Breast Lymphatics Research Articles

Localization of Central Breast Lymphatics and Predefined Separation of Lobes along the Horizontal Septum

Background: The predictable localization of the neurovascular supply along the ligamentous suspension, composed of the horizontal septum, vertical ligaments, and superficial fascia, has increased safety in breast reduction. Lymphatic drainage of the breast has always been described as running close to vascular supply. However, the correlation between the lymphatic course and ligamentous suspension has not yet been considered. This study aimed to visualize the relationship, direction of lymphatic flow, and predefined separation of lobes along the horizontal septum. Methods: To investigate central drainage, methylene blue was injected subareolarly in five breasts of female cadavers before blunt preparation of the horizontal septum in anatomical studies. To visualize central and peripheral drainage, lymphographin was injected into one of the three different sites in 14 breasts; the dynamic flow of drainage was observed during subsequent septum-based breast reductions in clinical settings. Results: In all anatomical studies, a predefined section of the glandular layers allowed access to central clearance along the stained horizontal septum. Clinical investigations similarly showed clearance along the corresponding part of the ligamentous suspension, most reliably along the horizontal septum. The affected quadrant of the breast, its relation to the nipple-areola complex, and the anterior-posterior axis toward the thoracic wall mainly determine the direction of lymphatic flow. Interconnections along the ligaments may explain the unpredictability of final clearance directions. Conclusions: This study shows the horizontal septum as a guiding structure for central mammary drainage. This may encourage a septum-based approach for refinement of procedures such as oncoplastic, irradiation, and lymphedema treatments.

Abstract P1-10-08: Incidence of rib fracture following treatment with proton therapy for breast cancer

Abstract Purpose: To determine the rate of rib fracture in a cohort of patients with breast cancer treated with proton therapy and enrolled on a prospective registry. In series investigating photon therapy for breast cancer, the rib fracture rate ranges from 0.3-13% Methods: From a prospective database, we identified patients treated with proton therapy for breast cancer at our institution between January 1, 2012 and December 31, 2020. Clinical and dosimetric data was extracted from the electronic medical record. The cumulative incidence method assessed rib fracture rate; the Fine-Gray test statistic assessed prognostic significance select variables. Results: 225 patients were identified, 223 women and 2 men. Median age at the time of radiation was 57.8 years (range, 25 – 87). 26% of patients were black, 69% white and 5% were other races or race not disclosed. 5% were Hispanic. 74% of patients had left-sided breast cancer, 5% bilateral, and 21% right-sided. DEXA scan was normal in 20%, showed osteopenia in 34%, osteoporosis in 6%, and not performed in 40%. 57% of patients received antiendocrine therapy with an aromatase inhibitor. For 16% of patients, the breast +/- internal mammary nodes (IMN) were treated, while 32% underwent proton therapy to treat the breast and comprehensive regional lymphatics. 1% of patients had chest wall +/- IMN treated, while 51% underwent proton therapy to treat the chest wall and comprehensive regional lymphatics. 41% of patients were treated with passive scatter proton therapy (n=92); 52% of these had mastectomy. 58% of patients received treatment with pencil beam scanning (PBS) proton therapy (n=131); 53% of these had mastectomy. A combination of passive scatter and PBS was used for 2 patients (1%). 85% of patients received a boost. Median follow-up was 3.1 years (range, 0.2 – 9.1). 97% of patients had > 12 months of follow-up. The 3 year cumulative incidence of in-field rib fracture was 3.7% (95%CI: 1.6%-7.1%). In total, 8 patients developed in-field rib fractures, one symptomatic and 7 identified incidentally on surveillance imaging, for a 0.4% rate of symptomatic rib fracture. Median time from completion of radiation to identification of rib fracture was 1.8 years (range, 0.4-7.4 years). Rib fractures occurred within 2.2 years from radiotherapy completion for seven of the eight patients who experienced this side effect. Three patients developed rib fractures outside of the radiation field, for a cumulative incidence of out-of-field rib fracture of 0.9% (95%CI: 0.2-3.0%). No variables were associated with rib fracture on univariate analysis. Of those with in-field rib fractures, 3 had low and 3 had normal bone density while 2 had not undergone testing; of those with out-of-field rib fractures, 2 had osteopenia and 1 did not have testing. Conclusions: Although a proton beam has higher biological dose deposition at end-of-range than the assumed constant RBE=1.1, the 3 year rate of any in-field rib fractures in our series remains low at 3.7%, with a 0.4% rate of symptomatic rib fracture. Contouring ribs as an OAR and establishing a dose constraint warrants further investigation as a means to maintaining a low rate of rib fracture with proton therapy. Citation Format: Julie Bradley, Xiaoying Liang, Raymond Mailhot Vega, Chunbo Liu, Eric Brooks, Teena Burchianti, Emma Viviers, Roi Dagan, Oluwadamilola Oladeru, Christopher Morris, Nancy Mendenhall. Incidence of rib fracture following treatment with proton therapy for breast cancer [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 P1-10-08.

