High Tumor Cell Proliferation Research Articles

Abstract 2124: Comprehensive analysis of hormone receptor status during prostate cancer progression: A novel prognostic role for pAR and ERbeta

Abstract Aims: Patients with advanced prostate cancer (PrCa) are usually treated with androgen withdrawal. Although this therapy is effective at the beginning, nearly all prostate cancers become refractory to it. Hormone receptors play a crucial role during this progression. Aim of this study was to analyze the genomic and expression status of hormone receptors in prostate cancer and the evolution of these markers in the development of castration-resistance. Methods: We selected 915 transurethral resection (TURP) specimens from 107 untreated PrCa and from 101 castration-resistant (CR) PrCa. These samples were used for the construction of a tissue microarray (TMA). In addition, we included 56 distant metastases. We analyzed the androgen receptor (AR) gene copy number by fluorescence in-situ hybridization and the expression profiles of AR, phosphorylated AR (pAR), ERβ, ERα and the proliferation marker Ki67 by immunohistochemistry. Results: AR gene amplification was almost restricted to PrCa and was significantly associated with increased AR protein expression (p<0.0001) and higher tumor cell proliferation (p=0.001). Interestingly, although pAR was predominantly found in the CR PrCa (p=0.003), pAR expression in untreated PrCa patients identified a subgroup of patients with poor survival (p<0.05). In contrast to ERα expression, which was restricted to CR PrCA cells (10%), ERβ was found in 40% of the PrCa, independent of treatment status. Similar to pAR, the presence of ERβ in untreated patients was significantly associated with adverse prognosis (p<0.005). Conclusions: This is the first comprehensive study comparing the hormonal receptor status in a large cohort of hormone-naïve and CR PrCa. Our results strongly suggest a major role for pAR and ERβ in untreated PrCa. Expression of these markers might be a mechanism of CR tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2124. doi:10.1158/1538-7445.AM2011-2124

A Gene Expression Signature from Human Breast Cancer Cells with Acquired Hormone Independence Identifies MYC as a Mediator of Antiestrogen Resistance

Although most patients with estrogen receptor α (ER)-positive breast cancer initially respond to endocrine therapy, many ultimately develop resistance to antiestrogens. However, mechanisms of antiestrogen resistance and biomarkers predictive of such resistance are underdeveloped. We adapted four ER(+) human breast cancer cell lines to grow in an estrogen-depleted medium. A gene signature of estrogen independence was developed by comparing expression profiles of long-term estrogen-deprived (LTED) cells to their parental counterparts. We evaluated the ability of the LTED signature to predict tumor response to neoadjuvant therapy with an aromatase inhibitor and disease outcome following adjuvant tamoxifen. We utilized Gene Set Analysis (GSA) of LTED cell gene expression profiles and a loss-of-function approach to identify pathways causally associated with resistance to endocrine therapy. The LTED gene expression signature was predictive of high tumor cell proliferation following neoadjuvant therapy with anastrozole and letrozole, each in different patient cohorts. This signature was also predictive of poor recurrence-free survival in two studies of patients treated with adjuvant tamoxifen. Bioinformatic interrogation of expression profiles in LTED cells revealed a signature of MYC activation. The MYC activation signature and high MYC protein levels were both predictive of poor outcome following tamoxifen therapy. Finally, knockdown of MYC inhibited LTED cell growth. A gene expression signature derived from ER(+) breast cancer cells with acquired hormone independence predicted tumor response to aromatase inhibitors and associated with clinical markers of resistance to tamoxifen. Activation of the MYC pathway was associated with this resistance.

