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Bland KI, Konstadoulakis MM, Vezeridis MP, Wanebo HJ.. Oncogene protein co-expression. Value of Ha-ras, c-myc, c-fos, and p53 as prognostic discriminants for breast carcinoma. Ann Surg 221: 706-718
Abstract
A refinement of prognostic variables using traditional pathologic markers integrated with oncogene proteins, enzymes, and hormonal factors may enhance the ability to predict for recurrence or survival in patients with mammary carcinoma. Although various oncogenes and oncogene products have been identified in human breast carcinoma, their relationship to disease outcome remains controversial.
Using the monoclonal antibodies cS93.1, 9E1.0, F235-1.7.1, and PAb 1801 against each oncogene protein studied, the avidin-biotin complex immunoperoxidase method provided immunohistochemical staining of bound oncogene protein for c-fos, c-myc, Ha-ras, and p53, respectively. Analyses were made on archival pathology tissues of 85 breast cancer patients (stages I, IIA, and IIB). Forty patients (47%) had recurrence of disease; 45 remained free of local-regional or distant disease at mean follow-up of 48 months (range 6-180 months). Molecular biological data were merged with clinicopathologic demographics 1) to determine the frequency of single or co-expression of oncogenes in this patient population; 2) to evaluate the value of these molecular protein markers to predict probability of recurrence; and 3) to determine worth of the studied oncogenes to correlate with traditional clinical pathologic parameters and overall survival.
In this study, oncogene expression had statistical correlation for recurrence with increasing co-expression: one oncogene 17.2%, two oncogenes 56.3%, three or four oncogenes, 100% (p = 0.001). Increasing oncogene or co-oncogene expression correlated with statistically significant reduction in disease-free and overall survival; with no expression of oncogenes, disease-free survival was 30 (SE +/- 5.7) months and overall survival was 56.4 (SE +/- 4.57) months. With expression of three oncogenes, disease-free survival was 12 (SE +/- 1.23) months (p = 0.0018) and overall survival was 23.4 (SE +/- 3.38) months (p = 0.0025). In univariate Wilcoxon analysis, oncogene expression was the most significant variable to determine survival (p = 0.035); in multivariate analysis, age and oncogene co-expression each emerged as the most significant variables for overall survival. For the proportional hazards regression model, oncogene co-expression was significant (p = 0.0104, risk-ratio 1.914) and correlated with age and tumor size as significant variables. Ha-ras and c-fos both emerged as important individual oncogene proteins to affect survival (p = 0.0925, risk-ratio 3.517 and p = 0.025, risk-ratio 4.214, respectively). The proto-oncogene c-myc and the antitumor suppressor gene p53 did not have significant effects as individual oncogenes to influence survival.
Approximately one fifth of the breast cancer patients in this analysis (disease-free and recurrent) expressed only a single oncogene marker (c-fos, c-myc, Ha-ras, or p53); one quarter of patients with recurrent disease expressed only one oncogene protein. Single oncogene expression did not possess independent prognostic significance for prediction of recurrence. Further, p53 mutations did not function as independent correlates for prognosis. The co-expression of the studied proto-oncogenes (c-myc, Ha-ras) and the nuclear transcriptional protein (c-fos) functioned as a strong prognostic correlate for recurrence and survival; the effect of individual oncogenes to predict survival was greatest for Ha-ras and c-fos. Immediate or early co-expression of three oncogene proteins in neoplastic transformation endowed cells of invasive carcinoma with an aggressive phenotype. This aggressive phenotype was evident in a small percentage of the studied population (11%) and predicted adverse disease-free and overall survival. These findings suggest that oncogene co-expression possesses significant prognostic and potential therapeutic value; incorporation of this molecular technology into future prospective randomized trials is advisable.
... It has been well known that c-fos (the human homolog of the retroviral oncogene v-Fos), one of major components of activator protein (AP)-1 complex, mediates cancer growth, angiogenesis, invasion and metastasis, by regulating expressions of down-stream genes [11]. The findings that c-fos expression was significantly associated with recurrence, metastasis and poor prognosis in squamous lung cancer, breast cancer and osteosarcoma provided its potential as a proto-oncogene [12][13][14]. However, other data suggested that patients with high c-fos expression carried favorable survival in colorectal, ovarian and gastric cancers [15][16][17][18]. ...
... In the future, detailed explorations for phenotypes and relative mechanisms worth to be done. It was shown that c-fos was linked to poor or good prognosis in many types of cancers [12][13][14][15][16][17][18]. However, its impact on outcome of PC has not been elucidated. ...
... The results that c-fos was of independent prognostic significance in some subsets of PC strengthen its prognostic role. These findings, supported by the positive relationship between c-fos expression and malignant phenotypes of PC established in the present study, are consistent with those derived from osteosarcoma, lung and breast cancers [12][13][14]. No doubt these survival analysis results also indicate the function of c-fos as a proto-oncogene. ...
It has long been regarded that pancreatic cancer (PC) is a life-threatening malignant tumor. Thus, much attention has been paid for factors, especially relative molecules, predictive for prognosis of PC. However, c-fos expression in PC was less investigated. In addition, its association with clinicopathologic variables and prognosis remains unknown. In the present study, expression of c-fos was detected by tissue microarray-based immunohistochemical staining in cancer and adjacent tissues from 333 patients with PC. The staining results were correlated with clinicopathologic parameters and overall survival. Furthermore, prognostic significance of c-fos in subsets of PC was also evaluated. It was shown that low expression of c-fos was more often in cancer than in adjacent tissues of PC (P<0.001). Besides, high cancerous c-fos expression was significantly associated with tumor site and T stage, whereas peri-neural invasion was of a borderline significant relevance. Log-rank test revealed that high expression of c-fos in cancer tissues was a significant marker of poor overall survival, accompanied by some conventional clinicopathologic variables, such as sex, grade, peri-neural invasion, T and N stages. More importantly, cancerous c-fos expression was identified as an independent prognosticator in multivariate analysis. Finally, the prognostic implication of c-fos expression was proven in four subsets of patients with PC. These data suggested that c-fos expression was of relationships with progression and dismal prognosis of PC.
... AP-1 deals with different biological processes such as neoplastic transformation, cell proliferation, and invasiveness (46). Indeed, the role of FOS and the Jun complex in tumor pathogenesis has been demonstrated in a number of malignancies such as ovarian cancer (46,47), breast cancer (48), and lymphoma (49). Furthermore, the overexpression of AP-1 along with the stimulation of cyclin D2, CCR7, and proto-oncogene c-met on the one hand and cooperation of AP-1 with NF-κB on the other hand were demonstrated as critical events that occur in lymphoma pathogenesis (49). ...
... Mahner et al. (2008) (47) reported c-FOS as a prognostic factor in ovarian carcinoma in human beings and a decrease in the c-FOS expression was detected with the progression of the tumor. Furthermore, c-FOS was considered a predictor of decreased survival in patients with breast cancer (48). However, the prognostic efficacy of the FOS proteins or AP-1 complex in lymphoma or other hematopoietic malignancy has not yet been reviewed in the literature. ...
Canine B-cell lymphoma GRN was reconstructed from gene expression data in the STRING and MiMI databases. Critical genes of networks were identified and correlations of critical genes with overall survival (OS) and progression-free survival (PFS) were evaluated. Significant changes were detected in the expressions of GLUL, CD44, CD79A, ARF3, FOS, BLOC1S1, FYN, GZMB, GALNT3, IFI44, CD3G, GNG2, ESRP1, and CCND1 in the STRING network and of PECAM1, GLUL, CD44, GDI1, E2F4, TLE1, CD79A, UCP2, CCND1, FYN, RHOQ, BIN1, and A2M in the MiMI network. Final survival analysis highlighted CCND1 and FOS as genes with significant correlations with OS and PFS.
... Given the key role of FOS in cell transformation, it is not surprising that it is typically highly expressed in different cancers, and is a prognostic marker of cancer progression. Examples include cervical (Prusty & Das, 2005), heptacarcinoma (Yuen et al., 2001), pancreatic (Wakita et al., 1992), breast (Bland et al., 1995), osteosarcoma (Gamberi et al., 1998), and endometrial cancer (Bamberger et al., 2001). In each study, FOS is found to be positively correlated with a negative prognosis and survival rate, and is concluded to be an excellent marker for cancer progression. ...
... Therefore, it may well be that the predominant role or roles of FOS in cancer progression can vary with tissue type, though this remains to be studied in greater detail. FOS is proposed to be an effective prognostic and regression marker in breast cancer studies, and upregulation of FOS is indicative of poor prognosis (Bland et al., 1995). Importantly, while there is increased amount of FOS expression in tumorigenic cells, FOS itself is rarely mutated in cancer, requiring instead dysfunction of upstream signalling pathways for abnormal activity. ...
Cancer has been described as not one disease, but several, each with unique characteristics, symptoms, prognostics and outcomes. Underlying this complexity is a differential expression of genes, leading to a motley of phenotypes which orchestrate the hallmarks of cancer. The idea of treating, suppressing or even preventing all forms of cancer with a single form of therapy seems untenable given the complexities of these gene expression profiles. However, recent advances in the study of immediate early genes, a family of genes that are rapidly and transiently upregulated following an external stimulus such as growth factors, hormones or stress, and their ubiquitous involvement in regulating oncogenomic responses may lend itself to new and unique therapies. At the very least, understanding and targeting immediate early gene expression and function remains an untapped area in cancer prevention research, and could very well provide new resources in cancer treatment and new perspectives in directed cancer suppression. In this review, we will discuss the critical role immediate early genes play in cancer progression, and provide specific examples of immediate early gene function and inhibition.
... It quantifies the correlation between two or more proteins that are simultaneously expressed within a specific region. This is of biological importance as co-expression of protein markers can be a significant variable for overall survival [32,33]. ...
Pathological assessment of Hematoxylin & Eosin (H&E) stained tissue samples is a well-established clinical routine for cancer diagnosis. While providing rich morphological data, it lacks information on protein expression patterns which is crucial for cancer prognosis and treatment recommendations. Imaging Mass Cytometry (IMC) excels in highly multiplexed protein profiling but faces challenges like high operational cost and a restrictive focus on small Regions-of-Interest. Addressing this, we introduce Multi-V-Stain, a novel image-to-image translation method for multiplexed IMC virtual staining. Our method effectively utilizes the rich morphological features from H&E images to predict multiplexed protein expressions at a Whole-Slide Image level. In evaluations using an in-house melanoma dataset, Multi-V-Stain consistently outperforms existing methods in terms of image quality and biological relevance of the generated stains.
... FOS is a transcription factor that plays an important role in cell proliferation, differentiation, and tumor formation [78]. According to these studies [79,80], elevated Fos protein expression in breast cancer has potential signi cance as a marker of poor prognosis. Based on in silico predictions using The [22]. ...
Background
Breast cancer (BC) is the most common and aggressive type of cancer in females, and exploring the mechanisms of disease progression is playing a crucial role in the development of potential therapeutics. Recently, systems biology approaches such as network strategies have been successfully applied to reveal the interaction mechanisms between genes. The main objective of the current study was to investigate potential biomarkers for BC patients at different stages by constructing differential regulatory networks (DRNs).
Method
In the present study, clinical information and RNA-seq data from patients with BC were obtained from The Cancer Genome Atlas (TCGA). According to the clinical staging information, the gene expression data of TCGA-BRCA was divided into different stages (stages I–IV) and analyzed separately. The differentially co-expressed genes and links (DCGL) package in R was used to identify differentially co-expressed genes (DCGs) and differentially co-expressed links (DCLs) in different stages (I–IV) of BC patients compared to normal samples. A q < 0.25 was considered the cut-off criterion. Besides, differentially-regulated genes (DRGs) and differentially-regulated links (DRLs) were identified by DCGs, DCLs, and TF-to-target knowledge. Stage-specific gene regulatory networks (GRNs) were further analyzed with Cytoscape to explore the core TFs. Afterward, Kaplan-Meier (K-M) analysis was utilized to explore the prognostic value of the core TFs. Cancer-related pathway analysis of candidate hub TF was done through the GSCALite database. Finally, the relationship between candidate transcription factors expression and tumor-infiltrating lymphocytes was analyzed using TCGA-BRCA data and the TIMER database.
Results
From DRNs of stages I–IV, 29 unique core TFs were screened. Survival analysis indicated that the expression of KLF12, FOS, BACH2 EPAS1, PPARA, and MRPL36 had significant effects on the survival of breast cancer patients (P < 0.05). Hub genes were responsible for the infiltration levels of immunocytes. Based on the GSCALite database, these six TFs are significantly related to multiple signaling pathways, including RAS/MAPK, EMT, PI3K/AKT, and TSC/mTOR. These pathways play vital roles in oncogenesis, suggesting that these candidate hub TFs may participate in BC progression.
Conclusion
Our findings suggest these six TFs might play important roles in the pathogenesis of BC and could be used as therapeutic targets for BC. However, further studies at the molecular level are required to confirm these observations.
... Overexpression of FOS was reported in breast cancer with a poor prognosis, and it was confirmed as a therapeutic target in the treatment of this malignancy [121]. Bland et al. demonstrated that FOS is an independent predictor of worse survival in breast cancer [11]. Conducting a comparative analysis between all breast cancer subtypes, we found that FOS expression was significantly lower in the HER2 subtype and higher in the Lum A subtype. ...
