TITOLO DEL PROGETTO The immunohistochemical and microRNA profiles of adenocarcinoma of the esophagus and cardia.
DESCRIZIONE DEL PROGETTO
Background
In the Western countries, adenocarcinoma of the esophagus and cardia (ADECC) is the commonest type of esophageal cancer; it is the sixth leading cause of cancer death worldwide, with a variable geographic distribution, a global incidence of 461,000 cases and a lifetime risk of 2-5%. The, 7th edition AJCC-UICC staging system aimed to harmonize ADECC staging: ADECC are included in the “esophageal cancer staging” chapter, under the simplified definition “adenocarcinoma of the esophagus” which comprehends adenocarcinomas located in the distal thoracic esophagus, EGJ and within the first 5 cm of the stomach. This definition is recalled in the “ Gastric cancer “ staging chapter, where all cancers located in the stomach 5 cm or more below the EGJ or (ii) those arising within 5 cm of the EGJ but not extending into the EGJ or esophagus are considered gastric in origin. The 7th TNM Committee (AJCC 2010) decision has raised substantial controversies. It was argued that tumors located at the distal esophagus, the EGJ, and the proximal stomach may differ according to epidemiological, pathogenetic, clinical, immunohistochemical (IHC) and molecular features. It was demonstrated that different immunoprofiles could allegedly correspond to a Barrett’s epithelium–like type, a gastric cancer–like type, and a third undefined type, with a more cardio-pyloric-like histological appearance. The debate on possible etiologic factors is strictly topical and it is the core of the controversy that obviously extends to prevention, early diagnosis programs and modalities of surgical treatment for these tumors, firstly on the relationship between gastro-esophageal reflux and ADECC via the sequence Barret’s metaplasia, dysplasia and cancer.
Aims
Aim 1a: define the genetic identification of specific miRNA signatures in the different ADECC histotypes (based on IHC);
Aim b: to assess the relationships among histological features and molecular profiles, relapse, metastasis and survivals;
Aim 2: to identify biomarkers predictors of response to tumor therapy, or prognostic.
Phase1: 12 ADECC patients submitted to esophago-gastric resection will be selected. The RNA will be extracted from tissues samples and miRNA profiling will be executed for 754 human miRNAs.
Phase 2: a cohort of ADECC surgical patients will be enrolled. IHC and genetic analysis will be carried out on the target genes identified in the Phase 1 and it will be performed on tumoral surgical samples.
Methods:
Phase 1. We will retrospectively analyze in a group of 12 patients, the histo-morphological and immunohistochemical profiles of ADECCs, together with the patterns of expression of miRNA. Phase 2. With a prospective non-controlled, explorative, study we will: • focus on the analysis of the expression of miRNAs selected in phase 1, in relation to the histological, immunohistochemical and biological patterns, in order to identify specific variants of primitive and recurrent tumors; • evaluate the pros and cons of the clinical applicability of the above mentioned processes. •Follow up of patients enrolled in Phase 1 and 2: i) to link miRNAs patterns with cancer specific survival (CSS) and modalities of recurrence; ii) to compare IHC and miRNAs patterns of primitive and recurrent tumor. • Follow up of patients enrolled in Phase 1 and 2: the molecular analysis of recurrences will be performed in order to investigate cancer clones selection during/after therapy. In consideration of the fact that the Phase 2 study is observational and the clinical course will be dictated by the current guide lines, cancer recurrence miRNA patterns will be studied in tissue provided i) in course of redo surgery, ii) by diagnostic tissue or cytology sampling.
Materials:
IHC will be performed automatically with Benchmark ULTRA® immunostainer (Ventana/Roche, Ventana Medical Systems) or manually performed with NovoLink Polymer Detection Kit (Novocastra, Newcastle, UK). RNA will be manually extracted from paraffin-embedded tissues samples with RecoverAllTM Total Nucleic Acid Isolation kit (Ambion) kit.
