Patterns Of Allelic Loss Research Articles

Clonal divergence in lung cancer development is associated with allelic loss on chromosome 4

Patients who receive curative treatment for lung cancer can develop additional lung tumors that may or may not be related to the original tumor and thus require different clinical management. If a subsequent tumor has a pattern of allele loss, revealed by allelotype analysis, overlapping that of the first tumor, it is believed to be a local recurrence or metastasis. In this case history, we present loss of heterozygosity analyses of the original primary tumor, and two second primary tumors occurring in the ipsilateral and the contra-lateral lungs. The allelotyping suggests that these tumors are all clonally related but concordance is not complete. Our interpretation is that the original primary tumor and the two new primary tumors have developed to full malignancy independently, but are clonally related, possibly via a clone of motile progenitor cells. Deletion mapping of DNA from biopsies of this patient delineated a region in 4p16 that we had previously shown to be lost in the transition from carcinoma in situ to invasive tumor. We identified a minimally deleted region encompassing six genes including two candidate tumor suppressor genes, CRMP1 a lung cancer metastasis-suppressing gene and PPP2R2C a gene for a regulatory subunit of the PP2 complex known to suppress tumorigenesis, particularly viral induced transformation.

Frequent allelic loss at the FRA3B site in endemic nasopharyngeal carcinoma: association with clinical features and Epstein–Barr virus infection

In this study, we aimed to precisely define the patterns of allelic loss at the FRA3B site in endemic nasopharyngeal carcinoma and to determine whether an association exists between allelic loss, clinicopathological features and Epstein-Barr virus infection. We examined the loss of heterozygosity in 40 cases of nasopharyngeal carcinoma from an endemic area in southern China, using eight high dense, polymorphic, microsatellite markers within or flanking the FRA3B site. Loss of heterozygosity at the FRA3B region was shown in 31 (77.5 per cent) primary tumours. Loss of heterozygosity was found most frequently at the D3S1300 (55.6 per cent) and D3S2757 (50.0 per cent) loci. The common area of deletion was located between the D3S4103 and D3S4260 loci. In nasopharyngeal carcinoma, loss of heterozygosity at the FRA3B/fragile histidine triad locus correlated with the following clinicopathological parameters: tumour T-stage, lymph node status, clinical stage, tumour differentiation and serum antibody titres of immunoglobulin (Ig) A against Epstein-Barr virus capsid antigen. Significantly frequent loss of heterozygosity was observed in nasopharyngeal carcinoma with tumour stages T3 and T4, lymph node metastasis and advanced tumour-node-metastasis staging (III and IV). Very frequent loss of heterozygosity was also observed to correlate with World Health Organization type III nasopharyngeal carcinoma histopathology. We also found that nasopharyngeal carcinoma patients with high titres of IgA against Epstein-Barr virus capsid antigen showed very frequent loss of heterozygosity. Allelic loss at the FRA3B site occurs significantly more commonly in endemic nasopharyngeal carcinoma patients. This suggests that the region between D3S4103 and D3S4260 may represent a preferential molecular target in nasopharyngeal carcinogenesis.

Histogenesis of sarcomatoid urothelial carcinoma of the urinary bladder: evidence for a common clonal origin with divergent differentiation

The histogenesis of sarcomatoid urothelial carcinoma, a rare neoplasm with bidirectional epithelial and mesenchymal differentiation, has been a matter of controversy. To clarify its origin, we analysed the status of X-chromosome inactivation in sarcomatoid urothelial carcinomas from 10 female patients and examined losses of heterozygosity (LOH) in these specimens and in additional 20 tumours from male patients. Six polymorphic microsatellite markers where genetic alterations occur frequently in early or advanced stages of urothelial carcinomas, including D3S3050, D8S261, IFNA, D9S177, D11S569 and TP53, were investigated in the current study. The identical pattern of non-random X-chromosome inactivation in both carcinomatous and sarcomatous components was identified in five of eight informative female patients, and the remaining three informative cases showed a random, but concordant, pattern of X-chromosome inactivation. The concordant X-chromosome inactivation results in all eight informative cases support the concept of a monoclonal origin of both components of this biphasic neoplasm. Among the tumours demonstrating loss of heterozygosity, high incidences of an identical pattern of allelic loss between carcinomatous and sarcomatous components were identified in genetic alterations associated with early carcinogenesis: 86% at D8S261, 78% at D11S569, 75% at D9S177 and 57% at IFNA. In contrast, concordant LOH patterns were less frequently observed for microsatellites related to advanced carcinogenesis: only 40% at D3S3050 and 40% at TP53. The significant overlap of loss of heterozygosity supports a monoclonal cell origin and suggests that clonal divergence may occur during tumour progression and differentiation. Divergent patterns of discordant allelic loss of microsatellite markers imply that heterogeneous pathogenetic pathways may exist in the evolution of this enigmatic neoplasm.

