Differential effects of epidermal growth factor receptor inhibitors on glioblastoma multiforme

OBJECTIVE: Glioblastoma Multiforme (GBM), one of the most malignant forms of primary brain tumors, is characterized by its highly heterogenous genetic composition, aggressive infiltration of surrounding tissue, and resistance to current treatments. Gene expression analysis has characterized GBM into four main types, with a significant portion belonging to the Classical subtype, typified by overexpression and/or mutation of the epidermal growth factor receptor (EGFR). Also common to this subtype of GBM is the loss of crucial tumor suppressor genes Ink4A/ARF and PTEN, which contribute to the invasive nature and unregulated proliferation that underlie the GBM pathology. The high rate of tumor recurrence post treatment with surgical resection, chemotherapy, and radiation has driven the pursuit of more effective molecularly targeted therapies. This study was undertaken to determine the effects of two types of small molecule tyrosine kinase inhibitors on cells overexpressing wild-type EGFR in the context of their respective complements of tumor suppressor genes. METHODS: Several cell lines were established from mouse models of EGFR wild-type (EGFRWT) driven gliomagenesis and treated with 10 μM of type I tyrosine kinase inhibitors Gefitinib (Iressa®, Astra Zeneca), CI-1033 (Canertinib, Pfizer), or Dimethyl Sulfoxide vehicle. Cells were exposed to each drug treatment as part of a time course ranging from 0 to 24 hours and then evaluated by trypan blue exclusion and Western blot analysis for cell viability and molecular and biochemical effects respectively. RESULTS: Evaluation of cell viability indicated that CI-1033 caused a greater increase in cell death than gefitinib when compared to control treated cells regardless of the tumor suppressors lost. Gefitinib was found to cause cell death only in cells expressing the PTEN tumor suppressor whereas CI-1033 showed similar levels of cell death for cells deficient in Ink4A/ARF or both Ink4A/ARF and PTEN tumor suppressors. Western blot analysis revealed that CI-1033 more effectively inhibited EGFR compared to gefitinib. Treatment with both gefitinib and CI-1033 effectively blocked phosphorylation of EGFR, but this effect was less pronounced with gefitinib treatment. Further analysis of downstream signaling molecules showed a greater presence of cleaved caspase 3, a hallmark of apoptosis, in gefitinib treated cells expressing PTEN than in those cells treated with CI-1033. Cells deficient in both Ink4A/ARF and PTEN did not demonstrate any induction of cleaved caspase 3 following either treatment. CONCLUSIONS: Based on the significant differences in cell viability between treatments, CI-1033 is an overall more effective inhibitor of EGFRWT expressing cells lacking PTEN, while gefitinib and CI-1033 were found to be similarly effective in cells expressing PTEN. The results of western blot analysis indicate that total and irreversible EGFR inhibition may be necessary to induce cell death in a manner that effectively terminates downstream cell signaling. It is likely that CI-1033, unlike gefitinib, induces apoptosis in a caspase-independent manner, which may be one of the many differences in downstream effects produced by these two drugs. Further research is necessary to determine the extent to which each inhibitor shuts down proliferative cell signaling pathways such as PI3K-AKT and MEK-ERK signaling pathways downstream of EGFR. Overall, these data indicate that genotype plays an important role in the determination of therapeutic response and may aid in the evaluation of clinical prognoses

Autologous lymphokine-activated killer cell therapy of lymphoproliferative disorders arising in organ transplant recipients

Cell lymphomas which often contain the Epstein-Barr virus (EBV) make up the preponderance of tumors that collectively have been termed posttransplant lymphoproliferative disorders (PTLD). The unusual susceptibility of PTLD to immune surveillance was first demonstrated in organ allograft recipients following reduction (or discontinuance) of immunosuppression 1 with its attendant risk of precipitating allograft rejection. Restoration of tumor surveillance has recently been accomplished in bone marrow transplant recipients with PTLD by infusing naive cytotoxic T lymphocytes (CTL) obtained from the original donors into the tumor-bearing recipients. In these cases the tumors are invariably of donor origin, and HLA-restricted effector cells directed against viral targets are thought to be the main mediators of tumor regression. 2,3 The unavailability of naive pretransplant recipient leukocytes has precluded direct application of this technology to the PTLDs which develop after organ transplantation and which are almost always of recipient origin. 4 However, we report herein an approach whereby the anti-PTLD activity of the recipient's own cells can be intensified in vitro. Reinfusion of these cells has been associated with clinical tumor regression in several cases

Disseminated Intravascular Coagulation with Purpura Fulminans Presentation of Acute Promyelocytic Leukemia

