Tumor ER Research Articles

PIK3CA mutation is an independent indicator of malignant phenotype and prognosis in breast cancer

The phosphatidylinositol-3-kinase (PI3K)-AKT signaling pathway is considered to play an important role in tumorigenesis. Frequent somatic mutations in the PI3K subunit p110a (PIK3CA) occur in a variety of cancer types. The purpose of this study was to determine the relationship between PIK3CA mutation in breast cancer and pathological features and outcome of patients. The PIK3CA mutations in exons 7, 9, 20 were screened in 250 primary breast cancers using PCR and fluorescent (F)-SSCP, and the results were analyzed according to their cliniopathological data. The frequency of PIK3CA mutations among the 250 cases was 35.2% (88/250), point mutations in exon 7 were found in 8 (3.2%) cases,40 (16.0%) cases in exon 9 and 47 (18.8%) cases in exon 20. No significant correlation between PIK3CA mutation and age, histological type, differentiation, and lymph node metastasis was observed. Mutations were associated with larger tumor size (P = 0.004) and positive estrogen receptor status (P = 0.008). Patients with PIK3CA mutations showed a significantly worse survival (P = 0.004), particularly in those with positive estrogen receptor expression or non-amplified HER-2 (both P = 0.002). PIK3CA mutations may play an important role in the carcinogenesis and development of breast cancer. The association with large tumor size, ER+ and poor survival indicates that PIK3CA mutation could be an independent factor for tumor malignant phenotype and prognosis.

Cerebral metastases in metastatic breast cancer: disease-specific risk factors and survival

BackgroundSurvival of patients suffering from cerebral metastases (CM) is limited. Identification of patients with a high risk for CM is warranted to adjust follow-up care and to evaluate preventive strategies. Patients and methodsExploratory analysis of disease-specific parameter in patients with metastatic breast cancer (MBC) treated between 1998 and 2008 using cumulative incidences and Fine and Grays’ multivariable regression analyses. ResultsAfter a median follow-up of 4.0 years, 66 patients (10.5%) developed CM. The estimated probability for CM was 5%, 12% and 15% at 1, 5 and 10 years; in contrast, the probability of death without CM was 21%, 61% and 76%, respectively. A small tumor size, ER status, ductal histology, lung and lymph node metastases, human epidermal growth factor receptor 2 positive (HER2+) tumors, younger age and M0 were associated with CM in univariate analyses, the latter three being risk factors in the multivariable model. Survival was shortened in patient developing CM (24.0 months) compared with patients with no CM (33.6 months) in the course of MBC. ConclusionYoung patients, primary with non-metastatic disease and HER2+ tumors, have a high risk to develop CM in MBC. Survival of patients developing CM in the course of MBC is impaired compared with patients without CM.

A breast cancer meta-analysis of two expression measures of chromosomal instability reveals a relationship with younger age at diagnosis and high risk histopathological variables

Breast cancer in younger patients often presents with adverse histopathological features, including increased frequency of estrogen receptor negative and lymph node positive disease status. Chromosomal instability (CIN) is increasingly recognised as an important prognostic variable in solid tumours. In a breast cancer meta-analysis of 2423 patients we examine the relationship between clinicopathological parameters and two distinct chromosomal instability gene expression signatures in order to address whether younger age at diagnosis is associated with increased tumour genome instability. We find that CIN, assessed by the two independently derived CIN expression signatures, is significantly associated with increased tumour size, ER negative or HER2 positive disease, higher tumour grade and younger age at diagnosis in ER negative breast cancer. These data support the hypothesis that chromosomal instability may be a defining feature of breast cancer biology and clinical outcome.

High relapse rates and poor survival of native Hawaiian (NH) women with HER2-positive breast cancer.

e11116 Background: The Hawaii Tumor Registry reports of higher mortality rates and advanced disease at diagnosis for Native Hawaiian (NH) women with breast cancer may represent a biologically more aggressive cancer complimented by HER2 overexpression. This study investigates the association of HER2 overexpression potentiating NH breast cancer leading to early relapses and poor survival. Methods: This retrospective study analyzed data for women diagnosed with breast cancer from 2000-2005, and followed through 2007 (n=1488), at the Queen’s Medical Center in Honolulu, Hawaii. This study focused on three major ethnicities: NH (n=359), Japanese (JA; n=609) and Caucasian (CA; n=520) women. Demographics on tumor grade, stage, ER, PR, and HER2 expression were obtained from the hospital’s cancer registry. Cox regression models of 1) relapse; 2) death for these patients were used to evaluate the association of intrinsic breast cancer subtypes and ethnicities. Survival time was the interval between diagnosis and death or cessation of follow-up at 2007. Results: We compared the frequency distribution of tumor subtypes (Luminal A, Luminal B, HER2-positive, and Triple Negative). Triple Negative breast cancers were associated with increased risk of death for the NH women (Hazard Ratio (HR)=2.8, p=0.046) and JA (HR=2.2, p=0.052). HER2-positive breast cancers were associated with increased risk of death for NH (HR=2.6, p=0.044) and CA (HR=2.9, p= 0.018) women, but not for the JA women. In HER2+ tumors, we observed that there was a significant increase in the frequency of relapse for NH (23.6%) compared to JA (9.8%) or CA (10%) women. Cox regression analysis indicate that NH women are more likely to relapse (HR=3.28) compared to JA (HR=1.05) or CA (HR=1.2) women. Conclusions: These data illustrate the importance of cross-ethnic studies in understanding racial/ethnic based differences in breast cancer incidence and mortality. We observed that Her2 overexpression is associated with disproportionately higher relapse and mortality rates in NH women with breast cancer. An ethnicity - HER2 interaction is suggested by this data and further molecular studies are indicated.

