Shogo Hasegawa

PIK3CA mutation is an oncogenic aberration at advanced stages of oral squamous cell carcinoma

Phosphatidylinositol 3‐kinases (PI3K) are a group of heterodimeric lipid kinases that regulate many cellular processes. Gene amplification and somatic mutations mainly within the helical (exon 9) and kinase (exon 20) domains of PIK3CA, which encode the 110‐kDa catalytic subunit of PI3K and are mapped to 3q26, have been reported in various human cancers. Herein, 14 human oral squamous cell carcinoma (OSCC) cell lines and 108 primary OSCC tumors were investigated for activating mutations at exons 9 and 20 as well as amplifications in PIK3CA. PIK3CA missense mutations in exons 9 and 20 were identified in 21.4% (3/14) of OSCC cell lines and 7.4% (8/108) of OSCC tumors by genomic DNA sequencing. An increase in the copy number of PIK3CA, although small, was detected in 57.1% (8/14) of OSCC lines and 16.7% (18/108) of OSCC tumors using quantitative real‐time PCR. A significant correlation between somatic mutations of PIK3CA and disease stage was observed: the frequency of mutations was higher in stage IV (16.1%, 5/31) than in a subset of early stages (stages I–III) (3.9%, 3/77; P = 0.042, Fishers extract test). In contrast, the amplification of PIK3CA was observed at a similar frequency among all stages. AKT was highly phosphorylated in OSCC cell lines with PIK3CA mutations compared to those without mutations, despite the amplification. The results suggest that somatic mutations of the PIK3CA gene are likely to occur late in the development of OSCC, and play a crucial role through the PI3K–AKT signaling pathway in cancer progression. (Cancer Sci 2006; 97: 1351–1358)

Identification of a predictive gene expression signature of cervical lymph node metastasis in oral squamous cell carcinoma

An accurate assessment of the cervical lymph node metastasis status in oral cavity cancer not only helps predict the prognosis of patients, but also helps surgeons to perform the appropriate treatment. We investigated the utilization of microarray technology focusing on the differences in gene expression profiles between primary tumors of oral squamous cell carcinoma that had metastasized to cervical lymph nodes and those that had not metastasized in the hope of finding new biomarkers to serve for diagnosis and treatment of oral cavity cancer. To design this experiment, we prepared two groups: the learning case group with 30 patients and the test case group with 13 patients. All tissue samples were performed using laser captured microdissection to yield cancer cells, and RNA was isolated from purified cancer cells. To identify a predictive gene expression signature, the different gene expressions between the two groups with and without metastasis in the learning case (n = 30) were analyzed, and the 85 genes expressed differentially were selected. Subsequently, to construct a more accurate prediction model, we further selected the genes with a high power for prediction from the 85 genes using the AdaBoost algorithm. The eight candidate genes, DCTD, IL‐15, THBD, GSDML, SH3GL3, PTHLH, RP5‐1022P6 and C9orf46, were selected to achieve the minimum error rate. Quantitative reverse transcription–polymerase chain reaction was carried out to validate the selected genes. From these statistical methods, the prediction model was constructed including the eight genes and this model was evaluated by using the test case group. The results in 12 of 13 cases (∼92.3%) were predicted correctly. (Cancer Sci 2007; 98: 740–746)

Quantitative Analysis of Invasive Front in Tongue Cancer Using Ultrasonography

PURPOSE Ultrasonography is a useful diagnostic modality for the head and neck region. The mode of invasion is one of the predictive factors for the cervical lymph node metastasis. The purpose of this study is to evaluate the correlation between quantified invasive fronts from ultrasonographic images and the pathological malignancy grading of tongue squamous cell carcinoma. MATERIALS AND METHODS Forty-eight previously untreated T1-2N0 tongue squamous cell carcinoma patients were enrolled. The ultrasonographic images of intraoral lesions and the excised lesions were collected. The invasive front on the ultrasonographic image was analyzed by a multipurpose software package. The length of the invasive front and the line of smoothed invasive front were measured and the invasive front ratio was calculated. The hematoxylin and eosin stained sections were assessed according to modified Jakobssons malignancy grading as low and high malignancy grade groups. The correlation between invasive front ratio and the histological malignancy grade groups was evaluated. RESULTS The mean invasive front ratio for the excised lesions was 1.145 in low malignancy grade group and 1.248 in high malignancy grade group. These values as determined for the intraoral lesions were 1.074 in the low malignancy grade group and 1.174 in high malignancy grade group. Significant statistical differences were observed between the 2 groups by Mann-Whitneys U test in both images. CONCLUSIONS Invasive front ratio is related to the histological malignancy grade group. It can be considered to be a valuable diagnostic method which can predict neck node metastasis, and help surgeons to choose adequate neck treatment.

