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PURPOSE: The 21-gene recurrence score (RS) assay quantifies the likelihood of distant recurrence in women with estrogen receptor-positive, lymph node-negative breast cancer treated with adjuvant tamoxifen. The relationship between the RS and chemotherapy benefit is not known. METHODS: The RS was measured in tumors from the tamoxifen-treated and tamoxifen plus chemotherapy-treated patients in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B20 trial. Cox proportional hazards models were utilized to test for interaction between chemotherapy treatment and the RS. RESULTS: A total of 651 patients were assessable (227 randomly assigned to tamoxifen and 424 randomly assigned to tamoxifen plus chemotherapy). The test for interaction between chemotherapy treatment and RS was statistically significant (P = .038). Patients with high-RS (≥ 31) tumors (ie, high risk of recurrence) had a large benefit from chemotherapy (relative risk, 0.26; 95% CI, 0.13 to 0.53; absolute decrease in 10-year distant recurrence rate: mean, 27.6%; SE, 8.0%). Patients with low-RS (< 18) tumors derived minimal, if any, benefit from chemotherapy treatment (relative risk, 1.31; 95% CI, 0.46 to 3.78; absolute decrease in distant recurrence rate at 10 years: mean, -1.1%; SE, 2.2%). Patients with intermediate-RS tumors did not appear to have a large benefit, but the uncertainty in the estimate can not exclude a clinically important benefit. CONCLUSION: The RS assay not only quantifies the likelihood of breast cancer recurrence in women with node-negative, estrogen receptor-positive breast cancer, but also predicts the magnitude of chemotherapy benefit.
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The identification of gene fusions promises to play an important role in personalized cancer treatment decisions. Many rare gene fusion events have been identified in fresh frozen solid tumors from common cancers employing next-generation sequencing technology. However the ability to detect transcripts from gene fusions in RNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissues, which exist in very large sample repositories for which disease outcome is known, is still limited due to the low complexity of FFPE libraries and the lack of appropriate bioinformatics methods. We sought to develop a bioinformatics method, named gFuse, to detect fusion transcripts in FFPE tumor tissues. An integrated, cohort based strategy has been used in gFuse to examine single-end 50 base pair (bp) reads generated from FFPE RNA-Sequencing (RNA-Seq) datasets employing two breast cancer cohorts of 136 and 76 patients. In total, 118 fusion events were detected transcriptome-wide at base-pair resolution across the 212 samples. We selected 77 candidate fusions based on their biological relevance to cancer and supported 61% of these using TaqMan assays. Direct sequencing of 19 of the fusion sequences identified by TaqMan confirmed them. Three unique fused gene pairs were recurrent across the 212 patients with 6, 3, 2 individuals harboring these fusions respectively. We show here that a high frequency of fusion transcripts detected at the whole transcriptome level correlates with poor outcome (P<0.0005) in human breast cancer patients. This study demonstrates the ability to detect fusion transcripts as biomarkers from archival FFPE tissues, and the potential prognostic value of the fusion transcripts detected.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Formaldehído/química , Proteínas de Fusión Oncogénica/análisis , Adhesión en Parafina/métodos , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Técnicas In VitroRESUMEN
The phosphoinositide 3-kinase (PI3K) signaling pathway is significantly altered in a wide variety of human cancers, driving cancer cell growth and survival. Consequently, a large number of PI3K inhibitors are now in clinical development. To begin to improve the selection of patients for treatment with PI3K inhibitors and to identify de novo determinants of patient response, we sought to identify and characterize candidate genomic and phosphoproteomic biomarkers predictive of response to the selective PI3K inhibitor, GDC-0941, using the NCI-60 human tumor cell line collection. In this study, sixty diverse tumor cell lines were exposed to GDC-0941 and classified by GI(50) value as sensitive or resistant. The most sensitive and resistant cell lines were analyzed for their baseline levels of gene expression and phosphorylation of key signaling nodes. Phosphorylation or activation status of both the PI3K-Akt signaling axis and PARP were correlated with in vitro response to GDC-0941. A gene expression signature associated with in vitro sensitivity to GDC-0941 was also identified. Furthermore, in vitro siRNA-mediated silencing of two genes in this signature, OGT and DDN, validated their role in modulating sensitivity to GDC-0941 in numerous cell lines and begins to provide biological insights into their role as chemosensitizers. These candidate biomarkers will offer useful tools to begin a more thorough understanding of determinants of patient response to PI3K inhibitors and merit exploration in human cancer patients treated with PI3K inhibitors.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Indazoles/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Sulfonamidas/farmacología , Transcriptoma/efectos de los fármacos , Acetilglucosamina/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa de Punto de Control 2 , Análisis por Conglomerados , Técnicas de Silenciamiento del Gen , Humanos , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Receptor ErbB-3/metabolismo , Transducción de Señal , Estadísticas no ParamétricasRESUMEN
RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR <10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts.