Breast cancer is the most frequently diagnosed malignant tumor in women and a major public health concern. NRF2 axis is a cellular protector signaling pathway protecting both normal and cancer cells from oxidative damage. NRF2 is a transcription factor that binds to the gene promoters containing antioxidant response element-like sequences. In this report, differential expression of NRF2 signaling pathway elements, as well as the correlation of NRF2 pathway mRNAs with various clinicopathologic characteristics, including molecular subtypes, tumor grade, tumor stage, and methylation status, has been investigated in breast cancer using METABRIC and TCGA datasets. In the current report, our findings revealed the deregulation of several NRF2 signaling elements in breast cancer patients. Moreover, there were negative correlations between the methylation of NRF2 genes and mRNA expression. The expression of NRF2 genes significantly varied between different breast cancer subtypes. In conclusion, substantial deregulation of NRF2 signaling components suggests an important role of these genes in breast cancer. Because of the clear associations between mRNA expression and methylation status, DNA methylation could be one of the mechanisms that regulate the NRF2 pathway in breast cancer. Differential expression of Hippo genes among various breast cancer molecular subtypes suggests that NRF2 signaling may function differently in different subtypes of breast cancer. Our data also highlights an interesting link between NRF2 components' transcription and tumor grade/stage in breast cancer.
... 29 In BC, increased cFOS expression was related to weak prognosis. 30 The knockdown of cFos expression could increase patient survival and decrease the proliferation and invasiveness of BC xenografts. 31 The cFos protein contributes in normal development, cellular growth and apoptotic cell death in the proliferative conditions or in response to cellular injury. ...
Article history: Breast cancer (BC) is a significant cause of global mortality in women. This study was aimed to evaluate the immune-activation of malignant BC via the administration of attenuated Mycobacterium obuense. For this purpose, an in vivo model was developed with BALB/c mice. Mice were injected with 2.00 × 10 6 4T1 cells with breast tumor cell line. Forty-two mice were equally divided into control as well as low dose (0.20 mg 100 µL-1) and high dose (0.50 mg 100 µL-1) groups of M. obuense to investigate gene expression in the antitumor effects of M. obuense. In one group, paclitaxel was administrated as a choice drug in BC treatment. Antitumor manners were characterized by cytotoxicity against tumor target cells, size of the tumor and the expression of some BC metastatic genes together with pathology. The MTT assay demonstrated that different concentrations of both low and a high dose of bacteria did present no cytotoxicity effect on 4T1 cells. According to our findings, M. obuense significantly repressed tumor growth. M. obuense downregulated the expression of collagen type I alpha 1 (COLIA1), cFos, alkaline phosphatase (ALP), claudin 3 (cldn3), and conversely, activated transcription factor 4 (ATF4) and Twist related protein-1 (Twist1). All these alternations induced a decrease in the migratory and invasive capabilities of BC. The result of pathology was indicative of tumor regression in the paclitaxel and HK-M. obuense-recipient group. Thus, it seems most likely that M. obuense might impinge upon cell growth and metastatic behavior of malignant cells exerting anti-tumor activity in BC.
... Sotiriou et al. observed a similar breast cancer stratification to this work and reported that Basal-2 (the Basal-like Basal group) showed higher expression of FOS compared to Basal-1 (the Her2-like Basal) and other breast cancer subtypes [47]. FOS is a proto-oncogene and has been identified as a survival predictor [51] and a driver for breast cancer metastasis formation [52]. At the genetic level, gene FOS, JUN and NR4A2 are all classified as immediate early genes (IEGs) [53]. ...
Nuclear receptors are a class of transcriptional factors. Together with their co-regulators, they regulate development, homeostasis, and metabolism in a ligand-dependent manner. Their ability to respond to environmental stimuli rapidly makes them versatile cellular components. Their coordinated activities regulate essential pathways in normal physiology and in disease. Due to their complexity, the challenge remains in understanding their direct associations in cancer development. Basal-like breast cancer is an aggressive form of breast cancer that often lacks ER, PR and Her2. The absence of these receptors limits the treatment for patients to the non-selective cytotoxic and cytostatic drugs. To identify potential drug targets it is essential to identify the most important nuclear receptor association network motifs in Basal-like subtype progression. This research aimed to reveal the transcrip-tional network patterns, in the hope to capture the underlying molecular state driving Basal-like oncogenesis. In this work, we illustrate a multidisciplinary approach of integrating an unsupervised machine learning clustering method with network modelling to reveal unique transcriptional patterns (network motifs) underlying Basal-like breast cancer. The unsuper-vised clustering method provides a natural stratification of breast cancer patients, revealing the underlying heterogeneity in Basal-like. Identification of gene correlation networks (GCNs) from Basal-like patients in both the TCGA and METABRIC databases revealed three critical transcriptional regulatory constellations that are enriched in Basal-like. These represent critical NR components implicated in Basal-like breast cancer transcription. This approach is easily adaptable and applicable to reveal critical signalling relationships in other diseases.
... This gene is on a locus with AGAP2 and may function as a co-regulator of AGAP2 [8][9][10]. It is now widely accepted that AGAP2 regulates cell migration [11], and overexpression of AGAP2 causes intracellular redistribution of activator protein 1 (AP-1) [12], which is an important Ivyspring International Publisher modulator in several carcinomas, such as breast [13,14] and endometrial carcinomas [15], ovarian cancer [16], colorectal cancer [17], gastric cancer [18], acute myeloid leukemia [19] and anaplastic large cell lymphoma [20]. Despite these findings, however, the relationship between AGAP2-AS1 and AGAP2 has not been determined to date, and no published studies have investigated the clinical significance or biological function of AGAP2-AS1 in EOC. ...
Antisense long noncoding RNAs serve as important regulators of protein-coding genes and contribute to tumorigenesis and metastasis. AGAP2-AS1, an antisense lncRNA transcribed from AGAP2, is involved in various cancer types. However, the clinical significance, biological roles and regulatory mechanisms of AGAP2-AS1 in epithelial ovarian cancer (EOC) have not been thoroughly elucidated to date. In this study, we demonstrated the expression pattern and biological roles of AGAP2-AS1 in EOC. Clinically, AGAP2-AS1 expression was decreased in EOC tissues compared to that in the controls. Low expression of AGAP2-AS1 was associated with advanced FIGO stage, high histological grade, serous subtype and lymph node metastasis in patients with EOC. AGAP2-AS1 inhibited cell migration, invasion and proliferation in vitro. AGAP2-AS1 suppressed tumor growth in vivo. Mechanistically, AGAP2-AS1 inhibited cell metastasis and proliferation by downregulating KRAS, FGFR4, and CTSK and suppressing epithelial-mesenchymal transition. In conclusion, we provide the first evidence for the tumor-suppressing effect of AGAP2-AS1 in EOC and demonstrate that AGAP2-AS1 may represent a promising therapeutic target for EOC patients.
... FOS is one of 3 hub genes which plays a critical role in the development and progression of breast cancer by mediating the transcription of AP-1 target genes. 29,30 However, there are fewer studies to uncover FOS response to radiation in breast cancer. Recent studies indicated that silencing FOS could sensitize glioma cells to radiation by disturbing DNA damage repair and cell cycle arrest. ...
Radiotherapy is mainly a traditional treatment for breast cancer; however, the key genes and pathways in breast cancer associated with irradiation are not clear. In this study, we aimed to explore the messenger RNA expression changes between preradiation and postradiation breast cancer. The gene expression data set (GSE59733) was downloaded from Gene Expression Omnibus database. According to |log 2 FC (fold change) | ≥ 1 and with false discovery rate adjusted P value <.05, differentially expressed genes (DEGs) were screened and annotated by R programming software. The protein–protein interaction (PPI) network was conducted through STRING database, and subnetworks and hub genes were extracted by plug-in in Cytoscape. A total of 82 DEGs (74 upregulated and 8 downregulated genes) were identified. These DEGs mainly enriched in an intrinsic apoptotic signaling pathway and G-protein-coupled receptor binding. What’s more, tumor necrosis factor signaling pathway and interleukin 17 signaling pathway abnormally activated in postradiation tumor samples. Two characteristic subnetworks and 3 hub genes ( FOS, CCL2, and CXCL12) were strongly distinguished in PPI network. Moreover, the expression level of the hub genes was confirmed in irradiated MCF-7 cell and SUM-159 cell using quantitative real-time polymerase chain reaction assay. These findings imply that these hub genes may play momentous function in breast cancer to irradiation.
... c-Fos is a proto-oncogene which promotes malignant conversion, tumor formation, invasion, and metastasis [12,13]. Many studies have reported the overexpression of c-Fos in human cancers and its correlation with poor prognosis in patients [14][15][16]. Expression of c-Fos induces tumorigenesis, while deficiency of c-Fos can prevent cancer development and cancer progression [17,18]. Also, overexpression of c-Fos has been implicated in resistance to cancer therapy and enhancement of cancer stem cell stemness [14,19]. ...
Chili peppers are one of the most widely consumed spices worldwide. However, research on the health benefits of chili peppers and some of its constituents has raised controversy as to whether chili pepper compounds possess cancer-promoting or cancer-preventive effects. While ample studies have been carried out to examine the effect of capsaicin in carcinogenesis, the chemopreventive effect of other major components in chili pepper, including dihydrocapsaicin, capsiate, and capsanthin, is relatively unclear. Herein, we investigated the inhibitory effect of chili pepper components on malignant cell transformation. Among the tested chili pepper compounds, dihydrocapsaicin displayed the strongest inhibitory activity against epidermal growth factor (EGF)-induced neoplastic transformation. Dihydrocapsaicin specifically suppressed EGF-induced phosphorylations of the p70S6K1-S6 pathway and the expression of c-Fos. A reduction in c-Fos levels by dihydrocapsaicin led to a concomitant downregulation of AP-1 activation. Further analysis of the molecular mechanism responsible for the dihydrocapsaicin-mediated decrease in phospho-p70S6K1, revealed that dihydrocapsaicin can block amino acid-dependent mechanistic targets of rapamycin complex 1 (mTORC1)-p70S6K1-S6 signal activation. Additionally, dihydrocapsaicin was able to selectively augment amino acid deprivation-induced cell death in mTORC1-hyperactive cells. Collectively, dihydrocapsaicin exerted chemopreventive effects through inhibiting amino acid signaling and c-Fos pathways and, thus, might be a promising cancer preventive natural agent.
... c-fos is highly expressed in many cancers including breast cancer [36], endometrial cancer [37], pancreatic cancer [38], and hepatocellular carcinoma [39]. The oncogenic mechanism of AP-1 includes regulation of genes involved in invasion, metastasis, angiogenesis, hypoxia to name a few. ...
Nucleophosmin (NPM1) is a nucleolar protein that is frequently overexpressed in various types of solid tumors. NPM1 is involved in several cellular processes that might contribute significantly to the increased proliferation potential of cancers. Previous reports suggest that NPM1 expression is highly increased in response to mitogenic and oncogenic signals, the mechanisms of which have not been elucidated extensively. Using constructs incorporating different fragments of the NPM1 promoter upstream to a Luciferase reporter gene, we have identified the minimal promoter of NPM1 and candidate transcription factors regulating NPM1 promoter activity by luciferase reporter assays. We have validated the roles of a few candidate factors at the transcriptional and protein level by quantitative reverse transcriptase PCR, immunoblotting and immunohistochemistry, and explored the mechanism of regulation of NPM1 expression using immunoprecipitation and chromatin immunoprecipitation assays. We show here that the expression of NPM1 is regulated by transcription factor c‐fos, a protein that is strongly activated by growth factor signals. In addition, mutant p53 (R175H) overexpression also enhances NPM1 expression possibly through c‐myc and c‐fos. Moreover, both c‐fos and mutant p53 are overexpressed in oral tumor tissues that showed NPM1 overexpression. Collectively, our results suggest that c‐fos and mutant p53 R175H positively regulate NPM1 expression, possibly in synergism, that might lead to oncogenic manifestation. This article is protected by copyright. All rights reserved.
... However, these proteins were not affected by Eu in MCF-10A cells (Fig. 2B, Supplementary Fig. S2). Ras is essential for Myc protein stability, and the co-expression of H-ras and c-Myc contributes to neoplastic transformation with early mammary carcinogenesis 29,30 . Therefore, we examined the status of c-Myc, PGC-1β and ERRα following the H-ras depletion in MCF-10A-ras cells treated with Eu. ...
Alteration in cellular energy metabolism plays a critical role in the development and progression of cancer. Targeting metabolic pathways for cancer treatment has been investigated as potential preventive or therapeutic methods. Eugenol (Eu), a major volatile constituent of clove essential oil mainly obtained from Syzygium, has been reported as a potential chemopreventive drug. However, the mechanism by which Eu regulates cellular energy metabolism is still not well defined. This study was designed to determine the effect of Eu on cellular energy metabolism during early cancer progression employing untransformed and H-ras oncogene transfected MCF10A human breast epithelial cells. Eu showed dose-dependent selective cytotoxicity toward MCF10A-ras cells but exhibited no apparent cytotoxicity in MCF10A cells. Treatment with Eu also significantly reduced intracellular ATP levels in MCF10A-ras cells but not in MCF10A cells. This effect was mediated mainly through inhibiting oxidative phosphorylation (OXPHOS) complexs and the expression of fatty acid oxidation (FAO) proteins including PPARα, MCAD and CPT1C by downregulating c-Myc/PGC-1β/ERRα pathway and decreasing oxidative stress in MCF10A-ras cells. These results indicate a novel mechanism involving the regulation of cellular energy metabolism by which Eu may prevent breast cancer progression.
... Also, fosB was found to be expressed in T47D cells, which are receptor-positive, but not in MDA-MB 231 and HBL 100 cells, which are receptor-negative. These observations indicate that fosB expression, unlike that of c-fos, which was found to be a negative prognostic indicator in an immunohistochemical study on breast carcinomas (Bland et al., 1995), is significantly associated with the presence of a welldifferentiated, receptor-positive tumor phenotype. ...
