Epstein-Barr virus microRNAs and lung cancer.

BACKGROUND: We conducted the first analysis of viral microRNAs (miRNAs) in lung cancer, with a focus on Epstein-Barr virus (EBV). METHODS: We evaluated viral miRs with a two-channel oligo-array targeting mature, anti-sense miRNAs in 290 cases. In 48 cases, we compared microarray and real-time quantitative PCR (qPCR) expression for three EBV miRNAs. We tested for EBV DNA, RNA, and protein in tumour tissue from six cases with and six cases without strong qPCR-based evidence of EBV miRNAs. RESULTS: The EBV miRNAs strongly differentiated between adenocarcinoma and squamous cell carcinoma using the microarray (P<0.01 for 9 out of 16 EBV miRNAs). However, microarray and qPCR measurements of BART1, BART2, and BHRF1-3 expression were not significantly correlated (P=0.53, 0.94, and 0.47, respectively). Although qPCR provided substantial evidence of EBV miRNAs in 7 out of 48 cases, only 1 of these 7 cases had detectable EBV DNA in tumour tissue. None had detectable EBV RNA or protein by histochemical stains. CONCLUSION: In a comprehensive evaluation of EBV miRNA, DNA, RNA, and protein in lung cancer, we found little evidence of EBV in lung tumour tissue. Discrepancies between microarray- and qPCR-based strategies highlight the difficulty of validating molecular markers of disease. Our results do not support a role of EBV in lung cancer.

Signal pathways which promote invasion and metastasis: critical and distinct contributions of extracellular signal-regulated kinase and Ral-specific guanine exchange factor pathways.

Approximately 50% of metastatic tumors contain Ras mutations. Ras proteins can activate at least three downstream signaling cascades mediated by the Raf-MEK-extracellular signal-regulated kinase family, phosphatidylinositol-3 (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (RalGEFs). Here we investigated the contribution of RalGEF and ERK activation to the development of experimental metastasis in vivo and associated invasive properties in vitro. Each pathway contributes distinct properties to the metastatic phenotype. Following lateral tail vein injection, 3T3 cells transformed by constitutively active Raf or MEK produced lung metastasis that displayed circumscribed, noninfiltrating borders. In contrast, 3T3 cells transformed by Ras(12V,37G), a Ras effector mutant that activates RalGEF but not Raf or P13 kinase, formed aggressive, infiltrative metastasis. Dominant negative RalB inhibited Ras(12V,37G)-activated invasion and metastasis, demonstrating the necessity of the RalGEF pathway for a fully transformed phenotype. Moreover, 3T3 cells constitutively expressing a membrane-associated form of RalGEF (RalGDS-CAAX) formed invasive tumors as well, demonstrating that activation of a RalGEF pathway is sufficient to initiate the invasive phenotype. Despite the fact that Ras(12V,37G) expression does not elevate ERK activity, inhibition of this kinase by a conditionally expressed ERK phosphatase demonstrated that ERK activity was necessary for Ras(12V,37G)-transformed cells to express matrix-degrading activity in vitro and tissue invasiveness in vivo. Therefore, these experiments have revealed a hitherto-unknown but essential interaction of the RalGEF and ERK pathways to produce a malignant phenotype. The generality of the role of the RalGEF pathway in metastasis is supported by the finding that Ras(12V,37G) increased the invasiveness of epithelial cells as well as fibroblasts.

Neuroendocrine-specific protein (NSP)-reticulons as independent markers for non-small cell lung cancer with neuroendocrine differentiation. An in vitro histochemical study.

Neuroendocrine-specific protein (NSP)-reticulons have recently been discovered and were shown to exhibit a restricted, neuroendocrine/neural-specific expression pattern. These protein aggregates are anchored to the membranes of the endoplasmic reticulum and occur in small cell lung cancer (SCLC), but not in typical non-SCLC. In the current study we have examined the occurrence of NSP-reticulons in non-SCLC cell lines known to express neuroendocrine features (non-SCLC-NE). NSP-reticulon expression was observed in all three non-SCLC-NE cell lines studied, albeit with variable intensity and in varying numbers of cells. Western blot analysis confirmed the presence of NSP-reticulon expression in these non-SCLC-NE cell lines, and showed that they were predominantly of the NSP-A type. When compared to conventional neuroendocrine markers, NSP-reticulons revealed a distinct staining profile, showing only partial overlap with the other markers. The non-SCLC-NE cell lines combined these neuroendocrine characteristics with some features of non-SCLC. We conclude that NSP-reticulon expression is restricted to lung carcinoma cells with a neuroendocrine phenotype and predict that these constituents may become clinically relevant markers for the detection of neuroendocrine differentiation in solid tumours.

