Miia Suuriniemi

Array comparative genomic hybridization-based characterization of genetic alterations in pulmonary neuroendocrine tumors

The goal of this study was to characterize and classify pulmonary neuroendocrine tumors based on array comparative genomic hybridization (aCGH). Using aCGH, we performed karyotype analysis of 33 small cell lung cancer (SCLC) tumors, 13 SCLC cell lines, 19 bronchial carcinoids, and 9 gastrointestinal carcinoids. In contrast to the relatively conserved karyotypes of carcinoid tumors, the karyotypes of SCLC tumors and cell lines were highly aberrant. High copy number (CN) gains were detected in SCLC tumors and cell lines in cytogenetic bands encoding JAK2, FGFR1, and MYC family members. In some of those samples, the CN of these genes exceeded 100, suggesting that they could represent driver alterations and potential drug targets in subgroups of SCLC patients. In SCLC tumors, as well as bronchial carcinoids and carcinoids of gastrointestinal origin, recurrent CN alterations were observed in 203 genes, including the RB1 gene and 59 microRNAs of which 51 locate in the DLK1-DIO3 domain. These findings suggest the existence of partially shared CN alterations in these tumor types. In contrast, CN alterations of the TP53 gene and the MYC family members were predominantly observed in SCLC. Furthermore, we demonstrated that the aCGH profile of SCLC cell lines highly resembles that of clinical SCLC specimens. Finally, by analyzing potential drug targets, we provide a genomics-based rationale for targeting the AKT-mTOR and apoptosis pathways in SCLC.

Confirmation of a Positive Association between Prostate Cancer Risk and a Locus at Chromosome 8q24

Background: Family-based linkage studies, association studies, and studies of tumors have highlighted human chromosome 8q as a genomic region of interest for prostate cancer susceptibility loci. Recently, a locus at 8q24, characterized by both a single nucleotide polymorphism (SNP) and a microsatellite marker, was shown to be associated with prostate cancer risk in Icelandic, Swedish, and U.S. samples. Although the data were provocative, the U.S. samples were not population based, which precludes assessment of the potential contribution of this locus to prostate cancer incidence in the United States. Methods: We analyzed both markers in a population-based, case-control study of middle-aged men from King County, Washington. Results: Overall, there was a significant positive association between the A allele of the SNP rs1447295 and prostate cancer risk [odds ratio, 1.4; 95% confidence interval (95% CI), 1.1-2.0] but no significant association with the microsatellite DG8S737. However, significant associations were observed for both markers in men with high Gleason scores. Adjusting for age, first-degree family history of prostate cancer, and prostate cancer screening history, the adjusted odds ratios were 1.4 (95% CI, 1.1-1.8) for the A allele of the SNP and 1.9 (95% CI, 1.2-2.8) for the −10 allele of the microsatellite. Conclusions: These data suggest that the locus on chromosome 8q24 harbors a genetic variant associated with prostate cancer and that the microsatellite marker is a stronger risk factor for aggressive prostate cancers defined by poorly differentiated tumor morphology. (Cancer Epidemiol Biomarkers Prev 2007;16(4):809–14)

Archival Fine-Needle Aspiration Cytopathology (FNAC) Samples: Untapped Resource for Clinical Molecular Profiling

Microarray technologies provide high-resolution maps of chromosome imbalances and epigenomic aberrations in the cancer cell genome. Such assays are often sensitive to sample DNA integrity, voiding the utility of many archival pathology specimens and necessitating the special handling of prospective clinical specimens. We have identified the remarkable preservation of higher-molecular weight DNA in archival fine-needle aspiration cytopathology specimens from patients greater than 10 years of age. We further demonstrate the outstanding technical performance of 57 fine-needle aspiration cytopathology samples for aberration detection on high-resolution comparative genomic hybridization array, DNA methylation, and single nucleotide polymorphism genotyping platforms. Forty-four of 46 malignant aspirates in this study manifested unequivocal genomic aberrations. Importantly, matched Papanicolaou and Diff-Quik fine-needle aspiration cytopathology samples showed critical differences in DNA preservation and DNA integrity. Overall, this study identifies a largely untapped reserve of human pathology specimens for molecular profiling studies, with ramifications for the prospective collection of clinical biospecimens.

