Molecular determinants of enhanced response to somatostatin receptor ligands after debulking in large GH-producing adenomas.

OBJECTIVE: Large somatotrophic adenomas depict poor response to somatostatin receptor ligands (SRLs). Debulking has shown to enhance SRLs effect in some but not all cases and tumour volume reduction has been proposed as the main predictor of response. No biological studies have been performed so far in this matter. We aimed to identify molecular markers of response to SRLs after surgical debulking in GH-secreting adenomas. DESIGN: We performed a multicenter retrospective study. PATIENTS: 24 patients bearing large GH-producing tumours. MEASUREMENTS: Clinical data and SRLs response both before and after surgical debulking were collected, and 21 molecular biomarkers of SRLs response were studied in tumour samples by gene expression. RESULTS: From the 21 molecular markers studied, only two of them predicted enhanced SRLs response after surgery. Tumours with improved response to SRLs after surgical debulking showed lower levels of Ki-67 (MKI67, FC = 0.17 and P = .008) and higher levels of RAR-related orphan receptor C (RORC) (FC = 3.1 and P Ë‚ .001). When a cut-off of no detectable expression was used for Ki-67, the model provided a sensitivity of 100% and a specificity of 52.6% with an area under the curve of 65.8%. Using a cut-off of 2 units of relative expression of RORC, the prediction model showed 100% of sensitivity and specificity. CONCLUSIONS: High levels of RORC and low levels of Ki-67 identify improved SRLs response after surgical debulking in large somatotropic adenomas. To determine their expression would facilitate medical treatment decision-making after surgery.

Lack of cytomegalovirus detection in human glioma.

Gliomas are the most common brain tumors and include a variety of histologic types and grades of malignancy. They arise from glial cells and represent approximately 70% of the primary brain tumors. According to the criteria of the World Health Organization (WHO), the majority of gliomas can be classified into four grades of malignancy (I-IV). Virus infection, especially by DNA viruses and retroviruses, which may cause insertion of viral DNA sequences into the host genome, often triggers the host defense mechanisms. Particularly, the DNA methylation machinery can be activated to cause the methylation of foreign movable viral sequences and, therefore, silence viral gene expression. Several studies have shown the presence of Human Cytomegalovirus (HCMV) in glioblastoma, suggesting that the virus may participate in tumor pathogenesis. But this relationship is controversial because many other studies did not detect HCMV in these tumors. This study aims to detect the presence of HCMV in several samples of human glioma (94 formalin-fixed, paraffin-embedded samples and 28 snap-frozen samples) by different sensitive techniques. We have been unable to detect HCMV DNA and proteins in glioma samples. Therefore, arguments used so far to conclude that HCMV is an oncomodulator virus in gliomas must be, in our view, seriously reconsidered.

Association of Notch pathway down-regulation with Triple Negative/Basal-like breast carcinomas and high tumor-infiltrating FOXP3+ Tregs.

T regulatory cells (Tregs) are a lineage of lymphocytes involved in immune response suppression that are characterized by the expression of the forkhead box P3 (FOXP3) transcription factor. Notch pathway regulates FOXP3 transcription in Tregs, but its role in breast cancer is unknown. We aimed at studying whether Notch pathway regulates FOXP3 expression and Tregs content in breast cancer, and its association with luminal breast carcinomas. We analyzed by quantitative Real-Time PCR the mRNA levels of FOXP3, Notch pathway genes (Notch1, Notch2, Notch4 and Jagged1) and STAT3 in a series of 152 breast carcinomas including hormone receptor-positive and -negative phenotypes (luminal and Triple Negative/Basal-like). We also studied the protein expression of Notch1, STAT3 and FOXP3 by immunohistochemistry. High FOXP3 mRNA levels correlated with larger tumor size (p=0.010), histological grade 3 (p=0.008) and positive lymph-node status (p=0.031). Also, low levels of Notch pathway genes mRNA correlated with poor prognostic factors such as larger tumor size, positive lymph-node status, tumor phenotype and infiltrating tumor Tregs. A survival analysis for the patients showed that large tumor size, histological grade 3, vascular invasion, infiltrating Tregs and low Notch1 mRNA expression were significantly associated with a decreased patients' overall survival (p≤0.05). On a multivariate analysis, high Tregs content (HR=3.00, 95% CI 1.04-8.90, p=0.042) and low Notch1 mRNA levels (HR=3.33, 95% CI 1.02-10.86, p=0.046) were independent markers for overall survival. Our results support that the Notch pathway up-regulation promotes luminal breast carcinomas, whereas down-regulation correlates with the expression of FOXP3, favors tumor Tregs infiltration and associates with Triple Negative/Basal-like tumors.

