HER-2/neu (p185neu) protein expression in the natural or treated history of prostate cancer.

PURPOSE: Amplification of HER-2/neu gene and overexpression of its encoded product, the p185neu (HER-2/neu) tyrosine kinase membrane receptor, have been associated with tumor progression in certain neoplasms. We conducted this study to investigate patterns of HER-2/neu protein expression in prostate cancer, analyzing different points in the natural and treated history of the disease. EXPERIMENTAL DESIGN: Radical prostatectomy cases (83) and 20 metastatic lesions were studied for the association between HER-2/neu protein overexpression detected by immunohistochemistry and clinicopathological parameters, including time to prostate-specific antigen (PSA) relapse. RESULTS: HER-2/neu protein overexpression, defined as complete membrane staining in >10% of tumor cells using the Food and Drug Administration-approved Dako kit, was found in 9 of 45 (20%) of evaluable hormone naïve primary tumors and 23 of 34 (67%) primary tumors after androgen-deprivation therapy (P = 0.0001). Of the 20 metastatic lesions, positivity was noted in 16 (80%) of the cases. On univariate analysis, HER-2/neu overexpression was associated with pretreatment PSA (P = 0.011) and time to PSA relapse (P = 0.02). After controlling for pretreatment PSA, the association between hormone treatment and HER-2/neu was still observed. No association was found between HER-2/neu overexpression and Gleason score, capsular invasion, and tumor proliferative index determined by Ki67. CONCLUSIONS: These data suggest that there is significant HER-2/neu overexpression in primary tumors that persist after androgen deprivation. It also emphasizes the importance of characterizing tumors at determined points in the natural or treated history of prostate cancer when targeting treatment to specific biological processes.

Inhibition of transformed cell growth and induction of cellular differentiation by pyroxamide, an inhibitor of histone deacetylase.

PURPOSE: We have synthesized a series of hybrid polar compounds that induce differentiation and/or apoptosis of various transformed cells. These agents are also potent inhibitors of histone deacetylases (HDACs). Pyroxamide (suberoyl-3-aminopyridineamide hydroxamic acid) is a new member of this class of compounds that is currently under development as an anticancer agent. We investigated the activity of pyroxamide as an inducer of differentiation and/or apoptosis in transformed cells. EXPERIMENTAL DESIGN AND RESULTS: Pyroxamide, at micromolar concentrations, induced terminal differentiation in murine erythroleukemia (MEL) cells and caused growth inhibition by cell cycle arrest and/or apoptosis in MEL, prostate carcinoma, bladder carcinoma, and neuroblastoma cells. Administration of pyroxamide (100 or 200 mg/kg/day) to nude mice at doses that caused little evident toxicity significantly suppressed the growth of s.c. CWR22 prostate cancer xenografts. Despite the potent growth-inhibitory effects of pyroxamide in this tumor model, serum prostate-specific antigen levels in control versus pyroxamide-treated mice were not significantly different. Pyroxamide is a potent inhibitor of affinity-purified HDAC1 (ID(50) = 100 nM) and causes the accumulation of acetylated core histones in MEL cells cultured with the agent. Human CWR22 prostate tumor xenografts from mice treated with pyroxamide (100 or 200 mg/kg/day) showed increased levels of histone acetylation and increased expression of the cell cycle regulator p21/WAF1, compared with tumors from vehicle-treated control animals. CONCLUSIONS: The findings suggest that pyroxamide may be a useful agent for the treatment of malignancy and that induction of p21/WAF1 in transformed cells by pyroxamide may contribute to the antitumor effects of this agent.

Apoptosis, proliferation, and p27 expression during vessel wall healing: time course study in a mouse model of transluminal femoral artery injury.

