Clonal origin of lymph node metastases in bladder carcinoma

BACKGROUND: Evidence of genetic heterogeneity within urothelial carcinomas of the bladder has raised questions about the clonal origin of urothelial carcinoma and its metastases. High-grade urothelial carcinoma of the bladder frequently metastasizes to multiple regional lymph nodes in the pelvis. Whether or not these multiple lymph node metastases originate from the same tumor clone is uncertain. Molecular analysis of microsatellite alterations and X-chromosome inactivation status of distinct tumor cell populations from the same patient may further our understanding of the genetic basis of carcinoma progression and metastasis. METHODS: The authors examined 24 patients who underwent radical cystectomy for urothelial carcinoma. All patients had multiple (from two to four) lymph node metastases. Genomic DNA samples were prepared from formalin fixed, paraffin embedded tissue sections using laser-assisted microdissection. Loss of heterozygosity (LOH) assays for 3 microsatellite polymorphic markers on chromosome 9p21 (D9S171, region of putative tumor suppressor gene p16), 9q32 (D9S177, putative tumor suppressor gene involved in urothelial carcinoma tumorigenesis), and 17p13 (TP53, the p53 locus) were performed. In addition, X-chromosome inactivation analysis was performed in primary tumors and metastases from 10 female patients. RESULTS: In total, 79 tumors were analyzed. The overall frequency of allelic loss was 67% (16 of 24 tumors) in the primary urothelial carcinomas and 79% (19 of 24 tumors) in the metastatic carcinomas. The primary urothelial carcinoma showed LOH at the D9S171, D9S177, and TP53 loci in 39% (9 of 23 tumors), 30% (6 of 20 tumors), and 30% (7 of 23 tumors) of informative samples, respectively. LOH in > or = 1 lymph node metastases was seen at the D9S171, D9S177, and TP53 loci in 35% (8 of 23 tumors), 45% (9 of 20 tumors), and 48% (11 of 23 tumors) of informative samples, respectively. Eleven tumors demonstrated identical allelic loss patterns at all DNA loci both in the primary carcinoma and in all corresponding lymph node metastases. Three tumors showed allelic loss in the metastatic carcinoma but not in its matched primary carcinoma. Six tumors demonstrated a different LOH pattern in each of its lymph node metastases. Clonality analysis showed the same pattern of nonrandom X-chromosome inactivation both in the primary urothelial carcinoma and in all of the lymph node metastases in five of nine informative tumors studied. Four tumors showed a random pattern of X-chromosome inactivation in both the primary carcinoma and in the metastases. CONCLUSIONS: LOH and X-chromosome inactivation assays showed that multiple lymph node metastases and matched primary urothelial carcinomas of the bladder had the same clonal origin, suggesting that the capability for metastasis often arises in only a single clonal population in the primary tumor. The variable LOH patterns observed in some of the tumors likely reflect genetic divergence during the clonal evolution of urothelial carcinoma.     

Accumulated allelic losses in the development of invasive urothelial cancer

To investigate the roles of allelic loss in the development of urothelial cancer, loss of heterozygosity was examined on 7 chromosomal arms in 49 cases of urothelial cancer of various grades and stages. Loss of heterozygosity was found on alleles in order of frequency as follows: 9q (21/38, 55%), 11p (20/44, 45%), 17p (18/42,43%), 13q (10/39,26%), 3p (8/41, 20%), 10q 2/29, 7%) and 1p (1/36, 3%). lnvasive (high-grade or ≥pT2) tumors showed the loss of 17p (13/16,81%) and the loss of 13q (7/16, 44%) with significantly higher frequencies than non-invasive (grade 1-2 ≤_pTI ) tumors. Although the loss of 3p and the loss of 11p were also more frequently associated with the invasive phenotypes, the loss of 11p was detected in a considerable number (9 of 26,35%) of non-invasive tumors. Our results indicate that the loss of 11p might generally occur at an earlier stage before the loss of 3p, 13q or 17p in tumor progression. Since no correlation was found between the loss of 9q and the tumor grade or stage, this genetic alteration appears to be unrelated to invasiveness, and could be one of the initial events in tumorigenesis. Although accumulated allelic losses of 3p, 11p, 13q and 17p are considered to be involved in the development of the invasive type of urothelial cancers, these multiple genetic alterations may have already occurred in some pathologically non-invasive urothelial cancers. Furthermore, there appears to be some variation in the pattern of cumulative allelic loss.     

