Malgorzata Pienkowska

Excessive genomic DNA copy number variation in the Li-Fraumeni cancer predisposition syndrome

DNA copy number variations (CNVs) are a significant and ubiquitous source of inherited human genetic variation. However, the importance of CNVs to cancer susceptibility and tumor progression has not yet been explored. Li–Fraumeni syndrome (LFS) is an autosomal dominantly inherited disorder characterized by a strikingly increased risk of early-onset breast cancer, sarcomas, brain tumors and other neoplasms in individuals harboring germline TP53 mutations. Known genetic determinants of LFS do not fully explain the variable clinical phenotype in affected family members. As part of a wider study of CNVs and cancer, we conducted a genome-wide profile of germline CNVs in LFS families. Here, by examining DNA from a large healthy population and an LFS cohort using high-density oligonucleotide arrays, we show that the number of CNVs per genome is well conserved in the healthy population, but strikingly enriched in these cancer-prone individuals. We found a highly significant increase in CNVs among carriers of germline TP53 mutations with a familial cancer history. Furthermore, we identified a remarkable number of genomic regions in which known cancer-related genes coincide with CNVs, in both LFS families and healthy individuals. Germline CNVs may provide a foundation that enables the more dramatic chromosomal changes characteristic of TP53-related tumors to be established. Our results suggest that screening families predisposed to cancer for CNVs may identify individuals with an abnormally high number of these events.

TP53 Alterations Determine Clinical Subgroups and Survival of Patients With Choroid Plexus Tumors

PURPOSE Choroid plexus carcinomas are pediatric tumors with poor survival rates and a strong, but poorly understood, association with Li-Fraumeni syndrome (LFS). Currently, with lack of biologic predictors, most children are treated with aggressive chemoradiation protocols. PATIENTS AND METHODS We established a multi-institutional tissue and clinical database, which enabled the analysis of specific alterations of the TP53 tumor suppressor and its modifiers in choroid plexus tumors (CPTs). We conducted high-resolution copy-number analysis to correlate these genetic parameters with family history and outcome. Results We studied 64 patients with CPTs. All individuals with germline TP53 mutations fulfilled LFS criteria, whereas all patients not meeting these criteria harbored wild-type TP53 (P < .001). TP53 mutations were found in 50% of choroid plexus carcinomas (CPCs). Additionally, two sequence variants known to confer TP53 dysfunction, TP53 codon72 and MDM2 SNP309, coexisted in the majority of TP53 wild-type CPCs (92%) and not in TP53 mutated CPC (P = .04), which suggests a complementary mechanism of TP53 dysfunction in the absence of a TP53 mutation. High-resolution single nucleotide polymorphism (SNP) array analysis revealed extremely high total structural variation (TSV) in TP53-mutated CPC tumor genomes compared with TP53 wild-type tumors and choroid plexus papillomas (CPPs; P = .006 and .004, respectively). Moreover, high TSV was associated with significant risk of progression (P < .001). Five-year survival rates for patients with TP53-immunopositive and -immunonegative CPCs were 0% and 82 (+/- 9%), respectively (P < .001). Furthermore, 14 of 16 patients with TP53 wild-type CPCs are alive without having received radiation therapy. CONCLUSION Patients with CPC who have low tumor TSV and absence of TP53 dysfunction have a favorable prognosis and can be successfully treated without radiation therapy.

JNK1 determines the oncogenic or tumor-suppressive activity of the integrin-linked kinase in human rhabdomyosarcoma.

Although most reports describe the protein kinase integrin-linked kinase (ILK) as a proto-oncogene, occasional studies detail opposing functions in the regulation of normal and transformed cell proliferation, differentiation, and apoptosis. Here, we demonstrated that ILK functions as an oncogene in the highly aggressive pediatric sarcoma alveolar rhabdomyosarcoma (ARMS) and as a tumor suppressor in the related embryonal rhabdomyosarcoma (ERMS). These opposing functions hinge on signaling through a noncanonical ILK target, JNK1, to the proto-oncogene c-Jun. RNAi-mediated depletion of ILK induced activation of JNK and its target, c-Jun, resulting in growth of ERMS cells, whereas in ARMS cells, it led to loss of JNK/c-Jun signaling and suppression of growth both in vitro and in vivo. Ectopic expression of the fusion gene characteristic of ARMS (paired box 3-forkhead homolog in rhabdomyosarcoma [PAX3-FKHR]) in ERMS cells was sufficient to convert them to an ARMS signaling phenotype and render ILK activity oncogenic. Furthermore, restoration of JNK1 in ARMS reestablished a tumor-suppressive function for ILK. These findings indicate what we believe to be a novel effector pathway regulated by ILK, provide a mechanism for interconversion of oncogenic and tumor-suppressor functions of a single regulatory protein based on the genetic background of the tumor cells, and suggest a rationale for tailored therapy of rhabdomyosarcoma based on the different activities of ILK.

