From Formalin-fixed Paraffin-embedded Tissue Research Articles

Abstract 4880: Characterization of genetic concordance between primary tumor cells, circulating tumor cells, and metastatic tumor cells from patients with prostate cancer

Abstract Circulating tumor cells (CTCs) which disseminate from the primary tumor site have been found to be a major source of metastatic tumor. However, CTCs are heterogeneous and most CTC DNA is fragmented due to apoptosis. To better understand the molecular pathogenesis of metastasis, it is imperative to obtain single, non-apoptotic CTCs and investigate the genetic concordance between primary tumor cells, CTCs and metastatic tumor cells. Due to the abundance of normal cells present in the blood and in tumor tissue, isolation of single tumor cells or pure cell populations of these cells is extremely difficult. The DEPArray platform is designated to collect single CTCs after Cell-Search enrichment, and to sort pure intact tumor cells from formalin-fixed, paraffin-embedded (FFPE) tissues. Taking advantage of this technology, we have isolated single CTCs from prostate cancer patients. Whole genome amplification was performed and genomes with high integrity were used to evaluate the hotspot mutations in PIK3CA. For comparison, we also made single cell suspensions from FFPE tissue blocks of primary tumors and metastatic tumor in lymph nodes from the same patients. Immunofluorescence staining for cytokeratin (CK) and vimentin (Vim) was performed, followed by isolation of pure tumor cells (CK+), stromal cells (Vim+) and double-positive cells (CK+/Vim+) from both primary tumor and lymph node samples. We plan to do genetic analysis on these cells and compare the results to determine the genetic concordance between primary tumor cells, CTCs and metastatic tumor cells. By using the DEPArray, we have successfully isolated single CTCs, and populations of tumor cells, stromal cells and double-positive cells from primary and metastatic tumors. We will present new data on the genetic concordance between these cells, which will provide new insight into our understanding of metastasis in prostate cancer. Citation Format: Lixin Yang, Linling Chen, Yafan Wang, Jeremy Jones, Yun Yen, Sofia Loera, Raju Pillai, Peiguo Chu, Dennis Weisenburger. Characterization of genetic concordance between primary tumor cells, circulating tumor cells, and metastatic tumor cells from patients with prostate cancer. [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 4880. doi:10.1158/1538-7445.AM2015-4880

Molecular genotyping of Echinococcus granulosus using formalin-fixed paraffin-embedded preparations from human isolates in unusual tissue sites.

Cystic echinococcosis (CE) caused by Echinococcus granulosus remains a serious problem worldwide for issues relating to public health and the economy. The most predominantly affected sites are the liver and the lungs, but other organs such as the heart, the spleen and the peritoneum can also be infected. Access to cysts from uncommon sites has limited genomic and molecular investigations. In the present study, genotypes of E. granulosus sensu lato were identified from formalin-fixed paraffin-embedded tissues (FF-PETs) implicated in human CE. Tissue samples were obtained from 57 patients with histologically confirmed CE. DNA samples were analysed using Egss 1 polymerase chain reaction (PCR) specific to the mitochondrial 12S rRNA gene of E. granulosus sensu stricto. All cysts were typed as E. granulosus sensu stricto with up to 35% of the liver and 16.6% of lungs being the most frequently infected, and up to 48.4% of samples being from rare sites. No correlation was found between cyst site and either the gender or the age of patients. This study demonstrates the possibility of exploiting atypical cysts using FF-PET samples and highlights the predominance of E. granulosus sensu stricto species in the Tunisian population, even in unusual infection sites.

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

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

Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue

As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and “amplifiability” for aCGH and other downstream applications. Phenol–chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues.

Clinical Implications of Rabphillin-3A-Like Gene Alterations in Breast Cancer.

