Gender-Associated Genomic Differences in Colorectal Cancer: Clinical Insight from Feminization of Male Cancer Cells

Gender-related differences in colorectal cancer (CRC) are not fully understood. Recent studies have shown that CRC arising in females are significantly associated with CpG island methylator phenotype (CIMP-high). Using array comparative genomic hybridization, we analyzed a cohort of 116 CRCs (57 males, 59 females) for chromosomal copy number aberrations (CNA) and found that CRC in females had significantly higher numbers of gains involving chromosome arms 1q21.2–q21.3, 4q13.2, 6p21.1 and 16p11.2 and copy number losses of chromosome arm 11q25 compared to males. Interestingly, a subset of male CRCs (46%) exhibited a “feminization” phenomenon in the form of gains of X chromosomes (or an arm of X) and/or losses of the Y chromosome. Feminization of cancer cells was significantly associated with microsatellite-stable CRCs (p-value 0.003) and wild-type BRAF gene status (p-value 0.009). No significant association with other clinicopathological parameters was identified including disease-free survival. In summary, our data show that some CNAs in CRC may be gender specific and that male cancers characterized by feminization may constitute a specific subset of CRCs that warrants further investigation.     

High Concordance of Genomic Profiles between Primary and Metastatic Colorectal Cancer

The comparison of the genetic profiles between primary and metastatic colorectal cancer (CRC) is needed to enable the discovery of useful therapeutic targets against metastatic CRCs. We performed the targeted next generation sequencing assay of 170 cancer-associated genes for 142 metastatic CRCs, including 95 pairs of primary and metastatic CRCs, to reveal their genomic characteristics and to assess the genetic heterogeneity. The most frequently mutated gene in primary and metastatic CRCs was APC (71% vs. 65%), TP53 (54% vs. 57%), KRAS (45% vs. 44%), PIK3CA (16% vs. 19%), SMAD4 (15% vs. 14%) and FBXW7 (11% vs. 11%). The concordance in the top six frequently mutated genes was 85%, on average. The overall mutation frequencies were consistent with two sets of public data (TCGA and MSKCC). To the author’s knowledge, this is the first study to compare the genetic profiles of our cohort with that of the metastatic CRCs from MSKCC. Comparative sequencing analysis between primary and metastatic CRCs revealed a high degree of genetic concordance in the current clinically actionable genes. Therefore, the genetic investigation of archived primary tumor samples with the challenges of obtaining an adequate sample from metastatic sites appears to be sufficient for the application of cancer precision medicine in the metastatic setting.     

KRAS Mutation Detection in Paired Frozen and Formalin-Fixed Paraffin-Embedded (FFPE) Colorectal Cancer Tissues

KRAS mutation has been unambiguously identified as a marker of resistance to cetuximab-based treatment in metastatic colorectal cancer (mCRC) patients. However, most studies of KRAS mutation analysis have been performed using homogenously archived CRC specimens, and studies that compare freshly frozen specimens and formalin-fixed paraffin-embedded (FFPE) specimens of CRC are lacking. The aim of the present study was to evaluate the impact of tissue preservation on the determination of KRAS mutational status. A series of 131 mCRC fresh-frozen tissues were first analyzed using both high-resolution melting (HRM) and direct sequencing. KRAS mutations were found in 47/131 (35.8%) using both approaches. Out of the 47 samples that were positive for KRAS mutations, 33 had available matched FFPE specimens. Using HRM, 2/33 (6%) demonstrated suboptimal template amplification, and 2/33 (6%) expressed an erroneous wild-type KRAS profile. Using direct sequencing, 6/33 (18.1%) displayed a wild-type KRAS status, and 3/33 (9.1%) showed discordant mutations. Finally, the detection of KRAS mutations was lower among the FFPE samples compared with the freshly frozen samples, demonstrating that tissue processing clearly impacts the accuracy of KRAS genotyping.     

