Prognostic and Predictive Value of Immunoscore in Stage III Colorectal Cancer: Pooled Analysis of Cases From the SCOT and IDEA-HORG Studies.

PURPOSE: Immunoscore (IS) is prognostic in stage III colorectal cancer (CRC) and may predict benefit of duration (6 v 3 months) of adjuvant infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX) chemotherapy. We sought to determine IS prognostic and predictive value in stage-III CRC treated with adjuvant FOLFOX or oral capecitabine and infusional oxaliplatin (CAPOX) in the SCOT and IDEA-HORG trials. METHODS: Three thousand sixty-one cases had tumor samples, of which 2,643 (1,792 CAPOX) were eligible for IS testing. Predefined cutoffs (IS-Low and IS-High) were used to classify cases into two groups for analysis of disease-free survival (3-year DFS) and multivariable-adjusted hazard ratios (mvHRs) by Cox regression. RESULTS: IS was determined in 2,608 (99.5%) eligible cases, with 877 (33.7%) samples classified as IS-Low. IS-Low tumors were more commonly high-risk (T4 and/or N2; 52.9% IS-Low v 42.2% IS-High; P < .001) and in younger patients (P = .024). Patients with IS-Low tumors had significantly shorter DFS in the CAPOX, FOLFOX, and combined cohorts (mvHR, 1.52 [95% CI, 1.28 to 1.82]; mvHR, 1.58 [95% CI, 1.22 to 2.04]; and mvHR, 1.55 [95% CI, 1.34 to 1.79], respectively; P < .001 all comparisons), regardless of sex, BMI, clinical risk group, tumor location, treatment duration, or chemotherapy regimen. IS prognostic value was greater in younger (≤65 years) than older (>65 years) patients in the CAPOX cohort (mvHR, 1.92 [95% CI, 1.50 to 2.46] v 1.28 [95% CI, 1.01 to 1.63], PINTERACTION = .026), and in DNA mismatch repair proficient than deficient mismatch repair disease (mvHR, 1.68 [95% CI, 1.41 to 2.00] v 0.67 [95% CI, 0.30 to 1.49], PINTERACTION = .03), although these exploratory analyses were uncorrected for multiple testing. Adding IS to a model containing all clinical variables significantly improved prediction of DFS (likelihood ratio test, P < .001) regardless of MMR status. CONCLUSION: IS is prognostic in stage III CRC treated with FOLFOX or CAPOX, including within clinically relevant tumor subgroups. Possible variation in IS prognostic value by age and MMR status, and prediction of benefit from extended adjuvant therapy merit validation.

Multiplex analysis of intratumoural immune infiltrate and prognosis in patients with stage II-III colorectal cancer from the SCOT and QUASAR 2 trials: a retrospective analysis.

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 (CD8IE) 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 (FoxP3IS; 0·71 [0·64-0·78], p=1·5 × 10-13) but not as strongly in the epithelium (FoxP3IE; 0·89 [0·84-0·96], p=1·5 × 10-4). Associations of other markers with recurrence-free interval were moderate. CD8IE and FoxP3IS retained independent prognostic value in bivariable and multivariable analysis, and, compared with either marker alone, a composite marker including both markers (CD8IE-FoxP3IS) was superior when assessed as a continuous variable (adjusted [a]HR75 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 CD8IE-FoxP3IS was confirmed in cases from the QUASAR 2 trial, both as a continuous variable (aHR75 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 CD8IE and FoxP3IS could help to refine risk stratification in colorectal cancer. Investigation of FoxP3IS 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.

Accounting for intensity variation in image analysis of large-scale multiplexed clinical trial datasets.