Distinct Pattern of Allelic Loss and Inactivation of Cadherin 1 and 5 Genes in Mammary Carcinomas Arising in p53+/- Mice

p53 is one of the most frequently mutated genes in mammary carcinomas (MCs). To detect tumor suppressor genes cooperating with a hetero-deficient p53 gene in mammary carcinogenesis, we first examined allelotypes in MCs from (BALB/cHeA x MSM/Ms) F(1)- p53(+/-) and (BALB/cHeA x 129/SvEv) F(1)- p53(+/-) female mice, and then surveyed down-regulated genes in the allelic loss regions. Genome-wide screening at 42 loci identified frequent (more than 30%) loss of heterozygosity (LOH) on chromosomes 5, 8, 11, 12, 14 and 18 in the MCs from either of the F(1) mice. The MCs in the p53(+/- )mice indicated highly frequent LOH, especially on chromosomes 8, 11 and 12, distinct from other mouse tumors. More than 60% of the 38 MCs from (BALB/cHeA x MSM/Ms) F(1)- p53 (+/-) mice showed LOH in a region ranging from D8Mit85 (105.0 Mb from centromere) to D8Mit113 (111.8 Mb) on chromosome 8, a region syntenic to human chromosome 16q22.1, on which LOH has been found in breast cancers. RT-PCR analyses revealed that the LOH of chromosome 8 was associated with the reduced and/or complete loss of expression of Cdh1 and Cdh5 genes in 15 (58%) and 8 (31%) of 26 MCs derived from the F(1) mice, respectively. Thus, inactivation of Cdh1 and Cdh5 is likely to cooperate with the loss of p53, suggesting a possible tumor suppressive function of these genes in mammary carcinogenesis.

Association of Loss of Heterozygosity and PTEN Gene Abnormalities with Paraclinical, Clinical Modalities and Survival Time of Glioma Patients in Malaysia

The pattern of allelic loss of heterozygosity (LOH) and PTEN mutations appear to be associated with the progression of gliomas leading to a decrement in the survival rate of patients. This present study was carried out to determine the LOH and PTEN mutational status in glioma patients and its association with patients' survival. Thirty-seven Malaysian glioma patients of the Malay race were subject to PTEN mutational analysis and the presence of LOH using the cold single-strand conformation polymorphism method, and their clinical and paraclinical response were correlated. Among analysed glioma patients, seven (21.6%) cases with PTEN mutations were detected and 12 (32.4%) of 37 patients showed presence of LOH. Univariate analysis showed that tumour grade, vascularization, PTEN mutation, LOH and combination of both PTEN mutation and LOH were significantly associated with glioma patients' survival. Multivariate analysis revealed that no factors contributed to survival time. The results show that PTEN mutation and LOH are quite frequent in Malaysian glioma patients. However, they have no impact on the survival outcome of patients.