Expression and Prognostic Significance of CD47–SIRPA Macrophage Checkpoint Molecules in Colorectal Cancer

Despite the confirmed anti-cancer effects of T-cell immune checkpoint inhibitors, in colorectal cancer (CRC) they are only effective in a small subset of patients with microsatellite-unstable tumors. Thus, therapeutics targeting other types of CRCs or tumors refractory to T-cell checkpoint inhibitors are desired. The binding of aberrantly expressed CD47 on tumor cells to signal regulatory protein-alpha (SIRPA) on macrophages allows tumor cells to evade immune destruction. Based on these observations, drugs targeting the macrophage checkpoint have been developed with the expectation of anti-cancer effects against T-cell immune checkpoint inhibitor-refractory tumors. In the present study, 269 primary CRCs were evaluated immunohistochemically for CD47, SIRPA, CD68, and CD163 expression to assess their predictive utility and the applicability of CD47–SIRPA axis-modulating drugs. Thirty-five percent of the lesions (95/269) displayed CD47 expression on the cytomembrane of CRC cells. CRCs contained various numbers of tumor-associated immune cells (TAIs) with SIRPA, CD68, or CD163 expression. The log-rank test revealed that patients with CD47-positive CRCs had significantly worse survival than CD47-negative patients. Multivariate Cox hazards regression analysis identified tubular-forming histology (hazard ratio (R) = 0.23), age < 70 years (HR = 0.48), and high SIRPA-positive TAI counts (HR = 0.55) as potential favorable factors. High tumor CD47 expression (HR = 1.75), lymph node metastasis (HR = 2.26), and peritoneal metastasis (HR = 5.80) were cited as potential independent risk factors. Based on our observations, CD47–SIRPA pathway-modulating therapies may be effective in patients with CRC.     

Serrated Colorectal Lesions: An Up-to-Date Review from Histological Pattern to Molecular Pathogenesis

Until 2010, colorectal serrated lesions were generally considered as harmless lesions and reported as hyperplastic polyps (HPs) by pathologists and gastroenterologists. However, recent evidence showed that they may bear the potential to develop into colorectal carcinoma (CRC). Therefore, the World Health Organization (WHO) classification has identified four categories of serrated lesions: hyperplastic polyps (HPs), sessile serrated lesions (SSLs), traditional serrated adenoma (TSAs) and unclassified serrated adenomas. SSLs with dysplasia and TSAs are the most common precursors of CRC. CRCs arising from serrated lesions originate via two different molecular pathways, namely sporadic microsatellite instability (MSI) and the CpG island methylator phenotype (CIMP), the latter being considered as the major mechanism that drives the serrated pathway towards CRC. Unlike CRCs arising through the adenoma–carcinoma pathway, APC-inactivating mutations are rarely shown in the serrated neoplasia pathway.     

Heterogeneity of Mismatch Repair Status and Microsatellite Instability between Primary Tumour and Metastasis and Its Implications for Immunotherapy in Colorectal Cancers

Deficient mismatch repair system (dMMR)/microsatellite instability (MSI) is found in about 5% of metastatic colorectal cancers (mCRCs) with a major therapeutic impact for immune checkpoint inhibitor (ICI) use. We conducted a multicentre study including all consecutive patients with a dMMR/MSI mCRC. MSI status was determined using the Pentaplex panel and expression of the four MMR proteins was evaluated by immunohistochemistry (IHC). The primary endpoint was the rate of discordance of dMMR/MSI status between primary tumours and paired metastases. We included 99 patients with a dMMR/MSI primary CRC and 117 paired metastases. Only four discrepancies (3.4%) with a dMMR/MSI primary CRC and a pMMR/MSS metastasis were initially identified and reviewed by expert pathologists and molecular biologists. Two cases were false discrepancies due to human or technical errors. One discordant case could not be confirmed due to the low level of tumour cells. The last case had a confirmed discrepancy with a dMMR/MSI primary CRC and a pMMR/MSS peritoneal metastasis. Our study demonstrated a high concordance rate of dMMR/MSI status between primary CRCs and their metastases. The analysis of one sample, either from the primary tumour or metastasis, with consistent dMMR and MSI status seems to be sufficient prior to treatment with ICI.     

