PURPOSE:Neoadjuvant immune checkpoint inhibition has shown promise in localized deficient mismatch repair (dMMR) colorectal cancer, yet the optimal treatment duration, regimen, and methods for response evaluation remain undefined. This trial aimed to investigate the efficacy and safety of neoadjuvant pembrolizumab for patients with localized dMMR colon cancer. METHODS:Participants received a single cycle of pembrolizumab (4 mg/kg, max 400 mg, every-6-weeks dosing regimen), followed by a preoperative endoscopy with biopsies and surgery 3-5 weeks later. The primary end point was pathologic complete response (pCR); secondary and exploratory end points included major pathologic response (MPR), safety, survival, and endoscopic response assessment. RESULTS:From February 2023 to March 2024, 85 patients (median age 74 years) were enrolled. All received pembrolizumab; one patient declined surgery. Among 84 patients, pCR was achieved in 44% (37 of 84; 95% CI, 33 to 55), and MPR in 57% (48 of 84; 95% CI, 46 to 68). Two (2%) patients died of complications within 30 days of surgery. At a median follow-up of 18.4 months (IQR, 16.3-21.1), one patient had a recurrence, resulting in overall and disease-free survival rates of 98% and 96%. Grade 3 adverse events occurred in 11% of patients (9 of 85; 95% CI, 3 to 16), with three treatment-related events. In 81 patients, the sensitivity, specificity, and accuracy of the biopsies for predicting pCR were 68%, 75%, and 72%, respectively. In 76 patients, the sensitivity, specificity, and accuracy of the endoscopic images for predicting pCR were 77%, 93%, and 86%, respectively. CONCLUSION:A single cycle of neoadjuvant pembrolizumab led to pCR in nearly half of patients undergoing surgery for localized dMMR colon cancer. The utility of endoscopic evaluation may inform future strategies for patient selection in nonoperative management pathways.

Introduction PRELUCA is a randomised, intervention, non-inferiority study designed to use real-time, longitudinal circulating tumour DNA (ctDNA) measurements to evaluate the efficacy of immunotherapy in patients with advanced non-small cell lung cancer (NSCLC). The primary outcome is overall survival between the two groups: the standard of care group (computer tomography scan evaluation) and intervention group (ctDNA evaluation).Methods and analysis The inclusion and exclusion criteria align with European Society for Medical Oncology treatment guidelines and permit broad inclusion of NSCLC patients, ensuring ‘real-world’ representativeness. The study uses a tumour-informed method, using baseline next generation sequencing analyses to design patient-specific droplet digital PCR assays, which are run with collected blood samples 1 week prior to the intended treatment, enabling real-time evaluation via ctDNA Response Evaluation Criteria in Solid Tumours.Ethics and dissemination Inclusion began in July 2023 and patients are now being actively included in five locations across Denmark. Approval by The Committee on Health Research Ethics of Region Zealand was gained on 4 May 2023.Trial registration number NCT05889247.

Overall survival of patients in the OPSCC database compared to patients included in the project

Figure S3: Copies of HPV DNA per ml plasma in follow-up samples for patients with residual disease. A black dot denotes a cell-free HPV-DNA negative plasma sample, while a red dot denotes a cell-free HPV-DNA positive plasma sample measured by ddPCR. Missing samples are marked in grey. Size and placement of yellow rectangles and yellow lines indicates time and type of treatment. A red line indicates the date of the recurrence. RT: radiotherapy; OP: Operation; Re-OP: Re-resection and re-operation.

Comparing included patients to the OPSCC database

Figure S2: Copies of HPV DNA per ml plasma in the pretreatment sample in follow-up serial samples in patients with an HPV+ follow-up sample but with no clinical or radiological evidence of recurrence or residual disease. Red dots denote an HPV+ sample, black dots denote an HPV- sample measured by ddPCR. Size of yellow RT rectangle indicates time (days) of radiotherapy with curative intent.

HPV VisionArray scans of tumor samples in patients with HPV-negative pre-treatment plasma samples.

