Sulfurospirillum barnesii sp. nov. and Sulfurospirillum arsenophilum sp. nov., new members of the Sulfurospirillum clade of the epsilon Proteobacteria.

Two strains of dissimilatory arsenate-reducing vibrio-shaped bacteria are assigned to the genus Sulfurospirillum. These two new species, Sulfurospirillum barnesii strain SES-3T and Sulfurospirillum arsenophilum strain MIT-13T, in addition to Sulfurospirillum sp. SM-5, two strains of Sulfurospirillum deleyianum, and Sulfurospirillum arcachonense, form a distinct clade within the epsilon subclass of the Proteobacteria based on 16S rRNA analysis.

Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripigmentum sp. nov.

A newly discovered arsenate-reducing bacterium, strain OREX-4, differed significantly from strains MIT-13 and SES-3, the previously described arsenate-reducing isolates, which grew on nitrate but not on sulfate. In contrast, strain OREX-4 did not respire nitrate but grew on lactate, with either arsenate or sulfate serving as the electron acceptor, and even preferred arsenate. Both arsenate and sulfate reduction were inhibited by molybdate. Strain OREX-4, a gram-positive bacterium with a hexagonal S-layer on its cell wall, metabolized compounds commonly used by sulfate reducers. Scorodite (FeAsO42. H2O) an arsenate-containing mineral, provided micromolar concentrations of arsenate that supported cell growth. Physiologically and phylogenetically, strain OREX-4 was far-removed from strains MIT-13 and SES-3: strain OREX-4 grew on different electron donors and electron acceptors, and fell within the gram-positive group of the Bacteria, whereas MIT-13 and SES-3 fell together in the epsilon-subdivision of the Proteobacteria. Together, these results suggest that organisms spread among diverse bacterial phyla can use arsenate as a terminal electron acceptor, and that dissimilatory arsenate reduction might occur in the sulfidogenic zone at arsenate concentrations of environmental interest. 16S rRNA sequence analysis indicated that strain OREX-4 is a new species of the genus Desulfotomaculum, and accordingly, the name Desulfotomaculum auripigmentum is proposed.

Randomized, surgical adjuvant clinical trial of recombinant interferon alfa-2a in selected patients with malignant melanoma.

PURPOSE: We conducted a randomized prospective trial in selected patients with fully resected high-risk stage I and II malignant melanoma. PATIENTS AND METHODS: Interferon alfa-2a (IFN-alpha 2a) 20 x 10(6) U/m2 was administered three times each week for 12 weeks by the intramuscular route. Both the treatment group (n = 131) and the control group (n = 131) were evenly balanced with regard to relevant prognostic discriminants. RESULTS: The median disease-free survival (DFS) time was 2.4 years for the IFN-alpha 2a group and 2.0 years for the observation group (log-rank P = 0.19). The median survival times were 6.6 years for IFN-alpha 2a and 5.0 years for observation (log-rank P = .40). For stage I patients (n = 102), there was no apparent therapeutic advantage from IFN-alpha 2a therapy. The DFS for stage II patients was a median of 10.8 months in the control group versus 17 months in the treatment group. The overall survival time was 4.1 years for the treatment group versus 2.7 years for the control group. The differences in DFS for stage II patient were significant in a Cox model. These results must be interpreted cautiously because of subset analysis. A severe flu-like toxicity occurred in 44% of patients, 13% lost at least 10% of their baseline weight, and 45% experienced a worsening of Eastern Cooperative Oncology Group (ECOG) performance score. CONCLUSION: Our findings indicate trends that suggest a possible benefit for selected patients with high-risk malignant melanoma. The results will require further study in a larger patient population for confirmation.

Tumor necrosis factor-alpha and interleukin-1 beta in cerebrospinal fluid of patients with coccidioidal meningitis during therapy with fluconazole. National Institute of Allergy and Infectious Diseases Mycoses Study Group.

The levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 beta in the cerebrospinal fluid (CSF) of 66 patients with coccidioidal meningitis during therapy with fluconazole were measured by ELISA. The median concentration of TNF-alpha was 15.2 pg/mL for 322 samples; for IL-1 beta, it was 4.7 pg/mL for 316 samples. There were no significant changes in the level of either cytokine over 24 months of follow-up nor was there an association between the initial CSF concentrations of TNF-alpha and IL-1 beta and subsequent fluconazole treatment failure. Over time, concentrations of IL-1 beta were significantly associated with both clinical symptoms and white blood cell counts in CSF. These results indicate that CSF levels of TNF-alpha and IL-1 beta are relatively low compared with those associated with acute bacterial meningitis.

