Kerstin Heselmeyer-Haddad

Genomic changes defining the genesis, progression, and malignancy potential in solid human tumors: a phenotype/genotype correlation.

The transition of normal epithelium to invasive carcinoma occurs sequentially. In colorectal and cervical carcinogenesis, this transition is reflected by histomorphologically defined grades of increasing dysplasia that untreated may progress to invasive disease. In an attempt to understand the role of chromosomal aberrations during tumorigenesis we have applied comparative genomic hybridization using DNA extracted from defined stages of colorectal and cervical tumors, from low‐ and high‐grade astrocytic tumors and from diploid and aneuploid breast carcinomas. Genetic instability, as measured by the number of chromosomal copy alterations per case, increases significantly at the transition from precursor lesions to invasive carcinomas and continues to increase with tumor stage. Aggressive tumors have a higher number of copy alterations per case. High‐level copy number changes (amplifications) become more prevalent in advanced‐stage disease. Subtractive karyograms of chromosomal gains and losses were used to map tumor stage‐specific chromosomal aberrations and clearly showed that nonrandom chromosomal aberrations occur during disease progression. In colorectal and cervical tumors, chromosomal copy number changes were correlated with nuclear DNA content, proliferative activity, expression levels of the tumor suppressor gene TP53, and the cyclin‐dependent kinase inhibitor p21/WAF1, as well as the presence of viral genomes. Here we summarize and review the results of this comprehensive phenotype/genotype correlation and discuss the relevance of stage‐specific chromosomal aberrations with respect to diagnostic applications. Genes Chromosomes Cancer 25:195–204, 1999. Published 1999 Wiley‐Liss, Inc.

Genomic Amplification of the Human Telomerase Gene (TERC) in Pap Smears Predicts the Development of Cervical Cancer

Invasive cervical carcinomas almost invariably carry extra copies of chromosome arm 3q, resulting in a gain of the human telomerase gene (TERC). This provided the rationale for the development of a multicolor fluorescence in situ hybridization (FISH) probe set as a diagnostic tool for the direct detection of TERC gains in Pap smears. We previously used this probe set to show that cervical intraepithelial neoplasia (CIN) 2 and CIN3 lesions could be distinguished from normal samples, atypical squamous cell of undetermined significance (ASCUS) and CIN1, with a sensitivity and specificity exceeding 90%, independent of the cytomorphological assessment. In the current study, we explored whether gain of 3q and amplification of TERC could predict progression from CIN1/CIN2 to CIN3 and invasive carcinoma. We applied our probe set to a series of 59 previously stained Pap smears for which repeat Pap smears and clinical follow-up were available. The samples included CIN1/CIN2 lesions that progressed to CIN3 (progressors), CIN1/CIN2 lesions that regressed spontaneously (regressors), and normal Pap smears from women who subsequently developed CIN3 or cervical cancer. Here, we show that progressors displayed a gain of 3q whereas none of the regressors showed this genetic aberration. These data suggest that 3q gain is required for the transition from CIN1/CIN2 to CIN3 and that it predicts progression. Of note, 3q gain was found in 33% of cytologically normal Pap smears from women who were diagnosed with CIN3 or invasive cervical carcinoma after a short latency. The sensitivity of our test for predicting progression from CIN1/CIN2 to CIN3 was 100% and the specificity, ie, the prediction of regression, was 70%. We conclude that the detection of 3q gain and amplification of TERC in routinely collected Pap smears can assist in identifying low-grade lesions with a high progression risk and in decreasing false-negative cytological screenings.

Detection of Genomic Amplification of the Human Telomerase Gene (TERC) in Cytologic Specimens as a Genetic Test for the Diagnosis of Cervical Dysplasia

Invasive cervical carcinomas frequently reveal additional copies of the long arm of chromosome 3. The detection of this genetic aberration in diagnostic samples could therefore complement the morphological interpretation. We have developed a triple-color DNA probe set for the visualization of chromosomal copy number changes directly in thin-layer cervical cytology slides by fluorescence in situ hybridization. The probe set consists of a BAC contig that contains sequences for the RNA component of the human telomerase gene (TERC) on chromosome band 3q26, and repeat sequences specific for the centromeres of chromosomes 3 and 7 as controls. In a blinded study, we analyzed 57 thin-layer slides that had been rigorously screened and classified as normal (n = 13), atypical squamous cells (ASC, n = 5), low-grade squamous intraepithelial lesions (LSIL, n = 14), and high-grade squamous intraepithelial lesions (HSIL) grade 2 (CIN2, n = 8), and grade 3 (CIN3, n = 17). The percentage of tetraploid cells (P(Trend) < 0.0005) and cells with multiple 3q signals increased with the severity of the cytologic interpretation (P(Trend) < 0.0005). While only few normal samples, ASC and LSIL lesions, revealed copy number increases of 3q, 63% of the HSIL (CIN2) lesions and 76% of the HSIL (CIN3) lesions showed extra copies of 3q. We conclude that the visualization of chromosome 3q copy numbers in routinely prepared cytological material using BAC clones specific for TERC serves as an independent screening test for HSIL and may help to determine the progressive potential of individual lesions.

