Immunosuppressive plasma cells impede T-cell-dependent immunogenic chemotherapy.

Cancer-associated genetic alterations induce expression of tumour antigens that can activate CD8(+) cytotoxic T cells (CTLs), but the microenvironment of established tumours promotes immune tolerance through poorly understood mechanisms. Recently developed therapeutics that overcome tolerogenic mechanisms activate tumour-directed CTLs and are effective in some human cancers. Immune mechanisms also affect treatment outcome, and certain chemotherapeutic drugs stimulate cancer-specific immune responses by inducing immunogenic cell death and other effector mechanisms. Our previous studies revealed that B cells recruited by the chemokine CXCL13 into prostate cancer tumours promote the progression of castrate-resistant prostate cancer by producing lymphotoxin, which activates an IκB kinase α (IKKα)-BMI1 module in prostate cancer stem cells. Because castrate-resistant prostate cancer is refractory to most therapies, we examined B cell involvement in the acquisition of chemotherapy resistance. Here we focus on oxaliplatin, an immunogenic chemotherapeutic agent that is effective in aggressive prostate cancer. We show that mouse B cells modulate the response to low-dose oxaliplatin, which promotes tumour-directed CTL activation by inducing immunogenic cell death. Three different mouse prostate cancer models were refractory to oxaliplatin unless genetically or pharmacologically depleted of B cells. The crucial immunosuppressive B cells are plasmocytes that express IgA, interleukin (IL)-10 and programmed death ligand 1 (PD-L1), the appearance of which depends on TGFß receptor signalling. Elimination of these cells, which also infiltrate human-therapy-resistant prostate cancer, allows CTL-dependent eradication of oxaliplatin-treated tumours.

Analysis of the inflammatory tumor microenvironment in meningeal neoplasms.

BACKGROUND: Properties of the inflammatory tumor microenvironment are associated with disease subtype, grade, and prognosis in various cancer entities. As immune-modulatory therapies are currently being explored in patients with meningeal neoplasms, we investigated their inflammatory microenvironment (meningiomas and solitary fibrous tumor/hemangiopericytoma (SFT/HPC)). MATERIALS AND METHODS: 74 meningeal tumor specimens: (10/74 (13.5%) atypical meningioma; 8/74 (10.8%) anaplastic meningioma; 8/74 (10.8%) chordoid meningioma; 9/74 (12.2%) fibroblastic meningioma; 10/74 (13.5%) transitional meningioma; 3/74 (4.1%) rhabdoid meningioma; 7/74 (9.5%) meningothelial meningioma; SFT/HPC (19/74 (25.7%) were retrieved from the Neuro-Biobank, Medical University of Vienna, Austria. RESULTS: Tumor-infiltrating lymphocyte (TIL) infiltration could be observed in the majority of the investigated specimens (CD3+: 66/74 (89.2%); CD8+: 47/74 (63.5%); CD45RO+: 29/73 (39.2%); FOXP3+ 19/74 (25.7%); PD1+: 3/74 (4.1%). No difference in TIL infiltration was observed between SFT/HPC and meningioma cases. Higher density of FOXP3+ TILs was observed with increasing WHO grade in meningioma specimens (p = 0.005). Membranous programmed cell death ligand 1 (PD-L1) expression was observed in 4/74 (5.4%) specimens, with 3/74 (4.1%) presenting with 1% and 1/74 (1.4%) with 3% PD-L1 expressing tumor cells. Lymphatic vessels as identified by podoplanin immunohistochemistry were observed in 10/74 (13.5%) specimens and were significantly associated with presence of membranous PD-L1 expression on tumor cells (p = 0.003). CONCLUSION: Infiltration by various TIL subtypes can be observed in the majority of meningeal neoplasms, with enrichment of FOXP3-positive regulatory T-cells in higher-grade meningioma. PD-L1 expression on tumor cells was only infrequently found. A better understanding of the pathobiological role of the immune system in meningeal neoplasms may facilitate development of immunomodulatory treatment approaches in meningeal tumors.

Podoplanin expression in primary brain tumors induces platelet aggregation and increases risk of venous thromboembolism.

