Paraffin-embedded Breast Tumor Research Articles

Correlation of quantitative p95HER2, HER2, and HER3 protein expression with pathologic complete response (pCR) in HER2-positive breast cancer patients (pts) treated with neoadjuvant chemotherapy and trastuzumab.

137 Background: Elevated p95HER2 [HER2-M611-CTF (carboxy-terminal-fragment) also known as p95] expression has been correlated with poor outcomes in HER2+ pts with metastatic breast cancer treated with trastuzumab (T); however, limited data is available on the correlation between p95 and pCR to T in the neoadjuvant (NEO) setting, where p95 was measured by immunohistochemistry. The current study aims to determine whether quantitative p95, HER3 and HER2 expression correlated with pCR in pts treated with T + chemotherapy in the NEO setting. Methods: pCR data and quantitative HER2 (H2T), p95, and HER3 (H3T) results by HERmark/VeraTag assays were available in 45 patient cases with pre-therapy, formalin-fixed, paraffin-embedded breast tumors. pCR was defined as the absence of invasive disease in the breast. Quantitative biomarker data were correlated with pCR according to previously published or presented biomarker cutoffs. Results: The overall pCR rate was 46.7% (ER+: 14.3% vs. ER-: 75%; p<0.0001) and was significantly associated with higher H2T levels (p=0.02) and lower H3T levels (p=0.04). In ER- subjects (N=24), no difference in H2T levels was observed between pCR vs non-pCR groups (median H2T=111.5 vs 150.5, respectively; p=0.721). However, within the ER+ group (N=21), H2T levels were significantly higher in the pCR group vs non-pCR group (median H2T=254 vs 37.3; p=0.024). Using multivariate logistic regression, increasing log(H2T) (p = 0.012), ER-negativity (p = 0.027) and low p95 (p = 0.074) were found to correlate or trend with pCR. Conclusions: pCR was significantly associated with high H2T, particularly in ER+ HER2+ breast cancer pts who received NEO therapy with T + chemotherapy. Lower H3T was also associated with pCR. A trend towards pCR was seen in tumors with low p95. These data suggest that quantitative H2T, H3T and p95 may provide additional information on response to T-based regimens in breast cancer stratified by ER status. Additional investigation into the relationship between quantitative H2T, p95 and H3T expression and T response in the NEO setting in larger cohorts is warranted.

New insights into HER2 expression in breast cancer patients analyzed by qRT-PCR.

e11068 Background: HER2 gene status is a key determinant for anti-HER2 therapy with Herceptin. The common methods used for HER2 test in breast cancer patients are IHC and FISH, which are time-consuming and often generate variable results. Quantitative real-time PCR (qRT-PCR) has emerged as a new gold standard technique for quantifying gene expression in different cells and tissues. In this study, we explored the potential of utilizing this technique to determine HER2 expression status in breast cancer patients. Methods: We collected both freshly-frozen and formalin-fixed paraffin-embedded (FFPE) breast tumor samples at three locations. The total RNA was isolated with a RNA extraction kit and was quantified by RiboGreen method. Each sample was normalized against its total RNA concentration and analyzed by a real-time PCR method with inclusion of the Standards and RNA Control. The HER2 expression level was determined by the copy number, Ct value, as well as the ΔCt against RNA control. The significance of HER2 expression and correlation among the different groups were analyzed by statistical methods. Results: A total of 80 samples have been tested. We noticed that the RT-PCR method generated very consistent results with little intra- and inter-assay variance. We also noticed that HER2 expression in the fresh tumor samples exhibited two distinct populations, although HER2 expression in the normal fresh samples showed the continuous distribution. Similar results were obtained with the FFPE samples. Study of the other breast cancer-related genes, such as EGFR, ER-a, BRCA1, and BRCA2, revealed that their expression, unlike HER2, followed a pattern of continuous distribution. In comparison with FISH test, qRT-PCR method not only produced consistent results, but also able to differentiate breast tumors into two distinct groups, HER2 positive vs. HER2 negative. Conclusions: The qRT-PCR is a more reliable method than the currently used IHC and FISH for HER2 test. Showing two distinct groups of HER2 expression by qRT-PCR further warrants its application in testing HER2 gene status for breast cancer patients.

Highly sensitive determination of PIK3CA exon 9 and 20 hotspot mutations in breast tumors.

