CD34 immunohistochemistry in female genital tract carcinosarcoma (malignant mixed müllerian tumors) supports a dominant role of the carcinomatous component.

Female genital carcinosarcomas (FGTCSs) are aggressive biphasic malignant neoplasms with histologic features of both carcinomas and sarcomas. However, their behavior is dominated by the carcinomatous component. CD34 is useful in distinguishing carcinomas with sarcomatoid features from epithelioid sarcomas, as it is never expressed in carcinomas although often expressed in epithelioid sarcomas. This investigation hypothesizes that CD34 expression in FGTCSs will contribute useful histogenetic and clinical information. Paraffin sections from a surgical series of 43 FGTCSs were stained using anti-CD34 with an automated immunohistochemical stainer, antigen retrieval, and appropriate external/internal controls. Reactions were graded as follows: negative (N) or S1-3, strong reactivity in <10% = S1, 10-50% = S2, or >50% = S3 of cells, respectively. The series included 31 endometrial, 6 ovarian, 5 cervical, and 1 fallopian tubal FGTCSs, 70% exhibiting heterologous elements (22 rhabdomyosarcomas, 11 chondrosarcomas, 1 osteosarcoma, and 1 liposarcoma). The carcinomatous component included 19 endometrioid, 12 serous, 6 adenosquamous, 4 clear cell, and 2 poorly differentiated carcinomas: modified International Federation of Gynecologic Oncology grade I, 9%; II, 21%; and III, 70%. CD34 reactivity was 100% N in the carcinomatous components. CD 34 stained 21 % of sarcomatous components as follows: 2S1, 6S2, and 1S3 (three homologous and six heterologous). Of the heterologous components, only rhabdomyosarcoma stained with the following patterns: 1S1, 2S2, 1S3. Two of six heterologous FGTCSs exhibited staining of only the homologous sarcomatous component with the following pattern: 2S2, as the heterologous sarcomatous component (pure rhabdomyosarcoma in both cases) showed no staining. The staining pattern was unrelated to primary site of the FGTCSs. The pattern of CD34 expression (N in the carcinomatous component like carcinomas) supports the generally accepted opinion of dominance of FGTCS by the carcinomatous component. The staining of the sarcomatous component was rare (21%) and focal (only one of nine was S3). CD34 staining may help distinguish FGTCSs from epithelioid sarcomas, which strongly express CD34.

Transdermal estrogen replacement therapy in postmenopausal women previously treated for porphyria cutanea tarda.

Oral contraceptives and postmenopausal estrogen replacement therapy are recognized as risk factors for the development of porphyria cutanea tarda (PCT) in women. The recommended clinical practice is to withhold estrogen therapy in women who have had phlebotomy therapy for PCT and are clinically and biochemically normal. We tested the safety and efficacy of transdermal estrogen replacement therapy in 7 women previously treated for PCT and compared them with 19 non-porphyric control subjects treated with transdermal or oral estrogens. Gonadotrophic hormone levels, estrogen levels, liver function studies, body iron stores, urine porphyrin excretion, and cytochrome P4501A2 (CYP1A2) activity were monitored for 1 year. Four of the women previously treated for PCT completed the study. None had evidence of a porphyric relapse. CYP1A2 activity, measured by three different methods, did not differ between study subjects receiving estrogens, patients with active PCT, and non-porphyric control subjects, nor did CYP1A2 activity change during the study period. Gonadotrophic hormone levels fell and estrogen levels rose in all women receiving estrogens. The administration of estrogens by the transdermal route appeared to be safe in the small number of subjects we studied and should be considered for women previously treated for PCT.

Cell proliferation and apoptosis in stage III inoperable non-small cell lung carcinoma treated by radiotherapy.