Molecular Characterization of Choroid Plexus Tumors Reveals Novel Clinically Relevant Subgroups

Purpose: To investigate molecular alterations in choroid plexus tumors (CPT) using a genome-wide high-throughput approach to identify diagnostic and prognostic signatures that will refine tumor stratification and guide therapeutic options. Experimental Design: One hundred CPTs were obtained from a multi-institutional tissue and clinical database. Copy-number (CN), DNA methylation, and gene expression signatures were assessed for 74, 36, and 40 samples, respectively. Molecular subgroups were correlated with clinical parameters and outcomes. Results: Unique molecular signatures distinguished choroid plexus carcinomas (CPC) from choroid plexus papillomas (CPP) and atypical choroid plexus papillomas (aCPP); however, no significantly distinct molecular alterations between CPPs and aCPPs were observed. Allele-specific CN analysis of CPCs revealed two novel subgroups according to DNA content: hypodiploid and hyperdiploid CPCs. Hyperdiploid CPCs exhibited recurrent acquired uniparental disomy events. Somatic mutations in TP53 were observed in 60% of CPCs. Investigating the number of mutated copies of p53 per sample revealed a high-risk group of patients with CPC carrying two copies of mutant p53, who exhibited poor 5-year event-free (EFS) and overall survival (OS) compared with patients with CPC carrying one copy of mutant p53 (OS: 14.3%, 95% confidence interval, 0.71%–46.5% vs. 66.7%, 28.2%–87.8%, respectively, P = 0.04; EFS: 0% vs. 44.4%, 13.6%–71.9%, respectively, P = 0.03). CPPs and aCPPs exhibited favorable survival. Discussion: Our data demonstrate that differences in CN, gene expression, and DNA methylation signatures distinguish CPCs from CPPs and aCPPs; however, molecular similarities among the papillomas suggest that these two histologic subgroups are indeed a single molecular entity. A greater number of copies of mutated TP53 were significantly associated to increased tumor aggressiveness and a worse survival outcome in CPCs. Collectively, these findings will facilitate stratified approaches to the clinical management of CPTs. Clin Cancer Res; 21(1); 184–92. ©2014 AACR.

Identification of a novel zinc finger gene, zf5-3, as a potential mediator of neuroblastoma differentiation.

We established a unique parental neuroblastoma cell line, NUB‐7, which mimics the bipotentiality of neuroblastoma in vivo along neuronal and Schwann cell lineages following dibutyryl cAMP and retinoic acid treatments, respectively. Differential display identified a putative novel zinc finger gene as a potential differentiation‐responsive gene coincident with retinoic acid treatment of NUB‐7. This cDNA clone, now designated zf5‐3, was mapped to chromosome 19 using somatic cell hybrids, and a larger cDNA clone further localized this gene to band 13.1–13.2 by fluorescent in situ hybridization. zf5‐3 possesses 4 characteristic zinc finger DNA‐binding motifs as determined by its nucleic acid and proposed amino acid sequence. Expression of zf5‐3 is restricted to fetal neuronal, hepatic and renal tissues and their tumor‐derived cell lines, including 8/9 neuroblastomas and 2/2 malignant rhabdoid tumors of kidney. The restricted expression in the kidney of zf5‐3 to collecting tubules and ureter epithelium is suggestive of an ectodermal histogenesis of malignant rhabdoid tumors of kidney. During development of the fetal human brain, high levels of zf5‐3 mRNA are restricted to the mitotically active, undifferentiated neuroblasts. Morphological evidence of overt differentiation was generally accompanied by a marked loss in zf5‐3 expression. Therefore, the neuronal tissue expression profile and the down‐regulation coincident with retinoic acid–induced neuroblastoma maturation implicate zf5‐3 as a potential mediator of their differentiation. Int. J. Cancer 81:970–978, 1999.

