Dario Garcia-Carracedo

Distinctive clinicopathological associations of amplification of the cortactin gene at 11q13 in head and neck squamous cell carcinomas.

Amplification of the 11q13 region is a prevalent genetic alteration in head and neck squamous cell carcinoma (HNSCC). We investigated the clinical significance of cortactin (CTTN) and cyclin D1 (CCND1) amplification in both malignant transformation and tumour progression. CTTN and CCND1 amplification was analysed by differential and real‐time PCR in a prospective series of laryngeal/pharyngeal carcinomas and archival premalignant tissues. CTTN mRNA and protein expression were respectively determined by real‐time RT‐PCR and immunohistochemistry, and correlated with gene status. Molecular alterations were associated with clinicopathological parameters and disease outcome. CTTN and CCND1 amplifications were respectively found in 75 (37%) and 90 (45%) tumours. Both correlated with advanced disease; however, only CTTN amplification was associated with recurrence and reduced disease‐specific survival (p = 0.0022). Strikingly, CTTN amplification differentially influenced survival depending on tumour site (p = 0.0001 larynx versus p = 0.68 pharynx) and was an independent predictor of reduced survival in the larynx (p = 0.04). CCND1 amplification was detected in early tumourigenesis and increased with the severity of dysplasia. Importantly, CTTN amplification was only found in high‐grade dysplasias that progressed to invasive carcinoma. CTTN gene status strongly correlated with mRNA and protein expression. Furthermore, CTTN overexpression correlated significantly with reduced disease‐specific survival (p = 0.018). Taken together, these data indicate that CTTN may serve as a valuable biomarker to identify patients with laryngeal tumours at high risk of recurrence and poor outcome. Copyright

Podoplanin expression in the development and progression of laryngeal squamous cell carcinomas.

BackgroundPodoplanin expression is attracting interest as a marker for cancer diagnosis and prognosis. We therefore investigated the expression pattern and clinical significance of podoplanin during the development and progression of laryngeal carcinomas.ResultsPodoplanin expression was determined by immunohistochemistry in paraffin-embedded tissue specimens from 84 patients with laryngeal premalignancies and 53 patients with laryngeal squamous cell carcinomas. We found podoplanin expression extending from the basal to the suprabasal layer of the epithelium in 37 (44%) of 84 dysplastic lesions, whereas normal epithelium showed negligible expression. Patients carrying podoplanin-positive lesions had a higher laryngeal cancer incidence than those with negative expression reaching borderline statistical significance (51% versus 30%, P = 0.071). Podoplanin expression in laryngeal carcinomas exhibited two distinct patterns. 20 (38%) cases showed diffuse expression in most tumour cells and 33 (62%) focal expression at the proliferating periphery of tumour nests. High podoplanin expression was inversely correlated with T classification (P = 0.033), disease stage (P = 0.006), and pathological grade (P = 0.04). There was a trend, although not significant, towards reduced disease-specific survival for patients with low podoplanin levels (P = 0.31) and diffuse expression pattern (P = 0.08).ConclusionsPodoplanin expression increases in the early stages of laryngeal tumourigenesis and it seems to be associated with a higher laryngeal cancer risk. Podoplanin expression in laryngeal squamous cell carcinomas, however, diminishes during tumour progression. Taken together, these data support a role for podoplanin expression in the initiation but not in the progression of laryngeal cancers.

Loss of PTEN Expression Is Associated with Poor Prognosis in Patients with Intraductal Papillary Mucinous Neoplasms of the Pancreas

Purpose: Previously, we reported PIK3CA gene mutations in high-grade intraductal papillary mucinous neoplasms (IPMN). However, the contribution of phosphatidylinositol-3 kinase pathway (PI3K) dysregulation to pancreatic carcinogenesis is not fully understood and its prognostic value unknown. We investigated the dysregulation of the PI3K signaling pathway in IPMN and its clinical implication. Experimental Design: Thirty-six IPMN specimens were examined by novel mutant-enriched sequencing methods for hot-spot mutations in the PIK3CA and AKT1 genes. PIK3CA and AKT1 gene amplifications and loss of heterozygosity at the PTEN locus were also evaluated. In addition, the expression levels of PDPK1/PDK1, PTEN, and Ki67 were analyzed by immunohistochemistry. Results: Three cases carrying the E17K mutation in the AKT1 gene and one case harboring the H1047R mutation in the PIK3CA gene were detected among the 36 cases. PDK1 was significantly overexpressed in the high-grade IPMN versus low-grade IPMN (P = 0.034) and in pancreatic and intestinal-type of IPMN versus gastric-type of IPMN (P = 0.020). Loss of PTEN expression was strongly associated with presence of invasive carcinoma and poor survival in these IPMN patients (P = 0.014). Conclusion: This is the first report of AKT1 mutations in IPMN. Our data indicate that oncogenic activation of the PI3K pathway can contribute to the progression of IPMN, in particular loss of PTEN expression. This finding suggests the potential employment of PI3K pathway-targeted therapies for IPMN patients. The incorporation of PTEN expression status in making surgical decisions may also benefit IPMN patients and should warrant further investigation. Clin Cancer Res; 19(24); 6830–41. ©2013 AACR.