TPS10634 Background: Hotspot mutations in exons 9 (E542K and E545K) and 20 (H1047L and H1047R) of PIK3CA gene encoding for the p110α catalytic subunit of Phosphatidylinositol 3-kinases (PI3K) are found in approximately 25% of breast carcinomas. PIK3CA mutations cause the overactivation of PI3K/AKT/mTOR pathway and lead to resistance to anti-HER2 targeted therapies in breast cancers, are involved in response to anti-mTOR agents and are proposed as molecular diagnosis marker for PI3K inhibitors. A highly sensitive method is required to detect PIK3CA mutations in paraffin-embedded tumor tissues. Methods: We developed a real-time PCR assay using allele-specific scorpion primers and amplification refractory mutation system (PCR-ARMS) to detect hotspot mutations in exons 9 (E542K and E545K) and 20 (H1047L and H1047R) of PIK3CA of PI3K. Results: PIK3CA mutations were analyzed in 102 paraffin embedded breast tumors (88 invasive ductal carcinomas and 14 lobular ductal carcinomas). All specimens were validated by pathologist examination and processed for DNA extraction and PCR. PIK3CA exon 9 and 20 mutations were found in 22.5% of the specimens, 39.1% in exon 9 (26.1% for E542K, 13.0% for E545K) and 60.9% in exon 20 (17.4% for H1047L and 43.5% for H1047R). These results were comparable to the literature data (χ²=0.327 ; p<0.05). PCR ARMS was found to be highly sensitive, able to detect 0.5 % mutated DNA. Conclusion: PCR ARMS assay is highly sensitive for PIK3CA exon 9 and exon 20 hotspot mutations analysis in breast carcinomas as molecular marker for anti-HER2 and PI3K inhibitors response prediction.

Abstract 1254: FAK activity regulates αvβ5 integrin and osteopontin expression to control breast and ovarian cancer anchorage-independent growth

Abstract Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that controls cell motility and survival downstream of integrin receptors. FAK expression and activity are elevated during breast and ovarian cancer progression and this is associated with a poor clinical prognosis. Small molecule FAK inhibitors (FAK-I) block breast and ovarian tumor progression through a novel mechanism that is associated with the apoptosis under anchorage-deprived conditions. Metastatic cells can grow as anchorage-independent spheroids and we hypothesize that a spheroid micro-environment remains dependent on integrin- and FAK-associated signaling for survival. Here we show that intraperitoneal (ip) injection of murine ID8 ovarian carcinoma cells into mice, recovery of ascites-associated tumor cells (ID8ip) and growth ex vivo, yielded tumor cells with elevated levels of phosphorylated FAK (Y397 and Y576 phosphorylation), αvβ5 integrin, and osteopontin (OPN) matrix protein expression compared to parental ID8 cells. Analyses of paraffin-embedded breast and ovarian tumor sections revealed significantly higher pY397 FAK (p<0.05), β5 integrin (p<0.0001), and OPN expression (p<0.005) in stage II-III compared to stage I tumors. Treatment of ovarian ID8ip, human ovarian HEY carcinoma, or human MDA-MB-231 breast carcinoma cells with FAK-I inhibited cell growth, prevented FAK Y397 phosphorylation, reduced β5 integrin and OPN levels independent of effects on β1 or β3 integrins or changes in matrix proteins such as fibronectin. FAK-I impairs anchorage independent cell growth and orthotopic tumor growth but not growth of cells in plastic-adherent cell culture. Stable knockdown of either FAK or β5 integrin in HEY cells prevented anchorage-independent growth. Re-expression of wildtype or kinase-dead FAK within HEY cells verified the importance of FAK activity in promoting β5 integrin and OPN expression associated with enhanced tumor growth. Ongoing studies are focused on determining the molecular connections of FAK to the regulation of αvβ5 and OPN expression. Together, our studies support a novel role for a signaling loop involving OPN and αvβ5 integrin leading to FAK activation and reinforcement of OPN and β5 integrin expression within the spheroid and tumor micro-environment enhancing breast and ovarian cancer cell survival and tumor progression. Supported by Susan G. Komen for the Cure (KG111237) and NIH grant CA102310 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1254. doi:1538-7445.AM2012-1254

Abstract 3590: Use of four biomarkers to evaluate survival disparity between black women and white women 35 to 64 years of age at diagnosis with invasive breast cancer

Abstract Purpose: To evaluate the extent to which the black-white disparity in mortality following breast cancer diagnosis is explained by the expression status of four biomarkers - estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and p53 protein. Methods: The four biomarkers were assessed in paraffin-embedded breast tumor tissue for 1204 (523 black, 681 white) women, ages 35-64 years at diagnosis, who participated in the Women's CARE Study, a population-based case-control study of newly incident breast cancer, and who accrued a median of 10 years follow-up. The impact of these biomarkers on black-white disparity in breast cancer-specific and all-cause mortality was determined by fitting multivariable Cox proportional hazards regression models. Results: The black-white disparity in breast cancer-specific mortality was 54% in an age- and study site-adjusted model (relative risk [RR], 1.54; 95% confidence interval [CI], 1.21 to 1.97); it declined to a 36% greater risk for black women than white women after additionally controlling the status of the four markers (RR, 1.36; 95% CI, 1.06 to 1.75). Similar results were observed for all cause-mortality. Separate analyses for triple negative (ER-/PR-/HER2-) and luminal A (ER+ and/or PR+ plus HER2-) tumors showed that the black-white differences in mortality were predominately observed for older (50-64 years) women with luminal A tumors (breast cancer-specific RR, 2.07; 95% CI, 1.16 to 3.70; all-cause RR, 2.21; 95% CI, 1.40 to 3.47). For luminal A tumors, the greater mortality risk for older black women was attenuated after adjustment for stage, comorbidities, and education, and was most evident for all-cause mortality. Conclusions: Black-white differences in mortality following breast cancer diagnosis vary by tumor biomarker subtypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3590. doi:1538-7445.AM2012-3590

