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Background: A vaccine containing 6 melanoma-associated peptides to stimulate helper T cells (6MHP) is safe, immunogenic, and clinically active. A phase I/II trial was designed to evaluate safety and immunogenicity of 6MHP vaccines plus programmed death 1 (PD-1) blockade., Participants and Methods: Participants with advanced melanoma received 6MHP vaccines in an incomplete Freund's adjuvant (6 vaccines over 12 weeks). Pembrolizumab was administered intravenously every 3 weeks. Tumor biopsies at baseline and day 22 were analyzed by multiplex immunohistochemistry. Primary end points were safety (Common Terminology Criteria for Adverse Events V.4.03) and immunogenicity (ex vivo interferon-γ ELISpot assay). Additional end points included changes in the tumor microenvironment (TME) and clinical outcomes., Results: Twenty-two eligible participants were treated: 6 naïve to PD-1 antibody (Ab) and 16 PD-1 Ab-experienced. Median follow-up was 24.4 months. Most common treatment-related adverse events (any grade) included injection site reactions, fatigue, anemia, lymphopenia, fever, elevated aspartate aminotransferase, pruritus, and rash. Treatment-related dose-limiting toxicities were observed in 3 (14%) participants, which did not cross the study safety bound. A high durable T cell response (Rsp) to 6MHP was detected in only one participant, but twofold T cell Rsps to 6MHP were detected in 7/22 (32%; 90% CI (16% to 52%)) by week 13. Objective clinical responses were observed in 23% (1 complete response, 4 partial responses), including 4/6 PD-1 Ab-naïve (67%) and 1/16 PD-1 Ab-experienced (6%). Overall survival (OS) was longer for PD-1 Ab-naïve than Ab-experienced participants (HR 6.3 (90% CI (2.1 to 28.7)). In landmark analyses at 13 weeks, OS was also longer for those with T cell Rsps (HR 6.5 (90% CI (2.1 to 29.2)) and for those with objective clinical responses. TME evaluation revealed increased densities of CD8 + T cells, CD20 + B cells, and Tbet + cells by day 22., Conclusions: Treatment with the 6MHP vaccine plus pembrolizumab was safe, increased intratumoral lymphocytes, and induced T cell Rsps associated with prolonged OS. The low T cell Rsp rate in PD-1 Ab-experienced participants corroborates prior murine studies that caution against delaying cancer vaccines until after PD-1 blockade. The promising objective response rate and OS in PD-1 Ab-naïve participants support consideration of a larger study in that setting., Competing Interests: Competing interests: CLS has the following disclosures: research support to the University of Virginia from Celldex (funding, drug), GSK (funding), Merck (funding, drug), 3M (drug), Theraclion (device staff support); funding to the University of Virginia from Polynoma for PI role on the MAVIS Clinical Trial; funding to the University of Virginia for roles on Scientific Advisory Boards for Immatics and CureVac. CLS also receives licensing fee payments through the UVA Licensing and Ventures Group for patents for peptides used in cancer vaccines., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Experimental cancer vaccines are traditionally administered by injection in subcutaneous tissue or muscle, commonly with adjuvants that create chronic inflammatory depots. Injection of melanoma-derived peptides induces T cell responses; however, the depots that form following injection may inhibit optimization of the immune response. In skin, epidermal Langerhans cells (LC) are a dominant source of professional antigen presenting cells. We hypothesized that: (1) applying melanoma-derived peptides topically, in proximity to LC, could be immunogenic and safe, with low vaccine-site toxicity and (2) topical toll-like receptor 7 (TLR7) agonist would increase immunogenicity of the peptide vaccine., Methods: Twelve melanoma peptides plus a tetanus helper peptide were combined with granulocyte macrophage colony stimulating factor (GM-CSF) and were administered topically on days 1, 8, and 15, to 28 patients randomized to one of four adjuvant preparations: (1) incomplete Freund's adjuvant (IFA); (2) IFA plus a TLR7 agonist (imiquimod) administered on days 0, 7, 14; (3) dimethyl sulfoxide (DMSO) or (4) DMSO+ imiquimod administered on day 0, 7, 14. Every 3 weeks thereafter (x 6), the peptides were combined with GM-CSF and were injected into the dermis and subcutis in an emulsion with IFA. Toxicities were recorded and immune responses assayed by ELIspot., Results: CD8 + T cell responses to transdermal vaccination in DMSO occurred in 83% of participants in group 3 and 86% in group 4, and responses to vaccination in IFA were observed in 29% of participants in group 1 and 14% in group 2. Overall, 61% of participants had CD4 + T cell immune responses to the tetanus peptide, with large, durable responses in groups 3 and 4. Five of seven participants in group 4 had a severe rash, one that was dose limiting. Ten-year overall survival was 67% and disease-free survival was 44%., Conclusions: These data provide proof of principle for immunogenicity in humans of transdermal immunization using peptides in DMSO. Further study is warranted into the pharmacokinetics and immunobiology of TLR agonists as vaccine adjuvants during transcutaneous application. Overall survival is high, supporting further investigation of this immunization approach., Competing Interests: Competing interests: Several of the peptides used in the peptide vaccine reported in this manuscript were discovered at the University of Virginia, and have been licensed through the UVA Licensing and Ventures group; CLS is an inventor for these peptides and receives a portion of the licensing payments., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Peptide vaccines designed to stimulate melanoma-reactive CD4 + T cells can induce T cell and antibody (Ab) responses, associated with enhanced overall survival. We hypothesized that adding toll-like receptor 3 agonist polyICLC to an incomplete Freund's adjuvant (IFA) would be safe and would support strong, durable CD4 + T cell and Ab responses. We also hypothesized that oral low-dose metronomic cyclophosphamide (mCy) would be safe, would reduce circulating regulatory T cells (T-regs) and would further enhance immunogenicity., Participants and Methods: An adaptive design based on toxicity and durable CD4+ T cell immune response (dRsp) was used to assign participants with resected stage IIA-IV melanoma to one of four study regimens. The regimens included a vaccine comprising six melanoma peptides restricted by Class II MHC (6MHP) in an emulsion with IFA alone (Arm A), with IFA plus systemic mCy (Arm B), with IFA+ local polyICLC (Arm C), or with IFA+ polyICLC+ mCy (Arm D). Toxicities were recorded (CTCAE V.4.03). T cell responses were measured by interferon γ ELIspot assay ex vivo. Serum Ab responses to 6MHP were measured by ELISA. Circulating T-regs were assessed by flow cytometry., Results: Forty-eight eligible participants were enrolled and treated. Early data on safety and dRsp favored enrollment on arm D. Total enrollment on Arms A-D were 3, 7, 6, and 32, respectively. Treatment-related dose-limiting toxicities (DLTs) were observed in 1/7 (14%) participants on arm B and 2/32 (6%) on arm D. None exceeded the 25% DLT threshold for early closure to enrollment for any arm. Strong durable T cell responses to 6MHP were detected ex vivo in 0%, 29%, 67%, and 47% of participants on arms A-D, respectively. IgG Ab responses were greatest for arms C and D. Circulating T-regs frequencies were not altered by mCy., Conclusions: 6MHP vaccines administered with IFA, polyICLC, and mCy were well tolerated. The dRsp rate for arm D of 47% (90% CI 32 to 63) exceeded the 18% (90% CI 11 to 26) rate previously observed with 6MHP in IFA alone. Vaccination with IFA+ polyICLC (arm C) also showed promise for enhancing T cell and Ab responses., Competing Interests: Competing interests: CLS has the following disclosures: Research support to the University of Virginia from Celldex (funding, drug), Glaxo-Smith Kline (funding), Merck (funding, drug), 3M (drug), Theraclion (device staff support); Funding to the University of Virginia from Polynoma for PI role on the MAVIS Clinical Trial; funding to the University of Virginia for roles on Scientific Advisory Boards for Immatics and CureVac. Also CLS receives licensing fee payments through the UVA Licensing and Ventures Group for patents for peptides used in cancer vaccines., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Background: Cancer vaccines require adjuvants to induce effective immune responses; however, there is no consensus on optimal adjuvants. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC or TLR4 agonist lipopolysaccharide (LPS), combined with CD4 T cell activation, would support strong and durable CD8 + T cell responses, whereas addition of an incomplete Freund's adjuvant (IFA) would reduce magnitude and persistence of immune responses., Patients and Methods: Participants with resected stage IIB-IV melanoma received a vaccine comprised of 12 melanoma peptides restricted by Class I MHC (12MP), plus a tetanus helper peptide (Tet). Participants were randomly assigned 2:1 to cohort 1 (LPS dose-escalation) or cohort 2 (polyICLC). Each cohort included 3 subgroups (a-c), receiving 12MP + Tet + TLR agonist without IFA (0), or with IFA in vaccine one (V1), or all six vaccines (V6). Toxicities were recorded (CTCAE v4). T cell responses were measured with IFNγ ELIspot assay ex vivo or after one in vitro stimulation (IVS)., Results: Fifty-three eligible patients were enrolled, of which fifty-one were treated. Treatment-related dose-limiting toxicities (DLTs) were observed in 0/33 patients in cohort 1 and in 2/18 patients in cohort 2 (11%). CD8 T cell responses to 12MP were detected ex vivo in cohort 1 (42%) and in cohort 2 (56%) and in 18, 50, and 72% for subgroups V0, V1, and V6, respectively. T cell responses to melanoma peptides were more durable and of highest magnitude for IFA V6., Conclusions: LPS and polyICLC are safe and effective vaccine adjuvants when combined with IFA. Contrary to the central hypothesis, IFA enhanced T cell responses to peptide vaccines when added to TLR agonists. Future studies will aim to understand mechanisms underlying the favorable effects with IFA., Trial Registration: The clinical trial Mel58 was performed with IRB (#15781) and FDA approval and is registered with Clinicaltrials.gov on April 25, 2012 (NCT01585350). Patients provided written informed consent to participate. Enrollment started on June 24, 2012.

