From Formalin-fixed Paraffin-embedded Tissue Research Articles

Gene expression profiles in granuloma tissue reveal novel diagnostic markers in sarcoidosis

Sarcoidosis is an immune-mediated multisystem disease characterized by the formation of non-caseating granulomas. The pathogenesis of sarcoidosis is unclear, with proposed infectious or environmental antigens triggering an aberrant immune response in susceptible hosts. Multiple pro-inflammatory signaling pathways have been implicated in mediating macrophage activation and granuloma formation in sarcoidosis, including IFN-γ/STAT-1, IL-6/STAT-3, and NF-κB. It is difficult to distinguish sarcoidosis from other granulomatous diseases or assess disease severity and treatment response with histopathology alone. Therefore, development of improved diagnostic tools is imperative. Herein, we describe an efficient and reliable technique to classify granulomatous disease through selected gene expression and identify novel genes and cytokine pathways contributing to the pathogenesis of sarcoidosis. We quantified the expression of twenty selected mRNAs extracted from formalin-fixed paraffin embedded (FFPE) tissue (n=38) of normal lung, suture granulomas, sarcoid granulomas, and fungal granulomas. Utilizing quantitative real-time RT-PCR we analyzed the expression of several genes, including IL-6, COX-2, MCP-1, IFN-γ, T-bet, IRF-1, Nox2, IL-33, and eotaxin-1 and revealed differential regulation between suture, sarcoidosis, and fungal granulomas. This is the first study demonstrating that quantification of target gene expression in FFPE tissue biopsies is a potentially effective diagnostic and research tool in sarcoidosis.

TaqMan based real time PCR assay targeting EML4-ALK fusion transcripts in NSCLC

ObjectivesLung cancer with the ALK rearrangement constitutes only a small fraction of patients with non-small cell lung cancer (NSCLC). However, in the era of molecular-targeted therapy, efficient patient selection is crucial for successful treatment. In this context, an effective method for EML4-ALK detection is necessary. We developed a new highly sensitive variant specific TaqMan based real time PCR assay applicable to RNA from formalin-fixed paraffin-embedded tissue (FFPE). Materials and methodsThis assay was used to analyze the EML4-ALK gene in 96 non-selected NSCLC specimens and compared with two other methods (end-point PCR and break-apart FISH). ResultsEML4-ALK was detected in 33/96 (34%) specimens using variant specific real time PCR, whereas in only 23/96 (24%) using end-point PCR. All real time PCR positive samples were confirmed with direct sequencing. A total of 46 specimens were subsequently analyzed by all three detection methods. Using variant specific real time PCR we identified EML4-ALK transcript in 17/46 (37%) specimens, using end-point PCR in 13/46 (28%) specimens and positive ALK rearrangement by FISH was detected in 8/46 (17.4%) specimens. Moreover, using variant specific real time PCR, 5 specimens showed more than one EML4-ALK variant simultaneously (in 2 cases the variants 1+3a+3b, in 2 specimens the variants 1+3a and in 1 specimen the variant 1+3b). In one case of 96 EML4-ALK fusion gene and EGFR mutation were detected. All simultaneous genetic variants were confirmed using end-point PCR and direct sequencing. ConclusionOur variant specific real time PCR assay is highly sensitive, fast, financially acceptable, applicable to FFPE and seems to be a valuable tool for the rapid prescreening of NSCLC patients in clinical practice, so, that most patients able to benefit from targeted therapy could be identified.

Fusion transcript discovery in formalin-fixed paraffin-embedded human breast cancer tissues reveals a link to tumor progression.

