Impact of genomic stability on protein expression in endometrioid endometrial cancer.

BACKGROUND: Genomic stability is one of the crucial prognostic factors for patients with endometrioid endometrial cancer (EEC). The impact of genomic stability on the tumour tissue proteome of EEC is not yet well established. METHODS: Tissue lysates of EEC, squamous cervical cancer (SCC), normal endometrium and squamous cervical epithelium were subjected to two-dimensional (2D) gel electrophoresis and identification of proteins by MALDI TOF MS. Expression of selected proteins was analysed in independent samples by immunohistochemistry. RESULTS: Diploid and aneuploid genomically unstable EEC displayed similar patterns of protein expression. This was in contrast to diploid stable EEC, which displayed a protein expression profile similar to normal endometrium. Approximately 10% of the differentially expressed proteins in EEC were specific for this type of cancer with differential expression of other proteins observed in other types of malignancy (e.g., SCC). Selected proteins differentially expressed in 2D gels of EEC were further analysed in an EEC precursor lesion, that is, atypical hyperplasia of endometrium, and showed increased expression of CLIC1, EIF4A1 and PRDX6 and decreased expression of ENO1, ANXA4, EMD and Ku70. CONCLUSION: Protein expression in diploid and aneuploid genomically unstable EEC is different from the expression profile of proteins in diploid genomically stable EEC. We showed that changes in expression of proteins typical for EEC could already be detected in precursor lesions, that is, atypical hyperplasia of endometrium, highlighting their clinical potential for improving early diagnostics of EEC.

Differential expression of ANXA6, HSP27, PRDX2, NCF2, and TPM4 during uterine cervix carcinogenesis: diagnostic and prognostic value.

BACKGROUND: Cytology-based diagnostics of squamous cervical cancer (SCC) precursor lesions is subjective and can be improved by objective markers. METHODS: IHC-based analysis of ANXA6, HSP27, peroxiredoxin 2 (PRDX2), NCF2, and tropomyosin 4 (TPM4) during SCC carcinogenesis. RESULTS: Expression of ANXA6, HSP27, PRDX2, and NCF2 in the cytoplasm of dysplastic cells increased from cervical intraepithelial neoplasia 2/3 (CIN2/3) to microinvasive cancer. Invasive SCC showed lower expression of TPM4 than CIN and normal epithelium. CIN2/3 with the highest sensitivity and specificity differed from normal epithelium by cytoplasmic expression of HSP27. Patients with cytoplasmic HSP27 expression in SCC deviating from that observed in normal epithelium had worse relapse-free (P=0.019) and overall (P=0.014) survival. Invasive SCC with the highest sensitivity and specificity differed from normal epithelium by expression of PRDX2 and TPM4 in the cytoplasm, from CIN2/3 by the expression of ANXA6 and TPM4 in the cytoplasm, and from microinvasive SCC by the expression of PRDX2 and ANXA6 in the cytoplasm. The number of sporadic ANXA6+ cells between the atypical cells increased from CIN2/3 to invasive SCC. CONCLUSION: Detection of expression changes of the proteins ANXA6, HSP27, PRDX2, NCF2, and TPM4 in SCC precursor lesions may aid current cytological and pathological diagnostics and evaluation of prognosis.

Differential tissue-specific protein markers of vaginal carcinoma.

The objective was to identify proteins differentially expressed in vaginal cancer to elucidate relevant cancer-related proteins. A total of 16 fresh-frozen tissue biopsies, consisting of 5 biopsies from normal vaginal epithelium, 6 from primary vaginal carcinomas and 5 from primary cervical carcinomas, were analysed using two-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry. Of the 43 proteins identified with significant alterations in protein expression between non-tumourous and tumourous tissue, 26 were upregulated and 17 were downregulated. Some were similarly altered in vaginal and cervical carcinoma, including cytoskeletal proteins, tumour suppressor proteins, oncoproteins implicated in apoptosis and proteins in the ubiquitin-proteasome pathway. Three proteins were uniquely altered in vaginal carcinoma (DDX48, erbB3-binding protein and biliverdin reductase) and five in cervical carcinoma (peroxiredoxin 2, annexin A2, sarcomeric tropomyosin kappa, human ribonuclease inhibitor and prolyl-4-hydrolase beta). The identified proteins imply involvement of multiple different cellular pathways in the carcinogenesis of vaginal carcinoma. Similar protein alterations were found between vaginal and cervical carcinoma suggesting common tumourigenesis. However, the expression level of some of these proteins markedly differs among the three tissue specimens indicating that they might be useful molecular markers.

