Statistical controversies in clinical research: early-phase adaptive design for combination immunotherapies.

Background: In recent years, investigators have asserted that the 3 + 3 design lacks flexibility, making its use in modern early-phase trial settings, such as combinations and/or biological agents, inefficient. More innovative approaches are required to address contemporary research questions, such as those posed in trials involving immunotherapies. Design: We describe the implementation of an adaptive design for identifying an optimal treatment regimen, defined by low toxicity and high immune response, in an early-phase trial of a melanoma helper peptide vaccine plus novel adjuvant combinations. Results: Operating characteristics demonstrate the ability of the method to effectively recommend optimal regimens in a high percentage of trials with reasonable sample sizes. Conclusions: The proposed design is a practical, early-phase, adaptive method for use with combined immunotherapy regimens. This design can be applied more broadly to early-phase combination studies, as it was used in an ongoing study of two small molecule inhibitors in relapsed/refractory mantle cell lymphoma.

An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins.

T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum.

The class I antigen-processing pathway for the membrane protein tyrosinase involves translation in the endoplasmic reticulum and processing in the cytosol.

Formation of major histocompatibility complex class I-associated peptides from membrane proteins has not been thoroughly investigated. We examined the processing of an HLA-A*0201-associated epitope, YMDGTMSQV, that is derived from the membrane protein tyrosinase by posttranslational conversion of the sequence YMNGTMSQV. Only YMDGTMSQV and not YMNGTMSQV was presented by HLA-A*0201 on cells expressing full-length tyrosinase, although both peptides have similar affinities for HLA-A*0201 and are transported by TAP. In contrast, translation of YMNGTMSQV in the cytosol, as a minigene or a larger fragment of tyrosinase, led to the presentation of the unconverted YMNGTMSQV. This was not due to overexpression leading to saturation of the processing/conversion machinery, since presentation of the converted peptide, YMDGTMSQV, was low or undetectable. Thus, presentation of unconverted peptide was associated with translation in the cytosol, suggesting that processing of the full-length tyrosinase occurs after translation in the endoplasmic reticulum. Nevertheless, presentation of YMDGTMSQV in cells expressing full-length tyrosinase was TAP (transporter associated with antigen processing) and proteasome dependent. After inhibition of proteasome activity, tyrosinase species could be detected in the cytosol. We propose that processing of tyrosinase involves translation in the endoplasmic reticulum, export of full-length tyrosinase to the cytosol, and retransport of converted peptides by TAP for association with HLA-A*0201.

Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines.

Of several thousand peptides presented by the major histocompatibility molecule HLA-A2.1, at least nine are recognized by melanoma-specific cytotoxic T lymphocytes (CTLs). Tandem mass spectrometry was used to identify and to sequence one of these peptide epitopes. Melanoma-specific CTLs had an exceptionally high affinity for this nine-residue peptide, which reconstituted an epitope for CTL lines from each of five different melanoma patients tested. Recognition by multiple CTL lines suggests that this may be a promising candidate for use in peptide-based melanoma vaccines.

Direct analysis of tumor-associated peptide antigens.

Adoptive immunotherapy with tumor-specific cytotoxic T lymphocytes (CTLs) can induce tumor regressions in animals and in human cancer patients. Antigens recognized by CTLs from cancer patients are being sought as possible immunogens, a number of which have been identified during the past year. The ultimate result may be the development of novel peptide-based immunotherapies and a new understanding of the T-cell response to human cancer.

Human cytotoxic T cells specific for autologous melanoma cells: successful generation from lymph node cells in seven consecutive cases.

