Adenocarcinoma of the cervix and adenocarcinoma of the endometrium: distinction with PCR-mediated detection of HPV DNA.

The differential diagnosis between primary endocervical adenocarcinoma and adenocarcinoma originating in the endometrium may in some cases be difficult. The two cancer types have a different genesis, with human papillomavirus (HPV) as an important causal factor in the development of primary cervical carcinoma. In this study, the paraffin-embedded cervical tissues from 23 patients with stage II endometrial carcinoma and from 50 patients with primary cervical adenocarcinoma were examined for HPV DNA of types 16, 18, and 33. HPV DNA was demonstrated in 70% of the primary endocervical adenocarcinomas and in none of the endometrial carcinomas. It is concluded that HPV detection may be useful in the differential diagnosis of some cases of cervical and endometrial adenocarcinoma.

Prognosis of vulvar dysplasia and carcinoma in situ with special reference to histology and types of human papillomavirus (HPV).

Sixty-one women with vulvar dysplasia or carcinoma in situ were treated with local laser excision of the initial lesion and of the recurrences, and followed at intervals of from 3 increasing to 12 months. Recurrences were observed in 16 (26%) patients. No case of invasive carcinoma was seen. Patients with recurrences were significantly younger than those without (P < 0.02, median age 42.5 and 54 years, respectively). The resection borders were significantly more often involved in the initial lesions in the group with recurrences (36%) than in the group without (9%) (P < 0.014). All lesions were classified according to the WHO (mild, moderate, severe dysplasia or carcinoma in situ) and Toki et al. (1991) (warty, basaloid, combined warty/basaloid or mixed (warty, basaloid and simple). No pure types of Toki (1991) could be demonstrated. There were no differences regarding recurrences in any of these groups. HPV DNA was detected in the initial lesions by PCR in 50/56 (89%) (44 with HPV type 16 and 6 with HPV type 33) and by ISH in 23/61 (38%). The same type of HPV could be demonstrated in all first recurrences except in two, where HPV types 33 was shown in specimens harboring HPV type 16 in the initial lesions. In one of these cases, HPV type 16 could again be demonstrated in the second and final recurrence. In no specimen was more than one type of HPV detected. The results indicate that the most important parameter in predicting the recurrence of vulvar dysplasia or carcinoma in situ is the involvement of the resection borders. The location of the lesion, the degree and type of dysplasia, and the type of HPV seem to play a minor role. Local excision and subsequent intensive control with removal of any visible new lesion probably prevents development of vulvar invasive carcinoma.

Human papillomaviruses and multifocal genital neoplasia.

In 143 patients with vulvar carcinoma (76 cases) or vulvar intraepithelial neoplasia (VIN III, 67 cases), cervical cancer or cervical intraepithelial neoplasia CIN III lesions developed in 39 patients (27%) at some time during their life. In patients with classic keratinizing squamous cell carcinoma (KSC) of the vulva, cervical neoplasia developed in only one of 51 (2%), whereas the frequency was 10 of 25 (40%) in patients with vulvar carcinoma of the basaloid or warty type and 28 of 67 (42%) in patients with VIN III lesions. The original, paraffin-embedded surgical specimens were examined by polymerase chain reaction and type-specific molecular hybridization for human papillomavirus (HPV) DNA of the types 6, 11, 16, 18, and 33. DNA of the oncogenic types HPV 16 or HPV 33 was found in 4% of the KSCs, in 84% of the basaloid or warty carcinomas, in 90% of VIN III lesions, and in 89% of the cervical lesions. The same HPV type was found in both lesions in 81% of the patients with double primary tumors. The results support the concept that VIN III and a subgroup of vulvar carcinomas are HPV-related lesions, that they are frequently associated with another HPV-related genital primary tumor, and that these multiprimary tumors are secondary to an HPV infection involving the entire genital tract.

Use of tetranectin, CA-125 and CASA to predict residual tumor and survival at second- and third-look operations for ovarian cancer.

Tetranectin (TN), CA-125 and CASA were measured in serum prior to 63 second-look and 5 third-look operations for ovarian cancer. Patients with residual tumor had significantly lower levels of TN and higher levels of CASA and CA-125 compared with tumor-free patients. The predictive values PVPos = 100% and PVNeg = 50.9% were found for TN at 9.3 mg/l. For CASA, a predictive value PVPos = 100% was found at 10 U/ml with a corresponding PVNeg = 52.7%. At the cut-off 35 U/ml for CA-125, the PVPos was 100% and the PVNeg = 53.6%. By combining the markers, PVNeg increased to 61.7% with a PVPos on 100%. Significantly differences in survival were found by lifetable analysis between patients tested as positive and negative respectively for any of the markers. Using multivariate Cox analyses, it was found that every marker had an independent prognostic function for survival.

