Low Estrogen Receptor Research Articles

Erythropoietin-producing hepatocellular A6 overexpression is a novel biomarker of poor prognosis in patients with breast cancer.

Erythropoietin-producing hepatocellular A6 (EphA6) is a member of the Eph receptor tyrosine kinase family, which has been implicated in tumorigenesis. However, little is known about the expression and function of EphA6 in breast cancer. The aim of the present study was to investigate the expression of EphA6 and the possible association between EphA6 and clinicopathological characteristics in breast cancer. In the present study, EphA6 mRNA expression was measured in 26 paired breast cancer tissues and adjacent non-cancerous tissues by reverse transcription-quantitative polymerase chain reaction. Additionally, the protein expression of EphA6 in breast cancer tissues from 116 patients was examined by immunohistochemistry, and the prognostic value for patients with breast cancer was evaluated. The results of the present study indicated that EphA6 mRNA and protein expression in breast cancer was significantly higher than that in adjacent non-cancerous tissues (P<0.001). EphA6 overexpression was significantly associated with a high histological grade (P<0.001), overexpression of human epidermal growth factor 2 (HER-2; P=0.0106), low estrogen receptor expression (P=0.0247) and low progesterone receptor expression (P=0.0015). Furthermore, the increased expression of EphA6 was demonstrated to be associated with breast cancer subtypes (P=0.0164). Kaplan-Meier curves demonstrated that high EphA6 expression was associated with lower overall survival rates in patients with breast cancer (P=0.015). Univariate and multivariate analysis revealed that high EphA6 expression, Tumor-Node-Metastasis classification and subtype were independent prognostic factors for patients with breast cancer (all P<0.05). In conclusion, EphA6 may serve an important role in breast carcinogenesis and may pose as a novel prognostic indicator and therapeutic target for breast cancer, particularly in patients with steroid receptor negative expression and HER-2 overexpression.

ATX‑LPA axis facilitates estrogen‑induced endometrial cancer cell proliferation via MAPK/ERK signaling pathway.

Autotaxin (ATX) is a key enzyme that converts lysophosphatidylcholine to lysophosphatidic acid (LPA). ATX is a crucial factor that facilitates cancer progression; however, the effect of ATX on endometrial cancer has not been explored. The aim of the present study was to investigate the role of ATX in the progression of endometrial cancer. The immunohistochemical results revealed higher protein expression levels of ATX and LPA receptors (LPA 1, 2 and 3) in human endometrial cancer tissue than in non-carcinoma tissue. In addition, reverse transcription-quantitative polymerase chain reaction and western blotting analysis demonstrated that ATX and LPA receptor mRNA and protein expression was greater in Ishikawa cells, which are positive for estrogen receptor (ER), than in Hec-1A cells that exhibit low ER expression. Short interfering RNA knockdown of ATX in Ishikawa cells led to decreased cell proliferation and cell colony number, as determined by Cell Counting kit-8 and colony formation assays. Estrogen stimulated ATX mRNA expression. Inhibition of ATX decreased estrogen and LPA-induced cell proliferation. High LPA levels markedly elevated the phosphorylation levels of extracellular signal-regulated kinase (ERK). ATX downregulation moderately decreased estrogen- and LPA-induced phosphorylation of ERK. In addition, the ERK inhibitor, PD98059, reduced cell proliferation with estrogen, ATX and LPA treatment. The present study suggested that the ATX-LPA axis may facilitate estrogen-induced cell proliferation in endometrial cancer via the mitogen-activated protein kinase/ERK signaling pathway. The present study may provide ideas and an experimental basis for clinicians to identify new molecular targeted drugs for the treatment of endometrial cancer.

Evaluation of the Expression of Amine Oxidase Proteins in Breast Cancer

We aimed to evaluate the expression of amine oxidase proteins in breast cancer and their clinical implications. We performed immunohistochemical staining of amine oxidase proteins (LOX, lysyl oxidase, AOC3, amine oxidase, MAOA, monoamine oxidase A, MAOB, monoamine oxidase B). Based on their hormone receptors, such as estrogen receptor (ER) and progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), and Ki-67 immunohistochemical staining, breast cancer was divided into four molecular subtypes: luminal A, luminal B, HER-2 type, and triple-negative breast cancer (TNBC). Luminal A was observed in 380 cases (49.4%), luminal B in 224 (29.1%), HER-2 type in 68 (8.8%), and TNBC in 98 (12.7%). Stromal AOC3, MAO-A, and MAO-B expression varied according to molecular subtypes. Stromal AOC3 expression was high in luminal B and HER-2 type and MAO-A expression was high in luminal A and luminal B (p < 0.001). MAO-B expression was higher in TNBC than in other subtypes (p = 0.020). LOX positivity was associated with high histological grade (p < 0.001) and high Ki-67 labeling index (LI) (p = 0.009), and stromal AOC3 positivity was associated with high histological grade (p = 0.001), high Ki-67 LI (p < 0.001), and HER-2 positivity (p = 0.002). MAO-A positivity was related to low histological grade (p < 0.001), ER positivity, PR positivity (p < 0.001), and low Ki-67 LI (p < 0.001). In univariate analysis, MAO-A positivity was related to short disease-free survival in HER-2 type (p = 0.013), AOC3 negativity was related to short disease-free survival and overall survival in ER-positive breast cancer, PR-positive breast cancer, HER-2-negative breast cancer, and lymph node metastasis. In conclusion, the expression of amine oxidase proteins varies depending on the molecular subtype of breast cancer. Stromal AOC3 expression was high in luminal B and HER-2 type, and MAO-A expression was high in luminal A and luminal B.

