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Epstein–Barr virus (EBV)+ diffuse large B-cell lymphoma (DLBCL) has specific tumour cell characteristics, and these patients have worse outcomes than EBV-negative DLBCL patients. We compared 38 EBV+ DLBCL patients with 43 methotrexate-associated EBV+ B-cell lymphoproliferative disorders (MTX+/EBV+ BLPDs) and 30 non-germinal centre (GC) subtype DLBCL. Lymphoma cells of the EBV+ DLBCL group were positive for BCL2 in 17 patients (44.7%), CMYC in 23 patients (60.5%), and p53 in 33 patients (86.8%), which was significantly higher than in the MTX+/EBV+ BLPD group (P

Background: Methotrexate (MTX)-associated T/natural killer (NK)-cell lymphoproliferative disorders (TNKLPDs) are rare in patients with rheumatoid arthritis (RA), and patients generally recover after MTX withdrawal. Methods: We investigated the clinicopathological characteristics, genetic findings, therapeutic response, and prognostic factors in 21 RA patients with MTX-associated TNKLPDs, which were compared with those of 38 with MTX-associated B-cell LPDs (BLPDs). Results: Immunohistologically, 11 patients (52%) showed CD4+ TLPDs, and seven had a T follicular helper (TFH) phenotype. The other nine patients (43%) showed CD8+ TLPDs, and remaining one (5%) had features of CD3+ CD4− CD8− CD30+ nasal type TNK-cell lymphoma. CD30+, p53+, and CMYC+ atypical lymphocytes were identified in seven (33%), eight (38%), and five (24%) patients, respectively. In situ hybridisation detected Epstein–Barr virus (EBV)-encoded RNA (EBER)+ large atypical lymphocytes in five patients (24%). Nine of 17 patients (53%) showed clonal peaks of TCRγ by polymerase chain reaction. MTX withdrawal was effective in 12 patients (57%), and eight (38%) received chemotherapies. Two patients with TFH+ or EBV+ CD4+ CD30+ large cell peripheral T-cell lymphoma (PTCL), one of CD8+ systemic anaplastic large cell lymphoma, and two of systemic EBV+ CD8+ T-cell lymphoma showed a lethal progressive clinical course within 13 months. Over than 500 U/L LDH, large atypical lymphocytes, expressions of CD30, p53, CMYC, and EBER in atypical lymphocytes were significantly poor prognostic factors for overall survival (p < 0.05, respectively). Mean interval from RA onset to TNKLPDs was 72 months, which was shorter than 166 in BLPDs (p = 0.003). EBV+ atypical and reactive lymphocytes were frequently found in 15 patients with TNKLPDs (71%) and 27 with BLPDs (71%). Conclusions: RA patients with MTX-associated TNKLPDs occurred in early phase of RA, compared with those of the BLPDs, and occasionally showed a lethal progressive clinical course. Detection of MTX-associated TNKLPD patients with poor prognostic factors is necessary. EBV infection in the immunosuppressive states due to persistent RA, MTX and other biological drugs may play a role in forming the tumour microenvironment and lymphomagenesis of TNKLPDs.

Background Gastroenteric neuroendocrine carcinomas (NECs) account for 6.2% of gastroenteric neuroendocrine tumors (NETs), and only 1% or less of gastroenteric NETs occur in the ampulla of Vater (AoV). Clinical features of NEC of the AoV remain obscure. Case presentation A 65-year-old man visited a general practitioner because of jaundice, and an abdominal contrast-enhanced computed tomography scan revealed a tumor of 11 mm in diameter, which was enhanced in the arterial phase at the duodenal papilla, with dilation of the upstream bile duct. Gastrointestinal scope revealed an unexposed tumor of the AoV. Based on a biopsy of the site, a moderately differentiated tubular adenocarcinoma was suspected, and pancreatoduodenectomy was performed. Histopathological examination revealed dysplasia and highly proliferative small tumor cells, with solid and nodular formation at the AoV. Histological analysis showed a high mitotic count, and immunohistochemical staining revealed a Ki-67 index of 40–50% and cells positive for synaptophysin, chromogranin A, and p53. Small cell-type NEC was finally diagnosed. Four months post pancreatoduodenectomy, multiple liver metastases developed, and systemic chemotherapy was administered. Salvage liver resection for liver metastases was performed 14 months after the pancreatoduodenectomy. Unfortunately, multiple liver metastases developed 2 months after liver resection, and the patient died 18 months after the pancreatoduodenectomy. Conclusions Neuroendocrine carcinoma originating from the bile duct is very rare; therefore, in this article, we provide a review of the literature and a case report.

Background Other iatrogenic immunodeficiency-associated (OIIA) T- and natural killer (NK)-cell lymphoproliferative disorders (TNK-LPDs) are rare in patients with rheumatoid arthritis (RA). Methods We investigated the clinicopathological characteristics, Epstein–Barr virus (EBV) infection, genetic findings, therapeutic response, and prognostic factors in 21 RA patients with OIIA TNK-LPDs and compared these with those of 39 with OIIA B-cell LPDs (B-LPDs) and 22 with non-OIIA B-LPDs. Results Immunohistologically, 11 patients (52%) showed CD4+ T-LPDs, and 7 had a T follicular helper (TFH) phenotype. The other nine patients (43%) showed CD8+ T-LPDs, and the remaining one (5%) had features of CD3+ CD4− CD8− nasal type TNK-cell lymphoma. CD30+, p53+, and CMYC+ atypical lymphocytes were identified in seven (33%), eight (38%), and five (24%) patients, respectively. In situ hybridisation detected EBV-encoded RNA (EBER) + large atypical lymphocytes in five patients (24%). Nine of 17 patients (53%) showed clonal peaks of TCRγ by polymerase chain reaction. Withdrawal of MTX and biologic drugs was effective in 12 patients (57%), and 8 (38%) received chemotherapies. Two patients with TFH+ or EBV+ CD4+ CD30+ large cell peripheral T-cell lymphoma, one with CD8+ systemic anaplastic large cell lymphoma, and two with systemic EBV+ CD8+ T-cell lymphoma of childhood showed a lethal progressive clinical course within 13 months. Moreover, > 500 U/L LDH, large atypical lymphocytes, expression of CD30, p53, and CMYC, and EBER+ atypical lymphocytes were significantly poor prognostic factors for overall survival (p p = 0.003). EBV+ atypical and reactive lymphocytes were frequently found in 15 patients with OIIA TNK-LPDs (71%), in 27 with OIIA B-LPDs (69%), and only in 3 with non-OIIA B-LPDs (14%). Conclusions OIIA TNK-LPDs occurred in early phase of RA, compared with OIIA B-LPDs, and occasionally showed a lethal progressive clinical course. Detection of OIIA TNK-LPD patients with poor prognostic factors is necessary. EBV infection in immunosuppressed patients due to persistent RA, MTX, and biologic drugs may play a role in forming the tumour microenvironment and lymphomagenesis of TNK-LPDs.

