Littérature scientifique sur le sujet « BIOLOGIA APPLICATA MED »

L'articolo affronta il problema del rapporto tra sapere scientifico e pratica clinica. La medicina è una disciplina che persegue sia la conoscenza di numerosi fenomeni biologici che la possibilità di modificarli. Essa possiede, quindi, sia aspetti teorici che operativi. E questi, in realtà, sono solo apparentemente diversi, poiché il "fare" della tecnica non è un "fare" qualsiasi, ma un "fare efficace", che nasce dalla conoscenza di una serie di procedure operative progettate al fine di raggiungere determinati obiettivi. Dopo avere sottolineato che il "sapere razionale", nato durante la civiltà ellenica, trova un suo culmine nel XVII secolo con la nascita della scienza sperimentale, gli Autori iiiustrano la nascita della medicina moderna, quale scienza applicata, che è in grado - superando l'empirismo - di fornire le ragioni per le quali si agisce in un certo modo. Dopo avere argomentato sulla necessità dell'etica nella scienza, viene affrontata la medicina come scienza applicata, il cui fine è la salute dell'uomo. Pertanto il medico deve attenersi correttamente alle regole consolidate del procedimento clinico, metodologicamente e nei contenuti; inoltre è chiamato ad esercitare la scienza medica in modo da raggiungere gli scopi che costituiscono tale disciplina, adeguandosi alle conoscenze scientifiche più aggiornate e consolidate.

In questo articolo l’Autore ha voluto sottolineare l’importanza dell’Enciclica Evangelium Vitae nel risvegliare le coscienze contro uno dei più gravi capovolgimenti etici e giuridici della storia umana. L’intento è quello di esaminare tre questioni: 1) risalire alle basi sulle quali si fondava e si fonda il postulato giuridico e morale dell’inalienabilità del diritto alla vita dell’uomo innocente e, soprattutto, del concepito; 2) stabilire le cause che hanno portato ad una legislazione permissiva dell’aborto ed ad altri attentati contro la dignità dell’uomo e della vita umana (eutanasia, manipolazioni di geni ed embrioni…); 3) valutare quali siano i motivi filosofici e biologici la cui presa di coscienza sembri più necessaria per la tutela del diritto alla vita. Intento dell’Autore è, per ciò che riguarda il diritto alla vita, sottolineare l’importanza che il principio di non discriminazione, basato su quello dell’uguaglianza, venga applicato all’“essere umano”, all’“individuo umano” e non soltanto alla “persona giuridicamente riconosciuta”. L’articolo si sofferma, inoltre, ad illustrare la grande tradizione del diritto alla vita, il preoccupante regresso della civiltà giuridica, la necessità di un più stretto rapporto tra Diritto e Morale e Biologia e Morale (come campi di ricerca e di impegno intellettuale a difesa della vita).

La farmacogenetica è stata inizialmente definita come lo studio della variabilità di risposta individuale al farmaco legata all’ereditarietà e alle caratteristiche genetiche personali e familiari. Durante l’ultima decade il termine farmacogenomica ha ulteriormente affinato tale definizione delineando con più precisione gli ambiti e le finalità di questo nuovo ambito scientifico. L’obiettivo è ancora più affascinante e importante: sviluppare e utilizzare nuove terapie farmacologiche personalizzate, più efficaci e meno dannose, utilizzando le scoperte sul genoma umano. Dallo sviluppo di questa nuova applicazione della genetica probabilmente dipenderà anche un mutamento nella prassi medica, con nuovi criteri diagnostici e soprattutto nella possibilità di somministrare delle terapie personalizzate. La genomica applicata alla ricerca farmacologica, oltre alle diverse problematiche tecnico-scientifiche che ancora sono in via di risoluzione, impone anche, per l’ambito di applicazione - il genoma umano, quindi anche la persona umana, e le finalità che si prefigge cioè pervenire a nuove e ottimali soluzioni terapeutiche attraverso la ricerca clinica - un’ampia riflessione etica rispetto ad una pluralità di elementi in gioco. D’altra parte occorre anche considerare che ai vantaggi di una quota di popolazione potrebbero correlarsi svantaggi terapeutici per altre fasce di popolazione minoritarie, perciò è fondamentale che un’ampia e articolata riflessione e adeguate soluzioni vengano intraprese prima che la commercializzazione dei tests farmacogenetici possa causare discriminazioni tra i pazienti. Nell’articolo, gli Autori evidenziano le questioni etiche relative alla ricerca farmacogenetica di base, all’identificazione, alla collezione e alla brevettabilità dei dati, alla ricerca farmacogenetica applicata, con lo sviluppo di dispositivi diagnostici e il loro uso nelle sperimentazioni cliniche, alle procedure per la conservazione e dei campioni biologici e dei dati e per la tutela della riservatezza, al ruolo dei Comitati di Etica nella valutazione dei protocolli sperimentali di farmacogenetica, al consenso informato per i soggetti di sperimentazione.

Con ingegneria tessutale (IT) si fa riferimento a un campo disciplinare che applica i principi dell’ingegneria e delle scienze della vita per la realizzazione di sostituti biologici che ripristinino, mantengano o migliorino le funzioni di tessuti o organi. Questo nuovo settore di ricerca e applicazione clinica, che attualmente consente di realizzare principalmente cute, cartilagine e osso semiartificiali, può in prospettiva sostituire le tecnologie dei trapianti di organi naturali. Ma l’ingegneria dei tessuti pone dei quesiti bioetici: alcuni di tipo generale, implicati anche da altre questioni di interesse bioetico, altri ad essa peculiari. Quesiti generali sono, per esempio, i limiti della donazione di tessuti e i rapporti tra il mercato e la scienza. Un problema che l’ingegneria dei tessuti pone invece con sfumature inedite verte sullo statuto da riconoscere ai prodotti bioartificiali: entità che utilizzano (in alcuni casi) tessuti umani, ma che si collocano al confine tra naturale e artificiale. Interessante è, inoltre, nella ridefinizione della coppia naturale/artificiale, il ruolo assunto dal diritto, che in particolare con le norme sulla brevettabilità del biologico - integra ormai la scienza nella definizione delle stesse realtà scientifiche, e che diventa, quindi, al pari della scienza, un elemento fatturale da sottoporre al vaglio etico. Data la novità della materia, l’articolo ha un intento essenzialmente descrittivo: l’esposizione dei più importanti conseguimenti dell’ingegneria dei tessuti e dei temi di interesse bioetico che esigono un dibattito.

