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Patent
Indivumed GmbH | Date: 2017-06-07

Methods for assessing the quality of a tissue sample. In some aspects, methods are provided for determine the time that a biological sample has been exposed to cold ischemia condition by measuring the expression level of an mRNA and/or a phosphoprotein.


David K.A.,Indivumed GmbH | Unger F.T.,Indivumed GmbH | Uhlig P.,Indivumed GmbH | Juhl H.,Indivumed GmbH | And 6 more authors.
Oncotarget | Year: 2014

An understanding of tissue data variability in relation to processing techniques during and postsurgery would be desirable when testing surgical specimens for clinical diagnostics, drug development, or identification of predictive biomarkers. Specimens of normal and colorectal cancer (CRC) tissues removed during colon and liver resection surgery were obtained at the beginning of surgery and postsurgically, tissue was fixed at 10, 20, and 45 minutes. Specimens were analyzed from 50 patients with primary CRC and 43 with intrahepatic metastasis of CRC using a whole genome gene expression array. Additionally, we focused on the epidermal growth factor receptor pathway and quantified proteins and their phosphorylation status in relation to tissue processing timepoints. Gene and protein expression data obtained from colorectal and liver specimens were influenced by tissue handling during surgery and by postsurgical processing time. To obtain reliable expression data, tissue processing for research and diagnostic purposes needs to be highly standardized.


Wolf C.,Hoffmann-La Roche | Wolf C.,BioM BioTech Cluster Development GmbH | Jarutat T.,Hoffmann-La Roche | Vega Harring S.,Hoffmann-La Roche | And 6 more authors.
Histopathology | Year: 2014

Aims: For selection of patients who will benefit from targeted therapies, identification of biomarkers predictive of treatment response is desirable. Activation of the targeted pathway becomes apparent by protein phosphorylation. Determination of this phenomenon is therefore considered a promising biomarker approach. To date, however, it is unclear whether routinely collected tissue specimens allow determination of in-vivo phosphorylation states. Methods and results: To investigate whether routinely collected tissue specimens retain the true phosphorylation states of a tumour's proteins, we compared protein phosphorylation states between matched tumour samples that were subjected to different ischaemic times by immunohistochemistry. The influence of formalin fixation and paraffin-embedding on phosphorylation states was investigated by comparison of matched fresh frozen and formalin-fixed paraffin-embedded surgical specimens as well as small biopsies. We show that ischaemia influences protein phosphorylation in a tumour-specific, unpredictable manner. Formalin fixation and paraffin-embedding lead to a decrease in detectable protein phosphorylation in larger surgical specimens, but not in small biopsies. Conclusions: Determination of protein phosphorylation using routinely collected surgical specimens results in artefacts which do not reflect a tumour's true states of pathway activation. Valid measurement of phosphorylated biomarkers requires that tissue collection procedures are tightly controlled, avoiding ischaemia and large-specimen fixation. © 2013 John Wiley & Sons Ltd.


PubMed | NCI Inc, Indivumed GMBH, OBP DBRR III and Facility for Biotechnology Resources
Type: Journal Article | Journal: Clinical cancer research : an official journal of the American Association for Cancer Research | Year: 2016

To gain insight into factors involved in tumor progression and metastasis, we examined the role of noncoding RNAs in the biologic characteristics of colorectal carcinoma, in paired samples of tumor together with normal mucosa from the same colorectal carcinoma patient. The tumor and healthy tissue samples were collected and stored under stringent conditions, thereby minimizing warm ischemic time.We focused particularly on distinctions among high-stage tumors and tumors with known metastases, performing RNA-Seq analysis that quantifies transcript abundance and identifies novel transcripts.In comparing 35 colorectal carcinomas, including 9 metastatic tumors (metastases to lymph nodes and lymphatic vessels), with their matched healthy control mucosa, we found a distinct signature of mitochondrial transfer RNAs (MT-tRNA) and small nucleolar RNAs (snoRNA) for metastatic and high-stage colorectal carcinoma. We also found the following: (i) MT-TF (phenylalanine) and snord12B expression correlated with a substantial number of miRNAs and mRNAs in 14 colorectal carcinomas examined; (ii) an miRNA signature of oxidative stress, hypoxia, and a shift to glycolytic metabolism in 14 colorectal carcinomas, regardless of grade and stage; and (iii) heterogeneous MT-tRNA/snoRNA fingerprints for 35 pairs.These findings could potentially assist in more accurate and predictive staging of colorectal carcinoma, including identification of those colorectal carcinomas likely to metastasize.


