Formalin-fixed And Paraffin-embedded Tissue Research Articles

Novel Assay to Detect RNA Polymerase I Activity In Vivo.

This report develops an analytically validated chromogenic in situ hybridization (CISH) assay using branched DNA signal amplification (RNAscope) for detecting the expression of the 5' external transcribed spacer (ETS) of the 45S ribosomal (r) RNA precursor in formalin-fixed and paraffin-embedded (FFPE) human tissues. 5'ETS/45S CISH was performed on standard clinical specimens and tissue microarrays (TMA) from untreated prostate carcinomas, high-grade prostatic intraepithelial neoplasia (PIN), and matched benign prostatic tissues. Signals were quantified using image analysis software. The 5'ETS rRNA signal was restricted to the nucleolus. The signal was markedly attenuated in cell lines and in prostate tissue slices after pharmacologic inhibition of RNA polymerase I (Pol I) using BMH-21 or actinomycin D, and by RNAi depletion of Pol I, demonstrating validity as a measure of Pol I activity. Clinical human prostate FFPE tissue sections and TMAs showed a marked increase in the signal in the presumptive precursor lesion (high-grade PIN) and invasive adenocarcinoma lesions (P = 0.0001 and P = 0.0001, respectively) compared with non-neoplastic luminal epithelium. The increase in 5'ETS rRNA signal was present throughout all Gleason scores and pathologic stages at radical prostatectomy, with no marked difference among these. This precursor rRNA assay has potential utility for detection of increased rRNA production in various tumor types and as a novel companion diagnostic for clinical trials involving Pol I inhibition.Implications: Increased rRNA production, a possible therapeutic target for multiple cancers, can be detected with a new, validated assay that also serves as a pharmacodynamic marker for Pol I inhibitors. Mol Cancer Res; 15(5); 577-84. ©2017 AACR.

Unlocking Cancer Glycomes from Histopathological Formalin-fixed and Paraffin-embedded (FFPE) Tissue Microdissections

N- and O-glycans are attractive clinical biomarkers as glycosylation changes in response to diseases. The limited availability of defined clinical specimens impedes glyco-biomarker identification and validation in large patient cohorts. Formalin-fixed paraffin-embedded (FFPE) clinical specimens are the common form of sample preservation in clinical pathology, but qualitative and quantitative N- and O-glycomics of such samples has not been feasible to date. Here, we report a highly sensitive and glycan isomer selective method for simultaneous N- and O-glycomics from histopathological slides. As few as 2000 cells isolated from FFPE tissue sections by laser capture microdissection were sufficient for in-depth histopathology-glycomics using porous graphitized carbon nanoLC ESI-MS/MS. N- and O-glycan profiles were similar between unstained and hematoxylin and eosin stained FFPE samples but differed slightly compared with fresh tissue. This method provides the key to unlock glyco-biomarker information from FFPE histopathological tissues archived in pathology laboratories worldwide.

Choline Transporter Immunohistochemistry: An Effective Substitute for Acetylcholinesterase Histochemistry to Diagnose Hirschsprung Disease With Formalin-fixed Paraffin-embedded Rectal Biopsies.

Acetylcholinesterase enzymatic histochemistry (AChE EHC), which highlights abnormal cholinergic nerves in the mucosa of aganglionic bowel, has been used for decades to evaluate rectal biopsies for Hirschsprung disease (HSCR). While useful diagnostically, AChE EHC is not compatible with conventional formalin-fixed and paraffin-embedded (FFPE) tissues and is not widely available. The choline transporter (ChT) is a putative alternative marker of cholinergic nerves. ChT immunohistochemistry (IHC) was investigated using FFPE biopsies and resections from patients with confirmed HSCR, as well as appropriate non-HSCR controls. ChT immunostaining was effective at identifying cases with HSCR and qualitatively similar to AChE EHC on frozen section. Among 3 pathologists, the diagnostic positive and negative predictive values based on ChT IHC ranged from 0.84-0.94 and 0.85-0.89, respectively, with good inter-observer agreement (Cohen kappa = 0.70-0.90). ChT IHC was useful in unusual scenarios in which calretinin (CR) IHC failed to correctly identify patients with HSCR. In 10 cases of short-segment HSCR, abnormal ChT+ mucosal innervation was present through the entire aganglionic segment and into portions of the TZ with submucosal nerve hypertrophy. In contrast, mucosal CR IHC was retained in the TZ and adjacent aganglionic bowel, which could lead to misinterpretation of a biopsy as ganglionic bowel. Indeed, 6 such patients were identified with paradoxical CR-positive mucosal innervation in their diagnostic biopsies. ChT IHC was interpreted as unequivocal HSCR in these cases, and HSCR was confirmed on resection. In summary, ChT IHC in FFPE tissue demonstrates high positive and negative predictive values for HSCR, is superior to CR IHC in a subset of cases, and can be incorporated into routine practice without the need for specialized techniques.

