Ehrlich II –2nd World Conference on Magic Bullets

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Authors’ disclosure statement: The author is a previously employee of Dako Denmark A/S

Prognostic and predictive biomarkers in thyroid diseases: “magic bullets” for a personalized therapeutic strategy TROVATO M Department of Human Pathology, Policlinico Universitario, via Gazzi, 98100, Messina, Italy, [e-mail], University of Messina, Italy Background: Although a malignant conversion from benign thyroid nodules is rare, in worldwide population thyroid cancer incidence has increased. The majority of thyroid cancers show a very good outcome, are well-differentiated originating from the follicular epithelium, and are subdivided into papillary and follicular carcinomas. Undifferentiated carcinomas and medullary thyroid carcinomas have a poor prognosis, arise from C cells and are less common. Prognostic evaluation of thyroid cancer is determined by biomarkers of diseases such as histological type and stage of tumour at diagnosis. However, prediction for the outcome of the cancerous disease is strongly determined by specific characteristics of the patient such as age and response to initial treatment. Therefore, different scoring systems, based on multiple regression analysis of combined predictive factors, have been proposed for stratifying patients with thyroid cancer into low and high risk prognostic groups. Methods: In this issue a timely and detailed review of the most recent advances in prognostic and predictive biomarkers of thyroid cancers is provided, including their clinical impact for a personalized management of cancerous disease. Results: The most used scoring system for predicting survival is the TNM staging system but other scoring systems including AMES, AGES and MACIS, the Ohio State University Scoring System are associated for a correct prognostic evaluation of thyroid cancers. Prediction of thyroid cancer outcome is commonly based on circulating thyrogobulin and calcitonin measurement in complete absence of eutopic thyroid tissue. New possible prognostic biomarkers are levels of PA system components, over-expressions of met and BRAF mutations. Expressions of multidrug resistance 2 represent new predictive biomarkers in medullary thyroid carcinoma. Conclusions: Thyroid cancer management is based on total thyroidectomy, ablative doses of radioiodine and suppressive treatment. The follow-up is based on measuring serum thyroglobulin and imaging with radioiodine scans. New personalized treatments will use genetic biomarkers of thyroid cancer as therapeutic targets.	HIV Protease Inhibitor: An Antifungal Agent? TSANG CSP, HONG I The University of Hong Kong Background: Protease inhibitors were shown to inhibit Candida albicans adherence to epithelial cells but not endothelial cells. Whether protease inhibitors have any effect on C. albicans adherence to acrylic surface and can be used as an antifungal is still unknown. Aims: The current study aimed to investigate whether protease inhibitors attenuate Candida albicans adherence to acrylic surface. The effect of three protease inhibitors, namely Saquinavir, Ritonavir and Indinavir on adherence were compared. Methods: C. albicans suspensions were pre-treated with different concentrations (0.8, 4, 20, 100 and 500 µM) of Saquinavir, Ritonavir or Indinavir for one hour. The yeast cells were then allowed to adhere on acrylic strips treated with human pooled saliva for another hour (Group A). Adherence was determined by calculating the percentage of cell area over the acrylic surface using an image analyser. Another group with C. albicans not pre-treated with protease inhibitors (Group B) and a control group with no protease inhibitors added (Group C) were also included. Results: All three protease inhibitors significantly attenuated adherence of C. albicans to acrylic surface. Group B showed significant reduction in adhesion compared with Group C. 50% reduction in adherence occurred at concentrations of 100 µM, 100 µM and 20 µM, for Saquinavir, Ritonavir and Indinavir, respectively. A dose dependent inhibition of adhesion were observed for all the protease inhibitors in Group A, which was significantly higher in Indinvavir than in Saquinavir and Ritonavir. However, such difference disappeared at concentration of 500 µM. Conclusions: Protease inhibitor had a direct effect on C. albicans pathogenicity; it attenuated C. albicans adherence to acrylic surfaces in a dose related fashion. Moreover, different protease inhibitors exhibited different degrees of inhibition.
