Resistance to osimertinib in advanced EGFR-mutated NSCLC: a prospective study of molecular genotyping on tissue and liquid biopsies.

BACKGROUND: Resistance to osimertinib in advanced EGFR-mutated non-small cell lung cancer (NSCLC) constitutes a significant challenge for clinicians either in terms of molecular diagnosis and subsequent therapeutic implications. METHODS: This is a prospective single-centre study with the primary objective of characterising resistance mechanisms to osimertinib in advanced EGFR-mutated NSCLC patients treated both in first- and in second-line. Next-Generation Sequencing analysis was conducted on paired tissue biopsies and plasma samples. A concordance analysis between tissue and plasma was performed. RESULTS: Sixty-five advanced EGFR-mutated NSCLC patients treated with osimertinib in first- (n = 56) or in second-line (n = 9) were included. We managed to perform tissue and liquid biopsies in 65.5% and 89.7% of patients who experienced osimertinib progression, respectively. Acquired resistance mechanisms were identified in 80% of 25 patients with post-progression samples, with MET amplification (n = 8), EGFR C797S (n = 3), and SCLC transformation (n = 2) the most frequently identified. The mean concordance rates between tissue and plasma for the EGFR activating mutation and for the molecular resistance mechanisms were 87.5% and 22.7%, respectively. CONCLUSIONS: Resistance to osimertinib demonstrated to be highly heterogeneous, with MET amplification the main mechanism. Plasma genotyping is a relevant complementary tool which might integrate tissue analysis for the study of resistance mechanisms.

Three-dimensional assessment of umbilical vein deviation angle for prediction of liver herniation in left-sided congenital diaphragmatic hernia.

OBJECTIVES: To introduce a new sonographic marker of intrathoracic liver herniation in fetuses with left-sided congenital diaphragmatic hernia (CDH). METHODS: In a consecutive series of fetuses with isolated CDH, an ultrasound volume of the fetal abdomen was acquired. On this volume, offline calculation of the angle formed by the midline of the abdomen (joining the center of the vertebral body to the abdominal insertion of the umbilical cord) and a second line joining the center of the vertebral body to the intra-abdominal convexity of the umbilical vein was carried out to give the umbilical vein deviation angle (UVDA). The UVDA was measured in a group of normal fetuses selected as controls. At follow-up, the presence of liver herniation was investigated in all cases of CDH. UVDA values were compared between the CDH group and controls, and between CDH 'liver-up' vs 'liver-down' cases. A receiver-operating characteristics (ROC) curve was constructed to identify a cut-off value of the UVDA with the highest accuracy in predicting liver herniation in the CDH group. RESULTS: Between 2009 and 2015, 22 cases of left-sided CDH were included in the study group, of which nine cases had liver herniation. Eighty-eight normal fetuses were recruited as controls. The UVDA was significantly higher in the cases vs controls (15.25 ± 7.91° vs 7.68 ± 1.55°; P < 0.0001). Moreover, the UVDA was significantly increased in CDH fetuses with liver-up vs liver-down (21.77 ± 8.79° vs 10.75 ± 2.10°; P < 0.0001). On ROC curve analysis the UVDA showed good prediction of liver herniation (area under the ROC curve, 0.94; P < 0.0001) with the best cut-off of 15.2°, yielding a sensitivity of 89% and a specificity of 100% (P < 0.0001). CONCLUSIONS: In fetuses with CDH, umbilical vein bowing may be quantified by measuring the UVDA using three-dimensional ultrasound. This sonographic marker seems to be an accurate predictor of liver herniation in left-sided CDH. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Glutamine depletion by crisantaspase hinders the growth of human hepatocellular carcinoma xenografts.

BACKGROUND: A subset of human hepatocellular carcinomas (HCC) exhibit mutations of ß-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO). METHODS: Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1. RESULTS: Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro. CONCLUSIONS: The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth.

Comparison of HER2 status in primary and paired metastatic sites of gastric carcinoma.

BACKGROUND: Trastuzumab has recently shown efficacy in the treatment of HER2-positive advanced gastric adenocarcinoma. Although antibody-based therapies target the metastatic disease, HER2 status is usually evaluated in the primary tumour because metastatic sites are rarely biopsied. The aim of this study was to compare HER2 status in primary and paired metastatic sites of gastric adenocarcinoma. METHODS: The HER2 status was assessed by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) in 72 secondary lesions of gastric adenocarcinoma and in the corresponding primary tumours. RESULTS: Concordance of FISH results, evaluable in 68 primary and matched metastatic sites, was 98.5%. Concordance of IHC results, available in 39 of the 72 paired cases, was 94.9%. Only one case showed discordance between primary tumour and metastasis, being negative by both IHC and FISH in the primary and showing HER2 overexpression and amplification in the corresponding pancreatic lymph node metastasis. CONCLUSION: The high concordance observed between HER2 results obtained by both IHC and FISH on primary tumours and corresponding metastases suggests that in gastric cancer HER2 status is maintained in most cases unchanged during the metastatic process.

Chronic periaortitis associated with membranous nephropathy: clues to common pathogenetic mechanisms.