Background The clinicopathological characteristics and prognostic factors in nodal peripheral T-cell lymphomas (PTCLs) with two or more T follicular helper markers (TFH+) are not adequately investigated. Methods Immunohistologically, we selected 22 patients with TFH+ lymphoma (PTCL-TFH) in 47 of PTCL-not otherwise specified (NOS), and subclassified into large and small cell groups. We compared the two groups with 39 angioimmunoblastic T-cell lymphoma (AITL) and seven follicular T-cell lymphoma (F-TCL) patients. Prognostic factors were analysed by overall survival in patients with three types of TFH+ PTCLs. Results Thirteen large cell and nine small cell PTCL-TFH patients had more than two TFH markers including programmed cell death-1 (PD-1). Large cell PTCL-TFH showed frequent CMYC expression in 10 patients (77%), and four of 11 large cell group (36%) had somatic RHOA G17V gene mutation by Sanger sequencing. Large cell PTCL-TFH patients showed significantly worse prognosis than those of the small cell group, AITL, and F-TCL (p p p Conclusion Large cell PTCL-TFH indicated poor prognosis in TFH+ PTCLs. These data suggested that CMYC+ tumour cells and intense PD-L1+ cell reaction influenced tumour cell progression in TFH+ PTCLs, and PD-1+ tumour cell/intense PD-L1+ cell reactions may play a role in immune evasion.

Background The clinicopathological characteristics and prognostic factors in nodal T-cell lymphomas with T follicular helper markers (TFH+) are not adequately investigated. Methods Immunohistologically, we selected 22 patients with TFH + lymphoma in 47 of peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), and subclassified into large and small cell groups. We compared the two groups with 39 angioimmunoblastic T-cell lymphoma (AITL) and seven follicular T-cell lymphoma (F-TCL) patients. Prognostic factors were analysed by overall survival in patients with three types of TFH + lymphomas. Results Thirteen large cell and nine small cell PTCL patients had more than two TFH markers including programmed cell death-1 (PD-1). Large cell TFH + PTCL showed CMYC expression in 10 patients (76.9%), and four of 11 large cell group (36.4%) had somatic RHOA G17V gene mutation by Sanger sequencing. In TFH + lymphomas, large cell TFH + PTCL patients showed significantly worse prognosis than those of the small cell group, AITL, and F-TCL (p p p

Epstein-Barr virus (EBV)

OBJECTIVE Acute and lymphoma type adult T-cell leukaemia/lymphoma (ATLL) patients show an aggressive clinical course. While some clinical signs indicate good prognosis, definitive cytohistological prognostic factors have yet to be described. METHODS We classified 65 ATLL patients into three groups by tumour cell size and nuclear pleomorphism on fine-needle aspiration and tumour touch smear samples. Semi-quantitative analysis of background small lymphocytes, reactive CD20-positive B cells and CD8-positive T cells was performed. RESULTS Thirty-one patients had pleomorphic lymphoma with predominantly medium-sized cells and coarse granular nuclei. Another 24 patients showed pleomorphic large cell lymphoma with stippled chromatin. The remaining 10 demonstrated monomorphic large lymphoma cells with fine granular chromatin. Patients with pleomorphic lymphoma with medium-sized cells showed significantly higher serum lactate dehydrogenase and lower CD30 and C-MYC expression in lymphoma cells than the other two groups (P = .0216, P < 0.01, respectively). Patients with pleomorphic medium-sized ATLL had few usual small lymphocytes observed on routine morphological examination and showed less concurrent detection of CD20-positive B cells and CD8-positive T cells, both of which were lower than in the other two groups (P = .006, P = .019, respectively). Furthermore, ATLL patients with predominantly medium-sized lymphocytes exhibited a worse prognosis than patients with pleomorphic large cells (P = .0197). Background small lymphocytes and concurrent detection of CD20-positive B cells and CD8-positive T cells may thus be good prognostic factors (P = .011, P = .021, respectively). CONCLUSIONS Morphological features, size of neoplastic cells and background non-neoplastic lymphocyte (B cells and CD8-positive T cells) volume appear to influence the prognosis of patients with aggressive-type ATLL.

Background Pancreatic adenocarcinoma rarely metastasizes to the brain, and clinical features of brain metastasis in such cases remain elusive. To the best of our knowledge, only 21 cases of brain metastasis from pancreatic adenocarcinoma have been previously reported in the English language literature. Case Description A 61-year-old woman was diagnosed with pancreatic adenocarcinoma and began chemotherapy 1 year 4 months before the current admission. Three days before the current admission, she developed acute dysarthria. She was referred to a cancer center, where neuroradiologic examination showed multiple metastatic brain tumors, including a 30-mm-diameter tumor in the right cerebellar region. She was transferred to our institute. Three days after admission, she developed sudden-onset disturbance of consciousness and left hemiparesis. Computed tomography and magnetic resonance imaging of the head showed that the metastatic lesions had increased in size with development of intratumoral hemorrhage and obstructive hydrocephalus. She underwent urgent removal of the tumor in the cerebellum. Obstructive hydrocephalus was relieved, and her consciousness improved immediately after surgery. She was transferred to the palliative care unit of the cancer center and died under hospice care 3 weeks after surgery. Conclusions This case demonstrates that brain metastases from pancreatic adenocarcinoma can enlarge suddenly and simultaneously with intratumoral hemorrhage even without coagulation disorders, resulting in neurologic deterioration in a short time. Surgical resection of metastatic brain lesions from pancreatic adenocarcinoma has an extremely limited role, but such treatment can remove neurologic symptoms and temporarily improve the patient's quality of life in selected cases.

