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Treatment significantly affects the circulating immune compartment.

Representative gating strategy illustrating (A) CD4 and CD8 T cell identification (B) expression of ICOS on CD4 positive T cells (C) expression of PD-1 and Ki-67 within PD-1 positive cells and (D) expression of CD38/HLA-DR, PD-1 and KI-67 within CD8+ PD-1+ T cells is shown.

Treatment significantly affects the circulating immune compartment.

Purpose:Neoadjuvant immunotherapy may improve the clinical outcome of regionally advanced operable melanoma and allows for rapid clinical and pathologic assessment of response. We examined neoadjuvant pembrolizumab and high-dose IFNα-2b (HDI) therapy in patients with resectable advanced melanoma.Patients and Methods:Patients with resectable stage III/IV melanoma were treated with concurrent pembrolizumab 200 mg i.v. every 3 weeks and HDI 20 MU/m2/day i.v., 5 days per week for 4 weeks, then 10 MU/m2/day subcutaneously 3 days per week for 2 weeks. Definitive surgery followed, as did adjuvant combination immunotherapy, completing a year of treatment. Primary endpoint was safety of the combination. Secondary endpoints included overall response rate (ORR), pathologic complete response (pCR), recurrence-free survival (RFS), and overall survival (OS). Blood samples for correlative studies were collected throughout. Tumor tissue was assessed by IHC and flow cytometry at baseline and at surgery.Results:A total of 31 patients were enrolled, and 30 were evaluable. At data cutoff (October 2, 2019), median follow-up for OS was 37.87 months (range, 33.2–43.47). Median OS and RFS were not reached. Radiographic ORR was 73.3% [95% confidence interval (CI): 55.5–85.8], with a 43% (95% CI: 27.3–60.1) pCR rate. None of the patients with a pCR have had a recurrence. HDI and pembrolizumab were discontinued in 73% and 43% of patients, respectively. Correlative analyses suggested that intratumoral PD-1/PD-L1 interaction and HLA-DR expression are associated with pCR (P = 0.002 and P = 0.008, respectively).Conclusions:Neoadjuvant concurrent HDI and pembrolizumab demonstrated promising clinical activity despite high rates of treatment discontinuation. pCR is a prognostic indicator.See related commentary by Menzies et al., p. 4133

Introduction: Genetic information is highly relevant to the classification and risk assessment of myeloid neoplasms, especially myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Next generation sequencing (NGS) is increasingly used to track mutations and monitor measurable residual disease, but with the increased data comes a need to distinguish driver mutations from potentially unrelated variants caused by age-related clonal hematopoiesis (ARCH) or clonal hematopoiesis of indeterminant potential (CHIP). Herein, we evaluate a combined flow cytometry and NGS approach to identify oncogenic driver mutations within specific cell populations and increase variant accessibility by NGS. Methods: Proof of cell enrichment methodology was performed with a cell line expressing CD34 (hallmark of myeloid blasts). The cell line was spiked into healthy donor PBMCs at defined levels to create samples with varied tumor percentage. Purchased AML patient samples were also analyzed. CD34 expression of samples was confirmed by flow cytometry prior to- and following enrichment by anti-CD34 magnetic cell sorting technology. Sequencing libraries were prepared using the Archer VariantPlex Core Myeloid kit and sequenced on Illumina MiSeqs. Mutations were characterized using Archer software. The association between NGS and flow cytometry data was assessed through mapping the VAF data to the tumor population determined by flow cytometry. Results: Identified mutations in tested samples included known oncogenic mutations as well as mutations associated with ARCH/CHIP events. In a subset of samples, at least one mutation was identified at a VAF correlating to the tumor percentage determined by flow cytometry analysis; these mutations were enriched in the CD34+ cell fraction following magnetic cell sorting. Sequencing the enriched tumor fraction increased the VAF of these mutations, and simultaneously revealed additional tumor-associated mutations that were previously below the detection limit of the assay. Conversely, potential ARCH/CHIP mutations were present in both CD34+ and CD34- cell populations. Conclusion: Using a combination of flow cytometry, magnetic cell sorting, and NGS, variants specific to the tumor fraction of samples were identified. For patient specimens, the presence of ARCH/CHIP mutations in both enriched and residual cell populations indicate these variants evolved prior to the emergence of true leukemic progenitor cells; the identification of the flow-matched mutations only in the enriched population indicates the key role of these changes in the disease progression. Together, flow cytometry and cell-enriched NGS have the potential to enhance detection of disease-driving mutations earlier, thus, could be used as novel approaches to identify and treat myeloid neoplasms early with existing therapies and/or support development of new investigational agents. Citation Format: Sarah Johnson, Keegan Vaughan, Alexis Kurmis, Zeni Alfonso, Nathan Riccitelli. Assessing clonal evolution of myeloid neoplasms by flow cytometry guided, cell-enriched next generation sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3922.

