Key trend to look out for in 2023 liquid biopsy technology and market

1.From advanced stage cancer to early

In the 1970s, the U.S survival rates of NSCLC men and women patients were 20% and 30% respectively. Improvements in therapy led to an increase in survival rate around 40 and 50% by 2018.[1] Among different types of treatment, it is likely that treatment administered with companion diagnostics using molecular therapy had a positive impact on NSCLC patients' survival.[1] In the status quo, it is a common practice to administer specific inhibitors to advanced cancer based on the positivity to certain biomarkers. The update and innovation is ongoing to incorporate novel biomarkers and detect cancer by using liquid biopsy from the early to advanced cancer. Through this blog, we aim to explore the status quo of targeted therapy against advanced cancer suggested by different guidelines in the U.S and Europe, and new uprising early cancer detection technology such as minimal residual testing (MRD) and multi-cancer early detection (MCED) which use circulating free DNA as a key source. 

2.Targeted therapy & companion diagnostics targeting actionable mutation

A companion diagnostic refers to a medical device mainly in vitro diagnostics which is used to weigh the benefit and risk for identification of patients who are most likely to benefit from treatment.[2] In a clinical setting, actionable mutations are diagnosed to maximize the efficacy and minimize the side effects. Actionable mutations are mainly divided into 3 categories: point mutation, gene fusion, and amplification (table 1).[3] Among these biomarkers, EGFR, ALK, and KRAS are detected more frequently compared to the others.[3,4] EGFR is known to be mostly populated in East asians while in Caucasians, KRAS appeared most frequently as shown in table 2.[3,4] 

                                                                                                                 Table1. Different types of biomarker based on types of mutations [3]

2-a. Past, present, and future of EGFR TKI

Therapeutic treatment guideline for EGFR is the most established up to date. 1st generation to 3rd generation TKI has been developed to target exon 19 deletion of exon 21 L858R mutation which occurs in the vast majority.[5] Currently NCCN guideline recommends osimertinib the 3rd generation EGFR-TKI as the first-line therapy for exon 19 deletion and exon 21 L858R.[6] If patients show progression after 1st or 2nd generation EGFR-TKI, the treatment is decided based on the presence of T790M. If mutation is T790M negative, 1st or 2nd generation EGFR-TKI is recommended.[6] In ESMO guideline, osimertinib is rated as tier 1 and grade A which is the highest in curative setting while other EGFR-TKIs are considered grade B.[7] In both guidelines, without question, the osimertinib is considered preferable EGFR-TKIs.

The key issue with administration of EGFR TKI is acquired resistance. T790M is the first and widely known mutation occurring after gefitinib or erlotinib administration.[5] Osimertinib is administered to patients who are T790M positive. But osimertinib also has drawbacks which develop mutations such as C797S and are not effective in wild type EGFR and loss of T790M (Figure 1).[5] Unfortunately, there are no alternative EGFR-TKI treatment for patients who develop acquired resistance to osimertinib. The 4th generation TKI is currently under development to tackle this issue but it is at preclinical stage or early clinical trials.[8] Developing TKIs which are effective in C797S while it is still effective in exon 19 deletion, exon 21 L858R, and T790M will be the key mission to accomplish in the near future.

2-b. Uprising biomarker designated as EGFR TKI acquired resistance 

Regarding acquired resistance to EGFR, two biomarkers rose as a hot issue: MET and ERBB2. MET amplification is one of the known alternative pathway activation which causes primary resistance to EGFR TKI.[9] It was chosen as an emerging biomarker by 2023 NCCN NSCLC guidelines.[10] Capatinib, tepotinib, and Crizotinib are well-known first-line therapy for MET exon 14 skipping mutation but for its effect on MET amplification while negative to MET exon 14 is under investigation.[11, 12] 

ERBB2 also known as HER2 is another biomarker that is related to EGFR-TKI acquired resistance as well.[5] ERBB2 was an emerging biomarker in early 2022 but it has been included as part of the NCCN panel's recommendation for NSCLC treatment since the end of 2022.[13] For the treatment of ERBB2, Fam-trastuzumab deruxtecan-nxki is considered as subsequent therapy in NCCN NSCLC guideline.[14] In ESMO guideline, trastuzumab–emtansine is suggested however, it has not been approved by EMA yet.[7]