Genetic alterations related to lymph node metastasis and peritoneal dissemination in epithelial ovarian cancers

Genetic alterations are assumed to be necessary for the development and progression of ovarian cancer. However, the genetic alterations that occur during lymph node metastasis and peritoneal dissemination are poorly understood. In the present study, we used comparative genomic hybridization to detect genetic alterations in 30 tumors from patients with primary ovarian cancers and analyzed the associations of these genetic alterations with clinical stage and surgical pathological factors, such as histological grade, lymph node metastasis, and peritoneal dissemination. The total number of genetic alterations per tumor ranged from 0 to 39, with an average of 17.7 alterations per tumor. Among the genetic alterations in ovarian cancers, gains on chromosomes 8q and 3q and losses on chromosomes 17p, 18q, and 4q were observed frequently. Although the difference in total numbers of genetic alterations between early-stage tumors and advanced-stage tumors was not significant, the difference was significant when high-grade cancers were compared with low-grade cancers. Eight regions on seven chromosomes showed genetic alterations related to lymph node metastasis or peritoneal dissemination. Gain at 11q13-q14 and loss at 17q11.2-q21 were related not only to lymph node metastasis and peritoneal dissemination but also to clinical stage and histological grade.     

Genetic characterization of lung metastases in renal cell carcinoma

Prognosis of patients with renal cell carcinoma (RCC) is mainly determined by metastases. The understanding of the metastatic process will give the basis for a differential diagnosis leading to an individual prognosis and to new therapeutical strategies. In order to define specific genetic alterations which are common in renal cancer metastases of the lung, we performed comparative genomic hybridization (CGH) on metastases and in some cases on their related primary renal tumors. For CGH, DNA was isolated from 2 or 5 paraffin sections (5 micro m). Tumor and normal (control) DNAs were amplified by DOP-PCR and labeled with biotin-dUTP and digoxigenin-dUTP, respectively. Hybridization and detection were carried out according to standard protocols. In 33 out of 40 metastases, genetic alterations were detected, most frequently these were losses of chromosomes 3p (74%), 8p (31%), 9 or 9q (34%), 14 [26%, 18q (40%) and gains of chromosome 5/5q 34%], 7 (31%) and 12 (26%). Combination of loss of 8p and gain of 8q occurred frequently. The mean number of aberrations per tumor was 8.1 (1-11). The comparison of alterations in related primary and metastatic tumors showed identical alterations in 5 out of 8 cases. This study demonstrates, that lung metastases from renal cell carcinoma are characterized by an accumulation of specific genetic alterations which show a clonal relationship to the related primary tumors.     

A comparison of DNA copy number changes detected by comparative genomic hybridization in malignancies of the liver, biliary tract and pancreas

Tumors arising from the liver, biliary tract and pancreas, which originate in the foregut and are in close anatomical proximity to each other, sometimes show similar histological features. No studies have focused on genetic similarities and differences between tumors of these organs. To elucidate the similarities and differences in DNA copy number alterations between tumors of these organs, we applied comparative genomic hybridization (CGH) to cancers of the liver (31 cases), biliary tract (42 cases) and pancreas (27 cases). Some alterations were common to tumors of all three organs, and some were preferential in certain types of tumor. Gains of 1q and 8q and losses of 8p and 17p were common to all tumors. In contrast, 13q14 and 16q losses were detected exclusively in hepatocellular carcinomas (HCCs; p < 0.01). The incidence of 17q21 gain and 5q loss was higher in biliary tract cancers than in the other two types (p < 0.05). Pancreatic cancers exhibited higher incidence of 5q14-q23 gain and 19p loss than tumors of other organs (p < 0.01). Gains of 7p, 7q, 12p and 20q and losses of 3p, 6q, 9p and 18q were frequent in both biliary tract and pancreatic cancers but rare in HCCs (p < 0.05). The present results suggest that although genes located at 1q, 8p, 8q and 17p are frequently involved in HCC, biliary tract and pancreatic cancer, at least some of the genes implicated in carcinogenesis are different between these three types. It is also suggested that CGH analysis is useful as a potential adjunct for the diagnosis and management of these tumors of organs that are anatomically close to one another.     

