Relationship between stromal regulatory T cells and the response to neoadjuvant chemotherapy for locally advanced rectal cancer.

BACKGROUND: In addition to the direct power of anticancer drugs, the effectiveness of anticancer therapy depends on the host immune function. The present study investigated whether or not the reduction rate and histological response of preoperative chemotherapy were related to the immune microenvironment surrounding a primary tumor of the rectum. METHODS: Sixty-five patients received preoperative chemotherapy followed by resection from 2012 to 2014; all of these patients were retrospectively analyzed. CD3, CD8, and FoxP3 were immunohistochemically examined as markers for T lymphocytes, cytotoxic T lymphocytes, and regulatory T lymphocytes (Treg), respectively. The correlation between the tumor-infiltrating lymphocyte composition and the tumor reduction rate and histological response to neoadjuvant chemotherapy was investigated. RESULTS: The average tumor reduction rate was 41.5% ± 18.8%. According to RECIST, 47 patients (72.3%) achieved a partial response (PR), and 1 patient (1.5%) achieved a complete response (CR). Eight patients (12.3%) showed a grade 2 histological response, and 2 (3.1%) showed a grade 3 response. A multivariate analysis demonstrated that a low Treg infiltration in stromal cell areas was significantly associated with the achievement of a PR or CR [odds ratio (OR) 7.69; 95% confidence interval (CI) 1.96-33.33; p < 0.01] and a histological grade 2 or 3 response (OR 11.11; 95% CI 1.37-98.04; p = 0.02). CONCLUSION: A low Treg infiltration in the stromal cell areas may be a marker of a good response to neoadjuvant chemotherapy in patients with locally advanced rectal cancer.

A stem cell population at the anorectal junction maintains homeostasis and participates in tissue regeneration.

At numerous locations of the body, transition zones are localized at the crossroad between two types of epithelium and are frequently associated with neoplasia involving both type of tissues. These transition zones contain cells expressing markers of adult stem cells that can be the target of early transformation. The mere fact that transition zone cells can merge different architecture with separate functions implies for a unique plasticity that these cells must display in steady state. However, their roles during tissue regeneration in normal and injured state remain unknown. Here, by using in vivo lineage tracing, single-cell transcriptomics, computational modeling and a three-dimensional organoid culture system of transition zone cells, we identify a population of Krt17+ basal cells with multipotent properties at the squamo-columnar anorectal junction that maintain a squamous epithelium during normal homeostasis and can participate in the repair of a glandular epithelium following tissue injury.

A Human Cellular Model for Colorectal Anastomotic Repair: The Effect of Localization and Transforming Growth Factor-ß1 Treatment on Collagen Deposition and Biomarkers.

Anastomotic leakage (AL) is a devastating complication after colorectal surgery, possibly due to the loss of stabilizing collagen fibers in the submucosa. Our aim was to assess the formation of collagen in the colon versus the rectum with or without transforming growth factor (TGF)-ß1 exposure in a human cellular model of colorectal repair. Primary fibroblasts were isolated by an explant procedure from clinically resected tissue rings during anastomosis construction in 19 consecutive colorectal patients who underwent laparoscopy. The cells, identified as fibroblasts by morphologic characteristics and flow cytometry analysis (CD90+), were cultured for 8 days and in 12 patients in the presence of 1 ng/mL TGF-ß1. Total collagen deposition was measured colorimetrically after Sirius red staining of fixed cell layers, and type I, III, and VI collagen biosynthesis and degradation were specifically determined by the biomarkers PINP, PRO-C3, PRO-C6, and C3M in conditioned media by competitive enzyme-linked immunosorbent assays. Total collagen deposition by fibroblasts from the colon and rectum did not significantly differ. TGF-ß1 treatment increased PINP, PRO-C6, and total collagen deposition. Mechanistically, TGF-ß1 treatment increased COL1A1 and ACTA2 (encoding α-smooth muscle actin), and decreased COL6A1 and MMP2 mRNA levels in colorectal fibroblasts. In conclusion, we found no effect of anatomic localization on collagen production by fibroblasts derived from the large intestine. TGF-ß1 represents a potential therapeutic agent for the prevention of AL by increasing type I collagen synthesis and collagen deposition.

A Mechanistic In Vivo/Ex Vivo Pharmacokinetic-Pharmacodynamic Model of Tenofovir for HIV Prevention.

