PURPOSE:Two dominant neoadjuvant radiotherapy (RT) regimens are in common use for locally advanced rectal cancer (LARC): long-course RT (LCRT; 25 × 1.8 Gy) with concomitant chemotherapy and short-course RT (SCRT; 5 × 5 Gy), typically followed by systemic chemotherapy. This study uses serial sampling to investigate the evolution of systemic and local immune responses to different RT regimens in LARC. EXPERIMENTAL DESIGN:We conducted a serial sampling study involving patients receiving RT for LARC, in which longitudinal blood and tumor biospecimens were collected at baseline and at 2, 6, and 12 weeks after treatment initiation. Leukocyte concentrations via full blood count and a multiplex cytokine ELISA of plasma samples measured systemic changes. Multiplex immunofluorescence (CD8 and FOXP3) and RNA sequencing of tumor biopsies were used to assess local changes. RESULTS:Circulating lymphocyte concentrations tended to increase in SCRT patients and decrease in LCRT patients between week 2 and week 6 and were significantly lower in LCRT patients compared with SCRT patients at week 6 (P < 0.0001) and week 12 (P = 0.019). Additionally, we report higher densities of CD8+ and FOXP3+ tumoral lymphocytes from SCRT patients compared with LCRT patients at week 2 (CD8 P = 0.053; FOXP3 P = 0.023) and week 6 (CD8 P = 0.035; FOXP3 P = 0.0016). CONCLUSIONS:SCRT is less lymphodepleting and induces more frequent increases in intratumoral T-cell infiltration compared with LCRT. These results are relevant to the field of radiation-immune-oncology combination studies in LARC. Furthermore, these findings may underpin early trial results, in which higher rates of response to RT-immune checkpoint inhibitor combinations have been reported with SCRT-based regimens.

SpatialDecon derived immune cell counts aggregated by Region, KM grade and KRAS status with pairwise comparison between groups. Mann-Whitney test used to determine statistical significance between groups

Supplementary details regarding methods and materials

Topographic immune cell deconvolution primary colorectal cancer and CRLM. A, Images (from Fig. 4A) of primary colorectal cancer (bottom) and CRLM (top) with 48 ROIs superimposed. Abundance estimates as determined from transcriptome by SpatialDecon for 14 cell populations illustrated for each ROI with color coding detailing the annotated tumor region. Radius is proportional to the estimated cell counts within the ROI. The immune cell count per region was extracted and the square root of the ratio to the mean immune cell count per region (41.37) of all immune cells was calculated and is displayed. The square root of the ratio was calculated to minimize the skew caused by variance of highly expressed cell types. B, Box plots demonstrating the median and interquartile range for each cell type analyzed organized by cell type and topographic region and grouped by KM grade. All ROIs taken from primary colorectal cancer except TLR are grouped as primary. Dendritic cells were removed due to insignificant counts. The Mann–Whitney test was used to assess for statistical significance; *, P < 0.05.

Mutational characterization of primary colorectal cancer and CRLM. A, Venn diagram demonstrating primary colorectal cancer and CRLM that underwent genomic analysis. Co-Barplot illustrating most frequently mutated genes across 13 matched primary colorectal cancer and CRLM, including mutation type. Genes are ordered by mutational frequency. Sections were sequenced using GPOL (Glasgow Precision Oncology Laboratory) mutational panel. B, Oncoplot demonstrating concurrent mutations in the 13 matched lesions. Patients are ranked according to co-mutational burden on the y-axis and ranked according to KM grade on the x-axis. Blue, gene mutated in primary only; red, gene mutated in metastasis only; purple, mutated in primary and metastasis. The right-hand three columns denote the percentage of total patients with each mutation type. C, Correlation matrix demonstrating co-occurrence of mutations, with left of the blue demarcation line representing primary colorectal cancer, right of the blue demarcation line representing CRLM (pair-wise Fisher exact test; *, P < 0.05). Gene names are displayed along the x- and y-axes ordered by mutational frequency. Dark green boxes represent significant co-occurrence. D, Box plot illustrating mutational burden in primary colorectal cancer and CRLM according to KM grade using the Mann–Whitney test to assess for statistically significant difference between KM groups.