Amplification and overexpression of vinculin are associated with increased tumour cell proliferation and progression in advanced prostate cancer

Androgen withdrawal is the standard treatment for advanced prostate cancer. Although this therapy is initially effective, nearly all prostate cancers become refractory to it. Approximately 15% of these castration-resistant prostate cancers harbour a genomic amplification at 10q22. The aim of this study was to explore the structure of the 10q22 amplicon and to determine the major driving genes. Application of high-resolution array-CGH using the 244k Agilent microarrays to cell lines with 10q22 amplification allowed us to narrow down the common amplified region to a region of 5.8 megabases. We silenced each of the genes of this region by an RNAi screen in the prostate cancer cell lines PC-3 and 22Rv1. We selected genes with a significant growth reduction in the 10q22 amplified cell line PC-3, but not in the non-amplified 22Rv1 cells, as putative target genes of this amplicon. Immunohistochemical analysis of the protein expression of these candidate genes on a tissue microarray enriched for 10q22 amplified prostate cancers revealed vinculin as the most promising target of this amplicon. We found a strong association between vinculin gene amplification and overexpression (p < 0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that vinculin expression was highest in castration-resistant prostate cancers, but negative or very low in benign prostatic hyperplasia (p < 0.0001). Additionally, high tumour cell proliferation measured by Ki67 expression was significantly associated with high vinculin expression in prostate cancer (p < 0.0001). Our data suggest that vinculin is a major driving gene of the 10q22 amplification in prostate cancer and that vinculin overexpression might contribute to prostate cancer progression by enhancing tumour cell proliferation.

Abstract P2-09-29: Cyclin-Dependent Kinase-Based Risk Score Predicts Both Clinical and Pathological Response to Neoadjuvant Paclitaxel Followed by FEC in Early Breast Cancers

Abstract Background: We have reported that breast tumors with a high ratio of cyclin-dependent kinase (CDK) 2 to CDK 1 are associated with high tumor cell proliferation and poor clinical outcome in Japanese. In addition, we have developed risk score based on CDK2/1 ratio (CDK-RS), and have demonstrated a high risk group showed a significantly poor prognosis in Hollanders (van Nes, et al: Br J C 2009: 100: 494). The aim of the present study is to evaluate the correlation of CDK-RS with response to neoadjuvant paclitaxel followed by fluorouracil + epirubicin + cyclophosphamide (FEC) in breast cancers. Material and Methods: Of 131 primary breast cancer patients (age: 25-73y, mean: 51.7y), 126 were treated with paclitaxel (80 mg/m2, weekly) for 12 cycles followed by FEC (500/75/500 mg/m2, q3w) for four cycles and 5 were treated with paclitaxel-monotherapy (5 — 29 cycles) in the NAC or primary chemotherapy setting. Frozen tumor tissues were obtained from core needle biopsy before NAC, and CDK-RS was determined by the Cell Cycle Profiling (C2P) assay as previously reported. Clinical response was evaluated with MRI before NAC and after paclitaxel and FEC. Patients were classified into responders showing ≥80 % in reduction rates and non-responders showing &amp;lt; 80 %. Pathological CR (pCR) was defined as no residual invasive foci and no axillary lymph node metastasis. Results: Patients characteristics were as follows: menopausal status: pre-47%, post-53%; Stage: II 68%, III 27%, IV 7%; Tumor size (cm): ≥5 cm 73%, &amp;gt; 5 cm 27%; histologic grade (HG): I 16%, II 60%, III 24%; ER: (+) 56%, (-) 44%; PR: (+) 39%, (-) 61%; HER2: (+) 28%, (-) 72%. Of 131 patients, 22 (18%) attained pCR but 100 (82%) did not pCR (9 were excluded from evaluation of pathologic response because of stage IV diseases, no operations, and not available pathologic examination). In CDK-RS, 47% of 131 tumors were classified into high, 17% intermediate, and 37% low. In combination with high + intermediate risk score groups, that group had a tendency to show high HG (grade 2 or 3) (87% vs 78%), ER negativity (49% vs 35%), and PR negativity (65% vs 54%) compared with a low risk group, but their differences were not statistically significant. Tumors in the high + intermediate group were significantly more likely to show clinical response after the completion of not only paclitaxel (52% vs 27%, p=0.006) but also FEC (75% vs 52%, p=0.011) as well as to show pCR (24% vs 9%, p=0.037) compared with a low risk score group. In univariate analysis, CDK-RS showed a significant correlation with pCR (high + intermediate vs low, Odds ratio 4.03, 95%CI 1.03 — 10.3, p=0.045). Discussion: CDK-RS in tumor samples before NAC is significantly associated with clinical and pathological response to paclitaxel followed by FEC. Thus CDK-RS seems to be a novel and useful predictive factor for paclitaxel — FEC in breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-29.

