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Objectives: Targeted RNA-based Next-Generation Sequencing (tRNA-seq) is increasingly being used in molecular diagnostics for gene fusion detection in non-small cell lung cancer (NSCLC). However, few data support its clinical application for the detection of single nucleotide variants (SNVs) and small insertions/deletions. In this study, we evaluated the performance of tRNA-seq using Archer FusionPlex for simultaneous detection of actionable gene fusions, splice variants, SNVs and indels in formalin-fixed, paraffin-embedded NSCLC tissue., Materials and Methods: A total of 126 NSCLC samples, including 20 validation samples and 106 diagnostic cases, were analyzed by targeted DNA-based Next-Generation Sequencing (tDNA-seq) followed by tRNA-seq., Results: All 28 SNVs and indels in the validation set, and 34 out of 35 mutations in the diagnostic set were identified by tRNA-seq. The only mutation undetected by tRNA-seq, ERBB2 p.(Ser310Tyr), was not included in the current Archer panel design. tRNA-seq revealed one additional BRAF p.(Val600Glu) mutation not found by tDNA-seq. SNVs and indels were correctly called by the vendor supplied software, except for ERBB2 duplication p.(Tyr772_A775dup) which was only detected by an additional in-house developed bio-informatics pipeline. Variant allelic frequency (VAF) values were generally higher at the expression level compared to the genomic level (range 6-96% for tRNA-seq versus 6-61% for tDNA-seq) and low VAF mutations in DNA (6-8% VAF) were all confirmed by tRNA-seq. Finally, tRNA-seq additionally identified a driver fusion or splice variant in 10 diagnostic NSCLC samples including one MET exon 14 skipping variant not detected by tDNA-seq., Conclusion: Our results demonstrate that tRNA-seq can be implemented in a diagnostic setting as an efficient strategy for simultaneous detection of actionable gene fusions, splice variants, SNVs and indels in NSCLC provided that adequate RNA-seq analysis tools are available, especially for the detection of indels. This approach allows upfront identification of currently recommended targetable molecular alterations in NSCLC samples., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Detection of epidermal growth factor receptor (EGFR) mutations in exons 18-21 is recommended in all patients with advanced Non-small-cell lung carcinoma due to the demonstrated efficiency of the standard therapy with tyrosine kinase inhibitors in EGFR-mutated patients. Therefore, choosing a suitable technique to test EGFR mutational status is crucial to warrant a valid result in a short turnaround time using the lowest possible amount of tissue material. The Idylla™ EGFR Mutation Test is a simple, fast and reliable method designed for the detection of EGFR mutations from formalin-fixed paraffin-embedded samples. The aim of this study was the Clinical Performace Evaluation of the Idylla™ EGFR Mutation Test on the Idylla™ System., Methods: EGFR mutational status was determined on 132 archived formalin-fixed paraffin-embedded tissue sections with Idylla™ technology. Results were compared with the results previously obtained by routine method in the reference lab (Therascreen® EGFR RGQ PCR v2, Qiagen in Molecular Pathology lab, Hospital Universitario Virgen del Rocío de Sevilla)., Results: The overall agreement between results obtained with the Idylla™ EGFR Mutation Test and the Comparator test method was 95.38% (with 1-sided 95% lower limit of 91.7%) showing Positive Diagnostic Agreement of 93.22% and Negative Diagnostic Agreement of 97.18%, with a Limit Of Detection ≤5%., Conclusions: The Idylla™ EGFR Mutation Test passed its clinical validity performance characteristics for accuracy.

