High Frequency Of Allelic Loss Research Articles

High frequency of allelic loss at the BRCA1 locus in ovarian cancers: clinicopathologic and molecular associations

BRCA1 dysfunction may occur by different mechanisms that are rarely evaluated concomitantly. We aimed to analyze BRCA1 germline mutations, loss of heterozygosity (LOH) and promoter methylation in unselected ovarian carcinomas in the context of their clinicopathologic characteristics and other molecular changes. BRCA1 mutations were analyzed in 257 carcinomas using single-strand conformation polymorphism (SSCP), heteroduplex, and sequencing methods. LOH at the BRCA1 locus was screened for in 180 cancers. Methylation analysis was performed for 241 tumors using quantitative methylation specific PCR(qMSP). BRCA1 alterations, comprising germline mutations, allelic loss, and/or aberrant promoter methylation, were found in 77.6% (125/161) of ovarian carcinomas. Patients with germline mutations were younger than non-carriers (P < 0.0001). Germline mutations and LOH were associated with advanced stages (P=0.009, P < 0.0001), high tumor grade (P=0.005, P<0.0001), and TP53 mutations (P=0.003, P < 0.0001, for mutations and LOH, respectively). LOH was also associated with the serous histological type (P=0.004) and PIK3CA amplification (P=0.003). Aberrant promoter methylation was associated with LOH (P=0.017) and absence of germline mutations (P=0.037). The high frequency of LOH at the BRCA1 locus suggests that LOH may be an important mechanism of BRCA1 deficiency in ovarian carcinomas. Tumors with various BRCA1 alterations have a similar phenotype of high-grade, high-stage carcinomas with frequent TP53 mutations.

Tumor‐specific mutation and downregulation of ING5 detected in oral squamous cell carcinoma

Our previous study showed high frequency of allelic loss at chromosome 2q37 region in oral cancer. This location contains several candidate tumor suppressor genes such as PPP1R7, ILKAP, DTYMK and ING5. We previously showed 3 members of inhibitor of growth (ING) family, ING1, ING3 and ING4 as tumor suppressor gene in head and neck cancer. As ING5 shows high homology with other members of ING genes including highly conserved carboxy-terminal plant homeodomain and nuclear localization signal, we first picked up ING5 and examined it as a possible tumor suppressor in oral cancer. For this aim, mutation and mRNA expression status of ING5 in paired normal and oral squamous cell carcinoma samples were examined by reverse transcription polymerase chain reaction (RT-PCR) and sequencing. Three missense mutations located within leucine zipper like (LZL) finger and novel conserved region (NCR) domains in ING5 protein were detected, probably abrogating its normal function. We also found 5 different alternative splicing variants of ING5. Then, we examined mRNA level of ING5 by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) analysis, which demonstrated decreased expression of ING5 mRNA in 61% of the primary tumors as compared to the matched normal samples. In conclusion, tumor-specific mutation and downregulation of ING5 mRNA suggested it as a tumor suppressor gene in oral squamous cell carcinoma.

Reduced expression of EphB2 is significantly associated with nodal metastasis in Chinese patients with gastric cancer

EphB2 is a member of the Eph receptor tyrosine kinase family that has been involved in the regulation of cytoskeleton organization and cell migration in various cell types. Its role and regulation in carcinogenesis is controversial, especially in gastric cancer. We detected EphB2 expression and determined its clinical significance and explored its underlying molecular mechanism in gastric cancers. Tissue microarray blocks containing primary gastric cancer, lymph node metastases, and adjacent normal mucosa specimens obtained from 337 Chinese patients were constructed. Expression of EphB2 in these specimens was analyzed using immunohistochemistry. Mutation analysis at the A9 tract in exon 17 and loss of heterozygosity analysis at the EphB2 gene locus were carried out in 13 sporadic EphB2-negative gastric cancers. Complete loss of EphB2 expression was observed in 177 (52.5%) of the 337 primary tumor and 41 (82%) of the 50 nodal metastases. Loss of EphB2 expression was significantly associated with advanced T stage, nodal metastasis, advanced disease stage, and poor histological differentiation. Loss of EphB2 expression correlated significantly with poor survival rates in both univariate and multivariate analysis. No frameshift mutation, but a higher frequency of allelic loss, was found in EphB2-negative primary and metastatic tumor samples. Frequent deletion and decreased expression of EphB2 protein suggested it as a negative biomarker for gastric carcinogenesis and a potential predictor of the outcome of patients with gastric cancer.

