Colorectal cancer is the third most common cancer worldwide, and the proportion of individuals diagnosed under the age of 50 years, referred to as early-onset colorectal cancer (EOCRC), is increasing. The aim of this study was to evaluate how the demographic and clinical features of EOCR in northern Sweden and Finland have changed over time. All patient data were extracted from local hospital surgical department databases between 1995 and 2022. Two CRC cohorts, Study Cohort I (1995-2005) 1237 patients and Study Cohort II (2006-2022) 4526 patients, were compared for age, sex, disease stage, tumour grade, tumour location, and mismatch repair status. EOCRC patients comprised 7% of all CRCs in Study Cohort I and 4% in Study Cohort II. The mean ages were 42 and 43 years respectively, and 55% of patients were female. The vast part of EOCRC tumours were left-sided stage III-IV cancers. Most tumours (n = 204, 73%) were low grade, and 10% showed mismatch repair deficiency. No significant differences in demographic or tumour characteristics were seen over time. EOCRC in northern Sweden and Finland is characterised by advanced-stage, low tumour grade, a slight female predominance, and stable clinical and pathological features. These findings partly contrast with reports on EOCRC from other high-income countries, highlighting the need for further research on advanced molecular characteristics and potential gender differences in incidence and survival of this population.

BACKGROUND/AIM:Deficient mismatch repair (dMMR) colorectal cancer (CRC) arises from either sporadic epigenetic changes or hereditary Lynch syndrome. This retrospective multicenter cohort study is the first to evaluate the differences in risk for dMMR non-colorectal malignancy between patients with sporadic CRC and those with Lynch syndrome-associated CRC. PATIENTS AND METHODS:A cohort of 1,753 patients treated between 1996 and 2019 in Sweden, Finland, and the Czech Republic was evaluated for MMR status by immunohistochemistry and classified as either proficient (pMMR) or dMMR. The last one underwent BRAF V600E and MLH1 methylation testing to classify sporadic versus Lynch-associated cases. Non-CRC malignancies occurring within ±20 years of CRC diagnosis were identified via national cancer registries and medical records. Incidence rate ratios (IRRs) were estimated using Poisson regression adjusted for age, sex, tumor site, and stage. RESULTS:Among 277 dMMR cases (186 sporadic, 91 Lynch), 101 patients (36%) developed at least one non-CRC malignancy. Sporadic dMMR was associated with significantly lower risk compared to Lynch-associated dMMR [multivariable IRR=0.82; 95% confidence interval (CI)=0.51-0.91; p=0.014]. The reduced risk was consistent for malignancies occurring both before (IRR=0.48; p=0.047) and after CRC diagnosis (IRR=0.37; p=0.026). Age was an independent predictor of risk. CONCLUSION:Sporadic dMMR CRC confers a substantially lower risk of non-colorectal malignancy than Lynch syndrome-associated CRC. These findings underscore the importance of incorporating MMR etiology into personalized surveillance strategies.

Physical activity and immune cell infiltration of the tumor microenvironment are associated with improved prognosis in colon cancer. There is also evidence that physical activity may mobilize immune cells against colon tumors. One possible mechanism behind the positive effect of physical activity on colon cancer prognosis could thus be increased immune cell infiltration of the tumor microenvironment. There are few previous studies examining this association, and none has included an objective assessment of physical activity. This was a study on 69 patients enrolled in the Physical Activity and its impact on COlon cancer Surgery (PACOS) prospective cohort, all diagnosed with colon cancer stages I–III, all eligible for curative surgery, and with no previous treatment. Prior to surgery, the participants underwent physical tests, dual-energy X-ray absorptiometry, and wore an accelerometer to quantify their physical activity and physical fitness. Tumor infiltration by cytotoxic T cells, T-helper 1 cells, regulatory T cells, B cells, and macrophages was objectively assessed with multispectral quantitative automated pathology imaging. Odds ratios for associations between physical activity and immune cell infiltration were estimated. A higher number of repetitions in the 30-s sit-to-stand test was associated with a lower density of T-helper 1 cells in the stromal compartment of the tumor, in the model adjusted for age and sex. When correcting for multiple testing, the association was considered non-significant. No other significant association was seen for any of the physical tests or immune cell types. No consistent evidence was found to support the hypothesis of an association between physical activity or fitness and increased immune cell tumor infiltration in colon cancer. This study was registered at clinicaltrials.gov (ID NCT03947840), registration date 2019-05-19.

