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Vaccinia virus is a DNA poxvirus previously used as a vaccine to eradicate smallpox. The virus has a high efficiency of infection, replicates in the cytoplasm without chromosomal integration and can transport a large amount of recombinant DNA without losing infectivity. Therefore, it is an excellent choice as a vector for gene delivery in vivo. Large quantities of vaccinia have been injected into dermal, subcutaneous and peripheral lymph node melanoma metastases without significant side effects, and with efficient infection of the tumor cells and recombinant gene transfection. To determine if vaccinia, when given intravesically, can effectively infect bladder mucosa and tumor with acceptable toxicity, we performed a phase I trial of intravesical vaccinia in patients with muscle invasive transitional cell carcinoma before radical cystectomy.After documenting immune competence and demonstration of a major reaction after revaccination, patients received 3 increasing doses of intravesical Dryvax vaccinia virus (Wyeth-Ayerst Laboratories, Philadelphia, Pennsylvania) that was provided by the Centers for Disease Control. Approximately 24 hours after the third dose, cystectomy was performed and the tissue was examined microscopically.There were 4 patients who were treated. The 3 patients who received the highest doses (100 x 106 plaque forming units) had significant mucosal and submucosal inflammatory infiltration by lymphocytes, eosinophils, and plasma cells into tumor and normal tissue. Dendritic cells were recruited to the site after exposure to the vaccinia. Significant mucosal edema and vascular ectasia were seen. Tumor and normal urothelial cells showed evidence of viral infection, including enlarged vacuolated cells with cytoplasmic inclusions. There were no clinical or laboratory manifestations of vaccinia related toxicity except mild dysuria. Of the 4 patients 3 survived and were free of disease at 4-year followup.Our study demonstrates that vaccinia virus can be administered safely into the bladder with recruitment of lymphocytes and induction of a brisk local inflammatory response. To our knowledge, this is the first report of direct delivery of live virus into the human bladder. The role of wild type vaccinia as immunotherapy for bladder cancer warrants further study. Furthermore, these data support the exploration of recombinant vaccinia as a putative gene therapy vector for intravesical infection and transfection of bladder tumor cells with cytokine or other genes, an approach that our group pioneered and most recently studied in patients with superficial melanoma.

The authors provide an overview of laparoscopic dissecting instruments and discuss early development, surgical options, and special features. End effectors of different shapes and functions are described. A comparison of available energy sources for laparoscopic instruments includes discussion of thermal dissection, ultrasonic dissection, and water-jet dissection. The ergonomic risks and challenges inherent in the use of current laparoscopic instruments are outlined, as well as ergonomic issues for the design of future instruments. New directions that laparoscopic instrumentation may take are considered in connection with developing technology in robotics, haptic feedback, and MicroElectroMechanical Systems.

Perioperative preparations such as operating room setup, patient and equipment positioning, and operating port placement are essential to operative success in minimally invasive surgery. We developed an immersive virtual reality-based training system (REMIS) to provide residents (and other health professionals) with training and evaluation in these perioperative skills. Our program uses the qualities of immersive VR that are available today for inclusion in an ongoing training curriculum for surgical residents. The current application consists of a primary platform for patient positioning for a laparoscopic cholecystectomy. Having completed this module we can create many different simulated problems for other procedures. As a part of the simulation, we have devised a computer-driven real-time data collection system to help us in evaluating trainees and providing feedback during the simulation. The REMIS program trains and evaluates surgical residents and obviates the need to use expensive operating room and surgeon time. It also allows residents to train based on their schedule and does not put patients at increased risk. The method is standardized, allows for repetition if needed, evaluates individual performance, provides the possible complications of incorrect choices, provides training in 3-D environment, and has the capability of being used for various scenarios and professions.

For surgeons approaching minimally invasive donor nephrectomy it is important to identify variant anatomy preoperatively since this anatomy can vary significantly from patient to patient. The goal of this operation is to preserve the architecture and function of the organ so it can be transplanted and function successfully. The ability of the surgeon to navigate through an individual patient's anatomy in a virtual three-dimensional (3D) immersive environment augments understanding of anatomical relationships particular to that individual patient and facilitates conveying that information to other physicians and students. Utilizing automated 3D reconstruction of high contrast computed tomography (CT) scan files viewed in this way, surgeons reported a better preoperative understanding of the anatomical variations and encountered fewer surprises at the time of surgery.

