Molecular characterization of epithelial-mesenchymal transition and medical treatment related-genes in non-functioning pituitary neuroendocrine tumors.

Introduction: Different medical therapies have been developed for pituitary adenomas. However, Non-Functioning Pituitary Neuroendocrine Tumors (NF-PitNET) have shown little response to them. Furthermore, epithelial-mesenchymal transition (EMT) has been linked to resistance to medical treatment in a significant number of tumors, including pituitary adenomas. Methods: We aimed to evaluate the expression of EMT-related markers in 72 NF-PitNET and 16 non-tumoral pituitaries. To further explore the potential usefulness of medical treatment for NF-PitNET we assessed the expression of somatostatin receptors and dopamine-associated genes. Results: We found that SNAI1, SNAI2, Vimentin, KLK10, PEBP1, Ki-67 and SSTR2 were associated with invasive NF-PitNET. Furthermore, we found that the EMT phenomenon was more common in NF-PitNET than in GH-secreting pituitary tumors. Interestingly, PEBP1 was overexpressed in recurrent NF-PitNET, and could predict growth recurrence with 100% sensitivity but only 43% specificity. In parallel with previously reported studies, SSTR3 is highly expressed in our NF-PitNET cohort. However, SSTR3 expression is highly heterogeneous among the different histological variants of NF-PitNET with very low levels in silent corticotroph adenomas. Conclusion: NF-PitNET showed an enhanced EMT phenomenon. SSTR3 targeting could be a good therapeutic candidate in NF-PitNET except for silent corticotroph adenomas, which express very low levels of this receptor. In addition, PEBP1 could be an informative biomarker of tumor regrowth, useful for predictive medicine in NF-PitNET.

Implications of Heterogeneity of Epithelial-Mesenchymal States in Acromegaly Therapeutic Pharmacologic Response.

Acromegaly is caused by excess growth hormone (GH) produced by a pituitary tumor. First-generation somatostatin receptor ligands (SRLs) are the first-line treatment. Several studies have linked E-cadherin loss and epithelial-mesenchymal transition (EMT) with resistance to SRLs. Our aim was to study EMT and its relationship with SRLs resistance in GH-producing tumors. We analyzed the expression of EMT-related genes by RT-qPCR in 57 tumors. The postsurgical response to SRLs was categorized as complete response, partial response, or nonresponse if IGF-1 was normal, had decreased more than 30% without normalization, or neither of those, respectively. Most tumors showed a hybrid and variable EMT expression profile not specifically associated with SRL response instead of a defined epithelial or mesenchymal phenotype. However, high SNAI1 expression was related to invasive and SRL-nonresponsive tumors. RORC was overexpressed in tumors treated with SRLs before surgery, and this increased expression was more prominent in those cases that normalized postsurgical IGF-1 levels under SRL treatment. In conclusion, GH-producing tumors showed a heterogeneous expression pattern of EMT-related genes that would partly explain the heterogeneous response to SRLs. SNAI1 and RORC may be useful to predict response to SRLs and help medical treatment decision making.

Revisiting the UK Genetic Severity Score for NF2: a proposal for the addition of a functional genetic component.

BACKGROUND: Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterised by the development of multiple schwannomas, especially on vestibular nerves, and meningiomas. The UK NF2 Genetic Severity Score (GSS) is useful to predict the progression of the disease from germline NF2 pathogenic variants, which allows the clinical follow-up and the genetic counselling offered to affected families to be optimised. METHODS: 52 Spanish patients were classified using the GSS, and patients' clinical severity was measured and compared between GSS groups. The GSS was reviewed with the addition of phenotype quantification, genetic variant classification and functional assays of Merlin and its downstream pathways. Principal component analysis and regression models were used to evaluate the differences between severity and the effect of NF2 germline variants. RESULTS: The GSS was validated in the Spanish NF2 cohort. However, for 25% of mosaic patients and patients harbouring variants associated with mild and moderate phenotypes, it did not perform as well for predicting clinical outcomes as it did for pathogenic variants associated with severe phenotypes. We studied the possibility of modifying the mutation classification in the GSS by adding the impact of pathogenic variants on the function of Merlin in 27 cases. This revision helped to reduce variability within NF2 mutation classes and moderately enhanced the correlation between patient phenotype and the different prognosis parameters analysed (R2=0.38 vs R2=0.32, p>0001). CONCLUSIONS: We validated the UK NF2 GSS in a Spanish NF2 cohort, despite the significant phenotypic variability identified within it. The revision of the GSS, named Functional Genetic Severity Score, could add value for the classification of mosaic patients and patients showing mild and moderate phenotypes once it has been validated in other cohorts.

Early Genetic Diagnosis of Neurofibromatosis Type 2 From Skin Plaque Plexiform Schwannomas in Childhood.

