Analytical and pharmacological consequences of the in vivo deamidation of trastuzumab and pertuzumab.

A liquid chromatography-tandem mass spectrometry method is presented for the quantitative determination of the in vivo deamidation of the biopharmaceutical proteins trastuzumab and pertuzumab at an asparagine in their complementarity determining regions (CDRs). For each analyte, two surrogate peptides are quantified after tryptic digestion of the entire plasma protein content: one from a stable part of the molecule, representing the total concentration, and one containing the deamidation-sensitive asparagine, corresponding to the remaining non-deamidated concentration. Using a plasma volume of 10 µL and a 2-h digestion at pH 7, concentrations between 2 and 1000 µg/mL can be determined for the various protein forms with values for bias and CV below 15% and without unacceptable in vitro deamidation taking place. A considerable difference between the total and non-deamidated concentrations, and thus a substantial degree of deamidation, was observed in plasma for both trastuzumab and pertuzumab. After a 56-day forced deamidation test 40% of trastuzumab and 68% of pertuzumab was deamidated, while trastuzumab and pertuzumab showed up to 47% and 35% of deamidation, respectively, in samples collected from breast cancer patients during treatment with a combination of both drugs. A good correlation between the non-deamidated concentration results and those of a receptor binding assay indicate a loss of receptor binding for both trastuzumab and pertuzumab along with the deamidation in their CDRs. Deamidated trastuzumab also lost its capability to inhibit the growth of breast cancer cells in a cell-based viability assay, suggesting a relation between the degree of deamidation and pharmacological activity.

Genome-wide association study of cardiovascular disease in testicular cancer patients treated with platinum-based chemotherapy.

Genetic variation may mediate the increased risk of cardiovascular disease (CVD) in chemotherapy-treated testicular cancer (TC) patients compared to the general population. Involved single nucleotide polymorphisms (SNPs) might differ from known CVD-associated SNPs in the general population. We performed an explorative genome-wide association study (GWAS) in TC patients. TC patients treated with platinum-based chemotherapy between 1977 and 2011, age ≤55 years at diagnosis, and ≥3 years relapse-free follow-up were genotyped. Association between SNPs and CVD occurrence during treatment or follow-up was analyzed. Data-driven Expression Prioritized Integration for Complex Trait (DEPICT) provided insight into enriched gene sets, i.e., biological themes. During a median follow-up of 11 years (range 3-37), CVD occurred in 53 (14%) of 375 genotyped patients. Based on 179 SNPs associated at p ≤ 0.001, 141 independent genomic loci associated with CVD occurrence. Subsequent, DEPICT found ten biological themes, with the RAC2/RAC3 network (linked to endothelial activation) as the most prominent theme. Biology of this network was illustrated in a TC cohort (n = 60) by increased circulating endothelial cells during chemotherapy. In conclusion, the ten observed biological themes highlight possible pathways involved in CVD in chemotherapy-treated TC patients. Insight in the genetic susceptibility to CVD in TC patients can aid future intervention strategies.

Vascular aging in long-term survivors of testicular cancer more than 20 years after treatment with cisplatin-based chemotherapy.

BACKGROUND: Late effects of cisplatin-based chemotherapy in testicular cancer survivors (TCS) include cardiovascular morbidity, but little data is available beyond 20 years. The objective was to assess vascular damage in very long-term TCS. METHODS: TCS (treated with chemotherapy or orchiectomy only) and age-matched healthy controls were invited. Study assessment included vascular stiffness with ultrasound measurement of carotid-femoral pulse wave velocity (cf-PWV). RESULTS: We included 127 TCS consisting of a chemotherapy group (70 patients) and an orchiectomy group (57 patients) along with 70 controls. Median follow-up was 28 years (range: 20-42). The cf-PWV (m/s) was higher in TCS than in controls (geometrical mean 8.05 (SD 1.23) vs. 7.60 (SD 1.21), p = 0.04). The cf-PWV was higher in the chemotherapy group than in the orchiectomy group (geometrical mean 8.39 (SD 1.22) vs. 7.61 (SD 1.21), p < 0.01). In the chemotherapy group cf-PWV increased more rapidly as a function of age compared to controls (regression coefficient b 7.59 × 10-3 vs. 4.04 × 10-3; p = 0.03). CONCLUSION: Very long-term TCS treated with cisplatin-based chemotherapy show increased vascular damage compatible with "accelerated vascular aging" and continue to be at risk for cardiovascular morbidity, thus supporting the need for intensive cardiovascular risk management. CLINICAL TRIAL REGISTRATION: The clinical trial registration number is NCT02572934.