Background Brain tumor are a major etiology of secondary intracranial hemorrhage (ICH) because ICH in patients with cancer often occurs from an intratumoral hemorrhage. However, it is sometimes difficult to detect a tumor when it is tiny and buried, especially during initial examination. Case description A 65-year-old woman who was diagnosed with pulmonary small cell carcinoma 6 months previously developed sudden-onset consciousness disturbance and left hemiparesis. Head computed tomography (CT) showed a round, high-density lesion with a diameter of 31 mm in the right thalamus. There was no enhancement with administration of contrast agent. Five days later, CT revealed significant progression of the hematoma in the thalamus with perifocal edema. She underwent total removal of the hematoma. Histopathological examination revealed a tiny cluster of metastatic cancer tissue within the hematoma. Conclusions When cerebral hemorrhage occurs in a cancer patient, we must consider the possibility of hemorrhage due to a brain metastasis.

Fluorescence in situ hybridization (FISH) with centromeric probes is a method used to detect chromosomal instability (CIN), a hallmark of most cancers. However, no studies thus far have investigated the relationship between centromeric FISH signals and the cell cycle in cancer cells. In this study, the chromosome content in each cell cycle phase was evaluated with respect to the number of centromeric FISH signals in two breast cancer cell lines and eight surgically resected breast cancer specimens using image cytometry. Variations in chromosome number were detected at each phase of the cell cycle but were not associated with proliferative capacity in the cell lines. Furthermore, the chromosome doubling frequency differed in each cell line and clinical specimen. These results reveal two aspects of centromeric FISH signal variation in breast cancers that exhibit CIN, and suggest that chromosome doubling is a remarkable occurrence that may increase the heterogeneity of tumors.

Background The risk of transferring blood-borne infections during transfusion is continually increasing because of newly emerging and reemerging viruses. Development of a rapid screening method for emerging viruses that might be transmitted by transfusion is required to eliminate such pathogens during blood donor screening. Owing to increased use of human materials in organ transplants and cell therapy, the risk of donor-transmitted viral infections is also increasing. Although nucleic acid amplification technology (NAT) is dedicated to blood screening, a small, convenient detection system is needed at the laboratory and hospital level. Study Design and Methods We developed a new pathogen detection system that can detect multiple viruses simultaneously, using originally designed degenerate polymerase chain reaction primers to amplify a wide range of viral genotypes. Amplified samples were identified using a DNA microarray of pathogen-specific probes. Results We detected very low copy numbers of multiple subtypes of viruses, such as human hepatitis C virus (HCV), human hepatitis B virus (HBV), human parvovirus B19 (PVB19), and West Nile virus (WNV), using a single plate. We also detected all genotypes of human immunodeficiency virus (HIV) but sensitivity was less than for the other viruses. Conclusion We developed a microarray assay using novel primers for detection of a wide range of multiple pathogens and subtypes. Our NAT system was accurate and reliable for detection of HIV, HBV, HCV, PVB19, and WNV, with respect to specificity, sensitivity, and genotype inclusivity. Our system could be customized and extended for emerging pathogens and is suitable as a future NAT system.

Objective To investigate if detection of copy number aberrations of chromosomes 3, 7, 9p21, and 17 using multicolour fluorescence in situ hybridization (FISH) predicts patient outcome in non-muscle-invasive bladder cancer (NMIBC). Patients and Methods In all, 118 bladder wash samples were prospectively collected from patients who underwent transurethral resection of bladder tumour (median age 50.5 years, male/female: 91/27, tumour grade 1/2/3: 18/52/42, stage pTis/Ta/T1: 8/62/42) from 2007 to 2010. The 118 samples were analysed using the UroVysion® kit to detect the copy numbers of chromosomes 3, 7, 9p21, and 17. The variant fraction (VF; the sum of the non-modal copy number fraction of each chromosome) was defined as abnormal when the percentage was ≥16%. The percentage deletion of 9p21 (fraction of null or one copy number of the 9p21 locus) was defined as abnormal when the percentage was ≥12%. Maffezzini risk criteria were also analysed in our cohorts. Results There was recurrence in 57 (48.3%) patients and disease progression in 12 (10.1%), with a median follow-up of 35.7 months. Multivariate analysis showed that the percentage 9p21 loss (>12%) was an independent prognostic factor for recurrence (P < 0.001, odds ratio [OR] 3.24, 95% confidence interval [CI] 1.85–5.62). For disease progression, tumour grade, positive urine cytology, concurrent carcinoma in situ, and a mean VF of >16% were significant prognostic factors in univariate analysis. In multivariate analysis, a mean VF of >16% was a prognostic factor for disease progression (P = 0.048, OR 6.07, 95% CI 1.02–57.45). Conclusions Multicolour-FISH analysis using a commercially available kit could be a powerful tool not only for diagnosis, but also for prognostication in patients with NMIBC.

Objectives Proliferation of tetraploid cells (TCs) emerging from diploid cells is considered to be a critical event toward tumourigenesis, or cancer progression. Recently, several studies have reported that binuclear TCs emerging from normal cells are capable of mitosis, however, it has not been confirmed directly whether mononuclear TCs emerging from normal cells could proliferate, even cancer cells. The aim of this study is to detect mononuclear TCs in vitro, spontaneously emerging from diploid cells and to elucidate their proliferative capability directly. For this purpose, we have developed a novel method. Materials and methods In this study, two completely disomic cell lines were used, TIG-7, a fibroblast cell line and CAL-51, a breast cancer cell line. Cells were cultured on microscope slides and their DNA content was determined using an image cytometer. On the same slides, chromosome numbers were scored using centromere fluorescence in situ hybridization (FISH). For evaluating proliferative capability of TCs, bromodeoxyuridine (BrdUrd) incorporation and colony-forming ability were examined. Results Using our method, spontaneous emergence of mononuclear TCs was detected in both TIG-7 and CAL-51. Colonies of TIG-7 TCs were not observed, but were observed of CAL-51 TCs. Conclusions Our method enables detection of mononuclear TCs and elucidation of their proliferative capability, directly; this evidence reveals that mononuclear TIG-7 TCs do not proliferate but that mononuclear CAL-51 TCs are able to.

