Detecting cell-of-origin and cancer-specific methylation features of cell-free DNA from Nanopore sequencing

: The Oxford Nanopore (ONT) platform provides portable and rapid genome sequencing, and its ability to natively profile DNA methylation without complex sample processing is attractive for point-of-care real-time sequencing. We recently demonstrated ONT shallow whole-genome sequencing to detect copy number alterations (CNAs) from the circulating tumor DNA (ctDNA) of cancer patients. Here, we show that cell type and cancer-specific methylation changes can also be detected, as well as cancer-associated fragmentation signatures. This feasibility study suggests that ONT shallow WGS could be a powerful tool for liquid biopsy

乳兒に於ける化膿性骨髄炎の統計的觀察

Additional file 3: Figure S1. Methylation percentages of the two genes (CASP8 and SCGB3A1) in P, C and PCa samples that were differentially methylated in C and P samples in the training set. The figure highlights a higher methylation percentage for PCa than for healthy samples, but also a higher methylation percentage for P than for C samples in both genes

Sequencing-based approaches for the study of Lung-related diseases

My thesis is focused on sequencing-based methods for lung diseases monitoring. Modern sequencing techniques allow us to comprehensively characterize nucleic acids obtained from patient-derived biological material, with potential applications in both basic research and clinical practice. The thesis is divided in two sections: In Section 1: “Evidence for host-dependent RNA editing in the transcriptome of SARS-CoV-2” I describe the presence of RNA editing events in SARS-CoV-2, by analysing publicly available second generation RNA sequencing data from infected patients: Emerging viral infections represent a threat to global health, and the recent outbreak of novel coronavirus disease 2019 caused by SARS-CoV-2 exemplifies the risks. RNA editing is a physiological mechanism mediated by two enzyme families: APOBEC and ADAR, which introduce A-to-I and C-to-U mutations in double strand and single strand RNA respectively. RNA editing typically involves endogenous RNAs but, if targeting viral RNA, it is potentially deleterious for virus’ viability itself, by generating premature stop codons and missense mutations in the viral genome. On the other hand, RNA editing could fuel virus evolution by increasing the basal mutational rate. I have downloaded publicly available Illumina transcriptomic data, from BALF samples of infected patients; using a combination of published tools (Reditools2, JACUSA) I have detected an enrichment of APOBEC and ADAR related mutation on SARS-CoV-2 RNA. A similar enrichment was observed in genomic RNA SARS-CoV-2, SARS and MERS sequences downloaded from GISAID and NCBI virus. The evidence of RNA editing on SARS-CoV-2 suggests that APOBEC and ADAR can interact with viral RNAs, probably with an anti-viral purpose. C-to-U changes leading to stop codons are overrepresented in the transcriptomic data but—as expected—disappear in the genomic dataset. This might point—again—to an antiviral role for these editing enzymes. Also, the proportion of ADAR-related mutation was unexpectedly lower in genomic RNA sequences, compared to transcriptomic data. It is possible that A-to-I editing is somehow restricting viral propagation, thus reducing the number of viral progeny showing evidence of these changes. In Section 2: “Analysis of copy number variations from cell-free DNA of lung cancer patients via Nanopore sequencing” I have developed a customized workflow to exploit Nanopore sequence for the analysis of plasmatic cell-free DNA: Cancer is an extremely dynamic disease: malignant cells are constantly under selective pressure and the evolutionary path of each tumor can take different directions due to such pressure. It is hence important to monitor cancer development at multiple timepoints to closely follow its evolution; unfortunately, the risks and invasiveness of conventional biopsy make it unsuitable for repeated sampling. A valid and non-invasive alternative to tissue sampling is represented by the analysis of cfDNA from liquid biopsy samples (plasma). CNVs are an important class of genetic alterations that can affect tumor aggressivity and resistance to treatment. To date, the only reliable approach to obtain a whole-genome CNV profile from plasmatic cfDNA is Illumina sequencing. However the need for expensive sequencers is often an obstacle for smaller laboratories. Oxford Nanopore Technologies has recently released MinION: a fast and extremely inexpensive third generation sequencer based on the Nanopore technology. However, this technology is not designed for low quality DNA such as cfDNA (very fragmented, low concentration). I have modified Nanopore standard protocols to make them compatible with the characteristics of cfDNA. The technique has been tested on plasma samples obtained from lung cancer patients, with the aim of detecting tumor-specific copy number variations. The approach has been subsequently validated by comparing it with the current standard technique (Illumina). Nanopore and Illumina results strongly correlate (R = 0.96 – 0.99, p << 0.001), with concordant log2ratio values in 97-99% of genome positions. Nanopore features (i.e. reduced costs) represent advantages over current sequencing technologies, and might drive the adoption of molecular karyotyping from liquid biopsies as a tool for cancer monitoring in clinical settings