In the present study, the expression of members of the AP-1 family of transcription factors in breast tumors (n = 53) was investigated by Western blot with antibodies specific for each of the AP-1 family members (c-jun, junB, junD and c-fos, fosB, fra1 and fra2). The tumors were characterized with regard to grading, staging, histology, steroid-receptor-expression status and c-erbB2/neu expression. For comparison, normal breast-tissue samples, human breast-cancer cell lines (T47D and MDA-MB231) and the transformed human breast epithelial cell line HBL100 were also analyzed. For c-jun, junB, c-fos and fra2, a relatively uniform expression pattern without significant differences among tumors was observed. junD-protein amounts varied strongly in the tumor specimens. fosB-expression levels also varied strongly in the tumors, weak/absent expression being found in 47%, while 45% exhibited strong/very strong levels of expression. While none of the other AP-1 family members showed significant correlations with clinico-pathological tumor parameters or receptor status, expression of fosB was found to correlate significantly with positive steroid-hormone-receptor status (in the tumors and the cell lines) and a more differentiated tumor phenotype. Expression of 2 fra-1-specific bands of 33 and 36.5 kDa showed significant negative correlation with fosB expression, as well as with estrogen-receptor status and differentiation. We conclude that strong differences in the expression pattern of AP-1 family members are present in breast tumors, and that certain members of this family, such as fosB and fra-1, might be involved in the pathogenesis of these tumors. Int. J. Cancer (Pred. Oncol.) 84:533–538, 1999. © 1999 Wiley-Liss, Inc.
... The conclusions, however, were mixed. In human squamous cell lung carcinoma [16], breast carcinoma [17], human osteosarcoma [18], oral squamous cell carcinoma [19], and cutaneous squamous cell carcinoma [20], c-Fos overexpression was found to correlate with poor prognosis; while in refractory colorectal carcinoma [21] and epithelial ovarian carcinoma [22], elevated c-Fos expression was reported to be a good prognostic marker. There were additional studies from large numbers of patients with gastric cancer showed that loss of c-Fos expression correlated with shorter survival, advanced stage, lymph node metastasis, and lymphatic invasion [23,24]. ...
c-Fos is a major component of activator protein (AP)-1 complex. It has been implicated in cell differentiation, proliferation, angiogenesis, invasion, and metastasis. To investigate the role of c-Fos in glioma radiosensitivity and to understand the underlying molecular mechanisms, we downregulated c-Fos gene expression by lentivirus-mediated shRNA in glioma cell lines and subsequently analyzed the radiosensitivity, DNA damage repair capacity, and cell cycle distribution. Finally, we explored its prognostic value in 41 malignant glioma patients by immunohistochemistry. Our results showed that silencing c-Fos sensitized glioma cells to radiation by increasing radiation-induced DNA double strand breaks (DSBs), disturbing the DNA damage repair process, promoting G2/M cell cycle arrest, and enhancing apoptosis. c-Fos protein overexpression correlated with poor prognosis in malignant glioma patients treated with standard therapy. Our findings provide new insights into the mechanism of radioresistance in malignant glioma and identify c-Fos as a potentially novel therapeutic target for malignant glioma patients.
... Our study reports gene expression changes in the largest cohort of sequential samples from patients receiving no-intervening treatment yet assembled to demonstrate the molecular variation that occurs independent of treatment in the neoadjuvant and preoperative setting. Significant pairwise changes in gene expression were observed and a 50 gene signature identified comprised of genes associated with a number of cell growth, cell stress and cancer related signalling pathways, including ATF3, EGR1, FOS, FOSB and JUN, each of which have been previously implicated in prognostic discrimination and pathogenesis of breast cancer [39][40][41][42][43] as well as other cancers 44 . ...
Patient-matched transcriptomic studies using tumour samples before and after treatment allow inter-patient heterogeneity to be controlled, but tend not to include an untreated comparison. Here, Illumina BeadArray technology was used to measure dynamic changes in gene expression from thirty-seven paired diagnostic core and surgically excised breast cancer biopsies obtained from women receiving no treatment prior to surgery, to determine the impact of sampling method and tumour heterogeneity. Despite a lack of treatment and perhaps surprisingly, consistent changes in gene expression were identified during the diagnosis-surgery interval (48 up, 2 down; Siggenes FDR 0.05) in a manner independent of both subtype and sampling-interval length. Instead, tumour sampling method was seen to directly impact gene expression, with similar effects additionally identified in six published breast cancer datasets. In contrast with previous findings, our data does not support the concept of a significant wounding or immune response following biopsy in the absence of treatment and instead implicates a hypoxic response following the surgical biopsy. Whilst sampling-related gene expression changes are evident in treated samples, they are secondary to those associated with response to treatment. Nonetheless, sampling method remains a potential confounding factor for neoadjuvant study design.
... In a comparative analysis between precancerous lesion of the cervix uteri and invasive cervical cancer, c-Fos expression was significantly lower in precancerous lesions (Prusty and Das, 2005). C-Fos has also been identified as independent predictor of decreased survival in breast cancer (Bland et al, 1995). ...
Members of the Fos protein family dimerise with Jun proteins to form the AP-1 transcription factor complex. They have a central function in proliferation and differentiation of normal tissue as well as in oncogenic transformation and tumour progression. We analysed the expression of c-Fos, FosB, Fra-1 and Fra-2 to investigate the function of Fos transcription factors in ovarian cancer. A total of 101 patients were included in the study. Expression of Fos proteins was determined by western blot analysis, quantified by densitometry and verified by immunohistochemistry. Reduced c-Fos expression was independently associated with unfavourable progression-free survival (20.6, 31.6 and 51.2 months for patients with low, moderate and high c-Fos expression; P ¼ 0.003) as well as overall survival (23.8, 46.0 and 55.5 months for low, moderate and high c-Fos levels; P ¼ 0.003). No correlations were observed for FosB, Fra-1 and Fra-2. We conclude that loss of c-Fos expression is associated with tumour progression in ovarian carcinoma and that c-Fos may be a prognostic factor. These results are in contrast to the classic concept of c-Fos as an oncogene, but are supported by the recently discovered tumour-suppressing and proapoptotic function of c-Fos in various cancer types.
... A number of studies have investigated the oncogenic functions of FOS and found its target genes to be critical for tumorigenesis, responsible for invasive growth of tumor cells and inhibition of tumor suppressor activity [9,10]. In breast cancer, increased Fos protein expression was associated with a poor prognosis [11]. An immunohistochemical study of more than 600 patients with gastric carcinoma showed that loss of FOS expression was associated with adverse outcome [12]. ...
Effective suicide gene delivery and expression are crucial to achieving successful effects in gene therapy. An ideal tumor-specific promoter expresses therapeutic genes in tumor cells with minimal normal tissue expression. We compared the activity of the FOS (FBJ murine osteosarcoma viral oncogene homolog) promoter with five alternative tumor-specific promoters in glioma cells and non-malignant astrocytes. The FOS promoter caused significantly higher transcriptional activity in glioma cell lines than all alternative promoters with the exception of CMV. The FOS promoter showed 13.9%, 32.4%, and 70.8% of the transcriptional activity of CMV in three glioma cell lines (U87, U251, and U373). Importantly, however, the FOS promoter showed only 1.6% of the transcriptional activity of CMV in normal astrocytes. We also tested the biologic activity of recombinant adenovirus containing the suicide gene herpes simplex virus thymidine kinase (HSV-tk) driven by the FOS promoter, including selective killing efficacy in vitro and tumor inhibition rate in vivo. Adenoviral-mediated delivery of the HSV-tk gene controlled by the FOS promoter conferred a cytotoxic effect on human glioma cells in vitro and in vivo. This study suggests that use of the FOS-tk adenovirus system is a promising strategy for glioma-specific gene therapy but still much left for improvement.
... Biomarkers that are able to discriminate the fast progressing cancers from the slow growing cancers are most urgently needed not only to save on the cost of the therapy, but also to preserve the quality of life and to avoid unnecessary morbidity associated with therapy . A routinely [11,12] available method to successfully identified abnormal DNA markers will be important in modern oncology practice. ...
Prostate cancer is the most frequent malignancy and the second leading cause of cancer deaths among males in the Western world. A study of 40 cases of prostate cancer is conducted in an attempt to identify prognostic biomarkers that can distinguish aggressive cases that must be treated immediately. HER-2/neu oncogene amplification was initially studied because amplification of this gene has been reported in many other types of cancer. In this study, HER-2 gene amplification was assessed by fluorescence in situ hybridization (FISH) using a HER-2/neu gene probe with a chromosome 17 centromere control probe. The study was performed on formalin-fixed, paraffin-embedded tissues. FISH successfully analyzed all cases. Only 5 out of 40 (12.5%) were found to be amplified. This frequency was lower than the frequency of amplification found in other in other cancers studied. The level of amplification observed was correlated with the pathological grade. Our data indicate that HER-2/neu gene amplification status can be determined by FISH on archival prostate cancer specimens, significantly correlates with high tumor grade and is more frequently encountered in tumors with advanced pathological stage. Also, FISH is a sensitive technique for detection of abnormalities in the HER-2/neu gene and further studies should be undertaken to determine whether a FISH-based HER-2/neu detection method may prove of importance in the prediction of prognosis and planning of therapy in prostate cancer patients.
... Several investigations have found that higher expression of MYC protein correlated with poorer outcome (33)(34)(35), whereas other studies have shown positive associations between MYC mRNA levels and survival (36) and between MYC protein levels and survival, most notably for node-negative patients (15). Our findings are consistent with previous findings that showed that MYC protein expression alone was not related to recurrence (30) and are supported by a limited number of studies that did not find associations between MYC expression and prognosis (19,22). Importantly, however, we observed a benefit of concurrent trastuzumab in patients with or without MYC protein overexpression. ...
This study investigated the association between tumor MYC protein expression and disease-free survival (DFS) of patients randomized to receive chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C) in the N9831 (Alliance) adjuvant HER2+ trastuzumab breast cancer trial.
This analysis included 1736 patients randomized to Arms A, B, and C on N9831. Nuclear MYC protein expression was determined in tissue microarray (TMA) sections containing three biopsies per patient or whole tissue sections (WS) using standard immunohistochemistry (clone 9E10). A tumor was considered positive for MYC protein overexpression (MYC+) if the nuclear 3+ staining percentage was >30%.
574 (33%) tumors were MYC+. MYC+ was associated with hormone receptor positivity (χ2 p=0.006), tumors ≥ 2 cm (χ2 p=0.02), and a higher rate of nodal positivity (χ2 p<0.001). Hazard ratios (HRs) for DFS (median follow-up: 6.1 years) for Arm C versus A were 0.52 (p=0.006) and 0.65 (p=0.006) for patients with MYC+ and MYC- tumors, respectively (interaction p=0.40). For Arm B versus A, HRs for patients with MYC+ and MYC- tumors were 0.79 (p=0.21) and 0.74 (p=0.04), respectively (interaction p=0.71). For Arm C versus B, HRs for patients with MYC+ and MYC- tumors were 0.56 (p=0.02) and 0.89 (p=0.49), respectively (interaction p=0.17).
Our data do not support an impact of tumor MYC protein expression on differential benefit from adjuvant trastuzumab.
... Increased Fos protein expression in breast cancer has potential significance as a marker of poor prognosis (Bland et al., 1995), and XM6:antifos antisense strategies appear beneficial in prolonging subject survival and inhibiting the proliferation and invasiveness of breast cancer xenograft systems (Robinson-Benion et al., 1994;Arteaga and Holt, 1996). Furthermore, we have demonstrated significant associations between elevated Fos protein expression and increased proliferation, de novo endocrine insensitivity and a worsened prognosis in clinical breast cancer (Gee et al., 1995). ...
We have previously demonstrated that elevated Fos expression may be important in de novo endocrine resistance in breast cancer. However, changes in Fos expression during endocrine response and subsequently on acquisition of resistance are unknown. This study immunocytochemically monitors Fos protein within sequential biopsies from primary human breast cancer patients obtained pre-treatment (T1), during tamoxifen therapy (T2, T3) and on disease progression (T5), examining in parallel proliferation [i.e., MIB1 (Ki67) immunostaining, mitotic activity], cellularity and endocrine response. Significantly diminished Fos, proliferation and cellularity were observed after 6 weeks of therapy in patients exhibiting a better quality and/or duration of response, while modest Fos increases and a maintained proliferation and cellularity were seen in poorer responders. Decreases in Fos, proliferation and cellularity at 6 months similarly hallmarked better responders. We confirmed a significant association between de novo resistance and elevated Fos and proliferation. Additionally, however, these parameters increased at the time of disease relapse over pre-treatment and “on therapy” values. Our data indicate that tamoxifen response involves a reduction in both tumor cell proliferation and cell survival, potentially entailing diminished Fos protein expression in better-responding patients. Our data are also supportive of elevated Fos expression being involved in the departure from endocrine control inherent in both primary and acquired resistance. Int. J. Cancer (Pred. Oncol.) 84:54–61, 1999. © 1999 Wiley-Liss, Inc.
... A distinct mechanism apart from the point mutations in the ras family genes, overexpression of the wild-type allele, at both the RNA and the protein level has been reported (39,40) and in particular cases such as in squamous cell carcinomas of the head and neck was associated with favourable prognosis (41). The co-expression of the oncogenes H-ras, cmyc and the nuclear transcriptional protein c-fos functions as a strong prognostic correlate for recurrence and survival in breast carcinoma; the effect of individual oncogenes to predict survival is greatest for H-ras and c-fos (42). Furthermore, semi-quantitative immunohistochemical methods demonstrate that expression of the p21 antigen in prostate carcinoma strongly correlated with nuclear anaplasia and is inversely related to the degree of glandular differentiation (43). ...
