A novel 23-bp deletion in exon 5 of the p53 tumor suppressor gene.

We report a novel p53 deletion in a 63-year-old female with breast cancer. Mutation screening of DNA samples, obtained from tumor specimens from 98 individuals with breast cancer, by a combined polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis showed that the index case had a somatic mutation identified to be a 23-bp deletion in exon 5 of the p53 gene. This deletion would be expected to yield a truncated and functionally inactive p53 protein molecule, probably resulting in cell transformation. The existence of 6-bp palindromic-like sequences encompassing the deleted fragment suggests that the slipped mispairing mechanism is not involved in producing the deletion, which probably resulted from palindromic pairing during replication.

Plasticity of glomeruli and olfactory-mediated behavior in zebrafish following detergent lesioning of the olfactory epithelium.

The zebrafish olfactory system is a valuable model for examining neural regeneration after damage due to the remarkable plasticity of this sensory system and of fish species. We applied detergent to the olfactory organ and examined the effects on both morphology and function of the olfactory system in adult zebrafish. Olfactory organs were treated once with Triton X-100 unilaterally to study glomerular innervation patterns or bilaterally to study odor detection. Fish were allowed to recover for 4-10 days and were compared to untreated control fish. Axonal projections were analyzed using whole mount immunocytochemistry with anti-keyhole limpet hemocyanin, a marker of olfactory axons in teleosts. Chemical lesioning of the olfactory organ with a single dose of Triton X-100 had profound effects on glomerular distribution in the olfactory bulb at 4 days after treatment, with the most significant effects in the medial region of the bulb. Glomeruli had returned by 7 days post-treatment. Analysis of the ability of the fish to detect cocktails of amino acids or bile salts consisted of counting the number of turns the fish made before and after odorant delivery. Control fish turned more after exposure to both odorants. Fish tested 4 and 7 days after chemical lesioning made more turns in response to amino acids but did not respond to bile salts. At 10 days post-lesion, these fish had regained the ability to detect bile salts. Thus, the changes seen in bulbar innervation patterns correlated to odorant-mediated behavior. We show that the adult zebrafish brain has the capacity to recover rapidly from detergent damage of the olfactory epithelium, with both glomerular distribution and odorant-mediated behavior returning in 10 days.

Relationship of p53 gene alterations with tumor progression and recurrence in olfactory neuroblastoma.

Olfactory neuroblastoma (ONB) is a rare neuroectodermal tumor whose clinical course is not effectively predicted by initial stage or grade; p53 tumor suppressor gene alterations have not been determined concerning the ONB pathobiology and recurrence. We analyzed 18 formalin-fixed, paraffin-embedded ONB specimens (12 primary tumors and six recurrences or metastases) from 14 patients for p53 alterations using immunohistochemistry for p53 and WAF1 together with topographic genotyping (selection of minute tissue targets from unstained sections, PCR [polymerase chain reaction] amplification of exons 5-8 followed by direct DNA sequencing). Sequential material representing tumor recurrence or metastasis was available in four cases to compare genetic alterations over time in the same patient. None of the cases showed strong, diffuse p53 immunostaining. Focal weak to moderate intensity staining was evident in nine of 14 cases. Mutations in p53 were not detected in any of the cases, suggesting hyperexpression of p53 wild-type protein. Hyperexpression was further confirmed by correlation of WAF-1 and p53 immunopositivity. Importantly, in four cases with recurrence or metastasis, tumors manifested p53 wild-type hyperexpression. It appears that p53 point mutation does not play an important role in the initial development of ONB; however, p53 wild-type hyperexpression may occur in subsets of ONB likely to show local aggressive behavior and a tendency for recurrence. Wild-type p53 hyperexpression may be an important event in later stages of ONB growth and progression.

Prognostic significance of p53, bcl-2, vimentin, and S100 protein-positive Langerhans cells in endometrial carcinoma.

