Abstract
The emergence of multicancer early detection (MCED) tests holds promise for improving early cancer detection and public health outcomes. However, positive MCED test results require confirmation through recommended cancer diagnostic imaging modalities. To address these challenges, we have developed a consultation and work-up protocol for definitive diagnostic results post MCED testing, named SPOT-MAS. Developed through circulating tumor DNA (ctDNA) analysis and in line with professional guidelines and advisory board consensus, this protocol standardizes information to aid general practitioners in accessing, interpreting and managing SPOT-MAS results. Clinical effectiveness is demonstrated through a series of identified cancer cases. Our research indicates that the protocol could empower healthcare professionals to confidently interpret circulating tumor DNA test results for 5 common types of cancer, thereby facilitating the clinical integration of MCED tests.
Keywords: : case reports, consultation, ctDNA, MCED, SPOT-MAS, work-up diagnosis
Plain Language Summary
New tests can now screen for multiple types of cancer early, offering hope for better health outcomes. If one of these tests shows a positive result, doctors need to confirm it with imaging tests. We have developed a guide to help doctors understand and confirm these results. This guide could help healthcare professionals interpret results for five common types of cancer, making it easier to use these tests in regular medical practice.
Plain language summary
Article highlights.
Developing a standard diagnostic follow-up protocol, known as Screening for the Presence Of Tumor by Methylation And Size (SPOT-MAS), to evaluate the performance of our multicancer early detection test and improve the clinical utility of liquid biopsy is essential for its widespread adoption and effectiveness in cancer diagnosis and management.
The standard operating procedure ensures that appropriate steps are taken to achieve diagnostic resolution within a specified timeframe while providing comprehensive support and consultation for patients undergoing ctDNA analysis.
To demonstrate the utility of our standard operating procedure in providing definitive diagnostic results to participants undertaking the SPOT-MAS test, a series of clinical cases were recorded in the K-DETEK study.
This report presents a total of six clinical cases, including five true positive cases confirmed to have tumors at various stages in different organs (liver, lungs, breasts, colon and stomach) and one false positive case.
The protocol ensures that healthcare professionals can confidently provide post-test consultation to SPOT-MAS test-takers across various clinical scenarios, paving the way for the application of the SPOT-MAS test in clinical settings.
1. Introduction
The staggering global impact of cancer on individuals and healthcare systems cannot be overstated. With 19 million new cases and 20 million deaths in 2020 [1], the urgency for effective early detection and screening programs has never been more apparent. Early cancer detection plays an important role in improving treatment efficacy, increasing 5-year survival rate, and alleviating the economic burden of healthcare [2]. Hubbell et al., through modelling, proposed that the implementation of effective cancer screening programs have the potential to reduce the rate of late-stage diagnosis by 78%, the 5-year cancer mortality rate by 39% and the number of cancer deaths by 26% [2]. These statistics underscore the critical importance of early detection in the fight against cancer.
Current cancer screening methods are predominantly organ specific, utilizing diagnostic imaging tests with multiple modalities. In clinical practice, 71% of cancer deaths are caused by cancers without recommended screenings [3]. The limitation of current screening methods, such as low sensitivity and specificity, not only lead to missed cancers at intervenable stages – false negatives [4] or unnecessary interventions on cancer-free individuals – false positives [5] but also contribute to patient anxiety [4]. Moreover, the invasive nature of some screening methods can further deter patients from adhering to their recommended screening schedules [6].
Given the limitation of current cancer screening methods, researchers have been investigating alternative approaches to improve early cancer detection and reduce the psychological barriers associated with invasive screenings. One promising avenue of research involves the development of noninvasive liquid biopsies that can detect the presence of cancer biomarkers such as extracellular DNA fragments (cell-free DNA [cfDNA]) [7,8]. By offering a less invasive and more patient-friendly alternative to traditional imaging-based methods, these methods have the potential to revolutionize cancer screening, aligning with current cancer screening recommendations to broaden scope. Furthermore, liquid biopsies are able to detect a wide range of cancer types, resulting in reduced false positives, improved patients outcome, and enhance adherence to screening protocols [9]. Moreover, advancements in machine learning have brought about significant improvement in the accuracy and reliability of cancer screenings [2]. By analyzing complex datasets and identifying subtle patterns, these technologies aim to enhance the sensitivity and specificity of screening methods, ultimately reducing the incidence of false negatives and false positives.
