Differential deletions of chromosome 3p are associated with the development of uterine cervical carcinoma in Indian patients.

BACKGROUND: Deletions in chromosome 3 occur frequently in uterine cervical carcinoma (CA-CX). The common consensus regions deleted during CA-CX development are not well defined, and have not been correlated with tumour progression. AIMS: To define specific regions of chromosome 3 deleted during development of CA-CX and to correlate these with clinicopathological data. METHODS: Deletion mapping of chromosome 3 was done in seven cervical intraepithelial neoplasia (CIN) and 43 primary CA-CX samples using 20 highly polymorphic microsatellite markers. RESULTS: Deletions of chromosome 3 were significantly associated with tumour progression. High frequencies (33-53%) of loss of heterozygosity (LOH) were found in 3p26.1, 3p22.3, 3p21.2, and 3p13, suggesting the location of putative tumour suppressor genes (TSGs) in these regions. Among these four regions, deletions in 3p21.2 were suggested to occur early during CA-CX development. A significant correlation was found between LOH at 3p26.1 and 3p22.3 with tumour progression from stage I/IIB to stage III/IV. No association was found with the highly deleted regions and human papillomavirus positivity, parity, or menopausal status. Microsatellite size alteration was seen in only seven of the samples. However, rare biallelic alterations were seen in and around the highly deleted regions. Loss of normal copy of chromosome 3 and interstitial alterations in chromosome 3p were seen in some samples. CONCLUSION: These four regions on chromosome 3p may be differentially deleted during specific stages of CA-CX development. The putative TSGs located in these regions may have a cumulative effect on tumour progression.     

瘤体内DNA倍体异质性与食管癌组织学分级的关系

采用多点取材的方法，应用流式细胞术对８０例食管癌瘤体内ＤＮＡ倍体异质性进行研究。结果显示，倍体异质性肿瘤的检出率为４７．５％（３８／８０）；倍体异质性与组织学分级无关（Ｐ＞０．０５），而与患者的预后有关（Ｐ＜０．０５）。本研究提示，深入研究肿瘤ＤＮＡ倍体异质性将对判断患者预后，协助组织学分级具有重要的意义。     

Parental origin of de novo chromosome 9 deletions in del(9p) syndrome

Parental origin of de novo deletions in the short arm of chromosome 9 in patients with a clinical diagnosis of del(9p) syndrome was assessed in 13 patients using polymerase chain reaction (PCR) analysis of highly polymorphic dinucleotide repeat micro-satellite markers located in the putative deleted region. The deletion was found to be of paternal origin in 9 cases and of maternal origin in the remaining 4 cases, suggesting that the molecular event resulting in the deletion occurs in both male and female gametogenesis and that genomic imprinting does not appear to play a role in the patho-genesis of del(9p) syndrome. © 1995 Wiley-Liss, Inc.     

喉鳞状细胞癌MTS1／p16基因纯合子缺失的检测

为探讨MTS1／p16基因在喉癌中纯合子缺失情况及其与喉癌生物学行为的关系,本研究用比较PCR技术对59例喉鳞状细胞癌（LSCC）MTS1／p16基因纯合子缺失进行检测。剔除3例不可比样本,9／56 LSCC有MTS1／p16基因纯合子缺失,缺失率为16．07％。MTS1／p16基因纯合子缺失与LSCC病理分级无关;LSCC晚期（Ⅲ-Ⅳ）期的MTS1／p16基因纯合子缺失率（36．84％）明显高于早期（Ⅰ-Ⅱ期）肿瘤（5．4％）,P＜0．01,显示MTS1／p16基因纯合子缺失与LSCC的自然病程有关。认为MTS1／p16基因纯合子缺失是LSCC发生发展过程中的分子机制之一,可能与LSCC的恶性发展有关。     

Clinical heterogeneity associated with deletions in the long arm of chromosome 15: report of 3 new cases and their possible genetic significance

Deletions, duplications, and rearrangements of the long arm of chromosome 15 are frequently associated with the clinical diagnosis of the Prader-Willi syndrome. However, a number of other clinical entities have also been associated with similar, if not identical, cytogenetic defects, arguing for clinical heterogeneity associated with abnormalities in this region of chromosome 15. We present 3 patients who all appear to have deletions in 15q11-15q12, such as described for many patients with Prader-Willi syndrome; however, none of these patients has classical clinical features of the Prader-Willi syndrome. The first patient is a child with Williams syndrome, the second, Angelman (Happy Puppet) syndrome, and the third is a child with hypotonia of infancy, obesity, and developmental delay, but who does not meet specific diagnostic criteria for the Prader-Willi syndrome. It is proposed that different molecular abnormalities involving specific points or segments along the long arm of chromosome 15 might account for the clinical diversity seen among these and other patients.     

