Immunological and prognostic significance of tumour necrosis in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) causes the second most cancer deaths worldwide, but the disease course varies according to tumour characteristics and immunological factors. Our objective was to examine the associations of tumour necrosis with tumour characteristics, immune cell infiltrates, serum cytokine concentrations, as well as prognosis in CRC. METHODS: Three independent CRC cohorts, including 1413 patients, were analysed. Associations of the areal percentage of tumour necrosis with clinicopathologic parameters, tumour infiltrating immune cells, cytokine concentrations in systemic and mesenteric vein blood, and survival were examined. RESULTS: Higher tumour necrosis percentage associated with shorter colorectal cancer-specific survival independent of tumour grade, T, N or M-class, mismatch repair status, BRAF status, and other possible confounding factors. In the largest cohort (N = 1100), the HR for high tumour necrosis percentage (≥40% vs. <3%) was 3.22 (95% CI 1.68-6.17, Ptrend < 0.0001). Tumour necrosis percentage positively correlated with peripheral serum levels of CXCL8, a proinflammatory chemokine, and negatively correlated with mesenteric serum levels of CXCL10 and mast cell densities in the invasive margin of the tumour. CONCLUSIONS: Our results support the value of tumour necrosis as a prognostic factor in colorectal cancer. CXCL8 may have a role in the systemic effects of tumour necrosis.

A prognostic score based on B cell and plasma cell densities compared to T cell densities in colorectal cancer.

PURPOSE: The purpose of this study was to compare a B cell/plasma cell-based scoring system to T cell score and evaluate their prognostic value in colorectal cancer. METHODS: We used immunohistochemistry to analyze the expression of CD20, CD138, CD3, and CD8 in 221 colorectal cancer patients. CD20+ B cell and CD138+ plasma cell densities in the tumor center and invasive margin were calculated and converted into a B cell/plasma cell score. T cell score was defined similarly, using CD3+ and CD8+ T cell densities. Their associations with tumor and patient characteristics and survival were analyzed. RESULTS: Kaplan-Meier analysis showed a high B cell/plasma cell score was associated with a tendency towards longer survival (p = 0.089), but no statistically significant association was found. High T cell score associated with longer cancer-specific survival in Kaplan-Meier analysis and multivariable Cox regression analysis (p < 0.001). Additionally, high T cell score associated with lower disease stage (p < 0.001) and lesser lymphovascular invasion (p = 0.020). CONCLUSIONS: High T cell score is associated with longer survival and clinicopathological factors typical to less aggressive tumors. This study did not support the additional prognostic value of B cell/plasma cell quantification.

Perioperative oral nutritional support for patients diagnosed with primary colon adenocarcinoma undergoing radical surgical procedures -Peri-Nutri Trial: study protocol for a randomized controlled trial.

BACKGROUND: Colon cancer is one of the most common cancers in Finland and worldwide. Cancer-related malnutrition is a well-known risk factor for increased morbidity and mortality after surgery, and it is associated with complications and longer hospitalizations. There are no established recommendations on how to improve the nutritional status of colon cancer patients´ during the perioperative phase. Administration of simple oral nutritional supplements has been suggested to reduce complication rates, but evidence to support this practice is scarce. METHODS: The Peri-Nutri trial is a prospective, multicenter, randomized, controlled trial. Its primary endpoint is to evaluate whether perioperative oral nutritional support (ONS) decreases the number of complications during the 30-day follow-up after surgery. Secondary endpoints are to study the effect of ONS on quality of life after surgery, length of stay in institutional care, 90-day mortality rate, five-year disease-free survival and overall survival. The patients with a Nutritional risk screening 2002 (NRS-2002) questionnaire result between 2 and 5 (≥ 3 are classified at risk of malnutrition) will be randomized (1:1 ratio) into either the intervention or control group. The intervention group will receive preoperative ONS two weeks before the operation, and nutritional support will continue 10 days after the operation. The control group will not receive ONS. A total of 318 patients will be randomized into two groups and patients are followed five years. DISCUSSION: Peri-Nutri study evaluate the impact of ONS to short-term and long-term postoperative morbidity and mortality rates of colon cancer patients undergoing curative surgery. If ONS will decrease patients´ morbidity and mortality, that has a huge impact on patients´ quality of life and also to financial cost. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03863236 , Registered 25 February 2019.

