Subsequent Molecular Responses Research Articles

Effects of high stocking density on growth performance and expression of MyD88, and its temporal expression under the challenge of Vibrio parahaemolyticus in the noble scallop Chlamys nobilis

High stocking density has been regarded as an adverse factor in bivalve aquaculture. However, its subsequent molecular response to pathogenic bacteria has been little studied. In order to study the question, a novel MyD88 was first cloned using adult noble scallops Chlamys nobilis (CnMyD88), and its tissue distribution was investigated. Then, 1860 juvenile scallops were divided into two groups with two initial densities of high density (200 individuals/layer, HD) and normal density (110 individuals/layer, ND) and in-situ cultured for three months, in which their growth, survival, and the differential expression of CnMyD88 were examined, respectively. Finally, scallops were injected with the Vibrio parahaemolyticus to assess the temporal expression of CnMyD88. As the results show, CnMyD88 cDNA has a full length of 2241 bp and contains an 1107 bp ORF that encodes a 368-derived protein. It was widely expressed in examined tissues with a significantly higher level in hemolymph, intestine, mantle, and gonad than others. Besides, the HD group showed lower growth (0.39 ± 0.05 mm/day) and survival (37.00 ± 8.49%) than the ND group (0.55 ± 0.02 mm/day and 76.82 ± 5.78%). More importantly, the HD group exhibited significantly lower expression levels of CnMyD88 in their examined tissues than the ND group. After V. parahaemolyticus challenging, CnMyD88 had significantly lower expression levels in the scallops from the HD group than that of the scallops from the ND group at 6th, 24th, and 36th. The present results indicated that high stocking density not only made adverse impacts on growth and survival but also may induce immunosuppression in the noble scallop. Therefore, appropriate low stocking density may be worth considering to adopt in scallop aquaculture.

One-time Electromagnetic Irradiation Modifies Stress-sensitive Gene Expressions in Rice Plant.

Electromagnetic energy is utilized over multiple frequency bands to provide seamless wireless communication services. Plants can well perceive electromagnetic energy present in open environment due to reasonably high permittivity and electrical conductivity of constituent tissues. Moreover, higher surface-to-volume ratio of plant structure facilitates increased interaction with the incident electromagnetic waves. To date, a few well-designed studies have been conducted inside controlled electromagnetic reverberation chambers to investigate either short duration-low amplitude or long duration-periodic electromagnetic irradiation-induced molecular responses in plants. However, as far as is known, studies investigating molecular responses particularly at the mid-vegetative stage in plants following one-time (hours-long) electromagnetic irradiation have not been reported earlier. Hence, the present study aimed at investigating molecular responses in 40-day-old Swarnaprabha rice plants following one-time 1837.50 MHz, 2.75 mW/m2 electromagnetic irradiation of 2 h 30 min duration. Controlled electromagnetic irradiation inside a simple reverberation chamber was ensured to achieve pure electromagnetic environment at 1837.50 MHz with deterministic electromagnetic power density at selected position. Swarnaprabha rice plant was chosen for this investigation since the rice variety is widely cultivated and consumed in the Indian subcontinent. Subsequent alterations in some selected stress-sensitive gene expressions were assayed using real-time quantitative polymerase chain reaction technique-significant upregulation in calmodulin and phytochrome B gene expressions were noted. This investigation was purposefully focused on subsequent molecular responses immediately following electromagnetic irradiation so that the possible effects of secondary stimulations could be avoided. Observed molecular responses strongly suggested that plants perceive 1837.50 MHz, 2.75 mW/m2 electromagnetic irradiation similar to other injurious stimuli. © 2021 Bioelectromagnetics Society.

Any BCR-ABL Reduction Below 10% At 6 Months Of Therapy Significantly Improves Outcome For CML Patients With a Poor Response At 3 Months

