A retrospective study of neuroschistosomiasis in aquatic birds.

Background: The current project aims to explore the possibility to use formalin fixed paraffin embedded (FFPE) samples for exome sequencing, which would open up a large collection of tissue samples for molecular studies. Usage of formalin is known to degrade and modify the DNA both during treatment and storage resulting in problems in downstream molecular analyses. The aim of the present study is to evaluate exome sequencing of FFPE samples by comparing data from FFPE normal tissue to corresponding snap frozen (SF) blood controls with focus on data output such as amount of data removed due to low sequencing quality, amount of data mapped to the genome, GC-content, and duplicate levels. Methods: In the current study we used the western Swedish biobank of neural tumors archived at the Sahlgrenska Hospital, Gothenburg. We performed exome sequencing of FFPE normal adrenal tissue samples and corresponding SF blood samples from five patients diagnosed with pheochromocytoma/paraganglioma. Two of the patients had known heterozygous germline mutations and were included to determine if these mutations could be found in both sample types. Libraries for SF samples were prepared according to protocol using Agilent Technologies SureSelect Human All Exon 50 Mb library prep kit. FFPE samples were prepared with slight modifications in the protocol, with additional PCR cycles used to increase amplification. 75 bp paired end reads were generated on Illumina HiScan SQ according to manufacturer's protocol (v.3). Results: Between 92-99% of the raw data were kept after removal of data due to low sequencing quality independent of sample type. For the SF samples 99-100% of the trimmed data mapped to the genome, the number for FFPE samples were slightly lower ranging from 87-98%. The GC content in the exome enriched DNA from FFPE and SF tissue were similar, ranging between 45-51%. Looking at the mapped data we found higher levels of duplicates in the FFPE samples compared to the SF samples, 19-78% vs. 5-15%. For one of the two patients with a known germline mutation the variant was detected at approximately the same frequency in both FFPE and SF sample (39% in FFPE vs. 44% in SF). For the other patient the variant were found at higher frequency in the FFPE sample compared to SF sample (80% vs. 50%). Conclusions: We saw no difference in amount data removed due to low sequencing quality between the five FFPE and SF samples evaluated in this study. Also, we saw only slightly lower mapping frequencies for the FFPE samples. The GC-content for both FFPE and SF samples were within the range of what's expected for exome sequencing data for libraries prepared with this kit and sequenced on Illumina HiScan SQ platform. Due to the additional PCR cycles used for FFPE samples we saw higher duplicate levels in these samples, suggesting that deeper sequencing is necessary for FFPE samples to get the same amount of unique reads as for the SF samples. A probable explanation to the higher allele frequency of the variant observed in the FFPE sample from one of the patients with a known germline mutation is the presence of contaminant tumor cells in the normal tissue sample, resulting in a shifted allele frequency. To conclude, SF samples are to prefer for exome sequencing. However, if SF material is not available FFPE tissue is a good alternative to increase the sample cohort when studying rare and complex diseases. Citation Format: Annica Wilzen, Heidi Ottesen, Anna Larsson, Bo Wangberg, Andreas Muth, Ola Nilsson, Frida Abel. Exome sequencing of FFPE material: An evaluation. [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 A11.

Whole transcriptome profiling is a promising technique in adrenal studies; however, whole transcriptome profiling of adrenal disease using formalin-fixed paraffin-embedded (FFPE) samples has to be further explored. The aim of this study was to evaluate the utility of transcriptome data from FFPE samples of adrenocortical tumors. We performed whole transcriptome profiling of FFPE and fresh frozen samples of adrenocortical carcinoma (ACC, n = 3), aldosterone-producing adenoma (APA, n = 3), and cortisol-producing adenoma (CPA, n = 3), and examined the similarity between the transcriptome data. We further examined whether the transcriptome data of FFPE samples could be used to distinguish tumor types and detect marker genes. The number of read counts was smaller in FFPE samples than in fresh frozen samples (P < 0.01), while the number of genes detected was similar (P = 0.39). The gene expression profiles of FFPE and fresh frozen samples were highly correlated (r = 0.93, P < 0.01). Tumor types could be distinguished by consensus clustering and principal component analysis using transcriptome data from FFPE samples. In the differential expression analysis between ACC and APA-CPA, known marker genes of ACC (e.g., CCNB2, TOP2A, and MAD2L1) were detected in FFPE samples of ACC. In the differential expression analysis between APA and CPA, known marker genes of APA (e.g., CYP11B2, VSNL1, and KCNJ5) were detected in the APA of FFPE samples. The results suggest that FFPE samples may be a reliable alternative to fresh frozen samples for whole transcriptome profiling of adrenocortical tumors.