OBJECTIVE: The balance between cell death and proliferation in the vessel wall influences neointimal formation, remodeling, and eventual luminal narrowing after arterial injury. In this study, the time course of apoptosis, proliferation, and expression of p27-a critical regulator of cell-cycle progression-is characterized in a mouse model of transluminal arterial injury associated with substantial neointimal formation. METHODS: C57BL/6 mice underwent bilateral femoral artery injury by passage of an angioplasty guidewire. Expression of p27, Ki67 proliferative index, and apoptosis, as well as histomorphometry, were analyzed in cross sections of uninjured arteries and arteries harvested at different time intervals after injury. RESULTS: In the uninjured arteries, no apoptotic cells were detected, and p27 and Ki67 were expressed in less than 5% of medial cells. After injury, apoptosis increased markedly in medial smooth muscle cells from 1 to 24 hours and decreased gradually thereafter. Ki67 proliferative index increased after 1 week, peaked at 2 weeks in both the media and neointima, and decreased thereafter. p27 expression was undetectable from 1 to 48 hours, and increased gradually from 1 to 4 weeks. Well-developed neointima was present at 2 and 4 weeks. CONCLUSIONS: In vivo injury to the mouse femoral artery evokes a rapid apoptotic response and downregulation of p27, followed by gradual increase in proliferation. During later phases of arterial repair, p27 expression increases while a shift of proliferation rates toward baseline occurs. Future experiments using this model in genetically modified mice may help identify specific cell-cycle regulatory molecules as therapeutic targets for control of pathologic arterial healing.

Expression of c-kit and kit-ligand in benign and malignant prostatic tissues.

The tyrosine kinase receptor c-kit and its ligand [kit ligand (KL) or stem cell factor (SCF)] exert a broad range of biological activities during organogenesis and normal cell development. Recent studies have revealed that altered c-kit levels occur in a variety of malignancies and cancer cell lines. KL has also been shown to stimulate the growth of malignant cells, as well as to promote chemotaxis. We had previously reported expression of KL in stroma cells of normal human prostate. The present study was undertaken in order to analyze the patterns of expression of c-kit and KL in a well characterized set of prostatic tissues, including normal prostate (n=4), benign prostatic hyperplasia (BPH) (n=53) and adenocarcinoma (n=46) samples. The distribution of c-kit and KL proteins was studied by immunohistochemical analyses, while transcript levels were determined by in situ hybridization with specific RNA probes on a subset of the benign and malignant tissues referred above. In addition, reverse-transcriptase polymerase chain reaction (RT-PCR) was performed to determine levels of c-kit and KL expression in cultures of epithelial and stroma cells, as well as in the prostate cancer cell lines LNCaP, DU145 and PC3. c-kit protein in normal prostate was exclusively detected in mast cells by immunohistochemistry and in situ hybridization. However, c-kit transcripts, but not c-kit protein, were detected in low levels and with an heterogeneous pattern in basal epithelial cells of ducts and acini. c-kit in BPH was detected in epithelial cells in 9 of 53 (17%) specimens. c-kit protein expression in malignant epithelial cells was identified in 1 of 46 (2%) tumors. However, c-kit transcripts were detected in low levels by in situ hybridization in most of the tumors analyzed. KL protein and transcripts in normal prostate were detected in high levels in stroma cells. However, epithelial cells were unreactive for anti-KL antibody, but showed low levels of KL transcripts mainly in cells of the basal layer. Basal epithelial cells in hyperplastic glands showed KL expression in 13 of 53 (24%) specimens. KL protein in tumor cells was noted in 18 of 46 (39%) cases. c-kit transcripts were not found in normal prostate and in the 3 cancer cell lines analyzed by RT-PCR, however, it was present in cultured epithelial cells of BPH, and in cultures of stroma cells from both normal and BPH. The majority of cultured cell lines of epithelial and stromal origin displayed considerable levels of KL. In addition all prostate cell lines studied showed significant levels of KL transcripts. In summary, co-expression of c-kit and KL in a subset of BPH cases may suggest an autocrine mode of signaling. Data from this study reveals that altered patterns of c-kit and KL expression are associated with BPH and adenocarcinoma of prostate. It appears that KL induces mast cells proliferation and maturation and enhances their release of protease. This could explain the accumulation of mast cells at tumor sites, a phenomenon that was not observed in normal prostate or BPH samples.

Overexpression of cyclin D1 is associated with metastatic prostate cancer to bone.