Recent studies have revealed that the long-recognized link between the historically defined Fusobacterium nucleatum group and colorectal cancer is largely driven by Fusobacterium animalis. This species, along with two others (Fusobacterium polymorphum and Fusobacterium vincentii), was recently reclassified as distinct from F. nucleatum, highlighting functional divergence within this group. Due to their close genetic relatedness, traditional partial 16S rRNA gene sequencing lacks the resolution to reliably distinguish these species. Nevertheless, accurate species-level identification remains essential in cancer-associated microbiome research. Here, we demonstrate that full-length 16S rRNA sequencing using Oxford Nanopore Technology, combined with a novel custom demultiplexing software, enables robust species-level discrimination within the Fusobacterium genus. Our approach accurately classified clinically relevant Fusobacterium species and recovered their expected proportions from whole cells, DNA mixtures, and clinical CRC specimens. This method provides high-resolution profiling to elucidate species-specific roles of Fusobacterium in colorectal cancer.

Colorectal cancer (CRC) represents a major global disease burden with nearly 1 million cancer-related deaths annually. TNM staging has served as the foundation for predicting patient prognosis, despite variation across staging groups. The consensus molecular subtype (CMS) is a transcriptome-based system classifying CRC tumors into four subtypes with different characteristics: CMS1 (immune), CMS2 (canonical), CMS3 (metabolic), and CMS4 (mesenchymal). Transcriptomics is too complex and expensive for clinical implementation; therefore, an immunohistochemical method is needed. The prognostic impact of the immunohistochemistry-based four CMS-like subtypes remains unclear. Due to the complexity and costs associated with transcriptomics, we developed an immunohistochemistry (IHC)-based method supported by convolutional neural networks (CNNs) to define subgroups that resemble CMS biological characteristics. Building on previous IHC-classifiers and incorporating β-catenin to refine differentiation between CMS2- and CMS3-like profiles, we categorized CRC tumors in a cohort of 538 patients. Classification was successful in 89.4% and 15.9% of tumors were classified as CMS1-like, 35.1% as CMS2-like, 38.7% as CMS3-like, and 11.7% as CMS4-like. CMS2-like patients exhibited the best overall survival ( p = 0.018), including when local and metastasized disease were analyzed separately. Our method offers an accessible and clinically feasible CMS-inspired classification, although it does not serve as a replacement for transcriptomic CMS classification.

The recent introduction of immune checkpoint inhibitor therapy has significantly improved outcomes for patients with colorectal cancer (CRC). The most pronounced clinical benefits were observed in patients with immunogenic microsatellite instable (MSI)/deficient MMR (dMMR) tumors. However, emerging evidence indicates that a subset of patients with microsatellite stable tumors may also respond to therapy. Finding predictive markers to identify these patients is critical. In this study, we analyzed the immunohistochemical expression of immune checkpoints CTLA-4, PD-1, and PD-L1 using multispectral imaging in 151 CRC patients with defined molecular characteristics. Consistent with prior reports, MSI tumors had higher levels of all immune checkpoints analyzed than microsatellite stable tumors. Notably, distinct patterns of immune checkpoint expression were associated with KRAS and BRAF mutation status. KRAS-mutated tumors showed lower, and BRAF-mutated tumors higher, expression of immune checkpoints compared to wild-type/wild-type tumors. The strongest association with KRAS and BRAF mutations was observed for PD-L1 expression. The relationship between PD-L1 and KRAS/BRAF-mutational status was validated in a second cohort of 527 CRC patients, finding the association for PD-L1 expression in both stroma and in tumor cells. Furthermore, the role of BRAF mutation on immunity in CRC was found to be partly independent of MSI status. The strongest prognostic role was found for PD-L1 in stroma, underscoring the clinical significance of this marker. In conclusion, our findings suggest that KRAS and BRAF mutations, alongside MSI, may serve as valuable biomarkers for identifying CRC patient subgroups likely to benefit from immune checkpoint blockade in CRC.