The objective of this study was to evaluate the usefulness of resection of metastatic uveal melanoma and to analyze the characteristics of patients who may benefit from surgical intervention. PATIENTS AND METHODS Twelve patients underwent surgical removal of metastasis between 1976 and 1998. Data regarding primary uveal melanoma, systemic metastasis, surgical procedures, and outcomes were reviewed retrospectively.There were seven patients with liver metastases, two with lung metastases, one with brain metastasis, and two patients with metastases in the liver and other organs. Median time to systemic metastasis was 8 years. Seven of 12 patients were asymptomatic when they were found to have metastasis. Ten patients underwent complete resection of metastasis. No significant surgical complications were experienced. Median recurrence free and overall survival periods after complete resection were 19 months (range, 6-78 months) and greater than 27 months (range, 11-86 months), respectively. Recurrence free and overall 5-year survival rates of those patients were 15.6% and 53.3%, respectively. Three of these patients had no further systemic recurrence. All patients whose time to systemic metastasis was within 5 years developed further systemic recurrence within 2 years after surgery. In contrast, in 8 patients whose time to systemic metastases was greater than 5 years, 4 patients either were recurrence free or developed second metastasis more than 4 years after surgery.Complete surgical removal of metastatic uveal melanoma provided unexpectedly long survival without significant morbidity for the selected patients. These results are encouraging and justify a trial in which patients eligible for resection are randomized between standard treatment and surgery.

In early trials of paclitaxel administered as a 24-hour infusion, an overall response rate of 16% was reported for patients with metastatic melanoma. Paclitaxel is a natural product-based agent and is thus subject to the problem of multidrug resistance (MDR). Tamoxifen is an agent that can abrogate MDR and potentially enhance the effect of paclitaxel. A Phase II trial of the combination was undertaken with previously treated patients.Patients with metastatic cutaneous or mucosal melanoma who were previously treated with the Dartmouth chemotherapy regimen (dacarbazine, carmustine, cisplatin, and tamoxifen) were evaluated. Paclitaxel was administered at a dose of 225 mg/m(2) intravenously over 3 hours every 3 weeks. All patients also took tamoxifen 40 mg orally daily. Treatment continued until disease progression.Twenty-one patients completed at least two cycles of paclitaxel and were evaluable for response. Five responses were observed, 1 complete response, and 4 partial responses, for an overall response rate of 24%. The combination was well tolerated. The most common nonhematologic side effects were myalgia and paresthesia. Hematologic toxicity was mild. No patients developed neutropenic fever.This is the first report of a Phase II trial evaluating paclitaxel as a 3-hour infusion in melanoma patients. The 3-hour infusion is well tolerated and results in little myelosuppression and minimal neurotoxicity. The contribution of tamoxifen is difficult to evaluate because plasma levels were not measured. It is possible that a higher response rate might be observed with larger doses of tamoxifen. Further investigation of paclitaxel in the treatment of patients with metastatic melanoma is warranted.

The presence of several organ-specific molecules that could serve as immunogens or targets of an immune attack, the nonessential nature of the prostate gland, the substantial failure rate after treatment of the primary tumor, and the lack of effective chemotherapy for metastatic disease make prostate cancer an ideal candidate for immunotherapy. This report reviews the current status of two novel approaches to the treatment of prostate cancer. The first is an effort to induce antitumor immunity by enriching the cytokine environment within the primary cancer by intraprostatic injection of Leukocyte Interleukin (Cel-Sci Corp, Vienna, VA), a mixture of natural cytokines that includes interleukin-1 beta (IL-1beta), IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha). The second approach uses OncoVax-P (Jenner Biotherapies, Inc, San Ramon, CA), a vaccine consisting of liposome-encapsulated recombinant prostate-specific antigen (PSA) and lipid A. When administered as an emulsion or in association with bacillus Calmette-Guérin (BCG)/cyclophosphamide or GM-CSF with or without IL-2/cyclophosphamide, immunologic tolerance is broken as evidenced by the generation of humoral and cellular immunity. Both of these approaches have been shown to be feasible and safe, and are now being tested in patients with less advanced disease to determine if manipulation of the immune system can favorably influence clinical outcome.

We have devised a novel approach to active immunotherapy based on modification of autologous cancer cells with the hapten, dinitrophenyl (DNP). The treatment program consists of multiple intradermal injections of DNP-modified autologous tumor cells mixed with BCG. Administration of DNP-vaccine to patients with metastatic melanoma induces a unique reaction- the development of inflammation in metastatic masses. Histologically, this consists of infiltration of T lymphocytes, most of which are CD8+. These T cells usually produce interferon-gamma in situ. Moreover, they represent expansion of T-cell clones with novel T-cell receptor (TCR) structures. Occasionally, administration of DNP-vaccine results in regression of measurable metastases. The most common site of regression has been small lung metastases. Administration of DNP-vaccine to patients in the postsurgical adjuvant setting produces a more striking clinical effect. Of 62 patients with clinically evident stage III melanoma who had undergone lymphadenectomy, the 5-year relapse-free survival rate was 45% and the overall survival rate was 58%. These results appear to be better than those obtained with high-dose interferon, although a randomized phase III trial is required to prove that point. A recent phase I study suggests that this therapeutic approach is also applicable to stage III ovarian cancer. There appear to be no insurmountable impediments to applying this approach to much larger numbers of patients or to developing it as a standard cancer treatment.

