BM Samples Research Articles

Both PI3K p110α and p110δ Isoforms Control CD160-Mediated CLL Cell Viability and Cytokine Production

Expression of CD160 on normal immune cells is primarily observed on human and murine circulating cytotoxic lymphocytes (NK cells and a small subset of T-cells). CD160 exhibits a broad specificity for major histocompatibility complex molecules. While normal B-cells do not express CD160, malignant B-cells of CLL (and HCL) are CD160+.1 Triggering of CD160 on CLL cells mediates increased viability, PI3K dependent growth signals and marked IL-6 and IL-8 secretion.2 In this study, the role of the different PI3K subunits in CD160 effector functions in CLL have been investigated in terms of cytokine/chemokine secretion and cell viability.Unstimulated CLL cells underwent apoptosis over 5 days in culture, which was significantly inhibited by the anti-CD160 antibody, CL1-R2 (p<0.01). Similarly, the viability of purified CLL cells was enhanced by addition of a microenvironment milieu (CD19 negative fraction from BM samples) and further improved by CL1-R2 triggering of CD160 (p=0.03).Without stimulation, CLL cells secreted minimal levels of IL-2, IL-4, IL-10, TNF-alpha, INF-gamma, CCL2 and CCL5 (as assessed by cytokine bead array (BD Biosciences). At baseline, low levels of IL-6 (median: 23 pg/ml) and IL-8 (median: 114 pg/ml) were detected. Incubating CL1-R2 with CLL cells led to a significant increase in IL-6 (median: 1431 pg/ml, p<0.01), IL-8 (median: 7945 pg/ml, p<0.01), CCL2 (monocyte chemotactic protein-1, median: 813.3 pg/ml, p=0.01) and CCL5 (median: 53.61 pg/ml, p=0.03). These effects of CD160 triggering were suppressed, in a dose-dependent manner, by the pan PI3K inhibitors LY294002 (p=0.01) and Wortmannin (p<0.01, Table 1).Table 1(above): n= 12CD160 (10μg/ml) Median pg/ml (range)CD160 (10μg/ml) and LY294002 10μM Median pg/ml (range)CD160 (10μg/ml) and LY294002 20μM Median pg/ml (range)IL-61431 (147 - 8972)108 (4.0 - 5512) p=0.003929 (3.38-5250) p=0.0019IL-87945 (950 - 9665)4394 (71 - 7744) p=0.00171958 (28 - 7024) p=0.0075CCL2813 (84 - 879)7.0 (1.3 - 81) p=0.00631.8 (0.0 - 20) p=0.0045CCL554 (42 - 106)36 (24 - 72) p=0.001821 (6.5 -27) p=0.0024The roles of the different p110-catalytic subunits of PI3K in CD160 mediated activation were investigated. CLL cells from 5 different patients were incubated with the specific isoform-selective inhibitors PI103 (p110α), TGX-115 (p110β) and IC87114 (p110δ) at concentrations of 0.5, 1 and 10μM, in the presence of CL1-R2. CD160 induced cell viability was significantly reduced in a dose dependent manor by both PI103 (p=0.004) and IC87114 (p=0.002). Although the p110β inhibitor demonstrated a reduction in CD160 mediated cell viability it was not significant. Combining the p110α and p110δ inhibitors, a further decrease in CD160 mediated cell viability was observed (p<0.01), but not in a synergistic manner.Upon performing cytokine and chemokine bead array assays on the same patients, the CD160 induced cytokine and chemokine production of IL-6, IL-8, CCL2 and CCL5 were suppressed by all the inhibitors at a concentration of 10 μM (Table 2). However, only p110α and p110δ subunit inhibition resulted in significant reductions, with a further decrease in both CD160 mediated cytokine and chemokine secretion when both were inhibited together (IL-6: p<0.01, IL-8: p=0.03, CCL2: p=0.02, CCL5: p=0.03), but not with a synergistic or additive effect (Table 2).Table 2(Above): n=5CD160 (10μg/ml)CD160 (10μg/ml) and PI103 (p110a)CD160 (10μg/ml) and TGX-221 (p110b)CD160 (10μg/ml) and IC87114 (p110d)CD160 (10μg/ml) and PI103+IC87114IL-6457198 p=0.05365 p=NS189 p=0.02101 p<0.01IL-848613405 p=0.044066 p=NS3143 p=0.032625 p=0.03CCL2696130 p=0.04583 p=NS111 p=0.0246 p=0.02CCL59159 p=0.0573 p=NS61 p=0.0543 p=0.03These results demonstrate that CL1-R2 engagement of CD160 leads to: (1) a direct survival signal in CLL cells; and (2) production of IL-6, IL-8, CCL2 and CCL5. These functions are PI3K-dependent. These PI3K dependent responses signal largely through the p110α and p110δ isoform-selective catalytic subunits. From a clinical perspective, this data lends further support to targeting the p110α subunit, in addition to p110δ, as a therapeutic strategy. However, none of the inhibitors, alone or in combination, reduced cytokine secretion to basal levels, suggesting that CD160 is also signalling via other undefined pathways.

A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytomerty Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative strategy for acute myeloid leukemia (AML) patients in complete remission (CR) presenting poor prognostic factors or with relapsed/refractory disease. However, the risk for disease recurrence following allo-HSCT remains significant and associated with poor outcomes. After transplantation, the early detection of minimal residual disease (MRD) using immunophenotyping combined to chimerism documentation before morphological relapse may allow for immediate interventions and can lead to better results. Immunophenotyping using Multiparameter Flow Cytometry (MFC) and chimerism documentation by PCR have been widely used to track disease recurrence, although a validated consensus on the use of these techniques in the post-allo-HSCT follow-up has not been established yet. The aim of our study was to evaluate the impact of positive MRD by MFC associated to chimerism documentation at 3 months after allo-HSCT on patients overall and progression-free survival (OS, PFS). Patients and Methods We evaluated 137 AML patients who received allo-HSCT in a single center between January 2005 and October 2012 at our department and for whom a 3 months MRD evaluation and chimerism documentation has been performed using MFC, and PCR analysis. There were 71 (52%) males and 66 females with a median age of 47 years (range: 19-66), 77% had de novo AML, 20% had secondary AML and 3% had biphenotypic AML. According to cytogenetics, 40% were normal, 51% were unfavorable (9% classified as failure). According to molecular markers, 9% were favorable, 31% intermediate, 44% unfavourable and 16% had no molecular markers. At allo-HSCT, 46% of patients were in first complete remission (CR1), 25% were in CR 2 and 29% had active disease; 40% received a full intensity conditioning and 60% got reduced intensity one. As cell source, 35% were bone marrow, 53% peripheral blood and 12% cord blood cells. Donors were related in 53% of the cases (45% were 10/10 HLA matched) and unrelated in 47% of cases (20% were 10/10 HLA matched). MFC was performed using BM samples with a sensitivity of 0.01%. Chimerism analysis was performed on marrow and/or blood samples using polymerase chain reaction (PCR) based on informative polymorphic short tandem repeat with an accuracy of ± 5%, a mixed chimerismwas defined by having 5% or more of recipient cells. Results After transplantation, all patients engrafted, the cumulative incidence of acute GVHD at 3 months was 19.9% (95% CI: 16.2-20.6) while the cumulative incidence of chronic GVHD reached 26.7% (95% CI: 22.9-30.5) at 1 year. After a median follow-up of 16 months (range: 3-77), the median OS was 66 months (65-NR) with a 3 years probability of 64% (95% CI: 56-73), the median PFS was 32 months (13-NR) with a 3 years probability of 50% (95% CI: 37-58) while the transplant related mortality rate reached 13.6% (95% CI: 10-16) at 2 years. The 3 months chimerism evaluation (n=137) showed a mixed chimerism in 12 (9%) patients, while the MFC (n= 62) detected 15 patients with leukemic cells. Sixty eight patients showed morphological relapse after a median time of 4.8 months (1-34.7); the correlation study between MRD positivity, mixed chimerism detection and morphological relapse showed a higher correlation for both chimerism and MFC (correlation=0.69, p&lt;0.001) than if we consider chimerism or MFC alone. Multivariate analysis showed a significant worse OS for patients with 3 months positive MFC [1 year OS of 20% vs. 80%, HR= 4 (95% CI: 1.4-11.7), p=0.01] and patients with mixed chimerism [1 year OS of 21% vs. 70%, HR= 4 (95%CI: 1.3-12.1), p=0.01]; these results were still valid even after stratification on disease status at transplantation. These results applied also in terms of PFS for positive MFC [1 year PFS of 13% vs. 76%, HR= 3.6, p=0.02], and mixed chimerism[1 year PFS of 0% vs. 70%, HR= 7, p=0.001], Figure 1. Conclusion The 3 months MRD evaluation using MFC combined to chimerism documentation seems to be an independent prognostic factor on overall and progression-free survival for AML patients undergoing allo-HSCT. The standardisationof this evaluation may lead to the identification of patients with high relapse risk suggesting the need of early therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

Leukemic Pattern Of HOX Gene Expression Is Driven By Genetic Aberrations Through Epigenetic Modifiers

