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The translocation t(11;19)(q21;p13) results in the gene fusion of mucoepidermoid carcinoma translocated 1-mammalian mastermind like 2 genes that is the major chromosomal abnormality observed in mucoepidermoid carcinomas of salivary glands but has not been studied in bronchopulmonary mucoepidermoid carcinoma. To investigate the importance of the mammalian mastermind like 2 gene rearrangement and mucoepidermoid carcinoma translocated 1-mammalian mastermind like 2 fusion gene in bronchopulmonary mucoepidermoid carcinoma tumorigenesis and its differential diagnosis with primary pulmonary non-small-cell carcinomas, we evaluated the presence of the mammalian mastermind like 2 gene rearrangement and the mucoepidermoid carcinoma translocated 1-mammalian mastermind like 2 fusion in formalin-fixed, paraffin-embedded tissue sections from 17 adult bronchopulmonary mucoepidermoid carcinoma, 16 adenosquamous carcinomas, 24 squamous cell carcinomas, and 41 primary adenocarcinomas by fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction. We detected mammalian mastermind like 2 gene rearrangement by fluorescence in situ hybridization analysis in 13 (77%) of 17 bronchopulmonary mucoepidermoid carcinoma cases (10 of 10 being low grade and 3 of 7 being high grade). Reverse transcriptase polymerase chain reaction analysis confirmed positive fluorescence in situ hybridization results in 6 (43%) of 14 mucoepidermoid carcinoma cases. None of the squamous, adenosquamous, or adenocarcinoma cases revealed the mammalian mastermind like 2 gene rearrangement by fluorescence in situ hybridization, and the mucoepidermoid carcinoma translocated 1-mammalian mastermind like 2 fusion product by reverse transcriptase polymerase chain reaction was not identified specifically in our adenosquamous carcinoma cases. In conclusion, our study demonstrates that mammalian mastermind like 2 gene rearrangement and mucoepidermoid carcinoma translocated 1-mammalian mastermind like 2 fusion product can be detected by fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction analysis performed on low- and high-grade primary bronchopulmonary mucoepidermoid carcinoma and can be used to help discriminate low- and high-grade mucoepidermoid carcinoma from adenocarcinoma, adenosquamous carcinoma, and squamous cell carcinoma mimics in histologically challenging cases.

Homozygous deletion of p16/CDKN2A is the most common genetic abnormality in malignant mesotheliomas. The aim of this study was to determine prognostic significance of p16/CDKN2A loss in malignant pleural mesotheliomas (MPM) as defined by immunohistochemistry and fluorescence in situ hybridization (FISH). High-density tissue microarrays were constructed from archival formalin-fixed paraffin-embedded samples of 48 MPM. Long survival (LS) was defined as survival greater than 3 years from the time of diagnosis, and short survival was defined as less than 3 years from the time of diagnosis. Both loss of p16 protein expression by immunohistochemistry and homozygous deletion of p16 by FISH were associated with adverse prognosis. Female gender, positive p16 immunoexpression, and lack of p16/CDKN2A deletion significantly predicted the survival for the LS group. Statistical analysis showed a very strong correlation of immunohistochemistry and FISH data. Cases positive for p16 immunoexpression and negative for 9p21 deletion showed the best survival time. Our study is the first to demonstrate decreased frequency of homozygous deletion of 9p21 and loss of p16 immunoreactivity in pleural mesotheliomas from patients with long-term survival of greater than 3 years in contrast to patients with rapidly fatal mesotheliomas. A possible implementation of these tests into preoperative prognostication of MPM and therapeutic decisions should be considered.

Chordoid gliomas are rare, slow-growing neoplasms of the anterior third ventricle. We reported a case of chordoid glioma in a 41-year-old man with obstructive hydrocephalus. Histologically, the tumor consisted of polygonal epithelioid cells admixed with elongated cells in a myxoid stroma. A prominent lymphoplasmacytic infiltrate was present. The tumor cells expressed glial fibrillary acidic protein (GFAP), epithelial membrane antigen (EMA), vimentin, CD31, CD34, epidermal growth factor receptor (EGFR) and S100 but were negative for pankeratin and E-cadherin. The percentage of Ki67 positive cells was approximately 3%. Weak p53 immunoreactivity was seen in less than 10% of the cells. Array comparative genomic hybridization performed on this case, as well as on four other archived cases, showed losses at several loci. Fluorescence in situ hybridization (FISH) confirmed consistent genetic alterations at 9p21 and 11q13. These are the fifth through ninth reported cases of chordoid gliomas with molecular characterization suggesting a distinct genetic origin from other gliomas.

The hepatocyte growth factor (HGF)/c-Met signaling pathway is involved in lung tumor growth and progression, and agents that target this pathway have clinical potential for lung cancer treatment. L2G7, a single potent anti-human HGF neutralizing monoclonal antibody, showed profound inhibition of human HGF-induced phosphorylated mitogen-activated protein kinase induction, wound healing, and invasion in lung tumor cells in vitro. Transgenic mice that overexpress human HGF in the airways were used to study the therapeutic efficacy of L2G7 for lung cancer prevention. Mice were treated with the tobacco carcinogen, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, over 4 weeks. Beginning at week 3, i.p. treatment with 100 μg L2G7 or isotype-matched antibody control, 5G8, was initiated and continued through week 15. The mean number of tumors per mouse in the L2G7-treated group was significantly lower than in the control group (1.58 versus 3.19; P = 0.0005). Proliferative index was decreased by 48% (P = 0.013) in tumors from L2G7-treated mice versus 5G8-treated mice, whereas extent of apoptosis was increased in these same tumors by 5-fold (P = 0.0013). Phosphorylated mitogen-activated protein kinase expression was also significantly decreased by 84% in tumors from L2G7-treated mice versus 5G8-treated mice (P = 0.0003). Tumors that arose in HGF transgenic animals despite L2G7 treatment were more likely to contain mutant K-ras, suggesting that targeting the HGF/c-Met pathway may not be as effective if downstream signaling is activated by a K-ras mutation. These preclinical results show that blocking the HGF/c-Met interaction with a single monoclonal antibody delivered systemically can have profound inhibitory effects on development of lung tumors. [Mol Cancer Ther 2008;7(7):1913–22]

The development of small-molecule inhibitors of the epidermal growth factor receptor (EGFR) resulted in new therapeutic options for patients with advanced lung cancer. It was clear from the experience with targeted therapy for breast cancer that a new standardized assay procedure for assessing and predicting the effects of therapeutic agents must be developed. Three academic groups almost simultaneously reported the discovery of somatic mutations in the exons 18 to 21 of the tyrosine kinase (TK) domain of EGFR that correlated with a high likelihood of response to EGFR TK inhibitors. This observation revolutionized understanding of EGFR in lung carcinogenesis and resulted in numerous retrospective studies that correlated patient's response and molecular profile of the lung adenocarcinoma. The results of these studies indicate that clinical benefits from treatment with EGFR TK inhibitors are variable between the different subsets of patients. Multiple methodologic approaches were used including mutational analysis, fluorescence in situ hybridization, and immunohistochemistry. Conflicting results reflect the lack of standardization of the methodology and interpretation. Sample types, sample processing, and storage should also be taken into consideration as another potentially confounding factor. Therefore, it is important to standardize the approach and decide which assays are best to predict patient response to targeted therapies. It is also essential to determine the most cost-effective way to integrate EGFR molecular assays into clinical practice. This review will address practical aspects of each of the currently proposed assays. Difficulties in standardization of these assays in a clinical practice will be discussed.