Cyclin D1 is a key regulator of the G1 phase progression of the cell cycle. There is increasing evidence that deregulated cyclin D1 expression is implicated in tumorigenesis and tumor progression in certain neoplasms. Recently, it has been reported that cyclin D1 overexpression might be related to the evolution of androgen-independent disease in prostate cancer. This study was conducted to investigate patterns of cyclin D1 expression in prostate cancer samples representing different points in the natural history and treatment evolution of the disease. Association with clinical outcomes was also explored. Using immunohistochemistry, 86 radical prostatectomy specimens (53 naive and 33 after androgen deprivation) and 22 androgen-independent bone metastases were studied. We examined the difference in cyclin D1 expression in primary versus metastatic cases. In addition, we examined the association in primary cases between cyclin D1 expression and clinicopathological parameters of poor clinical outcome, including time to prostate-specific antigen relapse and Ki67 proliferative index. Cyclin D1-positive phenotype, defined as identification of positive immunoreactivity in the nuclei of > or =20% of tumor cells, was observed in 10 of 86 (11%) primary cases compared with 15 of 22 (68%) androgen-independent bone metastases (P = 0.001). There was no correlation between cyclin D1 overexpression and either Gleason score, neo-adjuvant hormone treatment, or prostate-specific antigen relapse We observed a statistical association between cyclin D1 overexpression and high Ki67 proliferative index, defined as > or =20% of positive tumor cells (P = 0.02). These data support the hypothesis that cyclin D1 overexpression may represent an oncogenic event in androgen-independent metastatic prostate cancer to the bone.

Modulation of apoptosis, proliferation, and p27 expression in a porcine coronary angioplasty model.

Smooth muscle cell (SMC) proliferation is a prominent feature of intimal hyperplasia after percutaneous coronary interventions. p27 is a critical regulator of cell proliferation. Our aims were to analyze the time course of p27 expression, Ki67 proliferative index, and apoptosis after angioplasty in the porcine coronary artery. We also investigated the effects of rapamycin--an antiproliferative drug--on these events. The expression of p27 and Ki67, and apoptosis were determined in porcine coronary arteries harvested at timed intervals from 1 h to 28 days after angioplasty. A gradual increase in p27 expression was observed from 7 to 28 days. Ki67 expression peaked by 7-14 days after angioplasty. By 21-28 days, Ki67 expression decreased, while p27 reached maximal levels. An early apoptotic response was found by 6 h, followed by a gradual return to baseline. Rapamycin induced a reduction in Ki67 proliferative index (2 +/- 0.5%) and an increase in apoptosis (7 +/- 1%) versus untreated animals at the 28-day time point (5 +/- 1 and 1 +/- 0.5%, respectively; P < 0.05). In summary, coronary angioplasty induced a rapid apoptotic response, followed by a progressive increase in proliferation. Later on, as p27 expression increased in the vessel wall, cell proliferation decreased. Modulation of cell cycle progression may be a useful therapeutic approach in the treatment of intimal hyperplasia after angioplasty.

Prostate cancer cell cycle regulators: response to androgen withdrawal and development of androgen independence.

BACKGROUND: Androgen withdrawal is a standard therapy for prostate cancer that results in a decrease in tumor volume and a decline in serum prostate-specific antigen in the majority of patients. To understand the factors associated with regression of prostate cancers after androgen withdrawal, we studied cell cycle regulator changes in the CWR22 human prostate cancer xenograft model. METHODS: Established tumors in nude athymic BALB/c mice were sampled at various times after androgen withdrawal and after the development of androgen independence. Changes in the expression of cell cycle regulators were categorized into early and mid-to-late events. RESULTS AND CONCLUSIONS: Early events included a decrease in androgen receptor expression, followed by a short-term increase in expression of the p53 and p21/WAF1 proteins and a marked decrease in the Ki67 proliferative index. Mid-to-late events included progressive and sustained increases in p27 and p16 protein expression, a decrease in retinoblastoma protein expression, and an increase in the transcription factor E2F1. Changes in apoptosis (programmed cell death) were not observed at any time after androgen withdrawal. These data suggest that androgen withdrawal results in a cell stress response, in which increased p53 protein produces a cell cycle arrest, without activation of p53-mediated apoptosis. The proliferative index is further decreased through the action of the cyclin-dependent kinase inhibitors p27 and p16. Androgen-independent sublines emerged 80-400 days after androgen withdrawal, and these sublines had variable growth phenotypes but were associated with mdm2 protein overexpression and increased expression of cyclin D1. These results indicate that tumor regression in this human prostate cancer model is due to cell cycle arrest rather than to apoptosis and that the emergence of androgen independence is associated with a release from cell cycle arrest.