Venous thromboembolism (VTE) is common in patients with brain tumors, and underlying mechanisms are unclear. We hypothesized that podoplanin, a sialomucin-like glycoprotein, increases the risk of VTE in primary brain tumors via its ability to induce platelet aggregation. Immunohistochemical staining against podoplanin and intratumoral platelet aggregates was performed in brain tumor specimens of 213 patients (mostly high-grade gliomas [89%]) included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study of patients with newly diagnosed cancer or progressive disease aimed at identifying patients at risk of VTE. Platelet aggregation in response to primary human glioblastoma cells was investigated in vitro. During 2-year follow-up, 29 (13.6%) patients developed VTE. One-hundred fifty-one tumor specimens stained positive for podoplanin (33 high expression, 47 medium expression, 71 low expression). Patients with podoplanin-positive tumors had lower peripheral blood platelet counts (P < .001) and higher D-dimer levels (P < .001). Podoplanin staining intensity was associated with increasing levels of intravascular platelet aggregates in tumor specimens (P < .001). High podoplanin expression was associated with an increased risk of VTE (hazard ratio for high vs no podoplanin expression: 5.71; 95% confidence interval, 1.52-21.26; P =010), independent of age, sex, and tumor type. Podoplanin-positive primary glioblastoma cells induced aggregation of human platelets in vitro, which could be abrogated by an antipodoplanin antibody. In conclusion, high podoplanin expression in primary brain tumors induces platelet aggregation, correlates with hypercoagulability, and is associated with increased risk of VTE. Our data indicate novel insights into the pathogenesis of VTE in primary brain tumors.

Survival prediction using temporal muscle thickness measurements on cranial magnetic resonance images in patients with newly diagnosed brain metastases.

OBJECTIVES: To evaluate the prognostic relevance of temporal muscle thickness (TMT) in brain metastasis patients. METHODS: We retrospectively analysed TMT on magnetic resonance (MR) images at diagnosis of brain metastasis in two independent cohorts of 188 breast cancer (BC) and 247 non-small cell lung cancer (NSCLC) patients (overall: 435 patients). RESULTS: Survival analysis using a Cox regression model showed a reduced risk of death by 19% with every additional millimetre of baseline TMT in the BC cohort and by 24% in the NSCLC cohort. Multivariate analysis included TMT and diagnosis-specific graded prognostic assessment (DS-GPA) as covariates in the BC cohort (TMT: HR 0.791/CI [0.703-0.889]/p < 0.001; DS-GPA: HR 1.433/CI [1.160-1.771]/p = 0.001), and TMT, gender and DS-GPA in the NSCLC cohort (TMT: HR 0.710/CI [0.646-0.780]/p < 0.001; gender: HR 0.516/CI [0.387-0.687]/p < 0.001; DS-GPA: HR 1.205/CI [1.018-1.426]/p = 0.030). CONCLUSION: TMT is easily and reproducibly assessable on routine MR images and is an independent predictor of survival in patients with newly diagnosed brain metastasis from BC and NSCLC. TMT may help to better define frail patient populations and thus facilitate patient selection for therapeutic measures or clinical trials. Further prospective studies are needed to correlate TMT with other clinical frailty parameters of patients. KEY POINTS: • TMT has an independent prognostic relevance in brain metastasis patients. • It is an easily and reproducibly parameter assessable on routine cranial MRI. • This parameter may aid in patient selection and stratification in clinical trials. • TMT may serve as surrogate marker for sarcopenia.

AID/APOBEC-network reconstruction identifies pathways associated with survival in ovarian cancer.

BACKGROUND: Building up of pathway-/disease-relevant signatures provides a persuasive tool for understanding the functional relevance of gene alterations and gene network associations in multifactorial human diseases. Ovarian cancer is a highly complex heterogeneous malignancy in respect of tumor anatomy, tumor microenvironment including pro-/antitumor immunity and inflammation; still, it is generally treated as single disease. Thus, further approaches to investigate novel aspects of ovarian cancer pathogenesis aiming to provide a personalized strategy to clinical decision making are of high priority. Herein we assessed the contribution of the AID/APOBEC family and their associated genes given the remarkable ability of AID and APOBECs to edit DNA/RNA, and as such, providing tools for genetic and epigenetic alterations potentially leading to reprogramming of tumor cells, stroma and immune cells. RESULTS: We structured the study by three consecutive analytical modules, which include the multigene-based expression profiling in a cohort of patients with primary serous ovarian cancer using a self-created AID/APOBEC-associated gene signature, building up of multivariable survival models with high predictive accuracy and nomination of top-ranked candidate/target genes according to their prognostic impact, and systems biology-based reconstruction of the AID/APOBEC-driven disease-relevant mechanisms using transcriptomics data from ovarian cancer samples. We demonstrated that inclusion of the AID/APOBEC signature-based variables significantly improves the clinicopathological variables-based survival prognostication allowing significant patient stratification. Furthermore, several of the profiling-derived variables such as ID3, PTPRC/CD45, AID, APOBEC3G, and ID2 exceed the prognostic impact of some clinicopathological variables. We next extended the signature-/modeling-based knowledge by extracting top genes co-regulated with target molecules in ovarian cancer tissues and dissected potential networks/pathways/regulators contributing to pathomechanisms. We thereby revealed that the AID/APOBEC-related network in ovarian cancer is particularly associated with remodeling/fibrotic pathways, altered immune response, and autoimmune disorders with inflammatory background. CONCLUSIONS: The herein study is, to our knowledge, the first one linking expression of entire AID/APOBECs and interacting genes with clinical outcome with respect to survival of cancer patients. Overall, data propose a novel AID/APOBEC-derived survival model for patient risk assessment and reconstitute mapping to molecular pathways. The established study algorithm can be applied further for any biologically relevant signature and any type of diseased tissue.

