Stanley D. Tucker

ESHAP--an effective chemotherapy regimen in refractory and relapsing lymphoma: a 4-year follow-up study.

PURPOSE This study attempted to determine the efficacy of the combination of etoposide (VP-16), methyl-prednisolone, and cytarabine (Ara-C) with or without cisplatin in relapsing and refractory adult lymphoma patients. PATIENTS AND METHODS The first 63 patients were randomized to receive VP-16 40 mg/m2/d for 4 days, methylprednisolone 500 mg intravenously daily for 5 days, and Ara-C 2 g/m2 intravenously over 2 to 3 hours on day 5 with or without cisplatin 25 mg/m2 IV administered by 24-hour infusion for 4 days (ESHA +/- P). Markedly different responses between ESHA (33%) and ESHAP (75%) led to deletion of the ESHA arm. A total of 122 patients on the ESHAP regimen were studied. RESULTS Forty-five patients (37%) attained a complete remission (CR) and 33 (27%) attained a partial remission (PR), for a total response rate of 64%. The median duration of CR was 20 months, with 28% of remitters still in CR at 3 years. The overall median survival duration was 14 months; the survival rate at 3 years was 31%. Overall time to treatment failure (TTF) showed 10% of all patients to be alive and disease-free at 40 months. Response and survival rates were similar in patients with low-grade (n = 34), intermediate-grade (n = 67), transformed (n = 18), and high-grade (n = 3) lymphoma. The most significant factors for response and survival by multivariate analysis were the serum lactic dehydrogenase (LDH) level, tumor burden, and age (when analyzed as a continuous variable), while prior CR was highly significant by univariate analysis. A significant difference in survival was noted for patients with normal LDH levels and low- or intermediate-tumor burden or patients with low tumor burden and elevated LDH levels (55% 3-year survival rate) versus patients with elevated LDH levels and intermediate or high tumor burden (< 20%). Major toxicities included myelosuppression, with a median granulocyte count of 500/microL and platelet count of 70,000/microL. CONCLUSION ESHAP was found to be an active, tolerable chemotherapy regimen for relapsing and refractory lymphoma. Applying a prognostic model based on tumor burden and serum LDH level shows significant differences in survival in this patient population.

Cationic liposome-mediated E1A gene transfer to human breast and ovarian cancer cells and its biologic effects : a phase I clinical trial

PURPOSE Preclinical studies have demonstrated that the adenovirus type 5 E1A gene is associated with antitumor activities by transcriptional repression of HER-2/neu and induction of apoptosis. Indeed, E1A gene therapy is known to induce regression of HER-2/neu-overexpressing breast and ovarian cancers in nude mice. Therefore, we evaluated the feasibility of intracavitary injection of E1A gene complexed with DC-Chol cationic liposome (DCC-E1A) in patients with both HER-2/neu-overexpressing and low HER-2/neu-expressing breast and ovarian cancers in a phase I clinical trial. PATIENTS AND METHODS An E1A gene complexed with DCC-E1A cationic liposome was injected once a week into the thoracic or peritoneal cavity of 18 patients with advanced cancer of the breast (n = 6) or ovary (n = 12). RESULTS E1A gene expression in tumor cells was detected by immunohistochemical staining and reverse transcriptase-polymerase chain reaction. This E1A gene expression was accompanied by HER-2/neu downregulation, increased apoptosis, and reduced proliferation. The most common treatment-related toxicities were fever, nausea, vomiting, and/or discomfort at the injection sites. CONCLUSION These results argue for the feasibility of intracavitary DCC-E1A administration, provide a clear proof of preclinical concept, and warrant phase II trials to determine the antitumor activity of the E1A gene.

A new serologic staging system for large-cell lymphomas based on initial beta 2-microglobulin and lactate dehydrogenase levels.

We report results of our investigation of prognostic factors for patients with large-cell lymphoma who were entered on the same treatment protocol and who had known pretreatment serum beta 2-microglobulin (beta 2M) and lactate dehydrogenase (LDH) levels. beta 2M and LDH levels were the most significant and independent variables for predicting time to treatment failure (TTF) and survival. The serum level of beta 2M correlated with tumor burden. These two serum markers defined three significantly different prognostic groups. All 27 patients in the low-risk group remain alive and in remission; in contrast, 22 of the 27 patients (81%) in the high-risk group have failed treatment, and only seven (26%) remain alive. In comparison with the Ann Arbor staging system, serum levels of beta 2M and LDH may provide a more precise system for defining risk groups and thereby allow a more rational approach to the development and analysis of treatment strategies.