PubMed | Indivumed GmbH, U.S. National Institutes of Health and Arizona State University
Type: | Journal: Journal of translational medicine | Year: 2016

Clinical diagnostic research relies upon the collection of tissue samples, and for those samples to be representative of the in vivo situation. Tissue collection procedures, including post-operative ischemia, can impact the molecular profile of the tissue at the genetic and proteomic level. Understanding the influence of factors such as ischemia on tissue samples is imperative in order to develop both markers of tissue quality and ultimately accurate diagnostic tests.Using NanoPro1000 technology, a rapid and highly sensitive immunoassay platform, the phosphorylation status of clinically relevant cancer-related biomarkers in response to ischemia was quantified in tissue samples from 20 patients with primary colorectal cancer. Tumor tissue and adjacent normal tissue samples were collected and subjected to cold ischemia prior to nanoproteomic analysis of AKT, ERK1/2, MEK1/2, and c-MET. Ischemia-induced relative changes in overall phosphorylation and phosphorylation of individual isoforms were calculated and statistical significance determined. Any differences in baseline levels of phosphorylation between tumor tissue and normal tissue were also analyzed.Changes in overall phosphorylation of the selected proteins in response to ischemia revealed minor variations in both normal and tumor tissue; however, significant changes were identified in the phosphorylation of individual isoforms. In normal tissue post-operative ischemia, phosphorylation was increased in two AKT isoforms, two ERK1/2 isoforms, and one MEK1/2 isoform and decreased in one MEK1/2 isoform and one c-MET isoform. Following ischemia in tumor tissue, one AKT isoform showed decreased phosphorylation and there was an overall increase in unphosphorylated ERK1/2, whereas an increase in the phosphorylation of two MEK1/2 isoforms was observed. There were no changes in c-MET phosphorylation in tumor tissue.This study provides insight into the influence of post-operative ischemia on tissue sample biology, which may inform the future development of markers of tissue quality and assist in the development of diagnostic tests.


Shivapurkar N.,Georgetown University | Weiner L.M.,Georgetown University | Marshall J.L.,Georgetown University | Madhavan S.,Georgetown University | And 4 more authors.
PLoS ONE | Year: 2014

Systemic treatment of patients with early-stage cancers attempts to eradicate occult metastatic disease to prevent recurrence and increased morbidity. However, prediction of recurrence from an analysis of the primary tumor is limited because disseminated cancer cells only represent a small subset of the primary lesion. Here we analyze the expression of circulating microRNAs (miRs) in serum obtained pre-surgically from patients with early stage colorectal cancers. Groups of five patients with and without disease recurrence were used to identify an informative panel of circulating miRs using quantitative PCR of genome-wide miR expression as well as a set of published candidate miRs. A panel of six informative miRs (miR-15a, mir-103, miR-148a, miR-320a, miR-451, miR-596) was derived from this analysis and evaluated in a separate validation set of thirty patients. Hierarchical clustering of the expression levels of these six circulating miRs and Kaplan-Meier analysis showed that the risk of disease recurrence of early stage colon cancer can be predicted by this panel of miRs that are measurable in the circulation at the time of diagnosis (P = 0.0026; Hazard Ratio 5.4; 95% CI of 1.9 to 15). © 2014 Shivapurkar et al.


