Melanoma: Houghton AN

Houghton AN, Coit DG, Daud A, Dilawari RA, Dimaio D, Gollob JA, Haas NB, Halpern A, Johnson TM, Kashani-Sabet M, Kraybill WG, Lange JR, Martini M, Ross MI, Samlowski WE, Sener SF, Tanabe KK, Thompson JA, Trisal V, Urist MM, Walker MJ, Anonymous00370. · No affiliation provided · J Natl Compr Canc Netw. · Pubmed #16884669 No free full text.

This publication has no abstract.

Uchi H, Stan R, Turk MJ, Engelhorn ME, Rizzuto GA, Goldberg SM, Wolchok JD, Houghton AN. · Swim Across America Laboratory, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York, USA. · Adv Immunol. · Pubmed #16730265 No free full text.

Abstract: A relationship between melanoma and vitiligo, a skin disorder characterized by the loss of melanocytes, has been postulated for many decades. In some cases, vitiligo is almost certainly a manifestation of autoimmune-mediated destruction of melanocytes. Melanocytes and melanoma cells share melanocyte differentiation antigens. Based on a number of observations, de novo vitiligo developing in patients with melanoma has been regarded as a sign of good prognosis. The immune system tolerates or ignores differentiation antigens because these antigens are self-derived. Therefore, immune tolerance or ignorance must be overcome to prime naive T and B cells to induce cancer immunity and autoimmunity against melanocyte differentiation antigens. Mouse models of concurrent melanoma and autoimmune vitiligo have revealed strategies to overcome immune ignorance or tolerance to melanocyte differentiation antigens: immunization with self-antigens as altered self (e.g., orthologues or mutated versions), expression in viral vectors, passive immunization with antibodies or T cells, incorporating potent adjuvants into active immunization, and blockade or removal of a downregulatory mechanism. Extensive investigations into the mechanisms that lead to tumor immunity and autoimmunity elicited by certain differentiation antigens have further revealed a variety of distinct cellular and molecular requirements, which are redundant and alternative.

Guevara-Patiño JA, Turk MJ, Wolchok JD, Houghton AN. · Memorial Sloan-Kettering Cancer Center and the Weill Graduate School of Medical Sciences and Medical School of Cornell University, 1275 York Avenue, New York, NY 10021, USA. · Adv Cancer Res. · Pubmed #14710950 No free full text.

Abstract: The adaptive immune system is capable of recognizing cancer through T- and B-cell receptors. However, priming adaptive immunity against self antigens is potentially a difficult task. Presentation of altered self to the immune system is a strategy to elicit immunity against poorly immunogenic antigens. We have shown that immunization with conserved paralogues of tumor antigens can induce adaptive immunity against self antigens expressed by cancer. Remarkably, cancer immunity elicited by closely related paralogues can generate distinct adaptive immune responses, either antibody or T-cell dependent. Cancer immunity induced by xenogeneic immunization follows multiple and alternative pathways. The effector phase of tumor immunity can be mediated by cytotoxic T cells or macrophages and perhaps natural killer cells for antibody-dependent immunity. Helper CD4+ T cells are typically, but not always, required to generate immunity. Autoimmunity is frequently observed following immunization. Cancer immunity and autoimmunity use overlapping mechanisms, and therefore they are difficult to uncouple, but distinct pathways can be discerned that open the eventual possibility of uncoupling tumor immunity from autoimmunity. Studies examining the molecular basis for immunogenicity of conserved paralogues are facilitating the development of new strategies to rationally design vaccines that trigger adaptive immune responses to cancer.

Ramirez-Montagut T, Turk MJ, Wolchok JD, Guevara-Patino JA, Houghton AN. · Memorial Sloan-Kettering Cancer Center and the Weill Graduate School of Medical Sciences of Cornell University, 1275 York Avenue, New York, NY 10021, USA. · Oncogene. · Pubmed #12789294 No free full text.

Abstract: Cancer cells express self-antigens that are weakly recognized by the immune system. Even though responses against autologous cells are difficult to induce, the immune system is still able to mount a response against cancer. The discovery of the molecular identity of antigens that are recognized by the immune system of melanoma patients has led to the elucidation of tumor immunity at a cellular and molecular level. Multiple pathways in both the priming and effector phases of melanoma rejection have been described. Animal models' active immunotherapies for melanoma show a requirement for the cellular compartment of the immune system in the priming phase, primarily CD4+T cells. More diverse elements are required for the effector phase, including components from the innate immune system (macrophages, complement and/or natural killer cells) and from the adaptive immune system (CD8+T cells and B cells). Minor differences in amino-acid sequences of antigens must determine the particular mechanisms involved in tumor rejection. Since the immune system contains T and B cells that recognize and reject autologous cells, a consequence of tumor immunity is potential autoimmunity. There are distinct pathways for tumor immunity and autoimmunity. The requirements for autoimmunity at the priming phase seem to be CD4+/IFN-gamma dependent while the effector mechanisms are alternative and redundant. Vitiligo (autoimmune hypopigmentation) can be mediated by T cells, FcgammaR+macrophages and/or complement.

