Genetic analysis of benign, low-grade, and high-grade ovarian tumors.

Genetic abnormalities were assessed in 56 benign, low-, and high-grade ovarian tumors using comparative genomic hybridization (CGH) and analysis of loss of heterozygosity (LOH). In addition, 95 epithelial tumors were analyzed for microsatellite repeat instability. DNA sequence copy number abnormalities (CNAs) were not detected in the benign tumors, and more were detected in high-grade than in low-grade cancers. Almost no microsatellite repeat instability was detected in these cancers. CNAs occurring in more than 30% of the cancers included increased copy number on 3q25-26 and 8q24 and reduced copy number on 16q and 17pter-q21. Another 14 CNAs occurred in more than 20% of the cancers. Increased copy number at 3q25-26 and 20q13 was the most frequent CNA in low-grade tumors, and increased copy number at 8q24 occurred preferentially in high-grade tumors. The presence of a large number of CNAs per tumor was significantly correlated with reduced patient survival duration. Reduced copy number on 17pter-q21 was most strongly associated with accumulation of a large number of CNAs. The overall concordance between LOH and reduced copy number detected by CGH was 84%, but only 31% of the LOH was associated with reduced copy number detected using CGH.

Expression of the chemokine CXCL14 and cetuximab-dependent tumour suppression in head and neck squamous cell carcinoma.

Cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR), has been successfully used to treat some patients with colorectal cancer and those with head and neck squamous cell carcinoma (HNSCC). For the effective treatment, it is essential to first identify cetuximab-responsive patients. The level of EGFR expression and/or the presence of mutations in signalling molecules downstream of the EGFR pathway have been reported to be determining factors for cetuximab responsiveness in colorectal cancer patients; however, limited data have been reported for HNSCC patients. We previously reported that the chemokine CXCL14 exhibits tumour-suppressive effects against xenografted HNSCC cells, which may be classified into two groups, CXCL14-expressing and non-expressing cells under serum-starved culture conditions. Here we employed CXCL14-expressing HSC-3 cells and CXCL14-non-expressing YCU-H891 cells as representatives of the two groups and compared their responses to cetuximab and their CXCL14 expression under various conditions. The growth of xenografted tumours initiated by HSC-3 cells, which expressed CXCL14 in vivo and in vitro, was suppressed by the injection of cetuximab into tumour-bearing mice; however, neither the expression of the chemokine nor the cetuximab-dependent suppression of xenograft tumour growth was observed for YCU-H891 cells. Both types of cells expressed EGFR and neither type harboured mutations in signalling molecules downstream of EGFR that have been reported in cetuximab-resistant colon cancer patients. The inhibition of the extracellular signal-regulated kinase (ERK) signalling increased the levels of CXCL14 messenger RNA (mRNA) in HSC-3 cells, but not in YCU-H891 cells. We also observed that the CXCL14 promoter region in YCU-H891 cells was hypermethylated, and that demethylation of the promoter by treatment with 5-aza-2'-deoxycytidine restored CXCL14 mRNA expression and in vivo cetuximab-mediated tumour growth suppression. Finally, we observed in vivo tumour growth suppression when YCU-H891 cells were engineered to express CXCL14 ectopically in the presence of doxycycline. These results indicate that CXCL14 expression may be a good predictive biomarker for cetuximab-dependent tumour suppression.

Myelodysplastic syndrome with Philadelphia-like chromosome without bcr rearrangement.

The case of a 62-year-old Japanese male with a myelodysplastic syndrome and a Philadelphia (Ph)-like chromosome, which probably involved bands 11q23 and 22q11, is presented. Cytogenetic analysis of bone marrow cells revealed a Ph chromosome as well as -5, -7, +8, +11, -16, and an extra Ph. Some of the cells had a normal karyotype. Molecular analysis using breakpoint cluster region probes (5' bcr and 3' bcr) did not detect a rearrangement within the bcr DNA sequences, indicating that the breakpoint at 22q11 occurred outside the bcr. Furthermore, the bone marrow cells from this patient did not express an 8.5-kb c-abl mRNA. Thus, the Ph chromosome in this case differs from that of Ph-positive chronic myelogenous leukemia, and the present case suggests that we should retain the term of "Ph-like chromosome" in such cases.

ETS1 gene in myelodysplastic syndrome with chromosome change at 11q23.

