There is an increasing demand for the evaluation of HER2 status in breast cancer. In this study, sections from fixed tissues and triton extracts of tissue homogenates were obtained from 163 malignant breast tumors and analyzed in parallel using immunohistochemistry combined with fluorescence in situ hybridization, as gold standard tests, and an ELISA test (c-erbB2/c-neu Rapid Format ELISA, Oncogene Research Products, USA). Tumor DNA was employed to evaluate two quantitative PCR methods: the HER2/neu DNA Quantification Kit (Roche Diagnostics GmbH, Germany), which uses the gastrin chromosome 17 reference gene, and our recently developed Oncolab qPCR assay, where both a chromosome 17 gene (somatostatin receptor type II (SSTR2)) and a non-chromosome 17 reference gene (glyceraldehyde-3-phosphate deshydrogenase (GAPDH)) were used to detect an increase in HER2 gene copy number and to evaluate the aneusomy of chromosome 17, respectively. By IHC/FISH and ELISA, HER2 was overexpressed in 27 (16.6%) and 24 (14.7%) samples, respectively. With the Roche and Oncolab qPCR assays, 29 (17.8%) samples showed a ratio of HER2/gastrin > or = 2.0 and 26 (16.0%) showed a ratio of HER2/SSTR2 > or = 2.0, respectively. In samples presenting HER2/SSTR2 <2.0 and HER2/GAPDH > or = 2.0, which was indicative of a chromosome 17 polysomy, we observed a modest increase in HER2 protein expression. Complete agreement between the four methods for HER2 status determination was obtained for 154 (94.5%) samples. Overall, these results demonstrate that quantitative PCR is a reliable method for analyzing HER2 status and chromosome 17 polysomy.

Abstract In human breast cancer, estrogen receptor-α (ERα), progesterone receptor (PR) and human epidermal growth factor receptor (ERBB2) status are currently determined using different techniques. We propose to assess the mRNA expression of these three clinically relevant markers using a unique technique, real-time nucleic acid sequence-based amplification (NASBA). Gene expression of hormone receptors was analyzed and compared to the cytosolic functional protein content as determined with a ligand binding assay (LBA), while ERBB2 mRNA expression was compared to quantitative PCR and ELISA. We observed that the three markers are significantly overexpressed at the mRNA level in positive tumors, as measured by DNA- or protein-based techniques. Biostatistical analysis of the receiver operating characteristic (ROC) curve demonstrated high concordance between NASBA and LBA [area under the curve (AUC) for ROC of 0.899] and showed that ERα status could be predicted using the molecular assay with a sensitivity of 72.7% and a specificity of 93.5%. Similar results were obtained for PR (AUC ROC 0.938, sensitivity 75.3%, specificity 100%). Moreover, excellent concordance was observed between NASBA, quantitative PCR and ELISA with respect to ERBB2 (AUC ROC 0.92, sensitivity 90%, specificity 89.7%; and AUC ROC 0.98, sensitivity 100%, specificity 91.5%, respectively). These results suggest that NASBA is well suited for assessing ER, PR and ERBB2 status in breast tumor samples. This approach is rapid, highly sensitive and a standardized method that could be complementary to the existing techniques, especially for small tumors.

The aim of this study was to evaluate, in a prospective study, the predictive role of p53 status analysed at four different levels in identifying the response to preoperative radiotherapy in rectal adenocarcinoma. Before treatment, 70 patients were staged and endoscopic forceps biopsies from the tumour area were taken. p53 status was assessed by total cDNA sequencing, allelic loss analysis, immunohistochemistry, and p53 antibodies. Neoadjuvant treatment was based on preoperative radiotherapy or radiochemotherapy. Response to therapy was evaluated after surgery by both pathologic downstaging and histologic tumour regression grade. In all, 35 patients (50.0%) had p53 gene mutations; 44.4% of patients had an allelic loss; nuclear p53 overexpression was observed in 39 patients (55.7%); and p53 antibodies were detected in 11 patients (16.7%). In the multilevel analysis of p53 status, gene mutations correlated with both nuclear protein overexpression (P<0.0001) and loss of heterozygosity (P=0.013). In all, 29 patients (41.4%) were downstaged by pathologic analysis, and 19 patients (29.2%) were classified as tumour regression grade 1. Whatever the method of evaluation of treatment response, no correlation between p53 alterations and response to radiotherapy was observed. Our results do not support the use of p53 alterations alone as a predictive marker for response to radiotherapy in rectal carcinoma.

