Expression Of HLA Antigens Research Articles

Cytokines in enucleated eyes

Abstract Purpose One of the prognostically bad parameters in uveal melanoma is the presence of an inflammatory phenotype, characterized by an increased expression of HLA antigens and an immunologic infiltrate. We wondered whether the presence of specific chemokines and cytokines in the aqueous humor (AqH) from uveal melanoma‐containing eyes is associated with this inflammatory phenotype, with the presence of macrophages, and/or with survival. Methods Directly following enucleation, AqH was obtained from 37 eyes containing uveal melanoma. Samples were stored at ‐80 °C till use. Using a multiplex bead array, 15 different cytokines were measured. Determination of intratumoral macrophages was performed by immunohistochemistry and immunofluorescence. The presence of specific cytokines was compared to histopathological, genetic and clinical tumor characteristics, as well as patient survival. Results Several cytokines showed a significantly higher expression in the AqH from uveal melanoma‐containing eyes compared to the AqH from eyes undergoing cataract surgery. Only MCP‐3 was associated with the presence of macrophages and the tumor promoting M2‐type macrophage in uveal melanoma patients. Hardly any correlations were found between cytokine levels and known prognostic factors for uveal melanoma. Also, cytokine levels were not of predictive value for survival. Conclusion Although increased levels of inflammation‐related cytokines are present in the AqH of uveal melanoma‐containing eyes, hardly any associations with the presence of macrophages and their subtypes, with clinical and histopathological parameters, and prognosis were found.

HLA haplotype in a patient with systemic lupus erythematosus triggered by hepatitis B vaccine

Find an association between hepatitis B vaccine-related systemic lupus erythematosus and HLA. A 27-year-old woman who developed a lupus nephritis after the administration of hepatitis B vaccine. We studied HLA antigen expression on lymphocytes and genomic haplotype. Class I-II HLA antigen typing was performed by the microlymphocytotoxicity test with the standard NIH method, and Class I-II HLA allele typing by polymerase chain reaction, using single-strand oligonucleotide dot-blot kits. The serological haplotype was HLA A24/25, B18 (Bw6)/-, C-/-, DQ7/-, DR11(5)/52. The genomic haplotype was A*2403/2504, B*1825/1825, C*1207/ 1207, DRB1*1102/1132, DRB3*0202/0202, DQA1*0505/0505, DQB1*0301/0301. Then we sought for analogies with haplotypes known to be related to other systemic AID. Since we have found HLA alleles typical both of systemic lupus erythematosus and Sjogren's syndrome, the persistence of ENA-SSA positivity was highly suspicious for a possible overlap syndrome. Hepatitis B vaccine can potentially trigger both the onset or the exacerbations of several autoimmune disorders, including systemic lupus erythematosus, by reduced immune complex clearance or molecular mimicry. This study represents the first report on the association between hepatitis B vaccine related systemic lupus erythematosus and HLA. Probably autoimmune reactions triggered by vaccines occur only in predisposed subjects, in which antigen presentation influenced by HLA haplotypes leads to the autoimmune cascade. More studies are needed to corroborate our hypothesis. They could disclose new pathways in the field of prevention.

Role of Major Histocompatibility Complex and Ethnicity in Acute Renal Graft Rejection

Background The major histocompatibility complex (MHC) plays a main role in antigen presentation. Class I, II, and III genes form defined “blocks” of conserved DNA sequences (conserved extended haplotypes) that are useful to follow the ancestry of a population. Each variant encodes a specific peptide that determines a particular individual's immune response. In addition, differential expression of HLA antigens in certain physiological and pathological conditions may participate in the pathogenesis of allograft rejection versus tolerance. Objectives The aim of this study was to determine whether the specific HLA ancestry was associated with acute renal graft rejection among the Mexican mestizo population. Materials and Methods We studied 544 Mexican mestizo renal donors and their respective recipients for their serologically determined HLA and based on antigens haplotype assignments. The acute rejection group was compared with the nonrejection group among donors and recipients, correspondingly. Results Frequent Mexican alleles were observed in this study. Moreover, HLA-B*61/-DR*04, HLA-A*35/-DR*06 (Amerindian ancestry), HLA-A*68/-DR*01, HLA-A*28/-B*65/-DR*01 (African ancestry), and HLA-A*33/-B*65 (Caucasian ancestry) in donors were associated with acute renal graft rejection episodes. Conclusion Knowing the ancestry of a donor's HLA molecules may help to individualize immunosuppressive therapy for postransplant surveillance, because they may be more membrane-exposed in parenchymal cells, making them more susceptible of being recognized by the recipient's immune system.