Comparison Of Single Nucleotide Mutations (SNVs) and Copy Number Variants (CNVs) Detection In Formalin Fixed Paraffin Embedded (FFPE) and Paired Frozen Tumor Tissues Using Target Capture and Sequencing Approach

e18556 Background: The results of comprehensive genome-wide characterization of lung cancer have recently been reported. However, the concordance of driver mutations between formalin-fixed paraffin-embedded (FFPE) and surgically resected snap-frozen samples is unclear. We conducted the Shizuoka Lung Cancer Mutation Study to analyze driver mutations in patients with lung cancer. Methods: Based on the biobanking system in conjunction with the clinic including the pathology lab, we developed a multiplexed mutational panel designed to assess 23 mutations in 9 genes (EGFR, KRAS, BRAF, PIK3CA, NRAS, MEK1, AKT1, PTEN and HER2), EGFR, MET, PIK3CA, FGFR1 and FGFR2 amplifications, and EML4-ALK translocations, using pyrosequencing plus capillary electrophoresis, qRT-PCR, and RT-PCR, respectively.DNA concentration in FFPE samples was analyzed by measuring absorbance at 260 nm (A260) in a spectrophotometer. Results: Between July 2011 and July 2012, sixty-five lung cancer patients with both FFPE and snap-frozen samples were included in this study. Patient characteristics were as follows: median age (range) 70 (38-92) years; female 68%; never-smoker 34%; histology adenocarcinoma/squamous cell carcinoma/small cell carcinoma/other 52/29/6/13 %. FFPE samples included 64 transbronchial biopsies and one pleural biopsy. We detected driver mutations in 58% of all cases. Mutations found: EGFR 28%, KRAS 12%, PIK3CA 6%, NRAS 2%, HER2 5%, EGFR amplification 5%, PIK3CA amplification 12%, FGFR1amplification 2%. Complete concordance of driver mutations between FFPE and snap-frozen samples was shown in 65%. Median DNA concentration in FFPE samples was 72.7 ng/ul (range; 2.4 - 472.4 ng/ul). Exploratory analyses using an ROC curve, done to evaluate the useful cutoff of DNA concentration in FFPE samples, showed low AUC (AUC; 0.5326). Conclusions: These results suggest that the rate of complete concordance of driver mutations between FFPE and snap-frozen samples might be acceptable, and DNA concentration in FFPE samples might not be correlated with concordance.

Accumulating evidence suggests that metabolic reprogramming has a critical role in carcinogenesis and tumor progression. The usefulness of formalin-fixed paraffin-embedded (FFPE) tissue material for metabolomics analysis as compared with fresh frozen tissue material remains unclear. LC/MS-MS-based metabolomics analysis was performed on 11 pairs of matched tumor and normal tissues in both FFPE and fresh frozen tissue materials from patients with colorectal carcinoma. Permutation t test was applied to identify metabolites with differential abundance between tumor and normal tissues. A total of 200 metabolites were detected in the FFPE samples and 536 in the fresh frozen samples. The preservation of metabolites in FFPE samples was diverse according to classes and chemical characteristics, ranging from 78% (energy) to 0% (peptides). Compared with the normal tissues, 34 (17%) and 174 (32%) metabolites were either accumulated or depleted in the tumor tissues derived from FFPE and fresh frozen samples, respectively. Among them, 15 metabolites were common in both FFPE and fresh frozen samples. Notably, branched chain amino acids were highly accumulated in tumor tissues. Using KEGG pathway analyses, glyoxylate and dicarboxylate metabolism, arginine and proline, glycerophospholipid, and glycine, serine, and threonine metabolism pathways distinguishing tumor from normal tissues were found in both FFPE and fresh frozen samples. This study demonstrates that informative data of metabolic profiles can be retrieved from FFPE tissue materials. IMPLICATIONS: Our findings suggest potential value of metabolic profiling using FFPE tumor tissues and may help to shape future translational studies through developing treatment strategies targeting metabolites.

