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.

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.

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.

Unique pulmonary presentation of an angiomyolipoma. Analysis of clinical, radiographic, and histopathologic features.

Extrarenal angiomyolipomas are rare lesions that have been described in the liver, hard palate, skin, uterus, vagina, penis, and spermatic cord. In this report we present the clinical, radiographic, and pathologic findings of an angiomyolipoma of the lung in a 68-year-old woman without tuberous sclerosis or lymphangioleiomyomatosis. To our knowledge, this report is the first description of pulmonary angiomyolipoma. Distinction from other benign and malignant pulmonary mesenchymal lesions depends on recognition of traditional histologic criteria. In contrast to renal angiomyolipomas, study of this case and review of prior reports reveals that extrarenal angiomyolipomas are most often well demarcated, easily resected, and not associated with tuberous sclerosis.

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.

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.

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.

The spectrum of immunohistochemical staining of small-cell lung carcinoma in specimens from transbronchial and open-lung biopsies.

Immunohistochemistry is increasingly used as an aid in the diagnosis of small-cell lung carcinoma (SCLC). Previous studies have investigated immunohistochemical staining of SCLC with small numbers of antibodies, but few have examined large series with a broad panel of antibodies. For this reason, the authors examined the distribution and intensity of staining of 20 open-lung biopsy (OLB) and 21 transbronchial biopsy (TBB) specimens of SCLC with a panel of epithelial, neuroendocrine, and hormonal markers. Small-cell lung carcinoma stained most frequently with epithelial markers, followed by neuroendocrine and hormonal markers. Similar percentages of OLB and TBB specimens stained for keratin (100% each) and epithelial membrane antigen (100% and 95%, respectively). Unexpectedly, BER-EP4 stained 100% of OLB specimens. Chromogranin A was the most frequent neuroendocrine marker in OLB and TBB specimens (60% and 47%, respectively) followed by neuron-specific enolase (60% and 33%), Leu-7 (40% and 24%), and synaptophysin (5% and 19%). No neuroendocrine immunohistochemical reactivity was found in 24% of TBB specimens and 20% of OLB specimens. Bombesin was the most sensitive hormonal marker (45% of OLB specimens). These results show that keratin, epithelial membrane antigen, and BER-EP4 are reliable epithelial markers for SCLC in both TBB and OLB specimens. In addition, negative staining for neuroendocrine markers, because it can occur in as many as 25% of cases, should not deter the diagnosis of SCLC.

Arachidonic acid and acetylcholine induced relaxation of bovine coronary artery: effect of indomethacin and methylene blue.

Endothelial intact bovine coronary artery (BCA) rings precontracted with U46619 relax to acetylcholine (Ach) and arachidonic acid (AA). Relaxation to Ach is blocked by atropine. In an attempt to elucidate the mechanisms of these responses, we found that preincubation with methylene blue, an inhibitor of soluble guanylate cyclase activity, inhibited both Ach and AA induced relaxation. Preincubation with indomethacin had no effect on Ach induced relaxation, but partially inhibited the relaxation of endothelial intact BCA rings to AA.

Pulmonary lymphangioleiomyomatosis. Diagnosis based on results of transbronchial biopsy and immunohistochemical studies and correlation with high-resolution computed tomography findings.

The diagnosis of lymphangioleiomyomatosis was established in a 35-year-old woman with hemoptysis, mild cough, and dyspnea based on histologic review of results of a transbronchial biopsy correlated with high-resolution computed tomographic scan findings. A chest x-ray film revealed diffuse interstitial lung disease, and a high-resolution computed tomographic scan showed diffuse cystic changes throughout both lungs. The transbronchial biopsy specimen revealed cystic changes and a patchy, sometimes nodular proliferation of smooth muscle that focally expanded the interstitium suggestive of lymphangioleiomyomatosis. The smooth-muscle nature of the cells was confirmed by positive immunohistochemical stains for actin and desmin; positive staining for HMB-45 was also observed. Although the diagnosis of lymphangioleiomyomatosis usually requires an open lung biopsy, this case shows that rarely, in the appropriate clinical setting, the diagnosis may be rendered based on results of a transbronchial biopsy in conjunction with findings from ancillary immunohistochemical studies and high-resolution computed tomography.

Clinically silent progressive renal tubulointerstitial disease during cisplatin chemotherapy.

