Proteogenomic characterization of difficult-to-treat breast cancer with tumor cells enriched through laser microdissection.

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors. METHODS: We retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers. RESULTS: We observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations in TP53, PLXNB3, Zinc finger genes, and fewer mutations in SDC2, CDH1, PIK3CA, SVIL, and PTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA-protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, including KIAA1522, DCK, FOXO3, MYO9B, ARID1A, EPRS, ZC3HAV1, and RBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors. CONCLUSIONS: This study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Association of clinicopathologic and molecular factors with the occurrence of positive margins in breast cancer.

PURPOSE: To explore the association of clinicopathologic and molecular factors with the occurrence of positive margins after first surgery in breast cancer. METHODS: The clinical and RNA-Seq data for 951 (75 positive and 876 negative margins) primary breast cancer patients from The Cancer Genome Atlas (TCGA) were used. The role of each clinicopathologic factor for margin prediction and also their impact on survival were evaluated using logistic regression, Fisher's exact test, and Cox proportional hazards regression models. In addition, differential expression analysis on a matched dataset (71 positive and 71 negative margins) was performed using Deseq2 and LASSO regression. RESULTS: Association studies showed that higher stage, larger tumor size (T), positive lymph nodes (N), and presence of distant metastasis (M) significantly contributed (p ≤ 0.05) to positive surgical margins. In case of surgery, lumpectomy was significantly associated with positive margin compared to mastectomy. Moreover, PAM50 Luminal A subtype had higher chance of positive margin resection compared to Basal-like subtype. Survival models demonstrated that positive margin status along with higher stage, higher TNM, and negative hormone receptor status was significant for disease progression. We also found that margin status might be a surrogate of tumor stage. In addition, 29 genes that could be potential positive margin predictors and 8 pathways were identified from molecular data analysis. CONCLUSION: The occurrence of positive margins after surgery was associated with various clinical factors, similar to the findings reported in earlier studies. In addition, we found that the PAM50 intrinsic subtype Luminal A has more chance of obtaining positive margins compared to Basal type. As the first effort to pursue molecular understanding of the margin status, a gene panel of 29 genes including 17 protein-coding genes was also identified for potential prediction of the margin status which needs to be validated using a larger sample set.

Assessing the quality of RNA isolated from human breast tissue after ambient room temperature exposure.

High quality human tissue is essential for molecular research, but pre-analytical conditions encountered during tissue collection could degrade tissue RNA. We evaluated how prolonged exposure of non-diseased breast tissue to ambient room temperature (22±1°C) impacted RNA quality. Breast tissue received between 70 to 190 minutes after excision was immediately flash frozen (FF) or embedded in Optimal Cutting Temperature (OCT) compound upon receipt (T0). Additional breast tissue pieces were further exposed to increments of 60 (T1 = T0+60 mins), 120 (T2 = T0+120 mins) and 180 (T3 = T0+180 mins) minutes of ambient room temperature before processing into FF and OCT. Total exposure, T3 (T0+180 mins) ranged from 250 minutes to 370 minutes. All samples (FF and OCT) were stored at -80°C before RNA isolation. The RNA quality assessment based on RNA Integrity Number (RIN) showed RINs for both FF and OCT samples were within the generally acceptable range (mean 7.88±0.90 to 8.52±0.66). No significant difference was observed when RIN at T0 was compared to RIN at T1, T2 and T3 (FF samples, p = 0.43, 0.56, 0.44; OCT samples, p = 0.25, 0.82, 1.0), or when RIN was compared between T1, T2 and T3. RNA quality assessed by quantitative real-time PCR (qRT-PCR) analysis of beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A (CYPA), and porphobilinogen deaminase (PBGD) transcripts showed threshold values (Ct) that indicate abundant and intact target nucleic acid in all samples (mean ranging from 14.1 to 25.3). The study shows that higher RIN values were obtained for non-diseased breast tissue up to 190 minutes after resection and prior to stabilization. Further experimental exposure up to 180 minutes had no significant effect on RIN values. This study strengthens the rationale for assessing RIN and specific gene transcript levels as an objective method for determining how suitable RNA will be for a specific research purpose ("fit-for purpose").

