Brad Love

Outer breast quadrants demonstrate increased levels of genomic instability.

AbstractBackground: Theory holds that the upper outer quadrant of the breast develops more malignancies because of increased tissue volume. This study evaluated genomic patterns of loss of heterozygosity (LOH) and allelic imbalance (AI) in non-neoplastic tissues from quadrants of diseased breasts following mastectomy to characterize relationships between genomic instability and the propensity for tumor development. Methods: Tissues from breast quadrants were collected from 21 patients with various stages of breast carcinoma. DNA was isolated from non-neoplastic tissues using standard methods and 26 chromosomal regions commonly deleted in breast cancer were examined to assess genomic instability. Results: Genomic instability was observed in breast quadrants from patients with ductal carcinomas in situ and advanced carcinomas. Levels of instability by quadrant were not predictive of primary tumor location (P = .363), but outer quadrants demonstrated significantly higher levels of genomic instability than did inner quadrants (P = .017). Marker D8S511 on chromosome 8p22– 21.3, one of the most frequently altered chromosomal regions in breast cancer, showed a significantly higher level of instability (P = .039) in outer compared with inner quadrants. Conclusions: Non-neoplastic breast tissues often harbor genetic changes that can be important to understanding the local breast environment within which cancer develops. Greater genomic instability in outer quadrants can partially explain the propensity for breast cancers to develop there, rather than simple volume-related concepts. Patterns of field cancerization in the breast appear to be complex and are not a simple function of distance from a developing tumor.

Identification of differentially expressed genes in breast tumors from African American compared with Caucasian women.

Breast tumors from African American women have less favorable pathological characteristics and higher mortality rates than those of Caucasian women. Although socioeconomic status may influence prognosis, biological factors are also likely to contribute to tumor behavior.

Combined histomorphometric and gene-expression profiling applied to toxicology

We have developed a unique methodology for the combined analysis of histomorphometric and gene-expression profiles amenable to intensive data mining and multisample comparison for a comprehensive approach to toxicology. This hybrid technology, termed extensible morphometric relational gene-expression analysis (EMeRGE), is applied in a toxicological study of time-varied vehicle- and carbon-tetrachloride (CCl4)-treated rats, and demonstrates correlations between specific genes and tissue structures that can augment interpretation of biological observations and diagnosis.

Amplification of HER2 is a marker for global genomic instability

BackgroundGenomic alterations of the proto-oncogene c-erbB-2 (HER-2/neu) are associated with aggressive behavior and poor prognosis in patients with breast cancer. The variable clinical outcomes seen in patients with similar HER2 status, given similar treatments, suggests that the effects of amplification of HER2 can be influenced by other genetic changes. To assess the broader genomic implications of structural changes at the HER2 locus, we investigated relationships between genomic instability and HER2 status in patients with invasive breast cancer.MethodsHER2 status was determined using the PathVysion® assay. DNA was extracted after laser microdissection from the 181 paraffin-embedded HER2 amplified (n = 39) or HER2 negative (n = 142) tumor specimens with sufficient tumor available to perform molecular analysis. Allelic imbalance (AI) was assessed using a panel of microsatellite markers representing 26 chromosomal regions commonly altered in breast cancer. Student t-tests and partial correlations were used to investigate relationships between genomic instability and HER2 status.ResultsThe frequency of AI was significantly higher (P < 0.005) in HER2 amplified (27%) compared to HER2 negative tumors (19%). Samples with HER2 amplification showed significantly higher levels of AI (P < 0.05) at chromosomes 11q23, 16q22-q24 and 18q21. Partial correlations including ER status and tumor grade supported associations between HER2 status and alterations at 11q13.1, 16q22-q24 and 18q21.ConclusionThe poor prognosis associated with HER2 amplification may be attributed to global genomic instability as cells with high frequencies of chromosomal alterations have been associated with increased cellular proliferation and aggressive behavior. In addition, high levels of DNA damage may render tumor cells refractory to treatment. In addition, specific alterations at chromosomes 11q13, 16q22-q24, and 18q21, all of which have been associated with aggressive tumor behavior, may serve as genetic modifiers to HER2 amplification. These data not only improve our understanding of HER in breast pathogenesis but may allow more accurate risk profiles and better treatment options to be developed.

