Breast Cancer Biomarkers: from Identification to Application to FFPE tissues

2011/2012Background: Breast carcinoma (BC) is the most common form of malignancy in women and the leading cause of cancer-related mortality among females internationally. BC includes a series of heterogeneous tumours with a great variability at histological level, biological level and clinical evolution. Because its complexity, BC treatment recommendations are continually changing with the new advances in this field. However there are still a significant number of patients with similar features that show distinct outcome. In order to detect the existing molecular differences and address patients to more personalized treatment, there is a need to find out new cancer biomarkers. Aim: The main goal of my PhD project is to investigate on the possibility of combining traditional clinical and pathological features with new candidate biomarkers for the prognostication of breast cancer. The first part concerns the prognostic role of molecular classification of primary tumours, according to luminal A, luminal B, HER-2+ and basal-like subtypes. In the second part, the prognostic value of nine candidate genes was investigated at mRNA level. The genes of interest belong to the RB pathway (CDK2, RB1), the RAS pathway (HER-2, PI3K, AKT1, AKT2, AKT3, RAF1) and cellular differentiation mechanism (CK8). Methods: This retrospective study comprises 305 BC patients, both lymph node negative (LN-) and lymph node positive (LN+). The molecular classification of primary tumours was performed by means of immunohistochemistry (IHC), using seven surrogate markers: ER, PR, HER-2, Ki67, CK8 and CK5/6, plus vimentin. Results of molecular classification were analysed with respect to morphologic and pathological features, and outcome. Moreover, the molecular characterization was also performed in a set of loco-regional metastatic lymph nodes, to compare the phenotype of primary tumour cells with their matched metastatic cells colonizing regional nodes. To the second purpose, gene’s expression was investigated in the entire cohort of primary tumours by means of real-time PCR, using the TaqMan chemistry. The expression of the nine genes was investigated in connection with the clinical-pathological factors, molecular classification and BC specific patient’s survival. Results: Regarding molecular classification of primary tumours, luminal A, luminal B, HER-2+ and basal-like accounted for 46%, 34%, 8% and 12% respectively. Luminal A tumours were mainly LN-, well differentiated and stage I, while luminal B and HER2+ showed higher tumour grade, nodal metastases as well as higher proliferation status and stage. Luminal A exhibited better survival in comparison to the other subtypes (p<0.001). HER2+ and basal-like showed a poorer outcome. Despite of the longer survival of patients with luminal tumours, they are the only one that underwent long-term recurrences. The molecular classification at the level of loco-regional metastasis, revealed that HER-2, Ki67, CK8 and vimentin positivity was significantly decreased with respect primary tumour, whereas CK5/6 positivity was increased. No significant differences for ER and PR positivity between primary and metastatic lesions were found. Regarding gene’s expression, we found a significant different distribution for all genes, except RAF-1, among LN- and LN+ groups. We also found specific pattern of genes’ expression among molecular classes for: CDK2, HER-2, PI3K, AKT2, AKT3 and CK8. Survival analysis revealed a significant and independent role on patient’s survival for one gene in luminal A tumours. Conclusions: We believe that the use of the traditional biomarkers ER, PR, HER-2 and Ki67 is essential for BC characterisation and prognostication in association with clinical pathological features. Nevertheless, we identified new molecular markers that could better distribute patients into more homogeneous subgroups of BC.XXV Ciclo198

“Analisi in vitro per la valutazione dell’effetto citotossico di nuovi inibitori di Akt in associazione con gemcitabina in linee cellulari tumorali di polmone”