Anti‐cancer Effects of Coumestrol in Triple‐Negative Inflammatory Breast Cancer 3D Models: Opportunities for In Vivo Studies

Inflammatory breast cancer (IBC) is the most aggressive subtype of breast cancer. Each molecular subtype is characterized by the presence or absence of two hormonal steroid receptors, estrogen receptor (ERα‐66) and progesterone receptor (PR), and a growth factor receptor called HER2. Particularly, a subtype of breast cancer that lacks these three biomarkers is known as triple‐negative breast cancer (TNBC). However, previous studies showed that TNBC can express alternate estrogen receptors known as ERα‐36 and GPR30, mediating a non‐genomic signaling upon estrogen (E2) treatment. Today, effective targeted therapeutics for triple‐negative inflammatory breast cancer (TN‐IBC) patients, accounting 20‐40% of TN‐IBC cases, is limited. Coumestrol (Cou), a plant derived compound structurally similar to estrogen has shown to induce a cytotoxic effect in TNBC. Till this day, there is no known study of the effects of Cou in three‐dimensional culture models using Matrigel, a solubilized membrane matrix that mimics the tissue microenvironment. Additionally, we want to test Cou’s inhibitory effect on tumor emboli formation, a distinct hallmark of IBC. This tumor cell clusters are known to infiltrate and block the breast lymphatics of patients, presenting a barrier for successful IBC treatment (Arora Etl. 2017). We hypothesize that Cou inhibits cell viability and tumor emboli formation in TN‐IBC cells. First, we performed 3D dose response curves to determine the half‐maximal inhibitory concentration (IC50) of Cou (50µM). Next, 3D proliferationassays were performed using 50µM of Cou. After 10 days, E2 showed a significant increase in colony size in comparison to vehicle control (DMSO). Also, proliferation decreased more than a half after treating the cells with Cou alone and in combination of both treatments. When testing the tumor emboli formation after 72 hours, Cou did not inhibit the formation of emboli, but it decreased the tumor emboli growth rate shown by the decreased in tumor emboli size in both culture conditions. In comparison, E2 promoted a significant increase in the size of the tumor emboli. Lastly, we used Western Blot analysis to validate the decrease in phosphorylation levels of ERK1/2 and AKT, two kinases involved in estrogen non‐genomic signaling, upon Cou treatment. Together, these data demonstrated that Cou decreases cell viability in 3D proliferation assays and decreased growth rate of tumor emboli in TN‐IBC cells. In summary, the anticancer effects of Coumestrol in IBC three‐dimensional models provide the basis for future in vivo studies using animal models.

457 Semaphorin7a expression in breast cancer promotes susceptibility to immune checkpoint blockade

OBJECTIVES/GOALS: The goal of this study is to decipher the mechanisms by which breast cancers evade the immune system to facilitate metastasis through the lymphatic vasculature. We believe this is caused, in part, by semaphorin 7A (SEMA7A) as its expression is associated with increased lymphatic vessels, tumor-associated macrophages, and metastasis in breast cancer. METHODS/STUDY POPULATION: We have observed that breast cancer cells, lymphatics, and macrophages exhibit SEMA7A-dependent expression of PD-L1 in vitro, which may make SEMA7A+ tumors more sensitive to anti-PD-1/PD-L1 immunotherapies. Therefore, we utilized multiple orthotopic, immunocompetent mouse models to determine if SEMA7A expression makes tumors more susceptible to immune checkpoint blockade. E0771 and 66cl4 mouse mammary carcinoma cells were injected orthotopically into the #4 mammary fat pads. Once tumors reached 50-100 mm^3 mice were injected intraperitoneally with 250ug of anti-PD-1, anti-PD-L1, or IgG control every third day. Tumors were measured with calipers every other day and harvested for flow cytometry to determine the effect of immune checkpoint blockade on the TME in SEMA7A+ tumors. RESULTS/ANTICIPATED RESULTS: We reveal that growth of SEMA7A overexpressing (OE) tumors, but not controls, was significantly slowed with both anti-PD-1 and anti-PD-L1 treatments. Flow cytometric analysis of cells from the TME revealed increased LECs, TAMs, and PoEMs in SEMA7A+ tumors, compared to controls – all populations had higher PD-L1 expression, which was decreased with both anti-PD-1 or anti-PD-L1. We also observed a decrease in PD-L1 expression on the tumor cells with treatment. Furthermore, LEC, TAM, and PoEM presence was decreased within the TME alongside increased presence of activated CD4 and CD8 T cells. Collectively, our results suggest that SEMA7A expression in breast cancers activates a major pathway associated with immune evasion and helps to establish a pro-tumor microenvironment. DISCUSSION/SIGNIFICANCE: SEMA7A expression establishes a pro-tumor microenvironment, which can be targeted with readily available FDA approved drugs such as immune checkpoint-based therapies. Since SEMA7A+ breast cancers have high rates of metastasis, more specific treatments for these patient populations should be explored.

Abstract P1-04-06: Semaphorin7a expression in breast cancers promotes susceptibility to immune checkpoint blockade