The identification of gene fusions promises to play an important role in personalized cancer treatment decisions. Many rare gene fusion events have been identified in fresh frozen solid tumors from common cancers employing next-generation sequencing technology. However the ability to detect transcripts from gene fusions in RNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissues, which exist in very large sample repositories for which disease outcome is known, is still limited due to the low complexity of FFPE libraries and the lack of appropriate bioinformatics methods. We sought to develop a bioinformatics method, named gFuse, to detect fusion transcripts in FFPE tumor tissues. An integrated, cohort based strategy has been used in gFuse to examine single-end 50 base pair (bp) reads generated from FFPE RNA-Sequencing (RNA-Seq) datasets employing two breast cancer cohorts of 136 and 76 patients. In total, 118 fusion events were detected transcriptome-wide at base-pair resolution across the 212 samples. We selected 77 candidate fusions based on their biological relevance to cancer and supported 61% of these using TaqMan assays. Direct sequencing of 19 of the fusion sequences identified by TaqMan confirmed them. Three unique fused gene pairs were recurrent across the 212 patients with 6, 3, 2 individuals harboring these fusions respectively. We show here that a high frequency of fusion transcripts detected at the whole transcriptome level correlates with poor outcome (P<0.0005) in human breast cancer patients. This study demonstrates the ability to detect fusion transcripts as biomarkers from archival FFPE tissues, and the potential prognostic value of the fusion transcripts detected.

Development of a prognostic breast cancer diagnostic: Quantitative analysis of HER2 status using a multiplex bead assay on homogenized formalin fixed paraffin embedded breast tumor tissue (LB484)

Immunohistochemistry is typically used to classify breast cancer into molecular subtypes based on the presence or absence of three protein biomarkers: estrogen receptor (ER), progesterone receptor (PR), and receptor tyrosine‐protein kinase erbB‐2 (HER2). Tumor cells that overexpress HER2 specifically respond to the drug trastuzumab (Herceptin). HER2 can be measured semi‐quantitatively by IHC, but in up to 20% of cases, IHC results are equivocal, meaning that additional testing (fluorescence in situ hybridization [FISH]) is required to distinguish clinically meaningful HER2 overexpression from normal HER2 levels. Aiming to ameliorate this situation, we developed a diagnostic assay that allows direct quantitative determination of HER2 protein concentration from formalin fixed paraffin embedded (FFPE) tumor tissue with a multiplex bead assay. We hypothesized that substantial preanalytical and analytical factors inherent in IHC methodology were contributing to the high rate of equivocal results, and that more accurate and precise quantitative analysis of HER2 protein concentration would serve as a gold standard method for HER2 assessment, obviating the need for reflexive FISH. In this method, tumor tissue from three 10‐micron histologic sections is first subjected to lysis and protein extraction using a buffer similar to that used in IHC for epitope retrieval. Numerous HER2 antibody clones were evaluated to identify a matched pair capable of detecting HER2 in these FFPE tissue lysates, using a sandwich immunoassay geometry. The final multiplex bead‐based assay was validated on tissue culture controls and clinical samples.

Genome‐wide DNA methylation analysis of formalin‐fixed paraffin embedded colorectal cancer tissue

Formalin fixation and embedding of clinical tissue samples in paraffin is a common method for archiving biological material. These samples are often well annotated and provide an invaluable resource for research. However, this process of fixation and storage of tissue leads to DNA damage and fragmentation. The use of DNA from formalin fixed, paraffin-embedded (FFPE) tissue to interrogate methylation levels on a genome-wide scale can pose challenges. We compared fresh and matched FFPE tissue DNA samples using the Illumina Infinium HD Human Methylation 450K BeadChip platform with a companion application for repair and "restoration" of DNA from FFPE tissue. Our results showed good correlation between fresh and FFPE sample data. FFPE DNA captured 99% of the CpG sites on the array on average. Significant cancer subgroups based on the CpG island methylator phenotype (CIMP) were clearly distinguished for both fresh and FFPE sample sets with cluster and scaling analysis. The DNA methylation status for the five standard CIMP panel genes which was evaluated for all samples by the MethyLight assay was correctly assigned in both fresh and FFPE samples by the array data. We conclude that the "restoration" method followed by assay on the Infinium HD Human Methylation 450K microarray can produce good quality data for DNA from FFPE samples.

Somatische Tumormutationen eröffnen neue Therapieoptionen

Prognostic or predictive somatic genetic biomarkers influence therapeutic decisions in oncology. With the availability of next-generation sequencing (NGS) technologies, large numbers of somatic and germ line mutations can be analysed in parallel both, from formalin-fixed, paraffin-embedded tissue (FFPE) and fresh frozen tissue at high quality. In this study we evaluated the feasibility, turn-around time and diagnostic usefulness of highthroughput NGS analyses in the clinical setting and offered two different diagnostic sequencing panels to 52 selected patients.