Juhl H.,Indivumed GmbH
Scandinavian Journal of Clinical and Laboratory Investigation | Year: 2010

Considerable research data is available that demonstrate that tissues are under tremendous biological stress when surgically separated from the body. This stress significantly changes gene and protein expression profiles, including activation or inhibition of signalling pathways and their receptors. Many of those are possibly involved in growth regulation and might serve as targets or stratification markers for new drugs. Factors that affect tissue dependent cancer research for target discovery and drug development include drug treatment and anesthesia of patients before surgical tissue removal, intrasurgical ischemia by ligation of main arteries, "cold" ischemia, i.e. the time interval between surgical removal and fixation of tissue, location of tumor biopsy within a given tumor, processing of tissue and fixation protocols and the availability of comprehensive clinical data. Controlled and rapid tissue processing is a prerequisite for understanding biological differences of patient tumors and to utilize these findings (e.g., cancer pathway activity) for targeted molecular therapies. © 2010 Informa UK Ltd.


PubMed | Indivumed GmbH
Type: Journal Article | Journal: Journal of clinical oncology : official journal of the American Society of Clinical Oncology | Year: 2016

22139 Background: The discovery of new anticancer drugs is currently performed by using common cancer cell lines. However, it has been shown that long term cultured cell lines are limited in forecasting in vivo drug sensitivities. Therefore, we developed a standardized procedure to isolate pure epithelial cells directly from gastrointestinal tumor tissues and used them to examine chemotherapeutic effects of various cancer agents.Primary epithelial cells were freshly derived from adenocarcinoma patients, cultivated for 0, 24, 48 and 72 hours and either treated with various single tumor therapeutics such as Oxaliplatin, Gemcitabine, Iressa and 5-FU or by using drug combinations (Leucovorin/5-FU; FOLFOX; IFOX; and GOLF). Drug responses were analysed by immunofluorescence, ATP measurement and electrochemiluminescence and compared to either similarly treated HT29 cells or short and long term cultivated primary cells.Application of the GOLF regiment showed in all cell cultures the strongest effect on cell proliferation and metabolism. We observed that compared to the single drug treatments, the different drugs composing GOLF were highly synergistic in inducing growth inhibition.The GOLF combination induced apoptosis in all colorectal cancer cells as measured by caspase-3 activation, PARP cleavage, as well as increased levels of total and phosphorylated p53. Since apoptosis onset in eukaryotic cells is substantially regulated by the balance between apoptotic and survival pathways the effects of the different drug treatments on expression and activity of the components of the Erk and Akt dependent pathways were also evaluated. In all cell cultures the induction of apoptosis due to GOLF treatment was paralleled by increased levels of total as well as phosphorylated ERK, whereas the expression and activation of EGFR, MEK, P70s6K, GSK3 and especially AKT were decreased. All these effects were also seen in cells treated with the single drugs or combinations thereof although less pronounced.Primary cells from gastrointestinal patients could provide a cellular platform beyond the common cancer cell lines for in vitro drug screening and might allow individualization of chemotherapies for patient treatments. No significant financial relationships to disclose.


Correlative studies have identified numerous biomarkers that are individualizing therapy across many medical specialties, including oncology. Accurate interpretation of these studies requires the collection of tissue samples of sufficient quality. Tissue quality can be measured by changes in levels of gene expression and can be influenced by many factors including pre-analytical conditions, ischemic effects and the surgical collection procedure itself. However, as yet there are no reliable biomarkers of tissue quality at researchers disposal. The aim of the current study was to identify genes with expression patterns that fluctuated reproducibly in response to typical post-surgical stress (ischemia) in order to identify a specific marker of tissue quality. All tissue samples were obtained from patients with primary colorectal carcinoma (CRC) (N = 40) either via colonoscopy prior to surgery, or by surgical resection. Surgically resected tissue samples were divided into three groups and subjected to cold ischemia for 10, 20 or 45 minutes. Normal colorectal tissue and CRC tissue was analyzed using microarray and quantitative real-time PCR (qPCR). Comparing changes in gene expression between pre- and post-surgical tissue using microarray analysis identified a list of potential tissue quality biomarkers and this list was validated using qPCR. Results revealed that post-operative ischemia significantly alters gene expression in normal and CRC tissue samples. Both microarray analysis and qPCR revealed regulator of G-protein signaling 1 (RGS1) as a potential marker of CRC tissue quality and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) as a potential reference gene of post-operative tissue quality. Larger studies with additional time points and endpoints will be needed to confirm these results.

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