Introduction: Methods to induce T cell responses to protein vaccines have not been optimized. The immunostimulant AS15 has been administered with the recombinant MAGE-A3 protein (recMAGE-A3) i.m. but not i.d. or s.c. This study tests hypotheses that the i.d./s.c. route is safe and will increase CD4(+) and CD8(+) T cell responses to MAGE-A3., Patients and Methods: Twenty-five patients with resected stage IIB-IV MAGE-A3(+) melanoma were randomized to immunization with recMAGE-A3 combined with AS15 immunostimulant (MAGE-A3 immunotherapeutic) either i.m. (group A, n = 13) or i.d./s.c. (group B, n = 12). Adverse events were recorded. Ab responses to MAGE-A3 were measured by ELISA. T cell responses to overlapping MAGE-A3 peptides were assessed in PBMC and a sentinel immunized node (SIN) after 1 in vitro stimulation with recMAGE-A3, by IFN-γ ELISPOT assay and by flow cytometry for multifunctional (TNF-α/IFN-γ) responses., Results: Both routes of immunization were well tolerated without treatment-related grade 3 adverse events. All patients had durable Ab responses. For all 25 patients, the T cell response rate by ELISPOT assay was 30 % in SIN (7/23) but only 4 % (1/25) in PBMC. By flow cytometry, multifunctional CD8(+) T cell responses were identified in one patient in each group; multifunctional CD4(+) T cell response rates for groups A and B, respectively, were 31 and 64 % in SIN and 31 and 50 % in PBMC., Conclusion: The MAGE-A3 immunotherapeutic was well tolerated after i.d./s.c. administration, with trends to higher CD4(+) T cell response rates than with i.m. administration. This study supports further study of AS15 by i.d./s.c. administration., Competing Interests: Dr. Slingluff has the following relationships directly related to this work: The recombinant MAGE-A3 protein, overlapping peptides, and AS15 were provided to the University of Virginia for this trial by GlaxoSmithKline, and the trial was funded largely by a grant to the University of Virginia from GlaxoSmithKline. The remaining authors have no conflicts.

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4(+) T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8(+) T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund's adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4(+) and CD8(+) T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4(+) T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3281-295 (49 %) and tyrosinase386-406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8(+) T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4(+) and CD8(+) T cell responses against melanoma antigens. CD4(+) T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8(+) T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.

Background: Cancers produce soluble and cell-associated molecules that can suppress or alter antitumor immunity. Preclinical studies suggest the disease burden may alter the cytokine profile of helper T cell responses to cancer antigens. We studied cytokine production by helper T cells responding to vaccination with 6 melanoma helper peptides (6MHP) in blood and lymph nodes., Methods: Twenty-three patients with stage IIIB-IV melanoma received a 6MHP vaccine. Antigen-reactive T cells from blood and draining lymph nodes were cultured, exposed to antigen, and then supernatants (days 2 and 5) were assayed for Th1 and Th2 cytokines. Results from 4 time points were compared to pre-vaccine levels., Results: Cytokine responses to vaccinating peptides were observed in 83% of patients. Th1 favoring responses were most common (17 of 19 responders). The most abundant cytokines produced were IFN-γ and IL-5 in the PBMC's. IL-2 responses predominated in cells obtained from draining lymph nodes in 2-day culture but not in 5-day cultures. Patients with clinically measurable disease produced similar levels of total cytokine and similar degree of Th1 polarization as patients with no evidence of disease (NED)., Conclusions: The MHC class II-associated peptides used in this study induced helper T cells with a Th1-biased cytokine response in both PBMC and sentinel immunized nodes. Most patients can mount a Th1 dominant response to these peptides. Future studies are needed to test newer vaccine adjuvants in combination with these peptides., Trial Registration: CDR0000378171, Clinicaltrials: NCT00089219.

