Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors.

Background: Lynch syndrome (LS) is an autosomal dominant multi-organ cancer syndrome with a high lifetime risk of cancer. The number of cumulative colorectal adenomas in LS does not generally exceed ten, and removal of adenomas via routine screening minimizes the cancer burden. However, abnormal phenotypes may mislead initial diagnosis and subsequently cause suboptimal treatment. Aim: Currently, there is no standard guide for the care of multiple colorectal adenomas in LS individuals. We aimed to shed insight into the molecular features and reasons for multiplicity of adenomas in LS patients. Methods: We applied whole exome sequencing on nine adenomas (ten samples) and three assumed primary carcinomas (five samples) of an LS patient developing the tumors during a 21-year follow-up period. We compared the findings to the tumor profiles of two additional LS cases ascertained through colorectal tumor multiplicity, as well as to ten adenomas and 15 carcinomas from 23 unrelated LS patients with no elevated adenoma burden from the same population. As LS associated cancers can arise via several molecular pathways, we also profiled the tumors for CpG Island Methylator Phenotype (CIMP), and LINE-1 methylation. Results: All tumors were microsatellite unstable (MSI), and MSI was present in several samples derived from normal mucosa as well. Interestingly, frequent frameshift variants in RNF43 were shared among substantial number of the tumors of our primary case and the tumors of LS cases with multiple tumors but almost absent in our control LS cases. The RNF43 variants were completely absent in the normal tissue, indicating tumor-associated mutational hotspots. The RNF43 status correlated with the mutational signature SBS96. Contrary to LS tumors from the reference set with no elevated colorectal tumor burden, the somatic variants occurred significantly more frequently at C>T in the CpG context, irrespective of CIMP or LINE-1 status, potentially indicating other, yet unknown methylation-related mechanisms. There were no signs of somatic mosaicism affecting the MMR genes. Somatic variants in APC and CTNNB1 were unique to each tumor. Conclusion: Frequent somatic RNF43 hot spot variants combined with SBS96 signature and increased tendency to DNA methylation may contribute to tumor multiplicity in LS.

High immune cell infiltration predicts improved survival in cholangiocarcinoma.

Background: Antitumoral immune response has a crucial role in constraining cancer. However, previous studies on cholangiocarcinoma (CCA), a rare and aggressive cancer, have reported contradictory findings on the prognostic impact of tumor-infiltrating T-lymphocytes. We aimed to clarify the effect of tumor-infiltrating CD3+ and CD8+ lymphocytes and PD-1/PD-L1 expression on CCA prognosis. Methods: CD3+, CD8+, and PD-1+ lymphocyte densities, as well as PD-L1 expression rate were analyzed from stained tissue microarray samples from the tumor center and invasive margin of 47 cholangiocarcinomas. The association of CD3+ and CD8+ based Immune cell score (ICS) and its components with overall survival was evaluated, adjusting for age, sex, TNM stage, radicality of surgery, tumor location, and PD-L1 expression on immune cells. Results: Low ICS was a strong independent prognostic factor for worse overall survival (Hazard ratio 9.27, 95% confidence interval 2.72-31.64, P<0.001). Among the ICS components, high CD8+ lymphocyte infiltration at the tumor center had the most evident impact on patient outcome. PD-1 and PD-L1 expression on immune cells did not have a significant impact on overall survival alone; however, PD-L1 positivity seemed to impair survival for ICSlow subgroup. Conclusion: Identifying patient subgroups that could benefit from immunotherapy with PD-1/PD-L1 pathway blockade may help improve treatment strategies for this aggressive cancer. Our findings highlight the importance of evaluating the immune contexture in cholangiocarcinoma, as ICS serves as a strong independent prognostic and selective factor for patients who might benefit from immunotherapy.

Mitotic abnormalities precede microsatellite instability in lynch syndrome-associated colorectal tumourigenesis.

