Cytological nuclear atypia classification can predict prognosis in patients with endometrial cancer.

OBJECTIVE: Endometrial cancer is one of the leading causes of malignancy in females. Nuclear findings are important for patients with cancer, and can provide valuable information to treating oncologists. We investigated whether nuclear findings were a useful prognostic factor in patients with endometrial cancer. METHOD: We investigated 71 cases of endometrial carcinoma with paired histology and cytology at Kurume University Hospital. We classified endometrial endometrioid adenocarcinoma (EEC) G1 and G2 as type I carcinomas, and uterine papillary serous carcinoma (UPSC), clear cell carcinoma (CC) and EEC G3 as type II carcinomas. For the establishment of the cytological nuclear atypia classification, we examined the following nuclear factors on the cytological smears: mitotic figures, prominent nucleoli, nuclear area and anisonucleosis. RESULTS: There was a significant difference in mitotic figures (P < 0.001) and anisonucleosis (P = 0.026) in cytological smears between type I and type II carcinomas. Based on these findings, we categorized cytological nuclear atypia into three groups, nuclear atypia-1 (57.7%), nuclear atypia-2 (19.7%) and nuclear atypia-3 (22.5%), and this classification system correlated well with prognosis in patients with endometrial cancer (P < 0.001). Furthermore, this classification system was able to extract patients with a good prognosis from those with high-grade carcinomas, such as UPSC+CC+EEC G3, and patients with a poor prognosis from those with EEC G1. CONCLUSIONS: Our system of cytological nuclear atypia classification based on endometrial cytology can predict patient prognosis. Cytological nuclear atypia classification and histological typing may be useful for the treatment and follow-up of patients with endometrial cancer, and should be routinely incorporated into cytological reports.

Phase III randomised controlled trial of neoadjuvant chemotherapy plus radical surgery vs radical surgery alone for stages IB2, IIA2, and IIB cervical cancer: a Japan Clinical Oncology Group trial (JCOG 0102).

BACKGROUND: A phase III trial was conducted to determine whether neoadjuvant chemotherapy (NACT) before radical surgery (RS) improves overall survival. METHODS: Patients with stage IB2, IIA2, or IIB squamous cell carcinoma of the uterine cervix were randomly assigned to receive either BOMP (bleomycin 7 mg days 1-5, vincristine 0.7 mg m(-2) day 5, mitomycin 7 mg m(-2) day 5, cisplatin 14 mg m(-2) days 1-5, every 3 weeks for 2 to 4 cycles) plus RS (NACT group) or RS alone (RS group). Patients with pathological high-risk factors received postoperative radiotherapy (RT). The primary end point was overall survival. RESULTS: A total of 134 patients were randomly assigned to treatment. This study was prematurely terminated at the first planned interim analysis because overall survival in the NACT group was inferior to that in the RS group. Patients who received postoperative RT were significantly lower in the NACT group (58%) than in the RS group (80%; P=0.015). The 5-year overall survival was 70.0% in the NACT group and 74.4% in the RS group (P=0.85). CONCLUSION: Neoadjuvant chemotherapy with BOMP regimen before RS did not improve overall survival, but reduced the number of patients who received postoperative RT.

Expression of activated signal transducer and activator of transcription-3 predicts poor prognosis in cervical squamous-cell carcinoma.

