Optimization of intracerebral tumour protection by active-specific immunization against murine melanoma B16/G3.12.

Development of brain metastases despite extracerebral response to systemic immunotherapy is a common problem in melanoma patients. We have previously described a murine melanoma vaccine of interferon-gamma (IFNgamma)-treated, irradiated syngeneic B16/G3.12 and allogeneic (Cloudman) melanoma cells, plus the adjuvant DETOX, that is protective against subcutaneous (93%) or intracerebral (69%) syngeneic challenge. This study aimed to optimize this vaccine. Groups of nine or 10 mice were immunized five times in 5 weeks with: (i) complete vaccine +/- IFNgamma (VAC+, VAC-); (ii) syngeneic 2 x 106 G3.12 cells plus DETOX (Syn+D), (iii) 2 x 106 allogeneic Cloudman cells plus DETOX (Allo+D); (iv) VAC+ without DETOX (no DETOX); (v) DETOX alone (DETOX); or (vi) phosphate buffered saline (PBS). Mice were challenged subcutaneously with 104 viable G3.12 (or Cloudman cells) and after 35 days intracerebrally with 104 G3.12 cells. Expression of H-2 antigens (measured using fluorescence-activated cell sorting), splenocyte cytotoxicity (measured using 51Cr release) and median overall survival (OAS) were analysed using the log-rank test. VAC+, VAC- and G3.12 mice were equally protected from subcutaneous (s.c.) and intracerebral (i.c.) melanoma challenge (OAS 65 days for s.c., 30 days for i.c.). Protection was less (P < 0.05) in DETOX mice (48 days for s.c.), PBS mice (47 days for s.c., 21 days for i.c.) or no DETOX mice (51 days for s.c.). Allo+D mice showed s.c. (59 days) but not i.c. protection (20 days). IFNgamma incubation did not increase the effect in either the challenge cells or the vaccine cells (P > 0.05). Specific cytotoxicity was seen with G3.12 targets in VAC+ (27%) but not PBS (2%; P < 0.05) mice with equal NK (YAC-1) lysis (10% versus 7%; P< 0.05). Optimal protection against s.c./i.c. experimental murine melanoma was yielded by irradiated syngeneic cells plus DETOX. DETOX alone was not active. Upregulation of H-2 antigens with IFNgamma under these conditions does not augment protection.

The N-terminal ATPase site in the large terminase protein gp17 is critically required for DNA packaging in bacteriophage T4.

Double-stranded DNA packaging in bacteriophages is apparently driven by the most powerful molecular motor ever measured. Although it is widely accepted that a translocating ATPase powers the DNA packaging machine, the identity of the ATPase that generates this driving force is unknown. Evidence suggests that the large terminase protein gp17, which possesses two consensus ATP binding motifs and an ATPase activity, is a strong candidate for the translocating ATPase in bacteriophage T4. This hypothesis was tested by a PCR-directed combinatorial mutagenesis approach in which mutant libraries consisting of all possible codon combinations were constructed at the signature residues of the ATP binding motifs. The impact on gp17 function of each randomly selected mutant was evaluated by phenotypic analysis following recombinational transfer into the viral genome. The precise mutation giving rise to a particular phenotype was determined by DNA sequencing. The data showed that the N-terminal ATP binding site I (SRQLGKT(161-167)), but not the ATP binding site II (TAAVEGKS(299-306)), is critical for gp17 function. Even conservative substitutions such as G165A, K166R, and T167A were not tolerated at the GKT signature residues, which are predicted to interact with the ATP substrate. Biochemical analyses of the mutants showed a complete loss of in vitro DNA packaging activity but not the terminase (DNA-cutting) activity. The purified K166G mutant showed a loss of gp17-ATPase activity. The data, for the first time, implicated a specific ATPase center in the viral dsDNA packaging.

A novel melanoma gene (MG50) encoding the interleukin 1 receptor antagonist and six epitopes recognized by human cytolytic T lymphocytes.