Il problema della formazione in bioetica è estremamente delicato e complesso e presenta due tipi di difficoltà, una di fatto, l’altra di principio. La difficoltà fattuale è legata alla giovane età di questa disciplina e, conseguentemente, alla mancanza di modelli consolidati di insegnamento; le difficoltà teoriche sono, invece, strettamente legate al carattere interdisciplinare (confronto e dialogo tra discipline diverse) e al pluralismo teoretico e pratico (pluralità di concezioni morali e giuridiche) che costituiscono la peculiarità della bioetica. La domanda che l’Autrice si pone è: quale formazione in bioetica? E soprattutto chi, come, quando, formare in bioetica? Ma soprattutto chi formare in bioetica? Occorre prima di tutto individuare i discenti ed operare una distinzione tra una formazione che tende al generale (ed è quindi diretta a tutta la società) e una formazione che tende allo specifico (rivolta a chi opera nel settore socio-sanitario e a chi non opera direttamente o indirettamente nella sanità). Il come formare in bioetica riguarda invece tre settori: il sapere (conoscenza dettagliata della ricerca scientifica e della tecnologia, applicata alla biologia e alla medicina, della struttura socio-sanitaria, della teologia e della filosofia), il saper fare e il saper essere (è importante sapersi calare dal piano teorico-conoscitivo a quello applicativo ed esperienziale, sia dell’agire, sia dell’essere). La questione del quando formare in bioetica non è stata ancora risolta. Anche quella del chi forma in bioetica è ancora in fase di sperimentazione: sarebbe auspicabile una équipe di docenti di materie diverse ma interagenti.

Il principio antropico afferma che l'Universo ha le caratteristiche che osserviamo perché noi siamo qui. Al di fuori di tutti i possibili universi, noi siamo in grado di sperimentare solamente la ristretta serie permessa agli osservatori. Il Principio può essere applicato allo studio delle connessioni tra alcune condizioni come la contingenza e l'indeterminatezza della materia da un lato e dall'altro la possibilità di esistenza dei soggetti liberi. Possedere la libertà sotto forma del controllo della realtà fisica da parte della volontà ed una Natura con relazioni causa-effetto puramente meccanicistiche potrebbe non essere adeguato. Ciò che è certamente necessario (sebbene non sufficiente) è che lo strumento materiale della libera volontà non dovrebbe essere rigorosamente deterministico. E' stata avanzata l'ipotesi (Eccles) che gli eventi mentali agiscono sugli eventi sinaptici probabilistici in maniera analoga ai campi di probabilità della meccanica quantistica. Anzi, l'attività caotica può essere parte della normale funzionalità del sistema nervoso. L'"hardware" mentale umano è così rappresentato da una struttura che in virtù della sua indeterminatezza (grossolanamente parlando) lascia libertà d'azione alla libertà. L'incompletezza, l'indeterminazione e la imprevedibilità algoritmica che garantisce la libertà e la creatività implica un mondo di relativa instabilità, precarietà ed errore, cioè dal punto di vista biologico la corruzione delle forme, il dolore e la morte. La radice del dolore è in tal modo correlata a quella della libertà, poiché il dolore rappresenta l'alto prezzo che la materia dell'Universo deve pagare in ordine alla predisposizione all'esistenza di esseri liberi. In virtù del Principio antropico, possiamo dire che l'Universo compatibile con la libera volontà deve essere un luogo di dolore e di morte.

BackgroundRecruitment of candidates is a cost- and effort-intensive aspect of rheumatology fellowship programs. For program leaders to efficiently use the available resources and improve recruitment outcomes, it is imperative to understand the attributes that influence the candidates’ choice of a program. Previous studies have examined the type and relative importance of the factors that candidates use in selecting other fellowship programs (1, 2). However, no such studies have been conducted in the field of rheumatology.ObjectivesTo examine the factors that influence the selection of fellowship programs by rheumatology applicants.MethodsAn anonymous, web-based survey comprised of 13 questions was shared with rheumatology fellowship applicants on messaging applications and online forums. The survey was open from 10/29/2021-11/06/2021. Participation was voluntary and informed consent was implied through the participants’ response. Three reminders to complete the survey were sent. Four domains of the applicant’s perception in relation to their preference of ranking rheumatology programs were assessed: (1) program prestige, (2) program structure, (3) interview day experience, and (4) career path of the alumni. The survey questions were devised in one of the following formats: (1) 5-point Likert scale, (2) rank order questions, (3) yes/no questions, (4) multiple choice questions, and (5) open-ended questions.ResultsThirty-two rheumatology applicants responded to the survey. The prestige of the program was reported to be extremely important by 16%, very important by 19%, somewhat important by 44%, and little or not important by 21% responders. The opportunity to see a diverse patient population was reported to be important by 97% respondents. The call schedule and higher number of fellows were considered important by 88% of the respondents. 66% preferred programs with higher number of faculty members. 69% favored programs with an ultrasound curriculum. The availability of clinician-educator track (18%), MCR/MPH (14%), and T32 grand (4%) were considered less important. 69% reported that the opportunity to train at a Veterans Administration hospital did not influence their choice. Regarding interview day experience, interaction with the faculty (63%) and the fellows (17%) were considered important factors influencing program ranking. Respondents preferred programs with alumni in academic clinician track (45%) and private practice (43%) compared to programs with alumni in academic research (13%) or industry pathway (4%). The geographical location of the program including the cost of living and location of significant others also influenced the applicants’ choice.ConclusionTo the best of our knowledge, this is the first survey to assess the attributes that influence a candidate’s choice of a rheumatology fellowship program. Our survey demonstrated that a positive interview day experience and program attributes including the opportunity to interact with a diverse patient population, relaxed call schedule, higher number of fellows and faculty, the presence of an ultrasound curriculum, and the location were the dominant factors influencing applicants’ choice of a program. The main limitation of our study is the lack of generalizability due to selection bias. Understanding the factors involved in decision making of the rheumatology fellowship applicants can provide valuable information for both the applicants and the programs and therefore lead to a better match.References[1]Kelm DJ, Skalski JH, Nelson DR, Kashani KB, Lee AS, Wesselius LJ, et al. Attributes Influencing the Selection of Fellowship Programs by Pulmonary and Critical Care Applicants: A Pilot Study. Ann Am Thorac Soc. 2016;13(4):572-4.[2]Caiola E, Litaker D. Factors influencing the selection of general internal medicine fellowship programs: a national survey. J Gen Intern Med. 2000;15(9):656-8.Disclosure of InterestsNone declared