Abstract Five-year survival rates for women diagnosed with stage II breast cancer are >85% on average; however, in women diagnosed with stage II breast cancer (BC) within 10 years of last childbirth, we observe a 5-fold increase in risk for developing distant metastasis when compared with nulliparous women1. Major driving factors for stage II diagnosis include increased tumor-associated lymphatic vessel density (LVD), lymphovascular invasion (LVI), and lymph node positivity (LN+) and are all associated with poor prognosis for breast cancer patients. Nonetheless, the mechanisms underlying development of LN metastasis, and how LN metastasis seed distant metastases, has remained elusive. LVD, LVI, and LN+ can be driven by tumor-associated macrophages (TAMs), which have been implicated in creating a pro-tumor tumor microenvironment (TME) in many types of cancer. Additionally, podoplanin (PDPN)-expressing macrophages (PoEMs), are a newly characterized type of TAM that specifically contribute to tumor-associated lymphatic vessel formation2,3. Our lab identified that semaphorin7A (SEMA7A)—a neuroimmune molecule—is significantly associated with increased metastasis in LN+, but not LN-, BC patients, as well as increased recurrence in cases of BCs diagnosed within 10 years of childbirth, designated postpartum breast cancers (PPBCs). In mouse models, SEMA7A expression is associated with increased LVD and TAM presence, including PoEMs3. Furthermore, we and others have shown that PoEMs intercalate into lymphatic vessels to form PoEM-LEC chimeric vessels, where tumor cells can often be found at the PoEM-LEC junctions2. We have also observed that breast cancer cells, lymphatics, macrophages, and PoEMs exhibit SEMA7A-dependent expression of PD-L1 in vitro, which may make SEMA7A+ tumors more sensitive to anti-PD-1/PD-L1 immunotherapies. Therefore, we utilized multiple orthotopic, immunocompetent mouse models to reveal that growth of SEMA7A overexpressing (OE) tumors, but not controls, was significantly slowed with both anti-PD-1 and anti-PD-L1 treatments. Flow cytometric analysis of cells from the TME revealed increased LECs, TAMs, and PoEMs in SEMA7A+ tumors, compared to controls—all populations had higher PD-L1 expression, which was decreased with both anti-PD-1 or anti-PD-L1. We also observed a decrease in PD-L1 expression on the tumor cells with treatment. Furthermore, LEC, TAM, and PoEM presence was decreased within the TME alongside increased presence of activated CD4 and CD8 T cells. Collectively, our results suggest that SEMA7A expression in breast cancers activates a major pathway associated with immune evasion and helps to establish a protumor microenvironment, which can be targeted with readily available FDA approved drugs such as immune checkpoint-based therapies. Since SEMA7A+ and PPBCs have high rates of metastasis, more specific treatments for these patient populations should be explored. 1.Goddard, E.T., et al. Association Between Postpartum Breast Cancer Diagnosis and Metastasis and the Clinical Features Underlying Risk. JAMA Netw Open 2, e186997 (2019). 2.Bieniasz-Krzywiec, P., et al. Podoplanin-Expressing Macrophages Promote Lymphangiogenesis and Lymphoinvasion in Breast Cancer. Cell Metab (2019). 3.Elder, A.M., et al. Semaphorin 7A Promotes Macrophage-Mediated Lymphatic Remodeling during Postpartum Mammary Gland Involution and in Breast Cancer. Cancer research 78, 6473-6485 (2018). Citation Format: Alan Elder, Alexander Stoller, Traci Lyons. Semaphorin7a expression in breast cancers promotes susceptibility to immune checkpoint blockade [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 P1-04-06.

Tangential Volumetric Modulated Arc Therapy for Locally Advanced Breast Cancer

Cardiovascular toxicity from breast radiation therapy (RT) is a concern to patients and providers. Herein, we present a cardiac-sparing strategy using tangential volumetric modulated arc therapy (tVMAT) in comparison with standard 3-dimensional conformal RT. Ten patients with left-sided breast cancer previously treated with adjuvant RT covering the breast, as well as the axillary and supraclavicular nodal regions, were selected for the study. For each patient, 2 plans were created: a dual-isocenter 3-field 3-dimensional conformal RT plan and a monoisocentric tVMAT plan. The prescription for both techniques was 50 Gy in 25 fractions to the breast and nodal target volumes. Compared with 3-dimensional conformal RT, tVMAT provided more uniform coverage to the breast and regional lymph nodes (mean conformity index: 1.42 for tVMAT vs 2.42 for 3-dimensional conformal RT; P < .01), and the maximum point dose for tVMAT was lower on average (112.8% for tVMAT vs 121.5% for 3-dimensional conformal RT; P < .001). Coverage to the lymph nodes was superior for tVMAT (average minimum coverage to 95% of entire nodal target volumes: 99.5% of prescribed dose for tVMAT vs 94.9% for 3-dimensional conformal RT; P < .001). Organ-at-risk sparing was improved with tVMAT, with a lower average V20Gy for the left lung (15.0% for tVMAT vs 24.6% for 3-dimensional conformal RT; P < .01) and lower mean heart dose (156 cGy for tVMAT vs 200 cGy for 3-dimensional conformal RT; P < .01). Tangential volumetric modulated arc therapy is a promising technique for the treatment of intact breast and regional lymphatics, and it may improve target coverage and organ-at-risk avoidance compared with 3-dimensional conformal techniques.

Modeling Tumor: Lymphatic Interactions in Lymphatic Metastasis of Triple Negative Breast Cancer.

Simple SummaryLymphatic metastasis is a critical prognostic factor of breast cancer aggressiveness and patient survival. Since existing therapeutic approaches have shown limited efficacy, new strategies to identify effective therapeutic targets for reducing breast cancer lymphatic metastasis are needed. We have used novel culture chambers, designed and fabricated by our group, to develop 3D models in which we can study spat ial interactions between breast cancer cells and lymphatic cells as they occur in real-time. This approach provides information on the complex cell–cell interactions involved in lymphatic metastasis of breast cancers. Factors in the secretome of the lymphatic cells promote invasive outgrowths from 3D cultures of breast cancer cells, suggesting that targeting interactions between breast cancer cells and lymphatic cells could be a potential therapeutic approach for the prevention of lymphatic metastasis.Breast cancer frequently metastasizes to lymphatics and the presence of breast cancer cells in regional lymph nodes is an important prognostic factor. Delineating the mechanisms by which breast cancer cells disseminate and spatiotemporal aspects of interactions between breast cancer cells and lymphatics is needed to design new therapies to prevent lymphatic metastases. As triple-negative breast cancer (TNBC) has a high incidence of lymphatic metastasis, we used a three-dimensional (3D) coculture model of human TNBC cells and human microvascular lymphatic endothelial cells (LECs) to analyze TNBC:LEC interactions. Non-invasive analyses such as live-cell imaging in real-time and collection of conditioned media for secretomic analysis were facilitated by our novel microfluidic chambers. The volumes of 3D structures formed in TNBC:LEC cocultures are greater than that of 3D structures formed by either LEC or TNBC monocultures. Over 4 days of culture there is an increase in multicellular invasive outgrowths from TNBC spheroids and an association of TNBC spheroids with LEC networks. The increase in invasive phenotype also occurred when TNBC spheroids were cultured in LEC-conditioned media and in wells linked to ones containing LEC networks. Our results suggest that modeling spatiotemporal interactions between TNBC and LECs may reveal paracrine signaling that could be targeted to reduce lymphatic metastasis.