Immunohistochemical expression of p53, Bc12, vimentin, and S100 protein-positive Langerhans cell was evaluated in 50 endometrial carcinomas (6 stage I, 14 stage II, 20 stage III, and 10 stage IV), in an attempt to use these markers as predictors of survival. Monoclonal antibodies to p53, Bcl-2, vimentin, and S100 proteins were applied to paraffin-embedded sections of endometrial adenocarcinoma, using the avidin-biotin peroxidase complex technique (ABC). All 20 patients with stage I and II carcinomas were alive with a mean follow-up of 3 years. Of 30 patients with stage III and IV carcinomas, 13 died of tumor (3-year survival, 57%; SE, 10%), eight were alive with tumor, and nine were alive with no evidence of tumor (mean follow-up, 46 months). Strong p53 positivity was present in 11 carcinomas (22%), including nine high-stage and two low-stage tumors. Bcl-2 positivity was identified in 33 tumors (66%). These tumors were mostly low stage; however, no correlation was found between Bcl-2 expression and prognosis. Vimentin positivity (P < .001), and tumor infiltration by a large number of S100 protein-positive Langerhans cells (P < .05) were associated with low-stage tumors. Vimentin was expressed in 23 carcinomas, including 70% of low-stage tumors and 20% of high-stage tumors. Most high-grade carcinomas were Langerhans cell depleted; most low-grade carcinomas showed >50 S100 protein-positive Langerhans cells/10 high-power fields. Our results indicate that Langerhans cell infiltration and vimentin positivity of tumor cells are favorable prognostic factors in endometrial carcinomas.

The role of p53 mutation and protein expression in primary and recurrent adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a malignant tumor of salivary gland origin having a propensity for spread by direct extension or perineural invasion with frequent recurrences. Previous reports have shown that tumor behavior is not always predicted by histological pattern or stage. Little is known of the role of p53 tumor suppressor gene mutation and altered protein expression with respect to ACC pathobiology and recurrence. The authors analyzed a group of 14 ACC specimens (seven primary; seven recurrent) from 13 patients treated between 1987 to 1993. Formalin-fixed, paraffin-embedded specimens were reviewed and subjected to, immunohistochemistry (p53, DO-7, DAKO, Nutley, NJ; and WAF-I, Ab-1, Oncogene Sciences, Uniondale, NY) on 4-microm-thick histological sections as a prelude to p53 genotyping. In one case, sequential material representing primary and recurrent tumor was analyzed. Each tumor specimen was topographically genotyped for p53 point mutational change. Minute tissue samples were removed from unstained sections, polymerase chain reaction (PCR) amplified for p53 exons 5 to 8, and then underwent direct DNA sequencing. Six of seven primary ACCs were p53 immunostain negative. Four of seven recurrent (57%) ACCs were p53 immunopositive. These tumors showed varying degrees of p53 immunopositivity ranging from diffuse, intense staining of most tumor cells (n = 1) to interspersed, strongly positive cells mixed with predominantly p53 immunonegative cells (n = 4). All tumors were WAF-I immunostain negative. Two of the most immunopositive recurrent tumors each manifested a single type of p53 point mutation detected by p53 DNA genotyping (p53 exon 5:codon 175 and p53 exon 6:codon 199). In the case in which both primary and recurrent tumor was available, only the recurrent tumor contained point mutational damage. Negative immunostaining for p53 in primary ACC suggests that p53 mutation is not important in early events involving development of this tumor. In contrast, the frequent presence of p53-positive cells and the detection of point mutations in recurrent ACC suggests that p53 alterations are involved in later stages of tumor progression, important in the phenomenon of ACC recurrence.

Carcinosarcomas (malignant mixed mullerian tumors) of the female genital tract: comparative molecular analysis of epithelial and mesenchymal components.

Female genital tract carcinosarcomas (FGTCS) are biphasic neoplasms composed of an admixture of malignant epithelial and mesenchymal elements. Histogenesis of FGTCS centers on two theories: (1) simultaneous formation of independent tumors (biclonal theory), (2) multidirectional differentiation of a single neoplasm (monoclonal theory). In an attempt to resolve this histogenetic controversy, we determined the presence, specific genotype, and timing of p53 mutational change in each component of FGTCS using a topographic genotyping (TG) approach. We selected 43 FGTCS from the files of Magee-Womens Hospital, Pittsburgh, and initially immunostained them for p53 protein. Strong p53 immunopositivity was detected in 35 (82%) of 43 tumors. Subsequently, topographic genotyping (TG) was performed on a subset of nine immunopositive tumors with sufficiently distinct malignant components to enable effective sampling. All nine tumors showed point mutations in p53 exons 5 through 8. In each case, the identical point mutational genotype was present in both components. Furthermore, in all nine cases mutations were present with loss of the wild-type allele. P53 gene mutation is a frequent event in progression of FGTCS. Of importance, both p53 mutation and allelic loss occur before the differentiation into separate epithelial and mesenchymal malignant components. These molecular findings strongly support monoclonal, multidirectional histogenesis of FGTCS.