IntroductionHomeobox (HOX) genes encode transcription factors crucial in embryogenesis. They are often dysregulated in malignancies including leukemias. The aberrant HOX gene expression and its regulation in leukemic cells is neither completely described nor understood. AimsOur main aim was to determine whether the leukemic HOX gene expression pattern is driven by differentiation stage of hematopoietic cells or determined de novo during the process of malignant transformation. Consequentially, we aimed to study the role epigenetic modifiers in regulation of HOX gene expression in normal and malignant hematopoiesis. MethodsThe expression pattern of HOX genes (cluster of HOX A and B) and epigenetic modifiers (DNMT1, DNMT3a, DNMT3b, EZH2, BMI-1, MLL, JMJD3, UTX) was assessed by qPCR in 8 FACS-sorted subpopulations of healthy BM representing stages of myeloid differentiation (each sample representing a pool of cells sorted from five individuals). The leukemic expression pattern of these genes was analyzed in diagnostic BM samples of childhood AML patients with typical genotypic and morphological (FAB classification) characteristics (N=46). In vitro experiments were performed using NB4 cell line. ResultsAs expected HOX genes were gradually downregulated during normal differentiation of granulocytic and monocytic lineages (assessed in four consecutive differentiation stages for each lineage). In AML samples, HOX gene expression patterns differed significantly among morphological subtypes. However, HOX gene expression did not correlate among subtypes of AML and their physiologically differentiated counterparts.Interestingly, unsupervised hierarchical clustering (HCA) divided AML patients into four main clusters characterized by the presence of prevalent gene rearrangement (PML-RARa, AML1-ETO, MLL rearrangements and NK-AML). The presence of PML/RARa rearrangement was strongly associated with the lowest expression of both HOXA and HOXB clusters, while the other groups had more variable expression of HOX genes. Moreover, the effect of genetic aberrations on HOX gene expression was clearly apparent within AML M2 and M4 subtypes, where AML1/ETO+ or CBFb/MYH11+ patients had significantly lower expression of HOX genes compared to patients with the same FAB classification but without the rearrangements.The expression pattern of epigenetic modifiers in sorted subpopulations of healthy BM followed their expected role in transcriptional regulation during differentiation. However, there was no relation of this pattern to HOX gene expression. On the contrary, in AML samples, the expression levels of epigenetic modifiers clearly correlated with expression profile of HOX genes. These results were supported by unsupervised HCA based on the expression of epigenetic modifiers that showed upregulation of histon demethylases JMJD3 and UTX together with downregulation of DNMT3b in concordance with high levels of HOX genes. Negative correlation between JMJD3 and DNMT3b expression was observed in all leukemic samples (p=0.03); most apparently in PML/RARa+ patients.Therefore we further studied the impact of genetic aberrations on the epigenetic regulation of HOX gene expression in vitrowith PML-RARa+ cell line. Treatment of NB4 cells with ATRA (8, 24hours, 1uM, 10uM) increased the levels of particular HOX genes (HOXA5, A7, B4, B7; FCA=2.8; 1.7; 4; 4 respectively) as well as JMJD3 (FCA=3) and UTX (FCA=1.6). Concordantly, the expression of DNMT3b (FCA=5) was downregulated.The hypothetical driving effect of PML-RARa on de novo determination of leukemic HOX gene expression is further supported by our Results. PML-RARa+ patients had the lowest HOX gene expression regardless of their FLT3/ITD status – previously shown to upregulate strongly HOX genes expression. ConclusionWe conclude that the leukemic expression pattern of HOX genes does not reflect the differentiation stages of malignant cells. Our data also demonstrate different contribution of epigenetic modifiers to the HOX gene expression in healthy and malignant hematopoiesis. Moreover, HCA and expression data together with the results of in vitro experiments suggest that the specific molecular aberrations (as exemplified by PML-RARa) participate in regulation of leukemic HOX gene expression through epigenetic changes.Supported by GACR P304/12/2214, GAUK 568213, 00064203. Disclosures:No relevant conflicts of interest to declare.

Functional Pathway Analysis Of JAK2 Mutated and Wild Type Myeloproliferative Neoplasms As a Tool For Patient Stratification and Therapeutic Selection

BackgroundJAK2 signaling is essential for normal development of multiple hematopoietic cell lineages. A JAK2 (V617F) gain of function mutation has been identified in 95% of polycythemia vera (PV) and 50-60% of essential thrombocythemia (ET) patients, providing a rationale for the development of small molecule JAK2 inhibitors. Clinically, JAK2 inhibitors have been shown to be effective in controlling hyperproliferation of hematopoietic cells in PV and ET in patients with (JAK2+) and without (JAK2 WT) the JAK2 V617F mutation. This suggests that deregulation of the JAK/STAT pathway is also involved in disease pathogenesis in JAK2 negative patients. SCNP, a multiparametric flow cytometry-based assay that quantitatively measures both extracellular surface markers and changes in intracellular signaling proteins in response to modulation, was used in this study to assess JAK/STAT pathway capacity in peripheral blood mononuclear cells (PBMCs) from a small subset of PV and ET patients with and without the JAK2 mutation to assess signaling differences between and within JAK2+/WT patient subgroups and the relevance of those findings to response to in vitro JAK inhibition. MethodsCryopreserved PBMCs from 9 patients with PV/ET (3 JAK2+, 6 JAK2 WT) and 2 healthy control subjects were evaluated. Using p-STAT1, 3, and 5 as readouts, SCNP analysis quantitatively measured basal and induced JAK-STAT pathway activation after modulation with a panel of cytokines including IL-2, IL-3, IL-6, IL-10, IL-27, IFNα, IFNγ, EPO, TPO, and GM-CSF, in the presence and absence of 3 JAK inhibitors (INCB-018424, TG-101348, CP-690550) simultaneously in multiple cell subsets (monocytes, B cells, CD4+ T cells, CD4- T cells, CD34+ cells, and nRBC subsets). ResultsTwo out of 3 JAK2+ patients, but only 2 out of 6 JAK2 WT patients, had circulating CD34+ cells comprising 8-10% of total PBMCs. Interestingly, nRBCs represented 35-73% of total PBMCs in those with a CD34+ population, but only 2-14% of total PBMCs in the patients who lacked circulating CD34+ cells suggesting a more pronounced myeloproliferative trait and/or differential homing of the affected cells in JAK2+ disease. Neither CD34+ cells nor nRBC were detected in the PBMC from the healthy control samples. Assessment of CD34+ JAK/STAT baseline and modulated signaling in the 4 samples with circulating CD34+ cells revealed functional differences between JAK2+ and WT samples. Specifically, modulated p-STAT5 responses in the two JAK2+ samples were generally lower than those observed in the WT samples, but were also distinctly different from each other. This demonstrates the presence of functional heterogeneity in otherwise molecularly defined subgroups. Importantly, examination of basal signaling levels in the presence of JAK inhibitors showed significant decrease in p-STAT5 in only one of the 2 JAK2+ samples, suggesting that the reduced modulated response observed in JAK2+/CD34+ patient samples was not due to elevated basal levels. Of note, signaling within monocytes simultaneously assessed in the same samples demonstrated a similar pattern with JAK2+/CD34+ samples having a reduced JAK2-dependent modulated signal (e.g. GM-CSF, IFNg, IL-3, IL-10) compared with both JAK2 WT/CD34+ and CD34- samples (Fig. 1A) thus suggesting the possibility of using this cell population (well represented in the peripheral blood samples) as a surrogate for CD34+ JAK/STAT signaling measurement. Finally, in vitro treatment of the CD34+ samples with different JAK inhibitors revealed different degrees of sensitivity between JAK2+ and WT samples (with the former being generally more sensitive) together with a marked heterogeneity within the JAK2+ samples themselves (Fig. 1B). [Display omitted] ConclusionsFunctional pathway analysis of a small myeloproliferative neoplasm sample set identified profound heterogeneity in JAK-STAT pathway activity in circulating CD34+ cells and monocytes from JAK2+ and WT patient samples, as well as heterogeneous responses to different JAK inhibitors. Future studies in larger sample cohorts (including BM samples) are needed to assess the potential clinical utility (e.g. biology-based classification of the disease to better inform treatment decision) of these findings. Disclosures:Westfall:Nodality, Inc.: Employment, Equity Ownership. Hawtin:Nodality, Inc.: Employment, Equity Ownership. Longo:Nodality, Inc.: Employment, Equity Ownership. Atallah:Nodality, Inc.: Employment. Cesano:Nodality, Inc.: Employment, Equity Ownership.

OP0139 A distinct subset of CD19-negative plasma cells resides in the human bone marrow

BackgroundPlasma cells (PC) are the unique source of protective and autoreactive immunoglobulin. Mature PC are resting and have downregulated CD20, rendering them refractory to almost all conventional and modern antirheumatic...

Use of flow cytometry during induction chemotherapy to determine outcomes in acute myeloid leukemia.

