Evaluation of Circulating Tumor DNA as a Liquid Biomarker in Uveal Melanoma.

Purpose: Uveal melanoma (UM) has a high propensity to metastasize. Prognosis is associated with specific driver mutations and copy number variations (CNVs), but limited primary tumor tissue is available for molecular characterization due to eye-sparing irradiation treatment. This study aimed to assess the rise in circulating tumor DNA (ctDNA) levels in UM and evaluate its efficacy for CNV-profiling of patients with UM. Methods: In a pilot study, we assessed ctDNA levels in the blood of patients with UM (n = 18) at various time points, including the time of diagnosis (n = 13), during fractionated stereotactic radiotherapy (fSRT) treatment (n = 6), and upon detection of metastatic disease (n = 13). Shallow whole-genome sequencing (sWGS) combined with in silico size-selection was used to identify prognostically relevant CNVs in patients with UM (n = 26) from peripheral blood retrieved at the time of diagnosis (n = 9), during fSRT (n = 5), during post-treatment follow-up (n = 4), metastasis detection (n = 6), and metastasis follow-up (n = 4). Results: A total of 34 patients had blood analyzed for ctDNA detection (n = 18) and/or CNV analysis (n = 26) at various time points. At the time of diagnosis, 5 of 13 patients (38%) had detectable ctDNA (median = 0 copies/mL). Upon detection of metastatic disease, ctDNA was detected in 10 of 13 patients (77%) and showed increased ctDNA levels (median = 24 copies/mL, P < 0.01). Among the six patients analyzed during fSRT, three (50%) patients had detectable ctDNA at baseline and three of six (50%) patients had undetectable levels of ctDNA. During the fSRT regimen, ctDNA levels remained unchanged (P > 0.05). The ctDNA fractions were undetectable to low in localized disease, and sWGS did not elucidate chromosome 3 status from blood samples. However, in 7 of 10 (70%) patients with metastases, the detection of chromosome 3 loss corresponded to the high metastatic-risk class. Conclusions: The rise in ctDNA levels observed in patients with UM harboring metastases suggests its potential utility for CNV profiling. These findings highlight the potential of using ctDNA for metastasis detection and patient inclusion in therapeutic studies targeting metastatic UM.

An 8q24 Gain in Pancreatic Juice Is a Candidate Biomarker for the Detection of Pancreatic Cancer.

Secretin-stimulated pancreatic juice (PJ), collected from the duodenum, presents a valuable biomarker source for the (earlier) detection of pancreatic cancer (PC). Here, we evaluate the feasibility and performance of shallow sequencing to detect copy number variations (CNVs) in cell-free DNA (cfDNA) from PJ for PC detection. First, we confirmed the feasibility of shallow sequencing in PJ (n = 4), matched plasma (n = 3) and tissue samples (n = 4, microarray). Subsequently, shallow sequencing was performed on cfDNA from PJ of 26 cases (25 sporadic PC, 1 high-grade dysplasia) and 19 controls with a hereditary or familial increased risk of PC. 40 of the 45 PJ samples met the quality criteria for cfDNA analysis. Nine individuals had an 8q24 gain (oncogene MYC; 23%; eight cases (33%) and one control (6%), p = 0.04); six had both a 2q gain (STAT1) and 5p loss (CDH10; 15%; four cases (7%) and two controls (13%), p = 0.72). The presence of an 8q24 gain differentiated the cases and controls, with a sensitivity of 33% (95% CI 16-55%) and specificity of 94% (95% CI 70-100%). The presence of either an 8q24 or 2q gain with a 5p loss was related to a sensitivity of 50% (95% CI 29-71%) and specificity of 81% (95% CI 54-96%). Shallow sequencing of PJ is feasible. The presence of an 8q24 gain in PJ shows promise as a biomarker for the detection of PC. Further research is required with a larger sample size and consecutively collected samples in high-risk individuals prior to implementation in a surveillance cohort.

Web-accessible application for identifying pathogenic transcripts with RNA-seq: Increased sensitivity in diagnosis of neurodevelopmental disorders.

For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals' cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual's specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.

Multiplex ligation-depending probe amplification is not suitable for detection of low-grade mosaicism.

'Apparent non-penetrance' occurs in several genetic disorders, including tuberous sclerosis complex and neurofibromatosis type 1: clinically unaffected parents may have multiple affected offspring. Germ line or somatic mosaicism in one of the parents of the index patient is the probable cause and results in an enhanced recurrence risk. Therefore, it is of great importance to use the most sensitive technology for testing DNA of the parents of the index patient for the presence/absence of the familial mutation. To detect large rearrangements multiplex ligation-depending probe amplification (MLPA) is often used. Here we show that MLPA is less sensitive in detecting low-grade somatic mosaicism than fluorescence in situ hybridization (FISH) or a mutation-specific PCR test. Therefore, we recommend FISH (if possible) or PCR analysis for the analysis of parental DNA.

Identification of a large rearrangement in CYLD as a cause of familial cylindromatosis.

Pathogenic mutations in CYLD can be identified in patients affected with Brooke-Spiegler syndrome, (Familial) Cylindromatosis or multiple familial trichoepithelioma. To date, only technologies which are able to identify small point mutations in CYLD, such as sequence and WAVE analysis, were used. Here we describe the identification of a larger rearrangement identified by Quantitative PCR analysis of CYLD, indicating that a combination of these technologies is necessary when searching for pathogenic mutations in CYLD.

Deletion of exons 1a-2 of BRCA1: a rather frequent pathogenic abnormality.

Women carrying a pathogenic mutation in either BRCA1 or BRCA2 have a major risk of developing breast and/or ovarian cancer. The majority of mutations in these genes are small point mutations. Since the development of multiplex ligation-dependent probe amplification, an increasing number of large genomic rearrangements have been detected. Here, we describe the characterization of pathogenic deletions of exons 1a-2 of BRCA1 in six families using loss of heterozygosity, array comparative genomic hybridization, and sequence analyses. Two families harbor a 37 kb deletion starting in intron 2 of psi BRCA1, encompassing NBR2, and exons 1a-2 of BRCA1, while the other four families have an 8 kb deletion with breakpoints in intron 2 of NBR2 and intron 2 of BRCA1. This observation, together with the previously described families with exon 1a-2 deletions of BRCA1, demonstrates that this type of deletions is relatively frequent in breast/ovarian cancer families.