IM Treatment Research Articles

The Level of Peroxiredoxin and Catalase On Single Cell Level Is Correlated with the Positivity of Ph Chromosome in the Bone Marrow of CML Patients During Imatinib Therapy.

Abstract 1114Poster Board I-136 Background: It has been proposed that treating cancer cells with vectors that cause the over expression of MnSOD and catalase could inhibit tumor cell growth by disrupting the redox signaling involved with cell division as well as, therapeutically, by providing protection for normal cells. These results support the hypotheses that increasing the dismutation of mitochondrial superoxide alters redox signaling in cancer cells that can result in growth suppression. Peroxiredoxins (Prxs) are a ubiquitous family of multifunctional antioxidant thioredoxin-dependent peroxidases that have roles in the reversible inactivation of PTPs and PTEN in cells stimulated by growth factors and protect cells against oxidative stress and modulate intracellular signaling cascades (Rhee et al, Cell Biology 17:183-189, 2005). In our study, we investigated the changes of the levels of H2O2-removing enzymes (ie Prxs, glutathione peroxidase 1 (Gpx1), and catalase) on a single cell during IM therapy in newly diagnosed CML. Patients and MethodsSix paired CML samples (two blast crisis, one accelerated phase, three chronic phases) were analyzed in a study was approved by the institutional review board. Patients gave written informed consent according to institutional guidelines. 400 mg or 600 mg of imatinib was given to the chronic phase and accelerated/blast crisis samples respectively. Following encircling cells from marrow BM aspiration from patients or normal individuals with the Dako Pen (Dako, Denmark) on a dry slides, cells were incubated with blocking solution (10% FBS in PBS) for 30minutes to block non-specific staining. Then, cells were incubated with specific primary antibodies to antioxidant enzymes in 0.2% TritonX-100/10% FBS/PBS solution overnight (1:100). After washing with PBS, cells were incubated with Alexa-488 or 594-conjugated goat antibodies to mouse or rabbit IgG, respectively for an hour. Confocal fluorescence images of cells were obtained with an LSM510 microscope (Carl Zeiss, Tornwood, NJ). ResultsSamples of newly diagnosed CML patients showed significantly decreased levels of Prx 2, but revealed increased level of catalase on single cell level. The immunohistochemical expression was well correlated with western blot results. As the level of Philadelphia chromosomes decreased with IM treatment, the expression levels of Prx II and catalase were restored to the levels of normal individuals. Using our technique, we are able to quantify the changes of Prx and catalase on each myeloid, erythroid and megakaryocytic lineages at the single cell level. ConclusionsDecreased Prx 2 and elevated catalase levels at the time of diagnosis are closely correlated with the elevated bcr/abl kinase level in CML at the each single cell level. The aberrant expression of those antioxidant enzymes were back to the level of normal individuals after IM treatment. This method might be accurate and convenient to assess the changes of the level of Prx and Catalase especially in the situation of cytopenia with IM treatment. Understanding the molecular mechanisms of changes on antioxidant might be potent tools to develop more effective new drugs in CML patients especially for Imatinib resistant patients. DisclosuresNo relevant conflicts of interest to declare.

Discontinuation of Imatinib Therapy After Achieving a Molecular Response in Chronic Myeloid Leukemia Patients.

Abstract 859▪▪This icon denotes an abstract that is clinically relevant. BackgroundImatinib (IM) has greatly improved survival rates in chronic myeloid leukemia* (*CML). However, all patients (pts) must continue treatment for an unknown period of time.A pilot study of the first pts who discontinued IM therapy was previously reported (Rousselot et al. Blood 2007;109:58–60). The multicentre study Stop Imatinib (STIM) was initiated in July 2007 in order to evaluate the persistence of complete molecular remission (CMR) after stopping IM, and to determine the factors that could be associated with CMR persistence. MethodsInclusion criteria were IM treatment duration of at least 3 years and sustained CMR. Sustained CMR was defined as BCR-ABL/ABL levels below a detection threshold corresponding to a 5-log reduction (undetectable signal using RQ-PCR) for at least 2 years. Molecular relapse, defined as RQ-PCR positivity, was taken into account if confirmed in two successive assessments. In cases of molecular relapse, pts were re-challenged with IM at 400 mg daily. ResultsFrom the pilot study, 8 among 15 patients are still in CMR with a median follow up of 42 months (range 37-49). The number of patients enrolled in the STIM study was 69. 34 patients had received interferon alpha (IFNa) prior to IM and 35 pts were de novo. Median follow-up (range) was 17 months (6-24). 37 pts relapsed (loss of CMR) within the first 6 months and two patient relapsed after more than 6 months (M7 ,M18). At M12, the probability of remaining in CMR was 45% (95% CI: 33-56%). For previously treated with IFN (n=34) this probability was 44% (95% CI: 27-59%) versus 46% (95% CI: 29-61%) for de novo pts (p=0.93, overall). All patients in molecular relapse were sensitive again after imatinib re-challenge (decreasing BCR-ABL level, achievement CMR again). Male pts had a better probability of survival without molecular relapse (p=0.02) and a trend was observed for the low Sokal risk group (p = 0.06). Peripheral NK cells counts prior to IM discontinuation were significantly lower in relapse pts (mainly cytotoxic cells CD56dim) as compared to the non relapse pts(p=0.005). ConclusionsWe have confirmed that CMR can be sustained after discontinuation of imatinib with a long follow-up, particularly in male patients and in pts with cytotoxic NK cells in their peripheral blood. Using stringent criteria, it is possible to stop treatment in patients with sustained CMR, even in those treated with IM as a single agent.(ClinicalTrials.gov number, NCT00478985 [ClinicalTrials.gov]) Disclosures:Mahon:Amgen: Honoraria; Novartis Pharma: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria. Guilhot:Novartis Pharma: Consultancy, Honoraria; BMS: Consultancy, Honoraria.

