Venkata Thodima

Neutrophil Chemokines Secreted by Tumor Cells Mount a Lung Antimetastatic Response during Renal Cell Carcinoma Progression

The mechanism by which renal cell carcinoma (RCC) colonizes the lung microenvironment during metastasis remains largely unknown. To investigate this process, we grafted human RCC cells with varying lung metastatic potential in mice. Gene expression profiling of the mouse lung stromal compartment revealed a signature enriched for neutrophil-specific functions that was induced preferentially by poorly metastatic cells. Analysis of the gene expression signatures of tumor cell lines showed an inverse correlation between metastatic activity and the levels of a number of chemokines, including CXCL5 and IL8. Enforced depletion of CXCL5 and IL8 in these cell lines enabled us to establish a functional link between lung neutrophil infiltration, secretion of chemokines by cancer cells and metastatic activity. We further show that human neutrophils display a higher cytotoxic activity against poorly metastatic cells compared with highly metastatic cells. Together, these results support a model in which neutrophils recruited to the lung by tumor-secreted chemokines build an antimetastatic barrier with loss of neutrophil chemokines in tumor cells acting as a critical rate-limiting step during lung metastatic seeding.

Genomic deregulation during metastasis of renal cell carcinoma implements a myofibroblast-like program of gene expression.

Clear cell renal cell carcinoma (RCC) is the most common and invasive adult kidney cancer. The genetic and biological mechanisms that drive metastatic spread of RCC remain largely unknown. We have investigated the molecular signatures and underlying genomic aberrations associated with RCC metastasis, using an approach that combines a human xenograft model; expression profiling of RNA, DNA, and microRNA (miRNA); functional verification; and clinical validation. We show that increased metastatic activity is associated with acquisition of a myofibroblast-like signature in both tumor cell lines and in metastatic tumor biopsies. Our results also show that the mesenchymal trait did not provide an invasive advantage to the metastatic tumor cells. We further show that some of the constituents of the mesenchymal signature, including the expression of the well-characterized myofibroblastic marker S100A4, are functionally relevant. Epigenetic silencing and miRNA-induced expression changes accounted for the change in expression of a significant number of genes, including S100A4, in the myofibroblastic signature; however, DNA copy number variation did not affect the same set of genes. These findings provide evidence that widespread genetic and epigenetic alterations can lead directly to global deregulation of gene expression and contribute to the development or progression of RCC metastasis culminating in a highly malignant myofibroblast-like cell.

Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia

Abstract Array comparative genomic hybridization (aCGH) has yet to be fully leveraged in a prognostic setting in chronic lymphocytic leukemia (CLL). Genomic imbalance was assessed in 288 CLL specimens using a targeted array. Based on 20 aberrations in a hierarchical manner, all 228 treatment-naive specimens were classified into a group with poor outcome (20.6%) exhibiting at least one aberration that was univariately associated with adverse outcome (gain: 2p, 3q, 8q, 17q, loss: 7q, 8p, 11q, 17p, 18p), good outcome (32.5%) showing 13q14 loss without any of the other 10 aberrations (gain: 1p, 7p, 12, 18p, 18q, 19, loss: 4p, 5p, 6q, 7p) or intermediate outcome (remainder). The three groups were significantly separated with respect to time to first treatment and overall survival (p < 0.001), and validation of the stratification scheme was performed in two independent datasets. Gain of 3q and 8q, and 17p loss were determined to be independent unfavorable prognostic biomarkers. TP53, NOTCH1 and SF3B1 mutations correlated with the presence of one poor outcome aCGH marker, at a considerably higher frequency than when only considering poor risk aberrations routinely detected by fluorescence in situ hybridization (FISH). These data support genomic imbalance evaluation in CLL by aCGH to assist in risk stratification.

