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Supplementary table2 Differentially methylated regions identified by bumphunter

Increasing evidence suggests that left-sided colon cancer (LCC) and right-sided colon cancer (RCC) are emerging as two different colorectal cancer types with distinct clinical characteristics. However, the discrepancy in the underlying molecular event between these types of cancer has not been thoroughly elucidated to date and warrants comprehensive investigation. To this end, an integrated dataset from The Cancer Genome Atlas was used to compare and contrast LCC and RCC, covering mutation, DNA methylation, gene expression, and miRNA. Briefly, the signaling pathway cross-talk is more prevalent in RCC than LCC, such as RCC-specific PI3K pathway, which often exhibits cross-talk with the RAS and P53 pathways. Meanwhile, methylation signatures revealed that RCC was hypermethylated relative to LCC. In addition, differentially expressed genes (n = 253) and differentially expressed miRNAs (n = 16) were determined between LCC and RCC. Especially for Prostate Cancer Susceptibility Candidate 1 (PRAC1), a gene that was closely associated with hypermethylation, was the top significantly downregulated gene in RCC. Multi-omics comparison of LCC and RCC suggests that there are more aggressive markers in RCC and that tumor heterogeneity occurs within the location-based subtypes of colon cancer. These results clarify the debate regarding the conflicting prognosis between LCC and RCC, as proposed by different studies.Implications: The underlying molecular features present in LCC and RCC identified in this study are beneficial for adopting reasonable therapeutic approaches to prolong overall survival and progression-free survival in colorectal cancer patients. Mol Cancer Res; 16(3); 476–85. ©2017 AACR.

IHC staining of selected LCC (A~C) and RCC (D~F) of PRAC

PDF file 145K, Fold change bar plot of differentially expressed genes between CRC tissues and adjacent normal ones; and diagnostic results in four batches of CRC samples and gastric cardiac cancer

PDF file 119K, This file includes supplementary text and figure legends for supporting figures

PDF file, 93K, High expression of SPARCL1 is associated with survival of patients with colon cancers and related to prognosis in both early and late stages.

PDF file 66K, Differentially expressed genes between CRC tissue and adjacent normal samples

Purpose: Metabolic phenotyping has provided important biomarker findings, which, unfortunately, are rarely replicated across different sample sets due to the variations from different analytical and clinical protocols used in the studies. To date, very few metabolic hallmarks in a given cancer type have been confirmed and validated by use of a metabolomic approach and other clinical modalities. Here, we report a metabolomics study to identify potential metabolite biomarkers of colorectal cancer with potential theranostic value.Experimental Design: Gas chromatography–time-of-flight mass spectrometry (GC–TOFMS)–based metabolomics was used to analyze 376 surgical specimens, which were collected from four independent cohorts of patients with colorectal cancer at three hospitals located in China and City of Hope Comprehensive Cancer Center in the United States. Differential metabolites were identified and evaluated as potential prognostic markers. A targeted transcriptomic analysis of 29 colorectal cancer and 27 adjacent nontumor tissues was applied to analyze the gene expression levels for key enzymes associated with these shared metabolites.Results: A panel of 15 significantly altered metabolites was identified, which demonstrates the ability to predict the rate of recurrence and survival for patients after surgery and chemotherapy. The targeted transcriptomic analysis suggests that the differential expression of these metabolites is due to robust metabolic adaptations in cancer cells to increased oxidative stress as well as demand for energy, and macromolecular substrates for cell growth and proliferation.Conclusions: These patients with colorectal cancer, despite their varied genetic background, mutations, pathologic stages, and geographic locations, shared a metabolic signature that is of great prognostic and therapeutic potential. Clin Cancer Res; 20(8); 2136–46. ©2014 AACR.

PDF file, 67K, Primer sequences in RT-PCR analysis.

