Snap-frozen Tissue Research Articles

Abstract B08: Patient-derived xenografts of oral squamous cell carcinoma: A model system for the identification of prognostic molecular signatures and for preclinical assessment of targeted therapies

Abstract Patients treated for oral squamous cell carcinoma (OSCC) continue to have a poor overall survival rate. The existence of tumor heterogeneity produces major challenges to successfully treating OSCC. Patient-derived xenografts (PDXs) provide an important tool for translational research and for improving our understanding of tumor heterogeneity in the treatment setting. PDXs of OSCCs recapitulate the histology, tumor behavior, and biomarker expression profile of the original patient tumor and our studies show that engraftment itself associates with poor patient outcome. This clinical relevance of engraftment demonstrates the use of PDXs as a powerful research tool in the advancement of personalized medicine. We have generated a large number of OSCC PDXs. Overall, 158 of 231 OSCC samples (68%) formed tumors when implanted into immune-compromised mice. The ability to engraft varied from patient to patient and these differences were associated to patient survival outcome and lymph node (LN) status. The clinical data on 108 patient samples with a minimum of 2 years follow-up demonstrated that OSCC patients whose tumors engrafted experienced a significantly worse overall survival (OS) and disease-free survival (DFS) compared to tumors that did not engraft (OS of 59% vs. 84%; DFS of 56% vs. 79%). When combined with nodal status, LN+ and LN- patients could be further stratified, wherein the 2-year OS probability for LN-, non-engrafted patients were 100%, vs. only 46% of patients that were both LN+ and engrafted. Next, given the observed clinical relevance of engrafters vs. non-engrafters, we sought to determine whether these two cohorts display distinct genetic alterations. We designed a custom in-solution hybrid capture targeted sequencing panel of 112 genes for both mutational and copy number alteration (CNA) analyses. DNA was extracted from 120 primary snap-frozen tissues (62 engrafters and 58 non-engrafters) and the targeted panel of genes was sequenced to identify gene and pathway alterations that correlate with engraftment and may be of prognostic value. Our repertoire of OSCC PDXs includes a viable tumor bank and fully annotated OSCC samples with known engraftment ability and genetic alterations. We will be able to use the molecular information obtained from our targeted sequencing results to tailor and test therapies in PDXs to the specific mutations and/or CNAs they bear. Overall, our OSCC PDXs provide a valuable system to identify prognostic and engraftment molecular signatures that can be studied for pre-clinical drug development and will contribute toward personalized medicine. Citation Format: Christina Karamboulas, Jeff Bruce, Xin Qiu, Jalna Meens, Keira Pereira, Ella Hyatt, Shao Hui Huang, Wei Xu, Trevor Pugh, Laurie Ailles. Patient-derived xenografts of oral squamous cell carcinoma: A model system for the identification of prognostic molecular signatures and for preclinical assessment of targeted therapies. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B08.

Abstract 1852: Multiple PI3K pathway mutations predominate in low stage endometrial carcinomas

Abstract Objectives: Endometrial cancers (ECs) frequently harbor mutations in PI3K pathway genes. Our objective was to determine the mutation frequency of the PIK3 pathway in the tumors of EC patients prospectively enrolled on an IRB-approved institutional tumor sequencing initiative (UNCseq) and correlate these with clinical factors. Methods: Snap-frozen and FFPE tissue samples were collected from patients enrolled on UNCseq (NCT01457196). DNA was isolated using the Puregene DNA purification system, and Illumina libraries were prepared separately from tumor and a matched normal sample from each patient. Relevant targets, were enriched by a custom designed Agilent SureSelect hybrid capture enrichment library using standard protocols. Samples were sequenced on Illumina HiSeq machines in a variety of formats. Statistical analysis of clinical correlates was performed in R (v 3.1.1). Results: Data was collected from 109 EC patients. Thirty-six tumors (33.0%), 34 of which were endometrioid, had >2 mutations between PIK3CA and PIK3R1. 20 patients had multiple PIK3CA mutations (18.3%) and 11 had multiple PIK3R1 mutations (10.1%). In all but 5 cases, multiple mutations of PIK3R1 or PIK3CA were coincident with PTEN mutation (p = 0.018). There were strong correlations between PI3K pathway mutations and tumor grade and stage. High stage tumors (stage >2) were less likely (5/36) than stage 1 tumors (31/73) to have multiple PI3K mutations (p = 0.0056). Though not reaching significance, grade 3 tumors (8/37) were less likely than low-grade tumors (28/72) to demonstrate multiple PI3K mutations (p = 0.11). Of the 10 serous ECs, none exhibited multiple PI3K mutations. Conclusions: There is a strong relationship between mutated PIK3 pathway genes and clinically relevant tumor biology in ECs. Multiple mutations in the PIK3 pathway are common, suggesting that PI3K pathway inhibition in EC may need to be directed against PIK3CA and PIK3R1 simultaneously. These multiply mutated tumors are strongly linked to lower stage and grade tumors with concurrent PTEN mutations. We posit that initially PTEN mutant clones may give rise to subclonal populations with various PI3K mutations which may be relatively indolent until additional driver mutations are acquired such that one subpopulation predominates, resulting in higher grade and stage ECs. Citation Format: Dario R. Roque, Will R. Jeck, David N. Hayes, Arthur M. Dizon, Leslie Clark, Stuart Pierce, Weiya Z. Wysham, Tara Castellano, Paola A. Gehrig, Victoria Bae-Jump. Multiple PI3K pathway mutations predominate in low stage endometrial carcinomas. [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 1852.

