Frequency of homozygous deletion at p16/CDKN2 in primary human tumours.

Many tumour types have been reported to have deletion of 9p21 (refs 1-6). A candidate target suppressor gene, p16 (p16INK4a/MTS-1/CDKN2), was recently identified within the commonly deleted region in tumour cell lines. An increasing and sometimes conflicting body of data has accumulated regarding the frequency of homozygous deletion and the importance of p16 in primary tumours. We tested 545 primary tumours by microsatellite analysis with existing and newly cloned markers around the p16 locus. We have now found that small homozygous deletions represent the predominant mechanism of inactivation at 9p21 in bladder tumours and are present in other tumour types, including breast and prostate cancer. Moreover, fine mapping of these deletions implicates a 170 kb minimal region that includes p16 and excludes p15.

Evidence for a prostate cancer susceptibility locus on the X chromosome.

Over 200,000 new prostate cancer cases are diagnosed in the United States each year, accounting for more than 35% of all cancer cases affecting men, and resulting in 40,000 deaths annually. Attempts to characterize genes predisposing to prostate cancer have been hampered by a high phenocopy rate, the late age of onset of the disease and, in the absence of distinguishing clinical features, the inability to stratify patients into subgroups relative to suspected genetic locus heterogeneity. We previously performed a genome-wide search for hereditary prostate cancer (HPC) genes, finding evidence of a prostate cancer susceptibility locus on chromosome 1 (termed HPC1; ref. 2). Here we present evidence for the location of a second prostate cancer susceptibility gene, which by heterogeneity estimates accounts for approximately 16% of HPC cases. This HPC locus resides on the X chromosome (Xq27-28), a finding consistent with results of previous population-based studies suggesting an X-linked mode of HPC inheritance. Linkage to Xq27-28 was observed in a combined study population of 360 prostate cancer families collected at four independent sites in North America, Finland and Sweden. A maximum two-point lod score of 4.60 was observed at DXS1113, theta=0.26, in the combined data set. Parametric multipoint and non-parametric analyses provided results consistent with the two-point analysis. Significant evidence for genetic locus heterogeneity was observed, with similar estimates of the proportion of linked families in each separate family collection. Genetic mapping of the locus represents an important initial step in the identification of an X-linked gene implicated in the aetiology of HPC.

Prostate cancer evolution from multilineage primary to single lineage metastases with implications for liquid biopsy.

The evolutionary progression from primary to metastatic prostate cancer is largely uncharted, and the implications for liquid biopsy are unexplored. We infer detailed reconstructions of tumor phylogenies in ten prostate cancer patients with fatal disease, and investigate them in conjunction with histopathology and tumor DNA extracted from blood and cerebrospinal fluid. Substantial evolution occurs within the prostate, resulting in branching into multiple spatially intermixed lineages. One dominant lineage emerges that initiates and drives systemic metastasis, where polyclonal seeding between sites is common. Routes to metastasis differ between patients, and likely genetic drivers of metastasis distinguish the metastatic lineage from the lineage that remains confined to the prostate within each patient. Body fluids capture features of the dominant lineage, and subclonal expansions that occur in the metastatic phase are non-uniformly represented. Cerebrospinal fluid analysis reveals lineages not detected in blood-borne DNA, suggesting possible clinical utility.

Major susceptibility locus for prostate cancer on chromosome 1 suggested by a genome-wide search.

Despite its high prevalence, very little is known regarding genetic predisposition to prostate cancer. A genome-wide scan performed in 66 high-risk prostate cancer families has provided evidence of linkage to the long arm of chromosome 1 (1q24-25). Analysis of an additional set of 25 North American and Swedish families with markers in this region resulted in significant evidence of linkage in the combined set of 91 families. The data provide strong evidence of a major prostate cancer susceptibility locus on chromosome 1.

Therapeutic implications of enhanced G(0)/G(1) checkpoint control induced by coculture of prostate cancer cells with osteoblasts.

