Mathias Krockenberger

Macrophage Migration Inhibitory Factor Contributes to the Immune Escape of Ovarian Cancer by Down-Regulating NKG2D

The proinflammatory cytokine macrophage migration inhibitory factor (MIF) stimulates tumor cell proliferation, migration, and metastasis; promotes tumor angiogenesis; suppresses p53-mediated apoptosis; and inhibits antitumor immunity by largely unknown mechanisms. We here describe an overexpression of MIF in ovarian cancer that correlates with malignancy and the presence of ascites. Functionally, we find that MIF may contribute to the immune escape of ovarian carcinoma by transcriptionally down-regulating NKG2D in vitro and in vivo which impairs NK cell cytotoxicity toward tumor cells. Together with the additional tumorigenic properties of MIF, this finding provides a rationale for novel small-molecule inhibitors of MIF to be used for the treatment of MIF-secreting cancers.

Ectonucleotidases CD39 and CD73 on OvCA cells are potent adenosine-generating enzymes responsible for adenosine receptor 2A-dependent suppression of T cell function and NK cell cytotoxicity

The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A2A adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (Treg) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4+ and CD8+ T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated Treg. Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4+ T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of Treg but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.

Transketolase‐like 1 expression correlates with subtypes of ovarian cancer and the presence of distant metastases

Tumorbiology of ovarian cancer remains unclear. However, it is known that ovarian tumors, especially carcinomas, show elevated expression of glucose membrane transporters for facilitated glucose uptake. It can be assumed that increased glucose uptake leads to higher glucose metabolism. The energy resources of fully malignant transformed carcinomas are mainly supplied by aerobic glycolysis, for which several pathways are known. A key role in aerobic glycolysis is described for the transketolase enzymes. Recently, a novel transketolase-like enzyme called transketolase-like 1 (TKTL1) has been described that links aerobic glycolysis to the synthesis of fatty acids via production of acetyl-CoA. In order to investigate the role of TKTL1 for the progression of ovarian carcinomas, we examined paraffin sections of normal ovarian tissues, ovarian borderline tumors, and mucinous or serous papillary ovarian adenocarcinomas with respect to their expression of TKTL1. We identified a significantly elevated expression of TKTL1 in serous papillary ovarian adenocarcinomas, which correlates with poor prognostic parameters in the examined study group. Therefore, it can be assumed that TKTL1 plays a crucial role in ovarian cancer metabolism and that its expression predicts poor prognosis. Further investigations should be performed in order to evaluate whether this new enzyme is important for ovarian cancer tumorbiology and to analyze the potential role of TKTL1 as new target for specific antitumoral therapy

Increased tartrate-resistant acid phosphatase (TRAP) expression in malignant breast, ovarian and melanoma tissue: an investigational study

BackgroundTartrate-resistant acid phosphatase (TRAP) is a metalloprotein enzyme that belongs to the acid phosphatases and is known to be expressed by osteoclasts. It has already been investigated as a marker of bone metastases in cancer patients. In this study, which examined the value of serum TRAP concentrations as a marker of bone disease in breast cancer patients, we observed high concentrations of TRAP even in patients without bone metastases. To elucidate this phenomenon, we examined the expression of TRAP in breast cancer cells and the cells of several other malignancies.MethodsTRAP concentrations in the serum of tumor patients were determined by ELISA. The expression of TRAP in breast, ovarian, and cervical cancer and malignant melanoma was analyzed by immunohistochemistry. RT-PCR and immunocytology were used to evaluate TRAP expression in cultured tumor cells.ResultsA marked increase in serum TRAP concentrations was observed in patients with breast and ovarian cancer, regardless of the presence or absence of bone disease. TRAP expression was found in breast and ovarian cancers and malignant melanoma, while cervical cancer showed only minimal expression of TRAP. Expression of TRAP was absent in benign tissue or was much less marked than in the corresponding malignant tissue. TRAP expression was also demonstrated in cultured primary cancer cells and in commercially available cell lines.ConclusionOverexpression of TRAP was detected in the cells of various different tumors. TRAP might be useful as a marker of progression of malignant disease. It could also be a potential target for future cancer therapies.

