ALBERT

Description: Eradication of minimal residual disease (MRD) during the first months of treatment for patients (pts) with ALL is associated with improved disease-free survival (DFS). We hypothesized that Alemtuzumab, a humanized monoclonal antibody directed against CD52, might be an effective, novel agent for eradication of MRD in ALL based on data demonstrating strong CD52 expression in other lymphoid malignancies and in several ALL cell lines, and from case reports of clinical activity in advanced ALL. In CALGB 10102, to define the percentage of CD52+ cases and to demonstrate feasibility, we tested dose escalation of Alemtuzumab in sequential cohorts to a target dose of 30 mg administered sc 3X/week for 4 weeks (12 doses) during post-remission therapy. Pts are eligible to receive Alemtuzumab if lymphoblast CD52 expression at diagnosis is ≥ 10% as determined in a CALGB reference laboratory. The 10102 therapy is composed of monthly treatment modules outlined below: Treatment module sequence is: A,B,C, D, A, B, C followed by maintenance therapy for a total of 2 years. Antimicrobial prophylaxis for cytomegalovirus (CMV) (e.g. acyclovir 800 mg qid) and pneumocystis carinii is mandated. Weekly quantitative monitoring for CMV viremia is performed. 150 pts with untreated ALL have enrolled: Median age is 48 yrs. 124 (83%) pts have precursor-B; 19(13%) have precursor T-ALL and 7 (4%) have biphenotypic or bilineal ALL. Of 139 evaluable pts, 95 (68%) had CD52 ≥ 10%. By immunophenotype, 72% of precursor B and 61% of precursor T pts were eligible to receive Alemtuzumab. Phase I dose escalation was recently completed. Dose limiting toxicity (DLT) for Phase I was defined as the inability to proceed with protocol treatment within 6 weeks of the last dose of Alemtuzumab. Non-heme toxicities have been mild and sc Alemtuzumab administration was well tolerated. Hematologic and infectious toxicities are summarized below: Myelosuppression was transient and use of G-CSF was permitted during Module D. 2 pts were treated for CMV viremia due to rising CMV titers in 2 sequential assays. 6 other pts had transient CMV elevations during or immediately following completion of Alemtuzumab that did not require treatment. 22/24 phase I pts received all 12 doses of Alemtuzumab. There were 2 DLTs reported: 1 pt in cohort 2 due to CMV viremia requiring gancyclovir following completion of Alemtuzumab; and 1 pt in cohort 3 due to ANC 〈 1500 six weeks after Alemtuzumab (pt was not given G-CSF). Based on these Phase I data, the targeted dose of 30 mg Alemtuzumab was recommended for further study in the ongoing Phase II study. In summary, we report for the first time that CD52 is expressed in the majority of ALL cases and demonstrate the feasibility of employing Alemtuzumab in front-line therapy. Ongoing accrual to the phase II study will evaluate the efficacy of Alemtuzumab in eradication of MRD in adult ALL. Module A Module B Module C Module D (Alemtuzumab) Maintenance Cytoxan Cytoxan Methotrexate (IV, PO, IT) 10 mg* cohort 1 Vincristine Daunorubicin Cytarabine Vincristine 20 mg* cohort 2 Dexamethasone Vincristine Vincrisitne 6-MP 30 mg* cohort 3 6-MP Dexamethasone L-asparaginase *Phase I dose escalation Methotrexate L-asparaginase IT-Methotrexate Alemtuzumab cohorts N Myelosuppression (grades 3 or 4) Lymphopenia CMV viremia 10 mg 6 2 1 2 20 mg 10 1 1 4 30 mg 8 1 0 2

Description: Mutations in the all-trans retinoic acid (ATRA)–targeted ligand binding domain of PML-RARα (PRα/LBD+) have been implicated in the passive selection of ATRA-resistant acute promyelocytic leukemia clones leading to disease relapse. Among 45 relapse patients from the ATRA/chemotherapy arm of intergroup protocol C9710, 18 patients harbored PRα/LBD+ (40%), 7 of whom (39%) relapsed Off-ATRA selection pressure, suggesting a possible active role of PRα/LBD+. Of 41 relapse patients coanalyzed, 15 (37%) had FMS-related tyrosine kinase 3 internal tandem duplication mutations (FLT3-ITD+), which were differentially associated with PRα/LBD+ depending on ATRA treatment status at relapse: positively, On-ATRA; negatively, Off-ATRA. Thirteen of 21 patients (62%) had additional chromosome abnormalities (ACAs); all coanalyzed PRα/LBD mutant patients who relapsed off-ATRA (n = 5) had associated ACA. After relapse Off-ATRA, ACA and FLT3-ITD+ were negatively associated and were oppositely associated with presenting white blood count and PML-RARα type: ACA, low, L-isoform; FLT3-ITD+, high, S-isoform. These exploratory results suggest that differing PRα/LBD+ activities may interact with FLT3-ITD+ or ACA, that FLT3-ITD+ and ACA are associated with different intrinsic disease progression pathways manifest at relapse Off-ATRA, and that these different pathways may be short-circuited by ATRA-selectable defects at relapse On-ATRA. ACA and certain PRα/LBD+ were also associated with reduced postrelapse survival.

