A fatal case of post endobronchial ultrasound transbronchial needle aspiration endobronchial haemorrhage: a case report and review of the literature.

Notwithstanding life-threatening haemorrhagic complications, endobronchial ultrasound-guided transbronchial needle aspiration represents a cornerstone in the evaluation of NSCLC patients. Due to its low invasiveness and satisfactory tolerability even in high-risk patients, it is usually scheduled in one-day surgery hospital stay. Moreover, EBUS-TBNA offers a viable alternative to other conventional endoscopic procedures such as mediastinoscopy with the addiction to gain access also to hilar nodes. We report an unexpected and rare event of post-bronchoscopy fatal endobronchial haemorrhage in a 67-year-old female patient with a right S1 solitary pulmonary nodule and concomitant multiple bilateral lymphadenopaties. According to clinical staging the patient was unfit for upfront surgery and endoscopic procedure for tissue diagnosis was scheduled. The immediate postoperative period was uneventful as no intraoperative injuries were reported. Twenty-four hours later, the patient claimed respiratory distress and, after admission to the Emergency Department, a massive and uncontrolled bleeding coming from the lower respiratory tract was diagnosed leading to a death. Although one-day surgery EBUS-TBNA is the standard of care in patients undergoing endoscopic exploration, current guidelines seem to be murky about proper case stratification in order to plan an exhaustive observation time especially in high-risk patients.

ROS1 Gene Fusion in Advanced Lung Cancer in Women: A Systematic Analysis, Review of the Literature, and Diagnostic Algorithm.

PURPOSE: Crizotinib, a mesenchymal-epithelial transition/anaplastic lymphoma kinase/c-ros oncogene 1 (ROS1) inhibitor, has recently been approved by the US Food and Drug Administration for the treatment of patients with advanced ROS1-positive non-small-cell lung cancer (NSCLC). Therefore, interest in ROS1 testing is growing. ROS1 gene fusions affect approximately 0.5% to 2% of unselected NSCLCs. Limited data are available on the prevalence and distribution of ROS1 fusions in patients with advanced-stage NSCLC. MATERIAL AND METHODS: A series of 727 lung adenocarcinomas from patients with stage IV disease, negative for epidermal growth factor receptor and anaplastic lymphoma kinase alterations, were tested for ROS1 fusions by fluorescent in situ hybridization analysis, with confirmation by immunohistochemistry. Results were correlated with clinicopathologic parameters and compared with data from the literature. RESULTS: ROS1 fusions were detected in 29 patients (4%), including 27 of 266 females (10.2%) and two of 461 males (0.4%; P = 1.2E-10). The mean age of patients with ROS1-positive disease was lower than that of patients with ROS1-negative disease (49.21 v 62.96 years, respectively; P = 1.1E-10). Eleven of 583 smokers (1.9%) and 18 of 144 nonsmokers (12.5%) showed ROS1 rearrangement (P = 4.05E-7). By logistic regression analysis, ROS1 fusions were independently associated with female sex, younger age at diagnosis, and absence of smoking history, (odds ratios, 12.4, 7.9, and 3.6, respectively). These data, integrated with those reported in the literature, indicate that the prevalence of ROS1 fusions in females and in nonsmokers was higher in patients with advanced disease than in patients with operable disease (11.2% v 3.1%, P < .001; 11.6% v 2.8%, P < .001, respectively). The mean age at diagnosis was significantly lower in patients with advanced disease (49.8 years) than in patients with operable disease (55.6 years; P < .001). CONCLUSION: Our data indicate that ROS1 fusions in patients with advanced-stage lung adenocarcinoma are more frequent in females, particularly if young and nonsmokers. A diagnostic algorithm for an accurate screening of ROS1 alterations was elaborated.

Validation of a new algorithm for a quick and easy RT-PCR-based ALK test in a large series of lung adenocarcinomas: Comparison with FISH, immunohistochemistry and next generation sequencing assays.

