Clinicopathological and molecular characterization of gastroesophageal junction (GEJ) adenocarcinoma before age of 40 years

Gastroesophageal junction (GEJ) adenocarcinoma are uncommon before age of 40 years. While certain clinical, pathological and molecular features of GEJ adenocarcinoma in older patients have been extensively studied, these characteristics in the younger population remain to be determined. In the recent literature, a high sensitivity and specificity for the detection of dysplasia and esophageal adenocarcinoma was demonstrated by using multicolor fluorescence in situ hybridization (FISH) DNA probe set specific for the locus specific regions 9p21 (p16), 20q13.2 and Y chromosome. We evaluated 663 patients with GEJ adenocarcinoma and further divided them into 2 age-groups of or= 50 years, rispectively. FISH with selected DNA probe for Y chromosome, locus 9p21 (p16), and locus 20q13.2 was investigated with formalin fixed and parassin embedded tissue from surgical resections of 17 younger and 11 older patients. Signals were counted in > 100 cells with each given histopathological category. The chromosomal aberrations were then compared in the 2 age-groups with the focus on uninvolved squamous and columnar epithelium, intestinal metaplasia (Barrett's mucosa), glandular dysplasia, and adenocarcinoma. Comparisons were performed by the X2 test, Fisher's exact test, Student's t-test and Mann-Whitney U-test as appropriate. Survival was estimated by the Kaplan-Meier method with univariate analysis by the log-rank. Significance was taken at the 5% level. There was no difference in the surgical technique applied in both age groups and most patients underwent Ivor Lewis esophagectomy. Among clinical variables there was a higher incidence of smocking history in older patient group. We identified a progressive loss of Y chromosome from benign squamos epithelium to Barrett's mucosa and glandular dysplasia, and, ultimately, to a near complete loss in adenocarcinoma in both age groups. The young group revealed significantly more losses of 9p21 in both benign and neoplastic cells when compared to the older patients group. In addition, we demonstrated an increase in the percentage of cells showing gain of locus 20q13.2 with progression from benign epithelium through dysplasia to adenocarcinoma with almost the same trend in both the young and the older patients. When compared with the older age-group, younger patients with GEJ adenocarcinoma possess similar known demographics, environmental factors, clinical, and pathologic characteristics. The most commonly detected genetic aberrations of progressive Y chromosomal loss, 9p21 locus loss, and 20q13 gains were similar in the younger and older patients. However the rate of loss of 9p21 is significantly higher in young patients, in both the benign and the neoplastic cells. The loss of 9p21, and possibly, the subsequent inactivation of p16 gene may be one of the molecular mechanisms responsible for the accelerated neoplastic process in young patients.

