Authors:	Haist, Maximilian
Title:	Hypoxie – assoziierte Faktoren als Prädiktoren für das Ansprechen auf eine kombinierte Radio – und Immuntherapie beim zerebral metastasierten malignen Melanom
Online publication date:	27-Jul-2021
Year of first publication:	2021
Language:	german
Abstract:	Malignant melanoma is among the five most common forms of cancer in the western world and has shown the fastest increase in incidence worldwide, presumably due to lifestyle changes. The mortality rate has been reduced by improvements in the therapy of locoregional disease stages. However, at an advanced stage, melanoma still represents a significant threat to public health, requiring various treatments in an interdisciplinary approach. Melanoma is considered a highly immunogenic tumor. Hence, immunotherapy is in the focus of attention to address this problem. Immunotherapy with the anti-CTLA-4 mAb Ipilimumab (IPI) has led to profound and durable tumor regressions in some patients with melanoma metastatic to the brain but failed to reproduce this effect in others. Here, we have investigated possible mechanisms that may mediate resistance to a combination of radio- and immunotherapy in such patients. We have established predictive markers for the response of patients with metastatic melanoma to combined "radioimmunotherapy." Among these mechanisms of resistance, hypoxia has been reported to tip the scale towards immune escape by the generation of immunosuppressive adenosine (ADO) via 5'-ectonucleotidase (CD73) on malignant cells. Conversely, radiotherapy causes immunogenic cell death and may stimulate anti-tumoral immune reactions, which can be further enhanced by the application of immune checkpoint inhibitors. We reasoned that melanomas with high baseline numbers of CD8+ cytotoxic T-lymphocytes, low hypoxia, and low CD73 expression might show a better clinical outcome even in an advanced stage of the disease. We have tested this hypothesis in a cohort of 35 patients who received radiotherapy (either whole-brain radiotherapy, WBRT, or stereotactic radiotherapy, STX, or both) for brain metastases of malignant melanomas in conjunction with IPI. We have used 4-plex fluorescence immunostains to analyze the number of CD8+ cytotoxic T-lymphocytes (CTL), the tumor immune phenotype – assessed by the infiltration pattern of CD8+ CTL – the expression of the hypoxia-associated marker glucose transporter (GLUT)-1, and the ADO-generating enzyme CD73 in various archived tumor tissues of these patients. Single-cell-based analyses were carried out using the DAPI channel for the segmentation of cell nuclei in the open-source software QuPath. Of note, IPI was administered within a median time interval of one month before or after irradiation in this cohort of patients. Since the CNS is an immunoprivileged site, we carried out a separate analysis excluding three patients for whom only CNS tissue was available for study (n = 33). A detailed spatial analysis of the relative localization of CD8+ CTL and GLUT-1high tumor cells was implemented using the ImageJ/Fiji Plugin MosaicIA. Results show that CD8+ cells regularly show clustering in the stroma and infiltrate melanoma cell aggregates to varying degrees. The expression of CD73, contrary to our expectations, was also predominantly found in stromal areas. CD73 expression was substantially weaker or even absent in the majority of melanoma cells. Conversely, expression of GLUT-1 in melanoma cells was widespread, intense, and followed a pattern which reflects the diffusion properties of oxygen in the tissue. Spatial analysis in MosaicIA revealed statistically significant clustering of CD8+ and CD73+ cells in stromal areas. Moreover, we could find evidence for a hypoxic inhibition of CD8+ CTL in the spatial analysis. Here, CD8+ CTL were excluded from hypoxic, GLUT-1 positive areas in most of the tumor tissues. Besides, correlation analysis also revealed a significant negative correlation between the amount of CD8+ CTL and hypoxic tumor cells among the investigated cohort. Furthermore, we could find that CD8+ CTL numbers above the median were correlated with a significantly longer median overall survival. When solely analyzing non-CNS tissue, this correlation became even stronger. As expected, better-oxygenated tumors had better outcomes, as assessed by GLUT-1 expression dichotomized at the median. Contrary, the extent of CD73 expression was not prognostic. However, a subpopulation of patients with a CD73high/GLUT-1high pattern showed a repulsive effect on CTL accumulation in spatial analyses. As reported earlier, patients receiving IPI after radiotherapy survived significantly longer than those who received IPI before brain irradiation. In conclusion, our data indicate that an "inflamed" tumor phenotype in initial, non-CNS tissue samples is a relatively stable trait that has prognostic relevance even after the disease has later progressed to an advanced metastatic state. The sequence of radiotherapy followed by immune checkpoint inhibition may reactivate an effective anti-tumor immune response. Hypoxia is a robust prognostic factor in these patients as well but may mediate a plethora of effects in addition to the immunosuppressive activity.
DDC:	610 Medizin 610 Medical sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 04 Medizin
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-6082
URN:	urn:nbn:de:hebis:77-openscience-43cacf9f-6c31-470c-86d1-e33cef385b994
Version:	Original work
Publication type:	Dissertation
License:	CC BY
Information on rights of use:	https://creativecommons.org/licenses/by/4.0/
Extent:	175 Seiten, Illustrationen, Diagramme
Appears in collections:	JGU-Publikationen