Annalisa Pession

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(1) Background: Human papillomaviruses (HPVs) are known to be related to the development of about 5% of all human cancers. The clinical relevance of HPV infection has been deeply investigated in carcinomas of the oropharyngeal area, uterine cervix, and anogenital area. To date, several different methods have been used for detecting HPV infection. The aim of the present study was to compare three different methods for the diagnosis of the presence of the HPV genome. (2) Methods: A total of 50 samples were analyzed. Twenty-five of them were tested using both next generation sequencing (NGS) and VisionArray^® technology, the other 25 were tested using Hybrid Capture (HC) II assay and VisionArray^® technology. (3) Results: A substantial agreement was obtained using NGS and VisionArray^® (κ = 0.802), as well as between HC II and VisionArray^® (κ = 0.606). In both analyses, the concordance increased if only high risk HPVs I(HR-HPVs) were considered as “positive”. (4) Conclusions: Our data highlighted the importance of technical choice in HPV characterization, which should be guided by the clinical aims, costs, starting material, and turnaround time for results.

Background: Non-canonical mutations of the isocitrate dehydrogenase (IDH) genes have been described in about 20–25% and 5–12% of patients with WHO grade II and III gliomas, respectively. To date, the prognostic value of these rare mutations is still a topic of debate. Methods: We selected patients with WHO grade II and III gliomas and IDH1 mutations with available tissue samples for next-generation sequencing. The clinical outcomes and baseline behaviors of patients with canonical IDH1 R132H and non-canonical IDH1 mutations were compared. Results: We evaluated 433 patients harboring IDH1 mutations. Three hundred and ninety patients (90.1%) had a canonical IDH1 R132H mutation while 43 patients (9.9%) had a non-canonical IDH1 mutation. Compared to those with the IDH1 canonical mutation, patients with non-canonical mutations were younger (p < 0.001) and less frequently presented the 1p19q codeletion (p = 0.017). Multivariate analysis confirmed that the extension of surgery (p = 0.003), the presence of the 1p19q codeletion (p = 0.001), and the presence of a non-canonical mutation (p = 0.041) were variables correlated with improved overall survival. Conclusion: the presence of non-canonical IDH1 mutations could be associated with improved survival among patients with IDH1 mutated grade II–III glioma.

Medulloblastoma is a highly aggressive brain tumor that typically affects children, while in adults it represents ~1% of all brain tumors. Little is known about microRNA expression profile of the rare adult medulloblastoma. The main aim of this study was to identify peculiar differences in microRNA expression between childhood and adult medulloblastoma. Medulloblastomas were profiled for microRNA expression using the Exiqon Human miRNome panel (I + II) analyzing 752 microRNAs in a training set of six adult and six childhood cases. Then, the most differentially expressed microRNAs were validated in a total of 21 adult and 19 childhood cases. Eight microRNAs (miR-196b-5p, miR-183-5p, miR-200b-3p, miR-196a-5p, miR-193a-3p, miR-29c-3p, miR-33b-5p, and miR-200a-3p) were differentially expressed in medulloblastoma of adults and children. Analysis of the validation set confirmed that miR-196b-5p and miR-200b-3p were significantly overexpressed in medulloblastoma of adults as compared with those of children. We followed an in silico approach to investigate direct targets and the pathways involved for the two microRNAs (miR-196b and miR-200b) differently expressed between adult and childhood medulloblastoma. Adult and childhood medulloblastoma have different miRNA expression profiles. In particular, the differential dysregulation of miR-196b-5p and miR-200b-3p characterizes the miRNA profile of adult medulloblastoma and suggests potential targets for novel diagnostic, prognostic, or therapeutic strategies.

Next generation sequencing (NGS) allows parallel sequencing of multiple genes at a very high depth of coverage. The need to analyze a variety of targets for diagnostic/prognostic/predictive purposes requires multi-gene characterization. Multi-gene panels are becoming standard approaches for the molecular analysis of solid lesions. We report a custom-designed 128 multi-gene panel engineered to cover the relevant targets in 22 oncogene/oncosuppressor genes for the analysis of the solid tumors most frequently subjected to routine genotyping. A total of 1695 solid tumors were analyzed for panel validation. The analytical sensitivity is 5%. Analytical validation: (i) Accuracy: sequencing results obtained using the multi-gene panel are concordant using two different NGS platforms and single-gene approach sequencing (100% of 83 cases); (ii) Precision: consistent results are obtained in the samples analyzed twice with the same platform (100% of 20 cases). Clinical validation: the frequency of mutations identified in different tumor types is consistent with the published literature. This custom-designed multi-gene panel allows to analyze with high sensitivity and throughput 22 oncogenes/oncosuppressor genes involved in diagnostic/prognostic/predictive characterization of central nervous system tumors, non-small-cell lung carcinomas, colorectal carcinomas, thyroid nodules, pancreatic lesions, melanoma, oral squamous carcinomas and gastrointestinal stromal tumors.