The use of polymerase chain reaction to detect metastatic cancer cells within lymph nodes in stage I cervical carcinoma.

The aim of the study was to determine whether human papillomavirus (HPV) in lymph nodes is a useful marker for the risk of recurrence in patients with HPV-related cervical cancer. The polymerase chain reaction and DNA-DNA hybridization techniques were used to examine 149 formalin-fixed, paraffin-embedded lymph nodes that had been resected from 24 patients undergoing radical hysterectomy for stage IB cervical carcinoma. The lymph nodes were examined for the HPV type, which in each case was found in the cervical tumor. Of 18 patients with histologically negative nodes, HPV DNA was found in a lymph node in two of 10 patients who later experienced a recurrence and in three of eight patients who were alive and well for > or = 5 years after surgery. In addition, HPV was detected in the lymph nodes of two of four patients with nodal metastases at the primary surgery; four of nine histologically positive lymph nodes in these patients contained HPV. It is concluded that detection of HPV in resected lymph nodes is probably not a useful means of identifying the cervical cancer patients who might benefit from adjuvant postoperative therapy.

Management of low-grade CIN: follow-up or treatment?

OBJECTIVE: To examine whether low-grade cervical dysplasia carries a higher risk of progression when associated with the cancer-related human papillomavirus types 16, 18, 31 or 33. STUDY DESIGN: Retrospective, with PCR-based HPV diagnosis on the original cervical biopsies from 71 patients with CIN I and II. CIN III developed in 34 lesions, and 37 showed complete regression during non-invasive follow-up. RESULTS: Progression occurred in 15/41 CIN I and in 19/30 CIN II lesions (P = 0.03). HPV DNA was detected in 43 specimens. CIN III developed in 25% of HPV-negative lesions, in 48% of HPV-positive CIN I lesions, and in 77% of HPV-positive CIN II lesions. CONCLUSION: Low-grade lesions are at higher risk of progression when associated with HPV types 16, 18, 31 or 33 (P = 0.002). HPV diagnosis can be useful in the triage of patients with low-grade CIN.

Human papillomavirus (HPV) in vulvar dysplasia and carcinoma in situ.

Surgical specimens from 62 patients with vulvar dysplasia and carcinoma in situ were morphologically investigated. Lesions were classified according to WHO (mild, moderate, severe dysplasia and carcinoma in situ) and according to Toki et al. (1991) (warty, basaloid, combined warty/basaloid or basaloid/warty types or mixed (warty, basaloid and simple) forms). Following the WHO classification, moderate dysplasia was shown in 4 cases, severe dysplasia in 47 and carcinoma in situ in 11 cases. Pure warty type was shown in 2 cases (both biopsy specimens). One case revealed pure simple dysplasia whereas no case of pure basaloid type was found. Various combinations of warty and basaloid types were shown in 52 cases and mixed forms in 7 cases. The results indicate that pure forms of warty and basaloid types probably do not exist. HPV DNA was detected by PCR in 51/58 cases (88%) (45 with HPV type 16 and 6 with HPV type 33) evenly distributed in all age groups and in all types of lesions (WHO and Toki et al. 1991). By ISH HPV was detected in 24/62 cases (39%) (21 with HPV type 16/18 and 3 with HPV type 31/33), nearly always in warty areas. All these cases were positive for the same virus type by PCR. No case revealed more than one type of HPV. HPV type 6, 11, 18, and 31 were not detected by PCR. The results indicate a correlation between HPV type 16 and 33 and dysplasia/carcinoma in situ in the vulva.

Ovarian carcinoma serum markers and ovarian steroid activity--is there a link in ovarian cancer? A correlation of inhibin, tetranectin and CA-125 to ovarian activity and the gonadotropin levels.