Abstract P6-09-23: SETER/PR - A robust 18-gene predictor of sensitivity to endocrine therapy in metastatic breast cancer

Abstract Rationale: A robust index for gene expression related to activity of estrogen (ESR1) and progesterone (PGR) receptors could predict sensitivity to endocrine therapy in metastatic breast cancer. Methods: Transcripts correlated with ESR1 and PGR expression in 389 hormone receptor-positive breast cancer samples (Affymetrix U133A microarrays) were ranked for reliability according to their pre-analytical (intratumoral heterogeneity, biopsy type) and analytical reproducibility. Eighteen target and ten reference genes were selected and summarized as the SETER/PR index. The SETER/PR index was evaluated in a different set of 140 biopsies from distant metastases of hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer, and in additional pre-analytical and analytical sample cohorts. Thereafter, SETER/PR was translated to a customized format for application to formalin-fixed and paraffin-embedded (FFPE) sections. Results: Higher SETER/PR in a metastasis was associated with longer progression-free survival (PFS, 9 vs. 2 months) and overall survival (OS, 50 vs. 19 months) following endocrine therapy in the cohort with metastatic breast cancer (MBC) and relapsed disease (n=79), so a cut point was defined in that cohort. SETER/PR was also significantly associated with PFS after adjusting for PR status of the metastasis, presence of visceral metastases, number of previous relapse events, and clinical history of previous sensitivity to endocrine therapy (HR 0.485, 95%CI 0.265 – 0.889, p = 0.019). Technically, SETER/PR was highly reproducible under different pre-analytical and analytical conditions, including host organ contamination. The translated SETER/PR assay used a single 10 µm FFPE tissue section, did not require RNA purification, and represented the microarray results from matched fresh samples with excellent agreement (correlation = 0.980, n = 31). Conclusion: The SETER/PR index is a new biomarker to predict PFS and OS for patients with HR+/HER2- MBC who receive endocrine therapy. The assay is applicable to FFPE tissue sections from small biopsies of metastases. Additional independent (blinded) validation studies will be necessary to confirm these results.Rationale: A robust index for gene expression related to activity of estrogen (ESR1) and progesterone (PGR) receptors could predict sensitivity to endocrine therapy in metastatic breast cancer. Methods: Transcripts correlated with ESR1 and PGR expression in 389 hormone receptor-positive breast cancer samples (Affymetrix U133A microarrays) were ranked for reliability according to their pre-analytical (intratumoral heterogeneity, biopsy type) and analytical reproducibility. Eighteen target and ten reference genes were selected and summarized as the SETER/PR index. The SETER/PR index was evaluated in a different set of 140 biopsies from distant metastases of hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer, and in additional pre-analytical and analytical sample cohorts. Thereafter, SETER/PR was translated to a customized format for application to formalin-fixed and paraffin-embedded (FFPE) sections. Results: Higher SETER/PR in a metastasis was associated with longer progression-free survival (PFS, 9 vs. 2 months) and overall survival (OS, 50 vs. 19 months) following endocrine therapy in the cohort with metastatic breast cancer (MBC) and relapsed disease (n=79), so a cut point was defined in that cohort. SETER/PR was also significantly associated with PFS after adjusting for PR status of the metastasis, presence of visceral metastases, number of previous relapse events, and clinical history of previous sensitivity to endocrine therapy (HR 0.485, 95%CI 0.265 – 0.889, p = 0.019). Technically, SETER/PR was highly reproducible under different pre-analytical and analytical conditions, including host organ contamination. The translated SETER/PR assay used a single 10 µm FFPE tissue section, did not require RNA purification, and represented the microarray results from matched fresh samples with excellent agreement (correlation = 0.980, n = 31). Conclusion: The SETER/PR index is a new biomarker to predict PFS and OS for patients with HR+/HER2- MBC who receive endocrine therapy. The assay is applicable to FFPE tissue sections from small biopsies of metastases. Additional independent (blinded) validation studies will be necessary to confirm these results. Citation Format: Sinn BV, Tsai T-H, Lau R, Fu C, Gould R, Murthy R, King TA, Hatzis C, Kwiatkowski DN, Valero V, Symmans WF. SETER/PR - A robust 18-gene predictor of sensitivity to endocrine therapy in metastatic breast cancer [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 P6-09-23.

A methodological study of genome-wide DNA methylation analyses using matched archival formalin-fixed paraffin embedded and fresh frozen breast tumors

BackgroundDNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples.ResultsFor 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER– tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types.Materials and MethodsRestored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (β-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER−) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort.ConclusionsFFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues.

