Renal oncocytomas are benign tumors characterized by a marked accumulation of mitochondria. We report a combined exome, transcriptome, and metabolome analysis of these tumors. Joint analysis of the ...nuclear and mitochondrial (mtDNA) genomes reveals loss-of-function mtDNA mutations occurring at high variant allele fractions, consistent with positive selection, in genes encoding complex I as the most frequent genetic events. A subset of these tumors also exhibits chromosome 1 loss and/or cyclin D1 overexpression, suggesting they follow complex I loss. Transcriptome data revealed that many pathways previously reported to be altered in renal oncocytoma were simply differentially expressed in the tumor’s cell of origin, the distal nephron, compared with other nephron segments. Using a heuristic approach to account for cell-of-origin bias we uncovered strong expression alterations in the gamma-glutamyl cycle, including glutathione synthesis (increased GCLC) and glutathione degradation. Moreover, the most striking changes in metabolite profiling were elevations in oxidized and reduced glutathione as well as γ-glutamyl-cysteine and cysteinyl-glycine, dipeptide intermediates in glutathione biosynthesis, and recycling, respectively. Biosynthesis of glutathione appears adaptive as blockade of GCLC impairs viability in cells cultured with a complex I inhibitor. Our data suggest that loss-of-function mutations in complex I are a candidate driver event in renal oncocytoma that is followed by frequent loss of chromosome 1, cyclin D1 overexpression, and adaptive up-regulation of glutathione biosynthesis.
The diversity of clinical tumor profiling approaches (small panels to whole exomes with matched or unmatched germline analysis) may engender uncertainty about their benefits and liabilities, ...particularly in light of reported germline false positives in tumor-only profiling and use of global mutational and/or neoantigen data. The goal of this study was to determine the impact of genomic analysis strategies on error rates and data interpretation across contexts and ancestries.
We modeled common tumor profiling modalities-large (n = 300 genes), medium (n = 48 genes), and small (n = 15 genes) panels-using clinical whole exomes (WES) from 157 patients with lung or colon adenocarcinoma. We created a tumor-only analysis algorithm to assess germline false positive rates, the impact of patient ancestry on tumor-only results, and neoantigen detection.
After optimizing a germline filtering strategy, the germline false positive rate with tumor-only large panel sequencing was 14 % (144/1012 variants). For patients whose tumor-only results underwent molecular pathologist review (n = 91), 50/54 (93 %) false positives were correctly interpreted as uncertain variants. Increased germline false positives were observed in tumor-only sequencing of non-European compared with European ancestry patients (p < 0.001; Fisher's exact) when basic germline filtering approaches were used; however, the ExAC database (60,706 germline exomes) mitigated this disparity (p = 0.53). Matched and unmatched large panel mutational load correlated with WES mutational load (r(2) = 0.99 and 0.93, respectively; p < 0.001). Neoantigen load also correlated (r(2) = 0.80; p < 0.001), though WES identified a broader spectrum of neoantigens. Small panels did not predict mutational or neoantigen load.
Large tumor-only targeted panels are sufficient for most somatic variant identification and mutational load prediction if paired with expanded germline analysis strategies and molecular pathologist review. Paired germline sequencing reduced overall false positive mutation calls and WES provided the most neoantigens. Without patient-matched germline data, large germline databases are needed to minimize false positive mutation calling and mitigate ethnic disparities.
African-American men have the highest incidence of and mortality from prostate cancer. Whether a biological basis exists for this disparity remains unclear. Exome sequencing (
= 102) and targeted ...validation (
= 90) of localized primary hormone-naïve prostate cancer in African-American men identified several gene mutations not previously observed in this context, including recurrent loss-of-function mutations in
, an ETS transcriptional repressor, in 5% of cases. Analysis of existing prostate cancer cohorts revealed
deletions in 3% of primary prostate cancers and mutations or deletions in
in 3% to 5% of lethal castration-resistant prostate cancers. Knockdown of
confers increased anchorage-independent growth and generates a gene expression signature associated with oncogenic ETS activation and androgen signaling. Together, these results suggest that
is a prostate cancer tumor-suppressor gene. More generally, our findings support the application of systematic cancer genomic characterization in settings of broader ancestral diversity to enhance discovery and, eventually, therapeutic applications.