Time to secondary resistance (TSR) after interruption of imatinib: Updated results of the prospective French Sarcoma Group randomized phase III trial on long-term survival

10508 Background: We previously demonstrated that imatinib mesylate (IM, Gleevec/Glivec; Novartis Pharma) must not be interrupted after 1 and 3 years (yr) in responding patients (pts) and has to be given continuously until disease progression (PD) or intolerance. The impact of IM re-introduction at progression remains unknown regarding the impact of the interruption on the TSR. Methods: This prospective national multicenter BFR14 study was initiated in June 2002. After 1, 3, and 5 yrs of IM 400mg/day, pts free from progression were randomly offered to continue (C arm) or interrupt (I arm) IM. Pts allocated to the I arm could restart IM (same dose) in case of PD. Primary endpoint was PFS. Pts declining randomization proceed with IM. Results: As of December 2008, 415 pts were included in this trial. Fifty-eight, 50 and 12 (ongoing) non progressive pts at 1, 3 and 5 yrs respectively were randomized in the I and C arm. Pt characteristics were well balanced between the two arms. The median time to progression (TTP) were 7.3 months (m) (rate of relapse: 91% of pts) and 9.4 m (rate of relapse: 84%) in the I arms after 1 and 3 years of treatment. In contrast the median TTP were 31.4 m and not reached in the C arms after 1 and 3 yrs of IM treatment respectively. IM reintroduction in the I arm after a re-progression allowed again a tumor control in 93% (43/46) of pts. The median follow-up from randomization is 56 m and 25 m at 1 and 3 yrs respectively. TSR after randomization to IM (first progression in the C arm, 2nd progression in the I arm) was not significantly different between the two arms (the 2-yrs TSR is similar in both arms 63% and 62% in the I and C arm respectively for the 1-yr randomization, 83.5% and 84.3% for the 3-yr randomization) but the rate of secondary resistance decrease over time in both arms: 40% or relapse in the 2 yrs following the 1 yr randomization vs less than 20% or relapse in the 2 yrs following the 3-yrs randomization. Conclusions: The majority of responding pts relapsed when IM was stopped after 1 and 3 yrs of treatment but response is reinduced in 93% of patients after IM reintroduction. TSR was not significantly affected by treatment interruption in this series of pts. [Table: see text]

A Distinctive Pattern of Different Gene Expression in Chronic Myelogenous Leukemia Patients

