Detection of ROS1-positive non-small cell lung cancer on cytological specimens using immunocytochemistry.

BACKGROUND: Rearrangements of the ROS1 oncogene are found in 1% to 2% of non-small cell lung cancers (NSCLC) and are regarded as mutually exclusive oncogenic driver mutations. Since the approval of targeted therapy for ROS1-positive NSCLC, ROS1 testing has become a part of the diagnostic routine. Fluorescence in situ hybridization (FISH), optionally selected for by immunohistochemistry on histological material, is a common practice for the detection of ROS1 rearrangements. However, NSCLC often is diagnosed by cytology alone, requiring predictive marker testing on cytological specimens. In the current study, the authors explored the accuracy of ROS1 immunocytochemistry (ICC) on non-cell block cytological specimens for the detection of ROS1 rearrangements. METHODS: ICC using the D4D6 antibody on an automated immunostainer was performed prospectively in the routine diagnostic setting on cytological specimens from 295 patients with NSCLC, including adenocarcinoma (241 patients), NSCLC not otherwise specified (50 patients), and other malignancies (4 patients). Any immunostaining was considered positive. RESULTS: ICC was positive in all 13 ROS1-rearranged NSCLC cases confirmed by FISH (12 cases) or next-generation sequencing (1 case). Confirmation of 282 ICC-negative cases was available for 208 patients. The sensitivity, specificity, and positive and negative predictive values for ROS1 ICC compared with the final ROS1 status all were 100%. CONCLUSIONS: ROS1 ICC is an accurate method for the detection of ROS1 rearrangements in NSCLC. Given the high costs and technical challenges of FISH and the rarity of ROS1 rearrangements, ICC is rapid and therefore well suited as a screening method. Cases with equivocal or positive findings on ICC can be confirmed by FISH or molecular tests. Cancer Cytopathol 2018;126:421-9. © 2018 American Cancer Society.

Culture and Drug Profiling of Patient Derived Malignant Pleural Effusions for Personalized Cancer Medicine.

INTRODUCTION: The use of patients' own cancer cells for in vitro selection of the most promising treatment is an attractive concept in personalized medicine. Human carcinoma cells from malignant pleural effusions (MPEs) are suited for this purpose since they have already adapted to the liquid environment in the patient and do not depend on a stromal cell compartment. Aim of this study was to develop a systematic approach for the in-vitro culture of MPEs to analyze the effect of chemotherapeutic as well as targeted drugs. METHODS: MPEs from patients with solid tumors were selected for this study. After morphological and molecular characterization, they were cultured in medium supplemented with patient-derived sterile-filtered effusion supernatant. Growth characteristics were monitored in real-time using the xCELLigence system. MPEs were treated with a targeted therapeutic (erlotinib) according to the mutational status or chemotherapeutics based on the recommendation of the oncologists. RESULTS: We have established a robust system for the ex-vivo culture of MPEs and the application of drug tests in-vitro. The use of an antibody based magnetic cell separation system for epithelial cells before culture allowed treatment of effusions with only moderate tumor cell proportion. Experiments using drugs and drug-combinations revealed dose-dependent and specific growth inhibitory effects of targeted drugs. CONCLUSIONS: We developed a new approach for the ex-vivo culture of MPEs and the application of drug tests in-vitro using real-time measuring of cell growth, which precisely reproduced the effect of clinically established treatments by standard chemotherapy and targeted drugs. This sets the stage for future studies testing agents against specific targets from genomic profiling of metastatic tumor cells and multiple drug-combinations in a personalized manner.

Screening for ALK in non-small cell lung carcinomas: 5A4 and D5F3 antibodies perform equally well, but combined use with FISH is recommended.