Osteoblastic metastases are common in lethal prostate cancer. Effective therapy for bone metastases is lacking. Thus, developing an appropriate in vitro screening system is critical to prioritize which of the newly developed agents should undergo additional expensive and time-consuming in vivo evaluation in bone metastases animal models. In the past, such in vitro screening evaluated the response of prostate cancer cells to chemotherapeutic agents in monoculture without the presence of osteoblasts. In such monoculture, prostate cancer cells have a high (i.e., >90%) proliferative growth fraction. In contrast, the growth fraction (i.e., mean: 7.1 +/- 0.8%; median: 3.1%) in 117 metastatic sites of prostate cancer obtained from 11 androgen ablation failing patients at "warm" autopsy was found to be >10-fold lower. To better mimic the lower growth fraction observed clinically, LNCaP human prostate cancer cells were cocultured with membrane-separated hFOB human osteoblasts. Such coculturing significantly lowered the growth fraction of the LNCaP cells (i.e., from >90 to <30%) without enhancing their low rate (i.e., <5%) of apoptosis. This lowering of the growth fraction was documented using flow cytometry, Ki-67 immunohistochemistry, and 5-bromo-2-deoxyuridine incorporation. Using RNase protection assays, it was documented that coculture with osteoblasts causes enhanced p53, p27, and p21 expression leading to a decrease in the number of LNCaP cells entering the cell cycle (i.e., enhanced number of LNCaP cells in G(0)-G(1) and a decrease in S and G(2)-M and thus the growth fraction). This osteoblast-induced enhanced G(0)-G(1) checkpoint control affected the chemosensitivity of LNCaP cells. This was documented by coculturing LNCaP cells with hFOB cells to condition the medium for 3 days to lower the growth fraction to <30% before exposing the LNCaP cells for 48 h to various concentrations of Taxol, doxorubicin, or thapsigargin (TG). In standard high (i.e., >90%) growth fraction cultures (i.e., cultures in the absence of osteoblast-conditioned medium), there was a dose-dependent and significant (P < 0.05) increase in apoptosis of LNCaP cells exposed to Taxol or doxorubicin. In contrast, even the highest dose of Taxol (1 microM) did not enhance apoptosis of lower growth fraction LNCaP cells cultured in osteoblast-conditioned medium. Similarly, only the highest concentration of doxorubicin (1 microM) enhanced apoptosis in lower growth fraction cells. In contrast, 100 nM TG induced high levels of apoptosis in both lower and high-growth fraction LNCaP cultures. These results demonstrate that the osteoblast/LNCaP coculture system is a better in vitro screen than monoculture to identify proliferation-independent agents for the treatment of prostate cancer bone metastases, and TG is such an agent.

Coding region of NKX3.1, a prostate-specific homeobox gene on 8p21, is not mutated in human prostate cancers.

Loss of heterozygosity at chromosome 8p21-22 is common in human prostate cancer, suggesting the presence of one or more tumor suppressor genes at this locus. A homeobox gene that is expressed specifically in adult human prostate, NKX3.1, the expression of which is regulated by androgen, maps to chromosome 8p21. Fine structure in situ mapping showed that NKX3.1 is proximal to MSR32 (macrophage scavenger receptor type II) and LPL (human lipoprotein lipase) and very close to NEFL (human neurofilament light chain) on 8p21. Single-strand conformational polymorphism analysis of 48 radical prostatectomy cancer specimens and 3 metastases for the entire coding region of NKX3.1 showed no tumor-specific sequence alterations in 50 specimens and total absence of the gene in 1 specimen known to have a biallelic deletion of 8p21. NKX3.1 was found to have a polymorphism at nucleotide 154 in codon 52 that resulted in a CGC-->TGC sequence change and an Arg-->Cys amino acid alteration (R52C). This polymorphism was present in 20% of DNA samples. If NKX3.1 is a target of the 8p21 LOH, it is not via disruption of the coding region of the gene.

Identification of 12p as a region of frequent deletion in advanced prostate cancer.

