Allelic imbalance of 12q22-23 associated with APAF-1 locus correlates with poor disease outcome in cutaneous melanoma

Cutaneous melanoma is a highly aggressive tumor that is relatively resistant to chemotherapy and radiotherapy. This resistance may be in part due to inhibition of apoptosis. Apoptotic protease activating factor-1(APAF-1), a candidate tumor suppressor gene, mediates p53-induced apoptosis, and its loss promotes oncogenic transformation. To determine whether loss of the APAF-1 locus influences tumor progression, we assessed loss of heterozygosity microsatellites on the APAF-1 locus (12q22-23) in 62 primary and 112 metastatic melanomas. We discovered that frequency of allelic imbalance was significantly higher in metastatic tumors (n = 36 of 98; 37%) than in primary melanomas (n = 10 of 54; 19%; P = 0.02). In metastatic melanomas, APAF-1 loss significantly correlated with a worse prognosis (P < 0.05) in the patients, and its loss during melanoma tumor progression suggests that APAF-1 is a tumor suppressor gene. Furthermore, loss of heterozygosity was frequent in the 12q22-23 chromosome region centromeric to the APAF-1 locus suggesting that other tumor-related genes may be present in the 12q22-23 region. In summary, the study demonstrates that allelic imbalance in the 12q22-23 region is a genomic surrogate of poor disease outcome for cutaneous melanoma patients.     

Allelic imbalance at NBS1 is frequent in both proximal and distal colorectal carcinoma

Nijmegen breakage syndrome (NBS) is a hereditary disorder involving chromosomal instability, cancer risk and radiosensitivity. NBS carriers have an increased risk of cancer, though the significance of mutations in the NBS1 gene in sporadic cancer has not yet been investigated. Because the loss of NBS1 is associated with increased chromosomal re-arrangements, and tumors of the colon are particularly prone to chromosomal anomalies, we have begun to study the NBS1 locus in colorectal cancer (CRC). DNA was isolated from 99 microdissected colorectal tumors, and microsatellite markers flanking the NBS1 locus at 8q21.3 as well as elsewhere on 8q were analyzed. Normal lymphocyte DNA from each patient served to normalize the amplification of each allele, and a reduction of at least 35% in the intensity of one allele was taken as evidence of allelic imbalance (AI). In proximal and distal CRCs we found 25.9 and 36.2% with AI at 8q21.3, respectively. AI in proximal CRC tended not to extend to marker D8S555 at 8q24.1, whereas in distal CRC the region of AI frequently included all the informative markers. AI of 8q21.3 was not associated with any clinical variable. These results suggest that 8q21.3 contains a tumor suppressor gene involved in proximal CRC, possibly NBS1. The large regions of AI make it difficult to determine the importance of AI at the NBS1 locus in distal CRC.     

Allelic imbalance at chromosome region 11q23 in cervical carcinomas

The long arm of chromosome 11 has received much scrutiny as a high frequency of deletions of various sites has been observed in different tumour types, indicating the presence of putative tumour suppressor genes. In the present study, 81 primary cervical carcinomas were examined for allelic imbalance (AI) using nine microsatellite markers, mapping to the chromosomal region 11q23.1 where the ATM gene is located. AI at any locus in the region was found in 34 of 81 (42%) tumours. AI frequencies varied from 12 to 31% for the different markers used, with the highest frequency at marker D11S1294. Based on the findings of 17 cases with restricted areas of deletions, four chromosomal regions of possible importance in cervical carcinomas could be distinguished. The first region is located between the markers D11S1325 and D11S1819, the second region between D11S2179 and D11S1294, the third region between D11S1778 and D11S1818 and the fourth region between D11S1818 and D11S1347. The second region may thus contain part of the ATM gene. No association between AI of any marker and histopathological or clinical parameters was seen. When comparing the AI findings of the different loci with TP53 protein overexpression, the only significant association found was with D11S2179 located within the ATM gene. The results indicate that a tumour suppressor gene (or genes) on chromosome 11q.23.1 may be involved in carcinogenesis of the cervix and the involvement of the ATM gene remains a possibility.     

