Search

Background: Subtyping of breast cancer has become an integral part of standard evaluation of breast cancer patients1. However, assaying of ER, PgR and Her-2/neu by immunohistochemistry (IHC) carries an up to 20% risk of erroneous results2,3. Moreover, reliable assessment of Ki-67 by IHC in grade 2 breast cancer is challenging due to high intra- and interobserver variations4,5. Interobserver concordance for ESR1, PGR, HER2, and KI67 determination on mRNA level using MammaTyper® reagents were 96.8%, 97.2%, 100%, 97.6%, respectively, based on predefined cutoffs (Laible et al., abstract submitted). Here we tested the clinical concordance and prognostic value of MammaTyper® determinations in the FinHer trial patient population. Methods: RNA was extracted from formalin-fixed paraffin-embedded (FFPE) breast tumor tissue, and candidate gene expression was analyzed using the RNXtract® IVD and MammaTyper® IVD kits (BioNTech Diagnostics GmbH, Mainz) from 791 patients who participated in the FinHer trial. RNA levels of ESR1, PGR, HER2, and KI67 mRNA expression were measured using RT-qPCR,normalized according to the 40-DDCT method and compared with IHC or CISH results. Associations with distant disease-free survival (DDFS) and overall survival (OS) were assessed using the log-rank test. Results: ESR1, PGR, HER2, and KI67 mRNA levels exhibited strong correlations with the respective clinical assays (each p-value 95%). Conclusions: Determination of ESR1, PGR, HER2, and KI67 without macrodissection of routine whole tissue FFPE specimens results in highly concordant results when compared to clinical assays. A significant minority of HER2 negative breast cancers expressed PGR mRNA despite low ESR1 mRNA levels and had superior outcome. HER2 mRNA levels differed substantially between ER-positive and ER negative tumors. This may explain why HER2 determination using a single cut-off for HER2 mRNA with the Oncotype DX assay frequently results in a false negative finding6. The MammaTyper-defined ESR1, PGR, HER2, and KI67 expression showed strong correlations with the corresponding clinical assays and survival. 1) Goldhirsch et al., Annals of Oncology 2013 2) Hammond et al., JCO 2010 3) Wolff et al., JCO 2007 4) Varga et al., PloS 2012 5) Polley et al., JNCI 2013. Citation Format: Ralph M Wirtz, Pirkko-Liisa Kellokumpu-Lehtinen, Jorma Isola, Vesa Kataja, Petri Bono, Taina Turpeenniemi-Hujanen, Sirkuu Jyrkkiö, Harri Sitho, Sebastian Eidt, Ugur Sahin, Heikki Joensuu. Comparison of ESR1, PGR, HER2 and KI67 expression by central IHC and MammaTyper® RT-qPCR kit in the FinHer trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-02-01.

Contains fulltext : 48736.pdf ( ) (Open Access) BACKGROUND: The molecular determinants of carcinogenesis, tumor progression and patient prognosis can be deduced from simultaneous comparison of thousands of genes by microarray analysis. However, the presence of stroma cells in surgically excised carcinoma tissues might obscure the tumor cell-specific gene expression profiles of these samples. To circumvent this complication, laser microdissection can be performed to separate tumor epithelium from the surrounding stroma and healthy tissue. In this report, we compared RNAs isolated from macrodissected, of which only surrounding healthy tissue had been removed, and microdissected rectal carcinoma samples by microarray analysis in order to determine the most reliable approach to detect the expression of tumor cell-derived genes by microarray analysis. RESULTS: As microdissection yielded low tissue and RNA quantities, extra rounds of mRNA amplification were necessary to obtain sufficient RNA for microarray experiments. These second rounds of amplification influenced the gene expression profiles. Moreover, the presence of stroma cells in macrodissected samples had a minor contribution to the tumor cell gene expression profiles, which can be explained by the observation that more RNA is extracted from tumor epithelial cells than from stroma. CONCLUSION: These data demonstrate that the more convenient procedure of macrodissection can be adequately used and yields reliable data regarding the identification of tumor cell-specific gene expression profiles.