The identification of homozygous deletions in malignant tissue has been a powerful tool for the localization of tumor suppressor genes. Representational difference analysis (RDA) uses selective hybridization and the PCR to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes such as BRCA2 and PTEN. Twenty RDA clones were generated by comparing genomic DNA from a prostate cancer xenograft to the same patient's normal kidney DNA. Southern blot analysis of the tester and driver and of normal and xenograft DNA, using the differential products as probes, showed the homozygous deletion in 16 of 20 RDA clones. The sequence of one of the differential products overlapped HSU59962, a genomic GenBank sequence on chromosome 12p12-13. Multiplex PCR of the xenograft DNA using polymorphic repeats mapped the deletion to a 1-5-cM region on 12p. Genomic DNA isolated from a panel of cryostat microdissected metastatic prostate adenocarcinomas/normal pairs was screened for loss of heterozygosity using the same polymorphic repeats. Loss of heterozygosity was demonstrated in 9 (47%) of 19 patients. This region may contain, or lie in close proximity to, tumor suppressor genes important in the progression and/or initiation of prostate cancer.

Methylation of the androgen receptor minimal promoter silences transcription in human prostate cancer.

Advanced hormone-independent prostate cancer is characterized by a significant loss of androgen receptor (AR) expression in 20-30% of the tumors. The transcriptional block underlying this phenomenon is not known, but we have proposed that methylation of CpG sites in the AR promoter may reversibly inactivate transcription of the AR (D. F. Jarrard et al, Cancer Res., 58: 5310-5314, 1998). In this study, detailed methylation analysis using bisulfite sequencing was performed on a series of AR expression-positive and -negative prostate cancer cells. We found that methylation of several consensus sequences in the AR promoter (from -131 to -121 and +44 to +54) are tightly linked to the loss of AR expression in metastatic hormone-independent prostate cancer cell lines. These consensus sites of methylation correlate with the minimal promoter region critical for AR transcription. In human tissues, no methylation was demonstrated in normal or primary prostate cancers that express the AR. Four of 15 tumors obtained from men who had died from hormone-independent prostate cancer demonstrated a significant loss of AR expression immunohistochemically and two (50%) of these AR-negative tumors contained AR methylation. We conclude that the AR promoter contains specific CpG methylation hot spots that are markers for gene silencing. Furthermore, AR methylation may represent a phenotype important in the development of hormone independence in a subset of advanced prostate cancer in which AR expression is lost. The finding of AR methylation also represents the first report of aberrant methylation on an X-linked gene associated with a somatic male cancer.

Telomerase activity: a prevalent marker of malignant human prostate tissue.

We urgently need biochemical markers to detect the malignant nature and pathological states of the human prostate. We report that telomerase activity is associated with prostate cancer but absent in the benign disease and normal gland. Telomerase is, therefore, a potential diagnostic marker for prostate cancer. Twenty-five human prostates resected at the time of radical prostatectomy were dissected to obtain matched adjacent areas of normal, central zone benign prostatic hyperplasia (BPH), and pathologically confirmed cancer tissue. These matched tissue samples were assayed for telomerase activity using a sensitive PCR technique. None of the normal tissues exhibited telomerase activity. In contrast, 21 of the 25 (84%) cancers were strongly positive. At the time of prostatectomy, four lymph nodes were positive for metastases and all were strongly positive for telomerase activity. In adjacent BPH tissues taken from the cancerous prostates, only 3 of the 25 tissues (12%) were weakly positive. Telomerase activity was not detected in ten BPH samples recovered from patients who underwent open surgery solely for BPH. All five available cell lines of human prostate cancer (DU145, LNCaP, PC3, PPC1, and TSU) were strongly positive. Short telomere lengths have been observed in several human cancers. We also measured the telomere lengths in 27 matched samples of normal, BPH, and cancer tissue taken from nine radical prostatectomies. The telomeres from cancer tissue were significantly and consistently shorter than either the adjacent normal or adjacent BPH tissues. Our results indicate that telomerase activity, as well as telomere lengths, may be markers for distinguishing prostate cancer from normal and benign prostate tissues.

Genetic alterations in untreated metastases and androgen-independent prostate cancer detected by comparative genomic hybridization and allelotyping.

