Location matters: spatial dynamics of tumor-infiltrating T cell subsets is prognostic in colon cancer.

Background: Colon cancer is a heterogeneous disease and consists of various molecular subtypes. Despite advances in high-throughput expression profiling, limitations remain in predicting clinical outcome and assigning specific treatment to individual cases. Tumor-immune interactions play a critical role, with tumors that activate the immune system having better outcome for the patient. The localization of T cells within tumor epithelium, to enable direct contact, is essential for antitumor function, but bulk DNA/RNA sequencing data lacks spatial distribution information. In this study, we provide spatial T cell tumor distribution and connect these data with previously determined genomic data in the AC-ICAM colon cancer patient cohort. Methods: Colon cancer patients (n=90) with transcriptome data available were selected. We used a custom multiplex immunofluorescence assay on colon tumor tissue sections for quantifying T cell subsets spatial distribution in the tumor microenvironment, in terms of cell number, location, mutual distance, and distance to tumor cells. Statistical analyses included the previously determined Immunologic Constant of Rejection (ICR) transcriptome correlation and patient survival, revealing potential prognostic value in T cell spatial distribution. Results: T cell phenotypes were characterized and CD3+CD8-FoxP3- T cells were found to be the predominant tumor-infiltrating subtype while CD3+FoxP3+ T cells and CD3+CD8+ T cells showed similar densities. Spatial distribution analysis elucidated that proliferative T cells, characterized by Ki67 expression, and Granzyme B-expressing T cells were predominantly located within the tumor epithelium. We demonstrated an increase in immune cell density and a decrease in the distance of CD3+CD8+ T cells to the nearest tumor cell, in the immune active, ICR High, immune subtypes. Higher densities of stromal CD3+FoxP3+ T cells showed enhanced survival outcomes, and patients exhibited superior clinical benefits when greater spatial distances were observed between CD3+CD8-FoxP3- or CD3+CD8+ T cells and CD3+FoxP3+ T cells. Conclusion: Our study's in-depth analysis of the spatial distribution and densities of major T cell subtypes within the tumor microenvironment has provided valuable information that paves the way for further research into the intricate relationships between immune cells and colon cancer development.

Longitudinal Serum Protein Analysis of Women with a High Risk of Developing Breast Cancer Reveals Large Interpatient Versus Small Intrapatient Variations: First Results from the TESTBREAST Study.

The prospective, multicenter TESTBREAST study was initiated with the aim of identifying a novel panel of blood-based protein biomarkers to enable early breast cancer detection for moderate-to-high-risk women. Serum samples were collected every (half) year up until diagnosis. Protein levels were longitudinally measured to determine intrapatient and interpatient variabilities. To this end, protein cluster patterns were evaluated to form a conceptual basis for further clinical analyses. Using a mass spectrometry-based bottom-up proteomics strategy, the protein abundance of 30 samples was analyzed: five sequential serum samples from six high-risk women; three who developed a breast malignancy (cases) and three who did not (controls). Serum samples were chromatographically fractionated and an in-depth serum proteome was acquired. Cluster analyses were applied to indicate differences between and within protein levels in serum samples of individuals. Statistical analyses were performed using ANOVA to select proteins with a high level of clustering. Cluster analyses on 30 serum samples revealed unique patterns of protein clustering for each patient, indicating a greater interpatient than intrapatient variability in protein levels of the longitudinally acquired samples. Moreover, the most distinctive proteins in the cluster analysis were identified. Strong clustering patterns within longitudinal intrapatient samples have demonstrated the importance of identifying small changes in protein levels for individuals over time. This underlines the significance of longitudinal serum measurements, that patients can serve as their own controls, and the relevance of the current study set-up for early detection. The TESTBREAST study will continue its pursuit toward establishing a protein panel for early breast cancer detection.

Cancer-immune interactions in ER-positive breast cancers: PI3K pathway alterations and tumor-infiltrating lymphocytes.