BACKGROUND: Lynch syndrome (LS) is one of the most common hereditary cancer syndromes worldwide. Dominantly inherited mutation in one of four DNA mismatch repair genes combined with somatic events leads to mismatch repair deficiency and microsatellite instability (MSI) in tumours. Due to a high lifetime risk of cancer, regular surveillance plays a key role in cancer prevention; yet the observation of frequent interval cancers points to insufficient cancer prevention by colonoscopy-based methods alone. This study aimed to identify precancerous functional changes in colonic mucosa that could facilitate the monitoring and prevention of cancer development in LS. METHODS: The study material comprised colon biopsy specimens (n = 71) collected during colonoscopy examinations from LS carriers (tumour-free, or diagnosed with adenoma, or diagnosed with carcinoma) and a control group, which included sporadic cases without LS or neoplasia. The majority (80%) of LS carriers had an inherited genetic MLH1 mutation. The remaining 20% included MSH2 mutation carriers (13%) and MSH6 mutation carriers (7%). The transcriptomes were first analysed with RNA-sequencing and followed up with Gorilla Ontology analysis and Reactome Knowledgebase and Ingenuity Pathway Analyses to detect functional changes that might be associated with the initiation of the neoplastic process in LS individuals. FINDINGS: With pathway and gene ontology analyses combined with measurement of mitotic perimeters from colonic mucosa and tumours, we found an increased tendency to chromosomal instability (CIN), already present in macroscopically normal LS mucosa. Our results suggest that CIN is an earlier aberration than MSI and may be the initial cancer driving aberration, whereas MSI accelerates tumour formation. Furthermore, our results suggest that MLH1 deficiency plays a significant role in the development of CIN. INTERPRETATION: The results validate our previous findings from mice and highlight early mitotic abnormalities as an important contributor and precancerous marker of colorectal tumourigenesis in LS. FUNDING: This work was supported by grants from the Jane and Aatos Erkko Foundation, the Academy of Finland (330606 and 331284), Cancer Foundation Finland sr, and the Sigrid Jusélius Foundation. Open access is funded by Helsinki University Library.

Circulating microRNA signature predicts cancer incidence in Lynch syndrome - a pilot study.

Lynch syndrome (LS) is the most common autosomal dominant cancer syndrome and is characterized by high genetic cancer risk modified by lifestyle factors. This study explored whether a circulating microRNA (c-miR) signature predicts LS cancer incidence within a 4-year prospective surveillance period. To gain insight how lifestyle behavior could affect LS cancer risk, we investigated whether the cancer-predicting c-miR signature correlates with known risk-reducing factors such as physical activity, body mass index (BMI), dietary fiber or non-steroidal anti-inflammatory drug usage. The study included 110 c-miR samples from LS carriers, 18 of whom were diagnosed with cancer during a 4-year prospective surveillance period. Lasso regression was utilized to find c-miRs associated with cancer risk. Individual risk sum derived from the chosen c-miRs was used to develop a model to predict LS cancer incidence. This model was validated using 5-fold cross-validation. Correlation and pathway analyses were applied to inspect biological functions of c-miRs. Pearson correlation was used to examine the associations of c-miR risk sum and lifestyle factors. Hsa-miR-10b-5p, hsa-miR-125b-5p, hsa-miR-200a-3p, hsa-miR-3613-5p and hsa-miR-3615 were identified as cancer predictors by Lasso, and their risk sum score associated with higher likelihood of cancer incidence (HR 2.72, 95% CI 1.64-4.52, C-index=0.72). In cross-validation, the model indicated good concordance with the average C-index of 0.75 (0.6-1.0). Co-regulated hsa-miR-10b-5p, hsa-miR-125b-5p and hsa-miR-200a-3p targeted genes involved in cancer-associated biological pathways. The c-miR risk sum score correlated with BMI (r=0.23, p<0.01). In summary, BMI-associated c-miRs predict LS cancer incidence within four years, although further validation is required.

Multiplexed analysis of macrophage polarisation in pulmonary metastases of microsatellite stable colorectal cancer.

