This Notice of Funding Opportunity (NOFO) will support extramural research to investigate and mitigate challenges in clinical assay development and subsequent analytical validation due to preanalytical variability in biospecimens as described in this NOFO. Research funded under this NOFO may include investigations of preanalytical variability associated with the procurement and study of small biopsies (core biopsies, small excision samples), blood utilized for "liquid biopsies", tissue swabs, tissue secretions, pleural and esophageal aspirates, feces, or bodily fluids like sweat, urine, CSF, breast milk and saliva. Investigator-designed experiments will explore how different biospecimen preanalytical conditions affect emerging and clinically relevant biomarkers quantified by a variety of testing platforms. The results from this research program will improve the understanding of how analytical quantification of clinically relevant biomarkers is affected by variation in biospecimen collection, processing, and storage procedures. The overall goal is to expedite biomarker clinical assay development through evidence-based standardization of biopsy handling practices.

Biospecimen preanalytical variability can affect the reproducibility of clinical research. Biospecimens are the essential starting materials for the biomarker assays that will enable precision medicine. Clinical assays used for diagnosis and therapeutic decision-making are based on assessment of biological molecules (e.g., DNA, RNA, proteins) from a patient’s biospecimen. Such assays are often based on the detection of one or more biomarkers and must be both accurate and reproducible. False positive or negative results from the evaluation of biomarkers in clinical assays can directly affect patient diagnosis, treatment, and outcomes and can lead to over-treatment, under-treatment, or incorrect treatment.

An increasing number of reports demonstrate that preanalytical factors such as the handling of a clinical biospecimen prior to analysis can have a significant impact on assay results, which can in turn affect patient care. Biospecimen preanalytical factors can directly influence molecular results from assays conducted for basic research, biomarker discovery, biomarker validation, and development of validated clinical assays. In fact, preanalytical processing has been highlighted as a significant impediment in the development of predictive biomarkers for oncology. Preanalytical variability has also been identified as a challenge in the implementation of next-generation technologies in the analysis of tissue biopsies. The methods used to collect, process and store small biopsy biospecimens such as core biopsies, fine needle aspirates (FNAs), and lung aspirates, for example, can vary widely within and across medical institutions and laboratories, introducing variability in such preanalytical factors as biopsy method, needle gauge, vacuum-assistance, biospecimen size/volume, duration and temperature of transport, preservation method, temperature and duration of storage, and number of freeze thaw cycles. A wide variety of preanalytical factors, alone and in combination, are known to affect the accurate assessment of biomarkers, adding to the complexity of biospecimen challenges for the clinical laboratory.

New knowledge about preanalytical effects can support evidence-based biospecimen procedures for cancer research activities including clinical trials. Smaller biopsies such as FNAs, core biopsies, and lung aspirates present a unique set of preanalytical factors and associated challenges for reliable biomarker assay results. Such biopsies are clinically valuable now and in the future for assessing current and recurrent disease state as well as for assessing the likely efficacy of therapeutic intervention. Research has shown discordant biomarker assay results when comparing such small biopsies with larger tumor resections.

Detection of biomarkers in blood analytes such as cfDNA (cell-free DNA) and CTCs (circulating tumor cells) is a promising approach for non-invasively tracking tumor behavior over time, potentially enhancing cancer management by assessing tumor burden, detecting recurrence, monitoring early response and identifying drug resistance. Preanalytical considerations are also challenging for such liquid biopsies, as inconsistency between sample handling protocols and lack of standardization among analytical techniques has created obstacles for translating cfDNA analysis to clinical practice. CTC analysis, including enumeration and characterization, is also affected by preanalytical factors. Challenges introduced by preanalytical variability also affect the isolation and analysis of exosomes and other extracellular vesicles, underscoring the need for rigorous analytical validation.

Bodily fluids such as urine and cerebrospinal fluid (CSF) are other examples of biospecimens that could be utilized for detection of biomarkers in clinical assays that may be helpful in patient diagnosis, treatment selection, and prediction of outcomes. Additional biospecimen types such as tissue swabs, tissue secretions, pleural and esophageal aspirates, feces, sweat, breast milk and saliva have potential application in clinical biomarker research and are included in this NOFO.