Interleukin-6 receptor alpha blockade improves skin lesions in a murine model of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease, characterized by antinuclear autoantibodies (ANA) and immunocomplexes, commonly affecting kidneys, skin, heart, lung or even the brain. We have shown that JunB(Δep) mice develop a SLE phenotype linked to increased epidermal Interleukin (IL)-6 secretion. Blocking of IL-6 receptor alpha (IL-6Rα) is considered as therapeutic strategy for the treatment of SLE. JunB(Δep) and wild-type mice were treated for short (5 weeks) or long term (21 weeks) with the IL-6Rα-blocking antibody MR16-1. Skin and kidney of mice were investigated by histology and immunofluorescence, and in addition, kidneys were analysed by electron microscopy. Furthermore, soluble IL-6R (sIL-6R), antihistone and antinucleosome antibodies levels were measured and associated with disease parameters. Treatment with MR16-1 resulted in significant improvement of SLE-like skin lesions in JunB(Δep) mice, compared to untreated mice. The sIL-6R amount upon long-term treatment with MR16-1 was significantly higher in JunB(Δep) versus untreated JunB(Δep) (P = 0.034) or wild-type mice (P = 0.034). MR16-1 treatment over these time spans did not significantly improve kidney pathology of immunoglobulin deposits causing impaired function. Significantly higher antihistone (P = 0.028) and antinucleosome antibody levels (P = 0.028) were measured in MR16-1-treated JunB(Δep) mice after treatment compared to levels before therapy. In conclusion, blockade of IL-6Rα improves skin lesions in a murine SLE model, but does not have a beneficial effect on autoimmune-mediated kidney pathology. Inhibition of IL-6R signalling might be helpful in lupus cases with predominant skin involvement, but combinatorial treatment might be required to restrain autoantibodies.

Enhanced expression of the stemness-related factors OCT4, SOX15 and TWIST1 in ectopic endometrium of endometriosis patients.

BACKGROUND: Current evidence suggests that endometrial-derived stem cells, spilled in the peritoneal cavity via retrograde menstruation, are key players in the establishment of endometriotic lesions. The aim of this study was to determine the presence and distribution of the stemness-related factors OCT4, SOX15, TWIST1 and DCAMLK1 in women with and without endometriosis. METHODS: Immunohistochemical analysis was used to determine stromal and epithelial expression of OCT4, SOX15, TWIST1 and DCAMLK1 in endometriosis patient (EP) endometrium (n = 69) and endometriotic tissue (n = 90) and in control endometrium (n = 50). Quantitative Real-Time PCR of OCT4, SOX15 TWIST1 and DCAMLK1 was performed in paired samples of EP endometrium and endometriotic tissue. Co-immunofluorescence staining was performed for OCT4 and SOX15. For statistical analyses we used unpaired t-test, Fisher combination test and Spearman test. For paired analyses, paired t-test and McNemar test were used. RESULTS: We detected a significant correlation between the expression of the established stem cell marker OCT4 and the stemness-related markers SOX15 (p < 0.001) and TWIST1 (p = 0.002) but not DCAMLK1. We showed a colocalization of SOX15 and OCT4 in epithelial and stromal cells of endometriotic tissue by coimmunofluorescence. A concordant expression of OCT4 and SOX15 in the same sample was observed in epithelial cells of the endometriotic tissue (71.7%). The expression of stemness-related factors was not associated with proliferative or secretory phase of the menstrual cycle in endometriosis patients but was found to be differentially expressed during the menstrual cycle in the control group. Increased expression of epithelial OCT4, SOX15 and TWIST1 was detected in endometriotic tissue compared to EP endometrium in paired (p = 0.021, p < 0.001 and p < 0.001) and unpaired analysis (p = 0.040, p < 0.001 and p = 0.001). CONCLUSION: Our findings support the hypothesis that upregulation of stem cell-related factors contribute to the establishment of endometriotic lesions. TRIAL REGISTRATION: The study was approved by the institutional review board (545/2010 on 6th of May 2014) of the Medical University of Vienna ( http://ethikkommission.meduniwien.ac.at/fileadmin/ethik/media/dokumente/register/alle_2010.pdf ).