Defining tissue and plasma-specific prophylactic drug concentrations is central to pre-exposure prophylaxis product development for sexual transmission of HIV-1. Pharmacokinetic (PK) data from study RMP-02/MTN-006 comparing single dose oral tenofovir disoproxil fumarate with single and multiple dose rectal tenofovir (TFV) gel administration in HIV-1 seronegative adults was used to construct a multicompartment plasma-rectal tissue population PK model for TFV and tenofovir-diphosphate (TFVdp) in plasma and rectal tissue. PK data were collected in five matrices: TFV (plasma, rectal tissue homogenate), TFVdp (peripheral blood mononuclear cells, rectal mononuclear cells (MMCs), rectal tissue homogenate). A viral growth compartment and a delayed effect compartment for p24 antigen expression measured from an ex vivo explant assay described HIV-1 infection and replication. Using a linear PK/pharmacodynamic model, MMC TFVdp levels over 9,000 fmol/million cells in the explant assay provided apparent viral replication suppression down to 1%. Parameters were estimated using NONMEM version 7.4.

G970R-CFTR Mutation (c.2908G>C) Results Predominantly in a Splicing Defect.

In previous work, participants with a G970R mutation in cystic fibrosis transmembrane conductance regulator (CFTR) (c.2908G>C) had numerically lower sweat chloride responses during ivacaftor treatment than participants with other CFTR gating mutations. The objective of this substudy was to characterize the molecular defect of the G970R mutation in vitro and assess the benefit of ivacaftor in participants with this mutation. This substudy assessed sweat chloride, spirometry findings, and nasal potential difference on and off ivacaftor treatment in three participants with a G970R/F508del genotype. Intestinal organoids derived from rectal biopsy specimens were used to assess ivacaftor response ex vivo and conduct messenger RNA splice and protein analyses. No consistent or meaningful trends were observed between on-treatment and off-treatment clinical assessments. Organoids did not respond to ivacaftor in forskolin-induced swelling assays; no mature CFTR protein was detected in Western blots. Organoid RNA analysis demonstrated that 3 novel splice variants were created by G970R-CFTR: exon 17 truncation, exons 13-15 and 17 skipping, and intron 17 retention. Functional and molecular analyses indicated that the c.2908G>C mutation caused a cryptic splicing defect. Organoids lacked an ex vivo response with ivacaftor and supported identification of the mechanism underlying the CFTR defect caused by c.2908G>C. Analysis of CFTR mutations indicated that cryptic splicing was a rare cause of mutation misclassification in engineered cell lines. This substudy used organoids as an alternative in vitro model for mutations, such as cryptic splice mutations that cannot be fully assessed using cDNA expressed in recombinant cell systems.

Butyrate enhances the efficacy of radiotherapy via FOXO3A in colorectal cancer patient­derived organoids.

Enhancing the radioresponsiveness of colorectal cancer (CRC) is essential for local control and prognosis. However, consequent damage to surrounding healthy cells can lead to treatment failure. We hypothesized that short­chain fatty acids (SCFAs) could act as radiosensitizers for cancer cells, allowing the administration of a lower and safer dose of radiation. To test this hypothesis, the responses of three­dimensional­cultured organoids, derived from CRC patients, to radiotherapy, as well as the effects of combined radiotherapy with the SCFAs butyrate, propionate and acetate as candidate radiosensitizers, were evaluated via reverse transcription­quantitative polymerase chain reaction, immunohistochemistry and organoid viability assay. Of the three SCFAs tested, only butyrate suppressed the proliferation of the organoids. Moreover, butyrate significantly enhanced radiation­induced cell death and enhanced treatment effects compared with administration of radiation alone. The radiation­butyrate combination reduced the proportion of Ki­67 (proliferation marker)­positive cells and decreased the number of S phase cells via FOXO3A. Meanwhile, 3/8 CRC organoids were found to be non­responsive to butyrate with lower expression levels of FOXO3A compared with the responsive cases. Notably, butyrate did not increase radiation­induced cell death and improved regeneration capacity after irradiation in normal organoids. These results suggest that butyrate could enhance the efficacy of radiotherapy while protecting the normal mucosa, thus highlighting a potential strategy for minimizing the associated toxicity of radiotherapy.

