Supplemental Figure from Trastuzumab Increases HER2 Uptake and Cross-Presentation by Dendritic Cells
Victor GallAnne V. PhilipsNa QiaoKaren Clise-DwyerAlexander A. PerakisMao ZhangGuy T. CliftonPariya SukhumalchandraQing MaSangeetha M. ReddyDihua YuJeffrey J. MolldremGeorge E. PeoplesGheath AlatrashElizabeth A. Mittendorf
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<p>SKBR3 breast cancer cells shed HER2 after treatment with chemotherapy or irradiation.</p>Keywords:
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Supplemental Figure from Trastuzumab Increases HER2 Uptake and Cross-Presentation by Dendritic Cells
<p>SKBR3 breast cancer cells shed HER2 after treatment with chemotherapy or irradiation.</p>
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<div>Abstract<p>Early-phase clinical trials evaluating CD8<sup>+</sup> T cell–eliciting, HER2-derived peptide vaccines administered to HER2<sup>+</sup> breast cancer patients in the adjuvant setting suggest synergy between the vaccines and trastuzumab, the mAb targeting the HER2 protein. Among 60 patients enrolled in clinical trials evaluating the E75 + GM-CSF and GP2 + GM-CSF vaccines, there have been no recurrences in patients vaccinated after receiving trastuzumab as part of standard therapy in the per treatment analyses conducted after a median follow-up of greater than 34 months. Here, we describe a mechanism by which this synergy may occur. Flow cytometry showed that trastuzumab facilitated uptake of HER2 by dendritic cells (DC), which was mediated by the Fc receptor and was specific to trastuzumab. <i>In vitro</i>, increased HER2 uptake by DC increased cross-presentation of E75, the immunodominant epitope derived from the HER2 protein, an observation confirmed in two <i>in vivo</i> mouse models. This increased E75 cross-presentation, mediated by trastuzumab treatment, enabled more efficient expansion of E75-specific cytotoxic T cells (E75-CTL). These results demonstrate a mechanism by which trastuzumab links innate and adaptive immunity by facilitating activation of antigen-specific T cells. On the basis of these data, we conclude that HER2-positive breast cancer patients that have been treated with trastuzumab may experience a more robust antitumor immune response by restimulation of T cells with the E75 peptide vaccine, thereby accounting for the improved disease-free survival observed with combination therapy. <i>Cancer Res; 77(19); 5374–83. ©2017 AACR</i>.</p></div>
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Abstract Early-phase clinical trials evaluating CD8+ T cell–eliciting, HER2-derived peptide vaccines administered to HER2+ breast cancer patients in the adjuvant setting suggest synergy between the vaccines and trastuzumab, the mAb targeting the HER2 protein. Among 60 patients enrolled in clinical trials evaluating the E75 + GM-CSF and GP2 + GM-CSF vaccines, there have been no recurrences in patients vaccinated after receiving trastuzumab as part of standard therapy in the per treatment analyses conducted after a median follow-up of greater than 34 months. Here, we describe a mechanism by which this synergy may occur. Flow cytometry showed that trastuzumab facilitated uptake of HER2 by dendritic cells (DC), which was mediated by the Fc receptor and was specific to trastuzumab. In vitro, increased HER2 uptake by DC increased cross-presentation of E75, the immunodominant epitope derived from the HER2 protein, an observation confirmed in two in vivo mouse models. This increased E75 cross-presentation, mediated by trastuzumab treatment, enabled more efficient expansion of E75-specific cytotoxic T cells (E75-CTL). These results demonstrate a mechanism by which trastuzumab links innate and adaptive immunity by facilitating activation of antigen-specific T cells. On the basis of these data, we conclude that HER2-positive breast cancer patients that have been treated with trastuzumab may experience a more robust antitumor immune response by restimulation of T cells with the E75 peptide vaccine, thereby accounting for the improved disease-free survival observed with combination therapy. Cancer Res; 77(19); 5374–83. ©2017 AACR.