A newly developed method of comparative genomic hybridization (CGH) employing quantitative statistical comparisons was applied to DNA from two different types of advanced prostate cancer tissue. Multiple CGH analyses were obtained for each chromosome in each tumor, and the results of point-by-point comparison of the mean tumor:normal color ratio to a control normal:normal color ratio in each of 1247 evenly distributed data channels constituting the entire human genome were interpreted as loss, gain, or no change in copy number in the tumor genome. Group I tissue was obtained from prostate cancer metastases from 20 patients, 19 of whom had received no prior prostate cancer treatment. This DNA also was analyzed by Southern and microsatellite allelotyping at 53 different loci on 20 different chromosome arms. CGH results agreed with allelotyping results at 92% of the informative loci studied. These samples, which contained highly enriched tumor DNA, showed the highest rates of alteration yet reported in several chromosomal regions known to be altered frequently in prostate cancer: 8q gain (85%), 8p loss (80%), 13q loss (75%), 16q loss (55%), 17p loss (50%), and 10q loss (50%). Group II tissue was obtained predominately from primary or recurrent tumor from 11 patients who had been treated with long-term androgen-deprivation therapy and developed androgen-independent metastatic disease. Quantitative CGH analysis on DNA from these tissues showed chromosomal alterations that were very similar to those found in group I, suggesting that untreated metastatic tumors contain the bulk of chromosomal alterations necessary for recurrence to occur during androgen deprivation. In the entire data set, a number of previously undetected regions of frequent loss or gain were identified, including losses of chromosomes 2q (42%), 5q (39%), 6q (39%), and 15q (39%) and gains of chromosomes 11p (52%), 1q (52%), 3q (52%), and 2p (45%). Chi-squared analysis showed a significantly higher frequency of gain of the 4q25-q28 region in tumors from African-American patients, indicating a possible oncogene whose activation may play a role in the higher rate of progression seen in this ethnic group. Additional study of these frequently altered regions may provide insight into the mechanism of prostate cancer progression and lead to important tools for tumor-specific prognosis and therapy.

Homozygous deletion and frequent allelic loss of chromosome 8p22 loci in human prostate cancer.

Allelic loss studies have been instrumental in identifying tumor suppressor gene loci in a variety of cancers. In this study we analyzed prostate cancer specimens from 52 patients for allelic loss using 8 polymorphic probes for the short arm of chromosome 8. Overall, 32 of 51 (63%) informative tumors showed loss of at least one locus on chromosome 8p. The most frequently deleted region is observed at chromosome 8p22-8p21.2. Loss of one allele is identified in 14 of 23 (61%) tumors at D8S163, in 15 of 32 (47%) tumors at lipoprotein lipase, and in 20 of 29 (69%) tumors at MSR, all on 8p22. Loss of one allele is identified in 16 of 27 (59%) tumors at D8S220 at 8p21.3-8p21.2. In addition to frequent loss of one allele at the MSR locus, one metastatic prostate cancer sample demonstrated homozygous deletion of MSR sequences. Loci telomeric and centromeric to this region are largely retained. A chromosome 8p deletion map is constructed and defines the smallest region of overlap to a 14-cM interval at 8p22 between D8S163 and lipoprotein lipase, flanking the MSR locus. Evidence of chromosome 8q multiplication at locus D8S39 was detected in 5 of 32 (16%) tumors, all of which demonstrated loss with at least one probe on chromosome 8p. This study extends the previous finding of frequent loss of chromosome 8p in prostate cancer by defining a common region of loss of heterozygosity at 8p22 and a homozygous deletion of the MSR locus contained within this region. This is the first homozygous deletion identified in the genome of a human prostate cancer and the highest rate of loss yet reported on chromosome 8p in cancer. These results strongly suggest the presence of a tumor suppressor gene in this region which is frequently inactivated in prostate cancer.

Down-regulation of the KAI1 metastasis suppressor gene during the progression of human prostatic cancer infrequently involves gene mutation or allelic loss.

