Impact of High-Grade Patterns in Early-Stage Lung Adenocarcinoma: A Multicentric Analysis.

OBJECTIVE: The presence of micropapillary and solid adenocarcinoma patterns leads to a worse survival and a significantly higher tendency to recur. This study aims to assess the impact of pT descriptor combined with the presence of high-grade components on long-term outcomes in early-stage lung adenocarcinomas. METHODS: We retrospectively collected data of consecutive resected pT1-T3N0 lung adenocarcinoma from nine European Thoracic Centers. All patients who underwent a radical resection with lymph-node dissection between 2014 and 2017 were included. Differences in Overall Survival (OS) and Disease-Free Survival (DFS) and possible prognostic factors associated with outcomes were evaluated also after performing a propensity score matching to compare tumors containing non-high-grade and high-grade patterns. RESULTS: Among 607 patients, the majority were male and received a lobectomy. At least one high-grade histological pattern was seen in 230 cases (37.9%), of which 169 solid and 75 micropapillary. T1a-b-c without high-grade pattern had a significant better prognosis compared to T1a-b-c with high-grade pattern (p = 0.020), but the latter had similar OS compared to T2a (p = 0.277). Concurrently, T1a-b-c without micropapillary or solid patterns had a significantly better DFS compared to those with high-grade patterns (p = 0.034), and it was similar to T2a (p = 0.839). Multivariable analysis confirms the role of T descriptor according to high-grade pattern both for OS (p = 0.024; HR 1.285 95% CI 1.033-1.599) and DFS (p = 0.003; HR 1.196, 95% CI 1.054-1.344, respectively). These results were confirmed after the propensity score matching analysis. CONCLUSIONS: pT1 lung adenocarcinomas with a high-grade component have similar prognosis of pT2a tumors.

Surgical management of tracheal chondrosarcoma.

Background: Tracheal chondrosarcoma is an extremely rare, slow-growing, malignant tumour. This study aims to analyze the cases of tracheal chondrosarcoma published in the literature and our case report, in order to better define tracheal chondrosarcoma management.Methods: A systematic review of the English literature was carried out for fully described tracheal chondrosarcoma cases. Additionally, we reported a new case of a 58-year-old man undergoing tracheal resection and reconstruction for tracheal chondrosarcoma.Results: To date, 30 cases were published. This tumour predominantly involved male patients (93%; median age: 65 years), generally conditioning dyspnoea and cough. Most of the patients underwent tracheal resection with end-to-end anastomosis, without recurrence (median follow-up: 2 years). Tumours endoscopically treated recurred in half cases.Conclusion: Tracheal resection is the treatment of choice for chondrosarcoma, with an excellent prognosis. Endoscopic treatment and/or radiotherapy should be indicated for patients unfit for surgery.

Prognostic impact of lung adenocarcinoma second predominant pattern from a large European database.

BACKGROUND AND OBJECTIVES: Adenocarcinoma patterns could be grouped based on clinical behaviors: low- (lepidic), intermediate- (papillary or acinar), and high-grade (micropapillary and solid). We analyzed the impact of the second predominant pattern (SPP) on disease-free survival (DFS). METHODS: We retrospectively collected data of surgically resected stage I and II adenocarcinoma. SELECTION CRITERIA: anatomical resection with lymphadenectomy and pathological N0. Pure adenocarcinomas and mucinous subtypes were excluded. Recurrence rate and factors affecting DFS were analyzed according to the SPP focusing on intermediate-grade predominant pattern adenocarcinomas. RESULTS: Among 270 patients, 55% were male. The mean age was 68.3 years. SPP pattern appeared as follows: lepidic 43.0%, papillary 23.0%, solid 14.4%, acinar 11.9%, and micropapillary 7.8%. The recurrence rate was 21.5% and 5-year DFS was 71.1%. No difference in DFS was found according to SPP (p = .522). In patients with high-grade SPP, the percentage of SPP, age, and tumor size significantly influenced DFS (p = .016). In patients with lepidic SPP, size, male gender, and lymph-node sampling (p = .005; p = .014; p = .038, respectively) significantly influenced DFS. CONCLUSIONS: The impact of SPP on DFS is not homogeneous in a subset of patients with the intermediate-grade predominant patterns. The influence of high-grade SPP on DFS is related to its proportion in the tumor.