pik3ca Mutations in Mucinous Cystic Neoplasms of the Pancreas

Objectives Mucinous cystic neoplasms (MCNs) are rare, potentially curable, mucin-producing neoplasms of the pancreas. We have previously reported PIK3CA (phosphoinositide-3-kinase catalytic subunit, p110&agr;) mutations in intraductal papillary mucinous neoplasms, another mucin-producing neoplasm of the pancreas. In this study, we analyzed the presence of PIK3CA and AKT1/PKB (V-akt murine thymoma viral oncogene homolog 1) hot-spot mutations in MCN specimens. Methods Using the genomic DNA sequencing of tumor tissues isolated by laser capture microdissection, we evaluated 15 well-characterized MCNs for the E542K, E545K (exon 9), and H1047R (exon 20) hot-spot mutations in the PIK3CA gene and the E17K mutation in the AKT1 gene. Results A hot-spot mutation (E545K) of the PIK3CA gene was detected in 1 of the 15 MCNs and further confirmed by a mutant-enriched method. Interestingly, this mutation was found to be present only in the high-grade but not in low-grade dysplastic epithelium obtained from this neoplasm and coexisted with a KRASG12D mutation. No mutations were identified in the AKT1 gene. Conclusions Our data, when combined with previous reports on intraductal papillary mucinous neoplasms, indicate that oncogenic activation of the PI3K pathway involving PIK3CA gene mutations can contribute to the progression of mucin-producing neoplasms but not pancreatic intraepithelial neoplasia. PIK3CA status could be useful for understanding their progression to malignancy.

Cortactin and Focal Adhesion Kinase as Predictors of Cancer Risk in Patients with Laryngeal Premalignancy

Novel markers are needed to accurately predict the risk of malignant transformation in laryngeal premalignancies. We therefore investigated the clinical significance of cortactin (CTTN) and focal adhesion kinase (FAK) during laryngeal tumorigenesis and their potential utility as cancer risk markers. CTTN and FAK protein expression and gene amplification were assessed in 82 patients with laryngeal dysplasia and correlated with clinicopathologic parameters and laryngeal cancer risk. Increased CTTN and FAK expression was found respectively in 41 (50%) and 40 (49%) of 82 laryngeal dysplasias; protein expression was maintained or further augmented in the corresponding patient-matched invasive tumors subsequently developed. CTTN and FAK/PTK2 gene amplifications were respectively detected in 10 (12%) and 26 (32%) laryngeal dysplasias. Both CTTN and FAK protein expression increased with the grade of dysplasia; however, CTTN and FAK expression but not histology correlated significantly with increased laryngeal cancer risk (P = 0.009 and P = 0.002, respectively). Patients carrying strong CTTN- or FAK-expressing dysplastic lesions experienced a significantly higher cancer incidence (P = 0.006 and P = 0.001, respectively; log-rank test). Furthermore, FAK expression was an independent predictor of laryngeal cancer development (HR = 3.706, 95% CI: 1.735–7.916; P = 0.001) and the combination of FAK and CTTN showed superior predictive value (HR = 5.042, 95% CI: 2.255–11.274; P < 0.001). Taken together, our findings support the involvement of CTTN and FAK in malignant transformation and provide original evidence for their potential clinical utility as biomarkers for the risk of developing laryngeal cancer. Cancer Prev Res; 4(8); 1333–41. ©2011 AACR.

Impact of PI3K/AKT/mTOR pathway activation on the prognosis of patients with head and neck squamous cell carcinomas

The PI3K/AKT/mTOR signaling pathway has emerged as one of the most frequently deregulated in head and neck squamous cell carcinomas (HNSCC). Numerous alterations of various upstream and downstream components have been described; however, their prognostic significance and impact on HNSCC patient survival remains to be established. This was addressed using an unbiased cohort of 93 consecutive and homogeneous surgically treated HNSCC patients and results confirmed in 432 HNSCC patients. Our findings reveal the high prevalence of S6 phosphorylation, a surrogate marker of mTORC1 activation, in HNSCC specimens (>70%) and, more importantly, demonstrate its relevance on clinical outcome. Phosphorylation of ribosomal protein S6 on either Ser235/236 or Ser240/244 was consistently and significantly correlated with favorable prognosis, although with differences depending on the tumor site. Thus, p-S6 expression was significantly correlated with better disease-specific survival specifically in the subgroup of laryngeal carcinoma patients (P< 0.001). In addition, multivariate regression models revealed p-S6 to be an inverse and independent predictor of lymph-node metastasis (P= 0.004) and distant metastasis (P= 0.006). Taken together, this study unveils an unprecedented correlation of mTOR activation with improved clinical outcome in patients with laryngeal carcinomas and uncovers the potential of p-S6 expression as a good prognostic biomarker and an inverse predictor of lymph node and distant metastases. These results should be of broad interest as immunohistochemical detection of p-S6 may help to stratify patients and guide treatment decisions.

