Iterative Motion Correction Technique with Deep Learning Reconstruction for Brain MRI: A Volunteer and Patient Study.

The aim of this study was to investigate the effect of iterative motion correction (IMC) on reducing artifacts in brain magnetic resonance imaging (MRI) with deep learning reconstruction (DLR). The study included 10 volunteers (between September 2023 and December 2023) and 30 patients (between June 2022 and July 2022) for quantitative and qualitative analyses, respectively. Volunteers were instructed to remain still during the first MRI with fluid-attenuated inversion recovery sequence (FLAIR) and to move during the second scan. IMCoff DLR images were reconstructed from the raw data of the former acquisition; IMCon and IMCoff DLR images were reconstructed from the latter acquisition. After registration of the motion images, the structural similarity index measure (SSIM) was calculated using motionless images as reference. For qualitative analyses, IMCon and IMCoff FLAIR DLR images of the patients were reconstructed and evaluated by three blinded readers in terms of motion artifacts, noise, and overall quality. SSIM for IMCon images was 0.952, higher than that for IMCoff images (0.949) (p < 0.001). In qualitative analyses, although noise in IMCon images was rated as increased by two of the three readers (both p < 0.001), all readers agreed that motion artifacts and overall quality were significantly better in IMCon images than in IMCoff images (all p < 0.001). In conclusion, IMC reduced motion artifacts in brain FLAIR DLR images while maintaining similarity to motionless images.

Metastasizing aneurysmal dermatofibroma initially diagnosed as angiosarcoma confirmed by CD63::PRKCD fusion gene detection with nanopore sequencing.

Dermatofibroma (DF) is a benign tumor that forms pedunculated lesions ranging in size from a few millimeters to 2 cm, usually affecting the extremities and trunks of young adults. Histopathologically, DF is characterized by the storiform proliferation of monomorphic fibroblast-like spindle cells. In addition to neoplastic cells, secondary elements such as foamy histiocytes, Touton-type giant cells, lymphoplasmacytes, and epidermal hyperplasia are characteristic histological features. Several histological variants, including atypical, cellular, aneurysmal, and lipidized variants, have been reported; cases with variant histologies are sometimes misdiagnosed as sarcomas. We present a case of metastasizing aneurysmal DF that was initially diagnosed as an angiosarcoma on biopsy. A 26-year-old woman was referred to our hospital with a gradually enlarging subcutaneous mass in her lower left leg. Positron emission tomography-computed tomography revealed high fluorodeoxyglucose uptake not only in the tumor but also in the left inguinal region. On biopsy, ERG and CD31-positive atypical spindle cells proliferated in slit-like spaces with extravasation, leading to the diagnosis of angiosarcoma. Histology of the wide-resection specimen was consistent with DF, and lymph node metastasis was also observed. Nanopore DNA sequencing detected CD63::PRKCD fusion and copy number gain, although CD63 was not included in the target region of adaptive sampling. This report highlights the importance of recognizing the unusual clinical, radiological, and pathological features of DF to avoid misdiagnosis, and the potential diagnostic utility of nanopore sequencer.

The diagnostic utility of cytology specimen in a case of EWSR1::NFATC2 sarcoma.

EWSR1::NFATC2 sarcoma, a rare round cell sarcoma constituting the majority of EWSR1::non-ETS sarcomas, has recently been defined in the latest WHO classification. To date, the cytological findings of EWSR1::NFATC2 sarcoma remain undocumented. We present the case of a 25-year-old man with a history of polyostotic fibrous dysplasia in the right leg, referred to our hospital with left thigh pain. Cytological findings included metachromasia, minimally pleomorphic round cells, and eosinophilic infiltration. There was no precursor fibrous dysplasia and the initial diagnosis was undifferentiated pleomorphic sarcoma. Following histologic review, we successfully performed immunocytochemistry and fluorescence in situ hybridization (FISH) on archival cytology specimens. The tumor cells were positive for NKX2-2, NKX3-1, and PAX7 and showed amplified 5' single signals of EWSR1 gene. Reverse transcriptase-polymerase chain reaction revealed an in-frame fusion of EWSR1 and NFATC2. This report describes the cytological features of EWSR1::NFATC2 sarcoma and highlights the diagnostic utility of archival cytology specimens.

High-risk Features for Recurrence in Patients With Stage III Colorectal Cancer: A Retrospective Cohort Study.