Inflammatory Cytokines in Eyes with Uveal Melanoma and Relation with Macrophage Infiltration

The presence of an inflammatory phenotype, characterized by an increased expression of HLA antigens and an immunologic infiltrate, carries a bad prognosis in uveal melanoma. This study was conducted to determine whether the aqueous humor (AqH) from eyes with uveal melanoma contains inflammatory cytokines and whether their presence is associated with inflammation. Immediately after enucleation, AqH was obtained from 37 eyes containing uveal melanoma. Samples were stored at -80°C until use. Fifteen different cytokines were measured with a multiplex bead array. Intratumoral macrophages were analyzed by immunohistochemistry and immunofluorescence staining. The presence of specific cytokines was compared with histopathologic, genetic, and clinical tumor characteristics, as well as patient survival. Several cytokines showed significantly higher expression in the AqH of uveal melanoma-containing eyes than in the AqH of eyes undergoing cataract surgery. MCP-3 was associated with the presence of CD68(+) macrophages. Correlations were found between some cytokine levels and a few known prognostic factors of uveal melanoma, but cytokine levels were not of predictive value for survival. Uveal melanoma-containing eyes often carry increased levels of inflammation-related cytokines in their AqH. However, the presence of most specific cytokines was not related to the presence of macrophages, clinical or histopathologic parameters, or prognosis.

Different HLA-DR expression in endometriotic and adenomyotic lesions: correlation with transvaginal ultrasonography findings

Human leukocyte antigen-DR (HLA-DR) has been implicated in eutopic and ectopic glandular epithelial cells in endometriosis. We investigated the expression of HLA-DR in endometriotic and adenomyotic tissues within the stromal and glandular cells. Moreover, we correlate the HLA-DR expression according the transvaginal ultrasonography findings. We studied operative and pathologic reports of 113 women who underwent laparoscopic or laparotomy treatment of endometrioma or adenomyosis. Tissues from 51 women with endometrioma and 62 women with adenomyosis were retrospectively evaluated. The distribution and intensity of the HLA-DR immunostaining was assessed using electron microscopy. Pathologic finding of the uterine junction zone and the size of endometrioma were evaluated with the laparoscopic results and the ultrasound findings. In adenomyosis tissues, the percentage of HLA-DR cells expression was significantly higher in stromal cells (83.9%) compared to glandular cells (25.8%), (p<0.001). The number of HLA-DR-positive endometriotic glandular cells was significantly higher than the total glandular adenomyotic cells (p<0.005). HLA-DR-positive cells was significantly different between stromal (p<0.016) and glandular cells (p<0.044) in each side of endometrioma. Finally, HLA-DR-positive percentage cells were significantly more frequent in the secretory phase than the proliferative in stromal and glandular cells in both groups. HLA-DR antigen expression in endometrium and adenomyotic tissues. However, HLA-DR expression is distributed preferentially in glandular epithelial cells in endometrioma and in the adenomyotic stroma. In both groups the HLA-DR expression was significantly higher in the secretory phase than the proliferative or glandular and stroma cells. Larger perspective studies are needed to establish the expression of HLA antigens in immune reactions which occur in adenomyosis and endometriosis.

Human Embryonic Stem Cells-Derived Hemangioblasts Express HLA Antigens and Genes Related to Hematopoeisis and Endopoiesis

