TYPE Original Research PUBLISHED 22 March 2023 DOI 10.3389/fendo.2023.1129213 Molecular characterization of OPEN ACCESS epithelial-mesenchymal EDITED BY Murat Aydin Sav, Yeditepe University, Türkiye transition and medical treatment REVIEWED BY Donatella Treppiedi, related-genes in non-functioning University of Milan, Italy Andrea Lania, Humanitas University, Italy pituitary neuroendocrine tumors *CORRESPONDENCE Manel Puig-Domingo Joan Gil 1,2,3†, Montserrat Marques-Pamies 1,4†, Elena Valassi 1,2,3, mpuigd@igtp.cat Guillermo Serra 5, Isabel Salinas 6, Gemma Xifra 7, † These authors share first authorship Paula Casano-Sancho 8, Cristina Carrato 9, Betina Biagetti 10, SPECIALTY SECTION This article was submitted to Gemma Sesmilo 11, Jennifer Marcos-Ruiz 1, Pituitary Endocrinology, Helena Rodriguez-Lloveras 1, Anna Rueda-Pujol 1, a section of the journal Frontiers in Endocrinology Anna Aulinas 2,3, Alberto Blanco 12, Cristina Hostalot 12, RECEIVED 21 December 2022 Andreu Simó-Servat 13, Fernando Muñoz 14, Marta Rico 14, 23 February 2023 ACCEPTED PUBLISHED 22 March 2023 Javier Ibáñez-Domı́nguez 15, Esteban Cordero 16, CITATION Susan M. Webb 2,3, Mireia Jordà 1 and Manel Puig-Domingo 1,3,4* Gil J, Marques-Pamies M, Valassi E, 1 Endocrine Research Unit, Germans Trias i Pujol Research Institute (IGTP), Badalona, Spain, Serra G, Salinas I, Xifra G, 2 Department of Endocrinology, Research Center for Pituitary Diseases, Hospital Sant Pau, IIB-SPau, Casano-Sancho P, Carrato C, Biagetti B, Department of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Centro de Sesmilo G, Marcos-Ruiz J, Investigación en Red de Enfermedades Raras, CIBERER, Unit 747, Instituto de Salud Carlos III, Rodriguez-Lloveras H, Rueda-Pujol A, Madrid, Spain, 4 Department of Endocrinology, Hospital Municipal de Badalona, Badalona, Aulinas A, Blanco A, Hostalot C, Catalonia, Spain, 5 Department of Endocrinology, Son Espases University Hospital, Palma de Simó-Servat A, Muñoz F, Rico M, Mallorca, Spain, 6 Department of Endocrinology and Nutrition, Germans Trias i Pujol University Ibáñez-Domı´nguez J, Cordero E, Hospital, Badalona, Spain, 7 Department of Endocrinology, Josep Trueta University Hospital, Webb SM, Jordà M and Puig-Domingo M Girona, Spain, 8 Centro de Investigación en Red de Diabetes y Enfermedades Metabólicas Asociadas (2023) Molecular characterization of (CIBERDEM), Pediatric Endocrinology Unit, Institut de Recerca SJS 39-57, Hospital Sant Joan de Déu, epithelial-mesenchymal transition and University of Barcelona, Esplugues, Spain, 9 Department of Pathology, Germans Trias i Pujol University medical treatment related-genes in Hospital, Badalona, Spain, 10Department of Endocrinology, University Hospital Vall d’Hebron, non-functioning pituitary Barcelona, Spain, 11 Department of Endocrinology, Dexeus University Hospital, Barcelona, Spain, neuroendocrine tumors. 12 Department of Neurosurgery, Germans Trias i Pujol University Hospital, Badalona, Spain, Front. Endocrinol. 14:1129213. doi: 10.3389/fendo.2023.1129213 13 Department of Endocrinology, Hospital Universitari Mútua de Terrassa, Terrassa, Spain, 14 Department of Neurosurgery, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 15 Department COPYRIGHT of Neurosurgery, Son Espases University Hospital, Palma de Mallorca, Spain, 16 Department of © 2023 Gil, Marques-Pamies, Valassi, Serra, Neurosurgery, University Hospital Vall d’Hebron, Barcelona, Spain Salinas, Xifra, Casano-Sancho, Carrato, Biagetti, Sesmilo, Marcos-Ruiz, Rodriguez-Lloveras, Rueda-Pujol, Aulinas, Blanco, Hostalot, Simó-Servat, Muñoz, Rico, Ibáñez-Domı´nguez, Cordero, Webb, Jordà Introduction: Different medical therapies have been developed for pituitary and Puig-Domingo. This is an open-access article distributed under the terms of the adenomas. However, Non-Functioning Pituitary Neuroendocrine Tumors (NF- Creative Commons Attribution License PitNET) have shown little response to them. Furthermore, epithelial- (CC BY). The use, distribution or reproduction in other forums is permitted, mesenchymal transition (EMT) has been linked to resistance to medical provided the original author(s) and the treatment in a significant number of tumors, including pituitary adenomas. copyright owner(s) are credited and that the original publication in this journal is Methods: We aimed to evaluate the expression of EMT-related markers in 72 NF- cited, in accordance with accepted academic practice. No use, distribution or PitNET and 16 non-tumoral pituitaries. To further explore the potential reproduction is permitted which does not usefulness of medical treatment for NF-PitNET we assessed the expression of comply with these terms. 