▪ BackgroundThe molecular response at 3 months (mo) after commencement of tyrosine kinase inhibitor (TKI) therapy for CML patients (pts) has prognostic significance and has been confirmed by many groups. Analyses by Marin, JCO 2012 and Neelakantan, Blood 2013 went further and suggest that measuring BCR-ABL at 3 mo is the only requirement to predict outcome, with BCR-ABL at later timepoints adding little useful information. However, Nazha (Haematologica 2013) using cytogenetic measurement suggest that assessing the 6 mo response for pts with a poor response at 3 mo may provide a better predictor of long-term outcome. AimIn this study, we aimed to dissect the prognostic importance of assessing both the 3 and 6 mo molecular response in our large cohort and determine whether a poor response at 3 mo identifies a group with high ongoing risk of treatment failure regardless of subsequent molecular response. MethodWe evaluated 528 pts enrolled in consecutive trials of first line imatinib from 2000-2009. Many pts were treated before alternative TKIs were available, but 89 switched. The utility of BCR-ABL as a predictor of death, progression (AP/BC/death: PFS) and treatment failure (according to ELN 2013: FFS) was assessed by ROC analysis, Kaplan-Meier survival with log rank test, and cumulative incidence with Fine & Gray regression for MMR. Pts were divided according to the 2013 ELN 3 and 6 mo molecular response definition: 3 mo, Optimal (Opt) ≤10% or Warning (Warn) >10%; 6 mo, Opt <1%, Warn 1-10% or Failure (Fail) >10%. The Warn category requires more frequent monitoring and Fail mandates a therapy change. ResultsOptimal BCR-ABL threshold values at 3 mo for outcome prediction were concordant with Marin, JCO 2012: death 16.1%; progression 9.5%; failure 8.4%; MMR 1.4%, demonstrating the commutability of the international reporting scale. Consistent with other studies, responses after 4 years were significantly superior for pts ≤10% (Opt) compared with >10% (Warn) at 3 mo: survival 97% v 90%, P = .003; PFS 96% v 84%, P < .0001; FFS 83% v 42%, P < .0001; and MMR 89% v 42%, P< .0001. 100 pts met the BCR-ABL Warn criterion at 3 mo: 14 of these progressed; 4 before 6 mo and a further 7 before 12 mo.When modeling BCR-ABL measurements at 0, 1, 2, 3, 6 and 12 mo as a time-dependent continuous covariate in Cox regression, a BCR-ABL decrease at any time significantly reduced the risk of death (P = .0002), progression (P < .0001) and treatment failure (P < .0001) indicating that response after 3 mo also has prognostic significance. Of the 528 pts, 45 had missing molecular analysis at 3 or 6 mo, including 13 who ceased before 6 mo. The remaining 483 pts were categorized according to the ELN response definition at 3 and 6 mo and included 89 pts >10% at 3 mo. The Table and Figure detail outcomes at 4 years. Any reduction below 10% at 6 mo for pts in the 3 mo Warn category significantly reduced the subsequent risk of death, progression and failure, and increased MMR rates. Notably, there was no significant difference in outcome for pts >10% at 3 mo and <1% at 6 mo (Warn / Opt) compared to pts with an optimal response at both timepoints. ConclusionOur data confirms the importance of achieving BCR-ABL values of 10% or below at 3 mo after starting therapy. Based on the 3 mo assessment, the prognosis was significantly superior for these pts. Progression before the 6 mo timepoint occurred in 4% of pts with BCR-ABL >10% at 3 mo. However, the vast majority of pts with >10% at 3 mo continued therapy and the 6 mo BCR-ABL assessment provided important long-term prognostic information. Any reduction below 10% at 6 mo led to significantly superior outcomes, approximating those with optimal response at 3 mo. The impact and optimal timing of therapeutic intervention for pts in the poor risk category at 3 mo still needs to be determined in prospective studies and may require large patient cohorts. However, pts who are >10% at both 3 and 6 mo have inferior outcomes and are undoubtedly in need of a therapy change. [Display omitted] Disclosures:Branford:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Honoraria, Research Funding. Yeung:Novartis: Honoraria, Research Funding; BMS: Honoraria. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Early Prediction of Subsequent Molecular Response to Nilotinib in Patients with Chronic Myeloid Leukemia: Comparison of the Quantification of BCR-ABL1 Ratios Using ABL1 or GUSB Control Genes

Molecular monitoring of BCR-ABL1 transcripts is a critical prognostic indicator of treatment response in chronic myeloid leukemia (CML). Quantification of BCR-ABL1 transcripts using ABL1 or GUSB as control genes on the early molecular response (MR) to frontline nilotinib was studied using data from 60 patients with chronic-phase CML from the Evaluating Nilotinib Efficacy and Safety in Clinical Trials as First-Line Treatment (ENEST1st) substudy. Effects of BCR-ABL1/ABL1 and BCR-ABL1/GUSB ratios at early time points as independent variables on subsequent MR were determined by logistic regression analyses and predictive cut-off values determined by receiver operating curve analyses. From day 45, concordance was found for both control genes' early transcript kinetics and ability to predict subsequent deep MR at 18 months. From baseline to 3 months, transcripts descended linearly with both control genes. Use of ABL1 allowed for an earlier prediction (2 months) of subsequent MR than with GUSB (3 months), with cut-off values of 1.5% and 0.19%, respectively. The dynamic determination of BCR-ABL1 transcripts using either internal control gene is valid and predictiveof subsequent MR. The use of GUSB to predict an earlier and more accurate response than ABL1 is not supported in the results. Accurate early indicators of MR are essential to identify patients likely to have inferior outcomes who may benefit from treatment with an alternative tyrosine kinase inhibitor.

