A single platform designed for the synchronous screening of multiple mutations can potentially enable molecular profiling in samples of limited tumor tissue. This approach is ideal for the assessment ...of advanced non–small-cell lung cancer (NSCLC) diagnostic specimens, which often comprise small biopsies. Therefore, we aimed in this study to validate the mass spectrometry-based Sequenom LungCarta panel and MassARRAY platform using DNA extracted from a single 5 μM formalin-fixed paraffin-embedded tissue section.
Mutations, including those with an equivocal spectrum, detected in 90 cases of NSCLC (72 lung biopsies, 13 metastatic tissue biopsies, three resections, and two cytology samples) were validated by a combination of standard sequencing techniques, immunohistochemical staining for p53 protein, and next-generation sequencing with the TruSight Tumor panel.
Fifty-five mutations were diagnosed in 47 cases (52%) in the following genes: TP53 (22), KRAS (15), EGFR (5), MET (3), PIK3CA (3), STK11 (2), NRF-2 (2), EPHA5 (1), EPHA3 (1), and MAP2K1 (1). Of the 90 samples, one failed testing due to poor quality DNA. An additional 7 TP53 mutations were detected by next-generation sequencing, which facilitated the interpretation of p53 immunohistochemistry but required 5 × 10 μM tumor sections per sample tested.
The LungCarta panel is a sensitive method of screening for multiple alterations (214 mutations across 26 genes) and which optimizes the use of limited amounts of tumor DNA isolated from small specimens.
Abstract The gold standard test for detection of epidermal growth factor receptor (EGFR) mutation is to genotype somatic DNA extracted from a tissue biopsy or cytology specimen. Yet, in at least 20% ...of patients this is not possible for various reasons including insufficient availability of neoplastic tissue, lack of fitness of the available tissue for a biopsy or that a biopsy is not technically feasible. Consequently, there has been intense investigation of circulating tumour DNA (ctDNA), released into the plasma fraction of blood from cancer cells during apoptosis/necrosis, as a minimally invasive ‘liquid biopsy’ and surrogate for cancer tissue. In 2014, the license for the EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib, was updated to allow the use of plasma to determine EGFR mutation status in patients where tissue was not available. Then in 2016 the United States Food and Drug Administration (US FDA) approved the first companion diagnostic plasma EGFR test. Herein, we review the evidence for ctDNA as a diagnostic in patients with non-small cell lung cancer (NSCLC) and describe steps needed to incorporate such ‘liquid biopsies’ into everyday routine practice.
Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) supports blood-based genomic profiling but is not yet routinely implemented in the setting of a phase I trials clinic. TARGET is a ...molecular profiling program with the primary aim to match patients with a broad range of advanced cancers to early phase clinical trials on the basis of analysis of both somatic mutations and copy number alterations (CNA) across a 641 cancer-associated-gene panel in a single ctDNA assay. For the first 100 TARGET patients, ctDNA data showed good concordance with matched tumor and results were turned round within a clinically acceptable timeframe for Molecular Tumor Board (MTB) review. When a 2.5% variant allele frequency (VAF) threshold was applied, actionable mutations were identified in 41 of 100 patients, and 11 of these patients received a matched therapy. These data support the application of ctDNA in this early phase trial setting where broad genomic profiling of contemporaneous tumor material enhances patient stratification to novel therapies and provides a practical template for bringing routinely applied blood-based analyses to the clinic.
Purpose
Brain metastases (BM) are an increasing clinical problem. This study aimed to assess paired primary breast cancers (BC) and BM for aberrations within TP53, PIK3CA, ESR1, ERBB2 and AKT ...utilising the MassARRAY® UltraSEEK® technology (Agena Bioscience, San Diego, USA).
Methods
DNA isolated from 32 paired primary BCs and BMs was screened using the custom UltraSEEK® Breast Cancer Panel. Data acquisition and analysis was performed by the Agena Bioscience Typer software v4.0.26.74.