Several multicancer early detection (MCED) methods have completed case–control research and clinical validation to ensure their effectiveness. The PATHFINDER study (ClinicalTrials.gov identifier: NCT04241796), which assessed the Galleri test, obtained a sensitivity of 51.5%, specificity of 99.5%, a positive predictive value (PPV) of 38.0%, a negative predictive value (NPV) of 98.6%. The Galleri test was able to predict where in the body the cancer signal originates (tumor-of-origin [TOO]) with an accuracy ranging from 87.0 to 100.0% [10,11]. Notably, the Galleri test has recently been clinically validated in symptomatic populations through the SYMPLIFY study (ISRCTN registry identifier: ISRCTN10226380), demonstrated a particularly high PPV of 75.5% and NPV of 97.6% [12]. Despite promising results, MCED methods demonstrated low sensitivity for detecting certain cancers (e.g., breast cancer) and early-stage tumors owing to low amount and high heterogeneity of ctDNA [13–15]. To improve the detection sensitivity of ctDNA, MCED screening methods tend to use high-depth sequencing, making it economically impractical for population-wide screening [7,13]. To address these limitations, we have recently developed a multimodal method, known as Screening for the Presence Of Tumor by Methylation And Size (SPOT-MAS). We further launched a prospective cohort study, K-DETEK (ClinicalTrials.gov identifier NCT05227261) to validate our previously developed multimodal liquid biopsy–based assay, SPOT-MAS. The study recruited 9,057 asymptomatic participants aged 40 years or older across 75 hospitals and one research institute in Vietnam from April 2022 to April 2023. All participants had SPOT-MAS testing and received results within a 30-day period following blood collection. SPOT-MAS provides two types of test results: “ctDNA signal not detected” (negative) or “ctDNA signal detected” (positive), with up to two prediction results for TOO. The participants with a positive result were consulted by physicians to undertake diagnostic imaging tests according to the TOO probability values. All participants were followed up after 6 months and 12 months to obtain information on possible cancer diagnosis. Our study showed that SPOT-MAS test achieved a sensitivity of 78.1%, specificity of 99.8%, a PPV of 58.1%, a NPV of 99.9% and predicted TOO accuracy of 84.0% [16]. These promising results demonstrate the potential of MCED tests in cancer screening and significantly improving early cancer detection prospects.
The current state of liquid biopsy research has made remarkable progress in term of accuracy. However, the translation of this research into clinical application is hindered by certain obstacles. One of the main challenges is that the nature of the test is primary for screening purposes rather than for diagnostic use. Positive ctDNA analysis results need confirmation through recommended cancer diagnostic imaging methods. In addition, post-test counseling and monitoring has revealed emerging situations that present contradictions between the results of ctDNA analysis and diagnostic imaging results [16]. These contradictions complicate the interpretation of results and raise questions about the reliability of liquid biopsy as a standard tool for diagnostic test in clinical practice. Developing a standard diagnostic follow-up protocol to evaluate the performance of an MCED test and improve the clinical utility of liquid biopsy will be esstential to its widespread adoption and effectiveness in cancer diagnosis and management.
Here, we develop a consultation protocol to achieve diagnosis resolution for those who undertake SPOT-MAS to screen five common types of cancer and to evaluate the clinical performance of SPOT-MAS. To demonstrate the feasibility of this protocol, we present a series of cases with both concordant and discordant results between SPOT-MAS and confirmatory diagnosis test. This will provide valuable insights into the practical application of this consultation and work-up protocol and its overall impact on clinical decision making.
2. Materials & methods
2.1. Study design
Clinical cases recorded in the K-DETEK study, officially registered with the US National Institutes of Health (ClinicalTrials.gov identifier: NCT05227261). The study underwent thorough review and approval by the institutional ethics and scientific committee of the University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam (approval number: 192/HĐĐĐ-ĐHYD).
