胰腺癌在首次切除后具有较高的复发风险，而辅助化疗（AC）能为患者带来一定的治疗效益。为了改善风险分层和/或实时指导AC获益，从而优化临床管理、加速试验进程，我们亟需一种生物标志物。先前的胰腺癌研究揭示，术后检测到ctDNA的患者复发风险显著上升，而AC结束后ctDNA的持续检出也与复发风险增加紧密相关。

研究在澳大利亚的26个医疗中心招募了接受首次切除手术的早期胰腺癌患者。这些患者ECOG评分为0~1，均适宜接受AC。采用基于肿瘤的ctDNA检测技术来识别体细胞突变，以追踪患者体内的游离循环DNA。ctDNA阳性的患者接受为期6个月的AC（具体药物由医生选择，如FOLFIRINOX或吉西他滨/卡培他滨），而ctDNA阴性的患者，在医生评估后，AC治疗时间可缩短至3个月。在AC治疗结束时再次评估患者的ctDNA状态。本研究的主要终点是ctDNA指导疗法的可行性，次要终点则包括探讨ctDNA与临床病理风险因素和生存结果的相关性。

自2019年3月~2023年11月，研究共招募了102例患者，中位年龄为68岁，其中男性占50%，ECOG评分为0~1的患者占95%，72%的患者肿瘤位于胰头。组织学检查显示T1（18%）、T2（50%）和T3（32%）肿瘤患者中71%有淋巴结转移，77%接受了R0切除术。术后CA19-9水平升高的患者占29%。40例（40%）患者在切除术后ctDNA阳性，54例（53%）ctDNA阴性，4例（4%）因肿瘤突变缺失而无结果，因此被视为不合格。

此外，ctDNA的存在与否与已知的临床病理风险因素无关。ctDNA收集的中位时间为5周，而开始AC的中位时间为6周。在54例ctDNA阴性患者中，有24例（44%）缩短治疗时间至计划的3个月AC。中位随访时间为36个月，ctDNA阳性患者的中位无复发生存期（RFS）为13个月，而ctDNA阴性患者的中位无复发生存期为22个月（HR 0.52，P= 0.003）。

DYNAMIC研究的前期结果显示，在Ⅱ期结肠癌（CC）的治疗中，与标准管理相比，采用ctDNA指导的疗法显著减少了辅助化疗（ACT）的使用，同时并未影响患者的2年无复发生存率（RFS）。鉴于MMR状态将Ⅱ期CC患者分为两个不同亚组，我们在此报告了ctDNA负荷、ACT结束时（EOT）ctDNA状态以及包括总生存期（OS）在内的最新生存数据所产生的影响。

DYNAMIC研究是一项多中心、随机的Ⅱ期临床试验。符合条件的患者（pts）需是已接受了Ⅱ期结肠癌（CC）的切除手术，且适合进行ACT的患者。将入组患者以2:1的比例随机分配至ctDNA指导管理组或标准管理组（基于传统标准的临床医生指导）。在ctDNA指导管理组中，若术后4周或7周的基于肿瘤的ctDNA测定结果为阳性，患者将接受基于奥沙利铂或氟嘧啶的ACT；而ctDNA阴性的患者则不接受治疗。在2015年8月至2019年8月期间，共有302例患者接受了ctDNA指导管理，153例患者则接受标准管理。该研究的主要终点是RFS率，其非劣效性界限设为8.5%。此外，预定的关键次要终点包括ACT的使用和OS，另一个次要终点是ctDNA的清除率。

中位随访时间为59.6个月，采用ctDNA指导管理和标准管理的患者5年RFS分别为88%和87%（差异为1.1%，95%CI：-5.8%~8.0%）。ctDNA指导管理组5年OS为93.8%，标准管理组为93.3%（HR 1.05，95%CI：0.47~2.37，P=0.887）。经过治疗的ctDNA阳性患者对比未治疗的ctDNA阴性患者的5年OS显著较差（85.6% vs. 95.3%，HR 3.30，95%CI：1.02~9.05，P=0.014）。ctDNA阴性的T3和T4分期的患者5年OS分别为96.0%和90.6%（HR 2.45，95%CI：0.65~9.25，P=0.171）。