BACKGROUND: Stat3 is a member of the Janus-activated kinase/STAT signalling pathway. It normally resides in the cytoplasm and can be activated through phosphorylation. Activated Stat3 (p-Stat3) translocates to the nucleus to activate the transcription of several molecules involved in cell survival and proliferation. The constitutive activation of Stat3 has been shown in various types of malignancies, and its expression has been reported to indicate a poor prognosis. However, the correlation between the constitutive activation of Stat3 and the prognosis of cervical cancer patients has not been reported. METHODS: The immunohistochemical analysis of p-Stat3 expression was performed on tissues from 125 cervical squamous-cell carcinoma patients who underwent extended hysterectomy and pelvic lymphadenectomy, and the association of p-Stat3 expression with several clinicopathological factors and survival was investigated. RESULTS: Positive p-Stat3 expression was observed in 71 of 125 (56.8%) cases and was significantly correlated with lymph node metastasis, lymph vascular space invasion, and large tumour diameter (>4 cm) by Fisher's exact test. Kaplan-Meier survival analysis showed that p-Stat3 expression was statistically indicative of a poor prognosis for overall survival (P=0.006) and disease-free survival (P=0.010) by log-rank test. CONCLUSION: These data showed that p-Stat3 expression in cervical cancer acts as a predictor of poor prognosis.

Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1.

Control of cyclin levels is critical for proper cell cycle regulation. In yeast, the stability of the G1 cyclin Cln1 is controlled by phosphorylation-dependent ubiquitination. Here it is shown that this reaction can be reconstituted in vitro with an SCF E3 ubiquitin ligase complex. Phosphorylated Cln1 was ubiquitinated by SCF (Skp1-Cdc53-F-box protein) complexes containing the F-box protein Grr1, Rbx1, and the E2 Cdc34. Rbx1 promotes association of Cdc34 with Cdc53 and stimulates Cdc34 auto-ubiquitination in the context of Cdc53 or SCF complexes. Rbx1, which is also a component of the von Hippel-Lindau tumor suppressor complex, may define a previously unrecognized class of E3-associated proteins.

Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase.

The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in most human kidney cancers. The VHL protein is part of a complex that includes Elongin B, Elongin C, and Cullin-2, proteins associated with transcriptional elongation and ubiquitination. Here it is shown that the endogenous VHL complex in rat liver also includes Rbx1, an evolutionarily conserved protein that contains a RING-H2 fingerlike motif and that interacts with Cullins. The yeast homolog of Rbx1 is a subunit and potent activator of the Cdc53-containing SCFCdc4 ubiquitin ligase required for ubiquitination of the cyclin-dependent kinase inhibitor Sic1 and for the G1 to S cell cycle transition. These findings provide a further link between VHL and the cellular ubiquitination machinery.

Deficiency of coagulation factor XIII A subunit caused by the dinucleotide deletion at the 5' end of exon III.

A congenital deficiency of the coagulation Factor XIII A subunit (F XIII A) is a rare autosomal recessive disorder that is characterized by a life-long bleeding tendency complicated by a difficulty in healing. Thus far, no molecular genetic analysis of this disorder has been reported. In this study, we demonstrate the molecular abnormalities in a family with this disorder. We performed Northern blot analysis of peripheral blood monocytes obtained from the propositus and found a 4-kb single band of F XIII A mRNA whose size was identical with that of normal subjects. Exons II-XV, which encode all the amino acids, were individually amplified by a polymerase chain reaction (PCR). All PCR products from the propositus had lengths indistinguishable from those of the wild type on agarose gel, suggesting that this defect results from either a point mutation or a short deletion/insertion. The sequencing of F XIII A cDNA from the propositus revealed a deletion of the dinucleotide AG within the AGAG repeat at the position of 210 to 213. Concerning the genomic sequence, a deletion of dinucleotide AG was also demonstrated in the intron B-exon III boundary. This deletion appeared to cause a frameshift mutation making a new stop codon shortly thereafter, and leading to a deficiency of plasma F XIII A. The heterozygosity of the F XIII A deficiency in the patient's offspring was documented by the nucleotide sequences of their exon III.

Regulation of hypoxia-inducible mRNAs by the von Hippel-Lindau tumor suppressor protein requires binding to complexes containing elongins B/C and Cul2.

