Effect of mutation order on myeloproliferative neoplasms.

BACKGROUND: Cancers result from the accumulation of somatic mutations, and their properties are thought to reflect the sum of these mutations. However, little is known about the effect of the order in which mutations are acquired. METHODS: We determined mutation order in patients with myeloproliferative neoplasms by genotyping hematopoietic colonies or by means of next-generation sequencing. Stem cells and progenitor cells were isolated to study the effect of mutation order on mature and immature hematopoietic cells. RESULTS: The age at which a patient presented with a myeloproliferative neoplasm, acquisition of JAK2 V617F homozygosity, and the balance of immature progenitors were all influenced by mutation order. As compared with patients in whom the TET2 mutation was acquired first (hereafter referred to as "TET2-first patients"), patients in whom the Janus kinase 2 (JAK2) mutation was acquired first ("JAK2-first patients") had a greater likelihood of presenting with polycythemia vera than with essential thrombocythemia, an increased risk of thrombosis, and an increased sensitivity of JAK2-mutant progenitors to ruxolitinib in vitro. Mutation order influenced the proliferative response to JAK2 V617F and the capacity of double-mutant hematopoietic cells and progenitor cells to generate colony-forming cells. Moreover, the hematopoietic stem-and-progenitor-cell compartment was dominated by TET2 single-mutant cells in TET2-first patients but by JAK2-TET2 double-mutant cells in JAK2-first patients. Prior mutation of TET2 altered the transcriptional consequences of JAK2 V617F in a cell-intrinsic manner and prevented JAK2 V617F from up-regulating genes associated with proliferation. CONCLUSIONS: The order in which JAK2 and TET2 mutations were acquired influenced clinical features, the response to targeted therapy, the biology of stem and progenitor cells, and clonal evolution in patients with myeloproliferative neoplasms. (Funded by Leukemia and Lymphoma Research and others.).

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis.

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

The developing role of receptors and adaptors.

The response of a cell to the myriad of signals that it receives is varied, and it is dependent on many different factors. The most-studied responses involve growth-factor signalling and these signalling cascades have become key targets for cancer therapy. Recent reports have indicated that growth-factor receptors and associated adaptors can accumulate in the nucleus. Are there novel functions for these proteins that might affect our understanding of their role in cancer and have implications for drug resistance?

Hypersynchrony in sarcomeric hypertrophic cardiomyopathy: description and mechanistic approach using multimodal electro-mechanical non-invasive cartography (HSYNC study).

Background: Little is known about left ventricular (LV) sequences of contraction and electrical activation in hypertrophic cardiomyopathy (HCM). A better understanding of the underlying relation between mechanical and electrical activation may allow the identification of predictive response criteria to right ventricular DDD pacing in obstructive patients. Objective: To describe LV mechanical and electrical activation sequences in HCM patients compared to controls. Materials and methods: We prospectively studied, in 40 HCM patients (20 obstructive and 20 non-obstructive) and 20 healthy controls: (1) mechanical activation using echocardiography at rest and cardiac magnetic resonance imaging, (2) electrical activation using 3-dimensional electrocardiographic mapping (ECM). Results: In echocardiography, healthy controls had a physiological apex-to-base delay (ABD) during contraction (23.8 ± 16.2 ms). Among the 40 HCM patients, 18 HCM patients presented a loss of this ABD (<10 ms, defining hypersynchrony) more frequently than controls (45% vs. 5%, p = 0.017). These patients had a lower LV end-diastolic volume (71.4 ± 9.7 ml/m2 vs. 82.4 ± 14.8 ml/m2, p = 0.01), lower native T1 values (988 ± 32 ms vs. 1,028 ± 39 ms, p = 0.001) and tended to have lower LV mass (80.7 ± 23.7 g/m2 vs. 94.5 ± 25.3 g/m2, p = 0.08) compared with HCM patients that had a physiological contraction sequence. There was no significant relation between ABD and LV outflow tract obstruction. While HCM patients with a physiological contraction sequence presented an ECM close to those encountered in controls, patients with a loss of ABD presented a particular pattern of ECM with the first potential more frequently occurring in the postero-basal region. Conclusion: The LV contraction sequence can be modified in HCM patients, with a loss of the physiological ABD, and is associated with smaller LV dimensions and a particular pattern of ECM. Further research is needed to determine whether this pattern is related to an electrical substrate or is the consequence of the hypertrophied heart's specific geometry. Clinical trial registration: ClinicalTrial.gov: NCT02559726.

