Comparison of breast fine-needle aspiration cytology and tissue sampling for high-throughput proteomic analysis and cancer biomarker detection.

BACKGROUND: Fine-needle aspiration cytology (FNAC) specimens are widely utilized for the diagnosis and molecular testing of various cancers. We performed a comparative proteomic analysis of three different sample types, including breast fine-needle aspiration cytology (FNAC), core needle biopsy (CNB), and surgically resection tissues. Our goal was to evaluate the suitability of FNAC for in-depth proteomic analysis and for identifying potential therapeutic biomarkers in breast cancer. METHODS: High-throughput proteomic analysis was conducted on matched FNAC, CNB, and surgical resection tissue samples obtained from breast cancer patients. The protein identification, including currently established or promising therapeutic targets, was compared among the three different sample types. Gene Ontology (GO) enrichment analysis was also performed on all matched samples. RESULTS: Compared to tissue samples, FNAC testing revealed a comparable number of proteins (7,179 in FNAC; 7,196 in CNB; 7,190 in resection samples). Around 85% of proteins were mutually identified in all sample types. FNAC along with CNB showed a positive correlation between the number of enrolled tumor cells and identified proteins. In the GO analysis, the FNAC samples demonstrated a higher number of genes for each pathway and GO terms than tissue samples. CCND1, CDK6, HER2, and IGF1R were found in higher quantities in the FNAC compared to tissue samples, while TUBB2A was only detected in the former. CONCLUSIONS: FNAC is suitable for high-throughput proteomic analysis, in addition to an emerging source that could be used to identify and quantify novel cancer biomarkers.

Concomitant expression patterns of CDX2 and SATB2 as prognostic factors in stage III colorectal cancers.

CDX2 and SATB2 are often used as biomarkers for identification of colorectal origin in primary or metastatic adenocarcinomas. Loss of CDX2 or SATB2 expression has been associated with poor prognosis in patients with colorectal cancer (CRC). However, little is known regarding clinicopathological features, including prognosis, of CRCs with concomitant loss of CDX2 and SATB2. A total of 431 stage III CRCs were analyzed for their expression status in CDX2 and SATB2 using tissue microarray-based immunohistochemistry and expression status was correlated with clinicopathological variables, molecular alterations, and survival. CDX2-negative (CDX2-) CRCs and SATB2-negative (SATB2-) CRCs were found in 8.1 % and 17.2 % of CRCs, respectively, whereas both CDX2-negative and SATB2-negative (CDX2-/SATB2-) CRCs comprised 3.2 % of the CRCs. On survival analysis, neither CDX2-/SATB2+ nor CDX2+/SABT2- CRCs but CDX2-/SATB2- CRCs were associated with poor prognosis. CDX2-/SATB2- CRCs showed significant associations with tumor subsite of right colon, poor differentiation, decreased expression of CK20, aberrant expression of CK7, CIMP-high, MSI-high, and BRAF mutation. In summary, our results suggest that concomitant loss of CDX2 and SATB2 is a prognostic biomarker but isolated loss of CDX2 or SATB2 is not a prognostic biomarker for stage III CRCs.

Probiotic Consortium Confers Synergistic Anti-Inflammatory Effects in Inflammatory Disorders.

The composition and diversity of gut microbiota significantly influence the immune system and are linked to various diseases, including inflammatory and allergy disorders. While considerable research has focused on exploring single bacterial species or consortia, the optimal strategies for microbiota-based therapeutics remain underexplored. Specifically, the comparative effectiveness of bacterial consortia versus individual species warrants further investigation. In our study, we assessed the impact of the bacterial consortium MPRO, comprising Lactiplantibacillus plantarum HY7712, Bifidobacterium animalis ssp. lactis HY8002, and Lacticaseibacillus casei HY2782, in comparison to its individual components. The administration of MPRO demonstrated enhanced therapeutic efficacy in experimental models of atopic dermatitis and inflammatory colitis when compared to single strains. MPRO exhibited the ability to dampen inflammatory responses and alter the gut microbial landscape significantly. Notably, MPRO administration led to an increase in intestinal CD103+CD11b+ dendritic cells, promoting the induction of regulatory T cells and the robust suppression of inflammation in experimental disease settings. Our findings advocate the preference for bacterial consortia over single strains in the treatment of inflammatory disorders, carrying potential clinical relevance.