7104 Background: The use of MRD to predict outcome in AML is controversial. We sought to determine concordance between FC and BM biopsy morphology on day 14 (D14BM) and CR bone marrow (CRBM) specimens and whether MRD detected by FC predicted for inferior outcomes. Methods: We performed a retrospective analysis of adult AML patients treated between 2005 and 2010 with standard induction chemotherapy. Based upon BM morphology and FC, patients were designated as BM+FC+, BM+FC-, BM-FC+, or BM-FC-. Outcomes assessed included induction failure, RFS and OS. Results were adjusted for age at diagnosis, NCCN risk classification, and secondary AML status. Results: Of 287 evaluable patients, 74 had D14 BM and FC results and 98 had CR BM and FC results. Using BM morphology as the gold standard, discordance rates for the presence and absence of disease for the D14 BM by FC were 29% and 23% respectively. Multivariate analysis revealed that patients categorized as BM-FC+ at D14 were more likely to experience induction failure (HR 8.62, CI 0.36-208) and had lower RFS and OS (HR 1.47, 1.95 CI 0.40-5.41, 0.52-7.36 respectively). Analysis of the CR BM samples showed similar results. Conclusions: In this retrospective study there was a high discordance rate between FC and BM morphology on D14 BM and CR BM. While there was a trend toward inferior patient outcomes when disease was detected by FC but not morphology, this was not statistically significant. Limitations of our study include the retrospective nature of the analysis, paucity of patient samples that had a FC evaluation, lack of data to see if positive FC at D14 or CR changed clinician therapy choices and interpretation bias by pathologists as a result of access to FC results during BM morphology interpretation. Larger prospective studies are needed to evaluate whether MRD detected by FC as early as D14 during AML therapy affects clinical outcomes.

EGFR, KRAS, BRAF, and HER‐2 molecular status in brain metastases from 77 NSCLC patients

The aim of this study was to determine the frequency of EGFR, KRAS, BRAF, and HER-2 mutations in brain metastases from non-small cell lung carcinomas (BM-NSCLC). A total of 77 samples of BM-NSCLC were included and 19 samples of BM from breast, kidney, and colorectal tumors were also studied as controls. These samples were collected from patients followed between 2008 and 2011 at Poitiers and Nice University Hospitals in France. The frequencies of EGFR, KRAS, BRAF, and HER-2 mutations in BM-NSCLC were 2.6, 38.5, 0, and 0% respectively. The incidence of KRAS mutation was significantly higher in female and younger patients (P < 0.05). No mutations of the four genes were found in BM from breast or kidney. However, among six BM from colorectal tumors, we identified KRAS mutations in three cases and BRAF mutations in two other cases. This study is the largest analysis on genetic alterations in BM-NSCLC performed to date. Our results suggest a low frequency of EGFR mutations in BM-NSCLC whereas KRAS mutations are as frequent in BM-NSCLC as in primitive NSCLC. These results raise the question of the variability of the brain metastatic potential of NSCLC cells in relation to the mutation pattern.

Impact of Dose Intensification of FluBu2 to FluBu4 on Transplant Related Mortality, Relapse, and Survival After Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia in Remission

Methods: A retrospective review of the patients who relapsed following an allogeneic HCT for a myeloid malignancy at Mayo Clinic in Arizona was performed. The BM sample closest in time prior to relapse was identified and the morphologic, flow cytometric, karyotypic, FISH, PCR and chimerism results were reviewed. Results: Out of 187 patients with a myeloid malignancy, 40 (31 AML, 4 MPN blast crisis, 4 MDS, 1 CML) relapsed at a median of 113 days (range 22-820 days) after HCT. Thirty percent of the relapses occurred after a myeloablative HCT. Donor type was equally distributed between matched related and mismatched/ matched unrelated donors. A cytogenetic and molecular marker with the diagnosis was identifiable in 22/40 and 6/40 patients respectively. 18/40 patients had residual disease present at HCT. 13/40 patients showed an early relapse within the first 90 days. In the remaining patients, time between the last BM and relapse ranged from 38-184 days (median 69). Abnormal results were found only in 9/27 patients in the preceding BM and included abnormal cytogenetic clone identical to diagnosis in 2 patients; abnormal RT-PCR for BCR/ABL in 1, cytogenetic abnormality in donor cell line in 3, and decreased chimerisms in unsorted marrow cells in 3 patients. Conclusions: A 3-month monitoring schedule of BM evaluation using the currently available tools had low sensitivity to predict relapse. Findings that may justify intervention were rare (less than 10% of patients), and were restricted to abnormal cytogenetic and abnormal RT-PCR results. The clinical significance of cytogenetic abnormalities observed in donor cells is not clear. Our findings emphasize that until the optimal surveillance technique and schedule can be defined and standardized, more frequent use of pre-emptive therapy in patients at high risk of relapse may be of benefit.

Single-Cell Network Profiling (SCNP)-Based Classifier to Predict Response to Induction Therapy in Elderly Patients with Acute Myeloid Leukemia (AML): Validation in Two Independent Sample Sets From ECOG and SWOG Trials.

AbstractAbstract 2489▪▪This icon denotes a clinically relevant abstract Purpose:Standard Induction chemotherapy (Ara-C/daunorubicin, 3+7 regimen) in elderly patients (pts) with AML results in approximately 35–45% complete remission (CR) rate, and pts with resistant disease (RD) have a median survival of only 1–3 months. Developing a test for accurate prediction of response to standard induction therapy at the time of diagnosis may help inform treatment selection and improve clinical trial design. Methods:Single cell network profiling (SCNP) is a multi-parameter flow cytometry based approach for simultaneous interrogation of intracellular signaling pathways at a single cell level. SCNP was used to evaluate signaling profiles in leukemic blasts and to develop a classifier (DXSCNP) of response to induction therapy (CR/CRi, i=incomplete) in a Training Set of cryopreserved diagnostic samples (57 PB and 43 BM) collected from 74 non-M3 AML pts, aged 56+ treated with 3+7-based regimens on 4 SWOG clinical trials. SCNP intracellular readouts quantifying apoptotic response after 24 hrs in vitro treatment with Ara-C/Daunorubicin formed the inputs for DXSCNP. Pt and disease characteristics available either at diagnosis, i.e. relevant to induction therapy choices (e.g., age, WBC counts, FAB class, secondary AML, performance status – CLINICAL1), or available after start of induction therapy (CLINICAL2) were used to develop 2 clinical predictors (DXCLINICAL1 and DXCLINICAL2) of CR/CRi in the Training Set. The performance characteristics of these 3 classifiers were then tested separately in BM and PB sample sets. Specifically, classifier validation was performed in 2 independent BM sample sets (A: n=24 BM samples from ECOG E3999 trial; B: n=42 independent BM samples from the same 4 SWOG trials from which the Training samples were derived) and in 1 PB sample set (C: n=53, from the 4 SWOG trials; notably only 24 patients were shared between Set B and C)). The area under the receiver operating characteristic curve (AUROC) was used to measure each classifier’s ability to predict response to 3+7 induction therapy. Out of bag estimates (OOB) of AUROC were calculated using the Training Set, and H0:AUROC=0.5 was tested against HA:AUROC>0.5 for each classifier in the Validation Sets. Results:As shown in Table 1, DXSCNP was a significant predictor of CR/CRi in BM samples. The AUROC for the DXSCNP classifier was 0.81 in the Training Set and 0.76 (p=0.01) and 0.72 (p=0.02) in Validation Sets A and B, respectively. No significant DXCLINICAL1 was identified from the 74 pts in the Training Set(OOB AUROC∼0.5). By contrast the AUROC of DXCLINICAL2 (which included inputs for cytogenetics, Flt3ITD, and NPM1 mutational status) was 0.63 (OOB) in the Training Set, and 0.61 (p=0.18) and 0.53 (p=0.38) in Validation Sets A and B, respectively. Of note, DXSCNP remained significant (p=0.03 and 0.04) when controlling for DXCLINICAL2 in both sample sets. Similar performance of DXSCNPwas observed in different pre-specified subgroups (although of small sample sizes) defined by pt, sample or disease characteristics.Using PB samples, the AUROCs for the DXSCNP classifier were, 0.87 (OOB) in the Training Set and 0.53 (p=ns) in the Validation Set C. However, the performance of DXSCNP in PB samples differed significantly between secondary vs. De novo AML i.e. AUROC= 0.24 vs 0.8 respectively. Of note, DXSCNPperformed similarly for the subset of de novo patients for whom paired BM with PB samples were tested (AUC=0.78 (PB) and 0.74 (BM)). Conclusion:This is the first study describing the successful validation of an SCNP-based classifier to predict response to standard induction chemotherapy in elderly AML pts with performance superior to clinical variables available at diagnosis. A similar SCNP classifier with overlapping cellular biology inputs was previously validated for pediatric AML. These results confirm the ability of quantitative SCNP readouts to provide independent and actionable information on disease biology and pt treatment choices. Independent validation in prospective studies is warranted.Table 1AUROC of DXSCNP, DXCLINICAL1, DXCLINICAL2 in elderly AML BM sample sets.ClassifierAUROCNameDescriptionTraining Set (OOB)Validation Set AValidation Set BDXSCNPSCNP0.810.760.72DXCLINICAL1Factors available at diagnosisxxxDXCLINICAL2CLINICAL1 plus cytogenetics, Flt3-ITD and NPM10.630.610.53x= no model limited to CLINICAL1 variables was significantly predictive in the Training Set. Disclosures:Cesano:Nodality, Inc: Employment, Equity Ownership. Gayko:Nodality, Inc.: Employment, Equity Ownership. Putta:Nodality, Inc.: Employment, Equity Ownership. Louie:Nodality, Inc.: Employment, Equity Ownership. Westfall:Nodality, Inc.: Employment, Equity Ownership. Purvis:Nodality, Inc.: Employment, Equity Ownership. Spellmeyer:Nodality, Inc.: Employment, Equity Ownership. Marimpietri:Nodality, Inc.: Employment, Equity Ownership. Hackett:Nodality, Inc.: Employment, Equity Ownership. Shi:Nodality, Inc.: Employment, Equity Ownership.