INTRODUCTION: The presence of tumor-infiltrating lymphocytes (TILs) is correlated with good prognosis and outcome after (immuno)therapy in triple-negative and HER2-positive breast cancer. However, the role of TILs in luminal breast cancer is less clear. Emerging evidence has now demonstrated that genetic aberrations in malignant cells influence the immune landscape of tumors. Phosphatidylinositol 3-kinase (PI3K) is the most common altered pathway in ER-positive breast cancer. It is unknown whether changes in the PI3K pathway result in a different composition of the breast tumor microenvironment. Here we present the retrospective analysis of a prospective randomized trial in ER-positive breast cancer on the prognostic and predictive value of specific tumor-associated lymphocytes in the context of PI3K alterations. METHODS: We included 563 ER-positive tumors from a multicenter trial for stage I to III postmenopausal breast cancer patients, who were randomized to tamoxifen or no adjuvant therapy. The amount of CD8-, CD4-, and FOXP3-positive cells was evaluated by immunohistochemistry and quantified by imaging-analysis software. We analyzed the associations between PIK3CA hotspot mutations, PTEN expression, phosphorylated proteins of the PI3K and MAPK pathway (p-AKT, p-ERK1/2, p-4EBP1, p-p70S6K), and recurrence-free interval after adjuvant tamoxifen or no adjuvant treatment. RESULTS: CD8-positive lymphocytes were significantly more abundant in PIK3CA-mutated tumors (OR = 1.65; 95% CI 1.03-2.68). While CD4 and FOXP3 were not significantly associated with prognosis, patients with tumors classified as CD8-high had increased risk of recurrence (HR = 1.98; 95% CI 1.14-3.41; multivariable model including PIK3CA status, treatment arm, and other standard clinicopathological variables). Lymphocytes were more often present in tumors with increased PI3K downstream phosphorylation. This was most pronounced for FOXP3-positive cells. CONCLUSION: These exploratory analyses of a prospective trial in luminal breast cancer suggest high CD8 infiltration is associated with unfavorable outcome and that PI3K pathway alterations might be associated with the composition of the tumor microenvironment.

A method for semi-automated image analysis of HLA class I tumour epithelium expression in rectal cancer.

Biomarkers may hold the key towards development and improvement of personalized cancer treatment. For instance, tumour expression of immune system-related proteins may reveal the tumour immune status and, accordingly, determine choice for type of immunotherapy. Therefore, objective evaluation of tumour biomarker expression is needed but often challenging. For instance, human leukocyte antigen (HLA) class I tumour epithelium expression is cumbersome to quantify by eye due to its presence on both tumour epithelial cells and tumour stromal cells, as well as tumour-infiltrating immune cells. In this study, we solved this problem by setting up an immunohistochemical (IHC) double staining using a tissue microarray (TMA) of rectal tumours wherein HLA class I expression was coloured with a blue chromogen, whereas non-epithelial tissue was visualized with a brown chromogen. We subsequently developed a semi-automated image analysis method that identified tumour epithelium as well as the percentage of HLA class I-positive tumour epithelium. Using this technique, we compared HCA2/HC10 and EMR8-5 antibodies for the assessment of HLA class I tumour expression and concluded that EMR8-5 is the superior antibody for this purpose. This IHC double staining can in principle be used for scoring of any biomarker expressed by tumour epithelium.

HLA-G protein expression in colorectal cancer evaluated by immunohistochemistry and western blot analysis: Its expression characteristics remain enigmatic.