Interleukin-34-regulated T-cell responses in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease with a multifaceted etiology, which primarily affects and results in the deterioration of the synovium of patients. While the exact etiology of RA is still largely unknown, there is growing interest in the cytokine interleukin-34 (IL-34) as a driver or modulator of RA pathogenesis on the grounds that IL-34 is drastically increased in the serum and synovium of RA patients. Several studies have so far revealed the relationship between IL-34 levels and RA disease progression. Nevertheless, the significance and role of IL-34 in RA have remained ambiguous, as illustrated by two most recent studies, which reported contrasting effects of genetic IL-34 deletion in RA. Of note, IL-34 is a macrophage growth factor and is increasingly perceived as a master regulator of T-cell responses in RA via macrophage-dependent as well as T cell-intrinsic mechanisms. In this regard, several studies have demonstrated that IL-34 potentiates helper T-cell (Th) responses in RA, whereas studies also suggested that IL-34 alleviates synovial inflammation, potentially by inducing regulatory T-cells (Treg). Herein, we provide an overview of the current understanding of IL-34 involvement in RA and outline IL-34-mediated mechanisms in regulating T-cell responses in RA.

Florid Reactive Periostitis of the Clavicle: A Case Report.

Florid reactive periostitis (FRP) is a rare benign fibro-osseous proliferation, occurring mostly in the short tubular bones of hands and rarely in the long tubular bones. We report a surgically confirmed case of FRP involving the clavicle in a 26-year-old male. On MRI scans, a soft tissue mass with T2 high signal intensity was found that originated from the periosteum of the clavicle and included surrounding a periosteal elevation and perilesional soft tissue edema. Strong contrast enhancement was noted inside the mass and along the periosteum involving more than half of the circumference of the clavicle. Serial radiographs revealed a soft tissue mass without mineralization that turned into an ossified mass with a solid periosteal reaction within a month.

The Therapeutic Potential of Anticoagulation in Organ Fibrosis.

Fibrosis, also known as organ scarring, describes a pathological stiffening of organs or tissues caused by increased synthesis of extracellular matrix (ECM) components. In the past decades, mounting evidence has accumulated showing that the coagulation cascade is directly associated with fibrotic development. Recent findings suggest that, under inflammatory conditions, various cell types (e.g., immune cells) participate in the coagulation process causing pathological outcomes, including fibrosis. These findings highlighted the potential of anticoagulation therapy as a strategy in organ fibrosis. Indeed, preclinical and clinical studies demonstrated that the inhibition of blood coagulation is a potential intervention for the treatment of fibrosis across all major organs (e.g., lung, liver, heart, and kidney). In this review, we aim to summarize our current knowledge on the impact of components of coagulation cascade on fibrosis of various organs and provide an update on the current development of anticoagulation therapy for fibrosis.

Combinatory statuses of tumor stromal percentage and tumor infiltrating lymphocytes as prognostic factors in stage III colorectal cancers.

