Tumour budding is associated with the mesenchymal colon cancer subtype and RAS/RAF mutations: a study of 1320 colorectal cancers with Consensus Molecular Subgroup (CMS) data.

BACKGROUND: Tumour budding is an important prognostic factor in colorectal cancer (CRC). Molecular profiling of tumour buds suggests (partial) epithelial-mesenchymal transition and cancer stem-cell phenotype, similarly described in the "mesenchymal" Consensus Molecular Subtype 4 (CMS4), which identifies a particularly poor prognostic subgroup. Here, we determine the association of tumour budding with CMS classification, prognosis, and response to therapy. METHODS: AMC-AJCCII-90 cohort (n = 76, stage II) was evaluated for peritumoural budding on H&E slides. LUMC (n = 270, stage I-IV), CAIRO (n = 504, metastatic CRC) and CAIRO2 (n = 472, metastatic CRC) cohorts were investigated for intratumoural budding using pan-cytokeratin-stained tissue microarrays. Budding was scored as count/area, then classified as <5 or ≥5 buds. For all cohorts, CMS classifications were available (gene-expression/immunohistochemistry-based classifiers). RESULTS: High (≥5) budding predicted a worse outcome in multivariate analysis in AMC-AJCCII-90 (p = 0.018), LUMC (p < 0.0001), and CAIRO (p = 0.03), and in CAIRO2 (continuous variable, p = 0.02). Tumour budding counts were higher in CMS4 compared to epithelial CMS2/3 cancers (p < 0.01, all), and associated with KRAS/BRAF mutations (p < 0.01, AMC-AJCCII-90, CAIRO, CAIRO2). CONCLUSION: Tumour budding is an adverse prognostic factor across all CRC stages and is associated with the mesenchymal CMS4 phenotype. KRAS/BRAF mutations are strongly correlated with tumour budding suggesting their involvement in the regulation of this process.

Statin Use After Diagnosis of Colon Cancer and Patient Survival.

BACKGROUND & AIMS: Statin use has been associated with a reduced incidence of colorectal cancer and might also affect survival of patients diagnosed with colon cancer. Statins are believed to inhibit Ras signaling and may also activate the bone morphogenetic protein (BMP) signaling pathway in colorectal cancer cells. We investigated the effects of statins on overall survival of patients with a diagnosis of colon cancer, and whether their effects were associated with changes in KRAS or the BMP signaling pathways. METHODS: Data were derived from the PHARMO database network (Netherlands) and linked to patients diagnosed with colon cancer from 2002 through 2007, listed in the Eindhoven Cancer Registry. We obtained information on causes of death from statistics Netherlands. We constructed a tissue microarray of 999 colon cancer specimens from patients who underwent surgical resection from 2002 through 2008. Survival was analyzed with statin user status after diagnosis as a time-dependent covariate. Multivariable Poisson regression survival models and Cox analyses were used to study the effect of statins on survival. Tumor tissues were analyzed by immunohistochemistry for levels of SMAD4, BMPR1A, BMPR1B, and BMPR2 proteins. Tumor tissues were considered to have intact BMP signaling if they contained SMAD4 plus BMPR1A, BMPR1B, or BMPR2. DNA was isolated from tumor tissues and analyzed by quantitative polymerase chain reaction to detect mutations in KRAS. The primary outcome measures were overall mortality and cancer-specific mortality. RESULTS: In this cohort, 21.0% of the patients (210/999) were defined as statin users after diagnosis of colon cancer. Statin use after diagnosis was significantly associated with reduced risk of death from any cause (adjusted relative risk [RR], 0.67; 95% confidence interval [CI], 0.51-0.87; P = .003) and death from cancer (adjusted RR, 0.66; 95% CI, 0.49-0.89; P = .007). Statin use after diagnosis was associated with reduced risk of death from any cause or from cancer for patients whose tumors had intact BMP signaling (adjusted RR, 0.39; 95% CI, 0.22-0.68; P = .001), but not for patients whose tumors did not have BMP signaling (adjusted RR, 0.81; 95% CI, 0.55-1.21; P = .106; P < .0001 for the interaction). Statin use after diagnosis was not associated with reduced risk of death from any cause or from cancer for patients whose tumors did not contain KRAS mutations (adjusted RR, 0.81; 95% CI, 0.56-1.18; P = .273) or whose tumors did have KRAS mutations (adjusted RR, 0.59; 95% CI 0.35-1.03; P = .062; P = .90 for the interaction). CONCLUSIONS: In an analysis of 999 patients with a diagnosis of colon cancer, we associated statin with reduced risk of death from any cause or from cancer. The benefit of statin use is greater for patients whose tumors have intact BMP signaling, independent of KRAS mutation status. Randomized controlled trials are required to confirm these results.