The KAI1 gene, located on human chromosome 11p11.2, suppresses tumor metastasis when expressed in certain cancer cells. To evaluate whether dysregulation of KAI1 occurs during the progression of human prostatic cancer, protein expression, mutation, and allelic loss of KAI1 were analyzed using a tissue bank of 98 primary cancers and 32 metastases. By immunohistochemical staining, high levels of KAI1 protein are detected in the epithelial but not stromal compartment of normal prostatic and benign prostatic hyperplasia tissue. In epithelial cells, KAI1 protein is expressed on the plasma membrane. KAI1 protein expression is downregulated in more than 70% of the 49 primary prostatic cancers from untreated patients. In 10 such untreated patients, down-regulation of KAI1 protein occurred in all of the lymph node metastases examined. In 15 patients with metastatic disease who had failed androgen ablation therapy, more than 90% of the primary prostatic cancers had downregulation, with 60% having no KAI1 protein expression. Primers derived from the sequences flanking each exon of KAI1 were used to analyze KAI1 mutation and allelic loss by the method of PLR-single-strand conformational polymorphism. Using this method, no point mutation or allelic loss was detected in metastases from 10 patients. No allelic loss was detected in an additional 34 primary and 12 lymph node metastases via microsatellite analysis using the marker D11S1344, which is located in the region of KAI1. These results demonstrate that KAI1 protein expression is consistently down-regulated during the progression of human prostatic cancer and that this down-regulation does not commonly involve either mutation or allelic loss of the KAI1 gene.

Methylation of the 5' CpG island of the endothelin B receptor gene is common in human prostate cancer.

Production of the potent vasoconstrictor endothelin-1 (ET-1) by human prostate cancer cells accompanies prostate cancer progression in vivo. The predominant endothelin receptor expressed by normal prostate epithelium, ETB, is not expressed by any of the established human prostate cancer cell lines, and ETB binding is decreased on prostate cancer tissues. ETB, which may mediate ET-1 clearance and may inhibit ET-1 secretion, is encoded by a gene that contains a 5' CpG island encompassing the transcriptional regulatory region. We examined this regulatory region of the ETB receptor gene (EDNRB) to determine whether hypermethylation of cytidine nucleotides accompanies decreased ETB expression in human prostate cancer. We found somatic methylation of CpG island sequences in EDNRB in 5 of 5 human prostate cancer cell lines, 15 of 21 primary prostate cancer tissues, and 8 of 14 prostate cancer metastases (70% of samples overall). Normal tissues contained only unmethylated EDNRB. Treatment of human prostatic carcinoma cell line cultures with 5-azacytidine induced ETB mRNA expression, suggesting that CpG island methylation changes might accompany the apparent transcriptional silencing of EDNRB in vivo.

Frequent inactivation of PTEN/MMAC1 in primary prostate cancer.

Sporadic prostate carcinoma is the most common male cancer in the Western world, yet many of the major genetic events involved in the progression of this often fatal cancer remain to be elucidated. Numerous cytogenetic and allelotype studies have reported frequent loss of heterozygosity on chromosomal arm 10q in sporadic prostate cancer. Deletion mapping studies have unambiguously identified a region of chromosome 10q23 to be the minimal area of loss. A new tumor suppressor gene, PTEN/MMAC1, was isolated recently at this region of chromosome 10q23 and found to be inactivated by mutation in three prostate cancer cell lines. We screened 80 prostate tumors by microsatellite analysis and found chromosome 10q23 to be deleted in 23 cases. We then proceeded with sequence analysis of the entire PTEN/MMAC1 coding region and tested for homozygous deletion with new intragenic markers in these 23 cases with 10q23 loss of heterozygosity. The identification of the second mutational event in 10 (43%) tumors establishes PTEN/MMAC1 as a main inactivation target of 10q loss in sporadic prostate cancer.

Interfocal heterogeneity of PTEN/MMAC1 gene alterations in multiple metastatic prostate cancer tissues.