HLA-G protein expression could play a role in evasion of tumor immune surveillance. Accumulating evidence demonstrates that HLA-G is expressed in different types of malignancies, including colorectal cancer (CRC). The purpose of the current study was to further unravel whether HLA-G protein expression could play a role in immune evasion of CRC. Therefore, to firmly establish HLA-G protein expression, eight early passage human CRC cell lines and five human rectal cancer tissues were analyzed by western blot analysis. The results obtained by western blot analysis were compared with immunohistochemistry on tumor tissue sections of the same patient. Furthermore, multiple monoclonal antibodies (mAbs), 4H84, MEM-G/1 and 5A6G7, targeting HLA-G were used to unravel staining patterns. We showed that results obtained with immunohistochemistry did not correlate with protein expression detected by western blot analysis, using three different HLA-G targeting mAbs. Furthermore, with respect to the specificity of the mAbs employed, additional immune reactivity was detected using the mAbs MEM-G/1 and 5A6G7 in western blot analysis with K562 control cell lines overexpressing HLA-A2 or HLA-G, all tumor tissues and in two out of eight CRC cell lines. Based on the current study and our previously reported results, we conclude that claiming HLA-G plays a role in immune modulation of CRC seems premature, as results from anti-body based detection of HLA-G protein remain inconclusive. Until the time that detection of HLA-G is sensitive enough to detect all aspects of HLA-G expression in biological samples, rather than transfected cells or long time cultured cell lines, conclusions should be drawn with great care.

Expression of HLA class I antigen, aspirin use, and survival after a diagnosis of colon cancer.

IMPORTANCE: Use of aspirin (which inhibits platelet function) after a colon cancer diagnosis is associated with improved overall survival. Identifying predictive biomarkers of this effect could individualize therapy and decrease toxic effects. OBJECTIVE: To demonstrate that survival benefit associated with low-dose aspirin use after a diagnosis of colorectal cancer might depend on HLA class I antigen expression. DESIGN, SETTING, AND PARTICIPANTS: A cohort study with tumor blocks from 999 patients with colon cancer (surgically resected between 2002 and 2008), analyzed for HLA class I antigen and prostaglandin endoperoxide synthase 2 (PTGS2) expression using a tissue microarray. Mutation analysis of PIK3CA was also performed. Data on aspirin use after diagnosis were obtained from a prescription database. Parametric survival models with exponential (Poisson) distribution were used to model the survival. MAIN OUTCOMES AND MEASURES: Overall survival. RESULTS: The overall survival benefit associated with aspirin use after a diagnosis of colon cancer had an adjusted rate ratio (RR) of 0.53 (95% CI, 0.38-0.74; P < .001) when tumors expressed HLA class I antigen compared with an RR of 1.03 (0.66-1.61; P = .91) when HLA antigen expression was lost. The benefit of aspirin was similar for tumors with strong PTGS2 expression (0.68; 0.48-0.97; P = .03), weak PTGS2 expression (0.59; 0.38-0.97; P = .02), and wild-type PIK3CA tumors (0.55; 0.40-0.75; P < .001). No association was observed with mutated PIK3CA tumors (0.73; 0.33-1.63; P = .44). CONCLUSIONS AND RELEVANCE: Contrary to the original hypothesis, aspirin use after colon cancer diagnosis was associated with improved survival if tumors expressed HLA class I antigen. Increased PTGS2 expression or the presence of mutated PIK3CA did not predict benefit from aspirin. HLA class I antigen might serve as a predictive biomarker for adjuvant aspirin therapy in colon cancer.

Four-color staining combining fluorescence and brightfield microscopy for simultaneous immune cell phenotyping and localization in tumor tissue sections.

Immune-cell infiltration is frequently seen within human solid tumors. A detailed phenotypic analysis of these cells may aid in the understanding of an antitumor immune response. Standard hematoxylin/eosin and conventional immunohistochemical stainings are helpful, but have major limitations in the number of markers that can be identified and localized per tissue section. Therefore, we developed a combined fluorescence and brightfield microscopic technique by using both immunofluorescence and immunogold-silver methods, thereby discriminating three different leukocyte markers plus one tumor marker simultaneously in a single section. This enabled us to study both phenotype and location of infiltrating immune cells in colorectal tumors. We used a two-step staining in which primary and secondary antibodies were selected for minimal cross-reactivity. Furthermore, the secondary fluorescent antibody conjugates were selected for minimal spectral overlap. For computer-assisted analysis the brightfield microscopy image was combined with the fluorescence microscopy images. This combination of techniques provides a powerful tool for detailed multiparameter microscopic analysis of formalin-fixed, paraffin-embedded tissue sections in general and for tumor-immune cell infiltration in particular.