Introduction: Molecular chaperones have many functions, such as protecting other proteins against aggregation, assisting in folding of nascent proteins/refolding of damaged proteins and targeting severely damaged proteins to degradation. As one of the molecular chaperones, Hsp90 functions to facilitate the folding of newly synthesized and denatured client proteins, including mutated p53, Bcr-Abl, p185ErbB2 and Raf-1. The Bcr-Abl fusion gene encodes for the p210Bcr-Abl tyrosine kinase (TK) implicated in the pathogenesis of chronic myelogenous leukemia (CML). Studies in cultured cells have identified many signal transduction pathways activated by Bcr-Abl, including activation of the Ras, MAPK, JNK/SAPK, phosphatidylinositol-3 kinase, nuclear factor-B and STAT pathways. Imatinib mesylate (imatinib IM) is a tyrosine kinase inhibitor that competitively inhibits ATP binding in the kinase domains of both the Bcr-Abl and c-Abl kinases. It has been suggested that resistance to imatinib stems from Bcr-Abl gene amplification, leading to overexpression of Bcr-Abl protein or point mutations in the Bcr-Abl gene However, several groups suggested that there might be other forms of Bcr-Abl-independent imatinib resistance Recently, it has been reported that changes in histone deacetylase (HDAC) expression in leukemic cells could be involved in mechanisms for abnormal cellular proliferation that operate through chromatin-independent pathways and thereby could lead to acquired drug resistance of the cellsIn the present study, we evaluated in primary leukemic blasts, obtained from chronic myelogenous leukemia patients at onset, patients in blast crisis and patients which were imatinib-resistant The espression the sirtuin members family and HSP70, HSP90 i-NOS and bcl-2 was evaluated by Nortern blot and Western blot analysis.Material and Methods: Primary leukaemia blastsWe harvested primary blast rich mononuclear cells were obtained by gradient centrifugation on ficoll-hypaque of bone marrow and peripheral blood cells after obtaining appropriate informed consent.Northern blotTotal RNAs from control or treated cells were isolated using Tri Reagent Aliquots of RNA were electrophoresed and blotted onto nylon membranes, that hybridized to 32P-labelled probe.Western BlotCells were lysed and. then were centrifugated. Protein concentration was determined by the Bradford assay.. Equivalent amounts of protein loaded and electrophoresed and were transferred to nitrocellulose membranes, that were incubated with the different primary antibodies:,Result and Discussion:. In the present study, we evaluated a pattern of different gene expression by Northern Blot and Western Blot analysis in bone marrow and peripheral blood cells from 16 CML patients at onset, from 2 patients in blast crisis evolved under IM treatment, and 14 imatinib-resistant patients. Some RNAs were underexpressed in most or all samples tested and never overexpressed (eg SIRT2, SIRT3, SIRT4 and SIRT5), while others were overexpressed in the great majority of samples and rarely, if ever, underexpressed (eg SIRT1, SIRT7, HSP90, iNOS)Furthermore, we examined the level of heat-shock related proteins HSP90 and bcl-2 in 2 patients during treatment with IM. and one patient IM-resistant by western Blot analysis: HSP90 and BCl-2 increased one patient during treatment with IM, while both protein levels was very high in one one patient IM-resistant These results suggest that the difference of genes expression might contribute to patterns of clinical response.

Early onset of antipsychotic response in the treatment of acutely agitated patients with psychotic disorders

Objective While the efficacy of antipsychotic medications for the treatment of psychosis is generally established, the speed of onset of antipsychotic action remains controversial. The objective of this study was to evaluate the early response (within the first 24 h) in psychosis with ziprasidone IM treatment, and to determine whether this early effect is distinct from a reduction in agitation symptoms. Methods In a 24-h, double-blind study, hospitalized patients with psychotic disorder and acute agitation were randomized to treatment with fixed doses of IM ziprasidone: 2 mg ( N = 38) or 20 mg ( N = 41). Efficacy assessments were based on the PANSS positive subscale and PANSS early psychosis factor score (conceptual disorganization, hallucinatory behavior, and unusual thought content) at 4 and 24 h. Results Ziprasidone IM demonstrated a significant dose-related effect (20 mg vs. 2 mg) on both the PANSS early psychosis factor score and the PANSS positive subscale at the first post-baseline time point (4 h), and at 24 h (all p < 0.05). There was a significant direct effect of ziprasidone IM on early psychosis at 24 h ( p < 0.05), distinct from improvement in agitation symptoms. Conclusions These findings suggest, in addition to an early reduction in acute agitation, ziprasidone IM may be associated with a more rapid improvement in psychotic symptoms than previously reported, thus lending further support to the emergent hypothesis of early psychosis improvement in antipsychotic treatment.

Effect of Erythromycin and Gentamicin on Abomasal Emptying Rate in Suckling Calves

Commonly used dosage protocols for antimicrobial agents may alter the rate of gastric emptying. Parenteral administration of erythromycin increases and gentamicin decreases the rate of abomasal emptying. Five male Holstein-Friesian calves (8-15 days of age). Calves received each of the following 4 IM treatments in random order: control, 2 mL of 0.9% NaCl; erythromycin, 8.8 mg/kg; low-dose gentamicin, 4.4 mg/kg; high-dose gentamicin, 6.6 mg/kg. Abomasal emptying rate was assessed by acetaminophen and glucose absorption. Calves were fed 2 L of cow's milk containing acetaminophen (50 mg/kg body weight) 30 minutes after each treatment was administered, and jugular venous blood samples were obtained periodically after suckling. The maximum observed plasma acetaminophen concentration (actual C(max)) and time of actual C(max) (actual T(max)) were determined, and pharmacokinetic modeling was used to calculate model C(max) and model T(max). Erythromycin increased abomasal emptying rate, as indicated by a shorter time to actual T(max) and model T(max) (P < .05). Abomasal emptying rate after injection of low-dose gentamicin was similar to that of control. Administration of high-dose gentamicin resulted in a longer time to actual T(max) (P= .021) but did not change model T(max) (P= .62). IM injection of erythromycin increased abomasal emptying rate in dairy calves, whereas low-dose and high-dose gentamicin did not alter the rate of abomasal emptying as measured by acetaminophen kinetics and glucose absorption. The clinical relevance of these findings remains to be determined.

