Next Generation Sequencing of Reactive Stroma and Residual Breast Cancer Cells in Tumor Bed after Neoadjuvant Chemotherapy.

Primary systemic or neoadjuvant chemotherapy of breast cancer has become a standard therapy option in locally advanced or predefined intrinsic subtypes such as triple negative or Her2 positive breast cancer. Neoadjuvant chemotherapy can result in complete pathological response without residual tumor cells (tumor bed) or partial response and non-response with different amounts of reactive stroma and residual tumor cells. The interaction between therapy regimens and tumoral driver mutations have been extensively studied, although the reactive stroma of the tumor bed received less attention. In this study, we characterized the mutational status of residual breast cancer cells and reactive tumor stroma devoid of residual tumor cells in partial or non-responders using next generation sequencing. Twenty-one post-therapeutic breast surgical specimens after neoadjuvant chemotherapy underwent pathogenic driver-mutation screening using microdissected residual breast cancer cells and in reactive stroma adjacent to tumor bed areas. In reactive stroma, no mutations could be validated. In residual breast cancer cells, mutations were detected in sixteen of twenty-one cases (76%). In nine of these twenty-one cases (43%), pathogenic driver mutations (PIK3CA, PTEN, TP53, FN1, PLAG1) were identified. Pathogenic driver-mutations are exclusively restricted to residual carcinoma cells and are absent in reactive stroma independently from intrinsic breast cancer subtypes or tumor stage. These data suggest that the absence of pathogenic mutations in a tumor bed without residual tumor cells may have prognostic implications after neoadjuvant chemotherapy.

MiR-99b-5p expression and response to tyrosine kinase inhibitor treatment in clear cell renal cell carcinoma patients.

A number of treatments targeting VEGF or mTOR pathways have been approved for metastatic clear cell Renal Cell Carcinoma (ccRCC), but the majority of patients show disease progression after first line therapy with a very low rate of complete or long-term responders. It has been shown that miRs may play a role in prediction of treatment response in various cancer types. The aim of our study was to identify a miR signature predictive for RCC patients' response to antiangiogenic tyrosine kinase inhibitor (TKI) treatment in the first line therapy. Sequencing of 40 paired normal/tumor formalin fixed and paraffin embedded ccRCC tissues revealed separate clustering via unsupervised dendrograms. With supervised analysis, the strongest differential expression was obtained with miR-99b-5p, which was significantly lower in patients with short progression free survival (<8 months) and TKI non-responders (progressive disease patients according to RECIST) (p<0.0001, each). Validation using RTqPCR and a second patient cohort compiled from three different hospitals (n=65) showed higher expression of miR-99b-5p in complete responders, but this trend did not reach statistical significance. It is concluded that low miR-99b-5p expression analyzed with sequencing methodology may correlate with tumor progression in TKI-treated ccRCC patients.

miR-30c-2-3p and miR-30a-3p: new pieces of the jigsaw puzzle in HIF2α regulation.

Clear cell renal cell carcinoma (ccRCC), the most frequent subtype of renal cell cancer, is characterized by mutation of the von Hippel-Lindau (VHL) tumor suppressor gene, which results in stabilization of hypoxia-inducible factor (HIF) α proteins. In this issue of Cancer Discovery, Mathew and colleagues report that miR-30c-2-3p and miR-30a-3p downregulation in ccRCC promotes increased expression of HIF2α.

Co-assessment of cell cycle and micronucleus frequencies demonstrates the influence of serum on the in vitro genotoxic response to amorphous monodisperse silica nanoparticles of varying sizes.