Expression of Topoisomerase I and IIα Protein in Primary Colorectal Cancer; is That the Culprit of Recurrent Disease Following 5-Fluorouracil-based Adjuvant Chemotherapy? TSAVARIS N^1,LAZARIS A^2, GOUVERIS P^1, KOSMAS C^3, KAVANTZAS N^2, KOPTERIDES P^1, PAPATHOMAS T^2, ARAPOGIANNIS G^2, ZORZOS H^2, KYRIAKOU V^2, PATSOURIS E^2 ^1Medical Oncology Unit, "Laikon" University General Hospital, Athens, Greece ^2First Department of Pathology, "Laikon" University General Hospital, Athens, Greece ^3"Metaxa" Cancer Hospital, Piraeus Background: Human DNA topoisomerases I and II (topo-I and -II) are essential for vital cellular processes such as DNA replication, transcription, translation, recombination and repair. Following a chain of observations and pilot studies, we present the findings of a pioneer study, in which topo-I and -II expression was correlated with outcome after chemotherapy in primary and relapsed colorectal cancer. Methods: Patients with colorectal cancer that had recurred, following surgery and adjuvant chemotherapy and underwent a second operation were included in the study. All had undergone surgical resection of the primary tumor and received post-operatively 5-FU-based (5FU+Leucovorin, Mayo Clinic regimen) adjuvant chemotherapy. Tumor tissue was collected at the initial operation from the primary tumor and at the time of recurrence (during the second operation following chemotherapy). All tissues samples were analyzed for levels of expression of both topo-I and topo-IIa using standard three-step immunohistochemistry on paraffin sections. Results: Forty patients were included in the study. Levels of expression of topo-I and topo-II were higher in malignant cells from tumor recurrences compared to primary tumors (p=0.0001 for both). There was a statistically significant positive relationship between patients' age and levels of topo-I (p=0.011) and topo-II (p=0.011) expression. Conclusions: The study results underscore the role of topoisomerase expression in colorectal cancer and suggest a potential role in tumor recurrence. This model could be further studied, to include other forms of neoplasia and infection, in an effort to elucidate the development of chemotherapy drug resistance, thus optimizing treatment strategies and improving cancer patient care.	Snake and Snail Neurotoxins – Magic Tools for Targeting Different Subtypes of Nicotinic Acetylcholine Receptors TSETLIN VI, UTKIN YUN, OSIPO VAV, KASHEVEROV IE Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia Background: Neurotoxins from snake venoms distinguish various nicotinic acetylcholine receptors (nAChR): α-bungarotoxin and other long-chain α-neurotoxins potently block muscle-type and neuronal α7 nAChRs, short α-neurotoxins target muscle-type nAChR, -bungarotoxin blocks α nAChR. Labeled α-neurotoxins are used to quantify nAChRs at different pathologies (muscle dystrophies, Alzheimer’s and Parkinson’s diseases, chronic pain). Some α-conotoxins, neurotoxic peptides from Conus snails, distinguish muscle-type nAChR, other block distinct neuronal nAChRs. However, new more potent and selective toxins are in need. Methods: combination of HPLC and mass-spectrometry is used for isolation and structure determination of new proteins from snake venoms. Naturally occurring α-conotoxins and their analogs were obtained by solid-phase peptide synthesis. Analysis of activity of toxins was performed in binding assays with Torpedo nAChR membranes, α7 nAChR cell line and water-soluble acetylcholine-binding proteins (AChBPs), excellent models of the nAChR extracellular ligand-binding domain, and by electrophysiology on distinct nAChRs expressed in Xenipus oocytes Results: New proteins were isolated from the N.kaouthia cobra venom. It was discovered that practically non-toxic weak toxin (WTX) blocks muscle-type and α7 nAChR and potently inhibits cell proliferation indicating its potential anticancer activity. For the first time a disulfide-bound dimeric α-cobratoxin was isolated and shown to interact not only with Torpedo and α7 nAChRs, but also with α3β2 nAChR, the latter activity found only in -bungarotoxin (collaboration with D.Bertrand, Geneva). Synthetic α-conotoxins bound with different affinity to AChBPs. The X-ray structure of α-conotoxin complexes with A.californica AChBP was determined (collaboration with A.Smit and T.Sixma, Amsterdam), unraveling the binding site architecture and providing a basis for design of novel more potent and selective antagonists and agonists of various nAChRs. Conclusions: Thus, the presented results from our and other laboratories show that protein and peptide neurotoxins serve not only as magnifying lenses accurately disclosing the details of the nAChR binding sites, but also open the ways for design of new drugs.
