Perspectives from an Expert-Guided Discussion on Maximizing the Research Potential of Small Biopsy Tissue

Abstract

Purpose

Tissue biopsy specimens, both remnant diagnostic specimens and those collected for ancillary study, are an invaluable resource for clinical oncology research. However, utilizing biopsy specimens for molecular research is associated with innate challenges, such as insufficient tissue and/or tumor content, and low nucleic acid yields as well as analyte degradation due to suboptimal preanalytical workflows.

Methods

The National Cancer Institute’s Biorepositories and Biospecimen Research Branch convened a meeting that included expert-guided discussions that centered on strategies to mitigate these challenges and their effects on molecular analysis.

Results

Participants, who included medical oncologists, interventional radiologists, pathologists, and molecular biologists, offered best practice guidance on biopsy collection, preservation, storage, and extraction techniques. Their recommendations were largely based on the optimized workflows that were implemented at their respective institutions, which improved the likelihood of producing reproducible molecular data. Pre- and post-collection techniques, such as clear cross-team communication, pre-biopsy scoring based on lesion- and patient-specific criteria, biopsy collection and handling practices, and tumor enrichment options were also discussed.

Conclusion

The proceedings revealed that increasing awareness of the challenges associated with research use of tissue biopsies is key to developing assay-specific strategies that ensure sufficient tumor specimens are available for molecular oncology research. The lessons shared here from large-scale and multicenter trials will, ideally, inform the design of new cancer research studies, thereby harnessing the full potential of valuable clinical biopsy specimens.

CONTEXT SUMMARY

Key Objective

What are the critical barriers to successfully using small biopsy specimens in cancer research and clinical trials, and how might they be mitigated?

Knowledge Generated

Summaries of expert-guided discussions distinguished the requirements of cancer research studies from routine diagnostic procedures, highlighting a critical lack of research-specific biopsy guidance. The expert panel identified inadequate tissue amount, insufficient tumor content, and analyte degradation as obstacles to the research study of diagnostic biopsies and established cross-disciplinary recommendations extrapolated from protocols of successful large-scale studies that included multi-core acquisition, rapid preservation, precision enrichment, and optimized nucleic acid extraction, within a fit-for-purpose context.

Relevance

These recommendations provide a roadmap for maximizing the research potential of limited biopsy tissue. These optimized workflows have improved assay success rates and data reproducibility in large-scale oncology trials while prioritizing ethical transparency.

Graphical Abstract

BACKGROUND

The molecular characterization of tumor tissues obtained for clinical purposes may be essential for diagnosis, treatment decision-making, and the identification of therapeutic targets. Such characterization is increasingly performed on small biopsy specimens (0.5–8 mm)^1–3, which are less invasive than surgical specimens and may be sampled longitudinally throughout treatment. In clinical trials, additional research biopsies may be obtained to support treatment decisions or ancillary research, making them an invaluable resource in both clinical and research contexts. Analyses of biopsy specimens have led to key insights into tumor biology, mechanisms of drug action and resistance, treatment response, and tumor progression^4–6.

Compared with diagnostic biopsies, research biopsies often necessitate more tissue and higher tumor content to enable complex molecular analyses. These stricter requirements contribute to lower success rates of research analysis (74%)⁷ compared with diagnostic biopsies (80–90%)^8–9. Large-scale studies such as the Adaptive Patient-Oriented Longitudinal Learning and Optimization (APOLLO) program and the National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial, have reported that 12.7%¹⁰ - 18.5%⁴ of biopsies were unsuitable for molecular analysis. Refinements to NCI-MATCH workflows, which included increasing the minimum tissue collected, reduced the percentage of unsuitable biopsies to 6.1%¹⁰.

Other major determinants of successful molecular characterization of biopsies include pre-analytical factors, defined as all steps, conditions, and characteristics that occur before analysis. Patient characteristics, lesion parameters, and variability in collection, processing, and storage can all affect tissue quantity, tumor cellularity, necrosis, and analyte degradation (Table 1). Biomarkers may, therefore, exhibit distinct degradation patterns based on their biochemical properties, specimen handling protocols, and conditions of data collection and processing^11–13 (Table 1), potentially compromising both clinical and research results from tumor biopsies^14–17.

Table 1.

