Rohini L. Kadle

A 35-Year Evolution of Free Flap-Based Breast Reconstruction at a Large Urban Academic Center.

BACKGROUND This study aims to characterize the evolution and trends in free flap breast reconstruction at our institution. METHODS The authors reviewed and analyzed a registry of free flap breast reconstructions performed at a large urban academic center. RESULTS Between 1979 and mid-2014, a total of 920 patients underwent breast reconstruction with 1,254 flaps. The mean age was 47.7 years (range, 16-79 years). Over the past 10 years, patients were older than all patients seen in the prior decade (average age 48.9 vs. 46.1 years, p = 0.002). Overall, 82% of flaps were performed at our university hospital, 17% at a major urban county hospital, and < 1% at other sites. A total of 99% patients received postmastectomy reconstruction for an existing cancer diagnosis or prophylaxis. There has been a significant increase in reconstructions, with 579 flaps performed over the past 5 years alone. There has been a fundamental shift in the predominant flap of choice over time. Perforator flaps have increased in popularity at our institution, with 74% of all reconstructions over this past 5 years being perforator based. Perforator flaps were more likely to be chosen over nonperforator flaps in older versus younger patients (p = 0.0008). There has been a steady increase in bilateral reconstructions since the first one was performed in 1987 (p = 0.002). CONCLUSIONS Over the past 35 years, our institution has seen a significant evolution in free flap-based breast reconstruction. Besides a massive increase in flap numbers we have seen a significant trend toward bilateral reconstructions and perforator-based flaps.

Microenvironmental cues enhance mesenchymal stem cell-mediated immunomodulation and regulatory T-cell expansion

Mesenchymal stem cells (MSCs) are known to both have powerful immunosuppressive properties and promote allograft tolerance. Determining the environmental oxygen tension and inflammatory conditions under which MSCs are optimally primed for this immunosuppressive function is essential to their utilization in promoting graft tolerance. Of particular interest is the mechanisms governing the interaction between MSCs and regulatory T cells (Tregs), which is relatively unknown. We performed our experiments utilizing rat bone marrow derived MSCs. We observed that priming MSCs in hypoxia promotes maintenance of stem-like characteristics, with greater expression of typical MSC cell-surface markers, increased proliferation, and maintenance of differentiation potential. Addition of autologous MSCs to CD4+/allogeneic endothelial cell (EC) co-culture increases regulatory T cell (Treg) proliferation, which is further enhanced when MSCs are primed in hypoxia. Furthermore, MSC-mediated Treg expansion does not require direct cell-cell contact. The expression of indolamine 2,3-dioxygenase, a mediator of MSC immunomodulation, increases when MSCs are primed in hypoxia, and inhibition of IDO significantly decreases the expansion of Tregs. Priming with inflammatory cytokines IFNγ and TNFα increases also expression of markers associated with MSC immunomodulatory function, but decreases MSC proliferation. The expression of IDO also increases when MSCs are primed with inflammatory cytokines. However, there is no increase in Treg expansion when MSCs are primed with IFNγ, suggesting an alternate mechanism for inflammatory-stimulated MSC immunomodulation. Overall, these results suggest that MSCs primed in hypoxia or inflammatory conditions are optimally primed for immunosuppressive function. These results provide a clearer picture of how to enhance MSC immunomodulation for clinical use.

Flap Reconstruction of Sarcoma Defects in the Setting of Neoadjuvant and Adjuvant Radiation

Background Limb‐sparing treatment of extremity soft tissue sarcomas requires wide resections and radiation therapy. The resulting complex composite defects necessitate reconstructions using either muscle or fasciocutaneous flaps, often in irradiated wound beds. Methods A retrospective chart review was performed of all limb‐sparing soft tissue sarcoma resections requiring immediate flap reconstruction from 2012 through 2016. Results Forty‐four patients with 51 flaps were identified: 25 fasciocutaneous and 26 muscle‐based flaps. Mean defect size, radiation treatment, and follow‐up length were similar between groups. More often, muscle‐based flaps were performed in younger patients and in the lower extremity. Seventeen flaps were exposed to neoadjuvant radiation, 12 to adjuvant radiation, 5 to both, and 17 to no radiation therapy. Regardless of radiation treatment, complication rates were comparable, with 28% in fasciocutaneous and 31% in muscle‐based groups (p < 0.775). Muscle‐based flaps performed within 6 weeks of undergoing radiotherapy were less likely to result in complications than those performed after greater than 6 weeks (p < 0.048). At time of follow‐up, Musculoskeletal Tumor Society scores for fasciocutaneous and muscle‐based reconstructions, with or without radiation, showed no significant differences between groups (mean [SD]: 91% [8%] vs. 89% [13%]). Conclusion The similar complication rates and functional outcomes in this study support the safety and efficacy of both fasciocutaneous flaps and muscle‐based flaps in reconstructing limb‐sparing sarcoma resection defects, with or without radiotherapy.