BACKGROUND AND AIM: Tumor stroma and tumor-infiltrating lymphocytes (TILs) are major constituents of the tumor microenvironment, although they have different effects on the prognosis of patients with colorectal cancer (CRC). Combinatory statuses of tumor-stromal percentage (TSP) and TILs are expected to provide more powerful prognostic information but have never been studied in CRCs. METHODS: Stage III CRCs from patients (n = 487) treated with adjuvant chemotherapy were assessed for their TSP and CD3-TIL or CD8-TIL densities using computer-aided methodology. With cut-off values set at median values for intraepithelial TIL (iTIL) and stromal TIL (sTIL) densities, CRCs were sorted into low and high iTIL or sTIL groups. CRCs were classified into five quintile (Q1-Q5) groups according to their TSP and divided into high TSP (Q5) and low TSP (Q1-4) groups. RESULTS: The combination of CD8 iTIL density and TSP was found to be an independent prognostic parameter in multivariate survival analysis in terms of cancer-specific survival and recurrence-free survival. CRCs with low CD8 iTIL density and high TSP showed the worst survival. The combinatory status showed more prognostic power than CD8 iTIL density or TSP alone. Multivariate survival analysis in an independent cohort of stage III CRC validated the prognostic power of the combinatory statuses. CONCLUSIONS: The findings suggest that the combinatory status might serve as a prognostic parameter in stage III CRCs. Further research in a large-scale cohort of patients with stage III CRC is needed to validate the prognostic power of the combinatory status.

Tumor microenvironment-adjusted prognostic implications of the KRAS mutation subtype in patients with stage III colorectal cancer treated with adjuvant FOLFOX.

Several studies have reported that the prognostic effect of KRAS mutations on colorectal cancers (CRCs) varies depending on the type of mutation. Considering the effect of KRAS mutations on tumor microenvironment, we analyzed the prognostic significance of KRAS mutation types after adjusting for the tumor-infiltrating lymphocytes (TIL) and tumor-stromal percentage (TSP) statuses. In two independent cohorts, KRAS mutations were analyzed by Sanger sequencing and/or next-generation sequencing. TIL density and the TSP were quantified from whole-slide immunohistochemical images. KRAS-mutant CRCs were divided into three subgroups (G12D/V, other codon 12 mutations and codon 13 mutations) to examine their differential effect on TIL density, the TSP and recurrence-free survival (RFS). Among the KRAS mutations, only the G12D/V subgroups showed significantly less TIL infiltration than the wild-type CRCs. According to survival analysis, G12D/V mutations were associated with short RFS; codon 13 mutations showed discordant trends in the two cohorts, and other codon 12 mutations showed no significant association. Multivariate analysis further supported the prognostic value of G12D/V mutations. This result is not only consistent with a recent study suggesting the immunosuppressive effect of mutant KRAS but also provides insight into the type-specific prognostic effect of KRAS mutations.

Is coronavirus disease (COVID-19) seasonal? A critical analysis of empirical and epidemiological studies at global and local scales.

Coronavirus disease (COVID-19) has infected more than 50 million people and killed more than one million, worldwide, during less than a year course. COVID-19, which has already become the worst pandemic in the last 100 years, is still spreading worldwide. Since the beginning of the outbreak, it has been of particular interest to understand whether COVID-19 is seasonal; the finding might help for better planning and preparation for the fight against the disease. Over the past 12 months, numerous empirical and epidemiological studies have been performed to define the distinct diffusion patterns of COVID-19. Thereby, a wealth of data has accumulated on the relationship between various seasonal meteorological factors and COVID-19 transmissibility at global and local scales. In this review, we aimed to discuss whether COVID-19 exhibits any seasonal features in a global and local perspective by collecting and providing summaries of the findings from empirical and epidemiological studies on the COVID-19 pandemic during its first seasonal cycle.

Targeted next-generation sequencing-based detection of microsatellite instability in colorectal carcinomas.