The systemic inflammatory response (SIR), defined as elevated levels of circulating C-reactive protein (CRP), is an important predictor of impaired survival in colorectal cancer. The aim of this study was to explore the prognostic role of SIR and its association with tumour mismatch repair status and the immune response. Immune activity profiles of mononuclear cells isolated from CRC tissues and blood in the U-CAN exploration cohort (n = 69), were analysed by flow cytometry. In the U-CAN validation cohort (n = 257), T-helper cells (T-bet(+)), cytotoxic T cells (CD8(+)), regulatory T cells (FoxP3(+)), B cells (CD20(+)), and macrophages (CD68(+)) were analysed by multispectral imaging. Microsatellite instability was determined using five mononucleotide-repeat microsatellite markers. Patients with high CRP levels (> 10 mg/l) were significantly more often diagnosed with high-grade tumours and tumours exhibiting microsatellite instability. However, some patients with high CRP levels were found to have microsatellite-stable tumours. Furthermore, high CRP levels were associated with specific tumour immune traits including an augmented macrophage response and were significantly linked to poorer cancer-specific survival, particularly in patients with microsatellite-stable tumours. In conclusion, our findings suggest an interplay between SIR and mismatch repair status in CRC prognosis which needs to be further explored.

Abstract Background Mounting evidence suggests a significant role of the gut microbiota in the development and progression of colorectal cancer (CRC). In particular, an over-representation of oral pathogens has been linked to CRC. The aim of this study was to further investigate the faecal microbial landscape of CRC patients, with a focus on the oral pathogens Parvimonas micra and Fusobacterium nucleatum. Methods In this study, 16S rRNA sequencing was conducted using faecal samples from CRC patients (n = 275) and controls without pathological findings (n = 95). Results We discovered a significant difference in microbial composition depending on tumour location and microsatellite instability (MSI) status, with P. micra, F. nucleatum, and Peptostreptococcus stomatis found to be more abundant in patients with MSI tumours. Moreover, P. micra and F. nucleatum were associated with a cluster of CRC-related bacteria including Bacteroides fragilis as well as with other oral pathogens such as P. stomatis and various Porphyromonas species. This cluster was distinctly different in the control group, suggesting its potential linkage with CRC. Conclusions Our results suggest a similar distribution of several CRC-associated bacteria within CRC patients, underscoring the importance of considering the concomitant presence of bacterial species in studies investigating the mechanisms of CRC development and progression.

Background The immune response has important clinical value in colorectal cancer (CRC) in both prognosis and response to immunotherapy. This study aims to explore tumour immune cell infiltration in relation to clinically well-established molecular markers of CRC. Methods Multiplex immunohistochemistry and multispectral imaging was used to evaluate tumour infiltration of cytotoxic T cells (CD8 + ), Th1 cells (T-bet + ), T regulatory cells (FoxP3 + ), B cells (CD20 + ), and macrophages (CD68 + ) in a cohort of 257 CRC patients. Results We found the expected association between higher immune-cell infiltration and microsatellite instability. Also, whereas BRAF -mutated tumours displayed increased immune-cell infiltration compared to BRAF wild-type tumours, the opposite was seen for KRAS -mutated tumours, differences that were most prominent for cytotoxic T cells and Th1 cells. The opposing relationships of BRAF and KRAS mutations with tumour infiltration of cytotoxic T cells was validated in an independent cohort of 608 CRC patients. A positive prognostic importance of cytotoxic T cells was found in wild-type as well as KRAS and BRAF -mutated CRCs in both cohorts. Conclusion A combined evaluation of MSI status, KRAS and BRAF mutational status, and immune infiltration (cytotoxic T cells) may provide important insights to prognosis and response to immunotherapy in CRC.