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 Frontiers in Endocrinology 01 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 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. KEYWORDS Epithelial-mesenchymal transition, non-functioning pituitary adenomas, somatostatin receptor ligands, dopamine agonists, somatostatin analogs 1 Introduction unpredictable. Tumor reduction has only been observed in 12% of patients after octreotide treatment and in 28% of patients treated Non-Functioning Pituitary Neuroendocrine Tumors (NF- with DA therapy (13). Some authors suggested that SRLs treatment PitNET) are defined as pituitary tumors that do not secrete in NF-PitNET could only be useful for the stabilization of tumor biologically active hormones depicting specific hormonal symptoms. growth without any significant effect on tumor shrinkage (14). These neoplasms are the most common pituitary tumors in humans Other authors have provided evidence favoring DA use when there and when they come to medical attention, it is usually due to a mass is post-surgical residual tumor (15), independently of any other effect and/or hypopituitarism (1–3). Histologically, NF-PitNET characteristic of the case. comprise a heterogeneous group of tumors arising from different Epithelial–mesenchymal transition (EMT) is a process that pituitary cell lineages (4). The new WHO Classification of Pituitary restructures the cell from an epithelial to a mesenchymal Tumors 2022 divides NF-PitNET according to the cell of origin; phenotype driven by a network of transcription factors (16). This however, it does not contain specific molecular information linked process produces changes in post-translational regulation to treatment response prediction (5–7). mechanisms and gene expression, leading to the loss of cell Nowadays, transsphenoidal surgery is the first-line treatment polarity and epithelial characteristics and the acquisition of for most of these patients, whereas medical therapy -mostly increased migratory and invasive properties. EMT is not a binary temozolomide in aggressive tumors- and irradiation are reserved process, and distinct intermediate cellular states have been reported for patients that are not good surgical candidates or that have (17). Epithelial-Mesenchymal Transition (EMT) has been linked to undergone unsuccessful surgery (5, 8). The recurrence rate of NF- both the clinical course of NF-PitNET (18, 19) and resistance to PitNET in patients who underwent complete surgical resection is medical therapy in pituitary tumors (20–23). Despite not being fully 20-25% at 10 years, while it rises up to 50-60% in case of partial understood, several mechanisms have been described to explain the removal (9, 10). association of EMT and the lack of response to therapy in PitNETs. Therefore, there is a need to identify molecular targets in NF- It has been proposed that EMT could disrupt alternative splicing in PitNETs that clarify their biological behavior leading to the GH-secreting tumors (21) and that this is associated with a lack of development of a more specific medical treatment. The current response to somatostatin receptor ligands (23, 24). Another possible available targeted medical treatments for pituitary tumors are first explanation is the involvement of the cytoskeleton, in particular, the and second generation somatostatin receptor ligands (SRLs), actin binding protein filamin A that regulates localization, dopamine receptor D2 agonists (DA) and the alkylating agent expression and signaling of SSTR2 and DRD2 in some PitNETs temozolomide, usually reserved for very aggressive tumors (11, (25–29). Moreover, b-Arrestin 2 mediates the downstream effects of 12). Tumor shrinkage during therapy with either DA or SRLs has DRD2 in NF-PitNETs (30). The aim of our study is to elucidate the been previously reported in a variable percentage of NF-PitNET molecular landscape of NF-PitNETs for EMT-associated genes and cases; however, response of NF-PitNET to medical treatment is still genes related to the response to medical therapy with SRLs considered poor, insufficiently understood and nowadays and DAs. Frontiers in Endocrinology 02 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 TABLE 1 Description of the cohort. 2 Materials and methods Patients Characteristics 2.1 Patients Cohort (N) 72 This retrospective study enrolled 72 adult patients with NF-PitNET Male/Female 37/35 from 26 tertiary centers from all over Spain who underwent pituitary Age (years) 60.2 ± 13.5 surgery and had tissue availability from 2014 to 2020. NF-PitNET was diagnosed by magnetic resonance image (MRI) of the sella turcica with Tumor Characteristics a tumor visualized, in the absence of symptoms suggesting hormone Max. Diameter (mm) 25.6 ± 8.9 hypersecretion and a biochemical confirmation of normal or Extrasellar Extension 59 (81.9%) hyposecretion of pituitary hormones. All tumors underwent surgical Sinus Invasion 34 (50.0%) treatment. Furthermore, the pathologist selected a surplus of tumor to be embedded in RNAlater (Invitrogen, Carlsbad, CA, USA) for Presurgical Comorbidities research purposes. All the tumors were naïve to medical treatment Hypopituitarism 33 (47.8%) and radiotherapy. NF-PitNET invasiveness and size were established according to the preoperative MRI following the conceptual Headache 31 (43.1%) classification by Raverot (31). Tumor recurrence was considered Visual Alterations 29 (40.3%) when a new tumor image was detected in patients with no apparent Surgery Outcome postsurgical remnants or when a regrowth of a known remnant tumor was of sufficient clinical entity that required a second surgical Cured 30 (41.6%) intervention. The minimum surveillance time for considering a Residual 36 (50%) recurrence was 2 years, with a median of 5.32 ± 2.05 years for a total Exitus 1 (1.4%) number of 58 patients. The clinical and neuropathogical characteristics of the cohort are described in Table 1. Sixteen non-tumoral pituitary Missing 5 (6.9%) samples from autopsies (8 samples) and organ donors (8 samples) were Postsurgical Comorbidities also analyzed as a non-pathological condition reference (mean age: 62.4 Hypopituitarism 36 (67.9%) years ± 10.9; 37.5% females). The molecular data of the GH-secreting adenomas used was Headache 6 (11.3%) obtained