Tumour-associated macrophages (TAMs) express a continuum of phenotypes ranging from an anti-tumoural M1-like phenotype to a pro-tumoural M2-like phenotype. During cancer progression, TAMs may shift to a more M2-like polarisation state, but the role of TAMs in CRC metastases is unclear. We conducted a comprehensive spatial and prognostic analysis of TAMs in CRC pulmonary metastases and corresponding primary tumours using multiplexed immunohistochemistry and machine learning-based image analysis. We obtained data from 106 resected pulmonary metastases and 74 corresponding primary tumours. TAMs in the resected pulmonary metastases were located closer to the cancer cells and presented a more M2-like polarised state in comparison to the primary tumours. Higher stromal M2-like macrophage densities in the invasive margin of pulmonary metastases were associated with worse 5-year overall survival (HR 3.19, 95% CI 1.35-7.55, p = 0.008). The results of this study highlight the value of multiplexed analysis of macrophage polarisation in cancer metastases and might have clinical implications in future cancer therapy.

Quantitative Multiplexed Analysis of Indoleamine 2,3-Dioxygenase (IDO) and Arginase-1 (ARG1) Expression and Myeloid Cell Infiltration in Colorectal Cancer.

Indoleamine 2,3-dioxygenase (IDO) and arginase-1 (ARG1) are amino acid-metabolizing enzymes, frequently highly expressed in cancer. Their expression may deplete essential amino acids, lead to immunosuppression, and promote cancer growth. Still, their expression patterns, prognostic significance, and spatial localization in the colorectal cancer microenvironment are incompletely understood. Using a custom 10-plex immunohistochemistry assay and supervised machine learning-based digital image analysis, we characterized IDO and ARG1 expression in monocytic cells, granulocytes, mast cells, and tumor cells in 833 colorectal cancer patients. We evaluated the prognostic value and spatial arrangement of IDO- and ARG1-expressing myeloid and tumor cells. IDO was mainly expressed not only by monocytic cells but also by some tumor cells, whereas ARG1 was predominantly expressed by granulocytes. Higher density of IDO+ monocytic cells was an independent prognostic factor for improved cancer-specific survival both in the tumor center (Ptrend = .0002; hazard ratio [HR] for the highest ordinal category Q4 [vs Q1], 0.51; 95% CI, 0.33-0.79) and the invasive margin (Ptrend = .0015). Higher density of granulocytes was associated with prolonged cancer-specific survival in univariable models, and higher FCGR3+ARG1+ neutrophil density in the tumor center also in multivariable analysis (Ptrend = .0020). Granulocytes were, on average, located closer to tumor cells than monocytic cells. Furthermore, IDO+ monocytic cells and ARG1- granulocytes were closer than IDO- monocytic cells and ARG1+ granulocytes, respectively. The mRNA expression of the IDO1 gene was assessed in myeloid and tumor cells using publicly available single-cell RNA sequencing data for 62 colorectal cancers. IDO1 was mainly expressed in monocytes and dendritic cells, and high IDO1 activity in monocytes was associated with enriched immunostimulatory pathways. Our findings provided in-depth information about the infiltration patterns and prognostic value of cells expressing IDO and/or ARG1 in the colorectal cancer microenvironment, highlighting the significance of host immune response in tumor progression.

Circulating metabolome landscape in Lynch syndrome.

