G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis.

Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.

Displaced granulosa cells in the fallopian tube mistaken for metastatic granulosa cell tumor.

A 44-yr-old woman underwent a total hysterectomy and bilateral salpingectomy secondary to uterine leiomyomas. Gross examination of the fallopian tubes revealed no masses or lesions; however, 2 small foci of granulosa cells were identified microscopically within one of the fallopian tubes. These foci were suspicious for granulosa cell tumor metastases. The patient subsequently underwent a bilateral oophorectomy, which revealed no primary granulosa cell tumor. Immunohistochemical studies were used to help support the benign nature of the granulosa cells within the fallopian tube. A review of the literature revealed only 1 similar case report of displaced benign granulosa cells within the fallopian tubes. The ovaries in both this case and the previous case report were found to contain multiple cystic follicles, suggesting ovulation as the likely mechanism of displacement. Knowledge of this rare occurrence and the use of immunohistochemical staining are paramount to making a correct diagnosis, thus preventing a misdiagnosis of malignancy and possible unnecessary treatment.

Effects of variations in serum estradiol concentrations on secretory endometrial development and function in experimentally induced cycles in normal women.

Eighteen normal women underwent pituitary down-regulation with leuprolide, followed by a 10-day treatment with 0.2 mg/d transdermal estradiol (E(2)) with subsequent allocation to one of two 10-day estradiol regimens plus 40 mg daily intramuscular P: supraphysiologic (0.2 mg/d transdermal E(2) mg/d vaginal micronized E(2)) or subphysiologic (no exogenous E(2) treatment). Average E(2) and P in the supraphysiologic, physiologic, and subphysiologic groups were 1,175.9 pg/mL and 17.5 ng/mL, 136.9 pg/mL and 21.2 pg/mL, and 23.8 ng/mL and 22.0 ng/mL, respectively, and there were no differences between groups in endometrial histology or expression of biomarkers of receptivity.

Endometrial development and function in experimentally induced luteal phase deficiency.

CONTEXT: It is generally assumed that delayed endometrial development observed in luteal phase deficiency (LPD) is the result of abnormally low progesterone (P) levels. This hypothesis has never been tested by direct experiment. OBJECTIVE: Our objective was to evaluate the effects of P concentrations on human endometrium. DESIGN AND SETTING: A randomized trial was conducted at an academic medical center. SUBJECTS: Twenty-nine healthy, ovulatory 18- to 35-yr-old women participated. INTERVENTION: Endometrial samples were obtained from women in natural cycles and two groups of experimentally modeled cycles. Women undergoing modeled cycles were treated with GnRH agonist and a fixed physiological dose of transdermal estradiol, followed by randomization to 10 or 40 mg daily im P administration to achieve either normal circulating luteal P or 4-fold lower P concentrations, the latter representing an experimental model of LPD. MAIN OUTCOME MEASURES: Tissue specimens, obtained after 10 days of P exposure, were analyzed by histological dating, immunohistochemistry, immunoblot, and real-time quantitative RT-PCR (qRT-PCR). RESULTS: Histological dating of endometrium, immunohistochemistry for endometrial integrins, and qRT-PCR analysis for nine putative functional markers showed no differences between the three groups. Preliminary data from Western analysis suggest that some proteins may be affected by low serum P concentrations. CONCLUSIONS: Histological endometrial dating does not reflect circulating P concentrations and cannot serve as a reliable bioassay of the quality of luteal function. Assessment of selected functional markers by either immunohistochemistry or qRT-PCR is similarly insensitive to decreased circulating P. Preliminary evidence suggests that abnormally low luteal phase serum P concentrations may have important functional consequences not otherwise detected.

Cyclic regulation of T-Bet and GATA-3 in human endometrium.

Endometrial cytokine expression is poorly understood. T-Bet and GATA-3 regulate cytokine expression in T-lymphocytes. Previous work has demonstrated expression of T-Bet in human endometrium. Changes in human endometrial T-Bet and GATA-3 mRNA and protein expression during the normal menstrual cycle were characterized. Human endometrium from each phase of the menstrual cycle underwent real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry to examine expression and localization. T-Bet and GATA-3 mRNA were increased in the late secretory phase. Progesterone receptor (PR) mRNA was increased during the proliferative and early secretory phases. T-Bet and GATA-3 proteins localized cytoplasmically in the late secretory phase. PR protein displayed nuclear localization and maximal immunostaining during the early secretory phase. T-Bet and GATA-3 are expressed in endometrial epithelium cyclically during the menstrual cycle. T-Bet and GATA-3 are both upregulated during the late secretory phase and in the same cell types. The expression patterns of T-Bet and GATA-3 oppose PR, suggesting antagonistic function and/or regulation between PR and T-Bet/GATA-3.