Il cancro del polmone rappresenta il tumore con il più alto tasso di mortalità e con la maggiore incidenza in tutto il mondo. Alla diagnosi oltre l’80% dei casi di carcinoma polmonare presenta il sottotipo istologico non a piccole cellule (NSCLC). Ancor oggi la diagnosi di tale tumore è sfortunatamente tardiva, in media 7 mesi dopo la comparsa dei sintomi, e il 75% dei pazienti presenta al momento della diagnosi uno stadio localmente avanzato o metastatico. Per questi casi lo standard terapeutico è rappresentato dalla chemioterapia, che ha come obiettivo primario la palliazione dei sintomi, il prolungamento della sopravvivenza e il miglioramento delle condizioni generali. Il farmaco chemioterapico gemcitabina è stato approvato come terapia di prima linea nei casi avanzati di NSCLC. Gemcitabina è un analogo nucleoside della deossicitidina, il suo meccanismo d’azione consiste nell’alterazione della sintesi degli acidi nucleici mediante meccanismi di competizione e incorporazione di falsi substrati. Inoltre, è stato dimostrato che tale farmaco è responsabile dell’induzione apoptotica in diverse linee cellulari, incluse quelle di carcinoma polmonare e, in relazione a questo, determina una riduzione dei livelli della proteina antiapoptotica Akt attiva. Negli ultimi anni nuove strategie terapeutiche hanno assunto una grande rilevanza, fra queste ci sono i farmaci bersaglio molecolare specifici. Questi composti sono in grado di bloccare la crescita tumorale attraverso l’inibizione di molecole implicate nei meccanismi di sopravvivenza cellulare. Recentemente è stato dimostrato che la via del segnale PI3K/PDK1/Akt risulta frequentemente alterata in diversi tipi di tumore nell’uomo. Questo pathway ricopre un ruolo fondamentale non soltanto nello sviluppo neoplastico, ma anche nella potenziale risposta del tumore al trattamento. La proteina Akt, una volta attivata ad opera degli enzimi PI3K e PDK1, svolge un ruolo chiave nella tumorigenesi in quanto promuove la crescita, la sopravvivenza e l’angiogenesi delle cellule tumorali. La glicogeno sintasi chinasi-3, i membri della famiglia Forkhead, la ciclina D, gli inibitori delle chinasi ciclina-dipendente p27 e p21, la pro-caspasi 9 e NF-kB sono solo alcuni dei target di Akt. Pertanto l’inibizione del segnale mediato da Akt rappresenta un interessante e valido strumento al fine di migliorare l’effetto delle terapie anticancro esistenti. In base a quanto appena riportato, si è ipotizzato che l’utilizzo di farmaci inibitori di Akt in associazione con gemcitabina favorisca l’inibizione del segnale di sopravvivenza evocato dalla proteina Akt, consentendo così una maggiore efficacia del farmaco standard anche nei casi di chemioresistenza. Lo scopo della presente tesi è stato analizzare l’eventuale effetto citotossico risultante dall’associazione del farmaco gemcitabina, con due composti nuovi inibitori di Akt, G22 e G24, su linee cellulari di cancro del polmone umano NSCLC. Nella prima parte del lavoro sperimentale è stata valutata l’azione antitumorale sia dei singoli composti inibitori di Akt, sia delle loro associazioni con gemcitabina, mediante test di citotossicità cellulare. L’effetto inibitore sulla crescita cellulare è stato analizzato mediante conteggio diretto delle cellule residue vive dopo i trattamenti chemioterapici. Sulla base della percentuale di inibizione, è stata poi calcolata per ogni trattamento la concentrazione inibente il 50% della crescita cellulare definita IC50. Successivamente, è stata condotta un’analisi dello stato fosforilativo della proteina Akt dopo ogni trattamento attraverso saggi immunoenzimatici ELISA, per verificare l’azione dei composti inibitori di Akt e l’eventuale effetto sinergico dell’associazione con gemcitabina. Successivamente è stata eseguita mediante Real Time PCR una discriminazione allelica per analizzare i due polimorfismi a singolo nucleotide o SNPs di Akt nelle linee cellulari in esame (SNP 3/4). L’aplotipo maggiore di entrambi gli SNPs, rispettivamente CC e GG, determina una maggiore quantità di Akt e pertanto una minore risposta apoptotica influenzando così l’eventuale risposta cellulare al trattamento farmacologico. Infine, per valutare le eventuali modulazioni del ciclo cellulare indotte da G22 e G24 è stata effettuata un’analisi con citofluorimetria a flusso che prevede l’utilizzo dello ioduro di propidio come marcatore del DNA. I risultati della citotossicità dei singoli composti inibitori di Akt mostrano un effetto citotossico dose dipendente. La successiva analisi delle combinazioni ha dimostrato che il trattamento in cui la gemcitabina precede entrambi i composti ha un effetto sinergico rispetto alla combinazione inversa, con una maggiore efficacia per il farmaco G24. Dall’analisi dello stato fosforilativo di Akt emerge che il composto G24 determina riduzioni consistenti della fosforilazione di tale proteina (da – 36,5% a -59,4%, rispettivamente nelle HCC827 e nelle A549), a differenza del composto G22 che mostra una riduzione del 22,39% solo nella linea A549. Lo studio degli SNP di akt1 ha riportato lo stesso genotipo per le due linee cellulari in esame, omozigoti per l’allele wild type dello SNP3 e eterozigoti per lo SNP4. Pertanto i polimorfismi analizzati non sono in grado di giustificare la diversa risposta delle linee cellulari al trattamento. Infine l’analisi del ciclo cellulare ha evidenziato un blocco delle cellule nella fase G1 dopo il trattamento con i composti G22 e G24 in entrambe le linee in studio. Questo dato potrebbe spiegare l’effetto di antagonismo delle combinazioni in cui i composti precedono la gemcitabina, dal momento che la gemcitabina viene incorporata nelle cellule in fase di attiva proliferazione (fase S). I risultati preliminari di questo studio evidenziano l’esistenza di un’interazione di tipo sinergica sequenza specifica tra questi nuovi inibitori di Akt, G22 e G24 e la gemcitabina, in parte giustificato dalla modulazione del ciclo cellulare e dallo stato fosforilativo della proteina Akt. Il progresso di queste indagini è finalizzato da un lato al potenziamento della risposta tumorale al trattamento farmacologico standard nei pazienti resitenti, e dall’altro alla riduzione degli effetti tossici collaterali causati dalle alte dosi dei farmaci chemioterapici