Molecular analysis of special type breast carcinomas

The project was developed into three parts: the analysis of p63 isoform in breast tumours; the study of intra-tumour eterogeneicity in metaplastic breast carcinoma; the analysis of oncocytic breast carcinoma. p63 is a sequence-specific DNA-binding factor, homologue of the tumour suppressor and transcription factor p53. The human p63 gene is composed of 15 exons and transcription can occur from two distinct promoters: the transactivating isoforms (TAp63) are generated by a promoter upstream of exon 1, while the alternative promoter located in intron 3 leads to the expression of N-terminal truncated isoforms (ΔNp63). It has been demonstrated that anti-p63 antibodies decorate the majority of squamous cell carcinomas of different organs; moreover tumours with myoepithelial differentiation of the breast show nuclear p63 expression. Two new isoforms have been described with the same sequence as TAp63 and ΔNp63 but lacking exon 4: d4TAp63 and ΔNp73L, respectively. Purpose of the study was to investigate the molecular expression of N-terminal p63 isoforms in benign and malignant breast tissues. In the present study 40 specimens from normal breast, benign lesions, DIN/DCIS, and invasive carcinomas were analyzed by immunohistochemistry and RT-PCR (Reverse Transcriptase-PCR) in order to disclose the patterns of p63 expression. We have observed that the full-length isoforms can be detected in non neoplastic and neoplastic lesions, while the short isoforms are only present in the neoplastic cells of invasive carcinomas. Metaplastic carcinomas of the breast are a heterogeneous group of neoplasms which exhibit varied patterns of metaplasia and differentiation. The existence of such non-modal populations harbouring distinct genetic aberrations may explain the phenotypic diversity observed within a given tumour. Intra-tumour morphological heterogeneity is not uncommon in breast cancer and it can often be appreciated in metaplastic breast carcinomas. Aim of this study was to determine the existence of intra-tumour genetic heterogeneity in metaplastic breast cancers and whether areas with distinct morphological features in a given tumour might be underpinned by distinct patterns of genetic aberrations. 47 cases of metaplastic breast carcinomas were retrieved. Out of the 47 cases, 9 had areas that were of sufficient dimensions to be independently microdissected. Our results indicate that at least some breast cancers are composed of multiple non-modal populations of clonally related cells and provide direct evidence that at least some types of metaplastic breast cancers are composed of multiple non-modal clones harbouring distinct genetic aberrations. Oncocytic tumours represent a distinctive set of lesions with typical granular cytoplasmatic eosinophilia of the neoplastic cells. Only rare example of breast oncocytic carcinomas have been reported in literature and the incidence is probably underestimated. In this study we have analysed 33 cases of oncocytic invasive breast carcinoma of the breast, selected according to morphological and immunohistochemical criteria. These tumours were morphologically classified and studied by immunohistochemistry and aCGH. We have concluded that oncocytic breast carcinoma is a morphologic entity with distinctive ultrastructural and histological features; immunohistochemically is characterized by a luminal profile, it has a frequency of 19.8%, has not distinctive clinical features and, at molecular level, shows a specific constellation of genetic aberration.

Adipose-derived stem cells and tissue revascularization: enhancing islet survival and performance for diabetes care.

Pancreatic islet transplantation represents a fascinating procedure that, at the moment, can be considered as alternative to standard insulin treatment or pancreas transplantation only for selected categories of patients with type 1 diabetes mellitus. Among the factors responsible for leading to poor islet engraftment, hypoxia plays an important role. Mesenchymal stem cells (MSCs) were recently used in animal models of islet transplantation not only to reduce allograft rejection, but also to promote revascularization. Currently adipose tissue represents a novel and good source of MSCs. Moreover, the capability of adipose-derived stem cells (ASCs) to improve islet graft revascularization was recently reported after hybrid transplantation in mice. Within this context, we have previously shown that hyaluronan esters of butyric and retinoic acids can significantly enhance the rescuing potential of human MSCs. Here we evaluated whether ex vivo preconditioning of human ASCs (hASCs) with a mixture of hyaluronic (HA), butyric (BU), and retinoic (RA) acids may result in optimization of graft revascularization after islet/stem cell intrahepatic cotransplantation in syngeneic diabetic rats. We demonstrated that hASCs exposed to the mixture of molecules are able to increase the secretion of vascular endothelial growth factor (VEGF), as well as the transcription of angiogenic genes, including VEGF, KDR (kinase insert domain receptor), and hepatocyte growth factor (HGF). Rats transplanted with islets cocultured with preconditioned hASCs exhibited a better glycemic control than rats transplanted with an equal volume of islets and control hASCs. Cotransplantation with preconditioned hASCs was also associated with enhanced islet revascularization in vivo, as highlighted by graft morphological analysis. The observed increase in islet graft revascularization and function suggests that our method of stem cell preconditioning may represent a novel strategy to remarkably improve the efficacy of islets-hMSCs cotransplantation.

