Contrasting effects of an Mdm2 functional polymorphism on tumor phenotypes.

MDM2, an E3 ubiquitin ligase, is a potent inhibitor of the p53 tumor suppressor and is elevated in many human cancers that retain wild-type p53. MDM2 SNP309G is a functional polymorphism that results in elevated levels of MDM2 (due to enhanced SP1 binding to the MDM2 promoter) thus decreasing p53 activity. Mdm2SNP309G/G mice are more prone to spontaneous tumor formation than Mdm2SNP309T/T mice, providing direct evidence for the impact of this SNP in tumor development. We asked whether environmental factors impact SNP309G function and show that SNP309G cooperates with ionizing radiation to exacerbate tumor development. Surprisingly, ultraviolet B light or Benzo(a)pyrene exposure of skin shows that SNP309G allele actually protects against squamous cell carcinoma susceptibility. These contrasting differences led us to interrogate the mechanism by which Mdm2 SNP309 regulates tumor susceptibility in a tissue-specific manner. Although basal Mdm2 levels were significantly higher in most tissues in Mdm2SNP309G/G mice compared with Mdm2SNP309T/T mice, they were significantly lower in Mdm2SNP309G/G keratinocytes, the cell-type susceptible to squamous cell carcinoma. The assessment of potential transcriptional regulators in ENCODE ChIP-seq database identified transcriptional repressor E2F6 as a possible negative regulator of MDM2 expression. Our data show that E2F6 suppresses Mdm2 expression in cells harboring the SNP309G allele but not the SNP309T allele. Thus, Mdm2 SNP309G exhibits tissue-specific regulation and differentially impacts cancer risk.

Loss of epithelial p53 and αv integrin cooperate through Akt to induce squamous cell carcinoma yet prevent remodeling of the tumor microenvironment.

Most of the squamous cell carcinomas (SCCs) of the skin and head and neck contain p53 mutations. The presence of p53 mutations in premalignant lesions suggests that they represent early events during tumor progression and additional alterations may be required for SCC development. Here we show that codeletion of the p53 and αv integrin genes in mouse stratified epithelia induced SCCs in 100% of the mice, more frequently and with much shorter latency than deletion of either gene alone. The SCCs that lacked p53 and αv in the epithelial tumor cells exhibited high Akt activity, lacked multiple types of infiltrating immune cells, contained a defective vasculature and grew slower than tumors that expressed p53 or αv. These results reveal that loss of αv in epithelial cells that lack p53 promotes SCC development, but also prevents remodeling of the tumor microenvironment and delays tumor growth. We observed that Akt inactivation in SCC cells that lack p53 and αv promoted anoikis. Thus, tumors may arise in these mice as a result of the increased cell survival induced by Akt activation triggered by loss of αv and p53, and by the defective recruitment of immune cells to these tumors, which may allow immune evasion. However, the defective vasculature and lack of a supportive stroma create a restrictive microenvironment in these SCCs that slows their growth. These mechanisms may underlie the rapid onset and slow growth of SCCs that lack p53 and αv.

Imaging findings of head and neck dermatofibrosarcoma protuberans.

BACKGROUND AND PURPOSE: Dermatofibrosarcoma protuberans is a rare, locally aggressive sarcoma of the skin in children and adults, usually involving the trunk and extremities and less commonly the head and neck. Despite clinical reports in the literature on the management of dermatofibrosarcoma protuberans, there are limited articles describing its imaging features. MATERIALS AND METHODS: We retrospectively reviewed the demographics and imaging findings in all 24 patients with pathologically proven dermatofibrosarcoma protuberans of the head and neck seen at a tertiary cancer center between 2001 and 2010. RESULTS: Twenty-two of the 24 lesions were nodular and well circumscribed; 19 of the 24 were located on the scalp. On imaging, all 24 lesions involved subcutaneous tissues. The lesions ranged in size from 0.6-9.5 cm (mean, 3.7 cm; standard deviation, 2.3 cm). Twelve lesions involved the soft tissues either at or extending directly to the midline. Thirteen lesions were associated with bulging of the skin surface. Fourteen lesions were imaged with CT and 14 with MR imaging. Whereas variable enhancement patterns were noted on CT and MR imaging, dermatofibrosarcoma protuberans was usually T2-hyperintense and demonstrated marked enhancement. None of the lesions was associated with bone invasion, perineural spread, or nodal/distant metastasis. CONCLUSIONS: Knowledge of the imaging characteristics of dermatofibrosarcoma protuberans may alert neuroradiologists to include dermatofibrosarcoma protuberans in the differential diagnosis of lesions about the head and neck with similar imaging characteristics.