Staging primary breast cancer. Are there tumour pathological features that correlate with a false-negative axillary ultrasound?

To investigate whether the histopathological characteristics of primary breast cancer tumours could predict the likelihood of false-negative axillary ultrasound. Screening and symptomatic patients were identified from pathology records and imaging and pathology records reviewed. True and false-negative axillary staging ultrasound groups were compared statistically in terms of tumour size, pathological type and grade, lymphovascular invasion, and oestrogen receptor (ER) status. Of 155 women with normal ultrasounds, 45 (29%) were node positive at axillary surgery. Breast tumour size was significantly different with the average size smaller in the true-negative group: 21 versus 30 mm (p < 0.02). The histological type varied significantly between the groups, with more lobular carcinomas in the false-negative group [6/110 (5%) versus 6/45 (13%), p < 0.001]. The false-negative group was also more likely to show lymphovascular invasion in the breast [6/110 (5%) versus 14/45 (31%), p < 0.001]. There was no significant difference in tumour grade or ER status. The present study has found significant differences in tumour characteristics between women with true-negative and false-negative axillary staging ultrasound in terms of size, primary tumour histological type and presence of lymphovascular invasion. In particular, axillary ultrasound in primary lobular carcinoma may be less accurate and a negative result is more likely to be spurious than with primary ductal carcinomas.

Morphological predictors of BRCA1 germline mutations in young women with breast cancer

Background:Knowing a young woman with newly diagnosed breast cancer has a germline BRCA1 mutation informs her clinical management and that of her relatives. We sought an optimal strategy for identifying carriers using family history, breast cancer morphology and hormone receptor status data.Methods:We studied a population-based sample of 452 Australian women with invasive breast cancer diagnosed before age 40 years for whom we conducted extensive germline mutation testing (29 carried a BRCA1 mutation) and a systematic pathology review, and collected three-generational family history and tumour ER and PR status. Predictors of mutation status were identified using multiple logistic regression. Areas under receiver operator characteristic (ROC) curves were estimated using five-fold stratified cross-validation.Results:The probability of being a BRCA1 mutation carrier increased with number of selected histology features even after adjusting for family history and ER and PR status (P<0.0001). From the most parsimonious multivariate model, the odds ratio for being a carrier were: 9.7 (95% confidence interval: 2.6–47.0) for trabecular growth pattern (P=0.001); 7.8 (2.7–25.7) for mitotic index over 50 mitoses per 10 high-powered field (P=0.0003); and 2.7 (1.3–5.9) for each first-degree relative with breast cancer diagnosed before age 60 years (P=0.01).The area under the ROC curve was 0.87 (0.83–0.90).Conclusion:Pathology review, with attention to a few specific morphological features of invasive breast cancers, can identify almost all BRCA1 germline mutation carriers among women with early-onset breast cancer without taking into account family history.

Analysis of predictors of axillary node metastases in 787 breast cancer patients

To explore the predictors of axillary nodal metastass in patients with breast cancer. A retrospective study was performed using the clinicopathological data of breast cancer cases diagnosed and treated in our Hospital between Dec 2006 and Nov 2008. Logistic regression analysis was used to determine the predictors of axillary node positivity. The total number of patients was 1133. 69.5% of them (787) had complete clinical and pathological data. The median age was 49 years old (range 20-85). The average number of lymph nodes removed was 14.6 per person. The average number of involved nodes was 3.5 per person. Increasing tumor size was associated with increased risk of lymph node metastases. Assessed by multivariate analysis, the tumor size, age, ER status, and pathological type were significantly associated with node metastasis. Axillary nodal metastases are significantly affected by the tumor size, ER status, age, and pathological type in breast cancer patients.