Circulating metabolites systemically reflect cellular processes and can modulate the tissue microenvironment in complex ways, potentially impacting cancer initiation processes. Genetic background increases cancer risk in individuals with Lynch syndrome; however, not all carriers develop cancer. Various lifestyle factors can influence Lynch syndrome cancer risk, and lifestyle choices actively shape systemic metabolism, with circulating metabolites potentially serving as the mechanical link between lifestyle and cancer risk. This study aims to characterize the circulating metabolome of Lynch syndrome carriers, shedding light on the energy metabolism status in this cancer predisposition syndrome.This study consists of a three-group cross-sectional analysis to compare the circulating metabolome of cancer-free Lynch syndrome carriers, sporadic colorectal cancer (CRC) patients, and healthy non-carrier controls. We detected elevated levels of circulating cholesterol, lipids, and lipoproteins in LS carriers. Furthermore, we unveiled that Lynch syndrome carriers and CRC patients displayed similar alterations compared to healthy non-carriers in circulating amino acid and ketone body profiles. Overall, cancer-free Lynch syndrome carriers showed a unique circulating metabolome landscape.This study provides valuable insights into the systemic metabolic landscape of Lynch syndrome individuals. The findings hint at shared metabolic patterns between cancer-free Lynch syndrome carriers and CRC patients.

Dominantly inherited micro-satellite instable cancer - the four Lynch syndromes - an EHTG, PLSD position statement.

The recognition of dominantly inherited micro-satellite instable (MSI) cancers caused by pathogenic variants in one of the four mismatch repair (MMR) genes MSH2, MLH1, MSH6 and PMS2 has modified our understanding of carcinogenesis. Inherited loss of function variants in each of these MMR genes cause four dominantly inherited cancer syndromes with different penetrance and expressivities: the four Lynch syndromes. No person has an "average sex "or a pathogenic variant in an "average Lynch syndrome gene" and results that are not stratified by gene and sex will be valid for no one. Carcinogenesis may be a linear process from increased cellular division to localized cancer to metastasis. In addition, in the Lynch syndromes (LS) we now recognize a dynamic balance between two stochastic processes: MSI producing abnormal cells, and the host's adaptive immune system's ability to remove them. The latter may explain why colonoscopy surveillance does not reduce the incidence of colorectal cancer in LS, while it may improve the prognosis. Most early onset colon, endometrial and ovarian cancers in LS are now cured and most cancer related deaths are after subsequent cancers in other organs. Aspirin reduces the incidence of colorectal and other cancers in LS. Immunotherapy increases the host immune system's capability to destroy MSI cancers. Colonoscopy surveillance, aspirin prevention and immunotherapy represent major steps forward in personalized precision medicine to prevent and cure inherited MSI cancer.

Tertiary lymphoid structures in pulmonary metastases of microsatellite stable colorectal cancer.

Tertiary lymphoid structures (TLSs) are ectopic lymphoid aggregates located at sites of chronic inflammation and recognized as prognosticators in several cancers. We aimed to analyse the prognostic effect of TLSs in colorectal cancer (CRC) pulmonary metastases and primary tumours, with a comparison to the CD3+ and CD8+ cell density-based immune cell score (ICS). For TLS density and TLS maximum diameter analysis, 67 pulmonary metastases and 63 primary tumours were stained with haematoxylin and eosin. For ICS scoring and analysis, CD3 and CD8 immunohistochemistry was performed. Excellent interobserver agreement was achieved in all TLS measurements. Of all patients, 36 patients had low TLS density (< 0.222 follicles/mm) and 31 patients had high TLS density (≥ 0.222 follicles/mm) in the first resected pulmonary metastases. TLS density (adjusted HR 0.91, 0.48-1.73) or maximum diameter (adjusted HR 0.78, 0.40-1.51) did not have prognostic value in pulmonary metastases. In primary tumours, higher TLS density (adjusted HR 0.39, 0.18-0.87) and maximum diameter (adjusted HR 0.28, 0.11-0.73) were associated with lower mortality. In the pulmonary metastases, ICS had superior prognostic value to TLSs; however, TLSs and ICS were significantly associated. In conclusion, TLSs in CRC pulmonary metastases had no prognostic value but correlated with the ICS. TLSs in primary tumours associated with favourable prognosis.