Abnormal activation of ras oncogenes by specific point mutations has extensively been reported to contribute to human tumorigenesis. Activating point mutations of H-ras, K-ras, N-ras and genes, are restricted to codon 12, 13 or 61 of their DNA sequences. Differential activation of the three ras genes at the transcription level has also been reported in cervical lesions, leukemias, bladder tumours, benign and malignant colorectal tumours, breast and ovarian carcinomas, hepatocellular carcinoma and in myelodysplastic tumours. Overexpression of the ras p21 protein has been detected both at the RNA and the protein level in several types of human cancers such as the squamous cell carcinomas of the head and neck, nasopharyngeal carcinomas or in bone marrow smears of children with acute leukemia. The discovery of new prognostic indicators is considered extremely important and detection of alterations in ras genes could serve as a crucial molecular marker for the early detection of human malignancies or alternatively for the discrimination between chronic non-malignant conditions and cancer. The research on ras genes in relation to human malignancies and its potential clinical significance in screening tests for early detection and accurate prognosis is reviewed and its potential application in future therapeutic treatment of human cancer is discussed.
... Furthermore, Hu et al (12) reported that c-fos is required for the expression of MMPs. In addition, c-fos overexpression has been identified as an independent predictor of survival in breast cancer (16). In contrast, some studies concluded that c-fos has tumor suppressor activity. ...
The transcription factor TWIST is an important factor in regulating epithelial-mesenchymal transition (EMT) and tumor metastasis. To explore the functions of TWIST in hypopharyngeal cancer, we investigated if overexpression of TWIST has an effect on FaDu cell morphology, and if alteration of TWIST has an effect on E-cadherin, N-cadherin, c-fos, MMP-9, as well as in cell migration, and the invasion ability of FaDu cells. Moreover, we also studied the relationship between TWIST overexpression and clinicopathological characteristics in hypopharyngeal cancer tissue samples by immunohistochemical assays. The results showed that overexpression of TWIST-induced morphological changes, such as occurrence of EMT. TWIST overexpression also increased cell migration and invasion ability, accompanied by an alteration of E-cadherin, N-cadherin, c-fos and MMP-9 expression. Furthermore, immunohistochemical assays showed that TWIST overexpression was related with tumor differentiation (P=0.038), tumor size (P=0.048) and lymph node metastasis (P=0.044). The data presented reveal that overexpression of TWIST plays a significant role in the metastasis of hypopharyngeal tumors, and alteration of TWIST has an effect on the EMT, c-fos and MMP-9 expression in FaDu cells. We conclude that TWIST promotes hypopharyngeal carcinoma metastasis, and the TWIST/c-fos/MMP-9 signaling pathway may play an important role in the metastasis of FaDu cells.
The mitogen- and stress-activated protein kinases activated by the extracellular-signal-regulated kinase 1/2 and/or stress-activated protein kinase 2/p38 mitogen-activated protein kinase pathways are recruited to the regulatory region of a subset of genes termed immediate-early genes, often leading to their induction. These genes, many of which code for transcription factors, have been directly linked to the phenotypic events in carcinogenesis. In this paper, we focus on the mitogen- and stress-activated protein kinases; their discovery, activation, H3 phosphorylation and recent discoveries in their roles in cancer.
Increased understanding of intrinsically disordered proteins (IDPs) and protein regions (IDRs) has revolutionized our view of the relationship between protein structure and function. Data now support that IDPs can be functional in the absence of a single, fixed, three-dimensional structure. Due to their dynamic morphology, IDPs have the ability to display a range of kinetics and affinity depending on what the system requires, as well as the potential for large-scale association. Although several studies have shed light on the functional properties of IDPs, the class of intrinsically disordered transcription factors (TFs) is still poorly characterized biophysically due to their combination of ordered and disordered sequences. In addition, TF modulation by small molecules has long been considered a difficult or even impossible task, limiting functional probe development. However, with evolving technology, it is becoming possible to characterize TF structure-function relationships in unprecedented detail and explore avenues not available or not considered in the past. Here we provide an introduction to the biophysical properties of intrinsically disordered TFs and we discuss recent computational and experimental efforts towards understanding the role of intrinsically disordered TFs in biology and disease. We describe a series of successful TF targeting strategies that have overcome the perception of the "undruggability" of TFs, providing new leads on drug development methodologies. Lastly, we discuss future challenges and opportunities to enhance our understanding of the structure-function relationship of intrinsically disordered TFs.
Analisis profil molekuler telah memberikan pengertian lebih dalam berkenaan dengan kompleksitas penyakit kanker di tingkat molekuler serta membuka potensi pengunaannya untuk mempelajari jalur onkogenik, hubungan jalur keganasan dengan sensitivitas terhadap terapi yang memberikan potensi yang besar sebagai petunjuk pengunaan terapi target. C-Fos merupakan proto-onkogen, berikatan dengan Jun membentuk komplek faktor transkripsi activating protein 1 (AP-1). Sebagai anggota dari AP-1, c-Fos memainkan peran merespon transduksi sinyal proliferasi dan diferensiasi sel yang menyebabkan pertumbuhan sel yang invasif. Sebaliknya laporan penelitian lainnya membuktikan bahwa c-Fos juga memiliki peran sebagai tumor supresor dengan keterlibatannya pada proses apoptosis. Mekanisme molekuler yang jelas tentang keterlibatan c-Fos baik sebagai tumor modulator proliferasi sel maupun supresor pertumbuhan tumor masih belum jelas. Penelitian lebih lanjut keterlibatan c-Fos di tingkat molekuler sangat penting untuk membuka jalan bagi target baru terapi kanker maupun sebagai marker prognostik penyakit kanker.
The proto-oncogene Myc serves as a paradigm for understanding the dynamics of transcriptional regulation. Myc protein has been linked to immune dysfunction, cancer development, and neoplastic transformation. We review recent research regarding functions of Myc as an important modulator in immune disorders, post-allogeneic hematopoietic stem cell transplantation (HSCT), and several cancers. Myc overexpression has been repeatedly linked to immune disorders and specific cancers: myasthenia gravis, psoriasis, pemphigus vulgaris, atherosclerosis, long-term allogeneic survival among HSCT patients, (primary) inflammatory breast cancer, (primary) ovarian carcinoma, and hematological malignancies: acute myeloid leukemia, chronic myelogenous leukemia, Hodgkin′s lymphoma, and diffuse large B-cell lymphoma. However, decreased expression of Myc has been observed in HSCT patients who did not survive. Understanding impaired or inappropriate expression of Myc may present a path for the discovery of new targets for therapeutic applications.
The discovery of new prognostic factors proceeds at a much more rapid pace than our knowledge of how to properly utilize this information in the management of patients with breast cancer, especially those with early breast cancer that has not metastasized to regional lymph nodes. Prognostic factors provide information on how the patient is likely to do regardless of treatment. Predictive factors provide information on whether a patient is likely to benefit from therapy. Most factors identified to date provide prognostic information, but relatively few provide information that is truly helpful in making a therapeutic decision in the management of individual patients. In large part this is because there has been insufficient study of the factor, especially prospective evaluations of the factor. Unfortunately this has resulted in the premature use of this information under the general rubric that patients with a poor prognosis deserve more treatment in spite of the fact that there may be no benefit from that therapy in the poor prognostic group.
The inflammatory response is known to have a significant role in certain autoimmune diseases and malignancies. We review current knowledge regarding the functions of activator protein 1 (AP-1) as an important modulator in several immune disorders and carcinomas. AP-1 is overexpressed in rheumatoid arthritis and in long-term allogeneic hematopoietic stem cell transplantation survivors; however, decreased expression of AP-1 has been observed in psoriasis, systematic lupus erythematosus and in patients who do not survive after hematopoietic stem cell transplantation. AP-1 also is implicated in the control of various cancer cells. Higher levels of AP-1 components are present in breast and endometrial carcinomas, colorectal cancer and in acute myeloid leukemia, Hodgkin׳s lymphoma and anaplastic large cell lymphoma, with downregulation in ovarian and gastric carcinomas and in patients with chronic myelogenous leukemia. AP-1 may enable the development of helpful markers to identify early-stage disease or to predict severity.
Antiestrogens are established as compounds which predominantly exert their actions by competing with estrogen for binding to the target steroid receptor. This is evidenced by the observations that their biological effects are most notably recognised in tissues that contain ER; they often structurally resemble estrogens in regions which are important for the binding of the steroid nucleus to the ER and their ER binding, while of differing efficiency, always displaces and/or prevents the association of estrogens (Nicholson, 1993). Simplistically, as a consequence of such binding, antiestrogens subsequently reduce estrogen signalling within responsive cells. In practice, however, they display a bewildering diversity of biological properties, with tissue-specific actions that are not easily reconciled with such a basic model (Furr and Jordan 1984; Nicholson et al. 1986). Thus, while the non-steroidal triph-enylethylene compound tamoxifen (the most widely prescribed antiestrogenic drug used in the therapy of breast cancer) promotes objective tumour remissions in approximately 30–50% of women (presumably an antiestrogenic response), it shows many estrogen-like characteristics on endometrium, bone and the cardiovascular system (Powles 1997). Indeed, long-term tamoxifen therapy, while delaying the recurrence of primary breast cancer and reducing the incidence of contralateral cancers (Early
Breast
Cancer
Trialist’s
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Group 1992), promotes a significant increase in the development of endometrial cancers (presumably an estrogenic response; Cohan 1997), with possible additional detrimental effects on the liver (Wogan 1997).
Breast cancer is the most common malignancy among women in the United States, accounting for nearly 32% ofall cancers and for nearly 20% ofall cancer deaths in women (1). Breast cancer is a multifactorial disease, and several factors are thought to influence the risk of breast cancer development, including geography, radiation exposure, and reproductive and family history. A family history of breast cancer constitutes the major risk factor, although familial breast cancer accounts for only 5% of all cases (1). Although breast cancer deaths declined by 5% between 1989 and 1992 (2), the incidence of breast cancer is expected to increase by 2–3% annually (1), and efforts to decrease mortality significantly, including new therapeutic approaches and early diagnosis, have been relatively unsuccessful. One of the critical factors in determining the therapeutic approach to breast cancer, along with the presence or absence of lymph node metastasis, is the estrogen and progesterone receptor (ER and PR) status of the tumor. Indeed, ER- and/or PR-positive tumors have been found to be more likely to respond to endocrine therapy and are associated with a better prognosis (1). An understanding of the genetic changes involved in breast cancer and of its biologic behavior, including possible interactions between hormones and oncogenes or tumor suppressor genes, will undoubtedly aid in the design of new therapies as well as in prevention and diagnosis of this disease.
The first part of this two-part review of established and emerging breast cancer biomarkers considers up-to-date knowledge and describes new types of prognostic and predictive biomarkers, with diagnostic and therapeutic applications in the experimental and clinical milieu. It also discusses new perspectives for utilizing cell cycle markers, growth factors, oncogenes, suppressor genes and cell adhesion markers as prognostic methods in breast cancer development. It outlines efforts for achieving high sensitivity and specificity of biomarkers permitting to detect high risk groups, evaluate the progress of the disease and treatment outcomes. Struggles against formal and technical setbacks hampering the full introduction of biomarkers into the clinic, as takes place in case of microarray techniques, are also described.
Epidemiology suggests a possible relationship between exposure to power frequency magnetic fields (EMF) and breast cancer. One mechanism through which EMF could stimulate breast cancer induction is via altered expression of oncogenes and/or tumor suppressor genes that regulate normal and neoplastic growth. To evaluate the hypothesis that EMF action in the breast is mediated by alterations in gene expression, transcript levels of c-myc and a battery of other cancer-associated genes were quantitated in human breast epithelial cells exposed to pure, linearly polarized 60 Hz EMF with low harmonic distortion. HBL-100 cells and normal (non-transformed) human mammary epithelial cells were exposed to EMF flux densities of 0.1, 1.0 and 10.0 Gauss (G) for periods ranging from 20 min to 24 h; concurrent sham controls were exposed to ambient fields (<0.001 G) only. Gene expression was quantitated using ribonuclease protection assays. EMF exposure had no statistically significant effect on basal levels of c-myc transcripts in either human breast cell model, and had no effect on alterations in c-myc expression induced by 12-O-tetradecanoylphorbol-13-acetate. Transcript levels of c-erbB-2, p53, p21, GADD45, bax, bcl-x, mcl-1, and c-fos were also unaffected by EMF exposure. These results suggest that EMF is unlikely to influence breast cancer induction through a mechanism involving altered expression of these genes.
Covering: 2000 to 2014Cancer is one of the leading causes of death worldwide. Ginseng, a key ingredient in traditional Chinese medicine, shows great promise as a new treatment option. As listed by the U.S. National Institutes of Health as a complementary and alternative medicine, its anti-cancer functions are being increasingly recognized. This review covers the mechanisms of action of ginsenosides and their metabolites, which can modulate signaling pathways associated with inflammation, oxidative stress, angiogenesis, metastasis, and stem/progenitor-like properties of cancer cells. The emerging use of structurally modified ginsenosides and recent clinical studies on the use of ginseng either alone or in combination with other herbs or Western medicines which are exploited as novel therapeutic strategies will also be explored.
Aberrant expression levels of the rns genes is a common event in human tumors. Transcriptional regulation of the H-ras proto-oncogene, the most well studied member of the ras family, occurs through nuclear factors that recognize elements in the promoter region of the gene, in the first and fourth intron and in the variable tandem repeat (VTR) region unit and involves alternative splicing and specific methylation patterns, as well. Overexpression of the Pas p21 protein is detected in a variety of human tumors, including neuroblastomas, esophageal, head and neck, laryngeal, thyroid, lung, liver, intestinal, gastric, colorectal, breast, bladder, endometrial, ovarian tumors and leukemias. Levels of the Ras p21 protein are often of great prognostic and clinical significance and controling the expression of ms Penes provides a useful target for gene therapy treatments.
Integrative functional genomics approaches are helpful in delineating the complex dysregulations in cancers. In the present study, in vitro activity profiling of 45 signaling pathway driven transcription factors in eight gastric cancer cell lines and direct comparison with genome-wide profiles of gastric tumors were performed and the integration resulted in the identification of three categories of factors/pathways: i) highly activated signaling pathways stem from mutations are the critical oncogenic drivers, ii) constitutively activated stress responsive pathways which are activated not due to genetic alterations, and iii) consistently down-regulated nuclear receptor responsive factors. This functional profiling helps discriminating therapeutic targets and signaling interactions.