Estrogen replacement therapy withdrawal and regression of metastatic breast cancer.

BACKGROUND: Data regarding the effects of estrogen replacement therapy (ERT) and its withdrawal in patients with breast cancer are limited. METHODS: Four cases were observed involving estrogen replacement therapy withdrawal as management of metastatic breast cancer. RESULTS: Three patients with a history of primary breast cancer developed metastatic disease while on ERT. A fourth patient presented with metastatic breast cancer while on ERT. Withdrawal of this "physiologic" ERT alone as the only therapeutic intervention resulted in regression of metastatic disease in all four patients. CONCLUSIONS: In patients receiving ERT who develop metastatic breast cancer, withdrawal of ERT alone is a therapeutic option. Awareness of this phenomenon may be of value in management of these patients. The role of ERT and value of ERT withdrawal in patients with breast cancer deserve further study.

Phase II trial of recombinant tumor necrosis factor in disseminated malignant melanoma.

Twenty-one patients with disseminated malignant melanoma received recombinant tumor necrosis factor (TNF), 150 micrograms/m2 intravenously on days 1-5 every 2 weeks for four cycles and then every 3 weeks. Recombinant TNF produced no meaningful palliation. One patient (5%) attained an objective response of nodal, but not visceral, disease, which lasted 3 weeks. The median time to progression was 4 weeks. The median survival was 7.7 months. Ninety percent of patients developed mild to severe cytokine "flu." Ten percent developed significant hepatic toxicity (AST greater than 3 times normal). As a single agent, recombinant TNF is not likely to palliate disseminated malignant melanoma. However, combinations of recombinant TNF and cytotoxic or immune modulatory agents, particularly gamma interferon, may merit further investigation.

DNA content flow cytometry as a prognostic factor for node-positive breast cancer. The role of multiparameter ploidy analysis and specimen sonication.

The DNA content was analyzed in paraffin-embedded material from 167 patients with node-positive breast cancer to learn whether specimen sonication and multiparameter ploidy analysis (MPPA) (using DNA content and light scatter) could improve the strength of ploidy as a prognostic variable. Sonicated specimens were found to have fewer aggregates, a lower percentage of cells in S-phase (%S) and G2M phase than the corresponding nonsonicated specimens. The results using MPPA predicted the prognosis better because they allowed detection of small aneuploid peaks in histograms classified as diploid or tetraploid using DNA content alone. Ploidy was a significant univariate factor, and patients with tetraploid tumors had the best survival. In the multivariate analysis, if other routine factors were examined preferentially, ploidy and %S did not provide additional prognostic information for survival. This study of paraffin-embedded breast cancers suggested that sonication and MPPA may improve the ploidy analysis in certain cases and that tetraploidy may be a favorable ploidy pattern in this group.

Rhizobium meliloti exopolysaccharides: genetic analyses and symbiotic importance.

Genetic experiments have indicated that succinoglycan (EPS I), the acidic Calcofluor-binding exopolysaccharide, of the nitrogen-fixing bacterium Rhizobium meliloti strain Rm1021 is required for nodule invasion and possibly for later events in nodule development on alfalfa and other hosts. Fourteen exo loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. We have identified two loci, exoR and exoS, that are involved in the regulation of EPS I synthesis in the free-living state. Certain exo mutations which completely abolish EPS I production are lethal in an exoR95 or exoS96 background. Histochemical analyses of the expression of exo genes during nodulation using exo::TnphoA fusions have indicated that the exo genes are expressed most strongly in the invasion zone. In addition, we have discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for exopolysaccharides in symbiosis are discussed.

A randomized trial of cyclophosphamide, doxorubicin, and prednisone versus cyclophosphamide, 5-fluorouracil, and prednisone in patients with metastatic breast cancer.

Ninety-four patients were entered in a clinical trial assessing the clinical activity of cyclophosphamide, doxorubicin, and prednisone (CAP) versus a combination of cyclophosphamide. 5-Fluorouracil, and prednisone (CFP) in patients with advanced breast cancer. Objective response rates were comparable, 49% for CFP and 46% for CAP. There was no statistical difference between the duration of response of the two regimens or in time to progression. Most importantly, survival differences were not apparent. Both regimens were clinically tolerable and toxicities, for the most part, were comparable. Thus, no therapeutic advantage existed for either of these polychemotherapy regimens in patients with advanced breast cancer.

Disseminated malignant melanoma and recombinant interferon: analysis of seven consecutive phase II investigations.