from our previous paper where the cohort is fully Visual Alterations 12 (22.6%) described (23). Briefly, a total of 57 (32 women) acromegaly Tumor Subtypes patients from the REMAH cohort who underwent pituitary surgery and had tissue availability was used. Mean age was 45.74 Silent ACTH + 8 (11.1%) ± 12.35, 82% of the tumors were macroadenomas, 68% presented Silent FSH +/LH + 21 (29.2%) extrasellar extension, with a mean tumor diameter of 19.49 ± 10.03. Silent PRL + 4 (5.6%) The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and implemented and Negative tumors 12 (16.7%) reported in accordance with the International Conference on Plurihormonal tumors 19 (26.4%) Harmonized Tripartite Guideline for Good Clinical Practice. The Missing 8 (11.1%) study was approved by the Germans Trias i Pujol Hospital Ethical Committee for Clinical Research (Ref.: EO-11-080 http:// www.ceicgermanstrias.cat/index.html). The protocol and protocols for diagnostic assessment. Hematoxylin-eosin and informed consent forms were approved by the institutional review reticulin staining, as well as cytokeratin, Ki-67, a-subunit and p53 board of the participating centers, independent ethics committee, immunolabeling, were also carried out in most of the cases as part of and/or research ethics board of each study site. All patients the standard pathological evaluation. Plurihormonal tumors were provided written informed consent to participate in the study. defined as tumors that showed immunoreactivity for more than one hormone that cannot be explained by normal cytophysiology or developmental mechanisms. We considered negative tumors those 2.2 Immunohistochemical analysis that did not express positivity for any of the hormones. Formalin-fixed paraffin-embedded tumor samples were used for anterior pituitary hormone expression assessment. Growth 2.3 RNA isolation and reverse transcription hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), luteinizing Representative fragments of the fresh tumor were selected by a hormone (LH) and follicle-stimulating hormone (FSH) expression pathologist and embedded in RNAlater (Invitrogen, Carlsbad, CA, were all tested with the corresponding antibodies, following local USA) for 24 h, after which the samples were frozen at −80°C. Total Frontiers in Endocrinology 03 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 RNA was isolated from pituitary adenomas using AllPrep DNA/RNA/ To quantify relative gene expression we calculated a normalization miRNA Universal Kit (Qiagen, Hilden, Germany). We removed factor for each sample based on the geometric mean of the selected contaminating genomic DNA from RNA by treating samples with reference genes, according to geNorm (RRID : SCR_006763, https:// RNAse-free DNAse twice: the first time during the extraction of RNA genorm.cmgg.be/) algorithms (33). following the manufacturer’s protocol and the second time, before the retrotranscription, for which ezDNase Enzyme (Invitrogen, Carlsbad, CA, USA) was used. The quantity and purity of DNA and RNA were 2.5 Statistical analysis measured using a NanoDrop™ 1000 spectrophotometer (RRID : SCR_016517, Thermo Fisher Scientific, Waltham, MA, USA). Descriptive results were expressed as mean ± standard Integrity of the RNA was checked by agarose gel electrophoresis. deviation. Unsupervised hierarchical clustering was used to Five hundred nanograms of total RNA were reverse transcribed investigate the potential identification of patient’s response using SuperScript IV reverse transcriptase (Invitrogen, Carlsbad, subgroups, based on their molecular expression profile. California, USA), and random hexamers in a final volume of 20 uL Differences between groups were compared using analysis of according to the manufacturer’s protocol. variance (Student’s t-test, Wilcoxon signed-rank test and Kruskal- Wallis analysis of variance as appropriate). Samples from all groups within an experiment were processed at the same time. The P values 2.4 Quantitative polymerase chain reaction were two-sided, and statistical significance was considered when P < 0.05. All statistical analyses were performed using R version 4.2.1 (R Gene expression was quantified using Taqman assays Project for Statistical Computing, RRID : SCR_001905). (Applied Biosystems, Fosters City, California, USA). The genes Unsupervised hierarchical clustering was performed using the R analyzed were Somatostatin Receptor Subtype 2 (SSTR2, package pheatmap (Pretty Heatmaps, https://CRAN.R-project.org/ Hs00990356_m1), Somatostatin Receptor Subtype 3 (SSTR3, package=pheatmap). The graphical representation was done using Hs00265633_s1), Somatostatin Receptor subtype 5 (SSTR5, package ggplot 2 (RRID : SCR_014601, Whickham https:// Hs00990408_s1), short Dopamine Receptor D2 (DRD2) Isoform CRAN.R-project.org/package=ggplot2) and the P values were (Hs01014210_m1), long DRD2 Isoform (Hs01024460_m1), Arrestin added using the ggpubr package (‘ggplot2’ Based Publication Beta 1 (ARRB1, Hs00930516_m1), Pleiomorphic Adenoma Gene-Like Ready Plots, https://CRAN.R-project.org/package=ggpubr). 