Novel Methylated Biomarkers and a Robust Assay to Detect Circulating Tumor DNA in Metastatic Breast Cancer

The ability to consistently detect cell-free tumor-specific DNA in peripheral blood of patients with metastatic breast cancer provides the opportunity to detect changes in tumor burden and to monitor response to treatment. We developed cMethDNA, a quantitative multiplexed methylation-specific PCR assay for a panel of ten genes, consisting of novel and known breast cancer hypermethylated markers identified by mining our previously reported study of DNA methylation patterns in breast tissue (103 cancer, 21 normal on the Illumina HumanMethylation27 Beadchip) and then validating the 10-gene panel in a TCGA breast cancer methylome database. For cMethDNA, a fixed physiological level (50 copies) of artificially constructed, standard non-human reference DNA specific for each gene is introduced into in a constant volume of serum (300 μl) prior to purification of the DNA, facilitating a sensitive, specific, robust and quantitative assay of tumor DNA, with broad dynamic range. Cancer-specific methylated DNA was detected in Training (28 normal, 24 cancer) and Test (27 normal, 33 cancer) sets of recurrent Stage 4 patient sera with a sensitivity of 91% and a specificity of 96% in the test set. In a pilot study, cMethDNA assay faithfully reflected patient response to chemotherapy (N = 29). A core methylation signature present in the primary breast cancer was retained in serum and metastatic tissues collected at autopsy 2–11 years after diagnosis of the disease. Together, our data suggest that the cMethDNA assay can detect advanced breast cancer, and monitor tumor burden and treatment response in women with metastatic breast cancer

Carcinomas mamários de tipo basal: estudo clínicopatológico e imunofenotípico em mulheres brasileiras

Exportado OPUSMade available in DSpace on 2019-08-14T16:01:57Z (GMT). No. of bitstreams: 1 marina_de_brot_andrade.pdf: 1169580 bytes, checksum: 8c68fa55f41d2235fef21ed5dfd1f72a (MD5) Previous issue date: 11Carcinomas mamários de tipo basal representam uma classe de tumores recentemente reconhecida, que apresentam perfis gênico e protéico semelhantes aos das células basais/mioepiteliais da mama normal, estando associados a pior prognóstico e comportamento biológico agressivo. Objetivo: Investigar a freqüência de carcinomas mamários de fenótipo basal em uma série de tumores triplo-negativos (TTN), definidos pela negatividade para receptores de estrógeno (RE), de progesterona (RP), e HER2. Métodos: Selecionamos 140 TTN, obtendo-se características clínico-patológicas e sobrevida. Microarranjo de tecido (2 cilindros de cada tumor) foi construído e submetido à imunoistoquímica para RE, RP, HER2, citoqueratinas (CK) 5 e 14,EGFR, p63, caveolina e p53. Consideramos carcinomas de fenótipo basal os tumores negativos para RE, RP e HER2, e positivos para CK5. Resultados: Encontramos 105 carcinomas de fenótipo basal entre 140 TTN (freqüência=75,0%). A idade média das pacientes foi de 54,8 anos, sendo que 34,3% estavam na pré-menopausa. A maioria dos tumores foi classificada como carcinoma ductal invasor de alto grau. Os TTN exibiram positividade para CK5 (75,0%), CK14 (29,0%), EGFR (36,4%), p63 (28,6%), caveolina (14,3%) e p53 (67,1%). Estadiamento avançado da doença foi observado em 52 pacientes (50,0%), com diâmetro tumoral maior que 5cm em 41 casos (39,0%) e metástases axilares em 61 casos (59,2%). Seguimento clínico foi obtido em 89 pacientes (média=51 meses). Destas, 45 pacientes (50,5%) evoluíram sem doença; 6 (6,7%) estavam vivas com doença; e 38 (42,6%) morreram pelo câncer. Recidiva sistêmica ocorreu em 42 pacientes (47,1%), sendo pulmões, cérebro e ossos os principais sítios de metástases. As médias das sobrevidas global e livre de doença foram de 36 e 28 meses, respectivamente. Identificouse pior sobrevida global nas pacientes com tumores basais, em comparação àquelas com tumores não basais, com risco de morte 2,4 vezes maior nas primeiras. Esta diferença, entretanto, não foi estatisticamente significativa (p=0,06). Conclusões: Nosso estudo confirma comportamento clínico agressivoe elevada freqüência dos carcinomas de fenótipo basal entre os TTN,semelhante ao descrito em casuísticas estrangeiras.Basal-like cancers (BLC) are a newly recognized subtype of breast cancer defined by the expression of genes and proteins characteristic of the myoepithelial/basal cells of normal breast, and have been associated with poor prognosis and agressive biological behavior. Aim: The aim of our study is to investigate basal phenotype in a series of triple-negative (estrogen and progesterone receptors-negative and HER2-negative) invasive mammary carcinomas. Methods: We selected 140 previously tested triple-negative tumors. Clinical, histopathological and survival data were obtained. A tissue microarray containing 2 cylinders from each tumor was constructed and immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), HER2, cytokeratins (CK) 5 and 14, EGFR, p63, caveolin, and p53 was performed. We considered BLC to be any tumors that were ER/PR/HER2- negative and CK5-positive. Results: We found 105 cases of BLC from 140 triple-negative tumors (frequency =75.0%). The mean age at diagnosis was 54.8 years-old and 34.3% were premenopausal women. The majority of tumors were high grade (83.7%) and of ductal/no-special-type (80.8%). Triple-negative tumors showed immunoreactivity for CK5 (75.0%), CK14 (29.0%), EGFR(28.6%), p63 (28.6%), caveolin (14.3%), and p53 (67.1%). Tumor size larger than 5cm was observed in 41 cases (39.0%) and axillary metastases were detected in 61 patients (59.2%). Follow-up was recorded for 89 patients (mean=51 months): 45 patients (50.5%) with no evidence of disease; 6 patients (6.7%) were alive with disease; and 38 patients (42.6%) died of the disease. Relapse was detected in 42 women (47.1%), lungs, brain, and bones being the most commom sites of metastases. The mean overall survival was 36 months and the mean disease-free interval was 28 months. Furthermore, the overall survival associated with BLC cases was shorter than that observed in non basaltumors (RR=2.4). However, this difference was not statistically significant(p=0.06). Conclusions: Our findings confirmed that BLC have a poor prognosis and are high-frequent carcinomas among triple-negative tumors, similar to the behavior previously reported in North American and European patients

