Andras Khoor

Atypical Protein Kinase Cι Is an Oncogene in Human Non–Small Cell Lung Cancer

Protein kinase C (PKC) isozymes have long been implicated in carcinogenesis. However, little is known about the functional significance of these enzymes in human cancer. We recently showed that the atypical PKC (aPKC) isozyme PKCiota is overexpressed in human non-small cell lung cancer (NSCLC) cells and that PKCiota plays a critical role in the transformed growth of the human lung adenocarcinoma A549 cell line in vitro and tumorigenicity in vivo. Here we provide compelling evidence that PKCiota is an oncogene in NSCLC based on the following criteria: (a) aPKCiota is overexpressed in the vast majority of primary NSCLC tumors; (b) tumor PKCiota expression levels predict poor survival in patients with NSCLC; (c) the PKCiota gene is frequently amplified in established NSCLC cell lines and primary NSCLC tumors; (d) gene amplification drives PKCiota expression in NSCLC cell lines and primary NSCLC tumors; and (e) disruption of PKCiota signaling with a dominant negative PKCiota allele blocks the transformed growth of human NSCLC cells harboring PKCiota gene amplification. Taken together, our data provide conclusive evidence that PKCiota is required for the transformed growth of NSCLC cells and that the PKCiota gene is a target for tumor-specific genetic alteration by amplification. Interestingly, PKCiota expression predicts poor survival in NSCLC patients independent of tumor stage. Therefore, PKCiota expression profiling may be useful in identifying early-stage NSCLC patients at elevated risk of relapse. Our functional data indicate that PKCiota is an attractive target for development of novel, mechanism-based therapeutics to treat NSCLC.

Diffuse Pulmonary Disease Caused by Nontuberculous Mycobacteria in Immunocompetent People (Hot Tub Lung)

The clinicopathologic spectrum of infections due to nontuberculous mycobacteria (NTM) includes cavitary disease, opportunistic infection, and nodular disease associated with bronchiectasis. We report a less well-described manifestation of NTM infection: 10 immunocompetent patients without preexisting bronchiectasis had radiographic evidence of diffuse infiltrative lung disease. The most common symptoms were dyspnea, cough, hypoxia, and fever. All 10 patients had used a hot tub. Histologic examination revealed exuberant nonnecrotizing, frequently bronchiolocentric, granulomatous inflammation in all cases. In 1 case, necrotizing granulomas were also noted. The inflammation often was associated with patchy chronic interstitial pneumonia and organization. Cultures revealed NTM in all cases (Mycobacterium avium complex in all but 1 case), but staining for acid-fast bacilli was positive in only 1 case. Four patients received corticosteroids alone for presumed hypersensitivity pneumonia, 4 were treated with antimycobacterial therapy, and 2 received both. All patients demonstrated significant improvement at the time of follow-up. These findings suggest that disease due to NTM may manifest as diffuse infiltrates in immunocompetent adults and that hot tub use may be an important risk factor for this disease pattern.

Atypical Protein Kinase Cι Is Required for Bronchioalveolar Stem Cell Expansion and Lung Tumorigenesis

Protein kinase Ciota (PKCiota) is an oncogene required for maintenance of the transformed phenotype of non-small cell lung cancer cells. However, the role of PKCiota in lung tumor development has not been investigated. To address this question, we established a mouse model in which oncogenic Kras(G12D) is activated by Cre-mediated recombination in the lung with or without simultaneous genetic loss of the mouse PKCiota gene, Prkci. Genetic loss of Prkci dramatically inhibits Kras-initiated hyperplasia and subsequent lung tumor formation in vivo. This effect correlates with a defect in the ability of Prkci-deficient bronchioalveolar stem cells to undergo Kras-mediated expansion and morphologic transformation in vitro and in vivo. Furthermore, the small molecule PKCiota inhibitor aurothiomalate inhibits Kras-mediated bronchioalveolar stem cell expansion and lung tumor growth in vivo. Thus, Prkci is required for oncogene-induced expansion and transformation of tumor-initiating lung stem cells. Furthermore, aurothiomalate is an effective antitumor agent that targets the tumor-initiating stem cell niche in vivo. These data have important implications for PKCiota as a therapeutic target and for the clinical use of aurothiomalate for lung cancer treatment.

