From Formalin-fixed Paraffin-embedded Tissue Samples Research Articles

B-231 Evaluation of the KingFisher Flex for DNA Isolation From FFPE Tissue Utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit

Abstract Background Our institution is facing increasing demand for oncology testing that requires high quality DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The current DNA extraction workflow involves a time and labor-intensive manual process that limits our ability to accommodate increasing downstream clinical testing volumes. We evaluated the KingFisher (KF) Flex utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit as a possible high-throughput, automated DNA isolation workflow option that would decrease hands-on time and be conducive to volume growth without the need for additional staff. Methods DNA from 52 FFPE tissue samples (n=24 unique cases previously extracted by the current “manual method”) were extracted using Autolys M tubes, the MagMAX FFPE DNA/RNA Ultra Kit, and the KF Flex (Thermo Fisher Scientific Waltham, MA) over 4 “automated method” runs. Method modifications to the published procedure were introduced over the runs to determine any effects on DNA quality/quantity. Timings were taken during each run to compare hands-on and total run time against that of the manual method. Precision experiments were performed by extracting 4 replicates of sample on a single run (intra-assay precision) and 2 samples from 2 cases over 2 runs (inter-assay precision). Three cases were processed using varying tissue input levels to assess input-yield correlation. DNA yield was measured using HS dsDNA Qubit (Thermo Fisher Scientific, Waltham, MA). A subset of samples was tested by digital droplet PCR (ddPCR) (n=10), chromosomal microsomal microarray (CMA) (n=1), and next-generation sequencing (NGS) (n=3). Variants and QC metrics of the KF DNA were compared to those of the manual method DNA. Results The average total DNA yield was 3% lower from samples extracted following the manufacturer recommended KF procedure compared to matched samples extracted using the manual method (n=28 samples from 23 unique cases). A CV of 2.8% was observed in an intra-assay experiment (n=4 replicates from 1 case) and 3.0% in an average inter-assay experiment (n=2 replicates from 2 cases). There was a direct correlation between the tissue input amount (4 amounts of varied tissue input) and the total DNA yield (n=3 samples). We observed concordant fractional abundance values for ddPCR samples and concordant variant detection for NGS and CMA samples. We did not see a significant difference in the quality metrics tracked for any of the downstream testing assays. Conclusions This evaluation demonstrated that, as compared to our manual method, the automated method resulted in comparable DNA yields and downstream assay results. The incorporation of this automation would significantly increase our FFPE DNA extraction capacity without requiring additional staff while reducing hands-on time per run by about six hours. We would also be able to reduce ergonomic hazards related to tube cap manipulation, eliminate use of hazardous reagents, and leverage the plate format to better integrate extracted DNA into our clinical sample preparation workflows. These operational benefits highlight the value of automating high-throughput workflows.

DNA Quantity and Quality Comparisons between Cryopreserved and FFPE Tumors from Matched Pan-Cancer Samples.

Personalized cancer care requires molecular characterization of neoplasms. While the research community accepts frozen tissues as the gold standard analyte for molecular assays, the source of tissue for testing in clinical cancer care comes almost universally from formalin-fixed, paraffin-embedded tissue (FFPE). As newer technologies emerge for DNA characterization that requires higher molecular weight DNA, it was necessary to compare the quality of DNA in terms of DNA length between FFPE and cryopreserved samples. We hypothesized that cryopreserved samples would yield higher quantity and superior quality DNA compared to FFPE samples. We analyzed DNA metrics by performing a head-to-head comparison between FFPE and cryopreserved samples from 38 human tumors representing various cancer types. DNA quantity and purity were measured by UV spectrophotometry, and DNA from cryopreserved tissue demonstrated a 4.2-fold increase in DNA yield per mg of tissue (p-value < 0.001). DNA quality was measured on a fragment microelectrophoresis analyzer, and again, DNA from cryopreserved tissue demonstrated a 223% increase in the DNA quality number and a 9-fold increase in DNA fragments > 40,000 bp (p-value < 0.0001). DNA from the cryopreserved tissues was superior to the DNA from FFPE samples in terms of DNA yield and quality.