IGF2 is highly expressed in pediatric undifferentiated sarcomas and reveals two distinct cytoplasmic trafficking patterns.

Pediatric undifferentiated soft tissue sarcomas (USTSs) are tumors composed of primitive mesenchymal cells. As such, they form an attractive model for studying the early events in sarcoma development. In an effort to better understand the critical molecular aberrations leading to sarcoma development, gene expression array analysis and post-array validation techniques were applied to several USTSs; the results were consistent with upregulation of the excitatory components of the insulin-like growth factor (IGF) pathway. Particularly high expression of the insulin-like growth factor 2 (IGF2) ligand was seen and confirmed by real-time reverse transcriptase-polymerase chain reaction. Immunohistochemistry performed using antibodies against IGF2 revealed overexpression of the IGF2 protein in 19 of 21 (90%) USTSs and revealed 2 distinct staining patterns, 1 in which there was diffuse cytoplasmic staining (16/19) and 1 in which there was punctate perinuclear positivity (3/19). Using ultrastructural immunogold localization of IGF2, it was determined that IGF2 was primarily localized to Golgi-derived vesicles and multivesicular bodies in tumor cells showing the punctate pattern, and to both the cytoplasm and plasma membrane of tumor cells showing the diffuse pattern. The results suggest that upregulation of the IGF pathway in pediatric USTSs is a critical early event in the development of sarcomas. Furthermore, findings from the immunocytochemical and immunogold analyses confirm the presence of 2 different cytoplasmic trafficking patterns and storage motifs of IGF2 within this type of tumor. Given that in one subcellular pattern the IGF2 protein does not appear to reach the membrane, these findings may have functional significance.

Genotoxic effects of thiram evaluated by sister-chromatid exchanges in human lymphocytes.

The purpose of this investigation was to study the genotoxic potential of thiram (CAS No. 137-26-8) using an in vitro sister-chromatid exchange (SCE) assay with human lymphocytes. The results indicate that thiram and its metabolites increase the SCE frequencies 2-fold over those observed in the negative controls. The standard inducers cyclophosphamide and ethyl methanesulfonate increased SCE frequencies 10- and 4-fold, respectively, over untreated levels.

Selection of probes for fluorescence in situ hybridization analysis by differential display polymerase chain reaction of mRNA from rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a malignancy of skeletal muscle derivation encompassing two major subtypes, embryonal and alveolar, which differ in clinical behavior and genetic markers. Because RMS is a relatively circumscribed tumor system for which the beginnings of a molecular genetic framework are in place, it becomes an ideal model for the application of improved methods of molecular genetic analysis. We have applied the technique known as differential display polymerase chain reaction (DD-PCR) to characterize expression of RNA in rhabdomyosarcoma subtypes. Our studies have shown that DD-PCR generates a characteristic electrophoretic profile that can be used to isolate subtype specific probes for fluorescence in situ hybridization (FISH) analysis. We have isolated two cDNA fragments and obtained clones suitable for FISH mapping to metaphase chromosomes. One probe was mapped to the centromeric region of human chromosome 22 and the other probe to the human chromosome band 6q25-26. This approach demonstrates the utility of DD-PCR as a technique for isolating novel cDNA expressed in tumors and their subsequent use as probes for FISH analysis. As more genes are identified by DD-PCR and their roles in tumorigenesis become defined, they are likely to provide novel targets for future molecular cytogenetic analysis.

Abstract 2789: Investigating the role of DNA methylation in pediatric choroid plexus tumors