Induction and maintenance for chronic myeloid leukemia-chronic phase: A novel treatment strategy.

e19526 Background: Imatinib remains the standard of care for CML-CP in many countries because of its affordability and safety with long term use. Given that upfront Nilotinib has higher rates of major molecular response and early deep responses predict long term responses, we hypothesized that upfront Nilotinib as induction followed by Imatinib maintenance may help to capitalize on the efficacy of Nilotinib without sacrificing the safety and efficacy of Imatinib. Methods: CML CP patients were divided into 3 groups at the time of diagnosis. Group 1 received Imatinib 400mg OD upfront, Group 2 and 3 received Nilotinib 300 mg BD for 1st 3 and 6 months respectively then switched over to Imatinib 400 OD. Quantitative real time PCR for BCR-ABL (RQ PCR for BCR-ABL) was done at 3, 6,12, 18, 24, 30, and 36 months to assess response. 3 early molecular responses were defined for analysis. EMR 1 was defined as Bcr-abl RQPCR &lt; 10% at 3 months, EMR 2 as &lt; 1% at 6 months and EMR 3 as &lt; 0.1% at 6 months. Results: Patients on Nilotinib induction had deeper responses than patients on Imatinib at 3 and 6 months. However, subsequent molecular responses were similar in all 3 groups. The rates of Early Molecular responses were significantly better in the Nilotinib group compared to the Imatinib group. Significantly lesser patients in Group 1 had EMR 1 compared to Groups 2 and 3 [80.49% vs 100% vs 95.56%, p value 0.001]. Results were similar for EMR 2 and 3 [EMR 2: 34.15% vs 42.11% vs 60%, p value 0.017, EMR 3: 25.71% vs 25.53% vs 44.44%, p value Group 1 vs Group 3: 0.030]. TKI failure/progression rates were significantly better amongst patients who achieved EMR vs those who did not achieve EMR for EMR 2 and 3 but not EMR 1. For EMR 2, 42.68% who did not achieve EMR eventually had TKI failure compared to 18.03% of those who achieved EMR (p value 0.002). Similarly, 32.95% who failed to achieve EMR 3 progressed vs 11.9% of those who achieved EMR 3 (p value 0.011). 3 patients in group 1 and 1 patient in group 2 developed T315I mutation while none did in Group 3. Conclusions: The early advantage achieved with Nilotinib Induction was not sustained after switching to Imatinib maintenance. Nevertheless, target responses were still achieved and similar failure rates were present in all 3 groups. Patients who attained an EMR &lt; 1 % at 3 months or &lt; 0.1% at 6 months tend to have higher EFS and lower rates of TKI failure/progression irrespective of the treatment arm.

Long Term Response Evaluation of Newly Diagnosed Chronic Myeloid Leukemia Patients on Imatinib Mesylate in Indian Population over 12 Years

INTRODUCTION There has been a quantum leap in the research of inhibitor drugs following the discovery of tyrosine kinase inhibitor (TKIs), Imatinib in the early 1990s. The development of Imatinib, the first BCR-ABL tyrosine kinase inhibitor revolutionized the long term outcome of Chronic Myeloid Leukemia (CML) patients. CML is one of the most common hematological malignancies diagnosed in India. The breathtaking success due to effectiveness and financial viability of Imatinib among Indian population even led to its sale in its generic formulations by pharmaceutical companies in India around early 2000s. METHODS This is a single-center retrospective observational study conducted among 232 CML patients diagnosed and followed up over 12 years from 2003 until 2015 at Kasturba Medical College and Hospital, Mangalore, Manipal University, Karnataka, India. We closely analyzed the handwritten files and the electronic medical records of each of 232 CML patients for their demographic details, symptoms, date of diagnosis, blood counts, bone marrow reports, spleen size at diagnosis, cytogenetic and molecular reports, date - dosage - change in dose - resistance - side-effects of Imatinib and outcome. Patients were followed up closely. Results Median age was 44 years (range, 10 -78 years). In the year of 2008-2012, 46.1% of patients diagnosed with CML. 66.4% were male and 33.6% were female. During the first evaluation of patients at the time of diagnosis, 90.9% of patients were in the chronic phase, 4.7% in accelerated phase and 4.3% in blast phase. 97.8% were started on Imatinib orally as first line. 176 (75.9) patients received 400 mg orally. Five patients chose not to be treated. The most common side effect of Imatinib among patients were as follows: abdominal pain (13.3%), fever (10.8%), general weakness (4.7%), blurred vision (4.7%), abdominal distention (3.4%) and joint pain (2.2%). Response to treatment with Imatinib was evaluated in terms of molecular response as measured by real-time RT Q-PCR. The 3.4% who failed to respond to Imatinib, 3.4% patients were started with second-generation TKI's (dasatinib, nilotinib, hyper CVAD cyclophosphamide, vincristine, Adriamycin and dexamethasone). The first choice for switching TKI therapy in 4 (1.7%) patients were dasatinib, in (3.1%) was nilotinib and only one patient had to use Hyper CVAD chemotherapy. Table has summarized the use of second-generation TKI's. One patient expired and 4 has progression. 181(78.0%) patients came throughout the year of 2015. Among all patients 200 (86.2%) are still alive with disease, 18 (7.8%) are alive without disease and 14 (6.0%) were expired. Overall survival (OAS) is 218 (96%) Progression free survival (86.78%). The median BCR ABL/ABL value at the end of the six months was 11% and at the end of the one year was 3.38%. Conclusion The introduction of Imatinib mesylate had phenomenal changes in the management of CML. Molecular response evaluation after six months can predict the subsequent molecular response and can also be used as a surrogate monitor of the marrow cytogenetic response to imatinib therapy in CML. We report a high overall (OAS) and progress free survival (PFR) rates with very less side effects. Table Disclosures No relevant conflicts of interest to declare.