Examples of Factors that Warrant Consideration for Their Potential Effects on the Successful Use of Biopsy Specimens in Research

Phase	Relevant Factors	References
Pre-analytical	Ethical Considerations	46–61–63
	Lesion Size	66
	Imaging Method	46–67
	Specimen Collection Method	7–16–42–44–45–68–70
	Needle Gauge	7–44–70–71
	Number of Cores	4–7
	Cold Ischemia Time	14–16–17–38–42–44–45–72
	Specimen Preparation Type (e.g., FFPE, direct smear, cytospin, liquid-based cytology, cell block, fresh-frozen, nitrocellulose imprint)	73–75
	Specimen Thickness at Time of Fixation	76–77
	Decalification Method	78–79
	Freezing Method	17–42–44–70–80
	Freezing Media (RNAlater)	81
	Freezing Media (OCT)	42–44–82
	Fixation Method	14–70–83
	Fixative Concentration	84
	Fixative Buffer Reagent	85
	Formalin-to-Tissue Ratio	13–86–87
	Time in Fixative	84
	Embedding Practices	20–76–88
	Trimming Practices	86
	Sectioning Practices	20
	Storage-Associated Oxidation	68–89–90
	FFPE Archival Block Age	91–93
	Temperature of Frozen Storage	17–42–44–94–95
	Freeze-Thaw Cycles	96
	Tumor Enrichment Method	97
	Extraction Method	17–42–44–45–71
	Thaw Temperature and Duration	17–42–44–70–98
	DNA Quality Assessment Method (DIN, Functional DNA Quantification)	12–99
	RNA Quality Assessment Method (RIN, DV200, TIN)	100–102
	Tumor Cellularity Estimation Method (Visual vs. AI-based)	103–104
	Tumor Purity	16–104–107
Analytical	Library Preparation Method	89
	Nucleic Acid Input Quantity and Quality Thresholds	102
	Sequencing Approach (e.g., Targeted vs. Whole Genome), Sequencing Platform and Chemistry	102–108
	Sequencing Depth / Coverage	109
	UMI-Based Error Correction and Deplex Sequencing	110–111
	Reagent Quality and Batch Variability	16
	Computational Image Segmentation	112
	Antibody clones; Platform; IHC Scoring Algorithm	17–40–41–44–45–70–113
Post-analytical	Variant Detection Method	114
	Bioinformatic Pipeline and Analysis Software	40–115–117
	Batch Effects and Inter-site Data Harmonization	118
	Reporting Criteria and Classification Thresholds	33–40–41–106
	Analytical Validation	17–33–40–42–44–70–119–120
	Interpretation and Reporting	7–14–16–41–43–46–106–121–122

Despite these challenges, biopsy-specific guidance for the pre-analytical phase remains limited. Existing biospecimen recommendations¹⁸ are not optimized for clinical biopsy specimens, and guidance is often insufficient or inconsistent across tissue types and analytical methods^19–21. As technologies evolve, there is an increasing need for evidence-based, harmonized approaches to optimize biopsy collection and handling practices within an ethical framework.

Approach

To identify critical aspects of the pre-analytical biopsy workflow and substantiated mitigation strategies for the research study of small tissue biopsy specimens, the NCI Biorepositories and Biospecimen Research Branch (BBRB) convened medical oncologists, interventional radiologists, pathologists, and molecular biologists. These experts shared challenges encountered in obtaining and analyzing small biopsy tissues, along with the institutional best practices that have improved specimen adequacy. Discussions also highlighted common misconceptions regarding biopsy collection and emphasized the importance of multidisciplinary communication and standardized procedures. Collectively, discussions provided insight into optimization approaches throughout the preanalytical phase to improve tissue quantity and quality for molecular analysis that may inform the design of future clinical studies in cancer research.

