Acute extracellular acidification reduces intracellular pH, 42 degrees C-induction of heat shock proteins and clonal survival of human melanoma cells grown at pH 6.7.

Two human melanoma cell lines, SK-Mel-28 and DB-1, were used for in vitro studies of the mechanisms underlying heat resistance of human tumour cells adapted to growth in acidic environments. Adaptation to growth at low pH was characterized by resistance to 42 degrees C cytotoxicity and accompanied by an increase in endogenous levels of Hsp70 and/or Hsp27. Acute extracellular acidification to levels below pH 6.5 was required to sensitize the melanoma cells to 42 degrees C. Furthermore, cells grown at low pH were more resistant to sensitization by acute acidification than cells grown at pH 7.3. The intracellular pH (pHi) of cells grown at pH 6.7 was less than the pHi of cells grown at pH 7.3 both before and after acute acidification. A pHi threshold existed for melanoma cells growing at pH 7.3 below which they became sensitized to 42 degrees C. This pHi threshold differed between the SK-Mel-28 and DB-1 cells. In contrast, a pHi threshold for heat sensitization did not exist for cells growing at pH 6.7: any reduction in pHi before heating resulted in increased cell killing. Since cells grown at low pH lack a pHi threshold for heat sensitization, they are sensitized more to 42 degrees C per unit decrease in pHi than cells grown at pH 7.3. Acute acidification abrogated the 42 degrees C-induction of Hsp70 and Hsp27 in the melanoma cells. The pHi thresholds for abrogation of these HSPs are slightly higher than or comparable with the thresholds for cytoxicity for each cell line grown at pH 7.3, but abrogation occurred over a narrower range of pHi compared with cytotoxicity. Abrogation of heat-induced expression of these HSPs correlates with cytotoxicity in both cell lines with the exception of Hsp27 expression in SK-Mel-28 cells. In conclusion, strategies that reduce pHi in melanoma cells growing at low pH, such as in acidotic regions of tumours, could selectively sensitize them to hyperthermia because they lack a pHi threshold for heat sensitization.

Acute extracellular acidification increases nuclear associated protein levels in human melanoma cells during 42 degrees C hyperthermia and enhances cell killing.

Acute acidification is being investigated as a strategy to sensitize human melanoma to 42 degrees C hyperthermia. The present study was conducted to determine the effect of hyperthermia and acute extracellular acidification on the nuclear associated protein (NAP) levels, heat shock protein (hsp) 70 and hsp27 content, and cell survival of human melanoma cells cultured at pH 7.3 or pH 6.7. It was observed that NAP levels increased slightly in both populations after 2 h of heating and then decreased to control levels with increasing time of heating at the growth pH. However, the NAP levels continued to increase in cells acutely acidified to pH 6.3 prior to and during heating. Hsp70 was induced to comparable levels in cells heated at their growth pH; however, the hsp27 levels were greater in cells cultured and heated at pH 6.7 than in cells cultured and heated at pH 7.3. Acute acidification to pH 6.3 prior to and during heating suppressed the 42 degrees C induction of hsp70 and hsp27 in both cell populations. The melanoma cells cultured and heated at pH 6.7 were more resistant to cell killing than cells cultured and heated at pH 7.3. Both populations were sensitized to cell killing by acute acidification to pH 6.3. The results suggest that hsps induced during 42 degrees C treatment associate with aggregating NAPs, enhancing their detergent solubility, and that abrogation of induced expression of hsps during heating at pH 6.3 contributes to increased levels of insoluble NAPS. In conclusion, acute extracellular acidification inhibits 42 degrees C induction of hsps, increases NAP levels, and decreases cell survival in DB-1 human melanoma cells.

Variability in glucose transporter-1 levels and hexokinase activity in human melanoma.