Tumor infiltrating lymphocytes and PD-L1 expression in brain metastases of small cell lung cancer (SCLC).

Brain metastases (BM) are frequent in small cell lung cancer (SCLC). Novel insights into their pathobiology are needed for development of better therapies. We investigated tumor-infiltrating lymphocyte (TIL) subsets (CD3+, CD8+, CD45RO+, FOXP3+ and PD-1+) and expression of PD-L1 in a series of 32 SCLC BM specimens and four matched primary tumor specimens using immunohistochemistry. 30/32 (93.8 %) BM specimens showed TIL infiltration. CD3+ TILs were observed in 30/32 (93.8 %) BM specimens, CD8+ TILs in 25/32 (78.1 %), CD45RO+ TILs in 15/32 (46.9 %), FOXP3+ TILs in 15/32 (46.9 %) and PD-1+ TILs in 1/32 (3.1 %) BM specimens. Patients with infiltration of CD45RO+ TILS had a significantly longer median survival time (11 months; 95 % CI 0.000-26.148) as compared to patients without the presence of CD45RO+ TILs (5 months; 95 % CI 0.966-9.034; p = 0.007; log rank test). Membranous PD-L1 on tumor cells was observed in 24/32 (75.0 %) BM specimens, with 11/32 (34.4 %) cases showing PD-L1 expression in over 5 % of viable BM tumor cells. PD-L1 expression on TILs was seen in 8/32 (25.0 %) and on tumor infiltrating macrophages in 9/32 (28.1 %) cases. Patients with PD-L1 expression on TILs presented with improved survival prognosis (6 versus 29 months; p = 0.002; log rank test). Among matched primary tumors, all (4/4; 100 %) specimens showed TIL infiltration, while PD-L1 expression found in only 1/4 (25.0 %) specimen. TIL infiltration and PD-L1 expression are commonly found in SCLC BM and presence of CD45RO+ memory T-cells and PD-L1+ TILs in SCLC BM seem to associate with favorable survival times. Our data suggest an active immune microenvironment in SCLC BM that may be targetable by immune-modulating drugs.

Mononuclear cell secretome protects from experimental autoimmune myocarditis.

AIMS: Supernatants of serum-free cultured mononuclear cells (MNC) contain a mix of immunomodulating factors (secretome), which have been shown to attenuate detrimental inflammatory responses following myocardial ischaemia. Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4+ T cell-dependent model, which mirrors important pathogenic aspects of iDCM. The aim of this study was to determine the influence of MNC secretome on myocardial inflammation in the EAM model. METHODS AND RESULTS: BALB/c mice were immunized twice with an alpha myosin heavy chain peptide together with Complete Freund adjuvant. Supernatants from mouse mononuclear cells were collected, dialysed, and injected i.p. at Day 0, Day 7, or Day 14, respectively. Myocarditis severity, T cell responses, and autoantibody formation were assessed at Day 21. The impact of MNC secretome on CD4+ T cell function and viability was evaluated using in vitro proliferation and cell viability assays. A single high-dose application of MNC secretome, injected at Day 14 after the first immunization, effectively attenuated myocardial inflammation. Mechanistically, MNC secretome induced caspase-8-dependent apoptosis in autoreactive CD4+ T cells. CONCLUSION: MNC secretome abrogated myocardial inflammation in a CD4+ T cell-dependent animal model of autoimmune myocarditis. This anti-inflammatory effect of MNC secretome suggests a novel and simple potential treatment concept for inflammatory heart diseases.

Evidence for serotonin as a relevant inducer of liver regeneration after liver resection in humans.