High incidence of allelic loss at 16q12.2 region spanning RB2/p130 gene in retinoblastoma

Retinoblastoma (Rb) is the most common intra-ocular tumor that manifests in early childhood. It is initiated by the inactivation of RB1/p105 gene, a prototype tumor suppressor gene. However, observed recurrent chromosomal aberrations accompanying RB1/p105 mutations suggest the involvement of additional mutational events. Chromosome 16q is one of the loci with recurrent losses which are likely to contain tumor suppressor genes. In this study, allelic loss was demonstrated at a second locus for retinoblastoma, RBL2/p130 on 16q12.2. Using intragenic single nucleotide polymorphisms (SNPs) (g.7085556A>C and g.7118919T>C) and flanking extragenic microsatellite markers (D16S411 and D16S408), 40 retinoblastoma tumor samples were analyzed. Loss of heterozygosity (LOH) of these markers was found in 11 (57.9%) out of 19 informative tumors at the RBL2/p130 gene locus and while a total of 15 (78.9%) tumors showed LOH in at least one marker. Deletions extending more than 13 cM across the pericentromeric region of 16q12.1-q13 were inferred from 4 tumors. Microsatellite instability was observed in two other tumors at the flanking markers. No mutations were found in RBL2/p130 exons 19-22 coding for the protein domain critical for biological activity. This is the first evidence of LOH within RBL2/p130 gene in retinoblastoma. The high frequency of allelic loss provides further evidence on the implication of this gene in retinoblastoma development and/or progression.

Molecular evidence for a clonal relationship between multiple lesions in patients with unknown primary adenocarcinoma

Unknown primary adenocarcinoma (UPA) comprises a group of heterogeneous cancers of great clinical and biological interest. UPA presents as metastatic disease without a detectable primary site after medical workup. Here we investigated whether or not a clonal relationship exists between multiple tumors within individual UPA patients. A molecular resemblance would argue for an early clonal outgrowth of tumor cells from the primary lesion, a mutual feature observed within this group of neoplasms. In 14 patients with UPA multiple tumors, obtained at autopsy, were analyzed by molecular allelotyping and immunohistochemistry. In addition, tumors of 4 patients could be analyzed by comparative genomic hybridization (CGH). Similar genetic and phenotypic profiles were used as indicator for a clonal relationship, whereas different profiles implicate independent tumors. The molecular data indicated that the multiple lesions in the 14 UPA patients, including the primary tumors, are clonally related. In agreement with the theory of tumor progression, some metastatic lesions showed additional genetic alterations besides the characteristics that were shared with the primary tumor. Furthermore, 8 UPA patients had tumors with a high frequency of allelic loss and/or imbalance (FALI; 43-71%), while 6 patients demonstrated a lower FALI (14-29%), suggesting the occurrence of chromosomal instability in the former group. Our data provide molecular evidence for a clonal relationship between multiple metastases and the primary tumor within individual UPA patients, independent of the anatomical origin of the cancer. This finding is in agreement with the suggestion that tumor progression is rapid in UPA patients, limiting the chance of clonal divergence. The identification of 2 groups of UPAs with either a high or low FALI indicates that chromosomal instability is not the only driving force behind early tumor cell dissemination. Thus, other molecular mechanisms must underlie the common biology of these tumors.