Phase II trial of fludarabine phosphate and interferon alfa-2a in advanced mycosis fungoides/Sézary syndrome.

PURPOSE: This phase II study was undertaken to assess the efficacy and toxicity of the addition of continuous low-dose interferon alfa-2a (IFN) to fludarabine in patients with advanced or refractory mycosis fungoides (MF) or the Sézary syndrome (SS). PATIENTS AND METHODS: Thirty-five patients were treated with fludarabine 25 mg/m2 intravenously (IV) on days 1 to 5 every 28 days along with IFN 5 x 10(6) U/m2 subcutaneously three times per week continuously for up to eight cycles. IFN doses were escalated to 7.5 x 10(6)/m2 at day 29 if constitutional toxicities were less than grade 3. Twenty-one patients had not responded to prior chemotherapy or total-skin electron-beam irradiation (TSEB), and 10 of these had received prior deoxycoformycin (pentostatin; DCF) and intermittent high-dose IFN; seven had received only topical therapies, and seven were untreated. RESULTS: Four patients achieved a complete response (CR) and 14 achieved a partial response (PR) for an overall response rate of 51% (95% confidence interval, 35% to 70%). Four of 11 patients with visceral involvement responded. The median progression-free survival duration of the patients who responded was 5.9 months, and three of the complete responders are in unmaintained response after 18 to 35 months. Grade 3 or 4 hematologic toxicity occurred in 21 patients, including two who developed persistent bone marrow aplasia. Eighteen patients developed infections during therapy, including five with herpes zoster, one with Pneumocystis carinii, one with extrapulmonary tuberculosis, and two with disseminated toxoplasmosis. CONCLUSION: The combination of fludarabine with continuous low-dose IFN is an active regimen in patients with advanced MF/SS, including patients with visceral involvement and patients who progressed after prior therapy with DCF and IFN. This regimen has induced unmaintained remissions in a small subset of patients.

Phase II study of pentostatin and intermittent high-dose recombinant interferon alfa-2a in advanced mycosis fungoides/Sézary syndrome.

PURPOSE: This phase II study was undertaken to assess the efficacy and toxicity of alternating administration of pentostatin (deoxycoformycin [DCF]) and interferon alfa-2a (IFN) in patients with advanced or refractory mycosis fungoides (MF) or the Sézary syndrome (SS). PATIENTS AND METHODS: Forty-one patients underwent therapy with alternating cycles of DCF 4 mg/m2 intravenously (IV) days 1 through 3 and IFN 10 million U/m2 intramuscularly (IM) day 22, and 50 million U/m2 intramuscularly (IM) days 23 through 26. Twenty-nine patients had not responded to prior chemotherapy or total-skin electron-beam irradiation (TSEB), six had not responded to topical therapies, and six had no previous treatment. RESULTS: Two patients achieved a complete response (CR) and 15 achieved a partial response (PR), for an overall response rate of 41% (95% confidence interval, 26% to 58%). No responses were observed in the seven patients with visceral involvement. The median progression-free survival of patients who responded was 13.1 months. IFN-related constitutional symptoms were reported in 39% of patients; severe toxicities included cardiomyopathy in one patient, acute and chronic pulmonary dysfunction in four, and reversible mental status changes in two. Seven patients developed herpes zoster during therapy and six had staphylococcal bacteremia. CONCLUSION: These results suggest that the combination of DCF and IFN is an active regimen in MF patients without visceral involvement.

Thymic carcinoid in association with MEN syndromes.

Although carcinoid tumors in association with multiple endocrine neoplasia syndrome (MEN) has been well described, thymic carcinoid in association with MEN is extremely rare (only 23 cases in the world literature). A patient with thymic carcinoid and MEN-I was treated with surgical resection and postoperative radiation therapy, which was later followed by subtotal parathyroidectomy for hyperparathyroidism. Four years later, a symptomatic recurrence of his thymic carcinoid was resected from below his right clavicle. Six years after his original operation, the patient came to the hospital with pancreatitis, and a 5 cm, distal, pancreatic metastasis was resected. He now has symptomatic paraspinal and pleural metastases and is receiving somatostatin. Review of the world's literature shows that the majority of patients with thymic carcinoid and MEN-I are men with an average age of 37 years. Their clinical course is indolent, and surgery represents the only means of cure. Adjuvant chemotherapy and radiation therapy confer no survival advantage. The surgical decision making involved in treating a patient with thymic carcinoid and hyperparathyroidism associated with MEN is also discussed.

