Stromal Signature Research Articles

Integrating Tumor and Stromal Gene Expression Signatures With Clinical Indices for Survival Stratification of Early-Stage Non-Small Cell Lung Cancer.

Accurate survival stratification in early-stage non-small cell lung cancer (NSCLC) could inform the use of adjuvant therapy. We developed a clinically implementable mortality risk score incorporating distinct tumor microenvironmental gene expression signatures and clinical variables. Gene expression profiles from 1106 nonsquamous NSCLCs were used for generation and internal validation of a nine-gene molecular prognostic index (MPI). A quantitative polymerase chain reaction (qPCR) assay was developed and validated on an independent cohort of formalin-fixed paraffin-embedded (FFPE) tissues (n = 98). A prognostic score using clinical variables was generated using Surveillance, Epidemiology, and End Results data and combined with the MPI. All statistical tests for survival were two-sided. The MPI stratified stage I patients into prognostic categories in three microarray and one FFPE qPCR validation cohorts (HR = 2.99, 95% CI = 1.55 to 5.76, P < .001 in stage IA patients of the largest microarray validation cohort; HR = 3.95, 95% CI = 1.24 to 12.64, P = .01 in stage IA of the qPCR cohort). Prognostic genes were expressed in distinct tumor cell subpopulations, and genes implicated in proliferation and stem cells portended poor outcomes, while genes involved in normal lung differentiation and immune infiltration were associated with superior survival. Integrating the MPI with clinical variables conferred greatest prognostic power (HR = 3.43, 95% CI = 2.18 to 5.39, P < .001 in stage I patients of the largest microarray cohort; HR = 3.99, 95% CI = 1.67 to 9.56, P < .001 in stage I patients of the qPCR cohort). Finally, the MPI was prognostic irrespective of somatic alterations in EGFR, KRAS, TP53, and ALK. The MPI incorporates genes expressed in the tumor and its microenvironment and can be implemented clinically using qPCR assays on FFPE tissues. A composite model integrating the MPI with clinical variables provides the most accurate risk stratification.

Abstract 4760: Stromal contribution to the colorectal cancer transcriptome

Abstract Recent studies have identified a stem/serrated/mesenchymal (SSM) molecular subtype of colorectal cancer (CRC) that is associated with poor prognosis. We noted that genes upregulated in this subtype are also prominently expressed by stromal cells. This led us to hypothesize that the SSM transcripts could derive from the tumor microenvironment, rather than being intrinsic to cancer cells. To test this hypothesis, we analyzed microarray and RNAseq expression data from patient-derived xenografts (PDXs) of CRC, where human cancer cells are supported by murine stroma. Species-specific expression analysis revealed that mRNA levels of SSM genes are mostly due to stromal expression. Considering only genes exclusively expressed by stromal cells in PDXs, We then built three expression signatures specifically reporting the abundance of cancer-associated fibroblasts (CAFs), leucocytes or endothelial cells. In human CRC samples, all stromal signatures were strongly associated with the SSM subtype. A high CAF signature was associated with poor prognosis in untreated CRC patients, while in rectal cancer high stromal signatures jointly predicted radioresistance. These data show that the distinctive transcriptional and clinical features of the SSM subtype can be ascribed to its particularly abundant stroma. Citation Format: Claudio Isella, Andrea Terrasi, Sara E. Bellomo, Consalvo Petti, Andrea Muratore, Alfredo Mellano, Mark De Ridder, Paola Cassoni, Guy Storme, Andrea Bertotti, Enzo Medico. Stromal contribution to the colorectal cancer transcriptome. [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 4760. doi:10.1158/1538-7445.AM2015-4760

Abstract 1924: Genes associated with histopathologic features of triple negative breast tumors predict molecular subtypes