Dysregulation of Nrf2/Keap1 Redox Pathway in Diabetes Affects Multipotency of Stromal Cells

The molecular and cellular level reaches of the metabolic dysregulations that characterize diabetes are yet to be fully discovered. As mechanisms underlying management of reactive oxygen species (ROS) gain interest as crucial factors in cell integrity, questions arise about the role of redox cues in the regulation and maintenance of bone marrow–derived multipotent stromal cells (BMSCs) that contribute to wound healing, particularly in diabetes. Through comparison of BMSCs from wild-type and diabetic mice, with a known redox and metabolic disorder, we found that the cytoprotective nuclear factor erythroid–related factor 2 (Nrf2)/kelch-like erythroid cell–derived protein 1 (Keap1) pathway is dysregulated and functionally insufficient in diabetic BMSCs (dBMSCs). Nrf2 is basally active, but in chronic ROS, we found irregular inhibition of Nrf2 by Keap1, altered metabolism, and limited BMSC multipotency. Forced upregulation of Nrf2-directed transcription, through knockdown of Keap1, restores redox homeostasis. Normalized Nrf2/Keap1 signaling restores multipotent cell properties in dBMSCs through Sox2 expression. These restored BMSCs can resume their role in regenerative tissue repair and promote healing of diabetic wounds. Knowledge of diabetes and hyperglycemia-induced deficits in BMSC regulation, and strategies to reverse them, offers translational promise. Our study establishes Nrf2/Keap1 as a cytoprotective pathway, as well as a metabolic rheostat, that affects cell maintenance and differentiation switches in BMSCs.

Ex vivo allotransplantation engineering: Delivery of mesenchymal stem cells prolongs rejection-free allograft survival

Current pharmacologic regimens in transplantation prevent allograft rejection through systemic recipient immunosuppression but are associated with severe morbidity and mortality. The ultimate goal of transplantation is the prevention of allograft rejection while maintaining recipient immunocompetence. We hypothesized that allografts could be engineered ex vivo (after allotransplant procurement but before transplantation) by using mesenchymal stem cell–based therapy to generate localized immunomodulation without affecting systemic recipient immunocompetence. To this end, we evaluated the therapeutic efficacy of bone marrow–derived mesenchymal stem cells in vitro and activated them toward an immunomodulatory fate by priming in inflammatory or hypoxic microenvironments. Using an established rat hindlimb model for allotransplantation, we were able to significantly prolong rejection‐free allograft survival with a single perioperative ex vivo infusion of bone marrow–derived mesenchymal stem cells through the allograft vasculature, in the absence of long‐term pharmacologic immunosuppression. Critically, transplanted rats rejected a second, nonengineered skin graft from the same donor species to the contralateral limb at a later date, demonstrating that recipient systemic immunocompetence remained intact. This study represents a novel approach in transplant immunology and highlights the significant therapeutic opportunity of the ex vivo period in transplant engineering.

2576: Optimization of a differential cytokine profile-based non-invasive diagnostic and predictive tool for reliable diagnosis of acute rejection in VCA

2576: Optimization of a differential cytokine profile-based non-invasive diagnostic and predictive tool for reliable diagnosis of acute rejection in VCA Piul S. Rabbani, PhD, Rohini L. Kadle, MD, Nakul Rao, Chin Park, and Daniel Ceradini New York University School of Medicine, New York, NY, USA Background Current methods of detection of early acute transplant rejection relies on invasive tissue biopsies and time-consuming histological analysis We propose an alternative method using adhesive discs to analyze molecular changes in cells sampled from the epidermis of a vascular composite allotransplant (VCA), to detect markers of acute rejection We aim to validate efficiency of skin stripping as a non-invasive predictor and sensitive diagnostic test for acute rejection in VCA. Methods Using an established VCA rat model, we transplanted composite flaps from donor Brown-Norway rats to age-matched recipient Lewis rats Following cyclosporine for 5 days, we inspected daily for clinical signs of rejection and sampled transplanted skin with adhesive CuDerm-discs at each time point up to rejection We performed QRT-PCR on sampled cells for cytokines associated with early rejection We sampled flaps for biopsies and histology to corroborate the disc data. Results CuDerm-disc-sourced PCR revealed that expressions of MCP1, MIP1̂I§, MIP1̂I and CXCL10 increased progressively with mild and advanced rejection, compared to the immunosuppressed stage (p < 005) MIP3̂I§ and CXCL9 showed significant upregulation at mild rejection (19-fold, 70-fold, respectively), but a downregulation during advanced rejection (4fold,20-fold, respectively, p < 005) Comparison of mild and advanced rejection showed highly significant differential cytokine expression (p < 001) We verified the sensitivity and validity of the CuDerm-disc method by comparison of mRNA expression in VCA biopsies and found cytokine detection comparable between both methods The mild and advanced rejection cytokine profiles from discs also corresponded with the Banff classification of cellular allograft rejection of the respective flap histologies. Conclusions Skin stripping, when compared to traditional tissue biopsy, is a comparable and reliable analytical tool The cytokine profiles gathered from skin stripping are distinct and capable of detecting the earliest stages of acute rejection, as well as distinguishing from advanced rejection, stages which are difficult to analyze definitively using traditional histology Our results clearly demonstrate the promise of skin stripping as a noninvasive tool in predicting and diagnosing early rejection, prior to onset of advanced rejection.

Abstract 119: Development of a Non-invasive Diagnostic and Predictive Tool for Acute Rejection in Vca Using Differential Cytokine Profiling

86 RESULTS: Herceptin administration increased the rate of motoneuron regeneration by 3x and sensory neuron regeneration by 3.7x compared to saline treated animals after the first week. However, the extent of motor and sensory regeneration was nearly complete in both groups by the end of the second week. In addition, the total number of myelinated fibers growing distally beyond the repair site was significantly increased in rats receiving Herceptin (2488 ± 154) compared to rats that received saline (1896 ± 251) (p < 0.05) four weeks after repair. When delayed repair was performed after a 3-month period of chronic denervation, Herceptin increased the number of acutely, but not chronically, axotomized motoneurons after two weeks. Interestingly, Western blot analysis revealed no change in ErbB2 activation with Herceptin administration. However, immunofluorescent imaging revealed decreased levels of activated EGFR on regenerating neurons, a factor known to be inhibitory to axon regeneration.