Inactivation of the p53 pathway in prostate cancer: impact on tumor progression.

To determine the potential role of p53 inactivation in prostate cancer, we studied a well characterized cohort of 86 patients treated with radical prostatectomy. We analyzed patterns of p53, mdm2, and p21/WAF1 expression by immunohistochemistry. Results were then correlated with clinicopathological parameters of poor outcome, including time to PSA relapse. In addition, data were also correlated with proliferative index, as assessed by Ki67 antigen detection. p53-positive phenotype, defined as identification of nuclear immunoreactivity in > 20% of tumor cells, was observed in 6 of 86 cases (7%). An association was observed between p53-positive phenotype and decreased time to PSA relapse (P < 0.01). mdm2-positive phenotype, defined as > or = 20% of tumor cells displaying nuclear immunoreactivity, was observed in 28 of 86 cases (32.5%). mdm-2-positive phenotype was found to be associated with advanced stage (P = 0.009). p21-positive phenotype, defined as > 5% of tumor cells with nuclear immunoreactivity, was observed in 28 of 86 cases (32.5%). An association was observed between p21-positive phenotype and high Ki67 proliferative index (P = 0.002). Patients with p21-positive phenotype had a significant association with decreased time to PSA relapse (P = 0.0165). In addition, a significant association was found between p21-positive phenotype and coexpression of mdm2 (P < 0.01). Forty-three of 86 cases (50%) were found to have one or more alterations, and patients with any alteration were found to have a higher rate of PSA relapse (P < 0.01). It is our hypothesis that a pathway of prostate cancer progression involves p53 inactivation caused by mdm2 overexpression and that p21 transactivation in this setting is due to an alternative signaling system, rather than through a p53-dependent mechanism.

Prognostic significance of transcription factor E2F-1 in bladder cancer: genotypic and phenotypic characterization.

BACKGROUND: We sought to identify and characterize potential alterations in E2F-1, a transcription factor that binds to the retinoblastoma protein (pRB), in bladder neoplasms and to elucidate a possible role for E2F-1 as an oncogene or a tumor suppressor gene. METHODS: Tumor samples from 133 evaluable patients with bladder cancer were analyzed for E2F-1 gene mutations by use of polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. In addition, tumors were studied for E2F-1 and pRB protein expression by use of immunohistochemistry. Results from the above analyses were correlated with clinicopathologic parameters and outcome. All P values are two-sided. RESULTS: A polymorphism, consisting of a nucleotide change at amino acid codon 393 in exon 7 (GGC-->AGC [Gly-->Ser]), was identified in seven of 133 case patients, being present in both tumor and corresponding normal tissues. No bandshifts were identified in the nuclear-localization or DNA-binding domains on PCR-SSCP analysis. On immunohistochemical analysis, E2F-1 nuclear reactivity was observed in less than 5% of the cells from 53 tumors and in 5%-75% of the cells from the remaining 80 tumors. The pattern of E2F-1 protein expression was not altered in relation to the identified polymorphism. pRB nuclear reactivity greater than 20% (of tumor cells stained) was present in 66% of the samples. E2F-1 nuclear reactivity correlated inversely with the percentage of cells showing pRB reactivity (Kendall tau(b) = -0.18; P = .019). On multivariate analysis, patients with lower E2F-1 reactivity had statistically significantly increased risks of progression to metastases (P = .001) and death (P = .02). CONCLUSIONS: E2F-1 alterations occur at the phenotypic level, rather than at the genotypic level, in bladder cancer. The adverse outcome for patients whose tumors exhibit low E2F-1 nuclear expression suggests a possible tumor suppressor role for E2F-1 in bladder cancer.

Alterations of INK4A and INK4B genes in adult soft tissue sarcomas: effect on survival.

