SARS-CoV-2 intra-host evolution during prolonged infection in an immunocompromised patient.

OBJECTIVES: Intra-host SARS-CoV-2 evolution during chronic infection in immunocompromised hosts has been suggested as being the possible trigger of the emergence of new variants. METHODS: Using a deep sequencing approach, we investigated the SARS-CoV-2 intra-host genetic evolution in a patient with HIV over a period of 109 days. RESULTS: Sequencing of nasopharyngeal swabs at three time points demonstrated dynamic changes in the viral population, with the emergence of 26 amino acid mutations and two deletions, 57% of them in the Spike protein. Such a combination of mutations has never been observed in other SARS-CoV-2 lineages detected so far. CONCLUSION: Our data confirm that persistent infection in certain immunocompromised individuals for a long time may favor the dangerous emergence of new SARS-CoV-2 variants with immune evasion properties.

Percutaneous radiofrequency ablation of hepatocellular carcinoma in cirrhosis: analysis of complications in a single centre over 20 years.

OBJECTIVE: To report on our 20 years' experience on complications after radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC) in patients with cirrhosis. METHODS: From 1994 to 2014, 1787 RFA procedures were performed percutaneously in 1162 patients with cirrhosis (852 Child A and 310 Child B) with HCC nodules (1.2-7 cm), prothrombin time >50%, platelet count of 50.000 mm3 and total bilirubin ranging from 0.80 to 4.5 mg dl-1. In 67 patients, RFA was performed on both intraparenchymal HCC nodule and tumour thrombus extended in the main portal vein and/or its branches. RESULTS: Four patients (0.3%) died after RFA. 39 patients (3.2%) changed in Child's class: 26 out of 28 Child A patients with cirrhosis changed to Child B and 2 changed to Child C class; 11 Child B patients changed to Child C class. On multivariate analysis, the total bilirubin pre-RFA was the only independent risk factor for impairment of liver function and death. Complications were hemoperitoneum, abscess and intrahepatic haematoma. CONCLUSION: RFA of HCC in patients with cirrhosis is safe, even in case of invasion of the portal venous system. Functional liver reserve should be strictly monitored, mainly when pre-RFA total bilirubin value is >2.5 mg dl-1. The study was approved by our institutional review board. Advances in knowledge: The total bilirubin value >2.5 mg dl-1 represents the main marker of functional liver reserve that predicts decompensation of liver cirrhosis in patients undergoing RFA for HCC.

Evaluation of the Ion Torrent PGM sequencing workflow for the routine rapid detection of BRCA1 and BRCA2 germline mutations.

PURPOSE: Conventional methods used to identify BRCA1/2 germline mutations in hereditary cancers are time-consuming and expensive, due to the large size of the genes. The recent introduction of next generation sequencing (NGS) benchtop platforms is a great promise, which is rapidly revolutionizing genetic screening in diagnostic and clinical applications. We recently transferred our methodology for routine BRCA1/2 mutation screening (denaturing High Performance Liquid Chromatography plus Sanger sequencing) to the Ion Torrent PGM platform with the Ion Ampliseq BRCA1 and BRCA2 panel and tested the performance of the system. METHODS: We first validated the NGS approach in a cohort of 33 patients who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we tested 29 newly diagnosed and uncharacterized patients by NGS, and Sanger sequencing was used to confirm results from the NGS platform. RESULTS: In the validation cohort, all previously identified single nucleotide variants, insertions and deletions (also composed of multiple bases and within complex homopolymeric stretches) were identified by NGS in their correct zygosity status except for variants in a complex multinucleotide region within intron 7 of BRCA1 gene. NGS approach was further able to identify previously undetected variants. In the prospective cohort, almost all (99.3%) called variants were confirmed by Sanger. In both cohorts, in addition to the false positive (31) and false negative (110) results in the intron 7 of BRCA1 gene, the NGS method detected 10 false positives, that were solved by Sanger. CONCLUSIONS: The Ion Torrent PGM NGS approach in BRCA1/2 germline mutation identification is highly sensitive, easy to use, faster and cheaper than traditional approaches. Therefore, according to other recently published works, we highly recommend this system for routine diagnostic testing on BRCA1/2 genes, along with Sanger confirmation of the called variants, and support the usefulness of the approach also in other routine genetic analysis.

