Hepatic metastases of colorectal cancer are rather homogeneous but differ from primary lesions in terms of immune cell infiltration.

The immune system plays an important role in shaping the clinical course of colorectal cancer (CRC). However, it is still unclear how the immune infiltrates of primary CRC lesions and distant metastases by immune effector cells are related to each other. To address this issue, we quantified CD3+, CD8+ and granzyme B+ lymphocytes in primary CRC samples and corresponding liver metastases. This analysis showed that the prognostic predictions that can be drawn from the infiltration of immune cells in primary CRCs and their metastases are heterogeneous. To investigate whether such heterogeneity would also be observed within CRC hepatic metastases, the density of the immune infiltrate and cytokine production were assessed in opposite sides of the same metastatic lesion. In addition, tumor-infiltrating lymphocytes were assessed in sequential sections of the same metastatic lesion, with a spacing of 30 µm. In summary, consistent cell counts and cytokine levels were detected within the same lesion. The study of a case of synchronous metastases, however, suggested that different metastatic lesions within the same patient may be heterogeneous, perhaps indicating a major impact for local causes on tumor infiltration by immune cells. In summary, our study demonstrates a consistent degree of heterogeneity between primary tumors and hepatic metastases but an excellent intra-lesional homogeneity. These findings may be of key importance for patient stratification and the development of personalized strategies against CRC.

Sequential metastases of colorectal cancer: Immunophenotypes and spatial distributions of infiltrating immune cells in relation to time and treatments.

The role of the immune system in the course of colorectal cancer has been elucidated in the last decade. While quantification of immune cell infiltrates within the resected specimen at diagnosis has a clear power to estimate the prognosis of the patient, the role of infiltrating immune cells within the metastatic situation and especially within the metastatic lesion itself requires further detailed analyses. Recent analyses of infiltrates in colorectal cancer liver metastases revealed a role for the infiltrate density not only for prognosis but also in the prediction of treatment response. This not only broadens the view on these infiltrates and indicates a systematic role of the local immunological microenvironment, but also raises the question how these infiltrates change during repeated courses of treatment (i.e., resection, chemotherapy, etc.). To address this question, sequential lung or sequential liver metastases of colorectal cancer patients were analyzed using whole slide image quantification after immunohistochemical staining against CD3, CD8, FOXP3, CD68 and Granzyme B. The clinical data and interventions were associated with each individual patient and the metastatic lesions. The resulting cell densities reveal a heterogeneous profile: after successful treatment of a metastatic lesion, the recurrent lesion can still have the same immunophenotype with similar cell distributions. In a situation of a favorable immune cell profile, this profile can return and apparently convey a similar favorable course throughout the disease. But also the opposite was found: the recurrent metastatic lesion could have a different profile with alterations in specific immune cell subsets over time. Further analyses are required to elucidate the different patterns and their associations to the treatment, the tumor cell phenotype and other dynamic factors. However, it is clear from this data however, that there is an immune cell plasticity that needs to be analyzed for individual patients.

Localization and density of immune cells in the invasive margin of human colorectal cancer liver metastases are prognostic for response to chemotherapy.

Analysis of tumor-infiltrating lymphocytes (TIL) in primary human colorectal cancer (CRC) by in situ immunohistochemical staining supports the hypothesis that the adaptive immune response influences the course of human CRC. Specifically, high densities of TILs in the primary tumor are associated with good prognosis independent of other prognostic markers. However, the prognostic role of TILs in metastatic CRC lesions is unknown, as is their role in response or resistance to conventional chemotherapy. We analyzed the association of TIL densities at the invasive margin of CRC liver metastases with response to chemotherapy and progression-free survival in a set of 101 large section samples. High-resolution automated microscopy on complete tissue sections was used to objectively generate cell densities for CD3, CD8, granzyme B, or FOXP3 positive immune cells. A predictive scoring system using TIL densities was developed in a training set and tested successfully in an independent validation set. TIL densities at the invasive margin of liver metastases allowed the prediction of response to chemotherapy with a sensitivity of 79% and specificity of 100%. The association of high density values with longer progression-free survival under chemotherapy was statistically significant. Overall, these findings extend the impact of the local immune response on the clinical course from the primary tumor also to metastatic lesions. Because detailed quantification of TILs in metastatic lesions revealed a strong association with chemotherapy efficacy and prognosis, we suggest that the developed scoring system may be used as a predictive tool for response to chemotherapy in metastatic CRC.