Differences in assay results can be a result of differences in preanalytical factors such as ischemia times, preservation method, and tumor heterogeneity. Such preanalytical factors may alter biomarker measurements and influence the performance of assay technologies. Variability and biases introduced in the early stages of biomarker assay development, if not addressed and understood, can increase the likelihood of irreproducible data and incorrect conclusions. This is of significant concern for NCI clinical research. Basic and applied biospecimen science investigation will provide valuable insights into how to limit variability in clinical assay results from small biopsies.

A classic example of how biospecimen preanalytical factors can have a dramatic influence on assay results is companion diagnostic HER2 assays that guide the therapeutic use of trastuzumab, a targeted therapy for breast cancer. Recommendations for the reliable assessment of HER2 status in patient biospecimens were established 14 years after the initiation of phase 1 clinical trials in 1992. These measures were developed in response to multiple issues with HER2 reproducibility including preanalytical variability. For example, one study showed that the number of patients reported to overexpress HER2 differed by approximately 20% between specimens processed in local pathology departments and those processed in centralized laboratories.

When investigating whether non-standardized approaches between laboratories directly influenced the clinical assay results, the American Society of Clinical Oncologists (ASCO) and the College of American Pathologists (CAP) concluded that several biospecimen preanalytical factors including fixative composition, delay time to tissue fixation, method of tissue processing, and time in fixative could influence HER2 assay results. A major issue for such guidelines is that there is often a lack of fundamental scientific data on the behavior of clinical biomarker assays in response to biospecimen preanalytical factors. Systematic evaluation and increased understanding of the impact of these factors in the context of particular assay platforms and tissue types, and how to mitigate their impact, are necessary to improve current clinical assays and the development of new biomarker assays.

Preanalytical factors also impact the reproducibility of highly sensitive liquid biopsy assays that require optimized conditions for identification of circulating tumor material. For example, blood collection tube type and a time-to-assay delay affect CTC enumeration and characterization of breast cancer patient samples using the high-definition single-cell analysis assay (HD-SCA). CTC recovery, EpCAM and cytokeratin immunostaining, and nuclear content are also affected by storage time and tube type. Concentration and fragmentation of circulating cell free DNA (cfDNA) are also influenced by differences in sample handling procedures. These examples highlight the crucial importance of biospecimen science in effective development and application of clinical diagnostic assays.

Past research funded by NCI included the Biospecimen Research Network (BRN) program, which advanced the field of biospecimen science through systematic investigation of the effects of different biospecimen collection, processing, and storage procedures on downstream molecular analyses. The NCI Biospecimen Preanalytical Variables (BPV) research program was a multi-site study of specific biospecimen handling issues, including cold ischemic time (delay to formalin fixation), time in formalin, freezing methods, and storage temperatures and durations.

Overall, results suggest that biospecimen preanalytical factors can significantly affect research and clinical assay results. The research also demonstrated that the downstream effects of different preanalytical factors can vary with respect to the specific analyte being evaluated and the analytical platforms and/or assays being used. Thus, new and emerging clinical biomarkers and assays could be affected to unknown extents.

The NCI continues to actively curate and maintain an online biospecimen science database, the Biospecimen Research Database (BRD). The BRD is a free and publicly accessible database that contains summaries of peer-reviewed primary and review articles as well as SOPs in the field of human Biospecimen Science. In addition, and as an adjunct to the NCI Best Practices for Biospecimen Resources, the NCI develops the Biospecimen Evidence-based Best Practices (BEBPs) series, an expanding collection of procedural guidelines developed using, and annotated with, evidence from primary research publications in the field of human Biospecimen Science.

Measurement and validation of clinically relevant biomarkers using diagnostic assays are increasingly challenging as reliance on smaller biospecimens and liquid biopsy increases and new biomarkers and analysis platforms emerge. For example, NCI-sponsored treatment trials need accurate assays for integral markers used for selection of patients, integrated markers for evaluation of the clinical correlation of biomarkers for new agents, and pharmacodynamic markers that indicate whether the target has been engaged.

To generate the biospecimen science data needed to address today’s clinical biomarker assay challenges, an integrated approach is required that addresses how preanalytical factors introduced during biospecimen handling affect clinical biomarker assay performance. The need is critical given that diminished reliability and effectiveness of clinical biomarker assays could adversely affect patient care and drug development.