Minimal Residual Disease Quantification in Acute Lymphoblastic Leukemia Using High-Throughput Sequencing of Multiple Immunoreceptor Genes Predicts Relapse and Survival After Allogeneic Hematopoietic Cell Transplantation.

AbstractAbstract 2512 Background:Tremendous progress has been made in the management of acute lymphoblastic leukemia (ALL) in children, in part, through the wide use of minimal residual disease (MRD) monitoring to guide therapeutic intensification before and after allotransplantation. Unfortunately, poor accessibility and the high costs of MRD testing have limited its use in the management of adult ALL. A universally applicable MRD quantification method has the potential to revolutionize the management of ALL in adults. Most B- and T-cell ALL patients exhibit clonal rearrangements of one or more immunoglobulin (heavy chain, IGH; light chain, IGK/IGL) or T-cell receptor (beta, B; delta, D; gamma, G) genes. Such rearrangements may be quantified in a mixture of polyclonal B or T cells by massively parallel high-throughput sequencing (HTS), enabling highly sensitive MRD quantification. Methods:Thirty-six allografted ALL patients were selected for this retrospective study based on availability of a diagnostic sample containing leukemic cells, which was necessary for validating the amplification and sequencing method with each disease clonotype. Using Sequenta’s LymphoSIGHT platform, we amplified and sequenced rearranged immunoreceptor (IR) loci from genomic DNA extracted from peripheral blood (PB) or bone marrow aspirates (BM) using V and J segment consensus primers for each IR gene (IGH, TCRB, TCRD, and TCRG) and, in some cases, D segment primers for incomplete IGH-DJ rearrangements. Sequences were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high prevalence in a PB or BM sample at a time of high disease burden. MRD levels were then determined in serial samples of PB or BM and quantified using spiked-in reference sequences. A total of 227 samples with a median 442,672 input genomes (range 8,038 – 7,162,715) were evaluated by IR-HTS. Results:A clonal IGH sequence was identified in 17/36 (47%) ALL patients. Amongst patients who did not have a detectable IGH clonotype, 14/19 (74%) had clonal sequences at one or more other loci, including partial IGH-DJ rearrangements (5/14; 36%), TCRB (4/14; 29%), TCRD (3/14; 21%), or TCRG (6/14; 43%). In total, 31 of 36 (86%) ALL patients had a clonal IR sequence suitable for MRD quantification. In 4 of 5 cases without an identified clonal sequence, only PB, but no BM samples, were available. Twenty patients achieved MRD negativity following HCT and 11 did not. In the MRD negative group, 12/20 patients (60%) ultimately relapsed with a median time to clinical progression of 231 days (range 77–889), whereas 11/11 patients (100%) in the MRD positive group relapsed with a median time to clinical progression of 139 days (range 60–304). The 12 patients who relapsed after achieving MRD negativity showed a median time to molecular disease progression of 93 days (range 59–689 days). Of the 8 patients who maintained MRD negativity following HCT, 7 remain alive at a median 1542 days (range 1133–2557 days). All 22 patients with MRD detected following HCT relapsed and 21 (95%) died (median survival 387 days; range 85–1991 days) (Figure 1). The lead time between molecular disease detection by IR-HTS and clinical relapse was a median 69 days (range 0–207 days) with significant likelihood of MRD detection in a PB sample at least one, three, and six months (each p<0.0001) prior to clinical relapse. Amongst 50 contemporaneous PB and BM samples, 25 (50%) were concordantly MRD negative, 7 (14%) were MRD positive in BM but negative in PB, and 15 (30%) exhibited significantly higher MRD in BM versus PB (median 41-fold higher, range 1.1 – 411-fold). Conclusions:We demonstrate a method for quantifying acute lymphoblastic leukemia MRD in the majority of patients using high-throughput sequencing of multiple immunoreceptor genes. IR-HTS MRD quantification provides valuable lead-time prior to clinical relapse that could provide an opportunity to apply additional therapeutic maneuvers while disease burden is low. Because MRD sensitivity in BM is significantly higher than in PB, marrow assessment may further improve the lead-time for predicting disease recurrence using IR-HTS. The dismal outcome of patients with ALL, particularly when disease burden persists after allogeneic transplant, demands further clinical study of both post-transplant MRD monitoring and novel methods for treating ALL relapse in evolution. [Display omitted] Disclosures:Faham:Sequentia Inc.: Employment. Carlton:Sequenta, Inc.: Employment. Zheng:Sequenta: Employment. Moorhead:Sequenta: Employment. Klinger:Sequenta, Inc.: Employment. Willis:Sequenta, Inc.: Employment.

Fulminant Onset of Acute Leukemia (FOAL) After Total Therapies (TT) for Multiple Myeloma (MM): Absence of MDS Pathological Criteria within 3 Months of Prior MM Follow-up

AbstractAbstract 1458 Background:Cytogenetic abnormalities (CA) in therapy-related myeloid neoplasms (t-MN) usually correlate with nature of previous therapy and clinical presentation. t-MN following prior therapy with alkylating agents and/or radiotherapy are associated with a long latency period (> 3 years) an antecedent myelodysplastic syndrome (MDS) phase, and unbalanced translocations or a complex karyotype. Majority of t-MNs that initially present as acute leukemia (AL) are associated with prior topoisomerase II therapy, a shorter latency period (1–3 years), lack of MDS phase and balanced translocations, commonly involving 11q23/MLL gene. In our experience the usual course of t-MN in MM patients is a prolonged MDS phase with progressive cytopenia, progressing to acute myeloid leukemia (AML) in a subset of patients. Here we report on 7 patients in complete remission (CR) for MM without cytopenia or morphologic evidence of dysplasia on bone marrow evaluation within 3 months prior to presenting with FOAL. One patient developed FOAL within 2.5 months of first documentation of MDS-associated CA (MDS-CA). Methods:Of 3890 patients at the University of Arkansas who had received melphalan-based autograft supported high dose therapy, 69 patients developed t-MN— 63 initially presented with MDS and 13 progressed to AML. Six of the seven patients had no prior MDS-CA or morphological dysplasia, and one with MDS-CA seen <2.5 months earlier, had fulminant onset of AL. All BM specimens at time of FOAL and within one-year period prior to FOAL were evaluated. Metaphase cytogenetic analysis was performed on unstimulated BM specimens using standard techniques. Interphase FISH was performed on BM samples using probes to detect CA commonly observed in MDS and AML. Results:All FOAL patients were treated on Total Therapy (TT) protocols for newly diagnosed MM including 3 on TT2 with thalidomide arm, 3 on TT3A with VTD maintenance, and 1 on TT4 with VRD maintenance. At the time of FOAL all patients were in CR for MM and on maintenance therapy with dexamethasone, thalidomide, lenalidomide, and bortezomib in combination or as single agents. The clinical and laboratory features prior to and at onset of AL are shown in the table.Patient No. Parameters1234567Karyotype at diagnosis62,XXX,-1,-2, +3,-4, -5, -6,-7, +9,+9,-13, -13, der(14)t(11;14) (q12∼13.1;q32) x2, -16, -17, +18, +19,-21, -22[7]/ 46,XX[cp11]42∼44,X,-X, add(6)(q?22), der(12)t(1;12) (q11∼12; q24.3),-13[cp9]/46, XX[cp11]46,XY[20]38∼40,X, -Y,+1, add(1)(p11), der(1;16)(q10; p10),-4,-6, t(8;14) (q24.1;q32),-8,-12, -13,-16 [cp4]/70∼75, idemx2, inc[cp3]/46, XY[cp13]46,XY[20]46,XY[20]46,XY[20]Interval from start of MM therapy126 months63.5 months9.5 months74.5 months114 months123 months66 monthsMM status at FOALStringent CRStringent CRStringent CRStringent CRStringent CRCRStringent CRKaryotype prior to FOAL53,XX,+X,+4,+4, +7,+8, inc[cp3]/ 46,XX[cp38]46,XX[20]46,XY[20]46,XY[20]46,XY[20]46,XY[20]46,XY[20]FISH+7, +8NDNormalNDNDNormalNDLeukemia subtypeALLALLAMLAMLAMLAMLAMLMDS-CA at time of FOAL55,XX,+X,+4,+4, +der(7;9)(q10;q10), +8, del(12)(q24.2), +14,+18,+21 [cp15]/ 46,XX[5]del(20q)46,XY[20]Complex with -5 and -7Complex with -5,-17, and del(11q23)Complex -5 and -746,XY[20]FISH+8, +21del(20q)Normal-5, del(5q),-7del(5q)del(5q), -7, del(7q)NDND= not done Conclusion:In this cohort, MDS-CA (+8, del20q, −7, del7q, −5, del5q and complex karyotype) were detected in leukemic blasts in 5 of 7 patients with abnormalities of chromosomes 5 and 7 most commonly seen. In contrast to clinical and cytogenetic associations described for t-MN initially presenting as AL, none of our patients showed balanced chromosomal translocations involving 11q23, and all but one (patient 3, 9.5m) had a long latency period of 5 to 10yr. Importantly, the absence of MDS-CA on prior bone marrow evaluations in 6 of 7 of our cases indicates that FOAL cannot be predicted based on surveillance BM metaphase cytogenetics or FISH. Studies using gene expression profile analysis of CD34 stem cells are underway to identify molecular alterations leading to FOAL. Results of these studies may improve t-MN surveillance. Disclosures:No relevant conflicts of interest to declare.