Valutazione dell'efficacia dell'infusione intraepatica di cellule staminali (SC) autologhe CD133+ in pazienti affetti da cirrosi ed insufficienza epatica di grado avanzato

Numerose evidenze sperimentali hanno dimostrato il contributo delle cellule staminali (SC) di derivazione midollare nei processi di rigenerazione epatica dopo danno tissutale. E’ cresciuto pertanto l’interesse sul loro potenziale impiego in pazienti con cirrosi. Questo studio si proponeva di valutare la fattibilità e la sicurezza della reinfusione intraepatica di cellule staminali midollari autologhe CD133+ in 12 pazienti con insufficienza epatica terminale. Previa mobilizzazione nel sangue periferico mediante somministrazione di granulocyte-colony stimulating factor (G-CSF) alla dose di 7,5 mcg/Kg/b.i.d. e raccolta per leucoaferesi (solo se la concentrazione di CD133 + SC era > 8/μL), le cellule CD133+ altamente purificate sono state reinfuse in arteria epatica a partire da 5x104/Kg fino a 1x106/kg. Nei tre giorni successivi è stato somministrato G-CSF per favorire l’espansione e l’attecchimento delle cellule. Durante la fase della mobilizzazione e quella della reinfusione sono stati eseguiti saggi biologici quali: caratterizzazione fenotipica delle SC circolanti, saggi clonogenici, valutazione della concentrazione sierica del Hepatocyte Growth Factor (HGF), Stromal-Derived Factor-1 (SDF-1) ed il Vascular-Endotelial Growth Factor (VEGF) e caratterizzazione fenotipica delle CD133+SC purificate. Fino ad oggi sono stati reinfusi 12 pazienti. Questi dati preliminari suggeriscono che è possibile mobilizzare e reinfondere un numero considerevole di SC autologhe CD133+ altamente purificate in pazienti con ESLD . Gli studi biologici mostrano che: il numero di progenitori ematopoietici ed endoteliali circolanti è aumentato dopo il trattamento con G–CSF; le SCs CD133+ altamente purificato esprimono marcatori emopoietici ed endoteliali; la concentrazione sierica di HGF, SDF-1, VEGF e la capacità clonogenica di progenitori emopoietici sono aumentati durante la mobilitazione e nelle fasi di reinfusione; il potenziale clonogenico dei progenitori endoteliali mostra espressione variabile.

Targeting epigenetic mechanisms in gastric cancer

Gastric cancer (GC) is a hard challenge for medical oncology, with globally over one million of new diagnoses each year and low survival rates. Gastric carcinogenesis is guided by the interaction of several risk factors, exerting through sequential histopathologic steps, including chronic gastritis, atrophic gastritis, intestinal metaplasia, dysplasia and cancer. GC is classified on the basis of anatomical, histological or molecular classification, reflecting the wide cancer heterogeneity, also highlighted by the inefficacy of the actual treatment schedules. Epigenetic mechanisms alterations affecting DNA methylation, histone methylation and acetylation, are a recognized hallmark of cancer and stand at the basis of gastric carcinogenesis and tumor development. The pharmacological targeting of these altered mechanisms is an attractive option for new cancer treatments. Aim of this study was to test the therapeutic potential of the compound CM-272 for GC, a selective and strong dual inhibitor of DNMT1 and EHMT2, which reached important results in pre-clinical models of other gastrointestinal malignancies. Moreover, in a GC patients case series, the expression of the target of the compound was tested, to prove the rationale for inhibition of DNMT1, EHMT2 and their functional adaptor were over-expressed in the majority of GC patients tissues. Through in-vitro testing of CM-272 alone and in combination with the most used chemotherapeutic treatments for GC in a panel of GC cell lines, this study demonstrated that the compound has a strong ability in inhibiting GC cells growth. Even though not directly inducing apoptosis, CM-272 was able to induce a senescent phenotype in GC cells, and to epigenetically reprogram the transcription of genes involved in phosphorylation cascades and mitochondria metabolism, thus affecting the growth and energetic machinery of cancer cells. In conclusion, the pharmacological targeting of epigenetic mechanisms demonstrated good potential pre-clinical models of GC, and further investigations to test in-vivo efficacy are needed.