BACKGROUND: Chronic cisplatin nephrotoxicity is well documented in animal models but not well characterized in humans. The authors report a 56-year-old woman who had end-stage chronic tubulointerstitial nephropathy develop during treatment with multiple courses of cisplatin chemotherapy for ovarian carcinoma. METHODS: A biopsy was performed to determine the etiology of renal failure, and the morphologic, immunofluorescent, and ultrastructural findings were analyzed to identify possible causes, other than cisplatin, of chronic renal disease. RESULTS: Morphologic studies showed extensive renal tubulointerstitial fibrosis with relative sparing of glomeruli. CONCLUSIONS: Profound, progressive renal injury occurred during cisplatin treatment despite adherence to treatment protocols designed to minimize such toxicity. Renal injury was undetected by pretreatment serum creatinine determinations. This case and others emphasize the relative insensitivity of this test for chronic renal damage during treatment with nephrotoxic drugs.

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.

Pleomorphic (spindle/giant cell) carcinoma of the lung. A clinicopathologic correlation of 78 cases.

BACKGROUND: The authors undertook this study to define the clinical and histologic characteristics of spindle and giant cell carcinomas of the lung and the survival and prognostic features of these tumors. METHODS: Seventy-eight cases of pleomorphic (spindle and/or giant cell) carcinoma of the lung were studied by light microscopy and immunohistochemistry to establish clinical, gross, and histologic parameters. Follow-up information was obtained from contributing physicians and analyzed by statistical means to determine prognostically significant parameters. RESULTS: The patient population consisted of 57 men and 21 women (male to female ratio, 2.7 to 1) between the ages of 35 and 83 years (mean, 62 years). Clinically, 58 patients (80%) presented with symptoms including thoracic pain, cough, and hemoptysis, whereas 14 (18%) were asymptomatic. At the time of diagnosis, 41% of the patients had clinical Stage I lesions, 6% Stage II lesions, 39% Stage III lesions, and 12% Stage IV lesions. Histologically, foci of squamous cell carcinoma were present in 8% of the tumors, large cell carcinoma in 25%, and adenocarcinoma in 45%. The remaining 22% of neoplasms were completely spindle and/or giant cell carcinomas. Spindle and giant cell carcinomas were found together in 38% of the patients. In the 69 patients for whom follow-up information was obtained, 53 (77%) died within 7 days to 6 years after diagnosis, with a 23-month mean survival (median, 10 months) (Kaplan-Meier method). There was a significant shortening of survival for patients with tumor size greater than 5 cm, clinical stage greater than 1, and lymph node involvement. The presence of nodal metastases was the most significant single prognostic factor, whereas the presence of squamous or adenocarcinomatous differentiation did not have an impact on length of survival. CONCLUSIONS: The frequency with which spindle and giant cell carcinomas are found together, their frequent association with other histologic subtypes of lung carcinoma, and the similar clinicopathologic features of these tumors suggest that they are best regarded as one type of lung cancer called pleomorphic carcinoma.

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.

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.

Gender comparisons in human lung cancer: analysis of p53 mutations, anti-p53 serum antibodies and C-erbB-2 expression.

Little is known about the molecular mechanisms of lung carcinogenesis in women. We initiated an investigation of the role of gender in pulmonary carcinogenesis by analysis of p53 mutations, immunohistochemistry, serum antibodies and c-erbB-2 expression in a series of 63 male and 44 female lung cancer patients whose tumors were resected at the Mayo Clinic between 1991 and 1992. There were 102 smokers and 5 never smoked. Adenocarcinoma was the more frequent histological type in women (62%) than in men (41%). Sequence analysis of exons 5-8 in 42 females and 49 males identified 44 p53 mutations in 42 tumors (46%). Base substitution mutations showed a preponderance of G:C-->T:A transversions, which were more frequent in women than men (40 versus 25%) and in individuals exposed to asbestos. c-erbB-2 immunohistochemical staining was identified more frequently in females (nine cases) than males (two cases). Marked immunohistochemical staining for p53 positively correlated with the presence of missense mutations in exons 5-8 (81%, P < 0.001). Seven missense mutations (four in exon 5, two in exon 6, one in exon 8) were identified in five of nine patients who had serum antibodies recognizing p53; tumors from these patients were also strongly positive for p53 by immunohistochemistry. These and other results indicate gender differences in the genetic and biochemical alterations in lung cancer and generate hypothesis regarding gender differences in lung cancer susceptibility.