Relationship of p53 Genotype to Second-look Recurrence and Survival in Ovarian Epithelial Malignancy.

Background: Clinical stage at presentation and tumor status at second-look laparotomy are currently the best predictors of patient survival in epithelial ovarian carcinoma (EOC). Methods and Results: To evaluate the predictive value of genetic analysis, the presence and type of p53 mutation (p53 genotype) was determined in 76 patients treated for EOC between 1987 and 1992 and subjected to second-look laparotomy following initial treatment. Mutational analysis of p53 was performed retrospectively by means of topographic genotyping (TG), using formalin-fixed, paraffin-embedded tissue of the primary and recurrent tumor. In TG, minute tissue samples were dissected from unstained histologic sections, amplified for p53 exons 5-8 with the polymerase chain reaction (PCR), and then direct sequenced. The p53 genotype was correlated with tumor stage, histologic grade and type, tumor status at second look, and survival at 3 and 5 years. Mutational change involving p53 exons 5-8 was found in 41 of 76 tumors (54%), consisting of 29 cases manifesting missense alterations and 12 cases having truncations (deletions, insertions, or stop codons). Tumor mutational change for each patient was strictly limited to a single type, being identical in all recurrences of an individual primary cancer. Mutations of p53 were distributed over exons 5-8, with certain "hot spots" being evident (codons 220, 245, and 273). Mutational change was present in all stages of primary EOC, but was relatively more frequent in tumors of advanced stage (III and IV). Epithelial ovarian carcinoma manifesting p53 mutational damage was significantly more likely to show recurrence at second-look laparotomy (P <.001) and to have shorter survival at the 3- and 5-year follow-up (P <.001) evaluation. The predictive value of p53 genotype was independent of stage at presentation, histologic grade, or histopathologic type. Conclusions: Genotyping of p53 provides useful information on tumor aggressiveness and is an informative predictive marker of biologic tumor behavior, treatment response, and survival in EOC.

Expression of bcl-2 and bax in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the uterine cervix.

Bcl-2 protein together with the pro-apoptotic protein bax, are thought to function by forming homo- and heterotypic dimers which control the progression to apoptosis. In this immunohistochemical study we investigated the expression of bcl-2 and bax apoptosis related proteins in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the uterine cervix. Twenty-four cervical intraepithelial neoplasias grade 1-2 (CIN I/II), 38 grade 3 (CIN III), and 53 invasive squamous cell carcinomas (ISCC) were investigated by immunohistochemical staining for bcl-2 and bax protein. Bcl-2 immunoreactivity was found in five of the 24 CIN I/II cases (20.8%), 18 of 38 CIN II cases (47.4%) and nine of 53 ISCC cases (17%). The positivity for CIN III was significantly higher than for CIN I/II or ISCC (p=0.0351 and p=0.0018, respectively). The percentage of bax immunopositivity was somewhat higher in CIN III than in CIN I/II but this slight difference was not statistically significant. Correlation of the immunostaining results with tumor grade revealed a significant difference for bcl-2 which was more frequently immunopositive in well-differentiated tumors than in poorly-differentiated tumors. There was no significant relation between bax expression and tumor differentiation. Our results suggest that alterations of bcl-2 and bax expression may occur as a relatively early event in cervical tumorigenesis.

The impact of adjuvant radiotherapy on carcinosarcoma of the uterus.

