Rb and TP53 pathway alterations in sporadic and NF1-related malignant peripheral nerve sheath tumors.

SUMMARY: Karyotypic complexities associated with frequent loss or rearrangement of a number of chromosome arms, deletions, and mutations affecting the TP53 region, and molecular alterations of the INK4A gene have been reported in sporadic and/or neurofibromatosis type I (NF1)-related malignant peripheral nerve sheath tumors (MPNSTs). However, no investigations addressing possible different pathogenetic pathways in sporadic and NF1-associated MPNSTs have been reported. This lack is unexpected because, despite similar morphologic and immunophenotypic features, NF1-related cases are, by definition, associated with NF1 gene defects. Thus, we investigated the occurrence of TP53 and p16(INK4A) gene deregulation and the presence of microsatellite alterations at markers located at 17p, 17q, 9p21, 22q, 11q, 1p, or 2q loci in MPNSTs and neurofibromas either related (14 cases) or unrelated (14 cases) to NF1. Our results indicate that, in MPNSTs, p16(INK4A) inactivation almost equally affects both groups. However, TP53 mutations and loss of heterozygosity involving the TP53 locus (43% versus 9%), and p53 wild type overexpression, related or not to mdm2 overexpression (71% versus 25%), seem to mainly be restricted to sporadic MPNSTs. In NF1-associated MPNSTs, our microsatellite results are consistent with the occurrence of somatic inactivation by loss of heterozygosity of the second NF1 allele.     

Loss of p16 (INK4A) expression is associated with allelic imbalance/loss of heterozygosity of chromosome 9p21 in microdissected malignant peripheral nerve sheath tumors.

The p16 is a tumor suppressor gene on the short arm of chromosome 9p21. The product of the p16 acts as a negative cell cycle regulator by inhibiting G1 cyclin-dependent kinases that phosphorylate the retinoblastoma protein. This study was designed to assess the frequency of genetic loss of 9p21 and to determine the role of p16 the pathogenesis of sporadic and neurofibromatosis 1 (NF1)-associated malignant peripheral nerve sheath tumors (MPNSTs). The authors examined 15 cases for p16 protein expression and 10 cases for allelic imbalance (AI)/loss of heterozygosity (LOH) of chromosome 9p. DNA was microdissected from normal and neoplastic tissues. AI/LOH analysis was performed using six microsatellite markers on the 9p region. On immunohistochemical analysis 80% of cases showed abnormal expression of p16. Similarly, 8 of 10 cases revealed genetic loss with at least one microsatellite marker. The most frequent deletion was that within the coding sequence. Of p16 at me D9S974 locus. These findings emphasize the role of loss of p16 in the development of both sporadic and NF1-associated MPNSTs.     

Frequent genomic imbalances in chromosomes 17, 19, and 22q in peripheral nerve sheath tumours detected by comparative genomic hybridization analysis

Comparative genomic hybridization (CGH) was used to detect changes in relative chromosome copy number in 50 cases of peripheral nerve sheath tumour (PNSTs), including nine malignant peripheral nerve sheath tumours (MPNSTs), 27 neurofibromas (with three plexiform neurofibromas) and 14 schwannomas. Chromosome imbalances were frequently detected in benign as well as malignant PNSTs. In both NF1-associated and sporadic MPNSTs, the number of gains was higher than the number of losses, suggesting proto-oncogene activation during MPNST progression. NF1-asociated MPNSTs exhibited gains of chromosomes 17q and X (2/4 cases each), whereas sporadic MPNSTs showed gains of chromosome 4q (3/5 cases). On the other hand, in benign neurofibromas and schwannomas, the number of losses was higher than the number of gains, suggesting a predominant role of tumour suppressor genes in tumourigenesis. Both sporadic and NF1-associated neurofibromas exhibited losses at chromosome 22q in more than 50% of cases. These chromosomal regions may contain common chromosomal abnormalities characteristic of both types of neurofibromas. In NF1-associated neurofibromas, most frequent losses were found in chromosomes 17 [17p11.2-p13 in nine cases (60%); 17q24-25 in 6 cases (40%)] and 19 [19p13.2 in eight cases (53%); 19q13.2-qter in eight cases (53%)], whereas in sporadic neurofibromas and schwannomas losses of chromosomes 17 and 19 were detected in less than 50% of cases. Since this 17p11.2-p13 region is known to contain the tumour suppressor gene TP53, patients with NF1 may be at high risk of malignant neoplasms including MPNSTs. Gains were more frequently detected in plexiform neurofibromas (2/3 cases) than other benign tumours, suggesting proto-oncogene activation in tumourigenesis of plexiform neurofibroma. The significance of the losses of chromosome 19 in these cases is not clear at present, but in NF1-associated neurofibromas, the presence of some as yet unknown tumour suppressor genes on chromosome 19 cannot be ruled out.     

