Analysis of Blood Plasma MicroRNAs to Enable Identification of Patients with Pancreatic Ductal Adenocarcinoma Who Will Benefit from Surgical Resection

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most common malignancy of pancreas, characterized by extremely poor prognosis largely due to problem with early diagnosis and lack of progress in personalization of therapy. Of all available treatment strategies, radical surgical resection of the tumour in its early stage remains the only possibility how to reach long-term survival. However, even a technically perfect surgical resection may still not provide a survival benefit for all PDAC patients. Appropriate selection of patients for surgical resection is one the important medical needs in management of PDAC patients. MATERIAL AND METHODS: To this study we enrolled 24 PDAC patients who underwent surgical resection and preoperatively collected their blood plasma specimends. Patients were divided into to two prognostic groups according to their overall survival - 12 patients with poor prognosis (median overall survival 10 months) and 12 patients with good prognosis (median overall survival 25 months). Small RNA sequencing technology was applied to screen for microRNAs (miRNA) with differential levels between both PDAC patients group. cDNA libraries were prepared using QIAseq miRNA Library Kit (Qiaqen) and sequencing by NextSeq500 instrument (Illumina). RESULTS: When miRNA expression profiles of the PDAC patients from good and poor prognostic groups were compared, 61 miRNAs were identified to have significantly different plasma levels between the two groups (p < 0.05). A total of 21 miRNAs showed increased expression and 40 miRNAs showed decreased expression in a group of patients with poor prognosis compared to patients with good prognosis. CONCLUSION: This study demonstrated differences in miRNA expression profiles in preoperative plasma specimens of PDAC patients with short and long overall survival. Our observations indicate that after independent validations plasma miRNAs might become useful biomarkers for identification of PDAC patients having clinical benefit from surgical resection of the tumour. This work was supported by Czech Ministry of Health, grant No. 16-31314A. All rights reserved. The authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 8. 3. 2019 Accepted: 9. 3. 2019.

[MicroRNA Analysis for Extramedullary Multiple Myeloma Relapse]. / Analýza mikroRNA u extramedulárního relapsu mnohocetného myelomu.

INTRODUCTION AND AIMS: Multiple myeloma (MM) is the second most common hematooncological disease. Patient survival has been greatly improved by the introduction of new drugs into clinical practice, but survival is negatively affected by the so-called extramedullary relapse (EM), caused by the loss of plasma cell dependence on the bone marrow microenvironment and their migration out of the bone marrow. The nature and causes of this process are currently unclear. MicroRNAs (miRNAs) are short, non-coding RNA molecules involved in many physiological and pathological processes. Their significance in the pathogenesis of MM has been demonstrated by several studies. We assume that they are also involved in the development of the EM. The aim of this study was to analyze different miRNA expression between MM and EM patients. MATERIAL AND METHODS: Using next generation sequencing, we analyzed 39 samples of bone marrow cells from MM patients at diagnosis and 9 bone marrow plasma samples of EM patients. RESULTS: In total, 2,278 miRNA were sequenced, but only 658 miRNAs were analyzed as they were expressed in all samples and had at least 20 reads. Expression data were generated using the Chimira tool from fastq data. All sequences were mapped using miRBase v20. Further analyses were performed using the R/Bioconductor package. The Bayesian procedure was used for normalization of expression. P values were adjusted using the Benjamini-Hochberg method. Analysis found 10 miRNA (p < 0.0005) that are statistically significantly expressed in EM vs. MM patients - these are miR-26a-5p, miR-26b-5p, miR-30e-5p, miR-424-3p, miR-503-5p, miR-767-5p, miR-105-5p, miR-5695-5p, miR-450b-5p and miR-92b-3p. These miRNAs will be further verified by qPCR method on a larger set of MM and EM patients. CONCLUSION: Our pilot study has shown that there are differentially expressed miRNAs between MM and EM patients.Key words: multiple myeloma - microRNA - carcinogenesis - next generation sequencing The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papersThis work was supported by grant MZ CR AZV 17- 29343A. Submitted: 17. 3. 2018Accepted: 20. 3. 2018.

Immune checkpoints in aggressive breast cancer subtypes.

Immune checkpoints are molecules referred to inhibitory pathways in the immune system that play a pivotal role in prevention of autoimmunity and oncogenesis. The aim of the study was to evaluate expression levels of selected immune checkpoints- PD-1 (programmed cell death protein 1), and PD-L1 (programmed cell death 1 ligand 1) in breast cancer patients, suitable for breast conservation and sentinel node biopsy and determine their associations with clinicopathological factors.Expression of the genes coding for PD-1 and PD-L1 was analyzed in formalin-fixed paraffin-embedded specimens using real-time PCR. mRNA expression levels were determined using beta actin (ACTB) as an endogenous control. There was a trend towards significance between higher PD-1 and PD-L1 levels in triple negative breast cancers (p=0.1). Higher PD-L1 expression was also found in aggressive breast cancer subtypes e.g. triple negative and HER2 (human epidermal growth factor receptor 2) -positive as compared with subtypes with better prognosis such as luminal A and luminal BHER2-negative (p=0.05). There was a trend towards significance in higher PD-1 levels in triple negative and HER-2 positive breast cancers (p=0.1). A statistically significant difference was found between PD-L1 expression and tumor grade (p=0.01). Elevated PD-L1 levels were noted in G3 tumors. Immunogenicity appears to be gaining importance in triple negative and HER2-positive molecular subtypes of breast cancer, and the results in this study provide a basis for further investigation into the role of immune checkpoints in breast cancer.