Different duration of parathyroid hormone exposure distinctively regulates primary response genes Nurr1 and RANKL in osteoblasts.

Parathyroid hormone (PTH) exerts dual effects, anabolic or catabolic, on bone when administrated intermittently or continuously, via mechanisms that remain largely unknown. PTH binding to cells induces PTH-responsive genes including primary response genes (PRGs). PRGs are rapidly induced without the need for de novo protein synthesis, thereby playing pivotal roles in directing subsequent molecular responses. In this study, to understand the role of PRGs in mediating osteoblastic cellular responses to PTH, we investigated whether various durations of PTH differentially induce PRGs in primary osteoblasts and MC3T3-E1. Nurr1 and RANKL, PRGs known for their anabolic and catabolic roles in bone metabolism respectively, presented distinctive transient vs. sustained induction kinetics. Corroborating their roles, maximum induction of Nurr1 was sufficiently achieved by brief PTH in as little as 30 minutes and continued beyond that, while maximum induction of RANKL was achieved only by prolonged PTH over 4 hours. Our data suggested distinctive regulatory mechanisms for Nurr1 and RANKL: PKA-mediated chromatin rearrangement for transcriptional regulation of both PRGs and ERK-mediated transcriptional regulation for RANKL but not Nurr1. Lastly, we classified PRGs into two groups based on the induction kinetics: The group that required brief PTH for maximum induction included Nur77, cox-2, and Nurr1, all of which are reported to play roles in bone formation. The other group that required prolonged PTH for maximum induction included IL-6 and RANKL, which play roles in bone resorption. Together, our data suggested the crucial role of PRG groups in mediating differential osteoblastic cellular responses to intermittent vs. continuous PTH. Continued research into the regulatory mechanisms of PKA and ERK for PRGs will help us better understand the molecular mechanisms underlying the dual effects of PTH, thereby optimizing the current therapeutic use of PTH for osteoporosis.

The Clinical Significance of Early Imatinib Induced ABCB1 Overexpression in Chronic Phase CML Patients: A TIDEL II Sub-Study

