Veronique Neumeister

Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features

Some breast cancers have been shown to contain a small fraction of cells characterized by CD44+/CD24−/low cell-surface antigen profile that have high tumor-initiating potential. In addition, breast cancer cells propagated in vitro as mammospheres (MSs) have also been shown to be enriched for cells capable of self-renewal. In this study, we have defined a gene expression signature common to both CD44+/CD24−/low and MS-forming cells. To examine its clinical significance, we determined whether tumor cells surviving after conventional treatments were enriched for cells bearing this CD44+/CD24−/low-MS signature. The CD44+/CD24−/low-MS signature was found mainly in human breast tumors of the recently identified “claudin-low” molecular subtype, which is characterized by expression of many epithelial-mesenchymal-transition (EMT)-associated genes. Both CD44+/CD24−/low-MS and claudin-low signatures were more pronounced in tumor tissue remaining after either endocrine therapy (letrozole) or chemotherapy (docetaxel), consistent with the selective survival of tumor-initiating cells posttreatment. We confirmed an increased expression of mesenchymal markers, including vimentin (VIM) in cytokeratin-positive epithelial cells metalloproteinase 2 (MMP2), in two separate sets of postletrozole vs. pretreatment specimens. Taken together, these data provide supporting evidence that the residual breast tumor cell populations surviving after conventional treatment may be enriched for subpopulations of cells with both tumor-initiating and mesenchymal features. Targeting proteins involved in EMT may provide a therapeutic strategy for eliminating surviving cells to prevent recurrence and improve long-term survival in breast cancer patients.

A Decade of Tissue Microarrays: Progress in the Discovery and Validation of Cancer Biomarkers

This year, 2008, marks the 10-year anniversary of the development of the modern tissue microarray (TMA). During the last decade, the use of TMAs has grown steadily and accounts for a small but increasing percentage of all cancer biomarker studies performed. The growing popularity of TMA-based studies attests to their benefits in the discovery and validation of new biomarkers. This review will focus on these benefits, but also on the faults of TMAs and the challenges of TMA studies that have been overcome in the last decade. We will also discuss the role of TMAs in the latest revolution in cancer treatment, the use of targeted drug therapy.

In Situ Identification of Putative Cancer Stem Cells by Multiplexing ALDH1, CD44, and Cytokeratin Identifies Breast Cancer Patients with Poor Prognosis

A subset of cells, tentatively called cancer stem cells (CSCs), in breast cancer have been associated with tumor initiation, drug resistance, and tumor persistence or aggressiveness. They are characterized by CD44 positivity, CD24 negativity, and/or ALDH1 positivity in flow cytometric studies. We hypothesized that the frequency or density of these cells may be associated with more aggressive tumor behavior. We borrowed these multiplexed, flow-based methods to develop an in situ method to define CSCs in formalin-fixed paraffin-embedded breast cancer tissue, with the goal of assessing the prognostic value of the presence of CSCs in breast cancer. Using a retrospective collection of 321 node-negative and 318 node-positive patients with a mean follow-up time of 12.6 years, we assessed TMAs using the AQUA method for quantitative immunofluorescence. Using a multiplexed assay for ALDH1, CD44, and cytokeratin to measure the coexpression of these proteins, putative CSCs appear in variable sized clusters and in 27 cases (of 490), which showed significantly worse outcome (log rank P = 0.0003). Multivariate analysis showed that this marker combination is independent of tumor size, histological grade, nodal status, ER-, PR,- and HER2-status. In this cohort, ALDH1 expression alone does not significantly predict outcome. We conclude that the multiplexed method of in situ identification of putative CSCs identifies high risk patients in breast cancer.

