Evidence of skewed X-chromosome inactivation in 47,XXY and 48,XXYY Klinefelter patients.

Klinefelter (47,XXY) syndrome occurs in approximately 1:800 male births and accounts for about 10-20% of males attending infertility clinics. Recent studies have shown no obvious phenotypic differences between subjects in which the extra X-chromosome is of paternal or maternal origin; however, a minority of Klinefelter patients are adversely affected clinically and intellectually to an exceptional level, and the underlying basis of this phenotypic variation is not known. We hypothesize that skewed X-inactivation and possibly parental origin of the X-chromosomes is involved. In this study, we determined parental origin and inactivation status of the X-chromosomes in 17 cytogenetically confirmed 47,XXY cases, two 48,XXYY cases and one mosaic 46,XY/47,XXY case. Eight highly polymorphic markers specific to the X-chromosome and the polymorphic human androgen-receptor (HUMARA) methylation assay were used to determine the parental origin and X-inactivation status of the X-chromosomes, respectively. Overall, 17 cases were fully informative, enabling parental origin to be assigned. In 59% of cases, both X-chromosomes were of maternal origin (Xm); in the remaining 41%, one X was of maternal (Xm) and one was of paternal origin (Xp). In 5 of 16 (31%) cases informative at the HUMARA locus, skewed X-inactivation was observed as defined by greater than 80% preferential inactivation involving one of the two X-chromosomes. The two 48,XmXpYY cases both showed preferential paternal X-chromosome (Xp) inactivation. Three 47,XmXmY cases also showed preferential inactivation in one of the two maternal X-chromosomes. These results suggest that skewed X-inactivation in Klinefelter (47,XXY and 48,XXYY) patients may be common and could explain the wide range of mental deficiency and phenotypic abnormalities observed in this disorder. Further studies are warranted to examine the role of X-inactivation and genetic imprinting in Klinefelter patients.

Subsequent pregnancy outcome in patients with spontaneous resolution of HCG after evacuation of hydatidiform mole: comparison between complete and partial mole.

This study compared subsequent pregnancy outcome in patients with complete and partial hydatidiform moles. Among 1052 patients with molar pregnancy (complete mole, 801; partial mole, 251) monitored at Chiba University Hospital between 1981 and 1999, 891 patients (84.7%) had spontaneous resolution of human chorionic gonadotrophin (HCG) after mole evacuation, and 161 patients (15.3%) required chemotherapy. Of the 891 patients, 438 (49.2%) had 650 subsequent pregnancies. The pregnancy outcome was not significantly different in patients with complete and partial moles, and was comparable with that in the general Japanese population. The incidence of repeat molar pregnancy in patients with complete and partial mole (1.3 and 1.5% respectively) was 5-fold higher than that of the general population, while no increased risk of persistent gestational trophoblastic tumour (GTT) associated with later molar pregnancy was observed. During HCG follow-up, 10 patients (1.1%) developed secondary high-risk GTT between 14 and 54 months after mole evacuation. The incidence of high-risk GTT in patients with and without subsequent pregnancies was 0.46% (2/438) and 1.8% (8/453) respectively (P = 0.1243). In conclusion, patients with complete and partial mole can anticipate a normal future reproductive outcome, and pregnancies after experiencing hydatidiform mole may not affect the development of high-risk GTT.

Adjuvant hysterectomy in low-risk gestational trophoblastic disease.

OBJECTIVE: To evaluate the efficacy of adjuvant hysterectomy with chemotherapy for women with low-risk gestational trophoblastic disease. METHODS: One hundred fifteen consecutive Japanese women (16-52 years old) with low-risk gestational trophoblastic disease (46 with metastatic disease and 69 without) were treated initially with single-agent chemotherapy (etoposide in 85, methotrexate in 27, and actinomycin D in three) with or without adjuvant hysterectomy, and 97 patients (84.3%) achieved primary remission with those treatments. Eight women (9.4%) treated with etoposide required other regimens because of drug resistance or toxicities. The total dose of etoposide given to achieve primary remission was analyzed in 77 women who received etoposide alone or with adjuvant hysterectomy. RESULTS: In 34 women with metastatic disease, the mean (+/- standard deviation [SD]) total dose of etoposide was not significantly different with and without adjuvant hysterectomy (2857 +/- 842 mg versus 2815 +/- 815 mg; P =.957; Mann-Whitney U test). However, in 43 women without metastases, the total dose of etoposide was significantly less in those who had adjuvant hysterectomies than in those who did not (1750 +/- 635 mg versus 2545 +/- 938 mg; P <.05; Mann-Whitney U test). CONCLUSION: Adjuvant hysterectomy decreased the total dose of etoposide given to achieve primary remission in women with nonmetastatic, low-risk gestational trophoblastic disease. If the lesions of gestational trophoblastic disease are confined to the uterus and the woman has no desire to preserve fertility, she should be informed of adjuvant hysterectomy as a treatment option.