Abstract 3201: Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 predicts poor prognosis and promotes cell proliferation in Ewing sarcoma

Abstract INTRODUCTION: Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 (IGF2BP3, also known as IMP3) is an oncofetal protein which binds RNA thus influencing the transcript target fate. IGF2BP3 is de novo synthesized in malignancies where it promotes proliferation, drug resistance and metastases. Moreover, IGF2BP3 represents a promising indicator of outcome in several cancers. Ewing sarcoma (EWS) is a rare bone and soft tissue tumor where the IGF system plays a pivotal role. This study aimed to investigate the relevance of IGF2BP3 in EWS and verify its value as a prognostic biomarker. MATERIAL AND METHODS: Total RNA was extracted from two independent cohorts of 30 and 109 snap-frozen tissues from localized primary EWS samples with more than 5 years follow-up. Microarray analysis (Affymetrix) was performed in the cohort of 30 patients while IGF2BP3 expression was analysed by qRT-PCR (Applied Biosystem) in the cohort of 109 cases. Prognostic value of IGF2BP3 was evaluated both in univariate and multivariate analysis. IGF2BP3 expression was assessed by qRT-PCR and western blot in a panel of 14 EWS cell lines. Plasmids for IGF2BP3 silencing or over-expression were transfected in A673 or LAP-35 and IOR/CAR EWS cells, respectively. Soft-agar growth of cell lines with differential IGF2BP3 expression was evaluated. catRAPID express human web server was employed for prediction of novel IGF2BP3/mRNAs interactions. IGF-2 and ABCF1 expression levels were analysed in vitro and in clinical specimens. RESULTS: Microarray results evidenced that IGF2BP3 higher expression correlated with a worse outcome (p-value 0.0002). This data was confirmed in the cohort analysed by qRT-PCR both considering univariate (p-value 0.001) and multivariate analysis (HR:2.83. IC95% [1.22-6.53].p = 0.015). In vitro, higher IGF2BP3 expression correlated with an increased capability of growth in anchorage-independent conditions and IGF2BP3 knockdown significantly decreased soft-agar growth of EWS cells. Oncogenic potential of IGF2BP3 was not found to be mediated through IGF-2-dependent mechanism but ABCF1, an ABC family member with a key role in translation initiation, was predicted as a target of IGF2BP3 by in silico analysis. Accordingly, IGF2BP3 loss- or gain-of-functions approaches induced down- or up-regulation of ABCF1 protein levels, respectively. In addition, a direct correlation between IGF2BP3 and ABCF1 was found in 109 EWS patients (p-value minor than 0.0001). Functional studies on the role of ABCF1 in EWS will follow. CONCLUSIONS: IGF2BP3 represents an indicator of prognosis in EWS therefore its detection may be of help to stratify patients according to risk. We also provided evidences for IGF2BP3 in the regulation of EWS aggressiveness and identified ABCF1 as a new putative mediator of IGF2BP3 malignant effects. (Grants: FIRB RBAP11884M_005 and AIRC IG2013_14049 to KS) Citation Format: Caterina Mancarella, Linda Calzolari, Stefano Ferrari, Davide Donati, Piero Picci, Katia Scotlandi. Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 predicts poor prognosis and promotes cell proliferation in Ewing sarcoma. [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 3201.