Anatomical Variation in Mesenteric Macrophage Phenotypes in Crohn's Disease.

INTRODUCTION: Clinical trials are currently investigating whether an extended mesenteric resection for ileocecal resections could reduce postoperative recurrence in Crohn's disease. Resection of the mesorectum, which contains proinflammatory macrophages, during proct(ocol)ectomy, is associated with reduced recurrent inflammation and improved wound healing. We aimed to characterize the macrophages in the ileocecal mesentery, which were compared with those in the mesorectum, to provide a biological rationale for the ongoing trials. METHODS: In 13 patients with Crohn's disease and 4 control patients undergoing a proctectomy, tissue specimens were sampled at 3 locations from the mesorectum: distal (rectum), middle, and proximal (sigmoid). In 38 patients with Crohn's disease and 7 control patients undergoing ileocecal resections, tissue specimens also obtained from 3 locations: adjacent to the inflamed terminal ileum, adjacent to the noninflamed ileal resection margin, and centrally along the ileocolic artery. Immune cells from these tissue specimens were analyzed by flow cytometry for expression of CD206 to determine their inflammatory status. RESULTS: In the mesorectum, a gradient from proinflammatory to regulatory macrophages from distal to proximal was observed, corresponding to the adjacent inflammation of the intestine. By contrast, the ileocecal mesentery did not contain high amounts of proinflammatory macrophages adjacent to the inflamed tissue, and a gradient toward a more proinflammatory phenotype was seen in the central mesenteric area. DISCUSSION: Although the mesentery is a continuous structure, the mesorectum and the ileocecal mesentery show different immunological characteristics. Therefore, currently, there is no basis to perform an extended ileocecal resection in patients with Crohn's disease.

Computational imaging modalities for multi-focal whole-slide imaging systems.

Whole-slide imaging systems can generate full-color image data of tissue slides efficiently, which are needed for digital pathology applications. This paper focuses on a scanner architecture that is based on a multi-line image sensor that is tilted with respect to the optical axis, such that every line of the sensor scans the tissue slide at a different focus level. This scanner platform is designed for imaging with continuous autofocus and inherent color registration at a throughput of the order of 400 MPx/s. Here, single-scan multi-focal whole-slide imaging, enabled by this platform, is explored. In particular, two computational imaging modalities based on multi-focal image data are studied. First, 3D imaging of thick absorption stained slides (∼60µm) is demonstrated in combination with deconvolution to ameliorate the inherently weak contrast in thick-tissue imaging. Second, quantitative phase tomography is demonstrated on unstained tissue slides and on fluorescently stained slides, revealing morphological features complementary to features made visible with conventional absorption or fluorescence stains. For both computational approaches simplified algorithms are proposed, targeted for straightforward parallel processing implementation at ∼GPx/s throughputs.

The Role of Cyclooxygenase-2 in Colorectal Cancer.

Colorectal cancer is the third common cancer in this world, accounting for more than 1 million cases each year. However, detailed etiology and mechanism of colorectal cancer have not been fully understood. For example, cyclooxygenase-2 (COX-2) and its product prostaglandin E2 (PGE2) have been closely linked to its occurrence, progression and prognosis. However, the mechanisms on how COX-2 and PGE2-mediate the pathogenesis of colorectal cancer are obscure. In this review, we have summarized recent advances in studies of pathogenesis and control in colorectal cancer to assist further advances in the research for the cure of the cancer. In addition, the knowledge gained may also guide the audiences for reduction of the risk and control of this deadly disease.

Resistant starch supplementation increases crypt cell proliferative state in the rectal mucosa of older healthy participants.