Allelic imbalance at 11q23-q24 chromosome associated with estrogen and radiation-induced breast cancer progression

Multiple genetic alterations are common in cancers including those of the breast. The mechanisms leading to these alterations such as point mutations, gene amplifications, deletions and replication error are often associated with frequent and consistent loss of heterozygosity (LOH) or microsatellite instability (MSI). Several cytological and molecular studies have shown high frequency loss of genetic information on the long arm of chromosome 11 (i.e., 11q) in various primary breast cancers. In the present study allelic alterations in a refined position on the long arm of chromosome 11 were studied to identify the spectrum of induced damage at different stages of malignant transformation of MCF-10F cell lines after exposure to high-LET radiation using alpha-particles and exposure to estradiol by using PCR-single strand conformation polymorphism (SSCP) and fluorescence in situ hybridization (FISH) analysis. Microsatellite markers were selected from chromosome 11 (11q23-q24 loci) and it was found that frequency of allelic imbalance occurs at different stages of tumor progression with a range of 15-45% depending on the marker studied. These results strongly suggested the presence of several tumor suppressor genes in this critical region of chromosome 11 (11q23-q24). It also represents the first indication of allele loss at these loci in human breast epithelial cells induced by radiation and estrogen treatment suggesting a potential interventional target in breast carcinogenesis.     

Allelic imbalance on 12q22-23 in serum circulating DNA of melanoma patients predicts disease outcome

Allelic imbalance (AI) encompassing the apoptotic protease-activating factor 1 (APAF-1) locus (12q22-23) is found frequently in metastatic melanoma. Circulating DNA with AI on 12q22-23 in serum was evaluated as a surrogate marker to predict biochemotherapy (BC) treatment response in melanoma patients. Sera were collected from 49 American Joint Committee on Cancer stage IV melanoma patients treated with BC. Serum AI of the 12q22-23 region was demonstrated to be present before and/or after BC. BC responders showed a significantly lower frequency of AI (5 of 24, 21%) compared with nonresponders (11 of 20, 55%; Fisher's exact test, P < 0.029). Serum AI on 12q22-23 was associated with worse prognosis (log-rank test, P < 0.046). These findings indicate that serial serum genetic analysis of tumor-related AI on 12q22-23 may have clinical use in predicting tumor response to therapy.     

Detailed deletion mapping at chromosome 11q23 in colorectal carcinoma

Loss of heterozygosity (LOH) is frequent at the chromosomal region 11q22-q23 in several types of tumours of diverse cell origin. Previous investigations of LOH at this chromosomal region in colorectal carcinoma have been contradictory in their findings, and have only included between 1-4 loci. In order to define any regions of LOH on 11q23, we investigated 16 loci between D11S940 and D11S934 on the long arm of chromosome 11 using microsatellite analysis. Of 57 colorectal carcinomas specimens, 36 (63.2%) demonstrated LOH at one or more marker, with the highest frequencies of LOH at D11S1340 (41.0%), located between 105.13-111.97 Mb from the centromere, and D11S924 (37.1%) and D11S4107 (40.5%), both located approximately 113 Mb from the centromere. No statistically significant associations between LOH and age-of-presentation or Dukes' stage were found. LOH was observed in colorectal tumours of all Dukes' stages, including Dukes' stages A and B, suggesting that the inactivation of a tumour suppressor gene(s) on 11q23 occurs in the early stages of colorectal carcinoma. These results confirm the presence of putative tumour suppressor gene(s) at chromosome 11q23, involved in the carcinogenesis of colorectal carcinoma, and will facilitate future identification of candidate genes.     

