Mark Ringhoffer

GRANULOCYTE-MACROPHAGE-COLONY-STIMULATING FACTOR ENHANCES IMMUNE RESPONSES TO MELANOMA-ASSOCIATED PEPTIDES IN VIVO

Peptide epitopes derived from differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for MHC‐restricted cytotoxic T lymphocytes (CTL). The characterization of multiple CTL‐defined antigenic determinants has opened possibilities of development of antigen‐targeted vaccines. In the present study, we determined CTL reactivity against melanoma‐associated peptides derived from Melan A/MART‐I, tyrosinase, and gp100/Pmel17 in 3 HLA‐A2+ melanoma patients. Then, we assessed the immune responses to synthetic melanoma‐associated peptides injected intradermally. After 3 cycles of immunization with peptide alone, we used systemic GM‐CSF as an adjuvant during the fourth cycle of immunization. Enhanced DTH reactions and CD8+ CTL responses were observed after treatment with systemic GM‐CSF. Immunohistochemical characterization of DTH‐constituting elements revealed infiltrates of CD4+ and CD8+ T lymphocytes and strong expression of IL‐2 and γIFN, suggesting the activation of CD4+ Thl and CD8+ CTL by peptides presented by MHC‐class‐I molecules of dermal APC. Objective tumor regression was documented in all patients. We conclude that systemic GM‐CSF enhances immune responses to melanoma‐associated peptides and supports CTL‐mediated tumor rejection in vivo.

Immunoselection in vivo: independent loss of MHC class I and melanocyte differentiation antigen expression in metastatic melanoma.

Peptides derived from melanocyte differentiation antigens have been identified as targets for MHC class I‐restricted cytolytic T lymphocytes (CTLs) in human melanoma. Regression of antigen‐expressing tumors as well as selection of antigen‐loss variants in the presence of antigen‐specific CTLs have previously been reported. In the present study, we determined the expression of the melanocyte differentiation antigens Melan A/MART‐I and tyrosinase by mRNA analysis and by immunohistochemical staining with the monoclonal antibodies (MAbs) A103 and T311. Co‐expression of Melan A/MART‐I and tyrosinase was detected by both methods in 18/20 melanomas tested. However, immunohistochemistry provided additional information on intensity and microheterogeneity of antigen expression that cannot be detected by mRNA analysis as a molecular basis for the escape from CTL recognition of antigen‐negative tumor cells. Comparative analysis of repeated biopsies of metastatic lesions in 5 HLA‐A2+ patients showed a gradual loss of Melan A/MART‐I expression in 4/5 and of tyrosinase in 2/5 samples in association with tumor progression. However, 3 of these patients had growing antigen‐positive tumors in the presence of antigen‐specific CTLs. This led us to assess the expression of MHC class I, the essential restriction element for CTL recognition, and of HLA‐A2. We found an unexpectedly high frequency of MHC class I‐negative tumors (9/20). Loss of MHC class I expression was detected in 3/5 progressive tumors and isolated loss of HLA‐A2 in 1/5 tumors. Our results suggest that strategies enhancing the expression of MHC class I and tumor‐associated antigens need to be considered in attempts at making vaccination more effective. Int. J. Cancer, 71:142–147, 1997.

Inverse relationship of melanocyte differentiation antigen expression in melanoma tissues and CD8+ cytotoxic‐T‐cell responses: Evidence for immunoselection of antigen‐loss variants in vivo

Antigenic peptides derived from differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for MHC‐restricted cytotoxic T lymphocytes (CTL). CTL directed against peptides derived from the Melan A/MART‐1, tyrosinase and gp100/Pmel17 antigens can be detected in melanoma patients and in healthy controls. The presence of defined antigenic peptides and corresponding precursor CTL in patients with metastatic melanoma opens perspectives for the development of antigen‐specific tumor vaccines. In this study, we examined the expression of Melan A/MART‐1, tyrosinase and gp100/Pmel17 in fresh melanoma tissues of HLA‐A2+ patients and the spontaneous CTL reactivity against antigenic peptides derived from these antigens. Our results demonstrate an inverse correlation of antigen expression and CTL response to Melan A/MART‐1 and tyrosinase were induced by intradermal immunization with synthetic nona‐ or deca‐peptides derived from these antigens. Metastases increasing in size over time showed a loss of Melan A/MART‐1 expression in the presence of CTL in one patient. The regression of a metastasis with persistent tyrosinase expression was observed in the other patient after the induction of CTL, reactive against tyrosinase. We conclude that CTL responses against melanocyte differentiation antigens may mediate regression of antigen‐positive tumors and select for antigen‐loss variants in vivo.