The purpose of this study was to describe the sonographic appearances of melanoma metastases of the skin, subcutaneous tissues, and superficial lymph nodes.Gray-scale sonography was performed on 31 superficial melanoma metastases in 18 patients. Discreteness of borders, contours, echogenicity, echotexture, and degree of acoustic through-transmission were evaluated for each lesion. Color Doppler sonography was also performed on 25 of the 31 lesions, by which the amount of internal color flow was qualitatively assessed.Twenty-eight (90%) of the 31 metastases had well-defined borders. Contours were smooth in 17 (55%), lobulated in 12 (39%), and spiculated in two (6%). Nineteen metastases (61%) were hypoechoic to muscle, 10 (32%) were isoechoic, and two (6%) were hyperechoic. Echotexture was homogeneous in six lesions (19%), mildly heterogeneous in 13 (42%), moderately heterogeneous in 11 (35%), and markedly heterogeneous in one (3%). Twenty-two lesions (71%) showed enhanced acoustic through-transmission. Of the 25 melanoma metastases for which we performed color Doppler sonography, 18 (72%) had internal arterial color flow and seven (28%) did not. The flow was characterized as mild in 13 (72%) of 18, moderate in four (22%), and marked in one (6%).On sonography, superficial melanoma metastases typically are well-defined hypoechoic lesions with smooth or lobulated contours, mild to moderate heterogeneity, and enhanced acoustic through-transmission. Internal flow revealed by color Doppler sonography is present in many, but not all, superficial melanoma metastases.

We conducted a phase II trial of bleomycin+vincristine+lomustine+dacarbazine (BOLD) with intercycle alpha interferon-2b in previously untreated patients with metastatic uveal melanoma. Objective tumor response and toxicity were assessed. Twenty-three patients with histologically verified metastatic uveal melanoma were enrolled into this study between November 1992 and August 1995. Chemotherapy was administered in the following fashion: dacarbazine (DTIC), 200 mg/m2 intravenously on days 1-5; vincristine, 1 mg/m2 (not to exceed 2 mg) intravenously on days 1 and 4; bleomycin, 15 mg intravenously on days 2 and 5; lomustine (CCNU), 80 mg orally on day 1; and alpha interferon-2b, 3 x 10(6) IU subcutaneously on days 8, 10, 12, 15, 17, 19. A cycle was 28 days, and patients were reevaluated after every 2 cycles. Among twenty evaluable patients, four objective responses were observed (RR = 20%). Hematologic toxicity was modest by comparison to some other combination chemotherapy regimens in common use. Neurotoxicity was frequently observed, but it was seldom severe. An unexpected and unpredictable severe pulmonary toxicity was observed in 3 patients, the etiology of which remains unclear. The regimen of BOLD+interferon is active in the treatment of metastatic uveal melanoma. The precise role of the regimen has to be defined in light of its toxicity, particularly the unpredictable pulmonary toxicity. The pattern of occurrence of these pulmonary events is most consistent with either an acquired hypersensitivity reaction or a cumulative toxic effect of 2 or more of the agents. Patients considered for treatment with this regimen must be judiciously selected. Those with no clear contraindications may benefit from a trial of this regimen, but they must be monitored closely.

The bonafide albeit infrequent examples of tumor regression observed with whole tumor cell vaccines give evidence for the hypothesis that active specific immunization can induce a therapeutically effective immune response in melanoma patients. A dinitrophenyl-conjugated autologous whole tumor cell plus bacille Calmette-Guérin (BCG) vaccine administered in conjunction with low dose cyclophosphamide has produced clinically significant prolongation of disease free survival when used as a postsurgical adjuvant in patients with stage III melanoma. However, tumor cell-based vaccines are cumbersome and consequently of limited applicability. Improvements in our understanding of the antimelanoma immune response and technological advances have allowed investigators to explore better defined immunogens and antigenic targets; these include anti-idiotypic antibodies, gangliosides, and tumor associated/specific proteins and derived peptides. The rationale for, promise of, and progress to date with these materials are reviewed.

Interleukin 10 (IL-10) has the physiological role of down-regulating cell-mediated immunity. We have recently reported that mRNA for IL-10 was present in most metastatic melanoma tissues. The purpose of this investigation was to determine whether melanoma metastases produce IL-10 protein. Single-cell suspensions were prepared by enzymatic dissociation of 28 lymph node metastases and 7 s.c. metastases and cryopreserved. Of these 35 samples, 30 produced IL-10 after a 24-h incubation (median, 125.1 pg/ml). IL-10 production was slightly diminished after 25 Gy irradiation but almost completely abrogated after modification with the hapten dinitrophenyl. After 7 or 14 days in tissue culture, melanoma cells continued to produce IL-10 but only at about 10% of the levels of freshly dissociated tissues. Moreover, of eight melanoma cell lines established from these cultures, only one produced IL-10 protein. To determine whether IL-10 was produced by melanoma cells or tumor-associated leukocytes, single-cell suspensions were fractionated with anti-CD45 antibody-conjugated magnetic beads. In four of five samples, IL-10 production was increased by depletion of leukocytes, suggesting that the primary source was the melanoma cells themselves. This was confirmed by immunohistochemical staining of cytospin preparations and frozen tissue sections. Finally, 10 of 55 patients with clinically evident metastases showed elevations of circulating IL-10; three patients who had been melanoma-free developed high serum IL-10 levels, concurrent with the appearance of distant metastases. These data indicate that production of IL-10 is characteristic of metastatic melanomas and raise the possibility that this cytokine allows tumors to avoid or to modulate immunological attack.