Systematic genomic sequencing of prostate cancer in African-American men revealed new insights into prostate cancer, including the identification of
as a prostate cancer gene; somatic copy-number alteration differences; and uncommon
and
alterations. This study highlights the importance of inclusion of underrepresented minorities in cancer sequencing studies.
.
Abstract
p50, the mature product of
NFKB1
, is constitutively produced from its precursor, p105. Here, we identify BARD1 as a p50-interacting factor. p50 directly associates with the BARD1 BRCT ...domains via a C-terminal phospho-serine motif. This interaction is induced by ATR and results in mono-ubiquitination of p50 by the BARD1/BRCA1 complex. During the cell cycle, p50 is mono-ubiquitinated in S phase and loss of this post-translational modification increases S phase progression and chromosomal breakage. Genome-wide studies reveal a substantial decrease in p50 chromatin enrichment in S phase and Cycln E is identified as a factor regulated by p50 during the G1 to S transition. Functionally, interaction with BARD1 promotes p50 protein stability and consistent with this, in human cancer specimens, low nuclear BARD1 protein strongly correlates with low nuclear p50. These data indicate that p50 mono-ubiquitination by BARD1/BRCA1 during the cell cycle regulates S phase progression to maintain genome integrity.
▪
Introduction. Minimal residual disease (MRD) is the most powerful prognostic factor in acute lymphoblastic leukemia (ALL). Currently, real-time quantitative PCR (RQ-PCR) is the most widely used ...molecular method for MRD assessment, rigorously standardized within the EuroMRD consortium. According to the EuroMRD guidelines (Van der Velden et al. Leukemia 2007), a non-negligible fraction of patients with very low MRD levels are classified as positive not-quantifiable (PNQ), a definition that may result problematic in the clinical practice. Digital-droplet-PCR (ddPCR) allows an absolute quantification without the need of a standard curve and has the potential to overcome some limitations of RQ-PCR. High degrees of efficiency, sensitivity and accuracy have been reported for ddPCR compared to RQ-PCR, but no established guidelines for ddPCR MRD analysis and interpretation have so far been defined and its ability to correctly evaluate very low MRD levels is still under investigation.
In the present study, we assessed MRD by ddPCR in pediatric ALL cases classified as PNQ and/or negative by RQ-PCR at days +33 and/or +78 of the AIEOP-BFM ALL 2000 trial, to evaluate the potential of ddPCR for low MRD quantification and patients' risk stratification.
Patients and Methods. A total of 211 pediatric ALL patients enrolled in the AIEOP-BFM ALL 2000 trial were included in the study. We analyzed 124 B-lineage ALL patients defined as intermediate risk (IR) who had high positive MRD at day +33 and at day +78 were either PNQ (n=45, Slow Early Responders (SER)) or negative (n=79). A case-control design was applied to 36 B- and T-lineage relapsed ALL patients (cases) who at day +33 had PNQ MRD (n=12, IR) or were negative (n=24, standard risk (SR)) and to matched controls (21 and 30 patients who did not present a relapse). ddPCR analysis was performed as previously published (Della Starza et al, BJH 174, 541-9, 2016), by using 1.5 μg and 3.0 μg DNA of the follow-up samples. In the absence of an international consensus, data have been analyzed using two alternative guidelines; results are reported according to Della Starza et al (BJH 2016).
Results. Among 45 SER patients, ddPCR performed on 1.5 μg DNA of PNQ samples at day +78 revealed that 13 were quantifiable (Q), 16 PNQ and 16 negative (NEG) . When 3.0 μg of DNA were used (41/45 samples due to material availability), 12 were Q, 19 PNQ and 10 NEG. Event-free survival (EFS) curves are shown in Fig. 1a.
Among the 79 patients with high positive MRD at day +33 but who were negative at day +78, ddPCR on 1.5 μg DNA of day +78 identified 5 as Q, 17 PNQ and 57 NEG. When 3.0 μg DNA was used (77/79 samples), 9 patients were Q, 27 PNQ and 41 NEG. EFS curves are reported in Fig. 1b.
