Robert J. Clifford

Deletion of IKZF1 and Prognosis in Acute Lymphoblastic Leukemia

BACKGROUND Despite best current therapy, up to 20% of pediatric patients with acute lymphoblastic leukemia (ALL) have a relapse. Recent genomewide analyses have identified a high frequency of DNA copy-number abnormalities in ALL, but the prognostic implications of these abnormalities have not been defined. METHODS We studied a cohort of 221 children with high-risk B-cell-progenitor ALL with the use of single-nucleotide-polymorphism microarrays, transcriptional profiling, and resequencing of samples obtained at diagnosis. Children with known very-high-risk ALL subtypes (i.e., BCR-ABL1-positive ALL, hypodiploid ALL, and ALL in infants) were excluded from this cohort. A copy-number abnormality was identified as a predictor of poor outcome, and it was then tested in an independent validation cohort of 258 patients with B-cell-progenitor ALL. RESULTS More than 50 recurring copy-number abnormalities were identified, most commonly involving genes that encode regulators of B-cell development (in 66.8% of patients in the original cohort); PAX5 was involved in 31.7% and IKZF1 in 28.6% of patients. Using copy-number abnormalities, we identified a predictor of poor outcome that was validated in the independent validation cohort. This predictor was strongly associated with alteration of IKZF1, a gene that encodes the lymphoid transcription factor IKAROS. The gene-expression signature of the group of patients with a poor outcome revealed increased expression of hematopoietic stem-cell genes and reduced expression of B-cell-lineage genes, and it was similar to the signature of BCR-ABL1-positive ALL, another high-risk subtype of ALL with a high frequency of IKZF1 deletion. CONCLUSIONS Genetic alteration of IKZF1 is associated with a very poor outcome in B-cell-progenitor ALL.

JAK mutations in high-risk childhood acute lymphoblastic leukemia

Pediatric acute lymphoblastic leukemia (ALL) is a heterogeneous disease consisting of distinct clinical and biological subtypes that are characterized by specific chromosomal abnormalities or gene mutations. Mutation of genes encoding tyrosine kinases is uncommon in ALL, with the exception of Philadelphia chromosome-positive ALL, where the t(9,22)(q34;q11) translocation encodes the constitutively active BCR-ABL1 tyrosine kinase. We recently identified a poor prognostic subgroup of pediatric BCR-ABL1-negative ALL patients characterized by deletion of IKZF1 (encoding the lymphoid transcription factor IKAROS) and a gene expression signature similar to BCR-ABL1-positive ALL, raising the possibility of activated tyrosine kinase signaling within this leukemia subtype. Here, we report activating mutations in the Janus kinases JAK1 (n = 3), JAK2 (n = 16), and JAK3 (n = 1) in 20 (10.7%) of 187 BCR-ABL1-negative, high-risk pediatric ALL cases. The JAK1 and JAK2 mutations involved highly conserved residues in the kinase and pseudokinase domains and resulted in constitutive JAK-STAT activation and growth factor independence of Ba/F3-EpoR cells. The presence of JAK mutations was significantly associated with alteration of IKZF1 (70% of all JAK-mutated cases and 87.5% of cases with JAK2 mutations; P = 0.001) and deletion of CDKN2A/B (70% of all JAK-mutated cases and 68.9% of JAK2-mutated cases). The JAK-mutated cases had a gene expression signature similar to BCR-ABL1 pediatric ALL, and they had a poor outcome. These results suggest that inhibition of JAK signaling is a logical target for therapeutic intervention in JAK mutated ALL.

Delineating Genetic Alterations for Tumor Progression in the MCF10A Series of Breast Cancer Cell Lines

To gain insight into the role of genomic alterations in breast cancer progression, we conducted a comprehensive genetic characterization of a series of four cell lines derived from MCF10A. MCF10A is an immortalized mammary epithelial cell line (MEC); MCF10AT is a premalignant cell line generated from MCF10A by transformation with an activated HRAS gene; MCF10CA1h and MCF10CA1a, both derived from MCF10AT xenografts, form well-differentiated and poorly-differentiated malignant tumors in the xenograft models, respectively. We analyzed DNA copy number variation using the Affymetrix 500 K SNP arrays with the goal of identifying gene-specific amplification and deletion events. In addition to a previously noted deletion in the CDKN2A locus, our studies identified MYC amplification in all four cell lines. Additionally, we found intragenic deletions in several genes, including LRP1B in MCF10CA1h and MCF10CA1a, FHIT and CDH13 in MCF10CA1h, and RUNX1 in MCF10CA1a. We confirmed the deletion of RUNX1 in MCF10CA1a by DNA and RNA analyses, as well as the absence of the RUNX1 protein in that cell line. Furthermore, we found that RUNX1 expression was reduced in high-grade primary breast tumors compared to low/mid-grade tumors. Mutational analysis identified an activating PIK3CA mutation, H1047R, in MCF10CA1h and MCF10CA1a, which correlates with an increase of AKT1 phosphorylation at Ser473 and Thr308. Furthermore, we showed increased expression levels for genes located in the genomic regions with copy number gain. Thus, our genetic analyses have uncovered sequential molecular events that delineate breast tumor progression. These events include CDKN2A deletion and MYC amplification in immortalization, HRAS activation in transformation, PIK3CA activation in the formation of malignant tumors, and RUNX1 deletion associated with poorly-differentiated malignant tumors.

