A Review of Liquid, Tissue-Based and Combined Next-Generation Sequencing

A Review of Liquid, Tissue-Based and Combined Next-Generation Sequencing
With a focus on NSCLC, Colorectal, Breast and Prostate Cancers

Next-generation sequencing (NGS) is an invaluable testing method offering clinically significant information to improve patient outcomes. In this literature review, we summarize the role of liquid and tissue NGS testing in cancer therapy with a focus on lung, breast, colorectal (CRC) and prostate cancer.

Guidelines for the use of NGS testing provide important resources for varying clinical scenarios in each specific cancer type. Some limitations of NGS testing can be compensated for through several different methods. NGS results can be used to enhance treatment decisions and track response to therapy. We aim to elaborate on NGS best practice recommendations and methods to decrease limitations in this review.

METHODS

This narrative literature review assesses NGS testing recommendations from both the National Comprehensive Cancer Network (NCCN) and the European Society for Medical Oncology (ESMO) through the Annals of Oncology journal. Data from relevant published peer-reviewed medical literature was also aggregated for comparison using predetermined rules.

Relevant literature searches were performed using peer-reviewed databases including PubMed and the National Library of Medicine. Search terms focused on “ESMO 2024 NGS recommendations,” “liquid NGS,” “tissue NGS” and “concurrent liquid and tissue NGS.”

Additional screening was performed based on specific cancer types with inclusion rules involving screening for recency, clinical relevance and scope. Exclusion criteria included outdated recommendations, study limitations including mention of bias, impractical applications and ongoing studies.

A total of 40 peer-reviewed published medical journal articles were reviewed and 27 were selected for data utilization. The key findings of selected literature were synthesized and assessed for similarities, differences, benefits and disadvantages, and the implications of these findings were summarized.

INTRODUCTION

a testing method using DNA and/ or RNA to determine the sequence of hundreds to thousands of genes associated with diseases, genetic variations and genomic features within a few hours or days.1 NGS testing advantages include its high throughput per run, speed, cost-efficiency and low sample requirements compared to other sequencing methods such as polymerase chain reaction (PCR)-based panels and Sanger Sequencing.2

Commercial availability of early NGS platforms began in 2005. Since then, NGS use has evolved in its capabilities. In oncology, NGS testing plays a significant role in identifying genetic variations associated with specific malignancies and provides valuable clinical and biomarker information supporting diagnoses, targeted therapy and immunotherapy, and response assessments.1

The advantages of NGS testing over other sequencing technologies include its ability to cost-effectively identify targetable and actionable mutations, such as single nucleotide variants, copy number alterations and structural variants simultaneously in a single sequencing run.2 This comprehensive approach is significantly more efficient than single-gene testing or small gene panels, which are limited in scope and require multiple assays to obtain similar information.

While the advantages of NGS largely depend on its broad genomic coverage and efficiency, the clinical utility of the results is also influenced by the source of the DNA or RNA being analyzed. The source of DNA and/or RNA provided for NGS testing varies between a liquid biopsy or tissue biopsy.

Tissue biopsies provide DNA and/or RNA directly from tumor cells, allowing for comprehensive genomic and transcriptomic analysis.

In contrast, liquid biopsies are obtained from blood or plasma samples, where tumors shed circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs) into the bloodstream.3

Each NGS testing platform and method has its own advantages and limitations. In this literature review, we summarize the role of liquid and tissue NGS testing in cancer therapy with a focus on lung, breast, colorectal and prostate cancer.

CURRENT GUIDELINES FOR NGS TESTING

alterations that have clinical and therapeutic relevance. Targeted therapy focuses on specific molecular targets unique to a cancer allowing for more precise and possibly less toxic treatment. The application of NGS results — which include actionable, resistant, emerging, predictive and prognostic alterations — offers valuable insights that can significantly improve patient outcomes.