MA17.02 Genome-Wide Copy Number and Mutational Analysis in Longitudinal Biopsies of Matched Primary and Metastatic Pulmonary Adenocarcinomas

There are still limited data on the extent of intratumoral heterogeneity of cancer gene mutations and genome-wide copy number aberrations between primary tumors and metastases in non-small cell lung cancers (NSCLC). Deconvolution of the intermixture of tumor and stromal components remains a major challenge for such analysis. To overcome these limitations, we applied a refined nuclei flow sorting approach on matched longitudinal biopsies (primary/metastasis) from pulmonary adenocarcinomas. Multiparameter Ploidy Profiling (MPP) comprises the isolation of nuclei from frozen or formalin-fixed and paraffin embedded (FFPE) tissues, followed by multiparameter flow sorting by DAPI for DNA content (ploidy) and TTF1 as a lineage marker to enrich for tumor cell nuclei. Homogenous TTF1 expression was ascertained by immunohistochemistry. Sorted populations were subjected to genomic profiling by high resolution aCGH and NGS with the Ion Torrent™ Comprehensive Cancer Panel. This approach allows for the detection of genome-wide copy number aberrations and provides all exon-coverage of 409 well-known cancer genes. Sequencing was performed with a mean depth of 965x. MPP was successfully applied on 44 frozen or FFPE tissue specimens from 19 patients. Clonally unrelated secondary primaries were found in three patients, defined by the absence of both shared copy number (CN) transition and somatic mutations. The concordance rate between primary tumor and corresponding metastases was 65.2% and reached 85.5% for mutations and copy number amplifications/deletions in the top 12 affected genes (including CDKN2A, KRAS, ATM, KEAP1, EGFR and STK11). The correlation of the allele frequencies between primary tumors and metastases was linear (r=0.87, p<0.001), irrespective of the time interval between the tissue resections. Overall, ploidy was not different between primary tumors and metastases. Additionally, the metastases did not bear a higher burden of private events (CN transitions and somatic mutations) than the primary tumors. MPP is a powerful method to increase the precision of downstream analysis due to unprecedented purity of tumor DNA. Our data argue for a high concordance rate of mutations and CN transitions between primary tumors and their corresponding metastases. Intriguingly, the ploidy remains remarkably stable during progression even after long time-periods, which suggests chromosomal stability with a limited degree of macroevolutionary shifts over time and space. Taken together, our data suggest the presence of at least two evolutionary patterns: 1) early/branched and 2) late/linear progression, with a continuum from high to low genetic divergence of the primary tumor and metastases to their most recent common ancestor.

MA02.02 A Novel 5-miR Signature Shows Promise as a Diagnostic Tool and as a Predictor of Cisplatin Response in NSCLC

MicroRNAs are a class of small non-coding RNAs that range in size from 19-25 nucleotides. They have been shown to regulate a number of processes within tumor biology, including metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemoresistance in solid tumors, including lung cancer. MicroRNA expression within an isogenic panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines was profiled using the 7th generation miRCURY LNA arrays (Exiqon). Significantly altered miRNAs within the CisR sublines were manipulated using antagomirs (Exiqon) and Pre-miRs (Ambion) and functional studies were carried out in the presence and absence of cisplatin. To examine the translational relevance of these miRNAs, their expression was examined in a cohort of chemo-naïve patient-matched normal and lung tumor tissue and serum from NSCLC patients of different histologies. To create a xenograft model of cisplatin resistance 1x103 cells H460 PT or CisR cells were injected into 5-7week old NOD/SCID mice. Tumor volume was measured over time and harvested once the tumor mass measured 500mm3 and formalin-fixed and paraffin embedded (FFPE). Expression of the 5-miR signature was analysed within FFPE murine tumors and cisplatin resistance was investigated relative to cisplatin sensitive controls. Profiling and subsequent validation revealed a 5-miR signature associated with our model of cisplatin resistance (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286). Inhibition of the miR-30 family and miR-34a-5p reduced clonogenic survival of CisR cells when treated cisplatin. Expression of the miRNA signature was significantly altered in both adenocarcinoma (AD) and squamous cell carcinoma (SCC) relative to matched normal lung tissue and between SCC and AD tissue. miR-4286 was significantly up-regulated in SCC sera compared to normal control and AD sera. Similarly to the cell line expression of the miRNAs, the miR-30 family members and miR-34a-5p were up-regulated in the CisR xenograft FFPE tissue relative to PT. A novel miRNA signature associated with cisplatin resistance was identified in vitro, genetic manipulation of which altered clonogenic response to cisplatin. The 5-miR signature shows both diagnostic and prognostic biomarker potential across a number of diagnostically relevant biological mediums.

