Sentinel lymph node biopsy in Japan

Similar to the practice in Western countries, intra-operative lymphatic mapping and selected lymphadenectomy (SLNB) have been validated and are widely performed for the staging of melanoma in Japan. Recent studies have shown that approximately 90% (73/81) of university hospitals and several cancer hospitals routinely perform SLNB, and half of all melanoma patients receive this examination. SLNB is performed according to a variation of the standard procedure described by Morton and Cochran. The most frequently used tracers are Tc-99m-tin colloid or Tc-99m-phytate for scintigraphy and patent blue violet or indigo carmine as a blue dye. Some institutions use indocyanine green, which is fluorescent and can be used to visualize sentinel lymph node(s) (SLNs) under an infrared camera. The recent detection rate of SLNs has increased to more than 95% with the method using blue dye, lymphoscintigraphy, and a handheld gamma probe. In a multicenter study, the rates of metastasis in SLN were as follows: pTis, 0% (0/36); pT1, 10.7% (6/56); pT2, 21.0% (13/63); pT3, 34.0% (35/103); and pT4, 62.4% (63/101). The metastasis rate was also significantly related to ulceration of the primary tumor. Here, we discuss data from Japanese patients and the present status of SLNB in Japan.ArticleINTERNATIONAL JOURNAL OF CLINICAL ONCOLOGY. 14(6):490-496 (2009)journal articl

Neural Activity Affects Distribution of Glutamate Receptors during Neuromuscular Junction Formation inDrosophilaEmbryos

AbstractChanges in the distribution and density of transmitter receptors in the postsynaptic cell are required steps for functional synapse formation. We raised antibodies againstDrosophilaglutamate receptors (DGluR-II) and visualized the distribution of receptors during neuromuscular junction formation in embryos. In wild-type embryos, embryonic development is complete within 22 hr after egg lying (AEL) and neuromuscular junction (NMJ) formation begins at 13 hr AEL. At the time of initial synapse formation, DGluR-IIs appeared as clusters closely associated with some muscle nuclei. Subsequently, these nonjunctional clusters dispersed while DGluR-IIs accumulated at the junctional region. In a paralytic temperature-sensitive mutant,parats1,neural activity decreases drastically at restrictive temperatures. When neural activity was blocked throughout synaptogenesis by rearing embryos at a restrictive temperature prior to the beginning of synaptogenesis, 12 hr AEL, the dispersal of extrajunctional clusters was significantly suppressed and no accumulation of receptors at the junction was observed at 22 hr AEL. However, when neural activity was blocked later, by rearing embryos at a restrictive temperature from 13 hr AEL, DGluR-IIs did not accumulate at the NMJ, although extrajunctional clusters dispersed normally. These findings suggest that the neural activity differentially regulates dissipation of receptor clusters in the nonjunctional region and accumulation of receptors at the junctional region

日本人の皮膚悪性黒色腫の遺伝子異常に関する研究

金沢大学医学部本研究では、日本人の皮膚悪性黒色腫患者から得られた検体を用いてその遺伝子異常を解析した。まず始めに、原発腫瘍46例を対象とし、癌抑制遺伝子p16の不活性化を系統的に検討した。その結果、46例中11例(24%)に9p21のLoss of heterozygosity(LOH)が検出されたが、これらの腫瘍ではp16遺伝子の体細胞変異やプロモーター領域のCpG islandのメチル化による転写の抑制は認められなかった。一方、p16蛋白の発現の消失が6例に、p16遺伝子のheterozygous変異が1例において認められた。以上の成績から、皮膚悪性黒色腫の原発巣におけるp16INK4a遺伝子の不活性化は、黒色腫細胞株に見い出されるほど高頻度ではないことが示され、さらに、9p21には黒色腫の発癌に重要な役割を演じるp16以外の未知の癌抑制遺伝子が存在する可能性が強く示唆された。次に、悪性黒色腫の転移に関与する遺伝子異常を明らかにするために、14例の患者から得られた原発腫瘍と転移を対象として、LOH解析,p16遺伝子変異およびp16蛋白発現の成績を比較検討した。その結果、p16蛋白の発現の消失と6q、11q、および7qのLOHが黒色腫の転移能の獲得に重要な役割を演じていることが示された。また、個々の症例における原発腫瘍と転移腫瘍の遺伝子異常の比較により、黒色腫はその進展に伴い、複雑なクローン展開を示すことが示唆された。さらに、microdissectionとLOH解析を併用したより詳細な解析により、黒色腫の原発腫瘍内には遺伝的に異なる複数の腫瘍細胞クローンが存在すること、転移は時に原発巣内の微小なサブクローンから発生すること、6qのLOHが転移に重要な役割を演じていることが示された。In a systematical analysis in 46 Japnese sporadic primary cutaneous melanomas, we detected loss of heterozygosity (LOH) of chromosome region 9p21 (where the p16 resides) in 11 (24%) tumors. Direct sequencing, however, revealed no somatic mutation of the p16 gene. Further sequencing analyses in 19 additional tumours with no evidence of LOH of 9p21 identified only one heterozygous C->T mutation at codon 81. De novo methylation of the promoter 5\u27CpG island of the p16 gene, which would lead to transcriptional silencing, was not demonstrated in any of these 12 tumours harboring 9p21 LOH or heterozygous p16 mutation by methylation-specific PCR assay. Nonetheless, complete loss of p16 protein, most likely due to homozygous deletion of the p16 gene, was observed in 6 (15%) out of 39 evaluable cases by immunohistochemical analyses on frozen sections. The results show that inactivation of p16 is not as frequent in primary melanoma as has been reported in cell lines, and warrant further search for another tumour suppressor on 9p21 which is likely to be more important in the initiation of melanoma. Simultaneous investigation examining corresponding metastases in 14 cases showed complete loss of p16 expression during matastatic progression in 4 cases, suggesting that inactivation of p16 plays an important role in progression (rather than initiation) of sporadic melanoma. This analysis also compared LOH of chromosome arms 6q, 9p, 9q, 10q, 11q and 18q, and provided clear evidence that in 3 cases clones of cells found in the sites of metastasis did not derive from the dominant subclone within the primary tumor, indicating that a linear model of melanoma progression is too simplistic, as there is likely to be considerable genetic heterogeneity at the earliest stages of tumourigenesis and that metastases from the same tumor may harbor different genetic change.研究課題/領域番号:09670868, 研究期間(年度):1997 – 1999出典：研究課題「日本人の皮膚悪性黒色腫の遺伝子異常に関する研究 」課題番号09670868（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-09670868/096708681999kenkyu_seika_hokoku_gaiyo/）を加工して作

