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Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma. Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying tFL are unclear. Here we performed the first profiling of expression of microRNAs (miRNAs) in paired samples of FL and tFL and identified 5 miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly downmodulated in all examined tFLs (∼3.5-fold), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins, which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as B-cell receptor and NF-κB signaling in malignant B cells. We revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its high-grade transformation.

The Czech National Diamond-Blackfan Anemia (DBA) Registry currently consists of 51 patients; in 36 of them ribosomal protein (RP) mutations have previously been identified. Targeted sequencing of genes encoding RP, exomes and transcriptomic sequencing and/or array CGH were performed in newly diagnosed DBA patients and in patients without a causative mutation. Two novel mutations in RPS7 and RPL11 were discovered in 5 individuals from two families. Aheterozygous mutation g[3580153G>T] in exon 6 of a gene coding for RPS7, leading to V134F substitution, was found in an 18-year-old female who was transfusion dependent during infancy, responded to steroids and is currently in remission. The identical mutation was detected in the patient's mother and older sister, who both are asymptomatic with borderline hemoglobin (Hb) levels, mild macrocytosis with no active treatment. A functional study using cellular models revealed that RPS7-deficient MRC-5 fibroblasts transiently expressing RPS7-V134F exhibit altered protein synthesis, defective ribosomal RNA processing, increased nucleolar and ribosomal stress. This was evidenced by overexpression and translocation of p53 protein to the nucleus in RPS7-V134F but not RPS7 wild type transfected cells. These data are consistent with previously reported cellular phenotypes in DBA. In addition, increased levels of erythrocyte adenosine deaminase (e-ADA) were detected in all three family members with V134F RPS7 mutation (patient: 4.2±0.8 IU/g Hb; mother: 3.5±0.2 IU/g Hb; sister: 4.6±0.3 IU/g Hb; reference range: 0.8-2.5 IU/g Hb). Recently it was shown that ribosomal insufficiency increases oxidative stress in red blood cells (RBC) of DBA patients. In this family, increased levels of reactive oxygen species (ROS) were detected by flow cytometry only in patient's RBC, but not in RBC of her asymptomatic mother and sister. Consistently, only the patient showed increased anti-oxidative defense parameters compared to the controls and her mother and sister. Markedly elevated levels of reduced glutathione (GSH) and glucose 6-phosphate dehydrogenase (G6PD), two essential antioxidants, were detected in patient's RBC (GSH: 4356±44 mM; G6PD: 10.3±0.6 IU/g Hb) compared to controls (GSH: 2434±454 mM; G6PD: 5.4-7.0 IU/g Hb) and patient's mother (GSH: 2125 mM; G6PD: 5.3±0.2 IU/g Hb) and sister (GSH: 2450±35 mM; G6PD: 6.5±0.1 IU/g Hb). Concomitant increase (approximately 2.5 times) in the activity of hexokinase (HK) and pyruvate kinase (PK) and in the levels of ATP in patient's RBC indicates augmented energy metabolism to sustain the integrity of RBC. Flow cytometry of Annexin V binding revealed increased exposure of phosphatidylserines on RBC membrane of the patient compared to controls and her mother and sister suggesting that the tendency towards redox balance restoration and membrane integrity does not completely prevent an enhanced recognition and destruction of patient's RBC by reticuloendothelial macrophages. The second c.281T>G point mutation in RPL11 was found in a 34-year-old woman with unspecified macrocytic anemia, who developed diffuse large B-cell lymphoma (DLBCL). She was transfusion dependent in infancy, responded to steroids and is currently in the anemia remission and second remission of DLBCL. Identical mutation was detected in her 56-year-old mother, followed for macrocytic anemia and gammopathy. In 10 DBA cases without the causative mutation an array CGH was performed, but no large deletions in RPS19, RPS17, RPS26, RPL5 and RPL11 genes were detected. In summary, we present the first case of a missense mutation in RPS7 as the cause of DBA; all to date published RPS7 mutations clinically associated with DBA were located in splice sites. Similarly, the co-occurrence of DBA and DLBCL has not been previously published. We also emphasize that the phenotype of family members with the same mutations could be different, including silent carriers. All family members should therefore be examined even if asymptomatic. Testing of eADA levels could serve as a sensitive screening method in apparently unaffected family members. Finally we suppose that altered RBC metabolism may affect the lifespan of RBC in DBA and thus contribute to the worsening of the blood condition, especially during infections. The analyses in a large cohort of DBA patients are ongoing. Grant Support: AZV16-32105A, GA15-13732S, MH CZ-DRO FNOL 00098892, NPU LO 1304, IGA UP LF_2016_014. Disclosures No relevant conflicts of interest to declare.