Clinical Study Are Breast Cancer Molecular Classes Predictive of Survival in Patients with Long Follow-Up?

In this study we investigate the clinical outcomes of 305 breast cancer (BC) patients, aged 55 years or younger, with long follow-up and according to intrinsic subtypes. The cohort included 151 lymph node negative (LN−) and 154 lymph node positive (LN+) patients. Luminal A tumors were mainly LN−, well differentiated, and of stage I; among them AR was an indicator of good prognosis. Luminal B and HER2 positive nonluminal cancers showed higher tumor grade and nodal metastases as well as higher proliferation status and stage. Among luminal tumors, those PR positive and vimentin negative showed a longer survival. HER2-positive nonluminal and TN patients showed a poorer outcome, with BC-specific death mostly occurring within 5 and 10 years. Only luminal tumor patients underwent BC death over 10 years. When patients were divided in to LN− and LN+ no differences in survival were observed in the luminal subgroups. LN− patients have good survival even after 20 years of follow-up (about 75%), while for LN+ patients survival at 20 years (around 40%) was comparable to HER2-positive nonluminal and TN groups. In conclusion, in our experience ER-positive breast tumors are better divided by classical clinical stage than molecular classification, and they need longer clinical follow-up especially in cases with lymph node involvement

Are Breast Cancer Molecular Classes Predictive of Survival in Patients with Long Follow-Up?

