Sequential profiling of plasma cell-free DNA (cfDNA) holds immense promise for early detection of patient progression. However, how to exploit the predictive power of cfDNA as a liquid biopsy in the clinic remains unclear. RAS pathway aberrations can be tracked in cfDNA to monitor resistance to anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer. In this prospective phase II clinical trial of single-agent cetuximab in wild-type patients, we combine genomic profiling of serial cfDNA and matched sequential tissue biopsies with imaging and mathematical modeling of cancer evolution. We show that a significant proportion of patients defined as wild-type based on diagnostic tissue analysis harbor aberrations in the RAS pathway in pretreatment cfDNA and, in fact, do not benefit from EGFR inhibition. We demonstrate that primary and acquired resistance to cetuximab are often of polyclonal nature, and these dynamics can be observed in tissue and plasma. Furthermore, evolutionary modeling combined with frequent serial sampling of cfDNA allows prediction of the expected time to treatment failure in individual patients. This study demonstrates how integrating frequently sampled longitudinal liquid biopsies with a mathematical framework of tumor evolution allows individualized quantitative forecasting of progression, providing novel opportunities for adaptive personalized therapies. Liquid biopsies capture spatial and temporal heterogeneity underpinning resistance to anti-EGFR monoclonal antibodies in colorectal cancer. Dense serial sampling is needed to predict the time to treatment failure and generate a window of opportunity for intervention. .

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Purpose: Anti-EGFR mAbs are effective in the treatment of metastatic colorectal cancer (mCRC) patients. RAS status and tumor location (sidedness) are predictive markers of patients' response to anti-EGFR mAbs. Recently, low miR-31-3p expression levels have been correlated with clinical benefit from the anti-EGFR mAb cetuximab. Here, we aimed to validate the predictive power of miR-31-3p in a prospective cohort of chemorefractory mCRC patients treated with single-agent anti-EGFR mAbs. Experimental Design: miR-31-3p was tested by in situ hybridization (ISH) in 91 pretreatment core biopsies from metastatic deposits of 45 patients with mCRC. Sequential tissue biopsies obtained before treatment, at the time of partial response, and at disease progression were tested to monitor changes in miR-31-3p expression overtreatment. miR-31-3p expression, sidedness, and RAS status in pretreatment cell-free DNA were combined in multivariable regression models to assess the predictive value of each variable alone or in combination. Results: Patients with low miR-31-3p expression in pretreatment biopsies showed better overall response rate, as well as better progression-free survival and overall survival, compared to those with high miR-31-3p expression. The prognostic effect of miR-31-3p was independent from age, gender, and sidedness. No significant changes in the expression of miR-31-3p were observed when sequential tissue biopsies were tested in long-term or poor responders to anti-EGFR mAbs. miR-31-3p scores were similar when pretreatment biopsies were compared with treatment-na€ ve archival tissues (often primary colorectal cancer). Conclusions: Our study validates the role of miR-31-3p as potential predictive biomarker of selection for anti-EGFR mAbs.