The early hematopoietic/endothelial progenitor (hemangioblast) has been recently described; however, HLA antigen expression of this progenitor was not characterized as yet. We have isolated blast-like cells (BLC) from ES/EB-based cultures and characterized their HLA-expression and ability to form hematopoietic and endothelial colonies. We used the two-step differentiation technology to obtain the bipotential blast cells from human embryonic stem cells (hES). This involved short differentiation in embryoid body (EB) system followed by differentiation in semisolid culture medium (Methocult H4436), supplemented with mixture of cytokines (BMP-4, VEGF, SCF, Flt3l, and TPO). First, we have shown that the occurrence of hemangioblast (BL-CFC) during EB differentiation (day 0–6) is transient and peaks on day 3. On this day about 125±35 out of 2400 EB cells form blast colonies. The emergence of this event was associated with the expression of T (early mesoderm gene) and was suppressed by the development of endoderm layer in subsequent days in culture. Similarly, the highest HB occurrence was associated with dramatic increase in expression of early hematopoietic/endothelial genes: CD34, CD31 and KDR. The similar pattern of expression of Patch1 and Gli1 genes suggests association with hedgehog (Hh) pathway activation during this process. The BL-CFC colonies were composed of 30–50 blast cells on day 6 of culture and had homogenous morphology in Wright-Giemsa stain with big nucleus containing disorganized chromatin and narrow rim of cytoplasm filled with large-size granules. However, as shown by FACS staining, they were quite heterogenous and expressed markers of both immature hematopoietic and endothelial progenitors (CD31, CD34, VE-cadherin, Flt-1) and mature differentiated cells (CD45, CD33, CD146). At least some of them expressed fetal and embryonic globin genes as shown by RT-PCR. Moreover, they could be characterized by high expression of HLA class I molecules by immunofluorescence, when compared to hES and EB cells. The ES cells, EB-derived cells and blast-like cells (BLC) were stained with FITC-labeled anti-HLA-A2 antibody BB7.2 and cells were observed under fluorescent microscope. While undifferentiated ES cells were negative for HLA-A2, the EB cells showed slight expression and the blast-like cells were strongly positive for HLA-A2 expression. These blast-like cells (BLC) could be successfully differentiated to hematopoietic cells in standard CFU assay. In these conditions, blast cells formed CFU-M colonies (63.4±0.8%) containing macrophages, BFU-E (19.5±3.5%) containing nucleated red blood cells and CFU-EM colonies (17.1±2.7%) composed of macrophages and nucleated erythrocytes. The cells of CFU-EM and BFU-E expressed both epsilon- and gamma-globin genes, but not adult-type beta-globin. When in endothelial cell culture conditions, the blast cells differentiated to endothelial cells which had the ability to uptake Dil-Ac-LDL and form complicated vascular networks in Matrigel. Concluding:Hemangioblasts transiently exist in early embryonic development and can form single cell-derived colonies,differentiation of hemangioblasts can be tracked by the use of chosen molecular markersblast-like colonies (BL-CFC) consist of cells having properties of endothelial and hematopoietic precursors, however, the issue if they can maintain dual properties over time needs to be further exploredthese blasts-like cells can be potentially used in regenerative medicine, however, strong expression of HLA molecules may limit their clinical applications in the future.

HLA antigen changes in malignant cells: epigenetic mechanisms and biologic significance

Changes in classical and nonclassical HLA class I as well as HLA class II antigens have been identified in malignant lesions. These changes, which are described in this review are believed to play a major role in the clinical course of the disease since both HLA class I and class II antigens are critical to the interaction between tumor cells and components of both innate and adaptive immune system. Abnormalities in HLA antigen expression in malignant cells, which range in frequency from 0-90%, are caused by distinct mechanisms. They include defects in beta(2)-microglobulin (beta(2)m) synthesis, loss of the gene(s) encoding HLA antigen heavy chain(s), mutations, which inhibit HLA antigen heavy chain transcription or translation, defects in the regulatory mechanisms, which control HLA antigen expression and/or abnormalities in one or more of the antigen processing, machinery (APM) components. More recently, epigenetic events associated with tumor development and progression have been found to underlie changes in HLA antigen, APM component, costimulatory molecule and tumor antigen (TA) expression in malignant cells. The types of epigenetic modifications that may occur in normal and malignant cells as well as their role in changes in HLA antigen expression by malignant cells have been reviewed. The epigenetic events associated with alterations in HLA antigen expression may be clinically relevant as, in some cases, they have been shown to impair the recognition of tumor cells by components of the adaptive immune system. The functional relevance and potential clinical significance of these epigenetic alterations have been addressed. Finally, unlike genetic alterations, epigenetic modifications can, in some cases, be reversed with pharmacologic agents that induce DNA hypomethylation or inhibit histone deacetylation. Therefore, strategies to overcome epigenetic modifications underlying changes in HLA antigen expression in malignant cells have been discussed.

