Cyclin A as a predictive factor for chemotherapy response in advanced head and neck cancer.

PURPOSE: Overall survival of head and neck squamous cell cancer (HNSCC) patients has not improved despite advances in our understanding of the biology and molecular features of this disease. In particular, patients with advanced HNSCC have the poorest prognosis. To understand more about the contribution of cell cycle alterations to HNSCC development and their possible value in predicting prognosis and response to chemotherapy, we evaluated the levels of proteins involved in cell cycle control in patients diagnosed with advanced HNSCC. EXPERIMENTAL DESIGN: A tissue microarray was made with 122 HNSCC specimens obtained from biopsy material. Protein expression was evaluated by immunohistochemistry and correlated with clinical and pathological characteristics. RESULTS: Multiple alterations at various checkpoints of cell cycle progression were observed. Loss of P16 protein was less common in oropharyngeal tumors than at other HNSCC locations (P = 0.02). Evaluation of the simultaneous expression of different proteins highlighted direct correlations (P < 0.05) such as that of the cyclin-dependent kinases with their cyclin-partners, and the Ki-67 protein with cyclin-dependent kinases 1, cyclin A (CA) and cyclin B1. Median overall survival and time-to-progression were longer in patients with CA-expressing tumors (not reached versus 34.4 months, P = 0.02) and (47.3 versus 14.6 months, P = 0.006), respectively. Moreover, expression of CA in tumors predicted a better response to chemotherapy. Positive expression of cyclin E in tumors was also associated with an increased median time-to-progression (14.6 versus 25.8 months, P = 0.04). Finally, patients with cyclin D1-expressing tumors had shorter median overall survival (29.6 months versus not reached, P = 0.05) and shorter median time-to-progression (21.5 months versus not reached, P = 0.06). However, in a multivariate analysis a CA-negative-expressing tumor was the only independent poor prognostic factor in the entire cohort of HNSCC patients [odds ratio, 2.3; 95% confidence interval (CI) = 1.2-4.5; P = 0.01]. CONCLUSIONS: Our results provide detailed information on the molecular profile of cell cycle components in HNSCCs and identify CA-negative-expressing tumors as an independent marker of tumor progression and poor response to chemotherapy in patients diagnosed with advanced HNSCC.     

Mutational and nonmutational activation of p21ras in rat colonic azoxymethane-induced tumors: effects on mitogen-activated protein kinase, cyclooxygenase-2, and cyclin D1

Azoxymethane (AOM)-induced colonic carcinogenesis involves a number of mutations, including those in the K-ras gene and CTNNB1, that codes for beta-catenin. Prior in vitro studies have also demonstrated that wild type p21(K-ras) can be activated by epigenetic events. We identified 15 K-ras mutations in 14 of 84 AOM-induced colonic tumors by three independent methods. By single strand conformational polymorphism, we also observed mutations in 22 of 68 tumors in exon 3 of CTNNB1. A highly sensitive method was then used to measure p21ras activation levels. All tumors assayed possessing K-ras mutations had significantly higher p21ras activation levels (8.8 +/- 1.5%; n = 13) compared with that of control colon (3.7 +/- 0.4; n = 6; P < 0.05) or tumors without such mutations (4.2 +/- 0.4%; n = 70; P < 0.05). Among tumors with wild-type K-ras, there was a subset of tumors (18 of 70) that had significantly higher p21ras activation levels (8.0 +/- 0.9%; n = 18) compared with control colons. In three of four tumors examined with activated wild-type p21ras, we observed increased c-erbB-2 receptor expression and decreased Ras-GAP expression. In contrast, only one of eight tumors examined with wild-type ras and nonactivated p21ras demonstrated these alterations. Mitogen-activated protein kinase (MAPK) activation and cyclooxygenase-2 (COX-2) expression were increased in tumors with mutated or activated wild-type p21ras, compared with their nonactivated counterparts. Although beta-catenin mutations did not alter COX-2 expression or MAPK activity, mutations in either K-ras or beta-catenin significantly increased cyclin D1 expression. In contrast, in tumors with wild-type but activated p21-ras, cyclin D1 expression was not enhanced. Thus, the spectrum of changes in MAPK, COX-2, and cyclin D1 is distinct among tumors with ras or beta-catenin mutations or nonmutational activation of p21ras.     

