Genomic copy number alterations in clear cell renal carcinoma: associations with case characteristics and mechanisms of VHL gene inactivation.

Array comparative genomic hybridization was used to identify copy number alterations in clear cell renal cell carcinoma (ccRCC) patient tumors to identify associations with patient/clinical characteristics. Of 763 ccRCC patients, 412 (54%) provided frozen biopsies. Clones were analyzed for significant copy number differences, adjusting for multiple comparisons and covariates in multivariate analyses. Frequent alterations included losses on: 3p (92.2%), 14q (46.8%), 8p (38.1%), 4q (35.4%), 9p (32.3%), 9q (31.8%), 6q (30.8%), 3q (29.4%), 10q (25.7%), 13q (24.5%), 1p (23.5%) and gains on 5q (60.2%), 7q (39.6%), 7p (30.6%), 5p (26.5%), 20q (25.5%), 12q (24.8%), 12p (22.8%). Stage and grade were associated with 1p, 9p, 9q, 13q and 14q loss and 12q gain. Males had more alterations compared with females, independent of stage and grade. Significant differences in the number/types of alterations were observed by family cancer history, age at diagnosis and smoking status. Von Hippel-Lindau (VHL) gene inactivation was associated with 3p loss (P

PI(3) kinase is associated with a mechanism of immunoresistance in breast and prostate cancer.

Immune escape describes a critical event whereby tumor cells adopt an immunoresistant phenotype to escape adaptive surveillance. We show that expression of a pivotal negative regulator of T-cell function, B7-H1, correlates with PI(3) kinase activation in breast and prostate cancer patients. B7-H1-mediated immunoresistance can be attenuated by inhibitors of the PI(3) kinase pathway, and is dependent on S6K1-mediated translational regulation of B7-H1 protein. Breast and prostate carcinoma cells with activated PI(3) kinase lose the immunoresistant phenotype after treatment with B7-H1 siRNA. Conversely, breast and prostate carcinoma cells with minimal PI(3) kinase activation adopt an immunoresistant phenotype when engineered to overexpress B7-H1 protein. These observations describe a mechanism for immune escape from tumor dormancy in humans that relates to oncogenesis.

Prognostic relevance of 20q13 gains in sporadic colorectal cancers: a FISH analysis.

BACKGROUND: Amplification of 20q13 is a frequent chromosomal alteration in solid tumors and harbors a number of putative oncogenes (CAS/CSE1-L, NABC1, or Aurora2). Amplifications on 20q13 have been identified as an independent prognostic marker indicating worse survival in breast and ovarian cancer. However, little is known about the prognostic significance of 20q13 gains in sporadic colorectal cancers. The aim of this study was to correlate 20q13 gains in sporadic colorectal cancers with other known prognostic factors, tumor progression, and overall survival. METHODS: Nuclei were extracted from 146 paraffin-embedded colorectal cancers of different UICC stages and used for fluorescence in situ hybridization (FISH) with a directly labeled probe for 20q13.2 (VYSIS). Signals were counted in 120 nuclei per sample. 20q13 was considered gained when > or =40% of the nuclei showed 3 or more FISH signals. Statistical correlations were tested with log-rank tests and Kaplan-Meier survival curves. RESULTS: Signal numbers for 20q13.2 were gained in 78 cases (53%). Cases with gains on 20q13.2 showed worse outcome than cases without: the gain of 20q13.2 was an independent prognostic marker for overall survival (P=0.006) as well as tumor progression (P=0.012) in univariate and multivariate analyses. Gains on 20q13.2 did not correlate with tumor stage. However, there was a significant association between 20q13.2 gains and tumor location in the left-sided colon and an inverse correlation between histologic grade and 20q13.2 gains. CONCLUSION: These data indicate that gains on 20q13.2 correlate with faster tumor progression and worse patient survival independent from tumor size and lymph node involvement. Therefore, alterations on 20q13 are an important biological event in colorectal tumor progression with independent prognostic relevance.

Genetic changes in paired atypical and usual ductal hyperplasia of the breast by comparative genomic hybridization.