Urban legends series: oral leukoplakia.

To date, the term oral leukoplakia (OL) should be used to recognize 'predominantly white plaques of questionable risk, having excluded (other) known diseases or disorders that carry no increased risk of cancer'. In this review, we addressed four controversial topics regarding oral leukoplakias (OLs): (i) Do tobacco and alcohol cause OLs?, (ii) What percentage of OLs transform into oral squamous cell carcinoma (OSCC)?, (iii) Can we distinguish between premalignant and innocent OLs?, and (iv) Is proliferative verrucous leukoplakia (PVL) a specific entity or just a form of multifocal leukoplakia? Results of extensive literature search suggest that (i) no definitive evidence for direct causal relationship between smoked tobacco and alcohol as causative factors of OLs, (ii and iii) the vast majority of OLs follow a benign course and do not progress into a cancer, and no widely accepted and/or validated clinical and/or biological factors can predict malignant transformation, and (iv) the distinction between multifocal/multiple leukoplakias and PVL in their early presentation is impossible; the temporal clinical progression and the high rate of recurrences and development of cancer of PVL are the most reliable features for diagnosis.

TrkC signaling is activated in adenoid cystic carcinoma and requires NT-3 to stimulate invasive behavior.

Treatment options for adenoid cystic carcinoma (ACC) of the salivary gland, a slowly growing tumor with propensity for neuroinvasion and late recurrence, are limited to surgery and radiotherapy. Based on expression analysis performed on clinical specimens of salivary cancers, we identified in ACC expression of the neurotrophin-3 receptor TrkC/NTRK3, neural crest marker SOX10, and other neurologic genes. Here, we characterize TrkC as a novel ACC marker, which was highly expressed in 17 out of 18 ACC primary-tumor specimens, but not in mucoepidermoid salivary carcinomas or head and neck squamous cell carcinoma. Expression of the TrkC ligand NT-3 and Tyr-phosphorylation of TrkC detected in our study suggested the existence of an autocrine signaling loop in ACC with potential therapeutic significance. NT-3 stimulation of U2OS cells with ectopic TrkC expression triggered TrkC phosphorylation and resulted in Ras, Erk 1/2 and Akt activation, as well as VEGFR1 phosphorylation. Without NT-3, TrkC remained unphosphorylated, stimulated accumulation of phospho-p53 and had opposite effects on p-Akt and p-Erk 1/2. NT-3 promoted motility, migration, invasion, soft-agar colony growth and cytoskeleton restructuring in TrkC-expressing U2OS cells. Immunohistochemical analysis demonstrated that TrkC-positive ACC specimens also show high expression of Bcl2, a Trk target regulated via Erk 1/2, in agreement with activation of the TrkC pathway in real tumors. In normal salivary gland tissue, both TrkC and Bcl2 were expressed in myoepithelial cells, suggesting a principal role for this cell lineage in the ACC origin and progression. Sub-micromolar concentrations of a novel potent Trk inhibitor AZD7451 completely blocked TrkC activation and associated tumorigenic behaviors. Pre-clinical studies on ACC tumors engrafted in mice showed efficacy and low toxicity of AZD7451, validating our in vitro data and stimulating more research into its clinical application. In summary, we describe in ACC a previously unrecognized pro-survival neurotrophin signaling pathway and link it with cancer progression.

A genetic mouse model for metastatic lung cancer with gender differences in survival.