Abstract 4957: DNA quality control metrics in cryopreserved tissue outperform DNA extracted from FFPE tissue

Abstract Personalized cancer care requires molecular characterization of neoplasms. While the research community accepts frozen tissues as the gold standard analyte for molecular assays, the source of tissue for testing of tumor tissue in clinical cancer care comes primarily from formalin-fixed, paraffin-embedded tissue (FFPE). Specific to genomics assays, numerous studies have shown significant discordance in genetic information obtained from FFPE samples and frozen samples. To explain the discordance between FFPE samples and cryopreserved samples, a head-to-head comparison between FFPE and cryopreserved tissues was performed to analyze the DNA yield, DNA purity and DNA quality in terms of DNA length. Additionally, short-read sequencing was performed on matched samples and quality control metrics were compared between the two sample types. Matched human tumors (n = 50) were processed either by formalin fixation and paraffin embedding or placed in cryovials containing cryopreservation medium. These cryovials were cooled slowly to -80°C and stored in liquid nitrogen. DNA was extracted using the same protocol for both tissue types with the exception that tissues embedded in paraffin were first dewaxed followed by a multistep rehydration protocol. Samples were weighed and calibrated for mass. After the column purification and elution, sample concentration and purity were measured. DNA fragment length was measured on a fragment microelectrophoresis analyzer. Graded amounts of tumor tissue were used to determine the lowest starting material needed to extract 200 ng of DNA. The average of all the samples reached the minimal threshold of 200 ng. However,74% of FFPE specimens failed to meet the minimum 40 ng/mg, whereas only 21% were below the threshold in the cryopreserved samples. In the cryopreserved group, the average DNA yield was 222.1 ng/mg, whereas 52.8 ng/mg was obtained from FFPE tissue. For DNA purity in cryopreserved tissues, the 260/280 ratio was 1.76, and in FFPE tissues the mean was 1.78. The DNA Quality Number (DQN) is a measurement of DNA fragment length and the percentage that exceeds the threshold of 300 bp. For FFPE, the DQN was 4.4 compared to a DQN of 9.8 for the cryopreserved samples. Setting a higher threshold of DNA length to 40,000 bp and measuring the area under the curve (AUC), it was observed that cryopreserved samples were 9-fold higher in fragments greater than 40,000 bp. Cryopreserved cancer tissue provides superior quality assurance measurements of DNA over FFFPE. Treatment decisions based on molecular results demand accuracy and validity. The pathology community should support efforts to cryopreserve cancer biospecimens in the clinical setting to provide valid molecular testing results. The automatic pickling of tumor specimens in formalin is no longer an acceptable default. Citation Format: Jared James Barrott, Nikole O'Neal, Jeffrey Okojie, David Booker, Ken Dixon. DNA quality control metrics in cryopreserved tissue outperform DNA extracted from FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4957.

Abstract 7627: First NGS-based companion diagnostic to aid in selecting non-small cell lung cancer patients with ERBB2 (HER2) activating mutations for treatment with trastuzumab deruxtecan