Increased expression of beta-catenin predicts better prognosis in nonsmall cell lung carcinomas

BACKGROUND: beta-Catenin has been shown to function as a Wnt signaling molecule to stimulate cyclin D1 expression and cell growth in several kinds of tumors. METHODS: The authors immunohistochemically examined specimens of 217 surgically resected primary nonsmall cell lung carcinomas (NSCLCs) for beta-catenin expression and classified them semiquantitatively into three categories, including those with high, moderate, and low scores of expression. RESULTS: High, moderate, and low scores of expression were found in 37 (17.1%), 145 (66.8%), and 35 (16.1%) tumors, respectively. beta-Catenin expression was not correlated with cyclin D1 expression, but was positively correlated with the Ki-67 cell growth fraction (P = 0.04). The direct sequencing analysis for the beta-catenin gene mutation of 13 specimens of 217 tumors for the current study revealed no mutations. The relation between survival and beta-catenin expression was evaluated in 148 potentially curatively resected tumors with pathologic Stages I-IIIA. A trend toward better survival was found in patients with tumors having higher scores. In multivariate analysis, high beta-catenin expression was a significant and independent favorable prognostic factor (hazards ratio, 0.31; P = 0.007) as was pathologic stage. Analyzed by cell type, in nonsquamous cell carcinomas, patients with tumors having high scores survived a significantly longer time than those with tumors having moderate or low scores (5-year survival rates, 84%, 55%, and 32%, respectively; P = 0.02), and high beta-catenin expression tended to be a favorable prognostic factor (hazards ratio, 0.32; P = 0.052). CONCLUSIONS: These results indicate that, in NSCLCs, increased expression of beta-catenin can predict favorable prognosis of patients with resected tumors, suggesting that accumulation of beta-catenin has no or little oncogenic effect via activation of the Wnt pathway, unlike in colon carcinomas or hepatomas.     

Cyclin B1 overexpression and resistance to radiotherapy in head and neck squamous cell carcinoma

Radiotherapy (RT) and surgery are the cornerstones in treating head and neck squamous cell carcinoma (HNSCC). RT is effective in the initial treatment of early to intermediate stage HNSCC but less effective for locally advanced disease, some cases of which are best managed using the combination of surgery and RT. Although various clinical/pathologic parameters have been used to classify patients according to their likelihood of responding to RT, they have generally low predictive value. We have shown previously that cyclin B1 is associated with a poor clinical outcome in HNSCC patients. In this study, we investigate the potential role of cyclin B1 in assessing RT response in patients with HNSCC. Tumor specimens obtained from 80 patients participated in a prospective Phase III clinical trial addressing the dose and fractionation regimen of postoperative RT were analyzed for cyclin B1 expression by immunohistochemistry. Patients were classified according to currently accepted clinical/pathologic parameters into three risk groups, i.e., low, intermediate, and high risk, and received surgery alone, surgery plus intermediate-dose RT, and surgery plus high-dose RT, respectively. The median follow-up duration was 4.9 years. Cyclin B1 overexpression was noted in 38 of the 80 (47%) HNSCC tumors. Interestingly, 11 of the 38 patients (29%) with cyclin B1-overexpressing tumors experienced local or nodal recurrence compared with only 3 of 42 patients (7%) having carcinomas with no or weak cyclin B1 expression (P = 0.01). When locoregional control was used as the end point for the high-risk group, patients whose tumors showed cyclin B1 overexpression had a statistically significant higher tumor recurrence and metastasis compared with patients whose tumors showed no cyclin B1 overexpression (P = 0.01). Our results indicate that tumors overexpressing cyclin B1 may be resistant to RT, and cyclin B1 may be an indicator of the risk of locoregional recurrence and metastasis in patients having HNSCC receiving RT.     

Cyclin E expression in breast cancer: predicting germline BRCA1 mutations, prognosis and response to treatment.