Melanoma exhibits heterogeneous growth patterns and widely varying sensitivities to multiple treatment modalities. This variability may reflect intrinsic genetic differences in factors giving rise to altered metabolism. Glucose is the primary energy source of tumours, including melanoma, and glucose transporter isoform 1 (Glut-1) and hexokinase are key rate-limiting factors in glucose metabolism. The levels of Glut-1 and total hexokinase activity were measured in 31 melanoma biopsies to determine the extent of tumour-to-tumour variability in these parameters. Relative Glut-1 levels were determined by Western immunoblot analysis using human anti-Glut-1 rabbit polyclonal antibody, and hexokinase activity was measured in the same samples by an enzymatic assay monitoring the reduction in the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) (in nmol NADP+ reduced/min per mg protein). All melanomas were from patients who had received no therapy prior to surgery. Immediately after excision, tumour biopsies were disaggregated to single cells by collagenase and DNase and frozen in liquid nitrogen. Thirty human melanomas exhibited a 22-fold variation in levels of Glut-1 and 29 exhibited a nine-fold variation in total cellular hexokinase activity. Glut-1 levels and hexokinase activity were not correlated with one another. The broad range in Glut-1 levels and hexokinase activity observed between melanomas suggests that these glycolytic rate-limiting parameters that influence the rate of glucose metabolism may contribute to the variability in melanoma response to treatment modalities.

Treatment of metastatic melanoma with autologous, hapten-modified melanoma vaccine: regression of pulmonary metastases.

A human cancer vaccine composed of autologous tumor cells modified with the hapten dinitrofluorobenzene (DNP) induces cell-mediated immunity to the tumor cells and the development of inflammatory responses within metastatic sites. In this study we determined whether DNP vaccine could induce regression of established metastases. Ninety-seven patients (83 evaluable) with surgically incurable metastatic melanoma were treated with DNP vaccine preceded by low-dose cyclophosphamide. Tumor regression was assessed by standard criteria. The development of cell-mediated immunity to melanoma-associated antigens was measured by delayed-type hypersensitivity (DTH) testing before and after DNP vaccine treatment. Survival analysis was performed by the Kaplan-Meier method. There were 11 antitumor responses: 2 complete, 4 partial and 5 mixed. Both complete responses and 2 of the 4 partial responses occurred in patients with lung metastases. Response durations were as follows: partial responses-5, 6, 8 and 47+ months; and complete responses-12 and 29 months. Tumor regression required at least 4 months to become evident and in 2 cases maximum regression was not observed until 1 year after beginning treatment. Patients who exhibited tumor regression survived longer than those who did not (median survival times: responders, 21.4 months; non-responders, 8.7 months; p = 0.010). DTH to DNP-modified and unmodified autologous melanoma cells was induced in 87% and 42% of patients, respectively. The DTH response to unmodified cells was significantly associated with prolonged survival. Autologous DNP-modified melanoma vaccine can induce clinically meaningful regression of metastases and small lung metastases appear to be unusually sensitive. The development of DTH to unmodified, autologous tumor cells may be an important indicator of the vaccine's efficacy.

Whole cell ELISA for detection of tumor antigen expression in tumor samples.

We have developed a whole cell enzyme-linked immunosorbent assay (ELISA) that detects the melanoma antigens gp100 and S-100 in tumor samples from patients with metastatic melanoma and the antigen CA 125 in tumor samples from patients with ovarian carcinoma. The assay is relatively simple to perform and interpret without specialized expertise in pathology. It is both sensitive and selective and is amenable to being automated. The results correlate very well with those obtained by flow cytometry and are in good accord with published values obtained by immunohistochemistry. We believe that this assay should be very helpful for characterizing autologous cell cancer vaccines and may also represent a useful alternative to immunohistochemistry for cancer diagnosis. It should be adaptable to other types of cancer where tumor antigens have been identified and good antibodies are available.

Autologous, hapten-modified vaccine as a treatment for human cancers.