Purpose:Neoadjuvant immunotherapy may improve the clinical outcome of regionally advanced operable melanoma and allows for rapid clinical and pathologic assessment of response. We examined neoadjuvant pembrolizumab and high-dose IFNα-2b (HDI) therapy in patients with resectable advanced melanoma.Patients and Methods:Patients with resectable stage III/IV melanoma were treated with concurrent pembrolizumab 200 mg i.v. every 3 weeks and HDI 20 MU/m2/day i.v., 5 days per week for 4 weeks, then 10 MU/m2/day subcutaneously 3 days per week for 2 weeks. Definitive surgery followed, as did adjuvant combination immunotherapy, completing a year of treatment. Primary endpoint was safety of the combination. Secondary endpoints included overall response rate (ORR), pathologic complete response (pCR), recurrence-free survival (RFS), and overall survival (OS). Blood samples for correlative studies were collected throughout. Tumor tissue was assessed by IHC and flow cytometry at baseline and at surgery.Results:A total of 31 patients were enrolled, and 30 were evaluable. At data cutoff (October 2, 2019), median follow-up for OS was 37.87 months (range, 33.2–43.47). Median OS and RFS were not reached. Radiographic ORR was 73.3% [95% confidence interval (CI): 55.5–85.8], with a 43% (95% CI: 27.3–60.1) pCR rate. None of the patients with a pCR have had a recurrence. HDI and pembrolizumab were discontinued in 73% and 43% of patients, respectively. Correlative analyses suggested that intratumoral PD-1/PD-L1 interaction and HLA-DR expression are associated with pCR (P = 0.002 and P = 0.008, respectively).Conclusions:Neoadjuvant concurrent HDI and pembrolizumab demonstrated promising clinical activity despite high rates of treatment discontinuation. pCR is a prognostic indicator.See related commentary by Menzies et al., p. 4133

e15036 Background: Patients with locally/regionally advanced melanoma were treated on a clinical trial with a neoadjuvant combination of ipilimumab (ipi) and high dose IFNα2b (HDI) (Tarhini et al, JITC 2018). In this study, immune cell composition in peripheral blood samples collected at various time points was measured to determine any correlation with clinical outcomes and investigate the immune modulating effect of the combination therapy. Methods: Patients were randomized to neoadjuvant ipi at 3 mg/kg or 10 mg/kg, both given in combination with HDI. Tumor radiologic responses were designated as complete (CR), partial (PR), stable disease (SD) or disease progression (PD). Pathologic complete response (pCR) was defined as absence of viable tumor on histologic assessment. Peripheral blood mononuclear cells (PBMC) from treated patients (N = 28) were tested at baseline (before initiating ipi-HDI), then at 6-weeks, 3-months and 12-months (following neoadjuvant ipi-HDI). High complexity (14-color) flow cytometry analysis was performed to detect key immunological biomarkers including myeloid derived suppressor cells (MDSCs), B cells, regulatory T cells (Tregs), PD-1 and TIM3 expression on T-cells, and differentiation of T-cells into Th1, Th2 or Th17 phenotype at different time points during systemic immunotherapy. Statistical significance was determined using R-package employing Kruskal’s test. Results: Lower levels of peripheral Tregs (p = 0.02), MDSCs (p = 0.05), and CD4 effector memory cells (p = 0.04) at 3-months post treatment correlated with radiologic response. In addition, lower change from baseline at 3 months in CD4/CD8 ratio (p = 0.04), levels of Tregs (p = 0.01) and CD4 effector memory cells (p = 0.02) was associated with radiologic response. Patients exhibiting pCR had significantly lower Tregs (p = 0.04) at 6-months post treatment and significantly higher CD8 central memory cells at both 3 months (p = 0.04) and 12 month time-points (p = 0.01) as compared to patients without pCR. Finally, patients without pCR had significantly lower change from baseline in CD19 B cells at 6 months (p = 0.01) and 12 months (p = 0.04) as compared to patients with pCR. Conclusions: Our data demonstrates that the levels of immunosuppressive cells including Tregs and MDSCs in periphery are negatively associated with response. Higher levels of CD8 memory cells and B cells on-treatment are associated with clinical benefit.

