The diagnostic work-up for non-small cell lung cancer (NSCLC) requires biomarker testing to guide therapy choices. This article is the second of a two-part series. In Part 1, we summarised evidence-based recommendations for obtaining and processing small specimen samples (i.e. pre-analytical steps) from patients with advanced NSCLC. Here, in Part 2, we summarise evidence-based recommendations relating to analytical steps of biomarker testing (and associated reporting and quality assessment) of small specimen samples in NSCLC. As the number of biomarkers for actionable (genetic) targets and approved targeted therapies continues to increase, simultaneous testing of multiple actionable oncogenic drivers using next-generation sequencing (NGS) becomes imperative, as set forth in European Society for Medical Oncology guidelines. This is particularly relevant in advanced NSCLC, where tissue specimens are typically limited and NGS may help avoid tissue exhaustion compared with sequential biomarker testing. Despite guideline recommendations, significant discrepancies in access to NGS persist across Europe, primarily due to reimbursement constraints. The use of increasingly complex testing methods also has implications for the reporting of results. Molecular testing reports should include clinical interpretation with additional commentary on sample adequacy as appropriate. Molecular tumour boards are recommended to facilitate the interpretation of complex genetic information arising from NGS, and to collaboratively determine the optimal treatment for patients with NSCLC. Finally, whichever testing modality is employed, it is essential that adequate internal and external validation and quality control measures are implemented.

• Keywords
• Best practice, External quality assessment, Liquid biopsy, Molecular diagnostics, Next-generation sequencing, Non-small cell lung carcinoma,
• MeSH
• Biomarkers MeSH
• Humans MeSH
• Mutation MeSH
• Lung Neoplasms * diagnosis   drug therapy   genetics   MeSH
• Carcinoma, Non-Small-Cell Lung * diagnosis   genetics   pathology   MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Biomarkers MeSH

Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) is accepted as a predictive biomarker for the selection of immune checkpoint inhibitors. We evaluated the staining quality and estimation of the tumor proportion score (TPS) in non-small-cell lung cancer during two external quality assessment (EQA) schemes by the European Society of Pathology. Participants received two tissue micro-arrays with three (2017) and four (2018) cases for PD-L1 IHC and a positive tonsil control, for staining by their routine protocol. After the participants returned stained slides to the EQA coordination center, three pathologists assessed each slide and awarded an expert staining score from 1 to 5 points based on the staining concordance. Expert scores significantly (p < 0.01) improved between EQA schemes from 3.8 (n = 67) to 4.3 (n = 74) on 5 points. Participants used 32 different protocols: the majority applied the 22C3 (56.7%) (Dako), SP263 (19.1%) (Ventana), and E1L3N (Cell Signaling) (7.1%) clones. Staining artifacts consisted mainly of very weak or weak antigen demonstration (63.0%) or excessive background staining (19.8%). Participants using CE-IVD kits reached a higher score compared with those using laboratory-developed tests (LDTs) (p < 0.05), mainly attributed to a better concordance of SP263. The TPS was under- and over-estimated in 20/423 (4.7%) and 24/423 (5.7%) cases, respectively, correlating to a lower expert score. Additional research is needed on the concordance of less common protocols, and on reasons for lower LDT concordance. Laboratories should carefully validate all test methods and regularly verify their performance. EQA participation should focus on both staining concordance and interpretation of PD-L1 IHC.

• Keywords
• External quality assessment, Immunohistochemistry, PD-L1, Tumor proportion score,
• MeSH
• B7-H1 Antigen analysis   antagonists & inhibitors   MeSH
• Artifacts MeSH
• Tissue Array Analysis MeSH
• Immunohistochemistry * MeSH
• Immune Checkpoint Inhibitors therapeutic use   MeSH
• Clinical Decision-Making MeSH
• Humans MeSH
• Biomarkers, Tumor analysis   antagonists & inhibitors   MeSH
• Lung Neoplasms drug therapy   immunology   pathology   MeSH
• Carcinoma, Non-Small-Cell Lung drug therapy   immunology   pathology   MeSH
• Observer Variation MeSH
• Predictive Value of Tests MeSH
• Reproducibility of Results MeSH
• Laboratory Proficiency Testing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Geographicals
• Europe MeSH
• Names of Substances
• B7-H1 Antigen MeSH
• CD274 protein, human MeSH Browser
• Immune Checkpoint Inhibitors MeSH
• Biomarkers, Tumor MeSH