Identification of an optimal Ncx binding sequence required for transcriptional activation.

The Ncx gene encodes a homeobox containing transcription factor that belongs to the Hox11 gene family. We determined specific Ncx protein binding consensus DNA sequences. Optimal Ncx binding sequences were 5'-CGGTAATTGG-3' (TAAT core) and 5'-CGGTAAGTGG-3' (TAAG core), which coincided with the Hox11 binding sequence. Both Ncx and Hox11 could bind to the TAAT and the TAAG core oligonucleotide in vitro. However, they could efficiently transactivate the reporter plasmid linked to the TAAT core sequence but not to the TAAG core sequence. Thus, Ncx and Hox11 act as transcriptional activators via their target sequence, 5'-CGGTAATTGG-3'.

Combination chemotherapy with methotrexate, etoposide, and actinomycin D for high-risk gestational trophoblastic tumors.

OBJECTIVES: The goal of this study was to evaluate the efficacy, toxicity, and survival of patients with high-risk gestational trophoblastic tumors (GTTs) treated with a methotrexate-etoposide-actinomycin D (MEA) regimen without cyclosphosphamide or vincristine. METHODS: Thirty-nine consecutive patients with high-risk GTTs (28 were defined high risk by WHO criteria) were treated with primarily the MEA regimen. Among them, 27 patients had received no prior chemotherapy and 12 had received prior chemotherapy. Survival, causes of treatment failure, and toxicity were analyzed retrospectively. RESULTS: After treatment with the MEA regimen, 29 of 39 patients achieved primary remission (74.4%), 8 developed resistance (20.5%), and 2 died of widespread metastases and chemotherapy-related toxicity. All 8 patients who developed resistance were treated with high-dose 5-fluorouracil and actinomycin D (FA); 6 were salvaged and 2 died of refractory disease. Three patients relapsed; 2 were controlled with FA or cisplatin-based chemotherapy and 1 who refused further treatment died. The disease-free survival rate was 87%. WHO grade 4 leukocytopenia and thrombocytopenia with the MEA regimen occurred in 5.3 and 6.4%, respectively, of the cycles; other toxic effects were acceptable and manageable. CONCLUSIONS: At present, MEA chemotherapy (without cyclophosphamide or vincristine) is our treatment of choice for patients with high-risk GTT. Its toxicity is predictable and manageable. For patients who become resistant to MEA, new salvage chemotherapy regimens are needed.

An enhancer element for expression of the Ncx (Enx, Hox11L1) gene in neural crest-derived cells.

The murine Ncx (Enx, Hox11L1) gene is specifically expressed in a neuronal subset of neural crest-derived tissues. In attempts to elucidate the regulatory DNA element of the tissue-specific expression, we sequenced the 5'-flanking region of the Ncx gene. The transcriptional initiation site was determined at 297 nucleotides (-297) upstream from the ATG start codon (+1). A retinoic acid response element was located on the region between -1163 and -1150. Transient transfection assays with the 5'-flanking sequences fused to the luciferase gene showed that the region between -1387 and -1368 was crucial for the tissue-specific enhancer activity. Furthermore, nuclear proteins extracted from neural crest-derived cells such as murine and human neuroblastoma cells bind to the DNA region between -1387 and -1368. This DNA element was also conserved in the 5'-flanking region of the human NCX gene. Our observations strongly suggest that the DNA element (between -1387 and -1368) contributes to tissue-specific expression of the Ncx gene in murine and human species.

Androgenetic complete mole coexistent with a twin live fetus.

OBJECTIVE: The aim of this study was to evaluate the clinical course and the management policy of complete mole coexistent with a twin live fetus confirmed with DNA polymorphism in a single hospital. METHODS: From 1981 to 1995, six patients with androgenetic complete hydatidiform mole coexistent with a twin live fetus were diagnosed by DNA polymorphism analysis. The clinical course of these six patients was analyzed. RESULTS: Two patients chose to terminate pregnancies and four patients desired to continue the pregnancy. However, the pregnancy had to be interrupted in two patients because of severe preeclampsia and sudden intrauterine fetal death. In two patients, fetuses were growing unremarkably and normal babies were delivered at term. The development of persistent trophoblastic tumor (PTT) in these rare pregnancies was higher (50.0%: 3/6) than that of single complete mole. In three patients, serum hCG titers during pregnancy were monitored. Although serum hCG levels progressively decreased during pregnancy in one patient without PTT, hCG levels initially decreased, but subsequently increased or showed a plateau with advancing gestational age in two patients with PTT. CONCLUSIONS: In patients with complete mole coexistent with a live fetus, the pregnancy may be allowed to continue when the fetal karyotype and development are normal and serum hCG titers are constantly falling with advancing gestational age.