The OncoLifeS data-biobank for oncology: a comprehensive repository of clinical data, biological samples, and the patient's perspective.

BACKGROUND: Understanding cancer heterogeneity, its temporal evolution over time, and the outcomes of guided treatment depend on accurate data collection in a context of routine clinical care. We have developed a hospital-based data-biobank for oncology, entitled OncoLifeS (Oncological Life Study: Living well as a cancer survivor), that links routine clinical data with preserved biological specimens and quality of life assessments. The aim of this study is to describe the organization and development of a data-biobank for cancer research. RESULTS: We have enrolled 3704 patients aged ≥ 18 years diagnosed with cancer, of which 45 with hereditary breast-ovarian cancer (70% participation rate) as of October 24th, 2019. The average age is 63.6 ± 14.2 years and 1892 (51.1%) are female. The following data are collected: clinical and treatment details, comorbidities, lifestyle, radiological and pathological findings, and long-term outcomes. We also collect and store various biomaterials of patients as well as information from quality of life assessments. CONCLUSION: Embedding a data-biobank in clinical care can ensure the collection of high-quality data. Moreover, the inclusion of longitudinal quality of life data allows us to incorporate patients' perspectives and inclusion of imaging data provides an opportunity for analyzing raw imaging data using artificial intelligence (AI) methods, thus adding new dimensions to the collected data.

Bleomycin-Induced Pulmonary Changes on Restaging Computed Tomography Scans in Two Thirds of Testicular Cancer Patients Show No Correlation With Fibrosis Markers.

BACKGROUND: In metastatic testicular cancer patients treated with bleomycin, etoposide, and cisplatin (BEP) chemotherapy, bleomycin-induced pneumonitis is a well-known and potentially fatal side effect. We sought to determine the prevalence of lesions as signs of bleomycin-induced pulmonary changes on restaging computed tomography (CT) scans after treatment and to ascertain whether fibrosis markers were predictive of these changes. PATIENTS AND METHODS: This prospective nonrandomized cohort study included metastatic testicular cancer patients, 18-50 years of age, treated with BEP chemotherapy. Restaging CT scans were examined for lesions as signs of bleomycin-induced pulmonary changes by two independent radiologists and graded as minor, moderate, or severe. Plasma samples were collected before, during, and after treatment and were quantified for transforming growth factor-ß1 (TGF-ß1), growth differentiation factor-15 (GDF-15), and high-sensitivity C-reactive protein (hs-CRP). RESULTS: In total, 66 patients were included: forty-five (68%) showed signs of bleomycin-induced pulmonary changes on the restaging CT scan, 37 of which were classified as minor and 8 as moderate. No differences in TGF-ß1, GDF-15, or hs-CRP plasma levels were found between these groups. CONCLUSION: Bleomycin-induced pulmonary changes are common on restaging CT scans after BEP chemotherapy for metastatic testicular cancer. Changes in TGF-ß1, GDF-15, and hs-CRP plasma levels do not differ between patients with and without radiological lesions as signs of bleomycin-induced pulmonary changes and are therefore not helpful as predictive biomarkers. IMPLICATIONS FOR PRACTICE: Bleomycin-induced pneumonitis (BIP) is a well-known and potentially fatal side effect in metastatic testicular cancer patients treated with bleomycin, etoposide, and cisplatin chemotherapy. Currently, the decision to discontinue bleomycin administration is made during treatment and is based on clinical signs. An upfront or early marker or biomarker that identifies patients likely to develop BIP would be preferable. This study found that bleomycin-induced pulmonary changes are common on restaging computed tomography scans and mostly resolve. No correlation was seen between these changes and fibrosis or inflammation markers (transforming growth factor-ß1, growth differentiation factor-15, and high-sensitivity C-reactive protein).

Dopamine and serotonin regulate tumor behavior by affecting angiogenesis.

