Absence of jaagsiekte sheep retrovirus DNA and RNA in bronchioloalveolar and conventional human pulmonary adenocarcinoma by PCR and RT-PCR analysis.

Bronchioloalveolar adenocarcinoma (BAC) morphologically resembles sheep pulmonary adenomatosis (SPA), a contagious ovine pulmonary adenocarcinoma caused by the jaagsiekte sheep retrovirus (JSRV). Previously, positivity for JSRV by immunostaining, reverse-transcription polymerase chain reaction (RT-PCR), and Western blot was reported in most nonmucinous BACs. Our objective in this study was to analyze additional BAC subtypes and conventional adenocarcinomas (CA) to further substantiate this association. Tumor tissue was microdissected from unstained paraffin sections of 26 cases of formalin-fixed, paraffin-embedded BAC (7 mucinous, 17 nonmucinous, 2 sclerosing) and 29 cases of CA. Positive controls consisted of 2 separate paraffin blocks of known SPA. Primer sequences were derived that were capable of hybridizing to all reported strain variants of both the DNA (endogenous) and RNA (exogenous) forms of JSRV. Each sample was tested using both PCR (DNA) and RT-PCR (RNA). All BAC and CA cases were negative for JSRV. Positive controls yielded PCR products that were sequenced and precisely matched the published prototype stain of JSRV. To control for negative effects of tissue fixation, dilutions of positive control tissue were added to BAC and CA samples. Detection of JSRV was evident at 1:50 dilution. Although the possibility of a viral association with BAC cannot be excluded, this study shows that the association with JSRV is probably very weak, if present at all.

Correlation between dysplasia and mutations of six tumor suppressor genes in Barrett's esophagus.

BACKGROUND: Barrett's esophagus (BE) may progress to adenocarcinoma through dysplastic progression. Classification of dysplasia in BE has significant interobserver variability. Our objective was to determine whether genetic alterations in BE correlate with degrees of histologic dysplasia. METHODS: Fixed tissue from 37 patients with BE and adenocarcinoma was studied for six tumor suppressor genes. Tissues were microdissected and analyzed for loss of heterozygosity. Microdissection of individual crypts showing metaplasia and dysplasia were performed and analyzed for 23 of the 37 patients whose tumors were heterozygous for at least four of the six genes studied. RESULTS: Frequency of alterations for MXI1, hOGG1, p53, MTS1, DCC, and APC were 7 of 32 (22%), 12 of 35 (34%), 12 of 26 (46%), 17 of 30 (57%), 17 of 27 (63%), and 23 of 36 (64%), respectively. Analysis of BE demonstrated that crypts with metaplasia, low-grade dysplasia, and high-grade dysplasia strongly correlated with alterations in tumor suppressor genes (p < 0.0001). CONCLUSIONS: This pilot study demonstrates that genetic analysis can be performed on individual crypts in patients with BE, and that alterations may facilitate objective classification of the severity of dysplasia.

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.

Molecular genotyping of BK and JC viruses in human polyomavirus-associated interstitial nephritis after renal transplantation.

The human polyomaviruses BK virus (BKV) and JC virus (JCV) have been linked to ureteric stenosis and allograft interstitial nephritis, but molecular characterization of the species involved has not been performed. We studied paraffin-embedded renal tissue from 19 cases of allograft viral interstitial nephritis. Histological sections were subjected to polymerase chain reaction amplification using consensus, BKV-, and JCV-specific primers, with subsequent DNA sequencing for strain determination. BKV was present in all (100%) interstitial nephritis kidneys and placed in genotypes corresponding to serological groups I (n = 11), II (n = 1), and IV (n = 5). Fourteen of 17 isolates (82%) showed sequence variations in the viral capsid protein-1 (VP1) capsid region, with predicted changes in the encoded amino acids and sometimes with potential implications for the secondary and tertiary structure of the corresponding protein molecules. An additional case showed a previously reported glutamine-->leucine T-antigen region mutation. JCV was seen in seven interstitial nephritis kidneys (37%), with types 4 (n = 3), 3A (n = 2), and 2A (n = 1) identified. Most white individuals with asymptomatic infection are reported to shed type 1 JCV in the urine. Simian 40 polyomavirus was not identified in any case. These observations may have pathogenic relevance to the development of an extremely refractory form of polyomavirus interstitial nephritis seen after kidney transplantation.

