Dermal Lymphatic Invasion Research Articles

Inflammatory breast cancer and the importance of skin punch biopsy

BackgroundThe oncologic outcomes of patients diagnosed with inflammatory breast cancer (IBC) based on clinical exam only versus those with dermal lymphatic invasion on skin punch biopsy may be different and are worth further investigation. MethodsPatients diagnosed from 2006 to 2021 with IBC at our institution were grouped according to clinical diagnosis or skin biopsy performed. Oncologic and survival outcomes among groups were compared. ResultsA total of 72 IBC patients were identified and grouped into 3 categories based on method of diagnosis: skin biopsy positive (n = 24), skin biopsy negative (n = 10) and no biopsy performed (n = 38). Skin biopsy positive patients had a higher incidence of lymphovascular invasion identified on final pathology and were more likely to experience a chest wall recurrence. At 5.1 yrs of follow-up, 40% of patients experienced recurrence, with 61% overall survival. ConclusionClinical diagnosis remains diagnostic for IBC, but skin punch biopsy allows for improved oncologic insight.

A Phase I Study of Adjuvant Niraparib Administered Concurrently with Postoperative Radiation Therapy in Patients with Localized Triple Negative Breast Cancer

<h3>Purpose/Objective(s)</h3> Residual disease after neoadjuvant therapy (NAT) is associated with higher risk of recurrence, including local failure (25%) in triple negative breast cancer (TNBC). Novel combinations that increase the efficacy of standard-of-care treatments on locoregional control are warranted. PARP inhibitors (PARPi) target the DNA damage repair pathway, which is often altered in TNBC, and show promise as radiosensitizers. The objective of this phase I trial (NCT03945721) was to determine the maximum tolerated dose (MTD) of a PARPi, niraparib, in pts with high risk TNBC receiving postoperative radiotherapy (RT). <h3>Materials/Methods</h3> Eligible pts had stage I-III TNBC with residual invasive disease following NAT or primary tumor ≥1 cm after upfront surgery. A 3+3 escalation design was used to test 2 dose levels of QD niraparib (100mg, 200mg) with concurrent RT to the chest wall or breast +/- regional lymph nodes over 4-6 weeks. Dose expansion followed at the MTD. A boost was delivered in pts who had lumpectomy. 2 pts received proton beam RT. Bolus was administered in postmastectomy RT or in cases of dermal lymphatic invasion. Circulating tumor DNA (ctDNA) was collected at baseline, mid-RT, end of RT, and 30 days post-RT to assess the kinetics of ctDNA levels during RT and the association with clinical outcomes. Adverse events (AEs) were assessed using CTCAE v5.0. <h3>Results</h3> 21 pts were enrolled from July 2019 – October 2021. The median age was 49 (31-69). 19/21 pts had residual disease after NAT (6 RCB I, 7 RCB II, 6 RCB III); among them, 2 underwent adjuvant chemotherapy prior to enrolling. The remaining 2/21 pts (pT2N0; pT1cN0) received upfront breast conserving surgery followed by adjuvant chemotherapy. In dose escalation, 6 pts received 100mg niraparib + RT and 2 pts received 200mg niraparib + RT. One DLT in the 100mg cohort (G3 dermatitis) was attributed to RT. Two DLTs in the 200mg cohort (G3 thrombocytopenia) were attributed to niraparib and study drug was discontinued. In dose expansion, 13 pts received 100mg niraparib + RT. G3 AEs were dermatitis (4/21), hematologic (4/21), consisting of thrombocytopenia, anemia & lymphopenia, and elevated alkaline phosphatase (1/21). There was one G4 thrombocytopenia. All skin AEs were attributed to RT and were not exacerbated by niraparib. No pts in dose expansion discontinued treatment due to toxicity. 20/21 went on to standard-of-care capecitabine after completing RT. Results of ctDNA will be reported at the meeting. <h3>Conclusion</h3> The MTD of niraparib + postop RT was determined to be 100mg QD and was associated with a 24% rate of G3-4 hematologic toxicities and 19% rate of G3 radiation dermatitis in pts with localized TNBC, which is within upper limits of normal for pts in whom chest wall/skin is targeted. These results will be informative for a future larger study testing the efficacy of niraparib + postop RT in high risk TNBC.

Abstract P1-24-04: Spatially resolved cell type heterogeneity uncovers the distinct biology of inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) is the most lethal form of breast cancer due to rapid progression, distinct morbidity due to pain, swelling, infection and eventual death. It accounts for 10% of all breast cancer caused deaths despite being only 2-4% of all breast cancer diagnosis. Histopathologically, scattered tumor emboli and dermal lymphatic invasion are typical histopathological findings of IBC, shown in about 75% of all IBC cases. However, the etiology of this disease remains largely unknown and its diagnostic difficult to establish.Using single cell transcriptomics, we profiled a total of 10 patient-derived needle biopsies from IBC-hormone receptor (IBC-HR), IBC-triple negative breast cancer (IBC-TNBC), TNBC and HR subtypes, and one normal breast sample. After data QC, we obtained a total of 36,000 cells representing immune, epithelial and stromal cell populations. We performed analysis of the spatial cell distribution through a transcriptomics-informed panel using highly multiplex imaging mass cytometry (IMC) on FFPE section obtained from the same patients.While we noted interpatient heterogeneity within cancer cells in our single cell dataset, immune and stromal cells did show similarity across IBC patients. We identified differences across the myeloid compartment between IBC and non-IBC cells, specifically affecting the dendritic cell subpopulations.The interferon gamma pathway was specifically elevated in several cell types across IBC patients, and particularly in immune cells such as T and myeloid cells. We used MX1 as a marker of interferon gamma response as part of the IMC panel and confirmed this signature in IBC samples. There was no alpha and beta interferon transcriptomic signature in the single cells. Through IMC, we were able to identify the cellular composition surrounding the tumor microemboli. Contrary to what has been postulated in the literature, these cells are not lymphatic cells, but appear to be of fibroblastic origin and express the fibroblast marker CD90. As well, we detected a non-overlapping distribution of Collagen 1 positive and CD90 positive fibroblasts throughout the IBC specimens, revealing heterogeneity within the fibroblast population and its organization. Spatial immune cell analysis also revealed the presence of tertiary lymphoid structures in IBC tumors. Citation Format: Elise Courtois, William Flynn, Santhosh Sivajothi, Martine Seignon, Diane Luo, Bora Lim, Paul Robson. Spatially resolved cell type heterogeneity uncovers the distinct biology of inflammatory breast cancer [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 P1-24-04.

