Genetic alterations of the tumour suppressor gene regions 3p, 11p, 13q, 17p, and 17q in human breast carcinomas.

Fifty-nine primary breast carcinomas and 11 metastases were examined to identify genetic alterations in the tumour suppressor gene regions 3p, 11p, 13q, 17p, and 17q. Loss of heterozygosity (LOH) was frequently observed on chromosome arms 17p (p144D6 lost in 75%, pYNZ22.1 in 55%, and TP53 in 48% of the primary tumours), 13q (RBI lost in 40% of the primary tumours), and 17q (pRMU3 lost in 35%, pTHH59 in 29%, and NM23HI in 26% of the primary tumours). Loss of all the markers except p144D6 was observed even more frequently in the metastases. Pairwise comparisons for concordance of allele losses on 17p indicated that there might be two genes on 17p implicated in breast cancer development; the TP53 gene and a gene located close to the p144D6 and pYNZ22.1 markers. LOH of the RBI gene was associated with LOH of pYNZ22.1 and p144D6, but not with LOH of TP53. LOH of RBI and TP53 was associated with occurrence of ductal carcinomas, RBI and p144D6 losses with tumour size, and p144D6 losses with positive node status as well. LOH of TP53 and the three 17q markers NM23HI, pTHH59, and pRMU3 was most frequently observed in tumours from postmenopausal women. p144D6 losses occurred most frequently in progesterone receptor-negative tumours, whereas pTHH59 losses occurred most frequently in oestrogen receptor-negative tumours. LOH of the investigated loci was not associated with ERBB2 protooncogene amplification, with positive family history of breast cancer, or with survival.     

Discordance of genetic alterations between primary head and neck tumors and corresponding metastases associated with mutational status of the TP53 gene.

Ample molecular data are available on the progression from normal mucosa to invasive head and neck squamous cell carcinoma (HNSCC), but information on further genetic progression to metastatic disease is scarce. To obtain insight into the metastatic process, we compared 23 primary HNSCCs with 25 corresponding lymph node metastases (LNMs) and 10 corresponding distant metastases (DMs) with respect to TP53 mutations and patterns of loss of heterozygosity (LOH) based on 26 microsatellite markers on six chromosome arms (3p, 9p, 17p, 13q, 8p, and 18q). In 18 of the 23 patients, a TP53 mutation was detected in the primary tumor, and in all cases the same TP53 mutation was present in the corresponding LNM or DM. In nine of 20 patients with LNMs and three of seven patients with DMs, the LOH pattern of metastasis differed from that of the corresponding primary tumor by at least one marker. Microsatellite markers located on chromosome arms 13q, 8p, and 18q were most frequently discordant, providing evidence that alterations at these chromosomes occur late in HNSCC carcinogenesis. Moreover, evidence was found that DMs had developed directly from the primary tumor and not from LNMs. Remarkably, we observed that the mutational status of the TP53 gene is associated significantly with the degree of genetic differences between primary HNSCCs and corresponding metastases. All patients with TP53 wild-type primary tumors showed significantly more discordant LOH patterns in the corresponding LNMs and DMs than patients with TP53-mutated tumors. The percentages were 100% versus 27% (LNMs) and 100% versus 0% (DMs), respectively (P = 0.008 and P = 0.029; two-sided Fisher exact test). This finding suggests that TP53-mutated tumors need fewer additional genetic alterations to develop metastases compared with TP53 wild-type primary tumors.     

Loss of heterozygosity at 11q23.3 in vasculoinvasive and metastatic squamous cell carcinoma of the cervix