There is strong evidence that foods containing dietary fibre protect against colorectal cancer, resulting at least in part from its anti-proliferative properties. This study aimed to investigate the effects of supplementation with two non-digestible carbohydrates, resistant starch (RS) and polydextrose (PD), on crypt cell proliferative state (CCPS) in the macroscopically normal rectal mucosa of healthy individuals. We also investigated relationships between expression of regulators of apoptosis and of the cell cycle on markers of CCPS. Seventy-five healthy participants were supplemented with RS and/or PD or placebo for 50 d in a 2 × 2 factorial design in a randomised, double-blind, placebo-controlled trial (the Dietary Intervention, Stem cells and Colorectal Cancer (DISC) Study). CCPS was assessed, and the expression of regulators of the cell cycle and of apoptosis was measured by quantitative PCR in rectal mucosal biopsies. SCFA concentrations were quantified in faecal samples collected pre- and post-intervention. Supplementation with RS increased the total number of mitotic cells within the crypt by 60 % (P = 0·001) compared with placebo. This effect was limited to older participants (aged ≥50 years). No other differences were observed for the treatments with PD or RS as compared with their respective controls. PD did not influence any of the measured variables. RS, however, increased cell proliferation in the crypts of the macroscopically-normal rectum of older adults. Our findings suggest that the effects of RS on CCPS are not only dose, type of RS and health status-specific but are also influenced by age.

High stromal nicotinamide N-methyltransferase (NNMT) indicates poor prognosis in colorectal cancer.

PURPOSE: Nicotinamide n-methyltransferase (NNMT) has good biochemical activity and epigenetic regulation, and has been reported as a major metabolic regulator of cancer. The goal of this study was to investigate the significance of stromal NNMT expression in colorectal cancer (CRC). PATIENTS AND METHODS: Stromal expression of NNMT in primary CRC, metastasis CRC, and their non-cancerous tissues from 1088 CRC patients was examined by immunohistochemistry. The associations between stromal NNMT expression and survival outcomes in 967 patients with stage I-III CRC were further evaluated with Kaplan-Meier curve and Cox model analyses. RESULTS: NNMT expression was mainly sourced from stromal compartments and also elevated in CRC. Patients with high stromal NNMT (IHC-score ≥ 106) have a worse survival than those patients with low stromal NNMT. In multiple Cox analyses, high expression of stromal NNMT remained as an independent risk factor in CRC for disease-free survival with a hazard ratio (HR) of 1.415 (95% confidence interval [CI], 1.015-1.972) and disease-specific survival with a HR of 5.004 (95% CI, 2.301-10.883). In addition, high stromal NNMT expression in CRC also indicates the poor survival outcomes in patients with early stage CRC (stage I and II) and in patients who undergo chemotherapy. CONCLUSION: NNMT is mainly located in CRC stromal compartment. High stromal NNMT expression predicts an unfavorable postoperative prognosis.

Timing of Antiretroviral Therapy Initiation Determines Rectal Natural Killer Cell Populations.

Despite antiretroviral therapy (ART), innate and adaptive immunologic damage persists in the periphery and gut. T memory stem cells (Tscm) and natural killer (NK) cells are pivotal for host defense. Tscm are memory cells capable of antigen response and self-renewal, and circulating and gut NK cell populations may facilitate HIV control. The impact of early ART on circulating and gut Tscm and NK cells is unknown. We enrolled participants who initiated ART during acute versus chronic HIV-1 infection versus no ART in chronic infection. We performed flow cytometry to identify NK and Tscm cells in the blood and rectum and polymerase chain reaction to quantify the HIV-1 reservoir in both sites. We used the Mann-Whitney U-test and Spearman correlation coefficients for analysis. Participants who started ART in acute infection had lower rectal CD56brightCD16dim cell frequencies than participants who started ART in chronic HIV-1 infection and lower CD56bright and CD56brightCD16- cell frequencies than participants with chronic infection without ART. Higher circulating NK cell, CD56-CD16bright, CD56dim, and CD56dimCD16bright frequencies correlated with higher HIV-1 DNA levels in rectal CD4+ T cells, whereas higher circulating CD4+ T cell counts correlated with higher rectal NK, CD56brightCD16dim, and CD56dimCD16bright frequencies. Peripheral CD56brightCD16- cells were inversely associated with rectal CD56-CD16bright cells. Rectal CD8+ Tscm frequencies were higher in participants without ART than participants with chronic infection on ART. Timing of ART initiation determines rectal NK cell populations, and ART may influence rectal Tscm populations. Whether the gut reservoir contributes to NK cell activation requires further study.

Impact of Q-Griffithsin anti-HIV microbicide gel in non-human primates: In situ analyses of epithelial and immune cell markers in rectal mucosa.

