Operationalizing Stakeholder Engagement Through the Stakeholder-Centric Engagement Charter (SCEC).

There is a need for clear strategies and procedures to operationalize stakeholder engagement in research studies. Clear guidelines that promote shared leadership among study investigators and research stakeholders are important for inclusive and sustainable partnerships. Such guidelines may take the form of a governance charter and can be a means for encouraging the participation and inclusion of stakeholders who may have little to no experience with research or are otherwise underrepresented in research. This perspective article presents the Stakeholder-Centric Engagement Charter (SCEC), one effort to operationalize a stakeholder engagement approach between researchers and an advisory committee as guided by the Patient-Centered Outcomes Research Institute's (PCORI) Research Engagement Principles (i.e., reciprocal relationships, partnerships, co-learning, transparency-honesty-trust). Building on the SCEC can help future investigators develop a study-specific, dynamic, governance document outlining advisory committee and research team preferences in areas such as role expectations, study governance, and decision-making procedures.

Exploring the Effects of Methylation on the CID of Protonated Lysine: A Combined Experimental and Computational Approach.

We report the results of experiments, simulations, and DFT calculations that focus on describing the reaction dynamics observed within the collision-induced dissociation of l-lysine-H+ and its side-chain methylated analogues, Nε-methyl-l-lysine-H+ (Me1-lysine-H+), Nε,Nε-dimethyl-l-lysine-H+ (Me2-lysine-H+), and Nε,Nε,Nε-trimethyl-l-lysine-H+ (Me3-lysine-H+). The major pathways observed in the experimental measurements were m/z 130 and 84, with the former dominant at low collision energies and the latter at intermediate to high collision energies. The m/z 130 peak corresponds to loss of N(CH3)nH3-n, while m/z 84 has the additional loss of H2CO2 likely in the form of H2O + CO. Within the time frame of the direct dynamics simulations, m/z 130 and 101 were the most populous peaks, with the latter identified as an intermediate to m/z 84. The simulations allowed for the determination of several reaction pathways that result in these products. A graph theory analysis enabled the elucidation of the significant structures that compose each peak. Methylation results in the preferential loss of the side-chain amide group and a reduction of cyclic structures within the m/z 84 peak population in simulations.

Modeling the Effects of O-Sulfonation on the CID of Serine.

We present the results of direct dynamics simulations and DFT calculations aimed at elucidating the effect of O-sulfonation on the collision-induced dissociation for serine. Toward this end, direct dynamics simulations of both serine and sulfoserine were performed at multiple collision energies and theoretical mass spectra obtained. Comparisons to experimental results are favorable for both systems. Peaks related to the sulfo group are identified and the reaction dynamics explored. In particular, three significant peaks (m/z 106, 88, and 81) seen in the theoretical mass spectrum directly related to the sulfo group are analyzed as well as major peaks shared by both systems. Our analysis shows that the m/z 106 peaks result from intramolecular rearrangements, intermolecular proton transfer among complexes composed of initial fragmentation products, and at high energy side-chain fragmentation. The m/z 88 peak was found to contain multiple constitutional isomers, including a previously unconsidered, low energy structure. It was also observed that the RM1 semiempirical method was not able to obtain all of the major peaks seen in experimens for sulfoserine. In contrast, PM6 did obtain all major experimental peaks.

Expansion of cancer germline antigen-specific cytotoxic T lymphocytes for immunotherapy.

The cancer germline antigens MAGE-A1, MAGE-A3, and NY-ESO-1 can be used to target relapsed or therapy-resistant malignant solid tumors, and previous studies have demonstrated that these antigens can be epigenetically upregulated on the surface of tumor cells following exposure to low-dose demethylating chemotherapy agents, such as decitabine. The extent to which cancer germline antigen cytotoxic T lymphocytes can be reliably expanded from healthy donors has not been well characterized, specifically in terms of whether these T cells consistently kill antigen-bearing targets or simply produce interferon-γ in the presence of the antigen. Cancer germline antigen cytotoxic T lymphocytes were generated using conventional method and high-density lymphocyte culture method. We demonstrate that there is no difference in the extent of antigen-specific killing with or without CD25 depletion when interleukin-21 is added to the cultures. Cancer germline antigen-specific killer cells could be expanded from 8/12 healthy donors using overlapping peptide mixes derived from MAGE-A1, MAGE-A3, and NY-ESO-1 and from 7/9 healthy donors using HLA-restricted epitopes. Furthermore, cytotoxic T lymphocyte derived from 4/5 patients displayed specific cytotoxicity of target cells expressing respective cancer germline antigen and HLA partially matched tumor lines. High-density lymphocyte culture prior to stimulation with cancer germline antigen peptides resulted in antigen-specific cytotoxic T lymphocyte from healthy donors and patients from whom cancer germline antigen cytotoxic T lymphocyte culture with conventional methods was not feasible. These data demonstrate that MAGE-A1-, MAGE-A3-, and NY-ESO-1-specific T cells with antigen-specific cytotoxicity can be cultured from healthy donors and patient-derived cells making adoptive immunotherapy with these cytotoxic T lymphocyte feasible.