Genetic analysis of multifocal superficial urothelial cancers by array-based comparative genomic hybridisation

The purpose of this study was to investigate the accumulation of genetic alterations during metachronous and/or synchronous development of multifocal low-grade superficial urothelial tumours in the same patient, by using array-based comparative genomic hybridisation (array-CGH) and FGFR mutation analysis. We analysed 24 tumours (pTa-1 G1-2) from five patients. We had previously identified a clonal relationship among the tumours of each patient by microsatellite analysis. This time, unsupervised hierarchical cluster analysis revealed that the tumours from each patient were clustered together independently of the tumours from the other patients. All of the tumours from a single patient showed a set of 2-7 identical regional or whole-arm chromosomal changes. In addition, several individual alterations were also found. Cladistic diagrams revealed that the accumulation of genetic alterations could not be explained by a linear model, and the existence of a hypothetical precursor cell was assumed in four patients. In some cases, FGFR mutation seemed to occur later during multifocal tumour development. Taken together, these findings suggest that low-grade superficial urothelial tumours accumulate minor genetic alterations during multifocal development, although these tumours are genetically stable.     

Isolation of disseminated neuroblastoma cells from bone marrow aspirates for pretreatment risk assessment by array comparative genomic hybridization

In neuroblastoma, tumor biopsies are used for prognostic evaluation and risk assessment by molecular genetic analyses such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (array CGH). Analysis of primary tumors by array CGH can be hampered by the lack of sufficient tumor cells due to small biopsy size or availability of invaded bone marrow only. Given the importance of accurate assessment of genetic alterations in the diagnostic work-up of patients with neuroblastoma, we evaluated the possibility to analyze bone marrow metastases in patients with disseminated disease. Disseminated neuroblastoma cells were isolated from bone marrow aspirates by using either laser capture microdissection (LCM) or magnetic activated cell sorting (MACS). The array CGH profiles of these isolated metastases were compared to array CGH profiles and/or FISH data of the corresponding primary tumor. Here, we show that the major recurrent DNA copy number alterations detected in primary neuroblastoma tumors (i.e., 1p, 3p and 11q deletion, 17q gain and MYCN amplification) can be detected, with high sensitivity and specificity, in the disseminated neuroblastoma cells isolated from the bone marrow aspirates, using an array platform with high coverage for these regions. Moreover, we demonstrate that for archived material, for example, for retrospective studies, LCM is the method of choice, while for fresh bone marrow aspirates, acquired at the time of diagnosis, MACS is superior.     

Molecular evidence supporting field effect in urothelial carcinogenesis.

PURPOSE: Human urothelial carcinoma is thought to arise from a field change that affects the entire urothelium. Multifocality of urothelial carcinoma is a common finding at endoscopy and surgery. Whether these coexisting tumors arise independently or are derived from the same tumor clone is uncertain. Molecular analysis of microsatellite alterations and X-chromosome inactivation status in the cells from each coexisting tumor may further our understanding of urothelial carcinogenesis. EXPERIMENTAL DESIGN: We examined 58 tumors from 21 patients who underwent surgical excision for urothelial carcinoma. All patients had multiple separate foci of urothelial carcinoma (two to four) within the urinary tract. Genomic DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-capture microdissection. Loss of heterozygosity (LOH) assays for three microsatellite polymorphic markers on chromosome 9p21 (IFNA and D9S171), regions of putative tumor suppressor gene p16, and on chromosome 17p13 (TP53), the p53 tumor suppressor gene locus, were done. X-chromosome inactivation analysis was done on the urothelial tumors from 11 female patients. RESULTS: Seventeen of 21 (81%) cases showed allelic loss in one or more of the urothelial tumors in at least one of the three polymorphic markers analyzed. Concordant allelic loss patterns between each coexisting urothelial tumor were seen in only 3 of 21 (14%) cases. A concordant pattern of nonrandom X-chromosome inactivation in the multiple coexisting urothelial tumors was seen in only 3 of 11 female patients; of these 3 cases, only one displayed an identical allelic loss pattern in all of the tumors on LOH analysis. CONCLUSION: LOH and X-chromosome inactivation assays show that the coexisting tumors in many cases of multifocal urothelial carcinoma have a unique clonal origin and arise from independently transformed progenitor urothelial cells, supporting the "field effect" theory for urothelial carcinogenesis.     