Turk MJ, Wolchok JD, Guevara-Patino JA, Goldberg SM, Houghton AN. · 1Weill Graduate School of Medical Sciences of Cornell University, New York, USA. · Immunol Rev. · Pubmed #12445286 No free full text.

Abstract: The immune repertoire contains T cells and B cells that can recognize autologous cancer cells. This repertoire is directed against self, and in some cases altered self (mutations). Priming immune responses against self antigens can be difficult. Strategies are presented using altered self to elicit immunity against self in poorly immunogenic tumor models. Mechanisms underlying immunity to self antigens on cancer cells show that the immune system can use diverse strategies for cancer immunity, in both the immunization and the effector phases. CD4+ T cells are typically, but not always, required for immunization. The effector phase of tumor immunity can involve cytotoxic T cells, macrophages with activating Fc receptors, and/or killer domain molecules. This diversity in the effector phase is observed even when immunizing with conserved paralogs. A consequence of tumor immunity is potentially autoimmunity, which may be undesirable. Autoimmunity uses similar mechanisms as tumor immunity, but tumor immunity and autoimmunity can uncouple. These studies open up strategies for active immunization against cancer.

Houghton AN, Polsky D. · Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. · Cancer Cell. · Pubmed #12398891 No free full text.

This publication has no abstract.

Houghton AN, Gold JS, Blachere NE. · Memorial Sloan-Kettering Cancer Center and Weill Medical School of Cornell University, 1275 York Avenue, New York, NY 10021, USA. · Curr Opin Immunol. · Pubmed #11228404 No free full text.

Abstract: Most major advances in human cancer immunology and immunotherapy have come from studies in melanoma. We are beginning to understand the immune repertoire of T cells and antibodies that are active against melanoma, with recent glimpses of the CD4(+) T cell repertoire. The view of what the immune system can see is extending to mutations and parts of the genome that are normally invisible.

Klimek VM, Wolchok JD, Chapman PB, Houghton AN, Hwu WJ. · Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. · Clin Plast Surg. · Pubmed #10941565 No free full text.

Abstract: The main use of systemic chemotherapy in metastatic melanoma remains palliative. Dacarbazine (dimethyl-1-triazeno imidazole-4-carboxamide [DTIC]) is the standard chemotherapy agent for advanced disease. The combination chemotherapy and biochemotherapy regimens have achieved higher response rates, but have not led to durable remission or improved survival. The field of systemic therapy remains in need of a more effective and less toxic treatment strategy.

Perales MA, Yuan J, Powel S, Gallardo HF, Rasalan TS, Gonzalez C, Manukian G, Wang J, Zhang Y, Chapman PB, Krown SE, Livingston PO, Ejadi S, Panageas KS, Engelhorn ME, Terzulli SL, Houghton AN, Wolchok JD. · Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. · Mol Ther. · Pubmed #18797450 No free full text.

Abstract: Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances immune responses by inducing proliferation, maturation, and migration of dendritic cells (DCs) as well as expansion and differentiation of B and T lymphocytes. The potency of DNA vaccines can be enhanced by the addition of DNA encoding cytokines, acting as molecular adjuvants. We conducted a phase I/II trial of human GM-CSF DNA in conjunction with a multipeptide vaccine (gp100 and tyrosinase) in stage III/IV melanoma patients. Nineteen human leukocyte antigen (HLA)-A*0201+ patients were treated. Three dose levels were studied: 100, 400, and 800 microg DNA/injection, administered subcutaneously every month with 500 microg of each peptide. In the dose-ranging study, three patients were treated at each dose level. The remaining patients were then treated at the highest dose. Most toxicities were grade 1 injection-site reactions. Eight patients (42%) developed CD8+ T-cell responses, defined by a > or =3 SD increase in baseline reactivity to tyrosinase or gp100 peptide in tetramer or intracellular cytokine staining (ICS) assays. There was no relationship between dose and T-cell response. Responding T cells had an effector memory cell phenotype. Polyfunctional T cells were also demonstrated. At a median of 31 months follow-up, median survival has not been reached. Human GM-CSF DNA was found to be a safe adjuvant.

Yuan J, Gallardo HF, Rasalan T, Ranganathan R, Wang J, Zhang Y, Panageas K, Stan R, Young JW, Houghton AN, Wolchok JD. · Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. · Cytotherapy. · Pubmed #17050255 No free full text.