We have previously demonstrated a strong relationship between loss of heterozygosity (LOH) at chromosome 11q23.3 and the presence of extensive tumor plugs in lymphvascular spaces (LVS) in stage 1B cervical carcinoma, suggesting that genes at this locus may regulate vasculoinvasion. This study examined LOH at 11q23.3 in microdissected tumor plugs within LVS and in metastatic foci in lymph nodes (MFLN), as well as corresponding invasive tumor and adjacent cervical intraepithelial neoplasia (CIN) 3 in stage 1B squamous cell carcinoma. Of 49 invasive carcinomas, 38.8% had LOH at 11q23.3. Of 36 tumor plugs in LVS, 39% had LOH at 11q23.3. Twenty percent of 15 MFLN demonstrated LOH at 11q23.3. Patients with LOH at 11q23.3 are significantly more likely to have disease recurrence than patients without LOH at 11q23.3 (P =.02). Of 10 foci of CIN 3, none showed LOH at 11q23.3. Although unlikely to have an impact early in carcinogenesis, tumor-suppressor genes located in the region of 11q23.3 appear to be important in tumor progression, facilitating lymphvascular space invasion and, by inference, spread to lymph nodes in squamous cell carcinoma of the cervix.     

Comparative genomic hybridization detects genetic alterations during early stages of cervical cancer progression.

Invasive cervical carcinoma is thought to arise from cervical intraepithelial neoplasm (CIN). Genetic changes that occur during progression of CIN to cervical carcinoma are poorly understood, although they appear to be directly involved in this process. We used comparative genomic hybridization (CGH) with precise microdissection and degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) to detect genetic alterations in normal epithelial, CIN, and invasive carcinoma tissues colocalized in tumors from 18 patients with squamous cell carcinoma of the uterine cervix. Gains on chromosome 1 and on 3q and losses on 2q, 3p, 4, 6p, 11q, and 17p were frequent alterations found in CIN and invasive carcinoma lesions. Interestingly, several of these genetic changes were observed in preinvasive carcinoma lesions. The frequency and average number of genetic alterations corresponded directly to the extent to which the cervical carcinoma had progressed. Frequent alterations were found in more than 90% of CIN III lesions. Gains on 3q and losses on 11q were the most prevalent genetic alterations found in association with uterine cervix carcinogenesis. The common regions of alteration were 3q26.1-q28 and 11q23-qter. The majority of tumor samples showed variability in genetic alterations across lesion types within a single specimen.     

Identification of chromosome 9 alterations and p53 accumulation in isolated carcinoma in situ of the urinary bladder versus carcinoma in situ associated with carcinoma

Carcinoma in situ (CIS) of the urinary bladder is a flat, aggressive lesion and may be the most common precursor of invasive bladder cancer. Although chromosome 9 alterations are among the earliest and most prevalent genetic alterations in bladder cancer, discrepancy exists about the frequency of chromosome 9 losses in CIS. We analyzed 22 patients with CIS of the bladder (15 patients with isolated CIS, 7 patients combined with synchronous pTa or pT1 carcinomas) for gains and losses of chromosome (peri)centromere loci 1q12, 7p11-q11, 9p11-q12, and 9p21 harboring the INK4A/ARF locus (p16(INK4A)/p14(ARF)) and INK4B (p15(INK4B)) by multiple-target fluorescence in situ hybridization, and for p53 protein accumulation by immunohistochemistry. In 15 of 20 (75%) CIS lesions analyzed p53 overexpression was detected, whereas aneusomy for chromosomes 1 and 7 was identified in 20 of 22 (91%) CIS. In 13 of 22 (60%) CIS cases analyzed, 12 of which were not associated with a synchronous pTa or pT1 carcinoma, no numerical losses for chromosome 9 (p11-q12 and 9p21) were detected as compared with chromosomes 1 and 7. Furthermore 6 of 12 (50%) patients showed a metachronous invasive carcinoma within 2 years. In the remaining nine biopsies CIS lesions (40%) were recognized that showed losses of chromosome 9p11-q12 and 9p21, six of these were associated with a synchronous pTa or pT1 carcinoma. Three of these carcinomas were pTa and exhibited loss of 9q12 as well as a homozygous deletion of 9p21. The others were invasive carcinomas in which CIS lesions were also recognized that showed no numerical loss of chromosome 9, but did show an accumulation of p53. In conclusion our data demonstrate that predominantly isolated CIS lesions contained cells with no specific loss of chromosome 9, as opposed to CIS lesions with synchronous carcinomas that showed evidence of chromosome 9 loss. Furthermore our data strengthen the proposition that p53 mutations (p53 overexpression) precede loss of chromosomes 9 and 9p21 in CIS as precursor for invasive bladder cancer, as opposed to noninvasive carcinomas where chromosome 9 (9p11-q12) losses are early and frequently combined with homozygous deletions of 9p21.     