When ddPCR was applied to 33 PNQ samples at day +33, 2 were Q, 9 PNQ and 22 NEG; when using 3.0 μg of DNA, 1 was Q, 15 were PNQ and 17 NEG. EFS curves are shown in Fig. 1c.
Lastly, ddPCR on 1.5 μg of day +33 DNA of 54 SR patients showed 5 PNQ and 49 NEG, whilst by using 3.0 μg on 53 sample, 7 were PNQ and 46 NEG.
Conclusions. Our data demonstrate that ddPCR is a very promising tool for the evaluation of MRD in ALL cases with very low or negative RQ-PCR MRD results. In particular, among SER patients most relapses occurred in cases with quantifiable ddPCR MRD at day +78, while patients with negative or PNQ MRD by ddPCR at day +78 had a better outcome. Based on these results, high-risk treatment could be offered only to ddPCR quantifiable cases. Among patients with highly positive MRD at day +33 and negative at day +78, the small number of cases with quantifiable disease by ddPCR at present does not allow to establish the impact of quantification; consistently with SER patients, the outcome was similar for patients with negative or PNQ MRD by ddPCR at day +78. Similarly, among patients with PNQ MRD by RQ-PCR at day +33, a similar outcome was observed for cases negative or PNQ by ddPCR. Lastly, in most SR patients ddPCR confirmed the negative results of RQ-PCR at day +33, associated with an extremely good kinetics of disease reduction, independently of the MRD PCR method. Overall, our data indicate that ddPCR is as sensitive as RQ-PCR and can provide a potentially more accurate prognostic stratification for cases defined as PNQ MRD by RQ-PCR, in view of its ability to quantify without a standard curve. The application of ddPCR in a prospective clinical protocol with international guidelines is needed to define whether it can result in an overall improvement of pediatric ALL patients' stratification and outcome.
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Foà:AMGEN: Other: ADVISORY BOARD; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD; INCYTE: Other: ADVISORY BOARD; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; NOVARTIS: Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; GILEAD: Speakers Bureau.
Minimal residual disease (MRD) is the most powerful prognostic factor in pediatric acute lymphoblastic leukemia (ALL). Real‐time quantitative polymerase chain reaction (RQ‐PCR) represents the gold ...standard for molecular MRD assessment and risk‐based stratification of front‐line treatment. In the protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP) and the Berlin‐Frankfurth‐Munschen (BFM) group AIEOP‐BFM ALL2009 and ALL2017, B‐lineage ALL patients with high RQ‐PCR‐MRD at day+33 and positive at day+78 are defined slow early responders (SERs). Based on results of the AIEOP‐BFM ALL2000 study, these patients are treated as high‐risk also when positive MRD signal at day +78 is below the lower limit of quantification of RQ‐PCR (“positive not‐quantifiable,” POS‐NQ). To assess whether droplet digital polymerase chain reaction (ddPCR) could improve patients’ risk definition, we analyzed MRD in 209 pediatric B‐lineage ALL cases classified by RQ‐PCR as POS‐NQ and/or negative (NEG) at days +33 and/or +78 in the AIEOP‐BFM ALL2000 trial. ddPCR MRD analysis was performed on 45 samples collected at day +78 from SER patients, who had RQ‐PCR MRD ≥ 5.0 × 10–4 at day+33 and POS‐NQ at day+78 and were treated as medium risk (MR). The analysis identified 13 of 45 positive quantifiable cases. Most relapses occurred in this patients’ subgroup, while ddPCR NEG or ddPCR‐POS‐NQ patients had a significantly better outcome (P < 0.001). Overall, in 112 MR cases and 52 standard‐risk patients, MRD negativity and POS‐NQ were confirmed by the ddPCR analysis except for a minority of cases, for whom no differences in outcome were registered. These data indicate that ddPCR is more accurate than RQ‐PCR in the measurement of MRD, particularly in late follow‐up time points, and may thus allow improving patients’ stratification in ALL protocols.