Results
Mutations were identified in 91% primary BCs and 88% BM cases. TP53, AKT1, ESR1, PIK3CA and ERBB2 genes were mutated in 68.8%, 37.5%, 31.3%, 28.1% and 3.1% respectively of primary BCs and in 59.4%, 37.5%, 28.1%, 28.1% and 3.1% respectively of BMs. Differences in the mutations within the 5 genes between BC and paired BM were identified in 62.5% of paired cases. In primary BCs, ER-positive/HER2-negative cases harboured the most mutations (70%), followed by ER-positive/HER2-positive (15%) and triple-negatives (13.4%), whereas in BMs, the highest number of mutations was observed in triple-negative (52.5%), followed by ER-positive/HER2-negative (35.6%) and ER-negative/HER2-positive (12%). There was a significant association between the number of mutations in the primary BC and breast-to-brain metastasis-free survival (
p
= 0.0001) but not with overall survival (
p
= 0.056).
Conclusion
These data demonstrate the discordancy between primary BC and BM, as well as the presence of clinically important, actionable mutations in BCBM. The UltraSEEK® Breast Cancer Panel provides a tool for BCBM that can be utilised to direct more tailored treatment decisions and for clinical studies investigating targeted agents.
We report a patient with a germline
and a mosaic
variant. The diagnosis of the RASopathy was confirmed by targeted sequencing following the identification of transient cardiomyopathy in a patient ...with
-related overgrowth spectrum (PROS). Our observation confirms that the
gain-of-function (GoF) variant effects dominate those of the RASopathy, and the resulting blended phenotype mostly resembles megalencephaly-capillary malformation syndrome (MCAP PROS). There appears to be interaction between RIT1 and PI3K-AKT because the latter pathway is needed for the growth-promoting activity of the first, at least in adenocarcinomas, but the details of this interaction are not known. If so, the
somatic variant may not be just a chance event. It could also be of etiological relevance that Rit activation mediates resistance to cellular stress-that is, promotes cell survival. This anti-apoptotic effect could also make it more likely that a cell that spontaneously acquires a
GoF variant will survive and proliferate. We aim to encourage clinicians to investigate atypical findings in individuals with PROS. If further similar cases are reported, this would suggest that the establishment of PROS mosaicism is facilitated by the background of a RASopathy.
We have studied the effect of a 13-bp deletion in the promoter of the von Willebrand factor (VWF) gene in a patient with type 1 von Willebrand disease. The index case has a VWF:Ag of 0.49 IU/mL and ...is heterozygous for the deletion. The deletion is located 48 bp 5′ of the transcription start site, and in silico analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies all predict aberrant binding of Ets transcription factors to the site of the deletion. Transduction of reporter gene constructs into blood outgrowth endothelial cells showed a 50.5% reduction in expression with the mutant promoter (n = 16, P < .001). A similar 40% loss of transactivation was documented in transduced HepG2 cells. A similar marked reduction of transgene expression was shown in the livers of mice injected with the mutant promoter construct (n = 8, P = .003). Finally, in studies of BOEC mRNA, the index case showed a 4.6-fold reduction of expression of the VWF transcript associated with the deletion mutation. These studies show that the 13-bp deletion mutation alters the binding of Ets (and possibly GATA) proteins to the VWF promoter and significantly reduces VWF expression, thus playing a central pathogenic role in the type 1 von Willebrand disease phenotype in the index case.