Association Between Micro-RNA and Epigenetic Modifiers DNA Methyltransferases (DNMTs), Histone Deacetylases (HDACs) in Multiple Myeloma (MM) and Monoclonal Gammmopathy with Undetermined Significance (MGUS)

AbstractAbstract 3942 Background:Micro RNAs (miRs) are small non-coding RNAs of 19–25 bases in length having the ability to modulate the expression of other genes. MiRs are frequently involved in carcinogenesis and its expression analysis to predict the phenotype of malignancies has been shown. Transcriptional silencing of tumor suppressor genes in cancer cells is often associated with hypermethylation of their promoter CpG islands and histone deacetylation. Three different DNMTs play major roles in establishing and maintaining DNA methylation patterns, DNMT1, 3a, 3b. Histone acetylation is carried out by histone acetyltransferases and acetyl groups can be removed by histone deacetylases (HDACs). Cancer cells often show high expression of epigenetic modifiers DNMTs and HDACs. It has been demonstrated that miR-15a, b miR-16, miR-29 family down-regulate DNMTs and Sp-1 regulating HDACs expression. Many studies evaluate miR expression and its association with chromosomal abnormalities and the prognosis of multiple myeloma (MM), but the relationship between miR and its target gene expression in the patients has not been reported. Methods:MM cell lines and BM samples obtained from 46 of MM patients, 23 of MGUS patients are subjected to the study after informed consent. Normal plasma cells are obtained from the bone marrow samples of 13 patients with malignant lymphoma. Sequential BM samples during the treatment were obtained from 6 patients. RNA and miR were extracted from plasma cells separated by CD138 antibody and magnetic beads. MiR15a, 15b, 16, 29a, 29b, 29c and mRNA expression of DNMT1, 3A, 3B, HDAC 1–11 were quantified by real time PCR with either Taqman-probe or SYBR green. To verify the role of these miRs, miR mimics and miR inhibitor were transduced to MM cell lines with lipofectamine, mRNA expressions of their target genes were analyzed with real time PCR. Results:The expression level of miR15a and 16 was reduced in plasma cells of MM (6.4±3.1, 19.5±7.5) than of MGUS (34.8±15.4, 72.1±36.5) and of normal subjects (74.4±1.5, 25.5±12.9)(p=0.02, p=0.02). MiR 15b, 29a, 29b, 29c expressions were not significantly different among MM, MGUS and normal subjects. Conversely, DNMT1, 3A were elevated in MM (7.2±4.5, 8.5±3.3) than in MGUS (0.8±0.2, 1.7±0.6) and normal subjects (0.4±0.3, 2.1±1.8) (p=0.03). DNMT3B expression was not different among subjects. HDAC3, 7, 9 expression were elevated in MM (0.9±0.4, 6.7±2.8, 138±59) than in MGUS (0.46±0.24, 1.4±0.7, 46.8±43.3) (p=0.03, p=0.001, p=0.001) and normal subjects (0.18±0.17, 1.1±0.97, 20.8±9.9) (p=0.01, p=0.017, p=0.012). HDACs expressions were not significantly different in between plasma cells of MGUS and of normal subjects. In the MM cell lines, miR 29a and 29b expression were inversely correlated with DNMT1 mRNA expression (r=0.96, p=0.0003: r=0.86, p=0.014). In the patient samples, DNMT1 was inversely correlated with miR15a, miR15b, miR16, miR29a, miR29b (r=-0.435, p=0.003; r=-0.341, p=0.02, r=-0.332, p=0.03, r=-0.419, p=0.005, r=-0.407, p=0.006), DNMT3A was inversely correlated with miR15a, miR29a, miR29b, miR29c (r=-0.365, p=0.02; r=-0.315, p=0.04; r=-0.371, p=0.01; r=-0.315, p=0.04), DNMT3B was inversely correlated with miR15a, miR15b, miR29a, miR29b (r=-0.418, p=0.005; r=-0.385, p=0.01; r=-0.353, p=0.02; r=-0.358, p=0.02). HDAC1, 3, 7 were inversely correlated only with miR15a (r=-0.25, p=0.039, r=-0.27, p=0.025, r=-0.25, p=0.038). Four of 6 patients’ plasma cells showed decreasing miR 15a, 15b, 16 and 29 families and increasing DNMT1 expression during the treatment. Those patients acquired drug resistance. One patient’s sample whose miR expression increased during the treatment showed reduced DNMT1 expression. The effects of transduction of miR mimics and inhibitors to MM cell lines were various among cell lines, miR16, 29a, b showed ability to reduce DNMTs expression. Conclusion:We found significant reduction of miRs expression and elevation DNMT1, 3A and HDAC3, 7, 9 in MM and possibly associated with MM progression. Negative correlation with the expression level of the DNMTs, HDACs and miRs in patients' samples were seen. The sequential samples during the treatment and transduction experiment in vitro suggest that miRs regulate epigenetic modifiers expression It has been reported that miRs expressions themselves are epigenetically controlled, thus these factors may affect each other and be related to MM progression. Disclosures:No relevant conflicts of interest to declare.

The Role of the ABL1/YAP1/P73 Axis in Prevention of DNA Damage-Mediated Apoptosis in Multiple Myeloma

AbstractAbstract 725 Background:Genome integrity plays a crucial role in the development of normal plasma cells to eliminate aberrant ones. Multiple myeloma (MM) is a plasma cell malignancy characterized by complex heterogeneous cytogenetic abnormalities. MM cells show constitutive DNA Damage Response (DDR) and activate compensatory mechanisms to prevent DNA-damage mediated apoptosis. Here we define the molecular mechanisms of these protective effects. Methods:A panel of 15 MM cell lines was used. Blood and BM samples from healthy volunteers and MM patients were obtained after informed consent and subjected to Ficoll-Paque density sedimentation to get mononuclear cells (MNCs). Patient MM cells were isolated from BM MNCs by CD138-positive selection. Lentiviral delivery system was used for expression and knock-down of YAP1 in KMS-18, KMS-20, MM.1S and UTMC-2 MM cell lines. The biologic impact of YAP1 phenotype was evaluated using cell growth, viability and apoptosis assays. Results:We confirmed that a wide range of MM patient cells and MM cell lines have markers of constitutive DDR, including phosphorylation of H2A.X, ATM, ATR, Chk2 and Chk1, assessed by western blot analysis and immunofluorescence. However, these MM cells do not show basal level of apoptosis. Specifically, cleaved forms of caspase 3 and PARP are lacking in non-treated cells, and the absence of co-expression of cleaved caspase 3 with phospho-H2A.X by immunofluorescence confirms that phospho-H2A.X positive cells are viable cells. Since DDR is present in both p53-wild-type (wt) and p53-mutated cell lines, we examined whether ABL1/YAP1/p73 axis represents an alternative and crucial pathway to avoid DNA-damage mediated apoptosis. Indeed, ABL1 is up-regulated and predominantly localizes in the nucleus, a potential apoptotic stimulus, in MM cells, as assessed by western blot and immunofluorescence. To define if ABL1 nuclear localization was triggered by DDR, we treated MM cells with a specific ATM inhibitor (Ku55933) and a DNA damaging agent, doxorubicin. Inhibition of DDR in both p53 wt cell lines (MM.1S and H929) and p53 mutated cell lines (UTMC-2, JJN-3 and KMS-20) causes ABL1 cytoplasmic retention, while doxorubicin increases ABL1 nuclear translocation. Co-treatment with doxorubicin and ABL1 inhibitor STI-571 rescues MM cells from doxorubicin-mediated cell death. In particular, apoptotic cells decrease from 47.2% to 21.5% in U266, from 55.3% to 12.4% in MM.1S, and from 57.9% to 19.1% in UTMC-2 cells in response to combination treatment. To delineate the molecular mechanisms whereby MM cells repress ABL1 pro-apoptotic function, we focused on YAP1, a downstream target of the Hippo pathway involved in ABL1 cascade. We explored a large dataset of aCGH data on MM patients and discovered that YAP1 genomic locus (chr. 11q22) is deleted, associated with BIRC2 and BIRC3 in 11% of patients. This genetic abnormality was also found in KMS-18 and KMS-20 cell lines. Although YAP1 expression was normal in peripheral blood MNCs (PBMCs), its expression was decreased in the majority of patient MM cells and MM cell lines regardless of the presence of focal deletion. Importantly, low expression of YAP1 is associated with poor prognosis of MM patients. To further delineate the biologic significance of YAP1 in MM cells, we re-expressed pLENTI4-YAP1-EGFP in MM cell lines with either YAP1 deletion (KMS-18, KMS-20) or YAP1 low expression (MM.1S). We also silenced YAP1 using two different specific shRNAs in UTMC-2 MM cell line. As expected, YAP1 re-expression reduces cellular growth and increases apoptosis in all cell lines tested (25.7%, 37.1% and 32.3% apoptotic KMS-20, KMS-18 and MM.1S cells, respectively), condition that was further enhanced by doxorubicin treatment. Previous studies have shown that P73 is expressed in MM even though at low levels and we here show that they inversely correlate with YAP1 protein expression. Importantly, YAP1 re-expression increases p73 stability and promotes transcription of p73-target genes including BAX, PUMA and p21. In contrast, UTMC-2-YAP1−/− cells show improved survival with lower levels of basal apoptosis and higher resistance to treatment with bortezomib or doxorubicin. Conclusion:YAP1 mediates a strong apoptotic signal for MM cells. Thus, activation and/or overexpression of YAP1 represent a novel therapeutic strategy to improve outcome of patients with MM. Disclosures:Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder and Scientific Founder, Scientific Founder and Scientific Founder Other.