Background: This pilot study explores the influence of preadmission data on podiatric medical school performance, specifically, the role of undergraduate institutional selectivity. This type of study has never been described in the podiatric medical education literature. We conducted a longitudinal analysis of preadmission data on 459 students from the graduating classes of 2000 to 2009 at the College of Podiatric Medicine and Surgery at Des Moines University. Methods: Multivariate linear regression was used to assess the relationship between performance during the first year of podiatric medical school and a set of independent variables that represent certain preadmission student characteristics. Student demographic characteristics, such as race/ethnicity and sex, were also included in the regression analysis as control variables. Results: The regression analysis revealed that ethnic origin, undergraduate grade point average, Medical College Admission Test biological science and verbal reasoning scores, and institutional selectivity together had a significant effect on the dependent variable (F = 18.3; P < .001). The variance for the independent variable/constant variables was 32%. Almost twice as many students were dismissed or withdrew in poor academic standing who attended undergraduate institutions in the lowest selectivity category. Conclusions: This analysis revealed that in the College of Podiatric Medicine and Surgery, some preadmission variables, such as institutional selectivity, undergraduate grade point average, ethnic origin, and Medical College Admission Test verbal reasoning and biological science scores, are statistically significant in predicting first-year podiatric medical school grade point average. The selectivity of a student’s undergraduate institution should be considered when screening potential podiatric medical school applicants. (J Am Podiatr Med Assoc 100(6): 479–486, 2010)

Abstract Objective: Neoplastic milieu is an integral part of all malignant diseases including multiple myeloma and plays variable role in their development, retention/adhesivity, resistency or sensitivity to therapeutic approach, homing and also paraneoplastic manifestations. Relatively genetically stable milieu may play an important role in new specific molecular therapeutic approaches and therefore should be contextually studied with neoplastic cells as complex neoplastic tissues. The expressions of 15 proteins with close relation to the development of myeloma bone disease (MBD) were analysed in consecutive multiple myeloma specimens. Methods: Bone marrow trephine biopsy specimens (n=57) with multiple myeloma were included in our prospective study. FFPE tissues were processed in app. 5microm sections and placed on charged slides. The indirect immunohistochemical staining was applicated after antigen retrieval and commercial primary antibodies were used for the detection of observed proteins. Standard secondary antibody and ABC method were included in visualisation. We analysed the expressions of MIP1alfa, Annexin A2, TRAP, DKK-1, RANK, RANKL, OPG, Sclerostin, Activin A, NFkappaB proteins (p50, p52, p65), p62 (sequestosome 1), MMP9 and RUNX2. Results: Bone marrow multiple myeloma specimens showed variable positivity of MIP1alfa in 60% (cut-off point 20%), Annexin A2 in 42% (myeloma cells, cut-off point 30%) and in 74% (stromal cells, cut-off point 5%), TRAP in 28% (cut-off point 5%), DKK-1 in 23% (cut-off point 30%), RANK in 53% (cut-off point 30%), RANKL in 70%, OPG in 39% (cut-off point 5%), Sclerostin in 95% (cut-off point 90%), Activin A in 35% (cut-off point 30%), cytoplasmic positivity of p50 in 5% (cut-off point 10%), p52 in 86% (cut-off point 10%), p62 in 91% (cut-off point 10%), p65 in 89% (cut-off point 10%), positivity of MMP9 in 22% (cut-off point 30%) and positivity of RUNX2 in 56% (cut-off point 30%). Conclusion: Our study showed variable expression of proteins related to MBD in multiple myeloma and its bone marrow microenvironment that imply biological heterogeneity, different development and stromal plasticity in this complex hemato-oncological disease. The exact and contextual knowledge of the engaged signaling pathways may suggest more specific or tailored therapeutic approaches (e.g. anti-RANKL, anti-DKK-1, anti-Sclerostin, anti-Activin A). Supported by the grant NT 14393. Disclosures No relevant conflicts of interest to declare.

Abstract Aim: Myeloma bone disease (MBD) is present in 80-90% patients with multiple myeloma (MM). Up to now, three signalling pathways have been described in the pathogenesis of MBD – receptor activator of nuclear factor kappa B and its ligand (RANK/RANKL) pathway, macrophage inflammatory proteins (MIP) pathway, and wingless type (Wnt) pathway. Moreover, several cytokines and parameters of bone microenvironment have been shown to interfere with bone homeostasis in MM. The aim of our study was to assess the activity of selected parameters in bone marrow of patients with MM and monoclonal gammopathy of undetermined significance (MGUS) in order to define the principal processess occuring within MBD. Materials and methods: We designed a prospective study aimed at signalling in MBD. Formaline-fixed, parafin-embedded diagnostic tissue of patients with MM and MGUS has been processed in routine tissue sections (app. 5 um) and placed on plus-charged slides. After the antigen retrieval (Table 1) indirect immunohistochemical evaluation has been processed with the use of commercial available primary antibody for particular detected protein (according to manufactor´s manual) in optimalised dilution. For the visualisation secondary antibody has been applicated with the use of the standard method avidin-biotin (ABC). Following parameters have been evaluated: RANK on myeloma and mononuclear stromal cells, RANKL and osteoprotegerin (OPG) on stromal cells, MIP-1α in plasma cells (both membrane and cytoplasm), sclerostin, MMP 9 and DKK-1 in the cytoplasm of plasma cells, Annexin A2 in plasma and stromal cells, tartrate resistant acid phosphatase (TRAP) in the cytoplasm of osteoclasts and precursor cells, Activin A in the nucleus of plasma cells, p50, p52, p62, p65, in nucleus and cytoplasm of plasma cells. Results: Activity of RANK varied between 0-100% in plasma cells with 0% activity in stromal cells. Positivity of RANKL was found on endosteum of stromal cells in 12/17 patients (71%). The activity of OPG on stromal cells was in all patients under 10%. Assessment of MIP-1α revealed 100% positivity in 9/17 patients (53%), in 13 patients (76%) the activity was more than 50%. The activity of sclerostin reached 90-100% in all patients. The levels of DKK reached in 3 patients more than 60%, in the rest it was under 10%. The levels of Annexin A2 in stromal cells were low, in 16/17 patients below 20%. In plasma cells, higher activity (above 60%) was found in 4 patients (24%). Activity of p50 above 70% was found in cytoplasm of 2 patients only (12%). The levels of p52 varied between 1-90%, majority of the patients (53%) having more than 80% activity. Similar results were found within the assessment of p65. The levels of p62 were with high activity above 70% in 16/17 patients. The activity of MMP-9 was in 9/17 patients above 70%, the rest of patients had the activity of MMP-9 under 20%. Conclusions: Patientswith monoclonal gammopathies displayed significant activation of all three signalling pathways of MBD. There were however, differences in the involvement of each individual pathway as well as in the levels of other cytokines participating on bone homeostasis, suggesting different mechanisms of the cascade occurring in patients with similar skeletal involvement. With support of the grant NT14393. Abstract 5679. Table 1 Primary antibodies Antibodies clone Antigen retrieval Dilution Source Anti- MIP1alfa C-16 MW 1:200 Santa Cruz Anti- RANK 9A725 MW 1:100 Santa Cruz Anti- RANKL N-19 MW 1:100 Santa Cruz Anti- Ann A2 Polyclonal MW 1:1000 Abcam Anti- TRAP Polyclonal MW 1:1000 GeneTex Anti- Act A Polyclonal MW 1:50 Sigma-Aldrich Anti- OPG N-20 MW 1:50 Santa Cruz Anti- p50 E-10 MW 1:50 Santa Cruz Anti- p52 C-5 MW 1:100 Santa Cruz Anti- p65 F-6 MW 1:100 Santa Cruz Anti- p62 SQSTM1 (ab56416) MW and methanol unblocking 1:50 Abcam Anti- Sclerotisin Polyclonal MW 1:100 Abcam Anti- MMP9 Polyclonal MW 1:50 Abcam Anti- Dkk-1 H-120 MW 1:100 Santa Cruz Abbreviations: MW – microwave oven Disclosures No relevant conflicts of interest to declare.