Bioactivity‐guided isolation of antiproliferative compounds from Centaurea arenaria

The antiproliferative effects of n-hexane, chloroform and aqueous methanol extracts prepared from the whole plant of Centaurea arenaria M.B. ex Willd. were investigated against cervix adenocarcinoma (HeLa), breast adenocarcinoma (MCF7) and skin epidermoid carcinoma (A431) cells, using the MTT assay. The chloroform extract displayed high tumour cell proliferation inhibitory activity (higher than 85% at 10 μg/mL concentration), and was therefore subjected to a bioassay-guided multistep separation procedure. Flavonoids (eupatilin, eupatorin, 3'-methyleupatorin, apigenin and isokaempferid), lignans (arctigenin, arctiin and matairesinol), the sesquiterpene cnicin, serotonin conjugates (moschamine and cis-moschamine), β-amyrin and β-sitosterin-β-D-glycopyranoside, identified by means of UV, MS and NMR spectroscopy, were obtained for the first time from this species. The isolated compounds were also evaluated for their tumour cell growth inhibitory activities on HeLa, MCF7 and A431 cells, and different types of secondary metabolites were found to be responsible for the antitumour effects of the extracts; in addition to moderately active compounds (isokaempferid and moschamine), especially apigenin, eupatorin, arctigenin, arctiin, matairesinol and cnicin exert marked antitumour effects against these cell lines.

Abstract 250: Vinculin is a putative amplification target gene at 10q22 in advanced prostate cancer

Abstract Patients with advanced prostate cancer are usually treated with androgen withdrawal. Although this therapy is effective at the beginning, nearly all prostate cancers become refractory to it. Approximately 15% of these hormone-refractory prostate cancers contain a genomic amplification at 10q22. Aim of this study was to explore the structure of the 10q22 amplicon and to determine the major driving gene. We applied high-resolution array-CGH using the 244k Agilent microarrays to cell lines harboring 10q22 amplification. We identified a common amplified region (CAR) and silenced each of the 26 genes in this region by an RNAi screen in the prostate cancer cell lines PC-3 and 22rv1. Genes with a significant growth reduction in the 10q22 amplified cell line PC-3 but not in the non-amplified 22rv1 cells were selected as putative candidate genes of this amplicon. They were further investigated in vivo by functional assays and in vitro by immunohistochemical analysis of the protein expression in more than 500 human prostate cancers on a tissue microarray (TMA). We could narrow down the CAR to a region of 5.8 Mb. The siRNA screening experiments revealed Vinculin (VCL) as the most promising candidate gene of this amplicon. Immunohistochemical analysis of the Vinculin protein expression on a TMA enriched for 10q22 amplified prostate cancers showed a strong association between VCL gene amplification and overexpression (p &amp;lt; 0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that Vinculin expression was highest in hormone-refractory prostate cancers, but negative or very low in benign prostatic hyperplasia (p &amp;lt; 0.0001). Notably, high tumor cell proliferation measured by Ki67 expression was significantly associated with high Vinculin expression in prostate cancer (p &amp;lt; 0.0001). Although there are countless reports on Vinculin as a cytoskeletal protein, its role in prostate cancer has previously not been investigated. Our data strongly suggest Vinculin as a major driving gene of the 10q22 amplification in prostate cancer and that Vinculin overexpression might contribute to prostate cancer progression by enhancing tumor cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 250.