Abstract Distinct subtypes of triple negative (TN) breast cancer have currently been identified by tumor expression profiling and are thought to be influenced by the tumor microenvironment. However, little is known about the relationship between distinguishing histopathologic features of TN tumors, which reflect aspects of both tumor behavior and tumor microenvironment, and molecular TN subtypes. We analyzed whole genome expression profiles of 593 TN tumors from the Triple Negative Breast Cancer Consortium (TNBCC) using the Illumina Whole Genome DASL array. Histopathologic review of mitotic index, grade, trabecular pattern, growth pattern, involution, presence of ductal carcinoma in situ, and degree of lymphoid infiltrate, necrosis, fibrosis, and lobulitis was completed for 310 of these tumors. Clusters were identified both agnostically and using genes associated with these histopathologic features, and subtypes were evaluated for enriched pathways and associations with histopathologic features. Agnostic clustering identified four stable clusters which represented a basal-like (BL) signature, a luminal signature with overexpression of androgen receptor and targets, an immune signature, and a stromal (STR) signature. Overall, the TN tumors tended to have little or no necrosis (91%), high grade (88%), a high mitotic index (80%), fibrosis (77%), an epicentric growth pattern (62%), incomplete lobular involution (55%), trabecular structures (37%), lymphocytic infiltration (25%), adjacent ductal carcinoma in situ (24%), and variable amounts of lobulitis. Using a combined total of 2,776 unique probes individually associated with the ten histopathologic features measured, we identified six distinct TN subtypes, with an additional BL and STR signature compared to the four agnostic subtypes. Our findings suggest that histopathologic features that reflect heterogeneity in tumor cell structure and behavior as well as the tumor microenvironment, particularly the presence of lymphocytic infiltrate, are critical for TN subtype identification. Further work is needed to validate these signatures and to better understand the immunologic pathways associated with each subtype. Citation Format: Kristen Purrington, Daniel Visscher, Drakoulis Yannoukakos, Jane Carpenter, Heli Nevanlinna, Arto Mannerma, Xianshu Wang, Graham Giles, Wei Zheng, Angela Cox, Hiltrud Brauch, Ute Hamann, Diana Eccles, Celine M. Vachon, Fergus J. Couch. Genes associated with histopathologic features of triple negative breast tumors predict molecular subtypes. [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 1924. doi:10.1158/1538-7445.AM2015-1924

Abstract 2885: Differential expression of long non-coding RNAs in ovarian cancer-associated fibroblasts versus normal ovarian fibroblasts

Abstract Ovarian cancer is the most lethal gynecological malignancy in women. Expression profiling of the high-grade serous subtype has revealed that patients exhibiting a “stromal expression signature” demonstrate the poorest prognosis1. However, genetic aberrations in ovarian cancer-associated fibroblasts (CAFs) are extremely rare2, raising the possibility that alternative mechanisms that regulate gene expression, such as long non-coding RNAs (lncRNAs), are present in CAFs. LncRNAs are polyadenylated RNA transcripts that do not encode for protein, but have been shown to correlate with cancer progression and outcome. Therefore, our aim was to investigate the presence of differentially expressed lncRNAs in ovarian CAFs versus normal ovarian fibroblasts. CAFs were laser-capture microdissected from 51 advanced-stage high-grade serous ovarian cancers and 10 normal ovaries removed from women for non-cancer reasons. RNA was extracted from the microdissected samples and expression analyzed using Affymetrix U133 Plus 2.0 Arrays. Probes previously identified as lncRNAs were used in this analysis3. Samples were normalized and background corrected using the robust multi-arrray average (RMA) method and expression values were log2 transformed. Differentially expressed probes between cancer versus normal samples were calculated using linear models for microarray data (limma) package from Bioconductor and a moderated t-statistic used to assess significance. Based on a significance cutoff of fold-change &amp;gt; 2 and a p-value &amp;lt; 0.05, 54 lncRNAs were identified as differentially expressed in cancer versus normal fibroblasts. These included lncRNAs previously implicated in ovarian cancer such as XIST and H19, the p53-regulated lncRNA TUG1, as well as several lncRNAs that are known to play a role in other cancer types such as NEAT1, GAS5 and CASC2. In addition, 16 lncRNAs were differentially expressed between patients with short (&amp;lt;15 months) versus patients with long (&amp;gt;15 months) overall survival. These included MALAT1, MEG3, TUG1, XIST and CRNDE. In summary, we have identified lncRNAs that are differentially expressed in ovarian CAFs compared to normal ovarian fibroblasts, as well as lncRNAs that are differentially expressed based on survival. Given that CAFs are known to promote tumor progression and metastasis, these lncRNAs present suitable candidates for their role in the tumor-promoting function of CAFs.

Abstract A82: Virtual microdissection reveals tumor specific heterogeneity in pancreatic cancer

