De novo uterine sarcoma with good response to neo-adjuvant chemotherapy.

We report here the extremely rare case of a 28-year-old woman with advanced stage uterine sarcoma arising soon after a cesarean section. She underwent an abdominal cesarean section because of a breech presentation. At the time of the procedure, there were no abnormal findings such as leiomyoma of the uterus in the abdominal cavity. One year later, she was referred to our hospital because of a large abdominal tumor. Transabdominal power Doppler ultrasonography and magnetic resonance imaging (MRI) showed a large hypervascular tumor in the abdominal cavity. Her serum levels, for the two tumor markers carbohydrate antigen CA125 and LDH, were elevated, at 219 U/ml (< 35 U/ml) and 862 IU/l (115 U/ml-217 U/ml), respectively. On the basis of a diagnosis of malignant tumor of gynecological origin, exploratory laparotomy was performed, and through biopsy, the tumor was found to be advanced undifferentiated uterine sarcoma. She exhibited a good response to neoadjuvant chemotherapy consisting of cisplatin, epirubicin, and dimethyltriazenoimidazole carboxamide (DTIC) every 28 days, which was successfully followed by a hysterectomy.

Electrophoretic characterization of heat-stable squamous cell carcinoma antigen.

The aim of this study was to investigate the heat stability of squamous cell carcinoma (SCC) antigen, a tumor-associated serine proteinase inhibitor (serpin), in tumor tissue extract by electrophoretic methods. After heat treatment at 70 degrees C for 2 h, the tumor tissue extract showed a single main protein band of 45 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which reacted with a monoclonal antibody specific for SCC antigen. The heat-stable SCC antigen was separated by two-dimensional electrophoresis (2-DE) into four spots with pI 6.4-5.9 and Mr 44500-45 000 of SCC antigen-1. Furthermore, the SCC antigen-1 still showed its inhibitory activity against a cysteine proteinase, papain, by gelatin zymography. These results suggest that heat treatment of protein sample at 70 degrees C for 2 h may be a useful method for a partial purification of SCC antigen-1 which can inhibit lysosomal cysteine proteinases such as cathepsin L, S, and K.

Novel forms of squamous cell carcinoma antigen transcripts produced by alternative splicing.

Squamous cell carcinoma antigen (SCCA) is a member of the ovalbumin serine protease inhibitor family, and the serum level of SCCA is a tumor marker of squamous cell carcinoma. Reverse transcription (RT)-PCR of the squamous cell carcinoma cell line showed the existence of a 156 base shorter transcript compared with that of SCCA1 cDNA. By inverse PCR, we cloned the full length cDNA of this SCCA (SCCA1b). Sequence analysis of the complete 1541 bp SCCA1b cDNA showed that it coded for 338 amino acids and had no typical signal sequence in the NH(2) terminus. The cDNA was expressed in Escherichia coli and the product was detected using Western blotting with antibodies against SCCA. Furthermore, RT-PCR of the full coding region of SCCA2 cDNA from cancer tissue showed the existence of a 63 base short transcript (SCCA2b). A comparison of SCCA1b and SCCA2b cDNA with the SCCA1 and SCCA2 genes showed that these messages were derived from each gene by an alternative splicing mechanism.

Squamous cell carcinoma antigen suppresses radiation-induced cell death.

Previous study has demonstrated that squamous cell carcinoma antigen (SCCA) 1 attenuates apoptosis induced by TNF alpha, 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.

Suppression of a squamous cell carcinoma (SCC)-related serpin, SCC antigen, inhibits tumor growth with increased intratumor infiltration of natural killer cells.