Validation of Next Generation Sequencing Cancer Panels for Clinical Somatic Mutation Profiling-- Identification of Source of Variations and Artifacts using FFPE Tissues

Use of a Next Generation Sequencing (NGS)-based test as a clinical trial assay requires that the test be analytically validated to CLIA/CAP regulations. During the course of analytically validating NGS cancer panels for use as patient enrollment assays, we assessed the repeatability, reproducibility and accuracy of commercially available Cancer Panels (Illumina TruSeq Cancer Panel on MiSeq and Life Technologies AmpliSeq v2 Cancer Panel on Ion Torrent PGM). We measured the repeatability and reproducibility by evaluating all variant calls among technical replicates and found in both platforms that variants with higher variant frequency (VF >30%) were called with much higher repeatability and reproducibility than those with lower VF (between 5 and 25%), a level at which many somatic mutations are found. Also, Illumina MiSeq run-to-run reproducibility was significantly higher than that of Ion Torrent PGM. However, Illumina TruSeq library preparation protocol resulted in much lower repeatability than those obtained from Ion Torrent AmpliSeq protocol at the low VF range. To determine the optimal variant call settings, we used different sets of more stringent filters (lower false positive rate, but higher false negative rate), each platform could achieve close to 95% reproducibility and repeatability. Sequenom MassArray was used as a tie-breaker assay for discordant calls between the two NGS platforms to establish the “truth”. Our data provide insight into the steps that contribute most to variability, such as the procedure of library preparation, the sequencingby- synthesis chemistry, the factors that impact mutation calls and sampling variation. We also found very high C to T mutation calls associated with Illumina Cancer Panel using TruSeq library preparation protocol (but not with Ion Torrent using AmpliSeq protocol) when a less stringent filter set was used. The C to T artifact mutation calls from formalin-fixed paraffin-embedded (FFPE) tissue samples observed in this study together with high C to A artifact mutation calls from acoustic shearing of intact DNA observed by others have the potential to negatively impact mutation profiling and mutation signature identification if not carefully addressed. Based on these results we conclude library preparation protocols that start with PCR amplification, such as the AmpliSeq protocol, provide higher repeatability on variant calls with low VF and therefore, are more suitable for mutation profiling and mutation signature studies where somatic mutations (including unknown mutations) are the focus and balanced false positive and false negative rates are critical to success.

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.

High miR-21 expression from FFPE tissues is associated with poor survival and response to adjuvant chemotherapy in colon cancer

Colon cancer (CC) is a leading cause of cancer mortality. Novel biomarkers are needed to identify CC patients at high risk of recurrence and those who may benefit from therapeutic intervention. The aim of this study is to investigate if miR-21 expression from RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue sections is associated with prognosis and therapeutic outcome for patients with CC. The expression of miR-21 was measured by quantitative reverse transcriptase-polymerase chain reaction in a Japanese cohort (stage I-IV, n = 156) and a German cohort (stage II, n = 145). High miR-21 expression in tumors was associated with poor survival in both the stage II/III Japanese (p = 0.0008) and stage II German (p = 0.047) cohorts. These associations were independent of other clinical covariates in multivariable models. Receipt of adjuvant chemotherapy was not beneficial in patients with high miR-21 in either cohort. In the Japanese cohort, high miR-21 expression was significantly associated with poor therapeutic outcome (p = 0.0001) and adjuvant therapy was associated with improved survival in patients with low miR-21 (p = 0.001). These results suggest that miR-21 is a promising biomarker to identify patients with poor prognosis and can be accurately measured in FFPE tissues. The expression of miR-21 may also identify patients who will benefit from adjuvant chemotherapy.