Purpose: Survival after amputation for melanoma is short; however, rare long-term survivors are reported. The mechanism for durable systemic tumor control in patients with regional failure is not known. To explore whether systemic tumor immunity may be implicated, tumor and circulating immune responses were examined in a patient who survived disease-free 14 years after hip disarticulation., Methods: A 71-year-old female with extensive regional metastases of melanoma in the left lower extremity underwent amputation for palliative reasons. Tumor was collected at surgery, and blood was collected during follow-up. Tumor sections were evaluated for lymphocytic infiltration and NY-ESO-1 expression by immunohistochemistry. Cellular immune responses to defined tumor antigens were evaluated by ELISPOT assay, and antibody responses to a panel of tumor antigens were assayed by ELISA., Results: The patient's tumor had minimal lymphocytic infiltrate (immunotype A). NY-ESO-1 was strongly expressed by the melanoma cells. Circulating T-cell responses to NY-ESO-1 peptides were observed 6 and 12 years postoperatively, and antibodies to NY-ESO-1 were detected 2-6 years after surgery., Conclusion: The patient described in this report experienced relentless regional tumor progression, with intravascular metastases, and then 14-year systemic disease-free survival after palliative resection, without evidence of melanoma recurrence before death from other causes. Her immune response to NY-ESO-1 likely failed to control established regional metastases because T cells were unable to infiltrate them. It is possible, however, that among other factors, the host immune response may have contributed to systemic protection.

Purpose: A CTEP-sponsored phase II trial was conducted to evaluate safety and clinical activity of combination therapy with CCI-779 (temsirolimus) and bevacizumab in patients with advanced melanoma., Experimental Design: Patients with unresectable stage III to IV melanoma were treated intravenously with temsirolimus 25 mg weekly and bevacizumab 10 mg every 2 weeks. Adverse events were recorded using CTCAE v3.0. Tumor response was assessed by Response Evaluation Criteria in Solid Tumors and overall survival was recorded. Correlative studies measured protein kinases and histology of tumor biopsies and immune function in peripheral blood., Results: Seventeen patients were treated. Most patients tolerated treatment well, but 2 had grade 4 lymphopenia and 1 developed reversible grade 2 leukoencephalopathy. Best clinical response was partial response (PR) in 3 patients [17.7%, 90% confidence interval (CI) 5, 0-39.6], stable disease at 8 weeks (SD) in 9 patients, progressive disease (PD) in 4 patients, and not evaluable in 1 patient. Maximal response duration for PR was 35 months. Ten evaluable patients had BRAF(WT) tumors, among whom 3 had PRs, 5 had SD, and 2 had PD. Correlative studies of tumor biopsies revealed decreased phospho-S6K (d2 and d23 vs. d1, P < 0.001), and decreased mitotic rate (Ki67(+)) among melanoma cells by d23 (P = 0.007). Effects on immune functions were mixed, with decreased alloreactive T-cell responses and decreased circulating CD4(+)FoxP3(+) cells., Conclusion: These data provide preliminary evidence for clinical activity of combination therapy with temsirolimus and bevacizumab, which may be greater in patients with BRAF(wt) melanoma. Mixed effects on immunologic function also support combination with immune therapies., (©2013 AACR.)

Purpose: We have previously shown that within tumors, recombinant interleukin-2 (rIL-2, aldesleukin) consistently activates tumor-associated macrophages and upregulates IFN-stimulated genes while inducing minimal migration, activation, or proliferation of T cells. These effects are independent of tumor response to treatment. Here, we prospectively evaluated transcriptional alterations induced by rIL-2 in peripheral blood mononuclear cells (PBMC) and within melanoma metastases., Experimental Design: We evaluated gene expression changes by serially comparing pre- to posttreatment samples in 13 patients and also compared transcriptional differences among lesions displaying different responsiveness to therapy, focusing on 2 lesions decreasing in size and 2 remaining stable (responding lesions) compared with nonresponding ones., Results: As previously described, the effects of rIL-2 were dramatic within PBMCs, whereas effects within the tumor microenvironment were lesion specific and limited. However, distinct signatures specific to response could be observed in responding lesions pretreatment that were amplified following rIL-2 administration. These signatures match the functional profile observed in other human or experimental models in which immune-mediated tissue-specific destruction (TSD) occurs, underscoring common pathways leading to rejection. Moreover, the signatures observed in pretreatment lesions were qualitatively similar to those associated with TSD, underlining a determinism to immune responsiveness that depends upon the genetic background of the host or the intrinsic genetic makeup of individual tumors., Conclusions: This is the first prospectively collected insight on global transcriptional events occurring during high-dose rIL-2 therapy in melanoma metastases responding to treatment., (©2011 AACR.)

Purpose: This multicenter randomized trial was designed to test whether melanoma-associated helper peptides augment CD8(+) T-cell responses to a melanoma vaccine and whether cyclophosphamide (CY) pretreatment augments CD4(+) or CD8(+) T-cell responses to that vaccine., Patients and Methods: In all, 167 eligible patients with resected stage IIB to IV melanoma were randomly assigned to four vaccination study arms. Patients were vaccinated with 12 class I major histocompatibility complex-restricted melanoma peptides (12MP) to stimulate CD8(+) T cells and were randomly assigned to receive a tetanus helper peptide or a mixture of six melanoma-associated helper peptides (6MHP) to stimulate CD4(+) T cells. Before vaccination, patients were also randomly assigned to receive CY pretreatment or not. T-cell responses were assessed by an ex vivo interferon gamma ELISpot assay. Clinical outcomes and toxicities were recorded., Results: Vaccination with 12MP plus tetanus induced CD8(+) T-cell responses in 78% of patients and CD4(+) T-cell responses to tetanus peptide in 93% of patients. Vaccination with 12MP plus 6MHP induced CD8(+) responses in 19% of patients and CD4(+) responses to 6MHP in 48% of patients. CY had no significant effect on T-cell responses. Overall 3-year survival was 79% (95% CI, 71% to 86%), with no significant differences (at this point) by study arm., Conclusion: Melanoma-associated helper peptides paradoxically decreased CD8(+) T-cell responses to a melanoma vaccine (P < .001), and CY pretreatment had no immunologic or clinical effect. Prior work showed immunologic and clinical activity of 6MHP alone. Possible explanations for negative effects on CD8 responses include modulation of homing receptor expression or induction of antigen-specific regulatory T cells.

An incomplete Freund's adjuvant (IFA) commonly used in experimental cancer vaccines has recently been reformulated. Oleic acid used in the surfactant was purified from a vegetable source (olives, IFA-VG) rather than an animal source (beef tallow, IFA-AN). To provide an insight into the adjuvant properties of the new formulation, we reviewed T-cell responses, by enzyme-linked immunospot assay, to multipeptide vaccines in 2 sequential clinical trials that spanned this transition of adjuvants. Analyses included 194 patients who received either IFA-AN or IFA-VG for all vaccines, and a subset of 93 patients best matched by study arm for vaccine antigens (12 melanoma peptides restricted by major histocompatibility complex class I, 12MP; plus a tetanus helper peptide, tet) administered with IFA but without granulocyte macrophage-colony stimulating factor. Inflammation was observed at vaccine sites clinically for almost all patients, even including ulceration in a subset with each IFA formulation. CD8 T-cell response rates to the 12 melanoma peptides were 53% [95% confidence interval (CI), 44%, 61%)] for IFA-AN and 46% [95% CI, 32%, 59%)] for IFA-VG. In the 93 patient subset, these rates were 73% [95% CI, 61%, 83%)] and 70% [95% CI, 47%, 87%)], respectively. CD4 T-cell responses to tetanus helper peptide were identified in 94% [95% CI, 86%, 98%)] and 96% [95% CI, 78%, 100%)], respectively. Responses to individual human leukocyte antigen (HLA)-A1, A2, and DR associated peptides were largely preserved, but reactivity trended lower for some HLA-A3 associated peptides. Despite the necessarily retrospective nature of the analysis and limitations of multiple comparisons, our summary data support the use of IFA-VG as an adjuvant with multipeptide vaccines in melanoma patients.

