A proof-of-concept study of sequential treatment with the HDAC inhibitor vorinostat following BRAF and MEK inhibitors in BRAFV600mutated melanoma.

PURPOSE: Development of resistance limits the clinical benefit of BRAF and MEK inhibitors (BRAFi/MEKi) in BRAFV600 mutated melanoma. It has been shown that short-term treatment (14 days) with vorinostat was able to initiate apoptosis of the resistant tumor cells. We aimed to assess the anti-tumor activity of sequential treatment with vorinostat following BRAFi/MEKi in patients with BRAFV600 melanoma who progressed after initial response to BRAFi/MEKi. PATIENTS AND METHODS: Patients with BRAFi/MEKi resistant BRAFV600 melanoma were treated with vorinostat 360 mg QD for 14 days followed by BRAFi/MEKi. The primary endpoint was an objective response rate of progressive lesions of at least 30% according to RECIST 1.1. Secondary endpoints included progression-free survival (PFS), overall survival (OS), safety, pharmacokinetics of vorinostat and translational molecular analyses using ctDNA and tumor biopsies. RESULTS: Twenty-six patients with progressive BRAFi/MEKi resistant BRAFV600 mutated melanoma received treatment with vorinostat. Twenty-two patients were evaluable for response. The ORR was 9% (one complete response for 31.2 months and one partial response for 14.9 months. Median PFS and OS were 1.4 and 5.4 months, respectively. Common adverse events were fatigue (23%) and nausea (19%). ctDNA analysis showed emerging secondary mutations in NRAS and MEK in eight patients at time of BRAFi/MEKi resistance. Elimination of these mutations by vorinostat treatment was observed in three patients. CONCLUSIONS: Intermittent treatment with vorinostat in patients with resistant BRAFV600mutated melanoma is well tolerated. Although the primary endpoint of this study was not met, durable anti-tumor responses were observed in a minority of patients (9%).

Comparison of tumor-informed and tumor-naïve sequencing assays for ctDNA detection in breast cancer.

Analysis of circulating tumor DNA (ctDNA) to monitor cancer dynamics and detect minimal residual disease has been an area of increasing interest. Multiple methods have been proposed but few studies have compared the performance of different approaches. Here, we compare detection of ctDNA in serial plasma samples from patients with breast cancer using different tumor-informed and tumor-naïve assays designed to detect structural variants (SVs), single nucleotide variants (SNVs), and/or somatic copy-number aberrations, by multiplex PCR, hybrid capture, and different depths of whole-genome sequencing. Our results demonstrate that the ctDNA dynamics and allele fractions (AFs) were highly concordant when analyzing the same patient samples using different assays. Tumor-informed assays showed the highest sensitivity for detection of ctDNA at low concentrations. Hybrid capture sequencing targeting between 1,347 and 7,491 tumor-identified mutations at high depth was the most sensitive assay, detecting ctDNA down to an AF of 0.00024% (2.4 parts per million, ppm). Multiplex PCR targeting 21-47 tumor-identified SVs per patient detected ctDNA down to 0.00047% AF (4.7 ppm) and has potential as a clinical assay.

Accurate detection of benign and malignant renal tumor subtypes with MethylBoostER: An epigenetic marker-driven learning framework.

Current gold standard diagnostic strategies are unable to accurately differentiate malignant from benign small renal masses preoperatively; consequently, 20% of patients undergo unnecessary surgery. Devising a more confident presurgical diagnosis is key to improving treatment decision-making. We therefore developed MethylBoostER, a machine learning model leveraging DNA methylation data from 1228 tissue samples, to classify pathological subtypes of renal tumors (benign oncocytoma, clear cell, papillary, and chromophobe RCC) and normal kidney. The prediction accuracy in the testing set was 0.960, with class-wise ROC AUCs >0.988 for all classes. External validation was performed on >500 samples from four independent datasets, achieving AUCs >0.89 for all classes and average accuracies of 0.824, 0.703, 0.875, and 0.894 for the four datasets. Furthermore, consistent classification of multiregion samples (N = 185) from the same patient demonstrates that methylation heterogeneity does not limit model applicability. Following further clinical studies, MethylBoostER could facilitate a more confident presurgical diagnosis to guide treatment decision-making in the future.