Hormone receptor and human epidermal growth factor receptor 2 status evaluation on ThinPrep specimens from breast carcinoma

Fine-needle aspiration cytology (FNAC) is a well-accepted procedure for the diagnosis and biological characterization of breast carcinoma. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status have a strong prognostic and predictive value in invasive breast carcinoma (IBC). ThinPrep (TP) cytology, which uses an alcohol-based fixative, is increasingly being used for immunocytochemistry. In this study, the authors compared the immunocytochemical evaluation of hormone receptors (HR) and HER2 on TP-processed FNAC with the immunohistochemical analysis performed on the corresponding formalin-fixed paraffin-embedded (FFPE) breast tumor specimens, which are considered the gold standard. FNACs were performed on 116 primary IBCs at the time of diagnosis and subjected to immunocytochemical evaluation of HR and HER2 using the TP method. The same markers were immunohistochemical evaluated on the corresponding FFPE tissue specimens. HER2 fluorescent in situ hybridization analysis was performed only on the equivocal immunohistochemical results. The HR results of the TP cytology specimens showed a very good agreement with those of the corresponding FFPE tissue samples (Cohen kappa test = 0.92; concordance rate = 98%) for estrogen receptor, and a good agreement (kappa = 0.76; concordance rate = 90.9%) for progesterone receptor. A perfect agreement (kappa = 1) was observed between TP and FFPE tissue samples in evaluating HER2 status. Alcohol-based fixation seems not to affect the immunocytochemical evaluation of HR and HER2. Considering the high levels of agreement between the evaluation of HR and HER2, on both cytology specimens and on the corresponding FFPE tissue samples, the authors concluded that the TP technique can be routinely used for the biological characterization of IBC.

Progranulin (GP88) tumor tissue expression is associated with increased risk of recurrence in breast cancer patients diagnosed with estrogen receptor positive invasive ductal carcinoma

IntroductionGP88 (progranulin) has been implicated in tumorigenesis and resistance to anti-estrogen therapies for estrogen receptor positive (ER+) breast cancer. Previous pathological studies showed that GP88 is expressed in invasive ductal carcinoma (IDC), but not in normal mammary epithelial tissue, benign lesions or lobular carcinoma. Based on these results, the present study examines GP88 prognostic significance in association with recurrence and death risks for ER+ IDC patients.MethodsTwo retrospective multi-site clinical studies examined GP88 expression by immunohistochemistry (IHC) analysis of paraffin-embedded breast tumor tissue sections from ER+ IDC patients (lymph node positive and negative, stage 1 to 3) in correlation with patients' survival outcomes. The training study established a GP88 cut-off value associated with decreased disease-free (DFS) and overall (OS) survivals. The validation study verified the GP88 cut-off value and compared GP88 prognostic information with other prognostic factors, particularly tumor size, grade, disease stage and lymph node status in multivariate analysis.ResultsGP88 expression is associated with a statistically significant increase in recurrence risk for ER+ IDC patients. The training study established that GP88 3+ score was associated with decreased DFS (P = 0.0004) and OS (P = 0.0036). The independent validation study verified that GP88 3+ score was associated with a 5.9-fold higher hazard of disease recurrence and a 2.5-fold higher mortality hazard compared to patients with tumor GP88 < 3+. GP88 remained an independent risk predictor after considering age, ethnicity, nodal status, tumor size, tumor grade, disease stage, progesterone receptor expression and treatments.ConclusionsThe survival factor GP88 is a novel prognostic biomarker, predictive of recurrence risk and increased mortality for non-metastatic ER+ IDC patients. Of importance, our data show that GP88 continues to be a prognostic factor even after five years. These results also provide evidence that GP88 provides prognostic information independent of tumor and clinical characteristics and would support prospective study to examine whether GP88 expression could help stratify patients with ER+ tumors for adjuvant therapy.