Could Maximum SUV be Used as Imaging Guidance in Large Lung Lesions Biopsies? Double Sampling Under PET-CT/XperGuide Fusion Imaging in Inhomogeneous Lung Uptaking Lesions to Show That it can Make a Difference.

Introduction: The purpose of this study is to evaluate the diagnostic value of positron emission computed tomography-cone beam computed tomography (PET/CT-CBCT) fusion guided percutaneous biopsy, targeted to the maximum standardized uptake value (SUVmax) and minimum standardized uptake value (SUVmin) of large lung lesions. Materials and Methods: Inside a larger cohort of PET/CT-CBCT guided percutaneous lung biopsies, 10 patients with large pulmonary lesions (diameter > 30 mm) were selected retrospectively. These patients have been subjected to double biopsy sampling respectively in the SUVmax area and in the SUVmin area of the lesion. Technical success has been calculated. For each sample, the percentage of neoplastic, inflammatory, and fibrotic cells was reported. Furthermore, the possibility of performing immunohistochemical or molecular biology investigations to specifically define the biomolecular tumor profile was analyzed. Results: Nine lesions were found to be malignant, one benign (inflammation). Technical success was 100% (10/10) in the SUVmax samples and 70% (7/10) in the SUVmin samples (P-value: .21). In the first group, higher percentages of neoplastic cells were found at pathologic evaluation, while in the second group areas of inflammation and fibrosis were more represented. The biomolecular profile was obtained in 100% of cases (9/9) of the first group, while in the second group only in 33.3% of cases (2/6), with a statistically significant difference between the 2 groups (P-value: .011). Conclusion: A correlation between the standardized uptake value value and the technical success of the biopsy sample has been identified. PET/CT-CBCT guidance allows to target the biopsy in the areas of the tumor which are richer in neoplastic cells, thus obtaining more useful information for the planning of patient-tailored cancer treatments.

Neuroendocrine Carcinoma of the Urinary Bladder: CT Findings and Radiomics Signature.

Background: We present a case series of Neuroendocrine Carcinoma of the Urinary Bladder (NECB) to analyse their radiologic appearance on CT, find a "Radiomic signature", and review the current literature. Methods: 14 CT cases of NECB were reviewed and compared with a control group of 42 patients with high-grade non-neuroendocrine bladder neoplasm for the following parameters: ring enhancement; implantation site; dimensions; density; margins; central necrosis; calcifications; number of lesions; wall thickness; depth of invasion in the soft tissue; invasion of fat tissue; invasion of adjacent organs; lymph-node involvement; abdominal organ metastasis. To extract radiomic features, volumes of interest of bladder lesions were manually delineated on the portal-venous phase. The radiomic features of the two groups were identified and compared. Results: Statistical differences among NECB and control group were found in the prevalence of male sex (100% vs. 69.0%), hydronephrosis (71.4% vs. 33.3%), mean density of the mass (51.01 ± 15.48 vs. 76.27 ± 22.26 HU); product of the maximum diameters on the axial plane (38.1 ± 59.3 vs. 14.44 ± 12.98 cm2) in the control group, trigonal region involvement (78.57% vs. 19.05%). About the radiomic features, Student's t-test showed significant correlation for the variables: "DependenceNonUniformity" (p: 0.048), "JointAverage" (p: 0.013), "LargeAreaLowGrayLevelEmphasis" (p: 0.014), "Maximum2DDiameterColumn" (p: 0.04), "Maximum 2DDiameterSlice" (p: 0.007), "MeanAbsoluteDeviation" (p: 0.021), "BoundingBoxA" (p: 0.022) and "CenterOfMassB" (p: 0.007). Conclusions: There is a typical pattern (male patient, large mass, trigonal area involvement) of NECB presentation on contrast-enhanced CT. Certain morphological characteristics and encouraging results about Radiomic features can help define the diagnosis.