Successful management of epithelial skin cancers with imiquimod 5% cream (Aldara), an immunomodulatory agent, led to speculation that it may promote an immune response against melanoma. Studies, mostly case reports, have assessed the value of imiquimod as a topical treatment for dermal melanoma metastases that prove difficult to manage surgically. The precise value of imiquimod, however, in treatment of dermal and subcutaneous metastases remains unclear. A case at our institution elucidates histopathologically that subcutaneous metastases may progress despite excellent treatment of superficial dermis in the same location. In preparation for a clinical trial using imiquimod to treat patients with dermal melanoma metastases, we have treated several patients off protocol. We present a case report in which the observed changes are documented photographically and histologically. The patient experienced dramatic improvement in the locally treated dermis with concurrent regional treatment failure in the subcutaneous space. Our experience supports growing evidence that imiquimod for some provides an effective option for dermal disease. The unique histological documentation we provide regarding the differential effectiveness of imiquimod in treating various tissue components may help guide future investigations regarding optimal clinical application of imiquimod therapy for melanoma metastases., (Copyright © 2009 John Wiley & Sons A/S.)

Purpose: Granulocyte/macrophage colony-stimulating factor (GM-CSF) administered locally together with vaccines can augment T-cell responses in animal models. Human experience has been limited to small and uncontrolled trials. Thus, a multicenter randomized phase II trial was done to determine whether local administration of GM-CSF augments immunogenicity of a multipeptide vaccine. It also assessed immunogenicity of administration in one versus two vaccine sites., Experimental Design: One hundred twenty-one eligible patients with resected stage IIB to IV melanoma were vaccinated with 12 MHC class I-restricted melanoma peptides to stimulate CD8+ T cells plus a HLA-DR-restricted tetanus helper peptide to stimulate CD4+ T cells, emulsified in incomplete Freund's adjuvant, with or without 110 microg GM-CSF. Among 119 evaluable patients, T-cell responses were assessed by IFN-gamma ELIspot assay and tetramer analysis. Clinical outcomes were recorded., Results: CD8+ T-cell response rates to the 12 MHC class I-restricted melanoma peptides (by day 50) with or without GM-CSF were 34% and 73%, respectively (P < 0.001), by direct ELIspot assay. Tetramer analyses corroborated the functional data. CD4+ T-cell responses to tetanus helper peptide were higher without GM-CSF (95% versus 77%; P = 0.005). There was no significant difference by number of vaccine sites. Three-year overall and disease-free survival estimates (95% confidence interval) were 76% (67-83%) and 52% (43-61%), respectively, with too few events to assess differences by study group., Conclusions: High immune response rates for this multipeptide vaccine were achieved, but CD8+ and CD4+ T-cell responses were lower when administered with GM-CSF. These data challenge the value of local GM-CSF as a vaccine adjuvant in humans.

Background: We hypothesized that lymph nodes draining sites of cutaneous vaccination could be identified by sentinel node biopsy techniques, and that measuring T-cell response with lymphocytes obtained from these lymph nodes would provide a more sensitive measure of immunogenicity than would the same measurement made with peripheral blood lymphocytes (PBL)., Methods: ELISpot analysis was used to determine the magnitude of vaccine-specific T-cell response in the sentinel immunized nodes (SIN), random lymph nodes, and peripheral blood lymphocytes (PBL) obtained from patients enrolled in clinical trials of experimental melanoma vaccines., Results: The SIN biopsy was successful in 97% of cases and morbidity was very low. The T-cell response to vaccination was detected with greater sensitivity in the SIN (57%) than in PBL (39%), and evaluation of T-cell responses in the SIN and the PBL together yielded T-cell responses in 63% of patients. When the T-cell responses from a SIN and a random lymph node were compared in four patients, immune responses were detected to one of the vaccine peptides in three of these four patients. In all of those cases, responses were present in the SIN but absent from the random lymph node., Conclusion: Measurements of T-cell responsiveness to cutaneous immunization are more frequently positive in the SIN than they are in the PBL, however evaluation of both the SIN and PBL permit a more sensitive measure of T-cell immunogenicity than use of either single source.

Purpose: Human melanoma cells express shared antigens recognized by CD8(+) T lymphocytes, the most common of which are melanocytic differentiation proteins and cancer-testis antigens. However, peptide vaccines for melanoma usually target only one or two MHC class I-associated peptide antigens. Because melanomas commonly evade immune recognition by selective antigen loss, optimization of melanoma vaccines may require development of more complex multipeptide vaccines., Experimental Design: In a prospective randomized clinical trial, we have evaluated the safety and immunogenicity of a vaccine containing a mixture of 12 peptides from melanocytic differentiation proteins and cancer-testis antigens, designed for human leukocyte antigen types that represent 80% of the melanoma patient population. This was compared with a four-peptide vaccine with only melanocytic differentiation peptides. Immune responses were assessed in peripheral blood and in vaccine-draining lymph nodes., Results: These data show that (a) the 12-peptide mixture is immunogenic in all treated patients; (b) immunogenicity of individual peptides is maintained despite competition with additional peptides for binding to MHC molecules; (c) a broader and more robust immune response is induced by vaccination with the more complex 12-peptide mixture; and (d) clinical outcome in this peptide vaccine trial correlates with immune responses measured in the peripheral blood lymphocytes., Conclusions: These data support continued investigation of complex multipeptide vaccines for melanoma.

The development of effective immune therapy for cancer is a central goal of immunologists in the 21st century. Our laboratories have been deeply involved in characterization of the immune response to melanoma and translation of laboratory discoveries into clinical trials. We have identified a cohort of peptide antigens presented by Major Histocompatibility Complex (MHC) molecules on melanoma cells and widely recognized by T cells from melanoma patients. These have been incorporated into peptide-based vaccines that induce CD8(+) and CD4(+) T-cell responses in 80-100% of patients. Major objective clinical tumor regressions have been observed in some patients, and overall survival in vaccinated patients exceeds expected stage-specific survival. New clinical trials will determine the value of combination of melanoma helper peptides (MHP) into multipeptide vaccines targeting CD8 cells. New trials will also evaluate new approaches to modulating the host-tumor relationship and will develop new combination therapies. Parallel investigations in murine models are elucidating the immunobiology of the melanoma-host relationship and addressing issues that are not feasible to approach in human trials. Based on the fact that the largest cohort of melanoma antigens are derived from normal proteins concerned with pigment production, we have evaluated the mechanisms of self-tolerance to tyrosinase (Tyr) and have determined how T cells in an environment of self-tolerance are impacted by immunization. Using peptide-pulsed dendritic cells as immunogens, we have also used the mouse model to establish strategies for quantitative and qualitative enhancement of antitumor immunity. This information creates opportunities for a new generation of therapeutic interventions using cancer vaccines.