Gli ormoni steroidei sono una classe di composti che regolano specifiche funzioni endocrine o non-endocrine dell'organismo quali, per esempio, i processi riproduttivi, il comportamento sessuale (progesterone, estrogeni, androgeni), o sono coinvolti in funzioni omeostatiche modulando equilibri idrico-salini (mineralcorticoidi), o i livelli ematici di glucosio, le risposte immunitarie, le risposte allo stress (glucocorticoidi), ecc. Nell'esplicare queste funzioni, gli ormoni steroidei agiscono a livello delle cellule bersaglio tramite proteine recettoriali specifiche, che modulano l'espressione di una grande varietà di geni specifici, agendo come fattori di trascrizione. I recettori per il progesterone, gli estrogeni, gli androgeni, i glucocorticoidi e i mineralcorticoidi sono stati caratterizzati sia biochimicamente che, più recentemente, con metodi di biologia molecolare che hanno permesso il clonaggio del DNA che codifica per le varie proteine. Attraverso i dati raccolti, si è definita una famiglia di proteine con caratteristiche comuni, sia in termini strutturali (per la presenza di caratteristici domini funzionali), sia per il meccanismo d'azione, che è generalmente conosciuta come "famiglia dei recettori degli steroidi"; questa super-famiglia comprende anche proteine recettoriali il cui legante è solo parzialmente correlato, dal punti di vista strutturale, agli steroidi, come la vitamina D, o la cui struttura è totalmente non-steroidea come gli ormoni tiroidei, o l'acido retinoico (Evans, 1988). Infine comprende anche una serie di proteine, caratterizzate durante il clonaggio dei vari recettori per gli steroidi che hanno alcune caratteristiche in comune con detti recettori, ma che vengono chiamate orfane in quanto non è stato per loro ancora identificato nessun tipo di legante (Carson-Jurica et al., 1990). Il lavoro qui descritto riguarda un aspetto particolare del meccanismo d'azione del recettore steroideo e cioè la sua fosforilazione. Verrà discusso con maggiore attenzione quello che avviene nel caso del recettore per il progesterone; si cercherà tuttavia di evidenziare, ove possibile, e sulla base della letteratura disponibile, i meccanismi comuni a tutta la famiglia dei recettori steroidei.

Introduction: Cellular retinoic acid metabolism and homeostasis; Epigenetic gene regulation by retinoic acid; Retinoic acid signalling in breast development and morphogenesis; Disruption of epigenetic retinoic acid signalling in breast tumorigenesis. Scope of the thesis Materials and Methods Experimental Work: An impaired RA signal leads to RARbeta2 epigenetic silencing and RA resistance; Impairment of RA-RARalfa signalling leads to the concerted epigenetic silencing of RARbeta2 and CRBP1 in untransformed human breast epithelial cells; Mechanistic relationship between RARbeta2 and CRBP1 transcription and different phenotypes of breast epithelial cell transformation: Implications for predicting breast cancer risk; CRABP2 downregulation results in epigenetic dysregulation of RA signalling and consequent breast epithelial cell transformation. General Discussion Summary References Curriculum Vitae

Background: The p.E318K variant of the microphthalmia-associated transcription factor (MITF) has been implicated in genetic predisposition to melanoma as an intermediate penetrance allele. However, the impact of this variant on clinico-phenotypic, as well as on dermoscopic patterns features of affected patients is not entirely defined. The purpose of our study was to assess the association between the p.E318K germline variant and clinic-phenotypical features of MITF+ compared to non-carriers (MITF-), including dermoscopic findings of melanomas and dysplastic nevi. Methods: we retrospectively analyzed a consecutive series of 1386 patients recruited between 2000 and 2017 who underwent genetic testing for CDKN2A, CDK4, MC1R and MITF germline variants in our laboratory for diagnostic/research purposes. The patients were probands of melanoma-prone families and apparently sporadic single or multiple primary melanoma patients. For all, we collected clinical, pathological information and dermoscopic images of the histopathologically diagnosed melanomas and dysplastic nevi, when available. Results: After excluding patients positive for CDKN2A/CDK4 pathogenic variants and those affected by non-cutaneous melanomas, our study cohort comprised 984 cutaneous melanoma patients, 22 MITF+ and 962 MITF-. MITF+ were more likely to develop dysplastic nevi and multiple primary melanomas. Nodular melanoma was more common in MITF+ patients (32% compared to 19% in MITF-). MITF+ patients showed more frequently dysplastic nevi and melanomas with uncommon dermoscopic patterns (unspecific), as opposed to MITF- patients, whose most prevalent pattern was the multicomponent. Conclusions: MITF+ patients tend to develop melanomas and dysplastic nevi with histopathological features, frequency and dermoscopic patterns often different from those prevalent in MITF- patients. Our results emphasize the importance of melanoma prevention programs for MITF+ patients, including dermatologic surveillance with digital follow-up.