PURPOSE: Breast cancer is thought to develop from noninvasive precursor lesions, although the earliest steps of neoplastic transformation are still undefined. Usual ductal hyperplasia (UDH) is considered to represent a benign proliferation of ductal epithelial cells, whereas atypical ductal hyperplasia (ADH) may represent the first clonal neoplastic expansion of these cells. The aim of this study was to examine genetic alterations in UDH and ADH and to determine the relationship between these lesions in the same breast biopsy. EXPERIMENTAL DESIGN: Comparative genomic hybridization analysis was used to define copy number alterations in DNA extracted from archival sections of 18 patients. Nine patients showed ADH with adjacent UDH, and nine showed pure UDH. None showed evidence of invasive cancer or ductal carcinoma in situ. RESULTS: Five of the nine ADH lesions showed chromosome copy number alterations. 16q loss (five cases) and 17p loss (two cases) were the most frequent changes. The associated UDH lesions in these five patients also showed copy number alterations, always a subset of the changes present in the paired ADH. In one other patient, the UDH showed eight chromosomal alterations, whereas the paired ADH showed no changes. Only one of nine cases with pure UDH showed comparative genomic hybridization abnormalities. CONCLUSIONS: These data support the likelihood that UDH is a precursor of ADH, at least in some cases representing neoplastic growth. The frequencies of 16q and 17p losses suggest that alterations of candidate genes located in these chromosomal regions may play a role early in breast carcinogenesis.

HER2 protein expression and gene amplification in androgen-independent prostate cancer.

The role of the HER2 receptor remains uncertain in the pathogenesis and progression of human prostate cancer. Previous studies have reported widely divergent rates for HER2 expression in primary prostate tumors, probably owing to significant methodologic differences in the studies. Few data exist about the frequency of HER2 protein overexpression and gene amplification in androgen-independent prostate cancer (AIPC), although recent xenograft models suggest HER2 expression may be up-regulated in the transition from androgen-dependent to androgen-independent disease. We studied the role of HER2 protein in AIPC by immunohistochemical and fluorescence in situ hybridization (FISH) analyses on AIPC specimens using well-characterized and validated reagents. Fourteen (36%) of 39 specimens expressed HER2; however, only 2 (5%) had moderate (2+) expression, and 2 (5%) had high-level (3+) expression. Two (6%) of 36 specimens had gene amplification by FISH. These data suggest that HER2 protein overexpression and gene amplification are relatively uncommon in AIPC.

Genomic alterations in tubular breast carcinomas.

Tubular carcinoma of the breast is a well-differentiated variant of invasive ductal carcinoma and has been shown to have an exceptionally favorable prognosis, as manifested by a low incidence of lymph node metastases and an excellent overall survival. It is unknown whether this subtype represents an early step along the continuum of development to a more aggressive, poorly differentiated ductal cancer, or whether these cancers are destined to remain well differentiated with limited metastatic potential. We undertook an analysis of 18 pure tubular carcinomas of the breast using comparative genomic hybridization to evaluate the chromosomal changes in these tumors. An average of 3.6 chromosomal alterations of the genome were identified per case. The most frequent change involved loss of 16q (in 78% of tumors) and gain of 1q (in 50% of tumors). All but one case with 1q gain also exhibited a concomitant 16q loss. Other frequent changes involved 16p gain in 7 of 18 cases (39%) and distal 8p loss in 5 of 18 cases (28%). Comparison with known genomic alterations in a mixed group of invasive cancers shows tubular cancer to have fewer overall chromosomal changes per tumor (P <.01), higher frequency of 16q loss (P <.001), and lower frequency of 17p loss (P =.007). These results strongly suggest that tubular carcinomas are a genetically distinct group of breast cancers.

Loss of chromosome 16q in lobular carcinoma in situ.