Recent studies have reported that lymphovascular invasion (LVI) is a predictor of patient prognosis in upper urinary tract urothelial carcinoma (UUTUC). DNA copy number aberrations (DCNAs) identified by array-based comparative genomic hybridization (aCGH) had not previously been examined in UUTUC. We therefore examined DCNAs in UUTUC and compared them with DCNAs in LVI. We applied aCGH technology using DNA chips spotted with 4,030 BAC clones to 32 UUTUC patients. Frequent copy number gains were detected on chromosomal regions 8p23.1 and 20q13.12, whereas frequent copy number losses were detected on chromosomal regions 13q21.1, 17p13.1, 6q16.3, and 17p11.2. DCNAs occurred more frequently in tumors with LVI than in those without it (P = 0.0002), and this parameter was more closely associated with LVI than with the tumor grade or pT stage. Disease-specific survival rate was higher in tumors without LVI than in those with it (P = 0.0120); however, tumor grade and stage were not significant prognostic factors of patient outcome. These data support our hypothesis that tumors with LVI have more genetic alterations in terms of total numbers of DCNAs than those without, and provide proof that aggressive adjuvant therapy should be considered for UUTUC patients with LVI.

Recent studies have reported that centrosome amplification is closely related to chromosomal instability and patient prognosis in human malignancies. The purpose of this study was to elucidate the relationship between centrosome amplification and genomic alterations in urothelial carcinomas. Centrosomes were evaluated by immunohistochemistry using anti-γ-tubulin antibody. Array-based comparative genomic hybridization technology using DNA chips spotted with 4030 bacterial artificial chromosome clones was applied to 70 urothelial carcinomas to examine DNA copy number aberrations. Studying aberrations in the number of chromosomes 7, 9, and 17 using fluorescence in situ hybridization allowed the estimation of the degree of chromosomal instability. DNA copy number gains at 20p12.2, 5p15.2, 5p15.31, and 17q25.3 and losses at 17p12, 8p22, 2q37.3, 5q31.1, and 2q37.3 were more frequent in tumors with centrosome amplification than in those without it. The total numbers of DNA copy number aberrations and frequency of chromosomal instability were also larger in tumors with centrosome amplification than in those without it (P = .0263 and P < .0001, respectively). These parameters were more closely associated with centrosome amplification than with the subjectively assigned tumor grade (P = .0405 and P = .0020, respectively). Thus, these data suggest that centrosome amplification may have great potential as a biomarker for improved objective classification of urothelial carcinoma and estimation of prognosis.

症例は17歳男性．10歳時から時々血便を認めていたため，17歳時に近医で下部消化管内視鏡検査を施行された．肛門縁に接して，ほぼ全周性に白色顆粒状隆起の集簇からなる腫瘍を認め，生検の結果serrated adenoma（SA）と診断された．CT，PETでは直腸以外に病変はなく，腫瘍マーカーの上昇は認めなかった．下部直腸に限局するSAと診断し，経肛門的粘膜下層切除術を施行した．病理組織診断は中等度異型のSAであり，術後８日で退院した．非常に稀な若年発症の下部直腸Serrated adenoma を経験し，経肛門的切除術を施行することにより肛門機能を温存した治療が可能であった．

The accuracy of comparative genomic hybridization (CGH) analysis is affected by hybridization efficiency. We describe here a simple method for enhancing hybridization efficiency. The hybridization procedure is essentially the same as that of conventional methods. Hybridization solution containing denatured DNA probe mixture was applied to a metaphase chromosome slide or DNA chip slide and covered with a coverslip. In the new method, however, the slide was inverted by turning the coverslip downward prior to hybridization. We termed this method the inverted slide method. To estimate the efficiency of the new method, metaphase chromosome slides and DNA chip slides were treated by both the conventional and inverted slide methods and incubated in a moist chamber at 37°C for 12, 24, 48, and 72 h. Hybridization signals were approximately 1.5 to 2 times brighter on the slides using the inverted slide method than those using the conventional method after 48 and 72 h of incubation. Furthermore, topographical differences in fluorescence intensity were smaller in slides using the inverted-slide method than in those prepared by the conventional method. The inverted slide method is methodologically very simple and improves the resolution of CGH.

症例は64歳女性．近医の上部消化管内視鏡検査にて，出血を伴う潰瘍を形成した腫瘍が十二指腸下行脚に認められたため山口大学第一内科へ紹介された．腹部CTおよびMRIで十二指腸前方に最大径85mm大の腫瘤性病変を認めた．内視鏡下生検にてGISTと診断し，外科的切除を施行した．腫瘍は線維性被膜によって覆われ，他の周囲臓器への浸潤は認めなかった．術後の病理組織所見から高リスク群と診断され，イマチニブの術後補助化学療法を行い，術後7ヵ月再発なく経過観察中である．

Centrosome amplification, which may accelerate tumor progression through chromosomal instability, is frequently observed in human malignancies. The intercellular relation between the number of centrosomes and chromosomes, however, is poorly understood. Therefore, the relationship between centrosomes and chromosomal copy number in the same cells was investigated in bladder cancer. Centrosomes were evaluated by immunohistochemistry, using anti–γ-tubulin antibody in eight bladder cancer cell lines. Fluorescence in situ hybridization with centromeric probes for chromosomes 7, 9, and 17 was then performed on the same cells stained with γ-tubulin. The number of centrosomes was directly proportional to the number of chromosomes in cells with centrosome amplification, while a large intercellular variation in chromosomal copy number was detected in cells with normal numbers of centrosomes. Cancer cells with centrosome amplification of even centrosome numbers had significantly more even numbers of chromosomes. In cancer cells with four centrosomes, even numbers of chromosomes were detected more frequently (87.5%). These bladder cancer cell lines showed Aurora-A and p53 overexpression. These data indicate the occurrence of centrosome amplification with the possible mechanism of cytokinesis failure, resulting in a doubling of the number of centrosomes and chromosomes.

