Ginesa Garcia-Rostan

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.

Assessment of RET/PTC Oncogene Activation and Clonality in Thyroid Nodules with Incomplete Morphological Evidence of Papillary Carcinoma : A Search for the Early Precursors of Papillary Cancer

Noninvasive thyroid nodules that exhibit borderline morphological signs of papillary cancer are difficult to diagnose and we do not know if they represent papillary carcinoma precursor lesions. Forty-six such nodules were analyzed for RET activation by immunohistochemistry and, in selected cases, by reverse transcriptase-polymerase chain reaction performed on RNA extracted after laser capture microdissection (LCM) of the tumor foci with and without papillary carcinoma features and positive RET immunoreactivity. RET immunoreactivity was identified, at least focally, in 30 of 46 (65.2%) of the nodules where it closely paralleled the morphological changes. Enough RNA was obtained after LCM in seven samples. RET/PTC1 or RET/PTC3 were detected in microscopic foci with papillary carcinoma features in most of the thyroid nodules (five of seven cases). No RET/PTC1 or RET/PTC3 rearrangements were detected in areas of the same tumors that lacked the cytological alterations. Analysis of clonality in the same nodules selected for LCM demonstrated that two were monoclonal and six were polyclonal. We conclude that RET activation closely parallels the morphological changes, that it is restricted to those areas of the tumor with the cytological alterations and that it is detectable in both mono- and polyclonal tumors. Although the finding of microscopic foci indicative of papillary carcinoma in a hyperplastic or adenomatous nodule does not justify the interpretation of the entire lesion as papillary carcinoma, it is possible that such foci may precede the development of invasive papillary cancer.

Downregulation of p27KIP1 and Ki67/Mib1 labeling index support the classification of thyroid carcinoma into prognostically relevant categories

The cyclin-dependent kinase inhibitor p27KIP1 has been proposed as a valuable prognostic indicator for a variety of human neoplasms. Immunohistochemical reactivity for p27KIP1 and the proliferation marker Ki67/Mib1 were investigated in 90 thyroid carcinomas of follicular cell origin. The neoplasms were divided into three prognostic groups on the basis of their morphologic features: group 1, well-differentiated papillary or follicular carcinomas with favorable pathologic features (43 papillary carcinomas and 4 minimally invasive follicular carcinomas); group 2, papillary or follicular carcinomas with unfavorable pathologic features (21 poorly differentiated carcinomas and 2 papillary carcinomas, tall cell variant); and group 3, undifferentiated, or anaplastic, carcinomas. p27KIP1 expression (p = 0.007) and Ki67/Mib1 labeling index (p = 0.02) showed a strong correlation with the subdivision of the thyroid carcinomas in the three prognostic groups with a significant linear trend for tumors with low p27KIP1 (p = 0.002) and high Ki67/Mib1 labeling index (p = 0.005) to segregate into the unfavorable categories (groups 2 and 3). Low p27KIP1 expression, but not cellular proliferation, was related to adverse prognostic factors, such as large tumor size (p = 0.03) and extrathyroidal extension (p = 0.01), but the correlation was not independent of the subdivision in the three groups. Low p27KIP1 expression (p = 0.03) and high proliferative rate (p = 0.02) were associated with poor survival, reflecting the close association between patient morbidity and mortality rates and tumor differentiation. No significant association could be seen between p27KIP1 or cellular proliferation and clinicopathologic parameters (e.g., age, sex, tumor size, extrathyroidal extension, vascular invasion, lymph node metastases, distant metastases, tumor stage, and survival rate) within any of the groups, or the histologic diagnosis of papillary versus follicular carcinoma irrespective of their degree of differentiation. Modulation of p27KIP1 and cellular proliferation patterns in thyroid carcinoma correlate with tumor differentiation and support the morphologic classification of thyroid carcinoma into prognostically relevant categories.

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.