Abstract Introduction The ERBB2 companion diagnostic program aims to expand the intended use of the Oncomine ™ Dx Target Test (ODxTT) to determine ERBB2 single nucleotide variants (SNVs) in exons 8, 17, 18, 19, 20, 21 and 22 and exon 20 insertions from formalin-fixed, paraffin-embedded (FFPE) tumor samples in non-small cell lung cancer (NSCLC) patients who may benefit from targeted drug therapy. Methods The ODxTT is a qualitative in vitro diagnostic test that uses targeted high throughput, parallel-sequencing technology to detect SNVs, deletions, insertions, and CNVs in genes from DNA and fusions from RNA isolated from FFPE tissue samples using the Ion PGM ™ Dx System. Analytical validation (AV) studies interrogated ODxTT’s ability to detect ERBB2 mutations. The clinical validation (CV) study involved a method comparison/concordance analysis of ERBB2 mutations between the ODxTT and the TruSight Tumor 170 (TST170) assay, as well as a series of clinical trial assays (CTAs). The CV study was conducted using clinical specimens from 91 patients enrolled in Destiny-Lung01 and 121 stage-matched commercially procured samples. The aim was to evaluate positive percent agreement (PPA), negative percent agreement (NPA) and overall percent agreement (OPA) for CTAs and TST170 assay. Results A total of 91 ERBB2 subjects were included in the full analysis of the Destiny-Lung01 clinical trial. 49 samples had valid ODxTT results available and 42 samples were unevaluable which included 13 unknowns and 29 test not performed. The 49 patient samples that were evaluable were used to assess the clinical efficacy based on ODxTT results. In the series of 121 samples that were used to assess clinical efficacy in the CV study, 109 had evaluable ODxTT results. The PPA, NPA and OPA of ODxTT to the CTAs when excluding unknowns were 98%, 100% and 99.4%, respectively, and based off the Clopper-Pearson method for binomial distribution a 95% lower bound confidence interval (CI) were 89.6%, 96.6% and 96.5%, respectively. There was one discordant sample with a negative ODxTT and a positive CTA result (false negative). The PPA, NPA and OPA of ODxTT to TST170 assay were 100%, 99.1% and 99.3% respectively with a 95% lower bound CI of 90.8%, 95.0% and 96.3%, respectively. There was one discordant sample with a negative TST170 and a positive ODxTT result (false positive). Conclusion AV and CV studies successfully verified and validated the ODxTT for the detection of ERBB2 mutations in NSCLC FFPE samples. The 6 AV studies met the necessary product requirements and acceptance criteria to detect clinical samples with ERBB2 mutations. Additionally, the CV study clinical accuracy and clinical efficacy results demonstrated the safety and effectiveness of the use of the ODxTT in FFPE samples as an aid to identify NSCLC patients eligible for treatment with the Daiichi Sankyo lung cancer therapeutic T-DXd. Citation Format: Thomas Ha. First NGS-based companion diagnostic to aid in selecting non-small cell lung cancer patients with ERBB2 (HER2) activating mutations for treatment with trastuzumab deruxtecan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7627.

The extraction of Peste Des Petits Ruminants Virus RNA from paraffin-embedded tissues using a modified extraction method

Peste des petits ruminants (PPR) which is caused by small ruminant morbillivirus (PPRV) has an important economic impact on small ruminant farming due to high mortality rates, weight loss and restrictions on the export of small ruminants products. Molecular assays are commonly used in the diagnosis of the disease. Extraction of RNA from formalin-fixed paraffin-embedded (FFPE) tissues is challenging because of the RNA is often degraded by formalin fixation process. Although commercial kits have been developed for extraction of nucleic acids from FFPE tissues, they are expensive than other extraction kits. In this study, a modified extraction method was evaluated for detection of PPRV from FFPE tissues. A total of 20 FFPE tissue samples including 15 PPRV positive and 5 PPRV negative FFPE tissue samples were used. Two years ago, these selected FFPE tissue samples were analysed by nucleoprotein gene based one step real time RT-PCR method before they were fixed with formalin and embedded in paraffin. FFPE tissue samples were extracted using modified extraction method and were tested by fusion (F) gene based one step RT-PCR. PPRV specific RNA was detected in 12 FFPE tissue samples whereas 3 positive samples were found negative by one-step RT-PCR. Furthermore, 5 negative FFPE tissue samples were also found negative. Three false negative results were from samples with high real-time RT-PCR cycle threshold. Therefore, false negative results could be related with lower viral loads which might be lower than detection limit of the one-step RT-PCR. The results of the study show that modified extraction method could be used for RNA extraction from FFPE tissues which had been stored for 2 years.

Identification of fusion transcripts in sarcoma from archival formalin-fixed paraffin-embedded tissues: A next-generation sequencing approach.

Most sarcomas are highly aggressive, and cause necrosis and hemorrhage. The diagnosis of sarcoma is challenging because of the lack of specificity of immunohistochemical staining; however, molecular biological approaches, such as genetic mutation, chromosomal translocation, and gene amplification, are promising. In this study, we extracted RNA from formalin-fixed paraffin-embedded (FFPE) tissue derived from surgically resected specimens of sarcoma stored for various periods and performed next-generation sequencing (NGS) analysis by MiniSeq using the Archer Fusion-Plex Sarcoma Panel. RNA was extracted from 63 FFPE tissue samples, and the degree of RNA degradation was assessed. The number of reads and fragment lengths were evaluated by NGS analysis. RNA extraction and cDNA synthesis were successful in 56 cases and library preparation was possible. Fusion genes were detected in 16 of 63 archived FFPE tissue samples in this study. However, in 18 cases, fragmentation was strong, and high-quality libraries could not be obtained. Nevertheless, comprehensive analysis of fusion genes with high sequence specificity by NGS can be a powerful alternative to reverse transcription-polymerase chain reaction and fluorescence in situ hybridization methods.