UNLABELLED: Liver regeneration (LR) involves a complex interplay of growth factors and antagonists. In this context, platelet-derived serotonin (5-HT) has been identified as a critical inducer of LR in mice. Clinical evidence for a role of 5-HT in LR in humans is lacking. Accordingly, serum and plasma 5-HT was monitored perioperatively in 60 patients undergoing liver resection, of which 35 served as exploration and 25 as validation sets. Intraplatelet (IP) levels of 5-HT were calculated by subtraction of plasma 5-HT from serum values. Serum markers of liver function were used to evaluate LR and liver dysfunction (LD). In the exploration setting, IP 5-HT levels significantly decreased after liver resection (P < 0.001) and gradually recovered during the first week. IP 5-HT measured before surgery specifically predicted LD in the subsequent 7 days (area under the curve: 0.721; P = 0.029). Patients suffering from postoperative LD and morbidity were found to have reduced IP 5-HT levels during the entire perioperative period. Furthermore, we validated that reduced preoperative IP 5-HT (<73 ng/mL) was associated with an increased incidence of postoperative LD and morbidity (P = 0.045 and P = 0.021) and were able to demonstrate that IP 5-HT levels were an independent predictor of poor clinical outcome. CONCLUSIONS: These findings provide evidence that IP 5-HT correlates with LR in humans: Patients with low IP 5-HT before liver resection suffered from delayed hepatic regeneration. Therefore, IP 5-HT levels may prove a helpful clinical marker to predict postoperative LD and clinical outcome before hepatic resection and initiate suitable interventions.

Differential role of angiogenesis and tumour cell proliferation in brain metastases according to primary tumour type: analysis of 639 cases.

AIM: We aimed to characterize angiogenesis and proliferation and their correlation with clinical characteristics in a large brain metastasis (BM) series. METHODS: Ki67 proliferation index, microvascular density (MVD) and hypoxia-inducible factor 1 alpha (HIF-1 alpha) index were determined by immunohistochemistry in BM and primary tumour specimens. RESULTS: Six hundred thirty-nine BM specimens of 639 patients with lung cancer (344/639; 53.8%), breast cancer (105/639; 16.4%), melanoma (67/639; 10.5%), renal cell carcinoma (RCC; 52/639; 8.1%) or colorectal cancer (CRC; 71/639; 11.1%) were available. Specimens of the corresponding primary tumour were available in 113/639 (17.7%) cases. Median Ki67 index was highest in CRC BM and lowest in RCC BM (P < 0.001). MVD and HIF-1 alpha index were both highest in RCC BM and lowest in melanoma BM (P < 0.001). Significantly higher Ki67 indices, MVD and HIF-1 alpha indices in the BM than in matched primary tumours were observed for breast cancer, non-small cell lung cancer (NSCLC) and CRC. Correlation of tissue-based parameters with overall survival in individual tumour types showed a favourable and independent prognostic impact of low Ki67 index [hazard ratio (HR) 1.015; P < 0.001] in NSCLC BM and of low Ki67 index (HR 1.027; P = 0.008) and high angiogenic activity (HR 1.877; P = 0.002) in RCC. CONCLUSION: Our data argue for differential pathobiological and clinical relevance of Ki67 index, HIF1-alpha index and MVD between primary tumour types in BM patients. An independent prognostic impact of tissue-based characteristics was observed in patients with BM from NSCLC and RCC, supporting the incorporation of these tissue-based parameters into diagnosis-specific prognostic scores.

Evaluation of tyrosine kinase receptors in brain metastases of clear cell renal cell carcinoma reveals cMet as a negative prognostic factor.

AIMS: Brain metastases (BMs) of clear cell renal cell carcinoma (ccRCC) are associated with a dismal prognosis, with limited treatment options. Tyrosine kinases are relevant 'druggable' biomarkers. The aim of this study was to evaluate the tyrosine kinase receptors anaplastic lymphoma kinase (ALK), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor-α (PDGFRA) and cMet in a large series of ccRCC BMs. METHODS AND RESULTS: ALK, EGFR, PDGFRA and cMet protein expression was determined by immunohistochemistry in 53 ccRCCs BMs and 12 matched primary tumours. ALK and MET gene status and copy number alterations of chromosome 7 were studied with fluorescence in-situ hybridization (FISH). Data on the expression of hypoxia-inducible factor 1α (HIF1α) and Ki67 and microvessel density were available from previous studies. ALK was negative in all analysed specimens. EGFR was overexpressed in 41 of 51 (80.4%) BMs and in seven of eight primary tumours, PDGFRA was overexpressed in all BMs except one and in all primary tumours, and cMet was expressed in 26 of 50 (52%) BMs and in two of seven primary tumours, and did not correlate with MET amplification or polysomy 7. cMet was the only parameter associated with significantly shorter BM-specific survival (median 8 months versus 33 months, P = 0.005, Cox regression). CONCLUSIONS: EGFR, PDGFRA and cMet are commonly overexpressed in ccRCC BMs. cMet overexpression correlates with significantly shorter BM-specific survival.