Abstract 4905 Modern molecular diagnostic techniques allow the detection of minimal residual disease (MRD) in hematological malignancies with high sensitivity, allowing patient-specific assays of MRD levels over the course of treatment. One challenge for these techniques is finding appropriate MRD marker targets. For instance, PCR-based MRD analysis requires unique sequence-specific information for the malignant clone of interest. In many acute leukemias, such detailed information is lacking, either because no abnormality is detected (e.g. in 40–50% of adult Acute myeloid leukemia-AML), or the resolution of cytogenetic methods is too low to precisely define newly-found abnormalities. We have used multicolor-FISH (mFISH) to perform karyotyping of 141 patients. Using this approach we have identified chromosomal abnormalities in about 35% of analyzed cases. The aberrations found involved the recurrent aberrations t(15;17), t(8;21), inv(16), t(9;22),−5q, −7, +8 but also an array of unique abnormalities, such as der(15)t(5;15)(q15;q24), t(6;12)(p22;q13), and der(17)t(12;17)(q14;pter), which may serve as possible targets for molecular MRD follow-up. To further characterize such targets, we seek to bridge the gap from cytogenetic resolution down to higher-resolution molecular techniques. For this, we first employ the mBAND technique, followed by multicolor FISH using DNA probes with known cytogenetic location. These probes are drawn from the Human Minimal Genomic Clone Set (Version 1.0, Source Bioscience LifeSciences) of over 25,000 bacterial artificial chromosome (BAC) clones with human DNA inserts. These are tiling clones covering almost 100% of the genome, and therefore allow us to develop FISH probes to regions containing almost any conceivable target breakpoint. Starting with breakpoint information from mFISH/mBAND, we perform consecutive multi-color/multi-BAC hybridizations around the region of interest, looking for breakpoint-spanning probes. Using this system, we are able to move from a resolution of around 2 Mbp (the limit of mBAND) to the resolution of individual BAC clones (average 150 kbp) in three hybridizations. To achieve further resolution, long-range PCR products are designed within the region defined by breakpoint-spanning BACs, with labeling and FISH-mapping of these probes. At present, we are starting to perform these hybridizations on stretched DNA (fiber-FISH or combed DNA), where one micrometer of a stretched DNA molecule represents about 2000 base pairs, allowing visualization of probe distance and spacing, and even higher-resolution breakpoint analysis. Chromosome microdissection followed by high-throughput sequencing (Roche GS Junior) is also being pursued to identify novel breakpoints by rapidly sequencing large chromosomal regions on both sides of a translocation. The final goal is to map genomic abnormalities to a resolution amenable to long-range PCR, yielding specific targets for MRD detection, and allowing clone-specific Real-Time PCR assays for sensitive and specific monitoring of MRD in hematooncological patients. Disclosures: Smolej: GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants; Roche: Honoraria, Travel Grants; Genzyme: Honoraria, Travel Grants.

