Leveraging Biospecimen Resources for Discovery or Validation of Markers for Early Cancer Detection

Validation of early detection cancer biomarkers has proven to be disappointing when initial promising claims have often not been reproducible in diagnostic samples or did not extend to prediagnostic samples. The previously reported lack of rigorous internal validity (systematic differences between compared groups) and external validity (lack of generalizability beyond compared groups) may be effectively addressed by utilizing blood specimens and data collected within well-conducted cohort studies. Cohort studies with prediagnostic specimens (eg, blood specimens collected prior to development of clinical symptoms) and clinical data have recently been used to assess the validity of some early detection biomarkers. With this background, the Division of Cancer Control and Population Sciences (DCCPS) and the Division of Cancer Prevention (DCP) of the National Cancer Institute (NCI) held a joint workshop in August 2013. The goal was to advance early detection cancer research by considering how the infrastructure of cohort studies that already exist or are being developed might be leveraged to include appropriate blood specimens, including prediagnostic specimens, ideally collected at periodic intervals, along with clinical data about symptom status and cancer diagnosis. Three overarching recommendations emerged from the discussions: 1) facilitate sharing of existing specimens and data, 2) encourage collaboration among scientists developing biomarkers and those conducting observational cohort studies or managing healthcare systems with cohorts followed over time, and 3) conduct pilot projects that identify and address key logistic and feasibility issues regarding how appropriate specimens and clinical data might be collected at reasonable effort and cost within existing or future cohorts

Mammalian tumour necrosis factor-α (TNF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) AU-rich elements (AREs)

<p><b>Copyright information:</b></p><p>Taken from "The tandem CCCH zinc finger protein tristetraprolin and its relevance to cytokine mRNA turnover and arthritis"</p><p>Arthritis Research & Therapy 2004;6(6):248-264.</p><p>Published online 8 Oct 2004</p><p>PMCID:PMC1064869.</p><p>Copyright © 2004 BioMed Central Ltd</p> The central ARE region of the TNF mRNA 3' untranslated region from all mammalian species for which this region of the mRNA has been deposited in GenBank. In most cases these were derived from EST sequences; note that the horse sequence has not been completed at the 3' end. The overlines indicate the nine-base tristetraprolin (TTP)-binding site 5'-UUAUUUAUU-3'. Sequences from the various mammals are divided into groups based on the pattern of these nonamers, with the top group of 10 mammals being the most common group. A similar approach was used to align the central ARE from the GM-CSF transcript, after alignment using the program ClustalW. The asterisks below the alignment represent base identity at that position; note that gaps were used to optimize the alignment. The overlines again represent the nonamer TTP-binding site. These data are modified from [37]

Robustness of RNA sequencing on older formalin-fixed paraffin-embedded tissue from high-grade ovarian serous adenocarcinomas.

Formalin-fixed paraffin-embedded (FFPE) tissues are among the most widely available clinical specimens. Their potential utility as a source of RNA for transcriptome studies would greatly enhance population-based cancer studies. Although preliminary studies suggest FFPE tissue may be used for RNA sequencing, the effect of storage time on these specimens needs to be determined. We conducted this study to determine whether RNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries was present in sufficient quantity and quality for RNA-Seq analysis. FFPE tissues, stored from 7 to 32 years, were obtained from three SEER sites. RNA was extracted, quantified, quality assessed, and subjected to RNA-Seq (a whole transcriptome sequencing technology). FFPE specimens stored for longer periods of time had poorer RNA sample quality as indicated by negative correlations between specimen storage time and fragment distribution values (DV). In addition, sample contamination was a common issue among the RNA, with 41 of 67 samples having 5% to 48% bacterial contamination. However, regardless of specimen storage time and bacterial contamination, 60% of the samples yielded data that enabled gene expression quantification, identifying more than 10,000 genes, with the correlations among most biological replicates above 0.7. This study demonstrates that FFPE high-grade ovarian serous adenocarcinomas specimens stored in repositories for up to 32 years and under varying storage conditions are a promising source of RNA for RNA-Seq. We also describe certain caveats to be considered when designing RNA-Seq studies using archived FFPE tissues

Immunogenetic risk and protective factors for juvenile dermatomyositis in Caucasians

Objective. To define the relative importance (RI) of class II major histocompatibility complex (MHC) alleles and peptide binding motifs as risk or protective factors for juvenile dermatomyositis (DM), and to compare these with HLA associations in adult DM. Methods. DRB1 and DQA1 typing was performed in 142 Caucasian patients with juvenile DM, and the results were compared with HLA typing data from 193 patients with adult DM and 797 race-matched controls. Random Forests classification and multiple logistic regression were used to assess the RI of the HLA associations. Results. The HLA-DRB1*0301 allele was a primary risk factor (odds ratio [OR] 3.9), while DQA1*0301 (OR 2.8), DQA1*0501 (OR 2.1), and homozygosity for DQA1*0501 (OR 3.2) were additional risk factors for juvenile DM. These risk factors were not present in patients with adult DM without defined autoantibodies. DQA1 alleles *0201 (OR 0.37), *0101 (OR 0.38), and *0102 (OR 0.51) were identified as novel protective factors for juvenile DM, the latter 2 also being protective factors in adult DM. The peptide binding motif DRB1 9EYSTS 13 was a risk factor, and DQA1 motifs F25, S26, and 45(V/A)W(R/K)47 were protective. Random Forests classification analysis revealed that among the identified risk factors for juvenile DM, DRB1*0301 had a higher RI (100%) than DQA1*0301 (RI 57%), DQA1*0501 (RI 42%), or the peptide binding motifs. In a logistic regression model, DRB1*0301 and DQA1*0201 were the strongest risk and protective factors, respectively, for juvenile DM. Conclusion. DRB1*0301 is ranked higher in RI than DQA1*0501 as a risk factor for juvenile DM. DQA1*0301 is a newly identified HLA risk factor for juvenile DM, while 3 of the DQA1 alleles studied are newly identified protective factors for juvenile DM. © 2006, American College of Rheumatology

