TP53 variants in p53 signatures and the clonality of STICs in RRSO samples.

OBJECTIVE: Precursor lesions may be identified in fallopian tube tissue after risk-reducing salpingo-oophorectomy (RRSO) in patients with pathogenic variants of BRCA1/2. Serous tubal intraepithelial carcinoma (STIC) is considered a precursor of high-grade serous carcinoma, whereas the significance of the p53 signature remains unclear. In this study, we investigated the relationship between the p53 signature and the risk of ovarian cancer. METHODS: We analyzed the clinicopathological findings and conducted DNA sequencing for TP53 variants of p53 signatures and STIC lesions isolated using laser capture microdissection in 13 patients with pathogenic variants of BRCA1/2 who underwent RRSO and 17 control patients with the benign gynecologic disease. RESULTS: TP53 pathogenic variants were detected significantly higher in RRSO group than control (p<0.001). No difference in the frequency of p53 signatures were observed between groups (53.8% vs 29.4%; p=0.17). TP53 sequencing and next-generation sequencing analysis in a patient with STIC and occult cancer revealed 2 TP53 mutations causing different p53 staining for STICs and another TP53 mutation shared between STIC and occult cancer. CONCLUSION: The sequence analysis for TP53 revealed 2 types of p53 signatures, one with a risk of progression to STIC and ovarian cancer with pathological variants in TP53 and the other with a low risk of progression without pathological variants in TP53 as seen in control.

Usefulness of Endoscopy for Female Genital Plastic Surgery: A Series of 4 Cases with Genital Malformations.

BACKGROUND Female genital plastic surgery is rare and requires specific patient anatomical information to successfully carry out the appropriate procedure for each female genital malformation. Performing endoscopic surgery can increase the amount of information intraoperatively. We report on our experience of proactively using endoscopy to obtain additional intraoperative information to perform the appropriate surgical procedure suited to each female genital malformation, and evaluate the usefulness of concurrent endoscopy for improved surgical outcomes. CASE REPORT In Case 1, an 18-year-old woman underwent colpoplasty for Mayer-Rokitansky-Küster-Hauser syndrome using the laparoscopic Davydov procedure, wherein the pelvic peritoneum was precisely dissected using light from a laparoscope as a guide. In Case 2, a 25-year-old woman presented with suspected Wunderlich syndrome. Since the junction of the affected uterus with the normal uterus was unclear on preoperative imaging, we performed total hysterectomy of the affected uterus after identifying the anatomical structure by hysteroscopy. In Case 3, a 12-year-old girl with obstructed hemivagina and ipsilateral renal anomaly (OHVIRA) syndrome underwent transvaginal fenestration of the obstructed vaginal wall to prevent future menstrual molimen. Simultaneous hysteroscopy and laparoscopy allowed us to determine the location of a unilateral vaginal wall cyst and drain it safely. In Case 4, a 29-year-old woman had OHVIRA syndrome, and we conducted a laparoscope-based fenestration of the cervical canal safely and accurately. CONCLUSIONS Our series showed how, even for gynecological malformations without established standards for surgical procedure, a safe and minimally invasive surgery can be ensured if the amount of anatomical information is increased via endoscopy.

Transcription Factor Homeobox D9 Drives the Malignant Phenotype of HPV18-Positive Cervical Cancer Cells via Binding to the Viral Early Promoter.

Persistent infections with two types of human papillomaviruses (HPV), HPV16 and HPV18, are the most common cause of cervical cancer (CC). Two viral early genes, E6 and E7, are associated with tumor development, and expressions of E6 and E7 are primarily regulated by a single viral promoter: P97 in HPV16 and P105 in HPV18. We previously demonstrated that the homeobox D9 (HOXD9) transcription factor is responsible for the malignancy of HPV16-positive CC cell lines via binding to the P97 promoter. Here, we investigated whether HOXD9 is also involved in the regulation of the P105 promoter using two HPV18-positive CC cell lines, SKG-I and HeLa. Following the HOXD9 knockdown, cell viability was significantly reduced, and E6 expression was suppressed and was accompanied by increased protein levels of P53, while mRNA levels of TP53 did not change. E7 expression was also downregulated and, while mRNA levels of RB1 and E2F were unchanged, mRNA levels of E2F-target genes, MCM2 and PCNA, were decreased, which indicates that the HOXD9 knockdown downregulates E7 expression, thus leading to an inactivation of E2F and the cell-cycle arrest. Chromatin immunoprecipitation and promoter reporter assays confirmed that HOXD9 is directly associated with the P105 promoter. Collectively, our results reveal that HOXD9 drives the HPV18 early promoter activity to promote proliferation and immortalization of the CC cells.