Aberrant c-MET activation impairs perinuclear actin cap organization with YAP1 cytosolic relocation

Growing evidence indicates that cell and nuclear deformability plays a crucial role in the determination of cancer cells tumorigenic and metastatic potential. The perinuclear actin cap, by wrapping the nucleus with a functional network of actomyosin cables, can modulate nuclear architecture and consequently cell/nuclear elasticity. The hepatocyte growth factor receptor (MET) stands out among other membrane receptors as crucial player of the actin filaments organization, but no data are available on a specific role for MET in the actin cap assembly and the overall nuclear architecture organization. In a cell system characterized by MET hyperactivation, we observed a strong rearrangement of the cellular actin caps, with a complete dismantling of apical stress fibers and a strikingly enhanced nuclear height. CRISPR/Cas9 silencing of MET completely reverted the aberrant phenotype, resulting in flattened cells with perfectly aligned perinuclear actomyosin bundles, as well as decreased MAPK and PI3K/AKT signaling, cell proliferation rate and aggressiveness. Interestingly, MET ablated cells acquired a remarkably directed and polarized migratory phenotype, contrarily to cells with MET sustained activation showing meandering random walk. A pathway enrichment analysis comparing MET-activated and MET-KO cells RNAseq data, unveiled the contribution of multiple pathways associated with cytoskeleton remodeling, regulation of cell shape and response to mechanical stimuli. In line, the co-transcriptional activator YAP1, playing a major role in cell mechanosensing and focal adhesions/actin stabilization, appeared the culprit of the genetic reassembling of KO cells. Indeed, MET silencing was shown to induce YAP1 nuclear shuttling and increased co-transcriptional activity. Finally, we were able to induce in a normal epithelial model a phenotype closer to MET activated cancer cells only by introducing a constitutive fusion protein of MET. Taken together, our results demonstrate a new mechanism of MET-mediated actin remodeling responsible for a tumor-initiating capacity and meandering random migration, which requires YAP1 inactivation.

Riscontri istologici in individui transgender AFAB in terapia con testosterone

CONTESTO: Il rischio oncologico dell’esposizione al testosterone (T) di organi genitali in transgender AFAB non è noto. SCOPO: valutazione istologica di utero, cervice, salpingi e ovaia asportati in corso di intervento chirurgico di affermazione di genere (GAS) in AFAB che assumevano testosterone. MATERIALI E METODI: valutazione dei dati istologici condotta retrospettivamente tramite la consultazione di 187 cartelle cliniche di soggetti transgender AFAB sottoposti a GAS presso la Ginecologia dell’IRCCS Sant’Orsola, Bologna. RISULTATI: 187 transgender AFAB sono stati sottoposti a isteroannessiectomia bilaterale. Nessun paziente sottoposto a ovariectomia, chemioterapia o radioterapia prima della chirurgia. La mediana della durata di assunzione di T era di 36 mesi (12 mesi-14 anni). 96/187 (51.4%) uteri presentavano endometrio ipoattivo/atrofico, mentre 1 caso di iperplasia senza atipie cellulari è stato identificato (0.5%), 8/187 (4.3%) endometrio polipoide e 4/187 (2.2%) secretivo. Il più comune riscontro istologico cervicale è stata la cervicite cronica (n=174, 93%) associata a metaplasia (n=131,76%). Le salpingi sono risultate indenni in 90/187 (48.1%) casi e con infiammazione cronica in 91/187 (48,7%) casi. La maggior parte delle ovaie analizzate mostravano follicoli in diversi stati di maturazione (n=117, 62.5%). In 20 analisi istologiche sono stati identificati corpi lutei/corpi lutei emorragici (10.7%). CONCLUSIONI: Nessuna lesione premaligna o maligna è stata riscontrata in questi 187 soggetti che assumevano testosterone fino a un massimo di 168 mesi prima della chirurgia. La presenza di follicoli in vari stadi di sviluppo e di corpi lutei suggerisce la possibilità di cicli ovulatori in corso di terapia con testosterone. I risultati di questo studio confermano la sicurezza dell'uso prolungato di T sugli organi genitali di transgender AFAB. Seppur ancora limitate, le evidenze suggeriscono sempre più la mancanza di necessità assoluta di rimuovere utero e ovaia nei soggetti trasgender in terapia con T con il solo fine di prevenire patologie oncologiche.