Putative anoikis resistant subpopulations are enriched in lymph node metastases and indicate adverse prognosis in colorectal carcinoma.

Anoikis refers to apoptosis induced by the loss of contact with the extracellular matrix. Anoikis resistance is essential for metastasis. We have recently shown that it is possible to quantitatively evaluate putative anoikis resistant (AR) subpopulations in colorectal carcinoma (CRC). Abundance of these multi-cell structures is an independent marker of adverse prognosis. Here, we have quantified putative AR subpopulations in lymph node (LN) metastases of CRC and evaluated their prognostic value and relationship with the characteristics of primary tumors. A case series included 137 unselected CRC patients, 54 with LN metastases. Areal densities (structures/mm2) of putative AR structures in primary tumors had been analyzed previously and now were determined from all nodal metastases (n = 183). Areal density of putative AR structures was higher in LN metastases than in primary tumors. Variation of the areal density within different LN metastases of a single patient was lower than between metastases of different patients. Abundance of putative AR structures in LN metastases was associated with shorter cancer specific survival (p = 0.013), and this association was independent of T and N stages. Abundance of putative AR structures in primary tumors and LN metastases had a cumulative adverse effect on prognosis. Enrichment of putative AR subpopulations in LN metastases suggest that in metastasis formation, there is a selection favoring cells capable of forming these structures. Higher intra-case constancy relative to inter-case variation suggests that such selection is stable in metastasis development. Our findings indirectly support the biological validity of our concept of putative AR structures.

Sarcopenia and Myosteatosis Are Associated with Neutrophil to Lymphocyte Ratio but Not Glasgow Prognostic Score in Colorectal Cancer Patients.

Cancer patients commonly present sarcopenia, myosteatosis, and systemic inflammation, which are risk factors of poor survival. In this study, sarcopenia and myosteatosis were defined from preoperative body computed tomography scans of 222 colorectal cancer (CRC) patients and analyzed in relation to tumor and patient characteristics, markers of systemic inflammation (modified Glasgow prognostic score (mGPS), neutrophil−lymphocyte ratio (NLR), serum levels of C-reactive protein (CRP), albumin, and 13 cytokines, and survival. Of the systemic inflammation markers, sarcopenia and/or myosteatosis associated with elevated NLR (p = 0.005) and low albumin levels (≤35 g/L) (p = 0.018), but not with mGPS or serum cytokine levels. In addition, myosteatosis was associated with a proximal tumor location (p = 0.039), serrated tumor subtype (p < 0.001), and severe comorbidities (p = 0.004). Multivariable analyses revealed that severe comorbidities and serrated histology were independent predictors of myosteatosis, and older age and elevated NLR were independent indicators of sarcopenia. Myosteatosis associated with shorter overall survival in univariable analysis (HR 1.959, 95% CI 1.24−3.10, p = 0.004) but not in multivariable analysis (p = 0.075). We conclude that sarcopenia and myosteatosis were associated with inflammatory marker NLR, but not with mGPS. Moreover, patients with serrated CRC may have an increased risk of myosteatosis. Myosteatosis or sarcopenia were not independent predictors of patient survival.

Global Methylome Scores Correlate with Histological Subtypes of Colorectal Carcinoma and Show Different Associations with Common Clinical and Molecular Features.

