CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor.

Malignant rhabdoid tumor (MRT) is a rare, highly aggressive pediatric malignancy that primarily develops during infancy and early childhood. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is dismal; therefore, a greater understanding of the biology of this disease is required to establish novel therapies. In this study, we identified a highly tumorigenic sub-population in MRT, based on the expression of CD146 (also known as melanoma cell adhesion molecule), a cell adhesion molecule expressed by neural crest cells and various derivatives. CD146+ cells isolated from four MRT cell lines by cell sorting exhibited enhanced self-renewal and invasive potential in vitro. In a xenograft model using immunodeficient NOD/Shi-scid IL-2Rγ-null mice, purified CD146+ cells obtained from MRT cell lines or a primary tumor exhibited the exclusive ability to form tumors in vivo. Blocking of CD146-related mechanisms, either by short hairpin RNA knockdown or treatment with a polyclonal antibody against CD146, effectively suppressed tumor growth of MRT cells both in vitro and in vivo via induction of apoptosis by inactivating Akt. Furthermore, CD146 positivity in immunohistological analysis of 11 MRT patient samples was associated with poor patient outcomes. These results suggest that CD146 defines a distinct sub-population in MRT with high tumorigenic capacity and that this marker represents a promising therapeutic target.

PAX3-NCOA2 fusion gene has a dual role in promoting the proliferation and inhibiting the myogenic differentiation of rhabdomyosarcoma cells.

We analyzed a complex chromosomal translocation in a case of embryonal rhabdomyosarcoma (RMS) and showed that it generates the fusion gene PAX3 (paired box 3)-NCOA2 (nuclear receptor coactivator 2). To understand the role of this translocation in RMS tumorigenesis, we established two types of stable mouse myoblast C2C12 cell lines expressing PAX3-NCOA2 and PAX3-FOXO1A (forkhead box O1A), respectively. Compared with control cells, PAX3-NCOA2 cells grew faster, were more motile, were less anchorage dependent, progressed more quickly through the G1/S phase of cell cycle and showed greater transcriptional activation of the PAX3 consensus-binding site. However, PAX3-NCOA2 cells proliferated more slowly and differentiated more weakly than did PAX3-FOXO1A cells. Both PAX3-NCOA2 cells and PAX3-FOXO1A cells formed tumors in nude mice, although the PAX3-NCOA2-induced tumors grew more slowly. Our results may explain why NCOA2 rearrangement is mainly found in embryonal rhabdomyosarcoma, which has a better prognosis than alveolar rhabdomyosarcoma, which expresses the PAX3-FOXO1A fusion gene. These results indicate that the PAX3-NCOA2 fusion gene has a dual role in the tumorigenesis of RMS: promotion of the proliferation and inhibition of the myogenic differentiation of RMS cells.

RASSF1A hypermethylation in pretreatment serum DNA of neuroblastoma patients: a prognostic marker.

The tumour suppressor gene RASSF1A is known to be frequently silenced by promoter hypermethylation in neuroblastoma tumours. Here we explored the possible prognostic significance of aberrant promoter hypermethylation of RASSF1A in serum DNA samples of patients with neuroblastoma as a surrogate marker for circulating tumour cells. We analysed the methylation status of the RASSF1A gene in matched tumour and pretreatment serum DNA obtained from 68 neuroblastoma patients. Hypermethylation of RASSF1A in tumour samples was found in 64 patients (94%). In contrast, serum methylation of RASSF1A was observed in 17 patients (25%). Serum methylation of RASSF1A was found to be statistically associated with age > or =12 months at diagnosis (P=0.002), stage 4 (P<0.001) and MYCN amplification (P<0.001). The influence of serum RASSF1A methylation on prognosis was found to be comparable with that of the currently most reliable marker, MYCN amplification on univariate analysis (hazard ratio, 9.2; 95% confidence interval (CI), 2.8-30.1; P<0.001). In multivariate analysis of survival, methylation of RASSF1A in serum had a hazard ratio of 2.4 (95% CI, 0.6-9.2), although this association did not reach statistical significance (P=0.194). These findings show that the methylation status of RASSF1A in the serum of patients with neuroblastoma has the potential to become a prognostic predictor of outcome.

Continuous remission in an infant with chest wall malignant rhabdoid tumor after relapse.

Malignant rhabdoid tumor (MRT) is a highly aggressive tumor that occurs in infancy or childhood. The prognosis, especially in infants, is very poor. Here we report the long-term survival of a 5-month-old boy with MRT that arose from the chest wall. After total resection of the tumor, the patient was given 4 cycles of doxorubicin, vincristine, and cyclophosphamide, alternating with ifosfamide and etoposide. After 18 months off therapy, he had a local recurrence at the same site. After a second total resection, he was given additional chemotherapy with 30.6-Gy local irradiation. No further recurrence has been observed for 5 years since the second complete remission. Currently, he is alive and well at 7.5 years post-onset. Our experience in this case suggests a fundamental strategy of successful treatment of this highly malignant pediatric tumor: (1) complete resection of the localized tumor, (2) intensive multiagent chemotherapy for the minimal disseminated disease, and (3) radiotherapy for local control of the disease.

MYCN gene amplification is a powerful prognostic factor even in infantile neuroblastoma detected by mass screening.