Clonal Origin of Metastatic Testicular Teratomas

Testicular teratomas in adult patients are histologically diverse tumors that frequently coexist with other germ cell tumor (GCT) components. These mixed GCTs often metastasize to retroperitoneal lymph nodes where multiple GCT elements are frequently present in the same metastatic lesion. Neither the genetic relationships among the different components in metastatic lesions nor the relationships between primary and metastatic GCT components have been elucidated. We examined metastases from 31 patients who underwent primary retroperitoneal lymph node dissection for metastatic testicular GCT. All patients had metastatic mature teratoma with one or more other GCT components. This study included a total of 72 metastatic GCT components and 16 primary GCT components from 31 patients. Genomic DNA samples from each component were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-assisted microdissection. Loss of heterozygosity (LOH) assays for seven microsatellite polymorphic markers on chromosomes 1p36 (D1S1646), 9p21 (D9S171 and IFNA), 9q21 (D9S303), 13q22-q31 (D13S317), 18q22 (D18S543), and 18q21 (D18S60) were done to assess clonality. Twenty-nine of 31 (94%) cases showed allelic loss in one or more components of the metastatic GCTs. Twenty-nine of 31 mature teratomas showed allelic loss in at least one of seven microsatellite polymorphic markers analyzed. The frequency of allelic loss in informative cases of metastatic mature teratoma was 27% (8 of 30) with D1S1646, 34% (10 of 29) with D9S171, 37% (10 of 27) with IFNA, 27% (8 of 30) with D9S303, 46% (13 of 28) with D13S317, 26% (7 of 27) with D18S543, and 36% (10 of 28) with D18S60. Completely concordant allelic loss patterns between the mature teratoma and all of the other metastatic GCT components were seen in 26 of 29 cases in which the mature teratoma component showed LOH. Nearly identical allelic loss patterns were seen in the three remaining cases. In six cases analyzed, LOH patterns of each metastatic component were compared with each GCT component of the primary testicular tumor. In all six cases, each primary and metastatic component showed an identical pattern of allelic loss. Our data support the common clonal origin of metastatic mature teratomas with other components of metastatic testicular GCTs and with each component of the primary tumor.

Genomic profiles of colorectal cancers differ based on patient smoking status

Human sporadic colorectal cancer is the result of a lengthy somatic evolutionary process facilitated by various forms of genomic instability. Such instability arises endogenously from mutations in genes whose role is to preserve genomic integrity, and exogenously from environmental agents that generate genomic damage. We have found that cigarette smoking shifts the genomic profiles and genomic instability patterns of colorectal carcinomas. The genomic profiles of 57 consecutive cancers were examined; 31 cases were current or former smokers and 26 were nonsmokers. Genome-wide allelotypes of 348 markers were examined, along with comparative genomic hybridization (CGH) on ordered BAC microarrays, microsatellite instability, and inter-(simple sequence repeat) polymerase chain reaction instability. Tumors from nonsmokers exhibited losses of heterozygosity, particularly on chromosomes 14 and 18, whereas tumors from smokers exhibited a more diffuse pattern of allelic losses. Tumors from smokers exhibited higher overall rates of loss of heterozygosity, but showed lower rates of background microsatellite instability (MSI-L). On BAC array CGH, higher levels of generalized amplifications and deletions were observed in tumors from smokers, differentially affecting male smokers. In the transforming growth factor-β signaling pathway, MADH4 mutations were more common in tumors from smokers, whereas transforming growth factor-β RII mutations were more common among nonsmokers.

Genetic and expression analysis of the KCNRG gene in hepatocellular carcinomas

The potassium channels are ubiquitous multisubunit membrane proteins, and potassium-dependent alterations in the membrane potential play an important role in the proliferation of many types of cells. This study analyzed the mutation, allelic loss and expression patterns of the KCNRG gene in 77 HCCs in order to determine if the KCNRG gene, which encodes the potassium channel regulating protein, is involved in the tumorigenesis of hepatocellular carcinoma (HCC). One KCNRG missense mutation, CGT CAT (Arg His) was found at codon 92 within the T1 domain. Hep3B hepatoma cells were transfected with the wild- or mutant-KCNRG to determine the effect of this mutation in KCNRG. Interestingly, the suppressive cell growth activity of the mutant-type KCNRG was significantly lower than that of the wild-type KCNRG. In addition, allelic loss was detected in 17 out of 64 (26.5%) informative HCC cases, and all were hepatitis B virus (HBV)-positive. Moreover, the allelic loss was closely related to an intrahepatic metastasis (P=0.0247), higher grade (P=0.0078) and clinical stage (P=0.0071). Expression analysis revealed 22 tumor tissues to have a loss of expression of the KCNRG transcript. These results suggest that genetic alterations and the expression of KCNRG might play an important role in the development and/or progression of a subset of HCCs.