Blockade of the Hedgehog Signaling Pathway by the Novel Agent NVP-LDE225 Induces Differentiation, Prevents De-Differentiation and Inhibits Proliferation of Multiple Myeloma Stem Cells in Vitro

AbstractAbstract 3950Chemotherapy-resistant cancer stem cells are a major cause of relapse. It is considered that the small population of CD38++ cells which also exhibit “side population” (SP) characteristics after Hoescht 33342 staining contain chemotherapy resistant multiple myeloma (MM) stem cells. The hedgehog (Hh) signalling pathway is a key regulator of stem cell proliferation and differentiation. A range of Hh inhibitors are currently under development as therapeutics. Our aim was to determine the effect that NVP-LDE225, a novel Hh pathway inhibitor has on MM stem cells.NVP-LDE225 was kindly supplied by Novartis. Side population (SP) cells, considered to contain the MM stem cells, were detected after Hoescht 33342 staining using a BD ARIA II flow cytometer, Proliferation was monitored with CFSE tracking and viability with propidium iodide. MM cell lines RPMI8226, KMS-11, OPM2, U266 and primary MM cells were included. Expression of Hh pathway proteins PTCH1, Smo and Gli1 was determined by flow cytometry. Bone marrow samples were obtained from patients with myeloma after informed consent.CD38++ SP cells were identified in all 4 MM cell lines tested and 90% (26/29) of MM BM samples with the percentage of SP cells ranging from 0 to 9.5% of CD38++ cells. There was no correlation between %SP and %CD38++ and no significant difference in %SP during therapy. PTCH1 expression was higher on SP cells of KMS-11, OPM2 and U266 than non-SP cells (80.5% vs 51.7%) suggesting that the canonical Hh pathway was upregulated in CD38++ SP cells. PTCH1 expression varied from 0.1 – 87% on patient’s CD38++ cells. In vitro dose response curves demonstrated increased killing of plasma cell lines above 1μM NVP-LDE225. The IC50 was 4.7uM for KMS-11 SP cells and 3.4uM for KMS-11 non-SP cells. During culture of flow-sorted CD38++ SP+ and non-SP cells (RPMI 8226 and KMS11), fluctuations in %SP suggest that SP cells differentiated to non-SP cells but also that the SP phenotype is unstable. This is supportive of the concept that the stem cell compartment is in a state of flux. The presence of NVP-LED225 caused a 90% inhibition of non-SP returning to SP and induced an additional 79% differentiation of SP to non-SP KMS11 cells on day 2. Proliferation studies using CFSE tracking of sorted SP cells demonstrated a 34% reduction in proliferation after exposure to NVP-LDE225. Studies with 9 primary human bone marrow samples from patients with MM confirmed the data from cell lines demonstrating that differentiation was significantly induced (t=2.78; p<0.0275) and de-differentiation was blocked.The studies confirm there is flux, to and from the SP and presumably MM stem cell compartment, that the canonical Hh pathway is upregulated in MM SP cells, and that the Hh pathway inhibitor NVP-LDE225 can induce SP differentiation, block de-differentiation, inhibit SP proliferation and at high concentrations is cytotoxic to all cells including MM stem cells. NVP-LDE25 could synergise with other maintenance therapy to reduce drug resistance by inducing the differentiation of chemo-resistant MM stem cells. Disclosures:No relevant conflicts of interest to declare.

Minimal Residual Disease After Allogeneic Stem Cell Transplantation: A Comparison Among Multiparameter Flow Cytometry, Wilms Tumor 1 Expression and Chimerism Status (Complete vs Low-Level Mixed Chimerism) in Patients with Acute Leukemia

AbstractAbstract 1971 Introduction.Relapse represents the main cause of treatment failure after allogeneic stem cell transplantation (allo-SCT). Thus, monitoring of minimal residual disease (MRD) in allografted patients allows an early detection of recurrence and a subsequent intervention prior to florid relapse. Multiparameter Flow Cytometry (MFC), chimerism, cytogenetics and molecular analysis have been widely used for this purpose, although the gold standard needs to be established yet. The evaluation of fusion transcripts represents the most sensitive method but more than 65% of patients do not demonstrate a molecular target. WT1 mRNA is over expressed in > 90% of patients with acute myeloid leukemia (AML) but it is not so expressed in acute lymphoblastic leukemia (ALL). On the MFC analysis instead, the complexity of maturational patterns and phenotypic shifts after therapy may underestimate residual leukemic cells. Finally, previous papers reported that a state of mixed chimerism (MC) in RT-PCR may identify higher risk of relapse in patients with AML. However, the test has a low sensitivity (from 0,1% to 1%) and recipient cells are not necessarily linked to disease recurrence. Hypothesizing the presence of blast cells in the low-level mixed chimerism (MC, 1%<MC<5% of autologous cells) but not in the complete chimerism (CC, no evidence of autologous cells) status, we used these two ranges as markers of positive and negative MRD, respectively. The aim of our study was twofold. Firstly, to assess the overall agreement among chimerism, MFC and WT1 mRNA methods in monitoring MRD. Secondly, to investigate whether such methods were associated to patient’ s relapse-free survival. Methods.Fresh BM samples from 24 patients (17 AML and 7 ALL) in both morphological CR and CC or low-level MC status after allo-SCT were investigated. MRD with MFC, WT1 mRNA expression and chimerism analysis was evaluated at different time points: +1, +3, +6,+12,+18,+24 months after allo-SCT. The immunophenotypic analysis was performed using a six-color combinations and acquiring 250.000 events. RQ- PCR to test WT1 mRNA expression was made according to the standardized and quality-controlled method. Chimerism studies were performed with a multiplex amplification of 16 (STR). The agreement between two methods in monitoring MRD after allo-SCT was assessed by Kappa statistic. Moreover, time-to-event analyses were performed using Cox proportional-hazard models with time-dependent covariates. Risks were reported as hazards ratios (HR) along with their 95% confidence interval (95% CI). Results.Comparisons among results of MFC, RQ-PCR for WT1 mRNA and Chimerism were performed in all 67 serial samples obtained from 24 patients. A significant moderate agreement between MFC and WT1 mRNA evaluations was found (k= 0.463, p<0.001) as well as fair agreement between chimerism and MFC (k= 0.284, p=0.009) and chimerism and WT1 mRNA (k= 0.197, p=0.073). These results suggested a concordance among the three investigated techniques. In particular, the low-level MC would well detect the presence of leukemic cells, since the proportion of positive samples for MFC was not statistically different to the proportion of positive samples for WT1 mRNA within samples with low-level MC. Indeed, among 12 samples with low-level MC, 9 samples (75.0%) were also positive for MFC and 7(58.3%) were also positive for WT1 mRNA. Cut-off of 0.1% and 0.01% for MFC, 83.5 × 104 ABL for WT1 mRNA and 96%-99% of donor cells for chimerism were selected. The median follow-up times for relapse-free was 12.8 mths and the overall estimated relapse-free survival after 36 mths was 66.5%. At the end of follow- up, 5 patients relapsed and 4 patients died. Although not significant, the detection of a positive MRD for all methods were associated to a higher incidence of relapse than the negative MRD. Similar HRs were observed in the analysis of MFC(HR = 6.55, 95%CI= 0.71–60.17, p=0.096) and WT1 mRNA (HR= 7.17, 95% CI=0.77–66.42, p=0.083) whilst a slighter HR was found for the chimerism analysis (HR= 0.40, 95%CI= 0.04–4.44, p=0.456). Conclusions.According to our study MFC, WT1 mRNA and CC or low-level MC displayed an overall agreement in monitoring MRD. In particular, the agreement on the low-level MC may suggest the presence of leukemic cells. The detection of a positive MRD by MFC and WT1 mRNA similarly identified higher risk of relapse in patients with acute leukemia (AL) undergone to allo-SCT. Disclosures:No relevant conflicts of interest to declare.

