ER-positive Invasive Ductal Carcinoma Research Articles

Cost-Effectiveness Analysis of Ultra-Hypofractionated Whole Breast Radiation Therapy Alone Versus Hormone Therapy Alone or Combined Treatment for Low-Risk ER-Positive Early Stage Breast Cancer in Women Aged 65 Years and Older

The optimal management of early-stage, low-risk, hormone-positive breast cancer in older women remains controversial. Recent trials have shown that 5-fraction ultrahypofractionated whole-breast irradiation (U-WBI) has similar outcomes to longer courses, reducing the cost and inconvenience of treatment. We performed a cost-utility analysis to compare U-WBI to hormone therapy alone or their combination. We simulated 3 different treatment approaches for women age 65 years or older with pT1-2N0 ER-positive invasive ductal carcinoma treated with lumpectomy with negative margins using a Markov microsimulation model. The strategies were U-WBI performed with a 3-dimensional conformal technique over 5 fractions without a boost ("radiation therapy [RT] alone"), adjuvant hormone therapy (anastrozole for 5 years) without RT ("aromatase-inhibitor [AI] alone"), or the combination of the 2. The combination strategy was calibrated to match trial results, and the relative effectiveness of the RT alone and AI alone strategies were inferred from previous randomized trials. The primary endpoint was the cost-effectiveness of the 3 strategies over a lifetime horizon as measured by the incremental cost-effectiveness ratio (ICER), with a value of $100,000/quality-adjusted life-year deemed "cost-effective." The model results compared with the prespecified target outcomes. On average, RT alone was the least expensive strategy ($14,775), with AI alone slightly more ($14,998), and combination therapy the costliest ($19,802). RT alone dominated AI alone (the incremental cost-effectiveness ratio [ICER] -$5089). Combination therapy, compared with RT alone, was slightly more expensive than our definition of cost-effective (ICER $113,468) but was cost-effective compared with AI alone (ICER $54,451). Probabilistic sensitivity analysis demonstrated RT alone to be cost-effective in 50% of trials, with combination therapy in 36% and AI alone in 14%. U-WBI alone appears the more cost-effective de-escalation strategy for these low-risk patients, compared with AI alone. Combining U-WBI and AI appears more costly but may be preferred by some patients.

Cost-Effectiveness Analysis of Ultra-Short Whole Breast Radiation Therapy Alone vs. Endocrine Only or Combined Treatment for Low-Risk ER-Positive Early-Stage Breast Cancer in Women Age 70 and Older

<h3>Purpose/Objective(s)</h3> The optimal management of early-stage, low-risk, hormone-positive breast cancer in women age 70 and older remains controversial. Recent trials have shown whole-breast radiation courses of only 5 fractions have similar outcome as longer courses, reducing the cost and inconvenience of treatment. We therefore performed a cost-utility analysis to compare ultra-short whole breast irradiation (U-WBI) to endocrine therapy (ET) alone and to combined ET and U-WBI. <h3>Materials/Methods</h3> We simulated the cost-effectiveness and acute and late effects of different treatment approaches for women age 70 years or older with pT1-2N0 ER-positive invasive ductal carcinoma treated with lumpectomy with negative margins using a Markov microsimulation model. The strategies were: U-WBI performed with a 3D technique over 5 fractions without a boost ("RT Alone"), adjuvant ET (anastrozole for 5 years) without RT ("AI Alone"), or the combination of the two. The FAST-Forward trial served as the basis for this analysis. The combination strategy was calibrated to match the trial results. The relative effectiveness of the RT Alone and AI Alone strategies were inferred from historical randomized trials. Costs were obtained from 2022 Medicare rates. Utilities and other costs were obtained from the literature. The primary endpoint was the cost-effectiveness of the 3 strategies over a 10-year horizon as measured by the incremental cost-effectiveness ratio (ICER), with a value of $100,000/QALY deemed "cost-effective". Deterministic and probabilistic sensitivity analyses were performed to evaluate parameter uncertainty. <h3>Results</h3> The model was validated using 500,000 simulated patients, each treated with the 3 strategies. The model results agreed well with the pre-specified target outcomes. On average, RT Alone was the least expensive strategy ($15,200), with AI Alone slightly more expensive, ($16,761), and combination therapy the most costly ($20,416). The most effective strategy was the combination (9.497 QALY), with small differences between RT Alone (9.453) and AI Alone (9.420). Therefore, the AI Alone strategy was more costly and less effective than RT Alone (dominated), and the combination therapy was slightly more expensive than the usual definition of "cost-effective" (ICER $117,721 relative to RT Alone). Probabilistic sensitivity analysis demonstrated RT Alone to be cost-effective in 52% of trials, with combination in 35% and AI Alone in 13%. <h3>Conclusion</h3> U-WBI without ET represents a cost-effective strategy for low-risk women in the United States, with slightly higher QALY outcome compared to AI Alone. Combination therapy improves outcome very modestly. Hence, many patients may prefer RT Alone in order to avoid the possible side effects of ET. Discussion of these options must account for patients' weighing of these issues.