High endogenous CCL2 expression promotes the aggressive phenotype of human inflammatory breast cancer

Inflammatory Breast Cancer (IBC) is a highly aggressive malignancy with distinct clinical and histopathological features whose molecular basis is unresolved. Here we describe a human IBC cell line, A3250, that recapitulates key IBC features in a mouse xenograft model, including skin erythema, diffuse tumor growth, dermal lymphatic invasion, and extensive metastases. A3250 cells express very high levels of the CCL2 chemokine and induce tumors enriched in macrophages. CCL2 knockdown leads to a striking reduction in macrophage densities, tumor proliferation, skin erythema, and metastasis. These results establish IBC-derived CCL2 as a key factor driving macrophage expansion, and indirectly tumor growth, with transcriptomic analysis demonstrating the activation of multiple inflammatory pathways. Finally, primary human IBCs exhibit macrophage infiltration and an enriched macrophage RNA signature. Thus, this human IBC model provides insight into the distinctive biology of IBC, and highlights potential therapeutic approaches to this deadly disease.

Dermal Lymphatic Invasion, Survival, and Time to Recurrence or Progression in Inflammatory Breast Cancer.

Dermal lymphatic invasion (DLI) with tumor emboli is a common pathologic characteristic of inflammatory breast cancer (IBC), although its presence is not required for diagnosis. We examined whether documented DLI on skin biopsy was associated with survival and time to recurrence or progression in IBC. A total of 340 women enrolled in the IBC Registry at Dana-Farber Cancer Institute between 1997 and 2019 were included in this study. Kaplan-Meier curves and multivariable Cox proportional hazards models were used to estimate hazard ratios and 95% confidence intervals for associations of DLI and overall survival, time to locoregional recurrence/progression, and distant metastasis by stage at presentation. DLI was detected in 215 (63.2%) of IBC cases overall. At disease presentation, IBC with DLI had a higher prevalence of de novo metastases (37.7% vs. 26.4%), breast skin ulceration (6.1% vs. 2.4%), and lymphovascular invasion within the breast parenchyma (52.9% vs. 25.5%) and a lower prevalence of palpable breast mass (48.2% vs. 70.6%) than IBC without DLI. Over a median follow-up of 2.0 years, 147 deaths occurred. DLI was not associated with survival or recurrence in multivariable models (all P ≥0.10). For example, among women with stage III disease, hazard ratios (95% confidence intervals) for DLI presence was 1.29 (0.77-2.15) for overall survival, 1.29 (0.56-3.00) for locoregional recurrence, and 1.71 (0.97-3.02) for distant metastasis. Although the extent of tumor emboli in dermal lymphatics may be associated with biological features of IBC, DLI was not an independent prognostic marker of clinical outcomes in this study.

Small extracellular vesicle-encapsulated miR-181b-5p, miR-222-3p and let-7a-5p: Next generation plasma biopsy-based diagnostic biomarkers for inflammatory breast cancer

Inflammatory breast cancer (IBC) is a rare, but aggressive entity of breast carcinoma with rapid dermal lymphatic invasion in young females. It is either poorly or misdiagnosed as mastitis because of the absence of a distinct lump. Small extracellular vesicles (sEVs) circulating in liquid biopsies are a novel class of minimally invasive diagnostic alternative to invasive tissue biopsies. They modulate cancer progression via shuttling their encapsulated cargo including microRNAs (miRNAs) into recipient cells to either trigger signaling or induce malignant transformation of targeted cells. Plasma sEVs < 200 nm were isolated using a modified cost-effective polyethylene glycol (PEG)-based precipitation method and compared to standard methods, namely ultracentrifugation and a commercial kit, where the successful isolation was verified by different approaches. We evaluated the expression levels of selected sEV-derived miR-181b-5p, miR-222-3p and let-7a-5p using quantitative real PCR (qPCR). Relative to non-IBC, our qPCR data showed that sEV-derived miR-181b-5p and miR-222-3p were significantly upregulated, whereas let-7a-5p was downregulated in IBC patients. Interestingly, receiver operating characteristic (ROC) curves analysis revealed that diagnostic accuracy of let-7a-5p alone was the highest for IBC with an area under curve (AUC) value of 0.9188, and when combined with miR-222-3p the AUC was improved to 0.973. Further, 38 hub genes were identified using bioinformatics analysis. Together, circulating sEV-derived miR-181b-5p, miR-222-3p and let-7a-5p serve as promising non-invasive diagnostic biomarkers for IBC.

