Preanalytical handling of tissue samples can influence bioanalyte quality and ultimately outcome of analytical results. The aim of this study was to compare RNA quality, performance in real time RT ...PCR and histology of formalin-fixed tissue to that of tissue fixed and stabilized with a formalin-free fixative, the PAXgene Tissue System (PAXgene), in an animal model under highly controlled preanalytical conditions. Samples of rat liver, kidney, spleen, intestine, lung, heart muscle, brain, and stomach tissue were either fixed in formalin or fixed in PAXgene or fresh frozen in liquid nitrogen. RNA was extracted from all samples, examined for integrity in microcapillary electrophoresis, and used in a series of quantitative RT PCR assays with increasing amplicon length. Histology of paraffin-embedded samples was determined by staining with hematoxylin and eosin.
Histology of all formalin-fixed and PAXgene fixed samples was comparable. RNA with acceptable integrity scores could be isolated from all embedded tissues, 4.0 to 7.2 for formalin and 6.4 to 7.7 for PAXgene, as compared to 8.0 to 9.2 for fresh frozen samples. While RNA with acceptable RINs (RNA integrity number) could be isolated from formalin-fixed samples, in microcapillary electrophoresis this RNA separated with a slower migration rate and displayed diffuse, less focused peaks for ribosomal RNA as compared to RNA from frozen or PAXgene fixed samples. Furthermore, RNA from formalin-fixed tissues exhibited inhibition in quantitative RT PCR assays which increased with increasing amplicon length, while RNA from PAXgene fixed samples did not show such inhibition.
In conclusion, our results demonstrate that excluding other preanalytical factors, PAXgene Tissue System preserves histology similarly to formalin, but unlike formalin, does not chemically modify RNA. RNA purified from PAXgene fixed tissues is of high integrity and performs as well as RNA from fresh frozen tissue in RT PCR regardless of amplicon length.
► Tissues treated with PAXgene Tissue or formalin are morphologically comparable. ► RNA with high integrity can be purified from tissue treated with PAXgene Tissue. ► RNA from formalin fixed tissue is chemically modified and inhibits RT PCR. ► RNA from PAXgene Tissue fixed samples is free of chemical modifications. ► RT PCR results of PAXgene Tissue RNA and frozen tissue RNA are identical.
Personalized medicine requires capabilities to detect and measure health-associated biomarkers with increasingly specific and sensitive methods, putting analytical chemists at the front lines of ...translational research. Analytical scientists must be upstream in the experimental design process because the analysis of a biospecimen (tissue, blood, etc.) presents technical and experimental design complexities. (To listen to a podcast about this feature, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html.).
Making precision (personalized) medicine a routine clinical reality will require a comprehensive inventory of validated biomarkers and molecular diagnostic tests to stratify disease subtypes and ...improve accuracy in diagnosis and treatment selection. Realization of this promise has been hindered by the poor productivity of biomarker identification and validation. This situation reflects deficiencies that are pervasive across the entire spectrum of biomarker R&D, from discovery to clinical validation and in the failure of regulatory and reimbursement policies to accommodate new classes of biomarkers. The launch of the National Biomarker Development Alliance is the culmination of a 2-year review and consultation process involving diverse stakeholders to advance standards, best practices and guidelines to enhance biomarker discovery and validation by adoption of systems-based approaches and trans-sector collaboration between academia, clinical medicine and relevant private and public sector stakeholders.
Despite prodigious advances in tumor biology research, few tumor-biomarker tests have been adopted as standard clinical practice. This lack of reliable tests stems from a vicious cycle of ...undervaluation, resulting from inconsistent regulatory standards and reimbursement, as well as insufficient investment in research and development, scrutiny of biomarker publications by journals, and evidence of analytical validity and clinical utility. We offer recommendations designed to serve as a roadmap to break this vicious cycle and call for a national dialogue, as changes in regulation, reimbursement, investment, peer review, and guidelines development require the participation of all stakeholders.
This report details the proceedings of the 2009 Biospecimen Research Network (BRN) Symposium that took place on March 16 to 18, 2009, the second in a series of annual symposia sponsored by the ...National Cancer Institute Office of Biorepositories and Biospecimen Research. The BRN Symposium is a public forum addressing the relevance of biospecimen quality to progress in cancer research and the systematic investigation needed to understand how different methods of collection, processing, and storage of human biospecimens affect subsequent molecular research results. More than 300 participants from industry, academia, and government attended the symposium, which featured both formal presentations and a day of workshops aimed at addressing several key issues in biospecimen science. An additional 100 individuals participated via a live webcast (archived at http://brnsymposium.com). The BRN Symposium is part of a larger program designed as a networked, multidisciplinary research approach to increase the knowledge base for biospecimen science. Biospecimens are generally understood to represent an accurate representation of a patient's disease biology, but can instead reflect a combination of disease biology and the biospecimen's response to a wide range of biological stresses. The molecular signatures of disease can thus be confounded by the signatures of biospecimen biological stress, with the potential to affect clinical and research outcomes through incorrect diagnosis of disease, improper use of a given therapy, and irreproducible research results that can lead to misinterpretation of artifacts as biomarkers. Biospecimen research represents the kind of bricks-and-mortar research that provides a solid scientific foundation for future advances that will directly help patients.
