DENEB: Development of new criteria for curability after local excision of pathological T1 colorectal cancer using liquid biopsy

Abstract According to the current international guidelines, high‐risk patients diagnosed with pathological T1 (pT1) colorectal cancer (CRC) who underwent complete local resection but may have risk of developing lymph node metastasis (LNM) are recommended additional intestinal resection with lymph node dissection. However, around 90% of the patients without LNM are exposed to the risk of being overtreated due to the insufficient pathological criteria for risk stratification of LNM. Circulating tumor DNA (ctDNA) is a noninvasive biomarker for molecular residual disease and relapse detection after treatments including surgical and endoscopic resection of solid tumors. The CIRCULATE‐Japan project includes a large‐scale patient‐screening registry of the GALAXY study to track ctDNA status of patients with stage II to IV or recurrent CRC that can be completely resected. Based on the CIRCULATE‐Japan platform, we launched DENEB, a new prospective study, within the GALAXY study for patients with pT1 CRC who underwent complete local resection and were scheduled for additional intestinal resection with lymph node dissection based on the standard pathologic risk stratification criteria for LNM. The aim of this study is to explore the ability of predicting LNM using ctDNA analysis compared with the standard pathological criteria. The ctDNA assay will build new evidence to establish a noninvasive personalized diagnosis in patients, which will facilitate tailored/optimal treatment strategies for CRC patients.


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Abstract According to the current international guidelines, high-risk patients diagnosed with pathological T1 (pT1) colorectal cancer (CRC) who underwent complete local resection but may have risk of developing lymph node metastasis (LNM) are recommended additional intestinal resection with lymph node dissection. However, around 90% of the patients without LNM are exposed to the risk of being overtreated due to the insufficient pathological criteria for risk stratification of LNM. Circulating tumor DNA (ctDNA) is a noninvasive biomarker for molecular residual disease and relapse detection after treatments including surgical and endoscopic resection of solid tumors. The CIRCULATE-Japan project includes a large-scale patient-screening registry of the GALAXY study to track ctDNA status of patients with stage II to IV or recurrent CRC that can be completely resected. Based on the CIRCULATE-Japan platform, we launched DENEB, a new prospective study, within the GALAXY study for patients with pT1 CRC who underwent complete local resection and were scheduled for additional intestinal resection with lymph node dissection based on the standard (pT1) CRC with a negative margin in the US, European, and Japanese guidelines include submucosal invasion (>1000 µm), lymphovascular invasion, high-grade tumor budding, and poorly differentiated histology. [1][2][3] However, the rate of LNM indicated by these guidelines is only 6%-16%. 4 Therefore, around 90% of the patients without LNM are exposed to the risk of being overtreated with a recommendation of additional intestinal resection because of the insufficient/ unclear pathological criteria for risk stratification of LNM. To reduce these unnecessary additional surgical resections, a new high-quality marker for determining the risk of LNM is necessary.
Circulating tumor DNA (ctDNA) is a noninvasive biomarker for molecular residual disease (MRD) and relapse detection after treatments including surgical and endoscopic resection of solid tumors. 5 Signatera™ (Natera, Inc) is an innovative, custom-built ctDNA monitoring assay for MRD detection via a personalized blood test tailored to fit the unique signature of somatic single-nucleotide variants found in the individual's tumor. 6 Signatera has been utilized for ctDNA detection in a prospective, multicenter cohort study for stage I to III CRC, wherein, preoperatively, ctDNA was detected in 108 of 122 samples with a sensitivity of 90% in patients with stage III CRC, and at postoperative day 30, ctDNA-positive patients were observed to be seven times more likely to relapse than ctDNA-negative patients, suggesting ctDNA as an effective biomarker that enables stratification of patients with high risk of recurrence. 6 Furthermore, the utility of ctDNA is currently being evaluated in a large platform called CIRCULATE-Japan project. Circulate-Japan encompasses an observational (GALAXY study) and two randomized phase III trials (VEGA and ALTAIR). 7 Based on the CIRCULATE-Japan platform, we launched DENEB, a new prospective study, within the GALAXY study for patients with pT1 CRC who underwent complete local resection and were scheduled for additional intestinal resection with lymph node dissection based on the standard pathologic risk stratification criteria for LNM. The DENEB study will determine the ability of ctDNA to help predict the risk of LNM in patients diagnosed with pT1 CRC after complete local resection compared with the standard pathological criteria.

| Study design
The DENEB study, in which a total of 200 patients will be enrolled, is a prospectively conducted nationwide registry designed to evaluate the relationship between preoperative ctDNA status and pathological factors, especially LNM for patients with pT1 CRC after complete local resection ( Figure 1). The method of local resection may be pathologic risk stratification criteria for LNM. The aim of this study is to explore the ability of predicting LNM using ctDNA analysis compared with the standard pathological criteria. The ctDNA assay will build new evidence to establish a noninvasive personalized diagnosis in patients, which will facilitate tailored/optimal treatment strategies for CRC patients. Group performance status of 0 or 1. Additionally, patients with histologically confirmed pT1 CRC who underwent complete local resection with a negative margin and have additional surgical resection scheduled based on the pathologic criteria for risk stratification of LNM will be included ( Table 1)

| Endpoints and statistical analyses
The endpoints of this study include concordance rates of diagnosis of ctDNA for the presence or absence of LNM in additional intestinal resection as well as sensitivity, specificity, false positive rate, and false negative rate of ctDNA. These endpoints will be summarized using the proportions and 95% exact (Clopper-Pearson) confidence interval. For the diagnostic concordance rate, a binomial test with a threshold of 80% will be performed at a one-sided significance level of 2.5%.

| DISCUSS ION
The CIRCULATE-Japan project includes a large-scale patientscreening registry of the GALAXY study for patients with clinical stage II to IV or recurrent CRC and two ctDNA-guided phase III trials: VEGA and ALTAIR. 7 The launch of the DENEB study within the GALAXY study, evaluating the ctDNA status of patients with pT1 CRC will help stratify high-risk patients who may benefit from additional treatment/intestinal resection, sparing low-risk patients from being exposed to unnecessary treatment. This will be achieved by examining the relationship between the locally resected specimens, the additional resected specimens, and ctDNA analysis. In the United States, Natera, Inc received Medicare coverage for Signatera TM MRD Test in Stage II-III CRC for its effectiveness in guiding adjuvant therapy decisions, detecting recurrence earlier than standard diagnostic tools, and monitoring treatment response. 6 In the DENEB study, we will further demonstrate the clinical usefulness of ctDNA for detecting MRD after local excision of CRC.