ALBERT

Description: Abstract 1104 Poster Board I-126 Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL), and is difficult to predict from information at diagnosis in the majority of cases. To explore the prognostic impact of recurrent copy number abnormalities on relapse in children diagnosed with precursor-B cell ALL, we performed genome-wide copy number profiling of 34 paired diagnosis-relapse samples. Lesions detected at diagnosis were often absent at relapse, including recurrent targets in precursor-B ALL like PAX5 (not preserved in 2 out of 7 cases with deletions at diagnosis), CDKN2A (not preserved in 1 out of 15 cases), and EBF (not preserved in 2 out of 5 cases), which illustrates that these lesions are often secondary events that are not present in the therapy-resistant progenitor that causes relapse. In contrast, deletions and nonsense mutations in IKZF1, which encodes the lymphoid differentiation factor IKAROS, were highly frequent (38%) and always preserved at time of relapse. Locus-specific copy number screening of IKZF1 in an additional cohort of diagnosis samples from children enrolled in the Dutch treatment protocol DCOG-ALL9 with (n=40) or without (n=51) relapse revealed that IKZF1 deletions were significantly enriched in relapse-prone cases (22.5% vs 3.9%; P=0.007). An independent and unbiased validation cohort of 150 DCOG-ALL9 cases was used to confirm these findings, which established that 28.6% of the cases with IKZF1 deletion at diagnosis developed a relapse. Together, we conclude that deletions of IKZF1 in DCOG-ALL9 treated pediatric precursor-B ALL patients provide a strong prognostic marker for relapse. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Several studies have shown the role of the immune system in the development of MM, but there is no systematic description of normal B-cell regeneration during treatment points. Recently, EuroFlow consortium has developed NGF panel with high sensitivity to evaluated MRD and potentially, to assess of the normal B-cell compartment of patients with MM. Aims: Here we evaluated the B cell regeneration pattern of bone marrow (BM) from patients with MM by Next Generation Flow (NGF) cytometry at diagnosis and at minimal residual disease (MRD) time points of treatment. Material and Methods: Overall 190 samples of MM patients (45% female and 55% male, median age of 65 years - 37 to 87 years, of which: 16 at diagnosis, 30 post-induction, 76 D+100, 59 maintenance/consolidation and 9 out of treatment, and 8 samples of BM healthy donor - HD (≥ 50 years). At the moment treatment, D+100 were included samples from two research centers: HCS/USAL (n=64) and HUCFF/UFRJ (n=12). Induction regimens were composed of triple protocols (PI + IMIDs + Steroids); followed by high doses of melphalan with ASCT, while patients in maintenance/consolidation followed second line treatment regimens - IP+IMIDs and/or PIs+Steroids and/or monoclonal antibodies or INF or IPs. All samples of BM were subjected to bulk cell lysis with ammonium chloride solution for 〉107 cell acquisition and labeled with a combination of 10 antibodies - Panel EuroFlow MM MRD.Results: Of the 174 post treatment samples, 36% presented MRD- and 64% MRD+. At diagnosis, patients exhibit a significant reduction of precursors B cells and normal plasma cells (nPC) relative to HD, probably a reflection of the occupation of their binding sites by cPC. At post-induction, there was an increase in precursors B, compared to MM patients at diagnosis, associated with a reduction of mature B-cell (transitional/naïve and memory), regardless of MRD status. Concerning the nPC compartment, a reduction was observed, relative to HD. During treatment, reduction of the tumor burden leaves these sites free for precursors B, which rapidly recover while leads to a drastic decrease in mature B cells and regeneration of the precursors to mature B-cells is slower. On the other hand, in D+100, independent of the MRD status, there was a post treatment medullary recovery, with an increase in B precursors and transitional/naïve B-cells, in contrast, with a reduction of memory B-cells. Out of treatment, we observed a recovery of precursors B, mature B-cells and increase of nPC, but the immune recovery of these nPC is not sufficient to reach the levels of a healthy individual. Conclusion: NGF emerges as an optimal approach for simultaneous assessment of the BM regeneration profile of B-cells and the MRD status. After starting therapy, MM patients re-establish the compartments of B-cell precursors and transitional/naïve B-lymphocytes; however, memory B cells and nPC do not recovery until the end of treatment. This study is a starting point for exploring the importance of the B cells of the medullary microenvironment in the MM. Its potential impact on patient outcome deserving further investigations Disclosures Puig: Takeda: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Mateos:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: BACKGROUND The CLL11 trial evaluated front line treatment with Clb vs. R-Clb vs. G-Clb in 781 physically unfit patients. Genomic aberrations, IGHV and TP53 mutation status are established prognostic factors in CLL, while NOTCH1, SF3B1, ATM and othersare recurrently mutated genes of potential prognostic and/or predictive value. METHODS We performed amplicon-based targeted next generation sequencing of all coding exons (TP53, ATM, MYD88, FBXW7, BIRC3, XPO1, POT1) or hotspots (NOTCH1 exons 33/34, SF3B1 exons 14-16/18) via Illumina TSCA™ in a representative subset of 689 (88.2%) patients of the CLL11 trial at baseline. Mean read depth was 1363x (range 636-1879) and a variant allele frequency 〉5% was determined as mutation. RESULTS Incidences of gene mutations were: ATM 19.6%, NOTCH1 18.6%, SF3B1 12.9%, TP53 10.7%, POT1 7.3%, XPO1 6.8%, BIRC3 5.2%, FBXW7 3.8%, and MYD88 3.3%. We found several significant associations with clinical and genetic baseline characteristics including most prominently IGHVunmut with ATMmut (p=0.013), NOTCH1mut (p

Description: Background Juvenile myelomonocytic leukemia (JMML) is a lethal myeloproliferative disease (MPD) of young childhood characterized by an overproduction of myelomonocytic cells and an increased in vitro sensitivity of hematopoietic progenitors to granulocyte-macrophage colony-stimulating factor [GM-CSF] (Emanuel PD et al, Blood 1991). Diagnostic criteria for JMML are currently well-established and based on clinical features and laboratory findings. However, in some patients diagnosis of JMML vs other overlapping disease entities, still remains a challenge, immunophenotyping being not part of the diagnostic work-up of JMML. Here, we aimed at detailed characterization of the CD34+ cell compartment in JMML bone marrow (BM) using the standardized EuroFlow myeloid panel in combination with innovative EuroFlow software maturation tools. Our major goal was to determine the potential utility of immunophenotyping of CD34 cells in the diagnostic work-up of JMML. Methods Overall, we analyzed BM cells from 10 JMML patients at diagnosis (age range: 0-7 years), 17 control subjects (age range: 0-15 years) and 5 patients (age range: 0-5 years) with a suspected diagnosis of JMML that was subsequently not confirmed following standardized EuroFlow antibody combinations: 1) cyCD3/ CD45/ cyMPO/ cyCD79a/ CD34/ CD19 / CD7/smCD3 (for early lineage assignement); 2) HLADR/CD45/CD16/CD13/CD34/CD117/CD11b/CD10 (neutrophilic maturation); 3) HLADR/CD45/CD35/CD64/CD34/CD117/CD300e (IREM2)/CD14 (monocytic maturation); 4) HLADR/CD45/CD36/CD105/CD34/CD117/CD33/CD71 (erythroid vs plasmacytoid dendritic cell maturation). Samples were processed and analyzed according to the Euroflow standard operating protocols (van Dongen JJM et al, Leukemia 2012, Kalina T et al, Leukemia 2012). Data analysis was specifically focused on the immunophenotypic profile of CD34+ gated cells. Results Within the CD34+ BM cell compartment the proportion (mean % ± 1SD) of granulocytic and monocytic precursors were not significantly different in JMML as compared to controls: 33% ± 15% vs 25% ± 12% (p = 0.16) and 14% ± 6.3% vs 12% ± 7.1% (p = 0.68) respectively. Otherwise we observed a slightly decreased in erythroid CD34+ progenitors in JMML vs controls (1.0% ± 1.2% vs 2.8% ± 1.7%, p

Description: Relapse is the most common cause of treatment failure in childhood acute lymphoblastic leukemia (ALL), and is difficult to predict in the majority of cases. Here, we performed genome-wide copy number profiling of 34 paired diagnosis-relapse samples from children diagnosed with precursor-B cell ALL. The majority of the copy number abnormalities were preserved between matched diagnosis and relapse samples, but lesions unique in either of the two samples were observed in 82% of the cases. In 68% of the cases lesions present at diagnosis were no longer detected in relapse samples (including recurrent lesions affecting the PAX5, CDKN2A, and EBF genes), indicating that these lesions were secondary events, absent in the original therapy-resistant progenitor clone. Deletions in the IKZF1 gene encoding the hematopoietic differentiation factor Ikaros were observed in 38% of the diagnosis samples, which is 〉6-fold the amount detected in an unselected group of pediatric ALLs. Tiling-resolution oligo arrays were used to map the breakpoints, which demonstrated that the protein-coding exons 3–6, encoding the DNA-binding Zn-finger domains, were most commonly deleted. Sequence analysis revealed that point mutations in IKZF1 do occur but are less frequent. Furthermore, IKZF1 deletions were always preserved in relapse. Together, we conclude that IKZF1 deletions are frequent events in therapy-resistant clones of relapse-prone pediatric precursor B-ALL.