To evaluate the impact of superficial ultrasonography (US) on clinical management of melanoma.Superficial US in areas at high risk for local recurrence or nodal metastases was performed in 33 patients with cutaneous melanoma. Impact categories were assigned to each US study (n = 55): category 3, US added information that altered clinical management; category 2, US added information that did not change management; category 1, no added information and management unchanged; category 0, not helpful or was misleading.Twenty-two US studies (40%) were category 3: detection of nonpalpable metastases altered surgical therapy (n = 2), demonstration of pharmacodynamic response to chemotherapy (n = 5), and determination of benignancy or malignancy (n = 15). Nine (16%) were category 2: identification of nonpalpable metastases did not alter management. Twenty-two (40%) were category 1: supported clinical impression of no metastases (n = 18) or helped confirm cutaneous, subcutaneous, or nodal metastases (n = 4). Two (4%) were category 0: missed proved metastases.Superficial US affected management of melanoma by allowing detection and characterization of masses, guidance of biopsy, and assessment of pharmacodynamic response.

Studies by a number of investigators have focused on inducing tumor-specific immunity as a therapeutic approach to cancer. Successful immunotherapeutic strategies have involved localized treatment with immune-active adjuvants, systemic administration using cytokines such as interleukin-2, and the use of whole tumor cells or tumor cell fragments as vaccines. With an increasing understanding of the requirements for the development of an immune response, immunotherapeutic strategies have focused on providing mechanistic requirements, such as tumor or accessory antigen expression and cytokine-based "immune help." Recent preclinical studies have shown that ex vivo cytokine gene transfection of tumor cells and their use as vaccines result in the enhanced development of antitumor immunity and in some cases can be used to successfully treat pre-existing tumors. Studies from our laboratory have explored the use of vaccinia virus recombinants to directly transfect tumor cells in situ with cytokine genes as a strategy for enhancing the development of antitumor immunity. We have demonstrated that vaccinia virus recombinants are highly efficient in transfecting a wide range of murine and human tumors in vitro and can be used with similar effects in in vivo murine models. In addition, we have found that vaccinia virus productively infects human melanoma cells following intratumoral injection in patients with accessible lesions. In situ transfection is highly efficient, and therapy with increasing doses of virus is safe with only minor side effects. The results of our studies support the use of cytokine-encoding recombinant vaccinia virus vectors for in situ transfection in patients with cancer.

Intratumoral gene transfer may be a significant tool in active immunotherapy. The ability to insert functional genes into a tumor in vitro and in vivo using recombinant vaccinia vectors was examined in the murine bladder tumor model. Vaccinia recombinants expressing the influenza hemagglutinin or nucleoprotein antigens infected/transfected murine (MB-49 and MBT-2) and human (T24) bladder tumor cell lines in vitro. Systemic vaccinia immunity was induced with as few as 10 plaque-forming units of recombinant vaccinia instilled intravesically, and the encoded protein was expressed in vivo in tumor and urothelium. However, preimmunity to vaccinia did not inhibit intravesical tumor transfection. Thus, recombinant vaccinia virus is effective in introducing foreign antigens locally into tumor in vivo, supporting its use in clinical immunotherapy.

8577 Background: The prognosis of metastatic uveal melanoma is poor; the median overall survival is reportedly less than 6 months. A significant proportion of metastatic uveal melanomas express C-k...

Twenty-four patients with metastatic melanoma were treated with a novel form of active immunotherapy, autologous tumor cell vaccine conjugated to the hapten, dinitrophenyl. This approach is based on the idea, well established in animal systems, that presentation of tumor antigens in the context of a strongly immunogenic hapten augments the development of immunity to those antigens. After being sensitized to dinitrophenyl, patients were given injections of dinitrophenyl-vaccine every 28 days following pretreatment with low dose cyclophosphamide. The vaccine induced a striking inflammatory response in superficial metastases in 14 of 24 patients, consisting of erythema, swelling, warmth, and tenderness over tumor masses. Immunohistochemistry and flow cytometric analysis of biopsy specimens showed marked infiltration with lymphocytes, the majority of which were CD8+, HLA-DR+ T-cells. These observations suggest that a T-cell-mediated immune response against melanoma-associated antigens was facilitated by the "helper" effect of the anti-hapten response.