A total of 211 cases of primary lung adenocarcinoma were tested for expression of gross cystic disease fluid protein-15 (GCDFP-15) and only 11 cases (5.2%) were positive. The cases occurred with an equal sex distribution in older individuals whose carcinomas were frequently identified on screening radiographs. The adenocarcinomas were peripheral lesions and had an average size of 2.9 cm (range, 1.1 to 7.0). Histologically, they were usually mixed acinar and papillary adenocarcinomas with abundant extracellular mucin production, with the neoplastic cells having a polygonal shape, round to oval nuclei, diffuse powdery chromatin, and abundant eosinophilic granular cytoplasm. Clear cells and apocrinelike cells with prominent central nucleoli were common. GCDFP-15 was expressed in conjunction with thyroid transcription factor-1 in 81% of cases and synaptophysin was seen in 65%. Estrogen and progesterone receptors were not expressed. EGFR gene amplification and mutations of exons 19 and 21 were rare. KRAS mutations and HER2 gene amplification were not seen. This report details the first 11 cases of pulmonary adenocarcinoma to express GCDFP-15 and their distinctive morphology with frequent mucin production and coexpression of thyroid transcription factor-1 and synaptophysin.

Context.—Improved screening techniques for lung cancer have resulted in detection of lesions that are considered to represent precursors of invasive lung carcinomas. These lesions may cause a diagnostic dilemma particularly on small biopsy or cytology specimens. Ancillary studies are usually not helpful, and diagnosis is based on morphology alone. Recognition of these lesions is very important to prevent potential diagnostic mistakes that may result in inadequate patient management. Future molecular studies may provide clinically useful diagnostic and prognostic gene markers. Objective.—To review currently proposed morphologic criteria for precursor lesions of non–small cell lung carcinomas including squamous dysplasias, atypical adenomatous hyperplasia, and diffuse idiopathic neuroendocrine cell hyperplasia. Major molecular abnormalities are briefly discussed. Data Sources.—Published literature and recent World Health Organization classification of lung tumors. Conclusions.—Practicing surgical pathologists must be familiar with morphology of recognized pulmonary preneoplastic lesions that are more frequently detected radiographically and subjected to diagnostic procedures. Future understanding of underlying molecular abnormalities associated with progression of these lesions into invasive lung carcinoma may result in a development of molecular assays with potential diagnostic and prognostic importance.

Solid pseudopapillary neoplasms of the pancreas are low-grade malignancies, but their biological behavior cannot be stratified solely on the basis of histopathologic criteria. Aside from mutations in beta-catenin and lack of genetic changes common to pancreatic ductal adenocarcinomas, little is known about the chromosomal alterations in solid pseudopapillary neoplasms. We applied array comparative genomic hybridization to a series of 12 patients. The average age was 31 years (range 12-52 years) with 10 female and 2 male patients. The average tumor size was 7.3 cm (range 2-24 cm) with five lesions greater than 5 cm. All cases had 'bland' cytology without significant pleomorphism or high nuclear grade, but seven cases demonstrated at least one of these potentially aggressive histopathologic features: size5 cm, tumor necrosis, lymphovascular invasion, perineural invasion and peripancreatic invasion. Clinically, one lesion demonstrated aggressive behavior. By array comparative genomic hybridization, chromosomal losses and/or gains were identified in eight cases; five cases had multiple (five or more) alterations. The most common alterations were gains at 13q, 17q, 1q and 8q. Six of the seven cases with at least one aggressive feature had genetic alterations, while only two cases without adverse features had genetic alterations (P=0.024). The single clinically aggressive tumor exhibited seven chromosomal gains and four aggressive histopathologic features. Our study demonstrates that genetic alterations detected by array comparative genomic hybridization are common in solid pseudopapillary neoplasms of the pancreas. Additional study and longer follow-up are needed to determine if these genetic abnormalities could help predict clinical behavior in these neoplasms.

To determine the mechanism underlying ribavirin induced amelioration of experimental autoimmune encephalomyelitis (EAE), cytokine profiles were evaluated in draining lymph node (DLN) cell culture supernatants and spinal cord obtained from EAE and/or ribavirin-treated EAE Dark Agouti rats. Administration of ribavirin to EAE rats markedly affected the production of pro-inflammatory cytokines IFN-gamma, IL-1 beta and TNF-alpha in DLN and spinal cord, thus shifting the balance towards the anti-inflammatory cytokines IL-10 and TGF-beta. These findings suggest that ribavirin attenuates EAE by limiting cytokine-mediated immunoinflammatory events leading to CNS destruction. The conducted experiments provide rationale for ribavirin to be considered as a candidate drug in the development of new therapeutic strategies for the treatment of autoimmune diseases in humans, such as multiple sclerosis. (c) 2008 Elsevier B.V. All rights reserved. null

Molecular testing in anatomic pathology is becoming increasingly important for most organ systems, including in the lung. Such tests are used both diagnostically and prognostically and are particularly important in the workup of neoplasia and for identification or subclassification for certain infectious processes. Fresh and frozen tissues are always considered to be the most optimal source of DNA and RNA that serves as the template for targeted molecular analysis. However, archival paraffin-embedded tissue is an attractive alternative source of tissue for clinical testing. Paraffin-embedded tissue can be a critical source of nucleic acid when unexpected diagnoses are rendered in the pathologic evaluation of tissue material. It also, however, provides the advantage of allowing for archival analysis with correlation to outcome.

Purpose: The goal of this study is to identify novel genes frequently silenced by promoter hypermethylation in lung cancer. Experimental Designs: Bioinformatic analysis was done to identify candidate genes significantly down-regulated in lung cancer. The effects of DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine on the expression of the candidate genes were determined. Methylated CpG sites in the promoters of the candidate genes were identified using bisulfite DNA sequencing. Methylation-specific PCR was developed and used to analyze DNA methylation in cell lines and clinical specimen. Pathologic and functional analyses were done to study the role of one candidate gene, receptor activity-modifying protein 2 (RAMP2), in suppressing lung cancer cell growth. Results: Among 54 candidate genes down-regulated in lung cancer, 31 were found to contain CpG islands in their promoters. Six of these 31 genes could be reactivated by 5-aza-2′-deoxycytidine in at least four of six lung cancer cell lines analyzed. Promoter hypermethylation of RAMP2, epidermal growth factor–containing fibulin-like extracellular matrix protein 1, and deleted in U Twenty Twenty cells was detected in 36% to 77% of 22 lung cancer cell lines and in 38% to 50% of 32 primary lung tumors, whereas hypermethylathion of these genes was rarely found in the matched normal samples. The methylation frequencies of these genes in lung cancer were similar to those of commonly used methylation markers, such as RAS association domain family protein 1A, p16, and methylguanine-DNA methyltransferase. Immunohistochemistry showed that RAMP2 was down-regulated in a majority of lung tumors, and RAMP2 down-regulation was correlated with high tumor grade. Ectopic expression of RAMP2 inhibited lung cancer cell growth and caused apoptotic cell death. Knockdown of RAMP2 by RNA interference stimulated cell proliferation. Conclusions: Studying the newly identified genes may provide new insight into lung tumorigenesis. These genes might be useful as molecular markers of lung cancer.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS) and the helpful tool in preclinical testing of various substances considered for treatment of this human CNS disease. Ribavirin (R) and tiazofurin (T) are purine nucleoside analogues, with the broad spectrum of anti-viral, anti-tumoral and anti-inflammatory properties. We proposed that combined treatment with RT, administrated during the effector phase of EAE, would attenuate disease severity, both clinically and pathologically. Ribavirin was given daily at a dosage of 30 mg/kg and tiazofurin was given at a dosage of 10 mg/kg every other day for 15 days. We detected amelioration of clinical signs and faster recovery in the RT group compared to the control group. Immunohistochemical analyses revealed that RT treatment decrease the number of T cells, macrophages and microglia. In the controls, we detected reactive type of microglia, while in the RT group we noticed ramified/resting form. Demyelination areas and axonal damage were not recorded in the RT group, in contrast to the control group where multiple areas of demyelination zones and axonal loss were found. RT combination treatment suppresses ongoing EAE, prevents demyelination and axonal loss, and therefore may well be the potential therapy for the treatment of MS. (C) 2007 Elsevier B.V. All rights reserved. null