Loss of APAF-1 expression is associated with tumour progression and adverse prognosis in colorectal cancer

The aim of this study was to determine the prognostic value of APAF-1 in colorectal cancer (CRC). Immunohistochemistry for APAF-1 was performed on a tissue microarray of 1015 mismatch-repair (MMR) proficient and 130 sporadic MLH1-negative CRCs. The association of APAF-1 with clinico-pathological features including 10-year survival time was analysed. Methylation specific PCR was performed on a subset of MMR-proficient and MLH1-negative CRC. Loss of APAF-1 was associated with advanced T stage (p-value=0.022), N stage (p-value=0.009), vascular invasion (p-value=0.001) and worse survival (p-value=0.017) in MMR-proficient CRC. In MLH1-negative CRC, loss of APAF-1 was associated with metastasis (p-value=0.041), worse prognosis (p-value<0.001) and independently predicted shorter survival time (p-value<0.001). No methylation was found in the selected region of APAF-1. APAF-1 is a marker of tumour progression in MMR-proficient CRC and an independent adverse prognostic factor in MLH1-negative CRC.     

Allelic deletion at 11q23 is common in MYCN single copy neuroblastomas.

Deletions of the long arm of chromosome 11 (11q) have been noted in primary neuroblastomas, but a comprehensive analysis has not been performed. Therefore, we analysed 331 neuroblastomas (295 sporadic, 15 familial and 21 tumor-derived cell lines) to determine the prevalence of 11q allelic deletions, to map the location of a putative tumor suppressor gene and to perform clinical correlative studies. Assays for loss of heterozygosity (LOH) were performed at 24 microsatellite loci spanning 11q. LOH was observed at multiple 11q loci in 129/295 (44%) sporadic neuroblastomas, 5/15 (33%) familial neuroblastomas, and 5/21 (24%) neuroblastoma cell lines. A single region of 2.1 cM within 11q23.3, flanked by markers D11S1340 and D11S1299, was deleted in all specimens with 11q LOH. Allelic loss at 11q23 was inversely related to MYCN amplification (P<0.001). Within the subset of cases with a single copy of MYCN, 11q LOH was associated with advanced stage disease (P=0.008), unfavorable histopathology (P=0.042), and decreased overall survival probability (P=0.008). However, 11q LOH was not independently prognostic in multivariate analyses. These data support the hypothesis that a tumor suppressor gene mapping within 11q23.3 is commonly inactivated during the malignant evolution of a large subset of neuroblastomas, especially those with unamplified MYCN.     

Allelic loss on chromosome 1 is associated with tumor progression of cervical carcinoma.

BACKGROUND: Alterations in chromosome 1 are common in human malignancies. The frequency of loss of heterozygosity (LOH) on chromosome 1 in cervical carcinoma and its clinical significance are not clearly understood. METHODS: LOH on chromosome 1 was studied in 100 cervical carcinomas by the polymerase chain reaction (PCR) using 29 highly polymorphic microsatellite markers spaced approximately 10 centimorgans apart. Loci with high frequencies of LOH were identified and the findings were correlated with clinicopathologic characteristics. RESULTS: LOH on chromosome 1 at 1 or more loci was detected in 93% of tumors. The frequencies of LOH at locus D1S2829 (1p31), D1S2663 (1p36.3), and D1S2725 (1q25) exceeded 30%, and 12 other loci exhibited frequencies of LOH of 20-30%. Advanced stage tumors had a significantly higher percentage of informative microsatellite markers with LOH than early stage tumors. Of the 29 microsatellite markers studied, 4 loci had a significantly higher frequency of LOH in Stage III and IV tumors than in earlier stage tumors. CONCLUSIONS: Frequent aberrations on chromosome 1 in cervical carcinoma suggest that inactivation of tumor suppressor genes is important in cervical tumorigenesis. Higher frequencies of LOH in Stage III and IV tumors suggest that chromosome 1 changes are late events in cervical carcinoma. The findings of this study are consistent with earlier reports that suggest that tumor suppressor genes are present at 1p36.3 and 1p31. To the authors' knowledge, the high frequency of LOH mapped to 1q25 has not been reported previously. Its significance awaits further clarification.     

