Early-Stage Breast Cancer Detection in Breast Milk.

Breast cancer occurring during pregnancy (PrBC) and postpartum (PPBC) is usually diagnosed at more advanced stages compared with other breast cancer, worsening its prognosis. PPBC is particularly aggressive, with increased metastatic risk and mortality. Thus, effective screening methods to detect early PrBC and PPBC are needed. We report for the first time that cell-free tumor DNA (ctDNA) is present in breast milk (BM) collected from patients with breast cancer. Analysis of ctDNA from BM detects tumor variants in 87% of the cases by droplet digital PCR, while variants remain undetected in 92% of matched plasma samples. Retrospective next-generation sequencing analysis in BM ctDNA recapitulates tumor variants, with an overall clinical sensitivity of 71.4% and specificity of 100%. In two cases, ctDNA was detectable in BM collected 18 and 6 months prior to standard diagnosis. Our results open up the potential use of BM as a new source for liquid biopsy for PPBC detection. SIGNIFICANCE: For the first time, we show that BM obtained from patients with breast cancer carries ctDNA, surpassing plasma-based liquid biopsy for detection and molecular profiling of early-stage breast cancer, even prior to diagnosis by image. See related commentary by Cunningham and Turner, p. 2125. This article is featured in Selected Articles from This Issue, p. 2109.

A pan-cancer clinical platform to predict immunotherapy outcomes and prioritize immuno-oncology combinations in early-phase trials.

BACKGROUND: Immunotherapy is effective, but current biomarkers for patient selection have proven modest sensitivity. Here, we developed VIGex, an optimized gene signature based on the expression level of 12 genes involved in immune response with RNA sequencing. METHODS: We implemented VIGex using the nCounter platform (Nanostring) on a large clinical cohort encompassing 909 tumor samples across 45 tumor types. VIGex was developed as a continuous variable, with cutoffs selected to detect three main categories (hot, intermediate-cold and cold) based on the different inflammatory status of the tumor microenvironment. FINDINGS: Hot tumors had the highest VIGex scores and exhibited an increased abundance of tumor-infiltrating lymphocytes as compared with the intermediate-cold and cold. VIGex scores varied depending on tumor origin and anatomic site of metastases, with liver metastases showing an immunosuppressive tumor microenvironment. The predictive power of VIGex-Hot was observed in a cohort of 98 refractory solid tumor from patients treated in early-phase immunotherapy trials and its clinical performance was confirmed through an extensive metanalysis across 13 clinically annotated gene expression datasets from 877 patients treated with immunotherapy agents. Last, we generated a pan-cancer biomarker platform that integrates VIGex categories with the expression levels of immunotherapy targets under development in early-phase clinical trials. CONCLUSIONS: Our results support the clinical utility of VIGex as a tool to aid clinicians for patient selection and personalized immunotherapy interventions. FUNDING: BBVA Foundation; 202-2021 Division of Medical Oncology and Hematology Fellowship award; Princess Margaret Cancer Center.

Expression of TILs and Patterns of Gene Expression from Paired Samples of Malignant Pleural Mesothelioma (MPM) Patients.

MPM is an aggressive disease with an immunosuppressive tumor microenvironment, and interest in exploring immunotherapy in this disease has been increasing. In the first line of treatment, the combination of nivolumab and ipilimumab demonstrated an improvement in survival over chemotherapy. The presence of TILs has been recognized as a marker of antitumor immune response to chemotherapy in solid tumors. The aim of our study is to identify the effect of treatment on immune cells and the immune gene profile in MPM. We investigated the changes in expression of TILs in 10 human MPM paired tumor tissues using immunohistochemistry and gene expression analysis from paired untreated and treated samples. In this small series, we demonstrated that during the evolution of disease without any treatment there was an increase in the inflammatory component in tumor samples. After systemic treatment there was a decrease in the number of TILs. We observed that after systemic treatment or disease progression immune gene signatures were suppressed. Our integrated analysis of paired samples with immune profile and genomic changes on MPM suggested that during the evolution of the disease the immune system tends to switch, turning off with treatment.

SWI/SNF and RSC remodeler complexes bind to MBF-dependent genes.