Atypical adenomatous hyperplasia (AAH) is considered to be a precursor lesion of the lung adenocarcinoma. Several genetic abnormalities have been reported in AAH associated with adenocarcinoma, but little is known about AAH associated with benign lung lesions. To address this we compared the molecular characteristics of AAH present in benign conditions to those coexisting with carcinoma. Seven cases of AAH from resected non-neoplastic lungs (AAH-B) and 12 cases from lungs resected for primary lung carcinoma (AAH-M) were analyzed for loss of heterozygosity (LOH) using 21 polymorphic microsatellite markers situated in proximity to known tumor suppressor genes on chromosomes 3p, 5q, 7p, 9p, 10q, and 17p. Direct DNA sequencing for K-ras mutation was also performed. There was a broad range of LOH in both groups. No LOH was identified in 3 cases (25%) of AAH-M, but all cases of AAH-B showed LOH (P=0.26). Six cases (50%) of AAH-M and 3 cases (43%) of AAH-B showed loss at 1 marker (P=0.99). LOH at 2 or more markers was identified in 3 (25%) cases of AAH-M and 4 (57%) cases of AAH-B (P=0.32). LOH was most frequently detected on chromosomes 3p and 10q in both groups. The difference in overall fractional allelic loss between the 2 groups did not reach statistical significance. K-ras mutations were not identified in either group. Our results showed a significant overlap in LOH patterns between AAH with or without coexistent lung malignancy. Therefore, AAH may represent a smoking induced low-grade neoplastic lesion that may be a precursor lesion of only a subset of invasive lung adenocarcinoma.

Differential microRNA (miR) expression is described in non–small cell lung carcinoma. miR biogenesis requires a set of proteins collectively referred to as the miR machinery. In the proposed multistep carcinogenesis model, peripheral adenocarcinoma of the lung develops from noninvasive precursor lesions known as atypical adenomatous hyperplasia (AAH) and bronchioloalveolar carcinoma (BAC). The gene array analysis of BAC and adenocarcinoma showed a transient up-regulation of Dicer (a key effector protein for small interfering RNA and miR function) and PACT along with down-regulation of most genes encoding miR machinery proteins. Immunohistochemically, Dicer was up-regulated in AAH and BAC and down-regulated in areas of invasion and in advanced adenocarcinoma. A fraction of adenocarcinomas lose Dicer as a result of deletions at the Dicer locus. Expanded immunohistochemical and Western blot analysis showed higher Dicer level in squamous cell carcinoma (SCC) of the lung when compared with adenocarcinoma. Other proteins of the RNA-induced silencing complex (RISC; SND1, PACT, and FXR1) were also present at higher levels in a SCC cell line when compared with an adenocarcinoma cell line. In conclusion, the stoichiometry of miR machinery and RISC depends on histologic subtype of lung carcinoma, varies along the AAH-BAC-adenocarcinoma sequence, and might explain the observed abnormal miR profile in lung cancer. The status of the endogenous miR machinery in various histologic subtypes and stages of lung cancer may help to predict the toxicity of and susceptibility to future RNA interference–based therapy. [Cancer Res 2007;67(5):2345–50]

Distribution of two enzymes involved in the ectonucleotidase enzyme chain, ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5'-nucleotidase, was assessed by immunohistochemistry in the rat hippocampus. Obtained results have shown co-expression of the enzymes in the hippocampal region, as well as wide and strikingly similar cellular distribution. Both enzymes were expressed at the surface of pyramidal neurons in the CA1 and CA2 sections, while cells in the CA3 section were faintly stained. The granule cell layer of the dentate gyrus was moderately stained for NTPDase1, as well as for ecto-5'-nucleotidase. Glial association for ecto-5'-nucleotidase was also observed, and fiber tracts were intensively stained for both enzymes. This is the first comparative study of NTPDase1 and ecto-5'-nucleotidase distribution in the rat hippocampus. Obtained results suggest that the broad overlapping distribution of these enzymes in neurons and glial cells reflects the functional importance of ectonucleotidase actions in the nervous system. null

The estrogen signaling pathway induces proliferation in non-small cell lung cancer (NSCLC) and represents a novel therapeutic target for lung cancer. Estrogen receptor β (ERβ) is the predominant ER isoform in lung cancer and we have previously shown that high cytoplasmic ERβ combined with low progesterone receptor (PR) expression is a poor prognostic marker for NSCLC patients. To further elucidate the biological differences between ERβ high/PR low versus ERβ low/PR high expressing lung tumors, a mRNA microarray analysis using the Illumina HT-12 V4 Bead Chip platform was performed using RNA extracted from 64 cases. 165 genes were differentially expressed between these two groups at P Citation Format: Laura P. Stabile, Natalie J. Rothenberger, Marjorie Romkes, Lisa Koodie, Mariya Farooqui, Sanja Dacic, Jill M. Siegfried. Estrogen modulation of fibroblast growth factor signaling in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1861. doi:10.1158/1538-7445.AM2015-1861

We evaluated epidermal growth factor receptor (EGFR) protein expression by immunohistochemical analysis and EGFR gene amplification by fluorescence in situ hybridization in 199 consecutive newly diagnosed and surgically treated patients with primary non-small cell lung carcinoma (NSCLC) and correlated results with clinicopathologic findings. EGFR protein expression was more common in squamous cell carcinoma (SCC; 17 [26.2%]) than in adenocarcinoma (14 [11.1%]; (P = .0076) and more frequently associated with EGFR amplification (8 [14.5%] vs 4 [3.6%] cases; P = .0208). Poor differentiation was associated with a higher average number of EGFR gene copies per cell (mean, 4.18; P = .0322) and a higher EGFR/chromosome 7 ratio (mean, 1.84; P = .0324). N0 disease showed a higher number of EGFR gene copies (mean, 4.196; P = .0163). SCCs demonstrated a higher EGFR/chromosome 7 ratio than adenocarcinomas (mean, 1.95 vs 1.47; P = .0324), particularly T1 tumors (mean, 1.79; P = .0243). Statistical analysis failed to show correlation between outcome and EGFR protein expression and gene amplification in early NSCLC. EGFR protein expression was uncoupled from gene amplification in most cases, although good correlation occurred in a subset of SCCs.

Distinction of multiple primary lung carcinomas from intrapulmonary metastases using empiric clinical and histopathologic criteria can be difficult. Recent advances have provided several molecular markers that can be used for clonal analysis of separate tumor nodules and enhance tumor staging and subsequent treatment and prognosis. To address this issue, we performed a microdissection-based allelotyping of 20 cases of histologically similar, pathologic stage T4 adenocarcinomas (ADCs). Loss of heterozygosity (LOH) analysis included a panel of 15 polymorphic microsatellite markers located on 1p, 3p, 5q, 9p, 9q, 10q, 17p, and 22q. The tumor size, visceral pleural and angiolymphatic invasion, lymph node status, outcome, and survival were assessed. Allelotypes of 60 cases of solitary primary non-small cell lung carcinomas (NSCLC) (stages I-II) were used to define the percentage of discordant LOH patterns within solitary primary lung carcinoma that would discriminate between survivors and nonsurvivors. These criteria were used in the analysis of pathologic stage T4 ADC. Two groups of stage T4 cases were created: molecularly homogenous (or = 40% discordances) (14 cases, 70%), and molecularly heterogenous (40% discordances) (6 cases, 30%). Molecularly homogenous tumors were more frequently associated with visceral pleural invasion (92% vs. 8%) (P = 0.018). Allelotype did not correlate with age, gender, tumor size, tumor differentiation, lymph node status, angiolymphatic invasion, survival, or outcome. Our study showed that discordant and concordant genotypic profiles exist in morphologically similar synchronous ADC of the lung.