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 > or = 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-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.

Max interacting protein 1: loss of heterozygosity is frequent in desmoplastic melanoma.

Max interacting protein 1 (MXI1), a negative regulator of myc oncoprotein with tumor suppressor properties, has been mapped to chromosome 10q24-25. MXI1 gene loss, demonstrated by loss of heterozygosity (LOH) analysis (allelic imbalance), is a frequent event in astrocytomas and other forms of glial neoplasia. Development and progression of malignant melanoma likely involves several cooperative oncogene/tumor suppressor gene alterations, many of which remain to be elucidated. We sought to discover whether desmoplastic melanoma (DM) exhibited MXI1 LOH. Archival fixative treated tissue was used for genotyping; this necessitated a microdissection-based molecular approach uniquely designed for minute tissue samples. Nineteen formalin-fixed tissue samples from 11 patients representing primary, locally recurrent, and metastatic DM were available for study. In each case, normal and neoplastic tissue was microdissected under stereomicroscopic observation as a basis for genetic analysis. We identified MXI1 LOH in neoplastic tissue by observing allelic imbalance for a microsatellite repeat polymorphism in the 3' nontranslated region of the MXI1 gene in subjects shown to be informative. Colorectal adenocarcinoma (n = 21) and astrocytomas (n = 19) were similarly analyzed, serving as negative and positive controls, respectively, for MXI1 LOH. Five of 11 DMs in subjects informative for the MXI1 microsatellite manifested MXI1 allelic imbalance consistent with tumor suppressor LOH. Genotype fidelity with respect to MXI1 status was present in two patients from whom primary and recurrent tumor was available for comparative analysis. We found that MXI1 LOH was independent of tumor stage and survival. MXI1 LOH seems to be a relatively frequent and early event in DM development and progression, consistent with neuroectodermal histogenesis of the neoplasm.

Cold-temperature plastic resin embedding of liver for DNA- and RNA-based genotyping.

The standard practice of tissue fixation in 10% formalin followed by embedding in paraffin wax preserves cellular morphology at the expense of availability and quality of DNA and RNA. The negative effect on cellular constituents results from a combination of extensive cross-linking and strand scission of DNA, RNA, and proteins induced by formaldehyde as well as RNA loss secondary to ubiquitous RNase activity and negative effects of high temperature exposure during paraffin melting, microscopic section collection, and tissue adherence to glass slides. An effective strategy to correlate cellular phenotype with molecular genotype involves microdissection of tissue sections based on specific histopathological features followed by genotyping of minute representative samples for specific underlying molecular alterations. Currently, this approach is limited to short-length polymerase chain reaction amplification (<250 bp) of DNA, due to the negative effects of standard tissue fixation and processing. To overcome this obstacle and permit both cellular morphology and nucleic acid content to be preserved to the fullest extent, we instituted a system of cold-temperature plastic resin embedding based on the use of the water-miscible methyl methacrylate polymer known as Immunobed (Polysciences, Warminster, PA). The system is simple, easy to adapt to clinical practice, and cost-effective. Immunobed tissue sections demonstrate a cellular appearance equivalent or even superior to that of standard tissue sections. Moreover, thin sectioning (0.5-1.0 microm thickness) renders ultrastructural evaluation feasible on plastic-embedded blocks. Tissue microdissection is readily performed, yielding high levels of long DNA and RNA for genomic and transcription-based correlative molecular analysis. We recommend the use of Immunobed or similar products for use in molecular anatomical pathology.