Multiplex immunofluorescence (mIF) imaging can provide comprehensive quantitative and spatial information for multiple immune markers for tumour immunoprofiling. However, application at scale to clinical trial samples sourced from multiple institutions is challenging due to pre-analytical heterogeneity. This study reports an analytical approach to the largest multi-parameter immunoprofiling study of clinical trial samples to date. We analysed 12,592 tissue microarray (TMA) spots from 3,545 colorectal cancers sourced from more than 240 institutions in two clinical trials (QUASAR 2 and SCOT) stained for CD4, CD8, CD20, CD68, FoxP3, pan-cytokeratin, and DAPI by mIF. TMA slides were multi-spectrally imaged and analysed by cell-based and pixel-based marker analysis. We developed an adaptive thresholding method to account for inter- and intra-slide intensity variation in TMA analysis. Applying this method effectively ameliorated inter- and intra-slide intensity variation improving the image analysis results compared with methods using a single global threshold. Correlation of CD8 data derived by our mIF analysis approach with single-plex chromogenic immunohistochemistry CD8 data derived from subsequent sections indicates the validity of our method (Spearman's rank correlation coefficients ρ between 0.63 and 0.66, p ≪ 0.01) as compared with the current gold standard analysis approach. Evaluation of correlation between cell-based and pixel-based analysis results confirms equivalency (ρ > 0.8, p ≪ 0.01, except for CD20 in the epithelial region) of both analytical approaches. These data suggest that our adaptive thresholding approach can enable analysis of mIF-stained clinical trial TMA datasets by digital pathology at scale for precision immunoprofiling.

Functional analysis reveals driver cooperativity and novel mechanisms in endometrial carcinogenesis.

High-risk endometrial cancer has poor prognosis and is increasing in incidence. However, understanding of the molecular mechanisms which drive this disease is limited. We used genetically engineered mouse models (GEMM) to determine the functional consequences of missense and loss of function mutations in Fbxw7, Pten and Tp53, which collectively occur in nearly 90% of high-risk endometrial cancers. We show that Trp53 deletion and missense mutation cause different phenotypes, with the latter a substantially stronger driver of endometrial carcinogenesis. We also show that Fbxw7 missense mutation does not cause endometrial neoplasia on its own, but potently accelerates carcinogenesis caused by Pten loss or Trp53 missense mutation. By transcriptomic analysis, we identify LEF1 signalling as upregulated in Fbxw7/FBXW7-mutant mouse and human endometrial cancers, and in human isogenic cell lines carrying FBXW7 mutation, and validate LEF1 and the additional Wnt pathway effector TCF7L2 as novel FBXW7 substrates. Our study provides new insights into the biology of high-risk endometrial cancer and suggests that targeting LEF1 may be worthy of investigation in this treatment-resistant cancer subgroup.

Transformer-based biomarker prediction from colorectal cancer histology: A large-scale multicentric study.

Deep learning (DL) can accelerate the prediction of prognostic biomarkers from routine pathology slides in colorectal cancer (CRC). However, current approaches rely on convolutional neural networks (CNNs) and have mostly been validated on small patient cohorts. Here, we develop a new transformer-based pipeline for end-to-end biomarker prediction from pathology slides by combining a pre-trained transformer encoder with a transformer network for patch aggregation. Our transformer-based approach substantially improves the performance, generalizability, data efficiency, and interpretability as compared with current state-of-the-art algorithms. After training and evaluating on a large multicenter cohort of over 13,000 patients from 16 colorectal cancer cohorts, we achieve a sensitivity of 0.99 with a negative predictive value of over 0.99 for prediction of microsatellite instability (MSI) on surgical resection specimens. We demonstrate that resection specimen-only training reaches clinical-grade performance on endoscopic biopsy tissue, solving a long-standing diagnostic problem.

Non-Canonical Activin A Signaling Stimulates Context-Dependent and Cellular-Specific Outcomes in CRC to Promote Tumor Cell Migration and Immune Tolerance.