The biogenic amines dopamine and serotonin are neurotransmitters and hormones, which are mainly produced in the central nervous system and in the gastro-intestinal tract. They execute local and systemic functions such as intestinal motility and tissue repair. Dopamine and serotonin are primarily stored in and transported by platelets. This review focuses on the recently recognized role of dopamine and serotonin in the regulation of tumor behavior by affecting angiogenesis and tumor cell proliferation. Preclinical studies demonstrate that dopamine inhibits tumor growth via activation of dopamine receptor D2 on endothelial and tumor cells. Serotonin stimulates tumor growth via activation of serotonin receptor 2B on endothelial cells and serotonin receptors on tumor cells. Drugs that stimulate dopamine receptor D2 or inhibit serotonin receptors are available and therefore clinical intervention studies for cancer patients are within reach.

Loss of CD44 and SOX2 expression is correlated with a poor prognosis in esophageal adenocarcinoma patients.

BACKGROUND: It has been suggested that markers associated with cancer stem cells (CSC) may play a role in esophageal cancer. Our aim was to investigate the expression pattern of proposed CSC markers ALDH1, Axin2, BMI1, CD44, and SOX2 in esophageal adenocarcinoma (EAC) and to relate their expression to survival. METHODS: In this study we included 94 EAC patients and examined the expression of the above-mentioned markers by using immunohistochemistry on tissue microarrays. Expression was scored as positive or negative or categorized as low or high in terms of an immunoreactivity score (IRS). Expression rates were related to clinicopathologic characteristics and overall and disease-free survival (DFS). RESULTS: In a multivariate analysis, negative expression of CD44 and of SOX2 were both significant prognostic factors for DFS [hazard ratio (HR), 1.73; 95 % confidence interval (CI), 1.00-2.96; P = 0.046 and HR, 2.06; 95 % CI 1.14-3.70 P = 0.016). When CD44 and SOX2 expression were analyzed together, negative SOX2 expression was an independent prognostic factor for DFS (HR, 1.91; 95 % CI 1.05-3.46; P = 0.034). Low IRS scores for ALDH1 or Axin2 were associated with a reduced median survival (12.8 vs. 28.7 and 12.1 vs. 25.5 months, respectively). However, these markers and BMI1 were not prognostic factors for survival. CONCLUSIONS: Loss of CD44 expression and loss of SOX2 expression are prognostic factors of poor survival in EAC patients. This suggests a role of these proteins in EAC that requires further investigation.

Change in telomere length and cardiovascular risk factors in testicular cancer survivors.

BACKGROUND: Testicular cancer (TC) survivors cured with chemotherapy (CT) are prone to develop cardiovascular diseases, as part of an accelerated aging phenotype. A mechanism contributing to these events can be telomere shortening. PATIENTS AND METHODS: In a prospective cohort of patients with disseminated TC who received cisplatin-based CT, mean absolute leukocyte telomere length (TL) was measured before and 1 year after start of treatment. Cardiovascular risk factors, including development of the metabolic syndrome and hypogonadism, were assessed before and up to 5 years after CT. RESULTS: For the whole group (n = 55), TL did not change 1 year after CT (5.7 (2.2-13.4) vs. 5.8 kb (1.6-19.2), P = 0.335). At baseline, patients with a BMI >30 kg/m2 (n = 12) had shorter TL (4.9 (2.2-13.4) vs. 6.3 kb (3.1-12.9), P = 0.045), while no age-dependent differences were measured. Patients with TL shortening after 1 year (n = 7) showed a significant increase in diastolic blood pressure (P = 0.007) and triglycerides (P = 0.003), compared to those with unchanged TL. There was no association between telomere shortening after 1 year or short TL at baseline (n = 7+11) and development of metabolic syndrome (25% vs. 21%; P = 0.777), or hypogonadism (38% vs. 17%; P = 0.120) after 5 years. CONCLUSIONS: A small subset of TC patients treated with cisplatin-based CT showed telomere shortening 1 year after treatment. This shortening was associated to a rise in diastolic blood pressure and triglycerides, but not to newly developed metabolic syndrome and hypogonadism after 5 years.

'Big'-insulin-like growth factor-II signaling is an autocrine survival pathway in gastrointestinal stromal tumors.