FOXA2 mRNA expression is associated with relapse in patients with Triple-Negative/Basal-like breast carcinoma.

The FOXA family of transcription factors regulates chromatin structure and gene expression especially during embryonic development. In normal breast tissue FOXA1 acts throughout mammary development; whereas in breast carcinoma its expression promotes luminal phenotype and correlates with good prognosis. However, the role of FOXA2 has not been previously studied in breast cancer. Our purpose was to analyze the expression of FOXA2 in breast cancer cells, to explore its role in breast cancer stem cells, and to correlate its mRNA expression with clinicopathological features and outcome in a series of patients diagnosed with breast carcinoma. We analyzed FOXA2 mRNA expression in a retrospective cohort of 230 breast cancer patients and in cell lines. We also knocked down FOXA2 mRNA expression by siRNA to determine the impact on cell proliferation and mammospheres formation using a cancer stem cells culture assay. In vitro studies demonstrated higher FOXA2 mRNA expression in Triple-Negative/Basal-like cells. Further, when it was knocked down, cells decreased proliferation and its capability of forming mammospheres. Similarly, FOXA2 mRNA expression was detected in 10% (23/230) of the tumors, especially in Triple-Negative/Basal-like phenotype (p < 0.001, Fisher's test). Patients whose tumors expressed FOXA2 had increased relapses (59 vs. 79%, p = 0.024, log-rank test) that revealed an independent prognostic value (HR = 3.29, C.I.95% = 1.45-7.45, p = 0.004, Cox regression). Our results suggest that FOXA2 promotes cell proliferation, maintains cancer stem cells, favors the development of Triple-Negative/Basal-like tumors, and is associated with increase relapses.

Identification of new targets for glioblastoma therapy based on a DNA expression microarray.

This study provides a comprehensive perspective on the deregulated pathways and impaired biological functions prevalent in human glioblastoma (GBM). In order to characterize differences in gene expression between individuals diagnosed with GBM and healthy brain tissue, we have designed and manufactured a specific, custom DNA microarray. The results obtained from differential gene expression analysis were validated by RT-qPCR. The datasets obtained from the analysis of common differential expressed genes in our cohort of patients were used to generate protein-protein interaction networks of functionally enriched genes and their biological functions. This network analysis, let us to identify 16 genes that exhibited either up-regulation (CDK4, MYC, FOXM1, FN1, E2F7, HDAC1, TNC, LAMC1, EIF4EBP1 and ITGB3) or down-regulation (PRKACB, MEF2C, CAMK2B, MAPK3, MAP2K1 and PENK) in all GBM patients. Further investigation of these genes and enriched pathways uncovered in this investigation promises to serve as a foundational step in advancing our comprehension of the molecular mechanisms underpinning GBM pathogenesis. Consequently, the present work emphasizes the critical role that the unveiled molecular pathways likely play in shaping innovative therapeutic approaches for GBM management. We finally proposed in this study a list of compounds that target hub of GBM-related genes, some of which are already in clinical use, underscoring the potential of those genes as targets for GBM treatment.

Integrative clinical, hormonal, and molecular data associate with invasiveness in acromegaly: REMAH study.