Peripheral blood lymphocytes (PBL), when cultured in vitro in the presence of autologous irradiated tumor and interleukin-2 (IL-2), become more restricted in the spectrum of their cytotoxicity. The cells continue to exhibit cytotoxicity for autologous tumor cells and major histocompatibility complex (MHC)-concordant allogeneic tumor cells of similar histologic type but not for the natural killer target cell line, K562. Furthermore, the addition of autologous tumor at different time points after the initiation with IL-2 alone of conventional lymphokine-activated killer cell cultures modifies both the specificity and the degree of cytotoxicity of these lymphocytes for tumor targets. By varying the culture conditions it may be possible to generate killer cells that will exhibit similarly enhanced and more restricted antitumor effects in vivo.

Twelve of 21 patients treated with BCG immunotherapy developed hepatic abnormalities. Percutaneous liver biopsy was performed in all twelve. A diffuse noncaseating granulomatous reaction with fatty metamorphosis and minute hepatic parenchymal necrosis was demonstrated in six. The development of hepatic granulomas appeared to be unrelated to total amount of BCG administered, duration of therapy, or immunologic status of the patients.

Human T cell clones cytotoxic for autologous sarcoma cell lines have been developed from patient JM with an osteogenic sarcoma, and from patients EG and RM with malignant fibrohistiocytoma. These clones were derived from the cocultivation of peripheral blood lymphocytes (PBL) with the respective patient's autologous irradiated established tumor cell lines (AIT). After two cycles of stimulation for 5 days in bulk culture, these "educated" lymphocytes were seeded at a density of 1 X 10(6) cells/well in 24-well plates and were cultured in the presence of highly purified natural IL 2 and AIT, the latter serving as a feeder layer. Cell numbers were reduced from the initial seeding density by one log each week until reaching a density of 10(2) cells. These cells were found to be stable in viability and cytotoxic activity, after which limiting dilution was then performed. Within 4 to 6 wk, clones were isolated with unique specificities. These clones were capable of proliferating to a total density of 10(9) cells/ml and maintained their specific cytotoxicity for more than 6 mo. Testing with a panel of target cells of various histotypes, cold-target inhibition assays, and blocking of cytotoxicity with anti-HLA monoclonal antibodies showed that the T cell clones recognize a common sarcoma-associated antigen and that the lysis is HLA restricted. Phenotypically, cytotoxic clones derived from JM were Leu-1+, Leu-2+, and Leu-3-, whereas those derived from EG exhibited either Leu-24 or Leu-3+ markers, the latter phenotype lacking cytotoxicity. RM exhibited mainly Leu-3+ clones with strong cytotoxicity. All were HNK-1- and HLA class II+, with less than 1% of cells of each clone stained by anti-TAC monoclonal antibody. The clones from each patient did not lyse autologous or allogeneic PBL, mitogen-induced T lymphoblasts, normal fibroblasts, cells isolated from benign neoplasms, carcinoma cells, Daudi B lymphoid cells, or K562 cells. With the exception of EG, all clones produced immune interferon in a range from 12 to 50 U/ml. The generation of long-term specific T cell clones can be used to further dissect the cellular immune response to sarcomas. Cytotoxic T cell clones have potential application for tumor immunotherapy.

We used flow cytometry to measure the expression of human melanoma antigens on cell suspensions dissociated from metastatic masses. The objective was to study the heterogeneity between tumor samples from different patients and between different tumors excised from a single patient. Fifty-three metastases excised from 34 melanoma patients were analyzed with a panel of nine murine monoclonal antibodies (MOABs). Melanoma cells were stained by an indirect fluorescent method and analyzed on a Coulter EPICS C flow cytometer after gating to exclude tumor-infiltrating leukocytes and dead cells. The most consistently and most strongly expressed antigen was the high-molecular-weight proteoglycan (detected by the MOAB 9.2.27), which was expressed on 95% of the melanoma specimens and by a high proportion of cells within each specimen (mean +/- SE, 79.2 +/- 5.5). However, strong expression of this antigen was limited to melanoma cells that had been dissociated mechanically and was markedly diminished by exposure to collagenase. Culture of collagenase-dissociated tumor cells for 24 to 48 h resulted in reexpression of the antigen. The expression of other melanoma-associated antigens was not affected by collagenase treatment, but for these antigens there was more variability between cells from an individual tumor and between tumors from different patients. The percentage of enzyme-dissociated tumors considered positive for MOAB binding (defined as at least 10% of cells positive) and the mean +/- SE of the percentage of positive cells within a tumor were as follows: MOAB ME-9-61 (antigen, p97) = 84% + (41.2 +/- 5.4%); MOAB ME-20.4 (antigen, nerve growth factor receptor) = 40% + (18.7 +/- 5.1%); MOAB ME-24 (antigen, ganglioside GD3) = 84% + (50.8 +/- 4.8%); MOAB ME-311 (antigen, ganglioside 9-O-acetyl-GD3) = 76% + (42.5 +/- 5.1%); MOAB ME-361 (antigen, mainly ganglioside GD2) = 3% + (1.9 +/- 0.8%); MOAB 3F8 (antigen, ganglioside GD2) = 36% (10.5 +/- 3.8%); MOAB 14G2a (antigen, ganglioside GD2) = 86% + (46.0 +/- 6.7%); MOAB L243 (antigen, HLA-DR) = 56% + (22.5 +/- 5.5%). In 19 cases, we were able to compare the antigenic profiles of two tumors excised from the same patient at different times. Analysis by nonindependent t test showed no significant differences in MOAB binding between the paired tumors. Moreover, linear regression analysis indicated that there was a linear relationship, with a slope approximately = 1, between the percentage of positive cells in Tumor 1 versus Tumor 2.(ABSTRACT TRUNCATED AT 400 WORDS)