Immunological and prognostic significance of tumour necrosis in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) causes the second most cancer deaths worldwide, but the disease course varies according to tumour characteristics and immunological factors. Our objective was to examine the associations of tumour necrosis with tumour characteristics, immune cell infiltrates, serum cytokine concentrations, as well as prognosis in CRC. METHODS: Three independent CRC cohorts, including 1413 patients, were analysed. Associations of the areal percentage of tumour necrosis with clinicopathologic parameters, tumour infiltrating immune cells, cytokine concentrations in systemic and mesenteric vein blood, and survival were examined. RESULTS: Higher tumour necrosis percentage associated with shorter colorectal cancer-specific survival independent of tumour grade, T, N or M-class, mismatch repair status, BRAF status, and other possible confounding factors. In the largest cohort (N = 1100), the HR for high tumour necrosis percentage (≥40% vs. <3%) was 3.22 (95% CI 1.68-6.17, Ptrend < 0.0001). Tumour necrosis percentage positively correlated with peripheral serum levels of CXCL8, a proinflammatory chemokine, and negatively correlated with mesenteric serum levels of CXCL10 and mast cell densities in the invasive margin of the tumour. CONCLUSIONS: Our results support the value of tumour necrosis as a prognostic factor in colorectal cancer. CXCL8 may have a role in the systemic effects of tumour necrosis.

Tumor-independent Detection of Inherited Mismatch Repair Deficiency for the Diagnosis of Lynch Syndrome with High Specificity and Sensitivity.

Lynch syndrome (LS) is the most common hereditary cancer syndrome. Early diagnosis improves prognosis and reduces health care costs, through existing cancer surveillance methods. The problem is finding and diagnosing the cancer predisposing genetic condition. The current workup involves a complex array of tests that combines family cancer history and clinical phenotypes with tumor characteristics and sequencing data, followed by a challenging task to interpret the found variant(s). On the basis of the knowledge that an inherited mismatch repair (MMR) deficiency is a hallmark of LS, we have developed and validated a functional MMR test, DiagMMR, that detects inherited MMR deficiency directly from healthy tissue without need of tumor and variant information. The validation included 119 skin biopsies collected from clinically pathogenic MMR variant carriers (MSH2, MSH6) and controls, and was followed by a small clinical pilot study. The repair reaction was performed on proteins extracted from primary fibroblasts and the interpretation was based on the MMR capability of the sample in relation to cutoff, which distinguishes MMR proficient (non-LS) from MMR deficient (LS) function. The results were compared with the reference standard (germline NGS). The test was shown to have exceptional specificity (100%) with high sensitivity (89%) and accuracy (97%). The ability to efficiently distinguish LS carriers from controls was further shown with a high area under the receiving operating characteristic (AUROC) value (0.97). This test offers an excellent tool for detecting inherited MMR deficiency linked to MSH2 or MSH6 and can be used alone or with conventional tests to recognize genetically predisposed individuals. Significance: Clinical validation of DiagMMR shows high accuracy in distinguishing individuals with hereditary MSH2 or MSH6 MMR deficiency (i.e., LS). The method presented overcomes challenges faced by the complexity of current methods and can be used alone or with conventional tests to improve the ability to recognize genetically predisposed individuals.

Spatially resolved multimarker evaluation of CD274 (PD-L1)/PDCD1 (PD-1) immune checkpoint expression and macrophage polarisation in colorectal cancer.

BACKGROUND: The CD274 (PD-L1)/PDCD1 (PD-1) immune checkpoint interaction may promote cancer progression, but the expression patterns and prognostic significance of PD-L1 and PD-1 in the colorectal cancer microenvironment are inadequately characterised. METHODS: We used a custom 9-plex immunohistochemistry assay to quantify the expression patterns of PD-L1 and PD-1 in macrophages, T cells, and tumour cells in 910 colorectal cancer patients. We evaluated cancer-specific mortality according to immune cell subset densities using multivariable Cox regression models. RESULTS: Compared to PD-L1- macrophages, PD-L1+ macrophages were more likely M1-polarised than M2-polarised and located closer to tumour cells. PD-L1+ macrophage density in the invasive margin associated with longer cancer-specific survival [Ptrend = 0.0004, HR for the highest vs. lowest quartile, 0.52; 95% CI: 0.34-0.78]. T cell densities associated with longer cancer-specific survival regardless of PD-1 expression (Ptrend < 0.005 for both PD-1+ and PD-1- subsets). Higher densities of PD-1+ T cell/PD-L1+ macrophage clusters associated with longer cancer-specific survival (Ptrend < 0.005). CONCLUSIONS: PD-L1+ macrophages show distinct polarisation profiles (more M1-like), spatial features (greater co-localisation with tumour cells and PD-1+ T cells), and associations with favourable clinical outcome. Our comprehensive multimarker assessment could enhance the understanding of immune checkpoints in the tumour microenvironment and promote the development of improved immunotherapies.