In the present study, we developed a computational method and panel markers to assess microsatellite instability (MSI) using a targeted next-generation sequencing (NGS) platform and compared the performance of our computational method, mSILICO, with that of mSINGS to detect MSI in CRCs. We evaluated 13 CRC cell lines, 84 fresh and 119 formalin-fixed CRC tissues (including 61 MSI-high CRCs and 155 microsatellite-stable CRCs) and tested the classification performance of the two methods on 23, 230, and 3,154 microsatellite markers. For the fresh tissue and cell line samples, mSILICO showed a sensitivity of 100% and a specificity of 100%, regardless of the number of panel markers, whereas for the formalin-fixed tissue samples, mSILICO exhibited a sensitivity of up to 100% and a specificity of up to 100% with three differently sized panels ranging from 23 to 3154. These results were similar to those of mSINGS. With the application of mSILICO, the small panel of 23 markers had a sensitivity of ≥95% and a specificity of 100% in cell lines/fresh tissues and formalin-fixed tissues of CRC. In conclusion, we developed a new computational method and microsatellite marker panels for the determination of MSI that does not require paired normal tissues. A small panel could be integrated into the targeted NGS panel for the concurrent analysis of single nucleotide variations and MSI.

Prognostic Value of Tumor Regression Grade on MR in Rectal Cancer: A Large-Scale, Single-Center Experience.

OBJECTIVE: To determine the prognostic value of MRI-based tumor regression grading (mrTRG) in rectal cancer compared with pathological tumor regression grading (pTRG), and to assess the effect of diffusion-weighted imaging (DWI) on interobserver agreement for evaluating mrTRG. MATERIALS AND METHODS: Between 2007 and 2016, we retrospectively enrolled 321 patients (male:female = 208:113; mean age, 60.2 years) with rectal cancer who underwent both pre-chemoradiotherapy (CRT) and post-CRT MRI. Two radiologists independently determined mrTRG using a 5-point grading system with and without DWI in a one-month interval. Two pathologists graded pTRG using a 5-point grading system in consensus. Kaplan-Meier estimation and Cox-proportional hazard models were used for survival analysis. Cohen's kappa analysis was used to determine interobserver agreement. RESULTS: According to mrTRG on MRI with DWI, there were 6 mrTRG 1, 48 mrTRG 2, 109 mrTRG 3, 152 mrTRG 4, and 6 mrTRG 5. By pTRG, there were 7 pTRG 1, 59 pTRG 2, 180 pTRG 3, 73 pTRG 4, and 2 pTRG 5. A 5-year overall survival (OS) was significantly different according to the 5-point grading mrTRG (p = 0.024) and pTRG (p = 0.038). The 5-year disease-free survival (DFS) was significantly different among the five mrTRG groups (p = 0.039), but not among the five pTRG groups (p = 0.072). OS and DFS were significantly different according to post-CRT MR variables: extramural venous invasion after CRT (hazard ratio = 2.259 for OS, hazard ratio = 5.011 for DFS) and extramesorectal lymph node (hazard ratio = 2.610 for DFS). For mrTRG, k value between the two radiologists was 0.309 (fair agreement) without DWI and slightly improved to 0.376 with DWI. CONCLUSION: mrTRG may predict OS and DFS comparably or even better compared to pTRG. The addition of DWI on T2-weighted MRI may improve interobserver agreement on mrTRG.

Whole-Slide Image Analysis Reveals Quantitative Landscape of Tumor-Immune Microenvironment in Colorectal Cancers.

PURPOSE: Despite the well-known prognostic value of the tumor-immune microenvironment (TIME) in colorectal cancers, objective and readily applicable methods for quantifying tumor-infiltrating lymphocytes (TIL) and the tumor-stroma ratio (TSR) are not yet available. EXPERIMENTAL DESIGN: We established an open-source software-based analytic pipeline for quantifying TILs and the TSR from whole-slide images obtained after CD3 and CD8 IHC staining. Using a random forest classifier, the method separately quantified intraepithelial TILs (iTIL) and stromal TILs (sTIL). We applied this method to discovery and validation cohorts of 578 and 283 stage III or high-risk stage II colorectal cancers patients, respectively, who were subjected to curative surgical resection and oxlaliplatin-based adjuvant chemotherapy. RESULTS: Automatic quantification of iTILs and sTILs showed a moderate concordance with that obtained after visual inspection by a pathologist. The K-means-based consensus clustering of 197 TIME parameters that showed robustness against interobserver variations caused colorectal cancers to be grouped into five distinctive subgroups, reminiscent of those for consensus molecular subtypes (CMS1-4 and mixed/intermediate group). In accordance with the original CMS report, the CMS4-like subgroup (cluster 4) was significantly associated with a worse 5-year relapse-free survival and proved to be an independent prognostic factor. The clinicopathologic and prognostic features of the TIME subgroups have been validated in an independent validation cohort. CONCLUSIONS: Machine-learning-based image analysis can be useful for extracting quantitative information about the TIME, using whole-slide histopathologic images. This information can classify colorectal cancers into clinicopathologically relevant subgroups without performing a molecular analysis of the tumors.