PDF file 133K, ROC and Kaplan-Meier curves for survival analysis in samples from Shanghai using the ratio of hypoxanthine to aspartate

PDF file 63K, VIP values from OPLS-DA models and p values in the samples the Hangzhou, Beijing and City of Hope cohorts

PDF file, 65K, Determine the specificity of SPARCL1 antibody.

PDF file 50K, Treatment information for stage II and stage III patients in Shanghai cohort

PDF file 126K, PCA and OPLS-DA scores plot for CRC samples and adjacent non-tumor tissues collected from Hangzhou, Beijing and City of Hope

Purpose:Secreted protein acidic and rich in cysteines-like 1 (SPARCL1) is an extracellular matrix glycoprotein with malignancy-suppressing potential. The hypothesis that SPARCL1 reduces cancer invasiveness and predicts better survival in colorectal cancers (CRC) was investigated.Experimental Design: Stable SPARCL1 transfectants, RKO-SPARCL1, and corresponding vector control were constructed and implanted into nude mice to generate a mouse xenograft model of liver metastasis. Also, a retrospective outcome study was conducted on the COH set (222 CRCs) and ZJU set (412 CRCs). The protein expression level of SPARCL1 was determined by immunohistochemistry. The Kaplan–Meier and Cox analyses were used for survival analysis. The association of SPARCL1 with mesenchymal–epithelial transition (MET) was examined by reverse transcription PCR (RT-PCR) and Western blot analysis.Results: The ectopic expression of SPARCL1 significantly reduced the potential for anchorage-independent growth, migration, invasion and induced cell differentiation in RKO and SW620 cells. In mouse xenograft model, the expression of SPARCL1 significantly reduced the liver metastasis (P < 0.01). The patient-based studies revealed that the expression of SPARCL1 was related to better differentiation (P < 0.01), less lymph node involvement [OR, 0.67; 95% confidence interval (CI), 0.45–1.00], and less distant metastasis (OR, 0.38; 95% CI, 0.18–0.79). The Kaplan–Meier and Cox analysis showed that the expression of SPARCL1 was associated with better overall survival (log-rank: P < 0.01; HR, 0.57; 95% CI, 0.39–0.84). Transfection of SPARCL1 induced MET of colon cancer cells.Conclusion:SPARCL1 functions as a tumor suppressor promoting differentiation possibly via MET, which inhibits the aggressiveness of CRCs. Clin Cancer Res; 18(19); 5438–48. ©2012 AACR.

PDF file, 38K, Study design.

PDF file, 49K, The quality of CRC database in COH set and ZJU set.

PDF file, 379K, Recombinant human SPARCL1 protein reversed malignant phenotypes colon cancer cells in vitro.

PDF file, 112K.

PDF file, 126K, Uni- and Multivariate COX analysis for SPARCL1 and overall survival of CRCs.

PDF file, 89K, Standard of cytoplasm staining of SPARCL1.

PDF file 104K, PCA scores plot, permutation test and representative chromatograms for samples collected from Shanghai

Background Alpha-2-glycoprotein 1, zinc-binding (AZGP1) is a 41 kDa secreted glycoprotein that has been considered as a potential prognostic biomarker. High expression of AZGP1 in serum is considered to be an indicator of poor prognosis in CRC, however further validation in tissue specimens is lacking. Methods We detected the expression level of AZGP1 in 859 CRC patients and 210 normal mucosa samples by immunohistochemistry and analyzed the relationship between AZGP1 expression and clinicopathological parameters of CRC patients. The relationship between AZGP1 and overall survival of patients was also analyzed to verify the prognostic value of AZGP1 in CRC patients. Multivariate analysis of prognostic factors in CRC patients using Cox proportional hazards regression models. Results The high expression rate of AZGP1 in colorectal adenocarcinoma was 48.4% higher than that in adjacent mucosal tissues and 13.3% (PPP=0.023), distant metastasis (P=0.002) and pTNM stage(P=0.003). Univariate analysis found that histological type, differentiation, invasion, lymph node metastasis, distant metastasis, pTNM stage and AZGP1 expression were associated with the prognosis of CRC patients (all P < 0.05). Multivariate analysis suggested lymph node metastasis (95% CI: 0.331-0.775, HR: 0.506, P=0.002), distant metastasis (95% CI: 0.126-0.293, HR: 0.192, PCI: 0.262-0.637, HR: 0.408, P

