Pathway Analysis of Selected Circulating miRNAs in Plasma of Breast Cancer Patients: A Preliminary Study.

MicroRNAs in the circulation of breast cancer (BC) patients have great potential for the early diagnosis, treatment and monitoring of breast cancer. The aim of this preliminary study was to obtain the expression profile of selected miRNAs in the plasma of BC patients that could discriminate BC patients from healthy volunteers and may be useful in early detection of BC. Significantly deregulated miRNAs were evaluated by pathway analysis with the prediction of potential miRNA targets. The study enrolled plasma samples from 65 BC patients and 34 healthy volunteers. Selected miRNAs were screened in pilot testing by the real-time PCR (qPCR) method, and the most appropriate reference genes were selected for normalisation by the geNorm algorithm. In the final testing, we detected miR-99a, miR-130a, miR-484 and miR-1260a (p < 0.05) as significantly up-regulated in the plasma of BC patients. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed that all significantly deregulated miRNAs are involved in the Hippo and Transforming Growth Factor-beta (TGF-beta) signalling pathways. Our study confirmed a different profile of selected circulating miRNAs in the plasma of BC patients with an emphasis on some critical points in the analysis process.

Molecular analysis of circulating tumor DNA from breast cancer patients before and after surgery and following adjuvant chemotherapy.

The primary aim of the present study is to provide a complex molecular profile of tumors using liquid biopsy and to monitor profile changes over time in association with surgery and administered adjuvant therapy. Our secondary aim was to compare the liquid biopsy profile with the tissue biopsy and assess concordance. A total of 27 samples of circulating tumor DNA (ctDNA) collected from 9 breast cancer patients at three different time points and their matched formalin-fixed and paraffin-embedded (FFPE) samples of primary tumor were analyzed with targeted next-generation sequencing. Somatic pathogenic variants were detected before surgery in samples from 5 patients (55.6%). The most frequently mutated genes were phosphatase and tensin homolog (4/9, 44.4%) and tumor protein 53 (4/9, 44.4%). Serial sampling of ctDNA enabled the detection of more variants compared with single-time tissue primary tumor biopsy. There were 17 ctDNA variants across all samples, but only 6 FFPE variants across all patients. In addition, the concordance between ctDNA and FFPE DNA was determined in only 1 patient, and this was connected with higher variant allele frequency. The findings of the present study suggest that liquid biopsy and tissue biopsy may be used as complementary analyses to adequately capture all tumor variants.

Comparison of Somatic Mutation Profiles Between Formalin-Fixed Paraffin Embedded Tissues and Plasma Cell-Free DNA from Ovarian Cancer Patients Before and After Surgery.

Ovarian carcinogenesis can be induced by a large number of somatic gene mutations. Circulating tumor DNA (ctDNA) released into peripheral blood can provide insights into the genomic landscape of cancer cells and monitor their dynamics. Our aim was to detect and compare the genetic profiles in tumor tissue and plasma before and after tumor resection in ovarian cancer patients. All three samples were collected from each patient. In this study, we used a commercial cancer panel to identify somatic mutations in 26 genes in seven selected patients through next-generation sequencing on the Illumina platform. Overall, 16 variants with pathogenic effect were identified in the TP53, PIK3CA, PTEN, APC, NRAS, KRAS, GNAS, and MET genes involved in important signaling pathways. The genetic alterations found in the presurgical plasma in six of seven ovarian cancer patients were no longer present in the plasma after tumor surgical removal. Identical variants in formalin-fixed paraffin embedded (FFPE) tissues and preoperative plasma specimens were observed in only two cases. These findings suggest that the detected presurgical pathogenic variants absent in postsurgery plasma are associated with the primary ovarian tumor. Finally, the low-identified concordance between FFPE and plasma can be due to various factors, but most likely to high tumor heterogeneity and low ctDNA level.

Detection of BRAFV600E Mutation in Melanoma Patients by Digital PCR of Circulating DNA.

AIMS: About 50% of melanomas have the BRAFV600E mutation. This mutation is an attractive therapeutic target. The aims of our study were to detect BRAFV600E mutations within circulating cell-free DNA in plasma ("liquid biopsy") by a droplet digital PCR (ddPCR) method, and to investigate how well the Breslow-Clark score can be predicted by ddPCR. MATERIALS AND METHODS: We analyzed 113 patients with malignant melanoma. ddPCR was performed using the QX200 system (BIO-RAD®, Hercules). All samples were tested in duplicate. Besides the results of the liquid biopsy, we have collected data on gender and age of the patients, as well as the mitotic activity of the tumor; the tumor subtype and localization, and the Breslow-Clark score. The limit of detection (LoD) was determined by the method of Tzonev. The LoD was found to be five events per well. RESULTS: The BRAFV600E mutation was detected in 37 of 113 samples. A moderate predictive accuracy of the Breslow-Clark score can be attained with the mitotic activity and the type of melanoma as the most important predictors. CONCLUSION: Our results show that ddPCR is a highly sensitive method and could be used for a routine laboratory detection of the BRAFV600E mutation as well as for follow-up monitoring to determine the treatment response in patients with malignant melanomas.

Droplet digital PCR for detection of BRAF V600E mutation in formalin-fixed, paraffin-embedded melanoma tissues: a comparison with Cobas® 4800, Sanger sequencing, and allele-specific PCR.

Cutaneous melanoma has the worst prognosis of all skin cancers. Although emerging targeted therapies, such as B-Raf kinase inhibitor vemurafenib, improve prognosis they require an accurate and sensitive means of detecting the pathogenic BRAF V600E mutation. We compared the sensitivity of four BRAF V600E detection methods in formalin-fixed, paraffin-embedded melanoma biopsies from 87 consecutive melanoma patients with Breslow stage I-V disease (staging based on the depth of tumor of invasion). The methods assessed were the widely used Cobas® 4800 system based on real-time PCR amplification, Sanger sequencing, allele-specific PCR (AS-PCR), and droplet digital PCR (ddPCR). The BRAF V600E mutation was found in 8 (9.2%), 23 (26.4%), 23 (26.4%) and 31 (35.6%) biopsies, respectively. The limit of detection (LoD) was determined by three different methods: Poisson confidence limits, calibration regression and Tzonev's method. Pair-wise agreement between the methods was as follows: Cobas vs. Sanger, P = 0.33; Cobas® 4800 vs. AS-PCR, P = 0.33; Cobas® 4800 vs. ddPCR, P = 0.65; Sanger vs. AS-PCR, P = 1; Sanger vs. ddPCR, P = 0.08; AS-PCR vs. ddPCR, P = 0.06. Multinomial logistic regression was used for predictive modeling of the Breslow-Clark score; ddPCR emerged as the best predictor, the other predictors were mitotic activity, type of malignant melanoma and patient's age. Our results demonstrate that ddPCR is the most sensitive method of detecting the BRAF V600E mutation.