采用对比基因杂交技术建立嗅神经母细胞瘤基因畸变模型

目的建立嗅神经母细胞瘤(ENB)的基因畸变模型并分析其特征。方法采用对比基因杂交(CGH)技术对12例原发、3例复发、7例转移ENB标本进行基因检测,用特殊软件系统对所采集照片的荧光信号进行量化分析,确定肿瘤DNA与正常对照DNA之间的基因差别。结果ENB常见DNA丢失主要见于1P、2q、3p／q、4p／q、5p／q、6q、8p／q、9p、10p／q、11P、12q、13q、18q和21q,DNA过度表达见于1p、7q、9q、1lq、14q、16p／q、17p／q、19p／q、20p／q和22p／q。ENB的1p21-p31DNA丢失与该类肿瘤患者预后差有明显相关性。死于ENB的患者均具有以下共性：1p21-p31DNA丢失、临床分期为C或D期、分化程度低(Ⅲ或Ⅳ级)。对4例发生转移、复发的患者,将检测结果与其原发病灶基因畸变情况进行对比分析表明,转移、复发病灶与其原发病灶间具有高度的克隆一致性。结论采用CGH能建立ENB的典型基因畸变模型,该模型有助于解释此肿瘤的生物学特性并对其预后进行评估,并与神经母细胞瘤、小细胞肺癌以及头颈部鳞癌进行鉴别。     

Comparative genomic hybridization detects genetic imbalances in primary ovarian carcinomas as correlated with grade of differentiation

BACKGROUND: In the current study the authors attempted to evaluate genetic alterations in a large set of primary ovarian carcinomas and to compare the genetic findings with clinical parameters such as grade of tumor differentiation. This strategy was applied to identify chromosomal regions containing genes associated with tumor progression. METHODS: Genetic imbalances were assessed in 106 primary ovarian carcinomas using comparative genomic hybridization (CGH). CGH was applied because it is a powerful tool with which to screen the entire genome of a tumor for genetic changes by highlighting regions of altered DNA sequence copy numbers (deletions and amplifications). Multivariate statistical standard procedures were used to determine an association between tumor grading and genetic alterations. RESULTS: One hundred three carcinomas showed aberrant CGH profiles. The most frequent alterations were amplifications of 8q, 1q, 20q, 3q, and 19p, which occurred in 69-53% of tumors, and underrepresentations of 13q, 4q, and 18q, which occurred in 54-50% of tumors. Undifferentiated ovarian carcinomas (World Health Organization Grade 3) were found to be correlated significantly with underrepresentation of 11p and 13q as well as with overrepresentation of 8q and 7p (P = 0.001, 0.001, 0.01, and 0.027, respectively). However, 12p underrepresentation and 18p overrepresentation were significantly more frequent in well and moderately differentiated tumors (P = 0.01 and 0.004, respectively). To facilitate the interpretation and clinical application of the results of the current study, the significant aberrations were translated into a score system. This score system can be used easily for the prediction of an undifferentiated phenotype with a specificity and sensitivity of 79% and 86%, respectively. CONCLUSIONS: The current study data show that primary ovarian carcinomas are based on consistent genetic alterations that most likely are important for the development of this tumor entity. The correlation between certain aberrations and undifferentiated carcinomas may help to discriminate between primary and secondary genetic events and may indicate the location of those genes involved in cellular functions associated with tumor progression and the development of anaplastic and aggressive phenotypes.     

Clonal and parallel evolution of primary lung cancers and their metastases revealed by molecular dissection of cancer cells.