Evaluating the importance of routine drainage following laparoscopic pelvic lymph node dissection for gynecological malignancies.

OBJECTIVE: To determine the efficacy of drainage following pelvic lymph node (PLN) dissection, especially for cases involving laparoscopic surgery. METHODS: In this retrospective study, 368 patients with malignant gynecological tumors who underwent systemic PLN dissection at Keio University Hospital between January 2012 and October 2018 were enrolled. Drainage tubes were placed in the retroperitoneal fossa in all patients. Medical records were used for data collection. RESULTS: Laparoscopy was performed on 81 patients, and laparotomy was performed on 287 patients. In the laparoscopy group, tubes were removed 1 day post surgery. In the laparotomy group, tubes were removed 1 day post surgery in 167 patients and 4 days post surgery in 120 patients. Compared with the laparotomy group, we determined the laparoscopy group to have a significantly lower prevalence of lymphocyst (6.2% vs 20.2%, p = 0.002) but a similar prevalence of lymphedema (4.9% vs 5.2%), and symptomatic lymphocyst (2.5% vs 4.5%). The two laparotomy groups did not differ significantly with respect to the prevalence of lymphedema (4.8% vs 5.8%), lymphocyst (20.4% vs 20.0%), or symptomatic lymphocyst (4.2% vs 5.0%). CONCLUSION: Our results suggest that routine drainage should be omitted, especially in cases involving laparoscopic surgery.

Evaluation of preoperative prediction of intestinal invasion in patients with ovarian cancer.

OBJECTIVE: To optimize prediction for intestinal invasion of epithelial ovarian cancer. It is important to achieve debulking surgery to improve prognosis in ovarian cancer; intestinal resection is adopted if the cancer is invaded and resectable, but the preoperative evaluation method of intestinal invasion is still controversial. METHODS: Patients (n = 174) who underwent primary debulking surgery for epithelial ovarian cancer were recruited for retrospective study; 28 and 146 patients were classified into the invasion and non-invasion groups, whether they needed intestinal resection or not. We collected clinical data including evaluation of computed tomography (CT), magnetic resonance imaging (MRI), and barium contrast radiography, and analyzed their accuracy. RESULTS: The sensitivity and specificity for intestinal invasion were 33.3% and 98.6%, 42.9% and 98.6%, and 66.7% and 93.9% in CT, MRI, and barium contrast radiography, respectively. CT and MRI combined showed a sensitivity of 58.3% and specificity of 96.9%; all three methods combined was the most sensitive combination, showing a sensitivity of 79.2% and specificity of 90.8%. CONCLUSION: Combination of CT, MRI, and barium contrast radiography predicts intestinal invasion with the highest sensitivity. These three modalities, however, could not predict all intestinal invasion. Patients should be informed of the possibility of unexpected extensive resection.

Retrospective evaluation of risk-reducing salpingo-oophorectomy for BRCA1/2 pathogenic variant carriers among a cohort study in a single institution.

BACKGROUND: Risk-reducing salpingo-oophorectomy is performed for the primary prevention of ovarian cancer in patients with hereditary breast-ovarian cancer syndrome. We performed risk-reducing salpingo-oophorectomy for the first time in Japan in 2008, and we experienced 20 cases of risk-reducing salpingo-oophorectomy through 2019. In the past, the use of risk-reducing salpingo-oophorectomy in Japan was restricted because it was not covered by a Japanese National Health Insurance. Since April 2020, risk-reducing salpingo-oophorectomy has been covered by insurance for patients with breast-ovarian cancer syndrome and pre-existing breast cancer, and this surgery is expected to become more widely implemented in Japan. METHODS: To contribute to the widespread use of risk-reducing salpingo-oophorectomy in the future, we retrospectively reviewed 20 cases of risk-reducing salpingo-oophorectomy at our hospital cohort study to clarify the issues in its implementation. RESULTS: The variant genes for which risk-reducing salpingo-oophorectomy was indicated were BRCA1 and BRCA2 in 13 (65%) and 7 patients (35%), respectively. The median age at which risk-reducing salpingo-oophorectomy was performed was 49 years (range, 38-58), 13 patients (65%) had gone through menopause, and 16 patients (80%) had a history of breast cancer. Of the five patients (25%) with vasomotor symptoms, four received Chinese medicine, and only one received hormone replacement therapy. Occult cancer was detected in the removed ovaries in two patients (10%), although no postoperative peritoneal carcinogenesis has been observed to date. CONCLUSIONS: Women who paid for risk-reducing salpingo-oophorectomy out of pocket were older than the recommended age at which the procedure should be performed, and this may explain the higher rate of occult cancers than previously reported. We need to perform risk-reducing salpingo-oophorectomy at the recommended age to ensure that the procedure is effective for primary prevention.