Molecular analysis of malignant triton tumors.

Triton tumors are rare variants of malignant peripheral nerve sheath tumor (MPNST) with muscle differentiation, often seen in patients with neurofibromatosis 1 (NF1). Individuals affected with NF1 harbor mutations in the NF1 tumor suppressor gene and develop neurofibromas and MPNSTs. The NF1 gene is expressed in Schwann cells and its expression is lost in schwannian neoplasms, suggesting a role in malignant development. Separately, there is evidence that p53 suppressor gene mutations are involved in MPNSTs. To determine the role of the NF1 and p53 genes in the development of the malignant Triton tumor we examined 2 such tumors, 1 from a 3-year-old boy without clinical manifestations of NF1 and another from a 24-year-old man with NF1. Histological analysis of these tumors showed both neural and muscle differentiation with S-100 and desmin immunoreactivity, respectively. Reverse transcribed RNA polymerase chain reaction (RT-PCR) of NF1 mRNA showed NF1 expression in the sporadic tumor. Strong nuclear immunoreactivity for p53 was observed throughout the malignant population in both tumors. This was confirmed by loss of heterozygosity for p53 in the non-NF1 patient, suggesting that p53 is involved in both hereditary and sporadic Triton tumors. The finding of preserved NF1 gene expression in the non-NF1-related Triton tumor suggests that different genetic events predispose to the development of this rare neoplasm in sporadic cases.     

Chromosome 17 loss-of-heterozygosity studies in benign and malignant tumors in neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a common autosomal dominant condition characterized by benign tumor (neurofibroma) growth and increased risk of malignancy. Dermal neurofibromas, arising from superficial nerves, are primarily of cosmetic significance, whereas plexiform neurofibromas, typically larger and associated with deeply placed nerves, extend into contiguous tissues and may cause serious functional impairment. Malignant peripheral nerve sheath tumors (MPNSTs) seem to arise from plexiform neurofibromas. The NF1 gene, on chromosome segment 17q11.2, encodes a protein that has tumor suppressor function. Loss of heterozygosity (LOH) for NF1 has been reported in some neurofibromas and NF1 malignancies, but plexiform tumors have been poorly represented. Also, the studies did not always employ the same markers, preventing simple comparison of the frequency and extent of LOH among different tumor types. Our chromosome 17 LOH analysis in a cohort of three tumor types was positive for NF1 allele loss in 2/15 (13%) dermal neurofibromas, 4/10 (40%) plexiform neurofibromas, and 3/5 (60%) MPNSTs. Although the region of loss varied, the p arm (including TP53) was lost only in malignant tumors. The losses in the plexiform tumors all included sequences distal to NF1. No subtle TP53 mutations were found in any tumors. This study also reports the identification of both NF1 "hits" in plexiform tumors, further supporting the tumor suppressor role of the NF1 gene in this tumor type.     

Losses in chromosomes 17, 19, and 22q in neurofibromatosis type 1 and sporadic neurofibromas: a comparative genomic hybridization analysis

Neurofibromatosis type 1 (von Recklinghausen's NF1) is an autosomal dominant disease associated with an increased risk of benign and malignant neoplasia including malignant peripheral nerve sheath tumors (MPNSTs). In this study, we employed comparative genomic hybridization (CGH) to determine changes in the relative chromosome copy number in 24 patients with neurofibromas, including 12 NF1-associated and 12 sporadic cases. Differences in the frequency and distribution of chromosomal imbalances were observed in both NF1-asociated and sporadic neurofibromas. Chromosomal imbalances were more common in NF1-associated tumors than in sporadic neurofibromas. In both groups, the number of losses was higher than the number of gains, suggesting a predominant role of tumor suppressor gene in tumorigenesis. A number of new chromosomal imbalances were noted including chromosomes 17, 19, and chromosome arm 22q, which may be related to oncogenes or tumor suppressor genes in neurofibromas. In NF1-associated neurofibromas, the most frequent losses were found in chromosome 17 (the minimal common regions were 17p11.2-->p13 in nine cases and 17q24-->q25 in six cases) and 19p (19p13.2 in nine cases). In addition, both NF1-associated and sporadic neurofibromas often exhibited losses at chromosome arms 19q and 22q (in NF1 tumors, the minimal common regions were 19q13.2-->qter in seven cases).     