Tumour-infiltrating lymphocytes and expression of programmed death ligand 1 (PD-L1) in melanoma brain metastases.

AIMS: In this study we aimed to characterize immune infiltrates and expression of programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) in a series of melanoma BM to provide a basis for experimental therapy using immune checkpoint inhibitors. METHODS AND RESULTS: We investigated expression of PD-1, PD-L1, CD3, CD8, CD45RO, forkhead box protein 3 (FoxP3), CD20 and BRAF V600E by immunohistochemistry in melanoma BM samples. Forty-three specimens [27 of which (62.8%) were BRAF V600E-positive] were available. CD3(+) tumour-infiltrating lymphocytes (TILs) were evident in 33 specimens (76.7%), CD8(+) in 39 (90.7%), CD45RO(+) in 32 (74.4%), PD-1(+) in 27 (62.8%), FoxP3(+) in 21 (48.8%) and CD20(+) TILs in 19 (44.2%). Tumour PD-L1 expression was observed in 22 specimens (51.1%), and in nine of these (40.9%) expression was observed in more than 5% of tumour cells. PD-L1 expression was associated with higher density of PD-1(+) (P < 0.001), CD3(+) (P = 0.014) and FoxP3(+) (P < 0.001) TIL infiltration. Density of CD3(+) TILs was associated with density of CD8(+) (P < 0.001), PD-1(+) (P < 0.001) and CD45RO(+) (P < 0.001) TILs. PD-L1 expression or PD-1(+) , CD3(+) , CD8(+) or CD45RO(+) TILs density did not correlate with BRAF V600E status, previous systemic therapy or survival (P > 0.05). CONCLUSIONS: Melanoma BM showed considerable lymphocytic infiltrates and expression of PD-L1 in the majority of investigated specimens, with high PD-L1 expression found predominantly in regions of abundant inflammation. Our data indicate that clinical studies should investigate the value of checkpoint inhibitors in patients with melanoma BMs.

microRNA-21 expression is elevated in esophageal adenocarcinoma after neoadjuvant chemotherapy.

We investigated whether microRNA-21 and microRNA-148a are predictive for neoadjuvant treatment in esophageal adenocarcinoma. Thirty-six patients with neoadjuvant therapy and surgical resection were included. FFPE tissue from biopsy and esophagectomy were analyzed using RT-qPCR. Results were correlated to histological tumor regression, histopathological variables, FDG-PET-CT and survival. MicroRNA-21 was significantly higher in esophagectomies than in corresponding biopsies (p = .027). No association of microRNA-21 or microRNA-148a expression in tissue specimens with other clinical parameters was present. Although no influence of microRNA-21 and microRNA-148a on the response to neoadjuvant therapy was seen, upregulation of microRNA-21 might represent an escape mechanism of tumor cells.

Enhanced epithelial to mesenchymal transition (EMT) and upregulated MYC in ectopic lesions contribute independently to endometriosis.