Design and Application of a Novel PNA Probe for the Detection At a Single Cell Level of BCR-ABL T315I Mutation in Chronic Myeloid Leukemia Patients

AbstractAbstract 3732 Background:The BCR-ABL T315I mutation at the gatekeeper residue is frequent in advanced phases of chronic myeloid leukemia (CML) and is one of the main cause of resistance to Tyrosine Kinase Inhibitors (TKI) by disrupting important contact points between the drugs and the enzyme. Although this mutation can be detected by different techniques and at different levels of the mutated clone, the prognostic significance of the absolute amount of the mutated allele is widely unknown.The aim of the study was to develope a novel assay based on peptide nucleic acid (PNA) technology coupled to immuno-fluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of T315I mutation thus improving both the diagnostic resolution and the knowledge on the behaviour of the mutated clone. Methods:We designed a fluorescently-labelled PNA probe, coupled to FISH technology, which allows to distinguish with a high degree of specificity between CD34+ progenitor stem cells harbouring T315I mutation or the wild type form of BCR-ABL. CD34+ cells were enriched from CML patients who were positive for T315I mutation, or from patients who lost T315I mutation after ponatinib treatment. In addition we tested CML patients without T315I mutation as control and BM samples from healthy donors to establish the specificity of the method. CD34+ progenitors cells were enriched by MACS and then cytospun onto slides and hybridized with human species-specific fluorescinated 15 base pairs (bp)-long oligo-PNA, specifically recognizing the BCR-ABL T315I sequence. Slides were analyzed by fluorescence confocal microscopy. Results:We found, with a rather wide variability occurring among patients, a percentage of mutated CD34+ cells ranging from 3 to 90%. In addition these data indicate that fluorescinated BCR-ABL T315I/PNA probe displays a very high specificity towards a single base-pair mismatch. Interestingly, when evaluating the presence of T315I positive cells collected from a patient who lost the mutation (evaluated by sequencing) after ponatinib therapy and acquired T317 mutation a small amount of T315I positive CD34+ cells were still present. This percent did not exceeded 2% of the total CD34+ cell population. Importantly, the lack of positivity detected in CD34 positive cells from CML patient without mutations or in 20 healthy subjects demonstrates a high specificity of this method. Conclusions:BCR/ABL T315I/PNA probe method displays high specificity and reliability in discriminating cell subpopulations harbouring the mutation. In addition, it allows to analyze the CD34+ population at the single cell level and to monitor the behaviour of the clone at the stem/progenitor cell level. This approach allows to monitor longitudinally the evolution of the mutated population over time, the response to specific drugs active against T315I mutants and to characterization the mutated stem/progenitor cell compartment in patients with CML. Disclosures:Saglio:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Combined Use of WT1 and Flow Cytometry Monitoring Can Promote Sensitivity of Predicting Relapse After Allogeneic HSCT without Affecting Specificity.

AbstractAbstract 3065 Introduction:Current studies suggest that monitoring the minimal residual disease (MRD) of patients with malignant hematological disease after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is helpful for screening patients at high risk of relapse. WT1 and leukemia-associated aberrant immune phenotypes (LAIPs) were the major MRD parameters used to predict leukemia relapse after allo-HSCT. The aim of this study was to first evaluate the clinical value of various positive MRD standards for accurately predicting relapse based on WT1 and flow cytometry (FCM) data in a large sample of adult patients with actue leukemia (AL) in a single transplant center. Methods:In total, 824 AL patients treated with non-T-cell-depleted allo-HSCT at the Peking University Institute of Hematology from January 1, 2006 to November 30, 2011 were enrolled in this study. BM samples from patients were obtained at regular time points: +1 month, +2 months, +3 months, +4.5 months, +6 months, +9 months, +12 months and every 6 months thereafter for the MRD investigation after HSCT. WT1 expression was evaluated using TaqMan-based RQ-PCR technology and LAIPs were detected using 4/7-color FCM. At least one positive MRD marker, including WT1 or LAIP, was detected before transplantation in 90.8% (748 of 824) of the subjects. Positive FCM could be found in all 748 patients. Both FCM+ and WT1+ were detected before transplantation in 58.7% (484/824) of subjects. We compared the sensitivity and specificity of diverse, multiple-criteria MRD forecasting standards including one WT1+, two consecutive WT1+, one FCM+, two consecutive FCM+, concurrent WT1+ & FCM+, concurrent WT11.0+ & FCM+, MRDco+ and MRDco1.0+, based on WT1 and FCM assays. Results:Higher sensitivity was achieved without a loss of specificity in total 824 patients as well as in 748 patients with WT1+ or FCM+ before transplant when MRDco+ was used as the positive MRD standard (Table 1). Similar results were observed, even in 484 patients with acute myeloid leukemia (AML, n=308) and acute lymphoblastic leukemia (ALL, n=176) who had both abnormal WT1 and LAIPs values before transplant (Table 1). A multivariate analysis that included age, sex, disease status (CR1/CR2), donor type, number of chemotherapy courses before CR1, and MRD status (including the different MRD standards mentioned above) after transplantation showed that MRDco+ was an independent risk factor to predict leukemia relapse after allo-HSCT; it had the most powerful contribution to leukemia recurrence after transplant in both AML (HR=12.54, 95% CI: 7.01–22.42, P<0.001) and ALL (HR=12.84, 95% CI: 6.05–27.25, P<0.001) compared with other MRD standards. Conclusions:The combined use of FCM and WT1 monitoring were able to distinguish between patients with low and high risk of relapse. Diverse standards of positive MRD raised in this work were conductive to guide different interventive measures. Further studies should focus on exploring the new better parameters with both high sensitivity and specificity in predicting the recurrence of leukemia after allo-HSCT.Table 1Sensitivity and specificity of combinative MRD criteria for predicting leukemia relapse in different groups after allo-HSCT.GroupsSensitivitySpecificityRelapse ratep valueYouden's indexAll subjectsMRDco+41.3%97.8%81.0% vs 12.2%<0.0010.391n = 824MRDco1.0+31.0%99.3%90.6% vs 13.9%<0.0010.303n = 748MRDco+43.5%97.9%82.2% vs 11.6%<0.0010.414MRDco1.0+34.1%99.2%90.4% vs 13.1%<0.0010.333AMLMRDco+50.0%97.2%79.4% vs 9.9%<0.0010.472n = 308MRDco1.0+35.2%99.2%90.5% vs 12.2%<0.0010.344ALLMRDco+37.8%99.3%93.3% vs 14.3%<0.0010.431n = 176MRDco1.0+35.1%99.3%92.9% vs 14.8%<0.0010.344 Disclosures:No relevant conflicts of interest to declare.

Analysis of Bone Marrow Microenviroment Factors As Early Markers of Response in Patients with Newly Diagnosed Bcr-Abl Positive CML in Chronic Phase Treated with Nilotinib.

AbstractAbstract 2795In the era of molecular target therapy, whereas Imatinib has shown an overall survival rate of 85% and an estimated event-free survival of 55% in a 8 year result update of IRIS trial, several in vitro data have confirmed that Ph+ stem cells housed within BM niches are resistant to TKI treatments. Second generation TKI Nilotinib has been designed with improved target specificity over Imatinib. Its efficacy in the treatment of patients with CML who are resistant to or intolerant to Imatinib led to the registration of a clinical trial CAMN107EIC01, a phase IIIb, multicentre, open-label study applying Nilotinib in the treatment of newly diagnosed CML. The aim of the current study (CAMN107EIC01 sub-study N10) is to define BM microenvironment markers that nurture and determine leukemic stem cell fate in the BM niches of Nilotinib treated patients. We enrolled 37 patients involving 21 Italian centers, from whom written informed consent has been obtained for sub-study N10. Patients have been monitored by Real Time RT-PCR (RT-qPCR) for the expression of the fusion mRNA BCR-ABL, as specified by the core protocol. Major Molecular Response (MMR) is defined as detectable disease ≤0.1% BCR-ABL according to the international scale (IS). Plasma and mononuclear cells have been collected from BM and PB samples of the enrolled patients at the screening visit (V0) and after 3 months of treatment (V4). We purified total RNA from BM and PB mononuclear cells of the enrolled patients to screen by RT-qPCR the expression of 10 genes (ARF, cKIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the regulation of the stemness and survival signaling of hematopoietic stem cells. RT-qPCR results were normalized by the expression of ABL mRNA (Normalized mRNA copy Number: NCN). Moreover, we evaluated by multiplex ELISA assay BioPlex, the BM plasma concentration level of 20 cytokines (IL1a, IL1b, IL3, IL6, IL7, IL8, IL10, IL12, IL15, G-CSF, M-CSF, SCF, SDF1, TRAIL, HGF, PDGFbb, GM-CSF, MIP-1a, TNFa and VEGF), known to be key factors in the interaction of Ph+ stem cells to BM microenvironment. The interim analysis of MMR until the 12th month is available in 26 out of 37 enrolled patients. We observed that MMR is achieved during the first 12 months of treatments in 17 out of 26 (65%) patients. Molecular analysis showed that the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) and cytokine signaling (SET) were significantly down-regulated at V0 in PB mononuclear cells of patients achieving MMR in comparison to cells from non-responding patients (4vs21 NANOg NCN, p=0.05; 8vs32 SET NCN, p=0.05). These data were also confirmed on BM patient’s sample. Moreover, we investigated the concentration level of the above-mentioned soluble factors in BM plasma samples of all 37 enrolled patients at both V0 and V4. We observed that the BM plasma level of several cytokines produced by leukemic cells significantly decreased during the first 3 months of Nilotinib treatment: IL3 (54vs3ng/ml; p=0.02), M-CSF (56vs12 ng/ml; p=0.005), SCF (170vs104 ng/ml; p=0.007), HGF (7565vs705 ng/ml; p<10-5), IL10 (67vs28 ng/ml; p=0.02), IL12 (167vs73 ng/ml; p=0.03) and VEGF (1326vs404 ng/ml; p= 0.0003). Whereas the plasma level of a stroma-derived cytokine SDF1, playing an important role in regulating the retention of hematopoietic progenitor cells within the BM microenvironment, was significantly increased at V4 respect to V0 (171vs390 ng/ml; p= 0.0004). Also, we observed that SCF and HGF concentration levels were significantly lower in the plasma of patients at V4 that achieved MMR compared to those in non- responding patients (71vs134 ng/ml, p=0.02; 421vs737 ng/ml, p=0.01; respectively). These data indicate that expression analysis of genes involved in cell pluripotency (NANOg) and cell signaling (SET), as well as measurement of the plasma concentration level of several soluble factors produced by leukemic progenitor cells and stroma cells (SCF and HGF) may assist in the early prediction of molecular response in patients treated with Nilotinib. Moreover, further studies need to be conducted to evaluate the functional role of stroma secreted cytokines, such as SDF1 and VEGF, in the regulation of TKI responsiveness in these subset of CML patients, since it may be plausible that the same factors that modulate hematopoiesis promote also leukemogenesis, enhance blast survival and make them resistant to treatment within the BM microenvironment. Disclosures:Paratore:Novartsi: Employment. Galimberti:Novartis: Employment. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Prenatal Origin of Monosomy 7 in Very Young Children.