Human T-cell populations specifically cytotoxic for autologous melanoma cells have been successfully generated from lymph node cells obtained from seven consecutive patients. The lymph node cells were stimulated in vitro with autologous irradiated melanoma cells; stimulation was repeated every 10-15 days at a tumor cell-to-lymphocyte ratio of approximately 1:20. Cytotoxic activity was assessed by a 4-hour 51Cr release assay. Mean lysis of autologous tumor cells was 47% at an effector-to-target cell ratio of 20:1, while mean lyses of the human myeloid leukemia cell line K562, allogeneic melanoma cells, and an osteosarcoma cell were 20%, 13%, and 11%, respectively. There was no lysis of autologous fibroblasts, fresh lymphocytes, or phytohemagglutinin-stimulated blasts. Three grades of specificity developed sequentially. In grade I, lysis of autologous tumor cells exceeded lysis of allogeneic tumor cells but did not exceed lysis of K562 cells. In grade II, lysis of autologous tumor cells exceeded lysis of K562 cells and all allogeneic tumor cells tested. In grade III, potent lysis of autologous tumor cells (greater than 40%) exceeded lysis of K562 cells and of all allogeneic tumor cells tested. All seven lymphocyte populations reached or exceeded grade I. Six reached or exceeded grade II. Two progressed to grade III. The generated cells were T cells, as determined by phenotypic analysis with flow cytometry. CD4+ cells and CD8+ cells accounted for 83%-100% of the cells. CD8+ T cells were separated from CD4+ T cells by panning with OKT8 and OKT4 antibodies. The resulting CD8-enriched and CD4-enriched populations were compared as effectors in cytotoxicity assays. The results suggest that the cell responsible for lysis of autologous tumor cells is CD8+. The methods used in this study have repeatedly resulted in the successful generation of cytotoxic T lymphocytes specifically cytotoxic for autologous melanoma cells; it is suggested that these cells have potential application for adoptive immunotherapy of melanoma.

Generation of human autologous melanoma-specific cytotoxic T-cells using HLA-A2-matched allogeneic melanomas.

Autologous tumor-specific cytotoxic T-lymphocytes (CTLs), generated by repeated stimulation with autologous melanoma and expanded in interleukin 2, are major histocompatibility complex restricted. These CTLs recognize a common tumor-associated antigen in the presence of HLA class I determinants, suggesting that allogeneic melanomas which express the restricting HLA-A region antigen could substitute for the autologous tumor in the generation of CTLs. This was investigated in the HLA-A2 system. Four T-cell lines were established by stimulation of lymphocytes with either autologous tumor or an HLA-A2-matched allogeneic melanoma. Allogeneic stimulated CTLs specifically lysed the autologous tumor and demonstrated an identical pattern of HLA-A2 restriction, when compared to the autologous stimulated CTLs. Lysis by the allogeneic stimulated CTLs was blocked by a monoclonal antibody to HLA class I antigens; lysis was also inhibited by both autologous tumor or HLA-A2 allogeneic melanomas when evaluated in cold target competition studies. The allogeneic stimulated CTLs proliferated in response to both autologous tumor and HLA-A2 melanomas, but not in response to HLA-A2 nonmelanomas. By phenotypic analysis these CTLs were CD3+ and predominantly CD8+ cells. We conclude that autologous tumor-specific CTLs can be generated using HLA-A region-matched allogeneic melanomas for stimulation. Since established, HLA-typed melanoma tumor lines can be used in the absence of autologous tumor; this procedure can be applied clinically to a broad patient population and may prove useful in the adoptive immunotherapy of melanoma.

Cytotoxic T-lymphocyte response to autologous human squamous cell cancer of the lung: epitope reconstitution with peptides extracted from HLA-Aw68.