6011 POSTER Karyotypic evolution during long-term IM treatment of CML patients

6011 POSTER Karyotypic evolution during long-term IM treatment of CML patients

Program for the control of subclinical mastitis in dairy Chios breed ewes during lactation

The present study was carried out to test the effectiveness of a standard treatment for the control of subclinical mastitis during lactating period in a modern, commercial dairy sheep farm. For this purpose, 140 Chios ewes were randomly selected and divided into two groups (control n = 70, treated n = 70) throughout their first lactating period. Milk yields were individually recorded and representative milk samples were collected from each mammary half, once monthly. Ewes giving apparently normal milk, but with Somatic Cell Counts (SCC) ≥ 500 × 10 3 cells/ml and bacteriologically positive were considered to have subclinical mastitis. Animals from the treated group suffering subclinical mastitis were subjected to a 3-day im treatment with Penethamate hydroiodide, while those of control group received no treatment. The percentage of animals from the treated group with SCC ≥ 500 × 10 3 cells/ml was lower compared to that of the control group throughout lactation, except from the last month when it was similar. Nevertheless, this difference was not significant ( P > 0.05). Prevalence of subclinical mastitis was unaffected, although treated animals showed lower rates throughout lactation ( P > 0.05). At the end of lactation 5.3% of the animals from the treated group and 15.6% from the control group had subclinical mastitis. Coagulase negative staphylococci were the only organisms isolated and they were generally sensitive to Penethamate hydroiodide. The predominant species identified was Staphylococcus epidermidis (60%). Ewes from the treated group produced more milk compared to those from the control group ( P < 0.05).

Imatinib Mesylate Inhibits Antigen-Specific Memory CD8 T Cell Responses In Vivo

Imatinib mesylate (IM) is effective at inducing complete cytogenetic remission in patients with chronic myelogenous leukemia. Because its influence on CD8 T cell responsiveness in vivo is unknown, we investigated the effects of IM by analyzing the response of OT-1 CD8 T cells to Listeria monocytogenes (LM) that express the cognate epitope OVA(257-264) (LM-OVA). In vitro, IM had no effect on Ag-specific expansion, cell division, cell cycle progression, or IFN-gamma expression in naive or memory OT-1 T cells. However, IM induced apoptosis of naive and memory OT-1 T cells at doses of >5 microM. At 15 microM IM, OT-1 T cells did not survive in in vitro cultures. The primary response of OT-1 T cells in vivo to LM-OVA infection was unaltered. In contrast, continuous IM treatment resulted in a diminished memory OT-1 response. The expression of IL-7Ralpha, a receptor required for memory cell survival, was lower (on OT-1 cells) in animals receiving IM. These results indicate that IM treatment affects the ability of the CD8 memory pool to respond to Ag and has the potential to increase susceptibility to infection.

Clonal evolution in a patient with CML detected by FISH precedes imatinib treatment failure

Case report A 61 year-old male was diagnosed on Sep/98 with CML chronic phase, he was treated with hydroxiurea, interferon and Ara-C. On July/01 the patient was on complete hematological response (CHR), but Ph positive in karyotyping (Ky) and fluorescence in-situ hybridization (FISH), there were not additional abnormalities. Patient began IM treatment, 400 mg/day. After 4 months of treatment, +der(22) was detected, but the patient was still on CHR. On subsequent visits, the patient had an increased frequency of chromosomal abnormalities, and after 2 years he lost CHR, regardless of the increase IM dose and the concomitant use of Ara-C. The patient was included on a dasatinib phase II protocol. 2 weeks after he had a profound cytopenia and 2 months after had CHR. Table 1 shows clinical and hematological and Ky and FISH follow-up.

Cancer et sclérose en plaques. Impact des traitements de fonds et présentation du projet CARIMS (Cancer Risk In Multiple Sclerosis)

Controversial results have been published on potential link between cancer and multiple sclerosis. Multiple sclerosis has been linked to reduced rates of cancer prior to the era of immunomodulating or immunosuppressive treatments and until today, only 9 studies can be found in the literature. New strategies and early use of IM or IS drugs in MS justify to study and follow patients to detect a potential increase of cancer's incidence in treated patients. It is important to follow and collect prospectively in MS centers, patients with history of cancer, to document histologies, and potential relations with repeated IM or IS treatments. A prospective study is in progress in French MS centers on behalf the Club Francophone de la SEP (CARIMS Project).

Dasatinib (SPRYCEL®) in Patients (pts) with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia Who Are Imatinib-Resistant (im-r) or -Intolerant (im-i): Updated Results from the CA180-015 ‘START-L' Study.