Characterization of the somatic mutational spectrum of the neurofibromatosis type 1 (NF1) gene in neurofibromatosis patients with benign and malignant tumors

One of the main features of neurofibromatosis type 1 (NF1) is benign neurofibromas, 10-20% of which become transformed into malignant peripheral nerve sheath tumors (MPNSTs). The molecular basis of NF1 tumorigenesis is, however, still unclear. Ninety-one tumors from 31 NF1 patients were screened for gross changes in the NF1 gene using microsatellite/restriction fragment length polymorphism (RFLP) markers; loss of heterozygosity (LOH) was found in 17 out of 91 (19%) tumors (including two out of seven MPNSTs). Denaturing high performance liquid chromatography (DHPLC) was then used to screen 43 LOH-negative and 10 LOH-positive tumors for NF1 microlesions at both RNA and DNA levels. Thirteen germline and 12 somatic mutations were identified, of which three germline (IVS7-2A>G, 3731delT, 6117delG) and eight somatic (1888delG, 4374-4375delCC, R2129S, 2088delG, 2341del18, IVS27b-5C>T, 4083insT, Q519P) were novel. A mosaic mutation (R2429X) was also identified in a neurofibroma by DHPLC analysis and cloning/sequencing. The observed somatic and germline mutational spectra were similar in terms of mutation type, relative frequency of occurrence, and putative underlying mechanisms of mutagenesis. Tumors lacking mutations were screened for NF1 gene promoter hypermethylation but none were found. Microsatellite instability (MSI) analysis revealed MSI in five out of 11 MPNSTs as compared to none out of 70 neurofibromas (p=1.8 x 10(-5)). The screening of seven MPNSTs for subtle mutations in the CDKN2A and TP53 genes proved negative, although the screening of 11 MPNSTs detected LOH involving either the TP53 or the CDKN2A gene in a total of four tumors. These findings are consistent with the view that NF1 tumorigenesis is a complex multistep process involving a variety of different types of genetic defect at multiple loci.     

Exploring the somatic NF1 mutational spectrum associated with NF1 cutaneous neurofibromas

Neurofibromatosis type-1 (NF1), caused by heterozygous inactivation of the NF1 tumour suppressor gene, is associated with the development of benign and malignant peripheral nerve sheath tumours (MPNSTs). Although numerous germline NF1 mutations have been identified, relatively few somatic NF1 mutations have been described in neurofibromas. Here we have screened 109 cutaneous neurofibromas, excised from 46 unrelated NF1 patients, for somatic NF1 mutations. NF1 mutation screening (involving loss-of-heterozygosity (LOH) analysis, multiplex ligation-dependent probe amplification and DNA sequencing) identified 77 somatic NF1 point mutations, of which 53 were novel. LOH spanning the NF1 gene region was evident in 25 neurofibromas, but in contrast to previous data from MPNSTs, it was absent at the TP53, CDKN2A and RB1 gene loci. Analysis of DNA/RNA from neurofibroma-derived Schwann cell cultures revealed NF1 mutations in four tumours whose presence had been overlooked in the tumour DNA. Bioinformatics analysis suggested that four of seven novel somatic NF1 missense mutations (p.A330T, p.Q519P, p.A776T, p.S1463F) could be of functional/clinical significance. Functional analysis confirmed this prediction for p.S1463F, located within the GTPase-activating protein-related domain, as this mutation resulted in a 150-fold increase in activated GTP-bound Ras. Comparison of the relative frequencies of the different types of somatic NF1 mutation observed with those of their previously reported germline counterparts revealed significant (P=0.001) differences. Although non-identical somatic mutations involving either the same or adjacent nucleotides were identified in three pairs of tumours from the same patients (P<0.0002), no association was noted between the type of germline and somatic NF1 lesion within the same individual.     