Antiproliferative effect of flavonoids and sesquiterpenoids from Achillea millefolium s.l. on cultured human tumour cell lines

The antiproliferative activities of n-hexane, chloroform, aqueous-methanol and aqueous extracts of the aerial parts of the Achillea millefolium aggregate on three human tumour cell lines were investigated by means of MTT assays. The chloroform-soluble extract exerted high tumour cell proliferation inhibitory activities on HeLa and MCF-7 cells, and a moderate effect on A431 cells; accordingly, it was subjected to detailed bioactivity-guided fractionation. As a result of the multistep chromatographic purifications (VLC, CPC, PLC, gel filtration), five flavonoids (apigenin, luteolin, centaureidin, casticin and artemetin) and five sesquiterpenoids (paulitin, isopaulitin, psilostachyin C, desacetylmatricarin and sintenin) were isolated and identified by spectroscopic methods. The antiproliferative assay demonstrated that centaureidin is the most effective constituent of the aerial parts of yarrow: high cell growth inhibitory activities were observed especially on HeLa (IC(50) 0.0819 microm) and MCF-7 (IC(50) 0.1250 microm) cells. Casticin and paulitin were also highly effective against all three tumour cell lines (IC(50) 1.286-4.76 microm), while apigenin, luteolin and isopaulitin proved to be moderately active (IC(50) 6.95-32.88 microm). Artemetin, psilostachyin C, desacetylmatricarin and sintenin did not display antiproliferative effects against these cell lines. This is the first report on the occurrence of seco-pseudoguaianolides (paulitin, isopaulitin and psilostachyin C) in the Achillea genus.

HER-2/neu expression is associated with high tumor cell proliferation and aggressive phenotype in a population based patient series of endometrial carcinomas

Gene alterations and overexpression of various oncogenes and cell-cycle regulators are important in tumor development. In a population based series of 316 endometrial carcinomas with long and complete follow-up we investigated the distribution of HER-2/neu and EGFR expression and copy number alteration in endometrial cancers. HER-2/ neu, EGFR and Ki-67 expression in curettage and hysterectomy specimens were studied immunohistochemically for expression in relation to molecular markers and clinical phenotype. Fresh tumor samples (n=76) were studied by global characterization of genetic alterations by single nucleotide polymorphism (SNP) array for detection of high level amplification for HER-2/neu and EGFR. Pathological expression of HER-2/neu in curettage was detected in 23% which significantly correlated to high FIGO stage, non-endometrioid subtype, high grade and aneuploidy. In hysterectomy specimens, pathological HER-2/neu staining was seen in 13% which correlated significantly with high FIGO stage, non-endometrioid subtype, high proliferation and poor survival (p=0.009). Expression of EGFR was examined with three different antibodies, but none showed significant correlation with molecular markers or clinical phenotype. High level amplification of HER-2/neu or EGFR was seen in only one out of 76 samples, respectively. High proliferation estimated in tumors from hysterectomy specimens showed independent prognostic impact and was superior to estimation in curettage specimens as a prognostic marker. In conclusion, high level amplification of HER-2/neu or EGFR is infrequent in endometrial cancer. Pathological HER-2/neu staining identifies endometrial carcinomas with an aggressive phenotype, high proliferation and patients with poor survival in a population based setting. These results motivate further clinical trials with trastuzumab based on HER-2/neu status in endometrial carcinomas.

Clarifying the Role of Rasburicase in Tumor Lysis Syndrome

Tumor lysis syndrome (TLS) is a complication of malignancies with high tumor cell proliferation, tumor burden, and chemosensitivity. It manifests with the release of intracellular components and results in hyperkalemia, hyperphosphatemia, hypocalcemia, and hyperuricemia. These biochemical abnormalities may lead to serious complications such as renal failure, cardiac dysrhythmias, and death. Rasburicase, a recombinant urate oxidase enzyme, is a new agent indicated in the treatment or prophylaxis of hyperuricemia in pediatric patients with cancer who are at high risk for TLS. We reviewed the evidence for treatment with this agent compared with standard therapy with allopurinol. Rasburicase may be considered for use in patients with hyperuricemia at presentation and in patients at high risk for TLS that would otherwise result in a delay in chemotherapy. However, randomized controlled trials are required to establish the comparative efficacy of rasburicase in the adult population. Preliminary evidence suggests that single-dose or reduced-dose rasburicase may be effective in the prophylaxis and the treatment of hyperuricemia and TLS. However, to our knowledge, there is no evidence comparing clinically relevant outcomes such as acute renal failure or dialysis.

