Added Value of 50-Gene Panel Sequencing to Distinguish Multiple Primary Lung Cancers from Pulmonary Metastases: A Systematic Investigation.

Differentiation between multiple primary lung cancers and pulmonary metastases (PM) has important implications in staging, prognosis, and treatment strategies. Clinical and immunohistopathologic criteria have been standardized; however, a substantial number of cases remain difficult to classify. Using next-generation sequencing, it is now possible to improve the classification of multiple lung cancer lesions. This study systematically investigated the value of routine morphologic and IHC characteristics, p53 protein expression, TP53 mutation analysis, and 50-gene panel sequencing (GPS) in 111 lesions from 50 patients with multiple lung lesions. Based on immunohistopathologic criteria, 32 paired lesions were classified as multiple primary lung cancer (MPLC) and 21 as PM. TP53 mutation analysis indicated MPLC in 23 and PM in 6 pairs, but in the majority of cases (n = 28, 49%) no mutation was observed and no conclusion could be drawn. In contrast, only 2 pairs were not conclusive using GPS. In a significant number of matching tumor samples (n = 19, 39%), sequencing results were contradictory to the initial immunohistopathology diagnosis. No separation in overall survival for classifications based on immunohistopathology was observed, while a clear but nonsignificant trend was observed concerning survival in MPLC patients (hazard ratio = 3.98) using 50-gene GPS. In about one-third of the patients, GPS provided additional information to improve the differentiation between MPLC and PM.

Molecular clonality assessment shows high performance to predict malignant B-cell non-Hodgkin's lymphoma using cytological smears.

AIMS: Molecular PCR-based clonality analysis is helpful for identification of monoclonal B-cell or T-cell populations and to distinguish malignant lymphoma from reactive lymphoid hyperplasia. Typically, clonality assessment on fine-needle aspiration cytology (FNAC) requires freshly obtained aspirates, but the collection and processing of these samples are often challenging in daily practice. In this study, we assessed the routine diagnostic value of the EuroClonality/BIOMED-2 assay for B-cell clonality on air-dried archived Giemsa-stained smears. METHODS: This study comprised a retrospective analysis of a consecutive diagnostic cohort of 192 FNAC samples from 184 patients with at least 2-year follow-up. The results from the clonality assay were integrated with cytomorphological assessment and evaluated for their accuracy in detecting malignant disease. EuroClonality expert re-evaluation was performed for all cases with ambiguous results and for cases in which the diagnosis did not match the follow-up data. RESULTS: The clonality assay showed a high accuracy of 93% for detection of malignancy, with a sensitivity of 93% and a specificity of 92%. All 64 cases with monoclonal Ig heavy chain (IGH)/Ig kappa chain (IGK) rearrangements were confirmed malignant by histology or clinical follow-up. Expert re-evaluation changed the definite diagnosis for five cases (3%), mainly because of low signals or no proper duplicate results. We discuss and elucidate all cases for which the clonality results did not match the disease follow-up. CONCLUSIONS: This study showed that EuroClonality/BIOMED-2 assay can successfully be performed on cytological Giemsa-stained smears and inclusion in daily practice can assist in better identification of malignant lymphoma.

EGFR and KRAS quality assurance schemes in pathology: generating normative data for molecular predictive marker analysis in targeted therapy.

INTRODUCTION: The aim of this study was to compare the reproducibility of epidermal growth factor receptor (EGFR) immunohistochemistry (IHC), EGFR gene amplification analysis, and EGFR and KRAS mutation analysis among different laboratories performing routine diagnostic analyses in pathology in The Netherlands, and to generate normative data. METHODS: In 2008, IHC, in-situ hybridisation (ISH) for EGFR, and mutation analysis for EGFR and KRAS were tested. Tissue microarray sections were distributed for IHC and ISH, and tissue sections and isolated DNA with known mutations were distributed for mutation analysis. In 2009, ISH and mutation analysis were evaluated. False-negative and false-positive results were defined as different from the consensus, and sensitivity and specificity were estimated. RESULTS: In 2008, eight laboratories participated in the IHC ring study. In only 4/17 cases (23%) a consensus score of ≥75% was reached, indicating that this analysis was not sufficiently reliable to be applied in clinical practice. For EGFR ISH, and EGFR and KRAS mutation analysis, an interpretable result (success rate) was obtained in ≥97% of the cases, with mean sensitivity ≥96% and specificity ≥95%. For small sample proficiency testing, a norm was established defining outlier laboratories with unsatisfactory performance. CONCLUSIONS: The result of EGFR IHC is not a suitable criterion for reliably selecting patients for anti-EGFR treatment. In contrast, molecular diagnostic methods for EGFR and KRAS mutation detection and EGFR ISH may be reliably performed with high accuracy, allowing treatment decisions for lung cancer.