2.2. SPOT-MAS assay
The SPOT-MAS assay methodology is detailed in a previous study [17]. In summary, the SPOT-MAS workflow involves three main steps. First, cfDNA is isolated from 10 ml of peripheral blood and then subjected to bisulfite conversion and adapter ligation to generate a whole-genome bisulfite cfDNA library. Next, the library undergoes hybridization with probes targeting 450 specific regions to capture the target fraction, while the whole-genome fraction is collected from the ‘flow-through’ and hybridized with probes specific to the adapter sequences. Both fractions are then analyzed using massive parallel sequencing. The resulting data are pre-processed into five distinct cfDNA features: target methylation, genome-wide methylation, fragment length profile, DNA copy number and End Motif. These features are input into a two-stage predictive model. In Stage 1, a stacked ensemble machine learning model is employed for binary classification of cancer versus healthy samples. Samples predicted as cancer are then passed to Stage 2, where a Graph Convolutional Neural Network (GCNN) is used to predict the tissue of origin.
2.3. Standard operating procedure (SOP)
SOP is proposed by the study team, including the principal investigator, subinvestigators and other study personnel (excluding the study sponsor), and tailored to each group of ctDNA result to facilitate diagnostic resolution during the 12-month follow-up period. All research team members at hospitals/institutions participating in the K-DETEK study are thoroughly trained for this SOP. The clinical care pathway is summarized by process diagrams outlining the consultation, support, and suggested diagnostic tests based on the standard of care and the SPOT-MAS results (Figure 1A) This approach ensures that the appropriate steps are taken to achieve diagnostic resolution within the specified timeframe while providing comprehensive support and consultation for the patients undergoing ctDNA analysis.
Figure 1.
The process of SPOT-MAS assay (A) Standard operating procedure for positive, and negative results (B) Clinical case series.
It is essential to acknowledge that SPOT-MAS is a screening test and not as a substitute for diagnostic cancer tests. Therefore, a positive result requires consultation from an oncologist or genetic specialist and confirmation through appropriate imaging diagnostics and biopsy methods. Consultations with both a genetic specialist and an oncologist are pivotal, as this helps participants correctly and comprehensively understand the results report and navigate the subsequent process of cancer diagnosis and treatment. First, clinicians advise test-takers with positive ctDNA signal and TOO prediction to conduct “on-site” diagnostic imaging tests which are suggested by The National Comprehensive Cancer Network to confirm the presence of tumor. Detailed procedures for confirming positive results for each cancer type are outlined in supplementary figures (Supplementary Figures S1–S5). A diagnostic biopsy becomes necessary to assess histopathological characteristics if imaging tests reveal suspected malignant lesions. The definitive diagnosis of cancer relies on confirmation from histopathological results. In scenarios where histopathological results fail to confirm cancer or imaging tests do not detect suspicious lesions, several possibilities should be considered: the imaging modality may lack sufficient resolution; the lesions may be too small to be detectable through imaging; incorrect prediction of the primary organ in cancer patients; false positive results due to the fact that the test specificity is 99.8% [16]. Considering these possibilities, we advise test-takers to undergo a repeat ctDNA analysis after 6 months. If the analysis continues to detect ctDNA signals, it is recommended that the individual undergo a whole-body CT scan (breast MRI included for females) to rule out the possibility of cancer lesions in organs beyond the current scope of the test. In cases where no suspicious lesions are found or suspected lesions yield inconclusive results with negative biopsy outcomes, regular surveillance (annual screening) is recommended.
A negative result of SPOT-MAS test indicates the absence of ctDNA signals. However, it is important to note that a negative result does not completely rule out the presence of cancer originating from organs outside the test’s scope or located in positions challenging for ctDNA release [18]. Given the test achieved a 12 month NPV of 99.9%, it is recommended to use this test as a supplementary screening tool and not as a substitute for conventional cancer screening methods [17]. Therefore, individuals should continue their routine health check-ups and cancer screening to ensure comprehensive health monitoring.
3. Results
To demonstrate the utility of our SOP in providing definitive diagnostic results to participants undertaking SPOT-MAS test, we present a series of clinical cases recorded in the K-DETEK study. Here, we report a total of six clinical cases across six participating hospitals, including five true positive cases who were confirmed to have tumors at varied stages in different organs including liver, lungs, breasts, colon and stomach (Patient 1–5, Figure 1B) and one false positive case (Patient 6, Figure 1B). The selection of six clinical cases in this manuscript illustrates the true positive and false positive scenarios during our study.