Abstract PS19-07: Plasma exosomal miRNAs: A minimally invasive diagnostic biomarker for inflammatory breast carcinoma

Abstract Background. Inflammatory Breast Cancer (IBC) is a rare, but aggressive subtype of breast carcinoma with dermal lymphatic invasion in young females. IBC is poorly diagnosed as it develops vast rapidly relative to other non-IBC and without distinct lumps formation, moreover, it is usually misdiagnosed with mastitis. Nanosized exosomes (20-200nm) circulating in liquid biopsies are a promising minimally invasive diagnostic alternative to standard biopsies. In cancer microenvironment, they modulate cancer progression via shuttling their encapsulated microRNAs (miRNAs) into recipient cells to either trigger signaling or induce malignant transformation of targeted cells. While small non-coding regulatory miRNAs are an already known cancer biomarkers, exosomal miRNAs serve as a novel class of diagnostic biomarkers. The present study aims to evaluate the expression levels of exosomal miRNAs relevant to IBC pathogenesis. Design/Materials/Methods. This is a prospective case-control study that includes 77 females; 57 were diagnosed with breast cancer (34 non-IBC and 23 IBC), while 20 were healthy volunteers. All have signed an informed consent to be enrolled in this investigation. Patients included were neither pregnant nor issued with bloodborne or autoimmune disease, while healthy subjects had no oncologic history. Plasma circulating exosomes were isolated using precipitation and ultracentrifugation methods. The successful isolation was verified by dynamic light scattering (DLS), transmission electron microscopy (TEM), Western blot (WB), and Dot blot (DB). The expression level of exosomes-derived miR-181b-5p, miR-222-5p and let-7a was quantified by qPCR. Afterwards, that expression level was verified in the human non-IBC MDA-MB-231 and IBC SUM149 cell lines. Statistics. Difference between groups was tested using Student’s t-test through IBM SPSS statistics software package, version 24. A p &amp;lt; 0.05 was considered significant. Results. DLS and TEM analysis revealed that nanovesicles with 146.65 nm were successfully isolated. Moreover, CD63, HSP70, Alix90 and GM130 antibodies used in DB and WB have further confirmed the successful isolation. Relative to non-IBC, our qPCR showed that plasma exosomes-derived miR-181b-5p and miR-222-5p were significantly (p &amp;lt;0.0001 and p &amp;lt;0.01, respectively) upregulated in IBC, whereas exosomal let-7a was significantly (p &amp;lt;0.0001) downregulated in IBC. Further, the expression levels of miR181-5p and miR-222 were upregulated in SUM-149-derived exosomes relative to those of MDA-MB-231-derived exosomes ( Both p &amp;lt; 0.001), while the expression levels of let-7a did not significantly change. Diagnostic accuracy of the candidate plasma exosomal miRNAs was assessed via receiver operating characteristic (ROC) curves analysis. Interestingly, Area Under Curve (AUC) was 0.826 for miR-181-5p (p &amp;lt;0.0001), 0.7212 for miR-222-5p (p &amp;lt;0.0001), and 0.9188 for let7a (p &amp;lt;0.01). Additionally, bioinformatical analysis for predicted targets of the identified exosomal miRNAs will be assessed. Conclusion. Exosomes derived miR-181-5p, miR-222-5p and let7-7a may act as non-invasive diagnostic biomarkers to discriminate IBC from non-IBC patients. Citation Format: Sarah Hamdy Ahmed, Ahmed El-Damen, Mohamed A. Badawy, Mohamed El-Shinawi, Martin Götte, Sherif Abdelaziz Ibrahim. Plasma exosomal miRNAs: A minimally invasive diagnostic biomarker for inflammatory breast carcinoma [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-07.

Epigenetics in Inflammatory Breast Cancer: Biological Features and Therapeutic Perspectives.

Evidence has emerged implicating epigenetic alterations in inflammatory breast cancer (IBC) origin and progression. IBC is a rare and rapidly progressing disease, considered the most aggressive type of breast cancer (BC). At clinical presentation, IBC is characterized by diffuse erythema, skin ridging, dermal lymphatic invasion, and peau d’orange aspect. The widespread distribution of the tumor as emboli throughout the breast and intra- and intertumor heterogeneity is associated with its poor prognosis. In this review, we highlighted studies documenting the essential roles of epigenetic mechanisms in remodeling chromatin and modulating gene expression during mammary gland differentiation and the development of IBC. Compiling evidence has emerged implicating epigenetic changes as a common denominator linking the main risk factors (socioeconomic status, environmental exposure to endocrine disruptors, racial disparities, and obesity) with IBC development. DNA methylation changes and their impact on the diagnosis, prognosis, and treatment of IBC are also described. Recent studies are focusing on the use of histone deacetylase inhibitors as promising epigenetic drugs for treating IBC. All efforts must be undertaken to unravel the epigenetic marks that drive this disease and how this knowledge could impact strategies to reduce the risk of IBC development and progression.

Inflammatory Breast Cancer in Pregnancy and Lactation.

Inflammatory breast cancer (IBC) represents only 1% to 5% of all breast malignancies and is an extremely aggressive subtype. At time of diagnosis, up to 85% of patients will present with regional nodal metastases and up to 30 % will have metastasis to distant organs. There is limited medical literature describing treatment guidelines for IBC during gestation. The best diagnostic tools are core needle and full-thickness skin punch biopsies to assess presence of dermal lymphatic invasion. Breast Ultrasound is preferred to mammogram, but mammography could still be done with proper fetal shielding. Ultrasound and Magnetic resonance imaging are used for staging. Pregnant patients should be managed with special attention to the health of the fetus by a multidisciplinary team. Treatment based on current guidelines consist of a sequence of systemic chemotherapy followed by mastectomy with axillary dissection (modified radical mastectomy), and even if good clinical nodal response to neoadjuvant therapy is obtained, sentinel node biopsy is not recommended. Radiation therapy is to be given once the baby has been delivered. Chemotherapy is not recommended in the first trimester, and anti-estrogen hormonal therapy, as well as targeted Her2-neu therapies are contraindicated during the length of the pregnancy. There is no evidence that early termination improves the outcome. However, given the poor prognosis of IBC, patients should be fully counseled on the risks and benefits of continuing or terminating an early pregnancy.

