Formalin-fixed Paraffin-embedded Patient Samples Research Articles

Quantitative multiplex immunohistochemistry reveals inter-patient lymphovascular and immune heterogeneity in primary cutaneous melanoma.

Quantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma. We established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naïve, primary cutaneous melanomas. Here we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naïve, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates. We describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for metastasis or immune infiltration. This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

Using global DNA-methylation and metabolome profiles to classify variants of unknown significance (VUS) in phaeochromocytoma and paraganglioma tumours

Abstract Background Phaeochromocytomas and paragangliomas (PPGLs) are rare but strongly heritable neuroendocrine tumours. Most patients undergo genetic testing and approximately 40% will have a germline causal mutation. In 10% of patients, genetic analysis identifies uncharacterised mutations, or variants of unknown significance (VUSs). Tests to distinguish between benign and pathogenic mutations would aid clinicians' and patients' decision-making. One test could exploit the biology of a subset of PPGLs. Mutations in genes controlling the Tricarboxylic Acid cycle cause a deranged metabolome, accumulation of oncometabolites and global DNA-hypermethylation. Data show that this stable chemical modification will be greatly elevated in pathogenic over benign mutations, and be present alongside a driver metabolome. Methods Clinical characteristics were compiled for 3 patient groups: causal germline mutations, no identified mutation (sporadic) and VUS. Archival Formalin Fixed Paraffin Embedded (FFPE) material was identified locally and also provided by the international ENS@T collaboration. Tissue analysis was optimised on mouse FFPE tissue. Laser microdisection allowed collection of precise tissue areas. DNA and metabolite extraction was optimised before analysis was undertaken by Liquid Chromatography Mass Spectrometry (LC-MS). Results 93 patients were recruited locally. Clinical characteristics were collected for 25 sporadic and 56 germline cases, including 14 VUS cases. Optimisation experiments in mouse tissue show that discrete areas can be extracted by laser microdissection. LC-MS identifies cytosine, 5'-methylcytosine and 5'-hydroxymethylcytosine in 1 μg of DNA at biologically relevant ratios. TCA cycle metabolites were detected using LC-MS: succinate, fumarate and malate were found in the metabolite extract from the same amount of tissue. Conclusions This study utilises DNA hypermethylation and metabolomics for clinical application. It represents a large repository of PPGL VUS samples nationally. Global DNA-methylation levels and metabolite profiles are shown to be quantifiable from existing FFPE patient samples, using reduced amounts of patient material. Legal entity responsible for the study Guy's and St Thomas' NHS Trust, London, UK; King's College London, UK. Funding National Institute of Health Research, King's College London, Isaac Shapera Research Trust, The Bernice Bibby Research Trust. Disclosure All authors have declared no conflicts of interest.

Abstract 799: Aberrant mitochondrial protein involvement through early PDAC initiation and progression using multiplexed DNA-PAINT and correlative histology

Abstract Prior to pancreatic ductal adenocarcinoma (PDAC), pancreatic acini cells change their morphology through pancreatic intraepithelial neoplasia (PanIN) stages, becoming increasingly dysplastic as stroma and tissue hypoxia increase. This increasing hypoxia forms a cancer promoting microenvironment, wherein we propose metabolic changes trigger aberrant mitochondrial networks form healthy tissue. We developed tissue superresolution imaging to directly quantify structural mitochondrial response through patient histology, whereby DNA-PAINT can provide super-resolution detail decoupled from photo bleaching to visualize mitochondria through tissue layers. Expanding from preliminary data, target mitochondrial dynamics proteins’ organization will also be correlated to PanIN stages. Introduction: Early oncogene involvement in PDAC progression links to metabolic and mitochondrial regulatory changes. Typically nutrient deprivation and hypoxia trigger cell death from increased reactive oxygen species production and organelle damage which trigger apoptosis, yet these prolonged effects can be cancer promoting when less severe. Mitochondrial dynamics and proteins related to mitochondrial fission and fusion can reduce apoptotic signaling, enhance aerobic glycolysis, and increase ROS to allow cancer progression. Materials and Methods: Adapting from previous proof of concept, mitochondria (TOM20) within formalin-fixed paraffin embedded (FFPE) tissue sections were imaged with stochastic optical reconstruction microscopy (STORM). In brief: cadaver healthy pancreas FFPE tissue samples underwent deparafinization, antigen retrieval, indirect immuno-labeling with AlexaFluor647, TIRF illumination with a 60x objective was used for data collection, whereby data processing was conducted using the open-source FIJI and custom MATLAB software. Results and Discussion: Preliminary data shows proof of principle that both STORM and DNA-PAINT can be correlated to histological staining of human pancreas. Conclusions: Superresolution imaging reveals ultrastructural details of mitochondria in FFPE patient samples not resolved via conventional fluorescence imaging. Through quantitative feature analysis, we would be able to correlate aberrant mitochondria structure and abundance to PDAC progression. Acknowledgements: Funding provided by the Cancer Early Detection Advanced Research (CEDAR) Center of OHSU Knight Cancer Institute. Citation Format: Matthew J. Rames, Fehmi Civitci, Ting Zheng, Josiah Wagner, Jason Link, Xiaolin Nan. Aberrant mitochondrial protein involvement through early PDAC initiation and progression using multiplexed DNA-PAINT and correlative histology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 799.

