Spatial variability of genomic aberrations in a large glioblastoma resection specimen.

In the present study, the distribution of genetic aberrations in a glioblastoma resection specimen of unusually large size (9x8x2 cm) was investigated using comparative genomic hybridization (CGH). CGH was performed on 20 samples taken from the specimen, and the genetic aberrations found were compared with the regional histology. The samples were histopathologically graded according to WHO criteria, and a division in high- and low-grade areas and infiltration rims was made. In high-grade areas, low-grade areas as well as infiltration rims, gains on 10p11.2-pter (14/20), 11q12-q22 (6/20) and losses on 4q13-qter (9/20), 10q22-qter (8/20), 11p14-pter (5/20), 13q12-qter (7/20) were revealed. Gains on 1q21-32 (2/4) and losses on 7p21-pter (3/4) were exclusively found in the high-grade areas. In the low-grade tumor samples and in the infiltration rim, gains on 16p11.2-pter (6/16), 17p11.2-pter (6/16), 17q11.2-qter (5/16), 20q11.2-q13 (3/16) and deletions on 5q31-qter (4/16) were detected. Gains on 7q21-qter (8/11) and 8q11.2-qter (6/11), and loss of chromosome 9 (4/11) and the Y-chromosome (4/11) were found in the high-grade and low-grade samples, not in the infiltration rims. The finding of a set of identical chromosomal aberrations throughout the resection specimen, most of which have been previously reported in gliomas, confirms a mechanism of clonal tumor proliferation operative in gliomas. The previously unreported genetic alterations which were predominantly traced in the tumor rims, might reflect either selection for properties related to infiltrating behavior, or genomic instability of subclones. The findings illustrate the importance of searching for high-grade genetic aberrations in low-grade tumor samples taken from cases in which sampling error is suspected.

Molecular cytogenetic evaluation of gastric cardia adenocarcinoma and precursor lesions.

Analyses of cancer incidence data in the United States and Western Europe revealed steadily rising rates over the past decades of adenocarcinomas of the esophagus and gastric cardia. Genetic information on gastric cardia adenocarcinoma and its preneoplasias is sparse. We have used comparative genomic hybridization to obtain a genome-wide overview of 20 archival gastric cardia adenocarcinomas and 10 adjacent preneoplastic lesions (4 metaplasias, 1 low-grade dysplasia, 5 high-grade dysplasias). Multiple genetic alterations were discriminated in all adenocarcinomas. Frequent loss (> or =25% of all tumors) was detected, in decreasing order of frequency, on 5q, 18q, 4q, 3p, 9p, 2q, 11q, 14q, 21q, 4p, 9q, 16q, 1p, and 8p. Frequent gain (> or =25% of all tumors) was disclosed, in decreasing order of frequency, on 20q, 7p, 8q, 1q, 7q, 20p, 17q, 13q, Xp, 6q, 8p, 19q, 5p, 6p, and Xq. Loss of the Y chromosome was found in 60% of male cases. High level amplification was frequently (>10% of all tumors) detected on 7q21, 8p22, 12p11.2, 17q12-q21, and 19q13.1-q13.2. The precursor lesions showed multiple aberrations in all high-grade dysplasias, whereas few genetic changes were discerned in LGD and metaplasias. High level amplifications were also found in high-grade dysplasias, ie, on 7q21, 8p22, and 17q12-q21. Moreover, the percentage of aberrations was not significantly different for invasive carcinomas or high-grade dysplasias. Approximately 70% of the precursor aberrations were also present in the adjacent carcinoma. Minimal overlapping regions in the preneoplasias included loss on 18q12-q21 and gains on 8q23 and 17q12-q21, suggesting involvement of genes residing in these regions. In conclusion, we have (i) created a map of genetic alterations in gastric cardia adenocarcinomas and (ii) provided evidence for the presence of a metaplasia-dysplasia-carcinoma sequence in this poorly understood type of cancer.

Evaluation of oncogene amplification in intact and truncated cell nuclei of gastro-esophageal cancer cell lines by DNA in situ hybridisation.

