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miRNAs are noncoding RNAs with abnormal expression in breast cancer; their expression in high-risk benign breast tissue may relate to breast cancer risk. We examined miRNA profiles in contralateral unaffected breasts (CUB) of patients with breast cancer and validated resulting candidates in two additional sample sets. Expression profiles of 754 mature miRNAs were examined using TaqMan Low Density Arrays in 30 breast cancer samples [15 estrogen receptor (ER)-positive and 15 ER-negative] and paired CUBs and 15 reduction mammoplasty controls. Pairwise comparisons identified miRNAs with significantly differential expression. Seven candidate miRNAs were examined using qRT-PCR in a second CUB sample set (40 cases, 20 ER+, 20 ER−) and 20 reduction mammoplasty controls. Further validation was performed in 80 benign breast biopsy (BBB) samples; 40 from cases who subsequently developed breast cancer and 40 from controls who did not. Logistic regression, using tertiles of miRNA expression, was used to discriminate cases from controls. Seven miRNAs were differentially expressed in tumors and CUBs versus reduction mammoplasty samples. Among them, miR-18a and miR-210 were validated in the second CUB set, showing significantly higher expression in tumor and CUBs than in reduction mammoplasty controls. The expression of miR-18a and miR-210 was also significantly higher in BBB cases than in BBB controls. When both miR-18a and miR-210 were expressed in the upper tertiles in BBB, OR for subsequent cancer was 3.20, P = 0.023. miR-18a and miR-210 are expressed at higher levels in CUBs of patients with breast cancer, and in BBB prior to cancer development, and are therefore candidate breast cancer risk biomarkers. Cancer Prev Res; 10(1); 89–97. ©2016 AACR.

Supplemental Figure 1. Validation of miR-214*, miR-124, miR-193a-3p, miR-485a-3p and miR-671-3p in independent sample set of tumor (ER+T and ER-T) and matching contralateral breast (ER+C, ER-C) compared to reduction mammoplasty controls. Supplemental Figure 2. Expression of ER in (A) CUB validation samples and (B) BBB samples. (A) There is no significant difference between ER+ CUB (ER+C) or ER- CUB (ER-C) and controls. Only the tumor samples (ER+T, ER-T) showed the significant difference vs controls. *** P

Supplemental Figure 5. Ki67 Change was not associated with drug concentration in Tumor samples. Tumor samples were available for quantitation from 22 women.

Supplemental Figure 2. Ki67 Change relative to treatment duration. There was no significant correlation between change in Ki67 and treatment duration, in either TPA or placebo arms.

Supplemental Figure 4. Plots showing changes in serum estradiol (left panel) and serum progesterone (right panel) in three subgroups: placebo, TPA-treated Ki67 responders and TPA-treated Ki67-nonresponders.

Supplemental Figure 3. Validation of RNA seq results of responders by Nanostring nCounter gene expression assay

Supplemental table 2. Differential gene expression and pathway enrichment analysis in placebo responders (excel file) -uploading separately.

Supplemental table 1. Differential gene expression and pathway enrichment analysis in TPA responders (excel file) -uploading separately.

Supplemental table 3. Nanostring nCounter Differential gene expression (excel file) -uploading separately.

Purpose:Selective progesterone receptor modulators (SPRMs) show preclinical activity against hormone-sensitive breast cancer, but have not been tested in patients with early, treatment-naïve tumors.Patients and Methods:In a double-blind presurgical window trial of oral telapristone acetate (TPA) 12 mg daily versus placebo, 70 patients with early-stage breast cancer were randomized 1:1 (stratified by menopause) and treated for 2 to 10 weeks. The primary endpoint was change in Ki67 between diagnostic biopsy and surgical specimens. Gene expression pre- and posttherapy was assessed using RNA-sequencing and gene set enrichment analysis was performed to determine pathways enriched in response to TPA and placebo treatments.Results:Among 61 evaluable women (29 placebo and 32 telapristone acetate), 91% of tumors were ER/PR positive. The mean Ki67 declined by 5.5% in all women treated with telapristone acetate (P = 0.003), and by 4.2% in all women treated with placebo (P = 0.04). After menopausal stratification, the Ki67 decline remained significant in 22 telapristone acetate–treated premenopausal women (P = 0.03). Differential gene expression analysis showed no significant modulation overall. However, in a subset of tumors that demonstrated ≥30% relative reduction in Ki67 in the telapristone acetate group, genes related to cell-cycle progression, and those in the HER2 amplicon were significantly downregulated. In contrast, no significantly enriched pathways were identified in the placebo group.Conclusions:Patients treated with telapristone acetate whose Ki67 decreased by ≥30% demonstrated a selective antiproliferative signal, with a potentially important effect on HER2 amplicon genes. Evaluation of SPRMs in a neoadjuvant trial is merited, with attention to predictors of response to SPRM therapy, and inclusion of pre- and postmenopausal women.

Supplemental Materials Supplemental Table 4. Changes in serum sex hormone concentrations according to the treatments. Supplemental Table 5. Summary of BESS Quality of Life Assessment by symptom clusters according to the treatments Supplemental Table 6. Summary of Adverse Effects.

Supplemental Figure 1. Validation of RNA seq results of responders by Nanostring nCounter gene expression assay.

Purpose: Selective progesterone receptor modulators (SPRMs) show preclinical activity against hormone-sensitive breast cancer, but have not been tested in patients with early, treatment-naïve tumors. Patients and Methods: In a double-blind presurgical window trial of oral telapristone acetate (TPA) 12 mg daily versus placebo, 70 patients with early-stage breast cancer were randomized 1:1 (stratified by menopause) and treated for 2 to 10 weeks. The primary endpoint was change in Ki67 between diagnostic biopsy and surgical specimens. Gene expression pre- and posttherapy was assessed using RNA-sequencing and gene set enrichment analysis was performed to determine pathways enriched in response to TPA and placebo treatments. Results: Among 61 evaluable women (29 placebo and 32 telapristone acetate), 91% of tumors were ER/PR positive. The mean Ki67 declined by 5.5% in all women treated with telapristone acetate (P = 0.003), and by 4.2% in all women treated with placebo (P = 0.04). After menopausal stratification, the Ki67 decline remained significant in 22 telapristone acetate–treated premenopausal women (P = 0.03). Differential gene expression analysis showed no significant modulation overall. However, in a subset of tumors that demonstrated ≥30% relative reduction in Ki67 in the telapristone acetate group, genes related to cell-cycle progression, and those in the HER2 amplicon were significantly downregulated. In contrast, no significantly enriched pathways were identified in the placebo group. Conclusions: Patients treated with telapristone acetate whose Ki67 decreased by ≥30% demonstrated a selective antiproliferative signal, with a potentially important effect on HER2 amplicon genes. Evaluation of SPRMs in a neoadjuvant trial is merited, with attention to predictors of response to SPRM therapy, and inclusion of pre- and postmenopausal women.