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Abstract Introduction: The purpose of this study was to identify the effect of trastuzumab on the uptake and presentation of HER2-derived peptides by dendritic cells (DCs). Clinical trials for HER2-derived peptide vaccines have shown improved outcomes for patients receiving a combination of vaccine and trastuzumab suggesting potential synergy between the two. We hypothesized that antigen processing and cross-presentation would be enhanced via trastuzumab facilitating antigen presenting cell (APC) uptake of HER2 shed from breast cancer cells. Methods: An ELISA assay was used to evaluate HER2 shedding in multiple cancer cell lines (SKBR3, SKOV3, BT474, MDA-MB-231). To look for uptake of HER2 shed from breast cancer cells into culture media by APCs, DCs were generated by incubating healthy donor monocytes in media supplemented with GM-CSF, IL-4, and TNF-α. These DCs were co-incubated with SKBR3, BT474, and SKOV3 (high HER2 expressers) as well as MDA-MB-231 (low HER2 expresser) in the presence of trastuzumab. Rituximab was used as an isotype control to determine trastuzumab specificity and an Fc blocker was used to evaluate the importance of the Fc receptor in the uptake mechanism. Cells were then stained for DC markers, including CD11c and HLA-DR, permeabilized and stained for HER2 to detect uptake, and then analyzed using flow cytometry. To evaluate antigen cross-presentation, antigen-specific T cells were expanded by co-culturing healthy donor PBMCs with DCs that have been pulsed with SKBR3 cells or trastuzumab-treated SKBR3 cells. DCs pulsed with E75 peptide (HER2, aa: 369-377) were used as a control. After T cell activation and expansion, the number of E75-specfic CD8+ T cells was enumerated using an E75-dextramer assay. Results: When compared to SKOV3 and MBA-MB-231 cells, SKBR3 and BT474 cells shed a significantly higher concentration of HER2 into the growth media. DCs took up HER2 when co-cultured with cell lines with higher levels of HER2 shedding (SKBR3, BT474). Trastuzumab treatment of SKBR3 resulted in a 50% increase in HER2 uptake by DCs at 24 hours (p = 0.0014). This finding persisted among all trastuzmab doses. Treatment with rituximab showed no significant increase in HER2 uptake when compared to untreated SKBR3. The effect of trastuzumab on uptake was abrogated by adding an Fc blocking agent. DCs treated with SKBR3 and trastuzumab also led to an increase in the generation of E75-CTLs as measured using a dextramer assay. Conclusions: Trastuzumab treatment leads to increased uptake of soluble HER2 by DCs in vitro. This effect is specific to trastuzumab and is Fc receptor mediated. Increased HER2 uptake led to increased E75-CTL generation secondary to increased cross-presentation of E75 by DCs. These data have global implications for HER2-targeting vaccine approaches. Specifically, by enhancing antigen presentation, trastuzumab can augment the immune response initiated by a peptide vaccine. Citation Format: Victor A. Gall, Anne V. Philips, Na Qiao, Karen C. Dwyer, Alexander A. Perakis, Mao Zhang, Pariya Sukhumalchandra, Jeffrey J. Molldrem, Gheath Alatrash, Elizabeth A. Mittendorf. Trastuzumab increases uptake and cross-presentation of HER2-derived antigens by dendritic cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 534.
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A 56-year-old man presented to the orthopedics department with chronic shoulder pain for 4 years not relieved by medication or physiotherapy. On physical examination, there was no restriction of movement, and there was focal tenderness over the lateral aspect of the clavicle. Magnetic resonance (MR) imaging, showed a well-defined T1 hypointense lesion [Figure 1a, arrow], which was hyperintense on short T1 inversion recovery images [Figure 1b, arrow]. The lesion was smaller than 1 cm, showed homogeneous enhancement in postcontrast T1-weighted images [Figure 1c, arrow] and was superior to the distal end of the clavicle in the subcutaneous plane. There was no obvious communication of the lesion with either the acromioclavicular or glenohumeral joint. There was no erosion, remodeling, or sclerosis of the superior clavicular surface. The fat planes with all 14 surrounding structures were maintained. Correlative ultrasound revealed a well-defined, homogenously hypoechoic lesion [Figure 1d, asterisk] with minimal intralesional vascularity [Figure 1d, arrow]. Surgical excision of the lesion resulted in complete disappearance of symptoms and follow-up imaging showed no recurrence.Figure 1: (a) T1-weighted Magnetic resonance image showing a well-defined hypointense lesion (arrow) superior to the distal end of the clavicle. (b) Well-defined lesion (arrow) showing short T1 inversion recovery hyperintensity. (c) Lesion (arrow) showing homogenous enhancement on postcontrast T1-weighted image. (d) Ultrasound grey scale and color Doppler images showing a well-defined, homogenously hypoechoic lesion (asterisk) with minimal intralesional vascularity (arrow)Glomus tumors are true benign neoplasms originating from the glomus bodies and have been reported to account for 1%–6% of all soft-tissue tumors [1]. Glomus tumor was first described by Wood in 1912, but the correct origin was clarified as hyperplasia of a normal glomus body by Masson in 1924 [2]. Classic glomus tumors are typically solitary. They are most commonly smaller than 1 cm and located in the upper extremities, especially the hands and subungual regions [3]. Glomus tumor is a hamartoma that develops from a neuromyoarterial glomus body and consists of dilated vascular channels surrounded by proliferating glomus and nerve cells. There is no sex predilection and the most common age at presentation is 20–40 years [4]. It is rare in certain locations such as the extremities, trunk, and viscera. The forearm is the most common extradigital location, with the shoulder and back the least common. Clinical manifestations include paroxysms of pain, cold sensitivity, and point tenderness with signs of hyperesthesia, muscle atrophy, or osteoporosis in the affected area. We presented imaging findings in a rare case of glomus tumor with chronic shoulder pain. Malignant transformation is rare. Hemangiomas are a close imaging differential for glomus tumors [5]. Ultrasound findings include a cystic/solid hypoechoic mass and rarely, erosion of the underlying bone. Intratumoral vascular shunts are noted in color Doppler imaging. MR imaging shows an intermediate or low-signal intensity on T1-weighted images, marked hyperintensity on T2-weighted images, and strong postcontrast imaging. MR angiography shows a focus of strong enhancement in the arterial phase and tumor blush, which progressively increases in size in the delayed phase. The treatment of choice for glomus tumors is complete excision, and even in cases of malignant transformation, no chemotherapy or radiation is needed [6]. Glomus tumors are neoplasms that arise from modified smooth muscle cells of the glomus body, which is a specialized form of an arteriovenous anastomosis that plays a significant role in the regulation of skin circulation. The present study reports a case of a glomus tumor in a man with chronic shoulder pain for 4 years. A correct clinical diagnosis was obtained from imaging modalities (ultrasound and MR imaging). Declaration of patient consent The authors certify that the patient consent form has been obtained. In the form, the patient has given his consent for his images and other clinical information to be reported in the journal. The patient understands that his name and initial will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.
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