We evaluated neoadjuvant ipilimumab in patients with surgically operable regionally advanced melanoma in order to define markers of activity in the blood and tumor as assessed at baseline (before ...ipilimumab) and early on-treatment. Patients were treated with ipilimumab (10 mg/kg intravenously every 3 weeks ×2 doses) bracketing surgery. Tumor and blood biospecimens were obtained at baseline and at surgery. Flow cytometry and immunohistochemistry for select biomarkers were performed. Thirty five patients were enrolled; IIIB (3; N2b), IIIC (32; N2c, N3), IV (2). Worst toxicities included Grade 3 diarrhea/colitis (5; 14%), hepatitis (2; 6%), rash (1; 3%), elevated lipase (3; 9%). Median follow up was 18 months: among 33 evaluable patients, median progression free survival (PFS) was 11 months, 95% CI (6.2-19.2). There was a significant decrease in circulating myeloid derived suppressor cells (MDSC). Greater decrease in circulating monocyte gate MDSC Lin1-/HLA-DR-/CD33⁺/CD11b⁺ was associated with improved PFS (p = 0.03). There was a significant increase in circulating regulatory T cells (Treg; CD4⁺CD25hi⁺Foxp3⁺) that, unexpectedly, was associated with improved PFS (HR = 0.57; p = 0.034). Baseline evidence of fully activated type I CD4⁺ and CD8⁺ antigen-specific T cell immunity against cancer-testis (NY-ESO-1) and melanocytic lineage (MART-1, gp100) antigens was detected and was significantly potentiated after ipilimumab. In tumor, there was a significant increase in CD8⁺ T cells after ipilimumab (p = 0.02). Ipilimumab induced increased tumor infiltration by fully activated (CD69⁺) CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T cells with evidence of induction/potentiation of memory T cells (CD45RO⁺). The change in Treg observed within the tumor showed an inverse relationship with clinical benefit and greater decrease in tumor MDSC subset Lin1-/HLA-DR-/CD33⁺/CD11b⁺ was associated with improved PFS at one year. Neoadjuvant evaluation revealed a significant immunomodulating role for ipilimumab on Treg, MDSC and effector T cells in the circulation and tumor microenvironment that warrants further pursuit in the quest for optimizing melanoma immunotherapy.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Purpose: Interferon (IFN)-α2b is the only Food and Drug Administration–approved treatment for operable high-risk melanoma that has
been shown to significantly and durably prolong relapse-free ...survival (RFS) of patients with stage IIB-III melanoma. Development
of reliable serum assays may contribute to the development of methods for earlier detection of melanoma and the selection
of patients who may be most susceptible to current available interventions with IFNα.
Experimental Design: A powerful high-throughput xMAP multiplex immunobead assay technology (Luminex Corp., Austin, TX) was used to simultaneously
test 29 cytokines, chemokines, angiogenic as well as growth factors, and soluble receptors in the sera of 179 patients with
high-risk melanoma and 378 healthy individuals.
Results: Serum concentrations of interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-12p40, IL-13, granulocyte colony-stimulating factor, monocyte
chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, IFNα, tumor necrosis factor (TNF)-α,
epidermal growth factor, vascular endothelial growth factor (VEGF), and TNF receptor II were found to be significantly higher
in patients with resected high-risk melanoma compared with healthy controls. Bayesian Network algorithm classification of
the data offered 90% sensitivity at 98% specificity with 96.5% of melanoma patients distinguished from healthy individuals.
IFN-α2b therapy resulted in a significant decrease of serum levels of immunosuppressive and tumor angiogenic/growth stimulatory
factors (VEGF, epidermal growth factor, and hepatocyte growth factor) and increased levels of antiangiogenic IFN-γ inducible
protein 10 (IP-10) and IFN-α. Pretreatment levels of proinflammatory cytokines IL-1β, IL-1α, IL-6, TNF-α, and chemokines MIP-1α
and MIP-1β were found to be significantly higher in the serum of patients with longer RFS values of 1 to 5 and >5 years when
compared with patients with shorter RFS of <1 year.
Conclusion: These data show that multiplexed analysis of serum biomarkers is useful for the evaluation of prognostic markers of clinical
outcome and potential predictive markers of response to IFN-α2b in patients with high-risk operable melanoma.
Adjuvant high-dose interferon-alfa-2b (HDI) improves disease-free and overall survival in patients with high-risk melanoma. However, its mechanism of action is largely unknown. Therefore, HDI was ...investigated in the neoadjuvant setting to assess clinical and pathologic responses after 4 weeks of HDI and to perform immunohistochemical evaluation of immune cell subsets and melanoma-associated antigens.