Abstract P6-09-10: Association of dermal lymphatic involvement and survival in inflammatory breast cancer

Abstract Purpose: The pathologic hallmark of inflammatory breast cancer (IBC) is the presence of tumor emboli within the papillary and reticular dermis of the skin, termed dermal lymphatic invasion (DLI). DLI can be confirmed with a skin-punch biopsy in approximately 75% of cases, but its presence is not required for the diagnosis of IBC because of variability of tumor emboli in affected areas. The impact of confirming DLI by skin biopsy on the clinical outcome of IBC is unknown. We hypothesize that the ability to confirm DLI by biopsy is associated with a higher tumor emboli load and consequently a poorer disease prognosis. Therefore, we examined whether documented DLI was associated with poorer overall survival (OS) in IBC. Methods: Clinical characteristics were evaluated among 286 women presenting with IBC between 1999 and 2016 and enrolled in the IRB-approved IBC registry at Dana-Farber Cancer Institute. Kaplan-Meier curves were used to estimate overall survival and the log-rank test to examine survival differences based upon the presence of DLI. Multivariable Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals for associations of DLI and risk of death among women with IBC. Results: A total of 102 deaths occurred in our study population over a median follow-up of 2.5 years (yr) (range 10 days to 16 yr). Compared to IBC without DLI, IBC with confirmed DLI was more likely to be associated with the presence of lymphovascular invasion in breast biopsies (73.1% vs. 37.7%; p-value &amp;lt;0.01) but less likely to be seen with edema of the skin (59.1% vs. 81.1%; p-value=0.02). Documented DLI was not related to the presence of de novo metastasis at presentation (11.5% vs. 28.7%; p-value=0.07). OS did not significantly differ among women with IBC based upon the presence of DLI (log-rank test p-value=0.68). In multivariable models, DLI was not independently associated with OS in inflammatory breast cancer, HR(95%CI) = 0.92(0.48-1.78). Conclusions: Our findings suggest that IBC presenting with documented DLI on skin biopsy may vary with regard to clinical characteristics at diagnosis, including lymphovascular invasion within the breast, edema of the skin of the breast, and the presence of de novo metastases. These clinical distinctions suggest potential differences in biology of IBC according to the presence or absence of DLI, and the extent of tumor emboli. However, in this study, DLI was not found to be an independent prognostic factor in IBC with respect to OS. Due to the variability in the clinical features of IBC at presentation and inherent complexities in selecting skin biopsy sites, studies to investigate the accuracy of determining DLI based on punch biopsy are necessary to more comprehensively assess the impact of DLI on clinical outcomes in IBC. Citation Format: Hirko KA, Schlossman J, Harrison BT, Yeh ED, Jacene HA, Nakhlis F, Schlosnagle E, Overmoyer BA. Association of dermal lymphatic involvement and survival in 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 P6-09-10.

Review of ultrasound appearance in inflammatory breast cancer: A pictorial essay.

Inflammatory breast cancer (IBC) is a rare malignancy accounting for 1-2% of breast cancers. It has an aggressive clinical presentation and poor prognosis. The sonographic findings in 41 patients with a clinical diagnosis of IBC and biopsy-proven breast malignancy are presented in this study. The most common finding was the presence of skin thickening (92%). Multiple small anechoic spaces within the dermis, correlating with the presence of dermal lymphatic invasion by tumour emboli on histopathology were noted in approximately one-third of cases. Other sonographic findings included single or multiple masses, parenchymal oedema, axillary lymphadenopathy, echogenic foci consistent with microcalcifications and increased vascularity.

Abstract P5-01-04: In vivo lymphatic imaging of a human inflammatory breast cancer model

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive but poorly understood type of breast cancer. IBC constitutes only appoximately 5% of all newly diagnosed breast cancers, yet responsible for 8-10% of breast cancer-related deaths. Unfortunately, there are no definitive molecular or pathological, early diagnostic criteria for IBC. IBC grows in nests or sheets, spreading outward into the skin and eventually distant sites through dermal lymphatic vessels; therefore, one of the key pathological characteristics of IBC is dermal lymphatic invasion by tumor emboli, which can lead to obstruction of the lymphatic drainage possibly causing the clinical inflammatory features of diffuse erythema, rapid breast enlargement and edema as indicated by peau d’orange (orange peel) appearance covering at least a third of the breast surface. However, relatively little is known about the role of lymphatic function in IBC growth and metastasis. The purpose of this study was to non-invasively and longitudinally image changes of lymphatic drainage patterns in mice bearing the triple negative, human IBC SUM149 cells, which were stably transfected with iRFP gene reporter (iRFP-SUM149) in order to better monitor tumor growth and metastasis. Methods: Eight to ten weeks old female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were housed and fed special sterilized pelleted food and sterilized water. Two weeks after feeding special diets, mice were subcutaneously injected with iRFP-SUM149 cells in the left hindlimb or orthotopically in the left inguinal mammary fat pad (MFP). Fluorescence images were acquired immediately after and for up to 10 mins after i.d. injection of indocyanine green (ICG) using a custom-built NIRF imaging system. To achieve a greater magnification, a macrolens was used. Results: Our NIRF imaging data showed gradual changes of lymphatic vessels around the tumor. In mice with an orthotopic tumor, ICG accumulation was observed in the distal peritumoral region at up to 3 weeks p.i., indicative of the start of tumor obstruction of normal lymphatic drainage. As tumors grew, the greater extent of lymph flow, but not all, was obstructed by a tumor, resulting in dermal backflow and thus staining more lymphatic capillaries and eventually rerouting of lymphatic drainage was detected due to complete obstruction of lymph flow by a tumor. Similar to an orthotopic model, mice with a s.c. tumor also showed altered lymphatic drainage patterns during tumor growth. Interestingly, extravasation of ICG into iRFP-SUM149 was detected at 3 weeks p.i., due to leaky lymphatic vessels. A similar lymphatic phenotype has also been observed in IBC patients. Previously, we have imaged atypical tortuous lymphatics and altered lymphatic drainage in the arm and affected breast of a patient with IBC using clinical NIRF imaging developed in our team to image as deep as 3-5 cm with a microdose of ICG. Conclusions: Longitudinal, non-invasive imaging of lymphatic functional and architectural changes provides a new method to dynamically monitor lymphatic response to IBC growth and metastasis. Citation Format: Sunkuk Kwon, Germaine Agollah, Grace Wu, Eva M Sevick-Muraca. In vivo lymphatic imaging of a human inflammatory breast cancer model [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 P5-01-04.