CD97 amplifies LPA receptor signaling and promotes thyroid cancer progression in a mouse model

CD97, a member of the adhesion family of G-protein-coupled receptors (GPCRs), complexes with and potentiates lysophosphatidic acid (LPA) receptor signaling to the downstream effector RHOA. We show here that CD97 was expressed in a majority of thyroid cancers but not normal thyroid epithelium and that the level of CD97 expression was further elevated with progression to poorly differentiated and undifferentiated carcinoma. Intratumoral progression also showed that CD97 expression correlates with invasiveness and dedifferentiation. To determine the functional role of CD97, we produced a transgenic model of thyroglobulin promoter-driven CD97 expression. Transgenic CD97 in combination with ThrbPV, an established mouse model of thyroid follicular cell carcinogenesis, significantly increased the occurrence of vascular invasion and lung metastasis. Expression of transgenic CD97 in thyroid epithelium led to elevated ERK phosphorylation and increased numbers of Ki67+ cells in developing tumors. In addition, tumor cell cultures derived from CD97 transgenic as compared with non-transgenic mice demonstrated enhanced, constitutive and LPA-stimulated ERK activation. In human thyroid cancer cell lines, CD97 depletion reduced RHO-GTP and decreased LPA-stimulated invasion but not EGF-stimulated invasion, further suggesting that CD97 influences an LPA-associated mechanism of progression. Consistent with the above, CD97 expression in human thyroid cancers correlated with LPA receptor and markers of aggressiveness including Ki67 and pAKT. This study shows an autonomous effect of CD97 on thyroid cancer progression and supports the investigation of this GPCR as a therapeutic target for these cancers.

TWIST1 plays a pleiotropic role in determining the anaplastic thyroid cancer phenotype.

CONTEXT Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human tumors; it is characterized by chemoresistance, local invasion, and distant metastases. ATC is invariably fatal. OBJECTIVE The aim was to study the role of TWIST1, a basic helix-loop-helix transcription factor, in ATC. DESIGN Expression of TWIST1 was studied by immunohistochemistry and real-time PCR in normal thyroids and well-differentiated, poorly differentiated, and ATC. The function of TWIST1 was studied by RNA interference in ATC cells and by ectopic expression in well-differentiated thyroid carcinoma cells. RESULTS ATCs up-regulate TWIST1 with respect to normal thyroids as well as to poorly and well-differentiated thyroid carcinomas. Knockdown of TWIST1 by RNA interference in ATC cells reduced cell migration and invasion and increased sensitivity to apoptosis. The ectopic expression of TWIST1 in thyroid cells induced resistance to apoptosis and increased cell migration and invasion. CONCLUSIONS TWIST1 plays a key role in determining malignant features of the anaplastic phenotype in vitro.

FOXM1 is a molecular determinant of the mitogenic and invasive phenotype of anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma (ATC) is a very aggressive thyroid cancer. forkhead box protein M1 (FOXM1) is a member of the forkhead box family of transcription factors involved in control of cell proliferation, chromosomal stability, angiogenesis, and invasion. Here, we show that FOXM1 is significantly increased in ATCs compared with normal thyroid, well-differentiated thyroid carcinomas (papillary and/or follicular), and poorly differentiated thyroid carcinomas (P=0.000002). Upregulation of FOXM1 levels in ATC cells was mechanistically linked to loss-of-function of p53 and to the hyperactivation of the phosphatidylinositol-3-kinase/AKT/FOXO3a pathway. Knockdown of FOXM1 by RNA interference inhibited cell proliferation by arresting cells in G2/M and reduced cell invasion and motility. This phenotype was associated with decreased expression of FOXM1 target genes, like cyclin B1 (CCNB1), polo-like kinase 1 (PLK1), Aurora B (AURKB), S-phase kinase-associated protein 2 (SKP2), and plasminogen activator, urokinase: uPA (PLAU). Pharmacological inhibition of FOXM1 in an orthotopic mouse model of ATC reduced tumor burden and metastasization. All together, these findings suggest that FOXM1 represents an important player in thyroid cancer progression to the anaplastic phenotype and a potential therapeutic target for this fatal cancer.

Abstract 5505: TERT promoter mutations in primary papillary thyroid carcinomas and matched local / distant metastases