In an effort to develop a new therapy for cancer and to improve antiprogrammed death inhibitor-1 (anti-PD-1) and anticytotoxic T lymphocyte-associated protein (anti-CTLA-4) responses, we have created a telomerase reverse transcriptase promoter-regulated oncolytic adenovirus rAd.sT containing a soluble transforming growth factor receptor II fused with human IgG Fc fragment (sTGFβRIIFc) gene. Infection of breast and renal tumor cells with rAd.sT produced sTGFβRIIFc protein with dose-dependent cytotoxicity. In immunocompetent mouse 4T1 breast tumor model, intratumoral delivery of rAd.sT inhibited both tumor growth and lung metastases. rAd.sT downregulated the expression of several transforming growth factor β (TGFβ) target genes involved in tumor growth and metastases, inhibited Th2 cytokine expression, and induced Th1 cytokines and chemokines, and granzyme B and perforin expression. rAd.sT treatment also increased the percentage of CD8(+) T lymphocytes, promoted the generation of CD4(+) T memory cells, reduced regulatory T lymphocytes (Tregs), and reduced bone marrow-derived suppressor cells. Importantly, rAd.sT treatment increased the percentage of CD4(+) T lymphocytes, and promoted differentiation and maturation of antigen-presenting dendritic cells in the spleen. In the immunocompetent mouse Renca renal tumor model, similar therapeutic effects and immune activation results were observed. In the 4T1 mammary tumor model, rAd.sT improved the inhibition of tumor growth and lung and liver metastases by anti-PD-1 and anti-CTLA-4 antibodies. Analysis of the human breast and kidney tumors showed that a significant number of tumor tissues expressed high levels of TGFβ and TGFβ-inducible genes. Therefore, rAd.sT could be a potential enhancer of anti-PD-1 and anti-CTLA-4 therapy for treating breast and kidney cancers.

We report here the development of oncolytic adenoviruses (Ads) that have reduced toxicity, enhanced tumor tropism, produce strong antitumor response, and can overcome resistance to immune checkpoint inhibitor therapy in breast cancer. We have shown that LyP-1 receptor (p32) is highly expressed on the surface of breast cancer cells and tumors from cancer patients, and that increased stromal expression of transforming growth factor β-1 (TGFβ-1) is associated with triple-negative breast cancer. Therefore, we constructed oncolytic Ads, AdLyp.sT and mHAdLyp.sT, in which the p32-binding LyP-1 peptide was genetically inserted into the adenoviral fiber protein. Both AdLyp.sT and mHAdLyp.sT express sTGFβRIIFc, a TGFβ decoy that can inhibit TGFβ pathways. mHAdLyp.sT is an Ad5/48 chimeric hexon virus in which hypervariable regions (HVRs 1-7) of Ad5 are replaced with the corresponding Ad48 HVRs. AdLyp.sT and mHAdLyp.sT exhibited better binding, replication, and produced higher sTGFβRIIFc protein levels in breast cancer cell lines compared with Ad.sT or mHAd.sT control viruses without LyP-1 peptide modification. Systemic delivery of mHAdLyp.sT in mice resulted in reduced hepatic/systemic toxicity compared with Ad.sT and AdLyp.sT. Intravenous delivery of AdLyp.sT and mHAdLyp.sT elicited a strong antitumor response in a human MDA-MB-231 bone metastasis model in mice, as indicated by bioluminescence imaging, radiographic tumor burden, serum TRACP 5b and calcium, and body weight analyses. Furthermore, intratumoral delivery of AdLyp.sT in 4T1 model in immunocompetent mice inhibited tumor growth and metastases, and augmented anti-PD-1 and anti-CTLA-4 therapy. Based on these studies, we believe that AdLyp.sT and mHAdLyp.sT can be developed as potential targeted immunotherapy agents for the treatment of breast cancer.

Approximately 0.5–1.4% of natal males and 0.2–0.3% of natal females meet DSM-5 criteria for gender dysphoria, with many of these individuals self-describing as transgender men or women. Despite recent improvements both in social acceptance of transgender individuals as well as access to gender affirming therapy, progress in both areas has been hampered by poor understanding of the etiology of gender dysphoria. Prior studies have suggested a genetic contribution to gender dysphoria, but previously proposed candidate genes have not yet been verified in follow-up investigation. In this study, we expand on the topic of gender identity genomics by identifying rare variants in genes associated with sexually dimorphic brain development and exploring how they could contribute to gender dysphoria. To accomplish this, we performed whole exome sequencing on the genomic DNA of 13 transgender males and 17 transgender females. Whole exome sequencing revealed 120,582 genetic variants. After filtering, 441 variants in 421 genes remained for further consideration, including 21 nonsense, 28 frameshift, 13 splice-region, and 225 missense variants. Of these, 21 variants in 19 genes were found to have associations with previously described estrogen receptor activated pathways of sexually dimorphic brain development. These variants were confirmed by Sanger Sequencing. Our findings suggest a new avenue for investigation of genes involved in estrogen signaling pathways related to sexually dimorphic brain development and their relationship to gender dysphoria.

Background Estrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities. Methods To better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology. Results Phospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate. Conclusions We conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0462-7) contains supplementary material, which is available to authorized users.

Risk biomarkers for estrogen receptor (ER)-negative breast cancer have clear value for breast cancer prevention. We previously reported a set of lipid metabolism (LiMe) genes with high expression in the contralateral unaffected breasts (CUBs) of ER-negative cancer cases. We now further examine LiMe gene expression in both tumor and CUB, and investigate the role of Pre-B-cell leukemia homeobox-1 (PBX1) as a candidate common transcription factor for LiMe gene expression. mRNA was extracted from laser-capture microdissected epithelium from tumor and CUB of 84 subjects (28 ER-positive cases, 28 ER-negative cases, 28 healthy controls). Gene expression was quantitated by qRT-PCR. Logistic regression models were generated to predict ER status of the contralateral cancer. Protein expression of HMGCS2 and PBX1 was measured using immunohistochemistry. The effect of PBX1 on LiMe gene expression was examined by overexpressing PBX1 in MCF10A cells with or without ER, and by suppressing PBX1 in MDA-MB-453 cells. The expression of DHRS2, HMGCS2, UGT2B7, UGT2B11, ALOX15B, HPGD, UGT2B28 and GLYATL1 was significantly higher in ER-negative versus ER-positive CUBs, and predicted ER status of the tumor in test and validation sets. In contrast, LiMe gene expression was significantly lower in ER-negative than ER-positive tumors. PBX1 overexpression in MCF10A cells up-regulated most LiMe genes, but not in MCF10A cells overexpressing ER. Suppressing PBX1 in MDA-MB-453 cells resulted in decrease of LiMe gene expression. Four binding sites of PBX1 and cofactor were identified in three lipid metabolism genes using ChIP-qPCR. These data suggest a novel role for PBX1 in the regulation of lipid metabolism genes in benign breast, which may contribute to ER-negative tumorigenesis.