Eribulin combined with radiation therapy in a young patient re‐irradiated for a new lesion of breast cancer

Eribulin is widely used in the treatment of metastatic breast cancer, with a manageable toxicity profile. This aggressive disease often requires systemic and local treatments, comprising surgery or radiotherapy. However, eribulin is usually discontinued during radiation therapy due to the lack of data concerning the safety of this combination, especially in the setting of repeat locoregional radiation therapy. Our patient was diagnosed with ER positive invasive ductal carcinoma of the left breast initially treated by surgery, radiation therapy, chemotherapy, and hormone therapy. She then received various lines of chemotherapy for multiple triple-negative relapses in the left axillary region. Since October 2020, she has been treated by eribulin. In order to improve local control, it was decided to add local radiation therapy to the region of recurrence in addition to systemic therapy. She underwent radiation therapy concomitantly with eribulin from February to March 2021. Treatment was very well tolerated, and no acute toxicity was reported. This is the first published case of repeat locoregional radiation therapy in combination with eribulin.

Omission of Surgical Axillary Lymph Node Staging in Patients with Tubular Breast Cancer.

With more effective screening and treatment strategies, there is debate over whether surgical axillary staging should be deescalated for patients with small favorable breast cancers, such as tubular carcinoma (TC). We identified patients with TC [defined as > 90% tubular tubules (angulated, not multilayered)] and known surgical axillary staging from our institutional database (2000-2018). Using the National Cancer Database (NCDB) (2004-2015), we identified patients with TC, ductal carcinoma in situ (DCIS), and pT1 estrogen receptor (ER)-positive invasive ductal carcinoma (IDC). We determined the rates of lymph node (LN) metastases, and the 5- and 10-year overall survival (OS) for patients with LN-negative versus LN-positive disease using the Kaplan-Meier method and propensity match analysis. In our institutional cohort, we identified 112 patients with T1 TC; only one (0.9%) patient had nodal involvement. In the NCDB cohort, we identified 6938 patients with T1 TC; 323 (4.7%) patients had axillary LN disease. The rate of axillary LN involvement for TC was comparable to that identified for patients with DCIS (4.2%), and much lower than that found for patients with grade I-III, T1, ER-positive IDC (20.5%), and patients with grade I, T1, ER-positive IDC (14.4%). There was no difference in 5-year (94.6% versus 95.4%, p = 0.67) and 10-year (83.9% versus 85.2%, p = 0.98) OS between TC patients with or without LN involvement. Kaplan-Meier survival curves even after propensity score matching suggest that tubular histology is independently associated with improved survival. T1 TC is an excellent starting point for deescalation of surgical axillary staging.

Abstract P3-06-07: Recurrent but not pathognomonic fusion genes in mucinous carcinomas of the breast

Abstract Introduction: Mucinous carcinoma of the breast (MCB) is a rare histologic subtype of estrogen receptor (ER)-positive invasive carcinoma, and is characterized by tumor cells floating in pools of mucin. Despite their characteristic histology, MCBs are heterogeneous at the genetic level and no driver genetic alterations have been identified. Notably, MCBs lack the common genetic alterations found in ER-positive invasive ductal carcinomas (i.e. 16q losses, 1q gains and PIK3CA mutations). Fusion genes have been reported in breast cancer, including highly recurrent or pathognomonic fusions genes, such as the ETV6-NTRK3 and MYB/MYBL1 rearrangements in secretory carcinoma and adenoid cystic carcinoma, respectively. In this study we sought to define whether MCBs would be underpinned by a pathognomonic fusion gene. Materials and methods: Seven pure mucinous A (hypocellular), seven pure mucinous B (hypercellular), and the mucinous component of a mixed MCB were microdissected, and subjected to RNA extraction followed by RNA-sequencing for fusion gene discovery. Read pairs supporting chimeric transcripts were identified using INTEGRATE, FusionCatcher and STARfusion. The Bayesian driver probability of the candidate fusion genes was annotated using OncoFuse. In-frame fusion gene candidates with a high driver probability were validated using orthogonal methods (RT-PCR and Sanger sequencing). Results: Our analysis identified fusion genes in 47% (7/15) of the MCBs analyzed (29% (2/7) of type A MCBs, 57% (4/7) type B MCBs and in the mucinous component of one mixed MCB). The OAZ1-CSNK1G2 and RFC4-LPP fusion genes were identified in 20% (3/15) and 13% (2/15) of the cases, respectively. The OAZ1-CSNK1G2 chimeric transcript results in the truncation of the kinase domain of CSNK1G2, which represses ER transactivation. The RFC4-LPP chimeric transcript leads to the fusion of exons 1-3 of RFC4 and exons 5-11 of LPP, where the LIM domains of LPP are conserved. LPP is a known partner of fusion genes identified in mesenchymal tumors and reported to mediate TGF-β induced breast oncogenesis via its LIM domain. Additional validated, potentially pathogenic fusion genes identified in MCBs involved kinases, phosphatases or regulators of tyrosine kinase receptor signaling, such as IRAK3-PPM1H (n=1), GIGYF2–GFRA3 (n=1) and PHF20-FAM217B (n=1). Conclusions: MCBs harbor fusion genes in almost half of the cases with two fusions being recurrent, involving primarily genes encoding kinases, phosphatases or receptor tyrosine kinase signaling regulators. Nevertheless, due to the lack of recurrence of a specific fusion gene, this special histologic type of breast cancer is unlikely to be underpinned by a highly recurrent/ pathognomonic pathogenic fusion gene. Citation Format: Pareja F, Gularte-Merida R, Da Cruz Paula A, Brown D, Geyer FC, Piscuoglio S, Marchio C, Vincent-Salomon A, Brogi E, Weigelt B, Reis-Filho JS. Recurrent but not pathognomonic fusion genes in mucinous carcinomas of the breast [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 P3-06-07.

Loss of function of NF1 is a mechanism of acquired resistance to endocrine therapy in lobular breast cancer.