Serum proteins have been shown to modulate the cytotoxic and genotoxic responses to nanomaterials. The aim was to investigate the influence of serum on the induction of micronuclei (MN) by nanoparticles (NPs) of different sizes. Therefore, A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles (SNPs) were used as models. Assessment of the cell viability, cell cycle changes and induction of MN by SNPs ranging from 12 to 174 nm was performed in presence or absence of serum, applying the in vitro flow cytometry-based MN assay. Here, it has been demonstrated that serum has an influence on these end points, with a lower cell viability in absence of serum compared with the presence of serum. Further, cell cycle changes, specifically, G1 and S-phase arrest, were observed in absence of serum for four out of six SNPs tested. A size-dependent MN induction was observed: larger SNPs being more active in absence of serum. In addition, the serum influence was characterised by a size-dependency for cytotoxic and genotoxic effects, with a higher influence of serum for smaller particles. The data indicate that the in vitro micronucleus assay in presence and absence of serum could be advised for hazard assessment because it demonstrates a higher sensitivity in serum-free conditions than in conditions with serum. However, this recommendation applies only if the cell line used is able to proliferate under serum-free conditions because cell division is a prerequisite for MN expression.

Flow cytometric determination of micronucleus frequency.

During the last two decades the micronucleus (MN) test has been extensively used as a genotoxicity screening tool of chemicals and in a variety of exploratory and mechanistic investigations. The MN is a biomarker for chromosomal damage or mitotic abnormalities, since it can originate from chromosome fragments or whole chromosomes that fail to be incorporated into daughter nuclei during mitosis (Fenech et al., Mutagenesis 26:125-132, 2011; Kirsch-Volders et al., Arch Toxicol 85:873-899, 2011). The simplicity of scoring, accuracy, amenability to automation by image analysis or flow cytometry, and readiness to be applied to a variety of cell types either in vitro or in vivo have made it a versatile tool that has contributed to a large extent in our understanding of key toxicological issues related to genotoxins and their effects at the cellular and organism levels. Recently, the final acceptance of the in vitro MN test guideline 487 (OECD Guideline for Testing of Chemicals, In vitro mammalian cell micronucleus test 487. In vitro mammalian cell micronucleus test (MNVIT). Organization for Economic Cooperation and Development, Paris, 2010) together with the standard in vivo MN test OECD guideline 474 (OECD Guideline for The Testing of Chemicals, Mammalian erythrocyte micronucleus test no. 474. Organization for Economic Cooperation and Development, Paris, 1997) will further position the assay as a key driver in the determination of the genotoxicity potential in exploratory research as well as in the regulatory environment. This chapter covers to some extent the protocol designs and experimental steps necessary for a successful performance of the MN test and an accurate analysis of the MN by the flow cytometry technique.

Flow cytometric 96-well microplate-based in vitro micronucleus assay with human TK6 cells: protocol optimization and transferability assessment.

An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96-well plate (Bryce SM et al. [2010]: Mutat Res 703:191-199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non-genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5- to 2-cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating ≥ 5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN-fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non-genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpoints-relative survival and quantification of ethidium monoazide-positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96-well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity.

Miniaturized flow cytometry-based in vitro primary human lymphocyte micronucleus assay-validation study.

Most in vitro mammalian genotoxicity assays show a low specificity (high rate of irrelevant positive results), and therefore, lead to an increase in follow-up in vivo genotoxicity testing. One of the sources of the high rate of in vitro irrelevant positive results that find no confirmation in in vivo studies may be the characteristics of the test system used. It has been shown that cells that are p53 deficient or carry an alteration in DNA repair genes may be more prone to produce high rate of false/irrelevant positive results. Primary human lymphocytes (HuLy) are considered to show a higher specificity in predicting the in vivo genotoxic potential of a tested compound. We recently developed a flow cytometry-based primary human T-lymphocyte micronucleus test (MNT) and showed that the technology is promising and reliable in detecting genotoxic compounds. The purpose of the present work was to develop and validate a miniaturized format of the assay. For validation purposes of the flow cytometry HuLy MNT a wide selection of compounds with different mechanisms of genotoxicity was used. The evaluation covered 30 compounds: 19 commercially available genotoxicants and nongenotoxicants and 11 early pharmaceutical development compounds. Being faster and less tedious than the microscopic analysis, the miniaturized flow cytometry-based methodology showed very promising results. Conveniently, cell division is verified within the same sample as the MN frequency. Moreover analysis of hypodiploid events may provide an indication for a mode of action, i.e. clastogenic versus aneugenic mechanism, for further follow-up testing.