Series Of Tissue Samples Research Articles

Abstract P2-01-15: Inflammatory breast cancer cells are characterized by attenuated SMAD dependent TGFβ signaling leading to impaired cell motility responses

Abstract Introduction.Inflammatory breast cancer (IBC) is an aggressive form of locally advanced breast cancer with elevated metastatic potential, characterized by the frequent presence of tumor emboli in dermal and parenchymal lymph vessels. In the past, evidence was provided that TGFβ signaling is part of the molecular biology of this disease. In this study, this relation was further examined. Materials and methods. TGFβ1 induced cell motility (i.e. XCELLigence, wound healing assays), gene expression (RNA-sequencing) and peptide phosphorylation (i.e. PAMGene technology) patterns were investigated in a panel of 3 IBC and 3 subtype-matched nIBC cell lines. In addition, a series of tissue samples from 75 and 135patients with and without IBC was investigated for nuclear expression of total SMAD2, SMAD3 and SMAD4 using immunohistochemistry. Finally, SMAD protein expression data were related to gene expression data from patients with available Affymetrix HGU133plus2 profiles. Results. The cell motility inducing capacity of TGFβ1 was strongly abrogated in all IBC cells independent of their molecular subtype (P=0.003). Genes differentially expressed between IBC and nIBC cells post 4 hours of TGFβ1 revealed attenuated expression of SMAD3 transcriptional regulators with concomitant overexpression of MYC target genes in IBC. Assessment of SMAD expression in patient samples demonstrated a near absence of nuclear SMAD3 expression in the primary tumors from patients with IBC (P<0.001) and a further reduction of SMAD3 staining intensity was observed in tumor emboli (P=0.019). Integrated analysis of gene and protein expression data revealed that a substantial fraction of the IBC signature genes correlated with SMAD3 and these genes (i.e. 21/24; P<0.001) carry evidence in favor of attenuated SMAD3 signaling in IBC. Discussion. It is demonstrated that IBC cells are characterized by attenuated SMAD3 protein expression and transcriptional activity that obliterates the cell motility inducing capacity of TGFβ1. Recent studies revealed that SMAD3 is essential for TGFβ1 induced cell motility through induction of epithelial to mesenchymal transition (EMT). In the absence of SMAD3 expression, a partial EMT is induced leading to collectively invading cancer cells that are gifted with a high metastatic potential and favor lymphatic dissemination, thereby providing an intriguing explanatory model of the biology of tumor emboli in IBC. Citation Format: Rypens C, Van Berckelaer C, Billet C, Hauspy J, Bertucci F, Peter V, Dirix L, Van Laere S. Inflammatory breast cancer cells are characterized by attenuated SMAD dependent TGFβ signaling leading to impaired cell motility responses [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-15.

Expression of STAT3 and IGF2 in adrenocortical carcinoma and its relationship with angiogenesis

The aim of this study was to determine the correlation between human adrenocortical carcinoma and the proteins involved in tumor angiogenesis, and to evaluate the angiogenic status of adrenocortical carcinoma. The expression of signal transducer and activator of transcription 3 and insulin-like growth factor 2 as well as microvessel density was measured in a series of tissue samples from 44 human sporadic adrenocortical tumors by immunohistochemistry. These specimens were classified as adenomas (n = 20) and carcinomas (n = 24) according to the histological criteria defined by Weiss. A total of 19 of 24 (79.17 %) malignant cases showed positive staining for signal transducer and activator of transcription 3 and 4 of 20 (20.00 %) benign cases showed positive, the difference of signal transducer and activator of transcription 3 expression between adrenocortical adenomas and adrenocortical carcinomas was statistically significant (P < 0.001). Similarly, insulin-like growth factor 2 staining was seen in 70.83 % (17/24) of the malignant cases versus 25.00 % (5/20) of the benign, the difference of insulin-like growth factor 2 expression among two groups was statistically significant (P = 0.002). Malignant cases showed higher microvessel density compared to benign tumors (84.70 ± 12.44 vs 21.05 ± 8.07, P < 0.001). Signal transducer and activator of transcription 3 and insulin-like growth factor 2 expression were positively correlated with microvessel density in all specimens (r_s = 0.832, P < 0.001; r_s = 0.703, P = 0.001). This study has confirmed that adrenocortical carcinoma overexpress signal transducer and activator of transcription 3 and insulin-like growth factor 2; these results suggest that angiogenesis of human adrenocortical carcinoma may be mediated by these proteins and they could represent selective targets for the molecularly targeted treatments of adrenocortical carcinoma.