A Phase II study of neoadjuvant axitinib for reducing the extent of venous tumour thrombus in clear cell renal cell cancer with venous invasion (NAXIVA).

BACKGROUND: Surgery for renal cell carcinoma (RCC) with venous tumour thrombus (VTT) extension into the renal vein (RV) and/or inferior vena cava (IVC) has high peri-surgical morbidity/mortality. NAXIVA assessed the response of VTT to axitinib, a potent tyrosine kinase inhibitor. METHODS: NAXIVA was a single-arm, multi-centre, Phase 2 study. In total, 20 patients with resectable clear cell RCC and VTT received upto 8 weeks of pre-surgical axitinib. The primary endpoint was percentage of evaluable patients with VTT improvement by Mayo level on MRI. Secondary endpoints were percentage change in surgical approach and VTT length, response rate (RECISTv1.1) and surgical morbidity. RESULTS: In all, 35% (7/20) patients with VTT had a reduction in Mayo level with axitinib: 37.5% (6/16) with IVC VTT and 25% (1/4) with RV-only VTT. No patients had an increase in Mayo level. In total, 75% (15/20) of patients had a reduction in VTT length. Overall, 41.2% (7/17) of patients who underwent surgery had less invasive surgery than originally planned. Non-responders exhibited lower baseline microvessel density (CD31), higher Ki67 and exhausted or regulatory T-cell phenotype. CONCLUSIONS: NAXIVA provides the first Level II evidence that axitinib downstages VTT in a significant proportion of patients leading to reduction in the extent of surgery. CLINICAL TRIAL REGISTRATION: NCT03494816.

Refined characterization of circulating tumor DNA through biological feature integration.

Circulating tumor DNA (ctDNA) in blood plasma is present at very low concentrations compared to cell-free DNA (cfDNA) of non-tumor origin. To enhance ctDNA detection, recent studies have been focused on understanding the non-random fragmentation pattern of cfDNA. These studies have investigated fragment sizes, genomic position of fragment end points, and fragment end motifs. Although these features have been described and shown to be aberrant in cancer patients, there is a lack of understanding of how the individual and integrated analysis of these features enrich ctDNA fraction and enhance ctDNA detection. Using whole genome sequencing and copy number analysis of plasma samples from 5 high grade serious ovarian cancer patients, we observed that (1) ctDNA is enriched not only in fragments shorter than mono-nucleosomes (~ 167 bp), but also in those shorter than di-nucleosomes (~ 240-330 bp) (28-159% enrichment). (2) fragments that start and end at the border or within the nucleosome core are enriched in ctDNA (5-46% enrichment). (3) certain DNA motifs conserved in regions 10 bp up- and down- stream of fragment ends (i.e. cleavage sites) could be used to detect tumor-derived fragments (10-44% enrichment). We further show that the integrated analysis of these three features resulted in a higher enrichment of ctDNA when compared to using fragment size alone (additional 7-25% enrichment after fragment size selection). We believe these genome wide features, which are independent of genetic mutational changes, could allow new ways to analyze and interpret cfDNA data, as significant aberrations of these features from a healthy state could improve its utility as a diagnostic biomarker.

Reloadable Stapler Use during Peripartum Hysterectomy for Placenta Accreta Spectrum: A Novel Surgical Technique and Case Series.

