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Biomedical subjects

R Holm

Publications and source records attributed to R Holm.

At least 55 records · Page 3Linked to original sources

Disruption of the E1 and E2 reading frames of HPV 16 in cervical carcinoma is associated with poor prognosis.

The E1 and E2 reading frames of 158 cervical carcinoma samples containing human papillomavirus (HPV) 16 were mapped using polymerase chain reaction (PCR). The reading frames were amplified using primers spanning the entire genes. Of the analyzed samples, 23% showed no amplification with the E1 primers and 29% showed no amplification with the E2 primers. There was an overlap, but not complete identity, between the E1- and E2-disrupted groups. All E1- and E2-negative samples were further analyzed with primers spanning subsections of the E1 and E2 reading frames, which together covered the entire genes. Of the 35 samples negative for E1, 11 were positive in specific amplification of the 3' end of the E1 gene. Several different subsections of E2 could be amplified from most samples negative for the entire gene (37/46). Five classes of patterns were found, in which either all subsections of the E2 gene or subsections in the 5', middle, or 3' end were disrupted. Although a variable pattern of disruption/deletion in the E1-E2 area of the HPV 16 genome was found in cervical carcinoma, the 5' end disruption was the most common one in both E1 and E2. Patients with carcinomas showing disruptions in E1/E2 had a poorer survival than those without such changes, and E1 disruptions were the most important prognostically.

DNA, Viral↗

TP53 protein accumulation and gene mutation in relation to overexpression of MDM2 protein in ovarian borderline tumours and stage I carcinomas.

Three hundred and seventy-four early-stage ovarian tumours, including 27 borderline tumours and 347 stage I carcinomas, were investigated immunohistochemically for overexpression of the TP53 and MDM2 proteins. TP53 (p53) and MDM2 alterations were detected in 15 and 4 per cent of borderline tumours, and in 50 and 13 per cent of stage I carcinomas, respectively. Mutations in the TP53 gene (exons 5-8) were demonstrated in 29 of the 50 stage I carcinomas studied, using denaturing gel electrophoresis followed by direct sequencing. TP53 overexpression was seen less often in tumours of mucinous and endometrioid type than in tumours of other histological types and more often in moderately and poorly differentiated than in well differentiated tumours. MDM2 protein overexpression was seen more often in clear cell carcinoma than in tumours of other histological types. These results indicate that TP53 abnormalities play a crucial role, and MDM2 abnormalities a minor role, in the development of early-stage ovarian carcinoma. There was no significant association between TP53 or MDM2 alterations and survival in multivariate analysis.

Adolescent↗

A novel grid polymerase chain reaction (G-PCR) approach at ultrastructural level to detect target DNA in cell cultures and tissues.

A novel grid polymerase chain reaction (G-PCR) method has been developed to be used at the ultrastructural level and with a high degree of resolution. Samples applied to test the method were fresh cell lines (CaSki, SiHa) and HPV-16 DNA-containing tissues rescued from routine paraffin blocks. The specimens were embedded in Epon-Araldite and/or hydrophilic-resin LRWhite. Ultrathin sections mounted on grids were subjected to G-PCR using an HPV-16-specific primer set. The amplified products were identified by auro-immunohistochemical labelling of the biotinylated nucleotide. The results indicated successful amplification of target DNA in both cell and tissue samples, being confined to the intranuclear region. The negative controls [HeLa cells, isolated mammary carcinoma cell cultures (MCF 7, and T47-D) (ATCC) (U.S.A.), normal thyroid tissue and steroid-producing tumour tissue] failed to exhibit any amplification of the target DNA sequences. The sensitivity of the G-PCR system was evaluated by performing a parallel in situ hybridization (ISH) of serial sections. The signals obtained from G-PCR were more intense than those of ISH and more informative as to the precise subcellular localization of amplicons.

Cell Line↗

Synovial sarcoma: immunohistochemical expression of P-glycoprotein and glutathione S transferase-pi and clinical drug resistance.

