Inka Zoernig

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. Clin Cancer Res; 17(4); 678–89. ©2011 AACR.

Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC

ABSTRACT Dendritic cell-derived exosomes (Dex) are small extracellular vesicles secreted by viable dendritic cells. In the two phase-I trials that we conducted using the first generation of Dex (IFN-γ-free) in end-stage cancer, we reported that Dex exerted natural killer (NK) cell effector functions in patients. A second generation of Dex (IFN-γ-Dex) was manufactured with the aim of boosting NK and T cell immune responses. We carried out a phase II clinical trial testing the clinical benefit of IFN-γ-Dex loaded with MHC class I- and class II-restricted cancer antigens as maintenance immunotherapy after induction chemotherapy in patients bearing inoperable non-small cell lung cancer (NSCLC) without tumor progression. The primary endpoint was to observe at least 50% of patients with progression-free survival (PFS) at 4 mo after chemotherapy cessation. Twenty-two patients received IFN-γ-Dex. One patient exhibited a grade three hepatotoxicity. The median time to progression was 2.2 mo and median overall survival (OS) was 15 mo. Seven patients (32%) experienced stabilization of >4 mo. The primary endpoint was not reached. An increase in NKp30-dependent NK cell functions were evidenced in a fraction of these NSCLC patients presenting with defective NKp30 expression. Importantly, MHC class II expression levels of the final IFN-γ-Dex product correlated with expression levels of the NKp30 ligand BAG6 on Dex, and with NKp30-dependent NK functions, the latter being associated with longer progression-free survival. This phase II trial confirmed the capacity of Dex to boost the NK cell arm of antitumor immunity in patients with advanced NSCLC.

Tumoral Immune Cell Exploitation in Colorectal Cancer Metastases Can Be Targeted Effectively by Anti-CCR5 Therapy in Cancer Patients

The immune response influences the clinical course of colorectal cancer (CRC). Analyzing the invasive margin of human CRC liver metastases, we identified a mechanism of immune cell exploitation by tumor cells. While two distinct subsets of myeloid cells induce an influx of T cells into the invasive margin via CXCL9/CXCL10, CCL5 is produced by these T cells and stimulates pro-tumoral effects via CCR5. CCR5 blockade in patient-derived functional in vitro organotypic culture models showed a macrophage repolarization with anti-tumoral effects. These anti-tumoral effects were then confirmed in a phase I trial with a CCR5 antagonist in patients with liver metastases of advanced refractory CRC. Mitigation of tumor-promoting inflammation within the tumor tissue and objective tumor responses in CRC were observed.

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 µm2) are counted directly, the number of cells contained in a conglomerate is estimated by dividing the area of the conglomerate in thin tissues sections (≤6 µm) by the median area covered by an isolated T cell which we determined as 58 µm2. 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/mm2 (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.

Advanced Malignant Melanoma: Immunologic and Multimodal Therapeutic Strategies

Immunologic treatment strategies are established in malignant melanoma treatment, mainly focusing on Interleukin-2 in advanced disease and interferon alpha in the adjuvant situation. In advanced disease, therapies with IL-2, interferon and different chemotherapeutic agents were not associated with better patient survival in the vast majority of patients. Therefore, an overview of novel immunological agents and combined therapeutic approaches is presented in this review, covering allogenic and autologous vaccine strategies, dendritic cell vaccination, strategies for adoptive immunotherapy and T cell receptor gene transfer, treatment with cytokines and monoclonal antibodies against the CTLA-4 antigen. As emerging treatment strategies are based on individual molecular and immunological characterization of individual tumors/patients, tailored targeted drug therapies move into the focus of treatment strategies. Multimodal combination therapies with considerable potential in altering the immune response in malignant melanoma patients are currently emerging. As oncology moves forward into the field of personalized therapies, a careful molecular and immunological characterization of patients is crucial to select patients for individual targeted treatment.

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.