INTRODUCTION: Growth hormone (GH)-secreting pituitary tumors (GHomas) are the most common acromegaly cause. At diagnosis, most of them are macroadenomas, and up to 56% display cavernous sinus invasion. Biomarker assessment associated with tumor growth and invasion is important to optimize their management. OBJECTIVES: The study aims to identify clinical/hormonal/molecular biomarkers associated with tumor size and invasiveness in GHomas and to analyze the influence of pre-treatment with somatostatin analogs (SSAs) or dopamine agonists (DAs) in key molecular biomarker expression. METHODS: Clinical/analytical/radiological variables were evaluated in 192 patients from the REMAH study (ambispective multicenter post-surgery study of the Spanish Society of Endocrinology and Nutrition). The expression of somatostatin/ghrelin/dopamine system components and key pituitary/proliferation markers was evaluated in GHomas after the first surgery. Univariate/multivariate regression studies were performed to identify association between variables. RESULTS: Eighty percent of patients harbor macroadenomas (63.8% with extrasellar growth). Associations between larger and more invasive GHomas with younger age, visual abnormalities, higher IGF1 levels, extrasellar/suprasellar growth, and/or cavernous sinus invasion were found. Higher GH1 and lower PRL/POMC/CGA/AVPR1B/DRD2T/DRD2L expression levels (P < .05) were associated with tumor invasiveness. Least Absolute Shrinkage and Selection Operator's penalized regression identified combinations of clinical and molecular features with areas under the curve between 0.67 and 0.82. Pre-operative therapy with DA or SSAs did not alter the expression of any of the markers analyzed except for DRD1/AVPR1B (up-regulated with DA) and FSHB/CRHR1 (down-regulated with SSAs). CONCLUSIONS: A specific combination of clinical/analytical/molecular variables was found to be associated with tumor invasiveness and growth capacity in GHomas. Pre-treatment with first-line drugs for acromegaly did not significantly modify the expression of the most relevant biomarkers in our association model. These findings provide valuable insights for risk stratification and personalized management of GHomas.

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.

Data mining analyses for precision medicine in acromegaly: a proof of concept.

Predicting which acromegaly patients could benefit from somatostatin receptor ligands (SRL) is a must for personalized medicine. Although many biomarkers linked to SRL response have been identified, there is no consensus criterion on how to assign this pharmacologic treatment according to biomarker levels. Our aim is to provide better predictive tools for an accurate acromegaly patient stratification regarding the ability to respond to SRL. We took advantage of a multicenter study of 71 acromegaly patients and we used advanced mathematical modelling to predict SRL response combining molecular and clinical information. Different models of patient stratification were obtained, with a much higher accuracy when the studied cohort is fragmented according to relevant clinical characteristics. Considering all the models, a patient stratification based on the extrasellar growth of the tumor, sex, age and the expression of E-cadherin, GHRL, IN1-GHRL, DRD2, SSTR5 and PEBP1 is proposed, with accuracies that stand between 71 to 95%. In conclusion, the use of data mining could be very useful for implementation of personalized medicine in acromegaly through an interdisciplinary work between computer science, mathematics, biology and medicine. This new methodology opens a door to more precise and personalized medicine for acromegaly patients.

Integrative Clinical, Radiological, and Molecular Analysis for Predicting Remission and Recurrence of Cushing Disease.

CONTEXT: Adrenocorticotropin (ACTH)-secreting pituitary tumors (ACTHomas) are associated with severe comorbidities and increased mortality. Current treatments mainly focus on remission and prevention of persistent disease and recurrence. However, there are still no useful biomarkers to accurately predict the clinical outcome after surgery, long-term remission, or disease relapse. OBJECTIVES: This work aimed to identify clinical, biochemical, and molecular markers for predicting long-term clinical outcome and remission in ACTHomas. METHODS: A retrospective multicenter study was performed with 60 ACTHomas patients diagnosed between 2004 and 2018 with at least 2 years' follow-up. Clinical/biochemical variables were evaluated yearly. Molecular expression profile of the somatostatin/ghrelin/dopamine regulatory systems components and of key pituitary factors and proliferation markers were evaluated in tumor samples after the first surgery. RESULTS: Clinical variables including tumor size, time until diagnosis/first surgery, serum prolactin, and postsurgery cortisol levels were associated with tumor remission and relapsed disease. The molecular markers analyzed were distinctly expressed in ACTHomas, with some components (ie, SSTR1, CRHR1, and MKI67) showing instructive associations with recurrence and/or remission. Notably, an integrative model including selected clinical variables (tumor size/postsurgery serum cortisol), and molecular markers (SSTR1/CRHR1) can accurately predict the clinical evolution and remission of patients with ACTHomas, generating a receiver operating characteristic curve with an area under the curve of 1 (P < .001). CONCLUSION: This study demonstrates that the combination of a set of clinical and molecular biomarkers in ACTHomas is able to accurately predict the clinical evolution and remission of patients. Consequently, the postsurgery molecular profile represents a valuable tool for clinical evaluation and follow-up of patients with ACTHomas.

