IncellDx Oncobreast 3Dx CSUPERB Poster
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This document describes a new multi-parametric assay being developed by IncellDx to simultaneously analyze genomic, proteomic, and cell cycle markers in breast cancer samples. The assay aims to measure 9 markers including ER, PR, HER2, E-cadherin, DNA content and proliferation. Initial studies mixing breast cancer cell lines expressing different markers demonstrated the ability to differentiate markers. Further studies on clinical breast cancer samples found the assay could simultaneously detect HER2 mRNA and protein levels that matched immunohistochemistry results. Future studies will evaluate the assay's clinical performance on 50 breast tumors and 10 normal samples compared to standard pathology.
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This document describes a new cell-based diagnostic technique called Cellular Multiplex that can simultaneously detect protein expression, mRNA expression, and cell cycle analysis from breast cancer biopsies. In a preliminary study of 16 breast tumors and 5 normal tissues, the technique identified heterogeneity in biomarker expression between tumors. It also established normal biomarker expression levels and cell cycle distributions in normal breast tissue. The approach has potential to provide a more comprehensive view of the tumor environment and limit inaccuracies of current methods, which could lead to better patient outcomes.
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2. Defects in cellular proliferation cause cancer cells to lose contact inhibition and divide indiscriminately on top of normal cells.
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1. Vermaat and colleagues compared the genetic profiles of 1,264 cancer-related genes in primary colorectal tumors and subsequent liver metastases in 21 patients. They found significant differences between primary and metastatic tumors that could impact treatment outcomes.
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The document describes a new ex vivo platform called CANScript that aims to predict patient responses to anticancer drugs. It does this by:
1. Engineering personalized tumour ecosystems from patient tumour biopsies that conserve tumour heterogeneity and microenvironment by culturing thin tumour sections on defined tumour-specific matrix proteins and in autologous patient serum.
2. Testing the response of over 100 such tumour ecosystems to anticancer drugs and correlating it to actual patient outcomes to train a machine learning algorithm.
3. Validating the algorithm on an independent group of 55 patients, where it achieved 100% sensitivity in predicting responses while maintaining high specificity.
The authors believe this platform, which contextualizes the native tumour environment
Pdx project
This document summarizes a study that analyzed whole transcriptome profiles of patient-derived xenograft (PDX) models of various cancer types using RNA sequencing. Specifically:
- Human tumor cells from 79 patients with cancers like breast, lung, GI, ovarian and leukemia were implanted in mouse models. RNA from both human tumor and mouse stromal cells was extracted and analyzed.
- Unsupervised analysis identified batch effects across samples and specific samples with high stromal or cancer expression. This helped distinguish tumor vs stromal signals and identify potential biomarkers.
- The study aims to better understand the relationship between human tumors and mouse stroma in PDX models and identify biomarkers for personalized cancer treatment.
Vu-2015_tissue-plasticity-PNET
1) The study cultured primitive neuroectodermal tumor (PNET) stem cells on soft polyacrylamide hydrogel plates that mimic brain tissue elasticity and compared gene expression to cells cultured on standard polystyrene plates.
2) Microarray analysis found more genes were downregulated on the soft hydrogel plates, suggesting tissue elasticity influences microRNA expression and gene silencing.
3) Bioinformatic analysis identified specific microRNA binding sites enriched on the soft plates, supporting the hypothesis that tissue elasticity regulates microRNA expression and downstream gene expression.
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1) Higher CCP scores, indicating more actively dividing cells, were associated with increased risk of biochemical recurrence after prostatectomy and death from prostate cancer in a conservatively managed cohort.
2) The CCP score was a strong univariate predictor of outcome and added significant prognostic information when combined with clinical factors like PSA and Gleason score in multivariate analyses.
3) The CCP score was robust across different patient populations and clinical settings and may help physicians select optimal treatment strategies at diagnosis.Kurrey_et_al-2009-STEM_CELLS
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An integrated approach to analyzing breast cancer at the proteomic and genomic level is presented using a cytometric readout. The approach analyzes fine needle aspirates to assess proteins, mRNA expression of HER2, and genomic integrity. Feasibility testing used a model system of mixed cell lines and analyzed 40 breast tumors and 10 normal tissues fixed in two solutions. The clinical performance relates to the model system and the cell-based assay could apply to xenograft models and circulating tumor cells.