Pathologic expression of p53 or p16 in preoperative curettage specimens identifies high-risk endometrial carcinomas

The purpose of this study was to investigate the prognostic impact of p53 and p16 expression in curettage material from patients with endometrial carcinoma. Preoperative curettage material from a population-based series of 236 endometrial carcinomas from Norway with long and complete follow-up was studied immunohistochemically for p53 and p16 expression. Pathologic expression of p53 and p16 was seen in 24% and 25%, respectively, and was significantly correlated with high International Federation of Gynecology and Obstetrics (FIGO) stage and serous/clear cell histologic subtypes. Pathologic p53 expression showed significant correlation with postmenopausal status, high grade, high tumor cell proliferation, and aneuploidy. Patients with normal expression had 85% 5-year survival compared with 51% and 50% when pathologic expression of p53 and p16, respectively. Five-year survival for patients with 2 pathologic markers was 13%, compared with 67% and 91% for 1 or no pathologic markers, respectively. Pathologic expression of p53 and p16 in curettage material identifies high-risk endometrial carcinoma patients with poor prognosis.

Prognostic and predictive value of changes in tumour cell proliferation in locally advanced breast cancer primarily treated with doxorubicin

We previously reported that high tumour cell proliferation evaluated by Ki-67 expression, high mitotic frequency and high histological grade were associated with resistance to primary doxorubicin monotherapy in locally advanced breast cancer harbouring wild-type (wt) TP53. The aim of our present study was to evaluate the predictive and prognostic impact of proliferation parameters assessed in tumour tissue obtained after chemotherapy, and alterations induced in tumour cell proliferation. While we found a significant reduction in Ki-67 expression and mitotic frequency in tumours with wtTP53 (p=0.001 and p=0.008, respectively), no significant change was recorded in tumours expressing mutant TP53. For histological grade there was no significant change in either group. There was a direct correlation between pre- and post-treatment values for Ki-67 and mitotic frequency in tumours harbouring wtTP53 (p=0.0001 for both), but no correlation in tumours harbouring mutated TP53. High post-treatment Ki-67 expression and mitotic frequency were found to predict doxorubicin resistance only in patients with wtTP53 (p=0.04 and p=0.03, respectively). The prognostic importance of proliferation markers and histological grade was found to be similar whether they were determined in the pre- or post-treatment samples (Ki-67; pre: p=0.02; post: p=0.03; mitotic frequency; p=0.002 and p=0.01, respectively; histological grade; p=0.0001 and p=0.002, respectively). While the reduction in mitotic frequency was associated with improved survival (p=0.03), no significant associations between changes in other parameters and outcome were recorded.

Loss of p21 expression is associated with p53 mutations and increased cell proliferation and p27 expression is associated with apoptosis in maxillary sinus squamous cell carcinoma

Conclusions. Loss of p21 expression dependent on the p53 mutation may be associated with higher tumor cell proliferation, and low p27 expression may be associated with decreased spontaneous apoptosis, resulting in poorer prognosis in patients with maxillary sinus squamous cell carcinoma (SCC). Objective. We have previously reported that p53 mutations and decreased spontaneous apoptosis were associated with poor prognosis in maxillary sinus SCC. However, whether p21 and p27 expression and cell proliferation correlate with either p53 status, spontaneous apoptosis or prognosis in maxillary sinus SCC has not been evaluated. Material and methods. Seventy patients with maxillary sinus SCC were analyzed. Tumor biopsy specimens were examined for p21 and p27 expression using an immunohistological method. The percentage of proliferating cells labeled by anti-Ki-67 mAb was expressed as the Ki-67 index (KI). Results. Loss of p21 expression correlated with p53 mutations (p=0.0072). The KIs in patients without p21 expression and with p53 mutations were significantly higher than those in patients with p21 expression (p=0.0119) and those without p53 mutations (p=0.0048). Patients with p27 expression showed a significantly higher apoptotic index than those without (p=0.0012). Kaplan–Meier analysis showed that p21 expression was closely associated with prolonged disease-free survival in the group with a normal p53 status (p=0.0472). Multivariate analysis identified high KI as an independent prognostic marker (p=0.047).

