Establishment and characterization of a novel MDM2/MYCN-co-amplified neuroblastoma cell line, NBN-SHIM, established from a late recurrent stage MS tumor.

The biological heterogeneity of neuroblastoma underscores the need for an in vitro model of each molecularly defined subgroup to investigate tumorigenesis and develop targeted therapies. We have established a permanently growing cell line from a 12-year-old girl who developed a late recurrent stage MS, MDM2-amplified neuroblastoma arising in the liver and performed histological, molecular, cytogenetic, exome, and telomere analyses of the recurrent tumor and the cell line. On histology, the recurrent tumor was immunoreactive for TP53, CDKN1A, and MDM2. A molecular cytogenetic study of the recurrent tumor revealed the amplification of MDM2 but no amplification of MYCN. The established cell line, NBM-SHIM, showed amplification of both MDM2 and MYCN on double-minute chromosomes. A copy number evaluation based on exome data confirmed the finding for MYCN and MDM2 and further identified high ploidy on CDK4 and GLI2 loci in the recurrent tumor and the cell line. The telomere maintenance mechanism on the cell line is unusual in terms of the low expression of TERT despite MYCN amplification and alternative lengthening of telomeres suggested by positive value for C-circle assay and telomere contents quantitative assay. The cell line is unique because it was established from a MYCN-nonamplified, MDM2-amplified, late-relapsed stage MS neuroblastoma, and MYCN amplification was acquired during cell culture. Therefore, the cell line is a valuable tool for investigating neuroblastoma tumorigenesis and new molecular targeted therapies for disrupted ARF-TP53-MDM2 pathway and amplification of MDM2 and CDK4.

Ligand Exchange Reactions of a Monomeric Zirconium Carbonate Complex with Carboxylic Acids Studied by Extended X-ray Absorption Fine Structure, UV Absorption and Raman Spectrophotometry.

Ligand exchange reactions of a monomeric zirconium carbonate complex with carboxylic acids were studied by means of extended X-ray absorption fine structure (EXAFS), UV absorption spectrophotometry and Raman spectrometry. Three carboxylic acids, gluconic acid, and L-tartaric acid and citric acid, which are mono-, di- and tri-carboxylic acids, respectively, were employed in this study. These three carboxylic acids gave different spectral signatures and concentration dependences, respectively. In the gluconic acid system, the peaks on Fourier transform of EXAFS spectrum and Raman spectrum caused by carbonate ion coordinating to zirconium atom were obviously decreased with increasing gluconic acid concentration compared to the other two carboxylic acid systems. This indicates the high association ability of gluconic acid to zirconium, which was revealed by UV spectrophotometric analysis.

A novel fluorescence in situ hybridization assay for synovial sarcoma.

Synovial sarcoma, which is difficult to diagnose precisely, is one of the most common childhood nonrhabdomyosarcoma soft-tissue sarcomas. The purpose of this study is to develop new molecular cytogenetic assay. We used two sets of two-color break-apart FISH probes, flanking either the SSX1/SSX4 or SSX2 locus. Each set of probes is composed of differentially labeled DNA fragments complementary to sequences proximal or distal to the break point within the SSX1/SSX4 or SSX2 locus, which are applied separately to histopathological sections. Interphase nuclei containing a translocation that disrupts either SSX1, SSX2, or SSX4 locus will display two single-color signals that have "broken apart" from each other. We applied it to two synovial sarcoma cell lines and clinical samples. This assay can detect translocation at either SSX1/SSX4, or SSX2 locus on interphase spread prepared from synovial sarcoma cell line and histopathological sections, which is sufficient to diagnose as synovial sarcoma. Our new FISH assay has several advantages, including its applicability to paraffin-embedded samples, discrimination of the SS18-SSX1 and SS18-SSX2 translocations particularly in cases with aneuploidy, and potentially detecting translocations in all cases of synovial sarcoma, even with variant translocations. Our strategy will improve the accuracy of diagnoses, thereby facilitating appropriate treatment planning.

Concurrent Ewing sarcoma family of tumors and fibrous dysplasia: possible diagnostic pitfall.

We present 2 patients with synchronous Ewing sarcoma family of tumors (ESFTs) and fibro-osseous lesion in the independent sites, possibly causing misjudgment in staging. Each patient showed another activity apart from the primary ESFT lesion on gallium and/or thallium scintigraphy at initial presentation. Of note is that such lesions showed no obvious radiologic change even though the primary ESFT lesions were mildly shrunken during chemotherapy. The biopsies confirmed fibrous dysplasia (FD) in the first patient and fibro-osseous lesion, possibly FD in the second patient. As far as we know, concurrent ESFT and FD in independent sites have never been described. However, this unusual combination emphasized the possibility of concurrent FD mimicking metastasis in a patient with malignancy and the view that exploratory biopsy should be performed in a critical case to make staging. Further investigation will be required about whether the co-occurrence of ESFD and FD in our patients is coincidence or genetic linkage.

Expression pattern of keratin subclasses in pancreatoblastoma with special emphasis on squamoid corpuscles.