Clinicopathological significance of loss of heterozygosity in intestinal- and solid-type gastric carcinomas: a comprehensive study using the crypt isolation technique.

The clinicopathological significance of loss of heterozygosity (LOH) in gastric carcinoma remains poorly understood. We and other researchers have previously demonstrated that LOH is fairly common in intestinal- and solid-type gastric carcinomas, but rare in diffuse-type tumors. In this study, we investigated the relationship between clinicopathological variables and LOH status in intestinal- and solid-type gastric carcinomas. The crypt isolation technique was utilized to analyze LOH at 1p36, 3p14, 4p15, 5q21-22, 8p11-12, 9p21, 13q22, 17p13.1 18q21 and 22q13.31 in 113 intestinal- and solid-type gastric carcinomas using a polymerase chain reaction assay. Immunostaining with D2-40 and Elastica van Gieson staining were used to detect lymphatic invasion and vessel invasion, respectively. High LOH rates (49-71%) were observed in all chromosomal regions tested. 1p36 loss was significantly associated with advanced tumors and lymph node metastasis. 8p11-12 loss was significantly associated with lymph node metastasis, lymphatic invasion, and vessel invasion. 17p13.1 (TP53) loss was significantly associated with vessel invasion. 22q13.31 loss was significantly associated with advanced tumors, lymph node metastasis, lymphatic invasion, vessel invasion and late TNM stage. No significant associations were observed between LOH at other chromosomal regions and aggressive behaviors. In addition, significantly higher LOH rates at 1p36, 9p21, 18q21 and 22q13.31 were observed in cardiac tumors compared with noncardiac tumors. These results suggest that in intestinal- and solid-type gastric carcinomas, LOH on 3p14, 4p15, 5q21-22, 9p21, 13q22 and 18q21 is associated with carcinogenesis, while LOH on 1p36, 8p11-12, 17p31.1 and 22q13.31 is associated with tumor progression.     

P53 immunohistochemical expression: messages in cervical carcinogenesis

AIMS: The pattern of p53 expression was studied in pre-invasive and invasive cervical carcinoma in an attempt to clarify its role in cervical carcinogenesis. METHODS: A total of 234 invasive cervical carcinomas (152 squamous cell carcinomas, 61 adenocarcinomas and 21 adenosquamous carcinomas) and 16 cervical intraepithelial neoplasia (CIN) I, six CIN II and 25 CIN III were immunohistochemically studied for p53. RESULTS: p53 was detected more frequently in CIN and invasive carcinoma (100% of CIN I, 74.2% CIN II + III and 70.1% invasive carcinoma) compared with benign cervices (P< 0.001); however, only three squamous cell carcinomas, 11 adenocarcinomas and two adenosquamous carcinomas exhibited p53 expression in >75% of tumour nuclei. Six of the 11 adenocarcinomas and both adenosquamous carcinomas were poorly differentiated compared with one of the three squamous carcinomas. p53 immunoreactive cells were randomly distributed in invasive carcinoma, confined to the lower third of the epithelium in CIN I, reached the middle third in 20% of CIN II and upper third in 16.6% of CIN III. CONCLUSIONS: Assuming that p53 immunoreactivity indicates gene mutation when the majority (> 75%) of neoplastic cells express p53, p53 mutations would seem uncommon in cervical carcinogenesis. Nonetheless, glandular malignancies, in particular poorly differentiated variants, may show a higher frequency of mutation. p53 was detected more frequently in CIN I compared with CIN II/III and invasive carcinoma which may be due to p53 protein degradation following interaction with high risk human papillomavirus E6 protein in CIN II/III and invasive carcinoma.     