The Prognostic Value of Microsatellite Instability, KRAS, BRAF and PIK3CA Mutations in Stage II Colon Cancer Patients.

In the era of personalized cancer medicine, identifying mutations within patient tumors plays an important role in defining high-risk stage II colon cancer patients. The prognostic role of BRAF V600E mutation, microsatellite instability (MSI) status, KRAS mutation and PIK3CA mutation in stage II colon cancer patients is not settled. We retrospectively analyzed 186 patients with stage II colon cancer who underwent an oncological resection but were not treated with adjuvant chemotherapy. KRAS mutations, PIK3CA mutation, V600E BRAF mutation and MSI status were determined. Survival analyses were performed. Mutations were found in the patients with each mutation in the following percentages: 23% (MSI), 35% (KRAS), 19% (BRAF) and 11% (PIK3CA). A trend toward worse overall survival (OS) was seen in patients with an MSI (5-year OS 74% versus 82%, adjusted hazard ratio [HR] 1.8, 95% confidence interval [CI] 0.6-4.9) and a KRAS-mutated tumor (5-year OS 77% versus 82%, adjusted HR 1.7, 95% CI 0.8-3.5). MSI and BRAF-mutated tumors tended to correlate with poorer disease-free survival (DFS) (5-year DFS 60% versus 78%, adjusted HR 1.6, 95% CI 0.5-2.1 and 5-year DFS 57% versus 77%, adjusted HR 1.1, 95% CI 0.4-2.6 respectively). In stage II colon cancer patients not treated with adjuvant chemotherapy, BRAF mutation and MSI status both tended to have a negative prognostic effect on disease-free survival. KRAS and MSI status also tended to be correlated with worse overall survival.

Prognostic value of HLA class I, HLA-E, HLA-G and Tregs in rectal cancer: a retrospective cohort study.

BACKGROUND: Evasion of immune surveillance and suppression of the immune system are important hallmarks of tumorigenesis. The goal of this study was to establish distinct patterns that reflect a rectal tumors' immune-phenotype and to determine their relation to patient outcome. METHODS: The study population consisted of 495 Stage I-IV non-preoperatively treated rectal cancer patients of which a tissue micro array (TMA) was available. Sections of this TMA were immunohistochemically stained and quantified for presence of Foxp3+ cells (Tregs) and tumor expression of HLA Class I and non-classical HLA-E and HLA-G. All markers were, separate and combined, analyzed for clinical prognostic value. RESULTS: Expression of HLA class I (DFS HR 0.637 (0.458-0.886), p = 0.013), Foxp3+ infiltration above median (OS HR 0.637 (0.500-0.813), p < 0.001 and DFS HR 0.624 (0.491-0.793), p < 0.001) and expression of HLA-G (DFS HR 0.753 (0.574-0.989), p = 0.042) were related to a better clinical prognosis. When these markers were combined, patients with 2 or 3 markers associated with poor prognosis (loss of HLA Class I, Foxp3+ below median, and weak HLA-G expression), showed a significantly worse survival (OS and DFS p < 0.001). This immune-phenotype was an independent predictor for DFS (HR 1.56 (1.14-2.14), p = 0.019). CONCLUSIONS: In conclusion, rectal tumors showing loss of HLA class I expression, Foxp3+ infiltration below median and weak HLA-G expression were related to a worse OS and DFS. Combining these immune markers lead to the creation of tumor immune-phenotypes , which related to patient outcome and were significant independent clinical prognostic markers in rectal cancer.

The Influence of BRAF and KRAS Mutation Status on the Association between Aspirin Use and Survival after Colon Cancer Diagnosis.