Generation of cytotoxic T‐cell responses with synthetic melanoma‐associated peptides in vivo: Implications for tumor vaccines with melanoma‐associated antigens

Peptide epitopes derived from differentiation antigens of the melanocyte lineage have been identified in human melanomas and normal cultured melanocytes as targets for MHC‐restricted cytotoxic T lymphocytes (CTL). Characterization of multiple CTL‐defined antigenic determinants and the presence of corresponding precursor CTL open perspectives for the development of antigen‐based vaccines. In the present study, we determined the CTL reactivity against melanoma‐associated peptides derived from Melan A/MART‐I, tyrosinase and gp100/Pme117 in 10 HLA‐A2* melanoma patients and 10 healthy individuals. Then, we examined the immunological effects and toxicity of intradermal inoculation of synthetic melanoma‐associated peptides. Six patients with advanced melanoma received weekly intradermal injections of 6 melanoma‐associated peptides and the influenza matrix peptide as a control for 4 consecutive weeks. DTH reactions were observed in 5/6 patients at the injection sites of the tyrosinase signal peptide and of the influenza matrix peptide. No toxic side effects were observed. Changes in CTL reactivity after peptide vaccination were assessed by an MLPC assay for each peptide. Generation of peptide‐specific CTL was documented against Melan A/MART‐I‐derived peptide epitopes, the tyrosinase signal peptide and the influenza matrix peptide after vaccination. A decreasing CTL response against the internal tyrosinase peptide was documented in 1 patient through the course of vaccination and a decrease in DTH reactions. No major tumor regressions were observed. Two patients with rapidly progressive disease before vaccination have shown disease stabilization since vaccinations started. In conclusion, our results demonstrate that peptide alone injected intradermally may generate antigen‐specific DTH reactions and an increase of antigen‐specific CTL reactivity.

Adoptive transfer and selective reconstitution of streptamer-selected cytomegalovirus-specific CD8+ T cells leads to virus clearance in patients after allogeneic peripheral blood stem cell transplantation

BACKGROUND: Cytomegalovirus (CMV) disease constitutes a serious complication after allogeneic stem cell transplantation. For the clearance of CMV, CD8+ T cells are pivotal.

mRNA expression of leukemia-associated antigens in patients with acute myeloid leukemia for the development of specific immunotherapies

Specific immunotherapies for patients with acute myeloid leukemia (AML) using leukemia‐associated antigens (LAA) as target structures might be a therapeutic option to enhance the graft‐vs.‐leukemia effect observed after allogeneic stem cell transplantation or to prolong a complete remission (CR) achieved by chemotherapy. Significant mRNA expression of LAA is a prerequisite for such immunotherapies. Here, previously characterized antigens associated with solid tumors (TAA) and newly characterized LAA were investigated for their expression in up to 60 AML patients and in leukemia cell lines. To investigate their specificity for leukemic blasts, the mRNA expression was also characterized in PBMN and CD34 positive cells of healthy volunteers and in a panel of normal tissues. The following antigens showed high mRNA expression in AML patients: MPP11 was detected in 43/50 (86%), RHAMM in 35/50 (70%), WT1 in 40/60 (67%), PRAME in 32/50 (64%), G250 in 18/35 (51%), hTERT in 7/25 (28%) and BAGE in 8/30 (27%) of AML patients. Real‐time RT‐PCR showed a tumor‐specific expression of the antigens BAGE, G250 and hTERT, as well as highly tumor‐restricted expression for RHAMM, PRAME and WT1. The antigen MPP11 was overexpressed. These antigens might be candidates for immunotherapies of leukemia patients and, because of their simultaneous expression, also for polyvalent vaccines.

Differential impact of allelic ratio and insertion site in FLT3-ITD-positive AML with respect to allogeneic transplantation.

The objective was to evaluate the prognostic and predictive impact of allelic ratio and insertion site (IS) of internal tandem duplications (ITDs), as well as concurrent gene mutations, with regard to postremission therapy in 323 patients with FLT3-ITD-positive acute myeloid leukemia (AML). Increasing FLT3-ITD allelic ratio (P = .004) and IS in the tyrosine kinase domain 1 (TKD1, P = .06) were associated with low complete remission (CR) rates. After postremission therapy including intensive chemotherapy (n = 121) or autologous hematopoietic stem cell transplantation (HSCT, n = 17), an allelic ratio ≥ 0.51 was associated with an unfavorable relapse-free (RFS, P = .0008) and overall survival (OS, P = .004); after allogeneic HSCT (n = 93), outcome was significantly improved in patients with a high allelic ratio (RFS, P = .02; OS, P = .03), whereas no benefit was seen in patients with a low allelic ratio (RFS, P = .38; OS, P = .64). Multivariable analyses revealed a high allelic ratio as a predictive factor for the beneficial effect of allogeneic HSCT; ITD IS in TKD1 remained an unfavorable factor, whereas no prognostic impact of concurrent gene mutations was observed. The clinical trials described herein were previously published or are registered as follows: AMLHD93 and AMLHD98A, previously published; AML SG 07-04, ClinicalTrials.gov identifier #NCT00151242.

