Detection of a major Lynch Syndrome-causing MLH1 founder variant in a large-scale genotyped cohort.

Some 50% of Finnish Lynch Syndrome (LS) cases are caused by a founder variant in MLH1, in which the entire exon 16 has been lost due to an Alu-mediated recombination event. We piloted detecting the variant in FinnGen, a large genotyped cohort comprising approximately 10% of the current Finnish population, and validated the MLH1 founder variant status of identified individuals residing in the Central Finland Biobank catchment area. A consensus sequence flanking the deletion was identified in whole genome sequences of six LS individuals with the founder variant. Genotype data of 212,196 individuals was queried for regional matches to the consensus sequence. Enrichment of cancer and age at cancer onset was compared between matching and non-matching individuals. Variant status was validated for a subset of the identified individuals using a polymerase chain reaction assay. Allelic matches in a chosen target region was detected in 348 individuals, with 89 having a cancer diagnosis (Bonferroni-adjusted p-value = 1), 20 a familial cancer history (p-adj. < .001), with mean age of onset of cancer being 53.6 years (p-adj. = .002). Eighteen of potential variant carriers had been sampled by the Central Finland Biobank, of which four (22%) were validated as true variant carriers. The workflow we have employed identifies MLH1 exon 16 deletion variant carriers from population-wide SNP genotyping data. An alternative design will be sought to limit false positive findings. Large genotyped cohorts provide a potential resource for identification and prevention of hereditary cancer.

TWINGEN: protocol for an observational clinical biobank recall and biomarker cohort study to identify Finnish individuals with high risk of Alzheimer's disease.

INTRODUCTION: A better understanding of the earliest stages of Alzheimer's disease (AD) could expedite the development or administration of treatments. Large population biobanks hold the promise to identify individuals at an elevated risk of AD and related dementias based on health registry information. Here, we establish the protocol for an observational clinical recall and biomarker study called TWINGEN with the aim to identify individuals at high risk of AD by assessing cognition, health and AD-related biomarkers. Suitable candidates were identified and invited to participate in the new study among THL Biobank donors according to TWINGEN study criteria. METHODS AND ANALYSIS: A multi-centre study (n=800) to obtain blood-based biomarkers, telephone-administered and web-based memory and cognitive parameters, questionnaire information on lifestyle, health and psychological factors, and accelerometer data for measures of physical activity, sedentary behaviour and sleep. A subcohort is being asked to participate in an in-person neuropsychological assessment (n=200) and wear an Oura ring (n=50). All participants in the TWINGEN study have genome-wide genotyping data and up to 48 years of follow-up data from the population-based older Finnish Twin Cohort (FTC) study of the University of Helsinki. The data collected in TWINGEN will be returned to THL Biobank from where it can later be requested for other biobank studies such as FinnGen that supported TWINGEN. ETHICS AND DISSEMINATION: This recall study consists of FTC/THL Biobank/FinnGen participants whose data were acquired in accordance with the Finnish Biobank Act. The recruitment protocols followed the biobank protocols approved by Finnish Medicines Agency. The TWINGEN study plan was approved by the Ethics Committee of Hospital District of Helsinki and Uusimaa (number 16831/2022). THL Biobank approved the research plan with the permission no: THLBB2022_83.

NGS of brush cytology samples improves the detection of high-grade dysplasia and cholangiocarcinoma in patients with primary sclerosing cholangitis: A retrospective and prospective study.