Aberrant signal transduction in Indian triple-negative breast cancer patients.

The aim of this study is to correlate the prominin-1 or CD133 association with functional pathway markers of cancer stemness in Indian triple-negative breast cancer (TNBC) patient samples. TNBC samples were confirmed for the absence of hormone receptors (estrogen receptor-ER/progesterone receptor) and human epidermal growth factor receptor-2 or proto-oncogene neu or erbB2 or CD340 by immunohistochemical analysis. Formalin-fixed paraffin-embedded samples of patients were used to collect the total RNA. Then, one-step reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the cancer stemness-related transcript levels in the different samples. The RT-PCR products were analyzed semi-quantitatively on agarose gels. The band intensities of respective samples for different transcripts were analyzed by densitometry. TNBC-confirmed samples had shown increased levels of CD133 transcript than control tissues. Further, elevated CD133 transcripts are correlated with higher transcript levels of NOTCH1/FZD7/transforming growth factor-beta receptor Type III R/patched-1 pathway mediators. This work has clearly indicated that there is a correlation between CD133 and functional pathways that control cancer stem cells in TNBC. These observations may indicate the possible association between cancer stemness and TNBC malignancy.

Evaluation and validation of HPV real-time PCR assay for the detection of HPV DNA in oral cytobrush and FFPE samples

Specific HPV genotypes have been recognized as risk factors inducing head and neck cancers (HNC). The aim of this study was to validate a real-time PCR assay to detect accurately High Risk HPV DNA in Formalin Fixed Paraffin Embedded (FFPE) and oral cytobrush samples and compare the results with conventional PCR. Repeatability, reproducibility and limit of detection of Cobas assay were estimated for oral cytobrush and FFPE samples of patients with HNC. 53 samples of patients with a HNC were then used for assay comparison with conventional PCR. Finally, 26 samples of patients with anogenital neoplasia cancer were analyzed as control and assays comparison. Among the 53 samples of patients with HNC, 12 (26.7%) were HPV positive, 33 (73.3%) were HPV negative and 8 (15.1%) were non contributive with the Cobas assay. Among the 26 samples of patients with anogenital neoplasia, 15 (57.7%) were HPV positive and 11 were HPV negative (42.3%). One sample was found with an HPV 16 and HPV 18 co-infection. Only 3 samples were found with discrepant results. Cobas assay was found suitable for routine HPV detection with a very good repeatability and reproducibility for all HPV genotypes (CV < 0.6% and <0.4% respectively). Sensitivity and specificity for Cobas assay were 91.7% [61.5%;99.8%] and 96.9% [83.8%;99.9%] respectively. Ten nanograms of DNA were sufficient for the detection of HPV 16, HPV 18 and HPV in FFPE and oral cytobrush samples. Cobas assay was found comparable to conventional PCR and can detect accurately and rapidly HPV DNA in FFPE and oral cytobrush samples for the management of HNC and other types of HPV-associated neoplasia.