Comparative analysis of differentially abundant proteins quantified by LC-MS/MS between flash frozen and laser microdissected OCT-embedded breast tumor samples.

BACKGROUND: Proteomic studies are typically conducted using flash-frozen (FF) samples utilizing tandem mass spectrometry (MS). However, FF specimens are comprised of multiple cell types, making it difficult to ascertain the proteomic profiles of specific cells. Conversely, OCT-embedded (Optimal Cutting Temperature compound) specimens can undergo laser microdissection (LMD) to capture and study specific cell types separately from the cell mixture. In the current study, we compared proteomic data obtained from FF and OCT samples to determine if samples that are stored and processed differently produce comparable results. METHODS: Proteins were extracted from FF and OCT-embedded invasive breast tumors from 5 female patients. FF specimens were lysed via homogenization (FF/HOM) while OCT-embedded specimens underwent LMD to collect only tumor cells (OCT/LMD-T) or both tumor and stromal cells (OCT/LMD-TS) followed by incubation at 37 °C. Proteins were extracted using the illustra triplePrep kit and then trypsin-digested, TMT-labeled, and processed by two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS). Proteins were identified and quantified with Proteome Discoverer v1.4 and comparative analyses performed to identify proteins that were significantly differentially expressed amongst the different processing methods. RESULTS: Among the 4,950 proteins consistently quantified across all samples, 216 and 171 proteins were significantly differentially expressed (adjusted p-value < 0.05; |log2 FC|> 1) between FF/HOM vs. OCT/LMD-T and FF/HOM vs. OCT/LMD-TS, respectively, with most proteins being more highly abundant in the FF/HOM samples. PCA and unsupervised hierarchical clustering analysis with these 216 and 171 proteins were able to distinguish FF/HOM from OCT/LMD-T and OCT/LMD-TS samples, respectively. Similar analyses using significantly differentially enriched GO terms also discriminated FF/HOM from OCT/LMD samples. No significantly differentially expressed proteins were detected between the OCT/LMD-T and OCT/LMD-TS samples but trended differences were detected. CONCLUSIONS: The proteomic profiles of the OCT/LMD-TS samples were more similar to those from OCT/LMD-T samples than FF/HOM samples, suggesting a strong influence from the sample processing methods. These results indicate that in LC-MS/MS proteomic studies, FF/HOM samples exhibit different protein expression profiles from OCT/LMD samples and thus, results from these two different methods cannot be directly compared.

The Impact of Hormonal Contraceptives on Breast Cancer Pathology.

This retrospective case series study, using data obtained through questionnaires and histopathological diagnoses from 656 patients enrolled in the Department of Defense (DoD) Clinical Breast Care Project (CBCP), evaluated associations between hormonal contraceptive use and breast cancer pathology including benign breast pathologies. Three combination hormonal contraceptive agents (COCs) Lo Ovral (LO), Ortho Novum (ON), and Ortho Tri-Cyclen (OTC) were evaluated as they represented the most commonly used hormonal contraceptives in our cohort. The results of this study suggest that the ever use of LO + ON + OTC does not influence the overall incidence of benign breast condition or malignant disease compared to other COCs; however, patients that have used OTC had an association with a diagnosis of benign or luminal A pathologies whereas ON was associated with a diagnosis of benign and DCIS; LO showed no association with any diagnosis-benign or malignant. Patients that have used LO or ON were more likely to be diagnosed with breast cancer at age ≥ 40 years whereas patients that had ever used OTC were likely to be diagnosed before the age of 40. Caucasians were less likely to have used OTC and more likely to have used ON; however, use of either hormonal agent positively correlated with premenopausal status at diagnosis and having a benign condition. Age at diagnosis, ethnicity, BMI, family history, menstruation status, and duration of use were all independent predictors of different histopathological subtypes. We conclude that patient-specific variables should be considered when deciding on which type of hormonal contraceptive to use to minimize the risk of developing breast cancer or a breast-related pathology.