Adenocarcinoma arising around the gastro-esophageal junction (GEJ) is a highly malignant form of cancer. Its incidence is rising sharply. The study of oncogenes in these carcinomas may give information concerning treatment and prognosis. In the present study, the fluorescence in situ hybridisation (FISH) technique was optimised for genetic characterisation of oncogenes in archival cancer specimens. Three cell lines derived from GEJ adenocarcinomas were investigated, i.e. JROECL 19, JROECL 33 and OACM5.1C, both in fresh and paraffin-embedded preparations. Furthermore, paraffin-embedded material of three xenografts was studied, i.e. JROECL 19, JROECL 33, and OACM4.1X. We focussed on the oncogenes MYC and HER2/neu, since they are frequently involved in intestinal cancers. Firstly, our results indicate that it is feasible to detect oncogene-specific probes with the FISH technique in formalin-fixed, paraffin-embedded material. Secondly, it appeared that the optimal section thickness for analysis was 2 microm. This thickness resulted in minimal nuclear overlap, which facilitates counting of FISH spots. Due to the truncation phenomenon, however, the sensitivity of the technique is less than FISH on intact nuclei. Importantly, (high level) oncogene amplifications were easily recognised in 2 microm thick sections. Finally, counting of the individual copy number of the MYC and HER2/neu oncogenes was feasible enabling an arbitrary assessment of low- and high-level amplification. In conclusion, FISH is an accurate technique for detecting amplification of oncogenes in paraffin-embedded patient material.

Genomic alterations in malignant transformation of Barrett's esophagus.

The incidence of adenocarcinoma in Barrett's esophagus has been increasing rapidly over the past decades. Neoplastic progression is characterized by three well-defined premalignant stages: metaplasia, low-grade dysplasia, and high-grade dysplasia. A genome-wide overview, based on comparative genomic hybridization, was performed, evaluating 30 Barrett's adenocarcinomas and 25 adjacent precursors, i.e., 6 metaplasias, 9 low-grade dysplasias, and 10 high-grade dysplasias. The frequency of losses and gains significantly increased in the subsequent stages of malignant transformation. Losses of 5q21-q23, 9p21, 17p12-13.1, 18q21, and Y were revealed in low-grade dysplasias. This was followed by loss of 7q33-q35 and gains of 7p12-p15, 7q21-q22, and 17q21 in high-grade dysplasias along with high-level amplification (HLA) of 7q21 and 17q21. In the invasive cancers, additional losses of 3p14-p21, 4p, 4q, 8p21, 13q14-q31, 14q24.3-q31, 16q21-q22, and 22q as well as gains of 3q25-q27, 8q23-24.1, 12p11.2-12, 15q22-q24, and 20q11.2-q13.1 were distinguished along with HLAs of 8p12-p22 and 20q11.2-q13.1. Approximately one-third of the alterations in the dysplasias were also found in the adjacent adenocarcinomas, illustrating that multiple clonal lineages can be present in Barrett's esophagus. Novel findings include loss on 7q, gain on 12p, and the observation of several HLAs in high-grade dysplasias. Furthermore, loss of 7q33-q35 was found to represent a significant distinction between low-grade and high-grade dysplasia (P = 0.01), whereas loss of 16q21-q22 and gain of 20q11.2-q13.1 were disclosed to significantly discriminate between high-grade dysplasia and adenocarcinoma (P = 0.02 and P = 0.03, respectively). This inventory of genetic aberrations increases our understanding of malignant transformation in Barrett's esophagus and might provide useful biomarkers for disease progression.

Characterization of complex chromosomal abnormalities in uveal melanoma by fluorescence in situ hybridization, spectral karyotyping, and comparative genomic hybridization.

Several nonrandom recurrent chromosomal changes are observed in uveal melanoma. Some of these abnormalities, e.g., loss of chromosome 3, gain of the q arm of chromosome 8, and chromosome 6 abnormalities, are of prognostic value. Cytogenetic analysis and/or fluorescence in situ hybridization (FISH) are used to detect these changes. In some cases, however, detailed cytogenetic analysis is not possible due to the presence of complex abnormalities. To define more accurately these cytogenetic changes, we have applied comparative genomic hybridization (CGH) and/or spectral karyotyping (SKY) to two uveal melanoma cell lines and five primary uveal melanomas, with partially defined and/or complex abnormalities. SKY provided additional information on 34/39 partially defined aberrant chromosomes and revealed a new abnormality, a der(17)t(7;17)(?;q?), that had not been recognized by conventional cytogenetics. Additionally, using SKY, abnormalities involving chromosome 6 or 8 were found to be twice as common as observed with cytogenetic analysis. CGH was especially useful in assigning the abnormalities identified by SKY to specific chromosomal regions and, in addition, resulted in the detection of a small deletion of chromosome region 3q13 approximately 21. We conclude that SKY and CGH, as methods complementary to cytogenetic and FISH analysis, provide more complete information on the chromosomal abnormalities occurring in uveal melanoma.