Our purpose was to study the role of the expression of P-glycoprotein (Pgp) and glutathione S transferase-pi (GST-pi) in predicting the response to chemotherapy, relapse-free interval, and survival of patients with synovial sarcoma (SS). Thirty-seven cases of primary SS, without regional lymph node or distant metastases, were studied. There were 17 females and 20 males, ranging in age from 7 to 81 years (median, 31 years) with tumors located in the lower extremity (n = 24) upper extremity (n = 5) and trunchus (n = 8). The cases were retrospectively studied without knowledge of clinical course to compare the immunohistochemical expression of Pgp and GST-pi, flow cytometry parameters (ploidy and % of cells in S+G2 phases), and PCNA and Ki-67 labeling of primary tumors before any therapy, with that observed in local recurrences and metastases after chemotherapy. The relationship of the aforementioned parameters with clinicopathological features (gender, age, and histo-blood group of the patients, size, location, histological subtype. TNM stage, and clinical response to chemotherapy of the tumors) was also evaluated. Results revealed that Pgp and GST-pi were expressed in 29.7% and 40.5% of the cases, respectively. In 48.6% of the tumors there was expression of a least one of the drug resistance markers. The markers were coexpressed in 25.0% of the tumors. The prevalence of Pgp expression was lower, but not significantly, in stage I-II (17.6%) than in stage III (40.0%) tumors, and also in cases without clinical progression (16.7%), than in cases with (36.0%). No such differences were observed for GST-pi expression. Pgp and GST-pi expressions were significantly associated with biphasic SS and were particularly noticeable in solid/glandular areas of biphasic SS. The expression of the drug resistance markers was not significantly associated with gender, age, and histo-blood group of the patients, dimension, location, and proliferative activity of the tumors; it was also not significantly related to relapse-free interval and survival of the patients. The expression of Pgp and GST-pi was not significantly associated either to response to chemotherapy or influenced by chemotherapy. We conclude that Pgp and GST-pi expressions are not good predictors response to of the chemotherapy in patients with localized SS. Other drug resistance mechanisms may be active in SS.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

A comparison of different modes for the detection of p53 protein accumulation. A study of bladder cancer.

The aim of the present study was to evaluate different techniques for the analysis of p53 protein accumulation in human bladder cancer. The accumulation was evaluated in 23 carcinomas by immunoblotting (IB), immunohistochemistry (IHC) and flow cytometry (FCM). The results revealed that six (26%), eight (35%) and ten (43%) of the tumours were p53 protein positive by IB, IHC and FCM, respectively. Mutation analysis of the TP53 gene confirmed mutations in 8 of 9 tumours which showed increased levels of p53 protein by FCM. Our results indicate that IHC could be applied for studies of p53 protein accumulation in archival formalin fixed, paraffin-embedded bladder tumours. However, FCM is a more sensitive and objective method for the detection of p53 protein than IHC and this should be taken into account when routinely evaluating the p53 protein accumulation by IHC.

Biopsy↗

HPV 16 infection in a patient with two primary squamous cell carcinomas: of the uterine cervix and the anal mucosa.

A 43-year-old woman presented with metrorrhagia and a polypoid anal tumor. Cervical dilatation and endocervical curettage and resection of the anal tumor revealed two primary squamous cell carcinomas: of the uterine cervix and the anal mucosa. HPV 16 was identified in both tumors using an in situ hybridization technique with probes against HPV 6, 11, 16, 18, 31 and 33. The cervical carcinoma was weakly positive for p53 protein, whereas the anal tumor was p53 protein negative. Five years later a recurrence of the cervical carcinoma was diagnosed and a hysterectomy was performed. Of 3,013 patients with squamous cell carcinoma of either the cervix or the anus registered in our files, only one had a primary carcinoma in both locations. It was concluded hat HPV-16-induced squamous cell carcinoma may occur simultaneously in the cervix and the anus, and carcinoma in either of the two locations has to be ruled out when dealing with HPV infection in the anogenital tract.

Adult↗

Diagnostic implications of p53 protein reactivity in nasal mucosa of nickel workers.

