Fresh Frozen Tissue Samples Research Articles

Identification of Protein Abundance Changes in Hepatocellular Carcinoma Tissues Using PCT-SWATH.

To rapidly identify protein abundance changes in biopsy-level fresh-frozen hepatocellular carcinoma (HCC). The pressure-cycling technology (PCT) is applied and sequential window acquisition of all theoretical mass spectra (SWATH-MS) workflow is optimized to analyze 38 biopsy-level tissue samples from 19 HCC patients. Each proteome is analyzed with 45 min LC gradient. MCM7 is validated using immunohistochemistry (IHC). A total of 11787 proteotypic peptides from 2579 SwissProt proteins are quantified with high confidence. The coefficient of variation (CV) of peptide yield using PCT is 32.9%, and the R2 of peptide quantification is 0.9729. Five hundred forty-one proteins showed significant abundance change between the tumor area and its adjacent benign area. From 24 upregulated pathways and 13 suppressed ones, enhanced biomolecule synthesis and suppressed small molecular metabolism in liver tumor tissues are observed. Protein changes based on α-fetoprotein expression and hepatitis B virus infection are further analyzed. The data altogether highlight 16 promising tumor marker candidates. The upregulation of minichromosome maintenance complex component 7 (MCM7) is further observed in multiple HCC tumor tissues by IHC. The practicality of rapid proteomic analysis of biopsy-level fresh-frozen HCC tissue samples with PCT-SWATH has been demonstrated and promising tumor marker candidates including MCM7 are identified.

Abstract 4627: Integrated multi-layer analysis reveals novel insights into the molecular landscape of colorectal cancer (CRC)

Abstract Metabolic reprogramming, rewired signalling cascades, altered microbiota composition and aberrations in the tumor micro-environment have been implicated in CRC progression and response to treatment. In this study, we integrated proteomics, metabolomics, gut microbiota and predictions from network-analysis in tumor and non-cancerous tissue to build a comprehensive atlas to decipher the associations among alterations that occur in the different molecular layers. We performed our molecular fingerprinting in fresh frozen tissue samples from both tumor (centre and/or junction) and matched mucosa (5 and/or 10 cm distance) prospectively collected from a cohort of n=53 stage 0-IV CRC patients. We quantified the protein expression of a panel of 86 cancer-related targets involved in DNA damage/repair mechanisms, cell cycle regulation, growth/angiogenesis signalling, apoptosis and bioenergetics by Reverse Phase Protein Array (RPPA) in n=157 samples (n=48 from tumor and n=109 from mucosa) from n=53 patients. RPPA profiling on both tumor and matched normal tissue was available for 34 out of 53 patients. We performed High Resolution Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy (1H HR-MAS NMR) to profile metabolites in n=332 samples from n=52 patients (n=161 and n=171 from tumor and normal tissue, respectively). We characterized gut microbiota features from 16S rRNA sequencing in n=54 samples from n=18 patients. We assessed bioenergetic fitness using an ordinary-differential equations based model of core carbon metabolism that we are developing. The model can simulate the network dynamic and response to fuels (such as glucose, pyruvate and lactate) from extracellular milieu and their utilization and conversion via reactions in the cytoplasm and mitochondrial compartments. We used RPPA-based protein expression for HKII, TIGAR, PGYM, LDHA, MCT4, ATP5A as a proxy for enzymatic activities as case-specific inputs to the model. We identified distinct phenotypic differences when comparing tumors with normal tissue at each data type. Tumors showed higher protein levels for CHK1, GSK3B and LDHA; were enriched with fusobacteria; had increased metabolic levels of glycerphosphocholine, isoglutamine, phosphocholine, taurine and lactate; and were predicted by the mathematical model to have a more glycolytic phenotype. Normal mucosa had higher protein levels for GAB1 and elevated isobutyrate and lipids content. We further analysed the NMR spectra and using a genetic algorithm we identified 10 features (ppm levels) that could classify the tissue type with 88% cross-validated accuracy. We presented a unified analysis connecting dysregulations affecting the proteome, metabolome and microbiome. We provided insights into the heterogeneity of CRC which may lead to more specific molecular classifications and ultimately more targeted treatments. Citation Format: Manuela Salvucci, Liam Poynter, Reza Minerzami, Steven Carberry, Robert O'Byrne, Mattia Cremona, Bryan T. Hennessy, Kirill Veselkov, James Kinross, Jochen H. Prehn. Integrated multi-layer analysis reveals novel insights into the molecular landscape of colorectal cancer (CRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4627.

Abstract 1428: Application of ONCO/REVEAL Lung & Colon Cancer Panel in CRC samples and effect of DNA damage in FFPE samples

