Similarities and differences in gene expression profiles of BRCA1 methylated and mutated epithelial ovarian cancers.

Introduction: BRCA1 methylated (BRCA1met) epithelial ovarian cancer (EOC) is a recently defined and not well-investigated subset of neoplasms. To date, no studies have focused on the transcriptional profiles of BRCA1met cases, and, as a matter of fact, we still do not know if this subset of EOCs is similar, and to what extent, to BRCA1 mutated (BRCA1mut) cases. Methods: We compared a group of 17 BRCA1met cases against 10 BRCA1mut cases using a subset of carefully selected 17 BRCAwt EOCs as a control group. Results: First, BRCA1met cases showed a downregulation of the relative transcript, while this association was not observed for BRCA1mut EOCs. The BRCA1met group exhibited a general upregulation of homologous recombination (HR)-related genes, as well as BRCA1mut. Overall, BRCA1met had a different gene expression profile, characterized by diffuse downregulation, whereas BRCA1mut showed a general upregulation (p < 0.0001). Both BRCA1-defective groups showed a slightly activated immune response mediated by interferon (IFN) gamma pathways. Discussion: In conclusion, even if the expression profile of many genes related to DNA damage and repair system is shared between BRCA1mut and BRCA1met EOCs supporting that BRCA1met EOCs may benefit from PARPi therapies, our data demonstrate that BRCA1mut and BRCA1met EOCs show different expression profiles, suggesting a different mechanism of carcinogenesis that can be reflected in different responses to therapies and disease recovery.

EGFR T790M detection rate in lung adenocarcinomas at baseline using droplet digital PCR and validation by ultra-deep next generation sequencing.

BACKGROUND: Routine testing of baseline EGFR T790M mutation may have important clinical impact but many discordant data have been reported regarding the diagnostic, prognostic and predictive role of this marker. In this study we aimed to assess T790M frequency in 164 untreated EGFR-mutated NSCLCs using methods with different sensitivity as well as to analyze the relationship between baseline T790M mutation status, patient's clinicopathologic features and tyrosine kinase inhibitors (TKI) treatment outcomes. METHODS: We compared the diagnostic performance, sensitivity and specificity of three methods, namely MALDI-TOF mass spectrometry (MS), Allele-Specific Real Time PCR (AS-PCR), droplet digital PCR (ddPCR). Ultra-deep next generation sequencing (NGS) validation of T790M-mutant NSCLCs was performed using SiRe® panel. RESULTS: Baseline T790M occurred in 17% of the tumors. Intermediately sensitive techniques such as MALDI-TOF MS (detection limit of T790M ≥5%) allow to detect T790M in 2% of cases exhibiting mutant-allele fractions ranging from 11.5% to 17%. Median overall survival (OS) in these patients was poor (7.3 months) and progression free survival (PFS) was of 3.3 months in patients treated with a 1st generation EGFR TKI. The remaining T790M-positive cases showed very low mutant-allele fractions ranging from 0.07% to 0.38% and required highly sensitive methods such as ddPCR and NGS to be identified. All these cases showed a concurrent sensitizing EGFR mutation (mainly exon 19 deletion), and clinicopathological features similar to those observed in EGFR mutant cancers. Median OS of these patients was 27 months while median PFS after TKI treatment was 20 months. CONCLUSIONS: Routine test of baseline EGFR T790M may have an important role in the prediction to EGFR TKI therapy response and should be performed using highly sensitive and quantitative methods, such as ddPCR and NGS, in order to reliably distinguish NSCLCs with high or very low T790M mutant-allele fraction.

Comprehensive analysis of HPV infection, EGFR exon 20 mutations and LINE1 hypomethylation as risk factors for malignant transformation of sinonasal-inverted papilloma to squamous cell carcinoma.

Different risk factors are suspected to be involved in malignant transformation of sinonasal papillomas and include HPV infection, tobacco smoking, occupational exposure, EGFR/KRAS mutations and DNA methylation alterations. In our study, 25 inverted sinonasal papillomas (ISPs), 5 oncocytic sinonasal papillomas (OSP) and 35 squamous cell carcinomas (SCCs) from 54 patients were genotyped for 10 genes involved in EGFR signalling. HPV-DNA detection was performed by in-situ hybridisation and LINE-1 methylation was quantitatively determined by bisulphite-pyrosequencing. High-risk HPV was observed only in 13% of ISP-associated SCC and in 8% of de novo-SCC patients. EGFR mutations occurred in 72% of ISPs, 30% of ISP-associated SCCs and 17% of de novo-SCCs. At 5-year follow-up, SCC arose in only 30% (6/20) of patients with EGFR-mutated ISPs compared to 76% (13/17) of patients with EGFR-wild-type ISP (p = 0.0044). LINE-1 hypomethylation significantly increased from papilloma/early stage SCC to advanced stage SCC (p = 0.03) and was associated with occupational exposure (p = 0.01) and worse prognosis (p = 0.09). In conclusion, our results suggest that a small subset of these tumours could be related to HPV infection; EGFR mutations characterise those ISPs with a lower risk of developing into SCC; LINE-1 hypomethylation is associated with occupational exposure and could identify more aggressive nasal SCC.

Harmonization of values for serum alkaline phosphatase catalytic activity concentration employing commutable calibration materials.

BACKGROUND: Harmonization of results allows a more effective utilization of laboratory tests; we verified the feasibility of harmonizing serum alkaline phosphatase results by two methods. METHODS: Patient sera (n=106) and candidate calibration materials (n=8) were analyzed by two methods, employing either diethanolamine (DEA) or 2-amino-2-methyl-1-propanol (AMP) as phosphate-accepting buffers. Results for patient sera by the DEA method were recalculated, with either a commutable or a non-commutable calibration material, both with values assigned by the AMP method. RESULTS: After calibration with the commutable material, the median intermethod difference (DEA-AMP) and ratio (DEA/AMP) dropped from 195 U/l to 0 U/l and from 2.47 to 1.00, respectively. When a non-commutable material was used the former became 124 U/l and the latter 1.94. After recalibration with the commutable material, linear regression and correlation analysis of DEA vs AMP values for the set of 106 patient sera gave: intercept=0.8 U/l; slope=0.997; and nonparametric correlation coefficient r=0.9995. CONCLUSIONS: Harmonization of alkaline phosphatase results by AMP and DEA methods is feasible when commutable calibration materials are used in the trueness transfer process.