BACKGROUND: Biliary dysplasia, a precursor of cholangiocarcinoma (CCA), is a common complication of primary sclerosing cholangitis. Patients with high-grade dysplasia (HGD) or early CCA who have received oncological treatment are candidates for liver transplantation. The preoperative diagnosis of CCA or HGD is challenging, and the sensitivity of biliary brush cytology (BC) is limited. METHODS: By using next-generation sequencing (NGS), we retrospectively analyzed archived tissue samples (n=62) obtained from explanted liver tissue and CCA samples to identify oncogenic mutations that occur during primary sclerosing cholangitis carcinogenesis. BC samples were prospectively collected from patients with primary sclerosing cholangitis (n=97) referred for endoscopic retrograde cholangiography to measure the diagnostic utility of NGS combined with BC compared with traditional cytology alone. RESULTS: Mutations in KRAS, GNAS, FLT3, RNF43, TP53, ATRX, and SMAD4 were detected in archived CCA or HGD samples. KRAS, GNAS, TP53, CDKN2A, FBXW7, BRAF, and ATM mutations were detected in prospectively collected brush samples from patients with histologically verified CCA or HGD. One patient with low-grade dysplasia in the explanted liver had KRAS and GNAS mutations in brush sample. No mutations were observed in brush samples or archived tissues in liver transplantation cases without biliary neoplasia. While KRAS mutations are common in biliary neoplasms, they were also observed in patients without biliary neoplasia during surveillance. CONCLUSIONS: In summary, NGS of BC samples increased the sensitivity of detecting biliary neoplasia compared with traditional cytology. Performing NGS on BC samples may help diagnose HGD or early CCA, benefiting the timing of liver transplantation.

Impact of Primary Tumor Location on Demographics, Resectability, Outcomes, and Quality of Life in Finnish Metastatic Colorectal Cancer Patients (Subgroup Analysis of the RAXO Study).

The primary tumor location (PTL) is associated with the phenotype, metastatic sites, mutations, and outcomes of metastatic colorectal cancer (mCRC) patients, but this has mostly been studied according to sidedness (right vs. left sided). We studied right colon vs. left colon vs. rectal PTL in a real-life study population (n = 1080). Health-related quality of life (HRQoL) was assessed multi-cross-sectionally with QLQ-C30, QLQ-CR29, EQ-5D, and 15D. A chi-square, Kaplan-Meier, and Cox regression were used to compare the groups. The PTL was in the right colon in 310 patients (29%), the left colon in 396 patients (37%), and the rectum in 375 patients (35%). The PTL was associated with distinct differences in metastatic sites during the disease trajectory. The resectability, conversion, and resection rates were lowest in the right colon, followed by the rectum, and were highest in the left colon. Overall survival was shortest for right colon compared with left colon or rectal PTL (median 21 vs. 35 vs. 36 months), with the same trends after metastasectomy or systemic therapy only. PTL also remained statistically significant in a multivariable model. The distribution of symptoms varied according to PTL, especially between the right colon (with general symptoms of metastases) and rectal PTL (with sexual- and bowel-related symptoms). mCRC, according to PTL, behaves differently regarding metastatic sites, resectability of the metastases, outcomes of treatment, and HRQoL.

Assessment of targeted therapy opportunities in sinonasal cancers using patient-derived functional tumor models.

Malignant tumors derived from the epithelium lining the nasal cavity region are termed sinonasal cancers, a highly heterogeneous group of rare tumors accounting for 3 - 5 % of all head and neck cancers. Progress with next-generation molecular profiling has improved our understanding of the complexity of sinonasal cancers and resulted in the identification of an increasing number of distinct tumor entities. Despite these significant developments, the treatment of sinonasal cancers has hardly evolved since the 1980s, and an advanced sinonasal cancer presents a poor prognosis as targeted therapies are usually not available. To gain insights into potential targeted therapeutic opportunities, we performed a multiomics profiling of patient-derived functional tumor models to identify molecular characteristics associated with pharmacological responses in the different subtypes of sinonasal cancer. METHODS: Patient-derived ex vivo tumor models representing four distinct sinonasal cancer subtypes: sinonasal intestinal-type adenocarcinoma, sinonasal neuroendocrine carcinoma, sinonasal undifferentiated carcinoma and SMARCB1 deficient sinonasal carcinoma were included in the analyses. Results of functional drug screens of 160 anti-cancer therapies were integrated with gene panel sequencing and histological analyses of the tumor tissues and the ex vivo cell cultures to establish associations between drug sensitivity and molecular characteristics including driver mutations. RESULTS: The different sinonasal cancer subtypes display considerable differential drug sensitivity. Underlying the drug sensitivity profiles, each subtype was associated with unique molecular features. The therapeutic vulnerabilities correlating with specific genomic background were extended and validated with in silico analyses of cancer cell lines representing different human cancers and with reported case studies of sinonasal cancers treated with targeted therapies. CONCLUSION: The results demonstrate the importance of understanding the differential biology and the molecular features associated with the different subtypes of sinonasal cancers. Patient-derived ex vivo tumor models can be a powerful tool for investigating these rare cancers and prioritizing targeted therapeutic strategies for future clinical development and personalized medicine.

