CT-guided fine-needle aspiration biopsy of solitary pulmonary nodules under 15 mm in diameter: time for an afterthought?

BACKGROUND: Many studies on fine-needle aspiration biopsy (FNAB) for undetermined pulmonary nodules reported that diagnostic accuracy tended to decline, whereas complication prevalence raised as the size of nodule decreased. Reconsideration on the effectiveness of FNAB would be appropriate considering the dramatic increase in the identification of small nodules with screening programs and new demands of target therapies. The aim of this study was to verify the efficacy of FNAB in pulmonary nodules smaller than 15 mm. METHODS: A retrospective, cohort study was conducted on patients with undetermined solitary pulmonary nodules (SPNs) who underwent computer tomography (CT) guided FNAB at our Institution from January 2012 to December 2014. Patients with SPNs with diameter up to 15 mm were considered; inclusion criteria comprised ASA 3, FEV1 <70% of predicted, cardiac comorbidity or previous chest surgery. FNAB diagnostic performance and clinical efficacy were calculated. RESULTS: Out of 225 patients referred for FNAB, 68 covered inclusion criteria. Forty-nine out of 68 smears (72%) were adequate for diagnosis. Specificity was 100% (95% CI: 77-100%), sensitivity was 100% (95% CI: 90-100%). Positive and negative predictive values were 1.0 (95% CI: 0.9-1.0) and 1.0 (95% CI: 0.77-1.0) respectively. A post-biopsy pneumothorax was detected in 27 cases (39%); the pneumothorax rate was significantly affected by the number of passages (P=0.01). CONCLUSIONS: The satisfactory results of our study lead to reconsidering FNAB in patients with pulmonary nodules below 15 mm in diameter, especially in order to avoid unnecessary surgery.

A HS6ST2 gene variant associated with X-linked intellectual disability and severe myopia in two male twins.

X-linked intellectual disability (XLID) refers to a clinically and genetically heterogeneous neurodevelopmental disorder, in which males are more heavily affected than females. Among the syndromic forms of XLID, identified by additional clinical signs as part of the disease spectrum, the association between XLID and severe myopia has been poorly characterized. We used whole exome sequencing (WES) to study two Italian male twins presenting impaired intellectual function and adaptive behavior, in association with severe myopia and mild facial dysmorphisms. WES analysis detected the novel, maternally inherited, mutation c.916G > C (G306R) in the X-linked heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) gene. HS6ST2 transfers sulfate from adenosine 3'-phosphate, 5'-phosphosulfate to the sixth position of the N-sulphoglucosamine residue in heparan sulfate (HS) proteoglycans. Low HS sulfation levels are associated with defective optic disc and stalk morphogenesis during mammalian visual system development. The c.916G>C variant affects the HS6ST2 substrate binding site, and its effect was considered "deleterious" by in-silico tools. An in-vitro enzymatic assay showed that the HS6ST2 mutant isoform had significantly reduced sulphotransferase activity. Taken together, the results suggest that mutant HS6ST2 is possibly involved in the development of myopia and cognitive impairment, characteristics of the probands reported here.

Molecular profiling of lung cancer specimens and liquid biopsies using MALDI-TOF mass spectrometry.

