Grant-in-Aid for Scientific Research (B).
|Research Institution||Ehime University|
HAMAKAWA Hiroyuki Faculty of Medicine, Ehime University, professor, 医学部, 教授 (20127905)
KAYAHARA Hiroaki University Hospital, Ehime University, Instructor, 医学部・附属病院, 助手 (50263942)
FUKUZUMI Masakuni University Hospital, Ehime University, Instructor, 医学部・附属病院, 助手 (60294815)
TANIOKA Hiroaki Faculty of Medicine, Ehime University, professor, 医学部, 教授 (10028748)
|Project Fiscal Year
1998 – 2001
Completed(Fiscal Year 2001)
|Budget Amount *help
¥11,500,000 (Direct Cost : ¥11,500,000)
Fiscal Year 2001 : ¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 2000 : ¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1999 : ¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1998 : ¥5,200,000 (Direct Cost : ¥5,200,000)
|Keywords||Oral cancer / Micrometastasis / Genetic diagnosis / Cervical lymph node / Circulating cancer cell / quantitative PCR / SCCA mRNA / Keratin 13 mRNA / 口腔癌 / 微小転移 / 遺伝子診断 / 準連続切片 / 頸部リンパ節 / 循環癌細胞 / CK13mRNA / CCAmRNA / 定量化PCR / SCCA / MASA法 / p53 / K13 / K20 / テロメラーゼ / K19 / SCC antigen|
Radical cure of primary oral squamous cell carcinoma (SCC) has improved owing to the development of diagnosis and treatment modalities, The 5-year survival rate of patients with multiple nodal metastases and/or distant metastasis is significantly low. Therefore, early detection of micrometastases in the cervical lymph node (LNs) and in the peripheral blood is expected to further improve their survival rate. We studied on several issueaas follows.
1. Microscopical detection of micrometastasis in the cervical lymph nodes of patients with oral cancer by means of semiserial sectionings.
2. Analysis of suitable tumor marker for detecting micometastasis of oral cancer cells.
3. Genetic diagnosis of micrometastasis in the cercical lymph nodes of clinical patients using conventional PCR and real time TaqMan PCR
4. Basic study for morphological detection of circulating cancer cells in the peripheral blood. -smear specimen, enrichment by cytospin, immunomagnetic separation by Dynabeads -
Five-hundred fifty-four nonmetastatic cervical lymph nodes taken from 73 patients with oral cancer were subjected to hematoxylin-eosin (HE) staining and keratin immunohistochemistry (IHC). Micrometastases, defined as foci 【less than or equal】 3 mm, were detected in 29 sites of 23 lymph nodes (4.2%) of 16 patients (21.9%). Nine patients (12.3%) showed pN upgrading, i.e., 6 from PNO to pN1, 1 from PNO to pN2b, and 2 from pN1 to pN2b. The remaining 13 lymph 【less than or equal】 nodes with occult metastasis were involved in 5 pN2b and 2 pN2c patients resulting in no pN upgrading. The occult metastasis was also detected in six small lymph nodes 5 mm in diameter. The average minor axis of the micrometastasis was 1.36 ± 0.85mm. We propose that the lymph nodes should be cut and examined in 1-mm intervals to detect micrometastatic foci and to evaluate the accurate pN classification.
2. Keratin 13 and SCCA mRNAs are promising tumor marker for detecting micrometastasis of oral cancer by means of conventional RT-PCR.
3. We examined the expression of SCCA mRNA in 12 primary tumors and 212 cervical LNs (101 LNs taken from 8 patients with tongue cancer, 71 from 7 patients with gingival cancer, 19 from 2 patients with laryngeal cancer, 9 from 2 patients with pharyngeal cancer, 7 from 1 patient with cancer of the buccal mucosa, and 5 from 1 patient with cancer of floor of the mouth). Detectability of metastatic LNs by nested and single reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with semiserial sections (hematoxylin-eosin staining and keratin immunostaining). All primary tumors expressed SCCA mRNA. Of 198 histologically metastasis-negative nodes, SCCA mRNA was detected in 37 (18.7%) by nested PCR. Eleven micrometastatic foci in 9 LNs (4.6%) were discovered by semiserial sectioning. This suggests that SCCA mRNA is a promising tumor marker for detecting the micrometastases in cervical LNs of head and neck cancer.
Real time quantitative PCR method was more sensitive than conventional PCR. One or several cacer cells are detectable by this method (unpublished data).
4. We contrived how to enrich whole mononuclear cells (MNCs) spiked with cancer cells on one slide specimen. Using this method and cytospin, cancer cells and MNCs from 1 ml of blood (without red blood cells) are easily checked on a 1.3 x 2.0 cm slide glass. This method allowed shortening of examination time. Next, we found that EOFR is a promising antibody for the immunomagnetic separation of oral cancer cells circulating in the peripheral blood and in the bone marrow. Less