Tumor quality and quantity terminology

The lack of uniform terminology for tumor quality and quantity in cancer research can confound the optimization of biopsy collection and handling methods, as superficially similar terms often carry nuanced differences that make them unique and/or applicable to specific stages of the biopsy workflow. For example, during the pre-analytical phase, tumor cellularity represents the number of tumor cells in a specimen, while incipient tumor content (incipient TN%) is an estimate of the proportion of tumor cells present at acquisition. Enriched tumor content (enriched TN%) represents the proportion of tumor cells following microdissection or another enrichment method. Tumor purity can be inferred via sequencing metric data during the analytical phase before (incipient tumor purity) and after (enriched tumor purity) enrichment, while Low Tumor Purity (LTP) conveys that the tumor threshold identified for a specific assay has not been met. Terms that describe specimen adequacy during the pre-analytical phase, such as Quantity Not Sufficient (QNS) and Tissue Insufficient for Analysis (TIFA), are often subjective, as they convey that analysis of a specimen is unlikely to be successful but do not address the underlying reason (ie, whether the specimen is too small or lacks sufficient tumor content). The terms Chimerism/Contamination indicate the presence of unintended DNA or cellular material, which can confound analysis. The nuanced details of these descriptive terms (outlined in Table 2) accurately reflect the specific metrics used in the respective studies discussed by each expert in the sections that follow.

Table 2.

Terms on Tumor Content–related Considerations Across the Pre-analytic and Analytic Workflow

Term	Workflow Stage	Basis of Assessment	Definition	Key Notes / Nuances
Tumor cellularity	Pre-analytic	Microscopy (pathologist estimate)	The number and ratio of viable tumor cells to the total number of viable cells in the sample or area (excludes necrotic areas)	Research biopsies often demand significantly higher tumor cellularity than diagnostic specimens to support complex molecular analysis and correlative studies.
Incipient tumor content (incipient TN%)	Pre-analytic	Histology (pathologist estimate)	Morphologic estimate of tumor nucleation or tumor content present in the collected specimen prior to any enrichment.	Specimen-intrinsic; assessed on H&E at acquisition. Sets the upper bound for what enrichment can achieve. Distinct from computational tumor purity.
Enriched tumor content (enriched TN%)	Pre-analytic	Histology (pathologist estimate)	Histology-based estimate of the intended or prescribed tumor content following microdissection or other enrichment approaches.	Represents an expected goal state, not a measured outcome. Dependent on tissue characteristics, tumor architecture, and confidence in enrichment feasibility and method performance.
Incipient tumor purity	Analytic	Computational (sequencing-based)	Tumor purity inferred from sequencing of the unenriched specimen using features such as aneuploidy and variant allele fraction (VAF) distributions.	Not routinely obtained when enrichment is prescribed, as unenriched sequencing may not be performed. Related to, but not interchangeable with, incipient tumor content.
Enriched tumor purity	Analytic	Computational (sequencing-based)	Tumor purity inferred from sequencing following enrichment.	Reflects the achieved tumor fraction. May differ from prescribed enriched TN% due to variability in enrichment methods and tissue heterogeneity.
Low tumor purity (LTP)	Analytic	Computational QC determination	Analytic determination that the tested specimen does not meet established tumor purity quality thresholds required to support validated assay performance.	Not equivalent to QNS or TIFA. LTP outcomes may still yield informative or actionable results in some contexts, or may constitute outright test failure depending on use case.
Tumor Fraction	Analytic	Computational (sequencing-based)	The proportion of tumor-derived DNA relative to total DNA.	Often used in the analytic phase to describe the achieved purity of a specimen.
Quantity Not Sufficient (QNS)	Pre-analytic (legacy term)	Context-dependent; often subjective	Umbrella term indicating specimen insufficiency without specifying the underlying failure mode.	May reflect inadequate tissue volume, inadequate tumor content, or a local determination that enrichment would be futile. Historically also used for extraction failures; reflects local evidence-based judgment.
Tissue Insufficient for Analysis (TIFA)	Pre-analytic (legacy term)	Context-dependent; often subjective	Nonspecific term used variably to describe insufficient tissue volume and/or insufficient tumor content.	Overlaps substantially with QNS and lacks specificity regarding the underlying cause of insufficiency.
Chimerism / Contamination	Pre-analytic and/or Analytic	Context-dependent (clinical, histologic, and/or computational)	Presence of DNA or cellular material from more than one individual or unintended source, resulting in admixture that confounds tumor content or purity assessment.	May be pre-analytic (e.g., transplant-related chimerism, specimen mix-up) or analytic (detected computationally). Outcomes may include suppressed reporting, limited interpretation, or test failure depending on context.