BACKGROUND: Identification of predictive molecular alterations in lung adenocarcinoma is essential for accurate therapeutic decisions. Although several molecular approaches are available, a number of issues, including tumor heterogeneity, frequent material scarcity, and the large number of loci to be investigated, must be taken into account in selecting the most appropriate technique. MALDI-TOF mass spectrometry (MS), which allows multiplexed genotyping, has been adopted in routine diagnostics as a sensitive, reliable, fast, and cost-effective method. Our aim was to test the reliability of this approach in detecting targetable mutations in non-small cell lung cancer (NSCLC). In addition, we also analyzed low-quality samples, such as cytologic specimens, that often, are the unique source of starting material in lung cancer cases, to test the sensitivity of the system. METHODS: We designed a MS-based assay for testing 158 mutations in the EGFR, KRAS, BRAF, ALK, PIK3CA, ERBB2, DDR2, AKT, and MEK1 genes and applied it to 92 NSCLC specimens and 13 liquid biopsies from another subset of NSCLC patients. We also tested the sensitivity of the method to distinguish low represented mutations using serial dilutions of mutated DNA. RESULTS: Our panel is able to detect the most common NSCLC mutations and the frequency of the mutations observed in our cohort was comparable to literature data. The assay identifies mutated alleles at frequencies of 2.5-10%. In addition, we found that the amount of DNA template was irrelevant to efficiently uncover mutated alleles present at high frequency. However, when using less than 10 ng of DNA, the assay can detect mutations present in at least 10% of the alleles. Finally, using MS and a commercial kit for RT-PCR we tested liquid biopsy from 13 patients with identified mutations in cancers and detected the mutations in 4 (MS) and in 5 samples (RT-PCR). CONCLUSIONS: MS is a powerful method for the routine predictive tests of lung cancer also using low quality and scant tissues. Finally, after appropriate validation and improvement, MS could represent a promising and cost-effective strategy for monitoring the presence and percentage of the mutations also in non-invasive sampling.

IMP3 expression in NSCLC brain metastases demonstrates its role as a prognostic factor in non-neuroendocrine phenotypes.

Brain metastases from NSCLC are associated with a poor prognosis, and local radiotherapy is the most effective therapeutic strategy. The oncofetal protein IMP3 has been studied extensively, and evidence suggests that its expression is related to shorter overall survival and a more aggressive phenotype in solid malignancies. Here, the prognostic role of IMP3 was investigated in a cohort of patients with NSCLC brain metastases in correlation with survival and tumor histotype. A series of 42 NSCLC brain metastases samples was analyzed by tissue microarray and immunohistochemical staining for IMP3. IMP3 expression was associated with shorter overall survival in the whole series and in subgroups of metastases from non-neuroendocrine pulmonary malignancies and adenocarcinoma metastases. These results indicated that IMP3 is a strong prognostic factor in non-neuroendocrine brain metastases and in particular in patients with adenocarcinoma metastases.

Ten-year experience with endobronchial ultrasound-guided transbronchial needle aspiration: single center results in mediastinal diagnostic and staging.

BACKGROUND: Endobronchial ultrasonography with transbronchial needle aspiration (EBUS-TBNA) is recognized as an accurate and minimal invasive procedure for diagnosis and staging of lung cancer and lymph nodal malignancies. EBUS is recommended as the first choice procedure for mediastinal staging in lung cancer in international guidelines. METHODS: A retrospective evaluation was performed on single center experience with EBUS-TBNA in our thoracic surgery department in a 10-year time frame. Main indication for the procedure was suspected non-lymphomatous malignancy in intrathoracic lymph-nodes on computed tomography (CT) or positron emission tomography (PET) scan. All procedures were performed under conscious sedation in a day-hospital setting. All the aspirated specimens were obtained with a 22-gauge needle and were fixed in 10% formalin and paraffin embedded. Sections of 3 micron in thickness were cut and hematoxylin-eosin stained. RESULTS: From October 2005 to August 2016, 496 patients were submitted to EBUS-TBNA. Number of nodal stations punctured was 592 with a mean of 2.25 punctures per patient. Diagnosis of malignancy was obtained in 291 patients (58.6%). In 25 cases a nodal metastasis from an extrathoracic primary tumor was diagnosed. Sensitivity, specificity and diagnostic accuracy were 95%, 100% and 96% respectively. Negative predictive value was 90% and positive predictive value (PPV) was 100%. When molecular tests were requested, mutational analysis was successfully performed on cell block derived material in 55 out of 56 cases (98.2%), and fluorescence in situ hybridization (FISH) analysis in 26 out of 27 cases (96.2%). CONCLUSIONS: EBUS-TBNA in our setting was an accurate and safe tool to diagnose non-lymphomatous nodal malignancies. Interestingly, in our series EBUS-TBNA has demonstrated to yield sufficient tissue for molecular analysis.