BackgroundInvasive lobular carcinoma (ILC) as a disease entity distinct from invasive ductal carcinoma (IDC) has merited focused studies of the genomic landscape, but those to date are largely limited to the assessment of early-stage cancers. Given that genomic alterations develop as acquired resistance to endocrine therapy, studies on refractory ILC are needed.Patients and methodsTissue from 336 primary-enriched, breast-biopsied ILC and 485 estrogen receptor (ER)-positive IDC and metastatic biopsy specimens from 180 ILC and 191 ER-positive IDC patients was assayed with hybrid-capture-based comprehensive genomic profiling for short variant, indel, copy number variants, and rearrangements in up to 395 cancer-related genes.ResultsWhereas ESR1 alterations are enriched in the metastases of both ILC and IDC compared with breast specimens, NF1 alterations are enriched only in ILC metastases (mILC). NF1 alterations are predominantly under loss of heterozygosity (11/14, 79%), are mutually exclusive with ESR1 mutations [odds ratio = 0.24, P < 0.027] and are frequently polyclonal in ctDNA assays. Assessment of paired specimens shows that NF1 alterations arise in the setting of acquired resistance. An in vitro model of CDH1 mutated ER-positive breast cancer demonstrates that NF1 knockdown confers a growth advantage in the presence of 4-hydroxy tamoxifen. Our study further identified a significant increase in tumor mutational burden (TMB) in mILCs relative to breast ILCs or metastatic IDCs (8.9% >20 mutations/mb; P < 0.001). Most TMB-high mILCs harbor an APOBEC trinucleotide signature (14/16; 88%).ConclusionsThis study identifies alteration of NF1 as enriched specifically in mILC. Mutual exclusivity with ESR1 alterations, polyclonality in relapsed ctDNA, and de novo acquisition suggest a role for NF1 loss in endocrine therapy resistance. Since NF1 loss leads to RAS/RAF kinase activation, patients may benefit from a matched inhibitor. Moreover, for an independent subset of mILC, TMB was elevated relative to breast ILC, suggesting possible benefit from immune checkpoint inhibitors.

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

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

Abstract P3-04-05: Invasive lobular carcinoma cell lines utilize WNT4 signaling to mediate estrogen-induced growth

Abstract Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer representing 10-15% of newly diagnosed breast tumors. Over 90% of ILC are ER-positive, however, endocrine response and estrogen signaling are not well described in ILC. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than similar invasive ductal carcinoma (IDC) patients, and that ILC patients may not benefit from adjuvant tamoxifen. Additionally, we recently identified ILC-specific ER-target genes and de novo tamoxifen resistance driven by ER in ILC model systems. Based on these observations, we hypothesize that ILC-specific signaling pathways driven by ER mediate growth and endocrine resistance in ILC cells. Among ILC-specific estrogen-regulated genes in the ILC cell lines MDA MB 134VI (MM134) and SUM44PE (SUM44), Wnt signaling genes were highly differentially expressed. The secreted ligand WNT4 was the most strongly estrogen-induced gene in ILC cells. The frizzled receptor FZD7 is also strongly induced in ILC cells, but only transiently induced in the ER-positive IDC cell line MCF-7. Among IDC cell lines, either WNT4 or FZD7 is over-expressed in ER-positive or ER-negative cells, respectively. Conversely, MM134 and SUM44 over-express both WNT4 and FZD7. Also, we identified an ILC-specific ER binding site at WNT4; located in intron 1, this site contains a predicted estrogen response element. Direct WNT4 regulation and parallel regulation of pathway genes suggests that ER controls a WNT4 signaling pathway in ILC cells. In samples from the Cancer Genome Atlas, WNT4 and FZD7 are each over-expressed in ER-positive ILC versus IDC; co-expression is also enriched only in ILC. These observations suggest that a WNT4 signaling pathway may be specifically active in ILC tumors. To assess whether WNT4 is necessary for estrogen-induced growth, we used siRNA to knock down WNT4. Using either of two siRNAs, WNT4 knockdown completely blocks estrogen-induced growth in ILC cells, but not IDC cells. Consistent with this, WNT4 knockdown abrogated estrogen-regulation of a subset of ER-target genes in MM134 cells; induction or repression was inhibited by WNT4 knockdown prior to estrogen treatment. Thus, a subset of estrogen-induced gene expression changes is mediated by WNT4 signaling. Though Wnt signaling typically acts via the canonical, β-catenin-dependent pathway, we observed that β-catenin signaling is dysfunctional in ILC cells. Additionally, WNT4 over-expression or recombinant protein cannot activate canonical Wnt signaling in breast cancer cell lines. This suggests that WNT4 signaling mediates estrogen-induced growth in ILC cells via a novel non-canonical signaling pathway. Wnt signaling pathway genes including WNT4 are uniquely regulated in ILC cell lines, and are over-expressed in ILC tumors, suggesting that a WNT4-driven pathway may be active specifically in ILC. WNT4 is necessary for estrogen-mediated growth in ILC cells, and likely signaling via a novel non-canonical signaling pathway. Targeting WNT4 signaling represents a novel approach to modulate endocrine response specifically for ILC patients. Future studies will focus on identifying the signaling pathway controlled by WNT4 in order to identify novel therapeutic targets. Citation Format: Matthew J Sikora, Amir Bahreini, Caroline M Alexander, Steffi Oesterreich. Invasive lobular carcinoma cell lines utilize WNT4 signaling to mediate estrogen-induced growth [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 P3-04-05.