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Neoplasias de la Mama/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Recurrencia Local de Neoplasia/genética , Adhesión en Parafina , Análisis de Secuencia de ARN , Fijación del Tejido , Secuencia de Bases , Biomarcadores de Tumor/genética , ADN Intergénico/genética , Femenino , Formaldehído , Humanos , Intrones/genética , Modelos de Riesgos Proporcionales , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico , Factores de RiesgoRESUMEN
PURPOSE: Several mechanisms have been proposed to explain tamoxifen resistance of estrogen receptor (ER) -positive tumors, but a clinically useful explanation for such resistance has not been described. Because the ER is the treatment target for tamoxifen, a linear association between ER expression levels and the degree of benefit from tamoxifen might be expected. However, such an association has never been demonstrated with conventional clinical ER assays, and the ER is currently used clinically as a dichotomous marker. We used gene expression profiling and ER protein assays to help elucidate molecular mechanism(s) responsible for tamoxifen resistance in breast tumors. PATIENTS AND METHODS: We performed gene expression profiling of paraffin-embedded tumors from National Surgical Adjuvant Breast and Bowel Project (NSABP) trials that tested the worth of tamoxifen as an adjuvant systemic therapy (B-14) and as a preventive agent (P-1). This was a retrospective subset analysis based on available materials. RESULTS: In B-14, ESR1 was the strongest linear predictor of tamoxifen benefit among 16 genes examined, including PGR and ERBB2. On the basis of these data, we hypothesized that, in the P-1 trial, a lower level of ESR1 mRNA in the tamoxifen arm was the main difference between the two study arms. Only ESR1 was downregulated by more than two-fold in ER-positive cancer events in the tamoxifen arm (P < .001). Tamoxifen did not prevent ER-positive tumors with low levels of ESR1 expression. CONCLUSION: These data suggest that low-level expression of ESR1 is a determinant of tamoxifen resistance in ER-positive breast cancer. Strategies should be developed to identify, treat, and prevent such tumors.