OBJECTIVE: This study aimed to describe a novel surgical technique for the management of antenatally suspected placenta accreta spectrum (PAS). STUDY DESIGN: This is a retrospective, case series of patients with suspected PAS undergoing peripartum hysterectomy with a reloadable articulating stapler at a tertiary care center. RESULTS: Eighteen patients with antenatally suspected PAS were identified and underwent peripartum hysterectomy with the aid of a reloadable stapler. Mean gestational age at delivery was 344/7 ± 11/7 weeks. Mean total operative time (skin-to-skin) was 117.3 ± 39.3 minutes, and 79.8 ± 19.8 minutes for the hysterectomy. Mean blood loss for the entire case was 1,809 ± 868 mL. Mean blood loss for the hysterectomy was 431 ± 421 mL. Mean units of intraoperative red blood cells transfused was 3 ± 1 units. Mean units of postoperative red blood cells transfused was 1 ± 0.5 units. Five cases were complicated by urological injury (two intentional cystotomies). Four patients were admitted to the intensive care unit (ICU) for a mean of ≤24 hours. Mean postoperative LOS was 4.11 ± 1.45 days. Three patients had final pathology that did not demonstrate PAS while four were consistent with accreta, six increta, and five percreta. CONCLUSION: Use of a reloadable articulating stapler device as part of the surgical management of antenatally suspected PAS results in a shorter operative time (117 ± 39 minutes vs. 140-254 minutes previously reported), lower average blood loss (1,809 ± 868 mL vs. 2,500-5,000 mL previously reported) and shorter LOS (4.11 ± 1.45 days vs. 9.8 ± 13.5 days previously reported) compared with traditional cesarean hysterectomy. The reloadable stapling device offers an advantage of more rapidly achieving hemostasis in the surgical management of PAS. KEY POINTS: · PAS is associated with severe maternal morbidity.. · Decreased operative time and blood loss have many clinical benefits.. · Reloadable stapler use for PAS decreases operative time.. · Reloadable stapler use for PAS decreases operative blood loss..

Characteristics, origin, and potential for cancer diagnostics of ultrashort plasma cell-free DNA.

Current evidence suggests that plasma cell-free DNA (cfDNA) is fragmented around a mode of 166 bp. Data supporting this view has been mainly acquired through the analysis of double-stranded cfDNA. The characteristics and diagnostic potential of single-stranded and damaged double-stranded cfDNA in healthy individuals and cancer patients remain unclear. Here, through a combination of high-affinity magnetic bead-based DNA extraction and single-stranded DNA sequencing library preparation (MB-ssDNA), we report the discovery of a large proportion of cfDNA fragments centered at ∼50 bp. We show that these "ultrashort" cfDNA fragments have a greater relative abundance in plasma of healthy individuals (median = 19.1% of all sequenced cfDNA fragments, n = 28) than in plasma of patients with cancer (median = 14.2%, n = 21, P < 0.0001). The ultrashort cfDNA fragments map to accessible chromatin regions of blood cells, particularly in promoter regions with the potential to adopt G-quadruplex (G4) DNA secondary structures. G4-positive promoter chromatin accessibility is significantly enriched in ultrashort plasma cfDNA fragments from healthy individuals relative to patients with cancers (P < 0.0001), in whom G4-cfDNA enrichment is inversely associated with copy number aberration-inferred tumor fractions. Our findings redraw the landscape of cfDNA fragmentation by identifying and characterizing a novel population of ultrashort plasma cfDNA fragments. Sequencing of MB-ssDNA libraries could facilitate the characterization of gene regulatory regions and DNA secondary structures via liquid biopsy. Our data underline the diagnostic potential of ultrashort cfDNA through classification for cancer patients.

Fragmentation patterns and personalized sequencing of cell-free DNA in urine and plasma of glioma patients.

Glioma-derived cell-free DNA (cfDNA) is challenging to detect using liquid biopsy because quantities in body fluids are low. We determined the glioma-derived DNA fraction in cerebrospinal fluid (CSF), plasma, and urine samples from patients using sequencing of personalized capture panels guided by analysis of matched tumor biopsies. By sequencing cfDNA across thousands of mutations, identified individually in each patient's tumor, we detected tumor-derived DNA in the majority of CSF (7/8), plasma (10/12), and urine samples (10/16), with a median tumor fraction of 6.4 × 10-3 , 3.1 × 10-5 , and 4.7 × 10-5 , respectively. We identified a shift in the size distribution of tumor-derived cfDNA fragments in these body fluids. We further analyzed cfDNA fragment sizes using whole-genome sequencing, in urine samples from 35 glioma patients, 27 individuals with non-malignant brain disorders, and 26 healthy individuals. cfDNA in urine of glioma patients was significantly more fragmented compared to urine from patients with non-malignant brain disorders (P = 1.7 × 10-2 ) and healthy individuals (P = 5.2 × 10-9 ). Machine learning models integrating fragment length could differentiate urine samples from glioma patients (AUC = 0.80-0.91) suggesting possibilities for truly non-invasive cancer detection.