We examined the c-ets1 gene (located at 11q23) in two myelodysplastic syndrome (MDS) patients displaying a chromosome change at band 11q23 to ascertain any association between this oncogene and the chromosome change. Besides the chromosome change at 11q23, the two MDS patients also showed other numerical and structural changes. Bone marrow cells from the first case showed a translocation between chromosomes 11 and 22, t(?;11;22)(?;p11 or q11----q23;q11), resulting in a Ph-like chromosome. Neither a transposition nor a rearrangement of the c-ets1 gene was detected. Bone marrow cells of the second case showed unidentified chromosomal material attached to bands 11q23 and 6q27. Southern blot study, however, revealed that these cells carried an amplified c-ets1 gene associated with the chromosomal rearrangement. In both MDS cases studied, the amount of c-ets1 related message was the same whether amplification of the c-ets1 gene was present or not, and the level of the c-ets1 gene in MDS cells was very low.

[Establishment and characterization of endometrial undifferentiated carcinoma cell line (TMCC-2.U)].

We established new cell line designed TMCC-2.U, which suggested transformation to undifferentiated carcinoma, derived from endometrial clear cell carcinoma cell line (TMCC-2). The monolayer culture cell showed a pavement arrangement and spindle like shape. A rough-endoplasmic reticulum, mitochondria etc. are well developed. But cytoplasmic endocrine granulosa were so poorly, it suggests functional developments are poor. The TMCC-2.U cells were transplanted to nude mice which showed no typical pattern suggested undifferentiated carcinoma. Their chromosome number varied and the mode is 78. Marker chromosome were found frequency. Growth pattern and production of tumor marker are clearly differentiate from TMCC-2. As mensioned above, TMCC-2.U cell line will be very valuable in basic research on mechanism of transformation and effects of patient's serum on hystogenesity.

Analysis of gene expression profiles associated with cisplatin resistance in human ovarian cancer cell lines and tissues using cDNA microarray.

Gene expression profiles were analyzed by using cDNA microarray for a cisplatin-sensitive cell line (KF), and three- and thirty-fold cisplatin-resistant ovarian cancer cell lines (KFr and KFrP200) both showing no p53 mutation within exon 5, 6, 7, 8 and no pglycoprotein overexpression. Expression of GST-pi mRNA increased as the level of resistance to cisplatin became high. Microarray analysis revealed that DNA repair associated genes, i.e., XRCC5, XRCC6, ERCC5, hMLH1 were over-expressed in three-fold cisplatin-resistant cell line, KFr as compared to cisplatin-sensitive parental cell line, KF. Apoptosis inhibitors, i.e., IGFR type I and II were over-expressed, and apoptosis inducer, i.e., caspase 3 and BAK were underexpressed in highly cisplatin-resistant cell line, KFrP200 as compared to KFr. As for clinical cases, cDNA microarray was used to compare gene expression profiles directly between two groups, i.e., the chemotherapy (CAP) sensitive group (n = 2) and the resistant group (n = 2). Six genes such as beta tubulin, high-mobility group (nonhistone chromosomal) protein 1, connective tissue growth factor, insulin-like growth factor binding protein 2, alpha tubulin, and RAS-related gene were overexpressed in CAP therapy resistance group, whereas seven genes such as CD9 antigen, alpha-2-macroglobulin, caveolin 2, interleukin 1 receptor antagonist, Rho GTPase activating protein 1, reticulon 3, cyclin-dependent kinase 10, keratin 7 were underexpressed in CAP therapy resistance group. By increasing clinical case number and gene number of microarray to be used in the analysis of expression profile of gene cluster affecting anticancer drug resistance and sensitivity of the ovarian cancer, it would be possible to apply microarray analysis to personalization of chemotherapy such as selection of effective chemotherapy protocol and prediction of therapeutic effect in the near future.

Photodynamic diagnosis in respiratory tract malignancy using an excimer dye laser system.

Equipment has been developed for the early-stage diagnosis and treatment of cancer using an excimer dye laser. The excimer laser beam is obtained by exciting XeCl. A 405 nm beam tuned by DPS dye is used for tumour localization and a 630 nm beam obtained with a rhodamine B dye is used for treatment. The equipment was applied clinically on the basis of extensive experimental research. Effectiveness for cancer localization was examined in 11 cases: four were early stage (three lung cancer and one vocal cord cancer), four were stage I, two were stage III and one was stage IV. All cases were squamous cell carcinoma except for one case of adenocarcinoma. Fluorescence was recognized in all lesions and the equipment was effective for localization.

Staining for dipeptidyl aminopeptidase IV activity in nodular thyroid diseases.

