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Amplicon-based next generation sequencing (NGS) approaches have been preferentially adopted by the clinical laboratories on the basis of a short turnaround time (TAT) and small DNA input needs. However, little work has been done to assess the amplicon-based NGS methods for copy number variation (CNV) detection in comparison with current standard methods like immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The correlation between NGS based CNV detection and the later standard methods has remained unexplored. We developed an amplicon-based panel to detect human epidermal receptor growth factor (HER2) amplification in formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 280 breast cancer and 50 gastric cancer patients. Assessment by IHC and FISH was conducted in parallel, and descriptive statistics were used to assess the concordance. The copy number detected by NGS was correlated with either the average HER2 copy number (signals/cell) (r = 0.844; p

Gene fusions play an important role in the carcinogenesis of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for precision medicine. We used formalin‐fixed, paraffin‐embedded tissue samples of non‐small cell lung cancer from 150 EGFR mutation‐negative cases and 10 fusion status‐known cases and compared the performance of the Oncomine Dx Fusion Transcript Test (ODxFT) with FISH break‐apart for the detection of ALK, RET, and ROS1 fusion genes. RNA was extracted from the paraffin‐embedded tissue samples with or without macrodissection under hematoxylin and eosin staining, and the ALK fusion gene was independently determined using these assays. Fusion detection analyses were successfully carried out using ODxFT in 150 cases, with only one invalid case. ALK fusion genes were detected at a frequency of 7.3% (11/150) in the lung cancer specimens. Concordance rate between the ODxFT and ALK‐FISH analyses was 99.3% (148/149). Sensitivity and specificity were 91.7% and 99.3%, respectively. All the samples with a known fusion status were accurately matched between the two assays. Our results show a high concordance rate between the ODxFT and ALK‐FISH analyses. ODxFT was thus validated as an effective method for detecting clinically significant ALK fusion genes in paraffin‐embedded tissue samples.

Background The advent of precision oncology has led a shift towards biomarker-driven clinical trial designs and molecular profiling of individual patients. Identification of patients with the target biomarker profile may be useful in guiding patient selection as an enrichment strategy for clinical trials. Targeted multiple reaction monitoring mass spectrometry (MRM-MS) analysis for multiplexed quantitation of biomarker proteins in FFPE tissue provides direct, more accurate and precise quantification over current ‘gold standard’ immunohistochemistry (IHC) methods. However, MRM-MS has not yet been broadly applied to clinical trials. In this study, we demonstrate the systematic development, optimization and analytical validation of quantitative, multiplexed MRM-MS assays for robust biomarker quantification in clinical FFPE tissues, including sample analysis under GCLP. Results from an MRM panel targeting 8 clinically relevant biomarker proteins will also be shown, including the measured HER2 levels in FFPE breast tumors classified by IHC as 0, 1+, 2+ or 3+. Methods MRM-MS biomarker panels were developed and optimized for multiplexed quantitation of ≤12 proteins, in which unique peptides derived from each target protein were monitored as a surrogate measure of protein levels. Tumor regions from FFPE tissue sections were dissected using laser capture or macrodissection, solubilized, digested with trypsin to generate peptides for analysis, spiked with fixed levels of stable isotope labeled (SIL) peptide standards, and analyzed by MRM-MS. Analytical validation was performed per NCI CPTAC guidelines, including response curves, assay repeatability, selectivity, stability, and reproducibility of endogenous detection. Clinical performance was assessed using commercially sourced FFPE-tumor tissues, including a cohort of breast tumor tissues with a wide range of HER2 expression. Results Assay performance results were protein/peptide dependent, with sensitivity in the low pg/µg total protein range. For HER2, assay linearity was demonstrated over 2.5 to 3 orders of magnitude, with a precision and accuracy of Conclusions GCLP-compliant quantitative multiplexed large-scale clinical analysis of protein biomarkers by MRM-MS in FFPE tissue is feasible and enables precise and accurate quantitation of proteins when IHC methods are unsuitable or unavailable. Data can be used for patient stratification, optimization of treatment outcomes, drug resistance prediction, and to support clinical development of novel therapies.

This study aimed to compare the performance of MilliSect dissection and manual dissection. Twenty-five formalin-fixed paraffin-embedded (FFPE) breast cancer tissue blocks were selected for comparison. Specific areas of interest (AOIs) in invasive carcinoma on tissue sections were transferred to dissection slides by manual macrodissection or the MilliSect instrument. The comparison criteria were 1) the time required for dissection; 2) RNA concentration and purity; 3) RNA quantity of 5 housekeeping genes (by RT-qPCR); and 4) ER, PR, HER2, Ki-67 and recurrence score (RS) values (by the 21-gene assay). Then, tumor-adjacent tissues, including fibrocollagenous and epithelial tissues, from the same selected tissue blocks of 8 of 25 patients were scraped using the mesodissection method, and their RS values were assessed to evaluate the influence of tumor-adjacent tissues on the target AOIs. Ultimately, 4 AOIs of invasive ductal carcinoma (IDC) from 1 tissue block of another 4 patients with lymph node (LN) metastases each, LN tissue and a mixture of IDC and LN tissue from the other tissue block of the same 4 patients were mesodissected to evaluate the influence of infiltrating lymphocyte levels on the RS values of AOIs. In our experience, the MilliSect instrument, which provides process management documentation, required more time than manual macrodissection (on average, approximately 9.1 min per sample versus 5.8 min per sample, respectively). The RNA yield and quality of the dissected tissues were comparable for the 2 methods. However, the tumor-adjacent tissues of the AOIs may influence the RS to some extent. Tumor-infiltrating lymphocytes (TILs) can dramatically increase RSs, far exceeding the influence of tumor-adjacent fibrocollagenous and epithelial tissues. In conclusion, MilliSect mesodissection is comparable to manual dissection. This mesodissection tool may facilitate AOI alignment and the dissection process for the 21-gene RS assay. Samples whose adjacent tissues are intermixed with TILs warrant special attention.