Objective: Metastasized non-small cell lung cancer (NSCLC) with an anaplastic lymphoma kinase (ALK) rearrangement is usually sensitive to a range of ALK-tyrosine kinase inhibitors. ALK-positive NSCLC have been identified in pivotal phase III trials with fluorescence in situ hybridization (ALK FISH+). These tumors are also expressing the fusion product (ALK immunohistochemistry (IHC)+). However, discrepant cases occur, including ALK IHC + FISH-. The aim of this study was to collect ALK IHC + cases and compare within this group response to crizotinib treatment of ALK FISH + cases with ALK FISH- cases., Materials and Methods: In this European prospective multicenter research study patients with Stage IV ALK IHC + NSCLC treated with crizotinib were enrolled. Tumor slides were validated centrally for ALK IHC and ALK FISH., Results: Registration of 3523 ALK IHC tests revealed a prevalence of 2.7% (n = 94) ALK IHC + cases. Local ALK FISH analysis resulted in 48 concordant (ALK IHC+/FISH+) and 16 discordant (ALK IHC+/FISH-) cases. Central validation revealed 37 concordant and 7 discordant cases, 5 of which had follow-up. Validation was hampered by limited amount of tissue in biopsy samples. The PFS at 1 year for ALK concordant and discordant was 58% and 20%, respectively (HR = 2.4; 95% CI: 0.78-7.3; p = 0.11). Overall survival was significantly better for concordant cases than discordant cases after central validation (HR=4.5; 95% CI= 1.2-15.9; p=0.010., Conclusion: ALK IHC + FISH- NSCLC is infrequent and associated with a worse outcome on personalized treatment. A suitable predictive testing strategy may be to screen first with IHC and then confirm with FISH instead of considering ALK IHC equivalent to ALK FISH according to the current guidelines., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: Molecular testing has become important in the biomarker program of clinical trials for advanced non-small lung cancer (NSCLC). These tissue samples often have to be analyzed in a central laboratory. We evaluated the turnaround time and possible delay in start of therapy in this process and how often testing resulted in inclusion in a clinical trial., Methods: We reviewed our prospective database on all molecular testing cases for clinical trial suitability in patients with advanced NSCLC between March 1, 2011 and October 31, 2014., Results: 250 patients were considered for biomarker-driven trials. Twenty-three cases did not have further analysis and 20 patients had failure of central biomarker analysis. Results were obtained for 207 (83%) patients. In 91 of 227 (40%) samples sent, a biomarker of interest was documented. This led to 34 (15%) clinical trial inclusions. The mean waiting time between informed consent and request for tissue sections from the pathology lab and receipt of biomarker result from central lab was 24.4 (SD 13.7) calendar days., Conclusion: While molecular biomarker testing is crucial in many NSCLC trials, our results show that waiting times for central laboratory analysis can cause an important delay in treatment initiation, and even ineligibility for the trial(s) under consideration. Start of therapy based on properly validated local testing, with a posteriori central biomarker testing to guarantee the integrity of the trial, would be more rewarding for quite some patients., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Introduction: Small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with no early detection strategy or targeted therapy currently available. We hypothesized that difference gel electrophoresis (DIGE) may identify membrane-associated proteins (MAPs) specific to SCLC, advance our understanding of SCLC biology, and discover new biomarkers of SCLC., Methods: MAP lysates were prepared from three SCLCs, three non-small-cell lung cancers, and three immortalized normal bronchial epithelial cell lines and coanalyzed by DIGE. Subsequent protein identification was performed by mass spectrometry. Proteins were submitted to Ingenuity Pathway Analysis. Candidate biomarkers were validated by Western blotting (WB) and immunohistochemistry (IHC)., Results: Principal component analysis on the global DIGE data set demonstrated that the four replicates derived from each of the nine cell lines clustered closely, as did samples within the same histological group. One hundred thirty-seven proteins were differentially expressed in SCLC compared with non-small-cell lung cancer and immortalized normal bronchial epithelial cells. These proteins were overrepresented in cellular/tissue morphology networks. Dihydropyrimidinase-related protein 2, guanine nucleotide-binding protein alpha-q, laminin receptor 1, pontin, and stathmin 1 were selected as candidate biomarkers among MAPs overexpressed in SCLC. Overexpression of all candidates but RSSA in SCLC was verified by WB and/or IHC on tissue microarrays. These proteins were significantly associated with SCLC histology and survival in univariables analyses., Conclusion: DIGE analysis of a membrane-associated subproteome discovered overexpression of dihydropyrimidinase-related protein 2, guanine nucleotide-binding protein alpha-q, RUVB1, and stathmin 1 in SCLC. Results were verified by WB and/or IHC in primary tumors, suggesting that investigating their functional relevance in SCLC progression is warranted. Association with survival requires further validation in larger clinical data sets.