Molecular genetic analysis of the BRCA2 tumor suppressor gene region in cutaneous squamous cell carcinomas

Germ line mutations of the BRCA2 tumor suppressor gene with subsequent loss of the remaining wild-type BRCA2 allele have been identified in up to 35% of familial breast cancer cases. A high frequency of allelic loss at the BRCA2 gene locus has also been reported in a variety of sporadic epithelial tumors including oesophageal squamous cell carcinomas (SCC), and sporadic head and neck SCC. The present study aimed to examine the integrity of the BRCA2 gene in cutaneous SCC. Allelic imbalance/loss of heterozygosity (AI/LOH) was examined in 22 histologically confirmed cutaneous SCC at two microsatellite markers, D13S260 (centromeric to the BRCA2 gene) and D13S267 (telomeric to the BRCA2 gene). Immunohistochemical analysis of BRCA2 protein expression was also examined in the cutaneous SCC. AI/LOH at the D13S260 locus was found in eight of the 19 informative SCC, and AI/LOH at the D13S267 locus was found in 12 of the 18 informative SCC. Seven SCC showed allelic loss at both markers, and six SCC showed retention of heterozygosity at both markers. Expression of BRCA2 protein was only detected in six of the normal epidermises and three of the 21 SCC examined. AI/LOH of the BRCA2 gene region was found to be common in the cutaneous SCC.

Tongue cancer patients have a high frequency of allelic loss at the von Hippel‐Lindau gene and other loci on 3p

Although genetic abnormalities on 3p have been suggested to be linked to the development of squamous cell carcinoma of the head and neck, to the authors' knowledge no study to date has examined such genetic abnormalities in patients with squamous cell carcinoma of the tongue. In the current study, loss of heterozygosity (LOH) was evaluated at several loci within 3p, including the von Hippel-Lindau gene (VHL), in samples of tongue squamous cell carcinoma. In addition, the coding region of the intact VHL allele was screened for sequence mutations. DNA was extracted from tumor and nontumor tissues collected from 28 patients with tongue squamous cell carcinoma. LOH was investigated by analysis of single nucleotide polymorphisms within exon 3 of VHL and by microsatellite analysis within another 10 loci. Mutation analysis of the VHL gene was performed by polymerase chain reaction (PCR) amplification and sequencing of the coding region of the gene. LOH within VHL was found at a high frequency (45.5%) within the tumor. However, mutations of the VHL gene were not detected in all tumor samples. LOH of other microsatellite markers on 3p was observed in 27.3% to 50% of tumor samples. Eleven (58%) of 19 samples that were informative at more than 2 loci exhibited LOH of at least 1 locus; 10 of these 11 cases exhibited LOH at multiple loci. A wide range of deletions in 3p, including at the VHL gene, may play a role in the development of tongue cancer.

Multiple splicing variants of Naf1/ABIN-1 transcripts and their alterations in hematopoietic tumors

Naf1 (Nef-associated factor 1)/TNIP1/ABIN-1 (A20-binding inhibitor of NF-kappaB activation) is a cellular protein that interacts and cooperates with the NFkappaB inhibiting protein A20. It is reported that Naf1 attenuates epidermal growth factor (EGF)/extracellular-signal-regulated kinase2 (ERK2) nuclear signaling. Naf1 also binds to Nef, which plays a key role in acquired immunodeficiency syndrome pathogenesis and HIV-1 virus replication. Naf1 mRNA consists of 18 exons and multiple splice variants have been reported; two isoforms for exon 1, deletion of exon 2 and isoforms alpha and beta for exon 18. Using specimens from 29 acute myeloid leukemia (AML) patients, we detected a high frequency of allelic loss on DNA at STS marker D5S2014 near the Naf1 gene. We therefore performed mutation and expression analyses using leukemia-lymphoma lines and 6 pairs of clinical AML samples. There was no mutation in the Naf1 coding region of any sample. As a result of expression analysis, we identified novel splice variants of the Naf1 gene; deletion of exon 16 (Naf1 alpha2, Naf1 beta2), deletion of exon 16 with an insertion (Naf1 alpha3, Naf1 beta3) and deletion of exons 16 and 17 (Naf1 alpha4). Naf1 alpha3 and beta3 showed premature termination. In peripheral blood mononucleocytes (PBMNCs) from healthy adults, almost no expression of full-length Naf1 (Naf1FL), Naf1 alpha3 and beta3 were observed. In contrast, their expression was clear in AML blasts and in the majority of leukemia-lymphoma lines investigated. Naf1 alpha2 was widely expressed in PBMNCs from healthy adults, AML blasts and cell lines, suggesting it is the main transcript of the Naf1 gene. Luciferase assay revealed that Naf1 alpha2 had equal NF-kappaB inhibitory effect to that of Naf1FL, while Naf1 alpha4 was less effective. In clinical AML patients, the expression of Naf1 alpha3 was much higher at diagnosis than on remission after chemotherapy, suggesting the possible dominant negative effect of Naf1 alpha3.