Frequent gain of the human telomerase gene TERC at 3q26 in cervical adenocarcinomas.

The level of genomic amplification of the human telomerase gene TERC, which maps to chromosome band 3q26, was determined in primary cervical adenocarcinomas. Interphase nuclei prepared from archival material of 12 primary cervical adenocarcinomas, eight of which were human papillomavirus positive, were hybridised with a triple colour probe set specific for centromeres of chromosomes 3 and 7 and the TERC gene. We observed high proportions of nuclei with increased absolute copy numbers for TERC in all tumours (mean 3.3; range 2.3-5.2). Amplification of the human telomerase gene TERC is a consistent aberration in cervical adenocarcinomas. Therefore, application of our probe set may provide an objective genetic test for the assessment of glandular cells in Pap smears and hence for the diagnosis of cervical adenocarcinomas.

Clinical and histopathologic factors related to prognosis in primary squamous cell carcinoma of the vagina.

The goal of this retrospective study concerning primary carcinoma of the vagina (PCV) was to analyze clinical and histopathologic prognostic factors in one of the largest known material, which comprised 314 patients. PCV is a rare disease, and the majority of published studies are based on small materials; therefore, the established knowledge concerning prognostic factors is insufficient. Routine treatment is based on irradiation with risk for undertreatment or overtreatment, which leads to unnecessary complications in the absence of prognostic factors. The overall 5-year disease-specific survival rate in this study was 45% and in stage I 75%. In the univariate statistical analysis, several factors correlated significantly with disease-specific survival. However, in the multivariate analysis, there were only three factors that independently could predict poor survival-high age at diagnosis, large tumors (> or =4 cm), and advanced stage. Common background factors with no prognostic significance were prior hysterectomy, other gynecological malignancies, and pelvic irradiation. In conclusion, this study has elucidated three strong prognostic factors that might be considered in the choice of therapy and also for modification of the FIGO guidelines. Increased knowledge concerning complementary biologic markers to discriminate between low- and high malignant tumors is however of great importance.

The carcinogenic role of oncogenic HPV and p53 gene mutation in cervical adenocarcinomas.

Thirty tumors were collected from our archive of cervical adenocarcinomas. They were examined with respect to the content of oncogenic HPV and presence of mutations in the p53 gene exons 5 through 8. Furthermore, available clinical information on the cases was reviewed. For the detection of p53 gene and presence of oncogenic HPV, PCR followed by direct sequence analysis of the amplified DNA was employed. Seventeen tumors were identified as HPV-positive, comprising both HPV types 18 and 16. Six cases showed a p53 gene mutation, of which five were of the missence and one of the silent type. No statistical correlation between the occurrence of oncogenic HPV and presence of p53 gene mutation (p = 0.67) was recorded. Among the tumors with p53 gene mutation, three were HPV-positive and three were HPV-negative. The determination of p53 gene mutations was not related to clinical findings such as the stage of the tumor or presence of metastases of the lymph nodes. However, p53 gene mutations were somewhat more prevalent in low differentiated tumors (p < 0.02). The results indicate that oncogenic HPV and p53 gene mutations have independent carcinogenic roles in cervical adenocarcinomas.

Parallel cyclin E and cyclin A expression in neoplastic lesions of the uterine cervix.

Cyclin E levels are high during late G1 and early S-phase in normal cells. The cyclin E expression over the cell cycle in tumours is not fully known. The impact on patient outcome by high cyclin E levels during other parts of the cell cycle than late G1- and early S-phase is unknown. We set out to study the expression of cyclin E over the cell cycle in cervical carcinomas. Using immunofluorescence staining of cyclin A, digital microscopy, and digital image analysis, we determined which cells in a tissue section that were in S- or G2-phase. M-phase cells were detected by morphology. By simultaneously staining for cyclin E, we investigated the variation in cyclin E levels over the cell cycle in cervical carcinoma lesions. In a case-control study, in which each deceased patient was matched with a patient still alive and well after >5 years of follow-up, we found that the deceased patients had a considerably higher fraction of cyclin A-positive cells staining for cyclin E than the survivors (n = 36). We conclude that parallel cyclin E and cyclin A expression is an indicator for poor outcome in cervical carcinomas. In addition, we investigated the expression pattern of cyclin E and cyclin A in consecutive biopsy samples from cervical carcinomas at different stages, as well as in human papillomavirus positive or negative adenocarcinomas in order to further study the cyclin E and cyclin A expression pattern in neoplastic lesions of the uterine cervix.