Abstract 3802 Poster Board III-738 Introduction Monosomy 7 or del(7q) are frequent cytogenetic abnormalities in children with myelodysplastic syndrome (MDS) and associates with poor prognosis. MDS globally affects all cellular subsets in bone marrow and in peripheral blood. We asked whether flow cytometry (FC) can separate individual subtypes of MDS from each other and from aplastic anemia (SAA) and whether in individual subtypes of childhood MDS can separate patients with and without monosomy 7. Patients/analyzed parameters In total we analyzed 94 children with centrally analyzed immunophenotype in the reference lab who were diagnosed and treated for MDS or SAA between 1998 and 2009. In total we analyzed 14 patients with refractory cytopenia, 37 patients with advanced forms of MDS (JMML 10, RAEB 25, CMML 2) and 43 patients with SAA. Monosomy 7/del(7q) was present in 17 patients (RC 6, JMML 3, RAEB 8). Analyzed parameters were as follows: B cells, CD10+CD19+, CD19+45dim/neg, CD19+34+, CD19/CD34 ratio, CD34+, CD117 cells, CD34+38dim/neg, CD3+, CD3+4+, CD3+8+, CD3+HLADR+. Statistics We analyzed all parameters using non parametric tests (Mann-Whitney, Kruskal Wallis) and principal component analysis (PCA). Results Principal component analysis of all analyzed patients together clearly separates advanced forms of MDS from RC and SAA, the most contributing factor being the number of CD34 and CD117+ cells. In non parametric statistics following factors significantly differ among MDS subtypes and SAA (Kruskal-Wallis): CD19, CD117, CD34, CD3, CD3+4+, CD8+ and CD3+HLADR+. RC and SAA patients are separated mainly by the number of B cells and the CD34:CD19 ratio. In addition, the following parameters differ between RC and SAA (Mann-Whitney): CD34, CD117 and CD3+HLADR+. Unlike the CD34:CD19 ratio, the number of CD19+34+ precursors does not differ between RC and SAA patients. Patients with monosomy 7 do not differ from the remaining patients when all MDS patients are analyzed together or separately in the respective subgroups (RC, non RC, JMML) by PCA or by non parametric statistics. Conclusion PCA separates advanced MDS forms from RC and SAA. Advanced forms of MDS are characterized by increased percentage of CD34+ and CD117+ cells compared to RC and SAA patients. The global reduction of B cell progenitor compartment is pronounced especially in non-JMML cases of MDS, whereas SAA patients typically present with isolated reduction of cells at early stages (CD19+34+) of B cell development. Patients with monosomy 7 cluster within the respective disease category, they do not form own cluster in PCA. Supported by MSMT VZ MSM0021620813, MZO 00064203 VZ FNM, MZO VFN2005, IGA NR/9531-3, NPV 2B06064. Disclosures: No relevant conflicts of interest to declare.

Measurement of imatinib plasma levels is recommended for CML patients on Glivec therapy with suboptimal response or failure. Recently, reports suggesting that imatinib plasma levels correlate both with cytogenetic and molecular response of the patient and may be used for routine management of the treatment were published. We have introduced the examination of imatinib plasma levels into our laboratory follow-up of CML patients in 2007. All patients have signed informed consent before sampling. New capillary electrophoretic method was developed for determination of imatinib plasma levels. Plasma samples were deproteinated by methanol, evaporated, resuspended and directly analyzed. Separation was performed in buffer consisting of 50 mmol/L citrate adjusted with γ-amino-n-butyric acid to pH 4.0. Imatinib was quantified by UV detection in the range 250 – 270 nm (limit of detection is 10 nmol/L). BCR-ABL transcript levels were monitored by TaqMan-based real-time quantitative PCR, the baseline value of BCR-ABL/ABL% was 60%. Here we report comparison of plasma drug levels in CML patients with optimal and suboptimal molecular response. Total 238 samples from 100 CML patients were examined. Patients with major and complete molecular response (n = 68) and patients with suboptimal molecular response (n = 35) were chosen for the final comparison. The actual sample was eligible for analysis providing patient had been treated with standard dose 400 mg daily for at least one year, there was no other evident cause of the suboptimal response (presence of additional cytogenetic abnormalities or BCR/ABL mutation) and the sample was taken 24±6 hours after the ingestion of the drug. Other medication, the actual duration of the therapy (providing it was longer than one year), time interval between the dose and the meal ingestion, weight, body-mass index, body surface area and co-morbidity of patients were not taken into the account. All patients in the suboptimal group have achieved the complete cytogenetic response. Three patients in the suboptimal group have progressed, while there were any such patients in the optimal group. The results showed considerable inter-individual variability between the patients. However, the imatinib plasma levels found in the optimal group significantly higher (median of 2.34 umol/l; range of 0.46 – 6.23) than in the suboptimal group (median of 1.75 umol/l; range of 0.84 – 4.63) p < 0.003. Moreover, patients with negativity in real-time quantitative PCR testing showed a trend for even higher drug plasma levels (median of 3.18 umol/l; range of 0.46 – 6.23, n = 36) p < 0.001. We can conclude that higher imatinib plasma levels are associated with better molecular response to therapy and laboratory measurements may be used for modification of the imatinib dosage.