We identified a novel 8.1-kb human melanoma gene, MG50, derived from subtractive hybridization with a squamous lung carcinoma cell line, LU-1. 6.8 kb containing an open reading frame were sequenced, and the structure of the encoded 1496 amino acid protein was deduced. With HLA-A2.1-transduced Drosophila cells as antigen-presenting cells, we identified six epitopes restricted by HLA-A2.1 that elicited CTLs in vitro. Reactivity of the CTLs to melanoma cells containing MG50 indicated that the epitopes were displayed naturally. Significant cross-reactivity of CTLs immunized against a melanoma cell line that lacked HLA-A2.1 indicated that at least four of the epitopes were also recognized in a different HLA class I context, most likely HLA-A*6802. By quantitative reverse transcription, MG50 message was found in one of two skin melanoma cell lines, an ocular melanoma cell line, two of four metastatic skin melanomas, two of three mammary carcinomas, one of two colon carcinomas, and an ovarian carcinoma. Of six normal tissues, MG50 was found only in a specimen of normal skin and was absent from a congenital nevus. It is likely that MG50 is the gene for the interleukin 1 receptor antagonist because a reported sequence of cDNA from the latter had a sequence of 528 bases in the 3' region, a long contiguous base sequence, and 176 encoded amino acids identical with those of MG50. MG50 is one of the few melanoma-associated antigens that is not a differentiation antigen or a mutated protein. Because of its nature, it may prove to be important in the pathogenesis of the tumors in which it is found, as well as an immunogen and target for immunotherapy.

Synthetic insertion signal sequences enhance MHC class I presentation of a peptide from the melanoma antigen MART-1.

Cytotoxic T lymphocytes (CTL) recognize minimal peptides of eight to ten residues which are the products of intracellularly processed proteins and are presented at the cell surface by MHC class I molecules. An important step in this process is the translocation of processed proteins from the cytosol across the endoplasmic reticulum membrane mediated by transporter associated with antigen processing (TAP) proteins, or as an alternative, by endoplasmic reticulum insertion signal sequences. We report here that the addition of synthetic signal sequences at the N terminus, but not at the C terminus, of an epitope from the human melanoma antigen MART-1 greatly enhances its presentation in both TAP-deficient and TAP-expressing cells. A newly designed peptide construct, composed of the epitope replacing the hydrophobic part of a natural signal sequence, was also very effective. Interestingly, an artificial signal sequence containing the same epitope was the most efficient construct for enhancing its presentation. These peptide constructs facilitated epitope presentation when loaded into the cytosol of TAP-deficient T2 cells, TAP-expressing melanoma cells and human dendritic cells. These findings may be of practical significance for the development of synthetic anti-cancer vaccines and in vitro immunization of CTL for adoptive immunotherapy.

The role of immunohistochemistry and fluorescence in situ hybridization for HER2/neu in assessing the prognosis of breast cancer.

Immunohistochemistry has been used in the past to identify overexpression of HER-2/neu protein on the cell membrane of breast cancer cells in fixed tissues. Most studies that have attempted to find an association between HER-2/neu expression and a poor prognosis have relied on this technique, which has intrinsic variability due to the antibody used and the degradation of surface proteins by fixation. Recent studies with fluorescence in situ hybridization have tended to confirm the purported association, showing that HER-2/neu overexpression causes a more aggressive, less responsive breast cancer. In many studies, the amplification of the HER-2/neu gene was the most important variable determining outcome, independent of other variables, such as tumor size and estrogen receptor status.

Cancer vaccines: novel approaches and new promise.

Cancer vaccines are a promising tool in the hands of the clinical oncologist. We have summarized the most recent findings and achievements in this exciting field. Tumor-associated antigens, as a basis for the new cancer vaccines, are reviewed. We emphasize novel approaches for the design of safe and more effective vaccines for cancer. We also discuss the possible clinical applications and the future prospects for vaccine development.

Perspective on allogeneic melanoma lysates in active specific immunotherapy.