PD-0013 Methylation Based Biomarkers for Non-Invasive Early Detection of Colorectal Cancer

ABSTRACT Introduction Colorectal cancer is a common disease with a world-wide annual incidence of 1.2 million. The mortality rate is high, especially for patients diagnosed with an advanced tumor stage. We recently identified DNA methylation based biomarkers for early detection of colorectal cancer (and unpublished). In the beginning of 2012, these markers were licensed to the British biotech company Oxford Gene Technology for development into a non-invasive test suitable for colorectal cancer screening (http://www.ogt.co.uk/news_events/news/659_ogt_signs_licensing_deal_for_colorectal_ cancer_biomarkers). Two tests based on cancer specific aberrant DNA methylation are currently commercially available, including analyses of VIM and SEPT9 in stool and blood samples, respectively. The aim of the present study was to compare the performance of the in house biomarker panel with previously published epigenetic biomarkers using a large series of tissue samples. Methods Quantitative methylation-specific polymerase chain reaction was used to analyze the promoter methylation status of ITGA4, NTRK2, OSMR, TUBG2, SEPT9, and VIM in test and validation series comprising altogether 169 primary colorectal cancers, 105 matching normal mucosa samples, 104 adenomas, and 107 normal samples from colorectal cancer-free individuals. Receiver operating characteristic (ROC) curves were used to evaluate the overall performance of the biomarkers in tissue samples and the results were compared with the performance of the in house biomarker panel. Results Across the test and validation series, ITGA4, SEPT9, OSMR, VIM, NTRK2, and TUBG2 were methylated in 91%, 82%, 79%, 67%, 7%, and 1% of the colorectal carcinomas, and in 79%, 88%, 78%, 54%, 3%, and 0% of the adenomas. The promoter methylation was highly cancer-specific and only 1%, 3%, 2%, 3%, 0%, and 0% of the normal mucosa samples were methylated for the same genes. Resulting areas under the ROC curves for separating colorectal carcinomas from normal mucosa samples were 0.95, 0.93, 0.92, and 0.80 for ITGA4, SEPT9, OSMR, and VIM, respectively. In comparison, the in house biomarker panel achieved an area under the ROC curve of 0.97. Conclusion In a direct comparison performed by a single lab, using the same protocol and the same series of tissue samples, the performance of our recently identified in house biomarker panel was shown to be better than previously identified DNA methylation based biomarkers, including VIM and SEPT9. This underscores the value of combining a manageable number of individually promising biomarkers into a panel, which can achieve more robustness in addition to a high sensitivity and specificity. From this comparison we conclude that the in house biomarker panel is highly suitable for developing into a non-invasive test for early detection of colorectal cancer.

Significance of heparanase-1 and vascular endothelial growth factor in adrenocortical carcinoma angiogenesis: potential for therapy

The purpose of this study was to determine the correlation between human adrenocortical carcinoma (ACC) and the proteins involved in tumor angiogenesis, and to evaluate the angiogenic status of ACC. The expression of heparanase-1 (HPA-1), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor-2 (VEGFR-2) as well as microvessel density (MVD) were measured in a series of tissue samples from 44 human sporadic adrenocortical tumors by immunohistochemistry. These specimens were classified as adenomas (n=20) and carcinomas (n=24) according to the histological criteria defined by Weiss. A total of 22 of 24 (91.67%) malignant cases showed positive staining for HPA-1 and 3 of 20 (15%) benign cases showed positive, the difference of HPA-1 expression between ACA and ACC was statistically significant (P<0.001). Similarly, VEGF staining was seen in 70.83% (17/24) of the malignant cases versus 25% (5/20) of the benign, the difference of VEGF expression among two groups was statistically significant (P=0.002). VEGFR-2 expressed highly in the ACC group (79.17%, 19/24) and lowly in the benign group (25%, 5/20), the two groups had extremely significant difference (P<0.001). Malignant cases showed higher MVD compared to benign tumors (84.70±12.44 vs. 21.05±8.07, P<0.001). HPA-1 and VEGF expression were positively correlated with MVD in all specimens (r_s=0.812, P=0.001; r_s=0.834, P<0.001). In conclusion, these results suggest that angiogenesis of human ACC maybe mediated by these proteins and they could represent selective targets for the molecularly targeted treatments of ACC.

Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity

Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. This cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues.

