Abstract Background: There is increasing evidence that the presence of a host inflammatory response to breast cancer may influence outcomes. Utilizing inflammation scores on the histology of breast cancer samples submitted for comprehensive molecular analyses for The Cancer Genome Atlas (TCGA), we provide an updated look at associations between the presence of host inflammation and breast cancer molecular and pathologic features. Design: Experts in breast pathology reviewed the digital slides of breast cancer samples submitted for comprehensive molecular profiling to the TCGA and scored each case for the level of inflammation present (high/moderate vs mild/minimal). We tested pairwise associations between host inflammation and molecular subtypes (DNA copy-number, RNA expression, RPPA defined subtypes, miRNA subtypes, methylation subtypes) and pathological features by performing Chi-Square analyses. Multiple hypothesis testing correction was performed using the Bonferroni method. Results: 598 breast cancer cases with TCGA molecular profiling data were scored by the expert breast pathologists for morphological features (including inflammation). 195 (33%) of these were scored as high/moderate inflammation. Cases with inflammation had a significantly higher rate of TP53 mutations (p = 9.0e-8) with 64 of 118 (54.2%) p53 mutant cases with inflammation. Inflammation was also significantly associated with PAM50 molecular subtypes (p = 2.2e-11), with the greatest enrichment among basal-like (64.5% of 70 basal-like cases had inflammation) and the greatest depletion among Luminal A (18.1% of 166 Luminal A cases had inflammation). Cases with inflammation were significantly less likely to be lobular (p = 1.5e-7), had less tubule formation (p = 0.0006), increased mitoses (p <2.2 e-16), increased nuclear pleomorphism (p = 2.9e-15), and increased necrosis (p = 2.9e-14). Conclusions: There are strong associations between breast cancer molecular and pathological features and the host inflammatory response. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-06.

Background: Aberrant signaling via the PI3K pathway is a common alteration in breast cancer (BC), with frequent activating mutations in the PIK3CA gene helical (exon 9) and catalytic (exon 20) domains. These mutations occur across all BC subtypes with an overall incidence of 36%, with the highest frequency (∼45%) in luminal A/ER+ tumors. Lobular phenotype is common among luminal A tumors. We examined associations between lobular histology and molecular features among BC samples submitted for comprehensive molecular analyses for The Cancer Genome Atlas (TCGA).Design: Experts in breast pathology reviewed digital slides of breast cancer samples submitted for comprehensive molecular profiling to the TCGA. Tumors were graded, subtyped and scored for additional histopathologic features. We tested pairwise associations between lobular features and components of grade, PAM50-derived molecular subtype and mutational status for BRAC1/2, PIK3CA, TP53 and CDH1 by performing Chi-Square analysis for comparisons with a categorical variable and the Mann-Whitney test for comparisons with an ordinal variableResults: A total of 1132 images were scored from 589 unique cases in TCGA. For cases with multiple scorers (43% of cases), we summarized scores by taking the median (for ordinal variables) or the consensus diagnosis (for categorical variables). A total of 567 cases had a consensus diagnosis for lobular features, all of which had pathological information on components of histologic grade and 540 of which had data for TP53, CDH1, and PIK3CA mutations. 110/567 (19%) of cases were classified as invasive lobular or invasive mammary carcinoma with lobular features. The lobular cases had significantly less nuclear pleomorphism (p = 3.3 e -12), lower mitotic index (p = 3.4e-16), less tubule formation (p = 3.9e-8), increased association with lobular carcinoma in situ (p < 2.2 e-16), decreased stromal inflammation (p = 1.5e-7), and decreased necrosis (p = 4.4e-11) compared with cases without lobular features. Cases with lobular features were highly enriched for CDH1 mutations with 19% of cases with lobular features having CDH1 mutations, compared with only 1% of cases without lobular features (p = 2.4 e-14). The lobular features cases were more likely to have PIK3CA mutations (p = 0.01), with 33% of the lobular features cases having PIK3CA mutations, compared with 21% of the non-lobular cases. The lobular features cases were less likely to have TP53 mutations (p = 0.02), with 13% of lobular features cases having TP53 mutations as compared with 24% of the non-lobular feature cases. Lobular status was associated with PAM50 molecular subtype (Chi-square p = 0.002) with the lobular cases significantly less likely to be basal molecular subtype and more likely to be Luminal-A.Conclusions: PIK3CA mutations are enriched in invasive lobular carcinomas and invasive mammary carcinomas with lobular features. These associations point to the possibility that PIK3CA mutations as well as CDH1 alterations are important drivers of invasive lobular carcinomas.Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-10.