Comparative Survival Analysis of Invasive Breast Cancer Patients Treated by a U.S. Military Medical Center and Matched Patients From the U.S. General Population.

OBJECTIVE: Many differences between U.S. military beneficiaries and the U.S. general population, including differences in health care access, are known factors affecting invasive breast cancer outcomes. Thus, comparing the two populations for any outcome differences and their contributing factors may provide insights to breast cancer prognosis. METHODS: Using a marginal Cox proportional hazards regression model, we compared disease-specific survival (DSS) and 5-year DSS rates between 418 patients from the Clinical Breast Care Project at the Walter Reed National Military Medical Center (CBCP-WR) and a set of 1:5 randomly matched patients from the Surveillance, Epidemiology, and End Results program. Patients were compared in the "demographic model" (adjusted by diagnosis year, age, and race) and the "overall model" (further adjusted by estrogen receptor, progesterone receptor, stage, and grade). RESULTS: In the "overall model," CBCP-WR patients were less likely overall to die from breast cancer (hazard ratio [HR] = 0.631, 95% confidence interval [CI] = 0.437-0.911; p = 0.014). This increase in survival was also significant in African American patients (HR = 0.524, 95% CI = 0.277-0.992; p = 0.047) and patients older than 50 (HR = 0.511, 95% CI = 0.306-0.854; p = 0.010). The advantage in 5-year DSS rate for CBCP-WR patients was 5.3% (93.1% vs. 87.8%; p < 0.001) in the "demographic model" and 3.4% (91.3% vs. 87.9%; p = 0.018) in the "overall model." CONCLUSION: CBCP-WR patients demonstrated significantly better DSS over matched SEER patients. Although a portion of the outcome disparity, i.e., 36% of the 5.3% DSS rate difference, could be explained by differences in tumor characteristics, the cause(s) behind the majority of the disparity has yet to be identified. Identification and further analysis of contributing factors to survival differences have the potential to improve clinical practice and outcomes for invasive breast cancer patients.

Molecular Heterogeneity in Primary Breast Carcinomas and Axillary Lymph Node Metastases Assessed by Genomic Fingerprinting Analysis.

Molecular heterogeneity within primary breast carcinomas and among axillary lymph node (LN) metastases may impact diagnosis and confound treatment. In this study, we used short tandem repeated sequences to assess genomic heterogeneity and to determine hereditary relationships among primary tumor areas and regional metastases from 30 breast cancer patients. We found that primary carcinomas were genetically heterogeneous and sampling multiple areas was necessary to adequately assess genomic variability. LN metastases appeared to originate at different time periods during disease progression from different sites of the primary tumor and the extent of genomic divergence among regional metastases was associated with a less favorable patient outcome (P = 0.009). In conclusion, metastasis is a complex process influenced by primary tumor heterogeneity and variability in the timing of dissemination. Genomic variation in primary breast tumors and regional metastases may negatively impact clinical diagnostics and contribute to therapeutic resistance.

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

Many environmental chemicals accumulate in human tissues and may contribute to cancer risk. Polychlorinated biphenyls (PCBs) are associated with adverse health effects, but relationships between PCB exposure and breast cancer are unclear. In this study, we sought to determine whether bioaccumulation of PCBs differs within regions of the human breast and whether PCB levels are associated with clinical and pathological characteristics in breast cancer patients. Tissue sections (n=245) were collected from breast quadrants from 51 women with a diagnosis ranging from disease-free to metastatic breast cancer. Ninety-seven PCB congeners were assayed by high resolution gas chromatography. ANOVA was used to examine PCB distribution within the breast and relationships with clinical/pathological variables. Pearson product-moment correlations assessed relationships between age at mastectomy and PCB levels. PCBs were abundant in breast tissues with a median concentration of 293.4ng/g lipid (range 15.4-1636.3ng/g). PCB levels in breast tissue were significantly different (p<0.001) among functional groupings of congeners defined by structure-activity properties: Group I (28.2ng/g), Group II (96.6ng/g), Group III (166.0ng/g). Total PCB concentration was highly correlated with age at mastectomy, but the distribution of PCBs did not differ by breast quadrant. PCB levels were not associated with patient status or tumor characteristics. In conclusion, PCB congeners with carcinogenic potential were present at high levels in the human breast, but were not associated with clinical or pathological characteristics in breast cancer patients.