2-c. ALK another established actionable target

Anaplastic lymphoma kinase (ALK) is one of the the only 2 biomarkers which are included as category 1 in NCCN guideline and tier 1 in ESMO guideline.[7,15] Alectinib, brigatinib, and lorlatinib are considered as the first-line therapy in both guidelines.[7,15] On certain occasions, ceritinib and crizotinib are recommended. Regardless, alectinib, brigatinib and lorlatinib hold stronger evidence therefore they are prioritized. Detection of ALK is considered important as a study suggests that patients who are ALK positive show low efficacy when treated with an immune checkpoint inhibitor.[16]

2-d. Will KRAS treatment guidelines be more established in 2023?

Contrast to the high frequency of kirsten rat sarcoma virus (KRAS), the development of KRAS faced challenge as KRAS was considered untargetable due to high affinity of KRAS for GTP and lack of binding sites once KRAS is bound to GTP.[17] Glycine substitution with cysteine at codon 12 known as KRAS G12C is the most frequent mutation.[17] Such perspective changed with FDA approval of KRAS G12C oral inhibitor, sotorasib in 2021.[18] Currently, the administration of sotorasib is recommended as subsequent therapy after standard treatment such as chemotherapy, immunotherapy in both NCCN and ESMO guideline.[19] In 2022, adagrasib received accelerated FDA approval diversifying choice of KRAS G12C inhibitor.[20] Both sotorasib and adagrasib work based on similar mode of action therefore, NCCN does not recommend switch.[19] NCCN guideline categorized both inhibitors as 2A. However, in ESMO guideline, sotorasib is categorized as tier 1 but adagrasib falls under tier 3, hypothetical target.[7, 19] 

3.The latest FDA approval status of molecular diagnostic using liquid biopsy 

Using an appropriate companion diagnostic is a prerequisite for successful targeted therapy. Molecular diagnostic is one of the widely used and approved tools of companion diagnostics techniques. Tissue is the preferred source for molecular diagnostic but liquid biopsy is used as a complementary specimen to offset the weakness of tissue. As of current date, over 154 companion diagnostics have been FDA approved.[21]

The approved status is either De Novo pathway and mostly premarket approval (PMA). Among 154 diagnostic kits, 17 molecular diagnostic kits are applicable for liquid biopsy. 12 molecular diagnostic kits are for EGFR detection approved for different types of EGFR-TKIs:gefitinib, erlotinib, afatinib, osimertinib. 2 molecular diagnostic kits are approved for KRAS G12C detection while each kit is approved for sotorasib and adagrasib, respectively. There are no FDA approved real time PCR kits yet for KRAS, ALK, ERBB2, and MET, only approved with next generation sequencing. For ALK detection, currently FISH is preferred tools for detection while some are detected through NGS.

3-a.Moving from late stage diagnosis to early screening: Status quo of MRD test and what to expect in 2023 

Liquid biopsy opened an opportunity for early-stage cancer screening. By using genetic information contained in DNA fragments called ctDNA, the detection of cancer became more convenient. There have been various efforts to use minimal residual disease (MRD) testing for the purpose of early screening, monitoring response after neoadjuvant or adjuvant therapy.

The MRD test has been gaining attention from the FDA. The FDA started the FDA breakthrough designation program to facilitate regulatory approval. In 2021, 3 MRD tests were granted FDA breakthrough designation and there are more MRD tests which achieved FDA breakthrough designation in 2023.[22,23] MRD test mainly utilizes circulating tumor DNA but its target is expanding and in search of better to increase sensitivity.[23]

In contrast to FDA’s effort to facilitate regulatory process, there is no MRD test that has received FDA approval yet for the purpose of early or advanced cancer detection. Currently, MRD tests can only be tested at CLIA-certified laboratories. In terms of reimbursement, MRD test received local coverage determination but this is only for advanced stage cancer not yet for early-stage cancer.[24] There is high interest in development but for MRD tests to be implemented in the early-stage cancer, further support and flexibility from both reimbursement and regulatory authorities may be necessary to establish the MRD test as part of clinical routine practice. 