Cytotoxic T-lymphocytes (CTLs) specific for autologous human squamous cell cancer of the lung were generated by stimulation of peripheral blood lymphocytes with autologous tumor cells in vitro. The CTL line was >97% CD3+, CD8+, CD16- and produced tumor necrosis factor-alpha, gamma-interferon, and granulocyte-macrophage colony-stimulating factor after stimulation with autologous tumor. The CTLs lysed autologous tumor but failed to recognize autologous or histocompatibility leukocyte antigen-matched lymphoid cells, K562, or allogeneic tumor cells of several histological types. Antibody-blocking studies suggested that the CTLs recognized one or more antigens presented by the class I major histocompatibility complex molecule Aw68. To characterize these antigens further, histocompatibility leukocyte antigen Aw68 molecules were extracted from the squamous cell cancer of the lung tumor line by immunoaffinity chromatography, and the associated peptides were eluted in acid and separated by reversed-phase high-performance liquid chromatography. Reconstitution of the CTL epitope was evaluated by adding these peptides to autologous Epstein-Barr virus-transformed B-cells. Two peaks of reconstituting activity were observed, suggesting that these CTLs recognize at least two Aw68-associated peptides. This study confirms the existence of a CTL response against autologous human squamous cell cancer of the lung and suggests that this CTL response is directed against peptide epitopes presented by the class I major histocompatibility complex molecules. It is anticipated that this approach will permit identification of peptide epitopes for lung cancer-specific CTLs.

The peptide recognized by HLA-A68.2-restricted, squamous cell carcinoma of the lung-specific cytotoxic T lymphocytes is derived from a mutated elongation factor 2 gene.

The identification of naturally processed tumor peptides that can stimulate a tumor-specific, CTL response is crucial to the development of a vaccine-based, immunotherapeutic approach to cancer treatment. One type of cancer in which a tumor-specific, CTL response has been observed is squamous cell carcinoma of the lung. In the system investigated here, the tumor-specific CTLs are HLA-A68.2 restricted. Immunoaffinity chromatography was used to isolate the HLA-A68.2 molecules from the tumor cell line, and peptide was eluted with acid from the HLA-A68.2 molecules and subjected to three rounds of separation by reversed phase-high performance liquid chromatography (RP-HPLC). To determine which fractions contained the peptide recognized by the tumor-specific CTLs, an aliquot of each RP-HPLC fraction was added to the autologous, B-lymphoblastoid cell line, and the cells were then tested as targets for tumor-specific CTLs. After the third round of RP-HPLC, mass spectrometry was used to sequence individual peptide candidates, and a peptide with a m/z of 497 was identified as the active peptide. Collision-activated dissociation of m/z 497 allowed identification of the peptide sequence as ETVSEQSNV. With the exception of a single amino acid difference (glutamic acid versus glutamine as the sixth position in the peptide), this peptide is identical to residues 581 to 589 of elongation factor 2. The PCR was used to amplify the elongation factor 2 gene in both the tumor cells and the autologous B cell line, and DNA sequencing of the products revealed the presence of a heterozygous mutation in the tumor cells that accounts for the difference between the two peptide sequences. Although a similar analysis did not reveal the presence of the mutation in three additional lung cell carcinomas, this does not rule out the possibility that a survey of a larger population of tumor cells would reveal the presence of the mutation at a low frequency. These results demonstrate the utility of this approach for identifying tumor-specific antigens that are the targets of a CTL response.

Patient preferences for adjuvant interferon alfa-2b treatment.

PURPOSE: Although trials of adjuvant interferon alfa-2b (IFN alpha-2b) in high-risk melanoma patients suggest improvement in disease-free survival, it is unclear whether treatment offers improvement in overall survival. Widespread use of adjuvant IFN alpha-2b has been tempered by its significant toxicity. To quantify the trade-offs between IFN alpha-2b toxicity and survival, we assessed patient utilities for health states associated with IFN therapy. Utilities are measures of preference for a particular health state on a scale of 0 (death) to 1 (perfect health). PATIENTS AND METHODS: We assessed utilities for health states associated with adjuvant IFN among 107 low-risk melanoma patients using the standard gamble technique. Health states described four IFN alpha-2b toxicity scenarios and the following three posttreatment outcomes: disease-free health and melanoma recurrence (with or without IFN alpha-2b) leading to cancer death. We also asked patients the improvement in 5-year disease-free survival they would require to tolerate IFN. RESULTS: Utilities for melanoma recurrence with or without IFN alpha-2b were significantly lower than utilities for all IFN alpha-2b toxicities but were not significantly different from each other. At least half of the patients were willing to tolerate mild-moderate and severe IFN alpha-2b toxicity for 4% and 10% improvements, respectively, in 5-year disease-free survival. CONCLUSION: On average, patients rate quality of life with melanoma recurrence much lower than even severe IFN alpha-2b toxicity. These results suggest that recurrence-free survival is highly valued by patients. The utilities measured in our study can be applied directly to quality-of-life determinations in clinical trials of adjuvant IFN alpha-2b to measure the net benefit of therapy.