In this study we investigate the clinical outcomes of 305 breast cancer (BC) patients, aged 55 years or younger, with long follow-up and according to intrinsic subtypes. The cohort included 151 lymph node negative (LN−) and 154 lymph node positive (LN+) patients. Luminal A tumors were mainly LN−, well differentiated, and of stage I; among them AR was an indicator of good prognosis. Luminal B and HER2 positive nonluminal cancers showed higher tumor grade and nodal metastases as well as higher proliferation status and stage. Among luminal tumors, those PR positive and vimentin negative showed a longer survival. HER2-positive nonluminal and TN patients showed a poorer outcome, with BC-specific death mostly occurring within 5 and 10 years. Only luminal tumor patients underwent BC death over 10 years. When patients were divided in to LN− and LN+ no differences in survival were observed in the luminal subgroups. LN− patients have good survival even after 20 years of follow-up (about 75%), while for LN+ patients survival at 20 years (around 40%) was comparable to HER2-positive nonluminal and TN groups. In conclusion, in our experience ER-positive breast tumors are better divided by classical clinical stage than molecular classification, and they need longer clinical follow-up especially in cases with lymph node involvement

Is Human Papillomavirus Associated with Prostate Cancer Survival?

The role of human papillomavirus (HPV) in prostate carcinogenesis is highly controversial: some studies suggest a positive association between HPV infection and an increased risk of prostate cancer (PCa), whereas others do not reveal any correlation. In this study, we investigated the prognostic impact of HPV infection on survival in 150 primary PCa patients. One hundred twelve (74.67%) patients had positive expression of HPV E7 protein, which was evaluated in tumour tissue by immunohistochemistry. DNA analysis on a subset of cases confirmed HPV infection and revealed the presence of genotype 16. In Kaplan-Meier analysis, HPV-positive cancer patients showed worse overall survival (OS) (median 4.59 years) compared to HPV-negative (median 8.24 years, P=0.0381). In multivariate analysis age (P<0.001), Gleason score (P<0.001), nuclear grading (P=0.002), and HPV status (P=0.034) were independent prognostic factors for OS. In our cohort, we observed high prevalence of HPV nuclear E7 oncoprotein and an association between HPV infection and PCa survival. In the debate about the oncogenic activity of HPV in PCa, our results further confirm the need for additional studies to clarify the possible role of HPV in prostate carcinogenesis

Expression of the Endocannabinoid Receptor 1 in Human Stroke: An Autoptic Study

Objective Stroke is one of the leading causes of disability and death in the world. The endocannabinoid (eCB) system is upregulated in several neurological diseases including stroke. A previous animal study demonstrated an increased expression of the endocannabinoid receptor 1 (CB1R) in the penumbra area surrounding the ischemic core, suggesting a crucial role in inflammation/reperfusion after stroke. Regarding the localization of CB1/CB2 receptors, animal studies showed that cortical neurons, activated microglia, and astroglia are involved. Our aim was to evaluate the cerebral expression of CB1R in the ischemic brain areas of 9 patients who died due to acute cerebral infarction in the middle cerebral artery territory. Methods The cerebral autoptic tissue was collected within 48\u2009hours since death. Ischemic and contralateral normal-appearing areas were identified. After tissue preprocessing, 4-\ub5m-thick cerebral sections were incubated with the primary CB1R antibodies (Cayman Chemical Company, Ann Arbor, MI). Thereafter, all cerebral sections were hematoxylin treated. In each section, the total cell number and CB1R-positive cells were counted and the CB1R-positive cell count ratio was calculated. For statistical analysis, Student's t-test was used. Results In normal tissue, CB1R-positive neurons were the majority; a few non-neuronal cells expressed CB1R. In the ischemic areas, a few neurons were detectable. A significant increase in total CB1R staining was found in the ischemic regions compared to contralateral areas. Conclusions We found an increase in CB1R expression in the ischemic region (neuronal and non-neuronal cell staining), suggesting the inflammatory reaction to the ischemic insult. Whether such response might mediate neuroprotective actions or excitotoxicity-related detrimental effects is still unclear