Abstract P6-04-16: Molecular Profiling of Stage II and III Breast Cancer in Latin American Women Receiving Standard-of-Care Treatment

Abstract Breast cancer, though genetically and clinically heterogeneous, still lacks reliable prognostic and predictive markers. Gene expression profiling studies have correlated with long-term survival and response to therapy. Multiple variables thought to modify gene expression (i.e., ancestry, socio-economical and cultural factors) are not always controlled when correlating molecular markers with clinical outcome. Breast cancer rates are lower in Latin America than in other countries; molecularly, variations in gene expression should reflect different genetic and epigenetic levels there. The United States-Latin America Cancer Research Network (US-LA CRN) launches its first breast cancer pilot study in 2010 in over 20 hospitals and research facilities across Argentina, Brazil, Chile, Mexico, and Uruguay, primarily to characterize molecular profile distribution (luminal A, luminal B, HER2-like, and basal) in Latin American women with non-metastatic stage II or III breast cancer. Molecular profiles determined by microarray will be correlated with epidemiological, histological, and clinical data, including pathologic response to standard neoadjuvant chemotherapy; a molecular signature that predicts response to neoadjuvant therapy in breast cancer will be defined. This prospective cohort study will not dispense investigational drugs. Part A characterizes molecular profiles of breast cancer in Latin America; Part B examines associations between response to neoadjuvant therapy and molecular profiles. Tissue samplesfor gene expression profiling will be collected during routine core biopsies or during surgery. Core biopsies and surgical resection specimens from participants will undergo histopathologic evaluation, tumor ER, PgR, HER2, and Ki67 status assessment. During Part B, patients will be sorted by hormone receptor and HER2 status and receive standard neoadjuvant chemotherapy. Tissue samples will be re-assessed to evaluate residual cancer burden for pathologic response to neoadjuvant treatment and gene expression. Five-year patient follow-up will determine associations between molecular profiles and disease evolution after standard treatment. Besides providing data on the effectiveness of standard chemotherapy in Latin American women, study infrastructure includes establishing biobanks in participating countries to conduct additional clinical trials incorporating complex genomic and pathologic biomarkers, including trials to evaluate investigational therapies. Studying the molecular profile of breast cancer in Latin America will improve diagnosis and treatment, correlate molecular subtypes with long-term survival and response to therapy, and identify indolent-disease subpopulations of cancer patients, perhaps enabling future personalized cancer management. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-16.

Abstract P4-07-01: Correlation of Breast Cancer Susceptibility Loci with Patient Characteristics, Metastasis-Free Survival and Expression of the Nearest Genes

Abstract Background: Genome-wide association studies has identified single nucleotide polymorphisms (SNPs) in several loci being associated with breast cancer risk: fibroblast growth factor receptor 2 (FGFR2), trinucleotide repeat containing 9 (TNRC9 or TOX3), mitogen-activated protein kinase kinase kinase 1 (MAP3K1), lymphocyte-specific protein 1 (LSP1) and the imprinted maternally expressed H19, and loci in gene deserts at 8q24 and 2q35. However, the mechanism by which these loci confer breast cancer risk in patients is still largely unknown. Here, we addressed this question by associating these SNPs with the mRNA expression of the genes located at or nearest to the SNP in the same linkage disequilibrium region. In addition, we associated the SNPs with clinical, pathological and patient characteristics and prognosis. Material and Methods : SNPs tagging breast cancer loci were genotyped in genomic tumor DNA samples of 2,480 breast cancer patients. All samples were collected between 1978-2004. The mean age was 55.6 years and median follow-up up was 106 months. The SNPs were correlated with patients and tumor characteristics. Of the 1,262 patients with lymph-node negative disease and who did not receive any adjuvant systemic therapy, SNP status was associated with distant metastasis-free survival (MFS) using Cox regression analysis. Finally, in a subset of 1,400 of the 2,480 patients, the mRNA expression of FGFR2, TNRC9, MAP3K1, LSP1 and H19 genes was determined by quantitative RT-PCR and correlated with SNP genotypes. Results: The SNP rs2981582 in FGFR2 was significantly associated with ER and PgR status of the tumors (both p=0.001). Besides weak associations with tumor grade (FGFR2, p=0.01), ER (MAP3K1, p=0.03; LSP1, p=0.03), and PgR (LSP1, p=0.05), no other association with any clinical or pathological variable for any of the SNPs was observed. Of the SNPs analyzed, only rs2107425 near H19 was significantly linked in uni-and multivariable analysis with MFS (Hazard ratio [HR]=1.44, 95% Confidence interval [CI]: 1.04-1.98; p=0.026; HR=1.53; 95% CI: 1.09-2.14; p=0.013, respectively) with the more aggressive minor allele displaying a recessive trade. Interestingly, the minor allele of SNP rs3803662 was significantly associated with lower mRNA expression of the 8 kb downstream TNRC9 gene (p=0.0019). However, none of the other risk alleles, including the one in FGFR2, had an association with mRNA expression of the nearest located gene. Conclusions: In agreement with previous studies, a clear correlation of the SNPs in FGFR2 with ER and PR status of tumors was observed. The lower level of TNRC9 mRNA in tumors having the minor allele genotype suggests that TNRC9 may act as a tumor suppressor gene and its expression might have a protective effect. A significant association of the SNP near H19 with poor outcome without apparent effect on H19 mRNA expression suggests that this prognostic SNP in a well-known imprinted region is not linked to prognosis through altering H19 gene expression. This study indicates that most of the studied SNPs that confer breast cancer risk are not linked to prognosis nor do they effect, in primary tumors mRNA expression of the nearest gene. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-07-01.