Abstract: BACKGROUND: Development of a practical and sensitive assay for evaluating immune responses against cancer Ag has been a challenge for immune monitoring of patients. We have established a reproducible method using peptide-pulsed K562-A*0201 cells as APC to expand Ag-specific T cells in vitro. This method may be applied for monitoring T-cell responses in cancer immunotherapy clinical trials. METHODS: Autologous PBMC from HLA-A*0201+ healthy donors and patients with melanoma were stimulated with peptide-pulsed K562-A*0201 cells under varying conditions. We investigated (1) different culture conditions, including the requirements for serum and cytokines for expansion of CD8+ T lymphocytes; (2) a range of peptide concentrations for Ag loading; (3) phenotypic characterization of responding T cells; and (4) APC:responder ratios and their effects on T-cell expansion. We validated these conditions by ELISPOT and intracellular cytokine staining (ICS) assays using peptides from influenza, Epslein-Barr Virus (EBV) and tyrosinase. RESULTS: Conditions for optimal T-cell expansion using K562-A*0201 APC included input of 2 x 10(6) PBMC, a 10 microg/mL peptide concentration to pulse K562-A*0201 cells, a 1:30 APC:responder T-cell ratio and culture in 10% autologous plasma supplemented with IL-2 and IL-15. In these conditions, Ag-specific T cells expanded >100-fold over a 10-day culture period (peak at day 12). DISCUSSION: This bulk culture method is simple and reliable for expanding human Ag-specific T cells using peptide-pulsed K562-A*0201 cells. This HLA-matched APC line can be adapted to other HLA haplotypes, and has advantages for monitoring clinical trials of immunotherapy with limited availability of autologous APC and PBMC from patients.

Krown SE, Niedzwiecki D, Hwu WJ, Hodgson L, Houghton AN, Haluska FG, Anonymous00220. · Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. · Cancer. · Pubmed #16986123 links to  free full text

Abstract: BACKGROUND: Preliminary studies suggesting that extended-dose temozolomide with thalidomide is safe and active in patients with metastatic melanoma have led to frequent use of this oral regimen. To confirm these observations the combination was tested in a multicenter Phase II trial in patients with melanoma brain metastases. METHODS: Eligible patients had melanoma brain metastases, with or without systemic metastases. The primary endpoint was response rate in brain metastases. Patients received temozolomide at a dose of 75 mg/m2/day for 6 weeks with a 2-week rest between cycles, and thalidomide (escalated to 400 mg/day for patients age < 70 years or to 200 mg/day for patients age > or = 70 years). A 2-stage design required > or = 3 responses in the first 21 patients before enrolling 29 additional patients in the second stage. RESULTS: Sixteen eligible patients were enrolled. No objective responses were observed. The median survival was 23.9 weeks. Seven patients withdrew because of tumor progression; 7 were removed during Cycle 1 because of adverse events, including allergic reaction (1 patient), severe fatigue (1 patient), sudden death (1 patient), and thromboembolic events (pulmonary embolism in 3 patients and deep vein thrombosis in 1 patient); 2 patients withdrew when the study was suspended and subsequently closed. No associations could be established between baseline characteristics and toxicity. CONCLUSIONS: The proportion of patients with lethal or potentially life-threatening adverse events was high (0.31, 95% confidence interval, 0.11-0.59), and the absence of objective responses made it unlikely that further accrual would demonstrate the efficacy of the regimen. These observations provide little support for the use of this combination for melanoma brain metastases unless safe and effective methods to prevent thrombosis are developed.

Hwu WJ, Panageas KS, Menell JH, Lamb LA, Aird S, Krown SE, Williams LJ, Chapman PB, Livingston PO, Wolchok JD, Houghton AN. · Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. · Cancer. · Pubmed #16639739 links to  free full text

Abstract: BACKGROUND: Temozolomide and interferon-alpha-2b (IFN-alpha-2b) are both active in melanoma. Therefore, the efficacy and safety of temozolomide in combination with pegylated IFN-alpha-2b in patients with metastatic melanoma without brain metastases was investigated. METHODS: Patients with histologically confirmed, unresectable, American Joint Committee on Cancer Stage IV melanoma were enrolled in an open-label, Phase II study. The primary endpoints were tumor response and safety. Patients received temozolomide (75 mg/m2/dayx6 weeks with a 2-week break between cycles) plus concomitant subcutaneous pegylated IFN-alpha-2b (0.5 microg/kg/wk, continuously). Treatment was continued until unacceptable toxicity or disease progression occurred. RESULTS: Thirty-five patients (median age, 55 years) with Stage IV melanoma and a median of 3 metastatic sites were enrolled and received a median of 1 cycle (i.e., 8 weeks) of therapy (range, 0-6 cycles). Eleven patients (31%) (95% confidence interval, 16% to 47%) had an objective tumor response, including 3 with clinical complete response durations of 6 months, 20 months, and 32+ months and 8 with partial responses. Three patients with a partial or mixed response were subsequently rendered free of clinically detectable disease with surgery. The median survival was 12 months with a median follow-up among survivors of 16 months. No patient developed brain metastases while receiving study treatment. Treatment was generally well tolerated. Hematologic toxicity consisted mainly of lymphopenia and leukopenia (National Cancer Institute Common Toxicity Criteria Grades 1-3); no Grade 4 hematologic toxicity was observed. CONCLUSIONS: The combination of temozolomide plus pegylated IFN-alpha-2b had antitumor activity and was well tolerated in patients with metastatic melanoma. Therefore, further study is warranted.

