Detection of circulating tumor DNA (ctDNA) after resection of stage II colon cancer may identify patients at the highest risk of recurrence and help inform adjuvant treatment decisions. We used massively parallel sequencing–based assays to evaluate the ability of ctDNA to detect minimal residual disease in 1046 plasma samples from a prospective cohort of 230 patients with resected stage II colon cancer. In patients not treated with adjuvant chemotherapy, ctDNA was detected postoperatively in 14 of 178 (7.9%) patients, 11 (79%) of whom had recurred at a median follow-up of 27 months; recurrence occurred in only 16 (9.8 %) of 164 patients with negative ctDNA [hazard ratio (HR), 18; 95% confidence interval (CI), 7.9 to 40; P < 0.001]. In patients treated with chemotherapy, the presence of ctDNA after completion of chemotherapy was also associated with an inferior recurrence-free survival (HR, 11; 95% CI, 1.8 to 68; P = 0.001). ctDNA detection after stage II colon cancer resection provides direct evidence of residual disease and identifies patients at very high risk of recurrence.

ctDNA is detectable in a high proportion of treatment naïve mCRC patients. Early changes in ctDNA during first-line chemotherapy predict the later radiologic response.

IMPORTANCEAdjuvant chemotherapy in patients with stage III colon cancer prevents recurrence by eradicating minimal residual disease. However, which patients remain at high risk of recurrence after completing standard adjuvant treatment cannot currently be determined. Postsurgical circulating tumor DNA (ctDNA) analysis can detect minimal residual disease and is associated with recurrence in colorectal cancers. OBJECTIVE To determine whether serial postsurgical and postchemotherapy ctDNA analysis could provide a real-time indication of adjuvant therapy efficacy in stage III colon cancer. DESIGN, SETTING, AND PARTICIPANTS This multicenter, Australian, population-based cohort biomarker study recruited 100 consecutive patients with newly diagnosed stage III colon cancer planned for 24 weeks of adjuvant chemotherapy from November 1, 2014, through May 31, 2017. Patients with another malignant neoplasm diagnosed within the last 3 years were excluded. Median duration of follow-up was 28.9 months (range, 11.6-46.4 months). Physicians were blinded to ctDNA results. Data were analyzed from December 10, 2018, through June 23, 2019.EXPOSURES Serial plasma samples were collected after surgery and after chemotherapy. Somatic mutations in individual patients' tumors were identified via massively parallel sequencing of 15 genes commonly mutated in colorectal cancer. Personalized assays were designed to quantify ctDNA. MAIN OUTCOMES AND MEASURES Detection of ctDNA and recurrence-free interval (RFI).RESULTS After 4 exclusions, 96 eligible patients were eligible; median patient age was 64 years (range, 26-82 years); 49 (51%) were men. At least 1 somatic mutation was identified in the tumor tissue of all 96 evaluable patients. Circulating tumor DNA was detectable in 20 of 96 (21%) postsurgical samples and was associated with inferior recurrence-free survival (hazard ratio [HR], 3.8; 95% CI, 2.4-21.0; P < .001). Circulating tumor DNA was detectable in 15 of 88 (17%) postchemotherapy samples. The estimated 3-year RFI was 30% when ctDNA was detectable after chemotherapy and 77% when ctDNA was undetectable (HR, 6.8; 95% CI, 11.0-157.0; P < .001). Postsurgical ctDNA status remained independently associated with RFI after adjusting for known clinicopathologic risk factors (HR, 7.5; 95% CI, 3.5-16.1; P < .001). CONCLUSIONS AND RELEVANCEResults suggest that ctDNA analysis after surgery is a promising prognostic marker in stage III colon cancer. Postchemotherapy ctDNA analysis may define a patient subset that remains at high risk of recurrence despite completing standard adjuvant treatment. This high-risk population presents a unique opportunity to explore additional therapeutic approaches.

Postoperative ctDNA analysis stratifies patients with LARC into subsets that are either at very high or at low risk of recurrence, independent of conventional clinicopathological risk factors. ctDNA analysis could potentially be used to guide patient selection for adjuvant chemotherapy.

Purpose: Oncogene mutations contribute to colorectal cancer development. We searched for differences in oncogene mutation profiles between colorectal cancer metastases from different sites and evaluated these as markers for site of relapse.Experimental Design: One hundred colorectal cancer metastases were screened for mutations in 19 oncogenes, and further 61 metastases and 87 matched primary cancers were analyzed for genes with identified mutations. Mutation prevalence was compared between (a) metastases from liver (n ¼ 65), lung (n ¼ 50), and brain (n ¼ 46), (b) metastases and matched primary cancers, and (c) metastases and an independent cohort of primary cancers (n ¼ 604). Mutations differing between metastasis sites were evaluated as markers for site of relapse in 859 patients from the VICTOR trial.Results: In colorectal cancer metastases, mutations were detected in 4 of 19 oncogenes: BRAF (3.1%), KRAS (48.4%), NRAS (6.2%), and PIK3CA (16.1%). KRAS mutation prevalence was significantly higher in lung (62.0%) and brain (56.5%) than in liver metastases (32.3%; P ¼ 0.003). Mutation status was highly concordant between primary cancer and metastasis from the same individual. Compared with independent primary cancers, KRAS mutations were more common in lung and brain metastases (P < 0.005), but similar in liver metastases. Correspondingly, KRAS mutation was associated with lung relapse (HR ¼ 2.1; 95% CI, 1.2 to 3.5, P ¼ 0.007) but not liver relapse in patients from the VICTOR trial.Conclusions: KRAS mutation seems to be associated with metastasis in specific sites, lung and brain, in colorectal cancer patients. Our data highlight the potential of somatic mutations for informing surveillance strategies.