Optimal Reference Genes for Normalization of qRT-PCR Data from Archival Formalin-fixed, Paraffin-embedded Breast Tumors Controlling for Tumor Cell Content and Decay of mRNA

Reliable determination of gene-expression levels from qRT-PCR requires accurate normalization of target genes to reference genes in order to remove nonbiological variation. Reference genes are ideally constitutively expressed in every cell, but many genes used for normalization has been shown to vary with tissue type, cellular proliferation, cancer progression, and degradation of nucleic acids. Gene-expression analysis is increasingly performed on degraded mRNA from formalin-fixed, paraffin-embedded tissue (FFPE), giving the option of examining retrospective cohorts. The aim of this study was to select robust reference genes showing stable expression over time in FFPE, controlling for various content of tumor tissue and decay of mRNA because of variable length of storage of the tissue. Sixteen reference genes were quantified by qRT-PCR in 40 FFPE breast tumor samples, stored for 1 to 29 years. Samples included 2 benign lesions and 38 carcinomas with varying tumor content. Stability of the reference genes were determined by the geNorm algorithm. mRNA was successfully extracted from all samples, and the 16 genes quantified in the majority of samples. Results showed 14% loss of amplifiable mRNA per year, corresponding to a half-life of 4.6 years. The 4 most stable expressed genes were CALM2, RPL37A, ACTB, and RPLP0. Several of the other examined genes showed considerably instability over time (GAPDH, PSMC4, OAZ1, IPO8). In conclusion, we identified 4 genes robustly expressed over time and independent of neoplastic tissue content in the FFPE block. Other widely used reference genes were concluded to be less suited for retrospective analysis of FFPE breast samples.

Evaluation of Mucin-1 protein and mRNA expression as prognostic and predictive markers after neoadjuvant chemotherapy for breast cancer

BackgroundMucin-1 (MUC1) is a promising antigen for the development of tumor vaccines. We evaluated the frequency of MUC1 expression and its impact on therapy response and survival after neoadjuvant chemotherapy for breast cancer. Patients and methodsPre-treatment core biopsies of patients from the GeparTrio neoadjuvant trial (NCT 00544765) were evaluated for MUC1 by immunohistochemistry (IHC; N = 691) and quantitative RT-PCR (qRT-PCR; N = 286) from formalin-fixed paraffin-embedded (FFPE) samples. ResultsMUC1 protein and mRNA was detectable in the majority of cases and was associated with hormone-receptor-positive status (P < 0.001). High MUC1 protein and mRNA expression were associated with lower probability of pathologic complete response (P = 0.017 and P < 0.001) and with longer patient survival (P = 0.03 and P < 0.001). In multivariable analysis, MUC1 protein and mRNA expression were independently predictive (P = 0.001 and P < 0.001). MUC1 protein and mRNA expression were independently prognostic for overall survival (P = 0.029 and P = 0.015). ConclusionsMUC1 is frequently expressed in breast cancer and detectable on mRNA and protein level from FFPE tissue. It provides independent predictive information for therapy response and survival after neoadjuvant chemotherapy. In clinical immunotherapy trials, MUC1 expression may serve as a predictive marker.

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Existence of feline morbillivirus infection in Japanese cat populations

Feline morbillivirus (FmoPV) is a member of a new virus species that has only been found in the Hong Kong cat population. For the first time, however, we have now detected nucleotide sequences similar to FmoPV in samples from Japanese cat populations. The positive rates for urine and blood samples from Japanese cats were 6.1 % (5/82) and 10 % (1/10), respectively. These sequences are similar to the previously reported FmoPV, with 92-94 % identity, and substantially different from all other morbilliviruses. Phylogenetic analysis of the identified Japanese FmoPVs and other morbilliviruses demonstrated a pattern similar to those previously published for the FmoPV viruses isolated in Hong Kong. FmoPV RNA was also detected from formalin-fixed paraffin-embedded (FFPE) kidney tissues of cats with nephritis, with a positive rate of 40 % (4/10). By using nested-set primers based on the FmoPV sequence and RNA from FFPE tissues, we demonstrated the existence of FmoPV infection in Japanese cats and established the method for detection of the FmoPV RNA from kidney tissues prepared for pathology examinations, which is useful for studies on the pathogenicity of the virus.