BRAF exon 15 mutations are the most common molecular alterations found in papillary thyroid carcinoma (PTC). To date, there is no information regarding BRAF alterations in the thyroid parenchyma surrounding the tumor. To explore the early events associated with the development of PTC, we used massively parallel sequencing to investigate BRAF exon 15 in 30 PTCs and in 100 samples from the thyroid parenchyma surrounding the tumor. BRAF p.V600E was identified in 19/30 PTCs (63.3%). BRAF p.V600E mutations were identified in the tissue adjacent the PTC only in samples containing psammoma bodies. The other samples were either BRAF wild type (WT) or carried BRAF non p.V600E mutations. Specifically, BRAF p.G593D, -p.A598T, -p.V600M, -p.R603Q, -p.S607F, and -p.S607P were identified in 4 of 36 (11.1%) samples with follicular cell atypia, in 2 of 16 (12.5%) with follicular cell hyperplasia, and in 1 of 33 (3.0%) histologically normal samples—Only in tissue surrounding BRAF p.V600E mutated PTCs. These mutations are predicted to affect protein function in silico but, in vitro, have kinase activity and BRAF phosphorylation levels similar to BRAF WT. No BRAF exon 15 mutations were identified in samples adjacent to PTCs that were BRAF WT. A mutagenic process affecting BRAF exon 15 occurs in a subset of thyroid glands that develop BRAF p.V600E mutated PTCs.

2017 Background: Molecular characterization of low grade gliomas (LGG) is essential for diagnosis and treatment of these diseases. LGG patients (pts) with IDH mutation and 1p19q codeletion (codel) are characterized by a median OS (mOS) longer than 10 years. Thus, the role of treatments and side effects should be carefully evaluated. Methods: We evaluated LGG pts from our data warehouse (n=679 pts) who received surgery and had sufficient tissue to assess biomarkers characterization. Pts with gliomatosis were excluded. IDH1/2 assessment was performed on formalin-fixed paraffin-embedded samples by qPCR. In wild type cases we performed NGS. 1p/19 codel analysis was performed by FISH. Results: 93 consecutive LGG with IDH mutation and codel were included. The median follow up (FU) was 96.1 months. Mean age was 40 yrs (range: 25-66); 8 pts (8.6%) underwent biopsy, 61 pts (65.6%) partial resection, 24 pts (25.8%) complete resection. 84 pts (90.3%) were considered high risk using RTOG criteria (>40 years and/or incomplete resection). Fifty pts (53.7%) received only FU, 17 pts (18.3%) received chemotherapy (CT), 18 pts (19.4%) received radiotherapy (RT), 8 pts (8.6%) received RT + CT. Median PFS (mPFS) was 59.6 months (95%CI: 41.8-77.4) and was significantly longer in pts who received postsurgical treatments (79.5 months, 95%CI: 66.4-92.7) than pts who received FU (46.3 months, 95%CI: 36.0-56.5; P=0.001). mPFS was 50.8 months (95%CI: 17.4-84.3), 103.6 months (95%CI: 11.7-195.6) and 120.2 months (95%CI: 40.5-199.8) in pts treated with CT alone, RT alone and RT + CT, respectively. Multivariate analysis showed that receiving a post-surgical treatment (P

2032 Background: Molecular characterization of low grade gliomas (LGG) has improved in recent years and is essential for diagnosis and treatment of these diseases. Moreover, clinical factors, such as age and the extent of surgery retain a prognostic role in LGG that were assessed for IDH1/2, 1p/19q codeletion, and MGMT methylation status. Methods: we retrospectively evaluated all adult LGG patients (pts) from our data warehouse who received surgery and had sufficient tissue to assess biomarkers characterization. IDH1/2 assessment was performed on formalin-fixed paraffin-embedded samples by PCR, MGMT by methylation specific PCR, 1p/19 codeletion by FISH. Results: 198 consecutive LGG were included. The median age was 38 (range:18–72). Median follow up was 74.0 months, 109 pts (55.1%) were < 40 years of age; 26 pts (13.1%) underwent biopsy, 119 pts (60.1%) partial resection, 53 pts (26.8%) complete resection. Twenty-eight pts (14.1%) were considered low risk (< 40 years with complete resection). IDH1/2 mutation was found in 79.8% of pts. 1p/19q codeletion was found in 41.4% of pts, MGMT methylation in 57.1% of pts. Median survival in low risk patients was 211.0 months (95%CI: 190.4–231.6) and was 145.3 months (95%CI: 108.5–182.2) in high risk patients (P = 0.006). Median survival for patients with IDH1/2 mutation was 159 months (95%CI: 103.3–214.7) and was 87.9 months in patients IDH1/2 wild type (95%CI: 61.1–114.6, P < 0.001). The impact of molecular biomarkers on survival is in the table. Multivariate analysis showed that clinical risk (P = 0.006), IDH1/2 mutation (P < 0.001) and 1p/19q codeletion (P = 0.03) were significantly correlated with overall survival. MGMT methylation was not significant. Conclusions: Molecular characteristic of LGG define the prognosis of these tumors. Moreover, clinical risk assessment retains a role even in the era of molecular characterization.

Background: The most appropriate management of recurrent glioblastoma is still controversial. In particular, the role of surgery at recurrence remains uncertain. Patients & methods: From our Institutional data warehouse we analyzed 270 consecutive patients who received second surgery for recurrent glioblastoma, to assess survival after second surgery, and to evaluate prognostic factors. Results: Complete resection was found in 128 (47.4%) and partial resection in 142 patients (52.6%). Median survival from second surgery was 11.4 months (95% CI: 10.0–12.7). Multivariate analysis showed that age (p = 0.001), MGMT methylation (p = 0.021) and extent of surgery (p < 0.001) are associated with better survival. Conclusion: A complete resection should be the goal for second resection and younger age and MGMT methylation status might be considered in the selection of patients.

MicroRNAs as possible biomarkers for diagnosis and prognosis of hepatitis B- and C-related-hepatocellular-carcinoma