A PCR-based protocol to accurately size C9orf72 intermediate-length alleles.

Although large expansions of the non-coding GGGGCC repeat in C9orf72 gene are clearly defined as pathogenic for Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), intermediate-length expansions have also been associated with those and other neurodegenerative diseases. Intermediate-length allele sizing is complicated by intrinsic properties of current PCR-based methodologies, in that somatic mosaicism could be suspected. We designed a protocol that allows the exact sizing of intermediate-length alleles, as well as the identification of large expansions.

Sorafenib Combined with Radio-frequency Ablation Compared with Sorafenib Alone in Treatment of Hepatocellular Carcinoma Invading Portal Vein: A Western Randomized Controlled Trial.

AIM: To compare in a randomized controlled trial (RCT) 3-year survival of cirrhotic patients with hepatocellular carcinoma (HCC) accompanied by portal vein tumor thrombus (PVTT) treated with sorafenib plus percutaneous radiofrequency ablation (RFA) of both intraparenchymal HCC and PVTT (combination Group) or sorafenib alone (sorafenib-alone Group). PATIENTS AND METHODS: Ninety-nine consecutive Child A cirrhotics were randomized to receive RFA of both HCC and main portal vein tumor thrombus (MPVTT) plus sorafenib (n=49) or sorafenib alone (n=50). RESULTS: One-, 2- and 3-year survival rates were 60%, 35% and 26%, respectively, in the combination group and 37% and 0 % at 1- and 2-year, respectively, in the sorafenib alone group. At multivariate analysis, the combination of RFA of both HCC and MPVTT was the only factor predicting survival. CONCLUSION: Use of RFA of both HCC and MPVTT plus sorafenib significantly increases 3-year survival compared to sorafenib alone.

Contrast-Enhanced Ultrasound: a Simple and Effective Tool in Defining a Rapid Diagnostic Work-up for Small Nodules Detected in Cirrhotic Patients during Surveillance.

BACKGROUND AND AIMS: Disappearance of portal blood flow and arterial vascularization is the hallmark of hepatocarcinogenesis. The capability of a dynamic imaging modality detecting arterial hypervascularization of small nodules is crucial to promote a rapid diagnostic and therapeutic work-up improving survival. We aimed to evaluate the capability of CEUS to detect arterial vascularization of ≤ 2 cm HCC nodules arising during surveillance so as to shorten the diagnostic and therapeutic work-up. METHODS: From October 2009 to September 2014, among 1757 consecutive cirrhotic patients under surveillance with ultrasound (US), 243 patients had new single nodules 7-20 mm; 229/243 had a conclusive histologic diagnosis and comprised the study group. All patients underwent CEUS followed by enhanced MRI and US guided percutaneous 18G needle core biopsy of the nodules. Of the 229 nodules, 27 were hyperechoic, 171 hypoechoic and 31 isoechoic lesions. RESULTS: The histology results revealed that 199/229 nodules were HCC and 30 were benign. Of 199 HCC, CEUS evidenced arterial hypervascularity in 190 nodules (95.5%) (sensitivity 94.48 %, specificity 100%, PPV 100%, NPV 76.92 %). Of the 39 CEUS arterial-unenhanced nodules, 30 were benign and 9 (23%) were well-differentiated HCC. eMRI showed arterial hypervascularity in 199 nodules (86,9%). Of these, only 193 (97%) were histologically HCCs while 6 were benign (sensitivity: 97%, specificity: 80%, PPV: 97%, NPV: 80%). CONCLUSIONS: CEUS has a great capability to detect arterial hypervascularity of small HCC. Because only 4.5% of new nodules escape the demonstration of arterial hyervascularity, CEUS must be performed immediately after conventional US to contrast the malignant fate of small lesions arising in a cirrhotic liver.