P1-01-17_poster
This study evaluates an integrative pathology system called Cellular Multiplex that can detect proteomic, genomic and DNA data from fine needle aspiration biopsy specimens of breast cancer tumors. In an interim analysis of the first 21 patients, the system showed almost perfect concordance with immunohistochemistry for detecting estrogen receptor, progesterone receptor and HER2 levels. When at least 100 epithelial cells were obtained, there were no significant differences in measurement results between samples taken from intact tumors versus excised tumors, suggesting the system may be useful as a diagnostic tool and for monitoring response to neoadjuvant therapy.
Low expression of N-myc downstream-regulated gene 2 in oesophageal squamous c...
Low expression of N-myc downstream-regulated gene 2 in oesophageal squamous c...Enrique Moreno Gonzalez
It is currently unclear whether a correlation exists between N-myc downstream-regulated gene 2 (NDRG2) expression and oesophageal squamous cell carcinoma (ESCC). The aim of this study was to examine the underlying clinical significance of NDRG2 expression in ESCC patients and to investigate the effects of NDRG2 up-regulation on ESCC cell growth in vitro and in vivo.Interrogating differences in expression of targeted gene sets to predict brea...
Interrogating differences in expression of targeted gene sets to predict brea...Enrique Moreno Gonzalez
Genomics provides opportunities to develop precise tests for diagnostics, therapy selection and monitoring. From analyses of our studies and those of published results, 32 candidate genes were identified, whose expression appears related to clinical outcome of breast cancer. Expression of these genes was validated by qPCR and correlated with clinical follow-up to identify a gene subset for development of a prognostic test.HETEROGENEITY OF C ERB B FAMILY MEMBERS EXPRESSION IS RELATED TO CELL MORPHOL...
This document summarizes a study examining the expression of HER2 and EGFR in pituitary adenomas and their relationship to hormone profiles. The study found that over one-third of pituitary adenomas expressed HER2, particularly prolactin-secreting adenomas. HER2 expression was also related to certain hormone co-expressions. Approximately half of adenomas expressed EGFR, which was correlated with GH-prolactin co-expression. The results suggest HER2 and EGFR expression may help define subclasses of pituitary adenomas and explain their clinical heterogeneity.
Bioinformatics
Now a day’s, pharma research is facing challenges in
deciphering molecular understanding of disease initiation,
progress and establishment as well as performance
assessment of drug molecule on such phases of disease
development. Emerging of next generation sequencing
bases molecular tools were found to be a key method for
creating genome wide genomics landscape of gene
mutations, gene expression and gene regulation events.
Although NGS is a powerful tool for molecular research but
same time it have its own technical challenges. Few major
challenges of NGS based pharmacogenomics is
summarized below
Journal club- breast ca basal like
1. The study aimed to validate immunohistochemical (IHC) surrogates for intrinsic breast cancer subtypes identified by gene expression profiling, using the PAM50 assay as the gold standard.
2. Among 46 biomarkers tested by IHC on a tissue microarray, loss of INPP4B expression showed the best combination of sensitivity (61.1%) and specificity (98.6%) for basal-like breast cancer defined by PAM50.
3. Nestin positivity also strongly correlated with basal-like subtype, consistent with other studies, and may be a useful positive basal marker. A multi-marker IHC panel may better define basal-like breast cancer than single biomarkers.
Breast Cancer MCF-7 Cell Spheroid Culture for Drug Discovery and Development.pdf
This document describes a method for culturing 3D spheroids of MCF-7 breast cancer cells using low-attachment 96-well plates. Key findings include:
1) MCF-7 cells formed spheroids over 1-4 days that continued growing for over a month, mimicking tumor growth.
2) Drugs targeting estrogen synthesis (STX64) and estrogen receptors (tamoxifen) inhibited spheroid growth, while quercetin killed spheroids, demonstrating the model's utility for drug screening.