Single nucleotide primer extension reaction has been widely used in DNA testing, and several detection methods based on this core allelic discrimination have been developed. Most of the reported formats are based on a two step protocol involving first, a liquid phase extension reaction, then a physical separation process (chromatography, electrophoresis, capture on solid support, mass spectrometry). Here we describe a new strategy based on homogeneous time-resolved fluorescence (HTRF), which does not involve any separation process and which allows a simple "mix and measure" protocol. In this approach, a 5'-(europium) cryptate-labeled primer is elongated by a biotinylated dideoxynucleoside-triphosphate, followed by the addition of a streptavidin-acceptor conjugate, which gives rise to a long-life fluorescence resonance energy transfer (FRET) signal between the cryptate donor and the acceptor. We present the development of HTRF technology as applied to the diagnosis of tumor suppressor gene p53 (TP53) mutations, and its application to the analysis of genomic DNA from human tumoral samples. The sensitivity of the reported method is compared to the corresponding fluorescent polarization assay.

Les differences phenotypiques et metaboliques que presentent les cellules tumorales ont ete mises a profit pour le diagnostic des metastases. Ainsi, les marqueurs tumoraux sont des molecules libereesdans la circulation generale et qui signalent apres exerese de la tumeur primaire une reprise silencieuse de la maladie. On considere que moins de 20 % des elevations initiales de marqueurs tumoraux sont accompagnes de signes cliniques ou radiologiques. Cette situation a conduit la communaute medicale a mettre en doute l'interet de marqueurs qui annoncent une maladie invisible. Le developpement de la tomographie par emission de positons au 1 8 fluorodeoxyglucose (TEP- 1 8 FDG) permet de visualiser des tumeurs a un stade precoce de developpement comme de detecter les metastases. La mise en oeuvre de cette exploration fonctionnelle ne peut se faire que sur un ensemble d'arguments informatifs dont les marqueurs tumoraux sont partie prenante. Examens precocement positifs, peu invasifs, et de faible cout, ils deviennent un complement et une aide a la prescription de TEP- 1 8 FDG en cancerologie.

