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Background Patients with glioblastoma (GBM) have a dramatically poor prognosis. The recent REGOMA trial suggested an overall survival (OS) benefit of regorafenib in recurrent GBM patients. Considering the extreme genetic heterogeneity of GBMs, we aimed to identify molecular biomarkers predictive of differential response to the drug. Methods Total RNA was extracted from tumor samples of patients enrolled in the REGOMA trial. Genome-wide transcriptome and micro (mi)RNA profiles were associated with patients’ OS and progression-free survival. Results In the first step, a set of 11 gene transcripts (HIF1A, CTSK, SLC2A1, KLHL12, CDKN1A, CA12, WDR1, CD53, CBR4, NIFK-AS1, RAB30-DT) and 10 miRNAs (miR-93-5p, miR-203a-3p, miR-17-5p, let-7c-3p, miR-101-3p, miR-3607-3p, miR-6516-3p, miR-301a-3p, miR-23b-3p, miR-222-3p) was filtered by comparing survival between regorafenib and lomustine arms. In the second step, a mini-signature of 2 gene transcripts (HIF1A, CDKN1A) and 3 miRNAs (miR-3607-3p, miR-301a-3p, miR-93-5p) identified a subgroup of patients showing prolonged survival after regorafenib administration (median OS range, 10.6–20.8 mo). Conclusions The study provides evidence that a signature based on the expression of 5 biomarkers could help identify a subgroup of GBM patients exhibiting a striking survival advantage when treated with regorafenib. Although the presented results must be confirmed in larger replication cohorts, the study highlights potential biomarker options to help guide the clinical decision among regorafenib and other treatments in patients with relapsing GBM.

Traditional methods for the analysis of repeat expansions, which underlie genetic disorders, such as fragile X syndrome (FXS), lack single-nucleotide resolution in repeat analysis and the ability to characterize causative variants outside the repeat array. These drawbacks can be overcome by long-read and short-read sequencing, respectively. However, the routine application of next-generation sequencing in the clinic requires target enrichment, and none of the available methods allows parallel analysis of long-DNA fragments using both sequencing technologies. In this study, we investigated the use of indirect sequence capture (Xdrop technology) coupled to Nanopore and Illumina sequencing to characterize FMR1, the gene responsible of FXS. We achieved the efficient enrichment (> 200×) of large target DNA fragments (~60–80 kbp) encompassing the entire FMR1 gene. The analysis of Xdrop-enriched samples by Nanopore long-read sequencing allowed the complete characterization of repeat lengths in samples with normal, pre-mutation, and full mutation status (> 1 kbp), and correctly identified repeat interruptions relevant for disease prognosis and transmission. Single-nucleotide variants (SNVs) and small insertions/deletions (indels) could be detected in the same samples by Illumina short-read sequencing, completing the mutational testing through the identification of pathogenic variants within the FMR1 gene, when no typical CGG repeat expansion is detected. The study successfully demonstrated the parallel analysis of repeat expansions and SNVs/indels in the FMR1 gene at single-nucleotide resolution by combining Xdrop enrichment with two next-generation sequencing approaches. With the appropriate optimization necessary for the clinical settings, the system could facilitate both the study of genotype–phenotype correlation in FXS and enable a more efficient diagnosis and genetic counseling for patients and their relatives.

Mummified remains of relevant historical figures are nowadays an important source of information to retrace data concerning their private life and health, especially when historical archives are not available. Next-generation-sequencing was proved to be a valuable tool to unravel the characteristics of these individuals through their genetic heritage. Using the strictest criteria currently available for the validation of ancient DNA sequences, whole-genome and whole-exome sequencing were generated from the mummy remains of an Italian nobleman died almost 700 years ago, Cangrande della Scala. While its genome sequencing could not yield sufficient coverage for in depth investigation, exome sequencing could overcome the limitations of this approach to achieve significantly high coverage on coding regions, thus allowing to perform the first extensive exome analysis of a mummy genome. Similar to a standard “clinical exome analysis” conducted on modern DNA, an in-depth variant annotation, high-quality filtering and interpretation was performed, leading to the identification of a genotype associated with late-onset Pompe disease (glycogen storage disease type II). This genetic diagnosis was concordant with the limited clinical history available for Cangrande della Scala, who likely represents the earliest known case of this autosomal recessive metabolic disorder.

Neuroblastoma (NB) is an aggressive infancy tumor, leading cause of death among preschool age diseases. Here we focused on characterization of exosomal DNA (exo-DNA) isolated from plasma cell-derived exosomes of neuroblastoma patients, and its potential use for detection of somatic mutations present in the parental tumor cells. Exosomes are small extracellular membrane vesicles secreted by most cells, playing an important role in intercellular communications. Using an enzymatic method, we provided evidence for the presence of double-stranded DNA in the NB exosomes. Moreover, by whole exome sequencing, we demonstrated that NB exo-DNA represents the entire exome and that it carries tumor-specific genetic mutations, including those occurring on known oncogenes and tumor suppressor genes in neuroblastoma (ALK, CHD5, SHANK2, PHOX2B, TERT, FGFR1, and BRAF). NB exo-DNA can be useful to identify variants responsible for acquired resistance, such as mutations of ALK, TP53, and RAS/MAPK genes that appear in relapsed patients. The possibility to isolate and to enrich NB derived exosomes from plasma using surface markers, and the quick and easy extraction of exo-DNA, gives this methodology a translational potential in the clinic. Exo-DNA can be an attractive non-invasive biomarker for NB molecular diagnostic, especially when tissue biopsy cannot be easily available.