PURPOSE: The purpose of this study was to assess the prognostic value of the expression of p53 and bcl-2, the apoptotic index and the expression of topoisomerase II alpha in patients with inoperable non-small cell lung cancer (NSCLC) treated with high dose radiotherapy. PATIENTS AND METHODS: A number of 161 patients with inoperable NSCLC treated with high dose radiotherapy (60 Gy) were included. Immunohistochemical analysis was used to assess the expression of nuclear p53-protein, topoisomerase II alpha and cytoplasmatic expression of bcl-2, while spontaneous apoptosis was assessed using in situ labeling. The minimal follow up period was 2 years. RESULTS: Local control did not only depend on the presence of p53 expression, but also on the proportion of p53 positive cells. The most important prognostic factor was the apoptotic index. A high apoptotic index was associated with worse local control, more distant metastases and a significantly worse overall survival. No association was noted between the expression of bcl-2 and topoisomerase II alpha with any of the endpoints. CONCLUSION: This study indicates that p53 expression and the apoptotic index are prognostic factors with regard to local control in patients with inoperable NSCLC treated with radiotherapy and by combining these 2 factors, a clinically relevant estimation of the local control probability can be made. The apoptotic index turned out to be the only factor significantly related to survival.

Topoisomerase II alpha: prognostic predictor and cell cycle marker in surface epithelial neoplasms of the ovary and peritoneum.

Immunohistochemistry for Topoisomerase II alpha (TopoIIa), a nuclear protein important for the separation of chromosomes and deoxyribonucleic acid replication, provides insight into the molecular events in the cell cycle and the response to chemotherapeutic agents, which target TopoIIa. We test the hypothesis that the percentage of TopoIIa immunoreactive nuclei (TopoIIaI) aids in the treatment and prognostic evaluation of ovarian and primary peritoneal surface epithelial neoplasms (SENs) and correlates with established cell cycle control markers: p53, p21WAF1/CIP1 (p21), and Ki67. Paraffin sections from a retrospective surgical series of 108 SENs were immunostained with anti-TopoIIa, anti-p53, anti-p21, and anti-Ki67. The TopoIIaI, the Ki67 proliferation index (Ki67PI), and the immunoreactivity score for p53 and p21 (IMS: S1, S2, S3 < 10%, 10 to 50%, > 50% of strong staining cells, respectively) were evaluated manually. TopoIIaI and Ki67PI ranged from 5 to 84% and 4 to 88% (mean/median: 31/30 and 44/46%), respectively, and were correlated (coefficient 0.62, p < 10(-11)). IMS of 108 SENs was as follows: p53 50% + (2S1, 52S3) and p21 66% + (38S1, 12S2, 21S3). The TopoIIaI associated directly with p53 (p < 10(-5) and inversely with p21 (p < 0.005) IMS. TopoIIaI correlated with SEN architectural/nuclear grade (p < 10(-5)/10(-7)), but not histologic type. Sixty-seven patients had disease at last follow-up, 55 were dead from disease at 2 to 67 months (mean/median 24/21), and 14 were alive with disease at 31 to 230 months (mean/median 73/59). Forty-one patients were disease free at 5 to 228 months (mean/median 75/54). TopoIIaI correlated with presence of disease (p < 0.01) and poor survival (p < 1 x 10(-9), even when only 93 invasive SEN cases are considered (p < 0.005). TopoIIaI correlates with poor prognosis and other cell cycle control markers. The patients in this retrospective series of SEN were treated primarily with platinum-based chemotherapy. These data may suggest further prospective studies in which patients with SENs exhibiting high TopoIIaI are treated with chemotherapy targeted against TopoIIa (e.g., etoposide). In this retrospective series, high SEN TopoIIaI predicted poor survival when treated with platinum-based chemotherapy, which does not target TopoIIa.

Hemochromatosis genes and other factors contributing to the pathogenesis of porphyria cutanea tarda.