Purpose: miRNAs are non-coding RNAs that are abnormally expressed in breast cancer, with critical roles in cancer due to their regulation of large gene networks. miRNA expression in benign high-risk breast tissue has never been evaluated, but it may provide information about early dysregulation events that contribute to breast cancer risk. The contralateral unaffected breast (CUB) of women with unilateral breast cancer is in high-risk for the second primary cancer. Thus, we examined miRNA expression profiles in tumor and the matching CUBs to seek potential miRNA biomarkers for breast cancer risk. Methods: FFPE tissues of breast cancer and their matching CUB tissues were sectioned. The areas of tumor and normal ductal epithelium were outlined and then dissected using laser microdissection system. Total RNA was extracted for miRNA profiling studies. Expression profiles of 754 mature miRNAs were examined using TaqMan Low Density Arrays assays in 30 paired breast cancer and CUB samples (15 with ER+ tumors, 15 with ER- tumors) and 15 reduction mammoplasty (RM) controls, matched by age, race and menopausal status. ANOVA test was performed to examine the differential expression among groups and pairwise comparison with Sidak adjustment was used for multiple comparison. Seven candidate miRNAs were then examined in an independent CUB sample set (20 with ER+ tumors, 20 with ER- tumors) and 20 RM controls. Further independent validation was performed using qRT-PCR in 80 benign breast biopsy (BBB) samples: 40 from women who subsequently developed breast cancer (cases) and 40 from those who did not (controls). Logistic regression analysis and receiver operating characteristic (ROC) analysis were performed using combinations of the expression of multiple miRNAs to establish models discriminating cases from controls. Results: Seven miRNAs (miR-18a, miR-210, miR-214*, miR-124, miR-193a-3p, miR485-3p and miR-671-3p) were found to be differentially expressed in breast cancer and CUB samples vs. RM samples in the discovery sample set. Among them, miR-18a and miR-210 were validated in a second, independent CUB sample set. The expression of miR-18a and miR-210 were significantly higher in tumor (regardless ER status) compared to CUBs and RM controls. The expression levels in CUBs were significantly higher than in RM. We then examined miR expression in case BBB samples, and confirmed that expression of miR-18a and miR-210 were increased compared with controls. ROC analysis using miR-18a and miR-210 discriminated high-risk cases from standard-risk controls with OR 2.44, P = 0.022. Conclusion: The expression of miR-18a and miR-210 were elevated in breast cancer, in matched CUBs, and in BBB predating cancer diagnosis. These data provide strong support for the hypothesis that miR-18a and miR-210 expression in BBB is an indicator of increased risk of breast cancer. Given the high expression in tumors, they are also potential cancer detection biomarkers. Citation Format: Wang J, Shidfar A, Costa FF, Scholtens D, Bischof JM, Sullivan ME, Ivancic D, Soares MB, Khan SA. Expression of miR-18a and miR-210 in normal breast tissue as candidate markers of breast cancer risk [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-07-02.

PurposeAlthough survival rates for patients with localized breast cancer have increased, patients with metastatic breast cancer still have poor prognosis. Understanding key factors involved in promoting breast cancer metastasis is imperative for better treatments. In this study, we investigated the role of syndecan-1 (Sdc1) in breast cancer metastasis.MethodsTo assess the role of Sdc1 in breast cancer metastasis, we silenced Sdc1 expression in the triple-negative breast cancer human MDA-MB-231 cell line and overexpressed it in the mouse mammary carcinoma 4T1 cell line. Intracardiac injections were performed in an experimental mouse metastasis model using both cell lines. In vitro transwell blood-brain barrier (BBB) and brain section adhesion assays were utilized to specifically investigate how Sdc1 promotes brain metastasis. A cytokine array was performed to evaluate differences in the breast cancer cell secretome when Sdc1 was silenced.ResultsSilencing expression of Sdc1 in breast cancer cells significantly reduced metastasis to the brain. Conversely, overexpression of Sdc1 increased metastasis to the brain. We found that the reduction in brain metastases with Sdc1 knockdown was likely due to reduced breast cancer cell migration across the BBB and adhesion to the perivascular regions of the brain. However, there was no change in attachment to brain endothelial cells or astrocytes. Loss of Sdc1 also led to changes in breast cancer cell-secreted cytokines, which may influence the BBB.ConclusionsTaken together, our study demonstrates a role for Sdc1 in promoting breast cancer metastasis to the brain. These findings suggest that Sdc1 supports breast cancer cell migration across the BBB through regulation of cytokines, which may modulate the BBB. Further elucidating this mechanism will allow for the development of therapeutic strategies to combat brain metastasis.

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040 . 1

Women at high risk of breast cancer and those with carcinoma in situ need non-toxic, well-tolerated preventive interventions. One promising approach is drug delivery through the breast skin (local transdermal therapy, LTT). Our goal was to test novel drugs for LTT, to establish that LTT is applicable to non-steroidal drugs. Athymic nude rats were treated with oral tamoxifen, transdermal 4-hydroxytamoxifen (4-OHT) or endoxifen gel applied daily to the axillary mammary gland for 6 weeks (Study 1). Study 2 was identical to Study 1, testing transdermal telapristone acetate (telapristone) gel versus subcutaneous implant. At euthanasia, mammary glands and blood were collected. In Study 3, consenting women requiring mastectomy were randomized to diclofenac patch applied to the abdomen or the breast for 3 days preoperatively. At surgery, eight tissue samples per breast were collected from predetermined locations, along with venous blood. Drug concentrations were measured using liquid chromatography–tandem mass spectroscopy. Mammary tissue concentrations of 4-OHT, endoxifen, and telapristone were significantly higher in the axillary glands of the gel-treated animals, compared to inguinal glands or to systemically treated animals. Plasma concentrations were similar in gel and systemically treated animals. The clinical trial showed significantly higher mammary concentrations when diclofenac was applied to the breast skin versus the abdominal skin, but concentrations were variable. These results demonstrate that lipophilic drugs can be developed for LTT; although the nude rat is suitable for testing drug permeability, delivery is systemic. In human, however, transdermal application to the breast skin provides local delivery.

Background: The identification of women at risk for ER- cancer would allow optimization of breast cancer prevention strategies by guiding their recruitment to studies of agents with efficacy against ER- cancer and sparing them the toxicity of prevention agents effective only against ER+ cancer. In our previous studies, we identified lipid metabolism (LiMe) gene set in rFNA samples from contralateral unaffected breast (CUB) that was associated with tumor ER status. In the current study, we further validate LiMe gene expression in tumor and CUB at the mRNA and protein levels. Methods: Tissue samples from 56 bilateral mastectomy cases (28 ER+ and 28 ER-) and 28 healthy reduction mammoplasty (RM) controls were used. The ER+ cases, ER- cases and controls were matched by age, race and menopausal status. We performed laser capture microdissection of epithelial cells in fresh frozen tissues from tumor and unaffected breast. Total RNA was extracted and LiMe genes were detected using Taqman low density gene expression arrays. The difference among groups was analyzed using ANOVA with Sidak multiple comparison adjustment. Three proteins (HMGCS2, ACSL3 and HPGD) were detected in FFPE sections of tumor and CUB tissues using immunohistochemistry. Results: Among the 13 LiMe genes, 6 genes (DHRS2, HMGCS2, UGT2B7, UGT2B11, UGT2B28 and GLYATL1) were significantly higher in CUB of ER- cases compared to CUB of ER+ cases (2.2-2.9 fold, P Conclusion: Differential expression of the LiMe genes in the CUB is associated with ER- index tumors and may characterize the environment leading to the development of ER- breast cancer. The converse patterns in tumor and CUB by ER status suggest that LiMe genes may be regulated by different mechanisms in benign and malignant tissues. These genes are potential risk biomarkers of ER- breast cancer and generate novel etiologic hypotheses regarding the development of ER- versus ER+ disease. GeneER-C vs ER+CER-T vs ER+TER-C vs RMER+C vs RMDHRS22.4 (0.034)*0.16 (0.042)*6.0 (0.014)*1.3HMGCS22.9 (0.050)*0.15 (0.0095)*6.5 (0.006)*2.2UGT2B72.2 (0.004)*0.771.81.0UGT2B112.4 (0.019)*0.11 (0.0068)*9.6 (0.013)*2.0UGT2B282.3 (0.009)*0.15 (0.018)*2.8 (0.029)*1.5GLYATL12.9 (0.010)*0.374.3 (0.026)*1.4GSTT21.10.37 (0.040)*1.51.3ALOX15B1.60.562.5 (0.016)*1.5SERHL1.80.294.9 (0.0047)*1.4ER-C: ER- CUB; ER+C: ER+ CUB; ER-T: ER- tumor; ER+T: ER+ tumor; RM: reduction mammoplasty Citation Format: Ali Shidfar, David Ivancic, Megan E Sullivan, Pranjal Patankar, Seema A Khan, Jun Wang. Expression of lipid metabolism genes in tumor and contralateral unaffected breast are conversely associated with tumor estrogen receptor status [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-08-02.