Endometrial expression of Cyr61: a marker of estrogenic activity in normal and abnormal endometrium.

OBJECTIVE: To compare the expression of Cyr61 in normal cycling endometrium with endometrium from women with polycystic ovarian syndrome (PCOS) and endometrial hyperplasia and adenocarcinoma. METHODS: This is a retrospective study of 59 samples of normal and abnormal endometrium. Endometrial biopsies were obtained from normal fertile controls throughout the menstrual cycle and compared with endometrium from ovulatory and anovulatory women with PCOS and complex endometrial hyperplasia and endometrioid adenocarcinoma. Cyr61 expression was evaluated by using immunohistochemistry and reverse transcription PCR for Cyr61, estrogen receptor (ER)-alpha, a marker of cell proliferation (Ki67), and another marker of early estrogen action, cFos. Regulation of Cyr61 protein was studied in a steroid-responsive endometrial carcinoma cell line, ECC1. RESULTS: Cyr61 protein was regulated by estrogen. In normal endometrium, Cyr61 was highest in the proliferative phase and lowest in the normal midsecretory phase. In contrast, elevated levels of Cyr61, ER-alpha, Ki67, and cFos were all found in the midsecretory endometrium of ovulatory PCOS patients, endometrial cancer patients, and hyperplasia patients. CONCLUSION: Cyr61 is overexpressed in PCOS endometrium, reflecting a heightened responsiveness to estrogen. As a unique marker of estrogen action, Cyr61 may be an early biomarker for the development of hyperplasia or adenocarcinoma in this group of women.

Estrogen receptor-alpha (ER-alpha) and defects in uterine receptivity in women.

Endometriosis is a disorder that affects 5% of the normal population but is present in up to 40% of women with pelvic pain and/or infertility. Recent evidence suggests that the endometrium of women with endometriosis exhibits progesterone insensitivity. One endometrial protein that fluctuates in response to progesterone is the estrogen receptor-alpha (ER alpha), being down-regulated at the time of peak progesterone secretion during the window of implantation. Here we demonstrate that the biomarker of uterine receptivity, beta 3 integrin subunit, is reduced or absent in some women with endometriosis and that such defects are accompanied by inappropriate over-expression of ER alpha during the mid-secretory phase. Using a well-differentiated endometrial cell line we showed that the beta 3 integrin protein is negatively regulated by estrogen and positively regulated by epidermal growth factor (EGF). By competing against estrogen with various selective estrogen receptor modulators (SERMs) and estrogen receptor agonists and antagonists, inhibition of expression of the beta 3 integrin by estrogen can be mitigated. In conclusion, we hypothesize that certain types of uterine receptivity defects may be caused by the loss of appropriate ER alpha down-regulation in the mid-secretory phase, leading to defects in uterine receptivity. Such changes might be effectively treated by timely administration of the appropriate anti-estrogens to artificially block ER alpha and restore normal patterns of gene expression. Such treatments will require further clinical studies.

Invasive carcinomas of the male breast: a morphologic study of the distribution of histologic subtypes and metastatic patterns in 778 cases.

The current investigation was conducted to evaluate the proportional distribution of the various histologic subtypes (including newly recognized variants) of male breast carcinomas, to determine whether any histologic subtypes occur with a frequency that is markedly discordant with the expected frequencies from published data on parallel female breast tumors. We also aimed to document the distribution of malignancies metastatic to the breast. Seven hundred fifty-nine archived cases of primary invasive carcinoma involving the male breast were retrieved and sub-categorized into histologic subtypes according to contemporary criteria. Six hundred forty-three (84.7%) tumors were pure infiltrating ductal carcinoma (IDC) not otherwise specified. The most common of the remainder included papillary carcinoma with invasion in the form of IDC (n = 34), mixed IDC and mucinous carcinoma (n = 26), and pure mucinous carcinoma (n = 21). In 19 cases, metastases from other sites involved the breast, most commonly (58%) cutaneous melanoma. Invasive carcinoma of the male breast appears to display a morphologic spectrum and distribution of histologic subtypes that is comparable to those of the female breast, with some expected variation. Compared with published experience on their female counterparts, there is a two-fold increase in the frequency of invasive papillary carcinoma in the male breast. Finally, the most common tumor metastatic to the male breast in this series was cutaneous melanoma.