Tissue adequacy challenges encountered and lessons learned from NCI-MATCH, a successful, large-scale, multicenter clinical trial

Dr. Stanley R. Hamilton, a pathologist and Professor at City of Hope National Medical Center and Comprehensive Cancer Center, shared the key challenges that were identified and mitigation strategies developed during the NCI-MATCH study (NCT02407405)²², a phase II precision medicine clinical trial aimed at evaluating the success of treatment approaches based not on tumor type, but upon targeted genomic alterations present²³. NCI-MATCH’s central tumor profiling used a clinical next-generation sequencing (NGS) assay that evaluated 143 genes and immunohistochemistry (IHC) assays for protein expression of specific markers²⁴. NCI-MATCH, which included over 1,000 clinical sites across the U.S. and a protocol-specified biospecimen management plan, provided invaluable insights into the practicality and challenges associated with collecting, processing, and analyzing small tumor biopsy specimens for molecular profiling in a clinical trial setting²⁴.

In NCI-MATCH, tissue acquisition and processing workflows were standardized across clinical sites by providing each center with comprehensive collection and shipping kits that included detailed specimen collection instructions with photographic illustrations; all required containers, fixatives, and tubes to support core needle biopsy (CNB, the primary tissue acquisition method) and fine-needle aspiration (FNA) specimen acquisition as well as cell block processing; and a pre-paid return shipping label. Typically, CNB specimens (collected using either a 20-gauge needle for lung lesions or an 18-gauge needle for all other lesion types) were placed into 10% neutral buffered formalin²⁵ as quickly as feasible to minimize nucleic acid degradation (Figure 1).

Figure 1.

Recommendations from the Expert-guided Discussion on Maximizing the Research Potential of Small Biopsy Tissue held by the National Cancer Institute.

The main challenges encountered during NCI-MATCH centered on tissue adequacy. An initial assay success rate of 87% increased to 93% at the end of the trial by implementing several strategic changes to increase the likelihood of collecting specimens with sufficient tumor content^24–26. While initial instructions specified that 4–6 cores were to be collected from each lesion, detailed changes to the instructions for radiologists and research coordinators included that collection should include 6 cores whenever possible and an FNA specimen (Figure 1). Including FNA as a backup improved the percentage of adequate specimens; further, FNA often provided better tumor cellularity, a higher tumor fraction with less stroma, and superior sequencing metrics compared to concurrently acquired CNBs²⁶. Increasing the expectations for research tissue allowed otherwise unevaluable specimens to be rescued by improving tumor tissue yield and taking action to ensure the presence of sufficient tumor cell numbers^24–26. Sample availability was also improved by permitting, in addition to fresh specimens, the inclusion of existing archived biopsy specimens collected within the previous 6 months if there was no response to treatment within that period²⁴. Although a threshold for incipient tumor content was not specified, specimens that were primarily comprised of stroma that lowered the percentage of tumor cell content were, in many instances, informative after tumor enrichment by microdissection²⁴. Notably, a high stromal component may be observed following cancer treatment²⁷; thus, the timing of biopsy collection relative to prior therapeutic modalities should be recorded as it may affect the successful use of research biopsy specimens.

The NCI-MATCH trial provided critical insights into the challenges associated with utilizing small tissue biopsy specimens for molecular research in a multi-center trial and the mitigation strategies that were applied successfully. The provision of a collection kit with pre-paid return shipping, an adaptive approach, and fluid communication among all members of the multidisciplinary team were key to identifying and addressing obstacles early in the clinical trial. Findings from NCI-MATCH confirmed recommendations from NCI’s Division of Cancer Treatment and Diagnosis (DCTD) that biopsy specimen requirements for cancer research often differ from those of diagnostic specimens, necessitating more tissue and greater tumor content⁷. In NCI-MATCH, these needs were addressed by collecting six CNB cores and an FNA specimen. Overall, patient safety was not compromised by these changes, as severe adverse events associated with sample acquisitions occurred in less than 1% of cases, which is in line with diagnostic procedures²⁴. However, the incidence of complications for intrathoracic biopsies was higher (17.1%), highlighting the need for biopsy site-specific risk stratification^5–24. The trial also revealed the potential of genomic sequencing to improve diagnosis and cancer treatment selection, underscoring the ethical need for better communication with trial participants about the benefits of research biopsies alongside the discussion of potential risks. The success of NCI-MATCH in generating molecular profiling results from small biopsy specimens provides a crucial framework for future precision oncology trials.