BACKGROUND Epidermal growth factor receptor (EGFR) overexpression and amplification are important prognostic factors in many solid tumors and anti-EGFR antibody-based therapy is now available as a promising therapeutic modality. There is little information in the literature regarding the biologic role of EGFR in thymomas that are characterized by variable clinical presentations, histologic heterogeneity, and unpredictable behavior. METHODS Protein expression and gene amplification of EGFR were investigated in 32 thymomas (9 World Health Organization [WHO] type A, 5 type AB, 7 type B2, 7 type B3, 4 type C) using immunohistochemistry and fluorescence in situ hybridization (FISH). FISH analysis included assessment of the average number of copies of the EGFR gene per cell, the average ratio of the EGFR gene to chromosome 7 copy numbers, and ploidy. RESULTS The results of FISH analysis showed statistically significant correlation with WHO histologic type, invasion, advanced clinical stage, but not with tumor size and outcome. Thymomas associated with myasthenia gravis more frequently showed hyperploidy when compared with sporadic tumors, but there was no difference in EGFR gene amplification. EGFR protein expression assessed by immunohistochemistry did not correlate with any studied clinicopathologic variables. There was poor correlation between the protein expression and gene amplification, only 7 of 23 specimens (30%). CONCLUSIONS The potential role of EGFR in the pathogenesis of advanced-stage thymomas indicated that evolving anti-EGFR antibody therapy may be considered as a treatment option. Cancer 2005. © 2005 American Cancer Society.

This report details the pulmonary pathologic findings in four patients with rheumatoid arthritis, who developed new onset of pulmonary signs and symptoms with alveolar infiltrates temporally related to the institution of etanercept therapy. Biopsy findings showed an interstitial and air space lymphohistiocytic infiltrate with non-necrotizing granulomas, in the setting of negative cultures and special stains for microorganisms. The association with etanercept therapy and granulomatous reactions is discussed along with the differential diagnosis.

Although the most frequently altered oncogenes and tumor suppressor genes in non-small cell lung carcinoma (NSCLC) have been recognized, the exact mechanisms responsible for the progression and phenotypic expression of carcinoma, particularly adenocarcinoma of the lung are uncertain. Fifty-six cases of adenocarcinoma of the lung (11 bronchioloalveolar carcinoma [BAC], 25 stage 1, 20 stage 2) and paired 19 lymph node metastases (LNM) of stage 2 adenocarcinomas were analyzed for loss of heterozygosity (LOH). Analysis included a panel of 14 polymorphic microsatellite markers located on 1p, 3p, 5q, 9p, 10q, and 17p. LOH on chromosomes 1p (P = 0.0209) and 17p (P = 0.0274) was more frequently present in stage 1 adenocarcinomas than in BAC. There was no significant difference between BAC, stage 1 and stage 2 adenocarcinoma in the frequency of LOH at individual chromosomal arms. The pattern of LOH in LNM of stage 2 adenocarcinoma was similar to the primary tumor. Overall fractional allelic loss (FAL) was significantly different between BAC and stage 1 invasive adenocarcinoma (P = 0.0013), and it was significantly higher in stage 1 adenocarcinoma than in stage 2 adenocarcinoma (P = 0.0062) and their LNM (P = 0.0001). Stage 2 adenocarcinomas showed significantly higher overall FAL than their LNM (P = 0.022). Our study failed to identify a single target gene responsible for progression of lung adenocarcinoma. A trend towards lower overall FAL in advanced stage tumors and in their metastases suggests that clonal selection may play a role in lung adenocarcinoma progression.

Reported studies show that the systemic form of Langerhans cell histiocytosis (LCH) is a clonal expansion of Langerhans cells (LC) associated with aberrant expression of several oncogenes or tumor-suppressor genes. LCH of the lung is a heterogenous group of lesions thought to be a reactive rather than neoplastic process. The histogenesis of the LCH of the lung is uncertain, and to date there are no studies investigating its underlying molecular abnormalities. We performed comparative genotypic analysis by using allelic loss (LOH) of polymorphic microsatellite markers associated with tumor suppressor genes. Fourteen cases of formalin-fixed, paraffin-embedded LCH of the lung were studied. Microdissection of a total of 26 nodules from 14 patients and paired reference lung tissue was performed under stereomicroscopic visualization. To evaluate allelic loss, we used a panel of 11 polymorphic microsatellite markers that were situated at or near tumor suppressor genes on chromosomes 1p, 1q, 3p, 5p, 9p, 17p, and 22q. The PCR products were analyzed by using capillary electrophoresis to identify germline heterozygous alleles and LOH. Allelic loss at 1 or more tumor suppressor gene loci was identified in 19 of 24 nodules. The total fractional allelic loss (FAL) ranged from 6% (1q) to 41% (22q), with a mean of 22%. The FAL in individual cases ranged from 0 (7 nodules) to 57% (1 nodule). Fifteen discordant allelic losses at 1 to 3 chromosomal loci were identified in 8 patients with multiple synchronous nodules. Our results show that LOH of tumor suppressor genes is present in the LCH of the lung, and they indicate that the putative tumor suppressor genes situated on chromosomes 9p and 22q may play a role in the development of a subset of the LCH of the lung.

The authors report 10 patients with a distinctive idiopathic bronchiolocentric interstitial pneumonia having some histologic similarities to hypersensitivity pneumonitis. Bronchiolocentric interstitial pneumonia has a marked predilection for women (80%) and occurs in middle age (40-50 years). Chest radiographs and pulmonary function tests show interstitial and restrictive lung disease, while the histologic appearance is that of a centrilobular inflammatory process with small airway fibrosis and inflammation that radiates into the interstitium of the distal acinus in a patchy fashion. Granulomas are not identified. At a mean followup of approximately 4 years in nine patients, 33% of patients were dead of disease and 56% had persistent or progressive disease suggesting a more aggressive course than hypersensitivity pneumonitis and nonspecific interstitial pneumonia, the two major disease processes in the differential diagnosis. Whether Bronchiolocentric interstitial pneumonia is a unique entity or not, the pattern of bronchiocentric injury to the lung in the absence of known causes and its clinical presentation as interstitial lung disease, warrants further investigation of this unusual interstitial process.

Pulmonary carcinosarcoma is a rare, biphasic tumor composed of malignant epithelial and mesenchymal elements. Its histogenesis is controversial in light of the presence of divergent cell lineages and the clonal nature of malignancy. To address these issues, we performed an extensive comparative genotypic analysis using microdissection to secure representative mesenchymal and epithelial components from each of six cases of pulmonary carcinosarcoma. Loss of heterozygosity was analyzed with a panel of 12 polymorphic microsatellite markers designed to indicate allelic loss and situated in proximity to known tumor suppressor genes located on 1p, 3p, 5q, 9p, 10q, and 17p. In accordance with the relatively greater biologic aggressiveness of this tumor type, both the epithelial and mesenchymal components showed extensive allelic loss, most notably for 3p, 5q, and 17p. More importantly, we found overall equivalent patterns of acquired allelic loss between the two components on an individual case basis, strongly supporting the monoclonal origin of these neoplasms. Minor differences in the allelic fingerprint between the two cell lineages could be explained by progressive accumulation of allelic loss alterations that appear to occur more frequently in the mesenchymal component of the tumor. The data support the efficacy of microdissection-based allelic fingerprinting to delineate the relationship between different morphologic components of a single neoplasm.

