Inflammatory Breast Cancer Tumor Research Articles

Abstract PO2-24-03: Soluble E-cadherin promotes inflammatory breast cancer tumorigenesis via PDIA4-mediated suppression of ferroptosis

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer known for its rapid progression and high metastatic potential without known distinctive drivers. Currently, there are no FDA-approved targeted therapies specifically for IBC patients, highlighting the urgent need for novel and effective treatments. Through our investigation of metastatic xenograft IBC sublines, we have identified soluble E-cadherin (sEcad), an extracellular proteolytic fragment of full-length E-cadherin, as a protein associated with IBC tumor progression. We hypothesize that sEcad promotes IBC tumorigenesis by suppressing ferroptosis. Methods: MDA-IBC3 (ER–/HER2+) and SUM149 (ER–/HER2–) IBC cell lines were used in this study. Stable overexpression of sEcad in IBC cell lines was achieved using lentiviral vectors. Cell death for ferroptosis was measured by propidium iodide staining using flow cytometry. Mass spectrometry and Bio-ID-based proteomics assays were used to identify sEcad-interacting proteins and potential mechanisms. For in vivo studies, control and sEcad -overexpressing SUM149 and MDA-IBC3 cells were injected into the cleared mammary fat pads of SCID/Beige mice and tumor growth was monitored via caliper measurements. Serum sEcad levels from IBC patients (n=301) were analyzed by ELISA. Results: High serum sEcad levels in IBC patients correlated with poorer OS (p=0.02) and earlier development of metastasis (p=0.006). Overexpression of sEcad in IBC cell lines enhanced cell proliferation and colony formation in vitro. Mice injected with sEcad-overexpressing SUM149 and MDA-IBC3 cells had significantly higher tumor growth rates compared to controls (SUM149: p=0.007; MDA-IBC3: p=0.006). Mechanistically, mass spectrometry and Bio-ID assays identified Protein Disulfide Isomerase Family A Member 4 (PDIA4) as a novel binding partner of sEcad, which was validated through co-immunoprecipitation. Additionally, sEcad increased the expression of PDIA4, an enzyme that plays a role in cellular redox regulation and protein folding. PDIA4 has been shown to regulate key players of ferroptosis such as ATF4, SLC7A11. Our results showed that, compared to controls, sEcad-overexpressing IBC cells displayed significant resistance to ferroptosis inducers, including RSL3, FIN56, and sulfasalazine. Knockdown of PDIA4 in sEcad high-expressing cells significantly increased their sensitivity to ferroptosis-promoting drugs. Moreover, we found that PDIA4 regulates ATF4 to impact ferroptosis-mediated cell death in IBC cells. Conclusions: Our study shows that sEcad affects ferroptosis through PDIA4, impacting tumorigenesis in IBC. These findings uncover a novel and pivotal role for sEcad in IBC tumor growth and progression, providing new insights and potential therapeutic targets for IBC patients. Citation Format: Xiaoding Hu, Yun Xiong, Emilly Villodre, Juhee Song, Debu Tripathy, Wendy Woodward, Savitri Krishnamurthy, Junjie Chen, Bisrat Debeb. Soluble E-cadherin promotes inflammatory breast cancer tumorigenesis via PDIA4-mediated suppression of ferroptosis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-03.

Abstract PO1-25-04: PTN-positive luminal progenitors induce angiogenesis and metastasis in inflammatory breast cancer

Abstract Background Inflammatory breast cancer (IBC) is a lethal subtype of breast cancer characterized by rapidly arising diffuse erythema and edema of the overlying skin that was thought to contribute to rapid metastasis. Dissecting the intricate cellular ecosystems within tumors and affected skin and their complex interplay may be pivotal in unraveling the aggressive nature of IBC. Methods Integrative analysis containing single-cell RNA sequencing, bulk RNA sequencing, and whole exome sequencing, was performed on paired tumor and skin samples from 17 treatment-naive IBC patients and 5 non-IBC patients from multiple centers. Meanwhile, paraffin sections and serum specimens from two centers were conducted to validate the omics results. Additionally, public single-cell transcriptomic data of non-IBC tumors and normal mammary glands, RNA microarray data from IBC cases, and the Cancer Genome Atlas (TCGA) database were utilized to validate the characteristics of IBC microenvironment. The regulatory mechanism was verified by in vitro experiments. Results We delineated the landscapes of IBC tumor and affected skin microenvironment at the single-cell level and demonstrated that the luminal progenitors (LP) cells highly expressing pleiotrophin (PTN) were most significantly enriched in IBC tumors compared to non-IBC tumors and normal mammary glands, in two independent cohorts. Moreover, the serum concentrations of PTN were significantly higher in IBC patients compared with non-IBC patients. Notably, PTN secreted by PTN+ LP cells interacted with Neuropilin 1(NRP1) receptor on endothelial tip cells in both tumor and affected skin of IBC. Moreover, in vitro assay demonstrated knocking down the expression of NRP1 on human umbilical vein endothelial cells significantly blocked PTN-induced migration, tube formation and sprouting, which might be independent of VEGFA. In addition, two immature phenotypes of PVL_MMP9 and PVL_CCL19 cells recruited by endothelial tip cells via PDGFB/PDGFRB axis were accumulated in affected skin of IBC, which might be associated with promotion of malignant cell metastasis. Finally, we revealed that malignant cells in affected skin of IBC had a higher ability of epithelial-mesenchymal transition than IBC intratumoral malignant cells and identified enhanced crosstalk between immature PVL cells and malignant cells in the skin affected with IBC , through TNFSF12-TNFRSF12A, TGFB-TGFBR1/TGFBR2, MDK-SDC1/4/NCL/(ITGA6+ITGB1). Conclusion We identified a unique, non-malignant epithelial cell subpopulation PTN+ LP cells, which induce massive angiogenesis via the PTN-NRP1 axis, recruit the immature phenotype PVL cells accumulated in affected skin of IBC thus promoting tumor progression. Our findings offer comprehensive insights into the pathogenesis of IBC and uncover a previously unknown, promising therapeutic target. Patient of discovery cohort details Clinical and pathology details for inflammatory breast cancer(IBC) and non-IBC patients analysed by single-cell RNA sequencing(scRNA-seq), bulk RNA sequencing(bulk RNA-seq) and whole exome sequencing(WES) in this study. Clinicalpathological feature of validation cohort Citation Format: Mengmeng Zhang, Kaiwen Zhou, Ting Liu, Filipa Lynce, Filipa Lynce, Wendy Chen, Qianjun Chen, Xiaoping Li, Zhongyu Yuan, Juan Xu, Zhenhai Cai, Jianping Guo, Nan Shao, Ying Lin. PTN-positive luminal progenitors induce angiogenesis and metastasis in inflammatory breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-04.