BACKGROUND: The typical methylation patterns associated with cancer are hypermethylation at gene promoters and global genome hypomethylation. Aberrant CpG island hypermethylation at promoter regions and global genome hypomethylation have not been associated with histological colorectal carcinomas (CRC) subsets. Using Illumina's 450 k Infinium Human Methylation beadchip, the methylome of 82 CRCs were analyzed, comprising different histological subtypes: 40 serrated adenocarcinomas (SAC), 32 conventional carcinomas (CC) and 10 CRCs showing histological and molecular features of microsatellite instability (hmMSI-H), and, additionally, 35 normal adjacent mucosae. Scores reflecting the overall methylation at 250 bp, 1 kb and 2 kb from the transcription starting site (TSS) were studied. RESULTS: SAC has an intermediate methylation pattern between CC and hmMSI-H for the three genome locations. In addition, the shift from promoter hypermethylation to genomic hypomethylation occurs at a small sequence between 250 bp and 1 Kb from the gene TSS, and an asymmetric distribution of methylation was observed between both sides of the CpG islands (N vs. S shores). CONCLUSION: These findings show that different histological subtypes of CRC have a particular global methylation pattern depending on sequence distance to TSS and highlight the so far underestimated importance of CpGs aberrantly hypomethylated in the clinical phenotype of CRCs.

SLC4A2 anion exchanger promotes tumour cell malignancy via enhancing net acid efflux across golgi membranes.

Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive "H+ leak" pathway. Here, we show that this proton leak across Golgi membranes is mediated by the AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on bicarbonate import (in exchange for chloride export) and the expression level of the Golgi-localized AE2a anion exchanger. In the acidic Golgi lumen, imported bicarbonate anions and protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The flattened morphology of the Golgi cisternae helps this process, as their high surface-volume ratio is optimal for water and gas exchange. Interestingly, this net acid efflux pathway is often upregulated in cancers and established cancer cell lines, and responsible for their markedly elevated Golgi resting pH and attenuated glycosylation potential. Accordingly, AE2 knockdown in SW-48 colorectal cancer cells was able to restore these two phenomena, and at the same time, reverse their invasive and anchorage-independent growth phenotype. These findings suggest a possibility to return malignant cells to a benign state by restoring Golgi resting pH.

Systemic inflammation is associated with circulating cell death released keratin 18 fragments in colorectal cancer.

Systemic inflammation is a stage-independent marker of poor prognosis in colorectal cancer (CRC), activated in a complex, multifactorial process. It has been proposed that one of the main factors driving systemic inflammation may be tumor necrosis. Keratin 18 (KRT18) fragments are released from dead cells and their serum levels are markers for apoptotic and necrotic cell death. In CRC, high KRT18 levels associate with advanced disease, but their relationship with tumor necrosis and systemic inflammation is unknown. In this study, serum total soluble KRT18 (tKRT18) and apoptosis-related, caspase-cleaved fragment (aKRT18) levels were measured preoperatively from 328 CRC patients, and their difference was calculated to assess necrosis related KRT18 (nKRT18) levels. The relationships of these markers with tumor necrosis, clinicopathologic features, systemic inflammation markers (C-reactive protein, albumin, and 13 cytokines), and survival were analyzed. High serum tKRT18, aKRT18, and nKRT18 levels showed association with a higher extent of tumor necrosis, distant metastasis, and increased levels of several markers of systemic inflammation, including CXCL8. High serum tKRT18 (multivariable HR 1.94, 95% CI 1.28-2.95, p = .002) and nKRT18 (multivariable HR 1.87, 95% CI 1.24-2.82, p = .003) levels were associated with poor overall survival independent of potential confounding factors. Our results show that tumor necrosis in CRC contributes to serum levels of KRT18 fragments, and both necrosis and KRT18 levels associate with systemic inflammation. Moreover, we show that serum tKRT18 and nKRT18 levels have independent prognostic value in CRC. Our observations confirm the link between cell death and systemic inflammation.

Putative anoikis-resistant subpopulations in colorectal carcinoma: a marker of adverse prognosis.

