Real-world EGFR testing practices for non-small-cell lung cancer by thoracic pathology laboratories across Europe.

BACKGROUND: Testing for epidermal growth factor receptor (EGFR) mutations is an essential recommendation in guidelines for metastatic non-squamous non-small-cell lung cancer, and is considered mandatory in European countries. However, in practice, challenges are often faced when carrying out routine biomarker testing, including access to testing, inadequate tissue samples and long turnaround times (TATs). MATERIALS AND METHODS: To evaluate the real-world EGFR testing practices of European pathology laboratories, an online survey was set up and validated by the Pulmonary Pathology Working Group of the European Society of Pathology and distributed to 64 expert testing laboratories. The retrospective survey focussed on laboratory organisation and daily EGFR testing practice of pathologists and molecular biologists between 2018 and 2021. RESULTS: TATs varied greatly both between and within countries. These discrepancies may be partly due to reflex testing practices, as 20.8% of laboratories carried out EGFR testing only at the request of the clinician. Many laboratories across Europe still favour single-test sequencing as a primary method of EGFR mutation identification; 32.7% indicated that they only used targeted techniques and 45.1% used single-gene testing followed by next-generation sequencing (NGS), depending on the case. Reported testing rates were consistent over time with no significant decrease in the number of EGFR tests carried out in 2020, despite the increased pressure faced by testing facilities during the COVID-19 pandemic. ISO 15189 accreditation was reported by 42.0% of molecular biology laboratories for single-test sequencing, and by 42.3% for NGS. 92.5% of laboratories indicated they regularly participate in an external quality assessment scheme. CONCLUSIONS: These results highlight the strong heterogeneity of EGFR testing that still occurs within thoracic pathology and molecular biology laboratories across Europe. Even among expert testing facilities there is variability in testing capabilities, TAT, reflex testing practice and laboratory accreditation, stressing the need to harmonise reimbursement technologies and decision-making algorithms in Europe.

Pan-cancer analysis of genomic scar patterns caused by homologous repair deficiency (HRD).

Homologous repair deficiency (HRD) is present in many cancer types at variable prevalence and can indicate response to platinum-based chemotherapy and PARP inhibition. We developed a tumor classification system based on the loss of function of genes in the homologous recombination repair (HRR) pathway. To this end, somatic and germline alterations in BRCA1/2 and 140 other HRR genes were included and assessed for the impact on gene function. Additionally, information on the allelic hit type and on BRCA1 promoter hypermethylation was included. The HRDsum score including LOH, LST, and TAI was calculated for 8847 tumors of the TCGA cohort starting from genotyping data and for the subcohort of ovarian cancer also starting from WES data. Pan-cancer, deleterious BRCA1/2 alterations were detected in 4% of the tumors, while 18% of the tumors were HRD-positive (HRDsum ≥ 42). Across 33 cancer types, both BRCA1/2 alterations and HRD-positivity were most prevalent in ovarian cancer (20% and 69%). Pan-cancer, tumors with biallelic deleterious alterations in BRCA1/2 were separated strongly from tumors without relevant alterations (AUC = 0.89), while separation for tumors with monoallelic deleterious BRCA1/2 alterations was weak (AUC = 0.53). Tumors with biallelic deleterious alterations in other HHR genes were separated moderately from tumors without relevant alterations (AUC = 0.63), while separation for tumors with such monoallelic alterations was weaker (AUC = 0.57). In ovarian cancer, HRDsum scores calculated from WES data correlated strongly with HRDsum scores calculated from genotyping data (R = 0.87) and were slightly (4%) higher. We comprehensively analyzed HRD scores and their association with mutations in HRR genes in common cancer types. Our study identifies important parameters influencing HRD measurement and argues for an integration of HRDsum score with specific mutational profiles.

The immune microenvironment in EGFR- and ERBB2-mutated lung adenocarcinoma.