Cytomegalovirus Treatment in Pediatric Hematopoietic Stem Cell Transplant Patients.

Cytomegalovirus (CMV) is a frequent complication of hematopoietic stem cell transplant in pediatric patients, with significant morbidity and mortality. Antiviral drugs are used as prophylactic, preemptive or therapeutic medicines; however, no uniform guidelines exist for the best strategy to prevent CMV disease. Resistance to standard antiviral therapies can lead to further difficulty in managing CMV disease. Studies for investigational therapies are underway and could provide options for treatment of resistant CMV, while limiting toxicities associated with currently used antiviral therapies.

Dendritic cell vaccines: A review of recent developments and their potential pediatric application.

For many cancers the use of conventional chemotherapy has been maximized, and further intensification of chemotherapy generally results in excess toxicity with little long-term benefit for cure. Many tumors become resistant to chemotherapy, making the investigation of novel approaches such as immunotherapy of interest. Because the tumor microenvironment is known to promote immune tolerance and down regulate the body's natural defense mechanisms, modulating the immune system with the use of dendritic cell (DC) therapy is an attractive approach. Thousands of patients with diverse tumor types have been treated with DC vaccines. While antigen specific immune responses have been reported, the duration and magnitude of these responses are typically weak, and objective clinical responses have been limited. DC vaccine generation and administration is a multi-step process with opportunities for improvement in source of DC for vaccine, selection of target antigen, and boosting effector cell response via administration of vaccine adjuvant or concomitant pharmacologic immunomodulation. In this review we will discuss recent developments in each of these areas and highlight elements that could be moved into pediatric clinical trials.

Cellular and Antibody Based Approaches for Pediatric Cancer Immunotherapy.

Progress in the use of traditional chemotherapy and radiation-based strategies for the treatment of pediatric malignancies has plateaued in the past decade, particularly for patients with relapsing or therapy refractory disease. As a result, cellular and humoral immunotherapy approaches have been investigated for several childhood cancers. Several monoclonal antibodies are now FDA approved and commercially available, some of which are currently considered standard of practice. There are also several new cellular immunotherapy approaches under investigation, including chimeric antigen receptor (CAR) modified T cells, cancer vaccines and adjuvants, and natural killer (NK) cell therapies. In this review, we will discuss previous studies on pediatric cancer immunotherapy and new approaches that are currently being investigated in clinical trials.

A phase I trial combining decitabine/dendritic cell vaccine targeting MAGE-A1, MAGE-A3 and NY-ESO-1 for children with relapsed or therapy-refractory neuroblastoma and sarcoma.

Antigen-specific immunotherapy was studied in a multi-institutional phase 1/2 study by combining decitabine (DAC) followed by an autologous dendritic cell (DC)/MAGE-A1, MAGE-A3 and NY-ESO-1 peptide vaccine in children with relapsed/refractory solid tumors. Patients aged 2.5-15 years with relapsed neuroblastoma, Ewing's sarcoma, osteosarcoma and rhabdomyosarcoma were eligible to receive DAC followed by DC pulsed with overlapping peptides derived from full-length MAGE-A1, MAGE-A3 and NY-ESO-1. The primary endpoints were to assess the feasibility and tolerability of this regimen. Each of four cycles consisted of week 1: DAC 10 mg/m(2)/day for 5 days and weeks 2 and 3: DC vaccine once weekly. Fifteen patients were enrolled in the study, of which 10 were evaluable. Generation of DC was highly feasible for all enrolled patients. The treatment regimen was generally well tolerated, with the major toxicity being DAC-related myelosuppression in 5/10 patients. Six of nine patients developed a response to MAGE-A1, MAGE-A3 or NY-ESO-1 peptides post-vaccine. Due to limitations in number of cells available for analysis, controls infected with a virus encoding relevant genes have not been performed. Objective responses were documented in 1/10 patients who had a complete response. Of the two patients who had no evidence of disease at the time of treatment, one remains disease-free 2 years post-therapy, while the other experienced a relapse 10 months post-therapy. The chemoimmunotherapy approach using DAC/DC-CT vaccine is feasible, well tolerated and results in antitumor activity in some patients. Future trials to maximize the likelihood of T cell responses post-vaccine are warranted.