Characterization of Genomic Rearrangements Involving CIITA and SOCS1 Using Targeted Capture Sequencing of Archival Tissue Specimens

Introduction: Malignant lymphomas account for 5% of all newly diagnosed cancer cases per year and affect patients of all ages. Genomic rearrangements represent a pathogenic hallmark of most B-cell lymphoma entities and some are associated with an unfavorable clinical outcome. Recurrent structural genomic aberrations involving the MHC class II transactivator CIITA (located on chromosome 16p13) have been identified in multiple lymphoma subtypes in which they contribute to an immune escape phenotype. Moreover, inactivating mutations of the tumor suppressor gene SOCS1, located in close proximity to CIITA on chromosome 16, result in constitutively active JAK-STAT-signaling across a spectrum of lymphomas. Preliminary data from our group indicate that SOCS1 is also involved in recurrent rearrangements. However, more detailed study of these rearrangements was hampered due to the lack of analytic methods and platforms applicable to archival formalin-fixed and paraffin-embedded tissue (FFPET) specimens. Here, using FFPET biopsies, we sought to characterize the comprehensive landscape of CIITA and SOCS1rearrangement partner genes, determine the exact breakpoint anatomy, and study the functional impact of individual alterations.Methods: In order to select cases for DNA extraction and subsequent sequencing analysis we revisited the results of previously conducted fluorescence in-situ hybridization (FISH) experiments performed on lymphoma cases arranged on tissue microarrays. Cases were included based on the presence of rearrangements in CIITA, SOCS1 and the 9p24.1 locus. 92 specimens met these criteria and DNA was extracted using the Qiagen AllPrep DNA/RNA FFPE kit. An optimized 96-well-plate protocol was used for whole genome library construction (FFPET small gap protocol) and 16 to 18 libraries were multiplexed and pooled prior to capture using a custom Agilent SureSelect design targeting the aforementioned loci. Sequencing was performed on an Illumina HiSeq 2500. Predicted structural variants (SV) were generated with the computational tools DELLY and deStruct, and subsequently filtered based on read support. High confidence predictions were validated by either PCR-amplification of the genomic DNA spanning the breakpoint sequence followed by Sanger Sequencing, or customized FISH assays.Results: Library construction was successful in 68 cases (74%) and the mean coverage across the target regions for these libraries was above 600x with 97.7% of bases having an average depth of > 100x. After filtering, a list of 82 rearrangement events was generated consisting of 16 translocations (12 affecting CIITA and 4 affecting SOCS1), 44 deletions, 19 inversions and 3 duplications. Mapping of the translocation breakpoints revealed two distinct breakpoint cluster regions within intron 1 of CIITA and exon 2 of the SOCS1 gene. None of the exact breakpoints were recurrent and identification of rearrangement partner genes confirmed that CIITA and SOCS1 rearrange promiscuously. Furthermore, we demonstrate that some translocation events result in the generation of novel in-frame fusion transcripts (e.g. CIITA-PRDM16), warranting further functional characterization. Consistent with previous studies, intra-chromosomal alterations were detected in intron 1 of CIITA in48% of the cases while SOCS1 was shown to be frequently deleted (20% of cases).Overall, the concordance between FISH and targeted capture sequencing was 63%, reflecting the differences in sensitivity to detect large- and small-scale genomic events.Conclusions: Targeted capture sequencing, in conjunction with FISH, represents a valuable tool to explore the spectrum of genomic structural variants and rearrangement partner genes in archival lymphoma FFPET specimens. Future studies are required to address the functional impact of specific alterations and the utility of capture-sequencing-based assays for clinical decision-making. DisclosuresScott:NanoString Technologies: Patents & Royalties: named inventor on a patent for molecular subtyping of DLBCL that has been licensed to NanoString Technologies.

Depth-Sensitive Raman Spectroscopy of Intact Formalin-Fixed and Paraffin-Embedded Tissue Blocks for Objective Diagnosis of Cancer- An Exploratory Study

Histopathology, the current “gold standard is prone to human errors as it depends on expert interpretation of the microscopically derived cellular and sub-cellular information for tissue diagnosis. Further, this light microscope based approach requires preparation of appropriately stained specimens of micro-thin tissue sections from the formalin-fixed and paraffin-embedded (FFPE) blocks of tissue samples. We report a method that provides quantitative feedback about tissue diagnosis by measuring depth-sensitive Raman spectra from the intact FFPE tissue blocks without requiring preparation of any thin tissue sections or any other processing. The FFPE blocks of pathologically certified cancerous and normal breast tissues were used for validating the approach. The measured depth-sensitive Raman spectra were mathematically de-paraffinized for retrieving the characteristic tissue Raman signatures using scaled-subtraction. A multivariate analysis of the scaled-subtracted, depth-sensitive Raman spectra employing a probability-based diagnostic algorithm developed using the framework of sparse multinomial logistic regression (SMLR) provided a sensitivity and specificity of up to 100% towards cancer based on leave-one-block-out cross validation. The results of this exploratory study suggest that depth-sensitive Raman spectroscopy along with a multivariate statistical algorithm can provide a valuable alternate diagnostic modality in clinical pathology setting for discriminating cancerous from normal FFPE tissue blocks.