The von Hippel-Lindau tumor suppressor protein (pVHL) binds to elongins B and C and posttranscriptionally regulates the accumulation of hypoxia-inducible mRNAs under normoxic (21% O2) conditions. Here we report that pVHL binds, via elongin C, to the human homolog of the Caenorhabditis elegans Cul2 protein. Coimmunoprecipitation and chromatographic copurification data suggest that pVHL-Cul2 complexes exist in native cells. pVHL mutants that were unable to bind to complexes containing elongin C and Cul2 were likewise unable to inhibit the accumulation of hypoxia-inducible mRNAs. A model for the regulation of hypoxia-inducible mRNAs by pVHL is presented based on the apparent similarity of elongin C and Cul2 to Skp1 and Cdc53, respectively. These latter proteins form complexes that target specific proteins for ubiquitin-dependent proteolysis.

Development of thermotolerance during fractionated hyperthermia in a solid tumor in vivo.

The effect of 43.5 degrees water bath heating on a C3H mammary carcinoma inoculated into the foot of BALB/c x DBA F1 (hereafter called CD2F1 mice was investigated. A single heat treatment resulted in a linear dose-response relationship between heating time and tumor growth time (i.e., the time for tumors to reach 5 times the initial volume of the first treatment day). Recovery from hyperthermic damage, demonstrated by two-dose fractionation experiments (30 min + 60 min at 43.5 degrees), increased with increasing fractionation interval and reached its maximum at a 16-hr interval. Preheating for 30 min at 43.5 degrees induced thermal resistance to a second heat treatment at 43.5 degrees (thermotolerance) which was evidenced by a decrease in the slope of the dose-response curves. This thermotolerance gradually increased with increasing interval and reached a maximum at a 16-hr interval with a thermotolerance ratio of 5.2. Subsequently, the thermotolerance gradually decayed and completely disappeared at a 120-hr interval. No detectable repair of hyperthermic damage was found in this tumor. In principle, there data confirm the observations on thermotolerance reported previously for cell cultures in vitro and for several normal tissues in vivo.

Identification of a new endoplasmic reticulum-resident protein recognized by HLA-A24-restricted tumor-infiltrating lymphocytes of lung cancer.

To help clarify the molecular basis of tumor immunology in lung cancer, we have investigated antigens recognized by HLA-A24-restricted CTLs established from T cells infiltrating into lung adenocarcinoma and report a new gene encoding tumor epitopes recognized by the CTLs. This gene was located on chromosome 4q31.22 and encoded an unreported endoplasmic reticulum-resident protein with 412 deduced amino acids. This protein had a molecular mass of 46 kDa and was expressed in the majority of malignant cells and tissues tested, with the exception of T-cell leukemia cells, but was not expressed in a panel of normal cells and tissues, except in those of the testis, placenta, and fetal liver. Two peptides at positions 13-20 and 75-84 were recognized by the CTLs and had an ability to induce HLA-A24-restricted and tumor-specific CTLs in peripheral blood mononuclear cells of lung cancer patients. Thus, these peptides might be appropriate molecules for use in the specific immunotherapy of HLA-A24+ patients with lung and other cancers.

Tumor-associated antigen 22-1-1 expression in the uterine cervical squamous neoplasias.

We have reported that a novel tumor-associated antigen (Ag), 22-1-1, was expressed in cancer cells derived mainly from the uterus and ovary [K. Sonoda et al., Cancer (Phila.), 77: 1501-1509, 1996]. The 22-1-1 Ag existed not only in adenocarcinomas but also in squamous cell carcinomas in the uterine cervix. Here, a relationship between tumor progression and invasion and 22-1-1 Ag expression was investigated in squamous cell neoplasms of the uterine cervix using immunohistochemical staining. The 22-1-1 Ag was not detected in normal uterine cervix (0 of 10 total cases) and dysplasias (0 of 47 total cases). However, 20% of carcinoma in situ (4 of 20 total cases) and 16.7% of microinvasive carcinomas (2 of 12 total cases) stained positively for 22-1-1 Ag. Moreover, areas depicting microinvasion on histology in uterine cancers (stage Ia) were more strongly stained than carcinoma in situ lesions. 22-1-1 Ag expression was found to be more frequent in invasive squamous cell carcinomas (82.6%; 57 of 69 total cases). The 22-1-1 Ag existed both in the cytoplasm and on the membrane of cancer cells. These findings suggest that 22-1-1 Ag expression might be related to tumor cell progression and invasion in the uterine cervical squamous cell epithelium.

