Anke Bakker

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.

p53 sequence analysis predicts treatment response and outcome of patients with esophageal carcinoma

The ability to predict biologic behavior and treatment responsiveness would be a valuable asset in the multimodality approach to esophageal carcinoma. The authors examined whether alterations of the p53 gene correlate with clinicopathologic parameters, response to preoperative chemotherapy/radiotherapy, and outcome in patients with esophageal carcinoma.

Prognostic value of TP53 and K-ras-2 mutational analysis in stage III carcinoma of the colon

Background Genetic mutations involving onccgenes and tumor-suppressor genes occur in carcinogenesis, and may affect biologic behavior of neoplasms. In this study, we analyzed the prognostic value of mutational analysis in Colon carcinoma. Patients and methods Archival pathology specimens from 70 consecutive patients, resected for stage III colon carcinoma, were analyzed for point mutations by amplification and direct sequencing of exons from the K- ras -2 and the TP53 genes (topographic genotyping). Mutations were compared with adverse histopathologic features (poor differentiation, vascular and lymphatic invasion, mucin production) as prognostic markers. Results Five-year survival was 75% in patients with nonmutated lesions, significantly lower (21%) with TP53 mutations ( P = 0.01), and intermediate with K- ras -2 only (45%) or both K- ras -2 and TP53 mutations (36%). A TP53 mutation carried the highest relative risk of death (2.39; 95% confidence interval, 1.29 to 4.42; P = 0.006). There was an additive effect on the risk of death between TP53 mutations and adverse histopathologic features. Conclusions The information derived from mutational analysis is creating new prognostic variables that may play a role in the choice of therapy for colorectal carcinoma.

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.

A microdissection and molecular genotyping assay to confirm the identity of tissue floaters in paraffin-embedded tissue blocks

CONTEXT A recurring problem in surgical pathology practice is specimen mix-up and floater contamination. While many cases can be resolved histologically, a significant number remain unclear and may have serious clinical and medicolegal implications. OBJECTIVES To design a microdissection and genotyping assay to identify contaminating floater tissues in paraffin-embedded tissues that is optimized for small samples, and to use the assay to resolve a series of clinical cases with floater tissues. MATERIALS AND METHODS Twenty-one cases of possible tissue floater contamination in paraffin-embedded tissue blocks were included. Using 4 unstained, 4-microm-thick histologic sections, multiple sites were microdissected under direct visualization either by hand or by laser capture microdissection. Nonneoplastic and neoplastic tissues were sampled. Polymerase chain reaction was performed for a panel of 10 polymorphic microsatellite markers at 1p34, 3p26, 5q21, 9p21, 10q23, and 17p13. Allele size and content were analyzed semiquantitatively by fluorescent capillary electrophoresis, and the genotypes for the tissues in the paraffin-embedded tissue blocks were compared for identity. RESULTS Tissue identification was successful in all cases, despite small tissue sample size and fixation effects. Comparative analysis of neoplastic tissue floaters and the presumptive source tumor was performed when possible to control for possible allelic loss or microsatellite instability. CONCLUSIONS Microdissection and genotyping are effective and reliable means to objectively resolve problems of possible floater contamination. Even minute tissue samples provide sufficient DNA template for polymerase chain reaction microsatellite analysis. Because of the potential clinical implications of floaters, we recommend that all suspected floaters that would change a diagnosis from benign to malignant be subjected to genotyping assay to confirm the identity of the floater tissue.

Multiple genetic alterations involved in the tumorigenesis of human cholangiocarcinoma: a molecular genetic and clinicopathological study