Gene expression differences in adipose tissue associated with breast tumorigenesis.

Long thought to function only as an inert energy storage depot, the role of adipose tissue in breast tumorigenesis has been largely ignored. In light of increasing rates of obesity and use of breast conserving therapy and autologous fat grafting, improved understanding of the role of adipose tissue in tumor etiology is crucial. Thus, adipose tissue adjacent to and distant from invasive breast tumors (n = 20), or adjacent to non-malignant diagnoses (n = 20) was laser microdissected from post-menopausal women. Gene expression data were generated using microarrays and data analyzed to identify significant patterns of differential expression between adipose tissue groups at the individual gene and molecular pathway level. Pathway analysis revealed significant differences in immune response between non-malignant, distant, and tumor-adjacent adipose tissue, with the highest response in tumor-adjacent and lowest in non-malignant adipose tissue. Adipose tissue from invasive breasts exhibits increased expression of anti-inflammatory genes such as MARCO and VSIG4 while genes differentially expressed between tumor-adjacent and distant adipose tissue such as SPP1, RRM2, and MMP9, are associated with increased cellular proliferation, invasion, and angiogenesis. These data suggest that molecular profiles of adipose tissue differ depending on presence of or proximity to tumor cells. Heightened immunotolerance in adipose tissue from invasive breasts provides a microenvironment favorable to tumorigenesis. In addition, tumor-adjacent adipose tissue demonstrates expression of genes associated with tumor growth and progression. Thus, adipose tissue is not an inert component of the breast microenvironment but plays an active role in tumorigenesis.

Effect of ASCO/CAP guidelines for determining ER status on molecular subtype.

BACKGROUND: Determination of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status is standard for predicting prognosis and determining treatment options for patients with breast cancer. In 2010, the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) issued guidelines that tumors with ≥1% positively staining cells should be considered ER positive. Here, we determined how this cutoff relates to molecular subtype. METHODS: Clinicopathological characteristics were compared between ER-negative, ER-positive, and low-ER-staining (1-10%) tumors using chi-square analysis with P<0.05 defining statistical significance. Gene expression data were generated for 26 low-ER-staining tumors, and their intrinsic subtype determined. Immunohistochemistry (IHC)-defined surrogate subtypes, using the threshold of positivity defined by ASCO/CAP guidelines, were compared with molecular subtypes. RESULTS: Low-ER-staining tumors were clinicopathologically more similar to ER-negative than to ER-positive tumors; 88% of low-staining tumors were basal like or HER2 enriched. Only those tumors expressing 10% ER-positive cells were classified as luminal A subtype. CONCLUSIONS: Under ASCO/CAP guidelines, tumors with 1-10% ER staining would be classified as ER positive, yet most are basal like or HER2 enriched and have pathological features similar to ER-negative tumors. Clinical trials seeking to treat tumors of ER-negative basal-like and/or HER2-enriched subtypes should thus not preclude enrollment based solely on results of ER immunohistochemistry. As ER status is a critical element in the choice of treatments for patients with breast cancer, it is imperative that the most effective method for classifying tumors be developed.

Tissue imprints: assessing their potential for routine biobanking collection.