The impact of human adipose tissue-derived stem cells on breast cancer cells: implications for cell-assisted lipotransfers in breast reconstruction

BackgroundIn this study we evaluated the interactions of human adipose tissue-derived stem cells (ADSCs) and different human breast cancer cell lines (BRCAs) with regard to the safety of cell-assisted lipotransfers for breast reconstruction and a thereby unintended co-localization of ADSCs and BRCAs.MethodsADSCs were co-cultured with five different human BRCAs (MCF-7, MDA-MB-231, SK-BR-3, ZR-75-30, and EVSA-T) and primary BRCAs from one patient in a transwell system, and cell-cell-interactions were analyzed by assessing doubling time, migration and invasion, angiogenesis, quantitative real-time polymerase chain reaction (PCR) of more than 300 tumor-associated genes, and multiplex protein assays of 20 chemokines and growth factors and eight matrix metalloproteinases (MMPs). Results of co-culture were compared to those of the respective monoculture.ResultsQuantitative real-time PCR revealed remarkable changes in the expression of multiple tumor-associated genes in co-culture compared to monocultures of both ADSCs and BRCAs. Concomitantly, the concentration of several tumor-associated proteins, such as cytokines and MMPs, were strongly increased in co-culture. Furthermore, exclusively in co-culture with ADSCs, the different BRCAs were exposed to several important tumor-modulating proteins, such as CCL2, HGF, or interleukins.Co-culture did not significantly affect cellular proliferation of either ADSCs or BRCAs (p > 0.05). The migration of MCF-7 and MDA-MB-231 BRCAs was significantly increased in co-culture with ADSCs by a mean of 11% and 23%, respectively (p = 0.04 and 0.012), as well as that of ADSCs in co-culture with MDA-MB-231, ZR-75-30, and EVSA-T (+11–15%, p = 0.035–0.045). Co-culture with MDA-MB-231, SK-BR-3, and EVSA-T BRCAs significantly increased the invasive behavior of ADSCs by a mean of 24–41% (p = 0.014–0.039). There were no significant differences in the in vitro invasive properties of BRCAs in co-culture compared to monoculture. An in vitro angiogenesis assay revealed an increased tube formation of conditioned media from co-cultured BRCAs and ADSCs compared to the respective monocultures.ConclusionThis study further elucidates the possible interactions of primary human ADSCs with human BRCAs, pointing towards a potential increased oncological risk which should not be neglected when considering a clinical use of cell-assisted lipoaspirates in breast reconstruction.

Detailed resolution analysis reveals spatial T cell heterogeneity in the invasive margin of colorectal cancer liver metastases associated with improved survival

ABSTRACT On a broader scale, T cell density and localization in colorectal cancer liver metastases have prognostic and predictive implications. As T cell distribution at higher resolutions has not been fully investigated, a detailed resolution analysis of T cell distribution was performed. Patient tissues were divided into 10 µm distance classes between the tumor border and adjacent normal liver. Thereby, distinct density patterns of T cell localization in relation to the malignant tissue could be detected. At a distance of 20 to 30 µm to the tumor, a decrease of CD3 T cells is common. Within this area, cytotoxic Granzyme B and CD8+ T cells were found to be significantly reduced as well as CD163 macrophages were increased and identified to be in close contact with T cells. Our data suggests a physical or functional border within this region. Survival analysis revealed improved overall survival in patients with high T cells numbers at the direct tumor border. Interestingly, the decreased T cells in the 20 to 30 µm region were also found to be significantly associated with improved survival. Consequently, the detailed localization of T cells, despite blockade, could be associated with improved clinical outcome. The high-resolution analysis represents new insights into relevant heterogenous T cell distributions especially related to clinical responses. As the paradoxical observation of localization-dependent prognostic relevance of T cell densities is only detectable by detailed spatial analyses, this investigation of spatial profiles at higher resolutions is suggested as a new biomarker for survival and response to therapies.