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DNA methylation alterations are early events during tumourigenesis, affecting genes involved in the crosstalk between cells and surroundings in colorectal cancer (CRC). Among these genes, GRIA4, Glutamate Ionotropic Receptor AMPA Type Subunit 4, displays hypermethylation in the promoter region, and is an early diagnostic biomarker. It is well known that methylation can also affect alternative transcription. The purpose of this study is to evaluate the expression, at transcript and protein level, of GRIA4 main isoforms (the canonical one and a short variant) in 23 CRC and matched normal samples, of which we previously verified the methylation status. We further predicted miRNA/transcript target interactions as a possible post-transcriptional regulation using bioinformatics tools. As expected, downregulation of both variants has been observed in tumours. Interestingly, in contrast to what observed at transcriptional level, the GluR4 protein short isoform displayed higher expression than the canonical one either in normal or tumoural tissues. This may be explained by miRNA specifically targeting the canonical isoform. Our study is the first one that shows the expression of both isoforms in colon tissues. To note, the evident expression of the short isoform suggests a functional role in intestinal cell biology.

Background Biliary tract cancers (BTC) are rare but highly aggressive tumours with poor prognosis, usually detected at advanced stages. Herein, we aimed at identifying BTC-specific DNA methylation alterations. Methods Study design included statistical power and sample size estimation. A genome-wide methylation study of an explorative cohort (50 BTC and ten matched non-tumoral tissue samples) has been performed. BTC-specific altered CpG islands were validated in over 180 samples (174 BTCs and 13 non-tumoral controls). The final biomarkers, selected by a machine-learning approach, were validated in independent tissue (18 BTCs, 14 matched non-tumoral samples) and bile (24 BTCs, five non-tumoral samples) replication series, using droplet digital PCR. Results We identified and successfully validated BTC-specific DNA methylation alterations in over 200 BTC samples. The two-biomarker panel, selected by an in-house algorithm, showed an AUC > 0.97. The best-performing biomarker (chr2:176993479-176995557), associated with HOXD8, a pivotal gene in cancer-related pathways, achieved 100% sensitivity and specificity in a new series of tissue and bile samples. Conclusions We identified a novel fully efficient BTC biomarker, associated with HOXD8 gene, detectable both in tissue and bile by a standardised assay ready-to-use in clinical trials also including samples from non-invasive matrices.

Colorectal cancer (CRC) develops through the accumulation of both genetic and epigenetic alterations. However, while the former are already used as prognostic and predictive biomarkers, the latter are less well characterized. Here, performing global methylation analysis on both CRCs and adenomas by Illumina Infinium HumanMethylation450 Bead Chips, we identified a panel of 74 altered CpG islands, demonstrating that the earliest methylation alterations affect genes coding for proteins involved in the crosstalk between cell and surrounding environment. The panel discriminates CRCs and adenomas from peritumoral and normal mucosa with very high specificity (100%) and sensitivity (99.9%). Interestingly, over 70% of the hypermethylated islands resulted in downregulation of gene expression. To establish the possible usefulness of these non-invasive markers for detection of colon cancer, we selected three biomarkers and identified the presence of altered methylation in stool DNA and plasma cell-free circulating DNA from CRC patients.

Background Clustered protocadherins (PCDHs) map in tandem at human chromosome 5q31 and comprise three multi-genes clusters: α-, β- and γ-PCDH. The expression of this cluster consists of a complex mechanism involving DNA hub formation through DNA-CCTC binding factor (CTCF) interaction. Methylation alterations can affect this interaction, leading to transcriptional dysregulation. In cancer, clustered PCDHs undergo a mechanism of long-range epigenetic silencing by hypermethylation. Results In this study, we detected frequent methylation alterations at CpG islands associated to these clustered PCDHs in all the solid tumours analysed (colorectal, gastric and biliary tract cancers, pilocytic astrocytoma), but not hematologic neoplasms such as chronic lymphocytic leukemia. Importantly, several altered CpG islands were associated with CTCF binding sites. Interestingly, our analysis revealed a hypomethylation event in pilocytic astrocytoma, suggesting that in neuronal tissue, where PCDHs are highly expressed, these genes become hypomethylated in this type of cancer. On the other hand, in tissues where PCDHs are lowly expressed, these CpG islands are targeted by DNA methylation. In fact, PCDH-associated CpG islands resulted hypermethylated in gastrointestinal tumours. Conclusions Our study highlighted a strong alteration of the clustered PCDHs methylation pattern in the analysed solid cancers and suggested these methylation aberrations in the CpG islands associated with PCDH genes as powerful diagnostic biomarkers. Electronic supplementary material The online version of this article (10.1186/s13148-019-0695-0) contains supplementary material, which is available to authorized users.