Prognostic genomic biomarkers that can be measured at diagnosis to aid choice of treatment options are unavailable for most common cancers. This is due in part to the poor quality and quantity of available diagnostic specimens for discovery research and to limitations in genomic technologies. Recent technical advances now enable high-density molecular analyses using suboptimal biological specimens. Here we describe the optimization of a transcriptome-specific protocol for use with formalin-fixed, paraffin-embedded (FFPE) diagnostic prostate cancer (PrCa) specimens. We applied the Ion AmpliSeq Transcriptome Human Gene Expression Kit (AmpliSeq Kit) to RNA samples extracted from 36 tumor-enriched and 16 adjacent normal tissues (ADJNT) from 37 FFPE PrCa specimens over a series of eight pilot studies, incorporating protocol modifications from Pilots 2 to 5. Data quality were measured by (1) the total number of mapped reads; (2) the percentage of reads that mapped to AmpliSeq target regions (OnTarget%); (3) the percentage of genes on the AmpliSeq panel with a read count ≥10 (TargetsDetected%); and (4) comparing the gene read-count distribution of the prostate tissue samples with the median gene read-count distribution of cell line-derived RNA samples. Modifications incorporated into Pilot study 5 provided gene expression data equivalent to cell line-derived RNA samples. These modifications included the use of freshly cut slides for macrodissection; increased tissue section thickness (8 µm); RNA extraction using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (ThermoFisher); 18 target amplification cycles; and processing six samples per Ion PI chip. This protocol will facilitate the discovery of prognostic biomarkers for cancer by allowing researchers to exploit previously underutilized diagnostic FFPE specimens.

Simple Summary The PAM50 risk of recurrence (ROR) score is predictive of the risk of distant recurrence and the benefits of adjuvant therapy in early breast cancer. The Prosigna assay utilizes RNA from tumor cells selected via macrodissection of formalin-fixed, paraffin-embedded (FFPE) tissue sections to measure the activity of the PAM50 genes. An alternative and widely used extraction method is RNA purification from fresh-frozen (FF) bulk tissue without enriching the tumor cellularity. However, the impact of the RNA preparation approach on ROR scores and subsequent treatment selection has not been systematically evaluated. We compared the different approaches and found high correlation between risk of recurrence scores estimated from macrodissected FFPE tissue and scores estimated from bulk FF tumor tissue. However, important discrepancies were revealed for luminal tumors, which may have consequences for treatment recommendations for these patients. Abstract The PAM50 gene expression subtypes and the associated risk of recurrence (ROR) score are used to predict the risk of recurrence and the benefits of adjuvant therapy in early-stage breast cancer. The Prosigna assay includes the PAM50 subtypes along with their clinicopathological features, and is approved for treatment recommendations for adjuvant hormonal therapy and chemotherapy in hormone-receptor-positive early breast cancer. The Prosigna test utilizes RNA extracted from macrodissected tumor cells obtained from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, RNA extracted from fresh-frozen (FF) bulk tissue without macrodissection is widely used for research purposes, and yields high-quality RNA for downstream analyses. To investigate the impact of the sample preparation approach on ROR scores, we analyzed 94 breast carcinomas included in an observational study that had available gene expression data from macrodissected FFPE tissue and FF bulk tumor tissue, along with the clinically approved Prosigna scores for the node-negative, hormone-receptor-positive, HER2-negative cases (n = 54). ROR scores were calculated in R; the resulting two sets of scores from FFPE and FF samples were compared, and treatment recommendations were evaluated. Overall, ROR scores calculated based on the macrodissected FFPE tissue were consistent with the Prosigna scores. However, analyses from bulk tissue yielded a higher proportion of cases classified as normal-like; these were samples with relatively low tumor cellularity, leading to lower ROR scores. When comparing ROR scores (low, intermediate, and high), discordant cases between the two preparation approaches were revealed among the luminal tumors; the recommended treatment would have changed in a minority of cases.

Breast cancer care in the developing world is limited by access to quality ER and HER2 IHC diagnostic assays needed to justify hormone and HER2 therapeutics. Shipping pathology specimens to a central testing site often out of country delays therapy and is costly. The Xpert Breast Cancer Stratifier assay makes quantitative measurements of ESR1, PGR, ERBB2, and MKi67 mRNAs from FFPE specimens in 83 breast tumor samples were chosen including those with low cellularity, small volume disease, unusual subtypes, ER- tumors with surrounding benign epithelium, and low level HER2+ tumors. mRNA, IHC and FISH assays were performed. Slides were tested following macrodissection of invasive carcinoma and as non-macrodissected whole sections. GX measurements for Ki67 were compared with mitotic rate as an alternative to Ki67 IHC. Overall percent agreement following macrodissection was 95% for ER, 89% for HER2, 76% for PR, and 80% for Ki67 (>20% positive cut), and using whole section, 99% for ER, 80% for PR, 92% for HER2, and 73% for Ki67. Concordance was 92% for both macrodissection and whole section using mitotic rate to assess proliferation. Ignoring HER2 2+ calls which represented low level amplified tumors by FISH, the concordance rates were 95% for macrodissection and 99% for whole section. Discordance when testing long-term stored 4μm sections was resolved in a number of cases by using a fresh cut from the FFPE block. Half the ER discrepancies were in very small volume tumors ≤25mm2 and 75% were classified as ER-ve by IHC, and positive by Stratifier. 80% of ER IHC- cases were appropriately identified as ER- by the Stratifier in the presence of benign breast epithelium. HER2+ DCIS adjacent to HER2- invasive tumor resulted in a discrepant HER2 mRNA result even with macrodissection. No ER or HER2 discrepancies occurred in low cellularity tumors (≤30% cellularity) nor in lobular and mucinous subtypes. In a study intended to challenge an mRNA breast biomarker assay, concordance between mRNA results and IHC was high for ER and HER2, the two most important prognostic markers needed for therapeutic decision making. Use of whole sections rather than tumor macrodissection did not decrease concordance. Discrepant ER cases were more prevalent when analyzing low volumes of tumor and in this setting were seen in ER IHC- tumors surrounded by ER+ normal epithelium, or with weak IHC expression, highlighting predictable limitations of the assay. Concordance was better between Ki67 mRNA and mitotic rate than with IHC. Re-test data suggested that a fresh cut of the FFPE block yields the best results by GX, perhaps due to mRNA degradation in stored 4μm sections. The Xpert Breast Cancer Stratifier may provide a rapid, cost-effective solution to the problem of obtaining accurate diagnostic results at the point-of-care in low resource settings, and deserves further evaluation in developing countries. Citation Format: Brock JE, Milner DA, Ho K, Natalie W, Victor C, Annaliza R, Teresa B, Kathryn G-F, Edwin LW, Jodi W, Wendy W, Michael B. Comparison of the Xpert breast cancer stratifier mRNA assay with central ER, PR, HER2, and Ki67 immunohistochemistry (IHC) for rapid biomarker analysis in developing countries [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-07.