Aberration of FHIT Gene is Associated with Increased Tumor Proliferation and Decreased Apoptosis—Clinical Evidence in Lung and Head and Neck Carcinomas

Human FHIT (fragile histidine triad) gene is highly conserved gene homologous to a group of genes identified in prokaryotes and eukaryotes. Loss of FHIT function may be important in the development and/or progression of various types of cancer. We undertook a clinical study to analyze the relation between aberrant function of FHIT gene, tumor cell proliferation, and intensity of apoptosis as well as prognostic output in lung and squamous cell head and neck carcinoma (HNSCC). Status of FHIT gene, expression of p21waf1, intensity of apoptosis, and cell proliferation were analyzed in HNSCC and lung carcinoma tissues by molecular genetic methods, immunohistochemistry, [3H]-thymidine labeling method, and FACScan analysis in frozen and paraffin-embedded tissue sections. The majority of the malignant lung and HNSCC lesions displayed aberrant expression of FHIT gene, followed by low or negative expression of p21waf1, and increased intensity of cell proliferation. Similar results were obtained on synchronous combinations of normal, precancerous, and cancerous head and neck tissues. The observed changes increased with progression of these lesions. We examined tumor and corresponding normal tissue samples for microsatellite markers D3S1300 and D3S4103 to evaluate the loss of heterozygosity (LOH) at the FHIT gene loci. We found high percentage of LOH in both lung tumors and HNSCC (75% for D3S1300 and 79% for D3S4103 in lung cancer, and 87% for D3S1300 and 78% for D3S4103 in HNSCC). The median survival time of the patients suffering from lung cancer without FHIT protein expression was 22.46 months and that of the patients with FHIT expression 36.04 months. FHIT-negative cases tended to correlate with a worse prognosis, but this was not statistically significant. Median survival time of HNSCC patients without FHIT protein expression was 30.86 months and that of the patients with FHIT expression was 64.04 months (p < 0.05). Our results show a correlation between aberrant FHIT expression, a low rate of apoptosis, and high tumor cell proliferation. Aberrant FHIT gene could be a prognostic marker in lung cancer.

Molecular cytogenetic alterations associated with rapid tumor cell proliferation in advanced urinary bladder cancer.

Invasive urinary bladder carcinomas are characterized by a high number of cytogenetic alterations which are thought to pinpoint the location of critical genes, some of which may be involved in cell cycle control. To identify genomic alterations that may affect such genes the proliferative activity (Ki67 labeling index) was assessed in 93 invasively growing bladder carcinomas analyzed by comparative genomic hybridization. Only a few changes were significantly associated with rapid tumor cell proliferation, including 3p+ (p=0.0357), 6p+ (p=0.003), 8q+ (p=0.0273), and 11q- (p=0.0329). Among these alterations 6p+ is of particular interest because high level 6p22 amplifications occur frequently in bladder cancer. The particular strong association between 6p+ and a high tumor cell proliferation being independent of grade and stage suggests that a putative oncogene on 6p22 involved in cell cycle regulation.

Decreased Programmed Cell Death in the Uterine Cervix Associated with High Risk Human Papillomavirus Infection