Genetic variations at loci involved in the immune response are risk factors for hepatocellular carcinoma.

Primary liver cancer is the third most common cause of cancer‐related death worldwide, with a rising incidence in Western countries. Little is known about the genetic etiology of this disease. To identify genetic factors associated with hepatocellular carcinoma (HCC) and liver cirrhosis (LC), we conducted a comprehensive, genome‐wide variation analysis in a population of unrelated Asian individuals. Copy number variation (CNV) and single nucleotide polymorphisms (SNPs) were assayed in peripheral blood with the high‐density Affymetrix SNP6.0 microarray platform. We used a two‐stage discovery and replication design to control for overfitting and to validate observed results. We identified a strong association with CNV at the T‐cell receptor gamma and alpha loci (P < 1 × 10−15) in HCC cases when contrasted with controls. This variation appears to be somatic in origin, reflecting differences between T‐cell receptor processing in lymphocytes from individuals with liver disease and healthy individuals that is not attributable to chronic hepatitis virus infection. Analysis of constitutional variation identified three susceptibility loci including the class II MHC complex, whose protein products present antigen to T‐cell receptors and mediate immune surveillance. Statistical analysis of biologic networks identified variation in the “antigen presentation and processing” pathway as being highly significantly associated with HCC (P = 1 × 10−11). SNP analysis identified two variants whose allele frequencies differ significantly between HCC and LC. One of these (P = 1.74 × 10−12) lies in the PTEN homolog TPTE2. Conclusion: Combined analysis of CNV, individual SNPs, and pathways suggest that HCC susceptibility is mediated by germline factors affecting the immune response and differences in T‐cell receptor processing. (HEPATOLOGY 2010)

Global Gene Expression Profiling and Validation in Esophageal Squamous Cell Carcinoma and Its Association with Clinical Phenotypes

Purpose: Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with poor prognosis. Understanding molecular changes in ESCC will enable identification of molecular subtypes and provide potential targets for early detection and therapy. Experimental Design: We followed up a previous array study with additional discovery and confirmatory studies in new ESCC cases by using alternative methods. We profiled global gene expression for discovery and confirmation, and validated selected dysregulated genes with additional RNA and protein studies. Results: A total of 159 genes showed differences with extreme statistical significance (P < E-15) and 2-fold differences or more in magnitude (tumor/normal RNA expression ratio, N = 53 cases), including 116 upregulated and 43 downregulated genes. Of 41 genes dysregulated in our prior array study, all but one showed the same fold change directional pattern in new array studies, including 29 with 2-fold changes or more. Alternative RNA expression methods validated array results: more than two thirds of 51 new cases examined by real-time PCR (RT-PCR) showed 2-fold differences or more for all seven genes assessed. Immunohistochemical protein expression results in 275 cases which were concordant with RNA for five of six genes. Conclusion: We identified an expanded panel of genes dysregulated in ESCC and confirmed previously identified differentially expressed genes. Microarray-based gene expression results were confirmed by RT-PCR and protein expression studies. These dysregulated genes will facilitate molecular categorization of tumor subtypes and identification of their risk factors, and serve as potential targets for early detection, outcome prediction, and therapy. Clin Cancer Res; 17(9); 2955–66. ©2011 AACR.

Genomic characterization of esophageal squamous cell carcinoma from a high-risk population in China.

Genomic instability plays an important role in most human cancers. To characterize genomic instability in esophageal squamous cell carcinoma (ESCC), we examined loss of heterozygosity (LOH), copy number (CN) loss, CN gain, and gene expression using the Affymetrix GeneChip Human Mapping 500K (n = 30 cases) and Human U133A (n = 17 cases) arrays in ESCC cases from a high-risk region of China. We found that genomic instability measures varied widely among cases and separated them into two groups: a high-frequency instability group (two-thirds of all cases with one or more instability category of > or =10%) and a low-frequency instability group (one-third of cases with instability of <10%). Genomic instability also varied widely across chromosomal arms, with the highest frequency of LOH on 9p (33% of informative single nucleotide polymorphisms), CN loss on 3p (33%), and CN gain on 3q (48%). Twenty-two LOH regions were identified: four on 9p, seven on 9q, four on 13q, two on 17p, and five on 17q. Three CN loss regions-3p12.3, 4p15.1, and 9p21.3-were detected. Twelve CN gain regions were found, including six on 3q, one on 7q, four on 8q, and one on 11q. One of the most gene-rich of these CN gain regions was 11q13.1-13.4, where 26 genes also had RNA expression data available. CN gain was significantly correlated with increased RNA expression in over 80% of these genes. Our findings show the potential utility of combining CN analysis and gene expression data to identify genes involved in esophageal carcinogenesis.