Background and ObjectivesDisparities between tumors arising via different sporadic carcinogenetic pathways have not been studied systematically. This retrospective multicenter cohort study evaluated the differences in the risk for non-colorectal malignancy between sporadic colorectal cancer (CRC) patients from different DNA mismatch repair status.MethodsA retrospective European multicenter cohort study including in total of 1706 CRC patients treated between 1996 and 2019 in three different countries. The proficiency (pMMR) or deficiency (dMMR) of mismatch repair was determined by immunohistochemistry. Cases were analyzed for tumor BRAFV600E mutation, and BRAF mutated tumors were further analyzed for hypermethylation status in the promoter region of MLH1 to distinguish between sporadic and hereditary cases. Swedish and Finish patients were matched with their respective National Cancer Registries. For the Czech cohort, thorough scrutiny of medical files was performed to identify any non-colorectal malignancy within 20 years before or after the diagnosis of CRC. Poisson regression analysis was performed to identify the incidence rates of non-colorectal malignancies. For validation purposes, standardized incidence ratios were calculated for the Swedish cases adjusted for age, year, and sex.ResultsOf the 1706 CRC patients included in the analysis, 819 were female [48%], median age at surgery was 67 years [interquartile range: 60-75], and sporadic dMMR was found in 188 patients (11%). Patients with sporadic dMMR CRC had a higher incidence rate ratio (IRR) for non-colorectal malignancy before and after diagnosis compared to patients with a pMMR tumor, in both uni- (IRR = 2.49, 95% confidence interval [CI] = 1.89-3.31, p = 0.003) and multivariable analysis (IRR = 2.24, 95% CI = 1.67-3.01, p = 0.004). This association applied whether or not the non-colorectal tumor developed before or after the diagnosis of CRC in both uni- (IRR = 1.91, 95% CI = 1.28-2.98, p = 0.004), (IRR = 2.45, 95% CI = 1.72-3.49, p = 0.004) and multivariable analysis (IRR = 1.67,95% CI = 1.05-2.65, p = 0.029), (IRR = 2.35, 95% CI = 1.63-3.42, p = 0.005), respectively.ConclusionIn this retrospective European multicenter cohort study, patients with sporadic dMMR CRC had a higher risk for non-colorectal malignancy than those with pMMR CRC. These findings indicate the need for further studies to establish the need for and design of surveillance strategies for patients with dMMR CRC.

ABSTRACTColorectal cancer (CRC) is caused by a sequence of somatic genomic alterations affecting driver genes in core cancer pathways1. To understand the functional and prognostic impact of cancer-causing somatic mutations, we analysed the whole genomes and transcriptomes of 1,063 primary CRCs in a population-based cohort with long-term follow-up. From the 96 mutated driver genes, 9 were novel to CRC and 24 to any cancer. Two distinct patterns of pathway co-mutations were observed, timing analyses identified 6 early and 3 late driver gene mutations, and several new signatures of CRC specific mutational processes were uncovered. Mutations in 10 protein-coding genes belonging to the WNT, EGFR, and TGF-β pathways, 2 mitochondrial DNA genes and 3 regulatory elements along with the COSMIC SBS44 signature impacted survival. Gene expression classification yielded 5 prognostic subtypes with distinct molecular features, in part explained by underlying genomic alterations. Microsatellite instable tumours could be divided in two classes with different levels of hypoxia and infiltration of immune and stromal cells. This study constitutes the largest integrated genome and transcriptome analysis of CRC to date, and links mutations, gene expressions and patient outcomes. The identification of prognostic mutations and expression subtypes can guide future efforts to individualize CRC therapy.

Mismatch repair deficient or microsatellite instable (MSI) tumors are often characterized by high mutational burdens and strong immune infiltration and have shown higher sensitivity to immune checkpoint inhibitors (ICIs) compared to microsatellite stable (MSS) tumors. However, only a portion of the patients with MSI colorectal cancer (CRC) gain survival benefits from ICIs treatment, suggesting unclear heterogeneity in MSI tumors. We analyzed the whole-genomes and transcriptomes of 223 ICIs treatment-naïve MSI CRC tumors from U-CAN project. Immune cell infiltration and cancer-relevant pathways were evaluated by CIBERSORT and PROGENy, respectively. An MSI subtyping model was developed based on ResNet50. Unsupervised classification based on gene expression data divided MSI tumors into two distinct classes (class1 and 2), with MSI class1 characterized by a lower level of chromosomal instability, a higher level of hypoxia, and more activated EGFR, JAK-STAT, TNFa, and NFkB pathways than class2. However, two MSI classes showed no differences in 5-year survival. Further investigation of the immune cell population showed that class1 had considerably higher immune cell infiltration compared to class2, specifically in M0, M1 and M2 macrophages, CD4 and CD8 T-cells, naïve B-cells, resting NK-cells, and neutrophiles. Class2 exhibited significantly higher loss in 10q23.31 (containing PTEN) and reduced expression of PTEN, which may lead to an immunosuppressive tumor microenvironment and partially explain the higher activation in PI3K and VEGF pathways in class2 compared to class1. Importantly, ICIs targets, such as CTLA4, PD-1, and PD-L1 all had higher expressions in calss1. We further developed an MSI subtyping model based on refined pathway features of normal tissue, tumors with MSS, class1 or 2. Ongoing analyses include validation of MSI classification in the TCGA dataset. We illustrated the heterogeneity in MSI CRCs and revealed two subclasses of MSI with distinct molecular features, which may be informative for immune-based treatments. The MSI subtyping model may further facilitate precision immunotherapy and allow patients to derive clinical benefits.