Abstract PD07-09: Hypoxia (VEGF-S) Signature and CRYAB Predict Response to Neoadjuvant Anthracycline/Taxane Containing Chemotherapy within Triple Negative and Unselected Breast Tumors

Abstract Background: Aggressive breast tumors are a clinically challenge to manage. A VEGF-signature (VEGF-s) developed in vivo (Hu et al. BMC Med. 09) and the profile of the anti-apoptotic gene and regulator of tumor angiogenesis, CRYAB (Moyano et al. J Clin Invest 06; Dimberg et al. Blood 08), are both associated with distant metastasis. In this study, we sought to test if these two mechanistically related biomarkers predict pathological complete response(pCR) to anthracyline/taxane-based neoadjuvant chemotherapy. Methods: Gene expression data of pre-treatment fresh frozen tumors were combined, 149 from the ISPY trial (Agilent 44K) and 225 from MD Anderson Cancer Center(MDACC) (Affymetrix HG U133A). Patients from I-SPY were treated with doxorubicin/cyclophosphamide followed by a taxane. Patients from MDACC were treated with paclitaxel followed by sequential fluorouracil-AC. Tumors were classified into intrinsic subtypes using PAM50(Parker et al. J Clin Oncol 09) and Claudin-low(CL) predictor(Prat et al., Submitted). Odds ratios of biomarkers to pCR were determined using logistic regression. Predictive values of multivariable models were estimated using receiver operating characteristic curve. Results: 319 patients had complete data; the pCR rate was similar between the datasets (ISPY 22%, MDACC 20%). Both the VEGF-s and CRYAB were highly expressed within Basal-like(BL) and CL (P&amp;lt;0.0001); these two biomarkers were positively correlated with each other (r: 0.4, P&amp;lt;0.001). In univariate analysis, both VEGF-s (P&amp;lt;0.001) and CRYAB (P&amp;lt;0.001) were significantly associated with pCR. A multivariable test including tumor size, grade, ER, Her2, intrinsic subtypes, VEGF-s and CRYAB, had an AUC value of 0.86 to predict pCR(Table 1). T stage, Her2, intrinsic subtypes and CRYABwere significant. 82 patients with triple-negative tumors within this combined cohort. In univariate analysis, T stage, grade or classifying tumors into BL vs not did not predict pCR; but VEGF-s (p= 0.04) and CRYAB (p = 0.02) were associated with pCR. Multivariable model, including VEGF-s, CRYAB and the clinical variables, had a predictive value of 0.71 to pCR(Table 1), which identified CRYAB as the most significant factor by likelihood ratio test. Table 1: Multivariable logistic regression models to predict pCR. Conclusion: VEGF-s and CRYAB expression are associated with aggressive subtypes and may possibly help to predict response of anthracycline/taxane-based neoadjuvant chemotherapy beyond ER, Her2, and intrinsic subtypes. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD07-09.

Abstract P1-11-01: ANZAC: A Randomised Neoadjuvant Biomarker Study Investigating the Anti-Tumour Activity of the Addition of Zoledronic Acid to Chemotherapy in Breast Cancer

Abstract Background: Pre-clinical studies have demonstrated sequence-dependent synergistic anti-tumour effects of chemotherapy (CT) and zoledronic acid (Z) in bone and soft tissue tumours. We have also reported that the addition of Z to neoadjuvant CT appears to improve pathological response in the surgical resection specimen. The ANZAC study aimed to investigate the short-term anti-tumour effects of neoadjuvant CT +/− Z in patients with invasive breast cancer, evaluating biological end-points including apoptosis, proliferation and angiogenesis. Methods: Forty patients were randomised to receive a single 4mg infusion of Z 24 hours after the first cycle of FE100C chemotherapy, as per the most effective schedule demonstrated pre-clinically, or CT alone (CT n=20, CT+Z n=20). Randomisation was stratified for tumour (T) stage, ER, HER2 status and menopausal status to minimise biological differences in the two treatment groups that may influence chemosensitivity. All patients had repeat breast core biopsy at Day 5 (D5) +/− Day 21 (D21). Apoptotic index (AI) and proliferation were measured on core biopsy specimens using TUNEL and Ki67 immunohistochemistry, counting a total of 2000 and 1000 tumour cells respectively. Serum VEGF was also measured at baseline prior to CT +/− Z, and on D5 and D21. Differences between the groups in change from baseline to subsequent timepoints were investigated using the Mann-Whitney U test. Results: Baseline clinico-pathological characteristics were well balanced between the two groups. For the different biological end-points, percentage change from baseline to D5 and from baseline to D21 are shown in Table 1. At D5, a greater reduction in serum VEGF occurred in patients treated with CT+Z compared to CT: median percentage change -23.8% (IQR -32.9, -15.8) vs. -8.4% (IQR -27.3, +8.9); p=0.02), but these effetcs were lost by D21. Cell turnover index fell at D5 in both groups (increased apoptosis and reduced proliferation) but recovered much more rapidly with CT+Z than CT alone by D21 to levels above baseline (p=0.006). Conclusions: No definite evidence of a direct anti-tumour effect of the addition of Z to CT was observed, but potentially relevant biological effects were seen in this small study. The clinical relevance of recovery of cell turnover by D21 with CT+Z is unclear. Studies with more frequent dosing of Z are warranted to exploit any potential anti-angiogenic effect of Z. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-11-01.