We have performed seven phase II trials with recombinant interferons (IFN) involving 191 patients with biopsy-proved, measurable disseminated malignant melanoma. The regimens and numbers of patients have included IFN-alpha 2A, 50 X 10(6) U/m2 subcutaneous (SQ) TIW (regimen A, 31 patients); IFN-alpha 2A, 12 X 10(6) U/m2 SQ TIW (regimen B, 30 patients); IFN-alpha 2A with cimetidine as an immunorestorative agent (regimen C, 35 patients); IFN-gamma (regimen E, 29 patients); IFN-alpha 2A with IFN gamma (Regimen E, 20 patients); IFN-alpha 2A with bis-chloroethylnitrosourea (BCNU) (regimen F, 30 patients); and IFN-alpha 2A with the biochemical modulator, difluoromethylornithine (DFMO) (regimen G, 16 patients). The objective regression rates were as follows: A, 23%; B, 20%; C, 23%; D, 10%; E, 5%; F, 7%; G, 0%. Despite the higher response rate from regimen A, there appeared to be no survival advantage from any of these programs. The median time to progression was 1 month with a median survival time of 6 months. Most regressions involved soft tissue disease, were partial, and occurred within 2-3 months of treatment. Four patients received IFN for approximately 6 months and have manifested extraordinarily durable regressions of greater than 4+ years. The alpha-regimens produced a flu-type illness and anorexia which were dose-related. Leukopenia was most noteworthy with regimens containing gamma-interferon. Ongoing trials involving alternative and improved immune-related modalities are awaited with keen interest.

Phase II assessment of recombinant leukocyte A interferon with difluoromethylornithine in disseminated malignant melanoma.

Sixteen patients with advanced melanoma received IFN-alpha 2A, 36 X 10(6) U/m2 i.m., on days 3-7 with 2.25 g/m2 DFMO p.o. on days 1-7. We observed no objective regressions. Median time to progression was 1.2 months with a median survival of 5.2 months. A flu-type syndrome was the predominant sequela. From the dose and schedule that we utilized, this regimen holds little promise against disseminated malignant melanoma.

Blood transfusion exposure does not influence survival in patients with carcinoma of the breast.

There is abundant evidence of immune modulation induced by exposure to blood transfusions. Some studies have demonstrated a detrimental effect of transfusion on the recurrence of malignant disease and survival. We retrospectively studied the impact of blood transfusion exposure on 229 patients with breast cancer who were seen from July 1973 to September 1980, had at least 5 years' follow-up and had been randomized by therapy at the time of diagnosis. The patients were divided into four groups according to transfusion history: Group 1 (111 patients), no transfusion; Group 2 (34 patients), first transfusion after mastectomy; Group 3 (41 patients), first transfusion at mastectomy; and Group 4 (43 patients), first transfusion before mastectomy. All transfused patients received red cells or whole blood or both. At the time of analysis, 124 (54%) of the patients had died. Only Group 2 was statistically associated with decreased survival; recurrence of disease was 85 percent in this group, compared with 53 percent to 61 percent in the other three groups (p = 0.006, log-rank test). In general, Group 2 patients received transfusions because of recurrent disease. We conclude that transfusions before or at mastectomy are not associated with increased recurrence or reduced survival in patients with breast cancer.

Results of salvage hormonal therapy and salvage chemotherapy in women failing adjuvant chemotherapy after mastectomy for breast cancer.

We have evaluated the results of salvage systemic therapy in 257 patients with breast cancer recurrent after surgical adjuvant treatment with cyclophosphamide, fluorouracil, and prednisone (CFP) with or without tamoxifen. The overall objective response rate to salvage hormonal therapy was 29% (47 responses in 161 patients) and to salvage chemotherapy was 28% (43 responses in 156 patients). Response rates to salvage chemotherapy were similar whether or not prior salvage hormonal therapy or local modalities had been administered. Retreatment with CFP as a salvage chemotherapy yielded responses in 11 of 44 patients (25%). Response rates were similar for patients who began salvage CFP less than or equal to 12 months or greater than 12 months after completion of adjuvant CFP. We conclude that when this unselected population of patients failing adjuvant CFP is considered, 1) response rates to salvage chemotherapy were low regardless of whether or not prior salvage hormonal or local therapies were given, 2) repeating adjuvant chemotherapy (CFP) following relapse produced a low response rate, and 3) response rates to salvage hormonal therapy were low, but on the order of those observed in patients with advanced disease unselected by estrogen receptor status who are treated with first line hormonal maneuvers.

Complete responses and long-term survivals after systemic chemotherapy for patients with advanced malignant melanoma.