Pilocytic astrocytoma (PA) is the most common glioma in pediatric patients and occurs in different locations. Chromosomal alterations are mostly located at chromosome 7q34 comprising the BRAF oncogene with consequent activation of the mitogen-activated protein kinase pathway. Although genetic and epigenetic alterations characterizing PA from different localizations have been reported, the role of epigenetic alterations in PA development is still not clear. The aim of this study was to investigate whether distinctive methylation patterns may define biologically relevant groups of PAs. Integrated DNA methylation analysis was performed on 20 PAs and 4 normal brain samples by Illumina Infinium HumanMethylation27 BeadChips. We identified distinct methylation profiles characterizing PAs from different locations (infratentorial vs supratentorial) and tumors with onset before and after 3 years of age. These results suggest that PA may be related to the specific brain site where the tumor arises from region-specific cells of origin. We identified and validated in silico the methylation alterations of some CpG islands. Furthermore, we evaluated the expression levels of selected differentially methylated genes and identified two biomarkers, one, IRX2, related to the tumor localization and the other, TOX2, as tumoral biomarker.

The KIAA1549:BRAF fusion gene is considered a driver genetic event in pilocytic astrocytoma. We investigated a series of 69 pediatric brain neoplasms of diverse histogenesis and grade using the RT-PCR and sequencing. We detected the KIAA1549:BRAF fusion gene in five of 34 non-PA tumors (14.7%), that is, one glioblastoma, one anaplastic astrocytoma, one anaplastic pleomorphic xanthoastrocytoma, 1 ependymoma, and 1 Atypical Teratoid Rhabdoid Tumor. Our study showed that the K-B, although uncommon, it can be detected in non-PA tumors of various histogenesis and grading.

KIAA1549-BRAF fusion gene and isocitrate dehydrogenase (IDH) mutations are considered two mutually exclusive genetic events in pilocytic astrocytomas and diffuse gliomas, respectively. We investigated the presence of theKIAA1549-BRAF fusion gene in conjunction with IDH mutations and 1p/19q loss in 185 adult diffuse gliomas. Moreover BRAF v600E mutation was also screened. TheKIAA1549-BRAF fusion gene was evaluated by reverse-transcription polymerase chain reaction (RT-PCR) and sequencing. We found IDH mutations in 125 out 175 cases (71.4%). There were KIAA1549-BRAF fusion gene in 17 out of 180 (9.4%) cases and BRAF v600E in 2 out of 133 (1.5%) cases. In 11 of these 17 cases, both IDH mutations and the KIAA1549-BRAF fusion were present, as independent molecular events. Moreover, 6 of 17 cases showed co-presence of 1p/19q loss, IDH mutations and KIAA1549-BRAF fusion. Among the 17 cases with KIAA1549-BRAF fusion gene 15 (88.2%) were oligodendroglial neoplasms. Similarly, the two cases with BRAF v600E mutation were both oligodendroglioma and one had IDH mutations and 1p/19q co-deletion. Our results suggest that in a small fraction of diffuse gliomas, KIAA1549-BRAF fusion gene and BRAF v600E mutation may be responsible for deregulation of the Ras-RAF-ERK signaling pathway. Such alterations are more frequent in oligodendroglial neoplasm and may be co-present with IDH mutations and 1p/19q loss.

Background Ganglioglioma (GG) and pilocytic astrocytoma (PA) represent the most frequent low-grade gliomas (LGG) occurring in paediatric age. LGGs not amenable of complete resection (CR) represent a challenging subgroup where traditional treatments often fail. Activation of the MAP Kinase (MAPK) pathway caused by the BRAFV600E mutation or the KIAA1549-BRAF fusion has been reported in pediatric GG and PA, respectively. Case presentation We report on a case of BRAFV600E mutated cervicomedullary GG treated with standard chemotherapy and surgery. After multiple relapse, BRAF status was analyzed by immunohistochemistry and sequencing showing a BRAFV600E mutation. Treatment with Vemurafenib as single agent was started. For the first time, a radiological and clinical response was obtained after 3 months of treatment and sustained after 6 months. Conclusion Our experience underline the importance of understanding the driver molecular alterations of LGG and suggests a role for Vemurafenib in the treatment of pediatric GG not amenable of complete surgical resection.