Activation of Src family of kinases (SFK) is associated with chronic myeloid leukemia (CML) disease progression and resistance to imatinib (IM) therapy. Activation of SFK can be either Bcr-Abl-dependent or Bcr-Abl-independent in IM-resistant CML patients and clinical importance of this phenomenon is not completely understood. Second generation of tyrosine kinase inhibitors (TKIs) such as dasatinib, targeting both Bcr-Abl kinase as well as SFK, could represent a logical alternative for treatment of patients with acquired IM resistance due to SFK activation. Recent studies raised question whether overexpression of SFK could lead to an acquired resistance to dasatinib. Sensitivity of individual patients’ leukemia cells to TKIs can be carried out by assessment of inhibition of phosphorylation of Crkl and SFK after incubation of patient’s leukocytes with the drug in vitro. We used 10 μM IM or 250 nM dasatinib in vitro and detection with western blot analysis with anti-Crkl 32H4 monoclonal antibody (Cell SignalingTechnology, Beverly, MA) and Phospho-Src Family (Tyr416) Antibody (Cell Signaling Technology Inc., Danvers, MA). By this functional approach we identified several IM-resistant CML cases, where IM failed to inhibit phospho-Crkl and phospho-SFK in vitro; these patients were either in blast crisis or carried a BCR-ABL mutation. In most cases, however, phospho-SFK were inhibited after incubation of the patients’ leukocytes with dasatinib. The exception was a patient with hematological resistance to IM and acquired resistance to dasatinib therapy due to Bcr-Abl-independent activation of SFK. This patient was a 56-year-old female diagnosed with CML in chronic phase. The cytogenetic examination at the time of diagnosis revealed Ph chromosome and an additional aberration involving derivative chromosomes 2, 3 and 5. After 10 months of IM therapy the BCR-ABL-positive clone was eradicated, but the clinical response to the treatment was unsatisfactory. At the same time the number of cells with the complex additional aberration remained high (>70%). Western blot analysis of the patient’s leukocytes revealed overexpression of phospho-SFK suggesting activation of an alternative signaling pathway not inhibited by IM and independent of Bcr-Abl. In vitro sensitivity to dasatinib provided a rationale for switch to dasatinib therapy. Therefore, dasatinib was administered to the patient (2×70 mg daily). However, a rapid development of resistance to dasatinib followed, which corresponded with a loss of inhibition of SFK in in vitro phosphorylation assay. Interestingly, the phenotype of the patient’s disease resembles a mouse model of v-src gene induced myeloproliferative disease, characterized by splenomegaly, anemia, and increased numbers of immature erythroid cells (Keller G and Wagner EF, Genes Dev.1989; 3: 827–37). Our data suggest that the oncogene driving myeloproliferative disease in this patient belongs to SFK. We demonstrate that the detection of activated SFK by immunoblot could lead to recognition of Bcr-Abl-independent and SFK-dependent resistance to dasatinib.