Purpose: To develop an approach that models the cutaneous healing that occurs in a patient with full thickness thermal burn injury complicated by total body radiation exposure sufficient to induce sub-lethal prodromal symptoms. An assessment of the effects of an autologous cell therapy on wound healing on thermal burn injury with concomitant radiation exposure was used to validate the utility of the model.Methods: Gottingen minipigs were subjected to a 1.2 Gy total body irradiation by exposure to a 6 MV X-ray linear accelerator followed by ∼10 cm2 full thickness burns (pre-heated brass block with calibrated spring). Three days after injury, wounds were excised to the underlying fascia and each animal was randomized to receive treatment with autologous adipose-derived regenerative cells (ADRC) delivered by local or intravenous injection, or vehicle control. Blood counts were used to assess radiation-induced marrow suppression. All animals were followed using digital imaging to assess wound healing....

Background. Acute kidney injury (AKI) represents a major clinical problem with high mortality and limited causal treatments. The use of cell therapy has been suggested as a potential modality to improve the course and outcome of AKI. Methods. We investigated the possible renoprotection of freshly isolated, uncultured adipose tissue-derived stem and regenerative cells (ADRCs) before and after cryopreservation in a rat ischemia–reperfusion (I–R) model of AKI. Results. We demonstrated that ADRC therapy drastically reduced mortality (survival 100% vs. 57%, ADRC vs. controls, respectively) and significantly reduced serum creatinine (sCr on Day 3: 3.03 ± 1.58 vs. 7.37 ± 2.32 mg/dL, ADRC vs. controls, respectively). Histological analysis further validated a significantly reduced intratubular cast formation, ameliorated acute tubular epithelial cell necrosis and mitigated macrophage infiltration. Furthermore, a reduced RNA expression of CXCL2 and IL-6 was found in the ADRC group which could explain the reduced macrophage recruitment. Use of cryopreserved ADRCs resulted in an equally high survival (90% vs. 33% in the control group) and similarly improved renal function (sCr on Day 3: 4.64 ± 2.43 vs. 7.24 ± 1.40 mg/dL in controls). Conclusions. Collectively, these results suggest a potential clinical role for ADRC therapy in patients with AKI. Importantly, cryopreservation of ADRCs could offer an autologous treatment strategy for patients who are at high risk for AKI during planned interventions.

Smooth muscle is a major component of human tissues and is essential for the normal function of a multitude of organs including the intestine, urinary tract and the vascular system. The use of stem cells for cell-based tissue engineering and regeneration strategies represents a promising alternative for smooth muscle repair. For such strategies to succeed, a reliable source of smooth muscle precursor cells must be identified. Adipose tissue provides an abundant source of multipotent cells. In this study, the capacity of processed lipoaspirate (PLA) and adipose-derived stem cells to differentiate into phenotypic and functional smooth muscle cells was evaluated. To induce differentiation, PLA cells were cultured in smooth muscle differentiation medium. Smooth muscle differentiation of PLA cells induced genetic expression of all smooth muscle markers and further confirmed by increased protein expression of smooth muscle cell-specific α actin (ASMA), calponin, caldesmon, SM22, myosin heavy chain (MHC), and smoothelin. Clonal studies of adipose derived multipotent cells demonstrated differentiation of these cells into smooth muscle cells in addition to trilineage differentiation capacity. Importantly, smooth muscle-differentiated cells, but not their precursors, exhibit the functional ability to contract and relax in direct response to pharmacologic agents. In conclusion, adipose-derived cells have the potential to differentiate into functional smooth muscle cells and, thus, adipose tissue can be a useful source of cells for treatment of injured tissues where smooth muscle plays an important role.