We have shown that activin A (activin), a TGF-ß superfamily member, has pro-metastatic effects in colorectal cancer (CRC). In lung cancer, activin activates pro-metastatic pathways to enhance tumor cell survival and migration while augmenting CD4+ to CD8+ communications to promote cytotoxicity. Here, we hypothesized that activin exerts cell-specific effects in the tumor microenvironment (TME) of CRC to promote anti-tumoral activity of immune cells and the pro-metastatic behavior of tumor cells in a cell-specific and context-dependent manner. We generated an Smad4 epithelial cell specific knockout (Smad4-/-) which was crossed with TS4-Cre mice to identify SMAD-specific changes in CRC. We also performed IHC and digital spatial profiling (DSP) of tissue microarrays (TMAs) obtained from 1055 stage II and III CRC patients in the QUASAR 2 clinical trial. We transfected the CRC cells to reduce their activin production and injected them into mice with intermittent tumor measurements to determine how cancer-derived activin alters tumor growth in vivo. In vivo, Smad4-/- mice displayed elevated colonic activin and pAKT expression and increased mortality. IHC analysis of the TMA samples revealed increased activin was required for TGF-ß-associated improved outcomes in CRC. DSP analysis identified that activin co-localization in the stroma was coupled with increases in T-cell exhaustion markers, activation markers of antigen presenting cells (APCs), and effectors of the PI3K/AKT pathway. Activin-stimulated PI3K-dependent CRC transwell migration, and the in vivo loss of activin lead to smaller CRC tumors. Taken together, activin is a targetable, highly context-dependent molecule with effects on CRC growth, migration, and TME immune plasticity.

IL7 genetic variation and toxicity to immune checkpoint blockade in patients with melanoma.

Treatment with immune checkpoint blockade (ICB) frequently triggers immune-related adverse events (irAEs), causing considerable morbidity. In 214 patients receiving ICB for melanoma, we observed increased severe irAE risk in minor allele carriers of rs16906115, intronic to IL7. We found that rs16906115 forms a B cell-specific expression quantitative trait locus (eQTL) to IL7 in patients. Patients carrying the risk allele demonstrate increased pre-treatment B cell IL7 expression, which independently associates with irAE risk, divergent immunoglobulin expression and more B cell receptor mutations. Consistent with the role of IL-7 in T cell development, risk allele carriers have distinct ICB-induced CD8+ T cell subset responses, skewing of T cell clonality and greater proportional repertoire occupancy by large clones. Finally, analysis of TCGA data suggests that risk allele carriers independently have improved melanoma survival. These observations highlight key roles for B cells and IL-7 in both ICB response and toxicity and clinical outcomes in melanoma.

A clinical decision support system optimising adjuvant chemotherapy for colorectal cancers by integrating deep learning and pathological staging markers: a development and validation study.

BACKGROUND: The DoMore-v1-CRC marker was recently developed using deep learning and conventional haematoxylin and eosin-stained tissue sections, and was observed to outperform established molecular and morphological markers of patient outcome after primary colorectal cancer resection. The aim of the present study was to develop a clinical decision support system based on DoMore-v1-CRC and pathological staging markers to facilitate individualised selection of adjuvant treatment. METHODS: We estimated cancer-specific survival in subgroups formed by pathological tumour stage (pT<4 or pT4), pathological nodal stage (pN0, pN1, or pN2), number of lymph nodes sampled (≤12 or >12) if not pN2, and DoMore-v1-CRC classification (good, uncertain, or poor prognosis) in 997 patients with stage II or III colorectal cancer considered to have no residual tumour (R0) from two community-based cohorts in Norway and the UK, and used these data to define three risk groups. An external cohort of 1075 patients with stage II or III R0 colorectal cancer from the QUASAR 2 trial was used for validation; these patients were treated with single-agent capecitabine. The proposed risk stratification system was evaluated using Cox regression analysis. We similarly evaluated a risk stratification system intended to reflect current guidelines and clinical practice. The primary outcome was cancer-specific survival. FINDINGS: The new risk stratification system provided a hazard ratio of 10·71 (95% CI 6·39-17·93; p<0·0001) for high-risk versus low-risk patients and 3·06 (1·73-5·42; p=0·0001) for intermediate versus low risk in the primary analysis of the validation cohort. Estimated 3-year cancer-specific survival was 97·2% (95% CI 95·1-98·4; n=445 [41%]) for the low-risk group, 94·8% (91·7-96·7; n=339 [32%]) for the intermediate-risk group, and 77·6% (72·1-82·1; n=291 [27%]) for the high-risk group. The guideline-based risk grouping was observed to be less prognostic and informative (the low-risk group comprised only 142 [13%] of the 1075 patients). INTERPRETATION: Integrating DoMore-v1-CRC and pathological staging markers provided a clinical decision support system that risk stratifies more accurately than its constituent elements, and identifies substantially more patients with stage II and III colorectal cancer with similarly good prognosis as the low-risk group in current guidelines. Avoiding adjuvant chemotherapy in these patients might be safe, and could reduce morbidity, mortality, and treatment costs. FUNDING: The Research Council of Norway.

