Karsten Schlüns

CD24 Is Expressed in Ovarian Cancer and Is a New Independent Prognostic Marker of Patient Survival

CD24 is a small heavily glycosylated glycosylphosphatidylinositol-linked cell surface protein, which is expressed in hematological malignancies as well as in a large variety of solid tumors. Very recently its expression in ovarian cancer has been found on RNA level by chip analysis. We evaluated CD24 protein expression by immunohistochemistry in 9 normal ovaries and 69 epithelial ovarian tumors (5 adenomas, 8 borderline tumors, and 56 carcinomas) with known follow-up data. Surface epithelium of normal ovaries as well as adenomas did not express CD24. In borderline tumors CD24 was expressed in membrane in 75% of cases, whereas cytoplasmic expression was detected in only one of nine cases. In invasive ovarian carcinomas, a membranous expression was detected in 84% and a cytoplasmic expression in 59% of cases. In univariate survival analysis of all invasive ovarian carcinomas, a highly significant association of increased cytoplasmic CD24 expression with shortened patient survival (mean 98 months versus 37 months, P = 0.0002, log rank test) was demonstrated. Other significant prognostic parameters were International Federation of Gynecology and Obstetrics (FIGO) stage, Silverberg grade, patient age, undifferentiated histological type, and metastatic disease. We did not detect a significant correlation of CD24 with these clinicopathological parameters. In multivariate analysis, only CD24 and FIGO stage were independent prognostic parameters. Our data suggest that the expression of CD24 as detected by immunohistochemistry is a new independent molecular marker for shortened survival time of patients with epithelial ovarian carcinomas.

Chromosomal alterations in the clonal evolution to the metastatic stage ofquamous cell carcinomas of the lung

Comparative genomic hybridization (CGH) was applied to squamous cellcarcinomas (SCC) of the lung to define chromosomal imbalances that are associated with the metastatic phenotype. In total, 64 lung SCC from 50 patients were investigated, 25 each with or without evidence of metastasis formation. The chromosomal imbalances summarized by a CGH histogram of the 50 cases revealed deletions most frequently on chromosomes 1p21–p31, 2q34–q36, 3p, 4p, 4q, 5q, 6q14–q24, 8p, 9p, 10q, 11p12–p14, 13q13–qter, 18q12–qter and 21q21. DNA over-representations were most pronounced for chromosomes 1q11–q25, 1q32–q41, 3q, 5p, 8q22–qter, 11q13, 12p, 17q21–q22, 17q24–q25, 19, 20q and 22q. In ten cases, paired samples of primaries and at least one metastasis were analysed. The comparison revealed a considerable chromosomal instability and genetic heterogeneity; however, the CGH pattern indicated a clonal relationship in each case. The difference in histograms from the metastatic and non-metastatic tumour groups was most useful in pinpointing chromosomal imbalances associated with the metastatic phenotype, indicating that the deletions at 3p12–p14, 3p21, 4p15–p16, 6q24–qter, 8p22–p23, 10q21–qter and 21q22, as well as the over-representations at 1q21–q25, 8q, 9q34, 14q12 and 15q12–q15, occurred significantly more often in the metastatic tumour group. The comparison of the paired samples confirmed these findings in individual cases and suggested distinct genetic changes, in particular the extension of small interstitial deletions, during tumour progression. Importantly, metastasis-associated lesions were frequently detectable in the primary tumour providing a method of identifying patients at risk for tumour dissemination. Individual profiles and histograms are accessible at our web site http://amba.charite.de/cgh.

Patterns of chromosomal imbalances in invasive breast cancer.