Involvement of 3p deletions in sporadic and hereditary forms of renal cell carcinoma.

Deletions of the short arm of chromosome 3 and associated allele losses have been reported in the majority of sporadic renal cell carcinomas (RCC). On the basis of the combined cytogenetic and molecular data, it is reasonable to assume that a putative RCC locus, which contributes to tumor development by its loss, is located telomerically of the D3F15S2 site. Using H3E4, a D3F15S2-specific probe, we have isolated a cDNA clone (cl.4-2), and a sequence comparison revealed that the cDNA clone corresponds to the human acyl-peptide hydrolase gene. The gene is fairly universally expressed, but in RCC biopsies its expression is severely reduced, compared to the normal kidney. Cl.4-2 was used for in situ hybridization on metaphase chromosomes prepared from an Epstein-Barr virus (EBV) transformed lymphoblastoid cell line, derived from a t(3;8) (p14.2;q24.1) carrying member of the RCC family described by Cohen et al. in 1979 (N Engl J Med: 301:592-595). Carriers of this translocation regularly develop RCC by middle age. We now report that D3F15S2 is localized on the telomeric side of the constitutional breakpoint, in 3p21. The region of 3p affected by this familial translocation is thus not identical with the region of 3p most frequently deleted in sporadic RCC.     

Differential deletions in 3p are associated with the development of head and neck squamous cell carcinoma in Indian patients

In this study we performed detailed deletion mapping of two broad regions in the short arm (p) of chromosome 3 (i.e., 3p21.2 approximately p22 and 3p12 approximately p13), which were shown to have a high rate of deletions in head and neck lesions in our previous study. Using 18 highly polymorphic microsatellite markers, the deletion mapping was done in 35 dysplastic lesions and 46 primary head and neck squamous cell carcinoma (HNSCC) samples from Indian patients. Within the 21.6-megabase (Mb) region of 3p21.1 approximately p21.33, we have identified four areas (D1, 3p21.33; D2, 3p21.32; D3, 3p21.31; D4, 3p21.1) that showed a high frequency (46%-69%) of deletions in our samples. In the 3p12 approximately p13 region, we narrowed down the deletion within the 0.7-Mb region (D5, 3p12.1). Among these five regions (D1-D5), deletion in D3 is suggested to be necessary for the development of early dysplastic lesions, whereas the deletion in D2 may be necessary for dysplastic lesions and tumor progression. On the other hand, the deletion in D5 is significantly associated with progression of the lesions from mild/moderate to severe dysplasia. The deletions in D1 and D4, however, are required for tumor progression. As in our previous study, microsatellite size alterations (MA) were observed to be high in and around the highly deleted regions and gradually increased during the progression of the tumor. Loss of normal copy/interstitial alterations of chromosome 3 in the late stages of the tumor as well as rare biallelic alterations around the highly deleted regions also were seen in our samples. Human papilloma virus infection has been found to be associated with the deletion in the D5 region and MA in the D1 region, whereas nodal involvement of the tumor correlated only with the MA in D1 and D5. Thus, this study indicates that multiple tumor suppressor genes whose differential deletions are associated with the development of HNSCC may be present in 3p.     

REPORTS: Linkage of Pfeiffer syndrome to chromosome 8 centromere and evidence for genetic heterogeneity

Pfeiffer syndrome (PS) Is an autosomal dominant disorder characterizedby cranlosynostosis, mldfaclal hypoplasia, and broad thumbsand great toes. We examined 129 Individuals from 11 familieswith PS and performed linkage studies using microsatellite markersspanning the entire genome. Strongest support for linkage waswith DNA markers (D8S255, GATA8G08) from chromosome 8. Obligatecrossovers exclude close linkage to this region in six families,and there was significant evidence for genetic heterogeneity.A multipoint lod score of 7.15 was obtained In five families.The 11 cM Interval between D8S278 and D8S285 contains one genefor PS and also spans the centromere of chromosome 8.     