Tertiary lymphoid structures critical for prognosis in endometrial cancer patients.

B-cells play a key role in cancer suppression, particularly when aggregated in tertiary lymphoid structures (TLS). Here, we investigate the role of B-cells and TLS in endometrial cancer (EC). Single cell RNA-sequencing of B-cells shows presence of naïve B-cells, cycling/germinal center B-cells and antibody-secreting cells. Differential gene expression analysis shows association of TLS with L1CAM overexpression. Immunohistochemistry and co-immunofluorescence show L1CAM expression in mature TLS, independent of L1CAM expression in the tumor. Using L1CAM as a marker, 378 of the 411 molecularly classified ECs from the PORTEC-3 biobank are evaluated, TLS are found in 19%. L1CAM expressing TLS are most common in mismatch-repair deficient (29/127, 23%) and polymerase-epsilon mutant EC (24/47, 51%). Multivariable Cox regression analysis shows strong favorable prognostic impact of TLS, independent of clinicopathological and molecular factors. Our data suggests a pivotal role of TLS in outcome of EC patients, and establishes L1CAM as a simple biomarker.

Discordant prognosis of mismatch repair deficiency in colorectal and endometrial cancer reflects variation in antitumour immune response and immune escape.

Defective DNA mismatch repair (dMMR) causes elevated tumour mutational burden (TMB) and microsatellite instability (MSI) in multiple cancer types. dMMR/MSI colorectal cancers (CRCs) have enhanced T-cell infiltrate and favourable outcome; however, this association has not been reliably detected in other tumour types, including endometrial cancer (EC). We sought to confirm this and explore the underpinning mechanisms. We first meta-analysed CRC and EC trials that have examined the prognostic value of dMMR/MSI and confirmed that dMMR/MSI predicts better prognosis in CRC, but not EC, with statistically significant variation between cancers (hazard ratio [HR] = 0.63, 95% confidence interval [CI] = 0.54-0.73 versus HR = 1.15, 95% CI = 0.72-1.58; PINT = 0.02). Next, we studied intratumoural immune infiltrate in CRCs and ECs of defined MMR status and found that while dMMR was associated with increased density of tumour-infiltrating CD3+ and CD8+ T-cells in both cancer types, the increases were substantially greater in CRC and significant only in this group (PINT = 4.3e-04 and 7.3e-03, respectively). Analysis of CRC and EC from the independent Cancer Genome Atlas (TCGA) series revealed similar variation and significant interactions in proportions of tumour-infiltrating lymphocytes, CD8+ , CD4+ , NK cells and immune checkpoint expression, confirming a more vigorous immune response to dMMR/MSI in CRC than EC. Agnostic analysis identified the IFNγ pathway activity as strongly upregulated by dMMR/MSI in CRC, but downregulated in EC by frequent JAK1 mutations, the impact of which on IFNγ response was confirmed by functional analyses. Collectively, our results confirm the discordant prognosis of dMMR/MSI in CRC and EC and suggest that this relates to differences in intratumoural immune infiltrate and tumour genome. Our study underscores the need for tissue-specific analysis of cancer biomarkers and may help inform immunotherapy use. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Histological and Somatic Mutational Profiles of Mismatch Repair Deficient Endometrial Tumours of Different Aetiologies.