Analysis of a natural immune response against tumor antigens in a melanoma survivor: lessons applicable to clinical trial evaluations.

The long-term survival of some patients with metastatic melanoma may be attributable in part to cellular immune responses to melanoma antigens. However, little is known about the level of CTL reactivity in vivo that is required for immunological control of tumor progression. In the present report, T-cell responses were evaluated with lymphocytes obtained from tumor-involved nodes and peripheral blood of a long-term melanoma survivor. Using an ELISPOT assay, naturally occurring functional T cells, which recognize the peptide ALLAVGATK (gp100(17-25)) plus two other HLA-A3 restricted peptides, were detected in a tumor-involved lymph node. The ALLAVGATK-reactive T cells were also evaluated by MHC-tetramers staining and were found to be CD8+ CD45RO+ L-selectin(-) CD11a+, suggesting that they are antigen experienced and have a memory phenotype. Unstimulated peripheral blood lymphocytes from the same patient demonstrated no detectable T-cell responses; however, a single stimulation with ALLAVGATK peptide in vitro resulted in a dramatic expansion of peptide-reactive CTLs. This patient, with evidence of tumor-reactive CTLs targeted to several tumor antigens in a tumor-involved lymph node and with evidence of a circulating memory T-cell response, has remained disease-free for 6 years, despite prior bulky nodal metastasis. In contrast, three HLA-A3+ patients with rapidly progressive metastatic melanoma had no detectable T-cell response in tumor-involved nodes or peripheral blood lymphocytes, even after peptide stimulation ex vivo. The presented data are consistent with a systemic polyvalent immune response against tumor in this long-term survivor. These data provide an estimate of the level of CTL response that may be associated with protection from tumor recurrence.

Phase I trial of a melanoma vaccine with gp100(280-288) peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes.

A melanoma vaccine composed of HLA-A2-restricted peptide YLEPGPVTA (gp100(280)), with or without a modified T-helper epitope from tetanus toxoid AQYIKANSKFIGITEL, has been evaluated in a Phase I trial to assess safety and immunological response. The vaccines were administered s.c. in either of two adjuvants, Montanide ISA-51 or QS-21, to 22 patients with high-risk resected melanoma (stage IIB-IV). Local and systemic toxicities were mild and transient. We detected CTL responses to the gp100(280) peptide in peripheral blood in 14% of patients. Helper T-cell responses to the tetanus helper peptide were detected in 79% of patients and had a Th1 cytokine profile. One patient with a CTL response to gp100 had a recurrence in a lymph node 2 years later; her nodes contained CD8+ cells reactive to gp100(280) (0.24%), which proliferated in response to peptide. The overall survival of patients is 75% (95% confidence interval, 57-94%) at 4.7 years follow-up, which compares favorably with expected survival. Four of 14 patients who completed at least six vaccines subsequently developed metastases, all of which were solitary and surgically resectable. They remain alive and clinically free of disease at last follow-up. Data from this trial demonstrate immunogenicity of the gp100(280) peptide and suggest that immune responses may persist long-term in some patients. The frequency and magnitude of the CTL response may be improved with more aggressive vaccination regimens. Although this Phase I study was not intended to evaluate clinical benefit, the excellent survival of patients on this protocol suggests the possibility of a benefit that should be assessed in future studies.

Multiple primary melanoma: incidence and risk factors in 283 patients.

