ctDNA from liquid biopsy can be used as a surrogate marker for T790M in tumour tissue.

Background: a-Selective phosphatidylinositol 3-kinase (PI3K) inhibitors improve outcome in patients with PIK3CAmutated, hormone receptor-positive (HRþ)/Her2À metastatic breast cancer (mBC). Nevertheless, it is still unclear how to integrate this new drug family in the treatment landscape. Patients and methods: A total of 649 patients with mBC from the SAFIR02 trial (NCT02299999), with available mutational profiles were selected for outcome analysis. PIK3CA mutations were prospectively determined by nextgeneration sequencing on metastatic samples. The mutational landscape of PIK3CA-mutated mBC was assessed by whole-exome sequencing (n ¼ 617). Finally, the prognostic value of PIK3CA mutations during chemotherapy was assessed in plasma samples (n ¼ 44) by next-generation sequencing and digital PCR. Results: Some 28% (104/364) of HRþ/Her2À tumors and 10% (27/255) of triple-negative breast cancer (TNBC) presented a PIK3CA mutation (P < 0.001). PIK3CA-mutated HRþ/Her2À mBC was less sensitive to chemotherapy [adjusted odds ratio: 0.40; 95% confidence interval (0.22e0.71); P ¼ 0.002], and presented a worse overall survival (OS) compared with PIK3CA wild-type [adjusted hazard ratio: 1.44; 95% confidence interval (1.02e2.03); P ¼ 0.04]. PIK3CA-mutated HRþ/Her2À mBC was enriched in MAP3K1 mutations (15% versus 5%, P ¼ 0.0005). In metastatic TNBC (mTNBC), the median OS in patients with PIK3CA mutation was 24 versus 14 months for PIK3CA wild-type (P ¼ 0.03). We further looked at the distribution of PIK3CA mutation in mTNBC according to HR expression on the primary tumor. Some 6% (9/138) of patients without HR expression on the primary and 36% (14/39) of patients with HRþ on the primary presented PIK3CA mutation (P < 0.001). The level of residual PIK3CA mutations in plasma after one to three cycles of chemotherapy was associated with a poor OS [continuous variable, hazard ratio: 1.03, 95% confidence interval (1.01e1.05), P ¼ 0.007]. Conclusion: PIK3CA-mutated HRþ/Her2À mBC patients present a poor outcome and resistance to chemotherapy. Patients with PIK3CA-mutated TNBC present a better OS. This could be explained by an enrichment of PIK3CA mutations in luminal BC which lost HR expression in the metastatic setting. Trial registration: SAFIR02 trial: NCT02299999.

Purpose: Liquid biopsies based on circulating cell-free DNA (cfDNA) analysis are described as surrogate samples for molecular analysis. We evaluated the concordance between tumor DNA (tDNA) and cfDNA analysis on a large cohort of patients with advanced or metastatic solid tumor, eligible for phase I trial and with good performance status, enrolled in MOSCATO 01 trial (clinical trial NCT01566019).Experimental Design: Blood samples were collected at inclusion and cfDNA was extracted from plasma for 334 patients. Hotspot mutations were screened using next-generation sequencing for 50 cancer genes.Results: Among the 283 patients with tDNA-cfDNA pairs, 121 had mutation in both, 99 in tumor only, 5 in cfDNA only, and for 58 patients no mutation was detected, leading to a 55.0% estimated sensitivity [95% confidence interval (CI), 48.4%-61.6%] at the patient level. Among the 220 patients with mutations in tDNA, the sensitivity of cfDNA analysis was significantly linked to the number of metastatic sites, albumin level, tumor type, and number of lines of treatment. A sensitivity prediction score could be derived from clinical parameters. Sensitivity is 83% in patients with a high score (!8). In addition, we analyzed cfDNA for 51 patients without available tissue sample. Mutations were detected for 22 patients, including 19 oncogenic variants and 8 actionable mutations.Conclusions: Detection of somatic mutations in cfDNA is feasible for prescreening phase I candidates with a satisfactory specificity; overall sensitivity can be improved by a sensitivity score allowing to select patients for whom cfDNA constitutes a reliable noninvasive surrogate to screen mutations.

Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation.

