Fumitaka Numa

Comparative genomic hybridization detects genetic alterations during early stages of cervical cancer progression.

Invasive cervical carcinoma is thought to arise from cervical intraepithelial neoplasm (CIN). Genetic changes that occur during progression of CIN to cervical carcinoma are poorly understood, although they appear to be directly involved in this process. We used comparative genomic hybridization (CGH) with precise microdissection and degenerate oligonucleotide primed‐polymerase chain reaction (DOP‐PCR) to detect genetic alterations in normal epithelial, CIN, and invasive carcinoma tissues colocalized in tumors from 18 patients with squamous cell carcinoma of the uterine cervix. Gains on chromosome 1 and on 3q and losses on 2q, 3p, 4, 6p, 11q, and 17p were frequent alterations found in CIN and invasive carcinoma lesions. Interestingly, several of these genetic changes were observed in preinvasive carcinoma lesions. The frequency and average number of genetic alterations corresponded directly to the extent to which the cervical carcinoma had progressed. Frequent alterations were found in more than 90% of CIN III lesions. Gains on 3q and losses on 11q were the most prevalent genetic alterations found in association with uterine cervix carcinogenesis. The common regions of alteration were 3q26.1‐q28 and 11q23‐qter. The majority of tumor samples showed variability in genetic alterations across lesion types within a single specimen.

Genetic Aberrations Detected by Comparative Genomic Hybridization in Ovarian Clear Cell Adenocarcinomas

Genetic abnormalities were detected by comparative genomic hybridization (CGH) in 12 ovarian clear cell adenocarcinomas. DNA sequence copy number abnormalities (CNAs) occurring in more than 20% of the cancers included increased copy numbers of 8q11-q13, 8q21-q22, 8q23, 8q24-qter, 17q25-qter, 20q13-qter and 21q22-qter and reduced copy numbers of 19p. Increases in copy numbers of 8q11-q13, 8q21-q22, 8q23 and 8q24-qter occurred more frequently in disease-free patients than in recurrent/non-surviving patients (p < 0.05). However, increases in copy numbers of 17q25-qter and 20q13-qter occurred more frequently in recurrent/non-surviving patients than in disease-free patients (p < 0.05). Furthermore, increases in copy numbers of 17q25-qter and 20q13-qter occurred together (p < 0.05). Additionally, there were negative correlations between increases in copy numbers of 8q21-q22 and 17q25-qter, and between 8q21-q22 and 20q13-qter (p < 0.05). It appears that ovarian clear cell adenocarcinomas can be classified into two subtypes, one being cancer with an increase in copy numbers of 8q and the other being cancer with increases in copy numbers of 17q25-qter and 20q13-qter.

Squamous cell carcinoma antigen suppresses radiation-induced cell death

Previous study has demonstrated that squamous cell carcinoma antigen (SCCA) 1 attenuates apoptosis induced by TNFα, NK cell or anticancer drug. In this study, we have examined the effect of SCCA2, which is highly homologous to SCCA1, but has different target specificity, against radiation-induced apoptosis, together with that of SCCA1. We demonstrated that cell death induced by radiation treatment was remarkably suppressed not only in SCCA1 cDNA-transfected cells, but also in SCCA2 cDNA-transfected cells. In these transfectants, caspase 3 activity and the expression of activated caspase 9 after radiation treatment were suppressed. Furthermore, the expression level of phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) was suppressed compared to that of the control cells. The expression level of upstream stimulator of p38 MAPK, phosphorylated MKK3/MKK6, was also suppressed in the radiation-treated cells. Thus, both SCCA1 and SCCA2 may contribute to survival of the squamous cells from radiation-induced apoptosis by regulating p38 MAPK pathway.

Genetic aberrations detected by comparative genomic hybridization predict outcome in patients with endometrioid carcinoma.

Endometrial cancer progression is determined by a complex pattern of multiple genetic aberrations, but how these aberrations affect prognosis is unknown. In this study, we undertook a genome‐wide screening to detect genetic changes by comparative genomic hybridization (CGH) in 51 tumors from patients with primary endometrioid carcinoma of the uterine corpus. The observed genetic changes were subsequently correlated with the progression of the disease and the clinical outcome in each case. The average number of genetic aberrations (copy number gains and losses) was significantly greater in non‐surviving patients than in disease‐free patients (12.6 vs. 2.7, P < 0.0001). According to multivariate analysis, lymph node metastasis (P = 0.015), cervical involvement (P = 0.007) and one or more copy number losses at 9q32–q34, 11q23, or Xq12–q24 (P = 0.023) were significantly predictive of death from the disease. Interestingly, lymph node metastasis was significantly associated with copy number gains at 8q22–q23 and 8q24–qter (P = 0.003 and P = 0.025, respectively). Moreover, cervical involvement was also correlated significantly not only with gains of 8q22–q23 and 8q24–qter but also with loss of 11q23 (P = 0.04, 0.0003, and P = 0.009, respectively). These results suggest that analysis of genetic changes may help predict clinical outcome and the presence of metastatic disease as well as assist in therapeutic decision making for patients with endometrioid carcinoma. Genes Chromosomes Cancer 29:75–82, 2000.

Aneuploidy Predicts Outcome in Patients with Endometrial Carcinoma and Is Related to Lack of CDH13 Hypermethylation

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.

Altered Proliferative and Metastatic Potential Associated with Increased Expression of Syndecan-1

The process of metastasis involves a series of sequential steps in which malignant cells are released from the primary tumor and metastasize to distant sites. Syndecan-1 is a cell surface proteoglycan that mediates cell adhesion and undergoes changes upon cell transformation of some cells that may contribute to the process of metastasis. To investigate the possible role of syndecan-1 in cell proliferation and metastatic potential, we employed a highly metastatic cell line (KLN 205) derived from mouse lung squamous cell carcinoma that expressed moderate amounts of syndecan-1. At first, endogenous syndecan-1 production was inhibited by an antisense oligodeoxynucleotides (ODNs). Since antisense ODNs of syndecan-1 inhibited cell growth, we established stable transfectants of syndecan-1 in this cell line to examine a proliferative advantage with the level of syndecan-1 expression. Overexpresser cells grew at a significantly faster rate than the vector-transfected control and showed greater incidence of tumor formation when injected subcutaneously into nude mice. Surprisingly, overexpresser cells enhanced pulmonary metastasis when injected intravenously. These results indicate that syndecan-1 expression plays a role in the control of cell proliferation and suggest that syndecan-1 expression may be involved in facilitating distant metastasis of tumor cells once they managed to enter the bloodstream (after intravasation steps).

Specific Detection and Quantitation of SCC Antigen 1 and SCC Antigen 2 mRNAs by Fluorescence-Based Asymmetric Semi-Nested Reverse Transcription PCR

Squamous cell carcinoma antigen (SCCA) is expressed in normal squamous epithelia and malignant squamous cell tissues. The serum level of SCCA has been used to evaluate treatment efficacy, clinical course of disease, and recurrence. SCCA is produced by at least two genes (SCCA1 and SCCA2); both of them have been located on chromosome 18q21.3. It has been difficult to examine the expression levels of SCCA1 and SCCA2 mRNAs separately because of their high homology at nucleotide level. In the present study, asymmetric semi-nested reverse transcription PCR, based on the principle of fluorescence energy transfer, enabled to quantitate the copy numbers of both SCCA1 and SCCA2 mRNAs. Using this method, the expression levels of these mRNAs were evaluated in normal and malignant squamous tissues. The copy number of SCCA2 mRNA was higher in malignant tissues than in normal tissues, while those of SCCA1 mRNA did not significantly differ between normal and malignant tissues. These data indicate that specific quantitation of the expression level of SCCA2 mRNA may be useful for the diagnosis and management of patients with squamous cell carcinoma.

Pelvic exenteration for the treatment of gynecological malignancies

Twenty-three patients undergoing pelvic exenteration for primary and recurrent gynecological malignancies from 1976 to 1994 are reported. Fifteen patients underwent total pelvic exenteration, 3 underwent anterior exenteration, and 5 underwent a posterior procedure. Eight patients had exenteration as their primary treatment (primary group), and 15 underwent exenteration as secondary treatment (recurrent group). In the primary group, two patients developed recurrence and died of it at 6 and 20 months after operation. Five patients are still being followed up and are alive without disease. Four of these 5 patients have survived more than 5 years. In the recurrent group, 12 patients were followed up and three died of complications during the early years. Seven patients died of cancer with the mean survival time of 16.6 months. The mean age, average operating time, and mean blood loss in the primary and recurrent groups were 57 vs. 53 years, 8 hours and 20 min vs. 8 hours and 10 min, and 4,120 vs. 4,190 ml, respectively. The overall cumulative 5-year survival rate was 34.7%, being 68.6% in the primary group and 16.7% in the recurrent group. It is noteworthy that the 5-year survival rate was 51.3% in the patients who had surgical margins free of disease. In conclusion, pelvic exenteration should be considered an acceptable therapeutic option when appropriately selected.

Phase II study of intraperitoneal carboplatin with intravenous paclitaxel in patients with suboptimal residual epithelial ovarian or primary peritoneal cancer: a Sankai Gynecology Cancer Study Group Study.

Purpose: To assess the antitumor efficacy and safety of 2 treatment modalities: intraperitoneal carboplatin combined with intravenous (IV) paclitaxel. Patients and Methods: Eligible patients were those with epithelial ovarian carcinoma or primary peritoneal carcinoma stages II to IV who underwent initial surgery and had a residual tumor size of 2 cm or larger. Patients received IV paclitaxel 175 mg/m2 followed by intraperitoneal carboplatin AUC6. The primary end point was a response. Secondary end points were toxicity, progression-free survival, and overall survival. Results: Twenty-six patients were enrolled, and 24 patients were eligible for assessment. The response rate was 83.3% (95% CI, 62.6%-95.3%; Table 4). The median progression-free survival was 25 months. The median overall survival had not been reached. Incidences of grade (G) 3/4 hematological toxicities were absolute neutrophil count, 96%; hemoglobin, 29%; and thrombocytopenia, 16%. Nonhematological toxicities included G2 liver function, 4%; G3 sensory neuropathy, 8%; and G3 myalgia and arthralgia, 4%. Conclusions: Intraperitoneal administration of carboplatin combined with IV paclitaxel was well tolerated and showed satisfactory response in the patients with bulky residual tumor. Large-scale phase III trial comparing with IV carboplatin is warranted in this patient population.

Tumor Necrosis Factor-α Stimulates the Production of Squamous Cell Carcinoma Antigen in Normal Squamous Cells

Squamous cell carcinoma (SCC) antigen, a tumor marker of squamous cell carcinoma, is also increased in several nonmalignant skin lesions, e.g. pemphigus. The aim of the present investigation was to determine if tumor necrosis factor-alpha (TNF-alpha), one of the important environmental factors, stimulated the production of SCC antigen in the normal squamous cells. The exposure of normal human epidermal keratinocytes to TNF-alpha (100 IU/ml) for 72 h greatly increased the SCC antigen production. The stimulatory effect of TNF-alpha (1,000 IU/ml) on the production of SCC antigen was also observed in the normal squamous epithelium tissue. These results would be helpful for understanding the increase of SCC antigen in several nonmalignant skin disorders.