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Antineoplásicos Hormonales/uso terapéutico , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/prevención & control , Receptor alfa de Estrógeno/metabolismo , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Neoplasias Hormono-Dependientes/prevención & control , Tamoxifeno/uso terapéutico , Adulto , Anciano , Biomarcadores de Tumor/genética , Neoplasias de la Mama/química , Regulación hacia Abajo , Receptor alfa de Estrógeno/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Lineales , Persona de Mediana Edad , Neoplasias Hormono-Dependientes/química , Análisis por Matrices de Proteínas , ARN Mensajero/metabolismo , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Oxaliplatin is widely used to treat colorectal cancer, as both adjuvant therapy for resected disease and palliative treatment of metastatic disease. However, a significant number of patients experience serious side effects, including prolonged neurotoxicity, from oxaliplatin treatment creating an urgent need for biomarkers of oxaliplatin response or resistance to direct therapy to those most likely to benefit. As a first step to improve selection of patients for oxaliplatin-based chemotherapy, we have conducted an in vitro cell-based small interfering RNA (siRNA) screen of 500 genes aimed at identifying genes whose loss of expression alters tumor cell response to oxaliplatin. The siRNA screen identified twenty-seven genes, which when silenced, significantly altered colon tumor cell line sensitivity to oxaliplatin. Silencing of a group of putative resistance genes increased the extent of oxaliplatin-mediated DNA damage and inhibited cell-cycle progression in oxaliplatin-treated cells. The activity of several signaling nodes, including AKT1 and MEK1, was also altered. We used cDNA transfection to overexpress two genes (LTBR and TMEM30A) that were identified in the siRNA screen as mediators of oxaliplatin sensitivity. In both instances, overexpression conferred resistance to oxaliplatin. In summary, this study identified numerous putative predictive biomarkers of response to oxaliplatin that should be studied further in patient specimens for potential clinical application. Diverse gene networks seem to influence tumor survival in response to DNA damage by oxaliplatin. Finally, those genes whose loss of expression (or function) is related to oxaliplatin sensitivity may be promising therapeutic targets to increase patient response to oxaliplatin.
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Fenómenos Biológicos/genética , Genómica/métodos , Neoplasias/genética , Neoplasias/patología , Compuestos Organoplatinos/farmacología , Fenómenos Biológicos/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Daño del ADN/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Ensayos de Selección de Medicamentos Antitumorales , Genes Relacionados con las Neoplasias/genética , Humanos , Modelos Biológicos , Oxaliplatino , ARN Interferente Pequeño/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
PURPOSE: These studies were conducted to determine the relationship between quantitative tumor gene expression and risk of cancer recurrence in patients with stage II or III colon cancer treated with surgery alone or surgery plus fluorouracil (FU) and leucovorin (LV) to develop multigene algorithms to quantify the risk of recurrence as well as the likelihood of differential treatment benefit of FU/LV adjuvant chemotherapy for individual patients. PATIENTS AND METHODS: We performed quantitative reverse transcription polymerase chain reaction (RT-qPCR) on RNA extracted from fixed, paraffin-embedded (FPE) tumor blocks from patients with stage II or III colon cancer who were treated with surgery alone (n = 270 from National Surgical Adjuvant Breast and Bowel Project [NSABP] C-01/C-02 and n = 765 from Cleveland Clinic [CC]) or surgery plus FU/LV (n = 308 from NSABP C-04 and n = 508 from NSABP C-06). Overall, 761 candidate genes were studied in C-01/C-02 and C-04, and a subset of 375 genes was studied in CC/C-06. RESULTS: A combined analysis of the four studies identified 48 genes significantly associated with risk of recurrence and 66 genes significantly associated with FU/LV benefit (with four genes in common). Seven recurrence-risk genes, six FU/LV-benefit genes, and five reference genes were selected, and algorithms were developed to identify groups of patients with low, intermediate, and high likelihood of recurrence and benefit from FU/LV. CONCLUSION: RT-qPCR of FPE colon cancer tissue applied to four large independent populations has been used to develop multigene algorithms for estimating recurrence risk and benefit from FU/LV. These algorithms are being independently validated, and their clinical utility is being evaluated in the Quick and Simple and Reliable (QUASAR) study.