Molecular characterisation of chromosome 4p deletions resulting in Wolf-Hirschhorn syndrome.

We present three patients with Wolf-Hirschhorn syndrome with small cytogenetic deletions of 4p16. One case is a de novo translocation and two cases represent de novo deletions. Using molecular techniques we determined the extent of these deletions and attempted to ascertain parental origin. Case 1 had a deletion of 4p16.3 with a breakpoint proximal to D4S10, case 2 had a larger deletion including D4S62 in 4p16.2, and case 3 had the largest deletion which included D4S240, but not the Raf2 locus in 4p16.1. The parental origin of the deletion in case 3 was paternal; the other two cases were indeterminable. Our results show that these three deletions include the currently proposed Wolf-Hirschhorn syndrome critical region within the most distal 2 Mb of 4p16.3 and offer supportive evidence for continuous terminal deletions.     

Metastasizing vulvar carcinosarcoma with squamous carcinomatous and leiomyosarcomatous differentiation: genetic evidence of clonal origin

In this report, we present the first case of a vulvar carcinosarcoma with squamous carcinomatous and leiomyosarcomatous differentiation. Comparative genomic hybridization was used to analyze clonality of the two tumor components. A widely identical pattern of genetic imbalances in the comparative genomic hybridization analysis in both the carcinomatous and the sarcomatous tumor component strongly supported the concept of a bidirectionally differentiated neoplasm. In both tumor components and two lymph node metastases, an amplicon was detected on chromosome 11q12-q13, homing the cyclin D1 gene locus. In contrast, exclusively in the sarcomatoid component, a characteristic amplicon on 12q13-q14 was found. The cytogenetic profile of the lymph node metastases revealed an increase in imbalances compared with the primary tumor. In summary, we found strong indications for a clonal origin of the two tumor components in a vulvar carcinosarcoma and a good correlation of the histological morphology with the pattern of genetic imbalances.     

p53 immunoreactivity in cervical intraepithelial neoplasia and non-neoplastic cervical squamous epithelium.

AIMS--To determine the pattern of p53 immunoreactivity in cervical squamous epithelium and to investigate the relation between p53 immunostaining and human papillomavirus (HPV) infection. METHODS--Immunocytochemistry for p53 was performed in 65 specimens of formalin fixed, paraffin wax embedded cervical tissue using a polyclonal antibody against recombinant p53. Microwave oven heating was used for antigen retrieval. Eight normal biopsy specimens, eight cases with histological features of HPV infection, and 49 cases of cervical intraepithelial neoplasia (CIN) were examined. Thirty one cases of CIN were examined. Thirty one cases of CIN were examined for evidence of HPV infection using in situ hybridisation with probes directed against wide spectrum HPV, HPV 16 and HPV 18. RESULTS--p53 immunoreactivity was seen in seven of eight (87%) of specimens with histological features of HPV infection, five of eight (62%) normal specimens, 13 of 22 (59%) CIN III, three of 14 (21%) CIN II and five of 13 (38%) CIN I specimens. The numbers of positive nuclei were small in cases of CIN and the location of positive nuclei within the epithelium paralleled the degree of dysplasia. Eleven of 15 (73%) CIN specimens which were immunoreactive for p53 yielded a positive signal for HPV by in situ hybridisation. A positive signal for HPV was also seen in 10 of 16 (63%) of CIN specimens in which p53 staining was absent. CONCLUSIONS--p53 immunoreactivity can be demonstrated in a small proportion of cells in the cervical squamous epithelium in a significant proportion of cases of CIN. This immunoreactivity seems to be independent of the presence of HPV, as assessed by in situ hybridisation. p53 immunoreactivity also occurs in non-neoplastic cervical squamous epithelium with a pattern of distribution within the epithelium which differs from that seen in CIN. Antigen retrieval by microwave oven heating enhances p53 immunostaining and may result in visualisation of cellular p53 in the absence of mutation.     

No evidence of genetic heterogeneity in dominant optic atrophy.