Abstract Using matrix factorization, we have removed confounding normal tissue gene expression from profiles of primary and metastatic tumors, facilitating the study of underlying tumor biology without the need for laser capture microdissection. Understanding molecular mechanisms of metastasis in pancreatic cancer has the potential to yield rationally designed therapies. Previous work has used gene expression arrays and next generation sequencing to look for potential targets, focusing on identifying differences between metastatic lesions and primary tumors. However, such analyses are hampered by the low cellularity of malignant epithelium in patient samples. Since profiles of primary and metastatic tumors often contain contributions from adjacent normal tissues from the tumor site, comparison of these samples are confounded by the differences between normal pancreas and normal distant organs. We seek to avoid this pitfall by leveraging the blind source separation technique, nonnegative matrix factorization (NMF)[1], to the analysis of gene expression data. Microarray data were obtained from matched primary (n=8) and metastatic tumors (n=31) as well as adjacent normal tissues (n=85) for both local and distant sites of patients who died of metastatic pancreatic cancer[2]. Pathological analysis showed that tumor cellularity varied with a mean of 48% and a standard deviation of 31% across all sites. We applied NMF to our cohort, thus computationally microdissecting tumors into the source tissues composing our samples. 200 iterations of 5-fold resampling were performed to achieve stable NMF results. Genes with expression ranked in the top 50 of any factor together were recorded in a consensus matrix. This consensus matrix was then used as the basis of a hierarchical clustering as to yield k gene clusters. These k gene-clusters were used to seed a supervised NMF using the MATLAB NMF multiplicative update solver until completion. Using an unbiased approach, we identified distinct molecular signatures associated with adjacent normal and tumor tissue in primary and metastatic pancreatic cancer samples. Specifically, we identified confounding liver, lung, lymph, and muscle tissue gene expression from our metastatic samples as well as both endocrine and exocrine pancreas gene expression from our primary tumors. Furthermore, we estimated the relative composition of each of our samples in terms of a weighted sum of these tissue-specific signatures. Both primary tumors and metastases appeared to be admixtures of pancreatic tumor epithelial expression and adjacent normal tissue. For example, in liver tissues histological assessment of tumor content correlated well with NMF predictions of tumor content, (n=39, R2=0.72, p&amp;lt;0.01). Prior to NMF, unsupervised clustering of all data caused samples to cluster by site of harvest. After digital subtraction of confounding tissue factors identified by NMF, samples grouped by patient of origin, suggesting intrinsic similarity among tumor sites within a patient. We have demonstrated a method to virtually microdissect tissues, thereby identifying tumor-specific gene expression data in pancreatic cancer. By applying a fresh computational approach to a large cohort of data, we can generate new insight into the complex nature of low-cellularity tumors such as pancreatic cancer, and facilitate the study of inter- and intra-patient heterogeneity. This approach may be further leveraged to study the role of tumor and stroma signatures in pancreatic cancer. 1. Brunet, J.P., et al., Metagenes and molecular pattern discovery using matrix factorization. Proceedings of the National Academy of Sciences, 2004. 101(12): p. 4164-4169. 2. Stratford, J.K., et al., A six-gene signature predicts survival of patients with localized pancreatic ductal adenocarcinoma. PLoS medicine, 2010. 7(7): p. e1000307. Citation Format: Richard A. Moffitt, Keith A. Volmar, Judy M. Anderson, Michael A. Hollingsworth, Jen Jen Yeh. Virtual microdissection reveals tumor specific heterogeneity in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A82.

Upregulation of Periostin and Reactive Stroma Is Associated with Primary Chemoresistance and Predicts Clinical Outcomes in Epithelial Ovarian Cancer.

Up to one third of ovarian cancer patients are intrinsically resistant to platinum-based treatment. However, predictive and therapeutic strategies are lacking due to a poor understanding of the underlying molecular mechanisms. This study aimed to identify key molecular characteristics that are associated with primary chemoresistance in epithelial ovarian cancers. Gene expression profiling was performed on a discovery set of 85 ovarian tumors with clinically well-defined response to chemotherapies as well as on an independent validation dataset containing 138 ovarian patients from the chemotreatment arm of the ICON7 trial. We identified a distinct "reactive stroma" gene signature that is specifically associated with primary chemoresistant tumors and was further upregulated in posttreatment recurrent tumors. Immunohistochemistry (IHC) and RNA in situ hybridization (RNA ISH) analyses on three of the highest-ranked signature genes (POSTN, LOX, and FAP) confirmed that modulation of the reactive stroma signature genes within the peritumoral stromal compartments was specifically associated with the clinical chemoresistance. Consistent with these findings, chemosensitive ovarian cells grown in the presence of recombinant POSTN promoted resistance to carboplatin and paclitaxel treatment in vitro. Finally, we validated the reactive stroma signature in an independent dataset and demonstrated that a high POSTN expression level predicts shorter progression-free survival following first-line chemotherapy. Our findings highlight the important interplay between cancer and the tumor microenvironment in ovarian cancer biology and treatment. The identified reactive stromal components in this study provide a molecular basis to the further development of novel diagnostic and therapeutic strategies for overcoming chemoresistance in ovarian cancer.

Tumor infiltration by Tbet+ effector T cells and CD20+ B cells is associated with survival in gastric cancer patients