Mutation analysis of the 8p22 candidate tumor suppressor gene ATIP/ MTUS1 in hepatocellular carcinoma

A high frequency of allelic loss affecting chromosome 8p and a minimal region of deletion at p21-22 have been previously reported in hepatocellular carcinoma (HCC), suggesting that at least one tumor suppressor gene is present in this region. In this study, we assessed whether the angiotensin II AT2 receptor interacting protein ( ATIP)/mitochondrial tumor suppressor gene ( MTUS1), a gene newly identified at position 8p22, may be a candidate tumor suppressor gene mutated in HCC. We searched for alterations in the 17 coding exons of ATIP/ MTUS1 by means of denaturating high-performance liquid chromatography and sequencing, in 51 HCC tumors and 58 cell lines for which loss of heterozygosity status was known. Five major nucleotide substitutions were identified, all located in exons used by the ATIP3 transcript which is the only ATIP transcript variant expressed in liver. These nucleotide variations result in amino-acid substitution or deletion of conserved structural motifs (nuclear localisation signal, polyproline motif, leucine zipper) and also affect exonic splicing enhancer motifs and physiological splice sites, suggesting potential deleterious effects on ATIP3 function and/or expression.

Localization of a susceptibility locus for hepatocellular carcinoma to chromosome 4q in a hepatitis B hyperendemic area

Chromosome 4q is one of the most common regions with a high frequency of allelic loss in hepatocellular carcinoma (HCC). To identify the HCC-susceptibility locus on chromosome 4q, we have performed linkage and family-based association analyses on Chinese families with HCC from Taiwan, where hepatitis B is hyperendemic. Using 77 microsatellite markers spanning chromosome 4q on 52 multiplex families, we found suggestive evidence of linkage to 4q22.3-28.1 with a maximum two-point heterogeneity LOD (HLOD) score of 2.55 at marker D4S3240 on chromosome 4q25. Multipoint analyses with microsatellite markers in the region 4q22.3-28.1 resulted in a maximum HLOD score of 3.12 and a maximum nonparametric linkage (NPL) Z score of 1.98 (pointwise P=0.0080; region-wide empirical P=0.021) for D4S3240. The evidence for linkage to D4S3240 was seen mostly in a subset of 28 families lacking affected parents, which showed multipoint HLOD and NPL scores of 3.25 and 2.79 (pointwise P=0.0028; region-wide empirical P=0.008), respectively. Family-based association analyses of the 77 microsatellite markers in 191 families (53 multiplex plus 138 singleton families) using the pedigree disequilibrium test provide further support for observed linkage. Additional genotyping in the 52 multiplex families informative for linkage analyses was performed for 29 single-nucleotide polymorphisms around D4S3240. A common haplotype (at markers rs7442180 and rs221330) positioned approximately 873 kb away from D4S3240 was associated with HCC, with P=0.0074.