Utilization of fluorescence in situ hybridization with cytokeratin discriminators in TOP2A assessment of chemotherapy-treated patients with breast cancer

Tumor biomarkers increasingly provide information for predicting outcomes with chemotherapeutic regimens (personalized medicine). Topo2A is a DNA helicase targeted by anthracyclines, cytotoxic therapeutics used in both adjuvant and palliative treatments of breast cancer. TOP2A gene amplification/deletion is implicated in response to anthracycline-based chemotherapy. We describe an approach for analyzing formalin-fixed, paraffin-embedded breast tumors on tissue microarrays with TOP2A fluorescence in situ hybridization coupled with cytokeratin immunofluorescence to target tumor cells. Stained tissue from patient specimens was imaged and analyzed using Metafer/Metacyte (Metasystems, Waltham, MA, USA), including customized image classifiers. TOP2A/CEN17 ratios of 2.0 or greater (amplified) and 0.8 or less (deleted) were observed for 10.0% and 6.1% of the patients, respectively. Patient outcomes for adjuvant chemotherapy (cyclophosphamide-epirubicin-fluorouracil, cyclophosphamide-methotrexate-fluorouracil, no chemotherapy) were evaluated. No statistical significance was achieved for clinical end points regarding TOP2A status in anthracycline-treated patients. However, patients with TOP2A aberrations receiving methotrexate-based therapy exhibited a significant decrease in 5-year distant disease-free survival and breast cancer-specific overall survival, especially for patients with TOP2A deletions (disease-free survival: hazard ratio, 5.31 [P = .001], and breast cancer-specific overall survival: hazard ratio, 6.45 [P ≤ .001]). No significant differences were seen in patients included in the no-chemotherapy group. Topo2A protein levels were assessed by immunohistochemistry with no correlative statistical relevance to immunofluorescence/fluorescence in situ hybridization-based prognosis for cyclophosphamide-epirubicin-fluorouracil or cyclophosphamide-methotrexate-fluorouracil groups. Interestingly, aberrant (under)expressing patients in the no-chemotherapy group exhibited better 5-year distant disease-free survival (hazard ratio, 0.39; P = .004), trending toward more favorable breast cancer-specific overall survival (hazard ratio, 0.61; P = .11). Our results indicate a strategy by which fluorescence in situ hybridization scoring targeted to cytokeratin-positive tumor cells may provide a tool for added precision and efficiency in TOP2A evaluation from tumor tissue.

Molecular characterisation of formalin-fixed paraffin-embedded (FFPE) breast tumour specimens using a custom 512-gene breast cancer bead array-based platform

Background:Formalin-fixed, paraffin-embedded (FFPE) tumour tissue represents an immense but mainly untapped resource with respect to molecular profiling. The DASL (cDNA-mediated Annealing, Selection, extension, and Ligation) assay is a recently described, RT–PCR-based, highly multiplexed high-throughput gene expression platform developed by Illumina specifically for fragmented RNA typically obtained from FFPE specimens, which enables expression profiling. In order to extend the utility of the DASL assay for breast cancer, we have custom designed and validated a 512-gene human breast cancer panel.Methods:The RNA from FFPE breast tumour specimens were analysed using the DASL assay. Breast cancer subtype was defined from pathology immunohistochemical (IHC) staining. Differentially expressed genes between the IHC-defined subtypes were assessed by prediction analysis of microarrays (PAM) and then used in the analysis of two published data sets with clinical outcome data.Results:Gene expression signatures on our custom breast cancer panel were very reproducible between replicates (average Pearson's R2=0.962) and the 152 genes common to both the standard cancer DASL panel (Illumina) and our breast cancer DASL panel were similarly expressed for samples run on both panels (average R2=0.877). Moreover, expression of ESR1, PGR and ERBB2 corresponded well with their respective pathology-defined IHC status. A 30-gene set indicative of IHC-defined breast cancer subtypes was found to segregate samples based on their subtype in our data sets and published data sets. Furthermore, several of these genes were significantly associated with overall survival (OS) and relapse-free survival (RFS) in these previously published data sets, indicating that they are biomarkers of the different breast cancer subtypes and the prognostic outcomes associated with these subtypes.Conclusion:We have demonstrated the ability to expression profile degraded RNA transcripts derived from FFPE tissues on the DASL platform. Importantly, we have identified a 30-biomarker gene set that can classify breast cancer into subtypes and have shown that a subset of these markers is prognostic of OS and RFS.

Identification of a subpopulation of metastatic breast cancer patients with very high HER2 expression levels and possible resistance to trastuzumab

BackgroundPatients with metastatic breast cancer (MBC) overexpressing HER2 (human epidermal growth factor receptor 2) are currently selected for treatment with trastuzumab, but not all patients respond. Patients and methodsUsing a novel assay, HER2 protein expression (H2T) was measured in formalin-fixed, paraffin-embedded primary breast tumors from 98 women treated with trastuzumab-based therapy for MBC. Using subpopulation treatment effect pattern plots, the population was divided into H2T low (H2T < 13.8), H2T high (H2T ≥ 68.5), and H2T intermediate (13.8 ≤ H2T < 68.5) subgroups. Kaplan–Meier (KM) analyses were carried out comparing the groups for time to progression (TTP) and overall survival (OS). Cox multivariate analyses were carried out to identify correlates of clinical outcome. Bootstrapping analyses were carried out to test the robustness of the results. ResultsTTP improved with increasing H2T until, at the highest levels of H2T, an abrupt decrease in the TTP was observed. KM analyses demonstrated that patients with H2T low tumors [median TTP 4.2 months, hazard ratio (HR) = 3.7, P < 0.0001] or H2T high tumors (median TTP 4.6 months, HR = 2.7, P = 0.008) had significantly shorter TTP than patients whose tumors were H2T intermediate (median TTP 12 months). OS analyses yielded similar results. ConclusionsMBC patients with very high levels of H2T may represent a subgroup with de novo resistance to trastuzumab. These results are preliminary and require confirmation in larger controlled clinical cohorts.