Allelotype study of esophageal carcinoma

To investigate genetic features of esophageal cancer, we have examined 93 squamous cell carcinomas of the esophagus for loss of heterozygosity (LOH), using 41 restriction fragment length polymorphism (RFLP) markers representing all autosomal chromosomes. Allelic losses at frequencies of at least 30% were observed at loci on chromosomal arms 3p (35%), 3q (30%), 5q (36%), 9p (57%), 9q (60%), 10p (33%), 13q (43%), 17p (62%), 17q (46%), 18q (38%), 19q (32%), and 21q (37%). These results suggest that several putative tumor suppressor genes, in addition to the cyclin D and TP53 genes that are sometimes mutated in esophageal carcinomas, may be associated with development and/or progression of esophageal cancer. By a comparison of LOH on each chromosomal arm with clinicopathological parameters, we have found a significant correlation between LOH on 19q and regional lymph node metastases. Interestingly, the frequency of LOH on 17q was significantly higher in tumors in female patients (12 of 14 cases) than in those in male patients (20 of 56 cases) (P = 0.0009 by Fisher's exact test). Furthermore, we examined for mutations of the APC gene on chromosome arm 5q. Screening of nearly one third of the APC coding region, including the MCR (mutation cluster region), revealed no alterations. Therefore, although allelic loss at the APC locus is frequent in squamous cell carcinomas of the esophagus, it is likely that a gene on 5q other than APC is involved in esophageal tumorigenesis. Genes Chromosom Cancer 10:177–182 (1994). © 1994 Wiley-Liss, Inc.     

Accumulation of genetic alterations during esophageal carcinogenesis

Using polymerase chain reaction amplification of microsatelliteregions in DNA from 11 epithelial dysplasias of the esophagusand 21 early squamous cell carcinomas, we were able to detectfrequent loss of heterozygosity (LOH) on chromosomes 3p21.3and 9q31 even in low-grade dysplasias. In contrast, we observedfrequent LOHs on chromosomes 9p22 and 17p13 (TP53 locus) onlyin high-grade dysplasias and carcinomas, but not in any low-gradedysplasias. Analysis of LOH at the same four chromosomal regionsIn DNA of five additional minimal carcinomas and accompanyingdysplastic lesions revealed loss of alleles at the loci on 3p21.3and 9q31 throughout various degrees of dysplasia and carcinoma;again, LOHs on 9q22 and 17p13 occurred only in high-grade dysplasiaand carcinoma in situ. Our results indicated that Inactivationof putative tumor suppressor genes on 3p21.3 and 9q31 may beearly genetic events during esophageal carcinogenesis, and thatadditional genetic alterations on 9p22 and 17p13 probably playroles in progression.     

Loss of heterozygosity is detected at chromosomes 1p35-36 (NB), 3p25 (VHL), 16p13 (TSC2/PKD1), and 17p13 (TP53) in microdissected apocrine carcinomas of the breast.

INTRODUCTION: Apocrine carcinomas of the breast are an unusual special category of predominantly AR+, ER-, and PR- breast cancer, characterized by cells with abundant, eosinophilic cytoplasm and nuclei with often prominent nucleoli. To further investigate these lesions, loss of heterozygosity (LOH) was evaluated at multiple chromosomal loci, including loci frequently mutated in breast cancer. MATERIALS AND METHODS: Twenty-five intraductal apocrine carcinomas, 11 invasive apocrine carcinomas, and six apocrine hyperplasias were retrieved from the files of the Armed Forces Institute of Pathology (Washington, DC) and Fairfax Hospital (Fairfax, VA). Cells from lesional as well as normal tissues were microdissected. LOH was performed at a number of chromosomal loci, including loci commonly altered in breast cancer: 1p35-36 (NB), 3p25.5 (VHL), 8p12 (D8S136), 9p21 (p16), 11p13 (D11S904), 11q13 (INT-2 and PYGM), 16p13.3 (TSC2/PKD1 gene region), 17p13 (TP53), 17q13 (NM23), and 22q12 (D22S683). RESULTS: Among informative in situ and invasive apocrine carcinomas, LOH was present in 33% of 15 cases for 17p13 (TP53), as well as 36% of 14 cases for 3p25 (VHL), 30% of 10 cases for 1p35-36 (NB), and 27% of 11 cases for 16p13.3 (TSC2/PKD1). A higher frequency of LOH was noted among invasive apocrine carcinomas (30 to 50%) compared with in situ apocrine carcinomas (23 to 33%) at these loci. LOH was present simultaneously for TP53 and either VHL or NB in five cases. Infrequent (< or =12%) or absent LOH was detected at the remaining loci, including several loci commonly mutated in breast cancer (i.e., INT2, PYGM, and NM23). LOH was not detected in any of the six apocrine hyperplasias. CONCLUSION: An intermediate frequency of allelic loss was detected at multiple tumor suppressor gene loci, including 17p13 (TP53), as well as 1p35-336 (NB), 3p25 (VHL), and 16p13 (PKD1/ TSC2), in apocrine carcinomas of the breast, with a higher overall frequency of LOH noted among invasive tumors compared with in situ tumors. Aside from LOH at p53, LOH was infrequent or absent at several other loci commonly mutated in breast cancer. This preliminary molecular evidence supports immunohistochemical data that apocrine carcinomas of the breast may possess unique mechanisms of carcinogenesis, compared with ordinary ductal carcinomas. However, further study is needed to support this assertion and to determine if the LOH detected is truly etiologic or if it is the result of genetic progression.     