Lot‐to‐Lot Variability in anti‐HLA Screening

BackgroundIdentifying antibodies to HLA (anti-HLA) by solid-phase assays is used to screen blood donors to mitigate transfusion-related acute lung injury risk. Various cutoffs for detection assays have been proposed in the literature; however, these do not take into consideration lot-to-lot variability of commercially available assays.Study design and methodsSamples from 93 nontransfused males were tested using five different lots of a multiplex bead-based anti-HLA detection kit. A subset of 17 samples was tested on 5 days using a single lot. An additional 96 samples from donations with varied anti-HLA levels were tested using kits from two different lots. Results were reported as a normalized background (NBG) ratio.ResultsFor the 93 nontransfused donors, NBG values generated using the reference lot were significantly higher than those obtained with three of the four comparator lots. However, for the 96 samples with low-, moderate-, and higher-level anti-HLA, Class I (CL-I) values were 1.4 times lower and Class II (CL-II) values were 1.2 times lower using the reference versus comparator lot. For CL-I antibodies the between-lot standard deviation (SD) was 1.36 (95% confidence interval [CI], 1.19-1.60), while the between-day SD was 1.27 (95% CI, 1.08-1.52). Similarly, for CL-II antibodies the between-lot SD was 0.81 (95% CI, 0.70-0.95), while the between-day SD was 0.50 (95% CI, 0.43-0.60).ConclusionsThere is interlot variability in the tested HLA detection assay as well as significant bias between lots. It may be reasonable to develop a new cutoff when a new lot is obtained

The Prostate, Lung, Colorectal and Ovarian Cancer (PLCO) Screening Trial Pathology Tissue Resource

BACKGROUND: Pathology tissue specimens with associated epidemiological and clinical data are valuable for cancer research. The Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial undertook a large-scale effort to create a public resource of pathology tissues from PLCO participants who developed a cancer during the trial. METHODS: Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks were obtained from pathology laboratories on a loan basis for central processing of tissue microarrays, with additional free-standing tissue cores collected for nucleic acid extraction. RESULTS: Pathology tissue specimens have been obtained for prostate cancer (n=1,052), lung cancer (n=434), colorectal cancer (n=675) and adenoma (n=658), ovarian cancer and borderline tumors (n=212), breast cancer (n=870) and bladder cancer (n=204). The process of creating this resource was complex, involving multi-disciplinary teams with expertise in pathology, epidemiology, information technology, project management and specialized laboratories. CONCLUSIONS: Creating the PLCO tissue resource required a multi-step process including obtaining medical records and contacting pathology departments where pathology materials were stored after obtaining necessary patient consent and authorization. The potential to link tissue biomarkers to prospectively collected epidemiological information, screening and clinical data, and matched blood or buccal samples offers valuable opportunities to study etiologic heterogeneity, mechanisms of carcinogenesis and biomarkers for early detection and prognosis. IMPACT: The methods and protocols developed for this effort, and the detailed description of this resource provided here will be useful for those seeking to use PLCO pathology tissue specimens for their research and may also inform future tissue collection efforts in other settings

The effect of previous pregnancy and transfusion on HLA alloimmunization in blood donors: implications for a transfusion-related acute lung injury risk reduction strategy.

BackgroundAntibodies to human leukocyte antigens (HLA) in donated blood have been implicated as a cause of transfusion-related acute lung injury (TRALI). A potential measure to reduce the risk of TRALI includes screening plateletpheresis donors for HLA antibodies. The prevalence of HLA antibodies and their relationship to previous transfusion or pregnancy in blood donors was determined.Study design and methodsA total of 8171 volunteer blood donors were prospectively recruited by six US blood centers from December 2006 to May 2007. Donors provided a detailed history of pregnancy and transfusion and a sample for HLA Class I and II antibody testing by multiantigen bead flow analysis.ResultsA total of 8171 donors were enrolled; 7920 (96.9%) had valid HLA antibody test results and 7841 (99%) of those had complete pregnancy and transfusion information. The prevalence of any HLA antibody was similar in nontransfused (n = 1138) and transfused (n = 895) men, 1.0% versus 1.7% (p = 0.16). HLA antibodies were detected in 17.3% of all female donors (n = 5834) and in 24.4% of those with a history of previous pregnancy (n = 3992). The prevalence of HLA antibodies increased in women with greater numbers of pregnancy: 1.7% (zero), 11.2% (one), 22.5% (two), 27.5% (three), and 32.2% (four or more pregnancies; p &lt; 0.0001).ConclusionHLA Class I and Class II antibodies are detectable at low prevalence in male donors regardless of transfusion and in female donors without known immunizing events. The prevalence of HLA antibodies increases significantly with more pregnancies. These data will allow blood centers to estimate the impact of HLA antibody testing as a potential TRALI risk reduction measure