BACKGROUND: Mismatch repair deficient (MMRd) tumours may arise from somatic events acquired during carcinogenesis or in the context of Lynch syndrome (LS), an inherited cancer predisposition condition caused by germline MMR pathogenic variants. Our aim was to explore whether sporadic and hereditary MMRd endometrial cancers (EC) display distinctive tumour biology. METHODS: Clinically annotated LS-EC were collected. Histological slide review was performed centrally by two specialist gynaecological pathologists. Mutational analysis was by a bespoke 75- gene next-generation sequencing panel. Comparisons were made with sporadic MMRd EC. Multiple correspondence analysis was used to explore similarities and differences between the cohorts. RESULTS: After exclusions, 135 LS-EC underwent independent histological review, and 64 underwent mutational analysis. Comparisons were made with 59 sporadic MMRd EC. Most tumours were of endometrioid histological subtype (92% LS-EC and 100% sporadic MMRd EC, respectively, p = NS). Sporadic MMRd tumours had significantly fewer tumour infiltrating lymphocytes (p ≤ 0.0001) and showed more squamous/mucinous differentiation than LS-EC (p = 0.04/p = 0.05). PTEN mutations were found in 88% sporadic MMRd and 61% LS-EC, respectively (p < 0.001). Sporadic MMRd tumours had significantly more mutations in PDGFRA, ALK, IDH1, CARD11, CIC, MED12, CCND1, PTPN11, RB1 and KRAS, while LS-EC showed more mutations affecting SMAD4 and ARAF. LS-EC showed a propensity for TGF-ß signalling disruption. Cluster analysis found that wild type PTEN associates predominantly with LS-EC, whilst co-occurring mutations in PTEN, PIK3CA and KRAS predict sporadic MMRd EC. CONCLUSIONS: Whilst MMRd EC of hereditary and sporadic aetiology may be difficult to distinguish by histology alone, differences in infiltrating immune cell counts and mutational profile may predict heterogenous responses to novel targeted therapies and warrant further study.

Automated assessment of CD8+ T-lymphocytes and stroma fractions complement conventional staging of colorectal cancer.

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.

Evaluation of treatment effects in patients with endometrial cancer and POLE mutations: An individual patient data meta-analysis.

BACKGROUND: Endometrial cancers (ECs) with somatic mutations in DNA polymerase epsilon (POLE) are characterized by unfavorable pathological features, which prompt adjuvant treatment. Paradoxically, women with POLE-mutated EC have outstanding clinical outcomes, and this raises concerns of overtreatment. The authors investigated whether favorable outcomes were independent of treatment. METHODS: A PubMed search for POLE and endometrial was restricted to articles published between March 1, 2012, and March 1, 2018, that provided individual patient data (IPD), adjuvant treatment, and survival. Following the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) reporting guidelines for IPD, the authors used univariate and multivariate one-stage meta-analyses with mixed effects Cox models (random effects for study cohorts) to infer the associations of treatment, traditional prognostic factors, and outcome, which was defined as the time from first diagnosis to any adverse event (progression/recurrence or death from EC). RESULTS: Three hundred fifty-nine women with POLE-mutated EC were identified; 294 (82%) had pathogenic mutations. Worse outcomes were demonstrated in patients with nonpathogenic POLE mutations (hazard ratio, 3.42; 95% confidence interval, 1.47-7.58; log-rank P < .01). Except for stage (P < .01), traditional prognosticators were not associated with progression/recurrence or death from disease. Adverse events were rare (11 progressions/recurrences and 3 disease-specific deaths). Salvage rates in patients who experienced recurrence were high and sustained, with 8 of 11 alive without evidence of disease (range, 5.5-14.2 years). Adjuvant treatment was not associated with outcome. CONCLUSIONS: Clinical outcomes for ECs with pathogenic POLE mutations are not associated with most traditional risk parameters, and patients do not appear to benefit from adjuvant therapy. The observed low rates of recurrence/progression and the high and sustained salvage rates raise the possibility of safely de-escalating treatment for these patients. LAY SUMMARY: Ten percent of all endometrial cancers have mutations in the DNA repair gene DNA polymerase epsilon (POLE). Women who have endometrial cancers with true POLE mutations experience almost no recurrences or deaths from their cancer even when their tumors appear to have very unfavorable characteristics. Additional therapy (radiation and chemotherapy) does not appear to improve outcomes for women with POLE-mutated endometrial cancer, and this supports the move to less therapy and less associated toxicity. Diligent classification of endometrial cancers by molecular features provides valuable information to inform prognosis and to direct treatment/no treatment.

Promises and challenges of adoptive T-cell therapies for solid tumours.