Mycosis fungoides (MF) and Sézary syndrome (SS) are two variants of cutaneous T-cell lymphomas (CTCL). These entities are considered two distinct diseases, based on phenotypic and genetic analyses. Neoplastic cells in MF seem to derive from T effector memory cells, instead, neoplastic SS cells have a central memory (TCM) profile. Moreover, evidence of a resident memory T cell (TRM) profile in early-stage MF, compared to migratory memory T cells (TMM) profile in advanced-stage MF, seems to explain the clinical behavior of MF during progression. Recent studies have shown more heterogeneity in CTCL phenotypes compared to that theory. Differences between MF and SS also involve the microenvironment, which predominantly expresses Th1 phenotype compared to Th2 phenotype in advanced stages and SS. A series of studies have identified multiple molecular changes in CTCL, showing a heterogeneous landscape of numerous genetic alterations without a strong differential signature between the two diseases. Molecular mutations could be found more frequently in the pathways of epigenetic and/or chromatin regulation, TCR and T-cell/ cytokine signaling, JAK/STAT signaling, and phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt) and NF-kB pathway. The aim of this project is to delineate the differences between MF and SS about the phenotypic and genetic point of view, taking into account all stages of the diseases. In the first phase of the project, 15 patients with a new diagnosis of MF and SS were selected, including 5 early-stage MF (IA-IIA), 3 advanced MF (2 III MF, 1 IIB MF), 5 classical SS, 2 non-erythrodermic SS. Neoplastic T-cell immune phenotypes were evaluated on paraffin-embedded formalin-fixed sections of skin biopsies and on CD4+CD7- sorted T cells of the peripheral blood by flow cytometry (except for two CD7+ cases). About the 3 patients with stage IB MF, two of them revealed a TRM phenotype (CD69+CD103+CCR7-CD62L-). In the third of them, T cells aberrantly showed expression of CD69 and also CD62L. One IIA-MF patient showed expression of CD69 and CD103 (TRM markers) but also a partial expression of CCR7 with the negativity of CD62L, as TMM phenotype. Erythrodermic and tumoral MF patients were characterized by an infiltrate with a TMM phenotype. In SS, 3 patients showed a typical TCM phenotype (CD45RO+CD27+CCR7+CD62L+). Out of 5 CD45RA+ cases, 4 of them evidenced a T naïve phenotype (CD62L+), including an early MF (IIA). One SS showed complete negativity of CD62L and CCR7, arguing a TEMRA phenotype. Immunophenotype of blood samples revealed a better correlation with the skin of SS patients compared to MF patients. Neoplastic T cells in MF mainly had a Th1 phenotype, also in advanced stages, compared to Th2 phenotype of SS patients. This first step of the project confirmed that MF and SS are characterized by heterogeneity of phenotypes with partial correlation to the clinical features. Evidence of the same TCM phenotype in patients with classical and atypical SS suggests that criteria of this disease should be revised, including also non-erythrodermic forms. In the second phase of the project, we performed a gene expression analysis using a 770-genes panel by NanoString technologies (PanCancer Immune Profiling Panel). To obtain more statically significant data, we included 95 FFPE slides of CTCL. Total RNA from 87 samples was extracted (36 SS and 51 MF at any stage). NanoString data were processed and statistical analysis was performed within the statistical environment R. A principal component analysis was performed on all samples together, but we found a homogeneous group without any evidence of clustering between MF and SS. So, we decided to study separately the two entities. About MF, 12 differentially expressed genes (DEGs) between advanced stages and early stages were found (p-value <0.01): 9 of them resulted upregulated (CCR3, PRAME, FPR2, PMCH, AMBP, TRL7, TNFRSF10C, CFI, HAVCR2) and 3 were down-regulated (KLRB1, CD1B, CD5). Gene ontology (GO) enrichment analysis showed that those genes were significantly enriched in the regulation of the immune system, macrophage activation and Toll-like receptor (TLR) signaling. (FDR <0.05) KEGG pathways analysis revealed a not significantly representation in any pathways. Then, MF cohort was divided into two groups based on the median expression for each gene and the effects of high or low expression levels on OS were examined using the Kaplan-Meier (KM) survival curve. A list of 39 genes was identified as significant associated with OS (p-value <0.05). The HRs and p-values of those selected 39 genes were calculated through Cox regression model, revealing a 9-genes signature, which did not match with DEGs but showed a high significance from KM survival analysis. The increased expression of the following genes is significantly associated with poor prognosis: CDK1 (HR=2.06), IL6ST (HR=1.49), CCR4 (HR=1.66), ITK (HR=1.78), NOS2A (HR=1.38), IL2RA (HR=2.06), LRRN3 (HR=1.39), DUSP4 (HR=1.74). Instead, the lower expression of CCL26 (HR=0.53, p-value 0.028) is significantly associated to poor prognosis. A prognostic score was developed based on the incidence of each gene of the signature on overall survival and was defined as the linear combination of logarithmically transformed gene expression levels weighted by average Cox regression co-efficient. Patients with a high score of expression of the signature had a significantly poorer prognosis compared to patients with a low score (p-value < 0.05). Similarly, considering advanced versus early stages, the two groups with high score confirmed to have a statistically significant poorer prognosis compared to low score (p-value <0.05). This result is particularly evident comparing the score in early stages. KEGG pathway analysis showed that the 9-genes signature was significantly enriched in cytokine and JAK-STAT signaling. For SS samples, we did not evaluate DEGs because of the absence of different stages like in MF, but we performed the same analyses based on OS. A 14-gene signature was found, characterized by the association of their high expression and poor prognosis. The genes identified were IL12A (HR=2.12), IL5RA (HR=2.80), IFNL2 (HR=2.21), NT5E (HR=1.97), IL18RAP (HR=1.89), ABCB1 (HR=1.74), CCL16 (HR=1.89), CCL1 (HR=1.95), IL22RA2 (HR=1.67), IFNA17 (HR=1.69), C9 (HR=1.79), CCR9 (HR=3.31), SPANXB1 (HR=1.62), TREM1 (HR=1.76). We applied the prognostic score, showing that patients with a high score had a worse prognosis compared to patients with a low score. GO analysis showed that this 14-gene signature was significantly involved in defense response and Th1 cytokine production (FDR <0.05). KEGG pathway analysis was able to confirm a statistically significant involvement of these genes in JAK-STAT and TLR signaling. In this second phase of the project, we were able to find a gene signature for each disease with a significant prognostic value that could be useful in clinical practice, especially in early stages. Evidence of a strong involvement of JAK-STAT pathway in analysis of both MF and SS is interesting because this pathway is well known to be involved in CTCL pathogenesis and its pharmaceutical inhibition is still studied.

2014 - 2015
The urokinase type plasminogen activator (uPAR) is a three domain GPI-anchored cell surface receptor. uPAR expression is strongly up-regulated and represents a negative prognostic factor in various tumors, including hematologic malignancies. uPAR expression is post-transcriptionally regulated by RNA binding proteins (RBPs). RBPs bind specific sequences in the 3’untranslated region (3’UTR) of uPAR-mRNA, stabilizing or destabilizing the transcript. The 3’UTR of transcripts from a large number of genes includes target sequences also for small translational repressors RNAs (miRNAs). miRNAs play key roles in many cellular pathways; their aberrant expression is a common feature of various malignancies. We selected three miRNAs miR-146a, miR-335 and miR-622 that could bind the 3’UTR of uPAR-mRNA; these three miRNAs, as reported in literature, are expressed in CD34+ HSC or in acute myeloid leukemia (AML) cells and can act as oncosuppressors by inhibiting oncogene expression. We found that selected miRNAs regulate uPAR expression by directly targeting its 3’UTR in AML cell lines. Indeed, uPAR expression is reduced by their overexpression and increased by their specific inhibitors. Overexpression of selected miRNAs impaired cell migration, invasion and proliferation of AML cell lines. Interestingly, we found an inverse relationship between uPAR expression and miR- 146a and miR-335 levels in AML blasts. This suggests their possible role in regulating uPAR expression also in vivo. We also investigated the capability of uPAR-3’UTR to act as competing endogenous RNA (ceRNA). We showed that uPAR-3’UTR overexpression up-regulates uPAR expression and expression of other targets of selected miRNAs; these results suggest that uPAR-3’UTR may recruit selected miRNAs, allowing translation of their targets, thus acting as ceRNA. [edited by Author]
XIV n.s.