Microbiota dysbiosis may affect both the development and progression of colorectal cancer (CRC). Large metagenomic studies have highlighted specific oral bacteria linked to CRC including Porphyromonas gingivalis. Few studies have however analysed the implications of this bacterium in CRC progression and survival. In this study, we investigated the intestinal presence of P. gingivalis by qPCR in both faecal and mucosal samples from two different patient cohorts, including patients with precancerous dysplasia or CRC, as well as controls. P. gingivalis was detected in 2.6-5.3% of CRC patients and significantly different levels of P. gingivalis were found in faeces of CRC patients compared to controls (P = 0.028). Furthermore, an association was found between the presence of P. gingivalis in faeces and tumour tissue (P < 0.001). Our findings further suggested a potential link between mucosal P. gingivalis and tumours of MSI subtype (P = 0.040). Last but not least, patients with faecal P. gingivalis were found to have a significantly decreased cancer-specific survival (P = 0.040). In conclusion, P. gingivalis could be linked to patients with CRC and to a worse patient prognosis. Further studies are needed to elucidate the role of P. gingivalis in CRC pathogenesis.

Colorectal cancer (CRC) is a heterogeneous disease with multiple subtypes that differ in their clinical and molecular features. The presence of neoantigens derived from somatic mutations is a promising avenue for immunotherapy in CRC. However, the heterogeneity of the neoantigen landscape among CRC subtypes remains unclear. We analyzed the whole-genomes and transcriptomes of 1063 CRC patients from a Swedish large cohort. HLA alleles were called by at least two HLA callers including HLA-HD, HLA-LA, Kourami, OptiType, PHLAT, Polysolver, xHLA for each patient. Neoantigens were predicted by pVACseq pipeline using the default settings. In total, 83,630 MHC class I neoantigens derived from 29,106 mutations (511,159 mutations were inputted) were predicted in 979 patients. Average 6.1 neoantigens were from 4,480 frameshift INDELs while only average 2.3 neoantigens were from 24,276 SNVs. Patients older than 80 years carried more neoantigens than those under 65 years, and women tended to have more neoantigens than men (Wilcoxon test, P<0.01). Tumor neoantigen burden was significantly higher in right-colon, stage II, and hypermutated (HM) tumors (P<0.0001). In total, 850 mutations were predicted to generate neoantigens in at least two patients, including KRAS G12D (6.3%), RNF43 G659X (6.1%), KRAS G12V (5.2%), TBC1D23 K647X (2.9%), and MSH3 K381X (2.8%). Neoantigen numbers from PODXL2 and CRAT mutations were significantly higher in consensus molecular subtype (CMS) 3 tumors, whereas neoantigens from JAG2 mutations were more common in CMS2 (chi-square test, P <0.05). Neoantigens derived from DNAH3, KRAS and ERBB2 mutations (37%, 47% and 52% respectively) in non-HM patients were significantly more than those (5%, 14% and 13% respectively) in HM patients. Furthermore, in stage I-III, nHM patients (n=11) with DNAH3 neoantigens have significantly longer overall survival than patients (n=24) with only DNAH3 mutations. Our study constitutes the largest neoantigen dataset from integrated genome and transcriptome of CRC to date and illustrates the heterogeneity of the neoantigen landscape among CRC subtypes. The identified neoantigens may contribute to future individualized CRC immunotherapies.