1 (PLAGL1, Hs00414677_m1), Raf Kinase Inhibitory Protein (RKIP/ PEBP1, Hs01110783_g1), E-cadherin (CDH1, Hs01023894_m1), Ki- 67 (MKI67, Hs01032443_m1), Ghrelin and Obestatin Prepropeptide 3 Results (GHRL, Hs01074053_m1), Aryl Hydrocarbon Receptor Interacting Protein (AIP, Hs00610222_m1), Snail Family Transcriptional 3.1 Clinical description of the Repressor 1 (SNAI1, Hs00195591_m1), Snail Family Transcriptional NF-PitNET cohort Repressor 2 (SNAI2, Hs00950344_m1), Epithelial Splicing Regulatory Protein 1 (ESRP1, Hs00214472_m1), RAR Related Orphan Receptor C We analyzed 72 patients with NF-PitNETs, 37 men and 35 women. (RORC, Hs01076112_m1), N-Cadherin (CDH2, Hs00983056_m1), The mean age was 60.3 years ± 13.5 and the mean follow-up was 915.7 Twist Family bHLH Transcription Factor 1 (TWIST1, ± 696.4 days. During follow-up, 6 patients with tumor remnants Hs00361186_m1) and Vimentin (VIM, Hs00958111_m1); presented a recurrence after a mean of 759.8 days and 3 patients furthermore, a custom assay was ordered for Intron 1 Ghrelin In1- without visible remnants after surgery presented a recurrence after a GHRL (AJ89KWC). We tested six reference genes to normalize gene mean of 783 days. Based on MRI, maximal tumor diameter did not expression: Hypoxanthine Phosphoribosyltransferase 1 (HPRT1, show significant differences in tumors with or without extrasellar Hs99999909_m1), Proteasome 26S Subunit ATPase 4 (PSMC4, extension (p=0.22). However, maximal tumor diameter showed Hs00197826_m1), Glucuronidase Beta (GUSB, Hs00939627_m1), differences among tumors with sinus invasion (mean: 28.3 mm) and TATA-Box Binding Protein (TBP, Hs00427621_m1), Mitochondrial tumors without (mean: 23.3 mm) (p=0.02). The proportion of patients Ribosomal Protein L19 (MRPL19, Hs01040217_m1) and presenting headache and visual alterations fell after surgery (43.1% to Phosphoglycerate Kinase 1 (PGK1, Hs00943178_g1), and selected 11.3%, and 40.3% to 22.6%, respectively in intrasellar and extrasellar the last three reference genes based on their stability in our samples tumors) while the proportion of patients presenting hypopituitarism according to Chainy software (available on: http://maplab.imppc.org/ increased after surgery (from 47.8% to 67.9%) (Table 1). chainy/) (32). Quantitative polymerase chain reactions (qPCR) were carried out in a 7900HT Fast Real-Time PCR System (RRID : SCR_018060; 3.2 Association of EMT markers with Applied Biosystems, Fosters City, California, USA). We used clinical variables in NF-PitNETs TaqMan Gene Expression Master Mix (Applied Biosystems, Fosters City, California, USA), and the amplification reactions were performed We analyzed the gene expression of different EMT-related in triplicate for each sample in a final volume of 10 uL in 384-well markers and their relationship with clinical features of NF- plates. To minimize the inter-assay variation, all the genes, including PitNETs (Table 2). We found that SNAI1 and vimentin the reference genes, for each sample were analyzed in the same plate. expression was associated with invasive tumors (p=0.049 and Frontiers in Endocrinology 04 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 TABLE 2 Association between the relative expression of each EMT marker and different tumor characteristics. Gene Relative expression mean ± SE P-value TUMOR SIZE < 2cm Ø > 2cm Ø n = 18 n = 48 SNAI1 0.169 ± 0.125 0.134 ± 0.043 0.367 SNAI2 0.301 ± 0.201 0.351± 0.122 0.835 ESRP1 0.662 ± 0.122 0.665 ± 0.202 0.989 E-cadherin 1.431 ± 0.234 1.240 ± 0.137 0.486 RORC 1.549 ± 0.322 1.970 ± 0.449 0.450 VIM 5.831 ± 2.940 7.003 ± 1.995 0.743 N-cadherin 1.686 ± 0.335 2.576 ± 0.576 0.187 TWIST 0.025 ± 0.014 0.053 ± 0.028 0.378 EXTRASELLAR GROWTH NO YES n = 11 n = 60 SNAI1 0.050 ± 0.019 0.152 ± 0.051 0.049 SNAI2 0.129 ± 0.044 0.356 ± 0.116 0.060 ESRP1 0.549 ± 0.118 0.665 ± 0.167 0.579 E-cadherin 2.068 ± 1.265 1.228 ± 0.919 0.047 RORC 1.889 ± 0.235 1.924 ± 0.377 0.938 VIM 2.795 ± 0.805 7.011 ± 1.836 0.039 N-cadherin 2.046 ± 0.208 2.414± 0.480 0.484 TWIST 0.016 ± 0.011 0.048 ± 0.023 0.209 SINUS INVASION NO YES n = 33 n = 34 SNAI1 0.160 ± 0.074 0.124 ± 0.054 0.693 SNAI2 0.323 ± 0.144 0.341 ± 0.147 0.933 ESRP1 0.482 ± 0.084 0.833 ± 0.279 0.236 E-cadherin 1.326 ± 0.198 1.379 ± 0.156 0.835 RORC 1.413 ± 0.206 2.378 ± 0.620 0.147 VIM 5.627 ± 2.502 7.188 ± 2.528 0.633 N-cadherin 1.729 ± 0.215 2.950 ± 0.801 0.149 TWIST 0.029 ± 0.014 0.059 ± 0.037 0.453 PRESURGICAL HEADACHE NO YES n = 41 n = 31 SNAI1 0.054 ± 0.021 0.238 ± 0.092 0.050 SNAI2 0.147 ± 0.056 0.536 ± 0.205 0.076 (Continued) Frontiers in Endocrinology 05 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 TABLE 2 Continued Gene Relative expression mean ± SE P-value ESRP1 0.720 ± 0.239 0.541 ± 0.093 0.488 E-cadherin 1.471 ± 0.165 1.263 ± 0.177 0.391 RORC 2.110 ± 0.499 1.663 ± 0.330 0.458 VIM 4.109 ± 1.523 9.084 ± 2.886 0.134 N-cadherin 2.529 ± 0.586 2.105 ± 0.525 0.592 TWIST 0.016 ± 0.009 0.076 ± 0.042 0.084 PRESURGICAL HYPOPITUITARISM NO YES n = 39 n = 33 SNAI1 0.066 ± 0.024 0.230 ± 0.092 0.095 SNAI2 0.154 ± 0.073 0.539 ± 0.200 0.078 ESRP1 0.553 ± 0.089 0.794 ± 0.304 0.453 E-cadherin 1.402 ± 0.177 1.299 ± 0.175 0.679 RORC 1.631 ± 0.284 2.274 ± 0.642 0.365 VIM 3.223 ± 1.092 10.092 ± 3.175 0.048 N-cadherin 1.987 ± 0.451 2.820 ± 0.742 0.346 TWIST 0.014 ± 0.005 0.080 ± 0.042 0.132 VISUAL ALTERATIONS NO YES n = 43 n = 29 SNAI1 0.069 ± 0.025 0.175 ± 0.067 0.145 SNAI2 0.274 ± 0.168 0.344 ± 0.119 0.737 ESRP1 0.620 ± 0.152 0.655 ± 0.207 0.895 E-cadherin 1.402 ± 0.177 1.299 ± 0.175 0.679 RORC 2.017 ± 0.667 1.852 ± 0.309 0.823 VIM 4.552 ± 1.709 7.342 ± 2.254 0.327 N-cadherin 2.081 ± 0.559 2.506 ± 0.550 0.590 TWIST 0.019 ± 0.009 0.057 ± 0.030 0.238 P-value was calculated for each tumor characteristic (Student’s t test or Mann–Whitney U test, as appropriate). Significant p-values are shown in bold. SE, Standard Error. p=0.039, respectively) while E-cadherin