Dataset Information

Mass spectrometry-based quantification of proteins in tumor samples from tissue preserved by different methods – a source of markers of urothelial bladder cancer

ABSTRACT: Tissue samples in biobanks preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature (OCT)-embedded and subsequently freezing are highly valuable in experimental studies where the results can be related to clinical parameters. Today, mass spectrometry (MS)-based proteomics is the method of choice for unbiased and relative comparison of the abundances of thousands of proteins present in biological samples. MS analysis of FFPE- and OCT-preserved tissue has been limited, but it is now approachable using newly developed protocols. However, it has not been clarified how the results from different preservation methods agrees and differ. In this study, we use a unique sample set of urinary bladder cancer tissues of two different tumor stages (Ta/T1 – non-muscle invasive and T2/T3 muscle invasive); upon sampling the tumors were divided and preserved by the two methods. Thereafter a protocol for parallel processing of the samples were applied after which the samples were analyzed with label free quantitative MS analysis. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Principal component analysis showed that the largest differences between samples are based on the preservation method, but this analysis could also classify the tumors into different stages in each preservation. The overlap of quantifiable proteins between the preservation methods were 84% when all proteins and full data set were taken into consideration, but only 41% on average when proteins from a specific tumor were analyzed. Proteins involved in mitochondrial function were overrepresented among proteins present in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Moreover, univariate analysis showed that HMGCS2 protein is uniquely quantified in Ta/T1 tumors in both FFPE and OCT data, and that LGALS1, ANXA5 and plastin proteins are upregulated in T2/T3 tumors compared to Ta/T1 tumors in both FFPE and OCT data, confirming the consistency between the data sets and suggesting these proteins as biomarkers. Finally, LGALS1 data were verified by immunohistochemistry staining of an independent data set.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urinary Bladder

DISEASE(S): Urinary Bladder Cancer

SUBMITTER: Sara Lind

LAB HEAD: Sara Lind

PROVIDER: PXD020194 | Pride | 2021-09-09

REPOSITORIES: Pride

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Proteomic comparison between different tissue preservation methods for identification of promising biomarkers of urothelial bladder cancer.

Valdés Alberto A Bitzios Athanasios A Kassa Eszter E Shevchenko Ganna G Falk Alexander A Malmström Per-Uno PU Dragomir Anca A Segersten Ulrika U Lind Sara Bergström SB

Scientific reports 20210407 1

Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1-non-muscle invasive bladder cancer ( ...[more]