Twenty-five patients with measurable metastatic melanoma refractory to DTIC and a nitrosourea were treated with dibromodulcitol (DBD). DBD was administered orally at bedtime at a dose of 100 mg/m2/day until hematologic toxicity (a greater than or equal to 50% decrease in the wbc or platelet count) was induced. Five patients experienced clinically useful objective remissions; responding lesions included both soft tissue metastases and visceral metastatic disease. It is concluded that DBD is useful in the treatment of patients with metastatic melanoma and thus joins DTIC and the nitrosoureas as single agents which are active against this malignancy.

The therapeutic efficacy of intralesional BCG (Bacillus Calmette-Guerin; one immunizing dose every 2 weeks for a minimum of five treatments) was studied in 19 melanoma patients. Of 15 patients evaluable for response, five experienced significant objective improvement (two complete and three partial remissions). Objective improvement was limited to those patients with dermal metastatic disease. In vitro cytotoxicity in the presence of patient's serum bore, on average, a relationship to the clinical disease. In certain individual cases, serum blocking and/or lymphocyte stimulation may have had prognostic significance.

Ten patients who survived for at least five years after the development of metastatic melanoma were compared with 45 other patients who died of disseminated disease. The difference between the groups was the persistent absence of internal organ involvement in the first group and its invariable presence in the latter group. In addition, it appeared that several of the primary tumors from the nonlethal group were thinner, located in a more favorable location and occurred with greater frequency in women. All of these features tend to be indicative of a lesser degree of biologic aggressiveness of these tumors. They may prove helpful in identifying those patients with metastatic disease who are potential long term survivors and, therefore, require continued aggressive treatment.

We administered anti-Leu 2a, a murine monoclonal antibody to the human suppressor-cytotoxic T-cell subset, to 20 patients with advanced cancer to determine the toxicity and its ability to deplete circulating Leu 2(+) lymphocytes. Four doses were tested, 1, 5, 25, and 100 mg, and the infusion rate varied from 2 to 24 h. Administration of anti-Leu 2a produced rapid (within 4 h) depletion of circulating Leu 2(+) lymphocytes, the magnitude and duration of which were dose dependent: the 1-mg dose caused a less than 50% fall in circulating Leu 2(+) lymphocytes, while the higher doses caused 75-98% depletion of those cells in 10 of 17 patients so treated. The duration of depletion of Leu 2(+) cells was 1-2 days after 5 mg of anti-Leu 2a, 3-4 days after 25 mg, and 4-30+ days after 100 mg. In seven patients (3-5, 3-25, and 1-100 mg), there was persistence of variable numbers of circulating mouse Ig-coated Leu 2(+) cells, possibly indicating impaired capacity to clear these cells. Modulation of the Leu 2a antigen after antibody treatment was not observed. Peak serum levels of anti-Leu 2a were (medians): 5-mg dose = 350 ng/ml, 25-mg dose = 5500 ng/ml, and 100-mg dose = 48,000 ng/ml; murine antibody was detectable in the serum for 7-14 days after the 100-mg dose. All patients had increased titers of antimouse antibody following treatment with peak titers on day 14. The most common toxicity was shaking chills. This occurred within 1.5 h of beginning the infusion, lasted for no more than 30 min, and did not require cessation of the treatment. Anti-Leu 2a administration caused a clinically unimportant, but statistically significant fall in hematocrit (mean = -7%) and platelet (mean = -23%) count and a transient (less than 1 day duration) fall in the total lymphocyte count. Thus, infusion of murine monoclonal anti-Leu 2a can cause substantial depletion of the suppressor-cytotoxic T-cell subset with modest toxicity.

A 77-year-old white man with 64 intracutaneous melanoma metastases and a pulmonary metastatic deposit was treated with immunotherapy. Over an 8-month period, 17 intracutaneous lesions were inoculated with BCG. All 17 injected lesions and all 47 uninjected intracutaneous lesions resolved; no new nodules appeared and the pulmonary metastasis regressed (greater than 50%). This is the first documented case of a pulmonary metastatic focus responding to intralesional BCG therapy of intracutaneous metastases.