Abstract Introduction: The ONCO/REVEAL Lung&Colon Caner Panel (LC103, Pillar Biosciences Inc.) interrogates regions in 22 genes that are frequently mutated in NSCLC and CRC. To evaluate the performance of ONCO/REVEAL Lung&Colon Cancer Panel, we assessed a cohort of 207 colorectal cancer FFPE samples collected by a top-tier hospital in Shanghai between 2015 and 2016. Among them, 27 FFPE samples showed abnormally high numbers of low frequency variants (<2%) and were further investigated to assess the effect of DNA damage in somatic variant detection. Materials and Methods: DNA library preparation and sequencing: 10-20 ng of FFPE DNA (Quantitated by Qubit, Life Tech.) was used to prepare libraries using the LC103 panel. All libraries were subsequently sequenced on Illumina MiSeq sequencer. FFPE DNA repair: DNA extracted from 27 FFPE samples with a high number of low VAF (<2%) variants was treated with NEBNext FFPE DNA Repair Mix prior to library preparation. FFPE and DNA repaired FFPE were compared to analyze the effect of DNA damage in variant detection. Matched fresh frozen tumor tissue samples were also tested. Data analysis: PiVATTM (Pillar Biosciences Inc.) was used for data analysis. PCR errors and sequencing errors are reduced to be well below 1% VAF through the PiVAT error correction algorithm. Results: 100% successful rate of library preparation and sequencing: All of 207 FFPE samples yielded high quality sequencing data that detected mutant alleles at frequencies as low as 1%. Variant detection: A total of 414 somatic variants, including SNV and small indels, were identified above 2% VAF in 193 out of 207 samples. TP53 (38%), KRAS (24%), PIK3CA (12%), FBXW7 (9%) and PTEN (4%) were the most frequently mutated genes. CNVs were identified in EGFR, MET, ERBB2, KRAS and FGFR1 genes. The effect of DNA damage: The majority of detected variants between 1% and 2% VAF, are not known hotspot mutations. C:G>T:A mutations account for 73.8% of variants between 1% and 2% VAF. DNA repair by NEBNext FFPE DNA Repair Mix or other UDG enzymes reduced 1-2% VAF calls significantly, indicating that these variants are false positive calls. However, DNA repair enzymes used in our study could not eliminate false positive calls completely. Other mechanisms are suspected to contribute to the remaining low frequency calls. Variant between 2% and 5% VAF: In total, 33 somatic variants were detected at 2-5% VAF. There are many clinically actionable mutations or common driver mutations, including KRAS G12C (2.10%), KRAS G12D (2.21%), KRAS Q61L (2.74%), BRAF G469E (2.51%), PIK3CA E545K (2.08%, 2.39%, 4.78%), PIK3CA E545G (4.61%), PIK3CA Q546K (4.51%), PIK3CA H1047R (2.80%). Conclusions: The ONCO/REVEAL Lung&Colon Caner Panel is a robust and sensitive NGS Assay for the detection of somatic variants. DNA damage confounds variant identification in FFPE samples between 1 and 5% frequency. Citation Format: Jianfeng Xiao, Jiajie Tang, Xiangzhi Liu, Xiaoyan Tian. Application of ONCO/REVEAL Lung & Colon Cancer Panel in CRC samples and effect of DNA damage in FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1428.

Aberrant expression of stress-induced phosphoprotein 1 in colorectal cancer and its clinicopathologic significance

Stress-induced phosphoprotein 1 (STIP1) is an adaptor protein that bridges HSP70 and HSP90 folding and a secretory protein that regulates malignant tumor progression. The aim of the present study was to demonstrate the clinicopathological significance and prognostic role of STIP1 in colorectal cancer (CRC). We used data from The Cancer Genome Atlas (TCGA) to analyze STIP1 expression in CRC and utilized 8 pairs of fresh-frozen tissue samples to investigate STIP1 expression in CRC tissues and adjacent normal tissues using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays. We also used immunohistochemical staining to detect STIP1 expression in 144 formalin-fixed, paraffin-embedded (FFPE) CRC tissue samples and determine the clinical significance of STIP1 expression in CRC. The results of bioinformatics analysis, qRT-PCR, and Western blot showed that STIP1 expression was higher in CRC tissues than in adjacent normal tissues. High STIP1 expression was significantly correlated with advanced T stage (P = .01), N stage (P = .001), M stage (P < .001), and TNM stage (P < .001). Moreover, Kaplan-Meier analyses indicated that higher STIP1 expression predicted a worse prognosis in patients with CRC, and Cox regression analysis revealed that STIP1 was an independent prognostic factor for overall survival and disease-free survival in patients with CRC. In conclusion, our results suggest that STIP1 acts as an oncogene in CRC and can therefore serve as a biomarker for the prognosis of patients with CRC.

P.048 Correlation of preoperative serum lactate, MR spectroscopy and frozen tissue lactate levels as a biomarker for giomas – a prospective clinical study

Background: Lactate, a by-product of glycolysis, has been well established as a marker of poor tissue perfusion. Elevated lactate production is observed in tumor glycolysis known as the Warburg effect. We have previously shown that serum lactate correlated with brain tumor grade. In this prospective study we aimed to determine if the preoperative serum lactate correlated with preoperative MR spectroscopy and in lactate levels in the fresh frozen tissue samples. Methods: Twenty-one glioma patients (13 male, 8 female) ages 34 – 86 underwent craniotomy at a single institution by lead author. Tumor pathology revealed a Glioblastoma (n=16), grade II (oligodendroglioma n=1) and Grade III Glioma (anaplastic astrocytoma n=4). Preoperative spectroscopy was performed on 18 patients. A fellowship trained neuro-radiologist (JPC) was blinded to the serum and tissue lactate levels and graded the spectroscopy lactate levels as low or elevated. Results: There was direct correlation of spectroscopy tissue lactate levels with serum lactate levels. Pre-operative serum lactate (range 6.6- 29.9 mg/dl) was directly correlated with the fresh frozen tissue lactate levels (range 0.1 – 0.39 ug/mg; Pearson r=0.6 p = 0.0021). Conclusions: This study supports that serum lactate correlates with spectroscopy and tissue lactate levels.

