Atypical (non-V600E) BRAF mutations in metastatic colorectal cancer in population and real-world cohorts.

BRAF-V600E mutation (mt) is a strong negative prognostic and predictive biomarker in metastatic colorectal cancer (mCRC). Non-V600Emt, designated atypical BRAFmt (aBRAFmt) are rare, and little is known about their frequency, co-mutations and prognostic and predictive role. These were compared between mutational groups of mCRC patients collected from three Nordic population-based or real-world cohorts. Pathology of aBRAFmt was studied. The study included 1449 mCRC patients with 51 (3%) aBRAFmt, 182 (13%) BRAF-V600Emt, 456 (31%) RAS&BRAF wild-type (wt) and 760 (52%) RASmt tumours. aBRAFmt were seen in 2% of real-world and 4% of population-based cohorts. Twenty-six different aBRAFmt were detected, 11 (22%) class 2 (serrated adenocarcinoma in 2/9 tested), 32 (64%) class 3 (serrated in 15/25) and 4 (8%) unclassified. aBRAFmt patients were predominantly male, had more rectal primaries, less peritoneal metastases, deficient mismatch repair in one (2%), and better survival after metastasectomy (89% 5-year overall survival [OS]-rate) compared with BRAF-V600Emt. aBRAFmt and BRAF-V600Emt had poorer performance status and received fewer treatment lines than RAS&BRAFwt and RASmt. OS among aBRAFmt (median 14.4 months) was longer than for BRAF-V600Emt (11.2 months), but shorter than for RAS&BRAFwt (30.5 months) and RASmt (23.4 months). Addition of bevacizumab trended for better OS for the aBRAFmt. Nine patients with aBRAFmt received cetuximab/panitumumab without response. aBRAFmt represents a distinct subgroup differing from other RAS/BRAF groups, with serrated adenocarcinoma in only half. OS for patients with aBRAFmt tumours was slightly better than for BRAF-V600Emt, but worse than for RASmt and RAS&BRAFwt. aBRAFmt should not be a contraindication for metastasectomy.

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.

Immunohistochemical Glucagon-like Peptide-1 Receptor Expression in Human Insulinomas.

Insulinomas are rare functional pancreatic neuroendocrine tumours, which metastasize in 10% of cases. As predicting the prognosis can be challenging, there is a need for the determination of clinicopathological factors associated with metastatic potential. The aim of this study is to evaluate the glucagon-like peptide-1 receptor (GLP-1R) expression in insulinomas and to analyse its association with clinicopathological features and patient outcome. This retrospective study involves pancreatic tumour tissue samples from fifty-two insulinoma patients. After histological re-evaluation, formalin-fixed paraffin-embedded tissue samples were processed into tissue microarrays and stained immunohistochemically with a monoclonal GLP-1R antibody. Forty-eight of the forty-nine (98%) non-metastatic tumours expressed GLP-1R, while one non-metastatic, multiple endocrine neoplasia type 1 (MEN1)-related tumour and all three of the metastatic tumours lacked GLP-1R expression. The lack of GLP-1R expression was associated with impaired overall survival, larger tumour diameter, higher Ki-67 PI and weaker insulin staining. Somatostatin receptor 1-5 expression did not differ between GLP-1R-positive and GLP-1R-negative insulinomas. In conclusion, the lack of GLP-1R expression is associated with metastatic disease and impaired survival in insulinoma patients. Thus, GLP-1R expression could be a useful biomarker in estimating the metastatic potential of the tumour and the prognosis of surgically treated patients.

Prognostic significance of spatial and density analysis of T lymphocytes in colorectal cancer.

BACKGROUND: Although high T cell density is a strong favourable prognostic factor in colorectal cancer, the significance of the spatial distribution of T cells is incompletely understood. We aimed to evaluate the prognostic significance of tumour cell-T cell co-localisation and T cell densities. METHODS: We analysed CD3 and CD8 immunohistochemistry in a study cohort of 983 colorectal cancer patients and a validation cohort (N = 246). Individual immune and tumour cells were identified to calculate T cell densities (to derive T cell density score) and G-cross function values, estimating the likelihood of tumour cells being co-located with T cells within 20 µm radius (to derive T cell proximity score). RESULTS: High T cell proximity score associated with longer cancer-specific survival in both the study cohort [adjusted HR for high (vs. low) 0.33, 95% CI 0.20-0.52, Ptrend < 0.0001] and the validation cohort [adjusted HR for high (vs. low) 0.15, 95% CI 0.05-0.45, Ptrend < 0.0001] and its prognostic value was independent of T cell density score. CONCLUSIONS: The spatial point pattern analysis of tumour cell-T cell co-localisation could provide detailed information on colorectal cancer prognosis, supporting the value of spatial measurement of T cell infiltrates as a novel, robust tumour-immune biomarker.