The specificities of monoclonal antibodies against melanoma cells were analyzed using radioimmunoassay, mixed-hemadsorption assay, and quantitative absorption on a variety of malignant and nonmalignant cells. Three of the six hybridoma-secreted antibodies bound to the majority of melanoma cell lines, melanoma tumors, and astrocytoma cell lines as well as to all normal and Epstein-Barr virus-transformed lymphocytes tested. The binding pattern coincides with the presence or absence of the DR antigen on human cells. Conversely, two other antibodies, 19-19 and Nu4B, detected two different antigens common to melanoma and astrocytoma cells only. Cloning of melanoma cells resulted in establishment of DR-positive and DR-negative clones, with the binding of Nu4B antibody retained in all.

We studied peripheral blood lymphocytes (PBL) from 42 patients with metastatic melanoma undergoing treatment with cyclophosphamide (CY) plus melanoma vaccine to determine whether CY immunopotentiation could be related to depletion of T-cells that function as inducers of suppression. Every 28 days, the patients were given CY, 300 mg/m2 i.v., followed 3 days later by the intradermal injection of autologous, irradiated melanoma cells mixed with Bacillus Calmette-Guérin. PBL were separated by density gradient centrifugation and cryopreserved until needed for testing. They were stained with monoclonal antibodies directly conjugated to fluorescein isothiocyanate or phycoerythrin and analyzed by two-color flow cytometry. At no time after the initiation of CY plus vaccine were there any significant changes in the percentages of helper-inducer T-cells (CD4+), suppressor-cytotoxic T-cells (CD8+), or the subpopulation of CD8+ cells expressing Leu 15, a marker for suppressor cells. Treatment of melanoma patients with CY plus vaccine resulted in a progressive fall in the proportion of CD4+ T-cells expressing the 2H4 (CD45) antigen, which identifies inducers of suppression. The reduction of CD4+, 2H4+ T-cells did not become apparent until day 28 after the first dose of CY and reached statistical significance only on days 49 (21 days after the second dose) and 105 (21 days after the fourth dose) (mean changes +/- SE: day 49, -5.4 +/- 1.4%, P less than 0.01; day 105, -9.1 +/- 2.2%, P less than 0.01; t test for nonindependent samples). In contrast, the proportion of CD4+ T-cells expressing the antigen 4B4 (CDw29), which are true helper cells, increased slightly, although not significantly, following the institution of CY plus vaccine (mean changes: day 49, +2.9 +/- 2.1%; day 105, +3.6 +/- 2.4%). Similar results were obtained when absolute numbers of circulating cells, rather than percentages, were analyzed. Thus the number of CD4+, 2H4+ T-cells fell from a mean of 395,000/ml on day 0 to 309,000/ml on day 49 (P less than 0.01) to 256,000/ml on day 105 (P less than 0.05). The absolute number of CD4+, 4B4+ cells remained unchanged at the same time points. These changes were not due to progression of metastatic disease, since a comparison of patients with progressive metastases with those who were rendered disease free by surgery showed no significant differences in the reduction of the percentage of CD4+, 2H4+ T-cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The oxazaphosphorine drug cyclophosphamide (CY), has significant immunopotentiating effects. Maguire and Ettore (21) were the first workers to describe CY-mediated immunopotentiation: They reported that guinea pigs treated with CY and then contact-sensitized developed much more intense and prolonged allergic contact dermatitis reactions than did control guinea pigs that had received sensitizer alone. Subsequently, CY was shown to immunopotentiate the acquisition of delayed-type hypersensitivity (DTH) to a variety of antigens and even to syngeneic tissue. The critical factor determining whether CY depresses or potentiates an immune response in experimental animals is the timing of administration of CY and antigen. The dose of CY seems to be much less important. Since 1982, we have performed immunological studies of 74 cancer patients treated with CY. We have tested two doses--1000 mg/M2 and 300 mg/M2, given by rapid intravenous infusion. Using the animal data as a guide, we have elected to administer CY 3 days prior to sensitization with antigen. Our initial two studies were performed with the primary antigen keyhole limpet hemocyanin (KLH): patients with advanced cancer were either sensitized with KLH alone or with KLH 3 days after administration of CY, 1000 mg/M2 ("high dose") or 300 mg/M2 ("low dose"). We found that pretreatment of patients with CY, at either "high" or "low" dose, significantly augmented the development of DTH to KLH. In contrast, the antibody response to KLH was potentiated by pretreatment with "low" dose CY, but not with "high" dose CY. DTH responses to microbial recall antigens (trichophyton, mumps, candida) were not augmented by CY administration.(ABSTRACT TRUNCATED AT 250 WORDS)

The incidence of cutaneous malignant melanoma is increasing by 10% a year and by as much as 30% a year among whites living in the sunbelt. If present trends continue, it will soon be one of the most common cancers occurring in fair-skinned individuals who have sustained severe sunburns. Although sunlight is the major cause of melanoma, several other factors, such as hormones, have also been implicated. Malignancy should be suspected in any pigmented lesion that changes in color or size, begins to itch, or bleeds or oozes spontaneously. Because of these characteristic and often highly visible changes, the disease can usually be diagnosed when surgically curable. Microstaging allows prognostic categorization of risk, with thickness the single most important prognostic factor for patients with disease clinically limited to the primary site. Staging also determines the treatment plan.