Abstract OT1-1-15: A phase II study of dovitinib as salvage therapy in patients with stage IV inflammatory breast cancer HER2-negative with local or distant relapse

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer that accounts for 3 to 5% of all invasive breast tumors in the United States. IBC possesses an increase of proangiogenic factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet derived growth factor (PDGF) as compared with non-IBC. In particular, FGF family receptors play a critical role in tumorigenesis, morphogenesis, and inducers of angiogenesis. Dovitinib (TKI258) is an oral tyrosine kinase inhibitor with in vitro IC50 values of approximately 10 nmol/L against FGFR1-3, VEGFR1-3, and PDGFR. These structurally related receptors are important for the growth and survival of endothelial cells during tumor angiogenesis. Trial Design: This is a single institution, single arm phase II study. Patients receive a single daily oral dose of dovitinib 500 mg for 5 consecutive days, followed by a 2-day rest period (5 days on/2 days off schedule). Eligibility: Patients have histological confirmation of breast carcinoma with a clinical diagnosis of IBC based on presence of inflammatory changes in the involved breast, including diffuse erythema and edema (peau d’orange). Pathological evidence of dermal lymphatic invasion should be noted but is not required for diagnosis. HER2-negative. ECOG PS 0-2. Baseline MUGA or echocardiogram scans with LVEF of &amp;gt; 50%. Normal hematology, liver and kidney function laboratory studies. Patients must have received at least 2 chemotherapy lines for metastatic disease and have relapsed. Research Hypothesis: Dovitinib has antitumor activity in patients with HER2-negative advanced IBC. Specific Aims: Primary objective: to determine the disease control rate (CR, PR, and SD). Secondary objective: to evaluate safety profile. Exploratory biomarkers: circulating tumor cells (CTC), CTC undergoing EMT, and cancer stem cells Statistical Methods: The primary endpoint is the six-month disease control rate (ORR) as defined by RECIST 1.1. A response is anyone who experiences SD, CR or PR in the first 6 months. We will conduct this study with Simon’s two-stage design using the mini-max criterion and the response rate will be estimated accordingly. It is assumed that dovitinib will have a target ORR of 30%. An ORR of 10% or lower is considered a failure based on the typical ORR with a second line regimen for IBC and the new regimen will be rejected under this circumstance. When the probability of accepting a "bad" regimen (i.e. response rate &amp;lt; 10%) is 0.05 and the probability of rejecting a "good" regimen (i.e. response rate &amp;gt; 30%) is also 0.10, Simon’s design to minimize the maximum sample size requires 22 patients in the first stage. If two or less patients respond to the treatment, the trial will be stopped and the regimen will be declared as ineffective. If at least three of the first 22 patients respond to the treatment, 11 additional patients will be entered in the study to reach a total of 33 patients. By the end of the study, the new regimen will be rejected if response rate is less than or equal to 6 out of 33 patients and will be accepted otherwise. Present Accrual and Target Accrual: A total of 22 patients were accrued. Target accrual is 33 patients. Citation Format: Ricardo H Alvarez, Mariana Chavez-MacGregor, Joe Ensor, James L Murray, Kimberly Koenig, Savitri Krishnamurty, Antony Lucci, Gildy V Babiera, Wendy Woodward, Gary J Whitman, Summer A Jackson, Michael Shi, Kenneth Culver, James L Reuben, Naoto T Ueno, Vicente Valero. A phase II study of dovitinib as salvage therapy in patients with stage IV inflammatory breast cancer HER2-negative with local or distant relapse [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 OT1-1-15.

Mesenchymal stem cells mediate the clinical phenotype of inflammatory breast cancer in a preclinical model.

IntroductionInflammatory breast cancer (IBC) is an aggressive type of breast cancer, characterized by very rapid progression, enlargement of the breast, skin edema causing an orange peel appearance (peau d’orange), erythema, thickening, and dermal lymphatic invasion. It is characterized by E-cadherin overexpression in the primary and metastatic disease, but to date no robust molecular features that specifically identify IBC have been reported. Further, models that recapitulate all of these clinical findings are limited and as a result no studies have demonstrated modulation of these clinical features as opposed to simply tumor cell growth.MethodsHypothesizing the clinical presentation of IBC may be mediated in part by the microenvironment, we examined the effect of co-injection of IBC xenografts with mesenchymal stem/stromal cells (MSCs).ResultsMSCs co-injection significantly increased the clinical features of skin invasion and metastasis in the SUM149 xenograft model. Primary tumors co-injected with MSCs expressed higher phospho-epidermal growth factor receptor (p-EGFR) and promoted metastasis development after tumor resection, effects that were abrogated by treatment with the epidermal growth factor receptor (EGFR) inhibitor, erlotinib. E-cadherin expression was maintained in primary tumor xenografts with MSCs co-injection compared to control and erlotinib treatment dramatically decreased this expression in control and MSCs co-injected tumors. Tumor samples from patients demonstrate correlation between stromal and tumor p-EGFR staining only in IBC tumors.ConclusionsOur findings demonstrate that the IBC clinical phenotype is promoted by signaling from the microenvironment perhaps in addition to tumor cell drivers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0549-4) contains supplementary material, which is available to authorized users.