Lobular carcinoma in situ (LCIS) and infiltrating lobular carcinoma may represent different forms of the same disease based on their frequent clinical association and similar histologic features. Patients with LCIS are at increased risk of multicentric and bilateral disease. Thus, LCIS may represent both a precursor to infiltrating lobular carcinoma and a marker of risk for breast cancer. To identify genomic alterations in LCIS, comparative genomic hybridization was performed on 17 cases without concurrent invasive carcinoma. Loss involving chromosome 16q was present in 88% of cases and was the sole detected alteration in 29%. Gain involving 1q was second in frequency, occurring in 41% of tumors, and in all cases was associated with loss of 16q. Other recurrent changes were loss involving 17p (18%), 8p (12%), and 12q24 (12%). E-cadherin immunohistochemistry was performed on all LCIS cases to evaluate the correlation of loss involving 16q22, the site of the E-cadherin gene, and altered protein expression. Most cases with 16q22 loss showed altered E-cadherin expression (12 of 13). These results in LCIS are similar to changes reported in infiltrating lobular cancer, confirming a genetic relationship between them. HUM PATHOL 32:292-296.

Altered distribution of beta-catenin, and its binding proteins E-cadherin and APC, in ulcerative colitis-related colorectal cancers.

The beta-catenin pathway plays a central role in transcriptional signaling and cell-cell interactions in colonic epithelium. Alterations of the expression of beta-catenin, and its binding partners E-cadherin and the adenomatous polyposis coli protein (APC), are frequent events in sporadic colorectal cancer. Ulcerative colitis (UC)-related cancers originate in a field of chronic inflammation and therefore may have different alterations in the beta-catenin pathway than sporadic cancers. To test this hypothesis, expression and subcellular localization of beta-catenin, E-cadherin, and APC were detected by immunohistochemistry in paraffin sections from 33 UC-related and 42 sporadic colorectal cancers. Although beta-catenin and E-cadherin expression were predominantly limited to the lateral cell membrane in normal colonic epithelium, both tumor groups showed an overall shift from membranous to cytoplasmic expression for these proteins. An increase in nuclear localization of beta-catenin and a decrease in cytoplasmic APC expression also were seen in both cancer groups compared with normal epithelium. Abnormal beta-catenin expression was more closely linked to E-cadherin alterations in UC-related cancers than in sporadic cancers. In contrast, abnormal beta-catenin expression was more closely linked to APC alterations in sporadic cancers than in UC-related cancers. These data suggest that alterations of the beta-catenin pathway are important in both UC-related and sporadic colorectal cancers. However, differences in the expression patterns of beta-catenin, E-cadherin, and APC between UC-related and sporadic colorectal cancers suggest that the specific alterations in this pathway may differ in these two cancer groups.

Comparative genomic hybridization.

Comparative Genomic Hybridization (CGH) is a powerful molecular cytogenetic technique that permits assessment of DNA copy number on a genome-wide scale. Of note, this methodology uses tumor DNA as a probe for fluorescence in situ hybridization (FISH) to normal metaphase chromosomes and does not require dividing cells from the tumor specimen. This unit provides protocols for CGH, for preparation of metaphase chromosomes, tumor and normal DNAs for FISH and for the microscopy and image analysis of CGH experiments.

Donor origin of neuroendocrine carcinoma in 2 transplant patients determined by molecular cytogenetics.

Organ transplant recipients have an increased tumor incidence owing to their immunocompromised state. The origin of such tumors, whether donor or recipient, will have a clinical impact on decision-making concerning immunosuppressive therapy, retransplantation, and for recipients of other organs from the same donors. We report molecular cytogenetic determination of donor origin in 2 cases of small-cell neuroendocrine carcinoma developing in sex-mismatched transplant recipients (kidney and liver). Fluorescence in situ hybridization (FISH) analysis was performed on liver core needle biopsy material from the liver transplant patient and on liver fine needle aspiration cytopreparations from the kidney transplant patient. The results for the liver transplant patient were confirmed with microsatellite allelic analysis and with comparative genomic hybridization. In both cases, FISH showed the presence of only X chromosomes within the tumor cells, indicating the donor origin of the neoplasms. FISH is an excellent method to determine neoplastic origin in sex-mismatched transplant patients. HUM PATHOL 31:1425-1429.

Comparison of genetic changes in schistosome-related transitional and squamous bladder cancers using comparative genomic hybridization.