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias del Colon/genética , Perfilación de la Expresión Génica , Algoritmos , Quimioterapia Adyuvante , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Neoplasias del Colon/cirugía , Terapia Combinada , Fluorouracilo/administración & dosificación , Humanos , Leucovorina/administración & dosificación , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Molecular tests for diagnosis of disease, particularly cancer, are gaining increased acceptance by physicians and their patients for disease prognosis and selection of treatment options. Gene expression profiles and genetic mutations are key parameters used for the molecular characterization of tumors. A variety of methods exist for mutation analysis but the development of assays with high selectivity tends to require a process of trial and error, and few are compatible with real-time PCR. We sought to develop a real-time PCR-based mutation assay methodology that successfully addresses these issues. METHODOLOGY/PRINCIPAL FINDINGS: The method we describe is based on the widely used TaqMan real-time PCR technology, and combines Allele-Specific PCR with a Blocking reagent (ASB-PCR) to suppress amplification of the wildype allele. ASB-PCR can be used for detection of germ line or somatic mutations in either DNA or RNA extracted from any type of tissue, including formalin-fixed paraffin-embedded tumor specimens. A set of reagent design rules was developed enabling sensitive and selective detection of single point substitutions, insertions, or deletions against a background of wild-type allele in thousand-fold or greater excess. CONCLUSIONS/SIGNIFICANCE: ASB-PCR is a simple and robust method for assaying single nucleotide mutations and polymorphisms within the widely used TaqMan protocol for real time RT-PCR. The ASB-PCR design rules consistently produce highly selective mutation assays while obviating the need for redesign and optimization of the assay reagents. The method is compatible with formalin-fixed tissue and simultaneous analysis of gene expression by RT-PCR on the same plate. No proprietary reagents other than those for TaqMan chemistry are required, so the method can be performed in any research laboratory with real-time PCR capability.
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Análisis Mutacional de ADN/métodos , Reacción en Cadena de la Polimerasa/métodos , Técnicas de Laboratorio Clínico , Análisis Mutacional de ADN/normas , Humanos , Métodos , Mutación , Reacción en Cadena de la Polimerasa/normas , Polimorfismo de Nucleótido Simple , Juego de Reactivos para DiagnósticoRESUMEN
Previously, we had identified gene expression patterns that predicted response to neoadjuvant docetaxel. Other studies have validated that a high Recurrence Score (RS) by the 21-gene RT-PCR assay is predictive of worse prognosis but better response to chemotherapy. We investigated whether tumor expression of these 21 genes and other candidate genes can predict response to docetaxel. Core biopsies from 97 patients were obtained before treatment with neoadjuvant docetaxel (4 cycles, 100 mg/m2 q3 weeks). Three 10-microm FFPE sections were submitted for quantitative RT-PCR assays of 192 genes that were selected from our previous work and the literature. Of the 97 patients, 81 (84%) had sufficient invasive cancer, 80 (82%) had sufficient RNA for QRTPCR assay, and 72 (74%) had clinical response data. Mean age was 48.5 years, and the median tumor size was 6 cm. Clinical complete responses (CR) were observed in 12 (17%), partial responses in 41 (57%), stable disease in 17 (24%), and progressive disease in 2 patients (3%). A significant relationship (P<0.05) between gene expression and CR was observed for 14 genes, including CYBA. CR was associated with lower expression of the ER gene group and higher expression of the proliferation gene group from the 21 gene assay. Of note, CR was more likely with a high RS (P=0.008). We have established molecular profiles of sensitivity to docetaxel. RT-PCR technology provides a potential platform for a predictive test of docetaxel chemosensitivity using small amounts of routinely processed material.
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Antineoplásicos/uso terapéutico , Neoplasias de la Mama , Fijadores , Formaldehído , Regulación Neoplásica de la Expresión Génica , Adhesión en Parafina , Taxoides/uso terapéutico , Fijación del Tejido/métodos , Biopsia , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Quimioterapia Adyuvante , Docetaxel , Femenino , Perfilación de la Expresión Génica/métodos , Pruebas Genéticas , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Terapia Neoadyuvante , Selección de Paciente , Valor Predictivo de las Pruebas , Receptor ErbB-2/análisis , Receptor ErbB-2/genética , Receptores de Estrógenos/análisis , Receptores de Estrógenos/genética , Receptores de Progesterona/análisis , Receptores de Progesterona/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resultado del Tratamiento , Reino Unido , Estados UnidosRESUMEN
BACKGROUND: Reverse transcription PCR (RT-PCR) is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE) clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis) and the likelihood of tumor response to standard chemotherapy regimens (prediction). We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application. RESULTS: RNA was extracted from formalin fixed paraffin embedded (FPE) tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery. CONCLUSION: We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity, sensitivity and reproducibility that are characteristics of RT-PCR. Biomarkers discovered using this approach can be transferred to a clinical reference laboratory setting without having to re-validate the assay on a second technology platform.