Inherited and acquired factors have been implicated in the pathogenesis of porphyria cutanea tarda (PCT), a disorder characterized by a photosensitive dermatosis and hepatic siderosis. This study, comprising 108 patients with PCT, was intended to define the role of hemochromatosis gene (HFE) mutations in the expression of PCT and to determine the contribution of acquired factors including alcohol, hepatitis C virus (HCV), and estrogen. The 2 known HFE mutations, cysteine 282 tyrosine (Cys282Tyr) and histidine 63 asparagine (His63Asp), were detected by polymerase chain reaction, and anti-HCV immunoglobulin G was detected serologically. Liver biopsies were graded for iron content, inflammation, and fibrosis. Estimates of alcohol and estrogen use were based on a questionnaire. Of the PCT patients tested, 19% were homozygous for the Cys282Tyr mutation; controls were equal to 0.5%. The compound heterozygous genotype was detected in 7% of the PCT patients; controls were less than 1%. The transferrin saturation, serum ferritin, and liver iron burden of all PCT patients were higher than those of nonporphyric controls. The highest values were found in PCT patients homozygous for the Cys282Tyr mutation. Of the patients studied, 59% were HCV positive (compared with 1.8% of the population), and 46% consumed more than 70 g of alcohol daily. Of the female patients, 63% were ingesting estrogens. Hepatic damage was most marked in patients with the Cys282Tyr/Cys282Tyr genotype who had HCV and drank heavily. Homozygosity for the Cys282Tyr mutation and HCV are the greatest risk factors for expression of PCT, and in most patients, more than 1 risk factor was identified. It was common for patients with HCV to consume alcohol. Patients with PCT should be screened for HFE mutations and for HCV. (Blood. 2000;95:1565-1571)

Proliferation, C-myc, and cyclin D1 expression in diffuse alveolar damage: potential roles in pathogenesis and implications for prognosis.

In this study we compared expression of DNA topoisomerase IIalpha, a marker of cellular proliferation, c-myc, and cyclin D1 in lung biopsy specimens showing diffuse alveolar damage (DAD) with control lung tissues. We subsequently correlated DNA topoisomerase IIalpha, c-myc, and cyclin D1 expression with survival. We hypothesized that poor outcome may correlate with a higher proliferation index, and that c-myc and cyclin D1 activation are potentially important regulators of both proliferation and apoptosis in DAD. Immnuohistochemical stains for c-myc, cyclin D1, and DNA topoisomerase IIalpha were performed on 10 cases of DAD (15 cases for DNA topoisomerase IIalpha) and 10 control lungs. A proliferation index for each case was calculated by dividing the number of nuclei expressing DNA topoisomerase IIalpha by the total number of nuclei counted. The percentages of alveolar pneumocytes and interstitial cells staining positively for c-myc and cyclin D1 were estimated. The average proliferation index (DNA topoisomerase IIalpha index) in DAD (0.16 +/- 0.06, n = 15) was significantly greater than in control lungs (0.00 +/- 0.01, n = 10) (P < .0001). The average proliferation index of patients with DAD who died of respiratory failure (0.18 +/- 0.05, n = 9) was significantly greater than the average proliferation index of patients whose respiratory disease resolved or stabilized (0.11 +/- 0.05, n = 5) (P < .03). Expression of c-myc in alveolar pneumocytes and interstitial cells was more intense and slightly more widespread in cases of DAD compared with control lungs. In 9 of 10 cases of DAD, cyclin D1 expression was present in up to 30% of alveolar pneumocytes and up to 10% of interstitial cells. No staining for cyclin D1 was present in control lungs. These results show that the proliferation index in DAD potentially correlates with patient survival. Furthermore, enhanced expression of c-myc and cyclin D1 may contribute to dysregulation of cellular proliferation and apoptosis observed in DAD.

Proliferation and cellular phenotype in lymphomatoid granulomatosis: implications of a higher proliferation index in B cells.

