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. The application of this genetic test to routinely collected cytological specimens proved that high-grade intraepithelial lesions (HSIL, which are comprised of CIN2 and CIN3) could be discerned from normal samples, ASCUS and low-grade intraepithelial lesions (LSIL, or CIN1) with a sensitivity and specificity exceeding 90%, independent of the cytomorphological assessment. In order to explore whether gain of 3q and genomic amplification of hTERC increases the propensity of progression from LSIL to HSIL and invasive carcinoma, we have applied the probe set to a series of previously stained PAP-smears. The samples included (i) CIN1 and CIN2 lesions that progressed to CIN3, (ii) CIN1 and CIN2 lesions that regressed spontaneously, and (iii) normal PAP-smears from women who subsequently developed CIN3 or cervical cancer. Our data suggest that genomic amplification of hTERC is required for the transition from CIN1 and CIN2 to CIN3 and that it predicts progression. Of note, hTERC amplification was found in 30% of cytologically normal PAP-smears from women who were diagnosed with CIN3 or invasive cervical carcinomas after a short latency. We conclude that the detection of genomic amplification of hTERC 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. In an independent study we were curious as to whether the amplification of the human telomerase gene could also be used to diagnose another type of cervical cancer, i.e., cervical adenocarcinomas. Interphase nuclei prepared from archival material of 12 primary cervical adenocarcinomas were hybridized with a triple colour probe-set specific for centromeres of chromosomes 3 and 7 and the TERC gene. The findings were correlated with HPV infection. We observed high proportions of nuclei with increased copy numbers for TERC in both HPV positive and HPV negative tumors. The absolute number of TERC signals was increased in HPV positive (mean 3.7) and HPV negative (mean 5.0) tumors. Amplification of the human telomerase gene TERC is a consistent aberration in cervical adenocarcinomas and is independent of the presence of HPV genomes. 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. In this study, we used a four-color FISH assay by Abbott/Vysis based on our previous results that included a biotinylated DNA probe for the detection of HPV (provided to us through a MTA with that company). In collaboration with the Karolinska Hospital in Stockholm, Sweden, we collected 78 liquid based cytology (LBC) samples from women referred to the Karolinska Hospital because of a suspicious Pap smear. At the time of LBC sampling, all patients were examined by colposcopy, at which time a biopsy was taken. For most patients a subsequent histological diagnosis afterloop electrosurgical excision procedure (LEEP) and a follow-up Pap smear were available. The LBC samples were also analyzed by several HPV tests (line blot, Quantovir HPV test, HPV mRNA detection using the NorChip assay), and for the expression levels of p16 using immunocytochemistry. The histological diagnosis revealed 14 benign cases, 17 samples rated as cervical intraepithelial neoplasia (CIN) grade 1, 22 CIN2, 23 CIN3, and two carcinomas. As expected, the number of TERC positive cases increased with the severity of the dysplasia. We then compared, using Receiver Operating Characteristic (ROC) curves, which of the tests rendered the highest sensitivity and specificity for the discernment of benign lesions and CIN1 from CIN2, CIN3, and cancer. We showed that detection of genomic amplification of TERC clearly outperforms any other test, including detection of HPV, as a diagnostic test for the presence of high-grade cervical dysplasia (CIN2 and CIN3) and cancer in routinely collected LBC samples. The correlation with the histological classification, the standard for a diagnosis, was excellent. For further validation, we have engaged in a prospective trial organized through NCIs Early Detection Research Network (EDRN) and the Centers for Disease Control and Prevention (CDC). 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. The gene expression signature of genomic instability in breast cancer is an independent predictor of clinical outcomeRecently, 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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010834-06
Application #
8552853
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2012
Total Cost
$615,247
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Gertz, E Michael; Chowdhury, Salim Akhter; Lee, Woei-Jyh et al. (2016) FISHtrees 3.0: Tumor Phylogenetics Using a Ploidy Probe. PLoS One 11:e0158569
Wangsa, Darawalee; Chowdhury, Salim Akhter; Ryott, Michael et al. (2016) Phylogenetic analysis of multiple FISH markers in oral tongue squamous cell carcinoma suggests that a diverse distribution of copy number changes is associated with poor prognosis. Int J Cancer 138:98-109
Hathcock, Karen S; Padilla-Nash, Hesed M; Camps, Jordi et al. (2015) ATM deficiency promotes development of murine B-cell lymphomas that resemble diffuse large B-cell lymphoma in humans. Blood 126:2291-301
Chowdhury, Salim Akhter; Gertz, E Michael; Wangsa, Darawalee et al. (2015) Inferring models of multiscale copy number evolution for single-tumor phylogenetics. Bioinformatics 31:i258-67
Gemoll, Timo; Auer, Gert; Ried, Thomas et al. (2015) Genetic Instability and Disease Prognostication. Recent Results Cancer Res 200:81-94
Chowdhury, Salim Akhter; Shackney, Stanley E; Heselmeyer-Haddad, Kerstin et al. (2014) Algorithms to model single gene, single chromosome, and whole genome copy number changes jointly in tumor phylogenetics. PLoS Comput Biol 10:e1003740
Heselmeyer-Haddad, Kerstin M; Berroa Garcia, Lissa Y; Bradley, Amanda et al. (2014) Single-cell genetic analysis reveals insights into clonal development of prostate cancers and indicates loss of PTEN as a marker of poor prognosis. Am J Pathol 184:2671-86
Kirshenbaum, Arnold S; Petrik, Amy; Walsh, Rosemary et al. (2014) A ten-year retrospective analysis of the distribution, use and phenotypic characteristics of the LAD2 human mast cell line. Int Arch Allergy Immunol 164:265-70
Akagi, Keiko; Li, Jingfeng; Broutian, Tatevik R et al. (2014) Genome-wide analysis of HPV integration in human cancers reveals recurrent, focal genomic instability. Genome Res 24:185-99
Winkler, Thomas; Hong, So Gun; Decker, Jake E et al. (2013) Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia iPSCs. J Clin Invest 123:1952-63

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