The biological behavior of prostate cancer is uncertain, and therefore, search for molecular prognostic markers associated with disease progression seems to be essential. We performed microsatellite allelotyping of DNA isolated from primary prostate tumors biopsies (prostate adenocarcinoma, PCa). We evaluated the frequency of allelic imbalance (AI), including loss of heterozygosity and/or microsatellite imbalance (LOH/MSI) as well as the association of these DNA alterations with clinicopathological variables. We assessed the significance of LOH/MSI alterations in selected imprinted and non-imprinted chromosomal regions (IR and NIR) in PCa. A total of 50 biopsies of prostate tumor (containing >75 % tumor cells) were histologically examined confirming prostate carcinoma. Microsatellite allelotyping using 16 microsatellite markers linked to the following chromosomal regions: 1p31.2, 3p21.3–25.3, 7q32.2, 9p21.3, 11p15.5, 12q23.2, and 16q22.1 was performed. The incidence of LOH/MSI alterations in prostate tumor cells was the highest for chromosomal regions 7q32.2 and 16q22.1 (31.25 and 26.60 %, respectively), followed by 1p31.2 and 3p21.3–25.3 (26.50 and 17.40 %, respectively). Statistically significant increase in LOH/MSI alterations has been observed for markers: D1S2137 (1p region; p = 0.00032), D9S974 (9p region; p = 0.0017), and D16S3025 (16q region; p = 0.0017). Statistically significant differences in frequency of LOH/MSI alterations in particular chromosomal regions have been found for 1p31.2, 7q32.2 and 16q22.1 (p = 0.027, p = 0.012 and p = 0.031, respectively). We documented statistically significant association between Fractional Allele Loss (FAL) index and advanced tumor stage (p < 0.05). We suggest that genomic instability of LOH/MSI type is a frequent event in prostate carcinogenesis and assessed as FAL index has clinical value for the molecular staging of prostate cancer in (TRUS)-guided prostate biopsy material.
Prostate cancer; LOH; MSI; Molecular markers; TRUS-guided prostate biopsy
The most frequent genetic alteration in transitional cell carcinoma of the urinary bladder (TCC) is loss of chromosome 9 which targets CDKN2A on 9p. The targets on 9q are not confirmed. Here, 81 advanced TCC specimens were investigated for loss of heterozygosity (LOH) and homozygous deletions (HD) on chromosome 9q using multiplex analysis of microsatellite markers. 41/81 tumours (51%) showed LOH on 9q, with LOH at all markers in 33 cases. Eight partial losses involved three regions in 9q12, 9q22.3, and 9q33– 9q34. No mutations were identified in the candidate tumour suppressor gene DBCCR1 in three tumours showing restricted LOH at 9q32-33. 22% of the specimens had HD at CDKN2A, but no HD was found on 9q. Two tumours had lost 9p only and five 9q only. 9q LOH was not related to tumour grade or stage and present or absent with equal frequency in recurrent TCC. LOH on 9q correlated with the extent of genome-wide hypomethylation (P < 0.0001) which extended into satellite sequences located in 9q12 juxtacentromeric heterochromatin. While the high frequency of chromosome 9q loss in TCC may reflect destabilization of the chromosome related to hypomethylation of repetitive DNA, the data are compatible with the existence of tumour suppressor genes on this chromosome arm. http://www.bjcancer.com © 2001 Cancer Research Campaign
bladder cancer; chromosome instability; DNA methylation; LINE-1; tumour suppressor genes
Allelic imbalance or loss of heterozygosity (LOH) has been widely used to assess genetic instability in tumours, and high LOH on chromosome arms 3p, 9p and 17p has been considered to be a common event in non-small-cell lung cancer (NSCLC). We have investigated allelic imbalance in 45 NSCLCs using 92 microsatellite markers on 38 chromosome arms. LOH of 38% was observed on 3p using nine markers, 58% on 9p using 15 markers and 38% on 17p using five markers. Fractional allele loss (FAL) has been calculated for each tumour (FAL is the number of chromosome arms showing LOH/number of informative chromosome arms) and a median FAL value of 0.09 was obtained in the 45 NSCLCs studied. The LOH data were examined on the basis of FAL scores: low FAL (LFAL) (0.00-0.04), medium FAL (MFAL) (0.05-0.13) and high FAL (HFAL) (0.14-0.45) based symmetrically around the median FAL value of 0.09. Tumours with HFAL values showed a very clear polarisation of the LOH data on chromosome arms 3p, 9p and 17p, such that 80% showed loss on 3p, 80% on 9p and 73% on 17p. These incidences of LOH were significantly higher than would be expected, since overall genetic instability in these HFAL tumours ranged from 14% to 45% LOH. Nine of the 14 patients in the LFAL group were found to have no LOH on 3p, 9p or 17p, but five of these had LOH at other sites: i.e. LOH on 5p, 5q, 8p, 13q, 16q and 19q. These results indicate that LFAL patients form a new subset of NSCLC tumours with distinct molecular-initating events, and may represent a discrete genetic population.