Background The discovery of novel biomarkers that predict treatment response in advanced cancer patients requires acquisition of high-quality tumor samples. As cancer evolves over time, tissue is ideally obtained before the start of each treatment. Preferably, samples are freshly frozen to allow analysis by next-generation DNA/RNA sequencing (NGS) but also for making other emerging systematic techniques such as proteomics and metabolomics possible. Here, we describe the first 469 image-guided biopsies collected in a large collaboration in The Netherlands (Center for Personalized Cancer Treatment) and show the utility of these specimens for NGS analysis. Patients and Methods Image-guided tumor biopsies were performed in advanced cancer patients. Samples were fresh frozen, vital tumor cellularity was estimated, and DNA was isolated after macrodissection of tumor-rich areas. Safety of the image-guided biopsy procedures was assessed by reporting of serious adverse events within 14 days after the biopsy procedure. Results Biopsy procedures were generally well tolerated. Major complications occurred in 2.1%, most frequently consisting of pain. In 7.3% of the percutaneous lung biopsies, pneumothorax requiring drainage occurred. The majority of samples (81%) contained a vital tumor percentage of at least 30%, from which at least 500 ng DNA could be isolated in 91%. Given our preset criteria, 74% of samples were of sufficient quality for biomarker discovery. The NGS results in this cohort were in line with those in other groups. Conclusion Image-guided biopsy procedures for biomarker discovery to enable personalized cancer treatment are safe and feasible and yield a highly valuable biobank.

To enable the widespread application of genomic medicine, the extraction of genomic DNA from thin sections of archived formalin-fixed and paraffin-embedded (FFPE) tissue blocks for next-generation sequencing (NGS) is often necessary. However, there are currently no guidelines available on which specific regions of the microtome sections to use for macrodissection with respect to the histopathological factors observed under microscopic examination. The aim of this study was to clarify the relationship between histopathological factors and DNA quality, and to standardize the macrodissection method for more efficient implementation of NGS. FFPE tissue specimens of 218 patients from the Biomarker Research for Anti-EGFR Monoclonal Antibodies by Comprehensive Cancer Genomics study were used to investigate the relationship between 15 histopathological factors and the quantitative ratio of double-stranded DNA (dsDNA) to total nucleic acids, as well as the ∆ crossing point value of each tissue specimen. Multivariate logistic regression analysis revealed that specimen storage of ≥3 years was negatively associated with dsDNA quality (P=0.0007, OR: 4.30, 95% CI: 1.85-10.04). In contrast, the presence of a mucus pool was positively associated with dsDNA quality (P=0.0308, OR: 0.23, 95% CI: 0.06-0.87). Metastatic tumors and longer specimen storage periods were significantly associated with lower ∆Cp values (P=0.0007, OR: 4.43, 95% CI: 1.87-10.49; and P=0.0003, OR: 5.51, 95% CI: 2.18-13.95, respectively). Therefore, macrodissection should not be performed on specimens exhibiting histopathological factors associated with poor DNA quality. In particular, the use of tissue blocks with a storage period of

// Matthias Christgen 1 , Jana L. van Luttikhuizen 2 , Mieke Raap 1 , Peter Braubach 1 , Lars Schmidt 1 , Danny Jonigk 1 , Friedrich Feuerhake 1 , Ulrich Lehmann 1 , Brigitte Schlegelberger 2 , Hans H. Kreipe 1 , Doris Steinemann 2 1 Institute of Pathology, Hannover Medical School, Hannover, Germany 2 Institute of Human Genetics, Hannover Medical School, Hannover, Germany Correspondence to: Doris Steinemann, email: Steinemann.Doris@MH-Hannover.de Keywords: HER2/ ERBB2 , OncoScan, breast cancer, copy number profiling, next generation sequencing Received: July 02, 2016 Accepted: September 19, 2016 Published: October 03, 2016 ABSTRACT HER2 /ERBB2 amplification/overexpression determines the eligibility of breast cancer patients to HER2-targeted therapy. This study evaluates the agreement between ERBB2 copy number assessment by fluorescence in situ hybridization, a standard method recommended by the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP), and newly available DNA extraction-based methods. A series of n=29 formalin-fixed paraffin-embedded breast cancers were subjected to ERBB2 copy number assessment by fluorescence in situ hybridization (FISH, Vysis, Abbott). Following macrodissection of invasive breast cancer tissue and DNA extraction, ERBB2 copy number was also determined by molecular inversion probe array analysis (MIP, OncoScan, Affymetrix) and next generation sequencing combined with normalized amplicon coverage analysis (NGS/NAC, AmpliSeq, Ion Torrent). ERBB2 copy number values obtained by MIP or NGS/NAC were tightly correlated with ERBB2 copy number values obtained by conventional FISH ( r s = 0.940 and r s = 0.894, P < 0.001). Using ASCO/CAP guideline-conform thresholds for categorization of breast cancers as HER2-negative, equivocal or positive, nearly perfect concordance was observed for HER2 classification by FISH and MIP (93% concordant classifications, κ = 0.87). Substantial concordance was observed for FISH and NGS/NAC (83% concordant classifications, κ = 0.62). In conclusion, MIP facilitates precise ERBB2 copy number detection and should be considered as an ancillary method for clinical HER2 testing.