Inhibition by all-trans-retinoic acid of tumor necrosis factor and nitric oxide production by peritoneal macrophages.

Besides their growth‐inhibiting and differentiation‐inducing properties, retinoids have been shown to exert immunomodulatory and anti‐inflammatory functions by mechanisms that are not well understood. Tumor necrosis factor‐α (TNF), a cytokine produced by mononuclear phagocytes, has been shown to be an important mediator of endotoxin‐induced septic shock, cachexia, bone resorption, and inflammation. Nitric oxide may also have a role in septic shock, hypotension, and vasodilatation. In this study, we examined the effects of retinoids on the production of TNF and nitric oxide by murine peritoneal macrophages. Of the various retinoids studied, all‐trans‐retinoic acid (RA) was most potent; it almost completely inhibited the production of TNF by macrophages activated with endotoxin and interferon‐γ. The inhibitory effect was dependent on the dose and duration of RA exposure to macrophages. RA also blocked phorbol ester‐induced TNF production in a macrophage cell line (RAW 264.7). Besides TNF, the retinoid suppressed the production of nitric oxide from activated peritoneal macrophages. The importance of these results in relation to controlling various harmful effects of cytokines released by activated macrophages is discussed. J. Leukoc. Biol. 55: 336–342; 1994.

Missense and nonsense suppressors can correct frameshift mutations

Missense and nonsense suppressor tRNAs, selected for their ability to read a new triplet codon, were observed to suppress one or more frameshift mutations in trpA of Escherichia coli. Two of the suppressible frameshift mutants, trpA8 and trpA46AspPR3, were cloned, sequenced, and found to be of the +1 type, resulting from the insertion of four nucleotides and one nucleotide, respectively. Twenty-two suppressor tRNAs were examined, 20 derived from one of the 3 glycine isoacceptor species, one from lysT, and one from trpT. The sequences of all but four of the mutant tRNAs are known, and two of those four were converted to suppressor tRNAs that were subsequently sequenced. Consideration of the coding specificities and anticodon sequences of the suppressor tRNAs does not suggest a unitary mechanism of frameshift suppression. Rather, the results indicate that different suppressors may shift frame according to different mechanisms. Examination of the suppression windows of the suppressible frameshift mutations indicates that some of the suppressors may work at cognate codons, either in the 0 frame or in the +1 frame, and others may act at noncognate codons (in either frame) by some as-yet-unspecified mechanism. Whatever the mechanisms, it is clear that some +1 frameshifting can occur at non-monotonous sequences. A striking example of a frameshifting missense suppressor is a mutant lysine tRNA that differs from wild-type lysine tRNA by only a single base in the amino acid acceptor stem, a C to U70 transition that results in a G.U base pair. It is suggested that when this mutant lysine tRNA reads its cognate codon, AAA, the presence of the G.U base pair sometimes leads either to a conformational change in the tRNA or to an altered interaction with some component of the translation machinery involved in translocation, resulting in a shift of reading frame. In general, the results indicate that translocation is not simply a function of anticodon loop size, that different frameshifting mechanisms may operate with different tRNAs, and that conformational features, some far removed from the anticodon region, are involved in maintaining fidelity in translocation.

Project Zero Delay: A Process for Accelerating the Activation of Cancer Clinical Trials

Drug development in cancer research is lengthy and expensive. One of the rate-limiting steps is the initiation of first-in-human (phase I) trials. Three to 6 months can elapse between investigational new drug (IND) approval by the US Food and Drug Administration and the entry of a first patient. Issues related to patient participation have been well analyzed, but the administrative processes relevant to implementing clinical trials have received less attention. While industry and academia often partner for the performance of phase I studies, their administrative processes are generally performed independently, and their timelines driven by different priorities: safety reviews, clinical operations, regulatory submissions, and contracting of clinical delivery vendors for industry; contracts, budgets, and institutional review board approval for academia. Both processes converge on US Food and Drug Administration approval of an IND. In the context of a strategic alliance between M. D. Anderson Cancer Center and AstraZeneca Pharmaceuticals LP, a concerted effort has been made to eliminate delays in implementing clinical trials. These efforts focused on close communications, identifying and matching key timelines, alignment of priorities, and tackling administrative processes in parallel, rather than sequentially. In a recent, first-in-human trial, the study was activated and the first patient identified in 46 days from completion of the final study protocol and about 48 hours after final US Food and Drug Administration IND approval, reducing the overall timeline by about 3 months, while meeting all clinical good practice guidelines. Eliminating administrative delays can accelerate the evaluation of new drugs without compromising patient safety or the quality of clinical research.