Cancer is a leading cause of death worldwide and, despite new targeted therapies and immunotherapies, many patients with advanced-stage- or high-risk cancers still die, owing to metastatic disease. Adoptive T-cell therapy, involving the autologous or allogeneic transplant of tumour-infiltrating lymphocytes or genetically modified T cells expressing novel T-cell receptors or chimeric antigen receptors, has shown promise in the treatment of cancer patients, leading to durable responses and, in some cases, cure. Technological advances in genomics, computational biology, immunology and cell manufacturing have brought the aspiration of individualised therapies for cancer patients closer to reality. This new era of cell-based individualised therapeutics challenges the traditional standards of therapeutic interventions and provides opportunities for a paradigm shift in our approach to cancer therapy. Invited speakers at a 2020 symposium discussed three areas-cancer genomics, cancer immunology and cell-therapy manufacturing-that are essential to the effective translation of T-cell therapies in the treatment of solid malignancies. Key advances have been made in understanding genetic intratumour heterogeneity, and strategies to accurately identify neoantigens, overcome T-cell exhaustion and circumvent tumour immunosuppression after cell-therapy infusion are being developed. Advances are being made in cell-manufacturing approaches that have the potential to establish cell-therapies as credible therapeutic options. T-cell therapies face many challenges but hold great promise for improving clinical outcomes for patients with solid tumours.

The Glasgow Microenvironment Score associates with prognosis and adjuvant chemotherapy response in colorectal cancer.

BACKGROUND: The Glasgow Microenvironment Score (GMS) combines peritumoural inflammation and tumour stroma percentage to assess interactions between tumour and microenvironment. This was previously demonstrated to associate with colorectal cancer (CRC) prognosis, and now requires validation and assessment of interactions with adjuvant therapy. METHODS: Two cohorts were utilised; 862 TNM I-III CRC validation cohort, and 2912 TNM II-III CRC adjuvant chemotherapy cohort (TransSCOT). Primary endpoints were disease-free survival (DFS) and relapse-free survival (RFS). Exploratory endpoint was adjuvant chemotherapy interaction. RESULTS: GMS independently associated with DFS (p = 0.001) and RFS (p < 0.001). GMS significantly stratified RFS for both low risk (GMS 0 v GMS 2: HR 3.24 95% CI 1.85-5.68, p < 0.001) and high-risk disease (GMS 0 v GMS 2: HR 2.18 95% CI 1.39-3.41, p = 0.001). In TransSCOT, chemotherapy type (pinteraction = 0.013), but not duration (p = 0.64) was dependent on GMS. Furthermore, GMS 0 significantly associated with improved DFS in patients receiving FOLFOX compared with CAPOX (HR 2.23 95% CI 1.19-4.16, p = 0.012). CONCLUSIONS: This study validates the GMS as a prognostic tool for patients with stage I-III colorectal cancer, independent of TNM, with the ability to stratify both low- and high-risk disease. Furthermore, GMS 0 could be employed to identify a subset of patients that benefit from FOLFOX over CAPOX.

Prediction of relapse-free survival according to adjuvant chemotherapy and regulator of chromosome condensation 2 (RCC2) expression in colorectal cancer.

BACKGROUND: There is a need for improved selection of patients for adjuvant chemotherapy after resection of non-metastatic colorectal cancer (CRC). Regulator of chromosome condensation 2 (RCC2) is a potential prognostic biomarker. We report on the establishment of a robust protocol for RCC2 expression analysis and prognostic tumour biomarker evaluation in patients who did and did not receive adjuvant chemotherapy. MATERIALS AND METHODS: RCC2 was analysed in 2916 primary CRCs from the QUASAR2 randomised trial and two single-hospital Norwegian series. A new protocol using fluorescent antibody staining and digital image analysis was optimised. Biomarker value for 5-year relapse-free survival was analysed in relation to tumour stage, adjuvant chemotherapy and the molecular markers microsatellite instability, KRAS/BRAFV600E/TP53 mutations and CDX2 expression. RESULTS: Low RCC2 expression was scored in 41% of 2696 evaluable samples. Among patients with stage I-III CRC who had not received adjuvant chemotherapy, low RCC2 expression was an independent marker of inferior 5-year relapse-free survival in multivariable Cox models including clinicopathological factors and molecular markers (HR 1.45, 95% CI 1.09 to 1.94, p=0.012, N=521). RCC2 was not prognostic in patients who had received adjuvant chemotherapy, neither in QUASAR2 nor the pooled Norwegian series. The interaction between RCC2 and adjuvant chemotherapy for prediction of patient outcome was significant in stage III, and strongest among patients with microsatellite stable tumours (pinteraction=0.028). CONCLUSIONS: Low expression of RCC2 is a biomarker for poor prognosis in patients with stage I-III CRC and seems to be a predictive biomarker for effect of adjuvant chemotherapy.