TERT promoter (TERTp) mutations represent a common oncogenic event in sporadic thyroid follicular cell carcinogenesis. Though TERTp mutations have been statistically associated with aggressiveness and metastatic spreading, their involvement in lymph node metastases (LNMs) and / or distant metastases (DMs) development among papillary thyroid carcinoma (PTC) patients remains to be defined. To evaluate the role of TERTp mutations on metastatic tumor expansion, primary tumors (Pt) and matched LNMs and/or DMs were genotyped by means of PCR-direct sequencing in a cohort of 33 patients diagnosed of PTC, which had been previously analyzed for BRAF and RAS mutations. Focal changes in the growth pattern or microscopic grade within the Pt or the metastases were separately genotyped to determine the clonal/subclonal nature of TERTp mutations, their association with particular histological variants of PTC or their presence in intra-tumoral PDC-like foci. Results were correlated with clinico-pathological parameters of pour outcome and survival. The analysis of 99 tumor samples obtained from 33 PTC cases revealed that TERTp mutations were quite common (42.4%).The mutation C228T was much more common than the C250T (78,6% vs 21,4%). TERTp mutations did not correlate with specific PTC subtypes [CL-PTC, FV-PTC or Mixed-PTC] and were subclonal in half of the cases. The mutations segregated to LNMs in 73% of cases [100% CL-PTC and FV-PTC; 57% Mixed PTC]. In 2 Mixed-PTC cases the mutation seemingly originated the novo in the LNM. TERTp mutations were present in all samples of DM. While 71% of the cases mutated at TERTp bore the BRAFV600E mutation, the coexistence of TERTp and RAS mutations was exceptional. TERTp mutations were found to be significantly correlated with age ≥ 45 years old, high grade poorly differentiated PTC foci or nesting-PDC-like foci, stage at diagnosis or at last follow-up and patient status. A trend of correlation with male sex, vascular invasion, tumor recurrence and development of LNM during the follow-up was also seen. Tumor multifocality was inversely correlated with TERTp mutations. The coexistence of TERTp and BRAFV600E mutants did not increase the prognostic power of TERTp mutations alone. All of the patients who died of disease displayed TERTp mutations. Kaplan-Meier analysis revealed that patients with PTC bearing TERTp mutations had a poor prognosis showing a higher tumor recurrence probability [p= 0.0085] and a reduced disease specific survival [p Citation Format: Noa Feas Rodriguez, Miram Corraliza Gomez, Tomas Alvarez Gago, Juan Jose Mateos Otero, Raquel Munoz Martinez, Ginesa Maria Garcia-Rostan. TERT promoter mutations in primary papillary thyroid carcinomas and matched local / distant metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5505. doi:10.1158/1538-7445.AM2017-5505

Abstract 4222: Usefulness of VE1 immunohistochemical detection of BRAFV600E in aggressive thyroid cancers (PDCs and UCs)

Activating BRAF mutations are frequent in thyroid follicular cell carcinogenesis. The BRAFV600E point mutation represents the most common oncogenic event in sporadic papillary thyroid cancer (PTC). Although at a much lower frequency the BRAFV600E mutation is also present among less differentiated, more aggressive, I131 resistant forms of thyroid cancer as poorly differentiated carcinomas (PDCs) and undifferentiated carcinomas (UCs). Small molecule inhibitors targeting either the BRAF V600E protein or upstream or downstream kinases involved in MAPK signalling are currently under pre-clinical or clinical investigation in advanced, metastatic, I131 resistant thyroid cancers. Recently, immunohistochemical (IHC) studies on PTCs, using the VE1 mouse anti-human BRAF V600E antibody have shown to be a reliable means for detecting the BRAFV600E mutation in a clinical setting without molecular genotyping units. In this study we sought to determine the usefulness of the VE1 antibody for detecting the BRAF V600E mutant protein in a series of 103 aggressive thyroid cancers (59 PDCs and 44 UCs) previously characterized by PCR-SSCP for the presence of BRAF mutations in exons 11 and 15. The BRAFV600E mutation was present in 10/59 PDCs (17%) and 11/44 UCs (25%). Immunohistochemistry revealed 9 mutated PDCs (sensitivity 90%) and 9 mutated UCs (82% sensitivity). The staining intensity in BRAF V600E mutated samples ranged from weak to strong. Non valuable results were found in 3 PDCs and 4 UCs. Overall the results indicate that immunohistochemistry with VE1 antibody may be an alternative to molecular biology approaches for the routine detection of BRAFV600E point mutations in clinical settings without molecular genotyping facilities. It may help clinicians in targeted therapy decision making. Citation Format: Noa Feas-Rodriguez, Angela M. Costa, Tomas Alvarez Gago, Juan Jose Mateos Otero, Jose Manuel Cameselle Teijeiro, Raquel Munoz, Ginesa Maria Garcia-Rostan. Usefulness of VE1 immunohistochemical detection of BRAFV600E in aggressive thyroid cancers (PDCs and UCs). [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 4222.