Adipose tissue can be harvested in large amounts with minimal morbidity. It contains numerous cells types, including adipocytes, preadipocytes, vascular endothelial cells and vascular smooth muscle cells; it also contains cells that have the ability to differentiate into several lineages, such as fat, bone, cartilage, skeletal, smooth, and cardiac muscle, endothelium, hematopoietic cells, hepatocytes and neuronal cells. Cloning studies have shown that some adipose-derived stem cells (ADSCs) have multilineage differentiation potential. ADSCs are also capable of expressing multiple growth factors, including vascular endothelial growth factor and hepatocyte growth factor. Early, uncontrolled, non-randomized clinical research, applying fresh adipose-derived cells into a cranial defect or undifferentiated ADSCs into fistulas in Crohn's disease, has shown healing and an absence of side effects. The combination of these properties, and the large quantity of cells that can be obtained from fat, suggests that this tissue will be a useful tool in biotechnology.

Purpose: We performed a pilot study to investigate the ability of human adipose derived, multipotent stem cells to be delivered to and survive within bladder and urethral smooth muscle. Materials and Methods: Lipoaspirate was acquired from female patients undergoing liposuction. The lipoaspirate was processed to yield a pluripotent population of processed lipoaspirate (PLA) cells. For tissue delivery PLA cells were fluorescent labeled and suspended in Hanks' balanced salt solution (Sigma Chemical Co., St. Louis, Missouri). To assess PLA viability in multiple animal models 8 Rnu athymic rats (Charles River, Wilmington, Massachusetts) and 6 SCID mice (Taconic Farms, Oxnard, California) underwent laparotomy and injection of PLA cells into the bladder and urethra. An additional 8 rats underwent sham injection of Hanks' balanced salt solution alone. Experimental and control animals were sacrificed 2, 4, 8 and 12 weeks after injection, and the bladders and urethras were analyzed. Results: Self-regenerating, pluripotent PLA cells were easily isolated from human adipose tissue. Evaluation 2, 4, 8 and 12 weeks after injection demonstrated PLA cell viability and incorporation into the recipient smooth muscle. Eight weeks following injection PLA cells demonstrated in vivo expression of α-smooth muscle actin, an early marker of smooth muscle differentiation. Conclusions: PLA cells are an easily accessible source of pluripotent cells, making them ideal for tissue regeneration. PLA cells remain viable up to 12 weeks in the lower urinary tract. Human PLA cells injected into the urinary tract show morphological and phenotypic evidence of smooth muscle incorporation and differentiation with time. PLA cells may provide a feasible and cost-effective cell source for urinary tract reconstruction.

Background—In diabetes or atherosclerosis, ectopic bone, fat, cartilage, and marrow often develop in arteries. However the mechanism is unknown. We have previously identified a subpopulation of vascular cells (calcifying vascular cells, CVC), derived by dilutional cloning of bovine aortic medial cells, and showed that they undergo osteoblastic differentiation and mineralization. We now show that CVC have the potential to differentiate along other mesenchymal lineages.Methods and Results—To determine the multilineage potential of CVC, molecular and functional markers of multiple mesenchymal lineages were assessed. Chondrogenic potential of CVC was evidenced by expression of types II and IX collagen and cytochemical staining for Alcian blue. Leiomyogenic potential of CVC was evidenced by the expression of smooth muscle-α actin, calponin, caldesmon, and myosin heavy chain. Stromogenic potential of CVC was evidenced by the ability to support growth of colony-forming units of hematopoietic progenitor cells from human CD34+umbilical cord blood cells for a period of 5 weeks. Adipogenic potential was not observed. CVC were immunopositive to antigens to CD29 and CD44 but not to CD14 or CD45, consistent with other mesenchymal stem cells. CVC retained multipotentiality despite passaging and expansion through more than 20 to 25 population triplings, indicating a capacity for self-renewal.Conclusions—These results suggest that the artery wall contains cells that have the potential for multiple lineages similar to mesenchymal stem cells but with a unique differentiation repertoire.