High-fat diet, but not duration of lactation, increases mammary gland lymphatic vessel function and subsequent growth of inflammatory breast cancer cells

Inflammatory breast cancer (IBC) presents as rapid-onset swelling and breast skin changes caused by tumor emboli in the breast and breast skin lymphatics. IBC has been linked with obesity and duration of breastfeeding, but how these factors affect IBC tumor progression is not clear. We modeled the simultaneous effects of diet and weaning in mice on in vivo lymphatic function; on IBC tumor growth; and on aspects of the mammary gland microenvironment before and after IBC (SUM149) xenograft inoculation. We hypothesized that weaning status and diet would have synergistic effects on lymphatic function and the breast microenvironment to enhance IBC tumor growth. Changes in lymphatic structure and function were characterized with in vivo near-infrared fluorescence (NIRF) imaging. Mice were fed either a high-fat diet (HFD; 60 kcal%) or a normal/low-fat diet (LFD; 10 kcal%), bred twice, and subjected to either normal-duration nursing (NW) or forced weaning (FW). SUM149 IBC tumors were implanted at 14 months; images were obtained before and after implantation. Multiparous mice fed HFD showed increased pre-tumor lymphatic pulsing in both the FW and NW groups relative to mice fed LFD. HFD promoted tumor growth independent of weaning time (P = 0.04). Pre-tumor lymphatic pulsing was associated with tumor volume at 8 weeks (P = 0.02) and was significantly correlated with expression of the lymphatic tracking ligand CCL21 (P = 0.05, Table 1). HFD significantly increased the numbers of monocyte-derived IBA1+, CD163+, and CD11c+ cells (P < 0.0001, P < 0.0001, P = 0.0005) in the contralateral, non-tumor-bearing mammary gland. Numbers of lymphangiogenic podoplanin+/IBA1+ macrophages were increased in the ducts of HFD and FW mice (all P < 0.003). HFD in nulliparous mice had a similar increase in lymphatic pulsing at 14 weeks (P = 0.006), indicating that this functional change was independent of parity. We conclude that HFD induced increases in mammary gland lymphatic function, assessed as pulsing rate before tumor initiation, and correlated with inflammation in the mammary gland and increased SUM149 tumor growth. The relationship between diet, lymphatic pulsing, and tumor growth warrants further investigation.

Abstract P3-08-06: Monitoring Lymphovascular Invasion and Tumor Growth of Inflammatory Breast Cancer in a Murine Lymphatic Reporter Window Chamber Model

Abstract Background: Lymphovascular invasion (LVI) is a major route of metastatic dissemination and recent studies indicate its value as an independent prognostic indicator for advanced breast, colorectal, squamous cell, prostate, brain cancers. LVI is a clinicopathological hallmark of inflammatory breast cancer (IBC), an understudied and most lethal breast cancer. IBC is often misdiagnosed due to an absence of a solid mass and its unique presentation of diffuse tumor cell clusters/emboli in the dermal lymphatics. Widely used mammary tumor implantation models coupled with bioluminescence or fluorescence imaging to monitor tumor growth kinetics are ineffective for evaluating spatial and temporal changes in growth and migration patterns of individual tumor cells and clusters within their microenvironmental context. The goal of this study was to develop a murine model to simulate the unique clinicopathological features of IBC patients and to assess both qualitatively and quantitatively local tumor growth, motility, and LVI. Methods: To specifically facilitate visualization of lymphatic and endothelial vessels along with tumor-vessel interactions, we generated a transgenic nude mice model (ProxTom RFP Nu/Nu) wherein, the mice exhibit red, fluorescent lymphatics [tdTomato fluorophore under control of a Prox1 promoter, which encodes a transcription factor (prospero-related homeobox 1) necessary for the formation and maintenance of lymphatic vessels]. Next, we employed a surgical technique, wherein a window chamber is placed on the dorsal skinfold of mice, which allows for microscopic examination of implanted tumor cells and ability to track dynamic changes of the tumor in its local microenvironment from the time of implantation up to 10 days. Patient-derived IBC or PDX stably transfected to express green, red fluorescent and/or dual tagged with luciferase reporters were transplanted in mice bearing window chambers. Intravital fluorescence microscopy and IVIS imaging were used to serially quantify local tumor growth, motility, length density of lymph and blood vessels, and degree of tumor cell lymphatic invasion over 0-140h. Results: Multichannel optical imaging of the window chamber in the ProxTom RFP Nu/Nu mice demonstrated co-localization of IBC tumor cells and lymphatics. Diffuse tumor cells were observed along regions of lymphatic vessels both proximal and distal to the primary tumor site. However, measurement of blood and lymph vessel density showed no significant change over time. Next, these datasets were used for quantitative analysis by setting the tumor cell channel (GFP) at a threshold to count any clusters greater than 50 pixels2 (~0.0013mm2) and with greater than the mean + 2 standard deviations of the background signal while avoiding noise/artifacts from very small regions (&amp;lt;~0.001mm2). This allowed for computation of the total tumor area including average area of each cluster. In addition, the area moment, which describes the basic directional growth pattern of the tumor cells, was quantified by multiplying a cluster distance from the center of mass by its area. Conclusions: A key novel finding from structured illumination imaging data was the observation of LVI occurring early, similar with the clinical presentation in IBC patients. This model was able to effectively track tumor cluster migration. This approach of short-term longitudinal imaging time frame in studying transient or dynamic events of diffuse, collectively migrating tumor cells in the local environment and quantitative analysis of the tumor area, motility and vessel characteristics is an innovation that can be used to investigate other cancer cell types exhibiting LVI. Funding in part by DoD W81XWH-17-1-0297; W81XWH201053 (GRD); ACS Mission Boost grant MBG-20-141-01 (GRD) and NIH grant P30-CA014236 (Imaging Core/GMP) Citation Format: Gayathri R Devi, Dorababu Sannareddy, Alexandra Bennion, Ashlyn Rickard, Douglas C Rouse, Mark W Dewhirst, Gregory M Palmer. Monitoring Lymphovascular Invasion and Tumor Growth of Inflammatory Breast Cancer in a Murine Lymphatic Reporter Window Chamber Model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-06.