Natural-product derived lectins can function as potent viral inhibitors with minimal toxicity as shown in vitro and in small animal models. We here assessed the effect of rectal application of an anti-HIV lectin-based microbicide Q-Griffithsin (Q-GRFT) in rectal tissue samples from rhesus macaques. E-cadherin+ cells, CD4+ cells and total mucosal cells were assessed using in situ staining combined with a novel customized digital image analysis platform. Variations in cell numbers between baseline, placebo and Q-GRFT treated samples were analyzed using random intercept linear mixed effect models. The frequencies of rectal E-cadherin+ cells remained stable despite multiple tissue samplings and Q-GRFT gel (0.1%, 0.3% and 1%, respectively) treatment. Whereas single dose application of Q-GRFT did not affect the frequencies of rectal CD4+ cells, multi-dose Q-GRFT caused a small, but significant increase of the frequencies of intra-epithelial CD4+ cells (placebo: median 4%; 1% Q-GRFT: median 7%) and of the CD4+ lamina propria cells (placebo: median 30%; 0.1-1% Q-GRFT: median 36-39%). The resting time between sampling points were further associated with minor changes in the total and CD4+ rectal mucosal cell levels. The results add to general knowledge of in vivo evaluation of anti-HIV microbicide application concerning cellular effects in rectal mucosa.

The landscape of somatic mutation in normal colorectal epithelial cells.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer1. However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.

A retrospective study on the transfer, persistence and recovery of sperm and epithelial cells in samples collected in sexual assault casework.

Data from all sexual assault cases analysed at the Section of Forensic Biology at Oslo University Hospital in the period 2013-2015 were reviewed to study transfer and persistence of cells deposited on the body. Data were recorded on detection of both sperm and epithelial cells. The final dataset consist of 2141 samples from 765 cases. In this study "positive findings" refer to evidence to support the proposition that the DNA profile was contributed by the POI and do not only correspond to detection of cell type, e.g. sperm cells. Positive findings from analysis of sperm cells could be detected in samples collected up to 72 h after deposition, and was less frequently detected in oral swabs were the longest observed persistence time was 12 h. Positive findings from analysis of epithelial cells were observed up to 43 h after deposition. A high success rate was observed from penile swabs collected within 24 h of the incidence demonstrating the importance of collecting and analysing such samples in cases where no semen is detected.

Moving tissue spectral window to the deep-ultraviolet via optical clearing.

The optical immersion clearing technique has been successfully applied through the last 30 years in the visible to near infrared spectral range, and has proven to be a promising method to promote the application of optical technologies in clinical practice. To investigate its potential in the ultraviolet range, collimated transmittance spectra from 200 to 1000 nm were measured from colorectal muscle samples under treatment with glycerol-water solutions. The treatments created two new optical windows with transmittance efficiency peaks at 230 and 300 nm, with magnitude increasing with glycerol concentration in the treating solution. Such discovery opens the opportunity to develop clinical procedures to perform diagnosis or treatments in the ultraviolet.

Analysis of glycoconjugates and morphological characterization of the descending colon and rectum of the plains viscacha, Lagostomus maximus.

Herbivores exhibit specializations at the intestinal level that facilitate the bacterial fermentation. The available information on the digestive physiology of Lagostomus maximus makes this rodent an interesting model to evaluate morpho-functional adaptations to herbivory. The general objective of this work was centered on the study of the morphology and histochemistry of the descending colon and rectum of L. maximus. To do so, a comparative analysis of the morphology, ultrastructure and glycosylation pattern of both anatomical regions was carried out. Histochemical results revealed that in both sectors of the large intestine, there are goblet cells with different glycosylation pattern within a morphologically homogeneous cell population. The main difference between both intestinal segments lay in the fact that the most distal region of the large intestine showed a greater proportion of sialomucins, characterized by being slightly O-acetylated. Further specific differences were revealed by lectin histochemistry. These data allowed to perform a functional interpretation of the cell types and secreted substances, thus contributing to a better understanding of the role of mucins in the intestinal tract functioning.