HER-2 Expression in Brain Metastases from Colorectal Cancer and Corresponding Primary Tumors: A Case Cohort Series

Brain metastases (BM) from colorectal cancer (CRC) are a rare but increasing event. Surgical resection of oligometastatic disease, including BM, may produce a survival benefit in selected patients. Previous studies described the HER-2 expression patterns in CRC patients, but its prognostic role still remains controversial. Information on the HER-2 expression in BM from CRC is currently lacking. Among the over 500 patients treated at our Department of Neurosurgery in the last 13 years (1999–2012), we identified a cohort of 50 consecutive CRC patients resected for BM. Clinical data were retrospectively reviewed using electronic hospital charts and surgical notes. Formalin-fixed, paraffin-embedded tissue samples were retrieved and histologically reviewed. HER-2 status was assessed on 4-μm sections by HerceptTest™, and scored by two pathologists according to gastric cancer HER-2 status guidelines. In score 2+ cases HER-2 gene copy number was analyzed by FISH, performed using the PathVysion HER-2 DNA Probe Kit. Median age at time of BM resection was 65 years (35–82); most patients were males (60%) with a good performance status. The majority of the BM were single (74%) and sited in the supratentorial area (64%); 2–4 lesions were diagnosed in 9 patients (18%), and >4 in 3 patients (6%). The rate of HER-2 positivity (defined as IHC score 3+ or IHC score 2+ and FISH gene amplification) was 8.1% for the primary CRC tumors and 12% for their corresponding BM. The concordance rate between primary tumors and matched BM was 89%. Median overall survival after neurosurgery was 6.5 months for HER-2 IHC score 0 vs. 4.6 months for HER-2 IHC score 1+/2+/3+; the difference was statistically significant (p = 0.01, Log-rank test). HER-2 positivity of our case cohort was low but comparable to literature. Concordance rate of HER-2 expression between BM and corresponding primary tumors is high and similar to those reported for breast and gastric cancers. Our data suggest a potential negative prognostic value of HER-2 expression in brain lesions from CRC.     

A Combination of Chemotherapy and Oncolytic Virotherapy Sensitizes Colorectal Adenocarcinoma to Immune Checkpoint Inhibitors in a cDC1-Dependent Manner

Immune checkpoint therapy has shown great promise in the treatment of cancers with a high mutational burden, such as mismatch repair-deficient colorectal carcinoma (dMMR CRC). However, many patients fail to respond to immune checkpoint therapy. Using a mouse model of dMMR CRC, we demonstrated that tumors can be further sensitized to immune checkpoint therapy by using a combination of low-dose chemotherapy and oncolytic HSV-1. This combination induced the infiltration of CD8⁺ and CD4⁺ T cells into the tumor and the upregulation of gene signatures associated with the chemoattraction of myeloid cell subsets. When combined with immune checkpoint therapy, the combination promoted the infiltration of activated type 1 conventional dendritic cells (cDC1s) into the tumor. Furthermore, we found this combination strategy to be dependent on cDC1s, and its therapeutic efficacy to be abrogated in cDC1-deficient Batf3^−/− mice. Thus, we demonstrated that the adjuvanticity of dMMR CRCs can be improved by combining low-dose chemotherapy and oncolytic HSV-1 in a cDC1-dependent manner.     

Natural Products of Marine Origin for the Treatment of Colorectal and Pancreatic Cancers: Mechanisms and Potential

Gastrointestinal cancer refers to malignancy of the accessory organs of digestion, and it includes colorectal cancer (CRC) and pancreatic cancer (PC). Worldwide, CRC is the second most common cancer among women and the third most common among men. PC has a poor prognosis and high mortality, with 5-year relative survival of approximately 11.5%. Conventional chemotherapy treatments for these cancers are limited due to severe side effects and the development of drug resistance. Therefore, there is an urgent need to develop new and safe drugs for effective treatment of PC and CRC. Historically, natural sources—plants in particular—have played a dominant role in traditional medicine used to treat a wide spectrum of diseases. In recent decades, marine natural products (MNPs) have shown great potential as drugs, but drug leads for treating various types of cancer, including CRC and PC, are scarce. To date, marine-based drugs have been used against leukemia, metastatic breast cancer, soft tissue sarcoma, and ovarian cancer. In this review, we summarized existing studies describing MNPs that were found to have an effect on CRC and PC, and we discussed the potential mechanisms of action of MNPs as well as future prospects for their use in treating these cancers.     