PO-375 Analysis of miRNA expression in non-small cell lung cancer

Introduction MicroRNAs (MiRNAs) can help understanding the carcinogenesis of lung cancer and serve as potential diagnostic biomarkers for differentiating adenocarcinoma (ADC) and squamous cell lung carcinoma (SCC). The aim of the present study is to analyse and compare the expression patterns of miR-21, miR-197, miR-205, miR-375, miR-495 in ADC and SCC samples. Material and methods Fresh frozen tissue samples from 50 non-small cell lung cancer (NSCLC) patients (25ADC, 25SCC) and adjacent normal tissues were examined. The expression of miR-21, miR-197, miR-205, miR-375, miR-495 were evaluated by RT-qPCR. The statistical analysis was performed by SPSSv20. Results and discussions We found overexpression of: miR-21 in 60% (12ADC and 18SCC), miR-205 in 62% (13ADC and 18SCC), miR-197 in 14% (5ADC and 2SCC), miR-375 in 30% (11ADC and 4SCC) and miR-495 in 40% (13ADC and 7SCC) of the samples. The following miRNAs demonstrated decreased expression: miR-21 in 10% (5ADC and 5SCC), miR-205 in 10% (7ADC and 3SCC), miR-197 in 42% (11ADC and 10SCC), miR-375 in 42% (6ADC and 15SCC) and miR-495 in 30% (9ADC and 6SCC) of the tumours. Statistically significant (p ROC analysis was performed for distinguishing ADC from SCC. Only miR-205 (AUC=0,672, p=0,036) and miR-375 (AUC=0,692, p=0,019) showed statistically significant results independently. The combination of these two miRNAs (AUC=0,771; p=0,001) could distinguish between ADC and SCC with 80% sensitivity and 76% specificity. With the same sensitivity and specificity the combination of miR-205 and miR-375 and miR-495 (AUC=0,776; p=0,001) and the combination of miR-205 and miR-197 and miR-375 (AUC=0,774; p=0,001) could also discriminate between ADC and SCC. Conclusion It has been suggested that miR-205 appeared to be a promising diagnostic biomarker for SCC. Our results show that different panels of miRNAs including miR-205, have better potential to discriminate between ADC and SCC. However further analysis of enlarged sample is necessary to ascertain their diagnostic potential in NSCLC. Acknowledgements This work was supported by Grants №D-138/Contract №8554/12.12.2016/MU-Sofia;DUNK01/2/2009/NSF.

The Association of BRAF V600E Mutation With Tissue Inhibitor of Metalloproteinase-3 Expression and Clinicopathological Features in Papillary Thyroid Cancer.

BackgroundPapillary thyroid cancer (PTC) is the most common endocrine malignancy. The aim of this study was to investigate the association of tissue inhibitor metalloproteinase-3 (TIMP3) mRNA and protein levels in thyroid tissues, based on BRAF V600E status with the clinicopathologic characteristics of PTC.MethodsA total of 60 fresh frozen tissue samples of PTC patients (15 male and 45 female) were collected during thyroidectomy. All clinicopathological information was obtained and samples were reviewed as well as confirmed by a pathologist; exon 15 of the BRAF gene was genotyped by sequencing, TIMP3 mRNA level was assessed using SYBR-Green Real-Time PCR, and TIMP3 protein level was measured using ELISA.ResultsOf 60 cases, BRAF mutation was found in 24 (40%). Larger tumor size and higher lymph node metastasis frequency were observed, significant in BRAF (+), compared to the BRAF (-) PTC group (P = 0.039 and P = 0.03, respectively). No significant difference was seen in the tumoral tissues of the TIMP3 mRNA level in BRAF (+), compared to BRAF (-) PTC samples. However, the mean TIMP3 protein level was significantly lower in tumoral tissues, compared to matched non-tumoral tissues in BRAF (+) PTC (P=0.003); TIMP3 protein level was significantly lower in tumoral tissues compared to matched non-tumoral tissues in BRAF (+), in subjects who had no lymph node metastasis and also in subjects with lymph node metastasis in both BRAF positive and negative PTC cases.ConclusionOur results showed that BRAF mutation was associated with a larger tumor size, higher frequency of lymph node metastasis, and lower TIMP3 protein levels. Lower TIMP3 protein level was associated with the lymph node metastasis in PTC patients.

Overexpression of ARID4B predicts poor survival in patients with hepatocellular carcinoma

AT-rich interaction domain 4B (ARID4B), which belongs to the ARID family, is heavily involved in cell growth and differentiation and is closely associated with many types of tumors. However, the role of this protein in hepatocellular carcinoma (HCC) remains unknown. In this study, we used data from The Cancer Genome Atlas and Gene Expression Omnibus to analyze ARID4B expression in HCC. We subjected 15 pairs of fresh-frozen tissue samples to quantitative real-time polymerase chain reaction and Western blotting analyses to investigate ARID4B expression. We also subjected 157 formalin-fixed, paraffin-embedded HCC tissue samples to immunohistochemical analysis to detect ARID4B expression and to determine the clinical significance of ARID4B expression in HCC. The bioinformatics analysis, quantitative real-time polymerase chain reaction, and Western blotting results showed that ARID4B was highly expressed in HCC tissues compared with adjacent normal liver tissues. High ARID4B expression was strongly correlated with tumor number (P = .02), vascular invasion (P = .004), Edmondson-Steiner grades (P = .000), and tumor-node-metastasis stages (P = .001). Moreover, Kaplan-Meier and Cox proportional-hazards analyses indicated that high ARID4B expression was significantly associated with poor survival in patients with HCC and that ARID4B was an independent prognostic factor for overall survival and disease-free survival in patients with HCC. In conclusion, our results suggest that ARID4B acts as an oncogene in HCC and can therefore serve as a biomarker for the prognoses of patients with HCC.