Until recently, the division of pulmonary carcinomas into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) was adequate for therapy selection. Due to the emergence of new treatment options subtyping of NSCLC and predictive testing have become mandatory. A practical approach to the new requirements involving interaction between pulmonologist, oncologist and molecular pathology to optimize patient care is described. The diagnosis of lung cancer involves (i) the identification and complete classification of malignancy, (ii) immunohistochemistry is used to predict the likely NSCLC subtype (squamous cell vs. adenocarcinoma), as in small diagnostic samples specific subtyping is frequently on morphological grounds alone not feasible (NSCLC-NOS), (iii) molecular testing. To allow the extended diagnostic and predictive examination (i) tissue sampling should be maximized whenever feasible and deemed clinically safe, reducing the need for re-biopsy for additional studies and (ii) tissue handling, processing and sectioning should be optimized. Complex diagnostic algorithms are emerging, which will require close dialogue and understanding between pulmonologists and others who are closely involved in tissue acquisition, pathologists and oncologists who will ultimately, with the patient, make treatment decisions. Personalized medicine not only means the choice of treatment tailored to the individual patient, but also reflects the need to consider how investigative and diagnostic strategies must also be planned according to individual tumour characteristics., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Purpose: The aim of this study was to validate a gradient-based segmentation method for GTV delineation on FDG-PET in NSCLC through surgical specimen, in comparison with threshold-based approaches and CT., Materials and Methods: Ten patients with stage I-II NSCLC were prospectively enrolled. Before lobectomy, all patients underwent contrast enhanced CT and gated FDG-PET. Next, the surgical specimen was removed, inflated with gelatin, frozen and sliced. The digitized slices were used to reconstruct the 3D macroscopic specimen. GTVs were manually delineated on the macroscopic specimen and on CT images. GTVs were automatically segmented on PET images using a gradient-based method, a source to background ratio method and fixed threshold values at 40% and 50% of SUV(max). All images were finally registered. Analyses of raw volumes and logarithmic differences between GTVs and GTV(macro) were performed on all patients and on a subgroup excluding the poorly defined tumors. A matching analysis between the different GTVs was also conducted using Dice's similarity index., Results: Considering all patients, both lung and mediastinal windowed CT overestimated the macroscopy, while FDG-PET provided closer values. Among various PET segmentation methods, the gradient-based technique best estimated the true tumor volume. When analysis was restricted to well defined tumors without lung fibrosis or atelectasis, the mediastinal windowed CT accurately assessed the macroscopic specimen. Finally, the matching analysis did not reveal significant difference between the different imaging modalities., Conclusions: FDG-PET improved the GTV definition in NSCLC including when the primary tumor was surrounded by modifications of the lung parenchyma. In this context, the gradient-based method outperformed the threshold-based ones in terms of accuracy and robustness. In other cases, the conventional mediastinal windowed CT remained appropriate., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Agents acting on signalling molecules of growth pathways have emerged as therapeutics for non-small cell lung cancer (NSCLC). Among them are inhibitors of the epidermal growth factor receptor (EGFR). To meet Belgian reimbursement criteria for erlotinib, patients must have an EGFR-positive tumour, defined as at least 10% of cells showing membranous staining on immunohistochemistry. This study compares results obtained with the Dako pharmDx kit versus other anti-EGFR antibodies in 634 NSCLC. More than 80% of patients qualify for erlotinib therapy using the Dako pharmDx kit or antibodies from Zymed or Novocastra; when NeoMarkers antibodies are used, less than 70% will do. Although immunohistochemical stainings for EGFR can be performed on biopsies, surgical and cytological samples from primary and metastatic NSCLC, highest positivity rates were obtained in biopsies from primary tumours. In conclusion, immunohistochemistry for patient selection for erlotinib therapy needs standardization in order to avoid results influenced by technical issues., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Objective: In recent studies focusing on the prognostic significance of histologic features of NSCLC tumors, vessel invasion was correlated to survival across all surgical stages. We similarly analyzed whether intra-tumoral permeation could affect survival in subgroups of stage I and II NSCLC., Methods: A retrospective single institution analysis of a prospectively computed database. Specimens were analyzed for intra-tumoral vascular, lymphatic and nervous permeation. Overall mortality was determined and for each stage, a Cox regression analysis of selected variables was performed. Detailed histologic information was available in all patients. Follow-up was 100% complete (median=69 months)., Results: From 1989 to 2004, out of 346 patients with stage I and II NSCLC, 253 patients with p stage I (75.7%) and 81 patients with p stage II (24.3%) underwent surgery with complete resection, for a completeness resection rate of 97% (334/346). We performed 70 pneumonectomies, 255 lobectomies and 9 lesser resections (respectively, 21%, 76.3% and 2.7%). In-hospital mortality was 2.1%. The incidence of intra-tumoral permeation was 14.4% (48/334). Permeation correlated both with T status (p=0.04), grade of differentiation (p=0.03) and stage (p=0.02). Median survival and overall 5-year survival for patients with and without permeation were 42.3 months (95% CI [20-64.6]) and 72.1 months (95% CI [56.9-87.2]), respectively; and 44% and 54%, respectively (p=NS). However, intra-tumoral permeation was not a significant predictor for overall death (HR=1.1 [95% CI=0.74-1.66)., Conclusion: In this large institutional study of early stage NSCLC, the presence of intra-tumoral permeation was correlated both to T, grade of differentiation, as well as to stage. However, in contrast to recent reports, we did not find that intra-tumoral permeation adversely affects long-term survival.