Dual-Layer Spectral CT as Innovative Imaging Guidance in Lung Biopsies: Could Color-Coded Z-Effective Images Allow More Diagnostic Samplings and Biomarkers Information?

The aim of the study was to try to obtain more information on diagnostic samplings and biomarkers using dual-layer spectral CT in lung biopsies. Lung biopsies were performed by merging images obtained with CBCT with those from spectral CT to use them as functional guidance, experimenting with double sampling to determine the difference between the area with a higher Z-effective number and that with a lower Z-effective number. Ten patients with large lung lesions on spectral CT were selected and underwent percutaneous transthoracic lung mass biopsy. Technical success was calculated. The percentage of neoplastic, inflammatory, fibrotic, necrotic cells, or non-neoplastic lung parenchyma was reported. The possibility of carrying out immunohistochemical or molecular biology investigations was analyzed. All lesions were results malignant in 10/10 samples in the Zmax areas; in the Zmin areas, malignant cells were found in 7/10 samples. Technical success was achieved in 100% of cases for Zmax sampling and in 70% for Zmin sampling (p-value: 0.2105). The biomolecular profile was detected in 9/10 (90%) cases in Zmax areas, while in 4/10 (40%) cases in Zmin areas (p-value: 0.0573). The advantage of Z-effective imaging would be to identify a region of the lesion that is highly vascularized and probably richer in neoplastic cells, thus decreasing the risk of obtaining a non-diagnostic biopsy sample.

Biphenotypic sinonasal sarcoma: European multicentre case-series and systematic literature review.

Objective: Biphenotypic sinonasal sarcoma (BSNS) is a rare low-grade cancer that was included from the 4th edition of WHO classification of head and neck tumours. The purpose of this study is to analyse clinical behaviour, pattern of recurrences and survival outcomes of this neoplasm. Methods: Retrospective review of patients affected by BSNS who were treated via an endoscopic-assisted approach in 6 European tertiary-care referral hospitals. Cases of BSNS described in literature since 2012 to date were fully reviewed, according to PRISMA guidelines. Results: A total of 15 patients were included. Seven patients were treated via an endoscopic endonasal approach, 4 with endoscopic transnasal craniectomy, and 4 via a cranio-endoscopic approach. Adjuvant treatment was delivered in 2 cases. After a mean follow-up of 27.3 months, systemic metastasis was observed in 1 case; the 5-year overall survival and disease-free survival rates were 100% and 80 ± 17.9%, respectively. Conclusions: BSNS is a locally aggressive tumour with a low recurrence rate and encouraging survival outcomes if properly treated with surgical resection and free margins followed by adjuvant radiotherapy for selected cases. Endoscopic-assisted surgery is safe and effective as an upfront treatment within a multidisciplinary care protocol.

Pathological and clinical features of multiple cancers and lung adenocarcinoma: a multicentre study.

OBJECTIVES: Lung cancer is increasingly diagnosed as a second cancer. Our goal was to analyse the characteristics and outcomes of early-stage resected lung adenocarcinomas in patients with previous cancers (PC) and correlations with adenocarcinoma subtypes. METHODS: We retrospectively reviewed data of patients radically operated on for stage I-II lung adenocarcinoma in 9 thoracic surgery departments between 2014 and 2017. Overall survival (OS) and time to disease relapse were evaluated between subgroups. RESULTS: We included 700 consecutive patients. PC were present in 260 (37.1%). Breast adenocarcinoma, lung cancer and prostate cancer were the most frequent (21.5%, 11.5% and 11.2%, respectively). No significant differences in OS were observed between the PC and non-PC groups (P = 0.378), with 31 and 75 deaths, respectively. Patients with PC had smaller tumours and were more likely to receive sublobar resection and to be operated on with a minimally invasive approach. Previous gastric cancer (P = 0.042) and synchronous PC (when diagnosed up to 6 months before lung adenocarcinoma; P = 0.044) were related, with a worse OS. Colon and breast adenocarcinomas and melanomas were significantly related to a lower incidence of high grade (solid or micropapillary, P = 0.0039, P = 0.005 and P = 0.028 respectively), whereas patients affected by a previous lymphoma had a higher incidence of a micropapillary pattern (P = 0.008). CONCLUSIONS: In patients with PC, we found smaller tumours more frequently treated with minimally invasive techniques and sublobar resection, probably due to a more careful follow-up. The impact on survival is not uniform and predictable; however, breast and colon cancers and melanoma showed a lower incidence of solid or micropapillary patterns whereas patients with lymphomas had a higher incidence of a micropapillary pattern.