Long-term stability of RNA from FFPE brian tissue post-sectioning on slides.

e22142 Background: Analysis of nucleic acids (NA) from formalin fixed paraffin embedded (FFPE) tissue can provide detailed information about gene sequence mutational status, which may be important for oncology treatment decisions. FFPE specimens also have utility for retrospective analyses. Potential degradation of NA during formalin fixation, paraffin embedding processes and possible continued deterioration during subsequent storage may diminish utility of FFPE tissue for these purposes. Using real-time PCR, this study investigated the functional stability of RNA from brain FFPE tissue sections on slides over an extended time period after sectioning. Methods: Brain biopsy specimens obtained from glioblastoma patients with informed consent were used to prepare blocks with standard formalin fixation and paraffin embedding techniques. Slides were made from the FFPE blocks and stored at room temperature until testing. RNA was extracted from sequential slides within one week of sectioning for a zero time and then at 4, 8 and 12 months. Reverse transcription PCR was performed, and real-time PCR was analyzed on the ABI7900 to detect EGFRvIII mutation and cABL gene. RNA Integrity Analysis was performed with an Agilent Bioanalyzer. Results: Consistent qualitative results were obtained with EGFRvIII mutant positive specimens (n =10) and wild type (wt) specimens (n =10) from slides stored up to twelve months at room temperature compared to the initial testing (95% agreement). One wt specimen showed negative results for the first three time points but a low positive result at 12 months, possibly due to tumor content change in the different sections of the FFPE block. Ct values for EGFR (wt and mutant) and cABL genes did not increase during the storage period. RNA integrity number (RIN) indicated the degradation of RNA during FFPE processing, although no further significant degradation occurred during the course of the experiment. Conclusions: The results of this study indicated that although the RNA was impacted by the tissue preparation, fixation, and processing steps, for the brain FFPE slide specimens, target genes with amplicon size up to 124bp could be detected with minimum degradation for up to 12 months when slides were stored at room temperature.

Micro-RNA signature differences in lung adenocarcinoma with specific driver alterations.

11066 Background: Oncogenic driving alterations define types of lung adenocarcinoma with different prognosis and sensitivity to targeted agents. MicroRNAs (miRNAs) are a new class of non-coding RNAs involved in gene expression regulation. How miRNAs are dysregulated in lung cancer with ALK translocation, EGFR or KRAS mutation is unknown. In this study we aimed to identify miRNA signatures according to the presence of specific driver and to correlate miRNAs deregulation with patient outcome. Methods: The study was conducted in a cohort of 70 lung cancer patients (pts) including 18 ALK+ tumors, 11 ALK-/EGFR mutation+, 15 ALK-/KRAS mutation+, 26 ALK-/EGFR and KRAS wild-type and defined as triple negative. Matched normal lung tissue from 18 cases representative of the entire cohort were also included onto the analysis. RNA was isolated from formalin-fixed paraffin-embedded tissue (FFPE), using the Recover ALL kit (Ambion). NanoString nCounter system platform was used to generate the miRNA profile. We used Limma to test for differential expression analysis of data. The miR-515 family expression between tissues was validated by RT-qPCRs, analyzed using the parametric t-test (unpaired, 2-tailed for validation). Results: miRNA expression profile clusters distinctly ALK+ pts from ALK- and normal lung tissue. Within the ALK- group we found specific miRNAs subsets able to sub-stratify KRAS versus EGFR careers clustering sharply triple negative versus EGFR mutation+ and triple negative versus KRAS mutation+. miRNAs belonging to the miR-515 family seems to be the most deregulated in the ALK+ versus ALK-. Although their expression is stably high in normal tissues and ALK+ class, they are highly downregulated in KRAS mutated versus EGFR mutated and versus triple negative (p-value &lt;0.001 for all comparisons). Conclusions: miRNAs profile significantly differs in pts with ALK translocation, EGFR mutations and KRAS mutations. Analysis of miR-515 family members is ongoing in order to correlate their expression levels with pts’ outcome. In vitro modulation of miR-515 family expression levels, together with drugs treatment are ongoing in order to find possible chemo-resistance/chemo-sensitivity miRNA dependent, in ALK+ and ALK- model.

Abstract 4133: Comparison of qNPA vs qPCR on FFPE tissue.