Antithrombotic Management and Outcomes of Anterior ST-Elevation Myocardial Infarction With New-Onset Wall Motion Abnormalities in Men and Women.

Background: In patients with anterior ST-elevation myocardial infarction (STEMI) and new-onset antero-apical wall motion abnormalities (WMAs), whether the rate of prophylaxis against left ventricular thrombus and outcomes differ between men and women is unknown. Methods: A multicentre retrospective cohort study of patients with STEMI and new-onset antero-apical WMAs treated with primary percutaneous coronary intervention was conducted. Patients with an established indication of oral anticoagulation (OAC) were excluded. The rates of triple therapy (double antiplatelet therapy + OAC) at discharge were compared for women vs men. The rates of net adverse clinical events, a composite of mortality, myocardial infarction, stroke or transient ischemic attack, systemic thromboembolism or Bleeding Academic Research Consortium (BARC) type 3 or 5 bleeding at 6 months were compared across sex using a multivariate logistic regression model. Results: A total of 1664 patients were included in the primary analysis, of whom 402 (24.2%) were women and 1262 (75.8%) were men. A total of 138 women (34.3%) and 489 men (38.7%) received a triple therapy prescription at discharge (P = 0.11). At 6 months, 33 women (8.2%) and 96 men (7.6%) experienced a net adverse clinical event (adjusted odds ratio 0.82; 95% confidence interval 0.49-1.37). No difference occurred in the risk of bleeding events and ischemic events between men and women, when these were analyzed separately. Conclusions: The rates of OAC prescription for left ventricular thrombus prophylaxis and clinical outcomes at 6 months were similar in women and men following anterior STEMI with new-onset antero-apical WMAs.

Contexte: On ignore si le taux de prophylaxie contre le thrombus ventriculaire gauche et les résultats thérapeutiques diffèrent entre les hommes et les femmes qui ont subi un infarctus du myocarde avec élévation du segment ST (STEMI) antérieur et ont des anomalies du mouvement pariétal (AMP) antéroapical d'apparition récente. Méthodes: Nous avons mené une étude de cohorte rétrospective multicentrique auprès de patients qui ont subi un STEMI et ont des AMP d'apparition récente traitées par une intervention coronarienne percutanée primaire. Nous avons exclu les patients chez lesquels il existait une indication établie à l'anticoagulation orale (ACO). Nous avons comparé les taux de trithérapie (bithérapie antiplaquettaire + ACO) à la sortie de l'hôpital entre les femmes et les hommes. Nous avons comparé les taux d'événements indésirables cliniques nets, le critère composite de mortalité, d'infarctus du myocarde, d'accident vasculaire cérébral ou d'accident ischémique transitoire, la thromboembolie systémique ou l'hémorragie de type 3 ou 5 selon le Bleeding Academic Research Consortium (BARC) après 6 mois entre les sexes au moyen du modèle de régression logistique multivariée. Résultats: Au sein des 1 664 patients de l'analyse principale, 402 (24,2 %) étaient des femmes et 1262 (75,8 %) étaient des hommes. Un total de 138 femmes (34,3 %) et de 489 hommes (38,7 %) ont reçu une ordonnance de trithérapie à la sortie de l'hôpital (P = 0,11). Après 6 mois, 33 femmes (8,2 %) et 96 hommes (7,6 %) ont subi un événement indésirable net (rapport de cotes ajusté 0,82 ; intervalle de confiance à 95 % 0,49-1,37). Aucune différence n'a été notée dans le risque d'événements hémorragiques et d'événements ischémiques entre les hommes et les femmes lorsque ces événements étaient analysés séparément. Conclusions: Les taux d'ordonnances d'ACO en prophylaxie du thrombus ventriculaire gauche et les résultats cliniques après 6 mois étaient similaires entre les femmes et les hommes à la suite du STEMI antérieur et des AMP antéroapicale d'apparition récente.