Biomedical research depends on the availability of good quality biospecimens. Unfortunately, certain specimens are scarce due to disease rarity or size restrictions of surgical materials. To increase access to limited surgical specimens, Biobanks need to reassess and adjust their collection programs. We evaluated the feasibility of adapting "touch imprints" to gain access to limited surgical specimens as well as to maximize the use of "precious" specimens. We utilized 12 kidney samples for touch imprints on defined areas of microscope glass slides and FTA paper. DNA was isolated from glass slides on the day of preparation, Day 0, and from glass slide and FTA paper preparations after two weeks of storage at room temperature and -80°C. Yield and purity of DNA from reference kidney samples were compared to DNA from the touch imprints and the quality determined by real-time PCR using the amplification of Cyclophilin A (Cyc A) as an index. DNA quality for glass slides at Day 0 was not significantly different from DNA after two weeks at room temperature (glass at room temperature; p=0.111 and 0.097, yield and purity, respectively) and after two weeks at -80°C (glass -80°C; p=0.358 and 0.281, yield and purity, respectively). Glass slide DNA at room temperature and -80°C were not significantly different (p=0.795 and 0.146 for yield and purity, respectively). DNA from FTA paper at room temperature and from FTA paper at -80°C were significantly different from glass at room temperature and glass at -80°C (p=0.002, respectively). Threshold values for Cyc A were ≤28 for the reference DNA and ≤32 for DNA from glass and FTA paper. This study demonstrates that touch preparations on microscope glass slides and FTA paper can provide sufficient and good quality DNA suitable for PCR. Touch imprints could therefore be adopted by biobanks to collect and bank biological materials from limited surgical specimens.

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

BACKGROUND: 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. METHODS: Patients with invasive breast cancer were matched by age, grade, and estrogen receptor status; patients with benign disease were matched by age and diagnosis type. RNA from laser microdissected tumors and whole-sectioned nonmalignant breast tissues was hybridized to HG U133A 2.0 microarrays. Data were analyzed using Partek Genomics Suite using a cutoff of P < .001, >1.5-fold change, and results were validated by quantitative real-time polymerase chain reaction. RESULTS: Clinicopathological factors did not differ significantly between groups for age at diagnosis, tumor size or stage, lymph node or human epidermal growth receptor 2 status, intrinsic subtype, or mortality. Two-way analysis of the tumor specimens revealed 25 probes representing 23 genes differentially expressed between populations; hierarchical clustering classified 24 of 26 African American women and 25 of 26 Caucasian women correctly. In the nonmalignant specimens, 15 probes representing 13 genes were differentially expressed, including 5 genes that also differed in the tumor specimens; these genes were able to correctly classify nonmalignant breast specimens from 20 of 22 of African American women and all of the Caucasian women. CONCLUSIONS: Despite matching of tumors by pathological characteristics, molecular profiles differed between African American women and Caucasian women in both invasive tumors and benign breast tissues. These differentially expressed genes, including CRYBB2, PSPHL, and SOS1, are involved in cellular growth and differentiation, invasion, metastasis, and immune response and thus may contribute to the poor outcome in African American women.

Laser microdissection for gene expression profiling.

Microarray-based gene expression profiling is revolutionizing biomedical research by allowing expression profiles of thousands of genes to be interrogated in a single experiment. In cancer research, the use of laser microdissection (LM) to isolate RNA from tissues provides the ability to accurately identify molecular profiles from different cell types that comprise the tumor and its surrounding microenvironment. Because RNA is an unstable molecule, the quality of RNA extracted from tissues can be affected by sample preparation and processing. Thus, special protocols have been developed to isolate research-quality RNA after LM. This chapter provides detailed descriptions of protocols used to generate micro-array data from high-quality frozen breast tissue specimens, as well as challenges associated with formalin-fixed paraffin-embedded specimens.

Chromosomal alterations in pure nonneoplastic breast lesions: implications for breast cancer progression.