The effect of recombinant interferon-alpha on lymphocyte subpopulations and HLA-DR expression on liver tissue of HBV-positive individuals

Interferon-alpha (IFN-alpha) has been reported to be beneficial in the treatment of chronic active hepatitis occurring as a result of hepatitis B virus (HBV) infection. Treatment with IFN-alpha has been proposed as a means of reducing the high rate of allograft infection in clinical liver transplantation in patients transplanted for HBV-related chronic active hepatitis and cirrhosis who are positive for hepatitis B surface antigen (HBsAg). We obtained resected whole livers from two groups of patients who received liver transplants. Group A consisted of 11 patients who were HBsAg+ but were not treated with IFN-alpha, and group B consisted of 10 patients who were also HBsAg+ but received IFN-alpha therapy for 29.4 +/- 5.6 days prior to orthotopic liver transplantation. No differences between the two groups existed in terms of a variety of demographic and clinical characteristics. The liver tissue was stained with monoclonal antibodies to cell surface antigens unique to different mononuclear cell populations by the avidin-biotin-immunoperoxidase technique to determine the effect of IFN-alpha on the lymphocyte subsets as well as HLA antigen expression on liver-infiltrating mononuclear cells. The number of HLA-DR+ lymphocytes in the liver was significantly increased (P less than 0.005) within the portal areas in group B compared with that found in group A (84 +/- 14 versus 33 +/- 5 per one high-power field). Moreover, the intensity of the HLA-DR antigen expression on lymphocytes in the portal areas (P less than 0.02) and in the hepatic lobule (P less than 0.05) was greater in group B than in group A. The number of natural killer (NK) cells was increased in the portal areas (P less than 0.05) of group B compared with group A. These alterations in the lymphocyte and NK cell populations present in the liver in response to IFN-alpha therapy presumably reflect an IFN-alpha-induced enhancement of the immune response to virus-infected cells.

Expression of MHC antigens by intestinal epithelial cells. Effect of transforming growth factor-beta 2 (TGF-beta 2).

The effect of interferon-gamma (IFN-gamma) and TGF-beta 2 on expression of MHC antigens by the human intestinal epithelial cell line HT-29 was examined by immunohistochemistry and flow cytometry. Untreated HT-29 cells constitutively expressed HLA-ABC but little HLA-DR. Expression of both molecules was increased by IFN-gamma (100 U/ml, 24 h). TGF-beta 2 at concentrations > or = 0.5 ng/ml given before or simultaneously with IFN-gamma, inhibited the IFN-induced expression of HLA-DR. Small increases in HLA-ABC expression by IFN-gamma were further increased by pretreatment with TGF-beta 2, while a strong induction of HLA-ABC was inhibited by the TGF-beta 2 pretreatment. Our results suggest that the inhibitory action of the TGF-beta 2 on both HLA-ABC and HLA-DR correlates with the degree of induction following IFN-gamma treatment. Since TGF-beta 2 is present in milk and is produced by gut epithelial cells, one of its possible functions may be to regulate expression of HLA antigens in the neonatal intestine and/or diseased intestine.

Cytokine regulation of HLA on thyroid epithelial cells

The regulation of class I and class II HLA expression in human thyroid follicular cells was studied in vitro. Tumour necrosis factor-alpha (TNF-alpha) enhanced the expression of class I antigen on thyrocytes, but these cytokines had little effect on the expression of class II antigen. Interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) did not affect class I and class II antigen expression. The combination of interferon-gamma (IFN-gamma) with TNF-alpha or IL-1 beta enhanced the induction of class I and class II antigens, compared with the effect of IFN-gamma alone. Neither class I nor class II expression was induced by IL-6 alone or in combination with IFN-gamma. These findings suggest that TNF-alpha and IL-1 beta may have an important role in inappropriate expression of HLA antigens on thyrocytes in thyroid gland.