▪Recent data from the TIDEL II trial indicate first-line imatinib (IM) treatment, with selective switching to nilotinib (NIL) for failure to meet specific molecular targets or for intolerance, results in excellent molecular response and overall survival in chronic phase Chronic Myeloid Leukaemia (CP-CML) patients. Drug transporters, particularly OCT-1 and ABCB1, have previously been demonstrated to impact IM response. Furthermore, ABCB1 overexpression has been implicated in IM and NIL resistance in vitro. In this TIDEL II sub-study the level of ABCB1 mRNA expression was retrospectively assessed by PCR at day 0 and day 22 post the start of IM therapy and where relevant, at cessation. A change in the level of expression of ABCB1 compared with baseline after 22 days exposure to IM was observed in all patients (Range: 0.6-12.3 fold; median: 2 fold), suggesting IM exposure resulted in altered ABCB1 expression that was variable between patients; this change was not related to the level of BCR-ABL1 expression (P =0.29 at day 0 and P =0.84 at 1 mo). Assessing the impact of >2 fold rise in ABCB1 expression after 22 days IM exposure on subsequent molecular response, and comparing this with patients where a <2 fold rise was observed revealed significant differences in outcome between the 2 groups (Table 1). Table 1% of patients achieving:Over the first 22 days of treatmentEarly Molecular ResponseMMR by 12 moEvent Free SurvivalMR4.5<2 fold rise in ABCB1 (n=22)86%64%77%64%>2 fold rise in ABCB1 (n=22)60%14%37%5%p -value0.0060.0010.018<0.001Importantly, change in ABCB1 mRNA over the first 22 days of treatment was predictive of MMR by 24 mo (Figure 1).These data support previous in vitro findings of ABCB1-mediated IM export and provide the first in vivo evidence for up regulation of ABCB1 in response to IM therapy. Because ABCB1-mediated export of NIL has also been demonstrated, we assessed the correlation between response to NIL therapy and changes in ABCB1 expression following 22 days IM exposure. Of the patients who demonstrated a >2 fold rise in ABCB1 compared with baseline, 16 of the remaining 20 patients (2/22 died at 3.2 mo (Cardiac event) and 3.7 mo (Blast Crisis)) switched to sequential NIL therapy because of sub-optimal response to IM (no prior MMR). Importantly, 0/16 patients receiving NIL subsequently achieved confirmed MMR, suggesting that NIL may provide a poor therapeutic option for patients with up-regulation of ABCB1. These data highlight the potential importance for monitoring increases in ABCB1 expression early in therapy in order to predict those patients likely to achieve poor molecular responses. We next determined the impact of ABCB1 expression on Event Free Survival (EFS) and demonstrated that the fold change in ABCB1 levels at day 22 was predictive for achieving EFS: 77% of patients with <2 rise in ABCB1 vs 37% of patients with >2 rise in ABCB1; p =0.018. Furthermore a sustained >2 fold rise in ABCB1 mRNA compared with levels at diagnosis was observed in 3/3 patients prior to progression to blast crisis and ABCB1 mRNA increased by >2 fold post therapy in 9/12 patients prior to development of KD mutations.Previous studies investigating the correlation between ABCB1 expression and response to therapy have focussed on absolute levels of ABCB1, usually at 12 mo post-therapy initiation when leukaemic cell burden would be low, and have observed no significant difference in ABCB1 expression in optimal vs sub-optimal responders. Conversely, our rapid PCR based assay performed pre and 22 days post the start of IM therapy in CP-CML patients provides a potent early predictor of subsequent IM response. The findings detailed here also suggest that NIL is likely a poor subsequent therapeutic option for patients with up-regulated ABCB1 expression in response to IM. Importantly, these data highlight the fact that better understanding of the effects of TKIs on factors with the potential to influence treatment outcome (such as drug transporter expression) can be used to personalise therapy. Overarchingly, these data exemplify the importance of drug transporters in the setting of TKI therapy and patient response, and, following validation in further studies, provide a new, effective and easily translatable prognostic biomarker based on ABCB1. [Display omitted] DisclosuresHughes:Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

High Peroxisome Proliferator-Activated Receptor-Gamma (PPARγ) Transcriptional Activity Reduces Active Influx of Imatinib and Kinase Inhibition in CML Cells

▪The human organic cation transporter-1 (hOCT-1) is the primary active influx protein for imatinib in BCR-ABL1+ cells. A low functional OCT-1 activity (OA) in peripheral blood mononuclear cells (MNCs) at diagnosis is associated with poorer subsequent molecular response and inferior outcomes in chronic phase chronic myeloid leukaemia (CP-CML) patients. We have previously demonstrated that diclofenac, an antagonist of peroxisome proliferator-activated receptor-γ (PPARγ), can significantly increase OA in BCR-ABL1+ cells. In the current study, we systematically assess the correlation between PPARγ and OA using leukemic cell lines and primary MNCs from untreated de novo CP-CML patients. Given the critical role of PPARγ in cell differentiation and the varying levels of PPARγ expression in different cell types, the present study also explored the relationship between the OA and myeloid-specific markers (CD14, CD15 and CD16) in CP-CML patient MNCs.Our results demonstrate that OA in BCR-ABL1 positive and negative leukemic cell lines (KU812, HL60 and HL60-BCRABL1) were significantly decreased with PPARγ agonists (GW1929, rosiglitazone) and increased with antagonists (GW9662, T0070907) (Table 1). Similarly, OA in PPARγ-transduced K562 cells (16.03 ng/200,000 cells) was significantly reduced when compared with empty vector control (22.73 ng/200,000 cells, p=0.028). In primary MNCs isolated from untreated CP-CML patients with high OA, a consistent inhibitory effect on OA was observed following the treatment with PPARγ agonists in all 5 cases. The average OA was significantly reduced by GW1929 (from 7.88 to 5.21 ng/200,000 cells, p=0.0075) or rosiglitazone (to 4.65 ng/200,000 cells, p<0.0001). In contrast, following treatment with PPARγ antagonists, OA was increased in all but one of the low OA patient samples (n=6). The average OA in these patients was increased from 2.71 to 5.07 ng per 200,000 cells by GW9662 (p=0.0129) and to 4.71 ng per 200,000 cells by T0070907 (p=0.0155). In addition, in combination with imatinib, these effects on OA resulted in corresponding changes in Bcr-Abl kinase inhibition and cell viability. While there was no correlation between OA and the expression of the PPARG gene or total PPARγ protein, there was a negative association between OA and PPARγ transcriptional activity in MNCs samples isolated from 84 untreated CP-CML patients (r=-0.5677, p<0.0001, Figure 1). Interestingly, in primary CML samples, the percentage of CD15+ CD14- CD16high MNC cells was significantly higher in patients with low OA (41.70%, n=10) than those with high OA (21.74%, n=11, p=0.0126), which may contribute to the higher PPARγ activity observed in these cases.In summary, the findings presented in this study highlight for the first time the association between active influx of imatinib and PPARγ. The consistent OA changes in both in BCR-ABL1 positive and negative leukemic cell lines indicate that treatment with PPARγ ligands significantly alter OA via a likely Bcr-Abl independent mechanism. In CP-CML patients at diagnosis, there is a strong negative correlation between OA and PPARγ activity. By up-regulating OA, PPARγ antagonists significantly increased intracellular imatinib and Bcr-Abl kinase inhibition, that subsequently resulted in increased imatinib induced apoptosis. Therefore, reagents repressing PPARγ activity may improve responses for de novo CP-CML patients with low OA. In addition, the CD15+ CD14- CD16high subset of MNCs is significantly enriched in low OA samples providing a potential readily translatable biomarker for a patientÕs molecular response to imatinib.Table 1OCT-1 activity changes in cell lines and MNC from CP-CML patients after treatment with PPARγ ligands (percentage of vehicle control)PPARγ agonistsPPARγγ antagonistGW1929RosiglitazoneGW9662T0070907KU81267%**49%**139%**151%**HL6075%**77%*123%*123%*HL60-BCRABL174%*79%*118%*136%***, p<0.05; **, p<0.01; compared with vehicle control. [Display omitted] DisclosuresHughes:ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