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.

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.

Atypical (non-V600E) BRAF mutations in metastatic colorectal cancer in population and real-world cohorts.

BRAF-V600E mutation (mt) is a strong negative prognostic and predictive biomarker in metastatic colorectal cancer (mCRC). Non-V600Emt, designated atypical BRAFmt (aBRAFmt) are rare, and little is known about their frequency, co-mutations and prognostic and predictive role. These were compared between mutational groups of mCRC patients collected from three Nordic population-based or real-world cohorts. Pathology of aBRAFmt was studied. The study included 1449 mCRC patients with 51 (3%) aBRAFmt, 182 (13%) BRAF-V600Emt, 456 (31%) RAS&BRAF wild-type (wt) and 760 (52%) RASmt tumours. aBRAFmt were seen in 2% of real-world and 4% of population-based cohorts. Twenty-six different aBRAFmt were detected, 11 (22%) class 2 (serrated adenocarcinoma in 2/9 tested), 32 (64%) class 3 (serrated in 15/25) and 4 (8%) unclassified. aBRAFmt patients were predominantly male, had more rectal primaries, less peritoneal metastases, deficient mismatch repair in one (2%), and better survival after metastasectomy (89% 5-year overall survival [OS]-rate) compared with BRAF-V600Emt. aBRAFmt and BRAF-V600Emt had poorer performance status and received fewer treatment lines than RAS&BRAFwt and RASmt. OS among aBRAFmt (median 14.4 months) was longer than for BRAF-V600Emt (11.2 months), but shorter than for RAS&BRAFwt (30.5 months) and RASmt (23.4 months). Addition of bevacizumab trended for better OS for the aBRAFmt. Nine patients with aBRAFmt received cetuximab/panitumumab without response. aBRAFmt represents a distinct subgroup differing from other RAS/BRAF groups, with serrated adenocarcinoma in only half. OS for patients with aBRAFmt tumours was slightly better than for BRAF-V600Emt, but worse than for RASmt and RAS&BRAFwt. aBRAFmt should not be a contraindication for metastasectomy.

TWINGEN - protocol for an observational clinical biobank recall and biomarker study to identify individuals with high risk of Alzheimer's disease.

Introduction: A better understanding of the earliest stages of Alzheimer's disease (AD) could expedite the development or administration of treatments. Large population biobanks hold the promise to identify individuals at an elevated risk of AD and related dementias based on health registry information. Here, we establish the protocol for an observational clinical recall and biomarker study called TWINGEN with the aim to identify individuals at high risk of AD by assessing cognition, health and AD-related biomarkers. Suitable candidates were identified and invited to participate in the new study among Finnish biobank donors according to TWINGEN study criteria. Methods and analysis: A multi-center study (n=800) to obtain blood-based biomarkers, telephone-administered and web-based memory and cognitive parameters, questionnaire information on lifestyle, health and psychological factors, and accelerometer data for measures of physical activity, sedentary behavior and sleep. A sub-cohort are being asked to participate in an in-person neuropsychological assessment (n=200) and wear an Oura ring (n=50). All participants in the TWINGEN study have genome-wide genotyping data and up to 48 years of follow-up data from the population-based older Finnish Twin Cohort (FTC) study of the University of Helsinki. TWINGEN data will be transferred to Finnish Institute of Health and Welfare (THL) biobank and we aim to further to transfer it to the FinnGen study where it will be combined with health registry data for prediction of AD. Ethics and dissemination: This recall study consists of FTC/THL/FinnGen participants whose data were acquired in accordance with the Finnish Biobank Act. The recruitment protocols followed the biobank protocols approved by Finnish Medicines Agency. The TWINGEN study plan was approved by the Ethics Committee of Hospital District of Helsinki and Uusimaa (number 16831/2022). THL Biobank approved the research plan with the permission no: THLBB2022_83.

