Molecular pathology testing for non-small cell lung cancer: an observational study of elements currently present in request forms and result reports and the opinion of different stakeholders.

BACKGROUND: For patients with non-small cell lung cancer (NSCLC), targeted therapies are becoming part of the standard treatment. It is of question which information the clinicians provide on test requests and how the laboratories adapt test conclusions to this knowledge and regulations. METHODS: This study consisted of two components; 1) checking the presence of pre-defined elements (administrative and key for therapy-choice) on completed requests and corresponding reports in Belgian laboratories, both for tissue- and liquid biopsy (LB)-testing and b) opinion analysis from Belgian pathologists/molecular biologists and clinicians during national pathology/oncology meetings. RESULTS: Data from 4 out of 6 Belgian laboratories with ISO-accreditation for LB-testing were analyzed, of which 75% were university hospitals. On the scored requests (N = 4), 12 out of 19 ISO-required elements were present for tissue and 11 for LB-testing. Especially relevant patient history, such as line of therapy (for LB), tumor histology and the reason for testing were lacking. Similarly, 11 and 9 out of 18 elements were present in the reports (N = 4) for tissue and LB, respectively. Elements that pathologists/molecular biologists (N = 18) were missing on the request were the initial activating mutation, previous therapies, a clinical question and testing-related information. For reporting, an item considered important by both groups is the clinical interpretation of the test result. In addition, clinicians (N = 28) indicated that they also wish to read the percentage of neoplastic cells. CONCLUSIONS: Communication flows between the laboratory and the clinician, together with possible pitfalls were identified. Based on the study results, templates for complete requesting and reporting were proposed.

Variation in nomenclature of somatic variants for selection of oncological therapies: Can we reach a consensus soon?

A standardized nomenclature for reporting oncology biomarker variants is key to avoid misinterpretation of results and unambiguous registration in clinical databases. External quality assessment (EQA) schemes have revealed a need for more consistent nomenclature use in clinical genetics. We evaluated the propensity of EQA for improvement of compliance with Human Genome Variation Society (HGVS) recommendations for reporting of predictive somatic variants in lung and colorectal cancer. Variant entries between 2012 and 2018 were collected from written reports and electronic results sheets. In total, 4,053 variants were assessed, of which 12.1% complied with HGVS recommendations. Compliance improved over time from 2.1% (2012) to 22.3% (2018), especially when laboratories participated in multiple EQA schemes. Compliance was better for next-generation sequencing (20.9%) compared with targeted techniques (9.8%). In the 1792 reports, HGVS recommendations for reference sequences were met for 31.9% of reports, for 36.0% of noncommercial, and 26.5% of commercial test methods. Compliance improved from 16.7% (2012) to 33.1% (2018), and after repeated EQA participation. EQA participation improves compliance with HGVS recommendations. The residual percentage of errors in the most recent schemes suggests that laboratories, companies, and EQA providers need to collaborate for additional improvement of harmonization in clinical test reporting.

Sensitive detection methods are key to identify secondary EGFR c.2369C>T p.(Thr790Met) in non-small cell lung cancer tissue samples.

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.

Neoplastic cell percentage estimation in tissue samples for molecular oncology: recommendations from a modified Delphi study.

AIMS: Results from external quality assessment revealed considerable variation in neoplastic cell percentages (NCP) estimation in samples for biomarker testing. As molecular biology tests require a minimal NCP, overestimations may lead to false negative test results. We aimed to develop recommendations to improve the NCP determination in a prototypical entity - colorectal carcinoma - that can be adapted for other cancer types. METHODS AND RESULTS: A modified Delphi study was conducted to reach consensus by 10 pathologists from 10 countries with experience in determining the NCP for colorectal adenocarcinoma. This study included two online surveys and a decision-making meeting. Consensus was defined a priori as an agreement of > 80%. All pathologists completed both surveys. Consensus was reached for 8 out of 19 and 2 out of 13 questions in the first and second surveys, respectively. Remaining issues were resolved during the meeting. Twenty-four recommendations were formulated. Major recommendations resulted as follows: only pathologists should conduct the morphological evaluation; nevertheless molecular biologists/technicians may estimate the NCP, if specific training has been performed and a pathologist is available for feedback. The estimation should be determined in the area with the highest density of viable neoplastic cells and lowest density of inflammatory cells. Other recommendations concerned: the determination protocol itself, needs for micro- and macro-dissection, reporting and interpreting, referral practices and applicability to other cancer types. CONCLUSION: We believe these recommendations may lead to more accurate NCP estimates, ensuring the correct interpretation of test results, and might help in validating digital algorithms in the future.