Myostatin Inhibiting Peptide Works as a Magic Bullet to Increase Skeletal Muscle Mass and to Ameliorate Muscle Pathology in Muscular Diseases by Transgenic Expression TSUCHIDA K, NAKATANI M, UEZUMI A Institute for Comprehensive Medical Science, Fujita Health Univ., Toyoake, Aichi, Japan Background: Increase of skeletal muscle mass and prevention of muscle atrophy have tremendous medical needs. Myostatin inhibition is capable of increasing skeletal muscle mass and preventing muscle atrophy. It is a promising therapeutic strategy for muscle wasting diseases such as muscular dystrophy and aging. It is also possible that myostatin inhibition will become a novel doping strategy that is difficult to detect in urine or blood tests. Methods: Myostatin inhibitors include myostatin propeptide, follistatin, and myostatin antibodies. We have developed the myostatin-specific inhibitors derived from follistatin by domain deletion and shuffling. Transgenic mice expressing the myostatin inhibitor, called FSI-I, under the control of a skeletal muscle-specific promoter showed increased skeletal muscle mass and strength (Faseb J. 2008). We crossed FS I-I Tg with mdx mice, a model for Duchenne muscular dystrophy. Microarray analysis was performed. Results: The skeletal muscles in the mdx/FS I-I mice showed enlargement and reduced cell infiltration. The muscle strength was recovered in the mdx/FS I-I mice. Our data indicate that myostatin inhibition by FS I-I has a therapeutic potential for muscular dystrophy. Microarray analysis showed the remarkable changes of expression of enzymes for fatty acid metabolism such as acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase (CPT-1) in skeletal muscle in FS I-I Tg. Expression of molecular markers for mitochondria such as cytochrome C and uncoupling proteins increased. Conclusions: 1) Myostatin inhibitors derived from follistatin are possible candidates of “Magic Bullets” that will increase skeletal muscle mass and may change the nature of our body composition and quality of life. 2) Microarray analysis suggests the changes of expression of metabolic enzymes. 3) Mitochondoria numbers in muscles and adipose tissues increase by myostatin inhibition by follistatin-derived molecules	Revolutionary impacts of caffeine-potentiated chemotherapy on osteosarcoma treatment TSUCHIYA H^1, NISHIDA H^1,2, KAWANO M^1, TOMITAK^1 ^1 Kanazawa Univ., Kanazawa, Japan; ^2Fujii Hosp., Kanazawa,Japan Background: We developed innovative caffeine-potentiated chemotherapy for musculoskeletal sarcomas and good results were obtained for limited number of patients with various carcinomas. Caffeine can enhance cytocidal effects of anticancer agents in terms of its DNA repair inhibiting effect. Caffeine never allows the cell cycle delay which is mandatory for tumor cells to repair damaged DNA, leading to tumor cell death eventually. We report on a long-term result of nonrandomized prospective study about caffeine-potentiated chemotherapy and function-preserving limb saving surgery for osteosarcoma. Methods: Fifty-one patients with non-metastatic osteosarcoma were treated with neoadjuvant chemotherapy consisting of cisplatin, adriamycin, and caffeine. Intraarterial chemotherapy was performed 3 to 5 times preoperatively and then intravenous chemotherapy 6 times postoperatively. Surgical tumor margin was reduced for chemo-responders. Results: Total tumor necrosis was histologically demonstrated in 41 patients (80%), more than 90% tumor necrosis in 6, and no response in 4. Overall effectiveness rate was 92%. Overall 5-year survival rate was 93% and event-free survival 81% with a mean follow-up of 105 months. Lung metastases newly developed in 9 patients. Seven of those have still no evidence of disease after metastasectomy and chemotherapy. Important anatomical structures such as epiphysis, muscle, tendon, ligament, tendon, and neurovascular bundles were mostly preserved in 47 chemo-responders to improve limb function. Namely, minimizing tumor resection margin was feasible under the umbrella of caffeine-potentiated chemotherapy. Most of patients with excellent or good limb function have retuned to normal athletic activities. Conclusions: Introduction of caffeine to osteosarcoma chemotherapy led to revolutionary advancement of survival and surgical treatment in patients with osteosarcoma. Randomized trials of caffeine-potentiated chemotherapy will provide much more clear evidence in musculoskeletal sarcomas and other cancers.