The Depth Of In Vivo Kinase Inhibition Achieved Over The First Month Of Nilotinib Therapy Predicts For Subsequent Molecular Response, and Is Closely Related To Nilotinib Plasma Levels

We have previously demonstrated that the degree of in vivo kinase inhibition achieved over the first month of imatinib therapy in newly diagnosed, chronic phase chronic myeloid leukaemia (CP-CML) patients, is an excellent predictor of subsequent molecular response. Patients achieving >50% in vivo kinase inhibition all achieved major molecular response (MMR; ≤0.1% BCR-ABL1 (IS)) by 24 months compared to 56% of patients with <50% in vivo kinase inhibition (p<0.001). (White et alJCO 2007). To date, no biomarker which accurately predicts response to nilotinib has been reported.Achievement of <10% BCR-ABL1 by 3 months (EMR) is predictive of subsequent molecular outcome in imatinib treated patients. Only 1 of the 70 CP-CML recipients of first-line nilotinib (300mg bid) monitored in Adelaide failed to achieve <10% by 3 months, and this patient withdrew from treatment due to toxicity 7 months after the start of nilotinib therapy. Failure to achieve <1% BCR-ABL1 (EMR 1%) by 3 months was strongly associated with a significantly lower rate of MMR by 12 months when compared to patients achieving <1% BCR-ABL in the 65 of 70 patients with molecular response data examinable at 12 months. (55% MMR (n=11) vs 93% MMR (n=54) p=0.002).The degree of in vivo kinase inhibition was measured by calculating the % reduction in the p-Crkl level of mononuclear cells collected at days 7 and 29 after the start of nilotinib therapy. A significantly higher proportion of nilotinib treated patients achieved 50% or greater in vivo kinase inhibition over the first month of treatment compared to our historical cohort of imatinib treated patients (75% vs 45% of patients. p<0.001). Dividing patients into those with low and those with high in vivo kinase inhibition revealed a significantly higher proportion of nilotinib treated patients with high in vivo kinase inhibition achieved EMR1% (p=0.012) and MMR by 12 months when compared to patients with low in vivo kinase inhibition (p<0.001) Table 1.Table 1The predictive value of in vivo kinase inhibition in nilotinib treated patients.The % of patients achievingEMR1%MMR by 12 monthslow in vivo kinase inhibition7171high in vivo kinase inhibition8994P value0.012<0.001Correlating drug levels achieved over the first month of therapy, with in vivo kinase inhibition revealed a strong correlation for nilotinib (p<0.001), but not imatinib (p>0.05), suggesting that early dose intensity may be the key factor for nilotinib response. In contrast, while dose is clearly important for imatinib other factors, such as variable drug influx also play a pivotal role. Previous studies have demonstrated that the pharmacokinetics of nilotinib are similar to that of imatinib, and that trough plasma levels of imatinib >1000ng/ml are associated with a higher rate of MMR. Grouping patients into those <1000ng/ml by day 29 and those >1000ng/ml revealed that early trough nilotinib plasma levels were also strongly predictive of the achievement of MMR by 12 mo (<1000ng/ml – 79% (n=38) versus >1000ng/ml – 96% (n=25). p=0.009). Of note two patients have developed kinase domain mutations and both patients failed to achieve high in vivo kinase inhibition or drug levels >1000ng/ml.Preliminary analysis of this cohort of nilotinib treated patients strongly suggests that the in vivo kinase inhibition achieved is significantly higher than observed with imatinib at equivalent timepoints, and is a significant factor for subsequent molecular response. Importantly the plasma levels achieved over the first month of nilotinib therapy appear to be a key factor in the achievement of both early in vivo kinase inhibition and subsequent molecular response. Disclosures:White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; CSL: Research Funding. David:Novartis: Honoraria, Research Funding; BMS: Research Funding. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL: Research Funding.