Comparison of allelic losses in chondroblastoma and primary chondrosarcoma of bone and correlation with fluorescence in situ hybridization analysis

Chondroblastoma (CBL) is a benign neoplasm of bone for which the genomic characteristics remain unclear. We compared the status of allelic losses of CBL with that seen in a set of chondrosarcomas (CS) to determine whether differences in their natural history and behavior are also reflected genetically. Eleven cases of CBL and 10 cases of CS of different grades were included. Tumors were subjected to microdissection and polymerase chain reaction using 17 markers located near genes on chromosomes 5, 9, 11, 13, 17, and 19. The selected chromosomes are known to be involved in several mesenchymal neoplasms. Fluorescence in situ hybridization was also performed on tumors displaying allelic losses, with dual-color probes for 9p, 17p, and 13q. Fractional allelic losses per gene ranged from 18.2% to 63.7% in CBLs and from 28.6% to 66.7% in CSs. Loss of heterozygosity (LOH) of 5q, 9p, 11p, 13q, and 19q occurred in both CBLs and CSs. Loss of heterozygosity of 17p (p53 locus) occurred in 7 of 11 CBLs and in only 1 case of recurrent CS. The pattern of allelic loss was similar in low-grade CSs and CBLs. Loci with LOH in both tumor types suggest possible involvement of the genes p53, RB1, CDKN2/p16, ERC, and XRCC in tumorigenesis. Overall correlation between LOH and fluorescence in situ hybridization results was 90% with 17p13, 80% with 9p, and 60% with 13q. The role of p53 in CBL is uncertain; however, given the benign behavior of this tumor, it is probably unrelated to tumor progression.

Molecular Evidence for Independent Origin of Multifocal Neuroendocrine Tumors of the Enteropancreatic Axis

Neuroendocrine tumors of the enteropancreatic axis are often multifocal. We have investigated whether multifocal intestinal carcinoid tumors and multifocal pancreatic endocrine tumors arise independently or whether they originate from a single clone with subsequent intramural or intrapancreatic spread. Twenty-four cases, including 16 multifocal intestinal carcinoid tumors and eight multifocal pancreatic endocrine tumors, were studied. Genomic DNA samples were prepared from 72 distinct tumor nodules using laser capture microdissection. Loss of heterozygosity (LOH) assays were done using markers for putative tumor suppressor genes located on chromosomes 9p21 (p16), 11q13 (MEN1), 11q23 (SDHD), 16q21, 18q21, and 18q22-23. In addition, X chromosome inactivation analysis was done on the tumors from eight female patients. Twenty-two of 24 (92%) cases showed allelic loss in at least one tumor focus, including 15 of 16 (94%) cases of multifocal carcinoid tumors and 7 of 8 (88%) cases of multifocal pancreatic endocrine tumors. Eleven of 24 (46%) cases exhibited a different LOH pattern for each tumor. Additionally, 9 of 24 (38%) cases showed different LOH patterns among some of the coexisting tumors, whereas other coexisting tumors displayed the same allelic loss pattern. Two of 24 (8%) cases showed the same LOH pattern in every individual tumor. X chromosome inactivation analysis showed a discordant pattern of nonrandom X chromosome inactivation in two of six informative cases and concordant pattern of nonrandom X chromosome inactivation in the four remaining informative cases. Our data suggest that some multifocal neuroendocrine tumors of the enteropancreatic axis arise independently, whereas others originate as a single clone with subsequent local and discontinuous metastasis.

Molecular Evidence for the Same Clonal Origin of Multifocal Papillary Thyroid Carcinomas

Patients with papillary thyroid carcinoma often have two or more distinct papillary tumors at thyroidectomy. Whether these multifocal papillary lesions are clonally related or whether they arise independently is unknown as previous studies have shown conflicting results. Molecular analysis of microsatellite alterations and X-chromosome inactivation status in separate tumors from the same patient can be used to define the genetic relationships among the multiple coexisting tumors. We examined 64 separate tumors from 22 female patients who underwent thyroidectomy for thyroid carcinoma. All patients had multiple and separate papillary carcinomas (range, two to six). Genomic DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-capture microdissection. Loss of heterozygosity assays for three microsatellite polymorphic markers for putative tumor suppressor genes on chromosomes 3p25 (D3S1597), 9p21 (D9S161), and 18p11.22-p11 (D18S53) were done. In addition, X-chromosome inactivation analysis was done on the tumors from all patients. Twenty of 22 (91%) cases showed allelic loss in one or more of the papillary lesions in at least one of the three polymorphic markers analyzed. Concordant allelic loss patterns between coexisting papillary tumors were seen in 20 of 23 (87%) cases. A concordant pattern of nonrandom X-chromosome inactivation in the multiple coexisting papillary lesions was seen in all informative cases. Our data suggest that the multifocal tumors in patients with papillary thyroid carcinoma often arise from the same clone. Thus, intrathryoid metastasis may play an important role in the spread of papillary thyroid carcinoma, a finding that has important therapeutic, diagnostic, and prognostic implications.