BACKGROUND: Elevated levels of the cell cycle protein cyclin E, and low levels of its inhibitor, p27(Kip1), have been associated with a poor prognosis following breast cancer. Some studies have found that germline mutations in the breast cancer susceptibility gene, BRCA1, are also associated with an inferior survival rate. The relationship between cyclin E/p27(Kip1) levels, BRCA1 status and outcome has not been studied in detail. PATIENTS AND METHODS: We analyzed a historical cohort of 288 Ashkenazi Jewish women who were diagnosed with breast cancer between 1980 and 1995 and were previously tested for BRCA1/2 mutations. Protein levels of cyclin E and p27(Kip1) were assessed by immunohistochemistry. Breast cancer-specific survival (BCSS) was the main outcome measured. RESULTS: The median follow-up was 8 years. Thirty tumors carried germline BRCA1 mutations. These tumors were more likely to have high cyclin E protein levels [odds ratio (OR) 9.5; P <0.001] and low p27(Kip1) protein levels (OR 2.8; P=0.03) than tumors from patients without BRCA1/2 mutations. High cyclin E expression level was the strongest predictor of BRCA1 germline mutations (multivariate OR 4.7; P=0.004). On univariate analysis, high cyclin E protein levels [relative risk (RR) 2.6; P <0.001] and low p27(Kip1) protein levels (RR 2.3; P=0.006) were significant prognostic factors for a poorer BCSS. In Cox multivariate models, high cyclin E levels remained an independent indicator of poor outcome only in the subgroup of patients who did not receive chemotherapy (P=0.002). CONCLUSIONS: In this ethnically restricted cohort, a high level of cyclin E is a characteristic of BRCA1-related breast cancer, and is a marker of poor prognosis following breast cancer, particularly in the absence of adjuvant chemotherapy.     

Expression and prognostic roles of beta-catenin in hepatocellular carcinoma: correlation with tumor progression and postoperative survival.

PURPOSE: Although hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the human liver, the molecular changes and mechanisms that regulate its development and progression remain unclear. In the present study, we investigated the correlation between beta-catenin expression and clinical outcome in 51 patients with relatively small (maximal diameter < 30 mm), solitary HCCs. EXPERIMENTAL DESIGN: The tumors were classified according to histological tumor differentiation (grade I, 11 tumors; grade II, 28 tumors; grade III, 12 tumors). Using immunohistochemical methods to detect nuclear accumulation of beta-catenin, we investigated the correlation between beta-catenin expression and clinical outcome and compared the correlation with cyclin D1, Ki-67, and E-cadherin. RESULTS: Focal or generalized nuclear beta-catenin expression was observed in 36.4% (4 of 11) of the grade I tumors, 39.3% (11 of 28) of the grade II tumors, and 25% (3 of 12) of the grade III tumors. Nuclear beta-catenin-positive grade III tumors were associated with significantly poorer survival (P = 0.004), whereas none of the patients with nuclear beta-catenin-negative grade I tumors died. With regard to proliferative activity, positive nuclear beta-catenin staining correlated significantly with an increased Ki-67 labeling index in grade I (P < 0.0001) and grade III (P = 0.0045) tumors and with reduced epithelial cadherin expression in the cell membrane (P < 0.001). In contrast, no association with the expression of cyclin D1, one of the target factors of beta-catenin, was detected. CONCLUSIONS: Our present data suggest that beta-catenin plays important roles in promoting tumor progression by stimulating tumor cell proliferation and reducing the activity of cell adhesion systems and is associated with a poor prognosis, especially in patients with poorly differentiated HCCs.     

Inactivation of human mutL homolog 1 and mutS homolog 2 genes in head and neck squamous cell carcinoma tumors and leukoplakia samples by promoter hypermethylation and its relation with microsatellite instability phenotype

BACKGROUND: A subset of head and neck squamous cell carcinoma (HNSCC) exhibits a microsatellite instability (MIN) phenotype. The authors correlated alterations in the mismatch-repair genes human mutL homolog 1 (hMLH1) and human mutS homolog 2 (hMSH2) in primary head and neck squamous cell carcinoma (HNSCC) tumors and in samples of leukoplakia with the MIN phenotype. METHODS: One hundred twenty-three paired HNSCC normal and tumor tissues and 27 leukoplakia samples were examined for hypermethylation of hMLH1 and hMSH2 promoters. The hypermethylation status of the tissues was confirmed by expression studies. Sixty-three of 123 randomly selected tumors and all 27 leukplakia samples were genotyped with 8 microsatellite markers to determine MIN. RESULTS: Fifty percent of HNSCC tumors and 63% of leukoplakia samples harbored hypermethylation at either or both hMLH1 and hMSH2 promoters. Normal tissues adjacent to methylation-positive tumors also demonstrated hypermethylation of both promoters at a high frequency (25%). A positive correlation between tobacco habit and promoter hypermethylation was observed (P = .001). A correlation was observed between MIN and the frequency of promoter hypermethylation in the leukoplakia samples, but no such trend was observed in the HNSCC tumors. It is noteworthy that patients who had a high frequency of MIN-positive tumors exhibited hypermethylation in both the affected tissues and the adjacent normal tissues (P = .007). Patients with a tobacco habit who had promoter hypermethylation at both the affected tissues and the adjacent normal tissues had tumors that mostly were MIN positive (P = .047). CONCLUSIONS: The current results suggested that tobacco-addicted individuals are more susceptible to promoter hypermethylation of hMLH1 and hMSH2 and that, if such hypermethylation occurs in the normal squamous epithelium of the head and neck region, then those tissues are likely to develop into tumors that involve the MIN pathway.     

