Phase III randomized study of second line ADI-PEG 20 plus best supportive care versus placebo plus best supportive care in patients with advanced hepatocellular carcinoma.

Background: Arginine depletion is a putative target in hepatocellular carcinoma (HCC). HCC often lacks argininosuccinate synthetase, a citrulline to arginine-repleting enzyme. ADI-PEG 20 is a cloned arginine degrading enzyme-arginine deiminase-conjugated with polyethylene glycol. The goal of this study was to evaluate this agent as a potential novel therapeutic for HCC after first line systemic therapy. Methods and patients: Patients with histologically proven advanced HCC and Child-Pugh up to B7 with prior systemic therapy, were randomized 2 : 1 to ADI-PEG 20 18 mg/m2 versus placebo intramuscular injection weekly. The primary end point was overall survival (OS), with 93% power to detect a 4-5.6 months increase in median OS (one-sided α = 0.025). Secondary end points included progression-free survival, safety, and arginine correlatives. Results: A total of 635 patients were enrolled: median age 61, 82% male, 60% Asian, 52% hepatitis B, 26% hepatitis C, 76% stage IV, 91% Child-Pugh A, 70% progressed on sorafenib and 16% were intolerant. Median OS was 7.8 months for ADI-PEG 20 versus 7.4 for placebo (P = 0.88, HR = 1.02) and median progression-free survival 2.6 months versus 2.6 (P = 0.07, HR = 1.17). Grade 3 fatigue and decreased appetite occurred in <5% of patients. Two patients on ADI-PEG 20 had ≥grade 3 anaphylactic reaction. Death rate within 30 days of end of treatment was 15.2% on ADI-PEG 20 versus 10.4% on placebo, none related to therapy. Post hoc analyses of arginine assessment at 4, 8, 12 and 16 weeks, demonstrated a trend of improved OS for those with more prolonged arginine depletion. Conclusion: ADI-PEG 20 monotherapy did not demonstrate an OS benefit in second line setting for HCC. It was well tolerated. Strategies to enhance prolonged arginine depletion and synergize the effect of ADI-PEG 20 are underway. Clinical Trial number: www.clinicaltrials.gov (NCT 01287585).

Arginine deiminase PEG20 inhibits growth of small cell lung cancers lacking expression of argininosuccinate synthetase.

BACKGROUND: Some cancers have been shown to lack expression of argininosuccinate synthetase (ASS), an enzyme required for the synthesis of arginine and a possible biomarker of sensitivity to arginine deprivation. Arginine deiminase (ADI) is a microbial enzyme capable of efficiently depleting peripheral blood arginine. METHODS: Argininosuccinate synthetase expression was assessed in human small cell lung cancer (SCLC) by immunohistochemistry (IHC), with expression also assessed in a panel of 10 human SCLC by qRT-PCR and western blot. Proliferation assays and analyses of apoptosis and autophagy assessed the effect of pegylated ADI (ADI-PEG20) in vitro. The in vivo efficacy of ADI-PEG20 was determined in mice bearing SCLC xenografts. RESULTS: Approximately 45% of SCLC tumours and 50% of cell lines assessed were negative for ASS. Argininosuccinate synthetase-deficient SCLC cells demonstrated sensitivity to ADI-PEG20, which was associated with the induction of autophagy and caspase-independent cell death. Arginine deiminase-PEG20 treatment of ASS-negative SCLC xenografts caused significant, dose-dependent inhibition of tumour growth of both small and established tumours. CONCLUSION: These results suggest a role for ADI-PEG20 in the treatment of SCLC, and a clinical trial exploring this therapeutic approach in patients with ASS-negative SCLC by IHC has now been initiated.

Negative argininosuccinate synthetase expression in melanoma tumours may predict clinical benefit from arginine-depleting therapy with pegylated arginine deiminase.