The widespread use of screening mammography as well as advances in imaging techniques has resulted in increased detection of high-risk breast lesions. These same imaging advances have also opened up the discussion regarding the necessity of routine surgical excisions for all types of breast atypia. In this chapter, the microscopic appearance and diagnostic criteria for flat epithelial atypia (FEA), atypical ductal hyperplasia (ADH), and lobular neoplasia will be described, highlighting the important clinical and pathologic differences between classic and variants of lobular carcinoma in situ (LCIS). The risk of upgrade to invasive carcinoma and/or ductal carcinoma in situ (DCIS) and its impact on the current management recommendations for each category of atypia will be reviewed with an emphasis on the importance of pathologic-radiologic concordance. Finally, the impact of breast magnetic response imaging (MRI) on diagnosis and management decisions will be addressed.

Yilmaz, Elanur/0000-0001-7045-5068; Alper, Ozgul/0000-0003-1536-2111 WOS: 000424131300001 PubMed: 29434669 Background: Little is known about the genetic contribution to Mullerian aplasia, better known to patients as Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome. Mutations in two genes (WNT4 and HNF1B) account for a small number of patients, but heterozygous copy number variants (CNVs) have been described. However, the significance of these CNVs in the pathogenesis of MRKH is unknown, but suggests possible autosomal dominant inheritance. We are not aware of CNV studies in consanguineous patients, which could pinpoint genes important in autosomal recessive MRKH. We therefore utilized SNP/CGH microarrays to identify CNVs and define regions of homozygosity (ROH) in Anatolian Turkish MRKH patients. Result(s): Five different CNVs were detected in 4/19 patients (21%), one of which is a previously reported 16p11.2 deletion containing 32 genes, while four involved smaller regions each containing only one gene. Fourteen of 19 (74%) of patients had parents that were third degree relatives or closer. There were 42 regions of homozygosity shared by at least two MRKH patients which was spread throughout most chromosomes. Of interest, eight candidate genes suggested by human or animal studies (RBM8A, CMTM7, CCR4, TRIM71, CNOT10, TP63, EMX2, and CFTR) reside within these ROH. Conclusion(s): CNVs were found in about 20% of Turkish MRKH patients, and as in other studies, proof of causation is lacking. The 16p11.2 deletion seen in mixed populations is also identified in Turkish MRKH patients. Turkish MRKH patients have a higher likelihood of being consanguineous than the general Anatolian Turkish population. Although identified single gene mutations and heterozygous CNVs suggest autosomal dominant inheritance for MRKH in much of the western world, regions of homozygosity, which could contain shared mutant alleles, make it more likely that autosomal recessively inherited causes will be manifested in Turkish women with MRKH. NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [HD33004]; Department of Ob/Gyn at Augusta University; Research Funds Office of Akdeniz University, Antalya, Turkey [2012. 03.0122.001] LCL was funded by NIH HD33004 and the Department of Ob/Gyn at Augusta University and OMA was funded by the Research Funds Office of Akdeniz University, Antalya, Turkey (Grant #2012. 03.0122.001).

Objective To study the genetic cause of Mayer-Rokitansky-Kuster-Hauser syndrome (MRKH). Although a few candidate genes and genomic domains for have been reported for MRKH, the genetic underpinnings remain largely unknown. Some of the top candidate genes are WNT4 , HNF1B , and LHX1 . The goals of this study were to: 1) determine the prevalence of WNT4 , HNF1B , and LHX1 point mutations, as well as new copy number variants (CNVs) in people with MRKH; and 2) identify and characterize MRKH cohorts. Design Laboratory- and community-based study. Setting Academic medical centers. Patient(s) A total of 147 MRKH probands and available family members. Interventions(s) DNA sequencing of WNT4 , HNF1B , and LHX1 in 100 MRKH patients, chromosomal microarray analysis in 31 North American MRKH patients, and characterization and sample collection of 147 North American and Turkish MRKH probands and their families. Main Outcome Measure(s) DNA sequence variants and CNVs; pedigree structural analysis. Result(s) We report finding CNVs in 6/31 people (∼19%) with MRKH, but no point mutations or small indels in WNT4 , HNF1B , or LHX1 in 100 MRKH patients. Our MRKH families included 43 quads, 26 trios, and 30 duos. Of our MRKH probands, 87/147 (59%) had MRKH type 1 and 60/147 (41%) had type 2 with additional anomalies. Conclusion(s) Although the prevalence of WNT4 , HNF1B , and LHX1 point mutations is low in people with MRKH, the prevalence of CNVs was ∼19%. Further analysis of our large familial cohort of patients will facilitate gene discovery to better understand the complex etiology of MRKH.

Intraductal papillomas (IDPs) of the breast can be associated with a variety of clinical symptoms and radiologic findings. Surgical excision is often recommended based on the possibility of an associated high-grade lesion. Although the rate of upgrades has been extensively evaluated for IDPs, many studies are hindered by broad inclusion criteria, a lack of pathologic-radiologic concordance, and no standard definition of what constitutes an upgrade. In the current study, we evaluate the risk of upgrade for a specific subset of IDPs: non-mass-associated IDPs. We identified all breast needle core biopsies with a diagnosis of IDP between 2003 and 2010. Patients with associated masses, architectural distortion, or ipsilateral breast cancer were excluded. All needle core biopsy slides and relevant imaging studies were reviewed to ensure pathologic-radiologic concordance. Excision pathology was also reviewed; an upgrade was defined as the presence of ductal carcinoma in situ or invasive carcinoma in the excision. Seventy-nine IDPs that met inclusion criteria were identified and were further divided into 3 histologic categories: micropapilloma, fragmented IDP, and atypical IDP. Micropapillomas and fragmented IDPs had no upgrades (0/37). In patients who did not undergo excision, none subsequently developed ipsilateral breast cancer (follow-up, 50-61 months). This is in contrast to atypical IDPs that had a 33% upgrade rate. One patient with an unexcised atypical IDP developed ipsilateral breast cancer within 2 years. Our data suggest that conservative follow-up is reasonable for non-mass-associated IDPs without atypia regardless of microscopic size, provided that careful pathologic-radiologic correlation is achieved.