Small bowel adenocarcinoma (SBA) is a rare malignancy with a poor prognosis and limited treatment options. Despite prior studies, molecular characterization of this disease is not well defined, and little is known regarding Chinese SBA patients. In this study, we conducted multigene next‐generation sequencing and 16S ribosomal RNA gene sequencing on samples from 76 Chinese patients with surgically resected primary SBA. Compared with colorectal cancer and Western SBA cohorts, a distinctive genomic profile was revealed in Chinese SBA cohorts. According to the levels of clinical actionability to targetable alterations stratified by OncoKB system, 75% of patients harbored targetable alterations, of which ERBB2, BRCA1/2, and C‐KIT mutations were the most common targets of highest‐level actionable alterations. In DNA mismatch repair–proficient (pMMR) patients, significant associations between high tumor mutational burden and specific genetic alterations were identified. Moreover, KRAS mutations/TP53 wild‐type/nondisruptive mutations (KRASmut/TP53wt/non‐dis ) were independently associated with an inferior recurrence‐free survival (hazard ratio [HR] = 4.21, 95% confidence interval [CI] = 1.94‐9.14, P, Molecular characterization of small bowel adenocarcinoma (SBA) is not well defined, and little is known regarding Chinese SBA patients. Compared with colorectal cancer and Western SBA cohorts, a distinctive genomic profile was revealed in Chinese SBA cohorts. A molecular subtype of KRAS mutations/TP53 wild‐type/nondisruptive mutations was found to be associated with an inferior recurrence‐free survival in SBA.

Lay abstract The heterogeneity of colorectal cancer is largely based on their primary tumor locations. For example, patients with left-sided colon cancer generally have better prognoses and different treatment strategies in comparison with patients with right-sided colon cancer. Research is required to uncover the mechanisms underlying these differences. The authors showed that the addition of methyl groups (DNA methylation) to DNA encoding SPARCL1 (a protein involved in cell adhesion) could inhibit regular colorectal cell motility. Furthermore, SPARCL1 protein expression was higher and positively correlated with better prognosis specifically in patients with left-sided colon cancer. The present study provides an evidential basis for the different molecular biological features between left- and right-sided colon cancer. SPARCL1 is a promising candidate for predicting colon cancer prognosis and potential therapeutic intervention. Further in-depth study of SPARCL1 would be of great value for clinical application.

Background Complete remission is observed in less than half of hypermutated (HM) tumours after immune checkpoint blockade therapy, indicating that HM tumours are very heterogeneous. Thus, there is an urgent requirement to decipher the unknown intrinsic HM tumour subtypes. Methods Statistical analysis was performed on somatic mutation data from 5519 tumours across 11 cancer types obtained from The Cancer Genome Atlas and 338 colorectal cancer (CRC) samples obtained from an Asian cohort. Samples with a tumour mutation burden >10 mut/Mb were classified as HM. A total of 1040 HM samples harbouring corresponding transcriptomes were used for non-negative matrix factorisation clustering. Tumour mutational burden, neoantigens, T cell receptor (TCR) diversity, stromal score and immune score were compared between the subtypes. Results HM tumours fell into three distinct immune subtypes: HM1, HM2 and HM3. HM3 tumours were correlated with increased CD8 T cell infiltration, high TCR diversity, a high immune score and prolonged survival. HM2 tumours were correlated with an abundant stromal component, epithelial–mesenchymal transition, TGFβ, angiogenesis hallmarks and poor outcomes. The infiltration of more CD8 T cells and increased chemokine expression in HM3 were validated in CRC by immunofluorescence. Conclusions These findings will facilitate the development of a subtype-oriented therapy strategy to enhance the treatment effect in the near future.