CpG Island Methylation in Sessile Serrated Adenoma/Polyp of the Colorectum: Implications for Differential Diagnosis of Molecularly High-Risk Lesions among Non-dysplastic Sessile Serrated Adenomas/Polyps.

BACKGROUND: Although colorectal sessile serrated adenomas/polyps (SSA/Ps) with morphologic dysplasia are regarded as definite high-risk premalignant lesions, no reliable grading or risk-stratifying system exists for non-dysplastic SSA/Ps. The accumulation of CpG island methylation is a molecular hallmark of progression of SSA/Ps. Thus, we decided to classify non-dysplastic SSA/Ps into risk subgroups based on the extent of CpG island methylation. METHODS: The CpG island methylator phenotype (CIMP) status of 132 non-dysplastic SSA/Ps was determined using eight CIMP-specific promoter markers. SSA/Ps with CIMP-high and/or MLH1 promoter methylation were regarded as a high-risk subgroup. RESULTS: Based on the CIMP analysis results, methylation frequency of each CIMP marker suggested a sequential pattern of CpG island methylation during progression of SSA/P, indicating MLH1 as a late-methylated marker. Among the 132 non-dysplastic SSA/Ps, 34 (26%) were determined to be high-risk lesions (33 CIMP-high and 8 MLH1-methylated cases; seven cases overlapped). All 34 high-risk SSA/Ps were located exclusively in the proximal colon (100%, p = .001) and were significantly associated with older age (≥ 50 years, 100%; p = .003) and a larger histologically measured lesion size (> 5 mm, 100%; p = .004). In addition, the high-risk SSA/Ps were characterized by a relatively higher number of typical base-dilated serrated crypts. CONCLUSIONS: Both CIMP-high and MLH1 methylation are late-step molecular events during progression of SSA/Ps and rarely occur in SSA/Ps of young patients. Comprehensive consideration of age (≥ 50), location (proximal colon), and histologic size (> 5 mm) may be important for the prediction of high-risk lesions among non-dysplastic SSA/Ps.

Prognostic value of MRI in assessing extramural venous invasion in rectal cancer: multi-readers' diagnostic performance.

OBJECTIVES: This study was conducted in order to determine the prognostic value of MRI for extramural venous invasion (EMVI) in rectal cancer compared to pathology and to assess the diagnostic performance of multireaders. METHODS: We retrospectively enrolled 222 patients (M:F = 148:74; mean age ± standard deviation, 61.5 ± 12 years) with histopathologically proven rectal cancers who underwent preoperative MRI between 2007 and 2016. Among them, 74 patients had positive EMVI on pathology (pEMVI) and 148 patients had negative pEMVI. Three radiologists with 7 (reviewer 1), 3 (reviewer 2), and 1 (reviewer 3) year of experience in rectal MR imaging determined the presence of EMVI on MRI (mrEMVI) using a 5-point grading system. Using histopathologic results as the reference standard, radiologists' performances were analyzed and compared with receiver operating characteristic (ROC) analysis. For assessment of interobserver variation, intraclass correlation coefficients (ICC) were used. Lastly, Kaplan-Meier estimation and Cox proportional hazard models were used for survival analysis. RESULTS: The area under the ROC curve (AUC) was highest in reviewer 1 (0.829), followed by reviewer 2 (0.798) and reviewer 3 (0.658). Differences in AUCs between reviewer 1 or 2 and reviewer 3 were statistically significant (p < 0.001). ICC was substantial between reviewers 1 and 2. Overall survival (OS) was significantly different according to the positive circumferential resection margin, adjuvant treatment, and the presence of mrEMVI, but not by the presence of pEMVI. CONCLUSIONS: For experienced radiologists, the diagnostic performance of mrEMVI was good, resulting in better prediction of OS than with pEMVI, with substantial interobserver agreement. KEY POINTS: • When read by experienced radiologists, MR can provide reliable diagnostic performance in assessing EMVI for patients with rectal cancer. • Positive mrEMVI is an adverse prognostic factor of overall survival and may influence the clinical decision-making.