Background Techniques for the accurate identification of activating mutations of BRAF in metastatic melanoma are of great clinical importance, due to the availability of targeted therapies for these tumors. There is uncertainty regarding the frequency with which BRAF status differs between primary and metastatic sites. Methods Between 2011 and 2016, 219 melanoma cases underwent BRAF testing in our institution. In 53 of these cases, paired primary and metastatic specimens were available for polymerase chain reaction (PCR) and immunohistochemical evaluation. Results Fifty-two out of 53 cases (98%) showed concordant BRAF status between primary and metastatic site by immunohistochemistry (IHC). In one case, a metastasis and its matched primary were positive by IHC, but the metastasis was negative on PCR. On further investigation, PCR was positive in the primary, and repeat PCR in the metastasis was positive, following macrodissection. Conclusions Our results suggest that discordance of BRAF mutational status between primaries and metastases is a rare occurrence. In one case, IHC provided strong evidence that initial PCR testing had provided a false-negative result due to low tumor volume. Thus, in cases where tissue is difficult to obtain from a metastasis or unavailable, the primary tumor can be used with confidence.

Background Tissue heterogeneity in formalin-fixed paraffin-embedded (FFPE) breast cancer specimens may affect the accuracy of reverse transcription quantitative real-time PCR (RT-qPCR). Herein, we tested the impact of tissue heterogeneity of breast cancer specimen on the RT-qPCR-based gene expression assay MammaTyper®. Methods MammaTyper® quantifies the mRNA expression of the four biomarkers ERBB2, ESR1, PGR, and MKI67. Based on pre-defined cut-off values, this molecular in vitro diagnostic assay permits binary marker classification and determination of breast cancer subtypes as defined by St Gallen 2013. In this study, we compared data from whole FFPE sections with data obtained in paired RNA samples after enrichment for invasive carcinoma via macro- or laser-capture micro-dissection. Results Compared to whole sections, removal of surrounding adipose tissue by macrodissection generated mean absolute 40-ddCq differences of 0.28–0.32 cycles for all four markers, with ≥90% concordant binary classifications. The mean raw marker Cq values in the adipose tissue were delayed by 6 to 7 cycles compared with the tumor-enriched sections, adding a trivial linear fold change of 1.0078 to 1.0156. Comparison of specimens enriched for invasive tumor with whole sections with as few as 20% tumor cell content resulted in mean absolute differences that remained on average below 0.59 Cq. The mean absolute difference between whole sections containing up to 60% ductal carcinoma in situ (DCIS) and specimens after dissection of DCIS was only 0.16–0.25 cycles, although there was a tendency for higher gene expression in DCIS. Observed variations were related to small size of samples and proximity of values to the limit of detection. Conclusion Expression of ESR1, PGR, ERBB2 and MKI67 by MammaTyper® is robust in clinical FFPE samples. Assay performance was unaffected by adipose tissue and was stable in samples with as few as 20% tumor cell content and up to 60% DCIS. Electronic supplementary material The online version of this article (10.1186/s13000-018-0760-6) contains supplementary material, which is available to authorized users.

Formalin-fixed paraffin embedded samples for actionable somatic pathogenic variant (SPV) testing should undergo review in order to ensure that the appropriate material has been submitted and the neoplastic cell content (NCC) is sufficient for SPV detection. In samples with suboptimal NCC, light microscopy identification of areas of higher NCC and the provision of a marked H&E slide for precise macrodissection increase the likelihood of detecting an actionable SPV. While this should reflect common practice, despite requesting this information, our experience is that a significant number of referrals arrive at the laboratory with no indication of NCC and/or are unmarked for macrodissection, necessary on samples with an NCC of less than 10%. This increases the risk of a false-negative result. Using malignant melanoma as a representative for actionable SPV testing, we audited 231 samples obtained by the laboratory between August and October 2018. Eight per cent (n=19) of the samples were obtained with no estimate of NCC and 7% (n=17) of the samples stated to have

BACKGROUND Data generated by next-generation sequencing technologies have a pivotal role in precision medicine. These high-throughput techniques are preferentially performed on fresh tissue, but there is an increasing need for protocols adapted to materials derived from formalin-fixed, paraffin-embedded tissue and cytology specimens. METHODS The aim of this work was to show that cytological material collected from archival smears processed for routine diagnoses could be used for massively parallel sequencing and array-based genomic analysis for further studies. RESULTS As a proof of concept, data obtained from May-Grunwald Giemsa– and Diff-Quik–stained archival smears were shown to be in keeping with those obtained from matched frozen controls. CONCLUSIONS The quality of DNA extracted from routinely processed smears is compatible with the multitargeted sequencing of a large series of genes of interest with methods such as array-based genomic analysis and whole-exome sequencing. Cancer Cytopathol 2016;124:241–53. © 2015 American Cancer Society.

The discovery and clinical application of molecular biomarkers in solid tumors, increasingly relies on nucleic acid extraction from FFPE tissue sections and subsequent molecular profiling. This in turn requires the pathological review of haematoxylin & eosin (H&E) stained slides, to ensure sample quality, tumor DNA sufficiency by visually estimating the percentage tumor nuclei and tumor annotation for manual macrodissection. In this study on NSCLC, we demonstrate considerable variation in tumor nuclei percentage between pathologists, potentially undermining the precision of NSCLC molecular evaluation and emphasising the need for quantitative tumor evaluation. We subsequently describe the development and validation of a system called TissueMark for automated tumor annotation and percentage tumor nuclei measurement in NSCLC using computerized image analysis. Evaluation of 245 NSCLC slides showed precise automated tumor annotation of cases using Tissuemark, strong concordance with manually drawn boundaries and identical EGFR mutational status, following manual macrodissection from the image analysis generated tumor boundaries. Automated analysis of cell counts for % tumor measurements by Tissuemark showed reduced variability and significant correlation (p < 0.001) with benchmark tumor cell counts. This study demonstrates a robust image analysis technology that can facilitate the automated quantitative analysis of tissue samples for molecular profiling in discovery and diagnostics.