Chronic hyperandrogenemia in the presence and absence of a western-style diet impairs ovarian and uterine structure/function in young adult rhesus monkeys.

Does chronic hyperandrogenemia beginning at menarche, in the absence and presence of a western-style diet (WSD), alter ovarian and uterine structure-function in young adult rhesus monkeys? Phenotypic alterations in ovarian and uterine structure/function were induced by exogenous testosterone (T), and compounded in the presence of a WSD (T+WSD). Hyperandrogenemia is a well-established component of PCOS and is observed in adolescent girls, indicating a potential pubertal onset of disease symptoms. Obesity is often associated with hyperandrogenemia and it is hypothesized that metabolic dysfunction exacerbates PCOS symptoms. Macaque females (n = 40) near the onset of menarche (~2.5 years of age) were assigned to a 2 by 2 factorial cohort design. Effects on reproductive characteristics were evaluated after 3 years of treatment. Rhesus macaques (Macaca mulatta) were fed either a normal balanced diet (n = 20) or a WSD (n = 20). Additionally, implants containing cholesterol (n = 20) or T (n = 20) were implanted subcutaneously to elevate serum T approximately 5-fold. This resulted in treatment groups of controls (C), T, WSD and T+WSD (n = 10/group). Vaginal swabbing was performed daily to detect menses. After 3 years of treatment, daily serum samples from one menstrual cycle were assayed for hormone levels. Ovarian structure was evaluated in the early follicular phase by 3D/4D ultrasound. Uterine endometrial size and ovarian/luteal vascular function was also evaluated in subgroups (n = 6/group) in the late follicular and mid-luteal phases by 3D/4D ultrasound and contrast-enhanced ultrasound, respectively. Expression of steroid hormone receptors and markers of decidualization and endometrial receptivity were assessed in endometrial biopsies at mid-luteal phase. Approximately 90% of menstrual cycles appeared ovulatory with no differences in frequency or duration between groups. Serum estradiol (E2) levels during the early follicular phase were greatest in the T alone group, but reduced in T+WSD (P < 0.02). Serum LH was elevated in the T group (P < 0.04); however, there were no differences among groups in FSH levels (P > 0.13). Ovarian size at menses tended to be greater in the WSD groups (P < 0.07) and antral follicles ≥1 mm were more numerous in the T+WSD group (P < 0.05). Also, females in T and T+WSD groups displayed polycystic ovarian morphology (PCOM) at greater frequency than C or WSD groups (P < 0.01). Progesterone (P4) levels during the luteal phase were reduced in the T+WSD group compared to C and T groups (P < 0.05). Blood volume (BV) and vascular flow (VF) within the corpus luteum was reduced in all treatment groups compared to C (P < 0.01, P = 0.03), with the WSD alone group displaying the slowest BV and VF (P < 0.05). C and WSD groups displayed endometrial glands at mid-luteal phase with low estrogen receptor 1 (ESR1) and progesterone receptor (PGR) mRNA and immunohistochemical staining in the functionalis zone, but appreciable PGR in the stroma. In contrast, T and T+WSD treatment resulted in glands with less secretory morphology, high ESR1 expression in the glandular epithelium and low PGR in the stroma. Endometrial levels of TIMP3 and MMP26 mRNA and immunostaining were also decreased in the T and T+WSD groups, whereas AR expression was unchanged. None. Females are young adults, so effects could change as they reach prime reproductive age. The T level generated for hyperandrogenemia may be somewhat greater than the 3-4-fold increase observed in adolescent girls, but markedly less than those observed in male monkeys or adolescent boys. Alterations to ovarian and uterine structure-function observed in T and, in particular, T+WSD-treated female macaques are consistent with some of the features observed in women diagnosed with polycystic ovary syndrome (PCOS), and suggest impaired fertility. Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH) under Award Number P50HD071836 (to RLS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Additional funding was provided by Office of the Director, NIH under Award Number P51OD011092 (Support for National Primate Research Center). Authors declare no competing interests.