Molecular cytogenetic analysis of prostatic adenocarcinomas from screening studies : early cancers may contain aggressive genetic features.

No objective parameters have been found so far that can predict the biological behavior of early stages of prostatic cancer, which are encountered frequently nowadays due to surveillance and screening programs. We have applied comparative genomic hybridization to routinely processed, paraffin-embedded radical prostatectomy specimens derived from patients who participated in the European Randomized Study of Screening for Prostate Cancer. We defined a panel consisting of 36 early cancer specimens: 13 small (total tumor volume (Tv) < 0.5 ml) carcinomas and 23 intermediate (Tv between 0.5-1.0 ml) tumors. These samples were compared with a set of 16 locally advanced, large (Tv > 2.0 ml) tumor samples, not derived from the European Randomized Study of Screening for Prostate Cancer. Chromosome arms that frequently (ie, > or = 15%) showed loss in the small tumors included 13q (31%), 6q (23%), and Y (15%), whereas frequent (ie, > or = 15%) gain was seen of 20q (15%). In the intermediate cancers, loss was detected of 8p (35%), 16q (30%), 5q (26%), Y (22%), 6q, and 18q (both 17%). No consistent gains were found in this group. In the large tumors, loss was seen of 13q (69%), 8p (50%), 5q, 6q (both 31%), and Y (15%). Gains were observed of 8q (37%), 3q (25%), 7p, 7q, 9q, and Xq (all 19%). Comparison of these early, localized tumors with large adenocarcinomas showed a significant increase in the number of aberrant chromosomes per case (Rs = 0.36, P = 0.009). The same was true for the number of lost or gained chromosomes per case (Rs = 0.27, P: = 0.05; Rs = 0.48, respectively; P < 0.001). Interestingly, chromosomal alterations that were found in previous studies to be potential biomarkers for tumor aggressiveness, ie, gain of 7pq and/or 8q, were already distinguished in the small and intermediate cancers. In conclusion, our data show that chromosomal losses, more specifically of 6q and 13q, are early events in prostatic tumorigenesis, whereas chromosomal gains, especially of 8q, appear to be late events in prostatic tumor development. Finally, early localized tumors, as detected by screening programs, harbor cancers with aggressive genetic characteristics.

Involvement of cancer-activating genes on chromosomes 7 and 8 in esophageal (Barrett's) and gastric cardia adenocarcinoma.

UNLABELLED: The incidence of adenocarcinomas of the distal esophagus (Barrett's esophagus) and proximal stomach (gastric cardia) has increased rapidly over the past decades. In contrast to this dramatic increase, genetic knowledge is sparse. MATERIALS AND METHODS: We investigated genomic amplification on chromosomes 7 and 8 by comparative genomic hybridization (CGH) and protein expression of relevant oncogenes (EGFR, HGF, MET, CTSB, MYC) by immunohistochemistry (IHC) in 22 esophageal and 22 gastric cardia carcinomas. RESULTS: The CGH and IHC patterns were very similar for the two cancer locations. IHC showed positive immunostaining in 93% of the adenocarcinomas for at least one of the investigated genes, whereas CGH disclosed genomic gains on chromosome 7 and/or 8 in 80%. CONCLUSION: Cancer-activating genes on chromosomes 7 and 8 are frequently involved in gastro-esophageal junction adenocarcinomas. Moreover, the similarities in chromosomal changes and protein expression patterns strongly suggest that esophageal and gastric cardia adenocarcinomas have a shared etiology. This is in agreement with studies addressing gastroesophageal reflux disease and intestinal metaplasia at these locations.

Molecular cytogenetic evaluation of virus-associated and non-viral hepatocellular carcinoma: analysis of 26 carcinomas and 12 concurrent dysplasias.