Tumor-infiltrating T and B lymphocytes could have the potential to affect cancer prognosis. The objective of this study was to investigate the prognostic significance of tumor infiltration by CD8 and CD4 T cells, and B lymphocytes in patients with localized gastric cancer. In a retrospective cohort of 82 patients with localized gastric cancer and treated by surgery we quantitatively assessed by immunohistochemistry on surgical specimen, immune infiltrates of IL-17+, CD8+, Foxp3+, Tbet+ T cells and CD20+ B cells both in the tumor core and at the invasive margin via immunohistochemical analyses of surgical specimens. We observed that CD8+ and IL17+ T-cell densities were not significantly associated with gastric cancer prognosis. In contrast, high infiltration of Tbet+ T cells, high numbers of CD20+ B-cell follicles, and low infiltration of Foxp3+ T cells, were associated with better relapse-free survival. Interestingly, treatment with neoadjuvant chemotherapy or histological tumor type (diffuse versus intestinal) did not influence type and density of immune infiltrates or their prognostic value. Immunohistochemical analysis of the gastric cancer stromal microenvironment revealed organized T and B cell aggregates, with strong structural analogies to normal secondary lymphoid organs and which could be considered as tertiary lymphoid structures. Using transcriptomic data from an independent cohort of 365 localized gastric cancer, we confirmed that a coordinated Th1, and B cell stromal gene signature is associated with better outcome. Altogether, these data suggest that tumor infiltration by B and Th1 T cells could affect gastric cancer prognosis and may be used to better define the outcome of patients with localized gastric cancer.

OT-08 * DISTINCT BRAIN LINEAGE AND STROMAL GENE EXPRESSION PATTERNS IN WNT, SHH, GROUP 3 AND GROUP 4 MOLECULAR SUBGROUPS OF PEDIATRIC MEDULLOBLASTOMA

Medulloblastomas are the most common malignant brain tumors in children, comprising distinct molecular subgroups that differ in neuroanatomical predilection, clinical characteristics and overall survival. Here we compare genetic brain lineage and stromal immune profiling in N = 558 pediatric medulloblastomas against N = 426 adult glioblastomas. The medulloblastoma cohort included N = 114 SHH (20%), N = 34 WNT (6%), N = 156 Group 3 (28%), and N = 254 Group 4 (46%). Genetic brain lineage analyses revealed WNT and SHH medulloblastoma to be distinctly astrocytic and astroglial; more similar to adult mesenchymal/classical glioblastoma than other pediatric medulloblastoma. In contrast, Group 4 was distinctly neuronal and oligodendrocytic; more similar to adult proneural/neural glioblastoma than other pediatric medulloblastoma. Interestingly, Group 3 demonstrated no brain lineage enrichment-unlike any other pediatric or adult brain tumor subtype-suggesting a non-CNS etiology for this subgroup known for distinctive large-cell anaplastic histology, aggressive behavior, and uniquely high incidence of metastatic disease. Analysis of stromal immune signatures revealed significant macrophage-specific enrichment in WNT and SHH, but not Group 3 and 4 tumors. These myeloid signatures were characteristic of M2-polarized macrophages, unlike adult glioblastomas, which enriched in both M1 and M2 signatures. While myeloid responses in adult glioblastoma were predictive of poor overall survival, the opposite was true in pediatric medulloblastoma-with improved survival in children with robust immune responses. These data demonstrate that medulloblastoma subtypes display distinct brain lineages and share greater similarities to adult glioblastomas than other pediatric medulloblastomas, further supporting distinct cellular origins for each subtype. Furthermore, innate immune responses were distinctive between subtypes and, in contrast to adult tumors, predictive of good outcomes, suggesting prognostic significance of immune responses differs in children versus adults. Taken together, these findings highlight the distinctiveness of medulloblastoma subtypes with important clinical implications for the design of future targeted molecular therapy and immunotherapy trials in pediatric and adult brain tumor patients.

Diffuse large B-cell lymphoma classification system that associates normal B-cell subset phenotypes with prognosis.

Current diagnostic tests for diffuse large B-cell lymphoma use the updated WHO criteria based on biologic, morphologic, and clinical heterogeneity. We propose a refined classification system based on subset-specific B-cell-associated gene signatures (BAGS) in the normal B-cell hierarchy, hypothesizing that it can provide new biologic insight and diagnostic and prognostic value. We combined fluorescence-activated cell sorting, gene expression profiling, and statistical modeling to generate BAGS for naive, centrocyte, centroblast, memory, and plasmablast B cells from normal human tonsils. The impact of BAGS-assigned subtyping was analyzed using five clinical cohorts (treated with cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP], n = 270; treated with rituximab plus CHOP [R-CHOP], n = 869) gathered across geographic regions, time eras, and sampling methods. The analysis estimated subtype frequencies and drug-specific resistance and included a prognostic meta-analysis of patients treated with first-line R-CHOP therapy. Similar BAGS subtype frequencies were assigned across 1,139 samples from five different cohorts. Among R-CHOP-treated patients, BAGS assignment was significantly associated with overall survival and progression-free survival within the germinal center B-cell-like subclass; the centrocyte subtype had a superior prognosis compared with the centroblast subtype. In agreement with the observed therapeutic outcome, centrocyte subtypes were estimated as being less resistant than the centroblast subtype to doxorubicin and vincristine. The centroblast subtype had a complex genotype, whereas the centrocyte subtype had high TP53 mutation and insertion/deletion frequencies and expressed LMO2, CD58, and stromal-1-signature and major histocompatibility complex class II-signature genes, which are known to have a positive impact on prognosis. Further development of a diagnostic platform using BAGS-assigned subtypes may allow pathogenetic studies to improve disease management.

