Purpose: Recent studies have highlighted the existence of subclones in tumors. Lymph nodes are generally the first location of metastasis for most solid epithelial tumors, including colorectal cancer. We sought to understand the genetic origin of lymph node metastasis in colorectal cancer by evaluating the relationship between colorectal cancer subclones present in primary tumors and lymph nodes. Experimental Design: A total of 33 samples from seven colorectal cancers, including two or three spatially disparate regions from each primary tumor and one to four matched lymph nodes for each tumor, underwent next-generation whole-exome DNA sequencing, Affymetrix OncoScan SNP arrays, and targeted deep confirmatory sequencing. We performed mapping between SNPs and copy number events from the primary tumor and matched lymph node samples, allowing us to profile heterogeneity and the mutational origin of lymph node metastases. The computational method PyClone was used to define subclones within each tumor. The method Clonality Inference in Tumors Using Phylogeny (CITUP) was subsequently used to infer phylogenetic relationships among subclones. Results: We found that there was substantial heterogeneity in mutations and copy number changes among all samples from any given patient. For each patient, the primary tumor regions and matched lymph node metastases were each polyclonal, and the clonal populations differed from one lymph node to another. In some patients, the cancer cell populations in a given lymph node originated from multiple distinct regions of a tumor. Conclusions: Our data support a model of lymph node metastatic spread in colorectal cancer whereby metastases originate from multiple waves of seeding from the primary tumor over time. Clin Cancer Res; 24(9); 2214–24. ©2017 AACR. See related commentary by Gerlinger, p. 2032

Colorectal cancer arises in part from the cumulative effects of multiple gene lesions. Recent studies in selected cancer types have revealed significant intra-tumor genetic heterogeneity and highlighted its potential role in disease progression and resistance to therapy. We hypothesized the existence of significant intra-tumor genetic heterogeneity in rectal cancers involving variations in localized somatic mutations and copy number abnormalities. Two or three spatially disparate regions from each of six rectal tumors were dissected and subjected to the next-generation whole-exome DNA sequencing, Oncoscan SNP arrays, and targeted confirmatory sequencing and analysis. The resulting data were integrated to define subclones using SciClone. Mutant-allele tumor heterogeneity (MATH) scores, mutant allele frequency correlation, and mutation percent concordance were calculated, and copy number analysis including measurement of correlation between samples was performed. Somatic mutations profiles in individual cancers were similar to prior studies, with some variants found in previously reported significantly mutated genes and many patient-specific mutations in each tumor. Significant intra-tumor heterogeneity was identified in the spatially disparate regions of individual cancers. All tumors had some heterogeneity but the degree of heterogeneity was quite variable in the samples studied. We found that 67–97% of exonic somatic mutations were shared among all regions of an individual’s tumor. The SciClone computational method identified 2–8 shared and unshared subclones in the spatially disparate areas in each tumor. MATH scores ranged from 7 to 41. Allele frequency correlation scores ranged from R²=0.69–0.96. Measurements of correlation between samples for copy number changes varied from R²=0.74–0.93. All tumors had some heterogeneity, but the degree was highly variable in the samples studied. The occurrence of significant intra-tumor heterogeneity may allow selected tumors to have a genetic reservoir to draw from in their evolutionary response to therapy and other challenges.

BACKGROUND: With improving survival from colorectal cancer, there is a growing population of patients undergoing surveillance. National accreditation organizations have increasingly endorsed formal survivorship care planning. To effectively design patient-centered survivorship programs, an understanding of the prevalence of unmet psychosocial and symptomatic needs is required. OBJECTIVE: The aim of this study is to understand the breadth of unmet needs among survivors of colorectal cancer. DESIGN: This is a cross-sectional survey of patients undergoing surveillance after curative-intent therapy for colorectal cancer. SETTING: This study was conducted June 2017 to January 2018 at an academic cancer center. PATIENTS: There were 99 patients (58 with colon cancer, 41 with rectal cancer). MAIN OUTCOME MEASURES: We measured patient-reported unmet needs by using a modification of the Cancer Survivor Unmet Needs instrument, within domains of emotional (stress, concerns about recurrence), relationship (fertility, interpersonal), logistical (need for accessible parking, case management), financial, treatment-related (neuropathy, bowel function), and surveillance-related needs. RESULTS: The mean (±SD) age was 58 (±12), and the time from diagnosis was 34 (±18) months. Overall, 74% of patients reported at least one unmet need, 49% reported emotional needs, 24% relationship needs, 24% financial needs, 25% logistical needs, and 33% surveillance needs. Thirty-six (62%) patients with colon cancer and 37 (90%) patients with rectal cancer reported at least one ongoing problem (p = 0.002). Thirty-five (82%) patients with rectal cancer reported an unmet treatment-related need in comparison with 23 (40%) patients with colon cancer (p < 0.001). The median (interquartile range) number of ongoing needs were 1 (0–5) in patients with colon cancer and 4 (2–8) in patients with rectal cancer (p = 0.007). LIMITATIONS: This study was limited by its small sample size and lack of generalizability, given the tertiary care setting. CONCLUSIONS: The majority of colorectal cancer survivors reported unmet needs years after completion of curative-intent therapy. Patients with rectal cancer were significantly more likely to have unmet needs and may benefit from additional care during survivorship. Colorectal cancer survivorship programs should incorporate psychosocial and symptomatic care in addition to cancer surveillance. See Video Abstract at http://links.lww.com/DCR/A885.

Familial adenomatous polyposis (FAP) is often due to adenomatous polyposis coli (APC) gene germline mutations. Somatic APC defects are found in about 80% of colorectal cancers (CRCs) and adenomas. Rapamycin inhibits mammalian target of rapamycin (mTOR) protein, which is often expressed in human adenomas and CRCs. We sought to assess the effects of rapamycin in a mouse polyposis model in which both Apc alleles were conditionally inactivated in colon epithelium. Two days after inactivating Apc, mice were given rapamycin or vehicle in cycles of two weeks on and two weeks off. Polyps were scored endoscopically. Mice were euthanized at time points or when moribund, and tissue analyses were performed. In other studies, mice with demonstrable Apc-defective colon polyps were given rapamycin, followed by analysis of their colon tissues. The median survival of mice receiving rapamycin treatment cycles was 21.5 versus 6.5 weeks in control mice (p = 0.03), and rapamycin-treated mice had a significantly lower percentage of their colon covered with polyps (4.3+/− 2 vs 56.5+/− 10.8 percent, p = 0.001). Mice with Apc-deficient colon tissues that developed high grade dysplasia treated with rapamycin underwent treatment for significantly longer than mice treated with vehicle (15.8 vs 5.1 weeks, p = 0.003). In Apc-defective colon tissues, rapamycin treatment was linked to decreased levels of β-catenin and Sox9 at 7 weeks. Other effects of rapamycin in Apc-defectivecolon tissues included decreased proliferation and increased numbers of differentiated goblet cells at 7 weeks. Rapamycin did not affect β-catenin-regulated gene expression in cultured intestinal epithelial cells. Rapamycin has potent inhibitory effects in a mouse colon polyposis model, and mTOR inhibition is linked to decreased proliferation and increased expression of differentiation markers in Apc-mutant colon epithelium and delays development of dysplasia. Our findings highlight the possibility that mTOR inhibitors may have relevance for polyposis inhibition approaches in FAP patients.