Despite growing adoption of NGS, standardized approaches to validation, analysis and interpretation continue to vary, with guidance provided by several professional groups such as the U.S. Food and Drug Administration (FDA), College of American Pathologists, The Canadian College of Medical Geneticists and International Quality Network of Pathology.4

Although the clinical use of NGS initially advanced ahead of standardized guidelines, formal recommendations from professional societies such as the NCCN and ESMO have since been established, reflecting the growing evidence base supporting its utility in oncology. The benefits and limitations of liquid and tissue NGS testing provide insight into NCCN and ESMO recommendations:

〉Tissue Biopsies: Tissue biopsies have the advantage of being obtained directly from the tumor of origin or a site of metastasis. For tissue-based NGS, only a small sample size is required to test numerous genes simultaneously and has a high sensitivity for point mutations, copy number variants and gene fusions.5 However, the utilization of tissue testing can depend on both the sample obtained and the patient condition.

〉Liquid Biopsies: The use of liquid biopsy is highlighted when the tissue sample is difficult or impossible to acquire due to location or when a noninvasive approach is preferred. Liquid biopsies are minimally invasive, convenient, require less sample preparation, offer ease of repeat testing, especially in frail patients, and provide more molecular heterogeneity by capturing DNA shed from multiple sites rather than a single biopsy site.6,7 Of note, the NCCN Guidelines® for NSCLC, version 5.2025, stated that many but not all liquid biopsy tests use NGS. Caution should be taken in observing methods of testing at different labs.3

COMPARATIVE STRENGTHS AND LIMITATIONS

Liquid NGS testing offers a faster turnaround time compared to tissue NGS testing. In general, liquid biopsy turnaround times are seven to 10 business days, while tissue NGS can take two to four weeks. In a non-small cell lung cancer (NSCLC) study, liquid NGS results returned on average 26.8 days faster than tissue samples.5 In breast cancer biopsies, liquid biopsy results took 10-13 days compared to tissue NGS of up to 33 days.8

Tissue biopsy has higher sensitivity than liquid biopsy in early-stage malignancies. Because early-stage tumors shed little or no circulating tumor DNA (ctDNA), detection rates are limited. Circulating tumor DNA is detectable in only about 50% of early-stage NSCLC cases and early prostate cancer are typically confined and do not release DNA into the bloodstream, both leading to a higher risk of false-negative results with liquid biopsy, especially for variants present at very low allele factions (<0.1%).9,10

Some plasma-based liquid biopsy assays include a measure of tumor fraction (TF), which can assist with identification of low ctDNA content. Samples with low TF, especially less than 1%, should be interpreted with caution due to sensitivities.3

Additional factors such as pre-analytical variability (e.g., sample handling, timing of collection), biological variability in ctDNA shedding between tumor types and technical limitations of certain platforms also contribute to reduced detection sensitivity in liquid biopsy.11

Therefore, liquid testing is more reliable in advanced or metastatic disease, where ctDNA levels are typically higher.

Tissue NGS testing limitations include invasive procedures with the possibility of complications, insufficient samples with the possibility of requiring additional biopsies, low tumor cellularity, poor specimen quality, deteriorated DNA quality and impractical repeat biopsies for surveillance.12

Liquid biopsy has a lower sensitivity, accuracy can be challenged by tumor heterogeneity and evolution, false negatives in low shedding tumors and false positives from clonal hematopoiesis of indeterminate potential (CHIP) may occur.12

NCCN Guidelines prefer tissue biopsies over liquid biopsies for initial biomarker testing and treatment decisions in metastatic solid tumors. However, they include a statement on use of liquid biopsy NGS when tissue is unavailable.3

CHIP AND FALSE POSITIVES

CHIP is characterized by acquired mutations in blood cells, specifically in genes known to be involved in hematologic cancers. The frequency of CHIP variants increases with age, previous cancer treatment and smoking.

These variants are detected in ctDNA and can lead to false-positive results such as in TP53 and ATM mutations. These variants may also represent true biological positives associated with secondary malignancies.