Hwu WJ, Lis E, Menell JH, Panageas KS, Lamb LA, Merrell J, Williams LJ, Krown SE, Chapman PB, Livingston PO, Wolchok JD, Houghton AN. · Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. · Cancer. · Pubmed #15861414 links to  free full text

Abstract: BACKGROUND: Temozolomide plus thalidomide is a promising oral combination regimen for the treatment of metastatic melanoma. The current Phase II study examined the efficacy and safety of this combination in chemotherapy-naive patients with brain metastases. METHODS: Patients with histologically confirmed metastatic melanoma and measurable brain metastases received temozolomide (75 mg/m2 per day for 6 weeks with a 2-week break between cycles) plus concomitant thalidomide (200 mg/day escalating to 400 mg/day for patients < 70 years or 100 mg/day escalating to 250 mg/day for patients > or = 70 years). The primary end point was tumor response in the brain assessed every 8 weeks. RESULTS: Twenty-six patients with a median age of 60 years were treated. All patients had progressive brain metastases: 16 were symptomatic and 25 had extensive extracranial metastases. Eight patients had received whole-brain radiotherapy, 4 had received stereotactic radiotherapy, and 8 had received craniotomy with resection of hemorrhagic lesions. Fifteen patients completed > or = 1 cycle (median, 1 cycle; range, 0-4 cycles), and 11 discontinued treatment before completing 1 cycle (7 for intracranial hemorrhage, 2 for pulmonary embolism, 1 for deep vein thrombosis, and 1 for Grade 3 rash). Of 15 patients assessable for response, 3 had a complete or partial response (12% intent to treat) and 7 had minor response or stable disease in the brain. However, 5 of these 10 patients had disease progression at extracranial sites. The median survival period was 5 months for all 26 patients and 6 months for the 15 assessable patients. CONCLUSIONS: Temozolomide plus thalidomide was an active oral regimen for patients with brain metastases from malignant melanoma.

Hwu WJ, Krown SE, Menell JH, Panageas KS, Merrell J, Lamb LA, Williams LJ, Quinn CJ, Foster T, Chapman PB, Livingston PO, Wolchok JD, Houghton AN. · Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021, USA. · J Clin Oncol. · Pubmed #12947072 No free full text.

Abstract: PURPOSE: To further investigate the efficacy and safety of temozolomide plus thalidomide in patients with metastatic melanoma without brain metastases. PATIENTS AND METHODS: Patients with histologically confirmed advanced-stage metastatic melanoma were enrolled in an open-label, phase II study. The primary end point was response rate. Patients received temozolomide (75 mg/m2/d x 6 weeks with a 2-week rest between cycles) plus concomitant thalidomide (200 mg/d with dose escalation to 400 mg/d for patients < 70 years old, or 100 mg/d with dose escalation to 250 mg/d for patients >/= 70 years old). Treatment was continued until unacceptable toxicity or disease progression occurred. RESULTS: Thirty-eight patients (median age, 62 years) with stage IV (three patients with M1a, eight with M1b, and 26 with M1c) or stage IIIc (one patient) melanoma and a median of four metastatic sites were enrolled, and received a median of two cycles of therapy. Twelve patients (32%) had an objective tumor response, including one with an ongoing complete response of 25+ months' duration and 11 with partial responses. Five patients achieving partial response with a more than 90% reduction of disease were converted to a complete response with surgery. Treatment was generally well tolerated. Median survival was 9.5 months (95% confidence interval, 6.05 to 19.38 months), with a median follow-up among survivors of 24.3 months. CONCLUSION: The combination of temozolomide plus thalidomide seems to be a promising and well-tolerated oral regimen for metastatic melanoma that merits further study.