Analysis of Allelic Loss as an Adjuvant Tool in Evaluation of Malignancy in Uterine Smooth Muscle Tumors

Uterine smooth muscle tumors of uncertain malignant potential (STUMPs) are difficult both from the diagnostic and patient management standpoint because they cannot be classified as benign or malignant by conventional histologic criteria. This study's aim was to determine the diagnostic utility of allelic imbalance (AI) analysis in uterine smooth muscle tumors. Using microdissection and genotyping, we tested 5 leiomyomas, 6 STUMPs, and 10 leiomyosarcomas with follow-up for AI across a panel of seven tumor suppressor genes (p16, p21, p53, VHL, XRCC3, RB, and NM-23). None of the 6 patients with STUMP experienced recurrent disease, whereas 8 of the 10 patients diagnosed with leiomyosarcoma died of disease at follow-up. The mean frequency of allelic loss (FAL) for leiomyomas (18%) was not significantly different from that of STUMPs (21%) (P = 1), whereas leiomyosarcomas displayed a significantly higher FAL (52%) than both leiomyomas (P = 0.001) and STUMPs (P = 0.002). Loss of NM-23, a reported tumor metastasis suppressor gene, was found only in leiomyosarcomas (5 of 9, or 56%), and 4 of 5 (80%) of these were the only cases that demonstrated distant metastases (P = 0.04). Additionally, an FAL of >50% correlated with both NM-23 loss (P = 0.008) and distant metastatic disease (P = 0.04). In conclusion, leiomyomas and STUMPs displayed similar mean FALs and all were clinically benign, whereas uterine leiomyosarcomas had significantly higher frequencies of allelic loss than both leiomyomas and STUMPs. Molecular profiling may thus provide a valuable tool in assessment of malignancy in uterine smooth muscle tumors. Additionally, NM-23 is a promising candidate gene for determination of metastatic potential in these tumors.

A novel region of deletion on 13q33-q34 in esophageal squamous cell carcinoma

Chromosome 13 presents frequent allelic loss in esophageal squamous cell carcinomas (ESCC). However, no ESCC suppressor gene has been identified from this chromosome. To define common deletion regions that possibly contain the ESCC suppressor gene(s), we performed a mapping of allelic loss in 50 esophageal squamous cell carcinomas using a panel of 25 microsatellite markers on chromosome 13q21-qter, which has rarely been studied for allelic loss. Loss of heterozygosity (LOH) with high frequencies (> or = 50%) was observed at markers D13S1494, D13S1323, D13S248, D13S1315, D13S285, and D13S1295, in which the peak LOH (69.2%) was at locus D13S248. Seven cases presented LOH at three consecutive markers D13S248, D13S1315 and D13S285, 4 of which also displayed LOH at another adjacent marker D13S1295. This overlapping region of deletion covers an interval of 6.36 Mb at 13q33.1-q34, whose deletion has not previously been reported in ESCC. Tumors of grade II showed significantly more frequent LOH at D13S248 than those of grade I. A significantly higher frequency of allelic loss at D13S152 was also found in tumors with lymph node metastasis compared to those without lymph node metastasis. The present study defined a novel region of allelic loss in 13q33-q34. LOH at D13S248 and D13S152 are associated with higher tumor grade and metastasis, respectively.

Aberrant expressions of annexin A10 short isoform, osteopontin and α-fetoprotein at chromosome 4q cooperatively contribute to progression and poor prognosis of hepatocellular carcinoma