Collection, processing, and coordination approaches for improving small biopsy yield and quality for molecular testing

Dr. Alda Tam, an interventional radiologist at the University of Texas M.D. Anderson Cancer Center (MDACC) reported that obtaining adequate tissue with sufficient tumor content was challenging in the Adaptive Patient-Oriented Longitudinal Learning and Optimization (APOLLO) program and the Biomarker-integrated Approaches for Targeted Therapy for Lung Cancer Elimination (BATTLE) trial. Biopsy cores with a “quantity not sufficient” (QNS) for analysis increase the monetary costs of a research study as a sunk cost or due to repeat testing, contributing to delays that also may adversely affect patient health^6–28.

Experiences from the APOLLO program and the multicenter BATTLE trial highlight persistent challenges in obtaining adequate tissue with sufficient tumor content²⁹. In APOLLO, 55.8% of biopsies contained at least one inadequate core, and 18.5% were entirely QNS, often due to low tumor content (< 10%)⁴. In the BATTLE trial, 17% of biopsies were QNS for molecular testing, with sampling error and lesion selection identified as key contributors³⁰. A post-study analysis of BATTLE that included interventional radiologists showed that a subset of patients (4.3%) could not/should not have been biopsied because either the lesion was unsuitable (i.e., necrotic) or there was an elevated risk to the patient³⁰. In the APOLLO program, additional factors that affected the likelihood of obtaining ≥1 inadequate core(s) for next-generation sequencing included lesion size (<2 cm), prior systemic therapy (> 3 months prior to biopsy), tumor pathology⁴, and patient age (younger)(Figure 1). Tumor histology also played a role, as breast cancer lesions had lower odds for adequacy for NGS than melanoma, potentially reflecting differences in stromal composition and fibrosis³¹. To address these sampling challenges, MDACC improved communication practices between interventional radiologists, oncologists, and research teams. Providing detailed sampling instruction sheets to radiologists that included research goals and sample requirements (e.g., number of cores, fixation methods, preferred tumor regions) was crucial (Figure 1) to achieving a compliance rate of >96%) within a practice (20 interventional radiologists)³².

Standardized collection practices at MDACC now include the routine collection of 4–6 cores (20-gauge for lung, 18-gauge for other tissues)³³ based on evidence that collecting 3–6 cores per procedure significantly increases the likelihood of obtaining adequate tissue^4–30–34 without increasing patient risk^4–35. Importantly, core order does not reliably predict tissue adequacy, and the assumption that earlier cores are superior is not supported. Factors that influence genomic adequacy include the site of biopsy and the lesion size, with larger lesions (≥2 cm) more likely to yield sufficient tissue⁴.

MDACC interventional radiologists also developed a lesion scoring system to estimate the likelihood of adequate yield based on tumor viability, aggressive sampling, and procedural risk, excluding patients from clinical trials that are unlikely to provide adequate tumor biopsies³². When multiple lesions are present, functional imaging (i.e., PET/CT) can ensure that metabolically active lesions are sampled³⁰. Point-of-care tissue assessment tools, such as Rapid On-Site Evaluation (ROSE)(recommended by the College of American Pathologists³⁶) and emerging imaging tools like confocal fluorescence microscopy can confirm malignancy, improve diagnostic yield and core adequacy²⁸, and reduce unnecessary sampling (Figure 1).

MDACC has established a multidisciplinary biopsy team infrastructure that facilitates early and consistent communication of research goals and sample requirements for each study/trial among oncologists, interventional radiologists, pathologists, and research coordinators²⁶ to improve the outcome of tumor biopsies in clinical trials^26–31–37.

Analyzing small biopsies for labile analytes in pharmacodynamic (PD) studies

Dr. Ralph E. Parchment, Managing Director of the Clinical Pharmacodynamics Program at the Frederick National Laboratory for Cancer Research, outlined the challenges encountered when evaluating labile analytes in tissue biopsies in pharmacodynamic (PD) studies, which primarily focus on the response of the intended molecular target of a drug, the biochemical and cellular consequences of target response, and, ultimately, its biological and physiological effects. By design, these longitudinal studies use longitudinal biopsies from individual patients to quantify molecular response to drug therapy.