MicroRNA analysis of colorectal cancer using fecal and tissue samples

Recently, several microRNAs (miRNAs) have been reported as promising biomarkers for cancer detection and tumor recurrence risk. Due to its stability, miRNA can be accessed from samples stored in severe conditions, such as feces or formalin-fixed paraffin-embedded (FFPE) tissue. Fecal miRNA extracted from the residuum of fecal occult blood tests (FOBTs) was assessed to determine whether a combination of this fecal miRNA test (FmiRT) with FOBT could improve the false-negative rate of colorectal cancer (CRC) screening compared with FOBT alone. Expression of miR-106a in patients with both positive and negative FOBTs was significantly higher than in healthy volunteers. To identify a high-risk group for recurrence, miRNAs were extracted from FFPE samples of patients with stage II CRC. Tumor recurrence occurred at a significantly higher rate in patients with increased miR-181c expression than in those with lower expression. The recurrence rate in patients with stage II CRC with higher expression of miR-181c was similar to that of patients with stage III CRC who had been treated by surgical resection alone. As miRNAs are stable in several severe storage conditions, such as in fecal and FFPE samples, they could be valuable, accessible biomarkers for CRC, for use both in cancer screening and as predictors of recurrence.

Abstract S5-01: DCIS biological risk profile predicts risk of recurrence after breast conserving surgery in a Kaiser Permanente NW population

Abstract Background: Patients with DCIS and their physicians need tools that provide better information about the individual patient's biological risk profile to help make treatment decisions. Prelude and the Kaiser Permanente Northwest Center for Health Research (KPCHR) validated a biological risk signature based test to assess ipsilateral breast event (IBE) risk after breast conserving surgery (BCS) with radiation (+RT) or without radiation therapy (-RT). Methods: The Prelude DCIS test was independently validated in a retrospective cohort from the Kaiser Permanente Northwest (KPNW) integrated healthcare system in patients diagnosed with DCIS from 1990- 2007 and treated with BCS±RT(n=608). KPCHR performed central pathology review to identify patients meeting study eligibility criteria with formalin fixed paraffin embedded (FFPE) tissue samples (n=475); KPCHR also reviewed medical records to collect patient, treatment, and outcome data. FFPE patient samples were provided to Prelude for testing. REMARK guidelines were followed. A panel of biomarkers (HER2, PR, Ki-67, COX2, p16/INK4A, FOXA1 and SIAH2) were assayed by the Prelude CLIA lab and scored by board-certified pathologists (n=455). Prelude's DCIS test was executed independently using biomarker and clinicopathologic data while blinded to patient outcome data. The risk results were provided to KPCHR under a Data Transfer Authority. KPCHR biostatisticians executed a predefined and co-developed statistical analysis plan. IBE rates were assessed using Kaplan-Meier survival analysis. Hazard ratios (HR) were determined using Cox proportional hazards analysis, with RT as a covariate. Results: The Prelude DCIS test score was statistically associated with total IBE as a continuous linear variable (0-10 unit scale) on a per unit basis, HR of 1.12, 95% CI [1.03,1.23], p=0.01. The DCIS test score (0-10) corresponded to recurrence risks ranging from 10% to 42% (≤2, &amp;gt;7) for patients treated with BCS-RT and ranging from 4% to 11% (≤2, &amp;gt;7) for patients treated with BCS+RT. Patients treated with BCS ±RT with an elevated test score (≤3 vs &amp;gt;3) had a higher recurrence risk, n=455, HR=1.87 [1.03 - 3.38], p=0.04. In patients treated with BCS-RT in this sample, patients with a higher DCIS signature had an elevated recurrence risk, n=78, HR=2.37, 95% CI [0.82, 6.85], p=0.11. The 10-year contralateral breast event rate was 4%, 95% CI [2%, 6%]. Median follow-up time was 10.4 years. Discussion: Patients diagnosed with DCIS and treated with BCS ±RT, were stratified into clinically relevant low and elevated risk groups (≤3 vs &amp;gt;3) in an independent validation of the Prelude DCIS test. Patients in the elevated risk group had substantially higher likelihood of 10-year total IBE. The number of patients treated with BCS -RT was limited and while the stratification by risk group for BCS -RT was in the expected direction, it did not reach statistical significance. Two additional validation studies are scheduled to be completed in 2016. 10-YEAR IBE RISKBCS –RTBCS +RTRisk, [95% CI]PrevalenceNRisk, [95% CI]PrevalenceNBaseline Total Risk20%, [12%, 32%]100%788%, [5%, 11%]100%377Low Risk Group (≤3)10% [3%, 29%]53%415%, [2%, 10%]40%149Elevated Risk Group (&amp;gt;3)30%, [17%, 51%]47%3710%, [6%, 15%]60%228 Citation Format: Bremer T, Whitworth P, Leo M, Barry T, Goldstein N, Ganders C, Francisco M, Leesman G, Linke S, Patel R, Pellicane J, Weinmann S. DCIS biological risk profile predicts risk of recurrence after breast conserving surgery in a Kaiser Permanente NW population [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 S5-01.