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

BACKGROUND: 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. METHODS. Histopathologic/genetic analysis of p53 was performed on formalin fixed, paraffin embedded tissues. Tissue sections were stained immunohistochemically for p53 protein followed by topographic genotyping comprised of polymerase chain reaction amplification and direct sequencing of p53 exons 5-8. All patients received induction chemotherapy (5-fluorouracil, cisplatin, and alpha-interferon) and concurrent external beam radiotherapy (4500 centigrays) followed by resection. RESULTS: p53 analysis performed on 42 tumors from patients with potentially resectable esophageal carcinoma revealed 25 of the 42 tumors (59.5%) to be p53 immunopositive; however, only 17 of the 42 tumors (40.5%) were proven to contain p53 point mutational damage in exons 8 (n=5), 5 (n=5), 7 (n=4), and 6 (n=3). Eight cases were weakly immunopositive and had no genotype mutation suggesting hyperexpression of normal wild-type p53. Genotyping also identified two immunonegative cases with deletion-type mutations (exons 5 and 6). Tissue samples collected before and after chemotherapy/radiotherapy exhibited fidelity in p53 mutational genotype in all cases. The presence of a p53 point mutation positively correlated with pTNM stage (P=0.003) and residual disease in the resected specimen (P=0.01). Moreover, survival of patients with p53 mutations was significantly lower than that of patients without mutations (overall survival of 21.6 months vs. 40 months; P=0.0038; and disease free survival of 14.1 months vs. 38 months; P=0.0004). CONCLUSIONS: Histopathologic/genetic analysis is a better determinant of p53 mutational damage than immunohistochemistry alone and can be used as a prognostic marker for esophageal carcinoma. p53 genotyping may define a subset of patients who respond to chemotherapy/radiotherapy and may predict who potentially benefits from multimodality therapy.

K-ras and p53 mutations in aberrant crypt foci and colonic tumors from colon cancer patients.

Aberrant crypt foci (ACF) are microscopic lesions which can be detected, after methylene blue staining, in the overtly normal looking colonic mucosa of cancer patients. ACF have been postulated to be precursor lesions which develop into colorectal cancer. Mutations of K-ras and p53 are two important genetic events implicated in colon carcinogenesis. Mutations in K-ras are detectable at earlier stages, while mutations in p53 are detectable at later stages of colon carcinogenesis. Our objective was to compare the nature of genetic alterations in K-ras (codon 12 and 13) and in p53 (exon 4-9) between ACF and corresponding colonic tumors from cancer patients. ACF with > or =20 crypts/focus were harvested from overtly normal looking colonic mucosa of cancer patients at a distance of (approx.) 5 cm from the site of colonic tumors. The colonic tumors and ACF samples were compared for K-ras codon 12 and 13 base pair sequence, using DNA sequencing and for p53 (exon 5-9) allelic types, using PCR-SSCP and DNA sequencing. The results demonstrated a perfect correlation in terms of the type of K-ras allele (wild or mutated) between the ACF (> or =20 crypts/focus) and corresponding colonic tumors in 11/13 cancer patients. Analyses of p53 mutations demonstrated the presence of p53 mutations in colonic carcinomas from 10/13 patients. However, p53 mutations could be detected in an ACF from only 1/13 patient. The results provides further evidence to the role of ACF as precursor to colon cancer. The presence of an identical K-ras as well as p53 mutation in an ACF and the corresponding colonic carcinoma in a patient suggests the possibility of existence of ACF that may be at a more advanced stage in the sequence of colonic tumorigenesis than others. In conclusion, the results suggest that a subset of ACF with higher multiplicity might be considered more likely to progress to more advanced lesions and should be explored as markers of colon cancer risk.

Sporadic and Thorotrast-induced angiosarcomas of the liver manifest frequent and multiple point mutations in K-ras-2.