Expression of Human Endogenous Retrovirus Type K Envelope Protein is a Novel Candidate Prognostic Marker for Human Breast Cancer

We previously observed that the HERV type K (HERV-K) envelope (env) protein was expressed in the majority of human breast tumors from a U.S. cohort of women from Texas. We also made the preliminary observation that the expression of HERV-K env transcripts was associated with markers of disease progression. In this follow-up study, env protein expression was evaluated immunohistochemically in an additional 195 paraffin-embedded breast tumors from a second U.S. patient cohort (Baltimore, Maryland) and in 110 tumors from Chinese patients. Moreover, we compared env transcript expression between fresh-frozen normal and cancerous breast tissues. We observed that while env mRNA and protein expression was undetectable in normal breast tissue and in a subset of uninvolved normal-appearing tissue adjacent to the tumor epithelium, it was overexpressed in most tumors. Furthermore, env expression was associated with breast cancer progression. In Baltimore cohort women, HERV-K tumor positivity was significantly associated with disease stage and lymph node metastasis. In Chinese women, HERV-K env positivity was significantly associated with tumor size, TNM stage, and lymph node metastases, which is consistent with the observations in the U.S. cohort. We also found that Chinese breast cancer patients with a high expression of HERV-K had a decreased overall survival compared with patients who had either a moderate or low HERV-K expression in their tumors (P = 0.049, χ(2) log rank test). In conclusion, the HERV-K env gene is expressed in the majority of breast cancers from U.S. or Chinese women but not in normal breast tissue. High expression of HERV-K env protein in breast cancer patients is associated with markers of disease progression and poor disease outcome, indicating that HERV-K env protein is a novel candidate prognostic marker for breast cancer.

Association of BRCA1 promoter methylation in triple-negative breast cancer (TNBC) with resistance to standard anthracyline-based adjuvant chemotherapy.

1123 Background: Methylation of the BRCA1 promoter is one of the mechanisms contributing to loss of BRCA1 expression in sporadic TNBC. Relative to other classes of chemotherapeutic agents, BRCA1-deficient cancers appear to be intrinsically sensitive to DNA damaging agents. However, it is not known if presence of BRCA1 promoter methylation (PM) in sporadic TNBC impacts response to chemotherapy. Aim: To investigate the prognostic significance of BRCA1 PM with respect to RFS and OS in patients receiving chemotherapy for stage I-III TNBC. Methods: TNBC patients undergoing chemotherapy b/w 1996-2008 were identified and their formalin-fixed paraffin-embedded (FFPE) breast tumor specimens were retrieved. Genomic DNA was isolated, bisulfite converted and then subjected to BRCA1 promoter methylation-specific PCR. RFS and OS were calculated by Kaplan-Meier method. Cox proportional hazards models were fit to determine the association of BRCA1 PM with the risk of death. Results: DNA was isolated from primary tumor specimens of 39 subjects. BRCA1 PM specific PCR was successful in 95% (37/39) of specimens and was detected in 30% (11/37) of subjects. The median age was 52 yrs (33-80), median tumor size: 2 cm, LN+: 51%, Stage III: 32%. All patients received neo/adjuvant chemotherapy (anthracycline: 90%, taxane: 73%). At a median follow up of 6 yrs, there have been 18 (48%) recurrences and 14 (37%) deaths. Node positivity, higher tumor stage and presence of BRCA1 PM were significantly associated with worse RFS and OS. 5-year RFS was 27% and 61% respectively for patients with and without BRCA1 PM (p =0.034). Similarly, 5-year OS was 36% and 81% respectively for patients with and without BRCA1 PM (p =0.004). After adjustment for stage and N status presence of BRCA1 PM was significantly associated with poor OS (p=0.002). Conclusions: BRCA1 PM methylation was detected in approximately 30% of TNBC patients and was associated with significantly poor outcomes using standard anthracycline chemotherapy. Alternative therapeutic modalities (platinum/PARP inhibitors) which are more likely to be effective against BRCA1-deficient tumors should be explored for patients with sporadic TNBC and BRCA1 PM.