PD studies face unique pre-analytical challenges because they quantify labile analytes, such as phosphorylated proteins, that are highly sensitive to ischemia and specimen handling. Degradation can occur minutes after tissue procurement; phosphorylated sites pY1234/1235 in the c-MET protein decreased by 50% in CNB after 3 min of cold ischemia¹⁷, and phospho-Ser473-Akt (p-Akt) has a half-life of 20 minutes at room temperature in xenografts awaiting processing³⁸. The global phosphotyrosine proteome of ovarian and colon tumors exhibited substantial changes 5 minutes after resection under conditions of controlled ischemia¹⁴. While diagnostic biopsies are often fixed in formalin, PD analysis may be confounded by formalin fixation at room temperature, as phosphatase activity can persist in the interior of solid tissue specimens long after the recorded time of collection and placement in fixative due to the slow penetration rate of formalin (e.g., 1.0 mm/h)³⁹. Diagnostic biopsy practices conflict with preferred methodology for stabilizing labile analytes in other ways by (a) sometimes prolonging cold ischemia time by delaying fixation while touch preparations and other cytology procedures are completed; (b) accepting a specimen that contains just a small number of distinctive cells that definitively establish a disease diagnosis, rather than intentionally collecting a specimen containing thousands of evaluable cells so a complete biological profile, pharmacodynamic response, and inter-cellular heterogeneity can be described^40–41; (c) halting phosphatase activity only at the exterior surface of the specimen but not in its interior by fixing at ambient temperature instead of under refrigeration; and (d) long-term storage as paraffin blocks instead of as cryopreserved specimens while awaiting laboratory analysis. These conflicts cause variable degrees of cumulative loss of labile analytes, especially in the interior of the biopsy specimen, which significantly confounds molecular analysis of highly labile phosphoproteins with half-lives of only a few minutes. Pre-analytical factors that affect labile analytes must be controlled within established limits that assure the quality and reliability of the planned analysis – especially when patient biopsies from clinical trials are being used.

Standard operating procedures (SOPs) specifically designed for the collection, handling, and processing of research biopsies for PD analysis solve these problems and preserve post-translationally modified phosphoproteins^17–40–45. SOPs for the collection of research biopsies for PD analysis of labile analytes utilize point of collection, snap-freezing of the biopsy specimens in the clinical procedure area within 1–2 minutes of collection followed by frozen storage (performed by staff dedicated in a designated area of the procedure room to maintain sterility), thawing specimens under cold fixative containing enzyme inhibitors, and then routine paraffin embedding that have been developed, analytically validated, and verified by clinical and preclinical studies to enable pharmacodynamic studies of changes in labile phosphoprotein patterns^17–40–45. Root cause analysis of unsuitable biopsies for PD studies has led to additional recommendations that further increase the likelihood of evaluable biopsies for labile analytes^7–16–46 that include using 18-gauge needles and collecting multiple cores, when possible, to increase the probability of obtaining adequate tumor content; rapidly snap-freezing cores within 3 minutes using a vial pre-cooled in liquid nitrogen to immediately halt enzyme activity; and thawing cores in extraction buffer containing phosphatase inhibitors at 4°C or in neutral buffered to maintain tissue and morphological integrity and preserve phosphorylation (Figure 1). Multidisciplinary communication between oncologists, interventional radiologists, surgeons, pathologists, and research scientists is vital to ensuring that biopsy specimens are of sufficient quantity, quality, and tumor content for research study; for example, the input of a pathologist can be essential when excluding areas with artifacts (necrosis, hemorrhage, etc.) by microdissection, a strategy employed during the NCI-MATCH trial. To address intratumoral heterogeneity, algorithms can also be used to restrict image analysis to malignant cells. While implementing SOPs for specimen handling and storage is crucial for reproducible and reliable biomarker levels, PD biomarker assays must also be rigorously validated for analytical performance, reproducibility, reliability, sensitivity, and accuracy. Futhermore, clinical readiness of the assay should be proven by a fitness-for-purpose study that demonstrates that the anticipated results from a first-in-human use of the test can be obtained in a non-clinical setting that simulates the clinical setting to the greatest extent possible^{17–42–44–46}. Applying these approaches, Dr. Parchment noted that his group has reduced the threshold for viable tumor content to 5–10%. Several fit-for-purpose SOPs on biopsy collection and handling and validated biomarker assays and procedures, including PD assays, are available on NCI’s website^47–49.

Target enrichment from small biopsies for clinical molecular profiling

Dr. Jonathan Keith Killian, the Associate Medical Director of Foundation Medicine, discussed the challenges his molecular testing laboratory faces when receiving small biopsies with insufficient tumor content, often due to low tumor purity or a small focus of neoplastic cells⁶, and the strategies he employs to enable genomic analysis.

