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Objective: To explore the impact of molecular subtype in endometrial cancer (EC) on CD8+T cell densities. Furthermore, this work will test the assumption that all mismatch repair deficient (MMRd) tumours are immunologically similar which would enable current trial data to be generalised to all MMRd ECs., Methods and Analysis: All tumours were characterised into the four clinical molecular subtypes. For analysis, the TP53 mutant and no-specific molecular profile tumours were grouped together and described as the low mutational burden (LMB) cohort. CD8+T cell counts were taken from four regions of interest which sampled the tumour-stromal interface and the tumour core. CD8+T cell counts were analysed as mean averages., Results: In total, 607 ECs contributed to the analysis. CD8+T cell counts in confirmed Lynch syndrome (LS) ECs were significantly higher than MLH1 -methylated ECs in all tumour locations excluding the tumour stroma. Confirmed LS and path_ POLE ECs had significantly higher CD8+T cell counts across all tumour locations when compared with LMB ECs. There were limited significant differences in CD8+T cell counts between path_ POLE versus confirmed LS ECs. There was no significant difference in the CD8+T cells counts and gene ( MLH1 , MSH2 , MSH6 , PMS2 ) in which the LS pathogenic variant was found; however, this analysis was limited by small numbers., Conclusion: These data indicate that CD8+T cell numbers and distribution is not equal between MLH1 -methylated and confirmed LS ECs. This is relevant when interpreting current trial data looking to the application of checkpoint inhibition treatments in MMRd cancers., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Background: Tumour-infiltrating CD8 + cytotoxic T cells confer favourable prognosis in colorectal cancer. The added prognostic value of other infiltrating immune cells is unclear and so we sought to investigate their prognostic value in two large clinical trial cohorts., Methods: We used multiplex immunofluorescent staining of tissue microarrays to assess the densities of CD8 + , CD20 + , FoxP3 + , and CD68 + cells in the intraepithelial and intrastromal compartments from tumour samples of patients with stage II-III colorectal cancer from the SCOT trial (ISRCTN59757862), which examined 3 months versus 6 months of adjuvant oxaliplatin-based chemotherapy, and from the QUASAR 2 trial (ISRCTN45133151), which compared adjuvant capecitabine with or without bevacizumab. Both trials included patients aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0-1. Immune marker predictors were analysed by multiple regression, and the prognostic and predictive values of markers for colorectal cancer recurrence-free interval by Cox regression were assessed using the SCOT cohort for discovery and QUASAR 2 cohort for validation., Findings: After exclusion of cases without tissue microarrays and with technical failures, and following quality control, we included 2340 cases from the SCOT trial and 1069 from the QUASAR 2 trial in our analysis. Univariable analysis of associations with recurrence-free interval in cases from the SCOT trial showed a strong prognostic value of intraepithelial CD8 (CD8 IE ) as a continuous variable (hazard ratio [HR] for 75th vs 25th percentile [75vs25] 0·73 [95% CI 0·68-0·79], p=2·5 × 10 -16 ), and of intrastromal FoxP3 (FoxP3 IS ; 0·71 [0·64-0·78], p=1·5 × 10 -13 ) but not as strongly in the epithelium (FoxP3 IE ; 0·89 [0·84-0·96], p=1·5 × 10 -4 ). Associations of other markers with recurrence-free interval were moderate. CD8 IE and FoxP3 IS retained independent prognostic value in bivariable and multivariable analysis, and, compared with either marker alone, a composite marker including both markers (CD8 IE -FoxP3 IS ) was superior when assessed as a continuous variable (adjusted [a]HR 75 vs 25 0·70 [95% CI 0·63-0·78], p=5·1 × 10 -11 ) and when categorised into low, intermediate, and high density groups using previously published cutpoints (aHR for intermediate vs high 1·68 [95% CI 1·29-2·20], p=1·3 × 10 -4 ; low vs high 2·58 [1·91-3·49], p=7·9 × 10 -10 ), with performance similar to the gold-standard Immunoscore. The prognostic value of CD8 IE -FoxP3 IS was confirmed in cases from the QUASAR 