EGFR expression and HER2/neu overexpression/amplification in endometrial carcinosarcoma.

OBJECTIVE: Endometrial carcinosarcomas are aggressive biphasic neoplasms traditionally treated as a high-grade uterine sarcoma. Epidermal growth factor receptor (EGFR) and HER2/neu (HER2) tyrosine kinases have been implicated in the development and progression of several human cancers and are targets for therapeutic intervention. The aim of this study was to evaluate for HER2 and EGFR expression in cases of endometrial carcinosarcoma. METHODS: Formalin-fixed, paraffin-embedded sections from 55 cases of confirmed endometrial carcinosarcoma were immunostained with commercially available antibodies to EGFR and HER2. Fluorescent in situ hybridization for HER2 gene amplification was performed on all cases showing 2+ or 3+ HER2 staining by immunohistochemistry. HER2 gene amplification and EGFR expression were correlated with several prognostic variables. RESULTS: EGFR expression was identified in the majority of tumors (45/55, 82%). HER2 overexpression (3+) was seen in 14/55 (25%) cases and HER2 gene amplification was seen in 11 (20%) cases. EGFR expression and HER2 gene amplification did not show significant correlation with disease progression, disease-free survival or overall survival. The carcinomatous component of tumors more frequently showed HER2 overexpression as compared to the sarcomatous component (25% vs. 4%, P = 0.008). The sarcomatous component of tumors more frequently showed EGFR overexpression as compared to the carcinomatous component (44% vs. 24%, P = 0.04). CONCLUSIONS: EGFR and HER2 appear to play a role in the carcinogenesis of endometrial carcinosarcomas. The carcinomatous and sarcomatous elements of these tumors showed consistent differences in HER2 and EGFR expression patterns supporting biologic differences between these components. Studies evaluating the clinical utility of HER2 or EGFR targeted therapy in these tumors appear warranted.

Morphologically similar epithelial and stromal cells in primary bilateral breast tumors display different genetic profiles: implications for treatment.

The morphologic features of primary bilateral breast carcinoma have been well elucidated, but it is not known whether tumors at two sides share a common genetic profile and undergo the same clinical course. To address this issue, morphologically comparable epithelial and stromal cells in 18 paired primary bilateral breast tumors were microdissected and subjected to comparisons for the frequency and pattern of loss of heterozygosity (LOH) and microsatellite instability (MI), as well as the profiles of comparative genomic hybridization. Of 18 paired bilateral epithelial samples assessed with 10 DNA markers at five chromosomes, 78 altered loci were found; of these, 23 (29.5%) displayed concurrent and 55 (70.5%) showed independent LOH, MI, or both. Of 18 paired bilateral stromal samples assessed with the same markers, 70 altered loci were seen; of these, 9 (12.9%) displayed concurrent and 61 (87.1%) showed independent LOH, MI, or both. Collectively, all the markers and 30 (83.3%) of 36 paired bilateral epithelial and stromal cells displayed significantly more (P < 0.01) independent than concurrent LOH, MI, or both. In contrast, the epithelial cells of a pulmonary small cell carcinoma metastasized to both breasts displayed concurrent LOH at each of the four altered loci. Of seven selected cases for comparative genomic hybridization, six (86%) displayed chromosomal changes, but none showed an identical pattern and frequency of changes in both breasts. The significantly higher rate of independent genetic alterations in morphologically comparable cells of paired bilateral primary breast tumors supports the notion that the development and clinical course of tumors in two sides differ substantially; consequently, different interventions might be needed for the optimal management of bilateral breast tumors.

Focal adhesion kinase overexpression in endometrial neoplasia.