OBJECTIVE: To investigate whether the quantitation of p53 protein reactivity in nasal biopsies could be related to nickel exposure by comparing nickel workers with various control groups. STUDY DESIGN: Nasal biopsies taken from nickel workers (n = 93) were compared immunohistochemically to various controls, including office staff members (n = 34) and hospital attendants (n = 6). The material was studied immunohistochemically with p53 antibody DO-1. p53 Protein-positive cells were counted at 400x magnification. RESULTS: p53 Protein reactivity was found in 54% (49/93) of nickel workers, 50% (17/34) of office staff members, 67% (4/6) of hospital attendants. No differences were seen between roasting/smelting, electrolysis and other workers in the refinery. The positive cells were present predominantly in the basal layer of the epithelium. The number of positive nasal cells per field in the hospital attendants on the average was half of that in the workers and the office staff in the refinery. In no case in the control group were more than 10 cells per field seen. No significant differences in p53 protein positivity were observed between the three nickel worker groups and between production workers and office staff members. CONCLUSION: Accumulation of p53 protein in nickel workers seems not to be attributable to nickel exposure. The lack of p53 protein positivity in fetal tissues shows that the accumulation of p53 protein is an event taking place after birth. Stimuli in the natural environment during life may explain p53 protein positivity.

Adolescent↗

[Ultrasound biometry of the fetal lung--measurement planes and reference values].

The aim of the study is to present biometrical data of the fetal lung measured in real-time sonography in mid-pregnancy. In 313 healthy fetuses of singleton pregnancies between the 15th and 30th week of gestation the fetal lung was measured in the horizontal, sagittal and frontal cross-sectional planes. In each plane the lung was measured in the following levels: clavicula, heart at the atrioventricular level (four chamber view) and diaphragm, as well as the lung length. During observation time all parameters showed a significant linear increase (r2 > 0.9; p < 0.001). Furthermore we found a high correlation between the same lung measurements collected in the different planes. The intraobserver variability was 3-8%. Such references of different planes could be helpful for differentiation of a small (hypoplasia) or a large lung (malformation).

Female↗

Evaluation of the prognostic significance of cathepsin D, epidermal growth factor receptor, and c-erbB-2 in early cervical squamous cell carcinoma. An immunohistochemical study.

BACKGROUND: This study evaluated the prognostic significance of immunohistochemical staining for cathepsin D, epidermal growth factor receptor (EGFR), and c-erbB-2 in patients with early cervical squamous cell carcinoma. METHODS: This retrospective analysis comprised 132 patients, all subjected to radical hysterectomy with bilateral pelvic lymphadenectomy for International Federation of Gynecology and Obstetrics (FIGO) Stage IB cervical squamous cell carcinoma. Immunohistochemical staining was correlated with various histopathologic and morphologic characteristics (i.e., tumor size, grade of differentiation, vessel invasion, invasion into parametria, and lymph node metastasis) and relapse free survival. RESULTS: Positive staining for cathepsin D was observed in 47% of tumors, more frequent in tumors giving rise to lymph node metastases. The relapse free survival was lower for patients with cathepsin D positive tumors. Overexpression of EGFR was observed in 25.8% of the tumors. There was no correlation with any of the histopathologic variables investigated. Relapse free survival was lower for patients with tumors overexpressing EGFR. Immunohistochemical staining for c-erbB-2 was observed in 12.1% of tumors with no correlation with relapse free survival. In multivariate analysis, immunostaining of cathepsin D and EGFR obtained independent prognostic significance, and considered together (both negative, one positive, or both positive) was the strongest prognostic factor after tumor size. CONCLUSIONS: Immunohistochemical staining for cathepsin D and EGFR is useful as a tool for evaluation of tumor aggressiveness in patients with early cervical squamous cell carcinoma.

Carcinoma, Squamous Cell↗

Evaluation of the prognostic significance of nm23/NDP kinase protein expression in cervical carcinoma: an immunohistochemical study.