Anoikis is a form of apoptosis induced when a cell loses contact with the extracellular matrix (ECM). Anoikis resistance is essential for metastasis formation, yet only detectable by in vitro experiments. We present a method for quantitation of putative anoikis-resistant (AR) subpopulations in colorectal carcinoma (CRC) and evaluate their prognostic significance. We studied 137 CRC cases and identified cell subpopulations with and without stromal or extracellular matrix (ECM) contact with hematoxylin-and-eosin-stained sections and immunohistochemistry for laminin and type IV collagen. Suprabasal cells of micropapillary structures and inner cells of cribriform and solid structures lacked both stromal contact and contact with ECM proteins. Apoptosis rate (M30) was lower in these subpopulations than in the other carcinoma cells, consistent with putative AR subpopulation. We determined the areal density of these subpopulations (number/mm2 tumor tissue), and their high areal density independently indicates low cancer-specific survival. In conclusion, we show evidence that subpopulations of carcinoma cells in micropapillary, cribriform, and solid structures are resistant to anoikis as shown by lack of ECM contact and low apoptosis rate. Abundance of these subpopulations is a new independent indicator of poor prognosis in CRC, consistent with the importance of anoikis resistance in the formation of metastasis.

ColPortal, an integrative multiomic platform for analysing epigenetic interactions in colorectal cancer.

Colorectal cancer (CRC) is the third leading cause of cancer mortality worldwide. Different pathological pathways and molecular drivers have been described and some of the associated markers are used to select effective anti-neoplastic therapy. More recent evidence points to a causal role of microbiota and altered microRNA expression in CRC carcinogenesis, but their relationship with pathological drivers or molecular phenotypes is not clearly established. Joint analysis of clinical and omics data can help clarify such relations. We present ColPortal, a platform that integrates transcriptomic, microtranscriptomic, methylomic and microbiota data of patients with colorectal cancer. ColPortal also includes detailed information of histological features and digital histological slides from the study cases, since histology is a morphological manifestation of a complex molecular change. The current cohort consists of Caucasian patients from Europe. For each patient, demographic information, location, histology, tumor staging, tissue prognostic factors, molecular biomarker status and clinical outcomes are integrated with omics data. ColPortal allows one to perform multiomics analyses for groups of patients selected by their clinical data.

Systemic inflammation in colorectal cancer: Underlying factors, effects, and prognostic significance.

Systemic inflammation is a marker of poor prognosis preoperatively present in around 20%-40% of colorectal cancer patients. The hallmarks of systemic inflammation include an increased production of proinflammatory cytokines and acute phase proteins that enter the circulation. While the low-level systemic inflammation is often clinically silent, its consequences are many and may ultimately lead to chronic cancer-associated wasting, cachexia. In this review, we discuss the pathogenesis of cancer-related systemic inflammation, explore the role of systemic inflammation in promoting cancer growth, escaping antitumor defense, and shifting metabolic pathways, and how these changes are related to less favorable outcome.

KRAS and BRAF mutations induce anoikis resistance and characteristic 3D phenotypes in Caco­2 cells.