&lt;div&gt;Abstract&lt;p&gt;Accumulating evidence suggests that metabolic reprogramming has a critical role in carcinogenesis and tumor progression. The usefulness of formalin-fixed paraffin-embedded (FFPE) tissue material for metabolomics analysis as compared with fresh frozen tissue material remains unclear. LC/MS-MS–based metabolomics analysis was performed on 11 pairs of matched tumor and normal tissues in both FFPE and fresh frozen tissue materials from patients with colorectal carcinoma. Permutation &lt;i&gt;t&lt;/i&gt; test was applied to identify metabolites with differential abundance between tumor and normal tissues. A total of 200 metabolites were detected in the FFPE samples and 536 in the fresh frozen samples. The preservation of metabolites in FFPE samples was diverse according to classes and chemical characteristics, ranging from 78% (energy) to 0% (peptides). Compared with the normal tissues, 34 (17%) and 174 (32%) metabolites were either accumulated or depleted in the tumor tissues derived from FFPE and fresh frozen samples, respectively. Among them, 15 metabolites were common in both FFPE and fresh frozen samples. Notably, branched chain amino acids were highly accumulated in tumor tissues. Using KEGG pathway analyses, glyoxylate and dicarboxylate metabolism, arginine and proline, glycerophospholipid, and glycine, serine, and threonine metabolism pathways distinguishing tumor from normal tissues were found in both FFPE and fresh frozen samples. This study demonstrates that informative data of metabolic profiles can be retrieved from FFPE tissue materials.&lt;/p&gt;Implications:&lt;p&gt;Our findings suggest potential value of metabolic profiling using FFPE tumor tissues and may help to shape future translational studies through developing treatment strategies targeting metabolites.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Accumulating evidence suggests that metabolic reprogramming has a critical role in carcinogenesis and tumor progression. The usefulness of formalin-fixed paraffin-embedded (FFPE) tissue material for metabolomics analysis as compared with fresh frozen tissue material remains unclear. LC/MS-MS–based metabolomics analysis was performed on 11 pairs of matched tumor and normal tissues in both FFPE and fresh frozen tissue materials from patients with colorectal carcinoma. Permutation &lt;i&gt;t&lt;/i&gt; test was applied to identify metabolites with differential abundance between tumor and normal tissues. A total of 200 metabolites were detected in the FFPE samples and 536 in the fresh frozen samples. The preservation of metabolites in FFPE samples was diverse according to classes and chemical characteristics, ranging from 78% (energy) to 0% (peptides). Compared with the normal tissues, 34 (17%) and 174 (32%) metabolites were either accumulated or depleted in the tumor tissues derived from FFPE and fresh frozen samples, respectively. Among them, 15 metabolites were common in both FFPE and fresh frozen samples. Notably, branched chain amino acids were highly accumulated in tumor tissues. Using KEGG pathway analyses, glyoxylate and dicarboxylate metabolism, arginine and proline, glycerophospholipid, and glycine, serine, and threonine metabolism pathways distinguishing tumor from normal tissues were found in both FFPE and fresh frozen samples. This study demonstrates that informative data of metabolic profiles can be retrieved from FFPE tissue materials.&lt;/p&gt;Implications:&lt;p&gt;Our findings suggest potential value of metabolic profiling using FFPE tumor tissues and may help to shape future translational studies through developing treatment strategies targeting metabolites.&lt;/p&gt;&lt;/div&gt;