INTRODUCTION: Columnar cell lesions (CCL) and atypical ductal hyperplasia (ADH) frequently coexist and share molecular changes with in situ and invasive components, suggesting that CCL and ADH may be precursors to breast cancer. These conclusions are largely based on studies examining CCL and/or ADH from patients diagnosed with more advanced disease. We assessed allelic imbalance (AI) in pure CCL or ADH specimens to characterize molecular changes in nonneoplastic breast lesions. METHODS: DNA samples were obtained from laser-microdissected pure CCL (n = 42) or ADH (n = 31). AI was assessed at 26 chromosomal regions commonly altered in breast cancer. Data were analyzed using Fisher's exact and Student's t-tests using a cutoff of P < 0.05. RESULTS: The average AI frequency was 6.2% in CCL and 6.1% in ADH; approximately 33% of nonneoplastic lesions had no detectable genetic changes. Levels of AI in CCL and ADH were significantly (P < 0.0001) lower than observed in either low- or high-grade ductal carcinoma in situ (DCIS) lesions. Genetic changes characteristic of in situ and invasive disease, especially on chromosomes 16q and 17p, were infrequent in pure nonneoplastic lesions. CONCLUSIONS: Pure CCL and ADH lesions demonstrate lower levels of genetic alterations than DCIS, invasive carcinomas or CCL/ADH lesions from cancerous breasts; alterations of chromosomes 16q and 17p were not detected. Pure CCL and ADH lesions are not genetically advanced, and molecular profiles do not support these lesions as obligatory precursors to more advanced disease. Molecular differences between pure and synchronous lesions support re-evaluation of current models of disease initiation, progression, and risk.

Amplification of HER2 is a marker for global genomic instability.

BACKGROUND: Genomic 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. METHODS: HER2 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. RESULTS: The 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. CONCLUSION: The 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.

Genomic alterations associated with early stages of breast tumor metastasis.

BACKGROUND: Molecular studies suggest that acquisition of metastatic potential occurs early in the development of breast cancer; mechanisms by which cells disseminate from the primary carcinomas and successfully colonize foreign tissues are, however, largely unknown. Thus, we examined levels and patterns of chromosomal alterations in primary breast tumors from node-negative (n = 114) and node-positive (n = 115) patients to determine whether specific genomic changes are associated with tumor metastasis. METHODS: Fifty-two genetic markers representing 26 chromosomal regions commonly altered in breast cancer were examined in laser microdissected tumor samples to assess levels and patterns of allelic imbalance (AI). Real time-PCR (RT-PCR) was performed to determine expression levels of candidate genes. Data was analyzed using exact unconditional and Student's t-tests with significance values of P < 0.05 and P < 0.002 used for the clinicopathological and genomic analyses, respectively. RESULTS: Overall levels of AI in primary breast tumors from node-negative (20.8%) and node-positive (21.9%) patients did not differ significantly (P = 0.291). When data were examined by chromosomal region, only chromosome 8q24 showed significantly higher levels (P < 0.0005) of AI in node-positive primary tumors (23%) versus node-negative samples (6%). c-MYC showed significantly higher levels of gene expression in primary breast tumors from patients with lymph node metastasis. CONCLUSIONS: Higher frequencies of AI at chromosome 8q24 in patients with positive lymph nodes suggest that genetic changes in this region are important to the process of metastasis. Because overexpression of c-MYC has been associated with cellular dissemination as well as development of the premetastatic niche, alterations of the 8q24 region, including c-MYC, may be key determinants in the development of lymph node metastasis.

The genomic heritage of lymph node metastases: implications for clinical management of patients with breast cancer.

BACKGROUND: Metastatic breast cancer is an aggressive disease associated with recurrence and decreased survival. To improve outcomes and develop more effective treatment strategies for patients with breast cancer, it is important to understand the molecular mechanisms underlying metastasis. METHODS: We used allelic imbalance (AI) to determine the molecular heritage of primary breast tumors and corresponding metastases to the axillary lymph nodes. Paraffin-embedded samples from primary breast tumors and matched metastases (n = 146) were collected from 26 patients with node-positive breast cancer involving multiple axillary nodes. Hierarchical clustering was used to assess overall differences in the patterns of AI, and phylogenetic analysis inferred the molecular heritage of axillary lymph node metastases. RESULTS: Overall frequencies of AI were significantly higher (P < 0.01) in primary breast tumors (23%) than in lymph node metastases (15%), and there was a high degree of discordance in patterns of AI between primary breast carcinomas and the metastases. Metastatic tumors in the axillary nodes showed different patterns of chromosomal changes, suggesting that multiple molecular mechanisms may govern the process of metastasis in individual patients. Some metastases progressed with few genomic alterations, while others harbored many chromosomal alterations present in the primary tumor. CONCLUSIONS: The extent of genomic heterogeneity in axillary lymph node metastases differs markedly among individual patients. Genomic diversity may be associated with response to adjuvant therapy, recurrence, and survival, and thus may be important in improving clinical management of breast cancer patients.