Micro RNA Profiling

MicroRNAs (miRNAs) are small regulatory RNAs varying in length between 20 and 24 nucleotides. They are thought to play a key role during development by negative gene regulation at the post-transcriptional level. Recent studies using quantitative polymerase chain reaction (QPCR) and northern blot analysis have reported the presence of several miRNA unique to specific cell types. The NCode multispecies miRNA array provides a means for simultaneously profiling the expression patterns of hundreds of known miRNAs in a given cell type or biological sample. Using this method, miRNA expression patterns in embryonic and adult stem cell lines can be characterized and compared with each other. The accuracy of NCode miRNA array data can be further confirmed by QPCR analysis of putative array hits. This array-based screening platform is a fast and easy to use analytical tool that allows one to asses the state of stem cell lines following multiple passages in culture as well as a discovery tool that eliminates the need to screen large numbers of candidate regulatory miRNAs by northern blot or PCR. In this chapter, we describe in detail the method to carry out miRNA array analysis in human embryonal carcinoma cells and confirm the array results using QPCR.

Timing of Critical Genetic Changes in Human Breast Disease

BackgroundBreast cancer development has been characterized as a nonobligatory sequence of histological changes from normal epithelium through invasive malignancy. Although genetic alterations are thought to accumulate stochastically during tumorigenesis, little is known about the timing of critical mutations. This study examined allelic imbalance (AI) in tissue samples representing a continuum of breast cancer development to examine the evolution of genomic instability.MethodsLaser-microdissected DNA samples were collected from histologically normal breast specimens (n = 25), atypical ductal hyperplasia (ADH, n = 16), ductal carcinoma-in-situ (DCIS, n = 37), and stage I to III invasive carcinomas (n = 72). Fifty-two microsatellite markers representing 26 chromosomal regions commonly deleted in breast cancer were used to assess patterns of AI.ResultsAI frequencies were <5% in histologically normal and ADH specimens, 20% in DCIS lesions, and approximately 25% in invasive tumors. Mann-Whitney tests showed (1) that levels of AI in ADH samples did not differ significantly from those in histologically normal tissues and (2) that AI frequencies in DCIS lesions were not significantly different from those in invasive carcinomas. ADH and DCIS samples, however, differed significantly (P < .0001).ConclusionsDCIS lesions contain levels of genomic instability that are characteristic of advanced invasive tumors, and this suggests that the biology of a developing carcinoma may already be predetermined by the in situ stage. Observations that levels of AI in ADH lesions are similar to those in disease-free tissues provide a genomic rationale for why prevention strategies at the ADH level are successful and why cases with ADH involving surgical margins do not require further resection.

Differential Gene Expression in Primary Breast Tumors Associated with Lymph Node Metastasis

Lymph node status remains one of the most useful prognostic indicators in breast cancer; however, current methods to assess nodal status disrupt the lymphatic system and may lead to secondary complications. Identification of molecular signatures discriminating lymph node-positive from lymph node-negative primary tumors would allow for stratification of patients requiring surgical assesment of lymph nodes. Primary breast tumors from women with negative (n = 41) and positive (n = 35) lymph node status matched for possible confounding factors were subjected to laser microdissection and gene expression data generated. Although ANOVA analysis (P < .001, fold-change >1.5) revealed 13 differentially expressed genes, hierarchical clustering classified 90% of node-negative but only 66% of node-positive tumors correctly. The inability to derive molecular profiles of metastasis in primary tumors may reflect tumor heterogeneity, paucity of cells within the primary tumor with metastatic potential, influence of the microenvironment, or inherited host susceptibility to metastasis.

Molecular Changes in Primary Breast Tumors and the Nottingham Histologic Score

Pathological grade is routinely used to stratify breast cancer patients into favorable and less favorable outcome groups. Mechanisms by which genomic changes in breast tumors specifically contribute to the underlying components of tumor grade – tubule formation, nuclear pleomorphism, and mitoses — are unknown. This study examined 26 chromosomal regions known to be altered in breast cancer in 256 invasive breast carcinomas. Differences in overall levels and patterns of allelic imbalance (AI) at each chromosomal region were compared for tumors with favorable (=1) and unfavorable (=3) scores for tubule formation, nuclear pleomorphism and mitotic count. Levels of AI were significantly different between samples with high and low scores for tubule formation (P < 0.001), nuclear pleomorphism (P < 0.001) and mitotic count (P < 0.05). Significantly higher levels of AI were detected at regions 11q23 and 13q12 for tumors with reduced tubule formation, chromosomes 9p21, 11q23, 13q14, 17p13 and 17q12 for those with high levels of nuclear atypia, and chromosomes 1p36, 11q23, and 13q14 for those with high mitotic counts. Region 16q11-q22 showed significantly more AI events in samples with low nuclear atypia. Patterns of genetic changes associated with poorly-differentiated breast tumors were recapitulated by the individual components of the Nottingham Histologic Score. While frequent alteration of 11q23 is common for reduced tubule formation, high nuclear atypia and high mitotic counts, suggesting that this is an early genetic change in the development of poorly-differentiated breast tumors, alterations at the other seven loci associated with poorly-differentiated tumors may specifically influence cell structure, nuclear morphology and cellular proliferation.