对于接受治疗的ctDNA阳性患者，在EOT观察到87.5%的患者ctDNA清除。EOT时ctDNA清除患者与ctDNA持续存在的患者5年RFS分别为85.2%和20.0%（HR 15.4，95%CI：3.91~61.0，P<0.001）。突变肿瘤分子（MTM /ml）≥ 0.38（中位数）的患者与< 0.38 MTM /ml的患者相比，ctDNA清除率较低（75% vs. 100%，P=0.047）且RFS较差（5年RFS：58.9% vs. 95.2%，HR 10.62，P= 0.005）。在59例错配修复缺陷（dMMR）和235例错配修复正常（pMMR）患者中，分别有5/59（8.5%）和40/235（17%）检测到术后ctDNA。在探索性分析中，dMMR和pMMR病例中分别有3/4（75%）和32/36（89%）观察到ctDNA清除。

肛门癌在美国的发病率持续上升，已有充分研究证实，超过90%的肛门癌病例与HPV感染密切相关。当前，针对局部肛门癌的标准治疗手段为同步放化疗（cRT）。尽管在其他实体瘤中，术后数周内ctDNA水平与复发风险存在关联，但针对HPV相关的肛门生殖器癌症，在cRT治疗后，尚无法明确ctDNA作为预后生物标志物的最佳检测时间点。因此，本研究采用了一种新型、高灵敏度的HPV ctDNA检测方法，旨在评估接受cRT治疗的局部肛门癌患者临床结果与HPV ctDNA存在状态之间的关联。

根据IRB（机构审查委员会）批准的协议，研究者从在本院接受cRT治疗的Ⅰ~Ⅲ期肛门癌患者身上，在治疗前、治疗结束时（第5周）以及治疗后第3、6、9和12个月，分别分离出ctDNA。采用微滴式数字PCR检测HPV ctDNA，对13种致癌型HPV的E6和E7致癌基因进行评估，以量化HPV ctDNA。设定HPV ctDNA检测限为16 HPV拷贝 /ml。通过Kaplan-Meier方法估算HPV ctDNA状态与中位RFS的关系，并使用对数秩检验进行比较。采用卡方检验评估选定临床因素与复发之间的关系，以单侧P<0.05为显著性水平。

在接受cRT后，第5周和治疗后第3、6、12个月，分别在9/41（22%）、4/30（13%）、2/20（10%）和0/12（0%）的患者中检测到HPV ctDNA。与HPV ctDNA阴性状态相比，cRT后3个月检测到HPV ctDNA与临床复发（100% vs. 8%，OR 88，95%CI：4~2000，P=0.006）和较差的RFS（5.9个月 vs. 未达到，HR 24，95%CI：1.2~475，P<0.001）相关。

在第5周治疗结束时，根据HPV ctDNA状态观察到的RFS差异并不显著（两者中位RFS均未达到，HR 2.4，95%CI：5~10，P=0.15）。

在治疗后第3个月，HPV ctDNA检测对复发的敏感性和特异性分别为57%和100%，阳性预测值（PPV）和阴性预测值（NPV）分别为100%和89%。基线临床分期、T分期、N分期、年龄和性别与cRT后的临床复发无关（所有P>0.25）。

通过这种新型且高度灵敏的检测方法，我们发现在cRT（化学放射治疗）后3个月检测到的HPV ctDNA与RFS之间存在显著相关性。此外，在这项回顾性系列研究中，HPV ctDNA的阳性状态在预测cRT后肛门癌复发方面展现出了比基线临床特征更为优越的预测能力。因此，未来的临床试验应当考虑纳入治疗后3个月的时间点作为评估节点，以便基于HPV ctDNA的状态来精准识别HPV阳性肛门癌患者中复发风险较高的个体。我们机构即将启动一项临床试验，旨在评估抗PD-L1与抗TIGIT免疫疗法在cRT后HPV ctDNA呈阳性的HPV相关癌症患者中的疗效。摘要原文

（一）The potential role of serial circulating tumor DNA (ctDNA) testing after upfront surgery to guide adjuvant chemotherapy for early stage pancreatic cancer: The AGITG DYNAMIC-Pancreas trial.

Background:Recurrence rates following upfront resection of pancreatic adenocarcinoma are high, with some benefit from adjuvant chemotherapy (AC). A biomarker that improves risk stratification and/or provides real time indication of AC benefit could improve routine clinical management and accelerate trial progress. Previous studies in pancreas cancer suggest that patients with detectable ctDNA post surgery are at an elevated risk of recurrence. Detectable ctDNA at the completion of AC may also be associated with an elevated recurrence risk.

