Analytical validation of HER2DX genomic test for early-stage HER2-positive breast cancer.

BACKGROUND: HER2DX, a multianalyte genomic test, has been clinically validated to predict breast cancer recurrence risk (relapse risk score), the probability of achieving pathological complete response post-neoadjuvant therapy (pCR likelihood score), and individual ERBB2 messenger RNA (mRNA) expression levels in patients with early-stage human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This study delves into the comprehensive analysis of HER2DX's analytical performance. MATERIALS AND METHODS: Precision and reproducibility of HER2DX risk, pCR, and ERBB2 mRNA scores were assessed within and between laboratories using formalin-fixed paraffin-embedded (FFPE) tumor tissues and purified RNA. Robustness was appraised by analyzing the impact of tumor cell content and protocol variations including different instruments, reagent lots, and different RNA extraction kits. Variability was evaluated across intratumor biopsies and genomic platforms [RNA sequencing (RNAseq) versus nCounter], and according to protocol variations. RESULTS: Precision analysis of 10 FFPE tumor samples yielded a maximal standard error of 0.94 across HER2DX scores (1-99 scale). High reproducibility of HER2DX scores across 29 FFPE tumors and 20 RNAs between laboratories was evident (correlation coefficients >0.98). The probability of identifying score differences >5 units was ≤5.2%. No significant variability emerged based on platform instruments, reagent lots, RNA extraction kits, or TagSet thaw/freeze cycles. Moreover, HER2DX displayed robustness at low tumor cell content (10%). Intratumor variability across 212 biopsies (106 tumors) was <4.0%. Concordance between HER2DX scores from 30 RNAs on RNAseq and nCounter platforms exceeded 90.0% (Cohen's κ coefficients >0.80). CONCLUSIONS: The HER2DX assay is highly reproducible and robust for the quantification of recurrence risk, pCR likelihood, and ERBB2 mRNA expression in early-stage HER2-positive breast cancer.

Plasmatic BRAF-V600E allele fraction as a prognostic factor in metastatic colorectal cancer treated with BRAF combinatorial treatments.

BACKGROUND: Combination of a BRAF inhibitor (BRAFi) and an anti-epidermal growth factor receptor (EGFR), with or without a MEK inhibitor (MEKi), improves survival in BRAF-V600E-mutant metastatic colorectal cancer (mCRC) over standard chemotherapy. However, responses are heterogeneous and there are no available biomarkers to assess patient prognosis or guide doublet- or triplet-based regimens. In order to better characterize the clinical heterogeneity observed, we assessed the prognostic and predictive role of the plasmatic BRAF allele fraction (AF) for these combinations. PATIENTS AND METHODS: A prospective discovery cohort including 47 BRAF-V600E-mutant patients treated with BRAFi-anti-EGFR ± MEKi in clinical trials and real-world practice was evaluated. Results were validated in an independent multicenter cohort (n= 29). Plasmatic BRAF-V600E AF cut-off at baseline was defined in the discovery cohort with droplet digital PCR (ddPCR). All patients had tissue-confirmed BRAF-V600E mutations. RESULTS: Patients with high AF have major frequency of liver metastases and more metastatic sites. In the discovery cohort, median progression-free survival (PFS) and overall survival (OS) were 4.4 and 10.1 months, respectively. Patients with high BRAF AF (≥2%, n = 23) showed worse PFS [hazard ratio (HR) 2.97, 95% confidence interval (CI) 1.55-5.69; P = 0.001] and worse OS (HR 3.28, 95% CI 1.58-6.81; P = 0.001) than low-BRAF AF patients (<2%, n = 24). In the multivariable analysis, BRAF AF levels maintained independent significance. In the validation cohort, high BRAF AF was associated with worse PFS (HR 3.83, 95% CI 1.60-9.17; P = 0.002) and a trend toward worse OS was observed (HR 1.86, 95% CI 0.80-4.34; P = 0.15). An exploratory analysis of predictive value showed that high-BRAF AF patients (n = 35) benefited more from triplet therapy than low-BRAF AF patients (n = 41; PFS and OS interaction tests, P < 0.01). CONCLUSIONS: Plasmatic BRAF AF determined by ddPCR is a reliable surrogate of tumor burden and aggressiveness in BRAF-V600E-mutant mCRC treated with a BRAFi plus an anti-EGFR with or without a MEKi and identifies patients who may benefit from treatment intensification. Our results warrant further validation of plasmatic BRAF AF to refine clinical stratification and guide treatment strategies.

