Alexander Knuth

Phase III Trial in Metastatic Gastroesophageal Adenocarcinoma with Fluorouracil, Leucovorin Plus Either Oxaliplatin or Cisplatin: A Study of the Arbeitsgemeinschaft Internistische Onkologie

PURPOSE This study was designed to compare fluorouracil, leucovorin, and oxaliplatin with fluorouracil, leucovorin, and cisplatin in patients with advanced gastric cancer. PATIENTS AND METHODS Patients with previously untreated advanced adenocarcinoma of the stomach or esophagogastric junction were randomly assigned to receive either fluorouracil 2,600 mg/m(2) via 24-hour infusion, leucovorin 200 mg/m(2), and oxaliplatin 85 mg/m(2) (FLO) every 2 weeks or fluorouracil 2,000 mg/m(2) via 24-hour infusion, leucovorin 200 mg/m(2) weekly, and cisplatin 50 mg/m(2) every 2 weeks (FLP). The primary end point was progression-free survival (PFS). RESULTS Two hundred twenty patients (median age, 64 years; metastatic, 94%) were randomly assigned. FLO was associated with significantly less (any grade) anemia (54% v 72%), nausea (53% v 70%), vomiting (31% v 52%), alopecia (22% v 39%), fatigue (19% v 34%), renal toxicity (11% v 34%), thromboembolic events (0.9% v 7.8%), and serious adverse events related to the treatment (9% v 19%). FLP was associated with significantly less peripheral neuropathy (22% v 63%). There was a trend toward improved median PFS with FLO versus FLP (5.8 v 3.9 months, respectively; P = .077) and no significant difference in median overall survival (10.7 v 8.8 months, respectively). However, in patients older than 65 years (n = 94), treatment with FLO resulted in significantly superior response rates (41.3% v 16.7%; P = .012), time to treatment failure (5.4 v 2.3 months; P < .001), and PFS (6.0 v 3.1 month; P = .029) and an improved OS (13.9 v 7.2 months) as compared with FLP, respectively. CONCLUSION FLO reduced toxicity as compared with FLP. In older adult patients, FLO also seemed to be associated with improved efficacy.

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.

Selective Survival of Naturally Occurring Human CD4+CD25+Foxp3+ Regulatory T Cells Cultured with Rapamycin

Naturally occurring CD4+CD25+ regulatory T (nTreg) cells are essential for maintaining T cell tolerance to self Ags. We show that discrimination of human Treg from effector CD4+CD25+ non-nTreg cells and their selective survival and proliferation can now be achieved using rapamycin (sirolimus). Human purified CD4+CD25high T cell subsets stimulated via TCR and CD28 or by IL-2 survived and expanded up to 40-fold in the presence of 1 nM rapamycin, while CD4+CD25low or CD4+CD25− T cells did not. The expanding pure populations of CD4+CD25high T cells were resistant to rapamycin-accelerated apoptosis. In contrast, proliferation of CD4+CD25− T cells was blocked by rapamycin, which induced their apoptosis. The rapamycin-expanded CD4+CD25high T cell populations retained a broad TCR repertoire and, like CD4+ CD25+ T cells freshly obtained from the peripheral circulation, constitutively expressed CD25, Foxp3, CD62L, glucocorticoid-induced TNFR family related protein, CTLA-4, and CCR-7. The rapamycin-expanded T cells suppressed proliferation and effector functions of allogeneic or autologous CD4+ and CD8+ T cells in vitro. They equally suppressed Ag-specific and nonspecific responses. Our studies have defined ex vivo conditions for robust expansion of pure populations of human nTreg cells with potent suppressive activity. It is expected that the availability of this otherwise rare T cell subset for further studies will help define the molecular basis of Treg-mediated suppression in humans.

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.

SSX: A multigene family with several members transcribed in normal testis and human cancer

Analysis of t(X;18) translocation in synovial sarcoma had previously led to the definition of the SSX2 gene, the fusion partner on chromosome X. Subsequent screening of testicular cDNA libraries identified 2 highly homologous genes, SSX1 and SSX3. Among these 3 genes, SSX2 has been found to be identical to HOM‐MEL‐40, which codes for an immunogenic tumor antigen expressed in various human cancers. SSX2 thus belongs to the family of cancer/testis (CT) antigens, i.e., immunogenic protein antigens with characteristic mRNA expression in normal testis and in cancer. To define additional CT antigens, we have immuno‐screened a testicular cDNA expression library with an allogeneic serum from a melanoma patient, and both SSX2 and SSX3 were isolated. Further studies using testicular cDNA and SSX probes defined 2 new members of this gene family, SSX4 and SSX5, while a shorter cDNA variant of SSX4 was also identified. All 5 members of the SSX family shared strong sequence homology, with nucleotide homology ranging from 88 to 95% and amino acid homology ranging from 77 to 91%. Genomic cloning of a prototype SSX gene (SSX2) showed that its coding region is encoded by 6 exons, and the shortened form of SSX4 cDNA represents an alternatively spliced product lacking the 5th exon. Analysis of SSX mRNA expression by gene‐specific RT‐PCR confirmed that all 5 SSX genes are expressed in testis. In addition, analysis of a panel of 12 melanoma cell lines showed strong mRNA expression of either SSX1 (3/12), SSX2 (3/12), SSX4 (1/12), or SSX5 (1/12), indicating variable activation of the genes in malignant cells. Int. J. Cancer 72:965–971, 1997.