Amyloid-like Pulmonary Nodules, Including Localized Light-Chain Deposition Clinicopathologic Analysis of Three Cases

Amyloid-like pulmonary nodules have been described in patients with systemic light-chain deposition disease, but their significance in other clinical contexts is unknown. We examined biopsy specimens of amyloid-like pulmonary nodules from 3 women without systemic light-chain deposition disease. Patient 1 (aged 62 years) had multiple pulmonary nodules and underwent 2 separate lung biopsies, the first showing nodules composed of kappa light-chain deposits accompanied by low-grade lymphoplasmacytic lymphoma limited to the lung and the second, obtained after chemotherapy 9 months later, showing only residual nodules without persistent lymphoma. Patients 2 (aged 65 years) and 3 (aged 69 years) had asymptomatic solitary pulmonary nodules. In all cases, electron microscopic examination showed dense granular extracellular deposits without the fibrillary characteristics of amyloid. Amyloid-like nodules should be distinguished from nodular amyloidosis and, in some patients, might represent a localized form of light-chain deposition.

Matrix Metalloproteinase-10 Promotes Kras-Mediated Bronchio-Alveolar Stem Cell Expansion and Lung Cancer Formation

Matrix metalloproteinase 10 (MMP-10; stromelysin 2) is a member of a large family of structurally related matrix metalloproteinases, many of which have been implicated in tumor progression, invasion and metastasis. We recently identified Mmp10 as a gene that is highly induced in tumor-initiating lung bronchioalveolar stem cells (BASCs) upon activation of oncogenic Kras in a mouse model of lung adenocarcinoma. However, the potential role of Mmp10 in lung tumorigenesis has not been addressed. Here, we demonstrate that Mmp10 is overexpressed in lung tumors induced by either the smoke carcinogen urethane or oncogenic Kras. In addition, we report a significant reduction in lung tumor number and size after urethane exposure or genetic activation of oncogenic Kras in Mmp10 null (Mmp10−/−) mice. This inhibitory effect is reflected in a defect in the ability of Mmp10-deficient BASCs to expand and undergo transformation in response to urethane or oncogenic Kras in vivo and in vitro, demonstrating a role for Mmp10 in the tumor-initiating activity of Kras-transformed lung stem cells. To determine the potential relevance of MMP10 in human cancer we analyzed Mmp10 expression in publicly-available gene expression profiles of human cancers. Our analysis reveals that MMP10 is highly overexpressed in human lung tumors. Gene set enhancement analysis (GSEA) demonstrates that elevated MMP10 expression correlates with both cancer stem cell and tumor metastasis genomic signatures in human lung cancer. Finally, Mmp10 is elevated in many human tumor types suggesting a widespread role for Mmp10 in human malignancy. We conclude that Mmp10 plays an important role in lung tumor initiation via maintenance of a highly tumorigenic, cancer-initiating, stem-like cell population, and that Mmp10 expression is associated with stem-like, highly metastatic genotypes in human lung cancers. These results indicate that Mmp10 may represent a novel therapeutic approach to target lung cancer stem cells.

SMARCB1 protein and mRNA loss is not caused by promoter and histone hypermethylation in epithelioid sarcoma

About 10% of epithelioid sarcomas have biallelic mutation of the SMARCB1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1) gene resulting in a lack of this nuclear protein. It has been suggested that SMARCB1 may be silenced by epigenetic changes in the remaining 90% of tumors. Thus, we hypothesized that the promoter of SMARCB1 is hypermethylated. We also examined SMARCB1 mRNA level to determine if a post-translational change was possible. Thirty-six cases of epithelioid sarcomas were studied. Immunohistochemistry and mutation analysis of the SMARCB1 gene were performed to select appropriate cases. Methylation status was assessed by methylation-specific PCR. Laser capture microdissection of tumor cells followed by real-time PCR was applied to examine the expression of SMARCB1 mRNA. Of 36 epithelioid sarcomas, 31 (86%) displayed a lack of SMARCB1 nuclear protein. In all, 4 (13%) of 31 SMARCB1-negative cases harbored biallelic deletion while 9 (33%) cases showed single-allelic deletion. One (4%) frameshift deletion of exon 3 and one point mutation of exon 7 were also found. In 16 (59%) cases, both alleles were intact. Altogether, 25/31 (81%) SMARCB1-negative cases had at least one intact allele. None of these cases demonstrated promoter hypermethylation. Low levels of SMARCB1 mRNA were found in all cases with tumor tissue extracted RNA (because of the minimal normal cell contamination) but no mRNA could be detected in laser dissected cases (containing only tumor cells). Enhancer of zeste homolog 2 (EZH2) overexpression was not characteristic of epithelioid sarcoma. Thus, loss of SMARCB1 expression in epithelioid sarcoma is caused neither by DNA hypermethylation nor by post-translational modifications. Most likely it is the microRNA destruction of SMARCB1 mRNA but further investigations are needed to elucidate this issue.