expression was associated clear binarial epithelial or mesenchymal phenotype (23). Taking with non-invasive intrasellar tumors (p=0.047). Moreover, these data into account, we compared the expression of EMT- vimentin and SNAI1 overexpression showed an association with related markers in GH-secreting tumors, NF-PitNETs and normal tumors presenting, respectively, headache and hypopituitarism pituitaries. Unsupervised hierarchical clustering analysis revealed before surgery (p=0.050 and p=0.048). multiple EMT transition states, with normal pituitaries forming a subcluster associated with high levels of the epithelial marker E- cadherin while NF-PitNETs and GH-secreting tumors were mixed 3.3 NF-PitNETs showed a more in several different subclusters. Interestingly, a cluster associated mesenchymal expression profile than GH- with a mesenchymal expression profile was found in 1 GH-secreting secreting tumors adenoma and 6 NF-PitNETs (Figure 1). This result showed that 12.5% of NF-PitNETs harbored an expression profile compatible We recently reported that most GH-secreting pituitary tumors with an advanced EMT transformation compared to 3.6% of GH- showed a hybrid and variable EMT expression profile rather than a secreting tumors. Frontiers in Endocrinology 06 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 3.4 Association of SRLs and dopaminergic response markers with clinical variables in NF-PitNETs: PEBP1 as a predictor of recurrence We also analyzed the expression of SRLs response biomarkers in NF-PitNETs. In those patients in which clinical information was available, gene expression was correlated with clinical parameters (Table 3). Tumor size showed a negative association with the DRD2 long isoform (Pearson’s r = -0.25, p = 0.05). Extrasellar extension was related to SSTR2, PEBP1, Ki-67 and KLK10 (p = 0.031, p=0.033, p =0.042 and p = 0.008, respectively). Differences were found also in SRLs biomarkers between NF- PitNETs, GH-secreting pituitary adenomas and non-tumoral pituitaries. GH-secreting tumors showed higher levels of SSTR2 and SSTR5, whereas SSTR3 was more expressed in NF-PitNETs (Figures 2A–C). ARRB1 levels were higher in normal tissue (Figure 2D). On the other hand, NF-PitNETs seem to be FIGURE 1 characterized by high levels of KLK10 and PLAGL1 (Figures 2E, Dendrogram and unsupervised hierarchical clustering heatmap of F). Finally, non-tumoral tissue presented high levels of E-cadherin the EMT-related genes in NF-PitNETs (NFPT), somatotropinomas (ACRO) and healthy pituitary tissue (NORMAL) using Ward’s and DRD2 (Figures 2G–I). minimum variance method and Minkowski distance. Furthermore, we wanted to investigate if some of these genes could be used to predict patient’s outcome. We investigated the TABLE 3 Association between the relative expression of each SRLs response marker and different tumor characteristics. Gene Relative expression mean ± SE P-value TUMOR SIZE < 2cm Ø > 2cm Ø n = 18 n = 48 SSTR2 0.144 ± 0.049 0.119 ± 0.034 0.660 SSTR3 0.621 ± 0.372 0.571± 0.129 0.911 SSTR5 0.258 ± 0.229 0.007 ± 0.004 0.288 DRD2 short isoform 2.706 ± 0.928 1.434 ± 0.171 0.096 DRD2 long isoform 2.908 ± 0.738 1.692 ± 0.170 0.046 ARRB1 0.845 ± 0.211 0.743 ± 0.113 0.716 PLAGL1 0.578 ± 0.181 0.498 ± 0.193 0.752 PEBP1 18.016 ± 1.411 16.920 ± 1.139 0.577 Ki-67 0.034 ± 0.009 0.060 ± 0.016 0.081 AIP 1.913 ± 0.017 2.066 ± 0.097 0.398 IN1-GHRL 0.080 ± 0.057 0.024 ± 0.002 0.348 KLK10 0.010 ± 0.007 0.017 ± 0.004 0.413 GHRL 0.055 ± 0.017 0.032 ± 0.005 0.232 EXTRASELLAR GROWTH NO YES n = 11 n = 60 SSTR2 0.054 ± 0.016 0.132 ± 0.031 0.031 (Continued) Frontiers in Endocrinology 07 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 TABLE 3 Continued Gene Relative expression mean ± SE P-value SSTR3 0.454 ± 0.092 0.611± 0.152 0.379 SSTR5 0.010 ± 0.009 0.083 ± 0.070 0.306 DRD2 short isoform 1.683 ± 0.360 1.850 ± 0.322 0.731 DRD2 long isoform 2.212 ± 0.459 2.107 ± 0.459 0.848 ARRB1 0.605 ± 0.090 0.786 ± 0.111 0.209 PLAGL1 0.412 ± 0.235 0.500 ± 0.160 0.760 PEBP1 14.673 ± 1.372 17.977 ± 1.026 0.033 Ki-67 0.028 ± 0.007 0.054 ± 0.013 0.042 AIP 2.041 ± 0.145 2.050 ± 0.088 0.959 IN1-GHRL 0.025 ± 0.005 0.041 ± 0.018 0.365 KLK10 0.005 ± 0.001 0.017 ± 0.004 0.008 GHRL 0.029 ± 0.008 0.039 ± 0.007 0.347 SINUS INVASION NO YES n = 33 n = 34 SSTR2 0.115 ± 0.038 0.133 ± 0.039 0.748 SSTR3 0.523 ± 0.198 0.672 ± 0.176 0.576 SSTR5 0.135 ± 0.122 0.012 ± 0.006 0.317 DRD2 short isoform 1.967 ± 0.516 1.585 ± 0.214 0.497 DRD2 long isoform 2.292 ± 0.416 1.796 ± 0.218 0.295 ARRB1 0.783 ± 0.155 0.706 ± 0.110 0.686 PLAGL1 0.561 ± 0.230 0.425 ± 0.176 0.642 PEBP1 17.495 ± 1.484 16.523 ± 0.978 0.587 Ki-67 0.040 ± 0.015 0.059 ± 0.018 0.422 AIP 2.141 ± 0.125 1.941 ± 0.094 0.207 IN1-GHRL 0.054 ± 0.030 0.023 ± 0.002 0.319 KLK10 0.011 ± 0.004 0.020 ± 0.006 0.194 GHRL 0.031 ± 0.008 0.038 ± 0.007 0.475 PRESURGICAL HEADACHE NO YES n = 41 n = 31 SSTR2 0.094 ± 0.024 0.147 ± 0.051 0.350 SSTR3 0.468 ± 0.076 0.731 ± 0.273 0.360 SSTR5 0.010 ± 0.004 0.148 ± 0.133 0.307 DRD2 short isoform 1.958 ± 0.231 1.676 ± 0.533 0.641 DRD2 long isoform 2.385 ± 0.275 1.816 ± 0.424 0.265 ARRB1 0.590 ± 0.067 0.975 ± 0.189 0.063 PLAGL1 0.367 ± 0.257 0.644 ± 0.257 0.349 PEBP1 17.877 ± 1.098 16.731 ± 1.455 0.532 (Continued) Frontiers in Endocrinology 08 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 TABLE 3 Continued Gene Relative expression mean ± SE P-value Ki-67 0.036 ± 0.008 0.068 ± 0.023 0.203 AIP 2.047 ± 0.093 2.042 ± 0.127 0.974 IN1-GHRL 0.023 ± 0.002 0.059 ± 0.033 0.289 