MGMT-Methylated Alleles Are Distributed Heterogeneously Within Glioma Samples Irrespective of IDH Status and Chromosome 10q Deletion.

Several molecular markers drive diagnostic classification, prognostic stratification, and/or prediction of response to therapy in patients with gliomas. Among them, IDH gene mutations are valuable markers for defining subtypes and are strongly associated with epigenetic silencing of the methylguanine DNA methyltransferase (MGMT) gene. However, little is known about the percentage of MGMT-methylated alleles in IDH-mutated cells or the potential association between MGMT methylation and deletion of chromosome 10q, which encompasses the MGMT locus. Here, we quantitatively assessed MGMT methylation and IDH1 mutation in 208 primary glioma samples to explore possible differences associated with the IDH genotype. We also explored a potential association between MGMT methylation and loss of chromosome 10q. We observed that MGMT methylation was heterogeneously distributed within glioma samples irrespective of IDH status suggesting an incomplete overlap between IDH1-mutated and MGMT-methylated alleles and indicating a partial association between these two events. Moreover, loss of one MGMT allele did not affect the methylation level of the remaining allele. MGMT was methylated in about half of gliomas harboring a 10q deletion; in those cases, loss of heterozygosity might be considered a second hit leading to complete inactivation of MGMT and further contributing to tumor progression.

Detection of Loss of Imprinting by Pyrosequencing®.

Genomic imprinting is an epigenetically regulated process determining allele-specific expression in a parent-of-origin dependent manner. Altered expression of imprinted genes characterizes numerous congenital diseases including Beckwith-Wiedemann, Silver-Russell, Angelman, and Prader-Willi syndromes as well as acquired disorders such as cancer. The detection of imprinting alterations has important translational implications in clinics and the application of the Pyrosequencing(®) technology offers the possibility to identify accurately also subtle modifications in allele-specific expression and in DNA methylation levels.Here, we describe two methods to investigate genomic imprinting defects (loss of imprinting, LOI) using Pyrosequencing: (1) Allele-specific expression analysis based on single nucleotide polymorphism (SNP), and (2) quantification of DNA methylation.The protocol for the quantification of the allele-specific expression is carried out by analyzing an informative SNP located within the transcribed portion of an imprinted gene. The method includes the cDNA amplification of the region containing the SNP and the Pyrosequencing-based analysis for the quantitative allelic discrimination comparing the ratio of the two alleles.The second protocol allows the accurate quantification of the DNA methylation levels at the Imprinting Control Regions (ICRs). Imprinted genes are clustered in chromosomal regions and their expression is mainly regulated by DNA methylation at CpG sites located within the ICRs. After bisulfite modification of the genomic DNA, the region of interest is amplified by PCR and analyzed by Pyrosequencing. The methylation value at each CpG site is calculated by the CpG software, which determines the ratio of the incorporation of "C" and "T" and converts the value in methylation percentage.

PDGFB hypomethylation is a favourable prognostic biomarker in primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterised by the clonal proliferation of the haematopoietic precursors together with the progressive development of bone marrow fibrosis. This stromal alteration is an important clinical issue and specific prognostic markers are not currently available. In bone marrow biopsies from 58 PMF patients, we explored the methylation pattern of genes encoding cytokines involved in the stromal reaction, namely platelet-derived growth factor-beta (PDGFB), transforming growth factor-beta (TGFB) and basic fibroblast growth factor (FGF2). We also evaluated the methylation profile of the Long Interspersed Nucleotide Element 1 (LINE-1). PDGFB, FGF2 and LINE-1, but not TGFB, were significantly differently methylated in PMF compared to controls. Significantly, PDGFB hypomethylation (<16%) was correlated with a favourable PMF prognosis (grade of marrow fibrosis, p=0.03; International Prognostic Scoring Systems p=0.01 and Dynamic International Prognostic Scoring Systems, p=0.02). Although the basis of the association of PDGFB hypomethylation with favourable prognosis remains to be clarified, we speculate that hypomethylation in PMF could represent the effect of acquired somatic mutations in genes involved in epigenetic regulation of the genome.