Proposal of a clinically relevant working classification of pituitary neuroendocrine tumors based on pituitary transcription factors.

The immunohistochemistry (IHC) characterization of pituitary transcription factors (PTFs) PIT1, TPIT, and SF1, which enable the identification of three different adenohypophyseal cell lines, has been incorporated into the latest classification system of the World Health Organization (WHO) for pituitary adenomas. This change overturns the concept of the adenoma as solely a hormone producer and classifies these tumors based on their cell lineage. The aim of the study was to provide a diagnostic algorithm, based on IHC expression of hypophyseal hormones with potential use in diagnostic practice, contributing to an improved classification of pituitary adenomas. Our sample included 146 pituitary adenomas previously classified based on hormonal subtypes by IHC (former 2004 WHO criteria) and re-evaluated after the IHC quantification of PIT1, TPIT, and SF1 expression, under WHO 2017 recommendations. We assessed the correlation between expression of PTFs and the classification as per hormonal IHC and correlated clinicopathological profiles based on PTFs. The IHC study of PTFs allowed reclassification of 82% of tumors that were negative for all pituitary hormones, with 21 positive cases for SF1 (reclassified as gonadotroph tumors), 1 positive case for TPIT (reclassified as a corticotroph tumor), and 4 positive cases for PIT1. Using SF1 enabled detection of a substantial portion of gonadotroph tumors, reducing the estimated prevalence of null cell tumors to less than 5%, and identification of plurihormonal pituitary neuroendocrine tumors with PIT1-SF1 coexpression and hormone-negative PIT1s, a group in which we did not observe differences in the clinical behavior compared with the rest of the tumors of the same cell lineage.Our results suggest that applying a diagnostic algorithm based on the study of PTFs could contribute to improving the classification of pituitary adenomas. By adding TPIT assessment, we propose a two-step algorithm, with hypophyseal hormones being used in a selective modality, depending on initial results.

Hedgehog gene expression patterns among intrinsic subtypes of breast cancer: Prognostic relevance.

PURPOSE: Hedgehog (Hh) signaling is a crucial developmental regulatory pathway recognized as a primary oncogenesis driver in various human cancers. However, its role in breast carcinoma (BC) has been underexplored. METHODS: We analyzed the expression of several Hh associated genes in a clinical series and breast cancer cell lines. We included 193 BC stratified according to intrinsic immunophenotypes. Gene expression profiling ofBOC, PTCH, SMO, GLI1, GLI2, and GLI3 was performed by qRT-PCR. Results were correlated with clinical-pathological variables and outcome. RESULTS: We observed expression ofGLI2 in triple-negative/basal-like (TN/BL) and GLI3 in luminal cells. In samples, BOC, GLI1, GLI2, and GLI3 expression correlated significantly with luminal tumors and good prognostic factors. In contrast, PTCH and SMO correlated with TN/BL phenotype and nodal involvement. Patients whose tumors expressed SMO had a poorer outcome, especially those with HER2 phenotype. Positive lymph-node status and high SMO remained independent poor prognostic factors. CONCLUSION: Our results support a differential Hh pathway activation in BC phenotypes.SMO levels stratified patients at risk of recurrence and death in HER2 phenotype, and it showed an independent prognostic value. Therefore, SMO could be a potential therapeutic target for a subset of BC patients.