Gene Set Enrichment Analysis results obtained by interrogating ranked list of differentially expressed KM high vs KM low genes against the REACTOME curated gene set database using ClusterProfiler package

IHC characterization of matched primary colorectal cancer and CRLM with integration of morphological and mutational features. A, Representative images of CD3 and CD66b immunohistochemical staining (Patient B). Whole section demonstrated at ×0.5 magnifications; ROIs corresponding to tumor center (TC) and invasive edge (IE) of primary and CRLM are shown at ×6 and ×10 (black box). Scale bar, 100 μm. B, Intrapatient comparison between primary and metastasis of CD3 and CD66b cell counts at tumor center and invasive edge. P values calculated using the Mann–Whitney test. C, Kaplan–Meier survival plots (log-rank test, P values displayed) demonstrating the prognostic impact of CD3 and CD66b cell density at CRLM IE identified by IHC for IE of CRLM. High and low values determined according to median expression. D, Comparison of CD3 and CD66b cell density at (i) primary IE, (ii) CRLM IE, and (iii) CD3 primary IE and metastatic TC. Spearman Rho analysis. E, Box plots illustrating the relationship between KM grade and CD3 and CD66b cell counts at the IE and TC of primary colorectal cancer and CRLM. The P value was calculated using the Mann–Whitney test. F, Box plot representing relationship between mutational features (APC, TP53, KRAS, Serrated) and CD3 and CD66b cell density at TC and IE of primary colorectal cancer and CRLM. The P value was calculated using the Mann–Whitney test. Lesions of serrated origin were defined as APCwild + KRAS/BRAFmutation.

Clinicopathological, morphological, and treatment characteristics for synchronously resected primary colorectal cancer and paired CRLM.

Spatially resolved transcriptomic analysis using Nanostring Cancer Transcriptome Atlas gene sets. A, Representative images of mIF staining of four matched primary colorectal cancer and CRLM. DAPI, blue; Pancytokeratin, green; CD45, pink; αSMA, yellow. Topographic regions are annotated and each box represents hand-selected area of tumor. Eight regions were taken from CRLM and four from primary colorectal cancer per patient. Patient A: KMhigh, KRAS-wt, good prognosis. Patient B: KMhigh, KRAS-mt, good prognosis. Patient C: KMlow, KRAS/TP53 co-mutation, poor prognosis. Patient D: KM-low, KRAS-wt, BRAF-mt high-mutational burden lesion. B, PCA plot of all ROIs selected. The patient from whom the lesion originated is represented by shape. A red border indicates region arises from CRLM and white border represents primary colorectal cancer. The topographical region within the lesion is illustrated by the innermost color of the shape. KM high metastatic edges, green circle; KMlow metastatic edges, red circle; dashed blue line, epithelial regions of primary colorectal cancer and CRLM. C, Heatmap demonstrating single sample GSEA for every ROI, ordered on the x-axis by patient and ROI. Key presented to aid patient identification. The y-axis represents annotated gene sets from Cancer Transcriptome Atlas ordered by and clustered within modules of Immune Response, Adaptive Immune, Innate Immune, Signaling Pathways. Cell Function, Metabolism. Each cell represents the NES scaled by pathway. D, Heatmap demonstrating GSEA providing interpatient comparison of selected areas between KMhigh and KMlow patients and intrapatient comparison between primary and metastatic sites and intralesional comparison between tumor center and immune edge. Subset of regions filtered before GSEA is demonstrated in subgroup. Subsequent groups compared in GSEA identified as groups A and B. Group A annotated at top of diagram and represented by green. Group B annotated at bottom of diagram and represented by orange. Cells of heatmap represent −log10(Padj) for comparison; cell is tinted green if pathway is upregulated in group A and orange if upregulated in group B.

Immune cell spatial deconvolution. A, Representative images from 1 CRLM (Patient A, Fig. 4A) showing cell detection from the Qupath package used on a CD3 and CD66b IHC-stained liver metastasis to count number of CD3 and CD66b-positive cells from 21 regions from a total of three CRLM. This count was compared with the SpatialDecon-derived count that uses the transcriptomic data from the corresponding ROI in the GeoMx mIF-stained matched sample (See Supplementary Fig. S5). B, Bland Altman plot comparing transcriptome SpatialDecon-derived cell count versus the IHC-derived cell count. C, Correlation plot comparing transcriptome SpatialDecon-derived cell count versus the IHC-derived cell count.