Severing the Gordian Knot: Are Glycolipids the solution for Effective Vaccines against Malaria and HIV? LI X*, LIANG PH, WU D, IMAMURA M, FUJIO M, WONG CH, TSUJI M* *HIV and Malaria Vaccine program, Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY, USA: #The Scripps Research Institute, La Jolla, CA, USA: +The Genome Research Center, Academia Sinica, Taipei, Taiwan. The eradication of global pathogens responsible for endemic and pandemic diseases hinges upon the development of effective vaccines. This is certainly the case for HIV and malaria. However, our inability to elicit “strong and long-lasting” protective T cell responses, particularly CD8+ T cell responses, has been a major obstacle to successful vaccine development. Accordingly, adjuvant technologies will likely be critical not only to overcome pre-existing immunity to viral vaccine vectors but also to further enhance vaccine immunogenicity. In our previous studies, a glycolipid, called alpha-galactosylceramide (alpha-GalCer), which binds to CD1d molecules and stimulates natural killer T (NKT) cells, was found to display an adjuvant activity and enhance protective CD8+ T cell responses elicited by HIV and malaria vaccines. In search of a more potent adjuvant, we have evaluated a compound library of approximately 100 new glycolipids and have identified several alpha-GalCer analogues that act as powerful NKT cell ligands. The evaluation process includes the testing of ability for glycolipids to stimulate human invariant NKT cells in vitro, as well as their ability to stimulate homologous dendritic cells (DCs). The selected compounds were then inoculated into mice in order to determine their ability to stimulate invariant NKT cells and DCs in vivo. By a series of assays, a few compounds were selected based on their potent biological activity. Finally, we have successfully identified a lead compound that displays a superb adjuvant effect against HIV and malaria vaccine platforms in a mouse model. Since CD1d molecules and NKT cells are very much conserved between mice and humans, we anticipate that the lead compound that we identified will improve the efficacy of various vaccines in humans in the near future.	Protective effects of minocycline on methamphetamine-induced dopaminergic neuronal damage: a positron emission tomography (PET) study with conscious monkeys TSUKADA H, NISHIYAMA S, FUKUMOTO D, KAKIUCHI T PET Center, Central Research Laboratory, Hamamatsu Photonics, Hamamatsu, JAPAN Background: Previous PET studies of methamphetamine (METH) abusers suggest that psychotic symptoms may be attributable to the reduced dopamine (DA) transporters (DAT) in the human brain. To search the effective treatments, the neuroprotective effects of minocycline, a second-generation tetracycline, on METH-induced DA neuronal damages in the striatum (Str) were evaluated with animal PET with conscious monkeys. Aims: 1) To develop monkey model of impaired Str DA functions. 2) To assess the METH-induced damages of Str DA system with PET. 3) To evaluate the neuroprotective effects of minocycline on damaged Str DA system by METH. Methods: This study included 10 male adult rhesus monkeys (Macaca mulatta,weight: 4.5 – 6.4 kg,). To induce dysfunction of Str DA system, METH (2 mg/kg, i.m.) was injected three times with 3-hours intervals, and minocycline (200 mg/kg, s.c.) was administered 0.5 hr before 1st METH injection or 0.5 hr post 3rd METH injection in 1st day, followed by minocycline administration 2 times a day from 2nd day to 7th day. PET scans with ligands of [^11C]2- carbomethoxy-3-(4-fluorophenyl)tropane (-CFT) for presynaptic DAT and [^11C]SCH 23390 for postsynaptic D1 receptors (D1R) were performed on pre-treatment, 2nd and 6th days of chronic administration of minocycline (200 mg/kg) under conscious condition. Results: Repeated METH injection drastically reduced [^11C]-CFT availability for DAT in Str, while while no significant changes in [^11C]SCH23390 binding to D1R. Pretreatment and subsequent administration of minocycline significantly attenuated the reduction of DAT in Str of monkeys treated with METH. Furthermore, posttreatment and subsequent administration of minocycline also significantly protected the reduction of DAT. In contrast, repeated administration of minocycline alone did not alter the density of DAT in Str. These results demonstrated that minocycline protects against METH-induced neurotoxicity in the monkey brain. Conclusions: 1) Drug abuse model of monkey was developed by repeated METH injection. 2) METH injection reduced DAT in Str. 3) Subsequent minocycline administration significantly protected the reduction of DAT.