Perifosine inhibits growth of human experimental endometrial cancers by blockade of AKT phosphorylation

OBJECTIVE Perifosine is an orally active alkylphospholipid analog, which has shown anti-tumor activity in a variety of cancers by inhibition of AKT phosphorylation. The objective of the current study was to evaluate its efficacy in in vitro models of human endometrial cancer. STUDY DESIGN The effect of 10microM and 40microM perifosine on AKT phophorylation in human endometrial cancer cell lines Ishikawa and HEC 1A was determined by Western blotting. To screen for a putative anti-tumor effect, HEC 1A and Ishikawa cells were incubated with increasing concentrations of perifosine for 24h, 48h and 72h and the number of viable cells was determined by crystal violet staining. Also the effect of a combined treatment with cisplatin and perifosine was investigated in Ishikawa cells. Flow cytometric analysis of DNA content was used to determine the effect of perifosine on the cell cycle distribution of HEC 1A and Ishikawa cells and to assess potential toxic side effects of perifosine on peripheral blood lymphocytes (PBL). RESULTS AKT phosphorylation was dose-dependently inhibited by perifosine. Concomitantly, perifosine displayed anti-tumor activity in both cell lines at concentrations that showed no effect on peripheral blood lymphocytes. Growth inhibitory effects became more pronounced with increasing treatment time. While IC 50 values at 24h were >40microM, IC 50 values after 48h were approximately 7microM in Ishikawa and 25microM in HEC 1A cells. After 72h, the IC 50 was below 1.25microM for Ishikawa and about 6microM for HEC 1A cells. Perifosine cotreatment substantially increased cytotoxic effects of cisplatin in human Ishikawa endometrial cancer cells. Of note, the anti-tumor activity of perifosine was not confined to a specific phase of the cell cycle. CONCLUSIONS The small molecule AKT inhibitor perifosine showed substantial anti-tumor activity in human endometrial cancer cell lines. Since these effects were increased with cisplatin, perifosine seems to be a good candidate for treatment combinations with classical cytostatic compounds. Thus, perifosine should be further evaluated in clinical studies in endometrial cancer.

Macrophage migration inhibitory factor expression in cervical cancer.

PurposeThe glycoprotein macrophage migration inhibitory factor (MIF) is a cytokine that has been shown to promote tumor progression and tumor immune escape in ovarian cancer. The present study investigates MIF in uterine cervical cancer.MethodsEighty surgical biopsies (32 cervical dysplasias, 23 in situ carcinomas and 25 invasive carcinomas) of uterine cervical tissue were evaluated immunohistochemically for MIF expression. In uterine cervical cancer cell lines SiHa and CaSki and their respective supernatants, MIF protein expression was analyzed by Western blotting, enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR).ResultsImmunohistochemical analysis shows that MIF is clearly overexpressed on the protein level in invasive cervical cancer compared to cervical dysplasias. MIF overexpression was confirmed by RT-PCR in surgical biopsies of invasive cervical cancer. Western blotting reveals that the MIF protein is overexpressed in SiHA und CaSki cervical cancer cell lines, whereas the ELISA reveals that cervical cancer cells secrete MIF.ConclusionsMIF has been shown to promote tumor immune escape mechanisms in other cancer entities, which makes it an interesting target for cancer therapy, given the known significance of immune mechanisms for uterine cervical cancer. The overexpression of MIF on the protein and mRNA level, as well as its secretion by cervical cancer cells points to a critical role of the protein for the pathogenesis of uterine cervical cancer.

Effects of lobaplatin as a single agent and in combination with TRAIL on the growth of triple-negative p53-mutated breast cancers in vitro.

Lobaplatin as a single agent and in combination with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is investigated in in-vitro models of p53-negative triple-negative breast cancers (TNBCs) and compared with a model of oestrogen receptor-positive p53-positive breast cancer. In addition, the induction of programmed cell death by lobaplatin is further explored. By using cell viability assays and western blotting, the cytotoxic effects of lobaplatin alone and in combination with TRAIL are compared with cisplatin in HCC 1806, HCC 1937, and MCF 7 cells. The multicaspase inhibitor z-VAD-fmk and necrostatin, an inhibitor of necroptosis, are used to demonstrate the mechanism of cell death caused by lobaplatin. Lobaplatin displayed antitumour activity in all three cell lines, which increased time dependently. Cotreatment of lobaplatin and TRAIL induced an increase in cytotoxicity by 30–50% in the different cell lines. The pan-caspase inhibitor z-VAD-fmk as well as necrostatin could weaken but not abolish the cytotoxic effect of lobaplatin and cisplatin. Lobaplatin showed substantial cytotoxic effects in two in-vitro models of p53-mutated TNBC. Cotreatment with TRAIL and platinum agents resulted in increased antitumour activity in the TNBC cell lines investigated. Cell death subsequent to treatment with cisplatin and lobaplatin occurred because of apoptosis. However, caspase-independent mechanisms of programmed cell death were also involved. It was also demonstrated that platinum compounds could induce necroptosis, although to a minor extent.