Chronic periaortitis (CP) is a rare disease hallmarked by the presence of a periaortic retroperitoneal fibro-inflammatory tissue which can often cause obstructive uropathy. CP is isolated in most cases but it may also be associated with other sclerosing inflammatory and immune-mediated diseases. We here present the case of a patient who was initially diagnosed as having CP and subsequently developed membranous nephropathy and chronic sclerosing sialoadenitis of the right parotid gland. As these conditions were all characterized by either pronounced infiltration of IgG4-positive plasma cells or marked IgG4 tissue deposition, we hypothesize that they are part of the same disease spectrum, and discuss the immune-mediated pathogenetic mechanisms potentially shared by these conditions. In particular, we consider the role of Th2-mediated immune reactions and of immunogenetic factors such as HLA genotype as common determinants of these disorders.

Prognostic impact of interval breast cancer detection in women with pT1a N0M0 breast cancer with HER2-positive status: Results from a multicentre population-based cancer registry study.

BACKGROUND: Although human epidermal growth factor receptor 2 (HER2) overexpression is associated with poor prognosis, patients (pts) with pT1a N0M0 breast cancers (BCs) have an excellent outcome across all subtypes. Interval cancers (ICs) have poorer survival than screen-detected (SD) tumours, and an association has been reported between ICs and HER2 overexpression. We aimed to determine, in a general population of pT1a N0M0 BCs with known screening status, whether HER2-positive ICs have a poorer outcome than HER2-positive SD cancers. METHODS: We evaluated all incident pT1a N0M0 BCs (n = 874) collected in the Emilia-Romagna region (Italy) from 2003 to 2009 and diagnosed in women aged 50-69. Pts unexposed to screening, with unknown HER2 status and/or treated with adjuvant trastuzumab were excluded from analysis. RESULTS: Sixty-one percent of the BCs were SD, whereas 19% were ICs. BCs with high histologic grade, hormone receptor-negative or HER2-positive status (odds ratio=1.7; 95% confidence interval [CI]: 1.1-2.7) were more likely ICs. Median follow-up was 115 months. The 10-year invasive disease-free survival (iDFS) for HER2-positive ICs was lower than that for HER2-positive SD cancers: 75.0% (95% CI: 55.5%-94.5%) versus 93.8% (95% CI: 86.5%-100%). An interaction between ICs and HER2-positive status was found for poorer iDFS after adjusting for prognostic variables (HR = 5.3; 95% CI: 1.6-16.7). CONCLUSIONS: IC detection may identify pts with HER2-positive pT1a N0M0 tumours in whom the rate of recurrence justifies consideration for conventional, anti-HER2, adjuvant treatment.

Placental lesions associated with oligohydramnios in fetal growth restricted (FGR) pregnancies.

INTRODUCTION: Aim of the study was to investigate the association between placental pathology and oligohydramnios in pregnancies complicated by fetal growth restriction (FGR). METHODS: Placentas from 221 consecutive FGR pregnancies and 63 healthy controls were studied. Pathological lesions were described according to consensus nomenclature and standardized criteria; both elementary lesions and constellations of lesions (patterns) were considered. Statistics included analysis of linear trends and multinomial logistic regression. RESULTS: Amniotic fluid index (AFI) was normal in 56 (25.3%) FGR pregnancies, whereas mild, moderate and severe oligohydramnios were diagnosed in 32 (14.5%), 44 (19.9%) and 89 (40.3%) subjects, respectively. In FGR pregnancies, after adjustment for potential confounders, membrane meconium staining (chi-square = 28.6, p < 0.001), chronic villous hypoxia pattern (chi-square = 18.8, p < 0.001) and fetal thrombotic vasculopathy pattern (chi-square = 9.2, p = 0.002) were positively and linearly correlated to AFI decrease. Odds ratios of meconium and chronic villous hypoxia were 9.2 (95% CI = 2.6-32.9) and 4.2 (95% CI = 1.3-13.6) in FGR pregnancies with normal AFI and 25.2 (95% CI = 6.9-91.8) and 9.7 (95% CI = 3-31.5) in those with severe oligohydramnios (p = 0.005 and p = 0.023 compared to normal AFI, respectively). DISCUSSION: In FGR pregnancies, reduction of amniotic fluid volume is directly correlated to histological features of placental under-perfusion, meconium staining of membranes and fetal vascular damage. These findings support the clinical notion that in FGR pregnancies oligohydramnios is a risk factor of fetal hypoxia and possibly of increased adverse neonatal outcomes.

K-ras gene mutations: an unfavorable prognostic marker in stage I lung adenocarcinoma.