ESX1 mRNA expression in seminal fluid is an indicator of residual spermatogenesis in non-obstructive azoospermic men.

STUDY QUESTION: Is the presence of ESX1 mRNA in seminal fluid (SF) an indicator of residual spermatogenesis in men with non-obstructive azoospermic (NOA)? SUMMARY ANSWER: ESX1 mRNA in SF is a suitable molecular marker for predicting the presence of residual spermatogenesis in testis. WHAT IS KNOWN ALREADY: ESX1 is an X-linked homeobox gene whose expression in testis is restricted to germ cells. We previously reported, in the testicular biopsies from azoospermic men, a positive correlation between the presence of ESX1 mRNA and residual spermatogenesis. STUDY DESIGN, SIZE, DURATION: We investigated ESX1 mRNA expression in 70 testicular fragments (TF) and 56 (SF) of 70 NOA men. As controls, we analyzed 8 TF from men with obstructive azoospermic (OA) and 9 SF from normozoospermic men. For all patients we considered the histological classification of testis biopsies and the recovery of spermatozoa by surgical procedures. PARTICIPANTS/MATERIALS, SETTING, METHODS: Relative ESX1 mRNA expression was evaluated by quantitative RT-PCR using the ΔΔCt method. The results were compared with the recovery of spermatozoa at surgery. MAIN RESULTS AND THE ROLE OF CHANCE: In TF from NOA patients we found that: (i) ESX1 mRNA level was significantly decreased as the severity of spermatogenic defects increased (P < 0.0001, one-way analysis of variance); (ii) the presence of ESX1 mRNA can predict the success of sperm retrieval (sensitivity: 80%). In SF from NOA patients we found that: (i) ESX1 mRNA was present in 78.5% of NOA men; (ii) the presence of ESX1 mRNA could predict the success of sperm retrieval (sensitivity: 84%). LIMITATIONS, REASONS FOR CAUTION: Spermatozoa were recovered at surgery in 5 out of 12 patients whose SF was negative for ESX1 mRNA expression. We think that discrepancies between molecular and clinical results could be reduced by analyzing more than one ejaculate from each man. WIDER IMPLICATIONS OF THE FINDINGS: The data confirm that the ESX1 transcript in the semen of men with NOA is a suitable molecular marker for predicting the presence of residual foci of spermatogenesis in the testis. The implication of these results is that some patients 'with azoospermia', although having a severe impairment of spermatogenesis, could still maintain residual foci of spermatogenesis in limited areas of the testes, not always recovered by surgery. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico: Ricerca Corrente [grant number RC2014/519-02] to M.M. and from ASM onlus 2010-2011 to M.M. The authors declare that they have no conflict of interest.

Synchronous pleural and peritoneal malignant mesothelioma: a case report and review of literature.

The coexistence of mesothelioma and other primary malignancies has been previously reported in literature, but the finding of a pleural mesothelioma with a synchronous peritoneal mesothelioma has not been reported so far. We report a case of a 58-years-old woman that came to our attention for the incidental finding of an inguinal mass. Fine-needle biopsies of the mass and a thoracoscopy with pleural biopsies were performed, after imaging studies showed pleural thickenings suspicious for malignancy. Histological morphology and growth pattern were similar in both cases. Both tumors stained for calretinin, but only the pleural mesothelioma showed positivity for Wilms-Tumor 1 antibody. We tried to demonstrate with molecular biology techniques whether they were synchronous or one was the metastasis of the other, but our studies did not give informative results. The prognosis in this case is poor, and after 6 months the patient is still following a chemotherapy regimen, which is the only practicable approach given the extent of the disease.