Chromosome 4q exhibits high frequency of allelic loss in hepatocellular carcinoma (HCC). This study aimed to elucidate the interaction of the frequent aberrant mRNA expression of alpha-fetoprotein (AFP), osteopontin (OPN) and a novel short isoform of annexin A10 (ANXA10S) at 4q in the tumor progression among 294 patients who received surgical resection of unifocal primary HCC. AFP overexpression, OPN overexpression and ANXA10S down-regulation correlated with high-grade and high-stage tumors, early tumor recurrence (all P<0.0001), and lower 10-year survival (all P=0.000001). The AFP overexpression correlated with OPN overexpression (P=0.0026) and ANXA10S down-regulation (P=0.00001), while OPN overexpression correlated with ANXA10S down-regulation (P=0.00001). Pair-wise combinations revealed interactive effects between these genetic variants for tumor grade, tumor stage and early recurrence (all P<0.0001). HCCs with more genetic aberrations had more frequent high tumor grade, portal vein invasion (stage IIIB-IV) and early recurrence (all P<0.0001). The 10-year survival rate for HCCs with all three genetic alterations was the lowest (7%), followed by those with two (22%) or one event (29%), and the highest for those without these changes (43%), P=0.000001. The prognostic stratification using these molecular factors was similar to that of histopathological staging. These three genetic alterations also helped to identify different subgroups of patients of stage II HCC but with different prognosis (P=0.015). In conclusion, the aberrant expressions of AFP, OPN and ANXA10S cooperatively contribute to tumor progression and poor prognosis, and are useful for molecular staging of HCC and the subclassification of stage II HCC without vascular invasion.

Evidence for a p27 tumor suppressive function independent of its role regulating cell proliferation in the prostate

Reduced p27 levels correlate with poor prognosis in a wide spectrum of human tumors and can accelerate tumorigenesis in mouse tissues. To determine whether p27 deficiency can accelerate tumorigenesis in tissues with inactive Rb and p53 pathways, we examined the effect of p27 status on prostate tumorigenesis in mice expressing simian virus 40 large T antigen (LT). In p27-deficient mice expressing LT, tumors progressed from high-grade prostatic intraepithelial neoplasia to poorly differentiated carcinoma at a greatly accelerated rate. p27 deficiency could not collaborate with a mutant of LT that fails to inactivate the Rb pathway alone. Furthermore, p27 deficiency does not increase the proliferation index, reduce the apoptotic index, or affect the expression of E2F-dependent genes in cells expressing LT at any stage of the disease. Expression of LT alone leads to maximal proliferation, but p27 deficiency still increases the amount of cyclin A and cyclin-dependent kinase 2-associated kinase activity in tissues. Interestingly, this model recapitulates an important feature of the human disease, specifically a high frequency of allelic loss of chromosome 16q, which is syntenic to mouse chromosome 8. Loss of heterozygosity may accelerate the inactivation of other tumor suppressors, such as E-cadherin, which are located in this interval. These experiments provide direct physiological and causal evidence that p27 has tumor suppressive functions independent of its role regulating cell proliferation.

Comprehensive characterization of annexin I alterations in esophageal squamous cell carcinoma.

The purpose is to characterize alterations of the annexin I gene, its mRNA, and protein expression in esophageal squamous cell carcinoma. Fifty-six cases of esophageal squamous cell carcinoma were analyzed using four microsatellite markers flanking the annexin I gene (9q11-q21) to identify loss of heterozygosity. In addition, we performed (a) single-strand conformation polymorphism and DNA sequencing along the entire promoter sequence and coding region to identify mutations, (b) real-time quantitative reverse transcription-PCR of RNA from frozen esophageal squamous cell carcinoma tissue (n = 37) and in situ hybridization (n = 5) on selected cases to assess mRNA expression, and (c) immunohistochemistry (n = 44) to evaluate protein expression. The prevalence of the allelic variants identified in the first 56 patients was refined in 80 additional esophageal squamous cell carcinoma patients and 232 healthy individuals. Forty-six of 56 (82%) esophageal squamous cell carcinoma patients showed loss of an allele at one or more of the four microsatellite markers; however, only one (silent) mutation was seen. Two intragenic variants were identified with high frequency of allelic loss (A58G, 64%; L109L, 69%). Thirty of 37 (81%) esophageal squamous cell carcinoma patients showed reduced annexin I mRNA expression, which was confirmed by in situ hybridization, whereas annexin I protein expression was reduced in 79% of poorly differentiated tumor cell foci but in only 5% of well-differentiated tumor foci, although allelic loss on chromosome 9 was found in both tumor grades. Allelic loss of annexin I occurs frequently, whereas somatic mutations are rare, suggesting that annexin I is not inactivated in esophageal squamous cell carcinoma via a two-hit mechanism. A decrease in annexin I protein expression was confirmed, consistent with a quantitative decrease in mRNA expression, and appeared to be related to tumor cell differentiation. We conclude that annexin I is not the tumor suppressor gene corresponding to the high levels of loss of heterozygosity observed on chromosome 9 in esophageal squamous cell carcinoma; however, dysregulation of mRNA and protein levels is associated with this tumor type.