BACKGROUND: Epithelial to mesenchymal transition (EMT) is a process in which epithelial cells lose polarity and cell-to-cell contacts and acquire the migratory and invasive abilities of mesenchymal cells. These abilities are thought to be prerequisites for the establishment of endometriotic lesions. A hallmark of EMT is the functional loss of E-cadherin (CDH1) expression in epithelial cells. TWIST1, a transcription factor that represses E-cadherin transcription, is among the EMT inducers. SNAIL, a zinc-finger transcription factor, and its close relative SLUG have similar properties to TWIST1 and are thus also EMT inducers. MYC, which is upregulated by estrogens in the uterus by an estrogen response cis-acting element (ERE) in its promoter, is associated with proliferation in endometriosis. The role of EMT and proliferation in the pathogenesis of endometriosis was evaluated by analyzing TWIST1, CDH1 and MYC expression. METHODS: CDH1, TWIST1, SNAIL and SLUG mRNA expression was analyzed by qRT-PCR from 47 controls and 74 patients with endometriosis. Approximately 42 ectopic and 62 eutopic endometrial tissues, of which 30 were matched samples, were collected during the same surgical procedure. We evaluated TWIST1 and MYC protein expression by immunohistochemistry (IHC) in the epithelial and stromal tissue of 69 eutopic and 90 ectopic endometrium samples, of which 49 matched samples were analyzed from the same patient. Concordant expression of TWIST1/SNAIL/SLUG and CDH1 but also of TWIST1 and MYC was analyzed. RESULTS: We found that TWIST1, SNAIL and SLUG are overexpressed (p < 0.001, p = 0.016 and p < 0.001) in endometriosis, while CDH1 expression was concordantly reduced in these samples (p < 0.001). Similar to TWIST1, the epithelial expression of MYC was also significantly enhanced in ectopic endometrium compared to eutopic tissues (p = 0.008). We found exclusive expression of either TWIST1 or MYC in the same samples (p = 0.003). CONCLUSIONS: Epithelial TWIST1 is overexpressed in endometriosis and may contribute to the formation of endometriotic lesions by inducing epithelial to mesenchymal transition, as CDH1 was reduced in ectopic lesions. We found exclusive expression of either TWIST1 or MYC in the same samples, indicating that EMT and proliferation contribute independently of each other to the formation of endometriotic lesions.

Mutant IDH1 inhibits PI3K/Akt signaling in human glioma.

BACKGROUND: Recently, isocitrate dehydrogenase 1 (IDH1) was identified as a major participant in glioma pathogenesis. At present, the enzymatic activity of the protein has been the main topic in investigating its physiological function, but its signaling pathway allocation was unsuccessful. Interestingly, proteins regulated by phosphoinositide 3-kinase (PI3K)/Akt signaling, are among the top downregulated genes in gliomas associated with high percentage of IDH1 and IDH2 mutations. The aim of this study was to investigate a hypothetical relation between IDH1 and PI3K signaling. METHODS: The presence of mutant IDH1 and markers for active PI3K/Akt signaling, present as phosphorylated Akt and podoplanin (PDPN), were investigated in a discovery cohort of 354 patients with glioma. In vitro experiments were used to confirm functional links. RESULTS: This study shows an inverse correlation between mutant IDH1 and markers for active PI3K/Akt signaling. In support of a functional link between these molecules, in vitro expression of mutant IDH1 inhibited Akt phosphorylation in a 2-hydroxyglutarate-dependent manner. CONCLUSIONS: This study provides patient tumor and in vitro evidence suggesting that mutant IDH1 inhibits PI3K/Akt signaling.

Improved molecular classification of serrated lesions of the colon by immunohistochemical detection of BRAF V600E.

BRAF V600E mutation in serrated lesions of the colon has been implicated as an important mutation and as a specific marker for the serrated carcinogenic pathway. Recent findings point to microvesicular hyperplastic polyps that have similar histologic and molecular features to sessile serrated adenomas/polyps, as potential colorectal carcinoma precursors. The aim of this study was to evaluate BRAF V600E mutation status by immunohistochemistry in serrated lesions of the colon with regard to histomorphology. We investigated 194 serrated lesions of the colon, comprising 42 sessile serrated adenomas/polyps, 16 traditional serrated adenomas, 136 hyperplastic polyps and 20 tubular/tubulovillous adenomas (conventional adenomas) with the novel BRAF V600E mutation-specific antibody VE1. In addition, BRAF exon 15 and KRAS exon 2 status was investigated by capillary sequencing in selected cases. All sessile serrated adenomas/polyps (42/42, 100%), 15/16 (94%) traditional serrated adenomas and 84/136 (62%) hyperplastic polyps were VE1+. None of the VE1- serrated lesions showed BRAF V600E mutation. Forty out of 42 (95%) sessile serrated adenomas/polyps displayed areas with microvesicular hyperplastic polyp-like features. In microvesicular hyperplastic polyps, VE1 positivity was significantly associated with nuclear atypia (P=0.003); however, nuclear atypia was also present in VE1- cases. Immunostaining with VE1 allows not only the detection of BRAF V600E mutation but also the correlation with histomorphology on a cellular level in serrated lesions. VE1 enables a subclassification of microvesicular hyperplastic polyps according to the mutation status. This improved classification of serrated lesions including immunohistochemical evaluation of BRAF V600E mutation may be the key to identify lesions with higher potential to progression into sessile serrated adenoma/polyp, and further to BRAF V600E-mutated colorectal cancer.