Stromal contribution to the colorectal cancer transcriptome.

Recent studies identified a poor-prognosis stem/serrated/mesenchymal (SSM) transcriptional subtype of colorectal cancer (CRC). We noted that genes upregulated in this subtype are also prominently expressed by stromal cells, suggesting that SSM transcripts could derive from stromal rather than epithelial cancer cells. To test this hypothesis, we analyzed CRC expression data from patient-derived xenografts, where mouse stroma supports human cancer cells. Species-specific expression analysis showed that the mRNA levels of SSM genes were mostly due to stromal expression. Transcriptional signatures built to specifically report the abundance of cancer-associated fibroblasts (CAFs), leukocytes or endothelial cells all had significantly higher expression in human CRC samples of the SSM subtype. High expression of the CAF signature was associated with poor prognosis in untreated CRC, and joint high expression of the stromal signatures predicted resistance to radiotherapy in rectal cancer. These data show that the distinctive transcriptional and clinical features of the SSM subtype can be ascribed to its particularly abundant stromal component.

Prognostic stromal gene signatures in breast cancer.

IntroductionGlobal gene expression analysis of tumor samples has been a valuable tool to subgroup tumors and has the potential to be of prognostic and predictive value. However, tumors are heterogeneous, and homogenates will consist of several different cell types. This study was designed to obtain more refined expression data representing different compartments of the tumor.MethodsFormalin-fixed paraffin-embedded stroma-rich triple-negative breast cancer tumors were laser-microdissected, and RNA was extracted and processed to enable microarray hybridization. Genes enriched in stroma were identified and used to generate signatures by identifying correlating genes in publicly available data sets. The prognostic implications of the signature were analyzed.ResultsComparison of the expression pattern from stromal and cancer cell compartments from three tumors revealed a number of genes that were essentially specifically expressed in the respective compartments. The stroma-specific genes indicated contribution from fibroblasts, endothelial cells, and immune/inflammatory cells. The gene set was expanded by identifying correlating mRNAs using breast cancer mRNA expression data from The Cancer Genome Atlas. By iterative analyses, 16 gene signatures of highly correlating genes were characterized. Based on the gene composition, they seem to represent different cell types. In multivariate Cox proportional hazard models, two immune/inflammatory signatures had opposing hazard ratios for breast cancer recurrence also after adjusting for clinicopathological variables and molecular subgroup. The signature associated with poor prognosis consisted mainly of C1Q genes and the one associated with good prognosis contained HLA genes. This association with prognosis was seen for other cancers as well as in other breast cancer data sets.ConclusionsOur data indicate that the molecular composition of the immune response in a tumor may be a powerful predictor of cancer prognosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0530-2) contains supplementary material, which is available to authorized users.

Clinical Implications of the New Molecular Knowledge for Treatment of Diffuse Large B-Cell Lymphoma

For more than 35 years, the standard induction therapy for patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL) consists of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). Until recently, efforts to improve upon this regimen were empiric and without success, and consisted of adding additional cytotoxic drugs, intensifying doses and schedules of drugs, and consolidating initial responses with high dose therapy and autologous stem cell support. The addition of rituximab to standard CHOP therapy (RCHOP) more than a decade ago improved progression-free and overall survival, and now the majority of patients presenting with DLBCL are cured with induction therapy. Biological insights from gene expression profile (GEP) studies indicate substantial heterogeneity of DLBCL, which is largely responsible for treatment failure. We are now at the cusp of being able to utilize rational targeted therapies in biologically-defined subgroups of patients of DLBCL. In addition to cell-of-origin defined subgroups, GEP has defined stromal signatures and pathway-specific signatures in DLBCL which also predict outcome and potentially define a group of patients for a biologically-targeted therapy approach. At present, clinical development strategies include combining novel agents with standard RCHOP treatment and/or evaluating novel agents as maintenance or consolidation after RCHOP treatment. Two recent failures illustrate the challenges inherent in these approaches: RCHOP + bevacizumab (targeting angiogenesis) and R-CHOP followed by enzastaurin (targeting protein kinase C-b). Ongoing randomized trials are evaluating R-CHOP + bortezomib (proteasome inhibitor targeting NFkB), R-CHOP + ibrutinib (Bruton’s tyrosine kinase inhibitor targeting B-cell receptor signaling pathway), and R-CHOP + lenalidomide (immunomodulator with pleotropic effects) in specific GEP (or immunohistochemistry surrogates for GEP)-defined groups of patients. Finally, the majority of treatment failures in DLBCL appear to occur in patients with overexpression of c-MYC oncogene (often in combination with BCL-2 protein), which represents up to one third of patients when defined using immunohistochemistry. This unique subgroup occurs across cell-of-origin designations, and represents a substantial opportunity to improve outcome in DLBCL with the addition of novel therapeutics. This talk will review the considerable challenges in translating the exciting biology defined by GEP into the clinic for patients with DLBCL, provide an update on current and future trial designs, and conclude with a forward looking time to when GEP, or an accurate surrogate, is a component of the standard diagnostic algorithm to define optimal therapeutic approach to DLBCL. Disclosures Off Label Use: Novel agents for DLBCL.