Pulmonary involvement by lymphomatoid granulomatosis (LYG) is characterized by nodules of a polymorphous lymphoreticular infiltrate with necrosis, angioinvasion, and variable numbers of large, atypical cells. Using combined immunohistochemistry, the authors compared the expression of a marker of proliferation (DNA topoisomerase IIalpha) between B cells, T cells, and histiocytes. Sixteen cases of LYG were stained by combined immunohistochemistry for DNA topoisomerase IIalpha and CD-20, CD-3, CD-68, and CD-57. A proliferation index was determined for B cells, T cells, histiocytes, and natural killer cells by dividing the number of cells with coexpression of DNA topoisomerase IIalpha and CD-20, CD-3, CD-68, or CD-57 by the total number of CD-20+, CD-3+, CD-68+, or CD-57+ cells, respectively. A significantly higher proliferation index was present in B cells compared to T cells, histiocytes, or natural killer cells (p < 0.002). The average proliferation index for B cells was 0.25+/-0.24 (range, 0.00-0.76), for T cells was 0.02+/-0.01 (range, 0.00-0.04), for histiocytes was 0.00+/-0.01 (range, 0-0.02), and for natural killer cells was 0.00+/-0.00 (range, 0.0-0.02). The average proliferation index of CD-20+ cells was greater in grade III LYG (0.36) than in grade II LYG (0.17) or the single case of grade I LYG (0.00). The authors conclude that (1) there is a spectrum of B-cell proliferation in LYG that roughly correlates with histologic grade, (2) T cells, histiocytes, and natural killer cells do not proliferate but are recruited, and (3) the average B-cell proliferation index in grade III LYG is similar to that observed in large cell non-Hodgkin's B-cell lymphomas. These observations provide a possible rationale for the use of chemotherapy for grade III LYG and observation or immunologic adjuvants for LYG with grade I or grade II histology.

Bcl-2 and bax protein expression in indolent versus aggressive B-cell non-Hodgkin's lymphomas.

Bcl-2 and bax are cellular proteins that are important in the regulation of apoptosis. Overexpression of bcl-2 protein is associated with prolonged cell survival, whereas overexpression of bax correlates with increased apoptosis after injury. It has been suggested that the ratio of bcl-2 and bax determines a cell's susceptibility to apoptosis. We studied bcl-2 and bax expression by immunohistochemical methods in 46 cases of B-cel non-Hodgkin's lymphoma characterized by the Revised European-American Lymphoma (REAL) classification to determine whether expression of these two proteins correlated with the histological subtype or the predicted clinical behavior (indolent v aggressive). For each case, both the percentage of cells staining as well as the intensity of staining of bcl-2 and bax were recorded, and a bcl-2-bax protein ratio (BBPR) was calculated. Bax staining was identified in 100% of the lymphomas studied. In contrast, bcl-2 staining was seen in only 67%. Bcl-2 expression correlated with the subtype of lymphoma with positive staining in 100% of small lymphocytic lymphomas, 80% of follicle center lymphomas, 38% of diffuse large cell lymphomas, 33% of high-grade B-cell Burkitt's-like lymphomas, 0% of Burkitt's lymphomas, and 0% of B-cell lymphoblastic lymphomas. The BBPR of indolent lymphomas (mean, 1.8) was significantly greater than the BBPR of aggressive lymphomas (mean, 0.6) (P < or = .002). This suggests that bax and bcl-2 expression may be linked to biological behavior in non-Hodgkin's B-cell lymphomas.

Allelic deletion analysis of the FHIT gene predicts poor survival in non-small cell lung cancer.

The fragile histidine triad (FHIT) gene at chromosome 3p14.2 is a candidate tumor suppressor gene linked to cancers of the lung, breast, colon, pancreas, and head and neck. Reports of frequent allelic deletion and abnormal transcripts in primary lung tumors plus recent evidence that it is targeted by tobacco smoke carcinogens suggest that it plays an important role in lung carcinogenesis. Non-small cell lung carcinoma still maintains a poor 5-year survival rate with the stage of disease at presentation as a major determinant of prognosis. We examined for allelic deletion at the FHIT locus in a series of 106 non-small cell lung carcinomas for which a full clinical, epidemiological, and 5-year survival profile was available. We found an allelic deletion frequency of 38% at one or two intragenic microsatellites. Allelic deletion of FHIT was related to tumor histology with 4 of 20 adenocarcinomas (20%) displaying loss of heterozygosity (LOH) compared with 12 of 22 (55%) nonadenocarcinomas (P = 0.03). We found that 63% of tumors with LOH of FHIT also had p53 missense mutations whereas only 26% with LOH had wild type p53 negative sequence (P = 0.02). We also found a significant trend toward poorer survival in patients with LOH of at least one locus of the FHIT gene (log rank, P = 0.01). This survival correlation is independent of tumor stage, size, histological subtype, degree of differentiation, and p53 mutation status. Our data support the hypothesis that the loss of the FHIT contributes to the molecular pathogenesis of human lung cancer and is an indicator of poor prognosis.

