Charting differentially methylated regions in cancer with Rocker-meth

Matteo Benelli et al. present Rocker-meth, a new Hidden Markov Model (HMM)-based method, to robustly identify differentially methylated regions (DMRs). They use Rocker-meth to analyse more than 6000 methylation profiles across 14 cancer types, providing a catalog of tumor-specific and shared DMRs

Glucose Metabolic Reprogramming of ER Breast Cancer in Acquired Resistance to the CDK4/6 Inhibitor Palbociclib

The majority of breast cancers express the estrogen receptor (ER) and are dependent on estrogen for their growth and survival. Endocrine therapy (ET) is the standard of care for these tumors. However, a superior outcome is achieved in a subset of ER positive (ER+)/human epidermal growth factor receptor 2 negative (HER2−) metastatic breast cancer patients when ET is administrated in combination with a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor, such as palbociclib. Moreover, CDK4/6 inhibitors are currently being tested in ER+/HER2+ breast cancer and reported encouraging results. Despite the clinical advances of a combinatorial therapy using ET plus CDK4/6 inhibitors, potential limitations (i.e., resistance) could emerge and the metabolic adaptations underlying such resistance warrant further elucidation. Here we investigate the glucose-dependent catabolism in a series of isogenic ER+ breast cancer cell lines sensitive to palbociclib and in their derivatives with acquired resistance to the drug. Importantly, ER+/HER2− and ER+/HER2+ cell lines show a different degree of glucose dependency. While ER+/HER2− breast cancer cells are characterized by enhanced aerobic glycolysis at the time of palbociclib sensitivity, ER+/HER2+ cells enhance their glycolytic catabolism at resistance. This metabolic phenotype was shown to have prognostic value and was targeted with multiple approaches offering a series of potential scenarios that could be of clinical relevance

p27(Kip1 )is expressed in proliferating cells in its form phosphorylated on threonine 187

BACKGROUND: G1/S cell cycle progression requires p27(Kip1 )(p27) proteolysis, which is triggered by its phosphorylation on threonine (Thr) 187. Since its levels are abundant in quiescent and scarce in cycling cells, p27 is an approved marker for quiescent cells, extensively used in histopathology and cancer research. METHODS: However here we showed that by using a specific phosphorylation site (pThr187) antibody, p27 is detectable also in proliferative compartments of normal, dysplastic and neoplastic tissues. RESULTS: In fact, whereas un-phosphorylated p27 and MIB-1 showed a significant inverse correlation (Spearman R = -0.55; p < 0,001), pThr187-p27 was positively and significantly correlated with MIB-1 expression (Spearman R = 0.88; p < 0,001). Thus proliferating cells only stain for pThr187-p27, whereas they are un-reactive with the regular p27 antibodies. However increasing the sensitivity of the immunocytochemistry (ICH) by the use of an ultra sensitive detection system based on tiramide signal amplification, simultaneous expression and colocalisation of both forms of p27 was shown in proliferating compartments nuclei by double immunofluorescence and laser scanning confocal microscopy studies. CONCLUSION: Overall, our data suggest that p27 expression also occurs in proliferating cells compartments and the combined use of both regular and phospho- p27 antibodies is suggested

Estrogen receptor co-regulators as prognostic and predictive markers of endocrine therapy in early breast cancer: the role of SMRT and p160 family

Background: The estrogen receptor (ER) signaling pathway is the dominant driver of cell proliferation and survival in the majority of human breast cancers. Not surprisingly, endocrine treatment, such as the anti-estrogen tamoxifen, represents the most effective and widely used therapy for ER positive breast cancer patients. Unfortunately not all patients respond to endocrine treatment and a wide proportion of patients ultimately develop resistance and die. Selecting patients with an increased risk of recurrence and identifying those that might benefit from a particular therapy is of great value in order to personalize breast cancer therapies. A minority of breast cancers does not express ER and displays features of aggressiveness and poor prognosis. Prognostic markers are urgently needed for this subset of patients as well. The p160 family of ER co-activator is composed of three different members: SRC1, SRC2 and AIB1. SRC1 and AIB1 are frequently overexpressed in breast cancer and appear to be linked to hormone resistance, particularly in HER2 positive breast cancer. SMRT is an ER co-repressor that has been implicated in tamoxifen resistance. Data on p160 family members and SMRT expression in human breast cancer samples and its prognostic and predictive significance in endocrine treated patients are controversial or lacking altogether. Moreover, the role of these co-regulators in ER negative disease is poorly understood. Methods: SRC1, SRC2, AIB1 and SMRT expression was determined by immunohistochemistry on tissue microarrays derived from two fully documented cohorts of 1812 and 1424 patients. Results: HER2 and AIB1 dual-positive tumors were associated with markedly worse outcome compared to tumors overexpressing either HER2 or AIB1 alone, irrespective of ER status. In ER negative disease both SRC1 and AIB1 were linked to early relapse and death. Additionally, we found that co-expression of two or more SRCs were significantly associated with worse outcome in ER positive endocrine-treated patients. However, expression of any SRC alone was not a significant predictor of resistance to endocrine therapy. Low nuclear SMRT expression was associated with a significantly better outcome in untreated patients but not in tamoxifen-treated patients. Conclusions: The SRC family of ER co-activators and nuclear SMRT are markers of early relapse in both ER negative and ER positive breast cancer. Evaluation of multiple markers co-expression (i.e. AIB1/HER2, multiple SRCs) rather than single markers allows a better assessment of breast cancer prognosis