DNA methylation profiling of esophageal adenocarcinoma using Methylation Ligation-dependent Macroarray (MLM)

Most types of cancer cells are characterized by aberrant methylation of promoter genes. In this study, we described a rapid, reproducible, and relatively inexpensive approach allowing the detection of multiple human methylated promoter genes from many tissue samples, without the need of bisulfite conversion. The Methylation Ligation-dependent Macroarray (MLM), an array-based analysis, was designed in order to measure methylation levels of 58 genes previously described as putative biomarkers of cancer. The performance of the design was proven by screening the methylation profile of DNA from esophageal cell lines, as well as microdissected formalin-fixed and paraffin-embedded (FFPE) tissues from esophageal adenocarcinoma (EAC). Using the MLM approach, we identified 32 (55%) hypermethylated promoters in EAC, and not or rarely methylated in normal tissues. Among them, 21promoters were found aberrantly methylated in more than half of tumors. Moreover, seven of them (ADAMTS18, APC, DKK2, FOXL2, GPX3, TIMP3 and WIF1) were found aberrantly methylated in all or almost all the tumor samples, suggesting an important role for these genes in EAC. In addition, dysregulation of the Wnt pathway with hypermethylation of several Wnt antagonist genes was frequently observed. MLM revealed a homogeneous pattern of methylation for a majority of tumors which were associated with an advanced stage at presentation and a poor prognosis. Interestingly, the few tumors presenting less methylation changes had a lower pathological stage. In conclusion, this study demonstrated the feasibility and accuracy of MLM for DNA methylation profiling of FFPE tissue samples.

Single-strand DNA library preparation improves sequencing of formalin-fixed and paraffin-embedded (FFPE) cancer DNA.

DNA derived from formalin-fixed and paraffin-embedded (FFPE) tissue has been a challenge to large-scale genomic sequencing, due to its low quality and quantities. Improved techniques enabling the genome-wide analysis of FFPE material would be of great value, both from a research and clinical perspective.Comparing a single-strand DNA library preparation method originally developed for ancient DNA to conventional protocols using double-stranded DNA derived from FFPE material we obtain on average 900-fold more library molecules and improved sequence complexity from as little as 5 ng input DNA. FFPE DNA is highly fragmented, usually below 100bp, and up to 60% of reads start after or end prior to adenine residues, suggesting that crosslinks predominate at adenine residues. Similar to ancient DNA, C > T substitutions are slightly increased with maximum rates up to 3% at the ends of molecules. In whole exome sequencing of single-strand libraries from lung, breast, colorectal, prostate and skin cancers we identify known cancer mutations. In summary, we show that single-strand library preparation enables genomic sequencing, even from low amounts of degraded FFPE DNA. This method provides a clear advantage both in research and clinical settings, where FFPE material (e.g. from biopsies) often is the only source of DNA available. Improving the genetic characterization that can be performed on conventional archived FFPE tissue, the single-strand library preparation allows scarce samples to be used in personalized medicine and enables larger sample sizes in future sequencing studies.

HPV16 detection by qPCR method in relation to quantity and quality of DNA extracted from archival formalin fixed and paraffin embedded head and neck cancer tissues by three commercially available kits

The aim of the present study was to compare HPV16 detection by quantitative polymerase chain reaction (qPCR) in relation to the quantity and quality of DNA isolated from 21 formalin fixed and paraffin embedded (FFPE) head and neck cancer tissues by three commercially available kits: EX-WAX™ DNA Extraction Kit (M) (Merck Millipore, Darmstadt, Germany), QIAamp® DNA FFPE Tissue (Q) (Qiagen, Hilden, Germany) and ReliaPrep™ FFPE gDNA Miniprep System (P) (Promega, Madison, USA). Quantity of extracted DNA was assessed spectrophometrically and fluorometrically. Its quality was analyzed using A260/280 and A260/230 ratios and the β-actin fragment amplifiability in qPCR. HPV16 presence was detected by qPCR, using specific primers and TaqMan probe. HPV infection was found in 8 DNA samples extracted with M kit (38.1%) and in 7 (33.3%) isolated with Q and P kits. Three samples from M and Q kits were characterized by HPV16 positivity and lack of β-actin amplifiability. They had significantly lower A260/280 ratio (M: 1.6±0.0, p=0.044 and Q: 1.7±0.0, p=0.016) compared to samples with both fragments amplification (M: 1.7±0.0 and Q: 1.9±0.0). Therefore, for HPV detection by qPCR in FFPE tissues we recommend ReliaPrep™ FFPE gDNA Miniprep System.