Long-Term Arrhythmic Follow-Up and Risk Stratification of Patients With Desmoplakin-Associated Arrhythmogenic Right Ventricular Cardiomyopathy.

Background: Patients with likely pathogenic/pathogenic desmoplakin (DSP) variants are poorly characterized. Some of them meet diagnostic criteria for arrhythmogenic right ventricular cardiomyopathy (ARVC), but it is unclear how risk stratification strategies for ARVC perform in this setting. Objectives: The purpose of this study was to characterize arrhythmic outcomes and to test the performance of the recently validated ARVC risk calculator in patients with DSP likely pathogenic/pathogenic variants fulfilling definite 2010 ARVC Task Force Criteria (DSP-TFC+). Methods: DSP-TFC+ patients were enrolled from 20 institutions across 3 continents. Ventricular arrhythmias (VA), defined as a composite of sustained ventricular tachycardia (VT), appropriate implantable cardioverter defibrillator therapies, and ventricular fibrillation/sudden cardiac death events in follow-up, were reported as the primary outcome. We tested the performance of the ARVC risk calculator for VA prediction, reporting c-statistics. Results: Among 252 DSP-TFC+ patients (age 39.6 ± 16.9 years, 35.3% male), 94 (37.3%) experienced VA over 44.5 [IQR: 19.6-78.3] months. Patients with left ventricle involvement (n = 194) were at higher VA risk (log-rank P = 0.0239). History of nonsustained VT (aHR 2.097; P = 0.004) showed the strongest association with VA occurrence during the first 5-year follow-up. Neither age (P = 0.723) nor male sex (P = 0.200) was associated with VAs at follow-up. In 204 patients without VA at diagnosis, incident VA rate was high (32.8%; 7.37%/y). The ARVC risk calculator performed poorly overall (c-statistic 0.604 [0.594-0.614]) and very poorly in patients with left ventricular disease (c-statistic 0.558 [0.556-0.560]). Conclusions: DSP-TFC+ patients are at substantial risk for VAs. The ARVC risk calculator performs poorly in DSP-TFC+ patients suggesting need for a gene-specific risk algorithm. Meanwhile, DSP-TFC+ patients with nonsustained VT should be considered as high-risk.

OPCML at 11q25 is epigenetically inactivated and has tumor-suppressor function in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC), the leading cause of death from gynecological malignancy, is a poorly understood disease. The typically advanced presentation of EOC with loco-regional dissemination in the peritoneal cavity and the rare incidence of visceral metastases are hallmarks of the disease. These features relate to the biology of the disease, which is a principal determinant of outcome. EOC arises as a result of genetic alterations sustained by the ovarian surface epithelium (OSE; ref. 3). The causes of these changes are unknown but are manifest by activation of oncogenes and inactivation of tumor-suppressor genes (TSGs). Our analysis of loss of heterozygosity at 11q25 identified OPCML (also called OBCAM), a member of the IgLON family of immunoglobulin (Ig) domain-containing glycosylphosphatidylinositol (GPI)-anchored cell adhesion molecules, as a candidate TSG in EOC. OPCML is frequently somatically inactivated in EOC by allele loss and by CpG island methylation. OPCML has functional characteristics consistent with TSG properties both in vitro and in vivo. A somatic missense mutation from an individual with EOC shows clear evidence of loss of function. These findings suggest that OPCML is an excellent candidate for the 11q25 ovarian cancer TSG. This is the first description to our knowledge of the involvement of the IgLON family in cancer.

Automated and semi-automated 3D echocardiographic software for aortic annulus sizing in transcatheter aortic valve implantation helps bridge the gap between expert and novice operators.