Importance: Neurofibromatosis type 2 (NF2) is a devastating genetic condition characterized by the development of multiple tumors of the nervous system. An early diagnosis of individuals with NF2 would facilitate treatment and reduction of disease impact because most severe effects of the disease do not usually develop before adolescence. Little attention has traditionally been paid to dermatological signs in NF2. However, skin plaques are commonly seen in patients with NF2, normally appearing either at birth or early childhood, providing an opportunity for early NF2 detection and testing. Objective: To determine the clinical utility of skin plaque identification and characterization in children for reaching an early diagnosis of patients with NF2 and to evaluate their molecular pathogenesis and their use in the genetic diagnostics of NF2. Design, Setting, and Participants: Diagnostic test study by the histological and genetic characterization of skin plaques from patients with NF2. Patients were 7 individuals with NF2 or clinical suspicion of NF2 treated at the Spanish Reference Center on Phakomatoses. Main Outcomes and Measures: Histological evaluation of all skin plaques was performed. Fresh skin plaques were cultured to obtain Schwann cells and the NF2 gene was genetically analyzed. For all 7 patients, NF2 clinical history was reviewed. Results: In all 7 patients (4 male and 3 female), all skin plaques analyzed were histologically characterized as plexiform schwannomas. Genetic analysis of primary Schwann cell cultures derived from them allowed the identification of a constitutional and a somatic NF2 mutation. Genetic testing allowed the early diagnosis of NF2 in a child only exhibiting the presence of skin plaques. Most of the patients with NF2 analyzed had an early presentation of skin plaques and a severe NF2 phenotype. Conclusions and Relevance: This work emphasizes the clinical utility of a careful dermatological inspection and the correct identification of skin plaques in children for an early diagnosis of NF2. We show for the first time that Schwann cells derived from skin plaque plexiform schwannomas bear the double inactivation of the NF2 gene and thus constitute an excellent source of tissue for genetic testing, especially in the context of mosaicism.

Segmental neurofibromatosis type 2: discriminating two hit from four hit in a patient presenting multiple schwannomas confined to one limb.

BACKGROUND: A clinical overlap exists between mosaic Neurofibromatosis Type 2 and sporadic Schwannomatosis conditions. In these cases a molecular analysis of tumors is recommended for a proper genetic diagnostics. This analysis is challenged by the fact that schwannomas in both conditions bear a somatic double inactivation of the NF2 gene. However, SMARCB1-associated schwannomas follow a four-hit, three-step model, in which both alleles of SMARCB1 and NF2 genes are inactivated in the tumor, with one of the steps being always the loss of a big part of chromosome 22 involving both loci. CASE PRESENTATION: Here we report a 36-year-old woman who only presented multiple subcutaneous schwannomas on her right leg. To help discriminate between both possible diagnoses, an exhaustive molecular genetic and genomic analysis was performed on two schwannomas of the patient, consisting in cDNA and DNA sequencing, MLPA, microsatellite multiplex PCR and SNP-array analyses. The loss of a big part of chromosome 22 (22q12.1q13.33) was identified in both tumors. However, this loss involved the NF2 but not the SMARCB1 locus. SNP-array analysis revealed the presence of the same deletion breakpoint in both schwannomas, indicating that this alteration was actually the first NF2 inactivating hit. In addition, a distinct NF2 point mutation in each tumor was identified, representing independent second hits. In accordance with these results, no deletions or point mutations in the SMARCB1 gene were identified. None of the mutations were present in the blood. Two of the patient's children inherited chromosome 22 deleted in schwannomas of the mother, but in its wild type form. CONCLUSIONS: These results conclusively confirm the segmental mosaic NF2 nature of the clinical phenotype presented.

Neoadjuvant cisplatin plus temozolomide versus standard treatment in patients with unresectable glioblastoma or anaplastic astrocytoma: a differential effect of MGMT methylation.

Patients with unresectable glioblastoma or anaplastic astrocytoma have a dismal prognosis. The role of neoadjuvant chemotherapy prior to irradiation in these patients has been studied primarily in non-randomized studies. We have compared the effect of neoadjuvant chemotherapy plus radiotherapy versus concomitant radiotherapy plus temozolomide in a retrospective analysis of two consecutive series of patients in whom surgery consisted of biopsy only. From 2003 to 2005, 23 patients received two cycles of temozolomide plus cisplatin followed by radiotherapy (Cohort 1), and from 2006 to 2010, 23 additional patients received concomitant radiotherapy and temozolomide followed by adjuvant temozolomide (Cohort 2). In Cohort 1, 91.3 % of patients received all planned chemotherapy cycles. Progression-free and overall survival were 3.3 and 8.5 months, respectively. In Cohort 2, progression-free and overall survival were 5.1 and 11.2 months, respectively. No differences between the two groups were observed in rate of completion of radiotherapy, progression-free or overall survival. MGMT methylation was assessed in 91.3 % of patients. In Cohort 1, patients without MGMT methylation showed a trend towards shorter progression-free survival (P = 0.09), while in Cohort 2, patients without MGMT methylation had longer progression-free survival (P = 0.04). In the overall patient population, neoadjuvant temozolomide plus cisplatin had neither a positive nor negative influence on outcome. However, our findings indicate that patients with methylated MGMT may derive greater benefit from neoadjuvant temozolomide than those with unmethylated MGMT.