We previously reported the development of a mill based instrument for dissection of slide-mounted tissue sections. This instrument employs a specialized disposable mill bit that simultaneously dispenses liquid, cuts tissue from the slide surface using rotary motion, and aspirates the liquid along with the displaced tissue fragments. An accompanying software package is capable of transferring digital annotations between images of serially cut tissue sections to guide dissection and generate an electronic record of the process. Since the original report, a new instrument and software package has been developed with a computer controlled stage for automatic dissection of tissue. Here we report three applications using this mesodissection system. The first application investigated the relationship between dissection resolution and percent tumor tissue recovered. Areas of high tumor content were indicated on digital images of lung tumor samples. The areas of highest concentration were dissected at 400 micron resolution, the remaining areas of tumor dissected at 800 micron resolution, and then surrounding areas that would typically be recovered using manual macrodissection were dissected at 2 mm resolution. The percent mutant DNA, determined using the Sequenom OncoCarta panel, was greater than 60% for the 400 micron resolution dissections, about 45% for the 800 micron resolution dissections, and less than 10% for the remaining regions. The second application investigated the effect of various dissection related parameters on expression analysis, as assayed by Reverse Transcription and Quantitative PCR (RT-qPCR). These parameters included placing freshly microtomed FFPE tissue sections directly on slides vs. first floating on water, dissection of deparaffinized vs. paraffinized tissue, the use of crude lysates vs. purified RNA for the reverse transcription reactions, and manual dissection vs. mesodissection of the same areas on serial sections. RT-qPCR results were robust and equivalent for all parameters except those derived from crude lysates, which were equivalent to the no template controls. The third application investigated if mesodissected tissue fragments could be re-adhered to glass slides and used for FISH analysis. This application has the potential to automate and increase the throughput of FISH on tissue sections while reducing sample consumption. The preliminary results demonstrate that tissue fragments can produce good FISH signals, but a more detailed comparison to undissected tissue sections is ongoing. A significant concern is the loss of context of these fragments. This concern can be somewhat minimized by adjusting dissection conditions to obtain fragments larger than 1 mm in diameter and by mapping the dissected area onto the corresponding area a serial H&E stained tissue section using the mesodissection software. Citation Format: Nils Adey, Dale Emery, Derek Bosh, Robert Parry. Mesodissection of slide mounted tissue: Applications including tumor tissue enrichment, expression analysis, and FISH on tissue fragments. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3472. doi:10.1158/1538-7445.AM2014-3472

Background: Mutations of the PIK3CA gene encoding for the p110α catalytic subunit of Phosphatidylinositol 3-kinases (PI3K) are found in approximately 25% of breast carcinomas. Most of PIK3CA mutations are reported as activators of the PI3K/AKT/mTOR pathway and lead to resistance to anti-HER2 therapies, and have an interest in molecular diagnosis for anti-mTOR and/or anti-PI3K therapies. Highly sensitive methods are required to detect PIK3CA mutations in paraffin-embedded tumor tissues. Methods: Forty-seven formalin-fixed paraffin embedded breast carcinomas tissues samples were assessed using two allele specific assays (Cobas® PIK3CA Mutation Test and PCR ARMS Scorpions®) and one non-specific real-time high resolution melting PCR assay (HRM) after macrodissection and DNA extraction. Cobas® allow the detection of 17 mutations on exons 1, 4, 7, 9 and 20 of PIK3CA, ARMS focuses on the detection on the four common hotspots (E542K, E545K/D, H1047R and H1047L) representing 90% of PIK3CA mutations and HRM allows the detection of all mutations located on the whole exons 9 and 20 of PIK3CA. Kappa statistics were used to compare the three assays. Results: Among the 47 samples, 18 (38.3%) were found to bear a PIK3CA mutation with Cobas®, 13 (27.7%) with ARMS and 19 (40.4%) with HRM. One sample was found to bear 3 mutations of PIK3CA (E542K, H1047X and H1049R). Calculated kappa were 0.893 (p Conclusion: Cobas® and HRM assays allowed to detect more extensively PIK3CA mutations than ARMS assay. ARMS assay only focusing on the 4 hotspots PIK3CA mutations seems to be inadequate with routine use since generating false negative PIK3CA wild-type results. Cobas® and HRM assays are significantly comparable for the detection of PIK3CA mutations, but Cobas® enables the characterization of the mutation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C38. Citation Format: Alexandre Harlé, Maëva Lion, Marie Rouyer, Nicola Lozano, Jean-Louis Merlin. Comparative evaluation of three real-time PCR assays for the detection of PIK3CA somatic mutations in formalin-fixed paraffin embedded breast carcinomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C38.