148 - The binding profile of proteins is maintained in snap-frozen tissue

148 - The binding profile of proteins is maintained in snap-frozen tissue

Impact of BMI on genetic variation in ovarian cancer.

e17090Background: We examined the effect of body mass index (BMI) on genetic mutations in ovarian cancer (OC) patients enrolled on UNCseq. Methods: Snap-frozen and FFPE tissue samples were collecte...

MP85-10 IDENTIFICATION OF WDR20 AS A NEW TUMOR SUPPRESSOR GENE PREFERENTIALLY ALTERED IN HIGH-GRADE CLEAR CELL RENAL CELL CARCINOMA.

INTRODUCTION AND OBJECTIVES: There are more than 200,000 cases of renal cell carcinoma (RCC) each year in the world, accounting for approximately 2% of all cancers. RCC clear cell type (ccRCC) is the most common subtype of RCC and accounts for 75 to 80% of the cases with highest rates of local invasion, development of metastasis and mortality. New target therapy is based on antiangiogenic antibodies and molecules, changing the course of the disease, but the results so far are disappointing. Our aim is to study miRNAs and their target genes related to angiogenesis in ccRCC trying to bring some new knowledge to the molecular pathways related to the disease METHODS: The expression levels of miRNAs miR-99a, 99b, 100; 199a; 106a; 106b; 29a; 29b; 29c; 126; 200a, 200b and their respective target genes: mTOR, HIF1-a, VHL, PDGF, VEGF, VEGFR1 and VEGFR2 were evaluated using qRT-PCR.in snap-frozen tumor tissue samples from 56 patients diagnosed with ccRCC and 5 samples of benign renal tissue as control. The results were related to tumor size, Fuhrman nuclear grade and microvascular invasion, considering the risk criteria proposed by Dall’Oglio et al. (2007). RESULTS: We found an underexpression of the most genes except the VEGFA and PDGF, while the analysis of miRNAs showed underexpression only the miR 199a, 100 and 126. We compared the expression of genes with their possible regulatory miRNAs, and we found that mTOR was underexpressed while miR99a was overexpressed in most samples. This relationship also occurs between VEGFA and miR126 and between miRNAs 106a, 106b, and their target gene VHL. Considering the risk groups the overexpression of miR200b was associated with high-risk patients (p 1⁄4 0.01) and the overexpression of miR126 was associated with lower Fuhrman grade (I-II) (p 1⁄4 0.03). CONCLUSIONS: Our results show that in ccRCC there is an unbalance in the expression of genes and miRNAs related to angiogenesis and cell proliferation and survival. Furthermore with our findings we can speculate the role of miR200b and miR126 in the prognostic of ccRCC. We believe that the relationship between miRNAs and their respective genes should be more profoundly searched as markers and possible therapeutic agents in this neoplasia.

MP85-11 GENETIC DELETION OF SAV1 ENHANCES PROLIFERATION AND NUCLEAR IRREGULARITY IN RENAL TUBULAR EPITHELIAL CELLS

INTRODUCTION AND OBJECTIVES: There are more than 200,000 cases of renal cell carcinoma (RCC) each year in the world, accounting for approximately 2% of all cancers. RCC clear cell type (ccRCC) is the most common subtype of RCC and accounts for 75 to 80% of the cases with highest rates of local invasion, development of metastasis and mortality. New target therapy is based on antiangiogenic antibodies and molecules, changing the course of the disease, but the results so far are disappointing. Our aim is to study miRNAs and their target genes related to angiogenesis in ccRCC trying to bring some new knowledge to the molecular pathways related to the disease METHODS: The expression levels of miRNAs miR-99a, 99b, 100; 199a; 106a; 106b; 29a; 29b; 29c; 126; 200a, 200b and their respective target genes: mTOR, HIF1-a, VHL, PDGF, VEGF, VEGFR1 and VEGFR2 were evaluated using qRT-PCR.in snap-frozen tumor tissue samples from 56 patients diagnosed with ccRCC and 5 samples of benign renal tissue as control. The results were related to tumor size, Fuhrman nuclear grade and microvascular invasion, considering the risk criteria proposed by Dall’Oglio et al. (2007). RESULTS: We found an underexpression of the most genes except the VEGFA and PDGF, while the analysis of miRNAs showed underexpression only the miR 199a, 100 and 126. We compared the expression of genes with their possible regulatory miRNAs, and we found that mTOR was underexpressed while miR99a was overexpressed in most samples. This relationship also occurs between VEGFA and miR126 and between miRNAs 106a, 106b, and their target gene VHL. Considering the risk groups the overexpression of miR200b was associated with high-risk patients (p 1⁄4 0.01) and the overexpression of miR126 was associated with lower Fuhrman grade (I-II) (p 1⁄4 0.03). CONCLUSIONS: Our results show that in ccRCC there is an unbalance in the expression of genes and miRNAs related to angiogenesis and cell proliferation and survival. Furthermore with our findings we can speculate the role of miR200b and miR126 in the prognostic of ccRCC. We believe that the relationship between miRNAs and their respective genes should be more profoundly searched as markers and possible therapeutic agents in this neoplasia.