Laparoscopic Surgery for Ovarian Cyst Infection with Avoidance of Ureteral Injury and Uterine Perforation following Intrauterine Insemination after Abdominal Modified Radical Trachelectomy.

Pelvic inflammatory disease (PID) sometimes develops after intrauterine insemination (IUI). We herein present a case of PID which developed after IUI performed after abdominal modified radical trachelectomy (AmRT) and was treated with laparoscopic surgery. To our knowledge, this is the first case report of laparoscopic surgery for PID that occurred after AmRT in Japan. A 39-year-old woman who was diagnosed with cervical cancer stage IA1 with lymphovascular invasion underwent AmRT and pelvic lymphadenectomy. At 3 years and 6 months after the surgery, she had fever and pain in her left lower abdomen 10 days after IUI. She was diagnosed with PID with left ovarian cyst infection and underwent laparoscopic left ovarian cystectomy. Before surgery, bilateral ureteral catheters were inserted because of possible difficulty identifying the ureters. During surgery, severe adhesion was seen in the pelvic cavity. By moving the catheters manually back and forth from outside the body, we were able to identify the ureters visually. A uterine manipulator was inserted during surgery, rather than before surgery, to avoid the risk of uterine perforation. Laparoscopic surgery with ureteral catheters and a uterine manipulator can be applied safely for such cases after AmRT even when severe intraperitoneal adhesion is present.

Local anatomy around terminal ureter related to the anterior leaf of the vesicouterine ligament in radical hysterectomy.

OBJECTIVES: Radical hysterectomy is performed for invasive cervical cancer. In this surgery, separation of the anterior leaf and posterior leaves of the vesicouterine ligament (VUL) is important. We studied the local anatomy of the anterior leaf of the VUL, especially the branches of the umbilical artery from the view point of surgery and cadaver dissection. STUDY DESIGN: We observed the cervicovesical blood vessels and the connective tissue layer continued from umbilical artery and searched for the origin of the cervicovesical blood vessels in radical hysterectomy. We also dissected a formalin-fixed female cadaver, and observed the same points. RESULTS: After separation of the connective tissue of urinary bladder from the cervical fascia, we could discern the outline of the distal ureter near the ureterovesical junction. We separate the connective tissue of the so-called anterior leaf of the VUL enwrapping the ureter gently, and then the ureter with the connective tissue is completely rolled out laterally. We identified a cervicovesical vessel crossing over the ureter. We looked for the central side of the cervicovesical vessel and found that cervicovesical vessel was a branch of the superior vesical artery. And, during cadaver dissection, we found that the connective tissue and the branches of the superior vesical artery were similarly observed. CONCLUSIONS: We found the precise anatomy of the connective tissue layer from umbilical artery to urinary bladder and the superior vesical artery. Our procedure based on the precise anatomy obtained in this study is reasonable anatomically as a method for separation of the vesicouterine ligament during radical hysterectomy.

Synchronous endometrial and ovarian cancer in Lynch syndrome with a MSH2 germline mutation: A case report.

Synchronous endometrial and ovarian cancer (SEOC) is a rare entity among gynecological cancers, which exhibits endometrioid histology in its early stages and generally has a good prognosis. However, diagnosis is difficult and recent reports have demonstrated that most clinically diagnosed cases of SEOC have clonally related cancers, indicating metastatic cancer. The association of SEOC with Lynch syndrome is also not clearly understood. We herein present the case of a 41-year-old SEOC patient with MSH2 mutation. The endometrial cancer was an endometrioid adenocarcinoma and the ovarian cancer was mainly endometrioid, but also included a clear cell carcinoma with a borderline clear cell adenofibromatous component, indicating primary ovarian cancer. Both tumors exhibited microsatellite instability (MSI) and loss of expression of MSH2 and MSH6. The patient had a family history of colorectal and gastric cancers. Genetic analysis revealed a germline mutation in exon 6 of MSH2 (c.1042C>T, p.Gln348*) and the patient was diagnosed with Lynch syndrome. This MSH2 mutation has only been registered in one case in the InSiGHT variant databases and has not been reported in a gynecological tumor or SEOC to date. This case is a rare example of a patient with genetically diagnosed Lynch syndrome who also developed SEOC. This synchronous cancer is not common, but it may be caused by Lynch syndrome. Testing for MSI and immunohistochemistry for mismatch repair deficiency is necessary in cases with suspected SEOC.