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Biomarcadores de Tumor/genética , Neoplasias del Colon/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Neoplasias del Colon/diagnóstico , ADN Complementario , Humanos , Familia de Multigenes , Reproducibilidad de los Resultados , Moldes Genéticos , Transcripción GenéticaRESUMEN
PURPOSE: Primary chemotherapy provides an ideal opportunity to correlate gene expression with response to treatment. We used paraffin-embedded core biopsies from a completed phase II trial to identify genes that correlate with response to primary chemotherapy. PATIENTS AND METHODS: Patients with newly diagnosed stage II or III breast cancer were treated with sequential doxorubicin 75 mg/M2 q2 wks x 3 and docetaxel 40 mg/M2 weekly x 6; treatment order was randomly assigned. Pretreatment core biopsy samples were interrogated for genes that might correlate with pathologic complete response (pCR). In addition to the individual genes, the correlation of the Oncotype DX Recurrence Score with pCR was examined. RESULTS: Of 70 patients enrolled in the parent trial, core biopsies samples with sufficient RNA for gene analyses were available from 45 patients; 9 (20%) had inflammatory breast cancer (IBC). Six (14%) patients achieved a pCR. Twenty-two of the 274 candidate genes assessed correlated with pCR (p < 0.05). Genes correlating with pCR could be grouped into three large clusters: angiogenesis-related genes, proliferation related genes, and invasion-related genes. Expression of estrogen receptor (ER)-related genes and Recurrence Score did not correlate with pCR. In an exploratory analysis we compared gene expression in IBC to non-inflammatory breast cancer; twenty-four (9%) of the genes were differentially expressed (p < 0.05), 5 were upregulated and 19 were downregulated in IBC. CONCLUSION: Gene expression analysis on core biopsy samples is feasible and identifies candidate genes that correlate with pCR to primary chemotherapy. Gene expression in IBC differs significantly from noninflammatory breast cancer.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Perfilación de la Expresión Génica , Adulto , Anciano , Biopsia , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/genética , Femenino , Humanos , Persona de Mediana Edad , Adhesión en ParafinaRESUMEN
INTRODUCTION: The Oncotype DX assay was recently reported to predict risk for distant recurrence among a clinical trial population of tamoxifen-treated patients with lymph node-negative, estrogen receptor (ER)-positive breast cancer. To confirm and extend these findings, we evaluated the performance of this 21-gene assay among node-negative patients from a community hospital setting. METHODS: A case-control study was conducted among 4,964 Kaiser Permanente patients diagnosed with node-negative invasive breast cancer from 1985 to 1994 and not treated with adjuvant chemotherapy. Cases (n = 220) were patients who died from breast cancer. Controls (n = 570) were breast cancer patients who were individually matched to cases with respect to age, race, adjuvant tamoxifen, medical facility and diagnosis year, and were alive at the date of death of their matched case. Using an RT-PCR assay, archived tumor tissues were analyzed for expression levels of 16 cancer-related and five reference genes, and a summary risk score (the Recurrence Score) was calculated for each patient. Conditional logistic regression methods were used to estimate the association between risk of breast cancer death and Recurrence Score. RESULTS: After adjusting for tumor size and grade, the Recurrence Score was associated with risk of breast cancer death in ER-positive, tamoxifen-treated and -untreated patients (P = 0.003 and P = 0.03, respectively). At 10 years, the risks for breast cancer death in ER-positive, tamoxifen-treated patients were 2.8% (95% confidence interval [CI] 1.7-3.9%), 10.7% (95% CI 6.3-14.9%), and 15.5% (95% CI 7.6-22.8%) for those in the low, intermediate and high risk Recurrence Score groups, respectively. They were 6.2% (95% CI 4.5-7.9%), 17.8% (95% CI 11.8-23.3%), and 19.9% (95% CI 14.2-25.2%) for ER-positive patients not treated with tamoxifen. In both the tamoxifen-treated and -untreated groups, approximately 50% of patients had low risk Recurrence Score values. CONCLUSION: In this large, population-based study of lymph node-negative patients not treated with chemotherapy, the Recurrence Score was strongly associated with risk of breast cancer death among ER-positive, tamoxifen-treated and -untreated patients.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Estudios de Casos y Controles , Femenino , Humanos , Persona de Mediana Edad , Pronóstico , Receptores de Estrógenos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Tamoxifeno/uso terapéuticoRESUMEN