BACKGROUND: Targeted therapies have improved survival and quality of life for patients with non-small-cell lung cancer with actionable driver mutations. However, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 gene (HER2, also known as ERBB2) exon 20 insertions (Ex20mut) are characterized by a poor response to currently approved tyrosine kinase inhibitors and immunotherapies. The underlying immune biology is not well understood. MATERIALS AND METHODS: We carried out messenger RNA expression profiling of lung adenocarcinomas (ADCs) with ERBB2 (n = 19) and EGFR exon 20-insertion mutations (n = 13) and compared these to tumors with classical EGFR mutations (n = 40, affecting EGFR exons 18, 19 or 21) and EGFR/ERBB2 mutation-negative lung ADC (EGFR/ERBB2wt, n = 26) focusing on immunologically relevant transcripts. Tumor-infiltrating immune cells were estimated from gene expression profiles. RESULTS: Cytotoxic cells were significantly lower in EGFR-mutated tumors regardless of the affected exon, while Th1 cells were significantly lower in EGFR-Ex20mut compared to EGFR/ERBB2wt tumors. We assessed the differentially expressed genes of ERBB2-Ex20mut and EGFR-Ex20mut tumors compared to EGFR-Ex18/19/21mut and EGFR/ERBB2wt tumors. Of these, the genes GUSB, HDAC11, IFNGR2, PUM1, RASGRF1 and RBL2 were up-regulated, while a lower expression of CBLC, GBP1, GBP2, GBP4 and MYC was observed in all three comparison groups. The omnibus test revealed 185 significantly (FDR = 5%) differentially expressed genes and we found these four most significant gene expression changes in the study cohort: VHL and JAK1 were overexpressed in ERBB2-Ex20mut and EGFR-Ex20mut tumors compared to both EGFR-Ex18/19/21mut and EGFR/ERBB2wt tumors. RIPK1 and STK11IP showed the highest expression in ERBB2-Ex20mut tumors. CONCLUSIONS: Targeted gene expression profiling is a promising tool to read out the characteristics of the tumor microenvironment from routine diagnostic lung cancer biopsies. Significant immune reactivity and specific immunosuppressive characteristics in ERBB2-Ex20mut and EGFR-Ex20mut lung ADC with at least some degree of immune infiltration support further clinical evaluation of immune-modulators as partners of immune checkpoint inhibitors in such tumors.

Effect of timing, technique and molecular features on brain control with local therapies in oncogene-driven lung cancer.

BACKGROUND: The improved efficacy of tyrosine kinase inhibitors (TKI) mandates reappraisal of local therapy (LT) for brain metastases (BM) of oncogene-driven non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: This study included all epidermal growth factor receptor-mutated (EGFR+, n = 108) and anaplastic lymphoma kinase-rearranged (ALK+, n = 33) TKI-naive NSCLC patients diagnosed with BM in the Thoraxklinik Heidelberg between 2009 and 2019. Eighty-seven patients (62%) received early LT, while 54 (38%) received delayed (n = 34; 24%) or no LT (n = 20; 14%). LT comprised stereotactic (SRT; n = 40; 34%) or whole-brain radiotherapy (WBRT; n = 77; 66%), while neurosurgical resection was carried out in 19 cases. RESULTS: Median overall survival (OS) was 49.1 months for ALK+ and 19.5 months for EGFR+ patients (P = 0.001), with similar median intracranial progression-free survival (icPFS) (15.7 versus 14.0 months, respectively; P = 0.80). Despite the larger and more symptomatic BM (P < 0.001) of patients undergoing early LT, these experienced longer icPFS [hazard ratio (HR) 0.52; P = 0.024], but not OS (HR 1.63; P = 0.12), regardless of the radiotherapy technique (SRT versus WBRT) and number of lesions. High-risk oncogene variants, i.e. non-del19 EGFR mutations and 'short' EML4-ALK fusions (mainly variant 3, E6:A20), were associated with earlier intracranial progression (HR 2.97; P = 0.001). The longer icPFS with early LT was also evident in separate analyses of the EGFR+ and ALK+ subsets. CONCLUSIONS: Despite preferential use for cases with poor prognostic factors, early LT prolongs the icPFS, but not OS, in TKI-treated EGFR+/ALK+ NSCLC. Considering the lack of survival benefit, and the neurocognitive effects of WBRT, patients presenting with polytopic BM may benefit from delaying radiotherapy, or from radiosurgery of multiple or selected lesions. For SRT candidates, the improved tumor control with earlier radiotherapy should be weighed against the potential toxicity and the enhanced intracranial activity of newer TKI. High-risk EGFR/ALK variants are associated with earlier intracranial failure and identify patients who could benefit from more aggressive management.

Risk stratification of EGFR+ lung cancer diagnosed with panel-based next-generation sequencing.