BACKGROUND/AIM: Risk classification for recurrence in stage III colorectal cancer (CRC) is not as well established as it is for stage II. This study aimed to identify high-risk factors for stage III colorectal cancer and to investigate their clinical significance. PATIENTS AND METHODS: We retrospectively analyzed data from 120 patients with stage III CRC who had undergone curative colectomy at our institution between 2014 and 2020. We used logistic regression analysis to identify risk factors for recurrence and subsequently explored their clinical significance. RESULTS: We identified three high-risk factors in stage III CRC: preoperative bowel obstruction [odds ratio (OR)=5.39; 95% confidence interval (CI)=1.61-18.03; p=0.007], N2 disease (OR=3.12; 95%CI=1.05-9.29; p=0.041), and having fewer than 17 examined lymph nodes (OR=3.17; 95%CI=1.11-8.99; p=0.031). The prognosis of patients was clearly stratified by the number of these risk factors, and furthermore, the effectiveness of adjuvant therapy depended on their number. CONCLUSION: Tumor obstruction, N-stage, and the number of lymph nodes examined are important high-risk features for recurrence. This study provides clinicians with valuable insights to predict and stratify patient outcomes in stage III CRC.

Relationship between stress levels and endolymphatic space volume in Meniere's disease.

OBJECTIVES: Since the first report by Hallpike and Yamakawa in 1938, many more patients with Meniere's disease (MD) with endolymphatic hydrops (EHs) have been described. Mental/physical stress and a subsequent increase in the release of the anti-diuretic hormone (ADH) supposedly triggers MD. In the present study, to assess the relationship between stress and EHs, we conducted a series of stress-related questionnaires as well as a 3D endolymphatic space (ELS) analysis in patients with unilateral MD. METHODS: We enrolled 76 patients with unilateral MD (uMD) as the active group and 75 patients with unilateral benign paroxysmal positional vertigo (uBPPV) as the control group; both underwent examinations between June 2014 and November 2019. All patients underwent 3-T magnetic resonance imaging (MRI) 4 h after intravenous gadolinium injection. We used the total fluid space (TFS), ELS, and ELS rate (ELS/TFS × 100), which is the percentage of the volume of the ELS relative to that of the TFS, for a precise evaluation of the ELS and EHs in MD. Stress was evaluated using the Self-Rating Depression Scale (SDS), the psychological Stress Response Scale (SRS), and the modified Dizziness Handicap Inventory (mDHI). Stress scores and blood ADH levels were compared across patient groups. RESULTS: In patients with uMD, ELS rates significantly correlated with SRS scores on both the affected and the healthy side and with mDHI scores on the affected side, while the SDS and ADH showed no significant correlation with the ELS rates. Correlations were much stronger in the group with severe SDS and one with low ADH levels. CONCLUSIONS: The present results indicate that stress may be involved in EHs development in uMD, not only in the ipsilateral but also the contralateral ear. They also suggest that patients with neuropsychiatric tendencies may develop EHs and MD in response to a stressful lifestyle.

Motion correction in MR image for analysis of VSRAD using generative adversarial network.

Voxel-based specific region analysis systems for Alzheimer's disease (VSRAD) are clinically used to measure the atrophied hippocampus captured by magnetic resonance imaging (MRI). However, motion artifacts during acquisition of images may distort the results of the analysis. This study aims to evaluate the usefulness of the Pix2Pix network in motion correction for the input image of VSRAD analysis. Seventy-three patients examined with MRI were distinguished into the training group (n = 51) and the test group (n = 22). To create artifact images, the k-space images were manipulated. Supervised deep learning was employed to obtain a Pix2Pix that generates motion-corrected images, with artifact images as the input data and original images as the reference data. The results of the VSRAD analysis (severity of voxel of interest (VOI) atrophy, the extent of gray matter (GM) atrophy, and extent of VOI atrophy) were recorded for artifact images and motion-corrected images, and were then compared with the original images. For comparison, the image quality of Pix2Pix generated motion-corrected image was also compared with that of U-Net. The Bland-Altman analysis showed that the mean of the limits of agreement was smaller for the motion-corrected images compared to the artifact images, suggesting successful motion correction by the Pix2Pix. The Spearman's rank correlation coefficients between original and motion-corrected images were almost perfect for all results (severity of VOI atrophy: 0.87-0.99, extent of GM atrophy: 0.88-00.98, extent of VOI atrophy: 0.90-1.00). Pix2Pix generated motion-corrected images that showed generally improved quantitative and qualitative image qualities compared with the U-net generated motion-corrected images. Our findings suggest that motion correction using Pix2Pix is a useful method for VSRAD analysis.