Description: Recruitment of histone deacetylases and DNA hypermethylation of promoter regions of specific genes are two mechanisms of transcriptional repression and gene silencing which have been linked, and are implicated in differentiation block in AML. We hypothesized that the histone deacetylase inhibitor (HDI) depsipeptide could result in transcriptional de-repression, upregulation of specific target genes and differentiation of the leukemic clone in AML. Eighteen patients (pts), median age 60 years (range 25–77) with relapsed or refractory AML were enrolled on a multicenter Phase II study of depsipeptide in AML. Patients were stratified into 2 groups on study entry: Group A (n=14) included patients without specific chromosomal abnormalities known to recruit histone deacetylases. Group B (n=4) included patients with chromosomal aberrations such as the t(8;21), inv 16 and t(15;17) known to recruit histone deacetylases. Depsipeptide was administered intravenously at a dose of 18mg/m2/d on days 1, 8 and 15 of a 28 day cycle. Peripheral blood mononuclear cells were obtained prior to (hour 0), and after 4 (hr 4) and 24 hrs (hr 24), on days 1 and 8 of the first cycle of therapy for evaluation of histone acetylation by flow cytometry, and gene re-expression by REAL-time RT-PCR. Target genes of interest include MDR1, a target of HDI mediated upregulation, and p15INK4B (p15), a target of DNA hypermethylation in AML. MDR1 and p15 copy numbers are expressed as a normalized quotient of MDR1 and p15, respectively, to the housekeeping gene ABL. The drug has been well tolerated. The most common adverse effects noted included grade 1/2 nausea, vomiting and fatigue. No objective evidence of response (CR or PR) or other evidence of antileukemic activity has been seen in group A. In contrast, 2 of 4 pts (50%) in Group B, have had a disappearance of bone marrow blasts (blast percentage 〈 5%) in the setting of a normocellular marrow, with concomitant recovery of near-normal hematopoiesis following 1 and 2 cycles of therapy respectively. This anti-leukemic effect was short-lived, with both pts developing an increase in bone marrow blasts within 30 days of the initial response. Both of these patients also had translocations involving the AML1 gene {1 had t(8;21) and the other had a novel translocation t(4;21)}. Interestingly both of these responding pts and one other pt (75%) in cohort B demonstrated an increase in H3 acetylation at 4 and/or 24 hrs, in contrast to 4 of 14 pts (28%) in cohort A. There was an overall mean increase of 41% in MDR1 expression at hr 4 on days 1 and 8 (p=0.04). p15 expression was also upregulated at hr 4 on days 1 and 8 (91% mean increase, p=0.01). We conclude that the HDI, depsipeptide, may have anti-leukemic activity in specific cytogenetic subsets of AML known to recruit histone deacetylases, and this is associated with a concomitant increase in histone acetylation. In addition, upregulation of specific target genes occurred in patient derived mononuclear cells, following depsipeptide treatment. The study remains open to accrual for pts with specific chromosomal abnormalities known to recruit histone deacetylases.