Lung cancer is a devastating disease with poor prognosis. The design of better therapies for lung cancer patients would be greatly aided by good mouse models that closely resemble the human disease. Unfortunately, current models for lung adenocarcinoma are inadequate due to the absence of metastases. In this study, we incorporated both K-ras and p53 missense mutations into the mouse genome and established a more faithful genetic model for human lung adenocarcinoma, the most common type of lung cancer. Mice with both mutations developed advanced lung adenocarcinomas that were highly aggressive and metastasized to multiple intrathoracic and extrathoracic sites in a pattern similar to that of human lung cancer. These mice also showed a gender difference in cancer-related death. Additionally, the presence of both mutations induced pleural mesotheliomas in 23% of these mice. This mouse model recapitulates the metastatic nature of human lung cancer and will be invaluable to further probe the molecular basis of metastatic lung cancer and for translational studies.

ICAM-5 (telencephalin) gene expression in head and neck squamous carcinoma tumorigenesis and perineural invasion!

ICAM-5 (telencephalin) is an intercellular adhesion molecule reported to be expressed only in the somatodendritic membrane of telencephalic neurons. We recently identified high ICAM-5 expression in a cDNA array study of head and neck neoplasms with a propensity for perineural invasion. To determine the association of this gene in tumorigenesis and perineural invasion, we analyzed the expression and functional status of ICAM-5 mRNA transcripts in 30 different human cancer cell lines and 25 head and neck squamous carcinoma specimens by reverse-transcriptase polymerase chain reaction (cell lines and specimens) and in vitro functional assays (cell lines). ICAM-5 transcripts were detected in 28 (93%) of 30 cell lines derived from primary head and neck, colon, thyroid, cervical, pancreatic, skin, and adenoid cystic carcinomas. In cell lines, small interfering RNA blocked ICAM-5 expression and inhibited cell proliferation. Treatment with the phosphatidylinositol 3'-kinase (PBK) inhibitor LY294002 resulted in ICAM-5 down-regulation. In tissue specimens, none of the 25 histologically normal oral mucosal specimens had detectable ICAM-5 level, whereas 16 (64%) of the 25 matched primary squamous carcinomas showed expression. Carcinoma specimens high ICAM-5 expression had a high incidence of perineural invasion. Our study indicates that ICAM-5 may play a role in tumorigenesis and perineural invasion, most likely through the P13K/Akt-signaling pathway.

The Akt inhibitor KP372-1 suppresses Akt activity and cell proliferation and induces apoptosis in thyroid cancer cells.

The phosphatidylinositol 3' kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten/Akt pathway, which is a critical regulator of cell proliferation and survival, is mutated or activated in a wide variety of cancers. Akt appears to be a key central node in this pathway and thus is an attractive target for targeted molecular therapy. We demonstrated that Akt is highly phosphorylated in thyroid cancer cell lines and human thyroid cancer specimens, and hypothesised that KP372-1, an Akt inhibitor, would block signalling through the PI3K pathway and inhibit cell proliferation while inducing apoptosis of thyroid cancer cells. KP372-1 blocked signalling downstream of Akt in thyroid tumour cells, leading to inhibition of cell proliferation and increased apoptosis. As thyroid cancer consistently expresses phosphorylated Akt and KP372-1 effectively blocks Akt signalling, further preclinical evaluation of this compound for treatment of thyroid cancer is warranted.

Detection and diagnosis of oral neoplasia with an optical coherence microscope.

The use of high resolution, in vivo optical imaging may offer a clinically useful adjunct to standard histopathologic techniques. A pilot study was performed to investigate the diagnostic capabilities of optical coherence microscopy (OCM) to discriminate between normal and abnormal oral tissue. Our objective is to determine whether OCM, a technique combining the subcellular resolution of confocal microscopy with the coherence gating and heterodyne detection of optical coherence tomography, has the same ability as confocal microscopy to detect morphological changes present in precancers of the epithelium while providing superior penetration depths. We report our results using OCM to characterize the features of normal and neoplastic oral mucosa excised from 13 subjects. Specifically, we use optical coherence and confocal microscopic images obtained from human oral biopsy specimens at various depths from the mucosal surface to examine the optical properties that distinguish normal and neoplastic oral mucosa. An analysis of penetration depths achieved by the OCM and its associated confocal arm found that the OCM consistently imaged more deeply. Extraction of scattering coefficients from reflected nuclear intensity is successful in nonhyperkeratotic layers and shows differentiation between scattering properties of normal and dysplastic epithelium and invasive cancer.