OBJECTIVES: Anaplastic Lymphoma Kinase (ALK) gene rearrangements have been described in 3-5% of lung adenocarcinomas (ADC) and their identification is essential to select patients for treatment with ALK tyrosine kinase inhibitors. For several years, fluorescent in situ hybridization (FISH) has been considered as the only validated diagnostic assay. Currently, alternative methods are commercially available as diagnostic tests. MATERIAL AND METHODS: A series of 217 ADC comprising 196 consecutive resected tumors and 21 ALK FISH-positive cases from an independent series of 702 ADC were investigated. All specimens were screened by IHC (ALK-D5F3-CDx-Ventana), FISH (Vysis ALK Break-Apart-Abbott) and RT-PCR (ALK RGQ RT-PCR-Qiagen). Results were compared and discordant cases subjected to Next Generation Sequencing. RESULTS: Thirty-nine of 217 samples were positive by the ALK RGQ RT-PCR assay, using a threshold cycle (Ct) cut-off ≤35.9, as recommended. Of these positive samples, 14 were negative by IHC and 12 by FISH. ALK RGQ RT-PCR/FISH discordant cases were analyzed by the NGS assay with results concordant with FISH data. In order to obtain the maximum level of agreement between FISH and ALK RGQ RT-PCR data, we introduced a new scoring algorithm based on the ΔCt value. A ΔCt cut-off level ≤3.5 was used in a pilot series. Then the algorithm was tested on a completely independent validation series. By using the new scoring algorithm and FISH as reference standard, the sensitivity and the specificity of the ALK RGQ RT-PCR(ΔCt) assay were 100% and 100%, respectively. CONCLUSIONS: Our results suggest that the ALK RGQ RT-PCR test could be useful in clinical practice as a complementary assay in multi-test diagnostic algorithms or even, if our data will be confirmed in independent studies, as a standalone or screening test for the selection of patients to be treated with ALK inhibitors.

ALK Protein Analysis by IHC Staining after Recent Regulatory Changes: A Comparison of Two Widely Used Approaches, Revision of the Literature, and a New Testing Algorithm.

INTRODUCTION: Recent regulatory changes have allowed the diagnostic use of immunohistochemical (IHC) analysis for the identification of patients with non-small cell lung cancer who are eligible for treatment with anaplastic lymphoma receptor tyrosine kinase (ALK) inhibitors. The U.S. Food and Drug Administration has approved the VENTANA ALK (D5F3) CDx Assay (Ventana Medical Systems, Tucson, AZ) as companion diagnostics, and the Italian Medicines Agency has recognized IHC analysis as a diagnostic test indicating an algorithm for patient selection. METHODS: On the basis of the new regulations, we compared two commonly used IHC assays on 1031 lung adenocarcinomas: the VENTANA ALK (D5F3) CDx Assay with the OptiView Amplification Kit (Ventana Medical Systems) and a standard IHC test with the clone 5A4 (Novocastra, Leica Biosystems, Newcastle Upon Tyne, United Kingdom) along with their interpretative algorithms. Fluorescence in situ hybridization (FISH) was performed in all cases. Next-generation sequencing was performed in FISH/IHC analysis-discordant samples. RESULTS: FISH gave positive results in 33 (3.2%) cases. When FISH was used as a reference, the VENTANA ALK (D5F3) CDx assay had a sensitivity of 90.9% ± 2.6%, a specificity of 99.8% ± 0.6%, and positive and negative predictive values of 93.8% ± 2.1% and 99.7% ± 0.6%, respectively. The clone 5A4-based IHC test showed a sensitivity of 90.9% ± 2.6%, a specificity of 98.3% ± 1.3%, and positive and negative predictive values of 63.8% ± 4.2% and 99.7% ± 0.6%, respectively. Five cases with IHC analysis/FISH-discordant results in our series were analyzed together with those previously reported in the literature. Overall, data from 35 patients indicate a response rate to ALK inhibitors in 100% of FISH-negative/IHC analysis-positive cases (seven of seven) and 46% of FISH-positive/IHC analysis-negative cases (13 of 28), respectively. CONCLUSIONS: Our results confirm the difficulty in managing an IHC test without amplification in the absence of confirmatory FISH analysis, as well as the possibility of performing a direct diagnosis in approximately 90% of patients by the VENTANA ALK (D5F3) CDx Assay. On the basis of the recent regulatory changes, the data that have emerged from the literature, and the results of the present study, a new algorithm for ALK assessment in non-small cell lung cancer has been devised.

Morbidity, mortality and survival after 110 consecutive bilobectomies over 12 years.