In a number of types of cancer, anoikis, a form of apoptosis induced by loss of extracellular matrix (ECM) attachment, is disturbed. Anoikis resistance is essential in the formation of metastases. A recent study identified carcinoma cell subpopulations surviving without ECM contact in pathological specimens of colorectal cancer. The occurrence of these subpopulations indicated anoikis resistance. In the present study, it is demonstrated that KRAS and BRAF mutations induce anoikis resistance in colon cancer (Caco­2) cells. In 3D cultures, Caco­2 cells transfected with mutated KRAS or BRAF formed multicellular structures analogous to anoikis­resistant subpopulations in actual carcinomas, and serve as an in vitro model for anoikis resistance. Caco­2 cell lines were constructed, with KRAS or BRAF mutations, using retroviral delivery. The current study investigated anoikis resistance using an Annexin V apoptosis test from suspension cultures. 3D in vitro cultures, which were generated in collagen­matrigel mixtures, were assessed using confocal microscopy. 3D cultures embedded in paraffin were analyzed using conventional histopathology. In suspension cultures, Caco­2 cells with KRAS or BRAF mutations indicated a significantly lower proportion of Annexin positivity than the native Caco­2 cells, indicating that these mutations induce anoikis resistance in Caco­2 cells. 3D cultures displayed native Caco­2 cells forming polarized cysts with a single layer thick epithelium, whereas Caco­2 cells with KRAS or BRAF mutations formed partially filled cystic structures or solid round structures where only the outermost layer was in contact with the ECM. Additionally, KRAS mutations induced reversed polarity to Caco­2 cells along with the emergence of solid growth. The present study demonstrated that KRAS and BRAF mutations induce anoikis resistance in Caco­2 colorectal cancer cells. The growth patterns generated from the KRAS and BRAF mutated cells in 3D cultures revealed a resemblance to the putative anoikis­resistant subpopulations in actual carcinomas, including micropapillary structures and solid tumor cell islands. Additionally, KRAS mutation induced the emergence of inverted polarity. In conclusion, 3D cultures with modified Caco­2 cells serve as a valid in vitro model for anoikis resistance and inverted polarity.

Serum enterolactone concentrations are low in colon but not in rectal cancer patients.

The dietary lignan metabolite, enterolactone, has been suggested to have anti-cancer functions, and high serum enterolactone concentrations have been associated with decreased risk of breast and prostate cancers. We hypothesized that serum enterolactone concentrations as a marker of plant-based foods are associated with decreased risk in colorectal cancer (CRC). We measured serum enterolactone glucuronide and sulfate concentrations by liquid chromatography-tandem mass spectrometry in 115 CRC patients and 76 sex- and age-matched controls and analyzed the results with respect to tumor parameters, clinical parameters, and systemic inflammatory markers. Patients with colon cancer had significant lower serum enterolactone glucuronide and sulfate concentrations than controls (glucuronide: median 3.14 nM vs. 6.32 nM, P < 0.001; sulfate: median 0.13 nM vs. 0.17 nM, P = 0.002), whereas rectal cancer patients had similar enterolactone levels as controls (glucuronide: median 5.39 nM vs. 6.32 nM, P = 0.357; sulfate: median 0.19 nM vs. 0.17 nM, P = 0.452). High serum enterolactone concentrations were associated with low tumor grade, high serum creatinine levels, and concomitant diabetes. In summary, our results suggest that serum enterolactone concentrations are decreased in colon but not in rectal cancer. Further investigations are required to assess whether this reflects an altered lignan metabolism by the colon microbiome.

Platelet count, aspirin use, and characteristics of host inflammatory responses in colorectal cancer.

BACKGROUND: Platelets not only contribute to hemostasis but also to the regulation of inflammatory reactions and cancer pathogenesis. We hypothesized that blood platelet count would be associated with systemic inflammation, the densities of tumor infiltrating immune cells, and survival in colorectal cancer (CRC), and these relationships could be altered by aspirin use. METHODS: We measured blood platelet count in a cohort of 356 CRC patients and analyzed its relationships with tumor and patient characteristics including aspirin use, markers of systemic inflammation (modified Glasgow Prognostic Score, mGPS; serum levels of CRP, albumin, and 13 cytokines), blood hemoglobin levels, five types of tumor infiltrating immune cells (CD3, CD8, FoxP3, Neutrophil elastase, mast cell tryptase), and survival. RESULTS: Platelet count inversely correlated with blood hemoglobin levels (p < 0.001) and positively correlated with serum levels of CRP and multiple cytokines including IL-1RA, IL-4, IL-6, IL-7, IL-8, IL-12, IFNγ, and PDGF-BB (p < 0.001 for all), while aspirin use was not associated with the levels of systemic inflammatory markers. High platelet count was also associated with high mGPS (p < 0.001) but did not show statistically significant multivariable adjusted associations with the densities of tumor infiltrating immune cells. Higher platelet counts were observed in higher tumor stage (p < 0.001), but platelet count or aspirin use were not associated with patient survival. CONCLUSIONS: High platelet count is associated with systemic inflammation in CRC. This study could not demonstrate statistically significant associations between platelet count, aspirin use, and the densities of tumor infiltrating immune cells.