A questionnaire on biomarker testing previously used in central European countries was extended and distributed in Western and Central European countries to the pathologists participating at the Pulmonary Pathology Society meeting 26-28 June 2019 in Dubrovnik, Croatia. Each country was represented by one responder. For recent biomarkers the availability and reimbursement of diagnoses of molecular alterations in non-small cell lung carcinoma varies widely between different, also western European, countries. Reimbursement of such assessments varies widely between unavailability and payments by the health care system or even pharmaceutical companies. The support for testing from alternative sources, such as the pharmaceutical industry, is no doubt partly compensating for the lack of public health system support, but it is not a viable or long-term solution. Ideally, a structured access to testing and reimbursement should be the aim in order to provide patients with appropriate therapeutic options. As biomarker enabled therapies deliver a 50% better probability of outcome success, improved and unbiased reimbursement remains a major challenge for the future.

• Keywords
• Europe, Lung cancer, health care, predictive testing, therapy,
• Publication type
• Journal Article MeSH
• Review MeSH

BACKGROUND: Correct identification of the EGFR c.2369C>T p.(Thr790Met) variant is key to decide on a targeted therapeutic strategy for patients with acquired EGFR TKI resistance in non-small cell lung cancer. The aim of this study was to evaluate the correct detection of this variant in 12 tumor tissue specimens tested by 324 laboratories participating in External Quality Assessment (EQA) schemes. METHODS: Data from EQA schemes were evaluated between 2013 and 2018 from cell lines (6) and resections (6) containing the EGFR c.2369C>T p.(Thr790Met) mutation. Adequate performance was defined as the percentage of tests for which an outcome was available and correct. Additional data on the used test method were collected from the participants. Chi-squared tests on contingency tables and a biserial rank correlation were applied by IBM SPSS Statistics version 25 (IBM, Armonk, NY, USA). RESULTS: In 26 of the 1190 tests (2.2%) a technical failure occurred. For the remaining 1164 results, 1008 (86.6%) were correct, 151 (12.9%) were false-negative and 5 (0.4%) included incorrect mutations. Correct p.(Thr790Met) detection improved over time and for repeated scheme participations. In-house non-next-generation sequencing (NGS) techniques performed worse (81.1%, n = 293) compared to non-NGS commercial kits (85.2%, n = 656) and NGS (97.0%, n = 239). Over time there was an increase in the users of NGS. Resection specimens performed worse (82.6%, n = 610 tests) compared to cell line material (90.9%, n = 578 tests), except for NGS (96.3%, n = 344 for resections and 98.6%, n = 312 for cell lines). Samples with multiple mutations were more difficult compared to samples with the single p.(Thr790Met) variant. A change of the test method was shown beneficial to reduce errors but introduced additional analysis failures. CONCLUSIONS: A significant number of laboratories that offer p.(Thr790Met) testing did not detect this relevant mutation compared to the other EQA participants. However, correct identification of this variant is improving over time and was higher for NGS users. Revising the methodology might be useful to resolve errors, especially for resection specimens with low frequency or multiple variants. EQA providers should include challenging resections in the scheme.

• Keywords
• EGFR, External quality assessment, Non-small cell lung cancer, Osimertinib, Predictive biomarker, Resistance, c.2369C>T p.(Thr790Met),
• MeSH
• ErbB Receptors genetics   MeSH
• Genetic Testing methods   standards   MeSH
• Polymorphism, Single Nucleotide MeSH
• Humans MeSH
• Longitudinal Studies MeSH
• Mutation * MeSH
• Tumor Cells, Cultured MeSH
• Lung Neoplasms diagnosis   enzymology   genetics   MeSH
• Follow-Up Studies MeSH
• Carcinoma, Non-Small-Cell Lung diagnosis   enzymology   genetics   MeSH
• Quality Control MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• EGFR protein, human MeSH Browser
• ErbB Receptors MeSH