Loss of heterozygosity in dysplasia and carcinoma of the gallbladder.

The loss or inactivation of genes at specific chromosomal loci is one of the important mechanisms during the tumor development in humans. To investigate the role of genetic alterations in the carcinogenesis of gallbladder carcinoma, 32 carcinoma cases and 11 dysplasia cases of gallbladder were analyzed for loss of heterozygosity (LOH) and microsatellite instability (MI) on chromosomal regions 3p, 5q, 8p, 9p, 13q, 17p, and 18q with 17 microsatellite markers. Loss of one allele was identified on chromosomes 5q (55%) and 17p (40%) in dysplasias and on chromosomes 3p (52%), 5q (66%), 9p (52%), and 17p (58%) in carcinomas. LOH on chromosomes 13q and 18q was frequent only in advanced stage (III and IV) carcinomas (40% and 31%, respectively). LOH on chromosome 17p was correlated with intranuclear p53 accumulation. LOH on multiple chromosomes was more frequent in advanced carcinomas with metastasis than in cases without metastasis (P < .05). A widespread MI was observed in only one case of carcinoma. We conclude that LOH on 5q is an early change of carcinogenesis in gallbladder and that LOH on 3p and 9p is related to the progression of gallbladder carcinoma LOH on 13q and 18q is likely to be a late event. LOH on 17p occurs not only in dysplasia but also increases during the subsequent stages. Accumulation of LOH may be associated with carcinogenesis of the gallbladder, but the role of MI may not be significant.     

Inverted papilloma of the urinary bladder: a molecular genetic appraisal.

Inverted papilloma of urinary bladder is an uncommon urothelial neoplasm. Its relationship to urothelial carcinoma is controversial. Little is known of the genetic abnormalities of inverted papilloma. To better understand its genetics, we analyzed 39 inverted papillomas, including 36 from men and three from women, for loss of heterozygosity (LOH). We examined four polymorphic microsatellite markers located on chromosome 9q32-33(D9S177), chromosome 9p22 (IFNA), chromosome 3p14.2 (D3S1300) and chromosome 17p13.1 (TP53), where genetic alterations occur frequently in urothelial carcinomas. Additionally, the status of inactivation of X-chromosome was examined in three female patients. The frequency of LOH in informative cases was 8% (3 of 37) for D9S177, 10% (4 of 38) for TP53, 8% (3 of 37) for IFNA and 8% (3 of 36) for D3S1300. In the analysis of X-chromosome inactivation, all three cases yielded informative results and one had nonrandom inactivation of X-chromosomes. The monoclonal origin demonstrated in the study of X-chromosome inactivation indicates the clonal process of inverted papilloma; however, the low incidence of LOH supports the view that inverted papilloma in urinary bladder is a benign neoplasm with molecular genetic abnormalities different from those of urothelial carcinoma.     