BACKGROUND: Arginine-depleting therapy with pegylated arginine deiminase (ADI-PEG20) was reported to have activity in advanced melanoma in early phase I-II trial, and clinical trials are currently underway in other cancers. However, the optimal patient population who benefit from this treatment is unknown. METHODS: Advanced melanoma patients with accessible tumours had biopsy performed before the start of treatment with ADI-PEG20 and at the time of progression or relapse when amenable to determine whether argininosuccinate synthetase (ASS) expression in tumour was predictive of response to ADI-PEG20. RESULTS: Twenty-seven of thirty-eight patients treated had melanoma tumours assessable for ASS staining before treatment. Clinical benefit rate (CBR) and longer time to progression were associated with negative expression of tumour ASS. Only 1 of 10 patients with ASS-positive tumours (ASS+) had stable disease, whereas 4 of 17 (24%) had partial response and 5 had stable disease, when ASS expression was negative (ASS-), giving CBR rates of 52.9 vs 10%, P=0.041. Two responding patients with negative ASS expression before therapy had rebiopsy after tumour progression and the ASS expression became positive. The survival of ASS- patients receiving at least four doses at 320 IU m(-2) was significantly better than the ASS+ group at 26.5 vs 8.5 months, P=0.024. CONCLUSION: ADI-PEG20 is safe and the drug is only efficacious in melanoma patients whose tumour has negative ASS expression. Argininosuccinate synthetase tumour positivity is associated with drug resistance and tumour progression.

Responses of purified phospholipases A2 to phospholipase A2 activating protein (PLAP) and melittin.

The role of the phospholipase A2 (PLA2) stimulating protein PLAP in the regulation of PLA2 activity was assessed by determination of the effects of PLAP on two purified PLA2s. An approx. 14 kDa enzyme was purified from mouse thymoma cells, EL-4 cells, by cation ion exchange HPLC and immunoaffinity HPLC (with antiserum to the N-terminal sequence of an inflammatory exudate PLA2). An approx. 110 kDa enzyme was purified from mouse mammary carcinoma derived cells by sequential hydrophobic, anion exchange, hydroxyapatite and gel filtration HPLC. Neither PLAP nor melittin, an immunologically related PLA2 stimulating peptide from bee venom, increased the activity of the high molecular weight enzyme. In contrast, there was more than a 20-fold stimulation of the low molecular weight PLA2 by PLAP and an approx. 5-fold stimulation by melittin. The stimulation of enzyme activity by PLAP was observed at a protein to phospholipid ratio of 1:10(6) while the ratio of melittin to phospholipid was 1:3. Thus, PLAP mediated stimulation of PLA2 activity may include an interaction between PLAP and the enzyme, in contrast to melittin stimulation, which involves interactions between melittin and phospholipid.

The role of fatty acid metabolites in the differentiation of the human monocyte-like cell line U937.

The human monocyte cell line, U937, can be induced to terminally differentiate into macrophage-like cells when treated with gamma-interferon. However, if these cells were treated with gamma-interferon and esculetin, an inhibitor of the lipoxygenase pathway, or BW755C, an inhibitor of both the lipoxygenase and the cyclooxygenase pathways, a marked inhibition in cellular differentiation occurred. In contrast, inhibitors of only the cyclooxygenase pathway had no effect on differentiation. These studies suggest a role for lipoxygenase products of arachidonic acid in the differentiation of the human U937 cell line. Arachidonic acid utilized in the production of eicosanoids is derived from phospholipids by the action of phospholipase A2 and phospholipase C. When U937 cells were cultured in medium supplemented with gamma-interferon, there was a striking increase in the level of phosphatidylcholine and phosphatidylethanolamine-specific phospholipase A2 activities and phosphatidylinositol-specific phospholipase C activity as compared to control cells. More ever, although there was not a significant difference in the incorporation of labeled arachidonic acid or linoleic acid into the major phospholipids of differentiated U937 cells as compared to undifferentiated control cells, there was a marked increase in the relative amount of the labeled arachidonic acid released from the differentiated cells as lipoxygenase products compared to cyclooxygenase products. These data suggest that lipoxygenase products may be essential in the differentiation process of U937 cells and that enhanced phospholipase enzyme activities that occur during differentiation help explain how arachidonic acid becomes available to form lipoxygenase products.

Leukotriene B4 modulates phospholipid methylation and chemotaxis in human polymorphonuclear leukocytes.