To evaluate the MR appearance of noncalcified ductal carcinoma in situ (DCIS), with comparison to calcified DCIS. A retrospective, IRB-approved review of all DCIS diagnosed via MR biopsy between 2007 and 2011 was performed. DCIS was categorized as noncalcified based on the absence of calcifications on mammography and specimen radiography. MR morphology (focus, mass, nonmass enhancement [NME]) and enhancement kinetics (initial and delayed) for noncalcified DCIS were recorded and compared based on nuclear grade (1-3), size (1.5 cm, 1.5-5 cm,5 cm), and presence of necrosis. Imaging features of noncalcified and calcified DCIS were also compared. 115 cases of MR biopsy-proven DCIS were identified: 65 (56%) noncalcified and 50 (44%) calcified. For noncalcified DCIS, NME morphology was more common than mass or focus (60% vs 30.8% and 9.2%). There was a significant association between morphology and enhancement kinetics, with NME more likely demonstrating medium and persistent kinetics, and foci or masses demonstrating rapid and plateau or washout kinetics (P .05). There was also a significant association between morphology and nuclear grade, with NME more likely seen with grade 3 DCIS (P = .024), and between size and initial enhancement, with lesions1.5 cm more likely to have rapid initial enhancement (P = .0036). No significant difference was identified between calcified and noncalcified DCIS in terms of morphology, enhancement characteristics, nuclear grade, or presence of necrosis. The MR appearance of noncalcified DCIS closely mirrors that of calcified DCIS. Recognizing these imaging features may allow for improved identification of this MRI-detected abnormality, even in the absence of calcifications.

The genetic basis is unknown for ∼60% of normosmic hypogonadotropic hypogonadism (nHH)/Kallmann syndrome (KS). DNAs from (17 male and 31 female) nHH/KS patients were analyzed by targeted next generation sequencing (NGS) of 261 genes involved in hypothalamic, pituitary, and/or olfactory pathways, or suggested by chromosome rearrangements. Selected variants were subjected to Sanger DNA sequencing, the gold standard. The frequency of Sanger-confirmed variants was determined using the ExAC database. Variants were classified as likely pathogenic (frameshift, nonsense, and splice site) or predicted pathogenic (nonsynonymous missense). Two novel FGFR1 mutations were identified, as were 18 new candidate genes including: AMN1, CCKBR, CRY1, CXCR4, FGF13, GAP43, GLI3, JAG1, NOS1, MASTL, NOTCH1, NRP2, PALM2, PDE3A, PLEKHA5, RD3, and TRAPPC9, and TSPAN11. Digenic and trigenic variants were found in 8/48 (16.7%) and 1/48 (2.1%) patients, respectively. NGS with confirmation by Sanger sequencing resulted in the identification of new causative FGFR1 gene mutations and suggested 18 new candidate genes in nHH/KS.

Background: In vitro and preclinical data indicate that TPA, a selective PR modulator, has activity against hormone-sensitive early breast cancer. We conducted a pre-surgical window trial of oral TPA in Stage 0-II breast cancer to assess the effect of TPA on suppression of cell proliferation (Ki67), and on differential gene expression in responsive and non-responsive tumors. Methods: We enrolled 70 pre and postmenopausal women into a 1:1 randomized, double-blind, placebo-controlled trial of oral TPA 12mg (Repros Therapeutics Inc.) for 2-10 weeks. The primary endpoint was Ki67 labelling, comparing diagnostic core needle biopsy to post-therapy surgical specimens. Ki67 changes were quantitated by dual immunohistochemistry (Ki67/pan-cytokeratin) and image analysis (Aperio ImageScope and Definiens Tissue Studio®). RNA-sequencing (using RNA extracted from the paraffin blocks) was performed with Illumina TruSeq RNA Coding Access method. Differential gene expression pre-post therapy was assessed, followed by Gene Set Enrichment Analysis for pathway analysis. Ki67 changes from baseline were tested with Paired signed-rank test. For gene expression analysis, p values were calculated by Wald test and adjusted for multiple comparisons by Benjamini-Hochberg method (adjusted p Results: Among 61 evaluable women, (29 placebo and 32 TPA) 97% of tumors were ER or PR positive and 91% were ER and PR positive (balanced across arms). A significant 6% decrease in mean %Ki67 was seen in the TPA arm (p= 0.003). When stratified by menopause, the significance held in premenopausal women (n= 22, p= 0.03) but not in postmenopausal women (n=10, p= 0.08). However, a Ki67 decrease (4%) was also observed in placebo group (p = 0.04); this was non-significant after pre- postmenopausal stratification. Overall, differential gene expression analysis showed no significant modulation of genes in either group. Using a pre-specified response parameter (50% relative reduction in Ki67), we identified 12/32 (38%) “responders” in the TPA, and 9/29 (31%) in the placebo arm. In sub-group analysis of these responders, we found 103 genes to be significantly modulated by treatment in the TPA “responders”, but saw no significant change in any gene expression in placebo “responders”. Gene set enrichment analysis for the 103 genes showed that TPA blocked the progression of cell cycle genes (PTTG1, PLK1, UBE2C, HIST1H3F, PSMD3, and etc.) and suppressed PGR and ERBB2 expression. In a pre-planned pooled analysis, these results will be combined with NCT02314156, reported in SABCS abstract 851790. Conclusions: An anti-proliferative (Ki67) signal of TPA was observed in early stage breast cancer patients, but interpretation was limited by placebo group changes. The TPA group demonstrated differential suppression of proliferation-related genes among Ki67 responders, but the placebo group did not. Ongoing analysis will examine signatures related to stemness, metastasis, and immune suppression (potentially better endpoints in trials targeting P signaling). These analyses may help us select the right population and the right biomarkers for future trials. Citation Format: Lee O, Sullivan ME, Xu Y, Shidfar A, Ivancic D, Zeng Z, Singhal H, Helenowski I, Jovanovic B, Hansen N, Bethke K, Gann P, Gradishar WJ, Clare SE, Khan SA. Progesterone receptor (PR) antagonism by telapristone acetate (TPA): A randomized, placebo-controlled phase IIB pre-surgical window trial in women with stage 0-II breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-02.

Background: CDB4124, anti-progesterone suppresses the development of carcinogen-induced ER+/PR+ mammary tumors in rats, and may have implications for prevention and treatment of human breast cancer. We hypothesize that progesterone (P4) and medroxyprogesterone acetate (MPA) will accelerate mammary carcinogenesis induced by MNU, however CDB4124 will efficiently suppress tumor formation stimulated by progesterone. Methods: ovary intact female Sprague Dawley rats received a single intraperitoneal injection of 50mg/kg MNU at 4-5 weeks of age. 30mg of CDB4124 and 25mg of P4 or MPA (90 release pellets, Innovative research of America, Inc) were implanted in dorsal area at 3 weeks and 4 weeks after MNU injection, respectively. 10-11 rats were used for each treatment group. Tumor incidence, latency, multiplicity, and burden were recorded weekly. 9 weeks after MNU injection all the mice were euthanized and mammary tumors and glands were fixed in 10% (v/v) neutral buffered formalin. Plasma concentrations of CDB4124 and its metabolite CDB4453 were determined by LC-MS/MS. Results: The first tumor appeared in the control group at 5 weeks, and in the P4 and MPA treated groups at 6 weeks after MNU injection. 7 weeks after MNU injection, mammary tumor incidence of MPA and P4 treated groups were 80% and 50%, respectively compared to 30% in the control group. 9 weeks after MNU injection all MPA treated mice, 80% of P4 treated mice, and 60% of control mice developed tumors. Tumor incidence, latency, multiplicity, and tumor weight were summarized as mean ± SD in Table 1. [Table 1] Tumor latency, incidence, multiplicity, and burden in mammary tumorsTreatmentsLatency (days)Incidence(%)MultiplicityBurden (g)Control53.7 ± 12.9602.34.25 ± 7.02P453.6 ± 9.6802.64.11 ± 5.32MPA50.8 ± 7.71002.56.02 ± 4.85P4 + CDB412459.3 ± 11.53621.38 ± 0.61MPA + CDB412454.3 ± 10.7732.84.19 ± 4.39 Tumor latency of CDB4124 treated groups was increased; tumor incidence and burden (g) of CDB4124 treated groups were decreased compared to P4 and MPA treated groups. In particular, tumor incidence and burden of CDB4124 + P4 treated group were significantly lower than those of the control group. Plasma CDB4124 and CDB4453 were 11.6 ±5.88 ng/mL and 3.4±1.68 ng/mL, respectively. Histopathology of tumors and mammary glands and immuno-histochemical evaluations of Ki67, activated caspase-3, CD34, ER, and PR are currently underway. Conclusions: Our results indicated that natural progesterone promotes MNU- induced mammary tumor formation similar to synthetic progesterone, MPA in rats. Under this tumor permissive environment, CDB4124 provided excellent prevention efficacy, suggesting good potential as breast cancer prevention agent. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-11-02.