European follow-up of incorrect biomarker results for colorectal cancer demonstrates the importance of quality improvement projects.

Biomarker analysis for colorectal cancer has been shown to be reliable in Europe with 97% of samples tested by EQA participants to be correctly classified. This study focuses on errors during the annual EQA assessment. The aim was to explore the causes and actions related to the observed errors and to provide feedback and assess any improvement between 2016 and 2017. An electronic survey was sent to all laboratories with minimum one genotyping error or technical failure on ten tumor samples. A workshop was organized based on 2016 survey responses. Improvement of performance in 2017 was assessed for returning participants (n = 76), survey respondents (n = 13) and workshop participants (n = 4). Survey respondents and workshop participants improved in terms of (maximum) analysis score, successful participation, and genotyping errors compared to all returning participants. In 2016, mostly pre- and post-analytical errors (both 25%) were observed caused by unsuitability of the tumor tissue for molecular analysis. In 2017, most errors were due to analytical problems (50.0%) caused by methodological problems. The most common actions taken (n = 58) were protocol revisions (34.5%) and staff training (15.5%). In 24.1% of issues identified no action was performed. Corrective actions were linked to an improved performance, especially if performed by the pathologist. Although biomarker testing has improved over time, error occurrence at different phases stresses the need for quality improvement throughout the test process. Participation to quality improvement projects and a close collaboration with the pathologist can have a positive influence on performance.

External Quality Assessment Identifies Training Needs to Determine the Neoplastic Cell Content for Biomarker Testing.

Neoplastic cell content determination is crucial for biomarker testing. It is known that interobserver variation exists, but largescale data are missing about variation in tumor delineation and cell content determination. Results were obtained from the external quality assessment program for metastatic colorectal cancer from the European Society of Pathology (N = 5776 observations). The study included three parts: current practices were surveyed, neoplastic cell content estimations and delineations were retrieved from stained slides, and clinical reports were analyzed. Seventeen of 43 pathologists determined the neoplastic cell content in a tumor-rich area for DNA extraction and took immune cells (n = 37), tumor cell distribution (n = 33), desmoplastic stroma (n = 30), necrosis (n = 29), and mucus (n = 23) into account. The selected area was highly variable, and the average difference between the highest and lowest estimation ranged between 51% and 78% (2011 to 2017). The number of overestimations was alarmingly high in samples containing <30% tumor cells. Of concern is that 33 of 105 laboratories reported a wild-type result in a sample without tumor in 2017. Standardization of neoplastic cell content determination is needed for test outcome interpretation. The authors' data show variation in estimation practices, tumor delineations and estimations, and interpretation problems (n = 226 reports). Further training for selecting the most suitable block and creating clear reports is urgently needed.

The ins and outs of molecular pathology reporting.

The raid evolution in molecular pathology resulting in an increasing complexity requires careful reporting. The need for standardisation is clearer than ever. While synoptic reporting was first used for reporting hereditary genetic diseases, it is becoming more frequent in pathology, especially molecular pathology reports too. The narrative approach is no longer feasible with the growing amount of essential data present on the report, although narrative components are still necessary for interpretation in molecular pathology. On the way towards standardisation of reports, guidelines can be a helpful tool. There are several guidelines that focus on reporting in the field of hereditary diseases, but it is not always feasible to extrapolate these to the reporting of somatic variants in molecular pathology. The rise of multi-gene testing causes challenges for the laboratories. In order to provide a continuous optimisation of the laboratory testing process, including reporting, external quality assessment is essential and has already proven to improve the quality of reports. In general, a clear and concise report for molecular pathology can be created by including elements deemed important by different guidelines, adapting the report to the process flows of the laboratory and integrating the report with the laboratory information management system and the patient record.