Abstract 4617: Targeting interferon-induced transmembrane protein 1: a novel strategy to treat inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) is a very rare and aggressive form of breast cancer that accounts for about 1 to 5% of all breast cancers diagnosed in the United States. IBC is characterized by absence of a discrete mass, coupled with very rapid progression and dermal lymphatic invasion, which makes it the most lethal of the breast cancers. IBC is currently treated by a multimodal approach involving systemic chemotherapy, surgery, followed by radiation therapy; however, this strategy has yielded a 5-year survival outcome of only 20 to 30%. There is an urgent need to better understand the molecular mechanism that contributes to the development of IBC and to use that knowledge to identify novel targets that can be manipulated to enhance treatment options for IBC patients. In the present study, we have examined the role of human interferon-inducible transmembrane protein 1 (IFITM1) in the proliferation and invasion of inflammatory breast cancer. IFITM1 is a member of the interferon-inducible transmembrane protein family, which is involved in many cellular functions, including the transduction of antiproliferative and homotypic adhesion signals in lymphocytes, inhibition of viral replication, and promotion of cell invasion. At present, however, very little is known about the function of IFITM1 in inflammatory breast cancer. Our in vitro studies were performed in human inflammatory breast cancer cell lines; SUM149, SUM190, MDA-IBC-3, and non-inflammatory MCF-7 breast cancer cells. We found that IFITM1 mRNA and protein levels were constitutively overexpressed in SUM149 and MDA-IBC-3 cell lines but not expressed in non-inflammatory MCF-7 cells. Functional analysis of IFITM1 by silencing of its expression with small-interfering RNA showed that the loss of IFITM1 significantly inhibited the proliferation and invasiveness of SUM149 and MDA-IBC-3 cells and it completely blocked the ability of these cells to form colonies in soft agar assays. Interestingly, we found that interferon (IFN-α) significantly induced IFITM1 expression in all three of the IBC cell lines; however, IFN-α treatment inhibited the proliferation and invasiveness of the IBC cells. Our data also showed that several other interferon-regulated genes, including STAT1, PLSCR1, and STAT6 were elevated in inflammatory SUM149, MDA-IBC-3, and SUM190 cells but not in non-inflammatory MCF-7 cells. Overall, these results demonstrate that IFITM1 plays an important role in the proliferation and invasiveness of IBC cells and they suggest that targeting IFITM1 might be a novel strategy to treat inflammatory breast cancer. Citation Format: Joshua W. Ogony, Joan Lewis-Wambi. Targeting interferon-induced transmembrane protein 1: a novel strategy to treat inflammatory breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4617. doi:10.1158/1538-7445.AM2014-4617

Initial Response to Chemotherapy, Not Delay in Diagnosis, Predicts Overall Survival in Inflammatory Breast Cancer Cases

To determine whether chemotherapy response and diagnostic delay affect overall survival (OS) of classic inflammatory breast cancer (IBC) cases receiving chemotherapy as initial treatment and to determine whether OS differs between classic and "atypical" IBC cases. This is a prospective cohort study of 155 patients enrolled in the IBC Registry. "Classic" IBC cases met AJCC or SEER case definitions. "Atypical" IBC cases exhibited classic features but involved <1/2 breast without documented dermal lymphatic invasion. Variables included OS (years from initial chemotherapy treatment until death or last contact), chemotherapy response (complete, partial, or none), diagnostic delay (days from first medical contact for signs/symptoms of abnormal breast to definitive pathologic IBC diagnosis), age at diagnosis (y), and triple-negative status (yes or no). OS curves stratified by individual predictors were estimated and compared using Kaplan-Meier methods and log-rank tests. Associations between OS and predictors were examined collectively using Cox proportional hazards regression. Classic IBC cases with complete, partial, or no response had respective median (95% confidence interval [CI]) OS times of 10.30 (6.78, +), 6.27 (4.42, +), and 2.86 (1.11, 11.42) years (P=0.0072). Chemotherapy response was significantly associated with OS after controlling for covariates (P=0.003). Women not responding to chemotherapy had a significantly higher hazard of death compared with women with complete (hazard ratio [HR]=5.76; 95% CI, 2.09-15.84) or partial (HR=3.40; 95% CI, 1.27-9.10) response. Diagnostic delay was not significantly associated with OS (HR=1.003; 95% CI, 0.999-1.007). OS did not differ significantly between classic and "atypical" IBC cases (P=0.60). Response to standard IBC chemotherapy is a dominant prognostic factor in determining patient outcomes. In our study, with limited statistical power, delay in diagnosis defined as >60 days from the time of first physician contact did not seem to affect patient outcomes. Data support similarities between classic and "atypical" IBC.