Intrauterine Growth Restriction (IUGR) is a pregnancy-related pathology characterized by a placental insufficiency phenotype and a multifactorial etiology that still needs to be completely clarified. IUGR is associated with increased risk of maternal and neonatal perinatal mortality and morbidity and a tendency to develop cardiovascular and metabolic pathologies in the adulthood. A deeper knowledge of the alterations occurring in IUGR has therefore become essential to find therapeutic tools to prevent fetal, neonatal and future adult complications. A specific placental phenotype has been associated with IUGR, characterized by placentation defects, altered transport of oxygen and nutrients to the fetus, impaired mitochondria content and increased oxidative stress (OxS). Mitochondria (mt) are eukaryotic ubiquitous organelles whose number range from hundreds to thousands of copies per cell. As they are the fuel stations of all cells, more than 95% of ATP is synthesized in these organelles Besides this well-known function, many essential pathways involve mitochondria, such as mt biogenesis. Mt biogenesis is a complex of mechanisms needed to mitochondria ex-novo creation: mt DNA duplication and translation of mt factors controlling the transcription machinery that produce all respiratory chain complexes (RCC). IUGR hypoxic features, and the consequent higher OxS, affect mitochondria as showed by in vivo models increased mt oxygen consumption trigger by hypoxia or in vitro downregulation of mt biogenesis. The aim of this study was to investigate, by ex vivo experiments and in vitro models, different types of placental cells to deeper characterize the placental insufficiency features of IUGR, with specific attention to the consequences of its hypoxic environment. IUGR and physiological placenta bioenergetics were first examined, by analyzing both mitochondrial (mt) content and function in whole placental tissue and in several placental cell types (cytotrophoblast and mesenchymal stromal cells). Mt DNA content resulted higher in IUGR placentas compared to controls, as well as NRF1 (biogenesis activator) mRNA levels. Oppositely, both mtDNA and NRF1 expression levels were significantly lower in cytotrophoblast cells isolated from IUGR placentas compared to controls. The observed divergence between placental tissue and cytotrophoblast cells may suggest that other placental cell types (e.g. syncytiotrophoblast, endothelial cells and mesenchymal stromal cells), that are subjected to different oxygen - and consequently oxidative stress - levels may be responsible for the mt content increase in the whole placental tissue. Moreover, a different exposure to progesterone may also explain this mt content divergence, since progesterone, regulating mt biogenesis, is produced by syncytio but not in cytotrophoblast cells. In IUGR cytotrophoblast cells, respiratory chain complexes (RCC) showed lower, though not significantly, gene expression levels and no differences in their protein expression compared to controls. In contrast, mt bioenergetics - represented by cellular O2 consumption - was higher in IUGR versus controls, especially in more severe IUGR cases. Thus, despite the protein content of RCC was not altered, their activity was significantly increased in IUGR cytotrophoblast cells, possibly due to a more efficient RCC assembly. Finally, as O2 consumption resulted inversely correlated to mtDNA in cytotrophoblast cells, a functional (respiration) compensatory effect to the decreased mitochondrial content might be hypothesized. Estrogen-Related Receptor (ERRγ) is a very interesting transcriptional factor involved both in mt biogenesis and function and in estradiol production (through CYP19 aromatase up-regulation). ERRγ and CYP19 mRNA levels were therefore analyzed, for the first time in human IUGR placentas. In whole placental tissue CYP19 showed higher expression in IUGR compared to controls, progressively increasing with IUGR severity. Higher ERRγ expression in IUGR cases was also found, though not significantly. These data are consistent with mtDNA and NRF1 results, thus confirming altered mt biogenesis and content in IUGR and strengthening the hypothesis of a restore attempt made through the stimulation of mt biogenesis. An additional effect of ERRγ increase is CYP19 upregulation. The observed higher CYP19 expression may indicate a protective mechanism exerted through estradiol against oxidative stress. Opposite to their placental tissue expression, ERRγ levels in cytotrophoblast cells significantly decreased in the IUGR group compared to controls. This is consistent with literature evidences of O2-dependent ERRγ gene expression in trophoblast cells. As well as for mt DNA and NRF1 levels, other cell types could be responsible for ERRγ increase in the whole placental tissue. CYP19 expression was not significantly different between IUGR and controls in cytotrophoblast cells, though it positively correlates with ERRγ levels, but low CYP19 levels are reported for cytotrophoblast cells, and this might complicate the detection of any difference. Interestingly, a significant positive correlation linked maternal BMI and expression of both ERRγ and CYP19 genes (in whole placental tissue: positive trend/cytotrophoblast cells: negative trend). An estradiol-dependent regulation of leptin production through ER (Estrogen Receptor) – ERR is known. Leptin, an anti-obesity hormone produced also by placenta, increase during. The future measure of plasmatic levels of both leptin and 17β estradiol in maternal blood will verify this speculation. Then in vitro experiments were performed to assess possible biomolecular mechanisms regulating mithocondrial content in Intrauterine Growth Restriction, by culturing primary placental cells under normal oxygen conditions and hypoxia, a typical feature of IUGR. Fluctuations in placental oxygen concentration may generate oxidative stress (OxS), that is enhanced in Intrauterine Growth Restriction condition. As mitochondria are the major producers of intracellular reactive oxygen (O2) species through free radicals generated by the mt oxidative phosphorylation, altered intrauterine O2 conditions might affect mt DNA content and function, leading to increased oxidative stress in IUGR placental cells. Using trophoblast primary cell lines could help to understand O2 conditions that placentas may be exposed to in IUGR pregnancy. Exposure of trophoblast cultures to hypoxia is an in vitro model commonly used in the last few years. Preliminary data from performed experiments show that the oxygen lack in cytotrophoblast cells leads to increased mt DNA levels. The evidence that O2 levels may regulate mt biogenesis in cytotrophoblast cells highlights their deep sensitivity to O2 conditions. However, further data are needed to confirm these preliminary results, also considering the implied difficulties in adapting the primary cytotrophoblast cultures, very sensitive to O2 concentration, to an in vitro model. A future goal will be reproduce particularly hypoxia/re-oxygenation intervals characterizing placental insufficiency and generating OxS and measuring cell apoptosis levels and autophagy markers (e.g. TNF-α, p53, caspases). Finally, in vitro experiments were performed to isolate and characterized p-MSCs from physiological and affected by IUGR placentas. p-MSCs have never been investigated before in IUGR pregnancies, but their role have been recently studied in preeclamptic placentas. PE p-MSCs show pro-inflammatory and anti-angiogenic features, that may result in abnormal placental development. In the performed p-MSCs cultures, mesenchymal markers enrichment and multipotent differentiation abilities confirm the successful isolation and selection of a mesenchymal stromal cell from placental membranes and basal disc of both physiological and IUGR placentas. As attested by flow cytometry data, the p-MSC population is earlier selected in IUGR placentas: this faster selection might represent a compensatory mechanism to metabolic alterations occurring in IUGR placental cells and/or to the adverse IUGR placental environment. During placenta development, the lower proliferation rate characterizing IUGR pMSCs could impair the primary villi formation and consequently trophoblast development, since MSCs both serve as structural of trophoblast cells. Moreover, IUGR p-MSCs population display lower endothelial and higher adipogenic differentiation potentials compared to controls. During pregnancy, pMSCs usually contribute to both vasculogenesis and angiogenesis Interestingly, several studies report some alterations in maternal and fetal endothelial progenitor or in the angiogenic capacity of IUGR placental cells. Opposite to endothelial differentiation ability, the adipogenic potential in pMSCs from IUGR is increased compared to controls: as these changes are evident early in life, the predisposition to obesity may be programmed in utero. To further characterize IUGR pMSCs, their mitochondrial (mt) content was investigated by measuring NRF1 and Respiratory Chain UQCRC1 and COX4I1 gene expression levels. Mesenchymal stem cell metabolism is known to be mainly anaerobic, with a shift towards an aerobic mitochondrial metabolism reported during differentiation. Interestingly, p-MSCs cultured with no differentiating medium present a trend towards higher NRF1, UQCRC1 and COX4I1 expression levels in IUGR basal disc samples compared to controls and higher COX4I1 levels in IUGR placental membranes; these differences are not statistically significant likely because of the low sample number. Nevertheless, they might account for metabolic alterations in IUGR p-MSCs, showing a possible shift to aerobic metabolism, with the loss of the metabolic characteristics that are typical of multipotent and undifferentiated cells. The different gestational age between cases and controls, typical of all IUGR versus term-placentas studies, is a possible limit that associate all the performed experiments. However, any significant correlation between gestational age (ge) and the O2 consumption of CIV (which presents the highest significance between IUGR and controls), ge and mt DNA levels, ge and ERRy/CYP19 expression, ge and p-MSCs. CYP19 gene expression have been analyzed assuming that it may represent an index of aromatase content in placental tissue. However, post-translational modifications (glycosylation and phosphorylation) may occur, affecting its functional activity. Finally, a potential limitation of placental mesenchymal stromal cells is that the analysis was performed on IUGR placentas at delivery, whereas placental abnormal development of IUGR pathology is supposed to start already at the beginning of placentation. Taken together, reported data highlight mitochondrial alterations occurring in placentas of Intrauterine Growth Restricted pregnancies, through ex vivo and in vitro approaches. These results shed genuine new data into the complex physiology of placental oxygenation in IUGR fetuses. Mitochondrial content is higher in IUGR total placental tissue compared with normal pregnancies at term. This difference is reversed in cytotrophoblast cells of IUGR fetuses, which instead present higher mitochondrial functionality. These findings suggest different mitochondrial features depending on the placental cell lineage. Indeed, our results on placental Mesenchymal Stromal Cells, showed higher levels of genes accounting for mitohcondrial content and function. The increased placental O2 consumption by placental tissue may represent a limiting step in fetal growth restriction, preventing adequate O2 delivery to the fetus. This limitation has potential consequences on fetal O2 consumption both in animal models and in human IUGR.