New treatment targets need to be identified in gastrointestinal stromal tumors (GISTs) to extend the treatment options for patients experiencing failure with small-molecule tyrosine kinase inhibitors, such as imatinib. Insulin-like growth factor (IGF)-II acts as an autocrine factor in several tumor types by binding to IGF receptor type 1 (IGF-1R) and/or the insulin receptor (IR) isoform A. The aim of the present study was to investigate the putative role of unprocessed pro-IGF-II, called 'big'-IGF-II, in GISTs. The imatinib-sensitive GIST882 and imatinib-resistant GIST48 cell lines secrete high levels of big-IGF-II as demonstrated by ELISA and Western blotting analyses. IR isoform A mRNA and protein expression, but not that of IGF-1R, was found in these KIT mutant cell lines and in KIT and platelet-derived growth factor receptor α-mutant GIST specimens. Down-regulation of either big-IGF-II or IR affected AKT and MAPK signaling and reduced survival in both cell lines. Disruption of big-IGF-II signaling in combination with imatinib had additive cytotoxic effects on GIST882 cells. IGF-II mRNA as determined by in situ hybridization was present in 91% of 60 primary GISTs. Immunohistochemical analysis of big-IGF-II protein expression was associated with moderate- to high-risk tumors compared with tumors with a lower risk classification (P < 0.028). Our data put forth the big-IGF-II/IR isoform A axis as an autocrine survival pathway and potential therapeutic target in GISTs.

Physical exercise in patients with testicular cancer treated with bleomycin, etoposide and cisplatin chemotherapy: pulmonary and vascular endothelial function-an exploratory analysis.

PURPOSE: Bleomycin, etoposide, and cisplatin combination chemotherapy (BEP) improves the survival of patients with testicular cancer, but is associated with potentially life-threatening toxicities like pneumonitis and thromboembolic events. This study explored the effects of physical exercise in patients with testicular cancer during or after BEP-chemotherapy on pulmonary and vascular endothelial toxicity. METHODS: In this post hoc analysis of a multicenter randomized clinical trial (NCT01642680), patients with metastatic testicular cancer scheduled to receive BEP-chemotherapy were randomized to a 24-week exercise intervention, initiated during (group A) or after BEP-chemotherapy (group B). Endpoints were pulmonary function (forced vital capacity (FVC), forced expiratory volume in one second (FEV1), lung transfer-coefficient and transfer factor for carbon monoxide (KCO, DLCO) and markers of vascular endothelial dysfunction (von Willebrand factor (vWF) and factor VIII). RESULTS: Thirty patients were included. Post-chemotherapy, patients declined less in FVC, FEV1 and DLCO in group A compared to group B. Post-chemotherapy, vWF and factor VIII were significantly lower in group A compared to group B. After completion of exercise, started either during BEP-chemotherapy or thereafter, no between-group differences were found. At 1-year post-intervention, significant between-group differences were found in favour of group A in DLCO and KCO. CONCLUSIONS: Patients who exercised during BEP-chemotherapy better preserved FVC, FEV1 and DLCO, measured directly post-chemotherapy and 1-year post-intervention (DLCO, KCO). This coincided with less increase in vWF and factor VIII measured directly post-chemotherapy. These data support a beneficial role of a physical exercise intervention during BEP-chemotherapy on pulmonary and vascular damage in patients with testicular cancer. TRIAL REGISTRY: Optimal Timing of Physical Activity in Cancer Treatment (ACT) Registry URL: https://clinicaltrials.gov/ct2/show/NCT01642680 . TRIAL REGISTRATION NUMBER: NCT01642680.

Association Study between Polymorphisms in DNA Methylation-Related Genes and Testicular Germ Cell Tumor Risk.