3.1. Confirmation of precancerous & malignant tumors in true positive cases post SPOT-MAS test
In this section, we presented five true positive cases to illustrate the efficiency of our SOP in confirming the precancerous and cancerous lesions in ctDNA positive cases, while also validating the prediction of tissue of origin for early cancer stages. These cases provide compelling evidence of the accuracy and reliability of our SOP in identifying and confirming the presence of cancer in individuals with positive ctDNA findings.
3.1.1. Patient 1: hepatocellular carcinoma stage IB (T1bN0M0)
A 60-year-old male patient with no personal or familial cancer history presented with a positive SPOT-MAS result, indicating the potential presence of ctDNA originating from the liver and colorectum. We advised the paticipants to perform diagnostic imaging tests for both organs simultaneously to save time and quickly obtain diagnostic resolution. Subsequent triple-phase abdominal CT scans revealed a 3.3 cm lesion in segment VIII, raising suspicions of hepatocellular carcinoma (HCC). After careful review and discussion of the findings, the patient underwent treatment with Transarterial chemoembolization (TACE) combined with Radiofrequency ablation (RFA). This nonsurgical approach has demonstrated favorable efficacy and safety outcomes, particularly in early-stage tumors. As part of the post-treatment follow-up protocol, the patient was scheduled for a 12-month assessment from the study baseline to evaluate treatment response. This case report exemplifies a true positive detection of ctDNA signals associated with stage IB HCC (refer to Figure 2).
Figure 2.
True positive case with stage IB hepatocellular carcinoma.
3.1.2. Patient 2: ductal mammary carcinoma stage II (T2N0M0)
A 68-year-old female patient was diagnosed with a positive SPOT-MAS result, indicating the primary origin of the tumor in the breast. Subsequent mammography identified suspected right breast cancer. Fine-needle aspiration (FNA) results confirmed the presence of ductal mammary carcinoma. Consequently, the patient underwent surgical intervention for tumor removal (refer to Supplementary Figure S6).
3.1.3. Patient 3: bronchial adenocarcinoma, stage IIB (T3N0M0)
A 54-year-old female patient, with a history of cervical cancer 5 years prior and a sister diagnosed with lung cancer, tested positive for the primary origin of the tumor in the breast and lung. Upon consultation, the patient was recommended to undergo a comprehensive diagnostic workup, including breast ultrasound, mammography and contrast-enhanced chest CT scan. However, she consented only to breast ultrasound and non-contrast-enhanced chest CT scan. The breast ultrasound identified a Breast Imaging Reporting & Data System 3 (BI-RADS 3) lesion in the left breast, while the CT scan revealed a 2.2 cm lesion in the left lung with indistinct characteristics. Subsequently, the patient was referred to a specialized oncology hospital for further evaluation. A subsequent PET-CT scan confirmed the presence of a 1.6 × 2.3 cm lesion in the S10 segment of the left lung, exhibiting increased FDG uptake, raising suspicion of malignancy. Following this, the patient underwent laparoscopic surgery for the resection of the affected lung lobe with lymph node dissection. Postoperative pathological examination confirmed the presence of bronchial adenocarcinoma (refer to Figure 3).
Figure 3.
True positive case with stage IIB bronchial adenocarcinoma.
3.1.4. Patient 4: gastric antrum cancer stage IIIA (T4aN2M0)
A 64-year-old male patient tested positive in the SPOT-MAS assay, predicting the primary origin of the tumor in the stomach and lung. Subsequently, the patient received recommendations to undergo diagnostic assessments, including upper endoscopy and contrast-enhanced chest CT scan. Upper endoscopy revealed suspicious lesions in the antrum leading to pyloric stenosis, and biopsy results suggested features consistent with poorly differentiated signet ring cell adenocarcinoma. Further evaluation through contrast-enhanced abdominal-chest CT scan indicated potential gastric antrum cancer, with no observable lesions in the chest. Consequently, the patient underwent total gastrectomy and D2 lymph node dissection. Postoperative biopsy results confirmed the presence of lymph node metastasis of adenocarcinoma (refer to Supplementary Figure S7).