Increased E2F1 mRNA and miR-17-5p Expression Is Correlated to Invasiveness and Proliferation of Pituitary Neuroendocrine Tumours.

miR-17-5p and E2F1 have been described as deregulated in cancer, but they have scarcely been studied in pituitary neuroendocrine tumours (PitNETs). This study evaluates the relationship of E2F1 and miR-17-5p with the invasiveness and proliferation of PitNETs. In this cross-sectional descriptive study, we evaluated the expression of E2F1, MYC, and miR-17-5p by quantitative real time PCR analysis in 60 PitNETs: 29 gonadotroph (GT), 15 functioning somatotroph (ST), and 16 corticotroph (CT) tumours, of which 8 were silent (sCT). The clinical data were collected from the Spanish Molecular Register of Pituitary Adenomas (REMAH) database. We defined invasiveness according to the Knosp classification and proliferation according to a molecular expression of Ki-67 ≥ 2.59. E2F1 was more expressed in invasive than in non-invasive tumours in the whole series (p = 0.004) and in STs (p = 0.01). In addition, it was overexpressed in the silent subtypes (GTs and sCTs; all macroadenomas) and normoexpressed in the functioning ones (fCTs and STs; some microadenomas). miR-17-5p was more expressed in proliferative than in non-proliferative tumours (p = 0.041) in the whole series but not by subtypes. Conclusions: Our study suggests that in PitNETs, E2F1 could be a good biomarker of invasiveness, and miR-17-5p of proliferation, helping the clinical management of these tumours.

Differential Expression of MicroRNAs in Silent and Functioning Corticotroph Tumors.

The potential role of miRNAs in the silencing mechanisms of pituitary neuroendocrine tumors (PitNETs) has not been addressed. The aim of the present study was to evaluate the expression levels and the potential associated role of some miRNAs, pathways, and transcription factors in the silencing mechanisms of corticotroph tumors (CTs). Accordingly, the expression of miR-375, miR-383, miR-488, miR-200a and miR-103; of PKA, MAP3K8, MEK, MAPK3, NGFIB, NURR1, PITX1, and STAT3 were analyzed via qRT-PCR in 23 silent and 24 functioning CTs. miR-200a and miR-103 showed significantly higher expression in silent than in functioning CTs, even after eliminating the bias of tumor size, therefore enabling the differentiation between the two variants. Additionally, miR-383 correlated negatively with TBX19 in silent CTs, a transcription factor related with the processing of POMC that can participate in the silencing mechanisms of CTs. Finally, the gene expression levels of miR-488, miR-200a, and miR-103 were significantly higher in macroadenomas (functioning and silent) than in microadenomas. The evidence from this study indicates that miRNAs could be involved in the pathophysiology of CTs. The translational implications of these findings suggest that pharmacological treatments specifically targeting these miRNAs could become a promising therapeutic option for these patients.

Molecular profiling for acromegaly treatment: a validation study.

Pharmacologic treatment of acromegaly is currently based upon assay-error strategy, the first-generation somatostatin receptor ligands (SRL) being the first-line treatment. However, about 50% of patients do not respond adequately to SRL. Our objective was to evaluate the potential usefulness of different molecular markers as predictors of response to SRL. We used somatotropinoma tissue obtained after surgery from a national cohort of 100 acromegalic patients. Seventy-one patients were treated with SRL during at least 6 months under maximal therapeutic doses according to IGF1 values. We analyzed the expression of SSTR2, SSTR5, AIP, CDH1 (E-cadherin), MKI67 (Ki-67), KLK10, DRD2, ARRB1, GHRL, In1-Ghrelin, PLAGL1 and PEBP1 (RKIP) by RT-qPCR and mutations in GNAS gene by Sanger sequencing. The response to SRL was categorized as complete response (CR), partial (PR) or non-response (NR) if IGF1 was normal, between >2<3 SDS or >3 SDS IGF1 at 6 months of follow-up, respectively. From the 71 patients treated, there were 27 CR (38%), 18 PR (25%) and 26 NR (37%). SSTR2, Ki-67 and E-cadherin were associated with SRL response (P < 0.03, P < 0.01 and P < 0.003, respectively). E-cadherin was the best discriminator for response prediction (AUC = 0.74, P < 0.02, PPV of 83.7%, NPV of 72.6%), which was validated at protein level. SSTR5 expression was higher in patients pre-treated with SRL before surgery. We conclude that somatotropinomas showed heterogeneity in the expression of genes associated with SRL response. E-cadherin was the best molecular predictor of response to SRL. Thus, the inclusion of E-cadherin in subsequent treatment-decision after surgical failure may be useful in acromegaly.