Improved accuracy in colorectal cancer tissue decomposition through refinement of established deep learning solutions.

Hematoxylin and eosin-stained biopsy slides are regularly available for colorectal cancer patients. These slides are often not used to define objective biomarkers for patient stratification and treatment selection. Standard biomarkers often pertain to costly and slow genetic tests. However, recent work has shown that relevant biomarkers can be extracted from these images using convolutional neural networks (CNNs). The CNN-based biomarkers predicted colorectal cancer patient outcomes comparably to gold standards. Extracting CNN-biomarkers is fast, automatic, and of minimal cost. CNN-based biomarkers rely on the ability of CNNs to recognize distinct tissue types from microscope whole slide images. The quality of these biomarkers (coined 'Deep Stroma') depends on the accuracy of CNNs in decomposing all relevant tissue classes. Improving tissue decomposition accuracy is essential for improving the prognostic potential of CNN-biomarkers. In this study, we implemented a novel training strategy to refine an established CNN model, which then surpassed all previous solutions . We obtained a 95.6% average accuracy in the external test set and 99.5% in the internal test set. Our approach reduced errors in biomarker-relevant classes, such as Lymphocytes, and was the first to include interpretability methods. These methods were used to better apprehend our model's limitations and capabilities.

The prognostic and predictive role of tumor-infiltrating lymphocytes (FoxP3 + and CD8 +) and tumor-associated macrophages in early HER2 + breast cancer.

PURPOSE: In HER2-positive (HER2 +) breast cancer, tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) may influence the efficacy of the HER2-antibody trastuzumab and the patient's outcome. In this HER2 + patient cohort, our aim was to study the numbers of FoxP3 + regulatory TILs and CD8 + cytotoxic TILs, their correlations with CD68 + and CD163 + TAMs, and the prognostic and predictive value of the studied factors. METHODS: We evaluated 139 non-metastatic HER2 + breast cancer patients operated between 2001 and 2008. The FoxP3+TIL count (FoxP3+TILs) was assessed using the hotspot method, and the CD8 + TIL count (CD8+mTILs) utilizing a digital image analysis from invasive margin areas. The ratios between CD8+mTILs and FoxP3+TILs as well as CD8+mTILs and TAMs were calculated. RESULTS: FoxP3 + TILs and CD8 + mTILs correlated positively with each other (p<0.001). FoxP3+TILs had a positive correlation with CD68+and CD163+TAMs (p≤0.038), while CD8 + mTILs correlated only with CD68+TAMs (p<0.001). In the HER2 + and hormone receptor-positive Luminal B subgroup, high numbers of FoxP3+TILs were associated with shorter disease-free survival (DFS) (54% vs. 79%, p = 0.040). The benefit from adjuvant trastuzumab was extremely significant among patients with a high CD8 + mTILs/CD68 + TAMs ratio, with overall survival (OS) 84% vs. 33% (p = 0.003) and breast cancer-specific survival (BCSS) 88% vs. 48% (p = 0.009) among patients treated with or without trastuzumab, respectively. CONCLUSION: In the HER2 + Luminal B subgroup, high FoxP3 + TILs were associated with shorter DFS. A high CD8 + mTILs/CD68 + TAMs ratio seems to associate with impressive efficacy of trastuzumab.

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.

Domain-specific transfer learning in the automated scoring of tumor-stroma ratio from histopathological images of colorectal cancer.