The clinicopathologic significance of laminin-5 gamma2 chain expression in cervical squamous carcinoma and adenocarcinoma.

Carcinoma of the uterine cervix is one of the most prevalent malignancies among women in developing countries and the third most common type worldwide. Squamous cell carcinoma predominates in the cervix uteri, while adenocarcinoma and adenosquamous carcinomas represent about 10-15% of all cervical cancers. Many studies have confirmed that the human papillomavirus (HPV) is the most important etiologic factor in the development of cervical cancer. The aim of our study was to investigate the expression of the laminin-5 gamma2 chain in primary malignancies of the cervix uteri and to focus on the clinicopathologic significance of the expression of the laminin-5 gamma2 chain in cervical squamous carcinoma and adenocarcinoma with respect to age and survival of the patients. The study consisted of a total of 89 cases of invasive cervical cancer (54 squamous carcinomas and 35 adenocarcinomas). The laminin-5 gamma2 chain was found in 80% of all the squamous carcinoma and in 66% of cervical adenocarcinoma. There was no correlation of the high expression of laminin-5 with survival. The univariate analysis in squamous cell carcinoma showed that factors such as the stage of the disease and positive lymph nodes had an impact on the survival of the patients, whereas in the multivariate analysis, only age at diagnosis was an independent prognostic factor. However, in cases with cervical adenocarcinoma, only the stage of the disease was an independent prognostic factor. There was no difference between HPV-positive and HPV-negative tumors concerning the high expression of laminin-5 gamma2 chain. Our results indicate that the majority of the primary cervical tumors, especially squamous cell carcinoma, showed expression of laminin-5 gamma2 chain immunoreactivity. Independent prognostic values for the survival of the patients were age and stage of the disease.

Primary carcinoma of the vagina: factors influencing the age at diagnosis. The Radiumhemmet series 1956-96.

The objective to this retrospective study of 341 cases of primary carcinoma of vagina (PCV) diagnosed between 1956 and 1996 was to find whether epidemiological, clinical, and histopathological variables were related to the age at diagnosis of patients with PCV. The univariate statistical analysis showed that younger age at diagnosis significantly correlated with a history of cervical dysplasia, hysterectomy, gynecological infections, and tumors located in the upper part of the vagina, whereas older age at diagnosis significantly correlated with late menarche and exophytically growing tumors. In the multivariate regression analysis, the remaining independent predictors were a history of cervical dysplasia and age at menarche. Further, parity >/=4 as well as nulliparity, smoking, and unstable marital status were more common among patients with PCV than among those in the general Swedish female population. This study indicates that the etiology of vaginal carcinoma may be age related. In young patients, the disease seems to be etiologically related to cervical neoplasia and thus human papillomavirus (HPV) dependent. However, in the most common age group, the older patients, there might be another (probably non-HPV-related) etiology associated with hormonal factors and trauma to the vagina.

Protein expression patterns in primary carcinoma of the vagina.

Protein patterns in six samples from primary vaginal cancers, in five from normal vaginal tissue and in five primary cervical cancers, were analysed using two-dimensional polyacrylamide gel electrophoresis (2-DE). Protein expression profile was evaluated by computer-assisted image analysis (PDQUEST) and proteins were subsequently identified using matrix-assisted laser desorption/ionisation mass spectrometry. The aim was to analyse the protein expression profiles using the hierarchical clustering method in vaginal carcinoma and to compare them with the protein pattern in cervical carcinoma in order to find a helpful tool for correct classification and for increased biomedical knowledge. Protein expression data of a distinct set of 33 protein spots were differentially expressed. These differences were statistically significant (Mann-Whitney signed-Ranked Test, P<0.05) between normal tissue, vaginal and cervical cancer. Furthermore, protein profiles of pairs of primary vaginal and cervical cancers were found to be very similar. Some of the protein spots that have so far been identified include Tropomyosin 1, cytokeratin 5, 15 and 17, Apolipoprotein A1, Annexin V, Glutathione-S-transferase. Others are the stress-related proteins, calreticulin, HSP 27 and HSP 70. We conclude that cluster analysis of proteomics data allows accurate discrimination between normal vaginal mucosa, primary vaginal and primary cervical cancer. However, vaginal and cervical carcinomas also appear to be relatively homogeneous in their gene expression, indicating similar carcinogenic pathways. There might, further, be a possibility to identify tumour-specific markers among the proteins that are differentially expressed. The results from this study have to be confirmed by more comprehensive studies in the future.