PURPOSE: The 21-gene recurrence score (RS) assay quantifies the likelihood of distant recurrence in women with estrogen receptor-positive, lymph node-negative breast cancer treated with adjuvant tamoxifen. The relationship between the RS and chemotherapy benefit is not known. METHODS: The RS was measured in tumors from the tamoxifen-treated and tamoxifen plus chemotherapy-treated patients in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B20 trial. Cox proportional hazards models were utilized to test for interaction between chemotherapy treatment and the RS. RESULTS: A total of 651 patients were assessable (227 randomly assigned to tamoxifen and 424 randomly assigned to tamoxifen plus chemotherapy). The test for interaction between chemotherapy treatment and RS was statistically significant (P = .038). Patients with high-RS (> or = 31) tumors (ie, high risk of recurrence) had a large benefit from chemotherapy (relative risk, 0.26; 95% CI, 0.13 to 0.53; absolute decrease in 10-year distant recurrence rate: mean, 27.6%; SE, 8.0%). Patients with low-RS (< 18) tumors derived minimal, if any, benefit from chemotherapy treatment (relative risk, 1.31; 95% CI, 0.46 to 3.78; absolute decrease in distant recurrence rate at 10 years: mean, -1.1%; SE, 2.2%). Patients with intermediate-RS tumors did not appear to have a large benefit, but the uncertainty in the estimate can not exclude a clinically important benefit. CONCLUSION: The RS assay not only quantifies the likelihood of breast cancer recurrence in women with node-negative, estrogen receptor-positive breast cancer, but also predicts the magnitude of chemotherapy benefit.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Receptores de Estrógenos/metabolismo , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/prevención & control , Cisplatino/administración & dosificación , Femenino , Fluorouracilo/administración & dosificación , Humanos , Modelos Lineales , Metástasis Linfática , Metotrexato/administración & dosificación , Persona de Mediana Edad , Mitomicinas/administración & dosificación , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/prevención & control , Oportunidad Relativa , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Ensayos Clínicos Controlados Aleatorios como Asunto , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Medición de Riesgo , Factores de Riesgo , Prevención Secundaria , Tamoxifeno/administración & dosificaciónRESUMEN
PURPOSE: This study, along with two others, was done to develop the 21-gene Recurrence Score assay (Oncotype DX) that was validated in a subsequent independent study and is used to aid decision making about chemotherapy in estrogen receptor (ER)-positive, node-negative breast cancer patients. EXPERIMENTAL DESIGN: Patients with >or=10 nodes diagnosed from 1979 to 1999 were identified. RNA was extracted from paraffin blocks, and expression of 203 candidate genes was quantified using reverse transcription-PCR (RT-PCR). RESULTS: Seventy-eight patients were studied. As of August 2002, 77% of patients had distant recurrence or breast cancer death. Univariate Cox analysis of clinical and immunohistochemistry variables indicated that HER2/immunohistochemistry, number of involved nodes, progesterone receptor (PR)/immunohistochemistry (% cells), and ER/immunohistochemistry (% cells) were significantly associated with distant recurrence-free survival (DRFS). Univariate Cox analysis identified 22 genes associated with DRFS. Higher expression correlated with shorter DRFS for the HER2 adaptor GRB7 and the macrophage marker CD68. Higher expression correlated with longer DRFS for tumor protein p53-binding protein 2 (TP53BP2) and the ER axis genes PR and Bcl2. Multivariate methods, including stepwise variable selection and bootstrap resampling of the Cox proportional hazards regression model, identified several genes, including TP53BP2 and Bcl2, as significant predictors of DRFS. CONCLUSION: Tumor gene expression profiles of archival tissues, some more than 20 years old, provide significant information about risk of distant recurrence even among patients with 10 or more nodes.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Antígeno Ki-67/genética , Ganglios Linfáticos/patología , Metástasis Linfática , Persona de Mediana Edad , Pronóstico , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptor ErbB-2/análisis , Receptor ErbB-2/genética , Receptores de Estrógenos/análisis , Receptores de Estrógenos/genética , Receptores de Progesterona/análisis , Receptores de Progesterona/genética , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la EnfermedadRESUMEN