Bergman PJ, McKnight J, Novosad A, Charney S, Farrelly J, Craft D, Wulderk M, Jeffers Y, Sadelain M, Hohenhaus AE, Segal N, Gregor P, Engelhorn M, Riviere I, Houghton AN, Wolchok JD. · Donaldson-Atwood Cancer Clinic and Flaherty Comparative Oncology Laboratory, The E&M Bobst Hospital of the Animal Medical Center, New York, New York 10021, USA. · Clin Cancer Res. · Pubmed #12684396 links to  free full text

Abstract: PURPOSE: Canine malignant melanoma (CMM) is a spontaneous, aggressive, and metastatic neoplasm. Preclinical mouse studies have shown that xenogeneic DNA vaccination with genes encoding tyrosinase family members can induce antibody and cytotoxic T-cell responses, resulting in tumor rejection. These studies provided the rationale for a trial of xenogeneic DNA vaccination in CMM using the human tyrosinase gene. EXPERIMENTAL DESIGN: Three cohorts of three dogs each with advanced (WHO stage II, III, or IV) CMM received four biweekly i.m. injections (dose levels 100, 500, or 1500 micro g, respectively/vaccination) of human tyrosinase plasmid DNA i.m. via the Biojector2000 delivery device. RESULTS: Mild local reactions at injection sites were the only toxicities observed, with no signs of autoimmunity. One dog with stage IV disease had a complete clinical response in multiple lung metastases for 329 days. Two dogs with stage IV disease had long-term survivals (421 and 588+ days) in the face of significant bulky metastatic disease, and two other dogs with locally controlled stage II/III disease had long-term survivals (501 and 496 days) with no evidence of melanoma on necropsy. Four other dogs were euthanized because of progression of the primary tumor. The Kaplan-Meier median survival time for all nine dogs was 389 days. CONCLUSIONS: The results of this trial demonstrate that xenogeneic DNA vaccination of dogs with advanced malignant melanoma is a safe and potentially therapeutic modality. On the basis of these results, additional evaluation of this novel therapeutic is warranted in locally controlled CMM and advanced human melanoma.

Hwu WJ, Krown SE, Panageas KS, Menell JH, Chapman PB, Livingston PO, Williams LJ, Quinn CJ, Houghton AN. · Departments of Medicine, Epidemiology and Biostatistics, and Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. · J Clin Oncol. · Pubmed #12039921 No free full text.

Abstract: PURPOSE: To establish a safe and tolerated regimen of an oral cytotoxic agent, temozolomide, and a cytostatic agent, thalidomide, in patients with unresectable stage III or IV malignant melanoma. PATIENTS AND METHODS: Patients with unresectable stage III or IV melanoma without brain metastases were entered successively onto four treatment cohorts: level 1, temozolomide 50 mg/m(2)/d for 6 weeks followed by a 4-week break; levels 2, 3, and 4, temozolomide 75 mg/m(2)/d for 6 weeks followed, respectively, by breaks of 4, 3, and 2 weeks. Thalidomide was started at 200 mg/d, and escalated to a maximum dose of 400 mg/d. Safety was assessed at weeks 2 and 4 and every 4 weeks thereafter; tumor response was evaluated every 8 to 10 weeks. RESULTS: Twelve patients were enrolled, three on each cohort. Therapy was generally well tolerated on all of the treatment schedules. Thalidomide at a dose of 400 mg/d was well tolerated in patients younger than 70, and 200 mg/d was well tolerated in older patients. The most common adverse events were grade 2 or 3 constipation and neuropathy, which were attributed to thalidomide. Five major responses (one complete, four partial) were documented, all at dose levels 2 to 4. Three of the five responding patients were in the over-70 age group. The median duration of response was 6 months (range, 4 to 17+ months), and the median overall survival was 12.3 months (range, 4 to 19+ months). CONCLUSION: The combination of temozolomide and thalidomide was well tolerated and had antitumor activity in patients with advanced melanoma, including elderly patients over 70 years old.

Schaed SG, Klimek VM, Panageas KS, Musselli CM, Butterworth L, Hwu WJ, Livingston PO, Williams L, Lewis JJ, Houghton AN, Chapman PB. · Swim Across America Laboratory of Tumor Immunology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. · Clin Cancer Res. · Pubmed #12006508 links to  free full text

Abstract: We conducted a randomized trial in HLA*A0201+ patientswith American Joint Committee on Cancer stage III or IV melanoma immunized with tyrosinase 368-376(370D) peptide and gp100 209-217(210M) peptide to compare the potency of three different adjuvants. Patients received 3 monthly immunizations with 500 microg of each peptide either with incomplete Freund's adjuvant (IFA), QS-21, or granulocyte macrophage colony-stimulating factor (GM-CSF). The primary end point was induction of IFN-gamma release by CD8+ T cells against tyrosinase and gp100 peptides measured by enzyme-linked immunospot assays without in vitro prestimulation measured pretreatment, 2 and 8 weeks after the third vaccination. Four of 9 and 4 of 8 patients immunized using QS-21 and GM-CSF, respectively, developed increased frequencies of CD8+ T cells against tyrosinase 370D peptide compared with 0 of 9 patients immunized using IFA (P = 0.045). T-cell responses against a gp100-related peptide showed similar results, but their relevance to T-cell reactivity against native gp100 209-217 is uncertain. These results show that: (a) QS-21 and GM-CSF are superior to IFA as immunological adjuvants for vaccination against tyrosinase 370D peptide; and (b) with appropriate adjuvants, increased frequencies of peptide-specific T cells after vaccination can be detected by enzyme-linked immunospot without prolonged prestimulation in vitro.