Patients with palpable regional lymph node metastases from melanoma (American Joint Committee on Cancer stage IIIB-C) underwent surgical biopsy at study entry and then received standard intravenous HDI (20 million units/m2, 5 days per week) for 4 weeks followed by complete lymphadenectomy and standard maintenance subcutaneous HDI (10 million units/m2 3 times per week) for 48 weeks. Biopsy samples were obtained before and after intravenous HDI and subjected to immunohistochemical analysis as well as routine pathologic study.
Twenty patients were enrolled, and biopsy samples were informative for 17. Eleven patients (55%) demonstrated objective clinical response, and 3 patients (15%) had complete pathologic response. At a median follow-up of 18.5 months (range, 7 months to 50 months) 10 patients had no evidence of recurrent disease. Clinical responders had significantly greater increases in endotumoral CD11c+ and CD3+ cells and significantly greater decreases in endotumoral CD83+ cells compared with nonresponders. No changes in the expression of melanoma-associated lineage antigens, tumor cell proliferation, angiogenesis, or apoptosis were evident.
Neoadjuvant HDI is highly effective for the treatment of palpable stage IIIB-C melanoma, and the findings of this study implicate an indirect immunomodulatory mechanism rather than a direct antitumor mechanism.
PURPOSE: Interferon (IFN)- alpha 2b is the only Food and Drug Administration-approved treatment for operable high-risk melanoma that has been shown to significantly and durably prolong relapse-free ...survival (RFS) of patients with stage IIB-III melanoma. Development of reliable serum assays may contribute to the development of methods for earlier detection of melanoma and the selection of patients who may be most susceptible to current available interventions with IFN alpha . Experimental Design: A powerful high-throughput xMAP multiplex immunobead assay technology (Luminex Corp., Austin, TX) was used to simultaneously test 29 cytokines, chemokines, angiogenic as well as growth factors, and soluble receptors in the sera of 179 patients with high-risk melanoma and 378 healthy individuals. RESULTS: Serum concentrations of interleukin (IL)-1 alpha , IL-1{szligbeta}, IL-6, IL-8, IL-12p40, IL-13, granulocyte colony-stimulating factor, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1 alpha , MIP-1{szligbeta}, IFN alpha , tumor necrosis factor (TNF)- alpha , epidermal growth factor, vascular endothelial growth factor (VEGF), and TNF receptor II were found to be significantly higher in patients with resected high-risk melanoma compared with healthy controls. Bayesian Network algorithm classification of the data offered 90% sensitivity at 98% specificity with 96.5% of melanoma patients distinguished from healthy individuals. IFN- alpha 2b therapy resulted in a significant decrease of serum levels of immunosuppressive and tumor angiogenic/growth stimulatory factors (VEGF, epidermal growth factor, and hepatocyte growth factor) and increased levels of antiangiogenic IFN- gamma inducible protein 10 (IP-10) and IFN- alpha . Pretreatment levels of proinflammatory cytokines IL-1{szligbeta}, IL-1 alpha , IL-6, TNF- alpha , and chemokines MIP-1 alpha and MIP-1{szligbeta} were found to be significantly higher in the serum of patients with longer RFS values of 1 to 5 and >5 years when compared with patients with shorter RFS of <1 year. CONCLUSION: These data show that multiplexed analysis of serum biomarkers is useful for the evaluation of prognostic markers of clinical outcome and potential predictive markers of response to IFN- alpha 2b in patients with high-risk operable melanoma.
The prognosis for malignant gliomas remains dismal. We addressed the safety, feasibility and preliminary clinical activity of the vaccinations using autologous glioma cells and interleukin (IL)-4 ...gene transfected fibroblasts.
In University of Pittsburgh Cancer Institute (UPCI) protocol 95-033, adult participants with recurrent glioblastoma multiforme (GBM) or anaplastic astrocytoma (AA) received gross total resection (GTR) of the recurrent tumors, followed by two vaccinations with autologous fibroblasts retrovirally transfected with TFG-IL4-Neo-TK vector admixed with irradiated autologous glioma cells. In UPCI 99-111, adult participants with newly diagnosed GBM or AA, following GTR and radiation therapy, received two intradermal vaccinations with the TFG-IL4-Neo-TK-transfected fibroblasts admixed with type-1 dendritic cells (DC) loaded with autologous tumor lysate. The participants were evaluated for occurrence of adverse events, immune response, and clinical response by radiological imaging.