BULK IO360 transcriptomic characterization of matched primary colorectal cancer and CRLM. A, PCA plot demonstrating two principal components of minimal variance for all samples. Primary colorectal cancer samples are demonstrated by circles and yellow outline. CRLM are demonstrated by triangular points and brown outline. KMhigh, KMlow Stromalow and KMlow Stromahigh samples are depicted by color. B, Unsupervised analysis using gene expression correlation matrix for all samples. Patient, site, KM grade, and TSP are depicted by key. Spearman correlation of all expressed genes performed between each sample sequenced and plotted on the heatmap. k-means clustering of heatmap to demonstrate correlated samples. Red, strong correlation. Blue, negative correlation. C, Volcano plots demonstrating differential gene expression results and clustered heatmap of significant genes for (i) all primary colorectal cancer versus CRLM; (ii) KM grade: KM high versus KM low primary colorectal cancer; (iii) KM grade: KM high versus KM low CRLM. The x-axis of volcano plot demonstrates log2-fold change; y-axis demonstrates –log10P. Colored points demonstrate significant changes in gene expression between groups (P < 0.05 and logFC > 1.5). Volcano plots demonstrate top 20 differentially expressed genes for each group. D, Heatmap demonstrating GSEA results comparing the different tumors grouped according with KM grade and TSP using io360-curated gene sets annotated on the right of the diagram. Heatmap squares represent log10-adjusted P value. Green, upregulation in group 1; orange, upregulation in group 2. The heatmap is clustered by y-axis only to demonstrate frequently upregulated gene sets. E, Box plot comparisons of immune cell populations and selected cell:cell ratios between KM grade and TSP segregated groups using deconvolution software included in the nCounter package. Annotated subgroups are: Kmhigh, Kmlow Stromalow, Kmlow Stromahigh. The y-axis represents log10 of estimated cell count. Primary colorectal cancer is represented in i and CRLM in ii.

All supplementary figures and supplementary tables 1 and 2 from the manuscript

Abstract Introduction/Aims: Colorectal cancer (CRC) remains a leading cause of cancer-related mortality. The canonical NF-κB pathway driven by IKKβ has been implicated in CRC development and progression, however less research has focused on non-canonical NF-κB signaling, regulated by inhibitory-κB kinase alpha (IKKα). This project aimed to assess IKKα, phospho-IKKα (pIKKαs176) and IKKβ expression in a retrospective CRC cohort to establish association with survival outcomes. Subsequently, this project aimed to determine the effect of abrogating IKKα activity without perturbing the canonical NF-κB pathway controlled by IKKβ using a first-in-class selective IKKα inhibitor in vitro/ex vivo. Methods: Immunohistochemical staining for IKKα/pIKKα s176/IKKβ was performed using tissue microarrays from a CRC patient cohort (n=787). QuPath® software was used to semi-quantitively assess expression levels and scores were analyzed for association with cancer-specific survival (CSS). CRC cell lines (n=3), patient-derived organoids (PDOs; n=5) and patient-derived explants (n=5) were treated with novel IKKα inhibitor SU1644 and assessed for cell viability and proliferation. Results: Positive staining for IKKα, pIKKαs176 and IKKβ was detected in tumor specimens. High cytoplasmic expression of IKKα within tumor cells was associated with reduced CSS in patients with right-sided colon cancer (HR=3.091, 95%CI; 1.128-9.467, log rank p=0.021). Conversely, high IKKβ expression was a marker of good prognosis (HR=0.627, 95%CI; 0.457-0.861, log rank p=0.004). A high ratio of IKKα to IKKβ was associated with reduced CSS in the full cohort (HR=1.404, 95%CI; 1.050-1.879, log rank p=0.021) and this was potentiated in right-sided colon cases (HR=1.772, 95%CI; 1.107-2.837, log rank p=0.016). Selective inhibition of IKKα using 1µM SU1644 in vitro caused a significant reduction in HT29 colony formation (p=0.012) and cell viability (p=0.039). PDOs showed altered cell morphology, reduced cell viability and proliferation when treated with SU1644. In the patient-derived explant model, Ki67 expression was reduced in tumor explants treated with SU1644. Conclusions: These data establish high cytoplasmic IKKα expression within tumor cells as a marker of poor prognosis in CRC and conversely high IKKβ expression as a marker of good prognosis. This highlights the importance of development of selective IKKα inhibitor SU1644. IKKα inhibition using SU1644 demonstrated anti-cancer activity in recapitulative CRC disease models. Citation Format: Kathryn A. Pennel, Molly McKenzie, Guang-Yu Lian, Jean A. Quinn, Sara Samir Foad Al-Badran, Campbell S. Roxburgh, Simon P. Mackay, Joanna Birch, Joanne Edwards. Inhibitory-κB kinase alpha as a biomarker and therapeutic target in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4634.