An on-demand, closed RT-qPCR, the GeneXpert (GX) system, has the potential to provide biomarker information in low-resourced settings and elsewhere. We used this system with a research use only version of the Breast Cancer STRAT4 cartridge that measures the mRNA expression levels of ERBB2, ESR1, PGR, and MKi67. Here we evaluated the impact of non-macrodissected (non m-d) versus macrodissected (m-d) samples using STRAT4 on formalin-fixed, paraffin-embedded (FFPE) core needle biopsies. Two cohorts were assessed: (1) 60 FFPE infiltrating ductal carcinoma (IDCA) cases and (2) 20 FFPE IDCA cases with ductal carcinoma in situ (DCIS) with a range of HER2 expression as determined by clinical immunohistochemistry and fluorescence in situ hybridization (IHC/FISH). We observed about half of the core needle biopsy area as invasive tumor in both IDCA (mean = 51.5%) and IDCA with DCIS (mean = 53.5%) cohorts, but also found the mRNA levels were independent of tumor area. We found excellent agreement of the mRNA transcript level between the paired samples, m-d versus non m-d, for ERBB2, ESR1, PGR, and MKi67 for both the IDCA and IDCA with DCIS cohorts. No significant difference (P > 0.99) was observed when we compared the mRNA transcript level between the paired samples m-d versus non m-d. In addition, we noted a significant concordance (P

Background Aberrations in histone post-translational modifications (hPTMs) have been linked with various pathologies, including cancer, and could not only represent useful biomarkers but also suggest possible targetable epigenetic mechanisms. We have recently developed an approach, termed pathology tissue analysis of histones by mass spectrometry (PAT-H-MS), that allows performing a comprehensive and quantitative analysis of histone PTMs from formalin-fixed paraffin-embedded pathology samples. Despite its great potential, the application of this technique is limited by tissue heterogeneity. Methods In this study, we further implemented the PAT-H-MS approach by coupling it with techniques aimed at reducing sample heterogeneity and selecting specific portions or cell populations within the samples, such as manual macrodissection and laser microdissection (LMD). Results When applied to the analysis of a small set of breast cancer samples, LMD-PAT-H-MS allowed detecting more marked changes between luminal A-like and triple negative patients as compared with the classical approach. These changes included not only the already known H3 K27me3 and K9me3 marks, but also H3 K36me1, which was found increased in triple negative samples and validated on a larger cohort of patients, and could represent a potential novel marker distinguishing breast cancer subtypes. Conclusions These results show the feasibility of applying techniques to reduce sample heterogeneity, including laser microdissection, to the PAT-H-MS protocol, providing new tools in clinical epigenetics and opening new avenues for the comprehensive analysis of histone post-translational modifications in selected cell populations. Electronic supplementary material The online version of this article (doi:10.1186/s13148-017-0369-8) contains supplementary material, which is available to authorized users.

Background During the diagnostic work-up of breast carcinomas, immunohistochemistry (IHC) is the currently used method for assessing the expression of estrogen- (ER) and progesterone-receptors (PR), human epidermal growth factor receptor 2 (HER2) as well as of Ki-67 as a marker of tumor cell proliferation. In this study, we analyzed the concordance of these four breast cancer biomarkers between the RT-qPCR- and IHC-based (evaluated by three independent pathologists) determinations. Methods The expression of ER/ESR1, PR/PGR, HER2/ERBB2 and Ki-67/MKI67 was determined in 269 FFPE breast cancer samples with tumor content >20% from Chinese patients. For IHC, the samples were freshly cut, stained and assessed by three independent pathologists using the same scoring methods in a blinded fashion (positivity defined as: ER/PR ≥1%, HER2 >2+ and Ki-67 ≥20%). Measurement of the markers on the mRNA level was done on total RNA extracts prepared from whole tissue sections from the same FFPE blocks using the CE-marked RT-qPCR based IVD MammaTyper® on a Cobas® z480 qPCR cycler. IHC assessments of the three pathologists were compared to each other with regard to concordance of positive/negative results. Subsequently, agreement of RT-qPCR and IHC results for each marker and in samples in which the three pathologists had a consensus positive/negative IHC result was determined. Furthermore, we compared the MammaTyper® assessments from a subset of whole FFPE sections to data obtained from paired samples enriched for invasive carcinoma via macrodissection. Results From the 269 samples, 256 were available for final analysis. When excluding cases with discordant IHC callings between the three pathologists (6.0% for ER; 7.4% PR; 4.1% Her2; 17.1% Ki-67)) the concordance to the RT-qPCR determination and consensus IHC-based analysis displayed an excellent agreement for ER (OPA: 95.4%, PPA: 97.5%, NPA: 91.5%, Kappa: 0.897), PR (OPA: 91.1%, PPA: 89.6%, NPA: 93.1%, Kappa: 0.820) and HER2 (OPA: 97.1%, PPA: 91.9%, NPA: 100.0%, Kappa: 0.936). For cancer MKI67 mRNA and Ki-67 protein expression, a lower but still good concordance was found (OPA: 90.1%, PPA: 91.8%, NPA: 83.3%, Kappa: 0.707). In addition, we could demonstrate an excellent agreement of quantitative RT-qPCR measurements between whole surface and paired tumor-enriched samples in 99 Chinese breast cancer patients with R2 of 0.927 for ER, 0.926 for PR, 0.923 for HER2 and 0.908 for KI67. Even under highly standardized IHC scoring conditions, the discordance rates in the RT-qPCR marker callings with 0.0% for ESR1, 5.0% for PGR, 3.0% for ERBB2, 13.1% for MKI67 were lower than disagreements by three pathologists on the identical slide. Conclusion Standardized determination of the breast cancer biomarkers ER, PR, HER2 and Ki-67 on the mRNA level shows high concordance to a consensus IHC determined by three experienced pathologists indicating that RT-qPCR may be a valid alternative for determining the four breast cancer biomarkers. In line with previous research we could show on a large set of samples that macrodissection is not required for reliable assessment of the four breast cancer markers in clinical FFPE samples. Citation Format: Teng X, Li X, Xu S, Zhang J, Hartmann K, Laible M, Hipfel R, Bai Y, Ba X, Wu Z, Wirtz RM, Liu S, Ugur S. Comparison of RT-qPCR with consensus immunohistochemistry by three pathologists for ER, PR, HER2 and Ki-67 in Chinese breast cancer patients [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 P4-02-12.