Functional axillary dissection based on lymphatic drainage for breast cancer: a single center randomized clinical trial

Objective: To evaluate the effectiveness and safety of functional axillary dissection based on lymphatic drainage (FUND) in decreasing breast cancer-related lymphedema (BCRL) events. Methods: A total of 168 eligible patients in Zhongnan Hospital of Wuhan University from July 2018 to February 2019 were randomly assigned to the FUND group or axillary lymph node dissection (ALND) group using random number table generated by SPSS. In the FUND group, methylene blue (MB) was adopted to reveal the sentinel lymph node (SLN) for all patients; 0.1 ml MB was injected into the SLNs before resection to reveal the efferent lymphatic channels and subsequent-echelon lymph node. The blue-stained lymphatic channels were mapped by bluntly dissecting along the lymphatic drainage channels from the breast to the axilla. Then, the SLNs were removed and pathologically analyzed by immediate frozen sectioning (FS); if the SLNs were positive, the blue-stained bALNs in breast lymphatic level (BLL) Ⅱ were removed and sent for immediate FS; if the blue-stained ALNs in BLL Ⅱ were confirmed negative, the tissues in BLL Ⅱ were removed'en bloc'. Clinicopathologic information for all the patients in the two groups were collected. The fixed-point circumference volume measurement method and the Norman questionnaire scoring method were used to evaluate the arm lymphedema between the two groups. Clinicopathological characteristics, incidences of arm lymphedema, locoregional recurrence, and distant metastasis between the two groups were compared. Results: The mean age were (50.3±8.0) in the FUND group and (51.1±9.0) in the ALND group. Seventy-four cases (88.1%) in the FUND group successfully underwent FUND surgery, and patients whose breast lymphatics failed to be stained blue underwent standard ALND. There was no statistically significant difference in terms of age, BMI, histological types, surgical approaches and adjunct therapy between the FUND group (n=74) and ALND group (n=84) (P>0.05). The average operation time of the FUND group and the stand ALND group were (169±15) and (123±12) min respectively (range: 145-198, 103-146 min) (P<0.001), and the number of lymph nodes removed [M (Q1, Q3)] were 8.3 (6, 15) and 12.9 (7, 18) (P=0.019). The cumulative BCRL rate, within a median follow-up of 24 months and 23 months respectively for FUND and ALND group, were 10.8% (8/74) vs 23.8% (20/84) (P=0.033) measured by fixed-point circumference volume measurement method, and was 12.2% (9/74) vs 27.4% (23/84) by Norman questionnaire (P=0.018). There were no local regional recurrence events during the follow-up period between the two groups. Conclusion: For breast cancer patients with clinically node-positive axilla or positive SLN, FUND based on lymphatic drainage was a less radical axillary surgery, with which eliminating the risk of BCRL might be achieved.

Axillary Reverse Mapping in Patients Undergoing Axillary Lymph Node Dissection: A Single Institution Experience From India

IntroductionAxillary lymph node dissection (ALND) remains the gold standard for clinically node-positive and sentinel node biopsy (SLNB) positive breast cancer patients, but it is associated with the debilitating morbidity of lymphedema. Recently, a new technique of axillary reverse mapping (ARM) has been described which helps in differentiating arm lymphatics from breast lymphatics.AimTo evaluate the applicability of the ARM technique with blue dye and the incidence of metastases in ARM nodes in the Indian population.MethodA total of 120 patients underwent ARM during ALND. Blue lymphatic channels and lymph nodes were noted. All axillary nodes along with ARM nodes were dissected and sent separately for pathological evaluation for metastases.ResultsARM nodes or lymphatics were identified in 65 (54.17%) out of 120 patients. The mean ARM lymph node yield was 1.4. The patients in whom ARM lymph nodes or lymphatics were not identified had significantly higher T stage and N stage (p <0.00001) than in whom it was identified. There was no significant correlation between ARM identification with BMI, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/neu), and neoadjuvant chemotherapy (NACT) status. ARM nodes were found metastatic in three patients (7.5%). All these patients had clinically N2 disease and all had pathologically more than ten nodes involved in the axilla.ConclusionThe identification rate of ARM nodes and lymphatics with blue dye is lower in Indian patients who present with higher clinical T and N stage disease. Other clinicopathological parameters were not associated with the identification rate. The rate of metastasis in ARM nodes is high in patients with a high axillary tumor burden. Hence, preserving ARM nodes may not be oncologically safe in higher N stage disease.