This Notice of Funding Opportunity (NOFO) will support extramural research to investigate and mitigate challenges in clinical assay development and subsequent analytical validation due to preanalytical variability in tumor biopsies, blood biospecimens utilized as liquid biopsies, and other biospecimen types as described in this NOFO. This NOFO will fund research to support clinical assay development and validation through the identification and mitigation of preanalytical factors associated with collection, processing, and storage that negatively influence clinical assay results and reproducibility.

Using a cooperative agreement mechanism, the research will be conducted by a collaborative, interdisciplinary network that integrates clinical researchers with academic and private sector scientists in projects that focus on preanalytical challenges specific to:

Potential preanalytical issues to address in solid tumors (FNA, Core biopsies, Excisions) may include but are not limited to biopsy methodology in a medical setting, tumor heterogeneity, preservation method (formalin, RNAlater, frozen, other), tissue processing, timing and temperature considerations, tradeoffs (morphology vs. molecular preservation), and shipping and storage considerations. Similarly, potential preanalytical issues to address in liquid biopsies may include but are not limited to collection device in a medical setting, collection methodology in a medical setting, timing and temperature considerations, sample processing, and shipping and storage considerations.

The NOFO intends to fund dedicated studies in biospecimen science to increase the success of clinical biomarker assays in cancer research. The research will align with current efforts to optimize clinical biomarker assays utilized in clinical trials networks and produce data needed for evidence-based biospecimen practices to support clinical trials. Applications to improve standardization or harmonization of assays targeted for use in clinical trials may be appropriate for this NOFO.

The work of the program will be centered on emerging and clinically relevant biomarker assays for a variety of molecular analysis platforms. The NOFO intends to fund clinically relevant emerging/established biomarkers (not discovery approaches) and may involve existing and emerging analysis platforms. Investigators will propose and design experiments to test assay performance and limits of detection of biomarkers under different preanalytical conditions. The work will be conducted by multidisciplinary scientific teams that may include pathologists, oncologists, radiologists, molecular biologists, and statisticians, as the work requires expertise and perspectives in clinical medicine, knowledge of the unique issues related to obtaining biopsy biospecimens, biology and technology platforms, and experimental design and analysis. A particular focus of this funding program is to address biomarker assay challenges that arise across early phase treatment trials. To best understand and address these challenges, applicant teams may wish to include investigators currently involved in Phase I and Phase II treatment trials.

This NOFO will fund multidisciplinary teams that together will constitute a U01 collaborative network. These teams may include but are not limited to oncologists, pathologists, radiologists, statisticians, molecular biologists etc. Each team’s application will focus on specific biomarker assays and associated questions in biospecimen science and present a research plan for acquiring the needed biospecimens. The assay may measure a single biomarker or may be a multiplex assay and must be amenable to conversion to a clinical assay that can be used in a clinical trial setting. Preliminary data should define the current status of the assay as well as justify support for optimization and usability in a clinical trial. Research ideas, experimental design approaches, biospecimens, and molecular analysis platforms will be shared across the network as possible to maximize research productivity.

Projects will involve defining workable biospecimen conditions for trials that utilize a particular type of tissue and/or liquid biopsy (e.g. lung aspirates, liver biopsies) in a biomarker assay system (e.g., Next Generation sequencing for mutation-targeted drug trials; multi-analyte IHC/IFA or targeted mass spec for pharmacodynamic assays/pathway analysis) to detect emerging clinical biomarkers. Projects may address biospecimen challenges that are relevant to multiple trials with assays utilizing similar platforms, and/or challenges relevant to specific assays in specific trials. Applications must propose biospecimen science research for assays that are relevant today and for the five-year funding period, with the understanding that new biomarkers and assay platforms may emerge within this time frame.

The program will address the following research questions in biospecimen science to enable clinical assay development utilizing small tissue biopsies and liquid biopsies:

Research Projects that are not responsive to this NOFO :

See Section VIII. Other Information for award authorities and regulations.

SHIPPING AND STORAG

Dates

Supporting links

Secure Grant Funding 10x Faster

Get opportunities sent to your inbox