We have tested allogeneic melanoma cell lysates as active immunotherapy, originally in stage IV patients to determine their safety and immunologic effectiveness. Surprisingly, phase I and II trials with frozen lysates showed a 20% objective response rate, with 8% long-term survivors. Melacine (Ribi ImmunoChem Research, Hamilton, MT), a lyophilized preparation from the same two cell lines, has been tested nationally and has caused regressions in approximately 10% of patients. Long-term stabilization of disease was noted in 10% to 20% of patients in all trials. A multicenter phase III comparison of low-dose cyclophosphamide plus Melacine versus four-drug chemotherapy showed no difference in response rates and survival, with fewer and milder side effects due to Melacine. In our single-arm trial in resected stage III disease, the overall survival rate (66-month median follow-up) is 66%, with a median relapse-free survival time of 36 months. Interferon-alfa 2b (IFN-alpha) given to patients failing to respond to Melacine elicited major objective responses in a larger proportion than anticipated with IFN-alpha alone. These results stimulated current multicenter trials in stage IV and resected stage III melanoma of Melacine and IFN-alpha in combination versus IFN-alpha alone. Of scientific note were (I) identification of a new melanoma antigen from a gene (MG50) isolated from one of the immunizing cell lines, and (2) demonstration that a new melanoma arising in 1995 in a long-term survivor was immunologically and genetically distinct from her original 1986 tumor. While it is important to define which epitopes are involved, multiepitopic (polyvalent) mixtures have established the therapeutic effect of melanoma vaccines.

Anti-MUC1 class I restricted CTLs in metastatic breast cancer patients immunized with a synthetic MUC1 peptide.

Sixteen metastatic breast cancer patients were immunized with a low dose (5 micrograms) of a 16 amino acid MUC1 peptide (GVTSAPDTRPAPGSTA) conjugated to KLH (BP16-KLH) plus DETOX adjuvant and evaluated for antibody titers against MUC1 peptide and KLH and for cytotoxic lymphocyte (CTL) activity using class 1 HLA-matched MUC1-positive tumor targets. All patients generated strong anti-KLH IgG responses. Only 3 patients developed an anti-MUC1 IgG response, which was weak in magnitude. As it is controversial whether human cancer patients generate class-1-restricted CTL against MUC1, we examined anti-MUC1 CTL activity of PBLs following 4 immunizations with BP16-KLH. The generation of MUC1-specific CTLs required only a 6-day in vitro stimulation of patients' T-cells with synthetic MUC1-peptide-pulsed autologous APCs. The assay for CTL activity was a 4 hour 51Cr release from labeled adenocarcinoma target cells. Eleven of the 16 immunized patients were tested for CTL activity using class-1-matched adenocarcinoma target cell lines. Evidence for class-1-restricted killing of MUC1-expressing tumor cell lines was obtained in 7 of these 11 patients.

The effect of bioburden on in-depth disinfection of denture base acrylic resin.

This study evaluated the effectiveness of three different disinfectant solutions against denture bioburden absorbed within the depth of acrylic resin. Specimens were taken from dentures that had been worn by the patients for 15 to 20 years and were scheduled for replacement.

New trends in the development of cancer vaccines.

Recent advances in understanding of the molecular mechanisms of antigen processing and presentation, and the identification of tumor-associated antigens in melanoma and other cancers, have stimulated the development of a new generation cancer vaccines. This review summarizes the most recent approaches for the design of safe and more effective vaccines for cancer. Peptide-based vaccines are safe and can be synthesized with high purity and reproducibility. Recombinant viruses encoding tumor-associated antigens allow efficient delivery and precise control over the form and the quantity of the delivered antigens. DNA-based vaccines induce long-lasting immune responses and are considered very safe. Antigen-loaded dendritic cells, and the use of newly developed adjuvants are also very promising new approaches. In this review, we also discuss the possible clinical applications and future directions for vaccine development.

Active specific immunotherapy of metastatic melanoma with an antiidiotype vaccine: a phase I/II trial of I-Mel-2 plus SAF-m.