The worldwide incidence of hepatocellular carcinoma (HCC) is approximately one million cases a year. This makes HCC one of the most frequent human malignancies, especially in Asia and Africa, although the incidence is increasing also in the western world. HCC is a complication of chronic liver disease, with cirrhosis as the most important risk factor. Viral co-pathogenesis makes cirrhosis due to hepatitis B (HBV) and hepatitis C virus (HCV) infection a very important factor in the development of HCC. As curative therapy is often ruled out due to the late detection of HCC, it would be attractive to find parameters which predict malignant transformation in HBV- and HCV-infected livers. This study has used comparative genomic hybridization (CGH) to analyse 26 HCCs (11 non-viral, nine HBV, six HCV) and 12 concurrent dysplasias (five non-viral, five HBV, two HCV). Frequent gain (> or =25% of all tumours) was detected, in decreasing order of frequency, on 8q (69%), 1q (46%), 17q (46%), 12q (42%), 20q (31%), 5p (27%), 6q (27%), and Xq (27%). Frequent loss (> or =25% of all tumours) was found, in decreasing order of frequency, on 8p (58%), 16q (54%), 4q (42%), 13q (39%), 1p (35%), 4p (35%), 16p (35%), 18q (35%), 14q (31%), 17p (31%), 9p (27%), and 9q (27%). Minimal overlapping regions could be determined at multiple locations (candidate genes in parentheses). Minimal regions of overlap for deletions were assigned to 4p14-15 (PCDH7), 8p21-22 (FEZ1), 9p12-13, 13q14-31 (RB1), 14q31 (TSHR), 16p12-13.1 (GSPT1), 16q21-23 (CDH1), 17p12-13 (TP53), and 18q21-22 (DPC4, DCC). Minimal overlapping amplified sites could be seen at 8q24 (MYC), 12q15-21 (MDM2), 17q22-25 (SSTR2, GH1), and 20q12-13.2 (MYBL2, PTPN1). A single high level amplification was seen on 5q21 in an HBV-related tumour. Aberrations appeared more frequent in HBV-related HCCs than in HCV-associated tumours (p=0.008). This was most prominent with respect to losses (p=0.004), specifically loss on 4p (p=0.007), 16q (p=0.04), 17p (p=0.04), and 18q (p=0.03). In addition, loss on 17p was significantly lower in non-viral cancers than in HBV-related HCC (p<0.001). Furthermore, loss on 13q was more prevalent in HCCs in non-cirrhotic livers (p=0.02), thus suggesting a different, potentially more aggressive, pathway in neoplastic progression. A tendency (p=0.07) was observed for loss on 9q in high-stage tumours; no specific changes were found in relation to tumour grade. A subset of the HCC-associated genetic changes was disclosed in the preneoplastic stage, i.e. liver cell dysplasia. This group of dysplasias showed frequent gain on 17q (25%) and frequent loss on 16q (33%), 4q (25%), and 17p (25%). The majority of the dysplasias with alterations revealed genetic changes that were also present in the primary tumour. In conclusion, firstly, this study has provided a detailed map of genomic changes occurring in HCC of viral and non-viral origin, and has suggested candidate genes. Loss on 17p, including the TP53 region, appeared significantly more prevalent in HBV-associated liver cancers, whereas loss on 13q, with possible involvement of RB1, was distinguished as a possible genetic biomarker. Secondly, CGH analysis of liver cell dysplasia, both viral and non-viral, has revealed HCC-specific early genetic changes, thereby confirming its preneoplastic nature. Finally, genes residing in these early altered regions, such as CDH1 or TP53, might be associated with hepatocellular carcinogenesis.

Identification of genetic markers for prostatic cancer progression.

Despite the high incidence of prostate cancer, only limited data are available on genes or chromosomes specifically involved in its initiation and progression. We have applied comparative genomic hybridization to routinely processed, paraffin-embedded, tissues at different times in prostatic tumor progression to screen the tumor genome for gains and losses. Our panel included specimens derived from 56 different patients: 23 patients with primary, prostate-confined carcinomas; 18 patients with regional lymph node metastases; and 15 patients with distant metastases. Chromosome arms that most frequently showed losses, included 13q (55%), 8p (48%), 6q (43%), 5q (32%), 16q (25%), 18q (20%), 2q (18%), 4q (18%), 10q (18%), and Y (16%). Gains were often seen of chromosome arms 8q (36%), 17q (23%), Xq (23%), 7q (21%), 3q (18%), 9q (18%), 1q (16%), Xp (16%). Furthermore, specific high-level amplifications, eg, of 1q21, 1q25, and Xq12 to q13, were found in metastatic cancers. A significant accumulation of genetic changes in distant metastases was observed, eg, loss of 10q (p = 0.03) and gain of 7q (p = 0.03) sequences. In addition, investigation of a potential biomarker identified in previous studies by our group, ie, extra copies of #7 and/or #8, revealed a high prevalence of 7pq and/or 8q gain in the distant metastases (p = 0.02). Importantly, gains were observed more frequently in tumors derived from progressors after radical prostatectomy, than in nonprogressors (mean time of follow-up, 74 months). Specifically, gain of chromosome 7pq and/or 8q sequences appeared an accurate discriminator between the progressors and nonprogressors. Multivariate analysis showed a significant correlation between progressive disease and the number of chromosomes with gains. This correlation also held true when stage (p = 0.007) or grade (p = 0.002) were taken into account. Likewise, this applied for gain of chromosome 7pq and/or 8q sequences (p = 0.03 and p = 0.005 for stage or grade, respectively). Additionally, an increase in the number of chromosomes with gains per case was related to a decrease in biochemical progression-free survival (Ptrend <0.001). More specifically, the gain of 7pq and/or 8q sequences markedly reduced the biochemical progression-free survival (p < 0.001). In conclusion, this study has, firstly, documented the spectrum of chromosomal alterations in subsequent stages of prostate cancer, a number of which had not been described previously. It allowed us to identify chromosomal regions related to advanced tumor stage, ie, loss of 10q24 and gain of 7q11.2 and/or 7q31 sequences. Secondly, gain of 7pq and/or 8q was identified as a potential genetic discriminator between progressors and nonprogressors after radical surgery.