Risk biomarkers that are specific to estrogen receptor (ER) subtypes of breast cancer would aid the development and implementation of distinct prevention strategies. The contralateral unaffected breast of women with unilateral breast cancer (cases) is a good model for defining subtype-specific risk because women with ER-negative (ER−) index primaries are at high risk for subsequent ER-negative primary cancers. We conducted random fine needle aspiration of the unaffected breasts of cases. Samples from 30 subjects [15 ER-positive (ER+) and 15 ER− cases matched for age, race and menopausal status] were used for Illumina expression array analysis. Findings were confirmed using quantitative real-time PCR (qRT-PCR) in the same samples. A validation set consisting of 36 subjects (12 ER+, 12 ER− and 12 standard-risk healthy controls) was used to compare gene expression across groups. ER− case samples displayed significantly higher expression of 18 genes/transcripts, 8 of which were associated with lipid metabolism on gene ontology analysis (GO: 0006629). This pattern was confirmed by qRT-PCR in the same samples, and in the 24 cases of the validation set. When compared to the healthy controls in the validation set, significant overexpression of 4 genes (DHRS2, HMGCS2, HPGD and ACSL3) was observed in ER− cases, with significantly lower expression of UGT2B11 and APOD in ER+ cases, and decreased expression of UGT2B7 in both subtypes. These data suggest that differential expression of lipid metabolism genes may be involved in the risk for subtypes of breast cancer, and are potential biomarkers of ER-specific breast cancer risk. Cancer Prev Res; 6(4); 321–30. ©2013 AACR.

Mucocele-like lesion (MLL) is a rare mucinous lesion of the breast with highly variable upgrade rates to atypia or malignancy on excision. This spectrum of data has led to differing opinions on the need for surgical excision. We evaluated 50 core biopsy specimens diagnosed as having MLLs and correlated the findings with those of excision pathology. Thirty-eight patients underwent surgical excision and 29 were benign (76%), 4 had atypical ductal hyperplasia (11%), and 5 had ductal carcinoma in situ (13%), with an overall upgrade rate of 13%. However, the risk of upgrade was exclusively associated with the presence of atypia as seen on the needle core biopsy. All 22 MLLs without atypia had benign excisions, while 5 (31%) of the 16 patients with MLLs with atypia were upgraded to ductal carcinoma in situ on excision. No invasive carcinoma was identified. We believe it is reasonable that women with the core biopsy diagnosis of MLL without atypia and no associated mass be offered close clinical follow-up as an alternative to surgery.

miRNAs are noncoding RNAs with abnormal expression in breast cancer; their expression in high-risk benign breast tissue may relate to breast cancer risk. We examined miRNA profiles in contralateral unaffected breasts (CUB) of patients with breast cancer and validated resulting candidates in two additional sample sets. Expression profiles of 754 mature miRNAs were examined using TaqMan Low Density Arrays in 30 breast cancer samples [15 estrogen receptor (ER)-positive and 15 ER-negative] and paired CUBs and 15 reduction mammoplasty controls. Pairwise comparisons identified miRNAs with significantly differential expression. Seven candidate miRNAs were examined using qRT-PCR in a second CUB sample set (40 cases, 20 ER+, 20 ER−) and 20 reduction mammoplasty controls. Further validation was performed in 80 benign breast biopsy (BBB) samples; 40 from cases who subsequently developed breast cancer and 40 from controls who did not. Logistic regression, using tertiles of miRNA expression, was used to discriminate cases from controls. Seven miRNAs were differentially expressed in tumors and CUBs versus reduction mammoplasty samples. Among them, miR-18a and miR-210 were validated in the second CUB set, showing significantly higher expression in tumor and CUBs than in reduction mammoplasty controls. The expression of miR-18a and miR-210 was also significantly higher in BBB cases than in BBB controls. When both miR-18a and miR-210 were expressed in the upper tertiles in BBB, OR for subsequent cancer was 3.20, P = 0.023. miR-18a and miR-210 are expressed at higher levels in CUBs of patients with breast cancer, and in BBB prior to cancer development, and are therefore candidate breast cancer risk biomarkers. Cancer Prev Res; 10(1); 89–97. ©2016 AACR.

Risk biomarkers for estrogen receptor (ER)-negative breast cancer have clear value for breast cancer prevention. We previously reported a set of lipid metabolism (LiMe) genes with high expression in the contralateral unaffected breasts (CUBs) of ER-negative cancer cases. We now further examine LiMe gene expression in both tumor and CUB, and investigate the role of Pre-B-cell leukemia homeobox-1 (PBX1) as a candidate common transcription factor for LiMe gene expression. mRNA was extracted from laser-capture microdissected epithelium from tumor and CUB of 84 subjects (28 ER-positive cases, 28 ER-negative cases, 28 healthy controls). Gene expression was quantitated by qRT-PCR. Logistic regression models were generated to predict ER status of the contralateral cancer. Protein expression of HMGCS2 and PBX1 was measured using immunohistochemistry. The effect of PBX1 on LiMe gene expression was examined by overexpressing PBX1 in MCF10A cells with or without ER, and by suppressing PBX1 in MDA-MB-453 cells. The expression of DHRS2, HMGCS2, UGT2B7, UGT2B11, ALOX15B, HPGD, UGT2B28 and GLYATL1 was significantly higher in ER-negative versus ER-positive CUBs, and predicted ER status of the tumor in test and validation sets. In contrast, LiMe gene expression was significantly lower in ER-negative than ER-positive tumors. PBX1 overexpression in MCF10A cells up-regulated most LiMe genes, but not in MCF10A cells overexpressing ER. Suppressing PBX1 in MDA-MB-453 cells resulted in decrease of LiMe gene expression. Four binding sites of PBX1 and cofactor were identified in three lipid metabolism genes using ChIP-qPCR. These data suggest a novel role for PBX1 in the regulation of lipid metabolism genes in benign breast, which may contribute to ER-negative tumorigenesis.