INTRODUCTION:Patients with metastatic colorectal cancer (mCRC) respond differently to first-line chemotherapy. Early identification of patients with limited or no clinical benefit could prompt a timelier introduction of second-line therapy and potentially lead to improved overall outcomes. Carcinoembryonic antigen (CEA) is currently the only blood-based marker in clinical use for disease control monitoring in mCRC. Circulating cell-free DNA (cfDNA), including circulating tumor DNA (ctDNA) could become a useful surrogate for oncological outcomes. MATERIALS AND METHODS:Forty patients with RAS-/BRAF-mutated mCRC from the prospective NORDIC-VII trial (NCT00145314) were included. An exploratory model system was made to describe the early on-treatment kinetics of CEA, cfDNA and ctDNA during first-line oxaliplatin-based chemotherapy, and investigate the associations with radiological response, progression-free survival (PFS) and overall survival (OS). RESULTS:Summary metrics were made, representing percentage change from treatment start to time-grid day 7 (P7), day 14 (P14), and day 49 (P49); slope from time-grid day 0 to 7 (S7), day 8 to 14 (S14), and day 15 to 49 (S49); and area under the curve from time-grid day 0 to 49 (AUC). Notably P49 and S49 for ctDNA and CEA were associated with radiological response and/or PFS. The early dynamics of the two markers differed substantially, with faster and more marked changes in ctDNA compared with CEA. Nine patients did not reach complete/near complete molecular ctDNA response close to first evaluation (∼week 8), a state associated with a short PFS (HR 2.72; 95% CI, 1.22-6.06; P = .01) and OS (HR 3.12; 95% CI, 1.35-7.23; P < .01). Contrary, twenty-two patients did not reach radiological response (i.e., complete or partial response) at first evaluation, but this was not associated with PFS (HR 1.21; 95% CI, 0.64-2.30; P = .55) nor OS (HR 1.37; 95% CI, 0.70-2.68; P = .37). CONCLUSION:Early dynamics of ctDNA during first-line oxaliplatin-based chemotherapy hold prognostic value, supporting the idea of prospectively validating a ctDNA-RECIST framework in the early care pathway of mCRC patients. TRIAL REGISTRATION:ClinicalTrials.gov, NCT00145314.

Background and study aims Colorectal cancer is one of the most common malignancies, with approximately 20 % of patients having metastatic disease. Local symptoms from the tumor remain a common issue and affect quality of life. Electroporation is a method to permeabilize cell membranes with high-voltage pulses, allowing increased passage of otherwise poorly permeating substances such as calcium. The aim of this study was to determine the safety of calcium electroporation for advanced colorectal cancer.Patients and methods Six patients with inoperable rectal and sigmoid colon cancer were included, all presenting with local symptoms. Patients were offered endoscopic calcium electroporation and were followed up with endoscopy and computed tomography/magnetic resonance scans. Biopsies and blood samples were collected at baseline and at follow-up, 4, 8, and 12 weeks after treatment. Biopsies were examined for histological changes and immunohistochemically with CD3/CD8 and PD-L1. In addition, blood samples were examined for circulating cell-free DNA (cfDNA).Results A total of 10 procedures were performed and no serious adverse events occurred. Prior to inclusion, patients reported local symptoms, such as bleeding (N = 3), pain (N = 2), and stenosis (N = 5). Five of six patients reported symptom relief. In one patient, also receiving systemic chemotherapy, clinical complete response of primary tumor was seen. Immunohistochemistry found no significant changes in CD3 /CD8 levels or cfDNA levels after treatment.Conclusions This first study of calcium electroporation for colorectal tumors shows that calcium electroporation is a safe and feasible treatment modality for colorectal cancer. It can be performed as an outpatient treatment and may potentially be of great value for fragile patients with limited treatment options.

Patients with non-conclusive scan evaluations

Representativeness of study participants

OS and PFS – substantial/no substantial increase

Before conducting randomized clinical trials in a new research field, feasibility must be tested. We evaluated feasibility of mutational testing on cell-free DNA for selecting primary systemic therapy for patients with metastatic colorectal cancer and for response evaluation during therapy. The setup was tested on 48 patients, proved feasible, and can be applied in randomized clinical trials evaluating clinical utility of circulating tumor DNA.Introduction: Both quantitative and molecular changes in ctDNA can hold important information when treating metastatic colorectal cancer (mCRC), but its clinical utility is yet to be established. Before conducting a large-scale randomized trial, it is essential to test feasibility. This study investigates whether ctDNA is feasible for detecting patients who will benefit from treatment with epidermal growth factor receptor inhibitors and the prognostic value of circulating tumor DNA (ctDNA) response. Materials and methods: Patients with mCRC, who were considered for systemic palliative treatment and were eligible for ctDNA analysis. Mutational testing on cell-free DNA (cfDNA) was done by ddPCR. ctDNA response from baseline to the third treatment cycle was evaluated in patients with detectable ctDNA at baseline. ctDNA maximum response was defined as undetectable ctDNA at the third treatment cycle, ctDNA partial response as any decrease in the ctDNA level, and ctDNA progression as any increase in the ctDNA level. Results: Forty-nine patients were included. The time to test results for mutational testing on cfDNA was significantly shorter than on tumor tissue ( p < .001). Progression-free survival were 11.2 months (reference group), 7.5 months (HR = 10.7, p = .02), and 4.6 months (HR = 11.4, p = .02) in patients with ctDNA maximum response, partial response, and progression, respectively. Overall survival was 31.2 months (reference group), 15.2 months (HR = 4.1, p = .03), and 9.0 months (HR = 2.6, p = .03) in patients with ctDNA maximum response, partial response, and progression, respectivelyConclusion: Pretreatment mutational testing on cfDNA in daily clinic is feasible and can be applied in randomized clinical trials evaluating the clinical utility of ctDNA. Early dynamics in ctDNA during systemic treatment hold prognostic value.