Wild-type but not mutant p53 suppresses the growth of human lung cancer cells bearing multiple genetic lesions.

Accumulating evidence indicates that lung cancer arises due to multiple genetic changes in both dominant oncogenes, such as ras, and tumor suppressor genes, such as p53. In this report we examined whether the wild-type p53 gene is able to suppress in vitro and/or in vivo cellular growth of lung cancer cell lines which carry multiple genetic abnormalities. Introduction of a wild-type p53 complementary DNA expression vector into lung cancer cell lines carrying either a homozygous deletion (NCI-H358) or a missense mutation (NCI-H23) in the p53 gene greatly suppressed tumor cell growth. In contrast, p53 expression vectors bearing lung cancer derived mutations affecting single amino acids had lost this growth suppressing ability.

Nonrandom structural and numerical chromosome changes in non-small-cell lung cancer.

Cytogenetic studies were performed on 27 tumor cell lines (most of which were derived from metastatic lesions) and four fresh malignant pleural and pericardial effusions from 30 patients with non-small-cell lung cancer (non-SCLC). Many clonal structural (deletions and nonreciprocal translocations) and numerical abnormalities were found in each specimen. Statistical analysis revealed these changes were nonrandomly distributed among the chromosomes. A statistically significant number of chromosomal breakpoints were seen in regions 1q1, 1q3, 3p1, 3p2, 3q1, 3q2, 7q1, 13p1, 14p1, 15p1, and 17q1 when the regions were compared to the total haploid complement. In addition, when a given region was compared to other regions within the same chromosome, statistically significant numbers of breakpoints were noted for regions 1q3, 5q1, 7q1, 13p1, 14p1, 15p1, 16q2, 17q1, and 21p1. Specific chromosome bands showing the most frequent involvement in structural abnormalities were (in descending order) 3p14.2, 3q21, 19q13, 11p15, 1q11, 7q11, 1q21, 3p23, and 3p21. The breakpoints indicate areas to look for new dominant oncogenes activated by translocations, while the areas of deletions and loss of material by nonreciprocal translocations highlight areas to search for recessive oncogenes. These cytogenetic studies represent strong evidence that multiple genetic lesions are associated with the development of metastatic lung cancer, and provide a roadmap to search for new genes involved in the pathogenesis of lung cancer.

Pathology of non-small cell lung cancer. New diagnostic approaches.

Non-small cell lung cancers (NSCLC) comprise 75% of all lung cancers and consist of three major histologic types: squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. The histopathology of lung cancer appears to be changing: The incidence of squamous cell carcinoma in the United States is declining, accompanied by the increase in the incidence of adenocarcinoma. Carcinoma of the lung is thought to arise from a pluripotent epithelial cell capable of expressing a variety of phenotypes. Malignant transformation is the end result of multiple events involving the growth control of bronchopulmonary epithelium. It is well known that squamous cell carcinomas are preceded by many years of progressive mucosal changes including squamous metaplasia, dysplasia, and carcinoma in situ. Premalignant changes associated with the other types of NSCLC are less well understood. Recently characterized markers for peripheral airway cell differentiation and selected monoclonal antibodies may be helpful. It is conceivable to identify specific genetic events at the cellular level using in situ hybridization or polymerase chain reaction. Biologic and genetic studies have renewed the awareness of the pleomorphism of NSCLC. Potentially interesting subsets include the following: (1) The expression of neuroendocrine (NE) markers has been demonstrated in selected NSCLC (NSCLC-NE), mostly in adeno- and large cell carcinomas. (2) The presence of K-ras mutations in surgically resected adenocarcinomas has been associated with shortened survival times. (3) Also, the neu gene encoded protein p185 has been associated with a more aggressive clinical course in adenocarcinomas. Further studies are needed to confirm such results and correlate the findings with the current WHO NSCLC classification. Rapid validation of relevant new diagnostic approaches is an enormous challenge. Although selected immunohistochemical and molecular biologic techniques may work on routinely processed paraffin-embedded material obtained from the pathology archives, many of the newest applications require fresh or freshly frozen specimens from large numbers of patients with a computerized clinical data base for adequate clinicopathologic correlations. Establishing such a resource is obviously a team effort requiring close collaboration of the oncologist, pathologist, surgeon, and technicians.