BACKGROUND: The role of adjuvant radiotherapy in the setting of uterine carcinosarcoma has not been clearly established. METHODS: A retrospective review of 60 patients receiving definitive therapy for carcinosarcoma of the uterus was undertaken at a single institution. Twenty-nine of 60 patients were treated with adjuvant radiotherapy. RESULTS: The addition of radiotherapy significantly reduced the local recurrence rate from 55% (17 patients) to 3% (1 patient). Adjuvant radiotherapy reduced the risk of distant failure and death in patients with disease confined to the uterus but did not impact distant recurrence or survival in stage III patients. Increasing stage and depth of myometrial tumor invasion were negatively associated with overall survival and disease-free survival but had no impact on local recurrence rates. The nuclear grade of the epithelial component was predictive of local recurrence (P = 0.0592), but epithelial architectural grade, grade of stromal component, and stromal versus epithelial predominance did not provide prognostic information. The relative risk of local recurrence of unirradiated patients versus irradiated patients was 17.54 (P = 0.0055) after adjusting for nuclear grade of the epithelial component. CONCLUSIONS: Local failure represents a significant site of failure in the absence of adjuvant radiotherapy. The improvement in local failure rates with the addition of radiotherapy translates into an improvement in distant failure rates and survival only for patients with stage I/II disease. Epithelial nuclear grade, in addition to depth of myometrial invasion and stage, provides important prognostic information. Epithelial architectural grade, stromal grade, type of stromal component (homologous versus heterologous), and predominance of either stromal or epithelial component were not found to be significant prognostic factors.

Immunohistochemical profile of endometrial adenocarcinoma: a study of 61 cases and review of the literature.

The differences in immunohistochemical expression of p53, bcl-2, bax, estrogen receptor (ER), and progesterone receptor (PR) were evaluated in 40 endometrioid and 21 papillary serous carcinomas of endometrium and correlated with known predictors of survival, such as grade and stage. Uterine papillary serous adenocarcinomas (UPSA) showed significantly higher p53 expression than did uterine endometrioid adenocarcinomas (UEA) (76.2% versus 35%), whereas both ER and PR were more often positive in endometrioid than in serous tumors (p = .005 and .0005). No significant difference was found in bcl-2 and bax expression between both histologic types. However, there was definite decrease in intensity of bcl-2 in UPSA compared with UEA. In endometrioid carcinoma, p53 overexpression was associated with high-grade and advanced-stage tumors (p = .0006 and .006), whereas ER and PR expression was associated with low-grade and early-stage tumors (p = .0006 and .0001; p = .003 and .0006). Bcl-2 immunopositivity was more common in low-grade, early-stage rather than in high-grade, advanced-stage adenocarcinomas, but the difference was not statistically significant (p = .24 and .07). Bax immunopositivity was associated with well-differentiated (p = .04) and early-stage tumors. Furthermore, a significant inverse relationship between bax and p53 reactivity was defined (p = .05), especially in tumors of endometrioid type. Bax and PR immunoexpression correlated near the limit of statistical significance (p = .08), whereas no relationship was found among bax, bcl-2, and ER immunopositivity. Our results indicate that the differences in immunohistochemical profiles of endometrioid and serous carcinomas support the existence of different molecular pathways of their development. The correlation of immunohistochemical findings with histologic grade and clinical stage could help in predicting biologic behavior and planning treatment in patients who are diagnosed as having these tumors.

Well-differentiated villoglandular adenocarcinoma of the uterine cervix: oncogene/tumor suppressor gene alterations and human papillomavirus genotyping.

Twelve well-differentiated villoglandular adenocarcinomas (WDVAs) of the uterine cervix were retrospectively analyzed for the presence and specific genotype of human papillomavirus (HPV), tumor suppressor loss (p53, MCC, APC, BRCA1), cancer gene mutation (K-ras-2, exons 1 and 2, p53 exons 5 to 8), and oncogene amplification (c-erbB-2/HER-2/neu, int-2). Tissue for genetic evaluation was obtained by microdissection, using 4-micron-thick histology sections of archival, formalin-fixed, paraffin-embedded specimens. Genotyping involved nucleic acid amplification and DNA sequencing with gene-specific oligonucleotides and L1 region consensus primers for common strains of HPV. Point mutation and HPV strain determination were accomplished by DNA sequence analysis. Tumor suppressor gene loss and oncogene amplification were performed by allelic imbalance analysis in informative subjects based on DNA sequence and microsatellite-length polymorphisms. HPV was present in all tumors and consisted of type 16 (n = 5, 42%) and type 18 (n = 7, 58%) strains, which have been closely associated with cervical neoplasia. K-ras-2 and p53 genes did not manifest point mutational damage. There was no evidence of oncogene amplification or tumor suppressor gene loss. The presence of HPV in all 12 tumors supports the role of HPV infection in the molecular pathogenesis of this uncommon neoplasm. The absence of associated oncogene or tumor suppressor gene damage is consistent with indolent biological behavior and the favorable prognosis of this unusual tumor.