The importance of the tumour microbiome in different aspects of colorectal cancer (CRC) has been increasingly recognised, but many questions remain. The aim of this study was to explore the effect of specific CRC associated microbes on the tumour immune response, which has a considerable prognostic value in CRC. We applied specific qPCR to detect Parvimonas micra and Fusobacterium nucleatum in tumour tissues from an immunologically well-characterised cohort of 69 CRC patients. This cohort included detailed analyses of immune profiles based on flow cytometry and transcriptomics in tumour tissue and blood, along with comprehensive analyses of molecular subtypes. P. micra and F. nucleatum were detected in 24% and 64% of tumour tissues, respectively. We found a significant association of P. micra with high-grade tumours and tumours of CMS1 subtype. F. nucleatum was significantly associated with right-sided tumours, microsatellite instability, and CMS1 tumours. The immunological analyses revealed significant associations of P. micra with activated CD69 + T lymphocytes and increased antigen-presenting HLA-DR + B lymphocytes. P. micra was also positively associated with M1 and M2 macrophage traits. The impact of P. micra tumour colonisation on the immune response was further assessed using transcriptomics in validation of our findings. No associations were found between F. nucleatum and immune profiles in this study. Our findings support novel associations between P. micra and the immune response in CRC. A better understanding of these interactions might help to identify important predictive and prognostic tools as well as new targets for therapy.

Increasing evidence suggests that the gut microbiota may impact colorectal cancer (CRC) development and progression. In this study, the tumour colonisation of two CRC-associated bacteria, Parvimonas micra and Fusobacterium nucleatum, was studied in relation to patient survival in a cohort of 257 CRC patients. Colonisation of P. micra and F. nucleatum was analysed in fresh frozen tumour tissue (n = 112) and in faeces (n = 250) by qPCR. When analysing tumour tissues, both P. micra and F. nucleatum were found to be associated with decreased five-year cancer-specific survival, an association that remained significant in multivariable analysis for P. micra. Furthermore, we found significant associations of high levels of P. micra and F. nucleatum with tumour molecular characteristics, i.e., tumours mutated in BRAFV600E, and tumours of the MSI subtype. The analysis of faecal samples showed weaker associations with prognosis and tumour molecular characteristics. In conclusion, our findings support a novel association of tumour colonisation of P. micra with decreased patient survival. A better understanding of the role of the gut microbiota in CRC might contribute to the advancement of prognostic tools and new targets for therapy.

Background: The effect of the genetic imprint on the emergency presentation of colon cancer remains unclear. The disparity between tumours evolving along different carcinogenetic pathways has not been studied systematically. This retrospective multicenter cohort study evaluates the association between mismatch repair status and the risk for acute surgery of colon cancer. Patients and methods: A retrospective multicenter cohort study including in total 870 patients from three different countries. Scandinavian cohort (Finland and Sweden), including a total of 412 patients operated between January 1, 1995 and December 31, 2010, was validated against a cohort from the Czech Republic, including a total of 458 patients, operated between January 1, 2018 and December 31, 2019. The proficiency or deficiency of mismatch repair was determined by immunohistochemistry. Primary outcome was the risk for acute colon cancer surgery given as the Odds Ratio (OR) in the univariable and multivariable analyses. Acute colon cancer surgery was defined as surgery performed during the same hospital admission as when the diagnosis of colon cancer was made. Results: Of the 870 patients (399 females [46%]) included in the analyses, median age at surgery was 69 [interquartile range, 61-76] years, deficient Mismatch Repair (dMMR) status was found in 190 patients (22%), and 179 patients (21%) underwent acute surgery during the same hospital admission as when the diagnosis of colon cancer was made. In the Scandinavian cohort, a significant association between dMMR status and acute surgery was seen in both the univariable (OR 1.82, 95% CI 1.11-3.02, P = 0.017) and the multivariable (OR = 2.21, 95% CI 1.28-3.95, P = 0.005) analyses. This was confirmed in the Czech validation cohort in both the univariable (OR = 1.94, 95% CI 1.09-3.26, P = 0.022) and the multivariable (OR = 1.77, 95% CI 1.15-3.18, P = 0.021) analyses. Conclusion: This multicenter study reveals a strong association between acute colon cancer surgery and dMMR tumour status. 2021 Elsevier Ltd. All rights reserved.