Live mRNA detection allows real time monitoring of specific transcripts and genetic alterations. The main challenge of live genetic detection is overcoming the high background generated by unbound probes and reaching high level of specificity with minimal off target effects. The use of Fluorescence Resonance Energy Transfer (FRET) probes allows differentiation between bound and unbound probes thus decreasing background. Probe specificity can be optimized by adjusting the length and through use of chemical modifications that alter binding affinity. Herein, we report the use of two oligonucleotide FRET probe system to detect a single nucleotide polymorphism (SNP) in murine Hras mRNA, which is associated with malignant transformations. The FRET oligonucleotides were modified with phosphorothioate (PS) bonds, 2'OMe RNA and LNA residues to enhance nuclease stability and improve SNP discrimination. Our results show that a point mutation in Hras can be detected in endogenous RNA of living cells. As determined by an Acceptor Photobleaching method, FRET levels were higher in cells transfected with perfect match FRET probes whereas a single mismatch showed decreased FRET signal. This approach promotes in vivo molecular imaging methods and could further be applied in cancer diagnosis and theranostic strategies.
This chapter describes the biology of high risk benign breast lesions. The natural history of breast cancer indicates that it develops over years—even decades—and may progress through recognizable stages of proliferative breast disease. Although breast cancers are slow growing, they metastasize early. The ability to recognize and treat high-risk, precursor breast lesions is desirable. In the human breast, a spectrum of microscopic changes is termed as proliferative breast disease (PBD). The progression of histopathological features of PBD is correlated with increased risk for the development of invasive carcinoma, but the focal and microscopic lesions of PBD provide scant tissue for genetic or other biological analyses. Carcinoma in situ (CIS), with an associated tenfold increased risk for breast cancer, is accepted as a precursor lesion because subsequent development of invasive cancer is frequently in the same breast in which CIS was detected earlier. Patterns of loss of heterozygosity (LOH) and gene expression in cancer and in experimental cell models support the notion that breast cancer can develop from a number of pathways of interdependent genetic changes.
La pathologie mammaire offre de nombreuses applications à l'immunohistochimie. Ses indications sont discutées au fil de différentes situations pratiques regroupées sous quatre rubriques : l'aide au diagnostic histologique, l'apport au pronostic, la prédiction de la réponse à l'hormonothérapie ou à la chimiothérapie et l'aide à la surveillance des patientes. L'immunohistochimie est appelée à se développer dès lors que les indications sont validées que les techniques sont standardisées et qu'un suivi dans la qualité est assuré.
Tumor-specific chromosomal abnormalities have been shown to characterize certain subgroups of mesenchymal neoplasms. Identification of these abnormalities has proven useful diagnostically and has provided direction for molecular studies of pathogenetically important genes. Recent studies focusing on cytogenetic and molecular cytogenetic findings in osteosarcoma have expanded our knowledge of chromosomal alterations in this neoplasm and are pointing to recurrent regions of interest. In our study, the cytogenetic findings of 36 osteosarcoma specimens and a review of the literature (for a total of 161 specimens) are provided. Molecular cytogenetic studies were performed on two specimens. Clonal chromosomal abnormalities were detected in 25 of 36 specimens and included 17 near-diploid, 8 near-triploid, 2 near-tetraploid, and 8 specimens with multiple clones of different ploidy levels. Examination of the present data and previously published results reveals that chromosomal bands or regions Ip11-13, Iq11-12, Iq21-22, 11p14-15, 14p11-13, 15p11-13, 17p, and 19q13 are most frequently rearranged, and the most common numerical abnormalities are +1, −9, −10, −13, and −17. The majority of osteosarcomas examined were characterized by complex karyotypes.
Estrogens play important roles in both the onset and malignant progression of breast cancer. The content of estrogen receptors in breast tumors is a valuable predictor of whether a patient will respond to therapy with antiestrogens, such as tamoxifen and fulvestrant (ICI 182,780). Expression and activity of ER can be lost or impaired in antiestrogen-resistant breast cancer. The proposed studies are designed to test the overall hypothesis that the ubiquitin-like NEDD8 protein modification pathway represses estrogen action by facilitating degradation of ER protein. Perturbation of this pathway may prove instrumental in breast tumor progression; alternatively, activation of this pathway may prove to be a valid target for novel therapeutics. This study on mechanisms that regulate ER levels and activity are highly relevant to the development and progression breast cancer, including tumor progression to states of hormone independence and antiestrogen resistance. Thus, understanding how the estrogen receptor is regulated is an area of research critical to understanding the tissue selective pharmacology of estrogens. In addition, tamoxifen and other selective estrogen receptor modulators target the estrogen receptor, and this study is of the utmost relevance to those important therapies.
Tumor-specific chromosomal abnormalities have been identified in several histologic subtypes of sarcomas. Characterization of recurrent chromosomal abnormalities has provided direction for molecular investigations of pathogenetically important genes. Cytogenetic reports of osteosarcoma, the most common primary malignant bone tumor, are relatively rare. In this study, 73 osteosarcoma specimens from 51 patients were cytogenetically analyzed following short-term culture. Clonal chromosomal abnormalities were detected in 47 and included one haploid specimen, 18 near-diploid specimens, 17 near-triploid, 8 near-tetraploid, 1 near-hexaploid, and 2 specimens with multiple clones of different ploidy levels. Examination of the present data and previously published data (111 clonally abnormal osteosarcoma specimens) reveals that chromosomal bands or regions 1p11-13, 1q10-12, 1q21-22, 11p15, 12p13,17p12-13, 19813, and 22ql1-13 are most frequently rearranged and the most common numerical abnormalities are +1, −9, −10, −13, and −17. Partial or complete loss of the long arm of chromosome 6 also was seen in all cases of the present study and all previously published cases describing structural abnormalities of 6q. Parosteal osteosarcoma, a prognostically favorable osteosarcoma subtype, was characterized by the presence of a ring chromosome accompanied by no or few other abnormalities. Complex karyotypes were seen nearly exclusively in the high-grade lesions. These findings indicate that specific chromosomal bands and/or regions are nonrandomly involved in osteosarcoma and may provide useful clinical information.
Ductal carcinoma in situ with microinvasion (DCISM) is a separate clinical and pathological entity, distinct from pure ductal carcinoma in situ (DCIS), with a low but well-known metastatic potential. Due to the low rate of axillary metastases in DCISM, there is controversy regarding the indication for complete axillary dissection (CAD) to stage the axilla. Sentinel lymph node biopsy (SNLB) could be routinely proposed to accurately stage the axilla avoiding the morbidity of a CAD. From March 1996 to December 2002, out of 4602 SLNBs performed for invasive carcinoma of the breast, 41 patients with DCISM in the definitive diagnosis were selected. Metastasis in the SLN were detected in 4 of 41 (9.7%) patients. Two of the 4 patients had only micrometastasis in the SLN. In three of these patients, the SLN was the only positive node after CAD. SLN biopsy should be considered as a standard procedure in DCISM patients. Complete AD may not be mandatory if only the SLN contains micrometastatic disease.
Inactivation of the deleted in colon cancer (DCC) gene on chromosome 18 is known to be associated with the tumorigenesis and metastasis of colorectal cancer. In the present study, we investigated the expression of DCC and the c-erbB-2 product in surgical specimens from 45 patients with breast cancer by immunohistochemical staining, and found the expression of DCC to be decreased in 23 (51%) tumors. In 8 years of follow-up, 11 of 22 (50%) patients with DCC-positive staining tumors, and 17 of 23 (74%) patients with DCC-negative tumors developed recurrence. The stratified analysis, according to the status of axillary lymph node metastasis, showed the same tendency. Overexpression of erbB-2 was detected in 13 (29%) of the 45 breast cancer specimens, but there were no differences in the relapse rate between patients with erbB-2 positive and those with erbB-2 negative tumors. Although the individual alteration of DCC or erbB-2 did not possess independent prognostic significance for the prediction of recurrence, patients with tumors having the double alteration of DCC-negative and erbB-2-positive showed adverse relapse-free survival (0.025PDCC expression and erbB-2 overexpression may influence the progression of breast carcinoma.
Estrogen, progesterone, and HER2 receptor-negative triple-negative breast cancers encompass the most clinically challenging subtype for which targeted therapeutics are lacking. We find that triple-negative tumors exhibit elevated MYC expression, as well as altered expression of MYC regulatory genes, resulting in increased activity of the MYC pathway. In primary breast tumors, MYC signaling did not predict response to neoadjuvant chemotherapy but was associated with poor prognosis. We exploit the increased MYC expression found in triple-negative breast cancers by using a synthetic-lethal approach dependent on cyclin-dependent kinase (CDK) inhibition. CDK inhibition effectively induced tumor regression in triple-negative tumor xenografts. The proapoptotic BCL-2 family member BIM is up-regulated after CDK inhibition and contributes to this synthetic-lethal mechanism. These results indicate that aggressive breast tumors with elevated MYC are uniquely sensitive to CDK inhibitors.
Although axillary lymph nodes status, tumour size, hormonal-receptor status and histological grade at diagnosis are frequently used to orient the treatment of breast cancer patients, some tumours recur in patients with early stage disease. Pre-operative assessment of individual tumour characteristics, based on oncogenes and growth factors related to tumour growth, invasion or metastasis, may guide the treatment for patients with breast carcinomas.
We examine here the prognostic significance of cyclin D1, urokinase type plasminogen activator, vascular endothelial growth factor (VEGF), platelet-derived growth factor, and c-erbB2 expression in pre-operatively obtained fine-needle aspirates from breast carcinomas less than or equal to 3 cm in size. Correlation between mRNA expression of these factors and clinicopathological characteristics was analysed.
The level of c-erbB2 mRNA expression was significantly higher in tumours with lymph node metastases than in those without lymph node metastases. VEGF mRNA expression positively correlated with the degree of angiogenesis as quantitated by immunohistological staining with a CD31 monoclonal antibody.
Analysis of c-erbB2 and VEGF mRNA expression in fine-needle aspirates may be useful in assessing the malignant potential of individual breast carcinomas, leading to a pre-operative discrimination of a high-risk group.
The expression of the c-erbB-2 oncogene has been evaluated using an immunohistochemical technique with the 21N polyclonal antibody in paraffin embedded tissue from 465 patients treated between the years 1975-1981 for Stage I and II breast cancer. One hundred and four (22%) patients exhibited positive staining. This was not associated with any other variables. Expression of the oncogene was associated with significantly poorer survival which was independent of other tumour variables.
The c-erbB-2 gene is overexpressed in about 20% of human breast cancers. Four hundred and eighty-three cases previously examined by immunohistochemical staining for c-erbB-2 expression were analysed to assess the risk associated with the elevated protein expression. Oncoprotein expression was correlated with increasing tumour grade but not with oestrogen receptor status, nodal involvement, tumour size or age. There was an increased risk of relapse and death associated with c-erbB-2 expression irrespective of nodal involvement. This marker thus appears to be a significant prognostic factor in the early as well as the late stages of breast cancer.
Immunohistological staining of primary colorectal carcinomas with antibodies specific to p53 demonstrated gross overexpression of the protein in approximately 50% of the malignant tumors examined. Benign adenomas were all negative for p53 overexpression. To determine the molecular basis for this overexpression we examined p53 protein expression in 10 colorectal cancer cell lines. Six of the cell lines expressed high levels of p53 in ELISA, cell-staining, and immunoprecipitation studies. Direct sequencing and chemical-mismatch-cleavage analysis of p53 cDNA by using the polymerase chain reaction in these cell lines showed that all cell lines that expressed high levels of p53 were synthesizing mRNAs that encoded mutant p53 proteins. In two of those four cell lines where p53 expression was lower, point mutations were still detected. Thus, we conclude that overexpression of p53 is synonymous with mutation, but some mutations would not be detected by a simple immunohistochemical analysis. Mutation of the p53 gene is one of the commonest genetic changes in the development of human colorectal cancer.
The tumour suppressor gene p53 has been found to be mutated or inactivated at high frequency in several common human tumours. We have examined a series of exocrine pancreatic carcinomas for over-expression of mutant forms of p53 by immunohistochemistry with a panel of specific antibodies. We found immunodetectable p53 in 13 of 22 (60%) frozen pancreatic cancers and seven of 13 pancreatic cell lines. One of the antibodies, CM1, recognises p53 in formalin-fixed, paraffin-embedded archival material and using this reagent we found immunodetectable p53 in 28 of 124 (23%) pancreatic cancers. We have successfully demonstrated the presence of point mutations by direct sequencing of genomic DNA extracted from archival tissue showing CM1 immunoreactivity. We conclude that p53 activation is an important event in human pancreatic tumorigenesis and that the CM1 antibody can detect a proportion of cases of overexpression of mutant p53 in archival pathological material.
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In the period from September 1980 to December 1987, our laboratory measured estrogen (ER) and progesterone receptor (PgR) levels in 960 patients with primary breast cancer. At presentation, 918 of these had no distant metastases. ER as well as PgR were considered positive at values above 10 fmol/mg cytosol protein. All the patients included had been operated on at one of two participating hospitals in the country of North Jutland, and all patients had been checked up in a uniform way at one oncological out-patient department. By applying test for interaction, the PgR was found to be dependent on nodal status. Separate multivariate analyses were carried out for node positive and node negative patients. By this method, size of tumor, histologic grading, and age turned out to be independent prognostic factors for the node negative patients. Independent prognostic parameters for the node positive patients were histologic grading, PgR and postoperative x-ray therapy. The results support the theory that PgR is a better predictor of disease-free survival than ER.