The occurrence of significant hematologic toxicity in patients with CML during the treatment with standard continuous dosing of imatinib (IM) is associated with inferior prognosis. In order to avoid prolonged interruptions of therapy in such patients and, possibly, to prevent induction of resistance we adopted a strategy of intermittent treatment (I-T): the daily dose of 300–600 mg of IM was given once to five times weekly. Here we report the experience with I-T in 10 patients (7 women and 3 men) aged 25 to 74 (me=49,3) years treated with I-T for median of 19,5 months (range: 4–26). Patients have 4–152 (me=23,5) months long history of CML at the initiation of IM therapy that was indicated for accelerated phase (in 4 patients), cytogenetic (3) or hematologic (2) resistance or toxicity after interferon (1). 7 of them were previously treated with interferon, 2 with hydroxyurea and one with busulfan only. Two patients have the history of autologous peripheral stem cell transplantation. Additional cytogenetic aberration (+8) was present before IM therapy in one patient and in other two cases interstitial deletions of 9q were identified. 5 patients had high and 5 intermediate Sokal score, while the value of Euro score was high in each case. All patients received initially the standard treatment (300–600mg daily) with IM for 1–6,5 (m=2,5) months resulting in hematologic remission with various degree of myelosupression in each of them. Hematologic toxicity was the reason for the discontinuing of therapy in 7 patients for 0,5–4 months. One patient was treated before introduction of I-T with daily dose of 100–150mg of IM for 11 months. In 7 cases complete hematologic remission was maintained by I-T and in one of them steady regeneration in blood counts allowed reintroduction of full dose standard therapy. Hematologic progression occurred in two patients. Both of them were successfully switched to full daily dosing of IM. Two patients improved their cytogenetic response during I-T. In four cases major cytogenetic remission achieved with standard dosing was maintained. Using interphase FISH the level of cytogenetic response was below 10% in 5 patients at the latest examination. In one patient the development of +8 in BCR/ABL negative cells was observed. In some cases in vitro tests for evaluating the sensitivity to IM and screening for BCR/ABL tyrosine-kinase mutation were performed. We conclude that the I-T with IM might serve as a reasonable and cost-effective alternative in heavily pre-treated or in high-risk patients with CML who could not be offered allogeneic stem cell transplantation or experimental therapy. Using this strategy, major cytogenetic remissions was achieved in two and maintained in other four patients. I-T allows safe tailoring the actual dose of IM in order to avoid excessive hematologic toxicity. Our observations need to be validated in larger cohort of patients during longer follow-up.

While deletions of the long arm of chromosome 6 (6q) are common in patients with ALL, translocations are rare, most are considered sporadic and none have been cloned. Among myeloid malignancies, the recurrent translocations: t(6;11)(q27;q23), t(6;11)(q21;q23) and t(6;8)(q27;p11) give rise to known fusion oncogenes. Other uncharacterised balanced or unbalanced 6q abnormalities have occasionally been reported. Using fluorescence in-situ hybridisation (FISH) with HGMP mapped PAC and BAC clones, we investigated chromosomal rearrangements of 6q in acute leukemia. Previously we reported 24 cases with deletions of 6q (Sinclair et al 2004, Can Res 64; 4089), here we describe the analysis of 11 patients with novel balanced rearrangements. Six of seven breakpoints in ALL and two in a single case of AML were localised to a 10.5 Mb ‘hotspot’ within 6q22–q23. Of these, five were analysed down to the level of a single clone. In two cases of childhood T ALL, both carrying a t(6;7)(q23;q31~36), split FISH signals were produced by adjacent PACs, RP1–32B1 and RP3–388E23, mapping the breakpoints to within a ~150Kb region. A search of published karyotypes identified five similar rearrangements, four also in pediatric T ALL, all present as a sole abnormality or represented in the major clone. Of genes in the region, two (c-MYB and AHI1) are tightly regulated during normal hemopoietic development and over-expression of both has been reported to occur in hemopoietic malignancies. Moreover, clusters of retroviral insertion sites occur within both genes and immediately proximal to c-MYB in murine and feline leukemias and lymphomas. Thus we have defined a new primary recurrent translocation in T ALL that is likely to target either c-MYB and/or AHI1.Three other non recurrent rearrangements of 6q22–q23, mapped in detail, interrupted regions without known genes.. Therefore, these abnormalities were unlikely to have promoted leukemia through classical oncogene activation, but may have influenced expression of one or more common target genes through a long-range position effect. Alternatively these clustered rearrangements may mark the position of a new fragile site on 6q. Additional 6q breakpoints in three cases of AML and one of ALL mapped to 6q15, 6q21, 6q25 and 6q26–q27, and were coincident with the positions of a known oncogene partner AF6q21, the haemopoietic transcription factor BACH2 and two cloned fragile sites FRA6E and FRA6F. Putative partner genes of four translocations were investigated by FISH and the involvement of MLL was demonstrated in one case with t(6;11)(q15;q23).