In order to characterize the molecular events in the carcinogenesis of esophageal cancer and to identify biomarkers for the early detection of the disease, matched precancerous and cancerous tissues resected from 34 esophageal cancer patients in Chongqing of southern China were compared for the extent of loss of heterozygosity (LOH). Sixteen microsatellite markers on nine chromosome regions were used for the PCR-based LOH analysis. The overall frequency of LOH at the 16 microsatellite loci was significantly increased as the pathological status of the resection specimens changed from low-grade dysplasia (LGD) to high-grade dysplasia (HGD) and squamous cell carcinoma (SCC) (P < 0.001), indicating that tumorigenesis of the esophageal squamous epithelia is a progressive process involving accumulative changes of LOH. A total of eight markers showed LOH in the LGD samples, suggesting that these loci may be involved in the early-stage tumorigenesis of esophageal squamous cell carcinoma (ESCC) and that LOH analysis at these loci may help improve the early detection of this disease. In addition, heterozygosity was regained at four loci in the SCC samples of four patients compared with the HGD samples, suggesting the possibility of genetic heterogeneity in the tumorigenesis of esophageal cancer.
esophageal squamous cell carcinoma; microsatellite; loss of heterozygosity
Most human cancers are characterized by genomic instability, the accumulation of multiple genetic alterations and allelic imbalance throughout the genome. Loss of heterozygosity (LOH) is a common form of allelic imbalance and the detection of LOH has been used to identify genomic regions that harbor tumor suppressor genes and to characterize tumor stages and progression. Here we describe the use of high-density oligonucleotide arrays for genome-wide scans for LOH and allelic imbalance in human tumors. The arrays contain redundant sets of probes for 600 genetic loci that are distributed across all human chromosomes. The arrays were used to detect allelic imbalance in two types of human tumors, and a subset of the results was confirmed using conventional gel-based methods. We also tested the ability to study heterogeneous cell populations and found that allelic imbalance can be detected in the presence of a substantial background of normal cells. The detection of LOH and other chromosomal changes using large numbers of single nucleotide polymorphism (SNP) markers should enable identification of patterns of allelic imbalance with potential prognostic and diagnostic utility.
The objective of this study was to characterize the molecular events in the carcinogenesis of esophageal squamous cell carcinoma (ESCC) and to identify biomarkers for early detection of the disease. Matched precancerous and cancerous tissues resected from 34 esophageal cancer patients from Chongqing, southern China, were compared to evaluate the extent of loss of heterozygosity (LOH). Sixteen microsatellite markers on chromosome regions 3p, 4p, 5q, 8p, 9p, 9q, 11p, 13q and 17p were used for PCR-based LOH analysis. The overall frequency of LOH at the 16 microsatellite loci was significantly increased as the pathological status of the resection specimens changed from low-grade dysplasia (LGD) to high-grade dysplasia (HGD) and SCC (P<0.001). A total of 8 markers showed LOH in the LGD samples. In addition, heterozygosity was regained at 4 loci in the SCC samples of 4 patients, respectively, in comparison to the results for these loci in the HGD samples. The overall rate of LOH increased significantly with the deterioration of the lesions, indicating that tumorigenesis of the esophageal squamous epithelia is a progressive process involving accumulative changes in LOH. The 8 loci showing allelic loss in the LGD samples may be involved in the early-stage tumorigenesis of ESCC, and LOH analysis at these loci may help improve the early detection of this disease. Regain of heterozygosity found in certain patients suggests the possibility of genetic heterogeneity in the tumori-genesis of esophageal cancer.