3.1.5. Patient 5: a high-grade dysplastic tubular adenoma
On the same day, a 73-year-old female patient with no history of cancer and no family history related to cancer underwent SPOT-MAS sample collection and a colorectal endoscopy. The endoscopic examination revealed no abnormalities. However, after 30 days, the patient received a positive SPOT-MAS result indicating the potential origin of the tumor in both the colorectal and gastric regions. Consequently, the patient was advised to perform further diagnostic procedures, including gastric and colorectal endoscopies administered by an experienced endoscopy specialist. Subsequent endoscopic evaluations demonstrated congestive inflammation in the gastric antrum and the presence of a wart-like polyp measuring D#15 mm in the mucosal area adjacent to the cecum of the right colon. The polyp was removed, followed by biopsy and pathological analysis confirmed the presence of a high-grade dysplastic tubular adenoma (refer to Figure 4).
Figure 4.
True positive case with high-grade dysplasia tubular adenomas colon polyp.
3.2. Detection of false positive ctDNA signal by SPOT-MAS test
SPOT-MAS test presents significant potential in revolutionizing cancer screening, offering non-invasiveness, convenience and high accuracy in multiorgan screening. Despite its considerable advantages in clinical application, it is crucial to recognize the technical limitations that exist in cancer screening. One significant challenge is the potential for false positive results in subjects with benign lesions that might exhibit methylation changes similar to cancerous lesions [19]. Here, we show that a false positive case related to benign lesions could be identified by our consultation and work-up diagnosis protocol.
3.2.1. Patient 6: liver hemangioma
A 60-year-old female patient, with a mother’s history of ovarian cancer, received a positive result in the SPOT-MAS assay indicating the primary origin of the tumor in the liver. Subsequent triple-phase abdominal CT imaging revealed the presence of multiple scattered liver lesions, consistent with a diagnosis of hemangioma. Following this finding, the patient was advised to undergo a reassessment of ctDNA analysis after a 6-month interval, which again indicated a positive result for TOO being the liver. Subsequently, the patient was recommended to undergo a comprehensive diagnostic assessment according to our SOP guidelines, including a whole-body CT scan and breast MRI. Importantly, the patient was informed about the potential for false positive test outcomes attributable to the presence of liver hemangioma. Despite recommendations, the patient opted not to proceed with the diagnostic imaging tests. Upon re-evaluation of clinical information after 6 months, no cancer related abnormalities were documented (refer to Figure 5).
Figure 5.
Pseudo ctDNA signal by liver hemangioma.
4. Discussion
The MCED tests have shown promising results through case–control investigations and prospective clinical validation, indicating their potential application in clinical practice as a complementary screening tool for current screening recommendations. The implementation process from research to practical application requires the establishment of consultation-monitoring-guidance protocols tailored to address diverse scenarios encountered post-ctDNA analysis and subsequent diagnostic determinations. It provides flowcharts for individualized approaches to various groups with different ctDNA results and diagnostic outcomes at different follow-up time points. This procedure is essential for the successful integration of MCED tests into clinical practice.
Based on the analysis of ctDNA, our SOP has provided a rational approach for integrating SPOT-MAS results into clinical practice, facilitating comprehension and navigation of subsequent steps for both patients and physician. The clinical cases of patients 1, 2, 3 and 4 illustrated the actual positive results with confirmed I–IIIA staged cancers by diagnostic tests based on our SOP recommendation. These findings strengthen the effectiveness of SPOT-MAS in early-stage detection of multiple cancers, thereby enhancing treatment efficacy and improving patient outcomes. It is estimated that the cancer death rate would be reduced at least 15% if all cancers were diagnosed at earlier stages than stage IV [20,21].
The implementation of our SOP has demonstrated its efficacy in recommending high-resolution diagnostic procedures, such as contrast-enhanced CT scans, in consultation with radiologists and oncologists [22]. This approach has proven to aid in characterizing early-stage lesions in the presented clinical case of patient 3. The clinical case of patient 5 demonstrates the significance of identifying precancerous lesions in asymptomatic individuals to enable proactive intervention in high-risk lesions, a concept referred to as cancer interception [23]. The necessity for repeated colonoscopy under the supervision of an experienced specialist to detect lesions highlights the limitations of traditional endoscopy screening. Despite being recommended for age-based regular screening, traditional endoscopy remains an invasive procedure with variable accuracy influenced by factors such as tumor size, anatomical location and the colonoscopist’s experience [24]. These factors underscore the important role of healthcare professionals in ensuring reliable outcomes.