Abstract Background: Previous studies have found that particular pathologic features are more common in breast cancers arising in BRCA mutation carriers. However, the biologic and molecular bases for the morphologic associations are not clear. This study is conducted to analyze pathologic and molecular features in tumors stratified by BRCA1 or BRCA2 mutation status using the breast cancer samples that have comprehensive molecular portraits characterized by the Cancer Genome Atlas (TCGA). Methods: The digital slides of breast cancer samples submitted for comprehensive molecular profiling to the TCGA were reviewed by expert breast pathologists, who were unaware of the BRCA status or other molecular signatures. Each tumor was evaluated and scored for histologic type, nuclear pleomorphism, tubule formation, mitosis, stromal inflammation, and necrosis. 562 cases had both pathology and tumor exome sequencing data available and constituted the current study population. We determined the association of somatic BRCA1 and BRCA2 mutation status with pathologic features and molecular characteristics (mutation of PIK3CA and TP53, and molecular subtypes defined by PAM50 mRNA data) using the Fisher exact test for categorical variables and the Wilcoxon test for ordinal variables. Results: Of the 562 tumors, 514 had no BRCA1 or BRCA2 mutation, while 48 (8.5%) of tumors were found to harbor a BRCA1 mutation (n = 16, 3%), BRCA2 mutation (n = 30, 5%), or mutation in both (n = 2, 0.3%). BRCA1 and BRCA2 mutational status showed no significant association with lobular features, tubule formation, nuclear pleomorphism, or stromal inflammation (all p > 0.05), although there was a trend for increased nuclear pleomorphism in BRCA2 mutant cases (p = 0.07). The lack of significant association of BRCA1/2 mutational status with these features may be due to our study's relatively small number of BRCA1/2 mutant cases. Both BRCA1 and BRCA2 mutations were associated with a higher mitotic count (p = 0.03 and 0.04, respectively). BRCA2 mutation showed no association with necrosis (p = 1), while BRCA1 mutation status was associated with increased necrosis (OR = 2.7, p = 0.04). BRCA2 mutation status showed no significant association with PAM50 subtype (p = 0.37), while BRCA1 mutation status was significantly associated with PAM50 molecular subtype (p = 0.005), with the greatest enrichment among Basal-like (7/70 Basal-like with BRCA1 mutation, 10%) and depletion among Luminal-B (0/79 Luminal-B with BRCA1 mutation, 0%). Neither BRCA1 nor BRCA2 mutations were significantly association with PIK3CA mutations (p = 0.39, 0.08, respectively). BRCA2 mutation status was not associated with TP53 mutations (p = 0.65), while BRCA1 mutation status was associated with increased TP53 mutations (OR = 4.0, p = 0.005). Conclusion: Tumors with BRCA1 and BRCA2 alterations are associated with specific pathologic and molecular features. However, there is molecular and morphologic heterogeneity within these cancers. These factors need to be considered when designing algorithms for BRCA testing and targeted therapy in BRCA-related cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-15.

PURPOSE:Epidermal growth factor receptor (EGFR) mutations have been associated with tumor response to treatment with single-agent EGFR inhibitors in patients with relapsed non-small-cell lung cancer (NSCLC). The implications of EGFR mutations in patients treated with EGFR inhibitors plus first-line chemotherapy are unknown. KRAS is frequently activated in NSCLC. The relationship of KRAS mutations to outcome after EGFR inhibitor treatment has not been described.PATIENTS AND METHODS:Previously untreated patients with advanced NSCLC in the phase III TRIBUTE study who were randomly assigned to carboplatin and paclitaxel with erlotinib or placebo were assessed for survival, response, and time to progression (TTP). EGFR exons 18 through 21 and KRAS exon 2 were sequenced in tumors from 274 patients. Outcomes were correlated with EGFR and KRAS mutations in retrospective subset analyses.RESULTS:EGFR mutations were detected in 13% of tumors and were associated with longer survival, irrespective of treatment (P < .001). Among erlotinib-treated patients, EGFR mutations were associated with improved response rate (P < .05) and there was a trend toward an erlotinib benefit on TTP (P = .092), but not improved survival (P = .96). KRAS mutations (21% of tumors) were associated with significantly decreased TTP and survival in erlotinib plus chemotherapy-treated patients.CONCLUSION:EGFR mutations may be a positive prognostic factor for survival in advanced NSCLC patients treated with chemotherapy with or without erlotinib, and may predict greater likelihood of response. Patients with KRAS-mutant NSCLC showed poorer clinical outcomes when treated with erlotinib and chemotherapy. Further studies are needed to confirm the findings of this retrospective subset analysis.