In a previous study, we have demonstrated that inhibin-production may be associated with improved survival and, also, that tetranectin (TN) is a valuable prognostic marker in ovarian epithelial cancer. We investigated the possible correlation between inhibin, tetranectin, CA-125, ovarian steroid activity and the gonadotropin levels. Preoperative serum levels of the tumor markers inhibin, tetranectin (TN) and CA-125 were measured and related to ovarian steroid function and the pituitary-gonadal axis (gonadotropin levels) in 28 postmenopausal ovarian cancer patients. The following median levels and 95% confidence limits were demonstrated for the tumor markers: Inhibin 0.4 U/l (0.2-0.9), TN 8.9 mg/l (6.8-9.2), CA-125 160 kU/l (75-687). A significant inverse correlation was demonstrated between inhibin and the gonadotropins. The Spearman correlation coefficients showed a highly significant correlation of inhibin with the examined ovarian steroid hormones except DHEAS which also has a suprarenal component. This indicates a synthesis of inhibin and the steroid hormones from the same cell compartment as known from the normal ovary and an apparently intact negative feed back mechanism. Inhibin may be produced in the normal ovary as a defense mechanism against an elevated gonadotropin level and inhibin acts by lowering the gonadotropins or by altering their biological activity. Elevated values of the tumor markers TN and CA-125 due to gonadotropin stimulation could not be demonstrated but a significant inverse correlation between TN and CA-125 was confirmed.

Vulvar intraepithelial neoplasia III: a viral disease of undetermined progressive potential.

Seventy-three patients with vulvar intraepithelial neoplasia (VIN) grade III were followed for a median of 5 years after primary treatment. Thirty women also had a diagnosis of cervical neoplasia. During the follow-up 26 patients (36%) had one or more vulvar recurrences. Recurrences were seen significantly more often in the patients who also had cervical neoplasia, indicating a common etiology. Microinvasive carcinoma developed in 12 patients, 3 of whom later developed frankly invasive vulvar cancer. The original, paraffin-embedded vulvar specimens were examined by the polymerase chain reaction for human papillomavirus DNA of the types HPV 6, 11, 16, 18, and 33. HPV types 16 and 33 were found in 90% of the VIN lesions. It is concluded that VIN III is an HPV-related disease in all or almost all cases, and that a generalized genital HPV infection may be a factor in the development of multicentric genital neoplasia. No association was observed between the specific HPV type and the risk of recurrent vulvar disease, cervical neoplasia, or malignant progression.

Serum tetranectin and CA-125 used to monitor the course of treatment in ovarian cancer patients.

Serum tetranectin (Se-TN) and CA-125 were measured in serum samples obtained before primary surgery, before start of chemotherapy and monthly during chemotherapy in 8 patients with ovarian cancer. The median Se-TN level before chemotherapy (7.3 mg/l) increased significantly after the start of chemotherapy (13.7 mg/l), with the highest increase for one survivor (145%). Five patients who died of cancer in the study period, had pronounced decreases (30-50%) in Se-TN with a maximal concentration during chemotherapy to the lowest concentration in the last sample. One patient who died 10 months after closure of the study had continuously normal Se-TN values, but the last CA-125 value elevated. The lead time could be calculated in four patients for Se-TN and six patients for CA-125. Median lead times of 3.6 months and 3.8 months were found for Se-TN and CA-125 respectively. In conclusion, chemotherapy induces significant increases in Se-TN levels. A decrease in Se-TN during chemotherapy is highly suspect for recurrence and a poor outcome. Measurements of TN should therefore be included in other comparative studies.

Epstein-Barr virus and Hodgkin's disease: a comparative immunological, in situ hybridization, and polymerase chain reaction study.

During recent years numerous studies have demonstrated the presence of Epstein-Barr virus (EBV) in tissues affected by Hodgkin's disease (HD). The percentage of cases with evidence of EBV infection has varied among the different studies, a positive result being highly dependent on the sensitivity of the method employed. In this study three different methods of detecting EBV in 48 cases of 'classical' HD (33 cases of nodular sclerosis and 15 cases of mixed cellularity) were compared: Immunohistochemistry (IH) for detection of latent membrane protein-1 (LMP-1), in situ hybridization (ISH) for detection of Epstein-Barr virus early RNAs (EBER 1 and 2), and polymerase chain reaction (PCR) for detection of a reiterated 110 base-pair EBV genomic sequence of the BamHI region. In 14 cases (29%) Hodgkin's (H) and Reed-Sternberg (RS) cells were positive for LMP-1 using IH, and in 21 cases (44%) positive signals were seen in H-RS cells with EBER 1 and 2 probes using ISH. A few EBER-positive non-malignant lymphocytes were seen in 17 cases. Thirty-two cases (71%) were EBV-positive by PCR. It is concluded that the PCR technique is the most sensitive method for detecting EBV in HD. However, this method cannot provide information about the cellular localization of EBV. ISH with EBER 1 and 2 probes is superior to immunohistochemical detection of LMP-1 with regard to sensitivity. The advantage that the latter two methods have over the PCR techniques is that it is possible to analyse whether the EBV infection occurs in the H-RS cells or in the admixed non-malignant cell population. Furthermore, this study supports the observation that EBV is associated with a considerable number of HD cases.