Clinical performance of quantitative PCR for the molecular identification of skeletal tuberculosis from formalin-fixed paraffin-embedded tissues

BackgroundAt present, skeletal tuberculosis (TB) diagnosis is mostly by histopathology, but the positivity rate is low. There is a need to develop new methods for the molecular identification of this disorder. Therefore, we aimed to investigate the clinical utility of quantitative PCR (qPCR)-based diagnosis of skeletal TB from formalin-fixed paraffin-embedded (FFPE) tissues and its comparative evaluation with acid-fast bacillus staining (AFS).MethodsWe detected Mycobacterium tuberculosis (M. tuberculosis/MTB) DNA using qPCR and AFS in FFPE tissue samples from 129 patients suspected of having skeletal TB. The sensitivity, specificity as well as area under the curve (AUC) of qPCR and AFS were calculated. Meanwhile, some factors potentially affecting qPCR and AFS results were investigated.ResultsOverall, qPCR outperformed AFS in detecting M. tuberculosis. The AUC of qPCR was higher than that of AFS (0.744 vs.0.561, p < 0.001). Furthermore, decalcification of bone tissues did not affect the sensitivity and specificity of qPCR tests. Whereas it impacted the performance of AFS, decalcification increased AFS's specificity and decreased its sensitivity (p < 0.05). Moreover, qPCR had a significantly larger AUC than AFS in decalcified and non-decalcified groups (0.735/0.756 vs. 0.582/0.534, p < 0.001) respectively. Similarly, the AUC of PCR was more extensive than that of AFS regardless of skeletal TB patients with concomitant pulmonary TB or not (0.929 vs. 0.762; 0.688 vs. 0.524, p < 0.01).ConclusionsOur data demonstrate that qPCR offers superior accuracy for the detection of mycobacteria in FFPE tissues compared to traditional AFS, indicating its clinical value in osteoarticular TB diagnosis.

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4',6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

Abstract 1836: Global gene expression profiles from bladder tumor FFPE samples

Abstract Cancer is a disease characterized by uncontrolled cell growth and proliferation. Recent advances in molecular medicine and cancer biology have changed the way clinicians evaluate and consider treatment. Selected tumor biomarkers have been utilized as targets for drug therapy leading to better more effective treatment. Gene expression profiling has been used for identifying new biomarkers for tumor classification and driving decision making for better patient outcome in different tumor types. DNA microarrays have become a key method to acquire a comparative snapshot of the gene expression profile from test samples in a high throughput manner. Quantitative PCR and newer sequencing techniques are popular alternatives offering highly accurate gene expression measurements, but with limitations due to cost, complex instrumentation and analysis needs. RNA extracted from formalin fixed paraffin embedded tissue (FFPE) creates considerable additional challenges in acquiring accurate gene expression measurements due to the highly fragmented and compromised integrity of FFPE RNA due to the fixation process. To address the challenges of current sequencing based methods and take advantage of the simplicity of analysis that comes with using technologies such as microarrays; we have tested the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit using RNA isolated from bladder tumor FFPE specimens. This targeted RNA sequencing approach allows profiling the global mRNA expression of human RNA in a highly multiplexed fashion using the Ion AmpliSeq™ technology. 10ng of total RNA extracted from FFPE tissue was reverse transcribed followed by automated library preparation on the Ion Chef™ system using the new Ion AmpliSeq™ Kit for Chef and the Ion AmpliSeq™ Transcriptome Human Gene Expression Panel. Eight pooled libraries were then sequenced on the Ion S5™XL System with Ion 540™ Chip. Libraries were also prepared with well characterized control RNAs, Universal Human Reference RNA (UHR) and First Choice Human Brain Reference RNA (HBR) using both the manual and automated library generation protocol for validation and comparison studies. The results show detection of more genes than popular microarray platforms with comparable differential gene expression measurements to quantitative PCR (r = 0.96) and RNA-Seq methods (r = 0.94). Gene expression values correlated with R&amp;gt;0.99 for all technical replicates and R&amp;gt;0.95 between manual and automated library preparation methods using well characterized samples. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit is a simple method to measure global gene expression profiles from human RNA samples in a timely, cost effective, and high throughput manner resulting in sensitive and accurate gene expression measurements. The new S5™XL System combined with automated library and template preparation on the Ion Chef™ system enable a simple RNA to gene expression data workflow requiring only 45 minutes of hands on time from 10ng of FFPE RNA. Citation Format: Varun Bagai, Jeoffrey Schageman, Kelli Bramlett, David J. McConkey, Woonyoung Choi. Global gene expression profiles from bladder tumor FFPE samples. [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 1836.