In UPCI 95-033, only 2 of 6 participants received the vaccinations. Four other participants were withdrawn from the trial because of tumor progression prior to production of the cellular vaccine. However, both participants who received two vaccinations demonstrated encouraging immunological and clinical responses. Biopsies from the local vaccine sites from one participant displayed IL-4 dose-dependent infiltration of CD4+ as well as CD8+ T cells. Interferon (IFN)-gamma Enzyme-Linked Immuno-SPOT (ELISPOT) assay in another human leukocyte antigen (HLA)-A2+ participant demonstrated systemic T-cell responses against an HLA-A2-restricted glioma-associated antigen (GAA) epitope EphA2883-891. Moreover, both participants demonstrated clinical and radiological improvement with no evidence of allergic encephalitis, although both participants eventually succumbed with the tumor recurrence. In 99-111, 5 of 6 enrolled participants received scheduled vaccinations with no incidence of major adverse events. Monocyte-derived DCs produced high levels of IL-12 p70. Treatment was well tolerated; however, we were unable to observe detectable IFN-gamma post-vaccine responses or prolonged progression-free survival in these participants.
Feasibility challenges inherent in the generation of a patient-specific gene transfection-based vaccine strongly suggests the need for more practical formulations that would allow for the timely administration of vaccines. Nevertheless, successful generation of type-1 DCs and preliminary safety in the current study provide a strong rationale for further efforts to develop novel glioma vaccines.
Purpose: The Janus-activated kinase/signal transducers and activators of transcription (STAT) pathway of IFN signaling is important
to immunoregulation and tumor progression. STAT1 plays a prominent ...role in the effector immune response, whereas STAT3 is
implicated in tumor progression and down-regulation of the response to type I IFNs. The goal of this study was to understand
the effects of high-dose IFNα2b (HDI) in relation to the balance of pSTAT1 and pSTAT3.
Experimental Design: We evaluated STAT1 and STAT3 jointly as mediators of IFNα effects in the setting of a prospective neoadjuvant trial of HDI,
in which tissue samples were obtained before and after 20 doses of HDI therapy. Double immunohistochemistry for pSTAT1 and
pSTAT3 was done on paired fixed (9 patients) or frozen (12 patients) biopsies.
Results: HDI was found to up-regulate pSTAT1, whereas it down-regulates pSTAT3 and total STAT3 levels in both tumor cells and lymphocytes.
Higher pSTAT1/pSTAT3 ratios in tumor cells pretreatment were associated with longer overall survival ( P = 0.032). The pSTAT1/pSTAT3 ratios were augmented by HDI both in melanoma cells ( P = 0.005) and in lymphocytes ( P = 0.022). Of the immunologic mediators and markers tested, TAP2 was augmented by HDI (but not TAP1 and MHC class I/II).
Conclusion: IFNα2b significantly modulates the balance of STAT1/STAT3 in tumor cells and host lymphocytes, leading to up-regulation of
TAP2 and augmented host antitumor response. The pSTAT1/pSTAT3 ratio in tumor cells at baseline may serve as a useful predictor
of clinical outcome in cutaneous melanoma; the modulation of this ratio may serve as a predictor of therapeutic effect.
1 Department of Surgery and
2 Center for Biologic Imaging,
University of Pittsburgh, Pittsburgh, Pennsylvania 15261
A role for nitric oxide (NO) in wound healing
has been proposed; however, the ...absolute requirement of NO for wound
healing in vivo and the contribution of endothelial NO synthase (eNOS) have not been determined. Experiments were carried out using eNOS gene
knockout (KO) mice to determine the requirement for eNOS on wound
closure and wound strength. Excisional wound closure was
significantly delayed in the eNOS KO mice (29.4 ± 2.2 days) compared with wild-type (WT) controls (20.2 ± 0.4 days). At 10 days, incisional wound tensile strength demonstrated a 38% reduction in the eNOS KO mice. Because effective wound repair requires growth factor-stimulated angiogenesis, in vitro and in vivo angiogenesis assays were performed in the mice to assess the effects of eNOS deficiency on angiogenesis. Endothelial cell sprouting assays confirmed
in vitro that eNOS is required for proper endothelial cell migration,
proliferation, and differentiation. Aortic segments harvested from eNOS
KO mice cultured with Matrigel demonstrated a significant reduction in
endothelial cell sprouting and
3 Hthymidine
incorporation compared with WT mice at 5 days. Capillary ingrowth into
subcutaneously implanted Matrigel plugs was significantly reduced in
eNOS KO mice (2.67 ± 0.33 vessels/plug) compared with WT mice
(10.17 ± 0.79 vessels/plug). These results clearly show that eNOS
plays a significant role in facilitating wound repair and growth
factor-stimulated angiogenesis.