The long arm of chromosome 10 is frequently affected by allelic loss in prostate cancer. PTEN/MMAC1, a candidate tumor suppressor gene located at 10q23.3, a region commonly deleted in prostate cancer, was recently identified and found to be deleted or mutated in cancer cell lines derived from a variety of human tissues including prostate. To examine the role of PTEN/MMAC1 in the progression of prostate cancer, we screened a unique set of 50 metastatic prostate cancer tissues from 19 cancer-death patients for alterations in the PTEN/MMAC1 gene, using single-strand conformational polymorphism analysis and direct sequencing to identify sequence changes and microsatellite analysis to examine allelic loss in the vicinity of PTEN/MMAC1. Overall, gene alterations (deletions or point mutations) were observed in at least 1 metastatic site in 12 of the 19 patients studied. Two cases had homozygous deletions that were confirmed by fluorescence in situ hybridization analysis. Four patients harbored point mutations, with one mutation being found in all four tumors (a primary lesion and three different metastases) from the same patient. The remaining three mutations were detected in only one of multiple metastases. Loss of heterozygosity was found in 10 of 18 informative cases, with 1 case showing a unique pattern of microsatellite instability in each of six different metastases examined. Loss of the same allele was found in all metastases in a given patient in 9 of 10 cases. These results indicate that PTEN/MMAC1 gene alterations occur frequently in lethal prostate cancer, although a substantial amount of mutational heterogeneity is found among different metastatic sites within the same patient. These latter findings emphasize the potentially complex genetic relationship that can exist between various clonal lineages of prostate cancer cells as they evolve during the metastatic process and suggest a molecular basis for phenotypic heterogeneity of different prostate cancer foci in patients with disseminated disease.

An uncertain role for p53 gene alterations in human prostate cancers.

Inactivation of the p53 gene has been implicated in prostate cancer progression. To determine the role of p53 inactivation in the progression of clinical prostatic carcinomas, we assessed 67 tumors derived from patients with clinically localized disease for chromosome 17p and p53 gene allelic loss, p53 gene mutations using single-strand conformational polymorphism and direct sequencing, and p53 protein expression using immunohistochemical staining. Of 55 informative tumors, 10 demonstrated loss of 17p or the p53 gene; however, only a single tumor had a mutation in its remaining p53 allele. Significant p53 overexpression was observed in 2 of 38 tumors, and 9 others had faint staining of a few nuclei ( < 1%). p53 overexpression occurred in no informative tumor with allelic loss or mutation. In a 1-7-year follow-up, positive immunohistochemical staining did not confer an increased risk of recurrence (risk of recurrence, 0.86, P = 0.78), whereas allelic loss of chromosome 17p appeared to be highly correlated with recurrence (risk of recurrence, 3.7, P = 0.003). In an unrelated group of 42 patients with metastatic prostate cancer, p53 overexpression was found in 26 tumors (62%), and 15(36%) had high grade staining. Neither the presence nor the degree of expression correlated with time to progression or time to death. This series suggests that p53 gene inactivation is rare in primary prostatic tumors, not essential to the development of prostate cancer metastases, and of limited use as a prognostic marker in patients with primary or metastatic disease. Another gene or genes on chromosome 17p may be involved in prostate cancer progression.

Endothelin-1 production and decreased endothelin B receptor expression in advanced prostate cancer.

The potent vasoconstrictor endothelin-1 (ET-1) is at its highest concentration in the normal human ejaculate and is associated with the progression of metastatic prostate cancer. ET-1 protein expression is detected in situ in 14 of 14 primary cancers and 14 of 16 metastatic sites of human prostatic carcinoma. Exogenous ET-1 induces prostate cancer proliferation directly and enhances the mitogenic effects of insulin-like growth factor I, insulin-like growth factor II, platelet-derived growth factor, basic fibroblast growth factor, and epidermal growth factor in serum-free conditions in vitro. The ETA-selective receptor antagonist A-127722 inhibits ET-1-stimulated growth, but the ETB-selective receptor antagonist BQ-788 does not. ET-3, an ETB-selective agonist, also had no effect on prostate cancer growth. No specific ETB-binding sites could be demonstrated in any established human prostate cancer cell line tested, and ETB mRNA, detected by reverse transcription PCR, was reduced. The predominance of ETB binding on human benign prostatic epithelial tissue is not present in metastatic prostate cancer by autoradiography. In human prostate cancer progression to metastases, ET-1 and ETA expression are retained, whereas ETB receptor expression is reduced.

Early age at diagnosis in families providing evidence of linkage to the hereditary prostate cancer locus (HPC1) on chromosome 1.