Comparison of estrogen receptor, progesterone receptor and HER2 results in concurrent ipsilateral samples with invasive breast carcinoma: a retrospective study of 246 biopsies from 119 patients

There is no consensus regarding biomarker testing on the ipsilateral breast carcinomas present in separate biopsies, irrespective of whether the biopsies are performed concurrently or consecutively. We aimed to investigate estrogen receptor (ER), progesterone receptor (PR) and HER2 concordance in ipsilateral concurrent biopsies with invasive breast tumors. Consecutive ipsilateral concurrent biopsies with invasive breast tumors were identified retrospectively. Biomarker results, histologic grade and histologic subtype among the tumors in concurrent samples were compared. ER, PR, and HER2 expression was different in 3 (2.5%), 11 (9.2%) and 7 (5.9%) cases, respectively. All ER-discordant cases were sets of ER-negative (ER-) and weak-low ER-positive (ER+), ductal subtype and histologic grade 2 or 3 tumors. All PR-discordant cases were ER+, and comprised of histologic grades 1 to 3 ductal as well as lobular tumors. All HER2 discordant cases were histologic grade 2 to 3 ductal tumors. Biomarker discordance was independent of grade and subtype discordance. We found very low biomarker discordance among tumors in concurrent samples from ipsilateral breast. Our results suggest that ER and HER2 discordance in concurrent samples is predictable. ER discordance is present only in a setting of low ER+ tumors. Low-grade ductal and/or lobular tumors are ER and HER2 concordant. HER2 discordance is noted in grade 2 to 3 ductal tumors only. Histologic subtype and grade may guide extent of biomarker testing in concurrent ipsilateral breast biopsies.

Prognostic and predictive value of low estrogen receptor expression in breast cancer.

Anti-hormonal therapy (tamoxifen) is recommended for estrogen receptor (er)-positive breast cancer (bca); however, its effect on low-receptor cancers is unclear. We retrospectively evaluated the effect of adjuvant tamoxifen in patients with weakly er-positive bca. We identified 2221 bca patients who had been er-tested by ligand-based assay (lba) during 1976-1995 and who had been treated and followed until 2008. Cox proportional hazards models adjusted for age, body mass index, tumour size, nodal status, surgery, and chemotherapy were used to assess the effect of er level on bca survival in patients who received tamoxifen. Overall, 17% (383) of patients were within 0-3 fmol/mg cytosol protein, and 12% (266) were within 4-9 fmol/mg cytosol protein. Patients with er levels of 0-3, 4-9, 10-19, 20-49, and 50 fmol/mg or more cytosol protein had 20-year bca survival rates of 56%, 56%, 63%, 71%, and 60% respectively. Of the 2221 patients studied, 661 (29.8%) received anti-hormonal therapy. Within the latter group, er levels of 0-3, 4-9, 10-19, 20-49, and 50 fmol/mg or more cytosol protein were associated with a hazard ratio for lower bca mortality: respectively, 1.00 (reference), 0.59 (p = 0.09), 0.19 (p < 0.0001), 0.26 (p < 0.0001), and 0.31 (p < 0.0001)-the risk reduction being significant only for er levels of 10 fmol/mg or more cytosol protein. Tamoxifen use in bca patients with a weakly positive er status (4-9 fmol/mg cytosol protein), compared with those having higher er levels (≥10 fmol/mg cytosol protein), is not associated with a significantly lower bca-specific mortality. Our results do not support treatment with anti-hormonal therapy for bca patients with a weakly positive er status as identified by lba.

EZH2 protein expression in normal breast epithelium and risk of breast cancer: results from the Nurses\u2019 Health Studies

BackgroundEnhancer of zeste homolog 2 (EZH2) is a polycomb-group protein that is involved in stem cell renewal and carcinogenesis. In breast cancer, increased EZH2 expression is associated with aggressiveness and has been suggested to identify normal breast epithelium at increased risk of breast cancer development. However, the association between EZH2 expression in benign breast tissue and breast cancer risk has not previously been evaluated in a large prospective cohort.MethodsWe examined the association between EZH2 protein expression and subsequent breast cancer risk using logistic regression in a nested case-control study of benign breast disease (BBD) and breast cancer within the Nurses’ Health Studies. EZH2 immunohistochemical expression in normal breast epithelium and stroma was evaluated by computational image analysis and its association with breast cancer risk was analyzed after adjusting for matching factors between cases and controls, the concomitant BBD diagnosis, and the Ki67 proliferation index.ResultsWomen with a breast biopsy in which more than 20% of normal epithelial cells expressed EZH2 had a significantly increased risk of developing breast cancer (odds ratio (OR) 2.95, 95% confidence interval (CI) 1.11–7.84) compared to women with less than 10% EZH2 epithelial expression. The risk of developing breast cancer increased for each 5% increase in EZH2 expression (OR 1.22, 95% CI 1.02–1.46, p value 0.026). Additionally, women with high EZH2 expression and low estrogen receptor (ER) expression had a 4-fold higher risk of breast cancer compared to women with low EZH2 and low ER expression (OR 4.02, 95% CI 1.29–12.59).ConclusionsThese results provide further evidence that EZH2 expression in the normal breast epithelium is independently associated with breast cancer risk and might be used to assist in risk stratification for women with benign breast biopsies.

