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

Erb-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.

CD25high Effector Regulatory T Cells Hamper Responses to PD-1 Blockade in Triple-Negative Breast Cancer.

Regulatory T cells (Treg) impede effective antitumor immunity. However, the role of Tregs in the clinical outcomes of patients with triple-negative breast cancer (TNBC) remains controversial. Here, we found that an immunosuppressive TNBC microenvironment is marked by an imbalance between effector αßCD8+ T cells and Tregs harboring hallmarks of highly suppressive effector Tregs (eTreg). Intratumoral eTregs strongly expressed PD-1 and persisted in patients with TNBC resistant to PD-1 blockade. Importantly, CD25 was the most selective surface marker of eTregs in primary TNBC and metastases compared with other candidate targets for eTreg depletion currently being evaluated in trials for patients with advanced TNBC. In a syngeneic TNBC model, the use of Fc-optimized, IL2 sparing, anti-CD25 antibodies synergized with PD-1 blockade to promote systemic antitumor immunity and durable tumor growth control by increasing effector αßCD8+ T-cell/Treg ratios in tumors and in the periphery. Together, this study provides the rationale for the clinical translation of anti-CD25 therapy to improve PD-1 blockade responses in patients with TNBC. SIGNIFICANCE: An imbalance between effector CD8+ T cells and CD25high effector Tregs marks immunosuppressive microenvironments in αPD-1-resistant TNBC and can be reversed through effector Treg depletion to increase αPD-1 efficacy.

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

BACKGROUND: 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.

Therapeutic Targeting of Tumor-Infiltrating Regulatory T Cells in Breast Cancer.

Regulatory T cells (Treg) are an immunosuppressive subtype of CD4+ T cells essential for maintaining self-tolerance in physiological settings. Tregs also abundantly infiltrate inflamed tumor tissues, impeding the host's antitumor immune response and contributing to tumor growth and metastasis. In breast cancers, subsets of Tregs express highly immunosuppressive effector phenotypes that favor tumorigenesis, progression, and resistance to immune-checkpoint inhibitor therapies. Tregs share phenotypic features with cytotoxic lymphocytes, rendering them difficult to inhibit without compromising productive antitumor immunity. In addition, systemic targeting of Tregs causes serious autoimmune adverse events in patients with cancer. Hence, the identification of candidate targets or methodologies allowing the specific elimination of tumor antigen-specific Tregs, including tumor-infiltrating Tregs, is a prerequisite for developing efficient and safe combinatorial immunotherapeutic strategies in breast cancers. To date, numerous preclinical studies have demonstrated that specific targeting of breast tumor-infiltrating Tregs restores a competent antitumor immune response and improves responses to immune-checkpoint inhibitors such as PD-1/PD-L1 blockade. Herein, we discuss major candidate molecules for Treg-targeted therapeutic strategies in breast cancers, detailing the pros and cons of various approaches, including mAb-mediated depletion, homeostasis destabilization, and functional blockade.

Development of multiplex digital PCR assays for the detection of PIK3CA mutations in the plasma of metastatic breast cancer patients.

With the approval of new therapies targeting the PI3K pathway, the detection of PIK3CA mutations has become a key factor in treatment management for HR+/HER2- metastatic breast cancer (MBC). We developed multiplex digital PCR (dPCR) assays to detect and quantify PIK3CA mutations. A first screening assay allows the detection of 21 mutations, with a drop-off system targeting the 542-546 hotspot mutations combined with the simultaneous detection of N345K, C420R, H1047L and H1047R mutations. In the case of a positive result, a sequential strategy based on other assays that we have developped allows for precise mutation identification. Clinical validity was determined by analyzing plasma circulating free DNA (cfDNA) from 213 HR+/HER2- MBC samples, as well as DNA extracted from 97 available matched tumors from 89 patients. Our assays have shown reliable specificity, accuracy and reproducibility, with limits of blank of three and four droplets for the screening assay. Sixty-eight patients (32%) had at least one PIK3CA mutation detectable in their plasma, and we obtained 83.1% agreement between the cfDNA analysis and the corresponding tumors. The high sensitivity and robustness of these new dPCR assays make them well-suited for rapid and cost-effective detection of PIK3CA mutations in the plasma of MBC patients.

Conserved functions for Mos in eumetazoan oocyte maturation revealed by studies in a cnidarian.

The kinase Mos, which activates intracellularly the MAP kinase pathway, is a key regulator of animal oocyte meiotic maturation. In vertebrate and echinoderm models, Mos RNA translation upon oocyte hormonal stimulation mediates "cytostatic" arrest of the egg after meiosis, as well as diverse earlier events [1-5]. Our phylogenetic survey has revealed that MOS genes are conserved in cnidarians and ctenophores, but not found outside the metazoa or in sponges. We demonstrated MAP kinase-mediated cytostatic activity for Mos orthologs from Pleurobrachia (ctenophore) and Clytia (cnidarian) by RNA injection into Xenopus blastomeres. Analyses of endogenous Mos in Clytia with morpholino antisense oligonucleotides and pharmacological inhibition demonstrated that Mos/MAP kinase function in postmeiotic arrest is conserved. They also revealed additional roles in spindle formation and positioning, strongly reminiscent of observations in starfish, mouse, and Xenopus. Unusually, cnidarians were found to possess multiple Mos paralogs. In Clytia, one of two maternally expressed paralogs accounted for the majority MAP kinase activation during maturation, whereas the other may be subject to differential translational regulation and have additional roles. Our findings indicate that Mos appeared early during animal evolution as an oocyte-expressed kinase and functioned ancestrally in regulating core specializations of female meiosis.