nitric oxide; nitric oxide synthase; endothelial nitric oxide
synthase; wound repair; endothelial nitric oxide synthase knockout; endothelial cell
PURPOSE: The Janus-activated kinase/signal transducers and activators of transcription (STAT) pathway of IFN signaling is important to immunoregulation and tumor progression. STAT1 plays a prominent ...role in the effector immune response, whereas STAT3 is implicated in tumor progression and down-regulation of the response to type I IFNs. The goal of this study was to understand the effects of high-dose IFN alpha 2b (HDI) in relation to the balance of pSTAT1 and pSTAT3. Experimental Design: We evaluated STAT1 and STAT3 jointly as mediators of IFN alpha effects in the setting of a prospective neoadjuvant trial of HDI, in which tissue samples were obtained before and after 20 doses of HDI therapy. Double immunohistochemistry for pSTAT1 and pSTAT3 was done on paired fixed (9 patients) or frozen (12 patients) biopsies. RESULTS: HDI was found to up-regulate pSTAT1, whereas it down-regulates pSTAT3 and total STAT3 levels in both tumor cells and lymphocytes. Higher pSTAT1/pSTAT3 ratios in tumor cells pretreatment were associated with longer overall survival (P = 0.032). The pSTAT1/pSTAT3 ratios were augmented by HDI both in melanoma cells (P = 0.005) and in lymphocytes (P = 0.022). Of the immunologic mediators and markers tested, TAP2 was augmented by HDI (but not TAP1 and MHC class I/II). CONCLUSION: IFN alpha 2b significantly modulates the balance of STAT1/STAT3 in tumor cells and host lymphocytes, leading to up-regulation of TAP2 and augmented host antitumor response. The pSTAT1/pSTAT3 ratio in tumor cells at baseline may serve as a useful predictor of clinical outcome in cutaneous melanoma; the modulation of this ratio may serve as a predictor of therapeutic effect.
Reconstruction of massive abdominal wall defects has long been a vexing clinical problem. A landmark development for the autogenous tissue reconstruction of these difficult wounds was the ...introduction of "components of anatomic separation" technique by Ramirez et al. This method uses bilateral, innervated, bipedicle, rectus abdominis-transversus abdominis-internal oblique muscle flap complexes transposed medially to reconstruct the central abdominal wall. Enamored with this concept, this institution sought to define the limitations and complications and to quantify functional outcome with the use of this technique. During a 4-year period (July of 1991 to 1995), 22 patients underwent reconstruction of massive midline abdominal wounds. The defects varied in size from 6 to 14 cm in width and from 10 to 24 cm in height. Causes included removal of infected synthetic mesh material (n = 7), recurrent hernia (n = 4), removal of split-thickness skin graft and dense abdominal wall cicatrix (n = 4), parastomal hernia (n = 2), primary incisional hernia (n = 2), trauma/enteric sepsis (n = 2), and tumor resection (abdominal wall desmoid tumor involving the right rectus abdominis muscle) (n = 1). Twenty patients were treated with mobilization of both rectus abdominis muscles, and in two patients one muscle complex was used. The plane of "separation" was the interface between the external and internal oblique muscles. A quantitative dynamic assessment of the abdominal wall was performed in two patients by using a Cybex TEF machine, with analysis of truncal flexion strength being undertaken preoperatively and at 6 months after surgery. Patients achieved wound healing in all cases with one operation. Minor complications included superficial infection in two patients and a wound seroma in one. One patient developed a recurrent incisional hernia 8 months postoperatively. There was one postoperative death caused by multisystem organ failure. One patient required the addition of synthetic mesh to achieve abdominal closure. This case involved a thin patient whose defect exceeded 16 cm in width. There has been no clinically apparent muscle weakness in the abdomen over that present preoperatively. Analysis of preoperative and postoperative truncal force generation revealed a 40 percent increase in strength in the two patients tested on a Cybex machine. Reoperation was possible through the reconstructed abdominal wall in two patients without untoward sequela. This operation is an effective method for autogenous reconstruction of massive midline abdominal wall defects. It can be used either as a primary mode of defect closure or to treat the complications of trauma, surgery, or various diseases.
Surgical meshes have become the standard procedure for a variety of surgical applications with 20 million meshes being implanted each year. The popularity of mesh usage among surgeons is backed by ...the multiple studies that support its functionality as a tool for improving surgical outcomes. However, their use has also been associated with infectious surgical complications and many surgeons have turned to biologic meshes. While there have been several studies investigating synthetic meshes, there is limited data comparing synthetic and biologic meshes in vitro in an infection model. This study evaluates the in vitro susceptibility of both synthetic and biologic meshes to single-species methicillin-resistant Staphylococcus aureus (MRSA) biofilms. This research compares biofilm biomass, average thickness, and coverage between the three meshes through florescent in situ hybridization (FISH), confocal scanning microscopy (CSLM), and image analysis. We also report the varying levels of planktonic and attached bacteria through sonication and cfu counts. While the data illustrates increased biofilm formation on biologic mesh in vitro, the study must further be investigated in vivo to confirm the study observations.
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