Abstract S6-06: The genomic landscape of male breast cancers

Abstract Background: Male breast cancer (MaBC) accounts for &amp;lt;1% of all breast cancers and its genomic landscape has yet to be characterized. The majority of MaBCs are ER-positive invasive ductal carcinomas of no special type and, unlike female breast cancers (FBC), rarely display HER2 gene amplification or a triple-negative phenotype. Given the relative rarity of MaBCs, treatment decisions for MaBC patients are often extrapolated from trials carried out with FBC patients. Here we employed targeted capture massively parallel sequencing to define the repertoire of somatic mutations and gene copy number alterations (CNAs) in MaBCs. Methods: Subtyping of the 64 MaBCs included in this study was performed by means of immunohistochemistry using the definitions described in the latest St. Gallen’s consensus report. DNA extracted from microdissected tumor and adjacent normal tissue were subjected to massively parallel targeting sequencing of all exons of 273 genes most frequently mutated in FBCs or directly related to DNA repair. Somatic mutations were defined using a combination of MuTect, SomaticSniper, MutationSeq and Haplotype Caller. Selected mutations were validated with Sequenom MassARRAY. CNAs were identified using Varscan2 and GISTIC2.0. Pathway and network analysis of mutations/CNAs was performed using Ingenuity Pathway Analysis and HOTNET. The genomic landscape of MaBCs was compared with that of FBCs of the same subtype analyzed as part of The Cancer Genome Atlas project. Results: All MaBCs were ER-positive and HER2-negative. Using the St. Gallen’s criteria, 37.5% and 62.5% were classified as luminal A-like or luminal B-like, respectively. The genes most frequently mutated in MaBCs were PIK3CA, GATA3, FLG and PLEC, with PIK3CA being the only significantly mutated gene as defined by MutSigCV (q=0.003). CNA analysis revealed recurrent gains of 1q and 8q and loss of 16q. GISTIC2.0 identified significantly recurrent high-level amplifications in 1q25.3, 8p11 (FGFR1, ZNF703), 8q24.3 (DEPTOR), 17q23 (PPM1D) and 15q26 (IGF1R) and deletions in 11q22 (ATM) and 21q22.12 (RUNX1). HOTNET analysis of the genes mutated and/or targeted by gene amplifications in MaBCs revealed significantly altered subnetworks involving genes related to DNA repair, PI3K and FGF signaling pathways. The most frequently mutated genes in luminal A-like MaBCs were PIK3CA and MLL3, while those of luminal B-like MaBCs were PIK3CA and GATA3. MLL3 and GATA3 mutations were only found in luminal A-like MaBCs (p=0.039) and luminal B-like MaBCs (p=0.048), respectively. Although the mutational landscapes of MaBCs and luminal FBCs were qualitatively similar, PIK3CA and TP53 were less frequently mutated in MaBCs (p&amp;lt;0.001), whereas genes found to be recurrently mutated in FBCs, such as MAP2K4 and NCOR1, were not mutated in MaBCs. Conclusions: MaBCs are preferentially of luminal subtype and are characterized by recurrent mutations in PIK3CA, GATA3, FLG and PLEC. Genetic alterations in MaBCs often target DNA repair and FGF signaling pathways. The known drivers of luminal FBCs appear to be less frequently altered in MaBCs. Given these important differences between MaBCs and FBCs, caution should be exercised in the extrapolation of biologic and clinical implications from studies in FBCs to the management of MaBCs. Citation Format: Salvatore Piscuoglio, Melissa Murray, Charlotte KY Ng, Elena Guerini Rocco, Luciano G Martelotto, Francois-Clement Bidard, Carey A Eberle, Nicola Fusco, Rita A Sakr, Leticia De Mattos-Arruda, Raymond Lim, Timour Baslan, James Hicks, Tari A King, Edi Brogi, Larry Norton, Britta Weigelt, Clifford A Hudis, Jorge S Reis-Filho. The genomic landscape of male breast cancers [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 S6-06.

Immunolocalization of thymidylate synthase as a favorable prognostic marker in estrogen receptor-positive breast carcinoma.

Thymidylate synthase (TS) is an enzyme involved in DNA synthesis, and it is a target for 5-fluorouracil. Previous studies have demonstrated that TS is a potent estrogen-induced gene in breast carcinoma cells, suggesting the importance of TS in estrogen-receptor (ER)-positive breast carcinoma. TS immunolocalization has been reported previously, but the clinicopathological significance of TS in ER-positive breast carcinoma still remains unclear. We immunolocalized TS in 178 breast carcinoma tissues in total, and examined its significance according to the ER-status. TS status was positive in 58% of ER-positive ductal carcinoma in situ (DCIS) cases, and it was significantly associated with the Ki-67 and progesterone receptor (PR). Moreover, in ER-positive DCIS patients who received aromatase inhibitor (AI) before surgery, TS immunoreactivity was significantly decreased after AI treatment. In ER-positive invasive ductal carcinoma (IDC) cases, TS status was significantly associated with PR, and it turned out an independent favorable prognostic factor for recurrence of the patients by multivariate analysis. On the other hand, TS status was positively correlated with pathological T factor in ER-negative IDC cases, and tended to have a worse prognosis for disease-free survival of the patients. These results suggest that TS expression is mainly regulated by estrogen in ER-positive breast carcinoma and is associated with estrogen-mediated proliferation. TS status is a favorable prognostic factor in ER-positive IDC patients, which is different from the ER-negative cases.