The purpose of this investigation was to evaluate the occurrence of autoimmune toxicities associated with the administration of low-dose IL-2 in conjunction with vaccines for melanoma. Ninety-three patients with stage IIB, III, or IV melanoma were enrolled in three clinical trials and received anti-melanoma vaccines on days 1, 8, 15, 29, 36, and 43. The vaccines comprised peptide-pulsed dendritic cells, autologous tumor cells with GM-CSF in Montanide ISA-51, or synthetic peptides with GM-CSF in Montanide ISA-51. In conjunction with the vaccines, all patients were administered 3 x 10(6) IU/m2/d IL-2 subcutaneously for 42 days, either days 8 to 49 or 29 to 70. Clinical and laboratory data from these studies were reviewed in aggregate to evaluate the occurrence of autoimmune toxicities. Of 91 evaluable patients, vitiligo was documented in 6 patients (7%). In addition, one patient experienced transient severe insulin-dependent diabetes that resolved after discontinuing IL-2, and another experienced an exacerbation of his pre-existing diabetes; these occurrences are consistent with an autoimmune insulitis. Four occurrences (4%) of transient minor ocular toxicity were documented, but no autoimmune ocular toxicities or changes in visual acuity were found. Of 55 evaluable patients, 14 experienced thyroid abnormalities (25%). These were attributed to an autoimmune thyroiditis, which was supported by findings of antithyroid antibodies in three of the seven patients evaluated. Overall, autoimmune toxicities affecting several organ systems were observed in patients receiving melanoma vaccines in conjunction with low-dose IL-2. None of these toxicities caused major long-term effects, though one was acutely life-threatening and others contributed to treatment-related morbidity. Peptide- or cell-based vaccines administered in combination with low-dose IL-2 appear to be capable of breaking tolerance to self-antigens; despite the associated toxicities, these combinations may still be useful to administer as an immunotherapy for cancer. However, careful monitoring for autoimmune toxicities should be incorporated in future clinical studies incorporating low-dose IL-2.

Twelve peptides derived from melanocyte differentiation proteins and cancer-testis Ags were combined and administered in a single mixture to patients with resected stage IIB, III, or IV melanoma. Five of the 12 peptides included in this mixture had not previously been evaluated for their immunogenicity in vivo following vaccination. We report in this study that at least three of these five peptides (MAGE-A1(96-104), MAGE-A10(254-262), and gp100(614-622)) are immunogenic when administered with GM-CSF in Montanide ISA-51 adjuvant. T cells secreting IFN-gamma in response to peptide-pulsed target cells were detected in peripheral blood and in the sentinel immunized node, the node draining a vaccine site, after three weekly injections. The magnitude of response typically reached a maximum after two vaccines, and though sometimes diminished thereafter, those responses typically were still detectable 6 wks after the last vaccines. Most importantly, tumor cell lines expressing the appropriate HLA-A restriction element and MAGE-A1, MAGE-A10, or gp100 proteins were lysed by corresponding CTL. This report supports the continued use of the MAGE-A1(96-104), MAGE-A10(254-262), and gp100(614-622) epitopes in peptide-based melanoma vaccines and thus expands the list of immunogenic peptide Ags available for human use. Cancer-testis Ags are expressed in multiple types of cancer; thus the MAGE-A1(96-104) and MAGE-A10(254-262) peptides may be considered for inclusion in vaccines against cancers of other histologic types, in addition to melanoma.

Purpose: A phase II trial was performed to test whether systemic low-dose interleukin-2 (IL-2) augments T-cell immune responses to a multipeptide melanoma vaccine. Forty patients with resected stage IIB-IV melanoma were randomly assigned to vaccination with four gp100- and tyrosinase-derived peptides restricted by human leukocyte antigen (HLA) -A1, HLA-A2, and HLA-A3, and a tetanus helper peptide plus IL-2 administered daily either beginning day 7 (group 1), or beginning day 28 (group 2)., Patients and Methods: T-cell responses were assessed by an interferon gamma ELIspot assay in peripheral blood lymphocytes (PBL) and in a lymph node draining a vaccination site (sentinel immunized node [SIN]). Patients were followed for disease-free and overall survival., Results: T-cell responses to the melanoma peptides were observed in 37% of PBL and 38% of SINs in group 1, and in 53% of PBL and 83% of SINs in group 2. The magnitude of T-cell response was higher in group 2. The tyrosinase peptides DAEKSDICTDEY and YMDGTMSQV were more immunogenic than the gp100 peptides YLEPGPVTA and ALLAVGATK. T-cell responses were detected in the SINs more frequently, and with higher magnitude, than responses in the PBL. Disease-free survival estimates at 2 years were 39% (95% CI, 18% to 61%) for group 1, and 50% (95% CI, 28% to 72%) for group 2 (P = .32)., Conclusion: The results of this study support the safety and immunogenicity of a vaccine composed of four peptides derived from gp100 and tyrosinase. The low-dose IL-2 regimen used for group 1 paradoxically diminishes the magnitude and frequency of cytotoxic T lymphocyte responses to these peptides.

In this phase 2 study, the authors assessed the hematologic and clinical toxicities of a melanoma peptide vaccine administered in conjunction with low-dose interleukin-2 (IL-2) therapy. Forty patients were randomized to receive a weekly vaccine paired with a regimen of subcutaneous IL-2 (3 x 10(6) IU/m2/day) administered daily for 6 weeks beginning either at week 1 or at week 4 of vaccine therapy. The differences in the time course of the IL-2 between the two groups permitted assessment of the cause of the toxicities, due either to IL-2 or to vaccine components. Both treatment regimens were well tolerated in the outpatient setting. Toxicities attributable to the vaccine components were principally limited to grade 1 injection site reactions. Systemic clinical toxicities correlated with the initiation of IL-2 therapy. These toxicities coincided temporally and in magnitude with changes in circulating eosinophil counts, suggesting that systemic clinical toxicities and eosinophilia may have common etiologic pathways. Other minor toxicities attributable to this low-dose IL-2 regimen were clinically insignificant hepatic toxicity, mild anemia, and mild thrombocytosis. The hematologic effects of this therapy were delayed in time between the two treatment groups, without dramatic differences in magnitude, which suggests minimal modulation of the IL-2 toxicity by components of the vaccine., (Copyright 2004 Lippincott Williams & Wilkins)

Purpose: To determine clinical and immunologic responses to a multipeptide melanoma vaccine regimen, a randomized phase II trial was performed., Patients and Methods: Twenty-six patients with advanced melanoma were randomly assigned to vaccination with a mixture of four gp100 and tyrosinase peptides restricted by HLA-A1, HLA-A2, and HLA-A3, plus a tetanus helper peptide, either in an emulsion with granulocyte-macrophage colony-stimulating factor (GM-CSF) and Montanide ISA-51 adjuvant (Seppic Inc, Fairfield, NJ), or pulsed on monocyte-derived dendritic cells (DCs). Systemic low-dose interleukin-2 (Chiron, Emeryville, CA) was given to both groups. T-lymphocyte responses were assessed, by interferon gamma ELIspot assay (Chiron, Emeryville, CA), in peripheral-blood lymphocytes (PBLs) and in a lymph node draining a vaccine site (sentinel immunized node [SIN])., Results: In patients vaccinated with GM-CSF in adjuvant, T-cell responses to melanoma peptides were observed in 42% of PBLs and 80% of SINs, but in patients vaccinated with DCs, they were observed in only 11% and 13%, respectively. The overall immune response was greater in the GM-CSF arm (P <.02). Vitiligo developed in two of 13 patients in the GM-CSF arm but in no patients in the DC arm. Helper T-cell responses to the tetanus peptide were detected in PBLs after vaccination and correlated with T-cell reactivity to the melanoma peptides. Objective clinical responses were observed in two patients in the GM-CSF arm and one patient in the DC arm. Stable disease was observed in two patients in the GM-CSF arm and one patient in the DC arm., Conclusion: The high frequency of cytotoxic T-lymphocyte responses and the occurrence of clinical tumor regressions support continued investigation of multipeptide vaccines administered with GM-CSF in adjuvant.