Identification of fungal pathogens in Formalin-fixed, Paraffin-embedded tissue samples by molecular methods

The etiology of invasive fungal infections (IFI) is incompletely understood due to diagnostic limitations including insensitivity of cultures and failure of histopathology to discriminate between different species. This diagnostic gap precludes the optimal use of antifungals, leading to adverse patient outcomes. The identification of fungal pathogens from Formalin-fixed, Paraffin-embedded tissue (FFPE) blocks by molecular methods is emerging as an alternative approach to study the etiology of IFI.PCR assays, including species specific- and broadrange fungal tests are used with FFPE samples from patients with proven IFI. Fungal species identification is achieved in 15–90% of the samples. This heterogeneity may be explained by the samples studied. However, comparison of different studies is impaired, as controls ruling out false positive-, false negative test results or PCR inhibition are frequently not reported.Studies using in situ hybridization also vary in the clinical samples included and the targeted fungi. In addition, target sequences, the probe chemistry and the detection of hybridization signals also account for the differences in diagnostic sensitivity.Using both approaches in parallel yields additive insights, potentially leading to a superior identification of fungal etiology and awareness of the limitations of both molecular diagnostic approaches.

Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors?

BackgroundDetection of fungal DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is challenging due to degradation of DNA and presence of PCR inhibitors in these samples. We analyzed FFPE samples of 26 patients by panfungal PCR and compared the results to the composite diagnosis according to the European Organization for Research and Treatment of Cancer (EORTC) criteria. Additionally we analyzed the quality of human and fungal DNA and their level of age-dependent degradation, as well as the existence of PCR inhibition in these tissue samples.MethodsWe evaluated two 45-cycle panfungal PCR tests that target the internal transcribed spacer 2 (ITS2) as well as the ITS1-5.8S-ITS2 (ITS1-2) region. The PCRs were applied to 27 FFPE specimens from 26 patients with proven invasive fungal disease (IFD), and one patient with culture and histologically negative but PCR-positive fungal infection collected at our institution from 2003 to 2010. Quality of DNA in FFPE tissue samples was evaluated using fragments of the beta-globin gene for multiplex PCR, inhibition of PCR amplification was evaluated by spiking of C. krusei DNA to each PCR premix.ResultsIn 27 FFPE samples the ITS2 PCR targeting the shorter fragment showed a higher detection rate with a sensitivity of 53.8% compared to the ITS1-2 fragment (sensitivity 38%). Significant time-dependent degradation of human DNA in FFPE sample extracts was detected based on partial beta-globin gene amplification which was not in correlation to successful panfungal PCR identification of fungal organisms. The analytical sensitivity of both assays compared with culture was 60 CFU/ml of a Candida krusei reference strain. The performance of the two tests in an Aspergillus proficiency panel of an international external quality assessment programme showed considerable sensitivity.ConclusionPanfungal diagnostic PCR assays applied on FFPE specimens provide accurate identification of molds in highly degraded tissue samples and correct identification in samples stored up to 7 years despite sensitivity limitations, mainly caused by partial PCR inhibition and DNA degradation by formalin.

Abstract P1-08-06: Low tumor CD68 mRNA content (intratumoral macrophages) is predictive for benefit from adjuvant trastuzumab in HER2-positive breast cancer: An analysis of the FinHER trial