Abstract A102: The role of socioeconomic status, pathology and genetic biomarkers in explaining racial disparities in breast cancer survival

Abstract Background and Objective: The current body of evidence suggests that while a portion of the racial disparity in breast cancer survival can be attributed to socioeconomic factors an independent effect of race remains, which may be partially due to differences between African American and Caucasian women in phenotypes of gene expression for markers that either promote or prevent tumor growth. While these data suggest a strong genetic pathway through which race may influence breast cancer survival, no studies to date have been able to simultaneously study race, socioeconomic status (SES), pathology in addition to biomarker status. This analysis builds upon earlier work with a breast cancer registry by adding measures of SES to permit a more detailed assessment of the relationship between race and SES on breast cancer survival in light of pathological and biomarker status. Methods: With IRB approval, retrospective data were compiled from the Thomas Jefferson University Hospital (TJUH) tumor registry of African-American and Caucasian breast cancer patients including tumor stage, histologic grade, ER and PR positivity, Ki-67 proliferation index, and c-erb-B2 and p53 expression. Patient overall survival data were assessed through the end of 2007. The tumor registry data were then merged to the 2000 Census data, matching on zip codes. The federal poverty level (FPL) and median household income of each zip code were used as proxies for individual patient SES. A multiple imputation modeling approach was utilized to increase the power of the results and minimize potential bias due to missing data. Multivariable Cox proportional hazards models were used to model association of demographic, clinical, pathological, and socioeconomic measures with breast cancer patient survival. Results: Registry data from 2,230 patients diagnosed between 1994-2002 were available. Approximately 13% of the patients in the registry were African-American, with the remaining 87% Caucasian. Median income appears to explain a greater portion of the race effect than does the FPL, as the hazard ratio for race decreases from 1.40 to 1.21 in the presence of median income but only decreases to 1.31 in the presence of the FPL. When pathology factors are added to the median income model, the hazard ratio for race decreases again to 1.17. Finally, the addition of biomarkers to the median income model further reduces the hazard ratio for race to 1.1. Conclusions: The analyses presented here provide evidence that although a portion of the impact of race on breast cancer survival is explained by differences in SES between African-American and Caucasian women, the race effect is also partially explained by racial variations in breast cancer phenotypes. Furthermore, the results of this study indicate that the relationships among race, SES, pathology and biomarker status are all intertwined, rendering typical statistical methodologies for multivariate models inadequate for fully describing the relationships among these important variables. Understanding what proportion of racial disparities is attributed to SES factors versus biological factors has implications for determining the most efficient interventions for reducing the disparity. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A102.

Transcriptional Analysis of Novel Hormone Receptors PGRMC1 and PGRMC2 as Potential Biomarkers of Breast Adenocarcinoma Staging

The expression of progesterone receptor membrane component 1 (PGRMC1) in breast cancer has generated interest in this recently discovered protein because of its role in tumorigenesis. However, correlations between patient age, PGRMC1 gene expression, breast cancer morphology, and breast cancer stage have not been adequately studied. Furthermore, very little is known about possible roles for other PGRMC isoforms in breast cancer, like PGRMC2. Thus, we examined the expression of PGRMC1 and PGRMC2 mRNA by relative quantitative PCR (RelqPCR) and determined whether transcript levels correlate with age, breast cancer staging, estrogen receptor alpha (ERα) status, and other morphometric features routinely used during the pathological examination of breast ductal adenocarcinomas. Twenty-eight frozen or paraffin embedded breast cancer samples (ductal carcinoma in situ and stages I thru IV invasive ductal adenocarcinoma) and 10 control benign breast tissue samples were randomly selected and interrogated by RelqPCR to determine PGRMC1, 2, and ERα mRNA transcript levels. To control for slight variations in sample preparation, receptor transcript was normalized to the housekeeping gene phosphoglycerate kinase 1 (PGK1). Descriptive statistics and ANOVA of multiparametric datasets were used to correlate transcript levels with pathological staging parameters. PGRMC1 mRNA levels decreased significantly with patient age (Pearson's correlation -0.369; P=0.035), whereas PGRMC2 levels did not. Although the mean relative expression of PGRMC1 significantly decreased in stage II breast cancer compared with controls (P=0.050), it was no longer significant when age was considered a covariance (P=0.371). On the other hand, PGRMC2 mRNA transcript was significantly decreased in stage II breast cancer when compared to stage III cancer (P=0.028) in a manner independent of age (corrected model Bonferroni pair wise comparison, P=0.036). Furthermore, PGRMC2 levels positively correlated with ERα mRNA transcripts in patients with ER positive tumors (Pearson's correlation 0.503, P=0.096). Decreases in PGRMC1 mRNA are partially explained by increasing patient age. On the other hand, compared to stage III, PCRMC2 mRNA was significantly decreased in stage II adenocarcinoma of the breast in an age-independent manner. Additionally, PGRMC2 mRNA levels displayed a positive correlation with ERα transcripts. Thus, in addition to morphometric pathologic staging criteria, measurements of PGRMC2 mRNA may be useful for distinguishing low stage tumors from higher stages that require more aggressive clinical management, and may be a useful test when tumor ER IHC results are equivocal.