Coinfection with Epstein-Barr Virus (EBV), Human Papilloma Virus (HPV) and Polyoma BK Virus (BKPyV) in Laryngeal, Oropharyngeal and Oral Cavity Cancer.

Most research providing evidence for the role of oncogenic viruses in head and neck squamous cell carcinoma (SCC) development is focused on one type of virus without analyzing possible interactions between two or more types of viruses. The aim of this study was to analyse the prevalence of co-infection with human papillomavirus (HPV), Epstein–Barr virus (EBV) and polyoma BK virus (BKPyV) in oral, oropharyngeal and laryngeal squamous cell carcinomas in Polish patients. The correlations between viral infection, SCC, demographic parameters, evidence of metastases and grading were also investigated. Fresh-frozen tumour tissue samples were collected from 146 patients with laryngeal, oropharyngeal and oral cancer. After DNA extraction, the DNA of the studied viruses was detected using polymerase chain rection (PCR) assay. Males (87.7%) with a history of smoking (70.6%) and alcohol abuse (59.6%) prevailed in the studied group. Histological type G2 was recognized in 64.4% cases. The patients were most frequently diagnosed with T2 stage (36.3%) and with N1 stage (45.8%). Infection with at least two viruses was detected in 56.2% of patients. In this group, co-infection with HPV/EBV was identified in 34.1% of cases, EBV/BKV in 23.2%, HPV/BKV in 22.0%, and HPV/EBV/BKV in 20.7%. No difference of multiple infection in different locations of cancer was observed. The prevalence of poorly differentiated tumours (G3) was more frequent in co-infection with all three viruses than EBV or BKV alone. A significant correlation was observed between tumour dimensions (T) and lymph-node involvement (N) in co-infected patients compared to single infection. Further studies are necessary to clarify whether co-infection plays an important role in the initiation and/or progression of oncogenic transformation of oral, oropharyngeal and laryngeal epithelial cells.

Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis

ABSTRACT The purpose of the present work was to evaluate the accuracy of quantitative polymerase chain reaction (qPCR) performed on samples of fresh frozen tissue (FT) and formalin-fixed, paraffin-embedded (FFPE) healthy skin. This is a validation study conducted with samples from 46 dogs from an endemic area in Brazil. After sample collection, DNA extractions were conducted using commercial kits and qPCR was oriented to kinetoplast DNA (kDNA) targets of the Leishmania infantum species. The results obtained for the FFPE samples showed 63.6% sensitivity and 77.1% specificity, whereas those obtained for the FT samples showed 100% and 48.6%, respectively. Poor agreement was observed for the results of the qPCR technique with FT and FFPE samples. Our results suggest freezing as the most suitable conservation method for the formation of sample databases considering DNA recovery

Identifying actionable variants using next generation sequencing in patients with a historical diagnosis of undifferentiated pleomorphic sarcoma.

There are limited data regarding the molecular characterization of undifferentiated pleomorphic sarcomas (UPS; formerly malignant fibrous histiocytoma). This study aimed to investigate the utility of next generation sequencing (NGS) in UPS to identify subsets of patients who harbour actionable mutations. Patients diagnosed with UPS underwent pathological re-evaluation by a pathologist specializing in sarcoma. Tumor DNA was isolated from archived fresh frozen tissue samples and genotyped using NGS with the Illumina MiSeq TruSeq Amplicon Cancer Panel (48 genes, 212 amplicons). In total, 95 patients initially classified with UPS were identified. Following pathology re-review the histological subtypes were reclassified to include: Myxofibrosarcoma (MFS, N = 44); UPS(N = 18); and Others (N = 27; including undifferentiated spindle cell sarcoma (N = 15) and dedifferentiated liposarcoma (N = 6)). Seven cases were excluded from further analysis for other reasons. Baseline demographics of the finalized cohort (N = 88) showed a median age of 66 years (32-95), primarily with stage I-III disease (92%) and high-grade (86%) lesions. Somatic mutations were identified in 31 cases (35%)(Total mutations = 36: solitary mutation(n = 27); two mutations( =n = 3); three mutations(n = 1)). The most commonly identified mutations were in TP53 (n = 24), ATM (n = 3) and PIK3CA (n = 2). Three of 43 patients with MFS and one of 18 patients with UPS had clinically relevant mutations, mainly related to biomarkers of prediction of response; however few had targetable driver mutations. Somatic mutation status did not influence disease free or overall survival. Based on the small number of clinically relevant mutations, these data do not support the routine use of targeted NGS panels outside of research protocols in UPS.