Receptor for hyaluronan acid–mediated motility (RHAMM) is a new immunogenic leukemia-associated antigen in acute and chronic myeloid leukemia

OBJECTIVE Identification of leukemia-associated antigens (LAA) eliciting an immune response in patients is a prerequisite for specific immunotherapy of leukemias. To identify new LAA, we used the method of serologic screening of cDNA expression libraries (SEREX). MATERIALS AND METHODS A SEREX library of the cell line K562 was subjected to allogeneic screening with sera from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML) vs sera from healthy volunteers. RESULTS The receptor for hyaluronan acid-mediated motility (RHAMM) involved in cell growth and metastasis was identified as a new LAA. Serologic responses to RHAMM were observed in patients with AML (42%), CML (31%), melanoma (83%), renal cell carcinoma (40%), breast cancer (67%), and ovarian carcinoma (50%), but not in HV or patients with autoimmune diseases. RHAMM mRNA was detectable in peripheral blood mononuclear cells (PBMN) of 60% of newly diagnosed AML patients. Western blotting stained positive for RHAMM protein in 70% of AML patients. mRNA expression of RHAMM also was found in patients with CML (40%), renal cell carcinoma (73%), breast carcinoma (60%), and ovarian carcinoma (50%). In melanoma, RHAMM mRNA expression was detected in metastases (80%) but not in primary tumors. RHAMM is differentially expressed: significant mRNA expression was not found in normal tissues, except from testis, placenta, and thymus, or in PBMN- and CD34-separated cell samples of healthy volunteers. CONCLUSIONS RHAMM is an immunogenic antigen in leukemias and solid tumors and might be a potential target structure for cellular immunotherapies and antibody therapies.

Clinical impact of DNMT3A mutations in younger adult patients with acute myeloid leukemia: results of the AML Study Group (AMLSG).

In this study, we evaluated the frequency and prognostic impact of DNMT3A mutations (DNMT3A(mut)) in 1770 younger adult patients with acute myeloid leukemia (AML) in the context of other genetic alterations and the European LeukemiaNet (ELN) classification. DNMT3A(mut) were found in 20.9% of AMLs and were associated with older age (P < .0001), higher white blood cell counts (P < .0001), cytogenetically normal AML (CN-AML; P < .0001), NPM1 mutations (P < .0001), FLT3 internal tandem duplications (P < .0001), and IDH1/2 mutations (P < .0001). In univariable and multivariable analyses, DNMT3A(mut) did not impact event-free, relapse-free (RFS), or overall survival (OS) in either the entire cohort or in CN-AML; a negative prognostic effect was found only in the ELN unfavorable CN-AML subset (OS, P = .011). In addition, R882 mutations vs non-R882 mutations showed opposite clinical effects-unfavorable for R882 on RFS (all: hazard ratio [HR], 1.29 [P = .026]; CN-AML: HR, 1.38 [P = .018]) and favorable for non-R882 on OS (all: HR, 0.77 [P = .057]; CN-AML: HR, 0.73 [P = .083]). In our statistically high-powered study with minimized selection bias, DNMT3A(mut) represent a frequent genetic lesion in younger adults with AML but have no significant impact on survival end points; only moderate effects on outcome were found, depending on molecular subgroup and DNMT3A(mut) type.

CHARACTERIZATION OF SEVERAL LEUKEMIA-ASSOCIATED ANTIGENS INDUCING HUMORAL IMMUNE RESPONSES IN ACUTE AND CHRONIC MYELOID LEUKEMIA

To design a specific immunotherapy for leukemia patients, the identification of leukemia‐associated antigens (LAAs) is a pivotal step. Antileukemic effects after hematopoetic stem cell transplantation for myeloid leukemias are observed and might be related to the recognition of LAAs. Using the serological screening of an expression library (SEREX) of K562 cells, we identified 16 different clones encoding LAAs eliciting a humoral immune response, among them the heat shock proteins HSJ2 and HSP70, the M‐phase phosphoprotein 11 (MPP11), the BRCA1‐associated protein (BRAP), the Jκ recombination binding protein (RBPJκ) and the receptor for hyaluronic acid mediated motility (RHAMM). Serological responses to MPP11 were observed in 7/19 (37%) of patients with acute myeloid leukemia (AML) and 6/16 (38%) of patients with chronic myeloid leukemia (CML), but not in healthy volunteers (0/20). IgG antibodies directed against MPP11 were also detected in 25–50% of the sera of patients with solid tumors such as melanoma, renal cell, ovarian and breast carcinoma. mRNA expression of MPP11 was detected in 20/20 AML patients and 7/10 patients with CML. In normal tissues, strong mRNA expression of MPP11 was only detected in testis. By real‐time PCR, we detected upregulation of MPP11 in leukemic blasts. Simultaneous humoral immune responses to 2 or more of the 16 LAAs identified here was observed, suggesting the feasibility of a polyvalent vaccination as an option for immunotherapies in leukemia patients.