3) Antagonists of G protein-coupled estrogen receptor (GPER; G-1 and G15) strongly inhibited spheroids, suggesting GPER critically supports MCF-7 growth.
IHC in breast pathology
This document discusses the use of immunohistochemistry in breast pathology. It covers several topics:
1. Analyzing prognostic markers like hormone receptors in breast cancer and their predictive value.
2. Using myoepithelial cell markers to help solve diagnostic dilemmas and distinguish lesions.
3. Identifying tumor subtypes and assessing diagnoses using markers like luminal vs basal.
4. Evaluating cell populations in proliferative breast lesions and assessing neoplasia vs hyperplasia.
Amy_Wood_CV _2015
Amy Wood has over 10 years of experience in molecular biology, protein production and purification, and cancer research. She holds a PhD in Molecular Biology/Immunology from the University of Birmingham and has worked in both academic and industrial settings. Her career has focused on developing cancer therapies through protein expression, structural analysis, and investigating protein-protein interactions.
Identifying novel and druggable targets in a triple negative breast cancer ce...
Identifying novel and druggable targets in a triple negative breast cancer ce...Thermo Fisher Scientific
In this study, we developed a CRISPR/Cas9-based high throughput loss-of-function screen for identifying target genes responsible for the tumor proliferation and growth in TNBC. Our initial focus was to identify essential kinases in MDA-MB-231 cell line using the Invitrogen™ LentiArray™ Human Kinase CRISPR Library, which targets 840 kinases with up to 4 different gRNAs per protein kinase for complete gene knockout. This functional screen identified over 90 protein kinases that are essential for cell viability and cell proliferation. Ten of these hits (CDK1, CDK2, CDK8, CDK10, CDK11A, CDK19, CDK19, CDC7, EPHA2 and WEE1) are well-known targets validated in the literature. Currently, we are in the process validating the novel hits through target gene sequencing, western blotting and target specific small molecule kinase inhibitors.Importance of Clinical and Pathological Characteristics of Meningiomas and Th...
Importance of Clinical and Pathological Characteristics of Meningiomas and Th...ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY
This study investigated the relationship between tumor characteristics and somatostatin receptor 2 (SSTR2) expression in 81 patients with meningioma. Univariate and multivariate analyses found that older age (>65), larger tumor size (>3 cm), and female sex were associated with poorer overall survival. However, there was no association found between SSTR2 expression levels (negative, weakly positive, strongly positive) and overall survival or other tumor characteristics. Therefore, further research is needed to understand the role of SSTR2 receptor expression in meningiomas beyond its diagnostic value.Gene expression profiling reveals molecularly and clinically distinct subtype...
This study used gene expression profiling to analyze molecular subtypes of glioblastoma multiforme (GBM), the most common and aggressive form of brain cancer. The analysis revealed:
1) Distinct gene expression patterns between GBM tumors and normal brain tissue, as well as between GBMs and lower-grade oligodendroglial tumors.
2) Significant differences in gene expression among individual GBM tumors, particularly in genes related to angiogenesis, immune response, and extracellular matrix remodeling.
3) Gene expression patterns of samples from the same GBM tumor were more similar to each other than to other tumors, even when the samples had divergent histologies.
4) A set of 70 genes associated
Effect of miR-21 on Oral Squamous Cell Carcinoma Cell Proliferation and Apopt...
Effect of miR-21 on Oral Squamous Cell Carcinoma Cell Proliferation and Apopt...ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY
Objective: To probe into the influence of miR-21 on the proliferation as well as apoptosis of oral squamous cell carcinoma (OSCC) and its causative role.
Study Design: We adopted microarray for detecting the differentially expressed genes in OSCC tumor tis-sues and paracancerous tissues. We assessed the link of miR-21 expression with tumor size, lymph node metastasis, and tumor differentiation. We employed CCK-8 and EdU assay for detecting the impact of miR-21 inhibitor and miR-21 mimic on Cal-27 cell proliferation, as well as TUNEL and AnnexinV-FITC/PI double staining for detecting miR-21 expression on cell apoptosis. We forecasted the possible target of miR-21 via TargetScan, as well as detected the interaction of miR-21 with PTEN via luciferase reporter experiment. The function of miR-21 expression in PTEN signaling pathway was monitored via western blot. We constructed PTEN overexpression plasmid and conducted rescue experiment to evaluate overexpressed PTEN on miR-21–induced proliferation.