Invasive breast carcinomas are characterized by a complex pattern of chromosomal alterations. We applied comparative genomic hybridization (CGH) to analyze 105 primary breast carcinomas using histograms to indicate the incidence of DNA imbalances of tumor subgroups and difference histograms to compare invasive ductal carcinomas (IDC) with lobular carcinomas (ILC), well and poorly differentiated carcinomas (G1/G3) and estrogen receptor‐positive and ‐negative tumors (ER+/ER−). Only single imbalances showed a higher incidence in ILC compared with IDC, i.e., gains on chromosomes 4 and 5q13–q23 as well as deletions on chromosomes 6q, 11q14‐qter, 12p12‐pter, 16q, 17p, 18q, 19, and 22q. Of these, particularly gains of 4 and losses at 16q21–q23, and 18q12–q21 were statistically significant. For most loci, IDC showed more alterations providing a genetic correlate to the fact that ductal carcinoma overall is associated with a worse prognosis than ILC. Of these, many imbalances showing statistical significance were also observed in G3 and ER− tumors, i.e., deletions at 2q35–q37, 3p12—p14, 4p15–p16, 5q, 7p15, 8p22–p23, 10q, 11p, 14q21–q31, 15q, and gains at 2p, 3q21–qter, 6p, 8q21‐qter, 10p, 18p11–q11, and 20q, suggesting that they contribute to a more aggressive tumor phenotype. By contrast, gains on chromosome 5q13–q23 as well as deletions at 6q, 16q and 22q were more prevalent in G1 and ER+ tumors. The ratio profiles of all cases as well as histograms are accessible at our CGH online tumor database at http://amba.charite.de/cgh. Our results highlight distinct chromosomal subregions for cancer‐associated genes. In addition, these imbalances may serve as markers for a genetic classification of invasive breast cancer. Int. J. Cancer 89:305–310, 2000.

Identification of biology-based breast cancer types with distinct predictive and prognostic features: role of steroid hormone and HER2 receptor expression in patients treated with neoadjuvant anthracycline/taxane-based chemotherapy

IntroductionReliable predictive and prognostic markers for routine diagnostic purposes are needed for breast cancer patients treated with neoadjuvant chemotherapy. We evaluated protein biomarkers in a cohort of 116 participants of the GeparDuo study on anthracycline/taxane-based neoadjuvant chemotherapy for operable breast cancer to test for associations with pathological complete response (pCR) and disease-free survival (DFS). Particularly, we evaluated if interactions between hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) expression might lead to a different clinical behavior of HR+/HER2+ co-expressing and HR+/HER2- tumors and whether subgroups of triple negative tumors might be identified by the help of Ki67 labeling index, cytokeratin 5/6 (CK5/6), as well as cyclooxygenase-2 (COX-2), and Y-box binding protein 1 (YB-1) expression.MethodsExpression analysis was performed using immunohistochemistry and silver-enhanced in situ hybridization on tissue microarrays (TMAs) of pretherapeutic core biopsies.ResultspCR rates were significantly different between the biology-based tumor types (P = 0.044) with HR+/HER2+ and HR-/HER2- tumors having higher pCR rates than HR+/HER2- tumors. Ki67 labeling index, confirmed as significant predictor of pCR in the whole cohort (P = 0.001), identified HR-/HER- (triple negative) carcinomas with a higher chance for a pCR (P = 0.006). Biology-based tumor type (P = 0.046 for HR+/HER2+ vs. HR+/HER2-), Ki67 labeling index (P = 0.028), and treatment arm (P = 0.036) were independent predictors of pCR in a multivariate model. DFS was different in the biology-based tumor types (P < 0.0001) with HR+/HER2- and HR+/HER2+ tumors having the best prognosis and HR-/HER2+ tumors showing the worst outcome. Biology-based tumor type was an independent prognostic factor for DFS in multivariate analysis (P < 0.001).ConclusionsOur data demonstrate that a biology-based breast cancer classification using estrogen receptor (ER), progesterone receptor (PgR), and HER2 bears independent predictive and prognostic potential. The HR+/HER2+ co-expressing carcinomas emerged as a group of tumors with a good response rate to neoadjuvant chemotherapy and a favorable prognosis. HR+/HER2- tumors had a good prognosis irrespective of a pCR, whereas patients with HR-/HER- and HR-/HER+ tumors, especially if they had not achieved a pCR, had an unfavorable prognosis and are in need of additional treatment options.Trial registrationClinicalTrials.gov identifier: NCT00793377

Incidence of chromosomal imbalances in advanced colorectal carcinomas and their metastases.