Synchrotron Fourier-Transform Infrared Microspectroscopy: Characterization of in vitro polarized tumor-associated macrophages stimulated by the secretome of inflammatory and non-inflammatory breast cancer cells

Studies suggested that the pathogenesis of inflammatory breast cancer (IBC) is related to inflammatory manifestations accompanied by specific cellular and molecular mechanisms in the IBC tumor microenvironment (TME). IBC is characterized by significantly higher infiltration of tumor-associated macrophages (TAMs) that contribute to its metastatic process via secreting many cytokines such as TNF, IL-6, IL-8, and IL-10 that enhance invasion and angiogenesis. Thus, there is a need to first understand how IBC-TME modulates the polarization of TAMs to better understand the role of TAMs in IBC. Herein, we used gene expression signature and Synchrotron Fourier-Transform Infrared Microspectroscopy (SR-μFTIR) to study the molecular and biochemical changes, respectively of in vitro polarized TAMs stimulated by the secretome of IBC and non-IBC cells. The gene expression signature showed significant differences in the macrophage's polarization-related genes between stimulated TAMs. FTIR spectra showed absorption bands in the region of 1700–1500 cm−1 attributed to the amide I ν(C=O), & νAS (CN), δ (NH), and amide II ν(CN), δ (NH) proteins bands. Moreover, three peaks of different intensities and areas were detected in the lipid region of the νCH2 and νCH3 stretching modes positioned within the 3000–2800 cm−1 range. The PCA analysis for the second derivative spectra of the amide regions discriminates between stimulated IBC and non-IBC TAMs. This study showed that IBC and non-IBC TMEs differentially modulate the polarization of TAMs and SR-μFTIR can determine these biochemical changes which will help to better understand the potential role of TAMs in IBC.

Florid Lymphovascular Invasion of Inflammatory Breast Cancer is Due to Geometric Embolic Budding

The sina qua non of inflammatory breast cancer (IBC) is numerous tumor emboli especially within overlying dermal lymphatics. The explanation remains a mystery. In human studies comparing IBC with non‐IBC, although common tumor parameters such as Ki‐67 index, mitotic count and nuclear size show a close overlap, there is a dramatic exponential difference in embolic density in IBC v non‐IBC. Using contrasting properties of two IBC xenografts, Mary‐X and Karen‐X, this study offers a novel explanation and proposed mechanism to explain the very high embolic numbers in IBC. The explanation is geometric budding of emboli. Mary‐X exhibits florid lymphovascular invasion in vivo which give rise to high numbers of CTCs and pulmonary metastases whereas Karen‐X lacks these features. Mary‐X also gives rise to compact spheroids in vitrowhich exhibit dramatic budding whereas Karen‐X exhibits only loose non‐budding aggregates. Furthermore Mary‐X emboli also bud into daughter emboli in vivo. The mechanism that regulates the compactness of the spheroids as well as the emboli involves the generation of E‐cad/NTF1, a calpain‐mediated cleavage product of membrane E‐cadherin. Inhibiting the generation of E‐cad/NTF1 by blocking either the calpain site of cleavage (SC) or the site of binding (SB) with specific decapeptides both reduces spheroid compactness and decreases budding. Since E‐cad/NFT1 retains the p120ctn binding site but loses both the β‐catenin and α‐binding sites, promoting its 360° distribution around the cell’s membrane, the varying levels of expression of this truncated molecule may trigger budding of both the spheroids as well as the emboli. Recurrent and geometric budding of parental emboli into daughter emboli then would account for the plethora of IBC tumor emboli seen in patients.

Abstract PD10-08: Remodeling the inflammatory breast cancer tumor microenvironment to enhance immunotherapy: Novel therapeutic development

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer, yet no targeted therapy has been approved specifically for this disease. There is a critical need for innovative treatment approaches for patients with IBC. There is a paucity of data on immune checkpoint inhibitors (ICIs) in IBC, yet a strong biological rationale exists to lay the groundwork for testing the efficacy of ICIs in IBC. We have previously shown that an anti–epidermal growth factor receptor (EGFR) antibody, panitumumab (sponsored by Amgen), combined with Abraxane (sponsored by Bristol Myers Squibb), and carboplatin followed by 5-fluorouracil, epirubicin, and cyclophosphamide achieves a very high pathologic complete response (42%) in patients with triple-negative receptor status (TN-IBC) (NCT01036087). We hypothesize that inducing a broad shift in the IBC tumor microenvironment (TME), a critical driver of the IBC clinical phenotype and metastasis, from an immunosuppressive to an immunoreactive phenotype can enhance the efficacy of ICIs. Here we report the impact of targeting EGFR on ICI effectiveness by modulating the immunosuppressive TME in TN-IBC. Methods: We examined the effects of panitumumab on components of the immune TME in IBC patient tissues collected before and after panitumumab treatment. Furthermore, we analyzed panitumumab effects in an IBC SUM149 humanized mouse model (SUM149-hu-NSG-SGM3) by using multiplex immunofluorescence staining (mIF) of single-cell RNA-sequencing (scRNA-seq) and multicolor flow cytometry. We examined the changes in chemokines in humanized mouse tissue after panitumumab treatment using a cytokine antibody array and enzyme-linked immunosorbent assay (ELISA). We also studied the mechanism of EGFR regulation of immunosuppressive chemokine expression via transcription factor EGR1 by using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and chromatin immunoprecipitation (ChIP) assays. In addition, we tested the efficacy of panitumumab combined with anti-PD-L1 antibody (Bio X Cell, clone 29E.2A3) in reducing IBC tumor growth in a humanized IBC SUM149 mouse model. Results: IBC patients who achieved pathologic complete response after receiving panitumumab/neoadjuvant chemotherapy had increased levels of CD8+ T-cells and decreased levels of M2 macrophages and T-reg cells. In the humanized mouse model, panitumumab treatment reduced IBC tumor growth and increased immune response that favors an antitumor effect, as shown by scRNA-seq, flow cytometry, and mIF analyses. In addition, we observed that panitumumab treatment increased the expression of chemokines that function as a chemoattractant for CD8+ T-cells, including CXCL10, CCL4, and CXCL9. We also observed that panitumumab treatment reduced EGR1 expression, leading to the downregulation of chemokines involved in the recruitment of immunosuppressive M2 macrophages and T-reg cells, including CCL2, CCL20 CXCL5, and IL-8. Finally, panitumumab enhanced the reduction in tumor growth by anti-PD-L1 antibody treatment; furthermore, the combination of panitumumab and anti-PD-L1 antibody increased the presence of CD8+ T-cells and reduced the presence of T-reg cells and M2 macrophages more effectively than either treatment alone. Conclusion: Panitumumab treatment converts the immunosuppressive IBC TME to an immunoreactive status by modulating the global expression of chemokines by downregulating the EGR1 transcription factor. This modulation of the TME by panitumumab improves the inhibition of IBC tumor growth by an ICI. Our study is the first to demonstrate that targeting the EGFR/EGR1 axis remodels the IBC TME by regulating chemokine expression, thus boosting the antitumor immune response in IBC and response to ICI. Citation Format: Xiaoping Wang, Takashi Semba, Ganiraju C. Manyam, Jing Wang, Shan Shao, Francois Bertucci, Pascal Finetti, Savitri Krishnamurthy, Lan ThiHanh Phi, Troy Pearson, Jared K. Burks, Evan N. Cohen, James M. Reuben, Fei Yang, Hu Min, Nicholas Navin, Toshiaki Iwase, Yichao Shen, Xiang Zhang, Debu Tripathy, Naoto T. Ueno. Remodeling the inflammatory breast cancer tumor microenvironment to enhance immunotherapy: Novel therapeutic development [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD10-08.