PMID: 33828141

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Project description:The heterotransplantation of human tumors to immune-deprived rodents has become an important preclinical tool for evaluating human tumor biology and responsiveness to therapeutic agents, based on the supposition that they reflect their original human origin. However, we demonstrate that such tumors can in fact change genetically, either by becoming tumors of the animal recipient or by becoming a hybrid of the human tumor and the animal hostâs cells. Three human tumors transplanted to the cheek pouch of golden hamsters resulted in permanently transplantable tumors that metastasize in the hamsters. Based on diverse lines of evidence, these transplants from a human glioblastoma multiforme and two Hodgkin lymphomas were malignant hybrids, showing both human and hamster genes in the malignant tumor cells, while retaining the morphology of the original human tumors. We demonstrate that even after being preserved for over 40 years in paraffin blocks, microarray analysis of RNA from these transplants disclosed over 3000 human genes derived from all 23 human chromosome pairs amongst these tumor transplants. A total of 3759 probe sets (ranging from 1040 to 1303 in each transplant) detected human gene transcripts in formalin-fixed, paraffin-embedded sections of the 3 hybrid tumors stored for over 40 years; the probe sets unambiguously map to 3107 unique Human Entrez Gene IDs and are representative of all human chromosomes, although, by karyology, one of the hybrid tumors (GB-749) had a total of 15 human chromosomes in its cells. Among the genes mapped, 39 probe sets, representing transcripts from 33 human genes , were detected in all hybrid tumor samples. Five of these 33 genes encode transcription factors that regulate cell growth and differentiation, five encode cell adhesion and transmigration-associated proteins known to participate in oncogenesis and/or metastasis and invasion, and additional genes encode components of pathways involved with signal transduction, regulation of apoptosis, DNA repair, and multidrug resistance. We posit that in-vivo fusion may disclose genes implicated in tumor progression, as well as gene families coding for the organoid phenotype. Thus, cancer cells can transduce adjacent stromal cells, with the resulting progeny having permanently transcribed genes with malignant and other functions of the donor DNA. Human gene expression profiles were determined by microarray analysis for 3 different Human-Hamster hybrid tumors, two Hodgkin lymphomas (GW-532, GW-584) and a glioblastoma multiforme (GB-749), that were first generated in the hamster cheek pouch after human tumor grafting and then propagated in hamsters and in cell cultures for years. Human gene expression was assessed using total RNA isolated from FFPE samples from GW-532 generations 2 and 34, GW-584 generation 28, and GB-749, in comparison to that for a Hamster control RNA sample isolated from a Hamster melanoma cell line (ATCC CCL-49). Expressed human genes were identified using MAS 5.0 signal expression values and detection P-values in the following manner: 1) Unannotated probe sets, as well as probe sets with no signal value greater than the median signal for AFFX spike-in controls with all Absent Detection Calls, were omitted from further analysis; 2) All remaining signal values for the hamster cell line sample were multiplied by the ratio of the median signal in all FFPE hybrid samples for AFFX spike-in control probe sets called present in all samples divided by the median signal for the same probe sets in the hamster CCL-49 sample; 3) Human transcripts were considered positive in a human-hamster hybrid FFPE sample if (a) a probe set signal exhibited a 2-fold or greater increase in any FFPE hybrid sample compared to the CCL-49 coontrol sample, (b) the fold change was greater than 2 standard deviations for that probe set across the FFPE samples, and (c) was called present (P) or marginal (M) for at least one or more FFPE samples.

Project description:Background and Aims Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly available form of archived clinical specimens, which are often stored as thin sections on glass slides. RNA isolated from such archived section (AS) of FFPE tissue is more degraded compared to freshly cut (FC) FFPE section because of prolonged air exposure. In this study, we evaluated performance of transcriptome profiling-based disease classification in AS-FFPE tissue. Methods Genome-wide gene-expression profiles of AS-FFPE tissues of 83 hepatocellular carcinoma (HCC) and 47 liver cirrhosis samples were generated by using whole-genome DASL assay (Illumina), and compared with the profiles previously produced by using FC tissue sections from the same FFPE blocks. Quality of the profiles and performance of gene signature-based class prediction were systematically evaluated. Results RNA quality and assay reproducibility of AS-FFPE RNA were comparable to intermediate ~ poor quality FC-FFPE samples (R2 as high as 0.93). Gene-expression signal was detected in lower number of probes in AS FFPE samples compared to FC-FFPE samples (proportion of probes with present signal (%P-call): 10%~60% and 70%~90% in AS- and FC-FFPE profiles, respectively). Based on %P-call quality threshold of 20%, 64/88 (77%) HCC and 37/48 (77%) liver profiles were judged as having relatively good quality data with comparable inter-sample correlation. Inter-sample correlation coefficient, as a measure to detect outlier profiles due to poor RNA quality, was also lower in AS-FFPE (0.4~0.9) compared to FC-FFPE (0.6~1.0). In the genome-wide profiling analysis, previously identified molecular subclasses of HCC tumors were reproduced in 67/83 (81%) samples, which was improved to 43/48 (90%) samples when we focused on statistically confident predictions (p<0.05). A 186-gene prognostic signature in liver cirrhosis was reproduced in 32/47 (68%) samples, which was slightly improved to 11/16 (69%) when focused on statistically significant predictions. Conclusions We observed decay of genome-wide transcriptional profiles in AS-FFPE tissues in quantitative manner. However, disease classification was still possible, which suggests potential of AS-FFPE material for clinical diagnosis and prognosis. FFPE tissue sections (10 micron-thick) sliced from 5~16-year-old FFPE blocks and archived for 6~7 years on glass slide Gene-expression profiles of archived section of formalin-fixed paraffin-embedded (AS-FFPE) liver tissues from HCC patients: 47 samples Gene-expression profiles of archived section of formalin-fixed paraffin-embedded (AS-FFPE) tumor tissues from HCC patients: 83 samples

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