One such strategy is targeted tumor enrichment. Targeted enrichment is a selective strategy to increase tumor content and can include macro- and/or micro-enrichment approaches. Macroenrichment by scraping an area from a glass slide³⁷ requires a relatively large area and is often unsuccessful in specimens that are ≤1 mm; it also lacks precision and eliminates the possibility of post-enrichment histological evaluation. Microdissection, which includes manual and laser-based methods, can be performed on individual slides in conjunction with a microscope and was successfully utilized during the NCI-MATCH trial to salvage CNB with limited tumor content^24–26, although the approach does have disadvantages that include expense and the time required. Precision enrichment (PE) is a method of microdissection that involves using a blunt-tip fine needle to punch out specific, tumor-rich areas directly from the paraffin block. The entire needle punch containing the enriched tumor cells is then digested and used for analysis⁵⁰. PE is rapid, inexpensive, and eliminates specimen waste, as the residual tissue in the paraffin block remains available for analysis^50–52. In an analysis of 49 tumor types, PE resulted in a mean increase in tumor content of >20% compared to specimens that were not microdissected. PE also allowed researchers to accurately estimate the amount of DNA that could be obtained from a punch⁵⁰ (Figure 1). PE has been successfully employed in tissue specimens with low tumor purity to allow for the detection of several clinically relevant cancer biomarkers^53–57, including assessing genomic loss of heterozygosity (gLOH)^51–52 and tumor mutational burden⁵⁸.

Maximizing utilization of limited specimens: from tissue preservation to optimization of nucleic acid extraction using carrier RNA

Dr. Dan Merrick, from the Department of Pathology at the University of Colorado and co-Chair of the Clinical Biospecimen Working Group of the NCI-sponsored Human Tissue Atlas Network (HTAN) has faced challenges making the most of small biopsy tissue for research, including isolating nucleic acids of suitable quality at sufficient quantities. Effective approaches leading up to and during extraction have been implemented in his laboratory to maximize the use of these limited specimens for molecular research and clinical testing.

In agreement with the findings of other studies, several HTAN teams have observed that small biopsies need to be promptly and adequately preserved. The preferred preservation method is contingent on a number of factors, including the research question, accurate histologic classification, planned downstream assays, and specimen availability. For retrospective, longitudinal cohorts, several HTAN teams primarily use remnant formalin-fixed, paraffin-embedded (FFPE) lung tissues. However, for prospective cohorts, frozen tissues are preferred. For the detection of molecular signatures within the tumor microenvironment, HTAN employs multiplex immunofluorescence, 2D CODEX-spatial imaging, DNA and RNA Bulk Sequencing including Whole Transcriptome or Whole Exome Sequencing (WTS/WES), and a variety of other assay types that inlcude single cell sequencing, with the latter generally performed in frozen tissue. In addition to microdissected tissues, whole tissue scrolls are often used for analysis to retain informative signatures of normal/stromal tissue, which would otherwise be lost with laser capture microdissection (LCM) enrichment. During DNA extraction, several HTAN centers include a bacteriophage-derived carrier RNA, which significantly and consistently increases DNA yield from small forceps biopsy specimens and is predominantly removed at the exome capture step when preparing the library for WES. Carrier RNA improves nucleic acid extraction efficiency at several levels, including acting as a decoy for RNases and non-specific binding. Inclusion of carrier RNA improved yields by 2-fold in limited biopsy specimens, with even greater improvements in yield seen in the smallest samples. Using matched samples extracted with or without carrier RNA, comparisons of key sequencing quality metrics showed that DNA extraction with carrier RNA can also reduce the percentage of duplicate reads and increase the depth of sequencing coverage without introducing sequencing noise while maintaining the fidelity of variant calls (Figure 1). The only potentially adverse effect of the inclusion of carrier RNA is a small decrease in the mean insert size (i.e., enrichment of smaller fragments). Different carrier RNA preparations are available. Notably, the use of carrier RNA during DNA extraction carries no risk of DNA contamination. Others have similarly reported substantial improvements in DNA extraction efficiency in degraded tissue specimens when carrier RNA is included during DNA isolation^59–60. Although Dr. Merrick has not evaluated the use of carrier RNA for RNA extraction, the RNA quality number (RQN) 200 (also known as DV200), which represents the fraction of fragments longer than 200 base pairs, is an accurate quality control metric for samples extracted from small biopsies with low RNA yields.