Great advances in analytical technology coupled with accelerated new drug development and growing understanding of biological challenges, such as tumor heterogeneity, have required a change in the focus for biobanking. Most current banks contain samples of primary tumors, but linking molecular signatures to therapeutic questions requires serial biopsies in the setting of metastatic disease, next-generation of biobanking. Furthermore, an integration of multidimensional analysis of various molecular components, that is, RNA, DNA, methylome, microRNAome and post-translational modifications of the proteome, is necessary for a comprehensive view of a tumor's biology. While data using such biopsies are now regularly presented, the preanalytical variables in tissue procurement and processing in multicenter studies are seldom detailed and therefore are difficult to duplicate or standardize across sites and across studies. In the context of a biopsy-driven clinical trial, we generated a detailed protocol that includes morphological evaluation and isolation of high-quality nucleic acids from small needle core biopsies obtained from liver metastases. The protocol supports stable shipping of samples to a central laboratory, where biopsies are subsequently embedded in support media. Designated pathologists must evaluate all biopsies for tumor content and macrodissection can be performed if necessary to meet our criteria of >60% neoplastic cells and

Patients diagnosed with BRAF V600E mutated cutaneous melanoma show response to treatment with the BRAF inhibitor Vemurafenib. Different methods for BRAF mutation detection exist; however, only the Cobas 4800 BRAF V600 Mutation Test has been approved by the US Food and Drug Administration for patient selection. The results from this test depend on the percentage of tumor cells in the samples, which clinically may be estimated with substantial variation. We have evaluated five different methods: the Cobas test, Sanger sequencing, pyrosequencing, TaqMan-based allele-specific PCR, and Competitive Amplification of Differentially Melting Amplicons (CADMA), for detection of BRAF c.1799T>A (V600E) mutations in 28 formalin-fixed paraffin-embedded (FFPE) cutaneous melanoma samples. We show that the frequency of the BRAF V600E mutation is influenced by the analytical sensitivity of the applied method. However, a 100% consensus was observed among all five methods when the tumor tissue fraction was more than 10% of all tissue or more than 50% of cell-dense tissue. When using Sanger sequencing, pyrosequencing, or the Cobas test, it may be advisable to perform macrodissection before mutation testing if the tumor cell fraction is low. CADMA and TaqMan may not require macrodissections for a reliable test. Therefore, the use of more sensitive methods may have a future in testing for BRAF mutations in clinical settings.

In the frame of translational breast cancer research, eligibility criteria for formalin-fixed paraffin-embedded tissue (FFPE) material processing for gene expression studies include tumor cell content (TCC) and sample site (primary vs metastatic tumors). Herein we asked whether the observed differences in gene expression between paired samples with respect to TCC and sample site also have different clinical significance. We assessed ESR1, ERBB2, MAPT, MMP7, and RACGAP1 mRNA expression with real time PCR in paired samples before (NMD) and after macrodissection (MD) from 98 primary tumors (PMD, PNMD) and 72 metastatic lymph nodes (LNMD, LNNMD), as well as from 93 matched P (mP) and LN (mLN). TCC range was 2.5–75 % in the NMD series and 28–98 % in the MD and in the mP/mLN series. The prognostic effect of these markers, individually or in clusters, remained stable between paired PMD/NMD. In comparison, cluster classification failed in the LNNMD group with lower TCC. In the mP/mLN cohort, RACGAP1 mRNA expression was of prognostic significance when tested in mLN samples (p

Molecular genetic testing on formalin fixed, paraffin embedded (FFPE) tumors frequently requires dissection of tumor from tissue sections mounted on glass slides. In a process referred to as "manual macrodissection," the pathologist reviews an H&E stained slide at the light microscope and marks areas for dissection, and then the laboratory performs manual dissection from adjacent sections without the aid of a microscope, using the marked reference H&E slide as a guide. Manual macrodissection may be inadequate for tissue sections with low tumor content. We compared manual macrodissection to a new method, digitally guided microdissection, on a series of 32 FFPE pancreatic cancer samples. KRAS hotspot mutation profiling was performed using the Sequenom MassARRAY system (Agena Bioscience). Digitally guided microdissection was performed on multiple smaller areas of high tumor content selected from within the larger areas marked for manual macrodissection. The KRAS mutant allele fraction and estimated neoplastic cellularity were significantly higher in samples obtained by digitally guided microdissection (p < 0.01), and 7 of the 32 samples (22%) showed a detectable mutation only with digitally guided microdissection. DNA quality and yield per cubic millimeter of dissected tissue were similar for both dissection methods. These results indicate a significant improvement in tumor content achievable with digitally guided microdissection.

Precise identification and characterization of candidate neoantigens is important for the development of effective cancer vaccines, adoptive T-cell transfer, and prediction of response to checkpoint inhibitors. The candidate tumor neoantigens are actionable only when expressed, however, current prediction methods lack the capacity to evaluate neoantigen expression. Sequencing both DNA and RNA from a patient’s tumor tissue enables identification of mutations and evaluation of their expression leading to accurate identification of putative neoantigens. The purpose of this study was to develop and validate a methodology for co-extraction and sequencing of DNA and RNA from formalin-fixed paraffin-embedded (FFPE) samples to enable a robust neoantigen prediction protocol that integrates whole exome and transcriptome data to identify and prioritize tumor neoantigens for application in immuno-oncology research and clinical trials. In order to prepare high-quality sequencing libraries from FFPE specimens, the tissue was macrodissected to enrich for tumor-specific material, and improve the overall accuracy of next-generation sequencing for detection of somatic alterations. Total DNA and RNA was co-extracted and purified. The DNA was used to prepare whole exome sequencing (WES) libraries, while the co-extracted RNA was ribosome-depleted, and reverse-transcribed to prepare RNA sequencing (RNAseq) libraries. The WES and RNAseq data was then analyzed using a multi-algorithm HLA typing and neoantigen prediction protocol (ImmunoSelect-RTM). ImmunoSelect-R evaluates somatic genomic alterations identified from WES of tumor and matched normal tissue to ensure appropriate prediction of candidate neoantigens. The process of neoantigen prediction was then refined by integration of patient tumor-matched RNAseq data, which allowed for removal of non-expressed putative neoantigens. To further validate the approach, we applied the methodology to a set of experimentally validated neoantigens. In this setting, ImmunoSelect-R correctly classified 18 out of 19 as strong neoantigen candidates, suggesting a sensitivity of greater than 90%. Moreover, in a set of 10 patients, ImmunoSelect-R consistently ranked experimentally validated neoantigens within the top 20% of all neoantigen candidates derived from whole exome sequencing. In summary, our combined tissue processing, macrodissection, co-extraction, and neoantigen prediction methodology is able to identify and prioritize candidate neoantigens. Our approach is unique in combining high-fidelity sequencing (WES) and expression (RNAseq) data to accurately inform the selection of actionable tumor neoantigens for immuno-oncology applications. Citation Format: Marián Novak, Sam Angiuoli, Luis A. Diaz, Andrew Georgiadis, Sian Jones, Peter R. Loverso, Sonya Parpart-Li, Maria Sevdali, Victor E. Velculescu, Ellen L. Verner, James White, Theresa Zhang, Mark Sausen. Accurate identification and prioritization of candidate neoantigens from integrated cancer exome and transcriptome sequencing of FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 604. doi:10.1158/1538-7445.AM2017-604