The origin and molecular characterization of adenoid basal carcinoma of the uterine cervix.

Five cases of adenoid basal carcinoma (ABC) of the uterine cervix were examined for the presence of p53 tumor suppressor gene, K-ras-2 oncogene, and human papillomavirus (HPV). A topographic genotyping approach was used to search for point mutations in K-ras-2 (exon 1 and 2) and p53 (exons 5 to 8) in archival formalin-fixed tissue blocks. Minute target sites were selected from polymerase chain reaction (PCR) amplified and directly sequenced tissue sections. Tissue sections were additionally subjected to immunohistochemical staining for p53 and WAF-1 protein. Because wild type p53 induces WAF-1 gene expression, immunohistochemical staining for WAF-1 protein using monoclonal antibodies may serve as an indirect means to test for p53 mutational damage. Mutational genotype was compared to histopathologic features and immunohistochemical staining. To study the role of HPV, L1 region consensus primers were used to amplify topographic samples, followed by HPV genotyping by direct sequencing and comparison to known viral strains. ABC was found to contain HPV in all cases, proven by genotyping to be HPV type 16 in each case. The virus showed no evidence of genomic variation from prototype HPV type 16 in the L1 segment examined. No K-ras-2 point mutations were identified. p53 immunopositivity was present in all tumors, being weak and focal in 4 and strong and diffuse in 1. WAF-1 immunostaining was positive in two tumors showing weak focal p53 immunopositivity. The single strong and diffuse p53 immunopositive tumor was negative for WAF-1 and was shown to contain a missense p53 point mutation (exon 7-codon 248 tryptophan). In conclusion, ABC is characterized by the presence of HPV type 16. K-ras-2 point mutation appears to play no role in the development of this tumor. p53 gene alterations are common including wild type hyperexpression (weak focal p53 immunopositivity, WAF-1 positivity, no mutational change) and p53 point mutational damage.

Prognostic value of p53 and K-ras-2 topographic genotyping in endometrial carcinoma: a clinicopathologic and molecular comparison.

The predictive value of p53 and K-ras-2 mutational genotyping in determining tumor aggressiveness and survival in patients with endometrial carcinoma (EC) was retrospectively evaluated using a molecular genotyping approach on fixative treated tissue specimens. Two groups of patients with EC were selected based upon length of survival. Group A consisted of 14 patients that died within 3 years of initial diagnosis and treatment (mean survival of 1.1 years). Group B consisted of 18 patients that survived beyond 3 years (mean survival of 4.7 years). Clinicopathologic features including clinical stage, histologic type, and combined nuclear and architectural grade of each tumor were statistically analyzed with respect to oncogene/tumor suppressor gene alterations. The majority of carcinomas in group A were serous (57%), stage III or IV (93%), and high combined grade (93%). Group B consisted mostly of endometrioid (89%) and low-grade carcinomas (83%); 56.1% were stage III or IV. K-ras-2 point alterations were found in 2 (14%) and 4 (22%) patients from group A and B respectively; the spectrum of K-ras-2 genotypes was similar in both groups. p53 gene mutations were identified in 9 (64%) and 1 (6%) patient from group A and B respectively. p53 staining in group A tended to be of strong intensity and diffuse distribution, being associated with the presence of point mutations, mainly in exon 8. Only a single group B tumor exhibited point mutational change. The presence of p53 mutations strongly correlated with short survival (p <0.05) but the finding of K-ras-2 alterations did not. p53 genotyping has potential prognostic value in EC and can be used along with histopathologic type and histologic grade to identify subsets of more aggressive tumors and to guide the treatment.