Epidermal growth factor receptor expression in pretreatment biopsies from head and neck squamous cell carcinoma as a predictive factor for a benefit from accelerated radiation therapy in a randomized controlled trial.

PURPOSE: Accelerated repopulation is a main reason for locoregional failure after fractionated radiotherapy for head and neck squamous cell carcinoma (HNSCC). Epidermal growth factor receptor (EGFR) is a key controller of cellular proliferation in HNSCC, which stimulated the current study to look for a direct link between EGFR status and a possible clinical advantage of accelerated radiotherapy. PATIENTS AND METHODS: Immunohistochemical staining for EGFR was performed in 304 patients with available pretreatment tumor biopsy material among 918 patients randomized to receive continuous hyperfractionated accelerated radiotherapy versus conventionally fractionated radiotherapy. The EGFR index was estimated as the proportion of tumor cells with EGFR membrane staining. RESULTS: Significant benefit in locoregional tumor control from continuous hyperfractionated accelerated radiotherapy was seen in patients with HNSCC with high EGFR expression (2P = .010) but not in those with low EGFR expression (2P = .85). EGFR status had no significant effect on survival or rate of distant metastases. The EGFR index was significantly associated with histologic grade and microvessel density. There was moderate support for an association between EGFR status and subsite within the head and neck region but no significant association with Ki-67 index, Ki-67 pattern, p53 index, p53 intensity, bcl-2 expression, or cyclin D1 index. CONCLUSION: This study indicates a key role for the EGFR receptor in determining the proliferative cellular response to fractionated radiotherapy in HNSCC. It also shows that we can select the dose-fractionation regime that has the greatest chance of benefiting the patient. These results also encourage further development of EGFR targeting combined with fractionated radiotherapy in HNSCC.     

Head and neck tumor sites differ in prevalence and spectrum of p53 alterations but these have limited prognostic value

The tumor site is a strong clinical factor in head and neck squamous cell carcinoma (HNSCC). To clarify the biologic and clinical role of p53 alterations in HNSCC, we have examined the prevalence and the nature of p53 alterations in a large cohort of tumors from the different sites. For immunohistochemical analysis of p53 protein expression, we introduced tyramide signal amplification immunohistochemistry (TSA-IHC) on a tissue microarray. This allowed the discrimination between normal low-level expression and reduced or lost expression. Two hundred fifty-three tumors were subjected to mutational analysis by genomic DNA sequencing, employing also the p53 GeneChip from Affymetrix. The prevalence of all p53 alterations, i.e., mutations, overexpression and loss of expression, was significantly higher in hypopharyngeal tumors than in the other sites (p = 0.001). Laryngeal tumors showed the lowest rate of p53 alterations, but revealed a distinct mutation spectrum: most mutations affected exon 5 (p = 0.013) and the S2' domain (p = 0.002), and most hot-spot 248 mutations occurred in the larynx (p < 0.001). Sequencing by p53GeneChip technology was shown to be only insignificantly more sensitive than dideoxy sequencing. In agreement with p53 mutations occurring prior to invasiveness, their prevalence did not increase with tumor stage, and all mutation classes lacked prognostic significance. The large patient cohort of this study showed that p53 is differentially affected in the different tumor sites of the head and neck, but its mode of inactivation does not play a major role in tumor progression.     

High Pin1 expression is associated with tumor progression in colorectal cancer

BACKGROUND AND OBJECTIVES: Peptidyl prolyl cis-trans isomerase (Pin1) isomerizes only phosphorylated serine or threonine residues preceding proline in certain proteins and affects the protein function. Pin1 interacts with many signaling pathways, including Wnt signaling pathway that is crucial for colorectal tumorigenesis. Pin1 promotes cyclin D1 over-expression directly or through the stabilization of beta-catenin. Pin1 is over-expressed in some cancers such as prostate and breast cancers. This study aimed to determine whether Pin1 plays a role in colorectal tumorigenesis through the upregulation of beta-catenin and cyclin D1. METHODS: Immunohistochemical analyses were performed on 105 colorectal cancer tissue samples using anti-Pin1, anti-beta-catenin, and anti-cyclin D1 antibodies. We examined the relationships between Pin1 expression and clinicopathological factors, prognosis, and beta-catenin/cyclin D1 expression. RESULTS: High Pin1 expression was observed in 40 cases (38%) and positively correlated with histological type (P=0.0240), depth of invasion (P=0.0051), and staging (P=0.0027) of colorectal tumors. High Pin1 expression was also correlated with the over-expressions of both beta-catenin (P=0.0225) and cyclin D1 (P=0.0137). CONCLUSIONS: These results suggest that Pin1 plays an important role in colorectal tumorigenesis, presumably by increasing beta-catenin and cyclin D1 expressions.     