Human papillomavirus infection, centrosome aberration, and genetic stability in cervical lesions.

DNA replication and centrosome duplication have to be strictly synchronized to guarantee genomic stability. p53, pRb, cyclin E, and cyclin A are reported to be involved in the synchronizing process. We investigated the relationship between papillomavirus infection, centrosome aberration and aneuploidy during genesis of cervical carcinoma. The number of centrosomes found in cells from normal cervical epithelium (n = 5), condyloma acuminata (n = 5), cervical intraepithelial neoplasia (CIN) I, II, and III (n = 14) and invasive cervical carcinoma (n = 5) was analyzed by gamma tubulin immunofluorescence staining. The nuclear DNA content was investigated by image cytometry and human papillomavirus (HPV) infection was determined by polymerase chain reaction. Normal epithelia and condyloma acuminata showed cells with one or two centrosomes, whereas CIN lesions showed cells with an increasing number of centrosomes. This abnormality was found to be lowest in CIN I lesions, increased with advancing grade of CIN and was highest in lesions of invasive carcinomas. In parallel, an increasing number of cells with aberrant DNA content was seen. All carcinomas and all except one of the CIN III lesions showed aneuploidy. Three CIN II cases were aneuploid and two cases with CIN I were tetraploid. Normal epithelia and condyloma acuminata showed diploidy. All invasive carcinomas and lesions with CIN were positive for high-risk HPV types 16, 18, or 31, except one invasive carcinoma and one CIN II lesion positive for universal primers only. Three condyloma acuminata were HPV 16-positive and one HPV 6-positive. The results suggest that high-risk HPV infection is correlated to a progressive numerical disturbance of centrosome replication followed by progressive chromosomal aberrations in CIN lesions and invasive carcinomas.

Carcinoma of the cervical stump. The radiumhemmet series 1959-1987. Treatment and prognosis.

BACKGROUND: The purpose of this retrospective study is to evaluate the longterm prognosis for cervical stump cancer compared to matched controls with cancer in an intact uterus. METHODS: From 1959 to 1987, 145 patients were treated for an infiltrating carcinoma of the cervical stump at Radiumhemmet representing 2.2% of all cervical cancers. Three control cases to each case were selected from the cohort of cervical carcinoma cases - matched to year of treatment, stage, histology and age (plus, minus 2 years). Actuarial survival was calculated for cases and controls. Survival differences were analyzed with the Kaplan-Meier technique. The age distribution for cases ranged between 36 and 84 years with a mean age of 60.6 years. The mean age for the control series is 9 years of age (range 35-86 years). Among the cases 87.6% were squamous cell carcinoma and 12.4% were adenocarcinomas. Treatment of carcinoma of the uterine stump at Radiumhemmet followed the same modality as was practised for ordinary cervical cancer cases i.e. two brachyradium applications with 3 weeks interval followed by external irradiation. The dose of irradiation from the intracavitary application given to the stump cancers was lower than that given to comparable cases of the common cervical cases. RESULTS: No evidence was found of poorer longterm prognosis for radiologically treated squamous cell carcinoma of the uterine stump compared to that of the ordinary cervical carcinomas. Stump cancers of the adenocarcinoma type had a worse prognosis than adenocarcinomas in an intact uterus (p<0.07) and also compared with stump cancers of the squamous epithelial type (p=0.05). The complication rate was higher for the stump cancer cases compared with that for cervical cancers in intact uterus. The mean time interval from subtotal hysterectomy to the stump cancer diagnosis was 17.6 years with a range from 1 to 46 years. CONCLUSIONS: Recent discussions argue for a better sexual function after subtotal hysterectomy. Our study gave no convincing argument in terms of poorer prognosis for radiologically treated carcinoma of the uterine stump compared to that of the total cervical cancer series. It is thus necessary to weigh the possible gains with subtotal hysterectomy against the relatively low risk to fall victim of a stump cancer. Complications following surgery, as well as possible physiologic and sexual functions of the cervix, should be taken into account.