Immunohistochemical Phenotype of Breast Cancer during 25-Year Follow-up of the Royal Marsden Tamoxifen Prevention Trial.

The randomized, double-blinded Royal Marsden Tamoxifen Breast Cancer Prevention Trial in healthy high-risk women started in 1986 and is still blinded. Eligible participants (n = 2,471) were randomly assigned to tamoxifen (20 mg/d) or placebo for 8 years. Analysis in 2006 showed a 30% risk reduction of estrogen receptor (ER)-positive invasive breast cancer mostly in the posttreatment period. Biomarker analysis in this population may identify any subgroup-specific preventive effects tamoxifen. After a median follow-up of 18.4 years, 242 patients had developed invasive cancer, 134 on placebo and 108 on tamoxifen. From these, 180 tissue blocks were available and ER, progesterone receptor (PgR), Ki67, HER2, and EGFR were immunohistochemically analyzed. A 32% reduction in ER+ and PgR+ invasive cancers resulted after 8 years of treatment. Quantitative levels of ER and PgR were lower in the tamoxifen-treated group, significantly so for ER (P = 0.001). These lower ER levels were restricted to the posttreatment period (P = 0.018). Among the ER+ group, there was a similar proportional decrease in PgR+ and PgR- tumors by tamoxifen. The median levels of Ki67 were similar in both arms. The numbers of HER2-positive and EGFR-positive cancers were higher in the tamoxifen arm but not significantly so. In conclusion, the preventive effects of tamoxifen result in reduced ER-positive but not ER-negative tumors and reduced ER expression in the ER-positive cases largely confined to the posttreatment period. Overall reductions in PgR expression are explained by lower frequency of ER-positive cases. Impact on Ki67, HER2, and EGFR was modest. Cancer Prev Res; 10(3); 171-6. ©2017 AACR.

Abstract P6-09-12: A consecutive series of early breast cancers with a low estrogen receptor expression

Abstract P6-09-12: A consecutive series of early breast cancers with a low estrogen receptor expression

Hormone�-receptor expression status of epithelial ovarian cancer in Ibadan, South-western Nigeria

IntroductionEpidemiological evidence strongly suggests that steroid hormones are implicated in the pathogenesis of ovarian cancer. Estrogen receptor (ER) and Progesterone receptor (PR) are prognostic indicators for a number of epithelial tumors and may play the same role in ovarian cancers. This study aims to evaluate the expression of ER and PR in epithelial ovarian cancer (EOC) in an African population and compare it with other prognostic factors such as age, International Federation of Gynaecology and Obstetrics (FIGO) stage, grade and histological subtype.MethodsNinety cases of histologically confirmed EOC were reviewed. Immunohistochemistry was used to assess their ER and PR expression status and was then compared with other demographic variables using statistical methods, with level of significance set at p < 0.05.Results30.2% and 8.3% of serous and mucinous carcinomas respectively were ER positive while 41.2% and 22.5% of both tumour types were PR positive. One of the two endometrioid carcinomas showed PR expression but neither were positive for ER. The only case of Brenner tumour in the series was ER positive but negative for PR. There was a significant association between ER and the histological subtypes (p = 0.042) while no significant association was found between PR expression and histological subtypes (p = 0.650). No significant association was found between hormone receptor status, age and stage of the EOC.ConclusionThe study showed a lower ER expression in serous carcinoma compared to large cohorts from developed countries. Future translational studies could be used to determine response of EOC to endocrine therapy.