Mucinous cystadenoma of the lung. A report of two cases with immunohistochemical and ultrastructural analysis.

We describe two patients who presented with solitary pulmonary masses that consisted of unilocular cysts lined by columnar mucinous epithelium. The cysts contained copious mucus. The epithelial lining of the cysts showed foci of stratification and papillary infolding. Histologically identical lesions have previously been termed unusual mucous cysts or mucinous cystadenomas. We believe that these tumors are true neoplasms differentiating toward the respiratory epithelial mucous cell. They should be distinguished from a variety of pulmonary neoplasms including bronchoalveolar carcinoma, bronchial mucous gland adenoma, mucoepidermoid carcinoma, and metastatic adenocarcinoma.

Electron microscopic localization of enkephalin-like immunoreactivity in the human adrenal medulla.

Enkephalin-like immunoreactivity in human adrenomedullary cells was studied at the light and electron microscopic levels. Enkephalin immunostaining was associated with chromaffin granules and, in a few cells, with the rough endoplasmic reticulum as well. The relative number of stained granules varied from cell to cell, and a correlation with a particular granular population was not noted. Both large and small granules were labelled. It is concluded that in the human the ability to store enkephalin immunoreactive peptides is a general property of chromaffin granules and, furthermore, is not correlated with specific granular subpopulations or the particular type of catecholamine stored within the cell.

Substance P: the relationship between receptor distribution in rat lung and the capacity of substance P to stimulate vascular permeability.

The interaction of substance P (SP) with specific receptors in intact lung tissue was autoradiographically visualized, using slide-mounted tissue sections of rat lung tissue. SP receptors are highly concentrated in the central airways and are not detectable in peripheral bronchi, vessels, and alveoli. Within central airways, receptor distribution is most concentrated in the epithelium and small vessels in the lamina propria. Smooth muscle in airway or blood vessel walls expressed no detectable SP receptors. Immunohistochemical staining for SP revealed SP-containing nerves in the same areas where the receptors are localized. Displacement curves of SP bound to rat lung indicated that the C-terminal fragment was much more effective than the N-terminal fragment at competing for SP binding. Injection of 0.3 to 30 nmol/kg SP dramatically increased vascular permeability in the trachea and to a lesser extent in the hilus. Peripheral lung failed to respond to SP with increased vascular permeability unless toxic concentrations of SP were employed. SP increased the transudation of protein into the trachea within 5 min of injection, and the extravasated protein persisted through at least 2 h. Both SP and SP(3-11) were capable of stimulating increased vascular permeability, but SP(1-4) was inactive. SP caused mast cell degranulation as reflected in increased plasma histamine levels after SP or SP(3-11) injection, but SP(1-4) had no effect. In order to determine if histamine release caused by SP contributed to the vascular permeability response, the effects of H1 and H2 antihistamine treatment were studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies for the in vitro detection of small cell lung cancer metastases in human bone marrow.

Three rat monoclonal antibodies were selected for the immunodetection of small cell lung cancer metastases in bone marrow and other hematologic samples. By membrane immunofluorescence, they define three distinct surface antigens here termed lung cancer-associated antigens or LCAs. The latter are widely expressed on small cell lung cancer and non-small cell lung cancer cells/cell lines, but not detectable on a variety of normal and transformed bone marrow, blood and lymphoid cells. Anti-LCA1 (IgM) is similar to the many anti-lacto-N-fucopentaose III IgM antibodies rasied against human tumors. In contrast, anti-LCA2 (IgG2b) and anti-LCA3 (IgG2a) define surface proteins of 29, 32, 41 and 98 kilodaltons, respectively, that have not been reported earlier. These three reagents have immunodiagnostic potential, since in combination they label all 49 lung cancer cell lines tested. Their ability to detect lung cancer metastases in patient's bone marrow samples is documented in an accompanying paper.

Changes in the phenotype of human small cell lung cancer cell lines after transfection and expression of the c-myc proto-oncogene.