Abstract 3616: Fix the fixation: effect of formalin fixation on targeted sequencing, variant calling and gene expression

Abstract Tumor biopsies are often Formalin-Fixed and Paraffin-Embedded (FFPE) for histological staining, genetic testing and archival purposes. Formalin treatment preserves tissue by crosslinking proteins, but can lead to mutation of the nucleic acid bases that can be detected by next-generation sequencing (NGS) methods despite being unrelated to the cancer. Studies have shown that certain fixation protocols are compatible with high quality nucleic acid isolation and variant calling by NGS. These studies used tissue fixed with 10% neutral buffered formalin for 24 hours, a standard protocol in the pathology field. In practice, however, FFPE sample handling varies from site-to-site. As we processed FFPE samples from various sites, we found that the quality of the isolated nucleic acids and subsequent sequencing results varied substantially. We hypothesized that this is due to deviations from the standard protocol such as improperly buffered reagents, variation in the fixation time and temperature, microwaving, and varied storage conditions of the samples. To understand the role of formalin fixation on the quality of the isolated nucleic acids and subsequent NGS analyses, we subjected the widely studied cell line NA12878 to various formalin fixation conditions. We then performed an augmented target enrichment and sequencing assay on both the DNA and RNA isolated from fresh frozen (FF) and formalin fixed (FFPE) NA12878 samples. We assessed raw nucleic acid quality, library quality, alignment rate, duplication rate, on-target efficiency and variant concordance between FF and FFPE. We noted substantial negative effects on sequencing library quality associated with sample incubation in unbuffered formalin, at high temperatures, and long periods of time (e.g. 3 days rather than 1 day). These harshly treated samples also showed poor alignment qualities, higher duplication rates, and lower mapping qualities. At the small variant level, they showed an increase in global C&amp;gt;T deamination (CG-&amp;gt;TA, 50% FFPE vs 35% in FF) and oxidation (CG-&amp;gt;AT, 15% FFPE vs 10% FF) rates. Variants caused by formalin fixation were most commonly detected at low (&amp;lt;5%) allele frequencies (AFs). When compared to gold set mutations in NA12878, we observed 80% false negative and 60% false positive variants in harshly treated samples. We are performing a similar analysis with RNA isolated from FF and FFPE NA12878 samples to understand the role of formalin on RNA variant and gene expression assays. Using the results of this study, we intend to provide guidance on optimal fixation conditions for NGS applications. It is crucial to fix tissues in buffered formalin for less than 24hr at room temperature to preserve nucleic acid integrity and amenability for downstream NGS assays. We also demonstrate how a deeper understanding of the effects of formalin can improve variant calling from FFPE tissues, especially at lower AFs where formalin-related errors have the greatest impact. Citation Format: Ravi Alla, Shujun Luo, Elena Helman, Sean M. Boyle, Michael J. Clark, Kirk Scott, Parin Sripakdeevong, Mirian Karbelashvili, Deanna M. Church, Michael Snyder, John West, Richard Chen. Fix the fixation: effect of formalin fixation on targeted sequencing, variant calling and gene expression. [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 3616.

Abstract 4919: Evaluating the gene expression of frozen tissue-derived prognostic signatures in FFPE colorectal cancer samples

Abstract OBJECTIVE: Defining molecular features that can predict the recurrence of colorectal cancer (CRC) for stage II and stage III patients remains a challenging problem in cancer research. Most available clinical samples are Formalin Fixed and Paraffin Embedded (FFPE). Nanostring nCounter® and Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA) are the two platforms marketed for high throughput measurement of mRNA expression from FFPE tissue samples. In this study, to identify an optimal platform for the gene expression profiling of FFPE CRC samples, we compared the expression of genes that make up published frozen tissue-derived prognostic signatures measured by these two platforms. METHODS: FFPE primary tumor tissue blocks were identified from 500 patients with stage II or III CRC and complete pathological information. We removed samples with neoadjuvant therapy, inadequate clinical follow-up or tumor specimens, samples with failure of RNA extraction and highly degraded samples. We identified 194 eligible FFPE-derived CRC primary tumors with a 7.4-year mean follow up. To measure the gene expression of the 194 samples, we designed a custom nCounter codeset using 536 gene elements from multiple published frozen tissue-derived prognostic signatures for CRC. We also performed gene expression profiling using the HTA platform on a subset of 84 of the 194 samples. For the nCounter data, samples with low total average count or with more than 20% genes having lower average count than the synthetic negative control genes were omitted to ensure adequate data quality. RESULTS: In total, for CRC samples from 42 patients, the gene expression data of 516 common genes measured by both platforms were of sufficient quality for comparative analysis. Based on HTA platform-derived data, we found that (1) 36 and 97 of the 516 common genes are significantly associated with overall survival (OS) and disease-free survival (DFS) at the single gene level (FDR &amp;lt; 0.05), respectively; and (2) two of the nine reported multi-gene signatures, for which sufficient information from the original papers enabled such evaluation, can divide patients into high-risk and low-risk groups with significantly different DFS (p ≤ 0.05). Based on nCounter platform-derived data, no individual gene was found to be significantly associated with survival at the single gene level (FDR &amp;lt; 0.05), but one of the nine published multi-gene signatures can divide patients into two groups with significantly different OS (p ≤ 0.05). Our results also showed moderate correlations between paired FFPE samples measured by nCounter and HTA platforms (the median correlation coefficient is 0.52). CONCLUSION: Our data demonstrate that while both platforms may identify gene or gene set expression differences associated with survival outcomes, the HTA appears to provide a more robust gene expression analysis dataset when using genes selected from published gene signatures. Citation Format: Jing Zhu, Natasha G. Deane, Keeli B. Lewis, Chandrasekhar Padmanabhan, Mary K. Washington, Kristen K. Ciombor, Cynthia Timmers, Richard M. Goldberg, R. Daniel Beauchamp, Xi Chen. Evaluating the gene expression of frozen tissue-derived prognostic signatures in FFPE colorectal cancer 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 4919.