Description: Recent studies suggest that detection of subclinical, or minimal residual disease (MRD) in apheresis products used for autografting correlates with poor disease-free survival following ASCT for mantle cell lymphoma (MCL). To validate this observation and to gain insights into the kinetics of MRD during treatment, we are performing a prospective analysis of MRD using quantitative real-time PCR (Q-PCR) in patients (pts) undergoing treatment for MCL on a CALGB study (59909). Q-PCR of sequential paired bone marrow (BM) and blood (B) samples and of apheresis products was performed using either a patient-specific immunoglobulin heavy chain (IgH) or BCL-1 gene rearrangement. All samples were analyzed in triplicate using LightCycler technology and reported as a normalized ratio of IgH or BCL-1 copy number to GAPDH copy number. The sensitivity of the assay ranged from 1 X 104–1 X 105. To date, a clonal IgH or BCL-1 gene rearrangement was detected in 36 of 41 (88%) pts entered on study. Patient-specific primers and consensus probes were used for Q-PCR monitoring of MRD following two courses of intensive induction therapy, during stem cell mobilization, and 3 and 12 months after ASCT and post-transplant immunotherapy with Rituximab (R). 27 pts have completed all protocol treatment with a median follow-up of 7 months (range: 0–28). 26 pts were evaluated for MRD following two courses of induction therapy with cyclophosphamide, methotrexate, doxorubicin, vincristine, prednisone, and R. 10 of 26 became MRD negative (−) following induction while 16 remained MRD positive (+). Following mobilization with high-dose cytarabine, etoposide and R, apheresis products from 9 of 10 MRD- pts were evaluated and all products were MRD- (1 pt not evaluable). Of the 16 pts who were MRD+ prior to mobilization therapy, MRD- apheresis products were collected in 8; 5 had MRD+ stem cell collections, and 3 were not evaluable. In total, apheresis products were evaluable in 22 of 26 pts; 17 (77%) had MRD- stem cells collected prior to ASCT. None of these MRD- pts has relapsed to date although 2 pts with MRD- products became weakly MRD+ 12 months following ASCT and R. Of the 5 pts with MRD+ apheresis collections, 4 have remained persistently MRD+ following ASCT and R; 1 has relapsed 19 months after completion of all treatment. Rising levels of MRD in BM and B samples were noted in this patient 3 and 12 months post-ASCT. Statistical comparison of MRD values in paired BM and B samples prior to, and post-ASCT demonstrated good agreement with an intraclass correlation coefficient of.814 and.777, respectively. In conclusion, our results demonstrate that prospective MRD monitoring using Q-PCR provides important insights into the kinetics of response during treatment of MCL. MRD- apheresis products were collected in the majority of pts following intensive induction and mobilization chemo-immunotherapy with R on CALGB 59909. To date, no pts with MRD- apheresis products have relapsed following ASCT. In contrast, it appears that pts with MRD+ apheresis products remain MRD+ following ASCT and R and may be more likely to relapse. Longer clinical follow-up is needed to clarify the significance of the persistence of MRD in apheresis products and following ASCT for MCL.