OBJECTIVE: Panel-based next-generation sequencing (NGS) is increasingly used for the diagnosis of EGFR-mutated non-small-cell lung cancer (NSCLC) and could improve risk assessment in combination with clinical parameters. MATERIALS AND METHODS: To this end, we retrospectively analyzed the outcome of 400 tyrosine kinase inhibitor (TKI)-treated EGFR+ NSCLC patients with validation of results in an independent cohort (n = 130). RESULTS: EGFR alterations other than exon 19 deletions (non-del19), TP53 co-mutations, and brain metastases at baseline showed independent associations of similar strengths with progression-free (PFS hazard ratios [HR] 2.1-2.3) and overall survival (OS HR 1.7-2.2), in combination defining patient subgroups with distinct outcome (EGFR+NSCLC risk Score, "ENS", p < 0.001). Co-mutations beyond TP53 were rarely detected by our multigene panel (<5%) and not associated with clinical endpoints. Smoking did not affect outcome independently, but was associated with non-del19 EGFR mutations (p < 0.05) and comorbidities (p < 0.001). Laboratory parameters, like the blood lymphocyte-to-neutrophil ratio and serum LDH, correlated with the metastatic pattern (p < 0.01), but had no independent prognostic value. Reduced ECOG performance status (PS) was associated with comorbidities (p < 0.05) and shorter OS (p < 0.05), but preserved TKI efficacy. Non-adenocarcinoma histology was also associated with shorter OS (p < 0.05), but rare (2-3 %). The ECOG PS and non-adenocarcinoma histology could not be validated in our independent cohort, and did not increase the range of prognostication alongside the ENS. CONCLUSIONS: EGFR variant, TP53 status and brain metastases predict TKI efficacy and survival in EGFR+ NSCLC irrespective of other currently available parameters ("ENS"). Together, they constitute a practical and reproducible approach for risk stratification of newly diagnosed metastatic EGFR+ NSCLC.

Optimizing panel-based tumor mutational burden (TMB) measurement.

BACKGROUND: Panel sequencing based estimates of tumor mutational burden (psTMB) are increasingly replacing whole exome sequencing (WES) tumor mutational burden as predictive biomarker of immune checkpoint blockade (ICB). DESIGN: A mathematical law describing psTMB variability was derived using a random mutation model and complemented by the contributions of non-randomly mutated real-world cancer genomes and intratumoral heterogeneity through simulations in publicly available datasets. RESULTS: The coefficient of variation (CV) of psTMB decreased inversely proportional with the square root of the panel size and the square root of the TMB level. In silico simulations of all major commercially available panels in the TCGA pan-cancer cohort confirmed the validity of this mathematical law and demonstrated that the CV was 35% for TMB = 10 muts/Mbp for the largest panels of size 1.1-1.4 Mbp. Accordingly, misclassification rates (gold standard: WES) to separate 'TMBhigh' from 'TMBlow' using a cut-point of 199 mutations were 10%-12% in TCGA-LUAD and 17%-19% in TCGA-LUSC. A novel three-tier psTMB classification scheme which accounts for the likelihood of misclassification is proposed. Simulations in two WES datasets of immunotherapy treated patients revealed that small gene panels were poor predictors of ICB response. Moreover, we noted substantial intratumoral variance of psTMB scores in the TRACERx 100 cohort and identified indel burden as independent marker complementing missense mutation burden. CONCLUSIONS: A universal mathematical law describes accuracy limitations inherent to psTMB, which result in substantial misclassification rates. This scenario can be controlled by two measures: (i) a panel design that is based on the mathematical law described in this article: halving the CV requires a fourfold increase in panel size, (ii) a novel three-tier TMB classification scheme. Moreover, inclusion of indel burden can complement TMB reports. This work has substantial implications for panel design, TMB testing, clinical trials and patient management.

[From panel diagnostics to comprehensive genomic analysis : Infobesity or empowerment?] / Von der Paneldiagnostik zu umfassenden genomischen Analysen : Informationsüberfluss oder Zugewinn?

Precision oncology is obtaining a central role in the therapy of malignant diseases. The indication for targeted therapy is based on the identification of molecular targets for which next-generation sequencing (NGS) is commonly used nowadays. All approved predictive biomarkers and molecular targets, including gene fusions and copy number alterations, can be identified depending on panel design and method applied. Some clinical scenarios, however, may require more holistic genomic approaches, such as whole-genome/whole-exome and transcriptome analysis, which must be embedded in a clinical trial. Here, key aspects and applications of each method are summarized and discussed.