Prognostic Integrated Image-Based Immune and Molecular Profiling in Early-Stage Endometrial Cancer.

Optimum risk stratification in early-stage endometrial cancer combines clinicopathologic factors and the molecular endometrial cancer classification defined by The Cancer Genome Atlas (TCGA). It is unclear whether analysis of intratumoral immune infiltrate improves this. We developed a machine-learning, image-based algorithm to quantify density of CD8+ and CD103+ immune cells in tumor epithelium and stroma in 695 stage I endometrioid endometrial cancers from the PORTEC-1 and -2 trials. The relationship between immune cell density and clinicopathologic/molecular factors was analyzed by hierarchical clustering and multiple regression. The prognostic value of immune infiltrate by cell type and location was analyzed by univariable and multivariable Cox regression, incorporating the molecular endometrial cancer classification. Tumor-infiltrating immune cell density varied substantially between cases, and more modestly by immune cell type and location. Clustering revealed three groups with high, intermediate, and low densities, with highly significant variation in the proportion of molecular endometrial cancer subgroups between them. Univariable analysis revealed intraepithelial CD8+ cell density as the strongest predictor of endometrial cancer recurrence; multivariable analysis confirmed this was independent of pathologic factors and molecular subgroup. Exploratory analysis suggested this association was not uniform across molecular subgroups, but greatest in tumors with mutant p53 and absent in DNA mismatch repair-deficient cancers. Thus, this work identified that quantification of intraepithelial CD8+ cells improved upon the prognostic utility of the molecular endometrial cancer classification in early-stage endometrial cancer.

Histological phenotypic subtypes predict recurrence risk and response to adjuvant chemotherapy in patients with stage III colorectal cancer.

Histological 'phenotypic subtypes' that classify patients into four groups (immune, canonical, latent and stromal) have previously been demonstrated to stratify survival in a stage I-III colorectal cancer (CRC) pilot cohort. However, clinical utility has not yet been validated. Therefore, this study assessed prognostic value of these subtypes in additional patient cohorts along with associations with risk of recurrence and response to chemotherapy. Two independent stage I-III CRC patient cohorts (internal and external cohort) were utilised to investigate phenotypic subtypes. The primary endpoint was disease-free survival (DFS) and the secondary endpoint was recurrence risk (RR). Stage II-III patients, from the SCOT adjuvant chemotherapy trial, were utilised to further validate prognostic value and for exploratory analysis assessing associations with adjuvant chemotherapy. In an 893-patient internal cohort, phenotypic subtype independently associated with DFS (p = 0.025) and this was attenuated in stage III patients (p = 0.020). Phenotypic subtype also independently associated with RR (p < 0.001) in these patients. In a 146-patient external cohort, phenotypic subtype independently stratified patients by DFS (p = 0.028), validating their prognostic value. In 1343 SCOT trial patients, the effect of treatment type significantly depended on phenotypic subtype (pinteraction = 0.011). Phenotypic subtype independently associated with DFS in stage III patients receiving FOLFOX (p = 0.028). Furthermore, the immune subtype significantly associated with better response to FOLFOX compared to CAPOX adjuvant chemotherapy in stage III patients (p = 0.013). In conclusion, histological phenotypic subtypes are an effective prognostic classification in patients with stage III CRC that associates with risk of recurrence and response to FOLFOX adjuvant chemotherapy.