EXPRESSION LEVEL OF ESTROGEN RECEPTOR ß IN NON-SMALL CELL LUNG CANCER AND LUNG METASTSIS

Background. A search for pathogenetically grounded approaches to the treatment of non-small cell lung cancer is undoubtedly one of the top priorities, because of the results of its treatment cannot be regarded as satisfactory. Discovery of a new type of receptors, estrogen receptors ß, that expressed in non-small cell lung cancer, created the preconditions for the development of a new treatment strategy for non-small cell lung cancer, namely antiestrogen therapy. Objective was to answer to the clinically significant question of how many patients with non-small cell lung cancer and lung metastases are potential candidates for antiestrogen treatment. Materials and methods. A comparative quantitative estimation of the level and frequency of the estrogen receptors ß expression in non-small cell lung cancer tissues and also in lung metastases (74 in total) was carried out by flow cytometry. Results. The estrogen receptors ß expression was detected in the majority of the investigated tumors and lung metastases, in 92 and 86 % of patients, respectively. The average level of estrogen receptors ß expression in non-small cell lung cancer tissue was higher then in metastases (42 % and 34.6 % respectively). The differences was statistically significant (p = 0.03). Primary tumors with a high and low+moderate estrogen receptors ß expression levels were detected in 35 % and 65 % of cases respectively and metastases with such expression levels - in 14 % and 86 % of cases respectively. Conclusion. 1. The estrogen receptors ß expression in the tumor predicts a more favorable course of the disease. One of the reasons for this may be a greater metastatic potential of tumor cells with low estrogen receptors ß expression. 2. The group of potential candidates for carrying out the adjuvant antiestrogen therapy comprises about 70 % of patients with primary lung tumors and with metastatic lesions of lung.

The Relationships between HER2 Overexpression and DCIS Characteristics.

The aim of this study was to demonstrate the correlation between human epidermal growth factor receptor 2 (HER2) overexpression and some poor prognosis factors in patients affected by ductal carcinoma in situ (DCIS). We evaluated 48 cases of DCIS, divided into two groups according to HER2 amplification status. Nuclear grade and "cancerization of lobules" were determined within primary DCIS and Ki67, estrogen receptor (ER), PR, and HER2 expression was established using immunohistochemistry. The histopathological variables in HER2-positive and in HER2-negative patients were compared to determine the recurrence risk. We also considered the median age at the time of surgery according to HER2 status. There were 11 recurrences (23%), 6 DCIS (55%), and 5 invasive cancer (45%). In an 8-year-long median follow-up, we hypothesized high risk of recurrence in HER2-positive DCIS. Patients with HER2-positive DCIS were younger than HER2-negative ones (p = 0.002). HER2-positive DCIS was also related to histopathological predictors of recurrence such as high nuclear grade (p < 0.001), high Ki67 expression (p = 0.003), low ER and PgR levels (p < 0.001), and the presence of "cancerization of lobules" (p < 0.049). Our trial suggests that HER2 amplification in primary DCIS is identified more frequently in younger patients and hypothesizes high risk of recurrence in HER2-positive DCIS related to histopathological predictors of overall relapse as high nuclear grade, high Ki67 expression, low ER and PgR levels, and the presence of "cancerization of lobules." In HER2-positive DCIS, other variables of recurrence risk are compared to HER2-negative lesions, without statistical significance. Our results show that HER2 testing might suggest clinicians the optimal treatment of patients with DCIS.

Breast cancer in young women.

Breast cancer (BC) in young women is rare, affecting only 4-6% of women under the age of 40. Regardless, BC remains the most common malignancy among younger patients. Recently, a significant increase in BC rates has been observed among pre-menopausal subjects. Breast cancer in young women requires special attention due to its specific morphologic and prognostic characteristics and unique aspects, including fertility preservation and psychosocial issues (e.g. its impact on family life and career). Young women are more likely to have tumors with higher incidence of negative clinicopathologic features (higher histological grade, more lymph node positivity, lower estrogen receptor (ER) positivity, higher rates of Her2/neu overexpression). Also, they tend to be diagnosed at more advanced stages of the disease. That, in turn, contributes to less favorable prognosis as compared to older women. Young women are generally treated similarly to older patients. Surgical management includes mastectomy or breast-conserving surgery, followed by radiation therapy (younger women have higher local recurrence rates than older women, especially after breast-conserving therapy). Although the basics of chemotherapy are the same for patients of all ages, younger women have some special considerations. It is important to consider options for fertility preservation before starting systemic treatment. Patients should have access to genetic testing as their results may affect the choice of therapy. Younger women and their families should receive adequate psychological support and counselling.

Estrogen receptor expression and gene promoter methylation in non-small cell lung cancer - a short report.

In the past, anomalous estrogen receptor (ER) regulation has been associated with various lung pathologies, but so far its involvement in lung cancer initiation and/or progression has remained unclear. Here, we aimed at assessing in vivo and in vitro ER expression and its possible epigenetic regulation in non-small cell lung cancer (NSCLC) samples and their corresponding normal tissues and cells. ERα and ERβ gene expression levels were assessed using real time quantitative PCR (RT-qPCR), whereas ERα and ERβ gene promoter methylation levels were assessed using DNA bisulfite conversion followed by pyrosequencing. We included NSCLC (n=87) and adjacent histologically normal lung tissue samples from lung cancer patients (n=184), primary normal bronchial epithelial-derived cell cultures (n=11), immortalized bronchial epithelial-derived cell lines (n=3) and NSCLC derived cell lines (n=9). Using RT-qPCR we found significantly lower ERα and ERβ expression levels in the NSCLC tissue samples compared to their normal adjacent tissue samples. These lower ER expression levels were confirmed in vitro using primary normal bronchial epithelial-derived cell cultures, immortalized bronchial epithelial-derived cell lines and NSCLC-derived cell lines. By using this latter panelof cells, we found that ER gene promoter hypermethylation was associated with decreased ER expression. In addition we found that in tumor and normal lung tissues, smoking was associated with decreased ER expression and that normal lung tissues with a low ERβ expression level exhibited increased smoking-related DNA adducts. Taken together, our results indicate that decreased ER expression mediated by DNA methylation may play a role in NSCLC development.