Multiplicity of Advanced T Category-Tumors Is a Risk Factor for Survival in Patients with Colorectal Carcinoma.

BACKGROUND: Previous studies on synchronous colorectal carcinoma (SCRC) have reported inconsistent results about its clinicopathologic and molecular features and prognostic significance. METHODS: Forty-six patients with multiple advanced tumors (T2 or higher category) who did not receive neoadjuvant chemotherapy and/or radiotherapy and who are not associated with familial adenomatous polyposis were selected and 99 tumors from them were subjected to clinicopathologic and molecular analysis. Ninety-two cases of solitary colorectal carcinoma (CRC) were selected as a control considering the distributions of types of surgeries performed on patients with SCRC and T categories of individual tumors from SCRC. RESULTS: SCRC with multiple advanced tumors was significantly associated with more frequent nodal metastasis (p = .003) and distant metastasis (p = .001) than solitary CRC. KRAS mutation, microsatellite instability, and CpG island methylator phenotype statuses were not different between SCRC and solitary CRC groups. In univariate survival analysis, overall and recurrence-free survival were significantly lower in patients with SCRC than in patients with solitary CRC, even after adjusting for the extensiveness of surgical procedure, adjuvant chemotherapy, or staging. Multivariate Cox regression analysis revealed that tumor multiplicity was an independent prognostic factor for overall survival (hazard ratio, 4.618; 95% confidence interval, 2.126 to 10.030; p < .001), but not for recurrence-free survival (p = .151). CONCLUSIONS: Findings suggested that multiplicity of advanced T category-tumors might be associated with an increased risk of nodal metastasis and a risk factor for poor survival, which raises a concern about the guideline of American Joint Committee on Cancer's tumor-node-metastasis staging that T staging of an index tumor determines T staging of SCRC.

Clinicopathological and molecular implications of aberrant thyroid transcription factor-1 expression in colorectal carcinomas: an immunohistochemical analysis of 1319 cases using three different antibody clones.

AIMS: The precise profile of aberrant expression of thyroid transcription factor-1 (TTF-1) according to antibody clones in colorectal carcinomas (CRCs) has been controversial. Moreover, the detailed clinicopathological and molecular features of CRCs with TTF-1 expression have rarely been investigated. The aim of this study was to evaluate TTF-1 expression status in a large series of CRC cases by using three different antibody clones. METHODS AND RESULTS: Immunohistochemistry for TTF-1 with clones 8G7G3/1, SPT24 and SP141 was performed on tumour tissues of 1319 primary CRCs and 98 corresponding metastatic lesions. Among the 1319 CRCs, TTF-1 expression was detected in 68 cases by both clone SPT24 and clone SP141. TTF-1 expression was not detected in any of the cases when clone 8G7G3/1 was used. The 68 CRCs with TTF-1 expression detected by both clone SPT24 and clone SP141 were considered to be TTF-1-positive in this study. TTF-1 positivity was significantly associated with distal tumour location, non-mucinous histology, intact CDX2 expression and a low frequency of KRAS mutations in CRCs. Nearly all TTF-1-positive CRCs showed microsatellite-stable and CpG island methylator phenotype-negative statuses. TTF-1 positivity was also found in all metastatic lesions of the five TTF-1-positive primary CRCs. TTF-1 negativity was maintained in all metastatic lesions of the 93 TTF-1-negative primary CRCs. CONCLUSIONS: Our study confirmed that the frequency and characteristics of aberrant TTF-1 expression in CRCs vary according to the antibody clone. Aberrant TTF-1 expression detected by clone SPT24 or SP141 may be encountered preferentially in distally located, conventional pathway-type CRCs.