Abstract B19: RB function as a central component of osteosarcoma behavior: A comparative assessment in dogs and humans

Abstract Introduction: There remain major gaps in our understanding of the pathogenesis of osteosarcoma (OS). This diagnosis represents a group of highly heterogeneous tumors, and an unresolved problem in their management is the lack of robust prognostic and predictive factors. Neither topography nor morphology, nor other conventional laboratory and ancillary tests can distinguish between highly aggressive and more indolent types of OS. Spontaneous OS is commonly diagnosed in dogs, and we showed recently that human and canine OS achieve similar phenotypes by altering convergent molecular pathways. Specifically canine and human OS can be stratified into two prognostically significant subgroups with inversely correlated expression of two gene clusters. This pattern was revealed only by the use of canine samples that also eliminated stromal signatures, as it was masked in studies that used human tumors or that employed different analyses. These two prognostic groups have significantly different behavior defined by survival, as well as by their ability to engraft and metastasize in mice. Equally important, there is exquisite overlap with a predictive signature called CINSARC (Complexity Index in SARComa) of soft tissue sarcomas, suggesting that OS can be used as proof of concept to support the canine model more generally for studies of sarcoma pathogenesis and therapy. Experimental Procedures: We used bioinformatics and genetic complementation, respectively, to identify and validate driver events that are responsible for the biological behavior of canine and human OS. The MEME Suite and Ingenuity Pathway Analysis were used for de novo motif discovery and to identify transcription factors with significant target enrichment in the prognostic signature. Immunoblotting, dual luciferase reporter assays, mutagenesis assays, and qRT-PCR were used to confirmed differential expression and activity of RB protein in cell lines derived from aggressive and indolent OS. Co-immunoprecipitation and ChIP were used to characterize complexes associated with endogenous or ectopic RB. Gene expression microarrays and bioinformatics were used to assess changes in genome wide gene expression after restoration of RB in aggressive OS. New Unpublished Data: The promoter sequences of all of the genes in the prognostic signature contained variants of RB-E2F-DP-1 binding sites. The gene signature also was significantly enriched for targets regulated by the RB and E2F families. RB expression was not the sole determinant of RB function, but genetic restoration of functional RB reversed the phenotype of each aggressive cell line analyzed to one resembling indolent tumors or normal osteoblasts, and it inevitably led to cell death. In contrast, indolent tumors that retained endogenous RB function were highly tolerant of RB addition, showing no (or minimal) phenotypic changes upon expression of ectopic RB. Somewhat surprisingly, restoration of RB also corrected other gene defects associated with the aggressive OS phenotype, such as activity of TP53 pathway. Conclusions: Taken together, our results suggest that RB inactivation is not necessary for the development of OS, but the integrity of the RB pathway is an important factor that contributes to behavior, clinical progression, and outcome for dogs and humans with OS. Our ongoing work seeks to develop pharmacological agents that mimic RB-dependent gene expression and chromatin organization that can be used to improve outcomes for patients with the most aggressive forms of OS. Citation Format: Milcah C. Scott, Aaron L. Sarver, Frances Phan, Rachit Gupta, Venugopal Thayanithy, Subbaya Subramanian, Jaime F. Modiano. RB function as a central component of osteosarcoma behavior: A comparative assessment in dogs and humans. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B19.

Functional Relationship between Tumor-Associated Macrophages and Macrophage Colony-Stimulating Factor as Contributors to Cancer Progression.

The current review article describes the functional relationship between tumor-associated macrophages (TAM) as key cellular contributors to cancer malignancy on the one hand and macrophage-colony-stimulating factor (M-CSF or CSF-1) as an important molecular contributor on the other. We recapitulate the available data on expression of M-CSF and the M-CSF receptor (M-CSFR) in human tumor tissue as constituents of a stromal macrophage signature and on the limits of the predictive and prognostic value of plasma M-CSF levels. After providing an update on current insights into the nature of TAM heterogeneity at the level of M1/M2 phenotype and TAM subsets, we give an overview of experimental evidence, based on genetic, antibody-mediated, and pharmacological disruption of M-CSF/M-CSFR signaling, for the extent to which M-CSFR signaling can not only determine the TAM quantity, but can also contribute to shaping the phenotype and heterogeneity of TAM and other related tumor-infiltrating myeloid cells (TIM). Finally, we review the accumulating information on the – sometimes conflicting – effects blocking M-CSFR signaling may have on various aspects of cancer progression such as tumor growth, invasion, angiogenesis, metastasis, and resistance to therapy and we thereby discuss in how far these different effects actually reflect a contribution of TAM.