An example of this would be JAK2 mutations in an undiagnosed secondary myeloproliferative disorder.11 In CRC, TP53 mutation is the most commonly mutated gene in solid tumors. However, it is also a common CHIP mutation. Tissue NGS results can be used to determine whether a mutation is of tumor origin versus a CHIP mutation. Results demonstrating positive liquid TP53 that were previously wild-type TP53 on tissue biopsy could be CHIP mutations.

It is important to note that a tumor may also evolve new mutations not seen in the original tissue, so this scenario requires nuanced interpretation; not all discordances are CHIP.

Using both liquid and tissue results could potentially help identify a CHIP mutation. Many of the limitations of liquid- and tissue-based NGS testing can be minimized by concurrent testing.11

CLINICAL UTILITY OF CONCURRENT TISSUE AND LIQUID TESTING

Concurrent testing using both liquid- and tissue-based genomic sequencing in advanced or metastatic patients was found to identify more patients with targetable, actionable variants than tissue testing alone. Both liquid and tissue NGS testing can detect unique, clinically relevant mutations, and the NCCN Guidelines support their complementary use to reduce false-negative rates, particularly in NSCLC.

The NCCN further recommends plasma liquid testing when tissue is insufficient or unavailable, with follow-up tissue testing if no oncogenic driver is detected in the plasma sample.3

In a multicancer study published by the Journal of American Medical Association, 3,209 patients undergoing concurrent testing found 9.3% of patients had a clinically actionable variant detected by ctDNA that was not detected by tissue compared to 24.2% who had a variant detected by tissue testing but not ctDNA. Specifically, in NSCLC, 65.7% of variants were detected in both tissue and ctDNA, while 29% were identified exclusively in tissue and 5.3% were found only in ctDNA.12

KRAS variants were the most common ctDNA-unique variants and EGFR variants were the most common tissue-unique variants. The utilization of liquid biopsy alongside tissue biopsy was able to identify additional cases of KRAS, MET, ERBB2 and ALK alterations.

The study found that liquid plus tissue-based testing identified more patients with targetable, guideline-based variants than tissue testing alone.12 Combined testing enhances detection rates and sensitivity compared to tissue alone.

In a lung cancer trial, combining tissue with ctDNA testing also demonstrated an increased mutation detection rate from 66.19% in tissue alone to 77.3% and the sensitivity increased to 85.63% regardless of stage, grade and metastatic status.6

Another benefit supporting concurrent use is that if one type of testing is delayed or fails, the other type can provide results without delaying treatment. A study on time to treatment in advanced or metastatic NSCLC found that guideline-concordant treatment was initiated at the first oncology visit in 74% of patients receiving concurrent testing compared to 46% with tissue only. A shorter median Time to Treatment Initiation of 12 days versus 20 days was also found with the concurrent testing cohort.7

The ACCELERATE trial of non-squamous NSCLC patients also found 23% of patients receiving concurrent testing had treatment initiated prior to tissue result availability.13 By combining liquid NGS and tissue NGS, the yield of actionable drivers identified increased from 47% in tissue alone to 59% combined.7 The ability of concurrent testing to improve time to treatment initiation is noteworthy.

Other methods of enhancing variant detection include combined RNA and DNA panels, which specifically increase fusion detection compared to DNA NGS alone. A study of advanced NSCLC patients who received concurrent RNA-NGS and DNA-NGS identified 15.3% more patients with actionable structural variants compared to DNA alone.14

DNA-based NGS panels often target exonic regions to infer potential fusion breakpoints, whereas RNA-based NGS allows for direct detection of gene fusions and splice junctions by analyzing expressed transcripts. The study cites the NCCN Guidelines describing RNA-based NGS methods as the preferred option for detecting fusions and MET exon 14 skipping alterations in NSCLC.3

The ESMO Guidelines recommend RNA NGS as NSCLC criterion standard for detecting NTRK fusions, while the American Society of Clinical Oncology (ASCO) guidelines recommend RNA-based methods when no oncogenic drivers are detected in DNA or other standard methods. Combined RNA plus DNA panels offer the ability to increase diagnostic yield and provide a more comprehensive picture.14

TUMOR-SPECIFIC APPLICATIONS

Guidelines for NGS testing vary depending not only on type of malignancy but also on stage, timing in treatment, extent of disease, differences in assays, validation processes and technical considerations.