The objective of this study was to evaluate the prognostic significance of immunohistochemical staining for nm23/nucleoside diphosphate (NDP) kinase in cervical carcinoma. A retrospective analysis of 176 patients with cervical carcinoma FIGO stage IB treated with radical hysterectomy and pelvic lymphadenectomy from 1987 to 1990 was conducted. Immunohistochemical staining using the polyclonal nm23-H1/NDP kinase A antibody was correlated to various histopathological and morphological characteristics (tumor size, histologic type, grade of differentiation, vessel invasion, invasion into parametria, and lymph node metastasis) and relapse-free survival. For controls, sections were obtained from 10 hysterectomy specimens with normal cervical epithelium. Staining for nm23/NDP kinase was observed in 90% of control cases and in 70.5% of cases of cervical carcinoma, more frequent in squamous and adenosquamous cell carcinoma than in adenocarcinoma and more frequent in poorly differentiated than in more highly differentiated tumors. There were no differences related to size of tumor or invasion into vessels or parametria or occurrence of lymph node metastasis. The relapse-free survival was lower for patients with squamous cell and adenosquamous tumors with positive immunostaining for nm23/NDP kinase than for those with negative tumors when evaluated in univariate analysis. In multivariate analysis with tumor size, vessel invasion, invasion into parametria, grade of differentiation, and lymph node metastasis included, this difference was no longer significant. In patients with adenocarcinoma no difference was found. In conclusion, we did not find immunostaining for nm23/NDP kinase to be a useful indicator for prognosis in cancer of the uterine cervix.

Adenocarcinoma↗

Evaluation of the prognostic significance of nm23/NDP kinase and cathepsin D in anal carcinomas. An immunohistochemical study.

Reduced expression of nm23/NDP kinase and increased expression of cathepsin D seem to be correlated with a high metastatic potential for a variety of malignancies. Nm23/NDP kinase and cathepsin D have been correlated with several clinical variables, including survival in 96 patients with squamous cell carcinoma of the anal canal. Immunohistochemical methods were used on paraffin-embedded biopsies. Seventy-six (79%) anal carcinomas were nm23/NDP kinase positive, whereas 35 (36%) and 28 (29%) of the cases were cathepsin D positive in tumor cells and stromal cells, respectively. We have found no indication that the extent of cathepsin D staining has any prognostic significance. The overall survival of patients with tumours positive for nm23/NDP kinase in the cytoplasm was significantly shorter than that of patients with anal carcinomas negative for nm23/NDP kinase.

Adult↗

TP53 alterations in atypical ductal hyperplasia and ductal carcinoma in situ of the breast.

Abnormalities in the TP53 tumour suppressor gene in 75 atypical ductal hyperplasias and 62 ductalcarcinomas in situ (DCIS) of the breast were studied using immunohistochemistry and mutation analysis. Accumulation of p53 protein was detected in 10 out of 62 (16%) DCIS, whereas no cases of positive staining was observed in the atypical lesions. TP53 mutations were identified in four out of 30 (13%) DCIS by constant denaturant gel electrophoresis (CDGE). Two of these cases were positive and two negative for p53 protein. A total of 12 out of 62 DCIS (19%) carried TP53 mutation and/or p53 protein over-expression. The present results suggest that TP53 alterations may be important in the development of a subset of DCIS.

Breast↗

Involvement of the pRb/p16/cdk4/cyclin D1 pathway in the tumorigenesis of sporadic malignant melanomas.

Biopsies from 61 sporadic metastatic malignant melanomas and five melanoma cell lines were examined for homozygous deletions and mutations in the CDKN2 gene (p16). As the p16 protein is involved in a cell cycle regulatory pathway consisting of at least pRb, cdk4 and cyclin D1, the tumours were also screened for amplifications of the last two genes. Moreover, the transcript levels of the genes were determined and the results compared with the immunohistochemically assessed expression of pRb. Altogether, homozygous deletions of CDKN2 were found in seven tumours (11%) and two of five cell lines, whereas a mutation was detected in only one biopsy, indicating that in sporadic melanomas the former mechanism is predominant for inactivating this gene. Notably, in total 59% of the metastatic lesions lacked detectable expression of p16 mRNA, whereas all the biopsies were found to express pRb. In accordance with the postulated negative feedback loop between p16 and pRb, one melanoma cell line showed overexpression of CDKN2 mRNA together with very low levels of the Rb protein. Amplification of the other two genes may not be important in the tumorigenesis of melanomas, as only one CDK4 and no CCND1 amplification was observed. However, highly elevated CDK4 mRNA levels, compared with that seen in a panel of normal tissues, were observed in 76% of the tumours, accompanied in 71% of the cases by high expression of the CCND1 cyclin activator. Although a low frequency of CDKN2 DNA aberrations was observed, the high number of tumours that lacked CDKN2 expression but showed overexpression of CDK4 and/or CCND1, suggest that functional inactivation of pRb through this pathway may be involved in the development or progression of sporadic human melanomas.