Recurrent fallopian tube carcinoma: TP53 mutation and clinical course.

Primary fallopian tube carcinoma is a rare, aggressive gynecological cancer; little is known about its cause. Previous studies have indicated that p53 immunopositivity is correlated with short-term survival in primary fallopian tube carcinoma. We examined p53 and p21/WAF1 immunostaining and TP53 mutation in exons 5 to 8 by single-stranded conformation polymorphism and constant denaturant gel electrophoresis in nine cases of primary fallopian tube carcinoma and their metastases/recurrences from patients who survived for between a few months and more than 20 years after diagnosis. We found that 1.) p53 immunopositivity without detectable p21/WAF1 immunostaining did not correlate with TP53 mutations in the conserved domains; 2.) mutations in TP53 occurred in two metastases/recurrences but not in their corresponding primary tumors; 3.) in two cancers, a TP53 mutation was observed in the primary tumor but not in the metastases/recurrences; 4.) constant denaturant gel electrophoresis seems to be more sensitive than single-stranded conformation polymorphism in detecting TP53 mutations; and 5.) in the nine cases studied, p53 immunoreactivity and/or TP53 mutation analysis did not correlate with tumor progression, survival, or response to treatment.

Cancer of the vagina: Laminin-5gamma2 chain expression and prognosis.

The purpose of this experiment was to investigate the expression and the prognostic impact of the gamma2 subchain of laminin-5 in vaginal malignancies. The outcome of the rare disease primary carcinoma of the vagina is poor and little is known about prognostic markers. The gamma2 chain of laminin-5, an epithelial basement membrane protein, is thought to play a crucial role in tumor cell adhesion, migration, and proliferation, and may thus be an additive potential marker. Archival, paraffin-embedded sections were stained immunohistochemically with an antibody against the gamma2 chain of human laminin-5 protein. The material consisted of 59 cases of primary vaginal malignancies, subdivided into short- and long-time survivors. All invasive malignancies of epithelial origin were positively stained with the antibody against the gamma2 chain. High expression of the gamma2 chain correlated significantly in an univariate analysis with short-time survival (P = 0.041), but in the multivariate analysis only age and tumor size were independent prognostic factors. A significant intercorrelation between large tumors and high gamma2 chain immunoreactivity was found (P = 0.003). These results indicate that laminin-5gamma2 subchain expression in primary vaginal carcinomas is of prognostic impact. However, in a multivariate analysis only patient age and tumor size had independent prognostic value.

Malignant mixed müllerian tumors of the ovary: histopathologic and clinical review of 36 cases.

Among 2,895 malignant ovarian tumor cases referred to Radiumhemmet, Stockholm from 1975 through 1995, 36 were certified to be malignant mixed müllerian tumors. The overall prognosis was poor with only 18% five-year actuarial survival (median survival 16.6 months). Five patients are still surviving after 75, 68, 117, 121, and 168 months, respectively. Fifteen women treated with melphelan, doxorubicin (adriamycin) and cisplatin (MAP) had a five-year actuarial survival of 33.3% and a median survival of 19.8 months. In a multivariate analysis taking into account stage, age, radiation, type of chemotherapy, histopathologic type and completeness of surgery, the most important predictors for survival were stage (stages I-II vs stages III-IV, P < 0.05), histopathologic type (homologous vs heterologous, P < 0.05), and type of chemotherapy (MAP or CAP vs other types, P < 0.05). We concluded that homologous tumor and chemotherapy containing cisplatin, doxorubicin, and melphalan, as well as early stage of the tumor, provided the optimal survival rate.

Primary carcinoma of the fallopian tube: comparative genomic hybridization reveals high genetic instability and a specific, recurring pattern of chromosomal aberrations.