A deterministic model for estimating the reduction in colorectal cancer incidence due to endoscopic surveillance

There is evidence that the removal of adenomas, by endoscopy, from the large bowel can prevent the occurrence of colorectal cancer (CRC). However, the reduction in cancer incidence due to endoscopic surveillance is difficult to estimate. Studies of cohorts of adenoma patients typically rely on comparisons with groups of historical controls. We present a model for disease progression which enables estimation of this quantity without direct comparison to a reference group. Models are applied to data from the National Polyp Study. Rates of adenoma recurrence and progression to carcinoma are estimated based on study data and relevant literature. This allows calculation of the number of cancers expected in the absence of surveillance and, thus, the number of cancers prevented. Results are compared with the original analysis. Models estimate that surveillance reduced CRC incidence by at least 97% in this cohort. The majority of the effect was due to the initial removal of adenomas rather than the follow-up surveillance. These results are similar to those produced in the original analysis when using the most appropriate reference groups. They indicate that polypectomy and follow-up surveillance can lead to large reductions in cancer incidence which may have been under-estimated in previous studies.     

The Behavior of Matrix Metalloproteinases and Their Inhibitors in Colorectal Cancer

Matrix metalloproteinases (MMPs) play an important role in the degradation of extracellular matrix components crucial for tumor growth, invasion and metastasis. MMPs are controlled by natural inhibitors called tissue inhibitors of metalloproteinases (TIMPs). We and others have demonstrated that MMPs and TIMPs are especially important in the process of tumor invasion, progression and the metastasis of colorectal cancer (CRC). It has been proposed that MMPs and TIMPs might play a part not only in tumor invasion and initiation of metastasis but also in carcinogenesis from colorectal adenomas. Several recent studies demonstrated that high preoperative serum or plasma MMP-2, MMP-9 and TIMP-1 antigen levels are strong predictive factors for poor prognosis in patients with CRC and their determination might be useful for identification of patients with higher risk for cancer recurrence. MMP-9 and TIMP-1 have significant potential tumor marker impact in CRC. Their diagnostic sensitivity is consistently higher than those of conventional biomarkers. The pharmacological targeting of CRC by the development of a new generation of selective inhibitors of MMPs, that is highly specific for certain MMPs, is a promising and challenging area for the future.     

Scutellaria barbata D. Don Inhibits Tumor Angiogenesis via Suppression of Hedgehog Pathway in a Mouse Model of Colorectal Cancer

Angiogenesis, which plays a critical role during tumor development, is tightly regulated by the Sonic Hedgehog (SHH) pathway, which has been known to malfunction in many types of cancer. Therefore, inhibition of angiogenesis via modulation of the SHH signaling pathway has become very attractive for cancer chemotherapy. Scutellaria barbata D. Don (SB) has long been used in China to treat various cancers including colorectal cancer (CRC). Our published data suggested that the ethanol extract of SB (EESB) is able to induce apoptosis of colon cancer cells and inhibit angiogenesis in a chick embryo chorioallantoic membrane model. To further elucidate the precise mechanisms of its anti-tumor activity, in the present study we used a CRC mouse xenograft model to evaluate the effect of EESB on tumor growth and angiogenesis in vivo. Our current data indicated that EESB reduces tumor size without affecting on the body weight gain in CRC mice. In addition, EESB treatment suppresses the expression of key mediators of the SHH pathway in tumor tissues, which in turn resulted in the inhibition of tumor angiogenesis. Furthermore, EESB treatment inhibits the expression of vascular endothelial growth factor A (VEGF-A), an important target gene of SHH signaling and functioning as one of the strongest stimulators of angiogenesis. Our findings suggest that inhibition of tumor angiogenesis via suppression of the SHH pathway might be one of the mechanisms by which Scutellaria barbata D. Don can be effective in the treatment of cancers.     