Independence of the pattern of early cytokine release from autoregulation by nitric oxide

The L‐arginine‐dependent tumor cell cytotoxicity produced by activated macrophages (Mφ) may be mediated either directly by production of nitric oxide (NO), or by induction of NO synthesis in the tumor cell. The influence of Mφ NO synthesis on the release of soluble cytotoxic mediators was investigated in this study. The synthesis of Mφ NO, measured as nitrite, was detected 6 h after lipopolysaccharide (LPS)‐triggering and reached a peak level by 44 h. A concurrent decrease in Mφ viability beginning at 18 h after triggering was detected during a period of 72 h in culture. Both the production of NO and loss of viability correlated with the presence of L‐arginine in the incubation media and was inhibited by NG‐monomethyl‐L‐arginine (NMA). The medium in which LPS‐triggered adherent peritoneal exudate cells were incubated was examined for the presence of tumor necrosis factor (TNF), gamma interferon (IFN‐γ), and the soluble mediators that induce mitochondrial respiratory inhibition in tumor cells. All of these effector molecules were released at peak levels into the conditioned supernatants within 12 h after LPS‐triggering. The peak level obtained for each effector molecule was influenced by the media in which the Mφ was incubated; however, no correlation was detected between the level of cytokines produced and the synthesis of nitrite by the Mφ indicating that NO synthesis has no inhibiting effect on the initial burst of cytotoxic factors released.

Inhibition of tumor cell mitochondrial respiration by macrophage cytotoxic mediators distinct from interferon-gamma.

Macrophage‐mediated inhibition of mitochondrial respiration in EMT‐6 murine mammary adenocarcinoma cells can be mimicked in vitro by treatment of the cells with interferon‐γ (IFN‐γ) in combination with tumor necrosis factor, interleukin‐1, or lipopolysaccharide. Conditioned supernatants obtained from activated macrophages appear to contain interferon‐γ, suggesting that inhibition of mitochondrial respiration in tumor cells was caused by synergy of IFN‐γ with other cytokines. To further characterize monokines that cause inhibition of mitochondrial respiration in tumor cells, EA13.5 macrophage‐like cells were isolated and selected for inhibition of mitochondrial respiration in EMT‐6 tumor cells. After stimulation with IFN‐γ and lipopolysaccharide, the EA13.5 cells released into conditioned supernatants a cytotoxic mediator that induced nitric oxide synthesis and caused lesions in the electron transport chain of EMT‐6 cells similar to the lesions caused by activated peritoneal macrophages. Enzyme‐linked immunosorbent assay demonstrated that the conditioned supernatants produced by EA13.5 macrophage cells did not contain IFN‐γ. Treatment of the EA13.5 cell‐conditioned supernatants with neutralizing antibody against IFN‐γ did not abrogate the inhibition of mitochondrial respiration in EMT‐6 cells caused by these conditioned supernatants. This study demonstrated that unidentified macrophage cytotoxic mediators distinct from IFN‐γ are involved in the induction of nitric oxide synthesis and inhibition of mitochondrial respiration in tumor cells.

Reversion of trpA nonsense mutations by deletion of the chain-termination codons

This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of Escherichia coli. This mechanism, deletion of the nonsense codon, was discovered in the course of selecting for missense revertants of trpA(UGA211) and for catalytically active tryptophan synthetase alpha chain revertants of trpA(UAA234) and trpA(UAG234). Each type of revertant trpA was cloned and its DNA sequence determined. trpA(UGA211) gave rise to two previously unidentified types of missense revertant. The first type was expected, namely trpA(CGA211), the result of a base substitution event. The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211. It was found to be the result of a 21 base-pair deletion of a region containing codon 211. The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a frequency of approximately 2 per 10(9) viable cells. They were identical in that they both resulted from a 3 base-pair deletion, namely deletion of the chain-terminating codon at position 234. One of them, however, also displayed an A instead of the normal G in the third position of codon 235. The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts. These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model multienzyme complex, and for the study of mechanisms by which deletions can be generated.