Agustin Herrero

ras Mutations Are Associated With Aggressive Tumor Phenotypes and Poor Prognosis in Thyroid Cancer

PURPOSE ras oncogenic activation has long been demonstrated in thyroid carcinomas of follicular cell derivation, but no consistent relationship has been shown between mutations and clinicopathologic features. MATERIALS AND METHODS We analyzed H-, K-, and N-ras mutations by polymerase chain reaction-single-strand conformational polymorphism followed by DNA sequencing in 125 thyroid carcinoma specimens from 107 patients, to include tumors covering the entire spectrum of thyroid tumor differentiation. RESULTS Mutations were identified in four (8.2%) of 49 well-differentiated carcinomas (WDCs; two [6.7%] of 30 of the tumors were papillary carcinomas, two [10.5%] of 19 of them were follicular carcinomas), in 16 (55.2%) of 29 poorly differentiated carcinomas (PDCs), and in 15 (51.7%) of 29 undifferentiated carcinomas, with a significant association between ras mutation and poorly or undifferentiated tumors (P <.001). Twenty-six (74.3%) of 35 patients with ras-mutated tumors died as a result of disease as opposed to 23 (31.9%) of 72 patients with tumors lacking the mutations. Among patients with differentiated thyroid carcinomas (WDC and PDC), 11 (55.0%) of 20 patients with mutated tumors died as a result of disease as opposed to nine (15.5%) of 58 patients with wild-type ras tumors, and the correlation was independent of tumor differentiation and stage (P =.016). K-ras codon 13 mutations (all with G-A nucleotide transitions resulting in Gly>Asp substitution) and single activating mutations in any of the ras genes were also independent predictors of poor survival in differentiated thyroid carcinomas (P =.027 and P =.007, respectively). CONCLUSION These findings demonstrate that ras mutations are a marker for aggressive cancer behavior and indicate a possible role of ras genotyping to identify thyroid carcinoma subsets associated with poor prognosis.

β-Catenin Dysregulation in Thyroid Neoplasms: Down-Regulation, Aberrant Nuclear Expression, and CTNNB1 Exon 3 Mutations Are Markers for Aggressive Tumor Phenotypes and Poor Prognosis

β-catenin has a role in cell adhesion and Wnt signaling. It is mutated or otherwise dysregulated in a variety of human cancers. In this study we assess β-catenin alteration in 145 thyroid tumors samples from 127 patients. β-catenin was localized using immunofluorescence and mutational analysis was performed by single-strand conformational polymorphism. Membrane β-catenin expression was decreased in eight of 12 (66%) adenomas and in all 115 carcinomas ( P P CTNNB1 exon 3 mutations and nuclear β-catenin localization were restricted to poorly differentiated [7 of 28 (25%) and 6 of 28 cases (21.4%), respectively] or undifferentiated carcinomas [19 of 29 (65.5%) and 14 of 29 (48.3%) cases, respectively]. Poorly differentiated tumors always featured mutations involving Ser and Thr residues and were characterized by Thr to Ile amino acid substitutions ( P = 0.0283). The association between CTNNB1 exon 3 mutations and aberrant nuclear immunoreactivity ( P = 0.0020) is consistent with Wnt activation because of stabilizing β-catenin mutations. Low membrane β-catenin expression as well as its nuclear localization or CTNNB1 exon 3 mutations are significantly associated with poor prognosis, independent of conventional prognostic indicators for thyroid cancer but not of tumor differentiation. Analysis of β-catenin dysregulation may be useful to objectively subtype thyroid neoplasms and more accurately predict outcomes.

BRAF mutation associated with other genetic events identifies a subset of aggressive papillary thyroid carcinoma

Purpose  BRAF V600E mutation represents the most common oncogenic event in sporadic papillary thyroid cancer (PTC). There are, however, significant discrepancies regarding the overall frequency, its prevalence in PTC‐variants, and its relationship with clinico‐pathological parameters of poor outcome. Moreover, the impact of BRAFV600E mutants on tumour‐related patients death has not been evaluated.