SPDL1 Is an Independent Predictor of Patient Outcome in Colorectal Cancer

Spindle Apparatus Coiled-Coil Protein 1 (SPDL1) is a relatively recently identified coiled-coil domain containing protein and an important determinant of DNA fidelity by ensuring faithful mitosis. Hence, SPDL1 is suspected to underlie genomic (in-)stability in human cancers, yet its exact roles in these diseases remain largely underexplored. Given that genomic instability (GIN) is a crucial feature in colorectal cancer (CRC), we primarily asked whether the expression of this protein may account for differences in clinicopathological features and survival rates of CRC patients. Protein expression was evaluated by immunohistochemistry in the institutional tissue microarray (TMA), and gene expression by the analysis of publicly available datasets. To place the prognostic relevance in a predicted biological context, gene co-expression set around SPDL1 identified by public data mining was annotated and assessed for enrichment in gene ontology (GO) categories, BRITE hierarchies, and Reactome pathways. The comparison with adjacent normal tissue revealed a high expression of SPDL1 protein in a subset of tumor cases (48.84%), and these had better prognosis than the SPDL1-low expression counterpart even after adjustment for multiple confounders. SPDL1-high expression within tumors was associated with a median 56-month survival advantage, but not with any clinicopathological characteristics of our cohort. In the TCGA cohort, SPDL1 was overexpressed in tumor tissue and positively associated with improved survival, chromosome instability phenotype, and various GIN markers. In addition to the genes critically involved in the cell cycle and mitosis, a gene set co-expressed with SPDL1 contained checkpoint members of both chromosome segregation and DNA replication, as well as those associated with defective DNA repair, and retrograde vesicle-mediated transport. In conclusion, SPDL1 is an independent predictor of CRC patient survival in a possible connection with chromosomal instability.     

Significant Overexpression of DVL1 in Taiwanese Colorectal Cancer Patients with Liver Metastasis

Undetected micrometastasis plays a key role in the metastasis of cancer in colorectal cancer (CRC) patients. The aim of this study is to identify a biomarker of CRC patients with liver metastasis through the detection of circulating tumor cells (CTCs). Microarray and bioinformatics analysis of 10 CRC cancer tissue specimens compared with normal adjacent tissues revealed that 31 genes were up-regulated (gene expression ratio of cancer tissue to paired normal tissue > 2) in the cancer patients. We used a weighted enzymatic chip array (WEnCA) including 31 prognosis-related genes to investigate CTCs in 214 postoperative stage I–III CRC patients and to analyze the correlation between gene expression and clinico-pathological parameters. We employed the immunohistochemistry (IHC) method with polyclonal mouse antibody against DVL1 to detect DVL1 expression in 60 CRC patients. CRC liver metastasis occurred in 19.16% (41/214) of the patients. Using univariate analysis and multivariate proportional hazards regression analysis, we found that DVL1 mRNA overexpression had a significant, independent predictive value for liver metastasis in CRC patients (OR: 5.764; 95% CI: 2.588–12.837; p < 0.0001 on univariate analysis; OR: 3.768; 95% CI: 1.469–9.665; p = 0.006 on multivariate analysis). IHC staining of the immunoreactivity of DVL1 showed that DVL1 was localized in the cytoplasm of CRC cells. High expression of DVL1 was observed in 55% (33/60) of CRC tumor specimens and was associated significantly with tumor depth, perineural invasion and liver metastasis status (all p < 0.05). Our experimental results demonstrated that DVL1 is significantly overexpressed in CRC patients with liver metastasis, leading us to conclude that DVL1 could be a potential prognostic and predictive marker for CRC patients.     