Molecular analysis to demonstrate that odontogenic keratocysts are neoplastic.

Odontogenic keratocysts (OKCs) are unique odontogenic lesions that have the potential to behave aggressively, that can recur, and that can be associated with the nevoid basal cell carcinoma syndrome. Whether they are developmental or neoplastic continues to be debated. To identify loss of heterozygosity of tumor suppressor genes in OKCs and to suggest a pathogenetic origin for these lesions. We examined 10 OKCs for loss of heterozygosity of tumor suppressor genes, using a microdissection and semiquantitative genotyping analysis. The genes analyzed included 10 common tumor suppressor genes, as well as the PTCH gene, which is mutated in nevoid basal cell carcinoma syndrome. Loss of heterozygosity was seen in 7 of 10 cases, with a frequency between 11% and 80% of the genes studied. The genes that exhibited the most frequent allelic losses were p16, p53, PTCH, and MCC (75%, 66%, 60%, and 60%, respectively). Daughter cysts were associated with a higher frequency of allelic loss (P =.02), but epithelial budding was not. Our study indicates that a significant number of OKCs show clonal loss of heterozygosity of common tumor suppressor genes. The finding of clonal deletion mutations of genomic DNA in these cysts supports the hypothesis that they are neoplastic rather than developmental in origin.

Evaluation of BRCA2 in the genetic susceptibility of familial esophageal cancer

Previous studies of esophageal squamous cell carcinoma (ESCC) have shown a high frequency of allelic loss on chromosome 13q, infrequent somatic mutations in BRCA2, and a suggested association between a positive family history (FH+) of upper gastrointestinal cancer and germline BRCA2 mutations. In all, 70 ESCC patients (44 FH+ and 26 FH-) were examined by direct full sequencing of germline DNA for BRCA2 mutations. In addition, 28 family members of three of these patients and 232 unrelated healthy blood bank donor controls were examined for the mutations identified in the 70 ESCC patients. Five BRCA2 germline mutations, including three not previously reported (N1600del, A2054P, and V2109I), were identified in six of 44 FH+ patients, but none of 26 FH- patients (14 vs 0%, P=0.078), consistent with our previous findings (3/34 or 9% FH+ vs 0/22 or 0% FH-, P=0.27). The cumulative frequency of BRCA2 germline mutations in ESCC patients in this and our previous study combined is 12%, with all mutations found in FH+ as opposed to FH- cases (9/78 or 12% FH+ vs 0/48 or 0% FH-, P=0.013). We conclude that germline mutations in BRCA2 in ESCC patients from this high-risk area of China are more frequent in FH+ than FH- cases, suggesting that BRCA2 may play a role in genetic susceptibility to familial ESCC.

Granular cell astrocytomas show a high frequency of allelic loss but are not a genetically defined subset.