For the rabphillin-3A-like (RPH3AL) gene, a putative tumor suppressor, the clinical significance of genetic alterations in breast cancers was evaluated. DNA and RNA were extracted from formalin-fixed, paraffin-embedded (FFPE) cancers and matching normal tissues. DNA samples were assessed for loss of heterozygosity (LOH) at the 17p13.3 locus of RPH3AL and the 17p13.1 locus of the tumor suppressor, TP53. RPH3AL was sequenced, and single nucleotide polymorphisms (SNPs) were genotyped. RNA samples were evaluated for expression of RPH3AL, and FFPE tissues were profiled for its phenotypic expression. Alterations in RPH3AL were correlated with clinicopathological features, LOH of TP53, and patient survival. Of 121 cancers, 80 had LOH at one of the RPH3AL locus. LOH of RHP3AL was associated with nodal metastasis, advanced stage, large tumor size, and poor survival. Although ~50% were positive for LOH at the RPH3AL and TP53 loci, 19 of 105 exhibited LOH only at the RPH3AL locus. Of these, 12 were non-Hispanic Caucasians (Whites), 15 had large tumors, and 12 were older (>50 years). Patients exhibiting LOH at both loci had shorter survival than those without LOH at these loci (log-rank, P = 0.014). LOH at the TP53 locus alone was not associated with survival. Analyses of RPH3AL identified missense point mutations in 19 of 125 cases, a SNP (C>A) in the 5’untranslated region at -25 (5’UTR-25) in 26 of 104, and a SNP (G>T) in the intronic region at 43 bp downstream to exon-6 (intron-6-43) in 79 of 118. Genotype C/A or A/A of the SNP at 5’UTR-25 and genotype T/T of a SNP at intron-6-43 were predominantly in Whites. Low levels of RNA and protein expression of RPH3AL were present in cancers relative to normal tissues. Thus, genetic alterations in RPH3AL are associated with aggressive behavior of breast cancers and with short survival of patients.

Amplicon sequencing of colorectal cancer: variant calling in frozen and formalin-fixed samples.

Next generation sequencing (NGS) is an emerging technology becoming relevant for genotyping of clinical samples. Here, we assessed the stability of amplicon sequencing from formalin-fixed paraffin-embedded (FFPE) and paired frozen samples from colorectal cancer metastases with different analysis pipelines. 212 amplicon regions in 48 cancer related genes were sequenced with Illumina MiSeq using DNA isolated from resection specimens from 17 patients with colorectal cancer liver metastases. From ten of these patients, paired fresh frozen and routinely processed FFPE tissue was available for comparative study. Sample quality of FFPE tissues was determined by the amount of amplifiable DNA using qPCR, sequencing libraries were evaluated using Bioanalyzer. Three bioinformatic pipelines were compared for analysis of amplicon sequencing data. Selected hot spot mutations were reviewed using Sanger sequencing. In the sequenced samples from 16 patients, 29 non-synonymous coding mutations were identified in eleven genes. Most frequent were mutations in TP53 (10), APC (7), PIK3CA (3) and KRAS (2). A high concordance of FFPE and paired frozen tissue samples was observed in ten matched samples, revealing 21 identical mutation calls and only two mutations differing. Comparison of these results with two other commonly used variant calling tools, however, showed high discrepancies. Hence, amplicon sequencing can potentially be used to identify hot spot mutations in colorectal cancer metastases in frozen and FFPE tissue. However, remarkable differences exist among results of different variant calling tools, which are not only related to DNA sample quality. Our study highlights the need for standardization and benchmarking of variant calling pipelines, which will be required for translational and clinical applications.

Comparison of DNA and RNA analyte extraction and melanin removal methods from formalin-fixed, paraffin-embedded (FFPE) melanoma.

e20002 Background: The increasing availability of molecular profiling techniques amenable to FFPE tissue presents a key opportunity to study the molecular basis of melanoma. In order to optimize ou...

Abstract P4-11-29: Quantitative measurement of HER2 levels by multiplexed mass spectrometry from FFPE tissue predicts survival in patients treated with anti-HER2 based therapy