3D-transesophageal echocardiography (3D-TEE) is an alternative to multidetector row computed tomography (MDCT) for aortic annulus (AoA) sizing in preparation for Transcatheter aortic valve implantation (TAVI). We aim to evaluate how the fully automated (auto) and semi-automated (SA) TEE methods perform compared to conventional manual TEE method and the gold standard MDCT for annulus sizing both in expert and novice operators. In this prospective cohort study, eighty-nine patients with severe aortic stenosis underwent multimodality imaging with 3D-TEE and MDCT. Annular measurements were collected by expert echocardiographers using 3D auto, SA and manual methods and compared to MDCT. A novice in the field of echocardiography retrospectively measured the AoA for all patients using the same methods. TEE measurements, independently of the method used, had good to very good agreement to MDCT. They significantly underestimated aortic annular area and circumference vs. MDCT with the auto method underestimating it the most and the manual method the least (6.5% and 1.3% respectively for area and circumference). For experts, the manual TEE method offered the least systematic bias while the SA method had narrower limits of agreement (LOA). For the novice operator, SA method provided the least bias and narrower LOA vs. MDCT. There is good agreement between novice and experts for all 3 TEE methods but better agreement with auto and SA methods as opposed to manual one. Our study supports the use of 3D-TEE as a complementary method to MDCT for aortic annular sizing. The newer auto and SA software, that requires minimal operator intervention, is an easy to use, reliable and reproducible tool for aortic annulus sizing for experienced operators, and especially less experienced ones.

Venous dysfunction in obstructive hypertrophic cardiomyopathies: Results from the "HCM-Vein" pilot study.

Background: Hypertrophic cardiomyopathies (HCM) can be complicated by left ventricular outflow-tract obstruction (LVOTO) responsible for disabling exercise symptoms, a phenomenon influenced by hemodynamic factors including venous return. Methods: We aimed to evaluate venous dysfunction in obstructive HCM patients compared to healthy controls, and to investigate the relationship between venous dysfunction parameters and LVOTO in HCM. This is a clinical, monocentric, prospective, pilot study, in a tertiary care center. We investigated venous function using venous air plethysmography, and endothelial function. Results: Among the 30 symptomatic obstructive HCM patients, 30% (n = 9) presented abnormal venous residual volume fraction (RVFv) which translates in elevated ambulatory venous pressure vs. 0% in the 10 healthy controls (p < 0.05). Comparing obstructive HCM patients with abnormal RVFv (n = 9) to other obstructive HCM patients with normal RVFv (n = 21), there were no significant differences in terms of age, sex (67% male), and classical echocardiographic parameters both at rest and during exercise, except for left ventricular end-diastolic volume index which was significantly lower in the group with abnormal RVFv compared to the other HCM patients (40.1 ± 9.0 ml/m2 vs. 50.2 ± 10.6 ml/m2, p = 0.01). Fifty six percent of obstructive HCM patients with abnormal RVFv had an absolute increase in Willebrand factor (vs. 26% of other obstructive HCM patients, p < 0.05). Conclusions: In this pilot monocentric study, venous insufficiency was observed in about 30% of symptomatic obstructive HCM patients. Patients with venous insufficiency had more frequently a smaller LV cavity volume. Due to the small sample size, this study is only hypothesis-generating, and further investigations are needed.

Contemporary incidence and predictors of left ventricular thrombus in patients with anterior acute myocardial infarction.