Characterization of a variant of t(14;18) negative nodal diffuse follicular lymphoma with CD23 expression, 1p36/ TNFRSF14 abnormalities, and STAT6 mutations

A predominantly diffuse growth pattern and CD23 co-expression are uncommon findings in nodal follicular lymphoma and can create diagnostic challenges. A single case series in 2009 (Katzenberger et al) proposed a unique morphologic variant of nodal follicular lymphoma, characterized by a predominantly diffuse architecture, lack of the t(14;18) IGH/BCL2 translocation, presence of 1p36 deletion, frequent inguinal lymph node involvement, CD23 co-expression, and low clinical stage. Other studies on CD23+ follicular lymphoma, while associating inguinal location, have not specifically described this architecture. In addition, no follow-up studies have correlated the histopathologic and cytogenetic/molecular features of these cases, and they remain a diagnostic problem. We identified 11 cases of diffuse, CD23+ follicular lymphoma with histopathologic features similar to those described by Katzenberger et al. Along with pertinent clinical information, we detail their histopathology, IGH/BCL2 translocation status, lymphoma-associated chromosomal gains/losses, and assessment of mutations in 220 lymphoma-associated genes by massively parallel sequencing. All cases showed a diffuse growth pattern around well- to ill-defined residual germinal centers, uniform CD23 expression, mixed centrocytic/centroblastic cytology, and expression of at least one germinal center marker. Ten of 11 involved inguinal lymph nodes, 5 solely. By fluorescence in situ hybridization analysis, the vast majority lacked IGH/BCL2 translocation (9/11). Deletion of 1p36 was observed in five cases and included TNFRSF14. Of the six cases lacking 1p36 deletion, TNFRSF14 mutations were identified in three, highlighting the strong association of 1p36/TNFRSF14 abnormalities with this follicular lymphoma variant. In addition, 9 of the 11 cases tested (82%) had STAT6 mutations and nuclear P-STAT6 expression was detectable in the mutated cases by immunohistochemistry. The proportion of STAT6 mutations is higher than recently reported in conventional follicular lymphoma (11%). These findings lend support for a clinicopathologic variant of t(14;18) negative nodal follicular lymphoma and suggests importance of the interleukin (IL)-4/JAK/STAT6 pathway in this variant.

Mechanism and Role of SOX2 Repression in Seminoma: Relevance to Human Germline Specification

Summary Human male germ cell tumors (GCTs) are derived from primordial germ cells (PGCs). The master pluripotency regulator and neuroectodermal lineage effector transcription factor SOX2 is repressed in PGCs and the seminoma (SEM) subset of GCTs. The mechanism of SOX2 repression and its significance to GC and GCT development currently are not understood. Here, we show that SOX2 repression in SEM-derived TCam-2 cells is mediated by the Polycomb repressive complex (PcG) and the repressive H3K27me3 chromatin mark that are enriched at its promoter. Furthermore, SOX2 repression in TCam-2 cells can be abrogated by recruitment of the constitutively expressed H3K27 demethylase UTX to the SOX2 promoter through retinoid signaling, leading to expression of neuronal and other lineage genes. SOX17 has been shown to initiate human PGC specification, with its target PRDM1 suppressing mesendodermal genes. Our results are consistent with a role for SOX2 repression in normal germline development by suppressing neuroectodermal genes.

Cross-platform assessment of genomic imbalance confirms the clinical relevance of genomic complexity and reveals loci with potential pathogenic roles in diffuse large B-cell lymphoma

Abstract Genomic copy number alterations (CNAs) in diffuse large B-cell lymphoma (DLBCL) have roles in disease pathogenesis, but overall clinical relevance remains unclear. Herein, an unbiased algorithm was uniformly applied across three genome profiling datasets comprising 392 newly-diagnosed DLBCL specimens that defined 32 overlapping CNAs, involving 36 minimal common regions (MCRs). Scoring criteria were established for 50 aberrations within the MCRs while considering peak gains/losses. Application of these criteria to independent datasets revealed novel candidate genes with coordinated expression, such as CNOT2, potentially with pathogenic roles. No one single aberration significantly associated with patient outcome across datasets, but genomic complexity, defined by imbalance in more than one MCR, significantly portended adverse outcome in two of three independent datasets. Thus, the standardized scoring of CNAs currently developed can be uniformly applied across platforms, affording robust validation of genomic imbalance and complexity in DLBCL and overall clinical utility as biomarkers of patient outcome.