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is a critical mediator of signaling events between cells and their extracellular matrix. Elevations in FAK mRNA and protein overexpression have been linked to tumor cell capacity for invasion and metastasis. FAK expression has been shown to be elevated in a variety of solid tumors. The purpose of this study was to evaluate for FAK upregulation in endometrial neoplasia. Tissue microarray blocks were made from formalin-fixed, paraffin-embedded archival tissue, including 115 carcinoma (100 endometrioid, 10 serous, and 5 clear cell), 28 hyperplasia, and 38 normal specimens using 1-mm punches. The tissue was immunostained with monoclonal antibody for FAK and p53. Immunoreactivity was scored by intensity (0-4+ scale) and percent positive staining. FAK overexpression was categorized as 4+ cytoplasmic intensity in more than 90% of neoplastic cells. Positive p53 was categorized at least 2+ nuclear intensity in more than 10% of neoplastic cells. Higher rates of FAK upregulation were identified in endometrial hyperplasia (P = 0.025) and carcinoma (P < 0.001) versus normal endometrium. FAK overexpression in carcinoma correlated with higher FIGO grade (P = 0.025) and p53 overexpression (P < 0.001). FAK was consistently overexpressed in high-grade tumors regardless of subtype, including 8 of 10 serous tumors, 4 of 5 clear cell tumors, and 16 of 23 grade 3 endometrioid tumors. In conclusion, upregulation of FAK is seen in both endometrial hyperplasia and carcinoma, implying that FAK may play an important role in endometrial carcinogenesis. FAK overexpression in endometrial carcinoma correlates with higher FIGO grade and p53 overexpression.

Hypoxia and differentiation in squamous cell carcinomas of the uterine cervix: pimonidazole and involucrin.

PURPOSE: Pimonidazole binding (hypoxia) and involucrin expression (differentiation) overlap extensively in squamous cell carcinomas. This study asks whether involucrin might serve as an endogenous marker for tumor hypoxia. A second question is whether differentiation affects hypoxia-inducible metallothionein (MT) expression in normal human epithelia and squamous cell carcinomas as it does in rodent epithelia. EXPERIMENTAL DESIGN: Thirty-four patients with squamous cell carcinoma of the uterine cervix were infused with pimonidazole hydrochloride solution. The next day, multiple biopsies were formalin-fixed, paraffin-embedded and sectioned at 4 micro m. Qualitative and quantitative analyses for involucrin expression, pimonidazole binding, and human MT-IIa mRNA expression were performed. RESULTS: No overall correlation between the extent of involucrin expression and pimonidazole binding was observed. The lack of correlation was because of heterogeneous patterns of immunostaining for involucrin generally related to tumor grade. Colocalized immunostaining for involucrin and pimonidazole binding was observed in intermediate grade tumors but not in well-differentiated or poorly differentiated tumors. Human MT-IIa mRNA and MT protein were expressed in basal lamina of normal human epithelia and in the proliferative rims of tumor nests. CONCLUSIONS: Colocalization of immunostaining for involucrin and pimonidazole binding is consistent with oxygen regulation, but the lack of involucrin expression in hypoxic regions of poorly differentiated tumors indicates that its transcriptional status with respect to hypoxia induction is altered by cell differentiation. The localization of MT message and protein in the outer rims of most tumor nests indicates that the transcriptional status of metallothionein is also altered by differentiation.

Papillary endothelial hyperplasia of the breast: the great impostor for angiosarcoma: a clinicopathologic review of 17 cases.

Seventeen cases of papillary endothelial hyperplasia (PEH, Masson's vegetant intravascular hemangioendothelioma) involving breast or mammary subcutaneous tissues are described. The mean patient age was 59; 14 (82%) were female and 12 (71%) presented with a mass. Nine women had mammographic evaluation, 3 of whom had microcalcifications. Five neoplasms were discovered by routine mammography. Sixteen cases were 2.7 cm or less in greatest dimension, and 8 (47%) were associated with a thrombus and/or cavernous hemangioma. Follow-up in 10 cases (up to nearly 8 years) showed no recurrences. Fifty-nine percent of the cases were received at AFIP for consultation with a working diagnosis of angiosarcoma. Features that help distinguish PEH from angiosarcoma include circumscription of the lesion, location in a vessel or association with thrombus, and papillary architecture without significant cytologic atypia or areas of solid growth. The recognition of the morphologic features of this lesion and its inclusion in the differential diagnosis of vascular mammary tumors will reduce the likelihood of its misdiagnosis as an angiosarcoma and avoid unnecessary and aggressive therapy.

Steroid receptor coactivator expression throughout the menstrual cycle in normal and abnormal endometrium.