INTRODUCTION: Pediatric GBMs (pGBM) are histologically indistinguishable from adult GBM (aGBM) however they are molecularly distinct. Few molecular data exists on the mechanism underlying development and progression of pGBM, whereas a number of gene expression profiling analyses performed in aGBM helped to identify molecular events leading to oncogenesis and provided more accurate means for classification of subtypes, outcomes and even response to treatments. The KIAA1549:BRAF fusion (K-B) gene has been considered a driver of genetic events and a useful diagnostic marker in adult pilocytic astrocytoma (PA). The K-B fusion, on the other hand, is an unusual finding in pediatric diffuse astrocytomas and oligoastocytoma and not reported in pHGG On the other hand, it has been reported a low frequency of B_F fusion gene in adult diffuse gliomas in association with IDH1 mutations. METHODS: To further elucidate the possible occurrence of the K-B fusion gene in non-PA tumors, we investigated its presence in a series of 69 pediatric brain neoplasms of different histogenesis and grade (34 non PA and 35 PA). Fusion has been investigated through the analysis of messenger RNA by reverse-transcription polymerase chain reaction (RT-PCR) followed by sequencing. Selected cases were screened for mutational analysis for IDH1, BRAFv600 and H3F3A. Positive cases have undergone to confirmation by the interphase Fluorescence in situ Hybridization (FISH) analysis and quantitative PCR (Taqman). RESULTS: We detected the KIAA1549:BRAF fusion (K-B) gene in 5 out of 34 (14.7%) non-PA primary tumors, i.e. 1 glioblastoma, 1 anaplastic astrocytoma, 1 anaplastic PXA, 1 ependymoma and 1 AT/RT. The fusion gene was also found in 21 out of 35 PAs (63.6%) and in 1/3 (33%) WHO grade I gangliogliomas. Quantitative PCR and FISH analysis confirmed the presence of the K-B fusion gene. The frequency of fusion harboring nuclei in non-PA neoplasms was lower than in PAs. Screening for IDH1, BRAFv600, and H3F3A mutations disclosed one case with the IDH1 and one with the K27M-H3.3 mutation, both unassociated with the K-B gene. CONCLUSION: Our study indicates that: 1) the K-B fusion gene can occasionally be detected in non-PA tumors of various histogenesis; 2) its occurrence in these neoplasms is probably due rather to a random alteration related to genomic instability than to a driving molecular event, as in PAs; and 3) the K-B fusion gene's role as a diagnostic marker outside the histological spectrum of pediatric low-grade gliomas should be carefully considered.

BACKGROUND: The KIAA1549:BRAF fusion (K-B fusion) gene has been considered a driver genetic events and a useful diagnostic marker in pilocytic astrocytomas (PA). However B-K gene fusion has been reported at low frequency in adult diffuse gliomas in association with IDH mutations. To further evaluate the possible occurrence of the B-K gene in non-PA tumors, we investigated the presence of the fusion gene in a series of 69 pediatric brain neoplasms of different histogenesis and grading. METHODS: Reverse-transcription polymerase chain reaction (RT-PCR) and subsequent sequencing. To further validate our findings we performed DNA-based methods as CNV and FISH. For CNV analysis, we used BRAF and KIAA specific primers for quantitative PCR in order to demonstrate the specific gain of the two genes, and for FISH analysis we used a commercially available probe. RESULTS: We detected B-K fusion gene in 5 out 34 (14,7%) non-PA primary tumors, i.e, 1 glioblastomas, 1 anaplastic astrocytoma, 1 anaplastic PXA, 1 ependymoma and 1 ATRT. Moreover, the fusion gene was present in 21 of 35 (63.6%) PAs, and out 3 (33%) WHO grade I gangliogliomas, a percentage overlapping previous studies on pediatric low grade gliomas. Both methods confirmed the presence of the B-K fusion gene but also indicate that the cells harboring the alteration in non-PAs neoplasms were less numerous compared to PAs. Screening for IDH, BRAFv600, and H3F3A mutations showed only one case with K27M-H3.3 mutation, which was not associated with B-K gene. CONCLUSIONS: Our study indicates that: 1) K-B fusion gene can be occasionally detected at low frequency in non-PA tumor of different histogenesis; 2) its occurrence in these neoplasms, probably, represents a random alteration related to genomic instability more than a driver molecular event, 3) its role as diagnostic marker outside the histological spectrum of pediatric low grade gliomas needs to be cautious. SECONDARY CATEGORY: Pediatrics.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the clonal expansion of malignant B cells. To predict the clinical course of the disease, the identification of diagnostic biomarkers is urgently needed. Aberrant methylation patterns may predict CLL development and its course, being very early changes during carcinogenesis. Our aim was to identify CLL specific methylation patterns and to evaluate whether methylation aberrations in selected genes are associated with changes in gene expression. Here, by performing a genome-wide methylation analysis, we identified several CLL-specific methylation alterations. We focused on the most altered one, at a CpG island located in the body of SHANK1 gene, in our CLL cases compared to healthy controls. This methylation alteration was successfully validated in a larger cohort including 139 CLL and 20 control in silico samples. We also found a positive correlation between SHANK1 methylation level and absolute lymphocyte count, in particular CD19+ B cells, in CLL patients. Moreover, we were able to detect gains of methylation at SHANK1 in blood samples collected years prior to diagnosis. Overall, our results suggest methylation alteration at this SHANK1 CpG island as a biomarker for risk and diagnosis of CLL, and also in the personalized quantification of tumor aggressiveness.