Chronically stimulated human MAIT cells are unexpectedly potent IL-13 producers.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize antigens derived from riboflavin biosynthesis. In addition to anti-microbial functions, human MAIT cells are associated with cancers, autoimmunity, allergies and inflammatory disorders, although their role is poorly understood. Activated MAIT cells are well known for their rapid release of Th1 and Th17 cytokines, but we have discovered that chronic stimulation can also lead to potent interleukin (IL)-13 expression. We used RNA-seq and qRT-PCR to demonstrate high expression of the IL-13 gene in chronically stimulated MAIT cells, and directly identify IL-13 using intracellular flow cytometry and multiplex bead analysis of MAIT cell cultures. This unexpected finding has important implications for IL-13-dependent diseases, such as colorectal cancer (CRC), that occur in mucosal areas where MAIT cells are abundant. We identify MAIT cells near CRC tumors and show that these areas and precancerous polyps express high levels of the IL-13 receptor, which promotes tumor progression and metastasis. Our data suggest that MAIT cells have a more complicated role in CRC than currently realized and that they represent a promising new target for immunotherapies where IL-13 can be a critical factor.

Protocol for the long-term culture of human primary keratinocytes from the normal colorectal mucosa.

Procedures for in vitro culturing of human primary keratinocytes from normal colon mucosa specimens have not been fully feasible, thus far. The protocol described herein allows primary keratinocytes from small tissue fragments of colorectal mucosa biopsies to grow in vitro. The procedure develops in three steps: (a) the enzymatic digestion of the tissue biopsy; (b) the use of cloning rings to purify primary keratinocyte colonies, (c) a defined keratinocyte medium to grow these cells in long-term culture. Our cultural method enables normal primary keratinocytes to be obtained by simple and rapid techniques. In our culture condition, primary keratinocytes express specific epithelial markers. Colorectal mucosa keratinocyte colonies require approximately 2 weeks to grow. Compared with previous approaches, our protocol provides a valuable model of study for human primary keratinocytes from normal colorectal (NCR) mucosa both at the cellular and molecular levels. It is well known, that different mutations occurring during the multistep process of carcinogenesis in the NCR mucosa, are strictly associated to the onset/progression of the colorectal carcinoma. On this ground, normal keratinocytes grown with our protocol, may represent an innovative tool to investigate the mechanisms that lead to colorectal carcinoma and other diseases. Our innovative procedure may allow to perform comparative investigations between normal and pathological colorectal cells.

Neuroendocrine Differentiation, Microsatellite Instability, and Tumor-infiltrating Lymphocytes in Advanced Colorectal Cancer With BRAF Mutation.

BACKGROUND: Approximately 10% of metastatic colorectal cancer (mCRC) cases will harbor the BRAF p.V600E mutation (BRAF-mCRC) and have been associated with a poor prognosis. Although they are usually considered a unique clinical entity, biologic heterogeneity has been described. We performed an extensive clinicopathologic study of a multicenter series of BRAF-mCRC to highlight differences between tumors with microsatellite instability (MSI) and microsatellite stable tumors, focusing on both inflammatory profiles and neuroendocrine differentiation. METHODS: We included 59 BRAF-mCRC cases and collected the clinical data (ie, surgery, treatment, and follow-up). We evaluated MSI status, budding, lympho-angioinvasion, neuroinvasion, extent of active stroma, CD3+ and CD8+ intratumoral and peritumoral lymphocytes, programmed cell death ligand 1, p53, Ki-67, synaptophysin, and CDX2 expression. RESULTS: The 22 MSI BRAF-mCRC cases were associated with the right side (P < .0001), an expansive grown pattern (P < .01), programmed cell death ligand 1 expression (P < .0001), high CD8 T-cell content (P = .0001), and lymph node metastases (P < .029). The 37 MSS BRAF-mCRC cases were characterized by a greater stromal component (P = .0002), pulmonary metastases (P = .095), and p53 and synaptophysin immunoreactivity (P = .004 and P = .001, respectively). Univariate analysis demonstrated that MSI and a high CD8 T-cell content were associated with a 34% (hazard ratio [HR], 0.66; 95% confidence interval [CI], 0.34-1.28; P = .2) and 33% (HR, 0.67; 95% CI, 0.45-0.99; P = .04) reduction in the risk of death, respectively. The combined presence of MSI and CD8 T-cell content decreased the hazard of mortality ≤ 63% (HR, 0.37; 95% CI, 0.14-0.97; P = .2), which was slightly reduced after multivariate analysis. CONCLUSION: A simultaneous evaluation of MSI, CD8 T-cell content, and neuroendocrine markers could allow for the identification of subsets of BRAF-mCRC with a different prognosis and potential eligibility for specific treatments.