R. Ilona Linnoila

Induction of small cell lung cancer by somatic inactivation of both Trp53 and Rb1 in a conditional mouse model

Small cell lung cancer (SCLC) is a highly aggressive human tumor with a more than 95% mortality rate. Its ontogeny and molecular pathogenesis remains poorly understood. We established a mouse model for neuroendocrine (NE) lung tumors by conditional inactivation of Rb1 and Trp53 in mouse lung epithelial cells. Mice carrying conditional alleles for both Rb1 and Trp53 developed with high incidence aggressive lung tumors with striking morphologic and immunophenotypic similarities to SCLC. Most of these tumors, which we designate MSCLC (murine small cell lung carcinoma), diffusely spread through the lung and gave rise to extrapulmonary metastases. In our model, inactivation of both Rb1 and p53 was a prerequisite for the pathogenesis of SCLC.

Classification of Proliferative Pulmonary Lesions of the Mouse Recommendations of the Mouse Models of Human Cancers Consortium

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20–22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.

Functional facets of the pulmonary neuroendocrine system

Pulmonary neuroendocrine cells (PNECs) have been around for 60 years in the scientific literature, although phylogenetically they are ancient. Their traditionally ascribed functions include chemoreception and regulation of lung maturation and growth. There is recent evidence that neuroendocrine (NE) differentiation in the lung is regulated by genes and pathways that are conserved in the development of the nervous system from Drosophila to humans (such as achaete-scute homolog-1), or implicated in the carcinogenesis of the nervous or NE system (such as the retinoblastoma tumor suppressor gene). In addition, complex neural networks are in place to regulate chemosensory and other functions. Even solitary PNECs appear to be innervated. For the first time ever, we have mouse models for lung NE carcinomas, including the most common and virulent small cell lung carcinoma. Moreover, PNECs may be important for inflammatory responses, and pivotal for lung stem cell niches. These discoveries signify an exciting new era for PNECs and are likely to have therapeutic and diagnostic applications.

Clinical features of patients with stage IIIB and IV bronchioloalveolar carcinoma of the lung

The incidence of bronchioloalveolar carcinoma of the lung (BAC), a pathologically distinct type of nonsmall cell lung carcinoma (NSCLC), appears to be rising. In this study, the authors compared data on the clinical presentation and clinical courses of patients with Stage IIIB and IV BAC with data on other types of NSCLC.

Identification of a Highly Effective Rapamycin Schedule that Markedly Reduces the Size, Multiplicity, and Phenotypic Progression of Tobacco Carcinogen–Induced Murine Lung Tumors

Purpose: Human and murine preneoplastic lung lesions induced by tobacco exposure are characterized by increased activation of the Akt/mammalian target of rapamycin (mTOR) pathway, suggesting a role for this pathway in lung cancer development. To test this, we did studies with rapamycin, an inhibitor of mTOR, in A/J mice that had been exposed to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Experimental Design: Tumorigenesis was induced by i.p. injection of NNK, and rapamycin was administered 1 or 26 weeks after NNK administration. Biomarkers associated with mTOR inhibition were assessed in lung and/or surrogate tissues using immunohistochemistry and immunoblotting. Rapamycin levels were measured using mass spectroscopy. Results: Rapamycin was administered on a daily (5 of 7 days) regimen beginning 26 weeks after NNK decreased tumor size, proliferative rate, and mTOR activity. Multiplicity was not affected. Comparing this regimen with an every-other-day (qod) regimen revealed that rapamycin levels were better maintained with qod administration, reaching a nadir of 16.4 ng/mL, a level relevant in humans. When begun 1 week after NNK, this regimen was well tolerated and decreased tumor multiplicity by 90%. Tumors that did develop showed decreased phenotypic progression and a 74% decrease in size that correlated with decreased proliferation and inhibition of mTOR. Conclusions: Tobacco carcinogen–induced lung tumors in A/J mice are dependent upon mTOR activity because rapamycin markedly reduced the development and growth of tumors. Combined with the Food and Drug Administration approval of rapamycin and broad clinical experience, these studies provide a rationale to assess rapamycin in trials with smokers at high risk to develop lung cancer.

Randomized, Double-Blind, Placebo-Controlled Phase IIB Trial of the Cyclooxygenase Inhibitor Ketorolac as an Oral Rinse in Oropharyngeal Leukoplakia

Purpose: Nonselective cyclooxygenase (COX) inhibitors have been reported to decrease the frequency of upper aerodigestive cancers. Ketorolac tromethamine oral rinse has been shown to resolve another COX-dependent process, periodontal disease, without incurring gastrointestinal side effects. This trial evaluated if a topically delivered oral rinse containing ketorolac was as safe as and more effective than oral rinse alone in reducing the area of oral leukoplakia. Experimental Design: 57 patients were randomized (2:1 ratio) in a double-blind, placebo-controlled study of ketorolac (10 ml of a 0.1% ketorolac rinse solution; n = 38) or placebo (10 ml of rinse solution; n = 19) given twice daily for 30 s over 90 days. Primary end point was evaluated visually obtaining bidimensional measurement of the size of leukoplakia lesion(s) at entry and at 90 days. Secondary end point was histological assessment of the leukoplakia as sampled by serial punch biopsy and independently reviewed by three pathologists. Results: The patients included 67% males, 11% non-Caucasian, and 86% used tobacco with no significant differences between the two arms. Both rinses were well tolerated with good compliance, and there was no significant difference in adverse events (P = 0.27). Major response rate (complete response and partial response) was 30% for ketorolac and 32% for the placebo arm. There was no significant difference in change in histology between the two arms. Conclusion: Local delivery of a COX-containing oral rinse was well tolerated but produced no significant reduction in the extent of leukoplakia compared with the placebo. However, the favorable response rate to placebo arm remains unexplained and additional investigation of the tissue penetration with ketorolac is warranted.