Results: Microarray and RT-qPCR indicated that miR-21 expression increased demonstrably in OSCC. Subsequently, statistical analysis showed that miR-21 expression was plainly correlated with tumor size, lymph node metastasis, tumor differentiation, and smoking history. CCK-8 and EdU method exhibited that miR-21 mimics manifestly promoted Cal-27 cell proliferation, while miR-21 inhibitor blatantly inhibited Cal-27 cell proliferation. TUNEL and V-FITC/PI double staining assay showed that miR-21 inhibitor conspicuously promoted Cal-27 cell apoptosis. CCK-8 and EdU assay exhibited that overexpressed PTEN abolished the pro-proliferation influence of miR-21 mimic. TUNEL and V-FITC/PI experiments pointed out that knocking down PTEN abrogated the pro-apoptosis impact of miR-21 inhibitor.
Conclusion: miR-21 contributes to OSCC cell proliferation via targeting PTEN and inhibits its apoptosis.
Keywords: Akt/PKB signaling pathway; apoptosis; biomarkers, tumor; carcinoma, squamous cell; cell line, tumor; cell proliferation; microRNAs; miR-21; miRNA-21; mouth neoplasms; oral cancer; oral squamous cell carcinoma; proliferation; real time PCR1 s2.0-s0015028214018603-main
This document summarizes a study that developed a new microarray platform capable of simultaneously assessing aneuploidy, mitochondrial DNA content, and single-nucleotide polymorphisms in human polar bodies and embryos. The microarray was optimized and validated using cell lines and clinical samples. Results found the microarray detected aneuploidies with 97% accuracy and could accurately determine relative mitochondrial DNA quantities and genotypes, allowing confirmation of parental origin. The microarray provides information beyond chromosomal analysis alone that could improve embryo assessment and selection.
Mining Gene Expression Data Focusing Cancer Therapeutics: A Digest
An understanding towards genetics and epigenetics is essential to cope up with the paradigm shift which is underway. Personalized medicine and gene therapy will confluence the days to come.
This review highlights traditional approaches as well as current advancements in the analysis of the gene expression data from cancer perspective.
Due to improvements in biometric instrumentation and automation, it has become easier to collect a lot of experimental data in molecular biology.
Analysis of such data is extremely important as it leads to knowledge discovery that can be validated by experiments. Previously, the diagnosis of complex genetic diseases has conventionally been done based on the non-molecular characteristics like kind of tumor tissue, pathological characteristics, and clinical phase.
The microarray data can be well accounted for high dimensional space and noise. Same were the reasons for ineffective and imprecise results. Several machine learning and data mining techniques are presently applied for identifying cancer using gene expression data.
While differences in efficiency do exist, none of the well-established approaches is uniformly superior to others. The quality of algorithm is important, but is not in itself a guarantee of the quality of a specific data analysis.
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Molecular classification of endometrial cancer
In this topic we will discuss in detail the Molecular classification of endometrial cancer and its clinical application
Construction and Validation of Prognostic Signature Model Based on Metastatic...
Colorectal Cancer (CRC) is a common malignant cancer with a poor prognosis. Liver metastasis is the dominant cause of death in CRC patients, and it often involves changes in various gene expression profiling. This study proposed to construct and validate a risk model based on differentially expressed genes between the primary and liver metastatic tumors from CRC for prognostic prediction.
Study on Drug Resistance Mediated by Expression of HER-2 and LRP in Tumor Tis...