Abstract P1-24-06: Risk factor modeled microenvironment effects lymphatics activity and IBC invasiveness and progression

Abstract Background. Lymphovascular invasion (LVI) and breast tumor emboli within dermal and breast lymphatic vessels are prognostic for metastatic spread and poor outcomes, and are abundant in Inflammatory breast cancer (IBC). IBC is an aggressive breast cancer that presents suddenly with breast swelling and redness due to tumor emboli in lymphatics. Lack of breast-feeding and obesity are IBC risk factors. We sought to demonstrate the combinatorial effects of a high-fat diet and nursing on lymphatic function and compare these to IBC tumor induced changes in lymphatic function. We hypothesize that risk factors for aggressive breast cancer may alter lymphatic function in the normal gland prior to tumor initiation. Methods. Following two rounds of pregnancy in 20 multiparous SCID Beige immunocompromised mice, half of the mice were force weaned while half nursed pups. Prior to forced weaning, half of each of these groups were fed a high fat diet (HFD: 60 Kcal %, N = 10) while the other half received a low-fat diet (LFD: 10 Kcal %, N = 10). Consecutive dynamic near-infrared fluorescence (NIRF) lymphatic imaging was performed at 6-7 months (covid interruption) and 14 months after initiating the diet by injecting a near-IR fluorophore into the mammary fatpad and recording lymphatic pulsing over 8 minutes using V++. Matlab and ImageJ were used to quantify pulsing rates on the ventral lymphatics in each animal. Fatpads were subsequently inoculated with SUM149 IBC cells and imaging was repeated 16 months post diet initiation. Lymphatic imaging over time by HFD vs LFD was further studied in nulliparous animals. Tissues were collected for further analyses. ResultsData analysis prior to tumor injection, demonstrated lymphatic pulsing (pulses/4 minutes) increased over time in HFD force weaned (HFFW) and HFD nursing (HFN) animals only (65.5 vs 72.6, P=0.059; 60.1 vs 76.6, P=0.0099, respectively). Comparing HFFW and HFN to matched LFD groups (LFFW and LFN), at 14 weeks HFD was associated with increased pumping after forced weaning (62.3 vs. 72.6, P=0.074), and nursing (62.5 vs 76.6, P=0.0023). There was an increase in pulsing after tumor initiation (16 months after initiation of diet) in all groups (80.1, 84.1, 83.2, 82.4, P &amp;gt; 0.05 all comparisons to initial timepoint). In a separate experiment examining HFD (N=5) vs LFD (N=5) in nulliparous mice, lymphatic contractile activity increased in all animals over. time, average ventral lymphatic contractile frequency for LFD and HFD at week 8,11 and 14 weeks after diet initiation were 5, 8.64, 15.9 pumps/4 mins vs 11.8, 18.5, 28.2 pumps/4 mins, (P = 0.01, 0.05, and 0.0005 respectively). ConclusionsHFD increased lymphatic pulsing rate over time to a significantly greater extent than LFD continuing over 14 months independent of reproductive and nursing status. Tumor initiation prompted further increased pulsing rates beyond that observed after HFD across all groups. The magnitude of the effect of HFD on lymphatic pulsing approached the rate after tumor initiation, while reproductive variables did not impact lymphatic pulsing. Further studies are warranted to demonstrate the relationship if any between lymphatic pumping pre-initiation and LVI after tumor initiation and examine the role of intervention on reducing LVI. Citation Format: Wintana Balema, Janelle Morton, Richard Larson, Fred C Velasquez, Eva Sevick-Muraca, Wendy A Woodward. Risk factor modeled microenvironment effects lymphatics activity and IBC invasiveness and progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-24-06.

Abstract P3-14-18: Adaptive stress response genes associated with breast cancer subtypes and survival outcomes reveal race-related differences

Abstract Background &amp; Hypothesis: The death rate is higher in Black (B) patients (40%), and, in particular, is two-fold higher among young B women (&amp;lt;50 years) compared to White (W) women with breast cancer. Aggressive breast cancer variants, like hormone receptor-negative and inflammatory breast cancer (IBC), contribute to a race-related survival gap and poor clinical outcomes in African American (AA) patients compared to Whites (W) in the United States. We previously identified the dominant role of XIAP in a tumor cell adaptive stress response (ASR) that promotes nuclear transcription factor (NFκB)-mediated proliferative, invasive, and immunosuppressive signaling in breast cancer progression. Our current work sought to address the urgent need to interrogate molecular mechanisms driving the disparity in both AA and IBC tumor biology by investigating gene expression differences between AA and White tumors and Triple-negative breast cancer (TNBC) and non-TNBC tumors, thereby identifying common genes which may be modifiable risk factors and targets that can be modulated for therapeutic benefits. Methods: We used The Cancer Genome Atlas (TCGA) breast cancer data to conduct a comparative analysis of the expression of 226 ASR genes in breast cancer molecular subtypes, normal-adjacent tissues, and in AA and W patients. The ASR gene list includes 101 XIAP-related, 11 NFκB targets, 10 MNK targets, 33 Oxidative Stress Response (OSR)-related, 13 TGFβ-related, 6 JAG1-Notch targets, and 52 immune-related, in addition to 14 genes that belong to more than one ASR set. In the present study, we focused on primary tumor samples [1090 Primary Solid Tumor] and normal tissue samples adjacent to the tumors [113 Solid Tissue Normal]. Within these, our sample set included 559 lumA, 207 lumB, 82 Her2, 190 Basal, and 40 Normal-like. Racial designations in TCGA are based on patient self-identification. In the present study, the race-related analysis focused on AA and W breast cancer patient datasets from TCGA (179 AA and 744 W). It is important to note that among the 113 Normal-adjacent samples, there are 105 W and only 6 AA samples. Results: Analysis of the ASR gene score in different Breast cancer subtypes revealed that ASR genes associated with oxidative stress and immune response pathways score highly in the basal breast cancer subtype. We also observed differential scores between samples of AA or W. The differential gene expression analysis identified 46-88 genes in the ASR set to be differentially expressed (≥2 fold-change and adjusted p-value &amp;lt;0.05) in breast cancer subtypes when comparing each subtype to normal-adjacent tissue or comparing the subtypes to each other. Pathway analysis of the enriched genes revealed the involvement of the cell cycle, the DNA damage response, the signal transduction, and the regulation of cell death-related processes. Moreover, on average, 20% of the ASR genes showed race-related differential expression. Furthermore, a number of ASR that were differentially expressed in breast cancer subtypes and significantly associated (hazard ratio &amp;gt;1.5 and p-value &amp;lt;0.05) with AA and/or W patient survival. For example, CDC45, MCM3, and CDKN2D were uniquely associated with poor survival in AA, but not in W. Conclusion: Within invasive breast cancer, factors that regulate tumor cell ASR reveal subtype and race-specific differences in expression. Understanding their function and downstream signaling will elucidate the intrinsic mechanisms that drive racial disparities in breast cancer outcomes and have the potential to reveal race-related biomarkers and therapeutic targets. Support: NCI-P20 NCCU-Duke Cancer Disparities Translational Research Partnership (SP; GRD, KPW, JF), DoD-W81XWH-17-1-0297 (GRD), Duke Bridge Funds (GRD). Citation Format: Muthana Al Abo, Larisa Gearhart-Serna, Steven Van Laere, Jennifer Freedman, Steven Patierno, Eun-Sil Hwang, Savitri Krishnamurthy, Kevin Williams, Gayathri Devi. Adaptive stress response genes associated with breast cancer subtypes and survival outcomes reveal race-related differences [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-14-18.