Prognostic significance of E-cadherin and N-cadherin expression in Gliomas

BackgroundEpithelial-mesenchymal transition (EMT), principally involving an E-cadherin to N-cadherin shift, linked to tumor invasion or metastasis, and therapeutic resistance in various human cancer. A growing body of recent evidence has supported the hypothesis that EMT play a crucial role in the invasive phenotype of gliomas. To evaluate the prognostic connotation of EMT traits in glioma, expression of E-cadherin and N-cadherin was explored in a large series of glioma patients in relation to patient survival rate.MethodsExpressions of E- and N-cadherin were examined using immunohistochemical analysis in 92 glioma cases diagnosed at our hospital. These markers expressions were also explored in 21 cases of fresh frozen glioma samples and in glioma cell lines by Western blot analysis.ResultsExpression of E-cadherin was observed in eight cases (8.7%) with weak staining intensity in the majority of the immunoreactive cases (7/8). Expression of N-cadherin was identified in 81 cases (88.0%) with high expression in 64 cases (69.5%). Fresh frozen tissue samples and glioma cell lines showed similar results by Western blot analysis. There was no significant difference in either overall survival (OS) or progression-free survival (PFS) according to E-cadherin expression (P > 0.05). Although the OS rates were not affected by N-cadherin expression levels (P = 0.138), PFS increased in the low N-cadherin expression group with marginal significance (P = 0.058). The survival gains based on N-cadherin expression levels were significantly augmented in a larger series of publicly available REMBRANDT data (P < 0.001).ConclusionsE- and N-cadherin, as representative EMT markers, have limited prognostic value in glioma. Nonetheless, the EMT process in gliomas may be compounded by enhanced N-cadherin expression supported by unfavorable prognostic outcomes.

Traceability and distribution of Neisseria meningitidis DNA in archived post mortem tissue samples from patients with systemic meningococcal disease

BackgroundThe pathophysiology and outcome of meningococcal septic shock is closely associated with the plasma level of N. meningitidis lipopolysaccharides (LPS, endotoxin) and the circulating level of meningococcal DNA. The aim of the present study was to quantify the number of N. meningitidis in different formalin-fixed, paraffin-embedded (FFPE) tissue samples and fresh frozen (FF) tissue samples from patients with systemic meningococcal disease (SMD), to explore the distribution of N. meningitidis in the body.MethodsDNA in FFPE and FF tissue samples from heart, lungs, liver, kidneys, spleen and brain from patients with meningococcal shock and controls (lethal pneumococcal infection) stored at variable times, were isolated. The bacterial load of N. meningitidis DNA was analyzed using quantitative real-time PCR (qPCR) and primers for the capsule transport A (ctrA) gene (1 copy per N. meningitidis DNA). The human beta-hemoglobin (HBB) gene was quantified to evaluate effect of the storage times (2-28 years) and storage method in archived tissue.ResultsN. meningitidis DNA was detected in FFPE and FF tissue samples from heart, lung, liver, kidney, and spleen in all patients with severe shock. In FFPE brain, N. meningitidis DNA was only detected in the patient with the highest concentration of LPS in the blood at admission to hospital. The highest levels of N. meningitidis DNA were found in heart tissue (median value 3.6 × 107 copies N. meningitidis DNA/μg human DNA) and lung tissue (median value 3.1 × 107 copies N. meningitidis DNA/μg human DNA) in all five patients. N. meningitidis DNA was not detectable in any of the tissue samples from two patients with clinical meningitis and the controls (pneumococcal infection). The quantity of HBB declined over time in FFPE tissue stored at room temperature, suggesting degradation of DNA.ConclusionsHigh levels of N. meningitidis DNA were detected in the different tissue samples from meningococcal shock patients, particularly in the heart and lungs suggesting seeding and major proliferation of meningococci in these organs during the development of shock, probably contributing to the multiple organ failure. The age of archived tissue samples appear to have an impact on the amount of quantifiable N. meningitidis DNA.

Abstract 3492: Subtype-specific expression of small non-coding RNAs in breast cancer