Notably, any and all remnant tissue is a valuable resource that may be especially important for in situ analysis. HTAN research utilizes such remnant tissue to construct tissue microarrays (TMAs) that are analyzed by highly multiplexed assays, such as multi-plex immunofluorescence, spatial transcriptomics, and other in situ assays. Dr. Merrick’s team has found that remnant tissue >1.5 cm² is sufficient for TMA block construction, but that tissues smaller than 1.5 cm² commonly fail during micro resetting, the practice of extracting tissue cores from a donor block and embedding them in a recipient block.

Ethical considerations

The expert discussions underscored key ethical considerations surrounding the collection of research biopsies in clinical trials, emphasizing the importance of patient autonomy, transparent informed consent, and clear communication with patients about the potential risks and benefits. The expert participants supported ASCO’s ethical framework⁶¹, which permits mandatory biopsies only when the scientific utility of research findings is “expected” and the associated risk is low to moderate, underscoring concerns that patients may feel coerced into participation when facing limited treatment options. Despite a low incidence of serious adverse events (AEs), biopsy procedures pose both minor and serious risks and often yield no direct clinical benefit to participants. Experts stressed that informed consent must clearly communicate risks and the presence or absence of individual benefit. The need for improved reporting of biopsy-related AEs and inclusion of biopsy experts in oversight bodies was widely agreed upon^61–62, alongside calls for validated assay procedures and the establishment of assay-specific tissue requirements. Notably, the FDA recently released recommendations on the collection and processing of research biopsies, which emphasize the importance of ensuring that potential participants are fully informed about whether research biopsies are optional or mandatory for trial enrollment, as well as all associated risks⁶³. Overall, the discussion called for enhanced ethical rigor through transparency, multidisciplinary oversight, and comprehensive risk assessment to safeguard patient welfare while advancing scientific goals.

SUMMARY

Expert-guided discussions during a meeting convened by NCI identified several challenges when using biopsy tissue specimens for clinical oncology research as well as highlighted verified mitigation strategies. Key challenges experienced by expert participants included inadequate amounts of tissue, insufficient tumor content, and the isolation of nucleic acids of suitable quality at sufficient quantities for the intended assay(s). Strategies applied by expert participants at their respective institutions included the implementation of a pre-biopsy scoring system to assess QNS risk, establishing clear lines of communication within the multidisciplinary team that includes tissue size and tumor cellularity requirements, provision of a collection and shipping kit, rapid evaluation of collected specimens on-site to ensure tissue requirements have been met, approaches for tumor enrichment pre- and post-analysis, and techniques to improve nucleic acid yields (Figure 1). Notably, extracting meaningful PD information from small biopsy tissues, especially for labile analytes like phosphoproteins, requires a fundamentally different approach than routine diagnostic biopsies. This involves stringent, rapid preservation techniques at the point of collection, careful handling in the laboratory, validated assays, and robust multidisciplinary collaboration to ensure samples are indeed “fit-for-purpose”.

The discussions, drawing on extensive experience from initiatives such as NCI-MATCH, BATTLE, and APOLLO, highlighted persistent challenges and effective strategies to ensure that research biopsy specimens yield meaningful scientific insights while prioritizing patient well-being. By embracing multidisciplinary collaboration, employing SOPs, leveraging real-time assessment tools, and implementing tumor enrichment approaches to salvage low-purity specimens, molecular oncology researchers can ensure that small biopsy specimens yield their maximum scientific and clinical potential. Several resources are available through the NCI DCTD that support the collection and processing of high-quality biospecimens suitable for clinical research. These include DCTD SOPs that cover pre-analytic and analytical workflows^47–49 and BBRB SOPs for pre-analytical specimen collection and handling⁶⁴. Another resource, the NCI Biospecimen Research Database⁶⁵, includes a publicly accessible online SOP Library of protocols contributed by over 100 government, non-government, and international sources; internally developed, analyte-tailored evidence-based recommendations; and a repository of literature evaluating pre-analytical effects in human biospecimens. Fostering transparency and embracing evidence- and experience-based standardization can minimize pre-analytical variability and improve the reproducibility of results in translational research.

Data Sharing Statement

No new data were generated or analyzed in support of this study.