Calcium-dependent FAK/CREB/TNNC1 signalling mediates the effect of stromal MFAP5 on ovarian cancer metastatic potential.

Ovarian cancer is the most lethal gynecologic malignancy in the United States, and advanced serous ovarian adenocarcinoma is responsible for most ovarian cancer deaths. However, the stroma-derived molecular determinants that modulate patient survival have yet to be characterized. Here we identify a stromal gene signature for advanced high-grade serous ovarian cancer using microdissected stromal ovarian tumor samples and find that stromal microfibrillar-associated protein 5 (MFAP5) is a prognostic marker for poor survival. Further functional studies reveal that FAK/CREB/TNNC1 signaling pathways mediate the effect of MFAP5 on ovarian cancer cell motility and invasion potential. Targeting stromal MFAP5 using MFAP5 specific siRNA encapsulated in chitosan nanoparticles significantly decreases ovarian tumor growth and metastasis in vivo, suggesting that it may be a new modality of ovarian cancer treatment.

Abstract 1178: Gene and protein expression profiling identifies molecular signature of resistance to anti-VEGF therapy in ovarian cancer

Abstract Purpose: Angiogenesis plays an important role in ovarian cancer progression, and the addition of anti-angiogenic agents targeting the vascular endothelial growth factor (VEGF) signaling pathways to conventional chemotherapy improves progression-free survival in relapsed, platinum-resistant ovarian cancer when bevacizumab is given as concurrent or maintenance therapy. Unfortunately, the long-term clinical benefits of anti-VEGF therapy have been consistently limited by the rapid development of tumor resistance. Understanding of tumor's functional adaptations to anti-VEGF therapy and identifying early indicators of resistance are crucial for better exploiting anti-angiogenic therapies. The objective of this study was to perform gene and protein expression profiling of ovarian cancer to identify tumor and stroma signature of anti-VEGF treatment-resistance using preclinical resistance models. Methods: We developed phenotypic resistance xenograft models of anti-VEGF therapy that mimic the clinical feature of resistance. SKOV3 xenografted mice were treated with sorafenib or bevacizumab over two months until there were phenotypic differences between tumors that remained responsive to the treatment and that regained growth after initial response. We conducted Illumina next generation sequencing to compare the gene expression in resistant and sensitive tumors. Sequencing analysis was performed using Perkin Elmer's Genesifter software and alignment to the H. sapiens and M. musculus reference genomes. Further, Reverse phase protein array (RPPA) analysis was done to identify differentially expressed proteins between resistant and sensitive tumors. Results: Our sequencing analysis revealed significant differences in expression of various genes between sensitive and resistant tumors. Based on their role in angiogenesis and angiogenesis resistance, we chose a set of genes for follow-up studies. In sorafenib treatment group amphiregulin and heparanase expression was ≥4 fold higher (P ≤ 0.05) in resistant group compared to sensitive group. In bevacizumab resistant group prostaglandin E receptor 1 and keratin 24 ≥3 fold higher expression ((P ≤ 0.05) was observed. RPPA analysis indicated that proteins such as ER alpha, VEGFR-2, elF4G, E-cadherin, mTOR and beta-catenin expressed ≥ 2.8 fold (P ≤ 0.0001) higher in sorafenib resistant tumors compared to sensitive tumors. Neurofibromin 2 (NF2) protein is expressed 2 fold (P ≤ 0.0001) higher in bevacizumab resistant tumors compared to sensitive tumors. Conclusion: Using gene and protein profiling, we identified important tumor and stroma biomarkers of resistance to anti-VEGF therapy in ovarian cancer that may provide insight into ovarian tumor's adaption principles to VEGF inhibition. Citation Format: Bharat Kumar Devapatla, Pharavee Jaiprasart, Sukyung Woo. Gene and protein expression profiling identifies molecular signature of resistance to anti-VEGF therapy in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1178. doi:10.1158/1538-7445.AM2014-1178

A monotonic and prognostic genomic signature from fibroblasts for colorectal cancer initiation, progression, and metastasis.