2 trial, both as a continuous variable (aHR 75 vs 25 0·84 [95% CI 0·73-0·96], p=0·012) and as a categorical variable for low versus high density (aHR 1·80 [95% CI 1·17-2·75], p=0·0071) but not for intermediate versus high (1·30 [0·89-1·88], p=0·17)., Interpretation: Combined evaluation of CD8 IE and FoxP3 IS could help to refine risk stratification in colorectal cancer. Investigation of FoxP3 IS cells as an immunotherapy target in colorectal cancer might be merited., Funding: Medical Research Council, National Institute for Health Research, Cancer Research UK, Swedish Cancer Society, Roche, and Promedica Foundation., Competing Interests: Declaration of interests DK has patents related to use of digital pathology algorithms, and serves as an advisor and has stock in Oxford Cancer Biomarkers. MPS has received honoraria from Merck, Server, Bayer, Takeda, and Amgen for meetings or lectures. WK has received royalties from Room4. DNC has participated in advisory boards for MSD and has received research funding on behalf of the TransSCOT consortium from HalioDx for analyses independent of this study. VHK has served as an invited speaker on behalf of Sharing Progress in Cancer Care and Indica Labs, and has received project-based research funding from The Image Analysis Group and Roche outside of the submitted work. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Tumor development is critically dependent on the supporting stroma consisting of inflammatory cells and fibroblasts. This study intended to improve prognostic prediction for early colorectal cancer (CRC) by combined estimation of T-lymphocyte and stroma fractions with conventional markers., Methods: In total 509 and 1041 stage II/ΙΙΙ CRC from the VICTOR and QUASAR 2 trials were included as a training set and a validation set, respectively. Intratumoral CD8 + T-lymphocytes and stroma were identified and quantified by machine-based learning on digital sections. The primary endpoint was to evaluate the prognostic value of the combined marker for time to recurrence (TTR)., Findings: For low-risk patients (n = 598; stage Ⅱ, and stage ΙΙΙ pT1-3 pN1 with neither lymphatic (L - ) nor vascular (V - ) invasion), low stroma fraction (n = 511) identified a good prognostic subgroup with 5-year TTR of 86% (95% CI 83-89), versus the high stroma subgroup TTR of 78% (HR = 1.75, 95% CI 1.05-2.92; P = 0.029). For high-risk patients (n = 394; stage ΙΙΙ pT3 pN1 L + /V + , pT4, or pN2), combined low CD8 + and high stroma fraction identified a poor prognostic subgroup (n = 34) with 5-year TTR of 29% (95% CI 17-50), versus the high CD8 + fraction and low stroma fraction subgroup (n = 138) of 64% (HR = 2.86, 95% CI 1.75-4.69; P < 0.001)., Interpretation: Quantification of intratumoral CD8 + T-lymphocyte and stroma fractions can be combined with conventional prognostic markers to improve patient stratification., Competing Interests: Declaration of Competing Interest DK reports personal fees from Oxford Cancer Biomarkers (Oxford, UK), outside the submitted work. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The chemokine CXCL13 mediates recruitment of B cells to tumors and is essential for the formation of tertiary lymphoid structures (TLSs). TLSs are thought to support antitumor immunity and are associated with improved prognosis. However, it remains unknown whether TLSs are formed in response to the general inflammatory character of the tumor microenvironment, or rather, are induced by (neo)antigen-specific adaptive immunity. We here report on the finding that the TGFβ-dependent CD103 + CD8 + tumor-infiltrating T-cell (TIL) subpopulation expressed and produced CXCL13. Accordingly, CD8 + T cells from peripheral blood activated in the presence of TGFβ upregulated CD103 and secreted CXCL13. Conversely, inhibition of TGFβ receptor signaling abrogated CXCL13 production. CXCL13 + CD103 + CD8 + TILs correlated with B-cell recruitment, TLSs, and neoantigen burden in six cohorts of human tumors. Altogether, our findings indicated that TGFβ plays a noncanonical role in coordinating immune responses against human tumors and suggest a potential role for CXCL13 + CD103 + CD8 + TILs in mediating B-cell recruitment and TLS formation in human tumors., (©2019 American Association for Cancer Research.)