The high poloidal-beta (
β
P
) regime was first proposed as a high bootstrap current scenario for a steady-state fusion pilot plant (FPP) in the 1990s (Kikuchi in Nucl Fusion 30:265, 1990). Since ...then, there have been many theoretical, modeling, and experimental research activities on this topic. A joint DIII-D/EAST research team began exploring the high-
β
P
regime in 2013, focusing on addressing the needs of attractive FPP design by taking advantage of the extensive diagnostic set and sophisticated plasma control system on DIII-D and the well-developed integrated modeling capability at General Atomics. The ultimate goal is to demonstrate such a scenario on EAST with truly long pulse and metal wall compatibility. This paper summarizes the highlights of the research results on DIII-D by the joint team in the past decade. Experimental evidence and modeling analysis show the high-
β
P
scenario has great advantages in addressing key needs for an attractive FPP design, such as high-energy confinement quality at low rotation, excellent core-edge integration, high line-averaged density above the Greenwald limit, low disruption risk, and high bootstrap current fraction for steady-state operation. This provides a relatively safe and economical option to base an FPP design on that will lead to commercial fusion energy.
We report on the first direct comparisons of microtearing turbulence simulations to experimental measurements in a representative high bootstrap current fraction (f_{BS}) plasma. Previous studies of ...high f_{BS} plasmas carried out in DIII-D with large radius internal transport barriers (ITBs) have found that, while the ion energy transport is accurately reproduced by neoclassical theory, the electron transport remains anomalous and not well described by existing quasilinear transport models. A key feature of these plasmas is the large value of the normalized pressure gradient, which is shown to completely stabilize conventional drift-wave and kinetic ballooning mode instabilities in the ITB, but destabilizes the microtearing mode. Nonlinear gyrokinetic simulations of the ITB region performed with the cgyro code demonstrate that the microtearing modes are robustly unstable and capable of driving electron energy transport levels comparable to experimental levels for input parameters consistent with the experimental measurements. These simulations uniformly predict that the microtearing mode fluctuation and flux spectra extend to significantly shorter wavelengths than the range of linear instability, representing significantly different nonlinear dynamics and saturation mechanisms than conventional drift-wave turbulence, which is also consistent with the fundamental tearing nature of the instability. The predicted transport levels are found to be most sensitive to the magnetic shear, rather than the temperature gradients more typically identified as driving turbulent plasma transport.
Inherited mutations in DNA-repair genes such as BRCA2 are associated with increased risks of lethal prostate cancer. Although the prevalence of germline mutations in DNA-repair genes among men with ...localized prostate cancer who are unselected for family predisposition is insufficient to warrant routine testing, the frequency of such mutations in patients with metastatic prostate cancer has not been established.
We recruited 692 men with documented metastatic prostate cancer who were unselected for family history of cancer or age at diagnosis. We isolated germline DNA and used multiplex sequencing assays to assess mutations in 20 DNA-repair genes associated with autosomal dominant cancer-predisposition syndromes.
A total of 84 germline DNA-repair gene mutations that were presumed to be deleterious were identified in 82 men (11.8%); mutations were found in 16 genes, including BRCA2 (37 men 5.3%), ATM (11 1.6%), CHEK2 (10 1.9% of 534 men with data), BRCA1 (6 0.9%), RAD51D (3 0.4%), and PALB2 (3 0.4%). Mutation frequencies did not differ according to whether a family history of prostate cancer was present or according to age at diagnosis. Overall, the frequency of germline mutations in DNA-repair genes among men with metastatic prostate cancer significantly exceeded the prevalence of 4.6% among 499 men with localized prostate cancer (P<0.001), including men with high-risk disease, and the prevalence of 2.7% in the Exome Aggregation Consortium, which includes 53,105 persons without a known cancer diagnosis (P<0.001).
In our multicenter study, the incidence of germline mutations in genes mediating DNA-repair processes among men with metastatic prostate cancer was 11.8%, which was significantly higher than the incidence among men with localized prostate cancer. The frequencies of germline mutations in DNA-repair genes among men with metastatic disease did not differ significantly according to age at diagnosis or family history of prostate cancer. (Funded by Stand Up To Cancer and others.).
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