Pregnancy outcome after treatment with etoposide (VP-16) for low-risk gestational trophoblastic tumor.

The aim of this study was to evaluate subsequent fertility and pregnancy in patients treated for persistent trophoblastic tumors with single-agent VP-16. Records of all patients treated for persistent trophoblastic tumors at the Chiba University Hospital between January 1, 1986 and December 31, 1997 were reviewed. Of these, 85 patients were initially treated with single-agent VP-16. Subsequent pregnancy outcome of these patients was investigated. After remission with VP-16, 36 patients (92.3%) of those who wished for a pregnancy (45.9% of all patients studied) conceived, and 91.7% had at least one live birth. A total of 56 conceptions resulted in 42 (75.0%) term live births, seven (12.5%) first-trimester spontaneous abortions, one (1.8%) second-trimester spontaneous abortion, four (7.1%) therapeutic abortions, and two (3.6%) repeated moles. There were no congenital anomalies, no stillbirths, and the neonates' physical growth was comparable to that of the standard population in Japan. Single VP-16 regimen for patients with low-risk gestational trophoblastic tumor appears to have no adverse effects on fertility potential and pregnancy outcome.

Genetic differentiation of complete hydatidiform moles coexisting with normal fetuses by short tandem repeat-derived deoxyribonucleic acid polymorphism analysis.

OBJECTIVE: We applied deoxyribonucleic acid polymorphism analysis on the basis of differences in the number of short tandem repeat sequences to genetically differentiate dizygotic twins with complete hydatidiform moles and normal fetuses from partial moles presenting a similar appearance. STUDY DESIGN: Six pregnant women exhibiting apparent moles and coexisting fetuses were the subjects of this study. Eight polymorphic loci including short tandem repeat sequences were amplified by polymerase chain reaction from deoxyribonucleic acid of peripheral leukocytes of parents, umbilical cord, grossly normal placenta-villi, and molar tissue. The segregation of alleles among samples were determined by comparing band patterns on polyacrylamide gels. RESULTS: In all 6 cases amplifications of polymorphic loci provided sufficient information to determine the parental origin. At informative loci the alleles of cord and placenta-villi were transmitted from both patients and husbands whereas molar tissue had only paternal alleles. These allele segregations indicated 2 different genetic origins, namely, normal parental for a fetus and androgenetic for molar tissue, and thus the diagnosis of dizygotic twins with a complete hydatidiform mole and a normal fetus was made. Additionally, the molar component was defined as a heterozygous mole in 2 cases. CONCLUSION: Short tandem repeat-derived deoxyribonucleic acid polymorphism analysis was demonstrated to be a useful and precise procedure for the differential diagnosis of a complete hydatidiform coexisting with a normal fetus and the determination of its zygosity as well.

Comparison of chemotherapies with methotrexate, VP-16 and actinomycin-D in low-risk gestational trophoblastic disease. Remission rates and drug toxicities.

OBJECTIVE: To compare the efficiency and toxicity of four chemotherapeutic regimens in low-risk gestational trophoblastic disease. METHODS: Since 1974, 247 patients with low-risk gestational trophoblastic disease have been treated with 5-day intramuscular methotrexate (MTX) (conventional MTX), 5-day intravenous drip infusion of VP-16, 5-day intravenous actinomycin-D (Act-D) or 8-day alternating intramuscular MTX-folic acid (MTX-CF) at Chiba University School of Medicine. We compared the primary remission rate, the response of human chorionic gonadotropin and the prevalence of drug toxicities in these 4 regimens. RESULTS: The primary remission rate was 73.6% in the conventional MTX regimen, 90.1% in VP-16, 84.0% in Act-D, and 60.0% in MTX-CF. The primary remission rate was significantly higher in the VP-16 and Act-D regimens than in the conventional MTX and MTX-CF regimens. The response rate was significantly higher in the VP-16 regimen than in the other 3 regimens. The drug toxicity with VP-16 and Act-D was less than that with conventional MTX and MTX-CF regimens. CONCLUSIONS: The VP-16 regimen was highly effective and less toxic for gestational trophoblastic disease compared with other chemotherapeutic regimens.