Our laboratory has characterized a population of stromal cells obtained from adipose tissue termed processed lipoaspirate cells (PLAs). PLAs, like bone-marrow derived mesenchymal stem cells (BM-MSCs), have the capacity to differentiate along the adipogenic, osteogenic, chondrogenic, and myogenic lineages, In order to better characterize these two multi-lineage populations, we examined the surface phenotype of both bone marrow and adipose tissue-derived cells from five patients undergoing surgery. PLA and BM-MSC cells were isolated, subcultivated, and evaluated for cell surface marker expression using flow cytometry. PLA and BM-MSC cells both expressed CD13, CD29, CD44, CD90, CD105, SH-3, and STRO-1. Differences in expression were noted for cell adhesion molecules CD49d (Integrin alpha4), CD54 (ICAM-1), CD34, and CD106 (VCAM-1). While markedly similar, the surface phenotypes of PLA and BM-MSC cells are distinct for several cell adhesion molecules implicated in hematopoietic stem cell homing, mobilization, and proliferation.

Our laboratory has recently characterized a population of cells from adipose tissue, termed processed lipoaspirate (PLA) cells, which have multi-lineage potential similar to bone-marrow-derived mesenchymal stem cells (MSCs). This study is the first comparison of PLA cells and MSCs isolated from the same patient. No significant differences were observed for yield of adherent stromal cells, growth kinetics, cell senescence, multi-lineage differentiation capacity, and gene transduction efficiency. Adipose tissue is an abundant and easily procured source of PLA cells, which have a potential like MSCs for use in tissue-engineering applications and as gene delivery vehicles.

Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.

Adipose tissue sciences have rapidly expanded since the identification of regenerative cells contained within the stromal vascular fraction (SVF) of fat. Isolation of the SVF, containing adipose-derived stem cells (ADSC), can be accomplished efficiently in the operating room or in the laboratory through enzymatic digestion of the adipose tissue and concentration of SVF. Cells can be directly re-injected as a mesotherapeutic agent, recombined with a tissue scaffold (e.g., cell-enriched fat grafts) or expanded in culture for tissue-engineered cell therapeutics. The potential for cell therapy is under current investigation by researchers around the world. This chapter reviews laboratory methods for isolating ADSCs and the ongoing clinical trials evaluating cell therapeutic efficacy across many specialties, including cardiology, neurology, immunology, tissue engineering, sports medicine, and plastic and reconstructive surgery.

Adipose tissue sciences have rapidly expanded since the identification of regenerative cells contained within the stromal vascular fraction (SVF) of fat. Isolation of the SVF, containing adipose-derived stem cells (ADSC), can be accomplished efficiently in the operating room or in the laboratory through enzymatic digestion of the adipose tissue and concentration of SVF. Cells can be directly re-injected as a mesotherapeutic agent, recombined with a tissue scaffold (e.g., cell-enriched fat grafts) or expanded in culture for tissue-engineered cell therapeutics. The potential for cell therapy is under current investigation by researchers around the world. This chapter reviews laboratory methods for isolating ADSCs and the ongoing clinical trials evaluating cell therapeutic efficacy across many specialties, including cardiology, neurology, immunology, tissue engineering, sports medicine, and plastic and reconstructive surgery.