Quantitative Assessment of Effect of Preanalytic Cold Ischemic Time on Protein Expression in Breast Cancer Tissues

BACKGROUND Companion diagnostic tests can depend on accurate measurement of protein expression in tissues. Preanalytic variables, especially cold ischemic time (time from tissue removal to fixation in formalin) can affect the measurement and may cause false-negative results. We examined 23 proteins, including four commonly used breast cancer biomarker proteins, to quantify their sensitivity to cold ischemia in breast cancer tissues. METHODS A series of 93 breast cancer specimens with known time-to-fixation represented in a tissue microarray and a second series of 25 matched pairs of core needle biopsies and breast cancer resections were used to evaluate changes in antigenicity as a function of cold ischemic time. Estrogen receptor (ER), progesterone receptor (PgR), HER2 or Ki67, and 19 other antigens were tested. Each antigen was measured using the AQUA method of quantitative immunofluorescence on at least one series. All statistical tests were two-sided. RESULTS We found no evidence for loss of antigenicity with time-to-fixation for ER, PgR, HER2, or Ki67 in a 4-hour time window. However, with a bootstrapping analysis, we observed a trend toward loss for ER and PgR, a statistically significant loss of antigenicity for phosphorylated tyrosine (P = .0048), and trends toward loss for other proteins. There was evidence of increased antigenicity in acetylated lysine, AKAP13 (P = .009), and HIF1A (P = .046), which are proteins known to be expressed in conditions of hypoxia. The loss of antigenicity for phosphorylated tyrosine and increase in expression of AKAP13, and HIF1A were confirmed in the biopsy/resection series. CONCLUSIONS Key breast cancer biomarkers show no evidence of loss of antigenicity, although this dataset assesses the relatively short time beyond the 1-hour limit in recent guidelines. Other proteins show changes in antigenicity in both directions. Future studies that extend the time range and normalize for heterogeneity will provide more comprehensive information on preanalytic variation due to cold ischemic time.

A Quantitative Comparison of Antibodies to Programmed Cell Death 1 Ligand 1

Importance Assessment of PD-L1 (programmed cell death 1 ligand 1) expression by immunohistochemical analysis has been used as a predictive diagnostic test to identify responders and guide treatment in trials of the PD-1 (programmed cell death 1) axis inhibitors. The definition of PD-L1 positive lacks standardization, and prediction of response by immunohistochemical analysis is additionally limited by the subjective nature of this technique. Objective To examine whether PD-L1 antibody reagents are interchangeable by quantitatively comparing the expression of the PD-L1 protein. Design, Setting, and Participants In this immunohistochemistry standardization study, 30 randomly selected cases of lung cancer resected from January 1, 2008, through December 31, 2009, were obtained from Yale Pathology Archives with a range of expression of PD-L1. To test for protein measurement, rather than clinical utility, a PD-L1 index tissue microarray, including cell line and tissue controls, was used. The results were then validated on a commercially available, genetically defined PD-L1 engineered cell line array with a range of controlled protein-expressing cell lines using 6 monoclonal antibodies (SP142, E1L3N, 9A11, SP263, 22c3, and 28-8). Protein levels were measured by quantitative immunofluorescence and quantitative chromogenic assessment. Data analysis was performed from September 2015 through May 2016. Results Concordance between 4 antibodies revealed regression for tumor tissue cores (R2 = 0.42-0.91) and cell line cores (R2 = 0.83-0.97) by quantitative immunofluorescence in the PD-L1 index tissue microarray. All 6 antibodies had high levels of concordance (R2 = 0.76-0.99) when using chromogenic staining in isogenic cell lines. Conclusions and Relevance Because the antibodies are highly concordant, these results suggest that assays based on the use of these antibodies could yield concordant results. They further suggest that previously described differences in PD-L1 expression in tissue are independent of the antibody used and likely attributable to tumor heterogeneity, assay- or platform-specific variables, or other factors.

Lin28 regulates HER2 and promotes malignancy through multiple mechanisms

The RNA binding protein Lin28 and its paralog Lin28B are associated with advanced human malignancies. Blocking the biogenesis of let-7 miRNA, a tumor suppressor, by Lin28/Lin28B has been thought to underlie their roles in cancer. Here we report that the mRNA for the human epidermal growth factor receptor 2 (HER2), a HER-family receptor tyrosine kinase known to play a critical role in cell proliferation and survival and also a major therapeutic target in breast cancer, is among several targets of Lin28 regulation. We show that Lin28 stimulates HER2 expression at the posttranscriptional level, and that enforced Lin28 expression promotes cancer cell growth via multiple mechanisms. Consistent with its pleiotropic role in regulating gene expression, Lin28 overexpression in primary breast tumors is a powerful predictor of poor prognosis, representing the first report on the impact of Lin28 expression on clinical outcome in human cancer. While revealing another layer of regulation of HER2 expression in addition to gene amplification, our studies also suggest novel mechanistic insights linking Lin28 expression to disease outcome and imply that targeting multiple pathways is a common mechanistic theme of Lin28-mediated regulation in cancer.