Expression and pharmacological inhibition of TrkB and EGFR in glioblastoma.

A member of the Trk family of neurotrophin receptors, tropomyosin receptor kinase B (TrkB, encoded by the NTRK2 gene) is an increasingly important target in various cancer types, including glioblastoma (GBM). EGFR is among the most frequently altered oncogenes in GBM, and EGFR inhibition has been tested as an experimental therapy. Functional interactions between EGFR and TrkB have been demonstrated. In the present study, we investigated the role of TrkB and EGFR, and their interactions, in GBM. Analyses of NTRK2 and EGFR gene expression from The Cancer Genome Atlas (TCGA) datasets showed an increase in NTRK2 expression in the proneural subtype of GBM, and a strong correlation between NTRK2 and EGFR expression in glioma CpG island methylator phenotype (G-CIMP+) samples. We showed that when TrkB and EGFR inhibitors were combined, the inhibitory effect on A172 human GBM cells was more pronounced than when either inhibitor was given alone. When U87MG GBM cells were xenografted into the flank of nude mice, tumor growth was delayed by treatment with TrkB and EGFR inhibitors, given alone or combined, only at specific time points. Intracranial GBM growth in mice was not significantly affected by drug treatments. Our findings indicate that correlations between NTRK2 and EGFR expression occur in specific GBM subgroups. Also, our results using cultured cells suggest for the first time the potential of combining TrkB and EGFR inhibition for the treatment of GBM.

ctDNA monitoring using patient-specific sequencing and integration of variant reads.

Circulating tumor-derived DNA (ctDNA) can be used to monitor cancer dynamics noninvasively. Detection of ctDNA can be challenging in patients with low-volume or residual disease, where plasma contains very few tumor-derived DNA fragments. We show that sensitivity for ctDNA detection in plasma can be improved by analyzing hundreds to thousands of mutations that are first identified by tumor genotyping. We describe the INtegration of VAriant Reads (INVAR) pipeline, which combines custom error-suppression methods and signal-enrichment approaches based on biological features of ctDNA. With this approach, the detection limit in each sample can be estimated independently based on the number of informative reads sequenced across multiple patient-specific loci. We applied INVAR to custom hybrid-capture sequencing data from 176 plasma samples from 105 patients with melanoma, lung, renal, glioma, and breast cancer across both early and advanced disease. By integrating signal across a median of >105 informative reads, ctDNA was routinely quantified to 1 mutant molecule per 100,000, and in some cases with high tumor mutation burden and/or plasma input material, to parts per million. This resulted in median area under the curve (AUC) values of 0.98 in advanced cancers and 0.80 in early-stage and challenging settings for ctDNA detection. We generalized this method to whole-exome and whole-genome sequencing, showing that INVAR may be applied without requiring personalized sequencing panels so long as a tumor mutation list is available. As tumor sequencing becomes increasingly performed, such methods for personalized cancer monitoring may enhance the sensitivity of cancer liquid biopsies.

Detection of ctDNA from Dried Blood Spots after DNA Size Selection.