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of malignancies with courses ranging from indolent to potentially lethal. We recently studied in a 157 patient cohort gene expression profiles generated by the TruSeq targeted RNA gene expression sequencing. We observed that the sequencing library quality and depth from formalin-fixed paraffin-embedded (FFPE) skin samples were significantly lower when biopsies were obtained prior to 2009. We also observed that the fresh CTCL samples clustered together, even though they included stage I–IV disease. In this study, we compared TruSeq gene expression patterns in older (≤2008) vs. more recent (≥2009) FFPE samples to determine whether these clustering analyses and earlier described differentially expressed gene findings are robust when analyzed based on the year of biopsy. We also explored biases found in FFPE samples when subjected to the TruSeq analysis of gene expression. Our results showed that ≤2008 and ≥2009 samples clustered equally well to the full data set and, importantly, both analyses produced nearly identical trends and findings. Specifically, both analyses enriched nearly identical DEGs when comparing benign vs. (1) stage I–IV and (2) stage IV (alone) CTCL samples. Results obtained using either ≤2008 or ≥2009 samples were strongly correlated. Furthermore, by using subgroup analyses, we were able to identify additional novel differentially expressed genes (DEGs), which did not reach statistical significance in the prior full data set analysis. Those included CTCL-upregulated BCL11A, SELL, IRF1, SMAD1, CASP1, BIRC5, and MAX and CTCL-downregulated MDM4, SERPINB3, and THBS4 genes. With respect to sample biases, no matter if we performed subgroup analyses or full data set analysis, fresh samples tightly clustered together. While principal component analysis revealed that fresh samples were spatially closer together, indicating some preprocessing batch effect, they remained in the proximity to other normal/benign and FFPE CTCL samples and were not clustering as outliers by themselves. Notably, this did not affect the determination of DEGs when analyzing ≥2009 samples (fresh and FFPE biopsies) vs. ≥2009 FFPE samples alone.

Background: To provide a systematic analysis of formalin fixation artifacts on Illumina sequencing libraries and results, we generated two complementary sequencing libraries (target enrichment sequencing and whole exome sequencing) from 11 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tumor samples and two pairs of matched FFPE and FF germline samples. We also generated whole genome sequencing data from a single pair of FF/FFPE tumor samples. Results: The results indicate minimal variations in library fragment size, coverage, and PCR duplicates within FF/FFPE paired samples that are less than 1 year old; whereas, a large variation in these parameters were observed in FF/FFPE pairs in samples that are approximately 2 years old. No significant increase in global mismatch rates and C•G&amp;gt;T•A substitutions were observed in FFPE samples from the former group; whereas, a discernible increase in mismatch rates and C•G&amp;gt;T•A substitutions were observed in FFPE samples from the latter group. However, over 99.7% and 99.5% of concordant calls were observed between matched FF and FFPE pairs at reference and non-reference positions within the targeted regions, respectively. Although an increased rate of global mismatches and C•G&amp;gt;T•A substitutions were observed in some FFPE samples, discordant rates were low (&amp;lt;0.26) in all samples because most of the FFPE artifacts were filtered out using GATK workflow. Consistent with this analysis, C•G&amp;gt;T•A substitutions are comparable in non-reference positions in paired FF and FFPE samples. Conclusions: We developed upfront quality assessment and library preparation method that use low input DNA from FFPE samples to perform next-generation sequencing. The results from our studies indicate the suitability of FFPE samples in sequencing studies. Citation Format: Sarah Munchel, Yen Hoang, Yue Zhao, Joseph Cottrell, Brandy Klotzle, Andrew Godwin, Janelle Noel, Brooke Fridley, Peter Beyerlein, Jian-Bing Fan, Marina Bibikova, Jeremy R. Chien. Targeted or whole genome sequencing of formalin-fixed tissue samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4281. doi:10.1158/1538-7445.AM2014-4281