Phase I Study of S-1 Combined with Irinotecan (CPT-11) in Patients with Advanced Colorectal Cancer TSUNODA A, YASUDA N, NAKAO K, NARITA K, YAMAZAKI K,WATANABE M, SUZUKI N, KUSANO M Department of General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo , Japan Backgrounds: S-1 is a rationally developed combination of tegafur, a prodrug of 5-FU, 5-chloro-2,4-dihydroxypyrimidine, an inhibitor of 5-FU catabolism, and potassium oxonate, an inhibitor of 5-FU-induced diarrhea. S-1 is one of the most active single agents for outpatient treatment of patients with advanced colorectal cancer. A dose-escalation study of irinotecan (CPT-11) combined with S-1 was performed to determine the maximum-tolerated dose (MTD), recommended dose (RD) and dose-limiting toxicities (DLTs) in advanced colorectal cancer. Methods: S-1 was administered orally at 80 mg/m^2/day for 21 consecutive days followed by a 2 week rest. CPT-11 was given intravenously on days 1 and 15 of each course, at an initial dose of 60 mg/m^2/day, stepping up to 80, 100 or 120 mg/m^2/day, depending on the DLT. Courses were repeated every 5 weeks, unless disease progression or severe toxicities was observed. The European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 was administered at baseline and 1 week after each course to evaluate quality of life (QOL). Results: A total of 20 patients were entered in this study. The median number of courses delivered at the RD was 6, and the mean relative dose intensity of S-1 and CPT-11 was 0.97 and 0.96, respectively. Only 1 of 14 patients at the RD needed to reduce the dose of CPT-11. Eighty-five percent of the treatment courses with the RD were delivered in the outpatient clinic. The MTD of CPT-11 was considered to be 100 mg/m^2, because 2 of 3 patients developed DLTs, anorexia and diarrhea. Therefore, the RD of CPT-11 was set at the dose of 80 mg/m^2. The overall response rate (RR) was 50% (7/14) and the RR at the RD was 55% (6/11), suggesting a promising degree of clinical efficacy. There was no significant difference in the scores of most of QOL dimensions after treatment. In general, the scores of QOL dimensions at 1 to 3 time point were not worse, and those at 4 to 6 time point were not better than those at baseline, respectively. Conclusions: A combination of S-1 with CPT-11 can be recommended for further phase II studies in patients with advanced colorectal cancer.	Mechanism of Resistance in Non Hodgkin’s Lymphoma- Expression of Fas Ligand on Endothelial Cells Lining Blood Vessels TUCKER CA^1,2, HOFFMAN BG^1, KYLE AH^1,2, WILSON IM^1,2, WALKER DC^1,3, BALLY MB^1,2 ^1British Columbia Cancer Agency, Vancouver, BC, Canada; ^2University of British Columbia, Vancouver, BC, Canada; ^3St-Paul’s Hospital, Vancouver, BC, Canada Background: n this study we investigated how the abnormal expression of Fas/FasL influences rituximab-mediated cell death, to establish modes of resistance in Mantle Cell Lymphoma (MCL) cells to current treatment regimens.