PURPOSE: Several models of cancer progression, including clonal evolution, parallel evolution, and same-gene models, have been proposed to date. The purpose of this study is to investigate the authenticity of these models by comparison of accumulated genetic alterations between primary and corresponding metastatic lung cancers. EXPERIMENTAL DESIGN: A whole-genome allelic imbalance scanning using a high-resolution single nucleotide polymorphism array and mutational analysis of the p53, EGFR, and KRAS genes were done on eight sets of primary and metastatic lung cancers. Based on the genotype data, the natural history of each case was deduced, and candidate metastasis suppressor loci were determined. RESULTS: Five to 20 chromosomal regions showed allelic imbalance in each tumor. Accumulated genetic alterations were similar between primary and corresponding metastatic tumors, and the majority(>67%) of genetic alterations detected in metastatic tumors was also detected in the corresponding primary tumors. On the other hand, in seven of the eight cases, there were genetic alterations accumulated only in metastatic tumors. Among these alterations, allelic imbalances at chromosome 11p15 and 11p11-p13 regions were the most frequent ones (4 of 8, 50%). Likewise, four cases showed genetic alterations detected only in primary tumors. CONCLUSIONS: The natural history of each case indicated that the process of metastasis varies among cases, and that all three models are applicable to lung cancer progression. According to the clonal and parallel evolution models, it is possible that a metastasis suppressor gene(s) for lung cancer is present on chromosome 11p.     

Evidence for oligoclonality and tumor spread by intraluminal seeding in multifocal urothelial carcinomas of the upper and lower urinary tract

Multifocality and recurrence of urothelial carcinoma may result from either the field effect of carcinogens leading to oligoclonal tumors or monoclonal tumor spread. Previous molecular studies, favoring the monoclonality hypothesis, are mostly limited to the urinary bladder. We investigated genetic alterations in a total of 94 synchronous or metachronous multifocal tumors from 19 patients with at least one tumor both in the upper and lower urinary tract. Loss of heterozygosity (LOH) was determined using eight markers on chromosome 9 and one marker on 17p13 (p53). Microsatellite instability was investigated at six loci and protein expression of MSH2 and MLH1 was evaluated by immunohistochemistry. In addition, exons 5-9 of the p53 gene were sequenced. Deletions at chromosome 9 were found in 73% of tumors and at 17p13 in 18% of tumors. There was no significant difference in the frequency of LOH in the upper and lower urinary tract. Deletions at 9p21 were significantly correlated with invasive tumor growth. The pattern of deletion revealed monoclonality of all tumors in nine patients. In five patients there were at least two tumor clones with different genetic alterations. In four of these patients the different clones occurred in the bladder and subsequently in the ureter and renal pelvis. All four patients with p53 mutations revealed identical mutations in all tumors. Thus, multifocal urothelial carcinomas are frequently monoclonal, whereas others show oligoclonality, providing molecular evidence for field cancerization. Intraluminal tumor cell seeding appears to be an important mechanism of multifocal occurrence and recurrence of urothelial carcinomas.     

Superimposed histologic and genetic mapping of chromosome 9 in progression of human urinary bladder neoplasia: implications for a genetic model of multistep urothelial carcinogenesis and early detection of urinary bladder cancer

The evolution of alterations on chromosome 9, including the putative tumor suppressor genes mapped to the 9p21-22 region (the MTS genes), was studied in relation to the progression of human urinary bladder neoplasia by using whole organ superimposed histologic and genetic mapping in cystectomy specimens and was verified in urinary bladder tumors of various pathogenetic subsets with longterm follow-up. The applicability of chromosome 9 allelic losses as non-invasive markers of urothelial neoplasia was tested on voided urine and/or bladder washings of patients with urinary bladder cancer. Although sequential multiple hits in the MTS locus were documented in the development of intraurothelial precursor lesions, the MTS genes do not seem to represent a major target for p21-23 deletions in bladder cancer. Two additional tumor suppressor genes involved in bladder neoplasia located distally and proximally to the MTS locus within p22-23 and p11-13 regions respectively were identified. Several distinct putative tumor suppressor gene loci within the q12-13, q21-22, and q34 regions were identified on the q arm. In particular, the pericentromeric q12-13 area may contain the critical tumor suppressor gene or genes for the development of early urothelial neoplasia. Allelic losses of chromosome 9 were associated with expansion of the abnormal urothelial clone which frequently involved large areas of urinary bladder mucosa. These losses could be found in a high proportion of urothelial tumors and in voided urine or bladder washing samples of nearly all patients with urinary bladder carcinoma.     