Recent advances in 3D culture technology allow embryonic and adult mammalian stem cells to generate organoids in vitro, which reflect key structural and functional properties of organs they originate (Clevers H., Cell, 2016). For this reason, they represent a powerful tool to study human physiological and pathological processes, in particular to investigate complex processes like tumorigenesis and tumor growth, resembling the in vivo mechanisms. In particular, in tumor contest neoplastic cells activate several strategies to escape from immunesurveillance, such as the recruitment of immune cells with immunosuppressive functions. In this regard, CD4+ T regulatory cells (Tregs), physiologically engaged in the maintenance of immunological self-tolerance and immune homeostasis, are potent suppressors of effector cells and found at high frequencies in various types of cancer. A recent transcriptome analysis performed in our lab (De Simone M. et al., Immunity, 2016) revealed that tumor-infiltrating Tregs, isolated from CRC (colorectal cancer) and NSCLC (non-small cell lung cancer) patients, expressed a unique and specific gene signature, correlated with patients’ survival. In line with our findings, non-lymphoid tissue infiltrating Tregs can exhibit specific phenotypes and transcriptional profile involved in glucose metabolism, tissue repair and muscle regeneration, far from their well-established suppressive roles (Cipolletta D. et al., 2012; Arpaia N. et al., 2015). Thus, our work wants to evaluate the immune dependent and independent function of tissue- infiltrating Tregs, exploiting a co-culture model with normal and colon cancer- derived organoids. This approach could be suitable to recapitulate primary tumorigenesis, cancer microenvironment effect on Tregs recruitment and phenotype and, vice versa, infiltrating Tregs influence on tumor onset, growth and tissue homeostasis. In order to answer our biological questions by using organoid model, we first of all derived organoids starting from CRC patients’ biopsies, according to protocol published by Sato T. et al. (2011). We generated a biobank of 20 and 28 human- normal and tumoral colon- derived organoid lines, respectively, from tumoral biopsies and the adjacent normal mucosa of patients affected by colorectal cancer. In particular, these organoid lines can be propagated, frozen and defrosted like any tumour cell line, morphologically recapitulating the cellular composition and architecture of colon primary tissue and, importantly, representative of all CRC molecular subtypes. Moreover, our RNA-seq bulk analysis on our organoid lines unveiled that they are very stable from the transcriptional point of view during passages and preserve the inter-individual heterogeneity. Furthermore, our ChIP-seq data showed that our library resembled the same epigenetic landscape of colorectal primary tumour; in this context our study represents an innovative approach to investigate epigenetic processes leading CRC development and progression. Considering that Tumor-infiltrating- Tregs (TI- Tregs) were found to express a peculiar gene signature (De Simone et al., 2016; Plitas et al., 2016), we decided to exploit organoid model to better elucidate processes leading the development and the recruitment of these cells at the tumour site. To this end, we proceeded with the establishment of Tregs- PDO co-culture, assessing the feasibility of this system and evaluating Tregs viability in organoid culture conditions, without observing any significant differences in their survival. Moreover, we evaluated their ability to migrate into 3D organoid structure, revealing their capacity to overcome firstly the obstacle represented by Matrigel (which mimics extracellular matrix) and then creeping into the 3D architecture of organoids, recapitulating the same behaviour they have when recruited at the tumour site. An important evidence about the possible influence of tumoroids on Treg phenotype came from our preliminary co-culture experiment, revealing that PDO-Tregs co-culture upregulated the expression of PDL1 (one of the genes belonging to TI-Treg signature (De Simone et al., 2016)) specifically on Treg but not on Tconv cells. On the other hand, with our co-culture preliminary experiment we observed that expanded TI-Tregs enhanced PDL1 expression on tumoroid cells, which not happened when organoids were co-cultivated with Tconv cells, suggesting the possible influence of Tregs on the expression of specific molecules on cancer cell surface. In conclusion, the exploitation of TI-Treg-PDO co-culture could shed light on the interplay between tumor and TI-Tregs, giving the opportunity to potentially interfere in this crosstalk and design a peculiar anticancer therapy targeting TI-Tregs in a personalized manner.