Abstract Introduction Approximately 20% of breast cancer patients overexpress HER2 and are treated with anti-HER2 therapies. However, there is a great deal of disparity of HER2 levels in the patients that are classified as HER2 positive (IHC3+). Techniques like FISH or IHC do not allow for HER2 quantification and a significant proportion of patients are wrongly classified as HER2 positive. Liquid Tissue-Selected reaction monitoring (LT-SRM) is a multiplexed mass spectrometric technique that can objectively quantify levels of Her2 and other targets from formalin fixed paraffin embedded (FFPE) sections. Given the different available anti-HER2 therapies (trastuzumab, TDM1, lapatinib and pertuzumab) with different modes of action, it would be beneficial for a clinician to understand the levels EGFR and HER3 so as to personalize the therapy. In this work, we have used LT-SRM to quantitate HER2, EGFR and HER3 from FFPE samples (one slide) of patients treated with anti-HER2 agents and correlated the levels of these proteins to clinical outcome. Methods FFPE sections from 60 HER2 positive (IHC3+) primary breast cancer patients were microdissected and proteins were solubilized and digested by trypsin in Liquid Tissue® buffer. Of the 60 samples, 24 were from metastatic setting and 36 from adjuvant setting. After trypsin digestion, internal standards were added and absolute quantitation for multiple proteins was performed using selected reaction monitoring (SRM) mass spectrometry. In addition to LT-SRM, FISH for HER2 was also conducted. Results HER2 quantitation by LT-SRM revealed receptor level ranges from 283 to 14938 amol/µg. ROC analysis was conducted and a cut-off of 2758.75 amol/µg gave the optimal sensitivity and specificity. Survival analysis revealed statistically significant DFS (4.40 years vs 3.38 years; p=0.013) and OS (4.43 years vs 4.03 years; p= 0039) in patients expressing ≥ 2758.75 amol/µg in the adjuvant setting and also statistically significant OS (5.51 years vs 3.37 years; p=0.037) in the metastatic setting. Correlation of HER2 FISH and levels of HER2 is ongoing. Approximately 41% of samples expressed EGFR (range 45 to 2317 amol/µg) and similarly 51% of the samples expressed HER3 (range 84 to 360 amol/µg) with 18% of samples expressing all three targets. Correlation of EGFR/HER3 expression with clinical outcome is ongoing. Conclusion We used an objective multiplex non-antibody based method to quantify multiple targets from FFPE tissue. Clinical correlation analysis of HER2 revealed improved OS and DFS in samples with high HER2 protein levels. Currently, we are expanding these studies to a larger set of samples and taking into account also the expression of other markers such as EGFR and HER3. This approach can potentially identify those tumors that are more dependent on these receptors for survival and also those patients that are exquisitely sensitive to anti-HER2 therapy. Citation Format: Paolo Nuciforo, Sheeno Thyparambil, Claudia Aura, Ana Garrido-Castro, Vicente Peg, Jose Jimenez, William Hoos, Jon Burrows, Todd Hembrough, Jose Perez-Garcia, Javier Cortes, Maurizio Scaltriti. Quantitative measurement of HER2 levels by multiplexed mass spectrometry from FFPE tissue predicts survival in patients treated with anti-HER2 based therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-29.

KRAS gene mutation in a series of unselected colorectal carcinoma patients with prognostic morphological correlations: A pyrosequencing method improved by nested PCR

IntroductionInhibition of EGFR is a strategy for treating metastatic colorectal cancer (CRC) patients. KRAS sequencing is mandatory for selecting wild-type tumor patients who might benefit from this treatment. DNA from formalin-fixed paraffin-embedded (FFPE) tissues is commonly used for routine clinical detection of mutations, and its amplification succeeds only when all preanalytical histological processes have been controlled. In cases that are not properly processed, the DNA results can be poor, with low peak pyrosequencing findings. We designed and tested a pair of forward and reverse primers for a nested PCR method, followed by pyrosequencing, in a single Latin American institution series of 422 unselected CRC patients, correlating KRAS mutations with pathological and clinical data. Materials and methodsPatient DNA samples from tumors were obtained by scraping or laser microdissection of cells from FFPE tissue and extracted using a commercial kit. DNA was first amplified by PCR using 2 primers that we designed; then, nested PCR was performed with the amplicon from the preamplification PCR using the KRAS PyroMark™ Q96 V2.0 kit (Qiagen). Pathological data were retrieved from pathology reports. ResultsKRAS mutation was observed in 33% of 421 cases. Codon 12 was mutated in 76% of cases versus codon 13 in 24%. Right-sided CRCs harbored more KRAS mutations than left-sided tumors, as did tumors that presented with perineural invasion. ConclusionOur findings in this Latin American population are consistent with the literature regarding the frequency of KRAS mutations in CRC, their distribution between codons 12 and 13, and type of nucleotide substitution. By combining nested PCR and pyrosequencing, we achieved a high rate of conclusive results in testing KRAS mutations in CRC samples — a method that can be used as an ancillary test for failed assays by conventional PCR.

Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues

A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA.

Do Antigen Retrieval Techniques Improve DNA Yield from Formalin Fixed Paraffin Embedded Tissue?