Germline and somatic NF1 gene mutation spectrum in NF1-associated malignant peripheral nerve sheath tumors (MPNSTs)

About 10% of neurofibromatosis type 1 (NF1) patients develop malignant peripheral nerve sheath tumors (MPNSTs) and represent considerable patient morbidity and mortality. Elucidation of the genetic mechanisms by which inherited and acquired NF1 disease gene variants lead to MPNST development is important. A study was undertaken to identify the constitutional and somatic NF1 mutations in 34 MPNSTs from 27 NF1 patients. The NF1 germline mutations identified in 22 lymphocytes DNA from these patients included seven novel mutations and a large 1.4-Mb deletion. The NF1 germline mutation spectrum was similar to that previously identified in adult NF1 patients without MPNST. Somatic NF1 mutations were identified in tumor DNA from 31 out of 34 MPNSTs, of which 28 were large genomic deletions. The high prevalence (>90%) of such deletions in MPNST contrast with the =or<20% found in benign neurofibromas and is indicative of the involvement of different mutational mechanisms in these tumors. Coinactivation of the TP53 gene by deletion, or by point mutation along with NF1 gene inactivation, is known to exacerbate disease symptoms in NF1, therefore TP53 gene inactivation was screened. DNA from 20 tumors showed evidence for loss of heterozygosity (LOH) across the TP53 region in 11 samples, with novel TP53 point mutations in four tumors.     

PTEN expression in non-small-cell lung cancer: evaluating its relation to tumor characteristics, allelic loss, and epigenetic alteration

The tumor suppressor PTEN encodes a lipid phosphatase that negatively regulates the phosphatidylinositol 3-kinase/AKT cell survival pathway. Mutations of this gene are common in brain, prostate, endometrial, and gastric cancers but occur rarely in non-small-cell lung cancer (NSCLC), although the PTEN protein is often lost in lung tumors. We have studied hypermethylation of the PTEN promoter, loss of heterozygosity (LOH) at microsatellites in chromosome 10q23 (surrounding and intragenic to the PTEN locus), and hypermethylation of PTEN's highly homologous pseudogene, PTENP1, and their association with PTEN protein loss in a surgical case series study of primary NSCLC. PTEN protein expression was reduced or lost in 74% (86/117) of tumors, with loss occurring more often in well to moderately differentiated tumors. In squamous cell carcinomas, PTEN loss occurred significantly more often in early-stage (stage I or II) disease. PTEN protein loss also occurred more frequently in tumors with low to no aberrant TP53 staining. Methylation of PTEN occurred in 26% (39/151) of tumors, and LOH at 10q23 was rare, occurring in only 19% (17/90) of informative tumors. Neither methylation nor LOH was a significant predictor of PTEN protein expression, although LOH occurred exclusively in early-stage disease. In NSCLC, loss of PTEN protein expression occurs frequently, although the mechanism responsible for loss is not clearly attributable to deletion or epigenetic silencing. PTEN loss may also be a favorable prognostic marker, although further studies are needed to confirm this finding.     

Loss of DAL-1, a protein 4.1-related tumor suppressor, is an important early event in the pathogenesis of meningiomas

Meningiomas are common nervous system tumors, whose molecular pathogenesis is poorly understood. To date, the most frequent genetic alteration detected in these tumors is loss of heterozygosity (LOH) on chromosome 22q. This finding led to the identification of the neurofibromatosis 2 (NF2) tumor suppressor gene on 22q12, which is inactivated in 40% of sporadic meningiomas. The NF2 gene product, merlin (or schwannomin), is a member of the protein 4.1 family of membrane-associated proteins, which also includes ezrin, radixin and moesin. Recently, we identified another protein 4.1 gene, DAL-1 (differentially expressed in adenocarcinoma of the lung) located on chromosome 18p11.3, which is lost in approximately 60% of non-small cell lung carcinomas, and exhibits growth-suppressing properties in lung cancer cell lines. Given the homology between DAL-1 and NF2 and the identification of significant LOH in the region of DAL-1 in lung, breast and brain tumors, we investigated the possibility that loss of expression of DAL-1 was important for meningioma development. In this report, we demonstrate DAL-1 loss in 60% of sporadic meningiomas using LOH, RT-PCR, western blot and immunohistochemistry analyses. Analogous to merlin, we show that DAL-1 loss is an early event in meningioma tumorigenesis, suggesting that these two protein 4.1 family members are critical growth regulators in the pathogenesis of meningiomas. Furthermore, our work supports the emerging notion that membrane-associated alterations are important in the early stages of neoplastic transformation and the study of such alterations may elucidate the mechanism of tumorigenesis shared by other tumor types.     