Hepatoid adenocarcinoma of the stomach (HAS) is a rare malignancy with aggressive biological behavior. This study aimed to compare the genetic landscape of HAS with liver hepatocellular carcinoma (LIHC), gastric cancer (GC), and AFP-producing GC (AFPGC) and identify clinically actionable alterations. Thirty-eight cases of HAS were collected for whole-exome sequencing. Significantly mutated genes were identified. TP53 was the most frequently mutated gene (66%). Hypoxia, TNF-α/NFκB, mitotic spindle assembly, DNA repair, and p53 signaling pathways mutated frequently. Mutagenesis mechanisms in HAS were associated with spontaneous or enzymatic deamination of 5-methylcytosine to thymine and defective homologous recombination-related DNA damage repair. However, LIHC was characteristic of exposure to aflatoxin and aristolochic acid. The copy number variants (CNVs) in HAS was significantly different compared to LIHC, GC, and AFPGC. Aggressive behavior-related CNVs were identified, including local vascular invasion, advanced stages, and adverse prognosis. In 55.26% of HAS patients there existed at least one clinically actionable alteration, including ERBB2, FGFR1, CDK4, EGFR, MET, and MDM2 amplifications and BRCA1/2 mutations. MDM2 amplification with functional TP53 was detected in 5% of HAS patients, which was proved sensitive to MDM2 inhibitors. A total of 10.53% of HAS patients harbored TMB > 10 muts/Mb. These findings improve our understanding of the genomic features of HAS and provide potential therapeutic targets.

Background Alpha-2-glycoprotein 1, zinc-binding (AZGP1) is a 41 kDa secreted glycoprotein that has been considered as a potential prognostic biomarker. High expression of AZGP1 in serum is considered to be an indicator of poor prognosis in CRC, however further validation in tissue specimens is lacking. Methods We detected the expression level of AZGP1 in 859 CRC patients and 210 normal mucosa samples by immunohistochemistry and analyzed the relationship between AZGP1 expression and clinicopathological parameters of CRC patients. The relationship between AZGP1 and overall survival of patients was also analyzed to verify the prognostic value of AZGP1 in CRC patients. Multivariate analysis of prognostic factors in CRC patients using Cox proportional hazards regression models. Results The high expression rate of AZGP1 in colorectal adenocarcinoma was 48.4% higher than that in adjacent mucosal tissues and 13.3% (P P P = 0.023), distant metastasis (P = 0.002) and pTNM stage(P = 0.003). Univariate analysis found that histological type, differentiation, invasion, lymph node metastasis, distant metastasis, pTNM stage and AZGP1 expression were associated with the prognosis of CRC patients (all P CI: 0.331–0.775, HR: 0.506, P = 0.002), distant metastasis (95% CI: 0.126–0.293, HR: 0.192, P CI: 0.262–0.637, HR: 0.408, P

Background Src homology 2 domain-containing phosphatase 2 (SHP2) is hyper-activated in some solid tumors. Previous findings suggest that the expression of SHP2 in colorectal cancer (CRC) may be associated with prognosis. However, validation with large sample data is lacking. Methods Tissue microarrays containing 860 CRCs and 197 mucosal tissues adjacent to the tumors were constructed. Immunohistochemistry was used to evaluate the expression of SHP2. Differences between SHP2 expression and clinicopathological parameters were evaluated. Kaplan-Meier survival curves and log-rank tests were used to analyze the relationships between SHP2 expression and overall survival of patients. A Cox proportional hazard regression model was used for univariate and multivariate analyses of prognostic factors. Results SHP2 expression levels in CRCs tissues were significantly higher than those in adjacent mucosal tissues (P P P = 0.008). Univariate analysis showed that the expression level of SHP2 was a prognostic factor for CRC (P = 0.008). Multivariate analysis demonstrated that SHP2 remained an independent prognostic factor for CRC (P = 0.033). Conclusion The expression levels of SHP2 were significantly higher in CRC tissues than in adjacent normal tissues. High expression levels of SHP2 were associated with a promising outcome, suggesting that SHP2 may be a favorable prognostic indicator of CRC.

