Marina De Brot

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 The Cancer Genome Atlas project breast cancer methylome database. For cMethDNA, a fixed physiologic level (50 copies) of artificially constructed, standard nonhuman reference DNA specific for each gene is introduced in a constant volume of serum (300 μL) before 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 IV 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 two to 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.

Lobular Carcinoma in Situ: A 29-Year Longitudinal Experience Evaluating Clinicopathologic Features and Breast Cancer Risk

PURPOSE The increased breast cancer risk conferred by a diagnosis of lobular carcinoma in situ (LCIS) is poorly understood. Here, we review our 29-year longitudinal experience with LCIS to evaluate factors associated with breast cancer risk. PATIENTS AND METHODS Patients participating in surveillance after an LCIS diagnosis are observed in a prospectively maintained database. Comparisons were made among women choosing surveillance, with or without chemoprevention, and those undergoing bilateral prophylactic mastectomies between 1980 and 2009. RESULTS One thousand sixty patients with LCIS without concurrent breast cancer were identified. Median age at LCIS diagnosis was 50 years (range, 27 to 83 years). Fifty-six patients (5%) underwent bilateral prophylactic mastectomy; 1,004 chose surveillance with (n = 173) or without (n = 831) chemoprevention. At a median follow-up of 81 months (range, 6 to 368 months), 150 patients developed 168 breast cancers (63% ipsilateral, 25% contralateral, 12% bilateral), with no dominant histology (ductal carcinoma in situ, 35%; infiltrating ductal carcinoma, 29%; infiltrating lobular carcinoma, 27%; other, 9%). Breast cancer incidence was significantly reduced in women taking chemoprevention (10-year cumulative risk: 7% with chemoprevention; 21% with no chemoprevention; P < .001). In multivariable analysis, chemoprevention was the only clinical factor associated with breast cancer risk (hazard ratio, 0.27; 95% CI, 0.15 to 0.50). In a subgroup nested case-control analysis, volume of disease, which was defined as the ratio of slides with LCIS to total number of slides reviewed, was also associated with breast cancer development (P = .008). CONCLUSION We observed a 2% annual incidence of breast cancer among women with LCIS. Common clinical factors used for risk prediction, including age and family history, were not associated with breast cancer risk. The lower breast cancer incidence in women opting for chemoprevention highlights the potential for risk reduction in this population.

Prognostic impact of the cancer stem cell related markers ALDH1 and EZH2 in triple negative and basal-like breast cancers.

Aims: We assessed the expression of ALDH1 and EZH2, cancer stem cell (CSC) related markers, in triple negative and basal-like breast cancers, investigating their association with clinicopathological features and outcome. Methods: Clinicopathological data were obtained from 140 cases of triple negative breast cancer. A tissue microarray was constructed and immunohistochemistry for ER, PR, HER2, ALDH1, EZH2, CK5, CK14, EGFR, p63, caveolin, and p53 was performed. Tumour cell and stromal expression of ALDH1 were evaluated. Multivariate analysis was conducted, including all significant variables. Results: The majority of triple negative breast cancers were invasive ductal carcinomas of no special type (NST) (116/140). Tumour cells exhibited cytoplasmic expression of ALDH1 in 26 of 140 cases, while stromal expression was detected in 117 of 140 cases. Tumour cell expression did not correlate with any of the parameters. Conversely, stromal expression was associated with better overall survival (p=0.044). Assessment by Cox Regression Model showed a HR of 2.80 (HR = 1/0.357 = 2.80; 95%CI 0.178–0.714; p = 0.004) for breast cancer death when ALDH1 was not found in the stromal compartment of tumours, independent of age, histological type/grade, nodal status, stage, relapse, and expression of basal markers. High EZH2 expression was noted in 120 of 140 triple negative breast cancers and was not associated with other variables. Basal-like cancers comprised 75% (105/140) of triple negative breast cancers. Interestingly, we found association between EZH2 and CK14 expression (p = 0.041). Conclusions: ALDH1 expression is frequent in tumour-associated stromal cells of triple negative breast cancer and is associated with better outcome. Tumour microenvironment should be considered when studying prognostic impact of CSCs in breast cancer.