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.

Many peptide epitopes for cytotoxic T lymphocytes (CTLs) have been identified from melanocytic differentiation proteins. Vaccine trials with these peptides have been limited mostly to those associated with HLA-A2, and immune responses have been detected inconsistently. Cases of clinical regression have been observed after peptide vaccination in some trials, but melanoma regressions have not correlated well with T-cell responses measured in peripheral blood lymphocytes (PBLs). We vaccinated stage IV melanoma patients with a mixture of gp100 and tyrosinase peptides restricted by HLA-A1 (DAEKSDICTDEY), HLA-A2 (YLEPGPVTA and YMDGTMSQV) and HLA-A3 (ALLAVGATK) in an emulsion with GM-CSF and Montanide ISA-51 adjuvant. CTL responses were assessed in PBLs and in a lymph node draining a vaccine site (sentinel immunized node, SIN). We found CTL responses to vaccinating peptides in the SIN in 5/5 patients (100%). Equivalent assays detected peptide-reactive CTLs in PBLs of 2 of these 5 patients (40%). CTLs expanded from the SIN lysed melanoma cells naturally expressing tyrosinase or gp100. We demonstrated immunogenicity for peptides restricted by HLA-A1 and -A3 and for 1 HLA-A2 restricted peptide, YMDGTMSQV. Immune monitoring of clinical trials by evaluation of PBLs alone may under-estimate immunogenicity; evaluation of SIN provides a new and sensitive approach for defining responses to tumor vaccines and correlating these responses with clinical outcomes. This combination of an immunogenic vaccine strategy with a sensitive analysis of CTL responses demonstrates the potential for inducing and detecting anti-tumor immune responses in the majority of melanoma patients., (Copyright 2001 Wiley-Liss, Inc.)

Background: In 1872, Hartung was the first to describe the case of a fully formed mammary gland arising in the left labium majora of a 30-year-old woman. Since Hartung's initial report, 38 additional cases of ectopic vulvar breast tissue have been described. This case report describes the rare occurrence of primary mammary adenocarcinoma arising within the vulva., Case: A 64-year-old G4P4 white female presented with a 4-year history of a 2 x 1 cm firm, indurated, raised lesion of the left lateral mons. A wide local excision with ipsilateral inguinofemoral lymphadenectomy was performed. Given histological findings characteristic of both invasive ductal carcinoma and invasive lobular carcinoma, in conjunction with the presence of estrogen and progesterone receptors within the tumor, a diagnosis of infiltrating adenocarcinoma arising within ectopic breast tissue was made., Conclusions: Thirty-nine reported cases of ectopic breast tissue arising within the vulva have been reported in the world literature. Though the diagnosis of primary breast carcinoma arising within the vulva is based primarily upon histologic pattern, estrogen and progesterone receptor positivity provide supporting evidence. Given the rarity of this condition, guidelines for therapy are unavailable; we therefore suggest looking to the current management of breast cancer in order to establish a sensible approach., (Copyright 1999 Academic Press.)

A high level of circulating nucleated red blood cells (NRBC) in patients with chronic myeloproliferative syndromes could potentially complicate peripheral blood stem cell (PSC) collection. The mononuclear NRBC might comprise a significant fraction of the total mononuclear cells in the final product. We report a successful PSC collection in a patient with more NRBC than WBC in the peripheral blood. A 27-year-old man with chronic myelogenous leukemia underwent eight PSC collection procedures, seven using the Cobe Spectra (Spectra) and one using the Fenwal CS3000 Plus (CS). PSC product manipulations to remove NRBC were unnecessary. As assessed by post-collection NRBC: WBC ratio as a percent of the initial ratio, Spectra selectively harvested mononuclear leukocytes over NRBC. The collected products had a mean NRBC: WBC ratio that was 3.4% of the peripheral blood ratio. Adequate numbers of mononuclear leukocytes were collected with less than 6% NRBC contamination. The single CS procedure resulted in a comparable NRBC reduction efficiency as the Spectra. We conclude that PSC harvest using automated blood cell separators from patients with a high level of circulating NRBC may result in a product with an acceptable number of NRBC.

The hemolymphopoietic growth factors, including the colony-stimulating factors (CSF) and interleukins (IL), are described and categorized on the basis of their biological features in laboratory systems. Although these agents are varied and exceptions exist, in general they lack lineage specificity although they may express lineage-predominant activity. They act at multiple levels of hemolymphopoietic cell differentiation, demonstrate additive or synergistic effects when combined in vitro, require surface receptors on target cells to directly express their activity, and may be produced by a variety of cells. This framework of behavioral generalizations, completed by the specifics of each factor's activity, despite the artifactual and simplified nature of in vivo systems, forms the basis for concepts of in vitro activity and for clinical applications. Hemolymphopoietic growth factors studied in the clinic have demonstrated impressive and important activity, validating much of the in vitro data. Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have clearly reduced neutropenia and infection rates when administered following conventional chemotherapy and high-dose chemotherapy followed by autologous bone marrow transplantation. To a varying degree, similar results with G-CSF and/or GM-CSF have been described in other diseases including acute myelogenous leukemia (AML) treated following induction chemotherapy, myelodysplastic syndrome, hairy cell leukemia, aplastic anemia, and chronic neutropenias. In preliminary studies IL-3 has been shown to have similar qualitative activities. However, these agents have not demonstrated a reproducible salutary impact on platelet or red cell lineages. Adverse effects on platelet counts and/or platelet recovery have been noted. Additionally, hemolymphopoietic growth factor receptors have been identified on malignant cells, suggesting that these factors could stimulate neoplastic growth. Studies with GM-CSF and IL-3 have demonstrated blast proliferation in some cases of AML and myelodysplasia, underscoring the capacity of these agents to stimulate the growth of myeloid leukemia. No clinically evident impact of these factors upon the growth of solid tumors has been identified but this issue has not been adequately studied. The toxicity of these agents has been surprisingly limited and appears to be related to their biologic activities. Hemolymphopoietic growth factors as single agents have broad clinical applications in cytopenias. Several methods for enhancing the clinical activity of these agents are under study, including the use of combinations of growth factors synergistic in vitro.