Background & Aims: The CpG island methylator phenotype (CIMP) is one of the mechanisms involved in colorectal carcinogenesis (CRC). Although CIMP is probably the cause of high-frequency ...microsatellite instability (MSI-H) sporadic CRCs, its role in microsatellite stable (MSS) tumors is debated. The majority of MSS CRCs demonstrate chromosomal instability (CIN) with frequent loss of heterozygosity (LOH) at key tumor suppressor genes. We hypothesized that the majority of sporadic CRCs without CIN would be associated with CIMP. Methods: We tested 126 sporadic CRCs for MSI and LOH and categorized tumors into MSI, LOH, or MSI−/LOH− subgroups. Methylation status was evaluated using 6 CIMP-related markers (MINT1, MINT2, MINT31, p16 INK4 α , p14 ARF , and hMLH1 ) and 6 tumor suppressor genes ( PTEN, TIMP3, RUNX3, HIC1, APC , and RARβ2 ). BRAF V600E mutation analysis was performed using allele-specific polymerase chain reaction and DNA sequencing. Results: We observed frequent methylation at all 12 loci in all CRCs. BRAF V600E mutations correlated with the MSI ( P < .0001) and MSI−/LOH− ( P = .03) subgroups. MSI and MSI−/LOH− tumors exhibited more promoter methylation than CRCs with LOH ( P < .0001). We also found an inverse correlation between the frequencies of methylation and LOH (ρ = −0.36; P < .0001). Conclusions: The associations between methylation frequencies at CIMP-related markers and MSI or MSI−/LOH− sporadic CRCs suggest that the majority of these tumors evolve through CIMP. These findings suggest that CIN and CIMP represent 2 independent and inversely related mechanisms of genetic and epigenetic instability in sporadic CRCs and confirm that MSI cancers arise as a consequence of CIMP.
Biospecimens are recognized as critical components of biomedical research, from basic studies to clinical trials and epidemiologic investigations. Biorepositories have existed in various forms for ...more than 150 years, from early small collections in pathology laboratories to modern automated facilities managing millions of samples. As collaborative science has developed, it has been recognized that biospecimens must be of consistent quality. Recent years have seen a proliferation of best practices and the recognition of the field of "biospecimen science." The future of this field will depend on the development of more evidence-based practices in both the research and clinical settings. As the field matures, educating a new generation of biospecimen/biobanking scientists will be an important need.
Purpose
Tumor levels of thymidylate synthase (TS), a target of 5‐fluorouracil (5‐FU)‐based chemotherapy for colorectal cancer, have been studied as a predictive or prognostic biomarker with mixed ...results.
Patients and Methods
Tumor TS levels were prospectively evaluated in two adjuvant therapy trials for patients with resected stage II or III colon cancer. TS expression was determined by standard immunohistochemistry and by automated quantitative analysis. Tumor mismatch repair deficiency (MMR‐D) and BRAF c.1799T > A (p.V600E) mutation status were also examined. Relationships between tumor TS, MMR‐D, and BRAF mutation status, overall survival (OS), and disease‐free survival (DFS) were investigated in the subset of stage III patients.
Results
Patients whose tumors demonstrated high TS expression experienced better treatment outcomes, with DFS hazard ratio (HR) = 0.67, 95% confidence interval (CI) = 0.53, 0.84; and OS HR = 0.68, 95% CI = 0.53, 0.88, for high versus low TS expression, respectively. No significant interaction between TS expression and stage was observed (DFS: interaction HR = 0.94; OS: interaction HR = 0.94). Tumors with high TS expression were more likely to demonstrate MMR‐D (22.2% vs. 12.8%; p = .0003). Patients whose tumors demonstrated both high TS and MMR‐D had a 7‐year DFS of 77%, compared with 58% for those whose tumors had low TS and were non‐MMR‐D (log‐rank p = .0006). Tumor TS expression did not predict benefit of a particular therapeutic regimen.
Conclusion
This large prospective analysis showed that high tumor TS levels were associated with improved DFS and OS following adjuvant therapy for colon cancer, although tumor TS expression did not predict benefit of 5‐FU‐based chemotherapy.
Implications for Practice
This study finds that measurement of tumor levels of thymidylate synthase is not helpful in assigning specific adjuvant treatment for colorectal cancer. It also highlights the importance of using prospective analyses within treatment clinical trials as the optimal method of determining biomarker utility.
Tumor thymidylate synthase (TS) levels were prospectively evaluated in two adjuvant therapy trials for patients with resected stage II or III colon cancer. Results indicated that high tumor TS levels were associated with improved disease‐free survival and overall survival following adjuvant therapy for colon cancer, although tumor TS expression did not predict benefit of 5‐fluorouracil‐based chemotherapy.
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