Description: Mutations in the ligand binding domain (LBD) of the RARα-region of PML-RARα were detected in 33% of tested patients who relapsed after treatment with all-trans retinoic acid (ATRA) on the first North American intergroup APL trial INT0129 (E2491). We hypothesized that the incidence of these mutations at the time of first morphological relapse would be reduced on the successor trial C9710, since the new treatment regimen (concurrent ATRA and chemotherapy for induction, first consolidation with two cycles of arsenic trioxide [ATO] in 50% of patients, and 2 subsequent cycles of consolidation with ATRA and daunorubicin) might prevent the early selection of ATRA-resistant PML-RARα mutant subclones. Procedures for making this assessment from low-density bone marrow (BM) and peripheral blood (PB) cells are well-established. In patients found to have a PML-RARα mutation, pre-relapse BM and PB samples collected for monitoring minimal residual disease (MRD) are also being tested for the presence of the mutation, using a mutation-specific real-time PCR assay, in order to assess the dynamics of mutant subclone emergence. Reconstruction experiments in which first-round, allele-specific PCR products from relapse mutant cells were serially diluted in PCR product from non-mutant APL cells documented a capacity to detect 1 mutant template among 103 to 104 non-mutant templates. 9/18 (50%) relapse patients, representing about half of all C9710 relapses to date, tested positive for PML-RARα LBD mutations by DNA sequence analysis, usually with virtual replacement of non-mutant PML-RARα. 9/10 mutations (1 patient had a double mutation) were missense, 2 repeats of previously reported mutations (Arg276Trp, Gly289Arg) and 5 novel mutations (Leu224Pro, Lys238Glu, Ile273Phe, Arg276Gln x2, Gly289Glu x2); 1 was an in-frame 3-codon deletion (Δ412–414). Serial monitoring of post-consolidation samples for the relapse mutation in 3 patients did not detect mutations until or just prior to clinical relapse. The median time (months) to relapse from post-consolidation therapy assessment for MRD was 9.5 (range, 4–36) for mutant patients and 9 (range, 1.5–16) for non-mutant patients. Two of the serially monitored patients relapsed 7 and 24 months after the termination of 12-month maintenance therapy with ATRA ± methotrexate (MTX) and 6-mercaptopurine (6MP). This, together with the lack of a detectable mutant subclone at the conclusion of maintenance therapy, indicates that proximate ATRA selection was not involved in the emergence of the predominant PML-RARα mutant subclone in these 2 patients. Overall, these results support two conclusions: contrary to our pre-study hypothesis, the incidence of PML-RARα mutations was not reduced in patients who relapsed on protocol C9710, but, since the patient assignment to consolidation therapy with ATO and/or maintenance therapy with MTX/6MP has not been disclosed, a difference between the randomized treatment groups is possible and the late emergence of PML-RARα mutant subclones suggests that the mutations provide an intrinsic APL cell growth/survival advantage that likely contributes to the probability of disease recurrence.

Description: DNA hypermethylation of promoter-specific CpG islands is a well known mechanism of epigenetic silencing, and has been implicated in the pathogenesis and progression of disease in MMM. We are evaluating 5-aza-2′deoxycytidine (Decitabine), a potent DNA methyltransferase inhibitor in a Phase II trial in patients (pts) with MMM. Decitabine was administered subcutaneously at a dose of 0.3mg/kg/d on days 1–5, and days 8–12 and cycles were repeated every 6 weeks, in the absence of dose limiting toxicities. Response was determined every 12 weeks, and defined as an improvement in cytopenias and/or splenomegaly. Pts who had no response after 2 cycles were eligible for dose escalation to 0.4mg/kg/d. Elevated levels of circulating CD34+ cells are associated with advanced stage and evolution to the blast phase of the disease in MMM. Therefore, CD34+ cells were measured in peripheral blood at baseline, and at days 1, 5 and 12 of the first 2 cycles of therapy as a surrogate marker of disease activity. Seven pts (5 males, 2 females) have been enrolled, median age 71 (range 42–89), median baseline absolute CD34+ cell count 27× 106/L (11–4959), Dupriez score of 2, 1 and 0 in 72%, 14% and 14% respectively. Median number of cycles administered was 4 (range 1–7). Median WBC and platelet (plt) count at baseline were 3.6K/uL (range 1.5–29), and 188K/uL (range 62–446K/uL) and 3 pts were red cell transfusion dependent. Grade 4 neutropenia (ANC) occurred in all pts, and grade3/4 thrombocytopenia in 5 pts. Nadir ANC and plts occurred at a median of 31 days (range 24–44) and 23 days (range 17–31) respectively. Recovery to ANC 〉0.5K/uL and plts 〉50K/uL occurred at a median of 43 (range 35–58) and 26 days (23–36) respectively. Two pts required a dose reduction for prolonged myelosuppression, and in 1 pt the dose was escalated to 0.4mg/kg/d for lack of a response after 2 cycles. Two pts have developed febrile neutropenia; one of these pts had a documented infection. Grade 3–4 non-hematologic toxicities were rare and include a variceal bleed in a patient with baseline portal hypertension, occurring in the setting of a platelet count of 486K/uL. There have been no injection site reactions. Five pts are evaluable for response. Of these, two pts have had a response including 1 pt with a CR (normalization of blood counts including transfusion independence). One pt in the blast phase of the disease has had a hematological improvement as evidenced by a normalization in platelet counts (from 62K/uL to 200K/uL) associated with a significant decrease (from 2.58K/uL to 0.03K/uL) in peripheral circulating blasts. The other 3 pts have had stable disease; 2 of these remain on treatment and have received 4 and 7 cycles of treatment, respectively. A trend towards a decrease in spleen size has been observed in 3 of 4 patients with palpable splenomegaly at baseline. Overall, there was a significant reduction in circulating CD34+ levels, with a mean decrease of approximately 70% at day 12 of cycles 1 and 2 (p=0.01) We conclude that low dose decitabine administered subcutaneously is feasible in MMM and is associated with minimal non-hematologic toxicity. Myelosupression is significant, though reversible and requires close monitoring. To our knowledge this is the first report demonstrating the potential clinical activity of decitabine in MMM. This observation requires confirmation in a larger group of patients, and accrual is ongoing to this multi-center Phase II study.