Low Frequency of Loss and Heterozygosity At the Nevoid Basal Cell Carcinoma Locus and Other Selected Loci in Appendageal Tumors

Previous studies of loss of heterozygosity (LOH) have revealed distinct patterns of allelic loss in some skin tumors. In basal cell carcinomas (BCCs) loss of heterozygosity is virtually restricted to chromosome 9, whereas in squamous cell carcinomas (SCCs) and actinic keratoses loss is more widespread involving chromosomes 3, 9, 13, and 17. Because there are histological similarities between BCCs and some appendageal tumors, and because some lines of evidence suggest that BCCs are appendageal in origin, we carried out a limited allelotype in 41 appendageal tumors. The overall frequency of allelic loss was low (4 out of 247 informative loci; 1.6%). LOH was seen in a proliferating trichilemmal cyst (l7p), a sebaceous epithelioma (l7q), an eccrine porocarcinoma (17q), a trichoepithelioma (9q), and in two basal cell carcinomas showing eccrine or granular cell differentiation that were originally misdiagnosed (9q). The pattern of loss in this mixed group of appendageal tumors shows differences from both BCCs and SCCs, and further emphasizes the unique genetic profile and behavior of BCCs. The finding of 9q loss in BCCs with eccrine or granular cell differentiation shows that 9q loss occurs in different histological subtypes of BCCs

Development and evaluation of dose calculation algorithm with a combination of Monte Carlo and point-kernel methods for boron neutron capture therapy

We developed a 'hybrid algorithm' that combines the Monte Carlo (MC) and point-kernel methods for fast dose calculation in boron neutron capture therapy. The objectives of this study were to experimentally verify the hybrid algorithm and to verify the calculation accuracy and time of a 'complementary approach' adopting both the hybrid algorithm and the full-energy MC method. In the latter verification, the results were compared with those obtained using the full-energy MC method alone. In the hybrid algorithm, the moderation process of neutrons is simulated using only the MC method, and the thermalization process is modeled as a kernel. The thermal neutron fluxes calculated using only this algorithm were compared with those measured in a cubic phantom. In addition, a complementary approach was used for dose calculation in a geometry simulating the head region, and its computation time and accuracy were verified. The experimental verification indicated that the thermal neutron fluxes calculated using only the hybrid algorithm reproduced the measured values at depths exceeding a few centimeters, whereas they overestimated those at shallower depths. Compared with the calculation using only the full-energy MC method, the complementary approach reduced the computation time by approximately half, maintaining nearly same accuracy. When focusing on the calculation only using the hybrid algorithm only for the boron dose attributed to the reaction of thermal neutrons, the computation time was expected to reduce by 95% compared with the calculation using only the full-energy MC method. In conclusion, modeling the thermalization process as a kernel was effective for reducing the computation time

Intensity-modulated irradiation for superficial tumors by overlapping irradiation fields using intensity modulators in accelerator-based BNCT