Prognostic role of standard uptake value according to pathologic features of lung adenocarcinoma.

OBJECTIVE: To evaluate the influence of lung adenocarcinoma second predominant pattern on the maximal standard uptake value (SUVmax) and its prognostic effect in different histologic groups. METHODS: We retrospectively collected surgically resected pathologic stage I and II lung adenocarcinoma from nine European institutions. Only patients who underwent preoperative PET-CT and with available information regarding SUVmax of T (SUVmaxT) and N1 (SUVmaxN1) component were included. RESULTS: We enrolled 344 patients with lung adenocarcinoma. SUVmaxT did not show any significant relation according to the second predominant pattern (p = 0.139); this relationship remained nonsignificant in patients with similar predominant pattern. SUVmaxT influenced the disease-free survival in the whole cohort (p = 0.002) and in low- and intermediate-grade predominant pattern groups (p = 0.040 and p = 0.008, respectively). In the high-grade predominant pattern cohort and in the pathologic N1 cases, SUVmaxT lost its prognostic power. SUVmaxN1 did not show any significant correlation with predominant and second predominant patterns and did not have any prognostic impact on DFS. CONCLUSIONS: SUVmaxT is influenced only by the adenocarcinoma predominant pattern, but not by second predominant pattern. Concurrently, in high-grade predominant pattern and pN1 group the prognostic power of SUVmaxT becomes nonsignificant.

Different intracellular compartmentalization of TA and DeltaNp73 in non-small cell lung cancer.

The p53 homologue p73 is overexpressed in many tumors, including lung cancer. We have evaluated the differential expression and subcellular localization of the functionally distinct apoptotic (TA) and anti-apoptotic (DeltaN) isoforms of p73 in non-small cell lung cancer (NSCLC), their possible association with p53 expression and determined the methylation status of the two p73 gene promoters (P1 and P2) in this tumor type. Immunohistochemical analysis showed that both isoforms are expressed in the majority of cases. However, the oncogenic DeltaN variant, derived from the transcripts DeltaN'p73 (from P1) and/or DeltaNp73 (from P2), is localized mainly in the nucleus, while the anti-oncogenic TAp73 isoform (derived from a P1 transcript) is sequestered in the cytoplasm in almost all cases analyzed. Significant correlation was found between p53 and DeltaNp73 expression (p=0.041). Methylation analysis conducted on 41 tumor samples showed that the P1 promoter is almost invariably unmethylated (39/41 cases) whereas P2 was found completely methylated in 17 cases and partially or totally unmethylated in 24 samples. No correlation was found between the methylation status of P1 and P2 and p73 expression. Our results demonstrate that both isoforms contribute to p73 overexpression in NSCLC and suggest that their different intracellular localization may reflect an alteration of the functional p53-p73 network that might contribute to lung cancer development.

Osteopontin is not a specific marker in malignant pleural mesothelioma.