A patient with biopsy-proven dermal recurrent malignant melanoma who refused therapy, and who was observed to undergo clinical regression during the period of November 1972 through June 1974 was studied to define the histologic features of spontaneous remission, and to evaluate the immune response as measured by in vitro assays of lymphocyte cytotoxicity and serum effects during the course of regression. Biopsy of regressed areas showed the following histologic features: 1) absence of malignant melanoma cells in basal layers of epidermis with relative increase in basal layer clear cells; 2) dermal inflammatory reaction with lymphocytic infiltrate, melanophages, and degenerate malignant melanocytes; and 3) dermal reactive vascular proliferation and interstitial edema progressing to reparative dermal fibrosis. Using a microcytotoxicity assay with two established allogeneic melanoma cell cultures as target cells, a statistically significant (p less than 0.01) increase in lymphocyte cytotoxicity values was observed over the clinical time course of regression. No significant serum cytotoxic or serum blocking effects were detectable. These findings are consistent with an immunologic basis for the spontaneous remission of the dermal melanoma metastases present in this patient.

A low-dose, three-drug regimen, C.M.F. (cyclophosphamide 50 mg, p.o., days 1-14; methotrexate, 25 mg, and 5-fluorouracil, 500 mg, i.v., days 1 and 8; cycled every 28 days) was used in 46 consecutive chemtherapy-eligible women (41 previously hormonally treated) with recurrent breast cancer. Thirteen percent of the patients had complete regressions (C.R.); 33% had partial regressions (P.R.); 26% stabilized; and 28% progressed. In evaluating response by sites of metastases, lymph nodes (30%), lung nodules (22%), and subcutaneous deposits (2/3) had the highest incidence of C.R.; 46-71% of patients with lymph node, lung, subcutaneous, liver, breast, or peritoneal disease showed C.R. or P.R. Skin and pleural disease responded in 30% of patients whereas no patients had radiographic healing of bony metastases. The toxicity was minimal: 7% gastrointestinal, 26% marrow-suppressive, and 7% infectious. This low-dose C.M.F. regimen resulted in regression resulted in regression rates similar to higher dose C.M.F. protocols, which use approximately twice these drug dosages with commensurate toxicity.

The activity of a complement-dependent cytotoxic antibody in the sera of 21 melanoma patients was investigated using a microcytotoxicity assay. Heat-inactivated sera were caused to react against mechanically dispersed fresh tumor cells in the presence of exogenous blood group AB complement. Cytotoxicity was evaluated relative to pooled normal sera as a control. Sera were cytotoxic against autochthonous tumor cells in 9 of 10 patients with localized or regional melanoma and in 1 of 11 patients with disseminated metastases. Cytotoxicity of sera was unrelated to size of tumor burden. Six of 7 antibody-positive sera (autochthonous system) were noncytotoxic to between 2 and 7 different allogeneic melanoma tumor cell preparations. Immunological reactivity of the cytotoxic antibody-positive and -negative groups was similar with respect to their capacity to be sensitized to dinitrochlorobenzene, produce positive skin tests to microbial antigens, and produce antibodies to typhoid vaccination; serum immunoglobulins were comparable. These results support the reported findings of the presence of cytotoxic antibody in the sera of melanoma patients without disseminated metastases.

Fifty patients with metastatic malignant melanoma were randomized to treatment with either DTIC (2 mg/kg/day X 10 iv) or the combination of BCNU (150 mg/m2 iv) plus vincristine (VCR) (2 mg/m2 iv on Day 1 only). Treatment failures were crossed over to the alternate therapy. Primary, secondary, and cumulative response rates to DTIC were 29%, 9%, and 22%, respectively. Primary, secondary, and cumulative response rates to BCNU plus VCR were 23%, 29%, and 25%, respectively. Five of 26 patients (19%) experienced objective regression from secondary therapy after failure to respond to primary therapy. DTIC produced gastrointestinal and hematologic toxic effects; BCNU plus VCR produced gastrointestinal, hematologic, and neurologic toxic effects. VCR administered at a dose of 2 mg/m2 resulted in excessive neurologic toxic effects in 12 of 21 patients; a maximum VCR dose of 2 mg/injection was well tolerated by 15 subsequent patients without an adverse effect upon response rate. An analysis of tumor burden and organ involvement in responders and nonresponders suggests that DTIC is the first-choice treatment for patients with limited tumor burdens and nonvisceral metastases; BCNU plus VCR is the first-choice treatment for patients with extensive tumor burdens and visceral-predominant disease. However, failure to respond to primary therapy does not preclude response to secondary therapy with the alternate regimen.