Resectability, conversion, metastasectomy and outcome according to RAS and BRAF status for metastatic colorectal cancer in the prospective RAXO study.

BACKGROUND: Outcomes after metastasectomy for metastatic colorectal cancer (mCRC) vary with RAS and BRAF mutational status, but their effects on resectability and conversion rates have not been extensively studied. METHODS: This substudy of the prospective RAXO trial included 906 patients recruited between 2011 and 2018. We evaluated repeated centralised resectability assessment, conversion/resection rates and overall survival (OS), according to RAS and BRAF status. RESULTS: Patients included 289 with RAS and BRAF wild-type (RAS and BRAFwt), 529 with RAS mutated (RASmt) and 88 with BRAF mutated (BRAFmt) mCRC. Metastatic prevalence varied between the RAS and BRAFwt/RASmt/BRAFmt groups, for liver (78%/74%/61%), lung (24%/35%/28%) and peritoneal (15%/15%/32%) metastases, respectively. Upfront resectability (32%/29%/15%), conversion (16%/13%/7%) and resection/local ablative therapy (LAT) rates (45%/37%/17%) varied for RASa and BRAFwt/RASmt/BRAFmt, respectively. Median OS for patients treated with resection/LAT (n = 342) was 83/69/30 months, with 5-year OS-rates of 67%/60%/24%, while systemic therapy-only patients (n = 564) had OS of 29/21/15 months with 5-year OS-rates of 11%/6%/2% in RAS and BRAFwt/RASmt/BRAFmt, respectively. Resection/LAT was associated with improved OS in all subgroups. CONCLUSIONS: There were significant differences in resectability, conversion and resection/LAT rates according to RAS and BRAF status. OS was also significantly longer for RAS and BRAFwt versus either mutant. Patients only receiving systemic therapy had poorer long-term survival, with variation according to molecular status. CLINICAL TRIAL REGISTRATION: NCT01531621/EudraCT2011-003158-24.

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.

Seasonal Regulation of Metabolism: The Effect of Wintertime Fasting and Autumnal Fattening on Key Central Regulators of Metabolism and the Metabolic Profile of the Raccoon Dog (Nyctereutes Procyonoides).

Investigations into the mechanisms regulating obesity are frantic and novel translational approaches are needed. The raccoon dog (Nyctereutes procyonoides) is a canid species representing a promising model to study metabolic regulation in a species undergoing cycles of seasonal obesity and fasting. To understand the molecular mechanisms of metabolic regulation in seasonal adaptation, we analyzed key central nervous system and peripheral signals regulating food intake and metabolism from raccoon dogs after autumnal fattening and winter fasting. Expressions of neuropeptide Y (NPY), orexin-2 receptor (OX2R), pro-opiomelanocortin (POMC) and leptin receptor (ObRb) were analyzed as examples of orexigenic and anorexigenic signals using qRT-PCR from raccoon dog hypothalamus samples. Plasma metabolic profiles were measured with 1H NMR-spectroscopy and LC-MS. Circulating hormones and cytokines were determined with canine specific antibody assays. Surprisingly, NPY and POMC were not affected by the winter fasting nor autumn fattening and the metabolic profiles showed a remarkable equilibrium, indicating conserved homeostasis. However, OX2R and ObRb expression changes suggested seasonal regulation. Circulating cytokine levels were not increased, demonstrating that the autumn fattening did not induce subacute inflammation. Thus, the raccoon dog developed seasonal regulatory mechanisms to accommodate the autumnal fattening and prolonged fasting making the species unique in coping with the extreme environmental challenges.

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.

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.

Mlh1 deficiency in normal mouse colon mucosa associates with chromosomally unstable colon cancer.

Colorectal cancer (CRC) genome is unstable and different types of instabilities, such as chromosomal instability (CIN) and microsatellite instability (MSI) are thought to reflect distinct cancer initiating mechanisms. Although 85% of sporadic CRC reveal CIN, 15% reveal mismatch repair (MMR) malfunction and MSI, the hallmarks of Lynch syndrome with inherited heterozygous germline mutations in MMR genes. Our study was designed to comprehensively follow genome-wide expression changes and their implications during colon tumorigenesis. We conducted a long-term feeding experiment in the mouse to address expression changes arising in histologically normal colonic mucosa as putative cancer preceding events, and the effect of inherited predisposition (Mlh1+/-) and Western-style diet (WD) on those. During the 21-month experiment, carcinomas developed mainly in WD-fed mice and were evenly distributed between genotypes. Unexpectedly, the heterozygote (B6.129-Mlh1tm1Rak) mice did not show MSI in their CRCs. Instead, both wildtype and heterozygote CRC mice showed a distinct mRNA expression profile and shortage of several chromosomal segregation gene-specific transcripts (Mlh1, Bub1, Mis18a, Tpx2, Rad9a, Pms2, Cenpe, Ncapd3, Odf2 and Dclre1b) in their colon mucosa, as well as an increased mitotic activity and abundant numbers of unbalanced/atypical mitoses in tumours. Our genome-wide expression profiling experiment demonstrates that cancer preceding changes are already seen in histologically normal colon mucosa and that decreased expressions of Mlh1 and other chromosomal segregation genes may form a field-defect in mucosa, which trigger MMR-proficient, chromosomally unstable CRC.