Study on Drug Resistance Mediated by Expression of HER-2 and LRP in Tumor Tis...ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY
This study examined the expression of HER-2 and LRP in tumor tissue samples from 65 gastric cancer patients and 32 non-gastric cancer patients. Immunohistochemical staining and Western blot analysis were used to test for HER-2 and LRP expression and phosphorylation levels. The results showed that HER-2 and LRP expression in gastric cancer tissues were positively correlated and higher than in non-gastric cancer tissues. HER-2 phosphorylation was highest in gastric cancer patients with lymphatic metastasis. This suggests that LRP may cause primary resistance in gastric cancer and that HER-2 activation may mediate gastric cancer resistance by influencing LRP expression.Biotech 2012 spring-4_cell_function_mircroarrays
This study compared fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) for determining HER2/neu gene amplification status in 188 breast cancer samples using tissue microarray technology. The results showed a high concordance rate of 94.1% between FISH and CISH. Both methods identified HER2/neu amplification in around 24% of samples. The study concluded that CISH is a feasible and cost-effective alternative to FISH for assessing HER2/neu amplification in breast cancer.
Similar to IncellDx Oncobreast 3Dx CSUPERB Poster (20)
Low expression of N-myc downstream-regulated gene 2 in oesophageal squamous c...
Low expression of N-myc downstream-regulated gene 2 in oesophageal squamous c...
Interrogating differences in expression of targeted gene sets to predict brea...
Interrogating differences in expression of targeted gene sets to predict brea...
HETEROGENEITY OF C ERB B FAMILY MEMBERS EXPRESSION IS RELATED TO CELL MORPHOL...
HETEROGENEITY OF C ERB B FAMILY MEMBERS EXPRESSION IS RELATED TO CELL MORPHOL...
Breast Cancer MCF-7 Cell Spheroid Culture for Drug Discovery and Development.pdf
Breast Cancer MCF-7 Cell Spheroid Culture for Drug Discovery and Development.pdf
Identifying novel and druggable targets in a triple negative breast cancer ce...
Identifying novel and druggable targets in a triple negative breast cancer ce...
Importance of Clinical and Pathological Characteristics of Meningiomas and Th...
Importance of Clinical and Pathological Characteristics of Meningiomas and Th...
Gene expression profiling reveals molecularly and clinically distinct subtype...
Gene expression profiling reveals molecularly and clinically distinct subtype...
Effect of miR-21 on Oral Squamous Cell Carcinoma Cell Proliferation and Apopt...
Effect of miR-21 on Oral Squamous Cell Carcinoma Cell Proliferation and Apopt...
Mining Gene Expression Data Focusing Cancer Therapeutics: A Digest
Mining Gene Expression Data Focusing Cancer Therapeutics: A Digest
Construction and Validation of Prognostic Signature Model Based on Metastatic...
Construction and Validation of Prognostic Signature Model Based on Metastatic...
Study on Drug Resistance Mediated by Expression of HER-2 and LRP in Tumor Tis...
Study on Drug Resistance Mediated by Expression of HER-2 and LRP in Tumor Tis...
IncellDx Oncobreast 3Dx CSUPERB Poster
- 1. Multi-Parametric Analysis of Breast Tissue Utilizing Available Cytometric Systems Amanda Chargin1,2, Keith Shults2, Lily Chen1, Linda Chen1, Bruce Patterson MD2 1Department of Biology, San Francisco State University, San Francisco, CA 2IncellDx, Menlo Park, CA ABSTRACT According to the American Society of Clinical Oncology and the College of American Pathologists, approximately 20% of current HER2/PR/ER breast testing by immunohistochemistry or in situ hybridization may be inaccurate. This inaccuracy in current testing methods for HER2/PR/ER markers in breast cancer highlights the need for an improved method of testing. Currently, there is an absence of tests on the market that have the ability to combine detection of mRNA, protein, DNA content and cell cycle analysis in one assay on intact cells. IncellDx’s proprietary technology has the ability to provide an all-encompassing view of a cancer cell by quantifying multiple biomarkers on a cytometry platform that enables high throughput testing of fine needle aspirates (FNA). INTRODUCTION • The feasibility of this assay was tested using two breast cancer cell