HER-2/neu protooncogene amplification and protein expression were analyzed with slot blot and Western blot techniques, respectively, in more than 300 invasive primary breast tumors of all stages. Amplification (2- greater than 30 copies) was found in 17% of these tumors and high expression was seen in 19%. There was a striking coincidence between gene amplification and high expression. Tumors associated with many involved axillary lymph nodes or with Stage IV disease were more often HER-2/neu amplified or overexpressed. Furthermore, gene alteration was strongly correlated with the absence of steroid receptors and with larger tumor size. High expression without gene amplification was seen in a minor subset of tumors of less aggressive character. Neither amplification nor overexpression was correlated with disease outcome for patients with negative axillary lymph nodes. For node-positive patients, however, HER-2/neu amplification was a significant predictor of early relapse and death (median follow-up = 45 months), and a similar trend, although not significant, existed for high gene expression. Multivariate analyses indicated that HER-2/neu alterations were not independent predictors of patient outcome.
Although bladder cancers are very common, little is known about their molecular pathogenesis. In this study, invasive bladder
cancers were evaluated for the presence of gene mutations in the p53 suppressor gene. Of 18 tumors evaluated, 11 (61 percent)
were found to have genetic alterations of p53. The alterations included ten point mutations resulting in single amino acid
substitutions, and one 24-base pair deletion. In all but one case, the mutations were associated with chromosome 17p allelic
deletions, leaving the cells with only mutant forms of the p53 gene products. Through the use of the polymerase chain reaction
and oligomer-specific hybridization, p53 mutations were identified in 1 to 7 percent of the cells within the urine sediment
of each of three patients tested. The p53 mutations are the first genetic alterations demonstrated to occur in a high proportion
of primary invasive bladder cancers. Detection of such mutations ex vivo has clinical implications for monitoring individuals
whose tumor cells are shed extracorporeally.
In this study, we have investigated the expression of the proto-oncogene c-erbB2 in a total of 70 human primary breast tumours. In agreement with other workers, we observed c-erbB2 gene amplification in 17.5% of the tumours studied. In addition, we carried out a comprehensive analysis of c-erbB2 mRNA expression in the tumours using RNase mapping and in situ hybridisation techniques. Our results indicated a more frequent (30%) overexpression of c-erbB2 mRNA, which was associated only with breast carcinomas of a ductal origin. Furthermore, analysis of the c-erbB2 mRNA gene locus in the same tumours demonstrated that enhanced c-erbB2 expression could occur in the presence or absence of gene amplification, suggesting that additional molecular mechanisms may result in overexpression of c-erbB2 mRNA in human mammary tumours. In situ hybridisation showed that elevated levels of c-erbB2 mRNA were specific to malignant cells within the breast tumour. Analysis of the association between c-erbB2 mRNA overexpression and clinicopathological factors revealed a significant correlation with poor tumour grade, but not with steroid receptor status or patient menopausal status. No significant correlation was observed between overexpression of c-erbB2 mRNA and early disease recurrence in our group of patients, although there was a definite trend towards poorer prognosis.
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Cell cycle checkpoints appear to contribute to an increase in cell survival and a decrease in abnormal heritable genetic changes following exposure to DNA damaging agents. Though several radiation-sensitive yeast mutants have been identified, little is known about the genes that control these responses in mammalian cells. Recent studies from our laboratory have demonstrated a close correlation between expression of wild-type p53 genes in human hematopoietic cells and their ability to arrest in G1 phase after certain types of DNA damage. In the present study, this correlation was first generalized to nonhematopoietic mammalian cells as well. A cause and effect relationship between expression of wild-type p53 and the G1 arrest that occurs after gamma irradiation was then established by demonstrating (i) acquisition of the G1 arrest after gamma irradiation following transfection of wild-type p53 genes into cells lacking endogenous p53 genes and (ii) loss of the G1 arrest after irradiation following transfection of mutant p53 genes into cells with wild-type endogenous p53 genes. A defined role for p53 (the most commonly mutated gene in human cancers) in a physiologic pathway has, to our knowledge, not been reported previously. Furthermore, these experiments illustrate one way in which a mutant p53 gene product can function in a "dominant negative" manner. Participation of p53 in this pathway suggests a mechanism for the contribution of abnormalities in p53 to tumorigenesis and genetic instability and provides a useful model for studies of the molecular mechanisms of p53 involvement in controlling the cell cycle.
Prognostic predictors for node-negative breast carcinoma have not been clearly established. Immunostaining with Ki-67 antibody was performed on frozen sections of histologically proved node-negative breast carcinomas from 42 patients to examine its prognostic value and its association with other clinicopathologic and biochemical parameters, i.e., patient age and tumor size, histologic type, nuclear grade, mitotic rate, presence of vascular or lymphatic invasion, DNA ploidy, percentage of cells in S-phase, estrogen content, and c-erbB-2 amplification. Thirty-seven of the 42 tumors showed immunoreactivity with Ki-67 antibody in 1% to 55% of the tumor cells. A strongly significant correlation was observed between Ki-67 staining percentage and, respectively, nuclear grade, age, and mitotic rate. Nuclear grade 1 (the most anaplastic) tumors showed a significantly higher median percentage of cells stained (median, 14; range, 3 to 40) compared with nuclear grade 3 tumors (median, 0.5; range, 0 to 8). Thirteen patients developed recurrence; six of them died of disease. On univariate analysis, both 5-year disease-free and overall survivals were strongly associated with percentage of cells stained with Ki-67 antibody. Our results suggest that Ki-67 immunostaining correlates well with nuclear grade and clinical outcome in node-negative breast carcinoma. Because of small sample size analyzed in this study we were unable to do multivariate analysis. Therefore, further studies with larger number of cases are needed to determine whether tumor proliferative activity determined by Ki-67 immunostaining is an independent prognostic parameter or it merely reflects histopathologic features such as nuclear grade or mitotic activity.
The analysis of censored failure times is considered. It is assumed that on each individual are available values of one or more explanatory variables. The hazard function (age‐specific failure rate) is taken to be a function of the explanatory variables and unknown regression coefficients multiplied by an arbitrary and unknown function of time. A conditional likelihood is obtained, leading to inferences about the unknown regression coefficients. Some generalizations are outlined.
Breast cancer proliferative capacity as determined by the DNA thymidine labeling index, along with estrogen and progesterone receptor status, is highly predictive for risk of relapse and overall survival. Recently, DNA ploidy and proliferative capacity (S-phase fraction [SPF]) as determined by flow cytometry have also shown significant prognostic value. The authors have developed a technique which allows a 50 to 100 mg aliquot of the same frozen breast tumor specimen routinely employed in steroid receptor assays, to be assayed for both DNA ploidy and SPF by flow cytometry. Of the 1331 tumors examined, DNA histograms were evaluable for ploidy in 89% (1184) of specimens examined; 57% of these were aneuploid. Adapting a trapezoidal model to estimate SPF in both diploid and aneuploid tumors, the authors found 81% (1084) to be evaluable for SPF, with a median SPF of 5.8% for the entire population. The median SPF was significantly lower in diploid tumors (2.6%) than in aneuploid tumors (10.3%, P < 0.0001). Both aneuploidy and high SPF were strongly associated with absence of steroid receptors. Aneuploid tumors showed more striking differences in the frequency of high S-phase values with respect to receptor status and age or menopausal status, whereas diploid but not aneuploid tumors showed lower SPF in node-negative versus node-positive patients. Because it is particularly important to identify the high-risk minority of node-negative patients, the authors examined the node-negative group separately. High SPF subgroups appeared in each category of receptor status and age or menopausal status within the node-negative group, suggesting that SPF will be an independent prognostic factor. With the DNA flow cytometric methods used here, it is now practical to determine ploidy and SPF for nearly every breast cancer patient. These factors, which show associations with established prognostic factors, such as receptor status can now be fully evaluated for their prognostic significance in broad patient populations.
Abstract Immunohistological staining of primary colorectal carcinomas with antibodies
specific to p53 demonstrated gross overexpression of the protein in approximately 50% of
the malignant tumors examined. Benign adenomas were all negative for p53
overexpression. To determine the molecular basis for this overexpression we examined p53
protein expression in 10 colorectal cancer cell lines. Six of the cell lines expressed high
levels of p53 in ELISA, cell-staining, and immunoprecipitation studies. Direct sequencing
In lifetesting, medical follow-up, and other fields the observation of the time of occurrence of the event of interest (called a death) may be prevented for some of the items of the sample by the previous occurrence of some other event (called a loss). Losses may be either accidental or controlled, the latter resulting from a decision to terminate certain observations. In either case it is usually assumed in this paper that the lifetime (age at death) is independent of the potential loss time; in practice this assumption deserves careful scrutiny. Despite the resulting incompleteness of the data, it is desired to estimate the proportion P(t) of items in the population whose lifetimes would exceed t (in the absence of such losses), without making any assumption about the form of the function P(t). The observation for each item of a suitable initial event, marking the beginning of its lifetime, is presupposed.
For random samples of size N the product-limit (PL) estimate can be defined as follows: List and label the N observed lifetimes (whether to death or loss) in order of increasing magnitude, so that one has \(0 \leqslant t_1^\prime \leqslant t_2^\prime \leqslant \cdots \leqslant t_N^\prime .\) Then \(\hat P\left( t \right) = \Pi r\left[ {\left( {N - r} \right)/\left( {N - r + 1} \right)} \right]\), where r assumes those values for which \(t_r^\prime \leqslant t\) and for which \(t_r^\prime\) measures the time to death. This estimate is the distribution, unrestricted as to form, which maximizes the likelihood of the observations.
Other estimates that are discussed are the actuarial estimates (which are also products, but with the number of factors usually reduced by grouping); and reduced-sample (RS) estimates, which require that losses not be accidental, so that the limits of observation (potential loss times) are known even for those items whose deaths are observed. When no losses occur at ages less than t the estimate of P(t) in all cases reduces to the usual binomial estimate, namely, the observed proportion of survivors.
Proefschrift Universiteit van Amsterdam. Met lit. opg. - Met samenvatting in het Nederlands. Auteursnaam op het omslag: M.J. van de Vijver.
Background:
Mutations of the tumor suppressor gene p53 have been identified in breast cancer cell lines, and some breast carcinomas are detectable by immunohistochemical assay because of p53 protein accumulation.
Purpose:
This study was designed to determine whether p53 protein accumulation in breast cancers correlates with p53 gene mutation, with survival, and with five pathobiologic factors associated with prognosis.
Methods:
IgG1 monoclonal antibody to human p53 protein (PAb 1801) and immunohistochemical methods were used to detect p53 protein accumulation in archival formalin-fixed, paraffin-embedded, randomly selected carcinomas. We studied 295 invasive ductal carcinomas from the Massachusetts General Hospital; 151 were determined to be sporadic (not hereditary). We also studied 97 invasive ductal carcinomas--21 sporadic and 76 familial (hereditary)--from Creighton University. In addition, we examined 31 archival in situ carcinomas, 15 snap-frozen invasive ductal carcinomas, primary cell cultures from three benign breast tissue samples, and breast carcinoma cell lines MDA-MB-231 and MDA-MB-468.
Results:
Nuclear p53 protein was observed in 16% of the 31 in situ carcinomas, 22% of the 172 sporadic carcinomas, 34% of the 50 tumors from patients with familial breast cancer, 52% of the 23 tumors from patients with the familial breast and ovarian cancer syndrome, and all three tumors from two patients with the Li-Fraumeni syndrome. There was complete concordance between p53 gene mutation and p53 protein accumulation in the 15 snap-frozen carcinomas and in both breast carcinoma cell lines. Statistically significant associations of p53 protein accumulation with estrogen receptor negativity and with high nuclear grade were found. There were statistically significant associations, independent of other prognostic factors, between p53 protein accumulation and metastasis-free and overall survival, for randomly accrued and for both sporadic and familial tumors.
Conclusions:
Immunohistochemically detected p53 protein accumulation was an independent marker of shortened survival and was seen more often in familial than in sporadic carcinomas. Our findings also suggest a correlation between p53 protein accumulation and p53 gene mutation.
Cell cycle checkpoints appear to contribute to an increase in cell survival and a decrease in abnormal heritable genetic changes following exposure to DNA damaging agents. Though several radiation-sensitive yeast mutants have been identified, little is known about the genes that control these responses in mammalian cells. Recent studies from our laboratory have demonstrated a close correlation between expression of wild-type p53 genes in human hematopoietic cells and their ability to arrest in G1 phase after certain types of DNA damage. In the present study, this correlation was first generalized to nonhematopoietic mammalian cells as well. A cause and effect relationship between expression of wild-type p53 and the G1 arrest that occurs after gamma irradiation was then established by demonstrating (i) acquisition of the G1 arrest after gamma irradiation following transfection of wild-type p53 genes into cells lacking endogenous p53 genes and (ii) loss of the G1 arrest after irradiation following transfection of mutant p53 genes into cells with wild-type endogenous p53 genes. A defined role for p53 (the most commonly mutated gene in human cancers) in a physiologic pathway has, to our knowledge, not been reported previously. Furthermore, these experiments illustrate one way in which a mutant p53 gene product can function in a "dominant negative" manner. Participation of p53 in this pathway suggests a mechanism for the contribution of abnormalities in p53 to tumorigenesis and genetic instability and provides a useful model for studies of the molecular mechanisms of p53 involvement in controlling the cell cycle.
Amplification and overexpression of the HER2 (c-erbB-2) oncogene was assessed in paraffin-embedded specimens from 27 in situ carcinomas of the breast and from 122 stage II breast cancers. Gene amplification detected in these archival tissues by differential polymerase chain reaction (PCR) was found in 48% of in situ carcinomas and in 21% of stage II lesions (chi 2 = 7.62, p less than or equal to 0.01). In addition, the level of gene amplification correlated with the level of HER2 oncoprotein expression as measured by immunohistochemistry for both in situ cancers (p less than or equal to 0.025) and stage II cancers (p less than or equal to 0.0005). This high incidence of HER2 gene amplification with accompanying overexpression in non-invasive breast tumors suggests that perturbations of the HER2 oncogene are among the earliest and most common genetic lesions in human breast cancer.
Overexpression of c-erbB-2 occurs in 60% of in situ and 25% of infiltrating ductal carcinomas. We have previously found very strong associations between immunohistochemical staining for c-erbB-2 and histological pattern and nuclear size in ductal carcinoma in situ (DCIS) and less strong correlation with proliferative activity. In a further study of infiltrating ductal carcinomas we have found that, in addition to tumours arising from c-erbB-2 positive, large celled, rapidly proliferating, comedo carcinomas and c-erbB-2 negative small celled cribriform/micropapillary carcinomas with a low proliferative rate, there is a third group of c-erbB-2 negative tumours with large nuclei and variable proliferative activity. These latter tumours are not seen in pure DCIS suggesting that they have a very transient in situ stage. Therefore, although in pure DCIS c-erbB-2 positively appears to be associated with tumours with a greater invasive potential, and c-erbB-2 negativity with tumours having a more favourable prognosis, the latter is not necessarily true in infiltrating disease.
Recent evidence indicates that a subset of axillary node-negative (ANN) breast cancer patients can benefit from adjuvant therapy. Reliable prognostic markers are needed, however, to help clinicians identify these patients and arrive at more rational treatment decisions.
Mutations of the p53 tumor suppressor gene often result in overexpression of the p53 protein. In this study, we evaluated the prognostic significance of p53 protein overexpression in patients with ANN breast cancer. We also studied the association between the tumor cell proliferation rate and overexpression of the p53 and c-erbB-2 proteins, both of which have been implicated in cell cycle control. The c-erbB-2 protein is the product of the ERBB2 gene.
Two hundred eighty-nine ANN cases were randomly selected from a population-based cohort of patients who had not received any kind of adjuvant chemotherapy or endocrine therapy. Overexpression of the p53 and c-erbB-2 proteins was studied immunohistochemically in archival paraffin-embedded tumor samples, using the CM-1 polyclonal and the TAb 250 monoclonal antibodies, respectively. The tumor cell proliferation rate was measured as the S-phase fraction by DNA flow cytometry. Statistical analyses were performed using BMDP software.
High-level p53 protein overexpression, found in 41 of the 289 tumors, was most common in tumors with high histologic grade, negative estrogen receptor status, c-erbB-2 protein overexpression, DNA index greater than 1.3, or high S-phase fraction. The lowest S-phase levels were found in tumors with neither p53 nor c-erbB-2 protein overexpression; the highest levels were seen in tumors showing overexpression of both proteins (P less than .0001). Both p53 and c-erbB-2 overexpression, as well as tumor size, had independent prognostic value in multivariate analysis. Eight-year survival of patients with p53 protein overexpression was 56% compared with 81% in patients with no overexpression (relative risk, 3.7; P less than .0001). If the S-phase fraction was included in a Cox regression analysis, however, only the tumor size and the S-phase fraction emerged as independent predictors of survival.
Overexpression of the p53 and c-erbB-2 proteins indicates a high malignant potential in ANN breast cancer, but it is not a significant prognostic factor independent of the cell proliferation rate. The correlation between overexpression of these proteins and an increased S-phase fraction suggests that they may confer a proliferative advantage to cancer cells in vivo.
To study retrospectively the long-term prognostic significance of HER-2 oncoprotein expression in breast cancer (BC).
Two hundred nine consecutive female patients with invasive operable BC from a defined urban population were observed for a median of 30 years. Tissue expression of HER-2 oncoprotein was demonstrated by using an immunoperoxidase procedure.
Fifty-five (26%) patients had cancer and a positive HER-2 oncoprotein stain reaction. They had significantly worse 10- and 25-year survival rates than those patients who had a negative stain reaction in their cancer (31% v 48% and 31% v 39%, respectively; P = .004). HER-2 expression was also associated with a poorer survival among patients who had axillary nodal metastases (P = .003; n = 104), but not among those patients who did not have metastases. HER-2 expression was related to the ductal histologic type, poor histologic grade, and high mitotic count, but not to tumor size, axillary nodal status, DNA ploidy, or S-phase fraction (SPF). In a multivariate analysis among patients with nodal metastases, HER-2 expression was an independent prognostic factor (P = .04) that predicted poor survival. However, if the entire series was entered onto the analysis, it did not emerge as an independent factor.
HER-2 oncoprotein expression has long-term prognostic significance for predicting poor survival in BC, and it has an independent prognostic value among patients who presented with axillary nodal metastases. This result is contradictory to the argument that HER-2 expression is only a marker for drug resistance because the patients were not given adjuvant drug therapy.
Inactivation of tumour suppressor genes may be an important aetiological factor in many human cancers including breast. In a study of 197 breast cancer patients, tumour tissue was snap-frozen at the time of surgery and immunohistochemical labelling for p53 protein and retinoblastoma (Rb) gene product carried out using an indirect immunohistochemical technique. Tumours were scored by two independent observers for the intensity of nuclear staining for each antibody. Expression of p53 protein showed a significant association with a shorter time to relapse (P = 0.03) and death (P = 0.02) (log rank test). p53 expression did not correlate with nodal status but showed a significant association with high tumour grade (P = 0.001). Rb gene expression showed no relationship to relapse or survival but loss of expression showed a significant correlation with positive lymph node status. The manner by which these proteins might act to determine tumour behaviour remains to be established.
Loss of cell cycle control and acquisition of chromosomal rearrangements such as gene amplification often occur during tumor progression, suggesting that they may be correlated. We show here that the wild-type p53 allele is lost when fibroblasts from patients with the Li-Fraumeni syndrome (LFS) are passaged in vitro. Normal and LFS cells containing wild-type p53 arrested in G1 when challenged with the uridine biosynthesis inhibitor PALA and did not undergo PALA-selected gene amplification. The converse occurred in cells lacking wild-type p53 expression. Expression of wild-type p53 in transformants of immortal and tumor cells containing mutant p53 alleles restored G1 control and reduced the frequency of gene amplification to undetectable levels. These studies reveal that p53 contributes to a metabolically regulated G1 check-point, and they provide a model for understanding how abnormal cell cycle progression leads to the genetic rearrangements involved in tumor progression.
The p53 locus on the short arm of chromosome 17 at 17p 13.1 was examined for loss of heterozygosity, mutation, mRNA and protein expression in 60 primary breast cancers. Allele loss around the p53 locus was detected in 19/45 informative tumours (42%). p53 mutations in the evolutionarily conserved exons 5 to 9 were detected in 17/60 (28%) by amplification mismatch and confirmed by direct DNA sequencing. p53 mRNA expression was detected by Northern blot in 36/59 (61%) of tumours, and p53 protein expression using antibody 1801 on frozen-tissue sections in 13/44 of the tumours examined. p53 mutation was significantly associated with oestrogen-receptor-poor tumours (p less than 0.01) and hence with poor prognosis, but not with other clinical or pathological parameters. There was no statistical correlation between loss of heterozygosity around the p53 locus at 17p13.1 and p53 mutation. Furthermore, p53 mutation was not associated with p53 expression detected by immunohistochemical staining with antibody 1801 or as p53 mRNA. In addition, events on 17p (allele losses, p53 mutation, p53 expression) were independent of c-erbB-2 expression. In breast cancer, by contrast with colorectal, lung and ovarian cancer, there appears to be no clear association between p53 DNA abnormalities and p53 expression.
An international trial (formerly Ludwig Trial V) has been conducted in 1,275 subjects to ascertain if perioperative chemotherapy is beneficial for node-negative breast cancer patients and to identify subgroups of patients who benefit from this therapy.
Node-negative breast cancer patients were randomized to receive either one cycle of perioperative chemotherapy or no adjuvant treatment. A detailed pathology review was conducted in 1,203 of the 1,275 patients enrolled. Stepwise Cox regression analysis was used to search for factors either predicting chemotherapeutic responsiveness and/or influencing disease-free survival (DFS).
As expected, primary tumor size, grade, and the presence of peritumoral vascular invasion are the most important prognostic factors. Perioperative chemotherapy provides a DFS advantage at 5 years of median follow-up and such treatment is more effective for estrogen receptor-negative than for estrogen receptor-positive tumors, for histologic grade 2 and 3 than for grade 1 tumors, and for patients in whom no axillary lymph node metastases were found even after serial sectioning and review by the Central Pathology Laboratory.
Hormone receptor status and tumor grade are important factors for predicting responsiveness to perioperative chemotherapy in node-negative breast cancer.
c-erbB-2 and ras expression was measured on tumor extracts from 132 human primary breast carcinomas, by immunoblotting analysis. Expression of the c-erbB-2-encoded p185 protein was observed in 39% of the samples and found to correlate with c-erbB-2 gene amplification, detected by Southern analysis in 19 of the 77 available tumor DNAs. p185 expression was linked to the absence of progesterone receptors, but it was not related to lymph-node status or to other clinico-pathological parameters. Levels of the ras-encoded p21 proteins higher than in normal breast tissues were found in 71% of the samples. No significant correlation was seen between p21 level and the available clinical parameters. Conversely, there was a strong positive correlation between p21 and p185 levels. Analysis of follow-up data revealed that p185 expression was associated with a shorter time to relapse and death. Most notably, the contemporaneous expression of p185 and of high p21 levels was more effective than p185 expression alone in identifying cases with poor prognosis. The prognostic value of p185/p21 co-expression was particularly significant in progesterone-receptor-positive tumors. Our data suggest that c-erbB-2 and ras may act synergistically to endow breast-tumor cells with a highly aggressive phenotype.
In a retrospective multicenter study to investigate the correlation between estrogen (ER) and/or progesterone receptors (PgR) in primary breast cancer with patient prognosis, 3118 patients with operable breast cancer (International Union Against Cancer Stages I, II, and III) were investigated from ten hospitals in Japan who underwent surgery from October 1972 to December 1982; 3089 were evaluable. The ER-positive and PgR-positive cancers were found in 56% and 34% of patients, respectively. The positivities decreased as the tumor size increased but were independent on lymph node metastasis. There were no significant differences in relapse-free survival (RFS) in relation to receptor status (median follow-up, 89 months [ER], 84 months [PgR]). However, in patients with four or more positive nodes, those with PgR-positive cancer had a longer RFS. The patients with ER-positive cancer survived significantly longer than ER-negative ones, with the greatest difference seen in those with four or more positive nodes. There was a significantly longer postrelapse survival (PRS) for patients with ER-positive cancer because of the different distribution of the major metastasis and better responses to first-line and subsequent treatments. Cox's multivariate analysis showed that overall survival but not PRS was affected by ER (and more weakly by PgR) because of the longer PRS in patients with ER-positive cancer.
The purpose of this review is to incorporate recent discoveries into a general biochemical pathway by wich the protein products of the oncogenes send signals from the cell surface to the nucleus .The protein-tyrosine kinase oncogenes will be the primary focus of the review .However, biochemical connections between the protein tyrosine kinases and oncoproteins of the Ras,Raf,Fos,Jun,and Rel families as well as the protein kinase C family are also discussed .
Primary lung cancer samples of the major histological types were examined for expression of the tumor suppressor gene p53 by immunohistochemistry. Abnormalities in p53 expression were found in 28 of 40 carcinomas, 14 of 17 squamous tumours showing abnormal p53 expression, whereas no expression of p53 was detectable in 7 carcinoid tumours or in 10 normal lung samples. Direct evidence for homozygous expression of mutant p53 mRNA in representative carcinomas was obtained by means of an asymmetric polymerase chain reaction mRNA sequencing strategy, which allowed sequencing without any cloning step. All the mutations were G to T transversions resulting in mis-sense mutations in aminoacids highly conserved in evolution. Mutation of the p53 gene is the most frequently identified genetic change in human lung cancer; these findings suggest that simple immunohistological methods can provide strong evidence of such mutation.
A chimeric gene comprising the hydroxymethylglutaryl coenzyme-A reductase promoter and the human GH (hGH) genomic sequences was used to create transgenic mice expressing hGH in all tissues. In transgenic females, morphological development of the mammary gland and milk protein (WAP) expression commences at 3 weeks of age. At 8 weeks of age the mammary gland is morphologically and functionally comparable to that normally reached after 14-15 days of gestation. Precocious development correlated with local expression of hGH in mammary gland. Organ culture in the presence of different lactogenic hormones revealed that insulin and hydrocortisone are sufficient to maintain transcription of the WAP gene in transgenic mammary gland. In contrast, WAP transcription in normal gland required either hGH or PRL in addition to insulin and hydrocortisone. However, the effect of hGH on mammary differentiation does not appear to be solely mediated through an interaction with PRL receptors, since PRL, when added to cultured mammary tissues, did not elicit an equivalent response.
At present ,lesions in the proto-oncogenes and tumor suppressor genes seem almost equally prevalent among human cancers ,although the number of tumor suppressor genes afflicted in any given tumor may be greater ,and examples exist in wich only one (or even no) genetic lesion has been detected .It seems likely ,however thatmost malignancies arise from the collaborative effects of dominant and recessive lesions ,enumeration of wich is now in progress ;
Three monoclonal antibodies (MAbs) (DF3, F36/22, CU18) were used to monitor expression of distinct epitopes present within a family of mucin-like, breast carcinoma-associated molecules. Primary tumor specimens from more than 190 stage II breast cancer patients were evaluated for expression of the high molecular weight antigens. With a median follow-up of 6 years, patients whose tumors exhibited high immunoperoxidase staining scores (greater than 50% positive cells) with MAb DF3 had a superior disease-free survival ([DFS] 56% +/- 6% v 37% +/- 5% at 6 years; P = .0088) and overall survival ([OS] 72% +/- 5% v 59% +/- 5% at 6 years; P = .025). Staining scores with the other two antibodies did not correlate with improved prognosis. For MAbs DF3 and CU18, patients whose tumors exhibited predominantly apical cellular reactivity patterns had improved DFS, although differences reached conventional levels of statistical significance only with MAb CU18. In multivariate analyses, the prognostic value of MAb DF3 staining was independent of other identified prognostic factors. Furthermore, the concordance between primary and axillary lymph node metastases staining with each MAb was 73%, 80%, and 85% for MAbs DF3, F36/22, and CU18, respectively. These results suggest that staining with MAb DF3 identifies a group of node-positive women with a relatively favorable prognosis. Expression of the DF3 mucin-like glycoprotein is related to better differentiation, and staining with MAb DF3 provides an accurate and objective estimate of clinical outcome independent of histopathologic evaluation.
The promoter of the mammary specific murine whey acidic protein gene was used to direct Ha-ras expression in different lines of transgenic mice. We found that this promoter contains a tissue specific enhancer which directed expression in both orientations albeit to different levels. We used this feature to generate low and high ras expressing transgenic lines. The reversed orientation led to a weak expression in lines 3 and 58 and to a tumor frequency of 2%. In contrast, 72% of mice from line 25 showing high ras expression developed mammary tumors. Nulliparity is one risk factor for human breast cancer, suggesting a protective effect of post-lactational mammary regression. In order to investigate the effect of post-lactational regression, the low tumor frequency lines were crossed with mice expressing ubiquitously the human growth hormone gene, which induces permanent development of the mammary epithelium. Indeed, mammary tumors were observed in 76% of double transgenic females. Thus, the tumorigenic potential of the ras oncogene in mammary cells in vivo correlates with the level of its expression and with the developmental history of the mammary gland. Transformation coincides with the escape of oncogene expression from the regulation of the Wap promoter and the extinction of endogenous Wap gene expression.
In a study of 90 breast cancer patients, tumour p53 protein expression was determined by immunohistochemistry using the monoclonal antibody PAb1801. Patient lymph node status and Bloom's grade were determined, and both oestrogen and progesterone status assessed, also by immunohistochemistry. Lymph node status, tumour grade, and progesterone receptor status all had a significant influence on survival. Patients with p53-positive tumours showed poorer survival but this did not achieve significance. p53 protein expression showed a significant relationship to high tumour grade and a weak correlation with negative oestrogen receptor status. The data suggest that p53 protein expression may be a marker of more aggressive carcinomas but that the prognostic power of expression is likely to be weak and unlikely, therefore, to be of clinical value. The results do not resolve whether detectable p53 protein expression represents a random product of dedifferentiation, or an important feature of the malignant phenotype, playing a key role in tumour behaviour. The number of patients in our study is small, however, and investigation of a larger series is clearly indicated.
Five oncogenes have been implied as having a role in human breast tumorigenesis: int-2, c-erbB-2 (HER-2), c-myc, c-Ha-ras and the recessive Rb-1. As far as the function and biochemistry of these oncogenes have been studied, they act at different levels and have totally different functions in the cells. They are normally cellular genes, likely to have important functions in normal cell growth or differentiation. In the tumors their regulation or function is altered, due to a wide class of mutations. The oncogenes may cooperate to result in the malignant cell phenotype. However, different oncogenes are mutated in different tumors, so that the tumors show a variable pattern at the molecular level, underlining the individuality of these tumors already described as differences in histopathology, hormone receptor expression and clinical course. The main importance of the oncogene studies is still to reveal basic pathogenetic mechanisms. When appropriate it is important to test diagnostic or prognostic significance of the oncogene mutations.
Characterization of breast cancer cells by histology, flow cytometry, and steroid receptors was performed on 197 Stage II breast node positive cancer patients given adjuvant chemotherapy, plus tamoxifen for patients with positive hormone receptors. Histologic and steroid receptor assays were performed using standard techniques; flow cytometric analysis was performed from paraffin-embedded blocks obtained from the primary tumor. Quality control studies on reproducibility, tissue heterogeneity, and analysis procedures have been included. Of the 197 patients studied, aneuploidy was found in 102 (52%); the median %S value was 8% with a range of 0.4% to 38%. Our results demonstrated that number of positive nodes, receptor status, and grade were of prognostic value. Cell cycle kinetic data were not of independent prognostic value in this series. However, ploidy could differentiate in prognosis in the receptor-negative subgroup. Patients with receptor-negative tumors had a significantly better overall survival if the tumor was diploid in nature. Cell kinetics was not significantly prognostic for either receptor subgroup, although patients with higher %S tended to have better relapse-free and overall survival. This is in disagreement with other studies and may demonstrate that treatment has confounded our results and diminished the ability of flow cytometry data to help predict outcome.
We investigated the possibility that cathepsin D, an estrogen-induced lysosomal protease, might have value as a prognostic factor in breast cancer by studying frozen tissue specimens from 397 patients. We measured the 34-kd mature form of the enzyme by Western blot assay and densitometry. Among 199 patients with node-negative disease, but not among 198 with node-positive disease, high levels of cathepsin D proved to be a significant predictor of reduced disease-free survival (median follow-up, 64 months), either as a continuous variable (log cathepsin D; P = 0.018) or as a dichotomous variable with an optimized cutoff point (P = 0.0001). Results were similar for overall survival (P = 0.009 and 0.0001, respectively). Relating the level of cathepsin D to other prognostic factors in the patients with node-negative disease, we found an association with aneuploidy but none with estrogen or progesterone receptors, tumor size, or the age of the patient. In multivariate analyses, a high level of cathepsin D was the most important independent factor in predicting shorter disease-free and overall survival in patients with node-negative disease. As compared with the risk in women with low levels of cathepsin D, the relative risk of tumor recurrence was 2.6 (95 percent confidence interval, 1.6 to 4.4) and the relative risk of death was 3.9 (95 percent confidence interval, 2.1 to 7.3) among those with high levels of cathepsin D. For disease-free survival, cathepsin D status was predictive of outcome primarily among those with aneuploid tumors; the actuarial five-year recurrence rates of aneuploid tumors were 60 percent among women with high levels of cathepsin D and 29 percent among those with low levels, as compared with 22 percent for all diploid tumors. We conclude that cathepsin D may be an independent predictor of early recurrence and death in node-negative breast cancer.
The isolation and construction of a complete human p53 cDNA and subsequent expression in monkey cells is described. A set of new anti-(human p53) monoclonal antibodies has also been obtained and used to show the expression of the human p53 cDNA in cos-l cells. These antibodies enable the specific detection of human p53, which is synthesised in the presence of p53 from other species. Fusion proteins of p53 with beta-galactosidase were used firstly as antigen and secondly, in conjunction with competition assays, to localise the determinants recognized by the antibodies. At least two previously unrecognized epitopes are involved and two of the antibodies are human-p53-specific. The epitopes are denaturation-resistant and the antibodies are, therefore, valuable for immunoblotting as well as immunoprecipitation and enzyme-linked immunoassay. Transfection of plasmids containing complete human p53 cDNA into monkey (cos-l) cells cause expression of human p53 recognized by the monoclonal antibodies. Control plasmids did not induce immunoreactive protein.
Prognostic factors have been examined in 644 patients with tumor-node-metastasis (TNM) stage T1 breast carcinoma treated by mastectomy and followed for a median of 18.2 years. Overall, 148 patients (23%) died of recurrent breast carcinoma. Eighteen (3%) were alive with recurrent disease and 478 (74%) were alive or died of other causes without recurrence. Unfavorable clinicopathologic features were larger tumor size (1.1 to 2.0 cm v less than or equal to 1 cm), perimenopausal menstrual status, the number of axillary lymph node metastases, poorly differentiated grade, presence of lymphatic tumor emboli (LI) in breast tissue near the primary tumor, blood vessel invasion (BVI), and an intense lymphoplasmacytic reaction around the tumor. Median survival after recurrence for the entire series was 2 years. This was not significantly influenced by tumor size, the number of axillary nodal metastases, the type of treatment for recurrence, or the interval to recurrence. The proportions surviving 5 and 10 years after recurrence were 17% and 5%, respectively. Among T1N0M0 cases, the chance of a local recurrence was 2.8% within 20 years. Median survival of T1N0M0 cases after local recurrence (4.5 years) was significantly longer than after systemic recurrence (1.5 years). A similar trend (3.7 v 2.0 years), not statistically significant, was seen in T1N1M0 patients, who had a 6.5% chance of local recurrence within 20 years. Median survival following systemic recurrence detected 10 or more years after diagnosis in T1N0M0 and in T1N1M0 patients was significantly longer than the median survival for systemic recurrences found in the first decade of follow-up. This difference did not apply following local recurrence in either T1N0M0 or T1N1M0 cases. It is evident that patients with T1 breast carcinoma can be subdivided into differing prognostic groups and this must be taken into account when considering the role of adjuvant chemotherapy for stage I disease. Systemic adjuvant treatment may prove to be beneficial for patients with unfavorable prognostic factors, while women with an especially low risk for recurrence (eg, T1N0M0 tumor 1.0 cm or less) might be spared such treatment.
Amplification of c-myc, c-erbB-2, hst and int-2 proto-oncogenes was investigated in two independently collected breast tumor series comprising 292 carcinomas. Differences in the frequencies of amplification could be observed between these two series for c-myc (9.3% vs. 20.8%) and hst/int-2 (21.5% vs. 15.6%) whereas similar values were found for c-erbB-2 (22.5% vs. 20.3%). Statistical correlations between amplification and disease parameters were also dependent on population sampling. Therefore we performed our statistical analysis on the pooled populations and focused on the 219 primary breast carcinomas from patients without therapy prior to surgery. Amplification of c-erbB-2 was strongly correlated to the absence of either estrogen (ER-, P = 0.003) or progesterone (PR-, P = 0.004) receptors. An amplified c-myc was significantly associated with PR- (P = 0.005) and was prevalent in high grade tumors. On the contrary, hst/int-2 amplification was correlated to PR+ tumors (P = 0.01) and was more frequent in ER+ and low grade tumors, and was also correlated with lymph node involvement (P = 0.04). Our data suggest that amplification of each of these proto-oncogenes could be representative of a particular subset of breast tumors. Therefore, proto-oncogene amplification may be helpful in characterizing new biological subclasses in human breast cancer.
Amplification of the HER-2/neu oncogene was recently reported to predict poor clinical outcome in node-positive breast cancer patients. Since expression of the oncogene as its protein product might be even more closely related than gene amplification to disease progression, we have now examined levels of the HER-2/neu oncogene protein for its prognostic potential in both node-positive and node-negative breast cancer. Using Western blot analysis, levels of this protein were determined in 728 primary human breast tumor specimens. We examined relationships between this protein and other established markers of prognosis, as well as clinical outcome. In node-negative patients (n = 378), the HER-2/neu protein failed to predict disease outcome. However, in node-positive patients (n = 350), those patients with higher HER-2/neu protein had statistically shorter disease-free (P = .0014) and overall survival (P less than .0001) than patients with lower levels of the protein. Higher HER-2/neu protein was found in tumors without estrogen receptor (ER) (P = .02) or progesterone receptor (PgR) (P = .0003), and in patients with more than three positive lymph nodes (P = .04). A significant correlation between levels of the HER-2/neu gene protein and amplification of the gene itself was also found (n = 48, P less than .001). Multivariate analyses in these patients showed that the HER-2/neu protein is a significant independent predictor of both the disease-free and the overall survival in node-positive breast cancer, even when other prognostic factors are considered.
Studies of mammary tumorigenesis in mice infected with the mouse mammary tumor virus and in certain strains of transgenic mice with an activated oncogene have provided strong evidence that multiple mutations contribute to the initiation and progression of malignancies in the breast. The increasing availability of recombinant DNA probes that detect various proto-oncogenes, growth factor genes, and growth factor receptor genes, as well as restriction fragment length polymorphisms in the human population, have made possible a molecular approach for the identification of frequently occurring mutations in primary human breast tumor DNA. The aim of studies using this molecular approach has been to investigate whether specific mutations are highly associated with various clinical parameters, including disease prognosis. Eight mutations have been identified, including amplification of c-myc, c-erbB2, and int-2, as well as loss of heterozygosity on five chromosomes (1q, 3p, 11p, 13q, and 17p). Loss of heterozygosity is thought to unmask recessive mutations of tumor-suppressor genes. In some studies, amplification of either c-myc, c-erbB2, or int-2 has been found to have a significant association with high risk of relapse or poor survival. The current status of these mutations as potentially useful prognostic indicators for the management of the disease is controversial and points to the need for further research in this area.
This study correlates the disease-free survival (DFS), distant disease-free survival (DDFS), and survival (S) of 1,157 histologically node negative breast cancer patients with the estrogen and/or progesterone receptor (ER, PR) and with the nuclear or histologic grade (NG, HG) of their tumors. All were treated by operation without systemic adjuvant therapy. The DFS, DDFS, and S were significantly greater (P = .005, .004, less than .001) in patients with ER positive than ER negative tumors but the magnitude of the differences after 5 years of follow-up was slight (8% in both DFS and DDFS and 10% in S). Differences of that magnitude are insufficient to discriminate clearly between patients who should or should not receive systemic therapy. As with ER, there were outcome differences in favor of PR positive tumors but only in S was the difference significant (8% at 5 years; P = .002). When combined with ER, PR made no independent contribution in the outcome prediction. Regression analysis indicated that NG was the most important single marker of outcome. The prognosis of women with unknown ER or PR was equivalent to or better than that in those with ER or PR positive tumors. This finding seems to be related to tumor size in that a higher proportion of tumors with unknown receptors were less than 1.0 cm, thus having insufficient tissue for analysis. Our findings disclose that in node negative breast cancer patients, NG is a better marker of prognosis than is tumor ER, and that PR is of little or no value. Tumor NG may also be useful for selecting the type of systemic therapy to be used in these patients.