The development of bladder tumors has been associated with a number of causative agents, including schistosomiasis. Schistosome-related cancers show different clinical and pathological features compared with non-schistosome-related bladder cancers, occurring in younger patients, and being predominantly of squamous cell type. This study addresses the difference between squamous and transitional tumor types in the presence of schistosome infection as a measure of the relationship between tumor genotype and phenotype. We have used comparative genomic hybridization to analyze primary muscleinvasive schistosome-related bladder tumors in 54 patients. Twenty-six of these tumors were squamous cell carcinomas; the remaining 28 were of transitional cell type. On average, transitional cell tumors showed 1.8 times the number of chromosomal aberrations as squamous cell tumors (14.4 versus 8.2, P: < 0.001). For both groups combined, the most prevalent genetic alterations were losses of 8p and 18q, and gains of 8q. Transitional cell cancers also showed frequent losses involving 5q, 9p, 10q, 11p and 11q, and gains at 1q and 17q. Loss of 11p was significantly more frequent in TCC than in SCC tumors (50 versus 4%, P: = 0.01). Squamous cell cancers showed more frequent losses of 17p and 18p than transitional tumors, which was clearly significant given the overall reduced frequency of changes in squamous cancers (P: = 0.001 and P: = 0.03, respectively). These data show that different histologic subgroups of bladder tumors are characterized by distinct patterns of chromosomal alterations. The genetic changes found in the transitional cell group are similar to those reported in non-schistosome-related transitional cell tumors, but differ from tumors exhibiting squamous differentiation.

The prognostic value of proliferation indices: a study with in vivo bromodeoxyuridine and Ki-67.

Proliferation indices are intended to help patients and clinicians make treatment decisions. We have previously demonstrated that a proliferation index based on in vivo labeling of S-phase cells with bromodeoxyuridine (BrdUrd) correlates with Ki-67 labeling index (LI). We now compare the prognostic value of these indices. With written consent, we gave 129 women with biopsy confirmed breast cancer 200 mg/M2 BrdUrd during 30 min immediately preceding surgery. We used IU-4 anti BrdUrd antibody to count the immunohistochemical labeling index (LI) of DNA-incorporated BrdUrd in 2,000 cells and MIB-1 to count Ki-67 (118 cases). Patients received standard surgical and adjuvant treatment. No patients were lost to follow-up and patients were followed a minimum of 2 (median 5.1) years. We compared survival and recurrence in tumors with high vs low labeling indices. We found that women in the low BrdUrd LI group had better disease free survival (92% vs 67% 5-yr DFS p = 0.001) and overall survival (94% vs 70% 5-yr OS, p = 0.0001) than those with a high LI. In comparison, a low Ki-67 index predicted better OS (87% vs 80% 5-yr OS, p = 0.020) and a trend for better DFS (84% vs 72% DFS p = 0.055). The apparent superiority of BrdUrd LI over Ki-67 LI is likely due to chance (p = 0.18). In multivariate survival analyses we found that BrdUrd LI proliferative index significantly improves prediction of DFS or OS even when node status, age or tumor size is in the model. We conclude that markers of proliferation are useful adjuncts in predicting patient prognosis.

Reproducibility of p53 immunohistochemistry in bladder tumors. National Cancer Institute, Bladder Tumor Marker Network.

The National Cancer Institute Bladder Tumor Marker Network conducted a study to evaluate the reproducibility of immunohistochemistry for measuring p53 expression in bladder tumors. Fifty paraffin blocks (10 from each of the five network institutions) were chosen at random from among high-grade invasive primary bladder tumors. Two sections from each block were sent to each laboratory for staining and scoring, and then all sections were randomly redistributed among the laboratories for a second scoring. Intra- and interlaboratory reproducibility was assessed with regard to both staining and scoring. For overall assessments of p53 positivity, the results demonstrated that intralaboratory reproducibility was quite good. Concordance across the five participating laboratories was high for specimens exhibiting no or minimal nuclear immunostaining of tumor cells or high percentages of tumor cells with nuclear immunoreactivities. However, there was a reduced level of concordance on specimens with percentages of stained tumor cells in an intermediate range. The discordancies were due mainly to staining differences in one of the five laboratories and scoring differences in another laboratory. These results indicate that some caution must be used in comparing results across studies from different groups. Standardization of staining protocols and selection of a uniform threshold for binary interpretation of results may improve assay reproducibility between laboratories.

Validation of fluorescent SSCP analysis for sensitive detection of p53 mutations.