Currently, the practice of pathology is undergoing a transition in which the focus is on individual molecular tests for specific predictive biomarkers for various types of cancers. This current model is predominantly based on single tests for 1 or more biomarkers to predict possible response to 1 or more distinct targeted therapies for a cancer. However, an even more powerful technology looms on the horizon and is expected to eventually replace individual tests as the standard for personalized health care. During the past several years, the ability to map or sequence the entire genome of a cancer in a matter of minutes for a reasonable cost has become an increasingly realistic goal that could make this technology available for routine use in the evaluation of patients’ cancers. Known as whole genome sequencing or analysis or mapping, this global approach would provide a tremendous wealth of information—essentially disclosing the full picture of possible therapeutic targets for a patient’s tumor—in contrast to limited testing for a few separate genes, which might or might not prove of value as targets in a given case. Today, one might test a sample of a pulmonary adenocarcinoma for epidermal growth factor receptor (EGFR) mutations. Most likely the test would be mutational analysis of the EGFR gene, and, in up to 10% of Western patients, one might find an EGFR mutation that is potentially responsive to anti-EGFR therapy. As part of an algorithm, one might next test for ALK fusion genes using fluorescent in situ hybridization, and, in about 3% to 7% of adenocarcinomas, one might find ALK fusion genes that are potentially responsive to anti-ALK therapy. And so on, for a restricted number of genes. Once the roster of candidate biomarkers has been exhausted, one is left with adenocarcinomas that do not have any of the potential therapeutic targets and, for all cases, one does not know what other targets may be undiscovered in the vast majority of the genome that has not been examined. In contrast, by mapping the same adenocarcinoma’s entire genome, one can examine not only for EGFR mutations and ALK fusion genes at the same time, but also for other somatic changes throughout the genome, including previously unsuspected new genes, loci, and pathways. This would provide a more complete picture of all possible therapeutic targets with one test. Of course, the utility of whole genome sequencing is not limited to the therapy of cancer. This technology also has applications for infectious diseases, inherited diseases, and other types of diseases, so that its impact is expected to be felt across the entire spectrum of human disease. Indeed, some have speculated that every person may have his or her genome sequenced at birth to provide a baseline for future prediction/prevention, diagnosis, and treatment throughout the person’s lifetime. The reason that whole genome sequencing was not previously used for routine clinical practice was the large amount of time and the exorbitant cost of such testing. When the Human Genome Project finished sequencing the first entire human genome in 2003, the project took 13 years and cost nearly $2.7 billion. Not surprisingly, commercial companies have developed new generations of genome sequencing machines that today allow whole genome sequencing at a fraction of the time and cost of earlier sequencers. Companies now talk of sequencing the entire human genome in less than 15 minutes for a cost of $100 per genome. It is the ability to deliver this powerful technology in real time at a practical cost that has led to the expectation that this technology may one day replace the plethora of individual tests now performed in laboratories. Besides clinically acceptable turnaround time and affordable cost, this technology must be suitable for the analysis of routine formalin-fixed, paraffin-embedded specimens. It seems inevitable that this technology will become routine clinical practice in the future. The pathologist practicing today or in training today will want to know how soon to expect this technology and, of course, what steps he or she should be taking to address it. The answers are complex and challenging. Regarding the latter question, calls to action have recently been published, including, but not limited to, the proceedings of the meeting at the Banbury Conference Center on genome-era pathology and a call for a third track in genomic pathology by James M. Musser, MD, PhD, chair of The Methodist Hospital Department of Pathology and Laboratory Medicine, published in the Archives of Pathology & Accepted for publication April 13, 2011. From the Department of Pathology, Methodist Hospital, Houston, Texas (Dr Cagle); the Department of Pathology, University of Pittsburgh Medical Center–Presbyterian Hospital, Pittsburgh, Pennsylvania (Dr Dacic); and the Department of Pathology, University of Texas Health Science Center at Tyler (Dr Allen). The authors have no relevant financial interest in the products or companies described in this article. Reprints: Philip T. Cagle, MD, Department of Pathology, The Methodist Hospital, 6565 Fannin St, Main Bldg, Room 227, Houston, TX 77030 (e-mail: pcagle@tmhs.org). Editorial

8001 Background: c-Met-amplified NSCLC defines a subset of NSCLC that may be sensitive to the small-molecule tyrosine kinase inhibitor crizotinib, approved multinationally for the treatment of advanced ALK-positive NSCLC. Efficacy and safety data are presented for crizotinib in patients with advanced c-Met-amplified NSCLC within 3 categories of amplification MET/CEP7 ratio ≥1.8-≤2.2 (Low), >2.2

Accumulating evidence shows that lung tumors are responsive to the steroid hormones β-estradiol and progesterone, and variables related to hormone receptor status have been reported to affect lung cancer survival. These findings suggest that there may be similarities in how hormonal pathways control tumor progression between lung cancer and breast cancer. We examined the relationship between disease-free survival (DFS) and expression of genes included in three published breast cancer survival signatures in a cohort of 104 early-stage (IA and IB) non-small cell lung cancers. Cases were selected from the University of Pittsburgh Lung Cancer SPORE Tissue Bank based on the following criteria: tumor tissue originated from a completely resected primary T1N0 or T2N0 adenocarinoma or squamous cell lung cancer; fresh-frozen lung cancer tissue was available; no neo-adjuvant therapy given; clinical variables (smoking history, age at diagnosis, sex, size of tumor, pleural invasion, site of recurrence) and outcome were known. RNA isolated from banked frozen tumor tissue was used to assess mRNA expression with the Illumina Human HT-12 v4 BeadChip; all mRNA analyzed had passed quality checks. Data were also subjected to background subtraction and quantile normalization. The Illumina BeadChip contains 16 out of 16 published Oncotype DX genes, 61 out of 66 MammaPrint genes, and 49 of 50 PAM50 genes. Supervised Principal Component Analysis was conducted to evaluate the ability to predict disease-free survival (DFS) for each gene panel as a group and also by using the top genes from the gene panels that showed differential expression in the lung cancer cohort. All results were subjected to 10-fold cross-validation. Genes from the Oncotype DX panel showed no differential expression in the cohort and had no ability to separate cases based on DFS. For the MammaPrint genes, three probes (ALDH4A1, FLT1, RECQL5) showed significant differences in expression in the cohort (p < 0.05), and the gene panel showed a relationship to DFS (HR 1.72, p = 0.07). Examination of the top differentially expressed genes did not improve prediction for the MammaPrint genes. For the PAM50 gene panel, three probes (CXXC5, FGFR4, FOXC1) showed significant differential expression (p < 0.05) and 23 probes showed differential expression at p < 0.1. The PAM50 gene panel as a group was able to separate lung cancer cases based on DFS (HR 1.9, p = 0.03). The PAM50 genes with differential expression at p < 0.1 also separated the cohort into four prognosis groups (p = 0.008 for trend). The worst prognosis group had a HR of 4.76 (p = 0.002). These genes were then analyzed in a subset of 44 cases in the Stage I cohort for whom ERβ protein expression status was known from immunohistochemistry (IHC) analysis. Comparing ERβ high (Allred score of >4, N= 20) and ERβ low (Allred score of 4 or less, N = 24) cases, the top PAM50 genes were more informative in ERβ high cases (HR 11.7, p = 0.0007) compared to ERβ low cases (HR 3.38, p = 0.045). For ERβ high cases, Kaplan-Meier survival curves showed that 50% of the high-risk group relapsed by 22 months, while 50% relapse was not reached in the low-risk group at 60 months. Similar analyses in the MammaPrint and Oncotype DX gene panels did not improve DFS prediction. We next validated the top PAM50 genes in a different cohort of 64 NSCLC cases, which included all stages and histologies, and had available ERβ IHC data, expression data from the Illumina Human HT-12 v4 BeadChip, and survival information. The top PAM50 genes were also able to predict DFS in this cohort: HR 2.19, p = 0.034 in all cases and HR 3.24, p = 0.042 in ERβ high cases. Pathway analysis indicated that the informative PAM50 genes describe a network that contains the estrogen and progesterone receptors, HER2, and cyclin E. The top canonical pathways identified were HER2/HER3 signaling and estrogen signaling. These results suggest that genes involved in interactions between hormonal and HER signaling may be predictive of lung cancer survival, especially in early-stage lung cancer, and provide further evidence for the importance of hormonal pathways in the biology of lung cancer. Supported by NCI SPORE in Lung Cancer P50 040990 and by the University of Pittsburgh Medical Center Genomics Initiative. Citation Format: Jill M. Siegfried, Yan Lin, Sanja Dacic, Brenda Diergaarde, Laura P. Stabile, Hui-Min Lin, Marjorie Romkes. Prediction of lung cancer survival by genes in the PAM50 breast cancer panel. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B20.