Genomic profiling reveals alternative genetic pathways of prostate tumorigenesis

Prostate cancer is clinically heterogeneous, ranging from indolent to lethal disease. Expression profiling previously defined three subtypes of prostate cancer, one (subtype-1) linked to clinically favorable behavior, and the others (subtypes-2 and -3) linked with a more aggressive form of the disease. To explore disease heterogeneity at the genomic level, we carried out array-based comparative genomic hybridization (array CGH) on 64 prostate tumor specimens, including 55 primary tumors and 9 pelvic lymph node metastases. Unsupervised cluster analysis of DNA copy number alterations (CNA) identified recurrent aberrations, including a 6q15-deletion group associated with subtype-1 gene expression patterns and decreased tumor recurrence. Supervised analysis further disclosed distinct patterns of CNA among gene-expression subtypes, where subtype-1 tumors exhibited characteristic deletions at 5q21 and 6q15, and subtype-2 cases harbored deletions at 8p21 (NKX3-1) and 21q22 (resulting in TMPRSS2-ERG fusion). Lymph node metastases, predominantly subtype-3, displayed overall higher frequencies of CNA, and in particular gains at 8q24 (MYC) and 16p13, and loss at 10q23 (PTEN) and 16q23. Our findings reveal that prostate cancers develop via a limited number of alternative preferred genetic pathways. The resultant molecular genetic subtypes provide a new framework for investigating prostate cancer biology and explain in part the clinical heterogeneity of the disease.     

Array CGH demonstrates characteristic aberration signatures in human papillary thyroid carcinomas governed by RET/PTC

The aim of this study is to investigate additional genetic alterations in papillary thyroid carcinomas (PTCs) with known RET/PTC rearrangements. We applied array-based comparative genomic hybridization (array CGH) to 33 PTC (20 PTC from adults, 13 post-Chernobyl PTC from children) with known RET/PTC status. Principal component analysis and hierarchical cluster analysis identified cases with similar aberration patterns. Significant deviations between tumour-groups were obtained by statistical testing (Fisher's exact test in combination with Benjamini-Hochberg FDR-controlling procedure). FISH analysis on FFPE sections was applied to validate the array CGH data. Deletions were found more frequently in RET/PTC-positive and RET/PTC-negative tumours than amplifications. Specific aberration signatures were identified that discriminated between RET/PTC-positive and RET/PTC-negative cases (aberrations on chromosomes 1p, 3q, 4p, 7p, 9p/q, 10q, 12q, 13q and 21q). In addition, childhood and adult RET/PTC-positive cases differ significantly for a deletion on the distal part of chromosome 1p. There are additional alterations in RET/PTC-positive tumours, which may act as modifiers of RET activation. In contrast, alterations in RET/PTC-negative tumours indicate alternative routes of tumour development. The data presented serve as a starting point for further studies on gene expression and function of genes identified in this study.     

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.     

High-resolution analysis of DNA copy number alterations in colorectal cancer by array-based comparative genomic hybridization

Array-based comparative genomic hybridization (CGH) allows for the simultaneous examination of thousands of genomic loci at 1-2 Mb resolution. Copy number alterations detected by array-based CGH can aid in the identification and localization of cancer causing genes. Here we report the results of array-based CGH in a set of 125 primary colorectal tumors hybridized onto an array consisting of 2463 bacterial artificial chromosome clones. On average, 17.3% of the entire genome was altered in our samples (8.5 +/- 6.7% gained and 8.8 +/- 7.3% lost). Losses involving 8p, 17p, 18p or 18q occurred in 37, 46, 49 and 60% of cases, respectively. Gains involving 8q or 20q were observed 42 and 65% of the time, respectively. A transition from loss to gain occurred on chromosome 8 between 41 and 48 Mb, with 25% of cases demonstrating a gain of 8p11 (45-53 Mb). Chromosome 8 also contained four distinct loci demonstrating high-level amplifications, centering at 44.9, 60, 92.7 and 144.7 Mb. On 20q multiple high-level amplifications were observed, centering at 32.3, 37.8, 45.4, 54.7, 59.4 and 65 Mb. Few differences in DNA copy number alterations were associated with tumor stage, location, age and sex of the patient. Microsatellite stable and unstable (MSI-H) tumors differed significantly with respect to the frequency of alterations (20 versus 5%, respectively, P < 0.01). Interestingly, MSI-H tumors were also observed to have DNA copy number alterations, most commonly involving 8q. This high-resolution analysis of DNA copy number alterations in colorectal cancer by array-based CGH allowed for the identification of many small, previously uncharacterized, genomic regions, such as on chromosomes 8 and 20. Array-based CGH was also able to identify DNA copy number changes in MSI-H tumors.     