Abstract. Comparative genomic hybridization (CGH) was used to screen 54 advanced colon carcinomas, i.e., 24 primary tumors and 30 metastases, for chromosomal alterations. Using a sensitive statistical method for the determination of DNA imbalances and histograms for analysis of the incidence of changes, we identified the DNA over-representation of chromosome 20q as the most common alteration being present in 100% of cases. High incidence deletions were observed on 18q21–18q23 (96%), 4q27–4q28 (96%), 4p14 (87%), 5q21 (81%), 1p21–1p22 (72%), 21q21 (74%), 6q16 (72%), 3p12 (66%), 8p24–8p21 (66%), 9p21 (64%), 11q22 (64%), and 14q13–14q21 (64%). Further frequent over-representation was found on 7q12–7q11.2 (75%), 16p11–16p12 (70%), 19p13 (70%), 9q34 (67%), 19q13 (67%), 13q34 (64%), 13q13 (64%), 17q21 (59%), 22q11 (61%), 8q24 (57%), and 1q21 (57%). Pronounced DNA gains and losses being defined as regions in which the ratio profiles exceeded the values of 1.5 and 0.5, respectively, frequently colocalized with peaks of incidence curve. The use of difference histograms for the comparison of tumor subgroups as well as case-by-case histogram for the analysis of 15 paired tumor samples identified several of the above alterations as relevant for tumor progression and metastasis formation. The study identified additional loci and delineates more precisely those that have been previously reported. For comparative purposes, we have made our primary data (ratio profiles, clinicopathological parameters, histograms) available at the interactive web site http://amba.charite.de/cgh, where the incidence of changes can be determined at individual loci and additional parameters can be applied for the analysis of our CGH results.

Chromosomal alterations during lymphatic and liver metastasis formation of colorectal cancer.

Comparative genomic hybridization (CGH) was used to screen colorectal carcinomas for chromosomal aberrations that are associated with metastatic phenotype. In total, 63 tumor specimens from 40 patients were investigated, comprising 30 primary tumors, 22 systemic metastases (12 liver, 6 brain, and 4 abdominal wall metastases) and 11 lymph node tumors. Using statistical analysis and histograms to evaluate the chromosomal imbalances, overrepresentations were detected most frequently at 20q11.2-20q13.2, 7q11.1-7q12, 13q11.2-13q14, 16p12, 19p13, 9q34, and 19q13.1-19q13.2. Deletions were prominent at 18q12-18q23, 4q27-4q28, 4p14, 5q21, 1p21-1p22, 21q21, 6q16-6q21, 3p12, 8p22-8p23, 9p21, 11q22, and 14q13-14q21. Hematogenous metastases showed more alterations than lymph node tumors, particularly more deletions at 1p, 3, 4, 5q, 10q, 14, and 21q21 and gains at 1q, 7p, 12qter, 13, 16, and 22q. Comparing liver metastases with their corresponding primary tumors, particularly deletions at 2q, 5q, 8p, 9p, 10q, and 21q21 and gains at 1q, 11, 12qter, 17q12-q21, 19, and 22q were more often observed. The analysis suggested that the different pathways of tumor dissemination are reflected by a nonrandom accumulation of chromosomal alterations with specific changes being responsible for the different characteristics of the metastatic phenotype.

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) has potential tumour-suppressive activity in human lung cancer

The expression of insulin‐like growth factor binding protein‐related protein 1 (IGFBP‐rP1) is decreased in various tumours, but the role of IGFBP‐rP1 in lung cancer is not yet clear. In this study, IGFBP‐rP1 expression in lung cancer cell lines was evaluated and reduced expression of IGFBP‐rP1 was found. In tissue microarrays containing 138 primary tumours and 20 normal lung tissues analysed by immunohistochemistry, 58 tumours (42%) exhibited no expression of IGFBP‐rP1, while all 20 normal lung tissues showed high expression. In squamous cell lung cancer, low expression of IGFBP‐rP1 was significantly linked to high‐grade tumours. Treatment with 5‐aza‐2′‐deoxycytidine restored the expression of IGFBP‐rP1 in three of four lung cancer cell lines. Sequencing of PCR products of sodium bisulphite‐treated genomic DNA from the three lung cancer cell lines revealed a heterogeneous methylation pattern in the region of exon 1 and intron 1. Stable transfection of IGFBP‐rP1 full‐length cDNA into the H2170 lung cancer cell line led to increased expression of IGFBP‐rP1 protein. IGFBP‐rP1‐positive transfectants exhibited remarkably reduced colony‐forming ability in soft agar, suppression of tumour growth rate in nude mice, and increased apoptotic cell number as well as activated caspase‐3 expression level. The data suggest that IGFBP‐rP1 is a tumour suppressor inactivated by DNA methylation in human lung cancer. Copyright