Minimize the extent and morbidity of axillary dissection for node-positive breast cancer patients: implementation of axillary lymph node dissection based on breast lymphatics level

BackgroundBreast cancer-related lymphedema (BCRL) is associated with extensive axillary dissection. Axillary lymph node dissection (ALND) based on breast lymphatics level (BLL) was proposed to minimize the surgical extent for node-positive breast cancer patients.MethodsA total of 156 consecutive sentinel lymph node-positive (SLN+) or clinically node-positive (cN+) patients underwent sentinel lymph node biopsy (SLNB) with indocyanine green and methylene blue (MB). The SLNs were injected with 0.1 ml MB before removal, and a standard ALND was subsequently performed. The nodes adjacent to the blue-stained axillary lymph nodes from the breast (bALNs) were sent for pathological examination separately by resecting serial tissue every 0.5 cm away from the marginal blue-stained bALNs. Then, a pilot study comparing ALND based on BLL and standard ALND was performed.ResultsBLL were successfully identified in 20 SLN+ (100%) and 134 cN+ (98.5%) patients. The median number of BLL was four, ranging from three to six. A horizontal line 1.0 cm away from the superior blue-stained bALN and a vertical line 1.0 cm away from the medial blue-stained bALN formed BLL II, III, and IV. All of the additional positive nodes were within 1.0 cm of the blue-stained bALNs. The minimized axillary dissection should resect upwards from the lowest BLL that contains the first confirmed negative blue-stained bALNs. In the pilot study, no patient developed axillary recurrence.ConclusionThe ALND surgical procedure based on BLL could minimize the surgical extent for pathological node-positive breast cancer patients and potentially reduce the BCRL rate.Trial registrationChiCTR1800014247.

Abstract PS14-21: Refining loco-regional therapy for inflammatory breast cancer protocol in progress

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive locally advanced breast cancer subtype. It is associated with loco-regional recurrence rates of 12-25%, and neoadjuvant chemotherapy (NAC) followed by modified radical mastectomy and comprehensive chest wall and regional nodal radiotherapy remain the standard of care. As has been demonstrated in non-IBC, achievement of pathologic complete response (pCR) has been shown to be associated with improved loco-regional control, recurrence-free and overall survival. Advances in NAC for IBC have resulted in improved pCR rates in both the breast and the axilla, with overall axillary pCR rates of approximately 30%, reaching as high as 67% in patients with HER2-positive disease receiving HER2-directed therapy. Hypothesis: Sentinel lymph node biopsy (SLNB) may be feasible in IBC patients who experience a good clinical and pathologic response in the axilla to NAC. Primary Objective: To evaluate the sentinel lymph node (SLN) identification rate in stage III IBC patients who experience cN0 status at completion of NAC. Secondary Objective: To assess the incidence of lymphedema following standard local-regional therapy for IBC. Methods and Study procedures: In this feasibility study, 50 patients with cT4dN0-2M0 IBC will be enrolled in order to evaluate 40 patients whose axillary nodal status becomes cN0 upon completion of NAC. All patients will undergo a research breast biopsy and lymphoscintigram pre and post NAC to evaluate lymphatic drainage patterns and patency of breast and axillary lymphatics. Post NAC lymphoscintigraphy will be appropriately timed for pre-operative SLN mapping and all patients will undergo SLNB using dual tracers (Tc99 Sulfur colloid and blue dye) with immediate axillary lymph node dissection (ALND), at the time of mastectomy. The patient-reported Lymphedema Symptom Intensity and Distress Survey (LSIDS-A) will be collected at 6 timepoints. Patients will be followed for 2 years post-surgery for oncologic outcomes. Correlatives: We plan to evaluate genetic and phenotypic heterogeneity in IBC and to assess markers of angiogenesis and lymph-angiogenesis associated with IBC, as well as to explore immunologic aspects of the tumor microenvironment and their association with pCR. Statistics: The identification rate will be calculated as number of patients in whom SLNs were successfully identified over the number of patients with cN0 disease after NAC in whom SLN mapping was attempted. Using a Simon two-stage design (α=.10, β=.10), a SLN identification rate of ≥90% would result in considering this procedure feasible whereas an identification rate of ≤75% (null hypothesis) would lead to the conclusion that it is not feasible. In the first stage, if greater than 18 of 25 patients have SLNs identified, then a total of 40 patients will be enrolled. If fewer than 33 of 40 patients have SLN identified, then the null hypothesis is rejected. Citation Format: Faina Nakhlis, Meredith Regan, Heather Jacene, Beth Harrison, Jennifer Bellon, Jean Landry, Eren Yeh, Elizabeth Mittendorf, Beth Overmoyer, Tari King. Refining loco-regional therapy for inflammatory breast cancer protocol in progress [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 PS14-21.

Assessing breast lymphoedema following breast cancer treatment using indocyanine green lymphography.

Breast lymphoedema is a largely unrecognised survivorship issue for women following breast cancer treatment. While a few objective methods have previously been applied to assess breast lymphoedema, none are capable of imaging breast lymphatics or identifying lymphatic morphological changes indicative of breast lymphoedema. The purpose of this study was to determine if indocyanine green (ICG) lymphography, a validated assessment technique in breast cancer-related lymphoedema), can visualise breast lymphatics and identify breast lymphoedema. Additionally, ICG lymphography was utilised to investigate lymphatic drainage pathways of the affected breast following breast-conserving therapy. Twenty female participants (10 breast lymphoedema and 10 healthy controls) were recruited for this pilot study. All underwent a medical history, physical breast assessment, tissue dielectric constant measures of breast water content, and ICG lymphography. ICG lymphography identified lymphatic morphological changes in all breast lymphoedema participants (dermal backflow patterns = 10, collateral lymphatic drainage = 9) and none in the control group. The dominant lymphatic drainage pathway to the ipsilateral axilla was observed in all control participants but in only four breast lymphoedema participants. Collateral drainage pathways in the breast lymphoedema group were to: parasternal (6/10); contralateral axilla (4/10); intercostal (3/10); and clavicular (2/10) regions. These findings suggest ICG lymphography, through the identification of morphological lymphatic changes, is a potential qualitative objective assessment technique for breast lymphoedema. Furthermore, in this group of breast lymphoedema patients it identified changes to the normal drainage pathway of the breast. Understanding these changes will have implications for clinical management.