Background - aim: Translational research studies increasingly employ routine histologic material (FFPE) for the investigation of gene expression with extract-based methods, like quantitative real time PCR (QPCR). Statistically significant differences in the expression levels of mRNA transcripts in tumor vs neighbouring non-tumor tissues have led to the consensus of including as high as possible tumor cell rates in the extract. Herein, we investigated whether the same mRNA markers yield different clinically relevant values when examined in macrodissected (MD) and non-macrodissected (NMD) FFPE breast carcinoma tissues. Methods: Matched NMD and MD samples from primary tumors (P, n=98 sample pairs) and from metastatic lymph nodes (LN, n=72, overlapping but not matching P samples) were recruited from a cohort of 357 early high-risk breast cancer patients that had been adjuvantly treated with epirubicin-CMF regimens with or without paclitaxel. FFPE sections were evaluated for tumor cell content (TCC) and were used as whole sections and upon manual macrodissection for the same tumor. All samples were assessed for ESR1, ERBB2, MAPT, MMP7 and RACGAP1 mRNA expression with QPCR, and the obtained relative quantification (RQ) values were compared as continuous variables and upon hierarchical clustering in matched sample series with respect to patient outcome. Results: Mean, median (±SD) values for TCC% in P-NMD / P-MD samples were 27.0, 25.0 (±14.8) / 68.1, 67.5 (±20.5) and in LN-NMD / LN-MD samples 30.1, 35.0 (±18.0) / 82.0, 90.0 (±18.2), respectively. Minimum TCC% was 2.5 in P-NMD and in LN-NMD samples, 35.0 in P-MD and 27.5 in LN-MD. In comparison to matched NMD samples, mRNA expression of ESR1 (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5554. doi:1538-7445.AM2012-5554

Electrical resistance of the skin and subcutaneous tissue is one of the major problems of neurophysiological facial nerve testing. The tests are sometimes abandoned due to the pain and stimulating initial artifacts. To overcome this disadvantage, the authors have found that the facial nerve can be stimulated with far less current through Stensen’s duct.

Background Gastric cancer samples obtained by histologic macrodissection contain a relatively high stromal content that may significantly influence gene expression profiles. Differences between the gene expression signature derived from macrodissected gastric cancer samples and the signature obtained from isolated gastric cancer epithelial cells from the same biopsies using laser-capture microdissection (LCM) were evaluated for their potential experimental biases. Methods RNA was isolated from frozen tissue samples of gastric cancer biopsies from 20 patients using both histologic macrodissection and LCM techniques. RNA from LCM was subject to an additional round of T7 RNA amplification. Expression profiling was performed using Affymetrix HG-U133A arrays. Genes identified in the expression signatures from each tissue processing method were compared to the set of genes contained within chromosomal regions found to harbor copy number aberrations in the tumor samples by array CGH and to proteins previously identified as being overexpressed in gastric cancer. Results Genes shown to have increased copy number in gastric cancer were also found to be overexpressed in samples obtained by macrodissection (LS P value < 10-5), but not in array data generated using microdissection. A set of 58 previously identified genes overexpressed in gastric cancer was also enriched in the gene signature identified by macrodissection (LS P < 10-5), but not in the signature identified by microdissection (LS P = 0.013). In contrast, 66 genes previously reported to be underexpressed in gastric cancer were enriched in the gene signature identified by microdissection (LS P < 10-5), but not in the signature identified by macrodissection (LS P = 0.89). Conclusions The tumor sampling technique biases the microarray results. LCM may be a more sensitive collection and processing method for the identification of potential tumor suppressor gene candidates in gastric cancer using expression profiling.

4002 Background: We compared the incidence of molecular markers in stage II (SII) and III (SIII) colon cancer and tested their prognostic value per stage, using PETACC 3, an adjuvant trial with 3,278 patients. We included expression of P53, SMAD4, thymidylate synthetase (TS) and hTERT, mutations of KRAS and BRAF, microsatellite instability (MSI) and 18qLOH. Methods: 1,564 formalin fixed paraffin embedded tissue blocks were prospectively collected and DNA from normal and tumor tissue was extracted after macrodissection. High P53, TS and hTERT expression and SMAD4 loss were assessed by immunohistochemistry. MSI was studied with 10 markers. KRAS exon 2 and BRAF exon 15 mutations were analyzed by allele specific real time PCR. 18qLOH was studied by pyrosequencing 7 SNPs. Prognostic value of the markers was analysed per stage by Cox regression for Relapse Free Survival (RFS). Results: marker frequencies and stage specific p-values in prognostic models in 420 SII and 984 SIII patients are listed in the table . Significant differences in frequency per stage were found for all markers except KRAS and BRAF. An interaction test for differences between marker prognostic value for SII and SIII was significant for MSI (p=0.04) and 18qLOH (p=0.04) in SII. Multivariate analysis including markers, T stage, N stage (for SIII), Tu grade, age [Table: see text] [Table: see text]