Colorectal cancer (CRC) is a heterogeneous disease, with varying clinical presentations and patient prognosis. Different molecular subgroups of CRC should be treated differently and therefore, must be better characterized. Organoid culture has recently been suggested as a good model to reflect the heterogeneous nature of CRC. However, organoid cultures cannot be established from all CRC tumors. The study examines which CRC tumors are more likely to generate organoids and thus benefit from ex vivo organoid drug testing. Long-term organoid cultures from 22 out of 40 CRC tumor specimens were established. It was found that organoid cultures were more difficult to establish from tumors characterized as microsatellite instable (MSI), BRAF-mutated, poorly differentiated and/or of a mucinous type. This suggests that patients with such tumors are less likely to benefit from ex vivo organoid drug testing, but it may also suggest biological difference in tumor growth. RNA sequencing analysis of tumor sections revealed that the in vivo maintenance of these non-organoid-forming tumors depends on factors related to inflammation and pathogen exposure. Furthermore, using TCGA data we could show a trend towards a worse prognosis for patients with organoid-forming tumors, suggesting also clinical differences. Results suggest that organoids are more difficult to establish from tumors characterized as MSI, BRAF-mutated, poorly differentiated and/or of a mucinous type. We further suggest that the maintenance of cell growth of these tumors in vivo may be promoted by immune-related factors and other stromal components within the tumor microenvironment.

The local anti-tumour immune response has important prognostic value in colorectal cancer (CRC). In the era of immunotherapy, a better understanding of the immune response in molecular subgroups of CRC may lead to significant advances in personalised medicine. On this note, microsatellite instable (MSI) tumours have been characterised by increased immune infiltration, suggesting MSI as a marker for immune inhibitor checkpoint therapy. Here, we used flow cytometry to perform a comprehensive analysis of immune activity profiles in tumour tissues, adjacent non-malignant tissues and blood, from a cohort of 69 CRC patients. We found several signs of immune suppression in tumours compared to adjacent non-malignant tissues, including T cells more often expressing the immune checkpoint molecules programmed cell death protein (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). We further analysed immune cell infiltration in molecular subgroups of CRC. MSI tumours were indeed found to be associated with increased immune infiltration, including increased fractions of PD-1+ T cells. No correlation was, however, found between MSI and the fraction of CTLA-4+ T cells. Interestingly, within the group of patients with microsatellite stable (MSS) tumours, some also presented with increased immune infiltration, including comparably high portions of PD-1+ T cells, but also CTLA-4+ T cells. Furthermore, no correlation was found between PD-1+ and CTLA-4+ T cells, suggesting that different tumours may, to some extent, be regulated by different immune checkpoints. We further evaluated the distribution of immune activity profiles in the consensus molecular subtypes of CRC. In conclusion, our findings suggest that different immune checkpoint inhibitors may be beneficial for selected CRC patients irrespective of MSI status. Improved predictive tools are required to identify these patients.

The use of faecal microbial markers as non-invasive biomarkers for colorectal cancer (CRC) has been suggested, but not fully elucidated. Here, we have evaluated the importance of Parvimonas micra as a potential non-invasive faecal biomarker in CRC and its relation to other microbial biomarkers. The levels of P. micra, F. nucleatum and clbA+bacteria were quantified using qPCR in faecal samples from a population-based cohort of patients undergoing colonoscopy due to symptoms from the large bowel. The study included 38 CRC patients, 128 patients with dysplasia and 63 controls. The results were validated in a second consecutive CRC cohort including faecal samples from 238 CRC patients and 94 controls. We found significantly higher levels of P. micra in faecal samples from CRC patients compared to controls. A test for P. micra could detect CRC with a specificity of 87.3% and a sensitivity of 60.5%. In addition, we found that combining P. micra with other microbial markers, could further enhance test sensitivity. Our findings support the potential use of P. micra as a non-invasive biomarker for CRC. Together with other microbial faecal markers, P. micra may identify patients with "high risk" microbial patterns, indicating increased risk and incidence of cancer.