Epstein-Barr virus in exfoliated cells from the postnasal space. Viral detection by polymerase chain reaction is not a useful means of screening for nasopharyngeal carcinoma in high-risk populations.

There is a high incidence of undifferentiated nasopharyngeal carcinoma (NCP) in certain populations, including Greenland Eskimos. The cancer appears to have a causal association with Epstein-Barr virus (EBV), which is regularly found in NPC epithelial cells. With the aim of developing a method of screening or facilitating early diagnosis of NPC, we used the polymerase chain reaction to examine exfoliated nasopharyngeal cells for EBV in 54 Greenland and 17 white Danish subjects, none of whom was suspected of having NPC. EBV DNA was found in 81% of Greenland and 35% of Danish subjects. These findings support the concept of EBV infection leading in most cases to a chronic carrier state. It is concluded that EBV detection in nasopharyngeal cells is not at present a suitable method for identification of individuals at increased risk of developing NPC.

Detection of human papillomavirus (HPV) DNA in cervical swabs by the polymerase chain reaction: an evaluation of the sensitivity of the method in patients with HPV 16-harboring cervical lesions.

The polymerase chain reaction (PCR) was used to detect human papillomavirus (HPV) type 16 DNA in cervical swabs from 37 patients with HPV 16-harboring cervical lesions (15 carcinomas and 22 cervical intraepithelial neoplasias). Primers amplifying a sequence of the human beta-globin genome were used for internal control together with the HPV 16-specific primers. The cell samples were prepared for PCR analysis by two different methods: either by phenol/chloroform extraction or by boiling in the presence of a chelating agent. HPV 16 DNA was found in 27 swabs. The detection rates were identical with both methods of preparation. Four of the 10 false-negative swabs contained too little DNA to permit amplification with the genomic primers. Excluding these insufficient samples, the detection rate was 82%. Reasons for false-negative results may include low cell numbers or failure to obtain cells representative of the underlying lesion. In conclusion, the PCR offers a satisfactory method of HPV detection in cervical swabs. Cell preparation can be restricted to simple boiling with a chelating agent. For optimal results, samples containing less than 2 x 10(4) cells should be discarded, and genomic primers should be used for internal control.

Vulvar squamous cell carcinoma and papillomaviruses: indications for two different etiologies.

The vulvectomy specimens of 78 patients with vulvar squamous cell carcinoma were reviewed and examined for human papillomavirus (HPV) types 6, 11, 16, 18, and 33 by the polymerase chain reaction technique. The tumors were classified as keratinizing squamous cell carcinoma (KSC), as warty carcinoma (WC), and as basaloid carcinoma (BC). DNA of HPV types 16 and 33 was found in 2/51 KSC, in 12/17 WC, and in 10/10 BC. HPV types 6, 11, and 18 were not detected. Patients with WC and BC were younger, and 78% had VIN III lesions adjacent to the carcinoma. Patients with KSC were older and had a high incidence of dystrophic lesions, including lichen sclerosus, adjacent to the tumor. None of the KSC showed adjacent VIN III. In conclusion, vulvar carcinoma segregates into two categories, of which KSC seems to be the classic type, only rarely associated with HPV infection, and mostly affecting older women; WC and BC constitute an HPV-related subgroup of tumors occurring in younger patients and are associated with VIN III lesions from which they may emerge.

The value of CA 125 measurement before second-look laparotomy in patients with ovarian carcinoma.

Serum CA 125 levels were evaluated in 71 patients undergoing second-look laparotomy for primary epithelial ovarian cancer. With a cut-off limit of 35 U/ml, 17 of 45 patients with residual disease (38%) were CA 125-positive. All of 26 patients with pathological complete response were marker-negative. Second-look laparotomy may be avoided in a select group of patients with elevated CA 125 values.