Expression profiling of formalin-fixed paraffin-embedded primary breast tumors using cancer-specific and whole genome gene panels on the DASL®platform

BackgroundThe cDNA-mediated Annealing, extension, Selection and Ligation (DASL) assay has become a suitable gene expression profiling system for degraded RNA from paraffin-embedded tissue. We examined assay characteristics and the performance of the DASL 502-gene Cancer Panelv1 (1.5K) and 24,526-gene panel (24K) platforms at differentiating nine human epidermal growth factor receptor 2- positive (HER2+) and 11 HER2-negative (HER2-) paraffin-embedded breast tumors.MethodsBland-Altman plots and Spearman correlations evaluated intra/inter-panel agreement of normalized expression values. Unequal-variance t-statistics tested for differences in expression levels between HER2 + and HER2 - tumors. Regulatory network analysis was performed using Metacore (GeneGo Inc., St. Joseph, MI).ResultsTechnical replicate correlations ranged between 0.815-0.956 and 0.986-0.997 for the 1.5K and 24K panels, respectively. Inter-panel correlations of expression values for the common 498 genes across the two panels ranged between 0.485-0.573. Inter-panel correlations of expression values of 17 probes with base-pair sequence matches between the 1.5K and 24K panels ranged between 0.652-0.899. In both panels, erythroblastic leukemia viral oncogene homolog 2 (ERBB2) was the most differentially expressed gene between the HER2 + and HER2 - tumors and seven additional genes had p-values < 0.05 and log2 -fold changes > |0.5| in expression between HER2 + and HER2 - tumors: topoisomerase II alpha (TOP2A), cyclin a2 (CCNA2), v-fos fbj murine osteosarcoma viral oncogene homolog (FOS), wingless-type mmtv integration site family, member 5a (WNT5A), growth factor receptor-bound protein 7 (GRB7), cell division cycle 2 (CDC2), and baculoviral iap repeat-containing protein 5 (BIRC5). The top 52 discriminating probes from the 24K panel are enriched with genes belonging to the regulatory networks centered around v-myc avian myelocytomatosis viral oncogene homolog (MYC), tumor protein p53 (TP53), and estrogen receptor α (ESR1). Network analysis with a two-step extension also showed that the eight discriminating genes common to the 1.5K and 24K panels are functionally linked together through MYC, TP53, and ESR1.ConclusionsThe relative RNA abundance obtained from two highly differing density gene panels are correlated with eight common genes differentiating HER2 + and HER2 - breast tumors. Network analyses demonstrated biological consistency between the 1.5K and 24K gene panels.

B7–H3 Ligand Expression by Primary Breast Cancer and Associated With Regional Nodal Metastasis

: B7 ligand family members have been shown to be important immunoregulatory factors in host tumor immune responses. We hypothesized that B7-H3, a coinhibitory factor, is expressed by primary breast cancer cells and associated with metastasis to regional tumor-draining lymph nodes. : American Joint Committee on Cancer stage I to III primary breast cancers (n = 82) and normal breast specimens (n = 17) were assessed for B7-H3 expression using paraffin-embedded archival tissues. B7-H3 expression by breast cancer cells was assessed by a quantitative real-time reverse transcription-polymerase chain reaction, and B7-H3 protein expression was evaluated using immunohistochemistry. : B7-H3 mRNA expression was detected in 32 of 82 (39%) primary breast tumors but not in normal breast tissues (P = 0.0029). B7-H3 expression in primary tumors significantly correlated with increasing tumor size, American Joint Committee on Cancer stage, and lymphovascular invasion (P < 0.0001, P < 0.0001, P = 0.0071). B7-H3 expression was highly correlated to sentinel lymph node and overall number of lymph nodes with metastasis P = 0.003, and P = 0.004, respectively). In a multivariate analysis, B7-H3 mRNA expression of the primary tumor significantly predicted metastasis to regional lymph nodes (P = 0.021, and P = 0.023, respectively). Antibody staining analysis of paraffin-embedded archival tissue breast tumors and flow cytometry of breast cancer cell lines demonstrated B7-H3 protein expression. : B7-H3 protein expressed by primary breast cancer cells is a tumor progression factor and is associated with extent of regional nodal metastasis.

Topoisomerase II-α as a predictive factor of response to therapy with anthracyclines in locally advanced breast cancer