Abstract Background: Tumor immune cell infiltrates influence prognosis of node negative breast cancer (BC), and intratumoral B-cells and T-cells are of importance for achieving response to chemotherapy in triple negative BC. The role of tumor infiltrating macrophages remains unclear, but they might promote tumorigenesis. We investigated the prognostic value of CD68 mRNA levels within the luminal, HER2-positive and triple negative subtypes in the FinHer trial patient population, and evaluated their predictive value on survival outcomes achieved with adjuvant trastuzumab and chemotherapy in early breast cancer. Methods: RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue of 917 (90.8%) patients out of the 1010 patients who participated in the FinHer trial. Intratumoral macrophage infiltration was assessed by measuring breast tumor CD68 mRNA content using RT-qPCR from representative FFPE tissue samples. Breast cancer molecular subtypes (luminal, HER2 positive and triple-negative) were approximated using immunohistochemistry (IHC) and central CISH testing data obtained from the FinHer trial datafile. Prognostic significance of CD68 on distant disease-free survival (DDFS) was assessed using Kaplan-Meyer analysis and log-rank test. Results: Tumor CD68 mRNA expression was normally distributed with median expression of 33.56 (dCt). CD68 mRNA levels correlated weakly with estrogen receptor (ER) mRNA expression and ER protein levels (r = 0.11 and r = 0.15, respectively; p&amp;lt;0.0001), but not with tumor HER2 mRNA level or IHC/CISH status (r = -0.06, p = 0.12; and r = 0.01, p = 0.92, respectively). The median tumor CD68 mRNA content was not prognostic for DDFS in the subset of luminal cancers (ER+, HER2-; n = 533, 5-year DDFS 88% versus 88%; p = 0.92), but a lower than the median CD68 level tended to be associated with favorable DDFS in triple negative cancer (n = 143; 81% vs. 70%; p = 0.11) and was significantly associated with favorable DDFS in HER2-positive cancer (n = 191; 86% vs. 67%; p = 0.001). In the subset of HER2-positive cancer with tumor CD68 mRNA expression level lower than the median, patients treated with trastuzumab benefitted from addition of trastuzumab to chemotherapy (5-year DDFS 93% with trastuzumab vs. 79% without trastuzumab; p = 0.02), whereas no benefit from trastuzumab was observed when tumor CD68 content was higher than the median (67% vs. 68%; p = 0.92). Conclusions: The study validates tumor CD68 concentration as a prognostic biomarker in HER2-positive early breast cancer. Patients with HER2-positive cancer and few tumor macrophages (low tumor CD68 mRNA content) benefitted from addition of trastuzumab to chemotherapy, whereas patients with HER2-positive BC with high tumor macrophage content derived no benefit from adjuvant trastuzumab. Trastuzumab may be effective only for macrophage-poor HER2-positive cancers that are prone to antibody-dependent cellular cytotoxicity (ADCC), whereas it may have little efficacy for cancers that progress despite or due to high intratumoral macrophage content that interferes with ADCC. Other agents, such as T-DM1, might work better than trastuzumab in the subset of women who have HER2-positive BC with a high tumor macrophage content. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-06.

Complete solubilization of formalin‐fixed, paraffin‐embedded tissue may improve proteomic studies

Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues.

Duplex reverse-hybridization assay for the simultaneous detection of KRAS/BRAF mutations in FFPE-extracted genomic DNA from colorectal cancer specimens.

We report the performance evaluation of a non-quantitative reverse-hybridization assay (KRAS-BRAF StripAssay) designed for the simultaneous detection of 10 mutations in codons 12 and 13 of the KRAS gene and BRAF mutation V600E. Dilution experiments using DNA from tumor cell lines or from formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) tissue were performed to assess assay sensitivity. Using 50 ng of total DNA (mutant and wild-type), the KRAS-BRAF StripAssay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. With respect to BRAF V600E, the KRAS-BRAF StripAssay was evaluated using 60 FFPE CRC samples previously analyzed by high resolution melting (HRM). Test strip hybridization identified 2/60 (3%) samples to carry the BRAF V600E mutation, and results were in agreement with those obtained by HRM analysis. This work demonstrates the KRAS-BRAF StripAssay to be a robust and sensitive method for the detection of common KRAS/BRAF mutations in genomic DNA isolated from FFPE tissue samples.