lines for ability to measure differences in protein and mRNA expression as well as DNA content differences. • MCF-7 cells express E-cadherin and ER and have a modal chromosome number of 80. SK-BR-3 cells express HER2 and have a modal chromosome number of 84. • Mixtures of these two cell lines were permeabilized, hybridized with probes to her2 mRNA, and stained with antibodies to E-cadherin, ER and an A-T specific DNA dye. • Each biomarker is labeled by a fluorophore with a differing emission which makes it possible to differentiate each analyte on multiple cytometric systems. STUDY AIM & OBJECTIVE To develop a diagnostic test that combines simultaneous genomic, proteomic, and cell cycle analysis of breast tissue samples utilizing 9 independent variables (ER+/-, PR+/-, her2+/-, E-cadherin expression, DNA proliferation, and DNA content) in a single assay. To determine the clinical performance of this assay, standard pathological parameters and diagnosis will be compared to the cytometric readouts of the derived assay on excised breast tumors. SFSU Student Demographics in the Stem Cell Program ACKNOWLEDGEMENT This internship project is supported by IncellDx, National Science Foundation Science Master’s Program Award (DGE-1011717), Susan G Komen for the Cure®, and Department of Biology at San Francisco State University. CONCLUSION • Multiple mixing studies of MCF7, SK-BR-3, and normal human mammary epithelial cells (HMEC) have demonstrated the ability to simultaneously detect multiple protein targets and complete cell analysis on a cell by cell basis. • Comparisons of oligonucleotide cocktails using HMEC and frozen breast tumor pellets of known her2 immunohistochemical status demonstrate the ability to simultaneously detect differing protein and mRNA levels. • Combinations of the principles described in this poster produced a proteomic, genomic, morphologic and proliferative readout on multiple cytometric systems. FUTURE DIRECTION Using the established technology described in this poster, IncellDx has contracted to obtain IRB approved FNA samples excised from 40 breast tumors and 10 cases of normal tissue excised during breast reduction procedures. The study will compare the multi-parametric readout to standard pathological metrics in an effort to derive clinical performance data. This exciting platform promises to be adaptable to standard diagnostic procedures as well as a companion diagnostic for her2 and selective estrogen blockers. Since the core of the assay is single cell analysis, adaptation of circulating tumor cells and xenograft models is feasible. RESULTS METHODOLOGY • Proteomic analysis of Estrogen and Progesterone nuclear hormone receptors as well as E-cadherin surface receptor with antibodies to characterize receptor expression of cell lines. • Determine optimal DAPI concentration on cell lines for DNA content analysis. • Analysis of her2 mRNA expression of cell lines by designing and utilizing a fluorescently labeled oligonucleotide cocktail. • Determine optimal variable mixture on cell lines in combination. • Test optimal variable mixture on characterized clinical samples. RESULTS The ability to measure both her2 mRNA and protein simultaneously was confirmed on human tissues on a Sony i-Cyt flow cytometer: Figure 2: HER2+, ER/PR+ Invasive and Infiltrating Ductal Breast Cancer Tissue Figure 3: HER2-, ER/PR- Invasive and Infiltrating Ductal Breast Cancer Tissue Tissue Pathology her2 mRNA MFI HER2 protein MFI HER2+, ER/PR+ 753.16 5717.58 Normal Human Epithelial Mammary Cells 555.55 283.67 HER2-, ER/PR- 119.82 32.17 Figure 1: Output from a sample of MCF-7 cells (depicted in red) and SK-BR-3 cells (depicted in blue) with ER, HER2, E-cadherin antibody staining and DAPI A-T specific DNA dye combined in suspension. Results 1: The 3D graph shown above is a 1:1 mixture of MCF-7 and SK-BR-3 cells. This graph has been further broken down to show each metric individually. Both populations are easily resolved in all four graphs and differences in expression levels are characteristic of each cell type. Figure 1: ER, HER2, E-cadherin, and DNA quantification in combination on mixtures of MCF-7 and SK-BR-3 cells collected on the Amnis FlowSight Result 2: Fluorescence intensity her2 mRNA vs. protein E-cadherin Alexa 488 DAPI HER2 PE ER Alexa 647