BACKGROUND AND AIMS: Osteopontin (OPN) is an integrin-binding protein recently shown to be related to tumorigenesis, progression and metastasis in different experimental models of malignancy. Malignant pleural mesothelioma (MPM) is a fatal disease in which the prognosis remains very poor and the knowledge of predictive factors for outcome is insufficient. The identification of new molecules involved in cancer initiation and development is a fundamental step for improving the curability of this kind of tumor. The purpose of this study is to define the role of OPN in the diagnosis of MPM by determining its prognostic and diagnostic value. METHODS: A group of 24 surgically staged MPM subjects was compared with a group of 31 subjects with nonmalignant pulmonary diseases, and with 37 healthy controls. Tumor tissue was analyzed for OPN by immunohistochemical tests, and plasma OPN levels were measured by an enzyme-linked immunosorbent assay. RESULTS: Plasma OPN levels were not significantly higher in either of the patient groups compared with the control group. Immunohistochemical analysis revealed OPN staining of tumor cells in 21 of 24 MPMs. Receiver operating characteristic curve/area under the curve (ROC/AUC) analysis comparing the plasma OPN levels in the healthy group with those of MPM patients showed 40% sensitivity and 100% specificity at a cutoff value of 60.8 ng of OPN per milliliter (AUC 0.6). CONCLUSION: Plasma OPN levels do not discriminate between chronic inflammatory and malignant lung diseases and staining intensity in MPM specimens does not correlate with OPN plasma levels.

Beta1- and Beta2-Adrenoceptors Expression Patterns in Human Non-small Cell Lung Cancer: Relationship with Cancer Histology.

Assessment of Beta-AR protein expression on tumour tissues might be a plausible strategy to select cancer patients who can benefit from Beta-blockers therapy. The aim of this study is to evaluate the differences between resected tissue specimens from primary lung cancer (adenocarcinoma (ADC) and squamous cell carcinoma (SCC)) in terms of expression pattern of Beta1- and Beta2-AR in both tumour and adjacent surrounding non-tumour tissue. This retrospective study was based on the analysis of 80 patients with histologically confirmed diagnosis of primary Non-Small Cell Lung Cancer (NSCLC) who received surgical treatment. The cases were carefully selected in order to obtain the most homogeneous sample in terms of histologic subtype (40 ADCs and 40 SCCs) and clinical stage (10 each). Beta1- and Beta2-AR expression was determined by immunohistochemistry and the staining evaluated by semi-quantitative scoring using the H-score method. In our NSCLC series, Beta1- and Beta2-AR are differentially expressed. Beta1-AR expression is present at low levels in both SCC and ADC. Likewise, when compared with the matched surrounding non-tumour tissues, Beta1-AR expression level was significantly lower in both histologic subtypes. Conversely, Beta2-AR is highly expressed in both histologic subtypes, but clearly highly expressed in ADC when compared with SCC and with their matched surrounding non-tumour tissue. Overall, this clinicopathological study highlights the differential expression of Beta1- and Beta2-AR in ADC and SCC. Repurposing non-selective Beta-blockers in oncologic setting might be a suitable therapeutic strategy for lung ADC. Graphical abstract.

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.

BACE2 is stored in secretory granules of mouse and rat pancreatic beta cells.

BACE2 is a protease homologous to BACE1 protein, an enzyme involved in the amyloid formation of Alzheimer disease (AD). However, despite the high homology between these two proteins, the biological role of BACE2 is still controversial, even though a few studies have suggested a pathogenetic role in sporadic inclusion-body myositis and hereditary inclusion-body myopathy, which are characterized by vacuolization of muscular fibers with intracellular deposits of proteins similar to those found in the brain of AD patients. Although BACE2 has also been identified in the pancreas, its function remains unknown and its specific localization in different pancreatic cell types has not been definitively ascertained. For these reasons, the authors have investigated the cellular and subcellular localization of BACE2 in normal rodent pancreases. BACE2 immunoreactivity was found in secretory granules of beta cells, co-stored with insulin and IAPP, while it was lacking in the other endocrine and exocrine cell types. The presence of BACE2 in secretory granules of beta cells suggests that it may play a role in diabetes-associated amyloidogenesis.

Prognostic factors for ampullary adenocarcinomas: tumor stage, tumor histology, tumor location, immunohistochemistry and microsatellite instability.