Lewis JJ, Janetzki S, Schaed S, Panageas KS, Wang S, Williams L, Meyers M, Butterworth L, Livingston PO, Chapman PB, Houghton AN. · Swim Across America Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. · Int J Cancer. · Pubmed #10897045 No free full text.

Abstract: The lack of reproducible, quantitative assays for T-cell responses has been a limitation in the development of cancer vaccines to elicit T-cell immunity. We utilized the Elispot assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubations. CD8(+) T-cell reactivity was determined with an interferon (IFN)-gamma Elispot assay detecting T cells at the single cell level that secrete IFN-gamma. We studied both healthy individuals and patients with melanoma. Healthy HLA-A*0201-positive individuals showed a similar mean frequency of CD8(+) cells recognizing a tyrosinase peptide, YMDGTMSQV, when compared with melanoma patients prior to immunization. The frequencies of CD8(+) cells recognizing the tyrosinase peptide remained relatively constant over time in healthy individuals. Nine HLA-A*0201-positive patients with stage IV metastatic melanoma were immunized intradermally with the tyrosinase peptide together with the immune adjuvant QS-21 in a peptide dose escalation study with 3 patients per dose group. Two patients demonstrated a significant increase in the frequency of CD8(+) cells recognizing the tyrosinase peptide during the course of immunization, from approx. 1/16,000 CD8(+) T cells to approx. 1/4,000 in the first patient and from approx. 1/14,000 to approx. 1/2,000 in the second patient. These results demonstrate that modest expansion of peptide-specific CD8(+) T cells can be generated in vivo by immunization with peptide plus QS-21 in at least a subset of patients with melanoma.

Nasi ML, Lieberman P, Busam KJ, Prieto V, Panageas KS, Lewis JJ, Houghton AN, Chapman PB. · Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. · Cytokines Cell Mol Ther. · Pubmed #10641571 No free full text.

Abstract: Dendritic cells (DCs) are the main antigen-presenting cells in the skin. We hypothesized that intradermal (i.d.) injection of granulocyte-macrophage colony-stimulating factor (GM-CSF) would recruit DCs into melanoma skin metastases and enhance autologous melanoma antigen presentation to host T cells. Sixteen patients with cutaneous or subcutaneous melanoma metastases were treated with GM-CSF injected i.d. into a single dermal metastasis and into a normal skin site for 10 consecutive days at one of four dose levels (10, 20, 40, or 80 microg/injection). Pretreatment and post-treatment skin and tumor biopsies were stained for a panel of T-cell, B-cell, macrophage, and DC immunohistochemical markers. Positive cells were quantitated in a blinded fashion. There was a significant increase in the number of DCs (HLA-DR+, S100+, factor XIIIa+) and CD45R0+ T cells in the skin and in the tumors Injected with GM-CSF at all dose levels. Uninjected control tumors showed no increase in HLA-DR+ cells or T-cell infiltrate, but did show an Increase in S100+ and factor XIIIa+ cells, suggesting a non-DC population. ID GM-CSF administered in this manner recruited DCs into melanoma tumors and normal skin. Although no antitumor effects were seen, this represents a potential method of preparing skin sites for vaccine delivery.

Chapman PB, Einhorn LH, Meyers ML, Saxman S, Destro AN, Panageas KS, Begg CB, Agarwala SS, Schuchter LM, Ernstoff MS, Houghton AN, Kirkwood JM. · Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. · J Clin Oncol. · Pubmed #10561349 No free full text.