DNA in situ hybridization (interphase cytogenetics) versus comparative genomic hybridization (CGH) in human cancer: detection of numerical and structural chromosome aberrations.

DNA in situ hybridization techniques for cytogenetic analyses of human solid cancers are nowadays widely used for diagnostic and research purposes. The advantage of this methodology is that it can be applied to cells in the interphase state, thereby circumventing the need for high-quality metaphase preparations for karyotypic evaluation. In situ hybridization (ISH) with chromosome specific (peri)centromeric DNA probes, also termed "interphase cytogenetics", can be used to detect numerical changes, whereas comparative genomic hybridization (CGH) discloses chromosomal gains and losses, i.e. amplifications and deletions. We wanted to compare both methods in human solid tumors, and for this goal we evaluated ISH and CGH within a set of 20 selected prostatic adenocarcinomas. Chromosomes 7 and 8 were chosen for this analysis, since these chromosomes are frequently altered in prostate cancer. ISH with chromosome 7 and 8 specific centromeric DNA probes was applied to standard, formalin-fixed and paraffin-embedded, histological sections for numerical chromosome analysis. CGH with DNA's, extracted from the same histologic area of the archival specimens, was used for screening of gains and losses of 7 and 8. ISH with centromeric probes distinguished a total of 26 numerical aberrations of chromosome 7 and/or 8 in the set of 20 neoplasms. In the same set CGH revealed a total of 35 losses and gains. CGH alterations of 7 and 8 were seen in twenty-two of the 26 chromosomes (85%) that showed aberrations in the ISH analysis. Concordance between ISH and CGH was seen in 11 (of 26; 42%) chromosomes. Eight chromosomes were involved in gains (5 x #7, 3 x #8), three in losses (3 x #8). This included both complete (3/11) and partial (8/11) CGH confirmation of the numerical alteration. Partial CGH confirmation was defined as loss or gain of a chromosome arm with involvement of the centromeric region. In the majority of these cases it concerned a whole chromosome arm, mostly the long arm. We conclude that generally a fair correlation was found between ISH and CGH in interphase preparations of a series of prostate cancers. However, when specified in detail, most of the numerical ISH aberrations were only partly represented in the CGH analysis. On the one hand, it suggests that CGH does not adequately discriminate numerical abnormalities. On the other hand, it likely implies that not all numerical changes, as detected by interphase cytogenetics, are truly involving the whole chromosome. A part of these discrepancies might be caused by structural mechanisms, most notably isochromosome formation.

Genetic aberrations in oligodendroglial tumours: an analysis using comparative genomic hybridization (CGH).

Four low-grade oligodendrogliomas, nine anaplastic oligodendrogliomas and two mixed oligoastrocytomas were investigated for chromosomal aberrations by comparative genomic hybridization on formalin-fixed, paraffin-embedded tissue samples. The most frequent losses observed involved 1p, 9p, 10pq, 14q, 16p, 19q, while the most frequent gains were seen on 7pq, 11pq, 17p, 19pq, and Xp. In one oligodendroglioma, a highly specific amplification of 1q32.1 was seen. The frequent losses of 14q have not been reported previously. In the two cases of mixed oligoastrocytomas multiple gains and losses were found that did not show a clear overlap with the alterations found in the pure oligodendrogliomas.