Abundance and distribution of polychlorinated biphenyls (PCBs) in breast tissue.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #5039 Background: Breast cancer is a complex disease whose causative factors remain ambiguous and poorly defined. Many environmental chemicals accumulate in human tissues and may contribute to breast cancer risk. Polychlorinated biphenyls (PCBs), which constitute a class of chlorinated compounds that are ubiquitous in the environment, are believed to be associated with adverse health effects. Because the rising incidence of breast cancer may result from exposure to increasing levels of exogenous chemicals, there is keen interest in determining relationships between exposure to environmental chemicals and breast cancer risk. Material and Methods: Frozen sections of breast tissue were collected from each quadrant and the central region of the breast from 47 patients enrolled in the Clinical Breast Care Project at Walter Reed Army Medical Center. Pathological diagnoses ranged from disease free prophylactic mastectomy to metastatic breast cancer. From a portion of each sample, 98 PCB congeners were assayed by pressurized liquid extraction followed by high resolution gas chromatography. Results: PCBs were abundant in breast tissues. Only one congener was not observed in any patient and four (153, 138+163, 180, 206) were seen in all patients in at least one quadrant. Mean total PCB concentration in 202 breast quadrants was 602 ng/g lipid (range 0-4030), while for individual congeners, PCB 153 had an average level of 107 ng/g lipid (range 8-773 ng/g). Total PCB concentration (P<<0.001) and presence of 26 congeners (P<0.05) were highly correlated with patient age, particularly in the UIQ and LOQ, but not in the central region, suggesting that chemicals may accumulate at different rates in different regions of the breast. Similarly, no significant differences were observed for PCB concentrations among the four traditional quadrants, but 23 PCBs were present at significantly lower levels (P<0.05) in the central region. When examining PCB levels by disease status, seven individual congeners and median overall levels were significantly higher (P<0.05) in patients with invasive cancer compared to disease free patients. Discussion: Population monitoring is beginning to assess exposures of the general public to environmental chemicals to evaluate long-term health effects. This study showed that many PCB residues are present in human breast tissue, these chemicals are not distributed evenly, and several are correlated with age and presence of invasive disease. Improved understanding of the true carcinogenic potential of environmental chemicals may be useful in efforts to reduce individual risk for breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5039.

Fingerprinting genomic heterogeneity in primary breast carcinomas and among sentinel lymph node metastases: implications for clinical management of breast cancer patients.

Abstract #1059 Background: Sentinel lymph node (SLN) status is a key prognostic factor for breast cancer patients. The SLN hypothesis postulates that cancer cells initially spread from the primary tumor to the first-draining (sentinel) node(s) before reaching higher echelon nodes. Although SLN biopsy is clinically useful for staging patients, little is known about the genomic heritage of cells that define the genetic makeup and clinical behavior of nodal metastases. We used allelic imbalance (AI) coupled with high-density SNP genotyping to examine the extent of genetic heterogeneity in primary tumors and relationships among metastases in sentinel and non-sentinel axillary lymph nodes in 30 patients with node positive breast cancer.
 Material and Methods: Pathologically positive nodes were identified by HE genome-wide copy number variation was examined using Affymetrix GeneChip Human Mapping 500K arrays and the Genotyping Consol. The genomic heritage of sentinel and non-sentinel nodal metastases in relation to multiple areas of the primary tumor was assessed by hierarchical clustering and phylogenetic analyses.
 Results: Extensive genomic heterogeneity was observed in primary tumors and among nodal metastases (0-44% in primaries, 0-36% in metastases). Overall levels of variation were significantly higher (P Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1059.