Small cell lung cancer growing in cell culture possesses biologic properties that allow classification into two categories: classic and variant. Compared with classic small cell lung cancer cell lines, variant lines have altered large cell morphology, shorter doubling times, higher cloning efficiencies in soft agarose, and very low levels of L dopa decarboxylase production and bombesin-like immunoreactivity. C-myc is amplified and expressed in some small cell lung cancer cell lines and all c-myc amplified lines studied to date display the variant phenotype. To investigate if c-myc amplification and expression is responsible for the variant phenotype, a normal human c-myc gene was transfected into a cloned classic small cell lung cancer cell line not amplified for or expressing detectable c-myc messenger RNA (mRNA). Clones were isolated with one to six copies of c-myc stably integrated into DNA that expressed c-myc mRNA. In addition, one clone with an integrated neo gene but a deleted c-myc gene was isolated and in this case c-myc was not expressed. C-myc expression in transfected clones was associated with altered large cell morphology, a shorter doubling time, and increased cloning efficiency, but no difference in L dopa decarboxylase levels and bombesin-like immunoreactivity. We conclude increased c-myc expression observed here in transfected clones correlates with some of the phenotypic properties distinguishing c-myc amplified variants from unamplified classic small cell lung cancer lines.

Immunohistochemical evidence for the occurrence of vasoactive intestinal polypeptide (VIP)-containing nerve fibres in human fetal abdominal paraganglia.

The abdominal paraganglia in man represent a major source of catecholamines, and perhaps peptide hormones, during the fetal period. The nature of the innervation of the abdominal paraganglia was studied immunohistochemically by utilising antibodies to vasoactive intestinal polypeptide, enkephalin, substance-P and somatostatin. The paraganglia showed an abundant network of VIP-immunoreactive fibres, and similar nerve fibres were found within nerve bundles of the preaortic sympathetic plexus. Occasionally, VIP-immunoreactive fibres were seen within the prevertebral ganglia, but stained cell bodies were never observed. It may be suggested that VIP-containing nerves could regulate a secretory response from fetal human abdominal paraganglia.

Markedly decreased expression of class I histocompatibility antigens, protein, and mRNA in human small-cell lung cancer.

We have found markedly deficient expression of the class I major histocompatibility antigens HLA-A,B,C and beta 2m on human small-cell lung cancer (SCLC) lines and fresh tumor samples. The deficit of HLA-A,B,C and beta 2-microglobulin (beta 2m) antigen expression was demonstrated with both radiobinding assays and indirect immunofluorescence assays. Immunoprecipitation of metabolically labeled cells with antibodies to class I antigens showed most SCLC lines to have synthesized almost no beta 2m and HLA-A,B,C proteins. Northern blot analysis, using human HLA-A,B, and beta 2m cDNA probes, showed that almost all SCLC lines tested had markedly decreased amounts of HLA and beta 2m mRNA, but both gene products could be induced with interferon treatment of SCLC lines. We conclude that human SCLC, in contrast to other lung cancer types, is characterized by greatly reduced transcription of HLA-A,B,C and beta 2m genes, which suggests the existence of a mechanism for evading the host immune response to the tumor and of an E1a-like product in this type of tumor cell.

Small cell lung cancer, endocrine cells of the fetal bronchus, and other neuroendocrine cells express the Leu-7 antigenic determinant present on natural killer cells.

Small cell lung cancer is distinguished from other lung cancer histologic types by possessing a variety of neuroendocrine properties. Anti-Leu-7 is a monoclonal antibody that recognizes a 110,000-dalton molecular weight glycoprotein initially described on natural killer cells and subsequently reported on a variety of normal and malignant neural and neuroendocrine cell types. We have found intense anti-Leu-7 binding to a large number of small cell lung cancers, while other lung cancer types were negative or showed only weak and focal binding. Other antigens expressed by natural killer cells, lymphocytes, and monocytes were never or less often expressed on small cell lung cancer cells. In addition, we report for the first time anti-Leu-7 binding by carcinoids, carotid body tumors, pheochromocytomas, endocrine cells of the fetal bronchus and the adult intestine, and select pancreatic islet cells. Anti-Leu-7 binding by small cell lung cancer is consistent with a derivation from pulmonary precursor cells, and anti-Leu-7 staining is clinically useful for the identification of human neuroendocrine tumors of the amine precursor uptake and decarboxylation ("APUD") type.

Hepatocellular carcinoma with carcinoid features.

A primary hepatic neoplasm with histologic features suggestive of both hepatocellular carcinoma and carcinoid tumor was studied by light microscopy, electron microscopy, and immunocytochemistry. These methods revealed areas of hepatocellular carcinoma, areas of carcinoid tumor, and mixed areas within the same cell. This case provides one more example of the coexistence of carcinoma and carcinoid in the same neoplasm and thereby supports the hypothesis that a malignantly transformed stem cell can differentiate in both epithelial and amine precursor uptake and decarboxylation (APUD) directions.