Cervical cytology (Pap-smears) CGH analyses of human tumors have revealed that chromosomal aberrations result in genomic imbalances specific for diverse tissue types. Furthermore, these changes define discrete steps in the progression of epithelial tumors. More than 95% of cervical carcinomas carry extra copies of chromosome 3, which results in the genomic amplification of the human telomerase gene TERC. It is therefore logical to apply the visualization of these recurring and specific chromosomal aberrations to complement and enhance the cytomorphological diagnosis of human cancers and their precursor lesions. This can be achieved using interphase cytogenetics with fluorescently tagged DNA probes that recognize specific chromosomal target regions directly in interphase cells. We conclude that the detection of genomic amplification of TERC in routinely collected PAP-smears can assist in identifying low-grade lesions with a high progression risk and in reduction of false negative individual cytological screenings. Therefore, application of our probe set may provide an objective genetic test for the assessment of glandular cells in Pap smears and hence for the diagnosis of cervical adenocarcinomas. Based on these results, we designed a large-scale validation study in which the presence of TERC amplification in Pap smears was correlated to the histological diagnosis after colposcopy and biopsy. The goal is to ultimately establish whether detection of genomic amplification of chromosome 3q in Pap smears could change the way cervical cancer screening is conducted. One could entertain the possibility that HPV screening is the primary test, and that those Pap smears positive for 3q would then sequentially be tested for the presence of 3q gain. In collaboration with Pamela Paris (UCSF) we will also study prostate tissue sections for copy number changes of a high-risk diagnostic probe cocktail which was defined after aCGH analyses of prostate carcinomas associated with a high progression risk. 2) Breast Cancer: The gene expression signature of genomic instability in breast cancer is an independent predictor of clinical outcome. Recently, expression profiling of breast carcinomas has revealed gene signatures that predict clinical outcome, and discerned prognostically relevant breast cancer subtypes. Measurement of the degree of genomic instability provides a very similar stratification of prognostic groups. We therefore hypothesized that these features are linked. We used gene expression profiling of 48 breast cancer specimens that profoundly differed in their degree of genomic instability and identified a set of 12 genes that defines the two groups. The biological and prognostic significance of this gene set was established through survival prediction in published datasets from patients with breast cancer. Of note, the gene expression signatures that define specific prognostic subtypes in other breast cancer datasets, such as luminal A and B, basal, normal-like, and ERBB2+, and prognostic signatures including MammaPrint and Oncotype DX, predicted genomic instability in our samples. This remarkable congruence suggests a biological interdependence of poor-prognosis gene signatures, breast cancer subtypes, genomic instability, and clinical outcome and that the degree of genomic instability per se is the dominant breast cancer inherent prognostic trait. Ductal carcinoma in situ (DCIS) is a precursor lesion of invasive ductal carcinoma (IDC) of the breast. To understand the dynamics of genomic alterations in this progression, we have used four multicolor FISH probe panels consisting of the oncogenes COX2, MYC, HER2, CCND1 and ZNF217, and the tumor suppressor genes DBC2, CDH1 and TP53 to visualize copy number changes in 13 cases of synchronous DCIS and IDC based on single cell analyses. The DCIS had a lower degree of chromosomal instability than the IDC. Despite an enormous intercellular heterogeneity in both DCIS and IDC, we observed signal patterns consistent with a non-random distribution of genomic imbalances. CDH1 was most commonly lost, and gain of MYC emerged during progression from DCIS to IDC. Four of 13 DCIS showed identical clonal imbalances in the IDC. Six cases revealed a switch, and in four of those, the IDC had acquired a gain of MYC. In one case, the major clone in the IDC was one of several clones in the DCIS, and in another case, the major clone of the DCIS became one of the two major clones in the IDC. Our study shows that despite considerable chromosomal instability, in most instances the evolution from DCIS to IDC is determined by recurrent patterns of genomic imbalances, which is consistent with a biological continuum. 3) Genomic Instability in Recurring and Nonrecurring Adenomas of the Colon Adenomatous polyps of the colon are frequent pre-cancerous lesions that might undergo malignant transformation developing into an invasive carcinoma. Despite surgical removal using polypectomy many polyps do recur. The identification of gene copy number alterations can overcome the lack in prognosis and prediction of recurrence. Early genomic instabilities in colorectal adenomas might also shed light on tumor clonality and heterogeneity. This might improve patient-specific surveillance intervals for colonoscopy. Thirty-five colorectal adenomas with low-grade dysplasia (LGD) were collected, formalin fixed and paraffin embedded. Tissue sections were taken for macro-dissection and DNA isolation. DNA was analyzed using array CGH. To confirm the results and to analyze intratumor heterogeneity single layer cytospins (Hedley-method) were prepared. We then analyzed fourteen colorectal cancer-specific gene probes (COX2, PIK3CA, APC, CLIC1, EGFR, MYC, CCND1, CDX2, CDH1, TP53, ERBB2/HER2, SMAD7, SMAD4 and ZNF217) and the centromere probe CEP10 using multiplex fluorescence in situ hybridization (FISH) on single nuclei (300 per case) of the adenomas. We evaluated adenomas with (n=13) and without recurrence (n=12). Additionally, five matched pair samples, consisting of the primary adenoma and the relapse, were analyzed. aCGH revealed characteristic gains of 6p and 7q in more than 50% of all samples. In addition, the genome-wide patterns of gains and losses were heterogeneous. However, gains (P0.01) in 6p21.33, 7q22.1 and 20q13.33 existed predominantly in initial adenomas with recurrence, whereas these alterations were hardly seen in nonrecurring adenomas. We confirmed these findings using multicolor FISH and found generally high correspondence of both techniques. CLIC1, EGFR and ZNF217 were the most frequently amplified genes. Additionally, we identified CDX2 as a potential indicator for adenoma recurrence since this marker was exclusively found in adenomas with recurrence. Genomic instability was more prominent among adenomas with recurrence. We conclude that there are characteristic amplifications in early stage colorectal adenomas that drive tumor recurrence.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010834-10
Application #
9343720
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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