Abstract P1-03-03: Multichannel serial imaging of transgenic, preclinical murine models provides the first quantitative analysis of the unusual growth kinetics and lymph-vascular invasion of patient-derived inflammatory breast cancer cells and tumor emboli

Abstract Background: Lymphovascular invasion (LVI), a critical feature of advanced cancers, is a major route of metastatic dissemination. Of the clinically-distinct types of breast cancer, the most lethal variant is inflammatory breast cancer (IBC) where LVI is a histopathological hallmark. Majority of IBC patients lack a distinct, solid tumor and instead present with diffuse tumor cell clusters cells, also termed as tumor emboli, in the breast and dermal lymphatics, which contributes to the clinical breast-skin symptoms and postulated to provide an efficient path to metastatic spread. The steps in lymphatic dissemination are challenging to study in vitro, and in vivo models are limited. Our goal was to develop new in vivo models that would enable direct visualization and quantitation of local tumor cell growth and tumor-lymphatic vessel interactions for improved understanding of the unique IBC tumor biology and for drug discovery. Methods: mCherry/GFP-expressing non-IBC (4T1, E0771) and IBC cells derived from patient untreated primary tumors (SUM149/basal and SUM190/Her2+) were implanted into the fascia in the center of a window chamber in the skin-fold or mammary fat pad. In order to allow direct study of tumor cell–lymphatic vessel interaction, we generated transgenic nude mice with fluorescent lymphatics [tdTomato fluorophore under control of a Prox1 promoter, encodes a transcription factor (prospero-related homeobox 1) necessary for the formation and maintenance of lymphatic vessels]. Multichannel microscopy was employed to serially quantify tumor growth, tumor spread and lymph-tumor interactions. Using this model, we also optimized implantation and live imaging of pre-formed IBC-cell derived tumor emboli generated by simulating the lymphatic sheer stress in culture. Results: Compared to non-IBC cells, which formed solid tumors that increased in size over a 14 day period, IBC models exhibited a diffuse and disseminating phenotype, within 24h, rather than a centralized tumor mass, similar to the disease presentation in patients. Using a threshold analysis in ImageJ, we quantified tumor area (as a metric of tumor growth), the tumor motility, and the linear density of lymph and blood vessels. Briefly, we binarized each fluorescent channel image such that the tumor or lymph signal was positive; then, an algorithm drew regions of interest (ROIs) around each defined tumor-cell cluster. The ROIs were used to calculate the parameters describing tumor growth and motility (distance of the tumor from the center of mass (CM)), and the area moment (the movement of the clusters relative to the CM) at each timepoint. The linear vascular density was calculated from the distance of segments drawn through the center of large vessels for each mouse, which provided quantitative data describing tumor cell or IBC emboli and lymphovascular interactions, suggesting that there is a specific microenvironment necessary for the unique phenotype and dissemination pattern exhibited by IBC tumor cells. Conclusions: Despite the poor prognosis of IBC, the clinical implications of how and why tumor emboli form and how they survive to migrate into the lymphatics is not defined. These in vivo models provide in-depth imaging and quantitative measurements of locoregional invasion, tumor cell-lymphatic or endothelial vessel interactions of implanted tumor cells or tumor emboli. This model has the potential to be extended to study other cancer cell types exhibiting LVI as well as a screening metric for IBC therapies. Supported by DOD-Breakthrough-W81XWH-17-1-029, IBC Research Foundation, DCI pilot funds, IBC Network Foundation, Duke Surgery Gardner Award. Citation Format: Ashlyn Rickard, Pranalee Patel, Scott J Sauer, Mark W Dewhirst, Gregory M Palmer, Gayathri R Devi. Multichannel serial imaging of transgenic, preclinical murine models provides the first quantitative analysis of the unusual growth kinetics and lymph-vascular invasion of patient-derived inflammatory breast cancer cells and tumor emboli [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 P1-03-03.

Using Robust Optimization for Skin Flashing in Intensity Modulated Radiation Therapy for Breast Cancer Treatment: A Feasibility Study

PurposeTo study the feasibility and the effectiveness of a novel implementation of robust optimization on 2 sets of computed tomography (CT) data simultaneously for skin flashing in intensity modulated radiation therapy for breast cancer. Method and MaterialsFive patients who received treatment to the breast and regional lymphatics were selected for this study. For each patient, 3 plans were generated using 3 different skin-flashing methods, including (1) a manual flash plan with optimization on the nominal planning target volume (PTV) not extending beyond the skin that required manually postplanning the opening of the multi-leaf collimator and jaw to obtain flash; (2) an expanded PTV plan with optimization on an expanded PTV that included the target in the air beyond the skin; and (3) a robust-optimized (RO) plan using robust optimization that simultaneously optimizes on the nominal CT data set and a simulated geometry error CT data set. The feasibility and the effectiveness of the robust optimization approach was investigated by comparing it with the 2 other methods. The robustness of the plan against target position variations was studied by simulating 0-, 5-, 10-, and 15-mm geometry errors. ResultsThe RO plans were the only ones able to meet acceptable criteria for all patients in both the nominal and simulated geometry error scenarios. The expanded PTV plans developed major deviation on the maximum dose to the PTV for 1 patient. For the manual flash plans, every patient developed major deviation either on 95% of the dose to the PTV or the maximum dose to the PTV in the simulated geometry error scenarios. The RO plan demonstrated the best robustness against the target position variation among the 3 methods of skin flashing. The doses to the lung and heart were comparable for all 3 planning techniques. ConclusionUsing robust optimization for skin flash in breast intensity modulated radiation therapy planning is feasible. Further investigation is warranted to confirm the clinical effectiveness of this novel approach.