Abstract Accurate, sensitive and robust multiplexed measurements of gene expression from formalin fixed paraffin embedded (FFPE) tissue or paraformaldehyde fixed samples are ideally required for clinical diagnostic tests and retrospective analysis of archived samples. We evaluated the measurement of gene expression from FFPE tissues using HTG Molecular's qNPA™ assay and made comparisons to qPCR. The results show that qNPA provides a highly sensitive, accurate, quantitative, and robustly reliable automated multiplexed assay of gene expression from FFPE, permitting the rapid development and launch of new diagnostic and research assays. qNPA uses a lysis-only, extraction-free protocol which measures up to 47 genes/sample. In contrast, qPCR requires RNA extraction/reverse transcription. Accuracy was determined by correlating measurements from matched frozen and fixed samples, determining the R2 correlation coefficient. Comparing frozen/FFPE cell pellets qNPA R2 = 0.97. Comparing frozen/FFPE pancreas tissue qNPA R2= 0.97 with a present call rate of 91%, compared to qPCR R2= 0.86 and a present call rate of just 17%, even using 26 times more FFPE sample amount than qNPA. Comparing fresh/paraformaldehyde fixed Islet cells, qNPA R2= 0.98, qPCR R2= 0.02, after staining for sorting by cytometry qNPA R2= 0.96, qPCR R2= 0.12. Thus, qNPA fixed tissue measurements were much more accurate than qPCR. In an additional comparison using matched breast cancer frozen compared with FFPE samples, qNPA measurements averaged R2=0.81, with 6% CV for triplicate measurements of separately processed samples. For &amp;gt;95% of genes the expression levels measured by qNPA were independent of cold ischemic times of 0 to 16 hr. Measured levels are also independent of fixation from 4 to 72 hr. The robust performance of qNPA measurements from FFPE translated into quantitative consistency that has previously not been achievable using qPCR. Data from clinical FFPE samples submitted by a large number of HTG's clients was reviewed and for each set the number of samples tested, the average area tested/sample (cm2 area of a 5 micron thick section), the failure rate (FR), and the avg reproducibility (%CV for samples independently processed in triplicate) was determined for breast FFPE (300 samples, 0.3 cm2/sample, 1% FR, 10.5%CV), lung FFPE (700 samples, 0.3 cm2/sample, 0.5% FR, 9.5%CV), prostate FFPE (15 samples, 0.3 cm2/sample, 0% FR, 11%CV), colon FFPE (15 samples, 0.3 cm2/sample, 0% FR, 9%CV), ovary FFPE (15 samples, 0.3 cm2/sample, 0% FR, 10%CV) and lymphoma FFPE (300 samples, 0.3 cm2/sample, 0.5% FR, 8%CV). Thus, qNPA provides a highly accurate, reproducible, robust multiplexed measurement of gene expression from FFPE tissue that is not affected by wide variations in ischemic or fixation time. Fully automated qNPA on the EDGE platform is expected in Jan 2013. This system, with a turn-around time of less than 24 hours, will improve even further the robustness of extraction-free gene expression. Citation Format: Matthew B. Rounseville, Klaus Pechhold, Debrah Thompson, Mark Schwartz, Bruce Seligmann. Comparison of qNPA vs qPCR on FFPE tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4133. doi:10.1158/1538-7445.AM2013-4133

Abstract 3213: A rapid, sensitive and quantitative method to measure protein biomarkers MUC16 and MSLN from archived clinical samples using TaqMan proximity ligation assays.

Abstract MUC16 and MSLN are tumor-specific antigens highly prevalent in ovarian and pancreatic cancers and have been used as targets in developing antibody-drug conjugates (ADCs). Development of a sensitive method to quantitatively measure these protein biomarkers in archival clinical samples may enable potential companion diagnostics to these novel therapeutic agents. Here we report the development of a method to quantitatively measure these biomarkers from formalin fixed paraffin embedded (FFPE) tumor samples using TaqMan proximity ligation assays (PLA). Monoclonal antibodies that recognize the extracellular domains of MUC16 and MSLN were conjugated to amplicon forming oligonucleotides to generate proximity ligation probes for TaqMan quantification. Using purified MUC16 and MSLN recombinant protein the assay sensitivities were determined to be lower than single-digit ng/mL concentrations for both MUC16 and MSLN and the dynamic ranges were &amp;gt;3 logs and ∼2 logs, respectively. In addition, the assays were demonstrated to be highly selective by testing lysates from expressing and non-expressing cell lines as determined by qRT-PCR. For clinical application of these assays, we further developed and optimized a simple two-step method for extraction and antigen retrieval of protein from archival FFPE tissues. FFPE extracts were prepared from a set of 10um sections of ovarian tumor tissue blocks and analyzed to determine relative MUC16 and MSLN protein expression levels. We benchmarked these results from the FFPE protocol with PLA data generated from matched fresh frozen tumor tissue as well as expression level determined by immunohistochemistry (IHC) and gene expression level determined by qRT-PCR assays from the same FFPE tissue blocks. All three comparisons with FFPE PLA were remarkably well correlated. In conclusion, we have established the technical feasibility and clinical utility of TaqMan PLA-based protein assays for sensitive and quantitative measurement of protein biomarkers in archival clinical samples. The methodology developed in this study represents a promising approach in companion diagnostic development for quantitative protein biomarker assessment. Citation Format: Lisa Ryner, Aimee Fourie, Siao Ping Tsai, Suzie Scales, Kathy Kozak, Ron Firestein, Yinghui Guan, An Do, Yulei Wang. A rapid, sensitive and quantitative method to measure protein biomarkers MUC16 and MSLN from archived clinical samples using TaqMan proximity ligation assays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3213. doi:10.1158/1538-7445.AM2013-3213