This study investigated the use of pressure cooking with different retrieval solutions at variable pHs to determine the optimum protocol for the extraction of high quality DNA from formalin fixed paraffin embedded (FFPE) tissues. Further, the oxidation effect on the archived FFPE tissues in relation to the age of the tissue blocks was also explored. The main aim of the study was to investigate this technique on 11 gastric cancers. The design of our study was based on the principles of the antigen retrieval technique using a conventional pressure cooker to improve the quality of DNA extracted from FFPE tissues. In addition to the gastric cancers, lymphoma, breast, prostatic, and colorectal carcinomas were used to eliminate tissue bias. Statistical correlation was done using the paired t-test and Benjamini-Hochberg test. Our findings show that high DNA concentrations were obtained using the different retrieval solutions with pressure cooking, compared to the control samples that were not subjected to this procedure. The mean DNA concentration increased with all the solutions tested but DNA yield was significantly higher in 3 of the 4 retrieval solutions used. The use of antigen retrieval solutions at high pressure and temperature provided by pressure cooking may enable the reversal of the crosslinking effect of formalin in FFPE tissues. Further, the observation that the DNA extracted from recently processed blocks was of a superior yield as opposed to older tissue blocks, confirms that oxidation does have a deteriorating effect on DNA.

Expanding epigenomics to archived FFPE tissues: an evaluation of DNA repair methodologies.

Epigenome-wide association studies are emerging in the field of cancer epidemiology with the rapid development of large-scale methylation array platforms. Until recently, these methods were only valid for DNA from flash frozen (FF) tissues. Novel techniques for repairing DNA from formalin-fixed paraffin-embedded (FFPE) tissues have emerged; however, a direct comparison of FFPE DNA repair methods before analysis on genome-wide methylation array to matched FF tissues has not been conducted. We conducted a systematic performance comparison of two DNA repair methods (REPLI-g Ligase vs. Infinium HD Restore Kit) on FFPE-DNA compared with matched FF tissues on the Infinium 450K array. A threshold of discordant methylation between FF-FFPE pairs was set at Δβ > 0.3. The correlations of β-values from FF-FFPE pairs were compared across methods and experimental conditions. The Illumina Restore kit outperformed the REPLI-g ligation method with respect to reproducibility of replicates (R(2) > 0.970), highly correlated β-values between FF-FFPE (R(2) > 0.888), and fewest discordant loci between FF-FFPE (≤0.61%). The performance of the Restore kit was validated in an independent set of 121 FFPE tissues. The Restore kit outperformed RELPI-g ligation in restoring FFPE-derived DNA before analysis on the Infinium 450K methylation array. Our findings provide critical guidance that may significantly enhance the breadth of diseases that can be studied by methylomic profiling. Epigenomic studies using FFPE tissues should now be considered among cancers that have not been fully characterized from an epigenomic standpoint. These findings promote novel epigenome-wide studies focused on cancer etiology, identification of novel biomarkers, and developing targeted therapies. See all the articles in this CEBP Focus section, "Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology."

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

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

P-A1 HPV associated Squamous Cell Carcinoma of the Head and Neck (HNSCC) yet to emerge in Nigeria

HPV associated HNSCC has emerged as a rapidly growing virus associated cancer in recent years. Studies show that the burden of HPV positive HNSCC is lower in African Americans compared to White Americans and the increase in incidence over time has been statistically significant only for White Americans. Differences in prevalence of oral sex behaviors have been blamed for this. There has been no previous study of the secular trend in HPV associated HNSCC in African population. We used QIAamp DNA FFPE Tissue Kit to extract DNA from Formalin fixed, Paraffin-Embedded (FFPE) HNC cancers blocks from 3 tertiary hospitals in Nigeria. We identified HPV types using Roche LINEAR ARRAY® HPV Genotyping Test. We examined 115 Head and Neck Cancers collected over 22 years (1990–2011); 94 (82%) were eligible for DNA core analysis, DNA purification was successful in 84 (73%). PCR amplification was successful in 49 (43%), Linear Array amplification was successful in 17 of these cases resulting in an overall success rate of 15%. These were from the larynx (6), cervical lymph nodes (3), nasal cavity (2), parotid (1), palate (1), maxillary sinus (1) and mandible (1). Two were non-specific head and neck masses and none from the oropharynx. Forty-one percent (7) were squamous cell carcinomas (larynx 6, maxillary sinus 1). None were found to be positive for HPV of any type. HPV associated HNSCC seems low in this population, this may be a result of a birth cohort effect and also because oral sex rates are believed to be low in this population. Public health education on the role of oral sex with multiple partners in the etiology of HPV associated HNSCC may mitigate future rise in incidence of HNSCC in this population. Study limitations are the small sample size and the low success rate for PCR and Linear Array amplification due to poor sample quality. Future studies with large sample size and improved sampling methods are required to aid improved understanding of the epidemiology of HPV associated HNSCC and of disparities in incidence and outcome among different populations.