In fission yeast, MBF-dependent transcription is required for cells to complete S phase. The MBF transcription factor is regulated through a complex feedback mechanism that involves the co-repressors Yox1 and Nrm1 that are loaded onto MBF at the end of S phase, while positive transactivation is achieved through the constitutive binding of the co-activator Rep2. Here we show that Rep2 is required to fully recruit the chromatin remodelers SWI/SNF and RSC to MBF-regulated promoters. On the contrary, Nrm1 and Yox1, when bound to the MBF complex, block the approximation of these chromatin remodelers to MBF-regulated promoters. We propose that SWI/SNF and RSC are recruited to MBF-regulated genes, and RSC together with SAGA complex are important to regulate the G1-to-S transcriptional wave. Mutants of these remodeler complexes are highly sensitive when cells are exposed to insults that challenge DNA synthesis.

Gcn5-mediated acetylation at MBF-regulated promoters induces the G1/S transcriptional wave.

In fission yeast, MBF-dependent transcription is inactivated at the end of S phase through a negative feedback loop that involves the co-repressors, Yox1 and Nrm1. Although this repression system is well known, the molecular mechanisms involved in MBF activation remain largely unknown. Compacted chromatin constitutes a barrier to activators accessing promoters. Here, we show that chromatin regulation plays a key role in activating MBF-dependent transcription. Gcn5, a part of the SAGA complex, binds to MBF-regulated promoters through the MBF co-activator Rep2 in a cell cycle-dependent manner and in a reverse correlation to the binding of the MBF co-repressors, Nrm1 or Yox1. We propose that the co-repressors function as physical barriers to SAGA recruitment onto MBF promoters. We also show that Gcn5 acetylates specific lysine residues on histone H3 in a cell cycle-regulated manner. Furthermore, either in a gcn5 mutant or in a strain in which histone H3 is kept in an unacetylated form, MBF-dependent transcription is downregulated. In summary, Gcn5 is required for the full activation and correct timing of MBF-regulated gene transcription.

The INO80 complex activates the transcription of S-phase genes in a cell cycle-regulated manner.

Chromatin structure is an essential factor in the proper regulation of DNA repair, DNA replication and transcription. The INO80 complex and the SWR complex have been shown to play a fundamental role in transcription regulation through remodeling chromatin at specific genes and loci. Here, we report that the Schizosaccharomyces pombe INO80 complex physically interacts with the mlui-binding factor (MBF) complex. Furthermore, we are able to detect the INO80 complex in MBF-regulated promoters. Binding of INO80 to these genes is cell cycle regulated, with a maximum binding preceding their transcription and accumulation of their mRNAs. In fact, the INO80 complex is required to fully and timely activate the transcription of these genes. We also show that the accumulation of acetylated H2A.Z at the +1 nucleosome is cell cycle regulated. Cells in which H2A.Z acetylation is abolished still have some cell cycle-regulated transcription of MBF-dependent genes, although to a much lesser extent.

Timeless protection of telomeres.

The DNA replication machinery encounters problems at numerous genomic regions that are inherently difficult to replicate. These genomic regions include telomeres, which contain repetitive DNA and telomere-binding proteins. If not properly regulated, replication of such genomic regions can result in DNA damage, leading to genomic instability. Studies implicated a role of Timeless-related proteins at difficult-to-replicate genomic regions, including telomeres. However, how these proteins maintain telomeres was elusive. In a recent report, we described the role of Swi1, a Timeless-related protein, in telomere maintenance in fission yeast. We demonstrated that Swi1 is required for proper replication of repeat DNA sequences at telomeres. We also showed that Swi1-deficient cells utilize recombination-based ALT (alternative lengthening of telomeres)-like mechanisms to maintain telomeres in the absence of telomerase. Here, we highlight these findings and present additional data to discuss the role of Swi1Timeless in telomere protection and ALT prevention.

A functional genome-wide genetic screening identifies new pathways controlling the G1/S transcriptional wave.

The Schizosaccharomyces pombe MBF complex activates the transcription of genes required for DNA synthesis and S phase. The MBF complex contains several proteins, including the core components Cdc10, Res1 and Res2, the co-repressor proteins Yox1 and Nrm1 and the co-activator Rep2. It has recently been shown how MBF is regulated when either the DNA damage or the DNA synthesis checkpoints are activated. However, how MBF is regulated in a normal unperturbed cell cycle is still not well understood. We have set up a genome-wide genomic screen searching for global regulators of MBF. We have crossed our knock-out collection library with a reporter strain that allows the measurement of MBF activity in live cells by flow cytometry. We confirm previously known regulators of MBF and show that COP9/signalosome and tRNA methyltransferases also regulate MBF activity.