Universal linkage system: an improved method for labeling archival DNA for comparative genomic hybridization.

Comparative genomic hybridization (CGH) has become a powerful technique for studying gains and losses of DNA sequences in solid tumors. Importantly, DNA derived from archival tumor tissue is also applicable in CGH analysis. However, DNA isolated from routinely processed, formalin-fixed, paraffin-embedded tissue is often degraded, with the bulk of DNA showing fragment sizes of only 400-750 bp. Enzymatic labeling of archival DNA by standard nick translation (NT) decreases DNA size even further, until it becomes too small for CGH (<300 bp). This study presents application in CGH of a commercially available, non-enzymatic labeling method, called Universal Linkage System (ULS), that leaves the DNA fragment size intact. To compare the effect of chemical labeling of archival DNA by ULS vs. enzymatic by NT on the quality of CGH, DNA derived from 16 tumors was labeled by both ULS and NT. In those cases (n = 8), in which the bulk of DNA had a fragment size of 400-1,000 bp, CGH was successful with ULS-labeled probes, but not with NT-labeled probes. In the DNA samples (n = 6) with a fragment size > 1 kb, the intensity of CGH signals was comparable for both ULS- and NT-labeled probes, but CGH with ULS-labeled samples showed a high, speckled, background, which seriously hampered image analysis. In the remaining two cases, which had evenly distributed DNA fragment sizes (range 250-5,000 bp), CGH was successful with both labeling methods. Using DNA fragment size < 1 kb as a selection criterion for ULS labeling, we were able to obtain good quality CGH of a large panel (n = 77) of a variety of archival solid tumors. We conclude that ULS is an excellent labeling method for performing CGH on small-fragment-sized DNA.

Effect of bone decalcification procedures on DNA in situ hybridization and comparative genomic hybridization. EDTA is highly preferable to a routinely used acid decalcifier.

Decalcification is routinely performed for histological studies of bone-containing tissue. Although DNA in situ hybridization (ISH) and comparative genomic hybridization (CGH) have been successfully employed on archival material, little has been reported on the use of these techniques on archival decalcified bony material. In this study we compared the effects of two commonly used decalcifiers, i.e. , one proprietary, acid-based agent (RDO) and one chelating agent (EDTA), in relation to subsequent DNA ISH and CGH to bony tissues (two normal vertebrae, six prostate tumor bone metastases with one sample decalcified by both EDTA and RDO). We found that RDO-decalcified tissue was not suited for DNA ISH in tissue sections with centromere-specific probes, whereas we were able to adequately determine the chromosomal status of EDTA-decalcified material of both control and tumor material. Gel electrophoresis revealed that no DNA could be successfully retrieved from RDO-treated material. Moreover, in contrast to RDO-decalcified tumor material, we detected several chromosomal imbalances in the EDTA-decalcified tumor tissue by CGH analysis. Furthermore, it was possible to determine the DNA ploidy status of EDTA- but not of RDO-decalcified material by DNA flow cytometry. Decalcification of bony samples by EDTA is highly recommended for application in DNA ISH and CGH techniques.

Longitudinal evaluation of cytogenetic aberrations in prostatic cancer: tumours that recur in time display an intermediate genetic status between non-persistent and metastatic tumours.

Only limited data are available on chromosomes specifically involved in prostatic tumour progression. This study has evaluated the cytogenetic status of primary prostatic carcinomas, local tumour recurrences, and distant metastases, representing different time points in prostatic tumour progression. Interphase in situ hybridization (ISH) was applied with a set of (peri) centromeric DNA probes, specific for chromosomes 1, 7, 8 and Y, to routinely processed tissue sections of 73 tumour specimens from 32 patients. Longitudinal evaluation was possible in 11 cases with local recurrence and nine cases with distant metastases. The remaining 12 patients showed no evidence of local recurrence or distant metastasis after radical prostatectomy on follow-up (mean 60.5 months) and served as a reference. Numerical aberrations of at least one chromosome were found in 27 per cent of the local recurrences and 56 per cent of the distant metastases. In decreasing order of frequency, +8, +7, and -Y were observed in the recurrences and +8, +7, -Y, and +1 in the distant metastases. Evaluation of the corresponding primary tumour tissue of the recurrence group showed numerical aberrations in 45 per cent of cases. The aberrations found were, in decreasing order of frequency, -Y, +7, and +8. In the concomitant primary tumour tissue of the distant metastasis group, numerical aberrations were detected in 67 per cent of cases. The aberrations most frequently encountered were +8, -Y, followed by +7. In four cases, a concordance was found between the primary tumour and its recurrence or distant metastasis. Discrepancies might have been caused by cytogenetic heterogeneity. Comparison of the primary tumour tissue of the reference, the recurrence, and the distant metastasis groups showed a significant increase for the percentage of cases with numerical aberrations (Ptrend = 0.02). Likewise, a trend was seen for gain of chromosome 7 and/or 8 (Ptrend < 0.05). The number of DNA aneuploid tumours also increased in these different groups (Ptrend = 0.03). These data suggest that cancers which recur in time display an intermediate position between tumours of disease-free patients and metastatic cancers.