Intratumor genomic heterogeneity correlates with histological grade of advanced oral squamous cell carcinoma

To assess the difference in genetic aberration patterns among the invasive tumor front (ITF), center/superficiality and the stroma adjacent to oral squamous cell carcinoma (OSCC), we studied loss of heterozygosity (LOH) and microsatellite instability (MI) at chromosome 9p21 and 17p13 on the three regions by combining laser capture microdissection (LCM) and PCR. We studied 20 OSCC patients with TP53 on chromosome 17p13 and RPS6 on chromosome 9p21. Genomic DNA samples from the ITF, center/superficial and stromal cells adjacent to the tumor were prepared from cryosections using laser-assistant microdissection, then LOH and MI were determined. Cells at the ITF, center/superficiality and stroma showed a high frequency of LOH and MI on chromosomes 17p13 (TP53) and 9p21 (RPS6). Comparison of the patterns of allelic loss and MI encountered at the ITF, center/superficial and stromal cells revealed no concordance. The frequency of RPS6 and TP53 aberration at the epithelial compartment (both ITF and center, 64.7%, 11/17; 70.6%, 12/17) was statistically higher than the stroma (23.5%, 4/17; 43.8%, 7/16) (p<0.05). Furthermore, for the epithelial compartment, the aberrations proportions of TP53 rose from 60.0% (9/15) to 64.7% (11/17) between the center/superficial part and ITF. Also the rate of RPS6 increased from 29.4% (5/17) to 58.8% (10/17) between the center/superficial parts and ITF. The overall frequency of the two markers was statistically higher at the ITF (20/32) than the center/superficial part (15/34) (p<0.05). The current study revealed that intratumor genetic heterogeneity exists in the different histological areas of OSCCs and some particular tumor cell genotypes have correlation with histological patterns.

Molecular Genetic Evidence for the Independent Origin of Multifocal Papillary Tumors in Patients with Papillary Renal Cell Carcinomas

In patients with papillary renal cell carcinoma, it is not uncommon to find two or more anatomically distinct and histologically similar tumors at radical nephrectomy. Whether these multiple papillary lesions result from intrarenal metastasis or arise independently is unknown. Previous studies have shown that multifocal clear cell renal cell carcinomas express identical allelic loss and shift patterns in the different tumors within the same kidney, consistent with a clonal origin. However, similar clonality assays for multifocal papillary renal cell neoplasia have not been done. Molecular analysis of microsatellite and chromosome alterations and X-chromosome inactivation status in separate tumors in the same patient can be used to study the genetic relationships among the coexisting multiple tumors. We examined specimens from 21 patients who underwent radical nephrectomy for renal cell carcinoma. All patients had multiple separate papillary lesions (ranging from 2 to 5). Eighteen patients had multiple papillary renal cell carcinomas. Seven had one or more papillary renal cell carcinomas with coexisting papillary adenomas. Genomic DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-capture microdissection. Loss of heterozygosity assays were done for six microsatellite polymorphic markers for putative tumor suppressor genes on chromosomes 3p14 (D3S1285), 7q31 (D7S522), 9p21 (D9S171), 16q23 (D16S507), 17q21 (D17S1795), and 17p13 (TP53). X-chromosome inactivation analyses were done on the papillary kidney tumors from three female patients. Fluorescence in situ hybridization analysis was done on the tumors of selected patients showing allelic loss at loci on chromosome 7 and/or chromosome 17. Twenty of 21 (95%) cases showed allelic loss in one or more of the papillary lesions in at least one of the six polymorphic markers analyzed. A concordant allelic loss pattern between each coexisting kidney tumor was seen in only 1 of 21 (5%) cases. A concordant pattern of nonrandom X-chromosome inactivation in the coexisting multiple papillary lesions was seen in two of three female patients. A discordant pattern of X-chromosome inactivation was seen in the tumors of the other female patient. Fluorescence in situ hybridization showed that the majority of tumors analyzed had gains of chromosomes 7 and 17. Two patients had one tumor with chromosomal gain and another separate tumor that did not. Our data suggest that, unlike multifocal clear cell renal cell carcinomas, the multiple tumors in patients with papillary renal cell carcinoma arise independently. Thus, intrarenal metastasis does not seem to play an important role in the spread of papillary renal cell carcinoma, a finding that has surgical, therapeutic, and prognostic implications.