Clinical characteristics of pancreatic and biliary tract cancers in Lynch syndrome: A retrospective analysis from the Finnish National Lynch Syndrome Research Registry.

Introduction: Patients with Lynch syndrome (LS) have an increased lifetime risk of pancreatic cancer (PC) and biliary tract cancer (BTC). These cancers have a notoriously pessimistic prognosis due to late diagnosis and limited therapeutic options. There are limited data based on small cohorts reviewing PC and BTC in LS patients. Methods: In this retrospective study of the Lynch Syndrome Registry of Finland (LSRFi), records of genetically verified LS patients diagnosed with PC or BTC between 1982 and 2020 were analyzed. Results: Thirty-nine patients were included: tumor(s) were in the pancreas in 26 patients, in the biliary tract in 10, and in the ampulla of Vater in three. A pathogenic germline variant was found in MLH1 in 33 of 39 patients. Twenty-six patients with 28 tumors located in the pancreas were identified: 23 pancreatic ductal adenocarcinomas (PDACs) and five neuroendocrine tumors (NETs). The median age at diagnosis of PC was 64 years (range of 38-81). In PC, the 5-year overall survival (OS) rate was 20%, and in PDAC, it was 13.6%. Ten patients with BTC were diagnosed: two intrahepatic, five perihilar, two distal extrahepatic cholangiocarcinomas, and one gallbladder carcinoma. Eight patients were male, and the median age at diagnosis was 54 years (range of 34-82). The 5-year OS rate for BTC was 30%. Metachronous tumors were diagnosed in 28 patients (70%). Colorectal cancer was the most common metachronous tumor, diagnosed in 20 patients (51%), and diagnosed prior to PC or BTC in all cases. Curative surgery was attempted on 17 of 39 patients. For 30 patients (91%), the cause of death was PC or BTC; two patients died from another LS-associated cancer, and one died from a stroke. Conclusion: Although the survival of LS patients with PC or BTC is better than in sporadic cancers, it is still poor and may be reflected by the relatively higher surgical resectability accounted for by the earlier age of onset. More studies on analyses of the molecular and immune profile, screening, and management of LS-associated pancreaticobiliary cancers are warranted.

The prognostic role of tumor budding and tumor-stroma ratio in pulmonary metastasis of colorectal carcinoma.

OBJECTIVE: To evaluate the prognostic value of tumor budding and tumor-stroma ratio (TSR) in resected pulmonary metastases of colorectal carcinoma (CRC). METHODS: In total, 106 pulmonary metastasectomies were performed to 74 patients in two study hospitals during 2000-2020. All relevant clinical data were retrospectively collected. Tumor budding based on the International Tumor Budding Consensus Conference recommendations and TSR in the first resected pulmonary metastases and primary tumors were evaluated from diagnostic hematoxylin-eosin-stained histopathological slides. RESULTS: 60 patients (85.7%) had low tumor budding (≤5 buds/field) and 10 patients (14.3%) had high tumor budding (>5 buds/field) in their first pulmonary metastases of CRC. 5-year overall survival rates of pulmonary metastasectomy in low and high total tumor budding were 28.3% and 37.3% (p = 0.387), respectively. 19 patients (27.1%) had low TSR and 51 patients (72.9%) had high TSR. The 5-year overall survival rates were 32.9% in low and 28.6% in high TSR of first pulmonary metastases (p = 0.746). Tumor budding and TSR did not provide prognostic value in Cox multivariate analysis. Tumor budding and TSR in resected pulmonary metastases were not associated with those of the primary tumor. CONCLUSION: Tumor budding and TSR in the resected pulmonary metastases of CRC showed no statistically significant prognostic value, however, additional well-powered confirmatory studies are needed.