Complex Regulation of the Cyclin-Dependent Kinase Inhibitor p27kip1 in Thyroid Cancer Cells by the PI3K/AKT Pathway

Functional inactivation of the tumor suppressor p27kip1 in human cancer occurs either through loss of expression or through phosphorylation-dependent cytoplasmic sequestration. Here we demonstrate that dysregulation of the PI3K/AKT pathway is important in thyroid carcinogenesis and that p27kip1 is a key target of the growth-regulatory activity exerted by this pathway in thyroid cancer cells. Using specific PI3K inhibitors (LY294002, wortmannin, and PTEN) and a dominant active AKT construct (myrAKT), we demonstrated that the PI3K/AKT pathway controlled thyroid cell proliferation by regulating the expression and subcellular localization of p27. Results obtained with phospho-specific antibodies and with transfection of nonphosphorylable p27kip1 mutant constructs demonstrated that PI3K/AKT-dependent regulation of p27kip1 mislocalization in thyroid cancer cells occurred via phosphorylation of p27kip1 at T157 and T198 (but not at S10 or T187). Finally, we evaluated whether these results were applicable to human tumors. Analysis of 100 thyroid carcinomas indicated that p27kip1 phosphorylation at T157/T198 and cytoplasmic mislocalization were preferentially associated with activation of the PI3K/AKT pathway. Thus the PI3/AKT pathway and its effector p27kip1 play major roles in thyroid carcinogenesis

Cell-Free DNA-Methylation-Based Methods and Applications in Oncology

Liquid biopsy based on cell-free DNA (cfDNA) enables non-invasive dynamic assessment of disease status in patients with cancer, both in the early and advanced settings. The analysis of DNA-methylation (DNAm) from cfDNA samples holds great promise due to the intrinsic characteristics of DNAm being more prevalent, pervasive, and cell- and tumor-type specific than genomics, for which established cfDNA assays already exist. Herein, we report on recent advances on experimental strategies for the analysis of DNAm in cfDNA samples. We describe the main steps of DNAm-based analysis workflows, including pre-analytics of cfDNA samples, DNA treatment, assays for DNAm evaluation, and methods for data analysis. We report on protocols, biomolecular techniques, and computational strategies enabling DNAm evaluation in the context of cfDNA analysis, along with practical considerations on input sample requirements and costs. We provide an overview on existing studies exploiting cell-free DNAm biomarkers for the detection and monitoring of cancer in early and advanced settings, for the evaluation of drug resistance, and for the identification of the cell-of-origin of tumors. Finally, we report on DNAm-based tests approved for clinical use and summarize their performance in the context of liquid biopsy

ddSeeker: a tool for processing Bio-Rad ddSEQ single cell RNA-seq data

Abstract Background New single-cell isolation technologies are facilitating studies on the transcriptomics of individual cells. Bio-Rad ddSEQ is a droplet-based microfluidic system that, when coupled with downstream Illumina library preparation and sequencing, enables the monitoring of thousands of genes per cell. Sequenced reads show unique features that do not permit the use of freely available tools to perform single cell demultiplexing. Results We present ddSeeker, a tool to perform initial processing and quality metrics of reads generated through Bio-Rad ddSEQ/Illumina experiments. Its application to the Illumina test dataset demonstrates that ddSeeker performs better than Illumina BaseSpace software, enabling a higher recovery of valid reads. We also show its utility in the analysis of an in-house dataset including two read sets characterized by low and high sequencing quality. ddSeeker and its source code are available at https://github.com/cgplab/ddSeeker. Conclusions ddSeeker is a freely available tool to perform initial processing and quality metrics of reads generated through Bio-Rad ddSEQ/Illumina single cell transcriptomic experiments

A gene expression signature of Retinoblastoma loss-of-function predicts resistance to neoadjuvant chemotherapy in ER-positive/HER2-positive breast cancer patients

HER2-positive (HER2+) breast cancers show heterogeneous response to chemotherapy, with the ER-positive (ER+) subgroup deriving less benefit. Loss of retinoblastoma tumor suppressor gene (RB1) function has been suggested as a cardinal feature of breast cancers that are more sensitive to chemotherapy and conversely resistant to CDK4/6 inhibitors. We performed a retrospective analysis exploring RBsig, a gene signature of RB loss, as a potential predictive marker of response to neoadjuvant chemotherapy in ER+/HER2+ breast cancer patients.We selected clinical trials of neoadjuvant chemotherapy +/- anti-HER2 therapy in HER2+ breast cancer patients with available information on gene expression data, hormone receptor status, and pathological complete response (pCR) rates. RBsig expression was computed in silico and correlated with pCR.Ten studies fulfilled the inclusion criteria and were included in the analysis (514 patients). Overall, of 211 ER+/HER2+ breast cancer patients, 49 achieved pCR (23%). The pCR rate following chemotherapy +/- anti-HER2 drugs in patients with RBsig low expression was significantly lower compared to patients with RBsig high expression (16% vs. 30%, respectively; Fisher's exact test p = 0.015). The area under the ROC curve (AUC) was 0.62 (p = 0.005). In the 303 ER-negative (ER-)/HER2+ patients treated with chemotherapy +/- anti-HER2 drugs, the pCR rate was 43%. No correlation was found between RBsig expression and pCR rate in this group.Low expression of RBsig identifies a subset of ER+/HER2+ patients with low pCR rates following neoadjuvant chemotherapy +/- anti-HER2 therapy. These patients may potentially be spared chemotherapy in favor of anti-HER2, endocrine therapy, and CDK 4/6 inhibitor combinations