Dominant high expression of wild-type HSP110 defines a poor prognostic subgroup of colorectal carcinomas with microsatellite instability: a whole-section immunohistochemical analysis.

The aim of this study is to establish heat shock protein 110 (HSP110) as a prognostic biomarker of colorectal carcinomas (CRCs) with microsatellite instability-high (MSI-H) by considering the intratumoral heterogeneity of HSP110 expression. We performed whole-section immunohistochemistry (IHC) for wild-type HSP110 (HSP110wt) in 164 MSI-H CRCs. The intensity of the HSP110wt expression in tumor cells was semiquantitatively scored (0/1/2/3), and the HSP110wt expression status of each tumor was classified as low or high using the following four scoring criteria: H-score, dominant intensity score, lowest intensity score, and highest intensity score. Among the four criteria, only the dominant intensity score-based dichotomous classification of HSP110wt expression was significantly associated with a difference in disease-free survival (log-rank p = 0.035) in 164 MSI-H CRCs. The HSP110wt-low MSI-H CRCs were significantly correlated with larger deletions in the HSP110 T17 mononucleotide repeat (≥4 bp; p < 0.001). In conclusion, the dominant intensity score-based assessment of HSP110wt IHC can be a simple and useful method for the prognostic stratification of MSI-H CRCs. It is expected that HSP110wt IHC may be used to identify a subgroup of MSI-H CRCs with poor prognosis and/or candidates for further treatment, such as immunotherapy using immune checkpoint inhibitors in MSI-H CRCs.

Intratumoral Fusobacterium nucleatum abundance correlates with macrophage infiltration and CDKN2A methylation in microsatellite-unstable colorectal carcinoma.

Fusobacterium nucleatum (Fn), a specific species of gut microbiota, has been suggested to be enriched in the microsatellite instability-high (MSI-H) molecular subtype of colorectal carcinomas (CRCs). However, the clinicopathologic and molecular factors that interact with Fn in MSI-H CRCs are poorly understood. In this study, 16S ribosomal RNA gene DNA sequence of Fn was quantitatively measured by real-time polymerase chain reaction in tumor DNA samples from a total of 160 surgically resected MSI-H CRC tissues. Each case was classified into one of the three categories based on the Fn DNA amount: Fn-high, Fn-low, and Fn-negative. The clinicopathologic and molecular associations of Fn in MSI-H CRCs were statistically analyzed. Among the 160 MSI-H CRC samples, 15 (9%), 92 (58%), and 53 (33%) cases were Fn-high, Fn-low, and Fn-negative, respectively. Compared with Fn-low/negative tumors, Fn-high MSI-H CRCs were significantly associated with a high density of CD68+ tumor-infiltrating macrophages (P = 0.019) and promoter CpG island hypermethylation of the CDKN2A (p16) gene (P = 0.008). There were also tendencies toward associations of Fn-high with the BRAF V600E mutation (P = 0.047) and active Crohn-like lymphoid reactions (P = 0.052) in MSI-H CRCs. However, Fn-high was not significantly associated with CD3+ T cell density, CD163+ M2 macrophage density or PD-L1 expression status. In conclusion, high amounts of intratumoral Fn are correlated with increased macrophage infiltration and CDKN2A promoter methylation in MSI-H CRCs.

Downregulation of acetyl-CoA synthetase 2 is a metabolic hallmark of tumor progression and aggressiveness in colorectal carcinoma.