Decitabine facilitates immune recognition of sarcoma cells by upregulating CT antigens, MHC molecules, and ICAM-1.

Rhabdomyosarcoma, osteosarcoma, and Ewing's sarcoma are the most common types of sarcoma in children. Despite standard therapy, nearly one third of the patients with Ewing's sarcoma relapse, and there are limited options with curative potential. Immunotherapy is a promising approach as it can target tumor-specific antigens that are specifically expressed on tumors while sparing non-malignant cells. We have demonstrated that a demethylating chemotherapeutic drug, 5-aza-2'-deoxycytidine (decitabine, DAC) can upregulate the expression of cancer-testis (CT) antigens, MHC molecules, and intracellular cell adhesion molecule-1 on pediatric sarcoma cell lines, resulting in enhanced killing of tumor cells by CT antigen-specific cytotoxic T lymphocytes derived from pediatric sarcoma patients. A significant increase in the mRNA expression levels of MAGE-A1 and MAGE-A3 were found in 70 %, and NY-ESO-1 in 80 % of the sarcoma lines following exposure to pharmacological levels of DAC. The high expression levels of MAGE-A1, MAGE-A3, and NY-ESO-1 were sustained in sarcoma lines and primary tumor lines over 30 days after the cessation of DAC. Furthermore, DAC treatment induced upregulation of MAGE-A1, MAGE-A3, or NY-ESO-1 protein expression in seven of nine lines studied. These studies show that demethylating chemotherapy could be combined with CT antigen-directed immunotherapy for treating pediatric sarcoma.

Complete remission following decitabine/dendritic cell vaccine for relapsed neuroblastoma.

Patients with relapsed stage 4 neuroblastoma have an extremely poor long-term prognosis, making the investigation of new agents of interest. We report the outcome of the first patient treated in a phase 1 study for relapsed neuroblastoma, using the chemotherapy agent decitabine to upregulate cancer testis antigen expression, followed by a dendritic cell vaccine targeting the cancer testis antigens MAGE-A1, MAGE-A3, and NY-ESO-1. Our patient had persistent tumor in his bone marrow after completion of standard therapy for neuroblastoma, including multiagent chemotherapy, tumor resection, stem cell transplantation, radiation therapy, and anti-GD2 monoclonal antibodies. His marrow disease persisted despite chemotherapy, which was given while the vaccine was being produced. After 3 cycles of decitabine and vaccine, this patient achieved a complete remission and is now 1 year from his last treatment, with no evidence of tumor in his bone marrow or other sites. This patient was noted to have an increase in MAGE-A3-specific T cells. This is the first report combining demethylating chemotherapy to enhance tumor antigen expression followed by a cancer antigen vaccine.

Cancer testis antigen and immunotherapy.

The identification of cancer testis (CT) antigens has been an important advance in determining potential targets for cancer immunotherapy. Multiple previous studies have shown that CT antigen vaccines, using both peptides and dendritic cell vaccines, can elicit clinical and immunologic responses in several different tumors. This review details the expression of melanoma antigen family A, 1 (MAGE-A1), melanoma antigen family A, 3 (MAGE-A3), and New York esophageal squamous cell carcinoma-1 (NY-ESO-1) in various malignancies, and presents our current understanding of CT antigen based immunotherapy.

Adoptive immunotherapy with CMV-specific cytotoxic T lymphocytes for stem cell transplant patients with refractory CMV infections.

Adoptive immunotherapy with cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) is an effective strategy for preventing and treating viral reactivation after allogeneic stem cell transplantation (SCT). We have shown previously that CMV CTL can be generated in 1 to 2 weeks by stimulating donor lymphocytes with peptide mixes derived from full-length pp65 and IE1. We conducted a multi-institutional study of CMV-specific CTL for patients with persistent or anti-viral-resistant CMV infections after allogeneic SCT, to determine the safety, feasibility, and immunologic effects of this approach. We were successful in stimulating CTL from 10/10 donors with pooled CMV overlapping peptide mixes. Five of the 7 subjects who met infusion criteria had new onset CMV-specific CTL activity detected within 4 to 6 weeks after infusion. Of the 2 subjects who did not have immunologic responses after infusion, 1 received CTL with a low viability after thawing, and the other patient received cyclosporine A and systemic corticosteroids at the time of the infusion, achieving only a low, transient increase (10%) in pp65-specific activity. There was no graft-versus-host disease attributable to these infusions. These findings indicate that the infusion of CTL stimulated over 1 to 2 weeks with overlapping CMV peptides can result in virus-specific immune reconstitution in SCT recipients, without exacerbations of graft-versus-host disease.