Clonal origin of lymph node metastases in bladder carcinoma

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. 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. 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. 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.

Molecular Evidence Supporting Field Effect in Urothelial Carcinogenesis

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. 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. 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. 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.

Ovarian cancer is a heterogeneous disease

The purpose of this study was to systematically evaluate the molecular profiles of different histologic types of epithelial ovarian cancer before the disease has metastasized beyond the ovary. Stage 1 epithelial ovarian cancers were chosen for analysis of early genetic events associated with different cell types. Allelotyping of 47 cases was performed using 224 polymorphic markers. Analysis with Fisher's exact test found markers specific for grade 3 tumors and clear cell histology. Hierarchal clustering analysis using dChip software revealed that the pattern of allele loss in eight regions on four chromosomes led to grouping of grade 3 tumors, endometrioid (grades 1 and 2) tumors, and clear cell tumors. We conclude that ovarian cancer is a heterogeneous disease in which histologic phenotypes correlate with distinct genetic patterns.

Clonal divergence and genetic heterogeneity in clear cell renal cell carcinomas with sarcomatoid transformation

Approximately 5% of clear cell renal cell carcinomas contain components with sarcomatoid differentiation. It has been suggested that the sarcomatoid elements arise from the clear cell tumors as a consequence of clonal expansions of neoplastic cells with progressively more genetic alterations. Analysis of the pattern of allelic loss and X-chromosome inactivation in both the clear cell and sarcomatoid components of the same tumor allows assessment of the genetic relationship of these tumor elements. The authors of the current study examined the pattern of allelic loss in clear cell and sarcomatoid components of renal cell carcinomas from 22 patients who had tumors with both components. DNA samples were prepared from formalin-fixed, paraffin-embedded renal tissue sections using laser-capture microdissection. Five microsatellite polymorphic markers for putative tumor suppressor genes on 5 different chromosomes were analyzed. These included D3S1300 (3p14), D7S522 (7q31), D8S261 (8p21), D9S171 (9p21), and TP53 (17p13). In addition, X-chromosome inactivation analysis was performed in 14 tumors from female patients. The clear cell components showed loss of heterozygosity (LOH) at the D3S1300, D7S522, D8S261, D9S171, and TP53 loci in 18% (4/22), 18% (4/22), 50% (10/20), 15% (3/20), and 20% (4/20) of informative cases, respectively. LOH in the sarcomatoid components was seen at the D3S1300, D7S522, D8S261, D9S171, and TP53 loci in 18% (4/22), 41% (9/22), 70% (14/20), 35% (7/20), and 20% (4/20) of informative cases, respectively. Six cases demonstrated an LOH pattern in the clear cell component that was not seen in the sarcomatoid component. Different patterns of allelic loss were seen in the clear cell and sarcomatoid components in 15 cases. Clonality analysis showed the same pattern of nonrandom X-chromosome inactivation in both clear cell and sarcomatoid components in 13 of the 14 cases studied. One case showed a random pattern of X-chromosome inactivation. X-chromosome inactivation analysis data suggest that both clear cell and sarcomatoid components of renal cell carcinomas are derived from the same progenitor cell. Different patterns of allelic loss in multiple chromosomal regions were observed in clear cell and sarcomatoid components from the same patient. This genetic heterogeneity indicates genetic divergence during the clonal evolution of renal cell carcinoma.