In Situ Identification of Putative Cancer Stem Cells by Multiplexing ALDH1, CD44, and Cytokeratin Identifies Breast Cancer Patients with Poor Prognosis

A subset of cells, tentatively called cancer stem cells (CSCs), in breast cancer have been associated with tumor initiation, drug resistance, and tumor persistence or aggressiveness. They are characterized by CD44 positivity, CD24 negativity, and/or ALDH1 positivity in flow cytometric studies. We hypothesized that the frequency or density of these cells may be associated with more aggressive tumor behavior. We borrowed these multiplexed, flow-based methods to develop an in situ method to define CSCs in formalin-fixed paraffin-embedded breast cancer tissue, with the goal of assessing the prognostic value of the presence of CSCs in breast cancer. Using a retrospective collection of 321 node-negative and 318 node-positive patients with a mean follow-up time of 12.6 years, we assessed TMAs using the AQUA method for quantitative immunofluorescence. Using a multiplexed assay for ALDH1, CD44, and cytokeratin to measure the coexpression of these proteins, putative CSCs appear in variable sized clusters and in 27 cases (of 490), which showed significantly worse outcome (log rank P = 0.0003). Multivariate analysis showed that this marker combination is independent of tumor size, histological grade, nodal status, ER-, PR,- and HER2-status. In this cohort, ALDH1 expression alone does not significantly predict outcome. We conclude that the multiplexed method of in situ identification of putative CSCs identifies high risk patients in breast cancer.

COX2 expression in prognosis and in prediction to endocrine therapy in early breast cancer patients

In breast cancer, the prognostic impact of COX2 expression varies widely between studies. We examined the prognostic value of COX2 expression in a large cohort of breast cancer patients treated with primary surgery between 1985 and 1994 and explained the variable results of COX2 expression found in the literature. A tissue microarray was constructed of available tumour material, and ER, PgR, HER2, Ki67 and COX2 were examined by immunohistochemistry. Median follow-up was 19years. Fifty-five percent (n=369/677) of patients received no systemic treatment. COX2 was scored using a weighted histoscore. Analysis of COX2 expression in two groups based on the median (148; below vs. above) showed an increased hazard ratio (HR) of 1.35 (95% CI 1.05-1.75, P=0.021) for disease-free survival (DFS) and of 1.39 (95% CI 1.03-1.82, P=0.016) for overall survival (OS). However, COX2 did not remain independent in multivariate analysis. In patients with hormone receptor positive tumours, COX2 expression had a negative influence on outcome (low vs. high: DFS: HR 1.37, 95% CI 1.07-1.76, P=0.013). This effect disappeared when endocrine therapy was administered (low vs. high: DFS: HR 0.93, 95% CI 0.51-1.70, P=0.811) while it remained statistically significant when endocrine therapy was omitted (low vs. high: DFS: HR 1.48, 95% CI 1.12-1.94, P=0.005). Our results show that COX2 plays a role in hormonal pathways. Our results can explain the results found in previously published studies.