Formation of phosphatidylcholine from phosphatidylethanolamine via the S-adenosylmethionine (AdoMet) pathway has been shown to be required for signal transduction of receptor-ligand interactions in a variety of cells. These interactions result in the remodeling of phospholipid pools and phospholipase activation. To extend these observations and to explore the role of the phosphatidylcholine synthesis pathway in transduction of the leukotriene B4 (LTB4) receptor-ligand response, we examined phospholipid methylation in human polymorphonuclear leukocytes (PMN) following stimulation by LTB4, a potent chemotactic agent that is a metabolite of arachidonic acid. At early time points (approximately 3-10 min), formation of methylated phospholipids was enhanced following LTB4 stimulation. The LTB4 analogs 6-trans LTB4 as well as LTB4 epimers induced less methylation compared with LTB4, and the potencies of these analogs in inducing methylation correlated with their diminished ability to induce chemotaxis. Furthermore, the ability of these agonists to induce methylation also correlated with the binding affinity of these agents to the LTB4 receptors on these cells. Synthesis of phosphatidylcholine by the choline transferase pathway was not affected by LTB4. Inhibition of the AdoMet reaction with 3- deazaadenosine, L-homocysteine homolactone, or erythro-9-[2-hydroxy-3-nonyl] adenine (EHNA) abrogated LTB4-induced phospholipid methylation and the chemotactic response. The potencies of these inhibitors in blocking phospholipid methylation also correlated with their ability to abrogate the LTB4-induced chemotactic response. These data suggest that phospholipid methylation and phospholipase activation play an important role in transduction of the LTB4 receptor-ligand interaction in PMN, which results in chemotaxis.

Enhanced phospholipase A2 and C activities of peripheral blood polymorphonuclear leukocytes from patients with rheumatoid arthritis.

Increased concentrations of eicosanoids are found in synovial fluids (SF) from patients with rheumatoid arthritis (RA). SF polymorphonuclear leukocytes (PMN), which derive from peripheral blood, usually account for approximately 90% of the cells in RA SF. Since eicosanoid precursor fatty acids (FA) are liberated by phospholipases from membrane phospholipids, we examined phospholipase A2 and C activities in peripheral blood PMN from patients with RA. Peripheral blood PMN from patients with RA (RA-PMN) exhibit greater phospholipase A2 activities against phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and greater phospholipase C activities against PC, PE, and phosphatidylinositol (PI) than PMN obtained from normal volunteers (N-PMN).

Oculocutaneous albinism, platelet storage pool disease, and progressive lupus nephritis.

Systemic lupus erythematosus (SLE) was seen in a patient with oculocutaneous albinism and platelet storage pool disease (Hermansky Pudlak syndrome). Although the patient had severely compromised platelet function, lupus nephritis developed nonetheless, which progressed to end-stage renal disease. The role of platelets in the pathogenesis of the nephritic process is discussed, and it is concluded that release of platelet granular constituents, particularly serotonin, is not required for the microvascular damage of SLE.

Phospholipase A2-mediated inflammation induces regression of malignant gliomas.

An ideal form of cancer therapy is the harnessing of innate immunity to eradicate spontaneously arising clones of malignant cells. To date, attempts to develop effective immunotherapies have met with limited success. Prostaglandins and leukotrienes, collectively known as eicosanoids, are important mediators of immune and inflammatory responses. Harnessing these compounds could be a method to treat cancers. Eicosanoids are formed after cleavage of fatty acids from phospholipids by phospholipase enzymes. We have previously described, characterized and cloned a naturally occurring mammalian activator of phospholipase A2. Injection of a 24 amino acid peptide from this phospholipase A2 activating protein (PLAP), resulted in induction of an acute inflammatory response, and a concomitant regression of gliomas in rats. Administration of 500 micrograms of this protein resulted in a 50% decrease of the tumor mass within 72 h. Tumor regression coincided with a greater than twenty-fold increase in levels of prostaglandin E2(PGE2) and leukotriene B4(LTB4), and a marked infiltration of natural killer(NK) cells. These data suggest that activation of phospholipase A2 and modulation of the eicosanoid biosynthetic pathway may provide a novel therapeutic strategy for the successful treatment of malignant tumors of the nervous system.

Alteration of uptake and distribution of eicosanoid precursor fatty acids by aspirin.

Aspirin is an important drug in the treatment of numerous disorders, especially rheumatic diseases. Its several mechanisms of action include inhibition of prostaglandin production by acetylation of prostaglandin synthetase. To explore further the modulatory effect of aspirin on eicosanoid production, we examined its effect on uptake and incorporation of fatty acids into phospholipids of human peripheral blood monocytes. Aspirin ingestion by normal volunteers inhibited uptake of arachidonic acid and linoleic acid in monocytes cultured for 3 days. Similar inhibition was observed when cultured normal human peritoneal macrophages were treated with aspirin for 3 days. In contrast, monocytes cultured for 12 days from both normal volunteers who had ingested aspirin and normal cells treated with aspirin in vitro for the first 3 days of a 12-day culture period expressed an increased uptake of both arachidonic and linoleic acids. Similarly, incorporation of fatty acid into phosphatidylcholine was depressed in 3-day cultured cells but was increased in 12-day cultured cells. Thus, aspirin, whether administered in vivo or added in vitro, modulates cellular uptake and incorporation of eicosanoid precursor fatty acids and their insertion into membrane phospholipids in cultured human monocytes and macrophages.