High-mass-resolution MALDI mass spectrometry imaging of metabolites from formalin-fixed paraffin-embedded tissue.

Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected ∼1,500 m/z species in the mass range m/z 50-1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice.

How Suitable is Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight for Metabolite Imaging from Clinical Formalin-Fixed and Paraffin-Embedded Tissue Samples in Comparison to Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry?

In research and clinical settings, formalin-fixed and paraffin-embedded (FFPE) tissue specimens are collected routinely and therefore this material constitutes a highly valuable source to gather insight in metabolic changes of diseases. Among mass spectrometry techniques to examine the molecular content of FFPE tissue, mass spectrometry imaging (MSI) is the most appropriate when morphological and histological features are to be related to metabolic information. Currently, high-resolution mass spectrometers are widely used for metabolomics studies. However, with regards to matrix-assisted laser desorption/ionization (MALDI) MSI, no study has so far addressed the necessity of instrumental mass resolving power in terms of clinical diagnosis and prognosis using archived FFPE tissue. For this matter we performed for the first time a comprehensive comparison between a high mass resolution Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer and a time-of-flight (TOF) instrument with lower mass resolving power. Spectra analysis revealed that about one-third of the detected peaks remained unresolved by MALDI-TOF, which led to a 3-5 times lower number of m/z features compared to FTICR measurements. Overlaid peak information and background noise in TOF images made a precise assignment of molecular attributes to morphological features more difficult and limited classification approaches. This clearly demonstrates the need for high-mass resolution capabilities for metabolite imaging. Nevertheless, MALDI-TOF allowed reproducing and verifying individual markers identified previously by MALDI-FTICR MSI. The systematic comparison gives rise to a synergistic combination of the different MSI platforms for high-throughput discovery and validation of biomarkers.

Loop-mediated isothermal amplification combined with PCR and immunohistochemistry for detecting Porphyromonas gingivalis in periapical periodontitis.

Porphyromonas gingivalis is important in the development of marginal periodontitis. However, the precise role and localization of P. gingivalis in chronic periapical periodontitis remain unclear. Thus, methods that can detect P. gingivalis in formalin-fixed and paraffin-embedded (FFPE) tissue samples are needed. We assessed a technique combining loop-mediated isothermal amplification (LAMP) with PCR (PCR-LAMP) for detection of P. gingivalis, using 110 FFPE tissue samples of chronic apical periodontitis. PCR-LAMP specifically detected P. gingivalis with high sensitivity in FFPE tissue samples, and the sensitivity of the technique was higher than that of PCR or LAMP alone. The results of immunohistochemistry (IHC) confirmed the specificity of PCR-LAMP. IHC showed that P. gingivalis was localized in a granular layer of chronic apical periodontitis, a region that correlated with the localization of macrophages. This is the first study to describe the localization of P. gingivalis in human periapical periodontitis. In conclusion, PCR-LAMP was an effective tool for detecting P. gingivalis in periapical periodontitis. In addition, IHC results improve our understanding of the role of P. gingivalis in the progression of periapical periodontitis. (J Oral Sci 58, 163-169, 2016).

Comparative evaluation of the GP5 +/6 +, MY09/11 and PGMY09/11 primer sets for HPV detection by PCR in oral squamous cell carcinomas

The aim of this study was to evaluate the use of GP5+/6+, MY09/11 and PGMY09/11 primer sets for the detection of human papillomavirus (HPV) DNA by single step polymerase chain reaction (PCR) and nested PCR in formalin-fixed and paraffin-embedded (FFPE) tissues from oral squamous cell carcinomas (OSCCs). DNA extracted from FFPE tissues were tested for amplification of the human beta globin gene with PCO3/4 primers. Positive samples for this gene were tested for HPV DNA using single step PCR with GP5+/6+, MY09/11 and PGMY09/11 primer sets. All negative samples at single step PCR with MY09/11 and PGMY09/11 were subjected to a further PCR with GP5+/6+ primers using the non-amplified product in the previously reactions (nested PCR) as samples. Among 26 samples, 23 were positive for the human beta globin gene and were considered viable for HPV DNA detection by PCR. Single step PCR with GP5+/6+ and MY09/11 primers and MY/GP+ nested PCR did not amplify HPV DNA in any samples. PGMY09/11 primers detected HPV DNA in 13.0% of OSCC cases and this rate was raise to 17.4% with the use of PGMY/GP+ nested PCR. According to our results the PGMY/GP+ nested PCR is the most appropriate primer set for the detection of HPV DNA using FFPE samples from OSCC.