Expression and clinical significance of the Kv3.4 potassium channel subunit in the development and progression of head and neck squamous cell carcinomas.

The concept of ion channels as membrane therapeutic targets and diagnostic/prognostic biomarkers has attracted growing attention. We therefore investigated the expression pattern and clinical significance of the Kv3.4 potassium channel subunit during the development and progression of head and neck squamous cell carcinomas (HNSCCs). KCNC4 mRNA levels were determined by real‐time RT‐PCR in both HNSCC tissue specimens and derived cell lines. Kv3.4 protein expression was evaluated by immunohistochemistry in paraffin‐embedded tissue specimens from 84 patients with laryngeal/pharyngeal squamous cell carcinomas and 67 patients with laryngeal dysplasias. Molecular alterations were correlated with clinicopathological parameters and patient outcome. Increased KCNC4 mRNA levels were found in 15 (54%) of 28 tumours, compared to the corresponding normal epithelia and varied mRNA levels were detected in 12 HNSCC‐derived cell lines analysed. Increased Kv3.4 protein expression was observed in 34 (40%) of 84 carcinomas and also at early stages of HNSCC tumourigenesis. Thus, 35 (52%) of 67 laryngeal lesions displayed Kv3.4‐positive staining in the dysplastic areas, whereas both stromal cells and normal adjacent epithelia exhibited negligible expression. No significant correlations were found between Kv3.4‐positive expression in HNSCC and clinical data; however, Kv3.4 expression tended to diminish in advanced‐stage tumours. Interestingly, patients carrying Kv3.4‐positive dysplasias experienced a significantly higher laryngeal cancer incidence than did those with negative lesions (p = 0.0209). In addition, functional studies using HNSCC cells revealed that inhibition of Kv3.4 expression by siRNA leads to the inhibition of cell proliferation via selective cell cycle arrest at the G2/M phase without affecting apoptosis. Collectively, these data demonstrate for the first time that Kv3.4 expression is frequently increased during HNSCC tumourigenesis and correlated significantly with a higher cancer risk. Our findings support a role for Kv3.4 in malignant transformation and provide original evidence for the potential clinical utility of Kv3.4 expression as a biomarker for cancer risk assessment. Copyright

Prognostic significance of lymphangiogenesis in pharyngolaryngeal carcinoma patients.

BackgroundLymphatic vessel spread is considered a major route for head and neck squamous cell carcinoma metastasis. Formation of new lymphatic vessels could facilitate the process, raising the malignant potential of these tumours. Recent identification of lymphatic markers allows the study of the lymphangiogenesis phenomenon. We searched for molecular events involved in the lymphangiogenic process that could have prognostic value in laryngeal/pharyngeal carcinoma patients.Methods104 paraffin-embedded pharyngeal/laryngeal tumour samples were studied. Immunohistochemical analysis of podoplanin and double immunofluorescence analysis of Ki-67 and D2-40 were performed. Lymph vessel density (inside the tumour mass, at its periphery or considered as a whole) and the presence of tumour emboli inside lymphatics were recorded. The proliferative state of endothelial lymphatic cells was evaluated.ResultsLymphatic vessels were detected inside the tumour mass (75%) and in the surrounding tissue (80%); some of them in a proliferative state. Tumour emboli were detected in a high proportion of the cases (45%). Lymphatic vessel density was higher in the pharyngeal cases (p = 0.0029), in greater size (p = 0.039), more advanced stage primary tumours (p = 0.006) and in carcinomas of patients with affected nodes (p = 0.019). The presence of tumour emboli and a high global vessel density were indicators of poor prognosis (recorded as death from tumour) in the laryngeal group (p = 0.015 and p = 0.027, respectively), but notably not in the pharyngeal one. Interestingly, high global vessel density showed a negative prognostic value among pathologically staged N0 laryngeal carcinomas (p = 0.03).ConclusionsThe lymphangiogenic process correlated with aggressive tumour features (pN category, tumour size, tumour stage), but might play different roles in tumours arising from different anatomic sites.Our results suggest that detection of tumour emboli and assessment of global vessel density using the D2-40 antibody, may be useful in the clinical practice, as predictors of reduced survival among pN0 laryngeal carcinoma patients.