BackgroundTopoisomerase II-α is a molecular target of anthracyclines; several studies have suggested that topoisomerase II-α expression is related to response to anthracycline treatment. The objective of this study was to evaluate if topoisomerase II-α overexpression predicts response to anthracycline treatment in locally advanced breast cancer patients. Material and methodsTopoisomerase II-α, HER2, estrogen receptor (ER) and progesterone receptor (PR) expression were evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded breast tumors from 111 patients presenting with locally advanced breast cancer between 1995 and 2002. The prognostic value of these markers was analyzed using a multivariate proportional hazards regression model and an interaction analysis between topoisomerase II-α status and dose intensity. ResultsTumors from 40 patients (36%) showed topoisomerase II-α overexpression, 62 patients (56%) for ER, 39 (35%) for PR and 26 (23%) for HER2. There were no significant correlations between topoisomerase II-α expression and response to therapy, progression-free survival (PFS) or overall survival (OS). Anthracycline dose intensity had a significant impact on PFS and OS in patients overexpressing topoisomerase II-α (P=0.010 and 0.027, respectively). Negative PR (P=0.041), positive HER2 (P=0.013) were identified as risk factors in the multivariate model. The multivariate analysis in patients topoisomerase II-α negative shown no significance (HR=0.92, IC 95% 0.39–2.15, P=0.839) while the multivariate analysis in topoisomerase II-α positive, dose intensity shown to be statistically significant (HR=2.725, IC 95% 1.07–6.95, P=0.036). ConclusionsOur data do not support a correlation between topoisomerase II-α expression in breast cancer patients and improved clinical benefit with anthracycline therapy. However, they do suggest that tumors overexpressing topoisomerase II-α may experience better clinical benefit with higher anthracycline dose intensity.

41 NOTCH2 in breast cancer: association of SNP rs11249433 with gene expression in ER-positive breast tumours without TP53 mutations

It is now understood that epigenetic alterations occur frequently in sporadic breast carcinogenesis, but little is known about the epigenetic alterations associated with familial breast tumors. We performed genome-wide DNA-methylation profiling on familial breast cancers (n = 33) to identify patterns of methylation specific to the different mutation groups (BRCA1, BRCA2, and BRCAx) or intrinsic subtypes of breast cancer (basal, luminal A, luminal B, HER2-amplified, and normal-like). We used methylated DNA immunoprecipitation (MeDIP) on Affymetrix promoter chips to interrogate methylation profiles across 25,500 distinct transcripts. Using a support vector machine classification algorithm, we demonstrated that genome-wide methylation profiles predicted tumor mutation status with estimated error rates of 19% (BRCA1), 31% (BRCA2), and 36% (BRCAx) but did not accurately predict the intrinsic subtypes defined by gene expression. Furthermore, using unsupervised hierarchical clustering, we identified a distinct subgroup of BRCAx tumors defined by methylation profiles. We validated these findings in the 33 tumors in the test set, as well as in an independent validation set of 47 formalin-fixed, paraffin-embedded familial breast tumors, by pyrosequencing and Epityper. Finally, gene-expression profiling and SNP CGH array previously performed on the same samples allowed full integration of methylation, gene-expression, and copy-number data sets, revealing frequent hypermethylation of genes that also displayed loss of heterozygosity, as well as of genes that show copy-number gains, providing a potential mechanism for expression dosage compensation. Together, these data show that methylation profiles for familial breast cancers are defined by the mutation status and are distinct from the intrinsic subtypes.

Regulation of IGF2 transcript and protein expression by altered methylation in breast cancer

Breast Cancer is one of the leading causes of cancer deaths among women worldwide. The role of epigenetics as a distinct mechanism to alter gene expression in a tissue-specific manner has emerged as an important mechanism in the pathophysiology of cancer. Present study was carried out to assess the role of methylation in regulating transcription and protein expression of Insulin-like growth factor 2 (IGF2), an oncogene with parental imprinting. Paraffin-embedded archival breast tumor and adjacent normal tissue samples were used for carrying out PCR-based methylation assay, genomic PCR, immunohistochemistry and Real-Time Reverse transcriptase PCR. A significant loss of methylation in exon 9 CpG cluster of IGF2 in breast tumor tissues was observed when compared to normal tissue (P<0.0001). Expression of IGF2 by immunohistochemistry exhibited a mean twofold increase correlating with the hypomethylation of this specific CpG. Real-Time RT PCR showed increased transcripts in the tumor tissue supporting the IHC and methylation results. A total of 33% of tumor samples heterozygous for the ApaI IGF2 polymorphism exhibited biallelic IGF2 expression due to loss of imprinting; this was not seen in any of the normal breast tissues. Altered methylation of exonic CpG plays an important role in the enhanced transcription/expression of IGF2 in breast tumors. Methylation analysis of exon 9 CpG can be used as a biomarker for upregulation of IGF2 in breast tumor tissue and maybe developed as a diagnostic test in future.

Abstract 2684: Telomere length of carcinoma-associated fibroblasts as a biomarker for breast cancer local recurrence