Abstract 2557Monosomy 7 is a common chromosomal abnormality in adults with myelodysplasia (MDS) and AML, seen most frequently in older persons. It is believed to be one of a series of stochastically acquired genetic anomalies that accumulate over many years to complete the process of leukemogenesis. MDS in general and monosomy 7 in particular are rarely seen in children, although cases in very young children are reported. Monosomy 7 arising in very young children likely has a different etiology and may contribute differently to leukemogenesis, as the young age of the children does not allow sufficient time for stochastic acquisition of mutation in the same way as in elderly persons.A proportion of young children with monosomy 7 have an associated DNA instability disorder such as Fanconi anemia (FA) or Dyskeratosis Congenita, and this genetic background likely plays a key role in the evolution of malignancy. In a few reported cases, the occurrence of monosomy 7 is familial, and in a smaller number of cases yet, the clone may resolve spontaneously or wax and wane, as the associated pancytopenia waxes and wanes.We report 5 young children with monosomy 7 in whom we have explored the origins of monosomy 7. In case 1, who presented with MDS and monosomy 7 at 2 years of age, a stored cord blood was available for analysis. The sample was flow sorted and FISH for monosomy 7 was performed on different populations. The monosomy 7 clone was present in approximately 10% of the cord blood cells overall, and the same clone was found in the most primitive population, i.e. CD34+CD38- cells, suggesting in utero origin of this abnormal clone in a very early precursor or stem cell population (Table 1). Similar analyses done on BM or PB samples of 4 additional young patients with monosomy 7, with age range of 4 months to 42 months (3 ½ years)(median 22.5 months), also showed the abnormal clone to be present in CD34+ cells, supporting our hypothesis that monosomy 7 arises in early precursors or stem cells (Table 2, below). In fact, we were able to confirm the presence of monosomy 7 in the CD34+CD38- population in one of these additional cases for which sufficient material was available. Interestingly, in one patient (#4) where we had sequential samplings nine months apart, total BM showed a decrease in the percentage of cells with monosomy 7, from 2.6% to 1.1%. However, in sorted CD34+ cells, we detected a nearly 10 fold increase in cells with monosomy 7 (Table 2). Clinical significance of this finding remains to be determinedIn summary, our preliminary studies provide proof of concept that in children, the abnormal monosomy 7 clone is present in the hematopoietic stem cell compartment, and can arise in utero in the absence of any known genetic susceptibility syndrome.Table 1Patient 1 (UCB)FACS Analysis: % of Cord Blood Cells% Monosomy 7 by FISHCD3 (T cells)23.1%6/66 (9.1%)CD 19 (B cells)9.1%1/44 (0.02%)CD 14 (monocytes)4%1/8 (12.5%)CD 16 (granulocytes)21.5%0/20 (0%)CD341%70%CD34+CD38- (primitive progenitor)NA124/136 (91.2%)CD34+38+19-7- (myeloid progenitor)NA8/13 (61.5%)Table 2Patient # (BM or PB)% of total sample% Monosomy 7 by FISH% of total sample% Monosomy 7 by FISH% of total sample% Monosomy 7 by FISH% of total sample% Monosomy 7 by FISH% of total sample% Monosomy 7 by FISH% of total sample% Monosomy 7 by FISHCD3 (T cells)CD 19 (B cells)CD 14 (monocytes)CD 16 (granulocytes)CD34+38+ or CD34+ ( progenitor)CD34+CD38- (primitive progenitor)2 BM27.00.41.910.95.946.411.316.91.277 (34+)NANA3 BM40.32.56.61.72.49415.992.40.2478.3 (34+).01NA4-1 BM37.4013.303.75.915.03.83.91.31.72.84-2 BM13.029.540.4035.60.40.212.2 (34+)NANA5 PB21.81.53.4NA22.41009.1100NANANANA Disclosures:No relevant conflicts of interest to declare.

Risk Factors for Donor-Type Aplasia After Bone Marrow Transplant in Children with Acquired Bone Marrow Failure Syndrome

Abstract 3474Graft rejection is a major cause of aplasia after bone marrow transplant (BMT) in patients with bone marrow failure syndrome (BMFS). However, we have experienced several patients with acquired BMFS who developed persistent aplasia with full donor chimerism after BMT (donor-type aplasia).To clarify risk factors for donor-type aplasia, we reviewed the clinical data of 58 children (male, n = 36; female, n = 22) with acquired BMFS who received allogeneic BMT at Nagoya University Hospital and Japanese Red Cross Nagoya First Hospital between January 1997 and December 2010. The median age at diagnosis was 10 (range, 1 – 20) years. The median interval between diagnosis and BMT was 30 (range, 0.70 – 105) months. Twenty-five of the 58 patients received > 20 units of red blood cell (RBC) transfusions. We retrospectively reviewed BM smears and samples of trephine biopsies according to the 2008 WHO classification. Of the 58 patients, 26 and 32 were diagnosed with aplastic anemia (AA) and refractory cytopenia of childhood (RCC), respectively. Twenty, five and 33 patients received BMT from HLA-matched and mismatched related donors and unrelated donors, respectively. Conditioning regimens consisting of cyclophosphamide (CY), antithymocyte globulin (ATG) with or without TBI and of fludarabine, CY, ATG and TBI (fludarabine regimen) were administered to 38 and 18 patients, respectively. Marrow grafts (median dose, 4.2 ×108 nucleated cells/kg) were not T cell–depleted. Prophylaxis against graft versus host disease (GVHD) consisted of cyclosporine/methotrexate (MTX) for 26 patients and tacrolimus/MTX for 32 others. Donor-type aplasia was defined as hypocellular BM with persistent cytopenia (hemoglobin < 8 g/dl; neutrophils < 1 ×109/L or platelets < 50 ×109/L) for > 6 months after achieving engraftment with full donor chimerism. We evaluated the following variables to determine risk factors for donor type aplasia; age at BMT, sex, BM morphology at BMT, year of BMT, period between diagnosis and BMT, units of RBC transfusion, history of immunosuppressive therapy (IST) before BMT, HLA disparity, BM cell dose, conditioning regimen, GVHD and viral infection.Eleven (19%) of the 58 children developed donor-type aplasia. All received BMT after 2003 and had the BM morphology of RCC. Three and five of them received BMT from HLA-mismatched and unrelated donors, respectively. Fludarabine regimens were administered to eight patients. Seven patients received ganciclovir to treat CMV reactivation and two received rituximab to treat Epstein-Barr virus reactivation before aplasia developed. The onset of donor-type aplasia ranged from 46 – 710 days after BMT. Donor-type aplasia continued for over three years in five patients. Eight patients required transfusions with either RBC or platelets or granulocyte stimulating factor (G-CSF). Donor-type aplasia improved in five patients within 203 – 1789 days. All patients with donor-type aplasia remained alive for 20 – 103 months after developing donor-type aplasia. Univariate analysis revealed BM morphology, year of BMT and the fludarabine regimen as risk factors for donor-type aplasia and multivariate analysis confirmed these findings. The cumulative incidence (CI) of donor-type aplasia was significantly higher in children who received the fludarabine regimen (45%) or had RCC (35%) than in those who received a non-fludarabine regimen (5%) or had AA (0%) (p = 0.001, respectively). The CI of donor-type aplasia was significantly higher in children with RCC who received the fludarabine regimen (62%) than in those who did not (15%). In contrast, none of the children with AA developed donor-type aplasia irrespective of the fludarabine regimen.The fludarabine regimen was identified as a risk factor for donor-type aplasia in children with RCC. Children with RCC should not be conditioned with the regimens used for children with AA. Disclosures:No relevant conflicts of interest to declare.