Bioinformatics Tools for Single Nucleotide Polymorphism Discovery and Analysis

Abstract: Single nucleotide polymorphisms (SNPs) are a valuable resource for investigating the genetic basis of disease. These variants can serve as markers for fine‐scale genetic mapping experiments and genome‐wide association studies. Certain of these nucleotide polymorphisms may predispose individuals to illnesses such as diabetes, hypertension, or cancer, or affect disease progression. Bioinformatics techniques can play an important role in SNP discovery and analysis. We use computational methods to identify SNPs and to predict whether they are likely to be neutral or deleterious. We also use informatics to annotate genes that contain SNPs. To make this information available to the research community, we provide a variety of Internet‐accessible tools for data access and display. These tools allow researchers to retrieve data about SNPs based on gene of interest, genetic or physical map location, or expression pattern.

Novel Functions for TAF7, a Regulator of TAF1-independent Transcription

The transcription factor TFIID components TAF7 and TAF1 regulate eukaryotic transcription initiation. TAF7 regulates transcription initiation of TAF1-dependent genes by binding to the acetyltransferase (AT) domain of TAF1 and inhibiting the enzymatic activity that is essential for transcription. TAF7 is released from the TAF1-TFIID complex upon completion of preinitiation complex assembly, allowing transcription to initiate. However, not all transcription is TAF1-dependent, and the role of TAF7 in regulating TAF1-independent transcription has not been defined. The IFNγ-induced transcriptional co-activator CIITA activates MHC class I and II genes, which are vital for immune responses, in a TAF1-independent manner. Activation by CIITA depends on its intrinsic AT activity. We now show that TAF7 binds to CIITA and inhibits its AT activity, thereby repressing activated transcription. Consistent with this TAF7 function, siRNA-mediated depletion of TAF7 resulted in increased CIITA-dependent transcription. A more global role for TAF7 as a regulator of transcription was revealed by expression profiling analysis: expression of 30–40% of genes affected by TAF7 depletion was independent of either TAF1 or CIITA. Surprisingly, although TAF1-dependent transcripts were largely down-regulated by TAF7 depletion, TAF1-independent transcripts were predominantly up-regulated. We conclude that TAF7, until now considered only a TFIID component and regulator of TAF1-dependent transcription, also regulates TAF1-independent transcription.

Abstract B029: Dissecting out the tumor suppressor aspect of TGF-β in breast cancer using integrated genomics

The purpose of our study was to identify a gene expression signature that specifically reflects the tumor suppressive effects of transforming growth factor-β (TGF-β), for use as a negative selective biomarker in clinical trials using TGF-β antagonists. TGF-β effects are highly contextual, and the dogma is that tumor suppressive effects are active in the early stages of carcinogenesis, but that pro-progression effects come to dominate later on. Since TGF-β antagonists are in early phase clinical trials in cancer, it is important to know whether the tumor suppressive effects of TGF-β are still intact in any tumors at the time of diagnosis and treatment, as this would be a contraindication for anti-TGF-β therapy. Existing TGF-β-related gene expression signatures were not designed a priori to discriminate the tumor suppressive from the tumor promoting activities. To address this question, we applied integrated ChIP-chip and transcriptomic approaches in the MCF10A-based model of breast cancer progression. We have previously shown that TGF-β has tumor suppressor activity in the less malignant cell lines of the series (MCF10AT1k, MCF10Ca1h), but that this effect is lost in the most malignant cell line (MCF10Ca1a). Here we showed that the tumor suppressor activity in this model system is specifically dependent on the downstream signaling component Smad3 and not Smad2. Using promoter-wide ChIP-chip, we found that the genomic landscape of TGF-β induced Smad3 binding differed dramatically between the four cell lines, despite their close genetic relatedness and similar Smad3 levels and activation profiles. Interestingly, TGF-β induced Smad3 binding only at loci that were already transcriptionally active, suggesting that TGF-βs may primarily play a modulator rather than an instigator role in regulating transcription. This feature probably contributes importantly to the known contextuality of TGF-β activity. By focusing on the two malignant cell lines (MCF10CA1h and MCF10CA1a), we identified a core signature of 26 TGF-β/Smad3 regulated genes that was specifically associated with the tumor suppressor activity of TGF-β. Unexpectedly, the direction of regulation of 25% of these genes by TGF-β differed in vitro and in vivo, highlighting a further novel contribution to TGF-β contextuality. The in vivo weighted form of the TGF-β/Smad3 tumor suppressor signature (TSTSS) was associated with good outcome specifically in estrogen-receptor positive (ER+) breast cancer patients, suggesting that TGF-β tumor suppressive pathways are still active and influencing disease outcome in a subset of patients at the time of surgery. TGF-β is a potent growth inhibitor for most epithelial cells and the TSTSS was weakly inversely correlated with proliferation in ER+ breast cancer, but the signature prognosticated independently of proliferation in multivariate analysis. Instead, the TSTSS was enriched for genes involved in cellular differentiation and movement, and ephrin was the top enriched signaling pathway. Ephrin knockdown in MCF10Ca1h cells led to increased migration, reduced apoptosis and differentiation and enhanced tumorigenesis. Our demonstration that a subset of breast cancer patients still has an active TGF-β tumor suppressor pathway at the time of surgery has important implications for patient stratification in ongoing clinical trials with TGF-β antagonists. Citation Format: Misako Sato, Mitsutaka Kadota, Binwu Tang, Yu-an Yang, Mengge Shan, Jia Weng, Michael Welsh, Yoshiko Nagano, Aleksandra Michalowski, Howard Yang, Robert Clifford, Maxwell Lee, Lalage Wakefield. Dissecting out the tumor suppressor aspect of TGF-β in breast cancer using integrated genomics. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B029.