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 gamma interferon in situ. Moreover, they represent expansion of T cell clones with novel T cell receptor structures. Occasionally, administration of DNP-vaccine results in partial or complete regression of measurable metastases. The most common site of regression has been small lung metastases. Administration of DNP-vaccine to patients in the post-surgical adjuvant setting produces a more striking clinical effect. We have treated 214 patients with clinically evident stage III melanoma who had undergone lymphadenectomy. With a median follow-up time of 4.4 years (1.8-10.4 years) the 5-year overall survival (OS) rate is 47% (one nodal site = 51%, two nodal sites = 33%). These results appear to be comparable to those obtained with high dose interferon. More recent studies suggest that this therapeutic approach is also applicable to 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.

Protracted survival after resection of metastatic uveal melanoma.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Paclitaxel and tamoxifen: An active regimen for patients with metastatic melanoma.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Cancer vaccines: reborn or just recycled?

The field of human cancer vaccines is currently undergoing a revival. This volume describes a variety of approaches to active immunotherapy, some traditional and some more technologically sophisticated. Interpretation of their claims to clinical efficacy requires an understanding of some of the principles of tumor immunotherapy. These include the following: (1) human cancers are weakly immunogenic or not immunogenic at all; (2) cell-mediated immune responses, mainly those of T lymphocytes, are critical to tumor rejection; (3) almost nothing is known about the nature of the rejection antigens on human tumors; (4) it is not clear whether these antigens are different for each case of human cancer or whether there are clinically useful antigens that are shared by all or most tumors of a given histologic type; and (5) the effectiveness of vaccines is greatly limited by excessive tumor burden. The reader is invited to approach this collection of papers with a combination of optimism and skepticism.

Autologous, hapten-modified vaccine as a treatment for human cancers.

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.

Intralesional selection of T cell clonotypes in the immune response to melanoma antigens occurring during vaccination.

T cells infiltrating pre- and postvaccine metastases obtained from melanoma patients vaccinated with either dinitrophenyl (DNP)-modified autologous tumor or with the MAGE-3.A1 peptide display selective T cell receptor (TCR) beta chain variable region (BV) repertoire changes at the tumor site as a consequence of vaccination. Restricted sets of BV families expand in all postvaccine lesions when compared with prevaccine specimens and often contain dominant clones. A protocol devised to obtain T cell lines highly enriched for expression of a given BV region through the use of anti-BV monoclonal antibodies was used to understand whether responses to specific antigen(s) accounted for these clonal expansions. In one of the patients vaccinated with DNP-modified tumor cells, BV-driven selection of the T lymphocytes expanded in two infiltrated postvaccine metastases resulted in T cell lines able to exert HLA class I-restricted lysis of the autologous tumor. These results indicate that TCR repertoire analysis at the tumor site facilitates the detection of T cell responses elicited by a vaccine and potentially cytotoxic for the autologous tumor.

Phase I study of R24 in patients with metastatic melanoma including evaluation of immunologic parameters.

R24 is a mouse IgG3 monoclonal antibody with specificity for the disialoganglioside GD3. Most human melanomas have substantial surface GD3; in addition, a significant proportion of T lymphocytes display surface GD3. In a phase I study, we have investigated the toxicity and effect on selected immunological parameters of three dose levels of R24 given intravenously daily for five days (10 mg/m2/d, 30 mg/m2/d and 50 mg/m2/d) to patients with advanced melanoma. R24 administration neither consistently diminished nor augmented expression of delayed type hypersensitivity (DTH) skin reactions to anergy panel antigens or to a contact allergen dinitrofluorobenzene. R24 was infrequently found on tumor cells, or on lymphocytes from DTH biopsies, despite measurable serum levels of R24. The 30 mg/m2/d dose of R24 produced a statistically significant drop in peripheral blood lymphocytes on treatment Day 5. Likewise, on Day 5 there was a modest but statistically significant decrement in the proportion of circulating cells which were R24+. While there was one mixed response, there were no complete or partial tumor regressions in the R24 treated patients; there was no evident clinical benefit from the R24 therapy. The toxicity of the R24 at the higher dose levels can be very substantial. One patient, on the highest dose level, died on the 4th day of R24 treatment; in the absence of a plausible alternative explanation, a relationship of the death to the administered R24 must be considered. A precipitous drop in serum albumin coincident with R24 administration was found in all cases; this effect has not been previously reported with R24.