Five hundred three patients with advanced malignant melanoma were exposed to a number of clinical investigative chemotherapeutic regimens between 1971 and 1984 in an effort to assess the clinical activity of these regimens in this disease. Of the 503 patients participating in the studies, ten patients experienced a complete response. However, only three of these patients survived more than 5 years. Of this group of 503 patients, seven additional patients who did not experience a complete response survived more than five years. Of the ten patients surviving more than 5 years, two had immediate progression after institution of investigative regimens, whereas five remained stable for brief periods of time before progressive metastatic disease. Three patients experienced a complete response. It appeared that systemic therapeutic interventions in these trials were conspicuously ineffective for this large group of patients. A few long-term survivors attest to the capricious nature of this neoplasm and its association with likely spontaneous regressions. Although these long-term survivors did survive after institution of systemic chemotherapy, it is likely that this survival was related temporally, but perhaps not causally, to the institution of treatment.

A phase I-II trial of the combination of recombinant leukocyte A interferon and recombinant human interferon-gamma in patients with metastatic malignant melanoma.

Twenty patients with advanced malignant melanoma received daily intramuscular recombinant leukocyte A interferon (rIFN-alpha A, Roferon-A, Hoffmann-Laroche, Nutley, NJ) concomitant with recombinant human interferon-gamma (rIFN-gamma Genentech, South San Francisco, CA). During the first week alpha dose was 2 X 10(6) U/m2 and the gamma dose was 0.01 mg/m2 with escalations, if clinically tolerable, during the second week to 5 X 10(6) U/m2 and 0.025 mg/m2, respectively. Twelve patients received the escalated doses; subsequent granulocytopenia and a flu-type illness were severe in four of the 12. We observed one partial response of MRI-documented and biopsy-confirmed osseous metastases for 7+ months. For all study participants, the median time to progression was 1 month with a median survival of 6 months. From the dose and schedule which we utilized, concurrent rIFN-alpha A and rIFN-gamma provided little impact on advanced malignant melanoma.

Recombinant interferons in the management of advanced malignant melanoma. Updated review of five prospective clinical trials and long-term responders.

One hundred forty-five patients with disseminated malignant melanoma have participated in five Phase II clinical trials utilizing leukocyte A recombinant interferon (IFN-alpha 2A) (96 patients), recombinant interferon gamma (rIFN-gamma) (29), or IFN-alpha 2A concomitant with rIFN-gamma (20 patients). The overall response rate was 17%, with most regressions occurring with the IFN-alpha 2A regimens. The median times to progression (1 month) and survival (6 months) were generally similar to those from chemotherapeutic agents. However, a limited cohort of patients had complete regressions or durable partial responses even after treatment was discontinued or maintained at less than or equal to 25% of the starting dosage. Most objective regressions were partial, occurred in nonvisceral sites, and were detected within 2 months of the beginning of therapy. The most noteworthy sequelae from these regimens were predominantly constitutional, but without any obvious long-term complications. These interferon programs can be conveniently self-administered on an outpatient basis. Although single-agent interferon regimens for advanced malignant melanoma will probably not offer a substantive therapeutic advance, combinations of these molecules with other biological agents (tumor necrosis factor), biochemical modulators (difluoromethylornithine), and cytotoxic agents (bischloroethylnitrosourea, BCNU) offer innovative therapeutic dimensions in the design of future clinical investigations.

A design for phase II testing of anticancer agents within a phase III clinical trial.

A design for testing new anticancer agents is proposed such that the initial testing of new agents (phase II trials) is included within the framework of a comparative clinical trial (phase III). Randomization between phase II trials and the treatment groups of the phase III trial enforces consistency of patient selection and evaluation of response criteria. Patients who progress on the phase II trials of the new agents are randomized to one of the treatments of the phase III trial. Design issues, such as sample size and power, and analysis of the proposed design, are discussed. Advantages and disadvantages of the design are illustrated by sample size calculations for a current clinical trial in advanced breast cancer.

The effect on survival of initial chemotherapy in advanced breast cancer: polychemotherapy versus single drug.

Since current clinical trials assessing new agents occur in patients with advanced breast cancer having failed one and sometimes many polychemotherapy programs, these new agents may not be given a fair trial. In an effort to assess the possibility of using an alternative study design, we analyzed older clinical trials that used a controlled study design, randomizing between a single new drug and an established polychemotherapy program with a cross-over design upon failure. We were interested in noting that the pooled data did display a slight survival advantage (median 3.7 months) for the group receiving polychemotherapy as initial therapy. The survival distributions were clearly not significant using the log rank test, but did approach significance using the Smirnov. It is apparent that, while some slight advantage does occur for that group of patients receiving initial polychemotherapy, the magnitude of this effect is not great and is short in duration. Serious consideration should be given to the assessment of new agents as first-line therapy, particularly should they have a unique mode of action or lessened morbidities or toxicities.