Natural killer cells are scarce in colorectal carcinoma tissue despite high levels of chemokines and cytokines.

PURPOSE: Tumor infiltrating T lymphocytes in colorectal cancer (CRC) have prognostic impact, but the role of natural killer (NK) cells in CRC tissue is unclear. The contribution of intratumoral cytokines and chemokines in shaping the composition of the inflammatory lymphocytic infiltrate is also unclear. EXPERIMENTAL DESIGN: In this study, localization and densities of NK and T cells within primary CRC, CRC liver metastases, adenomas, and normal tissues were analyzed on whole tissue sections from 112 patients. In a subset of these patients, the most important 50 cytokines and chemokines were quantified in paired serum, primary CRC and adjacent mucosa samples and in CRC liver metastases and correlated with NK and T-cell infiltration, respectively. RESULTS: The various compartments displayed characteristic differences like significantly higher chemokine concentrations in CRC tissue. Most importantly, despite high local chemokine levels, NK cells were generally scarce within CRC tumor tissues, independent of human leukocyte antigen (HLA) class I expression. Adjacent normal mucosa contained normal levels of NK cells. In contrast, corresponding T-cell numbers varied substantially and were positively correlated with higher chemokine levels. CONCLUSIONS: Our findings indicate a distinct regulation of NK cells versus T cells in the CRC tumor microenvironment. NK-cell migration into CRC tumor tissue is obviously impaired early during tumor development by mechanisms that do not affect T-cell infiltration.

Quantification of prognostic immune cell markers in colorectal cancer using whole slide imaging tumor maps.

OBJECTIVE: To analyze intratumoral heterogeneity of immune cells and the resulting impact of heterogeneity on the level of individual patient prediction. STUDY DESIGN: Using whole slide imaging by virtual microscopy, we present the first spatial quantitative study of immune cells in a set of colorectal cancer primary tumors. We generated "tumor maps" based on cell densities in fields of 1 mm2, visualizing intratumoral heterogeneity. In this example, cutoffs of marker-based cell stains identified by tissue microarray (TMA) led to ambiguous decisions in 11 of the 20 patients studied. Classic TMA analysis can be used in large patient cohorts to generate clinically significant predictors. The transfer of these predictors from large-scale TMA to individualized predictions thus far has not been investigated. In colorectal cancer, TMA-based quantitative immune cell counts using immune cell surface molecules (CD3, CD8, Granzyme B, and CD45RO) have been shown to be potentially better predictors for patient survival than the classical TNM system. RESULTS: Our results make clear that for individualized prognostic evaluations, whole slide imaging by virtual microscopy is irreplaceable during identification of prognostic markers as well as in their subsequent application. CONCLUSION: In the future, spatial marker signatures could contribute to individual patient classifiers.

Estimation of immune cell densities in immune cell conglomerates: an approach for high-throughput quantification.

BACKGROUND: Determining the correct number of positive immune cells in immunohistological sections of colorectal cancer and other tumor entities is emerging as an important clinical predictor and therapy selector for an individual patient. This task is usually obstructed by cell conglomerates of various sizes. We here show that at least in colorectal cancer the inclusion of immune cell conglomerates is indispensable for estimating reliable patient cell counts. Integrating virtual microscopy and image processing principally allows the high-throughput evaluation of complete tissue slides. METHODOLOGY/PRINCIPAL FINDINGS: For such large-scale systems we demonstrate a robust quantitative image processing algorithm for the reproducible quantification of cell conglomerates on CD3 positive T cells in colorectal cancer. While isolated cells (28 to 80 microm(2)) are counted directly, the number of cells contained in a conglomerate is estimated by dividing the area of the conglomerate in thin tissues sections (< or =6 microm) by the median area covered by an isolated T cell which we determined as 58 microm(2). We applied our algorithm to large numbers of CD3 positive T cell conglomerates and compared the results to cell counts obtained manually by two independent observers. While especially for high cell counts, the manual counting showed a deviation of up to 400 cells/mm(2) (41% variation), algorithm-determined T cell numbers generally lay in between the manually observed cell numbers but with perfect reproducibility. CONCLUSION: In summary, we recommend our approach as an objective and robust strategy for quantifying immune cell densities in immunohistological sections which can be directly implemented into automated full slide image processing systems.