Frequent HLA class I loss is an early event in cervical carcinogenesis

Loss at chromosome 6p21.3, the human leukocyte antigen (HLA) region, is the main cause of HLA downregulation, occurring in the majority of invasive cervical carcinomas. To identify the stage of tumor development at which HLA class I aberrations occur, we selected 12 patients with cervical carcinoma and adjacent cervical intraepithelial neoplasia (CIN). We investigated HLA class I and beta2-microglobulin expression by immunohistochemistry in tumor and adjacent CIN. Loss of heterozygosity (LOH) was studied using microsatellite markers covering the HLA region. Fluorescent in situ hybridization with HLA class I probes was performed to investigate the mechanism of HLA loss. Immunohistochemistry showed absent or weak HLA class I expression in 11/12 cases. In 10 of these 11 cases, downregulation occurred in both tumor and CIN. Only in one case did the concomitant CIN lesion show normal expression. In 9/12 cases, LOH was present for at least one marker in both tumor and CIN, 1 case showed only LOH in the CIN lesion, and 1 case showed retention of heterozygosity for all markers in both tumor and CIN. We conclude that HLA class I aberrations occur early and frequently in cervical carcinogenesis. This might allow premalignant CIN lesions to escape immune surveillance and progress to invasive cancer.     

Microsatellite analysis of breast carcinoma and corresponding local recurrences

Local recurrence is a serious complication of breast carcinoma that reduces quality of life and influences prognosis. The aim of this study was to determine whether local recurrences of breast carcinoma are genetically related to the primary tumours. Forty cases of locally recurrent breast carcinomas (median onset: 3.6 years after primary surgery) were analysed: 22 patients had undergone breast-conserving therapy and 18 mastectomy. Eighteen microsatellites on chromosomes 2p, 3p, 5q, 10q, 11p, 11q, 13q, 17q, 17p, 18p were amplified by PCR using fluorescent-labelled primers, automatically detected after polyacrylamide gel electrophoresis and analysed for loss of heterozygosity (LOH) or microsatellite instability (MSI). Follow-up data were available for 39 cases with a median value of 89 months. All LOH and MSI found in the primary tumours were also present in the corresponding recurrences, indicating that they are genetically related to the primary tumours and not secondary malignancies in the same breast. MSI was found in three cases, of which one harboured MSI at more than two loci. The median value of LOH per case was significantly higher in the recurrent (four per case) compared to the primary tumours (two per case; p < 0.001, Mann-Whitney test), reflecting the genotype of tumour progression. Early local recurrence was associated with specific LOH for TP53.15 (p = 0.018, log-rank test) in the primary tumours. LOH on D13S1699 or D17S855 was associated with lymph node metastases (p = 0.024 and p = 0.019, respectively; chi-square test). In addition, tumour grade, lack of oestrogen or progesterone receptor expression, young patient age and early appearance of local recurrence significantly correlated with poor survival. The development of local recurrence despite clear resection margins may result from residual DCIS distant from the invasive carcinoma, homing of circulating tumour cells, or genetically altered, histologically normal breast tissue not immediately adjacent to the invasive carcinoma.     

Genetic progression and divergence in pancreatic carcinoma

Genetic alterations of pancreatic intraductal lesions adjacent to invasive ductal carcinoma were investigated. We submitted nine foci of ordinary epithelium, 12 foci of nonpapillary hyperplasia, 12 foci of papillary hyperplasia (pap HP), 66 foci of severe ductal dysplasia, and 27 invasive foci from a total of 10 pancreatic carcinomas for genetic analysis. All foci were individually microdissected and allelic losses of 3p, 4q, 5q, 6q, 8p, 9p, 10q, 11q, 13q, 16q, 17p, and 18q were studied. All invasive and severely dysplastic intraductal foci exhibited loss of heterozygosity (LOH) at more than one chromosomal locus. For each case, allelic loss was frequently observed on 9p (severe ductal dysplasia 90%, invasion 100%), 17p (severe ductal dysplasia 80%, invasion 80%), and 18q (severe ductal dysplasia 88%, invasion 88%). Ninety-four percent of severe ductal dysplasia and 96% of invasive foci had multiple LOH. Seventeen percent of nonpapillary hyperplasia and 33% of pap HP showed LOH. Only one focus of pap HP showed multiple LOH. The patterns of allelic loss identified in severe ductal dysplasia were generally conserved in synchronous infiltrating tumors, supporting the paradigm that infiltrating tumors are clonally derived from severe ductal dysplasia. In eight of 10 cases, however, we found frequent genetic heterogeneity in the intraductal lesion, suggestive of genetic progression or diversion. These findings indicate that invasive pancreatic carcinoma evolves through successive and divergent genetic changes with selection of aggressive subclones in the intraductal component.     