Development and verification of the PAM50-based Prosigna breast cancer gene signature assay.

BackgroundThe four intrinsic subtypes of breast cancer, defined by differential expression of 50 genes (PAM50), have been shown to be predictive of risk of recurrence and benefit of hormonal therapy and chemotherapy. Here we describe the development of Prosigna™, a PAM50-based subtype classifier and risk model on the NanoString nCounter Dx Analysis System intended for decentralized testing in clinical laboratories.Methods514 formalin-fixed, paraffin-embedded (FFPE) breast cancer patient samples were used to train prototypical centroids for each of the intrinsic subtypes of breast cancer on the NanoString platform. Hierarchical cluster analysis of gene expression data was used to identify the prototypical centroids defined in previous PAM50 algorithm training exercises. 304 FFPE patient samples from a well annotated clinical cohort in the absence of adjuvant systemic therapy were then used to train a subtype-based risk model (i.e. Prosigna ROR score). 232 samples from a tamoxifen-treated patient cohort were used to verify the prognostic accuracy of the algorithm prior to initiating clinical validation studies.ResultsThe gene expression profiles of each of the four Prosigna subtype centroids were consistent with those previously published using the PCR-based PAM50 method. Similar to previously published classifiers, tumor samples classified as Luminal A by Prosigna had the best prognosis compared to samples classified as one of the three higher-risk tumor subtypes. The Prosigna Risk of Recurrence (ROR) score model was verified to be significantly associated with prognosis as a continuous variable and to add significant information over both commonly available IHC markers and Adjuvant! Online.ConclusionsThe results from the training and verification data sets show that the FDA-cleared and CE marked Prosigna test provides an accurate estimate of the risk of distant recurrence in hormone receptor positive breast cancer and is also capable of identifying a tumor's intrinsic subtype that is consistent with the previously published PCR-based PAM50 assay. Subsequent analytical and clinical validation studies confirm the clinical accuracy and technical precision of the Prosigna PAM50 assay in a decentralized setting.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0129-6) contains supplementary material, which is available to authorized users.

Abstract 5545: MethyMeter® - A quantitative, bisulfite-free DNA methylation assay for improved clinical diagnostic and prognostic analysis of glioma biomarkers in FFPE tumor samples

Abstract This presentation describes the clinical diagnostic use of a novel Bisulfite-free, Abscription-Based DNA methylation assay, MethylMeter, for analysis of grade 2, 3, &amp; 4 gliomas. The proprietary 9 gene CpG-Island Methylation Phenotype (CIMP) profile was validated with bisulfite based MS-PCR and was then licensed by Castle Biosciences for commercialization as the DecisionDx-LGG Test. Very few CIMP assays have been transposed successfully into clinical diagnostic assays, since they depend on bisulfite-based technologies (MS-PCR or pyrosequencing). Bisulfite methods are not well suited to clinical applications which utilize formalin-fixed, paraffin-embedded (FFPE) tissues because of the large amount of sample that is required and the poor level of reproducibility that is inherent in bisulfite assays. We describe a more sensitive and quantitative alternative to MS-PCR for detection of the DecisionDx-LGG CIMP profile in FFPE tumors. The method, MethylMeter®, combines affinity separation of methylated and unmethylated DNA with a new target and signal amplification technology called CAP™ (Coupled Abscription®-PCR). Data will be presented demonstrating that MethylMeter® was able to successfully detect the appropriate DNA methylation patterns in FFPE patient samples for the 9 biomarkers utilized in the DecisionDx-LGG assay. Gliomas are stratified by histological grade, which is an indication of malignant potential and response to treatment. However, there is up to a 40% inter- and intra-observer histological grade variance rate in grading 2, 3, &amp; 4 gliomas which is clinically signicant as histological grade determines treatment plan. A DecisionDx-LGG CIMP “positive” result is associated with significantly longer survival in patients with grade 2, 3, &amp; 4 gliomas enabling improved treatment planning.I Initial attempts at CIMP profile analysis of glioma tumors using MS-PCR yielded clinically unacceptable results, substitution of the CAP-based MethylMeter assay enabled appropriate and clinically relevant analysis of CpG Island methylation levels in all 9 glioma tumor biomarkers. The MethylMeter assay results showed 100% concordance with the previous MS-PCR results. In contrast to the bisulfite-based assay, MethylMeter worked efficiently and reproducibly in FFPE patient samples, yielded increased levels of sensitivity, and was significantly faster. Data will also be presented showing that the quantitative data output provided by Abscription-based CAP assays provides a quantifiable readout of methylation levels that will enable future patient stratification, in contrast to the plus/minus readout provided by bisulfite assays. The data presented will be highly significant in paving the way for new MethylMeter-based CIMP pattern assays to be developed for diagnostic, theranostic, and companion diagnostic purposes. 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 5545. doi:1538-7445.AM2012-5545