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PURPOSE To assess the feasibility and utility of circulating tumor DNA (ctDNA) by amplicon-based next-generation sequencing (NGS) analysis in the daily clinical setting in a cohort of patients with advanced non–small-cell lung cancer (NSCLC), as an alternative approach to tissue molecular profiling. PATIENTS AND METHODS In this single-center prospective study, treatment-naïve and previously treated patients with advanced NSCLC were enrolled. Clinical validation of ctDNA using amplicon-based NGS analysis (with a 36-gene panel) was performed against standard-of-care tissue molecular analysis in treatment-naïve patients. The feasibility, utility, and prognostic value of ctDNA as a dynamic marker of treatment efficacy was evaluated. Results of tissue molecular profile were blinded during ctDNA analysis. RESULTS Of 214 patients with advanced NSCLC who were recruited, 156 were treatment-naïve patients and 58 were pretreated patients with unknown tissue molecular profile. ctDNA screening was successfully performed for 91% (n = 194) of all patients, and mutations were detected in 77% of these patients. Tissue molecular analysis was available for 111 patients (52%), and tissue somatic mutations were found for 78% (n = 87) of patients. For clinically relevant variants, concordance agreement between ctDNA and tumor tissue analysis was 95% among 94 treatment-naïve patients who had concurrent liquid and tumor biopsy molecular profiles. Sensitivity and specificity were 81% and 97%, respectively. Of the 103 patients with no tissue available, ctDNA detected potential actionable mutations in 17% of patients; of these, 10% received personalized treatment. ctDNA kinetics correlated with response rate and progression-free survival in 31 patients treated with first-line platinum-based chemotherapy. CONCLUSION These real-world data from a prospective study endorse ctDNA molecular profile by amplicon-based NGS as an accurate and reliable tool to detect and monitor clinically relevant molecular alterations in patients with advanced NSCLC.

PURPOSE Liquid biopsy specimen genomic profiling is integrated in non–small-cell lung cancer (NSCLC) guidelines; however, data on the clinical relevance for ALK /ROS1 alterations are scarce. We evaluated the clinical utility of a targeted amplicon-based assay in a large prospective cohort of patients with ALK/ROS1-positive NSCLC and its impact on outcomes. PATIENTS AND METHODS Patients with advanced ALK/ROS1-positive NSCLC were prospectively enrolled in the study by researchers at eight French institutions. Plasma samples were analyzed using InVisionFirst-Lung and correlated with clinical outcomes. RESULTS Of the 128 patients included in the study, 101 were positive for ALK and 27 for ROS1 alterations. Blood samples (N = 405) were collected from 29 patients naïve for treatment with tyrosine kinase inhibitors (TKI) or from 375 patients under treatment, including 105 samples collected at disease progression (PD). Sensitivity was 67% (n = 18 of 27) for ALK/ROS1 fusion detection. Higher detection was observed for ALK fusions at TKI failure (n = 33 of 74; 46%) versus in patients with therapeutic response (n = 12 of 109; 11%). ALK-resistance mutations were detected in 22% patients (n = 16 of 74) overall; 43% of the total ALK-resistance mutations identified occurred after next-generation TKI therapy. ALK G1202R was the most common mutation detected (n = 7 of 16). Heterogeneity of resistance was observed. ROS1 G2032R resistance was detected in 30% (n = 3 of 10). The absence of circulating tumor DNA mutations at TKI failure was associated with prolonged median overall survival (105.7 months). Complex ALK-resistance mutations correlated with poor overall survival (median, 26.9 months v NR for single mutation; P = .003) and progression-free survival to subsequent therapy (median 1.7 v 6.3 months; P = .003). CONCLUSION Next-generation, targeted, amplicon-based sequencing for liquid biopsy specimen profiling provides clinically relevant detection of ALK/ROS1 fusions in TKI-naïve patients and allows for the identification of resistance mutations in patients treated with TKIs. Liquid biopsy specimens from patients treated with TKIs may affect clinical outcomes and capture heterogeneity of TKI resistance, supporting their role in selecting sequential therapy.

Purpose: The limited knowledge on the molecular profile of patients with BRAF-mutant non-small cell lung cancer (NSCLC) who progress under BRAF-targeted therapies (BRAF-TT) has hampered the development of subsequent therapeutic strategies for these patients. Here, we evaluated the clinical utility of circulating tumor DNA (ctDNA)-targeted sequencing to identify canonical BRAF mutations and genomic alterations potentially related to resistance to BRAF-TT, in a large cohort of patients with BRAFmutant NSCLC.Experimental Design: This was a prospective study of 78 patients with advanced BRAF-mutant NSCLC, enrolled in 27 centers across France. Blood samples (n ¼ 208) were collected from BRAF-TT-naïve patients (n ¼ 47), patients nonprogressive under treatment (n ¼ 115), or patients at disease progression (PD) to BRAF-TT (24/46 on BRAF monotherapy and 22/46 on BRAF/ MEK combination therapy). ctDNA sequencing was performed using InVisionFirst-Lung. In silico structural modeling was used to predict the potential functional effect of the alterations found in ctDNA.Results: BRAF V600E ctDNA was detected in 74% of BRAF-TTnaïve patients, where alterations in genes related with the MAPK and PI3K pathways, signal transducers, and protein kinases were identified in 29% of the samples. ctDNA positivity at the first radiographic evaluation under treatment, as well as BRAF-mutant ctDNA positivity at PD were associated with poor survival. Potential drivers of resistance to either BRAF-TT monotherapy or BRAF/MEK combination were identified in 46% of patients and these included activating mutations in effectors of the MAPK and PI3K pathways, as well as alterations in U2AF1, IDH1, and CTNNB1.Conclusions: ctDNA sequencing is clinically relevant for the detection of BRAF-activating mutations and the identification of alterations potentially related to resistance to BRAF-TT in BRAFmutant NSCLC.