RESUMO: O objectivo da presente meta-análise foi determinar o valor prognóstico de níveis séricos pré-terapêuticos elevados de Cyfra 21-1, ajustados a co-variáveis clássicas no CPNPC.Baseou-se em elementos registados em bases de dados de estudos controlados (período de 1993 a 2001), publicados e não publicados, apresentados na forma de abstracts ou incluídos em palestras, conferências ou outras apresentações, que tinham como principal end point a determinação do valor prognóstico dos níveis séricos pré-terapêuticos elevados de Cyfra 21-1, cotejados com outras variáveis prognósticas no CPNPC (estádio TNM, PS, e outras) e em que se teve, também, em atenção o tipo de tratamento instituído (cirurgia versus não cirurgia).Foram seleccionadas nove instituições que seguiram 2063 doentes com CPNPC por um período compreendido entre 27 e 78 meses.A sobrevida foi definida como o tempo decorrido entre a data da determinação do valor sérico pré-terapêutico de Cyfra 21-1 até à data da morte do doente. Considerou-se como valor cut-off do mar cador os 3,6 ng/ml; os doentes foram divididos em dois grupos etários, acima e abaixo da mediana de idades, isto é, dos 63 anos.Os elementos colhidos foram analisados estatisticamente utilizando-se o método de Kaplan-Meir para a determinação da distribuição das sobrevidas, enquanto na análise univariada teve-se em conta o teste log-rank e no estudo da regressão multivariada o modelo de Cox.Na série apresentada predominou o sexo masculino (84%), estando 51% dos doentes com um PS de 0-1; 70% dos doentes estavam incluídos no estádio IIIB (27%) ou IV (43%); 50% eram carcinomas epidermóides, só 21% foram submetidos a terapêutica cirúrgica e 49% revelaram valores pré-terapêuticos de Cyfra 21-1 acima de 3,6 ng/ml.Pela análise univariada das sobrevidas, constatou-se que estas foram piores no grupo com Cyfra 21-1 aumentado, sendo outros factores de pior prognóstico o estádio avançado da doença (TNM), o PS≥2, e a idade superior a 63 anos; por esta avaliação, os grupos histológicos do tumor e o sexo dos doentes não se revelaram factores de prognóstico.A análise multivariada das sobrevidas de toda a população estudada confirmou o valor pré-terapêutico elevado de Cyfra 21-1 como factor prognóstico negativo, sendo também factores de pior prognóstico o estádio da doença e o PS; aqui, a idade não se mostrou como determinante prognóstico negativo, mas, no grupo acima dos 63 anos, houve a tendência para que tal acontecesse.Por essa mesma análise, observou-se que, nos doentes não cirúrgicos, eram factores de prognóstico negativos a idade, o aumento do valor sérico pré-terapêutico de Cyfra 21-1, o estádio da doença e o PS, enquanto nos doentes cirúrgicos tal não ocorria com a idade e o PS.Em face dos dados apresentados, os autores concluem que a determinação sérica pré-terapêutica dos valores de Cyfra 21-1 é uma variável a considerar na avaliação prognóstica dos doentes com CPNPC, independentemente da terapêutica instituída, existindo concordância entre a presente meta-análise e os dados individuais das diferentes instituições seleccionadas, o que é um forte argumento de que se trata de um verdadeiro determinante prognóstico. COMENTÁRIO: Os factores de prognóstico clássicos incluem o estádio anatómico da doença (TNM), o performance status (PS) e outras condições, como a idade, o sexo, a perda de peso (W) e o(s) local(ais) de metastisação 1.Contudo, os tumores malignos do pulmão apresentam uma grande heterogeneidade no seu comportamento evolutivo, pelo que são necessárias outras variáveis que permitam determinar os casos com pior prognóstico e que, de algum modo, possam influenciar a respectiva programação terapêutica.Das variáveis biológicas, com bem demonstrado valor prognóstico no cancro do pulmão, podem citar-se os níveis séricos elevados de fosfatase alcalina ou de desidrogenase láctica (LDH), a hiperleucocitose ou a hiponatremia 1. Mais recentemente 2, indicam-se também como factores de prognóstico a ploidia, as mutações p53, ou o aumento de expressão da proteína bcl-2, cuja aplicação, na prática clínica, ainda não é rotineira, pela complexidade (e custos) das técnicas envolvidas na sua manipulação, mas sobre os quais recai um interesse sucessivamente crescente pelas implicações que potencialmente acarretam na área dos chamados novos alvos terapêuticos.Dentro da problemática do estudo dos marcadores tumorais séricos, acessíveis na prática clínica diária, tem tomado um lugar de crescente interesse a determinação do valor sérico pré-terapêutico de alguns, como é o caso da citoqueratina Cyfra 21-1, cujo valor, como factor de prognóstico nos CPNPC, tem sido objecto de múltiplos trabalhos, de resultados nem sempre concordantes.Daí o relevo da presente meta-análise, que vem confirmar a importância desse marcador tumoral como determinante prognóstico nos CPNPC.Enquanto as recomendações da American Thoracic Society/European Respiratory Society (ATS/ERS) não indicam qualquer marcador tumoral na avaliação pré-terapêutica dos CPNPC 3, nas recomendações da Société de Pneumologie de Langue Française (SPLF) 4, o Cyfra 21-1 é apontado como tendo valor na avaliação pré-terapêutica deste grupo de tumores, onde revela valor prognóstico independente.Estas afirmações são feitas com base em estudos prospectivos de grupos de doentes, mas questionase o seu interesse clínico, caso a caso, não sendo indicado como factor de prognóstico isolado 4.Por outo lado, numa revisão de 500 trabalhos publicados 5, defende-se que os marcadores tumorais não têm lugar na avaliação prognóstica dos doentes com CPNPC, o que não está de acordo com outros que afirmam que o Cyfra 21-1 se correlaciona bem com o TNM e o PS, constituindo um factor de prognóstico independente, e recomendando-o, mesmo, como uma co-variável a incluir em futuros ensaios clínicos 6.A meta-análise que apresentámos vem reforçar a opinião de que os valores séricos pré-terapêuticos elevados de Cyfra 21-1, nos CPNPC, constituem um factor de prognóstico isolado, independentemente do estádio da doença e da terapêutica instituída, reforçando as conclusões de outro publicado recentemente 7, em que esse marcador tumoral se revelou também nesse grupo de tumores, em estádios IIIB/IV, como um factor de prognóstico, quer isoladamente, quer quando associado a dois outros, o antigénio carcinoembrionário (CEA) e a neuroenolase específica (NSE).Nos doente cirúrgicos, o valor pré-terapêutico elevado de Cyfra 21-1, eventualmente, poderá indicar a necessidade de quimioterapia adjuvante, o que poderá ser mais um argumento a reforçar as conclusões do projecto IALT 8, em que se defende que aquela abordagem terapêutica poderá ser útil, também, nos estádios mais localizados da doença.

O objectivo da presente meta-análise foi determinar o valor prognóstico de níveis séricos pré-terapêuticos elevados de Cyfra 21-1, ajustados a co-variáveis clássicas no CPNPC.

The NEDD4L gene encodes a ubiquitin ligase that targets the epithelial sodium channel for degradation. A search for transcripts whose levels increase following androgen treatment of LNCaP human prostate cancer cells led to the isolation of three new NEDD4L transcripts designated NEDD4Lf, NEDD4Lg and NEDD4Lh. The three transcripts encode different forms of the NEDD4L protein, two of which contain an N-terminal C2 domain. These transcripts were detected at high levels in human prostate and mammary gland, and at lower levels in brain and skeletal muscle. We also found that the previously described NEDD4La and NEDD4Lc (KIAA0439) transcripts are also expressed in prostate and LNCaP cells. However, only NEDD4Lf, NEDD4Lg and NEDD4Lh were up-regulated by androgen in LNCaP cells. These data provide new information on the structure and expression profile of NEDD4L-derived transcripts and identify specific isoforms of the NEDD4L ubiquitin ligase as proteins with potentially important roles in androgen action and prostate physiology.

In searching for androgen-responsive genes in human prostate cancer cells, we have isolated two cDNAs that encode alternate forms of a novel Src homology 3 domain-containing guanine nucleotide exchange factor (SGEF). The SGEF mRNA is widely expressed in human tissues, and the predicted 871-amino acid SGEF protein contains Dbl homology and pleckstrin homology domains as well as an N-terminal proline-rich domain, a C-terminal Src homology 3 domain, and two nuclear localization signals. The second cDNA encodes a 139-amino acid N-terminally truncated form of SGEF designated C-terminal SGEF (CSGEF). In contrast to SGEF, CSGEF mRNA expression is restricted to prostate and liver. Moreover, CSGEF expression is up-regulated by androgens in LNCaP cells, whereas that of SGEF is not. Up-regulation of CSGEF was sensitive to actinomycin D but did not require new protein synthesis. The SGEF gene is located on chromosome 3q25.2 and consists of at least 15 exons. Based on the structure of the SGEF and CSGEF cDNAs, we deduced that CSGEF expression is controlled by an alternate androgen-responsive promoter of the SGEF gene. We hypothesize that SGEF is a ubiquitous regulator of Rho guanosine triphosphatases, whereas CSGEF may function as an androgen-induced regulator of Rho guanosine triphosphatase activity in epithelial cells of the human prostate.

This retrospective study aimed at determining the prognostic significance of neuroendocrine markers chromogranin A (CgA), pro-gastrin releasing peptide (ProGRP) and neuron-specific enolase (NSE), together with the cytokeratin 19 marker CYFRA 21-1 in small cell lung cancer (SCLC). A total of 148 histologically proven and previously untreated SCLC patients were included. Among them 118 patients received a cisplatin-etoposide combination or cisplatin-etoposide-cyclophosphamide-4'-epidoxorubicin combination. All tumour markers were tested using immunoradiometric assays except for ProGRP which was tested using an enzyme-linked immunosorbent assay. The thresholds for marker serum titrations were 53 pg/ml, 65, 17, and 3.6 ng/ml for ProGRP, CgA, NSE and CYFRA 21-1 respectively. Univariate analysis showed that patients affected by one of the following characteristics proved to have a significant shorter survival in comparison with the opposite status of each variable: age over 63 years, extensive-stage, serum LDH level higher than 600 U/l, serum NSE level higher than 17 ng/ml, serum CgA level higher than 65 ng/ml and serum CYFRA 21-1 level higher than 3.6 ng/ml. In addition, there was a trend towards a statistical significance for a high serum alkaline phosphatase level and a performance status equal to or worse than two. The following variables were independent determinants of a poor outcome: a poor performance status (hazard ratio [95% confidence interval]: 1.51 [1.02-2.22]), a high CgA level (HR: 1.61 [1.06-2.45]), a high CYFRA 21-1 level (HR: 2.10 [1.40-3.14]) and an age older than 63 years (HR: 1.68 [1.14-2.48]). When the multivariate analysis was restricted to patients receiving a cisplatin-etoposide-based chemotherapy, the same variables were prognostic determinants with nearly similar hazard ratios. In conclusion, aside classical variables such as age and performance status, high serum CYFRA 21-1 and high serum CgA level in SCLC are both prognostic determinants of prognosis, in particular in patients receiving conventional chemotherapy consisting of cisplatin and etoposide-based combinations.