Rationale and design of the POLEM trial: avelumab plus fluoropyrimidine-based chemotherapy as adjuvant treatment for stage III mismatch repair deficient or POLE exonuclease domain mutant colon cancer: a phase III randomised study.

BACKGROUND: 10%-15% of early-stage colon cancers harbour either deficient mismatch repair (dMMR), microsatellite instability high (MSI-H) or POLE exonuclease domain mutations, and are characterised by high tumour mutational burden and increased lymphocytic infiltrate. Metastatic dMMR colon cancers are highly sensitive to immune checkpoint inhibition, and recent data show POLE-mutant tumours are similarly responsive. We are conducting a phase III randomised trial to determine if the addition of the anti-PD-L1 antibody avelumab following adjuvant chemotherapy improves disease-free survival (DFS) in patients with stage III dMMR/MSI-H or POLE mutant colon cancer and is a cost-effective approach for the UK National Health Service (NHS). METHODS: We are recruiting patients with completely resected, stage III colon cancer confirmed to have dMMR/MSI-H, locally or POLE exonuclease domain mutation on central testing. Eligible patients are randomised in a 1:1 ratio to standard fluoropyrimidine-based chemotherapy (capecitabine, oxaliplatin for 12 weeks or capecitabine for 24 weeks) or chemotherapy, followed by avelumab (10 mg/kg, 2 weekly for 24 weeks). Stratification is by chemotherapy received and MMR/MSI-H status. The primary endpoint is DFS. Secondary endpoints include overall survival, toxicity, quality of life and health resource use. The 3-year DFS rate in the control arm is expected to be ~75%. Avelumab is expected to improve the 3-year DFS rate by 12% (ie, 87%). Target accrual is 402 patients, which provides 80% power to detect an HR of 0.48 for DFS at a two-sided alpha of 0.05. This national, multicentre phase III trial is sponsored by the Royal Marsden NHS Foundation Trust and it is anticipated that approximately 40 centres in the UK will participate. This study opened to recruitment in August 2018. TRIAL REGISTRATION NUMBER: NCT03827044.

Deep learning for prediction of colorectal cancer outcome: a discovery and validation study.

BACKGROUND: Improved markers of prognosis are needed to stratify patients with early-stage colorectal cancer to refine selection of adjuvant therapy. The aim of the present study was to develop a biomarker of patient outcome after primary colorectal cancer resection by directly analysing scanned conventional haematoxylin and eosin stained sections using deep learning. METHODS: More than 12 000 000 image tiles from patients with a distinctly good or poor disease outcome from four cohorts were used to train a total of ten convolutional neural networks, purpose-built for classifying supersized heterogeneous images. A prognostic biomarker integrating the ten networks was determined using patients with a non-distinct outcome. The marker was tested on 920 patients with slides prepared in the UK, and then independently validated according to a predefined protocol in 1122 patients treated with single-agent capecitabine using slides prepared in Norway. All cohorts included only patients with resectable tumours, and a formalin-fixed, paraffin-embedded tumour tissue block available for analysis. The primary outcome was cancer-specific survival. FINDINGS: 828 patients from four cohorts had a distinct outcome and were used as a training cohort to obtain clear ground truth. 1645 patients had a non-distinct outcome and were used for tuning. The biomarker provided a hazard ratio for poor versus good prognosis of 3·84 (95% CI 2·72-5·43; p<0·0001) in the primary analysis of the validation cohort, and 3·04 (2·07-4·47; p<0·0001) after adjusting for established prognostic markers significant in univariable analyses of the same cohort, which were pN stage, pT stage, lymphatic invasion, and venous vascular invasion. INTERPRETATION: A clinically useful prognostic marker was developed using deep learning allied to digital scanning of conventional haematoxylin and eosin stained tumour tissue sections. The assay has been extensively evaluated in large, independent patient populations, correlates with and outperforms established molecular and morphological prognostic markers, and gives consistent results across tumour and nodal stage. The biomarker stratified stage II and III patients into sufficiently distinct prognostic groups that potentially could be used to guide selection of adjuvant treatment by avoiding therapy in very low risk groups and identifying patients who would benefit from more intensive treatment regimes. FUNDING: The Research Council of Norway.