KLK10 0.012 ± 0.004 0.017 ± 0.005 0.480 GHRL 0.036 ± 0.007 0.059 ± 0.009 0.784 PRESURGICAL HYPOPITUITARISM NO YES n = 39 n = 33 SSTR2 0.155 ± 0.048 0.087 ± 0.019 0.201 SSTR3 0.695 ± 0.090 0.454 ± 0.090 0.347 SSTR5 0.133 ± 0.004 0.006 ± 0.004 0.276 DRD2 short isoform 2.162 ± 0.489 1.547 ± 0.247 0.267 DRD2 long isoform 2.338 ± 0.414 1.983 ± 0.271 0.476 ARRB1 0.606 ± 0.086 0.571 ± 0.175 0.181 PLAGL1 0.482 ± 0.114 0.511 ± 0.280 0.924 PEBP1 18.383 ± 1.269 16.528 ± 1.360 0.322 Ki-67 0.042 ± 0.016 0.057 ± 0.018 0.526 AIP 2.084 ± 0.119 2.001 ± 0.104 0.599 IN1-GHRL 0.051 ± 0.029 0.026 ± 0.003 0.390 KLK10 0.011 ± 0.004 0.019 ± 0.006 0.256 GHRL 0.035 ± 0.008 0.036 ± 0.007 0.961 VISUAL ALTERATIONS NO YES n = 43 n = 29 SSTR2 0.134 ± 0.048 0.107 ± 0.030 0.635 SSTR3 0.343 ± 0.075 0.724 ± 0.193 0.071 SSTR5 0.013 ± 0.006 0.104 ± 0.092 0.328 DRD2 short isoform 1.642 ± 0.294 1.953 ± 0.396 0.531 DRD2 long isoform 2.061 ± 0.382 2.187 ± 0.313 0.799 ARRB1 0.662 ± 0.146 0.812 ± 0.119 0.428 PLAGL1 0.671 ± 0.289 0.375 ± 0.132 0.359 PEBP1 17.078 ± 1.162 17.566 ± 1.234 0.774 Ki-67 0.045 ± 0.019 0.052 ± 0.014 0.774 AIP 1.932 ± 0.110 2.113 ± 0.101 0.228 IN1-GHRL 0.021 ± 0.002 0.048 ± 0.023 0.249 KLK10 0.011 ± 0.006 0.017 ± 0.004 0.409 GHRL 0.039 ± 0.008 0.036 ± 0.007 0.828 P-value was calculated for each tumor characteristic (Student's t test or Mann–Whitney U test, as appropriate). Significant p-values are shown in bold. SE, Standard Error. Frontiers in Endocrinology 09 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 A B C D E F G H I FIGURE 2 Boxplots of SRL-related genes according to the different histological samples. Relative expression of SSTR2 (A), SSTR3 (B), SSTR5 (C), ARRB1 (D), KLK10 (E), PLAGL1 (F), E-cadherin (G), DRD2 short (H) and long (I) isoform. NT, Normal tissue. ability of these markers in predicting recurrence in NF-PitNETs 3.5 Relative expression of drug receptors: (Table 4), and found that only PEBP1 showed a difference between DRD2 is the most expressed receptor both tumors that recurred and tumors that did not (p=0.036) in GH-secreting tumors and NF-PitNETs (Figure 3A). When performing a binomial logistic regression, PEBP1 showed an AUC of 69.9%, for a cut-off of 14.0, with a We wanted to compare relative quantities of drug receptor sensitivity of 100% and a specificity of 42.6% (Figure 3B). expression in both NF-PitNETs and GH-secreting tumors. In NF- TABLE 4 Association between the relative expression of each gene and the development of a recurrence. Gene Relative expression mean ± SE P-value RECURRENCE NO YES n = 47 n = 12 SNAI1 0.128± 0.053 0.183 ± 0.137 0.713 SNAI2 0.364 ± 0.140 0.257± 0.141 0.594 ESRP1 0.553 ± 0.103 0.495 ± 0.100 0.688 E-cadherin 1.433 ± 0.161 1.571 ± 0.278 0.673 RORC 2.020 ± 0.466 1.600 ± 0.307 0.455 VIM 5.888 ± 1.719 8.037 ± 4.541 0.665 N-cadherin 2.586 ± 0.593 1.632 ± 0.307 0.158 TWIST 0.025 ± 0.011 0.120 ± 0.101 0.368 SSTR2 0.131 ± 0.036 0.095 ± 0.053 0.581 (Continued) Frontiers in Endocrinology 10 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 TABLE 4 Continued Gene Relative expression mean ± SE P-value SSTR3 0.645 ± 0.186 0.454 ± 0.141 0.418 SSTR5 0.098 ± 0.088 0.018 ± 0.016 0.374 DRD2 short isoform 1.834 ± 0.377 1.878 ± 0.545 0.948 DRD2 long isoform 2.075 ± 0.303 2.357 ± 0.714 0.721 ARRB1 0.775 ± 0.128 0.921 ± 0.206 0.554 PLAGL1 0.463 ± 0.173 0.328 ± 0.125 0.531 PEBP1 16.809 ± 1.153 20.827 ± 1.805 0.036 Ki-67 0.056 ± 0.016 0.038 ± 0.010 0.348 AIP 2.075 ± 0.104 2.184 ± 0.078 0.407 IN1-GHRL 0.045 ± 0.022 0.030 ± 0.006 0.509 KLK10 0.016 ± 0.005 0.014 ± 0.008 0.762 GHRL 0.034 ± 0.007 0.058 ± 0.017 0.215 P-value was calculated for each tumor characteristics (Student’s t test or Mann–Whitney U test, as appropriate). Significant p-values are shown in bold. SE, Standard Error. PitNETs the more prevalent receptor was DRD2, followed by 3.6 Specific expression profile associated SSTR3, while SSTR2 and SSTR5 presented a low expression with NF-PitNET cell subtype: low levels (Figure 4A). In the case of GH-secreting tumors DRD2 was also of SSTR3 and ARRB1 in silent the most quantitatively expressed, SSTR2 and SSTR5 were corticotroph adenomas expressed at the same level as the SSTR3 in NF-PitNETs, while SSTR3 showed a very low expression level (Figure 4B). We correlated levels of EMT and SRL response gene expression with immunohistochemical characteristics of NF-PitNETs (Table 5). We found differences in SSTR3 comparing ACTH-expressing NF- PitNETs versus FSH/LH-positive, plurihormonal NF-PitNETs and negative tumors (p = 0.001, p = 0.005 and p = 0.004, respectively) A (Figure 5A). We also found differences in ARRB1 comparing ACTH- expressing NF-PitNETs versus FSH/LH-positive and negative cell tumors (p=0.005 and p=0.012, respectively) (Figure 5B). 