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.

NY-ESO-1: review of an immunogenic tumor antigen.

In the 9 years since its discovery, cancer-testis antigen NY-ESO-1 has made one of the fastest transitions from molecular, cellular, and immunological description to vaccine and immunotherapy candidate, already tested in various formulations in more than 30 clinical trials worldwide. Its main characteristic resides in its capacity to elicit spontaneous antibody and T-cell responses in a proportion of cancer patients. An overview of immunological findings and immunotherapeutic approaches with NY-ESO-1, as well the role of regulation in NY-ESO-1 immunogenicity, is presented here.

Humoral immune responses of cancer patients against "Cancer-Testis" antigen NY-ESO-1: correlation with clinical events.

Humoral immune responses against the “Cancer‐Testis” (CT) antigen NY‐ESO‐1 are frequently observed in patients with NY‐ESO‐1 expressing tumors. This is in contrast to other known tumor antigens (TA) defined by antibody or cytotoxic T cell (CTL) reactivity, i.e., MAGE‐1, MAGE‐3, SSX2, Melan A, and tyrosinase. No NY‐ESO‐1 antibody has been detected in healthy controls and patients with NY‐ESO‐1 negative tumors. In this study, we have assessed the NY‐ESO‐1 serum antibody response in patients with NY‐ESO‐1 positive tumors of different histological types and stages using Western blotting and an ELISA. Of the 12 patients analyzed, 10 had demonstrable NY‐ESO‐1 antibodies at the start of the study. All patients were followed for changes in NY‐ESO‐1 antibody titers during the course of tumor treatment and clinical evolution. In 4 patients, an increase of NY‐ESO‐1 antibody titer was observed with progression of disease or extensive tumor necrosis under treatment. One patient showed a stable NY‐ESO‐1 antibody titer over 3 years along with gradual regression of a large tumor mass. In 5 patients, a decrease of NY‐ESO‐1 antibody was detected: in 1 patient after curative tumor resection, in 3 patients with partial regression of metastatic disease under chemo‐ and immunotherapy, and in another patient with a NY‐ESO‐1 negative tumor relapse. Our results indicate that the induction and maintenance of NY‐ESO‐1 antibody is dependent on the presence of NY‐ESO‐1 expressing tumors. Furthermore, changes in NY‐ESO‐1 antibody titers correlate with the evolution of NY‐ESO‐1 positive disease. Int. J. Cancer (Pred. Oncol.) 84:506–510, 1999.

Survey of naturally occurring CD4+ T cell responses against NY-ESO-1 in cancer patients: Correlation with antibody responses

NY-ESO-1 is one of the most immunogenic proteins described in human cancers, based on its capacity to elicit simultaneous antibody and CD8+ T cell responses in vivo. Although HLA class II restricted epitopes from NY-ESO-1 have been identified, no broad survey has yet established the status of natural CD4+ T cell responses in cancer patients in relation to CD8+ and antibody responses. We used a recently developed general strategy for monitoring CD4+ responses that overcomes the need for prior knowledge of epitope or HLA restriction to analyze a series of 31 cancer patients and healthy donors for the presence of CD4+ T cells to NY-ESO-1, and related this response to NY-ESO-1 expression in tumor cells and serum antibodies to NY-ESO-1. None of the 18 patients that tested seronegative for NY-ESO-1 had detectable CD4+ T cell responses. On the contrary, 11 of 13 cancer patients with serum antibodies to NY-ESO-1 had polyclonal CD4+ T cell responses directed against various known and previously undescribed NY-ESO-1 epitopes. NY-ESO-1 peptide 80–109 was the most immunogenic, with 10 of 11 patients responding to this peptide. We show here that 12-mer determinants from NY-ESO-1 eliciting a CD4+ T cell response were peptide 87–98 with promiscuous HLA class II presentation, peptide 108–119 restricted by HLA-DP4, and peptides 121–132 and 145–156, both shorter epitopes from previously described HLA-DR4 peptides, also presented by HLA-DR7. This study represents the next step in compiling a comprehensive picture of the adaptive immune response to NY-ESO-1, and provides a general strategy for analyzing the CD4+ T cell response to other tumor antigens eliciting a humoral immune response.