Abstract 4904: Developing a universal target retrieval solution and protocol for RNA in situ hybridization based on RNAscope technology

Abstract Heat induced target retrieval (HITR) is a widely adopted approach for Immunohistochemistry (IHC) and in situ hybridization (ISH) applications on formalin fixed and paraffin embedded (FFPE) tissues to facilitate target access and increase detection sensitivity. A variety of factors, such as fixative type, fixation time, tissue type, epitope abundance, antibody affinity, and probe length greatly affect the efficiency of HITR. Given such variability, over the decades, multiple HITR buffer formulations and protocols were invented to fit the needs in different experimental scenarios. We have developed a novel HITR solution that allows highly efficient target retrieval for RNA ISH application based on the RNAscope technology. When tested on 15 different tumor types, the new HITR solution resulted in significantly better sensitivity and/or tissue morphology comparing to using traditional HITR buffers, including Citrate, EDTA, and Tris based buffers with various pH. Furthermore, this new HITR solution is highly tolerant to sample variability. When used on multiple human tumor tissue microarrays (TMAs), including breast, colon, lung, and gastric cancers, the new HITR solution improved overall signal-to-noise ratio and reduced core disqualification rate, compared to traditional citrate based HITR buffer for ISH. Moreover, when tested on multiple mouse tissues fixed with different types of fixatives for varying periods of time (6 - 72 hours), a single experimental condition with the new HITR solution showed comparable results in both detection sensitivity and morphological integrity in all the samples. It also allowed detection of a target (Tbp) with extremely low abundance (&amp;lt;5 copies/cell). Given that all the RNAscope probes are synthetic oligonucleotide probes with length shorter than 60 bases, the new HITR solution, in combination with the RNAscope technology, will allow universal detection of any RNA targets with single molecule detection sensitivity in FFPE tissues with minimal requirement for analytical condition optimization. Citation Format: Li-chong Wang, Liuliu Pan, Kuang-Jung Chang, Daniel Kim, Xingyong Wu, Hongwei Wang, Casey Kernag, Bingqing Zhang, Mingxiao He, Nan Su, Xiao-Jun Ma, Yuling Luo. Developing a universal target retrieval solution and protocol for RNA in situ hybridization based on RNAscope technology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4904. doi:10.1158/1538-7445.AM2015-4904

Abstract 3416: IHC assessment of PBRM1 loss in colon and lung carcinomas

Abstract PBRM1 (also known as BAF 180) is a chromatin remodeling polybromodomain protein recently identified as the second most frequently mutated gene in renal cell carcinoma (RCC). Loss of PBRM1 protein expression is most frequently observed in clear cell RCC and correlated with tumor progression. Approximately 40-50% of patients with RCC have mutations in PBRM1, and loss of PBRM1 was also reported in 28% of melanocytic neoplasms. However, loss of PBRM1 has not been studied in other tumors using patient tissues. The purpose of this study was to assess the protein expression of PBRM1 in a wide-ranging collection of normal and tumor tissues. Here we report our findings of PBRM1 loss in colon and lung carcinomas using a newly developed rabbit monoclonal antibody (RabMAb), Clone EP342. The antibody was generated by immunization of rabbits with PBRM1 recombinant protein. Sera collected from immunized rabbits were screened by ELISA, WB and IHC. After fusion, antibody from final hybridoma cell line was characterized using ELISA and Western blot (WB). A 180 KDa PBRM1 protein was detected by WB in A431 and MCF7 cell lysates. The antibody was further characterized by extensive IHC testing using formalin-fixed and paraffin-embedded (FFPE) human normal and tumor tissue microarray (TMA). IHC analysis on human tissue microarray (TMA) comprising seventeen types of normal tissues showed that PBRM1 antibody stained positively in the nuclei of all normal cells except cardiac muscle cells. In tumor TMAs comprising of fifteen types of tumors (2-4 cases for each type), negative staining was observed in carcinomas of the lung and colon, as well as clear cell RCC. Further analysis of specific types of tumor TMAs showed that PBRM1 was lost in 5 out of 30 colon carcinomas (17%), 8 out of 35 (14%) lung carcinomas, and 8 out of 1844%) clear cell RCC. In summary, the RabMAb PBRM1 antibody, clone EP342 specifically labels normal cells and most tumor cells. It is a useful tool in identifying cancers with loss of PBRM1 expression on FFPE tissues. The rate of PBRM1 loss in clear cell RCC (44%) detected by this clone is consistent with literature reported. Our study showed that PBRM1 protein was lost in colon and lung carcinomas, but less frequent than that of clear cell RCC. Further studies on colon and lung carcinomas are needed to clarify the role of PBRM1 in tumor progression in these tumors. Citation Format: Aihua Li, Yongfu Gao, Yuekai Zhang, Hongyang Pan, Jackie K. Chan, Ximing J. Yang, Taiying Chen. IHC assessment of PBRM1 loss in colon and lung carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3416. doi:10.1158/1538-7445.AM2015-3416