Loss of PTEN expression as a prognostic marker for tongue cancer.

BACKGROUND: Abnormalities of PTEN, a candidate tumor suppressor gene located at 10q23.3, play an important role in the tumorigenesis of multiple tumor types. OBJECTIVES: To investigate the expression of PTEN and its clinical implication in squamous cell carcinoma of the tongue. DESIGN: Retrospective analysis of PTEN protein expression in archived primary oral tongue tumor samples. SETTING: Academic center. PATIENTS AND METHODS: PTEN expression was determined by immunohistochemical analysis in tissue samples from 41 patients with stage II, III, and IV squamous cell carcinoma of the tongue. All the patients underwent curative surgical treatment with a median follow-up of 81 months. The Kaplan-Meier method was used for survival analysis. Multivariate analysis was performed according to the Cox proportional hazards model. RESULTS: Lack of staining for PTEN was demonstrated in 12 (29%) of the 41 tumors. Patients whose tumors lacked PTEN expression had a significantly shorter overall survival time (P = .03) and event-free survival time (P = .01) than those patients with positive PTEN expression. Multivariate regression analysis demonstrated that PTEN expression is an independent predictor of poor outcome when compared with tumor stage and nodal status. CONCLUSIONS: Although genetic alterations of the PTEN gene are rare in head and neck squamous cell carcinoma, loss of PTEN is not an uncommon event in squamous cell carcinoma of the tongue. Lack of PTEN expression may be an independent prognostic indicator for clinical outcome in patients with this tumor type.

Phase II study of the antiangiogenesis agent thalidomide in recurrent or metastatic squamous cell carcinoma of the head and neck.

BACKGROUND: Thalidomide has been shown to have antiangiogenic effects in preclinical models as well as a significant antitumor effect in hematologic tumors such as multiple myeloma. The authors performed this Phase II study to determine the activity, toxicity profile, and antiangiogenic effect of thalidomide in patients with locoregionally recurrent or metastatic squamous cell carcinoma of the head and neck. METHODS: Twenty-one patients with recurrent or metastatic squamous cell carcinoma of the head and neck were treated with single-agent thalidomide. All patients had received radiation therapy, and most had undergone surgery (95%) and/or chemotherapy (90%). Thalidomide was initiated at 200 mg;3>daily and increased to a target dose of 1000 mg daily. Patients continued treatment until disease progression, unacceptable toxicity, or death occurred. RESULTS: All 21 patients eventually developed progressive disease. Median time to progression was 50 days (95% confidence interval, 28-70), with median overall survival time of 194 days (95% lower confidence boundary, 151), similar to the progression and survival times reported for this patient group with other agents. Thalidomide was generally well tolerated, with few patients experiencing Grades 3 to 4 toxicities. Serum vascular endothelial growth factor and basic fibroblast growth factor levels increased in six of seven patients, for whom paired serum samples were available and all of whom had progressive disease. CONCLUSIONS: In this heavily pretreated population of patients with advanced squamous cell carcinoma of the head and neck, thalidomide does not appear to have single-agent antitumor activity. Further evaluation of the mechanism of action of thalidomide is indicated. Potentially, future evaluations of thalidomide may be performed in combination with other antiangiogenic or cytotoxic agents in patients with earlier stage disease or in patients with minimal residual disease.

Genetic heterogeneity in saliva from patients with oral squamous carcinomas: implications in molecular diagnosis and screening.

