 genes implicated in pediatric solid tumors. Tumor RNA was assessed for fusions using a commercially available multiplex gene fusion panel (Archer Oncology Research panel) targeting 75 pediatric solid tumor genes. Sequencing was performed on an Illumina MiSeq. After completion of DNA/RNA mutation panel testing in all available cases, more comprehensive analyses (exome sequencing, capture transcriptome sequencing) will be performed on cases in which driver alterations are not identified. Putative driver alterations will be confirmed by targeted DNA/RNA testing. Results: A total of 41 pediatric PTC cases were prioritized for analysis. Of those, 40 were from patients of age 8-18 years, with a single sample from a 23-year-old. The majority of patients were metastatic to cervical lymph nodes (31/40, 77.5%), and just over one third of those patients were found to have additional pulmonary metastases (11/31, 35.5%). The male-to-female ratio was 1:3. Relapsed or refractory disease occurred in 18 patients (18/41, 44%). Nucleic acid extractions have been completed for 29 cases. DNA sequencing has been performed for 11 cases, revealing potentially targetable mutations in 6 tumors (54%): BRAF p.V600E x 4, PIK3R1 p.Y29fs, and PTPN11 p.S502T. RNA samples have been successfully sequenced for 16 cases, identifying potentially targetable fusions in 8 tumors (50%): NTRK3 x 4 (both known and novel fusions), BRAF x 2, and RET x 2. All driver alterations detected in cases successfully subjected to both DNA and RNA sequencing to date are mutually exclusive, with a single alteration per tumor. Conclusions: Frequent alterations were identified in known cancer genes in pediatric PTC cases, many of which are currently targetable with FDA-approved or investigational agents (e.g., MAPK pathway inhibitors; PI3K pathway inhibitors, other kinase inhibitors). Use of both DNA and RNA panel sequencing targeting genes known to be altered in pediatric solid tumors has the potential to identify alterations of clinical relevance in a majority of pediatric PTC patients. Citation Format: Samara L. Potter, Jacquelyn Reuther, Ilavarasi Gandhi, Faith Hollingsworth, Hadi Sayeed, Horatiu Voicu, Nipun Kakkar, Koel Sen Baksi, Stephen F. Sarabia, Monica E. Lopez, Daniel C. Chelius, Ioanna D. Athanassaki, Rajkumar Venkatramani, Norma M. Quintanilla, Dolores Lopez-Terrada, Angshumoy Roy, Donald Williams Parsons. Integrated sequencing of pediatric thyroid carcinoma reveals frequent targetable alterations [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A36.

The uphill energy transfer in photosystems implies input energy at higher wavelength leading to energy output at lower wavelength. Briefly, energy is uphill transported from photosystem I (PSI) to photosystem II (PSII), the latter having a lower wavelength emission. This uphill energy transport involves absorption of thermal energy from the surroundings. While such cooling effects have been reported in laser systems we report for the first time a white light driven cooling in thylakoid suspension. The cooling of the surrounding medium by appropriate illumination was illustrated using thermal measurements. Again cooling is inhibited by agents like 3-(3,4-Dichlorophenyl)-1,1-dimethylurea,that block the linear electron flow between the photocenters, implying a dependence of the cooling on interplay between such centers. Furthermore, it is possible to modulate the cooling pattern by addition of external agents like nanopaticles, some favoring further cooling (e.g., Ag nanoparticle) and some like Au or chlorophyll nanoparticles, showing insignificant or even reverse trends. Interestingly, the cooling is invariably associated with the 77K spectra of the thylakoid suspension. With reference to the dark control, an agent causing cooling always increases PSII to PSI ratio and vice versa i.e.,the uphill energy transport. Importantly, the cooling effect, apart from its import role in plant physiology can be exploited artificially for energy saving in post-harvest or food preservation.

Energy distribution between photosystems (PSI & PSII) under prolonged and continuous white light irradiance was assessed by monitoring the progress of their fluorescence emission (FPSI/FPSII) at 77 K. Our observations indicate FPSI/FPSII to oscillate with the progress of irradiance treatments at all intensities tested (100, 200, 500, and 800 μE m−2 S−1). The amplitude of the oscillation increased with the progress, whereas the periodicity of the oscillation increased with the intensity of the incident irradiance. Spectral analysis indicated fluctuation of FPSI to be the major determinant of the observed oscillation. The first rise and fall of FPSI/FPSII overlapped with phosphorylation and dephosphorylation of LHCII, but oscillation of FPSI/FPSII continued for several cycles without any further phosphorylation of LHCII. Moreover, in presence of DCMU where linear electron flow (LEF) is suppressed and LHCII phosphorylation is completely abolished, the oscillation of FPSI/FPSII was not abolished. These data indicated that LHCII phosphorylation was not essential for the observed oscillation of energy distribution between the photosystems. In contrast, in the presence of inhibitors of cyclic electron flow (CEF) like Antimycin A (AA) and rotenone, the oscillation of FPSI/FPSII was either abolished or severely dampened. Additionally, the oscillation was also abolished in presence of uncouplers like NH4Cl and nigericin that cancels the trans-thylakoid ∆pH. Thus, trans-thylakoid ∆pH, generated through CEF, appear to be an important determinant of oscillation of FPSI/FPSII in isolated thylakoids. The phenomenon of oscillation could be associated with a CEF mediated chromatic adaptation of PSI in presence of excess irradiance.