Anti-p53 antibodies have been detected in the sera of patients with various types of cancers. In this report, we describe the development of a new ELISA aimed at detecting anti-p53 antibodies using two peptides belonging to immunodominant epitopes of the p53 N-terminal region. We first tested the reactivity of the sera by an indirect ELISA using the peptides as a capture system. Then, the specificity of the reaction was confirmed by an inhibition assay. Two systems of peptide presentation, phage display and the streptavidin/biotin system, were evaluated. Using a panel of sera from cancer patients, both systems were found to be equally reliable, demonstrating that both peptide-based ELISAs can be used for the specific detection of anti-p53 antibodies. The presence of anti-p53 antibodies was associated with p53 alteration whether it be mutation or accumulation.

Androgens play an important role in the development and physiology of the normal prostate as well as in prostate cancer cell proliferation. Comparison of the mRNA expression profiles of control and R1881-treated cultures of LNCaP human prostate cancer cells using cDNA subtraction led to the identification of a novel transcription factor that we named Androgen-Induced bZIP (AIbZIP) protein. AIbZIP is a 395 aa protein with homology to cyclic AMP-responsive element binding protein/activating transcription factor transcription factors. It contains an NH(2)-terminal activation domain, a central bZIP domain, and a COOH-terminal transmembrane domain. The AIbZIP gene is localized on chromosome 1q21.3 and consists of 10 exons. A major 1.7-kb transcript was detected exclusively in the prostate as well as in breast and prostate cancer cell lines. Androgens up-regulate AIbZIP mRNA and protein levels in a dose-dependent manner. The kinetics of AIbZIP mRNA up-regulation and the results of experiments with cycloheximide suggest that AIbZIP may be a delayed response gene. Immunoreactive AIbZIP protein was primarily detected in the cytoplasm of prostatic luminal epithelial cells. Similarly, full-length AIbZIP-green fluorescent protein fusion proteins were localized in the cytoplasm of LNCaP cells, whereas a truncated form of AIbZIP lacking the putative transmembrane domain was exclusively nuclear. Examination of AIbZIP protein and mRNA expression in a series of transurethral resection of the prostate and needle biopsy specimens indicated that AIbZIP is expressed at higher levels in cancerous prostate cells compared with noncancerous prostate cells. The highly tissue-specific expression profile, androgen regulation, chromosomal localization, and expression profile of AIbZIP in prostate tumors suggest that AIbZIP may play an important role in prostate cancer and in androgen receptor signaling in prostate cells. Future studies will confirm a possible relationship between AIbZIP and prostate cancer.

Estrogen receptor (ER) content is the most useful parameter for predicting hormone response therapy in breast cancer. Assays available for detecting ER in breast tumor cytosol are ligand-binding assay (LBA), which detects both ER alpha and ERP, and the enzymatic immunoassay (EIA), in which monoclonal antibodies are directed against EP alpha, As shown in several studies, the 2 assays correlate and both are used routinely. However, some discrepancies between the 2 assays were found and explanations remain controversial. We evaluated ER alpha and ER beta mRNA coexpression in breast tumors in order to study whether the presence of ER beta could account for differences between LBA and EIA in the determination of ER protein level. Using HeLa cell lines transfected with either EP alpha or ER beta, we confirmed that EIA, using H222 and D547 monoclonal antibodies, recognizes only ERa expression, whereas LBA detects both isoforms. In 119 breast tumor cytosols, the correlation between ER-EIA and ER-LBA was high (r = 0.72), although some discrepancies were found. When analyzing ER mRNA expression of samples with higher LBA values, no overexpression of ER beta mRNA relatively to ER alpha mRNA were observed, There was a difference in ER beta/ ER alpha ratio between ER-negative and ER-positive samples, with a IO-fold increased median ratio in ER-negative samples (p = 0.01). We thus confirmed that the major form of ER in breast cancer is the ER alpha at both the protein and mRNA levels. Moreover, our data do not support the hypothesis that ERP expression could explain differences between LBA and EIA in the determination of ER protein level. (C) 2001 Wiley-Liss, Inc.

Despite numerous studies, the influence of timing at surgery in relation to the menstrual cycle on the prognosis of breast carcinoma is still controversial. Most studies are retrospective, and the reliability of the menstrual history data is limited by the lack of hormonal assessment at the time of surgery. The authors prospectively studied the influence of the menstrual cycle phase as determined by circulating hormones at the time of surgery on the outcome of breast carcinoma.