Pleural ascites without abdominal fluid: surgical considerations.

The presence of ascitic fluid in the pleural cavity in the absence of peritoneal fluid is rare. We have recently encountered two patients who presented with red-sided pleural effusions and no abdominal ascites. Both patients had diaphragmatic defects: One was an old traumatic diaphragmatic tear and the other a pinpoint spontaneous perforation. These cases are unique because the diagnosis of total ascitic fluid movement across the diaphragm was made during life, and the condition was surgically corrected. The literature concerning transdiaphragmatic movement of fluid is reviewed, and an operative approach is outlined.

Immunohistochemical study of HER-2/neu, epidermal growth factor receptor, and steroid receptor expression in normal and malignant endometrium.

HER-2/neu oncogene protein, epidermal growth factor receptor, progesterone receptor, and estrogen receptor were examined immunohistochemically in specimens of normal and neoplastic endometrium. Tissues obtained at the time of hysterectomy were snap-frozen at liquid nitrogen temperature and serially sectioned at 4 microns. Normal endometrial epithelial cells stained with anti-epidermal growth factor receptor and anti-HER-2/neu with intensities graded from 0 to 3+. Of the 49 endometrial malignancies studied, seven (14%) contained tissue exhibiting HER-2/neu staining in excess (4+) of any of the normal tissues or the other 42 cancer specimens. Expression of both HER-2/neu and steroid receptors was heterogeneous within these seven tumors. To examine this heterogeneity more closely, sections of these and other tumors were double-stained for HER-2/neu and progesterone receptor. It was found that the cells exhibiting 4+ HER-2/neu staining were progesterone receptor-negative. Conversely, cells that were progesterone receptor-positive within the same specimen exhibited HER-2/neu immunostaining equal to or less than 3+. All specimens containing 4+ HER-2/neu tissue were graded 1 or 2 adenocarcinomas, stage I. Thus, there is an inverse relationship between overexpression of HER-2/neu and progesterone receptor in endometrial cancer. On the other hand, overexpression of HER-2/neu in endometrial cancer does not seem to be related to loss of other differentiated characteristics. The prognostic value of these observations awaits continued study.

Phospholipase A2 activating protein induces tumor regression.

There has been increasing interest in attempts to harness the body's normal inflammatory response mediated through the eicosanoid pathway to treat tumors. Accumulating data indicate that the growth of several different cancers is modulated by a group of pro-inflammatory bioactive lipids, the best known of which are the eicosanoids. Eicosanoid pathway constituents modulate cell function in several important ways, and an agent that activates PLA(2) and up-regulates LTB(4) levels could be expected to be an effective cytotoxic tumor agent, especially if it stimulated NK cells. PLAP is a 28-kDa polypeptide that is a member of the WD-repeat protein, G-protein-transducin superfamily. The pro-inflammatory properties of PLAP have been elucidated using a number of different approaches. PLAP has been found in inflamed tissues and synovial fluid from patients with rheumatoid arthritis. Based on knowledge of PLAP as a pro-inflammatory agent, its capacity to modulate the immune response and the role of the inflammatory and immune responses in immune surveillance, the role of PLAP in cancer therapy was explored. Significant tumor regression was observed 72 hours following a single treatment with PLAP in an animal air pouch model of glioma. PEG-PLAP treatment increased the life expectancy of animals with Lewis lung cancer, and in preliminary studies in MTVL breast tumors in mice, PLAP treatment resulted in a similar increase in life expectancy. These findings suggest that PLAP holds promise as a potential therapy for cancer, and warrants further study.

Prostaglandins and the inflammatory response.

Prostaglandins not only mediate the classic signs of inflammation but also help to regulate the function of cellular constituents in an inflammatory reaction. Clinical and experimental evidence of the role of prostaglandins as mediators is highlighted.

Thrombocytopenia in a prospective, randomized, double-blind trial of bovine and porcine heparin.