esophageal squamous cell carcinoma; microsatellite; loss of heterozygosity
Approximately 2/3 of patients diagnosed with superficial transitional cell carcinoma of the urinary bladder (TCC) will recur within 2 years. Loss of chromosome 9 and loss of heterozygosity (LOH) at 9q34 in index TCCs identify a subset of patients at high risk of recurrence. This study explores genetic alterations on chromosomes 4, 8, 11 and 17 as predictors of recurrence. A total of 109 carcinomas were investigated at 26 loci. DNA was extracted from microdissected archival normal/tumour tissue and was analysed for loss of heterozygosity (LOH). Fluorescent PCR was performed and genotyping carried out on a Perkin Elmer ABI377 sequencer. LOH of D11S490 or D17S928 was significantly more frequent in index carcinomas of patients who experienced recurrence compared to those with no recurrence (P = 0.004 and 0.019 respectively). These results suggest that loss of these regions is associated with recurrence of TCC. Further investigation is required to identify genes in these regions, which might be responsible for driving recurrence in TCC of the urinary bladder. © 2001 Cancer Research Campaign http://www.bjcancer.com
LOH; TCC of the urinary bladder; chromosome 11 and 17
Identification of the key genetic alterations leading to ovarian cancer is in its infancy. Polymerase chain reaction (PCR)-based analysis of loss of heterozygosity (LOH) is a powerful method for detecting regions of altered tumour-suppressor genes. Focusing on chromosome 12, we examined 23 ovarian cancer samples for LOH using 31 highly polymorphic microsatellite markers and found the chromosomal localization of two putative tumour-suppressor genes. Two commonly deleted regions were 12p12.3-13.1 in 6/23 (26%) and 12q23-ter in 7/23 (30%) samples. LOH on chromosome 12 was more common in late-stage ovarian carcinomas. The region of LOH at 12p12.3-13.1 includes the genes that code for the ETS-family transcriptional factor, known as TEL, and the cyclin-dependent kinase inhibitor, known as p27Kip1. Mutational analysis of both TEL and p27Kip1 using single-strand conformation polymorphism (SSCP) showed no abnormalities, suggesting that the altered gene in this region is neither of these genes. Taken together, our data suggest that new tumour-suppressor genes in the region of chromosomes 12p12.3-13.1 and 12q23-ter may be involved in the development of ovarian cancer.
Previous molecular genetic studies of laryngeal squamous cell carcinoma (SCC)have shown certain chromosomal regions with recurring alterations. But studies of sequential molecular alterations and genetic progression model of laryngeal SCC have not been clearly defined. To identify the chromosomal alterations associated with the carcinogenesis of laryngeal SCC, we analyzed genomic DNA from microdissected squamous metaplasia, squamous dysplasia, invasive SCC, and metastatic carcinoma samples from 22 laryngeal SCC patients for loss of heterozygosity (LOH) at microsatellite loci. Ten microsatellite markers on chromosome 3p, 8p, 9p, and 17p were used. LOH at 9p21 was observed in the all stages including squamous metaplasia, squamous dysplasia, invasive SCC and metastatic carcinoma. LOH at 17p13.1, 3p25 and 3p14.2 was observed from the squamous dysplasia, invasive SCC and metastatic carcinoma. LOH at 8p21.3-p22 was observed mainly from the invasive SCC and metastatic carcinoma. The results suggest that 9p21 in the early event, 17p13.1, 3p25 and 3p14.2 in the intermediate event and 8p21.3-p22 in the late event may be involved in the laryngeal carcinogenesis.
Loss of Heterozygosity; Chromosomes; Laryngeal Neoplasms; Carcinoma, Squamous Cell
Allelic losses or loss of heterozygosity (LOH) at many chromosomal loci have been found in the cells of meningiomas. The objective of this study was to evaluate LOH at several loci of different chromosomes (1p32, 17p13, 7q21, 7q31, and 22q13) in different grades of meningiomas.
Forty surgical specimens were obtained and classified as benign, atypical, and anaplastic meningiomas. After DNA extraction, ten polymorphic microsatellite markers were used to detect LOH. Medical and surgical records, as well as pathologic findings, were reviewed retrospectively.
LOH at 1p32 was detected in 24%, 60%, and 60% in benign, atypical, and anaplastic meningiomas, respectively. Whereas LOH at 7q21 was found in only one atypical meningioma. LOH at 7q31 was found in one benign meningioma and one atypical meningioma. LOH at 17p13 was detected in 4%, 40%, and 80% in benign, atypical, and anaplastic meningiomas, respectively. LOH at 22q13 was seen in 48%, 60%, and 60% in benign, atypical, and anaplastic meningiomas, respectively. LOH results at 1p32 and 17p13 showed statistically significant differences between benign and non-benign meningiomas.
LOH at 1p32 and 17p13 showed a strong correlation with tumor progression. On the other hand, LOH at 7q21 and 7q31 may not contribute to the development of the meningiomas.
Chromosome; Loss of heterozygosity; Meningiomas
Although a loss of heterozygosity (LOH) is commonly observed using microsatellite markers in a cell-proliferating malignant disorder, controversial findings of psoriasis, a keratinocyte-outgrowth disease, remain to be explained. It was hypothesized that unstable natures of the microsatellite markers for the polymerase chain reaction (PCR) might give a rise to either a false-positive or -negative LOH. Twenty-one frozen skin tissues and 33 formalin-fixed paraffin-embedded archives were obtained from patients with psoriatic plaques and colorectal cancers, respectively. In the frozen psoriatic skin, two of the 17 microsatellite markers selected from 11 chromosomal arms were associated with artifact LOHs that were not reproduced in repeated PCRs. The remaining 15 stable microsatellite markers with few PCR artifacts demonstrated a borderline-level LOH in cases with an ambiguous heterozygosity such as a juxtaposed allelic band. Infrequent LOHs (3 out of 242 heterozygous markers, 1.2%) were detected in psoriatic cases with two separate alleles. In colorectal cancers, a set of the 15 stable microsatellite markers identified a minimal borderline-level LOH at the cut-off point that was same with that of psoriasis. These results indicate that the selection of reproducible microsatellite sequences and the cautious criteria for informative heterozygosity are required to obtain the reliable LOH results from variable genomic DNAs, and that psoriatic lesions harbor few LOH.
Loss of heterozygosity (LOH) for chromosome 10 is the most frequent genetic abnormality observed in high-grade gliomas. We have used fluorescent microsatellite markers to examine a series of 83 patients, 34 with anaplastic astrocytoma (grade 3) and 49 with glioblastoma multiforme (grade 4), for LOH of chromosome 10. Genotype analysis revealed LOH for all informative chromosome 10 markers in 12 (35%) of patients with grade 3 and 29 (59%) grade 4 tumours respectively, while partial LOH was found in a further eight (24%) grade 3 and ten (20%) grade 4 tumours. Partial LOH, was confined to the long arm (10q) in six and the short arm (10p) in three cases, while alleles from both arms were lost in four cases. Five tumours (one grade 3 and four grade 4) showed heterogeneity with respect to loss at different loci. There was a correlation between any chromosome 10 loss and poorer performance status at presentation (χ2P = 0.005) and with increasing age at diagnosis (Mann–Whitney U-test P = 0.034) but not with tumour grade (χ2P = 0.051). A Cox multivariate model for survival duration identified age (proportional hazards (PH), P = 0.004), grade (PH, P = 0.012) and any loss of chromosome 10 (PH, P = 0.009) as the only independent prognostic variables. Specifically, LOH for chromosome 10 was able to identify a subgroup of patients with grade 3 tumours who had a significantly shorter survival time. We conclude that LOH for chromosome 10 is an independent, adverse prognostic variable in high-grade glioma. © 1999 Cancer Research Campaign
glioma; brain tumour; loss of heterozygosity; microsatellite
The aim of the study was to investigate the influence of allelic imbalance (AI) in several loci of tumor suppressor genes in 3p region on the non-small cell lung cancer (NSCLC) development. We evaluated the frequency of loss of heterozygosity and/or microsatellite imbalance (LOH/MSI) and assessed their association with patients’ characteristics (age, gender, tobacco addiction) and NSCLC classification according to TNM/AJCC staging. To analyze the potential role of AI involved in NSCLC pathogenesis, we allelotyped a group of 74 NSCLC patients using 7 microsatellite markers. The highest frequency of LOH/MSI, however, not statistically significant, was observed in RARβ and MLH1 (p = 0.104 and p = 0.216, respectively) loci. The association between high LOH/MSI frequency in 3p region with male gender (p = 0.041) as well as with age (especially >60 years) for RARβ and MLH1 genes (p = 0.0001 and p = 0.020, respectively) was documented. Statistically significant increased frequency of MLH1 allelic loss in squamous cell carcinoma (SCC) versus non-squamous cell carcinoma (non-SCC) was observed (p = 0.01). Significant increase in LOH/MSI frequency in 3p region (mainly in FHIT and MLH1loci) in correlation with cigarette addiction in a lifetime (≥40 years and ≥40 Pack Years) was also documented (p < 0.05). The highest LOH/MSI was revealed in RARβ locus in IA tumors (p = 0.0001), while the similarly high allelic loss of MLH1 correlated with III A/B tumors (p = 0.0002), according to AJCC staging. The obtained results demonstrate that AI is influenced by tobacco smoking and seems to be vital in the molecular diagnosis of NSCLC, especially of SCC subtype.
Non-small cell lung carcinoma (NSCLC); Loss of heterozygosity (LOH); Microsatellite instability (MSI); Microsatellite markers; Molecular diagnosis
Detection of loss of heterozygosity (LOH) and DNA flow cytometry (FCM) were used to trace the origin of bilateral ovarian cancer from 16 patients. From each tumour the DNA index (DI) and LOH patterns for chromosomes 1, 3, 6, 11, 17, 18, 22 and X were determined with 36 microsatellite markers. Formalin-fixed, paraffin-embedded as well as frozen specimens were used. Flow cytometric cell sorting was used to enrich tumour cells for polymerase chain reaction (PCR)-driven LOH analysis. Analysis of the LOH data showed that in 12 of the 16 cases concordance was observed for all informative markers, namely retention of heterozygosity (ROH) or loss of identical alleles in both tumour samples. In four cases discordant LOH patterns were observed. In two cases the discordant LOH was found for one of the chromosomes tested while other LOH patterns clearly indicated a unifocal origin. This suggests limited clonal divergence. In the other two cases all LOH patterns were discordant, most likely indicating an independent origin. The number of chromosomes showing LOH ranged from 0 to 6. Comparison of DNA FCM and the LOH data showed that the latter technique has a higher sensitivity for the detection of a unifocal origin. In 14/16 cases evidence was found for a unifocal origin, while in two cases clonal divergence was found at LOH level and in two other cases clonal divergence at DNA ploidy level. In 12 cases the complete observed allelotype had developed before the formation of metastases, including the two cases showing a large DNA ploidy difference.
Copy neutral loss of heterozygosity (CN-LOH) refers to a special case of LOH occurring without any resulting loss in copy number. These alterations is sometimes seen in tumors as a way to inactivate a tumor suppressor gene and have been found to be important in several types of cancer.
We have used high density single nucleotide polymorphism arrays in order to investigate the frequency and distribution of CN-LOH and other allelic imbalances in neuroblastoma (NB) tumors and cell lines. Our results show that the frequency of these near-CN-LOH events is significantly higher in the cell lines compared to the primary tumors and that the types of CN-LOH differ between the groups. We also show that the low-risk neuroblastomas that are generally considered to have a "triploid karyotype" often present with a complex numerical karyotype (no segmental changes) with 2-5 copies of each chromosome. Furthermore a comparison has been made between the three related cell lines SK-N-SH, SH-EP and SH-SY5Y with respect to overall genetic aberrations, and several aberrations unique to each of the cell lines has been found.
We have shown that the NB tumors analyzed contain several interesting allelic imbalances that would either go unnoticed or be misinterpreted using other genome-wide techniques. These findings indicate that the genetics underlying NB might be even more complex than previously known and that SNP arrays are important analysis tools. We have also showed that these near-CN-LOH events are more frequently seen in NB cell lines compared to NB tumors and that a set of highly related cell lines have continued to evolve secondary to the subcloning event. Taken together our analysis highlights that cell lines in many cases differ substantially from the primary tumors they are thought to represent, and that caution should be taken when drawing conclusions from cell line-based studies.
We have used polymerase chain reaction (PCR) analysis to study the incidence of allelic imbalance at four polymorphic microsatellite markers on chromosome 6q25.1-27, three dinucleotide repeats and one trinucleotide repeat, for microdissected tumour foci from a group of 75 'early' breast carcinomas. The tumours comprised 16 preinvasive cases of ductal carcinoma in situ (DCIS) and 59 mammographically detected early invasive carcinomas. Loss of heterozygosity (LOH) was detected at all four loci and in all types and grade of disease. The frequency of LOH ranged from 23% to 50% depending on the marker studied. The highest frequency of LOH was observed at the D6S186 locus for the cases of DCIS and at the oestrogen receptor locus for the invasive carcinomas. These data suggest that the inactivation of tumour-suppressor genes within this region on chromosome 6q is important for the development of these early lesions.
AIM: To investigate the prognostic value of chromosome 18q microsatellite alterations (MA) in stage II colon cancer.
METHODS: One hundred and six patients with sporadic stage II colon cancer were enrolled in this study. DNA was extracted from formalin-fixed, paraffin-embedded tumor and adjacent normal mucosal tissue samples. MA, including loss of heterozygosity (LOH) and microsatellite instability (MSI), was analyzed by polymerase chain reaction, polyacrylamide gel-electrophoresis and DNA sequencing at 5 microsatellite loci on chromosome 18q (D18S474, D18S55, D18S58, D18S61 and D18S64).
RESULTS: Among the 102 patients eligible for MA information, the overall frequencies of LOH, high and low frequency MSI/microsatellite stable were 49.0%, 17.6% and 82.4%, respectively. The high frequency of 18q-LOH was significantly associated with the poor 5-year overall survival (OS) (P = 0.008) and disease free survival (P = 0.006). High levels of MSI were significantly associated with a longer 5-year OS (P = 0.045) while the higher frequency of 18q-LOH at the loci of D18S474 and D18S61 was significantly associated with a poorer 5-year OS (P = 0.010 and 0.005, respectively). But multivariate analysis showed that only the frequency of 18q-LOH was significantly associated with the prognosis of the disease.
CONCLUSION: High frequency of 18q-LOH is an independent prognostic factor indicating poor prognosis of the patients with stage II colon cancer.
Chromosome 18q; Loss of heterozygosity; Microsatellite instability; Stage II colon cancer; Prognosis
The neurofibromatosis 2 locus (NF2) is inactivated through mutation and loss of heterozygosity (LOH) in 40–65% of all sporadic meningiomas, while the role of the p53 tumor suppression pathway in meningioma initiation and progression is still unclear. This study aims to determine if a p53 codon 72 arginine-to-proline polymorphism, found to be correlated with cancer development and cancer patient survival in other tumors, is associated with sporadic meningioma initiation or progression. We investigated Pro72 incidence in a cohort of 92 sporadic meningiomas and analyzed its association with histological grade (WHO classification) and with NF2 LOH (determined using polymorphic microsatellite markers on 22q). The Pro72 allele was not found to be selected for in the cohort. However, in the subgroup of meningiomas with NF2 LOH and carrying Pro72, 50.0% had high grade tumors (WHO grades II and III) compared to only 14.3% of those without NF2 LOH (OR = 6.0, CI = 1.56–23.11, P = 0.012). The significant association occurred only when considering subgroups of meningiomas with or without NF2 LOH, suggesting that not including NF2 status when analyzing study cohorts may explain the variability seen in the literature where all meningiomas were grouped together. Our data suggests a role for the p53 pathway in the progression of meningiomas in which NF2 is inactivated, and highlights the importance of accounting for NF2 LOH in future studies of meningiomas and the p53 pathway.
NF2; Mdm2; p53; Pro72; Meningiomas
Chromosome 6q has been shown to be one of the most frequent sites for allelic loss in human breast cancer. The mannose 6-phosphate/insulin-like growth factor 2 receptor (IGF2R) gene, which maps to chromosome 6q26-27, functions in the activation of TGF-beta1, a potent growth inhibitor for most cell types, the degradation of the mitogen IGF2 and the intracellular trafficking of lysosomal enzymes. Loss of heterozygosity (LOH) at the IGF2R locus with mutations in the remaining allele have been reported in liver cancers and recently in two high-grade cases of ductal carcinoma in situ of the breast. We have sought to confirm that allelic loss of IGF2R is an early event in the aetiology of breast cancer by screening a group of 'early' lesions for LOH at a polymorphic microsatellite marker within the IGF2R gene using polymerase chain reaction (PCR). Several microdissected tumour foci were analysed for each of 40 mammographically detected invasive carcinomas and 22 cases of pure ductal carcinoma in situ (DCIS). None of 25 (62.5%) informative early invasive carcinomas showed any evidence of LOH. This group comprised predominantly of well- to moderately differentiated cases (95%). However, 4 out of 18 informative DCIS cases (22%) showed clear evidence of LOH. Three of these were poorly differentiated (high-grade) lesions. These data suggest that loss of heterozygosity at the IGF2R gene is associated with poor differentiation at this early stage of breast cancer development and progression.
Most human cancers are characterized by genetic instabilities. Chromosomal aberrations include segments of allelic imbalance identifiable by loss of heterozygosity (LOH) at polymorphic loci, which may be used to implicate regions harboring tumor suppressor genes. Here we performed whole genome LOH profiling on over 40 human head and neck squamous cell carcinoma (HNSCC) cell lines. Several frequent LOH regions have been identified on chromosomal arms 3p, 4p, 4q, 5q, 8p, 9p, 10p, 11q, and 17p. A genomic region of ∼7 Mb located at 8p22-p21.3 exhibits the most frequent LOH (87.9%), which suggested that this region harbors important tumor suppressor gene(s). Mitochondrial tumor suppressor gene 1 (MTUS1) is a recently identified candidate tumor suppressor gene that resides in this region. Consistent down-regulation in expression was observed in HNSCC for MTUS1 as measured by real-time quantitative RT-PCR. Sequence analysis of MTUS1 gene in HNSCC revealed several important sequence variants in the exon regions of this gene. Thus, our results suggested that MTUS1 is one of the candidate tumor suppressor gene(s) reside in 8p22-p21.3 for HNSCC. The identification of these candidate genes will facilitate the understanding of tumorigenesis of HNSCC. Further studies are needed to functionally evaluate those candidate genes.
Recent reports indicate that anaplastic oligodendrogliomas frequently show allelic losses on chromosome arms 1p and 19q, and that these deletions are associated with better chemotherapeutic response and overall patient survival. Because of the diversified genetic makeup of the population and the centralized provincial referral system for brain tumor patients in Manitoba, the epidemiological features of such tumors sometimes differ from the published data acquired from non-community based settings. In this study, we assessed the prevalence of allelic deletions for chromosome arms 1p and 19q in anaplastic and in low-grade oligodendrogliomas in the Manitoba population.
Loss of heterozygosity (LOH) analysis of brain tumors was carried out using 4 microsatellite markers (D1S508, D1S2734, D19S219 and D19S412) and a PCR based assay. The tumors were consecutively acquired during the period September 1999–March 2001 and a total of 63 tumors were assessed.
We found that allelic loss of chromosome 1p and 19q was higher in oligodendrogliomas than in other diffuse gliomas and that for anaplastic oligodendrogliomas, younger patients exhibited significantly more deletions than older patients (>60 years of age).
These studies suggest that age may be a factor in the genetic alterations of oligodendrogliomas. In addition, these studies demonstrate that this assay can easily be carried out in a cost-effective manner in a small tertiary center.
Microsatellite markers; loss of heterozygosity; PCR; gliomas; chromosomes 1p; 19q
High levels of loss of distal markers on 17p13.3 in breast cancer suggested the presence within the region of at least one tumour-suppressor gene. Here we describe the derivation of two biallelic polymorphisms from the 17p telomeric yeast artificial chromosome (YAC) TYAC98. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR analysis demonstrated that the high level of allelic imbalance observed in breast tumours represented loss of constitutional heterozygosity (LOH) and that this LOH extended to the telomere. Lung carcinoma (but not Wilms' tumour)-derived DNA again revealed a high level of loss of subtelomeric 17p sequences. Telomeric microsatellite polymorphisms from other chromosome arms did not show such elevated loss in either tumour type. This suggested that the 17p loss observed did not reflect a general telomeric instability and provided further evidence for the presence of a breast cancer tumour-suppressor gene in the distal region of 17p13.3.
Allelic imbalance or loss of heterozygosity (LOH) studies have been used extensively to identify regions on chromosomes that may contain putative tumour-suppressor genes. We have undertaken an extensive allelotype of 80 specimens of squamous cell carcinoma of the head and neck (SCCHN) using 145 polymorphic microsatellite markers on 39 chromosome arms. Allelic imbalances were found most frequently on chromosome arms 3p, 9p, 17p and 18q with over 45% LOH and imbalances on 1p, 1q, 2p, 5q, 6p, 6q, 8p, 8q, 9q, 11q, 13q, 17q and 19q were found in more than 20% of SCCHN. These LOH data were analysed against a range of clinicopathological parameters which included previously untreated and previously treated tumours; correlations were found between LOH on 9q and nodes at pathology (P = 0.02) and between histopathological grade and LOH on 12q (P = 0.02) and 13q (P = 0.01). In the group of previously untreated tumours, a correlation was found between site of tumour and LOH on 3p (P = 0.019), and 8p (P = 0.029), while TNM staging correlated with LOH on 3p (P = 0.019) and 17p (P = 0.016). Fractional allele loss (FAL) was calculated for 52 tumours with LOH data on nine or more chromosomal arms and found to have a median value of 0.22 (range 0.0-0.80). Correlations were found between FAL > median value and nodes at pathology (P = 0.01) and tumour grade (P = 0.06), demonstrating that advanced tumours with lymph node metastasis often had LOH at multiple sites. FAL > median value was found to correlate with a poor survival (P < 0.03) and, furthermore, FAL > median value correlated with poor survival in the previously untreated patients (P < 0.019). These results indicate that assessment of the accumulation of genetic damage, as provided by allelotype data, provides a useful molecular indicator of the tumour behaviour and clinical outcome.
Chromosomal losses involving the short arm of chromosome 8 are frequent in a variety of tumour types, including breast cancer, suggesting the presence of one or more tumour suppressor genes in this region. In this study, we have used 11 microsatellite markers to analyse loss of heterozygosity (LOH) at chromosome 8p in 151 sporadic breast tumours and 50 tumours from subjects carrying the BRCA2 999del5 mutation. Fifty percent of sporadic tumours compared to 78% of BRCA2 linked tumours exhibit LOH at one or more markers at 8p showing that chromosome 8p alterations in breast tumours from BRCA2 999del5 carriers are more pronounced than in sporadic breast tumours. The pattern of LOH is different in the two groups and a higher proportion of BRCA2 tumours have LOH in a large region of chromosome 8p. In the total patient material, LOH of 8p is associated with LOH at other chromosome regions, for example, 1p, 3p, 6q, 7q, 9p, 11p, 13q, 17p, and 20q, but no association is found between LOH at 8p and chromosome regions 11q, 16q, 17q, and 18q. Furthermore, an association is detected between LOH at 8p and positive node status, large tumour size, aneuploidy, and high S phase fraction. Breast cancer patients with LOH at chromosome 8p have a worse prognosis than patients without this defect. Multivariate analysis suggests that LOH at 8p is an independent prognostic factor. We conclude that chromosome 8p carries a tumour suppressor gene or genes, the loss of which results in growth advantage of breast tumour cells, especially in carriers of the BRCA2 999del5 mutation.
Keywords: chromosome 8; BRCA2; LOH; breast cancer
Loss of heterozygosity (LOH) at tumor suppressor loci is a major contributor to cancer initiation and progression. Both deletions and mitotic recombination can lead to LOH. Certain chromosomal loci known as common fragile sites are susceptible to DNA lesions under replication stress, and replication stress is prevalent in early stage tumor cells. There is extensive evidence for deletions stimulated by common fragile sites in tumors, but the role of fragile sites in stimulating mitotic recombination that causes LOH is unknown. Here, we have used the yeast model system to study the relationship between fragile site instability and mitotic recombination that results in LOH. A naturally occurring fragile site, FS2, exists on the right arm of yeast chromosome III, and we have analyzed LOH on this chromosome. We report that the frequency of spontaneous mitotic BIR events resulting in LOH on the right arm of yeast chromosome III is higher than expected, and that replication stress by low levels of polymerase alpha increases mitotic recombination 12-fold. Using single-nucleotide polymorphisms between the two chromosome III homologs, we mapped the locations of recombination events and determined that FS2 is a strong hotspot for both mitotic reciprocal crossovers and break-induced replication events under conditions of replication stress.
Loss of heterozygosity (LOH) at tumor-suppressor genes contributes to cancer, and deletions resulting in LOH are frequently observed in tumor cells at certain chromosomal regions known as common fragile sites. LOH can also result from repair of DNA damage by mitotic recombination, if the homologous chromosome rather than the sister chromatid is used as a repair template. The extent to which fragile site instability causes LOH by mitotic recombination with the homologous chromosome is unknown. We evaluated mitotic recombination on the yeast Saccharomyces cerevisiae chromosome III, which contains a naturally-occurring fragile site known as FS2. We report that yeast chromosome III has a high frequency of spontaneous mitotic recombination that involves the homologous chromosome. Under conditions that stimulate instability at the fragile site, LOH resulting from mitotic recombination on yeast chromosome III is increased 12-fold, and FS2 is a hotspot for initiating these events. These results suggest that instability at human common fragile sites may drive mitotic recombination repair pathways that cause LOH and promote tumorogenesis.