Gene expression profiles in human non-small and small-cell lung cancers

Suppression subtractive hybridisation (SSH) was performed comparing normal bronchial epithelial cells with a lung squamous cell carcinoma (SCC) and a metastatic small-cell lung carcinoma (SCLC). The sequence analysis of four cDNA libraries revealed 869 individual sequences. Of these, 342 were tested using northern blots of lung cancer cell lines representing the three major subtypes (SCC, adenocarcinoma, SCLC) which confirmed the differential expression of 236 cDNAs. The extended analysis of 31 randomly chosen fragments confirmed the validity of the approach to identify genes associated with lung cancer development. Additionally, five novel full-length cDNA were isolated encoding the microtubule-associated proteins 1A/1B light chain 3, the epithelial V-like antigen 1 (EVA1), the GTP-binding protein SAR1, a new member of the S100-type calcium binding protein family and a new homeobox-containing gene.

Chromosomal imbalances in brain metastases of solid tumors.

Metastases account for approximately 50% of the malignant tumors in the brain. In order to identify structural alterations that are associated with tumor dissemination into the central nervous system we used Comparative Genomic Hybridization (CGH) to investigate 42 brain metastases and 3 primary tumors of 40 patients. The metastases originated from lung cancer (14 cases), melanomas (7), carcinomas of breast (5), colon (5), kidney (5), adrenal gland (1) and thyroid (1). In addition, tumors of initially unknown primaries were assessed in 3 cases. The highest incidence of DNA gains were observed for the chromosomal regions 1q23, 8q24, 17q24‐q25, 20q13 (>80% of cases) followed by the gain on 7p12 (77%). DNA losses were slightly less frequent with 4q22, 4q26, 5q21, 9p21 being affected in at least 70% of the cases followed by deletions at 17p12, 4q32‐q34, 10q21, 10q23‐q24 and 18q21‐q22 in 67.5% of cases. Two unusual narrow regional peaks were observed for the gain on 17q24‐q25 and loss on 17p12.The incidence at individual loci can be viewed at our CGH online tumor database at http://www.amba.charite.de/cgh. The metastases of each tumor type showed a recurrent pattern of changes. In those cases with primary tumor and metastases available, the CGH pattern exhibited a high degree of conformity. In conclusion, our data suggests that specific genetic lesions are associated with tumor dissemination into the nervous system and that CGH analysis may be a useful supplementary tool for classification of metastases with unknown origin.

Core classification of lung cancer: Correlating nuclear size and mitoses with ploidy and clinicopathological parameters

We attempted to establish a microscopy based tumour characterization providing insight into the genetics of cancer cells and in particular their DNA ploidy. The core classification defined semi-quantitative criteria for scoring the nuclear size ranging from small (core score 1) to giant nuclei (core score 4). By listing all nuclear sizes according to their relative frequencies it provided a measure for core size variability as a correlate of nuclear pleomorphism. Additionally, the mitosis size, their variability and the presence/abundance of tripolar and tetrapolar mitoses were determined. This classification was applied to 155 lung cancer samples from all major histologic types and the results were correlated with the analysis by DNA image cytometry and patient survival. The morphological assessments correlated highly significantly with the DNA ploidy parameters, e.g. small cell lung carcinomas showed the smallest values for nuclear size (mean core score of 1.18) and DNA content (DNA index mean of 2.08c) being highly significantly different from adenocarcinomas (1.95/3.10c), large cell lung carcinoma (2.00/3.26c) and squamous cell carcinoma (2.20/3.42c). In non-small cell lung carcinoma (NSCLC) in general and adenocarcinoma in particular, the core size variability correlated significantly with grading and survival. Furthermore, parameters indicative for chromosomal variability, i.e. 2c deviation index and 5c exceeding rate, were predictors of poor survival in NSCLC patients. As a complement to histologic tumour diagnosis the core classification should help to better stratify cancer subtypes.