Wilms' tumor 1-specific cytotoxic T lymphocytes can be expanded from adult donors and cord blood.

The use of WT1-specific CTL is one potential strategy to treat leukemic relapse following allogeneic stem cell transplant (SCT). Previous studies have largely focused on generating WT1-CTL from adult donors by cloning. We demonstrate that WT1-CTL can be generated from healthy adult donors and from cord blood by stimulating with an overlapping pool of peptides derived from full length WT1 and selecting antigen-specific cells based on the expression of CD137. The rapid expansion with anti-CD3 and IL-2 resulted in a 100-200-fold expansion. These CTL lysed WT1 expressing targets, including leukemia lines, in a HLA restricted manner.

MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells to facilitate cytotoxic T lymphocyte-mediated tumor cell killing.

Approximately half of patients with stage IV neuroblastoma are expected to relapse despite current therapy, and when this occurs, there is little likelihood of achieving a cure. Very few clinical trials have been conducted to determine whether cellular immune responses could be harnessed to fight this tumor, largely because potential tumor antigens for cytotoxic T lymphocytes (CTL) are limited. MAGE-A1, MAGE-A3, and NY-ESO-1 are cancer-testis (CT) antigens expressed on a number of malignant solid tumors, including neuroblastoma, but many tumor cell lines down-regulate the expression of CT antigens as well as major histocompatibility (MHC) antigens, precluding recognition by antigen-specific T cells. If expression of cancer antigens on neuroblastoma could be enhanced pharmacologically, CT antigen-specific immunotherapy could be considered for this tumor. We have demonstrated that the expression of MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells following exposure to pharmacologic levels of the demethylating agent 5-aza-2'-deoxycytidine (decitabine, DAC). Expression of NY-ESO-1, MAGE-A1, or MAGE-A3 was induced in 10/10 neuroblastoma cell lines after 5 days of exposure to DAC. Culture of neuroblastoma cell lines with IFN-γ was also associated with an increased expression of either MHC Class I or II by cytofluorometry, as reported by other groups. MAGE-A1, MAGE-A3, and NY-ESO-1-specific CTL were cultured from volunteer donors by stimulating peripheral blood mononuclear cells with dendritic cells pulsed with overlapping peptide mixes derived from full-length proteins, and these CTL preferentially lysed HLA partially matched, DAC-treated neuroblastoma and glioblastoma cell lines. These studies show that demethylating chemotherapy can be combined with IFN-γ to increase the expression of CT antigens and MHC molecules on neuroblastoma cells, and pre-treatment with these agents makes tumor cell lines more susceptible to CTL-mediated killing. These data provide a basis to consider the use of demethylating chemotherapy in neuroblastoma patients, in conjunction with immune therapies that facilitate the expansion of CT antigen-specific CTL.

The detection of CMV pp65 and IE1 in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly lethal brain tumor affecting children and adults, with the majority of affected individuals dying from their disease by 2 years following diagnosis. Other groups have reported the association of cytomegalovirus (CMV) with GBM, and we sought to confirm these findings in a large series of patients with primary GBM from our institution. Immunohistochemical analysis of paraffin embedded tissue sections was performed on 49 newly diagnosed GBM tumors, the largest series reported to date. We confirmed the presence of CMV pp65 on 25/49 (51%) and of IE1 on 8/49 (16%) of these tumors. While pp65 and IE1 are generally found in the nucleus of cells that are permissibly infected by CMV, GBM in this series had mostly cytoplasmic staining, with only 16% having nuclear staining for one or both of these antigens. We infected GBM cell lines with a laboratory strain of CMV, and found that most of the staining was cytoplasmic, with some perinuclear localization of IE1. To test the potential for CMV infected GBM cells to be recognized by CMV pp65 and IE1 specific cytotoxic T lymphocytes (CTL), we used CMV infected GBM cell lines in cytotoxicity assays with human leukocyte antigen partially matched CMV CTL. Lysis of CMV infected GBM tumor cells was accentuated by pre-treating these cell lines with either the demethylating agent decitabine or interferon-γ, both of which were shown to increase MHC Class I and II expression on tumor cells in vitro. These studies confirm the presence of CMV pp65 or IE1 on approximately half of GBM, with the possibility that CMV positive tumor cells can be recognized by CMV pp65/IE1 specific T cells.