BRAFV600E mutations are found in 10% of colorectal cancers (CRCs). The low frequency of this mutation therefore makes it a challenging target for drug development, unless subsets of patients with higher rates of BRAF V600E can be defined. Knowledge of the concordance between primary-metastasis pairs and the impact of BRAF V600E on outcome would also assist in optimal drug development. We selected primary CRCs from 525 patients (stages I-IV) evenly matched for age (<70 and !70), gender and tumor location (right, left and rectum), and 81 primary-metastasis pairs. BRAF V600E , KRAS mutation and microsatellite instability (MSI) were determined and correlated with clinical features and patient outcomes. In multivariate analyses, increasing patient age (p 5 0.04), female gender (p 5 0.0005) and right-sided tumor location (p < 0.0001) were independently associated with BRAF V600E . The prevalence of BRAF V600E was considerably higher in older (age > 70) females with KRAS wild-type right-sided colon cancers (50%) compared to the unselected cohort (10%). BRAF V600E was associated with inferior overall survival in metastatic CRC (HR 5 2.02; 95% CI 1.26-3.26), particularly evident in patients treated with chemotherapy, and is independent of MSI status. BRAF status was concordant in all primary tumors and matched metastases (79 wild-type pairs and two mutant pairs). Clinicopathological and molecular features can identify CRC patients with a higher prevalence of BRAF V600E . Patients with BRAF V600E wild-type primary tumor do not appear to acquire the mutation in their metastases, and BRAF V600E is associated with poorer outcomes in metastatic patients. Our findings are timely and will help inform the rational development of BRAF V600E inhibitors in CRC.

Purpose: PIK3CA and PTEN mutations are prevalent in colorectal cancer and potential markers of response to mitogen-activated protein/extracellular signal-regulated kinase inhibitors and anti-EGF receptor antibody therapy. Relationships between phosphoinositide 3-kinase (PI3K) pathway mutation, clinicopathologic characteristics, molecular features, and prognosis remain controversial.Experimental Design: A total of 1,093 stage I-IV colorectal cancers were screened for PIK3CA (exons 9 and 20), KRAS (codons 12-13), BRAF (codon 600) mutations, and microsatellite instability (MSI). PTEN (exons 3-8) and CpG island methylator phenotype (CIMP) status were determined in 744 and 489 cases. PIK3CA data were integrated with 17 previous reports (n ¼ 5,594).Results: PIK3CA and PTEN mutations were identified in 11.9% and 5.8% of colorectal cancers. PTEN mutation was associated with proximal tumors, mucinous histology, MSI-high (MSI-H), CIMP-high (CIMP-H), and BRAF mutation (P < 0.02). PIK3CA mutation was related to older age, proximal tumors, mucinous histology, and KRAS mutation (P < 0.04). In integrated cohort analysis, PIK3CA exon 9 and 20 mutations were overrepresented in proximal, CIMP-low (CIMP-L), and KRAS-mutated cancers (P 0.011). Comparing PIK3CA exonic mutants, exon 20 mutation was associated with MSI-H, CIMP-H, and BRAF mutation, and exon 9 mutation was associated with KRAS mutation (P 0.027). Disease-free survival for stage II/III colorectal cancers did not differ by PI3K pathway status.Conclusion: PI3K pathway mutation is prominent in proximal colon cancers, with PIK3CA exon 20 and PTEN mutations associated with features of the sessile-serrated pathway (MSI-H/CIMP-H/BRAF mut ), and PIK3CA exon 9 (and to a lesser extent exon 20) mutation associated with features of the traditional serrated pathway (CIMP-L/KRAS mut ) of tumorigenesis. Our data highlight the PI3K pathway as a therapeutic target in distinct colorectal cancer subtypes.

Purpose: Patients with colorectal cancer with peritoneal metastases (CRPMs) have limited treatment options and the lowest colorectal cancer survival rates. We aimed to determine whether organoid testing could help guide precision treatment for patients with CRPMs, as the clinical utility of prospective, functional drug screening including nonstandard agents is unknown.Experimental Design: CRPM organoids (peritonoids) isolated from patients underwent parallel next-generation sequencing and medium-throughput drug panel testing ex vivo to identify specific drug sensitivities for each patient. We measured the utility of such a service including: success of peritonoid generation, time to cultivate peritonoids, reproducibility of the medium-throughput drug testing, and documented changes to clinical therapy as a result of the testing.Results: Peritonoids were successfully generated and validated from 68% (19/28) of patients undergoing standard care. Genomic and drug profiling was completed within 8 weeks and a formal report ranking drug sensitivities was provided to the medical oncology team upon failure of standard care treatment. This resulted in a treatment change for two patients, one of whom had a partial response despite previously progressing on multiple rounds of standard care chemotherapy. The barrier to implementing this technology in Australia is the need for drug access and funding for off-label indications.Conclusions: Our approach is feasible, reproducible, and can guide novel therapeutic choices in this poor prognosis cohort, where new treatment options are urgently needed. This platform is relevant to many solid organ malignancies.