3-b.Multi-Cancer Early Detection, game changer or Pandora's box?

If tissue specimens are used, detecting different types of cancer at once requires collection of different specimens from different tissues or organs which may be impossible in real clinical settings. Use of ctDNA is changing this landscape and MCED is trying to utilize its strength for multi screening by overcoming low levels of ctDNA in early-stage cancer.

Compared to the MRD test, the evidence is still lacking but methylation is one of the well-studied epigenetic modifications that is known to hold information on tumors.[25] DNA methylation is the process of addition of methyl group to cytosine at fifth carbon.[25] ctDNA methylation shows relatively stable alterations compared to somatic mutation which makes it an ideal source for early-stage cancer detection.[26]

Bisulfite sequencing is a conventional way to detect the methylation pattern. Bisulfite does not convert 5-methylcytosine to uracil, while cytosine is converted to uracil.[25] Such differences in patterns are analyzed using artificial intelligence. However, bisulfite sequencing has drawbacks as it cleaves the DNA fragments which may be critical to early-stage cancer whose ctDNA volume is likely to be low. MCED based on ELISA-seq which does not cause damage to DNA has received FDA breakthrough designation in 2023.[27] Innovation is ongoing but there is still concern with false positives and controversies on whether expanding medicare for MCED is beneficial for the healthcare community. [28]

Conclusion

Targeted therapy for advanced stage cancer is becoming more intricate and systematic. Existing biomarkers are more updated with appropriate TKIs based on response while new emerging biomarkers are gaining evidence to be established as part of guidelines. Many laboratories and companies are trying to surpass the limit and accomplish early-stage cancer detection. Diverse new methods are developed and accumulating evidence to be used for early detection, multicancer screening, and facilitate targeted therapy for late-stage cancer.

Fundamentally, liquid biopsy technology needs to find a way to increase sensitivity and specificity. Genecast is currently focusing on developing ADPSTM smart DNA polymerase which is used in real time polymerase chain reaction for targeted therapy against advanced cancer. This technology is cost-effective at 1/10 of a NGS cost with sensitivity reaching 0.0001%.

In addition to technology advancement, regulatory matters and practical reimbursement policy needs to be followed accordingly for successful implementation of liquid biopsy to outweigh the benefit over risk in aiding cancer patients for the right treatment. 

Reference                                                 

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19. 18. FDA/CEDR resources page. Food and Drug Administration Website. FDA grants accelerated approval to sotorasib for KRAS G12C mutated NSCLC. https://www.fda.gov/drugs/resources-information-approved-drugs/fda-grants-accelerated-approval-sotorasib-kras-g12c-mutated-nsclc
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21. 20. FDA/CEDR resources page. Food and Drug Administration Website. FDA grants accelerated approval to adagrasib for KRAS G12C mutated NSCLC. https://www.fda.gov/drugs/resources-information-approved-drugs/fda-grants-accelerated-approval-adagrasib-kras-g12c-mutated-nsclc
22. 21. FDA/CEDR resources page. List of Cleared or Approved Companion Diagnostic Devices (In Vitro and Imaging Tools) https://www.fda.gov/medical-devices/in-vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-in-vitro-and-imaging-tools
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https://www.cms.gov/Medicare-Coverage-Database/view/lcd.aspx?LCDId=38835

1. 25. Luo, H., et al. (2021). Liquid Biopsy of Methylation Biomarkers in Cell-Free DNA. Trends in Molecular Medicine, 27(5):482–500.
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3. 27. LabPulse.com. Burning Rock nabs FDA breakthrough device designation for multicancer detection blood test. https://www.labpulse.com/diagnostic-technologies/molecular-diagnostics/liquid-biopsy/article/15305013/burning-rock-nabs-fda-breakthrough-device-designation-for-multicancer-detection-blood-test
4. 28. CMS Repeals MCIT/R&N Rule; Will Consider Other Coverage Pathways to Enhance Access to Innovative Medical Devices https://www.cms.gov/newsroom/press-releases/cms-repeals-mcitrn-rule-will-consider-other-coverage-pathways-enhance-access-innovative-medical