PURPOSE: To determine the toxicity and immunologic activity of an antiidiotype melanoma vaccine that consists of monoclonal antibody I-Mel-2 (MELIMMUNE-2, IDEC Pharmaceuticals, La Jolla, CA) and an immunologic adjuvant SAF-m. PATIENTS AND METHODS: Twenty-six patients with metastatic melanoma, 17 of whom had previously received chemotherapy, were given 2 mg of I-Mel-2 and either 100 micrograms (n = 6) or 250 micrograms (n = 20) of SAF-m. Antiidiotype vaccine was given intramuscularly (IM) biweekly for 4 weeks, and then bimonthly until disease progression. Human antimurine antibodies (HAMA), anti-I-Mel-2 antibodies, and specific antibody (Ab)3 against the melanoma epitope mimicked by the vaccine were titrated before treatment, biweekly from weeks 4 to 12, and every 4 to 8 weeks thereafter. Computed tomographic (CT) scans of the chest, abdomen, and pelvis and magnetic resonance imaging (MRI) of the brain were obtained before and bimonthly during treatment to evaluate responses. RESULTS: Elevated titers of human antimouse antibodies and anti-I-Mel-2 antibodies were associated with clinical antitumor effect (P = .02 and P = .05, respectively). Ab3 was absent in most patients, but was found in the best clinical responder. Fever, myalgias/arthralgias, fatigue, nausea, and headaches were the most common toxicities. Grade III myalgias/arthralgias and headaches required dose reduction of SAF-m in eight patients at the 250-microgram dose. No treatment-related death occurred. Six patients had an antitumor effect: one complete response in liver and lung, two minor responses, and three stable disease. The patient with a complete response has survived nearly 5 years. CONCLUSION: I-Mel-2 antiidiotype vaccine was safe, tolerated best at the 100-microgram dose of SAF-m, and had immunologic and clinical activity.

A personal (biased) perspective on cancer "vaccines".

The use of active specific immunotherapy (ASI) for cancer (cancer "vaccines") is still in its scientific infancy despite several decades of clinical and basic research. What has been established is the principle that stimulation of the immune response by "crude" (i.e., whole cell-derived) vaccines has led, in a proportion of patients, to rejection of tumor masses, in some instances for 10 years or more. Scientific investigations into the nature of recognition of tumor antigenic determinants (epitopes) by cytolytic T cells have begun to elucidate the mechanisms underlying rejection, making more precise vaccines possible. Yet there should be caution about assuming that a single epitope or even a few epitopes combined will be as effective as the "crude" materials, which might better be thought of as "polyvalent." ASI in at least one instance may have cured melanoma in a patient with metastatic disease, but that patient developed another immunologically and genetically distinct melanoma. This may provide an example of both immunological surveillance against the emergence of new melanomas and immunological selection of an immunologically resistant tumor. Combinations of vaccines with cytokines, cytolytic T cell infusions, or chemotherapy may improve the response rates and durations of survival achievable with vaccines alone. The best rationale for synthetically derived vaccines may be for prophylaxis-that is, as a true vaccine-where the use of tumor-derived materials in normal individuals is difficult to justify ethically.

Therapeutic vaccines for melanoma: progress and problems.

Melanoma vaccines, which have been under development for many years, are being refined as a result of the knowledge gained from practical testing. We now have a more complete understanding of their mode of action and the problems that remain to be solved. There are points to recommend both crude and pure vaccines-each have specific advantages, but both require further development. Isolation of relevant peptide epitopes, addition of co-stimulatory molecules, the development of novel vehicles for vaccine delivery and improved vaccine adjuvants, and the problem of tumor-induced immunosuppression are among the issues for future study.

The addition of tamoxifen to dacarbazine and cisplatin in metastatic malignant melanoma. A phase II trial of the Southwest Oncology Group, (SWOG-8921).

Based on the reports of substantial improvement in the response rate w ith the addition of tamoxifen to a multiagent chemotherapy regimen for metastatic melanoma, Southwest Oncology Group (SWOG)-8921 was initiated. A prior regimen (SWOG-8804) of dacarbazine (DTIC) 750 mg/m(2) i.v. day 1 and cisplatin 100 mg/m(2) day 1 repeated every 3 weeks produced a 13% response rate in patients with metastatic melanoma without brain metastasis. SWOG-8921 using identical chemotherapy and schedule added tamoxifen 10 mg twice daily. There were 55 eligible patients registered, median age 52, with 37 men and 18 women. Fifty (91%) patients had evidence of visceral metastasis at registration. There were 10 responders (2 complete and 8 partial responses) for an 18% response rate (95% CI, 9-31%). The response rate in women was 28% (95% CI, 10-53%; in men, 14% (95% CI, 5-29%). Tamoxifen has produced a small increase in the response rate when added to the present combination and schedule of chemotherapy. Further Phase III trials will be necessary to assess whether there is a statistical advantage to the use of tamoxifen when combined with chemotherapy and whether there are statistical differences between men and women.