Prognostic factors for ampullary carcinomas (ACs) are poorly defined. Fifty three resected ACs were analyzed for CDX2, MUC1, MUC5AC, MUC6, MUC2, and for mismatch repair proteins (hMLH1, hMSH2, PMS2, hMSH6) using immunohistochemistry. Microsatellite instability (MSI) status was evaluated by fluorescently labeled PCR using an automated sequencer. Univariate and multivariate analysis was performed for clinicopathological, immunohistochemical and molecular parameters. CDX2 was found in 32 out of 53 (60%) ACs with a significantly higher frequency among intestinal ACs compared with biliopancreatic (BP) ACs. The MUC1, MUC5AC, MUC6, MUC2 apomucins were expressed in 75, 43, 39, and 28% of ACs, respectively, with a significantly higher coexpression of MUC1/MUC5AC in BP ACs. MSI and loss of expression of hMLH1/PMS2 or hMSH2/hMSH6 proteins were observed only in intestinal ACs. Factors significantly correlated with improved survival in the univariate analysis were: low stage, absence of lymph nodes metastases, negative surgical margins (R0 status), and presence of MSI. In the multivariate analysis, stage was the only independent prognostic factor of survival. We conclude that stage is the only independent prognostic factor of survival in the multivariate analysis, whereas histological criteria and the immunohistochemical expression of apomucins and CDX2 are helpful in the classification and understanding of the histogenesis of ACs.

Ultrastructural evidence of Tropheryma whippelii in PAS-negative granulomatous lymph nodes.

The presence of Tropheryma whippelii was demonstrated in the PAS-negative mesenteric granulomatous lymph nodes of a patient affected by Whipple disease. Ultrastructurally a few bacteria, enclosed by a membrane characteristic of Tropheryma whippelii, were found in the extracellular spaces and remnants of bacteria were found in the phagocytic vacuoles of macrophages. The scarce number of bacilli, probably due to the fact that the disease was at an initial phase, could explain the absence of PAS positivity. This case confirms the role of the electron microscopy in the diagnosis of Whipple disease, especially for extra-intestinal lesions and at the initial phase of the disease, when the characteristic PAS-positive macrophages can be absent.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA)-from morphology to molecular testing.

In recent years, endobronchial ultrasound-guided TBNA (EBUS-TBNA) has emerged as an innovative technique for diagnosis and staging of lung cancer and has been successfully introduced into daily clinical practice with several advantages including minimally invasive approach, safe, cost-effective, real time image guidance, broad sampling capability, and rapid on-site evaluation (ROSE). Both cytological and histological approach could be useful to have material for diagnosis, immunohistochemical and molecular analyses which may be very important for targeted therapy with successful rate ranging from 89% to 98%. The utility of ROSE during EBUS-TBNA has been matter of debate. Indeed, although some evidence concluded that ROSE does not increase the diagnostic efficacy of EBUS-TBNA, other demonstrated that it improves the diagnostic yield of the procedure up to 30%, allows to avoid repetition of additional diagnostic procedures and reduces risk of complications. Furthermore the sample preparation by cytopathologist is optimized with the aid of direct macroscopic inspection, optimal smearing techniques, and triage of the sample permitting to obtain adequate tissue for diagnosis, ancillary techniques and molecular testing, when needed. Some pathological issues on EBUS-TBNA are reviewed and discussed with particular focus on ROSE and molecular testing.

LINE-1 hypomethylation is associated to specific clinico-pathological features in Stage I non-small cell lung cancer.

OBJECTIVES: We hypothesize that selected genetic and/or epigenetic changes associated with advanced tumours may help identifying early non-small cell lung cancers (NSCLCs) that recur after resection. Among epigenetic changes, long interspersed nuclear element-1 (LINE-1) hypomethylation is seen early during carcinogenesis and may act in concert with genetic alterations to cancer progression. LINE-1 hypomethylation and gene mutations frequently involved in lung cancer, were analysed to evaluate their prognostic role in resected stage I NSCLC. METHODS: Gene mutations and LINE-1 methylation were analysed in 167 Caucasian patients with stage I NSCLC, namely 100 adenocarcinomas (ADC) and 67 squamous-cell carcinomas (SqCC), using mass-spectrometry and pyrosequencing. We evaluated the correlation between molecular results and clinico-pathological data: age, gender, smoking status, period of surgery, histology, grading, pathological stage, p53 expression, LINE-1 hypomethylation. These variables have been assessed as possible predictors of cancer related survival by regression analysis. RESULTS: Frequency and spectrum of gene mutations were significantly different in ADCs compared with SqCCs. p53 positivity was more common in SqCC, while EGFR or KRAS mutations were mainly detected in ADC. LINE1 hypomethylation was associated with SqCC histology, p53 immunoreactivity and smoking habit. Stage IB, LINE-1 hypomethylation and PIK3CA mutation independently predicted a worse cancer-related survival. When combined into a scoring system, their prognostic power was strengthened. CONCLUSIONS: In many stage I NSCLC a mutation pattern of advanced disease was observed. Stage IB, LINE-1 hypomethylation and PIK3CA mutation were associated to poor prognosis. Genetic and epigenetic events occurring in early carcinogenesis may help identifying stage I NSCLC patients who deserve adjuvant therapy.