Duplex Reverse-Hybridization Assay for The Simultaneous Detection ofKRAS/BRAFMutations in FFPE-extracted Genomic DNA from Colorectal Cancer Specimens

We report the performance evaluation of a non-quantitative reverse-hybridization assay (KRAS-BRAFStripAssay) designed for the simultaneous detection of 10 mutations in codons 12 and 13 of theKRASgene andBRAFmutation V600E. Dilution experiments using DNA from tumor cell lines or from formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) tissue were performed to assess assay sensitivity. Using 50 ng of total DNA (mutant and wild-type), theKRAS-BRAFStripAssay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. With respect toBRAFV600E, theKRAS-BRAFStripAssay was evaluated using 60 FFPE CRC samples previously analyzed by high resolution melting (HRM). Test strip hybridization identified 2/60 (3%) samples to carry theBRAFV600E mutation, and results were in agreement with those obtained by HRM analysis. This work demonstrates theKRAS-BRAFStripAssay to be a robust and sensitive method for the detection of commonKRAS/BRAFmutations in genomic DNA isolated from FFPE tissue samples.

Abstract 4553: Multiplexed, digital gene expression and fusion transcript analysis to screen for EML4-ALK positive lung cancer

Abstract Purpose: EML4-ALK fusions occur in approximately 5% of non-small cell lung carcinoma and define a subpopulation of lung cancer patients highly responsive to ALK kinase inhibitors. Current methodologies for detecting presence of ALK rearrangements are labor-intensive, costly, and not ideally suited for screening large number of patient samples. To allow for a sensitive, facile, and inexpensive methodology to detect EML4-ALK fusions, we developed a direct transcript profiling to detect common variants of EML4-ALK fusions. Experimental Design: Using a single multiplex assay, we simultaneously interrogated presence of EML4-ALK transcripts and ALK 3′ over-expression in 8 ALK-positive and 22 ALK-negative NSCLC samples previously tested by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). For controls, we also tested ALK-positive cancer cells lines NCI-H3122 and NCI-H2228, and an ALK-negative cell line A549. Purified RNA from formalin-fixed paraffin-embedded (FFPE) tissue sections was hybridized in duplicates to a biotinylated capture probe and a color-coded reporter probe designed to hybridize to sequences spanning EML4-ALK junction. In addition, multiple probes were created to hybridize within 5′ and 3′ regions of ALK transcript to detect discordant levels between the breakpoint, indicative of fusion event. Purified hybridization complexes are immobilized and aligned on a cartridge where a microscope CCD camera images and counts the molecular color-coded tags attached to reporter probes. The number of reporter counts corresponds to the number of transcripts being interrogated. Results: With this assay, we obtained 100% concordance in EML4-ALK fusion calls to results generated by FISH and IHC analyses. Similar findings were obtained for control cancer cell lines. The assay is highly reproducible and sensitive, detecting ALK-fusion transcripts even in samples with low tumor content. Using the combined strategy of fusion detection and ALK 5′ and 3′ transcript quantification, we were able to validate the status of one patient negative for ALK rearrangement by FISH but positive for ALK protein expression by IHC. Conclusions: We have developed a novel and sensitive methodology to screen for common EML4-ALK fusions in NSCLC. The assay is inexpensive, easy to perform, high-throughput and compatible with FFPE tissue samples. This is a promising technology highly suitable for screening large numbers of tumor samples without the need for cDNA synthesis and PCR amplification. 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 4553. doi:1538-7445.AM2012-4553

Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens

BackgroundIdentification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients.MethodsWe designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broad-range fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach.ResultsPCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background.ConclusionsWhile PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples.

Abstract LB-327: Analytical performance of the nCounter analysis system for gene expression cancer signatures