Aberrant expression of beta-catenin, Pin1 and cylin D1 in salivary adenoid cystic carcinoma: relation to tumor proliferation and metastasis

The aims of this study were to investigate the expression levels of beta-catenin, Pin1 and cyclin D1 in salivary adenoid cystic carcinomas (SACC ) and to evaluate its clinical importance, furthermore, to elucidate whether beta-catenin expression was aberrant in SACC and whether Pin1 was involved in aberrant beta-catenin and cyclin D1 expression. The expression of Pin1, beta-catenin and cyclin D1 were examined in the specimens of 65 patients with SACC by immunohistochemistry, protein and mRNA expressions were detected by western blotting and RT-PCR in four SACC cell lines. Pin1 was overexpressed in 51 cases of SACC (78%), and high levels of Pin1 expression correlated with cyclin D1 positive expression (p = 0.02). Fourteen (22%) cases showed positive immunoreactivity for beta-catenin protein in the nuclear/cytoplasmic fraction in tumor tissues, which was defined as cytoplasm/nucleus staining, among which quite evident nuclear expression of beta-catenin was detected in six cases (9%), while cyclin D1 positive expression was detected in 41 cases of SACC (63%). Reduced membranous expression of beta-catenin was detected in the cases with metastasis (11/14). Theses results suggest that Pin1 and Wnt signalling pathway are activated in SACC and may play a pivotal role in SACC carcinogenesis and metastasis.     

Cortactin expression predicts poor survival in laryngeal carcinoma

Amplification of the 11q13 region is one of the most frequent aberrations in squamous cell carcinomas of the head and neck region (HNSCC). Amplification of 11q13 has been shown to correlate with the presence of lymph node metastases and decreased survival. The 11q13.3 amplicon carries numerous genes including cyclin D1 and cortactin. Recently, we reported that FADD becomes overexpressed upon amplification and that FADD protein expression predicts for lymph node positivity and disease-specific mortality. However, the gene within the 11q13.3 amplicon responsible for this correlation is yet to be identified. In this paper, we compared, using immunohistochemical analysis for cyclin D1, FADD and cortactin in a series of 106 laryngeal carcinomas which gene correlates best with lymph node metastases and increased disease-specific mortality. Univariate Cox regression analysis revealed that high expression of cyclin D1 (P=0.016), FADD (P=0.003) and cortactin (P=0.0006) predict for increased risk to disease-specific mortality. Multivariate Cox analysis revealed that only high cortactin expression correlates with disease-specific mortality independent of cyclin D1 and/or FADD. Of genes located in the 11q13 amplicon, cortactin expression is the best predictor for shorter disease-specific survival in late stage laryngeal carcinomas.     

Clinicopathological significance of mitochondrial D-Loop mutations in head and neck carcinoma

Mitochondrial DNA mutations have been reported in several types of tumours, including head and neck squamous cell carcinoma (HNSCC). The noncoding region of the Displacement-Loop (D-Loop) has emerged as a mutational hotspot and we recently found that they were associated with prognosis and response to 5 fluorouracil (5FU) in colon cancers. In order to evaluate the frequence of D-Loop mutations in a large series of HNSCC and establish correlations with clinicopathologic parameters, we sequenced the D-Loop of 109 HNSCC before a treatment by neoadjuvant 5FU-cisplatin-based chemotherapy and surgery. Then, we correlated these mutations with prognosis and response to chemotherapy. A D-Loop mutation was identified in 21% of the tumors, the majority of them were located in a C-tract (D310). The prevalence of D310 mutations increased significantly with the number of cytosines in the matched normal tissue sequence (P=0.02). Hypopharyngeal cancer was significantly more frequent (P=0.03) and tobacco consumption more important (P=0.01) in the group of patients with D-Loop mutation. The presence of D-Loop mutation was not associated with prognosis or with response to neoadjuvant chemotherapy. These results suggest that D-Loop mutations should be considered as a cancer biomarker that may be useful for the early detection of HNSCC in individuals at risk of this cancer.