Allelic imbalance of chromosome 6q in ovarian tumours.

Previous work has implicated putative tumour-suppressor (ts) genes at 6q27 and a broad region at 6p12-q23. Here we report the results of a coded, randomised study of allelic imbalance at 12 loci on 6q on 40 pairs of coded tumour-blood pairs from patients with ovarian tumours. Our results provide clear evidence for the involvement of different regions of 6q in tumours of different histological subtypes. The involvement in serous tumours of a ts gene at the distal site is confirmed. However, proximal 6q presents a complex picture, with possibly three further ts genes: one at 6q21-23.3 involved at high frequency in benign and endometrioid tumours, another at 6q14-q15, also involved in endometrioid tumours, and a third suggested by a smallest region of deletion at 6q16.3-q21, between D6S275 and D6S300, that appears to be involved in early stage tumours. These observations point the way to a statistical study of the involvement of 6q in tumours of different histological type and staging performed on larger cohorts of samples.     

Allelic loss is heterogeneous throughout the tumor in colorectal carcinoma

BACKGROUND: Loss of heterozygosity (LOH) at 17p and 18q in colorectal carcinoma has been depicted as a potential prognostic marker for the disease. However, conclusions vary among reports, and evidence of clinically useful genetic prognostic markers is still lacking. As a rule, single biopsies are used. In this study, the authors hypothesized that an important cause of earlier contradictory results was the heterogeneity of colorectal neoplasms. METHODS: In this study, DNA originating in each quadrant of tumors from 64 patients with colorectal carcinoma was analyzed. Microsatellite markers for chromosome 18q and 17p were amplified by polymerase chain reaction and automatically analyzed. RESULTS: The authors found that, regardless of stage, LOH and non-LOH in both 17p and 18q varied among biopsies within the tumors in a random fashion. LOH in 18q was detected in all 4 quadrants in 22% and in 1 of 4, 2 of 4, or 3 of 4 quadrants in 56% of the tumors, whereas 22% of the tumors were homogeneously without LOH in 18q. LOH 17p was distributed similarly throughout the tumors and was present in 1 of 4, 2 of 4, or 3 of 4 of the quadrants in 44%. The authors also reexamined a subset of tumors by subdividing one biopsy from each into four. Analysis of the microsatellite markers then yielded identical results. No correlation between the degree of LOH status and patient survival was observed. CONCLUSIONS: LOH status within a colorectal tumor is extensively heterogeneous. However, it is more homologous on a lower macroscopic level. For relevant genetic analysis, multiple biopsies and DNA sampling preceded by careful morphologic examination must be standard in the preparation of DNA.     

APAF-1 is related to an undifferentiated state in the testicular germ cell tumor pathway

Apoptotic protease activating factor-1 (APAF-1) is a key regulator gene of apoptosis, located downstream from p53. Loss of APAF-1 expression is associated with chemorefractory malignant melanoma and neuronal cell differentiation. In order to make clear the function of APAF-1 in the carcinogenesis of germ cell tumors, we evaluated the expression levels of APAF-1 and several apoptosis and differentiation markers by immunohistochemistry in formalin-fixed paraffin-embedded samples from 43 cases of testicular germ cell tumor (TGCT) and six specimens of normal testis tissue. Expression of cleaved caspase-3, Oct-3/4, and Ki-67 were also examined by immunohistochemistry to evaluate apoptotic reactivity, tumor differentiation, and proliferation activity, respectively. APAF-1 was downregulated in two TGCT cell lines by siRNA transfection, and subsequent expression of the Ki-67 and Oct-3/4 genes and differentiation markers of three embryonic germ layers including keratin16 (KRT16) for ectoderm, vimentin (VIM) for mesoderm and GATA4 for endoderm were then tested. No significant relationship was found between APAF-1 expression and apoptotic activity in TGCTs. Expression of APAF-1, Oct-3/4, and Ki-67 was significantly higher in seminomas than in non-seminomas. In TGCTs, higher APAF-1 expression was correlated with higher proliferation (high Ki-67) and a lower degree of differentiation (high Oct-3/4). Interestingly, the expression of APAF-1 gradually decreased in accordance with tumor differentiation (seminoma and embryonal carcinoma > teratoma). Downregulation of APAF-1 in TGCT cell lines resulted in a decrease of Ki-67 and Oct-3/4 and an increase of VIM and KRT16 gene expression. These data show that higher expression of APAF-1 is related to an undifferentiated state in the TGCT pathway.     