BACKGROUND: Testicular germ cell tumors (TGCT), histologically classified as seminomas and nonseminomas, are believed to arise from primordial gonocytes, with the maturation process blocked when they are subjected to DNA methylation reprogramming. SNPs in DNA methylation machinery and folate-dependent one-carbon metabolism genes have been postulated to influence the proper establishment of DNA methylation. METHODS: In this pathway-focused investigation, we evaluated the association between 273 selected tag SNPs from 28 DNA methylation-related genes and TGCT risk. We carried out association analysis at individual SNP and gene-based level using summary statistics from the Genome Wide Association Study meta-analysis recently conducted by the international Testicular Cancer Consortium on 10,156 TGCT cases and 179,683 controls. RESULTS: In individual SNP analyses, seven SNPs, four mapping within MTHFR, were associated with TGCT risk after correction for multiple testing (q ≤ 0.05). Queries of public databases showed that three of these SNPs were associated with MTHFR changes in enzymatic activity (rs1801133) or expression level in testis tissue (rs12121543, rs1476413). Gene-based analyses revealed MTHFR (q = 8.4 × 10-4), methyl-CpG-binding protein 2 (MECP2; q = 2 × 10-3), and ZBTB4 (q = 0.03) as the top TGCT-associated genes. Stratifying by tumor histology, four MTHFR SNPs were associated with seminoma. In gene-based analysis MTHFR was associated with risk of seminoma (q = 2.8 × 10-4), but not with nonseminomatous tumors (q = 0.22). CONCLUSIONS: Genetic variants within MTHFR, potentially having an impact on the DNA methylation pattern, are associated with TGCT risk. IMPACT: This finding suggests that TGCT pathogenesis could be associated with the folate cycle status, and this relation could be partly due to hereditary factors.

A unique small cell lung carcinoma disease progression model shows progressive accumulation of cancer stem cell properties and CD44 as a potential diagnostic marker.

OBJECTIVES: Cancer stem cells (CSCs) have been implicated in disease progression of aggressive cancers including small cell lung carcinoma (SCLC). Here, we have examined the possible contribution of CSCs to SCLC progression and aggressiveness. MATERIALS AND METHODS: GLC-14, GLC-16 and GLC-19 SCLC cell lines derived from one patient, representing increasing progressive stages of disease were used. CSC marker expressions was determined by RT-qPCR and western blotting analyses, and heterogeneity was studied by CSC marker expression by immunofluorescence microscopy and flow cytometry. Colony formation assays were used to assess stem cell properties and therapy sensitivity. RESULTS: Increasing expression of stem cell markers MYC, SOX2 and particularly CD44 were found in association with advancing disease. Single and overlapping expression of these markers indicated the presence of different CSC populations. The accumulation of more homogeneous double- and triple-positive CSC populations evolved with disease progression. Functional characterization of CSC properties affirmed higher proficiency of colony forming ability and increased resistance to γ-irradiation in GLC-16 and GLC-19 compared to GLC-14. GLC-19 colony formation was significantly inhibited by a human anti-CD44 antibody. CONCLUSION: The progressive increase of MYC, SOX2 and particularly CD44 expression that was accompanied with enhanced colony forming capacity and resistance in the in vitro GLC disease progression model, supports the potential clinical relevance of CSC populations in malignancy and disease relapse of SCLC.

Cognitive Impairment in Long-Term Survivors of Testicular Cancer More Than 20 Years after Treatment.

BACKGROUND: Impaired cognition can be a late effect after treatment in long-term testicular cancer (TC) survivors, negatively affecting their daily life. However, little data is available beyond 20 years post-treatment. We assessed cognitive impairment in very long-term TC survivors after CT or RT and compared the results with stage I TC survivors and controls. METHODS: In this cross-sectional multicenter cohort study, we enrolled TC survivors (treated with orchiectomy followed by CT or RT or orchiectomy only)-with a follow-up duration ≥ 20 years-and age-matched healthy controls. Cognitive testing included the Auditory Verbal Learning Test, Letter Fluency Test, Category Fluency Test, and Trail Making Test. We used fasting blood samples to assess the presence of hypogonadism and measured cardiovascular aging parameters, including carotid pulse wave velocity (c-PWV) and advanced glycation end products (AGEs). RESULTS: We included 184 TC survivors (66 CT patients, 53 RT patients, and 65 orchiectomy-only patients) and 70 healthy controls. The median follow-up was 26 years (range: 20-42). TC survivors had a lower combined score of the cognitive tests (mean cumulative Z-score -0.85; 95% CI -1.39 to -0.33) compared to controls (mean 0.67; 95% CI -0.21 to 1.57, p < 0.01). In univariate analysis, the presence of hypogonadism (ß -1.50, p < 0.01), high c-PWV (ß -0.35, p = 0.09), and high AGEs (ß -1.27, p = 0.02) were associated with lower cognitive scores, while only AGEs (ß -1.17, p = 0.03) remained a significant predictor in multivariate analysis (Model R2 0.31, p < 0.01). CONCLUSIONS: Long-term TC survivors performed worse on cognitive tests compared to controls. Physicians and patients should be informed about timely cardiovascular risk management and testosterone supplementation therapy during follow-up to reduce the risk of cognitive impairment. TRIAL REGISTRATION: NCT02572934.