Early on-treatment transcriptional profiling as a tool for improving pathological response prediction in HER2-positive inflammatory breast cancer.

Background:Inflammatory breast cancer (IBC) is a rare and understudied disease, with 40% of cases presenting with human epidermal growth factor receptor 2 (HER2)-positive subtype. The goals of this study were to (i) assess the pathologic complete response (pCR) rate of short-term neoadjuvant dual-HER2-blockade and paclitaxel, (ii) contrast baseline and on-treatment transcriptional profiles of IBC tumor biopsies associated with pCR, and (iii) identify biological pathways that may explain the effect of neoadjuvant therapy on tumor response.Patients and Methods:A single-arm phase II trial of neoadjuvant trastuzumab (H), pertuzumab (P), and paclitaxel for 16 weeks was completed among patients with newly diagnosed HER2-positive IBC. Fresh-frozen tumor biopsies were obtained pretreatment (D1) and 8 days later (D8), following a single dose of HP, prior to adding paclitaxel. We performed RNA-sequencing on D1 and D8 tumor biopsies, identified genes associated with pCR using differential gene expression analysis, identified pathways associated with pCR using gene set enrichment and gene expression deconvolution methods, and compared the pCR predictive value of principal components derived from gene expression profiles by calculating and area under the curve for D1 and D8 subsets.Results:Twenty-three participants were enrolled, of whom 21 completed surgery following neoadjuvant therapy. Paired longitudinal fresh-frozen tumor samples (D1 and D8) were obtained from all patients. Among the 21 patients who underwent surgery, the pCR and the 4-year disease-free survival were 48% (90% CI 0.29–0.67) and 90% (95% CI 66–97%), respectively. The transcriptional profile of D8 biopsies was found to be more predictive of pCR (AUC = 0.91, 95% CI: 0.7993–1) than the D1 biopsies (AUC = 0.79, 95% CI: 0.5905–0.9822).Conclusions:In patients with HER2-positive IBC treated with neoadjuvant HP and paclitaxel for 16 weeks, gene expression patterns of tumor biopsies measured 1 week after treatment initiation not only offered different biological information but importantly served as a better predictor of pCR than baseline transcriptional analysis.Trial Registration:ClinicalTrials.gov identifier: NCT01796197 (https://clinicaltrials.gov/ct2/show/NCT01796197); registered on February 21, 2013.

Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion.

Inflammatory breast cancer (IBC) is an aggressive form of primary breast cancer characterized by rapid onset and high risk of metastasis and poor clinical outcomes. The biological basis for the aggressiveness of IBC is still not well understood and no IBC‐specific targeted therapies exist. In this study, we report that lipocalin 2 (LCN2), a small secreted glycoprotein belonging to the lipocalin superfamily, is expressed at significantly higher levels in IBC vs non‐IBC tumors, independently of molecular subtype. LCN2 levels were also significantly higher in IBC cell lines and in their culture media than in non‐IBC cell lines. High expression was associated with poor‐prognosis features and shorter overall survival in IBC patients. Depletion of LCN2 in IBC cell lines reduced colony formation, migration, and cancer stem cell populations in vitro and inhibited tumor growth, skin invasion, and brain metastasis in mouse models of IBC. Analysis of our proteomics data showed reduced expression of proteins involved in cell cycle and DNA repair in LCN2‐silenced IBC cells. Our findings support that LCN2 promotes IBC tumor aggressiveness and offer a new potential therapeutic target for IBC.

Ultrastructural Analysis of Inflammatory Breast Cancer Cell Clusters in an Ex Vivo Environment Mechanically Mimicking the Lymph Vascular System.

Background:Inflammatory breast cancer (IBC) is a rare form of breast cancer with a poor prognosis. IBC is characterized by florid lymphovascular tumor emboli in the skin and the parenchyma of the breast. We hypothesized that the formation of these emboli/clusters plays a pivotal role in IBC metastasis and its rapid progression, and that their structure and function may be a key to identifying molecular biological differences between IBC and non IBC.Methods:Mechanical methods were used to mimic the lymph fluid viscosity by adding 2.25% of PEG8000 to the media. Clusters were obtained for IBC tumor cell lines (SUM149 and IBC-3), non IBC tumor cell lines (MDA-MB-231, MDA-MB-468, and MCF7), and a non-tumorigenic human mammary epithelial cell line (MCF10A). Clusters were analyzed by light microscopy, and then prepared for and observed by transmission electron microscopy (TEM).Results:Significant differences were seen between IBC and non IBC clusters. The TEM analysis revealed that IBC cells harbored numerous microvilli and microvesicles, both on the free outer surface and inside the cluster. Microvilli from IBC cell clusters were noted at higher density and were longer than those of non IBC cell clusters.Conclusions:IBC tumor cell clusters exhibited distinct ultrastructural features characterized by the presence of long, crowded microvilli and numerous microvesicles. These microvilli may play an important role in the biology and aggressiveness of IBC.