Hepatic angiosarcoma (HA) is an uncommon neoplasm associated with known etiologic factors in 25% to 42% of cases. It is, however, one of the most common sarcomas found in the liver. The aim of this study was to find was to find mutations in the K-ras-2 oncogene in sporadic and Thorotrast (TT)-induced HA. Point mutations in K-ras-2 were sought in archival, formalin-fixed tissue blocks from 24 patients with angiosarcoma. Of these, 19 cases were sporadic and 5 were TT-induced. Mutational analysis was performed by topographic microdissection with PCR amplification followed by genotyping. Specific mutations were determined by two independent methods: (a) direct sequencing of the PCR product confirmed by rePCR and by using a different sequencing primer, and (b) PCR-based selective enrichment of mutant DNA by endonuclease digestion followed by heteroduplex DNA analysis using denaturing gradient gel electrophoresis. Eleven K-ras-2 point mutations were detected in 7 of 24 (29%) tumors, including 5 of 19 (26%) sporadic HA and 2 of 5 (40%) TT-induced HA. There were seven G:C > A:T and four G:C > T:A mutations. All seven mutated tumors contained a codon 12-aspartate amino acid substitution. In addition, a second codon 12-cysteine mutant cell population was present in one of two codon 12-aspartate mutated TT-induced HA and in three of five codon 12-aspartate sporadic tumors. Of these four tumors, three contained both aspartate and cysteine mutations and were composed of multiple nodules; the fourth was a single mass. Seventeen tumors had multiple nodules; whereas 5 had a K-ras-2 mutation, 12 were wild-type. The molecular pathology of both sporadic and TT-induced HA is characterized by a high rate of K-ras-2 mutations characteristic of oxidative damage (ie, G:C > A:T and G:C > T:A mutations) resulting in two mutated population sets: codon 12 GGT > GAT and GGT > TGT (glycine to aspartic acid and cysteine). This is, to date, the first study to characterize the K-ras-2 gene mutations within human sporadic and TT-induced HA by direct sequence analysis and denaturing gradient gel electrophoresis. These data further support the hypothesis linking adduct-forming vinyl chloride exposure to HA containing a much higher frequency of K-ras-2 mutations and a mutational spectrum characteristic of chloroethylene oxide, a carcinogenic metabolite of vinyl chloride.

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.

Morphologic, histologic, and molecular similarities between adenocarcinomas arising in the gastric stump and the intact stomach.

BACKGROUND: Patients with gastric remnants resulting from partial resections have an increased risk for carcinoma. It is unclear whether adenocarcinoma arising in the gastric stump (GSca) differs from intact stomach carcinoma (Gca). The goal of this study was to examine the pathologic and molecular features of GSca and compare them with Gca. METHODS: Adjacent nonmalignant areas and tumors from 14 patients who were 19-55 years postgastrectomy (mean, 32.1 years) were compared with 14 Gca by pathologic and molecular analysis. Formalin fixed, paraffin embedded specimens were immunohistochemically stained for p53 followed by topographic genotyping. Exons 5-8 were amplified by the polymerase chain reaction and directly sequenced. RESULTS: No differences were noted between the two groups regarding gender, types of metaplasia, dysplasia, morphology, or histologic tumor type. However, a higher incidence of cystic dilatation and foveolar hyperplasia were present in GSca. p53 gene point mutations occurred in 5 of 14 (35.7%) GSca patients. GSca p53 mutations included missense point mutations (G:A transitions in four patients and G:C transversion in one patient) with allelic loss. In four of the five patients with p53 mutations, the same mutation was also observed in the adjacent area. p53 point mutations were present in 4 of 14 Gca (28.6%), in exons 5, 6, and 8. In one case, the same mutation was also detected in the adjacent nonmalignant mucosa. CONCLUSIONS: Similarities in clinical, pathologic, and molecular features between GSca and Gca suggest the possibility that they share similar mechanisms of carcinogenesis. p53 gene alterations in premalignant areas may denote a possible early role of this gene in gastric carcinoma.

Pleomorphic (giant and spindle cell) carcinoma is genetically distinct from adenocarcinoma and squamous cell carcinoma by K-ras-2 and p53 analysis.

Pleomorphic carcinoma (PC) of lung is a poorly differentiated epithelial neoplasm predominantly composed of pleomorphic giant and/or spindle tumor cells. The WHO classification of lung cancer recognizes spindle cell carcinoma and giant cell carcinoma as separate neoplasms related to squamous cell carcinoma (SqC) and large cell carcinomas, respectively. Further, the presence of foci of SqC or adenocarcinoma (AdC) in, respectively, 10% and 45% of PC produces additional uncertainty as to the distinctive nature of this tumor type. In this study, the authors tested the hypothesis that PC is an entity separate from SqC or AdC by evaluating the mutational spectrum seen in these tumor types. This is performed by documenting and comparing mutation type and rate of K-ras-2 and p53 genes in PC, SqC, and AdC. Comparative DNA sequence and immunohistochemical analysis were performed on 22 PC, 42 SqC, and 97 AdC. Archival formalin-fixed, paraffin-embedded tissues formed the basis of the study. Immunohistochemical staining with p53 antibody (DO-7) revealed statistically significant differences in the intensity and frequency of staining of PC (weak, 86% of cases) versus SqC (strong, 52% of cases) and AdC (strong, 27% of cases) (P < .001). Topographic genotyping with subsequent polymerase chain reaction (PCR) and sequence analysis of K-ras-2 showed mutations in significantly fewer cases of PC (9%, 2 of 22 cases) than in AdC (36%, 35 of 97 cases) or SqC (0%, 0 of 42 cases) (P < .001). Pleomorphic carcinoma also showed significantly fewer p53 point mutations (14%, 3 of 22 cases) than did AdC (27%, 26 of 97 cases) of SqC (43%, 18 of 42 cases) (P < .01). Finally, the p53 mutations in PC were more common in exon 7, whereas those in SqC and AdC were more frequent in exon 8. These findings reveal significant differences in the pattern and frequency of genetic mutations between PC and pulmonary SqC and AdC and are in keeping with the separate histopathologic classification of these tumors.