Acetyl-CoA synthetase-2 is an emerging key enzyme for cancer metabolism, which supplies acetyl-CoA for tumor cells by capturing acetate as a carbon source under stressed conditions. However, implications of acetyl-CoA synthetase-2 in colorectal carcinoma may differ from other malignancies, because normal colonocytes use short-chain fatty acids as an energy source, which are supplied by fermentation of the intestinal flora. Here we analyzed acetyl-CoA synthetase-2 mRNA expression by reverse-transcription quantitative PCR in paired normal mucosa and tumor tissues of 12 colorectal carcinomas, and subsequently evaluated acetyl-CoA synthetase-2 protein expression by immunohistochemistry in 157 premalignant colorectal lesions, including 60 conventional adenomas and 97 serrated polyps, 1,106 surgically resected primary colorectal carcinomas, and 23 metastatic colorectal carcinomas in the liver. In reverse-transcription quantitative PCR analysis, acetyl-CoA synthetase-2 mRNA expression was significantly decreased in tumor tissues compared with corresponding normal mucosa tissues. In acetyl-CoA synthetase-2 immunohistochemistry analysis, all 157 colorectal polyps showed moderate-to-strong expression of acetyl-CoA synthetase-2. However, cytoplasmic acetyl-CoA synthetase-2 expression was downregulated (acetyl-CoA synthetase-2 low expression) in 771 (69.7%) of 1,106 colorectal carcinomas and 21 (91.3%) of 23 metastatic lesions. The colorectal carcinomas with acetyl-CoA synthetase-2-low expression were significantly associated with advanced TNM stage, poor differentiation, and frequent tumor budding. Regarding the molecular aspect, acetyl-CoA synthetase-2-low expression exhibited a tendency of frequent KRT7 expression and decreased KRT20 and CDX2 expression. In survival analysis, acetyl-CoA synthetase-2-low expression was an independent prognostic factor for poor 5-year progression-free survival (hazard ratio, 1.39; 95% confidence interval, 1.08-1.79; P=0.01). In conclusion, these findings suggest that downregulation of acetyl-CoA synthetase-2 expression is a metabolic hallmark of tumor progression and aggressive behavior in colorectal carcinoma.

Characterisation of PD-L1-positive subsets of microsatellite-unstable colorectal cancers.

BACKGROUND: The aim of this study was to reveal the clinicopathological and molecular characteristics of microsatellite instability-high (MSI-H) colorectal cancers (CRCs) showing programmed death ligand-1 (PD-L1) positivity, which are good candidates for anti-PD-1/PD-L1 immunotherapy. METHODS: The PD-L1 expression status of 208 MSI-H CRCs was retrospectively analysed using immunohistochemistry. PD-L1 positivity in tumour cells (PD-L1+(T)) and PD-L1 positivity in immune cells (PD-L1+(I)) were separately evaluated. RESULTS: Programmed death ligand-1 positivity in tumour cells and PD-L1+(I) were observed in 26 (12.5%) and 62 (29.8%) MSI-H CRCs, respectively, and occasionally overlapped (n=12; 5.8%). Programmed death ligand-1 positivity tumours in MSI-H CRCs were significantly associated with older age, female sex, non-mucinous-type poor differentiation, infiltrating growth, tumour budding, advanced stage, CpG island methylator phenotype-high, MLH1 promoter methylation, and BRAF V600E mutations. However, PD-L1+(I) MSI-H CRCs were characterised by high-density tumour-infiltrating immune cells, including T cells and macrophages, and intense peritumoural lymphoid reactions. In patients with stage IV MSI-H CRCs who had undergone metastatectomy (n=4), the PD-L1 status of primary tumours was maintained in corresponding distant metastatic lesions. CONCLUSIONS: In MSI-H CRCs, PD-L1+(T) and PD-L1+(I) are linked to a sporadic hypermethylated subtype and an immune cell-rich subtype, respectively. Potential differential therapeutic implications of PD-L1+(T) and PD-L1+(I) in CRCs should be further investigated.