Radiation-induced angiosarcoma of the omentum diagnosed by laparoscopy: A case report.

Angiosarcoma is a rare malignant tumor with an aggressive clinical course and a poor prognosis. Intraperitoneal angiosarcoma, especially originating from the omentum, is extremely rare. We report a case of radiation-induced angiosarcoma of the omentum that arose in a 38-year-old female seven years after concurrent chemoradiotherapy for cervical cancer. The primary tumor was unknown until diagnostic laparoscopy revealed an unresectable omental mass. Pathological examination revealed high-grade malignant cells positive for endothelial markers. Although the small number of cases limits the consensus on optimal therapy for advanced angiosarcoma, the patient was managed successfully by taxane-based chemotherapy, leading to complete response and consequent complete cytoreductive surgery. Our report is the fifth case of radiation-induced angiosarcoma of the omentum, and all have developed after treatment for gynecologic cancer. Although very rare, this complication should be considered after radiation therapy in cancer treatment, particularly given the increasing importance of this therapy.

Recent findings on epigenetic gene abnormalities involved in uterine cancer.

Selective aberrant genetic effects that do not depend on abnormal DNA sequences are referred to as epigenetic abnormalities and are involved in carcinogenesis. In uterine cancer, various genes involved in apoptosis, cell cycle, DNA repair, cell proliferation and cell adhesion are abnormally methylated, resulting in gene silencing. Reversal of such epigenetic abnormalities in cancer cells is a potential strategy for cancer therapy, and studies on epigenetic abnormalities and treatment methods in uterine cancer are in progress. These include the evaluation of 5-hydroxymethylcytosine, which is present in cancer tissues at lower levels compared with those in normal tissues, as a prognostic marker in cervical cancer; combination therapy with 5-azacytidine and cisplatin; combination treatment focusing on tumor necrosis factor-related apoptosis-inducing ligand in cervical cancer; studies focusing on DNA mismatch repair in endometrial cancer; and use of a demethylating agent to reactivate tumor suppressor genes and inhibit tumor proliferation. Detection of epigenetic changes using biomarkers may be used for histological classification, evaluation of disease progression and identification of compounds that are able to modulate epigenetic changes and may be useful for uterine cancer treatment.

Comparison Between Laparoscopy and Laparotomy in Systematic Para-Aortic Lymphadenectomy for Patients with Endometrial Cancer: A Retrospective Multicenter Study.

Objective: Laparoscopic surgery has been developed worldwide due to its minimal invasion as well as noninferiority, compared with laparotomy. However, whether or not laparoscopic systematic para-aortic lymphadenectomy for endometrial cancer is feasible and has advantages of various clinical factors, such as a short hospital stay, less blood loss, and faster recovery, compared with open surgery has not yet been clarified. The aim of this study was to compare a laparoscopic procedure with laparotomy for para-aortic lymphadenectomy for patients with endometrial cancer. Study Design: This was a retrospective multicenter study of laparoscopic systematic para-aortic lymphadenectomy for endometrial cancer in five institutions. Materials and Methods: The current authors conducted a retrospective multicenter study of laparoscopic systematic para-aortic lymphadenectomy for endometrial cancer. The study involved patients from five institutions in Japan between January 2008 and March 2016. Clinical data were compared with those of a laparotomic procedure performed around the same period. Results: A total of 54 patients in the laparoscopic group and 99 patients in the laparotomic group were analyzed. In the laparoscopic group, 21 patients had stage IA disease, 19 had stage IB disease, 5 had stage II disease, and 9 had stage III disease. In the laparotomic group, 35 patients had stage IA disease, 19 had stage IB disease, 9 had stage II disease, and 36 had stage III disease. There were no significant differences in characteristics between the groups, including age, body mass index, and histologic type. The mean operative time in the laparoscopic group was 483 ± 102 minutes, while that in the laparotomic group was 481 ± 106 minutes (p = 0.9). The laparoscopic group had less intraoperative blood loss (143 ± 253 versus 988 ± 694 mL; p < 0.01) and shorter hospital stays (8.4 ± 5.7 versus 16.1 ± 8.0 days; p < 0.01). The rates of intraoperative complications were not significantly different between the groups. No cases of ileus occurred in the laparoscopic group. Procedures for 2 of the 54 patients in the laparoscopic group were converted to laparotomy. The number of dissected pelvic lymph nodes (31.8 ± 10.1 versus 39.9 ± 15.9, p < 0.01) and para-aortic lymph nodes (26.2 ± 10.9 versus 31.1 ± 13.2; p = 0.02) were lower in the laparoscopic group than in the laparotomic group. The postoperative minimum level of hemoglobin was higher in the laparoscopic group than in the laparotomic group (10.4 ± 1.1 g/dL versus 9.9 ± 1.4 g/dL; p = 0.02). In contrast, the postoperative maximum level of C-reactive protein was lower in the laparoscopic group than in the laparotomic group (6.3 ± 3.8 mg/dL versus 10.2 ± 4.9 mg/dL; p < 0.01). The recurrence rate was not significantly different between the groups in the above time period (7.4% versus 14.3%; p = 0.2). Conclusions: Laparoscopic systematic para-aortic lymphadenectomy is feasible and can be substituted for laparotomic procedures for patients with early stage endometrial cancer. ( J GYNECOL SURG 33:105).