Primary mediastinal and testicular seminomas: a comparison of K-ras-2 gene sequence and p53 immunoperoxidase analysis of 26 cases.

Primary mediastinal seminomas (MS) are rare tumors. Histologically, they are similar to their counterpart in the gonads. The survival rate has varied from 50% to 85% in different series. However, large series of these tumors primarily in the mediastinum are lacking. At the molecular level, a few reports document K-ras mutations in up to 40% of testicular seminomas (TS), localized predominantly to codon 12. Reports on TS p53 immunohistochemistry (IHC) range from negative to overexpression approaching 90% of cases, and by sequence analysis one small series showed a 23% mutation rate. To date, no analyses have been performed for either K-ras mutations or p53 immunohistochemical expression in primary MS. The authors studied 13 cases each of primary MS and TS from archival formalin-fixed, paraffin-embedded sections in which adequate tumor sampling and clinical history, including serological studies, and histological, histochemical, and IHC staining, were performed to confirm the diagnosis. p53 immunoperoxidase staining using citrate buffer/microwave antigen retrieval was performed. Topographic genotyping was performed on 5-microns-thick tissue sections up to 17 years old, in which the neoplastic cell population was sampled. Additionally, multiple sites within a given cases were sampled to determine clonality of the tumor cell population. Polymerase chain reaction and subsequent sequence analysis of the K-ras-2 exon-1 gene was used for mutation analysis. Focal weak staining with p53 IHC was observed in 4 of 13 (31%) MS and 10 of 13 (77%) TS cases, with all remaining cases being negative (P < .05). Only one MS case (8%) showed K-ras mutation (codon 13 GGC > GAC; glycine > aspartate), which is in contrast to 2 of the TS cases (15%), showing codon 12 mutations. All the remaining cases were wild type. Therefore, primary mediastinal seminomas appear to be different in their K-ras sequence and p53 immunostain profile from TS. Codon mutation type may be useful in determining primary versus metastatic origin of a mediastinal seminoma.

Comparative p53 mutational analysis of multiple primary cancers of the upper aerodigestive tract.

BACKGROUND: Tumors arising in the upper aerodigestive tract (UAT) are often associated with predisposing factors that place the patient at risk for development of multiple synchronous or metachronous tumors. The aim of this study was to evaluate p53 as a susceptibility gene in UAT malignancy. METHODS: Seventeen patients with 41 separate primary tumors involving esophagus (n = 15), larynx (n = 14), pharynx (n = 6), lung (n = 2), mouth (n = 2), and tongue (n = 2) were analyzed for the presence and specific genotype of p53 point mutation. Immunohistochemical staining of p53 and topographic genotyping consisting of polymerase chain reaction amplification and direct sequencing of p53 exons to 5 to 8 were performed. RESULTS: Eleven tumors were metachronous (6 months to 11 years), and 11 were synchronous. We found p53 point mutations in 19 (46.3%) of 41 tumors in exons 8 (n = 11), 7 (n = 4), 5 (n = 3), and 6 (n = 1). Tumors possessed either wild-type p53 or a single type of point mutation. Metastases displayed the identical genotype of its primary tumor in all cases. Most importantly, p53 genotype was found to be completely discordant between separate primary tumors for the same patient. CONCLUSIONS: Complete discordance in p53 genotype between separate primary UAT cancers strongly indicates that p53 is not functioning as a susceptibility gene in this setting.

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.

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.