OBJECTIVE: To determine the effectiveness of staining for dipeptidyl aminopeptidase IV (DAP IV) activity in thyroid nodular diseases. STUDY DESIGN: Imprint smears were made in 76 cases of papillary carcinoma, 10 of follicular carcinoma, 32 of follicular adenoma and 48 of adenomatous goiter after surgery, and staining for DAP IV activity was performed in all cases. RESULTS: All papillary carcinomas stained positively, and most adenomatous goiter cases were negative. Follicular carcinoma and follicular adenoma cases exhibited various degrees of positivity, though the former tended to be stained more than the latter. Of the total 166 cases of thyroid nodules, 53 showed negative staining; of those 53, all except 1 were benign. Follicular carcinoma cases that had vascular invasion tended to show a high DAP IV staining pattern, but no statistically significant difference between it and follicular carcinoma that did not show vascular invasion was found. CONCLUSION: DAP IV activity staining might reveal malignant potential and could have some value in the preoperative diagnosis between thyroid nodular diseases. The DAP IV method is a reliable indicator of benign disease if negative results are obtained.

[A case of both lung giant bullae treated by thoracoscopic bilateral resection].

We experienced a case of multiple bilateral giant bullae of the lungs and treated by thoracoscopic bilateral resection. A 46-year-old male was admitted to our hospital on the diagnosis of bilateral giant bullae of the lungs. Chest CT scan and lung perfusion scintigraphy showed giant bullae at the apex of both lungs and at the left lower lobe. The border of the bullae was relatively clear, and the other lung was almost normal although it was compressed by the bullae. We evaluated this case and found that it was a good indication for a thoracoscopic bilateral resection. The patient was positioned on his back, and a successful thoracoscopic bilateral resection was performed. The patient was discharged from the hospital on the 10th day postoperatively. Chest CT scan, lung perfusion scintigraphy and respiratory function test performed 2 months postoperatively, showed remarkable improvement. We would recommend this surgical technique in patients with similar diagnosis.

[A case of vivax malaria with thrombocytopenia suggesting immunological mechanisms].

Recently, it has been shown that the thrombocytopenia that complicates some malarial infections is caused by immune mechanisms. We report a case of malaria associated with thrombocytopenia and increased platelet-associated IgG (PAIgG). In this case, anti-malarial therapy reduced the level of PAIgG to normal levels in association with normalization of the platelet count. This case suggests the immunological mechanisms of thrombocytopenia in malaria.

[Refractory idiopathic thrombocytopenic purpura which responded to protein A column].

Currently, there is no satisfactory therapy available for patients with chronic idiopathic thrombocytopenic purpura (ITP) who are unresponsive to conventional therapeutic modalities. In this report, we describe a patient with chronic refractory ITP treated with immobilized protein A in an extracorporeal system. The patient was a 74 years old male diagnosed as ITP in 1980. Despite steroid therapy, the disease progressed and the patient exhibited ecchymosis and gum bleeding which was unresponsive to intravenous gammaglobulin therapy. Severe gastrointestinal bleeding was evident and administration of danazol was discontinued due to liver dysfunction. The patient was treated with extracorporeal protein A immunoabsorption. The patient's whole blood (200-300 ml per treatment) was separated into plasma and cellular components and the plasma was passed through an immunoabsorption column containing 200 mg of covalently bound protein A. The treated plasma and cellular components were returned to the patient. After 4 immunoabsorption treatments, the platelet counts elevated and there was evidence of improvement in gastrointestinal bleeding. This report indicates that protein A immunoabsorption therapy should be considered in patients with chronic refractory ITP.

[Resection of recurrent lung cancer].

The resected recurrent lung cancer cases were evaluated retrospectively. Out of 1,060 cases who received an operation due to primary lung cancer, 21 (1.8%) had recurrent lung cancer which were resected subsequently. Stage I adenocarcinoma cases were found to be most frequent at the first operation (76%). Out of 21 cases, 7 received completion pneumonectomy, 5 had lobectomy and 8 had limited operation, respectively. The 5-year survival rate after the second operation was 36.6% in all the cases. There was no statistical difference in survival rate between the lobectomy group (including completion pneumonectomy) and the limited operation group. There was statistical difference in survival rate between cases who received a second operation in a time span of 2 or more years after the first operation and the cases who received it in less than 2 years. Good prognosis can be expected after the resection of recurrent lung cancer, but further analysis would be required in evaluation of respiratory function as well as biological malignancy of the tumor.

[A case of mandibular gingival cancer T4N0M0 which markedly responded to a combined therapy of nedaplatin with 5-fluorouracil].

We recently experienced a case of mandibular gingival cancer T4N0M0 which markedly responded to a combination therapy of nedaplatin (254-S) with 5-fluorouracil (5-FU). The patient was a 68-year-old male who visited our department with the main complaint of ulceration in the left mandibular gingiva. Biopsy revealed a moderately differentiated squamous cell carcinoma which extended to the mandible, mandibular gingiva, buccal mucosa, half tongue and oral floor on the left side of the face. As a neoadjuvant chemotherapy (NAC), 254-S at a dose of 100 mg/m2 was intravenously administered on day 1, while 5-FU at a dose of 700 mg/m2/day was intravenously administered from day 1 to 5 in succession. Hydration (2,000 ml/day) was performed from day 1 to 3. Adverse reactions observed included thrombocytopenia, anorexia, nausea, vomiting, stomatitis and SIADH, but no sign of renal dysfunction was observed. The clinical outcome was evaluated as CR. Surgery was performed later. Pathological examination of the extracted tissues showed tumor cells in the tongue only, indicating an excellent effect of this combination therapy of 254-S and 5-FU.

[Qualitative improvement of the surgical treatment of cancer using laser equipment--surgical technic after photodynamic therapy].

Nineteen patients with lung cancer were treated by combined preoperative photodynamic therapy (PDT) and surgery. Preoperative photodynamic therapy was performed for the purpose of either reducing the extent of resection or increasing operability. Clinically, nine patients had stage I disease, one had stage II, eight had stage III and one had stage IV. There were two cases of tracheal superficial invasion from primary lesions, three cases of intrabronchial polypoid tumor or superficial invasion of the carina by primary lesions, eleven cases of polypoid tumor or superficial invasion of the main bronchus, and three cases of double primary lesions. Argon dye laser was used in this study. Preoperative PDT was performed 48 to 72 hours after intravenous injection of hematoporphyrin derivative (HpD). Therapeutic conditions were 60 to 600 joules/cm2 for the superficial invasive areas and an additional 200 to 800 m W for 8 to 15 minutes for polypoid lesions. Surgical resection was performed 1 to 9 weeks after PDT. The initial purpose of PDT was achieved in 15 of the 19 patients treated. In five of six originally inoperable cases, conversion to an operable status was achieved. Thirteen patients were originally candidates for pneumonectomy, and it became possible to reduce the extent of resection to lobectomy in ten of them. This study suggests that PDT may have an important role in combination with surgery and other modalities in advanced lung cancers.

[Photodynamic therapy (PDT) for early cervical cancer].

The incidence of carcinoma in situ (CIS) and dysplasia of the uterine cervix has been increasing among young women in recent years. Most of these patients want to preserve their fertility. Also, to accommodate high-risk patients with complications, elderly patients, and those who refuse surgery, we perform PDT as a method to preserve fertility. The technique required for PDT is relatively simple, and can be performed without anesthesia, since it causes no pain or bleeding. PDT, with the use of Excimer Dye Laser (EDL), a type of low pulse laser, has a considerably higher degree of tissue penetration, even compared to PDT using Argon Dye Laser (ADL). Also, PDT using EDL can manage glandular involvement of CIN, and its special feature of selective destruction of malignant cells with almost no effect on normal tissues is noteworthy. Beginning in 1995, PDT using YAG-OPO Laser with a variable laser wavelength has been performed. PDT is performed 48 hours after intravenous injection of 1.5 mg/kg to 2 mg/kg photosensitizer Porfimer sodium (PHE) when the difference in density of PHE becomes greatest between malignant cells and normal tissue. The most advanced features of our method compared to conventional radiation which uses cut fiber are: First, by using colposcope with an optical path for the laser, it is possible to show a 10 mm circular spot at the focus of observation. With this method, cervical lesions can be observed and checked while receiving stable and precise photoradiation by using colposcope through direct observation. Second, for cervical canal treatment, by using a cervical probe to administer photoradiation in the forward direction in the cervical canal and to the side walls, 70% of the laser light is scattered to the side walls, so that all of the cervical canal can be radiated. Also, the cervical canal probe used to administer photoradiation, by inserting 2 cm to 3 cm depending on the conditions of the cervical canal and withdrawing the probe 1 mm, can be performed precisely and promptly by using the cervical probe manipulator feature of the colposcope. At the present time, studies using the PDT method have been conducted on 56 patients (39 CIS and 17 dysplasia patients). Out of these 56 patients, there were 54 CR (96.4%), only one NC, and one PR with very limited remnants but most of the lesions had disappeared. The NC was highly suspected to be invasive carcinoma and the PR was CIS. In the CIS case, some remnant was evident at the end of the cervical canal, and PDT was administered again. After this treatment, it became CR. This was 10 months ago, and no abnormal condition has been reported since. The first CR case was reported 6 years ago among the 56 cases studied, and no recurrence has been observed to date. Five patients became pregnant after the treatment. Four had normal deliveries and one had a cesarean section. PDT's side effect is similar to symptoms of sunburn such as minor skin irritation due to sensitive reaction to sunlight. Normally, it can be relieved by applying carmine lotion, and even cases that required treatment were cured completely within a few days after applying steroid ointment. Before hospitalization, if the patient gets a sunburn from being outside, the sensitive reaction to laser light is almost nonexistent. Thus, we advise patients to get some exposure to the sun before being hospitalized. Also, in cases where strict shading time is observed, side effects are not apparent at all, and no abnormal findings are recognized in the blood and urine due to using PHE. With almost no side effects, bleeding or pain, and with certain improvements in administration methods, a better choice of photosensitizer which would shorten the shading time, PDT is considered to be the best therapy for treating CIS and dysplasia while preserving fertility.

[Cancer-associated gene abnormalities and chemosensitivity].

One of the most important clinical issues in cancer chemotherapy is the presence of intrinsic resistance or the appearance of acquired resistance against chemotherapy. As for intrinsic resistance, we had to perform direct chemo-sensitivity testing, or had to rely on the knowledge empirically acquired from randomized clinical trials. However, molecular or genetic markers associated with chemo-sensitivity have been reported recently. For example, inactivation of p53 or GML gene has been reported to be associated with chemo-resistance. Overexpression of topo-isomerase I has been reported to be associated with chemo-sensitivity to Topo I inhibitor. Overexpression of Thymidine Phosphorylase has been found to be associated with chemo-sensitivity to prodrug of 5-FU. By checking the status of such chemo-sensitivity markers prior to chemotherapy, it would be possible to predict the chemotherapeutic effect and even the necessity of the chemotherapy in the near future. In this article, we review the chemo-sensitivity markers reported so far, and methodology contributing to the discovery of new chemo-sensitivity markers. As a clinical study, 11 cases of ovarian cancer with high sensitivity to cisplatin-based chemotherapy and 29 cases of ovarian cancer with chemoresistance were analyzed by Comparative Genomic Hybridization (CGH). Copy number decrease in Xp, and copy number increase in 19q were observed in 13, 12 out of 29 resistant cases (45, 41%) and zero, 1 out of 11 sensitive cases (0, 9%), suggesting that -Xp and +19q were likely to be a genetic event associated with intrinsic drug-resistance (p = 0.006, 0.05, respectively). This effort should contribute to the discovery of new chemo-sensitivity and resistance markers.

[Prognostic factors in patients with invasive differentiated thyroid carcinoma who underwent palliative resection].

In order to determine surgical strategy for locally advanced thyroid carcinoma, a multivariate analysis of prognostic factors was carried out with 70 patients of locally advanced thyroid carcinoma who underwent palliative surgery. Multivariate analysis was conducted by Cox's proportional hazard model. The patients have been followed up from 5 to 26 years. We have demonstrated the influence of age, sex, distant metastasis, pathological findings, numbers of adjacent organs, infiltrated, extent of resection, external irradiation and 131I therapy on survival. The result of this study showed that age had an impact on survival. The survival rate of the patients under 40 years was much better than that of the patients 40 years and over. The result implies that aggressive surgery and postoperative adjuvant therapy should be considered in the old patients, but not always be indication in the young patients.

[CGH (comparative genomic hybridization)].

Comparative Genomic Hybridization (CGH) is a powerful new method which allows genome-wide mapping of regions with DNA sequence copy number changes (both increases and decreases) in a single experiment without previous knowledge of the locations of the regions of abnormality. CGH is based on in situ hybridization of differentially labeled total genomic tumor DNA and normal DNA to normal human metaphase chromosomes. After hybridization copy number variations among the sequences in the tumor DNA are detected by measuring the tumor/normal fluorescence intensity ratio for each locus in the target chromosomes. Many previously unknown chromosomal regions with relative copy number changes have been detected in various tumors by CGH. Some changes have been identified as genetic markers associated with biological and clinico-pathological characteristics (i.e., histopathological grade, and clinical outcome). We review the published CGH articles and discuss briefly on current progress in CGH analysis to ovarian and uterine cervical cancer in our laboratory.