Shiho Hamano

Neuroblastoma oligo-capping cDNA project: toward the understanding of the genesis and biology of neuroblastoma

Neuroblastoma (NBL) is a common pediatric cancer originated from the neuronal precursor cells of sympathoadrenal lineage. NBLs show a variety of clinical phenotypes from spontaneous regression to malignant progression with acquirement of resistance to therapy. To understand the molecular mechanism of the genesis, progression, and regression of NBL, we need to identify key molecules determining the neuronal development of sympathoadrenal lineage. To this end, we have performed the NBL cDNA project. It includes (1) mass-cloning of the expressed genes from oligo-capping cDNA libraries derived from primary NBLs with different clinical and biological features; (2) mass-identification of differentially expressed genes between favorable and unfavorable subsets; and (3) molecular and functional analyses of the novel genes, which could be useful prognostic indicators. To date, 10,000 cDNA clones in total, approximately 40% of which contained novel sequences, were randomly picked up and DNA sequenced. We have identified approximately 500 differentially expressed genes between favorable and unfavorable subsets of NBL, among which more than 250 were the genes with unknown function.

Increased occurrence of caspase-dependent apoptosis in unfavorable neuroblastomas

Neuroblastoma frequently shows spontaneous regression in which two distinct types of programmed cell death, ie, caspase-dependent apoptosis and H-Ras-mediated autophagic degeneration, have been suggested to play a key role. The current study was conducted to determine which of these cell suicide pathways predominated in this tumor regression. Periodic acid-Schiff (PAS) staining and immunostaining for H-Ras and for the full-length and cleaved forms of caspase-3, poly (ADP-ribose) polymerase (PARP), and lamin A were carried out on 55 archival tumor specimens. The incidence of caspase-dependent apoptosis in each tumor was quantified by cleaved lamin A staining and compared with clinicopathologic prognostic factors. Although a recent report has shown that neuroblastic cells undergoing autophagic degeneration were readily detectable by PAS and H-Ras staining, we could not confirm this result in any of our samples with the exception of one tumor. Instead, many of our neuroblastoma samples showed nonspecific PAS and Ras staining in areas of necrosis, suggesting that autophagic “degeneration” indeed corresponds to coagulation necrosis or oncosis. Unexpectedly, the incidence of caspase-dependent apoptosis was significantly correlated with indicators of a poor prognosis in these tumors, including Shimadas unfavorable histology, MYCN amplification, and a higher mitosis-karyorrhexis index, but not with factors related to tumor regression such as clinical stage and mass screening. These results indicate that neither caspase-dependent apoptosis nor autophagic “degeneration” may be involved in spontaneous neuroblastoma regression. This suggests that other mechanisms, perhaps such as tumor maturation, may be responsible for this phenomenon.

The usefulness of laryngotracheal separation in the treatment of severe motor and intellectual disabilities

Intractable aspiration is a life-threatening medical problem in patients with severe motor and intellectual disabilities (SMID). Laryngotracheal separation (LTS) is a surgical procedure for the treatment of intractable aspiration which separates the upper respiratory tract from the digestive tract. We performed LTS for 14 patients with SMID to prevent intractable aspiration, performing two types of operation. The standard diversion procedure connected the upper trachea to the esophagus. The modified diversion includes closure of the proximal trachea and a high tracheostomy, avoiding a tracheoesophageal anastomosis. LTS was performed on 14 patients. Operations performed before the LTS included tracheostomy in four patients, fundoplication in six and gastrostomy in two. A standard diversion was performed in 11 patients and a modified diversion in 3. There were no operative complications. Eleven patients were safely transferred to home-care after their LTS. Twelve patients are still alive and two died some months after operation. One patient died from their primary disease and the other died a tracheo-innominate artery fistula (TIAF). We recently experienced a patient who was at high risk of developing a TIAF. LTS is an effective operation, preventing intractable aspiration in patients with severe motor and intellectual disabilities. The results are similar for the standard or modified diversion procedure with the procedure chosen being related to the initial tracheostomy site. The most serious complication is a lethal TIAF.

Analysis of loss of heterozygosity at 16p12-p13 (familial neuroblastoma locus) in 470 neuroblastomas including both sporadic and mass screening tumors

BACKGROUND Neuroblastoma (NBL) in children usually occurs in a sporadic form. However, it rarely occurs in families. Recently, the familial neuroblastoma (FNB) locus has been mapped to 16p12-p13 by linkage analysis. PROCEDURE Here we show the result of loss of heterozygosity in the region spanning 16p12-p13 (D16S406-D16S409, 46 cM) in 470 NBLs including both sporadic and mass screening cases. RESULTS Allelic loss was found in 61(13%) tumors. Deletion of 16p was associated with mass screening tumor (P = 0.035) and <1 year of age at diagnosis (P = 0.048). We found two commonly deleted regions: the sizes of the region were approximately 2 cM and approximately 6 cM. CONCLUSIONS Our data suggested that allelic loss of 16p was common in favorable NBLs, and there may be at least two candidate loci within the region of FNB.