Comparative genomic hybridization analysis of tonsillar cancer reveals a different pattern of genomic imbalances in human papillomavirus‐positive and ‐negative tumors

Our aim was to map and compare genomic imbalances in human papillomavirus (HPV)‐positive and ‐negative squamous cell carcinomas of the tonsil. Twenty‐five primary carcinomas were analyzed by comparative genomic hybridization. Fifteen (60%) were found to be HPV‐positive by PCR, and the majority were HPV‐16. There were statistically significant differences in the distribution of DNA gains and losses between the HPV‐positive and ‐negative samples. Eleven of 15 HPV‐positive samples (73%) showed gain on chromosome 3q24‐qter, while only 4/10 (40%) HPV‐negative samples had the same gain (p = 0.049). Furthermore, 4/10 (40%) HPV‐negative samples but no HPV‐positive samples had gain on chromosome 7q11.2‐q22 (p = 0.017). As expected, and similar to previous studies, patients with an HPV‐positive tumor had a statistically significantly better disease‐specific survival than patients with an HPV‐negative tumor (p = 0.002). The most common changes, e.g., gain on 3q or 8q, loss on 11q or 13 and loss on chromosome 7q in HPV‐negative tumors, did not have any influence on prognosis. However the number of cases in each subgroup was limited.

Jumping translocations are common in solid tumor cell lines and result in recurrent fusions of whole chromosome arms

Jumping translocations (JTs) and segmental jumping translocations (SJTs) are unbalanced translocations involving a donor chromosome arm or chromosome segment that has fused to multiple recipient chromosomes. In leukemia, where JTs have been predominantly observed, the donor segment (usually 1q) preferentially fuses to the telomere regions of recipient chromosomes. In this study, spectral karyotyping (SKY) and FISH analysis revealed 188 JTs and SJTs in 10 cell lines derived from carcinomas of the bladder, prostate, breast, cervix, and pancreas. Multiple JTs and SJTs were detected in each cell line and contributed to recurrent unbalanced whole‐arm translocations involving chromosome arms 5p, 14q, 15q, 20q, and 21q. Sixty percent (113/188) of JT breakpoints occurred within centromere or pericentromeric regions of the recipient chromosomes, whereas only 12% of the breakpoints were located in the telomere regions. JT breakpoints of both donor and recipient chromosomes coincided with numerous fragile sites as well as viral integration sites for human DNA viruses. The JTs within each tumor cell line promoted clonal progression, leading to the acquisition of extra copies of the donated chromosome segments that often contained oncogenes (MYC, ABL, HER2/NEU, etc.), consequently resulting in tumor‐specific genomic imbalances. Published 2001 Wiley‐Liss, Inc.

Single-cell genetic analysis of ductal carcinoma in situ and invasive breast cancer reveals enormous tumor heterogeneity yet conserved genomic imbalances and gain of MYC during progression.

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 used four multicolor fluorescence in situ hybridization 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 enormous intercellular heterogeneity in DCIS and IDC, we observed signal patterns consistent with a nonrandom distribution of genomic imbalances. CDH1 was most commonly lost, and gain of MYC emerged during progression from DCIS to IDC. Four of 13 DCISs showed identical clonal imbalances in the IDCs. 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 in the DCIS became one of the two major clones in the IDC. Despite considerable chromosomal instability, in most cases the evolution from DCIS to IDC is determined by recurrent patterns of genomic imbalances, consistent with a biological continuum.

Detection of Genomic Amplification of the Human Telomerase Gene TERC, a Potential Marker for Triage of Women with HPV-Positive, Abnormal Pap Smears

The vast majority of invasive cervical carcinomas harbor additional copies of the chromosome arm 3q, resulting in genomic amplification of the human telomerase gene TERC. Here, we evaluated TERC amplification in routinely collected liquid based cytology (LBC) samples with histologically confirmed diagnoses. A set of 78 LBC samples from a Swedish patient cohort were analyzed with a four-color fluorescence in situ hybridization probe panel that included TERC. Clinical follow-up included additional histological evaluation and Pap smears. Human papillomavirus status was available for all cases. The correlation of cytology, TERC amplification, human papillomavirus typing, and histological diagnosis showed that infection with high-risk human papillomavirus was detected in 64% of the LBC samples with normal histopathology, in 65% of the cervical intraepithelial neoplasia (CIN)1, 95% of the CIN2, 96% of the CIN3 lesions, and all carcinomas. Seven percent of the lesions with normal histopathology were positive for TERC amplification, 24% of the CIN1, 64% of the CIN2, 91% of the CIN3 lesions, and 100% of invasive carcinomas. This demonstrates that detection of genomic amplification of TERC in LBC samples can identify patients with histopathologically confirmed CIN3 or cancer. Indeed, the proportion of TERC-positive cases increases with the severity of dysplasia. Among the markers tested, detection of TERC amplification in cytological samples has the highest combined sensitivity and specificity for discernment of low-grade from high-grade dysplasia and cancer.