Targeting Signaling Pathways in Inflammatory Breast Cancer.

Simple SummaryInflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer; it is highly likely to spread to other sites in the body. There is an urgent need to establish novel treatment strategies to reduce IBC recurrence and metastasis. The aim of this work is to provide a comprehensive overview of signaling pathways in IBC, covering understanding of their function in IBC tumor cells and cells surrounding tumor, and clinical efforts to target these pathways for patients with IBC. The findings described in this work will help guide the development of effective therapies through preclinical and clinical research, eventually improving the treatment of patients with IBC.Inflammatory breast cancer (IBC), although rare, is the most aggressive type of breast cancer. Only 2–4% of breast cancer cases are classified as IBC, but—owing to its high rate of metastasis and poor prognosis—8% to 10% of breast cancer-related mortality occur in patients with IBC. Currently, IBC-specific targeted therapies are not available, and there is a critical need for novel therapies derived via understanding novel targets. In this review, we summarize the biological functions of critical signaling pathways in the progression of IBC and the preclinical and clinical studies of targeting these pathways in IBC. We also discuss studies of crosstalk between several signaling pathways and the IBC tumor microenvironment.

Abstract P5-04-04: The spatial interactions between FOXP3+ Tregs, CD8+ cytotoxic T cells and tumor cells predict response to therapy and prognosis in inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare and aggressive type of locally advanced breast cancer. A 79-gene signature, reported by our lab, is shaped by specific immune response programs and discriminates between IBC and non-IBC (nIBC). However, it remains an enigma how infiltrating immune cells are able to determine the IBC phenotype. Furthermore, the presence of immune cells like FOXP3+ Tregs and CD8+ cytotoxic T cells is associated with outcome in proliferative subtypes of breast cancer and the interaction between these cells plays a role in the functional immune response. Therefore, we aimed to assess the spatial associations between immune cells in IBC. Additionally, we used deep-learning to examine interactions between cancer and immune cells. Methodology: Immunostainings (Hematoxylin-DAB, H-DAB) were done according to well-validated protocols for CD8 (cytotoxic T-cells), FOXP3 (Tregs) and CD163 (tumor-associated macrophages, TAMs) in a large population of 134 IBC patients. All slides were digitalized and evaluated using VISIOPHARM® software, allowing virtual multiplexing. We quantified the number of DAB+ immune cells and each positive immune cell was located using XY coordinates. Spatial co-localization was examined using statistics developed for ecological studies based on point pattern and quadrant analysis. TILs were scored according to the TIL working group guidelines on H&amp;E slides. Tumor cell coordinates were collected using a deep-learning algorithm applied to the CD8 stained slide. To perform deep-learning, we aligned two consecutive slides: one PanCK stained slide and one slide stained for CD8 (H-DAB). Using virtual multiplexing and the PanCK staining, we determined the tumor regions on the H-DAB stained slide. Subsequently, 18 images were incorporated to train the algorithm with more than 150.000 iterations (Deeplabv3+), after which the algorithm was evaluated in a test set of 12 images, approved and applied to all images to locate the tumor cells. Results: Most of the patients presented with a hormone receptor (HR) positive carcinoma (60.6%, n= 82/132). The presence of distant disease, HR status and TIL score were associated with overall survival (OS), but the density of the different immune cells or the CD8/FOXP3 ratio was not. However, using an effector index we demonstrated that patients with more FOXP3+ cells in a radius of 30 μm surrounding a CD8+ cell had a significant worse outcome (Median OS: 2.7 vs. 6.3 years, P= .01) and this remained significant in a multivariate model (HR: 2.85, P&amp;lt; .001). Complete pathological response (pCR) after neo-adjuvant chemotherapy was achieved by 28.7% (n= 27/94) of the patients with initially localized disease. Infiltration with CD8+ T cells (P= .005), TAMs (P= .008) and TILs (P= .002) predicted pCR, but a likelihood ratio test showed no difference between a model using CD8+ cells, TAMs or TILs. Interestingly, pCR was less often achieved in patients that had colocalization of FOXP3+ cells near the tumor cells (P= .003). This colocalization was determined using a Morista-Horn index and independent of the number of Tregs, CD8+ cells or TILs. The deep-learning algorithm had a mean dice coefficient in the test set of 0.83, indicating a good overlap between the tumor area determined by the AI on the CD8 slides and the area on the PanCK stained slides. Conclusions: We have created a deep learning algorithm that adequately detects IBC tumor cells on H-DAB stained images and show, in a large cohort of 132 patients, that the negative impact of Tregs appears to depend on the spatial arrangement. While solely the number of Tregs is not associated with pCR or OS, patients with FOXP3+ Tregs that cluster together near CD8+ cytotoxic T cells had a worse outcome and pCR was achieved more often in patients with less Tregs near the tumor cells. Citation Format: Christophe Van Berckelaer, Charlotte Rypens, Steven Van Laere, Koen Marien, Pieter-Jan van Dam, Peter Vermeulen, Luc Dirix, Mark Kockx, Cecile Colpaert, Peter van Dam. The spatial interactions between FOXP3+ Tregs, CD8+ cytotoxic T cells and tumor cells predict response to therapy and prognosis in inflammatory breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-04.