102 Background: Numerous studies have shown the clinical utility of ctDNA, a non-invasive biomarker to detect MRD and stratify CRC patients who are more likely to relapse. We present an analysis of MRD detection in CRC patients from a prospective multicentre UK study, who were monitored pre- and post-surgery before adjuvant chemotherapy (ACT). Methods: The study recruited patients diagnosed with stage II-III CRC (n=122), including a subset of rectal patients who underwent tri-modality treatment (TMT). All patients had their primary tumor resected and 56% (68/122) received ACT. Paired plasma samples (n=244) were collected before surgery/neaodjuvant chemoradiotherapy and after surgery; median follow-up for survival was 15.48 months (0.16 - 42.1 months). Individual tumors and matched germline DNA were whole-exome sequenced and somatic single nucleotide variants (SNVs) identified. Multiplex PCR assays were designed to track tumor-specific SNVs (Signatera, bespoke mPCR NGS assay) in plasma samples. The study evaluated ctDNA status and clinical outcomes including radiologic imaging. Cox regression was used to calculate recurrence-free survival (RFS) in patients stratified by post-op ctDNA status. Patients were also stratified into low and high-risk groups based on the clinicopathological features. Multivariate analysis was performed with covariates: ctDNA, age, gender, laterality, stage, number of lymph node resected, MSI & TMB. Results: Pre-treatment ctDNA was detected in 93.4% (100/107) of patients. Post-operative ctDNA status prior to ACT was assessed in 107 patients, of whom, 13% (14/107) were MRD-positive (MRDpos). Of the MRDpos patients 42.9% (6/14) eventually relapsed. In contrast, only 8.6% (8/93) of MRD-negative (MRDneg) cases relapsed (HR: 10; 95% CI: 3.3-30; p<0.001). MRD rates stratified by risk features in each of the stages with respective recurrence rates are shown in Table. In stage III patients (n=64), 45.4% (5/11) of the MRDpos patients relapsed, whilst only 17% (9/53) of the MRDneg cases relapsed (HR: 9; 95% CI:2.6-32; p<0.0001). In the multivariate analysis, ctDNA status was the most significant prognostic factor associated with RFS (HR: 28.8, 95% CI: 3.5-234.1; p<0.001). Conclusions: Postoperative ctDNA analysis with tumor informed assay enables detection of CRC patients at high-risk of recurrence. Early detection of MRD could guide ACT decisions in intervention trials and is currently underway in TRACC. Clinical trial information: NCT04050345. [Table: see text]

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Drugs targeting the VEGF (vascular endothelial growth factor) signaling pathway are approved for several malignancies. Unfortunately, VEGF inhibitors lead to hypertension in 30% to 80% patients. Reduced nitric oxide synthase activity, microvascular rarefaction, and increased vascular resistance have been proposed as potential mechanisms. We aimed to assess these mechanisms in patients receiving the VEGF inhibitor, pazopanib, for cancer. Twenty-seven normotensive patients with advanced solid malignancies received pazopanib 800 mg od. Endothelial function was assessed using forearm plethysmography with intraarterial infusions of acetylcholine. Detailed hemodynamic measurements were taken. Density and diameter of the conjunctival and episcleral microvasculature were evaluated using hemoglobin video imaging. Measurements were taken at baseline, 2, and 12 weeks after initiation of pazopanib or earlier if patients became hypertensive. By the end of the trial, systolic blood pressure increased by 12 mm Hg (95% CI, 4–19 mm Hg; P =0.003), diastolic by 10 mm Hg (95% CI, 5–15 mm Hg; P <0.001), and peripheral vascular resistance by 888 dynes×s/cm 5 (95% CI, 616–1168 dynes×s/cm 5 ; P <0.001). Forearm blood flow improved: Ratio of acetylcholine response at end of trial/baseline was 2.8 (95% CI, 1.84–4.25; P <0.001). Microvascular density in the sclera was reduced by −15.5% (95% CI, −25.7% to −5.3%; P =0.003) and diameter by −2.09 µm (95% CI, −3.95 to −0.19 µm; P =0.03). A post hoc colorimetric assay revealed that pazopanib inhibited acetylcholinesterase activity by −56% (95% CI, −62% to −52%; P <0.001). Unexpectedly, pazopanib led to an increase in acetylcholine-mediated forearm blood flow response, likely due to the inhibition of acetylcholinesterase activity. Pazopanib increased peripheral vascular resistance and reduced microvascular density and diameter, suggesting that microvascular rarefaction could be one of the key mechanisms behind VEGF inhibition–induced hypertension. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01392352.

Targeting of RTKs in HER2-negative breast cancer presents a major therapeutic opportunity in breast cancer, although robust selection strategies will be required to identify cancers with activation of specific RTKs if this potential is to be realized.

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