Plasma-based analysis of ERBB2 mutational status by multiplex digital PCR in a large series of patients with metastatic breast cancer

International audienceErb‐b2 receptor tyrosine kinase 2 (ERBB2)‐activating mutations are therapeutically actionable alterations found in various cancers, including metastatic breast cancer (MBC). We developed multiplex digital PCR assays to detect and quantify ERBB2 mutations in circulating tumor DNA from liquid biopsies. We studied the plasma from 272 patients with hormone‐receptor‐positive, human epidermal growth factor receptor 2‐negative (HR+/HER2−) MBC to detect 17 ERBB2 mutations using a screening assay. The assay was developed on the three‐color Crystal dPCR™ naica® platform with a two‐step strategy for precise mutation identification. We found that nine patients (3.3%) harbored at least one ERBB2 mutation. The mutation rate was higher in patients with lobular histology (5.9%) compared to invasive breast carcinoma of no special type (2.6%). A total of 12 mutations were found with the following frequencies: L755S (25.00%), V777L (25.00%), S310Y (16.67%), L869R (16.67%), S310F (8.33%), and D769H (8.33%). Matched tumor samples from six patients identified the same mutations with an 83% concordance rate. In summary, our highly sensitive multiplex digital PCR assays are well suited for plasma‐based monitoring of ERBB2 mutational status in patients with MBC

Multiple metastatic clones assessed by an integrative multiomics strategy in clear cell renal carcinoma: a case study

International audienceThe dynamics of metastatic evolution in clear cell renal cell carcinoma (ccRCC) are complex. We report a case study where tumour heterogeneity resulting from clonal evolution is a frequent feature and could play a role in metastatic dissemination.We used an integrative multiomics strategy combining genomic and transcriptomic data to classify fourteen specimens from spatially different areas of a kidney tumour and three non-primary sites including a vein thrombus and two adrenal metastases.All sites were heterogeneous and polyclonal, each tumour site containing two different aggressive subclonal populations, with differentially expressed genes implicated in distinct biological functions. These are rare primary metastatic samples prior to any medical treatment, where we showed a multiple metastatic seeding of two subclonal populations.Multiple interdependent lineages could be the source of metastatic heterogeneity in ccRCC. By sampling metastases, patients with resistance to therapies could benefit a combination of targeted therapies based on more than one aggressive clone

Single-cell Deconvolution of a Specific Malignant Cell Population as a Poor Prognostic Biomarker in Low-risk Clear Cell Renal Cell Carcinoma Patients

International audienceBACKGROUND: Intratumor heterogeneity (ITH) is a key feature in clear cell renal cell carcinomas (ccRCCs) that impacts outcomes such as aggressiveness, response to treatments, or recurrence. In particular, it may explain tumor relapse after surgery in clinically low-risk patients who did not benefit from adjuvant therapy. Recently, single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool to unravel expression ITH (eITH) and might enable better assessment of clinical outcomes in ccRCC. OBJECTIVE: To explore eITH in ccRCC with a focus on malignant cells (MCs) and assess its relevance to improve prognosis for low-risk patients. DESIGN, SETTING, AND PARTICIPANTS: We performed scRNA-seq on tumor samples from five untreated ccRCC patients ranging from pT1a to pT3b. Data were complemented with a published dataset composed of pairs of matched normal and ccRCC samples. INTERVENTION: Radical or partial nephrectomy on untreated ccRCC patients. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Viability and cell type proportions were determined by flow cytometry. Following scRNA-seq, a functional analysis was performed and tumor progression trajectories were inferred. A deconvolution approach was applied on an external cohort, and Kaplan-Meier survival curves were estimated with respect to the prevalence of malignant clusters. RESULTS AND LIMITATIONS: We analyzed 54 812 cells and identified 35 cell subpopulations. The eITH analysis revealed that each tumor contained various degrees of clonal diversity. The transcriptomic signatures of MCs in one particularly heterogeneous sample were used to design a deconvolution-based strategy that allowed the risk stratification of 310 low-risk ccRCC patients. CONCLUSIONS: We described eITH in ccRCCs, and used this information to establish significant cell population-based prognostic signatures and better discriminate ccRCC patients. This approach has the potential to improve the stratification of clinically low-risk patients and their therapeutic management. PATIENT SUMMARY: We sequenced the RNA content of individual cell subpopulations composed of clear cell renal cell carcinomas and identified specific malignant cells the genetic information of which can be used to predict tumor progression

Development of sensitive and robust multiplex digital PCR assays for the detection of ESR1 mutations in the plasma of metastatic breast cancer patients

International audienceBACKGROUND: Early detection of ESR1 mutations is a key element for better personalization of the management of patients with HR+/HER2- Metastatic Breast Cancer (MBC). Analysis of circulating tumor DNA from liquid biopsies is a particularly well-suited strategy for longitudinal monitoring of such patients. MATERIALS AND METHODS: Using the naica® three-color digital PCR platform, we developed a screening assay allowing the detection of 11 ESR1 mutations and designed a sequential strategy for precise mutation identification. We then applied this strategy in the analysis of plasma circulating cell-free DNA from 109 HR+/HER2- MBC patients and performed a double-blind comparison study on a subset of patients with the multiplex assay used at the Institut Curie (IC) for the PADA-1 study. RESULTS: Thirty-one patients (28.4%) harboured at least one ESR1 mutation, with the following frequencies: D538G (41.03%), Y537S (25.64%), E380Q (10.26%), Y537N (10.26%), "(536-540)" (7.69%), Y537C (2.56%), and L536R (2.56%). The presence of ESR1 mutation(s) was significantly associated with liver metastases (p = 0.0091). A very good agreement (91%) was observed with the IC assay. CONCLUSION: Our assays have proven to be robust and highly sensitive and are very well-suited for monitoring ESR1 mutations in the plasma of MBC patients