Background: Patients with detectable ctDNA after radical-intent treatment of metastatic spread from colorectal cancer (mCRC) have a very high risk of recurrence, which may be prevented with intensified adjuvant chemotherapy (aCTh). In the OPTIMISE study, we investigate ctDNA-guided aCTh after radical-intent treatment of mCRC. Here we present results from the preplanned interim analysis.Material and methods: The study is an open-label 1:1 randomized clinical trial comparing ctDNA-guided aCTh against standard of care (SOC), with a run-in phase investigating feasibility measures. Key inclusion criteria; radical-intent treatment for mCRC and clinically eligible for triple-agent chemotherapy. Patients underwent a PET-CT scan before randomization. ctDNA analyses of plasma samples were done by ddPCR, detecting CRC-specific mutations and methylation of the NPY gene. In the ctDNA-guided arm, ctDNA positivity led to an escalation strategy with triple-agent chemotherapy, and conversely ctDNA negativity led to a de-escalation strategy by shared-decision making. Patients randomized to the standard arm were treated according to SOC. Feasibility measures for the run-in phase were; the inclusion of 30 patients over 12 months in two Danish hospitals, compliance with randomization >80%, rate of PET-CT-positive findings <20%, and eligibility for triple-agent chemotherapy >80%.Results: Thirty-two patients were included. The rate of PET-CT-positive cases was 22% (n = 7/32). Ninety-seven percent of the patients were randomized. Fourteen patients were randomly assigned to SOC and sixteen to ctDNA-guided adjuvant treatment and follow-up. All analyses of baseline plasma samples in the ctDNA-guided arm passed the quality control, and 19% were ctDNA positive. The median time to result was three working days. All ctDNA-positive patients were eligible for triple-agent chemotherapy.Conclusion: The study was proven to be feasible and continues in the planned large-scale phase II trial. Results from the OPTIMISE study will potentially optimize the adjuvant treatment of patients undergoing radical-intent treatment of mCRC, thereby improving survival and reducing chemotherapy-related toxicity.

AbstractPurpose: To evaluate the accuracy of cell-free human papillomavirus-DNA (cfHPV-DNA) measurements in liquid biopsies in predicting disease in patients with HPV-positive/p16-positive (HPV+/p16+) oropharyngeal squamous cell carcinoma (OPSCC). Experimental Design: This was a prospective cohort study. Plasma samples were collected before treatment, serially after curative intended therapy at follow-up visits 2 weeks, and 6, 9, 12, 18, 24, and 30 months after treatment. A droplet digital PCR assay comprising eight HPV genotypes was used. HPV genotypes found in plasma and tumor tissue were compared. We correlated biopsy- or imaging-verified tumor progression to cfHPV-DNA in follow-up samples. Results: We enrolled 72 patients with HPV+/p16+ OPSCC. Baseline sensitivity for cfHPV-DNA detection was 97.2% (95% confidence interval, 90.3%–99.6%). CfHPV-DNA copy number/milliliter plasma correlated with tumor stage. We found a 100% concordance between HPV genotype in tumor tissue and plasma. Fifty-four patients were followed with serial blood samples for a median of 19.7 months (interquartile range, 13.5–25.5 months). Forty-one patients had undetectable plasma cfHPV-DNA in all follow-up samples, and none developed recurrences. Thirteen patients were classified as cfHPV-DNA–positive in a follow-up plasma sample. Of these, five patients developed a recurrence, and three had residual cancer. It was possible to detect cfHPV-DNA in plasma 97 to 166 days prior to the proven recurrence. Conclusions: To our knowledge, to date, our study, comprising the largest study of patients with HPV+/p16+ OPSCC, using an ultrasensitive multiplex HPV gene panel, revealed a high sensitivity of cfHPV-DNA detection in the liquid biopsies. We recommend serial plasma HPV samples for clinical monitoring of patients with HPV+/p16+ OPSCC.