BACKGROUND: The INK4A and INK4B genes map to chromosome 9p21, with the INK4A gene encoding two protein products, p16 and pl9ARF. Alterations of the INK4A and INK4B genes occur frequently in certain primary malignant neoplasms. This study was undertaken to evaluate the frequency of INK4A and INK4B gene alterations in a cohort of adult soft tissue sarcomas. METHODS: The status of the INK4A and INK4B genes was determined in 46 soft tissue sarcomas by use of the following methods: Southern blotting, polymerase chain reaction (PCR), single-strand conformation polymorphism analysis, comparative multiplex PCR, and a methylation assay focusing on the p16 promoter. Associations between alterations of the INK4A and INK4B genes and clinicopathologic variables, as well as with p53 and pRB (retinoblastoma protein) status, were evaluated by use of the two-tailed Fisher's exact test. Disease-specific survival was evaluated by use of the Kaplan-Meier method and the logrank test. Proportional hazards analysis was used to obtain estimates of relative risks. All P values are two-sided. RESULTS: Homozygous and hemizygous deletions, but no point mutations, were observed in these two genes. The overall frequency of gene alteration (deletion or rearrangement) was approximately 15% for the INK4A and INK4B genes, with changes restricted to high-grade sarcomas. Statistically significant associations were observed between INK4A/INK4B deletions (P = .036) or alterations (P = .005) and poor survival. Alteration of the INK4A and INK4B genes was the only statistically significant predictor for poor survival when controlling for tumor grade and size (P = .03). CONCLUSION/IMPLICATIONS: Coincident homozygous deletion of the INK4A and INK4B genes occurs frequently in adult soft tissue sarcomas. Loss of p16 and pl9ARF function in primary tumors, although not equivalent to alterations in p53 and pRB function, appears to be associated with cancers that have an aggressive biologic behavior.

Distinct altered patterns of p27KIP1 gene expression in benign prostatic hyperplasia and prostatic carcinoma.

BACKGROUND: The p27KIP1 gene, whose protein product is a negative regulator of the cell cycle, is a potential tumor suppressor gene; however, no tumor-specific mutations of this gene have been found in humans. This study was undertaken to identify and to assess potential alterations of p27KIP1 gene expression in patients with benign prostatic hyperplasia (BPH) and patients with prostate cancer. METHODS: We analyzed 130 prostate carcinomas from primary and metastatic sites, as well as prostate samples from normal subjects and from patients with BPH. Immunohistochemistry and in situ hybridization were used to determine the levels of expression and the microanatomical localization of p27 protein and messenger RNA (mRNA), respectively. Immunoblotting and immunodepletion assays were performed on a subset of the prostate tumors. Associations between alterations in p27KIP1 expression and clinicopathologic variables were evaluated with a nonparametric test. The Kaplan-Meier method and the logrank test were used to compare disease-relapse-free survival. Prostate tissues of p27Kip1 null (i.e., knock-out) and wild-type mice were also evaluated. RESULTS: Normal human prostate tissue exhibited abundant amounts of p27 protein and high levels of p27KIP1 mRNA in both epithelial cells and stromal cells. However, p27 protein and p27KIP1 mRNA were almost undetectable in epithelial cells and stromal cells of BPH lesions. Furthermore, p27Kip1 null mice developed enlarged (hyperplastic) prostate glands. In contrast to BPH, prostate carcinomas were found to contain abundant p27KIP1 mRNA but either high or low to undetectable levels of p27 protein. Primary prostate carcinomas expressing lower levels of p27 protein appeared to be biologically more aggressive (two-sided P = .019 [Cox regression analysis]). CONCLUSIONS/IMPLICATIONS: On the basis of these results, we infer that loss of p27Kip1 expression in the human prostate may be causally linked to BPH and that BPH is not a precursor to prostate cancer.

Cooperative effects of p53 and pRB alterations in primary superficial bladder tumors.