Efficacy and safety of ex vivo cultured adult human mesenchymal stem cells (Prochymal™) in pediatric patients with severe refractory acute graft-versus-host disease in a compassionate use study.

Preliminary studies using directed-donor ex vivo expanded human mesenchymal stem cells (hMSCs) have shown promise in the treatment of acute graft-versus-host disease (aGVHD). However, their production is cumbersome and standardization is difficult. We describe the first experience of using a premanufactured, universal donor, formulation of hMSCs (Prochymal) in children (n = 12; 10 boys; 9 Caucasian; age range: 0.4-15 years) with treatment-resistant grade III and IV aGVHD who received therapy on compassionate use basis between July 2005 and June 2007 at 5 transplant centers. All patients had stage III or IV gut (GI) symptoms and half had additional liver and/or skin involvement. Disease was refractory to steroids in all cases and additionally to a median of 3 other immunosuppressive therapies. The hMSCs (8 × 10(6)cells/kg/dose in 2 patients and 2 × 10(6)cells/kg/dose in the rest) were infused intravenously over 1 hour twice a week for 4 weeks. Partial and mixed responders received subsequent weekly therapy for 4 weeks. HLA or other matching was not needed. The hMSCs were started at a median of 98 days (range: 45-237) posttransplant. A total of 124 doses were administered, with a median of 8 doses (range: 2-21) per patient. Overall, 7 (58%) patients had complete response, 2 (17%) partial response, and 3 (25%) mixed response. Complete resolution of GI symptoms occurred in 9 (75%) patients. Two patients relapsed after initial response and showed partial response to retreatment. The cumulative incidence of survival at 100 days from the initiation of Prochymal therapy was 58%. Five of 12 patients (42%) were still alive after a median follow-up of 611 days (range: 427-1111) in surviving patients. No infusional or other identifiable acute toxicity was seen in any patient. Multiple infusions of hMSCs were well tolerated and appeared to be safe in children. Clinical responses, particularly in the GI system, were seen in the majority of children with severe refractory aGVHD. Given the favorable results observed in a patient population with an otherwise grave prognosis, we conclude that hMSCs hold potential for the treatment of aGVHD, and should be further studied in phase III trials in pediatric and adult patients.

Current therapeutic approaches in metastatic and recurrent ewing sarcoma.

Ewing sarcoma (ES) is the second most common type of primary bone malignancy in children and young adults. Survival rates for localized ES have improved to upwards of 70% with aggressive chemotherapy and local control. On the other hand, there has been little improvement in survival rates for patients with metastatic or recurrent ES. Herein we review the different current therapeutic approaches available, including the different upfront and salvage chemotherapy regimens, the role for stem cell transplantation, and potential use of immunotherapy.

Matched unrelated umbilical cord blood transplantation for a patient with chemotherapy resistant Wilms tumor.

We report a patient with chemotherapy refractory Wilms tumor who underwent an unrelated donor cord blood transplant for chemotherapy refractory disease. The preparative regimen consisted of busulfan, melphalan, and anti-thymocyte globulin, and was well tolerated. This patient did not experience significant toxicity related to the chemotherapy regimen and did not develop any graft versus host disease from his HLA (A, B, DR) 6/6 matched cord blood transplant. Follow-up CT scans 2 years post-transplant have shown no evidence of disease progression, with only a few pulmonary nodules remaining, which are unchanged in size from his pre-transplant CT scan. It is possible that high-dose chemotherapy and stem cell transplantation can be curative in patients with tumors that are non-responsive to conventional chemotherapy.

Infusion of cytomegalovirus specific cytotoxic T lymphocytes from a sero-negative donor can facilitate resolution of infection and immune reconstitution.

We report a stem cell transplant patient with a therapy-refractory cytomegalovirus (CMV) infection who received CMV-specific T cells from his sero-negative stem cell donor. This donor received the Towne strain CMV vaccine, and T cells were expanded using monocytes pulsed with pp65 overlapping peptides. CMV DNA decreased after the CTL infusion, and CMV-specific cytotoxicity increased. This strategy could be implemented in similar situations or with persistent viremia post-transplant.