Genomic aberrations in pediatric diffuse intrinsic pontine gliomas

Diagnostic biopsy is not routinely performed for children with diffuse intrinsic pontine glioma (DIPG). Consequently, our understanding of DIPG biology is hindered by limited tissue availability. We performed comparative genomic hybridization (CGH) on autopsy specimens to examine the feasibility of determining DNA genomic copy number aberrations on formalin-fixed, paraffin-embedded (FFPE) blocks. Histology on FFPE blocks obtained from autopsy of pediatric patients with DIPG was reviewed. Regions were marked for processing, and DNA was extracted from the tissue core, labeled by chemical coupling with Cy5, and hybridized to 105K oligonucleotide CGH arrays. After hybridization and washing, arrays were scanned, and data segmented and processed with Nexus software. Twenty-two samples from 13 subjects were obtained. Histologic variability was noted. CGH was successfully performed on 18 of 22 samples, representing 11 of 13 subjects. All demonstrated DNA copy number abnormalities. High copy number amplification of known oncogenes and homozygous deletions of known tumor suppressor genes were observed. Additional regions of high copy number amplification and homozygous deletion and geographical variations in the CGH patterns were found. CGH performed on FFPE tissue obtained from autopsy yields satisfactory results. This sample set from patients with DIPG was highly informative, with the majority of specimens showing ≥1 abnormality related to a known cancer gene. Aberrations in candidate drug targets were observed. This study establishes the feasibility of genomic DNA analysis from DIPG autopsy material, identifies several targets for which molecular targeted therapy exists, and suggests significant heterogeneity among patients with DIPG.

Evaluation of a variant in the transcription factor 7‐like 2 (TCF7L2) gene and prostate cancer risk in a population‐based study

Transcription factor 7‐like 2 (TCF7L2) is a high mobility group‐box containing protein that is a critical member of the Wnt/β‐catenin canonical signaling pathway. In addition to its recently recognized role in diabetes, aberrant TCF7L2 expression has been implicated in cancer through regulation of cell proliferation and apoptosis by c‐MYC and cyclin D. It has been hypothesized that germline variants within the TCF7L2 gene previously associated with diabetes may affect cancer risk through the Wnt/β‐catenin signaling pathway. Specifically, the same risk allele of single nucleotide polymorphism (SNP) rs12255372 that is associated with diabetes (T allele) has recently been associated with an increased risk of breast cancer.

Abstract 1148: Array comparative genomic hybridization of diffuse intrinsic pontine glioma samples obtained from formalin fixed paraffin embedded autopsy specimens

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We have recently optimized the method of formalin fixed paraffin embedded (FFPE) tissue processing for high resolution array comparative genomic hybridization (aCGH). This will facilitate the copy number assessment of number archival clinical surgical pathology specimens as well as biopsies with debated utility. Molecular characterization of archival specimens can potentially clarify the biology of diseases with unclear etiology and poor prognosis such as diffuse intrinsic pontine glioma (DIPG). In the case of DIPG, which is seldom biopsied, at present autopsy samples provide the only practical approach to studying this disorder. Here we performed aCGH on FFPE autopsy specimens obtained from children with DIPG. DNA was extracted from FFPE blocks obtained from autopsy on 14 pediatric DIPG patients using a modified Qiagen DNeasy kit. Non-enzymatic Universal Linkage System (ULS) chemical labeling was used to directly couple fluorescent dyes with the sample and reference DNAs (1ug each), which were then hybridized to 105k oligonucleotide CGH microarrays (Agilent). Arrays were scanned and data analyzed in Nexus Copy Number software. Twenty-one samples from 14 subjects were available for the study from which CGH was successfully performed on 18 samples representing 12 patients. All samples demonstrated DNA aberrations. Frequent DNA copy number changes included gain of 1q, 7p, and loss of 10q. High copy number amplification of known oncogenes was observed, including PDGFRA, EGFR, CCND1, and MYCN. Homozygous deletions of known tumor suppressor genes included PTEN and CDKN2A. Our tissue processing method demonstrates the feasibility of successfully using DNA derived from FFPE autopsy material for aCGH. The pediatric DIPG sample set was highly informative with the majority of specimens showing at least 1 abnormality related to a known cancer gene. Significant heterogeneity among DIPG patients as well as aberrations in candidate drug targets (KIT and EGFR) were observed. 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 1148.