Recombinant chromosome 4 from a familial pericentric inversion: prenatal and adulthood wolf-hirschhorn phenotypes.

Pericentric inversion of chromosome 4 can give rise to recombinant chromosomes by duplication or deletion of 4p. We report on a familial case of Wolf-Hirschhorn Syndrome characterized by GTG-banding karyotypes, FISH, and array CGH analysis, caused by a recombinant chromosome 4 with terminal 4p16.3 deletion and terminal 4q35.2 duplication. This is an aneusomy due to a recombination which occurred during the meiosis of heterozygote carrier of cryptic pericentric inversion. We also describe the adulthood and prenatal phenotypes associated with the recombinant chromosome 4.

Genome-wide analysis of primary plasma cell leukemia identifies recurrent imbalances associated with changes in transcriptional profiles.

Primary plasma cell leukemia (pPCL) is a rare, yet aggressive form of de novo plasma cell tumor, distinct from secondary PCL (sPCL) which represents a leukemic transformation of pre-existing multiple myeloma (MM). Herein, we performed a comprehensive molecular analysis of a prospective series of pPCLs by means of FISH, single nucleotide polymorphism (SNP) array and gene expression profiling (GEP). IGH@ translocations were identified in 87% of pPCL cases, with prevalence of t(11;14) (40%) and t(14;16) (30.5%), whereas the most frequent numerical alterations involved 1p (38%), 1q (48%), 6q (29%), 8p (42%), 13q (74%), 14q (71%), 16q (53%), and 17p (35%). We identified a minimal biallelic deletion (1.5 Mb) in 8p21.2 encompassing the PPP2R2A gene, belonging to a family of putative tumor suppressors and found to be significantly down-regulated in deleted cases. Mutations of TP53 were identified in four cases, all but one associated with a monoallelic deletion of the gene, whereas activating mutations of the BRAF oncogene occurred in one case and were absent in N- and K-RAS. To evaluate the influence of allelic imbalances in transcriptional expression we performed an integrated genomic analysis with GEP data, showing a significant dosage effect of genes involved in transcription, translation, methyltransferase activity, apoptosis as well as Wnt and NF-kB signaling pathways. Overall, we provide a compendium of genomic alterations in a prospective series of pPCLs which may contribute to improve our understanding of the pathogenesis of this aggressive form of plasma cell dyscrasia and the mechanisms of tumor progression in MM.

Cytomegalovirus UL131-128 products promote gB conformational transition and gB-gH interaction during entry into endothelial cells.

Herpesviruses use gB and gH-gL glycoproteins to execute fusion. Other virus-specific glycoproteins are required for receptor binding and fusion activation. The human cytomegalovirus (HCMV) UL131-128 proteins are essential for the infection of leukocytes, endothelial cells (ECs), and many epithelial cell lines. Here we show that UL131-128 play a role in a chain of events involving gB and gH during HCMV entry into ECs. An HCMV strain bearing the wild-type (wt) UL131-128 locus exhibited a gB transition from a protease-resistant to protease-sensitive form, a conformational change that was suppressed by a thiourea inhibitor of fusion (WY1768); in contrast, gH was susceptible to proteolysis throughout entry. Moreover, gB and gH transiently interacted, and a lipid mixing assay showed that the wt strain had carried out fusion by 60 min postinfection. However, these events were greatly altered when UL131-128-defective strains were used for infection or when there was an excess of soluble pUL128 during wt infection: the gB conformational change became WY1768 resistant, the gB-gH complex was no longer observed, and fusion was prevented. Both gB and gH in this case showed late protease resistance, related to their endocytic uptake. Our data point to the involvement of UL131-128 proteins in driving gB through a WY1768-sensitive fold transition, thus promoting a short-lived gB-gH complex and fusion; they also suggest that HCMV fuses with the EC plasma membrane and that endocytosis ensues only when the virus cannot trigger UL131-128-dependent steps.