Autosomal dominant optic atrophy (OPA, MIM 165500) is an eye disease causing a variable reduction of visual acuity with an insidious onset in the first six years of life. It is associated with a central scotoma and an acquired blue-yellow dyschromatopsia. A gene for dominant optic atrophy (OPA1) has recently been mapped to chromosome 3q in three large Danish pedigrees. Here, we confirm the mapping of OPA1 to chromosome 3q28-qter by showing close linkage of the disease locus to three recently reported microsatellite DNA markers in the interval defined by loci D3S1314 and D3S1265 in four French families (Zmax = 5.13 at theta = 0 for probe AFM 308yf1 at locus D3S1601). Multipoint analysis supports the mapping of the disease gene to the genetic interval defined by loci D3S1314 and D3S1265. The present study provides three new markers closely linked to the disease gene for future genetic studies in OPA.     

Brachyolmia: radiographic and genetic evidence of heterogeneity

Brachyolmia refers to a form of skeletal dysplasia characterized by general platyspondyly without significant epiphyseal, metaphyseal, or diaphyseal changes in the long bones. As a result of a study of 11 patients from 7 different families and a review of the literature we propose that there are 3 and possibly 4 different types of brachyolmia: 1. Hobaek type--an autosomal recessive condition with universal platyspondyly, irregular, and reduced intervertebral spaces and marked extension of the lateral margins of the vertebrae. Rectangular and elongated vertebral bodies are seen on lateral views of the spine, which become more pronounced with age. Toledo type--radiographically similar to Hobaek type but also associated with corneal opacities and precocious ossification of costal cartilage. It is uncertain as to whether this represents variability or heterogeneity. 2. Maroteaux type--an autosomal recessive disorder that is distinguished from Hobaek type by rounding of the anterior and posterior vertebral borders with less elongation on lateral view and less lateral extension on A.P. view. This type may be associated with precocious calcification of the falx cerebri, and minor facial anomalies. 3. Dominant type--a previously underscribed form which we have observed in a mother and her son. This type has the most severe vertebral changes with flattening and irregularities of the cervical spine. These clinical, radiological, and genetic differences suggest genetic heterogeneity in this group of platyspondylic disorders.     

1p and 3p deletions in meningiomas without detectable aberrations of chromosome 22 identified by comparative genomic hybridization

Meningioma is a common tumor of the meninges covering the central nervous system. Although generally a benign tumor, meningioma often recurs and is malignant in 5–10% of all cases. Loss of chromosome 22 loci, and specifically inactivation of the NF2 tumor suppressor gene, is considered one of several critical steps in the tumorigenesis of meningioma. However, cytogenetic and molecular investigations have failed to detect either aberrations of chromosome 22 or mutations in the NF2 gene in approximately 40% of all tumors, thus making it apparent that an alternative mechanism(s) is responsible for the development of a large fraction of meningiomas. This subset of meningiomas is not distinct with regard to clinical and histopathological features from tumors showing deletions on chromosome 22. It is, therefore, important to attempt the elucidation of molecular pathway(s) that may operate in the tumorigenesis of these tumors. We used comparative genomic hybridization (CGH) to identify regions of the genome other than chromosome 22, contributing to the development of meningioma. We analyzed 25 tumors that had undergone detailed LOH analysis on chromosome 22 and were shown to contain no detectable deletions. Two benign, malignancy grade I, meningiomas showed concurrent deletion of 1p and 3p. These results suggest that loss of both 1p and 3p may contribute to meningioma tumorigenesis. This may represent genetic changes that are alternative to deletions on chromosome 22. Genes Chromosomes Cancer 20:419–424, 1997. © 1997 Wiley-Liss, Inc.     

Bloom's syndrome. XIX. Cytogenetic and population evidence for genetic heterogeneity

Cells with abnormally high rates of sister-chromatid exchange (SCE) are uniquely characteristic of Bloom's syndrome (BS). However, in one in five persons a minor population of cells with a low-SCE phenotype circulates in the blood. The origin and significance of the low-SCE cells in BS have never been understood, although they are assumed to arise by somatic mutation. In the present investigation, the enigmatic high-SCE/low-SCE mosaicism was investigated by comparing the incidence in several subpopulations of persons in the Bloom's Syndrome Registry who exhibit the two types of cells, and a striking negative correlation emerged: in persons with BS whose parents share a common ancestor, the case in approximately half of registered persons, low-SCE cells are found only rarely; conversely, the mosaicism occurs almost exclusively in persons with BS whose parents are not known to share a common ancestor. Because those who share a common ancestor are predominantly homozygous-by-descent at the mutated BS locus, the negative correlation is interpreted to mean that the emergence of low-SCE cells in BS in some way depends on the pre-existence of compound heterozygosity. A corollary to this is that BS is genetically heterogeneous.     