BACKGROUND: The incidence of left ventricular thrombus (LVT) after anterior acute myocardial infarction (AMI) has not been well established in the era of primary percutaneous coronary intervention (pPCI) and potent dual antiplatelet therapy. The objective of this study is to establish the contemporary incidence of LVT in this population, to identify their risk factors, and to examine their association with clinical outcomes. METHODS: A multicenter retrospective cohort study including AMI patients with new-onset antero-apical wall motion abnormalities treated with pPCI between 2009 and 2017 was conducted. The primary outcome was LVT during the index hospitalization. Predictors of LVT were identified using multivariate logistic regression. Net adverse clinical events (NACE), a composite of mortality, myocardial infarction, stroke or transient ischemic attack, systemic thromboembolism or BARC type 3 or 5 bleeding at 6 months were compared between the LVT and no LVT groups. RESULTS: Among the 2136 patients included, 83 (3.9%) patients developed a LVT during index hospitalization. A lower left ventricular ejection fraction (LVEF) [adjusted odds ratio (aOR) 0.97; 95% confidence intervals (CI) 0.94-0.99] and the degree of worse anterior WMA (aOR 4.34; 95% CI 2.24-8.40) were independent predictors of LVT. A NACE occurred in 5 (5.72 per 100 patient-year) patients in the LVT group and in 127 (6.71 per 100 patient-year) patients in the no LVT group at 6 months [adjusted hazard ratio (aHR): 0.87; 95% CI 0.35-2.14]. CONCLUSIONS: The risk of LVT after anterior AMI with new-onset wall motion abnormalities is low, but this complication remains present in the contemporary era of timely pPCI and potent dual antiplatelet therapy .

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.

Oral Anticoagulation for the Prevention of Thromboembolic Events in Patients With Anterior ST-Segment Elevation Myocardial Infarction.

OBJECTIVES: The objective is to assess the comparative effectiveness and safety of dual-antiplatelet therapy (DAPT) vs triple therapy (TT) with DAPT + oral anticoagulant (OAC) in patients with anterior ST-segment elevation myocardial infarction (STEMI) and with new-onset anterior/apical wall-motion abnormalities (WMAs) treated with primary percutaneous coronary intervention (PCI). BACKGROUND: Patients with STEMI and new-onset anterior/apical WMA may benefit from the addition of OAC to prevent left ventricular thrombus and cardioembolic events. METHODS: A multicenter, retrospective cohort study was conducted. Patients with a concomitant indication for OAC were excluded. Patients discharged on TT were compared with patients discharged on DAPT using adjusted Cox proportional hazards analysis and inverse probability of treatment weighting. The primary endpoint was the net adverse clinical event (NACE) rate at 6 months (composite of all-cause mortality, non-fatal MI, stroke, or transient ischemic attack, systemic thromboembolism or type 3 or 5 Bleeding Academic Research Consortium [BARC] bleeding). RESULTS: A total of 1666 patients were included, among which 627 were treated with TT and 1039 were treated with DAPT. A NACE occurred in 55 patients (6.03 per 100 patient-years) in the TT group and in 74 patients (7.18 per 100 patient-years) in the DAPT group (adjusted hazard ratio, 0.86; 95% confidence interval, 0.55-1.32). Adjusted risk of the individual components of the primary endpoint, ischemic events, and bleeding events were similar between both groups (P>.05 for all). CONCLUSIONS: The addition of OAC to DAPT in anterior STEMI patients with new-onset WMA treated with PCI was not associated with a significant reduction in NACE.

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.

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.

Chromatin immunoprecipitation (ChIP) methodology and readouts.

The identification of direct nuclear hormone receptor gene targets provides clues to their contribution to both development and cancer progression. Until recently, the identification of such direct target genes has relied on a combination of expression analysis and in silico searches for consensus binding motifs in gene promoters. Consensus binding motifs for transcription factors are often defined using in vitro DNA binding strategies. Such in vitro strategies fail to account for the many factors that contribute significantly to target selection by transcription factors in cells beyond the recognition of these short consensus DNA sequences. These factors include DNA methylation, chromatin structure, posttranslational modifications of transcription factors, and the cooperative recruitment of transcription factor complexes. Chromatin immunoprecipitation (ChIP) provides a means of isolating transcription factor complexes in the context of endogenous chromatin, allowing the identification of direct transcription factor targets. ChIP can be combined with site-specific PCR for candidate binding sites or alternatively with cloning, genomic microarrays or more recently direct high throughput sequencing to identify novel genomic targets. The application of ChIP-based approaches has redefined consensus binding motifs for transcription factors and provided important insights into transcription factor biology.

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.