The prognostic significance of tumor-associated antigen 22-1-1 expression in adenocarcinoma of the uterine cervix.

We previously established (K. Sonoda et al., Int. J. Oncol., 6: 1099-1104, 1995) a novel monoclonal antibody, 22-1-1, generated from adenocarcinoma of the uterine cervix, and 22-1-1 antigen (Ag) was expressed in cancer cells derived mainly from the uterus and ovary. In this report, a relationship between 22-1-1 Ag expression and clinicopathological variables and the prognostic significance of 22-1-1 Ag were immunohistochemically investigated in adenocarcinoma of the cervix. Of 56 cases, the 22-1-1 Ag was negative in 7, 1+ in 14, 2+ in 26 and 3+ in 9 instances. The 22-1-1 Ag existed both in the cytoplasm and on the membrane of cancer cells. There was no correlation between 22-1-1 Ag expression and age, stage, grade, myometrial invasion, lymph-vascular space invasion, lymph node metastasis, and parametrial invasion. The estimated 5-year overall survival (OS) of patients with low 22-1-1 Ag expression (-/+) and high 22-1-1 Ag expression (++/ ) were 90.5 and 71.4%, respectively. Patients with high 22-1-1 Ag expression had significantly worse OS than those with low 22-1-1 Ag expression (log-rank test, P = 0.0193). In addition, lymph-node metastasis, age, and clinical stage were significantly related to OS in univariate analysis. Multivariate analysis for OS revealed a prognostic significance in 22-1-1 Ag expression, stage, age, and grade. These data suggest that 22-1-1 Ag expression may be related to prognosis in adenocarcinoma of the cervix.

Sclerosing stromal tumor of the ovary: a clinicopathologic, immunohistochemical, ultrastructural, and cytogenetic analysis with special reference to its vasculature.

Sclerosing stromal tumor (SST) is a rare ovarian neoplasm occurring predominantly in young women and is histologically characterized by cellular heterogeneity, prominent vasculature, and a pseudolobular appearance composed of cellular and hypocellular areas. In the current study, three cases of SST were ultrastructurally examined and the tumors were found to be composed of several kinds of cells, i.e., luteinized thecalike cells, spindle-shaped fibroblastic cells, and primitive mesenchymal cells. These findings thus supported the ovarian stromal origin of SST. Twelve cases of SST also were analyzed immunohistochemically and demonstrated an expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) in the luteinized thecalike cells and its receptor, fms-like tyrosine kinase 1 (flt-1), in capillary to medium-sized blood vessels. Reverse transcription-polymerase chain reaction (RT-PCR) also showed an expression of VPF/VEGF messenger RNA in SSTs. Accordingly, the characteristic vasculature and edema of SSTs were considered to be associated with the expression of VPF/VEGF. In addition, a fluorescence in situ hybridization (FISH) analysis also showed cells with three copy number of chromosome 12 in 13-21% of all examined SST cells, which suggested the presence of chromosome 12 trisomy in SSTs as well as in other ovarian stromal tumors.

Diversity of primary structures of the carboxy-terminal regions of mammalian fibrinogen A alpha-chains. Characterization of the partial nucleotide and deduced amino acid sequences in five mammalian species; rhesus monkey, pig, dog, mouse and Syrian hamster.