Abstract 4310: An integrated genomic approach specifically dissects out the tumor suppressor aspect of TGF-β in breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Transforming growth factor-βs (TGF-βs) have complex roles in tumorigenesis, with context-dependent effects that can either suppress or promote tumor progression. The dogma is that tumor suppressive effects are active in the early stages of carcinogenesis, but that later pro-progression effects come to dominate. Since TGF-β antagonists of various types are in early phase clinical trials in cancer, it is important to know whether the tumor suppressive effects of TGF-β are still intact in any tumors at the time of diagnosis and treatment. Existing TGF-β-related gene expression signatures were not designed a priori to discriminate the tumor suppressive from the tumor promoting activities. To address this question, we applied integrated ChIP-chip and transcriptomic approaches in the MCF10A-based model of breast cancer progression. We have previously shown that TGF-β has tumor suppressor activity in the less malignant cell lines of the series (MCF10A, MCF10AT1k, MCF10Ca1h), but that this effect is lost in the most malignant cell line (MCF10Ca1a). The tumor suppressor activity is dependent on the downstream signaling component Smad3. Using promoter-wide ChIP-chip, we found that the genomic landscape of TGF-β induced Smad3 binding differed dramatically between the four cell lines, despite their close genetic relatedness. Interestingly, TGF-β induced Smad3 binding only at genetic loci that were already transcriptionally active, suggesting that TGF-βs may primarily play a modulator rather than an instigator role in regulating transcription. This feature probably contributes significantly to the known contextuality of TGF-β activity. By focusing on the two malignant cell lines (MCF10CA1h and MCF10CA1a), we identified a core signature of 26 TGF-β/Smad3 regulated genes that were specifically associated with the tumor suppressor activity of TGF-β. Unexpectedly, the direction of regulation of 25% of these genes by TGF-β differed in vitro and in vivo, highlighting a further novel contribution to TGF-β contextuality, and emphasizing the importance of including in vivo data in this type of analysis. The in vivo weighted form of the TGF-β/Smad3 tumor suppressor signature was associated with good outcome in estrogen-receptor positive breast cancer patients, suggesting that TGF-β tumor suppressive pathways are still active and influencing disease outcome in a subset of patients’ tumors at the time of surgery. TGF-β is a potent growth inhibitor for most epithelial cells, but anti-proliferative effects made only a minor contribution to the tumor suppressor activity in the breast cancer cohorts. Instead, novel tumor suppressor effects of TGF-β captured by this approach included the restoration of tumor suppressive EphrinA signaling, leading to increased tumor cell differentiation. The results have important implications for patient stratification in ongoing clinical trials with TGF-β antagonists. Citation Format: Misako Sato, Mitsutaka Kadota, Binwu Tang, Yu-an Yang, Mengge Shan, Jia Weng, Michael Welsh, Aleksandra Michalowski, Howard Yang, Robert Clifford, Maxwell Lee, Lalage M. Wakefield. An integrated genomic approach specifically dissects out the tumor suppressor aspect of TGF-β in breast cancer. [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 4310. doi:10.1158/1538-7445.AM2013-4310