Molecular Responses At 3 Months Are Associated With a Better Subsequent Molecular response and Outcome In Children and Adolescents With CML In Chronic Phase: Results Of a Retrospective Analysis From The French Glivec Phase 4 Study

Here we report a retrospective analysis based on BCR-ABL transcript level at 3 months after the start of imatinib assessing its impact on subsequent response and outcome in children and adolescents with chronic myeloid leukemia (CML) enrolled in the French prospective trial Glivec Phase 4 (Millot F et al, JCO 2011). Methods44 children were enrolled in the Glivec Phase 4 Study. The median age was 11.5 years (range: 10 months-17 years). The median follow-up was 49 months (range: 16 to 83). We retrospectively analyzed the rates of complete cytogenetic responses (CCR) and major molecular response (MMR) 1 year after the start of imatinib, the progression free survival (PFS) and the overall survival (OS). ResultsAt 3 months after the start of imatinib, 40/44 (91%) patients were evaluable for molecular response. BCR-ABL transcripts levels were: BCR-ABL> 10% in 15 (37%) pts and BCR-ABL<10% in 25 (63%) pts. Children and adolescents with BCR-ABL >10% at 3 months had similar Sokal score distribution but a larger spleen size and a higher leukocyte count at diagnosis compared with patients with BCR-ABL <10%. The median dose of imatinib administered within the first 3 months of treatment was similar in patients with BCR-ABL >10% and those with <10% at 3 months. Two patients progressed to blastic phase 25 and 42 months after the start of imatinib and 1 died. Both of them had a BCR-ABL transcript level of more than 10% at 3 months after the start of imatinib. A transcript level <10% correlated with a better PFS (Table 1). Patients with a BCR-ABL transcript level <10% 3 months after the start of imatinib had a higher rate of CCR and MMR 12 months after the start of imatinib compared to those with a BCR-ABL transcript level >10% (Table 1). However the difference is statistically significant only for the molecular response.Table 1BCR-ABL/ABL at 3 monthsN (%)CCyR at 12 monthsMMR at 12 monthsPFS at 36 months< 10%25 (63%)18 (72%)12 (92%)100%> 10%15 (37%)7 (28%)1 (8%)92%p=0.177p=0.0128p=0.034 ConclusionThe children and adolescents with >10% of BCR-ABL at 3 months after the start of imatinib are characterized by a higher propensity to fail the treatment and to progress. The value of a cut-off of 10% of BCR-ABL 3 months after the start of imatinib as a reliable surrogate marker of response at 1 year and outcome remains to be determined in a larger cohort of children and adolescents. Disclosures:No relevant conflicts of interest to declare.

OCT-1 Activity in CML CD34+ Cells Is Not Predictive of Molecular Response to Imatinib Treatment in CP-CML Patients, Despite the Strong Predictive Value of MNC OCT-1 Activity.