Primary fallopian tube carcinoma (PFTC) is a rare and highly aggressive tumor. Twelve cases of PFTC (stages IA to IV) were analyzed by comparative genomic hybridization. The most consistent DNA gain was mapped to chromosome arm 3q in 11 of 12 cases. In six cases, the gain of 3q was present as a high level copy number increase (amplification) with a consensus region mapped to 3q26.2-qter. In the 12 cases, other frequent gains were located on chromosome arms 1q (in 11 cases), 2q (in 10), 7q (in 9), 8q (in 9), 5p (in 8), 6p (in 7), 12p (in 7), and 14q (in 6). Frequent copy number losses occurred on chromosome arms 16q (in 8 cases), 22q (in7), 6q (in 6). 8p (in 6), 18q (in 6), Xq (in 6), 1p (in 5), and 17p (in 5). All chromosomes were involved in chromosomal aberrations and the average number of copy alterations per case was 19.7. None of the 12 carcinomas revealed the presence of human papillomavirus (HPV) genomes. All of the cases exhibited crude aneuploidy. Strong p53 immunoreactivity could be observed in 10 of 12 cases while p21/WAF1 expression was low or undetectable. These results indicate that PFTC is a genomically highly unstable cancer, an observation that is in agreement with the poor prognosis associated with this tumor. A high frequency of 3q-gains has also been observed in HPV-related carcinomas of the uterine cervix. However, none of the PFTC was HPV related, suggesting that the 3q-gain is independent from HPV DNA.

Advanced-stage cervical carcinomas are defined by a recurrent pattern of chromosomal aberrations revealing high genetic instability and a consistent gain of chromosome arm 3q.

We have analyzed 30 cases of advanced-stage cervical squamous cell carcinoma (stages IIb-IV) by comparative genomic hybridization (CGH). The most consistent chromosomal gain in the aneuploid tumors was mapped to chromosome arm 3q in 77% of the cases. Acquisition of genetic material also occurred frequently on Iq (47%), 5p (30%), 6p (27%), and 20 (23%). Recurrent losses were mapped on 2q (33%), 3p (50%), 4 (33%), 8p (23%), and 13q (27%). High-level copy number increases were mapped to chromosome 8, chromosome arms 3q, 5p, 8q, 12p, 14q, 17q, 19q, 20p, and 20q, and chromosomal bands 3q26-27, 9p23-24, 11q22-23, and 12p13. In the majority of the cases, the presence of high-risk human papilloma virus genomes was detected. High proliferative activity was accompanied by crude aneuploidy. Increased p21/WAF-I activity, but low or undetectable expression of TP53 were representative for the immunophenotype. This study confirms the importance of a gain of chromosome arm 3q in cervical carcinogenesis and identifies additional, recurrent chromosomal aberrations that are required for progression from stage I tumors to advanced-stage carcinomas.

Occupational dermatoses in nursery workers.

BACKGROUND: Several workers at a nursery complained of skin problems. An occupational allergic contact dermatitis from tulip was diagnosed in 2 of the workers. OBJECTIVE: The aim of this study was to survey the frequency of occupational dermatoses among nursery workers and to investigate whether the tulip sensitizer alpha-methylene-chi-butyrolactone traces all contact allergy to tulip. METHODS: A questionnaire was delivered to 41 employees, and everyone with a present or previous skin disease was offered a consultation including patch testing with the known tulip sensitizer as well as two types of tulip extracts and parts of the plant. RESULTS: Occupational dermatoses were diagnosed in 11 workers, allergic contact dermatitis in 9 workers (tulip and/or daffodil), and irritant contact dermatitis in 2 workers. CONCLUSION: Occupational dermatoses are common among nursery workers. All workers with contact allergy to tulip was traced by alpha-methylene-chi-butyrolactone.

Value of uterine artery Doppler in endometrial cancer.

Twenty-seven women with endometrial cancer were studied with Doppler ultrasound coupled with a vaginal probe. Pulsatility index of the flow velocity of the uterine artery was recorded and compared to that of a control group. The subjects and the controls did not differ in blood flow measurements. There was no correlation between severity of disease and flow velocimetry values. Eleven of the patients underwent brachytherapy prior to surgery. Administration of brachytherapy resulted in a decrease of the peripheral resistance. The results of this study indicate that Doppler velocimetry of the uterine artery is not a valuable tool in discriminating between malignant and benign endometrium.