Purpose: Cholangiocarcinoma (CC) is the second most common malignant tumor in the liver and the molecular genetic alterations involved in the tumorigenesis of CC have not been well studied. Patients and methods: The authors analyzed the loss of heterozygosity (LOH) in four tumor suppressor genes, including the adenomatous polyposis coli (APC) gene, the deleted in colon cancer (DCC) gene, the 8-hydroguanine-specific DNA glycosylase (OGG1) gene, and the p53 gene in 22 surgically resected primary CCs by using microdissection-based PCR amplification and direct DNA sequencing. Results: A total of 19 (86.4%) out of 22 CCs exhibited genetic alterations, of which 11 (57.9%) and eight (42.1%) cases showed one and more than one gene alterations, respectively. The frequency of genetic alterations of the four genes studied ranged in order from high to low as APC (68.8%) > DCC (46.2%) > OGG1 (41.7%) > p53 (37.5%). Based on the pattern of altered genes and their correlation with clinical and pathological parameters, the genetic alterations were classified into three groups: group I: no detectable genetic alterations (n=3, 13.6%); group II: LOH in APC and/or DCC (n=9, 40.9%); and group III: LOH in OGG1 and/or p53 occurred separately or combined with LOH in APC and/or DCC (n=10, 45.5%). The ≥3-year survival rates between group II and group III are 88.9% and 30%, respectively (P < 0.05). No significant differences were found between genetic alterations and tumor size, tumor type, tumor invasion, TNM staging, and tumor differentiation (P > 0.05). Conclusion: Accumulation of multiple genetic alterations are involved in the tumorigenesis of CC, of which, genetic alterations of APC and DCC occur at a relatively early stage, and of OGG1 and p53 occur at a relatively late stage during development of CC.

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-&mgr;m-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.

Nras loss induces metastatic conversion of Rb1-deficient neuroendocrine thyroid tumor

Mutations in the gene encoding the retinoblastoma tumor suppressor predispose humans and mice to tumor development. Here we have assessed the effect of Nras loss on tumor development in Rb1 heterozygous mice. Loss of one or two Nras alleles is shown to significantly reduce the severity of pituitary tumors arising in Rb1+/− animals by enhancing their differentiation. By contrast, C-cell thyroid adenomas occurring in Rb1+/− mice progress to metastatic medullary carcinomas after loss of Nras. In Rb1+/−Nras+/− animals, distant medullary thyroid carcinoma metastases are associated with loss of the remaining wild-type Nras allele. Loss of Nras in Rb1-deficient C cells results in elevated Ras homolog family A (RhoA) activity, and this is causally linked to the invasiveness and metastatic behavior of these cells. These findings suggest that the loss of the proto-oncogene Nras in certain cellular contexts can promote malignant tumor progression.

Frequent Allelic Imbalance and Loss of Protein Expression of the DNA Repair Gene hOGG1 in Head and Neck Squamous Cell Carcinoma

Reactive oxygen species produced by aerobic cellular metabolism or through exposure to environmental carcinogens can cause oxidative DNA damage by generating DNA base lesions and strand breakage. Prime among these base lesions is the conversion of guanine to 8-oxoguanine. Among 20 or so oxidative DNA base lesions, 8-oxoguanine is the most abundant and is critical in terms of mutagenesis because it is capable of mispairing with adenine, which, if not sufficiently repaired, may lead to G:C to T:A transversion upon DNA replication. The gene encoding human 8-oxoguanine DNA glycosylase 1 (hOGG1), capable of excision repair of 8-oxoguanine, has been recently cloned, characterized, and mapped to the short arm of chromosome 3 (3p25–26), a region showing frequent loss of heterozygosity (LOH) in head and neck squamous cell carcinoma (HNSCC). In the present study, we developed a tissue microdissection approach designed for use with formalin-fixed, paraffin-embedded specimens which is capable of detecting and characterizing the hOGG1 allelic loss using two highly informative, intragenic single nucleotide polymorphisms. Among 45 cases of HNSCC, 18 cases were informative. We analyzed these 18 cases and found that 11 showed evidence of hOGG1 allelic loss. By immunohistochemical staining on a total of 71 HNSCC cases using a commercially available anti-hOGG1 antibody, we showed that hOGG1 gene expression was markedly suppressed in up to 38% of the cases. The frequent allelic imbalance and suppression of the hOGG1 gene thus imply that repair for oxidative DNA damages may be relevant in future studies on head and neck squamous carcinogenesis.

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

SummaryFive 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-I gene expression, immunohistochemical staining for WAF-I 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, LI 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 LI 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.