BACKGROUND: Use of aspirin after diagnosis of colon cancer has been associated with improved survival. Identification of cancer subtypes that respond to aspirin treatment may help develop personalized treatment regimens. The aim of this study was to investigate the influence of BRAF and KRAS mutation status on the association between aspirin use and overall survival after colon cancer diagnosis. METHODS: A random selection of 599 patients with colon cancer were analyzed, selected from the Eindhoven Cancer Registry, and BRAF and KRAS mutation status was determined. Data on aspirin use (80 mg) were obtained from the PHARMO Database Network. Parametric survival models with exponential (Poisson) distribution were used. RESULTS: Aspirin use after colon cancer diagnosis was associated with improved overall survival in wild-type BRAF tumors, adjusted rate ratio (RR) of 0.60 (95% CI 0.44-0.83). In contrast, aspirin use in BRAF mutated tumors was not associated with an improved survival (RR 1.11, 95% CI 0.57-2.16). P-value for interaction was non-significant. KRAS mutational status did not differentiate in the association between aspirin use and survival. CONCLUSION: Low-dose aspirin use after colon cancer diagnosis was associated with improved survival in BRAF wild-type tumors only. However, the large confidence interval of the rate ratio for the use of aspirin in patients with BRAF mutation does not rule out a possible benefit. These results preclude BRAF and KRAS mutation status to be used as a marker for individualized treatment with aspirin, if aspirin becomes regular adjuvant treatment for colon cancer patients in the future.

Practical and Robust Identification of Molecular Subtypes in Colorectal Cancer by Immunohistochemistry.

PURPOSE: Recent transcriptomic analyses have identified four distinct molecular subtypes of colorectal cancer with evident clinical relevance. However, the requirement for sufficient quantities of bulk tumor and difficulties in obtaining high-quality genome-wide transcriptome data from formalin-fixed paraffin-embedded tissue are obstacles toward widespread adoption of this taxonomy. Here, we develop an immunohistochemistry-based classifier to validate the prognostic and predictive value of molecular colorectal cancer subtyping in a multicenter study. EXPERIMENTAL DESIGN: Tissue microarrays from 1,076 patients with colorectal cancer from four different cohorts were stained for five markers (CDX2, FRMD6, HTR2B, ZEB1, and KER) by immunohistochemistry and assessed for microsatellite instability. An automated classification system was trained on one cohort using quantitative image analysis or semiquantitative pathologist scoring of the cores as input and applied to three independent clinical cohorts. RESULTS: This classifier demonstrated 87% concordance with the gold-standard transcriptome-based classification. Application to three validation datasets confirmed the poor prognosis of the mesenchymal-like molecular colorectal cancer subtype. In addition, retrospective analysis demonstrated the benefit of adding cetuximab to bevacizumab and chemotherapy in patients with RAS wild-type metastatic cancers of the canonical epithelial-like subtypes. CONCLUSIONS: This study shows that a practical and robust immunohistochemical assay can be employed to identify molecular colorectal cancer subtypes and uncover subtype-specific therapeutic benefit. Finally, the described tool is available online for rapid classification of colorectal cancer samples, both in the format of an automated image analysis pipeline to score tumor core staining, and as a classifier based on semiquantitative pathology scoring. Clin Cancer Res; 23(2); 387-98. ©2016 AACR.

Influence of Individual Surgeon Volume on Oncological Outcome of Colorectal Cancer Surgery.

BACKGROUND: Surgery performed by a high-volume surgeon improves short-term outcomes. However, not much is known about long-term effects. Therefore we performed the current study to evaluate the impact of high-volume colorectal surgeons on survival. METHODS: We conducted a retrospective analysis of our prospectively collected colorectal cancer database between 2004 and 2011. Patients were divided into two groups: operated on by a high-volume surgeon (>25 cases/year) or by a low-volume surgeon (<25 cases/year). Perioperative data were collected as well as follow-up, recurrence rates, and survival data. RESULTS: 774 patients underwent resection for colorectal malignancies. Thirteen low-volume surgeons operated on 453 patients and 4 high-volume surgeons operated on 321 patients. Groups showed an equal distribution for preoperative characteristics, except a higher ASA-classification in the low-volume group. A high-volume surgeon proved to be an independent prognostic factor for disease-free survival in the multivariate analysis (P = 0.04). Although overall survival did show a significant difference in the univariate analysis (P < 0.001) it failed to reach statistical significance in the multivariate analysis (P = 0.09). CONCLUSIONS: In our study, a higher number of colorectal cases performed per surgeon were associated with longer disease-free survival. Implementing high-volume surgery results in improved long-term outcome following colorectal cancer.