Silent somatotropinomas.

Silent somatotroph pituitary neuroendocrine tumors (or silent growth hormone pituitary neuroendocrine tumors, SGH-PitNET) are neoplasias with positive immunostaining for growth hormone (GH), in patients with no signs and symptoms of acromegaly nor biochemical evidence of GH hypersecretion. From a clinical stand-point they are considered and managed as non-functioning pituitary tumors, since they usually come to evidence due to mass-effects (headache, visual impairment, hypopituitarism) or as asymptomatic pituitary incidentalomas. SGH-PitNET have deserved little attention in the medical literature, and no specific guidelines exist regarding their management. However, identification of a particular tumor lineage through immunostaining patterns of non-functioning pituitary tumors may determine postoperative medical therapy in the near future. This review updates the current knowledge about the epidemiologic, clinical, pathological and molecular characteristics of this particular type of pituitary tumors.

DNA Methylation of Tumor Suppressor Genes in Pituitary Neuroendocrine Tumors.

CONTEXT: Epigenetic alterations may play a role in the development and behavior of pituitary neuroendocrine tumors (PitNETs). OBJECTIVE: To evaluate the effect of methylation of tumor suppressor genes (TSGs) on their gene expression and on the behavior of PitNETs. MATERIAL AND METHODS: We used methylation-specific multiplex ligation-dependent probe amplification and quantitative real-time PCR techniques to analyze the DNA-promoter hypermethylation and gene expression of 35 TSGs in 105 PitNETs. We defined functionality, size, and invasiveness of tumors according to their clinical manifestations, Hardy's classification, and MRI invasiveness of the cavernous sinus, respectively. RESULTS: We observed different methylation patterns among PitNET subtypes. The methylation status of TP73 correlated negatively with its gene expression in the overall series (P = 0.013) and in some subtypes. MSH6 and CADM1 showed higher methylation frequency in macroadenomas than in microadenomas in the overall series and in corticotroph PitNETs (all P ≤ 0.053). ESR1 and RASSF1 were more highly methylated in noninvasive than in invasive tumors in the overall series (P = 0.054 and P = 0.031, respectively) and in the gonadotroph subtype (P = 0.055 and P = 0.050, respectively). ESR1 and CASP8 appeared more hypermethylated in functioning than in silent corticotroph tumors (P = 0.034 and P = 0.034, respectively). CONCLUSIONS: DNA methylation of TSGs has a selective effect on their gene expression and on the growth and invasiveness of PitNETs. Its involvement in their functionality is biased because all silent operated tumors are macroadenomas, whereas all operated microadenomas are functioning ones. Therefore, the subtypes of PitNETs should be considered different entities.

How Valuable Is the RT-qPCR of Pituitary-Specific Transcription Factors for Identifying Pituitary Neuroendocrine Tumor Subtypes According to the New WHO 2017 Criteria?

The classification of pituitary neuroendocrine tumors (PitNETs) subtypes continues generating interest. In 2017, the World Health Organization (WHO) proposed considering the immunohistochemical (IHC) analysis of pituitary-specific transcription factors (TF) for their typification. The present study targeted the quantification of pituitary-specific TF (TPIT, PIT-1, SF-1, GATA2, ESR1) gene expression by RT-qPCR to overcome the shortcomings of IHC and to complement it. We analyzed 251 tumors from our collection of PitNETs and performed additional IHC studies in a subset of 56 samples to analyze the concordance between gene and protein expression of the TF. The molecular and IHC studies allowed us to significantly reduce the percentage of null cell tumors in our series, most of which were reclassified as gonadotroph tumors. The concordance between the molecular and the immunohistochemical studies was good for tumors coming from the corticotroph and Pit-1 lineages but worsened for the rest of the tumors. Indeed, the RT-qPCR helped to improve the typification of plurihormonal Pit-1 and unusual tumors. Overall, our results suggest that the RT-qPCR of pituitary-specific TF and hormone genes could help pathologists, endocrinologists, and neurosurgeons to improve the management of patients with pituitary tumors.

Molecular determinants of the response to medical treatment of growth hormone secreting pituitary neuroendocrine tumors.