To identify patients with melanoma at risk for a second primary lesion and to assess the prognostic relevance of multiple primary lesions, 7816 patients treated for malignant melanoma have been reviewed. Two to nine primary lesions were identified in 283 patients (3.6%): two lesions were identified in 82% of these 283 patients, three lesions in 11%, and four lesions in 3%. Sixty-four percent were metachronous. Among patients with melanoma, the 10-year actuarial risk of a second primary lesion was 5%; a third of that risk was expressed within 3 months of the initial diagnosis plus a subsequent risk of 0.38% per year. Risk factors for multiple primary lesions were family history of melanoma, thin primaries, male sex, Celtic complexion, and a history of another cancer. Patients with a family history of melanoma had a 14% risk of a second primary lesion during the first 10 years after diagnosis. By univariate and multivariate analyses, there was no survival disadvantage for patients with multiple primary lesions. After median follow-ups of 3.7 and 4.8 years, respectively, 51.7% and 50.5% of the groups with single and multiple primaries were disease free. Mortality rates were 31% and 25%, respectively. It is appropriate to base therapeutic decisions and prognostic evaluations on the specific risk factors of each individual lesion. Identification of patients at high risk for multiple primary lesions may permit early diagnosis and improved outcomes.

Anorectal melanoma: clinical characteristics and results of surgical management in twenty-four patients.

Twenty-four patients with primary anorectal melanoma diagnosed since 1974 have been studied retrospectively. There was a predominance of women (2.4:1): Mean age was 64 years. The most common initial symptom was rectal bleeding, typically misdiagnosed as hemorrhoids. Seventeen patients had stage I cancer when first seen, four had stage II, and three had distant disease. Progressive disease most commonly started as a large pelvic mass, diffuse bilateral pulmonary nodules, or diffuse liver metastases. Twenty patients (83%) died of the disease; none survived more than 6 years. Among the patients who died of the disease, median survival was 1.9 years. Median survival for patients with stage I disease (29 months) was longer than that for patients with stage II disease (11 months; p less than 0.05) or stage III disease (9 months). Twelve patients were treated initially with abdominoperineal resection (APR), and three additional patients underwent APR to manage recurrent local or regional disease that developed later in the clinical course. Among evaluable patients with stage I disease initially managed with APR, 50% developed recurrent local-regional disease (mean disease-free interval, 23 months), compared with 100% of those who underwent more limited surgery (mean disease-free interval, 16 months). Even after APR, however, distant metastases were common, and there was no prolongation of survival for patients treated with APR. Primary melanoma of the anorectum has a high metastatic potential and carries a grave prognosis. APR appears to have some effect in controlling local and regional disease, but prolongation of survival will depend on both earlier diagnosis and development of more successful therapeutic approaches.

Human T cells specifically activated against autologous malignant melanoma.

Lymphocytes from ten patients with melanoma were specifically stimulated in vitro with autologous melanoma cells and expanded in interleukin 2. Significant lysis of autologous melanoma cells was demonstrated in T cells derived from six of these patients. The mean percent of lysis of autologous tumor cells at an effector-target ratio of 20:1 was 46% among these six patients. The T cells derived from two patients developed specificity in lysing autologous melanoma cells. In both cases, specificity was enhanced by the in vitro stimulation with autologous tumor cells. Restimulation with autologous melanoma cells was associated with increasing specificity over time. Whether derived from peripheral blood lymphocytes or from lymph node cells, T cells from one patient lysed fresh autologous melanoma cells more potently than K562, allogeneic melanoma cells, and nonmelanoma cells. On day 38, at an effector-target ratio of 10:1, cell lysis of K562, an osteosarcoma, a pancreatic cancer, and three allogeneic melanomas was 3%, 4%, 7%, 8%, 7%, and 2%, respectively, while lysis of autologous melanoma cells was 47%. Specificity was maintained beyond day 60. The T cells could be expanded over 50-fold within one month.