Lynch Syndrome Genetics and Clinical Implications.

Lynch syndrome (LS) is one of the most prevalent hereditary cancer syndromes in humans and accounts for some 3% of unselected patients with colorectal or endometrial cancer and 10%-15% of those with DNA mismatch repair-deficient tumors. Previous studies have established the genetic basis of LS predisposition, but there have been significant advances recently in the understanding of the molecular pathogenesis of LS tumors, which has important implications in clinical management. At the same time, immunotherapy has revolutionized the treatment of advanced cancers with DNA mismatch repair defects. We aim to review the recent progress in the LS field and discuss how the accumulating epidemiologic, clinical, and molecular information has contributed to a more accurate and complete picture of LS, resulting in genotype- and immunologic subtype-specific strategies for surveillance, cancer prevention, and treatment.

Systemic circulating microRNA landscape in Lynch syndrome.

Circulating microRNAs (c-miRs) are small noncoding RNA molecules that migrate throughout the body and regulate gene expression. Global c-miR expression patterns (c-miRnomes) change with sporadic carcinogenesis and have predictive potential in early detection of cancers. However, there are no studies that have assessed whether c-miRnomes display similar potential in carriers of inherited pathogenic mismatch-repair gene variants (path_MMR), known as Lynch syndrome (LS), who are predisposed to highly increased cancer risk. Using high-throughput sequencing and bioinformatic approaches, we conducted an exploratory analysis to characterize systemic c-miRnomes of path_MMR carriers, sporadic rectal cancer patients and non-LS controls. We showed for the first time that cancer-free path_MMR carriers have a systemic c-miRnome of 40 differentially expressed c-miRs that can distinguish them from non-LS controls. The systemic c-miRnome of cancer-free path_MMR carriers also resembles the systemic c-miRnomes of cancer patients with or without path_MMR. Our pathway analysis linked the found differentially expressed c-miRs to carcinogenesis. A total of 508 putative target genes were identified for 32 out of 40 differentially expressed c-miRs, and 238 of them were enriched in cancer-related pathways. The most enriched c-miR-target genes include well-known oncogenes and tumor suppressor genes such as BCL2, AKT3, PIK3CA, KRAS, NRAS, CDKN1A and PIK3R1. Taken together, our findings suggest that LS and sporadic carcinogenesis share common biological pathways and alterations in these pathways can produce a c-miR signature which can track potential oncogenic stress in cancer-free path_MMR carriers. Therefore, c-miRs hold potential in monitoring the LS risk stratification patterns during clinical surveillance or cancer management.

Is HLA type a possible cancer risk modifier in Lynch syndrome?

Lynch syndrome (LS) is the most common inherited cancer syndrome. It is inherited via a monoallelic germline variant in one of the DNA mismatch repair (MMR) genes. LS carriers have a broad 30% to 80% risk of developing various malignancies, and more precise, individual risk estimations would be of high clinical value, allowing tailored cancer prevention and surveillance. Due to MMR deficiency, LS cancers are characterized by the accumulation of frameshift mutations leading to highly immunogenic frameshift peptides (FSPs). Thus, immune surveillance is proposed to inhibit the outgrowth of MMR-deficient cell clones. Recent studies have shown that immunoediting during the evolution of MMR-deficient cancers leads to a counter-selection of highly immunogenic antigens. The immunogenicity of FSPs is dependent on the antigen presentation. One crucial factor determining antigen presentation is the HLA genotype. Hence, a LS carrier's HLA genotype plays an important role in the presentation of FSP antigens to the immune system, and may influence the likelihood of progression from precancerous lesions to cancer. To address the challenge of clarifying this possibility including diverse populations with different HLA types, we have established the INDICATE initiative (Individual cancer risk by HLA type, http://indicate-lynch.org/), an international network aiming at a systematic evaluation of the HLA genotype as a possible cancer risk modifier in LS. Here we summarize the current knowledge on the role of HLA type in cancer risk and outline future research directions to delineate possible association in the scenario of LS with genetically defined risk population and highly immunogenic tumors.