To study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of 14-3-3σ (Stratifin) was analyzed in skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of PTEN-mediated AKT regulation (K14.creP/Δ5PTENflx/flx). Consistent with 14-3-3σ roles in epidermal differentiation, HK1.ras hyperplasia and papillomas displayed elevated 14-3-3σ expression in supra-basal keratinocytes, paralleled by supra-basal p-MDM2166 activation and sporadic p-AKT473 expression. In bi-genic HK1.fos/Δ5PTENflx/flx hyperplasia, basal-layer 14-3-3σ expression appeared, and alongside p53/p21, was associated with keratinocyte differentiation and keratoacanthoma etiology. Tri-genic HK1.ras/fos-Δ5PTENflx/flx hyperplasia/papillomas initially displayed increased basal-layer 14-3-3σ, suggesting attempts to maintain supra-basal p-MDM2166 and protect basal-layer p53. However, HK1.ras/fos-Δ5PTENflx/flx papillomas exhibited increasing basal-layer p-MDM2166 activation that reduced p53, which coincided with malignant conversion. Despite p53 loss, 14-3-3σ expression persisted in well-differentiated squamous cell carcinomas (wdSCCs) and alongside elevated p21, limited malignant progression via inhibiting p-AKT1473 expression; until 14-3-3σ/p21 loss facilitated progression to aggressive SCC exhibiting uniform p-AKT1473. Analysis of TPA-promoted HK1.ras-Δ5PTENflx/flx mouse skin, demonstrated early loss of 14-3-3σ/p53/p21 in hyperplasia and papillomas, with increased p-MDM2166/p-AKT1473 that resulted in rapid malignant conversion and progression to poorly differentiated SCC. In 2D/3D cultures, membranous 14-3-3σ expression observed in normal HaCaT and SP1ras61 papilloma keratinocytes was unexpectedly detected in malignant T52ras61/v-fos SCC cells cultured in monolayers, but not invasive 3D-cells. Collectively, these data suggest 14-3-3σ/Stratifin exerts suppressive roles in papillomatogenesis via MDM2/p53-dependent mechanisms; while persistent p53-independent expression in early wdSCC may involve p21-mediated AKT1 inhibition to limit malignant progression.

Colorectal cancer is the third most diagnosed malignancy worldwide and survival outcomes remain poor. Research is focused on the identification of novel prognostic and predictive biomarkers to improve clinical practice. There is robust evidence in the literature that inflammatory cytokine interleukin-6 (IL6) is elevated systemically in CRC patients and that this phenomenon is a predictor of poor survival outcome. However, evidence is more limited for the role of IL6 and its cognate receptor, IL6R, within the tumour epithelium and microenvironment. This study aimed to investigate IL6 and IL6R expression in a large cohort of retrospectively collected patient tumour specimens and determine association with clinical outcomes and characteristics. High expression of IL6R in the tumour epithelium was associated with reduced cancer-specific survival in patients with right-sided colon cancer. In these patients, high IL6R expression was also associated with an increased systemic neutrophil-to-lymphocyte ratio. A high number of copies of IL6 mRNA within the tumour-associated stroma, but not epithelium, was associated with reduced cancer-specific survival. The results from this study have validated IL6R as a marker of poor prognosis in a subgroup of CRC patients and identified the spatially resolved prognostic nature of intra-tumoural IL6 expression. This study has also highlighted the need for investigation of IL6/IL6R-targeted therapies as novel treatment strategies for patients with colon cancer.