A region on human chromosome 4 (q35.1-->qter) induces senescence in cell hybrids and is involved in cervical carcinogenesis

Human papillomavirus (HPV) types 16 and 18 are known to play a major role in cervical carcinogenesis. Additional genetic alterations are required for the development and progression of cervical cancer. Previously, we showed that the introduction of an entire human chromosome 4 into HPV-immortalized cells by microcell-mediated chromosome transfer (MMCT) can induce senescence in cell hybrids. In the present study, we established eight new murine donor cell lines harboring different fragments of the human chromosome 4. These were tested for their ability to induce senescence by MMCT into HPV16-immortalized keratinocytes (HPK II) and cervical carcinoma cells (HeLa). By exclusion, we could identify a region for a putative senescence gene or genes at 4q35.1-->qter. Further evidence that this locus may be involved in cervical carcinogenesis was obtained by studying sections of high-grade cervical intraepithelial neoplasias (CIN2/3) and cervical cancers from 87 women using a combination of interphase fluorescence in situ hybridization (I-FISH) and microsatellite PCR. I-FISH indicated copy number loss at 4q34-->qter. Microsatellite analysis showed that loss of one or more alleles at chromosome 4 was more frequent in the cervical carcinomas than in the CINs. Loss of heterozygosity (LOH) affected four areas, D4S412 (4p16.3), D4S2394 (4q28.2), D4S3041 (4q32.3), and D4S408 (4q35.1), and was highest at D4S408. LOH at terminal 4q has been reported previously for cervical carcinomas and other human malignancies. This is the first report associating allelic loss at 4q34-->qter with high-grade intraepithelial neoplasia and cervical carcinoma, and the first experimental evidence that this locus or these loci can induce senescence in cervical carcinoma cells and HPV16-immortalized cells.     

DNA Copy Number Changes in Thyroid Carcinoma

The genetic changes leading to thyroid cancer are poorly characterized. We studied DNA copy number changes by comparative genomic hybridization (CGH) in 69 primary thyroid carcinomas. In papillary carcinoma, DNA copy number changes were rare (3 of 26, 12%). The changes were all gains, and they were associated with old age (P = 0.01) and the presence of cervical lymph node metastases at presentation (P = 0.08). DNA copy number changes were much more frequent in follicular carcinoma (16 of 20, 80%) than in papillary carcinoma (P < 0.0001), and follicular carcinomas had more often deletions (13/20 versus 0/26, P < 0.0001). Loss of chromosome 22 was common in follicular carcinoma (n = 7, 35%), it was more often seen in widely invasive than in minimally invasive follicular carcinoma (54% versus 0%, P = 0.04), and it was associated with old age at presentation (P = 0.01). In three of the four patients with follicular carcinoma who died of cancer, the tumor had loss of chromosome 22. DNA copy number changes were found in 5 (50%) of the 10 medullary carcinomas studied. Four of these five carcinomas had deletions, and in two of them there was deletion of chromosome 22. Eleven (85%) of the thirteen anaplastic carcinomas investigated had DNA copy number changes, of which five had deletions, and one had deletion of chromosome 22. The most common gains in anaplastic carcinoma were in chromosomes 7p (p22-pter, 31%), 8q (q22-qter, 23%), and 9q (q34-qter, 23%). We conclude that DNA copy number changes are frequent in follicular, medullary, and anaplastic thyroid carcinoma but rare in papillary carcinoma when studied by CGH. Loss of chromosome 22 is particularly common in follicular carcinoma, and it is associated with the widely invasive type.     