Prone Positioning in the Treatment of the Whole Breast and Regional Lymphatics Using a Mono-Isocentric Four Field Radiation Technique: A Dosimetric Comparison

Prone Positioning in the Treatment of the Whole Breast and Regional Lymphatics Using a Mono-Isocentric Four Field Radiation Technique: A Dosimetric Comparison

Inflammatory breast cancer tumor emboli express high levels of anti-apoptotic proteins: use of a quantitative high content and high-throughput 3D IBC spheroid assay to identify targeting strategies.

Inflammatory breast cancer (IBC) is one of the most lethal breast cancer variants; with existing therapy, 5-yr survival rate is only 35%. Current barriers to successful treatment of IBC include frequent infiltration and the presence of tumor cell clusters, termed tumor emboli, within the breast parenchyma and lymphatics. Prior studies have identified the role of anti-apoptotic signaling, in particular hyperactivation of NFκB and its target genes, in IBC pathobiology and therapeutic resistance. The objectives of this study were to: (1) determine if IBC tumor emboli express anti-apoptotic proteins and (2) develop a high content, multiparametric assay to assess the morphology of the IBC 3D spheroids and to optimize a high throughput format to screen for compounds that can inhibit the formation of the IBC tumor clusters/embolic structures. Immunohistochemical analysis of IBC patient tumor samples with documented tumor emboli revealed high NFκB (p65) staining along with expression of XIAP, a potent anti-apoptotic protein known to interact with NFκB signaling in enhancing survival of malignant cells. Subsequently, the high content assay developed allowed for simultaneous imaging and morphometric analysis, including count and viability of spheroids derived from SUM149, rSUM149 and SUM190 cells and its application to evaluate XIAP and NFκB inhibitory agents. We demonstrate the efficacy of the off-patent drug disulfiram when chelated with copper, which we had previously reported to inhibit NFκB signaling, was highly effective in disrupting both IBC spheroids and emboli grown in vitro. Taken together, these results identify a high-throughput approach to target tumor spheroid formation for drug discovery. Finally, disulfiram is a safe and approved drug for management of alcohol abuse, warranting its evaluation for repurposing in IBC therapy.

Multimodality Imaging-based Evaluation of Single-Lumen Silicone Breast Implants for Rupture.

Breast implants are frequently encountered on breast imaging studies, and it is essential for any radiologist interpreting these studies to be able to correctly assess implant integrity. Ruptures of silicone gel-filled implants often occur without becoming clinically obvious and are incidentally detected at imaging. Early diagnosis of implant rupture is important because surgical removal of extracapsular silicone in the breast parenchyma and lymphatics is difficult. Conversely, misdiagnosis of rupture may prompt a patient to undergo unnecessary additional surgery to remove the implant. Mammography is the most common breast imaging examination performed and can readily depict extracapsular free silicone, although it is insensitive for detection of intracapsular implant rupture. Ultrasonography (US) can be used to assess the internal structure of the implant and may provide an economical method for initial implant assessment. Common US signs of intracapsular rupture include the "keyhole" or "noose" sign, subcapsular line sign, and "stepladder" sign; extracapsular silicone has a distinctive "snowstorm" or echogenic noise appearance. Magnetic resonance (MR) imaging is the most accurate and reliable means for assessment of implant rupture and is highly sensitive for detection of both intracapsular and extracapsular rupture. MR imaging findings of intracapsular rupture include the keyhole or noose sign, subcapsular line sign, and "linguine" sign, and silicone-selective MR imaging sequences are highly sensitive to small amounts of extracapsular silicone. ©RSNA, 2017.

Systematic review of axillary reverse mapping in breast cancer.

Axillary reverse mapping (ARM) assesses the lymphatic drainage of the arm simultaneously with that of the breast, enabling preservation of arm lymphatics during axillary surgery for breast cancer. This article systematically reviews the evidence on the lymphoedema rate and oncological safety of the ARM technique. PubMed, Embase and the Cochrane Library were searched systematically for studies that addressed the use of ARM during axillary surgery in breast cancer. Studies were eligible if they performed ARM during sentinel node biopsy (SNB) or axillary node clearance (ANC) for breast cancer in prospective studies of more than 50 patients, with assessment of lymphoedema and oncological outcomes during a minimum follow-up of 6 months. Eight studies reported data on ARM in 1142 patients undergoing axillary surgery for breast cancer. Lymphoedema rates ranged from 0 to 6 per cent during ARM-assisted SNB, and from 5.9 to 24 per cent during ARM lymphatic preservation at ANC. Crossover nodes between the arm and breast lymphatics were identified in 0-10 per cent of patients, and metastases were present in 0-20 per cent of these patients. ARM nodes were not preserved in between 11 and 18 per cent of patients with ARM nodes identified, and metastases were detected in 0-19 per cent of these patients. ARM can achieve low rates of lymphoedema, but the risk of metastasis in crossover and clinically suspicious ARM nodes, or those in close proximity to an involved sentinel node, warrants their excision.