Davion S M, Siziopikou K P & Sullivan M E (2012) Histopathology 61, 660–666 Cytokeratin 7: a re-evaluation of the ‘tried and true’ in triple-negative breast cancers Aims: Triple-negative breast cancers (TNBCs) are often poorly differentiated tumours that can present clinically as metastases of an unknown primary. Immunohistochemical panels are frequently used to determine the likelihood of a breast primary, but in this tumour subset cytokeratin (CK)7 may be the only positive finding. In this study we aimed to evaluate a commonly employed immunohistochemical panel using a large group of TNBCs (both basal-like and unclassified), and to analyse the CK7 staining patterns. Methods and results: Tissue microarrays containing 138 TNBCs were stained with antibodies against CK7, CK20, gross cystic disease fluid protein 15 (GCDFP-15), and mammaglobin. CK5/6 staining was used to identify basal-like tumours. CK7 staining was notably heterogeneous, with 14.5% of all cases demonstrating ≤20% tumour cell staining. A greater proportion of basal-like TNBCs than of unclassified TNBCs showed focal staining. GCDFP-15 and mammaglobin were not expressed in the majority of TNBCs, and were less frequently positive in basal-like than in unclassified TNBCs. Conclusions: TNBCs are commonly negative for most immunomarkers indicative of breast origin, with the exception of CK7. As about one in five TNBCs showed only focal CK7 positivity, use of this marker must be interpreted with caution, especially in small samples, so that the possibility of a breast primary is not overlooked.

Triple negative breast carcinomas (TNBC), are defined by the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression. The majority of TNBC are “basal-like”, a group originally defined by studies of mRNA gene expression profiles, but increasingly defined in the clinic by using surrogate markers such as CK 5/6. However, not all TNBC are basal-like. It is postulated that these subcategories of TNBC have distinct underlying biologies that drive their ultimate behavior, and response to treatment, with important implications for designing appropriate targeted therapies. In this study we report that within our cohort of 197 TNBC, distinct groups were identified that varied by CK 5/6 and p53 expression based on age and race. We propose that awareness of CK5/6 and p53 expression in younger and AA TNBC patients may facilitate identifying patients with unique tumor subtypes and may lead to better use of targeted therapies in this group of aggressive breast cancers.

Objectives Bi- and multinucleated (B/M) cells are present in a variety of tumors. We evaluated lobular carcinoma in situ (classic and pleomorphic types) and ductal carcinoma in situ (DCIS) to determine if this objective morphologic feature aids the differential diagnosis. Methods The number of B/M cells was recorded in pleomorphic lobular carcinoma in situ (PLCIS) (n = 20), classic lobular carcinoma in situ (CLCIS) (n = 26), and DCIS (n = 37). Results Binucleated cells were significantly more frequent in PLCIS (100%) vs DCIS (43%; P < .0001) and CLCIS (54%; P = .0004). Multinucleated cells were present in 25% of PLCIS cases and 8% of DCIS cases, and they were absent in CLCIS. The quantity of B/M per high-power field (hpf) was less in DCIS (mean, 1.1) and CLCIS (mean, 2.5) compared with PLCIS (mean, 5.8). Thirty-five percent of PLCIS cases had more than five B/M per hpf. Conclusions Binucleated cells are significantly more frequent in PLCIS vs CLCIS and DCIS. Multinucleated cells were never identified in CLCIS. PLCIS should be considered as a diagnosis when B/M is noted.

Breast cancer is a leading cause of death for women, with mortality resulting from metastasis. Metastases are often detected once tumour cells affect the function of solid organs, with a high disease burden limiting effective treatment. Here we report a method for the early detection of metastasis using an implanted scaffold to recruit and capture metastatic cells in vivo, which achieves high cell densities and reduces the tumour burden within solid organs 10-fold. Recruitment is associated with infiltration of immune cells, which include Gr1(hi)CD11b(+) cells. We identify metastatic cells in the scaffold through a label-free detection system using inverse spectroscopic optical coherence tomography, which identifies changes to nanoscale tissue architecture associated with the presence of tumour cells. For patients at risk of recurrence, scaffold implantation following completion of primary therapy has the potential to identify metastatic disease at the earliest stage, enabling initiation of therapy while the disease burden is low.

Brain metastasis is a devastating, late-stage event affecting approximately 10-30% of breast cancer patients. However, it is not well understood how breast cancer cells migrate across the blood-brain barrier (BBB) and invade into the brain. Syndecans (Sdcs) are cell surface heparan sulfate proteoglycans (HSPGs) that have previously been linked to breast cancer progression and metastasis. Preliminary findings from our lab indicate that Sdc1 may be involved in breast cancer metastasis to the brain. In clinical samples from breast cancer patients with brain metastases, 67% of the brain metastasis samples stained positive for Sdc1 expression. In an experimental mouse model of metastasis, we found that silencing expression of Sdc1 in MDA-231 breast cancer cells greatly reduced metastasis specifically to the brain, with no difference in lung or bone metastases. Additionally, silencing expression of Sdc1 in MDA-231 cells resulted in a reduction in cancer cell migration across an in vitro BBB transwell model system. These findings prompted us to investigate the mechanism through which Sdc1 facilitates breast cancer cell migration across the BBB. Since BBB endothelial cell junctions have been reported to be disrupted in metastasis to the brain, immunofluorescence was performed to examine localization of junction proteins in our BBB model following addition of conditioned medium (CM) from MDA-231 non-silenced (NS1) control and Sdc1KD cells. We observed disruption in PECAM-1 localization at BBB endothelial cell junctions upon addition of CM from MDA-231 NS1 cells with markedly less disruption occurring upon treatment with CM from Sdc1KD cells. These results suggest that paracrine factors secreted from MDA-231 cells may facilitate breast cancer cell migration across the BBB by affecting PECAM-1 localization on BBB endothelial cells. By performing a cytokine array using CM from MDA-231 cells, we determined that Sdc1KD CM contained lower levels of IL-8 than MDA-231 NS1 cell CM. These findings were confirmed by ELISA, qRT-PCR, and multiplex analysis. We then went on to treat endothelial cells in the xCELLigence in vitro BBB model system with IL-8 and observed a sufficient decrease in cell index readings, suggesting that IL-8 affects BBB barrier permeability. Taken together, our results suggest that Sdc1 supports breast cancer cell migration across the BBB through a signaling mechanism involving IL-8 and PECAM-1. Elucidating this mechanism will allow for the development of therapeutic strategies to combat breast cancer cell metastasis to the brain. [S.M.W. and M.S. contributed equally to this work.] Citation Format: Sierra Mosticone Wangensteen, Megan Sayyad, Madhavi Puchalapalli, Megan Sullivan, Jamie Singh, Briana Ratchford, Jayda Abrams, Majid Jahromi, Bin Hu, Michael Idowu, Jennifer Koblinski. Syndecan-1 mediates breast cancer metastasis to the brain through IL-8 and PECAM-1 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3058. doi:10.1158/1538-7445.AM2017-3058

Puberty and reproduction require proper signaling of the hypothalamic-pituitary-gonadal axis controlled by gonadotropin-releasing hormone (GnRH) neurons, which arise in the olfactory placode region and migrate along olfactory axons to the hypothalamus. Factors adversely affecting GnRH neuron specification, migration, and function lead to delayed puberty and infertility. Nasal embryonic luteinizing hormone-releasing factor (NELF) is a predominantly nuclear protein. NELF mutations have been demonstrated in patients with hypogonadotropic hypogonadism, but biallelic mutations are rare and heterozygous NELF mutations typically co-exist with mutations in another gene. Our previous studies in immortalized GnRH neurons supported a role for NELF in GnRH neuron migration. To better understand the physiology of NELF, a homozygous Nelf knockout (KO) mouse model was generated. Our findings indicate that female Nelf KO mice have delayed vaginal opening but no delay in time to first estrus, decreased uterine weight, and reduced GnRH neuron number. In contrast, male mice were normal at puberty. Both sexes of mice had impaired fertility manifested as reduced mean litter size. These data support that NELF has important reproductive functions. The milder than expected phenotype of KO mice also recapitulates the human phenotype since heterozygous NELF mutations usually require an additional mutation in a second gene to result in hypogonadotropic hypogonadism.