Background . Second lung primaries occur at a rate of 1% to 3% per patient-year after complete resections for non–small cell lung carcinoma (NSCLC). Fluorescence bronchoscopy appears to be a sensitive tool for surveillance of the tracheobronchial tree for early neoplasias. Methods . Patients who were disease-free after complete resection of a NSCLC were entered into a fluorescence bronchoscopy surveillance program. All suspicious lesions were biopsied along with two areas of normal mucosa to serve as negative controls. Results . A total of 73 fluorescence bronchoscopies were performed after conventional bronchoscopy in 51 patients at a median of 13 months postresection. The majority (46 of 51) of patients had stage I or II NSCLC, whereas 10% (5 of 51) had stage IIIA. Three intraepithelial neoplasias and one invasive carcinoma were identified in 3 of 51 patients (6%), all current or former smokers. Of the four lesions identified, three were in the 20 patients with prior squamous cell carcinomas. No intraepithelial neoplasias were identified by white-light bronchoscopy, whereas two of three were detected by fluorescence examination. The one invasive cancer detected was apparent on both white-light and fluorescence bronchoscopic examinations. Conclusions . Surveillance with fluorescence bronchoscopy identified lesions in 6% of postoperative NSCLC patients thought to be disease-free. Patients with prior squamous cell carcinomas appear to be a population that may warrant future prospective study of postoperative fluorescence bronchoscopic surveillance.

Nodular amyloidomas (NA) of the lung are non-neoplastic inflammatory nodules containing eosinophilic amyloid deposits and a lymphoplasmacytic infiltrate. In some instances, the extensive amyloid deposits may obscure an underlying lymphoproliferative disorder. The histologic and immunohistologic features that discriminate these two differential diagnostic possibilities were studied in this series of six cases of NA and five cases of primary low-grade malignant lymphomas of lung with secondary amyloid deposits (ML). Two of lymphoma cases showed histopathologic and immunophenotypic features of B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-cell CLL/SLL), and three cases were low-grade B-cell lymphoma derived from mucosa associated lymphoid tissue (MALT lymphoma). Key discriminating morphologic features between NA and ML included lymphatic tracking of the cellular infiltrate (3/5 ML; 1/6 NA), pleural infiltration (3/5 ML; 0/6 NA), sheet-like masses of plasma cells (5/5 ML; 0/6 NA) and reactive follicles (4/5 ML; 1/6 NA). Lesional circumscription, vascular and bronchial destruction, lymphoepithelial lesions, and granulomas were not helpful discriminators. Immunohistochemical features indicating a dominant CD20+, CD79a+ B-cell population (5/5 ML; 0/6 NA), light chain restriction (4/5 ML; 0/6 NA), and aberrant antigen expression of CD20/CD43 (2/5 ML; 0/6 NA) were helpful. Amyloid tumors with a reactive lymphoplasmacytic infiltrate can be separated from low-grade malignant lymphomas utilizing both histologic and immunohistochemical features.

Second lung primaries occur at a rate of up to 3% per patient-year after curative resection for non-small-cell lung carcinoma. Postresection patients are often poor candidates for further curative surgery because of their diminished pulmonary reserve. The aim of this study was to evaluate the role of fluorescence bronchoscopy by using the Xillix® LIFE-Lung Fluorescence Endoscopy SystemTM to identify second lung primaries in patients who have had a previous curative resection of a non-small-cell lung cancer. Patients who had no evidence of disease status after resection of a non-small-cell lung cancer were identified from a prospectively collected data base and entered onto a fluorescence bronchoscopy surveillance protocol. All suspicious areas, as well as several areas of apparently normal mucosa, were sampled for biopsy. A single pathologist reviewed all biopsy specimens, with 10% of biopsies re-reviewed, for quality control, by a second pulmonary pathologist. A total of 31 surveillance fluorescence bronchoscopies were performed on 25 patients after conventional bronchoscopy. Four intraepithelial neoplasias or invasive carcinomas were identified in 3 (12%) of 25 patients screened. The addition of the LIFE examination to conventional bronchoscopy increased the sensitivity of screening from 25.0% to 75.0%, which yielded a relative sensitivity of 300% with a negative predictive value of .97. Use of postresection surveillance with fluorescence bronchoscopy identified intraepithelial or invasive lesions in 12% of non-small-cell lung cancer patients, and the system was three times more sensitive than conventional bronchoscopy to identify these early mucosal lesions. Fluorescence bronchoscopic surveillance of this high-risk, postresection population will help better define the true rate of occurrence and the natural history of second primaries and may assist in monitoring their response to newer, noninvasive treatment methods, such as photodynamic therapy or chemopreventive agents, in future trials.

Background Second lung primaries occur at a rate of 2% per patient per year after curative resection for non-small cell lung carcinoma (NSCLC). The aim of this study was to evaluate the role of fluorescence bronchoscopy using the Xillix® LIFE-Lung Fluorescent Endoscopy SystemTM (LIFE-Lung system) in the surveillance of patients for second NSCLC primaries after resection or curative photodynamic therapy (PDT).Methods NSCLC patients who were disease-free following resection or endobronchial PDT were identified and recruited to participate in a LIFE bronchoscopy surveillance program. All suspicious areas were biopsied; areas of apparent normal mucosa served as negative controls. Biopsy specimens were reviewed by a single pulmonary pathologist.Results Thirty-six patients underwent 53 surveillance LIFE bronchoscopies and 6/112 biopsies revealed intraepithelial neoplasia (IEN) or invasive carcinoma in 6/36 (17%) of patients. The overall relative sensitivity of LIFE versus conventional bronchoscopy was 165% with a negative predictive value of 0.96, for the post-resection subset of patients these values increased to 200% and 0.97, respectively.Conclusions Surveillance LIFE bronchoscopy identified intraepithelial or invasive lesions in 17% of patients previously thought to be disease-free. These data support future multicenter trials on fluorescence bronchoscopic surveillance of NSCLC patients after curative surgical resection or PDT.

s S55 differentiated NSCLC (PDNSCLC) can be challenging. Our aim was to correlate the cytomorphology in PDNSCLCs with their immunohistochemical (IHC) and molecular features. Materials and Methods: FNA cases of PDNSCLC with mutation or FISH studies were identified over a 5 year period and evaluated for cytology and ancillary study results. Results: 26 (16%) FNA cases of PDNSCLC were identified (F:M Z 1:1.2; average age: 65 years) from a total of 160 cytology cases of NSCLC with molecular testing, including 13 (50%) primary lung tumors and 13 (50%) metastases. The cytological features included a predominance of clustering (65%) or discohesion (35%). Marked pleomorphism was seen in 74% cases and necrosis was present in 57% cases. Focal features of adenocarcinoma (ADC) were seen in 52%, squamous cell carcinoma (SqCC) in 35%, and both squamous and glandular features in 13%. IHC results revealed that 35% of cases were focally positive for both TTF1 and p63/p40, 35% negative for both, 17% focally TTF1+ only, and 13% focally p63/p40+ only. A KRAS mutation was identified in 8 patients (8/ 21, 38%), while no EGFR mutation, ALK, FGF, or c-Met rearrangement was identified. Conclusions: PDNSCLC represented 16% of our cases with molecular testing, and tended to have marked nuclear pleomorphism, necrosis, focal glandular and/or squamous differentiation, and often inconclusive IHC findings. Several (38%) tumors were found to have a KRAS mutation. Although PDNSCLC can be difficult to subclassify, acquisition of material for molecular testing to detect mutations, particularly KRAS mutations, is important.