Composition of inflammatory infiltrate and its correlation with HBV/HCV antigen expression

To study the composition of liver inflammatory infiltrate in biopsy material from patients chronically infected with hepatotropic viruses and to evaluate the correlation of inflammatory infiltrate with hepatitis B virus (HBV) and hepatitis C virus (HCV) viral antigen expression in chronic B and C hepatitis. The phenotype of inflammatory cells was evaluated by the EnVision system, using a panel of monoclonal antibodies. HBV and HCV antigens were detected with the use of monoclonal anti-HBs, polyclonal anti-HBc and anti-HCV antibodies, respectively. The cellular composition of liver inflammatory infiltrate was similar in the patients with B and C hepatitis: approximately 50%-60% of cells were T helper lymphocytes. Approximately 25% were T cytotoxic lymphocytes; B lymphocytes comprised 15% of inflammatory infiltrate; other cells, including NK, totalled 10%. Expression of HLA antigens paralleled inflammatory activity. Portal lymphadenoplasia was found more often in hepatitis C (54.5%) than in hepatitis B (30.6%). Expression of HBcAg was found more often in chronic B hepatitis of moderate or severe activity. Overall inflammatory activity in HBV-infected cases did not correlate with the intensity of HBsAg expression in hepatocytes. Inflammatory infiltrates accompanied the focal expression of HCV antigens. A direct correlation between antigen expression and inflammatory reaction in situ was noted more often in hepatitis C than B. Irrespective of the etiology and activity of hepatitis, components of the inflammatory infiltrate in liver were similar. Overall inflammatory activity did not correlate with the expression of HBsAg and HCVAg; HBcAg expression, however, accompanied chronic hepatitis B of moderate and severe activity.

Expression of HLA Molecules on Peripheral Blood Lymphocytes: A Useful Monitoring Parameter in Cardiac Transplantation

During the rejection process of cardiac allografts, the expression of HLA antigens increases on various graft tissues, ie, the myocardium and the interstitial structures. However, in this type of transplant there is a paucity of knowledge about HLA expression on recipient cells, such as peripheral blood mononuclear cells. In the present study expression of HLA class I and class II antigens was monitored on peripheral blood lymphocytes prior to and during a 12-month follow-up, using flow cytometry. In our series, the frequency of acute rejection episodes was greater from the fourth to the ninth month after transplantation, coinciding with a reduction in cyclosporine blood levels. At the same time, expression of HLA class I and class II antigens significantly increased among recipients suffering from more severe acute rejection episodes compared with those showing acceptance of their grafts ( P < .01). In conclusion, acute rejection episodes in cardiac transplantation were associated with up-regulation of HLA molecules on recipient peripheral blood cells. Monitoring the expression of HLA molecules on peripheral blood lymphocytes may represent an easy, noninvasive practice to individualize immunosuppressive therapy.

Correlation of molecular HLA typing and outcome in high-risk melanoma patients receiving adjuvant interferon

8029 Background: Serological typing for both HLA class I and class II antigen expression, has previously shown association of specific HLA antigen expression with clinical response and survival in patients with metastatic melanoma treated with IL-2 (e.g. HLA-DQ1). Purpose: To evaluate the impact of HLA class I (low-resolution) and class II (high-resolution) expression, on the outcome of high-risk melanoma patients receiving adjuvant high-dose interferon. Methods: 181 stage IIB, IIC and III melanoma patients (88 female and 93 male), median age 52.1 years and 246 healthy controls were included in this study. DNA was used for the determination of HLA-A, HLA-B, HLA-Cw, HLA-DRB1 and HLA-DQB1 genotypes. Results: With a median follow-up of 37 months, 59 (group 1) patients have remained with no evidence of recurrence and 122 have recurred (group 2). Statistical significant differences between the two groups, were found in the following genotypes: HLA-A*02 (42% vs. 57.3%, p=0.08), HLA-A*33 (15.2% vs. 6.5%, p=0.05), HLA-B*51 (15.2% vs. 34.4%, p=0.01), HLA-B*57 (11.8% vs. 2.4%, p=0.02). Statistical significant differences between group 1 and healthy controls, were found in the following genotypes: HLA-A*33 (15.2% vs. 6.5%, p=0.05), HLA-B*51 (15.2% vs. 28.5%, p=0.05), HLA-B*57 (11.8% vs. 4.5%, p=0.05), HLA-Cw*03 (23.7% vs. 11%, p=0.01), HLA-Cw*06 (27.1% vs. 16.1%, p=0.06), HLA-DRB1*0701 (27.1% vs. 13.4%, p=0.01), HLA-DRB1*1601 (35.6% vs. 22.3%, p=0.01), HLA-DQB1*0202 (23.8% vs. 10.1%, p=0.09). Conclusions: Statistical significant differences were seen in HLA-A and HLA-B alleles between the patients with high-risk melanoma free of recurrence and those who recurred after treatment with adjuvant interferon. Additionally, differences were seen between healthy controls and melanoma patients free of recurrence. No significant financial relationships to disclose.