Gamma probe guided biopsy of the sentinel node in malignant melanoma: a multicentre study.

Sentinel lymph node biopsy was attempted in 336 patients with clinically node-negative cutaneous melanoma. All patients were injected with technetium-99m labelled radiocolloid, with 108 patients simultaneously receiving vital blue dye for sentinel node identification. Sentinel lymph nodes were identified in 329 patients, giving a technical success rate of 97.9%. Metastatic disease was identified in 39 (11.9%) of the patients in whom sentinel nodes were found. Patients with negative sentinel nodes were observed and patients with positive sentinel nodes underwent comprehensive lymph node dissection. The presence of metastatic disease in the sentinel nodes and primary tumour depth by Breslow or Clark levels were joint predictors of survival based on Cox proportional hazards modelling. Disease recurrences occurred in 26 (8.8%) patients with negative sentinel lymph nodes, with isolated regional recurrences as the first site in 10 (3.4%). No patients with Clark level II primary tumours were found to have positive sentinel nodes or disease recurrences. One patient with a thin (<0.75 mm) Clark level III primary had metastatic disease in a sentinel node. Patients with metastases confined to the sentinel nodes had similar survival rates regardless of the number of nodes involved.

Toxic shock syndrome after inguinal hernia repair. Report of a case with patient survival.

A 40-year-old man developed fulminant multisystem failure several days after elective repair of an inguinal hernia. Toxic shock syndrome (TSS) was diagnosed. There was, however, no evidence of wound infection at the time of multisystem failure. Only later in his hospital course did the wound drain. Staphylococcus aureus was cultured from the wound and was the presumed etiologic agent in the patient's life-threatening illness. The patient recovered fully with supportive care, antibiotics, and surgical debridement of the inguinal hernia site. This case is discussed in the context of existing literature on the toxic shock syndrome. The site of infection is typically nonsuppurative, but the systemic manifestations are typically life threatening. The responsible organism is commonly believed to be a strain of S. aureus that expresses a toxin (TSS toxin-1) that effects multisystem failure, but which also diminishes the local inflammatory response and explains the benign appearance of the wound. Although this is a rare clinical entity, elective surgical procedures complicated by fatal TSS have been reported. Surgeons should understand this disease and the management necessary to avert mortality.

Tumor antigens and tumor vaccines: peptides as immunogens.

Tumor antigens recognized by human cytotoxic T lymphocytes (CTL) have been identified for multiple types of solid tumors. These include both shared and unique antigens. Unique antigens are those expressed uniquely by one patient's tumor, and shared antigens are those present on tumor cells from many different patients. Many of the shared antigens are derived from tissue-specific differentiation antigens, oncogenes, or a set of antigens expressed only in tumors or in testis. In addition to advances in understanding tumor antigens that stimulate CTL and T-helper cell responses, there have been advances in understanding immunity in general, including the characterization of cytokines, the recognition of the dendritic cell as an optimal antigen-presenting cell (APC), and the characterization of costimulatory molecules as critical components of antigen presentation. Together, these developments have breathed new life into tumor immunology, and they promise to lead to a new generation of peptide- and cell-based tumor vaccines.

Targeting unique tumor antigens and modulating the cytokine environment may improve immunotherapy for tumors with immune escape mechanisms.

Cytotoxic T-cell responses to shared tumor antigens have been characterized for several tumor types, and the MHC-associated peptides that comprise these antigens have been defined at a molecular level. These provide new tools to determine whether immune responses can be generated with these tumor antigens, and there are data to suggest that such immune responses can be generated. However, it is also clear that tumor cells can evade immune responses directed against some shared antigens, by downregulating expression of MHC or of the antigenic protein(s), as well as by more active methods such as secretion of immunosuppressive cytokines. Awareness of these mechanisms of immune escape will help to direct development of the next generation of tumor vaccines. Targeting unique antigens and modulating the cytokine environment likely will be critical to comprehensive vaccine systems in the future.