Analysis of chromosomal imbalances in sporadic and NF1-associated peripheral nerve sheath tumors by comparative genomic hybridization.

Peripheral nerve sheath tumors arise either sporadically or in association with neurofibromatosis type 1 (von Recklinghausen's neurofibromatosis, NF1) or type 2. In this study, comprehensive screening for relative chromosome copy number changes was performed on 10 benign and 19 malignant peripheral nerve sheath tumors (MPNSTs) by applying comparative genomic hybridization (CGH). In benign tumors, no chromosomal imbalances were found by CGH, whereas in MPNSTs chromosomal gains and losses were frequently detected. No differences regarding the frequency and distribution of chromosomal imbalances were observed between the 13 sporadic and 6 NF1-associated MPNSTs analyzed. In both, the number of gains was significantly higher than the number of losses, suggesting a predominant role of proto-oncogene activation during MPNST progression. Candidate regions with potentially relevant proto-oncogenes included chromosomal bands 17q24-q25, 7p11-p13, 5p15, 8q22-q24, and 12q21-q24; those with putative tumor suppressor genes were 9p21-p24, 13q14-q22, and 1p. High-level amplifications were restricted to sporadic tumors and affected eight different chromosomal subregions. In three of these MPNSTs, identical subregions on chromosomal arms 5p and 12q were coamplified. This study revealed a number of new characteristic chromosomal imbalances and provides a basis for molecular identification of oncogenes and tumor suppressor genes of pathogenetic relevance in both sporadic and NF1-associated MPNSTs. Genes Chromosomes Cancer 25:362-369, 1999.     

Genetic and epigenetic alteration of the NF2 gene in sporadic meningiomas

The role of the NF2 gene in the development of meningiomas has recently been documented; inactivating mutations plus allelic loss at 22q, the site of this gene (at 22q12), have been identified in both sporadic and neurofibromatosis type 2-associated tumors. Although epigenetic inactivation through aberrant CpG island methylation of the NF2 5' flanking region has been documented in schwannoma (another NF2-associated neoplasm), data on participation of this epigenetic modification in meningiomas are not yet widely available. Using methylation-specific PCR (MSP) plus sequencing, we assessed the presence of aberrant promoter NF2 methylation in a series of 88 meningiomas (61 grade I, 24 grade II, and 3 grade III), in which the allelic constitution at 22q and the NF2 mutational status also were determined by RFLP/microsatellite and PCR-SSCP analyses. Chromosome 22 allelic loss, NF2 gene mutation, and aberrant NF2 promoter methylation were detected in 49%, 24%, and 26% of cases, respectively. Aberrant NF2 methylation with loss of heterozygosity (LOH) at 22q was found in five cases, and aberrant methylation with NF2 mutation in another; LOH 22q and the mutation were found in 16 samples. The aberrant methylation of the NF2 gene also was the sole alteration in 15 samples, most of which were from grade I tumors. These results indicate that aberrant NF2 hypermethylation may participate in the development of a significant proportion of sporadic meningiomas, primarily those of grade I.     

NF1 Deletions in S-100 Protein-Positive and Negative Cells of Sporadic and Neurofibromatosis 1 (NF1)-Associated Plexiform Neurofibromas and Malignant Peripheral Nerve Sheath Tumors