Lymph-node metastasis is a main factor causing poor prognosis of patients with gastric cancer (GC). In order to determine the genes involved in lymph-node metastasis, we compared primary tumors with their synchronous lymph-node metastases for DNA sequence copy number aberrations (DSCNAs) in 20 patients diagnosed as having intestinal-type GC using comparative genomic hybridization (CGH). The results showed that some DSCNAs (gains at 8q, 13q, 5p, 7 and X, and losses at 1p, 17p, 19, 21q and 22q) were frequently found in both primary tumors and their metastases. However, metastases often contained DSCNAs that were not found in corresponding primary tumors, and gain at 20q12-13 and losses at 21qcen-21, 4q and 14q22-ter were significantly more frequently observed in metastatic lesions than in their primary tumors (10:2, 9:0, 6:0, and 7:0 between metastases and corresponding primary tumors, respectively). Our data indicate that gain at 20q12-13 and losses at 21qcen-21, 4q, and 14q22-ter are involved in lymph-node metastases, and that these chromosomal regions may contain the genes related to lymph-node metastases in intestinal-type GC.

It is important to estimate the biological characteristics of tumors, including the nodal status at the time of diagnosis for optimal treatment of individual cancer patients. Array-based comparative genomic hybridization (aCGH) was performed on 77 sporadic colorectal adenocarcinomas using a chip spotted with 4030 BAC clones. The nodal status was compared with an aCGH profiles depicted using a combination of decision-tree classifier and a Self-Organizing Map (SOM) analysis. Node metastasis was not detected in any of the 6 poorly differentiated adenocarcinomas with a 3q loss. A SOM analysis following the decision-tree classification of the aCGH data allowed for the differentiation in chromosomal regions between high- and low-level decreases in the DNA copy number. Node metastasis was detected in all 5 tumors with the high-level decrease in DNA copy number at Xp, irrespective of the histological type. Node metastasis was also found exclusively in 6 tumors with increase in DNA copy number at the chromosomal region between 11q13.3 and 11q22.3. Copy number aberrations linked to nodal metastasis were identified more collectively by the combination of the decision-tree classifier and a SOM analysis than by the conventional analysis method in aCGH analysis.

Although lobular endocervical glandular hyperplasia (LEGH) was originally described as a distinct hyperplastic glandular lesion of the uterine cervix, recent studies have raised a question that LEGH may be a cancerous precursor of minimal deviation adenocarcinoma (MDA) and other mucinous adenocarcinomas (MACs) of the uterine cervix. In the present study, we studied LEGH, MDA, and MAC by using molecular-genetic and immunohistochemical methods for chromosomal imbalance, microsatellite instability, human papillomavirus (HPV) infection, and gastric pyloric-type mucin secretion to clarify their relationship. Comparative genomic hybridization revealed recurrent chromosomal imbalances, that is, gains of chromosome 3q and a loss of 1p, which were common to MDA and MAC, in 3 of 14 LEGHs analyzed (21%). LEGHs with chromosomal imbalances showed a degree of cellular atypia in the hyperplastic glandular epithelium. Dual-color fluorescence in situ hybridization confirmed a gain of chromosome 3 fragment in these cervical glandular lesions. HPV in situ hybridization revealed that high-risk HPV (types 16 and 18) was positive in over 80% of MACs, but negative in all LEGHs and MDAs examined. Microsatellite instability was rarely detected in these cervical glandular lesions. Our present study results demonstrated a molecular-genetic link between LEGH and cervical mucinous glandular malignancies including MDA and MAC, and are thought to support the hypothesis that a proportion of LEGHs are cancerous precursors of MDA and/or MAC.

Purpose: Many investigators have reported that aneuploidy detected by flow cytometry is a useful prognostic marker in patients with endometrial cancer. Laser scanning cytometry (LSC) is a technology similar to flow cytometry but is more feasible for clinical laboratory use. We evaluated the usefulness of DNA ploidy detected by LSC as a prognostic marker in patients with endometrial cancer and investigated genetic and epigenetic factors related to aneuploidy. Experimental Design: Endometrial cancer specimens from 106 patients were evaluated. The methylation status of CDH13, Rassf1, SFRP1, SFRP2, SFRP4, SFRP5, p16, hMLH1, MGMT, APC, ATM, and WIF1 and mutations in the p53 and CDC4 genes were investigated. LSC was carried out to determine DNA ploidy. Fluorescence in situ hybridization was done with chromosome-specific centromeric probes to assess chromosomal instability. Results: Univariate and multivariate analyses revealed that p53 mutation and lack of CDH13 hypermethylation associated positively with aneuploidy. Univariate analysis showed that aneuploidy, chromosomal instability, and lack of CDH13 hypermethylation as well as surgical stage were significantly predictive of death from endometrial cancer. Furthermore, multivariate analysis revealed that stage in combination with either DNA aneuploidy or lack of CDH13 hypermethylation was an independent prognostic factor. Conclusion: These results suggest that analysis of DNA ploidy and methylation status of CDH13 may help predict clinical outcome in patients with endometrial cancer. Prospective randomized trials are needed to confirm the validity of an individualized approach, including determination of tumor ploidy and methylation status of CDH13, to management of endometrial cancer patients.

目的 : 浸潤性乳管癌における分子細胞遺伝子学的診断の有用性とエストロゲン受容体 (ER), プロゲステロン受容体 (PgR) の発現とHER-2遺伝子の増幅および核grade分類との関連を明らかにする.方法 : 浸潤性乳管癌と診断された121例を対象とした. DNA ploidyはlaser scanning cytometer (LSC) にて測定し, 染色体不安定性 (CIN) は多くの腫瘍で染色体数の異常が高頻度に生じるとされる染色体7, 11, 17と18番セントロメアプローブ (CEP) を用いたfluorescence in situ hybridization (FISH) により評価した. また, FISHにてHER-2遺伝子増幅の有無を, さらに免疫組織化学的染色にてER, PgRとKi-67の発現を, 細胞診標本で核grade分類を評価した.成績 : DNA ploidyとCINとは有意に相関していた (p