Methods:Patients with early stage pancreatic adenocarcinoma were enrolled following upfront resection at 26 Australian centres. Patients were ECOG 0-1 and fit for AC. A tumour-informed ctDNA assay was used to identify somatic mutations for tracking in circulating cell-free DNA. ctDNA+ patients received 6 months of AC (FOLFIRINOX or gemcitabine/capecitabine selected at the clinician's discretion), while ctDNA- patients could de-escalate to 3 months at the clinician’s discretion. ctDNA was assessed again at the end of AC. The primary study endpoint was the feasibility of ctDNA-guided therapy. Secondary endpoints included the association of ctDNA with clinicopathologic risk factors and survival outcomes.

Results:A total of 102 patients were enrolled from March 2019 to Nov 2023. Median age was 68 years (range 41 – 86), with 50% male and 95% ECOG 0-1. Tumours were located in the head of pancreas in 72%. Histology revealed T1 (18%), T2 (50%), and T3 (32%) tumors, with nodal involvement in 71%, and an R0 resection in 77%. Post-operative Ca19-9 was elevated in 29%. Forty patients (40%) were ctDNA+ve post resection, 54 (53%) were ctDNA-ve, and no result was obtained in 4 (4%) due to the absence of tumor mutation, and as a result, they were considered ineligible. The presence or absence of ctDNA was not associated with known clinicopathologic risk factors. Median time to ctDNA collection was 5 weeks (range 3 – 9) and to commencing AC was 6 weeks (range 4-12). Of 54 ctDNA-ve patients, 24 (44%) were de-escalated to receive a planned 3 months of AC. With a median follow-up of 36 months (range 2-56) the median recurrence free survival (RFS) in ctDNA+ve patients was 13 months compared to 22 months for ctDNA-ve patients (HR 0.52, p = 0.003).

Conclusions:A tumor informed ctDNA approach to AC selection is feasible for patients undergoing upfront resection of pancreatic adenocarcinoma, with the first blood draw to be scheduled at week 5, allowing time for ctDNA analysis to determine AC selection. A high proportion of patients had detectable ctDNA, which appears independent of known prognostic markers. ctDNA detection was associated with earlier recurrence. Analyses of the impact of changes in ctDNA over time on survival are ongoing. Clinical trial information:?ACTN12618000335291.

（二）Circulating tumor DNA analysis guiding adjuvant therapy in stage II colon cancer: Overall survival and updated 5-year results from the randomized DYNAMIC trial.

Background:Previous results of the DYNAMIC study demonstrated that a ctDNA-guided approach versus standard management in stage II colon cancer (CC) reduced adjuvant chemotherapy (ACT) use without compromising 2-year recurrence-free survival (RFS). MMR status defines two distinct subsets of stage II CC. Here, we report the impact of ctDNA burden, end of ACT (EOT) ctDNA, and updated survival data including overall survival (OS).

Methods:DYNAMIC is a multi-center randomized phase II trial. Eligible patients (pts) had resected stage II CC and were suitable for ACT. Pts were randomly assigned 2:1 to ctDNA-guided management or standard management (clinician-guided based on conventional criteria). For ctDNA-guided management, a ctDNA-positive result at 4 or 7 weeks after surgery with a tumor-informed assay prompted oxaliplatin-based or fluoropyrimidine ACT; ctDNA-negative pts were not treated. Between Aug 2015 and Aug 2019, 302 received ctDNA-guided and 153 standard management. The primary endpoint was RFS, with a non-inferiority margin of 8.5%. Pre-specified key secondary endpoints were ACT use and OS, with an additional secondary endpoint of ctDNA clearance rate.