Abstract We have performed a small RNA-sequencing (RNA-Seq) experiment to find candidate small non-coding RNAs (ncRNAs) in breast cancer (BC) for the identification of signatures that aid in the prediction of therapy responsiveness, prognosis or patient outcome. We used 195 invasive BC tumor and 20 benign breast tissue samples from the Kuopio Breast Cancer Project (KBCP) study material. KBCP material is collected from a genetically homogeneous population of Eastern Finland, and includes fresh-frozen and FFPE breast tissue samples, EDTA blood samples and serum, as well as comprehensive background/lifestyle information, clinical, treatment and follow-up data extending over 20 years. This well-defined material enables the identification of factors associating with BC risk, different stages of disease progression and outcome. Total RNA was extracted from the fresh-frozen BC and benign breast tissue using the Ambion mirVana miRNA Isolation Kit. Small RNA-Seq was performed using the Illumina TruSeq Small RNA Library Prep kit and the Illumina MiSeq next-generation sequencing instrument. Analyses were concentrated in PIWI-interacting RNAs (piRNAs) and other non-miRNA small ncRNAs (sncRNAs). Bioinformatic analysis commenced with quality control and preprocessing steps including read quality assessment (FastQC), adapter trimming (TRIMMOMATIC), and removal of e.g. ribosomal RNA reads (Bowtie). Preprocessed reads were aligned (Tophat) to human reference transcriptomes (piRNABank and GENCODE non-miRNA sncRNAs), followed by data conversions e.g. for visualization (samtools, IGVtools). Gene-wise read counts were collected using summarizeOverlaps R package, and statistically differentially expressed (DE) genes were identified using DESeq2 R package. We first compared the small RNA expression in the benign breast tissue with invasive BC and observed in total 635 sncRNAs and 94 piRNAs with differential expression (PAdj&amp;lt;0.05). Of these 520 sncRNAs and 73 piRNAs were upregulated, and 115 sncRNAs and 21 piRNAs downregulated in the invasive tumors compared to benign breast tissue. When comparing the luminal BC subtype with triple-negative BC (TNBC) we observed 204 DE (PAdj&amp;lt;0.05) sncRNAs and 32 piRNAs. Of these, 103 sncRNAs and 18 piRNAs were upregulated, and 101 sncRNAs and 14 piRNAs downregulated in TNBC compared to luminal tumors. Differential expression was also observed between ER (estrogen receptor) negative and ER positive invasive tumors. Of the 242 DE sncRNAs and 32 piRNAs, 105 sncRNAs and 12 piRNAs were upregulated, and 137 sncRNAs and 20 piRNAs downregulated in ER positive BC compared to ER negative. Preliminary results of hierarchical clustering and principal component analysis of normalized log2 piRNA and sncRNA expression levels revealed the potential for finding differences between BC subtypes. The subsequent statistical analysis enabled us to identify individual piRNAs and other sncRNAs as candidate druggable biomarkers for e.g. TNBC patients. Citation Format: Jaana M. Hartikainen, Sami Heikkinen, Maria Tengström, Veli-Matti Kosma, Arto Mannermaa. Subtype-specific expression of small non-coding RNAs in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3492. doi:10.1158/1538-7445.AM2017-3492

Abstract 4424: Cancer stem cells-related microRNAs have prognostic implications in non-small cell lung cancer

Abstract Background: Lung cancer stem cells (CSCs) are a small population of stem-like cells that remains largely unknown. MicroRNAs (miRNAs) regulation of CSCs gene expression has been reported, constituting a promising therapeutic target. The aim of this study was to isolate and analyze differential expression of a set of miRNAs in tumorspheres from lung cancer cell lines and tumor tissue from resectable non-small cell lung cancer (NSCLC) patients and to determine the prognostic implications of these miRNAs in a cohort of resected-NSCLC patients. Methods: Lung CSC-related miRNAs (miR-145-5p, miR-188-5p, miR-218-5p, miR-34a-5p, miR-21-5p and miR-125a-5p) were analyzed in cells from seven NSCLC tumor samples and six cell lines (H1650, H1993, H480, H358, A549 and PC9) grown in monolayer and as spheroids by RTqPCR using TaqMan® microRNA assays. The expression levels of these six miRNAs were also analyzed in paired fresh-frozen tumor and normal adjacent lung tissue samples (N=178) from resected-NSCLC patients. Statistical analyses were considered significant at p&amp;lt;0.05. Results: miRNA expression analysis of cultured cells revealed an increased expression of miR-125a (p=0.04) and miR-188 (p=0.05) in lung tumorspheres from tumor samples and cell lines compared to their paired-adherent cells. Moreover, lungspheres from patients showed elevated expression levels of miR-21 when compared with their paired monolayer-cell cultures (p= 0.028). Interestingly, miR-125a, miR-188 and miR-21 had prognostic value when were analyzed in patients’ tissue samples. We found that those patients with higher levels (above the median) of miR-188 and miR-21 had a significant reduction in relapse-free survival (RFS, 23.67 vs 66.97 months; p= 0.009 and 24.03 vs 56.83 months, p= 0.042, respectively) and overall survival (OS, 42.9 vs NR months; p= 0.002 and 42.6 vs 82.60 months; p= 0.043, respectively), whereas the group of patients with higher levels of miR-125a had a worse outcome (OS 51.90 vs NR; p= 0.014). A signature combining the expression of miR-188 and miR-21 was able to give more significant prognostic information (RFS, 16.97 vs. 56.83 months; p= 0.006 and OS, 29.90 vs. NR; p&amp;lt;0.0001). The multivariate analysis including clinico-pathological and analytical variables revealed this miRNAs signature as an independent prognostic biomarker for RFS (HR 2.170 [1.372-3.431]; p= 0.001) and OS (HR 3.256 [1.907-5.561]; p&amp;lt;0.0001). Conclusions: Lung tumorspheres had increased levels of the CSC-related miRNAs miR-125a, miR-188 and miR-21, highlighting their role in the CSC biology. The analyses performed in a large cohort of resectable NSCLC patients show that a two miRNAs-signature (miR-188 and miR-21) was an independent prognostic marker for RFS and OS. Supported by grants RD06/0020/1024 and RD12/0036/0025 from RTICC-FEDER, PI12-02838 and PI15-00753 from ISCIII, TRACE (TRA09-0132) and Beca Roche Oncohematología. Note: This abstract was not presented at the meeting. Citation Format: Sandra Gallach Garcia, Alejandro Herreros-Pomares, Silvia Calabuig-Fariñas, Eva Escorihuela, Atilio Navarro, Aminta Isabel Martínez, Alberto Jacobo Cunquero, Eloisa Jantus-Lewintre, Carlos Camps. Cancer stem cells-related microRNAs have prognostic implications in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4424. doi:10.1158/1538-7445.AM2017-4424