Correlation between 18F-fluorodeoxyglucose Positron Emission Tomography/computed Tomography and Clinicopathological Features in Invasive Ductal Carcinoma of the Breast.

We evaluated the usefulness of preoperative 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) examinations to predict the pathological features in primary breast cancer. In particular, we evaluated the correlation between the maximum standardized uptake values (SUVmax) obtained by 18F-FDG PET/CT and the Ki67 expression in estrogen receptor (ER)-positive invasive ductal carcinoma (IDC). Primary IDC patients operated between March 2009 and July 2013 at Okayama University Hospital were enrolled. We evaluated the correlations between the SUVmax and age, postoperative pT, histological grade, lymph vascular invasion, status of hormone receptor, human epidermal growth factor receptor 2 (HER2), Ki67 expression and node status. The Ki67 expression was classified as high (＞ 14%) versus low (< 14%). We enrolled 138 patients with IDC. Their median SUVmax was 3.85 (range:0-52.57). In a univariate analysis, the SUVmax was significantly related to age, pT, histological grade, lymphovascular invasion, hormone receptor status, HER2 status, node status and Ki67. In the 113 patients with ER-positive IDC, there was a significant correlation between Ki67 and SUVmax (p ＝ 0.0030). The preoperative 18F-FDG PET/CT results of IDC patients had significant relationships with pathological status parameters. The determination of the preoperative SUVmax might help classify Luminal A and Luminal B patients among luminal-type breast cancer patients.

Identification of cellular and genetic drivers of breast cancer heterogeneity in genetically engineered mouse tumour models

The heterogeneous nature of mammary tumours may arise from different initiating genetic lesions occurring in distinct cells of origin. Here, we generated mice in which Brca2, Pten and p53 were depleted in either basal mammary epithelial cells or luminal oestrogen receptor (ER)-negative cells. Basal cell-origin tumours displayed similar histological phenotypes, regardless of the depleted gene. In contrast, luminal ER-negative cells gave rise to diverse phenotypes, depending on the initiating lesions, including both ER-negative and, strikingly, ER-positive invasive ductal carcinomas. Molecular profiling demonstrated that luminal ER-negative cell-origin tumours resembled a range of the molecular subtypes of human breast cancer, including basal-like, luminal B and 'normal-like'. Furthermore, a subset of these tumours resembled the 'claudin-low' tumour subtype. These findings demonstrate that not only do mammary tumour phenotypes depend on the interactions between cell of origin and driver genetic aberrations, but also multiple mammary tumour subtypes, including both ER-positive and -negative disease, can originate from a single epithelial cell type. This is a fundamental advance in our understanding of tumour aetiology.

Abstract P5-09-03: Endocrine response in invasive lobular carcinoma is characterized by unique estrogen-mediated gene expression and de novo tamoxifen resistance

Abstract Invasive lobular carcinoma (ILC) represents ∼10% of newly diagnosed breast tumors, or ∼30,000 cases annually in the US. However, ILC-specific signaling and endocrine responsiveness are not well characterized. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than invasive ductal carcinoma (IDC) patients with similar biomarkers, and that ILC patients may not benefit from adjuvant tamoxifen. We hypothesize that estrogen receptor-alpha (ER) regulated gene expression is unique in ILC cells and drives endocrine resistance. The ER-positive ILC cell lines MDA MB 134VI and SUM44PE were used as in vitro models of cell growth and ER-regulated gene expression in response to estradiol (E2). To examine the ER-regulated transcriptome, we performed gene expression microarray analyses and ER ChIP-Seq following E2 treatment. In parallel, E2 response was assessed in vivo in the primary ILC xenograft HCI-013. Response to endocrine therapies, tamoxifen (Tam), 4-hydroxytamoxifen (4OHT), endoxifen (Bx), and fulvestrant (ICI), were also examined in ILC cell lines. We observed that E2 induced growth and ER target gene expression in MDA MB 134VI and SUM44PE. We compared our ILC microarray data to published data from ER-positive IDC cell lines (MCF-7, T47D, BT474), and identified 254 genes that were E2-regulated in all 5 cell lines (e.g. GREB1, MYC). 915 genes were E2-regulated only in both ILC cell lines. Consistent with this, roughly half of ER binding sites identified in MDA MB 134VI ChIP-Seq were unique versus published MCF-7 data. We chose a subset of ILC-specific and common E2-regulated genes (n = 107) to assess in vivo using Nanostring gene expression analyses of HCI-013. E2-regulation was observed for 32/107 target genes (30%), suggesting that these genes may be E2-regulated in vivo in ILC patient tumors. Consistent with clinical data, both ILC cell lines presented de novo tamoxifen resistance. SUM44PE were growth-inhibited by ICI, but unaffected by Tam, 4OHT, or Bx. Similarly, ICI blocked E2-induced growth in MDA MB 134VI, but Tam, 4OHT, and Bx acted as partial agonists, inducing ∼25% growth. Partial agonism was not limited to tamoxifen, as other SERMs (e.g. raloxifene) also induced growth. We then measured ER-regulated gene expression in MDA MB 134VI following tamoxifen treatment. Tam, 4OHT, and Bx acted as agonists for 38/107 genes, whereas ICI acted as an antagonist. All 38 genes were E2-repressed targets (e.g. CCNG2), suggesting that ER-mediated gene repression may be critical to tamoxifen-resistance in ILC. Finally, we observed that FGFR1, frequently amplified in ILC, may be critical for ILC cell survival in the presence of tamoxifen. These data support the hypothesis that unique ER-mediated gene expression in ILC cells drives endocrine resistance. The de novo tamoxifen resistance observed in ILC cells may correlate with the worse outcomes in ILC patients recently reported. We hypothesize that genes regulated by tamoxifen as an agonist may play a role in tamoxifen-induced growth or serve as biomarkers of resistance. Targeting growth factor signaling using FGFR1 inhibitors may block survival pathways required by ILC cells and reverse tamoxifen resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-03.

