Cardio-pathogenic variants in unexplained intrauterine fetal death: a retrospective pilot study.

To describe the prevalence and spectrum of cardio-pathogenic variants in singleton fetuses after unexplained intrauterine fetal death (IUFD). DNA from post-mortem fibroblastic tissue samples of 16 fetuses after unexplained IUFD was retrieved at two tertiary university hospitals for clinical exome sequencing with subsequent filtering of 122 cardio-specific genes to elucidate underlying cardio-pathogenic variants. In total, we included 12 (75%) male and four (25%) female fetuses who were stillborn at a median gestational age of 34+6 (23+2-40+5) weeks. In two (12.5%) fetuses no cardio-pathogenic variants were found. In 14 (87.5%) fetuses, overall 33 variants were detected in 22 cardio-specific genes, involving 14 (63.63%) genes associated with cardiomyopathy, six (27.27%) arrhythmogenic susceptibility genes and two (9.09%) arrhythmia and cardiomyopathy associated genes. Among the 33 variants, five (15.2%) were classified as likely benign according to the American College of Medical Genetics and Genomics; 28 (84.8%) variants were considered as variants of uncertain significance. Compared to a cohort of explained IUFDs, the cases with and without fetal variants in cardiac genes differed not significantly regarding maternal age, previous history of stillbirth, time of stillbirth or fetal sex. Unexplained stillbirth may be caused by cardio-genetic pathologies, yet a high number of variants of uncertain significance merit a more detailed post-mortem examination including family segregation analysis.

Whole-genome plasma sequencing reveals focal amplifications as a driving force in metastatic prostate cancer.

Genomic alterations in metastatic prostate cancer remain incompletely characterized. Here we analyse 493 prostate cancer cases from the TCGA database and perform whole-genome plasma sequencing on 95 plasma samples derived from 43 patients with metastatic prostate cancer. From these samples, we identify established driver aberrations in a cancer-related gene in nearly all cases (97.7%), including driver gene fusions (TMPRSS2:ERG), driver focal deletions (PTEN, RYBP and SHQ1) and driver amplifications (AR and MYC). In serial plasma analyses, we observe changes in focal amplifications in 40% of cases. The mean time interval between new amplifications was 26.4 weeks (range: 5-52 weeks), suggesting that they represent rapid adaptations to selection pressure. An increase in neuron-specific enolase is accompanied by clonal pattern changes in the tumour genome, most consistent with subclonal diversification of the tumour. Our findings suggest a high plasticity of prostate cancer genomes with newly occurring focal amplifications as a driving force in progression.

The dynamic range of circulating tumor DNA in metastatic breast cancer.

INTRODUCTION: The management of metastatic breast cancer needs improvement. As clinical evaluation is not very accurate in determining the progression of disease, the analysis of circulating tumor DNA (ctDNA) has evolved to a promising noninvasive marker of disease evolution. Indeed, ctDNA was reported to represent a highly sensitive biomarker of metastatic cancer disease directly reflecting tumor burden and dynamics. However, at present little is known about the dynamic range of ctDNA in patients with metastatic breast cancer. METHODS: In this study, 74 plasma DNA samples from 58 patients with metastasized breast cancer were analyzed with a microfluidic device to determine the plasma DNA size distribution and copy number changes in the plasma were identified by whole-genome sequencing (plasma-Seq). Furthermore, in an index patient we conducted whole-genome, exome, or targeted deep sequencing of the primary tumor, metastases, and circulating tumor cells (CTCs). Deep sequencing was done to accurately determine the allele fraction (AFs) of mutated DNA fragments. RESULTS: Although all patients had metastatic disease, plasma analyses demonstrated highly variable AFs of mutant fragments. We analyzed an index patient with more than 100,000 CTCs in detail. We first conducted whole-genome, exome, or targeted deep sequencing of four different regions from the primary tumor and three metastatic lymph node regions, which enabled us to establish the phylogenetic relationships of these lesions, which were consistent with a genetically homogeneous cancer. Subsequent analyses of 551 CTCs confirmed the genetically homogeneous cancer in three serial blood analyses. However, the AFs of ctDNA were only 2% to 3% in each analysis, neither reflecting the tumor burden nor the dynamics of this progressive disease. These results together with high-resolution plasma DNA fragment sizing suggested that differences in phagocytosis and DNA degradation mechanisms likely explain the variable occurrence of mutated DNA fragments in the blood of patients with cancer. CONCLUSIONS: The dynamic range of ctDNA varies substantially in patients with metastatic breast cancer. This has important implications for the use of ctDNA as a predictive and prognostic biomarker.