Abstract P5-17-06: Immune modulation with humanized anti-EGFR antibody panitumumab in an immunocompetent mouse model for inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer and there are no approved targeted therapies specifically for IBC. Targeting the epidermal growth factor receptor (EGFR) pathway is a promising therapeutic target for patients with triple-negative IBC (TN-IBC) with a reported pathological complete response rate of 42% (JAMA Oncology, 2018). The tumor microenvironment (TME) is a critical contributor to the aggressiveness of IBC. Delineating cross-talk between EGFR-targeted therapies and TME components, which define IBC, could inform more efficient combination regimens and novel clinical trial designs for IBC. However, such studies have not been conducted due to the lack of a syngeneic IBC mouse model. Here we report the establishment of an IBC immunocompetent mouse model and the effects of panitumumab (PmAb) on IBC tumor growth and the TME. Methods: TN-IBC cell lines, SUM149 or FC-IBC-02, were mixed with 50% Matrigel and inoculated into mammary fat pads of hu-NSG-SGM3 mice engrafted with hematopoietic stem cells (The Jackson Laboratory). SUM149 tumor growth in hu-NSG-SGM3 mice treated with either IgG2 (isotype control, 4 mg/kg) or PmAb (1 mg/kg and 4 mg/kg) was measured. The percentages of TME components, including human CD4+ T, CD8+ T, regulatory T (Tregs), and natural killer (NK) cells, and M1 or M2 macrophages, in the peripheral blood and tumor tissues treated with IgG2 and PmAb for 7 weeks were measured by flow cytometry. Results: Hu-NSG-SGM3 mice supported the growth of TN-IBC SUM149 and FC-IBC-02 xenografts. These humanized mouse models were named SUM149-huSGM3 and FC-IBC-02-huSGM3, respectively. Analysis of the blood cells showed that SUM149-huSGM3 mice display human CD4+ T, CD8+ T, Tregs, M1 and M2 macrophages. T cell infiltration and M1 and M2 macrophages were also detected in SUM149-huSGM3 tumors. NK cells were not detected in both peripheral blood and tumors. PmAb treated SUM149-huSGM3 mice had significantly reduced SUM149 tumor growth, compared with mice that received the IgG2 control. PmAb treatment increased the percentage of CD8+ T cells and reduced the percentage of Tregs in peripheral blood. A similar analysis of tumor infiltrating lymphocytes isolated from each group showed an increase in percent CD8+ T cells in mice treated with PmAb. There were no significant changes of M1 or M2 macrophages following PmAb treatment. These results suggest that the increase in percentage of CD8+ T cells in peripheral blood and IBC tumors, and the decrease in percentage of Tregs in peripheral blood may contribute to the therapeutic efficacy of PmAb. Conclusion: We established the first immunocompetent mouse model to study the TME and immune response in IBC, which provides the premise for conducting a diversity of novel preclinical therapeutic studies. The mechanism of how immune responses of TN-IBC xenografts mediates the therapeutic efficacy of PmAb in IBC tumors needs to be further investigated. Our study also suggests that combination therapy with immune checkpoint inhibitors may potentiate the efficacy of anti-EGFR therapy in IBC. The therapeutic efficacy of PmAb and anti-PD-L1 combination in SUM149 humanized mice is in progress. Citation Format: Wang X, Shao S, Pearson T, Cheng Y, Reuben JM, Tripathy D, Ueno NT. Immune modulation with humanized anti-EGFR antibody panitumumab in an immunocompetent mouse model for inflammatory 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 P5-17-06.

Abstract P5-17-01: Patient-derived organoid models of inflammatory breast cancer

Abstract Background: Inflammatory Breast Cancer (IBC) is an aggressive subtype of breast cancer that is associated with an increased likelihood of early metastasis and a poor prognosis, accounting for approximately 10% of all breast cancer mortality. Currently, there is limited availability of cell line and patient-derived xenograft (PDX) models of IBC. As a result, there is an urgent need for additional model systems to facilitate our understanding of this disease. Organoids have been used to grow breast cancer cells with high efficiency, resulting in long-term cultures with genetic and phenotypic stability; however, the extent to which IBC can be modeled using this system is unknown. We undertook an evaluation of organoid culture technology for growing IBC patient samples, with the goal of generating a model of IBC that more faithfully recapitulates the key features of the disease. Methods: Tumor samples (taken from lumpectomy, mastectomy, PDX, or core biopsy samples), were digested to the organoid level using collagenase, and were grown in three dimensional cultures using a basement membrane extract and a fully-defined organoid medium, as described (Sachs et al. Cell 2018). First, we surveyed non-IBC metastatic breast cancer samples to determine the efficiency of growth of multiple breast cancer subtypes under these conditions. Then, we assessed the efficiency of growth of IBC samples taken after treatment, at the time of mastectomy. Co-culture experiments in which IBC organoids were grown in the presence of human endothelial cells were performed in three dimensional culture using a mixture of type I collagen and basement membrane extract. Results: Our success rate for establishing organoid cultures from breast cancer samples in general was 75% (33 out of 44 samples), and for triple-negative breast cancers, the success rate for establishment of organoid cultures was 100% (14 out of 14 samples). Expansion of ER+ tumor organoids (16 of 26 samples) was primarily restricted by a slow growth rate in culture, as compared to triple-negative tumor organoids. We were able to establish 12 breast cancer organoids from core biopsies of multiple metastatic sites, including brain, liver, bone, breast, and skin. We subsequently were able to establish organoid cultures from five out of five IBC tumor samples. Our IBC organoids preserved histopathologic features of the original tumor, and expressed key markers, such as E-cadherin, that are known to be associated with IBC. In addition, our IBC organoids interacted with human endothelial cells under co-culture conditions designed to model lymphovascular invasion, an essential feature of IBC. Conclusion: We have shown that organoid cultures can be established to propagate IBC tumors with high efficiency, preserve key pathologic features of IBC in vitro, and model interactions between tumor cells and the microenvironment. Thus, organoids are useful complements to existing IBC models, and can be used to identify potential therapeutic vulnerabilities associated with this particularly aggressive breast cancer subtype. Citation Format: Rosenbluth JM, Zervantonakis I, Boedicker M, Wagle N, Dillon D, Nakhlis F, Brugge JS, Overmoyer B. Patient-derived organoid models of inflammatory 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 P5-17-01.

Abstract P2-01-03: Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal form of primary breast cancer and accounts for a significant 10 % of breast cancer deaths in the USA owing to its aggressive proliferation and metastasis, and a lack of effective therapeutic options. Unraveling the underlying mechanisms of growth and metastasis of this aggressive disease could lead to effective therapeutic strategies for an improved outcome in IBC patients. We recently generated in vitro and in vivo IBC models for brain metastasis studies [Debeb et al. JNCI, 2016] and observed an upregulation of Lipocalin 2 (LCN2), a small, secreted iron-trafficking protein which plays a significant role in immune and inflammatory responses and the promotion of malignant progression. The purpose of this study was to investigate the function of LCN2 in IBC tumorigenesis and metastasis. Methods: Stable knockdown (KD) of LCN2 in IBC cell lines was achieved with lentiviral vectors. Proteomic and gene expression profiling were performed using RPPA and Affymetrix Clariom D microarray. For in vivo studies, control and LCN2 KD IBC cells were transplanted into the cleared mammary fat pad of SCID/Beige mice. Tumor-skin involvement was assessed visually during primary tumor growth and tumor excision. LCN2 gene expression levels in clinical samples were analyzed from the IBC Consortium as well as public data sets. LCN2 serum levels in IBC patients were measured using ELISA and were correlated with clinicopathological variables and outcome data. Results: LCN2 gene expression is higher in IBC versus non-IBC patients (p=0.00036), independently of the molecular subtypes, and higher in more aggressive (TNBC and HER2+) than hormone receptor-positive subtypes (p&amp;lt;0.00001). LCN2 expression in patient tissues is correlated with reduced overall survival (p&amp;lt;0.00001) and metastasis-free survival (p=0.04) in non-IBC; however, LCN2 was not associated with overall survival in IBC patient serum samples. LCN2 expression was also significantly higher in IBC cell lines, in their culture media, and in brain metastasis sublines compared to non-IBC cell lines (p=0.004). In IBC cell lines, LCN2 KD reduced proliferation, colony formation, migration, and cancer stem cell properties. In vivo silencing of LCN2 in SUM149 cells inhibited primary tumor growth (p=0.001)and resulted in a well-differentiated tumor histology. Additionally, SUM149 LCN2 KD significantly reduced skin invasion/recurrence (LCN2 control vs LCN2 KD: 88 % vs 25 %, p=0.01) suggesting LCN2 is a mediator of tumorigenesis. Analysis of proteomics data showed changes in major signaling pathways including PI3K-Akt signaling and EGF/EGFR signaling pathways. Mechanistically, LCN2 depletion in SUM149 abrogated EGF-induced EGFR phosphorylation and ERK activation. Conclusions: Our findings suggest that LCN2 may drive IBC tumor progression and skin invasion/recurrence potentially via the EGFR signaling pathway.Future studies will determine the role of LCN2 in metastasis and pinpoint the detailed mechanisms of LCN2-mediated IBC tumorigenesis and recurrence. Citation Format: Villodre ES, Larson R, Hu X, Stecklein SR, Gomez K, Finetti P, Krishnamurthy S, Ivan C, Su X, Ueno NT, Van Laere S, Bertucci F, Tripathy D, Vivas-Mejía P, A Woodward W, Debeb BG. Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion [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 P2-01-03.