PURPOSE: We sought to identify gene expression markers that predict the likelihood of chemotherapy response. We also tested whether chemotherapy response is correlated with the 21-gene Recurrence Score assay that quantifies recurrence risk. METHODS: Patients with locally advanced breast cancer received neoadjuvant paclitaxel and doxorubicin. RNA was extracted from the pretreatment formalin-fixed paraffin-embedded core biopsies. The expression of 384 genes was quantified using reverse transcriptase polymerase chain reaction and correlated with pathologic complete response (pCR). The performance of genes predicting for pCR was tested in patients from an independent neoadjuvant study where gene expression was obtained using DNA microarrays. RESULTS: Of 89 assessable patients (mean age, 49.9 years; mean tumor size, 6.4 cm), 11 (12%) had a pCR. Eighty-six genes correlated with pCR (unadjusted P < .05); pCR was more likely with higher expression of proliferation-related genes and immune-related genes, and with lower expression of estrogen receptor (ER) -related genes. In 82 independent patients treated with neoadjuvant paclitaxel and doxorubicin, DNA microarray data were available for 79 of the 86 genes. In univariate analysis, 24 genes correlated with pCR with P < .05 (false discovery, four genes) and 32 genes showed correlation with P < .1 (false discovery, eight genes). The Recurrence Score was positively associated with the likelihood of pCR (P = .005), suggesting that the patients who are at greatest recurrence risk are more likely to have chemotherapy benefit. CONCLUSION: Quantitative expression of ER-related genes, proliferation genes, and immune-related genes are strong predictors of pCR in women with locally advanced breast cancer receiving neoadjuvant anthracyclines and paclitaxel.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Mama/patología , Adulto , Anciano , Biopsia con Aguja , Neoplasias de la Mama/patología , Doxorrubicina/uso terapéutico , Femenino , Perfilación de la Expresión Génica , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Paclitaxel/uso terapéutico , Valor Predictivo de las Pruebas , Inducción de Remisión , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: To test the ability of a reverse transcriptase-PCR (RT-PCR) assay, based on gene expression profiles, to accurately determine the risk of recurrence in patients with node-negative breast cancer who did not receive systemic therapy using formalin-fixed, paraffin-embedded tissue. A secondary objective was to determine whether the quantitative RT-PCR data correlated with immunohistochemistry assay data regarding estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status. PATIENTS AND METHODS: We obtained archival paraffin-embedded tissue from patients with invasive breast cancer but no axillary lymph node involvement who had received no adjuvant systemic therapy and been followed for at least 5 years. RNA was extracted from three 10-microm-thick sections. The expression of 16 cancer-related genes and 5 reference genes was quantified using RT-PCR. A gene expression algorithm was used to calculate a recurrence score for each patient. We then assessed the ability of the test to accurately predict distant recurrence-free survival in this population. RESULTS: We identified 149 eligible patients. Median age at diagnosis was 59 years; mean tumor diameter was 2 cm; and 69% of tumors were estrogen receptor positive. Median follow-up was 18 years. The 5-year disease-free survival rate for the group was 80%. The 21 gene-based recurrence score was not predictive of distant disease recurrence. However, a high concordance between RT-PCR and immunohistochemical assays for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status was noted. CONCLUSIONS: RT-PCR can be done on paraffin-embedded tissue to validate the large numbers of genes associated with breast cancer recurrence. However, further work needs to be done to develop an assay to identify the likelihood of recurrent disease in patients with node-negative breast cancer who do not receive adjuvant tamoxifen or chemotherapy.