How does genome sequencing impact surgery?

Cancer is a leading cause of death worldwide. Great efforts are dedicated to the development of prognostic and predictive biomarkers to improve diagnosis and achieve optimal treatment selection, thereby, introducing precision medicine in the multimodality treatment of cancer. Genomic aberrations are the basis of tumour development, representing excellent candidates for the development of promising clinical biomarkers. Over the past decade, single-gene mutations and genomic profiling have been increasingly used in multidisciplinary consultations for risk-assessment and treatment planning for patients with cancer. We discuss the impact of such genetic-based information on surgical decision-making. Single-gene mutations have already influenced surgical decision-making in breast, colorectal and thyroid cancer. However, the direct impact of genomic profiling on surgical care has not yet been fully established. We discuss the direct and indirect influences of genomic profiling on surgery, and analyse the limitations and unresolved issues of a genotypic-approach to the surgical management of cancer.

Validation of the 12-gene colon cancer recurrence score as a predictor of recurrence risk in stage II and III rectal cancer patients.

BACKGROUND: The 12-gene Recurrence Score assay is a validated predictor of recurrence risk in stage II and III colon cancer patients. We conducted a prospectively designed study to validate this assay for prediction of recurrence risk in stage II and III rectal cancer patients from the Dutch Total Mesorectal Excision (TME) trial. METHODS: RNA was extracted from fixed paraffin-embedded primary rectal tumor tissue from stage II and III patients randomized to TME surgery alone, without (neo)adjuvant treatment. Recurrence Score was assessed by quantitative real time-polymerase chain reaction using previously validated colon cancer genes and algorithm. Data were analysed by Cox proportional hazards regression, adjusting for stage and resection margin status. All statistical tests were two-sided. RESULTS: Recurrence Score predicted risk of recurrence (hazard ratio [HR] = 1.57, 95% confidence interval [CI] = 1.11 to 2.21, P = .01), risk of distant recurrence (HR = 1.50, 95% CI = 1.04 to 2.17, P = .03), and rectal cancer-specific survival (HR = 1.64, 95% CI = 1.15 to 2.34, P = .007). The effect of Recurrence Score was most prominent in stage II patients and attenuated with more advanced stage (P(interaction) ≤ .007 for each endpoint). In stage II, five-year cumulative incidence of recurrence ranged from 11.1% in the predefined low Recurrence Score group (48.5% of patients) to 43.3% in the high Recurrence Score group (23.1% of patients). CONCLUSION: The 12-gene Recurrence Score is a predictor of recurrence risk and cancer-specific survival in rectal cancer patients treated with surgery alone, suggesting a similar underlying biology in colon and rectal cancers.

Expression of HLA class I antigen, aspirin use, and survival after a diagnosis of colon cancer.

IMPORTANCE: Use of aspirin (which inhibits platelet function) after a colon cancer diagnosis is associated with improved overall survival. Identifying predictive biomarkers of this effect could individualize therapy and decrease toxic effects. OBJECTIVE: To demonstrate that survival benefit associated with low-dose aspirin use after a diagnosis of colorectal cancer might depend on HLA class I antigen expression. DESIGN, SETTING, AND PARTICIPANTS: A cohort study with tumor blocks from 999 patients with colon cancer (surgically resected between 2002 and 2008), analyzed for HLA class I antigen and prostaglandin endoperoxide synthase 2 (PTGS2) expression using a tissue microarray. Mutation analysis of PIK3CA was also performed. Data on aspirin use after diagnosis were obtained from a prescription database. Parametric survival models with exponential (Poisson) distribution were used to model the survival. MAIN OUTCOMES AND MEASURES: Overall survival. RESULTS: The overall survival benefit associated with aspirin use after a diagnosis of colon cancer had an adjusted rate ratio (RR) of 0.53 (95% CI, 0.38-0.74; P < .001) when tumors expressed HLA class I antigen compared with an RR of 1.03 (0.66-1.61; P = .91) when HLA antigen expression was lost. The benefit of aspirin was similar for tumors with strong PTGS2 expression (0.68; 0.48-0.97; P = .03), weak PTGS2 expression (0.59; 0.38-0.97; P = .02), and wild-type PIK3CA tumors (0.55; 0.40-0.75; P < .001). No association was observed with mutated PIK3CA tumors (0.73; 0.33-1.63; P = .44). CONCLUSIONS AND RELEVANCE: Contrary to the original hypothesis, aspirin use after colon cancer diagnosis was associated with improved survival if tumors expressed HLA class I antigen. Increased PTGS2 expression or the presence of mutated PIK3CA did not predict benefit from aspirin. HLA class I antigen might serve as a predictive biomarker for adjuvant aspirin therapy in colon cancer.