Changes in colorectal carcinoma genomes under anti-EGFR therapy identified by whole-genome plasma DNA sequencing.

Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in four patients (40%), which occurred either as novel focal amplifications (n = 3) or as high level polysomy of 12p (n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2) and ERBB2 (n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression.

Multiplex genetic cancer testing identifies pathogenic mutations in TP53 and CDH1 in a patient with bilateral breast and endometrial adenocarcinoma.

BACKGROUND: Germline genetic testing for familial cancer syndromes is usually performed serially for the most likely genetic causes. In recent years the way genetic testing carried out has changed, as next generation sequencing now allows the simultaneous testing of multiple susceptibility genes at low costs. CASE PRESENTATION: Here, we present a female with bilateral breast cancer and endometrial adenocarcinoma. After simultaneous sequencing of 150 genes (890 kb) associated with hereditary cancer we identified pathogenic mutations in two high-penetrance genes, i.e. TP53 and CDH1 that would most likely not have been elucidated by serial screening of candidate genes. CONCLUSION: As the two mutated genes are located on different chromosomes and cause different cancer syndromes these findings had a tremendous impact not only on genetic counseling of the index patient and her family but also on subsequent surveillance strategies.

Outcome of coumarin-therapy in an outpatient setting over a 10-year period: coumarin-therapy in clinical practice.

OBJECTIVES: A retrospective study was conducted on an unselected sample of patients on vitamin K antagonists (VKAs; phenprocoumon, acenocoumarol) in an outpatient setting in Austria. The main objective was to determine whether bleeding and thromboembolic events reported in randomised trials are comparable to the experience in clinical practice. In addition, we focused on differences between the two VKAs and the particular indications for treatment and influences of risk factors. PARTICIPANTS: Total observation time was 10 years, the number of patients was 599 and the patient years-at-risk (pyr) was 1,856. RESULTS: Severe bleeding occurred in 1.1 % pyr and the bleeding-related mortality was 0.1 %. Severe thromboembolic events occurred in 2.8 % pyr, with a rate of fatal events of 0.3 %. A significant increase in risk was found in patients older than 75 years concerning bleeding as well as thromboembolic events. Acenocoumarol showed significantly higher rates in life-threatening and fatal bleeding episodes in comparison to phenprocoumon. CONCLUSIONS: The outcome of patients receiving VKA in an office setting is similar to that reported in the literature and the use of VKAs in this setting appears to be safe.

Spongious hypertrophic cardiomyopathy in patients with mutations in the four-and-a-half LIM domain 1 gene.

BACKGROUND: X-linked myopathy with postural muscle atrophy is a novel X-linked myopathy caused by mutations in the four-and-a-half LIM domain 1 gene (FHL1). Cardiac involvement was suspected in initial publications. We now systematically analyzed the association of the FHL1 genotype with the cardiac phenotype to establish a potential cardiac involvement in the disease. METHODS AND RESULTS: Seventeen male patients and 23 female mutation carriers were compared with healthy controls. Every patient underwent a comprehensive clinical and cardiovascular workup. ECG abnormalities occurred frequently in affected males and were less frequent in heterozygous females. Both male and female mutation carriers had increased myocardial mass (affected males=115.1±25.3 g/m(2); heterozygous females=95.1±19.6 g/m(2); controls=89.0±15.6 g/m(2) and 72.6±12.6 g/m(2); respectively) with increased wall thickness (typically midventricular and apical segments) mainly in affected males. Longitudinal systolic function was reduced in affected males (radial systolic strain: affected males=24.6±11.8%; male controls=43.2±14.8%; P=0.002). Diastolic dysfunction occurred in both affected males and heterozygous females. Cardiac MRI revealed a morphological hallmark of X-linked myopathy with postural muscle atrophy; a characteristic spongious structure and replacement fibrosis indicated by late enhancement could be detected in most affected males. X-linked myopathy with postural muscle atrophy was associated with reduced exercise capacity in affected males but not in heterozygous female mutation carriers. CONCLUSIONS: X-linked myopathy with postural muscle atrophy patients consistently showed electrical, functional, and characteristic morphological cardiac abnormalities that translate into reduced exercise capacity. Reduced systolic and diastolic function is associated with a novel type of spongious hypertrophic cardiomyopathy. An unexpected finding was that some cardiac abnormalities were also present in heterozygous female mutation carriers.