Prevalence of deleterious germline variants in risk genes including BRCA1/2 in consecutive ovarian cancer patients (AGO-TR-1)

Background Identification of families at risk for ovarian cancer offers the opportunity to consider prophylactic surgery thus reducing ovarian cancer mortality. So far, identification of potentially affected families in Germany was solely performed via family history and numbers of affected family members with breast or ovarian cancer. However, neither the prevalence of deleterious variants in BRCA1/2 in ovarian cancer in Germany nor the reliability of family history as trigger for genetic counselling has ever been evaluated. Methods Prospective counseling and germline testing of consecutive patients with primary diagnosis or with platinum-sensitive relapse of an invasive epithelial ovarian cancer. Testing included 25 candidate and established risk genes. Among these 25 genes, 16 genes (ATM, BRCA1, BRCA2, CDH1, CHEK2, MLH1, MSH2, MSH6, NBN, PMS2, PTEN, PALB2, RAD51C, RAD51D, STK11, TP53) were defined as established cancer risk genes. A positive family history was defined as at least one relative with breast cancer or ovarian cancer or breast cancer in personal history. Results In total, we analyzed 523 patients: 281 patients with primary diagnosis of ovarian cancer and 242 patients with relapsed disease. Median age at primary diagnosis was 58 years (range 16–93) and 406 patients (77.6%) had a high-grade serous ovarian cancer. In total, 27.9% of the patients showed at least one deleterious variant in all 25 investigated genes and 26.4% in the defined 16 risk genes. Deleterious variants were most prevalent in the BRCA1 (15.5%), BRCA2 (5.5%), RAD51C (2.5%) and PALB2 (1.1%) genes. The prevalence of deleterious variants did not differ significantly between patients at primary diagnosis and relapse. The prevalence of deleterious variants in BRCA1/2 (and in all 16 risk genes) in patients <60 years was 30.2% (33.2%) versus 10.6% (18.9%) in patients ≥60 years. Family history was positive in 43% of all patients. Patients with a positive family history had a prevalence of deleterious variants of 31.6% (36.0%) versus 11.4% (17.6%) and histologic subtype of high grade serous ovarian cancer versus other showed a prevalence of deleterious variants of 23.2% (29.1%) and 10.2% (14.8%), respectively. Testing only for BRCA1/2 would miss in our series more than 5% of the patients with a deleterious variant in established risk genes. Conclusions 26.4% of all patients harbor at least one deleterious variant in established risk genes. The threshold of 10% mutation rate which is accepted for reimbursement by health care providers in Germany was observed in all subgroups analyzed and neither age at primary diagnosis nor histo-type or family history sufficiently enough could identify a subgroup not eligible for genetic counselling and testing. Genetic testing should therefore be offered to every patient with invasive epithelial ovarian cancer and limiting testing to BRCA1/2 seems to be not sufficient.

CD33+/HLA-DRneg and CD33+/HLA-DR+/− Cells: Rare Populations in the Human Decidua with Characteristics of MDSC

Human pregnancy needs a remarkable local immune tolerance toward the conceptus. Myeloid‐derived suppressor cells (MDSC) are important players promoting cancer initiation and progression by suppressing T‐cell functions and thus inducing immune tolerance. Therefore, MDSC were expected within decidua.

The impact of breast cancer biological subtyping on tumor size assessment by ultrasound and mammography - a retrospective multicenter cohort study of 6543 primary breast cancer patients

BackgroundMammography and ultrasound are the gold standard imaging techniques for preoperative assessment and for monitoring the efficacy of neoadjuvant chemotherapy in breast cancer. Maximum accuracy in predicting pathological tumor size non-invasively is critical for individualized therapy and surgical planning. We therefore aimed to assess the accuracy of tumor size measurement by ultrasound and mammography in a multicentered health services research study.MethodsWe retrospectively analyzed data from 6543 patients with unifocal, unilateral primary breast cancer. The maximum tumor diameter was measured by ultrasound and/or mammographic imaging. All measurements were compared to final tumor diameter determined by postoperative histopathological examination. We compared the precision of each imaging method across different patient subgroups as well as the method-specific accuracy in each patient subgroup.ResultsOverall, the correlation with histology was 0.61 for mammography and 0.60 for ultrasound. Both correlations were higher in pT2 cancers than in pT1 and pT3. Ultrasound as well as mammography revealed a significantly higher correlation with histology in invasive ductal compared to lobular cancers (p < 0.01). For invasive lobular cancers, the mammography showed better correlation with histology than ultrasound (p = 0.01), whereas there was no such advantage for invasive ductal cancers. Ultrasound was significantly superior for HR negative cancers (p < 0.001). HER2/neu positive cancers were also more precisely assessed by ultrasound (p < 0.001). The size of HER2/neu negative cancers could be more accurately predicted by mammography (p < 0.001).ConclusionThis multicentered health services research approach demonstrates that predicting tumor size by mammography and ultrasound provides accurate results. Biological tumor features do, however, affect the diagnostic precision.