Activation of K-ras gene by point mutations, a common finding in lung adenocarcinomas, has been suggested to decrease patient survival. We investigated 109 lung adenocarcinomas, mostly small, peripheral, stage I tumours (81/109) for presence of K-ras gene mutations at codons 12 and 13. Mutations were detected by denaturing gradient gel electrophoresis analysis of specific sequences amplified by polymerase chain reaction from DNA extracted from archival pathological material. Thirty-three of 109 (30.3%) tumours showed mutations at codon 12 (28/33, 84.8%) or 13 (5/33, 15.2%) of the gene. Mutations and type of nucleotide substitutions were differently distributed among cytological subtypes, being more prevalent among less differentiated (G2 and G3) tumours and among bronchial than bronchiolo-alveolar type adenocarcinomas. Survival analysis showed an adverse effect of K-ras mutation on survival, restricted to stage I tumours. Median survival for 81 stage I patients was 30 months for non-mutated tumours versus 20 months for mutated tumours (p = 0.016). Multivariate analysis showed that age of patient (p = 0.001) and K-ras mutation status (p = 0.04) were the only independent factors influencing survival significantly. These data strengthen the hypothesis that K-ras gene mutations may be useful in identifying a subgroup of patients with poor outcome.

Hepatitis C virus genotypes and risk of cirrhosis in southern Italy.

Because hepatitis C virus (HCV) genotypes have raised considerable interest as variables that influence chronic hepatitis C progression, a case-control study was conducted to estimate their effects on patients with cirrhosis. Case patients (n = 46) had tested positive for anti-HCV antibody and HCV RNA and were residents of the study area who had cirrhosis recently diagnosed. Controls (n = 138) were drawn randomly from a residents' cohort from the same area. Demographic and other information were recorded. Presence of HCV infection, presence of HCV RNA, and HCV genotypes were assessed. Crude, stratified, and logistic regression analyses were performed. HCV genotype 2a/c occurred in 84 controls (60.9%) and 9 case patients (19.6%); HCV genotype 1b was found in 45 controls (32.6%) and 34 case patients (73.9%). HCV 1b genotype showed an independent effect on the risk of cirrhosis (odds ratio, 7.49; 95% confidence interval, 3.15--17.81). No significant effects related to other variables were observed. These results indicate that the genetic diversity of HCV phylogenetic variants may explain differences in biological behaviors.

Molecular epidemiology of hepatitis C virus infection in an area of hyperendemicity in southern Italy: a population-based study.

In a cohort of subjects from Italy, anti-hepatitis C virus (HCV) and HCV RNA [HCV(+) subgroup] prevalences were 24.6 and 79.6%, respectively. HCV types 1b and 2a/c accounted for 95% of infections. Adjusted alanine aminotransferase levels were higher in males than in females and in RNA-positive subjects than in RNA-negative subjects regardless of HCV type. Genotype distribution was unrelated to demographic variables.

Dynamics of hypervariable region 1 variation in hepatitis C virus infection and correlation with clinical and virological features of liver disease.

Hepatitis C virus (HCV) infection is a dynamic process during which molecular variants are continuously selected as the result of virus adaptation to the host. Understanding the nature of HCV genetic variation is central to current theories of pathogenesis and immune response. We prospectively studied hypervariable region 1 (HVR1) variation in the E2 gene of 36 hepatitis C patients, including 10 asymptomatic carriers, followed up for 1 to 2 years. Sequence changes in single and consecutive serum samples were assessed and correlated with clinical and virological parameters of liver disease. A region of the E1 gene was sequenced for comparison in 3 subjects. HVR1 heterogeneity at single time points widely varied in individual patients, did not increase cumulatively over the follow-up period, and did not correlate with HVR1 evolutionary rates. Conversely, the process of HVR1 sequence diversification, although differed considerably among patients, was stable over time and directly correlated with infections by HCV type 2, lower alanine aminotransferase (ALT) levels, and absence of cirrhosis. HCV carriers showed the highest HVR1 variation rates. Our findings indicate that HVR1 variation has an adaptive significance and is associated with favorable features of liver disease and suggest that prospective, rather than static, observations are required to model the process of HCV variation.

Human leukocyte antigen class II and III alleles and severity of hepatitis C virus-related chronic liver disease.

Hepatitis C outcome is likely related both to viral factors and host's immune responses. We correlated the severity of liver disease with human leukocyte antigen (HLA) genes (C4A, C4B, TNFA, TNFB, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, TAP1, and TAP2) in three groups of subjects: 99 patients with chronic hepatitis, 41 asymptomatic carriers, and 179 uninfected controls. Patients with grade/stage 3 to 4 hepatitis significantly differentiated for their low frequency of alleles TNFB*1, DRB1*1104, and DRB3*03, which had a protective role, and high frequency of allele DRB1*1001, which was associated with disease severity. HLA-DRB*11 subtypes were differentially distributed: DRB1*1104 was most frequent in carriers, whereas DRB1*1101 was more frequent in patients. The TAP1C,2A haplotype was also underrepresented in patients with respect to controls. Finally, a decrease of heterozygous subjects was observed in patients with respect to carriers at the DQB1 locus. Multivariate analysis by correspondence analysis and multiple logistic regression indicated that age, sex, and hepatitis C virus (HCV) type were the strongest risk factors; however, some immunogenetic variables (TNFB*1, DRB1*1104, and DRB3*03) showed an independent contribution, especially in comparing patients with extreme manifestations of disease. The involvement of different genes in various HLA subregions suggests that anti-HCV responses are modulated by a complex gene interplay rather than by single alleles.