Clinical next-generation sequencing in patients with non-small cell lung cancer.

A clinical assay was implemented to perform next-generation sequencing (NGS) of genes commonly mutated in multiple cancer types. This report describes the feasibility and diagnostic yield of this assay in 381 consecutive patients with non-small cell lung cancer (NSCLC). Clinical targeted sequencing of 23 genes was performed with DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The assay used Agilent SureSelect hybrid capture followed by Illumina HiSeq 2000, MiSeq, or HiSeq 2500 sequencing in a College of American Pathologists-accredited, Clinical Laboratory Improvement Amendments-certified laboratory. Single-nucleotide variants and insertion/deletion events were reported. This assay was performed before methods were developed to detect rearrangements by NGS. Two hundred nine of all requisitioned samples (55%) were successfully sequenced. The most common reason for not performing the sequencing was an insufficient quantity of tissue available in the blocks (29%). Excisional, endoscopic, and core biopsy specimens were sufficient for testing in 95%, 66%, and 40% of the cases, respectively. The median turnaround time (TAT) in the pathology laboratory was 21 days, and there was a trend of an improved TAT with more rapid sequencing platforms. Sequencing yielded a mean coverage of 1318×. Potentially actionable mutations (ie, predictive or prognostic) were identified in 46% of 209 samples and were most commonly found in KRAS (28%), epidermal growth factor receptor (14%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (4%), phosphatase and tensin homolog (1%), and BRAF (1%). Five percent of the samples had multiple actionable mutations. A targeted therapy was instituted on the basis of NGS in 11% of the sequenced patients or in 6% of all patients. NGS-based diagnostics are feasible in NSCLC and provide clinically relevant information from readily available FFPE tissue. The sample type is associated with the probability of successful testing.

Abstract B51: Extraction and analysis of genomic DNA from formalin-fixed paraffin-embedded neuroblastoma samples following laser capture microdissection

Abstract Introduction: Neuroblastoma (NB) is the most common extracranial solid tumor of infants. We constructed a tissue microarray (TMA) including 47 diagnostic NB cases and performed immunohistochemical (IHC) staining for a number of biomarkers. Differential IHC staining was identified between duplicate tissue cores for a proportion of NB cases. Differential IHC staining occurs frequently in cancer specimens, and can be due to technical factors, tissue artefacts, or underlying biological differences between tumor sub-regions. As laser capture microdissection (LCM) allows isolation of cells from formalin-fixed paraffin-embedded (FFPE) tissue slides, we applied LCM to FFPE NB samples, to investigate the basis of differential IHC staining. Material and Methods: NSE, NB84, ALK, TPD52 and MGMT proteins were detected within diagnostic NB TMA cores and whole NB sections using IHC. Cells were isolated from FFPE sections using LCM, and genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit. Extracted genomic DNA was quantified using a Nanodrop spectrophotometer, and DNA quality was assessed by a Bioanalyzer 2100. All DNA samples were tested using PCR with TPD52 and ALK primers. Results including clinical and sample variables were analysed using SPSS version 19. Results: Of 47 NB cases within the TMA, 35 cases were represented by duplicate tissue cores, whereas the remaining 12 cases were represented by single cores. IHC analysis of 5 biomarkers showed visually different IHC staining in tumour tissue between duplicate tissue cores in 15/35 (43%) cases. Whole tissue sections from 6 of these 15 cases were then examined, which reproduced differential IHC staining. Isolation of NB cells by LCM was successfully performed after substantial optimisation. A total of 14 regions from 8 NB cases were microdissected, producing DNA yields ranging from 0.12 - 4.05 pg/ µm2 tissue. There were no significant associations between total yields of extracted DNA, and microdissected tissue areas, or tissue sample ages. All DNA samples were successfully amplified by TPD52 but not by ALK PCR primers. Conclusions: Genomic DNA can be reliably extracted from microdissected FFPE NB tissues, although only one of 2 genes could be successfully PCR amplified. Downstream assays of DNA extracted from FFPE samples may therefore have particular design requirements. Further application of this approach may help improve our understanding of the level of tumour heterogeneity present in NB samples at diagnosis. Citation Format: Le M. Thwe, Laurence C. Cantrill, Daniel R. Catchpoole, Loretta Lau, Jennifer A. Byrne. Extraction and analysis of genomic DNA from formalin-fixed paraffin-embedded neuroblastoma samples following laser capture microdissection. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B51.