Abstract 91: The mutational landscape of mucinous carcinomas of the breast

Abstract Background: Mucinous breast carcinoma (MBC) is a special histologic type of breast cancer, comprising approximately 2% of all invasive breast carcinomas. Morphologically, MBCs are characterized by clusters of tumor cells floating in large amounts of extracellular mucin. MBCs are preferentially of low histologic grade and estrogen receptor (ER)-positive and HER2-negative. MBCs have been shown to lack the hallmark 1q gains and 16q losses found in low-grade ER-positive invasive ductal carcinomas of no special type (IDC-NSTs). Here we sought to characterize the mutational landscape and copy number alterations (CNA) of MBCs by whole exome sequencing analysis. Material and Methods: Frozen sections from 25 pure MBCs (n = 13 mucinous A (paucicellular), n = 12 mucinous B (hypercellular)) were subjected to microdissection to ensure a tumor cell content &amp;gt;85%. In addition, five cases of mixed mucinous carcinomas composed of mucinous and IDC-NST components were included in this study. In these cases, the MBC and IDC-NST components were microdissected separately. DNA samples extracted from microdissected tumors and matched normal counterparts were subjected to whole exome sequencing on an Illumina HiSeq2000. Somatic single nucleotide variants (SNVs) were identified using MuTect and somatic insertions and deletions (indels) were identified using Strelka and Varscan2. Somatic CNAs were defined using FACETS. Mutational frequencies in MBCs were compared to those of luminal IDC-NSTs from The Cancer Genome Atlas. Results: A median of 26 (range 5-71) non-synonymous SNVs and indels were identified per MBC. GATA3, mutated in 28% of cases, was the only significantly mutated gene as defined by MutSigCV (q&amp;lt;0.1). Compared to IDC-NSTs of luminal molecular subtype, MBCs were found to harbor significantly fewer mutations in TP53 (3% vs 19%, p&amp;lt;0.001) and PIK3CA (6% vs 41%, p&amp;lt;0.001). Mucinous subtypes A and B did not differ in terms of mutations and/ or CNAs, whereas MBCs of mixed phenotype harbored a greater extent of genomic instability. While MBC and IDC-NST components of mixed MBCs shared the majority of the somatic genetic alterations, indicating their clonal relatedness, mutations and CNAs restricted to either the MBC or the IDC-NST components were also identified. Furthermore, CNA analysis confirmed our previous observations that MBCs, contrary to low-grade ER-positive IDC-NSTs, lack concurrent copy number gains of 1q and 16p and losses of 16q. Conclusions: Our data suggest that the repertoires of somatic mutations and gene CNAs of MBCs are distinct from those of luminal IDC-NSTs, and that MBCs are an entity distinct from low-grade ER-positive IDC-NSTs not only at the morphologic but also at the genetic level. Citation Format: Salvatore Piscuoglio, Charlotte KY Ng, Felipe C. Geyer, Carey A. Eberle, Elena Guerini-Rocco, Caterina Marchio, Anne Vincent-Salomon, Jorge S. Reis-Filho, Britta Weigelt. The mutational landscape of mucinous carcinomas of the breast. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 91.

Abstract 4295: Androgen receptor expression in normal breast TDLUs and subsequent breast cancer risk