Polo-like kinase 1 (PLK1) participates in bipolar spindle formation and entry into mitosis. Chromosomal instability (CIN) is caused by abnormalities in spindle formation and chromosome segregation. In this study, we investigated the relationship of PLK1 overexpression to CIN, and compared the PLK1 status with clinicopathological parameters in 101 human urothelial carcinomas of the urinary bladder. Expression of PLK1 and the number of centrosomes were assessed by immunohistochemistry. Numerical aberrations of chromosomes 7, 9 and 17 spots that allowed estimation of CIN were evaluated by fluorescence in situhybridization, and DNA ploidy was assessed by laser scanning cytometry. Cancers with a large intercellular variation in centromere copy number were defined as CIN cancers.Tumors with PLK1 overexpression were associated more frequently with CIN (p < 0.0001), DNA aneuploidy (p = 0.0007) and centrosome amplification (p = 0.0013) than those without. Overexpression of PLK1 was significantly related to higher pathological grade (p = 0.0024), multiple tumors (p = 0.0241) and positive urine cytology (p = 0.0192). These data suggest that a high level expression of PLK1 confers tumor progression advantages to urothelial cancer cells, although other factors are also involved.

Purpose: Recent studies have reported that centrosome hyperamplification (CH) is closely related to chromosomal instability in bladder cancer. In this study, we investigated whether CH could be used as a prognostic biomarker for patients with bladder cancer. Experimental Design: CH was evaluated by immunohistochemistry in 50 bladder cancers (≤pT1: 43; ≥pT2: 7). In addition, numerical aberrations of chromosomes 7, 9, and 17 and gain of 20q13, on which the Aurora-A gene is located, were evaluated by fluorescence in situ hybridization, and DNA ploidy was assessed. Preliminary experiments on eight bladder cancer cell lines found that six had over 5% of CH cells associated with a gain of 20q13 and overexpression of Aurora-A; therefore, CH-positive cases (CH+) were defined as those having over 5% of cells with ≥3 centrosomes per cell. Results: CH+, 20q13 gain, chromosomal instability, and DNA aneuploidy were detected in 30 (60%), 18 (36%), 22 (44%), and 19 (38%) patients, respectively. There were significant differences in tumor number, grade, recurrence, and progression between the CH+ and CH− groups. The later had significantly higher recurrence-free and progression-free survivals than the former (P = 0.0028 and P = 0.0070, respectively, log-rank test). Multivariate analysis revealed that CH+ was the strongest predictor for tumor recurrence in nonmuscle invasive (pTa and pT1) bladder cancer (hazard ratio, 1.882; 95% confidence interval, 1.161–3.325; P = 0.0094). Conclusions: Detection of CH may provide crucial prognostic information about tumor recurrence in bladder cancer.

To clarify the molecular basis of acquired cisplatin (CDDP) resistance in esophageal squamous cell carcinoma (ESCC), we used cDNA microarray technology. A CDDP-resistant cell line (YES-2/CDDP), which shows a 7.5-fold increase in resistance to CDDP and a 3-fold decrease in CDDP accumulation compared with the parental YES-2 ESCC cell line, was generated from YES-2 by exposure to increased concentrations of CDDP. By cDNA microarray analysis, we identified 44 genes with significantly different expression levels between YES-2/CDDP and YES-2 cells. Interestingly, 15 of these 44 genes encoded ribosome-related proteins, almost all of which were underexpressed in YES-2/CDDP cells. Our present data suggest that many ribosome-related genes may be involved in the acquired resistance to CDDP in ESCC and that such information may allow us to better understand the mechanism of CDDP resistance.

To clarify the genetic aberrations involved in the development and progression of hepatitis C virus-associated hepatocellular carcinoma (HCV-HCC), we investigated DNA copy number aberrations (DCNAs) in 19 surgically resected HCCs by conventional CGH and array CGH. Conventional CGH revealed that increases of DNA copy number were frequent at 1q (79% of the cases), 8q (37%), 6p (32%), and 10p (32%) and that decreases were frequent at 17p (79%), 16q (58%), 4q (53%), 13q (42%), 10q (37%), 1p (32%), and 8p (32%). In general, genes that showed DCNAs by array CGH were usually located in chromosomal regions with DCNAs detected by conventional CGH analysis. Increases in copy numbers of the LAMC2, TGFB2, and AKT3 genes (located on 1q) and decreases in copy numbers of FGR/SRC2 and CYLD (located on 1p and 16q, respectively) were observed in more than 30% of tumors, including small, well-differentiated carcinomas. These findings suggest that these genes are associated with the development of HCV-HCC. Increases of MOS, MYC, EXT1, and PTK2 (located on 8q) were detected exclusively in moderately and poorly differentiated tumors, suggesting that these alterations contribute to tumor progression. In conclusion, chromosomal and array CGH technologies allow identification of genes involved in the development and progression of HCV-HCC.

Cat scratch disease, caused by Bartonella henselae, typically presents with a localized lymphadenopathy with a brief period of fever and general symptoms. However, there are atypical cases with a wide spectrum of clinical manifestations including prolonged fever (or =37.5 degrees C, for more than 7 days), or with systemic complication, or without lymphadenopathy. We analyzed relationships among those manifestations in children with cat scratch disease. A total of 127 patients were serologically diagnosed as having Bartonella infection between 1997 and 2003. Relationships among clinical manifestations were analyzed by use of multiple regression and multiple logistic regression analyses. Of the 127 seropositive cases, 75 (59.1%) had typical cat scratch disease and 52 (40.9%) had an atypical one. As atypical manifestations, 46 (36.2%) had prolonged fever, 23 (18.1%) had no lymphadenopathy, and 21 (16.5%) had complications: hepatic/splenic abscesses or low-echoic lesions, hepatic granuloma, and central nervous system involvements. Prolonged fever was observed in 20 (87%) of the 23 cases without lymphadenopathy and 16 (76.2%) of the 21 cases with complications. By multiple regression analysis, the duration of fever was significantly associated with both the absence of lymphadenopathy and the presence of complications. The child suffering from cat scratch disease without lymphadenopathy or with complication tends to have prolonged fever. Conversely, when a child has a prolonged fever of unknown origin, possibility of cat scratch disease should be considered, and a search for underlying systemic complications is recommended for prompt diagnosis and appropriate treatment.