The partial amino acid sequences of fibrinogen A alpha-chains from five mammalian species have been inferred by means of the polymerase chain reaction (PCR). From the genomic DNA of the rhesus monkey, pig, dog, mouse and Syrian hamster, the DNA fragments coding for alpha-C domains in the A alpha-chains were amplified and sequenced. In all species examined, four cysteine residues were always conserved at the homologous positions. The carboxy- and amino-terminal portions of the alpha-C domains showed a considerable homology among the species. However, the sizes of the middle portions, which corresponded to the internal repeat structures, showed an apparent variability because of several insertions and/or deletions. In the rhesus monkey, pig, mouse and Syrian hamster, 13 amino acid tandem repeats fundamentally similar to those in humans and the rat were identified. In the dog, however, tandem repeats were found to consist of 18 amino acids, suggesting an independent multiplication of the canine repeats. The sites of the alpha-chain cross-linking acceptor and alpha 2-plasmin inhibitor cross-linking donor were not always evolutionally conserved. The arginyl-glycyl-aspartic acid (RGD) sequence was not found in the amplified region of either the rhesus monkey or the pig. In the canine alpha-C domain, two RGD sequences were identified at the homologous positions to both rat and human RGDS. In the Syrian hamster, a single RGD sequence was found at the same position to that of the rat. Triplication of the RGD sequences was seen in the murine fibrinogen alpha-C domain around the homologous site to the rat RGDS sequence. These findings are of some interest from the point of view of structure-function and evolutionary relationships in the mammalian fibrinogen A alpha-chains.

The promotion of invasion through the basement membrane of cervical carcinoma cells by fibronectin as a chemoattractant.

An in vitro migration and invasion assay was used as the model system to study the effect of 3T3 fibroblast conditioned medium (FCM) and purified human fibronectin on the invasion of cervical carcinoma cells. The 3T3 FCM significantly enhanced both the migration and the invasion of a cervical carcinoma cell line, HeLa. This enhancement of migration and invasion was inhibited by anti-fibronectin antibody. Purified fibronectin alone enhanced the invasion in a dose-dependent manner for all cervical carcinoma cell lines, HeLa, CAC-1 and TMCC. The pretreatment of cells with cell binding aminosequences, GRGDSP and/or YIGSR blocked the enhancement of cell invasion. The implication of these findings for the invasion of cervical carcinoma is discussed.

An attempt to generate an antitumor effect in the regional lymph nodes against endometrial cancer cells by inducing antitumor cytokines.

Phytohemagglutinin-stimulated regional lymph node cells obtained from gynecological cancer patients exerted a significant antiproliferative activity against an endometrial cancer cell line, RL95-2 on a human tumor clonogenic assay, and released a high amount of tumor necrosis factor alpha (TNF alpha), and interferons. The activity was thought to be partly due to released TNF alpha, because RL95-2 was highly sensitive to recombinant TNF alpha. However, anti-TNF alpha failed to inhibit the activity, which indicated the probability of some as yet unclarified participation of cytokines. Therefore, the regional lymph nodes might be used as sites of endogenous cytokine therapy in endometrial cancer patients.

Successful treatment of two patients with recurrent endometrial cancer by weekly paclitaxel.

OBJECTIVE: The aim of this study was to evaluate the toxicity and efficacy of weekly paclitaxel in patients with recurrent endometrial cancer. METHODS: Paclitaxel (70 mg/m(2) by 1-h infusion weekly) was administered to two patients with recurrent endometrial cancer of the lung. RESULTS: After 5 cycles, both patients with platinum-resistant disease achieved clinical partial responses confirmed by computed tomography (CT) scan. The serum CA125 levels of case 1 decreased to cut-off level. The response duration of both patients was 4 months. The toxicity was acceptable and probably less pronounced than that characterize of the standard tri-weekly schedules. CONCLUSION: Although conclusions regarding survival are premature, weekly paclitaxel might offer better quality of life during treatment.

Conservative therapy for adenocarcinoma and atypical endometrial hyperplasia of the endometrium in young women: central pathologic review and treatment outcome.