The distribution of the thermal neutron flux has a significant impact on the treatment efficacy. We developed an irradiation method of overlapping irradiation fields using intensity modulators for the treatment of superficial tumors with the aim of expanding the indications for accelerator-based boron neutron capture therapy (BNCT). The shape of the intensity modulator was determined and Monte Carlo simulations were carried out to determine the uniformity of the resulting thermal neutron flux distribution. The intensity modulators were then fabricated and irradiation tests were conducted, which resulted in the formation of a uniform thermal neutron flux distribution. Finally, an evaluation of the tumor dose distribution showed that when two irradiation fields overlapped, the minimum tumor dose was 27.4 Gy-eq, which was higher than the tumor control dose of 20 Gy-eq. Furthermore, it was found that the uniformity of the treatment was improved 47% as compared to the treatment that uses a single irradiation field. This clearly demonstrates the effectiveness of this technique and the possibility of expanding the indications to superficially located tumors

Development of optimization method for uniform dose distribution on superficial tumor in an accelerator-based boron neutron capture therapy system

To treat superficial tumors using accelerator-based boron neutron capture therapy (ABBNCT), a technique was investigated, based on which, a single-neutron modulator was placed inside a collimator and was irradiated with thermal neutrons. In large tumors, the dose was reduced at their edges. The objective was to generate a uniform and therapeutic intensity dose distribution. In this study, we developed a method for optimizing the shape of the intensity modulator and irradiation time ratio to generate a uniform dose distribution to treat superficial tumors of various shapes. A computational tool was developed, which performed Monte Carlo simulations using 424 different source combinations. We determined the shape of the intensity modulator with the highest minimum tumor dose. The homogeneity index (HI), which evaluates uniformity, was also derived. To evaluate the efficacy of this method, the dose distribution of a tumor with a diameter of 100 mm and thickness of 10 mm was evaluated. Furthermore, irradiation experiments were conducted using an ABBNCT system. The thermal neutron flux distribution outcomes that have considerable impacts on the tumor’s dose confirmed a good agreement between experiments and calculations. Moreover, the minimum tumor dose and HI improved by 20 and 36%, respectively, compared with the irradiation case wherein a single-neutron modulator was used. The proposed method improves the minimum tumor volume and uniformity. The results demonstrate the method’s efficacy in ABBNCT for the treatment of superficial tumors

Polyclonality of BRAF Mutations in Acquired Melanocytic Nevi

Melanocytic nevi are thought to be senescent clones of melanocytes that have acquired an oncogenic BRAF mutation. BRAF mutation is considered to be a crucial step in the initiation of melanocyte transformation. However, using immunomagnetic separation or laser-capture microdissection, we examined BRAF mutations in sets of approximately 50 single cells isolated from acquired melanocytic nevi from 13 patients and found a substantial number of nevus cells that contained wild-type BRAF mixed with nevus cells that contained BRAF(V600E). Furthermore, we simultaneously amplified BRAF exon 15 and a neighboring single nucleotide polymorphism (SNP), rs7801086, from nevus cell samples obtained from four patients who were heterozygous for this SNP. Subcloning and sequencing of the polymerase chain reaction products showed that both SNP alleles harbored the BRAF(V600E) mutation, indicating that the same BRAF(V600E) mutation originated from different cells. The polyclonality of BRAF mutations in acquired melanocytic nevi suggests that mutation of BRAF may not be an initial event in melanocyte transformation.ArticleJOURNAL OF THE NATIONAL CANCER INSTITUTE. 101(20):1423-1427 (2009)journal articl

Assessment of Melanoma-Initiating Cell Markers and Conventional Parameters in Sentinel Lymph Nodes of Malignant Melanoma

Sentinel lymph node (SLN) biopsies have widely been used for the detection of occult LN metastasis of malignant melanoma (MM). In addition to conventional biomarkers, we assessed the diagnostic and prognostic significance of melanoma-initiating cell (MIC) markers in SLNs of MM. We examined the expressions of gp100, MART-1 and tyrosinase mRNA for routine diagnosis and those of ABCB5, CD133, nestin, KDM5B, NGFR and RANK mRNA as MIC markers. The presence of micrometastasis was confirmed immunohistochemically using antibodies to S-100, HMB-45, MART-1, and tyrosinase. Discordance between immunohistochemical and molecular data was observed in 14 of 70 (20.0%) patients, among whom five (7.1%) were positive for only molecular markers;two of these five patients tested positive for micrometastasis by repeated immunohistochemical stainings. The quantitative expression levels of gp100, MART-1, and tyrosinase mRNA were significantly higher in the metastatic LNs;the cut-off values remain to be elucidated. ABCB5 mRNA expression was detected more frequently in the metastatic SLNs (p＜0.05) and in the group of patients with recurrence. To make a definite diagnosis of metastasis, we still need a combination of immunohistochemical and molecular probes. ABCB5 might be a suitable molecular marker for the detection of melanoma-initiating cells in SLNs