Human adipose tissue has been shown to contain a population of cells that possesses extensive proliferative capacity and the ability to differentiate into multiple cell lineages. These cells are referred to as adipose tissue-derived stem cells (ADSCs) and are generally similar, though not identical, to mesenchymal stem cells (also referred to as marrow stromal cells). ADSCs for research are most conveniently extracted from tissue removed during an elective cosmetic liposuction procedure but may also be obtained from resected adipose tissue. This chapter describes surgical procedures associated with improved ADSC recovery and the processes by which aspirated adipose tissue is washed and digested with collagenase to yield a heterogeneous population from which ADSCs can be expanded. The large volume of tissue obtained from a liposuction procedure (average approximately 2 L), combined with the relatively high frequency of ADSC within the digestate, yields substantially more stem cells than can be realized from marrow without extensive expansion in culture.

Human adipose tissue has been shown to contain a population of cells that possesses extensive proliferative capacity and the ability to differentiate into multiple cell lineages. These cells are referred to as adipose tissue-derived stem cells (ADSCs) and are generally similar, though not identical, to mesenchymal stem cells (also referred to as marrow stromal cells). ADSCs for research are most conveniently extracted from tissue removed during an elective cosmetic liposuction procedure but may also be obtained from resected adipose tissue. This chapter describes surgical procedures associated with improved ADSC recovery and the processes by which aspirated adipose tissue is washed and digested with collagenase to yield a heterogeneous population from which ADSCs can be expanded. The large volume of tissue obtained from a liposuction procedure (average approximately 2 L), combined with the relatively high frequency of ADSC within the digestate, yields substantially more stem cells than can be realized from marrow without extensive expansion in culture.

Background Human adipose tissue has been shown to contain multipotent cells with properties similar to mesenchymal stromal cells. While there have been many studies of the biology of these cells, no study has yet evaluated issues associated with tissue harvest. Methods Adipose tissue was obtained from the subcutaneous space of the abdomen and hips of 10 donors using both syringe and pump-assisted liposuction. Tissue was digested with collagenase and then assayed for the presence of different stem and progenitor cell types using clonogenic culture assays, including fibroblast colony-forming unit (CFU-F) and alkaline phosphatase-positive colony-forming unit (CFU-AP). Paired analysis of samples obtained from the same individual was used to compare harvest method and site. Results Syringe suction provided significantly greater recovery of adipocytes and a non-significant trend towards improved recovery of cells in the adipocyte-depleted fraction. There was considerable donor-to-donor variation in stem cell recovery. However, paired analysis of tissue obtained from different subcutaneous sites in the same donor showed that tissue harvested from the hip yielded 2.3-fold more CFU-F/unit volume and a 7-fold higher frequency of CFU-AP than that obtained from the abdomen. These differences were statistically significant. Discussion Harvest site influences the stem and progenitor cell content of subcutaneous adipose tissue.

Tissue engineering offers considerable promise in the repair or replacement of diseased and/or damaged tissues. The cellular component of this regenerative approach will play a key role in bringing these tissue engineered constructs from the laboratory bench to the clinical bedside. However, the ideal source of cells still remains unclear and may differ depending upon the application. Current research for many applications is focused on the use of adult stem cells. The properties of adult stem cells that make them well-suited for regenerative medicine are (1) ease of harvest for autologous transplantation, (2) high proliferation rates for ex vivo expansion and (3) multilineage differentiation capacity. This review will highlight the use of adipose tissue as a reservoir of adult stem cells and draw conclusions based upon comparisons with bone marrow stromal cells. (Keio JM ed 54 (3): 132-141, September 2005)

Recent preclinical and clinical studies have suggested that adult stem cells have the ability to promote the retention or restoration of cardiac function in acute and chronic ischemia. Published clinical studies have used autologous donor cells, including skeletal muscle myoblasts, cultured peripheral blood cells, or bone marrow cells. However, our research and that of others indicates that human adipose tissue is an alternative source of cells with potential for cardiac cell therapy. These findings include the presence of cells within adipose tissue that can differentiate into cells expressing a cardiomyocytic or endothelial phenotype, as well as angiogenic and antiapoptotic growth factors. This potential is supported by preclinical studies in large animals.