Description: Recruitment of histone deacetylases and CpG island methylation at promoter regions of specific genes are two mechanisms of epigenetic silencing which have been linked and are implicated in differentiation block in AML. We hypothesized that the HDI depsipeptide could cause transcriptional de-repression and upregulation of specific target genes in AML, with subsequent differentiation of the leukemic clone. Twenty-one patients (pts), median age 60 years (range 25–77) were enrolled on a multicenter Phase II study of depsipeptide in AML. Pts were stratified into 2 groups on study entry: Group A (n= 14) included pts without specific chromosomal abnormalities known to recruit histone deacetylases. Group B (n=7) included pts with chromosomal aberrations such as the t(8;21) and inv 16 known to recruit histone deacetylases. All 7 pts in cohort B had translocations involving CBFα or AML1 (n=6) or CBFβ (n=1). Depsipeptide was administered intravenously at a dose of 13mg/m2/d on days 1, 8 and 15 of a 28 day cycle. Peripheral blood mononuclear cells were obtained prior to, and after 4 and 24 hrs, on days 1 and 8 of the first cycle of therapy for evaluation of histone (H3) acetylation by flow cytometry, and gene re-expression by Quantitatative real-time RT-PCR (RQ-PCR). Target genes of interest include MDR1, a target of HDI mediated upregulation, p15INK4B(p15) a target of DNA hypermethylation in AML, and p14ARF (p14), a target of AML1-ETO mediated transcriptional repression. H3 acetylation at the p15 and MDR1 promoters was analyzed by chromatin immunoprecipitation, followed by Q- PCR. The most common adverse effects noted included grade 1/2 nausea, vomiting and fatigue. No objective evidence of response (CR or PR) or other evidence of antileukemic activity has been seen in group A. In contrast, in group B, antileukemic activity has been observed in 4 of 7 (57%) of pts. These include 2 pts with clearance of bone marrow (BM) blasts in the setting of a normocellular marrow, and 2 other pts with a significant decrease (〉50% decrease) BM blasts. This effect was short-lived, with all 4 pts developing evidence of disease progression within 30 days of the initial response. Interestingly 5 of 7 pts (including all 4 pts with evidence of an antileukemic response) in cohort B demonstrated an increase in global H3 acetylation at 4 and/or 24 hrs, in contrast to 4 of 14 pts (28%) in cohort A. Furthermore, in cohort B, at 24hrs, there was a 75% mean increase in MDR1 expression (p=0.005), a 162% mean increase in p15 (p=0.01) and a 106% mean increase in p14 (p

Description: Abstract 1032 Poster Board I-54 MLL-AF9 translocation is the most common MLL fusion gene found in AML and has been associated with a relatively poor prognosis. The acquisition of MLL-AF9 is a crucial event in leukemogenesis but might require additional genetic events to induce AML. Mutations in cell-signaling molecules including FLT3 and RAS, which confer cell survival and proliferation advantages, occur frequently in MLL leukemias and point to a potential crucial pathogenetic role of the receptor tyrosine kinase (RTK) FLT3 and downstream PI3K/AKT, RAS/MAPK and STAT5 pathways. Targeted inhibition of these pathways can be achieved therapeutically, but efficacy of a single kinase inhibitor (KI) is often limited due to activation of compensatory survival pathways. To explore potential synergy and better efficacy, we have tested a number of KI combinations in MLL-AF9 murine leukemia cells. METHODS: AML was induced in MLL-AF9 knock-in mice using the murine leukemia virus MOL4070LTR, and 4 different cell lines have been generated and listed as 467D, 512C, 467B and 417B. The following KIs were tested in various dual combinations: rapamycin, a mTOR inhibitor; PI-103, a PI3K inhibitor; sunitinib, a RTK inhibitor; erlotinib, another