A quantitative proteomic analysis of FFPE melanoma

A quantitative proteomic analysis of FFPE melanoma

Single molecule RNA sequencing of formalin-fixed paraffin-embedded tissue derived from patients with lung cancer.

10550 Background: Next generation sequencing has revolutionized the study of cancer biology by providing an unprecedented view of cancer genomes. However, high throughput sequencing has conventionally utilized fresh frozen tissue collected for research prospectively. The ability to apply this tool to formalin-fixed, paraffin-embedded (FFPE) clinical specimens, where nucleic acid integrity is not optimal and where next generation sequencing has been challenging, would enable its use to improve patient care and clinical research. Methods: Total RNA was extracted from FFPE blocks of 7 lung adenocarcinomas and matching normal lung tissue for 6 of the 7 tumors. Genotype information for selected mutation alleles in EGFR, TP53, etc. was previously determined using the SNaPshot single nucleotide polymorphism genotyping platform. Ribosomal RNA was depleted using biotinylated oligos targeting 28S and 18S ribosomal subunits. cDNA was synthesized using random hexamers. Whole transcriptome sequencing was performed using the Helicos BioSciences Heliscope single molecule sequencer. Sequences were aligned to reference human transcriptome and genome as well as a reference of mutation alleles. Results: Ribosomal RNA depletion increased mRNA yield from ~20% to ~35% of RNA species sequenced. We obtained ~10 million aligned mRNA reads per sample. We re-identified clinically relevant EGFR exon 19 deletions in two samples. In another sample, we discovered an exon 20 insertion in EGFR that was not previously appreciated. In a fourth sample, we were able to detect the presence of an EML4-ALK fusion through the integration of sequence and expression information. Conclusions: This study shows that RNA sequencing from FFPE patient samples is feasible, and in cases where gene expression is sufficient, enables the determination of clinically relevant mutations. These findings represent the initial step towards leveraging the power of next generation sequencing into a comprehensive diagnostic and clinical research tool.

Gene expression profiling in formalin-fixed, paraffin-embedded (FFPE) benign and cancerous prostate tissues using universal bead arrays

9526 Background: Archival tissues from patients with known clinical history have become an invaluable resource for gene expression analysis for tumor classification and clinical outcome prediction. These samples may be studied using qPCR, but RNA extracted from FFPE tissues is often significantly degraded, making them difficult to use in microarray analyses. Methods: We have developed the DASL assay, a flexible, sensitive and reproducible expression profiling method for parallel analysis of hundreds of genes in FFPE samples using universal bead arrays. The method can tolerate partial RNA degradation because it does not depend on an intact poly-A tail for cDNA synthesis. Tissue- and cancer-specific expression profiles have been obtained with as little as 50 ng total RNA derived from FFPE tissues that had been stored up to 11 years (Bibikova, et al., Am. J. Pathol. 165:1790–1807, 2004). Results: We performed a profiling study of 174 benign or cancerous prostate FFPE tissues retrieved from the VA San Diego Healthcare System. A total of 512 prostate cancer-related genes selected from the literature were monitored at 3 probes per gene. Despite RNA degradation, highly reproducible expression profiles were obtained for these samples (average r2 = 0.98). Expression data were used to identify tumor- and stroma-specific genes, which were then included in a sample classification scheme. Resulting gene panels correlated with clinical parameters such as tumor type, tumor grade, therapeutic response, and patient survival. In additional studies using laser capture microdissection of FFPE tissues, increased expression of tumor- and stroma-specific genes were measured in the correct cell types, confirming the results based on heterogeneous tissues. Conclusions: Putative prostate cancer biomarkers were identified using the DASL expression assay and RNAs derived from FFPE patient samples. In combination with the high-throughput capability of the bead array-based platform, the DASL assay may be used to monitor hundreds of genes in hundreds of samples at once. These data suggest that FFPE tissue archives, with known patient outcomes, can now be accessed for microarray expression analysis. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Illumina Illumina