MOTIVATION:Expression profiling in diverse tissues is fundamental to understanding gene function as well as therapeutic target identification. The vast collection of expressed sequence tags (ESTs) and the associated tissue source information provides an attractive opportunity for studying gene expression.RESULTS:To facilitate EST-based expression analysis, we developed GEPIS (gene expression profiling in silico), a tool that integrates EST and tissue source information to compute gene expression patterns in a large panel of normal and tumor samples. We found EST-based expression patterns to be consistent with published papers as well as our own experimental results. We also built a GEPIS Regional Atlas that depicts expression characteristics of all genes in a selected genomic region. This program can be adapted for large-scale screening for genes with desirable expression patterns, as illustrated by our large-scale mining for tissue- and tumor-specific genes.AVAILABILITY:The email server version of the GEPIS application is freely available at http://share.gene.com/share/gepis. An interactive version of GEPIS will soon be freely available at http://www.cgl.ucsf.edu/Research/genentech/gepis/. The source code, modules, data and gene lists can be downloaded at http://share.gene.com/share/gepis.

Genetic defects in the Wnt-1 signaling pathway contribute to human tumor progression and are especially prevalent in colorectal cancer. We screened mouse C57MG cells to isolate mRNAs induced by Wnt-1 and identified Stra6, an mRNA known to be up-regulated by retinoic acid. Up-regulation of Stra6 mRNA was also observed in hyperplastic mammary tissue and mammary gland tumors from transgenic mice expressing Wnt-1 and in human tumors that frequently harbor defects in Wnt-1 signaling. Stimulation of C57MG cells with retinoic acid plus Wnt-1 resulted in expression of Stra6 transcript to levels greatly exceeding that observed with either stimulus alone. This synergy could be explained in part by the up-regulation of retinoic acid receptor-gamma that was observed in response to Wnt-1 signaling. Accordingly, treatment of human colorectal cancer cell lines with retinoic acid resulted in the up-regulation of Stra6 mRNA and accumulation of Stra6 protein at the cell membrane. The data support a model in which Wnt-1 signaling synergizes with retinoids to activate retinoic acid receptor-gamma-responsive genes in human cancers.

This study investigated mechanisms controlling the nuclear-cytoplasmic partitioning of annexin II (AnxII). AnxII and its ligand, p11, were localized by immunofluorescence to the cytoplasmic compartment of U1242MG cells, with minimal AnxII or p11 detected within nuclei. Similarly, GFP-AnxII and GFP-p11 chimeras localized to the endogenous proteins. Likewise, GFP-AnxII(1-22) was excluded from nuclei, whereas GFP-AnxII(23-338) and GFP alone were distributed throughout the cells. Immunoprecipitation and biochemical studies showed that GFP-AnxII did not form heteromeric complexes with endogenous p11 and AnxII. Thus, the AnxII N-tail is necessary and sufficient to cause nuclear exclusion of the GFP fusion protein but this does not involve p11 binding. A nuclear export signal consensus sequence was found in the AnxII 3-12 region. The consensus mutant GFP-AnxII(L10A/L12A) confirmed that these residues are necessary for nuclear exclusion. The nuclear exclusion of GFP-AnxII(1-22) was temperature-dependent and reversible, and the nuclear export inhibitor leptomycin B (LmB) caused GFP-AnxII or overexpressed AnxII monomer to accumulate in nuclei. Therefore, AnxII monomer can enter the nucleus and is actively exported. However, LmB had little effect on the localization of AnxII/p11 complex in U1242MG cells, indicating that the complex is sequestered in the cytoplasm. By contrast, LmB treatment of v-src-transformed fibroblasts caused endogenous AnxII to accumulate in nuclei. The LmB-induced nuclear accumulation of AnxII was accelerated by pervanadate and inhibited by genistein, suggesting that phosphorylation promotes nuclear entry of AnxII. Thus, nuclear exclusion of AnxII results from nuclear export of the monomer and sequestration of AnxII/p11 complex, and may be modulated by phosphorylation.

Growth of the human rhabdomyosarcoma A673 cell line in nude mice is substantially reduced but not completely suppressed after systemic administration of the antihuman vascular endothelial growth factor (VEGF) monoclonal antibody (Mab) A.4.6.1. Potentially, such escape might be attributable to incomplete local penetration of the antibody because of a diffusion barrier associated with tumor growth. Alternatively, it might reflect a compensatory up-regulation of murine VEGF, produced by the stroma of the host, or of other angiogenic factor genes. To test these potential mechanisms, systemic administration of Mab A.4.6.1, was performed in conjunction with intratumoral administration of an irrelevant antibody, an antihuman VEGF Fab or mFlt(1-3)-IgG that neutralizes both human and murine VEGF. Tumor growth in the systemic-plus-intratumoral anti-VEGF group was not different from that in the systemic anti-VEGF-plus-intratumoral-control antibody group, arguing against the possibility that bioavailability is the factor that limits the antitumor efficacy of Mab A.4.6.1. However, intratumoral mFlt(l-3)-IgG administration dramatically enhanced the activity of systemic anti-VEGF Mab and resulted in complete suppression of tumor growth, which indicated that host VEGF significantly contributes to tumor growth. Systemic administration of mFlt(1-3)-IgG alone replicated these findings. Histological analysis of residual tumor tissues revealed an almost complete absence of host-derived vasculature and massive tumor-cell necrosis in the mFlt(1-3)-IgG groups. Such extensive necrotic areas were not present in the other groups. Real-time reverse transcription-PCR analysis of total RNA derived from tumor tissues indicated strong up-regulation of both human and murine VEGF as well as other genes regulated by hypoxia. Our findings emphasize the need to completely block VEGF for maximal inhibition of tumor growth.