Genetic subtype classification using a simplified algorithm and mutational characteristics of diffuse large B-cell lymphoma in a Japanese cohort.

Recent large-scale genetic studies have proposed a new genetic classification of diffuse large B-cell lymphoma (DLBCL), which is clinically and biologically heterogeneous. However, the classification methods were complicated to be introduced into clinical practice. Here we retrospectively evaluated the mutational status and copy number changes of 144 genes in 177 Japanese patients with DLBCL, using targeted DNA sequencing. We developed a simplified algorithm for classifying four genetic subtypes-MYD88, NOTCH2, BCL2, and SGK1-by assessing alterations in 18 representative genes and BCL2 and BCL6 rearrangement status, integrating the significant genes from previous studies. In our cohort and another validation cohort from published data, the classification results in our algorithm showed close agreement with the other established algorithm. A differential prognosis among the four groups was observed. The NOTCH2 group showed a particularly poorer outcome than similar groups in previous reports. Furthermore, our study revealed unreported genetic features in the DLBCL subtypes that are mainly reported in Japanese patients, such as CD5-positive DLBCL and methotrexate-associated lymphoproliferative disorders. These results indicate the utility of our simplified method for DLBCL genetic subtype classification, which can facilitate the optimisation of treatment strategies. In addition, our study highlights the genetic features of Japanese patients with DLBCL.

Transformation of double-hit follicular lymphoma to plasmablastic lymphoma: a partial role of MYC gene rearrangement.

Follicular lymphoma (FL) is genetically characterized by BCL2/IGH translocation. Some FL cases histologically transform to high-grade lymphoma, and the majority of cases transform to diffuse large B-cell lymphoma. We report herein an unusual FL case that transformed to plasmablastic lymphoma (PBL) with MYC gene rearrangement as early as 12 months after FL diagnosis. IGH/MYC translocation, the most common cytogenetic abnormality seen in de novo PBL, was also detected in the transformed tumor (double-hit lymphoma). The patient became resistant to chemotherapy and died 4 months after transformation. We speculate that the "second hit" of MYC rearrangement played a crucial role in PBL transformation (PBL-T) in this case. Highly specific three-color FISH analysis demonstrated the presence of BCL2/IGH/MYC triple fusion signals on a single chromosome as we expected, but BCL2/IGH and IGH/MYC fusion signals also coexisted in a single nucleus. The PBL-T tumor was genetically heterogeneous, despite being histologically quite homogeneous PBL. Surprisingly, three-color FISH analysis revealed that the preceding FL tumor was also genetically heterogeneous, simultaneously harboring BCL2/IGH, IGH/MYC and BCL2/IGH/MYC fusion signals (i.e. double-hit lymphoma), despite being histologically quite homogeneous FL. This suggests that MYC rearrangement played a partial role in PBL-T. Genetic instability including MYC rearrangement in the preceding FL tumor would contribute to PBL-T and poor outcome in this case. This study will broaden our understanding of the pathogenesis of high-grade transformation of FL and help improve patient outcome.

Clinical significance of the 2016 WHO classification in Japanese patients with gliomas.

In this study, we retrospectively compared the prognostic value of the 2016 WHO classification with the former classification in 387 patients with glioma treated at our institution. According to the new classification, diagnoses included oligodendroglioma with isocitrate dehydrogenase (IDH) mutation and 1p/19q co-deletion (5.4%), anaplastic oligodendroglioma with IDH mutation and 1p/19q co-deletion (3.4%), diffuse astrocytoma IDH-mutated (3.9%), anaplastic astrocytoma IDH-mutated (2.8%), glioblastoma IDH-mutated (7.8%), glioblastoma IDH-wildtype (58.4%), diffuse midline glioma H3 K27M mutation (2.6%), oligodendroglioma NOS (1.3%), anaplastic oligodendroglioma NOS (0.8%), diffuse astrocytoma IDH-wildtype (2.8%), and anaplastic astrocytoma IDH-wildtype (10.9%). The prognoses of IDH-mutated astrocytomas clearly varied according to tumor grade. However, we identified no survival difference between IDH-wildtype anaplastic astrocytomas and glioblastomas; additionally, these tumors showed similar gene expression profiles. After exclusion of those without 1p/19q co-deletion, patients with oligodendroglial tumors showed excellent survival regardless of tumor grade. Our evaluation of chromosomal aberrations suggests that the MAPK/PI3K pathway plays a role in acquired malignancy of astrocytic tumors, whereas TP53 participates in tumorigenesis. We suspect the RB pathway also plays a role in tumorigenesis of IDH-mutated gliomas. The new WHO classification more clearly reflects the tumorigenesis of gliomas and improves the prognostic power of classification.