Dasatinib (SPRYCEL®, formerly BMS-354825) is a novel, oral, multi-targeted kinase inhibitor of BCR-ABL and SRC. Relapsing pts with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) who have been previously treated with chemotherapy including im have a very poor prognosis. START-L is an open label, multi-center, global phase-II study, which treated 46 im-r or im-i pts with Ph(+) ALL from January through June 2005. Dasatinib was given orally, 70 mg twice daily (BID) with escalation to 100 mg BID for poor response or reductions to 50 mg and 40 mg BID for toxicity. Response was assessed on weekly blood counts and monthly bone marrow exams, including cytogenetics. Mutation analysis was conducted at baseline and at end of study. Quantitative PCR was carried out pretreatment and at the time of complete cytogenetic response (CCyR). At the time of this update, all 46 pts had a minimum of 9 months (mo) of follow-up. Of these, 44 (96%) were im-r; due to the small number of im-i pts, data for all pts are presented. 59% of patients were male and median age was 48 years. The median time from initial Ph(+) ALL diagnosis to first dose was 18 mo. All patients had been pretreated with im including 46% with 600 mg per day or more, and 52% whose duration of im treatment was more than 12 mo. 37% of the pts had received a stem cell transplant. At baseline, 67% of pts had WBC <2000/mm3, 74% had platelets <100 × 103/mm3, and 24% had extramedullary disease outside of the spleen. In the 40 pts with baseline mutation data, im-resistant BCR-ABL mutations were observed in 78%, one with T315I. The median duration of therapy was 3.0 mo (range 0.03–16.5) for all pts and was 13.0 mo (9.2–16.5) for pts still on study. The overall complete hematologic response rate was 35%. The major cytogenetic response was 57%, including 54% CCyR. The major hematologic response (MHR) in the 31 pts with baseline mutations was 45%. The median duration of MHR was 11 mo and the median progression-free survival was 3.7 mo (95% CI 1.6–6.1). Grades 3 and 4 thrombocytopenia occurred in 13% and 67%, respectively and grades 3 and 4 neutropenia occurred in 27% and 52% of pts, respectively. Most frequent non-hematologic toxicities included diarrhea in 30% (grade 3–4, 7%), nausea in 22% (no grade 3–4), pyrexia in 22% (grade 3–4, 2%), and pleural effusion in 22% (grade 3–4, 7%) of pts. Dose was reduced in 30% and interrupted in 43% of pts, primarily due to non-hematologic toxicities. Dasatinib has important efficacy in this Ph(+) ALL pt population. Updated efficacy (including molecular response), safety, and mutational analysis data will be presented at the meeting.

Heterogeneous Prognostic Impact of 9q+ Deletions in Patients with Chronic Myelogenous Leukemia.

Deletions at the ABL-BCR reciprocal breakpoint on the derivative chromosome 9 are seen in 10–15% of patients with CML and have been associated with a poor prognosis, at least for cases treated with hydroxyurea or interferon alpha (IFN). Studies to date have used different fluorescent-in-situ-hybridization (FISH) probe sets to determine deletion status and thus the results are not always directly comparable. Furthermore, information concerning the extent of deletions is limited and no studies have been performed in the context of a prospective randomised controlled clinical trial. To provide more accurate information about deletion status, we developed a rapid DNA-based screen based on multiplex ligation-dependent probe amplification (MLPA). Der(9) deletions were detected at dilutions of 60% or more MC3 cell DNA in non-deleted DNA, indicating that the method was suitable for analysis of samples with up to 40% normal cells. We then investigated 348 chronic phase (CP) patients that were treated with IFN based therapies in three consecutive prospective controlled German studies. The median duration of IFN-based treatment was 17 months (range, 1–115) with a median observation time of seven years (range, 0.3–16); 160 patients (46%) were alive at the date of analysis. Therapy subsequent to IFN included allogeneic stem cell transplantation (SCT; n=130) or imatinib mesylate (IM; n=62). DNA extracted at diagnosis was tested by MLPA and 9q+ deletions, defined as the loss of at least two consecutive markers, were found in 59/348 (17%) patients. Of these 59 deletions, 21 spanned the ABL-BCR breakpoint and 38 were either upstream only (n=20) or downstream only (n=18) of the breakpoint. Analysis of the baseline parameters age, spleen size, leukocyte count, % eosinophils, basophils and blasts in peripheral blood, platelets, hemoglobin, and prognostic scores according to Hasford and Sokal did not reveal any significant difference between patients with and without deletions. There was no significant difference in overall survival between patients with or without deletions and also no difference in survival when patients were censored at SCT in CP or IM treatment. Patients with large ABL-BCR deletions had a poorer survival compared to all other cases (5.7 vs 8.8 years; p=0.0025) but this fell below the level of significance using censored survival (p=0.08). Patients with low risk Sokal or Hasford scores with breakpoint spanning deletions had a significantly worse prognosis than other cases (p=0.01 and p=0.04, respectively). Patients with ABL-BCR deletions also showed a trend towards a less favourable survival after allogeneic SCT in CP-CML (p=0.06). Surprisingly, patients with deletions that did not span the breakpoint showed a superior outcome (p=0.02). Univariate analysis of baseline data revealed that patients with either ABL or BCR deletions only were younger, presented with more circulating blasts and a larger spleen at diagnosis. Patients with large deletions spanning the breakpoint had fewer additional chromosomal abnormalities. In conclusion, MLPA is a reliable technique for detection of der(9) deletions in CML. Our analysis indicates that only those deletions that span the reciprocal ABL-BCR breakpoint are associated with adverse prognosis but the effect is relatively weak.