OBJECTIVES: To analyse statistical aspects of mortality, morbidity and survival after bilobectomy (BT), an operation rarely studied in the literature. METHODS: One hundred and ten cases were studied, comprising 58 upper-middle bilobectomies and 52 lower-middle bilobectomies performed between 1999 and 2010. Indications were of 9 benign diseases, 12 carcinoid tumours, 5 metastases and 84 non-small cell lung cancers (2 stage 0; 34 stage I; 22 stage II; 25 stage III and 1 stage IV). RESULTS: Mortality was nil. Twenty-six percent of patients experienced significant morbidity, influenced in multivariate analysis by the presence of three or more comorbidities (P = 0.03) and by a forced expiratory volume in 1 s of <60% (P = 0.01). Lower-middle BT was associated with more postoperative complications than upper-middle BT (P = 0.012). The 5-year survival rate of patients with non-small cell lung carcinoma was 82% in stage I, 59% in stage II and 20% in stage IIIA. Survival was significantly influenced by stage (P = 0.0018) and tobacco weaning (P = 0.0012). CONCLUSIONS: BT can be achieved with low mortality, and survival results that are comparable with those unregistered after standard lobectomy. However, almost one quarter of patients experienced significant postoperative complications. Surgical techniques aiming to reduce residual pleural space should be especially considered after lower-middle BT, due to the highest morbidity being associated with this procedure.

Complex mutations & subpopulations of deletions at exon 19 of EGFR in NSCLC revealed by next generation sequencing: potential clinical implications.

Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR) gene in non-small cell lung cancer (NSCLC) and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS), including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples), were subjected to deep next generation sequencing (NGS). All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88%) cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12%) NGS resolved deletions not accurately characterized by SS. In 21 (20%) cases the NGS showed presence of complex (double/multiple) frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43%) tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1% to 17% of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.

Clinical features and outcome of patients with non-small-cell lung cancer harboring BRAF mutations.

PURPOSE: To investigate the prevalence, distribution, and prognostic role of BRAF mutations in a large cohort of white patients with non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: A retrospective series of 1,046 NSCLCs-comprising 739 adenocarcinomas (ADCs) and 307 squamous cell carcinomas (SCCs)-was investigated for BRAF mutations. High-resolution melting analysis followed by sequencing and strip hybridization assay were used. All patients were also analyzed for KRAS and EGFR mutations. RESULTS: BRAF mutations were present in 36 ADCs (4.9%) and one SCC (0.3%; P = .001). Twenty-one of the mutations (56.8%) were V600E, and 16 (43.2%) were non-V600E. V600E mutations were significantly more prevalent in females (16 of 187 patients; 8.6%) than in males (five of 552 patients; 0.9%), as indicated by multivariate logistic regression analysis (hazard ratio [HR], 11.29; P < .001). V600E-mutated tumors showed an aggressive histotype characterized by micropapillary features in 80% of patients and were significantly associated with shorter disease-free and overall survival rates on both univariate (HR, 2.67; P < .001 and HR, 2.97; P < .001, respectively) and multivariate analyses (HR, 2.19; P = .011 and HR, 2.18; P = .014, respectively). All non-V600E mutations were found in smokers (P = .015) and were associated with neither clinicopathologic parameters nor prognosis. BRAF and EGFR were concomitantly mutated in two tumors. CONCLUSION: We report for the first time to our knowledge that V600E and non-V600E BRAF mutations affect different patients with NSCLC. V600E mutations are significantly associated with female sex and represent a negative prognostic factor. In addition, we identified a number of other clinicopathologic parameters potentially useful for the selection of patients carrying BRAF mutations.

A novel clinical prognostic score incorporating the number of resected lymph-nodes to predict recurrence and survival in non-small-cell lung cancer.

BACKGROUND: The number of resected lymph-nodes (#RNs) has proven prognostic in breast and colorectal cancer. Here we evaluated its prognostic impact in a series of resected NSCLC patients. METHODS: A panel of established prognostic factors plus (1) #RNs or (2) the ratio between the number of metastatic nodes and #RNs (NR) were correlated to overall- (OS), cancer-specific- (CSS), and disease-free-survival (DFS), using the Cox-model. Risk-classes according to hazard ratios (HR) were generated. Internal and external validation was accomplished. RESULTS: A dataset of 415 resected NSCLC patients was retrieved. At multivariate analysis, #RNs and NR were independent factor for longer OS, CSS and DFS (p<0.0001). Patients with a #RNs>10 (identified optimal cut-off) had a statistically significant OS (p=0.02) and DFS (p=0.0005) benefit. In node-positive patients, a NR<9% significantly correlated with better outcome. Stratification into High-, Medium-, and Low-Risk classes, based on High- (HRFs: stage, N-status, age, #RNs) and Intermediate-Risk Factors (IRFs: sex, grading, histology), efficiently predicted outcomes (p<0.0001). The risk class model performance was externally validated in and independent dataset of 297 patients. CONCLUSIONS: These results contribute to complete the panel of prognostic factors for resected NSCLC. A prospective larger validation and comparison with molecular prognostic tools is warranted.