Histopathological tumour microenvironment score independently predicts outcome in primary operable colorectal cancer.

Colorectal cancer (CRC) is a heterogenous malignancy and research is focused on identifying novel ways to subtype patients. In this study, a novel classification system, tumour microenvironment score (TMS), was devised based on Klintrup-Mäkinen grade (KMG), tumour stroma percentage (TSP), and tumour budding. TMS was performed using a haematoxylin and eosin (H&E)-stained section from retrospective CRC discovery and validation cohorts (n = 1,030, n = 787). TMS0 patients had high KMG, TMS1 were low for KMG, TSP, and budding, TMS2 were high for budding, or TSP and TMS3 were high for TSP and budding. Scores were assessed for association with survival and clinicopathological characteristics. Mutational landscaping and Templated Oligo-Sequencing (TempO-Seq) profiling were performed to establish differences in the underlying biology of TMS. TMS was independently prognostic in both cohorts (p < 0.001, p < 0.001), with TMS3 predictive of the shortest survival times. TMS3 was associated with adverse clinical features including sidedness, local and distant recurrence, higher T stage, higher N stage, and presence of margin involvement. Gene set enrichment analysis of TempO-Seq data showed higher expression of genes associated with hallmarks of cancer pathways including epithelial to mesenchymal transition (p < 0.001), IL2 STAT5 signalling (p = 0.007), and angiogenesis (p = 0.017) in TMS3. Additionally, enrichment of immunosuppressive immune signatures was associated with TMS3 classification. In conclusion, TMS represents a novel and clinically relevant method for subtyping CRC patients from a single H&E-stained tumour section.

JAK/STAT3 represents a therapeutic target for colorectal cancer patients with stromal-rich tumors.

Colorectal cancer (CRC) is a heterogenous malignancy underpinned by dysregulation of cellular signaling pathways. Previous literature has implicated aberrant JAK/STAT3 signal transduction in the development and progression of solid tumors. In this study we investigate the effectiveness of inhibiting JAK/STAT3 in diverse CRC models, establish in which contexts high pathway expression is prognostic and perform in depth analysis underlying phenotypes. In this study we investigated the use of JAK inhibitors for anti-cancer activity in CRC cell lines, mouse model organoids and patient-derived organoids. Immunohistochemical staining of the TransSCOT clinical trial cohort, and 2 independent large retrospective CRC patient cohorts was performed to assess the prognostic value of JAK/STAT3 expression. We performed mutational profiling, bulk RNASeq and NanoString GeoMx® spatial transcriptomics to unravel the underlying biology of aberrant signaling. Inhibition of signal transduction with JAK1/2 but not JAK2/3 inhibitors reduced cell viability in CRC cell lines, mouse, and patient derived organoids (PDOs). In PDOs, reduced Ki67 expression was observed post-treatment. A highly significant association between high JAK/STAT3 expression within tumor cells and reduced cancer-specific survival in patients with high stromal invasion (TSPhigh) was identified across 3 independent CRC patient cohorts, including the TrasnSCOT clinical trial cohort. Patients with high phosphorylated STAT3 (pSTAT3) within the TSPhigh group had higher influx of CD66b + cells and higher tumoral expression of PDL1. Bulk RNAseq of full section tumors showed enrichment of NFκB signaling and hypoxia in these cases. Spatial deconvolution through GeoMx® demonstrated higher expression of checkpoint and hypoxia-associated genes in the tumor (pan-cytokeratin positive) regions, and reduced lymphocyte receptor signaling in the TME (pan-cytokeratin- and αSMA-) and αSMA (pan-cytokeratin- and αSMA +) areas. Non-classical fibroblast signatures were detected across αSMA + regions in cases with high pSTAT3. Therefore, in this study we have shown that inhibition of JAK/STAT3 represents a promising therapeutic strategy for patients with stromal-rich CRC tumors. High expression of JAK/STAT3 proteins within both tumor and stromal cells predicts poor outcomes in CRC, and aberrant signaling is associated with distinct spatially-dependant differential gene expression.

CXCL8 expression is associated with advanced stage, right sidedness, and distinct histological features of colorectal cancer.

CXCL8 is an inflammatory chemokine elevated in the colorectal cancer (CRC) tumour microenvironment. CXCR2, the major receptor for CXCL8, is predominantly expressed by neutrophils. In the cancer setting, CXCL8 plays important roles in neutrophil chemotaxis, facilitating angiogenesis, invasion, and metastasis. This study aimed to assess the spatial distribution of CXCL8 mRNA expression in CRC specimens, explore associations with clinical characteristics, and investigate the underlying biology of aberrant CXCL8 levels. CXCR2 expression was also assessed in a second cohort of unique CRC primary tumours and synchronously resected matched liver metastases. A previously constructed tissue microarray consisting of a cohort of stage I-IV CRC patients undergoing surgical resection with curative intent (n = 438) was probed for CXCL8 via RNAscope®. Analysis was performed using HALO® digital pathology software to quantify expression in the tumour and stromal compartments. Scores were assessed for association with clinical characteristics. Mutational analyses were performed on a subset of these patients to determine genomic differences in patients with high CXCL8 expression. A second cohort of stage IV CRC patients with primary and matched metastatic liver tumours was stained via immunohistochemistry for CXCR2, and scores were assessed for clinical significance. CXCL8 expression within the stromal compartment was associated with reduced cancer-specific survival in the first cohort (p = 0.035), and this relationship was potentiated in right-sided colon cancer cases (p = 0.009). High CXCL8 within the stroma was associated with driving a more stromal-rich phenotype and the presence of metastases. When stromal CXCL8 scores were combined with tumour-infiltrating macrophage counts or systemic neutrophil counts, patients classified as high for both markers had significantly poorer prognosis. CXCR2+ immune cell infiltration was associated with increased stromal invasion in liver metastases (p = 0.037). These data indicate a role for CXCL8 in driving unfavourable tumour histological features and promoting metastases. This study suggests that inhibiting CXCL8/CXCR2 should be investigated in patients with right-sided colonic disease and stroma-rich tumours.