Abstract Background: The nCounter Analysis System is a platform for performing highly multiplexed, digital quantification of hundreds of different nucleic acid species in a single reaction. The system is being developed for use as a platform for in vitro diagnostic applications. The current study aimed to evaluate the analytical performance of the system by implementing a 50-gene signature used for determining the intrinsic subtype and prognostic risk of a breast cancer tumor from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The gene expression profiles can be used to divide breast cancer into four intrinsic subtypes: Basal-like, Luminal A, Luminal B, and HER2 enriched. A normal-like subtype identifies tumor specimens contaminated with a high percentage of normal tissue. A prognostic Risk-Of-Relapse (ROR) score is also calculated. Methods: The nCounter assay is performed by direct, multiplexed hybridization of molecular barcodes to target mRNAs. For this study, a CodeSet containing probes for 50 classification genes and 8 normalizing genes was developed. The intrinsic subtyping algorithm was trained by supervised hierarchical clustering of data from 538 samples. Prototypical centroids for tumor subtypes were chosen as statistically significant clusters, while the normal-like centroid was trained from reduction mammoplasty samples. All samples were correlated to the five prototypical centroids and assigned the subtype with the largest positive correlation. The analytical precision of the assay was measured by testing the same sample across replicates. The precision of the subtyping test was determined by assaying 40 different samples across 9 different combinations of reagent lots. The performance of the assay was also evaluated with varying input levels of RNA. Results: The precision of the nCounter assay was found to be driven by Poisson noise in the digital measurements at low expression levels. When samples were subtyped across multiple reagent lots, the correlations to each of the five subtypes were narrowly distributed. One sample varied in the categorical subtype call between Luminal B and HER2-enriched across all lots, however, this sample was similarly correlated between both Luminal B and HER2-enriched across all lot combinations. Not surprisingly, the variability for the ROR was minimal, even for the case where the categorical subtype call changed. Finally, the subtype call and ROR score were stable across a 10x range of RNA input. Conclusions: The NanoString nCounter system has the sensitivity and precision to be implemented as a distributed platform for multiplexed gene expression profiling of tumors. The sensitivity and direct digital detection without the need for enzymatic amplification make the technology compatible with FFPE tissue samples. The precision should make the technology robust under the many varied conditions seen in testing labs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-327. doi:10.1158/1538-7445.AM2011-LB-327

A High-Sensitivity, Medium-Density, and Target Amplification–Free Planar Waveguide Microarray System for Gene Expression Analysis of Formalin-Fixed and Paraffin-Embedded Tissue

Many microarray platforms and their associated assay chemistries do not work properly with RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue samples, a feature that severely hampers the use of microarrays in oncology applications, for which FFPE tissue is the routine specimen. Furthermore, the limited sensitivity of most microarray platforms requires time-consuming and costly amplification reactions of the target RNA, which negatively affects clinical laboratory work flow. We developed an approach for sensitively and reliably measuring mRNA abundances in FFPE tissue samples. This approach involves automated RNA extractions, direct hybridization of extracted RNA to immobilized capture probes, antibody-mediated labeling, and readout with an instrument applying the principle of planar waveguides (PWG). A 14-gene multiplex assay conducted with RNA isolated from 20 FFPE blocks was correlated to an analysis of the same with reverse-transcription quantitative real-time PCR (RT-qPCR). The assay sensitivity for gene expression analysis obtained for the PWG microarray platform was <10 fmol/L, eliminating the need for target preamplification. We observed a correlation coefficient of 0.87 to state-of-the-art RT-qPCR technology with RNA isolated from FFPE tissue, despite a compressed dynamic range for the PWG system (a 2.9-log dynamic range for PWG in our test system vs 5.0 logs for RT-qPCR). The precision of the PWG platform was comparable to RT-qPCR (Pearson correlation coefficient of 0.9851 for PWG vs 0.9896 for RT-qPCR) for technical replicates. The presented PWG platform demonstrated excellent sensitivity and precision and is especially well suited for any application for which fast, simple, and robust multiplex assays of RNA in FFPE tissue are required.

Reliability and Reproducibility of a RNA Preamplification Method for Low-density Array Analysis From Formalin-fixed Paraffin-embedded Breast Cancer Samples

Molecular signatures of tumors with prognostic and predictive value are now available. However, several technical problems prevent the performing of gene expression profiles in routine diagnostics. The highly sensitive fluorescence-based real-time quantitative reverse transcriptase-polymerase chain reaction procedure is able to analyze mRNA levels from formalin-fixed paraffin-embedded (FFPE) tissues, the most commonly available source of tumor samples with well-documented information. However, the time-dependent fragmentation and small amount of RNA extracted from FFPE requires a preliminary mRNA amplification step to amplify small amounts of mRNA without significantly distorting relative mRNA levels. The purpose of this study was to determine the performance of a commercial cDNA preamplification kit (TaqMan PreAmp Master Mix Kit) on RNA samples obtained from FFPE tissues, prepared, and archived for routine diagnosis. We tested the reliability and reproducibility of this procedure for performing low-density gene expression assay from FFPE tissue samples. The whole procedure reported herein is a powerful and reproducible approach for routine clinical purposes that can be performed even using poorer RNA quality samples and that allows the analysis of gene expression for 96 genes in stored samples.