Abstract Background Sex steroid hormone signaling is critical in the development and progression of breast cancers, though the role of androgens remains unspecified. Large epidemiologic studies have found a consistent association between circulating androgens and increased breast cancer risk, though it is unknown whether circulating androgens reflect the androgenic milieu in the breast. An interaction between androgen receptor (AR) and estrogen receptor (ER) signaling in the breast has been postulated, wherein AR signaling antagonizes ER signaling in estrogen-rich environments, and AR signaling induces proliferative effects in estrogen-deplete environments. Methods We evaluated the association between AR expression and subsequent breast cancer risk in a nested case-control study of women with benign breast disease (BBD) within the Nurses’ Health Studies. Cases were women with BBD that subsequently developed breast cancer (median 9 years later) while controls had BBD but did not develop breast cancer. Tissue microarrays were constructed containing normal terminal ductal lobular unit (TDLU) tissue from the BBD biopsy. AR expression was assessed by immunohistochemistry and the percent of positive-staining nuclei was digitally quantified for 61 breast cancer cases and 184 controls. Logistic regression models adjusting for the year of BBD biopsy, age at cancer diagnosis, years since BBD biopsy, and BBD lesion type were used to calculate odds ratios and 95% confidence intervals for the association between the tertile of AR expression and breast cancer risk. We further evaluated the impact of AR and ER co-expression, each dichotomized at the median, in a sub-analysis of 31 cases and 82 controls. Finally, we assessed AR expression as a predictor of subsequent ER tumor status using polytomous logistic regression. Results Overall, women in the highest tertile of AR expression experienced non-significant 1.32-fold increased odds of breast cancer (95% CI: 0.64-1.73, p-trend = 0.559) compared to the lowest tertile. A significant interaction was detected between AR and ER co-expression in normal breast TDLUs and subsequent breast cancer risk (p-interaction = 0.003). Among women with low ER expression, increased AR expression was associated with 2.52-fold increased odds (95% CI: 0.68-9.34) of developing breast cancer. In contrast, among women with high ER expression, high AR expression was associated with a 91% decrease in the odds (OR = 0.09, 95% CI: 0.01-0.62) of breast cancer. AR expression was not predictive of subsequent ER tumor status. Conclusions There was little evidence for an overall association between AR expression in normal breast tissue and breast cancer risk, though we observed a significant interaction between AR and ER expression. Our findings support the hypothesis that AR interacts with ER to promote cell proliferation in estrogen-deprived environments and inhibit growth in estrogen-rich environments. Citation Format: Kevin Kensler, Andrew Beck, Francisco Beca, Laura Collins, Stuart Schnitt, Aditi Hazra, Susan Hankinson, Myles Brown, Rulla Tamimi. Androgen receptor expression in normal breast TDLUs and subsequent breast cancer risk. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4295.

Abstract 1465: Analysis of tumor-infiltrating lymphocytes (TILs) reveals biologically different subgroups of breast ductal carcinoma in situ

Abstract Background: Treatment of breast ductal carcinoma in situ (DCIS) aims to prevent invasive recurrence. Recent studies have shown an important impact of TILs on the outcome of invasive breast cancer, however their influence on breast DCIS prognosis has not been fully explored. In this study we investigated whether the amount and/or phenotype of TILs can help recognizing DCIS subgroups of different biology and recurrence risk. Methods: The study included 134 patients, diagnosed and treated for a DCIS from 2001 to 2005 in our institution. Formalin-fixed and paraffin-embedded (FFPE) cancer tissue samples, taken before any treatment, were retrospectively collected. H&amp;E whole tissue sections served for assessment of the cancer size, grade, histotype, architecture, mitotic index and amount of stromal lymphocytic (Ly) infiltrate. The latter was semi-quantitatively graded into 4 grades (0 - absent, 1 - mild, 2 - moderate, 3 - intense). Ly-phenotype was assessed by immunohistochemistry (IHC) on tissue microarrays (TMAs), constructed by sampling each case at the area of the densest Ly-infiltration (3 cores of 0.6 mm diameter per case). The number/mm2 of the CD8+, CD4+, FOXP3+, CD20+ and CD38+ mononuclear cells was determined. TMAs served also for estrogen receptor (ER), progesterone receptor (PR), HER2 and Ki67 IHC staining, as well as for HER2 amplification status. Results: There were 97 DCIS and 37 microinvasive DCIS (micDCIS). The micDCIS displayed significantly more diffuse architecture, frequent HER2 amplification (HER2amp), higher grade, lower ER expression (0.029&amp;lt;p&amp;lt;0.044 for each) and more peritumoral Ly-infiltrate (grades 2+3, micDCIS vs DCIS, 51.5% vs. 39.0%, p = 0.036). All but CD20+ cells were more numerous in micDCIS than in DCIS (0.0016&amp;lt;p&amp;lt;0.05). Within the entire cohort, the cases having the (CD8+/CD4+):(CD20+/CD38+) ratio higher than 1 had a significantly greater risk of containing a microinvasive component (OR 3.47 (1.26-9.57), p = 0.029). Interestingly, that ratio was significantly higher (p = 0.012) in micDCIS than in the DCIS with grade 2 or 3 Ly-infiltrate (Ly-DCIS, n = 38). On the other side, there was no difference between micDCIS and Ly-DCIS in architecture, histograde, HER2amp rate and ER expression. Cluster analysis further confirmed significant similarities between micDCIS and Ly-DCIS, putting them both apart from non-Ly-DCIS (p = 0.0034). The overall 10-year recurrence rate was 13% (18/134 pts). No parameter significantly correlated with recurrence risk, however the micDCIS have received significantly more treatment than DCIS (axillary lymphadenectomy (p&amp;lt;10-7), chemotherapy (p = 0.031) or hormonal therapy (p = 5.4×10-6)). Conclusion: These results indicate that Ly-DCIS might be biologically and immunologically similar to micDCIS. TILs in DCIS are worth investigating in larger studies, as they could be a marker of microinvasion and help tailoring the initial treatment of the disease. Citation Format: Marie Beguinot-Cornillon, Marie-Melanie Dauplat, Fabrice Kwiatkowski, Guillaume Lebouedec, Lucie Tixier, Christophe Pomel, Frederique Penault-Llorca, Nina Radosevic-Robin. Analysis of tumor-infiltrating lymphocytes (TILs) reveals biologically different subgroups of breast ductal carcinoma in situ. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1465.