We treated 25 newly diagnosed patients with advanced epithelial ovarian cancer with an intensive induction chemotherapy regimen using high-dose cisplatin in combination with cyclophosphamide and doxorubicin. All patients had either stage IIIC or stage IV disease. Two intensive induction courses of chemotherapy were administered at 28-day intervals, which consisted of cisplatin 40 mg/m2 daily for 5 days, cyclophosphamide 500 mg/m2 day 1, and doxorubicin 40 mg/m2 day 1. Four courses of chemotherapy using cisplatin 60 mg/m2, doxorubicin 40 mg/m2, and cyclophosphamide 500 mg/m2 followed the high-dose induction therapy. Two of the first six patients died during high-dose induction therapy (one died of neutropenia and sepsis, one of intercurrent intracerebral hemorrhage). Doxorubicin was subsequently omitted from the induction therapy due to unacceptable myelosuppression; no deaths occurred in the remaining 19 patients, and myelosuppression was manageable. Peripheral neuropathy was the most severe side effect with this regimen. This complication was unpredictable, developed during the third or fourth month of treatment, and was disabling in five patients. Other toxicity included prolonged nausea and vomiting (eight patients), ototoxicity (five patients), and nephrotoxicity (two patients), but these did not compromise therapy. All 23 assessable patients had objective response to therapy. Four of 12 patients who underwent second-look laparotomy had pathologic complete response, while four additional patients had only microscopic residual disease. The median survival for the entire group was 25 months. Four patients remain continuously disease-free 23 to 48 months following completion of therapy. Although this regimen was tolerated by most patients, the unpredictable occurrence of disabling neuropathy may limit its usefulness.(ABSTRACT TRUNCATED AT 250 WORDS)

VP-16-213 (Etoposide) is an active antineoplastic agent which has undergone extensive evaluation of clinical dose escalation. To corroborate a putative dose-response relationship, we studied, in a modified clonogenic assay, various doses and durations of exposure. VP-16-213 at doses of 0.01, 0.05, 0.10, 0.50, 1.0, 5.0, and 10.0 micrograms/ml, each with exposure durations of 1, 3, 18, and 30 h, was studied in vitro against two human tumor cell lines, MOLT and 9812. The doses and durations of exposure were chosen to approximate some of the pharmacokinetic values achievable in either standard-dose or high-dose clinical studies. The results, summarized as linear regression lines, demonstrate with statistical significance (p less than 0.03) that there is correlation between dose and cytotoxicity and between dose X duration of exposure (representing the area under the concentration-time curve) and cytotoxicity. Our in vitro data thus support the concept of intensive use of VP-16-213 to maximize antitumor activity. However, how best to accomplish the manipulation of dose and duration of exposure is not yet clear and will be the subject of future clinical investigations.

Study Objective: To determine the efficacy of a 6-month course of combination chemotherapy with hexamethylmelamine, cyclophosphamide, doxorubicin, and cisplatin (H-CAP) in the treatment of advanced ovarian carcinoma., Design: Prospective, non-randomized, single-institution trial with a 6-month course of chemotherapy, followed by second-look laparotomy for restaging. Minimum follow-up after completion of therapy is 83 months., Patients: Fifty-five patients with advanced (stage III or IV), intermediate- or high-grade epithelial carcinoma of the ovary. Twenty patients had limited residual tumor (3 cm or less maximal tumor diameter) after initial cytoreductive surgery; 35 had extensive residual disease., Interventions: All patients received chemotherapy with hexamethylmelamine (150 mg/m2 body surface area orally on days 1 to 14), cyclophosphamide (350 mg/m2 intravenously on days 1 and 8), doxorubicin (20 mg/m2 intravenously on days 1 and 8), and cisplatin (60 mg/m2 intravenously on day 1). Courses were repeated at 4-week intervals; 41 patients (75%) received six courses; 10 patients received five courses, 3 patients received four courses, and 1 patients received three courses. Forty-seven patients underwent second-look laparotomy after completion of therapy; 8 had their disease restaged clinically., Results: Fifty-three of fifty-five patients (96%) had either partial or complete response to treatment. Nineteen of forty-seven patients who had a second-look laparotomy had a surgically documented complete response; 17 of these 19 patients began chemotherapy with limited residual tumor. Ten patients (18%) remain disease-free 83 to 108 months after therapy, whereas three additional patients died of other diseases without clinical evidence of recurrent ovarian cancer. Nine of twenty patients who began chemotherapy with limited residual tumor remain disease-free, as compared to only 1 of 35 patients with more extensive tumor (P less than 0.001). All long-term, disease-free survivors had surgically documented complete response at second-look laparotomy., Conclusions: Treatment with cisplatin-based combination chemotherapy after aggressive cytoreductive surgery should be considered standard treatment for advanced ovarian carcinoma. Our intensive, 6-month course of treatment produced results comparable to those previously reported with prolonged treatment.

Three patients with widely disseminated Merkel cell tumors of the skin are presented. In all three cases, neuron-specific enolase (NSE) was demonstrated in neoplastic tissue by immunohistochemical staining, and serum NSE levels were also elevated in all three patients. Serum NSE may prove to be a useful tumor marker in this and other malignancies of neuroendocrine origin.

Twenty patients with primary non-Hodgkin's lymphoma of the central nervous system (CNS) were seen at Vanderbilt and its affiliated hospitals between 1974 and 1986. Histologically, the most common subtypes were large, noncleaved cell lymphoma and immunoblastic lymphoma of B cells. However, multiple histologies were identified. Lesions most commonly involved the frontal lobes and/or deep nuclei. Positive cerebrospinal fluid cytology was rare at initial presentation. Seventeen patients were treated with surgical biopsy or resection followed by whole brain radiotherapy at a median dose of 5,000 cGy (range: 3,000-5,600 cGy). Seven patients have been followed for less than 12 months since diagnosis. Of the remaining patients, 7 (54%) survived at least 1 year. The extent of surgery performed, dose of radiotherapy administered, subclass of lymphoma diagnosed, or location(s) of involvement within the CNS did not influence survival. Treatment rarely caused a dramatic improvement in performance status despite objective signs of response. New treatment strategies are needed to improve the management of these tumors.

Twelve patients with malignant mixed mullerian tumors were treated with combination chemotherapy at Vanderbilt University Hospital from 1977 through 1981. Nine patients, all of whom received combination chemotherapy with hexamethylmelamine, cyclophosphamide, doxorubicin, and cisplatin (HCAP), were evaluable for response. Objective responses (all partial responses) were noted in 3 (33.3%) (response rate greater than 10% and less than 55% with 90% confidence limits), a minimal response was noted in one patient, and stable disease in four (50%) patients. Responders survived longer (calculated from the initiation of HCAP) than nonresponders (median 112 vs 19 weeks). These results are not at present statistically different from previous studies utilizing doxorubicin alone, cisplatin alone, the combination of doxorubicin and DTIC, or the combination of vincristine, actinomycin D, and cyclophosphamide.