Description: BACKGROUND Cytotoxic treatment in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients induces a dramatic decrease in B-cell precursor (BCP) and mature B-cell numbers, followed by regeneration of BCPs in the bone marrow (BM) and subsequent replenishment of mature B-cells in the peripheral blood (PB) in between treatment blocks and after stop of treatment. To understand the degree of B-cell recovery after such dramatic changes, we first evaluated the composition of the B-cell population in the BM and PB of pediatric BCP-ALL patients during and after therapy. Secondly, we investigated whether the immunophenotypic maturation of BCPs in regenerating BM is similar to normal BCP development or whether such regeneration induces immunophenotypic aberrancies, which could potentially hamper minimal residual disease (MRD) detection. Finally, we assessed whether compensatory proliferation plays a role during B-cell regeneration, since enhanced proliferation might limit the B-cell receptor diversity and consequently may affect susceptibility to infections during and after therapy. METHODS For immunophenotypic characterization of different B-cell subsets, 8-color flow cytometry was performed on fresh BM and PB samples at different time points after start of therapy (DCOG ALL11-protocol). To study BCP maturation, a multidimensional maturation pathway based on 5 backbone markers was designed and the expression pattern of several differentiation markers during this maturation pathway was evaluated. To assess proliferation in BCP subsets, BM samples were stained with subset-specific antibodies and DRAQ5 for cell cycle analysis. The proliferation history of sorted pre-B-II-small and immature subsets in BM and sorted mature B-cell subsets in PB was assessed by performing a kappa-deleting recombination excision circle (KREC)-assay. RESULTS BCP regeneration occurred mainly at day 78, month 5 and after stop of therapy. The BCP compartment in regenerating BM at time points during therapy showed a shift towards the most immature stages. In PB, mature B-cell numbers decreased after start of therapy and newly generated mature B-cells subsets reappeared at month 5 and after stop of therapy. Importantly, the BCP maturation pathway with its expression patterns of CD10, CD34, CD58, CD66c, CD38, CD123, CD9, CD81, CD24, TdT, Igκ and Igλ was comparable between regenerating BM and BM of healthy individuals, albeit that a shift in the relative BCP subset distribution was observed in regenerating BM. As expected, most proliferation in BM of healthy controls occurred in the pre-B-II-large subset (68% ±11% (mean ±SD) proliferating cells). Comparable percentages of proliferating pre-B-II-large cells were found in regenerating BM: 74%±10% at day 78, 72%±10% at month 5 and 63% (preliminary data, n=1) at one year after stop of therapy (month 36). Also pre-B-I cells showed some proliferation, with no significant differences between normal and regenerating BM (Figure 1). If present, the pre-B-II-small and immature BCP subsets showed no proliferation in regenerating and normal BM. KREC-analysis of sorted pre-B-II-small and immature subsets confirmed that no cell divisions had occurred after IGK-rearrangement in normal BM as well as regenerating BM at month 5 and month 36. Low numbers of pre-B-II-small and immature cells precluded KREC-analysis at day 78. KREC-analysis of the various mature B-cell subsets in PB showed no significant difference in proliferation history between PB of patients at different time points during or after therapy and PB of healthy controls. CONCLUSIONS In BCP-ALL patients, the B-cell compartment is drastically affected during treatment. Subsequent regeneration of BCPs and mature B-cells occurs at different time points during therapy and after stop of therapy. Immunophenotypically, BCP maturation in regenerating BM is similar to normal B-cell differentiation, indicating that MRD detection will not be hampered by aberrant immunophenotypes of regenerating BCPs. Importantly, no enhanced proliferation is observed in BCP subsets in BM and mature B-cells subsets in PB of patients during and after therapy. The lack of compensatory proliferation suggests that B-cell regeneration is due to a larger influx of non-committed stem cells into the B-cell lineage and indicates that a diverse immune repertoire will most likely be restored during recovery of the B-cell compartment. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: The tetraspan molecule CD81 is widely expressed on immune cells, such as B-, T-, NK-lymphocytes, monocytes and eosinophils, but also on most stromal and epithelial cells and on hepatocytes. In B-cells it is a member of the CD19 complex (CD19, CD21, CD81, CD225), which is required for signaling together with the B-cell antigen receptor upon antigen recognition. Its functions on other cells are unclear, but murine studies show an antiproliferative role for CD81. On hepatocytes, two different epitopes of CD81 act as a co-receptor for Hepatitis C virus and for Plasmodium infection. We evaluated a 4-year-old girl from consanguineous parents of Moroccan decent. She presented with recurrent infections and an acute nephrotic syndrome: 〉50% of glomeruli were affected due to focal mesangial hypercellularity. She showed poor weight gain (below 3rd percentile), but normal motor development. Her spleen and liver were enlarged, but function normally. Measurements of retina epithelium and CNS showed no signs of hypercellularity. Although serum IgG levels were strongly decreased (2.4 g/L) and IgM and IgA concentrations low within normal range, she tested positive for anti-platelet antibodies. Flow cytometric immunophenotyping of blood showed normal distribution and absolute numbers of granulocyte, monocyte and lymphocyte subsets; however, no CD19 expression was detected on the patient’s B-cells, whereas CD21 expression levels were normal. The patient carried no mutations in the CD19 and IFITM1 (CD225) genes. Additional immunophenotyping showed that all cells lacked CD81 expression. Sequencing of the CD81 gene showed a homozygous G〉A substitution immediately downstream of exon 6 (c.561+1G〉A). Spectratyping and quantitative PCR analysis showed clearly reduced total CD81 mRNA expression levels. Nearly all CD81 transcripts contained 13 additional nucleotides downstream of exon 6. This insertion results in a frame-shift and a premature stop (p.Glu188MetfsX13). The hypothetical protein lacks the fourth transmembrane domain. Similar to previously described CD19-deficient patients, our patient had reduced numbers CD5+ B-cells and Ig class switched and non-switched CD27+ memory-B cells. Whereas Vh-Cα and Vh-Cγ transcripts from Ig switched cells contained somatic hypermutations, the response of the patient’s B cells to in vitro stimulation through the B-cell receptor was impaired. The antibody response to rabies, tetanus and pneumococcal vaccinations is currently under investigation, as well as the potential impact of CD81 deficiency on the antigen-specific Th1 and Th2 cytokine production. In conclusion, the here presented CD81 deficiency is a new primary immunodeficiency, which leads to disruption of the CD19 complex and consequent hypogammaglobulinemia comparable to the CD19 deficiency. However, due to the broad tissue distribution, the clinical phenotype is not restricted to the B-cell system. Other organs are affected as well, most likely due to excessive proliferation and hypercellularity, with acute nephritic syndrome as dominant clinical problem. Currently, in vitro studies are being performed to identify whether the CD81 defect directly results in impaired B-cell and T-cell functions and abnormal proliferation of kidney and liver cells.

Description: The BCR-ABL fusion gene results from the translocation t(9;22). It is the hallmark of chronic myeloid leukemia (CML) and is present in a poor-risk subgroup of precursor B cell acute lymphoblastic leukemia (ALL), which represents 25–30% of adult ALL and 3–5% of childhood ALL. Consequently the detection of the BCR-ABL aberration is of utmost importance for diagnosis and classification of leukemia patients and can also be used as marker for monitoring of BCR-ABL+ leukemias to evaluate treatment effectiveness. So far, the BCR-ABL aberration has been detected by cytogenetics, FISH or PCR, all techniques that are time consuming and require special facilities. We developed a simple flow cytometric bead assay for detection of the BCR-ABL fusion protein in cell lysates, using a bead-bound catching antibody against one side of the fusion protein and a fluorochrome-conjugated detection antibody against the other side of the fusion protein. The anti-BCR antibody was developed against a non-homologous region of ~80 amino acids, encoded by exon 1 in order to detect all know BCR-ABL variants, including p190, p210, and p230. The assay appeared to be specific and sufficiently sensitive to detect BCR-ABL proteins in leukemic cell lysates. However, protein stability problems were encountered when cell samples contained high frequencies of mature myeloid cells with high levels of protease activity, such as CML cells and granulocyte fractions. This problem could be significantly reduced by adding protease inhibitors to several steps of the immunobead assay. The immunobead assay was further developed and standardized by BD Biosciences into the Cytometric Bead Array (CBA) assay (BCR-ABL Protein Kit; BD Biosciences, San Jose, CA) for Research Use Only. Large scale testing of this kit in 9 diagnostic laboratories of the EuroFlow Consortium showed that the results of the flow cytometric BCR-ABL immunobead assay were fully concordant with the PCR results in a series of 83 freshly collected samples from newly diagnosed (n=75) or relapsed (n=8) leukemia patients: 13 CML patients, 53 ALL patients, and 17 acute myeloid leukemia (AML) patients (see table). All blood samples tested from 61 healthy controls were negative in the immunobead assay. BCR-ABL PCR assay BCR-ABL immunobead assay negative p190 p210 negative* weak* medium* high* * negative: MFI value