Choroid plexus tumors (CPTs) are rare neoplasms of the central nervous system most commonly found in the pediatric population. CPTs represent 1- 4% of all childhood brain tumors, with 10- 20% occurring during the first year of life. Within this family of tumors, choroid plexus carcinoma (CPC) is the malignant neoplasm which is categorized as a grade III tumor by the WHO. Choroid plexus papilloma (CPP) is a benign form classified as a grade I tumor, and atypical choroid plexus papilloma (aCPP) as a grade II tumor. Distinction between these tumor subtypes is essential for treatment stratification. Previous studies performed in our laboratory suggest that CPTs are highly unstable and harbor unique patterns of chromosome-wide gains and losses. To better understand the complexities of tumor biology of CPTs as well as to identify better molecular biomarkers to distinguish between aggressive and benign forms of CPTs we performed a genome-wide DNA methylation study using Illumina Human Methylation450 BeadChip. We analyzed genome-wide DNA methylation profiles from 34 CPT (14 CPCs, 5 aCPPs and 15 CPPs) samples. Differential DNA methylation analysis did not identify significant differences between aCPPs and CPPs, therefore we explored CPC-specific DNA methylation signature in comparison to CPPs. Using a median beta value difference of 0.3 or greater and an FDR adjusted p-value Citation Format: Malgorzata Pienkowska, Sanaa Choufani, Andrei Turinsky, Diana Merino, Ana Novokmet, Michael Brudno, Rosanna Weksberg, Adam Shlien, Cynthia Hawkins, Eric Bouffet, Uri Tabori, Richard Gilbertson, David Malkin. Investigating the role of DNA methylation in pediatric choroid plexus tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2789.

Abstract 661: Identification of epigenomic markers on chromosome 11 that distinguish between choroid plexus carcinoma (CPC) and papilloma (CPP).

Choroid plexus tumors are rare epithelial brain tumors occurring predominantly in early childhood. Understanding molecular events leading to development and progression of these tumors is critical in determining patient treatment and increased survival. Loss of heterozygosity (LOH) is a common alteration observed during carcinogenesis. To investigate somatic genetic abnormalities in different subtypes of choroid plexus cancers and identify LOH hotspots, we utilized SNP 6.0 arrays (Affymetrix). Allele specific copy number analysis of 25 CPCs and 31 CPPs using Chromosome Analysis Suite (Affymetrix) revealed much higher frequency of LOH events throughout the genome in CPCs (8.4/case) compared with CPPs (1.2/case). One LOH hotspot, neutral copy loss of heterozygosity on chromosome 11, was identified in most CPCs but in no CPPs. Chromosome 11 is known to harbor an imprinted cluster that has been associated with growth dysregulation and pediatric cancer. Both maternal (KvDMR1) and paternal (H19DMR) differentially methylated regions overlap this imprinting cluster. Therefore, we investigated whether the observed LOH is related to uniparental disomy (UPD). Bisulfite pyrosequencing on a subset of CPP and CPC were studied for DNA methylation at both H19DMR and KvDMR1. Our data showed a reverse correlation in the degree of DNA methylation between the 2 DMRs in CPCs and CPPs. These data correlate well with the concept of UPD identified through our SNP arrays. Next, we investigated whether the observed higher expression of insulin like growth factor (IGF2) in CPP compared to CPC is associated with DNA methylation aberrations at the imprinted control region (H19 DMR) on 11p15.5. We identified higher DNA methylation at the H19DMR in CPC compared to CPP. Increased activity of the IGF2 gene has been associated with many types of cancer. To investigate the mechanism of CPC distinct IGF2 expression we examined the expression levels of microRNAs (miRNAs). MicroRNAs are often found to be misregulated in human cancer, and they can act posttranscriptionally as either potent oncogenes or tumor suppressor genes. We studied expression of miR-675-3p, miR-483-3p as well as miR-125-a-3p associated with IGF2/H19 imprinted locus. By quantitative RT-PCR, both miR-675 and miR-125 were found to be more significantly upregulated in CPCs than in CPPs whereas miR-483 was expressed at very low levels in CPCs and in higher levels in CPPs compared with normal control tissues . Misregulation of miR-483 was positively correlated with IGF2 levels in both groups. Our data suggest that dysregulation of epigenetic mechanisms contribute to the molecular events leading to tumor development and progression in CPC and may eventually facilitate identification of new diagnostic markers for CPC, as well as new therapeutic targets for this aggressive, lethal tumor. Citation Format: Malgorzata Pienkowska, Sanaa Choufani, Boleslaw Lach, Diana M. Merino, Ana Novokmet, Uri Tabori, Richard Gilbertson, David Malkin. Identification of epigenomic markers on chromosome 11 that distinguish between choroid plexus carcinoma (CPC) and papilloma (CPP). [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 661. doi:10.1158/1538-7445.AM2013-661