We have developed a fluorescence-based single strand conformation polymorphism (SSCP) method that offers fast and sensitive screening for mutations in exons 5-8 of the human p53 gene. The method uses an ABI 377 DNA sequencer for unique color detection of each strand, plus accurate alignment of lanes for better detection of mobility shifts. To validate the method, 21 cell lines with reported mutations in p53 exons 5-8 were analyzed by SSCP using various gel conditions. The sensitivity for mutation detection was 95% for all cell lines studied, and no false positives were seen in 10 normal DNA samples for all four exons. Experiments mixing known amounts of tumor and normal DNA showed that mutations were detected even when tumor DNA was mixed with 80% normal DNA. Fluorescent SSCP analysis using the ABI sequencer is a useful tool in cancer research, where screening large numbers of samples for p53 mutations is desired.

Chromosomal alterations in ductal carcinomas in situ and their in situ recurrences.

BACKGROUND: Ductal carcinoma in situ (DCIS) recurs in the same breast following breast-conserving surgery in 5%-25% of patients, with the rate influenced by the presence or absence of involved surgical margins, tumor size and nuclear grade, and whether or not radiation therapy was performed. A recurrent lesion arising soon after excision of an initial DCIS may reflect residual disease, whereas in situ tumors arising after longer periods are sometimes considered to be second independent events. The purpose of this study was to determine the clonal relationship between initial DCIS lesions and their recurrences. METHODS: Comparative genomic hybridization (CGH) was used to compare chromosomal alterations in 18 initial DCIS lesions (presenting in the absence of invasive disease) and in their subsequent ipsilateral DCIS recurrences (detected from 16 months to 9.3 years later). RESULTS: Of the 18 tumor pairs, 17 showed a high concordance in their chromosomal alterations (median = 81%; range = 65%-100%), while one case showed no agreement between the paired samples (having two and 20 alterations, respectively). Morphologic characterization of the DCIS pairs showed clear similarities. The mean number of CGH changes was greater in the recurrent tumors than in the initial lesions (10.7 versus 8.8; P =.019). The most common changes in both the initial and the recurrent in situ lesions were gains involving chromosome 17q and losses involving chromosomes 8p and 17p. The degree of concordance was independent of the time interval before recurrence and of the presence of positive surgical margins. CONCLUSIONS: In this study, DCIS recurrences were clonally related to their primary lesions in most cases. This finding is consistent with treatment paradigms requiring wide surgical margins and/or postoperative radiation therapy.

Chromosomal alterations in ulcerative colitis-related and sporadic colorectal cancers by comparative genomic hybridization.

Both ulcerative colitis (UC)-related and sporadic colorectal cancers are thought to evolve through a multistep process of genomic instability, accumulation of genomic alterations, and clonal expansion. This process may involve different genomic changes in UC-related cancers than in sporadic cancers because of the origin of UC-related cancers in an inflammatory field. This study was designed to define the specific genomic events occurring in UC-related cancers. Comparative genomic hybridization (CGH) was performed on 32 UC-related and 42 stage-matched sporadic colorectal cancers. The mean number of chromosomal alterations per case was similar in the UC-related and sporadic tumor groups (8.6 in UC, 8.1 in sporadic). The 2 tumor groups shared many chromosomal alterations: losses on 18q (78% UC v69% sporadic), 8p (53% v50%), 17p (44% v57%), and gains on 8q (63% v45%), 20q (44% UC v67%), and 13q (44% UC v38%). However, differences in the frequency and timing of specific alterations were observed. Chromosome 5q was lost in 56% of UC-related but in only 26% of sporadic cancers. Alterations of chromosome 8 were associated with stage progression in UC-related, but not in sporadic cancers. In contrast, 18q loss was associated with stage progression in sporadic cancers only. Thus, differences in the frequency and timing of individual chromosomal alterations suggest that genetic progression in these 2 tumor groups may follow multiple pathways.

Genomic instability is an early event during the progression pathway of ulcerative-colitis-related neoplasia.