Frequent allelic imbalance at the ATM locus in DNA multiploid colorectal carcinomas

DNA multiploidy may involve specific DNA ploidy states with respect to genetic alterations such as oncogenes, tumor suppressor gene mutation and microsatellite instability. To clarify the role of DNA multiploidy in colorectal cancer, we analysed allelic imbalance involving the ATM gene, localized to chromosome 11q22-23 and thought to be involved in genetic stability, in a series of multiploid colorectal carcinomas. In addition, p53 gene mutation (exons 5-8) and allelic imbalance at 11q24 loci distal to the ATM locus were also examined. The crypt isolation technique coupled with DNA cytometric sorting and polymerase chain reaction assay using 10 microsatellite markers tightly linked to the ATM gene were used to study ATM allelic imbalance in 55 colorectal carcinomas (15 diploid, 13 aneuploid, 27 multiploid). While allelic imbalance at the ATM locus was rarely observed in diploid and aneuploid carcinomas, multiploid carcinomas exhibited a high frequency of ATM allelic imbalance. In multiploid carcinoma samples, diploid subpopulations showed a smaller range of allelic imbalance at the loci tested compared to aneuploid subpopulations that demonstrated allelic imbalance over a relatively large region. Also, the frequency of AI at 11q24 showed a similar tendency to that at the ATM locus for each DNA ploidy state. An association between p53 gene mutation and ATM allelic imbalance in multiploid carcinoma was also observed. Our results suggest that ATM allelic imbalance and p53 gene mutations occur during the progression from diploid to aneuploid cell populations in multiploid colorectal carcinomas.     

Allelic imbalance in the clonal evolution of prostate carcinoma

BACKGROUND: To understand better the genetic basis of the clonal evolution of prostate carcinoma, the authors analyzed the pattern of allelic loss in 25 matched primary and metastatic prostate tumors. METHODS: Twenty-five cases were selected from the surgical pathology files of the Mayo Clinic from patients who had undergone radical retropubic prostatectomy and bilateral lymphadenectomy between 1987-1991. All patients had regional lymph node metastases at the time of surgery. DNA samples for the analysis of allelic loss pattern were prepared from primary tumors and matched synchronous lymph node metastases by tissue microdissection. The oligonucleotide primer pairs for the microsatellite DNA markers were D8S133, D8S136, D8S137, ANK1 on chromosome 8p12-21, LPLTET on chromosome 8p22, and D17S855 (intragenic to the BRCA1 gene) on chromosome 17q21. One case was not informative at any of the loci tested and was excluded from further analysis. RESULTS: The overall frequency of allelic imbalance was 79% in primary tumors and 88% in paired metastases. Of 24 informative cases, 14 patients (58%) showed the same pattern of allelic loss or retention in matched primary and metastatic tumors at all marker locus; discordant allelic loss was observed in the remaining 10 patients (42%). Four patients showed loss of the same allele at one or more marker loci in both primary and metastatic tumors, but discordant allelic loss was observed at other marker loci. Five patients showed allelic loss in at least one genetic marker in the metastatic tumor but not in its matched primary tumor. Five patients displayed loss of one allele at one or more marker loci in a primary tumor but not in the matched metastases. There was no significant difference in the frequency of allelic imbalance between primary and metastatic tumors at any marker analyzed (P>0.05). CONCLUSIONS: These data suggest that different patterns of allelic deletion may be acquired during cancer progression to metastases. The differences in genetic composition between primary prostate carcinoma and its metastases may be related to intrinsic cancer heterogeneity, overall genetic instability, and clonal divergence.     