Carcinosarcoma of the prostate: case report with molecular and histological characterization

We report a case of prostatic carcinosarcoma, a rare variant of prostatic cancer, which is composed of a mixture of epithelial and mesenchymal components with a generally poor outcome

GSTP1 Methylation and Protein Expression in Prostate Cancer: Diagnostic Implications

GSTP1 belongs to the GSTs family, a group of enzymes involved in detoxification of exogenous substances and it also plays an important role in cell cycle regulation. Its dysregulation correlates with a large variety of tumors, in particular with prostate cancer. We investigated GSTP1 methylation status with methylation specific PCR (MS-PCR) in prostate cancer (PCa) and in benign tissue of 56 prostatectomies. We also performed immunohistochemistry (IHC) so as to correlate gene methylation with gene silencing. GSTP1 appears methylated in PCa and not in healthy tissue; IHC confirmed that methylation leads to protein underexpression (p<0.001). GSTP1 is highly expressed in basal cell layer and luminal cells in benign glands while in prostatic intraepithelial neoplasia (PIN) it stains only basal cell layer, whereas PCa glands are completely negative. We demonstrated that methylation leads to underexpression of GSTP1. The progressive loss of GSTP1 expression from healthy glands to PIN and to PCa glands underlines its involvement in early carcinogenesis

Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients

Introduction. The detection of tumor-specific markers in urine has paved the way for new early noninvasive diagnostic approaches for prostate cancer. We evaluated the DNA integrity in urine supernatant to verify its capacity to discriminate between prostate cancer and benign diseases of the urogenital tract. Patients and Methods. A total of 131 individuals were enrolled: 67 prostate cancer patients and 64 patients with benign diseases of the urogenital tract (control group). Prostate-specific antigen (PSA) levels were determined. Urine cell-free (UCF) DNA was isolated and sequences longer than 250 bp corresponding to 3 genes (c-MYC, HER2, and AR) were quantified by Real-Time PCR to assess UCF-DNA integrity. Results. UCF-DNA was quantifiable in all samples, while UCF-DNA integrity was evaluable in all but 16 samples. Receiver operating characteristic analysis showed an area under the curve of 0.5048 for UCF-DNA integrity and 0.8423 for PSA. Sensitivity was 0.58 and 0.95 for UCF-DNA integrity and PSA, respectively. Specificity was 0.44 and 0.69, respectively. Conclusions. UCF-DNA integrity showed lower accuracy than PSA and would not seem to be a reliable marker for early prostate cancer diagnosis. Despite this, we believe that UCF-DNA could represent a source of other biomarkers and could detect gene alterations

Immunotherapy for Prostate Cancer: Where We Are Headed

Prostate cancer is one of the most common malignant neoplasms in men worldwide, and is the fifth cause of cancer-related death. In recent years, a new generation of therapies have been approved for the management of metastatic disease. Moreover, the development of new immunotherapeutic drugs has become a novel frontier for the treatment of several tumor types; to date, numerous studies have investigated their potential activity, including in prostate cancer. In this article, we discuss the role of emerging immunotherapeutic drugs in prostate cancer patients