Quantitative measurement of cancer tissue biomarkers in the lab and in the clinic

Detection of biomolecules in tissues provides contextual information and the possibility to assess the interaction of different cell types and markers. Routine qualitative assessment of immune- and oligonucleotide-based methods in research and the clinic has been associated with assay variability because of lack of stringent validation and subjective interpretation of results. As a result, the vast majority of in situ assays in clinical usage are nonquantitative and, although useful, often of questionable scientific validity. Here, we revisit the reporters and methods used for single- and multiplexed in situ visualization of protein and RNA. Then we examine methods for the use of quantitative platforms for in situ measurement of protein and mRNA levels. Finally, we discuss the challenges of the transition of these methods to the clinic and their potential role as tools for development of companion diagnostic tests.

Stathmin expression and its relationship to microtubule-associated protein tau and outcome in breast cancer

Microtubule‐associated proteins (MAPs) endogenously regulate microtubule stability. Here, the prognostic value of stathmin, a destabilizing protein, was assessed in combination with MAP‐tau, a stabilizing protein, in order to evaluate microtubule stabilization as a potential biomarker.

Measurement of Aldehyde Dehydrogenase 1 Expression Defines a Group with Better Prognosis in Patients with Non-Small Cell Lung Cancer

Aldehyde dehydrogenase 1 (ALDH1) has been suggested as a surrogate biomarker for cancer stem cells in breast cancer and other tumors. We quantitatively measured ALDH1 in two large cohorts of patients with non-small cell lung cancer (NSCLC) and investigated its prognostic value. The AQUA method of quantitative immunofluorescence was used to measure ALDH1 in 134 patients with NSCLC from Yale University and 296 patients with NSCLC from Sotiria and Patras University hospitals in Greece, using tissue microarrays. Patients were classified as positive or negative for ALDH1 based on the detection threshold for quantitative immunofluorescence. Patients with squamous cell carcinoma had higher scores than patients with adenocarcinoma. Detectable ALDH1 predicted better prognosis in both cohorts (P = 0.0035 for the Yale cohort; P = 0.0238 for the Sotiria/Patras cohorts). The effect of ALDH1 expression was independent of clinicopathologic factors in the Yale cohort (risk ratio = 3.2, P = 0.0008), but did not reach significance in the Sotiria/Patras cohort (hazard ratio = 1.51, P = 0.08). Among patients with adenocarcinoma, the ALDH1-negative group had shorter survival compared with the ALDH1-positive group in the Yale cohort (P = 0.00001), but not in the Sotiria/Patras cohort (P = 0.45). Unlike breast cancer, in which ALDH1 expression predicts poor outcome, in NSCLC our exploratory and retrospective study indicates that ALDH1 expression is associated with favorable outcome.

A tissue quality index: an intrinsic control for measurement of effects of preanalytical variables on FFPE tissue

While efforts are made to improve tissue quality and control preanalytical variables, pathologists are often confronted with the challenge of molecular analysis of patient samples of unknown quality. Here we describe a first attempt to construct a tissue quality index (TQI) or an intrinsic control that would allow a global assessment of protein status based on quantitative measurement of a small number of selected, informative epitopes. Quantitative immunofluorescence (QIF) of a number of proteins was performed on a series of 93 breast cancer cases where levels of expression were assessed as a function of delayed time to formalin fixation. A TQI was constructed based on the combination of proteins that most accurately reflect increased and decreased levels of expression in proportion to delay time. The TQI, defined by combinations of measurements of cytokeratin, ERK1/2 and pHSP-27 and their relationship to cold ischemic time were validated on a second build of the training series and on two independent breast tissue cohorts with recorded time to formalin fixation. We show an association of negative TQI values (an indicator for loss of tissue quality) with increasing cold ischemic time on both validation cohorts and an association with loss of ER expression levels on all three breast cohorts. Using expression levels of three epitopes, we can begin to assess the likelihood of delayed time to fixation or decreased tissue quality. This TQI represents a proof of concept for the use of epitope expression to provide a mechanism for monitoring tissue quality.