Allelic loss of 10q23, the PTEN tumour suppressor gene locus, in Barrett's oesophagus-associated adenocarcinoma

PTEN is a putative tumour suppressor gene located on chromosome band 10q23. Mutations in PTEN have been identified in numerous human malignancies, including cancers of the brain, endometrium, ovary, and prostate. In this study, we screened 80 Barrett's oesophagus-associated adenocarcinomas (BOAd) for loss of heterozygosity (LOH) at 10q23, using the microsatellite markers D10S541, D10S219, and D10S551. Tumours demonstrating LOH were then screened for the presence or absence of PTEN mutations. LOH at one or more loci was identified in 17/80 (21%) cases. In none of these cases did we detect mutations in PTEN. The presence of LOH did not correlate with patient age, tumour stage, degree of differentiation, presence of perineural or vascular invasion, or overall survival. We conclude that LOH at chromosome 10q23 is uncommon in BOAd, is not associated with mutations in the PTEN tumour suppressor gene, and does not correlate with the clinical or pathologic features of these tumours. It is possible that PTEN is inactivated through other mechanisms in BOAd.     

Allelic imbalance at rs6983267 suggests selection of the risk allele in somatic colorectal tumor evolution

A common single nucleotide polymorphism (SNP), rs6983267, at 8q24.21 has recently been shown to associate with colorectal cancer (CRC). Three independent SNP association studies showed that rs6983267 contributes to CRC with odds ratios (OR) of 1.17 to 1.22. Here, we genotyped a population-based series of 1,042 patients with CRC and 1,012 healthy controls for rs6983267 and determined the contribution of SNP to CRC in Finland, using germ line DNA, as well as the respective cancer DNA in heterozygous patients. The comprehensive clinical data available from the 1,042 patients and their first-degree relatives enabled us to thoroughly examine the possible association of this variant with different clinical features. As expected, a significant association between the G allele of rs6983267 and CRC [OR, 1.22; 95% confidence interval (CI), 1.08-1.38; P = 0.0018] was found, confirming the previous observations. A trend towards association of the G allele with microsatellite-stable cancer (OR, 1.37; 95% CI, 1.02-1.85; P = 0.04) and family history of cancers other than CRC was seen (OR, 1.20; 95% CI, 1-1.43; P = 0.05). Four hundred and sixty-six GT heterozygotes identified in this study were analyzed for allelic imbalance at rs6983267 in the respective cancer DNA. One hundred and one tumors showed allelic imbalance (22%). The risk allele G was favored in 67 versus 34 tumors (P = 0.0007). This finding implicates that the underlying germ line genetic defect in 8q24.21 is a target in the somatic evolution of CRC.     

Genetic mapping of a third Li-Fraumeni syndrome predisposition locus to human chromosome 1q23

Li-Fraumeni syndrome (LFS) is a clinically and genetically heterogeneous inherited cancer syndrome. Most cases ( approximately 70%) identified and characterized to date are associated with dominantly inherited germ line mutations in the tumor suppressor gene TP53 (p53) in chromosome 17p13.1. In a subset of non-p53 patients with LFS, CHEK2 in chromosome 22q11 has been identified as another predisposing locus. Studying a series of non-p53 LFS kindred, we have shown that there is additional genetic heterogeneity in LFS kindred with inherited predisposition at loci other than p53 or CHEK2. Using a genome-wide scan for linkage with complementing parametric and nonparametric analysis methods, we identified linkage to a region of approximately 4 cM in chromosome 1q23, a genomic region not previously implicated in this disease. Identification ofa third predisposing gene and its underlying mutation(s) should provide insight into other genetic events that predispose to the genesis of the diverse tumor types associated with LFS and its variants.