RTK inhibitor with off-target effects on STAT5; PD0325901, a MEK inhibitor; and P6, a JAK inhibitor. KIs concentrations for the combination treatments were based on available in vivo pharmacological data. Effects on cell cycle were measured using BrdU incorporation on all 4 cell lines and phospho-protein levels were measured on 512C using flow cytometry 24-hours after drug exposure. RESULTS: Each drug alone modestly affected cell proliferation, but some of the combinations exerted marked anti-proliferative and pro-apoptotic effects, suggesting synergy. Interestingly, some heterogeneity in response to the different combinations was seen in the 4 cell lines. Indeed, the most effective drug duets were PI-103 and P6 in 467D, rapamycin and PI-103 in 512C, and sunitinib and PD0325901 in 467B and 417B. The significant inhibitory effect of some of these combinations on cell cycle progression correlated with a decrease of phosphorylated histone 3 and an increase in cleaved caspase. Furthermore, we were able to demonstrate a correlation with intracellular signaling events, including strong inhibition of pS6, pmTOR, p4EBP1, peIF4E, pErk and Bcl-xl. No effect on pSTAT5 was observed. Despite the heterogeneity in response, 3 different drug combinations were identified as being the most effective for all 4 cell lines: rapamycin and PD0325901, rapamycin and sunitinib, and sunitinib and PD0325901 resulted in a mean decrease in S phase of 24.4 ± 6.4% and a mean increase in apoptosis of 7.2 ± 4.9% after 24 hours exposure in all 4 cell lines. Furthermore, the combination of sunitinib with PD0325901 resulted in a prolonged effect on proliferation and apoptosis after 48 and 72-hour exposure, while the other 2 combinations showed only a transient effect. CONCLUSIONS: The combination of sunitinib and PD0325901 was active in all 4 cell lines, overcoming the genetic heterogeneity, and will now be tested in vivo in these murine MLL-AF9 leukemias. Our results suggest that combined blockade of RTK and MEK signaling may be an effective and attractive therapeutic strategy for MLL-AF9 AML. Disclosures: Sher: invivoscribe: Employment.

Description: Preclinical models have demonstrated the efficacy of granulocyte-macrophage colony-stimulating factor-secreting cancer immunotherapies (GVAX platform) accompanied by immunotherapy-primed lymphocytes after autologous stem cell transplantation in hematologic malignancies. We conducted a phase 2 study of this combination in adult patients with acute myeloid leukemia. Immunotherapy consisted of autologous leukemia cells admixed with granulocyte-macrophage colony-stimulating factor-secreting K562 cells. “Primed” lymphocytes were collected after a single pretransplantation dose of immunotherapy and reinfused with the stem cell graft. Fifty-four subjects were enrolled; 46 (85%) achieved a complete remission, and 28 (52%) received the pretransplantation immunotherapy. For all patients who achieved complete remission, the 3-year relapse-free survival (RFS) rate was 47.4% and overall survival was 57.4%. For the 28 immunotherapy-treated patients, the RFS and overall survival rates were 61.8% and 73.4%, respectively. Posttreatment induction of delayed-type hypersensitivity reactions to autologous leukemia cells was associated with longer 3-year RFS rate (100% vs 48%). Minimal residual disease was monitored by quantitative analysis of Wilms tumor-1 (WT1), a leukemia-associated gene. A decrease in WT1 transcripts in blood was noted in 69% of patients after the first immunotherapy dose and was also associated with longer 3-year RFS (61% vs 0%). In conclusion, immunotherapy in combination with primed lymphocytes and autologous stem cell transplantation shows encouraging signals of potential activity in acute myeloid leukemia (ClinicalTrials.gov: NCT00116467).