Serum TLR2 and TLR4 levels in colorectal cancer and their association with systemic inflammatory markers, tumor characteristics, and disease outcome.

Toll-like receptors (TLRs) are involved in colorectal cancer (CRC) pathogenesis. However, the significance of serum TLR concentrations in CRC is unknown. We analyzed serum TLR2 and TLR4 concentrations with ELISA in preoperative samples from 118 patients with CRC and 88 matched controls. We also assessed tissue TLR expression with immunohistochemistry and by detecting serum determinants of systemic inflammation. Most participants (>70%) had undetectable serum TLR2. The mean serum TLR4 levels were lower in patients than in controls (1.1 vs 1.8 ng/mL; p = 0.015). Undetectable TLR4 was more common in stage I (39%) than in stages II-IV (11%, p < 0.001). TLR2 or TLR4 expression in tumor cells did not correlate with serum levels, but abundant TLR2 expression in normal colon epithelium was associated with detectable serum TLR2 (p = 0.034). Undetectable serum TLR2 was linked to high modified Glasgow prognostic scores (p = 0.010), high CRP levels (p = 0.013), blood vessel invasion (p = 0.013), and tended to be associated with worse 5-year survival (p = 0.052). In conclusion, serum TLR2 levels were inversely associated with systemic inflammation in patients with CRC. Moreover, serum TLR2 levels might depend more on normal colorectal mucosa contributions than on tumor tissue contributions. Further studies are required to assess the prognostic value of serum TLR2.

ß6 -integrin serves as a novel serum tumor marker for colorectal carcinoma.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide and the need for novel biomarkers and therapeutic strategies to improve diagnosis and surveillance is obvious. This study aims to identify ß6 -integrin (ITGB6) as a novel serum tumor marker for diagnosis, prognosis, and surveillance of CRC. ITGB6 serum levels were validated in retro- and prospective CRC patient cohorts. ITGB6 serum levels were analyzed by ELISA. Using an initial cohort of 60 CRC patients, we found that ITGB6 is present in the serum of CRC, but not in non-CRC control patients. A cut-off of ≥2 ng/mL ITGB6 reveals 100% specificity for the presence of metastatic CRC. In an enlarged study cohort of 269 CRC patients, ITGB6 predicted the onset of metastatic disease and was associated with poor prognosis. Those data were confirmed in an independent, prospective cohort consisting of 40 CRC patients. To investigate whether ITGB6 can also be used for tumor surveillance, serum ITGB6-levels were assessed in 26 CRC patients, pre- and post-surgery, as well as during follow-up visits. After complete tumor resection, ITGB6 serum levels declined completely. During follow-up, a new rise in ITGB6 serum levels indicated tumor recurrence or the onset of new metastasis as confirmed by CT scan. ITGB6 was more accurate for prognosis of advanced CRC and for tumor surveillance as the established marker carcinoembryonic antigen (CEA). Our findings identify ITGB6 as a novel serum marker for diagnosis, prognosis, and surveillance of advanced CRC. This might essentially contribute to an optimized patient care.

Alterations in serum amino-acid profile in the progression of colorectal cancer: associations with systemic inflammation, tumour stage and patient survival.