Rapid real-time PCR for detection of Mycobacterium tuberculosis complex DNA in formalin-fixed paraffin embedded tissues: 16% of histological ‘sarcoid’ may contain such DNA

AimsTo investigate the diagnostic accuracy of IS6110 real-time PCR for detection of Mycobacterium tuberculosis complex (MTBC) in DNA extracted from formalin-fixed paraffin embedded (FFPE) tissues using two different methods. In...

Imaging of N-linked glycans from formalin-fixed paraffin-embedded tissue sections using MALDI mass spectrometry.

Aberrant glycosylation is associated with most of the diseases. Direct imaging and profiling of N-glycans on tissue sections can reveal tissue-specific and/or disease-associated N-glycans, which not only could serve as molecular signatures for diagnosis but also shed light on the functional roles of these biomolecules. Mass spectrometry imaging (MSI) is a powerful tool that has been used to correlate peptides, proteins, lipids, and metabolites with their underlying histopathology in tissue sections. Here, we report an MSI technique for direct analysis of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissues. This technique consists of sectioning FFPE tissues, deparaffinization, and rehydration of the sections, denaturing tissue proteins, releasing N-linked glycans from proteins by printing peptide-N-glycosidase F over the sections, spray-coating the tissue with matrix, and analyzing N-glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Brain sections from a C57BL/6 mouse were imaged using this technique at a resolution of 100 μm. Forty-two N-glycans were analyzed from the mouse brain section. The mass spectrometry images were used to study the relative abundance of oligomannose, nonfucosylated, and fucosylated complex N-glycans in different brain areas including isocortex, hippocampal formation, and brainstem and specific glycans associated with different areas of the brain were identified. Furthermore, glioblastoma tumor xenografts in a NOD/SCID mouse were imaged. Several glycans with differential expression in tumor versus normal brain tissues were identified. The MSI technique allows for imaging of N-glycans directly from FFPE sections. This method can potentially identify tissue-specific and/or disease-associated glycans coexpressed with other molecular signatures or within certain histological structures.

A simple and cost-effective method of DNA extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing.

BackgroundExtraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column.MethodsThree DNA extraction methods were compared: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student’s t-test.ResultsThe combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods.ConclusionsUsing H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment.

A Comparison of Different Methods for Rna and Dna Extraction From Formalin – Fixed Paraffin-Embedded Tissues From Different Cancer Samples

RNA and DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. we compared quantity of RNA extracted by two different protocols from 14 recently archived from patients suffered from different cancers distributed among formalin-fixed paraffin-embedded (FFPE) breast cancer tissues ,thyroid cancer tissues and Cervical uterus carcinoma tissues by using Guanidine isothiocyanate (GTC)with phenol-chloroform (protocol-1) and Silica Gel Column(SGC) dependent on spin column purification-based ( protocol- 2), to assess, which technique is the most efficient and reproducible in terms of total yield and purity. The results showed RNA isolated with SGC technique was characterized by higher mean concentration in a range (80-180) µg/ml ,but it give positive results to 12 sample with degradation in comparison with RNA isolated by the (GTC) technique (protocol-1), comparison with total RNA extraction from human blood ( two distinguished bands ) .In this study comparative methods have been performed to analyze the efficiency for extraction and purification of Genomic DNA from six selective FFPE Tissues samples, revealing that the extraction of DNA by using extraction modified method give good result with yield higher mean concentration of DNA in a range (160-260) µg/ml.The lysis of FFPE Tissues was enhanced by increased the concentration of proteinase K to30 mg\ml for 2 hour at 65C, which considered the best time for lysis tissues and heated comparable with results that obtained from lysis tissues by using 20mg\ml for (24 -48 hours) at 55C