HLA-G Proteins in Cancer: Do They Provide Tumor Cells with an Escape Mechanism?

Convincing clinical evidence indicates that the limited success of T-cell-based immunotherapy of malignant diseases is caused, at least in part, by the ability of malignant cells to escape from immune recognition and destruction. Among the multiple escape mechanisms identified, a major role is played by changes in the expression and/or function of HLA antigens expressed by tumor cells, because they may markedly affect tumor cell-host's immune system interactions. In this article, we review the data about the aberrant expression of the nonclassical HLA class I antigen HLA-G by tumor cells. Furthermore, we discuss the possible reasons for the conflicting information in the literature about HLA-G antigen expression by malignant cells. Lastly, in light of the well-documented immunotolerant function of HLA-G, we discuss the potential role of these antigens in the escape of tumor cells from immune recognition and destruction and in the clinical course of malignant diseases.

The expression of HLA antigens on acute lymphoblastic leukemia cells and analysis the host immune function

目的 通过研究儿童急性淋巴细胞性白血病患者的白血病细胞及完全缓解后患者外周血人白细胞(HLA)Ⅰ类、Ⅱ类抗原表达情况,探讨儿童急性淋巴细胞性白血病发病机制及机体免疫状态.方法采用流式细胞术检测儿童急性淋巴细胞性白血病初发患者42例、完全缓解期患者36例、对照组34例患者HLA-B、HLA-DQ、HLA-DR抗原表达.结果 (1)初发患者组的白血病细胞表面HLA-B抗原表达明显下调,初发患者组与完全缓解患者组或对照组比较,差异有显著意义(P＜0.01).(2)初发患者组白血病细胞表面HLA-DQ、HLA-DR抗原表达明显升高,尤以HLA-DR升高更为明显,初发患者组与完全缓解组或对照组比较,差异有显著意义(P＜0.01).结论 (1)急性淋巴细胞白血病人白血病细胞表面HLA-B抗原表达下调,可能是白血病细胞逃脱机体免疫监视的主要机制之一.(2)急性淋巴细胞白血病人白血病细胞表面HLA-DQ和HLA-DR抗原表达异常升高,尤其HLA-DR升高更为明显;使白血病细胞处于去分化状态,不能刺激机体产生免疫应答,可能也是白血病细胞逃脱机体免疫监视机制之一。

Feto-maternal immune interaction at the placental level.

A special interaction is established during pregnancy between the maternal immune system and fetal cells to allow the survival and the normal growth of the fetus. Fetal cells expressing paternal alloantigens are not recognized as foreign by the mother because of an efficient anatomic barrier and a local immunosuppression determined by the interplay of locally produced cytokines, biologically active molecules and hormones. A special balance between TH1 and TH2 lymphocytes has also been observed at the feto-maternal barrier that contribute to control the immune response at this level. An important role is played by trophoblast cells that act as a physical barrier forming a continuous layer and exert immunomodulatory function. Trophoblast cells have also been shown to express regulators of the complement system and to downregulate the expression of HLA antigens. Dysfunction of these cells leads to morphological and functional alterations of the feto-maternal barrier as well as to hormonal and immune imbalance and may contribute to the development of pathologic conditions of pregnancy, such as recurrent spontaneous abortions. Efforts are still needed to better understand the physiology of the feto-maternal interaction and the pathogenetic mechanisms responsible for tissue damage in pathologic conditions of pregnancy.