The relationship between apoptosis, apoptosis regulatory proteins, cell proliferation and human papillomavirus infection during various phases of tumor progression in the uterine cervix was studied. Apoptosis was defined by morphological criteria and the TUNEL assay. Expression of p53, bcl-2, bax, cyclin D1, Ki 67 and E6 protein was evaluated by immunocytochemistry. Presence of mutant p53 was detected using a mutant specific ELISA. Type of HPV infection was determined by PCR using type specific primers. Apoptosis showed significant negative correlation with increasing histological abnormality (p=0.0005). Higher tumor cell proliferation was associated with increasing histological abnormality (p=0.001 for Ki 67 and cyclin D1). There was significant correlation between histological grade and immunoreactivity of p53 (p=0.0001) and bcl-2 (p=0.0002). However, mutant p53 was expressed by only 12 of the 230 samples. Expression of bax and the bax/bcl-2 ratio showed an inverse correlation to histological grade (p=0.0003 and 0.0001, respectively). There was also an inverse correlation between extent of apoptosis and immunoreactivity of p53 (p=0.0001) and bcl-2 (p=0.0001). A significant positive correlation between expression of the bax protein and apoptosis was evident (p=0.0001). HPV infection significantly correlated to the extent of histological abnormality (p=0.0001). High risk HPV-E6 protein also showed this significant correlation (p=0.0002). There was an inverse correlation between apoptosis and HPV infection (p=0.0002). High risk HPV infection was associated with decreased apoptosis and also increased human cell proliferation. Lowest levels of bax/bcl-2 ratio was also associated with HPV 16 and 18 infection (p=0.0001). Modulation of apoptosis and apoptotic regulatory proteins by high risk HPV infection may be an important factor in the development of cervical cancer. Copyright 1999 W.B. Saunders Company Ltd on behalf of the Arányi Lajos Foundation

Gene Amplification and Overexpression of CDK4 in Sporadic Breast Carcinomas Is Associated with High Tumor Cell Proliferation

Amplification of the cyclin-dependent kinase 4 (CDK4) gene, located at 12q13-q14, has been found as an alternative genetic alteration to CDKN2A inactivation in various human tumors including malignant gliomas and sarcomas. In the present study, we have evaluated the frequency of the CDK4 gene amplification in sporadic breast cancer by applying a nonradioactive quantitative differential polymerase chain reaction based on fluorescent DNA technology. Fluorescent-labeled polymerase chain reaction products were analyzed with an automated DNA sequencer. Amplification of CDK4 gene was detected in 15 (15.8%) of 95 breast cancers. All tumors with CDK4 gene amplification showed high CDK4 protein expression determined by immunohistochemistry. Furthermore, the mean Ki-67 labeling index in tumors with CDK4 gene amplification was significantly higher than in those without CDK4 gene amplification. No significant associations were observed between CDK4 gene amplification and any specific histopathological parameter. The findings of this study provide the first evidence of CDK4 gene amplification in breast cancer and suggest that CDK4 gene amplification appears to be of importance in the pathogenesis of a subset of sporadic breast cancer.

Angiogenesis during tumor progression in the oral cavity is related to reduced apoptosis and high tumor cell proliferation.

Angiogenesis, the growth of new blood vessels, is believed to aid tumor progression and metastasis. Tumor progression is also influenced by the extent of proliferation and apoptosis. This study, therefore, analyzed in lesions of the oral cavity, the significance of angiogenesis in relation to apoptosis, expression of apoptosis regulatory p53, bax and bcl-2 proteins as well as tissue proliferation defined by cyclin D1 expression. Results from this study suggest that angiogenesis increases as histological abnormality increases in the oral mucosa. The expression of apoptosis regulatory proteins also appears to be altered in a histologically dependent manner. The correlation seen between CD34 expression, cyclin D1 and TUNEL reactive cells suggests that increased angiogenesis, decreased apoptosis and deregulated proliferation occur simultaneously during tumor progression in the oral mucosa. Presence of a mutant p53, increased bcl-2 expression and altered bax expression are also involved in this complex process.

High telomerase activity in primary lung cancers: association with increased cell proliferation rates and advanced pathologic stage.