We compared the performance of the 3D-Gene® mutation assay (3D-Gene® KRAS mutation assay kit) with the Scorpion-ARMS (therascreen® KRAS RGQ PCR Kit) and Luminex (MEBGEN™ KRAS kit) assays for the detection of KRAS mutations in formalin-fixed, paraffin-embedded tissue samples from 150 patients diagnosed with colorectal cancer. DNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining and the KRAS mutation status was independently determined using these assays. Discordant results were re-analyzed by Sanger sequencing. Mutation detection analysis was successfully performed in all 150 specimens using the 3D-Gene® mutation assay without an invalid case. The concordance rate between the 3D-Gene® mutation assay and Scorpion-ARMS or Luminex was 98.7% (148/150). KRAS mutations were detected at a frequency of 35.3% (53/150) in colorectal cancer specimens. Three discrepant cases were found between the three assays. Overall, our results demonstrate a high concordance rate of between the 3D-Gene® mutation assay and the two existing in-vitro diagnostics kits. All three assays proved to be validated methods for detecting clinically significant KRAS mutations in paraffin-embedded tissue samples.

Background Dissection of specific Areas Of Interest (AOIs) of slide-mounted tumor samples is often used to enrich for cancer cells in order to generate better signal to noise ratios in subsequent biochemical characterization. Most clinical laboratories utilize manual dissection for practical reasons and to avoid the expense and difficulties of laser microdissection systems. Unfortunately, manual methods often lack resolution and process documentation. The goal of this project was to design a dissection system for slide-mounted tissue with better precision than manual methods that also provides digital image guidance and electronic process documentation. Methods An instrument that is essentially a micro tissue mill was developed. It employs a specialized disposable mill bit that simultaneously dispenses liquid, cuts tissue from the slide surface, and aspirates the liquid along with the displaced tissue fragments. A software package was also developed that is capable of transferring digitally annotated AOIs between images of serially cut tissue sections to guide dissection and generate an electronic record of the process. Results The performance of this “meso” dissection system was tested using post dissection visual examination for resolution and accuracy, fluorescence based DNA quantitation for recovery efficiency, and dissection of closely situated mouse-human tissue sections followed by PCR amplification for purity determination. The minimum resolution is a dissected circle smaller than 200 microns in diameter, edge dissection accuracy is tighter than 100 microns, recovery efficiency appears greater than 95%, and recovery purity is greater than 99% relative to a different tissue located 100 microns from the dissection boundary. The system can dissect from both paraffinized and deparaffinized FFPE tissue sections that are mounted on plain glass slides, and it is compatible with DNA, RNA, and protein isolation. Conclusions The mesodissection system is an effective alternative to manual dissection methods and is applicable for biomarker analysis of anatomical pathology samples, where enrichment of AOIs from the tissue section is helpful, but pure cell populations are not required.

Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin's lymphoma with two distinct molecular subtypes defined by Cell-of-Origin (COO): germinal center B-cell (GCB) and activated B-cell (ABC). COO is being evaluated as a predictive biomarker in multiple DLBCL clinical trials hence the need for an accurate and precise companion diagnostics (CDx). NanoString LST is an investigational multiplexed digital gene expression assay performed on the nCounter® Dx Analysis System that identifies COO from FFPE tissue with a turnaround time of 2-3 days (Wallden B et al, JCO 2015; Storhoff J et al, Blood 2015). The test is based on the Lymph2Cx gene expression assay which profiles 15 classifier genes and 5 housekeeping genes to compute a linear predictor score (LPS) and determine the COO subtype (Scott D et al, Blood 2014). To establish a CDx, both analytical and clinical validations are required using the locked algorithm and assay procedure. Clinical validity of the LST is currently being evaluated in a global Phase 3 study in which the assay is used to prospectively select patients with ABC-type DLBCL to receive in a randomized fashion R2-CHOP or R-CHOP (Nowakowski G et al, JCO 2016). The analytical validation studies described herein were designed to evaluate the analytical performance of the test across multiple laboratory sites, operators, instruments, reagent lots, and RNA input levels. Design: Reproducibility was measured across 3 sites and 6 operators by testing replicate tissue sections from 43 FFPE DLBCL tumor blocks following independent pathology review at each site. The testing included both core needle and surgical biopsy specimens. The standard deviation (SD) of the LPS output was analyzed as a continuous variable using a linear mixed model to estimate assay variance, and the site-to-site concordance was evaluated. Analytical precision was measured across 3 sites and 6 operators by testing 5 pooled DLBCL tumor RNA samples representing each COO subtype including samples near the subtype thresholds. The sensitivity of the assay was evaluated by testing 4 RNA levels within the recommended input range (62.5 ng, 125 ng, 500 ng, 1000 ng) and 2 input levels outside of this range (50 ng and 1250 ng) using 14 DLBCL tumor RNA samples and 2 reagent lots. The percentage of test samples passing all QC metrics was determined at each input level, and the subtype concordance was evaluated compared to the nominal test input level (500 ng). Interference of human genomic DNA was assessed by omitting DNase from the assay procedure. The impact of including non-tumor tissue was assessed by omitting the tissue macrodissection step from the assay. Results: In the multi-site reproducibility study, the average site-to-site LST subtype concordance with independent pathology review was >97% with no ABC-to-GCB discordances (or vice versa). The overall range of LPS spanned approximately 5000 units. The total SD was Conclusions: The investigational NanoString LST has been analytically validated for identifying COO subtypes across multiple testing laboratories. The test provides a highly precise, sensitive and rapid method for measuring COO on FFPE DLBCL tumor specimens, including both excisional and core needle biopsies. Disclosures Chen: NanoString Technologies, Inc.: Employment, Other: Stock option. Dennis:NanoString Technologies, Inc.: Employment, Other: Stock option. Danaher:NanoString Technologies, Inc.: Employment, Other: Stock option. Wallden:NanoString Technologies, Inc.: Employment. Hood:NanoString Technologies, Inc.: Employment, Other: Stock option. Ren:NanoString Technologies, Inc.: Employment, Other: Stock option. Liu:NanoString Technologies, Inc.: Employment, Other: Stock option. Dowidar:NanoString Technologies, Inc.: Employment, Other: Stock option. Sullivan:NanoString Technologies, Inc.: Employment, Other: Stock option. Haffner:NanoString Technologies, Inc.: Employment, Other: Stock option. Cesano:NanoString Technologies, Inc.: Employment, Other: Stock option. Ferree:NanoString Technologies, Inc.: Employment, Other: Stock option, Patents & Royalties: NanoString Technologies, Inc.. Storhoff:NanoString Technologies, Inc.: Employment, Other: Stock option.