OBJECTIVES: Immunohistochemistry (IHC) has become a promising method for pre-screening ALK-rearrangements in non-small cell lung carcinomas (NSCLC). Various ALK antibodies, detection systems and automated immunostainers are available. We therefore aimed to compare the performance of the monoclonal 5A4 (Novocastra, Leica) and D5F3 (Cell Signaling, Ventana) antibodies using two different immunostainers. Additionally we analyzed the accuracy of prospective ALK IHC-testing in routine diagnostics. MATERIALS AND METHODS: Seventy-two NSCLC with available ALK FISH results and enriched for FISH-positive carcinomas were retrospectively analyzed. IHC was performed on BenchMarkXT (Ventana) using 5A4 and D5F3, respectively, and additionally with 5A4 on Bond-MAX (Leica). Data from our routine diagnostics on prospective ALK-testing with parallel IHC, using 5A4, and FISH were available from 303 NSCLC. RESULTS: All three IHC protocols showed congruent results. Only 1/25 FISH-positive NSCLC (4%) was false negative by IHC. For all three IHC protocols the sensitivity, specificity, positive (PPV) and negative predictive values (NPV) compared to FISH were 96%, 100%, 100% and 97.8%, respectively. In the prospective cohort 3/32 FISH-positive (9.4%) and 2/271 FISH-negative (0.7%) NSCLC were false negative and false positive by IHC, respectively. In routine diagnostics the sensitivity, specificity, PPV and NPV of IHC compared to FISH were 90.6%, 99.3%, 93.5% and 98.9%, respectively. CONCLUSIONS: 5A4 and D5F3 are equally well suited for detecting ALK-rearranged NSCLC. BenchMark and BOND-MAX immunostainers can be used for IHC with 5A4. True discrepancies between IHC and FISH results do exist and need to be addressed when implementing IHC in an ALK-testing algorithm.

Detection of ALK-positive non-small-cell lung cancers on cytological specimens: high accuracy of immunocytochemistry with the 5A4 clone.

INTRODUCTION: Lung cancer is often diagnosed by cytology, necessitating predictive molecular marker analyses on cytological specimens. The gold standard for detection of predictive anaplastic lymphoma kinase (ALK)-rearrangements is fluorescence in situ hybridization (FISH), but FISH is both expensive and often challenging to interpret. The aim of our study was to investigate the accuracy of ALK immunocytochemistry (ICC) on cytological specimens of non-small-cell lung cancers (NSCLCs). METHODS: Forty-one cytological specimens with available ALK FISH results were retrospectively analyzed with the 5A4 monoclonal antibody (Novocastra; Leica Biosystems) on a fully automated slide stainer. The specimens were enriched for ALK FISH-positive NSCLCs (14 of 41; 34.1%). Evaluation of the ICC staining was performed blinded to the FISH results. The staining intensity and the percentage of stained cancer cells were recorded. Any ICC staining was regarded as a positive result. The ALK ICC results were compared with the FISH results. In case of a discrepancy the ICC-stained slide and the FISH signals were reviewed. RESULTS: ICC was evaluable on 40 of 41 specimens. Fifteen of 40 NSCLCs (37.5%) were ALK ICC-positive, with staining of the majority of cancer cells (median 100%; mean 82.3%). Twelve of the ICC-positive NSCLCs (80.0%) showed an intense staining (3+). Compared with the ALK FISH results, only one NSCLC was false-negative, and one false-positive by ICC, respectively. The sensitivity, specificity, and positive and negative predictive values for ALK ICC compared with ALK FISH were 93.3%, 96.0%, 93.3%, and 96%, respectively. CONCLUSION: ALK ICC is highly accurate for detecting ALK-rearranged NSCLCs.

Bronchoalveolar lavage quality influences the T4/T8 ratio in sarcoidosis.

BACKGROUND: Sarcoidosis is characterised by a T-lymphocytic alveolitis with a typically increased T4/T8 ratio. The diagnostic value of this ratio is under debate. AIM OF THE WORK: We prospectively evaluated the influence of BAL pre-lavage and the impact of bronchial contamination on BAL differential cell count in 108 BAL specimens obtained from patients with histologically confirmed sarcoidosis. METHODS: BAL was performed by instilling 150-300 ml normal saline either in the middle lobe or the lingula. Fifty-one patients (47%) underwent additional pre-lavage with 50 ml normal saline. Bronchial contamination was assessed by semi-quantitative analysis of mucus, ciliated and squamous cells in the untreated BAL recovery. RESULTS: Pre-lavage did neither influence the lavage cellularity nor extend of contamination of the BAL. Content of mucus and ciliated cells, indicating bronchial contamination, showed a high correlation (Kendal's tau=0.61). Presence of either mucus or ciliated cells in the BAL recovery was associated with a significant lower T4/T8 ratio (mucus: 4.9 vs. 8.0, p=0.009; ciliated cells: 4.1 vs. 7.4, p=0.001). Squamous cells in the BAL recovery representing oropharyngeal contamination did not significantly influence the T4/T8 ratio (7.7 vs. 5.6, p=0.10). CONCLUSION: Bronchial contamination of BAL as determined by the presence of mucus and ciliated cells in the recovery decreases the T4/T8 ratio of BAL in sarcoidosis.