Impact of laver treatment practices on the geoenvironmental properties of sediments in the Ariake Sea.

Since the 1970s, the catch of Tairagi and Agemaki shellfish that inhabit the shallow sediments of the Ariake Sea of Japan has fallen dramatically. This is partly accounted for by the Isahaya land reclamation dike project and by the increasingly frequent local red tides. A recent survey of local fisherman suggested that the decline in the shellfish harvest may also be due to the practice of laver treatment in the tidal flats of the Ariake Sea. We carried out field and laboratory investigations to determine whether the practice changes the geoenvironmental properties of the fine-grained sediments in the tidal flats. There were notable changes in the salt concentration, pH, and sulfide content between the sediments exposed to a laver treating agent and those without laver treatment. Based on these differences, we identified potential mechanisms by which the laver treating agent was transported into the sediments and influenced the sulfide levels.

Comparison of T2-weighted and contrast-enhanced T1-weighted MR imaging at 1.5 T for assessing the local extent of cervical carcinoma.

OBJECTIVE: To compare two MR sequences at 1.5 T-T2-weighted and contrast-enhanced T1-weighted images-by using macroscopic sections to determine which image type enables the most accurate assessment of cervical carcinoma. METHODS: Forty consecutive patients (mean age, 39.2 years) with biopsy-proven cervical carcinoma were included. Each MR sequence was assessed for tumour localisations, tumour margins, and cancer extent with the consensus of two readers, and tumour margins were rated on a five-point scale. MR findings were correlated with histopathological findings. Contrast-to-noise ratios (CNRs) obtained with each image were compared using nonparametric tests. RESULTS: Thirty-one of 40 patients underwent hysterectomies and nine of 40 underwent trachelectomies. In 36 patients, lesions were identified on at least one sequence. The tumours at stage 1B or higher were detected in 94.7% on contrast-enhanced T1-weighted images and in 76.3% on T2-weighted images (P < 0.05). Tumour margins appeared significantly more distinct on contrast-enhanced T1-weighted images than on T2-weighted images (P < 0.001). The CNRs obtained using contrast-enhanced T1-weighted images were significantly higher (P < 0.001) than those obtained using T2-weighted images. CONCLUSION: Contrast-enhanced T1-weighted imaging is more useful for assessing cervical carcinoma than T2-weighted imaging.

Cr(VI) concentration from batch contact/tank leaching and column percolation test using fly ash with additives.