Purpose: Local transdermal therapy to the breast may achieve effective target-organ drug delivery, while diminishing systemic effects. We conducted a randomized, double-blind, placebo-controlled phase II trial comparing transdermal 4-hydroxytamoxifen gel (4-OHT) to oral tamoxifen (oral-T) in women with ductal carcinoma in situ (DCIS). Methods: Twenty-seven pre- and postmenopausal women were randomized to 4-OHT (4 mg/day) or oral-T (20 mg/day) for 6 to 10 weeks before surgery. Plasma, nipple aspirate fluid, and breast adipose tissue concentrations of tamoxifen and its major metabolites were determined by liquid chromatography/tandem mass spectrometry. The primary endpoint was Ki67 labeling in DCIS lesions, measured by immunohistochemistry. In plasma, insulin-like growth factor-1 (IGFI), sex hormone–binding globulin (SHBG), and coagulation protein concentrations were determined. Results: Posttherapy Ki67 decreased by 3.4% in the 4-OHT and 5.1% in the oral-T group (P ≤ 0.03 in both, between-group P = 0. 99). Mean plasma 4-OHT was 0.2 and 1.1 ng/mL in 4-OHT and oral groups, respectively (P = 0.0003), whereas mean breast adipose tissue concentrations of 4-OHT were 5.8 ng/g in the 4-OHT group and 5.4 ng/g in the oral group (P = 0.88). There were significant increases in plasma SHBG, factor VIII, and von Willebrand factor and a significant decrease in plasma IGFI with oral-T, but not with 4-OHT. The incidence of hot flashes was similar in both groups. Conclusions: The antiproliferative effect of 4-OHT gel applied to breast skin was similar to that of oral-T, but effects on endocrine and coagulation parameters were reduced. These findings support the further evaluation of local transdermal therapy for DCIS and breast cancer prevention. Clin Cancer Res; 20(14); 3672–82. ©2014 AACR.

Erythrocyte exchange, a procedure in which blood is removed and replaced with donor cells, is often used to prevent or treat severe vasocclusion in patients with sickling hemoglobinopathies. This procedure increases the percentage of hemoglobin A without dramatically increasing the hematocrit or viscosity. The efficacy of erythrocyte exchange is often measured by hemoglobin electrophoresis and densitometry to determine the posttransfusion percentages of hemoglobin A and S. We performed a manual erythrocyte exchange on a 28-year-old pregnant woman with hemoglobin SD-Punjab. Five units of whole blood were withdrawn and replaced with packed erythrocytes and saline. The expected percentage of hemoglobin A in each unit withdrawn was calculated in an iterative fashion. We calculated the total volume of hemoglobin SD-Punjab erythrocytes using the patient’s total blood volume estimated by nomogram, measured hematocrit, and assuming 100% hemoglobin SD-Punjab. The volume of erythrocytes removed was subtracted from the hemoglobin SD-Punjab erythrocyte volume tally, and the volume of erythrocytes administered (assumed to be an average of 160 mL) …

Background: Breast cancer prevention is currently in a challenge due to the inability to identify high-risk women accurately only based on family history and pathologic evaluation of benign biopsy. Somatic molecular changes in clinically normal breasts have potential value for to improve the assessment of truly high-risk women. The identification of risk biomarkers in benign biopsy material will directly benefit the millions of women who undergo benign breast biopsy annually, adding precision to the risk implied by the histologic features of the benign biopsy. Therefore, we are pursuing robust biomarkers for breast cancer risk by sequencing pre-cancer benign biopsies, which may lead to discovery of genomic variation responsible for initiation and early progression of breast cancer. Methods: We are assembling a case-control set of benign breast biopsy matched by age, race, and duration of follow-up. The cases were the benign biopsies from women who subsequently developed breast cancer after at least one year, and the controls were the benign biopsy samples obtained from women who remain cancer free. 10-micron sections from formalin-fixed, paraffin-embedded tissue blocks are used for laser capture microdissection of selected epithelial areas and extraction of DNA. As a pilot experiment, we examined hotspot mutation in 4 cases and 4 control samples, using Ion AmpliSeq cancer hotspot panel consisting of 50 oncogenes/tumor suppressor and detecting 2855 hotspot mutation from COSMIC (Catalogue Of Somatic Mutations In Cancer). The average amplicon length was 154 (111-187) with average depth coverage of 1400x. SNP detection sensitivity was 98% for 5% variant frequency. Results: Among the 2855 hotspot mutation in 50 oncogenes/tumor suppressor detected, 153 mutation in 27 genes were identified in cases, and 62 mutations in 18 genes were found in controls. The most frequent mutanted gene was TP53 (71 mutation in cases, 24 mutation in controls). In 6 genes (CTNNB1, CDKN2A, ATM, KRAS, STK11 and SMARCB1), hotspot mutation was identified in at least two cases, but no hotspot mutation was identified in controls. Among the 4 cases, one cases had 95 hotspot mutation, much higher than the other 3 cases (5, 16, 37 mutation, respectively) and 4 controls (8, 11, 14, 29 mutation, respectively). This case progressed to triple negative (ER-, PR-, HER2-) breast cancer, a very aggressive subtype of cancer with poor prognosis. Conclusion: The pilot study of deep sequencing of breast benign biopsy suggested that the overall hotspot mutation frequency was higher in cases which progressed to malignant cancer compared to controls which did not progress to malignant cancer. Several genes were identified to be specifically associated with the progression. Citation Format: Jun Wang, Gang Feng, Nadereh Jafari, Ali Shidfar, David Ivanicic, Megan E. Sullivan, Seema A. Khan. Hotspot mutation in breast benign biopsy associated with subsequent progression to malignant breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-03.

Ductal carcinoma in situ (DCIS) is a noninvasive cancer that accounts for 25% of all breast cancers diagnosed in the United States. DCIS is a heterogeneous disease process with varied clinical manifestations and a broad spectrum of imaging findings. With advances in technology, the ability to detect early-stage cancers has improved, and understanding the role of ultrasonography (US) in the multimodality era of detection and diagnosis is paramount. When calcifications are identified at mammography, US can be performed to evaluate for an invasive component and to allow possible US-guided biopsy. Use of high-frequency transducers, spectral compounding, and speckle reduction algorithms can aid in the detection of calcifications. Calcified DCIS most commonly manifests as echogenic foci located within a mass or duct, associated with internal microlobulations, or distributed in a branch pattern. Noncalcified DCIS, which is more often identified in symptomatic patients, may manifest as a hypoechoic mass with microlobulated margins and no posterior acoustic features, or it may have a "pseudomicrocystic" appearance. Harmonic imaging and coronal reconstruction may improve detection of noncalcified DCIS. The appearance of DCIS at "second-look" US can be subtle and may warrant a lower threshold for detection, given a higher pretest probability of malignancy. US features are nonspecific, and careful correlation with respect to lesion location, size, shape, and depth is needed. The presence of internal vascularity can help increase the positive predictive value of US in this setting. US is a useful adjunct to mammography and magnetic resonance imaging, and recognizing the US appearance of DCIS will aid in the detection and diagnosis of this disease entity.