Immunotherapy of melanoma.

Melanoma is one of the first tumors for which several types of immunotherapy have been applied extensively. It is becoming clear that although only a minority of patients respond to immunotherapy, those who do have a major clinical response experience an improvement in survival. Melanoma vaccines (active specific immunotherapy) have elicited clinical remissions in advanced disease, in some instances for years. They may have extended survival in resected stage II or III disease, although the results of controlled, randomized trials in these stages are still pending. Perhaps their major advantage is their nearly total lack of significant systemic toxicity. Gene-modified whole-cell vaccines, newer constructs involving cell membranes on synthetic beads, and purified immunogenic peptides are interesting second-generation vaccines. Cytokines such as interferon-alpha and interleukin-2 also have had a beneficial effect in approximately 20% of patients with advanced disease, with a suggestion of higher rates of response when interferon is combined with a vaccine. A randomized, controlled Intergroup study has shown that interferon-alpha at 10 million U/m2/d three times weekly for 2 y is superior to observation alone in prolonging relapse-free and overall survival in resected stage III patients. Other such trials are necessary to prove the usefulness of immunotherapy in melanoma, but its promise as a new approach to a difficult disease is already evident.

Active specific immunotherapy of melanoma.

Active specific immunotherapy, or the use of tumor 'vaccines', attempts to stimulate the patient to reject his or her tumor. Nowhere has this approach been utilized more than in melanoma, often with encouraging results. The best results have occurred in the setting of minimal residual disease after resection of the primary tumor and involved lymph nodes, but responses have also been obtained in disseminated disease. Prolonged survivals of several years have been achieved in both settings, particularly the former, with little toxicity attributable to the treatment. Genetic and biochemical approaches promise considerably improved preparations of 'vaccines', with defined components and improved activity within the immediate future.

Fatigue in patients with cancer receiving interferon alpha.

Fatigue is the most frequently reported symptom of patients with cancer. The purpose of this study was to describe the experience of fatigue over time in patients with cancer receiving treatment with interferon alpha. Piper's Integrated Fatigue Model guided this study. A descriptive repeated-measures design was used. A convenience sample of 30 patients with malignant melanoma was drawn from a comprehensive cancer center in Southern California. Two instruments were used in data collection, the Symptom Distress Scale and the Piper Fatigue Scale. Study findings revealed descriptive data on patients' perceptions of the causes and remedies for fatigue while receiving active treatment for cancer. The pattern of fatigue was consistent over the five points of time during treatment, with the most extreme fatigue scores in the affective domain, followed by the sensory, temporal, total fatigue, and fatigue severity scores. The patterns and dimensions of fatigue provide implications for care of patients receiving interferon alpha, and for further investigation in the area of fatigue as a critical aspect of quality of life.

The management of depression. Part 2. The place of the new antidepressants.

Four years since the introduction of fluoxetine into Australian clinical practice, and two years since the availability of moclobemide, it is now possible to comment with some degree of certainty about the place of the new antidepressants. There do not appear to be any major complications or adverse effects associated with the recently marked antidepressants. While these medications clearly share their own similar and specific side effects, these problems are less frequent and severe than those experienced with the TCAs. Additionally, it has become obvious that they have fewer cardiac effects and possess a lower toxicity on overdose than the older classes of antidepressants. The differences in adverse effects between the various SSRIs appear to be minimal. Does this mean that the SSRIs, moclobemide and mianserin should be the treatment of first choice for depression? On balance, the evidence presented in this article would suggest rather that the new agents and TCAs should all be considered first-line agents. While discontinuation rates due to adverse effects are clearly less frequent with the new antidepressants, the currently available data do not indicate any difference in drop-out rates when discontinuation due to lack of efficacy is also incorporated into the equation. Irrespective of cost issues, the possible greater effectiveness of the TCAs in melancholic depression must also be taken into account. In conclusion, the advent of the SSRIs, moclobemide and mianserin has clearly been a major advance in the treatment of depression, and is already leading to major shifts in the pattern of antidepressant prescribing.(ABSTRACT TRUNCATED AT 250 WORDS)