Temozolomide suppresses MYC via activation of TAp63 to inhibit progression of human glioblastoma.

Glioblastoma multiforme (GBM) is a highly invasive and chemoradioresistant brain malignancy. Temozolomide (TMZ), a DNA-alkylating agent, is effective against GBM and has become the standard first-line drug. However, the mechanism by which TMZ regulates the progression of GBM remains elusive. Here, we demonstrate that TMZ targets TAp63, a p53 family member, inducing its expression to suppress the progression of human GBM. High levels of TAp63 expression in GBM tissues after TMZ treatment was an indicator of favourable prognosis. In human GBM cells, TMZ-induced TAp63 directly repressed MYC transcription. Activation of this TAp63-MYC pathway by TMZ inhibited human GBM progression both in vitro and in vivo. Furthermore, downregulation of MYC mRNA levels in recurrent GBMs after TMZ treatment correlated with better patient survival. Therefore, our results suggest that the TAp63-mediated transcriptional repression of MYC is a novel pathway regulating TMZ efficacy in GBM.

ALK fusion gene positive lung cancer and 3 cases treated with an inhibitor for ALK kinase activity.

BACKGROUND: Anaplastic lymphoma kinase (ALK) fusion gene-positive lung cancer accounts for 4-5% of non-small cell lung carcinoma. A clinical trial of the specific inhibitor of ALK fusion-type tyrosine kinase is currently under way. METHODS: ALK fusion gene products were analyzed immunohistochemically with the materials obtained by surgery or by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). The echinoderm microtubule-associated protein-like 4(EML4)-ALK or kinesin family member 5B (KIF5B)-ALK translocation was confirmed by the reverse transcription polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH). After eligibility criteria were met and informed consent was obtained, 3 patients were enrolled for the Pfizer Study of Crizotinib (PF02341066), Clinical Trial A8081001, conducted at Seoul National University. RESULTS: Out of 404 cases, there were 14 of EML4-ALK non-small cell carcinoma (NSCLC) and one KIF5B-ALK NSCLC case (8 men, 7 women; mean age, 61.9 years, range 48-82). Except for 2 light smokers, all patients were non-smokers. All cases were of adenocarcinoma with papillary or acinar subtypes. Three were of stage IA, 5 of stage IIIA, 1 of stage IIIB and 6 of stage IV. Ten patients underwent thoracotomy, 3 received chemotherapy and 2 only best supportive care (BSC). One BSC and 2 chemotherapy cases were enrolled for the clinical trial. Patients with advanced stages who received chemotherapy or best supportive care were younger (54.0±6.3) than those who were surgically treated (65.8±10.1) (p<0.05). The powerful effect of ALK inhibitor on EML4-ALK NSCLC was observed. Soon after its administration, almost all the multiple bone and lymph node metastases quickly disappeared. Nausea, diarrhea and the persistence of a light image were the main side effects, but they diminished within a few months. CONCLUSION: ALK-fusion gene was found in 3.7% (15/404) NSCLC cases and advanced disease with this fusion gene was correlated with younger generation. The ALK inhibitor presented in this study is effective in EML4-ALK NSCLC cases. A further study will be necessary to evaluate the clinical effectiveness of this drug.

Detection of epithelial growth factor receptor mutations in cerebrospinal fluid from patients with lung adenocarcinoma suspected of neoplastic meningitis.

