Paul Thiruchelvam

The estrogen receptor-α-induced microRNA signature regulates itself and its transcriptional response

Following estrogenic activation, the estrogen receptor-α (ERα) directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) modulated by ERα have the potential to fine tune these regulatory systems and also provide an alternate mechanism that could impact on estrogen-dependent developmental and pathological systems. Through a microarray approach, we identify the subset of microRNAs (miRNAs) modulated by ERα, which include upregulation of miRNAs derived from the processing of the paralogous primary transcripts (pri-) mir-17–92 and mir-106a-363. Characterization of the mir-17–92 locus confirms that the ERα target protein c-MYC binds its promoter in an estrogen-dependent manner. We observe that levels of pri-mir-17–92 increase earlier than the mature miRNAs derived from it, implicating precursor cleavage modulation after transcription. Pri-mir-17–92 is immediately cleaved by DROSHA to pre-miR-18a, indicating that its regulation occurs during the formation of the mature molecule from the precursor. The clinical implications of this novel regulatory system were confirmed by demonstrating that pre-miR-18a was significantly upregulated in ERα-positive compared to ERα-negative breast cancers. Mechanistically, miRNAs derived from these paralogous pri-miRNAs (miR-18a, miR-19b, and miR-20b) target and downregulate ERα, while a subset of pri-miRNA-derived miRNAs inhibit protein translation of the ERα transcriptional p160 coactivator, AIB1. Therefore, different subsets of miRNAs identified act as part of a negative autoregulatory feedback loop. We propose that ERα, c-MYC, and miRNA transcriptional programs invoke a sophisticated network of interactions able to provide the wide range of coordinated cellular responses to estrogen.

The liver receptor homolog-1 regulates estrogen receptor expression in breast cancer cells

Estrogen receptor-α (ER) is expressed in the great majority of breast cancers, and the inhibition of ER action is a key part of breast cancer treatment. The inhibition of ER action is achieved using anti-estrogens, primarily tamoxifen, and with aromatase inhibitors that inhibit estrogen biosynthesis, thereby preventing ER activation. However, resistance to these therapies is common. With the aim of identifying new molecular targets for breast cancer therapy, we have identified the liver receptor homolog-1 (LRH-1) as an estrogen-regulated gene. RNA interference and over-expression studies were used to investigate the role of the LRH-1 in regulating breast cancer growth and to identify the targets of an LRH-1 action. Promoter recruitment was determined using reporter gene and chromatin immunoprecipitation (ChIP) assays. We show that LRH-1 regulates breast cancer cell growth by regulating the ER expression. Reporter gene and in vitro DNA-binding assays identified an LRH-1-binding site in the ER gene promoter, and ChIP assays have demonstrated in vivo binding at this site. We also provide evidence for new LRH-1 variants in breast cancer cells arising from the use of alternative promoters. Previous studies have shown that LRH-1 functions in estrogen biosynthesis by regulating aromatase expression. Our findings extend this by highlighting LRH-1 as a key regulator of the estrogen response in breast cancer cells through the regulation of ER expression. Hence, inhibition of LRH-1 could provide a powerful new approach for the treatment of endocrine-resistant breast cancer.

Smartphone breast applications – What's the evidence?

INTRODUCTION There are around 40,000 healthcare applications (apps) available for smartphones. Apps have been reviewed in many specialties. Breast cancer is the most common malignancy in females with almost 1.38 million new cases a year worldwide. Despite the high prevalence of breast disease, apps in this field have not been reviewed to date. We have evaluated apps relevant to breast disease with an emphasis on their evidence base (EB) and medical professional involvement (MPI). METHODS Searching the major app stores (apple iTunes, Google Play, BlackBerry World, Windows Phone) using the most common breast symptoms and diseases identified relevant apps. Extracted data for each app included target consumer, disease focus, app function, documentation of any EB, documentation of MPI in development, and potential safety concerns. RESULTS One-hundred-and-eighty-five apps were reviewed. The majority focused on breast cancer (n = 139, 75.1%). Educational (n = 94) and self-assessment tools (n = 30) were the most common functions demonstrated. EB and MPI was identified in 14.2% and 12.8% of apps respectively. Potential safety concerns were identified in 29 (15.7%) apps. CONCLUSIONS There is a lack of EB and MPI in the development of current breast apps. Safety concerns highlight the need for regulation, full authorship disclosure and clinical trials. A robust framework for identifying high quality applications is necessary. This will address the current barrier pertaining to a lack of consumer confidence in their use and further aid to promote their widespread implementation within healthcare.

Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth

Estrogen receptor-α (ERα) positive breast cancer frequently responds to inhibitors of ERα activity, such as tamoxifen, and/or to aromatase inhibitors that block estrogen biosynthesis. However, many patients become resistant to these agents through mechanisms that remain unclear. Previous studies have shown that expression of ERα in ERα-negative breast cancer cell lines frequently inhibits their growth. In order to determine the consequence of ERα over-expression in ERα-positive breast cancer cells, we over-expressed ERα in the MCF-7 breast cancer cell line using adenovirus gene transduction. ERα over-expression led to ligand-independent expression of the estrogen-regulated genes pS2 and PR and growth in the absence of estrogen. Interestingly, prolonged culturing of these cells in estrogen-free conditions led to the outgrowth of cells capable of growth in cultures from ERα transduced, but not in control cultures. From these cultures a line, MLET5, was established which remained ERα-positive, but grew in an estrogen-independent manner. Moreover, MLET5 cells were inhibited by anti-estrogens showing that ERα remains important for their growth. Gene expression microarray analysis comparing MCF-7 cells with MLET5 highlighted apoptosis as a major functional grouping that is altered in MLET5 cells, such that cell survival would be favoured. This conclusion was further substantiated by the demonstration that MLET5 show resistance to etoposide-induced apoptosis. As the gene expression microarray analysis also shows that the apoptosis gene set differentially expressed in MLET5 is enriched for estrogen-regulated genes, our findings suggest that transient over-expression of ERα could lead to increased cell survival and the development of estrogen-independent growth, thereby contributing to resistance to endocrine therapies in breast cancer patients.

Considerations for Clinicians in the Diagnosis, Prevention, and Treatment of Breast Cancer-Related Lymphedema: Recommendations from a Multidisciplinary Expert ASBrS Panel : Part 1: Definitions, Assessments, Education, and Future Directions

Sarah A. McLaughlin, MD, Alicia C. Staley, MBA, MS, BS, Frank Vicini, MD, FACR, FABS, Paul Thiruchelvam, BSc, MD, PhD, FRCS, Nancy A. Hutchison, MD, CLT-LANA, Jane Mendez, MD, Fiona MacNeill, FRCS, MD, FEBS, Stanley G. Rockson, MD, Sarah M. DeSnyder, MD, Suzanne Klimberg, MD, PhD, Michael Alatriste, LMT, CLT, Francesco Boccardo, MD, PhD, FACS, Mark L. Smith, MD, FACS, and Sheldon M. Feldman, MD, FACS

Considerations for Clinicians in the Diagnosis, Prevention, and Treatment of Breast Cancer-Related Lymphedema, Recommendations from an Expert Panel: Part 2: Preventive and Therapeutic Options

Sarah A. McLaughlin, MD, Sarah M. DeSnyder, MD, Suzanne Klimberg, MD, PhD, Michael Alatriste, LMT, CLT, Francesco Boccardo, MD, PhD, FACS, Mark L. Smith, MD, FACS, Alicia C. Staley, MBA, MS, BS, Paul T. R. Thiruchelvam, BSc, MD, PhD, FRCS, Nancy A. Hutchison, MD, CLT-LANA, Jane Mendez, MD, Fiona MacNeill, FRCS, MD, FEBS, Frank Vicini, MD, FACR, FABS, Stanley G. Rockson, MD, and Sheldon M. Feldman, MD, FACS

NSQIP Analysis of Axillary Lymph Node Dissection Rates for Breast Cancer: Implications for Resident and Fellow Participation