Amplificación del gen de la ciclina D1 en los carcinomas adenoides quísticos de glándulas salivares menores

Resumen Introduccion/Objetivo El carcinoma adenoide quistico (CAQ) es un tumor de estirpe epitelial y representa el tumor maligno mas frecuente de las glandulas salivares menores. Sin embargo, se conoce poco de los genes implicados en el desarrollo y progresion de estos tumores. El gen de la ciclina D1 (CCND1) juega un papel clave en el control del ciclo celular, y su anomalia esta descrita en numerosos canceres. El objetivo de este estudio es determinar si existe amplificacion del CCND1 en los CAQ de glandulas salivares menores y su posible relacion con el pronostico Material y metodos Se realiza un estudio retrospectivo de 12 pacientes intervenidos de CAQ de fosas nasales y senos paranasales. Se determino la existencia de amplificacion del CCND1 mediante PCR multiple Resultados Se encontro amplificacion del CCND1 en 4 casos (33,3%). No se hallo relacion significativa con ninguno de los parametros clinico-patologicos analizados (localizacion, tipo histologico, estadio; p>0,05). La supervivencia fue menor en los pacientes que presentaban amplificacion de CCND1 Discusion/Conclusiones Nuestro estudio es el primero que demuestra la amplificacion del gen de la ciclina D1 en los CAQ. La amplificacion del CCND1 parece tener relacion con un peor pronostico en estos tumores, aunque es necesario confirmar esta relacion en estudios mas amplios

Abstract 2051: Tumor-treating fields (TTFields) intensity in the gross tumor volume and peritumoral brain zone: implications for local recurrence in glioblastoma

The purpose of this study was to simulate the intensity of TTFields delivered to the brain during the course of glioblastoma (GBM) treatment and to determine whether therapeutic intensities are delivered to the proximal peri-tumoral brain zone (PBZ). Background: TTFields are low-intensity (1-3 V/cm), intermediate frequency (200kHz), alternating electric fields delivered orthogonally in a localized manner during the course of GBM therapy. A recent phase 3 randomized, controlled trial conducted in patients newly diagnosed with GBM was stopped early for efficacy when the end points for progression-free survival (PFS) and overall survival (OS) were met at the interim analysis. Patients receiving TTFields in combination with temozolomide (TMZ) had a significantly longer PFS and OS compared with patients receiving TMZ alone. More than 90% of GBM recur at the margin of a resection cavity or within the PBZ where the presence of infiltrating tumor cells, inflammatory cells and tumorigenic stromal cells are thought to promote recurrence. Phantom model simulation studies suggest that field intensities >1V/cm are delivered to the brain in a non-uniform distribution, however the field distribution to the gross tumor volume (GTV) and PBZ have not been previously evaluated. Methods: Two MRI cases (frontal and posterior-parietal tumors) were used to generate TTFields treatment array layout maps using NovoTAL(TM) System planning software, targeting areas of contrast enhancement on T1 sequences. Simulations for the respective array layouts were created for solid tumors, resection cavities and for tumors with a necrotic core (modified Colin27 model, meshed and solved using the Sim4Life software solver package). Two orthogonal fields (left-right and antero-posterior) at a field frequency of 200 kHz were employed for all simulations. Field intensity was determined in the GTV, tumor margin(TM) and proximal PBZ (20mm) for all models. Results: Transducer array layout maps generated by the NovoTAL software deliver therapeutic intensities of TTFields in both L-R and A-P directions. Bi-directional intensities exceed therapeutic levels (>1 V/cm) in the GTV (median 1.84 V/cm), TM (median 1.9 V/cm) and PBZ (median 2.09 V/cm) in all solid tumors and in the PBZ (median 1.83 V/cm) surrounding a gross total resection (GTR) cavity. The highest areas of field intensity are observed directly adjacent to resection cavities and the ventricles. Conclusions: The delivery of therapeutic intensities of TTFields to patients who have undergone a GTR, subtotal resection or who have inoperable GBM, targets therapy to the area of active disease and importantly, to the PBZ. TTFields target residual tumor cells in the GTV and may also disrupt infiltrating tumor cells in the PBZ. Clinically, this may decrease local GBM recurrence rates and prospective clinical studies are warranted to explore this further. Citation Format: Aafia Chaudhry, Zeev Bomzon, Hadas Sara Hershkovich, Dario Garcia-Carracedo, Cornelia Wenger, Uri Weinberg, Yoram Palti. Tumor-treating fields (TTFields) intensity in the gross tumor volume and peritumoral brain zone: implications for local recurrence in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2051.