Objective: Malignant rhabdoid tumor (MRT) is a rare but aggressive malignancy. It has been a long time since data on this tumor have been updated. Methods: We retrospectively reviewed patients from the SEER database who were pathologically diagnosed with MRT and analyzed incidence rates, clinical features and survival using Stata 12.0. Results: In total, 544 patients were included in the epidemiological analysis. There were two peak periods of MRT incidence: patients younger than 4 years and those older than 70 years. Further survival analysis showed that the survival of children (especially younger than 1 year) was markedly worse than that of adults (P

POLE mutations, which lead to an ultramutated phenotype in colorectal cancer (CRC), have been reported as a promising marker in immunotherapy. We performed sequencing of CRC cases in Zhejiang University (ZJU) and extracted obtainable data from recently published results, including The Cancer Genome Atlas (TCGA), Japanese studies and clinical trials, to present clinical patterns of POLE driver‐mutated CRC and reveal its heterogeneity. The rate of somatic POLE driver mutations has been reported as 2.60% (ZJU cohort), 1.50% (TCGA cohort), 1.00% (Japan cohort), and 1.00% (Lancet cohort). POLE driver mutations show a clearly increased mutation burden (mean TMB: 217.98 mut/Mb in ZJU; 203.13 mut/Mb in TCGA). Based on pooled data, more than 70.00% of patients with POLE driver mutations were diagnosed before they were 55 years old and at an early disease stage (Stage 0–II >70.00%), and more than 70.00% were male. Among Asian patients, 68.40% developed POLE driver mutations in the left‐side colon, whereas 64.00% of non‐Asian patients developed them in the right‐side colon (p, POLE could be a promising marker for immunotherapy, and its heterogeneity should not be ignored. Here we present the clinical patterns and heterogeneity of POLE driver mutations so that investigators and physicians will be better equipped to design clinical trials and analyze their data.

Background The objective of this study was to determine the features of fragment length for circulating cell-free DNA (cfDNA) from plasma and serum samples. Methods Plasma and serum samples from different sources were randomly collected. Circulating cfDNA was extracted and purified by a precipitation-enriched and spin-column-based kit. The concentration of the purified DNA was immediately measured by a highly sensitive dsDNA quantitative assay, and then the fragment length was analyzed by capillary electrophoresis. The abundance of a specific fragment was estimated by the area under curve (AUC) for the fragment peak in the capillary electrophoresis. Results A total of 199 plasma and 117 serum samples were extracted and analyzed. The average yield of cfDNA from the serum samples (131.67 ng/mL) was significantly higher than that from the plasma samples (32.78 ng/mL, p Conclusions The fragment lengths of serum cfDNA and plasma cfDNA have very different features. Fragment size selection is suitable for plasma cfDNA but may not apply to serum cfDNA.

Background Obg-like ATPase 1(OLA1) is a member of the GTPase protein family but utilizes ATP over GTP in humans. OLA1 has been reported to be involved in lung and breast cancer progression, however, it remains unclear whether OLA1 plays a critical role in gastric cancer. Methods TCGA and GEO datasets data were collected to identify differential gene expression profiles in gastric cancer patient samples. Western blot and real-time polymerase chain reaction were performed to evaluate OLA1 expression in gastric cancer cells and normal gastric epithelium. Cell count Kit-8, Propidium iodide staining assay, colony formation assay, wound-healing and transwell assays were applied to analyze the effects of OLA1 on gastric cancer cell proliferation and metastasis in vitro. Moreover, the Subcutaneous gastric cancer xenograft tumor model was introduced to investigate the role of OLA1 in vivo. Results OLA1 demonstrated a higher expression in gastric cancer tissues with stage III-IV compared to paired normal tissue (25 paired sample; P Conclusions OLA1 inhibition could suppress gastric cancer progression and enhance the sensitivity to adjuvant chemotherapy with sparing normal gastric epithelium cells. Therefore, OLA1 could be a novel and promising target in treating gastric cancer.