INTRODUCTION Management of the axilla in invasive breast cancer (IBC) has shifted away from more radical surgery such as axillary lymph node dissection (ALND), towards less invasive procedures, such as sentinel lymph node biopsy. Because of this shift, we hypothesize that there has been a national downward trend in ALND procedures, subsequently impacting surgical trainee exposure to this procedure using the ACS-NSQIP database to evaluate this. METHODS Women with IBC were identified in the ACS-NSQIP database from 2007 to 2014. Procedures including ALND were identified using CPT codes. This number was divided by total cases, given a varying number of participating institutions each year. Next, cases involving resident participation were identified and divided by training level: junior (post graduate year-[PGY] 1-2), senior (PGY 3-5) and fellow (PGY ≥ 6). Two tailed z tests were used to compare proportions, with significance determined when p < 0.05. RESULTS A total of 128,372 women were identified with IBC with 36,844 ALND. ALND rates decreased by an average of 2.43% yearly from 2007 to 2014. Resident participation significantly drops in 2011, from 49.3% before to 29.4% after (p < 0.01). Junior residents experienced a significant decrease in participation rate (43.3%-32.2%, p < 0.05). Senior residents and fellows experienced an upward trend in their participation, although not significant (51.2%-56.3%, p = 0.35, and 5.6%-11.6%, p = 0.056, respectively). CONCLUSIONS Using the ACS-NSQIP database, we demonstrate the downward trend in rate of ALND for IBC with subsequent decrease in resident participation. Junior residents experienced a significant decrease in their participation with no significant change for senior or fellow-level trainees. Awareness of this trend is important when creating future surgical curriculum changes for general surgery and fellowship training programs.

Characterisation of Estrogen Responses in Breast Cancer Cells Highlights LRH-1 as a New Target for Breast Cancer Treatment.

Background: Liver receptor homolog-1 (LRH-1; NR5A2) is an orphan member of the Ftz-F1 family of nuclear receptors, which comprises four members (NR5A1-NR5A4). It is expressed in endodermal derived tissues and has been linked to a number of developmental, metabolic and proliferative processes. LRH-1 plays an important role in cholesterol and lipid homeostasis. Recent findings have shown that intestinal production of glucocorticoids is regulated by LRH-1, a deficiency of which sensitizes mice to colitis. Uncontrolled LRH-1 activity participates in the induction of intestinal tumours. LRH-1 protein expression has also been detected by immunohistochemistry in tumour cells of human mammary ductal carcinomas.Materials and Methods: Using a panel of breast cancer cell lines and human tissues we have studied the expression of LRH-1 together with another Ftz-F1 subfamily member steroidogenic factor 1 (SF-1) and the potent LRH-1 co-repressors small heterodimer partner (SHP) and DAX-1/Dax-1 (dosage-sensitive sex-reversal adrenal hypoplasia congenital critical region on the X chromosome 1). RNA ligase Mediated Rapid amplification of 59 complementary DNA ends (59RLM-RACE) was performed in order to map the estrogen regulated transcription start sites for the LRH-1 gene. No natural ligand for LRH-1 has been described. Using fluorescence resonance energy transfer (FRET)-based assays a series of unusual bicyclic compounds have been defined as potent LRH-1 ligands. The effects of these compounds on breast cancer cell growth and gene expression has been investigated using SRB growth assays and quantitative RT-PCR.Results & Discussion: LRH-1 expression was found to be highest in estrogen receptor positive cell lines and breast, colon,small intestine and liver tissue. Treatment with 17β-estradiol increased LRH-1 levels in estrogen receptor positive cell lines, while treatment with anti-estrogens Tamoxifen and ICI 182,780 (Fulvestrant) reduced LRH-1 levels. siRNA mediated RNA interference against LRH-1 significantly reduced the estrogen-dependent growth of estrogen receptor positive breast cancer cell lines. We have shown that LRH-1 regulates the expression of the LRH-1 target gene Inhibin-α (a member of the transforming growth factor-β superfamily) in breast cancer cell lines. 59 RACE identified two new variant forms of LRH-1, which we have named LRH-1 289 and LRH-1 325 respectively. These arise from a novel promoter region lying between exon 2 and exon 3. RT- PCR for the LRH-1 289 variant showed E2 regulation in MCF7 cells and was seen to account for the majority of LRH-1 expression in this line. SRB growth assays demonstrated that these bicyclical compounds stimulate the growth of estrogen receptor/LRH-1 positive breast cancer cell lines in a dose dependent manner. Luciferase assays have demonstrated a titratable increase in LRH-1 reporter activity following the addition of these compounds.Our studies show that LRH-1 is a potently estrogen regulated gene in breast cancer cells, which acts to regulate cell growth and gene expression. These results therefore help validate LRH-1 as a potential new target for breast cancer treatment Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4143.