A 47-year-old male presented to the emergency department with 12 hours of nausea, vomiting, abdominal pain, and a widespread skin eruption with mottled, irregular, purpuric lesions with subsequent rapid decompensation. Laboratory analysis revealed thrombocytopenia, bandemia, elevated metamyelocytes, abnormal coagulation panel, decreased fibrinogen, elevated fibrin split products, renal dysfunction, bacterial rods, dohle bodies, and toxic granulation. Acute promyelocytic leukemia (APML) was confirmed via bone marrow biopsy, flow cytometry, and fluorescence in situ hybridization analysis. Disseminated intravascular coagulation (DIC) may be the initial presentation of APML. When treated promptly, APML can achieve high remission rates; however, conditions such as DIC continue to increase mortality requiring early recognition to improve survival rates

Reprint of: B Cells in Chronic Graft-versus-Host Disease

AbstractChronic graft-versus-host disease (cGVHD) continues to be a common complication of allogeneic hematopoietic stem cell transplantation. Unlike acute graft-versus-host disease, which is mediated almost entirely by donor T cells, the immune pathology of cGVHD is more complex and donor B cells have also been found to play an important role. Recent studies from several laboratories have enhanced our understanding of how donor B cells contribute to this clinical syndrome and this has led to new therapeutic opportunities. Here, Dr Sarantopoulos reviews some of the important mechanisms responsible for persistent B cell activation and loss of B cell tolerance in patients with cGVHD. Dr Blazar describes recent studies in preclinical models that have identified novel B cell–directed agents that may be effective for prevention or treatment of cGVHD. Some B cell–directed therapies have already been tested in patients with cGVHD and Dr Cutler reviews the results of these studies documenting the potential efficacy of this approach. Supported by mechanistic studies in patients and preclinical models, new B cell–directed therapies for cGVHD will now be evaluated in clinical trials

Rorc restrains the potency of ST2+ regulatory T cells in ameliorating intestinal graft-versus-host disease

Soluble stimulation-2 (ST2) is increased during graft-versus-host disease (GVHD), while Tregs that express ST2 prevent GVHD through unknown mechanisms. Transplantation of Foxp3- T cells and Tregs that were collected and sorted from different Foxp3 reporter mice indicated that in mice that developed GVHD, ST2+ Tregs were thymus derived and predominantly localized to the intestine. ST2-/- Treg transplantation was associated with reduced total intestinal Treg frequency and activation. ST2-/- versus WT intestinal Treg transcriptomes showed decreased Treg functional markers and, reciprocally, increased Rorc expression. Rorc-/- T cells transplantation enhanced the frequency and function of intestinal ST2+ Tregs and reduced GVHD through decreased gut-infiltrating soluble ST2-producing type 1 and increased IL-4/IL-10-producing type 2 T cells. Cotransfer of ST2+ Tregs sorted from Rorc-/- mice with WT CD25-depleted T cells decreased GVHD severity and mortality, increased intestinal ST2+KLRG1+ Tregs, and decreased type 1 T cells after transplantation, indicating an intrinsic mechanism. Ex vivo IL-33-stimulated Tregs (TregIL-33) expressed higher amphiregulin and displayed better immunosuppression, and adoptive transfer prevented GVHD better than control Tregs or TregIL-33 cultured with IL-23/IL-17. Amphiregulin blockade by neutralizing antibody in vivo abolished the protective effect of TregIL-33. Our data show that inverse expression of ST2 and RORγt in intestinal Tregs determines GVHD and that TregIL-33 has potential as a cellular therapy avenue for preventing GVHD

Wrist and Hand Injuries in the Athlete

Athletes subject themselves to considerable disability. No matter the sport, the hand and upper extremity are among the most commonly injured sites. Frequently, the most debilitating complications of these conditions are the result of misdiagnosis or delayed diagnosis. Unfortunately, many patients with these delayed and misdiagnosis injuries need to be treated with surgical procedures. To compound the dilemma in treating hand and wrist injuries in this population, one needs to appreciate the athletic personality and the mentality that wishes to dismiss hand injuries as minor. It is essential to educate athletes by clearly communicating the risks and complications inherent to these injuries and the applicable therapy. The purpose of this chapter is to discuss the anatomy, mechanism of injury, diagnosis, and treatment of common athletic injuries as it relates to the fingers, wrist, and hand. Appreciation of the anatomy and mechanism of injury is extremely helpful in diagnosing the pathology. Early and accurate diagnosis minimizes the delayed problems of pain and dysfunction in hand injuries. As with any other sport injury the primary goal is to return the athlete to full participation as soon as possible without risking further injury or permanent disability. Common sense management of the injury is presented in regards to acute treatment, protective splinting and surgical intervention. Specific rehabilitation exercises are outlined at the end of the chapter to avoid repetition, since many of the same exercises are used in the various rehabilitation regimens described