Introduction: We have previously shown reliability in isolating pure populations of rare cells from complex tissues using the DEPArray™ system. Several molecular techniques can be applied to a variety of histological samples, for example Macrodissection is the gross manual dissection of FFPE samples used to isolate areas of interest within a specimen for optimal downstream analysis, while Fine Needle Aspiration (FNA) is a safe procedure routinely used to examine a lesion helping to make a diagnosis. These techniques are also used to assess the effect of treatment. Here we demonstrate preliminary results showing 100% efficiency in recovering pure tumor cell populations from different samples using the DEPArray™ platform to overcome the issue of tumor heterogeneity. Method: FFPE macrodissected sections (n = 9;) and FNA (n = 5;) originating from prostate, breast, pancreatic and lung tumors were evaluated. Each was processed using a tissue disassociation and staining procedure followed by DEPArray™ sorting based on cytokeratin (Ker), vimentin (Vim) and nuclear staining. The recovered cell populations were lysed in the collection tube prior to PCR-based target enrichment for next generation sequencing using IonTorrent™ AmpliSeq CHPv2. Results: DEPArray™ analysis allowed identification of well separated cell populations, including Tumor (Vim-/Ker+) and Stromal (Vim+/Ker-) cells in all the samples analyzed. In the Macrodissection samples we were able to estimate the% of tumor cells (mean 23% range 4-54%), demonstrating an unexpected low frequency of tumor cells remaining following macrodissection. In FNA specimens analyzed only 21% (4.3% to 42.7% range) of the total (mean of 6335) cells analyzed were of tumor (KER+) origin. For subsequent NGS analysis, groups of pure cells (mean 174 cells, range from 37 to 280) for each population were recovered. Among the tumor cells isolated from the macrodissected and FNA specimens, we observed non-synonymous somatic variants and LOH events for different genes. This situation can not be highlighted in unsorted population. Conclusions: DEPArray™ technology can be used to isolate pure tumor cells from heterogeneous FFPE samples used in diagnostic application, such as Macrodissection or FNA specimens. Thus, the DEPArray™ platform brings digital precision to detection, quantification and recovery of pure target cells for subsequent downstream molecular analysis that can improve cancer diagnosis and treatment decisions. Citation Format: Valeria Sero, Claudio Forcato, Chiara Bolognesi, Genny Buson, Giulio Signorini, Paola Tononi, Gianni Medoro, Nicolò Manaresi, Farideh Z Bischoff. Efficiency in recovery of pure tumor cell populations from limited tumor tissue specimens intended for clinical application. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2417.

PURPOSE: Previous mutational analyses have failed to identify a predictive or prognostic marker for pulmonary carcinoid tumors (PCT). The heterogeneity of PCTs coupled with a lack of detailed information about the tumor biology, impedes patient stratification into groups based on tumor phenotypes or treatment response. We sought to use high-throughput next-generation sequencing to improve prognostication. METHODS: We stratified 37 patients with resected PCTs as low risk (n = 17) and high risk (n = 20) based on a 5 year long follow up for recurrence. Patients with no recurrence within 5 years from initial treatment were deemed low risk. Macrodissection was followed by genomic DNA isolation and next-generation sequencing was performed using an Illumina Mate Pair library protocol. Sequence reads were mapped to the human genome, and primers spanning the fusion junctions were used for validation polymerase chain reaction. RESULTS: Carcinoids commonly harbor rearrangements (median 3.5, range 1-167). Large chromosomal gains or losses were found in 24 of the cases (64.8%). We developed a prognostic score that included the total amount of genetic alterations (deletions and translocations). ROC analysis revealed an AUC of 0.76. High risk patients had a mean score of 20 and low risk patients had a mean score of 5.45 (p value = 0.03, Welch Two Sample t-test). Further analysis of the specific genetic alterations harbored by high and low risk PCTs and correlation with the pathologic and immunohistochemical information is currently underway. CONCLUSION: Large chromosomal rearrangements and aneuploidy portend a poor prognosis for patients with PCTs. Mate Pair sequencing of PCTs provides valuable prognostic information for this highly heterogeneous group of cancers. Citation Format: Konstantinos Leventakos, Aaron S. Mansfield, Stephen J. Murphy, Sarah H. Johnson, Sarah E. Kerr, Marie Christine Aubry, George Vasmatzis, Julian R. Molina. A prognostic model for pulmonary carcinoid tumors based on large chromosomal alterations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4513.

BackgroundTesting for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations.Methodology/principal findingsTumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%).Conclusions/significanceDiagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making.

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

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.