Expression patterns of keratins (K), both simple epithelia-type (K7, K8, K18, K19) and complex/stratified epithelia-type (K1, K4, K5/6, K10, K13, K14, K15, K16, K17), and epithelial membrane antigen (EMA) were immunohistochemically studied in six pancreatoblastomas (PBL). In all six tumors, areas with overt acinar differentiation (AA), solid areas without any specific differentiation (SO), and squamoid corpuscles (SC) were diffusely positive for K8, K18, and K19. The AA and SO in all the tumors were diffusely positive for K7, but the SC were negative or displayed only scattered reactivity for K7. In three tumors, the AA and the SC showed scattered reactivity for K5/6. No reactivity for other complex/stratified epithelia-type K was found in any of the examined tumor. All tumors were reactive for EMA with consistent predominancy in the SC. Ultrastructurally, well-developed desmosome-tonofilament complexes were only partially observed in tumor cells comprising the SC. These results implied that (i) the SC usually lack a character of complete squamous metaplasia; and (ii) the SC have a characteristic phenotype (K8/K18/K19/EMA-positive, K7-negative or scatteredly positive) that can potentially be useful to delineate the SC in PBL.

Aberrations of the CHK2 gene are rare in pediatric solid tumors.

In pediatric solid tumors, such as neuroblastoma (NB), it has been reported that the frequency of TP53 gene alterations is lower than that in adult tumors, suggesting that other tumor suppressor genes may play more important roles in the development of pediatric solid tumors. The CHK2 gene, whose product is a checkpoint kinase that plays a central role in DNA damage response and acts upstream of TP53, has been found to be mutated in a subset of Li-Fraumeni syndrome without mutations of TP53 and in some other sporadic human tumors, earmarking this serine/threonine kinase as a candidate tumor suppressor gene. Thus, we analyzed the CHK2 gene to address whether it is a candidate tumor suppressor gene for pediatric solid tumors. We screened for mutations of the CHK2 gene in 25 NB, 8 rhbdomyosarcoma, 12 Ewing sarcoma, and 26 other pediatric solid tumor cell lines as well as 77 fresh tumors including two cases of multiple cancers. Using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis and reverse transcriptase (RT)-PCR-SSCP followed by direct sequence analysis, we detected only one missense mutation (S505T) in one NB cell line and two silent mutations in one NB cell line and one NB fresh tumor, respectively. Through RT-PCR and subcloning analysis, we detected a similar expression of the CHK2 gene in all of the NB cell lines and fresh tumors; however, we identified at least three isoforms of the CHK2 gene, two of which have not been reported previously. These results suggest that aberrations of the CHK2 gene are rare in pediatric solid tumors.

Gene expression profiling and identification of novel prognostic marker genes in neuroblastoma.

To investigate the various genetic characteristics of and differences between early- and advanced-stage neuroblastoma (NB) and to identify candidate genes involved in NB progression, we performed DNA microarray analysis on 20 primary tumors. Two-way clustering analysis based on the expression pattern of approximately 500 of 1,700 genes revealed genetic subgroups in these NB tumors. Although 9 of the 13 early-stage tumors (69%) and 4 of the 6 advanced-stage tumors (67%) were classified as being in the same cluster, the remaining tumors showed different expression profiles. This indicates that both the early- and advanced-stage tumors were heterogeneous. Based on the microarray data, we identified the BIRC, CDKN2D, and SMARCD3 genes as those that are predominantly expressed in either the early or the advanced stage of NB. These genes have been reported to be associated with apoptosis, cell cycles, and the transcriptional activator, respectively. To better assess the prognostic value of the expression of these genes in NB, real-time polymerase chain reaction was carried out on 50 primary tumors. The expression of both the BIRC3 and CDKN2D genes was significantly higher in the early-stage group than in the advanced-stage group (P = 0.002 and 0.003, respectively), whereas the expression of the SMARCD3 gene was significantly reduced in the early-stage group (P = 0.02). Therefore, the BIRC, CDKN2D, and SMARCD3 genes are possible candidates for being novel prognostic markers for NB.

Aberrations of p16INK4A, p14ARF and p15INK4B genes in pediatric solid tumors.

Expression of the p16INK4A (p16), p15INK4B (p15), and p14ARF genes, located at 9p21, was examined in pediatric neuroblastoma (NB), Ewing's sarcoma (ES), and rhabdomyosarcoma (RMS). p16 expression was absent in 4 of 5 ESs, and 2 of these 4 cases died. p16 expression was reduced or absent in 10 of 12 RMSs, and 4 of these 10 cases died. These results suggested the possibility that p16 expression was associated with the progression of ES and RMS. There has been no previous report on p14ARF in NB. Our investigation might indicate that abnormal expression of the p16 and p14ARF was associated with a poor prognosis in NB, although in some cases of NB normal p16 and abnormal p14ARF expression was seen. These findings suggest an important role of p14ARF gene in the tumorigenesis of NB. The different incidence of expression of the p16, p15, and p14ARF genes in these 3 tumor types may reflect differences of the molecular process through which the 3 tumors develop. Our results suggest that abnormal expression of the p16 and/or p14ARF may be associated with a poor prognosis in these 3 tumors.