Abstract The use of breast-conservative surgery (BCS) for the treatment of early-stage breast cancers has been increasing and the ability to identify patients at high risk of local recurrence is highly desirable for guiding treatment decisions. Currently, relatively few breast cancer local recurrent risk factors have been identified; those known risk factors, which are based on clinical and histological features of tumor tissue, have limited predictive power. It is known that molecular genetic changes often precede morphologic evidence of malignant transformation. Thus, early molecular genetic alterations in breast tumor and adjacent normal tissues are likely to be more sensitive tools for the identification of patients at increased risk of local recurrence than current histological criteria. A nested case-control study was carried out to evaluate whether telomere length in morphologically normal tissues adjacent to the tumor is predictive of breast cancer local recurrence. One hundred forty-two women who are diagnosed with breast cancer at Lombardi Comprehensive Cancer Center between 1998 and 2005 were included in the study. Cases (N = 42) are patients who had local recurrences of breast cancer after BCS. Controls (N = 100) are patient who had no local recurrence and are matched to cases on year of surgery, age at diagnosis, disease stage and type of surgery. Quantitative telomere fluorescent in situ hybridization (FISH) was used to determine the telomere length of four cell types: cancer cells, carcinoma-associated fibroblast cells (CAFs), adjacent normal epithelial cells and lymphocytes, using 5-micron sections of formalin fixed paraffin-embedded (FFPE) breast tumor tissues. Telomere length of lymphocytes was used to normalize the FISH hybridization variation and relative telomere length (RTL) was defined as telomere length in cells of interest divided by the telomere length of lymphocytes. Multivariate logistic regression was used to assess the association between RTL and breast cancer local recurrence. We found that mean RTL of CAFs was significantly longer in patients with breast cancer local recurrence (mean = 0.99) than in patients without local recurrence (mean = 0.74, p=0.022). When RTL of CAFs were dichotomized using median value of control subjects, patients who had longer RTL in CAFs had 2.4-fold (95% CI=0.86-6.56) increased risk of breast cancer local recurrence than women who had shorter RTL in CAFs. We observed no statistically significant case-control difference in mean RTL of normal epithelial cells adjacent to the tumor (p = 0.712) or in mean RTL of cancer cells (p = 0.285). This study is the first to report that telomere length in tumor stromal cells (CAFs) is associated with breast cancer local recurrence. If confirmed by future studies, telomere length in CAFs has the potential to become a novel molecular tool for the predicting breast cancer local recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2684.

DNA Methylome of Familial Breast Cancer Identifies Distinct Profiles Defined by Mutation Status

It is now understood that epigenetic alterations occur frequently in sporadic breast carcinogenesis, but little is known about the epigenetic alterations associated with familial breast tumors. We performed genome-wide DNA-methylation profiling on familial breast cancers (n = 33) to identify patterns of methylation specific to the different mutation groups (BRCA1, BRCA2, and BRCAx) or intrinsic subtypes of breast cancer (basal, luminal A, luminal B, HER2-amplified, and normal-like). We used methylated DNA immunoprecipitation (MeDIP) on Affymetrix promoter chips to interrogate methylation profiles across 25,500 distinct transcripts. Using a support vector machine classification algorithm, we demonstrated that genome-wide methylation profiles predicted tumor mutation status with estimated error rates of 19% (BRCA1), 31% (BRCA2), and 36% (BRCAx) but did not accurately predict the intrinsic subtypes defined by gene expression. Furthermore, using unsupervised hierarchical clustering, we identified a distinct subgroup of BRCAx tumors defined by methylation profiles. We validated these findings in the 33 tumors in the test set, as well as in an independent validation set of 47 formalin-fixed, paraffin-embedded familial breast tumors, by pyrosequencing and Epityper. Finally, gene-expression profiling and SNP CGH array previously performed on the same samples allowed full integration of methylation, gene-expression, and copy-number data sets, revealing frequent hypermethylation of genes that also displayed loss of heterozygosity, as well as of genes that show copy-number gains, providing a potential mechanism for expression dosage compensation. Together, these data show that methylation profiles for familial breast cancers are defined by the mutation status and are distinct from the intrinsic subtypes.

Multiplexed tandem polymerase chain reaction identifies strong expression of oestrogen receptor and Her-2 from single, formalin-fixed, paraffin-embedded breast cancer sections

To establish the suitability of multiplex tandem polymerase chain reaction (MT-PCR) for rapid identification of oestrogen receptor (ER) and Her-2 status using a single, formalin-fixed, paraffin-embedded (FFPE) breast tumour section. Tissue sections from 29 breast tumours were analysed by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). RNA extracted from 10 mum FFPE breast tumour sections from 24 of 29 tumours (14 ER positive and 5 Her-2 positive) was analysed by MT-PCR. After establishing a correlation between IHC and/or FISH and MT-PCR results, the ER/Her-2 status of a further 32 randomly selected, archival breast tumour specimens was established by MT-PCR in a blinded fashion, and compared to IHC/FISH results. MT-PCR levels of ER and Her-2 showed good concordance with IHC and FISH results. Furthermore, among the ER positive tumours, MT-PCR provided a quantitative score with a high dynamic range. Threshold values obtained from this data set applied to 32 archival tumour specimens showed that tumours strongly positive for ER and/or Her-2 expression were easily identified by MT-PCR. MT-PCR can provide rapid, sensitive and cost-effective analysis of FFPE material and may prove useful as triage to identify patients suited to endocrine or trastuzumab (Herceptin) treatment.