Relationship between immune checkpoint proteins, tumour microenvironment characteristics, and prognosis in primary operable colorectal cancer.

The tumour microenvironment is an important factor for colorectal cancer prognosis, affecting the patient's immune response. Immune checkpoints, which regulate the immune functions of lymphocytes, may provide prognostic power. This study aimed to investigate the prognostic value of the immune checkpoints TIM-3, LAG-3 and PD-1 in patients with stage I-III colorectal cancer. Immunohistochemistry was employed to detect TIM-3, LAG-3, PD-1 and PD-L1 in 773 patients with stage I-III colorectal cancer. Immune checkpoint protein expression was assessed in tumour cells using the weighted histoscore, and in immune cells within the stroma using point counting. Scores were analysed for associations with survival and clinical factors. High tumoural LAG-3 (hazard ratio [HR] 1.45 95% confidence interval [CI] 1.00-2.09, p = 0.049) and PD-1 (HR 1.34 95% CI 1.00-1.78, p = 0.047) associated with poor survival, whereas high TIM-3 (HR 0.60 95% CI 0.42-0.84, p = 0.003), LAG-3 (HR 0.58 95% CI 0.40-0.87, p = 0.006) and PD-1 (HR 0.65 95% CI 0.49-0.86, p = 0.002) on immune cells within the stroma associated with improved survival, while PD-L1 in the tumour (p = 0.487) or the immune cells within the stroma (p = 0.298) was not associated with survival. Furthermore, immune cell LAG-3 was independently associated with survival (p = 0.017). Checkpoint expression scores on stromal immune cells were combined into a Combined Immune Checkpoint Stromal Score (CICSS), where CICSS 3 denoted all high, CICSS 2 denoted any two high, and CICSS 1 denoted other combinations. CICSS 3 was associated with improved patient survival (HR 0.57 95% CI 0.42-0.78, p = 0.001). The results suggest that individual and combined high expression of TIM-3, LAG-3, and PD-1 on stromal immune cells are associated with better colorectal cancer prognosis, suggesting there is added value to investigating multiple immune checkpoints simultaneously.

Src family kinases, HCK and FGR, associate with local inflammation and tumour progression in colorectal cancer.

BACKGROUND: In colorectal cancer (CRC), inflammatory responses have been reported to associate with patient survival. However, the specific signalling pathways responsible for regulating inflammatory responses are not clear. Src family kinases (SFKs) impact tumourigenic processes, including inflammation. METHODS: The relationship between SFK expression, inflammatory responses and cancer specific survival (CSS) in stage I-III CRC patients was assessed using immunohistochemistry on a 272 patient discovery cohort and an extended 822 patient validation cohort. RESULTS: In the discovery cohort, cytoplasmic FGR associated with improved CSS (P = 0.019), with membrane HCK (p = 0.093) trending towards poorer CSS. In the validation cohort membrane FGR (p = 0.016), membrane HCK (p = 0.019), and cytoplasmic HCK (p = 0.030) all associated with poorer CSS. Both markers also associated with decreased proliferation and cytotoxic T-lymphocytes (all p < 0.05). Furthermore, cytoplasmic HCK was an independent prognostic marker compared to common clinical factors. To assess synergy a combine FGR + HCK score was assessed. The membrane FGR + HCK score strengthened associations with poor prognosis (p = 0.006), decreased proliferation (p < 0.001) and cytotoxic T-lymphocytes (p < 0.001). CONCLUSIONS: SFKs associate with prognosis and the local inflammatory response in patients with stage I-III CRC. Active membrane FGR and HCK work in parallel to promote tumour progression and down-regulation of the local inflammatory lymphocytic response.

Immunotherapy: enhancing the efficacy of this promising therapeutic in multiple cancers.

Cancer treatments often reach a refractory period leading to treatment failure and patients developing disease recurrence. This can be due to tumour cells escaping the immune response and creating an immunosuppressive microenvironment enhancing cancer progression. Immunotherapy has become a promising tool for cancer treatment as it restores the anti-tumour response of the patient's immune system. Immune checkpoint inhibitors are the most widely studied immunotherapies worldwide and are now approved for multiple cancers. However, chimeric antigen receptor (CAR)-T cell therapy has also shown promise by targeting T lymphocytes that are genetically modified ex vivo to express CARs and this is now approved to treat some haematological cancers. Although immunotherapy has shown successful treatment outcomes in multiple cancers, some patients do not respond to this treatment. Therefore, approaches to enhance the efficacy of immunotherapies are likely to be the key to improve their effectiveness. Therefore, combination therapies of checkpoint inhibitors +/- chemotherapy are at the forefront of current research. Furthermore, biomarkers that predict treatment response are now beginning to emerge. Additionally, utilising nanoparticles as a newly targeted drug delivery system to enhance CAR-T cell therapy may enhance the efficacy of the cells when re-infused within the patient. Even if efficacy is enhanced, severe immune-related adverse events (irAEs) occur that are life-threatening and could lead to therapy being stopped. Therefore, predictive biomarkers for toxicity are also needed to improve both the patient's quality of life and treatment outcomes. This review will look at the current immunotherapies in clinical trials and discuss how to enhance their efficacy.