Identification of 22 susceptibility loci associated with testicular germ cell tumors.

Testicular germ cell tumors (TGCT) are the most common tumor in young white men and have a high heritability. In this study, the international Testicular Cancer Consortium assemble 10,156 and 179,683 men with and without TGCT, respectively, for a genome-wide association study. This meta-analysis identifies 22 TGCT susceptibility loci, bringing the total to 78, which account for 44% of disease heritability. Men with a polygenic risk score (PRS) in the 95th percentile have a 6.8-fold increased risk of TGCT compared to men with median scores. Among men with independent TGCT risk factors such as cryptorchidism, the PRS may guide screening decisions with the goal of reducing treatment-related complications causing long-term morbidity in survivors. These findings emphasize the interconnected nature of two known pathways that promote TGCT susceptibility: male germ cell development within its somatic niche and regulation of chromosomal division and structure, and implicate an additional biological pathway, mRNA translation.

The insulin-like growth factor system and sarcomas.

Sarcomas are a diverse group of malignant mesenchymal tumours arising from bone and soft tissues. The identification of critical cellular signalling pathways in sarcomas is an important issue for the development of new targeted therapies. This review highlights the experimental and clinical evidence supporting the role of the insulin-like growth factor (IGF) signalling system in the cellular transformation and progression of several types of sarcoma, including rhabdomyosarcoma, synovial sarcoma, leiomyosarcoma, Ewing's sarcoma and osteosarcoma. Preclinical data suggest that the IGF system could be a promising target for therapy in these sarcomas. Currently, therapies interrupting IGF signalling have been or are being developed. In recent phase 1 clinical studies with humanized monoclonal antibodies directed against IGF receptor type 1 (IGF-1R), objective tumour responses were observed in several patients with Ewing's sarcoma, encouraging further clinical testing in Ewing's sarcoma and other sarcoma (sub)types. Moreover, the occasional occurrence of paraneoplastic hypoglycaemia as a result of the secretion of incompletely processed forms of pro-IGF-II by sarcomas is discussed.

Early ageing after cytotoxic treatment for testicular cancer and cellular senescence: Time to act.

Treatment of testicular cancer (TC) has an exceptionally high success rate compared to other cancer types; even in case of metastasized disease, 80-90 % of TC patients can be cured. Consequently, attention has been drawn to a potential downside of this treatment success: late adverse treatment effects such as the accelerated development of otherwise age-associated features like cardiovascular disease and second malignancies. Underlying mechanisms are poorly understood. Emerging data suggest that cytotoxic treatment induces cellular senescence, resulting in secretion of inflammatory factors contributing to this early ageing phenotype. Molecular and cellular characterization of this early ageing will enhance understanding the pathogenesis of TC treatment-induced morbidity and contribute to better recognition and prevention of late effects. In this review, we describe clinical manifestations of the early ageing phenotype among TC survivors, and subsequently focus on potential underlying mechanisms. We discuss the clinical implications and describe perspectives for future research and intervention strategies.

Serotonin and Dopamine Receptor Expression in Solid Tumours Including Rare Cancers.