The aim of the present study was to examine the prognostic significance of p27(Kip1) and cyclin E expression in patients with spindle-cell soft tissue sarcomas. In 46 cases of spindle-cell sarcoma including 17 pre-operative biopsy materials, the expression of p27(Kip1) and cyclin E was immunohistochemically examined. The expression of p27(Kip1) decreased in the nuclei of metastatic primary tumor cells (stage IV), whereas the expression of cyclin E increased in those lesions. On univariate analysis, when the expression of p27(Kip1) and cyclin E was analyzed together, patients with spindle-cell sarcoma exhibiting low expression of p27(Kip1) and high expression of cyclin E showed lower distant-metastasis-free survival (DMFS) and overall survival (OS) than those with other combinations of the two parameters (both P < 0.0001). Multivariate analysis revealed that patients with low p27(Kip1) and high cyclin E expression also showed a decrease in DMFS (P = 0.0007, relative risk = 21.3) and OS (P = 0.005, relative risk = 20.8). These results suggest that the combination analysis of p27(Kip1) and cyclin E expression even in biopsy specimens allows the prediction of the clinical behavior of spindle-cell sarcoma.

BACKGROUND Although recent experimental studies have implicated p27 as a regulator of apoptosis in a number of neoplasms, to the authors' knowledge the relation between p27 expression and apoptosis has not yet been established in synovial sarcoma. METHODS The relation between p27, apoptosis, and clinicopathologic features were examined in 62 synovial sarcoma cases. RESULTS p27 expression was found to be correlated inversely with the extent of apoptosis in synovial sarcomas. On survival analysis, p27 expression was found to be a significant and independent prognostic factor. Although the extent of apoptosis was determined to be prognostically significant, it failed to retain an independent and significant value on multivariate analysis. The combination of p27 expression and apoptosis allowed for the more accurate estimation of prognosis in patients with synovial sarcoma than either parameter alone. CONCLUSIONS A combination of p27 expression and extent of apoptosis permitted the more accurate estimation ofy the prognosis of patients with synovial sarcoma. Cancer 2002; 94:2712–8. © 2002 American Cancer Society. DOI 10.1002/cncr.10556

We evaluated sequential thallium scans on both early images (EI) and delayed images (DI) for 62 patients who had bone and soft tissue lesions. The purpose was to determine whether this technique could be used to ascertain accurately whether lesions were malignant or benign and to predict the response to chemotherapy. The thallium-201 chloride (201Tl) accumulation in malignant tumors and benign lesions was statistically different. Sensitivity, specificity, and accuracy for 201Tl scans in detecting malignant tumors was 94%, 65%, and 82%, respectively, for EI, and 94%, 85%, and 90%, respectively, for DI. On multivariate analysis, significant independent factors for 201Tl uptake were malignant lesions on EI and DI and high cellularity on EI. Thirteen patients with malignant bone and soft tissue tumors underwent 201Tl scans before and after preoperative chemotherapy. There was a good correlation between percentage of tumor necrosis and percentage change of accumulation in lesion-to-normal tissue ration, and the correlation coefficient was higher on EI ( r = 0.801) than on DI ( r = 0.664). These results support the notion that 201Tl scintigraphy, although showing some false-positive and false-negative findings, is a useful tool in the evaluation of either malignant tumors or benign lesions. Furthermore, 201Tl scans on EI provide benefit concerning the evaluation of chemotherapeutic response in patients with malignant bone and soft tissue tumors.

To evaluate the clinicopathological significance of matrix metalloproteinase-7 (MMP-7) expression in gastric carcinoma, we investigated immunohistochemically MMP-7 expression in 214 gastric carcinomas, and examined its relations with the clinicopathologic parameters including patient prognosis. MMP-7 expressed predominantly in cancer cells, and MMP-7-positive tumor cells were preferentially found in deeply invading nests, especially at the invasive front. The mean MMP-7 labeling index (LI) at the invasive front was significantly higher in tumors invading or penetrating the muscularis propria and in stages II - IV than within the submucosal layer and in stage I, respectively (P0.001). Statistical analysis revealed that MMP-7 LI at the invasive front was related to lymph node metastasis, vascular invasion, and lymphatic permeation, when all 214 cases were examined as one group (P0.05 for all), and the cases with high MMP-7 expression at the invasive front showed significantly more unfavorable prognosis as compared with that of low MMP-7 expression tumors (P0.01). Multivariate analysis revealed that TNM stage and MMP-7 expression status at the invasive front were independent prognostic factors (P = 0.0017, relative risk (RR) = 3.12; P = 0.0019, RR = 2.67, respectively). Our findings indicated that expression of MMP-7 at the invasive front is closely associated with local invasiveness, and might be a reliable prognostic marker for patients with gastric carcinoma.

To assess the potential of cell array technology, a cell array slide with 50 spots was designed specifically for automated analysis of changes in expression of cyclins A and B1 during the cell cycle at the cellular level. Cells harvested every 1 hour from 0 through 23 hours after synchronization by mitotic selection were spotted in duplicate on a glass slide. Each slide contained 48 spots representing 24 different cell cycle phases, and the remaining two spots were peripheral blood lymphocytes, which were included as negative control. This cell array provided temporal and spatial information related to changes in cyclin expression during the cell cycle in a single experiment. The present study indicates that expression analysis by cell array is novel approach for cell cycle studies. Furthermore, sophisticated multiplexed cell array technology has great potential for analyses of expression of specific genes during diverse cellular events at the cellular level.

The aim of this study was to elucidate cytogenetic changes in pancreatic cancers (PCs) and to examine their clinical implications. We screened for genetic alterations in 32 primary PCs including 4 cases with distant organ metastasis using comparative genomic hybridization coupled with tissue microdissection and degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). The present study revealed frequent gains of chromosomes 13q and 15q and a loss of Xq in addition to a high prevalence of chromosomal imbalances. The average number of total genetic alterations and gains tended to be higher in N1 tumors (TNM classification) than in N0 tumors. The average number of amplifications was significantly higher in M1 tumors than in M0 tumors (p = 0.024). Gain/amplification of 20q was more frequently observed in M1 tumors than in M0 tumors (p = 0.016), and this change was also detected in all of 4 distant metastatic lesions. Losses of 6q, 8p, 9p, 17p, and 18q were recurrent in N0 and M0 tumors, and these alterations were also retained in N1 and M1 tumors. These observations suggest that these genetic losses contribute to the development of PCs and that increases in the DNA copy number confer an aggressive character on cancer cells. Especially, gain/amplification of 20q was associated with the potential of distant organ metastasis of tumor cells.