Quantitative Imaging of D-2-Hydroxyglutarate in Selected Histological Tissue Areas by a Novel Bioluminescence Technique.

Patients with malignant gliomas have a poor prognosis with average survival of less than 1 year. Whereas in other tumor entities the characteristics of tumor metabolism are successfully used for therapeutic approaches, such developments are very rare in brain tumors, notably in gliomas. One metabolic feature characteristic of gliomas, in particular diffuse astrocytomas and oligodendroglial tumors, is the variable content of D-2-hydroxyglutarate (D2HG), a metabolite that was discovered first in this tumor entity. D2HG is generated in large amounts due to various “gain-of-function” mutations in the isocitrate dehydrogenases IDH1 and IDH2. Meanwhile, D2HG has been detected in several other tumor entities, including intrahepatic bile-duct cancer, chondrosarcoma, acute myeloid leukemia, and angioimmunoblastic T-cell lymphoma. D2HG is barely detectable in healthy tissue (<0.1 mM), but its concentration increases up to 35 mM in malignant tumor tissues. Consequently, the “oncometabolite” D2HG has gained increasing interest in the field of tumor metabolism. To facilitate its quantitative measurement without loss of spatial resolution at a microscopical level, we have developed a novel bioluminescence assay for determining D2HG in sections of snap-frozen tissue. The assay was verified independently by photometric tests and liquid chromatography/mass spectrometry. The novel technique allows the microscopically resolved determination of D2HG in a concentration range of 0–10 μmol/g tissue (wet weight). In combination with the already established bioluminescence imaging techniques for ATP, glucose, pyruvate, and lactate, the novel D2HG assay enables a comparative characterization of the metabolic profile of individual tumors in a further dimension.

Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples

Next-generation sequencing (NGS) of tumour samples is a critical component of personalised cancer treatment, but it requires high-quality DNA samples. Routine neutral-buffered formalin (NBF) fixation has detrimental effects on nucleic acids, causing low yields, as well as fragmentation and DNA base changes, leading to significant artefacts. We have carried out a detailed comparison of DNA quality from matched samples isolated from high-grade serous ovarian cancers from 16 patients fixed in methanol and NBF. These experiments use tumour fragments and mock biopsies to simulate routine practice, ensuring that results are applicable to standard clinical biopsies. Using matched snap-frozen tissue as gold standard comparator, we show that methanol-based fixation has significant benefits over NBF, with greater DNA yield, longer fragment size and more accurate copy-number calling using shallow whole-genome sequencing (WGS). These data also provide a new approach to understand and quantify artefactual effects of fixation using non-negative matrix factorisation to analyse mutational spectra from targeted and WGS data. We strongly recommend the adoption of methanol fixation for sample collection strategies in new clinical trials. This approach is immediately available, is logistically simple and can offer cheaper and more reliable mutation calling than traditional NBF fixation.

Abstract A27: Transcriptome analyses of human ampulla and fimbriae highlight similarities and differences.