11085 Background: The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) jointly initiated an effort to establish evidence-based recommendations for the molecular analysis of lung cancers required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Methods: Three co-chairs without relevant conflicts of interest were selected, one from each of the sponsoring societies: CAP (P.T.C.), IASLC (M.L.), and AMP (N.I.L.). Writing and advisory panels were formed from additional experts from these societies. Unbiased literature searches were performed to capture articles up to February 2012, yielding 1,533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for relevance. Evidence was formally graded for each of the recommendations first formulated by the co-chairs and panel members at a public meeting. Each guideline section was assigned to at least two panelists. Successive drafts were circulated for comments to the writing panel, the advisory panel, the public (online posting), and the three professional societies. Results: We generated 37 guideline items addressing 14 areas of EGFR and ALK testing. The major, evidence-based recommendations are to test for EGFR mutations and ALK fusions in all patients with advanced stage adenocarcinoma, regardless of sex, race, or smoking history, and to prioritize EGFR and ALK testing over other molecular predictive tests. Recommendations and expert consensus opinions were generated for all other key aspects of EGFR and ALK testing in lung cancer related to oncology and pathology practice and technical issues in molecular testing. Conclusions: As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards and thereby improving patient outcomes.

Background: EGFR inhibition is an established therapy for previously treated NSCLC. Estrogen receptors (ER) and aromatase are expressed in most NSCLC specimens in both men and women. In preclinical models, estrogen stimulates NSCLC growth, an effect blocked by F, a pure ER antagonist. Preclinical models show enhanced anti-tumor effects by combining EGFR inhibitors with F. Methods: We conducted a Phase II clinical study to evaluate whether addition of F enhances antitumor efficacy of E. Men and women with advanced NSCLC and > 1 prior chemotherapy regimen (unless patient refused) were randomized 2:1 to receive E (150 mg PO qd) + F (500 mg IM q2wk x 3, then q4wk) or E alone. Stratification for gender and ECOG (0, 1 vs. 2) was performed. Response rate (RR) was the primary endpoint. Secondary endpoints included progression free survival (PFS), overall survival (OS) and correlation between clinical endpoints and tumor tissue and blood-based biomarkers. Results: 106 patients (pts) were randomized from March 2006 to June 2010. 100 (evaluated population) received E +/- F. E + F was well tolerated, with adverse events well balanced between arms. For E + F and E respectively, RR (23.6% vs. 14.8%, p = 0.35), PFS [1.9 vs. 1.8 months, hazard ratio (HR) 0.85, 95% confidence interval 0.55, 1.33] and OS [9.4 vs. 5.7 months, HR 0.96 (0.6, 1.55)] were similar between arms. EGFR mutational data could be obtained on 69 pts. EGFR mutations were more prevalent in the E arm (35% vs. 20%). EGFR mutations strongly predicted best response, PFS and OS (p < 0.0002 for each). RR, PFS and OS were similar among EGFR mutants between arms in this small subset (17 patients). Among the 52 pts with EGFR WT tumors, 3 partial responses (PR) were seen with E + F vs. none for E alone. Clinical benefit rate [CBR (RR + stable disease): 54.8% vs. 8.3%, p = 0.0056] was significantly higher among pts with WT tumors treated with E + F. Trends were observed in favor of E + F in PFS [2.0 vs. 1.6 months, HR 0.56 (0.29, 1.07)] and OS [7.4 vs. 5.9 months, HR 0.69 (0.36, 1.31)]. Conclusion: E + F was well tolerated in previously treated NSCLC pts, including men and pre- and post-menopausal women. The study showed a high RR (23.6%) for E + F, that was not adequately explained by EGFR mutations. Among EGFR WT pts, a significantly higher CBR that included 3 PRs was seen with trends towards improved PFS and OS with E + F as compared to E alone. Evaluation of tumor tissue biomarkers (including ER-α and -β, aromatase), blood estrogen levels and EGFR ligands known to be induced by ER signaling is ongoing to determine NSCLC subpopulations most likely to benefit from antiestrogens. [Supported by 1K23CA149079, P50 CA090440, V Foundation for Cancer Research, Jonsson Comprehensive Cancer Center, Wolfen Family Lung Cancer Research Program, Stiles Program in Oncology, National Lung Cancer Partnership and One Ball Matt Memorial Golf Tournament] Citation Format: Edward B. Garon, Jill M. Siegfried, Steven M. Dubinett, Robert M. Elashoff, David J. Park, Rupesh J. Parikh, Ravi Patel, Eddie H. Hu, Karen L. Reckamp, Brad Adams, Diego Martinez, He-Jing Wang, Fairooz Kabbinavar, Sanja Dacic, Meghan Brennan, Isett Laux, Diana C. Marquez-Garban, Laura P. Stabile, Dennis J. Slamon, Richard J. Pietras. Result of TORI L-03, a randomized, multicenter phase II clinical trial of erlotinib (E) or E + fulvestrant (F) in previously treated advanced non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4664. doi:10.1158/1538-7445.AM2013-4664

Genome wide data has transformed disease diagnostic and prognostic model development through the identification of molecular profiles. Enhanced molecular insight can be achieved by multiple -omic profile integration. Accordingly, we used a novel multistep integrative unsupervised approach, the network phenotyping strategy (NPS), for discovery of disease diagnostic/prognostic panels in resected NSCLC frozen tumor samples (n=81; 29 Stage IA/52 Stage IB). Whole genome gene expression and DNA methylation data was generated using Illumina BeadChips. In NPS step 1, the most common (>18% of the cohort) hypo- and hyper-methylated genes (lowest/highest β-score; n=31) were identified. In step 2, a subset of these genes was found to reside in networked multiple loci, co-protected against genetic variation by extreme (high n=10 or low n=6) incorporation energy costs, based on an exome entromic analysis. In step 3, maximal spanning tree reduction of a network of all 120 possible expression level relationships for these 16 genes, weighted by the correlation coefficients quantifying co-regulation, was used to identify the most informative sub-network, an 8-partite graph K8. For each partition, cases were dichotomized into groups where gene 1 was expressed at least 1.5x higher or lower relative to gene 2. In step 4, K8 cycle-decomposition revealed only 8 distinct gene expression patterns (C1-C8) representing nominally different molecular NSCLC subtypes. Characterization of individual tumor expression patterns was computed as the patient's difference vector (ΔC) from C1-C8. Stage IA tumor gene expression patterns (overexpression of all 16 genes) most closely matched C1 (true positive rates 0.8 - 0.93 and false positive rates 0.2 - 0.07, ROC 0.9 in 10-fold cross-validation). For stage IB, these 16 gene expression levels were lower and heterogeneous. In step 5, we generated OS prediction models. The ΔCs were used in an alternating decision tree algorithm. Two rule sets were found, one for the high gene expression Stage IA tumors and the second for Stage IB. In a 10-fold cross-validation, the log-rank test for equality of survivor functions resulted in optimal OS separation at 1650 days (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4937. doi:1538-7445.AM2012-4937