Altered patterns of p53 and pRB expression have been reported to be frequent events and to have prognostic significance in bladder cancer. To assess the potential adverse consequences of having altered patterns of both p53 and pRB proteins in patients with bladder neoplasms compared with having one or neither abnormality, we have studied a cohort of superficial transitional cell carcinomas of the urinary bladder by immunohistochemical analysis. The present study included 59 well-characterized superficial transitional cell carcinomas (Ta, n = 28; T1, n = 31) for which clinicopathological variables were available. Nuclear overexpression of p53 was identified in 22 cases (37%). A statistically significant association was observed between the p53-positive phenotype and disease progression (P < 0.001), as well as reduced survival (P < 0.001). Undetectable levels of pRB were observed in 11 cases (19%). Patients with a pRB-negative phenotype had a more frequent disease progression (P = 0.014) and decreased overall survival (P = 0.014). We also observed a significant association between altered p53 and undetectable pRB expression patterns (P = 0.001). Nine tumors showed both a p53-positive and a pRB-negative phenotype. There was an even more marked increase in progression (P = 0.00005) and decreased overall survival (P = 0.0004) in patients whose tumors had both alterations after controlling for tumor stage, tumor grade, and suspicion of vascular invasion. These data suggest that alterations of p53 and pRB have a cooperative negative effect on both progression and survival in primary bladder cancer. It may be postulated that aberrant p53 and pRB expression deregulates cell cycle control at the G1 checkpoint and engenders tumor cells with reduced response to programmed cell death. The imbalance produced by an enhanced proliferative activity and a decreased apoptotic rate may determine the aggressive clinical course of the bladder tumors harboring both p53 and pRB alterations.

Altered patterns of retinoblastoma gene product expression in adult soft-tissue sarcomas.

Altered expression of the retinoblastoma (RB) tumour-suppressor gene product (pRB) has been detected in sporadic bone and soft-tissue sarcomas. Earlier studies, analysing small cohorts of sarcoma patients, have suggested that these alterations are more commonly associated with high-grade tumours, metastatic lesions and poorer survival. This study was designed to re-examine the prevalence and clinical significance of altered pRB expression in a large and selected group of soft-tissue sarcomas from 174 adult patients. Representative tissue sections from these sarcomas were analysed by immunohistochemistry using a well-characterised anti-pRB monoclonal antibody. Tumours were considered to have a positive pRB phenotype only when pure nuclear staining was demonstrated, and cases were segregated into one of three groups. Group 1 (n = 36) were patients whose tumours have minimal or undetectable pRB nuclear staining (< 20% of tumour cells) and were considered pRB negative. Patients with tumours staining in a heterogeneous pattern (20-79% of tumour cells) were classified as group 2 (n = 99). The staining of group 3 (n = 39) was strongly positive with a homogeneous pRB nuclear immunoreactivity (80-100% of tumour cells). pRB alterations were frequently observed in both low- and high-grade lesions. Altered pRB expression did not correlate with known predictors of survival and was not itself an independent predictor of outcome in the long-term follow-up. These findings support earlier observations that alterations of pRB expression are common events in soft-tissue sarcomas; nevertheless, long-term follow-up results indicate that altered patterns of pRB expression do not influence clinical outcome of patients affected with soft-tissue sarcomas. It is postulated that RB alterations are primary events in human sarcomas and may be involved in tumorigenesis or early phases of tumour progression in these neoplasias.

Gene transfer of wild-type p53 results in restoration of tumor-suppressor function in a medulloblastoma cell line.

The replacement of functional genes into cells that lack genes or have mutant genes is the basis of gene therapy. In cancer, where cells often have multiple genetic defects, the replacement of critical genes may suffice to suppress cell growth or induce cell death. The high frequency of mutations of the p53 tumor-suppressor gene in human cancers, including primary brain tumors, suggests that p53 plays a critical role in carcinogenesis and tumor progression. We report the successful transfer of the wild-type p53 gene using a defective herpes simplex viral vector into a human medulloblastoma cell line containing a mutant copy of p53. Upon gene transfer, we detected novel expression of wild-type p53 protein in the cells. In addition, the p53 protein was functionally active, since gene transfer resulted in increased levels of mdm2 proteins and induced cell cycle arrest of the majority of transduced cells. To our knowledge, this is the first report of the use of this vector system to carry wild-type p53. We conclude that defective herpes simplex viral vectors can transfer and express p53 in human primary brain tumor cells in vitro, restoring wild-type p53 tumor-suppressor functions.