Tumor-stroma ratio (TSR) is a prognostic factor for many types of solid tumors. In this study, we propose a method for automated estimation of TSR from histopathological images of colorectal cancer. The method is based on convolutional neural networks which were trained to classify colorectal cancer tissue in hematoxylin-eosin stained samples into three classes: stroma, tumor and other. The models were trained using a data set that consists of 1343 whole slide images. Three different training setups were applied with a transfer learning approach using domain-specific data i.e. an external colorectal cancer histopathological data set. The three most accurate models were chosen as a classifier, TSR values were predicted and the results were compared to a visual TSR estimation made by a pathologist. The results suggest that classification accuracy does not improve when domain-specific data are used in the pre-training of the convolutional neural network models in the task at hand. Classification accuracy for stroma, tumor and other reached 96.1% on an independent test set. Among the three classes the best model gained the highest accuracy (99.3%) for class tumor. When TSR was predicted with the best model, the correlation between the predicted values and values estimated by an experienced pathologist was 0.57. Further research is needed to study associations between computationally predicted TSR values and other clinicopathological factors of colorectal cancer and the overall survival of the patients.

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.

A pan-cancer analysis shows immunoevasive characteristics in NRF2 hyperactive squamous malignancies.

The NRF2 pathway is frequently activated in various cancer types, yet a comprehensive analysis of its effects across different malignancies is currently lacking. We developed a NRF2 activity metric and utilized it to conduct a pan-cancer analysis of oncogenic NRF2 signaling. We identified an immunoevasive phenotype where high NRF2 activity is associated with low interferon-gamma (IFNγ), HLA-I expression and T cell and macrophage infiltration in squamous malignancies of the lung, head and neck area, cervix and esophagus. Squamous NRF2 overactive tumors comprise a molecular phenotype with SOX2/TP63 amplification, TP53 mutation and CDKN2A loss. These immune cold NRF2 hyperactive diseases are associated with upregulation of immunomodulatory NAMPT, WNT5A, SPP1, SLC7A11, SLC2A1 and PD-L1. Based on our functional genomics analyses, these genes represent candidate NRF2 targets, suggesting direct modulation of the tumor immune milieu. Single-cell mRNA data shows that cancer cells of this subtype exhibit decreased expression of IFNγ responsive ligands, and increased expression of immunosuppressive ligands NAMPT, SPP1 and WNT5A that mediate signaling in intercellular crosstalk. In addition, we discovered that the negative relationship of NRF2 and immune cells are explained by stromal populations of lung squamous cell carcinoma, and this effect spans multiple squamous malignancies based on our molecular subtyping and deconvolution data.

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.

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.

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.

Predictive value of p53, Ki67 and TLR5 in neoplastic progression of Barrett's esophagus: a matched case-control study.

Barrett's esophagus progresses to high-grade dysplasia or cancer along the well-established metaplasia-dysplasia-adenocarcinoma sequence. The aim of this study was to evaluate the value of p53, Ki67, and toll-like receptor 5 (TLR5) in prediction of malignant progression of Barrett's metaplasia and low-grade dysplasia. This was a retrospective matched case-control study based on Northern and Central Finland population. Patients diagnosed with esophageal high-grade dysplasia or adenocarcinoma were included. From these patients, all previous endoscopy samples were obtained along with original diagnostic HE-slides and clinical data. Age- and sex-matched patients with non-progressing Barrett's metaplasia and low-grade dysplasia confirmed with follow-up endoscopies were used as controls. Two gastrointestinal pathologist re-reviewed all original HE-slides, and newly made sections to confirm representative tissue material blinded from clinical data. p53, Ki67, and TLR5 were immunohistochemically stained. Final cohort included 45 patients with progressive Barrett's metaplasia (n = 21) or low-grade dysplasia (n = 24), and 92 patients with non-progressive Barrett's metaplasia (n = 52) or low-grade dysplasia (n = 40). In Barrett's metaplasia, aberrant p53 expression was observed in 6% of samples in progressors and 0% in non-progressors. In low-grade dysplasia, aberrant p53 was seen in 56% of samples in progressors and 17% in non-progressors (Odd's ratio 6.7, 95% CI 1.8-24.6). Ki67 or TLR5 showed no association with disease progression. In this matched case-control study, p53 expression associated with a high risk of malignant progression in Barrett's low-grade dysplasia. Routine staining of p53 is indicated in expert confirmed low-grade dysplasia.