Abstract ED4-2: Functional imaging: Getting beyond FDG-PET

Abstract Molecular imaging of metabolism and drug targets in the tumor may well be of additional value in the treatment of cancer patients. It can potentially be used for screening, ((pre) operative) staging, restaging and guiding targeted therapies. Molecular imaging is still in its infancy. It can be performed with various imaging modalities. Tracers used for this purpose can be labeled with either a contrast agent for magnetic resonance imaging (MRI), a fluorescent dye for optical imaging, or a radioactive nuclide for positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging. We will focus on PET and SPECT imaging given their sensitivity and available results. Most data are gathered on the visualization of general processes such as glucose metabolism and DNA synthesis. The PET-tracer [18F]fluorodeoxyglucose (FDG) is a glucose analogue and is used to visualize glucose metabolism, which is often increased in cancer cells. Increased DNA synthesis can be imaged with the pyrimidine analogue PET-tracer [18F]fluoro-L-thymidine (FLT), where FLT tumor uptake reflects the proliferation rate of cancer cells. With the increasing knowledge of the molecular processes that drive cancer and the introduction of the molecular targeted drugs, interest has been raised to image characteristics of tumor cells such as hormone receptors, growth factors and growth factor receptors. Molecular imaging of these characteristics can provide serial non-invasive information about the status of these characteristics within the tumor and its micro-environment and across lesions in the entire body when whole body imaging is performed. This is of interest as these characteristics can change over time and vary across lesions. Molecular imaging of the ER with the PET tracer 16-α-[18F]fluoro-17-β-estradiol (FES) has shown a good correlation between FES tumor uptake and ER density. This tracer is currently being tested for various applications in clinical trials. The field of visualizing growth factors and growth factor receptors is evolving rapidly. We imaged HER2 in breast cancer patients with the SPECT tracer 111In-trastuzumab and recently also with the PET tracer 89Zr-trastuzumab. With 111In-trastuzumab SPECT we showed that in 13 out of 15 patients with metastatic HER2 overexpressing disease more lesions were detected than with conventional staging procedures. Additionally the PET tracer 89Zr-trastuzumab shows excellent, quantifiable, and specific tumor uptake. In addition, we have developed 111In-bevacizumab for SPECT and 89Zr-bevacizumab for PET-imaging of vascular endothelial growth factor (VEGF) activity as an angiogenic marker. In melanoma patients small tumor lesions proved to be clearly visible with 111In-bevacizumab. Apart from using these techniques for staging and determination of drug targets, it is possible to evaluate modulation of these targets by drugs. Both HER2 and VEGF are downregulated by inhibitors of the heat shock protein (HSP) 90 and imaging of their expression was preclinically in vivo an excellent readout for the early molecular tumor response to HSP90 inhibitors. This is currently translated to clinical trials. Lastly, tracers for the receptors EGFR, IGF-1R, and the ligand TGFβ and are under development. Supported by grants from Dutch Cancer Society, Pink Ribbon and Pink Ribbon Gala. Citation Information: Clin Cancer Res 2010;16(7 Suppl):ED4-2

Variable Specimen Handling Affects Hormone Receptor Test Results in Women With Breast Cancer: A Large Multihospital Retrospective Study

Intermountain Healthcare hospitals use a single, standardized laboratory and automated testing process for estrogen receptor/progesterone receptor (ER/PR) tests to minimize testing errors. To test the (1) variability in ER/PR negativity among hospitals and (2) association between specimen handling conditions and ER/PR negativity. Retrospective study of women who had breast cancer surgery at 7 Intermountain hospitals and ER/PR tests ordered between 1997 and 2003. Data were extracted from cancer registry. Frequency of ER/PR negativity was calculated for each surgery day and compared among hospitals and between 2 groups: regular (specimens obtained Sunday through Thursday, more likely to be tested within 24 hours of surgery) and prolonged (specimens obtained on Friday and Saturday, more likely to be tested more than 24 hours after surgery) specimen handling conditions. Five thousand seventy-seven women were tested for ER/PR. The frequency of ER and PR negativity was 20.9% and 27.9%, respectively. It increased with each day of the week for both ER (P = .03) and PR (P = .059) and tended to be higher for prolonged specimens for ER (23.6% versus 20.4%; P = .03) and for PR (30.1% versus 27.4%; P = .11) compared with regular specimens. After controlling for age and tumor size, both ER (P = .02) and PR (P = .02) negativity was significantly different among the hospitals and was associated with prolonged specimens for ER (P = .04) but not for PR (P = .09). Estrogen receptor and PR negativity remained highly variable among hospitals despite use of a single laboratory and tended to be significantly associated with prolonged specimen handling. More studies are needed to confirm these findings.

The pathobiological behaviors and prognosis associated with Japanese gastric adenocarcinomas of pure WHO histological subtypes.

Japan is a high-risk region for gastric carcinoma with a comparatively early stage and favorable prognosis. To clarify the pathobiological behaviors and prognosis of Japanese gastric adenocarcinoma, we analyzed the clinicopathological characteristics of different WHO subtypes of carcinomas. The expression of ki-67, CPP32, p53, FHIT, maspin, parafibromin, GRP78, GRP94, EMMPRIN, VEGF, P-GSK3beta-ser9, fascin, cortactin, Arp2, Arp3 MUC-2, MUC-5AC and MUC-6 was examined using immunohistochemistry and tissue microarrays. The majority of cases were well-, poorly-, or moderately-differentiated subtype, whereas the minority were papillary or signet ring cell carcinoma (SRC). Patients with poorly-differentiated or SRC carcinoma were predominantly young and female. Poorly-differentiated and mucinous carcinomas were larger, with deeper invasion, more venous or lymphatic invasion, frequent lymph node involvement and peritoneal dissemination, or higher staging. The SRC group exhibited weaker expression of ki-67, CPP32, p53, parafibromin, GRP78, GRP94, P-GSK3beta-ser9, VEGF or cortactin. The moderately-differentiated subtype exhibited lower expression of FHIT and Arp3 positivity. The poorly-differentiated group showed weaker expression of CPP32, EMMPRIN, MUC-2, MUC-5AC, and MUC-6. Survival analysis indicated that the patients with poorly-differentiated or mucinous subtypes had a lower cumulative survival rate than those with papillary, well-, moderately-differentiated, or SRC carcinomas (P<0.05). The age, invasive depth, lymphatic invasion, peritoneal dissemination, and WHO classification were independent prognostic factors for carcinoma patients (P<0.05). It was suggested that poorly-differentiated and mucinous subtypes are more aggressive and of unfavorable prognosis among Japanese gastric carcinomas. Lower levels of proliferation and apoptosis, as well as alterations in tumor suppressor genes, mucin production and ER stress protein played important roles in the pathogenesis of poorly-differentiated and SRC carcinomas.