Inflammatory breast cancer: a clinical diagnosis

I read the case report “Lactating breast abscess: a rare presentation of adenosquamous breast carcinoma” by Alipour et al(1) with great interest. The authors presented an interesting case of breast cancer in a lactating woman who presented with a longstanding breast abscess of two months’ duration. They have diligently reiterated the need to perform abscess wall biopsy in all atypical breast abscesses. They also ruled out the possibility of inflammatory breast cancer (IBC) on account of the absence of tumour emboli in the dermal lymphatics. However, I would like to stress that the presence of tumour emboli in the dermal lymphovascular spaces is neither sufficient nor required for the diagnosis of IBC. IBC is clinically diagnosed by a short duration of symptoms, typical clinical signs on examination of the affected breast, and pathological diagnosis of breast carcinoma either through skin biopsy or core/needle biopsy of the underlying lump, if present. Rapidly progressing diffuse erythema (pink or mottled pink hue) and oedema of more than one-third of the breast distinguishes IBC from noninflammatory locally advanced breast cancer (LABC) with skin involvement.(2,3) The breast oedema manifests as an increase in the size of the breast and exaggerated hair follicle pits, classically known as peau d’orange. These skin changes represent the dermal lymphatic obstruction, resulting from invasion by the tumour emboli; this phenomenon is considered a pathological hallmark of IBC.(4) It is necessary to highlight here why dermal lymphatic invasion by tumour emboli is not considered diagnostic of IBC. Firstly, dermal tumour emboli may not be identified in all cases of IBC despite adequate sampling of the skin, and secondly, dermal tumour emboli, though less numerous and smaller in size, may also be seen in noninflammatory LABC.(2) In a consensus statement, an international expert panel agreed that the following minimum criteria are required for the diagnosis of IBC: a history of rapid onset of breast erythema; a duration of history of no more than six months; oedema and/or peau d’orange and/or warm breast (with or without an underlying palpable mass); erythema occupying at least one-third of the breast; and pathological confirmation of invasive carcinoma.(4) However, the panel also strongly recommended that skin biopsy be done in all patients suspected to have IBC, as it may confirm the presence of dermal tumour emboli and may also give the pathological diagnosis of carcinoma if no underlying lump or regional metastatic lymphadenopathy is detected. A set of uniform diagnostic criteria for IBC, which can be used worldwide, will help medical practitioners obtain more accurate epidemiological data, and also provide better understanding of the clinicopathological profile and treatment outcomes of IBC across different geographical regions. Yours sincerely,

Abstract P5-14-14: Trimodality therapy for inflammatory breast cancer: The fox chase experience

Abstract Objective: To evaluate the outcomes and toxicity of inflammatory breast cancer (IBC) patients (pts) treated with trimodality therapy. Materials and Methods: A retrospective chart review of 71 non-metastatic women with IBC treated using once daily (QD) radiation fractionation between 1990 and 2011 was performed. Eighty-six% of pts underwent ER/PR receptor status testing, and 43% underwent HER-2 testing. All pts underwent a course of neoadjuvant chemotherapy. Forty-three pts had adjuvant hormonal therapy, and 7 had adjuvant trastuzumab. All pts underwent mastectomy with axillary lymph node dissection followed by chest wall irradiation to a median dose of 50 Gy (34-50), and 46 Gy to supraclavicular region in 2-Gy QD fractions. Internal mammary nodes were treated in 3 pts (4%) and a posterior axillary boost (PAB) was added in 32 (45%) cases. Scar boosts (SB) were employed in 6 patients. A univariate (UVA) analysis using Kaplan-Meier estimation method included cN, pN, pT stage, PAB, SB, dermal lymphatic invasion, lymphovascular invasion, body mass index, response to chemo (RC), close/positive surgical margins (SM), age, and triple negative (TN) receptor status. Significant predictors on UVA were included in multivariate (MVA) analysis using the Cox proportional hazard model. Primary endpoints included local-regional recurrence (LRR), distant metastasis (DM), overall survival (OS), cause specific (CSS) and disease-free survival (DFS). Acute and chronic skin reactions and lymphedema were evaluated. Results: Median follow-up was 34 months (2-265) and the median age was 56 years (36-82). Actuarial 3 and 5 year OS was 63% and 43%, and DFS was 51% and 43%, respectively. The median time to first failure, LRR, and DM was 20 mo (1.5-147), 13.5 mo (9-18), and 21 mo (1.5-147), respectively. The first site of failure was distant for 34 (48%), and local-regional for 4 pts (6%). Twenty-five pts (35%) were without failure at the time of last follow up. UVA analysis for LRR revealed SM (p = 0.05) and RC (p = 0.002) to be significant predictors of failure, however, these were not significant on MVA. UVA for DM identified SM (p = 0.03), TN (p&amp;lt;0.0001), and pN (p = 0.03) to be significant predictors, with TN (p = 0.002) being the only significant variable on MVA. Significant predictors for OS on UVA were TN (p = 0.04), pN1 vs pN2 disease (p = 0.02), SM (p = 0.0008) and RC (p = 0.008). None of these remained significant on MVA. Significant predictors for CSS on UVA were TN (p = 0.008), pN (p = 0.05), SM (p = 0.0004) and RC (p = 0.002). Only SM (p = 0.01) was predictive on MVA. Significant predictors for DFS on UVA were SM (p = 0.0003), pN1 vs pN2 (p = 0.05), RC (p = 0.0006) and TN (p = 0.0001). Only RC (p = 0.005) and TN (p = 0.001) remained significant on MVA. Twelve pts developed dry, and 14 developed moist desquamation. Three pts developed CTCAE v3 G3 lymphedema, and 5 pts experienced G3 fibrosis. Conclusions: We found that TN predicted for a higher rate of DM and worse DFS. Improved CR predicted for better DFS. Age &amp;lt; 45, SM, pN2 disease and poor RC were not predictive of LRR. Use of SB did not improve LRR, although this cohort was small. Overall, our results show encouraging 3 and 5 year OS rates and acceptable toxicity for IBC pts treated with trimodality therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-14-14.