The differential gene expression patterns between normal colonic fibroblasts (NCF), carcinoma-associated fibroblasts from primary tumors (CAF-PT), and CAFs from hepatic metastasis (CAF-LM) are hypothesized to be useful for predicting relapse in primary tumors. A transcriptomic profile of NCF (n = 9), CAF-PT (n = 14), and CAF-LM (n = 11) was derived. Prediction Analysis of Microarrays (PAM) was used to obtain molecular details for each fibroblast class, and differentially expressed transcripts were used to classify patients according to recurrence status. A number of transcripts (n = 277) were common to all three types of fibroblasts and whose expression level was sequentially deregulated according to the transition: NCF→CAF-PT→CAF-LM. Importantly, the gene signature was able to accurately classify patients with primary tumors according to their prognosis. This capacity was exploited to obtain a refined 19-gene classifier that predicted recurrence with high accuracy in two independent datasets of patients with colorectal cancer and correlates with fibroblast migratory potential. The prognostic power of this genomic signature is strong evidence of the link between the tumor-stroma microenvironment and cancer progression. Furthermore, the 19-gene classifier was able to identify low-risk patients very accurately, which is of particular importance for stage II patients, who would benefit from the omission of chemotherapy, especially T4N0 patients, who are clinically classified as being at high risk. A defined stromal gene expression signature predicts relapse in patients with colorectal cancer.

Epithelial and stromal microRNA signatures of columnar cell hyperplasia linking Let-7c to precancerous and cancerous breast cancer cell proliferation.

Columnar cell hyperplasia (CCH) is the earliest histologically identifiable breast lesion linked to cancer progression and is characterized by increased proliferation, decreased apoptosis and elevated oestrogen receptor α (ERα) expression. The mechanisms underlying the initiation of these lesions have not been clarified but might involve early and fundamental changes in cancer progression. MiRNAs are key regulators of several biological processes, acting by influencing the post-transcriptional regulation of numerous targets, thus making miRNAs potential candidates in cancer initiation. Here we have defined novel epithelial as well as stromal miRNA signatures from columnar cell hyperplasia lesions compared to normal terminal duct lobular units by using microdissection and miRNA microarrays. Let-7c were among the identified downregulated epithelial miRNAs and its functions were delineated in unique CCH derived cells and breast cancer cell line MCF-7 suggesting anti-proliferative traits potentially due to effects on Myb and ERα. MiR-132 was upregulated in the stroma surrounding CCH compared to stoma surrounding normal terminal duct lobular units (TDLUs), and overexpression of miR-132 in immortalized fibroblasts and in fibroblasts co-cultured with epithelial CCH cells caused substantial expression changes of genes involved in metabolism, DNA damage and cell motility. The miRNA signatures identified in CCH indicate early changes in the epithelial and stromal compartment of CCH and could represent early key alterations in breast cancer progression that potentially could be targeted in novel prevention or treatment schedules.

Stromal fibroblast-derived miR-409 promotes epithelial-to-mesenchymal transition and prostate tumorigenesis.

Tumor-stromal interaction is a dynamic process that promotes tumor growth and metastasis via cell-cell interaction and extracellular vesicles. Recent studies demonstrate that stromal fibroblast-derived molecular signatures can be used to predict disease progression and drug resistance. To identify the epigenetic role of stromal noncoding RNAs in tumor-stromal interactions in the tumor microenvironment, we performed microRNA profiling of patient cancer-associated prostate stromal fibroblasts isolated by laser capture dissection microscopy and in bone-associated stromal models. We found specific upregulation of miR-409-3p and miR-409-5p located within the embryonically and developmentally regulated DLK1-DIO3 (delta-like 1 homolog-deiodinase, iodothyronine 3) cluster on human chromosome 14. The findings in cell lines were further validated in human prostate cancer tissues. Strikingly, ectopic expression of miR-409 in normal prostate fibroblasts conferred a cancer-associated stroma-like phenotype and led to the release of miR-409 via extracellular vesicles to promote tumor induction and epithelial-to-mesenchymal transition in vitro and in vivo. miR-409 promoted tumorigenesis through repression of tumor suppressor genes such as Ras suppressor 1 and stromal antigen 2. Thus, stromal fibroblasts derived miR-409-induced tumorigenesis, epithelial-to-mesenchymal transition and stemness of the epithelial cancer cells in vivo. Therefore, miR-409 appears to be an attractive therapeutic target to block the vicious cycle of tumor-stromal interactions that plagues prostate cancer patients.

Association of stromal cell gene expression with response to neoadjuvant chemotherapy in locally advanced breast cancer.

11129 Background: There is increasing evidence that breast cancer behavior is a reflection of an interactive signaling between the malignant epithelial compartment and the surrounding microenvironment. A high stromal gene expression, characterizing a reactive stroma, was associated with resistance to neoadjuvant chemotherapy, in indirect analysis. Our aim was to detect a stromal cell signature associated with response to neoadjuvant chemotherapy in locally advanced breast cancer, using a direct approach, consisting of stromal cell selection, through laser microdissection. Methods: Twenty one patients treated at Hospital de Câncer de Barretos were included. Patients were diagnosed with invasive ductal carcinoma, except one (lobular invasive carcinoma); median age was 44.5 years; all presented stage III disease; mean tumor dimension was 6.89 cm. Eleven tumors were luminal HER2 negative; 5 HER2 positive [3 ER(+); 2 ER(-)] and 5 triple negative. All patients were submitted to neoadjuvant chemotherapy (4AC fol...