Description: Abstract 2742 Molecular monitoring of BCR/ABL transcripts by real time quantitative reverse transcription PCR (qRT-PCR) is an essential technique for clinical management of patients with BCR/ABL-positive CML and ALL. Though quantitative BCR/ABL assays are performed in hundreds of laboratories worldwide, results among these laboratories cannot be reliably compared due to heterogeneity in test methods, data analysis, reporting, and lack of quantitative standards. Recent efforts towards standardization have been limited in scope. Aliquots of RNA were sent to clinical test centers worldwide in order to evaluate methods and reporting for e1a2, b2a2, and b3a2 transcript levels using their own qRT-PCR assays. Total RNA was isolated from tissue culture cells that expressed each of the different BCR/ABL transcripts. Serial log dilutions were prepared, ranging from 100 to 10−5, in RNA isolated from HL60 cells. Laboratories performed 5 independent qRT-PCR reactions for each sample type at each dilution. In addition, 15 qRT-PCR reactions of the 10−3 b3a2 RNA dilution were run to assess reproducibility within and between laboratories. Participants were asked to run the samples following their standard protocols and to report cycle threshold (Ct), quantitative values for BCR/ABL and housekeeping genes, and ratios of BCR/ABL to housekeeping genes for each sample RNA. Thirty-seven (n=37) participants have submitted qRT-PCR results for analysis (36, 37, and 34 labs generated data for b2a2, b3a2, and e1a2, respectively). The limit of detection for this study was defined as the lowest dilution that a Ct value could be detected for all 5 replicates. For b2a2, 15, 16, 4, and 1 lab(s) showed a limit of detection at the 10−5, 10−4, 10−3, and 10−2 dilutions, respectively. For b3a2, 20, 13, and 4 labs showed a limit of detection at the 10−5, 10−4, and 10−3 dilutions, respectively. For e1a2, 10, 21, 2, and 1 lab(s) showed a limit of detection at the 10−5, 10-4, 10-3, and 10-2 dilutions, respectively. Log %BCR/ABL ratio values provided a method for comparing results between the different laboratories for each BCR/ABL dilution series. Linear regression analysis revealed concordance among the majority of participant data over the 10-1 to 10-4 dilutions. The overall slope values showed comparable results among the majority of b2a2 (mean=0.939; median=0.9627; range (0.399 – 1.1872)), b3a2 (mean=0.925; median=0.922; range (0.625 – 1.140)), and e1a2 (mean=0.897; median=0.909; range (0.5174 – 1.138)) laboratory results (Fig. 1–3)). Thirty-four (n=34) out of the 37 laboratories reported Ct values for all 15 replicates and only those with a complete data set were included in the inter-lab calculations. Eleven laboratories either did not report their copy number data or used other reporting units such as nanograms or cell numbers; therefore, only 26 laboratories were included in the overall analysis of copy numbers. The median copy number was 348.4, with a range from 15.6 to 547,000 copies (approximately a 4.5 log difference); the median intra-lab %CV was 19.2% with a range from 4.2% to 82.6%. While our international performance evaluation using serially diluted RNA samples has reinforced the fact that heterogeneity exists among clinical laboratories, it has also demonstrated that performance within a laboratory is overall very consistent. Accordingly, the availability of defined BCR/ABL RNAs may facilitate the validation of all phases of quantitative BCR/ABL analysis and may be extremely useful as a tool for monitoring assay performance. Ongoing analyses of these materials, along with the development of additional control materials, may solidify consensus around their application in routine laboratory testing and possible integration in worldwide efforts to standardize quantitative BCR/ABL testing. Disclosure: Sher: Invivoscribe Technologies Inc: Employment. Miller:Invivoscribe Technologies Inc: Employment. Huang:Invivoscribe Technologies Inc: Employment. Shaw:Invivoscribe Technologies Inc: Employment.