Expression of cIAP-1 correlates with nodal metastasis in squamous cell carcinoma of the tongue

Cellular inhibitor-of-apoptosis protein 1 (cIAP-1) is a member of the inhibitor-of- apoptosis protein family, which predominantly regulates apoptosis. It has been suggested that expression of cIAP-1 correlates with certain clinicopathological features. The possible significance of cIAP-1 expression in cervical lymph node metastasis of tongue squamous cell carcinoma (SCC) is investigated. Seventy-five tongue SCCs were analyzed by immunohistochemistry. cIAP-1 immunoreactivity patterns were nuclear in 38 (51%), cytoplasmic in 47 (63%), and concurrent in 37 (49%) cases. Nuclear, cytoplasmic and concurrent cIAP-1 immunoreactions were significantly correlated with lymph node metastasis in tongue SCCs (P=0.0011, 0.0012, and 0.0006, respectively). The cleaved caspase-3, which is a marker of tumor apoptosis, and Ki-67 index, which is a marker of tumor proliferation, were immunohistochemically examined in 21 tongue SCCs with concurrent nuclear and cytoplasmic cIAP-1 expression and with metastasis, and in 23 tongue SCCs without concurrent nuclear and cytoplasmic cIAP-1 expression and without metastasis. Concurrent cIAP-1 expression was inversely correlated with caspase-3 (P=0.0066), but was positively correlated with Ki-67 expression (P=0.0028). The mode of invasion was associated with lymph node metastasis (P=0.014) and differentiation (P=0.013), but was not correlated with cIAP-1 expression. There was no statistically significant correlation between nuclear or cytoplasmic cIAP-1 expression and the clinicopathological factors of gender, age, clinical stage or differentiation. These results suggest that both patterns of cIAP-1 are useful markers for predicting cervical lymph node metastasis in tongue SCC.

Uptake and kinetics of 5-aminolevulinic acid in oral squamous cell carcinoma

Photodynamic diagnosis (PDD) is a form of cancer detection based on the administration of an exogenous photo-activated compound that accumulates in malignant cells, followed by appropriate photo irradiation. The authors describe a spectroscopic study of 5-aminolevulinic acid (5-ALA)-generated photosensitizer protoporphyrin IX (PpIX) fluorescence in human oral squamous cell carcinoma (OSCC) cell lines to validate its clinical use. 5-ALA-induced PpIX fluorescence intensity was measured in the presence and absence of deferoxamine mesylate (DFO). Two, one and two cell lines produced poorly, moderately and well differentiated carcinomas, respectively, on transplantation in scid mice. The fluorescence intensity was high in the poorly differentiated cell lines, followed by the moderately differentiated cell line; the intensity of the well differentiated cell lines was low and not significantly different from that of normal keratinocytes in the absence of DFO. It was elevated to the level of poorly differentiated cell lines following DFO treatment. This elevation was not observed in normal keratinocytes. The results indicate that DFO enhances the photodynamic sensitivity of 5-ALA in non-responsive carcinoma cells as a result of increased cellular accumulation by inhibiting haeme biosynthesis. This PDD system can be applied clinically to the detection of OSCC irrespective of the degree of differentiation.

Effect of irradiation on malignant tumour invasion into bone

Abstract Objective: The periosteum acts as a protective barrier against the entry of tumour cells during direct bone invasion of malignant tumours. Injuries to the periosteum may cause impairment of its barrier function. The effect of irradiation on tumour bone invasion was examined using a mouse tumour transplantation model. Materials and Methods: Forty four C57BL/6J mice were used to evaluate the effect of irradiation. The calvarias of the mice were scratched and/or irradiated (dose, 10 Gy), and BI6F10 melanoma cells were inoculated. Mice from the irradiation only group were inoculated with BI6F10 cells l, 7 or 14 days after irradiation, and the association of irradiation effects with this duration was evaluated. Calvaria specimens were radiologically and histologically evaluated, and the quantity of bone resorption was examined. Results: Bone invasion was not evident in non-irradiated non-scratched control mice, even when the tumour size reached 15 to 20 mm. However, cortical bone resorption and tumour cell entry were observed in both irradiation only and scratch only groups. More aggressive invasion of B16F10 was found in irradiation plus scratch calvarias. Evaluation based on sagittal suture opening revealed no significant differences in bone invasion among these groups. Conclusions: Injuries caused by irradiation made the periosteum more prone to malignant tumour invasion. The effect of irradiation on bones might be long lasting.