Studies of nuclear organisation, most commonly determining the nuclear location of chromosome territories and individual loci, have furthered our understanding of nuclear function, differentiation ...and disease. In this study, by examining eight loci on different chromosomes, we tested hypotheses that: (1) totipotent human blastomeres adopt a nuclear organisation akin to that of committed cells; (2) nuclear organisation is different in chromosomally abnormal blastomeres; and (3) human blastomeres adopt a `chromocentre' pattern. Analysis of in vitro fertilisation (IVF) conceptuses permits valuable insight into the cell biology of totipotent human nuclei. Here, extrapolations from images of preimplantation genetic screening (PGS) cases were used to make comparisons between totipotent blastomeres and several committed cells, showing some differences and similarities. Comparisons between chromosomally abnormal nuclei and those with no detected abnormality (NDA) suggest that the former display a significant non-random pattern for all autosomal loci, but there is a less distinct, possibly random, pattern in `NDA' nuclei. No evidence was found that the presence of an extra chromosome is accompanied by an altered nuclear location for that chromosome. Centromeric loci on chromosomes 15 and 16 normally seen at the nuclear periphery were mostly centrally located in aneuploid cells, providing some evidence of a `chromocentre'; however, the chromosome-18 centromere was more peripheral, similar to committed cells. Our results provide clues to the nature of totipotency in human cells and might have future applications for preimplantation diagnosis and nuclear transfer.
To determine if patient aspirin exposure and timing affect bleeding risk after renal allograft biopsy.
Review of 6,700 renal allograft biopsies (in 2,362 unique patients) was performed. Median ...patient age was 53.0 years interquartile range 43.0, 62.0; 56.2% of patients were male. Of biopsies, 4,706 (70.2%) were performed in patients with no aspirin exposure within 10 days of biopsy; 664 (9.9%), were performed within 8-10 days of aspirin exposure; 855 (12.8%), within 4-7 days; and 475 (7.1%), within 0-3 days. Follow-up to 3 months after the procedure was completed in all patients. Biopsies were categorized as protocol or indication; 19.7% were indication biopsies. Bleeding complications were graded based on SIR criteria. Logistic regression models examined the association between aspirin use and bleeding events.
Rate 95% confidence interval of major bleeding complications was 0.24% 0.14, 0.39, and rate of any bleeding complication was 0.66% 0.46, 0.90. Bleeding events were significantly associated with patients undergoing indication biopsies compared with protocol biopsies (odds ratio OR 2.27, P = .012). Patient factors associated with major bleeding complications in multivariate models included estimated glomerular filtration rate (OR 0.61, P = .016) and platelet count (OR 0.64, P = .033). Aspirin use was not significantly associated with increased risk of bleeding complication except for use of 325 mg of aspirin within 3 days of biopsy (any complication OR 3.87 1.12, 13.4, P = .032; major complication OR 6.30 1.27, 31.3, P = .024).
Renal allograft biopsy bleeding complications are very rare, particularly for protocol biopsies. Use of 325 mg of aspirin within 3 days of renal allograft biopsy was associated with increased bleeding complications.
Aberrant androgen receptor (AR) signaling drives prostate cancer (PC), and it is a key therapeutic target. Although initially effective, the generation of alternatively spliced AR variants (AR-Vs) ...compromises efficacy of treatments. In contrast to full-length AR (AR-FL), AR-Vs constitutively activate androgenic signaling and are refractory to the current repertoire of AR-targeting therapies, which together drive disease progression. There is an unmet clinical need, therefore, to develop more durable PC therapies that can attenuate AR-V function. Exploiting the requirement of coregulatory proteins for AR-V function has the capacity to furnish tractable routes for attenuating persistent oncogenic AR signaling in advanced PC. DNA-PKcs regulates AR-FL transcriptional activity and is upregulated in both early and advanced PC. We hypothesized that DNA-PKcs is critical for AR-V function. Using a proximity biotinylation approach, we demonstrated that the DNA-PK holoenzyme is part of the AR-V7 interactome and is a key regulator of AR-V-mediated transcription and cell growth in models of advanced PC. Crucially, we provide evidence that DNA-PKcs controls global splicing and, via RBMX, regulates the maturation of AR-V and AR-FL transcripts. Ultimately, our data indicate that targeting DNA-PKcs attenuates AR-V signaling and provide evidence that DNA-PKcs blockade is an effective therapeutic option in advanced AR-V-positive patients with PC.
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