Abstract: PURPOSE: Several single-institution phase II trials have reported that the Dartmouth regimen (dacarbazine, cisplatin, carmustine, and tamoxifen) can induce major tumor responses in 40% to 50% of stage IV melanoma patients. This study was designed to compare the overall survival time, rate of objective tumor response, and toxicity of the Dartmouth regimen with standard dacarbazine treatment in stage IV melanoma patients. PATIENTS AND METHODS: In this multicenter phase III trial, 240 patients with measurable stage IV melanoma were randomized to receive the Dartmouth regimen (dacarbazine 220 mg/m(2) and cisplatin 25 mg/m(2) days 1 to 3, carmustine 150 mg/m(2) day 1 every other cycle, and tamoxifen 10 mg orally bid) or dacarbazine 1, 000 mg/m(2). Treatment was repeated every 3 weeks. Patients were observed for tumor response, survival time, and toxicity. RESULTS: Median survival time from randomization was 7 months; 25% of the patients survived > or = 1 year.There was no difference in survival time between the two treatment arms when analyzed on an intent-to-treat basis or when only the 231 patients who were both eligible and had received treatment were considered. Tumor response was assessable in 226 patients. The response rate to dacarbazine was 10.2% compared with 18.5% for the Dartmouth regimen (P =.09). Bone marrow suppression, nausea/vomiting, and fatigue were significantly more common in the Dartmouth arm. CONCLUSION: There was no difference in survival time and only a small, statistically nonsignificant increase in tumor response for stage IV melanoma patients treated with the Dartmouth regimen compared with dacarbazine. Dacarbazine remains the reference standard treatment for stage IV melanoma.

Yao TJ, Meyers M, Livingston PO, Houghton AN, Chapman PB. · Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. · Clin Cancer Res. · Pubmed #9918205 links to  free full text

Abstract: BEC2 is an anti-idiotypic mouse monoclonal antibody that mimics GD3 ganglioside. Previous clinical trials demonstrated that intradermal immunization using 2.5 mg of BEC2 with BCG or i.v. immunization with 10 mg of BEC2 can induce anti-GD3 antibodies in a subset of patients. We hypothesized that combining these two immunization strategies might be more effective in inducing anti-GD3 antibodies and that conjugation of BEC2 to keyhole limpet hemocyanin (KLH) would further enhance the immunogenicity of BEC2. In this clinical trial, 18 melanoma patients who were free of disease after complete surgical resection within 1-6 months received intradermal immunizations on weeks 0, 2, 4, 6, and 10 with 2.5 mg of BEC2 conjugated to KLH and mixed with BCG (BEC2-KLH/BCG). Booster immunizations of 10 mg of unconjugated BEC2 were administered i.v. on weeks 24, 37, and 50. Four of 18 patients (22%) developed IgM anti-GD3 antibodies. No IgG anti-GD3 antibodies were detected. All four responding patients developed anti-GD3 IgM during immunization with BEC2-KLH/BCG; only one patient demonstrated a reboost of the IgM anti-GD3 titer during the i.v. immunizations. Thirteen of the patients are free of melanoma (3 after undergoing re-resection for local relapse); 14 patients (78%) remain alive with a median follow-up of 28 months. These results confirm our previous trial, showing that BEC2 with BCG can induce anti-GD3 antibodies in patients. The data do not provide evidence that conjugation to KLH increases the immunogenicity of BEC2 when it is administered with BCG.

Yuan J, Ku GY, Gallardo HF, Orlandi F, Manukian G, Rasalan TS, Xu Y, Li H, Vyas S, Mu Z, Chapman PB, Krown SE, Panageas K, Terzulli SL, Old LJ, Houghton AN, Wolchok JD. · Ludwig Center for Cancer Immunotherapy, Immunology Program, Sloan-Kettering Institute, New York, NY, USA. · Cancer Immun. · Pubmed #19496531 links to  free full text

Abstract: A differentiation antigen commonly expressed on melanoma cells, gp100 is the target of infiltrating T cells. We conducted a phase I randomized cross-over trial of melanoma patients with either xenogeneic (mouse) or human gp100 plasmid DNA injected intramuscularly at three dosages (100, 500 or 1,500 microg) every three weeks for three doses. After the first three injections, patients were then immunized three times with gp100 from the other species. Peripheral blood samples were analyzed at various time points following 10-day culture with gp100 peptides using multi-parametric flow cytometry. A total of 19 patients were enrolled, with 18 assessable for immune function and survival. 14 (74%) were male, with a median age of 56 years (range, 20-82). All patients had no evidence of disease; 10 (53%) had stage III disease, 3 each (16%) had stage IIB and IV disease, 2 (11%) had choroidal and 1 (5%) had anal mucosal involvement. With a median follow-up of 30 months, median progression-free survival (PFS) is 44 months. Median survival is not reached. There was no grade 3/4 toxicity; the most common grade 1/2 toxicity was an injection site reaction in 12 patients (63%, all grade 1). Five patients developed CD8+ cells binding gp100(280-288) HLA-A2-restricted tetramer. One patient had an increase in CD8+ IFN-gamma+ cells. This xenogeneic immunization strategy was safe and associated with minimal toxicity. There was also evidence of immune response.