Patients with detectable ctDNA after radical intent treatment of metastatic spread from colorectal cancer (mCRC) have a very high risk of recurrence, which might be prevented with intensified adjuvant chemotherapy (aCTh). In low-stage CRC, a ctDNA-guided approach for aCTh resulted in less use of chemotherapy without compromising outcome. In OPTIMISE, we investigate ctDNA-guided aCTh after radical intent treatment of mCRC. The preplanned interim analysis aimed at testing feasibility at an early time, with the possibility of optimizing study and laboratory setup and sample size calculations, if needed, before continuing in a large-scale phase II, multicenter, randomized controlled trial. OPTIMISE is an open-label 1:1 randomized trial comparing ctDNA-guided aCTh against standard of care (SOC), with a run-in phase investigating feasibility measures. Analyses of plasma samples for ctDNA were done by ddPCR, detecting CRC-specific mutations and methylation of the NPY gene. Key inclusion criteria were radical intent treatment for mCRC and clinically eligible for triple-agent chemotherapy. All included patients underwent a PET/CT scan before randomization. Patients randomized to the standard arm were treated according to SOC. In the ctDNA-guided arm, ctDNA positivity led to an escalation strategy with triple-agent chemotherapy, and ctDNA negativity led to a de-escalation strategy by shared-decision making. Feasibility measures for the run-in phase were; inclusion of 30 patients over 12 months in two Danish hospitals, compliance with randomization >80%, rate of unexpected PET-CT-positive cases 80%. Thirty-two patients were included. The rate of PET-CT-positive cases was 22% (n=7/32). Five of the PET-CT-positive cases underwent radical intent treatment and hereafter randomization. One patient awaits randomization due to ongoing treatment of residual disease. 97% of the patients were randomized (n=30/31). Fourteen patients were randomly assigned to SOC and sixteen to ctDNA-guided adjuvant treatment and follow-up. All analyses of the baseline plasma samples in the ctDNA-guided arm passed the quality control process. The median time to result was 3 working days. 19% (n=3/16) had a ctDNA-positive baseline plasma sample. All ctDNA-positive patients were eligible for triple-agent chemotherapy. The median time from inclusion until the start of aCTh was 22 days. The delay from inclusion to the start of aCTh was primarily due to the study-specific PET/CT scan. To the best of our knowledge, this is the first randomized clinical trial evaluating ctDNA-guided adjuvant treatment after radical intent treatment for mCRC. Hence, there were significant reservations when planning the study, so it was of utmost importance to test feasibility early. When taking into account the low number of patients and the consequent uncertainty in the estimates, the feasibility measures were complied with. Furthermore, the median time to result and the quality of analyses were adequate. A minor modification of the study flow has been made to reduce the delay from inclusion to the start of aCTh; solely ctDNA-positive patients in the experimental arm will undergo a PET/CT scan. In conclusion, the study has proven feasible and continues with an optimized study flow in the planned large-scale phase II, multicenter, randomized controlled trial.

Abstract Background Various conditions with cellular decay are associated with elevated cell-free DNA (cfDNA). This study aimed to investigate if perioperatively measured cfDNA levels were associated with the surgical approach, complications, or recurrence. Methods Plasma was obtained from patients who underwent surgery for colon cancer at admission and at the time of discharge. Quantitative measurement of cfDNA was performed by amplifying two amplicons of 102 base pairs (bp) and 132 bp of Beta-2-Microglobulin (B2M) and Peptidyl-Prolyl cis–trans Isomerase A (PPIA), respectively. Results cfDNA was measured in 48 patients who underwent surgery for colonic cancer. Sixteen patients had recurrence during the follow-up period, fifteen developed a postoperative complication, and seventeen patients developed neither, acting as the control group. Postoperative cfDNA levels were significantly elevated from baseline samples, across all groups, with a median preoperatively B2M level of 48.3 alleles per mL and postoperatively of 220 alleles per mL and a median preoperatively level PPIA of 26.9 alleles per mL and postoperatively of 111.6 alleles per mL (p < 0.001 for B2M and p < 0.001 for PPIA). Postoperative levels of PPIA, but not B2M, were significantly higher in patients experiencing complications than in the control group (p = 0.036). However, a tendency towards an association between the surgical approach and the changes in cfDNA levels was found for PPIA (p = 0.058), and B2M (p = 0.087). Conclusions Plasma cfDNA was increased after surgery in all patients with colon cancer. Postoperative PPIA levels were significantly higher in patients experiencing surgical complications but not in B2M levels.

The aim was to investigate if mutational analysis of circulating DNA (cfDNA) in plasma is feasible for selection of patients with metastatic colorectal cancer (mCRC) for anti-EGFR treatment. Secondary, to investigate emergence of mutations in cfDNA during therapy, and to compare the fractions of mutations detected by the ddPCR and MassARRAY methods.