Timing of critical genetic changes in human breast disease.

BACKGROUND: Breast 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. METHODS: Laser-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. RESULTS: AI 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). CONCLUSIONS: DCIS 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.

Allelic imbalance in primary breast carcinomas and metastatic tumors of the axillary lymph nodes.

Axillary lymph node status is the most important prognostic factor in predicting disease outcome in women with breast cancer. A number of chromosomal aberrations in primary breast tumors have been correlated with lymph node status and clinical outcome, but chromosomal changes particular to metastatic lymph node tumors have not been well studied. DNA samples isolated from laser-microdissected primary breast and metastatic axillary lymph node tumors from 25 women with invasive breast cancer were amplified using 52 microsatellite markers defining 26 chromosomal regions commonly deleted in breast cancer. Levels and patterns of allelic imbalance (AI) within and between breast and lymph node tumors were assessed to identify chromosomal alterations unique to primary or metastatic tumors and to examine the timing of metastatic potential. The overall frequency of AI in primary breast tumors (0.24) was significantly greater (P < 0.001) than that in lymph node tumors (0.10), and congruent AI events were observed for < 20% of informative markers. AI at chromosomes 11q23.3 and 17p13.3 occurred significantly more frequently (P < 0.05) in primary breast tumors alone; no chromosomal regions showed a significantly higher AI frequency in lymph nodes. Higher rates of AI in primary versus metastatic lymph node tumors suggest that acquisition of metastatic potential may be an early event in carcinogenesis, occurring before significant levels of AI accumulate in the primary tumor. In addition, patterns of AI were highly discordant between tumor types, suggesting that additional genetic alterations accumulated independently in the two cell populations.

Global search for chromosomal abnormalities in infiltrating ductal carcinoma of the breast using array-comparative genomic hybridization.

Array-comparative genomic hybridization (a-CGH) is a molecular cytogenetic technique for detection of multiple chromosomal abnormalities in genomic DNA samples. Using an a-CGH with 287 probes, we examined 14 cases of breast infiltrating ductal carcinoma (IDCA) that had previously been classified by fluorescent in situ hybridization (FISH) as either human epidermal growth factor receptor-2 positive (HER2+) or HER2- and analyzed the data by hierarchical, K-means, and principal component analyses. The aim of the study was to identify the genetic abnormalities that are present in breast IDCAs and determine if the global status of 287 cytogenetic locations could be used as a more objective method for breast IDCA classification. Concordance between FISH and a-CGH at the HER2 locus was 78.6% (11/14). In general, a-CGH detected more abnormalities in HER2+ cases. In HER 2+ cases, chromosomes 1, 2, 3, 7, 9, 17, and 20 had more regions that showed statistically significant (P < or = 0.01) changes in DNA copy number. Among all the aberrant cytogenetic locations detected, 20q13, 7p12.3 approximately p12.1, and 17q23.2 approximately q25.3, which contain among others, genes for TNFRSF6B, EGFR, and TK1 showed statistically significant gains (P < or = 0.01) in 83, 66.7, and 50% of the HER2+ IDCA cases, respectively. Chromosome location 8q24.12 approximately q24.13 was the only region that showed consistent amplification in approximately 50% of the HER2- cases. Unsupervised hierarchical and K-means cluster analyses and principal component analysis using the DNA copy number status of 287 cytogenetic locations or the 177 cytogenetic locations that showed statistically significant differences revealed a cluster consisting of mainly HER2- IDCA cases. Even though this study demonstrates the usefulness of a-CGH in the rapid identification of aberrant DNA regions in tumor samples, we conclude that an array-CGH with more than 287 probes will be needed for a more precise mapping of DNA aberrations at the global level.