Squamous cell carcinoma (SCC) antigen (SCCA), a member of the ovalbumin serine proteinase inhibitor family, serves as a circulating marker of squamous cell carcinoma (SC). One of the SCCAs, SCCA1, has been suggested to play a role in the attenuation of apoptosis in vitro and in the augmentation of tumor growth in vivo. In the present study, the infection of a SCC cell line (SKG IIIa) with recombinant retrovirus that expressed the antisense SCCA mRNA suppressed expression of SCCA in vitro. Local administration of this retrovirus into tumors by inoculation in nude mice suppressed tumor growth. Treatment of tumor tissue in vivo is also associated with increased numbers of apoptotic tumor cells and large mononuclear cells in the tumor. To test the possible role of SCCA in the infiltration of large mononuclear cells, we analyzed the effect of SCCA1 on migration of natural killer (NK) cells induced by monocyte-chemoattractant protein-1 in vitro. SCCA1 suppressed migration of NK cells completely, and this inhibitory effect was lost by mutation of the reactive site loop of SCCA1. These results suggest that antisense SCCA may suppress the growth of SCC in vivo not only by the augmentation of intracellular apoptosis but also by the increased infiltration of NK cells into the tumor.

Serum anti-p53 antibodies in uterine and ovarian cancer: association with dna sequence copy number abnormalities.

The aim of the present study was to evaluate the clinical significance of the serum anti-p53 antibody in patients with uterine and ovarian cancer. Some of the ovarian patients were also evaluated for overexpression of p53 by immunohistochemistry and for cytogenetic alterations by comparative genomic hybridization (CGH). Serum anti-p53 antibodies were determined by an enzyme immunoassay kit. The antibody was detected in 8/30 (27%) of ovarian cancers, in 12/86 (14%) cancers of the uterine cervix, in 5/41 (12%) cancers of the uterine body, and 0/9 (0%) healthy women. The overall survival rate in patients with ovarian cancer was significantly worse in patients with anti-p53 antibody positivity than that in patients with anti-p53-antibody-negative cancers using the log rank test (p = 0.017). There was a significant correlation between the presence of anti-p53 antibody and tissue overexpression of p53 in ovarian cancers. CGH analysis showed that the aberrations in DNA sequence copy number in ovarian cancers were significantly increased in anti-p53-antibody-positive cases compared to antip53-antibody-negative cases including increased copy number on 20q and reduced copy number on 5q and 13q. Although the exact relationship between the presence of serum anti-p53 antibody (specific humoral response) and cytogenetic alterations is still unknown, these findings suggest that the measurement of serum anti-p53 antibody may be useful for the assessment of genetic instability and tumor biological aggressiveness.

Nondenaturing two-dimensional electrophoretic analysis of loop-sheet polymerization of serpin, squamous cell carcinoma antigen-2.

Two homologous serine proteinase inhibitors (serpins), squamous cell carcinoma (SCC) antigen-1 and -2 were separated by nondenaturing two-dimensional electrophoresis combined with immunostaining to acquire further information on these proteins under physiological conditions. Polymers of SCC antigen-2 were detected in cytosolic extracts prepared from tumor tissues. The polymer formation of SCC antigen-2 was apparently decreased and the SCC antigen-2-synthetic peptide binary complexes were newly formed by the addition of synthetic peptide with sequences corresponding to residues from P14 to P2 in the reactive center loop of SCC antigen-2. On the other hand, the incubation with synthetic peptides having the sequence of the reactive center loop of SCC antigen-1 or antithrombin had no effect on polymerization of SCC antigen-2. These data suggest that the polymerization of SCC antigen-2 may occur spontaneously in vivo by the loop-sheet mechanism of serpin.

New molecular tumor markers for endometrial cancer.