Clonal relatedness between lobular carcinoma in situ and synchronous malignant lesions

IntroductionLobular 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.MethodsPatients with prior LCIS undergoing mastectomy were identified preoperatively from 2003 to 2008. Specimens were widely sampled, and frozen blocks were screened for LCIS and co-existing malignant lesions, and were 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.ResultsLCIS 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 nine pairs of LCIS/invasive lobular cancer, four pairs of LCIS/DCIS, and four pairs of LCIS/invasive ductal cancer. Overall, seven pairs (41%) were judged to be clonally related; in five (29%) evidence suggested clonality but was equivocal, and five (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 invasive ductal cancer.ConclusionOur results support the role of LCIS as a precursor in the development of both high-grade and low-grade ductal and lobular cancers.

MGMT and PTEN as potential prognostic markers in breast cancer

AIM To evaluate the prognostic importance of MGMT and PTEN concerning their correlation with other prognostic factors evaluated by immunohistochemistry (IHC) and the molecular phenotype of breast cancers. METHODS IHC for estrogen and progesterone receptors, HER2, Ki67, p53, p63, e-cadherin, EGFR, CK5, CK14, MGMT and PTEN was performed on 200 breast tumors. Basal-like and luminal breast carcinomas were defined by the IHC evaluation of these markers. Fluorescent in situ hybridization (FISH) was performed for PTEN and HER2 analysis using the Vysis PTEN and HER2 DNA probe kits (Abbott™). RT-PCR was performed to evaluate gene expressions of MGMT and PTEN in frozen tissue of 59/200 cases. RESULTS 147/200 cases were triple-negative (73.5%), 47/147 were basal-like carcinomas (31.9%). 53 cases (26.5%) were luminal-like type A or B. 56 (93.3%) and 46 samples (76.6%) expressed lower levels of MGMT and PTEN mRNA, respectively, compared with normal breast (p<0.001). There was a positive correlation between the IHC results and the RT-PCR values for MGMT and PTEN. Tumors with homozygotic deletion of PTEN expressed little or no mRNA or protein. Positive p53, high Ki67, and basal-like tumors expressed significant lower MGMT and PTEN. CONCLUSIONS We hypothesize that MGMT and PTEN expressions have prognostic significance in breast cancer. Also, based on their predictive value of response to therapy, evaluating MGMT and PTEN and learning to interpret their patterns of immunoexpression will undoubtedly lead to a greater understanding of breast cancer and its treatment.

Vaccinia virus GLV-1h153 is effective in treating and preventing metastatic triple-negative breast cancer

Objective:This study aimed to investigate the therapeutic impact of a new oncolytic vaccinia virus in a triple-negative breast cancer (TNBC) murine model and its potential for treating distant metastatic disease. Background:TNBCs are aggressive tumors associated with a high metastatic rate. Their lack of targets for hormonal/biological therapy presents significant clinical challenges and a dire need for novel therapies. Methods:GLV-1h153, a replication-competent vaccinia virus, was tested against multiple cell lines. Cytotoxicity and viral replication were determined. Intratumoral (IT) or intravenous (IV) injection of GLV-1h153 (1 × 107 plaque-forming units) or phosphate buffered saline was tested in an orthotopic murine model, which reliably produces systemic metastasis. Tumors, lymph nodes, and metastatic organs (lung, liver, and brain) were harvested 5 and 8 weeks after treatment and prepared for histopathological review. Demonstration of metastasis was performed using immunofluorescence and hematoxylin and eosin (H&E) staining. Results:GLV-1h153 infected, replicated in, and killed all TNBC cell lines in vitro. In vivo, mean tumor volume 2 weeks after treatment was 22 (IT), 29 (IV) versus 245 mm3 (control; P < 0.002). Five weeks after treatment, all harvested lymph nodes and organs showed no evidence of metastatic cells. All harvested tumors showed complete response to treatment, with only necrosis and fibrosis on H&E staining 8 weeks after treatment. Conclusions:This is the first study to demonstrate that TNBCs are killed by a novel vaccinia virus both in vitro and in vivo. Our results suggest that GLV-1h153 is a promising therapeutic agent for preventing and treating metastatic TNBC and warrants further clinical testing in patients.