In a recent study of 91 families having at least three first degree relatives with prostate cancer, we reported the localization of a major susceptibility locus for prostate cancer (HPC1) to chromosome 1 [band q24; J. R. Smith et al., Science (Washington DC), 274: 1371-1373, 1996]. There was significant evidence for locus heterogeneity, with an estimate of 34% of the families being linked to this locus. In this report, we investigate the importance of age at diagnosis of prostate cancer and number of affected individuals within a family as variables in the linkage analysis of an expanded set of markers on 1q24. Under two different models for the prostate cancer locus, we find that the evidence for linkage to HPC1 is provided primarily by large (five or more members affected) families with an early average age at diagnosis. Specifically, for 40 North American families with an average age at diagnosis <65 years, the multipoint lod score is 3.96, whereas for 39 families with an older average age at diagnosis, this value is -0.84. Assuming heterogeneity, the proportion of families linked is 66% for the 14 families with the earliest average ages at diagnoses, but it decreases to 7% for the families with the latest ages at diagnoses. A similar age effect is observed in 12 Swedish pedigrees analyzed. To test the hypotheses generated by these analyses, we examined an additional group of 13 newly identified prostate cancer families. Overall, these families provided additional evidence for linkage to this region (nonparametric linkage Z = 1.91; P = 0.04 at marker D1S1660), contributed primarily by the families in this group with early age at diagnosis [nonparametric linkage Z = 2.50 (P = 0.01) at D1S422]. These results are consistent with the existence of a locus in this region that predisposes men to develop early-onset prostate cancer.

DPC4 gene in various tumor types.

We recently identified a novel tumor-suppressor gene, DPC4, at chromosome 18q21.1 and found that both alleles of DPC4 were inactivated in nearly one-half of the pancreatic carcinomas. Here, we analyzed 338 tumors, originating from 12 distinct anatomic sites, for alterations in the DPC4 gene. Sixty-four specimens were selected for the presence of the allelic loss of 18q and were further analyzed for DPC4 sequence alterations. An alteration of the DPC4 gene sequence was identified in one of eight breast carcinomas and one of eight ovarian carcinomas. These results indicate that whereas DPC4 inactivation is prevalent in pancreatic carcinoma (48%), it is distinctly uncommon (< 10%) in the other tumor types examined. The tissue restriction of alterations in DPC4, as in many other tumor-suppressor genes, emphasizes the complexity of rate-limiting checkpoints in human tumorigenesis.

Frequent mutation of the E2F-4 cell cycle gene in primary human gastrointestinal tumors.

The E2F group of transcription factors transactivates genes that promote progression through the G1-S transition of the cell cycle. Members of the retinoblastoma (Rb) family of proteins bind to E2Fs and inhibit this function. E2F-4, one example of the E2F group, functions as an oncogene when transfected into nontransformed cells in vitro. On the other hand, mice that are homozygously lacking a normal E2F-1 gene develop cancers, consistent with a tumor-suppressive role for this gene. The exact function of E2Fs has thus been unclear; moreover, direct involvement of this gene in primary human tumorigenesis has not been shown. We, therefore, investigated mutation within the E2F-4 coding region in 16 primary gastric adenocarcinomas, 12 ulcerative colitis-associated neoplasms, 46 sporadic colorectal carcinomas, 9 endometrial cancers, and 3 prostatic carcinomas. We limited our investigation to the serine repeat within E2F-4, reasoning that this tract might be altered in genetically unstable tumors (replication error-positive, or RER+). All tumors were RER+, with the exception of a control group of 15 RER- sporadic colorectal carcinomas. PCR with incorporation of [32P]dCTP was performed using primers flanking the serine trinucleotide (AGC) repeat. Twenty-two of 59 gastrointestinal tumors (37%) contained E2F-4 mutations; these comprised 5 of 16 gastric tumors (31%), 4 of 12 ulcerative colitis-associated neoplasms (33%, including 1 dysplastic lesion), and 13 of 31 sporadic colorectal cancers (42%). No mutation was present in any of the endometrial, prostate, or RER- colorectal tumors. Of note, homozygous mutations occurred in three cases, and two of seven informative patients showed loss of one E2F-4 allele in their tumors. Furthermore, the RER+ sporadic colorectal tumors were evaluated at trinucleotide repeats within the genes for N-cadherin and B-catenin; no tumors demonstrated mutation of these genes. These data suggest that E2F-4 is a target of defective DNA repair in these tumors.