BACKGROUND: Cancer cachexia is a complex wasting syndrome affecting patients with advanced cancer, with systemic inflammation as a key component in pathogenesis. Protein degradation and release of amino acids (AAs) in skeletal muscle are stimulated in cachexia. Here, we define factors contributing to serum AA levels in colorectal cancer (CRC). METHODS: Serum levels of nine AAs were characterised in 336 CRC patients and their relationships with 20 markers of systemic inflammatory reaction, clinicopathological features of cancers and patient survival were analysed. RESULTS: Low serum glutamine and histidine levels and high phenylalanine levels associated with indicators of systemic inflammation, including high modified Glasgow Prognostic Score, high blood neutrophil/lymphocyte ratio and high serum levels of CRP, IL-6 and IL-8. Low levels of serum glutamine, histidine, alanine and high glycine levels also associated with advanced cancer stage and with poor cancer-specific survival in univariate analysis. CONCLUSIONS: In CRC, serum AA levels are associated with systemic inflammation and disease stage. These findings may reflect muscle catabolism induced by systemic inflammation in CRC.

Two histologically colorectal carcinomas subsets from the serrated pathway show different methylome signatures and diagnostic biomarkers.

BACKGROUND: Altered methylation patterns are driving forces in colorectal carcinogenesis. The serrated adenocarcinoma (SAC) and sporadic colorectal carcinoma showing histological and molecular features of microsatellite instability (hmMSI-H) are two endpoints of the so-called serrated pathological route sharing some characteristics but displaying a totally different immune response and clinical outcome. However, there are no studies comparing the methylome of these two subtypes of colorectal carcinomas. The methylation status of 450,000 CpG sites using the Infinium Human Methylation 450 BeadChip array was investigated in 48 colorectal specimens, including 39 SACs and 9 matched hmMSI-H. RESULTS: Microarray data comparing SAC and hmMSI-H showed an enrichment in functions related to morphogenesis, neurogenesis, cytoskeleton, metabolism, vesicle transport and immune response and also significant differential methylation of 1540 genes, including CD14 and HLA-DOA which were more methylated in hmMSI-H than in SAC and subsequently validated at the CpG, mRNA and protein level using pyrosequencing, quantitative polymerase chain reaction (qPCR) and immunohistochemistry. CONCLUSIONS: These results demonstrate particular epigenetic regulation patterns in SAC which may help to define key molecules responsible for the characteristic weak immune response of SAC and identify potential targets for treating SAC, which lacks molecular targeted therapy.

High-serum MMP-8 levels are associated with decreased survival and systemic inflammation in colorectal cancer.

BACKGROUND: Matrix metalloproteinase-8 (MMP-8) is a protease mainly expressed by neutrophils that cleaves numerous substrates, including collagens and cytokines. We have previously shown that serum MMP-8 levels increase in colorectal cancer (CRC) and correlate with distant metastasis. However, short follow-up in our prospective cohort did not enable survival analyses at the time of the first publication. METHODS: Preoperative serum MMP-8 levels were measured by immunofluorometric assay in 271 CRC patients and related to clinicopathological parameters, markers of systemic inflammation (modified Glasgow Prognostic Score, mGPS; serum levels of C-reactive protein (CRP), albumin and 13 cytokines), the density of six types of tumour-infiltrating immune cells and survival. RESULTS: Increased MMP-8 levels associated with higher mGPS and higher serum levels of CRP and several cytokines, including IL-1ra, IL-7 and IL-8 (p < 0.001 for all). Serum MMP-8 negatively correlated with tumour-infiltrating mast cells (invasive margin: p = 0.005, tumour centre: p = 0.010). The patients with high-serum MMP-8 levels (>100 ng/mL) had poor cancer-specific survival, independent of tumour stage, grade, lymphatic invasion, patient age, BRAF VE1 immunohistochemistry, mismatch repair deficiency, Immunoscore and mGPS (multivariate HR 2.12, 95% CI 1.21-3.71, p = 0.009). CONCLUSIONS: High-serum MMP-8 levels are associated with systemic inflammation and adverse outcome in CRC.