Metabolic Characterization of Inflammatory Breast Cancer With Baseline FDG-PET/CT: Relationship With Pathologic Response After Neoadjuvant Chemotherapy, Receptor Status, and Tumor Grade

BackgroundThe aim of this study was to determine if, in inflammatory breast cancer (IBC), baseline metabolic activity (maximum standardized uptake value [SUVmax]) of primary tumor and involved regional lymph nodes (IRLN) are prognostic markers of response after neoadjuvant systemic therapy (NAS). Patients and MethodsBaseline 2-deoxy-2-[18F]fluoro-D-glucose (FDG) positron emission tomography/computed tomography scans were retrospectively reviewed among 61 women with IBC who received NAS, had mastectomy, and had available pathology reports. Primary tumor and IRLN SUVmax were compared between patients with a pathologic complete response (pCR) versus those with residual disease after NAS. A multivariate Cox model was fit to evaluate the effects of SUVmax on overall survival, adjusting for pCR and stratified by receptor status and disease stage. ResultsSUVmax in primary IBC tumors tended to increase with tumor grade (trend test P = .06) and was lower for stage III, non–triple-negative (TN) versus stage III, TN and stage IV, non-TN disease (P = .04). Neither primary tumor nor IRLN SUVmax was significantly different comparing pCR versus residual disease after NAS. Adjusting for pathology response in the overall survival model stratified by stage and receptor status, baseline SUVmax in primary IBC tumor was associated with an estimated hazard ratio of 1.10 (95% confidence interval, 0.97-1.25; P = .15) for patients with stage III, TN and stage IV, non-TN disease. This hazard ratio corresponded to a 1.74-fold risk of death with 1 standard deviation (SD = 5.9) increase in baseline SUVmax in primary IBC tumor. Conclusion2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography/computed tomography provides prognostic information for newly diagnosed IBC. Larger studies are needed to confirm these findings and assess how such early information could affect treatment choices for IBC in the neoadjuvant setting.

Abstract 4854: Targeting ribonucleotide reductase M2 using didox causes inhibition of estrogen receptor-negative, inflammatory breast cancer cell proliferation and tumor emboli formation in culture

Abstract Ribonucleotide reductase (RR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides during the rate-limiting step of DNA synthesis. Given its significant role in cell cycle regulation, RR and its catalytic subunits are significant molecular targets for inhibiting tumor cell growth and proliferation. Didox (3,4-dihydroxybenzohydroxamic acid) is a known inhibitor of the catalytic RR subunit of RRM2 and overall DNA synthesis. Previous meta-analyses have revealed that RRM2 is highly expressed in estrogen receptor-negative (ER-) breast cancer tumors. As ER negativity is frequent in inflammatory breast cancer (IBC), a distinct and the most aggressive breast cancer subtype, we proposed to evaluate efficacy of didox in IBC cell models. ER- inflammatory breast cancer (IBC) cell lines SUM149 (ER-, PR-, EGFR-activated), lapatinib-resistant SUM149 (rSUM149), and non-IBC BT474M1 metastatic subline of BT474 (ER+, PR+, HER2+) were compared for the cytotoxic and cytostatic effects of didox using cell viability (trypan blue exclusion) and proliferation (MTT) assays. Protein profiling was performed to evaluate changes in molecular expression of key proteins involved in cellular signaling pathways for apoptosis and proliferation. Only ER- cell lines exhibited decreased dose-dependent decrease in viability following didox treatment. In addition, didox inhibited tumor emboli formation in a novel ex vivo tumor emboli in culture assay that mimics the clinicopathologic hallmark of IBC disease in patients. Moreover, didox decreased proliferation in lapatinib-resistant rSUM149, suggesting that the drug may help circumvent lapatinib resistance. Studies are ongoing to evaluate the mechanism of didox in overcoming lapatinib resistance and evaluation in an in vivo IBC invasion model. IBC is rare but accounts for 10% of breast cancer deaths, and despite multimodal therapy the 5-yr survival is only 25-55%. Further, there is a need to identify new therapies that can inhibit IBC tumor emboli formation and migration. Didox has demonstrated minimal toxicity in human phase I/II cancer clinical trials and therefore is an attractive strategy for inflammatory breast cancer therapy. Support in part from the Development funds (GRD) of the Duke Cancer Institute as part of the P30 Cancer Center Support Grant NIH CA014236 and Department of Defense Breakthrough Level 2 W81XWH-17-1-0297 (GRD). Citation Format: Pranalee Patel, Ben Mirman, Renzhi Zhan, Gayathri R. Devi. Targeting ribonucleotide reductase M2 using didox causes inhibition of estrogen receptor-negative, inflammatory breast cancer cell proliferation and tumor emboli formation in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4854.

Mammary stem cell and macrophage markers are enriched in normal tissue adjacent to inflammatory breast cancer.

We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype. Normal breast parenchyma ≥ 5cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44+CD49f+CD133/2+ mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples. 8 of 8 IBC samples expressed isolated CD44+CD49f+CD133/2+ stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44+CD49f+CD133/2+ cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p < 0.001). Normal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.