4 Discussion NF-PitNET is a very unspecific tumor class in which by definition, no clinically detectable hypersecretory pituitary tumors are included. B Its main clinical feature is the mass effect upon the surrounding anatomical sellar structures. In the present work we aimed to explore the molecular knowledge of this tumor class and confirmed its very heterogeneous nature. Regarding tumors with extrasellar growth, we found that both EMT and SRL response genes presented differential expression compared to non-invasive tumors. It has already been described that EMT genes correlate with invasive pituitary tumors as well as with angiogenesis and extracellular matrix degrading genes (34). However, we also found that SSTR2 and PEBP1 were associated to extrasellar growth in some NF-PitNETs that, as far as we know, has not been previously reported. FIGURE 3 The measurement of DA receptor and SRL response genes in (A) Relative expression of PEBP1 in NF-PitNETs in tumors that recurred vs. tumors that did not. (B) ROC curve for predicting NF-PitNETs provides interesting information that could be useful recurrence using ARRB1 expression in NF-PitNETs. in the case that a pharmacological treatment is considered in this type of pituitary tumors (35); thus, it would be recommendable to Frontiers in Endocrinology 11 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 A B FIGURE 4 Histogram and density plot of the log10 relative expression of DRD2 long and short isoform, SSTR2, SSTR3 and SSTR5 in NF-PitNETs (A) and GH- secreting pituitary neuroendocrine tumors (B). Dashed line showed the mean expression of each gene. perform such studies rather than initiating a blind course of Another interesting finding of the present study, probably the treatment with unpredictable results. In the present study, we one of most clinical relevance for its application in personalized found a striking NF-PitNET subtype clustering by using the medicine, is the association of EMT-markers with local growth in expression of a few genes. Our results indicate the existence of NF-PitNETs. Although this association has been previously substantial differences in the SRLs biomarkers’ landscape when NF- reported (18, 39–41), we could observe that the EMT PitNETs are compared with somatotropinomas, the latter being an phenomenon is more present in the NF-PitNETs compared to archetype of medically treated pituitary tumors. We believe that this somatotropinomas. Furthermore, EMT has been described as a is a relevant finding, since usually the detection of differences in cause of resistance to medical treatment in pituitary tumors, in clustering between different types of pituitary tumors requires particular to SRLs (20); thus, the evaluation of these biomarkers in genome-wide data (36). NF-PitNETs would seem highly recommendable in case of Furthermore, the present work can shed light on the considering medical treatment. controversy over the quantitative analysis of dopamine receptors, The absence of SSTR2, SSTR5 and PLAGL1 has also been somatostatin receptor subtypes and downstream effectors in NF- proposed by other groups as an explanation for the lack of PitNETs (14, 35, 37, 38). response to SRLs in NF-PitNETs (35, 37). Furthermore, regarding Frontiers in Endocrinology 12 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 TABLE 5 Analysis of differences between the relative expression of each gene and the positive immunostaining for pituitary hormones. A Gene P-value SNAI1 0.379 SNAI2 0.098 ESRP1 0.621 E-cadherin 0.135 RORC 0.232 VIM 0.333 B N-cadherin 0.187 TWIST 0.284 SSTR2 0.356 SSTR3 0.034 SSTR5 0.754 DRD2 short isoform 0.359 DRD2 long isoform 0.670 FIGURE 5 ARRB1 0.018 (A) Relative expression of SSTR3 according to the immunohistochemical expression of the pituitary hormones. (B) Relative expression of ARRB1 PLAGL1 0.060 according to the immunohistochemical expression of the pituitary hormones. P-values for the different group comparisons regarding PEBP1 0.455 SSTR3 expression: ACTH+ vs. FSH/LH-+ (p=0.001), ACTH+ vs. PRL+ (p=0.418), ACTH+ vs. plurihormonal NF-PitNETs (p=0.005), ACTH+ vs. Ki-67 0.074 negative tumors (p=0.004), FSH/LH+ vs. PRL+ (p=0.418), FSH/LH+ vs. AIP 0.059 plurihormonal NF-PitNETs (p=0.429), FSH/LH+ vs. negative tumors (p=0.603), PRL+ vs. plurihormonal NF-PitNETs (p=1), PRL+ vs. negative IN1-GHRL 0.676 tumors (p=0.795) and plurihormonal NF-PitNETs vs. negative tumors (p=0.787). P-values for the different group comparisons regarding KLK10 0.230 ARRB1 expression: ACTH+ vs. FSH/LH-+ (p=0.005), ACTH+ vs. PRL+ (p=0.509), ACTH+ vs. plurihormonal NF-PitNETs (p=0.152), ACTH+ vs. GHRL 0.633 negative tumors (p=0.012), FSH/LH+ vs. PRL+ (p=0.119), FSH/LH+ vs. plurihormonal NF-PitNETs (p=0.085), FSH/LH+ vs. negative tumors P-value was calculated using Kruskal–Wallis test or ANOVA test, as appropriate. Significant (p=0.147), PRL+ vs. plurihormonal NF-PitNETs (p=0.535), PRL+ vs. P-values are shown in bold. negative tumors (p=0.051) and plurihormonal NF-PitNETs vs. negative tumors (p=0.026). * indicates the subtypes of NF-PitNETs that showed SSTRs, SSTR3 ligands could be a very interesting druggable target in significant differences compared to ACTH+ NF-PitNETs. NF-PitNETs and its pharmacologic activation may be of potential benefits regarding the prevention of tumor regrowth, since SSTR3 inhibits cell cycle dynamics and promotes apoptosis. A recent experimental study has demonstrated that SSTR3 monoligands activate signaling mechanisms that reduce NF-PitNET cell and therefore no positive results of SRLs treatment would be viability and inhibit pituitary tumor growth in animal models expectable in this tumor subtype. This is an important issue in expressing SSTR3, suggesting that it could be an efficacious order to identify those tumors presenting a worse prognosis, as is treatment for NF-PitNETs (42). the case for this corticotrophic subtype (45). However, the When comparing the relative expression of the drug receptors, assumption that the effects of drugs are correlated with the levels we found that DRD2 was the most expressed receptor in both NF- of expression of their target receptors is too naïve. The presence of PitNETs and GH-secreting tumors. However, dopaminergic