Unrelated cord blood transplantation for second hemopoietic stem cell transplantation.

BACKGROUND: The Kanagawa Cord Blood Bank (KCBB) reports the treatment of 12 patients who received umbilical cord blood transplantation (CBT) from unrelated donors as their second hemopoietic stem cell transplantation (HSCT). METHODS: Provided by the KCBB, between February 1997 and September 2000, 12 patients had unrelated CBT as a second HSCT. Six patients were male and six female; nine patients were in malignant, and three were in non-malignant conditions. The median age of the patients was 7.9 years (range, 2.2-28.0), and the median bodyweight was 22.5 kg (12.0-55.0). The HLA-A and -B serological and DR genotypical disparities between the patients and CBT donors were as follows: one patient was a 0-mismatch, six were 1-mismatches, and five were 2-mismatches. RESULTS: The median time between first and second HSCT was 14.0 months (1.0-47.0). The overall survival rate was 25.0%, three years after CBT (Kaplan-Meier estimate). Mortality after CBT as a second HSCT accounted for nine cases, six from infection and three from treatment-related mortality other than infection. CONCLUSION: Cord blood transplantation offers the advantage of rapid availability, absence of donor risk, and possibly less HLA restriction. In these contexts, unrelated CBT should be considered as a source of HSCT for a second transplant.

Low natural killer activity and central nervous system disease as a high-risk prognostic indicator in young patients with hemophagocytic lymphohistiocytosis.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis HLH (FHL) is fatal, unless patients are rescued with hematopoietic stem cell transplantation (SCT). Although the molecular identification of FHL now is possible at least in part from perforin gene study, many cases escape detection or never are tested due to the lack of specific hallmarks, making diagnosis difficult. To the authors' knowledge, it remains to be determined whether persistently low natural killer cell (NK) activity and a high incidence of central nervous system (CNS) disease increase the probability of FHL. METHODS: The authors analyzed 42 HLH patients age < 2 years, 13 of whom developed overt CNS disease and 5 of whom demonstrated persistently deficient NK activity (Group 1). The remaining 24 patients had no CNS disease and had NK activity of moderate decrease to within the normal range (Group 2). RESULTS: In Group 1, CNS symptoms were detected in 6 cases within 1 month and between 4.5-9 months in 6 other patients. In these cases, spotty lesions demonstrating a high T2 signal in the white matter were noted on brain magnetic resonance imaging. The survival was significantly poor for patients in Group 1 unless they were rescued with SCT, which was performed in 5 of the 13 patients with CNS disease and in all 5 patients with persistent NK activity deficiency. SCT was successful in 9 patients, with no CNS sequelae reported after the transplantation. Conversely, the prognosis of the 24 patients in Group 2 was better and only 1 patient required SCT. CONCLUSIONS: Very young HLH patients (age < 2 years) who are at high risk of fatal FHL with persistently deficient NK activity and/or overt CNS disease require appropriate SCT to reverse CNS disease and achieve a complete cure.

Increased Ras expression and caspase-independent neuroblastoma cell death: possible mechanism of spontaneous neuroblastoma regression.

BACKGROUND: Neuroblastoma undergoes spontaneous regression frequently during its natural course. Although programmed cell death (PCD) has been implicated in this process, accumulating evidence suggests that apoptosis, a form of PCD that is regulated by caspases, may not play a major role. We examined the mechanism(s) of spontaneous regression of neuroblastoma, focusing on the role of Ras, a favorable prognostic marker of neuroblastoma. METHODS: Tumor tissues were analyzed by light microscopy, electron microscopy, and immunohistochemistry to examine cell degeneration and expression of Ras and several indicators of PCD. Cell degeneration was also studied in vitro in neuroblastoma cells transfected with the H-ras gene. All statistical tests were two-sided. RESULTS: Immunohistochemical analyses revealed that Ras expression was increased in areas of cellular degeneration lacking apoptotic characteristics. The degenerating cells were fragmented without nuclear condensation and, essentially, lacked caspase-3 activation and apoptotic DNA fragmentation. These cells had ultrastructural features of autophagic degeneration, another form of PCD that is distinct from apoptosis. Focal areas of degeneration associated with Ras expression were seen more frequently in tumors from patients detected in a mass-screening program (53 [60.9%] of 87) than in tumors from clinically detected, advanced-stage patients over 1 year of age (7 [29.2%] of 24) (P =.006; chi-square test), suggesting a positive relationship between Ras-associated degeneration and probability of spontaneous regression/favorable prognosis. The characteristic features of Ras-associated nonapoptotic degeneration observed in tumor samples were recapitulated in vitro by transfection-mediated Ras expression, and Ras-mediated degeneration was augmented by TrkA, another favorable prognostic marker. CONCLUSIONS: High-level expression of H-Ras in neuroblastoma cells is associated with caspase cascade-independent, nonapoptotic PCD. This Ras-mediated nonapoptotic tumor cell death may play a key role in spontaneous regression of neuroblastoma.