Abstract Background: Recently described precursors of high-grade serous carcinoma (HGSC), the p53 signature, a latent precursor, and Serous Tubal Intraepithelial Carcinoma (STIC), a pre-malignant precursor, occur most frequently at the distal and fimbriated end of the fallopian tube (FTE). In 3 previous reports, we have demonstrated that the FTE of BRCA1 mutation carriers, at genetic risk of HGSC, have altered signaling pathways compared to controls. Ovarian production of ROS is released after the LH surge to induce ovulation. Reactive oxygen species (ROS) have been implicated in serous carcinogenesis. The objective of this study is to compare the transcriptome profiles of normal fimbria (high-risk epithelia prone to transformation) FTE and normal ampulla (low-risk epithelia) FTE which may lead to understanding the distal end of the fallopian tube as the preferential anatomic location of the fallopian to tube for cellular transformation. Method: Snap-frozen matched fimbria and ampulla tissues were controlled for age and ovarian cycle status. Cases included 12 luteal phase and 12 follicular phase women at no known risk for ovarian cancer. Laser capture microscopy was used to microdissect FTE cells, using 7-10 sections per case. Total RNA was isolated, RNA extracted and cDNA amplified. The expression profiles were generated using Affymetrix Human Genome HTA-2.0 Array. Bayes moderated t-statistics implemented in the R/Bioconductor package LIMMA to compare mRNA expression values for probes of samples based on their ‘phase’ and ‘anatomy’, respectively. t-statistics was generated from the aforementioned respective analyses, and ranked based Wilcoxon tests on gene sets from the Reactome, GO and Cytoband databases. Results: The cases clustered predominantly by ovarian cycle status rather than by their differences in anatomical origin or their matched pair. There were 75 unique probe IDs that were significantly different (p&amp;lt;0.05) between ampulla and fimbria which were independent of ovarian cycle status, – SALL1, ME1 – higher in the ampulla; GSTA1, GSTA2, PDK4, SERPINA3 -higher in the fimbria – genes involved in metabolic pathways) were observed between the ampulla and fimbria FTE. This gene signature when applied to transcriptome analyes of normal FTE and serous carcinoma, segregate the samples by cancer versus normal. Most of the genes were down-regulated in cancer. Conclusions: The epithelia of the anatomically high-risk fallopian tube – the fimbria, show few differences in gene expression profiles compared to the lower risk portion – the ampulla. Expression differences predominantly are in response to the hormonal milieu, post-ovulation with an antioxidant program. The increased anatomic risk of the fimbria is likely due to effects of the microenvironment, such as repeated exposure to follicular fluid at ovulation (higher ROS) and antioxidant genes and their interaction with DNA repair genes (like TP53), rather than intrinsic differences of the FTE in the two sites Citation Format: Sophia George, Anca Milea, Sowamber Ramlogan, Michael Considine, Leslie Cope, Noor Salman, Patricia Shaw. Transcriptome analyses of human ampulla and fimbriae highlight similarities and differences. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A27.

The Expression of miR-375 Is Associated with Carcinogenesis in Three Subtypes of Lung Cancer.

Many studies demonstrated unique microRNA profiles in lung cancer. Nonetheless, the role and related signal pathways of miR-375 in lung cancer are largely unknown. Our study investigated relationships between carcinogenesis and miR-375 in adenocarcinoma, squamous cell carcinoma and small cell lung carcinoma to identify new molecular targets for treatment. We evaluated 723 microRNAs in microdissected cancerous cells and adjacent normal cells from 126 snap-frozen lung specimens using microarrays. We validated the expression profiles of miR-375 and its 22 putative target mRNAs in an independent cohort of 78 snap-frozen lung cancer tissues using quantitative reverse-transcriptase PCR. Moreover, we performed dual luciferase reporter assay and Western blot on 6 targeted genes (FZD8, ITGA10, ITPKB, LRP5, PIAS1 andRUNX1) in small cell lung carcinoma cell line NCI-H82. We also detected the effect of miR-375 on cell proliferation in NCI-H82. We found that miR-375 expression was significantly up-regulated in adenocarcinoma and small cell lung carcinoma but down-regulated in squamous cell carcinoma. Among the 22 putative target genes, 11 showed significantly different expression levels in at least 2 of 3 pair-wise comparisons (adenocarcinoma vs. normal, squamous cell carcinoma vs. normal or small cell lung carcinoma vs. normal). Six targeted genes had strong negative correlation with the expression level of miR-375 in small cell lung carcinoma. Further investigation revealed that miR-375 directly targeted the 3’UTR of ITPKB mRNA and over-expression of miR-375 led to significantly decreased ITPKB protein level and promoted cell growth. Thus, our study demonstrates the differential expression profiles of miR-375 in 3 subtypes of lung carcinomas and finds thatmiR-375 directly targets ITPKB and promoted cell growth in SCLC cell line.

High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq.

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.

The clinical phenotype of YWHAE-NUTM2B/E positive pediatric clear cell sarcoma of the kidney.