Asunto(s)
Neoplasias de la Mama/patología , Perfilación de la Expresión Génica , Adulto , Anciano , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Quimioterapia Adyuvante , Análisis por Conglomerados , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Antígeno Ki-67/genética , Metástasis Linfática , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Pronóstico , Receptor ErbB-2/análisis , Receptor ErbB-2/genética , Receptores de Estrógenos/análisis , Receptores de Estrógenos/genética , Receptores de Progesterona/análisis , Receptores de Progesterona/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis de Supervivencia , Tamoxifeno/uso terapéuticoRESUMEN
BACKGROUND: The likelihood of distant recurrence in patients with breast cancer who have no involved lymph nodes and estrogen-receptor-positive tumors is poorly defined by clinical and histopathological measures. METHODS: We tested whether the results of a reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay of 21 prospectively selected genes in paraffin-embedded tumor tissue would correlate with the likelihood of distant recurrence in patients with node-negative, tamoxifen-treated breast cancer who were enrolled in the National Surgical Adjuvant Breast and Bowel Project clinical trial B-14. The levels of expression of 16 cancer-related genes and 5 reference genes were used in a prospectively defined algorithm to calculate a recurrence score and to determine a risk group (low, intermediate, or high) for each patient. RESULTS: Adequate RT-PCR profiles were obtained in 668 of 675 tumor blocks. The proportions of patients categorized as having a low, intermediate, or high risk by the RT-PCR assay were 51, 22, and 27 percent, respectively. The Kaplan-Meier estimates of the rates of distant recurrence at 10 years in the low-risk, intermediate-risk, and high-risk groups were 6.8 percent (95 percent confidence interval, 4.0 to 9.6), 14.3 percent (95 percent confidence interval, 8.3 to 20.3), and 30.5 percent (95 percent confidence interval, 23.6 to 37.4). The rate in the low-risk group was significantly lower than that in the high-risk group (P<0.001). In a multivariate Cox model, the recurrence score provided significant predictive power that was independent of age and tumor size (P<0.001). The recurrence score was also predictive of overall survival (P<0.001) and could be used as a continuous function to predict distant recurrence in individual patients. CONCLUSIONS: The recurrence score has been validated as quantifying the likelihood of distant recurrence in tamoxifen-treated patients with node-negative, estrogen-receptor-positive breast cancer.
Asunto(s)
Antineoplásicos Hormonales/uso terapéutico , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/genética , ADN de Neoplasias/metabolismo , Expresión Génica , Tamoxifeno/uso terapéutico , Algoritmos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , ADN de Neoplasias/análisis , Antagonistas de Estrógenos/uso terapéutico , Femenino , Estudios de Seguimiento , Genes erbB-2 , Humanos , Metástasis Linfática , Persona de Mediana Edad , Análisis Multivariante , Metástasis de la Neoplasia , Pronóstico , Modelos de Riesgos Proporcionales , Receptores de Estrógenos , Receptores de Progesterona , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Riesgo , Análisis de SupervivenciaRESUMEN
Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.