Pure autologous tumor cells (TC) and dendritic cells (DC) with GM-CSF: Patient-specific vaccine for metastatic melanoma

7547 Background: DC loaded with TC is a promising approach for vaccine therapy. The novel aspect of the product used in this trial is the use of irradiated melanoma TC from autologous cell lines incubated with interferon-gamma (IFN) to enhance expression of HLA and tumor associated antigens. Methods: Pure cultures of melanoma TC are expanded to 108 cells, incubated with 103 U/ml IFN for 72 hrs, irradiated, then cryopreserved. DC are generated from peripheral blood by incubating mononuclear cells in AIM V medium with 1000 U/ml GM-CSF and 1000 U/ml IL-4 for one week. DC and TC are co-incubated overnight, then cryopreserved in aliquots of about 107 cells. For treatment, vial contents are suspended in 500 mg GM-CSF for each s.c. injection. After a baseline delayed-type hypersensitivity (DTH) test to an i.d. injection of 106 irradiated TC at week-0, patients receive s.c. vaccinations weekly × 3, then monthly × 5. The DTH test to autologous TC is repeated at week-4. Results: Cell lines have been established for 75% of patients and vaccines prepared from 60% of the lines. As of 12/01/03, 21 patients had been entered. 16 had received prior i.v. IL-2 and/or chemotherapy; 7 had measurable disease at entry. 138 vaccine injections have been administered; 16 patients have completed the entire series. At the median follow-up of 8 mos, progression-free survival is 51%. All 21 patients were still alive with the longest followed more than 2.5 years since starting the vaccine. The only common adverse event is mild erythema at the injection site. One patient had an anaphylactoid reaction following her 8th and final injection. No patients were anergic at baseline, but all were DTH-. 8/21 (38%) subsequently exhibited an immune response to autologous tumor; 5 became DTH+ at 4 weeks and 3 more at 6 mos. Conclusions: This patient-specific DC/autologous TC vaccine is feasible and well tolerated. The DTH conversion rate is encouraging, as are the progression-free and overall survival rates. No significant financial relationships to disclose.

Pure autologous tumor cells (TC) and dendritic cells (DC) with GM-CSF: Patient-specific vaccine for metastatic melanoma

7547 Background: DC loaded with TC is a promising approach for vaccine therapy. The novel aspect of the product used in this trial is the use of irradiated melanoma TC from autologous cell lines incubated with interferon-gamma (IFN) to enhance expression of HLA and tumor associated antigens. Methods: Pure cultures of melanoma TC are expanded to 108 cells, incubated with 103 U/ml IFN for 72 hrs, irradiated, then cryopreserved. DC are generated from peripheral blood by incubating mononuclear cells in AIM V medium with 1000 U/ml GM-CSF and 1000 U/ml IL-4 for one week. DC and TC are co-incubated overnight, then cryopreserved in aliquots of about 107 cells. For treatment, vial contents are suspended in 500 mg GM-CSF for each s.c. injection. After a baseline delayed-type hypersensitivity (DTH) test to an i.d. injection of 106 irradiated TC at week-0, patients receive s.c. vaccinations weekly × 3, then monthly × 5. The DTH test to autologous TC is repeated at week-4. Results: Cell lines have been established for 75% of patients and vaccines prepared from 60% of the lines. As of 12/01/03, 21 patients had been entered. 16 had received prior i.v. IL-2 and/or chemotherapy; 7 had measurable disease at entry. 138 vaccine injections have been administered; 16 patients have completed the entire series. At the median follow-up of 8 mos, progression-free survival is 51%. All 21 patients were still alive with the longest followed more than 2.5 years since starting the vaccine. The only common adverse event is mild erythema at the injection site. One patient had an anaphylactoid reaction following her 8th and final injection. No patients were anergic at baseline, but all were DTH-. 8/21 (38%) subsequently exhibited an immune response to autologous tumor; 5 became DTH+ at 4 weeks and 3 more at 6 mos. Conclusions: This patient-specific DC/autologous TC vaccine is feasible and well tolerated. The DTH conversion rate is encouraging, as are the progression-free and overall survival rates. No significant financial relationships to disclose.