Abstract 5162: MicroRNA regulation of epidermal lineage differentiation of NT2/D1 embryonal carcinoma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Micro RNAs (miRNAs) are small non-coding RNAs that regulate gene expression by targeting the complementary mRNA sequence for post-transcriptional modification, or mRNA cleavage. MiRNAs regulate a wide range of biological processes such as nervous system and cardiac development, as well as stem cell maintenance and differentiation. However, their role in regulating cell fate choice still remains largely unexplored. Cell lines derived from human embryonal carcinoma (EC) comprise a valuable in vitro resource for study of molecular events regulating cell fate and lineage decisions. NT2/D1 EC cells differentiate into an epithelial/smooth muscle phenotype when induced with BMP-2 (Chadalavada R et al., Stem Cells 25: 771-8, 2007). We used this experimental system to discover miRNAs likely to regulate epidermal cell specification. In a time-course experiment treating NT2/D1 cells with BMP-2, we performed gene expression profiling (GEP) using Affymetrix HG-U133A+B arrays and miRNA expression profiling using Agilent Human V.2 arrays. A set of miRNAs were identified that were up- or down-regulated as cells differentiated towards the epidermal lineage. 53 miRNAs were specifically down-regulated (p<0.01), of which 36 had predicted target genes that were known to play a role in epidermal lineage development. In contrast, 24 miRNAs were specifically up-regulated (p<0.01), whose predicted target genes were mainly involved in suppression of nervous system and cardiac development. Our result also identified expression of several other miRNAs that have been reported to be highly expressed in murine skin epidermis such as mmu-miR-99b and mmu-miR-143 (Rui Yi et al., Nat. Genet. 38:356-362, 2006). Our main interest however was miRNAs that were down-regulated in the NT2/D1-BMP-2 time-course since they presumably targeted genes involved in epidermal development. Among the top 10 miRNAs that were down-regulated during the time-course, hsa-miR-18b and hsa-miR-518b was associated with the up-regulated expression of the highest number of their predicted targets in the time-course GEP (hsa-Mir18b: FZD6, JAG1, CDH13, DAG1, and ELF3; hsa-miR-518b: LAMC1, ATP2A2, SOX9, ACVR1B, and TCF12), known to function in the epidermal lineage. Other down-regulated miRNAs such as hsa-miR-141, hsa-miR-425, and hsa-miR-101 also have predicted target genes with roles in the epidermal lineage development, although the numbers of such target genes were less than those of hsa-miR-18b and hsa-miR-518b. Their predicted target genes were also up-regulated in the time-course GEP. To further validate their biological function, we are currently performing Antagomir or LNA-mediated silencing of these miRNAs to evaluate the phenotype changes and expression changes of their presumptive target genes with epidermal lineage-associated functions. 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 5162.

Metastasis-associated mutations in clear cell renal cell carcinoma.

600 Background: Over 30% of patients with clear cell renal cell carcinoma (ccRCC) exhibit metastasis at the time of diagnosis and exhibit poor outcome. About 20-50% of patients with localized disease eventually develop metastasis after nephrectomy. The goal of the current study was to identify target gene mutations associated with ccRCC metastasis. Methods: In this IRB approved study, genomic DNA from 128 ccRCC resected specimens (128 unique patients) were profiled using a custom targeted next-generation sequencing (NGS) panel comprising 70 frequently mutated genes and prognostic SNPs in renal cancer. The specimen cohort consisted of 78 primary (29 stage I-III, 30 stage IV, 19 unknown) and 50 metastatic (10 lung, 9 bone, 25 other sites, 6 unknown) lesions. Following hybrid capture, sequencing was performed (MiSeq, Illumina) and variants identified using CLCbio (Qiagen). Chi-square test was used to test for significance. Results: The median specimen had 4 non-synonymous mutations (123/128 samples had at le...