Abstract 1493: High-resolution comparative genomic hybridization of preneoplastic and invasive esophageal squamous cell carcinoma: Identification of the earliest preneoplastic changes

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Esophageal cancer causes > 400,000 deaths/year in the world. Clinically useful early detection methods must identify these cancers and their precursor lesions at more curable stages. We conducted comparative genomic hybridization analyses of preneoplastic and invasive esophageal squamous cell carcinoma to look for gene copy number patterns that might be useful as early detection markers. The current effort focuses on aberrations associated with mild squamous dysplasia, the earliest histologic form of squamous preneoplasia. Methods: DNA was extracted from either biopsies or surgical resections from 122 esophageal tissues spanning a spectrum including normal mucosa, squamous preneoplasia, and invasive squamous cell carcinoma. High-resolution micro-array based comparative genomic hybridization (Agilent) was used to measure regions of chromosomal gain or loss with an emphasis on identifying changes associated with the transition from normal to mild dysplasia. Results: Chromosomal number aberrations were identified that involve several chromosomes, occur early in the neoplastic process, and progress with disease severity. Specific chromosomal number aberrations associated with mild squamous dysplasia, the earliest form of squamous preneoplasia, were uniquely identified. Conclusions: Comparative genomic hybridization identifies patterns of chromosomal number aberrations that may distinguish patients with and without high-grade squamous dysplasia or cancer. The current effort also identifies specific changes associated with mild squamous dysplasia, the earliest form of preneoplasia, and this may help elucidate mechanisms related to the initiation of carcinogenesis. 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 1493.

Abstract B179: An improved method for array comparative genomic hybridization of formalin fixed paraffin embedded surgical pathology specimens

Array comparative genomic hybridization (aCGH) has been widely used on high quality DNA obtained from fresh tissue samples to investigate chromosomal abnormalities associated with different cancers. Formalin fixed paraffin embedded (FFPE) tissue samples are an ideal source for retrospective and prospective genomic profiling studies because they are abundant in the archives and are the most common derivative of clinical surgical pathology analyses. However, DNA degradation and cross linking due to tissue processing and storage conditions has limited their prior utility for molecular testing. Here, we report an improved and reliable method to isolate, purify, and label DNA from FFPE tissue samples for high resolution aCGH. Samples (n=67) were obtained from FFPE tissue blocks of >10yrs old and represented a variety of malignancies. Guided by hematoxylin and eosin stained sections, corresponding tissue block regions of maximal tumor cell density were targeted for extraction using a tissue microarray needle punch. Several changes to the Qiagen DNeasy kit, including buffers, temperatures, centrifugations, and elution conditions, were used to maximize DNA yield and produce a DNA derivative suitable for aCGH. Our method produced suitable DNA roughly 90% of the time, yet occasional impurities following Qiagen column purification persisted and manifested as a decreased 260/230 ratio, in which case we employed a simple additional column purification to render such samples usable. Non‐enzymatic Universal Linkage System chemical labeling was used to directly couple fluorescent dyes with the sample and reference DNAs (1 ug each), which were then applied to an Agilent microarray. Data was analyzed in Nexus Copy Number software. Average DNA yield was 1 ug per 0.6mm needle core. Our optional clean up step typically raised the 260/230 ratio from 1.5 to above 2. The majorities of samples (87%) were successfully labeled and produced good quality aCGH profiles with a median DLR score of 0.25 and signal‐to‐noise ratio of 17. We were also able to set up a threshold of the amount of label incorporation that reliably predicted the success of the aCGH. This will guide us in the future to test only samples expected to pass QC measures and thus not waste costly microarray components. Our extraction and labeling method demonstrates the feasibility of successfully using DNA derived from FFPE tissue samples for aCGH, and brings this technique to the level of routine testing. Thus, our methodology removes many prior technical obstacles from aCGH based sample classification for diagnosis and clinical decision making using archival, current, and prospective FFPE specimens. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B179.