Senescence and apoptosis in carcinogenesis of cervical squamous carcinoma.

Senescence and apoptosis are two key mechanisms that protect against cancer development. Many cell cycle regulators, such as p14(ARF), p15(INK4b) and p16(INK4a), are important in G1 cell cycle arrest and oncogene-induced senescence. The bcl-2 protein is one of the key components that control apoptosis, while the p53 protein plays key roles in both mechanisms. The genes of these key regulator proteins are often mutated or deleted in various malignancies. It is unknown how senescence and apoptosis are regulated in one of the most common tumors of the female genital tract, cervical squamous cell carcinoma (SCC). In this study the, expression of senescence, apoptosis and proliferation markers in normal cervical epithelium, cervical intraepithelial neoplasia (CIN) and SCC are characterized via immunohistochemical staining for p14(ARF), p15(INK4b), p16(INK4a), bcl-2, p53 and Ki-67 in tissue microarray blocks containing 20 samples each of normal cervix, moderate-to-severe cervical dysplasia (CIN II-III) and invasive SCC. Samples are derived from 60 total cases of cervical biopsies and cervical conizations. Results showed that the proliferation marker, Ki-67, is markedly increased, and the senescence markers, p15(INK4b), p16(INK4a) and p14(ARF) are overexpressed in both dysplasia and carcinoma. P53 immunostain is negative in all normal cervical tissue, and positive in dysplasia and carcinoma. Although the expression of bcl-2 is increased in dysplasia, this marker is negative in approximately half of SCC cases. These results suggest that some senescence pathways are activated and are still maintained in cervical dysplasia and carcinoma. However proliferation is increased and carcinogenesis is not thwarted, leading to eventual development of cervical cancer. Other mechanisms, such as those that account for the apparent overexpression of p53 and paradoxical loss of bcl-2 expression in some SCC cases, as well as additional senescence and apoptotic pathways, may play key roles carcinogenesis of cervical SCC.     

Alterations in exon 1 of c-myc and expression of p62c-myc in cervical squamous cell carcinoma.

AIMS: To examine human papillomavirus (HPV) positive and negative squamous cell carcinomas of the cervix for structural alterations in exon 1 c-myc; and to investigate the expression pattern of p62, the protein product of c-myc. MATERIAL: Archival paraffin wax embedded tissues of cervical squamous cell carcinomas, stage I and II, retrieved from the files of the department of pathology, University College Cork, Ireland: 40 cases were examined for alterations in exon 1 of c-myc; 57 cases were used for immunocytochemical p62 analysis. METHODS: c-myc exon 1 PCR on HPV positive and negative stage I and II cervical squamous cell carcinomas was performed using primers designed to fragile sites in exon 1 of the c-myc oncogene, which are frequently involved in translocation phenomena and deletions in other neoplasms. This region is bordered by two promoter sequences P1 and P2. In addition, the expression of p62 was evaluated using the monoclonal antibody Mycl-9E10. RESULTS: Alterations in exon 1 of c-myc were shown in 7.5% of squamous cell carcinomas of the cervix. Changes in exon 1 and 2 of c-myc were also found in COLO 320 cells and Raji cells. These alterations were due to small deletions within exon 1 of c-myc, but point polymorphisms occurring within the priming sites (in one case) may also have occurred. The alterations uncovered appeared "clonal," as replicate samples showed the same amplicon band pattern. Expression of c-myc was variable, with cytoplasmic staining patterns predominating. All cases which showed exon 1 alterations were HPV positive and had strong nuclear positivity on p62 immunocytochemistry. CONCLUSIONS: Alterations in exon 1 of c-myc occur in a minority of cervical cancers and there was increased expression of p62 in a cohort of HPV positive and negative cervical squamous cell carcinomas. Exon 1 alterations may provide an alternative route to c-myc activation in early squamous cell carcinoma.