Fluorescence in situ hybridization (FISH) with centromeric probes is a method used to detect chromosomal instability (CIN), a hallmark of most cancers. However, no studies thus far have investigated the relationship between centromeric FISH signals and the cell cycle in cancer cells. In this study, the chromosome content in each cell cycle phase was evaluated with respect to the number of centromeric FISH signals in two breast cancer cell lines and eight surgically resected breast cancer specimens using image cytometry. Variations in chromosome number were detected at each phase of the cell cycle but were not associated with proliferative capacity in the cell lines. Furthermore, the chromosome doubling frequency differed in each cell line and clinical specimen. These results reveal two aspects of centromeric FISH signal variation in breast cancers that exhibit CIN, and suggest that chromosome doubling is a remarkable occurrence that may increase the heterogeneity of tumors.

Combined examination of p27Kip1, p21Waf1/Cip1 and p53 expression allows precise estimation of prognosis in patients with gastric carcinoma Aims: In order to estimate the prognostic values of p27Kip1, p21Waf1/Cip1, and p53, alone and in combination, we investigated immunohistochemically the expression of p27Kip1, p21Waf1/Cip1, and p53 proteins in gastric carcinomas. Methods and results: The expression of p27Kip1, p21Waf1/Cip1, and p53 was immunohistochemically examined in 140 gastric carcinomas. Positive expression of p27Kip1 and p21Waf1/Cip1 correlated significantly with a favourable prognosis (P

To identify amplified oncogenes involved in hepatocellular carcinomas (HCC), we applied a genomic DNA microarray spotted with 57 oncogenes to 20 HCCs. Aberrations in DNA copy number also were analyzed by comparative genomic hybridization (CGH) using an aliquot of DNA samples. In 5 of 20 HCCs, only 6 oncogenes (CCND1, FGF3/FGF4, SAS/CDK4, TERC, MET, and MYC) were amplified, whereas in the remaining 15 tumors no oncogenes were amplified. A comparison of DNA microarray and conventional CGH analyses showed that, although 5 of 6 amplified oncogenes shown by microarray were located in chromosomal regions shown by CGH to have increased DNA copy numbers, not all genes located in such chromosomal regions were affected. One of the amplified oncogenes (SAS/CDK4) was found in a chromosomal region that was undetected by CGH. We, therefore, conclude that amplification of the oncogenes examined in this series is not directly implicated in hepatocellular carcinogenesis.

Biological characteristics of malignant tumors including head and neck squamous cell carcinoma (HNSCC) are greatly affected by genetic alterations. However, the relationship between chromosomal aberrations and pathologic tumor stage in HNSCC has not been elucidated. In 32 patients, DNA sequence copy number aberrations (DSCNA) were surveyed by comparative genomic hybridization (CGH) combined with a microdissection method. The average number of DSCNA was 15.3 per tumor and increased with tumor stage (P

We have analyzed DNA sequence copy number aberrations (DSCNAs) and DNA ploidy by using comparative genomic hybridization and laser scanning cytometer in gastric carcinomas (GCs) to elucidate the genomic aberrations in relation to clinicopathological parameters. Thirty-two out of 33 cases showed one or more DSCNAs with a mean number of 11.7 per tumor. High-level gains were detected at 2p, 3q, 6p, 7p, 7q, 8q, 12p, 13q, 19q, and 20q. Frequency of gross genomic abnormalities and chromosome regions that have genomic aberrations were similar in both intestinal- and diffuse-type GCs, except aberrations at 8p, 9p, 12q, and 20q. The overall number of DSCNAs was significantly greater in DNA aneuploid tumors than that in DNA diploid tumors. We detected genomic aberrations characterized by histological subtype, tumor location, and DNA ploidy status: gain of 20q and losses of 8p and 9p in intestinal-type GCs, gains of 8p and 12q in diffuse-type GCs, gain of 20q in the lower third GCs, and loss of 5q, 9p, 10q, 16q, and 18q in DNA aneuploid GCs. Furthermore, 5q loss is associated with DNA aneuploidy (P = 0.0001) or the total number of losses (P = 0.001), gain + losses (P = 0.004), and high-level gains (P = 0.001) in GCs. Among these loci, chromosome 8p was unique. Gain of 8p was more common in diffuse-type GC, whereas loss of 8p was more frequently detected in intestinal-type GC. In conclusion, we describe chromosomal regions of 5q, 8p, and 20q, which are of interest for further investigation of GCs.

BACKGROUND Low expression of p27kip1, a dominant cyclin-dependent kinase inhibitor involved in G1-S transition of the cell cycle, recently has been reported to be associated with aggressive tumor growth. It has been shown that active cell proliferation alludes to poor prognosis in patients with synovial sarcoma. However, to the authors' knowledge, little is known about the clinicopathologic significance of p27kip1 in synovial sarcoma. METHODS p27kip1 expression was examined immunohistochemically in 55 cases of primary synovial sarcoma, and the relations between p27kip1 expression and several cell proliferation markers, i.e., mitotic index (MI), Ki-67 labeling index (Ki-67 LI), and clinicopathologic parameters related to poor prognosis, were determined. Univariate and multivariate survival analyses were performed to evaluate the prognostic significance of p27kip1 expression in synovial sarcomas. RESULTS p27kip1 labeling index (p27kip1 LI) correlated inversely with MI (r = −0.44, P = 0.0007) and Ki-67 LI (r = −0.63, P < 0.0001). Of the clinicopathologic parameters examined, tumor necrosis (P = 0.019) and American Joint Committee on Cancer (AJCC) stage (P = 0.021) correlated significantly with p27kip1 LI. Survival analysis showed that p27kip1 LI was an independent prognostic factor for overall survival in patients with synovial sarcoma (P = 0.0031). CONCLUSIONS The study results suggested that low expression of p27kip1 may be useful as a marker of poor-prognosis synovial sarcoma. Cancer 2001;91:1005–12. © 2001 American Cancer Society.