Colorectal cancer (CRC) is the third most common malignancy cause of cancer-related mortality worldwide. Epithelial-mesenchymal transition (EMT) promotes cancer metastasis and a tumour-based Glasgow EMT score was associated with adverse clinical features and poor prognosis. In this study, the impact of using the established five tumour-based EMT markers consisting of E-cadherin (E-cad), β-catenin (β-cat), Snail, Zeb-1, and Fascin in combination with the stromal periostin (PN) on the prediction of CRC patients’ prognosis were invesigated. Formalin-fixed paraffin-embedded tissues of 202 CRC patients were studies the expressions of E-cad, β-cat, Snail, Zeb-1, Fascin, and PN by immunohistochemistry. Individually, cytoplasmic Fascin (Fc), cytoplasmic Snail (Sc), nuclear Snail (Sn), stromal Snail (Ss), and stromal PN (Ps) were significantly associated with reduced survival. A combination of Ps with Fc, Fs, and Sn was observed in 2 patterns including combined Fc, Fs, and Ps (FcFsPs) and Fc, Sn, and Ps (FcSnPs). These combinations enhanced the prognostic power compared to individual EMT markers and were independent prognostic markers. As the previously established scoring method required five markers and stringent criteria, its clinical use might be limited. Therefore, using these novel combined prognostic markers, either FcFsPs or FcSnPs, may be useful in predicting CRC patient outcomes.

Objectives Adenomas are known precursors to colorectal cancer (CRC). Current UK post-polypectomy surveillance guidelines use polyp size, numbers, and histology to stratify the risk of patients developing metachronous polyps or CRC. However, these risk guidelines suffer from poor predictive value, often leading to under/over surveillance. Design Adenomas removed from 1257 patients at bowel screening colonoscopy were retrospectively identified to investigate mutational profile and protein expression trends associated with the detection of metachronous polyps or CRC. The presence or absence of metachronous polyps or CRC was recorded 6 months to 6 years after index polypectomy. Results APC and KRAS were the most mutated genes in these patients (87% and 34% respectively), and both were significantly co-occurring with the 6th most mutated gene SOX9 (17% co-occurring with APC , p=0.047; 23% co-occurring with KRAS , p=0.012). High SOX9 cytoplasmic expression was significantly associated with the detection of metachronous polyps or CRC (HR 1.543, p=0.001) and improved high risk stratification when combined with BSG2020 guidelines versus guidelines alone (HR 2.626, p<0.0001). High cytoplasmic SOX9 alone and in combination with current guidelines was an independent predictor of metachronous polyps or CRC according to various regression models. This was validated in an independent test dataset, where high cytoplasmic expression was significantly associated with the detection of metachronous polyps or CRC (HR 1.654, p=0.012) and enhanced risk stratification when combined with BSG2020 guidelines versus guidelines alone (HR 2.473, p=0.0018). Conclusion High cytoplasmic SOX9 expression within adenomas is associated with shorter time to detection of metachronous polyps or CRC.   ### Competing Interest Statement DA was employed by BioClavis LTD at the time the work for this manuscript was carried out. All other authors declare no conflicts of interest. ### Funding Statement This work was funded by Cancer Research UK (Grant ID 105858), Innovate UK (Grants 105858 and 10054829), Medical Research Scotland (Grant OHD-50246-2020), and Cancer Research UK Scottish Cancer Centre (CTRQQR-2021/100006). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The West of Scotland Research Ethics Committee gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Mutational data used in this study are available at Datacite DOI: 10.5525/gla.researchdata.1223, Datacite DOI: 10.5525/gla.researchdata.1307, Datacite DOI: 10.5525/gla.researchdata.1498, and Datacite DOI: 10.5525/gla.researchdata.1602.Data used in this study is available upon reasonable request under condition of collaboration.