We performed microsatellite analysis at chromosomal regions frequently altered in head and neck squamous carcinoma on matched saliva and tumor samples from 37 patients who had oral squamous carcinoma. The results were correlated with the cytologic findings and traditional clinicopathologic factors to assess the diagnostic and biological potential of these markers. Our data showed that 18 (49%) of the saliva samples and 32 (86%) of the tumors had loss of heterozygosity (LOH) in at least one of the 25 markers studied. In saliva, the combination of markers D3S1234, D9S156, and D17S799 identified 13 (72.2%) of the 18 patients with LOH in saliva (P < 0.001). For tumors, markers D3S1234, D8S254, and D9S171 together identified 27 (84.3%) of the 32 tumors with LOH at any of the loci tested (P < 0.001). Eleven (55%) of the 20 saliva samples with cytologic atypia and seven (35%) of the 17 specimens without atypia had LOH. Significant correlation between LOH in tumor at certain markers and smoking and alcohol use was found. Our results indicate that: 1) epithelial cells in saliva from patients with head and neck squamous tumorigenesis provide suitable material for genetic analysis; 2) combined application of certain markers improves the detection of genetic alteration in these patients; 3) clonal heterogeneity between saliva and matching tumor supports genetic instability of the mucosal field in some of these patients; and 4) LOH at certain chromosomal loci appears to be associated with smoking and alcohol consumption.

Molecular and biomarker analyses of salivary duct carcinomas: comparison with mammary duct carcinoma.

Salivary duct carcinoma (SDC) is a rare high-grade aggressive neoplasm that manifests close histologic features with invasive ductal carcinoma of the breast (IDC). In contrast to SDC, extensive molecular studies have been performed on IDC and led to the identification of certain biological markers. To investigate the underlying molecular and biologic characteristics of SDC, we performed molecular analyses using microsatellite markers on chromosomal arms 6q, 16q, 17p, and 17q, DNA flow cytometry and immunohistochemical staining for androgen receptor (AR) and p53 expression on 28 examples of these tumors in comparison to 24 IDC cases. Our results show that generally similar allelic alterations, elevated p53 and androgen receptor expressions, and high frequency of DNA aneuploidy are manifested in both SDCs and IDCs. Differences at certain markers on 6q, 17p and 17q chromosomal loci, however, were observed between the two entities. Certain loci on 6q were more frequently altered in SDC than IDC which loci on chromosomes 17p and q arms were more seen in IDCs than SDCs. The majority of SDCs had high AR expression while most of IDCs were AR negative. Our study indicates that: i) SDC may share some genetic alterations with IDC, ii) high AR expression in SDC may play a role in tumor progression, and iii) p53 overexpression and DNA aneuploidy in both entities reflect their aggressive behavior.

Clinical significance of cyclin B1 protein expression in squamous cell carcinoma of the tongue.

PURPOSE: Cyclin B1 plays an important role in control of the G(2)-M transition of the cell cycle. We have shown recently that overexpression of cyclin B1 is associated with poor outcome in patients with early stage squamous cell carcinoma (SCC) of the lung. EXPERIMENTAL DESIGN: To determine the role of cyclin B1 in SCC of the tongue, we analyzed tumor specimens from 41 patients with stage II-IV SCC of the tongue who underwent curative surgery using immunohistochemistry. RESULTS: The median follow-up of all patients was 83 months. Overexpression of cyclin B1 was observed in 15 (37%) of the 41 tumors, a similar frequency to that found in SCC of the lung. Patients whose tumors showed overexpression of cyclin B1 had a poor event-free survival compared with those lacking this feature (P = 0.04 by Log-rank test). Multivariate analysis of traditional clinical/pathological factors showed that cyclin B1 overexpression was an independent prognostic indicator. CONCLUSIONS: Our study indicates that cyclin B1 is overexpressed in a subset of SCC of the tongue and is associated with a more aggressive biological behavior of the disease.

UV irradiation augments lymphoid malignancies in mice with one functional copy of wild-type p53.