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.

The relationships between serum cytokine levels and tumor infiltrating immune cells and their clinical significance in colorectal cancer.

Increased inflammatory cell infiltration correlates to improved survival in colorectal cancer (CRC). Development and progression of CRC is associated with alterations in serum cytokine levels but their significance is not well defined. In this study, we investigated the relationships between the serum levels of 13 cytokines and the densities of eight types of tumor infiltrating inflammatory cells and their impact on disease-free survival (DFS), cancer-specific survival (CSS) and overall survival (OS) in a prospectively recruited group of 147 CRC patients. There were strong positive correlations between the serum concentrations of different cytokines, as well as between the different types of tumor infiltrating immune cells, whereas the associations between serum cytokines and tumor infiltrating immune cells were generally weak. High serum IL-12 levels associated with increased densities of peritumoral CD8(+) T cells, intraepithelial CD3(+) T cells and intratumoral neutrophils, while high serum CCL4 levels associated with increased densities of peritumoral CD68(+) cells. In multivariate survival models, increased infiltration of intraepithelial CD3(+) T cells and increased serum CCL4 associated with improved DFS, whereas higher intratumoral CD83(+) dendritic cell density and increased serum interferon gamma levels associated with improved CSS and OS. Also high density of peritumoral CD3(+) T cells associated with improved CSS. In conclusion, serum cytokines and tumor infiltrating immune cells in CRC represent entities with high intragroup correlations but relatively weak intergroup correlations. The results suggest that tumor infiltrating CD3(+) T cells, CD83(+) dendritic cells, serum CCL4 and serum interferon gamma represent relevant markers of disease outcome.

Clinical impact and network of determinants of tumour necrosis in colorectal cancer.

BACKGROUND: The disease outcome in colorectal cancer (CRC) can vary in a wide range within the same tumour stage. The aim of this study was to clarify the prognostic value and the determinants of tumour necrosis in CRC. METHODS: The areal proportion (%) of tumour tissue showing coagulative necrosis was evaluated in a cohort of 147 CRC patients and correlated with basic clinicopathological characteristics, microvascular density (MVD), cell proliferation rate, KRAS and BRAF mutations, and survival. To validate the prognostic significance of tumour necrosis, an independent cohort of 418 CRC patients was analysed. RESULTS: Tumour necrosis positively correlated with tumour stage (P=8.5E-4)-especially with T class (4.0E-6)-and inversely correlated with serrated histology (P=0.014), but did not significantly associate with cell proliferation rate, MVD, and KRAS or BRAF mutation. Abundant (10% or more) tumour necrosis associated with worse disease-free survival independent of stage and other biological or clinicopathological characteristics in both cohorts, and the adverse effect was directly related to its extent. High CD105 MVD was also a stage independent marker for worse disease-free survival. CONCLUSIONS: Tumour necrosis percentage is a relevant histomorphological prognostic indicator in CRC. More studies are needed to disclose the mechanisms of tumour necrosis.

Gremlin1 expression associates with serrated pathway and favourable prognosis in colorectal cancer.

AIMS: Gremlin1 is a bone morphogenetic protein (BMP) antagonist with a suggested role in colorectal cancer (CRC) progression. We have analysed Gremlin1 protein expression in CRC and assessed its correlation with clinicopathological characteristics, including developmental pathway and prognosis. METHODS AND RESULTS: Material included a non-selected series of 148 surgically treated CRC cases. The tumour-node-metastasis (TNM) stage, histological grade and inflammatory infiltrate at the invasive margin were assessed, and tumours were classified to serrated or non-serrated types. Immunohistochemistry was conducted to evaluate Gremlin1 expression. Prognosis (60-month follow-up) was analysed by Kaplan-Meier methods and Cox regression analysis. Gremlin1 expression was detected in epithelial cells both in normal mucosa and in carcinomas. Abundant expression in carcinomas associated with low TNM stage (P = 0.044), low histological grade (P = 0.044), serrated histology (P = 0.033 or P = 0.053 depending on the classification cut-off) and intensive inflammatory infiltrate at the invasive margin (P = 0.044), and was a stage independent indicator of extended survival (P = 0.029). CONCLUSIONS: Gremlin1 protein expression in CRC associates with low tumour stage and extended survival independently of tumour stage, suggesting that it represents a relevant prognostic indicator in CRC. High expression in carcinomas with serrated histology suggests a potential role for Gremlin1 in the serrated pathway of CRC.