A novel pathogenesis of megacolon in Ncx/Hox11L.1 deficient mice.

The Ncx/Hox11L.1 gene, a member of the Hox11 homeobox gene family, is mainly expressed in neural crest-derived tissues. To elucidate the role of Ncx/Hox11L.1, the gene has been inactivated in embryonic stem cells by homologous recombination. The homozygous mutant mice were viable. These mice developed megacolon with enteric ganglia by age 3-5 wk. Histochemical analysis of the ganglia revealed that the enteric neurons hyperinnervated in the narrow segment of megacolon. Some of these neuronal cells degenerated and neuronal cell death occurred in later stages. We propose that Ncx/Hox11L.1 is required for maintenance of proper functions of the enteric nervous system. These mutant mice can be used to elucidate a novel pathogenesis for human neuronal intestinal dysplasia.

Ncx, a Hox11 related gene, is expressed in a variety of tissues derived from neural crest cells.

We have isolated the murine homeobox gene (Ncx) that belong to a Hox11 gene family. Expression of the Ncx gene was analyzed in total RNAs from embryos by reverse transcribed polymerase chain reaction (RT-PCR). The mRNA was detected in embryos after 9.5 days of embryogenesis (E9.5) and was maximal at E12.5. The RT-PCR also detected the message in total RNAs from adrenal glands and intestine in adult mice. The expression was further examined in various tissues from embryos by in situ hybridization. It was detected in dorsal root ganglia, cranial nerve ganglia (V, IX, X), enteric nerve ganglia and adrenal glands from embryos between E9.5 and E13.5. Since its expression is restricted to tissues derived from neural crest cells, Ncx may play a role in differentiation and proliferation of neural crest lineage cells.

Two forms of Hox11 a T cell leukemia oncogene, are expressed in fetal spleen but not in primary lymphocytes.

HOX11 is identified from the breakpoint of human T cell acute lymphoblastic leukemias with t(10;14). Since overexpression of HOX11 in T cells caused leukemias in transgenic mice, the endogenous HOX11 may play a role in proliferation and differentiation of T cells. In order to elucidate the role, we examined the expression of Hox11 in normal lymphocytes by a reverse transcriptase-polymerase chain reaction analysis. Two alternatively spliced Hox11 mRNAs were expressed in fetal spleens. However, lymphocytes did not express Hox11 mRNA during differentiation. Furthermore, it was not induced in primary lymphocytes after activation. These results suggest that ectopic expression of HOX11 in T cells is responsible for leukemogenesis.

A complete hydatidiform mole coexisting with a normal fetus was confirmed by variable number tandem repeat (VNTR) polymorphism analysis using polymerase chain reaction.

The diagnosis of a complete hydatidiform mole coexisting with a live fetus is often difficult because of the morphological similarity to a partial mole, but it is crucial to management in the postmolar course. We present a recent case in which DNA polymorphism analysis clearly revealed a different genetic origin for the fetal and molar parts. DNA polymorphisms on three different variable number tandem repeat loci, which were detected by polymerase chain reaction, indicated that the cord/placenta and molar tissue were parental and androgenetic, respectively. Subsequently, the patient was admitted for chemotherapy of metastatic trophoblastic disease.

The effects of hyperthermia on the cell cycle of Ehrlich ascites cancer cells in vivo.

The effects of hyperthermia on the cell cycle of Ehrlich ascites cancer cells were studied, and these effects simultaneously evaluated in terms of prolonging the survival of test mice inoculated with tumor cells from heat-treated mice. DDY mice bearing Ehrlich ascites cancer cells were placed in a water bath at 37 degrees C, 39 degrees C, 41 degrees C, 42 degrees C. The heating of mice at 41 degrees C, 42 degrees C and 43 degrees C induced the accumulation of cancer cells at the G2M phase of the cell cycle with many cells exhibiting polyploidy (16 C). The extent of accumulation increased as the temperature of incubation was raised, however the interrupted cell cycle resumed 120 hours after heating. The retransplantation of cells from the heat-treated mice revealed that the mice which were inoculated with Ehrlich ascites cancer cells from mice heated at 43 degrees C survived longer, while the mice which were inoculated with Ehrlich ascites cancer cells from mice heated at 39 degrees C survived for only a slightly shorter time than those which were inoculated with cells from mice heated at 37 degrees C.

Relationship between the cytologic characteristics of intraperitoneal free cancer cells and the prognosis in patients with gastric cancer.