In preclinical studies serotonin stimulates and dopamine inhibits tumour growth and angiogenesis. Information regarding serotonin and dopamine receptor (5-HTR and DRD) expression in human cancers is limited. Therefore, we screened a large tumour set for receptor mRNA overexpression using functional genomic mRNA (FGmRNA) profiling, and we analysed protein expression and location of 5-HTR1B, 5-HTR2B, DRD1, and DRD2 with immunohistochemistry in different tumour types. With FGmRNA profiling 11,756 samples representing 43 tumour types were compared to 3,520 normal tissue samples to analyse receptor overexpression. 5-HTR2B overexpression was present in many tumour types, most frequently in uveal melanomas (56%). Receptor overexpression in rare cancers included 5-HTR1B in nasopharyngeal carcinoma (17%), DRD1 in ependymoma (30%) and synovial sarcoma (21%), and DRD2 in astrocytoma (13%). Immunohistochemistry demonstrated high 5-HTR2B protein expression on melanoma and gastro-intestinal stromal tumour cells and endothelial cells of colon, ovarian, breast, renal and pancreatic tumours. 5-HTR1B expression was predominantly low. High DRD2 protein expression on tumour cells was observed in 48% of pheochromocytomas, and DRD1 expression ranged from 14% in melanoma to 57% in renal cell carcinoma. In conclusion, 5-HTR1B, 5-HTR2B, DRD1, and DRD2 show mRNA overexpression in a broad spectrum of common and rare cancers. 5-HTR2B protein is frequently highly expressed in human cancers, especially on endothelial cells. These findings support further investigation of especially 5HTR2B as a potential treatment target.

ER stress and UPR activation in glioblastoma: identification of a noncanonical PERK mechanism regulating GBM stem cells through SOX2 modulation.

Patients with aggressive brain tumors, named glioblastoma multiforme (GBM), have a poor prognoses. Here we explored if the ER stress/unfolded protein response (UPR) is involved in the pathophysiology of GBM and may provide novel therapeutic targets. Immunohistochemical analyses of a tissue microarray containing primary GBM specimens showed strong variability in expression of the UPR markers GRP78/BiP, XBP1, and ATF4. Interestingly, high ATF4 expression was associated with poor overall survival suggesting involvement of PERK signaling in GBM progression. In vitro experiments using patient-derived neurospheres, enriched for GBM stem cells (GSCs), showed high sensitivity for the ER stressor thapsigargin (Tg) mainly via PERK signaling. In contrast, neurospheres-derived differentiated GBM cells were less sensitive likely due to lower UPR activity as indicated by comparative transcriptional profiling. Tg and Tunicamycin strongly reduced neurosphere forming ability of GSCs that was linked with potent PERK-dependent downregulation of SOX2 protein. Interestingly, SOX2 downregulation occurred directly via PERK, not requiring downstream activation of the PERK-UPR pathway. Moreover, PERK inactivation resulted in aberrant serum-induced differentiation of GBM neurospheres accompanied by persistent SOX2 expression, delayed upregulation of GFAP and reduced cell adherence. In conclusion, we provide evidence that PERK signaling contributes to the prognoses of primary GBM patients and identified PERK as a novel regulator of SOX2 expression and GSC differentiation. The role of PERK appeared to be pleiotropic involving UPR-dependent, as well as novel identified noncanonical mechanisms regulating SOX2. ER stress and PERK modulation appear to provide promising therapeutic targets for therapy in GBM.

miR-371a-3p, miR-373-3p and miR-367-3p as Serum Biomarkers in Metastatic Testicular Germ Cell Cancers Before, During and After Chemotherapy.

BACKGROUND: LDH (lactate dehydrogenase), AFP (alpha-fetoprotein) and ß-HCG (human chorionic gonadotropin) are used in diagnosis and follow-up of testicular germ cell cancer (TGCC) patients. Our aim was to investigate the association between levels of miR-371a-3p, miR-373-3p and miR-367-3p and clinical features in metastatic TGCC. METHODS: relative levels of miR-371a-3p, miR-373-3p and miR-367-3p were evaluated in serum of metastatic TGCC patients. A prospectively included and a retrospectively selected cohort were studied (total patient number = 109). Blood samples were drawn at start of chemotherapy and during follow-up. Serum microRNA (miR) levels were determined using the ampTSmiR test. RESULTS: at start of chemotherapy, miR-371a-3p, miR-373-3p and miR-367-3p levels were positively correlated to LDH. The median level of these miRs was higher in patients who developed a relapse after complete biochemical remission (n = 34) than in those who had complete durable remission (n = 60). Higher levels of miR-367-3p were found in patients with refractory disease (n = 15) compared to those who had complete response. miR levels decreased during the first week of chemotherapy in patients with complete response and stayed below threshold after one year of treatment. CONCLUSION: high miR levels at start of chemotherapy are associated with worse clinical outcome and can assist in early diagnosing of relapses.