Hormone Receptor and c-ERBB2 Status in Distant Metastatic and Locally Recurrent Breast Cancer

Estrogen receptor (ER), progesterone receptor (PR), and c-ERBB2 (HER2/neu) are therapeutically and prognostically important markers in the management of breast carcinoma. They are not always analyzed in distant metastatic and locally recurrent breast cancers. We compared immunohistochemical expression in a series of primary breast carcinomas with their distant metastases (n = 72) and local recurrences (n = 45) and analyzed the impact of any changes on survival. Discordance rates between primary and metastatic and between primary and locally recurrent lesions, respectively, were 18% (13/72) and 13% (6/45) for ER, 42% (30/72) and 33% (15/45) for PR, and 7% (5/72) and 2% (1/45) for c-ERBB2. There was statistically significant discordance between primary and metastatic PR status (P = .017; kappa = 0.201). Among locally recurrent tumors, 15 (33%) of 45 revealed discordance for PR (P = .006; kappa = 0.366). We observed a trend for shorter survival among women with ER- metastatic and locally recurrent tumors regardless of the primary tumor ER status. Our findings suggest a benefit for routine evaluation of ER, PR, and c-ERBB2 status in distant metastatic and locally recurrent breast cancer for therapeutic and prognostic purposes.

Biomarkers ER, PR, HER-2 En Topoisomerase II alpha in Correlation with Response to Neoadjuvant Chemotherapy for Primary Breast Cancer.

Abstract Background: Neoadjuvant chemotherapy (CT) is widely accepted for patients with primary breast cancer (BC) not eligible for breast conservative surgery (BCS). Docetaxel, when used in combination with or when sequentially added to an anthracycline-based regimen, is active in the adjuvant setting. The objectives of this trial were to assess the activity of sequential anthracycline-docetaxel chemotherapy in the neo-adjuvant setting and to evaluate these markers as predictors of response.Patients and methods: Patients with unilateral BC &amp;gt; 2 cm (with or without positive sentinel lymph node (LN)), adequate liver, kidney and bone marrow function and no evidence of distant metastasis were eligible for enrolment. Patients with stage T4d or inflammatory BC were excluded. Treatment prior to surgery consisted of 4 cycles of Adriamycin (60 mg/m²) plus cyclophosphamide (600 mg/m²) (AC) administered intravenously every 3 weeks (q3wk), followed by 4 cycles of docetaxel (D) (100mg/m²) q3wk. Patients were clinically evaluated after 4 cycles of AC and again after 4 cycles of D. Pathological evaluation was performed after definitive surgery. Hormonal receptor, HER2 and topoisomerase II alpha (topoII) status and tumor grade were evaluated for their ability to predict response to treatment.Results: Fifty-three patients with a mean age of 49 years (range 31-69), were included in this analysis. Mean largest tumor diameter before treatment was 49.1 mm (range 25-90 mm). There were 12 grade 2 and 30 grade 3 tumours representing 46 ductal carcinomas, 5 lobular carcinomas and 2 mixed tumors. Eleven tumors (21%) were not evaluable (NE). LNs were positive in 62% of patients. Forty patients were evaluable for HER-2 and topoII. Thirteen patients (33%) were HER2-positive by immunohistochemistry (IHC) and 11 of these 13 were also HER2-positive by fluorescence in situ hybridization (FISH), 4 patients were topoII positive by FISH. The mean topoII level for FISH-positive patients was 1.83 (CI, 95%, 1.49 to 2.16). There was no increase in the topoII level without HER2 amplification by FISH. Forty-three percent of patients were oestrogen (E) and progesterone (P) receptor (R) positive, 28% were ER and PR negative, and 15% of patients were triple negative. After 4 cycles of AC, a complete reponse (CR) was seen in 17% of patients, partial response (PR) in 53% stable disease (SD) in 26%, and progressive disease (PD) in 4%. After 4 cycles of D the corresponding figures were 30%, 45%, 11% and 2% respectively. Eleven percent of patients were NE. Forty patients (75.5%) had BCS and 13 (24.5%) underwent mastectomy. A correlation was found between HER2 amplification by FISH and topoII amplification: r=0.576 (95% CI, 0.290 to 0.767). ER, PR, tumor grade, HER2 and topoII failed to predict response.Conclusions: Sequential AC followed by D is a highly effective neoadjuvant treatment and permits BCS in more than 75% of patients. There is a significant correlation between HER2 amplification by FISH and topoII levels. Tumor grade, ER, PR, HER2 or topoII were no predictors of response to AC nor D. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2045.