Abstract 3047: PRC2-mediated silencing of circRNA CDR1as drives miR-7-independent melanoma metastasis

Abstract Circular RNAs are a novel class of non-coding RNAs with functions that remain poorly characterized in normal and pathological conditions. CDR1as is a non-canonical circRNA observed to act as a sponge for miR-7 in brain tissues. Analysis of RNA-seq data of melanocytes and melanoma cell lines and short-term cultures revealed loss of CDR1as expression as a hallmark of melanoma cells. We confirmed silencing of CDR1as in melanoma cells and tissues by RT-qPCR using divergent primers. Clinically, we observed CDR1as loss associated with metastatic progression and poor patient outcomes in a cohort of fresh-frozen melanoma tissue samples. Depletion of CDR1as in melanoma cell lines enhanced invasion in vitro and lung metastasis in vivo, demonstrating functional significance of CDR1as silencing. Surprisingly, CDR1as depletion had no clear effect on miR-7 activity in melanoma cells, and miR-7 inhibition was insufficient to rescue CDR1as silencing-induced invasion. Moreover, GSEA analyses of proteomic profiling of melanoma cells depleted of CDR1as revealed reductions of proteins involved in oxidative phosphorylation (OXPHOS) and mitochondrial function, suggesting CDR1as loss may alter metabolism of melanoma cells. Mining of CLIP-Seq data sets and subsequent RIP-PCR revealed direct interactions of CDR1as with the IGF2BP family of proteins and TARDBP, each of which are involved in regulation of mitochondrial function. These data suggest that CDR1as could regulate the subcellular localization and/or function of proteins involved in OXPHOS and mitochondrial respiration. To understand the upstream mechanism driving CDR1as loss in melanoma, we sought to examine the primary transcript from which CDR1as arises. Interestingly, examination of the CDR1as locus revealed an upstream long non-coding RNA, LINC00632, as a plausible primary transcript of CDR1as. CDR1as and LINC00632 expression is strikingly correlated across human tissues and in melanoma cell lines (r&amp;gt;.65). Moreover, depletion of LINC00632 using GapmeRs reduced expression of CDR1as, suggesting they arise from the same precursor transcript. Intriguingly, from data mining of ChIP-Seq datasets, we observed that the PRC2-dependent repressive chromatin mark H3K27me3, is abundant in the genomic locus of LINC00632 in melanoma cells. We validated the presence of H3K27me3 at LINC00632 by ChIP-PCR in melanoma cells lacking CDR1as expression. Strikingly, treatment of such melanoma cell lines with the EZH2 inhibitor, GSK126, induced robust re-expression of both CDR1as and LINC00632, and removal of H3K27me3 at LINC00632. Our data document that CDR1as is epigenetically silenced in melanoma, and its loss promotes melanoma invasion and metastasis. Moreover, CDR1as loss could contribute to metabolic adaptations during metastasis through misregulation of RNA binding proteins. Citation Format: Doug Hanniford, Rana Moubarak, Jochen Imig, Alejandro Ulloa, Beatriz Sanchez Sendra, Alcida Karz, Iman Osman, Ioannis Aifantis, Eva Hernando. PRC2-mediated silencing of circRNA CDR1as drives miR-7-independent melanoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2017-3047

Assessment of Bone Morphogenetic Protein 3 Methylation in Iranian Patients with Colorectal Cancer.

BACKGROUND Colorectal cancer (CRC) is a common cancer that results in outstanding morbidity and mortality worldwide. DNA methylation is one of the most important epigenetic events that is thought to occur during the early stages of oncogenic transformation especially in CRC. The aim of this study was to investigate whether hypermethylation of bone morphogenetic protein 3 (BMP3) in tissue samples is implicated in Iranian patients with CRC. METHODS From fresh frozen tissue samples of 30 patients with CRC, the DNA was isolated, treated with sodium bisulfite and analyzed by methylation-specific polymerase chain reaction with primers specific for methylated or unmethylated promoter sequences of the BMP3 gene. Demographic characteristics of the patients including age, sex, tumor grade, location, stage, and TNM classification were evaluated and the relationship between hypermethylation of the gene and clinicopathological features was analyzed. RESULTS Methylation of the BMP3 promoter was often present in the DNA extracted from the tumoral tissues. A sensitivity of 56.66% and specificity of 93.3% were attained in the detection of colorectal neoplasia. CONCLUSION We assumed that solely BMP3 methylation analysis in our population is not sufficient to select the gene as a screening biomarker and it should be considered in combination with other markers to screen for detection of colorectal cancer.