Abstract 52: Identification of genomic alterations associated with metastasis in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most abundant and lethal form of RCC and about one third of patients exhibit metastasis with poor prognosis. At the molecular level, metastasis is accompanied by multiple genetic and epigenetic alterations including genomic copy number changes. Identification of novel genomic markers and underlying mechanisms associated with metastasis could result in better therapeutic management of ccRCC patients. In this study, aCGH was performed using Agilent 244K arrays on DNA from 83 primary and 64 metastatic unmatched ccRCC fresh-frozen specimens and profiles were compared using Fisher9s Exact test (FDR, p-value ≤ 0.05), with a minimum threshold difference of 15%. Significant copy number changes were found at 15 loci between primary and metastatic lesions, after excluding loci enriched in normal CNVs. Loss of 2p16.1-p15, 2q33.1-q33.3, 3q11.1-q13.2, 3q22.3, 10q21.3-q22.1 and 11p15.4 were more frequent in primary lesions, while nine aberrations (gain of 5q23.3-q31.1, 7q21.3-q22.1, 16p11.2, 20q11.1-q13.12, 21p11.2-q11.1 and loss of 4p15.2-p14, 4q, 9p24.3-p13.3 and 18q21.2-q21.31) were more frequent in metastatic lesions. While some of these aberrations have previously been implicated in metastasis, nine were novel. In order to further examine the association of these aberrations with metastasis, the frequencies of the 15 aberrations were determined in primary ccRCCs publicly available from TCGA, stratified into low (stages I and II, n=287) and high stage groups (III and IV, n=202). Eight of the nine aberrations that were more frequent in metastatic lesions, were also observed at a higher frequency in high stage TCGA primary ccRCC (four were significantly higher between Stages I and IV). Little or no trend was observed for the six aberrations more frequent in primary lesions. In order to identify alterations associated with specific metastatic sites, aCGH profiles were compared across metastatic lesions at sites represented by at least 6 specimens: 15 lung, 7 bone, 6 lymph node, 6 pancreas and 6 brain metastatic ccRCC specimens. Gain of 7q11 and 8p12-p11, and loss of 6q14-q22 and 10q were specifically observed in lung metastases. Gain of chr12 and loss of chr22 were frequent in bone, loss of 1q32-q41 and gain of 22q11 in lymph node, and loss of 2p22.3, 8p12, 8q12-q13 and 8q24 in pancreatic lesions. No specific aberrations were found in brain lesions. In conclusion, this study has confirmed known and identified novel genomic loci associated with metastasis in ccRCC. Determination of candidate genes of the aberrations will further our understanding of metastatic progression of ccRCC and together with examination of metastatic site-specific alterations, may assist in selecting patients with metastatic disease for appropriate targeted therapy and lead to improved overall survival. Citation Format: Venkata J. Thodima, Banumathy Gowrishankar, Ana Molina, Murielle Georges, RSK Chaganti, Robert J. Motzer, Jane Houldsworth. Identification of genomic alterations associated with metastasis in clear cell renal cell carcinoma. [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 52. doi:10.1158/1538-7445.AM2014-52

Abstract 2247: Deconstructing tumor-stroma interactions during renal cell carcinoma metastasis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC In the US, ∼52,000 individuals are expected to be diagnosed with kidney cancer each year, the majority being clear cell renal cell carcinoma (RCC). Of these, ∼13,000 die of the disease. A quarter of the patients presents with advanced disease, including locally invasive or metastatic RCC and have poor prognosis. However, the molecular mechanisms that underlie RCC metastasis are poorly understood. To investigate the mechanisms of lung colonization by tumor cells during RCC metastasis we utilized a human xenograft system that enabled selected cell subpopulations with increased metastatic activity to invade the lung in NOD/SCID mice. An in vivo selection strategy was used to isolate from the poorly metastatic SN12C RCC cell line a collection of cell variants capable of efficient spontaneous metastasis to the lung. Gene expression profiling of tumor cells and lung stroma at specific time points during metastatic progression revealed sequential enrichment of genes mediating discrete biologic functions in each tissue compartment. Increased metastatic activity was associated with a desmoplastic response signature in the tumor cells. Moreover, gene expression profiling of paired primary and metastatic in vivo RCC tumors confirmed the relevance of this signature to the human disease. Lentivirus-mediated shRNA silencing of selected genes such as FSP1 and COL11A1 from the desmoplastic response signature validated its direct role in metastasis progression. Using a mouse gene expression array, we found that the stromal compartment displayed markedly different patterns of gene expression during tumor progression. Gene sets enriched for innate immune response functions were repressed rapidly in the stroma, suggesting a link between tumor cell invasion and evasion of local immune response. Interestingly, genes associated with endothelial cell proliferation and angiogenesis were progressively induced at late stages of metastatic colonization. To characterize the signal transduction pathways altered in the lung stroma during tumor progression, we performed a canonical pathway analysis using Ingenuity Pathways Analysis (Ingenuity Systems, www.ingenuity.com) in each temporal gene expression cluster. This analysis highlighted a central role for inflammation, tissue remodeling and TGF-beta signaling. Thus, integrating human xenograft, temporal gene expression profiling, and network modeling provided a tractable platform to study tumor-stromal interactions in vivo. The analysis of the complex gene expression signatures arising in the stroma during tumor progression will allow the identification of mechanisms that metastatic cells utilize to colonize secondary organs and, therefore, novel strategies of therapeutic intervention. 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 2247.