Pathologic, radiologic and PET scan response of gastrointestinal stromal tumors after neoadjuvant treatment with imatinib mesylate

Aim The aim of this study is to review the radiologic, PET scan and pathologic response and the outcome of patients with advanced GIST treated with neoadjuvant IM followed by surgical resection. Materials and methods We report a case and review 36 patients reported in MEDLINE with advanced GIST treated with neoadjuvant IM followed by surgical resection. Results Thirty-seven patients with a median age of 56 years (range, 32–76 years) at presentation were treated with neoadjuvant IM. Radiologic response accurately predicted pathological response in 31/36 patients, whereas PET scan was accurate in predicting treatment response in only 6/23 patients. Conclusion This study demonstrates that the pathologic response of GIST to IM is usually incomplete and does not correlate with the complete response seen on PET scan. This finding suggests that surgical resection will continue to play a vital role in the treatment of patients with advanced disease responding to IM treatment.

Sunitinib (SU) response in imatinib-resistant (IM-R) GIST correlates with KIT and PDGFRA mutation status

9502 Background: IM resistance in advanced GIST represents a major clinical problem. SU (SU11248) is an oral multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activities related to KIT, PDGFRs, VEGFRs, RET and FLT3 inhibition. SU has demonstrated efficacy in clinical trials of pts with IM-R GIST. Methods: This study examined the relationship between tumor kinase genotypes and SU clinical activity in 97 pts with metastatic IM-R GIST treated as part of a phase I/II trial. Tumors were imaged by CT or MRI for RECIST-defined response assessment. Tumor specimens were obtained prior to (n=76) and following (n=64) IM therapy and analyzed for primary or secondary KIT and PDGFRA mutations, respectively. Results: Clinical benefit (CB; defined as PR or SD &gt;6 months) with SU was observed for all major molecular GIST subtypes: KIT exon 11 (42 pts, CB 36%), KIT exon 9 (19 pts, CB 42%), PDGFRA (4 pts, CB 25%), no KIT or PDGFRA mutation (wild-type [WT]; 9 pts, CB 56%). Notably, the PR rate for GISTS with primary KIT exon 9 mutations was 37%, vs 5% for KIT exon 11 mutations (P=0.003). PFS and OS were significantly longer for pts with either a primary KIT exon 9 mutation or WT KIT/PDGFRA compared with pts with a KIT exon 11 mutation (exon 9 vs 11: PFS P=0.0007, OS P=0.005; WT vs exon 11: PFS P=0.03, OS P=0.01). Secondary KIT mutations (in exons 13, 14, 17 or 18) were found in 62% of GISTs with a primary KIT exon 11 mutation, but only in 16% with a primary KIT exon 9 mutation (P=0.002). No secondary mutations were found in GISTs lacking a primary KIT/PDGFRA mutation. Biochemical profiling of secondary kinase mutations revealed in-vitro sensitivity of KIT exon 13 and 14 mutations to SU, while secondary KIT exon 17 and 18 mutations were resistant to SU in vitro. These in-vitro results were consistent with clinical results: CB was observed in 65% of pts with secondary KIT mutations in exon 13 or 14 and in only 9% of pts with secondary KIT exon 17 or 18 mutations (P=0.006). Conclusions: Our findings suggest that, similar to prior results observed using imatinib, CB of SU following failure of IM treatment is significantly influenced by both primary and secondary mutations in the predominant pathogenic kinase, the uncontrolled activity of which is a critical etiologic factor for GIST. [Table: see text]

L’interféron β1-a IM ne modifie pas le fonctionnement psychologique global de patients atteints de sclérose en plaques rémittente pendant les premiers mois de traitement

Psychological troubles are common in multiple sclerosis but their underlying etiology is still controversial. The objective of this open, non comparative, multicenter study was to assess changes in global psychological functioning in new multiple sclerosis patients during the first 3 months of treatment with intramuscular interferon beta-1a once weekly (Avonex). This functioning was rated every 4 weeks with the GAF (Global Assessment Functioning) scale. Depression measured on MADRS (Montgomery & Asberg Depression Rating Scale), clinical global impression (CGI) on patients'psychological status and clinical as well as biological tolerance were also assessed every 4 weeks. Five hundred and ninety-nine patients (71.4 percent women), aged 39.4 years were included. No clinically significant difference in mean GAF score between baseline and the end of the first 3 months of interferon beta-1a IM treatment (main evaluation outcome) was found. Similar results were obtained on MADRS scale. No clinically significant alteration of global psychological functioning, including symptoms of depression, was observed during the first 3 months of treatment with interferon beta-1a IM.