Clear cell sarcoma of the kidney (CCSK) although uncommon, is the second most frequent renal malignancy of childhood. Until now, the sole recurrent genetic aberration identified in CCSKs is t(10;17)(q22;p13), which gives rise to a fusion transcript of YWHAE and NUTM2B/E. So far, the clinical relevance of this fusion transcript is unknown. The aim of this descriptive study was to determine the clinical phenotype of t(10;17)(q22;p13) positive CCSKs. Snap-frozen tissues, formalin-fixed paraffin-embedded tissues or RNA previously extracted from CCSK samples throughout European, North-American and Japanese study groups were screened by RT-PCR for the YWHAE-NUTM2B/E transcript. Clinical characteristics, tumor characteristics, and outcome of patients with and without the fusion transcript were studied. The cohort comprised 51 previously published cases to which were added 139 internationally collected CCSK samples. RNA from 57 of these additionally collected cases was of sufficient quality to be successfully screened for the YWHAE-NUTM2B/E transcript. In total, seven of the 108 cases harbored the fusion transcript. Patients with tumors containing the fusion transcript were relatively young (median age 10 months), had associated low median tumor volumes and stage I disease was not observed in these patients. Two of seven patients relapsed and one of seven patients died of disease. Ranges of values were not overtly different between patients with and without the fusion transcript; however, the number of fusion transcript positive cases turned out to be too small to permit reliable statistical analysis. The current study did not identify an explicit clinical phenotype of CCSK cases harboring the YWHAE-NUTM2B/E fusion transcript.

Proteomic analysis of neurons microdissected from formalin-fixed, paraffin-embedded Alzheimer's disease brain tissue.

The vast majority of human tissue specimens are formalin-fixed, paraffin embedded (FFPE) archival samples, making this type of tissue a potential gold mine for medical research. It is now accepted that proteomics can be done using FFPE tissue and can generate similar results as snap-frozen tissue. However, the current methodology requires a large amount of starting protein, limiting the questions that can be answered in these types of proteomics studies and making cell-type specific proteomics studies difficult. Cell-type specific proteomics has the potential to greatly enhance understanding of cell functioning in both normal and disease states. Therefore, here we describe a new method that allows localized proteomics on individual cell populations isolated from FFPE tissue sections using laser capture microdissection. To demonstrate this technique we microdissected neurons from archived tissue blocks of the temporal cortex from patients with Alzheimer’s disease. Using this method we identified over 400 proteins in microdissected neurons; on average 78% that were neuronal and 50% that were associated with Alzheimer’s disease. Therefore, this technique is able to provide accurate and meaningful data and has great potential for any future study that wishes to perform localized proteomics using very small amounts of archived FFPE tissue.

The Impact of Different Preservation Conditions and Freezing-Thawing Cycles on Quality of RNA, DNA, and Proteins in Cancer Tissue.

High-quality cancer tissues are essential for future research, especially molecular research. For the sake of better quality of tissues, some storage methods are chosen according to lab conditions. But the impact of different storing conditions on the quality of RNA, DNA (especially the degree of DNA methylation), and protein of tissues that have undergone a thawing process, is not clear. We analyzed the influence of different storage conditions including in RNALater solution, normal saline, Opti-mum Cutting Temperature compound (OCT), and snap frozen with no protective reagent (as control) in paired tissue samples on the quality of RNA (RNA Integrity Number value and mRNA expression), DNA quality (DNA amplification and DNA methylation degree of gene RASSF1a), and protein quality. Further, we analyzed the RNA quality of tissues that underwent three freeze-thaw cycles. The RNALater-treated group retained good RNA quality as expected on three repeated freeze-thaw cycles (RIN>8), but the snap-frozen tissues showed relatively poor results after one freeze-thaw cycle (RIN<7) and three times repeated freeze-thaw cycles (RIN<6). RNA from saline- and OCT-treated groups also yielded good results when we repeated freezing and thawing one time (RIN>7) and two times (RIN>6). The impact of different storing conditions on DNA amplification is small. However, DNA methylation and protein quality are different with different storing conditions. OCT seems to be more secure and stable compared with other two experimental groups, and show a similar trend with control group. In consideration of budget and efficiency, we suggest OCT as the best storing method that not only preserves RNA quality during the freezing-thawing process well, but also ensures more secure and stable DNA and protein.

Comparing gene expression data from formalin-fixed, paraffin embedded tissues and qPCR with that from snap-frozen tissue and microarrays for modeling outcomes of patients with ovarian carcinoma.