Moreover, the clinical case of patient 6 demonstrates the necessity of re-analyzing ctDNA after 6 months to guarantee positive signals due to liver hemangioma benign tumor composed of proliferating blood vessels. This condition can induce cellular apoptosis in neighboring hepatocytes, leading to increased release of liver-derived DNA into the circulating plasma DNA pool [19]. These cases serve as valuable illustrations of how our SOP can be effectively applied in clinical settings, demonstrating its relevance in diverse patient populations and disease presentations.
Despite its advancements, our SOP demonstrates certain limitations. First, it focuses exclusively on five prevalent cancer types identified by the SPOT-MAS test, thereby restricting its applicability to less common cancer types. Second, the diagnostic protocols outlined in our SOP are specifically designed for these five cancer types, posing challenges for their adaptation to other MCED tests that evaluate more than five cancer types, such as the Galleri test, which assesses over 50 cancer types. Consequently, our SOP lacks standardization across all MCED tests. Furthermore, while our protocol serves as a proof-of-concept for the development of a diagnostic work-up protocol, a tumor board comprising cancer specialists from specialized hospitals, universities and experts in molecular biology and pathology laboratories is necessary to collaboratively devise a comprehensive protocol tailored to each MCED test.
5. Conclusion
Our report presents an extensive overview of a consultation and work-up protocol alongside a series of clinical cases, to highlight its applicability in providing definitive diagnostic results to participants enrolled in the K-DETEK study. The protocol ensures that healthcare professionals can confidently provide the post-test consultation to SPOT-MAS test-takers across various clinical scenarios, thereby paving the way for the application of SPOT-MAS test in clinical settings.
Supplementary Material
Acknowledgments
We thank all participants who agreed to take part in this study, and all the clinics and hospitals who assisted in patient consultation and sample collection.
Funding Statement
This work was supported by Gene Solutions.
Supplemental material
Supplemental data for this article can be accessed at https://doi.org/10.1080/20565623.2024.2395244
Author contributions
Formal analysis: LHD Nguyen, BL Tieu, TT Nguyen, NP Ha, THH Nguyen, NM Phan. Patient consultancy and screening: LHD Nguyen, BL Tieu, TT Nguyen, NP Ha, THH Nguyen, VH Le, VQ Bui, LH Nguyen, NH Pham, TH Phan, HT Nguyen, VS Tran, CV Bui, VK Vo, PTN Nguyen, HHP Dang, VD Pham, VT Cao, VT Nguyen, TLQ Le, TLA Luong, TKP Doan, TTT Do, CD Phan, TXN Nguyen, NT Pham, BT Nguyen, TTT Pham, HL Le, CT Truong, TX Jasmine, MC Le, VB Phan, QB Truong, THL Tran, MT Huynh, TQ Tran, ST Nguyen, V Tran, VK Tran, HN Nguyen, DS Nguyen, TV Phan, TTT Do, DK Truong, HS Tang. Methodology: TV Phan, TTT Do, DK Truong, MD Phan, H Giang, HN Nguyen, LS Nguyen, DS Nguyen, HS Tang. Conceptualization: HN Nguyen, DS Nguyen, TV Phan, TTT Do, DK Truong, MD Phan, H Giang, HN Nguyen. Writing-original draft: LHD Nguyen, BL Tieu, GTH Nguyen, HS Tang. Writing-Review and Editing: LHD Nguyen, BL Tieu, GTH Nguyen, HST Tang.
Financial disclosure
This work was supported by Gene Solutions.
Competing interests disclosure
LS Tran, HN Nguyen, H Giang, MD Phan, HN Nguyen and DS Nguyen hold equity in Gene Solutions. We confirm that this does not alter our adherence to the journal policies on sharing data and materials. The authors have no other competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript apart from those disclosed.
Writing disclosure
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research
This study was approved by the Ethics Committee of the Medic Medical Center, University of Medicine and Pharmacy and Medical Genetics Institute, Ho Chi Minh city, Vietnam.
Written informed consent was obtained from each participant in accordance with the Declaration of Helsinki.
Data availability statement
The study was registered with the US National Institutes of Health (ClinicalTrials.gov identifier: NCT05227261). The analytical data are available upon reasonable request by email to the corresponding author. Raw sequencing data are not publicly available due to ethical and regulatory restrictions.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The study was registered with the US National Institutes of Health (ClinicalTrials.gov identifier: NCT05227261). The analytical data are available upon reasonable request by email to the corresponding author. Raw sequencing data are not publicly available due to ethical and regulatory restrictions.