Hirschhorn-Cymerman D, Rizzuto GA, Merghoub T, Cohen AD, Avogadri F, Lesokhin AM, Weinberg AD, Wolchok JD, Houghton AN. · Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. · J Exp Med. · Pubmed #19414558 No free full text.

Abstract: Expansion and recruitment of CD4(+) Foxp3(+) regulatory T (T reg) cells are mechanisms used by growing tumors to evade immune elimination. In addition to expansion of effector T cells, successful therapeutic interventions may require reduction of T reg cells within the tumor microenvironment. We report that the combined use of the alkylating agent cyclophosphamide (CTX) and an agonist antibody targeting the co-stimulatory receptor OX40 (OX86) provides potent antitumor immunity capable of regressing established, poorly immunogenic B16 melanoma tumors. CTX administration resulted in tumor antigen release, which after OX86 treatment significantly enhanced the antitumor T cell response. We demonstrated that T reg cells are an important cellular target of the combination therapy. Paradoxically, the combination therapy led to an expansion of T reg cells in the periphery. In the tumor, however, the combination therapy induced a profound T reg cell depletion that was accompanied by an influx of effector CD8(+) T cells leading to a favorable T effector/T reg cell ratio. Closer examination revealed that diminished intratumoral T reg cell levels resulted from hyperactivation and T reg cell-specific apoptosis. Thus, we propose that CTX and OX40 engagement represents a novel and rational chemoimmunotherapy.

Duan F, Lin Y, Liu C, Engelhorn ME, Cohen AD, Curran M, Sakaguchi S, Merghoub T, Terzulli S, Wolchok JD, Houghton AN. · Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA. · Cancer Res. · Pubmed #19351857 No free full text.

Abstract: How the immune system recognizes and responds to mutations expressed by cancer cells is a critical issue for cancer immunology. Mutated self-polypeptides are particularly strong tumor-specific rejection antigens for natural tumor immunity, but we know remarkably little about T-cell responses to mutated self during tumor growth in vivo, including levels of response, kinetics, and correlates that predict tumor rejection. To address these questions, a mutated self-antigen, designated tyrosinase-related protein 1 (Tyrp1)-WM, derived from Tyrp1 was expressed in the poorly immunogenic, spontaneously arising B16 melanoma and the immunogenic, chemically induced LiHa fibrosarcoma. Syngeneic mice challenged with LiHa fibrosarcoma cells expressing Tyrp1-WM, but not native Tyrp1, induced specific CD8(+) and CD4(+) T-cell responses against defined mutated epitopes in tumor-draining lymph nodes and in tumors. Subsequently, specific CD8(+) T-cell responses contracted as a minority of tumors progressed. B16 melanomas expressing Tyrp1-WM induced minimal T-cell responses, and no tumor immunity was detected. Treatment with an agonist monoclonal antibody against glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) increased the level of CD8(+) T cells recognizing a peptide derived from the Tyrp1-WM sequence and the proportion of mice rejecting tumors. These results show that B16 tumors expressing mutations that generate strongly immunogenic epitopes naturally induce T-cell responses, which are insufficient to reject tumors. Immune modulation, such as inducing GITR signaling, is required to enhance CD8(+) T-cell responses to specific mutations and to lead to tumor rejection.

Rizzuto GA, Merghoub T, Hirschhorn-Cymerman D, Liu C, Lesokhin AM, Sahawneh D, Zhong H, Panageas KS, Perales MA, Altan-Bonnet G, Wolchok JD, Houghton AN. · Departments of Medicine and Immunology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. · J Exp Med. · Pubmed #19332877 No free full text.

Abstract: A primary goal of cancer immunotherapy is to improve the naturally occurring, but weak, immune response to tumors. Ineffective responses to cancer vaccines may be caused, in part, by low numbers of self-reactive lymphocytes surviving negative selection. Here, we estimated the frequency of CD8(+) T cells recognizing a self-antigen to be <0.0001% ( approximately 1 in 1 million CD8(+) T cells), which is so low as to preclude a strong immune response in some mice. Supplementing this repertoire with naive antigen-specific cells increased vaccine-elicited tumor immunity and autoimmunity, but a threshold was reached whereby the transfer of increased numbers of antigen-specific cells impaired functional benefit, most likely because of intraclonal competition in the irradiated host. We show that cells primed at precursor frequencies below this competitive threshold proliferate more, acquire polyfunctionality, and eradicate tumors more effectively. This work demonstrates the functional relevance of CD8(+) T cell precursor frequency to tumor immunity and autoimmunity. Transferring optimized numbers of naive tumor-specific T cells, followed by in vivo activation, is a new approach that can be applied to human cancer immunotherapy. Further, precursor frequency as an isolated variable can be exploited to augment efficacy of clinical vaccine strategies designed to activate any antigen-specific CD8(+) T cells.