BackgroundPreviously, we have used clinical and gene expression data from The Cancer Genome Atlas (TCGA) to model a pathway-based index predicting outcomes in ovarian carcinoma. This data were obtained from snap-frozen tissue measured with the Affymetrix U133 platform. In the current study, we correlate the data used to model with data derived from TaqMan qPCR both snap frozen and paraffin embedded (FFPE) samples.MethodsTo compare the effect of preservation methods on gene expression measured by qPCR, we assessed 18 patient and tumor sample matched snap-frozen and FFPE ovarian carcinoma samples. To compare gene measurement technologies, we correlated qPCR data from 10 patients with tumor sample matched snap-frozen ovarian carcinoma samples with the microarray data from TCGA. We normalized results to the average expression of three housekeeping genes. We scaled and centered the data for comparison to the Affymetrix output.ResultsFor the 18 specimens, gene expression data obtained from snap-frozen tissue correlated highly with that from FFPE samples in our TaqMan assay (r > 0.82). For the 10 duplicate TCGA specimens, the reported microarray data correlated well (r = 0.6) with our qPCR data, and ranges of expression along pathways were similar.ConclusionsGene expression data obtained by qPCR from FFPE serous ovarian carcinoma samples can be used to assess in the pathway-based predictive model. The normalization procedures described control variations in expression, and the range calculated along a specific pathway can be interpreted for a patient’s risk profile.Electronic supplementary materialThe online version of this article (doi:10.1186/s12907-015-0017-1) contains supplementary material, which is available to authorized users.

Expression profiling of angiogenesis-related genes in brain metastases of lung cancer and melanoma.

Brain metastases (BM) are the most common brain tumors of adults and are associated with fatal prognosis. Formation of new blood vessels, named angiogenesis, was proposed to be the main hallmark of the growth of BM. Previous preclinical evidence revealed that angiogenic blockage might be considered for treatment; however, there were varying responses. In this study, we aimed to characterize the expression pattern of angiogenesis-related genes in BM of lung cancer and melanoma, which might be of importance for the different responses against anti-angiogenic treatment. Fifteen snap-frozen tissues obtained from BM of non-small cell lung cancer (NSCLC), small-cell lung cancer (SCLC), and melanoma patients were analyzed for angiogenesis-related genes using a commercially available gene expression kit. Epilepsy tissue was used as control. Expression values were analyzed using hierarchical clustering investigating relative fold changes and mapping to Omicsnet protein interaction network. CXCL10, CEACAM1, PECAM1, KIT, COL4A2, COL1A1, and HSPG2 genes were more than 50-fold up-regulated in all diagnosis groups when compared to control, whereas genes such as ANGPT4, PDGFRB, and SERPINF1 were down-regulated only in SCLC and melanoma groups, respectively. Using hierarchical clustering, 12 out of 15 cases were allocated to the correct histological primary tumor type. We identified genes with consistent up-regulation in BM of lung cancer and melanoma and other genes with differential expression across BM of these tumor types. Our data may be of relevance for targeted therapy or prophylaxis of BM using anti-angiogenic agents.

Abstract 2080: Differential transcription profile of epithelia in fimbria versus the ampulla of the fallopian tube

Abstract Background: Recently described precursors of high-grade serous carcinoma (HGSC), the p53 signature, a latent precursor, and Serous Tubal Intraepithelial Carcinoma (STIC), a pre-malignant precursor, occur most frequently at the distal and fimbriated end of the fallopian tube (FTE). In 3 previous reports, we have demonstrated that the FTE of BRCA1 mutation carriers, at genetic risk of HGSC, have altered signaling pathways compared to controls. Ovarian production of ROS is released after the LH surge to induce ovulation. Reactive oxygen species (ROS) have been implicated in serous carcinogenesis. The objective of this study is to compare the transcriptome profiles of normal fimbria (high-risk epithelia prone to transformation) FTE and normal ampulla (low-risk epithelia) FTE which may lead to understanding the distal end of the fallopian tube as the preferential anatomic location of the fallopian to tube for cellular transformation. Method: Snap-frozen matched fimbria and ampulla tissues were controlled for age and ovarian cycle status. Cases included 12 luteal phase and 12 follicular phase women at no known risk for ovarian cancer. Laser capture microscopy was used to microdissect FTE cells, using 7-10 sections per case. Total RNA was isolated, RNA extracted and cDNA amplified. The expression profiles were generated using Affymetrix Human Genome HTA-2.0 Array. Results: Using gene level differential expression analysis with the Affymetrix Expression Console software, we performed unsupervised hierarchical clustering analysis with all 24 samples. We used a fold change of &amp;lt; -2 or &amp;gt; 2 and ANOVA p-value &amp;lt; 0.05 as a cut-off criteria for selecting genes. The cases clustered predominantly by ovarian cycle status rather than by their differences in anatomical origin or their matched pair. There were 427 genes differentially expressed amongst the 4 groups– Fim-Luteal, Fim-Follicular, FT-Luteal and FT-Follicular. Independent of ovarian cycle status, very few differences (35 genes– SALL1, ME1– higher in the ampulla; GSTA1, GSTA2, SERPINA3-higher in the fimbria– genes involved in metabolic pathways) were observed between the ampulla and fimbria FTE. Conclusions: The epithelia of the anatomically high-risk fallopian tube– the fimbria, show few differences in gene expression profiles compared to the lower risk portion– the ampulla. Expression differences predominantly are in response to the hormonal milieu, post-ovulation with an antioxidant program The increased anatomic risk of the fimbria is likely due to effects of the microenvironment, such as repeated exposure to follicular fluid at ovulation (higher ROS) and antioxidant genes and their interaction with DNA repair genes (like TP53), rather than intrinsic differences of the FTE in the two sites. Citation Format: Sophia H. George, Anca Milea, Noor Hera Salman, Patricia Ann Shaw. Differential transcription profile of epithelia in fimbria versus the ampulla of the fallopian tube. [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 2080. doi:10.1158/1538-7445.AM2015-2080