Granular cell astrocytomas (GCA) are an uncommon morphologic variant of infiltrative glioma that contains a prominent population of atypical granular cells. As a rule, they are biologically aggressive compared to similar tumors without granular features. We sought to determine whether GCAs possess distinct genotypic alterations that might reflect their unique morphology or clinical behavior. Eleven GCAs occurring in 7 men and 4 women ranging in age from 46 to 75 years were investigated for genetic alterations of known significance in glial tumorigenesis, including LOH at 1p, 9p, 10q, 17p, and 19q, point mutations of TP53, deletions of p16(CDKN2A) and p14ARF, as well as EGFR amplifications. Tumors included had an infiltrative growth pattern and consisted of large, round cells packed with eosinophilic, PAS-positive granules that varied in quantity, ranging from 30 to 100% of tumor cells. Three tumors were of WHO grade II, one was grade III, and 7 were grade IV lesions. Overall, the tumors showed higher frequencies of LOH at 1p, 9p, 10q, 17p, and 19q than typical infiltrating astrocytomas of similar grades. Losses on 9p and 10q occurred in nearly all cases, including low grade lesions. TP53 mutations were identified in 2 grade IV GCAs, while combined p14ARF and p16(CDKN2A) homozygous deletions were noted in only one grade IV lesion. None showed EGFR amplification. We found no genetic alterations specific for GCA. Instead, it appears that granular cell change occurs across genetic subsets. The high frequency of allelic loss, especially on 9p and 10q, may confer aggressive growth potential and be related to their rapid clinical progression.

A novel microdissection and genotyping of follicular-derived thyroid tumors to predict aggressiveness

Distinguishing thyroid follicular adenoma from minimally invasive or encapsulated angioinvasive carcinoma can be diagnostically challenging. In some cases, tumors are distorted, fragmented, or stripped of their capsule, and a definitive diagnosis becomes nearly impossible. In other cases, the foci of capsular and/or vascular invasion are subtle, thus making the diagnosis of carcinoma difficult. We developed a microdissection genotyping assay for assessing a panel of tumor-suppressor genes for loss of heterozygosity mutations. The frequency of allelic loss (FAL) in follicular-derived neoplasms correlates with the histologic aggressiveness of the tumor. Furthermore, we calculated the amount of genetic heterogeneity within each tumor, as a second important measure of a tumor's ability for clonal expansion and a surrogate marker for its malignant potential. The follicular adenomas had a low FAL (average 9%) and low intratumoral heterogeneity (5% variability). The minimally invasive and encapsulated angioinvasive carcinomas had an intermediate FAL (average 30%) and intermediate intratumoral heterogeneity (10% variability). The widely invasive carcinomas had a high FAL (average 53%) and high intratumoral heterogeneity (24% variability). Although a larger retrospective study is needed to correlate genotyping studies with patient outcome and prognosis, our results indicate that performing a mutational genotyping assay can stratify tumors into the histologically well-defined categories of adenomas, minimally invasive/angioinvasive carcinomas, and widely invasive follicular carcinomas. Hum Pathol 34:375-380. © 2003 Elsevier Inc. All rights reserved.

Allelic imbalance in selected chromosomal regions in ovarian cancer

Ovarian cancer (OC) is often asymptomatic at the initial stage. When diagnosed, up to 75% of the patients present grade III or IV tumors with metastasis in nearby organs of the abdomen. Genetic imbalance is abundant in OC, and allelic loss (AL) of specific chromosomal regions is considered an early event. To establish association between genetic markers for early diagnosis/prognosis of OC, our target was to define narrow specific regions of AL. We analyzed 65 ovarian carcinomas by using 19 microsatellite markers located in three different chromosomes. First, a 7.6-Mb region containing the estrogen receptor ( ESR1) and the tumor suppressor gene LATS1 was analyzed. Several chromosomal breakpoints flanking ESR1 affecting the region harboring LATS1 were found. Second, we found chromosomal breakpoints on 13q13.1∼q13.3 that defined two narrow regions flanking the BRCA2 locus. Third, our ovarian tumors exhibited a very high frequency of AL on 16q and chromosomal breakpoints defining two narrow regions within 16q22.2∼q24.3. In this article, we report three new polymorphic microsatellite markers and strong evidence of AL of narrow well-defined regions in hot spots on 6q, 13q, and 16q in ovarian tumors.