Telomerase enzyme activity is not detected in most normal cells, a phenomenon believed to be associated with limitations on cellular proliferation. Since this activity is detected in nearly all human tumors, including non-small-cell lung cancers, it has been suggested that telomerase activation may be coupled to acquisition of the malignant phenotype. In this study, we determined whether telomerase activity was associated with tumor pathologic stage, tumor cell proliferation rates, and clinical outcome in a cohort of patients with resected non-small-cell lung cancer for whom long-term follow-up was available. Primary tumor specimens from 99 patients treated with surgery alone and six patients treated with surgery after chemotherapy were analyzed. Telomerase activity was measured by means of a modified Telomeric Repeat Amplification Protocol (TRAP) assay. Southern blot analysis of terminal restriction fragments was used to evaluate telomere length. Immunohistochemical analysis of Ki-67, a proliferation-associated nuclear antigen, was used to assess tumor cell proliferation. Telomerase activity was detected in 84 of the 99 tumors treated with surgery alone; this activity was not detected in specimens of adjacent, benign lung tissue. Telomerase was detected in only three of six tumors resected after chemotherapy. For the surgery-alone group, statistically significant positive associations were found between the level of telomerase activity and tumor stage, lymph node metastasis, pathologic TNM (tumor-node-metastasis) stage, and Ki-67 immunostaining; a statistically significant inverse association was found between telomerase activity and patient age. No statistically significant differences in telomere length were found in relation telomerase activity or pathologic stage. Telomerase activity was not found to be associated with clinical outcome in a multivariate Cox proportional hazards analysis adjusted for tumor stage and lymph node status. High telomerase activity is detected frequently in primary non-small-cell lung cancers that exhibit high tumor cell proliferation rates and advanced pathologic stage.

Biologic and clinical correlations among ploidy, cell kinetics, and the tumor-associated glycoprotein-72 tissue expression in colorectal cancer

The aim of this study was to evaluate the possibility of identifying biologically aggressive subgroups of patients, combining several biologic parameters such as the tumor and normal mucosa values from the ploidy, the S-phase cell percentage, and tumor-associated glycoprotein-72 (TAG-72) expression. One hundred five colorectal cancer patients were studied to analyze the possible prognostic role of ploidy and cell kinetics in multiple fresh specimens from the tumor and normal mucosa. Since the presence of TAG-72 in the colonic mucosa has been correlated to neoplastic transformation, the correlations between these parameters and the quantitative tissue expression of the TAG-72 were analyzed in a subgroup of 53 cases. A significantly lower five-year disease-free survival rate (21.4 percent) was observed in patients with multiploid tumors, when compared with that observed in patients with diploid or single aneuploid tumors (67.5 percent) (P = 0.03). The quantitative tissue TAG-72 expression contributed in identifying a particular patient subgroup (20 percent), characterized by S-phase percentage and TAG-72 values from the normal mucosa that were unexpectedly higher than 12.1 percent and 7.5 U/mg of proteins, respectively. In particular, when the 25 Dukes B patients were analyzed, similar results were observed. In fact, 14 (56 percent) cases showed high tumor cell proliferation and, surprisingly, a high tissue TAG-72 content in the normal mucosa was found in 4 (28.6 percent) of these patients. Other than multiploidy, the biologic aggressiveness of colorectal cancer might be successfully assessed introducing the evaluation of new biologic parameters, such as the TAG-72 content and S-phase percentage values of the normal mucosa, suggesting the possibility of further stratifying this patient population.

Prognostic significance of lymphocyte density distribution profiles in adult non-Hodgkin lymphomas

Biopsy material from 24 adults with advanced stages of non-Hodgkin lymphomas (NHL) were examined for the distribution profiles of infiltrating cells following centrifugation to equilibrium on linear density gradients. Seven of these biopsies were predominantly composed of cells with high buoyant densities and 9 further biopsies predominantly of cells with intermediate buoyant densities. Both patterns were associated with favorable histologic features and with low proliferation of lymphoma cells. Intensive polychemotherapy was rarely required to achieve long-lasting disease control; in both groups, only 28% of patients died within a minimal observation period of 40 mo. In 8 biopsies, a predominance of light lymphoma cells was observed. This pattern was frequently associated with unfavorable histology and with high spontaneous tumor cell proliferation. Despite intensive polychemotherapy, rapid disease progression occurred in all cases, leading to death in 75% of the patients within a minimal observation period of 40 mo. Surface marker studies excluded the hypothesis that only the variable proportions of normal lymphocytes contaminating the lymphoma suspensions were responsible for the differences in the density distribution patterns described above; they rather suggested that these patterns reflect the individual capacity of a lymphoma to differentiate into dense tumor clones with low spontaneous proliferation.