BACKGROUND Improvements in meningioma risk stratification are needed to guide postoperative management. We previously developed and externally validated a targeted gene expression biomarker predicting meningioma outcomes and radiotherapy response using fresh frozen meningiomas. Here, we present the analytical validity, test-retest and cross-platform reproducibility, intra-tumor heterogeneity, and formalin fixed paraffin embedded (FFPE) concordance for this biomarker. METHODS Matched FFPE and fresh frozen samples from 50 meningiomas underwent RNA extraction for concordance testing using a custom Nanostring gene expression panel comprised of 34 meningioma genes and 7 housekeeping genes. Matched Nanostring gene expression profiling and RNA-sequencing was available from 173 fresh frozen meningiomas. Nanostring batch, machine, and technician variability was tested on 10 FFPE meningiomas using 2 independent batches of biomarker reagents that were processed at 2 independent laboratories. To study intra-tumor heterogeneity, 68 spatially distinct meningioma biopsies obtained under stereotactic guidance from 13 tumors were analyzed using the Nanostring gene expression biomarker. RESULTS Matched FFPE and fresh frozen meningioma samples (N=50) demonstrated high gene expression concordance (Pearson R=0.89, F-test P&amp;lt; 2.2e-16) and risk score concordance (R=0.78, F-test P&amp;lt; 1.8e-11, residual standard error 0.12). The biomarker achieved high performance on FFPE samples (local freedom from recurrence [LFFR] c-index 0.75±SEM 0.05, 5y LFFR AUC 0.84, 95% interval 0.67-0.96). Test-retest concordance on FFPE samples (N=10) across independent reagent batches, machines, and technicians was high (gene expression concordance: R=0.97; risk score concordance R=0.94). Transcript counts were concordant across Nanostring and RNA sequencing approaches (N=173 frozen samples, R=0.90). Stereotactic intra-operative sampling (N=68 samples from 13 patients) revealed low levels of intra-tumor heterogeneity (median inter-quartile risk score within tumor: 0.10±0.01). CONCLUSIONS We demonstrate analytical reproducibility and robustness of a gene expression biomarker predicting meningioma outcomes and radiotherapy responses in FFPE and frozen samples across multiple conditions and platforms.

Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery, but it has been a major challenge to study gene expression in these samples due to mRNA degradation and modification during fixation and processing. We have developed methods for small RNA and whole transcriptome analysis of FFPE samples on the Ion Torrent PGM™ System using Ion total RNA-Seq Kit v2 and RiboMinus Eukaryote System v2. Paired FFPE and fresh-frozen RNA samples from lung adenocarcinoma tissue were used in this study. Small RNA libraries were prepared using the Ion Total RNA-Seq kit v2 with modified protocol, followed by sequencing on PGM™ system. Results show that small RNA extracted from FFPE sample was successfully converted to small RNA library. Although percentage of reads mapped to miRBase was slightly lower when compared to fresh-frozen sample, the overall miRBase coverage and detection sensitivity were comparable. For whole transcriptome analysis, RiboMinus Eukaryote System v2 kit was used to deplete rRNA from FFPE samples, followed by library construction using the Ion Total RNA-Seq kit v2 with a modified protocol. Significant reduction in rRNA reads was observed after sequencing. High RefSeq correlations were observed between fresh-frozen and FFPE samples. In conclusion, a full spectrum of gene expression data, from both small RNA and whole transcriptome, were generated from FFPE samples using Ion Total RNA-Seq kit v2 with modified protocols. High correlations were observed between paired fresh-frozen and FFPE samples, demonstrating the reliability of the modified workflow for gene expression profiling on FFPE samples. This study provides feasibility for systematic gene expression analysis on FFPE samples from cancer and other diseases. Citation Format: Jian Gu. Expression profiling of paired formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissue samples on Ion Torrent PGMTM. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4127. doi:10.1158/1538-7445.AM2013-4127