Thirty-nine patients with endometrioid adenocarcinoma (EA) and atypical hyperplasia (AH) of the endometrium who received conservative treatment to preserve fertility were collected from member institutions of the Japan Gynecologic Oncology Study Group. Twenty-nine and ten were originally diagnosed with EA without myometrial invasion and AH, respectively. We performed a central pathological review to make definite diagnoses, and the diagnosis of EA in 29 cases was changed to AH in ten, complex hyperplasia in three and atypical polypoid adenomyoma in three, and AH in ten was changed to EA in one and simple hyperplasia in one. Nine of 12 women (75%) with EA and 15 of 18 women (83%) with AH had an initial response to medroxyprogesterone acetate (MPA) treatment. Two of nine responders with EA later developed relapse, and one of them had metastasis to the left obturator lymph node. Two became pregnant, and one delivered one full-term infant. One of the responders with AH had a relapse in the endometrium. Five became pregnant, and four delivered four normal infants. The young women with endometrial carcinoma localized in the endometrium who wish to preserve fertility may be treated as successfully with MPA as those with AH.

Establishment of a new human uterine cervical adenocarcinoma cell-line, siso, and its reactivity to anticancer reagents.

A human cell line, SiSo, was established from a patient afflicted with uterine cervical adenocarcinoma. The SiSo cell expresses MHC class I antigen, various kinds of adhesion molecules and tumor-associated antigens such as CA125, CEA and GA733. The secretion of CA125 antigen was markedly suppressed by anti-cancer reagents. However, their growth was not affected by any of the anti-cancer reagents tested, suggesting a discrepancy between inhibition of tumor growth and suppression of secretion of tumor-associated antigens after treatment with anti-cancer reagents. The SiSo cell line will provide a tool for investigation of uterine adenocarcinoma.

Emergence of higher levels of invasive and metastatic properties in the drug resistant cancer cell lines after the repeated administration of cisplatin in tumor-bearing mice.

To establish a more suitable model for reflecting biological aggressiveness in clinically recurrent cancers after chemotherapy, we made the in-vitro-established cisplatin-resistant cell lines, by exposing the parental tumor cell lines to cisplatin in a culture system, and also the in-vivo-established cisplatin-resistant cell lines by repeated cisplatin administration to parental tumor-bearing mice. Although both cell lines similarly demonstrated a clinically relevant low level of drug resistance (from 1.5 to 2.9 times more resistance to cisplatin than their parental cell lines), only the in-vivo-established cisplatin-resistant cell lines showed significantly enhanced metastatic properties with a 2.1- to 3.4-fold increase in the number of lung metastatic nodules. These enhanced metastatic properties were caused by tumor invasiveness in combination with various levels of enhancement of cell attachment, proteolytic enzyme activity and cell motility. We concluded that anticancer drugs such as cisplatin could promote tumor progression only in the drug-resistant cell lines established in vivo. As a result, these cell lines are considered to be a more faithful and useful model for expressing biological aggressiveness in clinically recurrent cancers after chemotherapy than the conventional drug-resistant cell lines established in vitro.

Reassessment of myometrial invasion in endometrial carcinoma.

OBJECTIVE: To assess different methods of measuring the depth of myometrial invasion in endometrial carcinoma as a prognostic factor. METHODS: Eighty-eight cases of stage I or II endometrial carcinoma treated initially by hysterectomy between 1979-1989 were reviewed histologically. Three methods of measuring myometrial invasion were evaluated: 1) percentage of invaded tumor thickness to whole thickness of myometrium, 2) percentage of whole tumor thickness to total thickness of tumor and myometrium, and 3) distance from the tumor-myometrial junction to the uterine serosa. We evaluated the effect of several factors on prognosis by multivariate analysis using Cox regression models. RESULTS: Myometrial invasion determined by these three measurement methods was associated significantly with survival in a univariate analysis. When myometrial invasion assessed by each method and other prognostic factors were entered into a multivariate model, the distance from the tumor-myometrial junction to the uterine serosa, lymph-vascular space invasion, and cervical stromal involvement were identified as independently significant prognostic factors. CONCLUSION: This method of evaluating myometrial invasion by measuring the distance from the tumor-myometrial junction to the uterine serosa was most useful as a correlate with survival.