Although plexiform neurofibroma (PN) is thought to represent a benign neoplasm with the potential for malignant transformation (malignant peripheral nerve sheath tumor; MPNST), its neoplastic nature has been difficult to prove due to cellular heterogeneity, which hampers standard molecular genetic analysis. Its mixed composition typically includes Schwann cells, fibroblasts, perineurial-like cells, and mast cells. Although NF1 loss of heterozygosity has been reported in subsets of PNs, it remains uncertain which cell type(s) harbor these alterations. Using a dual-color fluorescence in situ hybridization and immunohistochemistry technique, we studied NF1 gene status in S-100 protein-positive and -negative cell subpopulations in archival paraffin-embedded specimens from seven PNs, two atypical PNs, one cellular/atypical PN, and eight MPNSTs derived from 13 patients, seven of which had neurofibromatosis type 1 (NF1). NF1 loss was detected in four of seven PNs and one atypical PN, with deletions entirely restricted to S-100 protein-immunoreactive Schwann cells. In contrast, all eight MPNSTs harbored NF1 deletions, regardless of S-100 protein expression or NF1 clinical status. Our results suggest that the Schwann cell is the primary neoplastic component in PNs and that S-100 protein-negative cells in MPNST represent dedifferentiated Schwann cells, which harbor NF1 deletions in both NF1-associated and sporadic tumors.     

Genetic alterations in pediatric high-grade astrocytomas

High-grade astrocytomas are tumors that are uncommon in children. Relatively few studies have been performed on their molecular properties and so it is not certain whether they follow different genetic pathways from those described in adult diffuse astrocytomas. In this study, we evaluated 24 pediatric high-grade astrocytomas (11 anaplastic astrocytomas and 13 glioblastomas) all of which were sporadic and primary. We studied mutations of p53, phosphatase and tensin homolog (PTEN), loss of heterozygosity (LOH) of chromosomes 17p13, 9p21 and 10q23-25, amplification of epidermal growth factor receptor (EGFR), and overexpression of EGFR and p53 protein. In addition, we searched for microsatellite instability (MSI) by using MSI sensitive and specific microsatellite markers. p53 mutations were found in 38% (9/24) of the high-grade astrocytomas and all brain stem tumors except 2 (71%, 5/7) had p53 mutations. PTEN mutations were found in 8% (2/24) of high-grade astrocytomas. However, no EGFR amplification was found in any of them. LOH was found at 17p13.1 in 50% (3/6 informative tumors), 9p21 in 83% (5/6 informative tumors), and 10q23-25 in 78% (7/9 informative tumors). Four tumors showed MSI, and 2 of them that showed widespread MSI were regarded as tumors with replication error (RER+) phenotype. All 4 tumors with MSI showed concurrent LOH of 9p21 and 10q23-25. Combining gene alterations, LOH, MSI, and gene mutations, inactivation of both alleles of PTEN and p53 was found in 57% (4/7 informative tumors) and 50% (3/6 informative tumors) of the cases respectively. We conclude that development of pediatric high-grade astrocytomas may follow pathways different from the primary or secondary paradigm of adult glioblastomas. In a subset of these tumors, genomic instability was also implicated.     

Loss of heterozygosity of p53, BRCA1, VHL,and estrogen receptor genes in breast carcinoma: correlation with related protein products and morphologic features

Correlation of loss of heterozygosity (LOH) of p53, BRCA1, VHL, and estrogen receptor (ER) genes with the expression of related protein products and morphologic features predictive of aggressive biologic behavior was investigated to determine the significance of LOH in these genes. DNA from 35 formalin-fixed, paraffin-embedded breast carcinomas was obtained by microdissection of histologic sections. LOH was determined by polymerase chain reaction (PCR) using primers TP53, D3S1038, D17S855, and ESR for p53, VHL, BRCA1, and ER genes, respectively. p53, ER, and progesterone receptor (PR) protein expression was evaluated by immunohistochemistry. Morphologic evaluation included histologic type, and histologic and nuclear grades. TP53 LOH was identified in 13 (52%), BRCA1 LOH in 3 (17%), VHL LOH in 1 (4%), and ER LOH in 4 (21%) of 25, 17, 24, and 19 informative cases, respectively. p53 and ER protein expression was identified in 20 (57%) and 25 (71%) cases, respectively. TP53 LOH directly correlated with both high histologic and nuclear grade (P<0.01). BRCA1, VHL, and ER LOH was not frequent enough for correlation to morphologic features. Although 4 of 4 ER and 7 of 13 p53 LOH cases expressed related proteins, LOH did not correlate with protein expression. TP53 LOH may be an event contributing to aggressive biologic behavior since it is strongly associated with high histologic and nuclear grade. Missense or nonsense mutations may explain the absence of detectable p53 protein in 6 of 13 cases with p53 LOH. All 4 ER LOH cases expressed ER protein. BRCA1 and VHL LOH is infrequent in sporadic breast carcinoma.     