Best-practice care pathway for improving management of mastitis and breast abscess: Management of mastitis and breast abscess

Surgical subspecialization has resulted in mastitis and breast abscesses being managed with unnecessary admission to hospital, prolonged inpatient stay, variable antibiotic prescribing, incision and drainage rather than percutaneous aspiration, and loss to specialist follow‐up. The objective was to evaluate a best‐practice algorithm with the aim of improving management of mastitis and breast abscesses across a multisite NHS Trust. The focus was on uniformity of antibiotic prescribing, ultrasound assessment, admission rates, length of hospital stay, intervention by aspiration or incision and drainage, and specialist follow‐up.

Abstract P3-14-07: Neoadjuvant radiotherapy in mastectomy and immediate autologous free flap reconstruction. Findings from the primary radiotherapy and DIEP flap (PRADA) pilot study

Background: The need for post mastectomy radiotherapy (PMRT), may preclude reconstructive surgeons from offering patients immediate, autologous reconstruction. This is due to historical evidence suggesting high rates of short- and long-term complications as well as poorer aesthetic outcomes. As the indications for PMRT broaden this practice denies an ever-increasing number of women the benefit of an immediate reconstruction. Aim: This pilot study evaluates the safety of offering radiotherapy prior to mastectomy and immediate DIEP flap reconstruction. Methods: Women planned for neoadjuvant chemotherapy (NAcT), mastectomy (following unsuccessful breast conservation surgery (BCS) or upfront selection) and PMRT were offered a change in sequencing of RT at two academic breast surgery units in London, UK. Data was prospectively captured on 19 women, including: patient demographics, treatment details, tumour characteristics, oncological and post-operative outcomes. Operative parameters included unplanned return to theatre [RTT] Results: The cohort demonstrated a broad range of age, body mass index (BMI) and mastectomy weight [mean (range): age=46 years (28-72); BMI = 28.4 kg/m2 (23-37.6) and specimen weight=678gm (257-1040)]. The mean time from completion of NAcT to neoadjuvant radiotherapy (NART) was 31.1 days (9-49 days), and time from completion of NART to mastectomy and DIEP was 17.8 days (13-24 days). There was one unplanned RTT at 72 hours for an evacuation of haematoma, 1 revision of micro-vascular anastomosis, 1 clinical fat necrosis requiring formal excision and 1 wound debridement and primary closure for poor wound healing (vertical pattern skin reduction). There were no flap failures and no mastectomy envelope necrosis. With a mean follow-up of 16.2 months, there were no loco-regional recurrences, 5 distant relapses with mean presentation at 13.7 months and 2 breast-cancer related deaths at 13.9 and 22.2months respectively. Conclusion: This pilot study suggests that mastectomy and DIEP reconstruction is surgically feasible within 4 weeks of completing NART. In this small cohort of oncologically high-risk women with altered sequencing of RT we did not observe flap failure or post-mastectomy skin flap necrosis. A larger multicentre study with aesthetic assessment, PROMS and translational aspects is planned. Citation Format: Thiruchelvam P, Hadjiminas D, Cleator S, Wood S, Leff D, Jallali N, James S, MacNeill F. Neoadjuvant radiotherapy in mastectomy and immediate autologous free flap reconstruction. Findings from the primary radiotherapy and DIEP flap (PRADA) pilot study [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 P3-14-07.