Cancer Prevention with Resistant Starch in Lynch Syndrome Patients in the CAPP2-Randomized Placebo Controlled Trial: Planned 10-Year Follow-up.

ABSTRACT: The CAPP2 trial investigated the long-term effects of aspirin and resistant starch on cancer incidence in patients with Lynch syndrome (LS). Participants with LS were randomized double-blind to 30 g resistant starch (RS) daily or placebo for up to 4 years. We present long-term cancer outcomes based on the planned 10-year follow-up from recruitment, supplemented by National Cancer Registry data to 20 years in England, Wales, and Finland. Overall, 463 participants received RS and 455 participants received placebo. After up to 20 years follow-up, there was no difference in colorectal cancer incidence (n = 52 diagnosed with colorectal cancer among those randomized to RS against n = 53 on placebo) but fewer participants had non-colorectal LS cancers in those randomized to RS (n = 27) compared with placebo (n = 48); intention-to-treat (ITT) analysis [HR, 0.54; 95% confidence interval (CI), 0.33-0.86; P = 0.010]. In ITT analysis, allowing for multiple primary cancer diagnoses among participants by calculating incidence rate ratios (IRR) confirmed the protective effect of RS against non-colorectal cancer LS cancers (IRR, 0.52; 95% CI, 0.32-0.84; P = 0.0075). These effects are particularly pronounced for cancers of the upper GI tract; 5 diagnoses in those on RS versus 21 diagnoses on placebo. The reduction in non-colorectal cancer LS cancers was detectable in the first 10 years and continued in the next decade. For colorectal cancer, ITT analysis showed no effect of RS on colorectal cancer risk (HR, 0.92; 95% CI, 0.62-1.34; P = 0.63). There was no interaction between aspirin and RS treatments. In conclusion, 30 g daily RS appears to have a substantial protective effect against non-colorectal cancer cancers for patients with LS. PREVENTION RELEVANCE: Regular bowel screening and aspirin reduce colorectal cancer among patients with LS but extracolonic cancers are difficult to detect and manage. This study suggests that RS reduces morbidity associated with extracolonic cancers. See related Spotlight, p. 557.

Prognostic significance of spatial and density analysis of T lymphocytes in colorectal cancer.

BACKGROUND: Although high T cell density is a strong favourable prognostic factor in colorectal cancer, the significance of the spatial distribution of T cells is incompletely understood. We aimed to evaluate the prognostic significance of tumour cell-T cell co-localisation and T cell densities. METHODS: We analysed CD3 and CD8 immunohistochemistry in a study cohort of 983 colorectal cancer patients and a validation cohort (N = 246). Individual immune and tumour cells were identified to calculate T cell densities (to derive T cell density score) and G-cross function values, estimating the likelihood of tumour cells being co-located with T cells within 20 µm radius (to derive T cell proximity score). RESULTS: High T cell proximity score associated with longer cancer-specific survival in both the study cohort [adjusted HR for high (vs. low) 0.33, 95% CI 0.20-0.52, Ptrend < 0.0001] and the validation cohort [adjusted HR for high (vs. low) 0.15, 95% CI 0.05-0.45, Ptrend < 0.0001] and its prognostic value was independent of T cell density score. CONCLUSIONS: The spatial point pattern analysis of tumour cell-T cell co-localisation could provide detailed information on colorectal cancer prognosis, supporting the value of spatial measurement of T cell infiltrates as a novel, robust tumour-immune biomarker.