Abstract P3-10-03: Bartonella henselae Infection Detected in Patients with Inflammatory Breast Cancer.

Abstract Inflammatory breast cancer (IBC) is a very aggressive type of advanced breast cancer with a poor prognosis. Clinical symptoms involve a rapid onset of changes in the skin overlying the breast, including edema, redness and swelling including a wrinkled and orange peel appearance in the skin. This particular presentation is due to the invasion of the skin dermal lymphatics by breast cancer cells that obstructed the lymph channels producing the characteristic skin changes that mimic an inflammatory process. Mouse Mammary Tumor-associated Virus (MMTV) and other infectious agents have been consider as possible etiological agents of IBC particularly related to the initial description of higher incidence in women living in rural areas in North Africa. Although, the etiopathological role of bacteria in this disease has never been explored in spite of the evidence that chronic infections with certain bacteria can facilitate tumors development. Several bacterial infections promote cell proliferation and could increase the rate of cell transformation. Bartonella spp. is an emerging pathogen that can cause conditions which are characterized by the development of proliferative lesions. Bartonella might cause vasculoproliferative disorders by triggering the proliferation of endothelial cells and inducing the secretion of proliferative cytokines from infected host cells. It cause persistent infection of erythrocytes and endothelial cells in their mammalian hosts and their transmission occur mainly by blood-sucking arthropods. Bartonella are Gram-negative bacteria usually associated with cat-scratch disease, urban trench fever, bacillary angiomatosis-peliosis and endocarditis. Some reports have showed some similarities between cat scratch disease and inflammatory breast cancer (Povoski et al., Breast J 9: 497–500, 2003). Methods and Results: In the present work, we report the identification of Bartonella henselae in the pleural effusions of two patients with IBC. These patients had both estrogen-receptor negative (ER−) and progesterone-receptor negative (PR−) disease and have failed a number of previous chemotherapy regimens. Furthermore, they had metastatic disease to the pleura, skin, lymph nodes and lung. Cells from the pleural fluids of these patients stained positive for Bartonella sp. using the Warthin-Starry staining kit. Moreover, we used PCR and sequencing of the 16S–23S intergenic spacer (ITS) region to identify the etiological agent as Bartonella henselae. By immunofluorescence using an antibody that specifically recognized Bartonella henselae, the bacteria were found in the nucleus of IBC tumor cells confirming the infection and their intracellular localization. Conclusions: These results suggested that bacteria infections such as the one produced by Bartonella henselae might contribute to the clinical and pathological characteristics of IBC, associated with rapid spread of the breast tumor cells through the lymphatic system. The infection and activation of endothelial lymphatics by Bartonella might contribute to the highly metastatic process observed in IBC patients. An acute inflammatory reaction triggered by the Bartonella infected endothelium may be crucial for initiating the chronic inflammation in IBC patients and the rapid spread of tumor cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-10-03.

Abstract OT2-3-10: Phase II study of panitumumab, nab-paclitaxel, and carboplatin for patients with primary inflammatory breast cancer (IBC) without HER2 overexpression

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive form of primary breast cancer. The outcome for patients with IBC is bleak despite multimodality treatment approaches. 10-year disease-free survival rates after combined anthracycline and taxane-containing chemotherapy, surgery, and radiation are only 20%–25%. Our recent study found EGFR overexpression, a predictive factor of poor outcome, in 12 of 40 (30%) patients with IBC. Panitumumab has shown activity against EGFR overexpressing breast cancer xenograft model. Trial design: This is a single center, open-label, phase II study to evaluate the safety and efficacy of panitumumab in combination with preoperative chemotherapy. The treatment regimen consists panitumumab 2.5 mg/Kg given intravenously alone for the first week, followed by weekly panitumumab, nab-paclitaxel (100mg/m2) and carboplatin (2 AUC) (PNC) for 12 weeks. Patients then will receive 5-FU, epirubicin, and cyclophosphamide (FEC) every 3 weeks for 4 cycles prior to surgery. Eligibility criteria: 1) Histological confirmation of breast carcinoma with pathologic evidence of dermal lymphatic invasion and clinical diagnosis of IBC, including diffuse erythema, heat, ridging, and peau d'orange; 2) Normal HER2 expression; 3) No prior therapies for IBC; 4) Adequate hematologic, cardiac, renal and hepatic functions. Specific aims: 1) Primary objective is to determine the pathologic complete response (pCR) rate in patients with primary IBC without HER2 overexpression; 2) Secondary objectives are to determine the disease-free survival (DFS), overall survival (OS), the safety and tolerability of PNC regimens and the correlates of pathologic response rate and EGFR expression level. Statistical methods: 1) Previous studies have shown that this IBC patient population achieved a 13% pCR rate on the standard of care. We assume a beta (0.26, 1.74) prior distribution for the pCR rate. This prior distribution has a mean of 13% and a standard deviation of 19%. 2) We will stop the trial early if P (pCR rate &amp;gt;/= 13%) is &amp;lt; 0.01. If we determine that there is less than a 1% chance that the pCR rate is 13% or more we will consider stopping the trial. 3) Once we have completed the study we will estimate the pCR rate with a 90% credible interval. If we have pCR in 4 of the 40 patients (10%), then our 90% credible interval for the pCR rate will be 4.0–19.6%. If we have pCR in 8 of the 40 patients (20%), then our 90% credible interval for the pCR rate will be 10.6–30.4%. We will also report the posterior probability that the pCR rate is 13% or more. For example, if we have pCR in 8 of the 40 patients (20%), then the probability that the pCR rate is 13% or more is 0.869. Present accrual and target accrual: To date, 13 patients have been enrolled. Target accrual is 40 patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT2-3-10.