How to design and use a research database

The vast majority of histopathology research projects based on a series of tissue samples will require a database to store and organize the data. Most of these databases will be relatively simple standalone databases that can be created in a spreadsheet application. However, there are still some important considerations in the design of the database and coding of the data that will make its subsequent use much more time efficient. This article reviews the scope of databases for common histopathology research projects, the ethical and legal considerations, design of the database structure, coding of data items, and import and export of data from databases.

Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome.

Mutations cause or influence the prevalence of many diseases. In human tissues, somatic point mutations have been observed at fractions at or below 4/10,000 and 5/100,000 in mitochondrial and nuclear DNA, respectively. In human populations, fractions for the multiple alleles that code for recessive deleterious syndromes are not expected to exceed 5 x 10(-4). Both nuclear and mitochondrial point mutations have been measured in human cells and tissues at fractions approaching 10(-6) using constant denaturant capillary electrophoresis (CDCE) coupled with high-fidelity PCR (hifiPCR). However, this approach is only applicable to those target sequences (approximately 100 bp) juxtaposed with a 'clamp', a higher-melting-temperature sequence, in genomic DNA; such naturally clamped targets represent approximately 9% of the human genome. To open up most of the human genome to rare point-mutational analysis, a high-efficiency DNA ligation procedure was recently developed so that a clamp could be attached to any target of interest. We coupled this ligation procedure with prior CDCE/hifiPCR and achieved a sensitivity of 2 x 10(-5) in human cells for the first time using an externally attached clamp. At this sensitivity, somatic mutations, each representing an anatomically distinct cluster of cells (turnover unit) derived from a mutant stem cell, may be detected in a series of tissue samples, each containing as many as 5 x 10(4) turnover units. Additionally, rare inherited mutations may be scanned in pooled DNA samples, each derived from as many as 10(5) persons.

Nerve Growth Factor Receptor Immunoreactivity in Breast Cancer Patients

Nerve growth factor receptor (NGF-R) has been shown to have antiproliferative, differentiative, or apoptotic effects on some types of tumor cells, whereas in others it may have mitogenic activity. The immunohistochemical distribution of NGF-R was analyzed in a series of tissue samples from breast cancer patients and its relationship with other clinical and pathological parameters was studied. The distribution of NGF-R was evaluated by immunohistochemistry in frozen tissue samples of 46 breast cancer patients (ME20-4 monoclonal anti-NGF-R). NGF-R immunoreactivity was localized in the plasma membrane of myoepithelial cells, differentiated ducts, neoplastic cells, blood vessels, and nerve fibers in 26 patients (57%). Less differentiated neoplastic tissues were usually NGF-R negative. NGF-R immunoreactivity was associated with estrogen receptor (ER) status (p = 0.02), small tumor dimension (pT) (p = 0.04), low histologic grade (G1–G2) (p < 0.05), old age(p = 0.02), menopause (p = 0.02), and long disease-free survival (DFS) (median follow up 86 months; p = 0.03; independently from ER, pT, age, menopause by multivariate analysis, p = 0.0078). The expression of NGF-R immunoreactivity by breast cancer patients with long DFS may represent a crucial step both in the differentiation status of neoplasia and in the host immune mechanism controlling tumor growth and metastasization.

Ornithine decarboxylase and polyamines in silkmoth pupal tissues: Effects of ecdysone and injury

1. 1. Ornithine decarboxylase was almost undetectable in fat body and wing tissue from cecropia silkmoth pupae in diapause, but after injection of β-ecdysone, activity rose steeply to a maximum at about 18 h. Injection of water or the inactive analog 22-isoecdysone also induced the enzyme in both tissues, an effect attributable to injury, but in wings β-ecdysone induced a 10-fold greater increase. Cycloheximide blocked induction of the enzyme. 2. 2. Tissues incubated for 12–18 h in culture medium developed a certain level of ornithine decarboxylase. β-Ecdysone in the medium caused definite additional induction, while α-ecdysone had little or no effect. 3. 3. The levels of the polyamines putrescine, spermidine and spermine were determined in a series of tissue samples. Putrescine was relatively concentrated (2–8 mM) in hemolymph, and was elevated 48 h after injection of ecdysone; in fat body, levels were lower and changed less. Spermidine was elevated in fat body and wings 24–36 h after injection of β-ecdysone. Spermine showed no clear pattern of change. 4. 4. Among the consequences of ecdysone action in pupal fat body and wings, induction of ornithine decarboxylase coincides in time with a previously observed peak of RNA synthesis, and maximal spermidine levels appear somewhat later.