Abstract 2189Poster Board II-166 Introduction.The organic cation transporter 1 (OCT-1) is the major active influx transporter for imatinib in PB mononuclear cells (MNC) from CML patients. We have previously demonstrated that the functional activity of the OCT-1 protein (OCT-1 Activity, OA) in MNC from chronic phase (CP) CML patients is highly variable (mean: 7.2 ng/200,000 cells, standard deviation: 6.4). We also demonstrated that the MNC OA is significantly related to a patient's sensitivity to imatinib-induced kinase inhibition, and their subsequent molecular response. In addition, we have recently demonstrated that OA is predictive of event free and transformation free survival to 5 years (White ASH submitted 2009). This is of a greater importance for those patients on lower doses of imatinib (below 600mg). For these patients who have a low MNC OA, 82% failed to achieve a major molecular response (MMR) by 18 months of imatinib treatment, as opposed to 17% for patients with a high MNC OA (p=0.022). Recently, we have shown that the OA in CML CD34+ cells is significantly lower than that found in mature CML cells (mean CD34+: 3.9, CD34-: 11.6 ng/200,000 cells, p<0.001, n=36), however interpatient variation in CD34+ OA still remains (standard deviation: 2.65). Given the importance of low MNC OA as a prognostic indicator of poor molecular response, we sought to ascertain whether a patient's MNC OA was related to the OA in their CD34+ cells, and as such could be used as a surrogate indicator for efficient targeting of the primitive population. To do this, we firstly looked for a correlation between the OA in a patient's MNC and their CD34+ cells. Secondly, we assessed the predictive value of CD34+ OA for MMR in imatinib treated patients. Methods.MNC and CD34+ cells were isolated from imatinib naïve, newly diagnosed CP-CML patients using density gradient centrifugation and magnetic cell sorting, respectively. OA was determined using [14C]-labeled imatinib and the OCT-1 inhibitor prazosin. OA is calculated as the difference between the uptake and retention of imatinib in the absence and presence of prazosin. Statistical analyses were performed using the Mann-Whitney Rank Sum or Student's t-test, correlations were performed using the Spearman-Rank Order. Results.In 34 CML patients no correlation was observed between the OA in their MNC and the OA in their CD34+ cells (R=0.09, p=0.577). Furthermore, when these patients were divided as having high or low OA in their MNC (about a previously described median of 7.2 ng/200,000 cells), no difference was seen between the OA in their corresponding CD34+ cells (Table 1).Table 1The mean OCT-1 Activity (OA) in a patient's CD34+ cells expressed as ng/200,000 cells (Standard Deviation)nCD34+ OAP ValueLow MNC OA254.02 (2.78)0.854High MNC OA93.83 (2.53)Finally, in 19 patients where 12 month response data was available, patients were grouped according to the achievement or not of MMR by 12 months. MNC OA was found to be significantly associated with the achievement of MMR (Table 2). This suggests that patients with a high OA in their MNC achieve better molecular responses, which is in agreement with our previous findings. However, assessment of CD34+ OA failed to demonstrate a relationship between OA and the achievement of MMR (Table 2). Therefore, a low OA in patient's CD34+ cells (unlike MNC) does not appear to be associated with poor molecular responses.Table 2The mean OCT-1 Activity (OA) in patients MNC and CD34+ cells expressed as ng/200,000 cells (Standard Deviation)nMNC OAP ValueCD34+ OAP ValueNo MMR73.13 (2.90)0.0313.65 (2.29)0.979Yes MMR128.38 (5.44)3.63 (1.81) Conclusion.No relationship was identified between the OA measured in a patient's MNC and that in their CD34+ cells. Furthermore, CD34+ OA is not predictive of a patient's molecular response to imatinib treatment. This is in contrast to MNC OA, which is predictive of event free and transformation free survival to 5 years of imatinib treatment. This data suggests that the predictive value of the MNC OA may primarily reflect the effective targeting and subsequent eradication of mature CML cells. And that kinase inhibition in these mature cells (as opposed to CD34+ progenitor cells) may be the key determinant of MMR achievement in CP-CML.Supported by Novartis Oncology, Clinical Development, TOPS Correlative Studies Network Disclosures:Zannettino:Novartis Pharmaceuticals: Research Funding. White:Novartis Pharmaceuticals: Honoraria, Research Funding. Hughes:Novartis Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau.

Can short-term administration of dexamethasone abrogate radiation-induced acute cytokine gene response in lung and modify subsequent molecular responses?

Purpose: To investigate the effects of short-term administration of dexamethasone (DEX) on radiation-induced responses in the mouse lung, focusing on expression of pro-inflammatory cytokine and related genes.Methods and Materials: At indicated times after thoracic irradiation and/or drug treatment, mRNA expression levels of cytokines (mTNF-α, mIL-1α, mIL-1β, mIL-2, mIL-3, mIL-4, mIL-5, mIL-6, mIFN-γ) and related genes in the lungs of C3H/HeN mice were measured by RNase protection assay.Results: Radiation-induced pro-inflammatory cytokine mRNA expression levels in lung peak at 6 h after thoracic irradiation. DEX (5 mg/kg) suppresses both basal cytokine mRNA levels and this early response when given immediately after irradiation. However, by 24 h, in mice treated with DEX alone or DEX plus radiation, there was a strong rebound effect that lasted up to 3 days. Modification of the early radiation-induced response by DEX did not change the second wave of cytokine gene expression in the lung that occurs at 1 to 2 weeks, suggesting that early cytokine gene induction might not determine subsequent molecular events. A single dose of DEX attenuated, but did not completely suppress, increases in cytokine mRNA levels induced by lipopolysaccharide (2.5 mg/kg) treatment, but, unlike with radiation, no significant rebound effect was seen. Five days of dexamethasone treatment in the pneumonitic phase also inhibited pro-inflammatory cytokine gene expression and, again, there was a rebound effect after withdrawal of the drug.Conclusions: Our findings suggest that short-term use of dexamethasone can temporarily suppress radiation-induced pro-inflammatory cytokine gene expression, but there may be a rebound after drug withdrawal and the drug does little to change the essence and course of the pneumonitic process.