Stemness,Inflammation and Epithelial–Mesenchymal Transition in Colorectal Carcinoma: The Intricate Network

In global cancer statistics, colorectal carcinoma (CRC) ranks third by incidence and second by mortality, causing 10.0% of new cancer cases and 9.4% of oncological deaths worldwide. Despite the development of screening programs and preventive measures, there are still high numbers of advanced cases. Multiple problems compromise the treatment of metastatic colorectal cancer, one of these being cancer stem cells—a minor fraction of pluripotent, self-renewing malignant cells capable of maintaining steady, low proliferation and exhibiting an intriguing arsenal of treatment resistance mechanisms. Currently, there is an increasing body of evidence for intricate associations between inflammation, epithelial–mesenchymal transition and cancer stem cells. In this review, we focus on inflammation and its role in CRC stemness development through epithelial–mesenchymal transition.     

Identification of a Novel Five-Gene Signature as a Prognostic and Diagnostic Biomarker in Colorectal Cancers

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. The current TNM (Tumor, Node, and Metastasis) classification approach is suboptimal in determining the prognosis of CRC patients. The prognosis for CRC is affected by a variety of features that are present at the initial diagnosis. Herein, we performed a systematic exploration and established a novel five-panel gene signature as a prognostic and early diagnosis biomarker after performing differential gene expression analyses in five independent in silico CRCs cohort and independently validating it in one clinical cohort, using immunohistochemistry. Four genes (BDNF, PTGS2, GSK3B, and CTNNB1) were significantly upregulated and one gene (HPGD) was significantly downregulated in primary tumor tissues compared with adjacent normal tissues throughout all the five in silico datasets. The univariate CoxPH analysis yielded a five-gene signature that accurately predicted overall survival (OS) and recurrence-free survival (RFS) in the in silico training (AUC = 0.73 and 0.69, respectively) and one independent in silico validation cohort (AUC = 0.69 and 0.74, respectively). This five-gene signature demonstrated significant associations with poor OS in independent clinical validation cohorts of colon cancer (CC) patients (AUC = 0.82). Intriguingly, a risk stratification model comprising of the five-gene signature together with TNM stage and gender status achieved an even superior AUC of 0.89 in the clinical cohorts. On the other hand, the circulating mRNA expression of the upregulated four-gene signature achieved a robust AUC = 0.83 with high sensitivity and specificity as a diagnosis marker in plasma from CRC patients. We have identified a novel, five-gene signature as an independent predictor of OS, which in combination with TNM stage and gender offers an easy-to-translate and facile assay for the personalized risk-assessment in CRC patients.     

No Association between HMOX1 and Risk of Colorectal Cancer and No Interaction with Diet and Lifestyle Factors in a Prospective Danish Case-Cohort Study

Red meat is a risk factor for colorectal cancer (CRC). We wanted to evaluate whether a functional polymorphism in the HMOX1 gene encoding heme oxygenase modifies risk of CRC or interacts with diet or lifestyle factors because this would identify heme or heme iron as a risk factor of CRC. The HMOX1 A-413T (rs2071746) was assessed in relation to risk of colorectal cancer (CRC) and interactions with diet (red meat, fish, fiber, cereals, fruit and vegetables) and lifestyle (use of non-steroidal anti-inflammatory drug and smoking status) were assessed in a case-cohort study of 928 CRC cases and a comparison group of 1726 randomly selected participants from a prospective study of 57,053 persons. No association between HMOX1 A-413T and CRC risk was found (TT vs. AA + TA; IRR = 1.15, 95% CI: 0.98–1.36, p = 0.10 for the adjusted estimate). No interactions were found between diet or lifestyle and HMOX1 A-413T. HMOX1 A-413T was not associated with CRC risk and no interactions with diet or lifestyle were identified in this large, prospective cohort with high meat intake. The results reproduced the previous findings from the same cohort and did not support a link between heme or heme iron and colorectal cancer. These results should be sought and replicated in other well-characterized cohorts with high meat intake.