The miRNA profiling will be assessed by means of a two-card set of TaqMan® MicroRNA Array v3.0 (Applied Biosystems). A pre-amplification step with Megaplex™ RT Primers will be applied in order to improve the detection of the miRNAs. Reactions will be run on ABI PRISM 7900HT Sequence Detection System (Life Technologies). The analysis of the collected data will be performed according to the methods of descriptive statistics. Variables will be expressed as means±standard deviations, ranges and frequencies. The Spearman's test, the chi-square test, the Mann-Whitney test and the Kruskal Wallis test will be used when appropriate. MicroRNA Arrays expression will be analyzed with ExpressionSuite Software v1.0 (Life Technologies). Student’s t-test or Anova test will be applied to compare the means of the normalized Ct value. Multivariate analysis will be performed according to the methods of logistic regression and Cox regression. Statistical analysis will be performed with the software IBM SPSS Statistics®, version 21.0 and Prism 5® software (GraphPad software, Inc).
Expected results
1- An improved ability to discriminate between ADECC subtypes by means of immunomorphological and phenotypical markers.
2- The immunophenotypical characterization and miRNA profile will clarify the ADECC tumoral heterogeneity: for each histotypes a specific mirRNA signature will be validated
3- The correlation between IHC markers and miRNA profile to clinically relevant parameters can contribute to a better classification, prognosis and diagnosis of ADECC
DESCRIZIONE DELLE ATTIVITÀ DELL’ASSEGNISTA
Le competenze richieste all’assegnista sono:
- esperienza in tecniche di biologia molecolare e cellulare (estrazione e purificazione di acidi nucleici, retrotrascrizione, analisi di espressione genica tramite Real-Time PCR, ELISA test…)
- esperienza e ottimizzazione (dimostrata da pubblicazioni e esperienza lavorativa in laboratorio) di tecniche immunoistochimiche, colorazioni, immunofluorescenza. Conoscenza di Laboratory Automation Workstation.
- conoscenza delle tecniche di conservazione di campioni di tessuti freschi
- esperienza in studi clinici su marker di IHC e miRNA ai fini prognostici
- gestione e inserimento dati nel database
- analizzare ed elaborare i dati ottenuti sui markers isotologici e molecolari. Conoscenza di software statistici (SPSS, Prism, Exoression Suite) e di imaging (Photoshop, ImageJ.)
- capacità organizzative e di progettazione per la scrittura di proposals
- partecipazione attiva dell’assegnista a conferenze e seminari a livello nazionale e internazionale.
- conoscenza della lingua Inglese, la conoscenza di un’ulteriore lingua europea costituisce titolo preferenziale
- esperienza in strutture private/accademiche internazionali
L’attività sarà svolta presso l’Istituto "F. Addari" Pad 26, Laboratory of Molecular Oncologic and Transplantation Pathology, Bologna. L'assegnista dovrà occuparsi di coordinare le attività di laboratorio del progetto per il suo svolgimento, delle analisi di IHC e di espressione genica in microarray, l'eventuale validazione tramite saggi singoli dei miRNA più interessanti e le analisi statistiche per la correlazione clinica dei dati.
Scheda attività assistenziale (se prevista)
PIANO DELLE ATTIVITÀ ASSISTENZIALI DELL’ASSEGNISTAATTIVITÀ / N. ORE SETTIMANA
Arruolamento pazienti / 5
Processare e conservare i campioni biologici / 3
Coordinare le procedure di analisi / 4
Elaborazione dati e stesura articoli / 6
ALMA MATER STUDIORUM - UNIVERSITÀ DI BOLOGNA
SEDE AMMINISTRATIVA C/O AZIENDA OSPEDALIERO-UNIVERSITARIA – POLICLINICO S. ORSOLA–MALPIGHI
Via MASSARENTI 9, PADIGLIONE 11 - 40138 Bologna - Italia