Dinitrophenyl-modified autologous melanoma vaccine induces a T cell response to hapten-modified, melanoma peptides.

Active specific immunotherapy with dinitrophenyl (DNP)-modified autologous melanoma vaccine elicits inflammatory responses in metastatic tumor sites. Postsurgical adjuvant immunotherapy with this vaccine prolongs survival in stage III melanoma patients. We have reported that, after administration of DNP-modified melanoma vaccine, T cell responses to DNP-modified autologous tumor cells are demonstrable in vivo and in vitro. These responses are hapten specific and MHC restricted. To elucidate this phenomenon, we investigated the immune response to DNP-modified peptides eluted from autologous cells. Short peptides were extracted from DNP-modified and unmodified autologous melanoma cells by an acid elution technique and HPLC fractionation. Peptides were also extracted from DNP-modified and unmodified, EB virus-transformed, autologous B lymphoblasts. These various peptide fractions were loaded onto autologous B lymphoblasts and tested for ability to elicit a response by a DNP-specific T cell line as measured by IFN-gamma production. Unexpectedly, stimulatory activity of peptides from DNP-modified melanoma cells was confined to a single HPLC fraction. Spectrometric analysis of this fraction confirmed modification of peptides with DNP. A weaker T cell response was observed to a single HPLC fraction of DNP-modified peptides from the patient's B lymphoblasts. No T cell response was elicited by corresponding fractions of peptides eluted from unmodified melanoma cells or B lymphoblasts. These findings demonstrate the human T cell response to DNP-modified autologous melanoma cells is mediated by hapten-modified, MHC-associated peptides. Further investigation of these peptides could lead to a new strategy for peptide-based cancer immunotherapy.

Autologous hapten-modified melanoma vaccine as postsurgical adjuvant treatment after resection of nodal metastases.

PURPOSE: To determine whether treatment with an autologous whole-cell vaccine modified with the hapten dinitrophenyl (DNP vaccine) is an effective postsurgical adjuvant treatment for melanoma patients with clinically evident nodal metastases. PATIENTS AND METHODS: Eligible patients had regional nodal metastases that were large enough (> or = 3 cm diameter) to prepare vaccine. Following standard lymphadenectomy, patients were treated with DNP vaccine on a monthly or weekly schedule. RESULTS: Of 62 patients with metastasis in a single lymph node bed (stage III), 36 are alive after a median follow-up time of 55 months (range, 29 to 76); the projected 5-year relapse-free and overall survival rates are 45% and 58%, respectively. Of 15 patients with metastases in two nodal sites, five are alive with a median follow-up time of 73 months. An unexpected finding was the significantly better survival of older patients; the projected 5-year survival of patients greater than 50 versus < or = 50 years was 71% and 47%, respectively (P = .011, log-rank test). The development of a positive delayed-type hypersensitivity (DTH) response to unmodified autologous melanoma cells was associated with significantly longer 5-year survival (71% v 49%; P = .031). Finally, the median survival time from date of first recurrence was significantly longer for patients whose subcutaneous recurrence exhibited an inflammatory response (> 19.4 v 5.9 months; P < .001). CONCLUSION: Postsurgical adjuvant therapy with autologous DNP-modified vaccine appears to produce survival rates that are markedly higher than have been reported with surgery alone. Moreover, this approach has some intriguing immunobiologic features that might provide insights into the human tumor-host relationship.

BOLD+interferon in the treatment of metastatic uveal melanoma: first report of active systemic therapy.

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.