Heterogeneous methylation and deletion patterns of the INK4a/ARF locus within prostate carcinomas

To elucidate the role of p53/p16(INK4a)/RB1 pathways in prostate carcinogenesis, we analyzed the p14(ARF), p16(INK4a), RB1, p21(Waf1), p27(Kip1), PTEN, p73, p53, and MDM2 gene status of multiple areas within 16 histologically heterogeneous prostate carcinomas using methylation-specific polymerase chain reaction, differential polymerase chain reaction, and immunohistochemistry. All focal areas examined had Gleason scores ranging from 1 to 5. Methylation of either PTEN or p73 was undetected in any sample, whereas expression of MDM2 seemed to be an independent event within small foci of 4 of 16 tumors. Loss of p14(ARF), p16(INK4a), RB1, and p27(Kip1) expression correlated with homozygous deletion or promoter hypermethylation. One carcinoma showed co-deletion of both p14(ARF) and p16(INK4a) in two of five areas examined; two areas within another tumor demonstrated concurrent hypermethylation of the promoter regions of the same genes. Focal hypermethylation of RB1, p21(Waf1), and p27(Kip1) was detected within two, two, and three tumors, respectively. These findings indicate that both genetic and epigenetic events occur independently in intratumor foci and further suggest hypermethylation-induced loss of gene function may be as critical as specific genetic mutations in prostate carcinogenesis.     

Biallelic inactivation of TP53 rarely contributes to the development of malignant peripheral nerve sheath tumors

About 10% of the patients with neurofibromatosis type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNSTs), accounting for half of all MPNST cases. Several nonrandom chromosomal aberrations have been found, but the target genes remain mostly unrecognized. Mutations in the NF1 and TP53 genes have been found in some MPNSTs, and recent data from mouse models support a synergistic effect of these two genes in the development of MPNST. In the present study, we have analyzed 16 MPNSTs, including 11 from patients with NF1 and 5 sporadic cases, for mutations in the coding sequence of the TP53 gene (exons 2-11). We applied denaturing gradient gel electrophoresis and modifications of this technique for analyses of 12 genomic fragments, followed by direct sequencing for identification of the mutated base(s). None of the MPNSTs revealed mutations. The detection of control mutants for each fragment analyzed, the high sensitivity of the technique, the detection of polymorphisms in some samples, and the high content of tumor tissue in the biopsies imply that false negatives are highly unlikely. Although we cannot exclude that deletions including large parts of the gene remain undetected by the mutation analyses, previous comparative genomic hybridization (CGH), cytogenetic banding analysis, and/or loss of heterozygosity studies on 14 of the cases included here had revealed 17p deletions in only three. We thus conclude that TP53 biallelic inactivation is rare in MPNST, and that the potential impact of an altered TP53 pathway on the malignant transformation of a neurofibroma into an MPNST may more frequently occur by changes in other components of that pathway.     

Loss of heterozygosity of the retinoblastoma gene is correlated with the altered pRb expression in human endometrial cancer

The retinoblastoma (Rb) gene was the first tumor suppressor gene to be discovered; however, data on the influence of Rb inactivation on endometrial carcinogenesis are scarce. We investigated 46 paired primary human endometrial carcinomas and normal tissues to assess the frequency of loss of heterozygosity (LOH) in Rb and 20 tumor pairs to detect the frequency of p53 LOH. Moreover, expression of the retinoblastoma protein (pRb) was assessed immunohistochemically. Of 44 informative cases 8 showed loss of one allele in at least one Rb marker; Rb LOH frequency thus reached 18%. Two omental metastases of endometrial origin showed a heterogeneity pattern similar to that of the primary tumors. We did not find a significant correlation between Rb LOH and patient age, clinical stage, histological grade or muscle invasion of the tumor. Nevertheless, Rb LOH was demonstrated at early (stage I, 5/27, 18%) and advanced (stages II-IV; 3/9, 33%) clinical stages of the neoplasm, suggesting that LOH at the Rb locus occurs before the clonal expansion of the tumor. There was a significant correlation between Rb LOH and weak/absent pRb expression. We noted a single case of p53 LOH at intron 1, but no tumor showed both alterations simultaneously. Our data suggest that LOH at the Rb locus plays a role in the oncogenesis of a subset of uterine neoplasms and corresponds with the altered expression of the pRb.