Characteristics and significance of colorectal cancer associated lymphoid reaction.

A subset of colorectal cancers (CRCs) exhibits so-called Crohn's like lymphoid reaction (CLR), an inflammatory reaction pattern that consists of numerous transmural lymphoid aggregates. However, the composition of these aggregates, their biological mechanisms and their prognostic significance are not well-defined. We analyzed two CRC cohorts (418 and 149 patients) and determined clinicopathological features including survival. A new method for evaluating CLR based on counting the areal density of the lymphoid follicles (CLR density) was adopted. Immune cell densities at intratumoral and peritumoral regions, as well as the composition of the lymphoid follicles, were studied by immunohistochemistry. We found that CLR comprised of lymphoid aggregates with no evidence of granuloma formation. High CLR density associated with lower tumor stage, lack of preoperative radiotherapy or chemoradiotherapy and deficient mismatch repair enzyme expression. CLR density had positive correlations with peritumoral and intratumoral densities of CD83(+) mature dendritic cells and T cells. High CLR density associated with better survival and had prognostic value that was independent of stage, Klintrup-Mäkinen score for peritumoral inflammation and the numbers of tumor infiltrating T cells. CLR density evaluation had excellent intraobserver and interobserver agreement. In conclusion, the results suggest that CLR contributes to the adaptive antitumor immunity. Quantitative evaluation of CLR density is a relevant prognostic indicator in CRC.

Establishing Criteria for Tumor Necrosis as Prognostic Indicator in Colorectal Cancer.

Tumor necrosis has been reported to represent an independent prognostic factor in colorectal cancer, but its evaluation methods have not been described in sufficient detail to introduce tumor necrosis evaluation into clinical use. To study the potential of tumor necrosis as a prognostic indicator in colorectal cancer, criteria for 3 methods for its evaluation were defined: the average percentage method (tumor necrosis percentage of the whole tumor), the hotspot method (tumor necrosis percentage in a single hotspot), and the linear method (the diameter of the single largest necrotic focus). Cox regression models were used to calculate cancer-specific mortality hazard ratios (HRs) for tumor necrosis categories in 2 colorectal cancer cohorts with more than 1800 cases. For reproducibility assessment, 30 cases were evaluated by 9 investigators, and Spearman's rank correlation coefficients and Cohen's kappa coefficients were calculated. We found that all 3 methods predicted colorectal cancer-specific survival independent of other prognostic parameters, including disease stage, lymphovascular invasion, and tumor budding. The greatest multivariable HRs were observed for the average percentage method (cohort 1: HR for ≥ 40% vs. <3% 3.03, 95% CI, 1.93-4.78; cohort 2: HR for ≥ 40% vs. < 3% 2.97; 95% CI, 1.63-5.40). All 3 methods had high reproducibility, with the linear method showing the highest mean Spearman's correlation coefficient (0.91) and Cohen's kappa (0.70). In conclusion, detailed criteria for tumor necrosis evaluation were established. All 3 methods showed good reproducibility and predictive ability. The findings pave the way for the use of tumor necrosis as a prognostic factor in colorectal cancer.

Impact of Primary Tumor Location on Demographics, Resectability, Outcomes, and Quality of Life in Finnish Metastatic Colorectal Cancer Patients (Subgroup Analysis of the RAXO Study).

The primary tumor location (PTL) is associated with the phenotype, metastatic sites, mutations, and outcomes of metastatic colorectal cancer (mCRC) patients, but this has mostly been studied according to sidedness (right vs. left sided). We studied right colon vs. left colon vs. rectal PTL in a real-life study population (n = 1080). Health-related quality of life (HRQoL) was assessed multi-cross-sectionally with QLQ-C30, QLQ-CR29, EQ-5D, and 15D. A chi-square, Kaplan-Meier, and Cox regression were used to compare the groups. The PTL was in the right colon in 310 patients (29%), the left colon in 396 patients (37%), and the rectum in 375 patients (35%). The PTL was associated with distinct differences in metastatic sites during the disease trajectory. The resectability, conversion, and resection rates were lowest in the right colon, followed by the rectum, and were highest in the left colon. Overall survival was shortest for right colon compared with left colon or rectal PTL (median 21 vs. 35 vs. 36 months), with the same trends after metastasectomy or systemic therapy only. PTL also remained statistically significant in a multivariable model. The distribution of symptoms varied according to PTL, especially between the right colon (with general symptoms of metastases) and rectal PTL (with sexual- and bowel-related symptoms). mCRC, according to PTL, behaves differently regarding metastatic sites, resectability of the metastases, outcomes of treatment, and HRQoL.