Background: Colonic neuroendocrine carcinomas (co-NECs) are heterogeneous and aggressive, especially with regard to metastasis. Whether co-NECs on the right and left sides of the colon have different characteristics from colon adenocarcinoma is unknown. Methods: The co-NEC patients were selected from the 2010–2017 Surveillance, Epidemiology, and End Results Program (SEER) database. The right and left sides of the colon were separated by the splenic flexure. Coarsened exact matching (CEM) was performed to adjust for relevant factors before regression models were constructed. Results: A total of 669 pathologically diagnosed co-NEC patients with sufficient baseline data were identified from the SEER database. A total of 80.72% of the patients had co-NEC that originated from the right side of the colon, and their mean overall survival (mOS) was similar to that of the patients with left-sided co-NECs (right versus left: 22.30 m versus 22.55 m). A total of 44.84% of the patients were diagnosed with liver metastasis (46.68% right side versus 37.98% left side). In patients with liver metastasis, those with right-sided co-NECs had better survival than those with left-sided co-NECs (mOS right versus left: 15.37 m versus 9.62 m; adjusted hazard ratio (HR) = 0.69, 95% confidence interval (CI): 0.49–0.98, p = 0.035). To further investigate the survival benefits of primary site resection, we separated the patients who had liver metastasis according to the primary site and performed CEM to balance the groups (no patients underwent liver metastasis resection or intervention). The results suggested that primary surgery could benefit patients with both left- and right-sided co-NECs (adjusted HR = 0.50, 95% CI: 0.33–0.77, p = 0.001 on the right side; HR = 0.38, 95% CI: 0.16–0.89, p = 0.026 on the left side). Conclusions: Co-NECs frequently originate on the right side and commonly develop liver metastasis. Right-sided co-NECs are associated with better survival than left-sided co-NECs after liver metastasis has occurred. Primary site resection is associated with prolonged survival in co-NEC patients with liver metastasis, regardless of the side from which the co-NEC has originated.

Neuroendocrine tumors (NETs) are heterogeneous, and the incidence of NETs is rapidly increasing. We observed different survival in patients with rectal NETs and rectosigmoid junction NETs, which are treated similarly. We included patients with rectal and rectosigmoid junction NETs from the SEER database. The 5‐year survival was set as the end‐point. 6675 patients with rectal NETs and 329 patients with rectosigmoid junction NETs, were eligible for the analysis. Initially, the survival analyses suggested that the 5‐year survival significantly differed between the patients with rectal and rectosigmoid junction NETs (HR = 0.82, 95% CI 0.70‐0.95; P = .01). Tumor differentiation, an invasion deeper than T2, and lymph node and distant metastases were still important risk factors affecting survival for both location. While, the males showed better survival (HR = 0.69, 95% CI 0.55‐0.88; P 5, P 5, P, Survival and risk factors of rectal and rectosigmoid junction NET are different. Rectosigmoid junction NETs presented a better 5‐year survival. A clear and consistent definition of the rectosigmoid junction is urgently needed for future studies.

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Terahertz technology has been widely used in biomedical research. Herein, terahertz time-domain attenuated total reflection (THz TD-ATR) spectroscopy was employed to characterize and discriminate human cancer cell lines (DLD-1 and HT-29). Terahertz responses of the cell lines were measured and Savitzky-Golay algorithm was applied to smooth the spectra of refractive index, absorption coefficient and dielectric loss tangent in terahertz regime. Principal component analysis (PCA) was then adopted for feature extraction and cell characterization. Based on the processed data, cancer cell lines were discriminated by applying random forests (RF) method to analyze three characteristic parameters separately and the results from them were compared. Results indicate that absorption coefficient was the most sensitive parameter for cancer cell discrimination. Our study suggests great potential for human cancer cell recognition and provides experimental basis for liquid biopsy.