Background: Sequencing-based analysis has become a well-established approach to deciphering the composition of the gut microbiota. However, due to the complexity of accessing sufficient material from colonoscopic biopsy samples, most studies have focused on faecal microbiota analysis, even though it is recognised that differences exist between the microbial composition of colonic biopsies and faecal samples. We determined the suitability of colonic lavage samples to see if it had comparable microbial diversity composition to colonic biopsies as they are without the limitations associated with sample size. We collected paired colonic biopsies and lavage samples from subjects who were attending for colorectal cancer screening colonoscopy., Results: Next-generation sequencing and qPCR validation were performed with multiple bioinformatics analyses to determine the composition and predict function of the microbiota. Colonic lavage samples contained significantly higher numbers of operational taxonomic units (OTUs) compared to corresponding biopsy samples, however, diversity and evenness between lavage and biopsy samples were similar. The differences seen were driven by the presence of 12 OTUs which were in higher relative abundance in biopsies and were either not present or in low relative abundance in lavage samples, whilst a further 3 OTUs were present in higher amounts in the lavage samples compared to biopsy samples. However, predicted functional community profiling based on 16S ribosomal ribonucleic acid (rRNA) data indicated minimal differences between sample types., Conclusions: We propose that colonic lavage samples provide a relatively accurate representation of biopsy microbiota composition and should be considered where biopsy size is an issue.

Background: Precision cancer medicine depends on defining distinct tumour subgroups using biomarkers that may occur at very modest frequencies. One such subgroup comprises patients with exceptionally mutated (ultramutated) cancers caused by mutations that impair DNA polymerase epsilon (POLE) proofreading., Methods: We examined the association of POLE proofreading domain mutation with clinicopathological variables and immune response in colorectal cancers from clinical trials (VICTOR, QUASAR2, and PETACC-3) and colorectal cancer cohorts (Leiden University Medical Centre 1 and 2, Oslo 1 and 2, Bern, AMC-AJCC-II, and Epicolon-1). We subsequently investigated its association with prognosis in stage II/III colorectal cancer by Cox regression of pooled individual patient data from more than 4500 cases from these studies., Findings: Pathogenic somatic POLE mutations were detected in 66 (1·0%) of 6517 colorectal cancers, and were mutually exclusive with mismatch repair deficiency (MMR-D) in the 6277 cases for whom both markers were determined (none of 66 vs 833 [13·4%] of 6211; p<0·0001). Compared with cases with wild-type POLE, cases with POLE mutations were younger at diagnosis (median 54·5 years vs 67·2 years; p<0·0001), were more frequently male (50 [75·8%] of 66 vs 3577 [55·5%] of 6445; p=0·0010), more frequently had right-sided tumour location (44 [68·8%] of 64 vs 2463 [39·8%] of 6193; p<0·0001), and were diagnosed at an earlier disease stage (p=0·006, χ 2 test for trend). Compared with mismatch repair proficient (MMR-P) POLE wild-type tumours, POLE-mutant colorectal cancers displayed increased CD8+ lymphocyte infiltration and expression of cytotoxic T-cell markers and effector cytokines, similar in extent to that observed in immunogenic MMR-D cancers. Both POLE mutation and MMR-D were associated with significantly reduced risk of recurrence compared with MMR-P colorectal cancers in multivariable analysis (HR 0·34 [95% CI 0·11-0·76]; p=0·0060 and 0·72 [0·60-0·87]; p=0·00035), although the difference between the groups was not significant., Interpretation: POLE proofreading domain mutations identify a subset of immunogenic colorectal cancers with excellent prognosis. This association underscores the importance of rare biomarkers in precision cancer medicine, but also raises important questions about how to identify and implement them in practice., Funding: Cancer Research UK, Academy of Medical Sciences, Health Foundation, EU, ERC, NIHR, Wellcome Trust, Dutch Cancer Society, Dutch Digestive Foundation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs)., (Copyright © 2015 Elsevier Inc. All rights reserved.)