Significant impact of HLA class I allele expression on outcome in melanoma patients treated with an allogeneic melanoma cell lysate vaccine. Final analysis of SWOG-9035

7501 Background: SWOG-9035 compared 2 years of an allogeneic melanoma lysate (Melacine™) to observation in patients (pts) with stage II melanoma 1.5–4.0 mm thick. We also analyzed the effects of expression of certain HLA antigens (≥2 of HLA-A2, A28, B44, B45, C3 - called M5 - or either HLA-A2 and or C3). Now we report the mature overall survival (OS) analysis as planned. Methods: Primary analyses were conducted on the eligible pt population (intent to treat, ITT) using a Cox model adjusted for stratification and recognized prognostic factors. HLA analyses were \(\underline{\mathrm{prospective}}\), involved all typed pts and were adjusted for stratification and prognostic factors. Results: In total, 689 pts were entered over 4.5 years (598 eligible). Median follow-up time is 7.8 years (maximum 10.9 years). Ninety-one patients are ineligible, mostly (n=75) by pathology (tumor too thin or too thick). For the ITT analysis, the 5-year OS for the observation arm is 77% and for the vaccine arm is 81% (p=.98). For all randomized pts, the 5-year OS for the observation arm is 76% and for the vaccine arm is 82% (p=.49). We HLA typed 553 of the total 689 pts enrolled (80% of the total). There was a significant interaction of M5 status (≥2 vs 0–1) and treatment (p=.02). Among pts with 2 or more of the M5, 5-year OS for vaccine pts is 93% and for observation pts is 74% (p=.009). Among patients with 0–1 of the M5, 5-year OS for vaccine pts is 83% and for observation pts is 82% (p=.68). The interaction remained significant when only HLA A2/C3 status was considered (A2+ and/or C3+ vs A2- and C3-, p=.004). In the A2+ and/or C3+ group, 5-year OS for vaccine pts is 90% and for observation pts is 76% (p=.007). In the A2- and C3- group, 5-year OS for vaccine pts is 80% and for observation pts is 84% (p=.16). Conclusion: There was no significant benefit of vaccine therapy overall, but this prospective analysis indicates significant survival benefit of vaccine therapy among early-stage melanoma pts expressing ≥2 of the M5 alleles. HLA-A2 and C3 were the predominant alleles contributing to this effect. A prospective confirmatory trial in pts expressing HLA-A2 and/or HLA-C3 is planned. No significant financial relationships to disclose.

Significant impact of HLA class I allele expression on outcome in melanoma patients treated with an allogeneic melanoma cell lysate vaccine. Final analysis of SWOG-9035

7501 Background: SWOG-9035 compared 2 years of an allogeneic melanoma lysate (Melacine™) to observation in patients (pts) with stage II melanoma 1.5–4.0 mm thick. We also analyzed the effects of expression of certain HLA antigens (≥2 of HLA-A2, A28, B44, B45, C3 - called M5 - or either HLA-A2 and or C3). Now we report the mature overall survival (OS) analysis as planned. Methods: Primary analyses were conducted on the eligible pt population (intent to treat, ITT) using a Cox model adjusted for stratification and recognized prognostic factors. HLA analyses were \(\underline{\mathrm{prospective}}\), involved all typed pts and were adjusted for stratification and prognostic factors. Results: In total, 689 pts were entered over 4.5 years (598 eligible). Median follow-up time is 7.8 years (maximum 10.9 years). Ninety-one patients are ineligible, mostly (n=75) by pathology (tumor too thin or too thick). For the ITT analysis, the 5-year OS for the observation arm is 77% and for the vaccine arm is 81% (p=.98). For all randomized pts, the 5-year OS for the observation arm is 76% and for the vaccine arm is 82% (p=.49). We HLA typed 553 of the total 689 pts enrolled (80% of the total). There was a significant interaction of M5 status (≥2 vs 0–1) and treatment (p=.02). Among pts with 2 or more of the M5, 5-year OS for vaccine pts is 93% and for observation pts is 74% (p=.009). Among patients with 0–1 of the M5, 5-year OS for vaccine pts is 83% and for observation pts is 82% (p=.68). The interaction remained significant when only HLA A2/C3 status was considered (A2+ and/or C3+ vs A2- and C3-, p=.004). In the A2+ and/or C3+ group, 5-year OS for vaccine pts is 90% and for observation pts is 76% (p=.007). In the A2- and C3- group, 5-year OS for vaccine pts is 80% and for observation pts is 84% (p=.16). Conclusion: There was no significant benefit of vaccine therapy overall, but this prospective analysis indicates significant survival benefit of vaccine therapy among early-stage melanoma pts expressing ≥2 of the M5 alleles. HLA-A2 and C3 were the predominant alleles contributing to this effect. A prospective confirmatory trial in pts expressing HLA-A2 and/or HLA-C3 is planned. No significant financial relationships to disclose.