Acromegaly is a chronic systemic disease mainly caused by a growth hormone (GH)-secreting pituitary neuroendocrine tumor (PitNETs), which is associated with many health complications and increased mortality when not adequately treated. Transsphenoidal surgery is considered the treatment of choice in GH-secreting PitNETs, but patients in whom surgery cannot be considered or with persistent disease after surgery require medical therapy. Treatment with available synthetic somatostatin analogues (SSAs) is considered the mainstay in the medical management of acromegaly which exert their beneficial effects through the binding to a family of G-protein coupled receptors encoded by 5 genes (SSTR1-5). However, although it has been demonstrated that the SST1-5 receptors are physically present in tumor cells, SSAs are in many cases ineffective (i.e. approximately 10-30% of patients with GH-secreting PitNET are unresponsive to SSAs), suggesting that other cellular/molecular determinants could be essential for the response to the pharmacological treatment in patients with GH-secreting PitNETs. Therefore, the scrutiny of these determinants might be used for the identification of subgroups of patients in whom an appropriate pharmacological treatment can be successfully employed (responders vs. non-responders). In this review, we will describe some of the existing, classical and novel, genetic and molecular determinants involved in the response of patients with GH-secreting PitNETs to the available therapeutic treatments, as well as new molecular/therapeutic approaches that could be potentially useful for the treatment of GH-secreting PitNETs.

Is it time to consider the expression of specific-pituitary hormone genes when typifying pituitary tumours?

The aim of the present study is to check whether we can replicate, in an independent series, previous results showing that the molecular study of pituitary-specific gene expression complements the inmunohistochemical identification of pituitary neuroendocrine tumours. We selected 112 patients (51 (46.4%) women; mean age 51.4±16 years; 102 macroadenomas (91.9%), 9 microadenomas (8.1%)) with complete clinical, radiological, immunohistochemical and molecular data from our data set of pituitary neuroendocrine tumours. Patients were different from those previously studied. We measured the expression of the pituitary-specific hormone genes and type 1 corticotrophin-releasing hormone and arginine vasopressin 1b receptors, by quantitative real-time polymerase chain reaction using TaqMan probes. Afterwards, we identified the different pituitary neuroendocrine tumour subtypes following the 2017 World Health Organization classification of pituitary tumours, calculating the concordance between their molecular and immuhistochemical identification. The concordance between molecular and immunohistochemical identification of functioning pituitary neuroendocrine tumours with the clinical diagnosis was globally similar to the previous series, where the SYBR Green technique was used instead of TaqMan probes. Our results also corroborated the poor correlation between molecular and immunohistochemical detection of the silent pituitary neuroendocrine tumour variants. This discrepancy was more remarkable in lactotroph, null-cell and plurihormonal pituitary neuroendocrine tumours. In conclusion, this study validates the results previously published by our group, highlighting a complementary role for the molecular study of the pituitary-specific hormone genes in the typification of pituitary neuroendocrine tumours subtypes.

CD44 induces FOXP3 expression and is related with favorable outcome in breast carcinoma.

We studied the relationship between CD44 and Forkhead box P3 (FOXP3) gene expression in cell lines and breast carcinomas and their association with clinicopathological variables and patient outcome. We assessed messenger RNA (mRNA) expression of CD44 and FOXP3 by quantitative real-time PCR and determined the number of FOXP3+ Tregs by immunohistochemistry in 264 breast cancer specimens. CD44 was stimulated with hyaluronan treatment, and the accompanying changes in FOXP3 mRNA expression in breast cancer cell lines representing breast cancer subtype were assessed. We found that lower CD44 expression correlated with the presence of necrosis, lymph-vascular invasion, grade 3 tumors, and aggressive phenotype (HER2 and basal-like). FOXP3 mRNA correlated positively with CD44 mRNA expression and Treg content. Moreover, stimulation of CD44 expression by hyaluronan in cell lines increased FOXP3 expression, which supports that their regulation is associated. Survival analysis revealed that low CD44 expression is associated with higher frequency of recurrence. Our findings indicate that CD44 has a regulatory role in FOXP3 expression and is associated with good prognostic factors in breast cancer.