Biomarkers in precision therapy in colorectal cancer.

Colorectal cancer (CRC) is the most commonly diagnosed cancer in Europe. Because CRC is also a major cause of cancer-related deaths worldwide, a lot of research has been focused on the discovery and development of biomarkers to improve the diagnostic process and to predict treatment outcomes. Up till now only a few biomarkers are recommended by expert panels. Current TNM criteria, however, cause substantial under- and overtreatment of CRC patients. Consequently, there is a growing need for new and efficient biomarkers to ensure optimal treatment allocation. An ideal biomarker should be easily translated into clinical practice, to identify patients who can be spared from treatment or benefit from therapy, ultimately resulting in precision medicine in the future. In this review we aim to provide an overview of a number of frequently studied biomarkers in CRC and, at the same time, we will emphasize the challenges and controversies that withhold the clinical introduction of these biomarkers. We will discuss both prognostic and predictive markers of chemotherapy, aspirin therapy as well as overall therapy toxicity. Currently, only mutant KRAS, mutant BRAF, MSI and the Oncotype DX® Colon Cancer Assay are used in clinical practice. Other biomarker studies showed insufficient evidence to be introduced into clinical practice. Divergent patient selection criteria, absence of validation studies and a large number of single biomarker studies are possibly responsible. We therefore recommend that future studies focus on combining key markers, rather than analysing single markers, standardizing study protocols, and validate the results in independent study cohorts, followed by prospective clinical trials.

The prognostic value of the apoptosis pathway in colorectal cancer: a review of the literature on biomarkers identified by immunohistochemistry.

Research towards biomarkers that predict patient outcome in colorectal cancer (CRC) is rapidly expanding. However, none of these biomarkers have been recommended by the American Association of Clinical Oncology or the European Group on Tumor Markers. Current staging criteria result in substantial under-and over-treatment of CRC patients. Evasion of apoptosis, a characteristic feature of tumorigenesis, is known to correlate with patient outcome. We reviewed the literature on immunohistochemistry-based studies between 1998 and 2011 describing biomarkers in this pathway in CRC and identified 26 markers. Most frequently described were p53, Bcl-2, survivin, and the Fas and TRAILR1 receptors and their ligands. None of the studies reviewed provided sufficient support for implementing a single marker into current clinical practice. This is likely due to the complex biology of this pathway. We suggest focusing on the combination of key markers within the apoptosis pathway that together represent an 'apoptotic tumor profile', which better reflects the status of this pathway in a tumor.

Aspirin use after diagnosis improves survival in older adults with colon cancer: a retrospective cohort study.

OBJECTIVES: To assess survival in relation to aspirin use after diagnosis in older adults with colon cancer. DESIGN: Subgroup analysis of a previously published cohort and retrospective study. SETTING: Individuals registered in the Eindhoven Cancer Registry (ECR) between 1998 and 2007, linked to prescriptions of low-dose aspirin (80 mg) registered in a community pharmacy database. PARTICIPANTS: Five hundred thirty-six individuals aged 70 and older diagnosed with colon cancer with or without aspirin use after diagnosis. MEASUREMENTS: Survival was analyzed with user status as a time-dependent covariate. Multivariate Poisson regression survival models were used to study the effect of aspirin on overall survival. RESULTS: One hundred seven participants (20.0%) started aspirin after being diagnosed with colon cancer; 429 (80.0%) were not prescribed aspirin. Three hundred thirty-nine participants (63.2%) had died by the end of follow-up. Aspirin use after diagnosis was associated with longer overall survival (rate ratio (RR) = 0.51, 95% confidence interval (CI) = 0.38-0.70, P < .001). Multivariate proportional hazards regression analysis revealed that aspirin use was associated with longer overall survival (adjusted RR = 0.59, 95% CI = 0.44-0.81, P = .001). CONCLUSION: Aspirin use after the diagnosis of colon cancer in older adults was associated with longer survival. Low-dose aspirin could be used as an effective adjuvant therapy in older adults with colon cancer.