Interphase cytogenetics of prostatic tumor progression: specific chromosomal abnormalities are involved in metastasis to the bone.

Only limited data are available on chromosomes specifically involved in the multistep tumorigenesis of prostate cancer. To investigate the cytogenetic status at different stages of prostatic tumor development, we have applied interphase in situ hybridization (ISH) with a set of (peri) centromeric DNA probes--specific for chromosomes 1, 7, 8, and Y--to routinely processed tissue sections of prostatic specimens from 75 different individuals. Our panel consisted of: 16 normal/benign prostatic hyperplasia specimens; 23 primary, localized, prostatic tumors (N0M0 stage); 20 regional lymph node metastases (M0 stage); and 16 distant metastases. Numerical aberrations of at least one chromosome were not observed in normal/benign prostatic hyperplasia cases, but were present in localized tumors (39%), regional lymph node metastases (40%), and distant metastases (69%). Within the different pTNM groups, we observed the following aberrations (listed, within each series, in decreasing order of frequency): -Y, +8, -8, +7 in primary tumors; +8, +7, -Y, +Y, -8 in regional lymph node metastases; and +8, +7, +1, -Y, -8 in distant metastases. In primary tumors, the number of aberrant cases increased significantly with local tumor stage (p < 0.05). A significant increase in gain of chromosome 8 was also observed (p < 0.02). Gain of chromosome 7 and/or 8 showed a significant increase with progression of local tumor stage (p < 0.02). Specific involvement of chromosome 8 was seen in bone metastases, but not in hematogenous metastases to other sites (p = 0.02). Comparative genomic hybridization analysis of these bone metastases disclosed centromere 8 gains as amplifications of the (whole) 8q arm, whereas centromeric loss appeared to be due to loss of 8p sequences. With progression toward metastatic disease, an accumulation of genetic changes was seen as exemplified by gain of chromosome 1, which was solely observed in distant metastases. With tumor progression, gain of chromosomes 7 and/or 8 significantly increased (p = 0.03), whereas the number of cases with aberrations of the Y chromosome did not change. Furthermore, ploidy status determined by ISH revealed a significant increase in the number of aneuploid cases along with advancement of pTNM stage (p = 0.04). Collectively, the data strongly suggest that: (a) gain of chromosome 7 and/or 8 sequences is implicated in prostatic tumor progression; (b) gain of chromosome 8 sequences is related to local tumor growth; (c) overrepresentation of 8q sequences, most likely by isochromosome 8q formation, is involved in metastatic spread to the bone; and (d) changes in the centromeric copy number, as detected by interphase ISH, might in some cases represent structural alterations, such as an isochromosome.

Interphase cytogenetics and comparative genomic hybridization of human epithelial cancers and precursor lesions.

The accuracy of cytogenetic analyses of human solid cancers has improved enormously over the past decade by the introduction and refinement of DNA in situ hybridization (ISH) techniques. This methodology can be applied to cells in the interphase state, thereby making it an excellent tool for the delineation of chromosomal aberrations in solid tumors. The use of non-isotopic ISH to intact and disaggregated cancer specimens will be discussed, as well as comparative genomic hybridization (CGH) with tumor-derived DNAs. In this review we will focus on hybridocytochemical interphase approaches for the detection of chromosomal changes in frequently occurring human epithelial malignancies, e.g., breast, lung, and prostate carcinomas. We will further discuss the use of ISH procedures for the genetic analysis of precursor conditions leading to invasive carcinomas. Knowledge concerning these precancerous conditions is increasing, and its importance in cancer prevention has been recognized. Interphase cytogenetics by ISH, as well as CGH, with DNAs derived from microdissected, precancerous, dysplastic tissue areas will increase our understanding of these lesions, both at the investigative and diagnostic levels.