BACKGROUND: Recent advances in the study and clinical applications of circulating tumor DNA (ctDNA) are limited by practical considerations of sample collection. Whole-genome sequencing (WGS) is increasingly used for analysis of ctDNA, identifying copy-number alterations and fragmentation patterns. We hypothesized that low-depth/shallow WGS (sWGS) data may be generated from minute amounts of cell-free DNA, and that fragment-size selection may remove contaminating genomic DNA from small blood volumes. Dried blood spots have practical advantages for sample collection, may facilitate serial sampling, and could support novel study designs in humans and animal models. METHODS: We developed a protocol for the isolation and analysis of cell-free DNA from dried blood spots using filter paper cards and bead-based size selection. DNA extracted and size-selected from dried spots was analyzed using sWGS and polymerase chain reaction (PCR). RESULTS: Analyzing a 50 µL dried blood spot from frozen whole blood of a patient with melanoma, we identified ctDNA based on the presence of tumor-specific somatic copy-number alterations, and found a fragment-size profile similar to that observed in plasma DNA. We found alterations in different chromosomes in blood spots from 2 patients with high-grade serous ovarian carcinoma. Extending this approach to serial dried blood spots from mouse xenograft models, we detect tumor-derived cell-free DNA and identified ctDNA from the originally grafted ascites. CONCLUSION: Our data suggest that ctDNA can be detected and monitored in dried blood spots from archived and fresh blood samples, enabling new approaches for sample collection and novel study/trial designs for both patients and in vivo models.

Comprehensive characterization of cell-free tumor DNA in plasma and urine of patients with renal tumors.

BACKGROUND: Cell-free tumor-derived DNA (ctDNA) allows non-invasive monitoring of cancers, but its utility in renal cell cancer (RCC) has not been established. METHODS: Here, a combination of untargeted and targeted sequencing methods, applied to two independent cohorts of patients (n = 91) with various renal tumor subtypes, were used to determine ctDNA content in plasma and urine. RESULTS: Our data revealed lower plasma ctDNA levels in RCC relative to other cancers of similar size and stage, with untargeted detection in 27.5% of patients from both cohorts. A sensitive personalized approach, applied to plasma and urine from select patients (n = 22) improved detection to ~ 50%, including in patients with early-stage disease and even benign lesions. Detection in plasma, but not urine, was more frequent amongst patients with larger tumors and in those patients with venous tumor thrombus. With data from one extensively characterized patient, we observed that plasma and, for the first time, urine ctDNA may better represent tumor heterogeneity than a single tissue biopsy. Furthermore, in a subset of patients (n = 16), longitudinal sampling revealed that ctDNA can track disease course and may pre-empt radiological identification of minimal residual disease or disease progression on systemic therapy. Additional datasets will be required to validate these findings. CONCLUSIONS: These data highlight RCC as a ctDNA-low malignancy. The biological reasons for this are yet to be determined. Nonetheless, our findings indicate potential clinical utility in the management of patients with renal tumors, provided improvement in isolation and detection approaches.

Clinical Factors Associated with Longer Hospital Stay Following Ovarian Cancer Surgery.

Background: Ovarian cancer (OC) is the leading cause of death from gynecologic malignancy and is treated with a combination of cytoreductive surgery and platinum-based chemotherapy. Extended length of stay (LOS) after surgery can affect patient morbidity, overall costs, and hospital resource utilization. The primary objective of this study was to identify factors contributing to prolonged LOS for women undergoing surgery for ovarian cancer. Methods: The American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database was queried to identify women from 2012-2016 who underwent hysterectomy for ovarian, fallopian tube and peritoneal cancer. The primary outcome was LOS >50th percentile. Preoperative and intraoperative variables were examined to determine which were associated with prolonged LOS. Results: From 2012-2016, 1771 women underwent elective abdominal surgery for OC and were entered in the ACS-NSQIP database. The mean and median LOS was 4.6 and 4.0 days (IQR 0-38), respectively. On multivariate analysis, factors associated with prolonged LOS included: American Society of Anesthesiologists (ASA) Classification III (aOR 1.71, 95% CI 1.38-2.13) or IV (aOR 1.88, 95% CI 1.44-2.46), presence of ascites (aOR 1.88, 95% CI 1.44-2.46), older age (aOR 1.23, 95% CI 1.13-1.35), platelet count >400,000/mm3 (aOR 1.74, 95% CI 1.29-2.35), preoperative blood transfusion (aOR 11.00, 95% CI 1.28-94.77), disseminated cancer (aOR 1.28, 95% CI 1.03-1.60), increased length of operation (121-180 min, aOR 1.47, 95% CI 1.13-1.91; >180 min, aOR 2.78, 95% CI 2.13-3.64), and postoperative blood transfusion within 72 h of incision (aOR 2.04, 95% CI 1.59-2.62) (p < 0.05 for all). Conclusions: Longer length of hospital stay following surgery for OC is associated with many patient, disease, and treatment-related factors. The extent of surgery, as evidenced by perioperative blood transfusion and length of surgical procedure, is a factor that can potentially be modified to shorten LOS, improve patient outcomes, and reduce hospital costs.