Pyrosequencing for EGFR mutation detection: diagnostic accuracy and clinical implications.

EGFR-activating mutations predict responsiveness to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) patients. Mutation screening is crucial to support therapeutic decisions and is commonly conducted using dideoxy sequencing, although its sensitivity is suboptimal in clinical settings. To evaluate the diagnostic performance of pyrosequencing and dideoxy sequencing, we examined EGFR mutation status in a retrospective cohort of 53 patients with NSCLCs clinically selected for TKI therapy and whose clinical outcome was available. Moreover, pyrosequencing quantitative results were compared with EGFR amplification data. EGFR mutations were investigated by pyrosequencing and by dideoxy sequencing. Detection rates of both methods were determined by titration assays using NCI-H1975 and HCC-827 cell lines. Increased EGFR copy number was assessed by fluorescence in situ hybridization (FISH). Pyrosequencing showed a higher detection rate than dideoxy sequencing. Tumor control rate of cases with mutant and wild-type EGFR was 86% and 29%, respectively. EGFR amplification was significantly associated with EGFR mutation and a positive correlation between high percentages of mutant alleles and clinical response to TKI was observed. We concluded that pyrosequencing is more sensitive than dideoxy sequencing in mutation screening for EGFR mutations. Detection rate of dideoxy sequencing was suboptimal when low frequencies of mutant alleles or low tumor cell contents were observed. Pyrosequencing enables quantification of mutant alleles that correlates well with increased EGFR copy number assessed by FISH. Pyrosequencing should be used in molecular diagnostic of NSCLC to appropriately select patients who are likely to benefit from TKI therapy.

The proangiogenic phenotype of natural killer cells in patients with non-small cell lung cancer.

The tumor microenvironment can polarize innate immune cells to a proangiogenic phenotype. Decidual natural killer (dNK) cells show an angiogenic phenotype, yet the role for NK innate lymphoid cells in tumor angiogenesis remains to be defined. We investigated NK cells from patients with surgically resected non-small cell lung cancer (NSCLC) and controls using flow cytometric and functional analyses. The CD56(+)CD16(-) NK subset in NSCLC patients, which represents the predominant NK subset in tumors and a minor subset in adjacent lung and peripheral blood, was associated with vascular endothelial growth factor (VEGF), placental growth factor (PIGF), and interleukin-8 (IL-8)/CXCL8 production. Peripheral blood CD56(+)CD16(-) NK cells from patients with the squamous cell carcinoma (SCC) subtype showed higher VEGF and PlGF production compared to those from patients with adenocarcinoma (AdC) and controls. Higher IL-8 production was found for both SCC and AdC compared to controls. Supernatants derived from NSCLC CD56(+)CD16(-) NK cells induced endothelial cell chemotaxis and formation of capillary-like structures in vitro, particularly evident in SCC patients and absent from controls. Finally, exposure to transforming growth factor-ß(1) (TGFß(1)), a cytokine associated with dNK polarization, upregulated VEGF and PlGF in peripheral blood CD56(+)CD16(-) NK cells from healthy subjects. Our data suggest that NK cells in NSCLC act as proangiogenic cells, particularly evident for SCC and in part mediated by TGFß(1).