Results:With a median follow-up of 59.6 months (IQR 55.0–61.5), 5-year RFS were 88% and 87% with ctDNA-guided and standard management, respectively (difference 1.1%, 95% confidence interval, -5.8% to 8.0%). 5-year OS for ctDNA-guided treatment was 93.8% and standard management 93.3% (HR 1.05; 95% CI, 0.47 to 2.37; P = 0.887). 5-year OS was significantly worse in treated ctDNA-positive versus untreated ctDNA-negative pts (85.6% vs 95.3%, HR 3.30; 95% CI, 1.02 to 9.05; P = 0.014). The 5-year OS for ctDNA-negative T3 and T4 disease were 96.0% and 90.6%, respectively (HR 2.45; 95% CI, 0.65 to 9.25; P = 0.171). For treated ctDNA-positive pts, ctDNA clearance was observed at EOT in 35/40 (87.5%). The 5-year RFS for EOT ctDNA clearance vs ctDNA persistence were 85.2% and 20.0%, respectively (HR 15.4; 95% CI, 3.91 to 61.0; P < 0.001). Pts with ≥ 0.38 (the median) mutant tumor molecules (MTM /ml) had a lower ctDNA clearance rate and worse RFS than pts with < 0.38 MTM /ml (ctDNA clearance 75% vs 100%, P = 0.047; 5-year RFS 58.9% vs 95.2%, HR 10.62, P = 0.005). Post-op ctDNA was detected in 5/59 (8.5%) of dMMR and 40/235 (17%) of pMMR cases. In an exploratory analysis, ctDNA clearance was observed in 3/4 (75%) and 32/36 (89%) of dMMR and pMMR cases, respectively.

Conclusions:Mature outcome data confirms the previous finding of non-inferiority of RFS with a ctDNA-guided approach to ACT for stage II CC. For ctDNA-positive pts, the post-surgery mutation burden provides additional prognostic information, as does the EOT ctDNA result. Additional data is needed to define any differential impact of ACT by MMR status. This data supports a role for ctDNA analysis, including serial sampling, in the management of stage II CC. Clinical trial information:?ACTRN12615000381583.

（三）

Time dependency for HPV ctDNA detection as a prognostic biomarker for anal cancer.

Background:Prior HPV infection is associated with > 90% of anal cancers, a malignancy with rising incidence in the United States. Standard treatment for localized anal cancer is concurrent chemoradiation (cRT). While detection of circulating tumor DNA (ctDNA) within weeks after surgery is linked to recurrence in other solid tumors, the optimal time point for ctDNA detection as a prognostic biomarker following cRT for HPV-associated anogenital cancers is not well characterized. We utilized a novel, highly sensitive HPV ctDNA assay to evaluate clinical outcomes according to HPV ctDNA status among patients with localized anal cancer treated with cRT.

Methods:ctDNA was isolated from patients with stages I-III anal cancer treated at our institution with cRT prior to treatment, at the end of treatment (week 5), and at months 3, 6, 9, and 12 after treatment under an IRB-approved protocol. A droplet digital HPV ctDNA PCR assay evaluating HPV E6 and E7 oncogenes for 13 oncogenic HPV types was utilized for quantification of HPV ctDNA. A limit of quantification at 16 HPV copies /ml plasma was set for “HPV ctDNA detection.” Median recurrence-free survival (RFS) according to HPV ctDNA status was estimated via Kaplan-Meier and compared using a log-rank test. Associations between selected clinical factors and recurrence were evaluated with a Chi-square test, with a one-sided p < .05 considered significant.

Results:Following cRT, HPV ctDNA was detected in 9/41 (22%), 4/30 (13%), 2/20 (10%), and 0/12 (0%) patients at week 5 and months 3, 6, and 12, respectively. Detection of HPV ctDNA 3 months after cRT was associated with clinical recurrence (100% versus 8%; odds ratio 88, 95% CI 4-2000; p = .006) and inferior RFS (5.9 months vs not reached (NR); hazard ratio (HR) 24, 95% CI 1.2-475; p < .001) relative to HPV ctDNA-negative status. Differences in RFS according to HPV ctDNA status were not observed at week 5/end of treatment (median RFS NR for both; HR 2.4, 95% CI .5-10; p = .15). At month 3, sensitivity and specificity for recurrence according to HPV ctDNA detection were 57% and 100%, respectively, with a PPV and NPV of 100% and 89%, respectively. Baseline clinical stage, T stage, N stage, age, and gender were not associated with clinical recurrence after cRT (p > .25 for all).

Conclusions:With a novel, highly sensitive assay, detection of HPV ctDNA at 3 months after cRT was associated with RFS. Further, an HPV ctDNA-positive status outperformed baseline clinical features for prognosticating anal cancer recurrence after cRT in this retrospective series. Future clinical trials should incorporate the 3-month post-treatment time point for identification of patients with HPV-positive anal cancer at elevated risk for recurrence according to HPV ctDNA status. A clinical trial evaluating anti-PD-L1 + anti-TIGIT immunotherapy for patients with HPV-associated cancers with detected HPV ctDNA after cRT is forthcoming at our institution.