Expression of c-kit and kit ligand proteins in normal human tissues.

The c-kit receptor and its cognate ligand, KL, play a critical role in melanogenesis, gametogenesis, and hematopoiesis. Studies on the expression of c-kit and KL have been primarily focused on mouse development. We undertook the present study to characterize the pattern of expression of these molecules in normal adult human tissues. Using immunohistochemistry and consecutive tissue sections from the same block, we evaluated a variety of well-preserved normal tissues for c-kit and KL microanatomic distribution. c-kit protein was identified in tissue mast cells, melanocytes, glandular epithelial cells of breast, parotid, dermal sweat, and esophageal glands. Scattered c-kit immunoreactivity was also observed for testicular and ovarian interstitial cells. A striking regional distribution of c-kit was detected in the central nervous system, particularly in the cerebellum, hippocampus, and dorsal horn of the spinal cord. KL protein was identified in cells complementary to staining for the receptor, such as glandular myoepithelium of breast and sweat glands. Intense KL immunoreactivity was observed in smooth muscle cells of the bladder, cervix, uterus, and gastrointestinal tract, as well as in striated and cardiac muscle. Strong KL staining was also detected in prostate fibromuscular stroma cells. In the central nervous system, KL expression was confined to Golgi and Purkinje cells in the cerebellum. These results suggest a role for this receptor and its ligand in the maintenance of a variety of fully differentiated tissues.

Chromosome 17 abnormalities and TP53 mutations in adult soft tissue sarcomas.

This study was designed to determine the frequency of structural genetic abnormalities of chromosome 17 and the incidence of TP53 mutations as they relate to the biological behavior of adult soft tissue sarcomas. We analyzed a group of 73 soft tissue sarcomas of adults that were clinically and pathologically well characterized using molecular genetic techniques and expression studies. We then correlated genotype and phenotype with pathological parameters. Overall, allelic loss of 17p and 17q was identified in 53 and 29% of informative cases, respectively. p53 nuclear overexpression was detected in 34% of the tumors analyzed. We observed an association between 17p deletions and tumor presentation being more frequent in recurrent and metastatic tumors than primary lesion. p53 nuclear overexpression was associated with tumor grade, size, and more frequently detected in metastatic than primary sarcomas. The 11 intragenic mutations characterized included 10 cases of single base substitution and one single base deletion; 8 were of the missense type and 3 were nonsense. It is concluded that 17p deletions and TP53 mutations are common events in adult soft tissue sarcomas and that due to the trends observed with the cohort of patients analyzed they may become prognostic markers for patients affected with these tumors.

Prognostic implications of p53 nuclear overexpression and high proliferation index of Ki-67 in adult soft-tissue sarcomas.

BACKGROUND: Morphologically similar soft-tissue sarcomas may behave in very different fashions, making it difficult to predict clinical outcomes and to properly design therapeutic interventions. In a preliminary study, we observed that TP53 mutations and nuclear overexpression of p53 protein were frequent events in soft-tissue sarcoma, and we noticed an association between p53-positive phenotype and poor clinical outcome. PURPOSE: We examined the potential clinical relevance of p53 overexpression in adults with soft-tissue sarcomas. We also studied the clinical implications of a high proliferation index. METHODS: A cohort of 174 adults with soft-tissue sarcomas were analyzed using anti-p53 and anti-Ki-67 antibodies and immunohistochemical assays on consecutive fresh frozen tissue samples. RESULTS: We observed a significant association between p53 nuclear overexpression and tumor grade (P = .001) and tumor size (P = .01). Patients displaying a p53-positive phenotype had significantly reduced survival (P = .02). Similarly, a significant difference was observed between high proliferation index and tumor grade (P < .001) and reduced patient survival (P = .03). A high Ki-67 proliferation index was detected in association with p53 nuclear overexpression. CONCLUSIONS: Overexpression of p53 protein and a high proliferation index strongly correlate with poor clinical outcome and reduced survival in patients having soft-tissue sarcomas.

Molecular abnormalities of mdm2 and p53 genes in adult soft tissue sarcomas.