Objective: To assess the effectiveness of positron emission tomography with radiolabeled [18F]-2-fluoro-deoxy-D-glucose (PET-FDG) imaging in mediastinal lymph node (LN) staging for non-small cell lung carcinoma (NSCLC) and to compare it to conventional clinical and surgical staging., Methods: From June 1998 to February 2000, we enrolled 64 potentially resectable NSCLC patients in a prospective study of PET-FDG imaging of the mediastinum to assess LN involvement. Results of this technique were compared to conventional clinical and surgical staging. Diagnostic efficacy was determined by calculating sensitivity, specificity, overall accuracy, and positive and negative predictive values for each method., Results: PET-FDG imaging correctly identified nodal stage (N0-N1 vs. N2) in 50 out of 61 patients (82%), overstaging occurred in eight patients (13%), and understaging in three patients (4.9%). The sensitivity, specificity, accuracy, and positive and negative predictive values for PET-FDG scan imaging were 67, 85, 82, 43, and 93.6%, respectively. Conventional staging correctly identified nodal stage (N0-N1 vs. N2) in 51 out of 62 patients (82%), overstaging occurred in five patients (8.1%), and understaging in six patients (9.7%). The sensitivity, specificity, accuracy, and positive and negative predictive values for conventional staging were 33, 90.6, 82, 37, and 89%, respectively. With regard to N2 disease, conventional staging showed a poor sensitivity (33%). Indeed, six out of 64 patients were understaged for mediastinal LN involvement. Even though the improvement was not statistically significant (McNemar P=0.08), the combined use of PET-FDG scan and computerized tomography (CT) scan allowed a two-fold increase in the sensitivity of our clinical preoperative staging. Moreover, relying on the PET-scan high negative predictive value might have contributed to a three-fold decrease in the number of required surgical staging procedures., Conclusions: Our study shows that the PET-FDG imaging strength lies in its very high negative predictive value and increased sensitivity. In this study, the overall accuracy of PET-FDG scan (82%) was lower than previously reported. Combined with chest CT-scan preoperatively, it may alleviate the need for surgical staging when PET-FDG studies of the mediastinum are negative. However, with a positive PET-FDG scan result, further diagnostic procedures should be pursued in order to avoid overstaging and allow better surgical patient selection.

Objective: To determine the presence of occult micrometastasis (OM) in a selected population of surgically resectable patients presenting with non-small cell lung carcinoma (NSCLC) and to evaluate its prognostic value on relapses and survival., Methods: From February 1996 to December 1999, 99 patients undergoing surgical treatment for NSCLC were prospectively investigated for the presence of occult bone marrow micrometastasis. Tumor cells were detected with monoclonal primary antibodies directed against low molecular weight cytokeratins., Results: Median follow-up time was 14.3 months (range 0.2-45.6 months). Overall prevalence of OM was 22.2% (22 out of 99). The presence of OM was not correlated to pathology, T status, or N status. In survival analysis, the only independent predictors of overall survival were N0 status and Stage I (P=0.016 and 0.004, respectively), while T1 was a predictor of disease-free survival (P=0.044). Metastasis and loco-regional recurrence were observed at follow-up in 18.2 (four out of 22) and 9% (two out of 22) of patients OM(+) and in 14.3 (11 out of 77) and 7.8% (six out of 77) of patients OM(-), respectively (P=not significant). OM was a predictor neither of overall survival nor of disease-free survival (P=0.52 and 0.97, respectively). In Stage I patients, 1-year overall survival and 1-year disease-free survival were 89 and 98% for OM(-) patients and 88 and 90% for OM(+) patients, respectively (P=0.57 and P=0.75)., Conclusions: OM was present in >20% of surgically treated NSCLC patients and did not correlate to pathological variables. In contrast to previous published data, in this study the presence of OM had no influence on overall or disease-free survival.