BACKGROUND: Neoplastic meningitis (NM) is a devastating neurological complication of cancer that needs to be diagnosed in the early stages of disease. Polymerase chain reaction detection of epithelial growth factor receptor (EGFR) mutations in cerebrospinal fluid (CSF), which are predictive markers for EGFR tyrosine kinase inhibitor therapy in lung cancer, might be important to diagnose and to treat NM in patients with lung cancer. In this study, we attempted to detect EGFR mutations in CSF and to compare EGFR status between CSF and primary or metastatic lesions in patients with lung adenocarcinoma suspected of NM. METHODS: Twenty-nine patients with lung adenocarcinoma suspected of having NM underwent lumbar puncture. EGFR status of CSF was analyzed by direct DNA sequencing. EGFR mutations of primary or metastatic lesions (lymph nodes and bones) were analyzed in 20 cases. RESULTS: EGFR mutations were detected in CSF of 13 (45%) of 29 patients. In 5 (31%) of 16 patients with negative CSF cytology, EGFR mutations were detected. In four patients, EGFR mutations were shown in CSF, but not in primary or metastatic lesions. Conversely, in two patients, EGFR mutations were shown in primary or metastatic lesions, but not in CSF despite positive CSF cytology. CONCLUSIONS: EGFR mutations, suggesting the existence of malignant cells, were detected in CSF, even in patients with non-small cell lung cancer with negative cytological results. EGFR mutations in CSF do not always reflect the same status as in primary or metastatic lesions.

EML4-ALK fusion gene assessment using metastatic lymph node samples obtained by endobronchial ultrasound-guided transbronchial needle aspiration.

PURPOSE: Anaplastic lymphoma kinase (ALK) fusion genes represent novel oncogenes for non-small cell lung cancers (NSCLC). Several ALK inhibitors have been developed, and are now being evaluated in ALK-positive NSCLC. The feasibility of detecting ALK fusion genes in samples obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) was determined. The clinicopathologic characteristics of ALK-positive lung cancer were also analyzed. EXPERIMENTAL DESIGN: From April 2008 to July 2009, NSCLC cases with hilar/mediastinal lymph node metastases detected by EBUS-TBNA were enrolled. Positive expression of ALK fusion protein was determined using immunohistochemistry, and ALK gene rearrangements were further examined to verify the translocation between ALK and partner genes using fluorescent in situ hybridization and reverse transcription-PCR. Direct sequencing of PCR products was performed to identify ALK fusion variants. RESULTS: One hundred and nine cases were eligible for the analysis using re-sliced samples. Screening of these specimens with immunohistochemistry revealed ALK positivity in seven cases (6.4%), all of which possessed echinoderm microtubule-associated protein-like 4-ALK fusion genes as detected by fluorescent in situ hybridization and reverse transcription-PCR. All ALK-positive cases had an adenocarcinoma histology and possessed no EGFR mutations. Compared with ALK-negative cases, ALK-positive cases were more likely to have smaller primary tumors (P < 0.05), to occur at a younger age (<60 years; P < 0.05), and to occur in never/light smokers (smoking index < 400; P < 0.01). Mucin production was frequently observed in ALK-positive adenocarcinomas (29.4%; P < 0.01). CONCLUSIONS: EBUS-TBNA is a practical and feasible method for obtaining tissue from mediastinal and hilar lymph nodes that can be subjected to multimodal analysis of ALK fusion genes in NSCLC.

Polycomb group molecule PHC3 regulates polycomb complex composition and prognosis of osteosarcoma.

Polyhomeotic homolog 3 (PHC 3) is a member of the human polycomb complex and has been regarded as a candidate tumor suppressor of osteosarcoma. In the present paper, we performed a mutation survey and PHC3 expression analysis by quantitative real-time PCR using 10 osteosarcoma cell lines and 42 primary osteosarcoma samples. Relative PHC3 expression values of clinical samples were analyzed with clinical outcomes, and it was suggested that lower PHC3-expressing patients had significantly worse overall survival. Relative PHC3 values of clinical samples were less than those of normal bone tissues, whereas they were greater than those of cell lines. By denaturing high performance liquid chromatography analysis and direct sequencing, we found a PHC3 missense mutation in U2OS cells, which resulted in arginine56 to proline substitution. The same point mutation existed in four of 42 primary osteosarcoma samples. Regarding functional analysis, PHC3 expression significantly suppressed the colony formation of tumor cells. Intriguingly, polycomb repressive complex 1 members, Bmi1 and Ring1b proteins, were reduced in PHC3-expressing osteosarcoma cells. Deletion mutant PHC3 expression suggested that the carboxyl terminus of PHC3 has a role in suppression; the above-mentioned point mutation of PHC3 also lost inhibitory activities. Conversely, Bmi1 expression reduced PHC3 at the mRNA level and induced the proliferation of osteosarcoma cells. Taken together, we confirmed the role of PHC3 as a tumor suppressor in osteosarcoma cells and found that PHC3-dependent tumor suppression may be caused by modification of the composition of polycomb repressive complex 1 in cancer cells.