Human papillomavirus and Epstein-Barr virus in the etiology of testicular germ cell tumours.

Epidemiological features suggest that the risk of testicular cancer may be related to exposure to unknown infectious agents, including viruses. Therefore a series of twenty specimens of testicular germ cell tumours, including preinvasive carcinoma in-situ, were tested for the presence of DNA sequences of two viruses with known transforming abilities, human papillomavirus (HPV) and Epstein-Barr virus (EBV). The polymerase chain reaction (PCR) technique was used. In none of the 19 successfully amplified samples were DNA sequences of HPV type 16 or type 18 detected. In six cases a faint trace of EBV DNA was revealed in one of two experiments. These samples were examined by immunohistochemical staining with specific antibodies raised against the EBV protein products and in-situ hybridization with specific molecular probes, and were confirmed to be negative. The study indicates that a significant direct involvement of HPV and EBV in human testicular germ cell carcinogenesis is unlikely. However, a putative growth-stimulating role of EBV-transformed lymphocytes, which are frequently present in the stromal tissues of testicular tumours, cannot be excluded.

Human papillomavirus types 11 and 16 detected in nasopharyngeal carcinomas by the polymerase chain reaction.

Most nasopharyngeal carcinomas (NPCs) are of the nonkeratinizing or undifferentiated types, which are consistently associated with Epstein-Barr virus (EBV). The smaller group of highly differentiated, keratinizing NPCs seems to be only infrequently associated with EBV. In order to examine whether these rare tumors were related to another oncogenic virus, the authors used the polymerase chain reaction to examine paraffin-embedded sections of 15 keratinizing NPCs for human papillomavirus (HPV) types 6, 11, 16, and 18 genomic sequences. HPV DNA was found in 4 tumors (1 HPV-11-positive, and 3 HPV-16-positive tumors). None of 23 undifferentiated or nonkeratinizing NPCs harbored HPV DNA. The putatively oncogenic HPV type 16 may thus be involved in the carcinogenesis of some EBV-negative keratinizing squamous cell nasopharyngeal carcinomas.

Immunoreactive inhibin-production in post-menopausal women with malignant epithelial ovarian tumors.

In post-menopausal women with a malignant epithelial ovarian tumor the follicle stimulating hormone (FSH) level was found to be significantly lower compared with healthy controls. We demonstrated immunoreactive (i.r.) inhibin in 20% of controls which was elevated to 60% of women with an ovarian tumor and correlating strongly to FSH in the tumor group (P = 0.0002). Steroid hormone levels were comparable in the two groups. In women with ovarian tumors the survival time for the i.r. inhibin-producing women was found to be 4.6 years compared with 0.9 year, or 5.1 times longer than in the non-producing women (P = 0.002). The site of i.r. inhibin production in these post-menopausal women is unknown, but i.r. inhibin production by the developing ovarian tumor or by the post-menopausal ovary may be regarded as a defense mechanism against an elevated gonadotrophin level (the gonadotrophin theory) which would promote further tumor growth. The recent suggestion that the alpha subunit of inhibin is a tumor suppressor gene is consistent with these results. The serum i.r. inhibin or alpha subunit concentrations might be used as an aid to diagnosis or as a prognostic indicator of survival in women with an ovarian carcinoma.

The post-operative gonadotropin level in post-menopausal women with epithelial ovarian cancer.

Serial estimates of the post-operative hormone levels were made in 15 women subjected to oophorectomy because of ovarian carcinoma. All women were post-menopausal. Pre-operatively, they had significantly lower follicle stimulating hormone (FSH) levels compared with an age-matched control group. Blood samples were collected after a median time of 8 months (139-378 days). After oophorectomy, significantly higher FSH values were found (P = 0.0002), whereas the luteinizing hormone (LH) values were not significantly changed. The inhibin, estradiol and progesterone values were found to be significantly lowered compared with the pre-operative sample. Total and unbound testosterone levels were significantly lower while dehydroepiandrosterone sulphate (DHEAS) and androstenedione levels were unchanged compared with the original sample and compared with controls. Most likely, estradiol and progesterone are produced by the epithelial malignant tumors, as the post-operative values are completely comparable with the primarily included healthy controls. The FSH is suppressed by inhibin and only to a minor degree by the steroid hormones as indicated by the correlation coefficients. Of great interest is the question whether inhibin production is random, or defensive, lowering the gonadotropin levels or influencing tumor growth in some hitherto unknown fashion.