p21waf1/cip1 and transforming growth factor beta 1 protein expression correlate with survival in non-small cell lung cancer.

p21waf1/cip1 encodes a cyclin-dependent kinase inhibitor that is transcriptionally activated by the p53 tumor suppressor gene, transforming growth factor beta 1 (TGF-beta 1), AP2, and other pathways. Because p21waf1/cip1, p53, and TGF-beta 1 all regulate apoptosis and the cell cycle, we tested the hypothesis that their relative protein levels would correlate with biological features including the survival of non-small cell lung cancer (NSCLC) patients. We conducted an immunohistochemical analysis of p21waf1/cip1 and TGF-beta 1 and identified four patient groups with distinct survival outcomes. Concordant p21waf1/cip1 and TGF-beta 1 expression (i.e., either high p21waf1/cip1 and high TGF-beta 1 expression or low p21waf1/cip1 and low TGF-beta 1 expression) predicted 70% disease-free survival at 2000 days of follow-up. Discordant p21waf1/cip1 and TGF-beta 1 expression (i.e., either high p21waf1/cip1 and low TGF-beta 1 expression or low p21waf1/cip1 and high TGF-beta 1 expression) predicted 35% disease-free survival (P = 0.0003; log-rank test). These survival relationships were not attributable to differences in grade, stage, or p53 status. Although current models do not fully explain these complex interactions, most of these data fit a paradigm whereby TGF-beta 1 regulation determines NSCLC survival. In addition to the survival correlation, we found that high p21waf1/cip1 protein expression correlated with high tumor grade (P = 0.014). There is little evidence that p21waf1/cip1 protein levels accurately predict p53 mutation status in NSCLC; specifically, 20 of 48 (42%) tumors with p53 mutations contained high levels of p21waf1/cip1 protein. These findings indicate that p21waf1/cip1 immunohistochemical analysis may provide useful information concerning the biological properties of NSCLC.

Bcl-2 immunohistochemistry in a surgical series of non-small cell lung cancer patients.

The bcl-2 gene is implicated in oncogenesis by its ability to prolong cell survival through the inhibition of apoptosis, without increasing cell proliferation. An association between immunohistochemical staining for bcl-2 protein and the histological type and prognosis of non-small cell carcinoma was hypothesized by Pezzella et al. (N Engl J Med 329:690-694, 1993). In a case series, we stained formalin-fixed, paraffin-embedded tumor tissue from 106 surgical non-small cell lung cancer patients with an antibody to bcl-2 protein (DAKO clone 124, Carpinteria, CA). The resulting bcl-2 staining data were evaluated for associations with demographic, histological, immunohistochemical, and genetic features, including p53 mutations. Bcl-2 staining was observed in tumors from 29 of 106 (27%) of subjects, but was significantly less frequent in subjects' adenocarcinoma histology (8 of 55, 14.6%) (P = .007). This finding persisted after adjustment for age, gender, stage, grade, smoking history, and disease-free survival. In univariate analyses, no association was seen with age, weight, body mass index, gender, or pack-years smoking; tumor grade, stage, or patient performance status; p53 or c-erbB2 immunohistochemical staining, or p53 mutations. These data agree with earlier reports that bcl-2 staining is less common in adenocarcinomas; however, our data do not support the hypothesis that bcl-2 staining confers a better prognosis overall, in squamous cell carcinoma, or in an older patient population.

p53 mutation spectrum in relation to GSTM1, CYP1A1 and CYP2E1 in surgically treated patients with non-small cell lung cancer.

p53 mutation status was analysed in relation to DNA polymorphisms of GSTM1, CYP1A1 and CYP2E1 from 105 surgically resected non-small cell lung cancer cases. Demographic factors, smoking, occupation, family history, tumour histology, grade and stage were taken into account. p53 mutations, detected either directly by DNA sequencing (P = 0.04, adjusted for smoking) or indirectly by immunostaining (P = 0.06), were overrepresented among CYP1A1 variants. Mutations in exon 8 and transitions at CpG sites in the p53 gene were favoured in this subset. There was no relation between the individual gene polymorphisms or p53 mutations and disease-free survival by Kaplan-Meier analysis. The finding of excess CYP1A1 heterozygotes in individuals with p53 mutations after adjustment for smoking suggests that CYP1A1 activation contributes to lung cancer via p53 inactivation.