Dual MET and ERBB inhibition overcomes intratumor plasticity in osimertinib-resistant-advanced non-small-cell lung cancer (NSCLC).

BACKGROUND: Third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as osimertinib are the last line of targeted treatment of metastatic non-small-cell lung cancer (NSCLC) EGFR-mutant harboring T790M. Different mechanisms of acquired resistance to third-generation EGFR-TKIs have been proposed. It is therefore crucial to identify new and effective strategies to overcome successive acquired mechanisms of resistance. METHODS: For Amplicon-seq analysis, samples from the index patient (primary and metastasis lesions at different timepoints) as well as the patient-derived orthotopic xenograft tumors corresponding to the different treatment arms were used. All samples were formalin-fixed paraffin-embedded, selected and evaluated by a pathologist. For droplet digital PCR, 20 patients diagnosed with NSCLC at baseline or progression to different lines of TKI therapies were selected. Formalin-fixed paraffin-embedded blocks corresponding to either primary tumor or metastasis specimens were used for analysis. For single-cell analysis, orthotopically grown metastases were dissected from the brain of an athymic nu/nu mouse and cryopreserved at -80°C. RESULTS: In a brain metastasis lesion from a NSCLC patient presenting an EGFR T790M mutation, we detected MET gene amplification after prolonged treatment with osimertinib. Importantly, the combination of capmatinib (c-MET inhibitor) and afatinib (ErbB-1/2/4 inhibitor) completely suppressed tumor growth in mice orthotopically injected with cells derived from this brain metastasis. In those mice treated with capmatinib or afatinib as monotherapy, we observed the emergence of KRAS G12C clones. Single-cell gene expression analyses also revealed intratumor heterogeneity, indicating the presence of a KRAS-driven subclone. We also detected low-frequent KRAS G12C alleles in patients treated with various EGFR-TKIs. CONCLUSION: Acquired resistance to subsequent EGFR-TKI treatment lines in EGFR-mutant lung cancer patients may induce genetic plasticity. We assess the biological insights of tumor heterogeneity in an osimertinib-resistant tumor with acquired MET-amplification and propose new treatment strategies in this situation.

Concordance of blood- and tumor-based detection of RAS mutations to guide anti-EGFR therapy in metastatic colorectal cancer.

BACKGROUND: Circulating tumor DNA (ctDNA) is a potential source for tumor genome analysis. We explored the concordance between the mutational status of RAS in tumor tissue and ctDNA in metastatic colorectal cancer (mCRC) patients to establish eligibility for anti-epidermal growth factor receptor (EGFR) therapy. PATIENTS AND METHODS: A prospective-retrospective cohort study was carried out. Tumor tissue from 146 mCRC patients was tested for RAS status with standard of care (SoC) PCR techniques, and Digital PCR (BEAMing) was used both in plasma and tumor tissue. RESULTS: ctDNA BEAMing RAS testing showed 89.7% agreement with SoC (Kappa index 0.80; 95% CI 0.71 - 0.90) and BEAMing in tissue showed 90.9% agreement with SoC (Kappa index 0.83; 95% CI 0.74 - 0.92). Fifteen cases (10.3%) showed discordant tissue-plasma results. ctDNA analysis identified nine cases of low frequency RAS mutations that were not detected in tissue, possibly due to technical sensitivity or heterogeneity. In six cases, RAS mutations were not detected in plasma, potentially explained by low tumor burden or ctDNA shedding. Prediction of treatment benefit in patients receiving anti-EGFR plus irinotecan in second- or third-line was equivalent if tested with SoC PCR and ctDNA. Forty-eight percent of the patients showed mutant allele fractions in plasma below 1%. CONCLUSIONS: Plasma RAS determination showed high overall agreement and captured a mCRC population responsive to anti-EGFR therapy with the same predictive level as SoC tissue testing. The feasibility and practicality of ctDNA analysis may translate into an alternative tool for anti-EGFR treatment selection.