Batch contact, tank leaching and column percolation tests were conducted to investigate the Cr(VI) concentration in the solution/leachate from two fly ashes (fly ash A and B) with additives. The additives used were cement, low alkalinity additive and Ariake clay. There are several factors influencing Cr(VI) concentration in solution/leachate, namely (1) properties of solid/liquid mixture (chemical composition, pH value, etc.), (2) cementation effect, (3) amount of water in contact with the solid mass (solid/liquid ratio in case of batch contact test), and (4) adsorption characteristics of the solid particles to Cr ions. The test results indicate that fly ash A has less cementation component (CaO of 1.92%) and the amount of water in contact with the fly ash played an important role. As a result, Cr(VI) concentration from the column percolation test was much higher than that of the batch contact test. Adding Ariake clay had more effect on reducing Cr(VI) concentration for fly ash A than B because the pH value of the solution from fly ash A was lower, which provided a favorable condition for Cr(VI) ions to be reduced to Cr(III) and possibly to be adsorbed by clay particles. Fly ash B has more cementation component (7.15%) and for column percolation test, curing the sample for 1 week reduced Cr(VI) concentration significantly. The test results indicate that in engineering practice, a method which closely simulates the field condition should be selected to assess possible environmental effects and corresponding countermeasure methods.

Intratumor injection of small interfering RNA-targeting human papillomavirus 18 E6 and E7 successfully inhibits the growth of cervical cancer.

Human papillomavirus (HPV) 18 is related not only to squamous cell carcinoma of the cervix, but also to adenocarcinoma and small cell carcinoma of the cervix, in which prognosis is known to be poor. Small interfering RNA (siRNA) that targets HPV18 E6 and E7 was tested in HPV18-positive cell lines to investigate its effect and investigate its mechanism of action. Nude mice were also tested in a combination of siRNA and atelocollagen to determine whether it might be useful as a new molecule-targeting therapy for cervical cancer. siRNAs targeting HPV18 E6 and E7 were transfected into cervical cancer cells in vitro and they were investigated for cell growth inhibition, expression of E6 and E7 mRNA, expression of retinoblastoma protein, and senescence-associated beta-galactosidase staining. Sequence-specific siRNA inhibited cell growth. Decreased expression of E6 and E7 mRNA followed with E7 protein was observed in the transfected cells, but the expression of retinoblastoma protein and the beta-galactosidase staining increased, suggesting cell growth inhibitory effect through senescence. Treatment of xenografts established from SKG-II cells with siRNA specific for E6 and E7 obviously suppressed tumor growth in vivo. These results indicate that atelocollagen-mediated delivery of siRNA HPV18 E6 and E7 can be used as a novel therapeutic approach for cervical cancer.

A laboratory study on migration of K+ in a two-layer landfill liner system.

Contaminant transport through the clay liner and the underIying secondary leachate drain layer (SLDL) in landfills was studied through a laboratory test, and analysis method on the transport of K+ in a two-layer soil system. The soils used for this study were Ariake clay and the underlying layer, Shirasu soil from the Kyushu region of Japan, representing the clay liner material and SLDL material, respectively. The effective diffusion coefficients (De) of the selected target chemical species, potassium (K+) for the Ariake clay and Shirasu soil were back-calculated using a computer program, and it was found that values of De derived from this study were consistent with those previously published. The hypothesis that the mechanical dispersion process can be negligible has been proved to be reasonable based on both the observation that the predicted values fit the experimental data and the analyses of two dimensionless parameters. Parametric analysis showed the transport of K+ through the soils is controlled by advection-diffusion rather than diffusion only, whereas at low Darcy velocity (i.e. < or = 10(-9) m s(-1)), transport of K+ would be controlled by diffusion. The test results and parametric analysis may be applied in design of landfill liners and SLDLs, particularly in coastal areas.

Comparison between in situ hybridization and real-time PCR technique as a means of detecting the integrated form of human papillomavirus 16 in cervical neoplasia.

Integration of the human papillomavirus (HPV) genome is thought to be one of the causes of cancer progression. However, there is controversy concerning the physical status of HPV 16 in premalignant cervical lesions, and there have been no reports on the concordance between detection of the integrated form of HPV16 by real-time PCR and by in situ hybridization. We investigated specimens of cervical intraepithelial neoplasia (CIN) and invasive carcinomas for the physical status of HPV 16 by real-time PCR and in situ hybridization. The presence of the integrated form was detected by both real-time PCR and in situ hybridization in zero of four cases of CIN1, three of six cases of CIN2, nine of 27 cases of CIN3, and two of six cases of invasive carcinomas. Integrated HPV 16 was present in some premalignant lesions but was not always present in carcinomas. The concordance rate between the two methods for the detection of the presence of the integrated form was 37 of 43 (86%) cases. Real-time PCR and in situ hybridization were found to be complementary and convenient techniques for determining the physical status of the HPV genome. We conclude that a combination of both methods is a more reliable means of assessing the physical status of the HPV genome in cervical neoplasia.