Patterns of Allelic Loss of Synchronous Adenocarcinomas of the Lung

Distinction of multiple primary lung carcinomas from intrapulmonary metastases using empiric clinical and histopathologic criteria can be difficult. Recent advances have provided several molecular markers that can be used for clonal analysis of separate tumor nodules and enhance tumor staging and subsequent treatment and prognosis. To address this issue, we performed a microdissection-based allelotyping of 20 cases of histologically similar, pathologic stage T4 adenocarcinomas (ADCs). Loss of heterozygosity (LOH) analysis included a panel of 15 polymorphic microsatellite markers located on 1p, 3p, 5q, 9p, 9q, 10q, 17p, and 22q. The tumor size, visceral pleural and angiolymphatic invasion, lymph node status, outcome, and survival were assessed. Allelotypes of 60 cases of solitary primary non-small cell lung carcinomas (NSCLC) (stages I-II) were used to define the percentage of discordant LOH patterns within solitary primary lung carcinoma that would discriminate between survivors and nonsurvivors. These criteria were used in the analysis of pathologic stage T4 ADC. Two groups of stage T4 cases were created: molecularly homogenous (< or = 40% discordances) (14 cases, 70%), and molecularly heterogenous (>40% discordances) (6 cases, 30%). Molecularly homogenous tumors were more frequently associated with visceral pleural invasion (92% vs. 8%) (P = 0.018). Allelotype did not correlate with age, gender, tumor size, tumor differentiation, lymph node status, angiolymphatic invasion, survival, or outcome. Our study showed that discordant and concordant genotypic profiles exist in morphologically similar synchronous ADC of the lung.

Molecular Genetic Evidence for a Common Clonal Origin of Urinary Bladder Small Cell Carcinoma and Coexisting Urothelial Carcinoma

In most cases, small-cell carcinoma of the urinary bladder is admixed with other histological types of bladder carcinoma. To understand the pathogenetic relationship between the two tumor types, we analyzed histologically distinct tumor cell populations from the same patient for loss of heterozygosity (LOH) and X chromosome inactivation (in female patients). We examined five polymorphic microsatellite markers located on chromosome 3p25-26 (D3S3050), chromosome 9p21 (IFNA and D9S171), chromosome 9q32-33 (D9S177), and chromosome 17p13 (TP53) in 20 patients with small-cell carcinoma of the urinary bladder and concurrent urothelial carcinoma. DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-assisted microdissection. A nearly identical pattern of allelic loss was observed in the two tumor types in all cases, with an overall frequency of allelic loss of 90% (18 of 20 cases). Three patients showed different allelic loss patterns in the two tumor types at a single locus; however, the LOH patterns at the remaining loci were identical. Similarly, the same pattern of nonrandom X chromosome inactivation was present in both carcinoma components in the four cases analyzed. Concordant genetic alterations and X chromosome inactivation between small-cell carcinoma and coexisting urothelial carcinoma suggest that both tumor components originate from the same cells in the urothelium.

Comparative genetic analysis of metachronous anaplastic oligoastrocytomas with extended recurrence-free interval

Two metachronous anaplastic oligoastrocytomas with different cerebral locations were analyzed in a 51-year-old patient with an extended recurrence-free interval of 6 years and an a long survival of 9 years. Remarkably, the patient had not undergone adjuvant chemotherapy. Different cytogenetic and molecular techniques were performed including comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), allelic loss analysis, sequencing of p53, p16(INK4a)/CDKN2A and p14(ARF), EGFRamplification studies, investigation of the DNA mismatch repair system as well as tumor clonality. Using CGH and FISH a profile of low accumulation of cytogenetic aberrations was found in the second tumor, with no significant increase in the percentage of hyperdiploid nuclei. Microsatellite analysis showed a common pattern of allelic losses at 1p36, 19q13 and 9p21. Both specimens were also similar in that they retained heterozygosity at 10q23-q24 and 13q14 and that they harbor neither EGFR amplification nor mutations of p53, p16(INK4a)/CDKN2A or p14(ARF). The only further alteration in the second tumor was an allelic loss at p53. The X-chromosome inactivation (HUMARA) analysis revealed a polyclonal pattern in both samples. Our data strongly suggest that the second anaplastic oligoastrocytoma developed as a distant relapse of the first tumor. Whether the paucity of accumulation of the observed genetic alterations might be associated with the unusually extended relapse-free time of the patient remains to be elucidated.