Genetic alterations in the p53 and mdm2 genes have been reported to occur in soft tissue sarcomas. This study was designed to determine the prevalence and potential clinical value of detected molecular abnormalities and altered patterns of expression of mdm2 and p53 genes in adult soft tissue sarcomas. A cohort of 211 soft tissue sarcomas from adults that were both clinically and pathologically well characterized was analyzed. Monoclonal antibodies directed against mdm2 and p53 proteins were used to measure overexpression of these proteins in the nuclei of cells from sections of these tumors. Seventy-six of 207 tumors had abnormally high levels of mdm2 proteins and 56 of 211 tumors overexpressed p53 protein. Twenty-two cases had abnormally high levels of both mdm2 and p53 proteins based upon immunoreactivity with these antibodies. There was a striking statistically significant correlation between the overexpression of p53 and mdm2 proteins in the same tumor and poor survival (P < 0.05) of the patients. A group of 73 soft tissue sarcomas was chosen for analysis using Southern blots, single strand conformation polymorphisms, and direct DNA sequencing to confirm mdm2 gene amplifications and p53 mutations and correlate these with the results of the immunoreactivities. The overexpression of p53 and mdm2 proteins in the nuclei of tumor cells did not always correlate well with gene amplification at the mdm2 locus or mutation at the p53 gene. The possible reasons for these discrepancies are discussed.

P53 and rb alterations in primary breast-carcinoma - correlation with hormone receptor expression and lymph-node metastases.

Expression of Rb and mutant p53 nuclear phosphoproteins was analyzed immunohistochemically in 69 breast cancer patients. Results were correlated with hormone receptor ( ER and PR ) and lymph node ( LN ) status. There was a significant association between the immunohistochemical evidence of p53 and/or Rb alterations and loss of hormone receptor expression. Mutations in p53 and/or low level Rb expression were not associated with the presence of axillary lymph node metastases. However, in patients with hormone receptor positive tumors, there was statistically significant correlation between altered expression of p53 and/or Rb and the presence of LN metastases. These results indicate that: (i) loss of steroid hormone receptor expression (and thus loss of hormonal growth control) is accompanied by somatic inactivation of the p53 or Rb tumor suppressor genes; and (ii) in tumors that remain under hormonal growth regulation inactivation of p53 and/or Rb may play a role in tumor progression.

Expression of HLA-A,B,C antigens on primary and metastatic tumor cell populations of human carcinomas.

The expression of monomorphic determinants of the histocompatibility leukocyte antigens (HLA) class I antigens by human malignant tumor cells was studied in tissue specimens of 70 primary tumor lesions obtained from patients with carcinoma of the breast (41 patients), colon (8 patients), urinary bladder (8 patients), and kidney (13 patients), and in samples of either synchronous or metachronous lymph node, lung, or liver metastases available in 44 of the patients. The frequencies of HLA class I expressor and nonexpressor tumor cells were determined by immunohistochemical staining of histological sections of fresh frozen tissue samples with the W6/32 monoclonal antibody. The tumor cell populations in the majority of the primary lesions consisted predominantly of HLA-immunoreactive cells (observed in 38 of 70 patients; 54%), especially in those patients who did not have clinical evidence of metastatic disease (8 of 11 patients; 73%). Various degrees of loss of reactivity were observed in other primary lesions, although in only 8 (12%) tumors (7 of which were obtained from patients with metastatic disease), the neoplastic cells were nearly exclusively HLA-nonreactive. In contrast, the majority of metastatic lesions consisted of either predominantly HLA-negative cells (33 of 44 specimens; 75%) or mixed populations (10 of 44 specimens; 23%), whereas only one metastatic lesion manifested HLA class I antigen staining in more than 70% of its tumor cells (P = 0.0005). Intravascular clusters of tumor cells consisted predominantly of HLA class I nonexpressors. The observed patterns of distribution of HLA expressors and nonexpressor tumor cells are compatible with the notion that HLA-negative cells in human carcinomas manifest a selective advantage with regard to metastatic progression and growth. The suppressed expression of major histocompatibility complex class I antigens on metastatic cells may lead to failure of presentation of cell surface tumor specific epitopes to host cytotoxic T-lymphocytes. Such a process would enable tumor cells to evade host immune responses and would promote and enhance cell dissemination and metastatic growth.