BackgroundWe evaluated (a) the feasibility of whole genome cDNA-mediated Annealing, Selection, extension and Ligation (DASL) assay on formalin-fixed paraffin-embedded (FFPE) tissue and (b) whether similar conclusions can be drawn by examining FFPE samples as proxies for fresh frozen (FF) tissues. We used a whole genome DASL assay (addressing 18,391 genes) on a total of 72 samples from paired breast tumor and surrounding healthy tissues from both FF and FFPE samples.ResultsGene detection was very good with comparable success between the FFPE and FF samples. Reproducibility was also high (r2 = 0.98); however, concordance between the two types of samples was low. Only one-third of the differentially expressed genes in tumor tissues (compared to corresponding normal) from FF samples could be detected in FFPE samples and conversely only one-fourth of the differentially expressed genes from FFPE samples could be detected in FF samples. GO-enrichment analysis, gene set enrichment analysis (GSEA) and GO-ANOVA analyses also suggested small overlap between the lead functional groups that were differentially expressed in tumor detectable by examining FFPE and FF samples. In other words, FFPE samples may not be ideal for picking individual target gene(s), but may be used to identify some of the lead functional group(s) of genes that are differentially expressed in tumor. The differentially expressed genes in breast cancer found in our study were biologically meaningful. The "cell cycle" & "cell division" related genes were up-regulated and genes related to "regulation of epithelial cell proliferation" were down-regulated.ConclusionsGene expression experiments using the DASL assay can efficiently handle fragmentation issues in the FFPE tissues. However, formalin fixation seems to change RNA and consequently significantly alters gene expression in a number of genes which may not be uniform between tumor and normal tissues. Therefore, considerable caution needs to be taken when interpreting gene expression data from FFPE tissues, especially in relation to specific genes.

The aim of this study was to identify and quantify lncRNAs in formalin-fixed paraffin-embedded (FFPE) samples from stage II colorectal cancer (CRC) patients. Colorectal cancer (CRC) is the third most common cancer worldwide, and has high metastasis and recurrence rates. Use of adjuvant chemotherapy in stage II CRC patients is challenging, and new biomarkers are required to identify patients with high probability of relapse. Long noncoding RNAs (lncRNA) play an important role in cancer, awakening especial interest as potential novel cancer biomarkers and therapeutic targets. Resected tissues and biopsy preserved as formalin-fixed, paraffin-embedded (FFPE) samples are the most abundant supply for translational research. Unfortunately, long-chain RNA molecules, such as lncRNAs, may suffer degradation in those conditions. This fact coupled with their low expression levels challenges their detection with current techniques. Thus, in this study we developed a new approach which couples target enrichment and RNAseq to enhance detection of lncRNAs. Our three main goals are: I) evaluate the efficiency of this methodology on FFPE samples, II) investigate the lncRNAs involved in stage II CRC, and III) check for the presence of lncRNA differentially expressed in tumors of individuals who relapsed after surgery and not. We designed a custom-made target enrichment based on Nimblegen (Roche) technology to sequence a total of 8,048 lncRNAs (258 of interest for CRC). We tested 36 paired stage II CRC tumor FFPE tissues and their adjacent normal tissue, 12 from patients who relapsed after surgery. We quantified the expression of the targeted lncRNA and performed a differential expression analysis. Our approach detected expression of numerous lncRNA from FFPE samples. Importantly, we validated 5 target lncRNAs expression values by real time qPCR using two independent housekeeping genes, and found significant correlation between RNAseq (log2 (TPM)) to the qPCR (deltaCT) values. In addition, we identified a total of 83 lncRNAs which are differentially expressed between normal and tumoral samples. This list is enriched in genes previously reported as being involved in CRC, but includes many novel candidates of unknown function. Finally, the expression profile of tumors from patients with and without relapse seems quite similar, although we identified 5 lncRNAs that are significantly differentially expressed. Further validation in a larger cohort is needed to determine whether they could have prognostic value. In summary, our study shows that SeqCap Enrichment System is a good strategy to determine lncRNAs expression in FFPE tissue. Of note, we identified a list of new lncRNAs that are deregulated in stage II CRC tumor. Further research on these lncRNAs may lead to novel clinical applications in cancer biogenesis and prognosis. Citation Format: Anna Brunet-Vega, Cinta Pegueroles, Susana Iraola-Guzmán, Laia Vilà, Alex Casalots, Paula Ribera, Ismael Macias, Hrant Hovhannisyan, Ester Saus, Carles Pericay, Toni Gabaldón. A novel approach of target enriched next generation sequencing reveals differences in expression of lncRNAs in stage II colorectal cancer formalin fixed paraffin embedded (FFPE) tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1396.