Abstract B060: Invasive lobular carcinoma cells express unique estrogen-mediated genes and are de novo tamoxifen resistant

Abstract Invasive lobular carcinoma (ILC) represents ~10% of newly diagnosed breast tumors, or ~30,000 cases annually in the US. However, ILC-specific signaling and endocrine responsiveness are not well characterized. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than invasive ductal carcinoma (IDC) patients with similar biomarkers, and that ILC patients may not benefit from adjuvant tamoxifen. We hypothesize that estrogen receptor-alpha (ER) regulated gene expression is unique in ILC cells and drives endocrine resistance. The ER-positive ILC cell lines MDA MB 134VI and SUM44PE were used as in vitro models of cell growth and ER-regulated gene expression in response to estradiol (E2). To examine the ER-regulated transcriptome, we performed gene expression microarray analyses and ER ChIP-Seq following E2 treatment. In parallel, E2 response was assessed in vivo in the primary ILC xenograft HCI-013. Response to endocrine therapies, tamoxifen (Tam), 4-hydroxytamoxifen (4OHT), endoxifen (Bx), and fulvestrant (ICI), were also examined in ILC cell lines. We observed that E2 induced growth and ER target gene expression in MDA MB 134VI and SUM44PE. We compared our ILC microarray data to published data from ER-positive IDC cell lines (MCF-7, T47D, BT474), and identified 254 genes that were E2-regulated in all 5 cell lines (e.g. GREB1, MYC). 915 genes were E2-regulated only in both ILC cell lines. Consistent with this, roughly half of ER binding sites identified in MDA MB 134VI ChIP-Seq were unique versus published MCF-7 data. We chose a subset of ILC-specific and common E2-regulated genes (n=107) to assess in vivo using Nanostring gene expression analyses of HCI-013. E2-regulation was observed for 32/107 target genes (30%), suggesting that these genes may be E2-regulated in vivo in ILC patient tumors. Consistent with clinical data, both ILC cell lines presented de novo tamoxifen resistance. SUM44PE were growth-inhibited by ICI, but unaffected by Tam, 4OHT, or Bx. Similarly, ICI blocked E2-induced growth in MDA MB 134VI, but Tam, 4OHT, and Bx acted as partial agonists, inducing ~25% growth. Partial agonism was not limited to tamoxifen, as other SERMs (e.g. raloxifene) also induced growth. We then measured ER-regulated gene expression in MDA MB 134VI following tamoxifen treatment. 4OHT acted as an agonist for E2-induced genes including SNAI1 and MYC. Further, for the majority of E2-repressed genes assessed including FOXO1, 4OHT also repressed gene expression, suggesting that ER-mediated gene repression may be critical to tamoxifen-resistance in ILC. Finally, we observed that FGFR1, frequently amplified in ILC, may be critical for ILC cell survival in the presence of tamoxifen. These data support the hypothesis that unique ER-mediated gene expression in ILC cells drives endocrine resistance. The de novo tamoxifen resistance observed in ILC cells may correlate with the worse outcomes in ILC patients recently reported. We hypothesize that genes regulated by tamoxifen as an agonist may play a role in tamoxifen-induced growth or serve as biomarkers of resistance. Targeting growth factor signaling using FGFR1 inhibitors may block survival pathways required by ILC cells and reverse tamoxifen resistance. Citation Format: Matthew J. Sikora, Kristine L. Cooper, Amir Bahreini, Soumya Luthra, Uma R. Chandran, Guoying Wang, David J. Dabbs, Alana L. Welm, Steffi Oesterreich. Invasive lobular carcinoma cells express unique estrogen-mediated genes and are de novo tamoxifen resistant. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B060.

Loss of heterozygosity at 13q13 and 14q32 predicts BRCA2 inactivation in luminal breast carcinomas

BRCA2 is the major high-penetrance predisposition gene for luminal (estrogen receptor [ER] positive) breast cancers. However, many BRCA2 mutant carriers lack family history of breast/ovarian cancers and do not benefit from genetic testing. Specific genomic features associated with BRCA2 inactivation in tumors could help identify patients for whom a genetic test for BRCA2 may be proposed. A series of ER-positive invasive ductal carcinomas (IDCs) including 30 carriers of BRCA2 mutations and 215 control cases was studied by single-nucleotide polymorphism (SNP) arrays. Cases and controls were stratified by grade and HER2 status. Independently, 7 BRCA2 and 51 control cases were used for validation. Absolute copy number and Loss of heterozygosity (LOH) profiles were obtained from SNP arrays by the genome alteration print (GAP) method. BRCA2 tumors were observed to display a discriminatively greater number of chromosomal breaks calculated after filtering out and smoothing <3 Mb variations. This argues for a BRCA2-associated genomic instability responsible for long-segment aberrations. Co-occurrence of two genomic features-LOH of 13q13 and 14q32-was found to predict BRCA2 status with 90% of sensitivity and 87% of specificity in discovery series of high-grade HER2-negative IDCs and 100% of sensitivity and 88% of specificity in an independent series of 58 IDCs. Estimated positive predictive value was 17.2% (confidence interval: 6.7-33.5) in the whole series. In conclusion, the simplified BRCA2 classifier based on the co-occurrence of LOH at 13q13 and 14q32 could provide an indication to test for BRCA2 mutation in patients with ER-positive IDC.