Temperature mediates secondary dormancy in resting cysts of Pyrodinium bahamense (Dinophyceae).

High-biomass blooms of the toxic dinoflagellate Pyrodinium bahamense occur most summers in Tampa Bay, Florida, USA, posing a recurring threat to ecosystem health. Like many dinoflagellates, P. bahamense forms immobile resting cysts that can be deposited on the seafloor-creating a seed bank that can retain the organism within the ecosystem and initiate future blooms when cysts germinate. In this study, we examined changes in the dormancy status of cysts collected from Tampa Bay and applied lessons from plant ecology to explore dormancy controls. Pyrodinium bahamense cysts incubated immediately after field collection displayed a seasonal pattern in dormancy and germination that matched the pattern of cell abundance in the water column. Newly deposited (surface) cysts and older (buried) cysts exhibited similar germination patterns, suggesting that a common mechanism regulates dormancy expression in new and mature cysts. Extended cool- and warm-temperature conditioning of field-collected cysts altered the cycle of dormancy compared with that of cysts in nature, with the duration of cool temperature exposure being the best predictor of when cysts emerged from dormancy. Extended warm conditioning, on the other hand, elicited a return to dormancy, or secondary dormancy, in nondormant cysts. These results directly demonstrate environmental induction of secondary dormancy in dinoflagellates-a mechanism common and thoroughly documented in higher plants with seasonal growth cycles. Our findings support the hypothesis that a seasonal cycle in cyst germination drives P. bahamense bloom periodicity in Tampa Bay and point to environmentally induced secondary dormancy as an important regulatory factor of that cycle.

Characteristics Associated with Prolonged Length of Stay after Myomectomy for Uterine Myomas.

STUDY OBJECTIVE: To identify factors contributing to prolonged hospitalization for women undergoing myomectomy for uterine myomas. PATIENTS: Women undergoing myomectomy for uterine myomas during 2014 to 2016 were identified by the Current Procedural Terminology code. DESIGN: Retrospective population-based analysis of the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database. SETTING: Data from the American College of Surgeons National Surgical Quality Improvement Project. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was length of stay longer than the median (1 day). Preoperative, intraoperative, and postoperative variables were examined to determine predictors for prolonged length of stay (LOS). Seven thousand five hundred thirty-one women underwent abdominal or laparoscopic myomectomy for uterine myomas. Nonwhite race (black: odds ratio [OR] = 2.25; 95% confidence interval [CI], 2.01-2.51; Asian: OR = 1.54; 95% CI, 1.27-1.85; other/unknown: OR = 2.82; 95% CI, 2.43-3.27), preoperative hematocrit <38% (OR = 1.38; 95% CI, 1.26-1.52), body mass index ≥30.1 kg/m2 (OR = 1.36; 95% CI, 1.21-1.53), preoperative blood transfusion (OR = 3.70; 95% CI, 2.03-6.74), perioperative blood transfusion (OR = 6.64; 95% CI, 4.76-9.27), removal of ≥5 myomas (OR = 1.47; 95% CI, 1.28-1.70), and operative time >120 minutes (121-150 minutes: OR = 1.42; 95% CI, 1.15-1.77; 151-180 minutes: OR = 1.59; 95% CI, 1.24-2.03; ≥181 minutes: OR = 1.36; 95% CI, 1.10-1.69) predicted prolonged LOS. Laparoscopy protected against prolonged LOS (OR = 0.11; 95% CI, 0.09-0.13). CONCLUSIONS: Limited potentially modifiable perioperative factors contributing to prolonged LOS for abdominal or laparoscopic myomectomy were identified and suggest areas for targeted interventions.