Background: Digital polymerase chain reaction (PCR) is a new technology that enables detection and quantification of cancer DNA molecules from peripheral blood. Detecting tumor-specific mutations in circulating plasma DNA may potentially be useful to select systemic therapies for solid tumors. The aim of our study is to evaluate the feasibility of detecting PIK3CA mutations from plasma of breast cancer patients. Methods: We have designed an allele-specific PCR assay and used a digital PCR system for the detection of PIK3CA mutations H1047R and E545K. Formalin-fixed paraffin-embedded (FFPE) tumor samples were analyzed by COBAS and by digital PCR. The matched plasma samples were then analyzed by digital PCR. Kappa Cohen’s coefficient (κ) was used to test agreement between methods Results: 37 ER positive breast cancer and matched plasma were evaluated. 31 (84%) patients were stage IV disease. E545K mutation was detected in 4 out of 37 (11%) tumor specimens and H1047R mutation was identified in 8 out of 37 (22%) tumor samples. The proportion of observed agreement between H1047R and E545K detection in tumor samples by COBAS and digital PCR was 100%. Regarding E545K mutation assay we found that the proportion of observed agreement between mutation status in FFPE samples and circulating tumor DNA (ctDNA) was 94.6% (kappa= 0.770; Sensitivity=100%; Specificity=93.9%). In H1047R assay the proportion of observed agreement between FFPE samples and ctDNA was 83.78% (Kappa=0.561; Sensitivity=60%; Specificity=92.59%). Conclusions: The agreement between methodologies when assessing the PIK3CA status in tumor samples was perfect. However, a moderate-to-fair agreement was found between FFPE samples and ctDNA which might be due to the heterogeneity of the disease. The methodology presented in this study is a feasible approach for PIK3CA mutation detection in blood derived samples. Citation Format: Jose Angel Garcia-Saenz, Daniel Acosta, Fernando Moreno, Paticia Ayllon, Miguel Sotelo, Trinidad Caldes, Eduardo Diaz-Rubio, Atocha Romero. Detection of H1047R and E545K PIK3CA mutations from peripheral blood in ER positive breast cancer patients [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 P4-02-10.

In recent years, bacterial DNA in formalin-fixed paraffin-embedded (FFPE) samples has been recognized as a valuable bioresource for microbiota studies. This study aimed to examine the effect of the FFPE process on microbiota profiling to evaluate whether FFPE samples could serve as an alternative bioresource to fresh samples in oral microbiota studies. Fresh saliva was collected from nine subjects. The pellets obtained by centrifuging the collected saliva were fixed in formalin, then dehydrated and embedded in paraffin to prepare FFPE samples. The abundance of the hypervariable regions V1-9, V1-2, and V3-4 of the 16S rRNA gene in fresh and FFPE samples was relatively compared. In addition, microbiota profiling was performed to compare the results between the two sample types. The results showed that the FFPE process resulted in a certain degree of fragmentation of the 16S rRNA gene. However, the V1-2 region was relatively well-preserved compared to the V1-9 and V3-4 regions, suggesting that short regions are suitable targets for oral microbiota analysis. Importantly, there were no significant differences in alpha and beta diversity of microbiota between fresh and FFPE samples, and microbiota profiles were similar between the two sample types, suggesting that FFPE samples could be a valuable bioresource for oral microbiota studies.