Abstract 2005: Analysis of gene expression and copy number variation in breast tumors using both sequencing and hybridization-based platforms.

Abstract Next Generation Sequencing (NGS) technologies provide rapid genomic analyses of single nucleotide variants, RNA expression and DNA copy number. Application of these technologies to material isolated from formalin fixed paraffin embedded (FFPE) tissue and even degraded frozen material could provide powerful replication samples but remains challenging. We tested the nanoString platform to validate deep sequence analysis of gene expression and DNA copy number in degraded and FFPE material. Firstly, RNA from the Universal Human Reference RNA and a breast cancer cell line (MDA-MB-436) was artificially degraded to different degrees (RIN 1.2-6.8). We used the nanoString platform to simultaneously measure RNA expression across 226 genes in each degraded sample and the corresponding undegraded RNA. Secondly we isolated RNA and DNA from matched fresh frozen and FFPE tissues from nine breast cancer patients (3 HER2+/ER+/PR+, 2 HER2+/ER-/PR-, 2 HER2+/ER+/PR-, 2 HER2-/ER+/PR+) using the nanoString platform to compare expression and copy number across 226 and 86 genes respectively. Finally, we correlated expression and copy number data generated by nanoString with Illumina transcriptome and whole genome sequencing (WGS). NanoString log2 expression fold-change between all artificially degraded samples and their undegraded counterpart showed extremely high correlation (r2&amp;gt;0.91). NanoString DNA copy number between matched fresh-frozen and FFPE showed a high degree of correlation (r2=0.71). All gene amplifications with copy number ≥ 5 in DNA from fresh-frozen material (N=9) were successfully identified in DNA from FFPE material. We also observed good correlation of gene expression between whole transcriptome sequencing and the nanoString platform (r2 0.59 - 0.72) in FFPE and artificially degraded material and for DNA copy number between WGS and nanoString in DNA isolated from cancer cell lines (r2=0.96). The nanoString platform provides reliable data from highly degraded and FFPE material and correlates with sequence analysis of both expression and copy number from NGS platforms demonstrating potential for large-scale replication studies in FFPE material. Citation Format: Nadine Norton, Edith A. Perez, Yan W. Asmann, Jennifer M. Carr, Brian M. Necela, Jennifer M. Kachergus, Jin Jen, Bruce W. Eckloff, E Aubrey Thompson. Analysis of gene expression and copy number variation in breast tumors using both sequencing and hybridization-based platforms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2005. doi:10.1158/1538-7445.AM2013-2005

Abstract 1999: A targeted exome sequencing platform for routine clinical molecular profiling of advanced thoracic malignancies relevant to targeted therapy.