Detection of chromosomal changes by interphase cytogenetics in biopsies of recurrent astrocytomas and oligodendrogliomas.

Recently, lineage-specific genetic pathways of tumor progression have been suggested in both oligodendrogliomas and astrocytomas. Aberrations consistently reported in gliomas include chromosomes 1, 7, 10, 17 and 19. Identification of specific genetic damage may have important clinical consequences, because oligodendrogliomas, unlike astrocytomas, are responsive to chemotherapy. Genetic alterations specific for tumor type and tumor progression were investigated in 5 pairs of recurrent astrocytomas and 8 pairs of recurrent oligodendrogliomas by means of interphase in situ hybridization (ISH) to paraffin-embedded, formalin-fixed tissue sections. A set of DNA probes specific for the centromeric regions of chromosomes 1, 7, 10, 17, X and Y was applied. Since LOH studies on oligodendrogliomas have revealed losses in the region of 1p32-1p36, a DNA probe specific for the 1p36.3 locus was included. Hybridization with the 1p36.3 probe revealed loss of 1p in 5 of the 8 oligodendroglioma recurrences, the aberration being present in the primary tumors in 2 cases. In none of the astrocytomas was loss of 1p observed. Numerical aberrations were found in one astrocytoma pair (+7) and in the second biopsy of an oligodendroglioma (+7, -10). Aneuploidy was found by in situ hybridization in 8 of the 13 tumor pairs. Detection of aberrations in the 1p36.3 locus by interphase in situ hybridization to paraffin-embedded, formalin-fixed tumors may become a very useful tool in delineation of oligodendroglial from astrocytic genotypes, directing tumor specific therapy. The technique may be of crucial importance in tumor cases in which histologic criteria of lineage are not obvious.

Automated assessment of numerical chromosomal aberrations in paraffin embedded prostate tumor cells stained by in situ hybridization.

We investigated the feasibility of automated counting of in situ hybridization signals (ISH) in interphase cells isolated from paraffin embedded prostate tissue. In total, 34 specimens from 7 patients with prostate cancer were stained with probes specific for the centromeric regions of chromosomes Y, 1, 7, 8, 10, and 15, using an immunoperoxidase based technique suitable for bright-field microscopy. Enumeration of the number of ISH spots of 500 nuclei per specimen was performed (1) using an automatic system developed without any human intervention and (2) using the same system, but including verification of the counts based on visual inspection of the stored images. As reference from each specimen, 200 cell nuclei were evaluated manually, using conventional microscopy. A typical analysis procedure (including user verification) took 35 min. The difference (root mean error) between the automated counting and the counting after visual interaction was relatively small (15%). The percentage of cells with incorrect counts by automated analysis was 20.2%, a number that could easily be improved by user interaction. Detection of cells with aneusomy proved to be more sensitive compared to the routine manual counting, in cases where aberrant frequencies were low. Automated counting of samples with low frequencies (< 10%) resulted in a higher frequency of aberrant cells in 9 of 11 cases, probably due to the fact that an unbiased cell selection is guaranteed. Automated assessment of ISH signals is considered useful for the evaluation of chromosomal aberrations in prostate tumor cells, provided that the counts are visually confirmed.

Comparison of automated and manual analysis of interphase in situ hybridization signals in tissue sections and nuclear suspensions.

In this study we compared visual and automated analyses of interphase in situ hybridization (ISH) signals in five prostatic tumor specimens and one normal prostate sample, both in tissue sections and nuclear suspensions. The advantage of tissue sections is preservation of tissue morphology allowing precise analysis of tumor cells only. The advantage of nuclear suspensions is easier access to automated analysis, due to their disaggregated and dispersed cellular appearance. The samples were hybridized with probes for the (peri)centromeric regions of chromosome 1 and Y. The number of ISH signals per nucleus was counted both manually and automatically by means of a commercially available image analysis system. After image analysis the results were interactively corrected using a gallery display. The automatic and manual counts, before and after interactive correction, were then statistically evaluated. We found no significant differences in overall distributions between the automated and the manual counts, before as well as after correction. This was observed for both tissue sections and cellular suspensions. It is therefore concluded that automated analysis of ISH signals is feasible in both nuclear suspensions and in tissue sections, despite a low percentage of nuclei that could be measured on the latter.