Conserved genetic findings in metastatic bladder cancer: a possible utility of allelic loss of chromosomes 9p21 and 17p13 in diagnosis

CONTEXT: Molecular analysis of microsatellite alterations of biologically distinct tumor cell subpopulations from the same patient may aid in the determination of tumor origin and further our understanding of the genetic basis of cancer progression. DESIGN: The authors examined the pattern of allelic loss with polymorphic microsatellite markers on chromosome 9p21 (D9S161, D9S171, IFNA), regions of putative tumor suppressor gene p16, and on chromosome 17p13 (TP53), the p53 locus, in matched primary and metastatic bladder cancers from 9 patients. All patients underwent cystectomy for bladder cancer and had regional lymph node metastases at the time of surgery. Genomic DNA was prepared from primary cancers and matched synchronous lymph node metastases using a microdissection method. RESULTS: The overall frequency of allelic loss was 78% in primary cancer and 89% in paired metastatic cancer. The frequency of allelic loss in the primary cancer was 86% with D9S161, 67% with D9S171, 71% with IFNA, and 80% with TP53. The frequency of allelic loss in matched metastatic cancer was 100% with D9S161, 62% with D9S171, 71% with IFNA, and 80% with TP53. An identical pattern of allelic imbalance (allelic loss or retention) at multiple DNA loci was observed in matched primary and metastatic carcinoma in 8 (88%) cases. One case showed allelic loss in the metastasis, but not in the primary cancer. CONCLUSIONS: The pattern of allelic loss at chromosome 9p21 (p16) and 17p13 (p53) was generally maintained during cancer progression to metastasis, and identical allelic loss in primary cancer was conserved in paired metastatic carcinoma. These data suggest that these genetic changes may be useful in establishing a diagnosis and determining tumor origins in difficult cases.     

Genomic integration of oncogenic HPV and gain of the human telomerase gene TERC at 3q26 are strongly associated events in the progression of uterine cervical dysplasia to invasive cancer

Recently proposed events associated with the progression of cervical intraepithelial neoplasia (CIN) 2/3 to cervical carcinoma include integration of human papillomavirus (HPV) into the host genome, genomic instability, and an increase in chromosome 3q copy number. In particular, the gene coding for the RNA component of telomerase (TERC) at 3q26 has been implicated as a possible candidate gene. Since it is not known to date how these events are temporally related during cervical carcinogenesis, the aim of the present study was to assess the correlation between TERC gene copy number and the physical status of HPV during progression in cervical neoplasia. Solitary precursor lesions of the uterine cervix (CIN 2/3, n = 17), lesions associated with a micro-invasive carcinoma (CIN 3&mCA, n = 13), and advanced invasive carcinomas (invCA, n = 7) were analysed by fluorescence in situ hybridization (FISH) to determine the physical status of the virus and TERC gene copy number. The TERC gene was increasingly gained with progression of CIN 2/3 (3 of 17) through CIN 3&mCA (7 of 13) to invCA (5 of 7). In the lesions exhibiting gain of TERC, the virus was predominantly integrated. This was seen in eight of ten diploid lesions, indicating that these events can occur prior to aneuploidization and are strongly associated with the progression of CIN 3 to mCA and invCA (p < 0.001). With progression to carcinoma, a number of these lesions show polyploidization, resulting in aneuploidy and high TERC gene copy numbers. In conclusion, genomic integration of oncogenic HPV and gain of the human telomerase gene TERC appear to be important associated genetic events in the progression of uterine cervical dysplasia to invasive cancer.     

17q allele loss is associated with lymph node metastasis in locally aggressive human colorectal cancer

A consecutive series of 87 colorectal tumours were studied for loss of a polymorphic probe on chromosome 17q and of the 64 informative cases, 13 (20 per cent) showed loss of heterozygosity (LOH). Examples of LOH were found in carcinomas of all stages and in a large non-invasive adenoma. There was no correlation between 17q LOH and patient age, sex, standard clinicopathological variables (differentiation and nature of tumour margin), DNA ploidy, or tumour site, nor was 17q LOH associated with 17p LOH defined at four loci adjacent to p53. However, comparison of Dukes' B and C carcinomas revealed that tumours which had metastasized to regional lymph nodes at the time of primary surgery were significantly more likely to have lost this 17q allele. Clinical follow-up of this cohort of patients showed no significant difference in survival between patients whose tumours had lost or retained 17q. Thus, we conclude that 17q allele loss is associated with lymph node metastasis in locally aggressive colorectal tumours but probably not with blood-borne metastasis.