The presence of free cancer cells in the peritoneal cavity of 387 patients with gastric cancer was analyzed using specimens obtained by intraoperative lavage of the pouch of Douglas. If cancer cells were found in the specimen, the case was classified with respect to the number and arrangement (clustered or isolated) of the cancer cells found. Study of the relationship between the histologic type of cancer and the presence of free cancer cells showed that poorly differentiated adenocarcinomas and signet ring-cell carcinomas were associated with a higher incidence of free cancer cells than were other types of cancer. The relationships between the characteristics of the free cancer cells and the postoperative survival rate were also studied. Patients whose free cancer cells were present in clusters were associated with a more favorable prognosis than were those with no such clusters. Patients with small numbers of free cancer cells survived longer than did those with large numbers of free cancer cells.

Hyperthermic peritoneal perfusion combined with anticancer chemotherapy as prophylactic treatment of peritoneal recurrence of gastric cancer.

We have conducted a clinical trial to evaluate the effectiveness of continuous hyperthermic peritoneal perfusion with mitomycin C (CHPP-M) as prophylactic treatment of peritoneal recurrence of gastric cancer. Between January 1983 and October 1985, 82 patients with macroscopic serosal invasion but no macroscopic peritoneal metastasis undergoing potentially curative resection of gastric cancer, were divided into two groups by random sampling: 42 patients were scheduled to receive CHPP-M, while 40 would not receive such treatment. The CHPP-M was administered immediately after closure of the abdomen following gastric resection, while the patient was still on the operating table under general anesthesia. The 5-year survival rate (71.5% of the patients in the CHPP-M group was higher than that (59.7%) of those in the control group. Although postoperative follow-up periods are not long enough, the longer survival obtained in the patients treated with CHPP-M should be emphasized, since no effective means of preventing peritoneal recurrence of gastric cancer is so far available.

Relationship between area of serosal invasion and prognosis in patients with gastric carcinoma.

We examined the relationship between the spatial extent of invasion of the gastric serosa in patients with gastric carcinoma and their postoperative 5-year survival rate. At the time of surgical resection of gastric cancer, intraperitoneal free cancer cells were detected by lavage of the Douglas cavity in 135 of 309 (44%) patients with gross evidence of serosal invasion. Examination of the relationship between the presence of intraperitoneal free cancer cells and serosal area invaded by the tumor revealed that only 22% of cases with an area of serosal invasion 10 cm2 or less were positive for free cancer cells, but such cells were found in 72% of cases with an area of serosal invasion greater than 20 cm2. The 5-year survival rate was 31% in patients with an area of serosal invasion of less than 10 cm2, whereas the rate was only 8% in patients with an area of serosal invasion greater than 20 cm2. Not only the presence of serosal invasion by a tumor but also the spatial extent of the invasion are significant factors that influence the prognosis of patients with gastric carcinoma.

[Prognosis of gastric cancer patients with special reference to the area of serosal cancerous invasion].

The area of serosal cancerous invasion and intraperitoneal free cancer cells were examined in 360 gastric cancer cases. The positivity rate of intraperitoneal free cancer cells was 20% in the group with under 20 cm2 of serosal invasion, 59% with 20 cm2 to 30 cm2, and 76% over 30 cm. The postoperative survival rate was significantly lower in the group with free cancer cells than in the group without them. Prognosis was better in case with the area of serosal invasion was smaller. Hematogenous metastasis was frequent under 20 cm2, but over 20 cm2, peritoneal metastasis predominated.

Peritoneal mesothelial cell injury factors in rat cancerous ascites.

To elucidate the mechanism of the peritoneal dissemination of cancer, the influence of cancerous ascites on peritoneal mesothelial cells was studied by scanning electron microscopy and light microscopy. We inoculated normal Donryu rats with AH100B ascites hepatoma cells and studied the influence of the supernatant from cancerous ascites on the normal rat peritoneal surface by i.p. injection. The mesothelial cells were damaged and exfoliated markedly, which is supposed to be a profitable condition for cancer cells to proliferate on the peritoneal surface. Therefore, the presence of mesothelial cell injury factors was noted. Subsequently, we divided the supernatant from rat cancerous ascites into four fractions by gel filtration and revealed the distribution of mesothelial cell injury factors by studying the influence of each fraction on the normal rat peritoneal surface. Although Fraction I (fibrin fraction) and Fraction II (IgG fraction) made no changes on the peritoneal surface, Fraction III (albumin fraction) and Fraction IV provoked damages on the mesothelial cells. We found that the mesothelial cell injury factors are present in the albumin fraction and in the fraction containing low-molecular-weight substances.