Project Title: „Clinical utility of extracellular vesicle-enclosed human and microbial transcriptomes”

Funding: European Regional Development Fund (ERDF), Measure 1.1.1.1 “Support for applied research”

Project No.: 1.1.1.1/18/A/084

Period: 1 April 2019 – 31 March 2022

Project costs: 648 648 EUR

Principle Investigator: Dr. biol. Aija Linē

Cooperation partner: SIA “GenEra”

Project summary:

Extracellular vesicles (EVs) circulating in human blood are sampled from various cell types in the body, including cancer. EVs isolated from cancer patients’ blood have been shown to contain cancer-specific molecules, therefore they may reveal the presence of cancer, help to predict its behaviour and track the progression of cancer. This project is based on the results of our previous studies showing that EVs contain not only various classes of RNAs derived from human cells but also RNA fragments of microbial origin. The main aim of this project is to gain new knowledge about the composition and origin of RNA cargo of EVs circulating in cancer patients’ blood, and to assess the clinical utility of EV-enclosed human and microbial RNAs for the diagnosis, prognosis and tracking disease progression in three distinct types of cancer – prostate, breast and gastric cancer.

We plan to expand and update the longitudinal Cancer Biobank comprising detailed clinical information, tumour tissue specimens, stool and blood samples from cancer patients. EVs will be isolated from plasma and stool samples of cancer patients and cancer-free controls and their RNA content will be sequenced together with small RNAs from matched tumour and normal tissues. Multiple data analyses will be performed to assess the correlation of EV-enclosed RNAs with various clinical and pathological features and to assess their diagnostic and prognostic value. Finally, digital PCR assays for selected biomarkers will be established and used to assess their clinical utility.

We expect, this project will lead to the discovery of novel RNA biomarkers of human and microbial origin that are associated with the presence, progression or drug response of cancer and are suitable for the development of blood-based tests. Currently, this technology is at TRL2 and we expect that at the end of the project it will reach TRL4.

Information published 01.04.2019.

Progress of the project:

1 April 2019 – 30 June 2019

• Development of a cohort of healthy controls is in progress;
• An application for the permission for testing EV biomarkers in the clinical samples of prostate cancer patients has been prepared and submitted to CMEK;
• Isolation of EVs from plasma samples has been started;
• Optimisation of the protocol for the construction and sequencing of small RNA libraries has been started;
• Development of the protocol for the isolation of EVs from fecal samples is in progress.

Information published 28.06.2019.

Progress of the project:

1 July 2019 – 30 September 2019

• Isolation of EVs from plasma samples of cancer patients and controls is ongoing.
• We are comparing various kits for the construction of small RNA libraries from ultra-low amount of input RNA in order to choose the optimal technology for RNASeq.
• We have developed a method for the isolation of EVs from faeces and currently working on the characterisation of the isolated EVs.

Information published 30.09.2019.

Progress of the project:

1 October 2019 – 31 December 2019

• RNA was extracted from plasma and urinary EVs and normal and cancerous prostate tissues from 4 PC patients.
• EVs were isolated from 2 faecal samples and the work on the characterisation of the EVs is ongoing.
• Forty-eight small RNA libraries were constructed from plasma and urinary EVs and tumour and adjacent normal prostate tissues. The libraries were sequenced on Illumina Nextseq instrument.

Information published 30.12.2019.

Progress of the project:

1 January 2020 – 31 March 2020

We continue to work on the isolation of EVs from the plasma of patients with prostate and breast cancer, quantification of EVs, RNA extraction and building of RNASeq libraries, however the work is slowed down by the delays with the delivery of reagents and materials. In the meantime, we are trying to optimize our RNAseq data analysis pipeline.

Information published 31.03.2020.

Progress of the project:

1 April 2020 – 30 June 2020

We have completed the isolation EVs from the plasma and urine of prostate cancer patients and the construction of RNASeq libraries. Currently, we are processing and analyzing the obtained RNAseq data and have started to work on the preparation of a manuscript. In parallel, we continue to work on the isolation of EVs from the blood and feces of healthy individuals as well as plasma samples of breast cancer patients.