Prognostic Impact of 21-Gene Recurrence Score in Patients With Stage IV Breast Cancer: TBCRC 013

PURPOSE The objective of this study was to determine whether the 21-gene Recurrence Score (RS) provides clinically meaningful information in patients with de novo stage IV breast cancer enrolled in the Translational Breast Cancer Research Consortium (TBCRC) 013. PATIENTS AND METHODS TBCRC 013 was a multicenter prospective registry that evaluated the role of surgery of the primary tumor in patients with de novo stage IV breast cancer. From July 2009 to April 2012, 127 patients from 14 sites were enrolled; 109 (86%) patients had pretreatment primary tumor samples suitable for 21-gene RS analysis. Clinical variables, time to first progression (TTP), and 2-year overall survival (OS) were correlated with the 21-gene RS by using log-rank, Kaplan-Meier, and Cox regression. RESULTS Median patient age was 52 years (21 to 79 years); the majority had hormone receptor-positive/human epidermal growth factor receptor 2 (HER2)-negative (72 [66%]) or hormone receptor-positive/HER2-positive (20 [18%]) breast cancer. At a median follow-up of 29 months, median TTP was 20 months (95% CI, 16 to 26 months), and median survival was 49 months (95% CI, 40 months to not reached). An RS was generated for 101 (93%) primary tumor samples: 22 (23%) low risk (< 18), 29 (28%) intermediate risk (18 to 30); and 50 (49%) high risk (≥ 31). For all patients, RS was associated with TTP (P = .01) and 2-year OS (P = .04). In multivariable Cox regression models among 69 patients with estrogen receptor (ER)-positive/HER2-negative cancer, RS was independently prognostic for TTP (hazard ratio, 1.40; 95% CI, 1.05 to 1.86; P = .02) and 2-year OS (hazard ratio, 1.83; 95% CI, 1.14 to 2.95; P = .013). CONCLUSION The 21-gene RS is independently prognostic for both TTP and 2-year OS in ER-positive/HER2-negative de novo stage IV breast cancer. Prospective validation is needed to determine the potential role for this assay in the clinical management of this patient subset.

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

IntroductionSurgery 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.MethodsGLV-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 μCi of 124I-iodide.ResultsViral infectivity, measured by green fluorescent protein (GFP) expression, was time- and concentration-dependent. 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 mm3 versus 168 mm3 in untreated controls (P < 0.05).ConclusionsThis 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.

Clonal relationships between lobular carcinoma in situ and other breast malignancies

BackgroundRecent evidence suggests that lobular carcinoma in situ (LCIS) can be a clonal precursor of invasive breast cancers of both the ductal and lobular phenotypes. We sought to confirm these findings with an extensive study of fresh frozen breast specimens from women undergoing mastectomy.MethodsPatients with a history of LCIS presenting for therapeutic mastectomy were identified prospectively. Frozen tissue blocks were collected, screened for lesions of interest, and subjected to microdissection and DNA extraction. Copy number profiling, whole-exome sequencing, or both were performed. Clonal relatedness was assessed using specialized statistical techniques developed for this purpose.ResultsAfter exclusions for genotyping failure, a total of 84 lesions from 30 patients were evaluated successfully. Strong evidence of clonal relatedness was observed between an LCIS lesion and the invasive cancer for the preponderance of cases with lobular carcinoma. Anatomically distinct in situ lesions of both ductal and lobular histology were also shown to be frequently clonally related.ConclusionsThese data derived from women with LCIS with or without invasive cancer confirm that LCIS is commonly the clonal precursor of invasive lobular carcinoma and that distinct foci of LCIS frequently share a clonal origin, as do foci of LCIS and ductal carcinoma in situ.

Carcinomas mamários de tipo basal: perfil clínico-patológico e evolutivo

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.