The potential role of BAX and BCL-2 expression in diffuse alveolar damage.

Apoptosis of type II pneumocytes has been identified in diffuse alveolar damage (DAD), is associated with p53 and WAF1 expression, and may be of pathogenetic importance. BAX, a homologue of BCL-2, is induced by p53 and is a promoter of apoptosis. The proapoptotic effect of BAX is negatively regulated by its binding with BCL-2. In this study, we sought to investigate that role of BAX and BCL-2 in DAD. We hypothesized that alterations in BAX and BCL-2 expression may be important in determining the susceptibility of type II pneumocytes and interstitial cells to apoptosis. Twenty-eight cases of DAD and 16 control cases (i.e., lung tissues adjacent to resected tumors) were retrieved from the files of the University of Utah and the Armed Forces Institute of Pathology. Immunohistochemical stains were performed with antigen retrieval by microwave using antibodies recognizing BAX and BCL-2. The percentage of positively staining pneumocytes and interstitial cells was estimated in each case to the nearest 10%. BAX expression was markedly increased in pneumocytes and interstitial cells in DAD compared with control lung tissues. In DAD, BAX was identified on an average of 80% of alveolar pneumocytes (range 30 to 100%) and 70% of interstitial cells (range 20 to 90%). In control lungs, BAX was identified on an average of 10% of pneumocytes (range 0 to 20%) but not in interstitial cells. Focal BCL-2 staining was identified in interstitial myofibroblasts in 7 of 25 cases of DAD but was only identified in bronchiolar epithelium of control lungs. These results suggest that the induction of BAX in DAD may enhance the susceptibility of alveolar epithelial cells to apoptosis, whereas BCL-2 expression may contribute to the absence of apoptosis in interstitial myofibroblasts. Expression of BCL-2 in interstitial myofibroblasts may contribute to the development of pulmonary fibrosis in some patients.

Human DNA topoisomerase II-alpha: a new marker of cell proliferation in invasive breast cancer.

DNA topoisomerase II-alpha is the molecular target of doxorubicin, an active drug used in the therapy of breast cancer. From many in vitro studies, it is known that high levels of topo II-alpha expression correlate with drug sensitivity, and low levels of topo II-alpha correlate with drug resistance. In addition, the enzyme is known to be a marker of cell proliferation in normal tissues. Because the number of proliferating cells in a breast cancer has been shown to be prognostically important, and because doxorubicin is used in the treatment of breast cancer, we hypothesized that the measurement of topo II-alpha in breast cancer may not only give drug sensitivity information but also may yield important data on cell proliferation. In this study, formalin-fixed, paraffin-embedded tissue from 30 specimens of invasive breast cancer from 20 patients were immunohistochemically stained for topo II-alpha with a mouse monoclonal antibody. For each case, a topo II-alpha index was determined that represents the number of positive-staining tumor cells divided by the total number of tumor cells counted times 100. A similar index was determined for MIB1, a known cell proliferation marker. Each case was also graded according to the modified Bloom-Richardson criteria and evaluated for c-erbB-2 amplification, hormonal status, S-phase fraction, and mitotic index. The topo II-alpha index correlates better with the MIB1 index than with the S-phase fraction or mitotic index. The topo II-alpha expression in breast cancer ranges from low (topo II-alpha index <1) to high (topo II-alpha index = 86). Amplification of c-erbB-2 was observed in 4 of 28 cases (14%) but did not correlate with high topo II-alpha indices. We conclude that measurement of topo II-alpha in invasive breast cancer can be readily performed by immunohistochemical staining, and it gives information on the number of cycling tumor cells. In addition, because the enzyme is the molecular target of doxorubicin, the expression of the enzyme may relate also to the sensitivity or resistance of the tumor to doxorubicin-based chemotherapeutic protocols.