The endometrium of reproductive aged women undergoes cyclic developmental changes in preparation for implantation in response to estrogen and progesterone. These steroids and their receptors are tightly regulated throughout the menstrual cycle, and their actions are facilitated by the presence of steroid receptor coactivators of the p160 family. In this study using immunohistochemistry and Western blot analysis, we characterize the expression patterns of three coactivators, steroid receptor coactivator-1, amplified in breast cancer-1 (AIB1), and transcriptional intermediary factor-2 in human endometrium obtained prospectively from normal fertile women throughout the menstrual cycle. With the exception of glandular AIB1, which increased in the late secretory phase, none of the coactivators changed significantly during the menstrual cycle. We compared coactivator expression patterns in fertile endometrium to the endometrium of anovulatory (proliferative; n = 3) and clomiphene-induced ovulatory (secretory; n = 13) women with polycystic ovarian syndrome (PCOS), a group that have a higher likelihood of developing estrogen-induced endometrial hyperplasia and cancer. To control for the effect of clomiphene citrate, an additional group was included consisting of ovulatory women treated with clomiphene citrate for "male factor" infertility. Compared with both fertile and infertile controls, PCOS women exhibited elevated levels of AIB1 and transcriptional intermediary factor-2 expression in both epithelial and stromal cells. We postulate that increased coactivator expression may render the endometrium more sensitive to estrogen. In support of this, we describe an increased expression of ERalpha (an estrogen-induced gene product) during the menstrual cycle in PCOS endometrium compared with fertile controls. In summary, we demonstrate that the expression of p160 coactivators are regulated in endometrium during the menstrual cycle in normal fertile women but are overexpressed in the endometrium of women with PCOS. Based on these findings, we suggest a possible mechanism to explain the poor reproductive performance observed in PCOS and the increased incidence of endometrial hyperplasia and cancer noted in this group of women.

Androgen and estrogen receptor mRNA status in apocrine carcinomas.

Eleven pure apocrine carcinomas (8 breast, 2 vulvar, 1 axillary) were evaluated for the presence of androgen and estrogen receptor mRNA. These immunohistochemically androgen receptor positive and estrogen receptor negative cases were microdissected and analyzed by the reverse transcription polymerase chain reaction for the presence of receptor message. Nine of these 11 estrogen receptor (ER) negative lesions were found to contain the ER mRNA through the first intron splice region. In 4 of the 11 cases, the androgen receptor (AR) mRNA could not be shown even though the protein was detected immunohistochemically. In the other seven, AR mRNA was identified. This indicates that the mechanism for production of the estrogen receptor is intact and functional in most cases through the first transcriptional splice region. Therefore, the immunohistochemical absence of the estrogen receptor in apocrine cells cannot be explained by an abnormal message at these common sites and should be sought beyond these points.

Elevated endometrial androgen receptor expression in women with polycystic ovarian syndrome.

Androgen receptors (AR) have been identified in human endometrium; however, their role in endometrial cyclic development and function remains poorly understood. The objective of the present study was to investigate the profile of endometrial AR in normal menstrual cycles and in the endometrium of women with polycystic ovarian syndrome (PCOS). This syndrome is characterized by chronic hyperandrogenism and oligo-ovulation, and it is often associated with poor reproductive performance. Using immunohistochemistry and reverse transcription-polymerase chain reaction, we found that women with PCOS exhibited elevated endometrial AR expression compared to normal, fertile controls. This increase was most apparent in glandular and luminal epithelium. Furthermore, when compared to endometrium from fertile women, PCOS endometrium showed other abnormalities in endometrial development, including delay or absence of the alpha(v)beta3 integrin, a well-characterized biomarker of uterine receptivity described previously (Lessey et al., JCI 1992; 90:188-195). To better understand and to gain insights regarding these findings, we used in vitro cell-culture models to study the regulation of AR in primary endometrial stromal and the well-differentiated epithelial cell line (Ishikawa). Based on Western blot analysis, epithelial AR is up-regulated by estrogens and androgens and is inhibited by progestins and epidermal growth factor (EGF). On the other hand, EGF significantly induced the expression of alpha(v)beta3, whereas estrogen and androgen treatment inhibited its expression. Collectively, these results suggest that the poor reproductive performance observed in women with PCOS may be due, in part, to the concomitant increase in both serum androgens and elevations in endometrial AR. This combination may reduce endometrial receptivity as judged by the down-regulation of alpha(v)beta3 integrin.