EGFR protein overexpression and gene copy number increases in oral tongue squamous cell carcinoma

New promising therapeutic agents targeting epidermal growth factor receptor (EGFR) have been developed although clinical information concerning EGFR status in oral tongue squamous cell carcinoma (OTSCC) is limited. We investigated EGFR protein expression and gene copy numbers in 78 pretreatment OTSCC paraffin samples. EGFR protein expression was found in all 78 tumours, of which 72% showed an intense staining. Fifty-four percent of the tumours had high (> or =four gene copies) EGFR gene copy numbers. EGFR gene copy number was significantly associated with EGFR protein expression (P=0.002). Pretreatment EGFR staining intensity tended to be associated with non-pathological complete remission after preoperative radiotherapy for Stage II OTSCC. No correlation was found between EGFR status and survival. EGFR FISH results were significantly (P=0.003) higher in more advanced tumours (Stages II, III and IV) than in the tumours in Stage I. Non-smokers exhibited a significantly higher EGFR gene copy number and protein overexpression in Stages I and II OTSCC than smokers (P=0.001, P=0.009). In conclusion, EGFR was found to be overexpressed in all OTSCCs making this cancer type interesting for exploring new therapeutic agents targeting the EGFR receptor.

Multicentric dermatofibrosarcoma protuberans in patients with adenosine deaminase-deficient severe combined immune deficiency

BACKGROUND Dermatofibrosarcoma protuberans (DFSP) is a rare malignant skin tumor associated with a characteristic chromosomal translocation (t[17;22][q22;q13]) resulting in the COL1A1-platelet-derived growth factor β(PDGFB) fusion gene. This malignancy is rarely diagnosed in childhood. OBJECTIVE We observed an unexpected high incidence of this DFSP in children affected with adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) and set out to evaluate the association of these 2 clinical entities. METHODS Twelve patients with ADA-SCID were evaluated with a complete dermatologic examination and skin biopsy when indicated. Conventional cytogenetic and molecular analyses (fluorescence in situ hybridization, RT-PCR, or both) were performed when possible. RESULTS Eight patients were found to have DFSP. Six patients had multicentric involvement (4-15 lesions), primarily of the trunk and extremities. Most lesions presented as 2- to 15-mm, round atrophic plaques. Nodular lesions were present in 3 patients. In all cases CD34 expression was diffusely positive, and diagnosis was confirmed either by means of cytogenetic analysis, molecular testing, or both. The characteristic DFSP-associated translocation, t(17;22)(q22;q13), was identified in 6 patients; results of fluorescence in situ hybridization were positive for fusion of the COL1A1 and PDGFB loci in 7 patients; and RT-PCR showed the COL1A1-PDGFB fusion transcript in 6 patients. CONCLUSIONS We describe a previously unrecognized association between ADA-SCID and DFSP with unique features, such as multicentricity and occurrence in early age. We hypothesize that the t(17;22)(q22;q13) translocation that results in dermal overexpression of PDGFB and favors the development of fibrotic tumors might arise because of the known DNA repair defect in patients with ADA-SCID. Although the natural course of DFSP in the setting of ADA-SCID is unknown, this observation should prompt regular screening for DFSP in patients with ADA-SCID.

Phylogenetic analysis of multiprobe fluorescence in situ hybridization data from tumor cell populations.

Motivation: Development and progression of solid tumors can be attributed to a process of mutations, which typically includes changes in the number of copies of genes or genomic regions. Although comparisons of cells within single tumors show extensive heterogeneity, recurring features of their evolutionary process may be discerned by comparing multiple regions or cells of a tumor. A useful source of data for studying likely progression of individual tumors is fluorescence in situ hybridization (FISH), which allows one to count copy numbers of several genes in hundreds of single cells. Novel algorithms for interpreting such data phylogenetically are needed, however, to reconstruct likely evolutionary trajectories from states of single cells and facilitate analysis of tumor evolution. Results: In this article, we develop phylogenetic methods to infer likely models of tumor progression using FISH copy number data and apply them to a study of FISH data from two cancer types. Statistical analyses of topological characteristics of the tree-based model provide insights into likely tumor progression pathways consistent with the prior literature. Furthermore, tree statistics from the resulting phylogenies can be used as features for prediction methods. This results in improved accuracy, relative to unstructured gene copy number data, at predicting tumor state and future metastasis. Availability: Source code for software that does FISH tree building (FISHtrees) and the data on cervical and breast cancer examined here are available at ftp://ftp.ncbi.nlm.nih.gov/pub/FISHtrees. Contact: sachowdh@andrew.cmu.edu Supplementary information: Supplementary data are available at Bioinformatics online.