Carcinomas mamários de tipo basal: perfil clínico-patológico e evolutivo Basal-like breast cancers: clinicopathological features and outcome

OBJETIVO: Investigar a frequência de carcinomas mamários de fenótipo basal em uma série de tumores triplo-negativos (TTN), definidos pela negatividade para receptores de estrógeno (RE), de progesterona (RP) e HER2. MÉTODOS: Selecionamos 140 TTN, obtendo-se características clínico-patológicas e sobrevida. Microarranjo de tecido (2 cilindros de cada tumor) foi construído e submetido à imunoistoquímica para RE, RP, HER2, citoqueratinas (Cks) 5 e 14, EGFR, p63 e p53. Consideramos carcinomas de fenótipo basal os tumores negativos para RE, RP e HER2, e positivos para CK5. RESULTADOS: Encontramos 105 carcinomas de fenótipo basal entre 140 TTN (frequência=75%). A idade média das pacientes foi de 54,8 anos, sendo que 34,3% estavam na pré-menopausa. A maioria dos tumores foi classificada como carcinoma ductal invasor de alto grau. Os TTN exibiram positividade para CK5 (75,0%), CK14 (29%), EGFR (36,4%), p63 (28,6%) e p53 (67,1%). Estadiamento avançado da doença foi observado em 52 pacientes (50%), com diâmetro tumoral maior que 5 cm em 41 casos (39%) e metástases axilares em 61 casos (59,2%). Seguimento clínico foi obtido em 89 pacientes (média=51 meses). Destas, 45 pacientes (50,5%) evoluíram sem doença; 6 (6,7%) estavam vivas com doença e 38 (42,6%) morreram pelo câncer. Recidiva sistêmica ocorreu em 42 pacientes (47,1%), sendo pulmões, cérebro e ossos os principais sítios de metástases. As médias das sobrevidas global e livre de doença foram de 36 e 28 meses, respectivamente. CONCLUSÕES: Nosso estudo confirma comportamento clínico agressivo e elevada frequência dos carcinomas de fenótipo basal entre os TTN, semelhante ao descrito em casuísticas norte-americanas e europeias.<br>OBJECTIVE: The aim of our study was to investigate basal phenotype in a series of triple-negative (estrogen and progesterone receptors-negative and HER2-negative) invasive mammary carcinomas. METHODS: We selected 140 previously tested triple-negative tumors. Clinical, histopathological and survival data were obtained. A tissue microarray containing 2 cylinders from each tumor was constructed and immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), HER2, cytokeratins (CK) 5 and 14, EGFR, p63, and p53 was performed. We considered basal like-cancers (BLC) those tumors that were ER/PR/HER2-negative and CK5-positive. RESULTS: We found 105 cases of BLC from 140 triple-negative tumors (frequency=75.0%). The mean age at diagnosis was 54.8 years-old and 34.3% were premenopausal women. The majority of tumors were high grade (83.7%) and of ductal/no-special-type (80.8%). Triple-negative tumors showed immunoreactivity for CK5 (75.0%), CK14 (29.0%), EGFR (28.6%), p63 (28.6%), and p53 (67.1%). Tumor size larger than 5cm was observed in 41 cases (39.0%) and axillary metastases were detected in 61 patients (59.2%). Follow-up was recorded for 89 patients (mean=51 months): 45 patients (50.5%) with no evidence of disease; 6 patients (6.7%) were alive with disease; and 38 patients (42.6%) died of the disease. Relapse was detected in 42 women (47.1%), lungs, brain, and bones being the most common sites of metastasis. The mean overall survival was 36 months and the mean disease-free interval was 28 months. CONCLUSION: Our findings confirmed that BLC are poor prognosis and highly-frequent carcinomas among triple-negative tumors, similar to data previously reported in North American and European patients

Vaccinia virus GLV-1h153 is a novel agent for detection and effective local control of positive surgical margins for breast cancer

Introduction: Surgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model. Methods: GLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 mu Ci of I-124-iodide. Results: Viral infectivity, measured by green fluorescent protein (GFP) expression, was time-and concentrationdependent. All cell lines showed less than 10% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( < 10,000-fold increase from the initial viral dose) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm(3) versus 168 mm(3) in untreated controls (P < 0.05). Conclusions: This is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors

Data from: Clonal relatedness between lobular carcinoma in situ and synchronous malignant lesions

INTRODUCTION: Lobular carcinoma in situ (LCIS) has been accepted as a marker of risk for the development of invasive breast cancer, yet modern models of breast carcinogenesis include LCIS as a precursor of low-grade carcinomas. We provide evidence favoring a clonal origin for LCIS and synchronous estrogen receptor-positive malignant lesions of the ductal and lobular phenotype. METHODS: Patients with prior LCIS undergoing mastectomy were identified preoperatively from 2003-2008. Specimens were widely sampled, and frozen blocks were screened for LCIS and co-existing malignant lesions, and subject to microdissection. Samples from 65 patients were hybridized to the Affymetrix SNP 6.0 array platform. Cases with both an LCIS sample and an associated ductal carcinoma in situ (DCIS) or invasive tumor sample were evaluated for patterns of somatic copy number changes to assess evidence of clonal relatedness. RESULTS: LCIS was identified in 44 of the cases, and among these, a DCIS and/or invasive lesion was also identified in 21 cases. A total of 17 tumor pairs had adequate DNA/array data for analysis, including 9 pairs of LCIS/invasive lobular cancer (ILC), 4 pairs of LCIS/DCIS, and 4 LCIS/invasive ductal cancer (IDC). Overall, 7 pairs (41%) were judged to be clonally related; in 5 (29%), evidence suggested clonality but was equivocal, and 5 (29%) were considered independent. Clonal pairs were observed with all matched lesion types and low and high histological grades. We also show anecdotal evidence of clonality between a patient-matched triplet of LCIS, DCIS, and IDC. CONCLUSIONS: Our results support the role of LCIS as a precursor in the development of both high- and low-grade ductal and lobular cancers

KingClonalityData2

Copy number log-ratio data for the 31 samples in the manuscript. It is a gzipped text file. This is part 2. Use cat to concatenate all 6 parts