EGFR, BRAF and KRAS status in patients undergoing pulmonary metastasectomy from primary colorectal carcinoma: a prospective follow-up study.

BACKGROUND: Pulmonary metastasectomy is an integral part of the interdisciplinary treatment of patients with pulmonary metastases (PMs) from colorectal carcinoma (CRC). Although alterations in the epidermal growth factor receptor (EGFR) pathway are common in CRC, there is still insufficient data regarding PM. We hypothesized that EGFR expression and Kirsten rat sarcoma viral oncogene homolog (KRAS)/BRAF mutations (Mts) might be associated with clinicopathological variables and the outcome in patients undergoing pulmonary metastasectomy. METHODS: In this single-center study, 44 patients undergoing pulmonary metastasectomy from primary CRC were included and prospectively followed up. Tissue specimens of resected PMs were assessed. Restriction fragment length analysis was used for BRAF V600E and KRAS codons 12 and 13 Mt analyses. EGFR expression was evaluated by immunohistochemistry. Patients were followed up in 3-6-month intervals. RESULTS: EGFR expression was evident in 49 % of the PMs, whereas Mts in KRAS and BRAF were detected in 48 and 0 %, respectively. Time to lung-specific recurrence after metastasectomy was significantly decreased in patients with KRAS mutated PMs in univariate (p = 0.013) and multivariate analysis (p = 0.035), whereas EGFR expression had no impact on recurrence free survival. Moreover, KRAS Mts were associated with the number of PMs (p = 0.037) and with the lung as first site of recurrence after metastasectomy (p = 0.047). DISCUSSION: This is the first evaluation of EGFR pathway alterations in the setting of pulmonary metastasectomy. Our data suggest that patients with KRAS Mts are at high risk for early pulmonary recurrence and have a more diffuse pattern of metastasis. These findings may have impact on the therapeutic management of CRC patients with pulmonary spreading.

Predictive molecular markers in metastases to the central nervous system: recent advances and future avenues.

Metastases to the central nervous system (CNS) are common in several cancer types. For most primary tumors that commonly metastasize to the CNS, molecular biomarker analyses are recommended in the clinical setting for selection of appropriate targeted therapies. Therapeutic efficacy of some of these agents has been documented in patients with brain metastases, and molecular testing of CNS metastases should be considered in the clinical setting. Here, we summarize the clinically relevant biomarker tests that should be considered in neurosurgical specimens based on the current recommendations of the European Society of Medical Oncology (ESMO) or the National Comprehensive Cancer Network (NCCN) for the most relevant primary tumor types: lung cancer (EGFR mutations, ALK rearrangement, BRAF mutations), breast cancer (HER2 amplification, steroid receptor overexpression), melanoma (BRAF mutations), and colorectal cancer (RAS mutations). Furthermore, we discuss emerging therapeutic targets including novel oncogenic alterations (ROS1 rearrangements, FGFR1 amplifications, CMET amplifications, and others) and molecular features of the tumor microenvironment (including immune-checkpoint molecules such as CTLA4 and PD-1/PD-L1). We also discuss the potential role of advanced biomarker tests such as next-generation sequencing and "liquid biopsies" for patients with CNS metastases.

Amplification and overexpression of CMET is a common event in brain metastases of non-small cell lung cancer.

BACKGROUND: CMET represents an emerging therapy target for monoclonal antibodies and tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC). METHODS: We investigated CMET gene amplification status by fluorescence in-situ hybridization (FISH) and CMET protein expression by immunohistochemistry in a large series of 209 NSCLC brain metastases (BM; 165 adenocarcinoma, 20 squamous cell carcinoma, 11 adenosquamous carcinomas, 11 large cell carcinomas and two large cell neuroendocrine carcinomas) and correlated our results to clinic-pathological parameters and molecular data from previous studies. RESULTS: We found CMET gene amplification in 36/167 (21.6%) and CMET protein expression in 87/196 (44.4%) of evaluable BM. There was a strong correlation between the presence of CMET gene amplification and CMET protein expression (P < 0.001, chi-square test). Furthermore, presence of CMET amplification correlated positively with presence of ALK amplifications (P = 0.039, chi-square test) and high HIF1 alpha index (P = 0.013, Mann-Whitney U-test). Neither CMET expression nor CMET gene amplification status correlated with patient outcome parameters or known prognostic factors. CONCLUSIONS: CMET overexpression and CMET amplification are commonly found in NSCLC BM and may represent a promising therapeutic target.