Decreased expression of neuropeptides in malignant paragangliomas: An immunohistochemical study**

Paraffin-embedded sections of 99 human adrenal and extraadrenal paragangliomas were analyzed by the indirect immunoperoxidase technique for the presence of neuron-specific enolase (NSE) and 10 neuropeptides. Each showed diffuse staining for NSE. Most tumors were positive for [Leu5]-enkephalin (76 per cent), [Met5]-enkephalin (75 per cent), somatostatin (67 per cent), and pancreatic polypeptide (51 per cent), followed by vasoactive intestinal polypeptide (VIP) (43 per cent), substance P (31 per cent), ACTH (28 per cent), calcitonin (23 per cent), bombesin (15 per cent), and neurotensin (12 per cent). The neuropeptides paralleled to a large extent those normally found in the sympathetic nervous system. Clinically malignant paragangliomas (n = 25) with proven regional or distant metastases expressed considerably fewer neuropeptides, although the spectrum of those seen remained similar. Malignant paragangliomas contained an average of two neuropeptides per tumor, in contrast to five for the benign tumors (P less than 0.05). Logistic regression analysis of staining results revealed that the paucity of enkephalins, somatostatin, pancreatic polypeptide, and VIP along with the patients sex was predictive of clinical malignancy. Our results show a definite relationship between expression of neuropeptides and the biologic behavior of these paragangliomas.

The Role of CC10 in Pulmonary Carcinogenesis: From a Marker to Tumor Suppression

Abstract: CC10 is infrequently expressed in human non‐small cell lung cancers (NSCLCs), despite being abundantly produced by progenitor cells for normal and neoplastic epithelium. Many abnormalities in the surrounding lung associated with field carcinogenesis, which reflect prolonged exposure to such carcinogens as tobacco smoke, also revealed altered expression of CC10. Exposure of hamsters and mice to the tobacco‐specific carcinogen NNK led to reduced CC10 expression, which was partially reversible. Overexpression of CC10 in immortalized bronchial epithelial cells delayed the induction of anchorage‐independent growth in response to NNK. The data suggest that downregulation of CC10 contributes to carcinogenesis because CC10 antagonizes the neoplastic phenotype.

The Comparative Pathology of Genetically Engineered Mouse Models for Neuroendocrine Carcinomas of the Lung

Introduction: Because small-cell lung carcinomas (SCLC) are seldom resected, human materials for study are limited. Thus, genetically engineered mouse models (GEMMs) for SCLC and other high-grade lung neuroendocrine (NE) carcinomas are crucial for translational research. Methods: The pathologies of five GEMMs were studied in detail and consensus diagnoses reached by four lung cancer pathology experts. Hematoxylin and Eosin and immunostained slides of over 100 mice were obtained from the originating and other laboratories and digitalized. The GEMMs included the original Rb/p53 double knockout (Berns Laboratory) and triple knockouts from the Sage, MacPherson, and Jacks laboratories (double knockout model plus loss of p130 [Sage laboratory] or loss of Pten [MacPherson and Jacks laboratories]). In addition, a GEMM with constitutive co-expression of SV40 large T antigen and Ascl1 under the Scgb1a1 promoter from the Linnoila laboratory were included. Results: The lung tumors in all of the models had common as well as distinct pathological features. All three conditional knockout models resulted in multiple pulmonary tumors arising mainly from the central compartment (large bronchi) with foci of in situ carcinoma and NE cell hyperplasia. They consisted of inter- and intra-tumor mixtures of SCLC and large-cell NE cell carcinoma in varying proportions. Occasional adeno- or large-cell carcinomas were also seen. Extensive vascular and lymphatic invasion and metastases to the mediastinum and liver were noted, mainly of SCLC histology. In the Rb/p53/Pten triple knockout model from the MacPherson and Jacks laboratories and in the constitutive SV40/T antigen model many peripherally arising non–small-cell lung carcinoma tumors having varying degrees of NE marker expression were present (non–small-cell lung carcinoma-NE tumors). The resultant histological phenotypes were influenced by the introduction of specific genetic alterations, by inactivation of one or both alleles of specific genes, by time from Cre activation and by targeting of lung cells or NE cell subpopulations. Conclusion: The five GEMM models studied are representative for the entire spectrum of human high-grade NE carcinomas and are also useful for the study of multistage pathogenesis and the metastatic properties of these tumors. They represent one of the most advanced forms of currently available GEMM models for the study of human cancer.

Effects of Diethylnitrosamine on Lung Neuroendocrine Cells

Diethylnitrosamine is known to cause squamous cell carcinoma and adenocarcinoma of the lung in Syrian golden hamsters. Sections of lungs obtained from hamsters treated with the systemic carcinogen diethylnitrosamine revealed a significant increase in the number of argyrophilic cells of neuroepithelial bodies. These affected cells also exhibited enhanced survival in vitro. After 7 days in culture, argyrophilia, dense-core vesicles, and corticotropin-like immunoreactivity were observed in many of the cells derived from the lungs of carcinogen-exposed hamsters by dissociation with pronase. In addition, nuclei of argyrophilic cells in neuroepithelial bodies of the exposed hamsters were labeled at 60 min following administration of [3H]thymidine. This suggests that the carcinogen stimulates the pulmonary neuroendocrine-like cells to divide. Normally, the component cells of neuroepithelial bodies may originate from nonargyrophilic precursor cells in the surrounding epithelium, as in control hamsters the argyrophilic cells of neuroepithelial bodies appeared labeled only at 8 days after the administration of thymidine. The relationship of the diethylnitrosamine-induced reactions to bronchial carcinoid tumors or small-cell carcinomas of the lung remains to be established.