Clinical benefit from neoadjuvant chemotherapy in oestrogen receptor-positive invasive ductal and lobular carcinomas

Background:The aim of this study was to compare clinical and pathological outcomes after neoadjuvant chemotherapy between oestrogen receptor (ER)-positive invasive pure lobular carcinoma (ILC) and invasive ductal carcinoma (IDC).Methods:This analysis included 1895 patients (n=177 ILC; n=1718 IDC), with stage I–III breast cancer, who received neoadjuvant chemotherapy. Clinical and pathological response rates, the frequency of positive surgical margins and rate of breast-conserving surgery were compared.Results:There was a trend for fewer good clinical responses in ILC compared with IDC. Tumour downstaging was significantly less frequent in ILC. Positive or close surgical resection margins were more frequent in ILC, and breast-conserving surgery was less common (P<0.001). These outcome differences remained significant in multivariate analysis, including tumour size, nodal status, age, grade and type of chemotherapy. Invasive pure lobular carcinoma was also associated with a significantly lower pathological complete response (pCR) rate in univariate analysis, but this was no longer significant after adjusting for tumour size and grade.Conclusion:Neoadjuvant chemotherapy results in lower rates of clinical benefit, including less downstaging, more positive margins and fewer breast-conserving surgeries in ER-positive ILC compared with ER-positive IDC. Pathological complete responses are rare in both groups, but do not significantly differ after adjusting for other variables.

Abstract 75: NRG1 and RARβ hypermethylation in breast cancer progression

Abstract We have developed an in vitro model of breast cancer progression that consists of four derived ER-negative PR-negative cell lines: (a) the normal-like breast epithelial cell line MCF-10F; (b) the transformed trMCF cells; (c) the invasive bsMCF cells and; (d) the tumorigenic caMCFs cells which shown all characteristics of a fully malignant breast cancer cells [1,2]. Briefly, the normal human breast epithelial MCF-10F cells were treated with high concentration of 17β-estradiol (70 nM) giving origin to trMCF cells; trMCF cells were seeded in Boyden chambers and the cells that crossed the membrane were selected and expanded giving origin to bsMCF. The bsMCF cells were injected in the mammary fat pad of SCID mice and they induced tumors that were poorly differentiated adenocarcinomas that were ESR, progesterone receptor (PR) and ERBB2 negatives. Cells were isolated from 4 different tumors growth in four animals and they gave origin to the tumorigenic caMCFs cells (caMCF1, caMCF2, caMCF3 and caMCF4) [1,2]. This in vitro model of breast cancer progression was used to study DNA-methylation changes at the different stages using sensitive methylation specific PCR (MSP) and, we found that NRG1 and RARβ showed progressive changes in their methylation pattern. NRG1 and RARβ were unmethylated in MCF-10F and trMCF cells, becoming hypermethylated in the invasive (bsMCF) and tumor (caMCF) stages. The clinical relevance of these findings were pursued by studying NRG1 and RARβ methylation in 17 cases of invasive ductal carcinomas (IDC) and 11 normal breast tissue samples (controls). NRG1 was hypermethylated in 14 out of 17 IDC (82.4%) vs. 2 out of 10 (20%) controls. All the ER-negative PR-negative tumors (10/10) showed NRG1 hypermethylated. From the seven ER-positive IDC, only 4 (57%) showed NRG1 hypermethylated. RARβ was methylated in 9 out of 16 IDC (56.3%) vs. 2 out of 11 (18.2%) controls. Most of the ER-negative PR-negative invasive ductal tumors showed RARβ hypermethylated (9/10). None of the ER-positive IDC shown RARβ hypermethylated. Altogether our data shown that the majority (90%) of the ER-negative PR-negative invasive ductal carcinomas shown hypermethylation of both NRG1 and RARβ. None of the ER-positive invasive ductal carcinomas showed RARβ hypermethylation and, only 57% showed hypermethylation of NRG1. (This work was supported by The Pennsylvania Breast Cancer Coalition). REFERENCES [1] S.V. Fernandez, K.E. Snider, Y.Z. Wu, I.H. Russo, C. Plass and J. Russo DNA methylation changes in a human cell model of breast cancer progression, Mutat Res 688 (2010) 28-35. [2] Y. Huang, S.V. Fernandez, S. Goodwin, P.A. Russo, I.H. Russo, T.R. Sutter and J. Russo Epithelial to mesenchymal transition in human breast epithelial cells transformed by 17beta-estradiol, Cancer Res 67 (2007) 11147-11157. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 75. doi:10.1158/1538-7445.AM2011-75

Abstract P2-06-05: p27 Expression in Hormone Sensitive Breast Carcinomas, Prior and after Short Term Hormone Treatment: A Prospective Placebo Double-Blind Study