Conventional clinical and pathologic risk factors in stage I non small-cell lung cancer (NSCLC) provide limited prognostic information. Novel prognostic biomarkers are needed to identify patients with highest recurrence risk who will receive the greatest absolute risk reduction from adjuvant chemotherapy/radiation. Transcriptome, epigenome, and global genetic variation analyses of resected NSCLC frozen tumor samples (n=81; 29 stage 1A/52 stage 1B) were performed in order to develop an integrated prognostic signature. Only tumors that were resected within 3 months of the first biopsy-proven diagnosis were used to reduce lead time bias in timing of removal of the primary tumor. Here, the association of gene expression with disease free survival (DSF) is described. Whole genome expression data were quantile-normalized using the Illumina GenomeStudio 2011.1 software. Log-transformed data were then processed in Biometric Research Branch (BRB)-Array Tools. Prognostic models were developed with the top ranking genes using the supervised principal component survival algorithm (PCA). Risk group membership was then assigned based on this multivariate model using leave-one-out cross validation and class prediction modeling. Both models adjusted for age, gender and tumor histology. For stage 1A cases, a 113 gene signature selected by fitting a Cox proportional hazard model (Cox) was able to discriminate between poor and good DFS (α=0.001). Upon further analysis using a class prediction algorithm support vector machine (SVM), we identified an 18 gene classifier that predicted outcome SN=0.952, SP=0.571). A 10-fold cross validation ROC curve reported a value of 0.748. For stage 1B cases, the Cox model identified 141 genes (α=0.01), but the SVM prediction algorithm showed reduced SN/SP (0.75/0.25) for a 12 gene classifier, indicative of possible increased heterogeneity among the 1B cases. Nine genes overlapped between the PCA and class prediction model gene signatures. These nine are members of common lung tumorigenesis pathways, i.e. G-protein signaling regulation (KRAS/BRAF/RASSFI); tyrosine protein kinase signaling (EGFR/ERBB2); nucleotide excision repair (ERRC5 and ERCC6) and genes implicated in cell differentiation regulated by miR200B (HGD) or miR338 (TXNRD1), miRNAs independently found to be associated with prognosis in our cohort. These initial results demonstrate that a gene expression profile can distinguish stage I NCSLC tumors and predict prognoses, but require further validation. Supported in part by NIH 5P50 CA090440, P30CA047904, UPMC Institutional Funds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1722. doi:1538-7445.AM2012-1722

A prognostic 15-gene signature for NSCLC was recently published (J Clin Oncol 28, 4417, 2010). We used a novel integrated approach, the network phenotyping strategy (NPS), to evaluate the performance of this signature in our NSCLC patients and to gain insight into NSCLC heterogeneity by analyzing global mRNA/miRNA and DNA methylation microarray data (n=47 Stage IB tumors). The mRNA data was compared between cases with overall survival (OS) 650 days. The mRNA levels of the 15 individual genes are not associated with OS. We hypothesized that the networked gene expression pattern rather than the absolute level of any 1 gene accounts for the disease outcome association. Maximal spanning tree reduction of a graph of all 105 possible pairwise expression relationships, weighted by the correlation coefficients quantifying co-regulation levels, was used to identify the 7 most informative gene pairs. For each pair, cases were dichotomized into groups where gene 1 was expressed at least 1.5x higher or lower relative to gene 2. A 7-partite graph (K7) captured these trend patterns. K7 cycle-decomposition revealed 10 different gene expression patterns (C1-C10) representing nominally different molecular NSCLC subtypes. Characterization of individual tumor expression trend patterns was computed as the difference vector (≥C) from C1-C10. A rule-based algorithm used ΔC to classify cases into 2 OS groups. We also analyzed DNA methylation and miRNA data for these 15 genes. For 2 genes, hypomethylation (≥0.5) was associated with low expression. For 10 genes with low expression and intermediate methylation, the expression data indicated the involvement of additional regulatory mechanisms. For example, HEXIM1 expression (most commonly down-regulated relative to FAM64A), is regulated by miR-623, which is itself a prognostic biomarker of OS in our study. Validation of the published signature was observed in this independent NSCLC cohort using NPS analysis. Not only was this gene signature prognostic, confirming the signature robustness, but the NPS strategy was equally effective as the originally published principal component analysis, both resulting in a significant separation of the Kaplan-Meier OS curves (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1720. doi:1538-7445.AM2012-1720

There is increasing evidence for steroid hormone effects in lung cancer. Estrogens are involved in lung cancer proliferation and progression, and human lung tumors express estrogen receptor β (ERβ) as well as aromatase, the enzyme which catalyzes the final step in 17-β-estradiol synthesis. We have previously demonstrated that high ERβ expression in lung tumors was an independent negative prognostic predictor of survival, and the contribution of aromatase expression resulted in further effects on survival in both men and women. To determine if the aromatase inhibitor anastrozole prevents the development of lung tumors induced by the tobacco carcinogen, NNK, alone or in combination with the ER antagonist fulvestrant, ovariectomized FVB/N female mice were exposed to NNK along with daily supplementation of 0.1mg androstenedione, the substrate for aromatase. Placebo control, anastrozole (0.1mg/kg/day; oral gavage) and/or fulvestrant (30mg/kg, 2X/week, s.c.) were administered in both an initiation model and a progression model of lung tumorigenesis. In the initiation model, the combination of fulvestrant and anastrozole was administered during NNK exposure and resulted in significantly fewer NNK-induced lung tumors (mean=0.5; range 0-1) compared to placebo (mean=4.6; range 2–8, P

7019 Background: C, an anti-EGFR monoclonal antibody, enhances the efficacy of both RT and CT. We evaluated a novel strategy of RT plus C followed by consolidation CT plus C for the treatment of st...

The hepatoprotective effects of a newly synthesized 15 amino acid fragment code named BPC 157 was evaluated in comparison with the reference standards (bromocriptine, amantadine and somatostatin) in various experimental models of liver injury in rats: 24 h-bile duct + hepatic artery ligation 48 h-restraint stress and CCl4 administration. BPC 157 administered either intragastrically or intraperitoneally, significantly prevented the development of liver necrosis or fatty changes in rats subjected to 24 h bile duct + hepatic artery ligation, 48 h- restraint stress, CCl4 treatment (1 ml/kg i.p., sacrifice 48 h thereafter). The other reference drugs had either little or no protective actions in these models. Noteworthy, the laboratory test results for bilirubin, SGOT, SGPT fully correlated with the macro/microscopical findings. Thus, on the basis of consistent protective eefect of BPC 157, possible clinical application in liver diseases is now warranted.

Sex steroids may be targets for lung cancer intervention. Estrogen has been shown to stimulate, while progesterone has been shown to inhibit, lung cancer growth. Cross-talk between the estrogen and the epidermal growth factor (EGF) signaling pathways in lung cancer has been reported. To further clarify the roles of estrogen, progesterone and EGF receptors, we analyzed primary lung tumors and normal lung tissue from 183 patients (primarily non-small cell lung cancer histologies) for expression and localization of estrogen receptor α (ERα), estrogen receptor β (ERβ), aromatase, progesterone receptor (PR), and EGF receptor (EGFR) using immunohistochemistry. No differences were observed in any marker expression analyzed by sex or histology. ERα expression was positive only when using an antibody that also detects ERα deletion variants, consistent with mRNA profiles of lung tumor cell lines showing multiple ERα variants with no full-length mRNA. ERα expression showed no effect on survival. Tumor tissues were found to express significantly higher levels (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3667.