Epidemiological studies have suggested an association between exposure to solar UV radiation and the incidence of lymphoid malignancies, which has increased substantially worldwide during the last two decades. Findings from animal studies have raised the question of whether UV radiation might influence the development of lymphoid malignancies by means of its immunosuppressive effect. In this study, we examined the effect of UV irradiation on the development of lymphoid malignancies in mice with no or only one functional copy of p53. Mice that lack both copies of p53 spontaneously develop high frequency of lymphoid malignancies in the thymus and spleen. p53 heterozygous mice with only one copy of the wild-type allele also develop lymphoid malignancies, but with a much lower frequency and a long latent period. In our study using mice of the C57BL/6 background, only one of the unirradiated mice lacking one copy of p53 (p53(+/-)) spontaneously developed a lymphoid tumor (6%), whereas 88% of UV-irradiated p53(+/-) mice developed lymphoid tumors in the spleen or liver. None of the control or UV-irradiated p53 wild-type mice developed lymphoid tumors during the 60-week observation period. Both UV-irradiated and unirradiated mice lacking both copies of p53 (p53(-/-)) rapidly developed thymic lymphomas and/or lymphoid tumors in spleen or liver. All of the lymphoid tumors tested were of T cell type. The immune responses of the mice to contact sensitization were identical and were suppressed to the same extent by UV irradiation regardless of the genotype. These results indicate that differences in immune reactivity do not account for the different effects of UV radiation on lymphoid malignancies and, in addition, that p53 is not required for generation of T cell-mediated immunity. Interestingly, whereas p53 mutations or loss of heterozygosity did not account for the accelerated development of lymphoid tumors in UV-irradiated p53(+/-) mice, deletions in the p16(INK4a) gene were quite common. These data provide the experimental evidence that UV irradiation induces lymphoid neoplasms in genetically susceptible mice and support the hypothesis that extensive sunlight exposure contributes to the induction of lymphoma in humans.

An alternatively spliced HDM2 product increases p53 activity by inhibiting HDM2.

The human counterpart hdm2 of the murine double-minute 2 (mdm2) gene encodes a 90-kD protein (HDM2) that inhibits the function of the p53 tumor suppressor. Hdm2 is amplified in approximately 30% of sarcomas, leading to overproduction of HDM2 and inactivation of p53. Using immunohistochemistry to screen a panel of human tumors for HDM2 overproduction, we detected high levels of HDM2 in the cytoplasm in 25% of lung tumors as opposed to its normal localization in the nucleus. These samples contained full-length hdm2 and several alternate-splice forms of hdm2 mRNA. Sequence analysis revealed deletions in the alternate-splice forms of the p53 binding domain and absence of a nuclear localization signal. In transient transfection assays, one of the alternate-splice forms, HDM2(ALT1), bound and sequestered full-length HDM2 in the cytoplasm. In addition, the binding of HDM2(ALT1) to HDM2 inhibited the interaction of HDM2 with p53, thus enhancing p53 transcriptional activity. These data suggest the existence of another level of regulation of HDM2 which increases the activity of p53.

Comparative image and flow cytometric TUNEL analysis of fine needle samples of breast carcinoma.

The assessment of apoptosis in solid tumors is of interest because of its biological role in tumor evolution and response to therapy. A commonly used method for apoptosis measurement is the TUNEL 3' end-labeling technique, which has shown wide variations in results when applied to solid tumors. Thirty-one fine needle breast carcinoma samples were analyzed by fluorescent TUNEL assay and DNA content using image analysis and flow cytometry. TUNEL positivity, seen both in cells with apoptotic morphology and in a subset of morphologically normal cells, was categorized into five staining patterns and quantitated. Values for patterns of TUNEL-positive cells were compared with TUNEL positivity measured by flow cytometry. Flow cytometric quantitation showed a mean of 24.3% positive cells, which correlated (P < 0.02) with total positive cells (all patterns) measured by image (22.4%). Image analysis quantitation of morphologically apoptotic cells (4.2%) did not correlate with flow cytometric TUNEL positivity and the majority of TUNEL-stained cells were morphologically normal (17%). Image analysis allows discrimination of TUNEL-positive morphologically apoptotic and nonapoptotic cells, which are included in the total number of TUNEL-positive events measured by flow cytometry.