Abstract P2-01-06: In patients with breast cancer, pre-operative sentinel node biopsy using intradermal microbubbles and contrast enhanced ultrasound predicts volume of axillary metastases

Abstract Introduction In patients with early breast cancer, there is a trend towards conservative axillary surgery but there are concerns that patients with high volume axillary metastases may receive sub-optimal axillary treatment leaving them vulnerable to local recurrence. Determining the metastatic status of sentinel lymph nodes (SLN) in the pre-operative/ diagnostic period would allow focussed surgical planning and facilitate decisions regarding adjuvant therapy early in the patient pathway. Methods 654 patients with primary invasive breast cancer and a normal grey-scale axillary ultrasound were included in the analysis. Pre-operatively, patients received periareolar intra-dermal injection of microbubble contrast agent, breast lymphatics were visualised by ultrasound and followed to identify axillary SLN. Contrast-pulse sequencing and grey-scale ultrasound modalities were used to image SLN. Sentinel lymph nodes were then subjected to core biopsy. Patients subsequently underwent tumour excision and axillary node clearance (ANC) or surgical sentinel node biopsy (SNB) using blue-dye and isotope +/- completion ANC. Results Sentinel lymph nodes were clearly visualised in 605 patients and successfully (B2-B5) core biopsied in 555. The test identified 53% of all SLN metastases with 100% specificity. The negative predictive value was 88%. The prevalence of axillary lymph node metastases was 23%. Given a benign (B2) SLN biopsy result, the post-test probability that a patient had SLN metastases on subsequent surgical excision was 12%. Following ANC, 55% of patients with a malignant (B4/5) biopsy result were found to have high volume axillary disease (2 macro metastases or more) whereas 21% of patients with an initial benign (B2) biopsy result and metastatic cells found at SNB had high volume axillary disease identified after completion ANC (P value less than 0.001). In total, only 2% of patients with a benign (B2) SLN core biopsy result had high volume axillary metastases and half of these had either multifocal cancer or invasive lobular carcinoma. Conclusions SLN can be readily identified and biopsied in the breast clinic using intradermal microbubbles and CEUS. In patients with primary invasive breast cancer and a normal grey scale axillary ultrasound, a benign (B2) SLN core biopsy result may be highly predictive of either no metastases or low volume metastatic disease in the ipsilateral axilla. This group of patients is therefore likely to benefit from axillary conservation and in certain cases, it may be appropriate to completely omit a surgical SNB. Citation Format: Karina Cox, Jennifer Weeks, Ritchie Chalmers, Pippa Mills, David Fish, Ali Sever. In patients with breast cancer, pre-operative sentinel node biopsy using intradermal microbubbles and contrast enhanced ultrasound predicts volume of axillary metastases [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 P2-01-06.

Axillary reverse mapping: Is it feasible in locally advanced breast cancer patients?

Axillary dissection is associated with a high incidence of lymphedema, which has been brought down with the introduction of sentinel lymph node biopsy (SLNB) in patients with early breast cancer. However, sentinel lymph node biopsy is not widely accepted in patients of locally advanced breast cancer (LABC) [T3N1, Any T4, Any N2-3 with no distant metastasis] after neo-adjuvant chemotherapy (NACT) and these patients routinely undergo axillary lymph node clearance. Axillary reverse mapping (ARM) with blue dye has the potential to differentiate the arm lymphatics from the breast lymphatics and it can be used to decrease lymphedema in patients undergoing ALND by preserving these lymphatics. However, ARM in LABC patients is yet to be accepted as the standard of care. 51 patients of locally advanced breast carcinoma were included in the study from May 2011 to May 2012. All patients received neo-adjuvant chemotherapy followed by modified radical mastectomy. Axillary reverse mapping (ARM) was carried out using blue dye. 2 ml of methylene blue dye was injected intradermal, upper medial aspect of the ipsilateral arm. The number, size and site and distribution of lymph nodes identified were recorded and the nodes were labelled as ARM nodes and complete axillary dissection was carried out. Blue nodes were identified in 45 (88.2%) out of the 51 patients. The average number of ARM nodes identified was 4.03 ± 0.28 [range 1-8]. In majority (77.8%) of the cases, nodes were located in the triangle formed by axillary vein above, below by the first intercostobrachial nerve and medially by the chest wall/serratus anterior. In patients with complete or partial response to NACT, ARM and breast axillary LN were negative in 63.3% patients whereas 36.6% had positive breast but negative ARM nodes. In this study we did not intend to preserve any ARM nodes but in 90% of these cases, at least one ARM node had to be removed or was injured during axillary clearance. ARM nodes could be identified in 15 (83.3%) out of the 18 patients with stable or progressive disease following ARM. 12 (80%) out of these 15 cases demonstrated positive ARM and breast LN whereas 3 (20%) patients had positive breast but negative ARM nodes. Skin tattooing (82.3%) was the most common complication observed in our study. Identification rates of ARM nodes can be improved by injecting the blue dye in the upper medial aspect of the arm at the time of induction. Majority of the arm nodes lie between the axillary vein and the first intercostobrachial nerve. It is difficult to preserve the ARM nodes in patients of LABC, who have had good response to NACT and in patients of LABC with poor response to NACT, the incidence of metastasis in ARM nodes is quite high. Therefore, ARM is not a feasible option in patients with locally advanced breast cancer.