Enhanced Primary CML Stem Cell Elimination by Bryostatin Priming with Imatinib Mesylate In Vitro.

In chronic myeloid leukemia (CML), CD34+ quiescent stem cells are sensitive to the anti-proliferative but not the pro-apoptotic effects of imatinib mesylate (IM; Gleevec®, Glivec®). Bryostatin-1 (bryo), a Protein Kinase C modulator, was investigated to determine whether it would increase IM-mediated apoptosis either through initiation of cycling of G0/G1 Ph + cells or reversal of the IM-induced anti-proliferative response. The Ph+ K562 cell line and primary CD34+ CML cells were studied. Cell cycle status was assayed by propidium iodide uptake. Following exposure to IM, 86 ± 1 % (mean ± SEM) of surviving K562 cells was in G0/G1 as compared to 52 ± 2 % in the control (p < 0.001). After accounting for IM-induced cell kill, the absolute number of viable G0/G1 cells was significantly increased, confirming the anti-proliferative, anti-apoptotic effect. However, priming for 24 hours with bryo both increased K562 total cell kill and normalised the percentage of cells recovered in G0/G1 thus reducing their absolute number. To assess the effect of priming with bryo on primary cells, endpoints on two levels were investigated: firstly, at the total cell, and secondly, at the stem cell level. For total primary CML CD34+ cells, as compared to untreated control (= 100 %), 56 ± 2 % survived 96 hours treatment with 5 μM IM, whereas 37 ± 2 and 24 ± 2 % survived 1 and 10 nM bryo alone, respectively (n = 7). Priming with bryo prior to IM (5 μM) significantly enhanced bryo-induced total cell death (19 ± 2 and 12 ± 1 % surviving 1 and 10 nM bryo then IM, respectively; p < 0.03). Critically, within the G0/G1 CD34+ cells, 59 ± 3 % of input cells survived IM (5 μM) alone (n = 7). Bryo alone (1 or 10 nM) significantly reduced the proportion of cells recovered in G0/G1 (35 ± 3 and 23 ± 2 %) relative to both the untreated control and IM treatment alone (p < 0.02). Bryo in scheduled combination with IM, further significantly reduced the number of cells recovered in G0/G1 (16 ± 3 and 9 ± 1 %) with respect to IM alone (p < 0.001) and bryo alone (p < 0.05). Thus, it was the combination that was more potent than each individual agent. These results suggest that carefully scheduled drug combinations may prove more efficacious within the Ph+ stem cell compartment than IM monotherapy. Specifically, inclusion of an agent to antagonise the anti-proliferative effect of IM is warranted.

Functional ABCG2 Is Expressed on CML Stem Cells and Its Inhibition Selectively Depletes CML CD34+ Cells.

We have previously described a population of deeply, but reversibly, quiescent stem cells (qSC) found in patients with chronic phase (CP) CML at diagnosis. In vitro studies have proven this population to be highly insensitive to imatinib mesylate (IM; Gleevec, STI571) induced killing, and more worryingly shown that qSC are accumulated after CML CD34+ cells are treated with IM. As it is likely that CML qSC closely resemble normal HSC, we hypothesise that they too may express the stem cell-associated ABCG2 and have therefore examined the expression and function of this drug efflux pump on CML cells. In agreement with other studies we show the interaction between ABCG2 and IM. Using ABCG2 over-expressing cells (AML6.2 and HL60-BCRP) we found that ≥0.5μM IM reduced efflux of the ABCG2 substrate BODIPY-Prazosin by a similar degree as the inhibitor fumitremorgin C (FTC; 10μM). We have now examined expression and function of ABCG2 on primary CML cells taken from patients in chronic phase (CP) and prior to any treatment. Quantitative Taqman analysis of 8 CD34+ enriched (≥90%+) CML samples revealed that the level of expression is 2.46 fold higher than that in normal mobilised CD34+ cells (n=8 CML, n=4 normal). In addition, we undertook microarray analysis of normal or CML CP CD34+ cells fractionated according to cell cycle using Hoechst-Pyronin (G0, G1 and G2/S/M). These analyses (n=3 normal, n=5 CML) show that at all stages of the cycle CML cells express more ABCG2 than normal cells and that G0 CML cells express 2.48 fold more than those in G1 , confirming both the over-expression in CML and relationship to the most primitive subset of cells. Using the antibody BXP21 we found that 8 of 9 samples contain ABCG2+ve cells (5 of 9 ≥60% of cells ABCG2+). We also examined the function of ABCG2 on CML CD34+ cells by performing efflux assays, 4 of 6 showed efflux that was inhibited by 10μM FTC or ≥0.5μM IM, and this efflux capacity correlated with BXP21 staining. We therefore considered whether the combination of IM therapy and ABCG2 inhibition would overcome the accumulation of CML qSCs we have previously reported after treatment with IM. Using CFSE to track cell division we treated CD34+ enriched CML samples with 5μM IM +/− FTC or with 10μM FTC alone for 3 days. In comparison to untreated controls 5μM IM reduced the total number of cells to 31.9±9.2 % and the number of CD34+ cells to 43.2±17.6%. However, the non-cycling qSC significantly increased to 318±75.8% of control. In contrast, the ABCG2 inhibitor FTC did not effect a reduction in total cells (99.5±11.9%) but gave a significant reduction of CD34+ cells (58.6±8.4%; p=0.02) and no accumulation of qSC (104.6±33.8%) when used alone. We saw no cumulative effect when IM and FTC were given concurrently. These data suggest strongly that FTC may be used to deplete CD34+ ‘stem cells' from CML, as the total cell number is unchanged it is likely that this depletion is by the induction of differentiation. We propose that the expression of ABCG2 may be clinically significant in CP CML and that inhibition of this pump may result in a ‘stem cell targeted therapy' that could be followed by IM treatment to reduce the tumor load. Such reduction of CML stem cells would result in elimination of minimal residual disease and effect a lasting remission.