RUNX3 modulates DNA damage-mediated phosphorylation of tumor suppressor p53 at Ser-15 and acts as a co-activator for p53.

Although it has been shown that the gastric tumor suppressor RUNX3 has a growth inhibitory activity, the precise molecular mechanisms behind RUNX3-mediated tumor suppression remained unclear. In this study, we found that RUNX3 is closely involved in DNA damage-dependent phosphorylation of tumor suppressor p53 at Ser-15 and acts as a co-activator for p53. The small interference RNA-mediated knockdown of RUNX3 inhibited adriamycin (ADR)-dependent apoptosis in p53-proficient cells but not in p53-deficient cells in association with a significant reduction of p53-target gene expression as well as phosphorylation of p53 at Ser-15. In response to ADR, RUNX3 was induced to accumulate in the cell nucleus and co-localized with p53. Immunoprecipitation experiments demonstrated that RUNX3 forms a complex with p53 in cells. In vitro pulldown assays revealed that the COOH-terminal portion of p53 is required for the interaction with RUNX3. Forced expression of RUNX3 enhanced p53-mediated transcriptional activation. Additionally, RUNX3 had an ability to induce the phosphorylation of p53 at Ser-15, thereby promoting p53-dependent apoptosis. Intriguingly, RUNX3 interacted with phosphorylated forms of ataxia telangiectasia-mutated in response to ADR; however, it did not affect the extent of DNA damage. From the clinical point of view, coordinated p53 mutation and decreased expression of RUNX3 in 105 human lung adenocarcinomas were significantly associated with the poor outcome of patients (p = 0.0203). Thus, our present results strongly suggest that RUNX3 acts as a novel co-activator for p53 through regulating its DNA damage-induced phosphorylation at Ser-15 and also provide a clue to understanding the molecular mechanisms underlying RUNX3-mediated tumor suppression.

topors, a p53 and topoisomerase I-binding RING finger protein, is a coactivator of p53 in growth suppression induced by DNA damage.

The RING family zinc-finger protein topors (topoisomerase I-binding protein) binds not only topoisomerase I, but also p53 and the AAV-2 Rep78/68 proteins. topors maps to human chromosome 9p21, which contains candidate tumor suppressor genes implicated in small cell lung cancers. In this study, we isolated the murine counterpart of topors and investigated its impact on p53 function. The deduced amino-acid sequence of mouse topors exhibits extensive similarity to human topors. Overexpressed myc-tagged topors associates with and stabilizes p53, and enhances the p53-dependent transcriptional activities of p21(Waf1), MDM2 and Bax promoters and elevates endogenous p21(Waf1) mRNA levels. Overexpression of topors consequently results in the suppression of cell growth by cell cycle arrest and/or by the induction of apoptosis. Taken together, these studies identify topors as a positive regulator of p53. The expression of topors is induced by exposure to the genotoxic reagents cisplatin and camptothecin, a DNA topoisomerase I inhibitor. We therefore postulate that topors mediates p53-dependent cellular responses induced by DNA damage, suggesting its physiological role as a tumor suppressor.

Expression profiling and characterization of 4200 genes cloned from primary neuroblastomas: identification of 305 genes differentially expressed between favorable and unfavorable subsets.

Neuroblastoma (NBL), one of the most common childhood solid tumors, has a distinct nature in different prognostic subgroups: NBL in patients under 1 year of age usually regresses spontaneously, whereas that in patients over 1 year of age often grows aggressively and eventually kills the patient. To understand the molecular mechanism of biology and tumorigenesis of NBL, we decided to perform a comprehensive approach to unveil the gene expression profiles among the NBL subsets. We constructed the subset-specific oligo-capping cDNA libraries from the primary NBL tissues with favorable (F: stage 1, high expression of TrkA and a single copy of MYCN) and unfavorable (UF: stage 3 or 4, decreased expression of TrkA and MYCN amplification) characteristics and randomly cloned 4654 cDNAs. Among 4243 cDNAs sequenced successfully, 1799 (42.4%) were the genes with unknown function. Excluding the housekeeping genes, an expression profile of each subset was extremely different. To determine the genes expressed differentially between F and UF subsets, we performed semiquantitative reverse transcriptase (RT)-PCR for each of the 1842 independent genes using RNA obtained from 16 F and 16 UF NBLs as template. This revealed that 278 genes were highly expressed in the F subset as compared to the UF one, while, surprisingly, only 27 genes were expressed at higher levels in the UF rather than the F subset. These differentially expressed genes included 194 genes with unknown function. Many of the genes expressed at high levels in the F subset were related to catecholamine biosynthesis, small GTPases, synapse formation, synaptic vesicle transport, and transcription factors regulating differentiation of the neural crest-derived cells. On the other hand, the genes expressed at high levels in the UF subset included transcription factors and/or receptors that might regulate neuronal growth and differentiation. The chromosomal mapping of those genes showed some clusters. Thus, our mass-identification and characterization of the differentially expressed genes between the subsets may become a powerful tool for finding the important genes of NBL as well as developing new diagnostic and therapeutic strategies against aggressive NBL.

Chromosomes that show partial loss or gain in near-diploid tumors coincide with chromosomes that show whole loss or gain in near-triploid tumors: evidence suggesting the involvement of the same genes in the tumorigenesis of high- and low-risk neuroblastomas.

We performed two-color fluorescence in situ hybridization analysis to detect the numbers of chromosomes 1 and 17, 1p deletion, and 17q gain in 177 neuroblastomas, including 101 tumors that were found by a mass-screening program for infants. Sixty-eight tumors with disomy 1 or tetrasomy 1 were classified as the Dis1 group, and 109 tumors with trisomy 1, pentasomy 1, or a mixed population of cells with trisomy 1 and cells with tetrasomy 1 were classified as the Tris1 group. 17q gain was the most frequent genetic event, followed by 1p deletion, and MYCN amplification in both Dis1 and Tris1 tumors. However, the incidence of all the genetic events was higher in Dis1 tumors than in Tris1 tumors. These findings suggest that Tris1 tumors are more resistant to acquiring the genetic events than are Dis1 tumors. In addition, there was an accumulation of genetic events in more advanced stages, with the exception of a high incidence of 17q gain in the stage IVS Tris1 tumors. Comparative genomic hybridization analysis, which was performed in 59 of the 177 tumors, showed that chromosomes partially lost or gained in Dis1 tumors coincided with chromosomes totally lost or gained in Tris1 tumors. Dis1 and Tris1 tumors were considered to have near-diploid/tetraploid and near-triploid/pentaploid chromosome numbers, respectively. These findings suggest that the same tumor-suppressor genes or oncogenes may be involved in the development and progression of both high- and low-risk neuroblastomas, and that the ploidy state of the tumor plays a fundamental role in the heterogeneous behavior.

Identification of the small interstitial deletion at chromosome band 1p34-p35 and its association with poor outcome in oligodendroglial tumors.

To narrow down the putative tumor-suppressor gene locus and to assess the predictability of clinical courses by genomic alterations, we analyzed 46 oligodendroglial tumors for loss of heterozygosity (LOH) in the distal region of the short arm of chromosome 1. LOH at 1p was found in 43 tumors (93.5%), including all 28 oligodendrogliomas, all eight oligo-astrocytomas, six of eight anaplastic oligodendrogliomas, and in one of two anaplastic oligo-astrocytomas. Thirty-seven tumors showed LOH patterns consistent with a large terminal deletion, whereas six tumors showed LOH suggesting interstitial deletions. Our data also showed two small regions of overlap at 1p34-p35 (approximately 5.7 Mb) and at 1p36.1-p36.2 ( approximately 12 Mb). Among the six tumors with interstitial deletion, the proximal region was deleted in five tumors, whereas the distal region was deleted in only half of them. Overall, 91% of tumors showed deletion including this proximal region. To examine the clinical significance of the LOH pattern, the samples were classified into three groups: tumors without 1p LOH (Group 1, n = 3), tumors with an interstitial deletion (Group 2, n = 6), and tumors with a large terminal deletion (Group 3, n = 37). Both overall and progression-free survival of patients in Group 2 was extremely poor compared with those included in Group 3 (P = 0.0006 and P = 0.003, respectively). As to the clinical response to chemotherapy, nimustine prevented tumor recurrence in Group 3 (P = 0.034) but not in Group 2. Our results demonstrate that a putative tumor-suppressor gene(s) in oligodendroglial tumors is localized at 1p34-p35 and that small interstitial deletions, in contrast to large terminal deletions, are strongly predictive of both chemoresistance and aggressive characteristics of these tumors.