Measurement of Plasma Cell-Free Mitochondrial Tumor DNA Improves Detection of Glioblastoma in Patient-Derived Orthotopic Xenograft Models.

The factors responsible for the low detection rate of cell-free tumor DNA (ctDNA) in the plasma of patients with glioblastoma (GBM) are currently unknown. In this study, we measured circulating nucleic acids in patient-derived orthotopically implanted xenograft (PDOX) models of GBM (n = 64) and show that tumor size and cell proliferation, but not the integrity of the blood-brain barrier or cell death, affect the release of ctDNA in treatment-naïve GBM PDOX. Analysis of fragment length profiles by shallow genome-wide sequencing (<0.2× coverage) of host (rat) and tumor (human) circulating DNA identified a peak at 145 bp in the human DNA fragments, indicating a difference in the origin or processing of the ctDNA. The concentration of ctDNA correlated with cell death only after treatment with temozolomide and radiotherapy. Digital PCR detection of plasma tumor mitochondrial DNA (tmtDNA), an alternative to detection of nuclear ctDNA, improved plasma DNA detection rate (82% vs. 24%) and allowed detection in cerebrospinal fluid and urine. Mitochondrial mutations are prevalent across all cancers and can be detected with high sensitivity, at low cost, and without prior knowledge of tumor mutations via capture-panel sequencing. Coupled with the observation that mitochondrial copy number increases in glioma, these data suggest analyzing tmtDNA as a more sensitive method to detect and monitor tumor burden in cancer, specifically in GBM, where current methods have largely failed. SIGNIFICANCE: These findings show that detection of tumor mitochondrial DNA is more sensitive than circulating tumor DNA analysis to detect and monitor tumor burden in patient-derived orthotopic xenografts of glioblastoma.

Enhanced detection of circulating tumor DNA by fragment size analysis.

Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for noninvasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 patients with cancer using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90 and 150 bp and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90 and 150 bp improved detection of tumor DNA, with more than twofold median enrichment in >95% of cases and more than fourfold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with area under the curve (AUC) >0.99 compared to AUC <0.80 without fragmentation features. Increased identification of cfDNA from patients with glioma, renal, and pancreatic cancer was achieved with AUC > 0.91 compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cfDNA.

Detection of cell-free DNA fragmentation and copy number alterations in cerebrospinal fluid from glioma patients.

Glioma is difficult to detect or characterize using current liquid biopsy approaches. Detection of cell-free tumor DNA (cftDNA) in cerebrospinal fluid (CSF) has been proposed as an alternative to detection in plasma. We used shallow whole-genome sequencing (sWGS, at a coverage of < 0.4×) of cell-free DNA from the CSF of 13 patients with primary glioma to determine somatic copy number alterations and DNA fragmentation patterns. This allowed us to determine the presence of cftDNA in CSF without any prior knowledge of point mutations present in the tumor. We also showed that the fragmentation pattern of cell-free DNA in CSF is different from that in plasma. This low-cost screening method provides information on the tumor genome and can be used to target those patients with high levels of cftDNA for further larger-scale sequencing, such as by whole-exome and whole-genome sequencing.

Canine rhinitis caused by an uncommonly-diagnosed fungus, Scedosporium apiospermum.

A Golden Retriever cross was presented with a four week history of violent sneezing and licking at the nasal planum. Nasal mycosis was diagnosed and Aspergillus sp. presumed the causative agent, until culture, PCR and DNA sequencing showed that Scedosporium apiospermum, an uncommonly diagnosed, yet emerging, fungal pathogen, was the agent responsible. Debridement of the fungal plaques and systemic itraconazole therapy resulted in complete resolution of clinical disease. We discuss the current literature on S. apiospermum, review its clinical significance and question the validity of its 'complex' taxonomy.