ESMO and NCCN have considered all these factors in making recommendations. Recommendations from both ESMO and NCCN concur that NGS testing via tissue biopsy is the gold standard for sequencing in NSCLC and preference initially in colorectal cancers (CRC) and other metastatic solid tumors.3,6,11

During times of progression tumor rebiopsy may be challenging and liquid biopsies play an important role in determining mutational status of the tumor, identifying resistance mutations and monitoring response to treatment.

TUMOR MUTATIONAL BURDEN

Next-generation sequencing can also be used for predictive responses through tumor mutational burden (TMB) testing. TMB is a measure of total somatic nonsynonymous mutations in a tumor and can be quantified using NGS. TMB calculations require larger NGS panels which often include POLE/ POLD1 sequencing.

NCCN states performing a large NGS assay on CRC tumor tissue has the advantage of not only identifying POLE/POLD1 pathogenic variants but also provides direct evidence of loss of proofreading function and high tumor mutational burden (TMB-H). These patients have a favorable prognosis due to immune responses and respond well to immune checkpoint inhibitor therapy.22

However, concerns exist on whether TMB thresholds for predicting response to PD-1 blockade are equivalent across the spectrum of solid tumors, and there are scenarios where high TMB does not predict response.23

The FDA approved pembrolizumab in solid tumors with TMB greater than 10 mutations/megabase in tissue-based NGS testing, based on one commercially available NGS test used as a companion diagnostic.24 This is reflected in NCCN Guidelines. In breast cancers with TMB-H, the NCCN lists pembrolizumab as an approved treatment and notes that tissue testing is more sensitive than ctDNA for detecting this biomarker.25

MINIMAL RESIDUAL DISEASE

Another significant use for NGS testing, and specifically ctDNA, is in cancer surveillance. Tissue biopsies cannot be taken frequently enough to allow for surveillance, whereas liquid biopsy is much more feasible in this setting. Currently, surveillance with ctDNA is not used outside of clinical trials for solid tumors, although studies are promising.

The DYNAMIC trial in CRC demonstrated that using ctDNA to guide adjuvant chemotherapy decisions can achieve similar outcomes while reducing overtreatment. Patients with detectable ctDNA after surgery, indicating minimal residual disease (MRD), had a significantly higher risk of recurrence if left untreated. In contrast, patients who were ctDNA-negative had a much lower risk of recurrence, and many were safely spared chemotherapy.26

In NSCLC investigational post-op, ctDNA MRD was found to predict high risk of recurrence. Specificity was high in predicting early relapse; however sensitivity was low and may not detect disease resulting in late recurrences.27

ESMO notes MRD detection requires assays optimized for ultra-high sensitivity. Standard ctDNA tests for advanced cancer may not be sensitive enough to detect residual disease compared to MRD assays using personalized patient-specific mutation tracking.11

NCCN Guidelines have acknowledged ctDNA as an emerging prognostic marker and recognize that positive ctDNA results offer useful information about the likelihood of cancer recurrence. However, NCCN points out that the usefulness may be uncertain if there are no effective treatment options available for the patient.10,18

REIMBURSEMENT AND ACCESS

Despite the preference for use of NGS testing when deemed clinically important, commercial insurers often do not offer coverage that is equivalent with guidelines. Barriers in utility include complex reimbursement processes. A study on these barriers found only 29% of insurance policies cover what is recommended by NCCN Guidelines, and in 71% coverage is more restrictive than NCCN Guidelines.28

Medicare’s National Coverage Determination for NGS testing came into effect in 2018 and covers NGS for patients with advanced cancer. Payers often provide coverage for NGS only when it is medically necessary, which leads to issues with multi-gene testing where novel targets are often included in an entire panel that is deemed experimental therefore reimbursement may be denied.