Pulmonary Langerhans cell histiocytosis presenting as a solitary nodule

Solitary pulmonary nodules are an uncommon manifestation of pulmonary Langerhans cell histiocytosis (PLCH). We describe a 45-year-old male cigarette smoker who presented with an asymptomatic solitary pulmonary nodule that showed histologic and immunophenotypic characteristics of PLCH. Twenty-one years after excision of the nodule, at the age of 66 years, he is asymptomatic with a new contralateral lung nodule but no evidence of interstitial disease. The new nodule has remained unchanged after 36 months of observation. This case affirms that PLCH can occasionally cause solitary lesions, which should not be interpreted as a harbinger of interstitial lung disease. Isolated PLCH should be included in the differential diagnosis of unusual solitary pulmonary nodules.

Utility of tissue-specific transcription factors thyroid transcription factor 1 and Cdx2 in determining the primary site of metastatic adenocarcinomas to the brain.

CONTEXT Brain metastases of adenocarcinoma of unknown primary pose a diagnostic dilemma to the surgical pathologist. Although the most common source in these cases is the lung, determining a primary source is difficult on routinely stained slides. Immunohistochemical stain panels including differential cytokeratins, hormone receptors, and breast-specific proteins are commonly used in these cases. Recently, attention has turned to tissue-specific transcription factors, such as thyroid transcription factor 1 (TTF-1) and Cdx2, in the appraisal of metastatic adenocarcinomas. OBJECTIVE To characterize the previously unpublished immunohistochemical expression of the relatively new tissue-specific transcription factor Cdx2 in metastatic adenocarcinomas to the brain. DESIGN We reviewed the surgical pathology files of the H. Lee Moffitt Cancer Center and Research Institute, Tampa, Fla, and retrieved 38 consecutive cases of metastatic adenocarcinoma (22 pulmonary, 10 breast, 6 gastrointestinal [2 esophagus/gastroesophageal junction, 4 colorectal]) to the brain with confirmation of the primary site by chart review and histologic evaluation. Sections were immunohistochemically stained with antibodies to TTF-1, Cdx2, and cytokeratins 7 and 20 by standard methods. RESULTS Specificities and positive predictive values for Cdx2 and TTF-1 equaled 100% for metastatic gastrointestinal and pulmonary adenocarcinomas, respectively. The negative predictive value of Cdx2 was also very high at 97%. CONCLUSIONS Cdx2 is a specific and valuable tool for the surgical pathologist when faced with the common problem of metastatic adenocarcinoma of unknown primary. In conjunction with TTF-1, cytokeratin 7, and cytokeratin 20, Cdx2 can accurately differentiate the most common sources of metastatic adenocarcinoma to the brain.

A simple two-gene prognostic model for adenocarcinoma of the lung

OBJECTIVE We hypothesized that clinical outcome of resected early-stage adenocarcinoma of the lung can be predicted by the expression of a few critically important genes as measured by quantitative real-time reverse-transcriptase polymerase chain reaction in formalin-fixed paraffin-embedded primary tumors. METHODS Twenty-two prognostic genes for the metastatic phenotype were identified through complementary DNA microarray analysis of 4 cancer cell lines and bioinformatics analysis. Expression levels of a subset of these genes (n = 13) were measured by real-time time reverse-transcriptase polymerase chain reaction in formalin-fixed paraffin-embedded primary adenocarcinoma from patients whose disease recurred within 2 years (n = 9) and in patients who did not have a recurrence (n = 11). Receiver operating characteristic curves were analyzed to establish prognostic values of single genes. The most informative gene was combined with the remaining genes to determine whether there was a particular pair(s) that yielded high diagnostic accuracy. A small validation study was performed. RESULTS Receiver operating characteristic curve analysis of the single genes revealed that high expression of CK19 was associated with nonrecurrence (area under the curve = 0.859, confidence interval = 0.651-0.970). The CK19/EpCAM2 gene ratio had the most reproducible prognostic accuracy, followed by the CK19/P-cadherin ratio. A Kaplan-Meier survival analysis generated from the CK19/EpCAM2 ratio resulted in highly significant curves as a function of marker positivity (P = .0007; hazard ratio = 10.7). Significance declined but was maintained in the validation study. CONCLUSIONS This preliminary study provides evidence that the CK19/EpCAM2 and/or CK19/P-cadherin ratio(s) may be a simple and accurate prognostic indicator of clinical outcome in early-stage adenocarcinoma of the lung. If further validation studies from large patient cohorts confirm the results, adjuvant therapy could be targeted to this high-risk group.

Pulmonary microcrystalline cellulose deposition from intravenous injection of oral medication in a patient receiving parenteral nutrition

A 50-year-old man who had been dependent on home parenteral nutrition (HPN) for 24 years presented with shortness of breath. A computed tomography scan of the lungs revealed a diffuse micronodular parenchymal infiltrate. On bronchoscopy, a crystalloid material was identified. This organic material was determined to be consistent with codeine. The patient had been injecting codeine into his intravenous catheter.