MYCN is the most powerful prognostic factor in cases of older children. However, how MYCN is related to the prognosis of infantile cases is not clear. A mass screening program was carried out by measuring urinary catecholamine metabolites (VMA and HVA) from 6-month-old infants. Of 2084 cases detected by the screening program, MYCN amplification (MNA) was examined by Southern blot analyses in 1533 cases from 1987 to 2000. Of the 1533 cases examined, 1500 (97.8%) showed no MNA, 20 cases (1.3%) showed MNA from three to nine copies, and 13 (0.8%) cases showed more than 10 copies. The 4-year overall survival rates of these three groups (99, 89 and 53%, respectively) were significantly different (P<0.001), indicating that MYCN copy number correlates with the prognosis. Cases with MNA more than 10 copies were more advanced than those without amplification (stage III, IV vs I, II, IVs; P<0.001). Patients with MNA more than 10 copies had significantly higher serum levels of neuron-specific-enolase (NSE) and ferritin than non-amplified patients (P=0.049, P=0.025, respectively). MYCN amplification was strongly correlated with a poor prognosis in infantile neuroblastoma cases. Therefore, for the selection of appropriate treatment, an accurate determination of MNA is indispensable.

Experience with International Neuroblastoma Staging System and Pathology Classification.

The International Neuroblastoma Staging System and Pathology Classification were proposed in 1988 and in 1999, respectively, but their clinical value has not yet been fully studied in new patients. Six hundred and forty-four patients with neuroblastoma treated between January 1995 and December 1999 were analysed by these classifications. The 4-year overall survival rate of patients <12 months of age with INSS stages 1, 2A, 2B, 3 and 4S disease was 98.5%, which was significantly higher than the 73.1% rate in stage 4 patients <12 months (P<0.0001). When patients were > or = 12 months, the 4-year overall survival rate of patients with neuroblastoma at 1, 2A, 2B and 3 stages was 100% and that of patients at stage 4 was 48.5% (P<0.0001). As to the International Neuroblastoma Pathology Classification histology, the 4-year overall survival rate was 98.8% in patients with favourable histology and 60.7% in those with unfavourable histology in the <12 months group (P<0.0001). In the > or = 12 months group, the 4-year oral survival of patients with favourable histology was 95.3% and that of patients with unfavourable histology was 50.6% (P<0.0001). Among biological factors, MYCN amplification, DNA diploidy and 1p deletions were significantly associated with poor prognosis in patients <12 months, as were MYCN amplification and DNA diploidy in patients > or = 12 months of age. Multivariate analysis showed that the INSS stage (stage 4 vs other stages) and International Neuroblastoma Pathology Classification histology (unfavourable vs favourable) were significantly and independently associated with the survival of patients undergoing treatment, stratified by age, stage and MYCN amplification (P=0.0002 and P=0.0051, respectively).

Cytogenetic analysis of infantile neuroblastomas by comparative genomic hybridization.

Neuroblastomas are heterogeneous tumors. Their clinical behavior varies from spontaneous regression to malignant progression. To investigate the cytogenetic heterogeneity of infantile neuroblastomas, we employed comparative genomic hybridization (CGH). To characterize chromosomal imbalances in 35 infantile neuroblastomas, we performed CGH and compared our results with those of other clinical and biological studies. The most frequent genetic imbalances were found in chromosome 17 (43%), including whole chromosome 17 gains in eight patients (23%) and 17q gains in seven patients (20%). A 1p loss and a 2p gain were detected in six patients each (17%). Losses of 11q and 14q were detected in two patients (6%) and one (3%) patient, respectively. The number of gains of 17q were significantly higher in DNA diploid tumors than in aneuploid tumors (P=0.006). Conversely, whole chromosome 17 gains were not found in DNA diploid tumors and/or MYCN amplification. Interestingly, nine of 17 tumors that were histologically evaluated showed a spontaneous regression and did not demonstrate any partial chromosomal abnormalities (i.e. 17q gain, 1p loss, 2p gain, 11q loss and 14q loss). These results suggest that a gene on chromosome 17q is associated with neuroblastoma progression. Finally, our observations indicate that the chromosomal imbalances observed in infantile neuroblastomas are different from those observed in older patients.

Risk of unfavorable character among neuroblastomas detected through mass screening. The Japanese Infantile Neuroblastoma Cooperative Study.

Current study shows that about 50% of neuroblastomas (NBs) detected through mass screening had factor(s) indicating an unfavorable biological nature and that early intervention after the screening might improve clinical outcome of the patients. On the other hand, favorable properties were detected in the remaining half of the mass-screening NBs. Some of them might have the ability to regress spontaneously. Therapeutic modality should be determined according to their biological nature. Further investigation for their biologic properties is necessary to evaluate the benefits of the mass screening.

Miscibility of phosphatidylcholine binary mixtures in unilamellar vesicles: phase equilibria.

Miscibility among phospholipids with different lipid chain-lengths or with different head groups has attracted a number of research efforts because of its significance in biological membrane structure and function. The general consensus about the miscibility of phosphatidylcholines with varying lipid chain-lengths appears to be that binary mixtures of phospholipids with a difference of two carbon atoms in the lipid chain mix well at the main phase transition. Miscibility between phosphatidylcholines with differences of four carbon atoms appears to be inconclusive. Previous reports on the phase transition of binary phospholipid mixtures are concerned mainly with multilamellar vesicles and are mostly limited to the main transition. In the present study, unilamellar vesicles were used and miscibility in binary systems between dimyristoyl-, dipalmitoyl- and distearoyl-phosphatidylcholines at pretransition as well as main transition temperatures was evaluated by constructing phase diagrams. Two methods were used to monitor the phase transitions: differential scanning microcalorimetry and optical absorbance methods. The optical method has the advantage that unilamellar vesicles of dilute phospholipid concentrations can be used. The liquidus and solidus phase boundaries were determined by the onset temperature of heating and cooling scans, respectively, because the completion temperature of a phase transition has no meaning in binary solutions. Dimyristoyl- and distearoyl-phosphatidylcholines, where the difference in the lipid chain-length is four carbon atoms, mixed well even at pretransition temperature.