A total of 21 patients were treated in a phase I trial using the biological response modifier MVE-2, a low molecular weight component of pyran copolymer. All patients received weekly IV MVE-2 infused over 2 h. Proteinuria, sometimes of nephrotic proportions, was the dose limiting toxicity, and was seen with increasing incidence as the cumulative dose of MVE-2 exceeded 2500 mg. Other toxicity with MVE-2 was minimal. Biologic response modification at tolerable doses was inconsistent, although several assays, particularly natural cell-mediated cytotoxicity, indicated enhanced activity at higher dosages of MVE-2. No objective tumor responses were observed. MVE-2 is not useful as a biological response modifier using our initial method of administration, since the dose limiting toxicity occurred at lower levels than were necessary to induce consistent biologic response modification. Following completion of the phase I study, we administered MVE-2 by 30-min infusion to 8 additional patients and did not detect proteinuria, in spite of large cumulative doses. It is possible that alternate schedules of MVE-2 administration could minimize proteinuria and allow the administration of dosages necessary for immunologic modification.

Fourteen patients with advanced ovarian carcinoma previously treated with chemotherapeutic agents including cisplatin were treated with vinblastine 0.1 mg/kg intravenously every week. There were no responses in 13 evaluable patients. The median survival was 19+ weeks following the initiation of vinblastine (VBL) therapy. Toxicity consisted of minimal myelo-suppression (WBC count less than 2500/microliter in 8/78 courses, WBC count less than 1500/microliter in 0/78 courses, platelets less than 150,000/microliter in 0/78 courses), nausea (4/13 patients), vomiting (2/13 patients), neuropathy (4/13 patients), and weakness and fatigue (6/13 and 5/13 patients, respectively). Although data derived from the human tumor stem cell assay (HTSCA) suggest that VBL may be an active agent against previously treated ovarian carcinoma, this study in patients with refractory advanced disease suggests that VBL is inactive (less than 20% response rate with 90% confidence levels) in that setting. Whether significant durable benefit can be achieved with VBL therapy in patients whose tumor is sensitive in the HTSCA remains to be seen.

Preoperative abdominopelvic computed tomography results and operative findings were compared in 52 patients undergoing second-look laparotomy to confirm tumor status. Seventeen true-negative, 22 false-negative, and 13 true-positive scans were found. The sensitivity was 0.38, specificity was 1.0, and diagnostic accuracy was 0.58. Negative studies were associated with positive findings at laparotomy in 42% of all cases. Fourteen patients were identified who had computed tomography that would have enabled an attempt at the diagnosis of persistent cancer by computed tomography-directed needle aspiration or biopsy, thus avoiding laparotomy. Assuming 80% accuracy of needle aspiration, the cost of computed tomography in all 52 patients is considerably outweighed by the savings that could have been realized by eliminating the need for second-look surgery in these 11 women.

All IFN types--alpha, beta, and gamma--appear to have some antitumor activity against RCC. IFNa has been extensively studied and has demonstrated objective response rates between 15% and 20% when administered in a variety of doses, routes, and schedules. Intermediate dose levels may be associated with greater response rates than low dose levels, and high dose levels are poorly tolerated and usually require dose reduction because of toxicity. Among the means of administration, intramuscular or subcutaneous routes are favored because of logistic advantages; in the low- and intermediate-dose ranges chronic sequential administration (daily, three times a week, or five days per week) is tolerable and may ameliorate toxicity; none of these therapeutic recommendations can be proven to be superior, with respect to response, to several other alternatives. No survival advantage can yet be proven to result from IFN therapy for patients with RCC. Studies evaluating combinations of IFNa and other IFNs or cytotoxic agents have demonstrated increased toxicity. Although responses have been seen in the limited number of studies performed to date, these studies do not appear to support in vivo suggestions of dramatic synergism between these agents. Knowledge of the therapeutic use of IFN is in its infancy. Although the response rates described in this review are unimpressive, they are commensurate with the best available conventional therapy for RCC. As clinical strategies for the use of IFN improve, so too, might the therapeutic efficacy in RCC improve.

Seventeen ambulatory patients with extensive-stage small-cell lung cancer received one or two courses of high-dose induction chemotherapy consisting of cyclophosphamide (100 mg/kg) plus etopside (1,200 mg/m2) followed by four or five cycles of conventional-dose cyclophosphamide (1,000 mg/m2), doxorubicin (40 mg/m2), and vincristine (1.4 mg/m2) (CAV) given every 21 days. No additional chemotherapy was given until relapse or progression was documented. Response was assessed initially after high-dose induction therapy and again after completion of all chemotherapy. After induction therapy, 16/17 (94%) patients had achieved an objective response, including five (29%) complete responses and 11 (65%) partial responses. Two partially responding patients improved to a complete response after CAV, while one partial responder progressed and one patient died of an intercurrent illness while receiving CAV. Thus, the overall response after completing all chemotherapy was 15/16 (94%), including seven (44%) complete responses and eight (50%) partial responses. Median response duration was six months (range, 3 to 11 months), and overall median survival was ten months (range, 2 to 17 months). All 31 courses of induction therapy were associated with leukopenia (less than 1,000/microL), 81% with thrombocytopenia (less than 20,000/microL), and 77% with fever (greater than 38.5 degrees C). Seven episodes of bacteremia and one axillary abscess were documented, and there was one treatment-related death (6%). These toxicities are similar to that produced by high-dose etoposide alone. High-dose cyclophosphamide combined with high-dose etoposide can be administered to ambulatory patients with extensive-stage small-cell lung cancer without requiring bone marrow transplantation to reestablish hematopoiesis. Complete response and median survival rates, however, are similar to those obtained with less intensive therapy.

Four patients with disseminated Merkel cell tumors were treated with cyclophosphamide (C), doxorubicin (A), and vincristine (V). Two partial responses (PR) were noted and in one case disease remained stable. One of the patients who achieved a PR achieved a second PR when treated with VP-16 (E) and cis-platin (P) after progressive disease developed. Systemic chemotherapy appears capable of inducing objective responses in some patients with disseminated Merkel cell tumors.

Study Objective: To define the clinical features and results of systemic treatment in women with adenocarcinoma of unknown primary site involving predominantly the peritoneal surfaces., Design: Retrospective analysis of 18 patients treated at a single institution between 1978 and 1984., Patients: All 18 women had abdominal carcinomatosis and had no primary site identified at laparotomy. Nine patients had limited residual tumor (maximal tumor diameter, 3 cm or less) after initial cytoreductive surgery, and 9 patients had extensive residual disease., Interventions: In general, patients were treated according to standard guidelines for treatment of advanced ovarian carcinoma. All patients had initial laparotomy with attempted cytoreduction; of these 18 patients, 16 subsequently received cisplatin-based chemotherapy. Patients were restaged either clinically (10 patients) or with second-look surgery (8 patients)., Results: The median survival for all patients was 23 months. Five patients had complete response to chemotherapy, and three patients remain disease-free 41, 59, and 77 months after diagnosis. Patients with limited residual disease had longer median survival than did those with extensive residual disease (31 months compared with 11 months)., Conclusions: Women with adenocarcinoma of unknown primary site involving predominantly the peritoneal surface should be distinguished from other patients with adenocarcinoma of unknown primary site because they have a more indolent disease course, a higher response rate to systemic therapy, and a chance for long-term, disease-free survival after therapy. Although optimal treatment is undefined, we recommend that these patients be treated using the guidelines established for therapy of advanced ovarian carcinoma, including initial surgical cytoreduction followed by cisplatin-based combination chemotherapy.