Abstract Background: Mutations in genes that regulate the cell cycle are found in most human cancers. In addition, there are other cell cycle genes, including those encoding p27, that contribute to tumor progression but are rarely mutated. p27 is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors and it is present in high levels in quiescent cells, but its levels decline when cells proliferate in response to mitogenic signals. Various functions have been attributed to p27, including promotion of apoptosis and regulation of drug resistance. p27 is functionally inactivated in most of human cancers through enhanced p27 proteolysis, sequestration by cyclin D/cdk complexes, and by cytoplasmic mislocalization. There is evidence from preclinical studies that p27 is essential for cell cycle arrest by tamoxifen and other antiestrogenic drugs. Objectives: To compare the expression of nuclear and cytoplasmic p27, cyclin D1, Ki-67, p53, HER2/ErbB2, ER and PR in hormone sensitive invasive ductal carcinomas (IDC) in postmenopausal women, prior and after tamoxifen and anastrozole in a short term treatment. Methods: Seventy one patients were enrolled in this study and fifty-eight were elegible for analyses. All patients had palpable ER-positive IDC (stage II and III) and were double-blind randomized in a prospective placebo controlled study with short term hormone treatment (HT) groups for twenty-six days prior to surgical treatment: Anastrozole 1mg/day (n= 18), Placebo (n=25) and Tamoxifen 20mg/day (n= 15). Pre and post HT samples were disposed in tissue micro array blocks and submitted to immunohistochemical assay. Biomarkers status (nuclear and cytoplasmic p27, cyclin D1, Ki-67, p53, HER2/ErbB2, ER and PR) were obtained comparing each immunohistochemical evaluation of pre and post-surgery samples using semi-quantitative Allred's method for nuclear biomarkers, and scored based on a four-point intensity based system: negative (no stain), weakly positive (1+), mild positive (2+) and strongly positive (3+). Statistical analysis were performed using the GEE (General Estimation Equations) and ANOVA tests with significant p ≥0.05. Results: The patients mean age was 66.25 ± 9.95 years. The mean tumor size was 4.0 ± 1.06 cm. There was no statistically significant correlation between nuclear p27 expression and tumor size or histologic grade. Before HT, 38 (62.5%) patients had positive nuclear p27 expression, 5 (8.6%) patients had positive cytoplasmic p27 expression, and after treatment their expression was increased in all groups. There was a positive correlation between Ki-67, cyclin D1, p53 and nuclear p27 prior to HT. After HT, there was a positive correlation between Ki-67, cyclin D1, p53, HER2/ErbB2, ER, and nuclear p27. Conclusions: There was an increment of nuclear and cytoplasmic p27 after short term treatment, regardless of the drug used. The positive correlation between nuclear p27 and poor prognostic markers was unexpected. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-06-05.

Role of the BCA2 Ubiquitin E3 Ligase in Hormone Responsive Breast Cancer

The BCA2 protein contains a RING H2 finger and a Zn finger near the N-terminus and has E3 ligase activity. RING finger proteins play critical roles in mediating the transfer of ubiquitin and ubiquitin like modifiers to heterologous substrates as well as to the RING finger proteins themselves. Protein modification by ubiquitin and small ubiquitin-related modifier (SUMO) plays a pivotal role in protein homeostasis and is critical to regulating basic cellular processes such as proliferation, differentiation, apoptosis, intracellular signaling, and gene-transcriptional regulation. The addition of ubiquitin or SUMO can modulate the ability of proteins to interact with their partners, alter their patterns of sub-cellular localization and control their stability. It is clear that SUMO influences many different biological processes however recent data suggest that it is specifically important in the regulation of transcription. BCA2 is an E3 ligase that interacts with the SUMO conjugating enzyme Ubc9. It could therefore function as an E3 in the sumoylation of various transcription factors. We have found that the BCA2 is co-expressed with the estrogen receptor in 74% of ER-positive invasive ductal carcinomas from a 635 member breast cancer cohort (p = 0.004). At the cellular level, BCA2 co-localizes with ER and it appears that at the transcriptional level BCA2 mRNA expression is regulated by estrogen. Bioinformatic analysis of the BCA2 promoter region revealed ER and PR binding sites as well as that of other more general transcription factors. The data presented here provides an overview of the potential involvement of the BCA2 in hormone responsive breast cancer and opens up avenues that should be exploited to better understand the regulation of ER expression, growth of breast cancer cells, and the importance of BCA2.

Correlation Between Anthropometric Measures and Biomarker Changes After Neoadjuvant Therapy With Tamoxifen or Anastrozole in Postmenopausal Women With Breast Cancer.

BackgroundEpidemiological studies have reported positive associations between anthropometric measures and risk for developing breast cancers that express hormone receptors and associated mortality. However, the impact of nutritional status on the molecular response to endocrine therapy has yet to be described.MethodsBody mass index (BMI), waist circumference (WC), hip circumference (HP), and waist-to-hip ratio (WHR) were measured in patients with invasive ductal carcinoma (IDC) before and after neoadjuvant treatment with either tamoxifen or anastrozole, and a possible correlation with prognostic factors, as estrogen receptor (ER), progesterone receptor (PgR), and proliferative index (Ki-67), was analyzed. Fifty-seven patients with palpable ER-positive IDC were randomized into three neoadjuvant treatment groups and received anastrozole or placebo or tamoxifen for twenty-one days. Biomarker status was obtained by comparing the immunohistochemical evaluation of samples collected before and after treatment, using the Allred scoring system. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS).Results and ConclusionsAfter treatment, the anastrozole group showed reduced ER and PgR expression (p < 0.05), and both the anastrozole and tamoxifen groups showed lower Ki-67 status. A significant reduction in PgR positivity (p < 0.05) was found in women with large WC and HC who were treated with anastrozole. Reduction in PgR positivity also tended to be associated with BMI (p = 0.09) in the anastrozole group. BMI, WC, HC and WHR correlated neither with biomarker levels in the tamoxifen and placebo groups nor with ER and Ki-67 status in the anastrozole group after primary endocrine treatment.