In vitro antisense therapeutics for a deep intronic mutation causing Neurofibromatosis type 2.

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder affecting about 1:33 000 newborns, mainly characterized by the development of tumors of the nervous system and ocular abnormalities. Around 85% of germline NF2 mutations are point mutations. Among them, ∼25% affect splicing and are associated with a variable disease severity. In the context of our NF2 Multidisciplinary Clinics, we have identified a patient fulfilling clinical criteria for the disease and exhibiting a severe phenotype. The patient carries a deep intronic mutation (g. 74409T>A, NG_009057.1) that produces the insertion of a cryptic exon of 167pb in the mature mRNA between exons 13 and 14, resulting in a truncated merlin protein (p.Pro482Profs*39). A mutation-specific antisense phosphorodiamidate morpholino oligomer was designed and used in vitro to effectively restore normal NF2 splicing in patient-derived primary fibroblasts. In addition, merlin protein levels were greatly recovered after morpholino treatment, decreasing patient's fibroblasts in vitro proliferation capacity and restoring cytoeskeleton organization. To our knowledge, this is the first NF2 case caused by a deep intronic mutation in which an in vitro antisense therapeutic approximation has been tested. These results open the possibility of using this approach in vivo for this type of mutation causing NF2.

Tumour and serum MGMT promoter methylation and protein expression in glioblastoma patients.

INTRODUCTION: Methylation of the promoter of the MGMT gene and MGMT protein expression are recognized as predictive markers for response to alkylating chemotherapy in glioblastoma (GB). MATERIAL AND METHODS: We have assessed MGMT methylation with the methylation-specific polymerase chain reaction (MSP) in tumor samples from 70 GB patients and in serum samples from 37 of these patients. We have also assessed MGMT protein expression by immunohistochemical (IHC) analysis in tissue samples from 63 of these patients. RESULTS: We found concordance between MGMT methylation status in tissue and serum (Cohen's Kappa = 0.586; p<0.0001). MSP for detection of non-methylated MGMT promoter in serum showed a sensitivity of 95.4% and a specificity of 60%, while the IHC methylation test showed a low specificity (8.9%). Patients whose MGMT promoter was methylated in tissue attained longer progression-free and overall survival. In the multivariate analysis, serum MGMT promoter methylation emerged as an independent factor for longer progression-free and overall survival. CONCLUSION: Serum-based MGMT methylation analysis offers a promising alternative to tumor-based MGMT analysis in cases where tissue samples are unavailable.

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas.

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB(1) and CB(2) receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [(35)S]GTPgammaS binding. Western blot analysis showed that CB(1) receptor immunoreactivity was significantly lower in glioblastoma multiforme (-43%, n=10; p<0.05) than in normal post-mortem brain tissue (n=16). No significant differences were found for astrocytoma (n=6) and meningioma (n=8) samples. Conversely, CB(2) receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n=9; p<0.05) and astrocytoma (471%, n=4; p<0.05) than in control brain tissue (n=10). Finally, the maximal stimulation of [(35)S]GTPgammaS binding by WIN 55,212-2 was significantly lower in glioblastomas (134+/-4%) than in control membranes (183+/-2%; p<0.05). The basal [(35)S]GTPgammaS binding and the EC(50) values were not significantly different between both groups. The present results demonstrate opposite changes in CB(1) and CB(2) receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.

Monoamine oxidase B activity is increased in human gliomas.

Glial tumours are the most common type of brain neoplasm in humans. Tumour classification and grading represent key factors for patient management. However, current grading schemes are still limited by subjective histological criteria. In this context, gliosis has been linked to increases in monoamine oxidase B (MAO-B) activity. Thus, in the present study, MAO-B activity in membranes of glial tumours (n=20), meningiomas (n=12) and non-pathological human brains (n=15) was quantified by [14C]PEA oxidation. MAO-B activity was significantly greater in glioblastoma multiformes than in postmortem control brains (p<0.01) or meningiomas (p<0.001). There were no significant differences in MAO-B activity between glioblastoma multiformes (n=11) and low-grade astrocytomas (n=3) or anaplastic astrocytomas (n=6). In conclusion, the present results demonstrate a significant and selective increase in MAO-B activity in human gliomas when compared with meningiomas or non-tumoural tissue. These results suggest that the quantification of MAO-B activity may be a useful diagnostic tool for differentiating glial tumours from other types of brain tumours or surrounding normal brain tissue.