Base Sequence↗

Use of multiple PCR primer sets for optimal detection of human papillomavirus.

Using multiple PCR primer sets, we tried to optimize the detection of human papillomavirus (HPV) in DNA samples isolated from 361 frozen biopsy specimens from patients with invasive cervical carcinomas. The HPVs detected were placed into three distinct groups, including group I/Inex at Telelab (Skien, Norway) and group Ineg and group II at the Norwegian Radium Hospital (Oslo, Norway). The consensus primer sets were Oli-1b-oli-2i, My09-My11, Gp5-Gp6, and Gp(5+)-Gp6+ from the HPV L1 gene and CpI-CpIIG from the E1 gene. Using these consensus primers together with the type-specific primers from E6-E7, we found that 355 patients (98%) were HPV positive. Type-specific primers for HPV types 11, 16, 18, 31, 33, and 35 detected more HPV-infected patients than the most sensitive consensus primer set, while the three consensus primer sets My, Gp/Gp+, and Cp together detected more HPV-positive patients than the type-specific primers. Testing of sensitivity of the PCR with SiHa cells serially diluted in lymphocytes (HPV-negative cells) indicated a detection limit of 6,300 HPV type 16 DNA copies with consensus primers (My, Gp+, and Cp) and 126 original HPV type 16 DNA copies with type-specific primers. Comparison of the amplification results for consensus L1 primers and type-specific E6-E7 primers indicated the presence of L1 deletions in 23 of 56 samples. The conclusion is that in PCR detection systems, multiple consensus primers and type-specific primers should be used in order to detect all patients harboring HPV.

Carcinoma↗

Presence of human papillomavirus in squamous cell laryngeal carcinomas. A study of thirty-nine cases using polymerase chain reaction and in situ hybridization.

Human papillomavirus (HPV) has a well-established association with benign and malignant cervical tumours as well as with laryngeal papillomas, while its association with laryngeal carcinomas is still controversial. We have examined 39 squamous cell carcinomas of the larynx for HPV using PCR with three consensus primers (Cp, My 09/11 and Gp 5+/6+). Specimens positive for consensus primers were examined with specific PCR primers for HPV 6, 11, 16, 18, 31, 33 and 35 and with in situ hybridization (ISH). Three samples (8%) were found HPV positive for two or three consensus primers. One case was confirmed to be HPV 16 with both PCR and ISH. The findings of both a punctate and diffuse signal with the use of ISH indicated HPV to be in both integrated and episomal form. For the other two HPV positive specimens, the examination indicated a type related to HPV 6/11, but it could not be determined with certainty. This study strongly indicates that HPV 16 is associated with some squamous cell laryngeal carcinomas. The possibility of association with other HPV types or mutants of these needs further investigation.

Adult↗

Cyclin kinase inhibitor p21WAF1/CIP1 in malignant melanoma: reduced expression in metastatic lesions.

Immunohistochemical analysis of the expression of the cyclin kinase inhibitor p21WAF1/CIP1 in a panel of primary and metastatic human melanocytic tumors was performed. It was found that, independent of the p53 status, approximately 30% of the primary melanomas and 40% of the metastases completely lacked expression of this cell cycle inhibitor. Some tumors were also analyzed by Northern blotting, and in most of the cases a consistant correlation between mRNA and protein expression was observed. In four benign nevi studied, WAF1/CIP1 mRNA was expressed whereas the protein was not detected, suggesting a post-transcriptional regulation of the inhibitor in these cases. In superficial spreading melanomas, a significant correlation between protein expression and tumor thickness was found, with thin lesions showing low protein levels. Interestingly, by comparing primary and metastatic specimens obtained from the same patient, a reduction in p21WAF1/CIP1 antibody staining was observed in the latter, probably reflecting a more aggressive phenotype of the metastases. In conclusion, our results demonstrate the complexity in the relationship between p21WAF1/CIP1 expression and tumor phenotype and furthermore suggest that aberrant expression of the cyclin-dependent kinase inhibitor may be of importance in the development and progression of sporadic malignant melanoma.

Biomarkers, Tumor↗