Disseminated lipogranulomas and sudden death from self-administered mineral oil injection.

The cosmetic injection of exogenous lipids and more recently of polydimethyl siloxane (injectable silicone) into the scrotum has been described since 1899. Sclerosing lipogranuloma and paraffinoma are terms applied to a complication of this practice in which the injected oils or silicone elicit a marked granulomatous reaction with prominent surrounding fibrosis. Although this complication has been described as a localized process occurring mainly in the scrotal area and regional lymph nodes, few studies have documented systemic manifestations. In this report we describe the autopsy findings of a 48-year-old man who had scrotal and systemic lipogranulomas from repeated self-administered injection of mineral oil. In addition, severe acute pulmonary edema resulted in sudden unexpected death. To our knowledge, this fatal complication of exogenous lipogranuloma has not been previously reported.

p53 mutations and occupational exposures in a surgical series of lung cancers.

p53 mutations are frequent in malignant lung tumors. Of 88 surgically treated lung cancers from cigarette smokers previously evaluated for p53 mutations, 45 tumors (51.1%) had mutations in exons 5-8 (D. G. Guinee, Jr. et al., Carcinogenesis (Lond.), 16: 993-1002, 1995). We report here the examination of 13 occupational exposures and 13 high-risk occupations in relation to these p53 mutations. Two molecular abnormalities were associated with occupational exposures: (a) G:C-->T:A transversions on the coding (nontranscribed) strand (n = 13) were associated with chromate exposure and employment in the metal industry (P < 0.05) and marginally associated with nickel exposure (P = 0.056); and (b) G:C-->A:T transitions at non-CpG sites (n = 9) were associated with work in the petrochemical industry (P = 0.05). No association was found between p53 mutations and gender, cigarette pack-years, tumor histology, age at diagnosis, or family history of lung cancer. Because all three chromate-exposed subjects had large cell carcinomas exhibiting G: C-->T:A coding-strand transversions, follow-up of a cohort with this exposure should clarify the association with the p53 gene.

Comparison of DNA topoisomerase II alpha expression in small cell and nonsmall cell carcinoma of the lung. In search of a mechanism of chemotherapeutic response.

BACKGROUND: Small cell carcinoma of the lung (SCLC) is distinguished from nonsmall cell carcinoma (NSCLC) by its exquisite initial sensitivity to chemotherapy. Antineoplastic drugs effective against SCLC include doxorubicin, etoposide, and others. Recently, the molecular target of these drugs has been identified as the alpha form of DNA topoisomerase II, which is important in DNA replication and in the separation of chromosomes during normal cellular division. In this study we compared DNA topoisomerase II alpha expression in SCLC and NSCLC by immunohistochemistry. We hypothesized that the sensitivity of SCLC and relative insensitivity of NSCLC to these chemotherapeutic agents stem from different frequencies of DNA topoisomerase II alpha expression. METHODS: DNA topoisomerase II alpha expression was analyzed in 17 cases of SCLC and 24 cases of NSCLC by immunohistochemistry utilizing a monoclonal antibody recognizing the alpha isoform of DNA topoisomerase II. A topo II index was determined by dividing the number of tumor nuclei expressing DNA topoisomerase II by the total number of tumor nuclei counted. RESULTS: A significantly higher frequency of DNA topoisomerase II alpha expression was identified in SCLC (P < 0.001). The average topo II index for SCLC was 0.60 (range: 0.45-0.76) compared with NSCLC, 0.31 (range: 0.05-0.75). CONCLUSIONS: We conclude that DNA topoisomerase II alpha is expressed at a higher frequency in SCLC than in NSCLC, and that this expression is possibly involved in the response of SCLC to chemotherapeutic agents.

Association of p53 and WAF1 expression with apoptosis in diffuse alveolar damage.