agents target receptors for both DA and SRLs that act via the MAPK are only useful in 30% of tumors in attaining a clinically significant pathway are a sine quanon requirement for a positive therapy volume reduction (13), while in GH-secreting pituitary tumors, outcome. However, the precise molecular mechanisms and the SRLs induce tumor shrinkage in more than 50% of the cases (43, modulatory effect of different transcription factors, intermediate 44). If we assume that the effects of the drugs are correlated with the effectors and even cytoskeleton proteins are still not clear (46). The expression of their target receptors, we would expect beneficial assumption that the presence of more target receptors means more results from targeting SSTR3 in NF-PitNETs at least at similar levels drug efficacy is not so straightforward (47). as observed when targeting SSTR2 in acromegaly. However, if Moreover, we found an association between KLK10 expression in according to our results SSTR3 could be an interesting target for NF-PitNETs and extrasellar extension. KLK10 is a gene encoding for most NF-PitNETs, silent corticotroph tumors should be excluded a serine protease, member of the tissue kallikrein proteins (KLKs) for such a treatment, because of the low receptor levels they express, (48). KLKs are widely recognized as cancer biomarkers (49) and are Frontiers in Endocrinology 13 frontiersin.org Gil et al. 10.3389/fendo.2023.1129213 considered interesting targets for the development of novel drugs Clinical Research (Ref.: EO-11-080 http://www.ceicgermanstrias.cat/ (50). KLK10 has been involved in the development of many cancers, index.html). The patients/participants provided their written such as ovarian (51), breast (52), prostate (53) and thyroid (54) informed consent to participate in this study. cancers, although the specific role of KLK10 in tumorigenesis is not yet sufficiently defined. Regarding human pituitary tumors, KLK10 has been found to be consistently expressed in prolactinomas, Author contributions thyrotropinomas, somatotrophinomas and corticotroph adenomas (55, 56). KLK10 could thus be an interesting target for NF-PitNETs, JG, MM-P, MJ and MP-D contributed to conception and design but currently more studies on its pathophysiologic and mechanistic of the study. MM-P organized the database. JG performed the implications should be performed, either silencing or overexpressing statistical analysis, performed the experiments and wrote the first its gene in order to clearly define its implications in tumor growth as draft of the manuscript. BB, MJ and MP-D wrote sections of the well as its potential therapeutic use. manuscript. The remaining authors provided patients and Finally, we also found that recurrent tumors expressed higher clinical information to the study and critically discussed the levels of PEBP1. PEBP1, also known as RKIP, is considered a results. All authors contributed to the article and approved the metastasis suppressor gene (45, 57). In addition, it has been submitted version. linked to poor response to SRLs in acromegaly (58). We found that PEBP1 is a potential biomarker for predicting recurrence, due to its high sensitivity, although its low specificity may limit its Funding usefulness in clinical practice. Thus, with the current lack of robust biomarkers for implementation of predictive medicine in NF- This research was funded by the Instituto de Salud Carlos PitNETs, PEBP1 may be a first step to classify a subgroup of III (PMP15/00027, co-funded by the European Union- these tumors requiring a more careful follow-up. A validation ERDF; and PMP22/00021, funded by the European Union study of this and the other biomarkers presented in the present -NextGenerationEU); and Novartis through the REMAH (Registro work is required. Moreover, drugs specifically designed to target Español Molecular de Adenomas Hipofisarios) consortium of the KLK10 and PEBP1 would be welcome, and would be useful for SEEN (Sociedad Española de Endocrinologı́a y Nutrició n). Novartis pituitary tumors currently lacking an efficient medical treatment. was not involved in the study design, analysis, interpretation of data, In summary, the lack of response to SRLs and DA in NF- the writing of this article or the decision to submit it for publication. PitNETs could be partly explained by a more common EMT phenomenon in this subset of pituitary tumors than in functioning ones (20). Moreover, despite the fact that SSTR3 Acknowledgments could be a good therapeutic target, it will presumably not be effective in silent corticotroph tumors. The absence of validated We want to acknowledge the efforts of the REMAH community. prognostic markers or a prognostic classification for NF-PitNETs limits the evaluation of medical strategies for these lesions. Different pathological markers have been suggested so far, including those of Conflict of interest the present study. Their prognostic value should be prospectively confirmed in a large multicenter study which would help to build MP-D, EV, GuS, IS, GX, PC-S, BB, GeS, SW declare to have the instrument to implement precision medicine also for received funds from Novartis, Ipsen, and Pfizer as lecturers. NF-PitNETs. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. Data availability statement The raw data supporting the conclusions of this article will be Publisher’s note made available by the authors, without undue reservation. 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