Background. Metabolic reprogramming is a key feature of neoplastic transformation and mitochondria are the most important organelles in such oncogenic process. Recent evidence suggests that TRAP1 is a key player in tumor-related metabolic rewiring. Most studies have addressed TRAP1- related oncogenesis by in vitro and in vivo analyses. Little is however known on the possible contribution of histology to such studies. Study aims. This study assessed the role of histology in the study of TRAP1-related metabolic reprogramming. Specifically, it aimed: (i) to integrate the results of in vitro and in vivo studies with the histological analysis of tumor samples; (ii) to verify the correspondence between primary human neoplasms and animal tumor models; (iv) to identify novel fields for the study of TRAP1-related oncogenic cascades. Materials and methods. This project considered the following neoplastic settings: (i) neurofibromatosis type 1 (NF1)-related benign and malignant nerve sheath tumors; and (ii) germinal center (GC)-derived lymphoproliferative disorders. For the NF1-related tumors, morphological analysis and phenotypic characterization (TRAP1, HIF1a and related metabolic markers) were performed on: (i) human samples of plexiform neurofibroma (PN) and malignant nerve sheath tumors (MPNST); (ii) engineered mouse models of NF1-related neoplasms; and (iii) xenografts of MPNST. For GC-derived lymphomas, TRAP1 expression was assessed in non-neoplastic lymphoid tissues and in Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and Hodgkin lymphoma (HL) samples. The immunohistochemical results were integrated with the results of in silico gene expression studies (Oncomine database). Results. Histological analysis of human PNs and MPNSTs documented the expression of TRAP1, HIF1a and downstream metabolic markers in both benign and malignant samples. A progressive increase in the positivity for such proteins was noted along the oncogenic cascade from nonneoplastic nerves to benign (PN) and malignant (MPNST) tumors. Similar expression patterns were observed in the animal tumor models. In this context, histological evaluation also proved instrumental: (i) to confirm the correspondence between human and animal tumors; (ii) to investigate the metastatic potential of MPNST xenografts; (iii) to detect the effects of TRAP1 knock-down on tumor cell growth and metabolic reprograming; and (iv) to highlight strongly versus minimally activated metabolic pathways in NF1-related oncogenic cascades. The histological characterization of reactive lymphoid tissues highlighted TRAP1 expression in subsets of GC blasts (i.e. differentiating immunoblasts and re-cycling centroblasts). The joint expression of TRAP1 and HIF1a in GC blasts confirmed the presence and activation of the TRAP1/HIF1a axis in GC physiology. In silico studies of GC-derived lymphomas showed very high TRAP1 mRNA levels in BL and (to a lesser extent) DLBCL and HL. Immunohistochemical analysis of primary tumor samples confirmed the in silico results. Conclusions. Histological analysis contributes to the understanding of tumor metabolism and integrates the results of in vitro and in vivo biochemical studies. In particular, it confirms the relevance of TRAP1 activation in NF1-related peripheral nerve sheath tumors and discloses a tight correspondence between primary human samples and animal tumor models. Immunohistochemical characterization of reactive lymphoid tissues and primary lymphoma samples also identifies specific TRAP1 expression profiles, possibly subtending tumor-related metabolic networks.
Background. Metabolic reprogramming is a key feature of neoplastic transformation and mitochondria are the most important organelles in such oncogenic process. Recent evidence suggests that TRAP1 is a key player in tumor-related metabolic rewiring. Most studies have addressed TRAP1- related oncogenesis by in vitro and in vivo analyses. Little is however known on the possible contribution of histology to such studies. Study aims. This study assessed the role of histology in the study of TRAP1-related metabolic reprogramming. Specifically, it aimed: (i) to integrate the results of in vitro and in vivo studies with the histological analysis of tumor samples; (ii) to verify the correspondence between primary human neoplasms and animal tumor models; (iv) to identify novel fields for the study of TRAP1-related oncogenic cascades. Materials and methods. This project considered the following neoplastic settings: (i) neurofibromatosis type 1 (NF1)-related benign and malignant nerve sheath tumors; and (ii) germinal center (GC)-derived lymphoproliferative disorders. For the NF1-related tumors, morphological analysis and phenotypic characterization (TRAP1, HIF1a and related metabolic markers) were performed on: (i) human samples of plexiform neurofibroma (PN) and malignant nerve sheath tumors (MPNST); (ii) engineered mouse models of NF1-related neoplasms; and (iii) xenografts of MPNST. For GC-derived lymphomas, TRAP1 expression was assessed in non-neoplastic lymphoid tissues and in Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and Hodgkin lymphoma (HL) samples. The immunohistochemical results were integrated with the results of in silico gene expression studies (Oncomine database). Results. Histological analysis of human PNs and MPNSTs documented the expression of TRAP1, HIF1a and downstream metabolic markers in both benign and malignant samples. A progressive increase in the positivity for such proteins was noted along the oncogenic cascade from nonneoplastic nerves to benign (PN) and malignant (MPNST) tumors. Similar expression patterns were observed in the animal tumor models. In this context, histological evaluation also proved instrumental: (i) to confirm the correspondence between human and animal tumors; (ii) to investigate the metastatic potential of MPNST xenografts; (iii) to detect the effects of TRAP1 knock-down on tumor cell growth and metabolic reprograming; and (iv) to highlight strongly versus minimally activated metabolic pathways in NF1-related oncogenic cascades. The histological characterization of reactive lymphoid tissues highlighted TRAP1 expression in subsets of GC blasts (i.e. differentiating immunoblasts and re-cycling centroblasts). The joint expression of TRAP1 and HIF1a in GC blasts confirmed the presence and activation of the TRAP1/HIF1a axis in GC physiology. In silico studies of GC-derived lymphomas showed very high TRAP1 mRNA levels in BL and (to a lesser extent) DLBCL and HL. Immunohistochemical analysis of primary tumor samples confirmed the in silico results. Conclusions. Histological analysis contributes to the understanding of tumor metabolism and integrates the results of in vitro and in vivo biochemical studies. In particular, it confirms the relevance of TRAP1 activation in NF1-related peripheral nerve sheath tumors and discloses a tight correspondence between primary human samples and animal tumor models. Immunohistochemical characterization of reactive lymphoid tissues and primary lymphoma samples also identifies specific TRAP1 expression profiles, possibly subtending tumor-related metabolic networks.