Overexpression of Focal Adhesion Kinase in Head and Neck Squamous Cell Carcinoma Is Independent of fak Gene Copy Number

The development of human malignancies can involve the aberrant regulation of intracellular signal transduction pathways that regulate cell-extracellular matrix interactions. Purpose: In the current study, we aimed to evaluate focal adhesion kinase (FAK) at both genetic and protein expression levels in head and neck squamous cell carcinomas (HNSCC) and to explore the prognostic significance of FAK. Experimental Design: A total of 211 tissue specimens, including 147 primary tumors, 56 lymph node metastases, 3 benign hyperplasias, and 5 dysplasias, were analyzed using immunohistochemistry. The fak gene dosage was determined in 33 tumors. Correlations among DNA, protein, and clinicopathologic variables were analyzed. Results: FAK protein was overexpressed in HNSCCs compared with corresponding normal mucosa. High expression levels were found in 62% of the samples. Positive immunostaining was also detected in benign hyperplasias and preinvasive dysplastic lesions. All lymph node metastases examined showed FAK overexpression, with significant correlation with the expression in matched primary tumor. DNA copy number ratios for fak were higher in 39% of the tumors compared with normal mucosa. However, elevated FAK expression did not correlate with gains on DNA level, and not all cases with an amplification of the fak gene displayed protein overexpression. Similar data were obtained in five HNSCC-derived cell lines, in which FAK mRNA levels were precisely correlated with FAK protein levels. FAK protein overexpression in tumors correlated with nodal metastases. Conclusions: These findings suggest an involvement of FAK in the onset and progression of HNSCC and provide an insight into a mechanism of FAK activation alternative to gene amplification.

Expression of E-cadherin in squamous cell carcinomas of the supraglottic larynx with correlations to clinicopathological features

The aim of this study was to investigate the prognostic significance of E-cadherin expression in squamous cell carcinomas of the supraglottic larynx. 101 primary carcinomas were retrospectively studied. The level of E-cadherin expression was determined by immunohistochemistry. There was a significant correlation between decreased E-cadherin expression and the presence of nodal metastases (P=0.007). T-stage (P=0.025) and histological grade (P=0.043) were also associated with nodal metastases. Multivariate analysis confirmed that these three parameters were independent predictors of nodal metastases. Decreased E-cadherin expression also correlated with an increase in recurrence rates (P=0.019). However, in multivariate analysis only pathological N-stage was significantly associated with disease-specific survival. We conclude that E-cadherin is an independent predictor of nodal metastases in supraglottic squamous cell carcinomas. Determination of E-cadherin expression levels might be useful in identifying patients with clinically negative lymph nodes who are at risk of occult metastases, allowing more effective treatment strategies to be implemented.

Annexin A1 expression in nasopharyngeal carcinoma correlates with squamous differentiation.

Background Alterations of annexin A1 (ANXA1) expression have been reported in various cancers. However, no data are available about the expression of this protein in nasopharyngeal carcinomas (NPCs). The objective of this study was to investigate the expression of ANXA1 in these tumors. Methods We examined noncancerous nasopharyngeal mucosa (4 cases) and NPC (20 cases) for ANXA1 expression using immunohistochemistry. Results All tumor tissues showed markedly reduced ANXA1 expression compared with a strong positive signal observed in the corresponding normal epithelia. We found that ANXA1 expression is associated with the histological type in NPC. Only squamous cell carcinomas presented a positive ANXA1 signal in differentiated areas whereas all poorly differentiated tumors exhibited negative staining. Conclusion Our data show for the first time that ANXA1 expression is down-regulated in NPC and that its expression seems to be related with the squamous differentiation status of these tumors.

Clinicopathologic Significance of Expression of CD44s and CD44v6 Isoforms in Squamous Cell Carcinoma of the Supraglottic Larynx

CD44 splice variants are assumed to have a critical role in the malignant progression of many human tumors. However, the clinical significance of CD44 expression is not yet understood. The aim of this study was to investigate the prognostic significance of expression of CD44s and CD44v6 isoforms in squamous cell carcinomas of the supraglottic larynx. CD44s and CD44v6 expression was determined by immunohistochemical analysis of paraffin-embedded tissue specimens from 101 patients. There was a significant correlation between decreased CD44s or CD44v6 expression and a poorer histologic differentiation. No relationship was observed with T stage or nodal metastasis. Decreased CD44s expression, but not CD44v6 expression, correlated with increased recurrence rates. There was no correlation between the decreased expression of any isoform tested and survival. These data confirm a reduction of CD44s and CD44v6 expression in poorly differentiated tumors. However, these changes do not offer a useful adjunct to current prognostic indicators.

Expression of E-cadherin, CD44s, and CD44v6 in laryngeal and pharyngeal carcinomas.