Little is known about alterations in cell cycle regulatory proteins such as p53 and WAF1 in diffuse alveolar damage (DAD). We hypothesized that up-regulation of p53 and WAF1 in type II pneumocytes in DAD is associated with underlying DNA damage and apoptosis. Twenty cases of DAD and twenty control specimens of lung adjacent to resected tumors were studied. Immunohistochemical stains with antibodies recognizing p53 and WAF1 were performed, and apoptosis was assessed in sixteen cases by the nick end-labeling method. We identified p53 expression and apoptosis in all cases of DAD but not in any of the control lungs. We detected WAF1 expression in nineteen of twenty cases of DAD and in sixteen of twenty control lungs. In general, the distribution and intensity of WAF1 staining were greater in DAD than in control lungs. Staining for both p53 and WAF1 and labeling of apoptotic cells in DAD were usually focal ( < 10% of cells) and predominantly localized in type II pneumocytes. We conclude that increased p53 and WAF1 expression in DAD reflects normal physiological up-regulation in response to cellular and DNA damage and is associated with apoptosis of type II pneumocytes. p53-dependent apoptosis may contribute to the pathogenesis of this disease.

Analysis of p53, K-ras-2, and C-raf-1 in pulmonary neuroendocrine tumors. Correlation with histological subtype and clinical outcome.

Neuroendocrine tumors of lung, including typical carcinoid (TC), atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC), and small cell lung carcinoma (SCLC) constitute a spectrum of malignancies in which the pathologist at times has difficulty in discerning tumor subtype and aggressiveness in a reproducible fashion. Therefore, 59 primary neuroendocrine lung tumors including 10 TCs, 26 ACs, 15 LCNECs, and 8 SCLCs were selected from cases collected from 1976 to 1988 and immunostained for p53 protein. All of these tumors were also genotyped for specific point mutational damage affecting p53 (exons 5, 7, and 8; with ACs additionally sequenced for p53 exon 6); 13 tumors for K-ras-2 (exon 1); and 31 tumors for c-raf-1 (exon 15) growth-regulatory genes. Genotyping was performed on topographically selected, minute tumor samples removed from unstained formalin-fixed, paraffin-embedded tissue sections (topographic genotyping) using polymerase chain reaction and direct sequencing. The distribution of p53 immunohistochemical staining had four patterns: negative in TCs, one-half of ACs, 3 of 15 LCNECs, and 1 of 8 SCLCs; less than 10% but more than five tumor cells per 10 high power fields (focal) in a subset (7 of 26) of aggressive ACs; 10 to 49% of tumor cells (patchy) in a subset (6 of 26) of ACs with a higher grade of aggressiveness; and 50 to 100% of tumor cells (diffuse), exclusively seen in LCNECs (12 of 15) and SCLCs (7 of 8). Three patterns of immunohistochemical staining intensity of p53 protein were seen: negative, weak or mild, and moderate to marked. SCLCs and LCNECs accounted for cases of moderate to marked staining and were the only ones to have mutations in p53 exons 5, 7, or 8. No mutations were found in AC and TC, showing absent to weak staining and no staining, respectively. The difference in distribution and staining intensities between LCNEC and SCLC compared with AC and TC was statistically significant (P < 0.001). Patients having AC with patchy p53 immunostaining usually had survival limited to 3 years, whereas those having AC with focal p53 immunostaining subsequently developed metastatic or recurrence of AC disease (P < 0.05). The absence of point mutations in cases with patchy or focal immunostaining suggests increased expression of wild-type p53 tumor suppressor protein likely in response to growth deregulation in a more aggressive subtype of AC. A novel hypothesis is presented in regard to these findings. K-ras-2 and c-raf-1 gene sequence analysis showed no evidence of point mutational change in any of the tumors studied. The TC and AC categories are therefore genetically distinct from the higher grade neuroendocrine SCLC and LCNEC. Immunohistochemistry for p53 on AC lung tumors may be helpful to delineate cases at higher risk for aggressive behavior. Additionally, although LCNEC is categorized as a non-small-cell carcinoma, it is more akin genetically and immunohistochemically to SCLC.