Prognostic differences of phenotypes in pT1-2N0 invasive breast cancer: a large cohort study with cluster analysis

To find phenotypic subgroups of patients with pT1-2N0 invasive breast cancer by means of cluster analysis and estimate the prognosis and clinicopathological features of these subgroups. From 1999 to 2013, 4979 patients with pT1-2N0 invasive breast cancer were recruited for hierarchical clustering analysis. Age (≤40, 41-70, 70+ years), size of primary tumor, pathological type, grade of differentiation, microvascular invasion, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) were chosen as distance metric between patients. Hierarchical cluster analysis was performed using Ward's method. Cophenetic correlation coefficient (CPCC) and Spearman correlation coefficient were used to validate clustering structures. The CPCC was 0.603. The Spearman correlation coefficient was 0.617 (P<0.001), which indicated a good fit of hierarchy to the data. A twelve-cluster model seemed to best illustrate our patient cohort. Patients in cluster 5, 9 and 12 had best prognosis and were characterized by age >40 years, smaller primary tumor, lower histologic grade, positive ER and PR status, and mainly negative HER-2. Patients in the cluster 1 and 11 had the worst prognosis, The cluster 1 was characterized by a larger tumor, higher grade and negative ER and PR status, while the cluster 11 was characterized by positive microvascular invasion. Patients in other 7 clusters had a moderate prognosis, and patients in each cluster had distinctive clinicopathological features and recurrent patterns. This study identified distinctive clinicopathologic phenotypes in a large cohort of patients with pT1-2N0 breast cancer through hierarchical clustering and revealed different prognosis. This integrative model may help physicians to make more personalized decisions regarding adjuvant therapy.

Low estrogen receptor (ER) positive breast cancer and neoadjuvant systemic therapy (NAT): Is response similar to ER+ or to ER- disease?

580Background: ER expression can be semi-quantitatively measured using a Histochemical or H-score that can range from 0 (negative) to 300 (diffuse strong positive).. We sought to demonstrate a difference in pathologic complete response (pCR) rates after NAT between strongly positive and weakly positive ER+ breast cancers. Methods: Retrospective chart review was conducted of patients diagnosed with invasive breast cancer who received NAT from 2010-2014 at our institution. Case inclusion required at least tumor ER status, HER2 status and pCR information. Low ER+ tumors were defined as tumors with ER H-score of 1-100 and their overall pCR rate was compared to that of ER negative (H-score 0), moderate ER+ (H-score 101-200) and high ER+ tumors (H-score 201-300). The data was further analyzed with respect to HER2 status. HER2 results were classified using the 2013 ASCO/CAP HER2 guidelines criteria. HER2 equivocal cases were excluded for this analysis due to small numbers. Univariate analyses were performed usin...

Biological differences between functionalis and basalis endometria in women with and without adenomyosis

ObjectiveEndometriosis is commonly believed to originate from functionalis endometrium and adenomyosis from basalis endometrium. Due to the lack of enough information, we investigated the biological differences between these two layers of endometrium in women with and without adenomyosis. Study designThis was a case-controlled study with 12 control women and 17 women with adenomyosis undergoing hysterectomy. Full thickness (extending from the endometrium to the myometrium) biopsy specimens were obtained after the hysterectomy. Based on the phases of the menstrual cycle, the expression patterns of estrogen receptor (ER), progesterone receptor (PR), Ki-67, and activated Caspase-3 were analyzed by immunohistochemistry in the functionalis and basalis endometria. Apoptotic cells were evaluated by TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay. ResultsA similar pattern of ER and PR expression was found in the functionalis and basalis endometria during the proliferative phase. In contrast, a significantly lower ER and PR expression was found in the basalis endometria than in the functionalis endometria during the secretory phase and the menstrual phase. This was equally observed in control women and in women with adenomyosis. Except Ki-67 indices, TUNEL-positive cells, and expression of activated Caspase-3 were significantly lower in the basalis endometria than in the functionalis endometria during the secretory phase. ConclusionA significant biological difference was found between the functionalis and the basalis endometria derived from women with adenomyosis.