A prospective, randomized, double-blind clinical trial of bovine and porcine heparin was conducted. One hundred forty-one patients were randomized, of whom 89 received heparin treatment for six or more days (mean, ten days). Two developed severe thrombocytopenia (platelet count less than 20,000 per microliter); both were randomized to the bovine heparin group, but one inadvertently received two doses of porcine heparin. Laboratory investigation suggested that the thrombocytopenia in these two patients was immunologically mediated, and platelet reactivity to both bovine and porcine heparin was demonstrated. Twenty patients had a decline in platelet count of greater than 50,000 from baseline, although the total count remained above 150,000 per microliter. In seven of these subjects, the platelet count returned to its original level while heparin therapy was continued. Of the 13 patients with a persistent decrease in platelet count, ten had received bovine heparin and their counts decreased by an average of 88,000 per microliter; the reduction in the three porcine-treated patients was 68,000; this difference was not statistically significant. In the remaining patients, the post-treatment platelet counts in both groups were significantly higher than pretreatment values (p less than .005), perhaps indicating a cessation of the platelet consumption that accompanied the original thrombotic event.(ABSTRACT TRUNCATED AT 250 WORDS)

Diagnosis and management of vulvar and vaginal intraepithelial neoplasia.

Vulvar intraepithelial neoplasia and VAIN present unique challenges to the practicing gynecologist. VIN may produce distressing symptoms and undergo malignant conversion. A high index of suspicion and liberal use of biopsy are required to make the diagnosis. The approach to therapy for VIN has been reviewed. Treatment should be tailored to each individual patient and may include a period of expectant observation. Variations and combinations are used whenever necessary to preserve normal function and anatomy. Frequent surveillance is a must because recurrence rates are high, especially with multifocal disease in young women. Although VAIN accounts for less than 0.5% of lower genital tract neoplasia, the frequency of its detection is increasing, especially in younger patients. These lesions are most commonly found in the upper third of the vagina and are often multifocal in nature. The close proximity of the upper vagina to the rectum, bladder, and ureters makes treatment difficult. The occult invasion rate may be as high as 28%, and a wide variety of therapies are available. As is true for VIN, recurrence is not uncommon.

Acute rheumatologic disorders in the elderly.

A variety of rheumatologic disorders affect the elderly. Some of these problems are seen almost exclusively in the elderly, such as temporal arteritis and pseudogout. Because of underlying chronic diseases, these patients are also at increased risk for joint infection and resultant sepsis. Evaluation of synovial fluid from the inflamed joint is important. Light microscopy evaluation with a red polarizing compensator can help diagnose crystal-mediated disease, such as gout or pseudogout. Examination of Gram stains can help diagnose infectious arthritis. Thus, appropriate processing of synovial fluid is mandatory for the diagnosis of many rheumatologic disorders that occur in the elderly. A variety of metabolic disorders are associated with pseudogout and should be searched for on laboratory evaluation. Appropriate laboratory evaluation and follow-up following the acute episode are important in the care of these patients. For example, temporal arteritis with resultant blindness is a feared disorder in the elderly. Transient blindness, headaches, jaw claudication, and an elevated Westergren sedimentation rate suggest this diagnosis. Aches and pain in the neck and shoulder area, especially in the morning, are typical of polymyalgia rheumatica. Polymyalgia rheumatica may also be a symptom of temporal arteritis.

Epigenetic status of argininosuccinate synthetase and argininosuccinate lyase modulates autophagy and cell death in glioblastoma.

Arginine deprivation, either by nutritional starvation or exposure to ADI-PEG20, induces adaptive transcriptional upregulation of ASS1 and ASL in glioblastoma multiforme ex vivo cultures and cell lines. This adaptive transcriptional upregulation is blocked by neoplasia-specific CpG island methylation in either gene, causing arginine auxotrophy and cell death. In cells with methylated ASS1 or ASL CpG islands, ADI-PEG20 initially induces a protective autophagic response, but abrogation of this by chloroquine accelerates and potentiates cytotoxicity. Concomitant methylation in the CpG islands of both ASS1 and ASL, observed in a subset of cases, confers hypersensitivity to ADI-PEG20. Cancer stem cells positive for CD133 and methylation in the ASL CpG island retain sensitivity to ADI-PEG20. Our results show for the first time that epigenetic changes occur in both of the two key genes of arginine biosynthesis in human cancer and confer sensitivity to therapeutic arginine deprivation. We demonstrate that methylation status of the CpG islands, rather than expression levels per se of the genes, predicts sensitivity to arginine deprivation. Our results suggest a novel therapeutic strategy for this invariably fatal central nervous system neoplasm for which we have identified robust biomarkers and which overcomes the limitations to conventional chemotherapy imposed by the blood/brain barrier.

Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis.

Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma.