Growth arrest-specific 5 lncRNA as a valuable biomarker of chemoresistance in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumour in children and teenagers, and it is characterised by drug resistance and high metastatic potential. Increasing studies have highlighted the critical roles of long noncoding RNAs (lncRNAs) as oncogenes or tumour suppressors as well as new biomarkers and therapeutic targets in osteosarcoma. The growth arrest-specific 5 (GAS5) lncRNA can function as a tumour suppressor in several cancers. The present study aimed to validate GAS5 and other chemoresistance-associated lncRNAs as biomarkers in a cohort of primary osteosarcoma samples, to obtain predictive information on resistance or sensitivity to treatment. The GAS5 and a panel of lncRNAs related to chemoresistance [SNGH1, FOXD2-AS1, deleted in lymphocytic leukemia (DLEU2) and LINC00963] were evaluated in a cohort of osteosarcoma patients enrolled at the Careggi University Hospital. Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue sections and the expression levels of the lncRNAs were quantified by qPCR. A bioinformatic analysis on deposited RNA-seq data was performed to validate the qPCR results. Clustering analysis shows that GAS5 could be linked to the expression of isoforms 02 and 04 of the lncRNA DLEU2, whereas the DLEU2 isoform 08 is linked to the lncRNA LINC00963. We found that GAS5 is significantly increased in patients with a good prognosis and is expressed differently between chemosensitive and chemoresistant osteosarcoma patients. However, the results obtained are not concordant with the in-silico analysis performed on the TARGET osteosarcoma dataset. In the future, we would enlarge the case series, including different disease settings.

Nanopore sequencing from liquid biopsy: analysis of copy number variations from cell-free DNA of lung cancer patients.

In the "precision oncology" era the characterization of tumor genetic features is a pivotal step in cancer patients' management. Liquid biopsy approaches, such as analysis of cell-free DNA from plasma, represent a powerful and noninvasive strategy to obtain information about the genomic status of the tumor. Sequencing-based analyses of cell-free DNA, currently performed with second generation sequencers, are extremely powerful but poorly scalable and not always accessible also due to instrumentation costs. Third generation sequencing platforms, such as Nanopore sequencers, aim at overcoming these obstacles but, unfortunately, are not designed for cell-free DNA analysis.Here we present a customized workflow to exploit low-coverage Nanopore sequencing for the detection of copy number variations from plasma of cancer patients. Whole genome molecular karyotypes of 6 lung cancer patients and 4 healthy subjects were successfully produced with as few as 2 million reads, and common lung-related copy number alterations were readily detected.This is the first successful use of Nanopore sequencing for copy number profiling from plasma DNA. In this context, Nanopore represents a reliable alternative to Illumina sequencing, with the advantages of minute instrumentation costs and extremely short analysis time.The availability of protocols for Nanopore-based cell-free DNA analysis will make this analysis finally accessible, exploiting the full potential of liquid biopsy both for research and clinical purposes.

GSTP1 methylation in cancer: a liquid biopsy biomarker?

The coding region of GSTP1 gene is preceded by a large CpG-rich region that is frequently affected by methylation. In many cancer types, GSTP1 is affected by hypermethylation and, as a consequence, it has a low expression. The aim of this review is to give an overview on GSTP1 methylation studies with a special focus on liquid biopsy, thus to summarize methods, results, sample types, different diseases, to have a complete information regarding this promising epigenetic biomarker. We used all the most valuable scientific search engines (PubMed, Medline, Scopus and Web of Science) searching the following keywords: GSTP1, methylation, cancer, urine, serum, plasma and blood. GSTP1 is a largely investigated tissue biomarker in several malignancies such as prostate, breast, lung and hepatocellular carcinoma with good performances especially for diagnostic purposes. As a liquid biopsy biomarker, it has been mainly investigated in prostate cancer (PCa) where it showed a high specificity but a low sensitivity; thus, it is recommended in combination with other biomarkers. Despite the large number of published papers and the promising results, GSTP1 has not yet entered the clinical practice even for PCa diagnosis. For this reason, further large and prospective studies are needed to validate this assay.

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.

Methylation pattern analysis in prostate cancer tissue: identification of biomarkers using an MS-MLPA approach.

BACKGROUND: Epigenetic silencing mediated by CpG island methylation is a common feature of many cancers. Characterizing aberrant DNA methylation changes associated with prostate carcinogenesis could potentially identify a tumour-specific methylation pattern, facilitating the early diagnosis of prostate cancer. The objective of the study was to assess the methylation status of 40 tumour suppressor genes in prostate cancer and healthy prostatic tissues. METHODS: We used methylation specific-multiplex ligation probe amplification (MS-MLPA) assay in two independent case series (training and validation set). The training set comprised samples of prostate cancer tissue (n = 40), healthy prostatic tissue adjacent to the tumor (n = 26), and healthy non prostatic tissue (n = 23), for a total of 89 DNA samples; the validation set was composed of 40 prostate cancer tissue samples and their adjacent healthy prostatic tissue, for a total of 80 DNA samples. Methylation specific-polymerase chain reaction (MSP) was used to confirm the results obtained in the validation set. RESULTS: We identified five highly methylated genes in prostate cancer: GSTP1, RARB, RASSF1, SCGB3A1, CCND2 (P < 0.0001), with an area under the ROC curve varying between 0.89 (95 % CI 0.82-0.97) and 0.95 (95 % CI 0.90-1.00). Diagnostic accuracy ranged from 80 % (95 % CI 70-88) to 90 % (95 % CI 81-96). Moreover, a concordance rate ranging from 83 % (95 % CI 72-90) to 89 % (95 % CI 80-95) was observed between MS-MLPA and MSP. CONCLUSIONS: Our preliminary results highlighted that hypermethylation of GSTP1, RARB, RASSF1, SCGB3A1 and CCND2 was highly tumour-specific in prostate cancer tissue.

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.

Analysis of Copy Number Variation in Urine: c-Myc Evaluation Using a Real-Time PCR Approach.

Urine cell-free DNA has been shown as an informative noninvasive source of biomarkers for a number of diseases, especially for urological cancers. Starting from the hypothesis that the gain of c-Myc gene is a frequent aberration in several cancer types, including prostate cancer, we analyzed c-Myc copy number variation in urine, studying a little case series of prostate cancer patients, to test its feasibility. Here we report a general protocol that may be considered to analyze gene copy number variation in the urine cell-free fraction.

Evidence for host-dependent RNA editing in the transcriptome of SARS-CoV-2.

The COVID-19 outbreak has become a global health risk, and understanding the response of the host to the SARS-CoV-2 virus will help to combat the disease. RNA editing by host deaminases is an innate restriction process to counter virus infection, but it is not yet known whether this process operates against coronaviruses. Here, we analyze RNA sequences from bronchoalveolar lavage fluids obtained from coronavirus-infected patients. We identify nucleotide changes that may be signatures of RNA editing: adenosine-to-inosine changes from ADAR deaminases and cytosine-to-uracil changes from APOBEC deaminases. Mutational analysis of genomes from different strains of Coronaviridae from human hosts reveals mutational patterns consistent with those observed in the transcriptomic data. However, the reduced ADAR signature in these data raises the possibility that ADARs might be more effective than APOBECs in restricting viral propagation. Our results thus suggest that both APOBECs and ADARs are involved in coronavirus genome editing, a process that may shape the fate of both virus and patient.

Cell-Free DNA: An Overview of Sample Types and Isolation Procedures.

The study of cell-free DNA (cfDNA) is often challenging due to genomic DNA contamination, low concentration, and high fragmentation. Therefore, it is important to optimize pre-analytical and analytical procedures in order to maximize the performance of cfDNA-based analyses.In this chapter, we report the most common methods for the correct collection, centrifugation, storage, and DNA isolation from cell-free biological sources such as plasma, urines, cerebrospinal fluid, and pleural effusion fluid.

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

BACKGROUND:: 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. AIMS AND METHODS:: We aim to identify molecular alterations, in particular copy number variations of AR and c -MYC genes, methylation and expression of glutathione S-transferase P1 (GSTP1), programmed death-ligand 1 (PD-L1), AR, and phosphorylated AR expression. RESULTS:: We found a distinct molecular pattern between adenocarcinoma and carcinosarcoma, which was characterized by high AR copy number variation gain; positive expression of PD-L1, AR, and phosphorylated AR; low espression of GSTP1 in epithelial component. The sarcomatoid component had a lower gain of the AR gene, and no expression of PD-L1, AR, phosphorylated AR, or GSTP1. Both components had a gain of c-MYC copy number variation. CONCLUSIONS:: Our findings suggest that carcinosarcoma has specific molecular characteristics that could be indicative for early diagnosis and treatment selection.

Cell-Free DNA Integrity Analysis in Urine Samples.

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals. A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

Serum and Plasma Copy Number Detection Using Real-time PCR.

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker. We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

Urinary RNA-based biomarkers for prostate cancer detection.

Prostate cancer (PCa) is the commonest malignancy in the male population worldwide. Serum prostate specific antigen (PSA) test is the most important biomarker for the detection, follow-up and therapeutic monitoring of PCa. Defects in PSA specificity have elicited research for new biomarkers to improve early diagnosis and avoid false-positive results. This review evaluates urinary RNA-based biomarkers. Urine is a versatile body fluid for non-invasive biomarker detection in case of urological malignancies. The importance of RNA-based biomarkers has been demonstrated by the current use of PCA3, a long non coding RNA biomarker already approved by the Food and Drugs Administration. Through the years, other urinary RNA biomarkers have been evaluated, including the well-known TMPRSS2:ERG transcript, as well as many messenger RNAs, long non coding RNAs and micro-RNA. Validation of a specific urinary RNA-based marker or an algorithm of different biomarkers levels as diagnostic markers for PCa could be useful to avoid unnecessary prostate biopsies.

Cell-free DNA detected by "liquid biopsy" as a potential prognostic biomarker in early breast cancer.

As conventional biomarkers for defining breast cancer (BC) subtypes are not always capable of predicting prognosis, search for new biomarkers which can be easily detected by liquid biopsy is ongoing. It has long been known that cell-free DNA (CF-DNA) could be a promising diagnostic and prognostic marker in different tumor types, although its prognostic value in BC is yet to be confirmed. This retrospective study evaluated the prognostic role of CF-DNA quantity and integrity of HER2, MYC, BCAS1 and PI3KCA, which are frequently altered in BC. We collected 79 serum samples before surgery from women at first diagnosis of BC at Forlì Hospital (Italy) from 2002 to 2010. Twenty-one relapsed and 58 non-relapsed patients were matched by subtype and age. Blood samples were also collected from 10 healthy donors. All samples were analyzed by Real Time PCR for CF-DNA quantity and integrity of all oncogenes. Except for MYC, BC patients showed significantly higher median values of CF-DNA quantity (ng) than healthy controls, who had higher integrity and lower apoptotic index. A difference nearing statistical significance was observed for HER2 short CF-DNA (p = 0.078, AUC value: 0.6305). HER2 short CF-DNA showed an odds ratio of 1.39 for disease recurrence with p = 0.056 (95% CI 0.991-1.973). Our study suggests that CF-DNA detected as liquid biopsy could have great potential in clinical practice once demonstration of its clinical validity and utility has been provided by prospective studies with robust assays.

Impact of Non-Pulmonary Visceral Metastases in the Prognosis and Practice of Metastatic Testicular Germ Cell Tumors.

Non-pulmonary visceral metastases, in bones, brain and liver, represent nearly the 10% of metastatic sites of advanced germ cell tumors and are associated with poor prognosis. This review article summarizes major evidences on the impact of different visceral sites on the prognosis, treatment and clinical outcome of patients with germ cell tumors. The clinic-biological mechanisms by which these metastatic sites are associated with poor clinical outcome remain unclear. The multimodality treatment showed a potential better survival, in particular in patients with relapsed disease. Patients with advanced germ cell tumors with visceral metastases should be referred to centers with high expertise in the clinical management of such disease.

The potential use of urine cell free DNA as a marker for cancer.

INTRODUCTION: Although the role of circulating cell free DNA in cancer has been widely demonstrated, less is known about the role of urine cell free DNA (UcfDNA). UcfDNA can serve as a 'liquid biopsy' for urological and non-urological tumors, as it carries information on DNA from cells exfoliated in urine and from circulation. Areas covered: We review the studies on UcfDNA as a source of biomarkers for cancer, focusing on the new techniques and the differences between urological and non-urological tumors. We searched Pubmed for articles published between 1998 and 2016 with the following key words and phrases: 'urine' and 'cell free DNA' or 'liquid biopsy' or 'cancer'. Expert commentary: Despite the few papers published on this topic, UcfDNA is an important component of 'liquid biopsy', a useful and non-invasive tool for cancer diagnosis, prognosis and treatment monitoring, containing a wide range of genetic information.

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.

Circulating AR copy number and outcome to enzalutamide in docetaxel-treated metastatic castration-resistant prostate cancer.

In the present study, we aimed to evaluate the association of circulating AR copy number (CN) and outcome in a cohort of patients with advanced castration-resistant prostate cancer (CRPC) treated with enzalutamide after docetaxel. Fifty-nine CRPC patients were evaluated. AR CN was analyzed with real-time and digital PCR in the serum collected at starting of treatment. Progressive disease was defined on the basis of Prostate Cancer Working Group 2 criteria. AR CN gain was found in 21 of 59 (36%) patients. Median baseline PSA, alkaline phosphatase and lactate dehydrogenase levels were higher in the AR CN gained group (p = 0.007, p = 0.003, p = 0.0009, respectively). Median PFS of patients with AR CN gain was 2.4 (95%CI: 1.9-3.2) vs. 4.0 months (95%CI: 3.0-6.5) of those with no gain (p = 0.0004). Median OS of patients with AR CN gain was 6.1 (95%CI: 3.4-8.6) vs. 14.1 months (95%CI: 8.2-20.5) of those with no gain (p = 0.0003). At multivariate analysis, PSA decline ≥ 50% and AR CN showed a significant association with PFS (p = 0.008 and p = 0.002, respectively) and OS (p = 0.009 and p = 0.001, respectively). These findings indicate that the detection of circulating AR CN gain is a promising non-invasive biomarker for outcome prediction to enzalutamide treatment in CRPC patients.

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.