vaRHC: an R package for semi-automation of variant classification in hereditary cancer genes according to ACMG/AMP and gene-specific ClinGen guidelines.

MOTIVATION: Germline variant classification allows accurate genetic diagnosis and risk assessment. However, it is a tedious iterative process integrating information from several sources and types of evidence. It should follow gene-specific (if available) or general updated international guidelines. Thus, it is the main burden of the incorporation of next-generation sequencing into the clinical setting. RESULTS: We created the vaRiants in HC (vaRHC) R package to assist the process of variant classification in hereditary cancer by: (i) collecting information from diverse databases; (ii) assigning or denying different types of evidence according to updated American College of Molecular Genetics and Genomics/Association of Molecular Pathologist gene-specific criteria for ATM, CDH1, CHEK2, MLH1, MSH2, MSH6, PMS2, PTEN, and TP53 and general criteria for other genes; (iii) providing an automated classification of variants using a Bayesian metastructure and considering CanVIG-UK recommendations; and (iv) optionally printing the output to an .xlsx file. A validation using 659 classified variants demonstrated the robustness of vaRHC, presenting a better criteria assignment than Cancer SIGVAR, an available similar tool. AVAILABILITY AND IMPLEMENTATION: The source code can be consulted in the GitHub repository (https://github.com/emunte/vaRHC) Additionally, it will be submitted to CRAN soon.

Obtaining miRNA from Saliva-Comparison of Sampling and Purification Methods.

The use of saliva as a biomarker source has advantages over other biofluids and imaging techniques, and miRNAs are ideal biomarker candidates. They are involved in numerous cellular processes, and their altered expression suggests that miRNAs play a crucial regulatory role in disease development. We wanted to find an easily reproducible and executable miRNA-obtaining methodology suitable for quantification. Three commercial miRNA extraction kits (mirVana, Nucleospin and miRNeasy) and three saliva collectors (50 mL tubes, Salimetrics and Oragene) were tested. Several features, including RNA quality and technical parameters, were evaluated. The expression of five synthetic spike-in controls and seven saliva-miRNAs was analyzed independently and grouped by the collectors and the extraction kits. The combination of Oragene and miRNeasy assured the most sensitive detection of all seven saliva miRNAs. Testing different combinations of saliva collectors and RNA purification kits permitted the establishment of combinations for different uses. The results of our study highlight that optimization of resources for biomarker studies is possible after careful planning of each study.

Expression Levels of an Alpha-Synuclein Transcript in Blood May Distinguish between Early Dementia with Lewy Bodies and Parkinson's Disease.

Lewy body diseases (LBD) including dementia with Lewy bodies (DLB) and Parkinson disease (PD) are characterized by alpha-synuclein pathology. DLB is difficult to diagnose and peripheral biomarkers are urgently needed. Therefore, we analyzed the expression of five alpha-synuclein gene (SNCA) transcripts, SNCAtv1, SNCAtv2, SNCAtv3, SNCA126, and SNCA112, in 45 LBD and control temporal cortex samples and in the blood of 72 DLB, 59 PD, and 54 control subjects. The results revealed overexpression of SNCAtv1 and SNCA112 in DLB, and SNCAtv2 in PD temporal cortices. In DLB blood, diminution of all SNCA transcripts was observed. SNCAtv1 and SNCAtv2 were diminished in PD with disease onset before 70 years. SNCAtv3, driven by its own promoter, showed opposite expression in early DLB and PD, suggesting that its amount may be an early, DLB specific biomarker. Correlation between blood transcript levels and disease duration was positive in DLB and negative in PD, possibly reflecting differences in brain alpha-synuclein aggregation rates associated with differences in disease courses. In conclusion, SNCA transcripts showed a disease-specific increase in the brain and were diminished in blood of LBD patients. SNCAtv3 expression was decreased in early DLB and increased in early PD and could be a biomarker for early DLB diagnosis.

Pre- and Post-Neoadjuvant Clinicopathological Parameters Can Help in the Prognosis and the Prediction of Response in HER2+ and Triple Negative Breast Cancer.

Neoadjuvant treatment (NAT) is one of the most widely used options for HER2+ and triple negative (TN) early breast cancer (BC). Since around half of the patients treated with NAT do not achieve a pathologically complete response (pCR), biomarkers to predict resistance are urgently needed. The correlation of clinicopathological factors with pCR was studied in 150 patients (HER2 = 81; TN = 69) and pre- and post-NAT differences in tumour biomarkers were compared. Low estrogen receptor (ER) expression, high tumour-infiltrating lymphocytes (TILs) and low cT-stage were associated with pCR in HER2+ tumours (p = 0.022; p = 0.032 and p = 0.005, respectively). Furthermore, ER expression was also associated with residual cancer burden (RCB; p = 0.046) in the HER2+ subtype. Similarly, pre-NAT, low progesterone receptor expression (PR; 1-10%) was associated with higher RCB (p < 0.001) in TN tumours. Only clinical and pathological T-stage (cpT-stage) had prognostic capacity in HER2+ tumours, whereas pre-NAT cpT-stage and post-NAT TILs had this capacity for the prognosis of TN tumours. We conclude that ER and PR expression may help predict response to NAT in HER2 and TN BC and should be taken into account in residual tumours. Also, changes observed in the phenotype after NAT suggest the need to reevaluate biomarkers in surviving residual tumour cells.

Peripheral α-synuclein isoforms are potential biomarkers for diagnosis and prognosis of isolated REM sleep behavior disorder.

INTRODUCTION: Isolated REM sleep behavior disorder (IRBD) represents an early manifestation of the synucleinopathies Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Aggregation of abnormal α-synuclein and its increased expression in the brain is crucial in the development of the synucleinopathies. Whereas α-synuclein gene (SNCA) transcripts are overexpressed in brain, a concomitant reduction occurs in blood of DLB patients. We assessed whether this decrease is also detectable in IRBD. METHODS: 108 IRBD patients and 149 controls were included of which 29 IRBD and 32 control cases were available for expression studies. Expression of SNCAtv1, SNCAtv2, SNCAtv3 and SNCA126 isoforms, and GBA were determined by real-time PCR. Genotype distribution of SNCA SNPs, rs356219 and rs2736990, and correlation with SNCA expression was analyzed. RESULTS: Expression of all SNCA transcripts was reduced in IRBD blood whereas GBA expression did not change. SNCAtv3 expression correlated inversely with IRBD duration, being lower in patients with longer follow-up. Rs356219-AA genotype frequency was increased in IRBD patients who later developed PD and DLB. Rs2736990-CC frequency was increased among IRBD cases who remained disease-free. No correlation was observed between rs356219 and rs2736990 genotypes and SNCA transcript levels. CONCLUSION: SNCA transcript expression is decreased in blood in IRBD, and levels decrease with IRBD duration. Our findings indicate that changes in SNCA expression occur in the earliest stages of the synucleinopathies before motor and cognitive symptoms become apparent.