Other barriers include a lack of knowledge around the clinical utility of NGS testing, complex processes for paying for testing, reimbursement challenges and restriction on tests to specific labs or testing providers. There is also no standard method for ordering NGS testing by different providers, which leads to confusion among providers.28

CONCLUSION

Guidelines for NGS testing vary depending not only on type of malignancy but also on stage, timing in treatment, extent of disease, differences in assays, validation processes and technical considerations.

ESMO and NCCN have considered all these factors in making recommendations. Recommendations from both ESMO and NCCN concur that NGS testing via tissue biopsy is strongly preferred in NSCLC and preference initially in CRC and other metastatic solid tumors.

The overall summary of NCCN and ESMO guidelines for NGS testing includes identification of actionable alterations, broad molecular testing for all patients with stage IV disease and recommends testing for advanced disease.

RNA-based NGS is also recommended to capture fusions when a driver oncogene is not initially identified.6,11

Both NCCN and ESMO support concurrent liquid and tissue NGS testing to assist treatment decisions, identify actionable mutations and resistance mechanisms.

In colorectal cancers, NGS is preferred by NCCN for patients with metastatic CRCs for determination of KRAS/ NRAS, BRAF, HER2 amplification and MSI/MMR status, if not previously tested. NCCN does not recommend ctDNA for surveillance, noting that it provides prognostic not predictive information.18

ESMO has a different preferred approach in metastatic colorectal cancers, which includes molecular testing on surgical tissue specimen for tissue RAS/ BRAF, then, if metastasis is confirmed, send for KRAS/NRAS/BRAF and MSI as standard of care. They also recommend liquid testing if tissue will be delayed or is not feasible with a warning that waiting weeks for tissue results could delay adding an EGFR inhibitor or immunotherapy for eligible patients. If testing is positive on liquid biopsy, then treatment can start while awaiting tissue confirmation.

ESMO further notes that the pattern of metastasis influences choices in CRCs. Liver metastases are known to correlate with higher ctDNA compared with single metastasis, therefore, diffuse liver metastases might be suitable for upfront plasma genotyping versus oligometastatic disease which should rely on tissue. They document that liquid biopsy is invaluable for patients who are too sick for a biopsy to prevent missing targeted therapy windows.

Finally, ESMO also notes liquid biopsy use in the post treatment setting as very informative for revealing resistance mutations that develop after EGFR antibodies.11

In prostate cancers, liquid biopsy assays are considered an option when metastatic biopsy is unsafe or unfeasible. NCCN notes that most NGS testing in prostate cancer is not designed or validated for germline assessments.10

In breast cancer, ESMO notes that tissue NGS testing is essential in initial breast cancer assessment to detect biomarkers with targeted therapy such as RET-fusions with selpercatinib, FGFR1-3 fusions and/or mutations with erdafitinib, and somatic BRCA mutations with olaparib. Also noted is the usefulness of NGS in recurrent unresectable or stage IV disease for PIK3CA activating mutation, AKT1 or PTEN alterations, and ESR1 mutation.11,25

Guidelines for the use of NGS testing provide an important resource for varying clinical scenarios in each specific cancer type. As research from clinical trials evolves it is important to remain up to date on changes in these guidelines. Some limitations of NGS testing can be compensated by high sensitivity optimized panels, timing of testing regarding disease and patient condition, and proper processing methods.

NGS results can be used to enhance treatment decisions through identification of actionable and targetable mutations, identification of resistance mechanisms, as well as providing a method for tracking response to therapy with dynamic monitoring.

However, results should be interpreted in clinical context, especially when findings have low variant allele frequency or when there is potential CHIP contamination. In summary, next-generation sequencing is an invaluable testing method offering clinically significant information to improve patient outcomes.