Although the International Federation of Gynecology and Obstetrics officially changed the classification system of endometrial cancer from a clinically staged to a surgically staged disease in 1988, optimal management of patients with endometrial cancer is still controversial. Gynecologists happen to experience that patients with tumors that are identical in grade and stage often have significantly different clinical outcomes or responses to therapy. In order to identify an objective biological factor correlating with tumor aggressiveness, many tumor markers have been investigated. So far, CA125 is one of the most reliable tumor marker for adenocarcinoma of the uterus and frequently used in a clinical setting. Recently, with the advent of molecular biological techniques, many genes and regions of the genome related to endometrial cancer have been identified. We undertook a genome-wide screening to detect genetic changes by comparative genomic hybridization (CGH) in primary endometrioid cancers, since CGH analysis provides comprehensive information concerning relative chromosomal losses and gains in tumors by a single hybridization. In this paper, the usefulness of serum tumor markers and the new promising molecular tumor markers for endometrial cancer are discussed.

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.

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.

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.

Identification of squamous cell carcinoma antigen-2 in tumor tissue by two-dimensional electrophoresis.

The aim of this study was to identify two homologous serine proteinase inhibitor (serpin) molecules, squamous cell carcinoma (SCC) antigen-1 and -2, by two-dimensional electrophoresis (2-DE), combined with immunoblotting, and examine their expression in tumor tissue. The recombinant SCC (rSCC) antigen-1 showed four spots with p/ 6.5, 6.4, 6.3 and 6.0, whereas rSCC antigen-2 showed a more acidic spot with p/5.95. SCC antigen in tumor tissue appeared in three new acidic spots (p/5.7-5.5, M(r) 44 500), numbered 5, 6 and 7, besides the previously reported four spots numbered 1 to 4. These new acidic spots of SCC antigen apparently increased in SCC tissue. Treatment of tissue extract by carboxymethyl (CM)-papain agarose matrix extinguished spots 1 to 4 encoded on the SCCA1 gene, but not 5 to 7 on the SCCA2 gene. Overexpression of the SCCA2 gene may play an important role in the malignant behavior of tumor cells.

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).

Alpha 1-antichymotrypsin inhibits chymotrypsin-induced apoptosis in rat hepatoma cells.

Increased serum levels of alpha1-antichymotrypsin (alpha 1ACT) are observed in some cancer patients, especially those with hepatocellular carcinoma. A possible role of alpha 1ACT in tumour growth has been suggested, but this remains uncertain. We have demonstrated that alpha 1ACT inhibited chymotrypsin-induced apoptosis in rat hepatoma H4 cells. Even low concentrations of chymotrypsin (but not trypsin) induce apoptosis in H4 cells with a minimum effective concentration of 2.4 x 10(-2) units/ml (0.5 microg/ml), and this apoptosis was inhibited by alpha 1ACT in a concentration-dependent manner. Furthermore, the concentrations of alpha 1ACT required to inhibit the apoptosis were lower than normal serum levels. These results may indicate that alpha 1ACT plays a role in the apoptosis of rat hepatoma cells.

Electrophoretic analysis of the "cross-class" interaction between novel inhibitory serpin, squamous cell carcinoma antigen-1 and cysteine proteinases.

We investigated the "cross-class" interaction between cysteine proteinases and a novel inhibitory serpin, recombinant squamous cell carcinoma (rSCC) antigen-1, which inhibits a serine proteinase, chymotrypsin. rSCC antigen-1 inhibited the cysteine proteinases, papain, papaya proteinase IV and cathepsin L. Interestingly, although rSCC antigen-1 formed sodium dodecyl sulfate (SDS)- and heat-stable complexes with chymotrypsin, rSCC antigen-1 gave the 40 kDa fragment and small molecular mass peptide by incubation with papain without forming an SDS- and heat-stable complex. The cleavage was observed between the Gly353-Ser354 bond, indicating that rSCC antigen-1 interacts with cysteine proteinases not at the predicted reactive site P1-P1' portion (Ser354-Ser355), but at the Gly353-Ser354 of the P2-P1 portion. These findings promote understanding of the "suicide inhibition" mechanism of SCC antigen-1 against cysteine proteinases.

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.

Penetration of cefpodoxime into uterine and vaginal secretions from postpartum women after a single oral dose of cefpodoxime proxetil.