Differential association of 5-hydroxymethylcytosine levels with clinical and histopathological characteristics in breast cancer tumors.

e23173 Background: Breast cancer is one of the top causes of cancer death worldwide. In Mexico, locally advanced breast cancer (LABC) comprises the majority of the breast cancer stages at diagnosis. Recent studies have suggested that 5-hydroxymethylcytosine (5hmC), could be a prognostic marker in breast cancer. However, the role of 5hmC on clinical and histopathologic characteristics in LABC has not been explored. Methods: From a cohort of locally advanced and advanced breast cancer patients treated at the National Cancer Institute in Mexico City with a 3-year follow-up, we measured 5hmC levels by immunodetection from fresh frozen tissue samples taken from the initial biopsy (N = 193). We determined the association between 5hmC levels and the most relevant clinical and histopathological characteristics. Results: From the full cohort analyzed; 42% were luminal A (n = 82), 33% were luminal B (N = 63), 9% were HER2-positive (N = 18), and 15% were triple-negative tumors. We found higher levels of global 5hmC in HER2 positive tumors vs. all other subtypes (p = 0.028, Kruskal-Wallis test). In luminal B tumors, 5hmC levels were associated with Ki67 (β = -0.04, 95%CI: -0.08, -0.01, p = 0.01 from multivariable model) but not in luminal A. Furthermore, we found that low 5hmC levels were associated with higher histological grade in HER2-positive tumors (p = 0.03, Kruskal-Wallis test). In subgroup analysis of LABC patients, we found higher 5hmC levels in non-ductal vs. ductal invasive tumors (β = 1.38, 95%CI: 0.049, 2.911, p = 0.043 from multivariable model). Also, lower 5hmC levels were associated with Ki67 (β = -2.53, 95%CI: -4.32, -0.74; p = 0.009 from multivariable model) and with histological type (p = 0.028, Kruskal-Wallis test) in LABC luminal B tumors. A borderline association with OS (p = 0.07, log-rank test) and RFS (p = 0.071, log-rank test) was observed in luminal A tumors in the LABC group. Conclusions: Our findings suggest that 5hmC levels are differentially associated with distinct clinical and histopathological characteristics in breast cancer, especially those linked to aggressiveness, including Ki67, histological type, and grade.

Molecular Evidence for Monoclonal Skip Progression in Main Duct Intraductal Papillary Mucinous Neoplasms of the Pancreas.

To clarify clonality of distinct multisegmental main duct (MD)-intraductal papillary mucinous neoplasms (IPMNs) using microarray analysis. IPMNs represent a pancreatic ductal cell field defect, which causes multiple occurrences of lesions. In addtion, it has been speculated that MD-IPMNs display features of monoclonal skip progression. Total RNA was extracted from fresh-frozen tissue samples of metachronous MD-IPMNs and nonneoplastic pancreas tissue from the same pancreas from two individuals, and whole human genome microarray analysis was performed. Formalin-fixed paraffin-embedded tissue specimens from 28 distinct IPMNs were then collected from 12 patients, genomic DNA was extracted, and GNAS/KRAS mutational status was investigated. Immunohistochemical analysis was performed to validate the expression pattern of the indicated proteins. Microarray analysis revealed that metachronous MD-IPMNs from the same individual displayed pair-wise correlation coefficients of 0.9523 and 0.9512. In contrast, MD-IPMNs of the same histological grade from different individuals displayed coefficients of 0.8092 and 0.8211. Scatter plot analysis revealed that metachronous MD-IPMNs from the same individual displayed a closer linear relationship. Furthermore, heat map and hierarchical cluster analyses revealed that metachronous MD-IPMNs from the same individual were classified in the same branch, and the gene expression patterns were similar. The GNAS/KRAS mutational statuses of distinct MD-IPMNs were consistent with each other. Immunohistochemical assessment of five specific proteins demonstrated that the same expression pattern between two lesions was observed in 95% of the samples. These findings using molecular analyses indicate that MD-IPMNs might display features of monoclonal skip progression.

Targeted DNA and RNA sequencing of paired urothelial and squamous bladder cancers to reveal discordant genomic and transcriptomic events and unique therapeutic opportunities.

296 Background: Expression-based molecular subtypes thought to be intrinsic in bladder cancer have been widely reported, carrying important potential clinical treatment implications. Histologically, bladder cancers are also heterogeneous diseases, with a large portion of urothelial carcinomas exhibiting divergent differentiation. Previous subtyping efforts have been carried out using predominantly fresh frozen tissue samples, potentially obscuring this known differentiation heterogeneity. Methods: Here we performed targeted multiplexed, amplicon-based DNA and RNA sequencing on 100 formalin-fixed paraffin-embedded (FFPE) bladder cancer samples (including 12 paired urothelial / squamous lesions). High-confidence somatic point mutations, short insertions/deletions (indels), and copy number alterations were detected using the DNA component of the Oncomine Comprehensive Assay (OCP). Targeted RNA sequencing was carried out using a custom Ampliseq panel comprised of 8 housekeeping genes and 103 target genes assessing major transcriptional programs as identified from publically available data. Results: By DNA analysis, we observe frequent TP53 (35%) and activating hotspot PIK3CA (23%) somatic mutations across the cohort, as well as targetable high-level (log-2 copy number ratio &gt; = 1.5) focal amplifications of ERBB2 (3%) or EGFR (3%) in a subset of samples. We report a novel approach for detecting sub-gene copy-number alterations, and confirm several detectable multi-exon losses using whole transcriptome RNA sequencing. Pairing targeted RNA expression analysis with DNA-based alterations, we show high level expression of EGFR and ERBB2 in focally-amplified samples. Most importantly, we show that despite identical prioritized somatic genomic alterations, we observe divergent expression-based profiles in 3 of 12 (25%) paired urothelial and squamous samples. Conclusions: Taken together, these results highlight the importance of molecular heterogeneity in bladder cancer and suggest important considerations for using existing expression-based clustering approaches to guide clinical treatment decisions.