Annexins are Ca2+-dependent phospholipid-binding proteins with anti-inflammatory properties that are present on the surfaces of, and released from, certain cell types, such as leukocytes and secretory epithelia. The present study investigated the possibility that annexins may bind directly to bacterial endotoxin, inhibiting its interactions with cellular receptors or accessory binding proteins. An enzyme-linked immunoassay demonstrated calcium-dependent binding of low nanomolar concentrations of annexin-I and annexin-II p36/p11 heterotetramer to lipid A. In contrast, little or no annexin binding to lipopolysaccharide (LPS) was detected under similar conditions. LPS-binding protein binding to lipid A was blocked by annexin-I, and the annexins inhibited nitrite generation in RAW 264.7 cells induced by lipid A but not that induced by LPS. The data suggest that direct binding of annexins to lipid A may represent a mechanism for suppressing cellular and systemic responses to endotoxin.

OBJECT The aim of this study was to evaluate the causes of treatment failure in patients with arteriovenous malformations (AVMs) who underwent radiosurgery, which is increasingly used as a treatment method for selected, surgically high-risk AVMs. Unfortunately, radiosurgical treatment fails in a small but significant percentage of patients. In the time period covered in this study, 72 patients attained angiographically confirmed cures after radiosurgery and 36 were retreated after the initial radiosurgical treatment failed. METHODS Using a computerized image fusion technique, the initial radiosurgical dosimetry plan was superimposed on the remaining AVM nidus at the time of retreatment. Twenty-six percent of the retreated cases were found to have AVM niduses outside the original treatment isodose line, which means that targeting error was a factor. The retreated group was also statistically compared with the cured group. CONCLUSIONS Multivariate analysis revealed that the following factors were statistically significant predictors of treatment failure: increasing AVM size, decreasing treatment dose, and increasing Spetzler-Martin grade.

Stereotactic radiosurgery effectively obliterates many arteriovenous malformations (AVMs). Hemodynamic changes in AVMs after radiosurgery have been illustrated using magnetic resonance imaging and angiography, but there have been no detailed reports describing the underlying histopathological changes. This study examines AVMs at various times after gamma knife radiosurgery (GKRS) and describes the histopathological changes that lead to vessel occlusion. The authors examined nine AVM specimens obtained 10 months to more than 5 years after GKRS, by using routine histopathological stains as well as immunohistochemical techniques to detect smooth-muscle actin, factor VIII, and type IV collagen. Blood vessels within the AVMs showed progressive changes leading to narrowing or obliteration of the lumen. The earliest changes after gamma knife irradiation appear to be damage to endothelial cells, followed by progressive thickening of the intimal layer caused by proliferation of smooth-muscle cells that elaborate an extracellular matrix that includes type IV collagen. Finally, cellular degeneration and hyaline transformation occur. For statistical correlation analyses, the specimens were graded according to the degree of histopathological change and the relative number of vessels showing such changes. Both of these parameters were significantly correlated with time after GKRS and with AVM size reduction shown on follow-up imaging studies. Gamma knife radiosurgery of AVMs causes endothelial damage, which induces the proliferation of smooth-muscle cells and the elaboration of extracellular collagen by these cells, which leads to progressive stenosis and obliteration of the AVM nidus. This series of pathological changes in AVMs after GKRS is essentially similar to the response-to-injury model of atherosclerosis.

Early diagnosis is essential for the effective management of primary angiitis of the CNS (PACNS), but the presence of cerebrovascular amyloid angiopathy (CAA) may complicate the pathologic diagnosis since nonvasculitic inflammatory reactions can accompany CAA. We report two patients with PACNS associated with CAA in whom the progression of symptoms ceased during combined corticosteroid/cyclophosphamide therapy. One patient had prominent eosinophilic vasculitis and eosinophilic CSF pleocytosis. Based on review of reported cases, features supporting the diagnosis of symptomatic vasculitis in these patients include subacute progression of mental status changes and multifocal deficits, elevated ESR and CSF protein, and multifocal nonhemorrhagic lesions on imaging studies. We conclude that combined disease (PACNS/CAA) is similar to PACNS and probably occurs more frequently than expected by coincidence. The presence of CAA should not alter the treatment strategy in patients presenting with symptoms and laboratory studies consistent with PACNS.