Contribution of root cap mucilage and presence of an intact root cap in maize (Zea mays) to the reduction of soil mechanical impedance.

The impedance to root growth imposed by soil can be decreased by both mucilage secretion and the sloughing of border cells from the root cap. The aim of this study is to quantify the contribution of these two factors for maize root growth in compact soil. These effects were evaluated by assessing growth after removing both mucilage (treatment I -- intact) and the root cap (treatment D -- decapped) from the root tip, and then by adding back 2 micro L of mucilage to both intact (treatment IM -- intact plus mucilage) and decapped (treatment DM -- decapped plus mucilage) roots. Roots were grown in either loose (0.9 Mg m(-3)) or compact (1.5 Mg m(-3)) loamy sand soils. Also examined were the effects of decapping on root penetration resistance at three soil bulk densities (1.3, 1.4 and 1.5 Mg m(-3)). In treatment I, mucilage was visible 12 h after transplanting to the compact soil. The decapping and mucilage treatments affected neither the root elongation nor the root widening rates when the plants were grown in loose soil for 12 h. Root growth pressures of seminal axes in D, DM, I and IM treatments were 0.328, 0.288, 0.272 and 0.222 MPa, respectively, when the roots were grown in compact soil (1.5 Mg m(-3) density; 1.59 MPa penetrometer resistance). The contributions of mucilage and presence of the intact root cap without mucilage to the lubricating effect of root cap (percentage decrease in root penetration resistance caused by decapping) were 43 % and 58 %, respectively. The lubricating effect of the root cap was about 30 % and unaffected by the degree of soil compaction (for penetrometer resistances of 0.52, 1.20 and 1.59 MPa).

Final data of the multicenter phase II study of cisplatin (CDDP) and 5-fluorouracil (5-FU) in combination with G17DT Immunogen in chemonaive patients with locally recurrent or metastatic gastric and gastroesophageal cancer

4048 Background: G17DT is an immunogen designed to raise antibodies to the growth factor gastrin 17 (G17). The purpose of the study was to determine the safety and efficacy of G17DT in combination with CDDP and 5-FU. Interim data indicated a correlation between survival and anti-G17 immune response, which was further explored in analyses of the final data. Methods: Main eligibility criteria for the open label study were: metastatic or locally recurrent gastric/gastroesophageal cancer; measurable disease; chemotherapy naive; intact organ function; KPS≥70. Patients received G17DT (500 μg, im) treatments at wks 2, 6, 10 and 26; CDDP 100 mg/m2 d1; continuous infusion 5-FU 1000 mg/m2 /d d1–5 q28 days. Endpoints: tumor response (per RECIST), survival, immune response and safety. Results: A total of 102 patients were enrolled. The best overall tumor response was 1 CR (1%), 39 PR (49%), 25 SD (32%) and 14 PD (18%). One patient, with PR, converted to CR after additional G17DT treatment in an extension protocol. Overall median survival was 8.7 months. The immune response occurred in 63% patients with anti-G17 titer ≥ 1 ELISA unit for 2 consecutive wks; 75% with at least 1 titer ≥ 1. Toxicity was consistent with CDDP+5-FU except for 3 NCI-CTC Grade-3 injection site reactions attributed to G17DT. Multivariate analysis showed the immune response as a predictor of tumor response and survival (Log rank: p=0.032 and p<0.001, respectively). Median survival was 10 months for anti-G17 responders and 2.3 months for non-responders. With the exception of performance status, no baseline parameters such as sex, age, race, location of metastases or organ systems involved with metastasis(es) significantly correlated with the anti-G17 response. After adjustment for KPS, the correlation between the anti-G17 response and survival remained statistically significant (p<0.001). Conclusion: The G17DT immune response predicts response and survival in untreated, advanced gastric cancer patients, offering promising prospects for an improved anti-cancer treatment regimen. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Aphton Corp. Aphton Corporation