We evaluated the usefulness of cefpodoxime proxetil (CPDX-PR) in the treatment of puerperal infection and obtained the following results. (i) The susceptibilities of 124 clinical isolates from 85 uterine lochia samples were determined. The MIC at which the growth of Streptococcus agalactiae, Escherichia coli, and Bacteroides fragilis isolates was inhibited by 90% (MIC90) was 0.39 micrograms/ml or less. The MIC90 for Staphylococcus aureus was 3.13 micrograms/ml. (ii) Seven puerperal women received 200 mg of CPDX-PR orally. The CPDX concentration in the lochias in the uterine cavity was not statistically different from that in the vagina, suggesting that the vaginal samples, which can be obtained more safely and aseptically, can be substituted for the uterine samples. The CPDX concentration in cubital venous blood reached a peak of 1.61 micrograms/ml at 3 hours after CPDX-PR administration. The CPDX concentration in the lochias gradually increased and reached a peak of 1.20 micrograms/ml in the uterine cavity and 1.27 micrograms/ml in the vagina at 5 h after drug administration and gradually declined thereafter. These results suggest that CPDX-PR, with its good transfer to the lochia and its potent antimicrobial activity, is a promising drug for the prophylactic and therapeutic treatment of puerperal infections caused by susceptible organisms.

A study of the luteolytic mechanism of the antiprogesterone RU486 during the late-luteal phase in pseudopregnant rats.

The purpose of this study was to examine the possible mechanism through which RU486 induces luteolysis during the late-luteal phase in pseudopregnant (PSP) rats. PSP rats received a subcutaneous injection of RU486 in sesame oil (5 mg/kg body weight) or sesame oil alone once a day between day 9 and day 11 of pseudopregnancy. Serial blood samples were collected on days 5, 9, 10, 11 and 12 and assayed for progesterone content. To examine the possible action of RU486 through a uterine and/or a pituitary (prolactin-dependent) mechanism, PSP rats and chronic hysterectomized PSP rats which had been hysterectomized before PSP induction received a subcutaneous injection of RU486 in sesame oil (5 mg/kg body weight), sesame oil alone, prolactin in 50% polyvinylpyrrolidone (15 IU/day), or RU486 and prolactin once a day between day 9 and day 11 of pseudopregnancy. Serial blood samples were collected on days 5, 9, 10 and 11 and assayed for progesterone content. Blood samples were also collected at 0400 h on day 12 and used for prolactin and progesterone determinations. To examine the direct effect of RU486 on corpus luteum and/or pituitary, hysterectomized rats underwent hypophysectomy and pituitary autotransplantation on dioestrus 1 and received a subcutaneous injection of RU486 in sesame oil or sesame oil alone for 3 days between day 21 and day 23 after surgery. Serial blood samples were collected on days 10, 21, 22, 23 and 24 and assayed for progesterone and prolactin contents. In ordinary PSP rats, serum progesterone levels were significantly (P < 0.01) lower in the RU486-treated group than in the control group (9 +/- 1 vs 53 +/- 7 ng/ml; mean +/- S.E.M.) on day 11. Serum prolactin levels at 0400 h on day 12 of pseudopregnancy were significantly (P < 0.05) lower in the RU486-treated group than in the control group (16 +/- 4 vs 154 +/- 44 ng/ml; mean +/- S.E.M.). The concomitant prolactin treatment reversed the luteolytic effects of RU486 on day 11 of pseudopregnancy. In hysterectomized PSP rats, RU486 also suppressed serum prolactin levels, and the concomitant prolactin treatment again reversed the luteolytic effects of RU486. In hysterectomized rats which were hypophysectomized and pituitary autotransplanted, RU486 treatment did not induce any significant changes in serum progesterone and prolactin levels. These results indicated that RU486 induced luteolysis during the late-luteal phase in PSP rats by suppressing prolactin secretion via a hypothalamic mechanism.