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon associated with a high risk of colorectal cancer. This increased cancer risk is thought to result from the cellular damage induced by the inflammatory field. The aim of this study was to determine the pattern and time course of genomic instability occurring in UC-related neoplasia. Sites of cancer, dysplasia, and nondysplasia from 14 UC colectomy cases containing cancer were analyzed for chromosomal alterations by comparative genomic hybridization (CGH) and for microsatellite instability using a series of 10 microsatellite markers. Clonal chromosomal alterations were present in 85% of cancer sites, 86% of dysplasia sites, and 36% of nondysplasia sites. Losses of chromosome 18 or 18q and chromosome 5 or 5q were common in cancer and dysplasia and were occasionally detected in nondysplasia. High-level microsatellite instability was detected in the cancer and dysplasia of two cases. Samples that demonstrated high-level microsatellite instability were unlikely to have chromosomal alterations demonstrable by CGH. These studies suggest that the predominant type of genomic instability in UC-related neoplasia is associated with chromosomal alterations and that this type of genomic instability frequently occurs before the development of histologically defined dysplasia.

p16/pRb pathway alterations are required for bypassing senescence in human prostate epithelial cells.

The cell cycle regulatory genes p16/CDKN2 and RB are frequently deleted in prostate cancers. In this study, we examined the role of alterations in p16 and pRb during growth, senescence, and immortalization in vitro of human prostate epithelial cells (HPECs). HPECs are established from normal prostate tissues and cultured on collagen-coated dishes. Our results show that p16 is reproducibly elevated at senescence in HPECs. HPECs are immortalized using human papilloma virus 16 E6 and/or E7 as molecular tools to inactivate p53 and/or pRb, respectively. Immortalization occurs infrequently in this system and only after a latent period during which additional genetic/epigenetic changes are thought to occur. Notably, all of the E6-immortalized HPEC lines but none of the E7 lines show inactivation of p16/CDKN2 (by deletion, methylation, or mutation) in association with immortalization. In contrast, E7 lines, in which pRb function is abrogated by E7 binding, retain the high levels of p16 observed at senescence. Thus, all lines show either a p16 or pRb inactivation. Analysis of six independent lines from metastatic prostate cancers reveals a similar loss of either p16 or pRb. Comparative genomic hybridization of HPECs shows that gains of chromosomes 5q, 8q, and 20 are nonrandomly associated with bypassing senescence (probability = 0.95). These results suggest that high levels of the cyclin-dependent kinase inhibitor p16 mediate senescence G1 arrest in HPECs and that bypassing this block by a p16/pRb pathway alteration is required for immortalization in vitro and possibly tumorigenesis in vivo. Our results further indicate that inactivation of the p16/pRb pathway alone is not sufficient to immortalize HPECs and that additional genetic alterations are required for this process.

Chromosome arm 20q gains and other genomic alterations in colorectal cancer metastatic to liver, as analyzed by comparative genomic hybridization and fluorescence in situ hybridization.

Comprehensive information about the molecular cytogenetic changes in metastases of colorectal cancer is not yet available. To define such changes in metastases, we measured relative DNA sequence copy numbers by comparative genomic hybridization (CGH). Samples from 27 liver metastases and 6 synchronous primary tumors were analyzed. An average of 9.9 aberrations per tumor was found in the metastases. Gains of chromosome arms 20q (85%), 13q (48%), 7p (44%), and 8q (44%) and losses of chromosome arms 18q (89%), 8p (59%), 1p (56%), and 18p (48%) were detected most frequently. Chromosomes 14 and 15 were lost in 26% and 30% of the metastases, respectively. No consistent differences were observed between primary tumors and synchronous metastases. Fluorescence in situ hybridization (FISH) was used for further characterization of gains of chromosome arm 20q. Touch preparations of 13 tumors that had demonstrated 20q gain with CGH were examined with FISH by use of a set of probes mapping to different parts of 20q. A probe for 20p was used as a reference. FISH showed relative gain of at least one 20q locus in 12 of the tumors. High-level gains were detected in 38% of the tumors, preferentially for probes mapping to band 20q13. Our CGH data indicate that colorectal metastases show chromosomal changes similar to those that have been reported for primary tumors. Chromosomal losses were seen at higher frequency, particularly for chromosomes 14 and 15. By FISH, we identified subregions on chromosome arm 20q that are frequently involved in DNA amplifications in colorectal cancer and that may harbor candidate proto-oncogenes.