Comparative genomic hybridization of fine needle aspirates from breast carcinomas

Detailed knowledge of chromosomal aberrations in a specific tumor may facilitate the development of individually tailored chemotherapy, hormone or gene therapy. Unfortunately, karyotype analysis requires living cells and is complicated by the low number of good metaphase spreads obtained. Comparative genomic hybridization (CGH), however, is capable of detecting and mapping genome-wide amplifications and deletions using an equimolar mixture of normal and tumor cell DNA. We show here that even the few cells from a fine needle aspirate of a tumor are sufficient for a direct CGH assay, independent of DNA amplification. Ten primary breast cancers were analyzed by CGH. A fresh frozen fine needle aspirate and a formalin-fixed and paraffin-embedded section were used for each tumor. Metaphases from each CGH reaction were imaged, and a sum ratio profile was determined for every chromosome. The ratio profiles of DNA isolated from the 2 material sources were then compared. Fine needle aspirates and the paraffin-embedded material of a single tumor yielded the same fluorescence ratio profiles, albeit with slightly different confidence intervals. Different tumors showed a variety of aberrations. The most frequently observed changes were 1q+, 8q+, 14q-, 16p+, 16q-, 17p-, 17q+, 19q+, 20q+, 21q- and 22q-. The ability of CGH to assess chromosomal changes in breast cancer from fine needle aspirates could facilitate genetic evaluation of tumors prior to surgery.     

Frequent genetic heterogeneity in the clonal evolution of gynecological carcinosarcoma and its influence on phenotypic diversity

Carcinosarcomas of the uterus, ovaries, and fallopian tubes are highly aggressive neoplasms with incompletely understood histogenesis. Although recent immunohistochemical, cell culture, and molecular genetic studies all favor these cancers to be monoclonal in origin, the extent of intratumoral genetic heterogeneity in these tumors with divergent histology has not been reported previously. For this study, we microdissected a total of 172 carcinomatous or sarcomatous foci from 17 gynecological carcinosarcomas and analyzed allelic status with 41 microsatellite markers on chromosomal arms 1p, 1q, 3p, 4q, 5q, 6q, 8p, 9p, 10q, 11p, 11q, 13q, 16q, 17p, 17q, 18q, and 22q. With the exception of a single case with microsatellite instability, we found shared allelic losses and retentions among multiple individually dissected foci of each case, strongly supportive of the concept of a monoclonal origin for these neoplasms. In eight of these cases, we also found heterogeneous patterns of allelic loss at limited numbers of chromosomal loci in either the carcinomatous or sarcomatous components of the neoplasms. These heterogeneous patterns of allelic losses were consistent with either genetic progression or genetic diversion occurring during the clonal evolution of these neoplasms. In two cases, we found the specific patterns of genetic progression to be consistent with sarcomatous components of the neoplasms arising from carcinomatous components. We conclude that most of the gynecological carcinosarcomas have a monoclonal origin, and that genetic progression and diversion parallel the development of divergent phenotypes in these tumors. Because phenotypically divergent areas of the tumors share numerous genetic alterations, this divergence most likely occurs relatively late in the evolution of these tumors.     

Correlation of allelic losses and clinicopathological factors in primary breast cancers

Human breast cancers frequently show allelic loss or loss of heterozygosity (LOH) at specific chromosomal regions. To understand the possible role of these genetic alterations in tumor development and progression, we examined LOH at loci on chromosomal arms lp, 3p, 11p, 13q, 16q, 18q, and 22q in 140 to 246 cases of primary breast cancers and compared it with lymph node metastasis, histological type, tumor stage, estrogen receptor (ER) and progesterone receptor (PgR) status. LOH at 1_p22-31 correlated with lymph node metastasis and a tumor size of greater than 2 cm. LOH at 13ql2-14 and 18q21 were most frequently observed in tumors of the solid-tubular type. LOH at 1p34-36 was more frequent in tumors of the scirrhous and solidtubular types than in other less aggressive histological types. Furthermore, a significant association was observed between LOH at 3pl4-21, 11p11-15 and 13ql2-14 and the absence of progesterone receptors. These results suggest that some clinical characteristics of breast cancers are determined by loss of tumor suppressor genes present at specific chromosomal regions.