Abstract Increasing number of oncogenic mutations important to the clinical management of advanced thoracic malignancies is being identified. To translate this into routine clinical testing environment, we developed and validated a targeted exome sequencing method using next generation sequencing (NGS) technology that is designed to detect both single nucleotide variants and short insertions or deletions (indels) by employing a custom designed target selection system. In each individual assay, 197 cancer related genes, including 12 clinically relevant genes (AKT1, AKT2, BRAF, EGFR, ERBB2, HRAS, KIT, KRAS, MEK1, NRAS, PIK3CA, and PTEN) are simultaneously sequenced from 100-500ng of total DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. To date, we have performed this assay on over 250 FFPE tissues from Non-Small-Cell Lung Cancers (NSCLC), Small-Cell Lung Cancers (SCLC), and thymic malignancies. Mutations in all 12 clinically relevant genes including EGFR L858R, KRAS codon 12 missense mutations as well as EGFR exon 19 deletions, EGFR exon 20 insertions, and ERBB2 exon 20 insertions have been identified and validated with alternate technology in approximately 30% of all samples tested. Compared with conventional capillary sequencing, which typically requires in excess of 25% mutant DNA, this assay can detect mutations with 5% - 10% mutant allele frequency. This is a robust, reliable and cost effective assay that not only is suitable for prospective molecular profiling of clinically relevant targets, but also provides a powerful tool for retrospective analysis of existing FFPE tissues from clinical trials. Citation Format: Christopher C. Lau, Keith Killian, Yuelin J. Zhu, Yonghong Wang, Marbin Pineda, Anish Thomas, Yisong Wang, Betsy Morrow, Liqiang Xi, Mark Raffeld, Giuseppe Giaccone, Paul S. Meltzer. A targeted exome sequencing platform for routine clinical molecular profiling of advanced thoracic malignancies relevant to targeted therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1999. doi:10.1158/1538-7445.AM2013-1999

Abstract 3061: Micro-RNA signature differences in lung cancer patients with ALK translocation, EGFR mutations and KRAS mutations.

Abstract Background: Oncogenic driving mutations identify lung adenocarcinoma with different prognosis and sensitivity to targeted therapies. miRNAs are a new class of non-coding RNA involved in gene expression regulation and recent studies have suggested that miRNAs could be useful for stratifying lung cancer subtypes. How miRNAs are dysregulated in lung cancer patients with ALK translocation, EGFR or KRAS mutation is unknown. Aim of the present study is to identify miRNA signature differences according to the presence of specific oncogenic driver and correlation between miRNAs deregulation and drug response. Materials and methods: The study was conducted in a cohort of 70 lung cancer patients including 68 primary cancer tissue (17 ALK positive tumors, 11 ALK negative EGFR positive tumors, 15 ALK negative KRAS positive tumors, 24 ALK negative EGFR and KRAS negative tumors defined as wild-type) with matched normal lung tissue (N=18) and 2 metastatic tissue. RNA was isolated from formalin-fixed paraffin-embedded tissue (FFPE), using the Recover ALL kit (Ambion). Data were processed according to manufacture guidelines. We used Limma to test for differential expression analysis of data. The miRNAs expression between tissues for all RT-qPCR was analyzed using the parametric t-test (unpaired, 2-tailed for validation). Results: miRNA expression profile, cluster distinctly ALK+ patients from ALK- and normal lung tissue. Furthermore we found specific miRNAs subsets, within the ALK- group, able to sub-stratify KRAS versus EGFR careers and cluster sharply wild-type versus EGFR and wild-type versus KRAS as well. miRNAs belonging to the miR-515 family seems to be the most deregulated in the ALK+ vs ALK- comparison. Although their expression is stably high in normal tissues and ALK+ class they are highly downregulated in KRAS mutated versus EGFR mutated and versus ALK-/KRAS wild type/EGFR wild type (p-value &amp;lt;0.001 for all comparisons). Conclusions: miRNAs profile significantly differs in lung cancer patients with ALK translocation, EGFR mutations and KRAS mutations. Molecular analysis of the pathways involving the miR-515 family is ongoing in order to correlate their expression levels with patients’ outcome. Since miRNAs can induce resistance or sensitize to chemotherapies, in vitro modulation of miR-515 family expression level together with drugs test are ongoing in order to find better targeted therapies for ALK+ and ALK- patients. Citation Format: Pierluigi Gasparini, Lorenza Landi, Stefania Carasi, Carmelo Tibaldi, Luciano Cascione, Greta Alì, Armida D'Incecco, Gabriele Minuti, Antonio Chella, Gabriella Fontanini, Federico Cappuzzo, Carlo M. Croce. Micro-RNA signature differences in lung cancer patients with ALK translocation, EGFR mutations and KRAS mutations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3061. doi:10.1158/1538-7445.AM2013-3061 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.