OBJECTIVE Tumors arising from different sites of the head and neck area have very different clinical behavior. Loss or reduction of expression of adhesion molecules has been assumed to play a critical role in the development of head and neck carcinomas. The aim of this study is to determine if there are differences in the expression of adhesion molecules E-cadherin, CD44s, and CD44v6 in pharyngeal and laryngeal squamous-cell carcinomas. MATERIALS AND METHODS E-cadherin, CD44s, and CD44v6 expression was determined by immunohistochemistry in paraffin-embedded tissue specimens from 72 patients with squamous-cell carcinoma, 37 of the pharynx and 35 of the larynx. RESULTS Expression of CD44s was significantly lower in pharyngeal than in laryngeal tumors (P =.01). No differences in the expression of E-cadherin and CD44v6 were observed between these sites. CONCLUSIONS These data suggest that there are some differences at molecular level between the different subsites of head and neck cancer.

Malignant fibrous histiocytoma of the nasal cavity and paranasal sinuses.

Malignant fibrous histiocytomas are uncommon in the head and neck, the sinonasal tract being the most common location. This report describes 5 cases in this area: two in the nasal cavity, two in the maxillary sinus, and one in the frontal sinus. Four were primary cases and one was secondary to previous irradiation. All patients received surgical treatment, one of them with postoperative irradiation. All five patients experienced local recurrences and three also experienced distant metastases. Only one of these recurrences was successfully salvaged and the patient is alive and free of disease 3 years after resection. A summary of knowledge about the entity is reviewed.

Abstract 3137: Oncogenic activation of PI3K and MAPK pathways in 95 aggressive thyroid carcinomas (PDC and ATC). Co-activation prevalence

The MAPK and the PI3K pathways are viewed as therapeutic targets of the future due to their pivotal role in cellular proliferation, growth regulation, survival, adhesion, motility and spreading. While genetic alterations of effectors along the MAPK pathway are early events in thyroid follicular cell carcinogenesis, being some of them (oncogenic BRAF and RAS activation) also involved in tumour dedifferentiation and progression, the genetic alterations of effectors along the PI3K pathway are normally associated with late stage, advanced thyroid cancers. So far only one study has addressed the prevalence of PIK3CA mutations in poorly differentiated thyroid carcinomas (PDC). Because conventional treatment is clearly ineffective among late stage, advanced thyroid cancers in this study we focused on genotyping “druggable” oncogenes encoding effectors of both pathways. Mutations at BRAF, H-, K-, N-RAS and PIK3CA were investigated by PCR-SSCP-direct sequencing in 40 PDC and 55 anaplastic thyroid carcinomas (ATC). Focal changes in the growth pattern or microscopic grade within the primary tumour, recurrences and metastases were separately genotyped (144 samples). Mutational status was correlated with the expression of downstream effectors such as p-ERK and p-AKT. While the overall prevalence of PIK3CA mutations and BRAF mutations in ATC was almost twice that of PDC [PIK3CA: 29% and 20% respectively; BRAF: 22% and 12.5% respectively], the overall prevalence of RAS mutations in PDC resulted considerably higher than in ATC [45% and 36% respectively]. Concurrent BRAF and RAS mutations were present in 2 PDC (5%) and 5 ATC (9%). Concurrent RAS and PIK3CA mutations were found in 4 PDC (10%) and 4 ATC (7%). Concurrent BRAF and PIK3CA mutations or concurrent BRAF + RAS + PIK3CA mutations were only identified among ATC (2 cases, 4%). Activation of AKT was observed in most PDC and ATC harbouring PIK3CA mutations [86% and 90% respectively]. Phospho-p44/42 MAPK, which specifically recognizes the dually phosphorylated and active forms of ERK1 and ERK2 was present in 50% of the PDC and 55.5% of the ATC bearing the BRAF V600E mutation. PDC and ATC with RAS mutations were found to signal preferentially via PI3K. Among the RAS mutated PDC 69% demonstrated activation of AKT and 31% activation of ERK. Among RAS mutated ATC 85% exhibited AKT activation and 46% ERK activation. Co-activation of both pathways (p-AKT and p-ERK) was seen in 2 PDC (12.5%, both cases RAS +) and 5 ATC [38%; 4 cases RAS + plus 1 case RAS + and BRAF +]. Strikingly, all 4 PDC mutated at RAS and PIK3CA had distant metastases at diagnosis and died of disease. These findings may significantly impact on the rationale of future, tailored gene targeted therapies applied to patients with ATC and PDC. Our results suggest that activation of the PI3K pathway is pivotal in disease dedifferentiation and progression which has important implications in PDC and ATC management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3137.