Abstract SY29-02: The use of innovative prostate biopsy tissue print techniques for molecular genomic, epigenomic and gene expression studies

Abstract Cancer health disparities can arise from research studies that use biospecimens that are not representative of the populations affected by the disease. In biorepositories at research oriented hospitals that support molecular biomarker research, an under-representation of important clinical subpopulations can result from practices that govern conventional biorepository collection of discarded tissues from surgical specimens. In prostate cancer biorepositories, most of the frozen samples are obtained from radical prostatectomy (RP) specimens. However, tissue collections based on RP specimens do not include patients who are diagnosed with relatively advanced disease and treated with first-line hormonal and/or radiation therapy rather than surgery; African Americans are more likely than European Americans to be diagnosed with this type of advanced prostate cancer. Moreover, RP collections have a limited representation of patient groups that preferentially choose non-surgical treatment for organ confined prostate cancer; such a preference for non-surgical treatment has been observed for African American prostate cancer patients in many areas of the US. Our collaborative group utilizes innovative prostate biopsy tissue print techniques to obtain a more complete representation of the men who are being evaluated for prostate cancer, including the patients with a biopsy diagnosis of “no cancer” and prostate cancer patients who chose active surveillance or non-surgical treatment rather than radical prostatectomy. Prostate biopsy tissue prints consist of nitrocellulose blots collected from each of the fresh tissue cores as it is transferred from the biopsy needle. This nitrocellulose blotting step is simple, inexpensive and results in no compromise of the biopsy tissue for surgical pathology. We have used snap-frozen tissue prints as the source of prostate tissues for multiple biomarker studies, including array-based and sequence-based profiling techniques that require high quality DNA and RNA. Because prostate biopsy tissue prints are easily collected in an outpatient office setting, this approach has allowed us to expand the Birmingham Area Prostate Cancer Biorepository (BAPrCAR) to include a multi-center urology practice that serves large numbers of African Americans and performs prostate biopsies on over 2000 patients per year. Prostate biopsy tissue prints have also been important in our studies of molecular marker correlations of magnetic resonance image (MRI) defined criteria of prostate cancer suspicion; our goal is to optimize the use of MRI/ultrasound guided biopsies for both African American and European American patients who are considering active surveillance. We have successfully used prostate biopsy tissue print samples to characterize multiple molecular biomarkers in parallel, including ancestry informative SNPs, mRNA and miRNA transcripts, genomic mutations and DNA methylation-marker patterns. In summary, our experience demonstrates that biopsy tissue print techniques provide an innovative, practical approach to overcoming disparities in prostate cancer research by significantly expanding patient representation in the frozen biorepository collections used for molecular biomarker discovery and translational studies. Citation Format: Sandra M. Gaston, William E. Grizzle, Rick Kittles, Jihad Hayek, Peter N. Kolettis, Soroush Rais-Bahrami, Jeffrey Nix, Jennifer Gordetsky, George W. Adams, Gary P. Kearney. The use of innovative prostate biopsy tissue print techniques for molecular genomic, epigenomic and gene expression studies. [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 SY29-02. doi:10.1158/1538-7445.AM2015-SY29-02

Characterisation of lymphocyte subpopulations in infantile haemangioma

AimsInterstitial CD45+ cells and T lymphocytes have previously been demonstrated within infantile haemangioma (IH). This study investigated the expression of B and T lymphocyte markers by the CD45+ population, and...