Information published 30.06.2020.

Progress of the project:

1 July 2020 – 30 September 2020

During this period we focused on the RNA-seq data analysis in prostate cancer dataset. After the quality check and trimming, all reads were mapped against the human genome and the catalogue of RNA types was generated for all the samples. Various correlation and differential expression analyses were performed and a number of RNA biomarker candidates were identified. Next, these candidates will be validated by ddPCR. In parallel, we continue to work on the isolation of EVs and RNA from the plasma of breast cancer patients, building RNA-seq libraries and sequencing. Two manuscripts are in preparation.

Information published 30.09.2020.

Progress of the project:

1 October 2020 – 31 December 2020

During this period the enrolment of new patients and the expansion of biobank were started. We continued to work on the RNA-seq data analysis in prostate cancer dataset – the full transcriptome libraries generated from cancerous and normal prostate tissues were mapped against the human genome and the differential expression analyses were carried out to identify mRNAs and lncRNAs that were overexpressed in tumour tissues and present in pre-operation plasma and urinary EVs. We also have started to work on the detection of somatic mutations in tumour tissue and EV RNA-seq libraries. Moreover, we continued to work on the isolation of EVs and RNA from the plasma of breast cancer patients and the construction of RNA-seq libraries. Results were presented at the virtual symposium on Extracellular Vesicles in Pathophysiology. A review on circulating biomarkers for the detection of gastric cancer was prepared and submitted for publication, whereas the original article on the RNA content of EVs in prostate cancer is in the preparation.

Information published 30.12.2020.

Progress of the project:

1 January 2021 – 31 March 2021

During this period the enrolment of new patients and the expansion of biobank was continued. A set of potential RNA biomarker candidates representing various RNA classes was selected from the prostate cancer RNAseq data set and testing of these candidates in an independent validation cohort has been started. We continued to work on the isolation of EVs and RNA from plasma of breast cancer patients and the construction and sequencing of RNA-seq libraries. A review article: M Leja, A Linē. Early detection of gastric cancer beyond endoscopy-new methods. Best Practice & Research Clinical Gastroenterology, 2021, has been published.

Information published 31.03.2021.

Progress of the project:

1 April 2021 – 30 June 2021

During this period the enrolment of new patients with gastric and breast was continued. Testing of the selected RNA biomarker candidates in plasma and urinary EVs of prostate cancer patients was carried out by ddPCR. The work on the isolation of EVs and RNA from the plasma of breast cancer patients and the construction and sequencing of RNA-seq libraries was continued. Recently, we have started to analyse the obtained results.

Information published 30.06.2021.

Progress of the project:

1 July 2021 – 30 September 2021

During this period, the enrolment of new patients with gastric and breast cancer was continued. We also continued to work on the isolation of EVs and RNA from plasma of breast cancer patients, and construction and sequencing of RNA-seq libraries. In parallel, we started to work on the isolation of RNA from the breast cancer tissues and construction of transcriptome libraries. A manuscript on prostate cancer biomarkers is in the preparation.

Information published 30.09.2021.

Progress of the project:

1 October 2021 – 31 December 2021

During this period, the main focus was on the analysis of plasma EV RNA content in breast cancer patients – we continued to work on the construction of RNAseq libraries, sequencing and data analysis. We tested various methods for the deconvolution analysis and detection of tumour somatic mutations in the EV RNA. The enrolment of new patients with gastric and breast cancer was also continued. Two manuscripts on prostate and breast cancer biomarkers are in preparation.

Information published 30.12.2021.

Progress of the project:

1 January 2022 – 31 March 2022

The enrolment of patients in this study has been completed. During this period, the construction and sequencing of RNA seq libraries from plasma EVs of breast and gastric cancer patients have been completed. Currently, we’re working on the analysis and interpretation of the obtained results. Results have been presented at four international conferences. Three original articles summarizing the main results of this project are in preparation.

Information published 31.03.2022.